Anti-ENTPD3 chimeric antigen receptor

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 ENTPD3 are provided.

Description

Technical Field

[0001] This disclosure generally relates 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 ENTPD3, which are expressed in immune cells (e.g., Tregs). Such immune cells have therapeutic use in diseases and conditions associated with cells that express ENTPD3 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, such as immune cells, to express the CAR. Background Technology

[0002] Immunotherapy is becoming an effective treatment for a variety of diseases, with applications covering cancer, autoimmune diseases, inflammatory diseases, and even the prevention of rejection in solid organ transplants. In particular, clinical research activity in the field of adoptive cell immunotherapy (ACT) is increasing, especially in the areas of autoimmune and inflammatory diseases, with research on regulatory T-cell (Treg) therapy being particularly prominent.

[0003] CD4 + FOXP3 + Regulatory T cells (Tregs) are a subset of lymphocytes that play a crucial role in maintaining dominant immune tolerance by suppressing the function of various effector immune cell subsets, such as T effector cells. Additionally, Tregs are known to promote tissue repair and regeneration. Tregs can confer immune tolerance through a variety of contact-dependent and contact-dependent mechanisms. These mechanisms include: producing anti-inflammatory soluble mediators such as IL-10, TGF-β, and IL-35; consuming IL-2; expressing negative regulatory cell surface receptors such as CTLA-4; and targeting T cells directly or indirectly via APCs. Importantly, once activated, Tregs can suppress immune responses in a non-antigen-specific manner (bystander suppression), meaning that once activated, Tregs can modulate the local immune microenvironment and suppress inflammation. Furthermore, Tregs can confer a suppressive phenotype to other cells of the immune system, a process known as "infectious 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 process is triggered by both genetic and environmental factors. The destruction of beta cells reduces or stops the body's insulin production and leads to inflammation of the pancreas. Insulin is the hormone needed to regulate glucose levels in the blood; before treatment, people with type 1 diabetes have excessively high blood sugar levels (hyperglycemia). Type 1 diabetes is a serious, lifelong condition. Currently, people with type 1 diabetes need to closely monitor their blood sugar levels and (e.g., through injections or pumps) take appropriate amounts of insulin. However, this treatment is not a cure and must be continued. In the long term, abnormal blood sugar levels, including large fluctuations, can lead to long-term complications such as damage to the heart, eyes, feet, and kidneys, and can also shorten life expectancy. Therefore, type 1 diabetes places a heavy burden on global healthcare systems. Furthermore, epidemiological data shows that the prevalence of autoimmune diseases such as type 1 diabetes has been steadily increasing in Western societies over the past few decades. In Western Europe and North America, the prevalence of type 1 diabetes is approximately 0.5%, with about 2 million people affected, and this number continues to rise.

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

[0006] The prospect of improving immunopathological states and restoring immune tolerance in inflammatory diseases has fueled increasing interest in the clinical development of Treg-based immunotherapies. However, for Treg immunotherapy to be successful, it is highly beneficial to develop strategies that can promote the migration of Tregs to sites of tissue damage and induce their in situ activation.

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

[0008] Tregs from transgenic mice expressing islet antigen-specific TCRs 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 restrictions, this transgenic model cannot be applied to patients because different patients 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 targeting the target antigen), a transmembrane domain, and an intracellular signaling domain. The intracellular signaling domain signals the cell to activate it upon binding to the extracellular antigen-binding domain. CAR-T therapy has been approved for the treatment of certain blood cancers. However, CARs are artificial molecules engineered and introduced into cells, and their sensitivity is lower than that of TCRs, partly because the TCR mechanism involves a greater number of molecules, namely the CD4 / CD8 co-receptor, the tyrosine-rich immune receptor activating motif (ITAM), and subunits within the receptor complex. For activation, CARs require 100 to 10,000 CAR molecules per target cell, while TCRs require only <10 molecules per target cell. Therefore, selecting suitable targets for CAR-T cell therapy is challenging; the target must be able to activate CAR-T cells to the appropriate level required to produce a therapeutic effect. For example, although insulin-specific CAR-Tregs can proliferate under insulin stimulation and have inhibitory effects in vitro, they cannot prevent the development of 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 autoimmune or inflammatory diseases can be developed by providing chimeric antigen receptors (CARs) containing an antigen recognition domain specific to ENTPD3 to a subset of immune cells. Given the bystander effect of Treg cells and their ability to attenuate immune responses and modulate the activation state of other immune cell subsets once activated, expressing such CARs on the surface of Treg cells provides a universal therapy for treating autoimmune or inflammatory diseases in which ENTPD3 is locally expressed at the disease site. Specifically, the inventors have developed an anti-ENTPD3 CAR for the treatment of type 1 diabetes.

[0011] Therefore, the inventors confirmed that ENTPD3 is a surprisingly effective target for CAR Treg therapy, particularly for T1D and other pancreatic autoimmune or inflammatory diseases. Specifically, the inventors found that cells expressing ENTPD3-specific CARs are activated in the presence of the antigen and are able to prevent the onset of cyclophosphamide-induced type 1 diabetes in NOD mice. In fact, the anti-ENTPD3 CAR developed by the inventors has a preventive effect on diabetes in every treated animal. Although studies have reported ENTPD3 expression in pancreatic tissue, its expression in other tissues (such as the digestive tract, kidneys, and brain) has also been reported in the art, making the selection of ENTPD3 as a specific migration target (thus activating Tregs to a level sufficient to treat type 1 diabetes) intuitively unreasonable. Therefore, the inventors' discovery that the ENTPD3 protein is an effective pancreatic target capable of sufficiently activating anti-ENTPD3 CAR Tregs to prevent the onset of diabetes is particularly surprising.

[0012] Therefore, in one aspect, the present invention provides a chimeric antigen receptor (CAR) comprising an antigen recognition domain that specifically binds to ENTPD3 (e.g., specifically binds to human ENTPD3).

[0013] In this regard, this article provides a CAR that includes:

[0014] a. Extracellular domains containing antigen recognition domains;

[0015] b. Transmembrane domains; and

[0016] c. Intracellular domains containing intracellular signal transduction domains.

[0017] CARs may also include hinge domains and / or one or more co-stimulatory domains.

[0018] The term "hinge domain" generally refers to the extracellular 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. Optionally, the CAR may include the CD8α or CH2CH3 hinge domain.

[0019] The co-stimulatory domain may be selected from the following: intracellular domains of CD28, ICOS, CD134 (OX40), CD137 (4-1BB), CD27, or TNFRSF25, or portions thereof or variants thereof. Optionally, the CAR may include the CD28 co-stimulatory domain.

[0020] The CAR may contain one or more transmembrane domains, which may be selected from the following: transmembrane domains of CD28, ICOS, CD8α, CD4, CD134(OX40), CD137(4-1BB), CD3ζ, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD154, CH2CH3, or portions thereof or variations thereof. Optionally, the CAR may include transmembrane domains of CD4, CD28, CD8α, or CH2CH3.

[0021] A CAR (specifically, the intracellular domain of a CAR) may comprise one or more intracellular signaling domains selected from the group consisting of: a CD3ζ signaling domain or any homolog thereof, a CD3 polypeptide, a syk family tyrosine kinase, a src family tyrosine kinase, CD2, CD5, and CD28, or portions or variants thereof. Optionally, a CAR may comprise a CD3ζ signaling domain.

[0022] 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 CH2CH3 transmembrane domain (i.e., a transmembrane domain derived from CD8α or CH2CH3); a CD28 costimulatory domain (i.e., a costimulatory domain derived from CD28); and a CD3ζ signal transduction domain (i.e., a signal transduction 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 CH2CH3 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 signal transduction domain. In a single implementation, the CAR may include a CH2CH3 hinge domain, a CH2CH3 transmembrane domain, a CD28 costimulatory domain, and a CD3ζ signaling domain. Furthermore, the CAR may include a CD28 transmembrane domain (i.e., a transmembrane domain derived from CD28), particularly in combination with a CD28 costimulatory domain.

[0023] The CAR of the present invention may comprise a signal peptide and / or a reporter peptide. In one embodiment, the polynucleotide sequence encoding the CAR of the present invention may comprise another polynucleotide sequence that encodes a reporter peptide linked by a self-cleaving or cleavable domain.

[0024] The antigen recognition domain of the CAR of the present invention can be an antibody, an antibody fragment, or an antibody-derived compound. Optionally, the antigen recognition domain is a single-chain antibody (scFv).

[0025] The CAR of the present invention may include:

[0026] An antigen recognition domain comprising the VH CDR1, 2, and 3 sequences listed in SEQ ID NO: 269, 2, and 270, and the VL CDR1, 2, and 3 sequences listed in any of the following: (i) SEQ ID NO: 4, 5, and 6; (ii) SEQ ID NO: 34, 35, and 36; (iii) SEQ ID NO: 40, 41, and 42; or (iv) SEQ ID NO: 52, 53, and 54, wherein one or more of the CDR sequences may optionally contain one to three amino acid modifications relative to the aforementioned CDR sequences, and in particular, one or more of the CDR sequences may optionally be modified by substituting one to three amino acids, adding one to three amino acids, or deleting one to three amino acids.

[0027] The CAR of the present invention may include:

[0028] An antigen recognition domain comprising the VHCDR1, 2, and 3 sequences listed in (i) SEQ ID No: 43, 44, and 45 or (ii) SEQ ID No: 61, 62, or 63, and the VLCDR1, 2, and 3 sequences listed in SEQ ID No: 271, 272, and 273, wherein one or more of the CDR sequences may optionally contain one to three amino acid modifications relative to the aforementioned CDR sequences, and in particular, one or more of the CDR sequences may optionally be modified by substituting one to three amino acids, adding one to three amino acids, or deleting one to three amino acids.

[0029] The CAR of the present invention may include:

[0030] (i) an antigen recognition domain comprising the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:1, 2 and 3, respectively, and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:4, 5 and 6, respectively;

[0031] (ii) an antigen recognition domain comprising the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:7, 8 and 9, respectively, and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:10, 11 and 12, respectively;

[0032] (iii) An antigen recognition domain comprising the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:13, 14 and 15, respectively, and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:16, 17 and 18, respectively;

[0033] (iv) An antigen recognition domain comprising the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:19, 20 and 21, respectively, and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:22, 23 and 24, respectively;

[0034] (v) An antigen recognition domain comprising the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:25, 26 and 27, respectively, and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:28, 29 and 30, respectively;

[0035] (vi) An antigen recognition domain comprising the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:31, 32 and 33, respectively, and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:34, 35 and 36, respectively;

[0036] (vii) An antigen recognition domain comprising the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:37, 38 and 39, respectively, and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:40, 41 and 42, respectively;

[0037] (viii) An antigen recognition domain comprising the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:43, 44 and 45, respectively, and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:46, 47 and 48, respectively;

[0038] (ix) An antigen recognition domain comprising the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:49, 50 and 51, respectively, and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:52, 53 and 54, respectively;

[0039] (x) An antigen recognition domain comprising the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:55, 56 and 57, respectively, and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:58, 59 and 60, respectively;

[0040] (xi) An antigen recognition domain comprising the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:61, 62 and 63, respectively, and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:64, 65 and 66, respectively;

[0041] (xii) An antigen recognition domain comprising the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:67, 68 and 69, respectively, and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:70, 71 and 72, respectively;

[0042] (xiii) An antigen recognition domain comprising the VH CDR1, 2, and 3 sequences listed in SEQ ID NO:73, 74, and 75, respectively, and the VL CDR1, 2, and 3 sequences listed in SEQ ID NO:76, 77, and 78, respectively; or

[0043] (xiv) An antigen recognition domain comprising the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:79, 80 and 81, respectively, and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:82, 83 and 84, respectively;

[0044] Wherein, one or more of the CDR sequences in (i) to (xiv) may optionally include one to three amino acid modifications relative to the aforementioned CDR sequences, and in particular, one or more of the CDR sequences may optionally be modified by substituting one to three amino acids, adding one to three amino acids, or deleting one to three amino acids.

[0045] Furthermore, in this respect, the antigen recognition domain of CAR can include:

[0046] (i) A VH domain containing the sequence listed in SEQ ID NO:85 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:86 or a sequence having at least 70% sequence identity with it.

[0047] (ii) A VH domain comprising the sequence listed in SEQ ID NO:87 or a sequence having at least 70% sequence identity with it, and a VL domain comprising the sequence listed in SEQ ID NO:88 or a sequence having at least 70% sequence identity with it.

[0048] (iii) A VH domain comprising the sequence listed in SEQ ID NO:89 or a sequence having at least 70% sequence identity with it, and a VL domain comprising the sequence listed in SEQ ID NO:90 or a sequence having at least 70% sequence identity with it.

[0049] (iv) A VH domain comprising the sequence listed in SEQ ID NO:91 or a sequence having at least 70% sequence identity with it, and a VL domain comprising the sequence listed in SEQ ID NO:92 or a sequence having at least 70% sequence identity with it.

[0050] (v) A VH domain containing the sequence listed in SEQ ID NO:93 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:94 or a sequence having at least 70% sequence identity with it.

[0051] (vi) A VH domain comprising the sequence listed in SEQ ID NO:95 or a sequence having at least 70% sequence identity with it, and a VL domain comprising the sequence listed in SEQ ID NO:96 or a sequence having at least 70% sequence identity with it.

[0052] (vii) A VH domain containing the sequence listed in SEQ ID NO:97 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:98 or a sequence having at least 70% sequence identity with it.

[0053] (viii) A VH domain comprising the sequence listed in SEQ ID NO:99 or a sequence having at least 70% sequence identity with it, and a VL domain comprising the sequence listed in SEQ ID NO:100 or a sequence having at least 70% sequence identity with it.

[0054] (ix) A VH domain comprising the sequence listed in SEQ ID NO:101 or a sequence having at least 70% sequence identity with it, and a VL domain comprising the sequence listed in SEQ ID NO:102 or a sequence having at least 70% sequence identity with it.

[0055] (x) A VH domain containing the sequence listed in SEQ ID NO:103 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:104 or a sequence having at least 70% sequence identity with it.

[0056] (xi) A VH domain containing the sequence listed in SEQ ID NO:105 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:106 or a sequence having at least 70% sequence identity with it.

[0057] (xii) A VH domain containing the sequence listed in SEQ ID NO:107 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:108 or a sequence having at least 70% sequence identity with it.

[0058] (xiii) A VH domain comprising the sequence listed in SEQ ID NO:109 or a sequence having at least 70% sequence identity with it, and a VL domain comprising the sequence listed in SEQ ID NO:110 or a sequence having at least 70% sequence identity with it; or

[0059] (xiv)VH domain containing the sequence listed in SEQ ID NO:111 or a sequence having at least 70% sequence identity with it, and VL domain containing the sequence listed in SEQ ID NO:112 or a sequence having at least 70% sequence identity with it.

[0060] For example, an antigen recognition domain may contain:

[0061] (i) a VH domain containing a sequence encoded by SEQ ID NO:205 or a sequence having at least 70% identity with a sequence encoded by SEQ ID NO:205, and a VL domain containing a sequence encoded by SEQ ID NO:206 or a sequence having at least 70% identity with a sequence encoded by SEQ ID NO:206;

[0062] (ii) A VH domain comprising a sequence encoded by SEQ ID NO:207 or a sequence having at least 70% identity with a sequence encoded by SEQ ID NO:207, and a VL domain comprising a sequence encoded by SEQ ID NO:208 or a sequence having at least 70% identity with a sequence encoded by SEQ ID NO:208;

[0063] (iii) A VH domain comprising a sequence encoded by SEQ ID NO:209 or a sequence having at least 70% identity with a sequence encoded by SEQ ID NO:209, and a VL domain comprising a sequence encoded by SEQ ID NO:210 or a sequence having at least 70% identity with a sequence encoded by SEQ ID NO:210;

[0064] (iv) A VH domain comprising a sequence encoded by SEQ ID NO:211 or a sequence having at least 70% identity with a sequence encoded by SEQ ID NO:211, and a VL domain comprising a sequence encoded by SEQ ID NO:212 or a sequence having at least 70% identity with a sequence encoded by SEQ ID NO:212;

[0065] (v) A VH domain comprising a sequence encoded by SEQ ID NO:213, or a sequence having at least 70% identity with a sequence encoded by SEQ ID NO:213, and a sequence encoded by SEQ ID NO:214, or a sequence having at least 70% identity with a sequence encoded by SEQ ID NO:214.

[0066] (vi) A VH domain containing a sequence encoded by SEQ ID NO:215 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:215, and a VL domain containing a sequence encoded by SEQ ID NO:216 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:216;

[0067] (vii) A VH domain containing a sequence encoded by SEQ ID NO:217 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:217, and a VL domain containing a sequence encoded by SEQ ID NO:218 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:218;

[0068] (viii) A VH domain comprising a sequence encoded by SEQ ID NO:219 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:219, and a VL domain comprising a sequence encoded by SEQ ID NO:220 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:220;

[0069] (ix) A VH domain containing a sequence encoded by SEQ ID NO:221 or a sequence having at least 70% identity with a sequence encoded by SEQ ID NO:221, and a VL domain containing a sequence encoded by SEQ ID NO:222 or a sequence having at least 70% identity with a sequence encoded by SEQ ID NO:222;

[0070] (x) A VH domain containing a sequence encoded by SEQ ID NO:223 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:223, and a VL domain containing a sequence encoded by SEQ ID NO:224 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:224;

[0071] (xi) A VH domain comprising a sequence encoded by SEQ ID NO:225 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:225, and a VL domain comprising a sequence encoded by SEQ ID NO:226 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:226;

[0072] (xii) A VH domain containing a sequence encoded by SEQ ID NO:227 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:227, and a VL domain containing a sequence encoded by SEQ ID NO:228 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:228;

[0073] (xiii) A VH domain comprising a sequence encoded by SEQ ID NO:229, or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:229, and a VL domain comprising a sequence encoded by SEQ ID NO:230, or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:230; or

[0074] (xiv) A VH domain containing a sequence encoded by SEQ ID NO:231 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:231, and a VL domain containing a sequence encoded by SEQ ID NO:232 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:232.

[0075] In addition, the antigen recognition domain of CAR can contain or be composed of the following:

[0076] (i) A sequence listed in SEQ ID NO:113 or a sequence having at least 80% sequence identity with it;

[0077] (ii) A sequence listed in SEQ ID NO:114 or a sequence having at least 80% sequence identity with it;

[0078] (iii) A sequence listed in SEQ ID NO:115 or a sequence having at least 80% sequence identity with it;

[0079] (iv) A sequence listed in SEQ ID NO:116 or a sequence having at least 80% sequence identity with it;

[0080] (v) A sequence listed in SEQ ID NO:117 or a sequence having at least 80% sequence identity with it;

[0081] (vi) A sequence listed in SEQ ID NO:118 or a sequence having at least 80% sequence identity with it;

[0082] (vii) A sequence listed in SEQ ID NO:119 or a sequence having at least 80% sequence identity with it;

[0083] (viii) A sequence listed in SEQ ID NO:120 or a sequence having at least 80% sequence identity with it;

[0084] (ix) A sequence listed in SEQ ID NO:121 or a sequence having at least 80% sequence identity with it;

[0085] (x) A sequence listed in SEQ ID NO:122 or a sequence having at least 80% sequence identity with it;

[0086] (xi) A sequence listed in SEQ ID NO:123 or a sequence having at least 80% sequence identity with it;

[0087] (xii) A sequence listed in SEQ ID NO:124 or a sequence having at least 80% sequence identity with it;

[0088] (xiii) A sequence listed in SEQ ID NO:125 or a sequence having at least 80% sequence identity with it; or

[0089] (xiv) The sequence listed in SEQ ID NO:126 or a sequence that has at least 80% sequence identity with it.

[0090] For example, an antigen recognition domain may contain or be composed of the following:

[0091] (i) A sequence encoded by the sequence listed in SEQ ID NO:191 or a sequence having at least 80% identity with the sequence encoded by the sequence listed in SEQ ID NO:191;

[0092] (ii) A sequence encoded by the sequence listed in SEQ ID NO:192 or a sequence having at least 80% identity with the sequence encoded by the sequence listed in SEQ ID NO:192;

[0093] (iii) A sequence encoded by the sequence listed in SEQ ID NO:193 or a sequence having at least 80% identity with the sequence encoded by the sequence listed in SEQ ID NO:193;

[0094] (iv) A sequence encoded by the sequence listed in SEQ ID NO:194 or a sequence having at least 80% identity with the sequence encoded by the sequence listed in SEQ ID NO:194;

[0095] (v) A sequence encoded by the sequence listed in SEQ ID NO:195 or a sequence having at least 80% identity with the sequence encoded by the sequence listed in SEQ ID NO:195;

[0096] (vi) A sequence encoded by the sequence listed in SEQ ID NO:196 or a sequence having at least 80% identity with the sequence encoded by the sequence listed in SEQ ID NO:196;

[0097] (vii) A sequence encoded by the sequence listed in SEQ ID NO:197 or a sequence having at least 80% identity with the sequence encoded by the sequence listed in SEQ ID NO:197;

[0098] (viii) A sequence encoded by the sequence listed in SEQ ID NO:198 or a sequence having at least 80% identity with the sequence encoded by the sequence listed in SEQ ID NO:198;

[0099] (ix) A sequence encoded by the sequence listed in SEQ ID NO:199 or a sequence having at least 80% identity with the sequence encoded by the sequence listed in SEQ ID NO:199;

[0100] (x) A sequence encoded by the sequence listed in SEQ ID NO:200 or a sequence having at least 80% identity with the sequence encoded by the sequence listed in SEQ ID NO:200;

[0101] (xi) A sequence encoded by the sequence listed in SEQ ID NO:201 or a sequence having at least 80% identity with the sequence encoded by the sequence listed in SEQ ID NO:201;

[0102] (xii) A sequence encoded by the sequence listed in SEQ ID NO:202 or a sequence having at least 80% identity with the sequence encoded by the sequence listed in SEQ ID NO:202;

[0103] (xiii) A sequence encoded by the sequence listed in SEQ ID NO:203 or a sequence having at least 80% identity with the sequence encoded by the sequence listed in SEQ ID NO:203; or

[0104] (xiv) A sequence encoded by the sequence listed in SEQ ID NO:204 or a sequence having at least 80% identity with the sequence encoded by the sequence listed in SEQ ID NO:204.

[0105] In a second aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a CAR according to the present invention.

[0106] Thirdly, 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.

[0107] In another 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. Optionally, the cell may be a T cell or its precursor, or a stem cell. In particular, the cell may be a Treg or its precursor, or an iPSC cell. The cell may be a production host cell. In one specific embodiment, the present invention provides a Treg comprising a CAR, wherein the antigen recognition domain of the CAR specifically binds to ENTPD3.

[0108] Cells can be provided in cell populations, which constitutes another aspect of the invention. In particular, the cell population can contain multiple cells according to the invention, especially multiple T cells (e.g., multiple Tregs) according to the invention. Specifically, the multiple T cells (especially Tregs) according to the invention have polyclonal TCRs. Specifically, the clonality of the TCRs of the multiple T cells (especially Tregs) according to the invention is not altered in vitro.

[0109] The present invention also provides a pharmaceutical composition comprising cells, cell populations or carriers according to the present invention.

[0110] In another aspect, the present invention provides a cell, cell population, or pharmaceutical composition according to the invention for use in treatment (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 treatment is adoptive cell transfer therapy.

[0111] In addition, 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 (particularly containing Treg cells) according to the present invention.

[0112] In this regard, the method may include the following steps:

[0113] (i) Isolate or provide Treg-enriched cell samples from the subject;

[0114] (ii) Introducing the nucleic acid molecule or vector of the present invention into Treg cells; and

[0115] (iii) Administer the Treg cells from (ii) to the subject.

[0116] The present invention also provides the use of cells, cell populations or pharmaceutical compositions according to the invention in the manufacture of medicaments for treating and / or preventing autoimmune or inflammatory diseases, or for inducing immunosuppression, or for promoting tissue repair and / or tissue regeneration in subjects, particularly wherein the cells are Treg cells.

[0117] Autoimmune or inflammatory diseases can be particularly type 1 diabetes (T1D).

[0118] In another aspect, the present invention provides a method for manufacturing 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 a nucleic acid molecule or vector according to the invention). The cells may be Treg cells, and the method may include: isolating or providing a cell-containing sample containing Tregs, and / or enriching or generating Tregs from the cell-containing sample before or after introducing the nucleic acid molecule or vector into the cells. The present invention also provides cells obtainable by this method, which constitutes another aspect of the invention.

[0119] In another aspect, the present invention provides the use of CAR-Treg to reduce (e.g., reduce mortality) or prevent the death of pancreatic β cells in a subject. In another aspect, the present invention provides the use of CAR-Treg to maintain or increase fasting insulin levels in a subject. In another aspect, the present invention provides the use of CAR-Treg to maintain or increase fasting C-peptide levels in a subject. In another aspect, the present invention provides the use of CAR-Treg to reduce or prevent hyperglycemia in a subject. In another aspect, the present invention provides the use of CAR-Treg to maintain or reduce fasting blood glucose levels in a subject. In another aspect, the present invention provides the use of CAR-Treg to maintain or reduce 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 ENTPD3 (e.g., human ENTPD3), and the CAR may have any of the characteristics of a CAR as disclosed herein. For example, the subject may have or be at risk of developing type 1 diabetes, particularly the subject may have recently developed type 1 diabetes. For example, the subject may not be administered exogenous insulin. Alternatively, the subject may be receiving a reduced dose of insulin compared to the insulin dose required before administering CAR-Treg cells. Attached Figure Description

[0120] Figure 1 The binding of various scFvs to HEK293T cells expressing human ENTPD3 (first and third rows) or mouse ENTPD3 (second and fourth rows) is shown.

[0121] Figure 2 Immunohistochemical staining of mouse pancreatic sections with mouse ENTPD3-specific scFV is shown.

[0122] Figure 3 This study demonstrates that murine hybridoma cells expressing murine ENTPD3-specific CARs are activated by HEK cells expressing murine ENTPD3.

[0123] Figure 4 An overview of the experimental protocol for inducing type 1 diabetes in NOD mice by administering T effector cells expressing ENTPD3-specific CARs is shown (Example 2).

[0124] Figure 5 An overview of the experimental protocol for observing the enrichment of T cells expressing ENTPD3-specific CARs in the pancreatic tissue of NOD mice is shown (Example 3).

[0125] Figure 6The enrichment of T cells expressing ENTPD3-specific CARs in the islets of NOD mice was shown compared to that in the spleen and lymph nodes.

[0126] Figure 7 An overview of an experimental protocol for the prevention of cyclophosphamide-induced type 1 diabetes in NOD mice (Example 4) is shown, in which the NOD mice were administered Tregs expressing ENTPD3-specific CARs.

[0127] Figure 8 This study demonstrates the use of Treg expressing ENTPD3-specific CARs to prevent cyclophosphamide-induced type 1 diabetes in NOD mice.

[0128] Figure 9 The enrichment of Tregs expressing ENTPD3-specific CARs in the islets of NOD mice was shown compared to that in the spleen and lymph nodes.

[0129] Figure 10 The construction of the rodent CAR used in Examples 1 to 6 is shown. Figure 10 (A) shows a schematic structure of a CAR with a mutant Fc-IgG hinge and a CD8 hinge. Figure 10 (B) shows a schematic structure of the construct used in a γ-retroviral vector for cell transduction.

[0130] Figure 11 An overview of the experimental protocol for observing the enrichment of Treg expressing ENTPD3-specific CARs in the pancreatic tissue of NOD mice is shown (Example 5).

[0131] Figure 12 The enrichment of Tregs expressing ENTPD3-specific CARs in the islets of NOD mice was shown compared to that in the spleen and lymph nodes.

[0132] Figure 13 The activation of Tregs exposed to the target (ENTPD3) is shown in the middle column compared to Tregs exposed to the control antigen (right column) or to anti-CD3 / CD28 beads (left column).

[0133] Figure 14 The proliferation of Tregs exposed to the target (ENTPD3) is shown in the middle column compared to Tregs exposed to the control antigen (right column) or to anti-CD3 / CD28 beads (left column).

[0134] Figure 15The activation status (% of NFAT GFP+ in the CAR+ population) of hybridoma cells expressing various ENTPD3-specific CARs is shown when exposed to human ENTPD3 protein, HEK cells expressing human or murine ENTPD3, untransduced HEK cells, and culture medium.

[0135] Figure 16 The construction of the human CAR used in Example 7 is shown. Figure 16 (A) shows a schematic structure of a CAR with a mutant Fc-IgG hinge and a CD8 hinge, and Figure 16 (B) shows a schematic structure of the construct used for cell transduction.

[0136] Figure 17 The transduction efficiency of Jurkat cells transduced with various ENTPD3-specific CAR constructs as described in Example 8 is shown.

[0137] Figure 18 The luminescence of Jurkat cells transduced with various ENTPD3-specific CAR constructs as described in Example 8 is shown after exposure to HEK293 cells expressing human ENTPD3, RT-4 cells, human ENTPD3 peptide, OKT3 anti-CD3 antibody, and unstimulated conditions.

[0138] Figure 19 It shows the relationship with Figure 18 Same results were obtained, but the positive control was removed to allow for a clearer observation of human ENTPD3-mediated activation.

[0139] Figure 20 The percentage of cells expressing FOXP3 is shown on days 7 and 12 after transduction.

[0140] Figure 21 The results of the Treg activation assay are shown, in particular the percentage of cells expressing activation markers CD69, CD137 and GARP.

[0141] Figure 22 The results of the Treg inhibition experiment are shown.

[0142] Figure 23 The results of the Treg proliferation experiment are shown.

[0143] Figure 24 The results of experiments on the secretion of cytokines IL-10, IL-13 and IFN-γ are shown.

[0144] Figure 25 , Figure 26 and Figure 27Representative in vivo images (2400 cells) of hIsMT co-cultured with various T cell populations for 24 hours, 48 ​​hours, and 72 hours are shown.

[0145] Figure 28 The study showed the effect of hIsMT on insulin secretion after co-culturing with various T cell populations for 48 hours and 72 hours (respectively). Figure 28 A and Figure 28 B), and the total insulin levels after 48 and 72 hours of co-culturing hIsMT with various T cell populations (respectively...). Figure 28 C and Figure 28 D).

[0146] Figure 29 The study showed the IFN-γ secretion of hIsMT after co-culturing with various T cell populations for 48 and 72 hours. Figure 29 A and Figure 29 C), and the secretion of IFN-γ by T cells cultured alone for 48 hours and 72 hours (C). Figure 29 B and Figure 29 D).

[0147] Figure 30 The study showed the TNF-α secretion after hIsMT was co-cultured with various T cell populations for 48 and 72 hours. Figure 30 A and Figure 30 C), and the secretion of TNF-α by T cells cultured alone for 48 hours and 72 hours (C). Figure 30 B and Figure 30 D).

[0148] Figure 31 The WPRE copy number / μg DNA in the pancreas and testes of NOD mice treated with PE-specific or ENTPD3-specific CAR-Treg and untreated NOD mice is shown. Detailed Implementation

[0149] This invention provides ENTPD3-specific CAR-Treg cells that are activated in the presence of the ENTPD3 antigen, which is specifically expressed on pancreatic β-cells. Therefore, these CAR-Tregs have therapeutic potential in treating autoimmune and inflammatory diseases where ENTPD3 is locally expressed at the disease site. In particular, these CAR-Treg cells have therapeutic potential in treating type 1 diabetes. Specifically, due to the bystander effect of Treg cells, the antigen (ENTPD3) only needs to be present and / or expressed at the site of inflammation or disease.

[0150] Therefore, the present invention provides a chimeric antigen receptor (CAR) comprising an antigen recognition domain that binds to ENTPD3, a cell or cell population expressing the CAR (e.g., a Treg or Treg population expressing the CAR), and the use of the cell or cell population in treating certain diseases (such as type 1 diabetes).

[0151] "Chimeric antigen receptor," "CAR," or "CAR construct" refers to an engineered receptor that endows cells (e.g., immune cells, such as Treg cells) with antigen specificity. Specifically, a CAR enables cells to specifically bind to a particular antigen, such as a target molecule, like a target protein, which is then signaled by the CAR's intracellular domains (containing intracellular signal transduction domains), such as signals that lead to cell activation. CARs are also known as artificial T-cell receptors, chimeric T-cell receptors, or chimeric immune receptors.

[0152] The structure of CARs is well known in the field, and several generations of CARs have been developed. For example, a CAR may contain at least an extracellular antigen-specific targeting region, an antigen-binding domain, a target-binding domain, or a ligand-binding domain, which are part of or constitute part of the extracellular domain (also called the extracellular domain or extracellular region) of the CAR, a transmembrane domain, and an intracellular signal transduction domain (which are part of or contained within the intracellular domain). However, CARs may also contain other domains to improve their function, such as one or more co-stimulatory domains to enhance T cell proliferation, cytokine secretion, anti-apoptosis, and in vivo persistence.

[0153] 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 in this document, binding domain, antigen recognition domain, antigen-binding domain, and ligand-binding domain are used interchangeably), an optional hinge domain that acts as a spacer to extend the binding domain beyond the plasma membrane of the cell expressing it (e.g., an immune cell), a transmembrane domain, an intracellular signaling domain (e.g., the signaling domain of the CD3 ζ chain (CD3ζ) in the TCR complex or an equivalent), and optional one or more co-stimulatory domains that assist the CAR-expressing cell in signal transduction or function. CARs may also include signaling or leader sequences or domains that function to target proteins to the membrane and can form part of the extracellular domain of the CAR. Different domains may be directly linked or linked via adapters, and / or may be present in different peptides, for example, in two mutually bound peptides.

[0154] When a CAR binds to its target antigen (i.e., ENTPD3), it delivers an activation signal to cells expressing that CAR. Thus, the CAR specifically directs engineered cells toward ENTPD3, especially to cells expressing ENTPD3.

[0155] The terms "directed towards" or "directed against" are synonymous with "specific for" or "anti." In other words, CARs recognize ENTPD3 target molecules. Therefore, this means that CARs can specifically bind to ENTPD3. Specifically, the antigen-binding domain of a CAR can specifically bind to ENTPD3 (more specifically, when the CAR is expressed on the cell surface, especially on the surface of immune effector cells). Specific binding can be distinguished from non-specific binding to non-target molecules or antigens. Therefore, cells expressing CARs are directed or redirected to specifically bind to target cells expressing ENTPD3, especially those expressing ENTPD3 on their cell surface. Specifically, "specific" binding means that binding occurs only or primarily to ENTPD3, and not 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 signaling. As described above, CARs can bind to ENTPD3 and transduce signals to cells expressing it. These cells can then be activated and can exert an inhibitory effect in the local environment. Following antigen binding, activation of CAR-expressing cells can be determined by an increase in CD69 levels compared to the same cells expressing CAR in the absence of the antigen. For example, CD69 levels are 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 and commercially available antibodies (e.g., FITC anti-human CD69 antibody, Biolegend). Therefore, CAR function in cells can be determined by the activation status of CAR-expressing cells, for example, by measuring CD69 expression.

[0156] ENTPD3 (exonucleoside triphosphate diphosphate hydrolase 3), also known as CD39L3, HB6, and NTPDase-3, is a membrane-bound enzyme similar to type E nucleotidases (NTPs) and expressed on pancreatic β cells. The amino acid sequence of human ENTPD3 is shown in SEQ ID NO:127, and the amino acid sequence of murine ENTPD3 is shown in SEQ ID NO:128.

[0157] The antigen-binding domain of a CAR can be derived from or obtained from any protein or polypeptide that can bind (i.e., has an affinity for) ENTPD3 (e.g., a protein or polypeptide that can bind any region or portion of ENTPD3, or any epitope within ENTPD3, whether ENTPD3 is present in isolated protein form or when expressed on a cell). Specifically, the antigen-binding domain of a CAR can be derived from or obtained from any protein or polypeptide that can bind (i.e., has an affinity for) the extracellular domain of ENTPD3. For example, it can be a ligand of ENTPD3, a physiologically binding protein of ENTPD3 or a portion thereof, or a synthetic or derived protein. The target molecule (i.e., ENTPD3) can typically be expressed on the surface of a cell, such as a target cell (e.g., pancreatic β-cell), or a cell near the target cell (producing a bystander effect), but is not required to be so.

[0158] Antigen-binding domains most commonly originate from antibody variable chains (e.g., which are typically in the form of scFv), but can also be generated by other molecules such as ligands or other binding molecules.

[0159] CARs are typically expressed as polypeptides that also contain a signal sequence (also called a leader sequence), specifically a signal sequence that targets the CAR to the cytoplasmic membrane. This signal sequence is usually located adjacent to or near the antigen-binding domain and is typically upstream of the antigen-binding domain. Therefore, the extracellular domain of a CAR can contain both a signal sequence and an antigen-binding domain, consist essentially of both a signal sequence and an antigen-binding domain, or consist of both a signal sequence and an antigen-binding domain.

[0160] As described above, the antigen-binding domain can be any protein or peptide capable of specifically recognizing and binding to ENTPD3. The antigen-binding domain includes any naturally occurring, synthetic, semi-synthetic, or recombinant binding partner targeting ENTPD3. Illustrative antigen-specific targeting domains include antibodies or antibody fragments or derivatives, or ligands, targeting soluble or membrane-bound ENTPD3.

[0161] In one implementation, the antigen-binding domain is an antibody or derived from an antibody.

[0162] As used herein, the term "antibody" broadly refers to any immunobinding agent or molecule containing an antigen-binding domain, including polyclonal and monoclonal antibodies. Depending on the type of constant domain in the heavy chain, intact antibodies are designated as one of five main classes: IgA, IgD, IgE, IgG, and IgM, and antibodies as described herein can be any of these classes. Some of these are further subdivided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, etc. Typically, IgG antibodies or IgM antibodies are the most commonly used antibodies in the physiological environment. 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 their fragments.

[0163] Antibody-derived binding domains can be fragments of the antibody or genetically engineered products of one or more fragments of the antibody that participate in binding to the antigen. Examples include variable regions (Fv), complementarity-determining regions (CDRs), Fab or F(ab')2, or light and heavy chain variable regions that can be single-chain (e.g., as scFv) and in either orientation (e.g., V). L -V H or V H -V L () are linked together. V can be modified. L and / or V H Sequence. In particular, the frame region can be modified (e.g., substituted, for example, with a humanized antigen-binding domain). Other examples include heavy chain variable regions (VH), light chain variable regions (VL), and single-domain antibodies (sAbs) (which may be referred to as nanobodies). An example of a single-domain antibody is a camel heavy chain antibody (HCAb), which has an antigen recognition site formed by a single domain called VHH.

[0164] In one implementation, the antigen-binding domain is a single-chain antibody (scFv). The scFv can be murine, human, or humanized scFv.

[0165] In alternative embodiments, the antigen-binding domain is derived from camel heavy chain antibodies (HCAbs), for example, the antigen-binding domain may be the VHH domain of an HCAb. The VHH has a structure similar to the VH domain from conventional IgG and includes three variable CDRs, although CDR 1 and CDR 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 sequences listed in SEQ ID NO: 1 to 84 as specified herein, or sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. For example, a VHH domain may contain one, two, or three VH CDRs of VHCDRs, wherein the VH CDRs contain or consist of sequences listed in SEQ ID NO:1 to 84 as specified herein, or sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with them. When a VHH domain contains two or three VH CDRs of SEQ ID NO:1 to 84, the CDRs may originate from the same conjugates described herein (e.g., all from conjugates A8, B2, B7, etc.).

[0166] 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's heavy chain containing three CDRs interposed between flanking segments called framework regions. Framework regions are more conserved than CDRs and form the backbone structure supporting the CDRs. The "light chain variable region" or "VL" refers to a segment of the antibody's light chain containing three CDRs interposed between framework regions.

[0167] "Fv" refers to the smallest fragment of an antibody that carries an intact antigen-binding site. An Fv fragment consists of a variable region of a single light chain that binds to a variable region of a single heavy chain. A "single-chain Fv antibody" or "scFv" refers to an engineered antibody composed of variable regions of a light chain and a variable region of a heavy chain, which are directly linked to each other in any direction or linked to each other via peptide linker sequences.

[0168] Antibodies that specifically bind to a predetermined antigen (i.e., ENTPD3) can be prepared using methods well 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 synthesized antibodies are available, and antibodies or fragments thereof capable of binding to the target molecule (i.e., ENTPD3) 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.

[0169] An antigen recognition domain can bind, appropriately and specifically, to one or more regions or epitopes within ENTPD3. An epitope, also known as an antigenic determinant, is a portion of an antigen 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, an antigen recognition domain binds, appropriately and specifically, to a region or epitope within ENTPD3.

[0170] The antigen recognition domain may contain at least one CDR (e.g., CDR3), which can be predicted from antibodies binding to the antigen (i.e., ENTPD3) (or variants of such predicted CDRs (e.g., variants 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) are 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 capable of binding target antigens, for example, in the form of microantibodies (Vaughan and Sollazzo, 2001, Combinational Chemistry & High Throughput Screening, 4, 417-430). Molecules containing a single CDR have been described, and such molecules can exhibit strong binding activity to the target (Nicaise et al, 2004, Protein Science, 13:1882-91).

[0171] In this respect, the antigen-binding domain may contain one or more variable heavy chain CDRs, such as one, two, or three variable heavy chain CDRs. Alternatively or additionally, the antigen-binding domain may contain one or more variable light chain CDRs, such as 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, heavy chain variable regions containing three CDRs and / or light chain variable regions containing three CDRs), wherein at least one CDR, and optionally all CDRs, may be derived from an antibody binding to ENTPD3.

[0172] The antigen-binding domain may comprise any combination of variable heavy chain CDRs and variable light chain CDRs, such as one variable heavy chain CDR with one variable light chain CDR, two variable heavy chain CDRs with one variable light chain CDR, two variable heavy chain CDRs with two or three variable light chain CDRs, three variable heavy chain CDRs with one or two variable light chain CDRs, one variable heavy chain CDR with two or three variable light chain CDRs, or three variable heavy chain CDRs with three variable light chain CDRs. Optionally, 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).

[0173] 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 from the heavy or light chain of an antibody binding ENTPD3, while another existing CDR can be predicted from different antibodies binding ENTPD3. Combinations of CDRs from different antibodies, particularly those binding the same desired region or epitope, can be used.

[0174] In one embodiment, the antigen-binding domain includes three CDRs predicted from the variable heavy chain sequence of an antibody binding to ENTPD3 and / or three CDRs predicted from the variable light chain sequence of an antibody binding to ENTPD3 (optionally, the same antibody).

[0175] In one embodiment, the antigen-binding domain comprises the VHCDR1, 2 and 3 sequences listed in SEQ ID NO:1, 2 and 3, respectively, and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:4, 5 and 6, respectively, or the CDR may comprise one to three, or more particularly one or two, amino acid sequence modifications from any of the aforementioned sequences.

[0176] More specifically, in such an embodiment, the antigen-binding domain of the CAR includes a VH domain containing the sequence listed in SEQ ID NO:85 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:86 or a sequence having at least 70% sequence identity with it.

[0177] For example, in such an embodiment, the antigen-binding domain of the CAR includes a VH domain containing a sequence encoded by SEQ ID NO:205 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:205, and a VL domain containing a sequence encoded by SEQ ID NO:206 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:206.

[0178] In one embodiment, the antigen-binding domain comprises the VHCDR1, 2, and 3 sequences listed in SEQ ID NO:7, 8, and 9, respectively, and the VL CDR1, 2, and 3 sequences listed in SEQ ID NO:10, 11, and 12, respectively, or the CDR may comprise one to three, or more particularly one or two, amino acid sequence modifications from any of the aforementioned sequences.

[0179] More specifically, in such an embodiment, the antigen-binding domain of the CAR includes a VH domain containing the sequence listed in SEQ ID NO:87 or a sequence having at least 70% sequence identity therewith, and a VL domain containing the sequence listed in SEQ ID NO:88 or a sequence having at least 70% sequence identity therewith.

[0180] For example, in such an embodiment, the antigen-binding domain of the CAR includes a VH domain containing a sequence encoded by SEQ ID NO:207 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:207, and a VL domain containing a sequence encoded by SEQ ID NO:208 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:208.

[0181] In one embodiment, the antigen-binding domain comprises the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:13, 14 and 15 and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:16, 17 and 18, respectively, or the CDR may comprise one to three, or more particularly one or two, amino acid sequences from any of the aforementioned sequences.

[0182] More specifically, in such an embodiment, the antigen-binding domain of the CAR includes a VH domain containing the sequence listed in SEQ ID NO:89 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:90 or a sequence having at least 70% sequence identity with it.

[0183] For example, in such an embodiment, the antigen-binding domain of the CAR includes a VH domain containing a sequence encoded by SEQ ID NO:209 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:209, and a VL domain containing a sequence encoded by SEQ ID NO:210 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:210.

[0184] In one embodiment, the antigen-binding domain comprises the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:19, 20 and 21 and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:22, 23 and 24, respectively, or the CDR may comprise one to three, or more particularly one or two, amino acid sequences from any of the aforementioned sequences.

[0185] More specifically, in such an embodiment, the antigen-binding domain of the CAR includes a VH domain containing the sequence listed in SEQ ID NO:91 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:92 or a sequence having at least 70% sequence identity with it.

[0186] For example, in such an embodiment, the antigen-binding domain of the CAR includes a VH domain containing a sequence encoded by SEQ ID NO:211 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:211, and a VL domain containing a sequence encoded by SEQ ID NO:212 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:212.

[0187] In one embodiment, the antigen-binding domain comprises the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:25, 26 and 27 and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:28, 29 and 30, respectively, or the CDR may comprise one to three, or more particularly one or two, amino acid sequences from any of the aforementioned sequences.

[0188] More specifically, in such an embodiment, the antigen-binding domain of the CAR includes a VH domain containing the sequence listed in SEQ ID NO:93 or a sequence having at least 70% sequence identity therewith, and a VL domain containing the sequence listed in SEQ ID NO:94 or a sequence having at least 70% sequence identity therewith.

[0189] For example, in such an embodiment, the antigen-binding domain of the CAR includes a VH domain containing a sequence encoded by SEQ ID NO:213 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:213, and a VL domain containing a sequence encoded by SEQ ID NO:214 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:214.

[0190] In one embodiment, the antigen-binding domain comprises the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:31, 32 and 33 and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:34, 35 and 36, respectively, or the CDR may comprise one to three, or more particularly one or two, amino acid sequences from any of the aforementioned sequences.

[0191] More specifically, in such an embodiment, the antigen-binding domain of the CAR includes a VH domain containing the sequence listed in SEQ ID NO:95 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:96 or a sequence having at least 70% sequence identity with it.

[0192] For example, in such an embodiment, the antigen-binding domain of the CAR includes a VH domain containing a sequence encoded by SEQ ID NO:215 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:215, and a VL domain containing a sequence encoded by SEQ ID NO:216 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:216.

[0193] In one embodiment, the antigen-binding domain comprises the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:37, 38 and 39, respectively, and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:40, 41 and 42, respectively, or the CDR may comprise one to three, or more particularly one or two, amino acid sequence modifications from any of the aforementioned sequences.

[0194] More specifically, in such an embodiment, the antigen-binding domain of the CAR includes a VH domain containing the sequence listed in SEQ ID NO:97 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:98 or a sequence having at least 70% sequence identity with it.

[0195] For example, in such an embodiment, the antigen-binding domain of the CAR includes a VH domain containing a sequence encoded by SEQ ID NO:217 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:217, and a VL domain containing a sequence encoded by SEQ ID NO:218 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:218.

[0196] In one embodiment, the antigen-binding domain comprises the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:43, 44 and 45 and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:46, 47 and 48, respectively, or the CDR may comprise one to three, or more particularly one or two, amino acid sequences modified from any of the aforementioned sequences.

[0197] More specifically, in such an embodiment, the antigen-binding domain of the CAR includes a VH domain containing the sequence listed in SEQ ID NO:99 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:100 or a sequence having at least 70% sequence identity with it.

[0198] For example, in such an embodiment, the antigen-binding domain of the CAR includes a VH domain containing a sequence encoded by SEQ ID NO:219 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:219, and a VL domain containing a sequence encoded by SEQ ID NO:220 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:220.

[0199] In one embodiment, the antigen-binding domain comprises the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:49, 50 and 51 and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:52, 53 and 54, respectively, or the CDR may comprise one to three, or more particularly one or two, amino acid sequences from any of the aforementioned sequences.

[0200] More specifically, in such an embodiment, the antigen-binding domain of the CAR includes a VH domain containing the sequence listed in SEQ ID NO:101 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:102 or a sequence having at least 70% sequence identity with it.

[0201] For example, in such an embodiment, the antigen-binding domain of the CAR includes a VH domain containing a sequence encoded by SEQ ID NO:221 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:221, and a VL domain containing a sequence encoded by SEQ ID NO:222 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:222.

[0202] In one embodiment, the antigen-binding domain comprises the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:55, 56 and 57 and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:58, 59 and 60, respectively, or the CDR may comprise one to three, or more particularly one or two, amino acid sequences from any of the aforementioned sequences.

[0203] More specifically, in such an embodiment, the antigen-binding domain of the CAR includes a VH domain containing the sequence listed in SEQ ID NO:103 or a sequence having at least 70% sequence identity therewith, and a VL domain containing the sequence listed in SEQ ID NO:104 or a sequence having at least 70% sequence identity therewith.

[0204] For example, in such an embodiment, the antigen-binding domain of the CAR includes a VH domain containing a sequence encoded by SEQ ID NO:223 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:223, and a VL domain containing a sequence encoded by SEQ ID NO:224 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:224.

[0205] In one embodiment, the antigen-binding domain comprises the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:61, 62 and 63 and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:64, 65 and 66, respectively, or the CDR may comprise one to three, or more particularly one or two, amino acid sequences from any of the aforementioned sequences.

[0206] More specifically, in such an embodiment, the antigen-binding domain of the CAR includes a VH domain containing the sequence listed in SEQ ID NO:105 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:106 or a sequence having at least 70% sequence identity with it.

[0207] For example, in such an embodiment, the antigen-binding domain of the CAR includes a VH domain containing a sequence encoded by SEQ ID NO:225 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:225, and a VL domain containing a sequence encoded by SEQ ID NO:226 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:226.

[0208] In one embodiment, the antigen-binding domain comprises the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:67, 68 and 69 and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:70, 71 and 72, respectively, or the CDR may comprise one to three, or more particularly one or two, amino acid sequences modified from any of the aforementioned sequences.

[0209] More specifically, in such an embodiment, the antigen-binding domain of the CAR includes a VH domain containing the sequence listed in SEQ ID NO:107 or a sequence having at least 70% sequence identity therewith, and a VL domain containing the sequence listed in SEQ ID NO:108 or a sequence having at least 70% sequence identity therewith.

[0210] For example, in such an embodiment, the antigen-binding domain of the CAR includes a VH domain containing a sequence encoded by SEQ ID NO:227 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:227, and a VL domain containing a sequence encoded by SEQ ID NO:228 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:228.

[0211] In one embodiment, the antigen-binding domain comprises the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:73, 74 and 75 and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:76, 77 and 78, respectively, or the CDR may comprise one to three, or more particularly one or two, amino acid sequences from any of the aforementioned sequences.

[0212] More specifically, in such an embodiment, the antigen-binding domain of the CAR includes a VH domain containing the sequence listed in SEQ ID NO:109 or a sequence having at least 70% sequence identity therewith, and a VL domain containing the sequence listed in SEQ ID NO:110 or a sequence having at least 70% sequence identity therewith.

[0213] For example, in such an embodiment, the antigen-binding domain of the CAR includes a VH domain containing a sequence encoded by SEQ ID NO:229 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:229, and a VL domain containing a sequence encoded by SEQ ID NO:230 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:230.

[0214] In one embodiment, the antigen-binding domain comprises the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:79, 80 and 81 and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:82, 83 and 84, respectively, or the CDR may comprise one to three, or more particularly one or two, amino acid sequences from any of the aforementioned sequences.

[0215] More specifically, in such an embodiment, the antigen-binding domain of the CAR includes a VH domain containing the sequence listed in SEQ ID NO:111 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:112 or a sequence having at least 70% sequence identity with it.

[0216] For example, in such an embodiment, the antigen-binding domain of the CAR includes a VH domain containing a sequence encoded by SEQ ID NO:231 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:231, and a VL domain containing a sequence encoded by SEQ ID NO:232 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:232.

[0217] When a CDR does contain amino acid sequence modifications, this can be the deletion, addition, or substitution of amino acid residues in the CDR sequence as listed in SEQ ID NO above. More specifically, the modification can be amino acid substitution, such as conserved amino acid substitution, as described above. Longer CDRs can tolerate more amino acid residue modifications. In the case of CDRs with a length of 5 or more, or 7 or more amino acid residues, the modification can be 0 residues, 1 residue, 2 residues, or 3 residues, for example, 2 residues. Typically, 0, 1, 2, or 3 modifications can be present for any given CDR sequence. Furthermore, in one embodiment, CDR1 and CDR2 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.

[0218] The antigen-binding domain can be in the form of an scFv, which contains the VH domain sequence and the VL domain sequence as described above in any order, such as VH-VL. The VH and VL sequences can be linked by an adapter sequence.

[0219] Suitable linkers can be easily selected and can have any suitable length, for example from 1 amino acid (e.g., Gly) to 30 amino acids, such as from 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 to 30, 5 to 25, 6 to 25, 10 to 15, 12 to 25, 15 to 25 amino acids, etc.

[0220] 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.

[0221] 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 connect or link proteins or protein domains to produce, for example, fusion proteins or chimeric proteins, or multifunctional proteins or peptides. Flexible linkers can have different properties, such as being 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 have also been 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.

[0222] Flexible joints are joints that allow a certain degree of movement between the linked domains or components. Flexible joints 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 for the mobility of the linked parts (domains or components). The incorporation of polar amino acids can maintain the stability of the joint in an aqueous environment by forming hydrogen bonds with water molecules.

[0223] 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 (see, for example, Chen et al., 2013), 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 and reported in the art can be used.

[0224] Although the length of the linker is not critical, a shorter linker sequence may be desirable in some implementations. For example, the length of the linker sequence may not exceed 25 amino acids, and optionally not exceed 24, 23, 22, or 21 amino acids.

[0225] In other embodiments, longer connector sequences may be required, such as connector sequences consisting of multiple repetitions of the GS domain or containing multiple repetitions of the GS domain.

[0226] In some embodiments, the length of the linker 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 length of the linker 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 length of the linker can be an intermediate range between these ranges, such as 6 to 21, 6 to 20, 7 to 20, 8 to 20, 9 to 20, 10 to 20, 8 to 18, 9 to 18, 10 to 18, 9 to 17, 10 to 17, 9 to 16, 10 to 16, etc. Therefore, the length of the linker can be within a range consisting of any integers listed above.

[0227] Using GS linkers, or more specifically, using GS (“Gly-Ser”) domains within the linker, allows for easy alteration of the linker length by varying the number of GS domain repeats; thus, such linkers represent a class of linkers. However, flexible linkers are not limited to those based on “GS” repeats, and other linkers containing Ser and Gly residues dispersed throughout the linker sequence have been reported, as described in the aforementioned Chen et al.

[0228] Therefore, in one embodiment, the linker sequence may contain at least 40% Gly residues or Gly residues and Ser residues.

[0229] In another embodiment, the linker sequence may contain Ser residues and / or Gly residues and contain no more than 15 other amino acid residues, optionally 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 can be any amino acid other than Ser or Gly.

[0230] Pro residues in the linker tend to impart rigidity, so in one implementation, 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.

[0231] In one embodiment, the linker sequence comprises at least one Gly-Ser domain consisting only of Ser and Gly residues. In such an embodiment, the linker may comprise no more than 15 other amino acid residues, optionally no more than 14, 13, 12, 11, 10, 9, 8, 6, 7, 5, or 4 other amino acid residues.

[0232] The Gly-Ser domain can have the following formula:

[0233] (S)q-[(G)m-(S)m]n-(G)p

[0234] Where q is 0 or 1; m is an integer from 1 to 8; n is an integer of at least 1 (e.g., 1 to 8, or more specifically 1 to 6); and p is 0 or an integer from 1 to 3.

[0235] More specifically, the Gly-Ser domain can have the following formula:

[0236] (i)S-[(G)mS]n;

[0237] (ii)[(G)mS]n; or

[0238] (iii)[(G)mS]n-(G)p

[0239] Where m is an integer from 2 to 8 (e.g., 3 to 4); n is an integer of at least 1 (e.g., 1 to 8, or more specifically 1 to 6); and p is 0 or an integer from 1 to 3.

[0240] In a representative example, the Gly-Ser domain can have the following formula:

[0241] S-[GGGGS]n

[0242] Where n is an integer of at least 1 (optionally, 1 to 8, or 1 to 6, 1 to 5, 1 to 4 or 1 to 3).

[0243] The representative sequence GGGS is shown in SEQ ID NO.163.

[0244] The linker sequence may consist of only one or more Gly-Ser domains as described above or as defined, or it may consist of one or more Gly-Ser domains. 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 at one or both ends of the Gly-Ser domains 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, additional amino acids may be located flanking the Gly-Ser domains. In other embodiments, additional amino acids may be located between the Gly-Ser domains. For example, two Gly-Ser domains may be located flanking a segment of other amino acids in the linker sequence. Furthermore, as described above, in other linkers, the GS domain does not need to be repeated, and G residues and / or S residues or short domains such as GS may simply be distributed along the length or sequence.

[0245] The following are representative exemplary connector sequences:

[0246] ETSGGGGSRL (SEQ ID NO.164)

[0247] SGGGGSGGGGSGGGGS((SEQ ID NO.165)

[0248] S(GGGGS) 1-5 (Where GGGGS is SEQ ID NO.166)

[0249] (GGGGS) 1-5 (Where GGGGS is SEQ ID NO.166)

[0250] S(GGGS) 1-5 (Where GGGS is SEQ ID NO.163)

[0251] (GGGS) 1-5 (Where GGGS is SEQ ID NO.163)

[0252] S(GGGGGS) 1-5 (Where GGGGGS is SEQ ID NO.167)

[0253] (GGGGGS) 1-5 (Where GGGGGS is SEQ ID NO.167)

[0254] S(GGGGGGS) 1-5 (Where GGGGGGS is SEQ ID NO.168)

[0255] (GGGGGGS) 1-5 (Where GGGGGGS is SEQ ID NO.168)

[0256] GGGGSGGGGSGGGGS(SEQ ID NO:169)

[0257] GGGGG (SEQ ID NO:170)

[0258] GGGGSGGGGS(SEQ ID NO:171)

[0259] GGGGSGGGGSGGGGSGGGGS(SEQ ID NO:172)

[0260] GGGGGGG(SEQ ID NO:173)

[0261] G6 (SEQ ID NO.174)

[0262] G8 (SEQ ID NO.175)

[0263] KESGSSVSSEQLAQFRSLD(SEQ ID NO.176)

[0264] EGKSSGSGSESKST(SEQ ID NO.177)

[0265] GSAGSAAGSGEF (SEQ ID NO.178)

[0266] SGGGGSAGSAAGSGEF(SEQ ID NO.179)

[0267] SGGGLLLLLLLLGGGS(SEQ ID NO.180)

[0268] SGGGAAAAAAAAGGGS(SEQ ID NO.181)

[0269] SGGGAAAAAAAAAAAAAAAAGGGS(SEQ ID NO.182)

[0270] SGALGGLALAGLLLAGLGLGAAGS(SEQ ID NO.183)

[0271] SLSLSPGGGGGPAR(SEQ ID NO.184)

[0272] SLSLSPGGGGGPARSLLSLSPGGGGG(SEQ ID NO.185)

[0273] GSSGSS (SEQ ID NO.186)

[0274] GSSSSSS(SEQ ID NO.187)

[0275] GGSSSS (SEQ ID NO.188)

[0276] GSSSSS (SEQ ID NO.189)

[0277] SGGGGS (SEQ ID NO.190).

[0278] In some embodiments, the connector has sequence (GGGGS)3 (SEQ ID NO:169).

[0279] Although the adapter sequence defined above is a flexible sequence, this disclosure also includes other polypeptides, including those that contain non-flexible adapters and / or do not meet the above definitions and requirements.

[0280] Another example of a linker that can be used to connect the VH and VL domains is KLEEGEFSEARV (SEQ ID NO: 233) or a sequence having at least about 60% identity with it, such as a sequence having at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% identity with it. Alternatively, the linker may have the sequence of SEQ ID NO: 233 or a sequence differing by no more than 6, for example, no more than 5, 4, 3, 2, or 1 amino acid. Specifically, the linker can be truncated or lengthened by one or more amino acids at the N-terminus and / or C-terminus (see, for example, Schmiedl A. et al. (Protein Eng. 2000 Oct; 13(10):725-34, wherein the linker is shortened by one amino acid at each end compared to SEQ ID NO:233). To improve the stability of the linker and remove the two putative trypsin cleavage sites (lysine and arginine), the linker can be modified by exchanging lysine (K) with isoleucine (I), arginine (R) with glycine (G), and valine (V) with cysteine ​​(C). The resulting linker has the amino acid sequence ILEEGEFSEAGC (SEQ ID NO:234). Any peptide linker carrying the shared amino acid sequence X1LEEGEFSEAX2X3 (SEQ ID NO:235) can also be used, wherein X1 is K or I, X2 is R or G, and X3 is V or C.

[0281] Therefore, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence listed in SEQ ID NO. 85, which is connected to the VL sequence of SEQ ID NO. 86 via a linker of sequence (X)n, wherein X is any amino acid and n is an integer between 15 and 25.

[0282] Therefore, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence listed in SEQ ID NO. 87, which is connected to the VL sequence of SEQ ID NO. 88 via a linker of sequence (X)n, wherein X is any amino acid and n is an integer between 15 and 25.

[0283] Therefore, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence listed in SEQ ID NO. 89, which is connected to the VL sequence of SEQ ID NO. 90 via a linker of sequence (X)n, wherein X is any amino acid and n is an integer between 15 and 25.

[0284] Therefore, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence listed in SEQ ID NO. 91, which is connected to the VL sequence of SEQ ID NO. 92 via a linker of sequence (X)n, wherein X is any amino acid and n is an integer between 15 and 25.

[0285] Therefore, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence listed in SEQ ID NO. 93, which is connected to the VL sequence of SEQ ID NO. 94 via a linker of sequence (X)n, wherein X is any amino acid and n is an integer between 15 and 25.

[0286] Therefore, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence listed in SEQ ID NO. 95, which is connected to the VL sequence of SEQ ID NO. 96 via a linker of sequence (X)n, wherein X is any amino acid and n is an integer between 15 and 25.

[0287] Therefore, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence listed in SEQ ID NO. 97, which is connected to the VL sequence of SEQ ID NO. 98 via a linker of sequence (X)n, wherein X is any amino acid and n is an integer between 15 and 25.

[0288] Therefore, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence listed in SEQ ID NO. 99, which is connected to the VL sequence of SEQ ID NO. 100 via a linker of sequence (X)n, wherein X is any amino acid and n is an integer between 15 and 25.

[0289] Therefore, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence listed in SEQ ID NO. 101, the VH sequence being linked to the VL sequence of SEQ ID NO. 102 via a linker of sequence (X)n, wherein X is any amino acid and n is an integer between 15 and 25.

[0290] Therefore, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence listed in SEQ ID NO. 103, which is connected to the VL sequence of SEQ ID NO. 104 via a linker of sequence (X)n, wherein X is any amino acid and n is an integer between 15 and 25.

[0291] Therefore, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence listed in SEQ ID NO. 105, the VH sequence being linked to the VL sequence of SEQ ID NO. 106 via a linker of sequence (X)n, wherein X is any amino acid and n is an integer between 15 and 25.

[0292] Therefore, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence listed in SEQ ID NO. 107, which is connected to the VL sequence of SEQ ID NO. 108 via a linker of sequence (X)n, wherein X is any amino acid and n is an integer between 15 and 25.

[0293] Therefore, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence listed in SEQ ID NO. 109, which is connected to the VL sequence of SEQ ID NO. 110 via a linker of sequence (X)n, wherein X is any amino acid and n is an integer between 15 and 25.

[0294] Therefore, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence listed in SEQ ID NO. 111, the VH sequence being linked to the VL sequence of SEQ ID NO. 112 via a linker of sequence (X)n, wherein X is any amino acid and n is an integer between 15 and 25.

[0295] In this regard, in one embodiment, the antigen-binding domain of the CAR comprises a sequence listed in SEQ ID NO:113 or a sequence having at least 80% identity with it, or consists of a sequence listed in SEQ ID NO:113 or a sequence having at least 80% identity with it.

[0296] In another embodiment, in one embodiment, the antigen-binding domain of the CAR comprises the sequence listed in SEQ ID NO:114 or a sequence having at least 80% identity with it, or consists of the sequence listed in SEQ ID NO:114 or a sequence having at least 80% identity with it.

[0297] In another embodiment, in one embodiment, the antigen-binding domain of the CAR comprises the sequence listed in SEQ ID NO:115 or a sequence having at least 80% identity with it, or consists of the sequence listed in SEQ ID NO:115 or a sequence having at least 80% identity with it.

[0298] In another embodiment, in one embodiment, the antigen-binding domain of the CAR comprises a sequence listed in SEQ ID NO:116 or a sequence having at least 80% identity with it, or consists of a sequence listed in SEQ ID NO:116 or a sequence having at least 80% identity with it.

[0299] In another embodiment, in one embodiment, the antigen-binding domain of the CAR comprises the sequence listed in SEQ ID NO:117 or a sequence having at least 80% identity with it, or consists of the sequence listed in SEQ ID NO:117 or a sequence having at least 80% identity with it.

[0300] In another embodiment, the antigen-binding domain of the CAR comprises a sequence listed in SEQ ID NO:118 or a sequence having at least 80% identity with it, or consists of a sequence listed in SEQ ID NO:118 or a sequence having at least 80% identity with it.

[0301] In another embodiment, in one embodiment, the antigen-binding domain of the CAR comprises a sequence listed in SEQ ID NO:119 or a sequence having at least 80% identity with it, or consists of a sequence listed in SEQ ID NO:119 or a sequence having at least 80% identity with it.

[0302] In another embodiment, in one embodiment, the antigen-binding domain of the CAR comprises the sequence listed in SEQ ID NO:120 or a sequence having at least 80% identity with it, or consists of the sequence listed in SEQ ID NO:120 or a sequence having at least 80% identity with it.

[0303] In another embodiment, in one embodiment, the antigen-binding domain of the CAR comprises a sequence listed in SEQ ID NO:121 or a sequence having at least 80% identity with it, or consists of a sequence listed in SEQ ID NO:121 or a sequence having at least 80% identity with it.

[0304] In another embodiment, in one embodiment, the antigen-binding domain of the CAR comprises a sequence listed in SEQ ID NO:122 or a sequence having at least 80% identity with it, or consists of a sequence listed in SEQ ID NO:122 or a sequence having at least 80% identity with it.

[0305] In another embodiment, in one embodiment, the antigen-binding domain of the CAR comprises a sequence listed in SEQ ID NO:123 or a sequence having at least 80% identity with it, or consists of a sequence listed in SEQ ID NO:123 or a sequence having at least 80% identity with it.

[0306] In another embodiment, in one embodiment, the antigen-binding domain of the CAR comprises the sequence listed in SEQ ID NO:124 or a sequence having at least 80% identity with it, or consists of the sequence listed in SEQ ID NO:124 or a sequence having at least 80% identity with it.

[0307] In another embodiment, in one embodiment, the antigen-binding domain of the CAR comprises the sequence listed in SEQ ID NO:125 or a sequence having at least 80% identity with it, or consists of the sequence listed in SEQ ID NO:125 or a sequence having at least 80% identity with it.

[0308] In another embodiment, in one embodiment, the antigen-binding domain of the CAR comprises the sequence listed in SEQ ID NO:126 or a sequence having at least 80% identity with it, or consists of the sequence listed in SEQ ID NO:126 or a sequence having at least 80% identity with it.

[0309] In one embodiment, the antigen-binding domain of the CAR comprises or consists of an amino acid sequence that is encoded by the sequence listed in SEQ ID NO:191 or has at least 80% identity with the amino acid sequence encoded by the sequence listed in SEQ ID NO:191. For example, the antigen-binding domain of the CAR may comprise or consist of an amino acid sequence that is encoded by the amino acid 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:191, or a variant thereof.

[0310] In one embodiment, the antigen-binding domain of the CAR comprises or consists of an amino acid sequence that is encoded by the sequence listed in SEQ ID NO:192 or has at least 80% identity with the amino acid sequence encoded by the sequence listed in SEQ ID NO:192. For example, the antigen-binding domain of the CAR may comprise or consist of an amino acid sequence that is encoded by the amino acid 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:192, or a variant thereof.

[0311] In one embodiment, the antigen-binding domain of the CAR comprises or consists of an amino acid sequence that is encoded by the sequence listed in SEQ ID NO:193 or has at least 80% identity with the amino acid sequence encoded by the sequence listed in SEQ ID NO:193. For example, the antigen-binding domain of the CAR may comprise or consist of an amino acid sequence that is encoded by the amino acid 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:193, or a variant thereof.

[0312] In one embodiment, the antigen-binding domain of the CAR comprises or consists of an amino acid sequence that is encoded by the sequence listed in SEQ ID NO:194 or has at least 80% identity with the amino acid sequence encoded by the sequence listed in SEQ ID NO:194. For example, the antigen-binding domain of the CAR may comprise or consist of an amino acid sequence that is encoded by the amino acid 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:194, or a variant thereof.

[0313] In one embodiment, the antigen-binding domain of the CAR comprises or consists of an amino acid sequence that is encoded by the sequence listed in SEQ ID NO:195 or has at least 80% identity with the amino acid sequence encoded by the sequence listed in SEQ ID NO:195. For example, the antigen-binding domain of the CAR may comprise or consist of an amino acid sequence that is encoded by the amino acid 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:195, or a variant thereof.

[0314] In one embodiment, the antigen-binding domain of the CAR comprises or consists of an amino acid sequence that is encoded by the sequence listed in SEQ ID NO:196 or has at least 80% identity with the amino acid sequence encoded by the sequence listed in SEQ ID NO:196. For example, the antigen-binding domain of the CAR may comprise or consist of an amino acid sequence that is encoded by the amino acid 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:196, or a variant thereof.

[0315] In one embodiment, the antigen-binding domain of the CAR comprises or consists of an amino acid sequence that is encoded by the sequence listed in SEQ ID NO:197 or has at least 80% identity with the amino acid sequence encoded by the sequence listed in SEQ ID NO:197. For example, the antigen-binding domain of the CAR may comprise or consist of a variant thereof, that is, an amino acid sequence encoded by the sequence remaining after removing the sequence encoding the signal sequence and / or any tag sequence (e.g., myc tag and / or his tag).

[0316] In one embodiment, the antigen-binding domain of the CAR comprises or consists of an amino acid sequence that is encoded by the sequence listed in SEQ ID NO:198 or has at least 80% identity with the amino acid sequence encoded by the sequence listed in SEQ ID NO:198. For example, the antigen-binding domain of the CAR may comprise or consist of an amino acid sequence that is encoded by the amino acid 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:198, or a variant thereof.

[0317] In one embodiment, the antigen-binding domain of the CAR comprises or consists of an amino acid sequence that is encoded by the sequence listed in SEQ ID NO:199 or has at least 80% identity with the amino acid sequence encoded by the sequence listed in SEQ ID NO:199. For example, the antigen-binding domain of the CAR may comprise or consist of an amino acid sequence that is encoded by the amino acid 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:199, or a variant thereof.

[0318] In one embodiment, the antigen-binding domain of the CAR comprises or consists of an amino acid sequence that is encoded by the sequence listed in SEQ ID NO:200 or has at least 80% identity with the amino acid sequence encoded by the sequence listed in SEQ ID NO:200. For example, the antigen-binding domain of the CAR may comprise or consist of an amino acid sequence that is encoded by the amino acid 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:200, or a variant thereof.

[0319] In one embodiment, the antigen-binding domain of the CAR comprises or consists of an amino acid sequence that is encoded by the sequence listed in SEQ ID NO:201 or has at least 80% identity with the amino acid sequence encoded by the sequence listed in SEQ ID NO:201. For example, the antigen-binding domain of the CAR may comprise or consist of an amino acid sequence that is encoded by the amino acid 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:201, or a variant thereof.

[0320] In one embodiment, the antigen-binding domain of the CAR comprises or consists of an amino acid sequence that is encoded by the sequence listed in SEQ ID NO:202 or has at least 80% identity with the amino acid sequence encoded by the sequence listed in SEQ ID NO:202. For example, the antigen-binding domain of the CAR may comprise or consist of an amino acid sequence that is encoded by the amino acid 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:202, or a variant thereof.

[0321] In one embodiment, the antigen-binding domain of the CAR comprises or consists of an amino acid sequence that is encoded by the sequence listed in SEQ ID NO:203 or has at least 80% identity with the amino acid sequence encoded by the sequence listed in SEQ ID NO:203. For example, the antigen-binding domain of the CAR may comprise or consist of an amino acid sequence that is encoded by the amino acid 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:203, or a variant thereof.

[0322] In one embodiment, the antigen-binding domain of the CAR comprises or consists of an amino acid sequence that is encoded by the sequence listed in SEQ ID NO:204 or has at least 80% identity with the amino acid sequence encoded by the sequence listed in SEQ ID NO:204. For example, the antigen-binding domain of the CAR may comprise or consist of an amino acid sequence that is encoded by the amino acid 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:204, or a variant thereof.

[0323] In another embodiment, the CAR construct may comprise a sequence or a variant thereof listed in any of SEQ ID NO:237 to 262, for example, a sequence having at least 80% identity with it, or consist of a sequence or a variant thereof listed in any of SEQ ID NO:237 to 262, for example, a sequence having at least 80% identity with it. In some embodiments, the CAR construct may comprise a sequence or a variant thereof listed in SEQ ID NO:238, for example, a sequence having at least 80% identity with it, or consist of a sequence or a variant thereof listed in SEQ ID NO:238, for example, a sequence having at least 80% identity with it. In some embodiments, the CAR construct may comprise a sequence or a variant thereof listed in SEQ ID NO:240, for example, a sequence having at least 80% identity with it, or consist of a sequence or a variant thereof listed in SEQ ID NO:240, for example, a sequence having at least 80% identity with it. In some embodiments, the CAR construct may comprise a sequence listed in SEQ ID NO:241 or a variant thereof, such as a sequence having at least 80% identity with it, or consist of a sequence listed in SEQ ID NO:241 or a variant thereof, such as a sequence having at least 80% identity with it. In some embodiments, the CAR construct may comprise a sequence listed in SEQ ID NO:243 or a variant thereof, such as a sequence having at least 80% identity with it, or consist of a sequence listed in SEQ ID NO:243 or a variant thereof, such as a sequence having at least 80% identity with it. In some embodiments, the CAR construct may comprise a sequence listed in SEQ ID NO:249 or a variant thereof, such as a sequence having at least 80% identity with it, or consist of a sequence listed in SEQ ID NO:249 or a variant thereof, such as a sequence having at least 80% identity with it. In some embodiments, the CAR construct may comprise a sequence listed in SEQ ID NO:253 or a variant thereof, such as a sequence having at least 80% identity with it, or consist of a sequence listed in SEQ ID NO:253 or a variant thereof, such as a sequence having at least 80% identity with it.

[0324] 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.

[0325] The CAR may also optionally include a hinge domain to keep the extracellular domain, particularly the antigen-binding domain, away from the cell surface, and also includes a transmembrane domain. The hinge and transmembrane domains may comprise hinge and transmembrane sequences from any protein having a hinge domain and / or a transmembrane domain, including any type I, type II, or type III transmembrane proteins. The hinge domain may be selected from CD28, CD8α, CD4, CD7, CH2CH3, the hinge region of an immunoglobulin, or portions or variants thereof. Typically, the hinge may be derived from CD8, particularly CD8α, or derived from CH2CH3. In one embodiment, the hinge may contain one or more cysteine ​​residues, for example, to allow disulfide bonds. For example, a CD8 hinge may contain one or more cysteine ​​residues, such as one cysteine ​​residue, two cysteine ​​residues, or three cysteine ​​residues.

[0326] The transmembrane domains of a CAR may also contain artificial hydrophobic sequences. The transmembrane domains of a CAR may be selected to be non-dimerized. Additional 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(18Pt 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(18Pt 1):5426-35); 4) CD3ζ TM region (Pule et al, Mol Ther, 2005, Nov; 12(5):933-41); 4) CD3ζ TM region (Pule et al, Mol Ther, 2005, Nov; 12(5):933-41); 5) CD28 ...6) CD3ζ TM region (Pule et al, Mol Ther, 2005, Nov; 12(5):933-41); 7) CD3ζ TM region (Pule et al, Mol Ther, 2005, Nov; 12(5):933-41); 8) CD3ζ TM region (Pule et al, Mol Ther, 2005, Nov; 12(5):933-41); 9) Ther, 2005, Nov; 12 (5): 933-41; Savoldo B, Blood, 2009, Jun 18; 113 (25): 6392-402.); 5) CD8αTM 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(18Pt 1):5426-35;Milone et al,Mol Ther,2009,Aug;17(8):1453-64.). Other transmembrane domains that can be used include those derived from ICOS, CD4, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD154, or CH2CH3. Alternatively, the transmembrane domain may be derived from CD4, CD28, CD8α, or CH2CH3.

[0327] In one embodiment, the CAR may not contain a dimerizing domain that binds to a regulatory molecule. A regulatory molecule is any molecule capable of binding to 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 another embodiment, dimerization with other CAR molecules may be uncontrolled. In yet another embodiment, monovalent binding of the CAR to an antigen may allow activation of cells expressing the CAR.

[0328] The hinge domain can be readily derived from the same protein as the transmembrane domain. In one embodiment, when the transmembrane domain is derived from the CD8α transmembrane domain, the hinge domain is derived from the CD8α hinge domain. In an alternative embodiment, when the transmembrane domain is derived from the CH2CH3 transmembrane domain, the hinge domain is derived from the CH2CH3 hinge domain.

[0329] Alternatively, hinge domains can be derived from proteins that are different from transmembrane domains. For example, hinge domains can be derived from CH2CH3 hinge domains, and transmembrane domains can be derived from CD28 transmembrane domains.

[0330] 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:236 or a variant having at least 80% identity with SEQ ID NO:236. Suitably, the variant may have at least 85%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO:236. An example of a modified CD8α hinge domain is shown in SEQ ID NO:132.

[0331] For example, the hinge domain may comprise an amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO:267 or SEQ ID NO:268, or a variant thereof having at least 80% identity with SEQ ID NO:267 or SEQ ID NO:268. Suitably, the variant may have at least 85%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO:267 or SEQ ID NO:268.

[0332] For example, the transmembrane domain may be derived from the CD8α transmembrane domain, and the transmembrane domain may contain the amino acid sequence shown in SEQ ID NO:129—representing amino acid positions 183 to 203 of human CD8α, or a variant having at least 80% identity with SEQ ID NO:129. Suitably, the variant may have at least 85%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO:129.

[0333] 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. 131 or SEQ ID NO. 133, or a variant thereof having at least 80% sequence identity with it. The variant may have at least 85%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO: 131 or SEQ ID NO. 133, respectively. SEQ ID NO. 131 comprises a modified hinge domain that contains an amino acid modification of two cysteine ​​residues relative to the wild-type CD8α hinge sequence. The modified CD8α hinge domain sequence is shown in SEQ ID NO. 132. The wild-type CD8α hinge and transmembrane domain sequence is shown in SEQ ID NO. 133. The terminal 6 amino acids of SEQ ID NO. 131 and SEQ ID NO. 133, when present, are not located in the membrane and form part of the intracellular domain of the CAR. Variants of such hinge sequences having at least 80% sequence identity with SEQ ID NO. 132 or SEQ ID NO. 133 may be used.

[0334] For example, the hinge domain may be derived from the CH2CH3 hinge domain, and the hinge domain may contain the sequence shown in SEQ ID NO. 130 or SEQ ID NO. 134, or variants thereof having at least 80% identity with SEQ ID NO. 130 or SEQ ID NO. 134, respectively. Variants may have at least 85%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO. 130 or SEQ ID NO. 134, respectively.

[0335] Alternatively, examples of CD28 hinge and transmembrane sequences that can be used are SEQ ID NO:135 or variants thereof that are 80% identical to SEQ ID NO:135. Variants may be at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:135.

[0336] In another example, the CAR may contain a natural or modified CD8α hinge domain and a CD28 transmembrane domain, or, for example, a CD28 hinge domain and a CD8α transmembrane domain based on the sequence given above.

[0337] In one embodiment, a CH2CH3 hinge sequence containing one or more cysteine ​​residues may be used, such as 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, such as one, two, three, four, or more cysteine ​​residues.

[0338] In one embodiment, the transmembrane domain may be derived from the CD4 transmembrane domain, and the transmembrane domain may contain the sequence shown in SEQ ID NO:263, or a variant thereof having at least 80% identity with SEQ ID NO:263. The variant may have at least 85%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO:263.

[0339] The CAR may also contain a signal (or leader) sequence that targets the endoplasmic reticulum pathway for expression on the cell surface. An exemplary signal / leader sequence is MALPVTALLLPLALLLHAAAP as shown in SEQ ID NO. 136. This comprises a single amino acid substitution compared to the wild-type CD8α sequence MALPVTALLLPLALLLHAARP shown in SEQ ID NO. 137. Any sequence or a variant sequence having at least 70% sequence identity with it can be used. For example, the variant sequence may have at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with it.

[0340] As described herein, the intracellular domains of CARs contain transduction effector functional signals and motifs necessary to guide CAR-expressing cells to perform their specialized functions upon antigen binding. Specifically, the intracellular domains may contain one or more (e.g., two or three) immune receptor tyrosine-based activation motifs (ITAMs), which typically contain 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 T cell receptors or any homolog thereof (e.g., η-chain, FcεR1γ and β-chains, MB1 (Igα) chain, B29 (Igβ) chain, etc.), CD3 polypeptide domains (Δ, δ, and ε), syk family tyrosine kinases (Syk, Zap70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transduction such as CD2, CD5, and CD28. Intracellular signal transduction domains may include human CD3ζ chain intracellular domains, FcγRIII, FcsRI, cytoplasmic tails of Fc receptors, immune receptor tyrosine-based activation motifs (ITAMs) carrying cytoplasmic receptors, or combinations thereof.

[0341] Typically, the intracellular signal transduction domain comprises the intracellular signal transduction domain of the human CD3ζ chain. The sequence of the intracellular signal transduction domain of the human CD3ζ chain is listed in SEQ ID NO. 138. The CAR may comprise a CD3ζ signal transduction domain comprising the sequence listed in SEQ ID NO. 138 or a sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO. 138, or consisting of the sequence listed in SEQ ID NO. 138 or a sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO. 138. In one embodiment, the signal transduction domain comprises or consists of SEQ ID NO. 138.

[0342] 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 may not include a co-stimulatory domain derived from 4-1BB within its intracellular domain.

[0343] This CAR may contain a complex intracellular domain comprising a fusion of the intracellular portion of a T cell costimulatory molecule with, 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 CD28 costimulatory domain. This provides the most potent costimulatory signal, namely immune signal 2, which triggers T cell proliferation. The CAR intracellular domain may also contain one or more TNF receptor family signaling domains, such as ICOS, (CD134)OX40, 4-1BB, CD27, or TNFRSF25 signaling domains, or portions or variants thereof, although optionally the CAR may not contain an intracellular domain containing both CD28 and 4-1BB signaling domains.

[0344] Intracellular signaling domains of CD28 that can be used as co-stimulatory domains are shown in SEQ ID NO. 140. Illustrative sequences of the OX40, 4-1BB, ICOS, and TNFRSF25 signaling domains are shown in SEQ ID NO: 141 to 144. CARs may comprise one or more co-stimulatory domains comprising sequences of any one of SEQ ID NO: 140, 141, 142, 143, and 144 or variants thereof having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with them, or consisting of sequences of any one of SEQ ID NO: 140, 141, 142, 143, and 144 or variants thereof having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with them.

[0345] 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 of the combination shown in SEQ ID NO:139, or variants thereof having at least 80% sequence identity with it. Variants may have at least 85%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO:139.

[0346] In one embodiment, the CAR includes a human CD8 hinge domain or a variant thereof, and a human CD8 transmembrane domain. Alternatively or additionally, the CAR includes an intracellular domain comprising, substantially composed of, or composed of, a human CD28 co-stimulatory domain and a human CD3ζ signaling domain.

[0347] In one implementation, the CAR includes the following hinge, transmembrane, and intracellular (or intracellular) domains:

[0348] (i) CH2CH3 hinge sequence, comprising a sequence listed in SEQ ID NO. 130 or SEQ ID NO. 134 or a sequence having at least 80% sequence identity with it, or consisting of a sequence listed in SEQ ID NO. 130 or SEQ ID NO. 134 or a sequence having at least 80% sequence identity with it;

[0349] (ii) a CD28 transmembrane and co-stimulatory domain comprising a sequence listed in SEQ ID NO. 139 or a sequence having at least 80% sequence identity with it, or consisting of a sequence listed in SEQ ID NO. 139 or a sequence having at least 80% sequence identity with it; and

[0350] (iii) CD3ζ signal transduction domain, which contains the sequence listed in SEQ ID NO.138 or a sequence having at least 80% sequence identity with it, or is composed of the sequence listed in SEQ ID NO.138 or a sequence having at least 80% sequence identity with it.

[0351] In an alternative implementation, the CAR includes the following hinge, transmembrane, and intracellular (or intracellular) domains:

[0352] (i) CD8α hinge and transmembrane domain sequence, comprising the sequence listed in SEQ ID NO. 131 or a sequence having at least 80% sequence identity with it, or consisting of the sequence listed in SEQ ID NO. 131 or a sequence having at least 80% sequence identity with it;

[0353] (ii) a CD28 co-stimulatory domain comprising a sequence listed in SEQ ID NO. 140 or a sequence having at least 80% sequence identity with it, or consisting of a sequence listed in SEQ ID NO. 140 or a sequence having at least 80% sequence identity with it; and

[0354] (iii) CD3ζ signal transduction domain, which contains the sequence listed in SEQ ID NO.138 or a sequence having at least 80% sequence identity with it, or is composed of the sequence listed in SEQ ID NO.138 or a sequence having at least 80% sequence identity with it.

[0355] In another embodiment, the CAR includes the following hinge, transmembrane, and intracellular (or intracellular) domains:

[0356] (i) CD8α hinge and transmembrane domain sequence, comprising the sequence listed in SEQ ID NO. 133 or a sequence having at least 80% sequence identity with it, or consisting of the sequence listed in SEQ ID NO. 133 or a sequence having at least 80% sequence identity with it;

[0357] (ii) a CD28 co-stimulatory domain comprising a sequence listed in SEQ ID NO. 140 or a sequence having at least 80% sequence identity with it, or consisting of a sequence listed in SEQ ID NO. 140 or a sequence having at least 80% sequence identity with it; and

[0358] (iii) CD3ζ signal transduction domain, which contains the sequence listed in SEQ ID NO.138 or a sequence having at least 80% sequence identity with it, or is composed of the sequence listed in SEQ ID NO.138 or a sequence having at least 80% sequence identity with it.

[0359] The CAR encoded and expressed may also include a leader sequence that comprises or has at least 80% sequence identity with a sequence listed in SEQ ID NO. 137, or is composed of or has at least 80% sequence identity with a sequence listed in SEQ ID NO. 137.

[0360] The antigen-binding domain of a CAR may comprise a sequence listed in SEQ ID NO. 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126 or a sequence having at least 80% sequence identity with the CAR and capable of binding ENTPD3, or may consist of a sequence listed in SEQ ID NO. 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126 or a sequence having at least 80% sequence identity with the CAR and capable of binding ENTPD3.

[0361] Therefore, in general, a representative CAR can include:

[0362] (i) A leader sequence comprising the sequence listed in SEQ ID NO. 137 or a sequence having at least 80% sequence identity with it, or consisting of the sequence listed in SEQ ID NO. 137 or a sequence having at least 80% sequence identity with it;

[0363] (ii) An antigen-binding domain comprising a sequence listed in SEQ ID NO. 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126 or a sequence having at least 80% sequence identity with such sequences, or consisting of a sequence listed in SEQ ID NO. 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126 or a sequence having at least 80% sequence identity with such sequences;

[0364] (iii) CH2CH3 hinge domain sequence, which includes the sequence listed in SEQ ID NO. 130 or 134 or a sequence having at least 80% sequence identity with it, or consists of the sequence listed in SEQ ID NO. 130 or 134 or a sequence having at least 80% sequence identity with it;

[0365] (iv) A CD28 transmembrane and costimulatory domain comprising a sequence listed in SEQ ID NO. 139 or a sequence having at least 80% sequence identity with it, or consisting of a sequence listed in SEQ ID NO. 139 or a sequence having at least 80% sequence identity with it; and

[0366] (v) CD3ζ signal transduction domain, which contains the sequence shown in SEQ ID NO. 138 or a sequence having at least 80% sequence identity with it, or is composed of the sequence shown in SEQ ID NO. 138 or a sequence having at least 80% sequence identity with it.

[0367] Alternative representative CARs may include:

[0368] (i) A leader sequence comprising the sequence listed in SEQ ID NO. 137 or a sequence having at least 80% sequence identity with it, or consisting of the sequence listed in SEQ ID NO. 137 or a sequence having at least 80% sequence identity with it;

[0369] (ii) An antigen-binding domain comprising a sequence listed in SEQ ID NO. 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126 or a sequence having at least 80% sequence identity with such sequences, or consisting of a sequence listed in SEQ ID NO. 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126 or a sequence having at least 80% sequence identity with such sequences;

[0370] (iii) CD8 hinge and transmembrane domain sequence, comprising a sequence listed in SEQ ID NO. 131 or 133 or a sequence having at least 80% sequence identity with it, or consisting of a sequence listed in SEQ ID NO. 131 or 133 or a sequence having at least 80% sequence identity with it;

[0371] (iv) A CD28 co-stimulatory domain comprising a sequence listed in SEQ ID NO. 140 or a sequence having at least 80% sequence identity with it, or consisting of a sequence listed in SEQ ID NO. 140 or a sequence having at least 80% sequence identity with it; and

[0372] (v) CD3ζ signal transduction domain, which contains the sequence shown in SEQ ID NO. 138 or a sequence having at least 80% sequence identity with it, or is composed of the sequence shown in SEQ ID NO. 138 or a sequence having at least 80% sequence identity with it.

[0373] The CAR of the present invention may comprise any one or more sequences listed in SEQ ID No. 237 to 262 or variants thereof having at least about 80% identity with them, or may consist of any one or more sequences listed in SEQ ID No. 237 to 262 or variants thereof having at least about 80% identity with them. For example, any variant may have at least about 85%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID No. 237 to 262. The CAR of SEQ ID No. 237 to 262 or variants thereof may also comprise a signal sequence, such as a signal sequence having a sequence listed in SEQ ID NO: 136 or 137.

[0374] CARs can bind to ENTPD3 and transduce signals to cells that express it.

[0375] 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.

[0376] The intracellular domains of the CAR described herein may include additional domains. For example, the CAR may include a domain that confers the ability to provide productive IL signaling to cells expressing it in an antigen-specific manner without the need for exogenous IL administration. For example, the CAR may include a domain containing a STAT5 association motif, a JAK1 and / or JAK2 binding motif, and optionally a JAK3 binding motif. In such embodiments, the intracellular domains may include one or more sequences of intracellular domains derived from cytokine receptors, such as interleukin receptor (IL) receptors. Such CARs are described in WO2020 / 044055 (also incorporated herein by reference). Exemplary amino acid sequences derived from the IL-2 receptor β containing the STAT5 association motif and the JAK binding motif are provided in SEQ ID NO:145-147. The inclusion of such domains confers the ability of the CAR to provide productive IL signaling to cells expressing it 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 a sequence corresponding to all or part of the β-chain intracellular domain of the IL receptor or its variants (such as the IL2 receptor), optionally combined with the γ-chain intracellular domain of the IL receptor or its variants (such as the IL2 receptor).

[0377] Alternatively or additionally, additional nucleic acid sequences or peptides can be introduced into cells or cell populations to improve cell persistence, 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, for example, involve the use of engineered or chimeric receptors capable of delivering 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 those described in WO 2020 / 183131, WO 2017 / 029512, WO 2012 / 138858, WO 2014 / 172584, WO 2017 / 068360, WO 2021 / 023987, WO 2020 / 180664, and WO The receptors described in 2020 / 044239; or engineered receptors with bound activating molecules, such as those described in WO 2017 / 201432 and WO 2019 / 183389.

[0378] 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, such as CAR, FOXP3, and safety switches, 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 independent or discrete components. This means that although these polypeptides are encoded by a single nucleic acid molecule, during or after translation, by “cleavage” at the encoded cleavage site, these polypeptides can be expressed or generated as independent polypeptides, and thus, at the end of the intracellular protein production process, these polypeptides may exist in the cell as independent entities or independent polypeptide chains. Alternatively, the additional exogenous polypeptides may be encoded by independent nucleic acid molecules or vectors.

[0379] "Discrete" or "independent" polypeptides refer to polypeptides that are not connected to each other and are physically distinct. In fact, after expression, these polypeptides are located at different or independent cellular locations. Therefore, CAR, FOXP3, and the safety switch polypeptide are ultimately expressed as single and independent components. CAR is expressed as a cell surface molecule. The safety switch polypeptide can be expressed intracellularly or on the cell surface. In a particular embodiment, the safety switch polypeptide and CAR are expressed on the cell surface for use in ACT. FOXP3 is expressed intracellularly, wherein FOXP3 can function as a transcription factor to regulate cell development and / or activity, as further described below.

[0380] Safety switch peptides provide a suicide component for the cells in which they are expressed, either intracellularly or on cells. This serves as a useful safety mechanism to remove cells that have been administered to a subject when needed, or more broadly, according to will or demand, such as when the cells have performed or completed their therapeutic function.

[0381] The suicide component has the ability to induce cell death, or more generally, cell elimination or clearance. An example of a suicide component is a suicide protein encoded by a suicide gene that can be expressed intracellularly or on a cell along with a target transgene—in this case, a CAR—and whose expression allows for cell clearance, thereby shutting down CAR expression. The suicide component in this article is a suicide peptide that, under permissive conditions—that are induced or activated—can lead to cell clearance.

[0382] The suicide moiety may be a polypeptide or amino acid sequence that can be activated by an activator administered to a subject to perform cell scavenging activity, or it may be active in the presence of a substrate that can be administered to a subject to perform cell scavenging activity. In a particular embodiment, the suicide moiety may represent a target of a separate cell scavenger administered to a subject. By binding to the suicide moiety, the cell scavenger can target cells to be scavenged. In particular, the suicide moiety may be recognized by an antibody, and when expressed on the cell surface, the binding of the antibody to the safety switch polypeptide results in the elimination or scavenging of the cell.

[0383] The suicide portion can be HSV-TK or iCasp9. However, the suicide portion can be an epitope recognized by a cell-clearing antibody or other binding molecules capable of inducing cell clearance, or contain an epitope recognized by a cell-clearing antibody or other binding molecules capable of inducing cell clearance. In such an embodiment, the safety switch peptide is expressed on the surface of the cell.

[0384] In this article, the term "cleansing" used in the context of cell clearance is synonymous with "removal," "ablation," or "elimination." This term is used to encompass cell killing or inhibition of cell proliferation, thereby reducing the number of cells in a subject's body. Ideally, 100% complete removal is achieved, but this is not always possible. Reducing the number of cells in a subject or inhibiting their proliferation may be sufficient to produce beneficial effects.

[0385] Specifically, the suicide portion can be a CD20 epitope recognized by the antibody rituximab. Therefore, in a safety switch peptide, the suicide portion can contain a minimal epitope based on a CD20 epitope recognized by the antibody rituximab. Biosimilars of rituximab are available and can be used. Those skilled in the art can readily prepare antibodies with rituximab binding specificity using its available amino acid sequence and conventional methods.

[0386] CAR-cells that are specific to ENTPD3 and also express a safety switch peptide containing this sequence can be selectively killed using rituximab or antibodies with rituximab binding specificity. The safety switch peptide is expressed on the cell surface, and cell death follows when the expressed peptide is exposed to or comes into contact with rituximab or antibodies with the same binding specificity.

[0387] Therefore, rituximab or antibodies with their binding specificity can be provided in combination 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.

[0388] For example, suicide constructs of WO2013 / 153391 or WO2021 / 239812 (both of which are incorporated herein by reference) can be used in cells or cell populations (e.g., Tregs or Treg populations) as described herein.

[0389] The nucleic acid molecule of the present invention can be designed to increase FOXP3 expression in cells (e.g., Treg cells) by introducing a nucleotide sequence encoding FOXP3 into the cell, where the term FOXP3 is synonymous with the term "FOXP3 polypeptide". Therefore, the nucleic acid molecule, as well as constructs and vectors comprising the nucleic acid molecule, provides a method for increasing FOXP3 in cells such as Treg or CD4+ cells. As discussed above, a single nucleic acid molecule can encode the CAR and FOXP3 polypeptide of the present invention, or the CAR and FOXP3 can also be encoded by independent or discrete nucleic acid molecules. Therefore, the present invention provides a cell comprising a nucleic acid molecule containing a nucleotide sequence encoding a CAR and a nucleic acid molecule containing a nucleotide sequence encoding FOXP3, the cell being particularly a pluripotent stem cell (e.g., iPSC), HPC cell (e.g., expressing CD34), CD4+ T cell, or Treg cell.

[0390] "FOXP3" is an abbreviation for Forkhead Box P3 protein. FOXP3 is a member of the FOX protein family of transcription factors and plays a key regulatory role in the development and function of regulatory T cells as a key regulator of this pathway. As used in this article, "FOXP3" includes variants, isotypes, and functional fragments of FOXP3.

[0391] "Increased FOXP3 expression" means an increase in 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 such cell populations) 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 such cell populations) not modified according to the present invention. Optionally, the cells are Tregs, or the cell population is a Treg population.

[0392] 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). Optionally, the cells are Treg cells or the cell population is a Treg population.

[0393] Techniques for measuring specific mRNA and protein levels are well known in the art. mRNA levels in cell populations, such as Treg populations, can be measured using techniques such as the Affymetrix eBioscience Prime Flow RNA assay, Northern blotting, Serial 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).

[0394] "FOXP3 polypeptide" is a polypeptide with FOXP3 activity, that is, a polypeptide capable of binding to FOXP3 target DNA and functioning as a transcription factor regulating Treg development and function. Specifically, the FOXP3 polypeptide may have the same or similar activity as wild-type FOXP3 (SEQ ID NO. 148), 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 the 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 proteins produced by the 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, incorporated herein by reference). As discussed in detail below, FOXP3 or FOXP3 peptides include their functional fragments, variants, and isotypes, such as the functional fragments, variants, and isotypes of SEQ ID NO. 148.

[0395] A “functional fragment of FOXP3” can refer to a portion or region of a FOXP3 polypeptide or a polynucleotide (i.e., a nucleotide sequence) encoding a FOXP3 polypeptide, having the same or similar activity as the full-length FOXP3 polypeptide or polynucleotide. The functional fragment may have at least 40%, 50%, 60%, 70%, 80%, 90%, 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 the known structural and functional characteristics of FOXP3. For example, these are described 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- and C-terminated FOXP3 fragments are described in WO2019 / 241549 (incorporated hereby by reference), for example, the N- and C-terminated FOXP3 fragment having the sequence SEQ ID NO. 149 as described below.

[0396] "FOXP3 variants" may comprise an amino acid or nucleotide sequence having at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, or 90%, optionally at least 95%, 97%, or 99% identity with the FOXP3 polypeptide or polynucleotide encoding the FOXP3 polypeptide (e.g., SEQ ID NO. 148). FOXP3 variants may have the same or similar activity as the wild-type FOXP3 polypeptide or polynucleotide, for example, they may have 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 turnover times (or degradation rates) in Treg cells, for example, at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the turnover time (or degradation rate) of wild-type FOXP3 in Tregs. Some FOXP3 variants may have reduced turnaround time (or degradation rate) compared to wild-type FOXP3, for example, FOXP3 variants with amino acid substitutions at amino acid position 418 and / or amino acid position 422 of SEQ ID NO. 148, such as S418E and / or S422A, as described in WO2019 / 241549 (incorporated herein by reference) and listed in SEQ ID NO. 150 to 152, which represent the aa418 mutant, aa422 mutant, aa418 mutant and aa422 mutant, respectively.

[0397] Suitable, the FOXP3 polypeptide encoded by the nucleic acid molecule, construct or vector described herein may comprise a polypeptide sequence of human FOXP3 (e.g., UniProtKB accession number Q9BZS1 (SEQ ID NO:148)) or a functional fragment or variant thereof, or may consist of a polypeptide sequence comprising human FOXP3 or a functional fragment or variant thereof.

[0398] In some embodiments of the present invention, the FOXP3 polypeptide comprises an amino acid sequence or a functional fragment thereof having at least 70% identity with SEQ ID NO: 148, or is composed of an amino acid sequence or a functional fragment thereof having at least 70% identity with SEQ ID NO: 148. Suitably, the FOXP3 polypeptide comprises an amino acid sequence or a functional fragment thereof 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: 148, or is composed of an amino acid sequence or a functional fragment thereof 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: 148. In some embodiments, the FOXP3 polypeptide comprises SEQ ID NO: 148 or a functional fragment thereof, or is composed of SEQ ID NO: 148 or a functional fragment thereof.

[0399] In some embodiments, as described above, the FOXP3 peptide may contain a mutation of residue 418 and / or residue 422 of SEQ ID NO. 148, as listed in SEQ ID NO. 150, SEQ ID NO. 151 or SEQ ID NO. 152.

[0400] In some embodiments of the present invention, the FOXP3 polypeptide may be truncated at its N and / or C ends to produce functional fragments. In particular, the N and C-terminal truncated functional fragments of FOXP3 may comprise or consist of the amino acid sequence of SEQ ID NO. 149 or a functional variant thereof having at least 80%, 85%, 90%, 95%, or 99% identity with it.

[0401] Suitably, the FOXP3 peptide may be a variant of SEQ ID NO:148, such as a native variant. Suitably, the FOXP3 peptide is an isotype of SEQ ID NO:148. For example, the FOXP3 peptide may contain a deletion of amino acids 72 to 106 relative to SEQ ID NO:148. Alternatively, the FOXP3 peptide may contain a deletion of amino acids 246 to 272 relative to SEQ ID NO:148.

[0402] Suitablely, the FOXP3 peptide comprises SEQ ID NO:153 or a functional fragment thereof. SEQ ID NO:153 represents an illustrative FOXP3 peptide.

[0403] Suitably, the FOXP3 polypeptide comprises an amino acid sequence or a functional fragment thereof having at least 70% identity with SEQ ID NO:153, or is composed of an amino acid sequence or a functional fragment thereof having at least 70% identity with SEQ ID NO:153. Suitably, the FOXP3 polypeptide comprises an amino acid sequence or a functional fragment thereof 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:153. In some embodiments, the FOXP3 polypeptide comprises SEQ ID NO:153 or a functional fragment thereof, or is composed of SEQ ID NO:153 or a functional fragment thereof.

[0404] Suitably, the FOXP3 peptide may be a variant of SEQ ID NO:153, such as a natural variant. Suitably, the FOXP3 peptide is an isotype of SEQ ID NO:153 or a functional fragment thereof. For example, the FOXP3 peptide may contain a deletion of amino acids 72 to 106 relative to SEQ ID NO:153. Alternatively, the FOXP3 peptide may contain a deletion of amino acids 246 to 272 relative to SEQ ID NO:153.

[0405] Suitablely, the polynucleotide encoding the FOXP3 polypeptide comprises or consists of the nucleotide sequence listed in SEQ ID NO:154, which represents the illustrative FOXP3 nucleotide sequence.

[0406] 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:154 or comprises 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:154 or comprises a fragment thereof encoding a functional FOXP3 polypeptide. In some embodiments of the invention, the polynucleotide encoding a FOXP3 polypeptide or variant comprises SEQ ID NO:154 or a fragment thereof encoding a functional FOXP3 polypeptide, or consists of SEQ ID NO:154 or a fragment thereof encoding a functional FOXP3 polypeptide.

[0407] Suitablely, the polynucleotide encoding the FOXP3 polypeptide comprises or consists of the polynucleotide sequence listed in SEQ ID NO:155, which represents another illustrative FOXP3 nucleotide.

[0408] 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:155 or comprises 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:155 or comprises a fragment thereof encoding a functional FOXP3 polypeptide. In some embodiments of the invention, the polynucleotide encoding a FOXP3 polypeptide or variant comprises SEQ ID NO:155 or a fragment thereof encoding a functional FOXP3 polypeptide, or consists of SEQ ID NO:155 or a fragment thereof encoding a functional FOXP3 polypeptide.

[0409] Those skilled in the art will understand that FOXP3 expression within Tregs can be indirectly increased by introducing a polynucleotide into the cell that encodes a protein that enhances FOXP3 transcription and / or translation, or prolongs the half-life of FOXP3 (e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%), or enhances FOXP3 function (e.g., as determined by the repressive capacity of the transduced Treg, as described above). 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 a non-coding sequence (CNS, such as CNS1, 2, or 3) found upstream of the coding region.

[0410] Suitablely, the polynucleotide encoding the FOXP3 polypeptide or a functional fragment or variant thereof may be codon-optimized. Suitablely, the polynucleotide encoding the FOXP3 polypeptide or a functional fragment or variant thereof may be codon-optimized for expression in human cells.

[0411] As described above, nucleic acid molecules can contain nucleotide sequences encoding self-cleaving sequences. In particular, 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, as described 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 jumping and result in a form of cleavage in which the ribosome skips the formation of a peptide bond between the C-terminus of the 2A peptide and the downstream amino acid sequence. The “cleavage” occurs between the glycine and proline residues at the C-terminus of the 2A peptide, meaning that an upstream cistron will be added to the terminal with some additional residues, while a downstream cistron will begin with proline. The term “cleavage” as used herein therefore includes the skipping of peptide bond formation.

[0412] Suitable self-cleaving domains include the P2A, T2A, E2A, and F2A sequences shown in SEQ ID NO:156 to 159, respectively. The sequences can be modified to include the amino acid GSG at the N-terminus of the 2A peptide. Therefore, possible alternatives also include sequences corresponding to SEQ ID NO:156-159, but with a GSG at the 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. (ibid.), such as the TaV sequence.

[0413] The self-cleaving sequences contained in nucleic acid molecules may be the same or different. In one embodiment, the self-cleaving sequences contained in nucleic acid molecules are all 2A sequences, particularly P2A and / or T2A sequences.

[0414] The self-cleaving sequence may include an additional cleavage site that can be cleaved by common enzymes present in the cell. This can facilitate complete removal of the 2A sequence post-translation. Such an additional cleavage site may, for example, include a furin protease cleavage site RXXR (SEQ ID NO:160), or RRKR (SEQ ID NO:161).

[0415] In a representative embodiment, the nucleic acid molecule may comprise: a nucleotide sequence encoding a CAR targeting ENTPD3 having a sequence of any one of SEQ ID NO. 237 to 262 or a variant thereof as described herein, a nucleotide sequence encoding a safety switch, and a nucleotide sequence encoding FOXP3.

[0416] In such an implementation, the CAR may include:

[0417] (a) A leader sequence comprising the sequence listed in SEQ ID NO. 137 or a sequence having at least 80% sequence identity with it, or consisting of the sequence listed in SEQ ID NO. 137 or a sequence having at least 80% sequence identity with it;

[0418] (b) An antigen-binding domain comprising a sequence listed in SEQ ID NO. 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126 or a sequence having at least 80% sequence identity with such sequences, or consisting of a sequence listed in SEQ ID NO. 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126 or a sequence having at least 80% sequence identity with such sequences;

[0419] (c) CD8α hinge and transmembrane domain sequence, comprising a sequence listed in SEQ ID NO. 131 or 133 or a sequence having at least 80% sequence identity with it, or consisting of a sequence listed in SEQ ID NO. 131 or 133 or a sequence having at least 80% sequence identity with it;

[0420] (d) A CD28 co-stimulatory domain comprising a sequence listed in SEQ ID NO. 140 or a sequence having at least 80% sequence identity with it, or consisting of a sequence listed in SEQ ID NO. 140 or a sequence having at least 80% sequence identity with it; and

[0421] (e) CD3ζ signal transduction domain, which contains the sequence listed in SEQ ID NO.138 or a sequence having at least 80% sequence identity with it, or is composed of the sequence listed in SEQ ID NO.138 or a sequence having at least 80% sequence identity with it.

[0422] In an alternative implementation, the CAR may include:

[0423] (a) A leader sequence comprising the sequence listed in SEQ ID NO. 137 or a sequence having at least 80% sequence identity with it, or consisting of the sequence listed in SEQ ID NO. 137 or a sequence having at least 80% sequence identity with it;

[0424] (b) An antigen-binding domain comprising a sequence listed in SEQ ID NO. 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126 or a sequence having at least 80% sequence identity with such sequences, or consisting of a sequence listed in SEQ ID NO. 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126 or a sequence having at least 80% sequence identity with such sequences;

[0425] (c) CH2CH3 hinge domain sequence, which includes the sequence listed in SEQ ID NO.134 or a sequence having at least 80% sequence identity with it, or consists of the sequence listed in SEQ ID NO.134 or a sequence having at least 80% sequence identity with it;

[0426] (d) The CD28 transmembrane and co-stimulatory domain, comprising a sequence listed in SEQ ID NO. 139 or a sequence having at least 80% sequence identity with it, or consisting of a sequence listed in SEQ ID NO. 139 or a sequence having at least 80% sequence identity with it; and

[0427] (e) CD3ζ signal transduction domain, which contains the sequence listed in SEQ ID NO.138 or a sequence having at least 80% sequence identity with it, or is composed of the sequence listed in SEQ ID NO.138 or a sequence having at least 80% sequence identity with it.

[0428] As can be clearly seen from the above description, in addition to the specific polypeptide and nucleotide sequences mentioned in this article, the uses of their variants or derivatives and fragments are also included.

[0429] The terms “derivative” or “variant”, used interchangeably herein, with respect to the protein or polypeptide of the present invention, include substitution, variation, modification, replacement, deletion, and / or addition of one (or more) amino acid residues in the sequence, provided that the resulting protein or polypeptide retains the desired function (e.g., if 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., an antigen-binding domain variant that binds ENTPD3 retains the ability to bind ENTPD3); if the derivative or variant is a signaling domain, the desired function may be the signal transduction ability of the domain (e.g., activation or inhibition of downstream molecules); if the derivative or variant is a transcription factor (e.g., FOXP3), the desired function may be the [missing information - likely a transcription factor or transcription factor]. The ability of a transcription factor to bind to target DNA and / or induce transcription; if the derivative or variant is a safety switch peptide, the desired function may be the peptide's ability to induce cell death (e.g., after binding to the molecule). Furthermore, all variants or derivatives described 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 function compared to the corresponding reference sequence. A variant or derivative may have similar or the same level of function compared to the corresponding reference sequence or may have an enhanced level of function (e.g., an enhancement of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%).

[0430] Typically, amino acid substitutions can be made, such as one, two, or three to ten or twenty substitutions, 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 the same level 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%).

[0431] Proteins or peptides can also have amino acid residue deletions, insertions, or substitutions, resulting in silencing changes and producing functionally equivalent proteins. Intentional amino acid substitutions can be made based on the similarity of residue polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties, as long as the endogenous 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 without polarized head groups with similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0432] Conservative substitution can be performed, for example, according to Table 1 below.

[0433] Table 1

[0434]

[0435] 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".

[0436] Homologous or variant sequences may include amino acid sequences that have at least 70%, 75%, 85%, or 90% identity with the host sequence, and optionally at least 95%, 96%, 97%, 98%, or 99%. Typically, variants will contain the same active sites as the host amino acid sequence. While homology can also be considered based on similarity (i.e., amino acid residues with similar chemical properties / functions), in the context of this paper, homology is expressed as sequence identity.

[0437] 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.

[0438] Homology or sequence identity percentage of consecutive sequences can be calculated, that is, by aligning one sequence with another, and directly comparing each amino acid in one sequence with the corresponding amino acid in the other sequence, one residue at a time. This is called "vacancy-free" alignment. Typically, this vacancy-free alignment is only performed on a relatively short number of residues.

[0439] While this is an extremely simple and consistent method, it fails to account for situations where, for example, in a pair of otherwise identical sequences, an insertion or deletion in one nucleotide sequence can cause subsequent codons to misalign, potentially leading to a significant drop in the percentage of homology during global alignment. Therefore, most sequence alignment methods aim to generate optimal alignments that account for possible insertions and deletions without unduly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment in an attempt to maximize local homology.

[0440] However, these more sophisticated methods assign a "vacancy penalty" to each vacancy that appears in the alignment, so that for the same number of identical amino acids, an alignment with as few vacancies as possible, reflecting a higher correlation between the two compared sequences, will receive a higher score than one with many vacancies. Affine vacancy cost is commonly used, which charges a relatively high cost for the presence of a vacancy and a smaller penalty for each subsequent residue within the vacancy. This is the most commonly used vacancy scoring system. A high vacancy penalty will naturally result in an optimized alignment with fewer vacancies. Most alignment programs allow modification of the vacancy penalty. However, when using such software for sequence alignment, default values ​​can be used. For example, when using the GGWisconsin Bestfit package, the default vacancy penalty for amino acid sequences is -12 for gaps and -4 for each extension.

[0441] 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 this alignment 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–Ch. 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 to 7-60). However, for certain applications, the GCG Bestfit program can be used. 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).

[0442] While the final percentage of homology can be measured by identity, the sequence 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. An example of such a matrix commonly used is the BLOSUM62 matrix—the default matrix in the BLAST program suite. The GCG Wisconsin program typically uses public defaults or a custom symbol lookup table (if provided) (see the user manual for more details). For some applications, the public defaults of the GCG package can be used, or in other software, a default matrix such as BLOSUM62 can be used. Appropriately, the percentage of identity is determined across the entire reference and / or query sequence.

[0443] Once the software produces the best alignment, it can calculate the percentage of homology, optionally including the percentage of sequence identity. The software typically includes this as part of the sequence comparison and generates numerical results.

[0444] A “fragment” typically refers to a selected region of a polypeptide or polynucleotide of functional interest, such as a functional or encoding 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.

[0445] 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 insert can be prepared, along with 5' and 3' flanking regions corresponding to the 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 number of standard techniques known in the art for manipulating DNA sequences, and other known techniques may also be used.

[0446] Nucleic acid molecules and polynucleotide / nucleotide / nucleic acid sequences as defined herein may comprise 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 on polypeptide sequences encoded by nucleic acid molecules / polynucleotide / nucleotide sequences as defined herein, without affecting the codon usage of any particular host organism in which the polypeptide of the invention will be expressed.

[0447] Nucleic acid molecules / polynucleotides / nucleotides 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.

[0448] Nucleic acid molecule / polynucleotide / nucleotide sequence ratios, such as DNA nucleic acid molecule / polynucleotide / sequence, can be recombinantly, synthetically, or by any means available to those skilled in the art. Nucleic acid molecules / polynucleotide / nucleotide sequences can also be cloned using standard techniques.

[0449] Longer nucleic acid molecules / polynucleotides / nucleotide sequences are typically generated using recombinant methods, such as polymerase chain reaction (PCR) cloning. This involves preparing a pair of primers (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 result in amplification of the desired region, and isolating the amplified fragment (e.g., by purifying the reaction mixture with an 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.

[0450] The nucleic acid molecules / polynucleotides / nucleotides of the present invention may also comprise a nucleic acid sequence encoding a selection marker. Suitable selection markers are well known in the art, and suitable selection markers include, but are not limited to, fluorescent proteins such as GFP. Suitably, the selection marker may be a fluorescent protein such as GFP, YFP, RFP, tdTomato, dsRed, or a variant thereof. In some embodiments, the fluorescent protein is GFP or a GFP variant. The nucleic acid sequence encoding the selection marker may be provided in combination with the nucleic acid molecules herein in the form of a nucleic acid construct. Such a nucleic acid construct may be provided in a vector.

[0451] Suitable, the selected marker / reporter domain can be based on luciferase reporter, PET reporter (e.g., sodium iodide cotransporter (NIS)), or membrane protein (e.g., CD34 or Thy1.1).

[0452] Nucleic acid sequences encoding one or more selectable 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 suitable co-expression sites include, for example, internal ribosome entry sites (IRES) and self-cleavage sites, such as those included in the nucleic acid molecule and as defined above. In one embodiment, this may be a 2A cleavage site as described above.

[0453] The use of selection markers is advantageous because selection markers can be used to screen and isolate cells (e.g., Tregs) from the starting cell population using conventional methods such as flow cytometry that have successfully incorporated the nucleic acid molecules, constructs, or vectors of the present invention (thereby expressing the encoded ENTPD3 CAR and other modules such as FOXP3 and safety switch peptides).

[0454] The nucleic acid molecules / polynucleotides / nucleotides used in this invention can be codon-optimized. Codon optimization has previously been described in WO1999 / 41397 and WO2001 / 79518. Different cells use specific codons differently. This codon bias corresponds to the bias of the relative abundance of a specific tRNA in the cell type. By changing codons in the sequence to match the relative abundance of the corresponding tRNA, expression can be increased. For the same reason, expression can be decreased by intentionally selecting codons known to be rare in a specific cell type for the corresponding tRNA. Therefore, translational control with additional degree is available.

[0455] 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. The promoter is located near the transcription start site of the gene and upstream of the DNA (towards the 5' region of the sense strand). Any suitable promoter can be used, and the selection of any suitable promoter can be readily made by those skilled in the art. The promoter can be from any source and can be a viral promoter or a eukaryotic promoter, including mammalian promoters or human promoters (i.e., physiological promoters). In one embodiment, the promoter is a viral promoter. Specific promoters include LTR promoters, EFS (or functionally truncated versions thereof), SFFV, PGK, and CMV. In one embodiment, the promoter is an SFFV or a viral LTR promoter. In particular, the SFFV promoter can be used within the nucleic acid molecule, construct, or vector of the present invention to allow transcription of the initiating nucleotide sequence. 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 CAR, FOXP3, and / or a safety switch.

[0456] The SFFV promoter may contain nucleotide sequences as listed in SEQ ID NO.162.

[0457] "Operationally linked to the same promoter" means that transcription of nucleic acid / polynucleotide / nucleotide sequences can begin from the same promoter (e.g., transcription of the first, second, and third polynucleotide sequences all begins from the same promoter), and these nucleotide sequences are positioned and oriented to initiate transcription from the promoter. Nucleic acid / polynucleotide / nucleotide sequences operably linked to a promoter are under the transcriptional regulation of that promoter.

[0458] In some embodiments of the invention, the nucleic acid / polynucleotide / nucleotide sequence is contained within an expression vector. As used herein, the term "expression vector" means a construct capable of expressing the CAR peptide and any additional peptides (such as the FOXP3 peptide or a safety switch peptide).

[0459] A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. As used herein and by way of example, some vectors used in recombinant nucleic acid technologies allow the transfer of entities such as nucleic acid segments (e.g., heterologous DNA segments, such as heterologous cDNA segments) into 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 target nucleotide.

[0460] 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.

[0461] In one implementation, the vector is a viral vector, such as a retrovirus, for example a lentiviral vector or a gamma retrovirus vector.

[0462] The vector may also contain 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 at either end of a retrotransposon or proviral DNA formed by reverse transcription of retroviral RNA. They are used by viruses to insert their genetic material into the host genome. Signals of gene expression are present in the LTR, which include enhancers, promoters (which may have transcriptional enhancers or regulatory elements), transcription initiation (e.g., capping), transcription terminators, and polyadenylation signals. Suitably, the vector may include 5' LTRs and 3' LTRs.

[0463] The vector may contain one or more additional regulatory sequences, which may function pre- or post-transcriptionally. A "regulatory sequence" is any sequence that promotes peptide expression, for example, by increasing transcript expression or enhancing mRNA stability. Suitable regulatory sequences include, for example, enhancer elements, post-transcriptional regulatory elements, and polyadenylation sites. Suitably, the additional regulatory sequences may be present in one or more LTRs.

[0464] Suitable, the vector may contain (e.g., operatively linked to a promoter) a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE).

[0465] The vector containing 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, including, for example, infection with recombinant viral vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, baculoviruses, and herpes simplex virus vectors; as well as direct injection of nucleic acids and bioprojectile transformation.

[0466] Non-viral delivery systems include, but are not limited to, DNA transfection methods. Here, transfection includes the process of delivering genes to target cells using non-viral vectors. Non-viral delivery systems may include liposomes or amphiphilic cell-penetrating peptides, optionally complexed with nucleic acid molecules or constructs.

[0467] Typical transfection methods include electroporation, DNA bioejection, lipid-mediated transfection, compressed DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic reagent-mediated transfection, cationic facial amphiphiles (CFA) (Nat. Biotechnol. (1996) 14: 556), and combinations thereof.

[0468] 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 the introduction of these nucleic acid molecules into cells in combination with other vectors, such as vectors encoding other polypeptides, which may also be intended for introduction into cells.

[0469] 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 with viral vectors and transfection with DNA or RNA.

[0470] 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).

[0471] The appropriate cells are discussed further below, but the cells can be derived from samples isolated from the subject. The subject can be a donor subject or a subject used for treatment (i.e., the cells can be autologous cells or donor cells used to introduce another recipient, such as allogeneic cells).

[0472] Cells can be produced by a method including the following steps:

[0473] (i) separating or providing cell-containing samples from or to the subject; and

[0474] (ii) Introducing (e.g., by transduction or transfection) a nucleic acid molecule, construct or vector as defined herein into a cell-containing sample to provide an engineered cell population.

[0475] Samples enriched with target cells can be isolated, enriched, and / or generated from cell-containing samples before and / or after step (ii) of the method. For example, Tregs (or other target cells) can be isolated, enriched, and / or generated before and / or after step (ii) to isolate, enrich, or generate samples enriched with Tregs. Separation and / or enrichment can be performed from cell-containing samples after step (ii) to enrich cells and / or Tregs (or other target cells) containing CARs, nucleic acid molecules / polynucleotides, constructs, and / or vectors as described herein.

[0476] Treg-enriched samples can be isolated or enriched by any method known to those skilled in the art, such as by FACS and / or magnetic bead sorting. Treg-enriched samples can be generated from cell-containing samples by any method known to those skilled in the art, such as 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 to those skilled in the art.

[0477] Suitable, engineered target cells can be generated by a method including the following steps:

[0478] (i) Separating or providing target cell-enriched samples from the subject; and

[0479] (ii) Introducing nucleic acids, constructs or vectors as defined herein into (e.g., by transduction or transfection) a sample enriched with target cells to provide an engineered target cell population.

[0480] Target cells can be Treg cells or their precursors or progenitors.

[0481] "Engineered cells" are cells that have been modified to contain or express polynucleotides not naturally encoded by the cell. Methods for engineered cells are known in the art and include, but are not limited to, genetic modifications of the cell, such as by transduction (e.g., retroviral or lentiviral transduction), transfection (e.g., transient DNA- or RNA-based transfection), including lipid transfection, polyethylene glycol, calcium phosphate, and electroporation, as described above. Nucleic acid sequences can be introduced into the cell using any suitable method. Non-viral techniques such as amphiphilic cell-penetrating peptides can be used for nucleic acid introduction. Cells can also be genetically modified, for example, using any known gene-editing technology such as CRISPR, TALEN, or Zn fingers, to insert nucleotide, polynucleotide, or nucleic acid sequences as described herein into the genome.

[0482] 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. Suitably, engineered cells are cells that have been modified, for example, by transduction or transfection. Suitably, engineered cells are cells that have been modified, for example, by transduction or transfection, or whose genome has been modified. Suitably, engineered cells are cells that have been modified by retroviral transduction, or whose genome has been modified. Suitably, engineered cells are cells that have been modified by lentiviral transduction, or whose genome has been modified.

[0483] As used herein, the term “introduction” refers to a method of inserting a foreign nucleic acid (e.g., DNA or RNA) into a cell. As used herein, the term introduction includes 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 in one of a variety of ways, including transduction with a viral vector and transfection with DNA or RNA. Cells can be activated and / or expanded before or after introducing 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. Suitablely, IL-2 can be replaced by 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, “activated” means that the cells have been stimulated to result in cell proliferation. As used herein, “amplified” means that cells or cell populations have been induced to proliferate. The expansion of a cell population can be measured, for example, by counting the number of cells present in a population. The phenotype of the cells can be determined by methods known in the art, such as flow cytometry.

[0484] 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 (CTLs; CD8+ T cells), helper T cells (HTLs; CD4+ T cells), and regulatory T cells (Tregs). Other T cell populations, such as naive T cells and memory T cells, may also be used in this study. Other immune cells include NK cells, NKT cells, tolerant 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 pluripotent stem cells (e.g., HPCs) or cells directed to a particular cell 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 the target immune cells.

[0485] Most notably, the immune cells can be NK cells, dendritic cells, MDSCs, or T cells, such as cytotoxic T lymphocytes (CTLs), helper T cells, or Treg cells.

[0486] In one implementation, 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.

[0487] As used herein, T cells are lymphocytes that include any type of T cell, such as αβ T cells (e.g., CD8 or CD4+), γδ T cells, memory T cells, and Treg cells.

[0488] Suitablely, immunosuppressive function can refer to the ability of Tregs to reduce or suppress one or more of a variety of physiological and cellular effects induced by the immune system in response to stimuli such as pathogens, allogeneic antigens, or autoantigens. 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 of Tconv in the presence of Tregs will indicate the ability of Tregs to suppress the immune response. For example, a relatively reduced secretion of cytokines will indicate a weaker immune response, and therefore the ability of Tregs to suppress the immune response. Tregs can also suppress the immune response by modulating 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 co-cultured activated Tregs in vitro.

[0489] Assays are known in the art for measuring the intensity of immune responses, thereby measuring the inhibitory capacity of Tregs. Specifically, antigen-specific Tconv cells can be co-cultured with Tregs, and peptides of the corresponding antigens can be added to the co-culture to stimulate responses from the Tconv cells. The degree of Tconv cell proliferation and / or the amount of the cytokine IL-2 secreted by them in response to the peptide addition can be used as indicators of the inhibitory capacity of the co-cultured Tregs.

[0490] The proliferation of antigen-specific Tconv cells co-cultured with the Tregs 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 containing nucleic acids, expression constructs, or vectors as defined herein (e.g., Tregs) can have increased inhibitory activity (e.g., an increase of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of inhibitory activity) compared to unengineered Tregs.

[0491] Antigen-specific Tconv cells co-cultured with the Tregs described herein may express at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% less effector cytokines than corresponding Tconv cells cultured in the absence of Tregs (e.g., in the presence of unengineered Tregs). 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. Suitably, effector cytokines may be selected from IL-2, IL-17, TNFα, GM-CSF, and IFN-γ.

[0492] Several distinct Treg subgroups have been identified that can express different or different levels of specific biomarkers. Tregs typically express the biomarkers CD4, CD25, and FOXP3 (CD4+). + CD25 + FOXP3 + T cells.

[0493] Treg cells can also express CTLA-4 (cytotoxic T-lymphocyte-associated molecule-4) or GITR (glucocorticoid-induced TNF receptor).

[0494] Treg cells are found in peripheral blood, lymph nodes, and tissues. The Tregs used in this article include natural Treg cells derived from the thymus (nTreg), peripherally generated Tregs, and induced Treg cells (iTreg).

[0495] The surface protein CD127 is absent or expressed at low levels (CD4). + CD25 + CD127 or CD4 + CD25 + In cases of low CD127 combinations, Tregs can be identified using the cell surface markers CD4 and CD25. The use of such markers for Treg identification is known in the art and is described, for example, in Liu et al. (JEM; 2006; 203; 7(10); 1701-1711).

[0496] Treg can be CD4 + CD25 + FOXP3 + T cells, CD4 + CD25 + CD127-T cells, or CD4 + CD25 + FOXP3 + CD127 - / 低 T cells.

[0497] 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 + Neuropilin 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).

[0498] Tregs can possess demethylated Treg-specific demethylation regions (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).

[0499] 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 γδFoxp3 + T cells.

[0500] It is known that different Treg subgroups exist, including the initial Treg (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 elevated levels of CD45RO (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more CD45RO) compared to the initial Tregs, and optionally do not express or have low levels of CD45RA (mRNA and / or protein) compared to the initial Tregs (e.g., at least 80%, 90%, or 95% less CD45RA than the initial Tregs). "Cytokine-producing Tregs" refers to Tregs that do not express or have very low levels of CD45RA (mRNA and / or protein) compared to the initial Tregs (e.g., at least 80%, 90%, or 95% less CD45RA than the initial Tregs), and "cytokine-producing Tregs" have low levels of FOXP3 compared to memory Tregs, such as 50%, 60%, 70%, 80%, or 90% less FOXP3 than memory Tregs. Cytokine-producing Tregs can produce interferon-gamma and may exhibit lower inhibitory activity compared to the naïve Treg (e.g., at least 50%, 60%, 70%, 80%, or 90% lower inhibitory activity than the naïve Treg). 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 levels can be determined by Western blotting, or optionally by using antibody-stained FACS to determine cell surface expression.

[0501] Specifically, Tregs can be naïve Tregs. As used interchangeably in this text, "naïve regulatory T cell," "naïve T regulatory cell," or "naïve Treg" refers to a Treg cell that expresses CD45RA (specifically, expresses CD45RA on its cell surface). Therefore, naïve Tregs are described as CD45RA-expressing Tregs. +Nascent Tregs typically represent Tregs that have not yet been activated by peptide / MHC via their endogenous TCR, while effector / memory Tregs refer to Tregs that have been activated by stimulation of their endogenous TCR. Typically, nascent Tregs may express at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more CD45RA than non-nascent Treg cells (e.g., memory Treg cells). Alternatively, nascent Treg cells may express at least 2, 3, 4, 5, 10, 50, or 100 times more CD45RA than non-nascent Treg cells (e.g., memory Treg cells). The expression level of CD45RA can be readily determined by methods in the art, such as flow cytometry using commercially available antibodies. Typically, non-nascent Treg cells do not express CD45RA or express low levels of CD45RA.

[0502] In particular, the initial Treg may not express CD45RO and can be considered as CD45RO. - Therefore, naive Tregs may express at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less CD45RO than memory Tregs, or alternatively, they may express at least 2, 3, 4, 5, 10, 50, or 100 times less CD45RO than memory Tregs.

[0503] Although initial Tregs express CD25 as described above, CD25 expression levels may be lower than those in memory Tregs, depending on the source of the initial Treg. For example, for initial 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 initial Tregs can be considered to express medium to low levels of CD25. However, those skilled in the art will understand that initial Tregs isolated from umbilical cord blood may not show this difference.

[0504] Typically, the initial Treg, as defined in this paper, can be CD4. + CD25 + FOXP3 + CD127 低 CD45RA + .

[0505] As used in this article, low expression of CD127 refers to the expression of CD4 from the same subject or donor. + Non-regulatory or Tcon cells showed lower CD127 expression levels compared to Tcon cells. Specifically, naïve Treg cells could express CD4+ compared to those from the same subject or donor.+ Non-regulatory or Tcon cells have less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% CD127 compared to other cells. CD127 levels can be assessed using standard methods in the art, including flow cytometry of cells stained with an anti-CD127 antibody.

[0506] Typically, naïve Tregs do not express or express low levels of CCR4, HLA-DR, CXCR3, and / or CCR6. Specifically, naïve Tregs may express even lower levels of CCR4, HLA-DR, CXCR3, and CCR6 than memory Tregs, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower expression levels than memory Tregs. Naïve Tregs may further express additional biomarkers, including CCR7. + and CD31 + .

[0507] The isolated initial Tregs can be identified by methods known in the art, which involve determining the presence or absence of any one or more of a set of markers discussed above on the cell surface of the isolated cells. Examples include CD45RA, CD4, CD25, and CD127. 低 This can be used to determine whether cells are naïve Tregs. Methods for determining whether isolated cells are naïve Tregs or have the desired phenotype can be performed as discussed below regarding 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.

[0508] Suitablely, cells, such as Tregs, are isolated from peripheral blood mononuclear cells (PBMCs) obtained from a subject. Suitablely, the subject from whom the PBMCs are obtained is a mammal, optionally a human. Suitablely, the cells are matched (e.g., HLA matched) with the subject to whom the engineered cells will be administered or are their own cells. Suitablely, the subject to be treated is a mammal, optionally a human. Cells can be generated ex vivo from the patient's own peripheral blood (first party), or from donor peripheral blood (second party) or from peripheral blood from an unrelated donor (third party) in the case of hematopoietic stem cell transplantation. Suitablely, the cells are the subject's own cells to whom the engineered cells will be administered.

[0509] Appropriately, Tregs are part of a cell population. Appropriately, a Treg population contains at least 70% Tregs, such as at least 75%, 85%, 90%, 95%, 97%, 98%, or 99% Tregs. Such a population may be referred to as an "enriched Treg population".

[0510] In some respects, Tregs can originate from the in vitro differentiation of induced progenitor cells (e.g., iPSCs) or embryonic progenitor cells into Tregs. The nucleic acid molecules or vectors described herein can be introduced into induced progenitor cells or embryonic progenitor cells before or after differentiation into Tregs. Suitable methods for differentiation are known in the art and include those disclosed in Haque et al., JVisExp., 2016, 117, 54720 (incorporated herein by reference).

[0511] 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 a co-receptor (which may be cluster 4 (CD4) or cluster 8 (CD8)) and does not possess immunosuppressive function. Conventional T cells are found in peripheral blood, lymph nodes, and tissues. Suitablely, 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.

[0512] 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 can be used for the production of nucleic acids (e.g., by cloning), vectors, or peptides.

[0513] This invention also provides cell populations comprising cells as defined or described herein. It should be understood that a cell population may comprise cells of the present invention containing nucleic acid molecules, expression constructs, or vectors as defined herein, as well as cells not containing nucleic acid molecules, expression constructs, or vectors of the present invention (e.g., untransduced or untransfected cells). Although in one embodiment, all cells in the population may contain the nucleic acids, expression constructs, or vectors of the present invention, cell populations are provided having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells containing the nucleic acids, expression constructs, or vectors of the present invention. Furthermore, a cell population may comprise more than one cell type, although in one 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 particularly Tregs. Additionally, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the T cells, particularly the Tregs, may contain the nucleic acids, expression constructs, or vectors of the present invention.

[0514] Specifically, the present invention provides a cell population comprising multiple cells, wherein the multiple cells contain a CAR, or a nucleic acid molecule or vector encoding the CAR, the CAR containing an antigen recognition domain that specifically binds to ENTPD3.

[0515] T cell populations (e.g., Treg populations) can be "educated" or "reprogrammed" by exposing T cells directly (e.g., via antigen-presenting cells) or indirectly to one or more specific antigens (e.g., self-antigens or non-self-antigens), causing these cells to be activated by the aforementioned specific antigens (e.g., in the case of regulatory T cells, to exhibit immunosuppressive or immune-tolerant properties towards these specific antigens) and proliferate. This can occur particularly 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 population with a larger proportion of cells possessing TCRs that bind to those 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 cells' TCRs) has been modified or increased.

[0516] However, this adds an extra step and 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 educated T cells are not used. Therefore, in some embodiments, the clonality of T cells (or the clonality of the cell's TCR or endogenous TCR) is not modified in vitro. For example, the clonality of Tregs (or the TCR clonality of Tregs) is not modified in vitro. Therefore, in some embodiments, the T cell population is not selected to have TCRs that bind pancreatic antigens. As used herein, "clonalness" refers to the diversity of antigens that the TCRs within a T cell population can bind. Therefore, an increase in clonality leads to a decrease in the diversity of antigens that can be bound, and a decrease in clonality leads to an increase in the diversity of antigens that can be bound.

[0517] Specifically, in some embodiments, the T cells are not exposed to one or more specific antigens in vitro, for example, not through antigen-presenting cells (such as dendritic cells). Specifically, in some embodiments, the T cells are not directly (e.g., through antigen-presenting cells) or indirectly exposed to or contacted in vitro with one or more specific antigens (e.g., self- or non-self antigens) such that the cells are activated against those specific antigens. Specifically, the T cells are not exposed to or contacted in vitro with one or more pancreatic antigens.

[0518] A particular approach that can be used for "educational" or "reprogramming" Tregs (specifically, TCRs of Tregs) may involve the following steps:

[0519] (a) Dendritic cells (DCs) are exposed to interleukin-10 (IL-10) to generate tolerant dendritic cells (tolDCs);

[0520] (b) Contacting the tolDC with extracellular vesicles (EVs) of pancreatic islet cells to generate antigen-loaded tolDCs; and

[0521] (c) Contacting Treg cells with the antigen-loaded tolDC to generate the reprogrammed Treg cells.

[0522] The method may also include (e.g., in the presence of IL-2) amplifying the reprogrammed Treg cells. 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, induced-tolerance dendritic cells are not used.

[0523] Therefore, in terms of their endogenous TCRs, the population of T cells (especially Tregs) can be described as polyclonal, i.e., possessing TCRs that are nonspecific to specific antigens, active against multiple antigens, and / or specific to unknown antigens. For example, a TCR can be specific to both pancreatic and non-pancreatic antigens. Tregs with polyclonal TCRs can be immune-tolerant to multiple antigens and / or immunosuppressive against multiple antigens (e.g., both pancreatic and non-pancreatic antigens). It should be noted that the ENTPD3-specific CAR described herein will provide T cells with antigen-specific polyclonal TCRs.

[0524] A pharmaceutical composition is also provided comprising cells or cell populations as defined or described herein, and 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 an introduced nucleic acid molecule. Vectors suitable for gene therapy are known in the art and include viral vectors.

[0525] Therefore, in another aspect, the present invention provides cells, cell populations or pharmaceutical compositions for treatment as defined herein.

[0526] A pharmaceutical composition is a composition comprising a therapeutically effective amount of a pharmaceutically active agent (i.e., cells (e.g., Treg), cell populations, or carriers) or composed thereof. A pharmaceutical composition may optionally include pharmaceutically acceptable carriers, diluents, or excipients (including combinations thereof). Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical industry and described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (ARGennaroedit. 1985). The choice of pharmaceutical carrier, excipient, or diluent can be based on the intended route of administration and standard pharmaceutical practice. As a carrier, excipient, or diluent, or in addition to a carrier, excipient, or diluent, a pharmaceutical composition may include any suitable binder, lubricant, suspending agent, coating agent, or solubilizer.

[0527] "Pharmaceutical acceptable" means that the formulation is sterile and pyrogen-free. The carrier, diluent, and / or excipients must be "acceptable," meaning they are compatible with the cells or carrier and harmless to the recipient. Typically, the carrier, diluent, and excipients will be sterile and pyrogen-free saline or infusion media; however, other acceptable carriers, diluents, and excipients may be used.

[0528] 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.

[0529] 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, although the appropriate dosage may be determined through clinical trials. Pharmaceutical compositions may be formulated accordingly.

[0530] The cells, cell populations, or pharmaceutical compositions described herein can be administered parenterally, such as intravenously or intrathecally, or via infusion techniques. The cells, cell populations, or pharmaceutical compositions can be administered in the form of 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 (optionally to a pH of 3 to 9). The pharmaceutical compositions can be formulated accordingly. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished using standard pharmaceutical techniques well known to those skilled in the art.

[0531] The pharmaceutical composition may contain cells in an infusion medium such as a sterile isotonic solution. The pharmaceutical composition may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0532] Cells, cell populations, or pharmaceutical compositions can be administered in single or multiple doses. In particular, cells, cell populations, or pharmaceutical compositions can be administered in a single, one-time dose. Pharmaceutical compositions can be formulated accordingly.

[0533] Depending on the disease / symptom and the subject to be treated, as well as the route of administration, cells, cell populations, or drug compositions can be administered at specific stages of the disease.

[0534] 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 have been destroyed, in order to maintain at least some functional 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, less than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of pancreatic beta cells may have been destroyed before administration of the cells, cell populations, or pharmaceutical compositions described herein.

[0535] 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., thymoglobin, alemtuzumab (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, TNFalpha, IL18).

[0536] Depending on the disease / condition to be treated, the subject, 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 will vary depending on the specific subject's age, weight, and response. However, typically, for the cells described herein, 5 x 10-1 cells may be administered per subject. 7 3x10 9 One cell or 10 8 2x109 The dose per cell.

[0537] 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.

[0538] The present invention also includes the use of kits comprising the cells, cell populations, and / or pharmaceutical compositions described herein. Optionally, the kit is used for the methods and uses described herein, such as the therapeutic methods described herein. Optionally, the kit includes instructions for use of the kit components.

[0539] The cells, cell populations, and pharmaceutical compositions of the present invention are particularly useful in treating conditions associated with cells expressing ENTPD3 or conditions in which ENTPD3 is located or near the disease site, especially in conditions in which the immunosuppressive or target-killing activity of the cells of the present invention can benefit.

[0540] 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 diseases or conditions treatable by or with CARs. The cells and cell-containing compositions 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, particularly the immunosuppressive effects of Treg cells. Therefore, the cells, cell populations, compositions, and vectors described herein can be used to induce or achieve immunosuppression in subjects. Treg cells administered or modified in vivo 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. Additionally, the cells, cell populations, compositions, and vectors described herein can be used to promote tissue repair and / or tissue regeneration.

[0541] Conditions requiring treatment or prevention include inflammation, or conditions related to or involving inflammation. Inflammation can be chronic or acute. Furthermore, inflammation can be low-level or systemic.

[0542] The term "target cell" refers to any cell expressing ENTPD3, to which the cells of the present invention will be directed to exert their therapeutic effect. In some embodiments, the target cell serves as a marker of the disease site, i.e., attracting the cells of the present invention to provide immunosuppressive effects. 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.

[0543] Specifically, the disease or condition to be treated can be type 1 diabetes. Other diseases or conditions that can be treated with the CAR described herein include, for example, autoimmune pancreatitis, type 2 diabetes, and insulinoma. Specifically, the CAR can be expressed in 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), as well as other cells that secrete immunomodulatory cytokines such as IL-10, TGFβ, IL-35, or bimodal proteins) to treat type 1 diabetes, autoimmune pancreatitis, or type 2 diabetes. The CAR can be expressed in cells with effector functions (e.g., T effector cells, NK cells, NKT cells) to treat insulinoma.

[0544] Furthermore, the CAR described herein can be used to prevent rejection of transplanted cells expressing ENTPD3 (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 immunosuppressive cells (e.g., CD4+ or CD8+ T regulatory cells, tolerant NK or NKT cells, γ-δ cells, and immunomodulatory 1 cells (Tr1), as well as other cells secreting immunomodulatory cytokines such as IL-10, TGFβ, IL-35, or bimodal proteins) to prevent rejection of transplanted cells expressing ENTPD3.

[0545] Engineered cells, such as Treg, can be administered to subjects with a disease to reduce, decrease, or improve at least one symptom of the disease, such as hyperglycemia. At least one symptom can be reduced, decreased, or improved by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%, or at least one symptom can be completely relieved.

[0546] Engineered cells, such as Treg, can be administered to subjects with a disease to slow, reduce, or block disease progression. Compared to subjects who have not received engineered cells, disease progression can be slowed, reduced, or blocked by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%, or disease progression can be completely stopped.

[0547] Specifically, the disease to be treated could be type 1 diabetes. As mentioned above, CAR-Tregs specific to ENTPD3 may be able to migrate to ENTPD3 expression sites and control inflammation through their bystander effect, thereby slowing the rate of pancreatic β-cell destruction.

[0548] 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 stops the body's production of insulin and leads to inflammation of the pancreas. Insulin is the 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, lifelong condition. People with type 1 diabetes currently require close monitoring of their blood sugar levels and ingestion (e.g., by injection or pump) of an appropriate dose of insulin. This treatment is not curable and must be continued. Over time, abnormal 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 glycemic control is particularly poor in younger subjects, especially those aged approximately 16 to 25 years, so the treatments described herein may be particularly suitable for this group.

[0549] Type 1 diabetes is a progressive disease that progresses sequentially through several identifiable stages at varying but predictable rates before the onset of symptoms. This is described in more detail in the literature Insel et al, Diabetes Care. 2015; 38(10):1964-1974. The ability to screen for risk and identify disease stages before the onset of symptoms in type 1 diabetes provides an opportunity for early intervention to delay and ultimately prevent the development of clinical symptoms.

[0550] Individuals at increased risk of developing type 1 diabetes can be identified through genetic screening. The HLA regions on chromosome 6 account for approximately 30% to 50% of the genetic risk for type 1 diabetes, with the HLA class II haplotypes RB1*0301-DQB1*0201 (DR3-DQ2) and DRB1*0401-DQB1*0302 (DR4-DQ8) showing the strongest associations. 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 come from the INS, PTPN22, CTLA4, and IL2RA genes. 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) can be prophylactically administered CAR-Treg as described herein.

[0551] Phase 1 represents individuals who have developed two or more type 1 diabetes-related islet autoantibodies (targeting insulin, GAD65, IA-2, and / or ZnT8) but have normal blood glucose levels.

[0552] 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 from loss of functional pancreatic β-cell clusters to glucose intolerance or abnormal blood glucose levels. Abnormal blood glucose levels 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 from a 75g oral glucose tolerance test (OGTT), high glucose levels at midpoints of the OGTT (30, 60, 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).

[0553] Stage 3 represents individuals with typical clinical symptoms and signs of diabetes, including, for example, polyuria, polydipsia, weight loss, fatigue, and diabetic ketoacidosis (DKA).

[0554] The CAR-Treg described herein can be used, for example, to treat subjects with disease in stages 1, 2, and / or 3. Alternatively, the CAR-Treg described herein can be used to treat subjects at risk of T1D, i.e., subjects in stages prior to stage 1.

[0555] When a subject is in stage 3 of the disease, CAR-Treg therapy should be started as soon as possible to minimize pancreatic beta cell destruction and maximize residual pancreatic beta cell function. This can be determined, for example, by measuring the subject's blood insulin or blood C-peptide levels.

[0556] For example, subjects could be asked to have a minimum stimulating C-peptide level of 0.2 pmol / mL, or, for example, 0.4 pmol / mL, from a mixed diet tolerance test at the time of CAR-Treg administration. These subjects could be considered to have “newly onset type 1 diabetes.”

[0557] For example, when a subject is in stage 3 of the disease, CAR-Treg treatment can begin no more than approximately 24 weeks after the onset of symptoms or diagnosis, such as no more than approximately 20 weeks, no more than approximately 16 weeks, no more than approximately 15 weeks, no more than approximately 14 weeks, no more than approximately 12 weeks, no more than approximately 8 weeks, or no more than approximately 6 weeks after the onset of symptoms or diagnosis. For example, CAR regulatory T-cell therapy can begin 100 days or less after the onset of symptoms or diagnosis.

[0558] 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. In particular, subjects can be between 8 and 30 years old, especially between 8 and 25 years old, 8 and 16 years old, or 16 and 25 years old.

[0559] Disease progression and pancreatic β-cell death can be monitored, for example, through a variety of methods.

[0560] For example, imaging of pancreatic β cells can be used to highlight β cell integrity. For example, the CAR-Treg described herein can maintain or increase the number of pancreatic β cells present in a subject after administration. This can be observed, for example, whether the subject is receiving a reduced dose of exogenous insulin or not receiving any dose of exogenous insulin.

[0561] For example, insulin levels in subjects 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-Tregs described herein can, for example, maintain or increase a subject's fasting blood insulin levels after administration, e.g., maintaining them within the normal range for fasting blood insulin. This can be observed, for example, whether the subject is receiving a reduced dose of exogenous insulin or not receiving any dose of exogenous insulin.

[0562] It can also detect cell-free insulin DNA (cfDNA) and unmethylated insulin.

[0563] Peptide levels in subjects can be monitored. Pancreatic beta cells first produce a protein called proinsulin. Each proinsulin breaks down into one insulin molecule and one C-peptide molecule. When blood glucose levels rise, both insulin and C-peptide molecules are released. The amount of insulin and C-peptide released is equal, 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-Tregs described herein can, for example, maintain or increase fasting C-peptide levels in subjects after administration, for example, maintaining them within the normal range for fasting C-peptides. This can be observed, for example, whether the subject is receiving a reduced dose of exogenous insulin or not receiving any dose of exogenous insulin.

[0564] In addition, blood glucose levels can be monitored in subjects. Normal fasting blood glucose levels (i.e., after fasting for at least 8 hours) can be considered to be approximately 3.9 mmol / L to 6.9 mmol / L. Alternatively or additionally, an oral glucose tolerance test can be performed and blood glucose levels measured (e.g., using 75g of oral glucose). Two hours after oral glucose administration, normal blood glucose levels can be considered to be less than approximately 7.8 mmol / L. At intermediate time points before 2 hours (e.g., 30 minutes, 60 minutes, 90 minutes), normal blood glucose levels can be considered to be less than 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, maintaining them within the normal range for fasting blood glucose and / or OGTT blood glucose. This can be observed, for example, when a subject is receiving a reduced dose of exogenous insulin or is not receiving any dose of exogenous insulin.

[0565] It can monitor hemoglobin (Hb)HbA1c levels in subjects. HbA1c is produced when glucose binds to hemoglobin and can be used to measure average blood glucose levels over the past 2 to 3 months. Normal HbA1c values ​​are considered to be approximately 4.0% to 5.6% (20 mmol / mol to 38 mmol / mol). For example, CAR-Treg can maintain or decrease HbA1c levels in subjects after administration, for example, maintaining them within the normal range. This can be observed, for example, whether the subject is receiving a reduced dose of exogenous insulin or not receiving any dose of exogenous insulin.

[0566] Appropriately, the subjects were mammals. Appropriately, the subjects were humans.

[0567] Suitablely, 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.

[0568] Suitable, the CAR may contain an antigen-binding domain capable of specifically binding to ENTPD3, i.e., the antigen is ENTPD3.

[0569] Methods of treating diseases or conditions involve the therapeutic use of the cells described herein. In this regard, the cells may be administered to a subject suffering from an existing disease or condition 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.

[0570] 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 medications (e.g., exogenous insulin) required by a subject suffering from the disease, or that allows the subject to discontinue existing medications.

[0571] 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 to reduce or prevent the development of at least one symptom associated with the disease or condition. Subjects may be susceptible to the disease or condition, or are considered to be at risk of developing the disease or condition (e.g., pre-stage 1 type 1 diabetes).

[0572] As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of a patient's disease within the context of clinicopathology. Ideal outcomes of treatment include slowing disease progression, improving or alleviating pathological conditions, and alleviating or improving the prognosis of a particular disease, disorder, or condition. For example, if one or more symptoms associated with a particular disease, disorder, or condition are reduced or eliminated, it indicates that the individual has received successful "treatment."

[0573] "Effective dose" refers to the amount that is effective in achieving the desired therapeutic or preventative effect within the necessary dosage and time period. An effective dose can be provided in one or more administrations.

[0574] A "therapeutic effective amount" is at least the minimum concentration required to induce a measurable improvement in a particular disease, disorder, or condition. The therapeutic effective amount as defined herein can vary depending on factors such as the patient's disease state, age, sex, weight, and the ability of chimeric receptors to elicit the desired response in an individual. A therapeutic effective amount is also the amount by which any toxic or adverse effects of the cells, cell populations, or pharmaceutical composition are exceeded by the therapeutically beneficial effects.

[0575] The terms “subject,” “patient,” and “individual” are used interchangeably herein and refer to mammals, optionally humans. In particular, the terms subject, patient, and individual refer to a person suffering from a disease or condition requiring treatment as defined herein.

[0576] In some embodiments of the invention, the patient may receive other treatments before, during, or after the treatment of the invention. For example, in some embodiments, the patient may be treated with other procedures for treating symptoms related to a disease or condition.

[0577] The medical applications or methods described herein may involve the following steps:

[0578] (i) Isolate or provide cell-containing samples;

[0579] (ii) Introducing nucleic acid molecules, constructs, or vectors as defined herein into cells; and

[0580] (iii) Administering cells from (ii) to the subject.

[0581] Cells may be Tregs as defined herein. Enriched populations of Tregs may be isolated and / or generated from cell-containing samples before and / or after step (ii) of the method. For example, isolation and / or generation may be performed before and / or after step (ii) to isolate and / or generate enriched Treg samples. Enrichment may be performed after step (ii) to enrich cells and / or Tregs containing CARs, polynucleotides, and / or vectors as described herein.

[0582] Appropriately, the cells can be autologous. Appropriately, the cells can be allogeneic.

[0583] Suitable, cells (e.g., engineered Tregs) may be administered in combination with one or more other therapeutic agents (e.g., lymphatic depletion agents, as described above). Engineered cells (e.g., Tregs) may be administered simultaneously or sequentially (i.e., before or after) with one or more other therapeutic agents.

[0584] Cells (e.g., Tregs) can be activated and / or expanded 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. The expansion protocol has been discussed above.

[0585] Cells, such as Tregs, can be washed after each step of the method, especially after amplification.

[0586] Engineered cell (e.g., Treg) populations can be further enriched by any method known to those skilled in the art, such as FACS or magnetic bead sorting.

[0587] The production process can be carried out in a closed and sterile cell culture system.

[0588] The present invention may also provide a method for increasing cell stability and / or inhibitory function, the method comprising the step of introducing the nucleic acid molecule, expression construct, or vector provided herein into cells. The increase in inhibitory function can be measured as described above, for example by co-culturing activated antigen-specific Tconv cells with the cells of the present invention, and, 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%.

[0589] Increased stability of cells (e.g., Tregs as defined herein) refers to an increase in the persistence or survival of these cells compared to unengineered Tregs, or an increase in the proportion of cells that retain the Treg phenotype over a period of time (e.g., cells that retain Treg markers such as FOXP3 and Helios).

[0590] 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 for Treg cell markers within the cell population and analysis by FACS.

[0591] This invention also provides the use of CAR-Treg in reducing the rate of pancreatic β-cell death or preventing pancreatic β-cell death, for example, in subjects with type 1 diabetes or at risk of developing type 1 diabetes, particularly subjects with newly developed 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 reducing the rate of pancreatic β-cell death or preventing pancreatic β-cell death. For example, this can be observed when the subject is receiving a reduced dose of exogenous insulin or is not receiving any dose of exogenous insulin.

[0592] 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, for subjects with type 1 diabetes or at risk of developing type 1 diabetes, particularly subjects with newly developed type 1 diabetes. This can be observed, for example, in subjects receiving a reduced dose of exogenous insulin or not receiving any dose of exogenous insulin.

[0593] This invention also provides the use of CAR-Treg in reducing or preventing hyperglycemia, for example, in subjects with type 1 diabetes or at risk of developing type 1 diabetes, particularly subjects with newly diagnosed 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 blood glucose and / or HbA1c levels in the subject can be considered as reducing or preventing hyperglycemia in the subject. This can be observed, for example, in subjects receiving a reduced dose of exogenous insulin or not receiving any dose of exogenous insulin.

[0594] 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, for subjects with type 1 diabetes or at risk of developing type 1 diabetes, particularly subjects with newly developed type 1 diabetes. This can be observed, for example, in subjects receiving a reduced dose of exogenous insulin or not receiving any dose of exogenous insulin.

[0595] The CAR of the present invention is that the CAR contains an antigen recognition domain that specifically binds to ENTPD3 (e.g., human ENTPD3), and the CAR may have any of the features of the CAR disclosed herein.

[0596] Those skilled in the art will also understand that the scFv described in this invention can also be used in forms other than CAR structures. Therefore, in another aspect of the invention, antibodies or antibody fragments that specifically bind to ENTPD3 are provided. The antibody or antibody fragment may, in particular, be an scFv. The antibody or antibody fragment (e.g., scFv) may comprise a CDR sequence, VH and VL sequences, or an scFv sequence as defined above with respect to the antigen-binding domain of CAR. Therefore, in this embodiment, all sequences listed above regarding CDR, VH and VL, and scFv are equivalently included within the antibody or antibody fragment. Specifically, the antibody or antibody fragment may comprise:

[0597] (a) VH CDR as listed in SEQ ID NO 1-3 and VL CDR as listed in SEQ ID NO 4-6;

[0598] (b) VH CDR as listed in SEQ ID NO 7-9 and VLCDR as listed in SEQ ID NO 10-12;

[0599] (c) VH CDR as listed in SEQ ID NO 13-15 and VLCDR as listed in SEQ ID NO 16-18;

[0600] (d) VH CDR as listed in SEQ ID NO 19-21 and VLCDR as listed in SEQ ID NO 22-24;

[0601] (e) VH CDR as listed in SEQ ID NO 25-27 and VLCDR as listed in SEQ ID NO 28-30;

[0602] (f) VH CDR as listed in SEQ ID NO 31-33 and VLCDR as listed in SEQ ID NO 34-36;

[0603] (g) VH CDR as listed in SEQ ID NO 37-39 and VLCDR as listed in SEQ ID NO 40-42;

[0604] (h) VH CDR as listed in SEQ ID NO 43-45 and VLCDR as listed in SEQ ID NO 46-48;

[0605] (i) VH CDR as listed in SEQ ID NO 49-51 and VLCDR as listed in SEQ ID NO 52-54;

[0606] (j) VH CDR as listed in SEQ ID NO 55-57 and VLCDR as listed in SEQ ID NO 58-60;

[0607] (k) VH CDR as listed in SEQ ID NO 61-63 and VLCDR as listed in SEQ ID NO 64-66;

[0608] (l) VH CDR as listed in SEQ ID NO 67-69 and VLCDR as listed in SEQ ID NO 70-72;

[0609] (m) VH CDR as listed in SEQ ID NO 73-75 and VLCDR as listed in SEQ ID NO 76-78; or

[0610] (n) VH CDR as listed in SEQ ID NO 79-81 and VLCDR as listed in SEQ ID NO 82-84;

[0611] Alternatively, the CDR may contain one to three amino acid sequence modifications in any of the aforementioned sequences, or more specifically one or two amino acid sequence modifications.

[0612] 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 alternatively be prepared by chemical synthesis (e.g., solid-phase synthesis or synthesis in a homogeneous solution) using techniques well known in protein chemistry.

[0613] It is possible to generate N-terminal or C-terminal fusion proteins comprising antibodies or antibody fragments as defined herein, or to conjugate antibodies to another molecule, such as a therapeutic molecule or a detectable molecule. Therefore, the invention further covers antibodies or antibody fragments as defined above conjugated to one or more additional molecules (e.g., immunoglobulins, hormones, growth factors, lectins, insulin, low-density lipoprotein, glucagon, endorphins, transferrin, tags, fluorescent dyes, radioisotopes, or therapeutic molecules (e.g., immunosuppressive drugs)).

[0614] It also provides nucleic acid molecules containing nucleotide sequences encoding antibodies, antibody fragments, or fusion proteins as defined herein, as well as vectors and cells containing such nucleic acid molecules.

[0615] These antibodies, antibody fragments, and fusion proteins / conjugates can be used to detect cells expressing ENTPD3 for identification and / or imaging (e.g., islet mass imaging or insulinoma detection). These antibodies, antibody fragments, and fusion proteins / conjugates can also be used to deliver therapeutic molecules to target sites (e.g., cells expressing ENTPD3).

[0616] Therefore, in this respect, the present invention provides a method for detecting or imaging cells expressing ENTPD3, particularly pancreatic islet cells, comprising incubating the cells with an antibody, an antibody fragment, or a 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 ENTPD3 in a subject, comprising administering an antibody, an antibody fragment, or a fusion protein / conjugate comprising an antibody or antibody fragment as defined herein to the subject and determining or detecting the binding of the antibody, antibody fragment, fusion protein / conjugate to the cells. In particular, 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.

[0617] Finally, the present invention provides a method for delivering a therapeutic molecule to a target site where ENTPD3 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.

[0618] This disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein may be used in the practice or testing of embodiments of this disclosure. Numerical ranges include numbers that define the range. Unless otherwise stated, any nucleic acid sequence is written from left to right in a 5' to 3' direction; amino acid sequences are written from left to right in the amino to carboxyl direction, respectively.

[0619] Where a numerical range is provided, it should be understood that, unless the context explicitly specifies otherwise, each intermediate value between the upper and lower limits of the range, up to one-tenth of the lower limit unit, is also specifically disclosed. Each smaller range between any stated value or intermediate value within the range and any other stated value or intermediate value within the range is included in this disclosure. The upper and lower limits of these smaller ranges may independently include or exclude them from the range, and each range within which any one, one, or both limits are included is also covered in this disclosure, subject to any specific exclusions from the range. Where the range includes one or two limits, ranges excluding one or both of those included limits are also included in this disclosure.

[0620] It should be noted that the singular forms “a,” “an,” and “the” used herein and in the appended claims include plural indicators unless the context clearly specifies otherwise.

[0621] The terms “comprising,” “containing,” and “including” as used herein are synonymous and are inclusive or open-ended, and do not exclude additional, unlisted components, elements, or method steps. The terms “comprising,” “including,” and “composed of” also include the meaning of the term “constituting from.”

[0622] The publications discussed herein are intended only to provide information about their public disclosure prior to the filing date of this application. Nothing herein should be construed as an admission that such publications constitute prior art as described in the appended claims.

[0623] Further embodiments of the present invention are provided in the following embodiments 1 to 49:

[0624] 1. A chimeric antigen receptor (CAR) comprising an antigen recognition domain that specifically binds to ENTPD3.

[0625] 2. The CAR according to Embodiment 1, wherein the antigen recognition domain binds to human ENTPD3.

[0626] 3. The CAR according to embodiment 1 or 2, comprising:

[0627] a. An extracellular domain containing the antigen recognition domain;

[0628] b. Transmembrane domains; and

[0629] c. Intracellular domains containing intracellular signal transduction domains.

[0630] 4. The CAR according to embodiment 3 further includes a hinge domain and / or one or more co-stimulatory domains.

[0631] 5. The CAR according to embodiment 4, wherein the hinge domain is selected from CD28, CD8α, CD4, CD7, CH2CH3, the hinge region of an immunoglobulin, or a portion or variant thereof, optionally wherein the CAR includes a CD8α or CH2CH3 hinge region.

[0632] 6. The CAR according to any one of embodiments 3 to 5, wherein the CAR comprises one or more transmembrane domains selected from the following: transmembrane domains of CD28, ICOS, CD8α, CD4, CD134(OX40), CD137(4-1BB), CD3ζ, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD154, CH2CH3, or portions or variations thereof, optionally wherein the CAR comprises a CD4, CD28, CD8α, or CH2CH3 transmembrane domain.

[0633] 7. The CAR according to any one of embodiments 4 to 6, wherein the co-stimulatory domain is selected from the intracellular domain of CD28, ICOS, CD134 (OX40), CD137 (4-1BB), CD27, or TNFRSF25, or portions or variants thereof, optionally wherein the CAR comprises a CD28 co-stimulatory domain.

[0634] 8. The CAR according to any one of embodiments 3 to 7, wherein the CAR comprises one or more intracellular signal transduction domains selected from the group consisting of: CD3ζ signal transduction domain or any homolog thereof, CD3 polypeptide, syk family tyrosine kinase, src family tyrosine kinase, CD2, CD5, CD28, or portions or variants thereof, optionally wherein the CAR comprises a CD3ζ signal transduction domain.

[0635] 9. The CAR according to any one of embodiments 3 to 8, wherein the CAR comprises: a CD8α or CH2CH3 hinge domain, a CD28, CD8α or CH2CH3 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 or CH2CH3.

[0636] 10. The CAR according to any one of embodiments 3 to 9, wherein the CAR comprises a signal peptide and / or a reporter peptide.

[0637] 11. The CAR according to any of the foregoing embodiments, wherein the antigen recognition domain is an antibody, an antibody fragment, or an antibody-derived domain.

[0638] 12. The CAR according to any of the foregoing embodiments, wherein the antigen recognition domain is a single-chain antibody (scFv).

[0639] 13. The CAR according to any of the foregoing embodiments, wherein the antigen recognition domain comprises:

[0640] (i) the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:1, 2 and 3 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:4, 5 and 6 respectively;

[0641] (ii) The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:7, 8 and 9 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:10, 11 and 12 respectively;

[0642] (iii) The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:13, 14 and 15 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:16, 17 and 18 respectively;

[0643] (iv) The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:19, 20 and 21 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:22, 23 and 24 respectively;

[0644] (v) The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:25, 26 and 27 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:28, 29 and 30 respectively;

[0645] (vi) The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:31, 32 and 33 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:34, 35 and 36 respectively;

[0646] (vii) The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:37, 38 and 39 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:40, 41 and 42 respectively;

[0647] (viii) The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:43, 44 and 45 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:46, 47 and 48 respectively;

[0648] (ix) the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:49, 50 and 51 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:52, 53 and 54 respectively;

[0649] (x) The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:55, 56 and 57 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:58, 59 and 60 respectively;

[0650] (xi) The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:61, 62 and 63 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:64, 65 and 66 respectively;

[0651] (xii) The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:67, 68 and 69 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:70, 71 and 72 respectively;

[0652] (xiii) The VH CDR1, 2, and 3 sequences listed in SEQ ID NO:73, 74, and 75, respectively, and the VL CDR1, 2, and 3 sequences listed in SEQ ID NO:76, 77, and 78, respectively; or

[0653] (xiv) The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:79, 80 and 81 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:82, 83 and 84 respectively;

[0654] Wherein, one or more of the CDR sequences in (i) to (xiv) may optionally include one to three amino acid modifications relative to the aforementioned CDR sequences, and in particular, one or more of the CDR sequences may optionally be modified by substituting one to three amino acids, adding one to three amino acids, or deleting one to three amino acids.

[0655] 14. The CAR according to any of the foregoing embodiments, wherein the antigen recognition domain comprises:

[0656] (i) A VH domain containing the sequence listed in SEQ ID NO:85 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:86 or a sequence having at least 70% sequence identity with it.

[0657] (ii) A VH domain comprising the sequence listed in SEQ ID NO:87 or a sequence having at least 70% sequence identity with it, and a VL domain comprising the sequence listed in SEQ ID NO:88 or a sequence having at least 70% sequence identity with it.

[0658] (iii) a VH domain comprising the sequence listed in SEQ ID NO:89 or a sequence having at least 70% sequence identity with it, and a VL domain comprising the sequence listed in SEQ ID NO:90 or a sequence having at least 70% sequence identity with it.

[0659] (iv) A VH domain comprising the sequence listed in SEQ ID NO:91 or a sequence having at least 70% sequence identity with it, and a VL domain comprising the sequence listed in SEQ ID NO:92 or a sequence having at least 70% sequence identity with it.

[0660] (v) A VH domain containing the sequence listed in SEQ ID NO:93 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:94 or a sequence having at least 70% sequence identity with it.

[0661] (vi) A VH domain containing the sequence listed in SEQ ID NO:95 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:96 or a sequence having at least 70% sequence identity with it.

[0662] (vii) A VH domain containing the sequence listed in SEQ ID NO:97 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:98 or a sequence having at least 70% sequence identity with it.

[0663] (viii) A VH domain comprising the sequence listed in SEQ ID NO:99 or a sequence having at least 70% sequence identity with it, and a VL domain comprising the sequence listed in SEQ ID NO:100 or a sequence having at least 70% sequence identity with it.

[0664] (ix) A VH domain comprising the sequence listed in SEQ ID NO:101 or a sequence having at least 70% sequence identity with it, and a VL domain comprising the sequence listed in SEQ ID NO:102 or a sequence having at least 70% sequence identity with it.

[0665] (x) A VH domain containing the sequence listed in SEQ ID NO:103 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:104 or a sequence having at least 70% sequence identity with it.

[0666] (xi) A VH domain containing the sequence listed in SEQ ID NO:105 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:106 or a sequence having at least 70% sequence identity with it.

[0667] (xii) A VH domain containing the sequence listed in SEQ ID NO:107 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:108 or a sequence having at least 70% sequence identity with it.

[0668] (xiii) A VH domain comprising the sequence listed in SEQ ID NO:109 or a sequence having at least 70% sequence identity with it, and a VL domain comprising the sequence listed in SEQ ID NO:110 or a sequence having at least 70% sequence identity with it; or

[0669] (xiv)VH domain containing the sequence listed in SEQ ID NO:111 or a sequence having at least 70% sequence identity with it, and VL domain containing the sequence listed in SEQ ID NO:112 or a sequence having at least 70% sequence identity with it.

[0670] 15. The CAR according to any of the foregoing embodiments, wherein the antigen recognition domain comprises:

[0671] (i) a VH domain containing a sequence encoded by SEQ ID NO:205 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:205, and a VL domain containing a sequence encoded by SEQ ID NO:206 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:206;

[0672] (ii) A VH domain comprising a sequence encoded by SEQ ID NO:207 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:207, and a VL domain comprising a sequence encoded by SEQ ID NO:208 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:208;

[0673] (iii) A VH domain comprising a sequence encoded by SEQ ID NO:209 or a sequence having at least 70% identity with a sequence encoded by SEQ ID NO:209, and a VL domain comprising a sequence encoded by SEQ ID NO:210 or a sequence having at least 70% identity with a sequence encoded by SEQ ID NO:210;

[0674] (iv) A VH domain containing a sequence encoded by SEQ ID NO:211 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:211, and a VL domain containing a sequence encoded by SEQ ID NO:212 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:212.

[0675] (v) A VH domain containing a sequence encoded by SEQ ID NO:213 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:213, and a VL domain containing a sequence encoded by SEQ ID NO:214 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:214;

[0676] (vi) A VH domain containing a sequence encoded by SEQ ID NO:215 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:215, and a VL domain containing a sequence encoded by SEQ ID NO:216 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:216.

[0677] (vii) A VH domain containing a sequence encoded by SEQ ID NO:217 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:217, and a VL domain containing a sequence encoded by SEQ ID NO:218 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:218.

[0678] (viii) A VH domain comprising a sequence encoded by SEQ ID NO:219 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:219, and a VL domain comprising a sequence encoded by SEQ ID NO:220 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:220;

[0679] (ix) A VH domain containing a sequence encoded by SEQ ID NO:221 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:221, and a VL domain containing a sequence encoded by SEQ ID NO:222 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:222;

[0680] (x) A VH domain containing a sequence encoded by SEQ ID NO:223 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:223, and a VL domain containing a sequence encoded by SEQ ID NO:224 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:224;

[0681] (xi) A VH domain containing a sequence encoded by SEQ ID NO:225 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:225, and a VL domain containing a sequence encoded by SEQ ID NO:226 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:226.

[0682] (xii) A VH domain containing a sequence encoded by SEQ ID NO:227 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:227, and a VL domain containing a sequence encoded by SEQ ID NO:228 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:228;

[0683] (xiii) A VH domain comprising a sequence encoded by SEQ ID NO:229 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:229, and a VL domain comprising a sequence encoded by SEQ ID NO:230 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:230; or

[0684] (xiv) A VH domain containing a sequence encoded by SEQ ID NO:231 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:231, and a VL domain containing a sequence encoded by SEQ ID NO:232 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO:232.

[0685] 16. The CAR according to any of the foregoing embodiments, wherein the antigen recognition domain comprises or is composed of the following sequences:

[0686] (i) A sequence listed in SEQ ID NO:113 or a sequence having at least 80% sequence identity with it;

[0687] (ii) A sequence listed in SEQ ID NO:114 or a sequence having at least 80% sequence identity with it;

[0688] (iii) A sequence listed in SEQ ID NO:115 or a sequence having at least 80% sequence identity with it;

[0689] (iv) A sequence listed in SEQ ID NO:116 or a sequence having at least 80% sequence identity with it;

[0690] (v) A sequence listed in SEQ ID NO:117 or a sequence having at least 80% sequence identity with it;

[0691] (vi) A sequence listed in SEQ ID NO:118 or a sequence having at least 80% sequence identity with it;

[0692] (vii) A sequence listed in SEQ ID NO:119 or a sequence having at least 80% sequence identity with it;

[0693] (viii) A sequence listed in SEQ ID NO:120 or a sequence having at least 80% sequence identity with it;

[0694] (ix) A sequence listed in SEQ ID NO:121 or a sequence having at least 80% sequence identity with it;

[0695] (x) A sequence listed in SEQ ID NO:122 or a sequence having at least 80% sequence identity with it;

[0696] (xi) A sequence listed in SEQ ID NO:123 or a sequence having at least 80% sequence identity with it;

[0697] (xii) The sequence listed in SEQ ID NO:124 or a sequence having at least 80% sequence identity with it;

[0698] (xiii) A sequence listed in SEQ ID NO:125 or a sequence having at least 80% sequence identity with it; or

[0699] (xiv) The sequence listed in SEQ ID NO:126 or a sequence having at least 80% sequence identity with it.

[0700] 17. The CAR according to any of the foregoing embodiments, wherein the antigen recognition domain comprises the following

[0701] The sequence may consist of the following sequences:

[0702] (i) A sequence encoded by the sequence listed in SEQ ID NO:191 or a sequence having at least 80% identity with the sequence encoded by the sequence listed in SEQ ID NO:191;

[0703] (ii) A sequence encoded by the sequence listed in SEQ ID NO:192 or a sequence having at least 80% identity with the sequence encoded by the sequence listed in SEQ ID NO:192;

[0704] (iii) A sequence encoded by the sequence listed in SEQ ID NO:193 or a sequence having at least 80% identity with the sequence encoded by the sequence listed in SEQ ID NO:193;

[0705] (iv) A sequence encoded by the sequence listed in SEQ ID NO:194 or a sequence having at least 80% identity with the sequence encoded by the sequence listed in SEQ ID NO:194;

[0706] (v) A sequence encoded by the sequence listed in SEQ ID NO:195 or a sequence having at least 80% identity with the sequence encoded by the sequence listed in SEQ ID NO:195;

[0707] (vi) A sequence encoded by the sequence listed in SEQ ID NO:196 or a sequence having at least 80% identity with the sequence encoded by the sequence listed in SEQ ID NO:196;

[0708] (vii) A sequence encoded by the sequence listed in SEQ ID NO:197 or a sequence having at least 80% identity with the sequence encoded by the sequence listed in SEQ ID NO:197;

[0709] (viii) A sequence encoded by the sequence listed in SEQ ID NO:198 or a sequence having at least 80% identity with the sequence encoded by the sequence listed in SEQ ID NO:198;

[0710] (ix) A sequence encoded by the sequence listed in SEQ ID NO:199 or a sequence having at least 80% identity with the sequence encoded by the sequence listed in SEQ ID NO:199;

[0711] (x) A sequence encoded by a sequence listed in SEQ ID NO:200 or a sequence having at least 80% identity with a sequence encoded by a sequence listed in SEQ ID NO:200;

[0712] (xi) A sequence encoded by the sequence listed in SEQ ID NO:201 or a sequence having at least 80% identity with the sequence encoded by the sequence listed in SEQ ID NO:201;

[0713] (xii) A sequence encoded by the sequence listed in SEQ ID NO:202 or a sequence having at least 80% identity with the sequence encoded by the sequence listed in SEQ ID NO:202;

[0714] (xiii) A sequence encoded by the sequence listed in SEQ ID NO:203 or a sequence having at least 80% identity with the sequence encoded by the sequence listed in SEQ ID NO:203; or

[0715] (xiv) A sequence encoded by the sequence listed in SEQ ID NO:204 or a sequence having at least 80% identity with the sequence encoded by the sequence listed in SEQ ID NO:204.

[0716] 18. The CAR according to any of the foregoing embodiments, wherein the CAR comprises a sequence listed in any of SEQ ID NO:237 to 262 or a sequence having at least 80% identity with it, or is composed of a sequence listed in any of SEQ ID NO:237 to 262 or a sequence having at least 80% identity with it.

[0717] 19. A nucleic acid molecule comprising a nucleotide sequence encoding a CAR as described in any of the foregoing embodiments.

[0718] 20. A carrier comprising a nucleic acid molecule according to embodiment 19.

[0719] 21. The vector according to embodiment 20 further comprises a nucleic acid molecule encoding the FOXP3 polypeptide.

[0720] 22. A cell comprising a CAR according to any one of embodiments 1 to 18, a nucleic acid molecule according to embodiment 19, or a vector according to embodiment 20 or 21.

[0721] 23. The cell according to embodiment 22, wherein the cell is a production host cell.

[0722] 24. The cell according to embodiment 22, wherein the cell is an immune cell or its progenitor cell or precursor, optionally a T cell or its precursor, or a stem cell.

[0723] 25. The cell according to embodiment 22 or 24, wherein the cell is a regulatory T cell (Treg) or its precursor, or an iPSC cell, and particularly wherein the cell further comprises exogenous nucleic acid containing a nucleotide sequence encoding a FOXP3 polypeptide.

[0724] 26. A cell population comprising cells according to any one of embodiments 22, 24 or 25.

[0725] 27. A cell population according to embodiment 26, comprising a plurality of cells according to any one of embodiments 22, 24 or 25, particularly a plurality of T cells according to embodiment 24, and more particularly a plurality of Tregs according to embodiment 25.

[0726] 28. The cell population according to embodiment 27, wherein the clonality of the plurality of T cells, particularly Tregs, has not been modified in vitro.

[0727] 29. The cell population according to embodiment 27 or 28, wherein the plurality of T cells, particularly Tregs, have polyclonal endogenous TCRs.

[0728] 30. A pharmaceutical composition comprising cells according to any one of embodiments 22, 24 or 25, a cell population according to any one of embodiments 26 to 29, or a carrier according to embodiment 20 or 21.

[0729] 31. Use in therapeutic applications of the cells described in any one of embodiments 22, 24 or 25, the cell populations described in any one of embodiments 26 to 29, or the pharmaceutical composition described in embodiment 30.

[0730] 32. The cells, cell populations, or pharmaceutical composition used for the purposes described in embodiment 31, wherein the treatment is adoptive cell transfer therapy.

[0731] 33. The use of the cells according to embodiment 22, 24 or 25, the cell population according to any one of embodiments 26 to 29, or the pharmaceutical composition according to embodiment 30 for the treatment or prevention of autoimmune diseases or inflammatory diseases, or for the induction of immunosuppression, or for the promotion of tissue repair and / or tissue regeneration, particularly wherein the cells are Tregs.

[0732] 34. The cells, cell populations, or pharmaceutical compositions used according to embodiment 33, wherein the autoimmune disease or the inflammatory disease is type 1 diabetes, such as newly diagnosed type 1 diabetes.

[0733] 35. 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 according to any one of embodiments 22, 24 or 25, particularly Tregs, or cell populations according to any one of embodiments 26 to 29, or a pharmaceutical composition according to embodiment 30, particularly comprising Tregs.

[0734] 36. The method according to embodiment 35 includes the following steps:

[0735] (i) Isolate or provide Treg-enriched cell samples from the subject;

[0736] (ii) Introducing the nucleic acid molecule according to Embodiment 19 or the vector according to Embodiment 20 or 21 into Treg cells; and

[0737] (iii) Administer the Treg cells from (ii) to the subject.

[0738] 37. Use of the cells according to embodiment 22, 24 or 25, the cell population according to any one of embodiments 26 to 29, or the pharmaceutical composition according to embodiment 30 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, in particular, the cells are Tregs.

[0739] 38. A method for manufacturing cells according to embodiment 22, 24 or 25, comprising the step of introducing (e.g. transducing or transfecting cells therewith) a nucleic acid molecule according to embodiment 19 or a vector according to embodiment 20 or 21 into the cells.

[0740] 39. The method according to embodiment 38, wherein the cell is a Treg cell, and the method comprises: 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 the vector into the cell.

[0741] 40. A cell that can be obtained by the method according to embodiment 38 or 39.

[0742] 41. Use of the cells according to embodiment 22, 24 or 25, the cell population according to any one of embodiments 26 to 29, or the pharmaceutical composition according to embodiment 30 for preventing or reducing the mortality rate of pancreatic β cells in a subject.

[0743] 42. Use of the cells according to embodiment 22, 24 or 25, the cell population according to any one of embodiments 26 to 29, or the pharmaceutical composition according to embodiment 30 for maintaining or increasing fasting blood insulin levels in a subject.

[0744] 43. Use of the cells according to embodiment 22, 24 or 25, the cell population according to any one of embodiments 26 to 29, or the pharmaceutical composition according to embodiment 30 for maintaining or increasing the fasting C-peptide level of a subject.

[0745] 44. Use of the cells according to embodiment 22, 24 or 25, the cell population according to any one of embodiments 26 to 29, or the pharmaceutical composition according to embodiment 30 for reducing or preventing hyperglycemia in a subject.

[0746] 45. Use of the cells according to embodiment 22, 24 or 25, the cell population according to any one of embodiments 26 to 29, or the pharmaceutical composition according to embodiment 30 for maintaining or reducing the fasting blood glucose level of a subject.

[0747] 46. ​​Use of the cells according to embodiment 22, 24 or 25, the cell population according to any one of embodiments 26 to 29, or the pharmaceutical composition according to embodiment 30 for maintaining or reducing the HbA1c level of a subject.

[0748] 47. A cell according to embodiment 22, 24 or 25, a cell population according to embodiment 26 to 29, or a pharmaceutical composition according to embodiment 30, for use according to any one of embodiments 41 to 46, wherein the subject has or is at risk of developing type 1 diabetes, for example, wherein the subject has recently developed type 1 diabetes.

[0749] 48. A cell according to embodiment 22, 24 or 25, a cell population according to embodiment 26 to 29, or a pharmaceutical composition according to embodiment 30, for use according to any one of embodiments 41 to 47, wherein the subject has not been administered exogenous insulin.

[0750] 49. A cell according to embodiment 22, 24 or 25, a cell population according to embodiment 26 to 29, or a pharmaceutical composition according to embodiment 30, for use according to any one of embodiments 41 to 47, wherein the subject is receiving a reduced dose of insulin compared to the insulin dose required prior to administration of CAR-Treg.

[0751] The present invention will now be further described by way of examples, which are intended to help those skilled in the art to carry out the invention, and are not intended to limit the scope of the invention in any way.

[0752] sequence list

[0753] The amino acid sequence of VH CDR1 in SEQ ID NO:1 is:

[0754] GFTFDDYA

[0755] The amino acid sequence of VH CDR2 of SEQ ID NO:2 is:

[0756] ISWNSGSI

[0757] The amino acid sequence of VH CDR3 of SEQ ID NO:3 is:

[0758] AKGANYDILTGYRKDNWFDP

[0759] The amino acid sequence of VL CDR1 of SEQ ID NO:4 is:

[0760] SSNIGSNY

[0761] The amino acid sequence of VL CDR2 of SEQ ID NO:5 is A8:

[0762] SSN

[0763] The amino acid sequence of VL CDR3 of SEQ ID NO:6 is:

[0764] AAWDDSLSGWV

[0765] SEQ ID NO:7 is the amino acid sequence of VH CDR1 of B2:

[0766] GFTFSSYA

[0767] SEQ ID NO:8 is the amino acid sequence of VH CDR2 of B2:

[0768] ISGSGGNT

[0769] SEQ ID NO:9 is the amino acid sequence of VH CDR3 of B2:

[0770] AKGGSTSSGYRFDY

[0771] SEQ ID NO:10 is the amino acid sequence of VL CDR1 of B2:

[0772] SGHSNYA

[0773] SEQ ID NO:11 is the amino acid sequence of VL CDR2 of B2:

[0774] VNRDGSY

[0775] SEQ ID NO:12 is the amino acid sequence of VL CDR3 of B2:

[0776] QTWGTGVQV

[0777] SEQ ID NO:13 is the amino acid sequence of VH CDR1 of B7:

[0778] GYTFTSYA

[0779] SEQ ID NO:14 is the amino acid sequence of VH CDR2 of B7:

[0780] INAGNGNT

[0781] The amino acid sequence of VH CDR3 of SEQ ID NO:15 is B7:

[0782] ARDSGYDLFDY

[0783] The amino acid sequence of VL CDR1 of SEQ ID NO:16 is B7:

[0784] SGSVSTTYY

[0785] The amino acid sequence of VL CDR2 of SEQ ID NO:17 is B7:

[0786] KTN

[0787] The amino acid sequence of VL CDR3 of SEQ ID NO:18 is B7:

[0788] LLYMGSGVWV

[0789] SEQ ID NO:19 is the amino acid sequence of VH CDR1 of C1:

[0790] GYSFTSYW

[0791] SEQ ID NO:20 is the amino acid sequence of VH CDR2 of C1:

[0792] IYPGDSGT

[0793] SEQ ID NO:21 is the amino acid sequence of VH CDR3 of C1:

[0794] ARQQGAGAFDI

[0795] SEQ ID NO:22 is the amino acid sequence of VL CDR1 of C1:

[0796] SRDVGGYNY

[0797] SEQ ID NO:23 is the amino acid sequence of VL CDR2 of C1:

[0798] EVT

[0799] SEQ ID NO:24 is the amino acid sequence of VL CDR3 of C1:

[0800] SSYTSSSTVV

[0801] SEQ ID NO:25 is the amino acid sequence of VH CDR1 of C6:

[0802] GFTFGDYA

[0803] SEQ ID NO:26 is the amino acid sequence of VH CDR2 of C6:

[0804] IRSKAYGGTT

[0805] SEQ ID NO:27 is the amino acid sequence of VH CDR3 of C6:

[0806] TRVPVVAGWYNWFDP

[0807] SEQ ID NO:28 is the amino acid sequence of VL CDR1 of C6:

[0808] SSNIGSNY

[0809] SEQ ID NO:29 is the amino acid sequence of VL CDR2 of C6:

[0810] RNN

[0811] SEQ ID NO:30 is the amino acid sequence of VL CDR3 of C6:

[0812] SSYAGSNNVV

[0813] SEQ ID NO:31 is the amino acid sequence of VH CDR1 in C8:

[0814] GFTFDDYA

[0815] SEQ ID NO:32 is the amino acid sequence of VH CDR2 at C8:

[0816] ISWNSGSI

[0817] SEQ ID NO:33 is the amino acid sequence of VH CDR3 of C8:

[0818] AKGANYDILTGYRKDNWFDP

[0819] SEQ ID NO:34 is the amino acid sequence of VL CDR1 of C8:

[0820] QTISNW

[0821] SEQ ID NO:35 is the amino acid sequence of VL CDR2 of C8:

[0822] KAS

[0823] SEQ ID NO:36 is the amino acid sequence of VL CDR3 of C8:

[0824] QQYHSYSRT

[0825] SEQ ID NO:37 is the amino acid sequence of VH CDR1 of D3:

[0826] GFTFDDYA

[0827] SEQ ID NO:38 is the amino acid sequence of VH CDR2 of D3:

[0828] ISWNSGSI

[0829] SEQ ID NO:39 is the amino acid sequence of VH CDR3 of D3:

[0830] AKGANYDILTGYEY

[0831] SEQ ID NO:40 is the amino acid sequence of VL CDR1 of D3:

[0832] TGAVTSDHH

[0833] SEQ ID NO:41 is the amino acid sequence of VL CDR2 of D3:

[0834] DTS

[0835] SEQ ID NO:42 is the amino acid sequence of VL CDR3 of D3:

[0836] FLYYSGTAI

[0837] SEQ ID NO:43 is the amino acid sequence of VH CDR1 of E11:

[0838] GFTFSSYA

[0839] SEQ ID NO:44 is the amino acid sequence of VH CDR2 of E11:

[0840] ISGSGGST

[0841] SEQ ID NO:45 is the amino acid sequence of VH CDR3 of E11:

[0842] AKDDYDFWSGSLGNY

[0843] SEQ ID NO:46 is the amino acid sequence of VL CDR1 of E11:

[0844] SSNIGSNY

[0845] SEQ ID NO:47 is the amino acid sequence of VL CDR2 of E11:

[0846] ENN

[0847] SEQ ID NO:48 is the amino acid sequence of VL CDR3 of E11:

[0848] AAWDDTLNAWV

[0849] SEQ ID NO:49 is the amino acid sequence of VH CDR1 of E4:

[0850] GFTFDDYA

[0851] The amino acid sequence of VH CDR2 of E4 is SEQ ID NO:50:

[0852] ISWNSGSI

[0853] SEQ ID NO:51 is the amino acid sequence of VH CDR3 of E4:

[0854] AKGANYDILTGYRKDNWFDP

[0855] SEQ ID NO:52 is the amino acid sequence of VL CDR1 of E4:

[0856] QSVGSS

[0857] SEQ ID NO:53 is the amino acid sequence of VL CDR2 of E4:

[0858] DAS

[0859] SEQ ID NO:54 is the amino acid sequence of VL CDR3 of E4:

[0860] QQRSNWPPYT

[0861] SEQ ID NO:55 is the amino acid sequence of VH CDR1 of F10:

[0862] GFTFSSYG

[0863] SEQ ID NO:56 is the amino acid sequence of VH CDR2 of F10:

[0864] ISYDGSNK

[0865] SEQ ID NO:57 is the amino acid sequence of VH CDR3 of F10:

[0866] AKDHHPYGSSDSFDY

[0867] SEQ ID NO:58 is the amino acid sequence of VL CDR1 of F10:

[0868] TGAVTRGHY

[0869] SEQ ID NO:59 is the amino acid sequence of VL CDR2 of F10:

[0870] DTV

[0871] SEQ ID NO:60 is the amino acid sequence of VL CDR3 of F10:

[0872] LLSFIDTRYPARYV

[0873] SEQ ID NO:61 is the amino acid sequence of VH CDR1 of A10:

[0874] GYTFTSYG

[0875] SEQ ID NO:62 is the amino acid sequence of VH CDR2 of A10:

[0876] ISAYNGNT

[0877] SEQ ID NO:63 is the amino acid sequence of VH CDR3 of A10:

[0878] ARDDPWGSYRPRPFDY

[0879] SEQ ID NO:64 is the amino acid sequence of VL CDR1 of A10:

[0880] SSNIGSNT

[0881] SEQ ID NO:65 is the amino acid sequence of VL CDR2 of A10:

[0882] SNN

[0883] SEQ ID NO:66 is the amino acid sequence of VL CDR3 of A10:

[0884] AAWDDSLNGWV

[0885] SEQ ID NO:67 is the amino acid sequence of VH CDR1 of B4:

[0886] GGTFSSYA

[0887] SEQ ID NO:68 is the amino acid sequence of VH CDR2 of B4:

[0888] IIPIFGTA

[0889] SEQ ID NO:69 is the amino acid sequence of VH CDR3 of B4:

[0890] ARGASGYDWSLDY

[0891] The amino acid sequence of VL CDR1 of SEQ ID NO:70 is B4:

[0892] QSLVYSDGNTY

[0893] SEQ ID NO:71 is the amino acid sequence of VL CDR2 of B4:

[0894] KVS

[0895] SEQ ID NO:72 is the amino acid sequence of VL CDR3 of B4:

[0896] MQGSRWPPT

[0897] SEQ ID NO:73 is the amino acid sequence of VH CDR1 of F8:

[0898] GYSFISHW

[0899] SEQ ID NO:74 is the amino acid sequence of VH CDR2 of F8:

[0900] IYPGDSGT

[0901] SEQ ID NO:75 is the amino acid sequence of VH CDR3 of F8:

[0902] ARLADGGLQFDH

[0903] SEQ ID NO:76 is the amino acid sequence of VL CDR1 of F8:

[0904] SSDVGGYNY

[0905] SEQ ID NO:77 is the amino acid sequence of VL CDR2 of F8:

[0906] GVS

[0907] SEQ ID NO:78 is the amino acid sequence of VL CDR3 of F8:

[0908] NSYTSSSTYV

[0909] SEQ ID NO:79 is the amino acid sequence of VH CDR1 of E1:

[0910] GFTFSSYW

[0911] SEQ ID NO:80 is the amino acid sequence of VH CDR2 of E1:

[0912] IKQDGSEK

[0913] SEQ ID NO:81 is the amino acid sequence of VH CDR3 of E1:

[0914] ARVPNYYDSSGTV

[0915] SEQ ID NO:82 is the amino acid sequence of VL CDR1 of E1:

[0916] SGSIASNY

[0917] SEQ ID NO:83 is the amino acid sequence of VL CDR2 of E1:

[0918] EDN

[0919] SEQ ID NO:84 is the amino acid sequence of VL CDR3 of E1:

[0920] QSYDSTLLV

[0921] SEQ ID NO:85 is the amino acid sequence of the VH domain of A8 (CDR is underlined).

[0922] QVTLKESGGGLVQPGRSLRLSCAAS GFTFDDYA MHWVRQAPGKGLEWVSG ISWNSGSI GYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYC AKGANYDILTGYRKDNWFDP WGQGTLVTVSS

[0923] SEQ ID NO:86 is the amino acid sequence of the VL domain of A8 (CDR is underlined).

[0924] LPVLTQPPSASGTPGQKVTISCSGS SSNIGSNY VFWYEQLPGAAPKLLMY SSN QRPSGVPDRFSGSSKSGTSASLAISGLRSEDEADYYC AAWDDSLSGWVFGGGTKLTVLSEQ ID NO:87 is the amino acid sequence of the VH domain of B2 (CDR is underlined).

[0925] QVQLVQSGGGVVQPGRSLRLSCAAS GFTFSSYA MSWVRQAPGKGLEWVSA ISGSGGNT NYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC AKGGSTSSGYRFDY WGQGTLVTVSS

[0926] SEQ ID NO:88 is the amino acid sequence of the VL domain of B2 (CDR is underlined).

[0927] QLVLTQSPSASASLGASVKLTCTLS SGHSNYA IAWHQQQPEKGPRFLMN VNRDGSY TKGDGIPDRFSGSSSGAERYLTISSLQSDDEADYYC QTWGTGVQV FGGGTKLTVL

[0928] SEQ ID NO:89 is the amino acid sequence of the VH domain of B7 (underlined in CDR).

[0929] QVQLQQSGAEVKKPGASVKVSCKAS GYTFTSYA MHWVRQAPGQRLEWMGW INAGNGNT KYSQKLQGRVTITRDTSASTAYMELSSLRSEDTAVYYC ARDSGYDLFDY WGQGTLVTVSS

[0930] SEQ ID NO:90 is the amino acid sequence of the VL domain of B7 (CDR is underlined).

[0931] QAVVTQEPSFSVSPGGTVTLTCGLT SGSVSTTYY PSWYQQTPGQTPRTLIY KTN LRSPGVPDRFSGSILGNKAALTITGAQADDDSDYYC LLYMGSGVWV FGGGTRLTVL

[0932] SEQ ID NO:91 is the amino acid sequence of the VH domain of C1 (CDR is underlined).

[0933] QVQLVQSGAEVKKPGESLKISCKGS GYSFTSYWIGWVRQMPGKGLEWMGI IYPGDSGT RYSPSFQGQVTISADKSISTAYLQWSSLKASNTAMYYC ARQQGAGAFDI WGQGTMVTVSS

[0934] SEQ ID NO:92 is the amino acid sequence of the VL domain of C1 (CDR is underlined).

[0935] QSALTQPPSASGSPGQSVTISCTGT SRDVGGYNY VSWYQQHPGKAPKLILY EVT KRPSGVPDRFSGSSKSGNTASLTISGLQAEDEADYYC SSYTSSSTVV FGGGTKLTVL

[0936] SEQ ID NO:93 is the amino acid sequence of the VH domain of C6 (CDR is underlined).

[0937] EVQLVQSGGGLVQPGRSLRLSCTAS GFTFGDYA MSWVRQAPGKGLEWVGF IRSKAYGGTT EYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYC TRVPVVAGWYNWFDP WGQGTLVTVSS

[0938] SEQ ID NO:94 is the amino acid sequence of the VL domain of C6 (CDR is underlined).

[0939] QSVLTQPPSASGTPGQRVTISCSGS SSNIGSNY VYWYQQLPGTapLLIY RNN QRPSGVPDRFSGSKSGNTASLTVSGLQAGDEADYYC SSYAGSNNVV FGGGTKLAVL

[0940] SEQ ID NO:95 is the amino acid sequence of the VH domain of C8 (CDR is underlined).

[0941] QVTLKESGGGLVQPGRSLRLSCAAS GFTFDDYA MHWVRQAPGKGLEWVSG ISWNSGSI GYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYC AKGANYDILTGYRKDNWFDP WGQGTLVTVSS

[0942] SEQ ID NO:96 is the amino acid sequence of the VL domain of C8 (CDR is underlined).

[0943] DIVMTQTPSTLSASVGDRVTITCRAS QTISNW LAWYQQKPGKAPKLLIY KAS TLESGVPSRFSGTGSGTEFTLTISSLQPDDFATYFC QQYHSYSRT FGQGTKVDV

[0944] SEQ ID NO:97 is the amino acid sequence of the VH domain of D3 (CDR is underlined).

[0945] QVQLVQSGGGLVQPGRSLRLSCAAS GFTFDDYA MHWVRQAPGKGLEWVSG ISWNSGSI GYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYC AKGANYDILTGYEY WGQGTLVTVSS

[0946] SEQ ID NO:98 is the amino acid sequence of the VL domain of D3 (CDR is underlined).

[0947] QAVVTQEPSLTVSPGETVTLTCGSS TGAVTSDHH PYWFQQKPGQVPRTLMH DTS TRYSWTPARFSGSIVGGKAALTLSGAQPEDEAEYHC FLYYSGTAI FGGGTKLTVL

[0948] SEQ ID NO:99 is the amino acid sequence of the VH domain of E11 (CDR is underlined).

[0949] EVQLVESGGGLVQPGGSLRLSCAAS GFTFSSYA MSWVRQAPGKGLEWVSA ISGSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC AKDDYDFWSGSLGNY WGQGTLVTVSS

[0950] SEQ ID NO:100 is the amino acid sequence of the VL domain of E11 (CDR is underlined).

[0951] QSVLTQPPSASGTPGQRVTISCSGR SSNIGSNY VYWYQQPPGTapLLIYENN HRPSGVPDRFSASKSGTSASLAISGLRSEDEADYYC AAWDDTLNAWV FGGGTKLTVL

[0952] SEQ ID NO:101 is the amino acid sequence of the VH domain of E4 (CDR is underlined).

[0953] QVTLKESGGGLVQPGRSLRLSCAAS GFTFDDYA MHWVRQAPGKGLEWVSG ISWNSGSI GYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYC AKGANYDILTGYRKDNWFDP WGQGTLVTVSS

[0954] SEQ ID NO:102 is the amino acid sequence of the VL domain of E4 (CDR is underlined).

[0955] DIVMTQSPATLSLSPGERATLSCRAS QSVGSS LAWFQQKPGQAPRLLIY DAS NRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPPYT FGQGTKLEIK

[0956] SEQ ID NO:103 is the amino acid sequence of the VH domain of F10 (CDR is underlined).

[0957] QMQLVQSGGGVVQPGRSLRLSCAAS GFTFSSYG MHWVRQAPGKGLERVAV ISYDGSNK YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTALYYC AKDHHPYGSSDSFDY WGQGTLVTVSS

[0958] SEQ ID NO:104 is the amino acid sequence of the VL domain of F10 (CDR is underlined).

[0959] QAVVTQEPSLTVSPGGTVTLTCGSS TGAVTRGHY PYWLQQKPGQAPRTLIY DTV KKHSWTPARFSGSLVGGKAALTLSGAQPEDEADYYC LLSFIDTRYPARYV FGTGTKVTVL

[0960] SEQ ID NO:105 is the amino acid sequence of the VH domain of A10 (CDR is underlined).

[0961] QVQLVQSGAEVKKPGASVKVSCKAS GYTFTSYG ISWVRQAPGQGLEWMGW ISAYNGNT NYAQKLQGRVTMTTDTSSTAYMELRSLRSDDTAVYYC ARDDPWGSYRPRPFDY WGQGTLVTVSS

[0962] SEQ ID NO:106 is the amino acid sequence of the VL domain of A10 (CDR is underlined).

[0963] QSVLTQPPSASGTPGQRVTISCSGS SSNIGSNT VNWYQQLPGTTPKLLIY SNN QRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYC AAWDDSLNGWV FGGGTKLTVL

[0964] SEQ ID NO:107 is the amino acid sequence of the VH domain of B4 (underlined in CDR).

[0965] EVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYC ARGASGYDWSLDY WGQGTLVTVSS

[0966] SEQ ID NO:108 is the amino acid sequence of the VL domain of B4 (CDR is underlined).

[0967] KIVMTQSPLSLPVTLGQPASISCRSS QSLVYSDGNTY LNWFQQRPGQSPRRLIY KVS NRHSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFC MQGSRWPPT FGPGTKVEIK

[0968] SEQ ID NO:109 is the amino acid sequence of the VH domain of F8 (CDR is underlined).

[0969] QIQLVQSGTEVKKPGESLKISCKGS GYSFISHW IGWVRQMPGKGLEWMGIIYPGDSGT RYSPSFQGQATISADKSISTAYLQWSSLKASDTAMYYC ARLADGGLQFDH WGQGTLVTVSS

[0970] SEQ ID NO:110 is the amino acid sequence of the VH domain of F8 (underlined in CDR).

[0971] QSALTQPASVSGSPGQSITISCTGT SSDVGGYNY VSWYQQHPGKAPKLMIY GVS NRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYC NSYTSSSTYV FGTGTKVTVL

[0972] SEQ ID NO:111 is the amino acid sequence of the VH domain of E1 (CDR is underlined).

[0973] EVQLVQSGGGLVQPGGSLRLSCAAS GFTFSSYW MSWVRQAPGKGLEWVAN IKQDGSEK YYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARVPNYYDSSGTV WGQGTLVTVSS

[0974] SEQ ID NO:112 is the amino acid sequence of the VL domain of E1 (CDR is underlined).

[0975] NFMLTQPHSVSESPGKTVTISCTRS SGSIASNY VQWYQQRPGSAPTTVIY EDN QRPSGVPDRFSGSIDSSSNSASLTISGLEPEDEADYYC QSYDSTLLV FGGGTKLTVL

[0976] SEQ ID NO:113 is the amino acid sequence of the antigen recognition domain containing the VH and VL sequences of A8.

[0977] QVTLKESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKGANYDILTGYRKDNWFDPWGQGTLVTVS SGGGGSGGGGSGGGGSLPVLTQPPSASGTPGQKVTISCSGSSSNIGSNYVFWYEQLPGAAPKLLMYSSNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTKLTVL

[0978] SEQ ID NO:114 is the amino acid sequence of the antigen recognition domain containing the VH and VL sequences of B2.

[0979] QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGNTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGSTSSGYRFDYWGQGTLVTVSSGG GGSGGGGSGGGGSQLVLTQSPSASASLGASVKLTCTLSSGHSNYAIAWHQQQPEKGPRFLMNVNRDGSYTKGDGIPDRFSGSSSGAERYLTISSLQSDDEADYYCQTWGTGVQVFGGGTKLTVL

[0980] SEQ ID NO:115 is the amino acid sequence containing the antigen recognition domains of the VH and VL sequences of B7.

[0981] QVQLQQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGWINAGNGNTKYSQKLQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDSGYDLFDYWGQGTLVTVSSGGG GSGGGGSGGGGSQAVVTQEPSFSVSPGGTVTLTCGLTSGSVSTTYYPSWYQQTPGQTPRTLIYKTNLRSPGVPDRFSGSILGNKAALTITGAQADDDSDYYCLLYMGSGVWVFGGGTRLTVL

[0982] SEQ ID NO:116 is the amino acid sequence of the antigen recognition domain containing the VH and VL sequences of C1.

[0983] QVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSGTRYSPSFQGQVTISADKSISTAYLQWSSLKASNTAMYYCARQQGAGAFDIWGQGTMVTVSSGGG GSGGGGSGGGGSQSALTQPPSASGSPGQSVTISCTGTSRDVGGYNYVSWYQQHPGKAPKLILYEVTKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTVVFGGGTKLTVL

[0984] SEQ ID NO:117 is the amino acid sequence of the antigen recognition domain containing the VH and VL sequences of C6.

[0985] EVQLVQSGGGLVQPGRSLRLSCTASGFTFGDYAMSWVRQAPGKGLEWVGFIRSKAYGGTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRVPVVAGWYNWFDPWGQGTLVTVSS GGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGNTASLTVSGLQAGDEADYYCSSYAGSNNVVFGGGTKLAVL

[0986] SEQ ID NO:118 is the amino acid sequence containing the antigen recognition domains of the VH and VL sequences of C8.

[0987] QVTLKESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKGANYDILTGYRKDNWFDPWGQGTLVT VSSGGGGSGGGGSGGGGSDIVMTQTPSTLSASVGDRVTITCRASQTISNWLAWYQQKPGKAPKLLIYKASTLESGVPSRFSGTGSGTEFTLTISSLQPDDFATYFCQQYHSYSRTFGQGTKVDV

[0988] SEQ ID NO:119 is the amino acid sequence of the antigen recognition domain containing the VH and VL sequences of D3.

[0989] QVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKGANYDILTGYEYWGQGTLVTVSSG GGGSGGGGSGGGGSQAVVTQEPSLTVSPGETVTTLTCGSSTGAVTSDHHPYWFQQKPGQVPRTLMHDTSTRYSWTPARFSGSIVGGKAALTLSGAQPEDEAEYHCFLYYSGTAIFGGGTKLTVL

[0990] SEQ ID NO:120 is the amino acid sequence of the antigen recognition domain containing the VH and VL sequences of E11.

[0991] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDDYDFWSGSLGNYWGQGTLVTVSSG GGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGRSSNIGSNYVYWYQQPPGTAPKLLIYENNHRPSGVPDRFSASKSGTSASLAISGLRSEDEADYYCAAWDDTLNAWVFGGGTKLTVL

[0992] SEQ ID NO:121 is the amino acid sequence of the antigen recognition domain containing the VH and VL sequences of E4.

[0993] QVTLKESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKGANYDILTGYRKDNWFDPWGQGTLVTV SSGGGGSGGGGSGGGGSDIVMTQSPATLSLSPGERATLSCRASQSVGSSLAWFQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPYTFGQGTKLEIK

[0994] SEQ ID NO:122 is the amino acid sequence of the antigen recognition domain containing the VH and VL sequences of F10.

[0995] QMQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLERVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTALYYCAKDHHPYGSSDSFDYWGQGTLVTVSSGGG GSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTRGHYPYWLQQKPGQAPRTLIYDTVKKHSWTPARFSGSLVGGKAALTLSGAQPEDEADYYCLLSFIDTRYPARYVFGTGTKVTVL

[0996] SEQ ID NO:123 is the amino acid sequence of the antigen recognition domain containing the VH and VL sequences of A10.

[0997] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSSTAYMELRSLRSDDTAVYYCARDDPWGSYRPRPFDYWGQGTLVTVSSG GGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISSCSGSSSNIGSNTVNWYQQLPGTTPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKLTVL

[0998] SEQ ID NO:124 is the amino acid sequence containing the antigen recognition domains of the VH and VL sequences of B4.

[0999] EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGASGYDWSLDYWGQGTLVTVSSGGG GSGGGGSGGGGSKIVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPGQSPRRLIYKVSNRHSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCMQGSRWPPTFGPGTKVEIK

[1000] SEQ ID NO:125 is the amino acid sequence of the antigen recognition domain containing the VH and VL sequences of F8.

[1001] QIQLVQSGTEVKKPGESLKISCKGSGYSFISHWIGWVRQMPGKGLEWMGIIYPGDSGTRYSPSFQGQATISADKSISTAYLQWSSLKASDTAMYYCARLADGGLQFDHWGQGTLVTVSSGGG GSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYGVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCNSYTSSSTYVFGTGTKVTVL

[1002] SEQ ID NO:126 is the amino acid sequence of the antigen recognition domain containing the VH and VL sequences of E1.

[1003] EVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVPNYYDSSGTVWGQGTLVTVSSGG GGSGGGGSGGGGSNFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSAPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLEPEDEADYYCQSYDSTLLVFGGGTKLTVL

[1004] SEQ ID NO:127 is the amino acid sequence of human ENTPD3 (two transmembrane domains are underlined, an extracellular domain is located between the two transmembrane domains, and a cytoplasmic domain is located at each end of the polypeptide).

[1005] MFTVLTRQPCEQAGLKALYRTP TIIALVVLLVSIVVLVSITVI QIHKQEVLPPGLKYGIVLDAGSSRTTVYVYQWPAEKENNTGVVSQTFKCSVKGSGISSYGNNPQDVPRAFEECMQKVKGQVPSHLHGSTPIHLGATAGMRLLRLQNETAA NEVLESIQSYFKSQPFDFRGAQIISGQEEGVYGWITANYLMGNFLEKNLWHMWVHPHGVETTGALDLGGASTQISFVAGEKMDLNTSDIMQVSLYGYVYTLYTHSFQCYGR NEAEKKFLAMLLQNSPTKNHLTNPCYPRDYSISFTMGHVFDSLCTVDQRPESYNPNDVITFEGTGDPSLCKEKVASIFDFKACHDQETCSFDGVYQPKIKGPFVAFAGFY YTASALNLSGFSLDTFNSSTWNFCSQNWSQLPLLLPKFDEVYARSYCFSANYIYHLFVNGYKFTEETWPQIHFEKEVGNSSIAWSLGYMLSLTNQIPAESPLIRLPIEPP VFVGTLAFFTAAALLCLAFLA YLCSATRRKRHSEHAFDHAVDSD

[1006] SEQ ID NO:128 is the amino acid sequence of mouse ENTPD3 (two transmembrane domains are underlined, an extracellular domain is located between the two transmembrane domains, and a cytoplasmic domain is located at each end of the polypeptide).

[1007] MFTVMTRQPCEQAGFRALSRTP AIVTLVVLLVSIVVLVTLTLIQIRHPQVLPPGLKYGVVLDAGSSRTTVYVYQWPAEKENNTGVVSQTFRCSVKGSGISSYENNPQDAPKAFEDCILKVKEQVPEHLHGSTRIYLGATAGMRLLRLQNETAA REVLESIQSYFKSQPFDFRGAQIISGQEEGVYGWITANYIMGNFLEKNLWHMWVHPHGVDTTGALDLGGASTQISFVAGEKMEPNASDTVQVSLYGYTYTLYTHSFQCYGQ NEAEKKFLAMLLQSPSTEANISNPCYPQGYSTAFTLGHVFGSLCTEKQRPESYNSSKSVTFMGTGDPRLCREKVASVFDFNACQEQDACSFDGIYQPKVQGPFVAFAGFY YTASALNLSGFSLTSFNDSSWDFCRHTWSELPALLSRFDETYARSYCFSAHYIYHLLVNGYKFTEETWPQIRFEKEVGNSSIAWSLGYMLSLTNQIPAGSPLIHLPIQPP VFMGVLAFFTAIALLCLAFLL YLCSSFRTKERSENAFDQAVDSD

[1008] SEQ ID NO:129 is the amino acid sequence of the transmembrane domain of human CD8α, representing amino acids 183 to 203 of human CD8α.

[1009] IYIWAPLAGTCGVLLLSLVIT

[1010] SEQ ID NO:130 is the amino acid sequence of the CH2CH3 hinge domain.

[1011] PCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1012] SEQ ID NO:131 is the amino acid sequence of the modified combination of the CD8α hinge and transmembrane domain (the transmembrane domain is underlined).

[1013] FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDDFAD IYIWAPLAGTCGVLL LSLVIT LYCNHR

[1014] SEQ ID NO:132 is the amino acid sequence of the modified CD8α hinge domain.

[1015] FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFADSEQ ID NO:133 is the amino acid sequence of the wild-type CD8α combined hinge and transmembrane domain (the transmembrane domain is underlined).

[1016] FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD IYIWAPLAGTCGVL LLSLVIT LYCNHR

[1017] SEQ ID NO:134 is the amino acid sequence of the CH2CH3 hinge domain.

[1018] EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1019] SEQ ID NO:135 is the amino acid sequence of the CD28 hinge and transmembrane domain (the transmembrane domain is underlined).

[1020] IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP FWVLVVVGGVLACYSLLVTVAFIIFWV

[1021] SEQ ID NO:136 is the amino acid sequence of the signal / leader sequence.

[1022] MALPVTALLLPLALLLHAAAP

[1023] SEQ ID NO:137 is the amino acid sequence of the wild-type CD8α leader sequence.

[1024] MALPVTALLLPLALLLHAARP

[1025] SEQ ID NO:138 is the amino acid sequence of the intracellular signal transduction domain of the CD3ζ chain.

[1026] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[1027] SEQ ID NO:139 is the amino acid sequence of the CD28 combined transmembrane and intracellular signal transduction domains (the transmembrane domains are underlined).

[1028] FWVLVVVGGVLACYSLLVTVAFIIFWV RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[1029] SEQ ID NO:140 is the amino acid sequence of the CD28 intracellular signal transduction domain.

[1030] WVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[1031] SEQ ID NO:141 is an exemplary amino acid sequence of the intracellular signal transduction domain of OX40.

[1032] ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI

[1033] SEQ ID NO:142 is an exemplary amino acid sequence of the intracellular signal transduction domain of 4-1BB.

[1034] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[1035] SEQ ID NO:143 is an exemplary amino acid sequence of the intracellular signal transduction domain of ICOS.

[1036] CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL

[1037] SEQ ID NO:144 is an exemplary amino acid sequence of the intracellular signal transduction domain of TNFRSF25.

[1038] TYTYRHCWPHKPLVTADEAGMEALTPPPATHLSPLDSAHTLLAPPDSSEKICTVQLVGNSWTPGYPETQEALCPQVTWSWDQLPSRALGPAAAPTLSPESPAGSPAMMLQPGPQLYDVMDAVPARRWKEFVRTLGLREAEIEAVEVEIGRFRDQQYEMLKRWRQQQPAGLGAVYAALERMGLDGCVEDLRSRLQRGP

[1039] SEQ ID NO:145 is the amino acid sequence representing amino acids 266 to 551 of the human IL-2 receptor β chain.

[1040] NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPL QPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV

[1041] SEQ ID NO:146 is the amino acid sequence representing a truncated and sequence-modified variant of SEQ ID NO:145 (Y510).

[1042] NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLPLNTDAYLSLQELQGQDPTHLV

[1043] SEQ ID NO:147 is the amino acid sequence representing a truncated and sequence-modified variant of SEQ ID NO:145 (Y510 and Y392).

[1044] NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV

[1045] SEQ ID NO:148 is the amino acid sequence of wild-type FOXP3 (UniProtKB accession number Q9BZS1).

[1046] MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFL KHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPE FLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGP

[1047] SEQ ID NO:149 is the amino acid sequence of the N- and C-terminal truncated FOXP3 fragment described in WO2019 / 241549.

[1048] 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

[1049] SEQ ID NO:150 is the amino acid sequence of a FOXP3 variant with a mutation at amino acid position 418.

[1050] MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVF EEPEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHL WGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKR E QRPSRCSNPTPGP

[1051] SEQ ID NO:151 is the amino acid sequence of a FOXP3 variant with a mutation at amino acid position 422.

[1052] MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRP A RCSNPTPGP

[1053] SEQ ID NO:152 is the amino acid sequence of a FOXP3 variant having mutations at amino acid positions 418 and 422

[1054] MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKR E QRPA RCSNPTPGP

[1055] SEQ ID NO:153 is the amino acid sequence of an exemplary FOXP3 polypeptide.

[1056] MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQA DHLLDEKGRAQCLLQREMVQSLEQVEELSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHN MRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGPEGRGSLLTCGDVEEN

[1057] SEQ ID NO:154 is the nucleotide sequence encoding an exemplary FOXP3 polypeptide.

[1058]

[1059] SEQ ID NO:155 is the nucleotide sequence encoding an exemplary FOXP3 polypeptide.

[1060]

[1061] SEQ ID NO:156 is the amino acid sequence of the P2A domain.

[1062] ATNFSLLKQAGDVEENPGP

[1063] SEQ ID NO:157 is the amino acid sequence of the T2A domain.

[1064] EGRGSLLTCGDVEENPGP

[1065] SEQ ID NO:158 is the amino acid sequence of the E2A domain.

[1066] QCTNYALLKLAGDVESNPGP

[1067] SEQ ID NO:159 is the amino acid sequence of the F2A domain.

[1068] VKQTLNFDLLKLAGDVESNPGP

[1069] SEQ ID NO:160 is the amino acid sequence of the furin protease cleavage site.

[1070] RXXR

[1071] SEQ ID NO:161 is the amino acid sequence of the furin protease cleavage site.

[1072] RRKR

[1073] SEQ ID NO:162 is the nucleotide sequence of the SFFV promoter.

[1074] GTAACGCCATTTTGCAAGGCATGGAAAAATACCAAACCAAGAATAGAGAAGTTCAGATCAAGGGCGGGTACATGAAAATAGCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTTTCGGCCCCGGCCCGGGGCCAAGAACAGATTTTCGGCCCCGGCCCGAGGCCAAGAACAGATGGTCCCCAGATA TGGCCCAACCCTCAGCAGTTTCTTAAGACCCATCAGATGTTTCCAGGCTCCCCCAAGGACCTGAAATGACCCTGCGCCTTATTTGAATTAACCAATCAGCCTGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTTCCCGAGCTCTATAAAAGAGCTCACAACCCCTCACTCGGCGCGCCAGTCCTCCGACAGACTGAGTCGGCCGG

[1075] SEQ ID NO:163 – Representative Connector Sequence

[1076] GGGS

[1077] SEQ ID NO:164 – Representative Connector Sequence

[1078] ETSGGGGSRL

[1079] SEQ ID NO:165 – Representative Connector Sequence

[1080] SGGGGSGGGGSGGGGS

[1081] SEQ ID NO:166 – Representative Connector Sequence

[1082] GGGGS

[1083] SEQ ID NO:167 – Representative Connector Sequence

[1084] GGGGGS

[1085] SEQ ID NO:168 – Representative Connector Sequence

[1086] GGGGGGS

[1087] SEQ ID NO:169 – Representative Connector Sequence

[1088] GGGGSGGGGSGGGGS

[1089] SEQ ID NO:170 – Representative Connector Sequence

[1090] GGGGG

[1091] SEQ ID NO:171 – Representative Connector Sequence

[1092] GGGGSGGGGS

[1093] SEQ ID NO:172 – Representative Connector Sequence

[1094] GGGGSGGGGSGGGGSGGGGS

[1095] SEQ ID NO:173 – Representative Connector Sequence

[1096] GGGGGGG

[1097] SEQ ID NO:174 – Representative Connector Sequence

[1098] G6

[1099] SEQ ID NO:175 – Representative Connector Sequence

[1100] G8

[1101] SEQ ID NO:176 – Representative Connector Sequence

[1102] KESGSVSSEQLAQFRSLD

[1103] SEQ ID NO:177 – Representative Connector Sequence

[1104] EGKSSGSGSESKST

[1105] SEQ ID NO:178 – Representative Connector Sequence

[1106] GSAGSAAGSGEF

[1107] SEQ ID NO:179 – Representative Connector Sequence

[1108] SGGGGSAGSAAGSGEF

[1109] SEQ ID NO:180 – Representative Connector Sequence

[1110] SGGGLLLLLLLLGGGS

[1111] SEQ ID NO:181 – Representative Connector Sequence

[1112] SGGGAAAAAAAAGGGS

[1113] SEQ ID NO:182 – Representative Connector Sequence

[1114] SGGGAAAAAAAAAAAAAAAAAAAGGGS

[1115] SEQ ID NO:183 – Representative Connector Sequence

[1116] SGALGGLALAGLLLAGLGLGAAGS

[1117] SEQ ID NO:184 – Representative Connector Sequence

[1118] SLSLSPGGGGGPAR

[1119] SEQ ID NO:185 – Representative Connector Sequence

[1120] SLSLSPGGGGGPARSLSLSPGGGGG

[1121] SEQ ID NO:186 – Representative Connector Sequence

[1122] GSSGSS

[1123] SEQ ID NO:187 – Representative Connector Sequence

[1124] GSSSSSS

[1125] SEQ ID NO:188 – Representative Connector Sequence

[1126] GGSSSS

[1127] SEQ ID NO:189 – Representative Connector Sequence

[1128] GSSSSS

[1129] SEQ ID NO:190 – Representative Connector Sequence

[1130] SGGGGS

[1131] SEQ ID NO:191 is the nucleotide sequence encoding A8, the signal sequence, and the tag (the sequence encoding the signal sequence and the tag is underlined).

[1132] ATGAAATACCTATTGCCTACGGCAGCCGCTGGCTTGCTGCTGCTGGCAGCTCAGCCGGCCATGGCCCAGGTCACCTTGAAGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTAGCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGGGGCTAATTACGATATTTTGACTGGTTATCGGAAGGATAACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTGCATCCGCCCCAAAGCTTGAAGAAGGTGAATTTTCAGAAGCACGCGTACTGCCTGTGCTGACTCAGCCCCCCTCAGCGTCTGGGACCCCCGGGCAGAAGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATTATGTATTCTGGTACGAGCAGCTCCCAGGAGCGGCCCCCAAGCTCCTCATGTATAGCAGTAATCAGAGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGACTATTATTGTGCAGCATGGGATGACAGCCTGAGTGGTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCACCGTCCTCT GCGGCCGCAGGTTCTGAACAAAAGCTGATCTCAGAAG AAGATCTATCCCATCATCACCATCATCAT

[1133] SEQ ID NO:192 is the nucleotide sequence encoding B2, as well as the signal sequence and the tag (the sequences encoding the signal sequence and the tag are underlined)

[1134] ATGAAATACCTATTGCCTACGGCAGCCGCTGGCTTGCTGCTGCTGGCAGCTCAGCCGGCCATGGCCCAGGTCCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAACACAAACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGGGGGAGTACTAGTAGTGGTTATCGTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTGCATCCGCCCCAAAGCTTGAAGAAGGTGAATTTTCAGAAGCACGCGTACAGCTTGTGCTGACTCAATCGCCCTCTGCCTCTGCCTCCCTGGGAGCCTCGGTCAAGCTCACCTGCACTCTGAGCAGTGGGCACAGCAACTACGCCATCGCATGGCATCAGCAGCAGCCAGAGAAGGGCCCTCGGTTCTTGATGAACGTTAATCGTGATGGCAGCTACACCAAGGGGGACGGGATCCCTGATCGCTTCTCAGGCTCCAGCTCTGGGGCTGAGCGCTACCTCACCATCTCCAGCCTCCAGTCTGACGATGAGGCTGACTATTACTGTCAGACCTGGGGCACTGGCGTTCAAGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAAGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCT GCGGCCGCAGGTTCTGAACAAAAGCTGATCTCAGAAGAAGATCTATCCCATC ATCACCATCATCAT

[1135] SEQ ID NO:193 is a nucleotide sequence encoding B7, as well as a signal sequence and a tag (the sequences encoding the signal sequence and the tag are underlined).

[1136] ATGAAATACCTATTGCCTACGGCAGCCGCTGGCTTGCTGCTGCTGGCAGCTCAGCCGGCCATGGCCCAGGTACAGCTGCAGCAGTCAGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCTTCTGGATACACCTTCACTAGCTATGCTATGCATTGGGTGCGCCAGGCCCCCGGACAAAGGCTTGAGTGGATGGGATGGATCAACGCTGGCAATGGTAACACAAAATATTCACAGAAGCTCCAGGGCAGAGTCACCATTACCAGGGACACATCCGCGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAAGACACGGCTGTGTATTACTGTGCGAGAGATAGTGGCTACGACCTCTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTGCATCCGCCCCAAAGCTTGAAGAAGGTGAATTTTCAGAAGCACGCGTACAGGCTGTGGTGACTCAGGAGCCATCGTTCTCAGTGTCCCCTGGAGGGACAGTCACACTTACTTGTGGCTTGACCTCTGGCTCAGTCTCTACTACTTACTACCCCAGCTGGTACCAGCAGACCCCAGGCCAGACTCCACGCACACTCATCTACAAAACAAATCTTCGCTCTCCTGGGGTCCCTGATCGCTTCTCTGGCTCCATCCTTGGGAACAAAGCTGCCCTCACCATTACGGGGGCCCAGGCAGACGACGACTCTGATTACTACTGTCTGCTGTATATGGGTAGTGGCGTTTGGGTGTTCGGCGGAGGGACCAGGTTGACCGTCCTGGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCT GCGGCCGCAGGTTCTGAACAAAAGCTGATCTCAGAAGAAGATCTATCCCATCATCACCATCATC AT

[1137] SEQ ID NO:194 is the nucleotide sequence encoding C1, as well as the signal sequence and...

Claims

1. A chimeric antigen receptor (CAR) comprising an antigen recognition domain that specifically binds to ENTPD3.

2. The CAR according to claim 1, comprising: a. An extracellular domain containing the antigen recognition domain; b. Transmembrane domains; and c. Intracellular domains containing intracellular signal transduction domains.

3. The CAR according to claim 2, further comprising a hinge domain and / or one or more co-stimulatory domains.

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

5. The CAR according to any one of claims 2 to 4, wherein, The CAR comprises one or more transmembrane domains selected from the following: transmembrane domains of CD28, ICOS, CD8α, CD4, CD134(OX40), CD137(4-1BB), CD3ζ, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD154, CH2CH3, or portions or variants thereof, optionally wherein... The CAR includes a CD4, CD28, CD8α, or CH2CH3 transmembrane domain; and / or The CAR comprises one or more intracellular signal transduction domains selected from the group consisting of: CD3ζ signal transduction domain or any homolog thereof, CD3 polypeptide, syk family tyrosine kinase, src family tyrosine kinase, CD2, CD5, CD28, or portions or variants thereof, optionally wherein the CAR comprises a CD3ζ signal transduction domain.

6. The CAR according to any one of claims 2 to 5, wherein, The CAR includes: a CD8α or CH2CH3 hinge domain, a CD28, CD8α or CH2CH3 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 or CH2CH3.

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

8. The CAR according to any one of the preceding claims, wherein, The antigen recognition domain includes: i. The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:7, 8 and 9 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:10, 11 and 12 respectively; ii. The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:19, 20 and 21 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:22, 23 and 24 respectively; iii. The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:73, 74 and 75 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:76, 77 and 78 respectively; iv. The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:25, 26 and 27 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:28, 29 and 30 respectively; v. The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:37, 38 and 39 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:40, 41 and 42 respectively; vi. The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:1, 2 and 3 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:4, 5 and 6 respectively; vii. The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:13, 14 and 15 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:16, 17 and 18 respectively; viii. The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:31, 32 and 33 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:34, 35 and 36 respectively; ix. The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:43, 44 and 45 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:46, 47 and 48 respectively; x. The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:49, 50 and 51 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:52, 53 and 54 respectively; xi. The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:55, 56 and 57 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:58, 59 and 60 respectively; xii. The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:61, 62 and 63 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:64, 65 and 66 respectively; xiii. The VH CDR1, 2, and 3 sequences listed in SEQ ID NO: 67, 68, and 69, respectively, and the VL CDR1, 2, and 3 sequences listed in SEQ ID NO: 70, 71, and 72, respectively; or xiv. The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:79, 80 and 81 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:82, 83 and 84 respectively; Wherein, one or more of the CDR sequences in (i) to (xiv) may optionally contain one to three amino acid modifications relative to the aforementioned CDR sequences, and in particular, one or more of the CDR sequences may optionally be modified by substituting one to three amino acids, adding one to three amino acids, or deleting one to three amino acids.

9. The CAR according to any one of the preceding claims, wherein, The antigen recognition domain includes: (i) a VH domain containing the sequence listed in SEQ ID NO:87 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:88 or a sequence having at least 70% sequence identity with it. (ii) A VH domain comprising the sequence listed in SEQ ID NO:91 or a sequence having at least 70% sequence identity with it, and a VL domain comprising the sequence listed in SEQ ID NO:92 or a sequence having at least 70% sequence identity with it. (iii) A VH domain comprising the sequence listed in SEQ ID NO:109 or a sequence having at least 70% sequence identity with it, and a VL domain comprising the sequence listed in SEQ ID NO:110 or a sequence having at least 70% sequence identity with it. (iv) A VH domain comprising the sequence listed in SEQ ID NO:93 or a sequence having at least 70% sequence identity with it, and a VL domain comprising the sequence listed in SEQ ID NO:94 or a sequence having at least 70% sequence identity with it. (v) A VH domain containing the sequence listed in SEQ ID NO:97 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:98 or a sequence having at least 70% sequence identity with it. (vi) A VH domain containing the sequence listed in SEQ ID NO:85 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:86 or a sequence having at least 70% sequence identity with it. (vii) A VH domain containing the sequence listed in SEQ ID NO:89 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:90 or a sequence having at least 70% sequence identity with it. (viii) A VH domain comprising the sequence listed in SEQ ID NO:95 or a sequence having at least 70% sequence identity with it, and a VL domain comprising the sequence listed in SEQ ID NO:96 or a sequence having at least 70% sequence identity with it. (ix) A VH domain comprising the sequence listed in SEQ ID NO:99 or a sequence having at least 70% sequence identity with it, and a VL domain comprising the sequence listed in SEQ ID NO:100 or a sequence having at least 70% sequence identity with it. (x) A VH domain containing the sequence listed in SEQ ID NO:101 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:102 or a sequence having at least 70% sequence identity with it. (xi) A VH domain containing the sequence listed in SEQ ID NO:103 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:104 or a sequence having at least 70% sequence identity with it. (xii) A VH domain containing the sequence listed in SEQ ID NO:105 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:106 or a sequence having at least 70% sequence identity with it. (xiii) A VH domain comprising the sequence listed in SEQ ID NO:107 or a sequence having at least 70% sequence identity with it, and a VL domain comprising the sequence listed in SEQ ID NO:108 or a sequence having at least 70% sequence identity with it; or (xiv)VH domain containing the sequence listed in SEQ ID NO:111 or a sequence having at least 70% sequence identity with it, and VL domain containing the sequence listed in SEQ ID NO:112 or a sequence having at least 70% sequence identity with it.

10. The CAR according to any one of the preceding claims, wherein, The antigen recognition domain comprises or is composed of the following sequence: (i) A sequence listed in SEQ ID NO:114 or a sequence having at least 80% sequence identity with it; (ii) A sequence listed in SEQ ID NO:116 or a sequence having at least 80% sequence identity with it; (iii) A sequence listed in SEQ ID NO:125 or a sequence having at least 80% sequence identity with it; (iv) A sequence listed in SEQ ID NO:117 or a sequence having at least 80% sequence identity with it; (v) A sequence listed in SEQ ID NO:119 or a sequence having at least 80% sequence identity with it; (vi) A sequence listed in SEQ ID NO:113 or a sequence having at least 80% sequence identity with it; (vii) A sequence listed in SEQ ID NO:115 or a sequence having at least 80% sequence identity with it; (viii) A sequence listed in SEQ ID NO:118 or a sequence having at least 80% sequence identity with it; (ix) A sequence listed in SEQ ID NO:120 or a sequence having at least 80% sequence identity with it; (x) A sequence listed in SEQ ID NO:121 or a sequence having at least 80% sequence identity with it; (xi) A sequence listed in SEQ ID NO:122 or a sequence having at least 80% sequence identity with it; (xii) The sequence listed in SEQ ID NO:123 or a sequence having at least 80% sequence identity with it; (xiii) A sequence listed in SEQ ID NO:124 or a sequence having at least 80% sequence identity with it; or (xiv) The sequence listed in SEQ ID NO:126 or a sequence having at least 80% sequence identity with it.

11. The CAR according to any one of the preceding claims, wherein, The CAR comprises or consists of any of the sequences listed in SEQ ID NO: 237 to 262, and in particular, the CAR comprises or consists of any of the sequences listed in SEQ ID NO: 238, 240, 249, 241 or 243.

12. A nucleic acid molecule comprising a nucleotide sequence encoding a CAR as described in any one of the preceding claims.

13. A vector comprising the nucleic acid molecule of claim 12, optionally further comprising a nucleic acid molecule encoding a FOXP3 polypeptide.

14. A cell comprising a CAR according to any one of claims 1 to 11, a nucleic acid molecule according to claim 12, or a vector according to claim 13.

15. The cell according to claim 14, wherein, The cells are regulatory T cells (Tregs) or their precursors, or iPSCs, particularly among them, The cell also contains exogenous nucleic acid, which contains a nucleotide sequence encoding the FOXP3 polypeptide.

16. A population of cells comprising the cells according to claim 14 or 15.

17. The cell population according to claim 16, wherein, The multiple cells are multiple T cells, particularly Tregs, and said multiple T cells have polyclonal endogenous TCRs.

18. A pharmaceutical composition comprising the cells according to claim 14 or 15, the cell population according to claim 16 or 17, or the carrier according to claim 13.

19. Use in a therapeutic context for the cells of claim 14 or 15, the cell population of claim 16 or 17, or the pharmaceutical composition of claim 18.

20. The cells, cell populations, or pharmaceutical composition used for the purposes described in claim 19, wherein, The treatment is adoptive cell transfer therapy.

21. Use of the cells according to claim 14 or 15, the cell population according to claim 16 or 17, or the pharmaceutical composition according to claim 18 for the treatment or prevention of autoimmune or inflammatory diseases, for the induction of immunosuppression, or for the promotion of tissue repair and / or tissue regeneration, particularly wherein, The cells in question are Treg cells.

22. The cells, cell population, or pharmaceutical composition used for the purpose according to claim 21, wherein, The autoimmune disease or the inflammatory disease is type 1 diabetes, such as newly diagnosed type 1 diabetes.

23. A method for preparing cells according to claim 14 or 15, comprising the step of introducing a nucleic acid molecule according to claim 12 or a vector according to claim 13 into the cells.

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

25. An antibody or antibody fragment, wherein, The antibody or antibody fragment specifically binds to ENTPD3, optionally as a single-chain antibody (scFv), comprising: (i) the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:7, 8 and 9 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:10, 11 and 12 respectively; (ii) The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:19, 20 and 21 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:22, 23 and 24 respectively; (iii) The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:73, 74 and 75 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:76, 77 and 78 respectively; (iv) The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:25, 26 and 27 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:28, 29 and 30 respectively; (v) The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:37, 38 and 39 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:40, 41 and 42 respectively; (vi) The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:1, 2 and 3 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:4, 5 and 6 respectively; (vii) The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:13, 14 and 15 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:16, 17 and 18 respectively; (viii) The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:31, 32 and 33 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:34, 35 and 36 respectively; (ix) the VH CDR1, 2 and 3 sequences listed in SEQ ID NO:43, 44 and 45 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:46, 47 and 48 respectively; (x) The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:49, 50 and 51 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:52, 53 and 54 respectively; (xi) The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:55, 56 and 57 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:58, 59 and 60 respectively; (xii) The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:61, 62 and 63 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:64, 65 and 66 respectively; (xiii) The VH CDR1, 2, and 3 sequences listed in SEQ ID NO: 67, 68, and 69, respectively, and the VL CDR1, 2, and 3 sequences listed in SEQ ID NO: 70, 71, and 72, respectively; or (xiv) The VH CDR1, 2 and 3 sequences listed in SEQ ID NO:79, 80 and 81 respectively and the VL CDR1, 2 and 3 sequences listed in SEQ ID NO:82, 83 and 84 respectively; Wherein, one or more of the CDR sequences in (i) to (xiv) may optionally contain one to three amino acid modifications relative to the aforementioned CDR sequences, and in particular, one or more of the CDR sequences may optionally be modified by substituting one to three amino acids, adding one to three amino acids, or deleting one to three amino acids.

26. The antibody or antibody fragment of claim 25, wherein the antibody or antibody fragment comprises: (i) a VH domain containing the sequence listed in SEQ ID NO:87 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:88 or a sequence having at least 70% sequence identity with it. (ii) A VH domain comprising the sequence listed in SEQ ID NO:91 or a sequence having at least 70% sequence identity with it, and a VL domain comprising the sequence listed in SEQ ID NO:92 or a sequence having at least 70% sequence identity with it. (iii) A VH domain comprising the sequence listed in SEQ ID NO:109 or a sequence having at least 70% sequence identity with it, and a VL domain comprising the sequence listed in SEQ ID NO:110 or a sequence having at least 70% sequence identity with it. (iv) A VH domain comprising the sequence listed in SEQ ID NO:93 or a sequence having at least 70% sequence identity with it, and a VL domain comprising the sequence listed in SEQ ID NO:94 or a sequence having at least 70% sequence identity with it. (v) A VH domain containing the sequence listed in SEQ ID NO:97 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:98 or a sequence having at least 70% sequence identity with it. (vi) A VH domain containing the sequence listed in SEQ ID NO:85 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:86 or a sequence having at least 70% sequence identity with it. (vii) A VH domain containing the sequence listed in SEQ ID NO:89 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:90 or a sequence having at least 70% sequence identity with it. (viii) A VH domain comprising a sequence listed in SEQ ID NO:95 or a sequence having at least 70% sequence identity with it, and a sequence listed in SEQ ID NO:96 or a sequence having at least 70% sequence identity with it; (ix) A VH domain comprising the sequence listed in SEQ ID NO:99 or a sequence having at least 70% sequence identity with it, and a VL domain comprising the sequence listed in SEQ ID NO:100 or a sequence having at least 70% sequence identity with it. (x) A VH domain containing the sequence listed in SEQ ID NO:101 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:102 or a sequence having at least 70% sequence identity with it. (xi) A VH domain containing the sequence listed in SEQ ID NO:103 or a sequence having at least 70% sequence identity with it, and a VL domain containing the sequence listed in SEQ ID NO:104 or a sequence having at least 70% sequence identity with it. (xii) VH domain, which contains the sequence listed in SEQ ID NO:105 or a sequence having at least 70% sequence identity with it, and contains the sequence listed in SEQ ID NO:106 or a sequence having at least 70% sequence identity with it; (xiii) A VH domain comprising the sequence listed in SEQ ID NO:107 or a sequence having at least 70% sequence identity with it, and a VL domain comprising the sequence listed in SEQ ID NO:108 or a sequence having at least 70% sequence identity with it; or (xiv)VH domain containing the sequence listed in SEQ ID NO:111 or a sequence having at least 70% sequence identity with it, and VL domain containing the sequence listed in SEQ ID NO:112 or a sequence having at least 70% sequence identity with it.

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