Chimeric Antigen Receptor

Abstract

This specification discloses chimeric antigen receptors (CARs), cells expressing the CARs (particularly immune cells such as regulatory T cells), nucleic acids or vectors encoding the CARs, and various uses of the CARs, cells, nucleic acids or vectors.In particular, a chimeric antigen receptor (CAR) is provided that comprises an antigen recognition domain that specifically binds to ENTPD3.

Description

[Technical Field]

[0001] Technical Field The present 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, the present disclosure provides CARs that contain an antigen recognition domain that binds to ENTPD3 and are expressed in immune cells (e.g., Tregs). Such immune cells have therapeutic applications for diseases and conditions associated with cells that express ENTPD3 on their surface. The present disclosure further provides nucleic acid molecules encoding such CARs and vectors containing them, which can be used to modify host cells, such as immune cells, to express the CARs. [Background technology]

[0002] background Immunotherapy is emerging as a valuable therapeutic tool for many conditions, from the treatment of cancer, autoimmune, and inflammatory diseases to the prevention of solid organ transplant rejection. In particular, the field of adoptive cellular immunotherapy (ACT), particularly cell therapy using regulatory T cells (Tregs), is seeing increased clinical activity across the spectrum of autoimmune and inflammatory diseases. CD4+FOXP3+ regulatory T cells (Tregs) are a lymphocyte subset essential for maintaining predominant immune tolerance by inhibiting the function of various effector immune cell subsets, including T effector cells. Additionally, Tregs are known to promote tissue repair and regeneration. Tregs can confer immune tolerance through multiple contact-dependent and contact-independent mechanisms. These include the production of anti-inflammatory soluble mediators such as IL-10, TGF-β, and IL-35, IL-2 consumption, expression of negative regulatory cell surface receptors such as CTLA-4, and direct targeting of T cells or indirect targeting via APCs. Importantly, once activated, Tregs can suppress immune responses in a non-antigen-specific manner (bystander suppression). That is, once activated, Tregs have the ability to regulate the local immune microenvironment and suppress inflammation. Furthermore, they can confer an inhibitory phenotype on other cells of the immune system, a process known as "infectious tolerance." Type 1 diabetes is a chronic autoimmune disease in which the immune system destroys pancreatic beta cells, which are responsible for producing insulin. This is caused by genetic and environmental factors. The destruction of beta cells reduces or eliminates insulin production in the body and causes inflammation in the pancreatic islets. Insulin is a hormone necessary for regulating glucose levels in the bloodstream. Before treatment, individuals with type 1 diabetes have excessively high blood glucose levels (hyperglycemia). Type 1 diabetes is a serious and lifelong condition. Currently, type 1 diabetes patients must carefully monitor their blood glucose levels and administer appropriate amounts of insulin (e.g., via injections or pumps). This treatment is not a cure and must be administered continuously. Over time, irregular blood glucose levels (e.g., large fluctuations in blood glucose levels) can lead to long-term complications such as damage to the heart, eyes, feet, and kidneys, as well as reduced life expectancy. Therefore, type 1 diabetes places a significant burden on healthcare systems worldwide. Furthermore, epidemiological data show that the prevalence of autoimmune diseases such as type 1 diabetes has been steadily increasing in Western societies over the past few decades. The prevalence of type 1 diabetes in Western Europe and North America is approximately 0.5%, with approximately 2 million people suffering from the disease, and there is a continuing trend toward an increase. Increasing evidence suggests that dysregulation of Treg-mediated suppression of effector T cells (Teff) contributes to disease pathogenesis in mouse models of type 1 diabetes and other autoimmune disorders (see Visperas and Vignali, J Immunol. 2016; 197(10): 3762-3770). For example, abnormalities in Treg phenotype and suppressive capacity have been observed in specimens from type 1 diabetes patients. Furthermore, depletion of Tregs has been shown to accelerate the onset of autoimmune diabetes in mouse models of type 1 diabetes. The potential for ameliorating immunopathology and reestablishing immune tolerance in inflammatory diseases has spurred interest in the clinical development of Treg-based immunotherapy. However, for Treg immunotherapy to be successful, it would be beneficial to develop methods to promote Treg trafficking to sites of tissue injury and induce their in situ activation. Autologous polyclonal Tregs have been administered to patients with type 1 diabetes with promising results, demonstrating the safety and feasibility of adoptive Treg therapy in this disease setting (Marek-Trzonkowski et al, Clin Immunol. 2014;153:23-30 and Bluestone et al, Sci Transl Med. 2015;7:315ra189). However, polyclonal Tregs lack specificity and may be associated with undesirable effects, such as systemic immunosuppression. Furthermore, transporting sufficient numbers of polyclonal Tregs to the disease site can be difficult. A recent large-scale clinical trial using polyclonal Tregs (the Sanford Project), conducted by Caladrius Biosciences, failed to demonstrate efficacy in type 1 diabetes. Tregs derived from transgenic mice expressing TCRs specific for pancreatic islet antigens 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 TCR-mediated activation of Tregs, and because of MHC restriction, this transgenic model cannot be applied to patients, as different TCRs would be required for each patient. Artificial chimeric antigen receptors (CARs) have been used to confer antigen specificity to cells. CARs generally consist of an extracellular antigen-binding domain (e.g., an scFv specific for the target antigen), a transmembrane domain, and an intracellular signaling domain that activates cells upon binding of an antigen to the extracellular antigen-binding domain. CAR-Teff cell therapy has been approved for the treatment of certain hematological cancers. However, CARs are artificial molecules integrated into cells and have lower sensitivity than TCRs. This is partly due to the molecules involved in the TCR mechanism, namely, the CD4 / CD8 co-receptor, the immunoreceptor tyrosinase-rich activation motif (ITAM), and the number of subunits within the receptor complex. For activation, CARs require 100–10,000 molecules per target cell, whereas TCRs require fewer than 10 molecules per target cell. Therefore, it is difficult to select the appropriate target for CAR-T cell therapy that activates CAR-T cells at the necessary and appropriate levels for therapeutic efficacy. For example, insulin-specific CAR-Tregs proliferated in response to insulin and exhibited suppressive activity in vitro, but failed to prevent spontaneous diabetes in NOD mice (Tenspolde et al, J Autoimmunity. 2019;103:102289). Summary of the Invention

[0003] overview The present inventors have discovered that therapies for the treatment of autoimmune or inflammatory diseases can be developed by delivering chimeric antigen receptors (CARs) containing an antigen-recognition domain specific for ENTPD3 to immune cell subsets. Expression of such CARs on the surface of Tregs provides a versatile therapeutic approach for the treatment of autoimmune or inflammatory diseases in which ENTPD3 is expressed locally at the disease site. This takes into account the well-known bystander effect of Tregs and their ability to suppress immune responses and regulate the activation state of other immune cell subsets upon activation. In particular, the present inventors have developed anti-ENTPD3 CARs for the treatment of type 1 diabetes.

[0004] Thus, the present inventors have identified ENTPD3 as a surprisingly effective target for CAR Treg therapy, particularly for type 1 diabetes (T1D) and other autoimmune or inflammatory disorders of the pancreas. In particular, the present inventors found that cells expressing ENTPD3-specific CARs can be activated in the presence of antigen and prevent the onset of cyclophosphamide-induced type 1 diabetes in NOD mice. Indeed, the anti-ENTPD3 CAR developed by the present inventors was able to prevent diabetes in all animals administered. While ENTPD3 has been reported to be expressed in pancreatic tissue, the art also reports ENTPD3 expression in other tissues (such as the gut, kidney, and brain). Therefore, selecting ENTPD3 as a specific target for transport (which would enable Tregs to be activated to a level sufficient to treat type 1 diabetes) is not intuitive. Therefore, the present inventors' finding that the ENTPD3 protein is an effective target in the pancreas, enabling the activation of anti-ENTPD3 CAR Tregs sufficient to prevent the onset of diabetes, is particularly surprising.

[0005] Thus, in one aspect, the present invention provides a chimeric antigen receptor (CAR) comprising an antigen recognition domain that specifically binds to ENTPD3 (e.g., that specifically binds to human ENTPD3).

[0006] In this regard, in this specification: a. an exodomain comprising an antigen recognition domain; b. a transmembrane domain; c. an endodomain comprising an intracellular signaling domain.

[0007] The CAR may further comprise a hinge domain and / or one or more costimulatory domains.

[0008] The term "hinge domain" typically refers to the portion of the exodomain that connects the antigen-recognition domain and the transmembrane domain. The hinge domain may be selected from the hinge region of CD28, CD8α, CD4, CD7, CH2CH3, or an immunoglobulin, or a portion or variant thereof. Optionally, the CAR may include a CD8α hinge domain or a CH2CH3 hinge domain. The costimulatory domain may be selected from the intracellular domains of CD28, ICOS, CD134 (OX40), CD137 (4-1BB), CD27, or TNFRSF25, or portions or variants thereof. Optionally, the CAR may include a CD28 costimulatory domain.

[0009] The CAR may comprise one or more transmembrane domains, which may be selected from the transmembrane domains of CD28, ICOS, CD8α, CD4, CD134 (OX40), CD137 (4-1BB), CD3ζ, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD154, or CH2CH3, or portions or variants thereof. Optionally, the CAR comprises a CD4 transmembrane domain, a CD28 transmembrane domain, a CD8α transmembrane domain, or a CH2CH3 transmembrane domain.

[0010] The CAR (particularly the endodomain of the CAR) may comprise one or more intracellular signaling domains selected from the group consisting of a CD3ζ signaling domain or any of its homologs, a CD3 polypeptide, a syk family tyrosine kinase, a src family tyrosine kinase, CD2, CD5, and CD28, or portions or variants thereof. Optionally, the CAR may comprise a CD3ζ signaling domain.

[0011] In one embodiment, the CAR may comprise 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 signaling domain (i.e., a signaling domain derived from CD3ζ); 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. Viewed another way, in one embodiment, the CAR may comprise a CD8α hinge domain, a CD8α transmembrane domain, a CD28 costimulatory domain, and a CD3ζ signaling domain. In another embodiment, the CAR may comprise a CH2CH3 hinge domain, a CH2CH3 transmembrane domain, a CD28 costimulatory domain, and a CD3ζ signaling domain. Additionally, the CAR may comprise a CD28 transmembrane domain (i.e., a transmembrane domain derived from CD28), particularly in combination with a CD28 costimulatory domain.

[0012] A CAR of the invention may comprise a signal peptide and / or a reporter peptide. In one embodiment, the polynucleotide sequence encoding a CAR of the invention may comprise an additional polynucleotide sequence encoding a reporter peptide linked by a self-cleavage domain or a cleavage domain.

[0013] The antigen recognition domain of the CAR of the invention may be an antibody, an antibody fragment, or derived from an antibody. Optionally, the antigen recognition domain is a single chain antibody (scFv).

[0014] The CAR of the present invention is The antigen recognition domain may comprise the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 269, 2, and 270, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in any of (i) SEQ ID NOs: 4, 5, and 6, respectively; (ii) SEQ ID NOs: 34, 35, and 36, respectively; (iii) SEQ ID NOs: 40, 41, and 42, respectively; or (iv) SEQ ID NOs: 52, 53, and 54, respectively. One or more of the CDR sequences may optionally contain one to three amino acid modifications relative to the CDR sequence. In particular, one or more of the CDR sequences may optionally be modified by substitution, addition, or deletion of one to three amino acids.

[0015] The CAR of the present invention is The antigen recognition domain may comprise (i) the VH CDR1 sequence, VH CDR2 sequence, and VH CDR3 sequence set forth in SEQ ID NOs: 43, 44, and 45, respectively, or (ii) the VL CDR1 sequence, VL CDR2 sequence, and VL CDR3 sequence set forth in SEQ ID NOs: 61, 62, and 63, respectively, and the VL CDR1 sequence, VL CDR2 sequence, and VL CDR3 sequence set forth in SEQ ID NOs: 271, 272, and 273, respectively, and one or more of the CDR sequences may optionally contain one to three amino acid modifications relative to the CDR sequence, and in particular, one or more of the CDR sequences may optionally be modified by substitution, addition, or deletion of one to three amino acids.

[0016] The CAR of the present invention is (i) an antigen recognition domain comprising the VH CDR1 sequence, VH CDR2 sequence, and VH CDR3 sequence set forth in SEQ ID NOs: 1, 2, and 3, respectively, and the VL CDR1 sequence, VL CDR2 sequence, and VL CDR3 sequence set forth in SEQ ID NOs: 4, 5, and 6, respectively; (ii) an antigen recognition domain comprising the VH CDR1 sequence, VH CDR2 sequence, and VH CDR3 sequence set forth in SEQ ID NOs: 7, 8, and 9, respectively, and the VL CDR1 sequence, VL CDR2 sequence, and VL CDR3 sequence set forth in SEQ ID NOs: 10, 11, and 12, respectively; (iii) an antigen recognition domain comprising the VH CDR1 sequence, VH CDR2 sequence, and VH CDR3 sequence set forth in SEQ ID NOs: 13, 14, and 15, respectively, and the VL CDR1 sequence, VL CDR2 sequence, and VL CDR3 sequence set forth in SEQ ID NOs: 16, 17, and 18, respectively; (iv) an antigen recognition domain comprising the VH CDR1 sequence, VH CDR2 sequence, and VH CDR3 sequence set forth in SEQ ID NOs: 19, 20, and 21, respectively, and the VL CDR1 sequence, VL CDR2 sequence, and VL CDR3 sequence set forth in SEQ ID NOs: 22, 23, and 24, respectively; (v) an antigen recognition domain comprising the VH CDR1 sequence, VH CDR2 sequence, and VH CDR3 sequence set forth in SEQ ID NOs: 25, 26, and 27, respectively, and the VL CDR1 sequence, VL CDR2 sequence, and VL CDR3 sequence set forth in SEQ ID NOs: 28, 29, and 30, respectively; (vi) an antigen recognition domain comprising the VH CDR1 sequence, VH CDR2 sequence, and VH CDR3 sequence set forth in SEQ ID NOs: 31, 32, and 33, respectively, and the VL CDR1 sequence, VL CDR2 sequence, and VL CDR3 sequence set forth in SEQ ID NOs: 34, 35, and 36, respectively; (vii) an antigen recognition domain comprising the VH CDR1 sequence, VH CDR2 sequence, and VH CDR3 sequence set forth in SEQ ID NOs: 37, 38, and 39, respectively, and the VL CDR1 sequence, VL CDR2 sequence, and VL CDR3 sequence set forth in SEQ ID NOs: 40, 41, and 42, respectively; (viii) an antigen recognition domain comprising the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence set forth in SEQ ID NOs: 43, 44, and 45, respectively, and the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence set forth in SEQ ID NOs: 46, 47, and 48, respectively; (ix) an antigen recognition domain comprising the VH CDR1 sequence, VH CDR2 sequence, and VH CDR3 sequence set forth in SEQ ID NOs: 49, 50, and 51, respectively, and the VL CDR1 sequence, VL CDR2 sequence, and VL CDR3 sequence set forth in SEQ ID NOs: 52, 53, and 54, respectively; (x) an antigen recognition domain comprising the VH CDR1 sequence, VH CDR2 sequence, and VH CDR3 sequence set forth in SEQ ID NOs: 55, 56, and 57, respectively, and the VL CDR1 sequence, VL CDR2 sequence, and VL CDR3 sequence set forth in SEQ ID NOs: 58, 59, and 60, respectively; (xi) an antigen recognition domain comprising the VH CDR1 sequence, VH CDR2 sequence, and VH CDR3 sequence set forth in SEQ ID NOs: 61, 62, and 63, respectively, and the VL CDR1 sequence, VL CDR2 sequence, and VL CDR3 sequence set forth in SEQ ID NOs: 64, 65, and 66, respectively; (xii) an antigen recognition domain comprising the VH CDR1 sequence, VH CDR2 sequence, and VH CDR3 sequence set forth in SEQ ID NOs: 67, 68, and 69, respectively, and the VL CDR1 sequence, VL CDR2 sequence, and VL CDR3 sequence set forth in SEQ ID NOs: 70, 71, and 72, respectively; (xiii) an antigen recognition domain comprising the VH CDR1 sequence, VH CDR2 sequence, and VH CDR3 sequence set forth in SEQ ID NOs: 73, 74, and 75, respectively, and the VL CDR1 sequence, VL CDR2 sequence, and VL CDR3 sequence set forth in SEQ ID NOs: 76, 77, and 78, respectively; or (xiv) an antigen recognition domain comprising the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence set forth in SEQ ID NOs: 79, 80, and 81, respectively, and the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence set forth in SEQ ID NOs: 82, 83, and 84, respectively; One or more of the CDR sequences (i) to (xiv) above may optionally contain one to three amino acid modifications to the CDR sequence, and in particular, one or more of the CDR sequences may optionally be modified by substitution, addition, or deletion of one to three amino acids.

[0017] In this regard, the antigen recognition domain of the CAR is (i) a VH domain comprising the sequence set forth in SEQ ID NO: 85, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 86, or a sequence having at least 70% sequence identity thereto; (ii) a VH domain comprising the sequence set forth in SEQ ID NO: 87, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 88, or a sequence having at least 70% sequence identity thereto; (iii) a VH domain comprising the sequence set forth in SEQ ID NO: 89, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 90, or a sequence having at least 70% sequence identity thereto; (iv) a VH domain comprising the sequence set forth in SEQ ID NO: 91, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 92, or a sequence having at least 70% sequence identity thereto; (v) a VH domain comprising the sequence set forth in SEQ ID NO: 93, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 94, or a sequence having at least 70% sequence identity thereto; (vi) a VH domain comprising the sequence set forth in SEQ ID NO: 95, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 96, or a sequence having at least 70% sequence identity thereto; (vii) a VH domain comprising the sequence set forth in SEQ ID NO: 97, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 98, or a sequence having at least 70% sequence identity thereto; (viii) a VH domain comprising the sequence set forth in SEQ ID NO: 99, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 100, or a sequence having at least 70% sequence identity thereto; (ix) a VH domain comprising the sequence set forth in SEQ ID NO: 101, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 102, or a sequence having at least 70% sequence identity thereto; (x) a VH domain comprising the sequence set forth in SEQ ID NO: 103, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 104, or a sequence having at least 70% sequence identity thereto; (xi) a VH domain comprising the sequence set forth in SEQ ID NO: 105, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 106, or a sequence having at least 70% sequence identity thereto; (xii) a VH domain comprising the sequence set forth in SEQ ID NO: 107, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 108, or a sequence having at least 70% sequence identity thereto; (xiii) a VH domain comprising the sequence set forth in SEQ ID NO: 109, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 110, or a sequence having at least 70% sequence identity thereto; or (xiv) may comprise a VH domain comprising the sequence set forth in SEQ ID NO: 111, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 112, or a sequence having at least 70% sequence identity thereto.

[0018] The antigen recognition domain may be, for example, (i) a VH domain comprising a sequence encoded by SEQ ID NO: 205, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 205, and a VL domain comprising a sequence encoded by SEQ ID NO: 206, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 206; (ii) a VH domain comprising a sequence encoded by SEQ ID NO: 207, or a sequence having at least 70% identity to 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 to the sequence encoded by SEQ ID NO: 208; (iii) a VH domain comprising a sequence encoded by SEQ ID NO: 209, or a sequence having at least 70% identity to the 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 to the sequence encoded by SEQ ID NO: 210; (iv) a VH domain comprising a sequence encoded by SEQ ID NO:211, or a sequence having at least 70% identity to the 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 to the sequence encoded by SEQ ID NO:212; (v) a VH domain comprising a sequence encoded by SEQ ID NO:213, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO:213, and a VL domain comprising a sequence encoded by SEQ ID NO:214, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO:214; (vi) a VH domain comprising a sequence encoded by SEQ ID NO: 215, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 215, and a VL domain comprising a sequence encoded by SEQ ID NO: 216, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 216; (vii) a VH domain comprising a sequence encoded by SEQ ID NO: 217, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 217, and a VL domain comprising a sequence encoded by SEQ ID NO: 218, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 218; (viii) a VH domain comprising a sequence encoded by SEQ ID NO: 219, or a sequence having at least 70% identity to 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 to the sequence encoded by SEQ ID NO: 220; (ix) a VH domain comprising a sequence encoded by SEQ ID NO: 221, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO: 221, and a VL domain comprising a sequence encoded by SEQ ID NO: 222, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO: 222; (x) a VH domain comprising a sequence encoded by SEQ ID NO: 223, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 223, and a VL domain comprising a sequence encoded by SEQ ID NO: 224, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 224; (xi) a VH domain comprising a sequence encoded by SEQ ID NO: 225, or a sequence having at least 70% identity to 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 to the sequence encoded by SEQ ID NO: 226; (xii) a VH domain comprising a sequence encoded by SEQ ID NO: 227, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 227, and a VL domain comprising a sequence encoded by SEQ ID NO: 228, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 228; (xiii) a VH domain comprising a sequence encoded by SEQ ID NO: 229, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO: 229, and a VL domain comprising a sequence encoded by SEQ ID NO: 230, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO: 230; or (xiv) a VH domain comprising a sequence encoded by SEQ ID NO: 231, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO: 231, and a VL domain comprising a sequence encoded by SEQ ID NO: 232, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO: 232.

[0019] Furthermore, the antigen recognition domain of the CAR is (i) the sequence set forth in SEQ ID NO: 113, or a sequence having at least 80% sequence identity thereto; (ii) the sequence set forth in SEQ ID NO: 114, or a sequence having at least 80% sequence identity thereto; (iii) the sequence set forth in SEQ ID NO: 115, or a sequence having at least 80% sequence identity thereto; (iv) the sequence set forth in SEQ ID NO: 116, or a sequence having at least 80% sequence identity thereto; (v) the sequence set forth in SEQ ID NO: 117, or a sequence having at least 80% sequence identity thereto; (vi) the sequence set forth in SEQ ID NO: 118, or a sequence having at least 80% sequence identity thereto; (vii) the sequence set forth in SEQ ID NO: 119, or a sequence having at least 80% sequence identity thereto; (viii) the sequence set forth in SEQ ID NO: 120, or a sequence having at least 80% sequence identity thereto; (ix) the sequence set forth in SEQ ID NO: 121, or a sequence having at least 80% sequence identity thereto; (x) the sequence set forth in SEQ ID NO: 122, or a sequence having at least 80% sequence identity thereto; (xi) the sequence set forth in SEQ ID NO: 123, or a sequence having at least 80% sequence identity thereto; (xii) the sequence set forth in SEQ ID NO: 124, or a sequence having at least 80% sequence identity thereto; (xiii) the sequence set forth in SEQ ID NO: 125, or a sequence having at least 80% sequence identity thereto; or (xiv) may comprise or consist of the sequence set forth in SEQ ID NO: 126, or a sequence having at least 80% sequence identity thereto.

[0020] The antigen recognition domain may be, for example, (i) a sequence encoded by the sequence set forth in SEQ ID NO: 191 or a sequence having at least 80% identity to the sequence encoded by the sequence set forth in SEQ ID NO: 191; (ii) a sequence encoded by the sequence set forth in SEQ ID NO: 192 or a sequence having at least 80% identity to the sequence encoded by the sequence set forth in SEQ ID NO: 192; (iii) a sequence encoded by the sequence set forth in SEQ ID NO: 193 or a sequence having at least 80% identity to the sequence encoded by the sequence set forth in SEQ ID NO: 193; (iv) a sequence encoded by the sequence set forth in SEQ ID NO: 194 or a sequence having at least 80% identity to the sequence encoded by the sequence set forth in SEQ ID NO: 194; (v) a sequence encoded by the sequence set forth in SEQ ID NO: 195 or a sequence having at least 80% identity to the sequence encoded by the sequence set forth in SEQ ID NO: 195; (vi) a sequence encoded by the sequence set forth in SEQ ID NO: 196 or a sequence having at least 80% identity to the sequence encoded by the sequence set forth in SEQ ID NO: 196; (vii) a sequence encoded by the sequence set forth in SEQ ID NO: 197 or a sequence having at least 80% identity to the sequence encoded by the sequence set forth in SEQ ID NO: 197; (viii) a sequence encoded by the sequence set forth in SEQ ID NO: 198 or a sequence having at least 80% identity to the sequence encoded by the sequence set forth in SEQ ID NO: 198; (ix) a sequence encoded by the sequence set forth in SEQ ID NO: 199 or a sequence having at least 80% identity to the sequence encoded by the sequence set forth in SEQ ID NO: 199; (x) a sequence encoded by the sequence set forth in SEQ ID NO: 200 or a sequence having at least 80% identity to the sequence encoded by the sequence set forth in SEQ ID NO: 200; (xi) a sequence encoded by the sequence set forth in SEQ ID NO: 201 or a sequence having at least 80% identity to the sequence encoded by the sequence set forth in SEQ ID NO: 201; (xii) a sequence encoded by the sequence set forth in SEQ ID NO: 202 or a sequence having at least 80% identity to the sequence encoded by the sequence set forth in SEQ ID NO: 202; (xiii) a sequence encoded by the sequence set forth in SEQ ID NO: 203 or a sequence having at least 80% identity to the sequence encoded by the sequence set forth in SEQ ID NO: 203; or (xiv) may comprise or consist of a sequence encoded by the sequence set forth in SEQ ID NO: 204 or a sequence having at least 80% identity to the sequence encoded by the sequence set forth in SEQ ID NO: 204.

[0021] In a second aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a CAR according to the present invention.

[0022] In a third aspect, the present invention provides a vector comprising a nucleic acid molecule according to the present invention, which may further comprise a nucleic acid molecule comprising a nucleotide sequence encoding a FOXP3 polypeptide, or a derivative or variant thereof.

[0023] In a further aspect, the present invention provides a cell comprising a CAR, nucleic acid molecule, or vector of the present invention. The cell may further comprise an exogenous FOXP3 polypeptide or an exogenous nucleic acid encoding FOXP3. The cell may be an immune cell, or a progenitor or precursor thereof. Optionally, the cell may be a T cell, or a precursor thereof, or a stem cell. In particular, the cell may be a Treg, or a precursor thereof, or an iPSC cell. The cell may be a producer host cell. In a specific embodiment, the present invention provides a Treg comprising a CAR, wherein the antigen recognition domain of the CAR specifically binds to ENTPD3.

[0024] The cells may be provided as a cell population, which constitutes a further aspect of the present invention. In particular, the cell population may comprise a plurality of cells according to the present invention, and in particular may comprise a plurality of T cells (e.g., Tregs) according to the present invention. In particular, the plurality of T cells (e.g., Tregs) according to the present invention have polyclonal TCRs. In particular, the clonality of the TCRs possessed by the plurality of T cells (e.g., Tregs) according to the present invention has not been modified ex vivo.

[0025] The present invention also provides a pharmaceutical composition comprising a cell, a cell population, or a vector according to the present invention.

[0026] In another aspect, the invention provides a cell, cell population, or pharmaceutical composition of the invention for use in therapy (e.g., for use in the treatment and / or prevention of an autoimmune or inflammatory disease, for use in inducing immunosuppression, or for use in promoting tissue repair and / or regeneration), which may be adoptive cell transfer therapy.

[0027] Viewed another way, the present invention provides a method for treating and / or preventing an autoimmune or inflammatory disease, for inducing immunosuppression or for promoting tissue repair and / or tissue regeneration, comprising administering a cell, in particular a Treg cell, cell population or pharmaceutical composition according to the present invention, in particular a pharmaceutical composition containing Treg.

[0028] In this regard, the method comprises: (i) isolating or providing a Treg-enriched cell sample from a subject; (ii) introducing the nucleic acid molecule or vector of the present invention into the Treg cells; (iii) administering the Treg cells obtained in step (ii) to the subject.

[0029] The present invention also provides the use of a cell, cell population or pharmaceutical composition according to the invention in the manufacture of a medicament for treating and / or preventing an autoimmune or inflammatory disease, inducing immunosuppression or promoting tissue repair and / or regeneration in a subject, in particular wherein the cell is a Treg cell. The autoimmune or inflammatory disease may in particular be type 1 diabetes (T1D).

[0030] In another aspect, the present invention provides a method for producing a cell of the present invention, the method comprising the step of introducing (e.g., transducing or transfecting) a nucleic acid molecule or vector of the present invention into a cell. The cell may be a Treg cell, and the method may comprise the step of isolating or providing a cell-containing sample comprising Tregs, and / or may comprise the step of enriching for or generating Tregs from the cell-containing sample before or after introducing the nucleic acid molecule or vector into the cell. The present invention also provides a cell obtained by this method, which constitutes a further aspect of the present invention.

[0031] In a further embodiment, the present invention provides the use of CAR-Tregs for reducing (e.g., reducing the cell death rate) or preventing pancreatic β-cell death in a subject. In a further embodiment, the present invention provides the use of CAR-Tregs for maintaining or increasing fasting blood insulin levels in a subject. In a further embodiment, the present invention provides the use of CAR-Tregs for maintaining or increasing fasting C-peptide levels in a subject. In a further embodiment, the present invention provides the use of CAR-Tregs for reducing or preventing hyperglycemia in a subject. In a further embodiment, the present invention provides the use of CAR-Tregs for maintaining or reducing fasting blood glucose levels in a subject. In a further embodiment, the present invention provides the use of CAR-Tregs for maintaining or reducing HbA1c levels in a subject. The CAR is a CAR of the present invention. That is, it contains an antigen recognition domain that specifically binds to ENTPD3 (e.g., human ENTPD3) and may have any of the characteristics of a CAR disclosed herein. The subject may, for example, have or be at risk of developing type 1 diabetes, particularly recently developed type 1 diabetes. The subject may, for example, not be receiving exogenous insulin. Alternatively, the subject may be receiving a reduced dose of insulin compared to the dose of insulin required prior to administration of the CAR-Tregs. [Brief explanation of the drawings]

[0032] [Figure 1] FIG. 1 shows the binding of various scFvs to HEK293T cells expressing human ENTPD3 (lines 1 and 3) or mouse ENTPD3 (lines 2 and 4). [Figure 2] FIG. 2 shows immunohistochemical staining of mouse pancreatic sections using mouse ENTPD3-specific scFV. [Figure 3] Figure 3 shows activation of mouse hybridoma cells expressing a mouse ENTPD3-specific CAR by HEK cells expressing mouse ENTPD3. [Figure 4] FIG. 4 shows an overview of the experimental protocol used for the induction of type 1 diabetes in NOD mice administered T effector cells expressing an ENTPD3-specific CAR (Example 2). [Figure 5] FIG. 5 shows an overview of the experimental protocol used to observe the enrichment of T cells expressing ENTPD3-specific CAR in NOD mouse pancreatic tissue (Example 3). [Figure 6] FIG. 6 shows enrichment of T cells expressing ENTPD3-specific CAR within pancreatic islets of NOD mice compared to the spleen and lymph nodes. [Figure 7] FIG. 7 shows an outline of the experimental protocol used for the prevention of cyclophosphamide-induced type 1 diabetes in NOD mice administered Tregs expressing an ENTPD3-specific CAR (Example 4). [Figure 8] FIG. 8 shows prevention of cyclophosphamide-induced type 1 diabetes in NOD mice using Tregs expressing an ENTPD3-specific CAR. [Figure 9] FIG. 9 shows enrichment of Treg cells expressing ENTPD3-specific CAR within the pancreatic islets of NOD mice compared to the spleen and lymph nodes. [Figure 10]Figure 10 shows the construction of the murine CAR used in Examples 1 to 6. (A) shows the schematic structure of the CAR with a mutated Fc-IgG hinge and CD8 hinge, and (B) shows the schematic structure of the construct used in the gamma retroviral vector for transduction into cells. [Figure 11] FIG. 11 shows an outline of the experimental protocol used to observe the enrichment of Tregs expressing ENTPD3-specific CAR in the pancreatic tissue of NOD mice (Example 5). [Figure 12] FIG. 12 shows the enrichment of Tregs expressing ENTPD3-specific CARs within the pancreatic islets of NOD mice compared to the spleen and lymph nodes. [Figure 13] FIG. 13 shows activation of Tregs exposed to target (ENTPD3) (middle bar) compared to Tregs exposed to control antigen (right bar) or anti-CD3 / CD28 beads (left bar). [Figure 14] FIG. 14 shows the proliferation of Tregs exposed to the target (ENTPD3) (middle bar) compared to Tregs exposed to a control antigen (right bar) or anti-CD3 / CD28 beads (left bar). [Figure 15] Figure 15 shows the activation (percentage of NFAT GFP+ in the CAR+ population) of human ENTPD3 protein, HEK cells expressing human or mouse ENTPD3, non-transduced HEK cells, and hybridoma cells expressing various ENTPD3-specific CARs when exposed to culture medium. [Figure 16] Figure 16 shows the construction of the human CAR used in Example 7, where (A) shows a schematic structure of the CAR with a mutated Fc-IgG hinge and a CD8 hinge, and (B) shows a schematic structure of the construct used for transduction into cells. [Figure 17] FIG. 17 shows the transduction efficiency of Jurkat cells transduced with various ENTPD3-specific CAR constructs described in Example 8. [Figure 18]Figure 18 shows the luminescence of Jurkat cells transduced with various ENTPD3-specific CAR constructs described in Example 8 after exposure to HEK293 cells expressing huENTPD3, RT-4 cells, huENTPD3 peptide, OKT3 anti-CD3 antibody, and non-stimulated conditions. [Figure 19] FIG. 19 shows the same results as FIG. 18, except that the positive control was omitted so that activation by huENTPD3 could be more clearly seen. [Figure 20] FIG. 20 shows the percentage of FOXP3-expressing cells at 7 and 12 days after transduction. [Figure 21] FIG. 21 shows the results of the Treg activation assay, specifically showing the percentage of cells expressing the activation markers CD69, CD137, and GARP. [Figure 22] FIG. 22 shows the results of a Treg suppression assay. [Figure 23] FIG. 23 shows the results of a Treg proliferation assay. [Figure 24] FIG. 24 shows the results of cytokine secretion assays for IL-10, IL-13, and INF-γ. [Figure 25] FIG. 25 shows representative live images of hIsMT co-cultured with various T cell populations at 24, 48, and 72 hours (2400 cells). [Figure 26] FIG. 26 shows representative live images of hIsMT co-cultured with various T cell populations at 24, 48, and 72 hours (2400 cells). [Figure 27] FIG. 27 shows representative live images of hIsMT co-cultured with various T cell populations at 24, 48, and 72 hours (2400 cells). [Figure 28]Figure 28 shows the stimulation-induced insulin secretion of hIsMT co-cultured with various T cell populations at 48 and 72 hours of co-culture (A and B, respectively), and the total insulin levels of hIsMT co-cultured with various T cell populations at 48 and 72 hours of co-culture (C and D). [Figure 29] Figure 29 shows IFN-γ secretion by hIsMT co-cultured with various T cell populations at 48 and 72 hours of co-culture (A and C), and by T cells alone at 48 and 72 hours of culture (B and D). [Figure 30] Figure 30 shows TNF-α secretion by hIsMT co-cultured with various T cell populations at 48 and 72 hours of co-culture (A and C), and TNF-α secretion by T cells alone at 48 and 72 hours of culture (B and D). [Figure 31] Figure 31 shows the number of WPRE copies per μg of DNA in the pancreas and testes of NOD mice administered PE- or ENTPD3-specific CAR-Tregs or untreated NOD mice.

[0033] Detailed Description The present invention provides ENTPD3-specific CAR-Tregs that are activated in the presence of the ENTPD3 antigen, which is particularly expressed in pancreatic β cells. Thus, these CAR-Tregs have therapeutic potential in the treatment of autoimmune and inflammatory disorders in which ENTPD3 is locally expressed at the disease site. In particular, these CAR-Tregs have therapeutic potential for type 1 diabetes. In particular, due to the bystander effect of Treg cells, the antigen (ENTPD3) may simply be present and / or expressed at the site of inflammation or disease.

[0034] Thus, the present invention provides chimeric antigen receptors (CARs) comprising an antigen recognition domain that binds to ENTPD3, cells or cell populations expressing the CARs (e.g., Tregs or populations of Tregs expressing the CARs), and uses of the cells or cell populations in the treatment of certain diseases, such as type 1 diabetes.

[0035] "Chimeric antigen receptor," "CAR," or "CAR construct" refers to an engineered receptor capable of conferring antigen specificity to a cell (e.g., an immune cell such as a Treg). In particular, a CAR enables a cell to specifically bind to a particular antigen, e.g., a target molecule such as a target protein, and upon binding, a signal is generated by the endodomain of the CAR (including an intracellular signaling domain), e.g., a signal that causes cell activation. CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors. The structure of CAR is well known in the art, and several generations of CAR have been produced so far.For example, CAR can at least comprise an extracellular antigen-specific targeting region, an antigen binding domain, a target binding domain or a ligand binding domain that is the exodomain (also called extracellular domain or ectodomain) of CAR or constitutes a part thereof, a transmembrane domain, and an intracellular signaling domain (that is an endodomain or is contained in the endodomain).However, CAR can also comprise additional domains to improve its functionality, such as one or more costimulatory domains to improve T cell proliferation, cytokine secretion, resistance to apoptosis, and persistence in vivo.

[0036] Thus, a chimeric receptor or CAR construct generally comprises a binding domain (which can be considered an antigen (i.e., target) or ligand-binding domain. The terms binding domain, antigen recognition domain, antigen-binding domain, and ligand-binding domain are used interchangeably herein), optionally a hinge domain (which functions as a spacer to position the binding domain away from the plasma membrane of the cell (e.g., immune cell) expressing the CAR), a transmembrane domain, an intracellular signaling domain (e.g., the signaling domain from the zeta chain of the CD3 molecule (CD3ζ) of the TCR complex, or its equivalent), and optionally one or more costimulatory domains (which may assist in signaling or functionality of the cell expressing the CAR). CARs may also comprise a signal or leader sequence, or a domain corresponding thereto, that functions to target the protein to the membrane; such a sequence or domain may constitute part of the exodomain of the CAR. These different domains may be linked directly or via a linker and / or may be contained in different polypeptides, e.g., two polypeptides that associate with each other.

[0037] Binding of the CAR to the target antigen (i.e., ENTPD3) delivers an activating signal to the cell expressing the CAR, thus directing the specificity of the engineered cell toward ENTPD3, and in particular, toward cells expressing ENTPD3.

[0038] The terms "directed toward" or "directed against" are synonymous with "specific for" or "anti." In other words, the CAR recognizes the ENTPD3 target molecule. Therefore, this term means that the CAR can specifically bind to ENTPD3. In particular, the antigen-binding domain of the CAR can specifically bind to ENTPD3 (more particularly, this is evident when the CAR is expressed on the surface of a cell, particularly an immune effector cell). Specific binding can be distinguished from nonspecific binding to a non-target molecule or a non-target antigen. Thus, a cell expressing a CAR is directed or redirected to specifically bind to a target cell expressing ENTPD3, particularly a target cell expressing ENTPD3 on its cell surface. In particular, "specific" binding means that binding occurs only or primarily to ENTPD3, and not to other proteins or polypeptides (i.e., binding to other proteins or polypeptides is negligible or weaker). Although some cross-reactivity with other proteins may occur, the binding level can be considered background. As described above, CAR can bind to ENTPD3 and transmit a signal into the cells expressing the CAR. The cells can then be activated and exhibit suppressive effects in the local environment. Activation of CAR-expressing cells after antigen binding can be determined by an increase in CD69 levels compared to the same CAR-expressing cells in the absence of antigen. For example, an increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% in CD69 expression is considered. The CD69 expression level can be measured using standard techniques, such as FACS, using commercially available antibodies (e.g., FITC anti-human CD69 antibody, Biolegend). Therefore, the function of CAR in cells can be determined by the activation state of CAR-expressing cells, for example, by measuring the amount of CD69 expression.

[0039] ENTPD3 (ectonucleoside triphosphate diphosphohydrolase 3) is also known as CD39L3, HB6, and NTPDase-3. ENTPD3 is a membrane-bound enzyme similar to E-type nucleotidase (NTPase) and is expressed in pancreatic beta cells. The amino acid sequence of human ENTPD3 is shown in SEQ ID NO: 127, and the amino acid sequence of mouse ENTPD3 is shown in SEQ ID NO: 128.

[0040] The antigen-binding domain of the CAR may be derived from or obtained from any protein or polypeptide that binds to (i.e., has affinity for) ENTPD3 (e.g., such a protein or polypeptide binds to any region or portion of ENTPD3, or binds to any epitope within ENTPD3 when present as an isolated protein or expressed on a cell). In particular, the antigen-binding domain of the CAR may be derived from or obtained from any protein or polypeptide that binds to (i.e., has affinity for) the extracellular domain of ENTPD3. For example, it may be a ligand of ENTPD3, or a physiological binding protein for ENTPD3, a portion thereof, or a synthetic or derivative protein. The target molecule (i.e., ENTPD3) may generally, but need not necessarily, be expressed on the cell surface, for example, on the surface of a target cell (e.g., pancreatic beta cell) or a cell near the target cell (due to bystander effect).

[0041] Antigen-binding domains are most commonly derived from antibody variable chains (e.g., commonly in the form of scFvs), but may also be generated from other molecules, such as ligands or other binding molecules.

[0042] CAR is typically expressed as a polypeptide that also contains a signal sequence (also known as a leader sequence), particularly a signal sequence that targets CAR to the plasma membrane of a cell. This signal sequence is generally located adjacent to or near the antigen binding domain, usually upstream of the antigen binding domain. Thus, the extracellular domain of CAR, i.e., the ectodomain, can comprise, consist essentially of, or consist of a signal sequence and an antigen binding domain.

[0043] As mentioned above, the antigen-binding domain can be any protein or peptide that can specifically recognize and bind to ENTPD3.The antigen-binding domain can include any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for ENTPD3.Examples of antigen-specific targeting domains include antibodies, antibody fragments or derivatives, or ligands for soluble or membrane-bound ENTPD3.

[0044] In one embodiment, the antigen-binding domain is an antibody or is derived from an antibody. As used herein, the term "antibody" broadly refers to any immunological binding agent or molecule that contains an antigen-binding domain, including polyclonal and monoclonal antibodies. Depending on the type of constant domain in the heavy chain, whole antibodies are classified into five major classes: IgA, IgD, IgE, IgG, and IgM. The antibodies described herein may belong to any one of these classes. Some of these classes are further subdivided into subclasses or isotypes, such as IgG1, IgG2, IgG3, and IgG4. Generally, IgG or IgM antibodies are the most common antibodies utilized under physiological conditions. As will be understood by those skilled in the art, the term "antibody" encompasses all antibodies, including whole, dimeric, trimeric, and multimeric antibodies; bispecific antibodies; chimeric antibodies; recombinant and engineered antibodies; and fragments thereof. An antibody-derived binding domain may be a fragment of an antibody or the product of genetic engineering of one or more fragments of an antibody, which are responsible for binding to an antigen. Examples include variable regions (Fv), complementarity determining regions (CDRs), Fab, F(ab')2, etc. Alternatively, the variable regions of the light and heavy chains may be linked into a single chain (e.g., scFv), in either orientation (e.g., V L -V H or V H -V L ). V L Sequence and / or V H The sequences may be modified. In particular, the framework regions may be modified (e.g., substituted for the purpose of humanizing the antigen-binding domain). Other examples include heavy chain variable regions (VH), light chain variable regions (VL), and single domain antibodies (sAb) (sometimes called nanobodies). An example of a single domain antibody is a camelid heavy chain antibody (HCAb), which has an antigen recognition site formed by a single domain called VHH.

[0045] In one embodiment, the antigen-binding domain is a single-chain antibody (scFv). The scFv may be a murine scFv, a human scFv, or a humanized scFv. In alternative embodiments, the antigen-binding domain is derived from a camelid heavy chain antibody (HCAb), for example, the antigen-binding domain may be a VHH domain of an HCAb. VHHs have a structure similar to that of VH domains derived from conventional IgGs and contain three variable CDRs, although CDR1 and CDR3 often have more amino acids than those of VH domains. In certain embodiments, the VHH domain may comprise one or more CDRs comprising or consisting of a sequence set forth in SEQ ID NOS: 1-84 set forth herein, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. For example, a VHH domain may comprise one, two, or three VH CDRs comprising or consisting of a sequence set forth in SEQ ID NOs: 1-84 set forth herein, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. When a VHH domain comprises two or three VH CDRs of SEQ ID NOs: 1-84, these CDRs may be derived from the same binder described herein (e.g., all from the A8, B2, or B7 binder).

[0046] A "complementarity-determining region" or "CDR" of an antibody or antigen-binding fragment thereof refers to the hypervariable loops in the variable region of the heavy or light chain of the antibody. CDRs interact with the conformation of the antigen and may largely determine antigen binding (although some framework regions are known to be involved in binding). The heavy and light chain variable regions each contain three CDRs. A "heavy chain variable region" or "VH" refers to a fragment of an antibody heavy chain in which the three CDRs are flanked by adjacent contiguous sequences called framework regions, which are more conserved than the CDRs and form a scaffold to support the CDRs. A "light chain variable region" or "VL" refers to a fragment of an antibody light chain in which the three CDRs are flanked by framework regions.

[0047] "Fv" refers to the smallest antibody fragment that contains a complete antigen-binding site. An Fv fragment consists of the variable region of one light chain bound to the variable region of one heavy chain. A "single-chain Fv antibody" or "scFv" refers to an engineered antibody in which the variable regions of the light and heavy chains are connected to each other, in either orientation, either directly or via a peptide linker sequence.

[0048] 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 producing human or humanized antibodies, or methods using transgenic animals or plants engineered to produce human antibodies. Phage display libraries of partially or fully synthetic antibodies are available, and these libraries can be screened for antibodies or fragments thereof that can bind to the target molecule, i.e., ENTPD3. Phage display libraries of human antibodies are also available. Once an antibody of interest is identified, the amino acid sequence or polynucleotide sequence encoding the antibody can be isolated and / or determined.

[0049] The antigen recognition domain can bind to, and preferably specifically bind to, one or more regions or epitopes in ENTPD3. An epitope, also known as an antigenic determinant, is a part of an antigen that is recognized by an antigen recognition domain (e.g., an antibody). In other words, an epitope is a specific part of an antigen that an antibody binds to. Preferably, the antigen recognition domain binds to, and preferably specifically binds to, one region or epitope in ENTPD3. The antigen recognition domain may comprise at least one CDR (e.g., CDR3) predicted from an antibody that binds to the antigen, i.e., ENTPD3 (or a variant of such a predicted CDR, e.g., a variant with one, two, or three amino acid substitutions). It will be understood that a molecule comprising three or fewer CDR regions (e.g., one CDR, or a portion thereof) may retain the antigen-binding activity of the antibody from which the CDR is derived. It has been described in the art that molecules comprising two CDR regions can bind to target antigens, for example, in the form of minibodies (Vaughan and Sollazzo, 2001, Combinational Chemistry & High Throughput Screening, 4, 417-430). It has also been described that molecules comprising one CDR can exhibit strong binding activity to targets (Nicaise et al., 2004, Protein Science, 13: 1882-91).

[0050] In this regard, the antigen-binding domain may comprise one or more variable heavy chain CDRs, for example, one, two, or three variable heavy chain CDRs. Alternatively, or in addition, the antigen-binding domain may comprise one or more variable light chain CDRs, for example, one, two, or three variable light chain CDRs. The antigen-binding domain may comprise three heavy chain CDRs and / or three light chain CDRs (more specifically, a heavy chain variable region comprising three CDRs and / or a light chain variable region comprising three CDRs), where at least one CDR, and optionally all CDRs, may be derived from an antibody that binds to ENTPD3.

[0051] The antigen-binding domain may comprise any combination of variable heavy chain CDRs and variable light chain CDRs. For example, it may comprise a combination of one variable heavy chain CDR and one variable light chain CDR, two variable heavy chain CDRs and one variable light chain CDR, two variable heavy chain CDRs and two or three variable light chain CDRs, three variable heavy chain CDRs and one or two variable light chain CDRs, one variable heavy chain CDR and two or three variable light chain CDRs, or three variable heavy chain CDRs and 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).

[0052] The one or more CDRs present in an antigen-binding domain do not all need to be derived from the same antibody, as long as the domain has the desired binding activity. Thus, one CDR may be predicted from the heavy or light chain of an antibody that binds to ENTPD3, while another CDR may be predicted from a different antibody that binds to ENTPD3. CDRs from different antibodies, particularly CDRs from antibodies that bind to the same desired region or epitope, may also be used in combination.

[0053] In one embodiment, the antigen-binding domain comprises three CDRs predicted from the variable heavy chain sequence of an antibody that binds to ENTPD3 and / or three CDRs predicted from the variable light chain sequence of an antibody that binds to ENTPD3 (optionally the same antibody).

[0054] In one embodiment, the antigen-binding domain comprises the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively. Alternatively, the CDRs may comprise one to three, more specifically one or two, amino acid sequence modifications in any of the above sequences. More specifically, in such embodiments, the antigen binding domain of the CAR comprises a VH domain comprising the sequence set forth in SEQ ID NO: 85, or a sequence with at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 86, or a sequence with at least 70% identity thereto. For example, in such embodiments, the antigen binding domain of the CAR comprises a VH domain comprising a sequence encoded by SEQ ID NO:205, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO:205, and a VL domain comprising a sequence encoded by SEQ ID NO:206, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO:206.

[0055] In one embodiment, the antigen-binding domain comprises the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively. Alternatively, the CDRs may comprise one to three, more specifically one or two, amino acid sequence modifications in any of the above sequences. More specifically, in such embodiments, the antigen binding domain of the CAR comprises a VH domain comprising the sequence set forth in SEQ ID NO: 87, or a sequence with at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 88, or a sequence with at least 70% sequence identity thereto. For example, in such embodiments, the antigen binding domain of the CAR comprises a VH domain comprising a sequence encoded by SEQ ID NO:207, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO:207, and a VL domain comprising a sequence encoded by SEQ ID NO:208, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO:208.

[0056] In one embodiment, the antigen-binding domain comprises the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively. Alternatively, the CDRs may comprise one to three, more specifically one or two, amino acid sequence modifications in any of the above sequences. More specifically, in such embodiments, the antigen binding domain of the CAR comprises a VH domain comprising the sequence set forth in SEQ ID NO: 89, or a sequence with at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 90, or a sequence with at least 70% sequence identity thereto. For example, in such embodiments, the antigen binding domain of the CAR comprises a VH domain comprising a sequence encoded by SEQ ID NO:209, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO:209, and a VL domain comprising a sequence encoded by SEQ ID NO:210, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO:210.

[0057] In one embodiment, the antigen-binding domain comprises the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively. Alternatively, the CDRs may comprise one to three, more specifically one or two, amino acid sequence modifications in any of the above sequences. More specifically, in such embodiments, the antigen binding domain of the CAR comprises a VH domain comprising the sequence set forth in SEQ ID NO: 91, or a sequence with at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 92, or a sequence with at least 70% sequence identity thereto. For example, in such embodiments, the antigen binding domain of the CAR comprises a VH domain comprising a sequence encoded by SEQ ID NO:211, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO:11, and a VL domain comprising a sequence encoded by SEQ ID NO:212, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO:212.

[0058] In one embodiment, the antigen-binding domain comprises the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 25, 26, and 27, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively. Alternatively, the CDRs may comprise one to three, more specifically one or two, amino acid sequence modifications in any of the above sequences. More specifically, in such embodiments, the antigen binding domain of the CAR comprises a VH domain comprising the sequence set forth in SEQ ID NO: 93, or a sequence with at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 94, or a sequence with at least 70% sequence identity thereto. For example, in such embodiments, the antigen binding domain of the CAR comprises a VH domain comprising a sequence encoded by SEQ ID NO:213, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO:213, and a VL domain comprising a sequence encoded by SEQ ID NO:214, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO:214.

[0059] In one embodiment, the antigen-binding domain comprises the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively. Alternatively, the CDRs may comprise one to three, more specifically one or two, amino acid sequence modifications in any of the above sequences. More specifically, in such embodiments, the antigen binding domain of the CAR comprises a VH domain comprising the sequence set forth in SEQ ID NO: 95, or a sequence with at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 96, or a sequence with at least 70% sequence identity thereto. For example, in such embodiments, the antigen binding domain of the CAR comprises a VH domain comprising a sequence encoded by SEQ ID NO:215, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO:215, and a VL domain comprising a sequence encoded by SEQ ID NO:216, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO:216.

[0060] In one embodiment, the antigen-binding domain comprises the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 37, 38, and 39, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 40, 41, and 42, respectively. Alternatively, the CDRs may comprise one to three, more specifically one or two, amino acid sequence modifications in any of the above sequences. More specifically, in such embodiments, the antigen binding domain of the CAR comprises a VH domain comprising the sequence set forth in SEQ ID NO: 97, or a sequence with at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 98, or a sequence with at least 70% sequence identity thereto. For example, in such embodiments, the antigen binding domain of the CAR comprises a VH domain comprising a sequence encoded by SEQ ID NO:217, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO:217, and a VL domain comprising a sequence encoded by SEQ ID NO:218, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO:218.

[0061] In one embodiment, the antigen-binding domain comprises the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 43, 44, and 45, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 46, 47, and 48, respectively. Alternatively, the CDRs may comprise one to three, more specifically one or two, amino acid sequence modifications in any of the above sequences. More specifically, in such embodiments, the antigen binding domain of the CAR comprises a VH domain comprising the sequence set forth in SEQ ID NO: 99, or a sequence with at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 100, or a sequence with at least 70% sequence identity thereto. For example, in such embodiments, the antigen binding domain of the CAR comprises a VH domain comprising a sequence encoded by SEQ ID NO:219, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO:219, and a VL domain comprising a sequence encoded by SEQ ID NO:220, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO:220.

[0062] In one embodiment, the antigen-binding domain comprises the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 52, 53, and 54, respectively. Alternatively, the CDRs may comprise one to three, more specifically one or two, amino acid sequence modifications in any of the above sequences. More specifically, in such embodiments, the antigen binding domain of the CAR comprises a VH domain comprising the sequence set forth in SEQ ID NO: 101, or a sequence with at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 102, or a sequence with at least 70% sequence identity thereto. For example, in such embodiments, the antigen binding domain of the CAR comprises a VH domain comprising a sequence encoded by SEQ ID NO:221, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO:221, and a VL domain comprising a sequence encoded by SEQ ID NO:222, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO:222.

[0063] In one embodiment, the antigen-binding domain comprises the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 55, 56, and 57, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 58, 59, and 60, respectively. Alternatively, the CDRs may comprise one to three, more specifically one or two, amino acid sequence modifications in any of the above sequences. More specifically, in such embodiments, the antigen binding domain of the CAR comprises a VH domain comprising the sequence set forth in SEQ ID NO: 103, or a sequence with at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 104, or a sequence with at least 70% sequence identity thereto. For example, in such embodiments, the antigen binding domain of the CAR comprises a VH domain comprising a sequence encoded by SEQ ID NO:223, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO:223, and a VL domain comprising a sequence encoded by SEQ ID NO:224, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO:224.

[0064] In one embodiment, the antigen-binding domain comprises the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 61, 62, and 63, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 64, 65, and 66, respectively. Alternatively, the CDRs may comprise one to three, more specifically one or two, amino acid sequence modifications in any of the above sequences. More specifically, in such embodiments, the antigen binding domain of the CAR comprises a VH domain comprising the sequence set forth in SEQ ID NO: 105, or a sequence with at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 106, or a sequence with at least 70% sequence identity thereto. For example, in such embodiments, the antigen binding domain of the CAR comprises a VH domain comprising a sequence encoded by SEQ ID NO:225, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO:225, and a VL domain comprising a sequence encoded by SEQ ID NO:226, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO:226.

[0065] In one embodiment, the antigen-binding domain comprises the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 67, 68, and 69, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 70, 71, and 72, respectively. Alternatively, the CDRs may comprise one to three, more specifically one or two, amino acid sequence modifications in any of the above sequences. More specifically, in such embodiments, the antigen binding domain of the CAR comprises a VH domain comprising the sequence set forth in SEQ ID NO: 107, or a sequence with at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 108, or a sequence with at least 70% sequence identity thereto. For example, in such embodiments, the antigen binding domain of the CAR comprises a VH domain comprising a sequence encoded by SEQ ID NO:227, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO:227, and a VL domain comprising a sequence encoded by SEQ ID NO:228, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO:228.

[0066] In one embodiment, the antigen-binding domain comprises the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 73, 74, and 75, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 76, 77, and 78, respectively. Alternatively, the CDRs may comprise one to three, more specifically one or two, amino acid sequence modifications in any of the above sequences. More specifically, in such embodiments, the antigen binding domain of the CAR comprises a VH domain comprising the sequence set forth in SEQ ID NO: 109, or a sequence with at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 110, or a sequence with at least 70% sequence identity thereto. For example, in such embodiments, the antigen binding domain of the CAR comprises a VH domain comprising a sequence encoded by SEQ ID NO:229, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO:229, and a VL domain comprising a sequence encoded by SEQ ID NO:230, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO:230.

[0067] In one embodiment, the antigen-binding domain comprises the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 79, 80, and 81, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 82, 83, and 84, respectively. Alternatively, the CDRs may comprise one to three, more specifically one or two, amino acid sequence modifications in any of the above sequences. More specifically, in such embodiments, the antigen binding domain of the CAR comprises a VH domain comprising the sequence set forth in SEQ ID NO: 111, or a sequence with at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 112, or a sequence with at least 70% sequence identity thereto. For example, in such embodiments, the antigen binding domain of the CAR comprises a VH domain comprising a sequence encoded by SEQ ID NO:231, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO:231, and a VL domain comprising a sequence encoded by SEQ ID NO:232, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO:232.

[0068] When a CDR contains an amino acid sequence modification, the modification may be a deletion, addition, or substitution of an amino acid residue in the CDR sequence set forth in the above SEQ ID NO. More specifically, the modification may be an amino acid substitution, for example, a conservative amino acid substitution as described above. Longer CDRs can tolerate more amino acid residue modifications. For CDRs having a length of 5 or more amino acid residues, or 7 or more amino acid residues, the modification may be 0, 1, 2, or 3 residues, for example, 2 residues. Generally, 0, 1, 2, or 3 modifications may be made to any particular CDR sequence. Furthermore, in one embodiment, CDR1 and CDR2 may be modified, while CDR3 may be unmodified. In another embodiment, all three CDRs may be modified. In yet another embodiment, the CDRs are unmodified.

[0069] The antigen-binding domain may be in the form of an scFv comprising the above VH and VL domain sequences in either order, for example, in the order VH-VL. The VH and VL sequences may be linked by a linker sequence. A suitable linker can be easily selected and may be of any suitable length, such as from 1 amino acid (e.g., Gly) to 30 amino acids. For example, the linker may be from 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids to 12, 15, 18, 20, 21, 25, or 30 amino acids in length, and examples include 5 to 30 amino acids, 5 to 25 amino acids, 6 to 25 amino acids, 10 to 15 amino acids, 12 to 25 amino acids, and 15 to 25 amino acids. Exemplary linkers include glycine polymers (G), glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art, as described above. The linker may also include one or more "GS" domains, as described above.

[0070] The linker sequence may be a flexible linker sequence. Flexible linkers are a category of linker sequences that are well known and described in the art. Linker sequences are generally known as sequences that can be used to connect or link proteins or protein domains together, for example, to create fusion proteins, chimeric proteins, or multifunctional proteins or polypeptides. They can have different properties, for example, flexibility, rigidity, and cleavability. Protein linkers are, for example, reviewed in Chen et al., 2013, Advanced Drug Delivery Reviews 65, 1357-1369, where the category of flexible linkers is compared with the category of rigid linkers and cleavable linkers. Flexible linkers are also described in Klein et al., 2014, Protein Engineering Design and Selection, 27(10), 325-330, van Rosmalen et al., 2017, Biochemistry, 56, 6565-6574, and Chichili et al., 2013, Protein Science, 22, 153-167.

[0071] A flexible linker is a linker that allows some degree of movement between the linked domains or components. Generally, flexible linkers are composed of small non-polar (e.g., Gly) or polar (e.g., Ser, Thr) amino acid residues. The small size of the amino acids allows for flexibility and mobility of the connected moieties (domains or components). The incorporation of polar amino acids allows for the formation of hydrogen bonds with water molecules, maintaining the stability of the linker in an aqueous environment. The most commonly used flexible linkers have sequences consisting mainly of Ser and Gly residues (so-called "GS linkers"). However, many other flexible linkers have been described (see, e.g., Chen et al., 2013, supra), which may contain additional amino acids (e.g., Thr and / or Ala and / or Lys and / or Glu) that may improve solubility. Any flexible linker known and reported in the art may be used. The length of the linker is not critical, although in some embodiments, shorter linker sequences may be desirable, for example, the length of the linker sequence may be 25 amino acids or less, and optionally 24 amino acids or less, 23 amino acids or less, 22 amino acids or less, or 21 amino acids or less.

[0072] In other embodiments, longer linker sequences may be desired, for example consisting of or including multiple repeats of GS domains.

[0073] In some embodiments, the linker length may be from 2, 3, 4, 5, or 6 amino acids to 24, 23, 22, or 21 amino acids. In other embodiments, the linker length may be from 2, 3, 4, 5, or 6 amino acids to 21, 20, 19, 18, 17, 16, or 15 amino acids. In other embodiments, the linker length may be intermediate between these ranges, such as 6-21, 6-20, 7-20, 8-20, 9-20, 10-20, 8-18, 9-18, 10-18, 9-17, 10-17, 9-16, or 10-16. Thus, the linker length may be within a range consisting of any of the above integers.

[0074] GS linkers, more specifically the use of GS ("Gly-Ser") domains in linkers, constitute a class of linkers because the length of the linker can be easily varied by changing the number of repeats of the GS domain. However, flexible linkers are not limited to those based on "GS" repeats; other linkers containing Ser and Gly residues dispersed throughout the linker sequence have been reported, including by Chen et al. (supra).

[0075] Thus, in one embodiment, the linker sequence may comprise at least 40% Gly residues, or Gly and Ser residues.

[0076] In another embodiment, the linker sequence may include Ser and / or Gly residues and 15 or fewer other amino acid residues, for example, 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 6 or fewer, 7 or fewer, 5 or fewer, or 4 or fewer other amino acid residues. It will be understood that the "other" amino acid residues may be any amino acid other than Ser and Gly. Pro residues in a linker tend to confer rigidity, so in one embodiment the linker sequence does not contain any Pro residues, however this is not absolute and a flexible linker sequence may contain one or more Pro residues depending on the sequence context.

[0077] In one embodiment, the linker sequence includes at least one Gly-Ser domain consisting solely of Ser and Gly residues, and may include 15 or fewer other amino acid residues, e.g., 14 or fewer, 13 or fewer, 12 or fewer, 11 or fewer, 10 or fewer, 9 or fewer, 8 or fewer, 6 or fewer, 7 or fewer, 5 or fewer, or 4 or fewer other amino acid residues. The Gly-Ser domain may have the formula: (S)q-[(G)m-(S)m]n-(G)p Here, q is 0 or 1, m is an integer of 1 to 8, n is an integer of 1 or greater (for example, 1 to 8, more specifically, 1 to 6), and p is 0 or an integer of 1 to 3. More specifically, the Gly-Ser domain may have any of the following formulas: (i)S-[(G)mS]n (ii) [(G)mS]n (iii) [(G)mS]n-(G)p Here, m is an integer of 2 to 8 (for example, 3 to 4), n is an integer of 1 or more (for example, 1 to 8, more specifically, 1 to 6), and p is 0 or an integer of 1 to 3. In a representative example, the Gly-Ser domain may have the formula: S-[GGGGS]n Here, n is an integer of 1 or more (1 to 8, or 1 to 6, 1 to 5, 1 to 4, or 1 to 3, as appropriate). A representative sequence, GGGS, is shown in SEQ ID NO:163.

[0078] A linker sequence may consist solely of, i.e., consist of, one or more Gly-Ser domains as described or defined above. However, as described above, a linker sequence may contain additional amino acids in addition to one or more Gly-Ser domains. The additional amino acids may be located at one or both ends of the Gly-Ser domain or at one or both ends of the repeated sequences of Gly-Ser domains. Thus, additional amino acids, which may be other amino acids, may be located at one or both ends of the linker sequence, for example, flanking the Gly-Ser domain(s). In other embodiments, additional amino acids may be located between Gly-Ser domains. For example, two Gly-Ser domains may be flanked by a stretch of other amino acids within the linker sequence. Furthermore, as noted above, repeating GS domains are not necessarily required in other linkers; G and / or S residues, or short domains such as GS, may simply be distributed along the length or sequence. Representative and exemplary linker sequences are listed below. ETSGGGGSRL (SEQ ID NO: 164) SGGGGSGGGGSGGGGS ((SEQ ID NO: 165) S(GGGGS) 1-5 (GGGGS is SEQ ID NO: 166) (GGGGS) 1-5 (GGGGS is SEQ ID NO: 166) S(GGGS) 1-5 (GGGS is SEQ ID NO: 163) (GGGS) 1-5 (GGGS is SEQ ID NO: 163) S(GGGGGS) 1-5 (GGGGGS is SEQ ID NO: 167) (GGGGGS) 1-5 (GGGGGS is SEQ ID NO: 167) S(GGGGGGS) 1-5 (GGGGGGS is SEQ ID NO: 168) (GGGGGGS) 1-5(GGGGGGS is SEQ ID NO: 168) GGGGSGGGGSGGGGS (SEQ ID NO: 169) GGGGG (SEQ ID NO: 170) GGGGSGGGGS (SEQ ID NO: 171) GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 172) GGGGGGG (SEQ ID NO: 173) G6 (SEQ ID NO: 174) G8 (SEQ ID NO: 175) KESGSVSSEQLAQFRSLD (SEQ ID NO: 176) EGKSSGSGSESKST (SEQ ID NO: 177) GSAGSAAGSGEF (SEQ ID NO: 178) SGGGGSAGSAAGSGEF (SEQ ID NO: 179) SGGGLLLLLLLLGGGS (SEQ ID NO: 180) SGGGAAAAAAAAGGGS (SEQ ID NO: 181) SGGGAAAAAAAAAAAAAAAAGGGS (SEQ ID NO: 182) SGALGGLALAGLLLAGLGLGAAGS (SEQ ID NO: 183) SLSLSPGGGGGPAR (SEQ ID NO: 184) SLSLSPGGGGGPARSLSLSPGGGGG (SEQ ID NO: 185) GSSGSS (SEQ ID NO: 186) GSSSSSS (SEQ ID NO: 187) GGSSSS (SEQ ID NO: 188) GSSSSS (SEQ ID NO: 189) SGGGGS (SEQ ID NO: 190) In certain embodiments, the linker has the sequence (GGGGS)3 (SEQ ID NO: 169).

[0079] Although the linker sequences defined above are flexible sequences, the present disclosure also includes other polypeptides that include linkers that are not flexible and / or that do not meet the above definitions and requirements.

[0080] Further examples of linkers that can be used to link the VH and VL domains include KLEEGEFSEARV (SEQ ID NO: 233), or a sequence having at least about 60% identity thereto, for example, 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 thereto. Alternatively, the linker may be the sequence of SEQ ID NO: 233, or a sequence that differs from the sequence by 6 amino acids or less, for example, 5 amino acids or less, 4 amino acids or less, 3 amino acids or less, 2 amino acids or less, or 1 amino acid or less. In particular, the linker may be shortened or lengthened by one or more amino acids at the N-terminus and / or C-terminus (see, e.g., Schmiedl A. et al (Protein Eng. 2000 Oct; 13(10): See, e.g., pp. 725-34, in which the linker is shortened by one amino acid at each end compared to SEQ ID NO: 233. To improve the stability of this linker and remove two putative trypsin cleavage sites (lysine and arginine), the linker may be modified by substituting lysine (K) with isoleucine (I), arginine (R) with glycine (G), and valine (V) with cysteine ​​(C). The resulting amino acid sequence of the linker is ILEEGEFSEAGC (SEQ ID NO: 234). Any peptide linker having the consensus amino acid sequence X1LEEGEFSEAX2X3 (SEQ ID NO: 235), where X1 is K or I, X2 is R or G, and X3 is V or C, may also be used.

[0081] Thus, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence of SEQ ID NO: 85 linked to the VL sequence of SEQ ID NO: 86 via a linker of sequence (X)n, where X is any amino acid and n is an integer between 15 and 25.

[0082] Thus, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence of SEQ ID NO: 87 linked to the VL sequence of SEQ ID NO: 88 via a linker of sequence (X)n (where X is any amino acid and n is an integer between 15 and 25).

[0083] Thus, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence of SEQ ID NO: 89 linked to the VL sequence of SEQ ID NO: 90 via a linker of sequence (X)n (where X is any amino acid and n is an integer between 15 and 25).

[0084] Thus, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence of SEQ ID NO: 91 linked to the VL sequence of SEQ ID NO: 92 via a linker of sequence (X)n, where X is any amino acid and n is an integer between 15 and 25.

[0085] Thus, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence of SEQ ID NO: 93 linked to the VL sequence of SEQ ID NO: 94 via a linker of sequence (X)n, where X is any amino acid and n is an integer between 15 and 25.

[0086] Thus, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence of SEQ ID NO: 95 linked to the VL sequence of SEQ ID NO: 96 via a linker of sequence (X)n, where X is any amino acid and n is an integer between 15 and 25.

[0087] Thus, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence of SEQ ID NO: 97 linked to the VL sequence of SEQ ID NO: 98 via a linker of sequence (X)n, where X is any amino acid and n is an integer between 15 and 25.

[0088] Thus, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence of SEQ ID NO: 99 linked to the VL sequence of SEQ ID NO: 100 via a linker of sequence (X)n, where X is any amino acid and n is an integer between 15 and 25.

[0089] Thus, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence of SEQ ID NO: 101 linked to the VL sequence of SEQ ID NO: 102 via a linker of sequence (X)n, where X is any amino acid and n is an integer between 15 and 25.

[0090] Thus, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence of SEQ ID NO: 103 linked to the VL sequence of SEQ ID NO: 104 via a linker of sequence (X)n, where X is any amino acid and n is an integer between 15 and 25.

[0091] Thus, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence of SEQ ID NO: 105 linked to the VL sequence of SEQ ID NO: 106 via a linker of sequence (X)n, where X is any amino acid and n is an integer between 15 and 25.

[0092] Thus, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence of SEQ ID NO: 107 linked to the VL sequence of SEQ ID NO: 108 via a linker of sequence (X)n, where X is any amino acid and n is an integer between 15 and 25.

[0093] Thus, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence of SEQ ID NO: 109 linked to the VL sequence of SEQ ID NO: 110 via a linker of sequence (X)n, where X is any amino acid and n is an integer between 15 and 25.

[0094] Thus, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence of SEQ ID NO: 111 linked to the VL sequence of SEQ ID NO: 112 via a linker of sequence (X)n, where X is any amino acid and n is an integer between 15 and 25.

[0095] In this regard, in one embodiment, the antigen-binding domain of the CAR comprises or consists of the sequence set forth in SEQ ID NO: 113, or a sequence having at least 80% identity thereto.

[0096] In another embodiment, in one embodiment, the antigen binding domain of the CAR comprises or consists of the sequence set forth in SEQ ID NO: 114, or a sequence having at least 80% identity thereto.

[0097] In a further embodiment, in one embodiment, the antigen binding domain of the CAR comprises or consists of the sequence set forth in SEQ ID NO: 115, or a sequence having at least 80% identity thereto.

[0098] In a further embodiment, in one embodiment, the antigen binding domain of the CAR comprises or consists of the sequence set forth in SEQ ID NO: 116, or a sequence having at least 80% identity thereto.

[0099] In a further embodiment, in one embodiment, the antigen binding domain of the CAR comprises or consists of the sequence set forth in SEQ ID NO: 117, or a sequence having at least 80% identity thereto.

[0100] In a further embodiment, in one embodiment, the antigen binding domain of the CAR comprises or consists of the sequence set forth in SEQ ID NO: 118, or a sequence having at least 80% identity thereto.

[0101] In a further embodiment, in one embodiment, the antigen binding domain of the CAR comprises or consists of the sequence set forth in SEQ ID NO: 119, or a sequence having at least 80% identity thereto.

[0102] In a further embodiment, in one embodiment, the antigen binding domain of the CAR comprises or consists of the sequence set forth in SEQ ID NO: 120, or a sequence having at least 80% identity thereto.

[0103] In a further embodiment, in one embodiment, the antigen binding domain of the CAR comprises or consists of the sequence set forth in SEQ ID NO: 121, or a sequence having at least 80% identity thereto.

[0104] In a further embodiment, in one embodiment, the antigen binding domain of the CAR comprises or consists of the sequence set forth in SEQ ID NO: 122, or a sequence having at least 80% identity thereto.

[0105] In a further embodiment, in one embodiment, the antigen binding domain of the CAR comprises or consists of the sequence set forth in SEQ ID NO: 123, or a sequence having at least 80% identity thereto.

[0106] In a further embodiment, in one embodiment, the antigen binding domain of the CAR comprises or consists of the sequence set forth in SEQ ID NO: 124, or a sequence having at least 80% identity thereto.

[0107] In a further embodiment, in one embodiment, the antigen binding domain of the CAR comprises or consists of the sequence set forth in SEQ ID NO: 125, or a sequence having at least 80% identity thereto.

[0108] In a further embodiment, in one embodiment, the antigen binding domain of the CAR comprises or consists of the sequence set forth in SEQ ID NO: 126, or a sequence having at least 80% identity thereto.

[0109] In one embodiment, the antigen binding domain of a CAR comprises or consists of the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 191, or an amino acid sequence having at least 80% identity to the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 191. For example, the antigen binding domain of a CAR may comprise or consist of the amino acid sequence encoded by the sequence remaining after removal of the sequence encoding the signal sequence and / or any tag sequence (e.g., myc tag and / or his tag) from SEQ ID NO: 191 or a variant thereof.

[0110] In one embodiment, the antigen binding domain of a CAR comprises or consists of the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 192, or an amino acid sequence having at least 80% identity to the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 192. For example, the antigen binding domain of a CAR may comprise or consist of the amino acid sequence encoded by the sequence remaining after the sequence encoding the signal sequence and / or any tag sequence (e.g., myc tag and / or his tag) is removed from SEQ ID NO: 192 or a variant thereof.

[0111] In one embodiment, the antigen binding domain of the CAR comprises or consists of the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 193, or an amino acid sequence having at least 80% identity to the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 193. For example, the antigen binding domain of the CAR may comprise or consist of the amino acid sequence encoded by the sequence remaining after removal of the sequence encoding the signal sequence and / or any tag sequence (e.g., myc tag and / or his tag) from SEQ ID NO: 193 or a variant thereof.

[0112] In one embodiment, the antigen binding domain of a CAR comprises or consists of the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 194, or an amino acid sequence having at least 80% identity to the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 194. For example, the antigen binding domain of a CAR may comprise or consist of the amino acid sequence encoded by the sequence remaining after removal of the sequence encoding the signal sequence and / or any tag sequence (e.g., myc tag and / or his tag) from SEQ ID NO: 194.

[0113] In one embodiment, the antigen binding domain of a CAR comprises or consists of the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 195, or an amino acid sequence having at least 80% identity to the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 195. For example, the antigen binding domain of a CAR may comprise or consist of the amino acid sequence encoded by the sequence remaining after removal of the sequence encoding the signal sequence and / or any tag sequence (e.g., myc tag and / or his tag) from SEQ ID NO: 195 or a variant thereof.

[0114] In one embodiment, the antigen binding domain of the CAR comprises or consists of the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 196, or an amino acid sequence having at least 80% identity to the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 196. For example, the antigen binding domain of the CAR may comprise or consist of the amino acid sequence encoded by the sequence remaining after the sequence encoding the signal sequence and / or any tag sequence (e.g., myc tag and / or his tag) is removed from SEQ ID NO: 196 or a variant thereof.

[0115] In one embodiment, the antigen binding domain of a CAR comprises or consists of the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 197, or an amino acid sequence having at least 80% identity to the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 197. For example, the antigen binding domain of a CAR may comprise or consist of the amino acid sequence encoded by the sequence remaining after removal of the sequence encoding the signal sequence and / or any tag sequence (e.g., myc tag and / or his tag) from SEQ ID NO: 197 or a variant thereof.

[0116] In one embodiment, the antigen binding domain of a CAR comprises or consists of the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 198, or an amino acid sequence having at least 80% identity to the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 198. For example, the antigen binding domain of a CAR may comprise or consist of the amino acid sequence encoded by the sequence remaining after removal of the sequence encoding the signal sequence and / or any tag sequence (e.g., myc tag and / or his tag) from SEQ ID NO: 198 or a variant thereof.

[0117] In one embodiment, the antigen binding domain of a CAR comprises or consists of the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 199, or an amino acid sequence having at least 80% identity to the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 199. For example, the antigen binding domain of a CAR may comprise or consist of the amino acid sequence encoded by the sequence remaining after removal of the sequence encoding the signal sequence and / or any tag sequence (e.g., myc tag and / or his tag) from SEQ ID NO: 199 or a variant thereof.

[0118] In one embodiment, the antigen binding domain of the CAR comprises or consists of the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 200, or an amino acid sequence having at least 80% identity to the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 200. For example, the antigen binding domain of the CAR may comprise or consist of the amino acid sequence encoded by the sequence remaining after removal of the sequence encoding the signal sequence and / or any tag sequence (e.g., myc tag and / or his tag) from SEQ ID NO: 200, or a variant thereof.

[0119] In one embodiment, the antigen binding domain of the CAR comprises or consists of the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 201, or an amino acid sequence having at least 80% identity to the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 201. For example, the antigen binding domain of the CAR may comprise or consist of the amino acid sequence encoded by the sequence remaining after the sequence encoding the signal sequence and / or any tag sequence (e.g., myc tag and / or his tag) is removed from SEQ ID NO: 201 or a variant thereof.

[0120] In one embodiment, the antigen binding domain of the CAR comprises or consists of the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 202, or an amino acid sequence having at least 80% identity to the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 202. For example, the antigen binding domain of the CAR may comprise or consist of the amino acid sequence encoded by the sequence remaining after removal of the sequence encoding the signal sequence and / or any tag sequence (e.g., myc tag and / or his tag) from SEQ ID NO: 202 or a variant thereof.

[0121] In one embodiment, the antigen binding domain of the CAR comprises or consists of the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 203, or an amino acid sequence having at least 80% identity to the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 203. For example, the antigen binding domain of the CAR may comprise or consist of the amino acid sequence encoded by the sequence remaining after removal of the sequence encoding the signal sequence and / or any tag sequence (e.g., myc tag and / or his tag) from SEQ ID NO: 203 or a variant thereof.

[0122] In one embodiment, the antigen binding domain of the CAR comprises or consists of the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 204, or an amino acid sequence having at least 80% identity to the amino acid sequence encoded by the sequence set forth in SEQ ID NO: 204. For example, the antigen binding domain of the CAR may comprise or consist of the amino acid sequence encoded by the sequence remaining after the sequence encoding the signal sequence and / or any tag sequence (e.g., myc tag and / or his tag) is removed from SEQ ID NO: 204 or a variant thereof.

[0123] In further embodiments, the CAR construct may comprise or consist of the sequence set forth in any one of SEQ ID NOs: 237-262, or a variant thereof (e.g., a sequence having at least 80% identity thereto). In certain embodiments, the CAR construct may comprise or consist of the sequence set forth in SEQ ID NO: 238, or a variant thereof (e.g., a sequence having at least 80% identity thereto). In certain embodiments, the CAR construct may comprise or consist of the sequence set forth in SEQ ID NO: 240, or a variant thereof (e.g., a sequence having at least 80% identity thereto). In certain embodiments, the CAR construct may comprise or consist of the sequence set forth in SEQ ID NO: 241, or a variant thereof (e.g., a sequence having at least 80% identity thereto). In certain embodiments, the CAR construct may comprise or consist of the sequence set forth in SEQ ID NO: 243, or a variant thereof (e.g., a sequence having at least 80% identity thereto). In certain embodiments, the CAR construct may comprise or consist of the sequence set forth in SEQ ID NO: 249, or a variant thereof (e.g., a sequence having at least 80% identity thereto). In certain embodiments, the CAR construct may comprise or consist of the sequence set forth in SEQ ID NO: 253, or a variant thereof (e.g., a sequence having at least 80% identity thereto).

[0124] The variant sequences disclosed and described herein (including variant CAR sequences, variant VH sequences, variant VL sequences, variant antigen-binding domain sequences) may have at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity to the designated SEQ ID NO:

[0125] The CAR also optionally includes a hinge domain to keep the extracellular domain, particularly the antigen-binding domain, away from the cell surface, and further includes a transmembrane domain. The hinge and transmembrane domain may include a hinge sequence and a transmembrane sequence derived from any protein having a hinge and / or transmembrane domain (including type I, type II, or type III transmembrane proteins). The hinge domain may be selected from CD28, CD8α, CD4, CD7, CH2CH3, an immunoglobulin hinge region, or a portion or variant thereof. Typically, the hinge may be derived from CD8, particularly CD8α, or CH2CH3. In one embodiment, the hinge may include one or more cysteine ​​residues, for example, to allow disulfide bonding. For example, a CD8 hinge may include one or more cysteine ​​residues, for example, one cysteine ​​residue, two cysteine ​​residues, or three cysteine ​​residues.

[0126] The transmembrane domain of CAR may also comprise an artificial hydrophobic sequence. The transmembrane domain of CAR may be selected so as not to dimerize. Additional transmembrane domains will be apparent to those skilled in the art. Examples of transmembrane (TM) regions used in CAR constructs include: 1) TM region of CD28 (Pule et al, Mol Ther, 2005, Nov;12(5):933-41, Brentjens et al, CCR, 2007, Sep 15;13(18 Pt 1):5426-35, Casucci et al, Blood, 2013, Nov 14;122(20):3461-72.), 2) TM region of OX40 (Pule et al, Mol Ther, 2005, Nov;12(5):933-41), 3) TM region of 4-1BB (Brentjens et al, CCR, 2007, Sep 15;13(18 Pt 1):5426-35);4) TM region of CD3ζ (Pule et al, Mol Ther, 2005, Nov;12(5):933-41, Savoldo B, Blood, 2009, Jun 18;113(25):6392-402.), 5) the TM region of CD8α (Maher et al., Nat Biotechnol, 2002, Jan;20(1):70-5. Imai C, Leukemia, 2004, Apr;18(4):676-84. Brentjens et al., CCR, 2007, Sep 15;13(18 Pt 1):5426-35. Milone et al., Mol Ther, 2009, Aug;17(8):1453-64.). Other transmembrane domains that can be used include those derived from ICOS, CD4, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD154, or CH2CH3. Optionally, the transmembrane domain may be derived from CD4, CD28, CD8α, or CH2CH3.

[0127] In one embodiment, CAR may not contain a dimerization domain that binds to a regulatory molecule. A regulatory molecule is any molecule that can bind to at least one dimerization domain in a CAR and prevent or cause the interaction of a pair of dimerization domains. Examples of regulatory molecules include soluble proteins (e.g., cytokines, TGF-β, VEGF) or small molecules. In a further embodiment, when dimerization with other CAR molecules occurs, the dimerization may not be controlled. In another embodiment, the monovalent binding of a CAR to an antigen may enable the activation of cells expressing the CAR.

[0128] The hinge domain can be easily obtained 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.

[0129] Alternatively, the hinge domain may be derived from a different protein than the transmembrane domain, for example, the hinge domain may be derived from a CH2CH3 hinge domain and the transmembrane domain may be derived from the CD28 transmembrane domain.

[0130] For example, the hinge domain may be derived from the CD8α hinge domain and comprise the amino acid sequence set forth in SEQ ID NO: 236, or a variant having at least 80% identity to SEQ ID NO: 236. Suitably, the variant may have at least 85%, 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 236. An example of a modified CD8α hinge domain is set forth in SEQ ID NO: 132. For example, the hinge domain may comprise the amino acid sequence set forth in SEQ ID NO: 267, or the amino acid sequence encoded by the nucleotide sequence set forth in SEQ ID NO: 268, or a variant having at least 80% identity to SEQ ID NO: 267 or SEQ ID NO: 268. Suitably, the variant may have at least 85%, 90%, 95%, 97%, 98% or 99% identity to SEQ ID NO: 267 or SEQ ID NO: 268.

[0131] For example, the transmembrane domain may be derived from the CD8α transmembrane domain and may comprise the amino acid sequence set forth in SEQ ID NO: 129, which represents amino acids 183 to 203 of human CD8α, or a variant having at least 80% identity to SEQ ID NO: 129. Suitably, the variant may have at least 85%, 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 129.

[0132] The CD8α transmembrane domain may be combined with a CD8α hinge domain. In one embodiment, the CAR comprises a sequence of the combined hinge and transmembrane domain of CD8α as set forth in SEQ ID NO: 131 or SEQ ID NO: 133, or a variant having at least 80% sequence identity to said sequence. The variant may have at least 85%, 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 131 or SEQ ID NO: 133. SEQ ID NO: 131 comprises a modified hinge domain with two amino acid modifications at cysteine ​​residues compared to the wild-type CD8α hinge sequence. This modified CD8α hinge domain sequence is set forth in SEQ ID NO: 132. The sequences of the hinge and transmembrane domain of wild-type CD8α are set forth in SEQ ID NO: 133. The six amino acids located at the ends of SEQ ID NOs: 131 and 133, if present, are not located in the membrane and form part of the endodomain of the CAR. Variants of such hinge sequences having at least 80% sequence identity to SEQ ID NO: 132 or 133 may be used.

[0133] For example, the hinge domain may be derived from a CH2CH3 hinge domain and may comprise the amino acid sequence set forth in SEQ ID NO: 130 or 134, or a variant thereof having at least 80% identity to SEQ ID NO: 130 or 134. The variant may have at least 85%, 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 130 or 134.

[0134] Alternatively, CD28 hinge and transmembrane sequences that may be used include, for example, SEQ ID NO: 135, or a variant having at least 80% identity to SEQ ID NO: 135. The variant may have at least 85%, 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 135.

[0135] As a further example, the CAR may comprise a native or modified CD8α hinge domain and a CD28 transmembrane domain, or a CD28 hinge domain and a CD8α transmembrane domain, for example, based on the sequences above.

[0136] In one embodiment, a CH2CH3 hinge sequence containing one or more cysteine ​​residues may be used. For example, a CH2CH3 hinge sequence containing one, two, three, four, or more cysteine ​​residues may be used. Other hinge domains that may be used include hinge domains derived from CD4, CD7, or immunoglobulins, or portions or variants thereof. These hinge domains may contain one or more cysteine ​​residues, for example, one, two, three, four, or more cysteine ​​residues.

[0137] In one embodiment, the transmembrane domain may be derived from the CD4 transmembrane domain and may comprise the sequence set forth in SEQ ID NO: 263, or a variant having at least 80% identity to SEQ ID NO: 263. The variant may have at least 85%, 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 263.

[0138] The CAR may further comprise a signal sequence (or leader sequence) that targets the CAR to the endoplasmic reticulum pathway for expression on the cell surface. An exemplary signal / leader sequence is MALPVTALLLPLALLLHAAAP, as set forth in SEQ ID NO: 136, which contains one amino acid substitution compared to the wild-type CD8α sequence MALPVTALLLPLALLLHAARP, as set forth in SEQ ID NO: 137. Either sequence, or a variant sequence having at least 70% sequence identity thereto, may be used. For example, the variant sequence may have at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity thereto.

[0139] The endodomain of the CAR described herein contains motifs necessary to transmit effector function signals and direct cells expressing the CAR to exert their specific functions upon antigen binding. In particular, the endodomain may contain one or more (e.g., two or three) immunoreceptor tyrosine-based activation motifs (ITAMs), which typically comprise 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 zeta chain endodomain of the T cell receptor or any of its homologs (e.g., eta chain, FcεR1γ chain and FcεR1β chain, MB1 (Igα) chain, B29 (Igβ) chain, etc.), CD3 polypeptide domains (Δ, δ, and ε), Syk family tyrosine kinases (e.g., Syk, ZAP 70, etc.), Src family tyrosine kinases (e.g., Lck, Fyn, Lyn, etc.), and other molecules involved in T cell signaling (e.g., CD2, CD5, and CD28). The intracellular signaling domain may comprise a human CD3 zeta chain endodomain, FcyRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine-based activation motif (ITAM), or a combination thereof.

[0140] Typically, the intracellular signaling domain comprises the intracellular signaling domain of the human CD3 zeta chain. The sequence of the intracellular signaling domain of the human CD3 zeta chain is set forth in SEQ ID NO: 138. The CAR may comprise a CD3 zeta signaling domain comprising or consisting of the sequence set forth in SEQ ID NO: 138, or a sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 138. In one embodiment, the signaling domain comprises or consists of SEQ ID NO: 138.

[0141] Other signaling domains that can be used include the signaling domain of CD28 or CD27, or variants thereof. Additional intracellular signaling domains will be apparent to those skilled in the art and can be used in connection with alternative embodiments of the present invention. In one embodiment, the CAR may not include the costimulatory domain derived from 4-1BB in the endodomain.

[0142] The CAR may also contain a composite endodomain comprising a fusion of the intracellular portion of a T cell costimulatory molecule with, for example, the intracellular portion of CD3ζ. Such composite endodomains are sometimes referred to as second-generation CARs, which can simultaneously transmit activation and costimulatory signals after antigen recognition. The most commonly used costimulatory domain is derived from CD28, which provides the most potent costimulatory signal, i.e., immune signal 2, that triggers T cell proliferation. The endodomain of the CAR may also contain one or more TNF receptor family signaling domains, or portions or variants thereof, such as the signaling domains of ICOS, (CD134)OX40, 4-1BB, CD27, or TNFRSF25. However, if desired, the CAR may not contain an endodomain containing both the CD28 and 4-1BB signaling domains.

[0143] The intracellular signaling domain of CD28 that can be used as a costimulatory domain is set forth in SEQ ID NO: 140. Exemplary sequences of the signaling domains of OX40, 4-1BB, ICOS, and TNFRSF25 are set forth in SEQ ID NOs: 141-144. A CAR may comprise one or more costimulatory domains that comprise or consist of the sequence of any one of SEQ ID NOs: 140, 141, 142, 143, 144, or a variant having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity thereto.

[0144] In certain embodiments, the transmembrane domain and the intracellular signaling domain derived from a T cell costimulatory molecule may be derived from the same protein. For example, in certain embodiments, the transmembrane domain and the intracellular signaling domain derived from a T cell costimulatory molecule may be derived from CD28. For example, a CAR may comprise a combination of the CD28 transmembrane domain and the CD28 intracellular signaling domain set forth in SEQ ID NO: 139, or a variant having at least 80% sequence identity to said sequence. The variant may have at least 85%, 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 139.

[0145] In one embodiment, the CAR comprises a human CD8 hinge domain or a variant thereof and a human CD8 transmembrane domain. Alternatively, or in addition, the CAR comprises an endodomain comprising, consisting essentially of, or consisting of a human CD28 costimulatory domain and a human CD3ζ signaling domain.

[0146] In one embodiment, the CAR comprises a hinge domain, a transmembrane domain, and an intracellular (or endo) domain as follows: (i) a CH2CH3 hinge sequence comprising or consisting of the sequence set forth in SEQ ID NO: 130 or SEQ ID NO: 134, or a sequence having at least 80% sequence identity thereto; (ii) a CD28 transmembrane and costimulatory domain comprising or consisting of the sequence set forth in SEQ ID NO: 139, or a sequence having at least 80% sequence identity thereto; (iii) A CD3ζ signaling domain comprising or consisting of the sequence set forth in SEQ ID NO: 138, or a sequence having at least 80% sequence identity thereto.

[0147] In an alternative embodiment, the CAR comprises a hinge domain, a transmembrane domain, and an intracellular (or endo) domain as follows: (i) a CD8α hinge and transmembrane domain sequence comprising or consisting of the sequence set forth in SEQ ID NO: 131, or a sequence having at least 80% sequence identity thereto; (ii) a CD28 costimulatory domain comprising or consisting of the sequence set forth in SEQ ID NO: 140, or a sequence having at least 80% sequence identity thereto; (iii) A CD3ζ signaling domain comprising or consisting of the sequence set forth in SEQ ID NO: 138, or a sequence having at least 80% sequence identity thereto.

[0148] In a further embodiment, the CAR comprises a hinge domain, a transmembrane domain, and an intracellular (or endo) domain as follows: (i) a CD8α hinge and transmembrane domain sequence comprising or consisting of the sequence set forth in SEQ ID NO: 133, or a sequence having at least 80% sequence identity thereto; (ii) a CD28 costimulatory domain comprising or consisting of the sequence set forth in SEQ ID NO: 140, or a sequence having at least 80% sequence identity thereto; (iii) A CD3ζ signaling domain comprising or consisting of the sequence set forth in SEQ ID NO: 138, or a sequence having at least 80% sequence identity thereto.

[0149] The encoded and expressed CAR may further comprise a leader sequence comprising or consisting of the sequence set forth in SEQ ID NO: 137, or a sequence having at least 80% sequence identity thereto.

[0150] The antigen binding domain of the CAR may comprise or consist of a sequence set forth in SEQ ID NO: 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, or 126, or a sequence having at least 80% sequence identity thereto and capable of binding to ENTPD3.

[0151] Thus, overall, one representative CAR may include: (i) a leader sequence comprising or consisting of the sequence set forth in SEQ ID NO: 137, or a sequence having at least 80% sequence identity thereto; (ii) an antigen-binding domain comprising or consisting of a sequence set forth in SEQ ID NO: 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, or 126, or a sequence having at least 80% sequence identity thereto; (iii) a CH2CH3 hinge domain sequence comprising or consisting of the sequence set forth in SEQ ID NO: 130 or 134, or a sequence having at least 80% sequence identity thereto; (iv) a CD28 transmembrane and costimulatory domain comprising or consisting of the sequence set forth in SEQ ID NO: 139, or a sequence having at least 80% sequence identity thereto; (v) A CD3ζ signaling domain comprising or consisting of the sequence set forth in SEQ ID NO: 138, or a sequence having at least 80% sequence identity thereto.

[0152] Another exemplary CAR may include: (i) a leader sequence comprising or consisting of the sequence set forth in SEQ ID NO: 137, or a sequence having at least 80% sequence identity thereto; (ii) an antigen-binding domain comprising or consisting of a sequence set forth in SEQ ID NO: 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, or 126, or a sequence having at least 80% sequence identity thereto; (iii) a CD8 hinge and transmembrane domain sequence comprising or consisting of the sequence set forth in SEQ ID NO: 131 or 133, or a sequence having at least 80% sequence identity thereto; (iv) a CD28 costimulatory domain comprising or consisting of the sequence set forth in SEQ ID NO: 140, or a sequence having at least 80% sequence identity thereto; (v) A CD3ζ signaling domain comprising or consisting of the sequence set forth in SEQ ID NO: 138, or a sequence having at least 80% sequence identity thereto.

[0153] The CARs of the present invention may comprise or consist of any one or more of the sequences set forth in SEQ ID NOs: 237-262, or variants having at least 80% identity thereto. For example, any variant may have at least about 85%, about 90%, about 95%, about 97%, about 98%, or about 99% identity thereto. The CARs of SEQ ID NOs: 237-262 or variants thereof may further comprise a signal sequence, for example, a signal sequence having the sequence set forth in SEQ ID NO: 136 or 137.

[0154] CARs are capable of binding to ENTPD3 and transmitting signals within cells expressing the CAR.

[0155] The cell may express only one type of CAR, i.e., when the cell expresses multiple CAR molecules, the amino acid sequences of each of these expressed molecules may be identical to each other.

[0156] The endodomain of the CAR herein may contain additional domains. For example, a CAR may contain a domain that confers to the CAR the ability to provide an effective IL signal to cells expressing the CAR in an antigen-specific manner without the need for exogenous IL administration. For example, a CAR may contain a domain including a STAT5 association motif, a JAK1 and / or JAK2 binding motif, and optionally a JAK3 binding motif. In such embodiments, the endodomain may include one or more sequences derived from the endodomain of a cytokine receptor, such as an interleukin receptor (IL) receptor. Such CARs are described in WO 2020 / 044055 (also incorporated herein by reference). Exemplary amino acid sequences derived from IL-2 receptor β, including a STAT5 association motif and each JAK binding motif, are set forth in SEQ ID NOS: 145-147. The inclusion of such domains confers to the CAR the ability to provide an effective IL signal to cells expressing the CAR in an antigen-specific manner without the need for exogenous IL administration. For example, IL-2 is important for the survival, proliferation, and persistence of Treg cells, but IL-2 levels are often reduced or impaired in patients requiring treatment. Thus, a CAR may comprise a sequence corresponding to all or part of the beta chain endodomain of an IL receptor or a variant thereof (e.g., the IL2 receptor), optionally in combination with the gamma chain endodomain of an IL receptor or a variant thereof (e.g., the IL2 receptor).

[0157] Alternatively, or in addition, additional nucleic acid sequences or polypeptides may be introduced into the cells or cell population to improve cell persistence, e.g., to provide the cells with an effective IL signal without the need for exogenous IL administration. Such IL signals may be constitutive or inducible. Exemplary techniques may include, for example, the use of engineered or chimeric receptors capable of transmitting IL signals without the need for exogenous IL administration. For example, the use of 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 2017 / 029512, WO 2012 / 138858, WO 2014 / 172584, WO 2017 / 068360, WO 2021 / 023987, WO 2020 / 180664, and WO 2020 / 044239; or engineered receptors with tethered activation molecules, such as those described in WO 2017 / 201432 and WO 2019 / 183389.

[0158] As described above, the cells or cell populations of the present invention may further comprise additional polypeptides, particularly exogenous polypeptides such as FOXP3 polypeptides and / or safety switch polypeptides. The polypeptides of the present invention, e.g., CAR, FOXP3, and safety switch, may be encoded by a single nucleic acid molecule. The nucleic acid molecule may contain a nucleotide sequence encoding a self-cleaving sequence between the encoded polypeptides, allowing the polypeptides to be expressed and / or produced as separate or independent components. That is, even if these polypeptides are encoded by a single nucleic acid molecule, they may be expressed or produced as separate polypeptides by being "cleaved" at the encoded cleavage site during or after translation. Thus, they may exist within the cell as separate entities or separate polypeptide chains at the end of the intracellular protein production process. Alternatively, the additional exogenous polypeptides may be encoded by separate nucleic acid molecules or vectors.

[0159] "Independent" or "separated" polypeptides refer to polypeptides that are not linked to each other and are physically distinguishable. Indeed, after expression, these polypeptides are present in different, i.e., separate, intracellular locations. Thus, CAR, FOXP3, and the safety switch polypeptide are ultimately expressed as separate and distinct components. CAR is expressed as a cell surface molecule. The safety switch polypeptide may be expressed intracellularly or on the cell surface. In certain embodiments, the safety switch polypeptide and CAR are expressed on the surface of cells intended for ACT. FOXP3 is expressed intracellularly and can act as a transcription factor to control the development and / or activity of cells, as described in more detail below.

[0160] The safety switch polypeptide provides a suicide moiety to the cells in which it is expressed, which is useful as a safety mechanism to allow cells administered to a subject to be removed if the need arises, or indeed more generally, as desired or necessary, for example, after the cells have exerted or completed their therapeutic effect.

[0161] A suicide moiety is inducible to cause cell death, or more generally, cell elimination or removal. An example of a suicide moiety is a suicide protein encoded by a suicide gene. The suicide protein can be expressed in or on a cell together with a desired transgene (in this example, a CAR), and upon expression, allows the cell to be eliminated, thereby blocking expression of the transgene (CAR). As used herein, a suicide moiety is a suicide polypeptide, which is a polypeptide that can cause a cell to be eliminated under permissive conditions, i.e., induced or activated conditions.

[0162] The suicide moiety may be a polypeptide or amino acid sequence that can be activated to exert cell-eliminating activity by an activating agent administered to a subject, or that is active to exert cell-eliminating activity in the presence of a substrate that can be administered to a subject. In certain embodiments, the suicide moiety can be targeted by a separate cell-eliminating agent administered to a subject. The cell-eliminating agent can be targeted to the cells to be eliminated by binding to the suicide moiety. In particular, the suicide moiety can be recognized by an antibody, and upon binding of the antibody to a safety switch polypeptide expressed on the cell surface, the cell is eliminated or eliminated.

[0163] The suicide moiety may be HSV-TK or iCasp9. However, the suicide moiety may also be or contain an epitope recognized by a cell-depleting antibody or other binding molecule capable of inducing cell elimination. In such embodiments, the safety switch polypeptide is expressed on the cell surface.

[0164] As used herein, the term "delete" when used in reference to the removal of cells is synonymous with "remove" or "ablate" or "eliminate." This term is used to encompass killing cells or inhibiting cell proliferation, which may reduce the number of cells in a subject. While 100% complete removal may be desirable, it is not always achieved. Reducing the number of cells in a subject or inhibiting cell proliferation may be sufficient to produce a beneficial effect.

[0165] In particular, the suicide moiety may be a CD20 epitope recognized by the antibody rituximab. Thus, in the safety switch polypeptide, the suicide moiety may comprise a minimal epitope based on the CD20-derived epitope recognized by the antibody rituximab. Biosimilars of rituximab are available and can be used. Those skilled in the art can easily prepare antibodies with the binding specificity of rituximab using available amino acid sequences by conventional methods.

[0166] ENTPD3-specific CAR cells that also express a safety switch polypeptide comprising the sequence can be selectively killed using the antibody rituximab or an antibody with the binding specificity of rituximab. The safety switch polypeptide is expressed on the cell surface, and exposure or contact of the expressed polypeptide with rituximab or an antibody with the same binding specificity as rituximab triggers cell death. Thus, rituximab, or an antibody having its binding specificity, can be provided for use in ACT in combination with the cells of the invention. The cells, or nucleic acids, vectors, or constructs used to produce the cells, and rituximab or an equivalent antibody can be provided as a kit or combination product.

[0167] For example, the suicide constructs of WO 2013 / 153391 or WO 2021 / 239812 (both of which are incorporated herein by reference) may be used in cells or cell populations (e.g., Tregs or Treg populations) as described herein.

[0168] The nucleic acid molecules of the present invention can be designed to increase FOXP3 expression in cells (e.g., Tregs) by introducing a nucleotide sequence encoding FOXP3 (this term is synonymous with the term "FOXP3 polypeptide") into the cells. Thus, the nucleic acid molecules, and constructs and vectors containing them, provide a means for increasing FOXP3 in cells, such as Treg cells or CD4+ cells. As described above, a single nucleic acid molecule may encode the CAR and FOXP3 polypeptide of the present invention, or the CAR and FOXP3 may each be encoded by separate, i.e., independent, nucleic acid molecules. Thus, the present invention provides cells, particularly pluripotent cells (e.g., iPSCs), HPC cells (e.g., expressing CD34), CD4+ T cells, or Treg cells, comprising a nucleic acid molecule comprising a nucleotide sequence encoding a CAR and a nucleic acid molecule comprising a nucleotide sequence encoding FOXP3.

[0169] "FOXP3" is an abbreviation for Forkhead Box P3 protein. FOXP3 is a member of the FOX protein family of transcription factors and functions as a key regulator of regulatory pathways in the development and function of regulatory T cells. As used herein, "FOXP3" encompasses variants, isoforms, and functional fragments of FOXP3.

[0170] "Increasing FOXP3 expression" means increasing the level of FOXP3 mRNA and / or protein in a cell (or a population of cells) compared to a corresponding cell (or a population of cells) that has not been modified by the introduction of a nucleic acid molecule, construct, or vector. For example, the level of FOXP3 mRNA and / or protein in a cell (or a population of such cells) modified according to the present invention may be increased by at least 1.5-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, or at least 150-fold over the level in a corresponding cell (or a population of such cells) that has not been modified according to the present invention. Optionally, the cell is a Treg, and the population of cells is a population of Tregs.

[0171] Suitably, the level of FOXP3 mRNA and / or protein in the modified cells (or population of such cells) may be increased by at least 1.5-fold, 2-fold, or 5-fold over the level in the corresponding non-modified cells (or population of such cells). Optionally, the cells are Tregs and the population of cells is a population of Tregs.

[0172] The technology for measuring the level of specific mRNA and protein is well known in the art.The mRNA level in a population of cells such as Treg can be measured by techniques such as Affymetrix's ebioscience prime flow RNA assay, Northern blotting, serial analysis of gene expression (SAGE) or quantitative polymerase chain reaction (qPCR).The protein level in a population of cells can be measured by techniques such as flow cytometry, high performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LC / MS), Western blotting or enzyme-linked immunosorbent assay (ELISA).

[0173] "FOXP3 polypeptide" refers to a polypeptide having FOXP3 activity, i.e., a polypeptide that can bind to FOXP3 target DNA and function as a transcription factor regulating the development and function of Tregs. In particular, FOXP3 polypeptide may have the same or similar activity as wild-type FOXP3 (SEQ ID NO: 148), for example, at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, or 150% of the activity of wild-type FOXP3 polypeptide. Therefore, the FOXP3 polypeptide encoded by the nucleotide sequence in the nucleic acid, construct, or vector described herein may have increased or decreased activity compared to wild-type FOXP3. Techniques for measuring transcription factor activity are well known in the art. For example, transcription factor DNA binding activity can be measured by ChIP. The transcriptional regulatory activity of a transcription factor can be measured by quantifying the expression level of the gene regulated by the transcription factor. Gene expression can be quantified by measuring the levels of mRNA and / or protein 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 described in more detail below, FOXP3 or FOXP3 polypeptides include functional fragments, variants, and isoforms, for example, SEQ ID NO: 148.

[0174] A "functional fragment of FOXP3" may refer to a portion or region of a FOXP3 polypeptide, or a portion or region of a polynucleotide (i.e., a nucleotide sequence) encoding a FOXP3 polypeptide, which has the same or similar activity as a full-length FOXP3 polypeptide or polynucleotide. The functional fragment may have at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the activity of a full-length FOXP3 polypeptide or polynucleotide. A person skilled in the art would be able to generate functional fragments based on the known structural and functional characteristics of FOXP3. These are described, for example, in Song, X. et al., 2012. Cell reports, 1(6), pp. 665-675, Lopes, JE et al., 2006. The Journal of Immunology, 177(5), pp. 3133-3142, and Lozano, T. et al., 2013. Frontiers in oncology, 3, p. 294. Additionally, N-terminally and C-terminally truncated FOXP3 fragments are described in WO 2019 / 241549 (incorporated herein by reference), such as those having the sequence of SEQ ID NO: 149, as described below.

[0175] A "FOXP3 variant" may refer to an amino acid or nucleotide sequence that has at least 50%, at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identity, and optionally at least 95%, at least 97%, or at least 99% identity, to a FOXP3 polypeptide or polynucleotide encoding a FOXP3 polypeptide, e.g., to SEQ ID NO: 148. A FOXP3 variant may have the same or similar activity as a wild-type FOXP3 polypeptide or polynucleotide, e.g., at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, or 150% of the activity of a 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 conservative substitutions. The FOXP3 variant may have a similar or the same turnover time (or degradation rate) in Treg cells as wild-type FOXP3, 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 Treg cells. Some FOXP3 variants may have a reduced turnover time (or degradation rate) compared to wild-type FOXP3, such as FOXP3 variants with amino acid substitutions at amino acids 418 and / or 422 of SEQ ID NO: 148, e.g., S418E and / or S422A, as described in WO 2019 / 241549 (incorporated herein by reference), and are set forth in SEQ ID NOs: 150-152. These represent the aa418 mutant, the aa422 mutant, and the aa418 and aa422 mutants, respectively.

[0176] Suitably, the FOXP3 polypeptide encoded by the nucleic acid molecules, constructs or vectors described herein may comprise or consist of the polypeptide sequence of human FOXP3, such as UniProtKB Accession No. Q9BZS1 (SEQ ID NO: 148), or a functional fragment or variant thereof.

[0177] In some embodiments of the invention, the FOXP3 polypeptide comprises or consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 148, or a functional fragment thereof. Suitably, the FOXP3 polypeptide comprises or consists of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 148, or a functional fragment thereof. In some embodiments, the FOXP3 polypeptide comprises or consists of SEQ ID NO: 148 or a functional fragment thereof.

[0178] In some embodiments, as described above, the FOXP3 polypeptide may include a mutation at residues 418 and / or 422 of SEQ ID NO: 148, as set forth in SEQ ID NO: 150, SEQ ID NO: 151, or SEQ ID NO: 152.

[0179] In some embodiments of the invention, FOXP3 polypeptides may be truncated at the N-terminus and / or C-terminus to produce functional fragments. In particular, N- and C-terminally truncated functional fragments of FOXP3 may comprise or consist of the amino acid sequence of SEQ ID NO: 149, or a functional variant having at least 80%, 85%, 90%, 95%, or 99% identity thereto.

[0180] Preferably, the FOXP3 polypeptide may be a variant of SEQ ID NO: 148, such as a naturally occurring variant. Preferably, the FOXP3 polypeptide is an isoform of SEQ ID NO: 148. For example, the FOXP3 polypeptide may comprise a deletion of amino acids 72 to 106 relative to SEQ ID NO: 148. Alternatively, the FOXP3 polypeptide may comprise a deletion of amino acids 246 to 272 relative to SEQ ID NO: 148.

[0181] Suitably, the FOXP3 polypeptide comprises SEQ ID NO: 153 or a functional fragment thereof. SEQ ID NO: 153 represents an exemplary FOXP3 polypeptide.

[0182] Suitably, the FOXP3 polypeptide comprises or consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 153, or a functional fragment thereof. Suitably, the FOXP3 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity to SEQ ID NO: 153, or a functional fragment thereof. In some embodiments, the FOXP3 polypeptide comprises or consists of SEQ ID NO: 153, or a functional fragment thereof.

[0183] Preferably, the FOXP3 polypeptide may be a variant of SEQ ID NO: 153, such as a naturally occurring variant. Preferably, the FOXP3 polypeptide is an isoform of SEQ ID NO: 153 or a functional fragment thereof. For example, the FOXP3 polypeptide may comprise a deletion of amino acids 72 to 106 relative to SEQ ID NO: 153. Alternatively, the FOXP3 polypeptide may comprise a deletion of amino acids 246 to 272 relative to SEQ ID NO: 153.

[0184] Suitably, the polynucleotide encoding the FOXP3 polypeptide comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 154. SEQ ID NO: 154 represents an exemplary FOXP3 nucleotide sequence.

[0185] In some embodiments of the invention, the polynucleotide encoding a FOXP3 polypeptide or variant thereof comprises a nucleotide sequence having at least 70% identity to SEQ ID NO: 154, or a fragment thereof that encodes a functional FOXP3 polypeptide. Suitably, the polynucleotide encoding a FOXP3 polypeptide or variant thereof 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 to SEQ ID NO: 154, or a fragment thereof that encodes a functional FOXP3 polypeptide. In some embodiments of the invention, the polynucleotide encoding a FOXP3 polypeptide or variant thereof comprises or consists of SEQ ID NO: 154, or a fragment thereof that encodes a functional FOXP3 polypeptide.

[0186] Suitably, the polynucleotide encoding the FOXP3 polypeptide comprises or consists of the polynucleotide sequence set forth in SEQ ID NO: 155. SEQ ID NO: 155 represents another exemplary FOXP3 nucleotide sequence.

[0187] In some embodiments of the invention, the polynucleotide encoding a FOXP3 polypeptide or variant thereof comprises a nucleotide sequence having at least 70% identity to SEQ ID NO: 155, or a fragment thereof that encodes a functional FOXP3 polypeptide. Suitably, the polynucleotide encoding a FOXP3 polypeptide or variant thereof 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 to SEQ ID NO: 155, or a fragment thereof that encodes a functional FOXP3 polypeptide. In some embodiments of the invention, the polynucleotide encoding a FOXP3 polypeptide or variant thereof comprises or consists of SEQ ID NO: 155, or a fragment thereof that encodes a functional FOXP3 polypeptide.

[0188] Those skilled in the art will understand that FOXP3 expression in Tregs can be indirectly increased by introducing into cells a polynucleotide encoding a protein that increases the transcription and / or translation of FOXP3, or that prolongs the half-life of FOXP3 (e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) or increases the function of FOXP3 (e.g., as determined by the suppressive ability of transduced Tregs, measured as described above). For example, it may be possible to introduce into Tregs a polynucleotide that increases the transcription of endogenous FOXP3 by interacting with a non-coding sequence (CNS, e.g., CNS1, CNS2, or CNS3) present upstream of the endogenous FOXP3 promoter or coding region.

[0189] Suitably, a polynucleotide encoding a FOXP3 polypeptide or functional fragment or variant thereof may be codon-optimised. Suitably, a polynucleotide encoding a FOXP3 polypeptide or functional fragment or variant thereof may be codon-optimised to be suitable for expression in human cells.

[0190] As described above, the nucleic acid molecule may contain a nucleotide sequence encoding a self-cleaving sequence. In particular, the self-cleaving sequence is a self-cleaving peptide. Such a sequence is automatically cleaved during protein production. Self-cleaving peptides that can be used include 2A peptides or 2A-like peptides, which are known in the art and are described, for example, in Donnelly et al., Journal of General Virology, 2001, 82, 1027-1041 (incorporated herein by reference). 2A peptides and 2A-like peptides are thought to cause ribosome skipping, resulting in a form of cleavage in which the ribosome skips the formation of a peptide bond between the end of the 2A peptide and the downstream amino acid sequence. "Cleavage" occurs between the glycine and proline residues at the C-terminus of the 2A peptide. In other words, the upstream cistron has several additional residues added to its end, and the downstream cistron starts with a proline. Therefore, the term "cleavage" in the present invention includes skipping the formation of a peptide bond.

[0191] Suitable self-cleaving domains include the P2A, T2A, E2A, and F2A sequences shown in SEQ ID NOs: 156-159, respectively. These sequences may be modified to include the amino acid GSG at the N-terminus of the 2A peptide. Thus, sequences corresponding to SEQ ID NOs: 156-159 but with GSG at the N-terminus are also possible options. 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. (supra), e.g., TaV sequences.

[0192] The self-cleaving sequences contained in the nucleic acid molecule may be the same or different, hi one embodiment both are 2A sequences, in particular P2A and / or T2A sequences.

[0193] The self-cleavage sequence may contain additional cleavage sites that can be cleaved by common enzymes present in cells. This can help achieve complete removal of the 2A sequence after translation. Such additional cleavage sites may include, for example, furin cleavage sites RXXR (SEQ ID NO: 160), such as RRKR (SEQ ID NO: 161).

[0194] In an exemplary embodiment, the nucleic acid molecule may comprise a nucleotide sequence encoding a CAR directed against ENTPD3 (having the sequence of any one of SEQ ID NOs: 237-262 described herein or a variant thereof), a nucleotide sequence encoding a safety switch, and a nucleotide sequence encoding FOXP3.

[0195] In such embodiments, the CAR may comprise: (a) a leader sequence comprising or consisting of the sequence set forth in SEQ ID NO: 137, or a sequence having at least 80% sequence identity thereto; (b) an antigen-binding domain comprising or consisting of a sequence set forth in SEQ ID NO: 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, or 126, or a sequence having at least 80% sequence identity thereto; (c) a CD8α hinge and transmembrane domain sequence comprising or consisting of the sequence set forth in SEQ ID NO: 131 or 133, or a sequence having at least 80% sequence identity thereto; (d) a CD28 costimulatory domain comprising or consisting of the sequence set forth in SEQ ID NO: 140, or a sequence having at least 80% sequence identity thereto; (e) A CD3ζ signaling domain comprising or consisting of the sequence set forth in SEQ ID NO: 138, or a sequence having at least 80% sequence identity thereto.

[0196] In an alternative embodiment, the CAR may include: (a) a leader sequence comprising or consisting of the sequence set forth in SEQ ID NO: 137, or a sequence having at least 80% sequence identity thereto; (b) an antigen-binding domain comprising or consisting of a sequence set forth in SEQ ID NO: 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, or 126, or a sequence having at least 80% sequence identity thereto; (c) a CH2CH3 hinge domain sequence comprising or consisting of the sequence set forth in SEQ ID NO: 134, or a sequence having at least 80% sequence identity thereto; (d) a CD28 transmembrane and costimulatory domain comprising or consisting of the sequence set forth in SEQ ID NO: 139, or a sequence having at least 80% sequence identity thereto; (e) A CD3ζ signaling domain comprising or consisting of the sequence set forth in SEQ ID NO: 138, or a sequence having at least 80% sequence identity thereto.

[0197] As is clear from the above description, in addition to the specific polypeptide and nucleotide sequences described herein, the use of variants, or derivatives and fragments thereof, is also encompassed.

[0198] The terms "derivative" or "variant," as used interchangeably herein, in reference to a protein or polypeptide of the invention, include substitution, mutation, modification, replacement, deletion, and / or addition of one or more amino acid residues to a sequence, provided that the resulting protein or polypeptide retains the desired function. (For example, if the derivative or variant is an antigen-binding domain, the desired function may be the ability of the antigen-binding domain to bind to its target antigen (e.g., a variant of an antigen-binding domain that binds to ENTPD3 retains the ability to bind to ENTPD3). If the derivative or variant is a signaling domain, the desired function may be the signaling ability of the domain (e.g., activation or inactivation of a downstream molecule). If the derivative or variant is a transcription factor (e.g., FOXP3), the desired function may be the ability of the transcription factor to bind to target DNA and / or induce transcription. Alternatively, if the derivative or variant is a safety switch polypeptide, the desired function may be, for example, the ability of the polypeptide to bind to a target DNA when a molecule binds to the polypeptide. It can be the ability to induce cell death.In other words, the variant or derivative referred to herein is a functional variant or functional derivative.For example, compared with corresponding reference sequence, variant or derivative can have at least 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% function.Compared with corresponding reference sequence, variant or derivative can have similar or the same level of function, or can have a higher level (for example, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% higher level) than corresponding reference sequence.

[0199] Typically, amino acid substitutions can be made, for example, from one, two, or three to ten or twenty, provided that the modified sequence retains the desired activity or potency. Amino acid substitutions can also include the use of non-naturally occurring analogs. For example, a variant or derivative can 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 potency of a corresponding reference sequence. A variant or derivative can have a similar or the same level of activity or potency as a corresponding reference sequence, or it can have a higher level of activity or potency (e.g., at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% higher) than a corresponding reference sequence.

[0200] Proteins or peptides may have deletions, insertions, or substitutions of amino acid residues, resulting in silent changes and resulting in functionally equivalent proteins. Deliberate amino acid substitutions may be made based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues, so long as the intrinsic function is maintained. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with uncharged polar head groups with similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine. Conservative substitutions may be made, for example, according to Table 1 below. [Table 1]

[0201] A derivative may be a homologue. As used herein, the term "homologue" refers to an entity that has a certain degree of homology with a wild-type amino acid sequence and a wild-type nucleotide sequence. The term "homology" can be used synonymously with "identity".

[0202] A homologous or variant sequence may comprise an amino acid sequence having at least 70%, 75%, 85%, or 90% identity to the subject sequence, and optionally at least 95%, 96%, 97%, 98%, or 99% identity. Typically, the variant contains the same active site, etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e., amino acid residues having similar chemical properties / functions), in the context of this specification, homology is sometimes expressed in terms of sequence identity.

[0203] Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percent homology or identity between two or more sequences.

[0204] Percent homology or sequence identity may be calculated over a contiguous sequence. That is, one sequence is aligned with the other and each amino acid in one sequence is directly compared, residue by residue, with the corresponding amino acid in the other sequence. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

[0205] Although this is a very simple and consistent method, it does not take into account that, for example, in a pair of otherwise identical sequences, a single insertion or deletion in a nucleotide sequence can cause subsequent codons to be misaligned, which can significantly reduce the homology rate when a global alignment is performed. Therefore, most sequence comparison methods are designed to generate an optimal alignment that takes into account possible insertions and deletions without unduly penalizing the overall homology score. This is achieved by inserting "gaps" into the sequence alignment to maximize local homology.

[0206] However, these more complex methods assign a "gap penalty" to each gap that occurs in the alignment, so that a sequence alignment with as few gaps as possible (reflecting a greater degree of relatedness between the two sequences being compared) will achieve a higher score than a sequence alignment with more gaps, for the same number of identical amino acids. An "affine gap cost" is typically used, which imposes a relatively high cost for the presence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. A higher gap penalty naturally results in an optimized alignment with fewer gaps. Most alignment programs allow the gap penalty to be modified; however, the default values ​​may be used when using such software for sequence comparison. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 per gap and -4 for each extension.

[0207] Therefore, to calculate the maximum percentage homology / sequence identity, it is first necessary to create an optimal alignment taking into account gap penalties. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit package (Devereux et al. (1984) Nucleic Acids Res. 12: 387, University of Wisconsin, USA). Examples of other software capable of performing sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. (1999) ibid, Chapter 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410), and the GENEWORKS comparison tool suite. Both BLAST and FASTA are available for offline and online searches (see Ausubel et al. (1999) ibid, pp. 7-58-7-60). However, for some applications, the GCG Bestfit program may be used. Another tool called BLAST 2 Sequences is also available for comparing protein and nucleotide sequences (see FEMS Microbiol. Lett. (1999) 174: 247-50, FEMS Microbiol. Lett. (1999) 177: 187-8).

[0208] Although the final percentage homology may be measured in terms of identity, the alignment process itself is typically not based on pairwise comparisons of exact matches. Instead, a scaled similarity score matrix is ​​typically used, which assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. One example of such a matrix commonly used is the BLOSUM62 matrix (the default matrix for the BLAST suite of programs). GCG Wisconsin programs typically use either the public default values ​​or, if supplied, a custom symbol comparison table (see user manual for details). For some applications, the public default values ​​for the GCG package may be used. For other software, a default matrix such as BLOSUM62 may be used. Preferably, the percentage identity is determined across the entire reference and / or query sequence.

[0209] Once the software has produced an optimal alignment, it is possible to calculate percent homology, and, if desired, percent sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

[0210] A "fragment" typically refers to a selected region of a polypeptide or polynucleotide that is of functional interest, e.g., a functional region or a region that encodes a functional fragment. Thus, a "fragment" refers to an amino acid sequence or nucleic acid sequence that is a portion (or part) of a full-length polypeptide or polynucleotide.

[0211] Such variants, derivatives, and fragments may be prepared using standard recombinant DNA techniques, such as site-directed mutagenesis. If an insertion is to be made, synthetic DNA encoding the insertion can be generated, along with 5' and 3' flanking regions corresponding to the native sequence flanking the insertion site. The flanking regions contain appropriate restriction enzyme sites corresponding to each site in the native sequence, allowing the sequence to be cleaved with the appropriate enzyme(s) and the synthetic DNA to be ligated to the cleavage site. The DNA is then expressed in accordance with the invention to produce the encoded protein. These methods are merely illustrative of the many standard techniques known in the art for manipulating DNA sequences; other known techniques may also be used.

[0212] The nucleic acid molecules and polynucleotides / nucleotides / nucleic acid sequences defined herein may comprise DNA or RNA. They may be single-stranded or double-stranded. Those skilled in the art will understand that, as a result of 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 routine techniques to make nucleotide substitutions without affecting the polypeptide sequence encoded by the nucleic acid molecules / polynucleotides / nucleotide sequences defined herein to reflect the codon usage of the specific host organism in which the polypeptide of the present invention is expressed.

[0213] The nucleic acid molecules / polynucleotides / nucleotides described above may be modified by any method available in the art, and such modifications may be carried out with the aim of enhancing the in vivo activity or lifespan of the nucleic acid molecules / polynucleotides defined herein.

[0214] Nucleic acid molecules / polynucleotides / nucleotide sequences (e.g., DNA nucleic acid molecules / DNA polynucleotides / DNA sequences) may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned using standard techniques.

[0215] Longer nucleic acid molecules / polynucleotides / nucleotide sequences are generally generated using recombinant DNA techniques, such as polymerase chain reaction (PCR) cloning techniques. This involves generating a pair of primers (e.g., about 15-30 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, isolating the amplified fragment (e.g., by purifying the reaction mixture on an agarose gel), and recovering the amplified DNA. Primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable vector.

[0216] The nucleic acid molecule / polynucleotide / nucleotide may further comprise a nucleic acid sequence encoding a selectable marker. Suitable selectable markers are well known in the art and include, but are not limited to, fluorescent proteins such as GFP. Preferably, the selectable 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 selectable marker may be provided in the form of a nucleic acid construct in combination with the nucleic acid molecule herein. Such a nucleic acid construct may be provided within a vector.

[0217] Suitably, the selectable marker / reporter domain may be a luciferase-based reporter, a PET reporter (eg, sodium iodide symporter (NIS)), or a membrane protein (eg, CD34 or Thy1.1). The nucleic acid sequences encoding one or more selectable markers may be separated from the nucleic acid molecule and / or from each other by one or more co-expression sites, allowing each polypeptide to be expressed as an independent entity. Suitable co-expression sites are known in the art and include, for example, internal ribosome entry sites (IRES) and self-cleavage sites, such as those contained in the nucleic acid molecule as defined above. In one embodiment, this may be a 2A cleavage site, as described above.

[0218] The use of a selectable marker is advantageous because it allows cells (e.g., Tregs) that have been successfully transfected with a nucleic acid molecule, construct, or vector of the invention (and thereby express the encoded ENTPD3 CAR and other modules, e.g., FOXP3 and safety switch polypeptides) to be selected and isolated from the starting cell population using conventional methods, such as flow cytometry.

[0219] The nucleic acid molecules / polynucleotides / nucleotides used in the present invention may be codon-optimized. Codon optimization has been previously described in International Publication Nos. 1999 / 41397 and 2001 / 79518. Different cells use specific codons differently. This codon bias corresponds to a bias in the relative abundance of certain tRNAs in a cell type. By changing the codons in the sequence to match the relative abundance of the corresponding tRNA, expression can be increased. Similarly, expression can be decreased by deliberately selecting codons whose corresponding tRNAs are known to be scarce in a particular cell type. In this way, a greater degree of translational control can be achieved.

[0220] The constructs of the present invention may contain one or more regulatory sequences, such as promoters. A "promoter" is a region of DNA that directs the initiation of transcription of a gene. A promoter is located upstream (toward the 5' region of the sense strand) in the DNA near the transcription start site of a gene. Any suitable promoter can be used, and its selection can be readily made by one of ordinary skill in the art. The promoter may be of any origin, including viral promoters and eukaryotic promoters, including mammalian or human promoters (i.e., physiological promoters). In one embodiment, the promoter is a viral promoter. Specific promoters include the LTR promoter, EFS (or functional truncates thereof), SFFV, PGK, and CMV. In one embodiment, the promoter is an SFFV or viral LTR promoter. In particular, the SFFV promoter can be used in the nucleic acid molecules, constructs, or vectors of the present invention to enable transcription initiation of the nucleotide sequence. Thus, this promoter can control the expression of the CAR of the present invention. When multiple nucleotide sequences are present, each sequence may be operably linked to the same promoter, such as, for example, nucleotide sequences encoding a CAR, FOXP3, and / or a safety switch. The SFFV promoter may comprise the nucleotide sequence set forth in SEQ ID NO:162. "Operably linked to the same promoter" means that transcription of nucleic acids / polynucleotides / nucleotide sequences can be initiated from the same promoter (e.g., transcription of a first, second, and third polynucleotide sequence is initiated from the same promoter), and that each nucleotide sequence is positioned and oriented so that transcription is initiated from the promoter. A nucleic acid / polynucleotide / nucleotide sequence that is operably linked to a promoter is under the transcriptional control of that promoter.

[0221] In some embodiments of the present invention, the nucleic acid / polynucleotide / nucleotide sequence is present in an expression vector. As used herein, the term "expression vector" refers to a construct that allows for the expression of a CAR polypeptide and any additional polypeptides, such as a FOXP3 polypeptide or a safety switch polypeptide.

[0222] A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. Exemplary vectors used herein include those used in recombinant nucleic acid technology, which allow an entity, such as a nucleic acid segment (e.g., a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a target cell. A vector can be non-viral or viral. Examples of vectors used in recombinant nucleic acid technology include, but are not limited to, plasmids, mRNA molecules (e.g., in vitro transcribed mRNA), chromosomes, artificial chromosomes, and viruses. A vector can also be, for example, a naked nucleic acid (e.g., DNA). In its simplest form, a vector can itself be the nucleotide of interest.

[0223] The vectors used herein may be, for example, plasmid, mRNA, or viral vectors, and may contain a promoter (described above) for expression of the nucleic acid molecule / polynucleotide, and may optionally contain a regulator of the promoter.

[0224] In one embodiment, the vector is a viral vector, e.g., a retroviral vector, e.g., a lentiviral vector or a gammaretroviral vector.

[0225] The vector may further comprise an additional promoter; for example, in one embodiment, the promoter may be an LTR, such as a retroviral LTR or lentiviral LTR. Long terminal repeats (LTRs) are identical DNA sequences that are repeated hundreds or thousands of times at both ends of retrotransposons or proviral DNA formed by reverse transcription of retroviral RNA. LTRs are used by viruses to insert their genetic material into the host genome. The following gene expression signals are present in the LTR: enhancers, promoters (which may have both transcription enhancers or regulatory elements), transcription initiation (such as capping), transcription termination, and polyadenylation signals. Preferably, the vector may comprise a 5'LTR and a 3'LTR.

[0226] The vector may contain one or more additional regulatory sequences that can act before or after transcription. "Regulatory sequence" refers to any sequence that promotes the expression of a polypeptide, including, for example, a sequence that acts to increase the expression of transcripts or enhance the stability of mRNA. Suitable regulatory sequences include, for example, enhancer elements, post-transcriptional regulatory elements, and polyadenylation sites. Preferably, the additional regulatory sequence may be present in the LTR. Suitably, the vector may include, for example, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) operably linked to the promoter.

[0227] The vectors containing the nucleic acid molecules / polynucleotides can be introduced into cells using various techniques known in the art, such as transformation and transduction, including infection with recombinant viral vectors such as retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, baculoviral vectors, and herpes simplex viral vectors, direct injection of nucleic acids, and biolistic transformation.

[0228] 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, which may be complexed with nucleic acid molecules or constructs as needed.

[0229] Typical transfection methods include electroporation, DNA biolistic methods, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic drug-mediated transfection, cationic facial amphiphiles (CFAs) (Nat. Biotechnol. (1996) 14: 556), and combinations thereof.

[0230] The present nucleic acid molecule is designed to be used as a single construct, which will be contained in a single vector, although it is not excluded that it may be introduced into a cell in conjunction with other vectors, for example vectors encoding other polypeptides that may also be desired to be introduced into the cell.

[0231] Engineered cells can be produced by introducing a nucleic acid molecule, construct, or vector as defined herein by one of a number of means, such as transduction with a viral vector or transfection with DNA or RNA.

[0232] The cells may be produced by introducing (eg, by transduction or transfection) a nucleic acid molecule / polynucleotide, construct, or vector as defined herein into the cell.

[0233] Suitable cells are described further below, but the cells may be derived from a sample isolated from a subject, which may be a donor subject or the subject of treatment (i.e., the cells may be autologous cells or donor cells, e.g., allogeneic cells, for introduction into another recipient).

[0234] The cells may be produced by a method comprising the steps of: (i) isolating or providing a cell-containing sample from a subject; and (ii) introducing (e.g., by transduction or transfection) a nucleic acid molecule, construct, or vector as defined herein into a cell-containing sample to obtain a population of engineered cells. Before and / or after step (ii) of the above method, a target cell-enriched sample may be isolated, enriched, and / or generated from the cell-containing sample. For example, isolation, enrichment, and / or generation of Tregs (or other target cells) may be performed before and / or after step (ii) to isolate, enrich, or generate a Treg-enriched sample. Isolation and / or enrichment from the cell-containing sample may be performed after step (ii) to enrich cells and / or Tregs (or other target cells) comprising the CAR, nucleic acid molecule / polynucleotide, construct, and / or vector described herein.

[0235] The Treg-enriched sample can be isolated or enriched by any method known to those skilled in the art, for example, by FACS and / or magnetic bead separation.The Treg-enriched sample can be generated from a cell-containing sample by any method known to those skilled in the art, for example, by introducing DNA or RNA encoding FOXP3 into Tcon cells, and / or by ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells.Methods for isolating and / or enriching other target cells are known in the art.

[0236] Preferably, the engineered cells may be produced by a method comprising the steps of: (i) isolating or providing a target cell enriched sample from a subject; and (ii) introducing (e.g., by transduction or transfection) a nucleic acid, construct, or vector as defined herein into the target cell enriched sample to obtain a population of engineered cells.

[0237] The target cells may be Treg cells, or their precursor or progenitor cells.

[0238] "Engineered cells" refer to cells that have been modified to contain or express a polynucleotide not naturally encoded by the cell. Methods for engineering cells are known in the art and include, but are not limited to, genetic modification of cells, which can be achieved by transduction methods such as retroviral or lentiviral transduction, or transfection methods (e.g., DNA- or RNA-based transient transfection), including lipofection, polyethylene glycol, calcium phosphate, and electroporation (these methods are described above). Any suitable method can be used to introduce a nucleic acid sequence into a cell. Non-viral techniques, such as amphipathic cell-penetrating peptides, can also be used to introduce nucleic acids. Cells can also be genetically modified using any known gene editing technology, such as CRISPR, TALEN, or zinc finger, to insert nucleotides, polynucleotides, or nucleic acid sequences described herein into the genome.

[0239] Therefore, the nucleic acid molecules described herein are not naturally expressed by corresponding unmodified cells.In fact, the nucleic acid molecule encoding CAR is an artificial construct, and in one embodiment, the safety switch polypeptide is also an artificial construct, so that they are not naturally present or naturally expressed.Preferably, the engineered cell is a cell that has been modified, for example, by transduction or transfection.Preferably, the engineered cell is a cell that has been modified, for example, by transduction or transfection, or a cell whose genome has been modified.Preferably, the engineered cell is a cell that has been modified by retroviral transduction, or a cell whose genome has been modified.Preferably, the engineered cell is a cell that has been modified by lentiviral transduction, or a cell whose genome has been modified.

[0240] As used herein, the term "introduced" refers to a method for inserting exogenous nucleic acid, e.g., DNA or RNA, into a cell. As used herein, the term "introduced" includes both transduction and transfection methods. Transfection is the process of introducing nucleic acid into a cell by non-viral methods. Transduction is the process of introducing exogenous DNA or RNA into a cell via a viral vector. Engineered cells can be generated by introducing the nucleic acid described herein by one of several means, such as transduction with a viral vector or transfection with DNA or RNA. Cells may be activated and / or expanded before or after introduction of the nucleic acid described herein, for example, by treatment with an anti-CD3 monoclonal antibody or with both an anti-CD3 monoclonal antibody and an anti-CD28 monoclonal antibody. Cells may also be expanded in the presence of a combination of an anti-CD3 monoclonal antibody and an anti-CD28 monoclonal antibody and IL-2. IL-2 may be suitably replaced with IL-15. Other components that may 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 cells have been stimulated to cause cell proliferation. As used herein, "expanded" means that a cell or population of cells has been induced to proliferate. The proliferation of a population of cells can be measured, for example, by counting the number of cells present in the population. The phenotype of a cell can be determined by methods known in the art, such as flow cytometry.

[0241] The cells may be immune cells or their precursors. Precursor cells may also be progenitor cells. Thus, 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, are also useful herein. Other immune cells include NK cells, NKT cells, tolerogenic NK or NKT cells, dendritic cells, MDSCs, neutrophils, and macrophages. Immune cell precursors include pluripotent stem cells (e.g., induced pluripotent stem cells (iPSCs)), or more differentiated precursor cells, including multipotent stem cells (e.g., HPCs), as well as cells differentiated into specific lineages. Precursor cells can be induced to differentiate into immune cells in vivo or in vitro. In one embodiment, the precursor cells may be somatic cells capable of transdifferentiation into the immune cells of interest.

[0242] Most notably, the immune cells may be NK cells, dendritic cells, MDSCs, or T cells such as cytotoxic T lymphocytes (CTLs), helper T cells, or Treg cells.

[0243] In one embodiment, the immune cells are Treg cells. "Regulatory T cells (Treg) or T regulatory cells" are immune cells with immunosuppressive function, which control cytopathic immune responses and are essential for maintaining immune tolerance. As used herein, the term Treg refers to T cells with immunosuppressive function.

[0244] As used herein, a T cell is a lymphocyte and includes any type of T cell, for example, an αβ T cell (e.g., CD8 or CD4+), a γδ T cell, a memory T cell, or a Treg cell.

[0245] Preferably, the immunosuppressive function refers to the ability of Tregs to reduce or inhibit one or more of several physiological and cellular actions promoted by the immune system in response to stimuli such as pathogens, alloantigens, or autoantigens. Such actions include, for example, increased proliferation of normal T cells (Tconv) and secretion of inflammatory cytokines. Either of these actions can be used as an indicator of the strength of the immune response. A relative weakening of the Tconv-mediated immune response in the presence of Tregs indicates the ability of Tregs to suppress the immune response. For example, a relative decrease in cytokine secretion indicates a weakened immune response, thus suggesting the ability of Tregs to suppress the immune response. Tregs can also suppress the immune response by regulating the expression of costimulatory molecules in 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 suppressive ability of activated Tregs in vitro after coculture.

[0246] Assays for measuring the strength of immune responses and thereby measuring the suppressive ability of Tregs are known in the art. In particular, antigen-specific Tconv cells can be co-cultured with Tregs, and the corresponding antigen peptide can be added to the co-culture system to stimulate responses from Tconv cells. The degree of proliferation of Tconv cells and / or the amount of cytokine IL-2 secreted by Tconv cells in response to the addition of peptides can be used as indicators of the suppressive ability of the co-cultured Tregs.

[0247] Antigen-specific Tconv cells co-cultured with Tregs disclosed herein may proliferate 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 95%, or 99% less than the same Tconv cells cultured in the absence of the Tregs. For example, antigen-specific Tconv cells co-cultured with the Tregs may proliferate 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 95%, or 99% less than the same Tconv cells cultured in the presence of unmanipulated Tregs. Cells, e.g., Tregs, comprising a nucleic acid, expression construct, or vector defined herein may have increased suppressive activity compared to unmanipulated Tregs (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% increased suppressive activity).

[0248] Antigen-specific Tconv cells co-cultured with Tregs 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 unmanipulated Tregs). The effector cytokines may be selected from IL-2, IL-17, TNFα, GM-CSF, IFN-γ, IL-4, IL-5, IL-9, IL-10, and IL-13. Preferably, the effector cytokines may be selected from IL-2, IL-17, TNFα, GM-CSF, and IFN-γ.

[0249] Several different subpopulations of Tregs have been identified, which may express different types or levels of specific markers. Tregs are generally characterized by CD4, CD25, and FOXP3 (CD4 + CD25 + FOXP3 + ) T cells that express a marker called

[0250] Tregs may also express CTLA-4 (cytotoxic T lymphocyte-associated molecule 4) or GITR (glucocorticoid-induced TNF receptor).

[0251] Treg cells are present in peripheral blood, lymph nodes, and tissues, and as used herein, Tregs include thymus-derived endogenous Treg (nTreg) cells, peripherally generated Tregs, and inducible Treg (iTreg) cells.

[0252] Tregs express the cell surface markers CD4 and CD25 in combination with the absence or low expression of the surface protein CD127 (CD4 + CD25 + CD127 - or CD4 + CD25 + CD127 low ) can be used to identify Tregs. The use of such markers for the identification of Tregs is known in the art and is described, for example, in Liu et al. (JEM; 2006; 203; 7(10); 1701-1711). Tregs are CD4 + CD25 + FOXP3 + T cells, CD4 + CD25 + CD127 - T cells, or CD4 + CD25 + FOXP3 + CD127 - / low It may be a T cell. Preferably, Tregs may be endogenous Tregs (nTregs). As used herein, the term "endogenous Tregs (natural Tregs)" refers to Tregs derived from the thymus. Endogenous Tregs are CD4 + CD25 + FOXP3 + Helios + Neuropilin 1 +Compared to iTregs, nTregs highly express PD-1 (programmed cell death-1, pdcd1), neuropilin 1 (Nrp1), Helios (Ikzf2), and CD73. nTregs can be distinguished from iTregs based on the individual expression of Helios protein or neuropilin 1 (Nrp1).

[0253] Tregs may have a demethylated Treg-specific demethylation region (TSDR), which is a key methylation-sensitive element that controls Foxp3 expression (Polansky, JK, et al., 2008. European journal of immunology, 38(6), pp. 1654-1663).

[0254] Further preferred Tregs include Tr1 cells (which do not express Foxp3 and produce high levels of IL-10), CD8 + FOXP3 + T cells, and γδFOXP3 + These include, but are not limited to, T cells.

[0255] Tregs include naive Tregs (CD45RA + FoxP3 low ), effector / memory Treg (CD45RA - FoxP3 high ), and cytokine-producing Tregs (CD45RA - FoxP3 low It is known that there are various subpopulations, such as "memory Tregs" that express CD45RO and +These cells have elevated levels of CD45RO compared to naive Tregs (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more CD45RO), and optionally do not express CD45RA (mRNA and / or protein) or have reduced levels of CD45RA compared to naive Tregs (e.g., at least 80%, 90%, or 95% less CD45RA compared to naive Tregs). "Cytokine-producing Tregs" are Tregs that do not express CD45RA (mRNA and / or protein) or have very low levels of CD45RA compared to naive Tregs (e.g., at least 80%, 90%, or 95% less CD45RA compared to naive Tregs) and low levels of FOXP3 compared to memory Tregs (e.g., less than 50%, 60%, 70%, 80%, or 90% FOXP3 compared to memory Tregs). Cytokine-producing Tregs may produce interferon-γ and may have lower in vitro suppressive properties compared to naive Tregs (e.g., less than 50%, 60%, 70%, 80%, or 90% suppressive properties compared to naive Tregs). As used herein, expression level may refer to mRNA or protein expression. In particular, for cell surface markers such as CD45RA, CD25, CD4, CD45RO, expression may refer to cell surface expression, i.e., the amount or relative amount of marker protein expressed on the cell surface.Expression level can be measured by any method known in the art.For example, mRNA expression level can be measured by Northern blotting / array analysis, and protein expression can be measured by Western blotting or, if necessary, by FACS using antibody staining for cell surface expression.

[0256] In particular, Tregs may be naive Tregs. As used interchangeably herein, "naive regulatory T cells, naive T regulatory cells, or naive Tregs" refer to Treg cells that express CD45RA (particularly, express CD45RA on the cell surface). Thus, naive Tregs are Treg cells that express CD45RA + Naive Tregs generally refer to Tregs that have not been activated via endogenous TCR by peptide / MHC, while effector / memory Tregs refer to Tregs that have been activated by stimulation via endogenous TCR. Typically, naive Tregs may express at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more CD45RA than non-naive Treg cells (e.g., memory Treg cells). In other words, naive Treg cells may express at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, or 100-fold more CD45RA than non-naive Treg cells (e.g., memory Treg cells). The expression level of CD45RA can be easily measured by methods in the art, for example, by flow cytometry using commercially available antibodies. Typically, non-naive Treg cells do not express CD45RA or express low levels of CD45RA.

[0257] In particular, naive Tregs may not express CD45RO, and CD45RO - Thus, naive Tregs may express at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less CD45RO than memory Tregs, or, viewed another way, may express at least 2-, 3-, 4-, 5-, 10-, 50-, or 100-fold less CD45RO than memory Treg cells.

[0258] As mentioned above, naive Tregs express CD25, but depending on the source of naive Tregs, the CD25 expression level may be lower than that of memory Tregs. For example, for naive Tregs isolated from peripheral blood, the CD25 expression level may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower than that of memory Tregs. Such naive Tregs may be considered to have a medium to low level of CD25 expression. However, those skilled in the art will understand that naive Tregs isolated from umbilical cord blood may not show such differences.

[0259] Typically, naive Tregs as defined herein are CD4 + , CD25 + , FOXP3 + , CD127 low , CD45RA + It could be.

[0260] As used herein, low expression of CD127 refers to CD4 expression from the same subject or donor. + Naive Tregs express lower levels of CD127 than non-regulatory or Tcon cells. In particular, naive Tregs express lower levels of CD4 T cells from the same subject or donor. + Compared to non-regulatory or Tcon cells, CD127 expression may be less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%. CD127 levels can be assessed by methods standard in the art, including flow cytometry of cells stained with anti-CD127 antibodies.

[0261] Typically, naive Tregs do not express or express at low levels CCR4, HLA-DR, CXCR3, and / or CCR6. In particular, naive Tregs may express CCR4, HLA-DR, CXCR3, and CCR6 at lower levels than memory Tregs, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower levels. Naive Tregs express CCR7 + and CD31 + The gene may further express additional markers, including:

[0262] Isolated naive Tregs can be identified by methods known in the art, including by determining the presence or absence of a panel of any one or more of the above-mentioned markers on the cell surface of isolated cells.For example, low expression of CD45RA, CD4, CD25, and CD127 can be used to determine whether cells are naive Tregs.Methods for determining whether isolated cells are naive Tregs or have a desired phenotype can be performed as described below in connection with additional steps that may be performed, and methods for determining the presence and / or expression level of cell markers are well known in the art, including, for example, flow cytometry using commercially available antibodies.

[0263] Preferably, cells such as Tregs are isolated from peripheral blood mononuclear cells (PBMCs) obtained from a subject. Preferably, the subject from which the PBMCs are obtained is a mammal, optionally a human. Preferably, the cells are matched (e.g., HLA-matched) or autologous to the subject to whom the engineered cells are administered. Preferably, the subject to be treated is a mammal, optionally a human. The cells may be generated ex vivo from either the patient's own peripheral blood (first party), donor peripheral blood in the context of a hematopoietic stem cell transplant (second party), or unrelated donor peripheral blood (third party). Preferably, the cells are autologous to the subject to whom the engineered cells are administered.

[0264] Preferably, the Tregs are part of a cell population. Preferably, the population of Tregs comprises at least 70% Tregs, for example, at least 75%, 85%, 90%, 95%, 97%, 98%, or 99% Tregs. Such a population may be referred to as an "enriched Treg population."

[0265] In some embodiments, Tregs can be obtained by ex vivo differentiation of inducible progenitor cells (e.g., iPSCs) or embryonic progenitor cells into Tregs. The nucleic acid molecules or vectors described herein can be introduced into inducible progenitor cells or embryonic progenitor cells before or after differentiation into Tregs. Suitable methods for differentiation are known in the art, including those disclosed in Haque et al., J Vis Exp., 2016, 117, 54720 (incorporated herein by reference).

[0266] As used herein, the term "normal T cells," i.e., Tcon or Tconv (which are used interchangeably herein), refers to T lymphocyte cells that express the αβ T cell receptor (TCR) and a co-receptor, which may be cluster of differentiation 4 (CD4) or cluster of differentiation 8 (CD8), and do not have immunosuppressive function. Normal T cells are present in peripheral blood, lymph nodes, and tissues. Preferably, engineered Tregs may be generated from Tcon by introducing a nucleic acid containing a sequence encoding FOXP3. Alternatively, engineered Tregs may be generated from Tcon by in vitro culture of CD4+CD25-FOXP3- cells in the presence of IL-2 and TGF-β. In another embodiment, the target cell into which the nucleic acid molecule, construct, or vector is introduced is not a cell intended for therapeutic use. In one embodiment, the cell is a production host cell. The cell may be a cell intended for nucleic acid production (e.g., cloning), vector production, or polypeptide production.

[0267] The present invention also provides cell populations comprising cells as defined or described herein. It will be understood that a cell population may include both cells of the invention comprising a nucleic acid molecule, expression construct, or vector as defined herein and cells that do not comprise a nucleic acid molecule, expression construct, or vector of the invention (e.g., untransduced or untransfected cells). In one embodiment, while all cells in the population may comprise a nucleic acid, expression construct, or vector of the invention, a cell population is provided in which at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells comprise a nucleic acid, expression construct, or vector of the invention. Furthermore, while a cell population may comprise multiple cell types, in one embodiment, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells are of the same cell type. In particular, the cell population may comprise at least 70%, 80%, 90%, 95%, or 99% T cells, more particularly Tregs. Furthermore, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the T cells, particularly Tregs, may comprise a nucleic acid, expression construct, or vector of the invention.

[0268] In particular, the present invention provides a cell population comprising a plurality of cells comprising a CAR comprising an antigen recognition domain that specifically binds to ENTPD3, or a nucleic acid molecule or vector encoding the CAR.

[0269] A population of T cells (e.g., a population of Tregs) can be "educated" or "reprogrammed" by directly (e.g., via antigen-presenting cells) or indirectly exposing or contacting the T cells with one or more specific antigens (e.g., self- or non-self-antigens), thereby activating the cells (e.g., in the case of Tregs, becoming immunosuppressive or tolerant to the specific antigens) and proliferating. This may be performed ex vivo. Specifically, binding of the T cells' endogenous TCRs to their specific antigens triggers activation-preferential proliferation, resulting in a population with a higher proportion of T cells activated by those specific antigens (i.e., a population with a higher proportion of cells bearing TCRs that bind to those specific antigens). Therefore, the TCR diversity in the resulting cell population is reduced. Generally, it can be said that the clonality (or TCR clonality) of the cells has been altered or increased.

[0270] However, this adds an additional step and layer of complexity to the manufacturing process. Furthermore, it is difficult to control the exact antigens and epitopes on which the TCRs are active. Therefore, in some embodiments of the present invention, reprogrammed or educated T cells are not used. Therefore, in certain embodiments, the clonality of T cells (or, viewed another way, the TCR clonality or endogenous TCR clonality of cells) is not modified ex vivo. For example, the clonality of Tregs (or the TCR clonality of Tregs) is not modified ex vivo. Thus, in certain embodiments, the T cell population is not selected to have a TCR that binds to a pancreatic antigen. As used herein, "clonality" refers to the diversity of antigens that can be bound by TCRs within a T cell population. Thus, an increase in clonality results in a decrease in the diversity of antigens that can be bound, and a decrease in clonality results in an increase in the diversity of antigens that can be bound.

[0271] In particular, in certain embodiments, the T cells are not exposed to or contacted ex vivo with one or more specific antigens, e.g., via antigen-presenting cells, such as dendritic cells. In particular, in certain embodiments, the T cells are not exposed to or contacted ex vivo with one or more specific antigens (e.g., autoantigens or non-autoantigens), either directly (e.g., via antigen-presenting cells) or indirectly, such that the cells are not activated against the specific antigens. In particular, the T cells are not exposed to or contacted ex vivo with one or more pancreatic antigens.

[0272] One method that can be used to "educate" or "reprogram" Tregs (particularly the TCR of Tregs) can include the following steps: (a) contacting dendritic cells (DCs) with interleukin-10 (IL-10), thereby generating tolerogenic dendritic cells (tolDCs); (b) contacting the tolDCs with pancreatic islet extracellular vesicles (EVs), thereby generating antigen-loaded tolDCs; and (c) contacting Treg cells with the antigen-loaded tolDCs, thereby generating the reprogrammed Treg cells.

[0273] The method may further comprise expanding the reprogrammed Treg cells, e.g., in the presence of IL-2. In certain embodiments of the invention, Tregs are not generated by this method and / or the method for generating Tregs (e.g., a population of Tregs) does not include these steps, or any one of these steps. In other words, in some such embodiments, tolerogenic dendritic cells are not used.

[0274] Therefore, a population of T cells (especially Tregs) can be described as polyclonal with respect to their endogenous TCR. That is, they can have TCRs that are not specific for a particular antigen, that are active against multiple antigens, and / or that have specificity for an unknown antigen. For example, a TCR may have specificity for both pancreatic and non-pancreatic antigens. Tregs with polyclonal TCRs can exhibit immune tolerance to multiple antigens and / or immunosuppressive properties against multiple antigens. For example, they can exhibit such properties against both pancreatic and non-pancreatic antigens. Note that the ENTPD3-specific CAR described herein confers antigen specificity to T cells with polyclonal TCRs.

[0275] Also provided are pharmaceutical compositions comprising the cells or cell populations defined or described herein, and the vectors defined herein. The vectors can be used in gene therapy. Thus, instead of administering cells, endogenous cells in a subject can be modified to express the introduced nucleic acid molecule by administering a vector. Vectors suitable for use in gene therapy are known in the art and include viral vectors.

[0276] Thus, in a further aspect, the present invention provides a cell, cell population, or pharmaceutical composition as defined herein for use in therapy.

[0277] A pharmaceutical composition is a composition comprising or consisting of a therapeutically effective amount of a pharmaceutically active agent, i.e., the above-mentioned cells (e.g., Tregs), cell populations, or vectors. Optionally, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent, or excipient (including combinations thereof). Carriers or diluents acceptable for therapeutic use are well known in the pharmaceutical arts and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro, ed. 1985). The choice of pharmaceutical carrier, excipient, or diluent can be determined taking into account the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may comprise any suitable binder, lubricant, suspending agent, coating agent, or solubilizing agent as, or in addition to, the carrier, excipient, or diluent.

[0278] "Pharmaceutically acceptable" implies that the formulation is sterile and pyrogen-free. Carriers, diluents, and / or excipients must be "acceptable" in the sense of being compatible with the cells or vectors and not harmful to the recipient. Typically, the carriers, diluents, and / or excipients will be sterile, pyrogen-free saline or infusion media, although other acceptable carriers, diluents, and excipients may be used.

[0279] Examples of pharmaceutically acceptable carriers include, for example, water, saline, alcohol, silicone, wax, petrolatum, vegetable oils, polyethylene glycol, propylene glycol, liposomes, sugar, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oils, fatty acid monoglycerides, fatty acid diglycerides, petroethral fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

[0280] The cells, cell populations, or pharmaceutical compositions can be administered in a manner suitable for treating and / or preventing the desired disease or condition. The dosage and frequency of administration are determined by factors such as the subject's condition and the type and severity of the subject's disease or condition, and the appropriate dosage may be determined by clinical trials. The pharmaceutical compositions can be formulated accordingly.

[0281] The cells, cell populations, or pharmaceutical compositions described herein may be administered parenterally (e.g., intravenously or intrathecally) or by infusion techniques. The cells, cell populations, or pharmaceutical compositions may be administered in the form of a sterile aqueous solution, which may contain other substances, for example, sufficient salts or glucose to make the solution isotonic with blood. The aqueous solution may be suitably buffered (adjusted to a pH of 3 to 9, if necessary). The pharmaceutical composition may be formulated accordingly. The preparation of suitable parenteral formulations under sterile conditions may be readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

[0282] The pharmaceutical composition may include cells in an infusion medium, such as a sterile isotonic solution, and may be enclosed in an ampoule, disposable syringe, or multiple dose vial made of glass or plastic.

[0283] The cells, cell populations, or pharmaceutical compositions may be administered in a single or multiple doses. In particular, the cells, cell populations, or pharmaceutical compositions may be administered in a single, one-off dose. The pharmaceutical compositions may be formulated accordingly.

[0284] Depending on the disease / condition and subject to be treated, and the route of administration, the cells, cell populations, or pharmaceutical compositions may be administered at a particular stage of the disease.

[0285] For example, in type 1 diabetes, pancreatic beta cells are destroyed and are unable to produce insulin. Therefore, the optimal time to administer the cells, cell populations, or pharmaceutical compositions of the present invention may be in the early stages of the disease before all pancreatic beta cells are destroyed, in order to maintain at least some functioning pancreatic beta cells (with residual pancreatic beta cell function) and maintain insulin production. In particular, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of pancreatic beta cells may be present upon administration of the cells, cell populations, or pharmaceutical compositions described herein. Alternatively, less than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of pancreatic beta cells may be destroyed before administration of the cells, cell populations, or pharmaceutical compositions described herein.

[0286] The pharmaceutical composition may further comprise one or more other active agents, such as lymphodepleting agents (e.g., thymoglobulin, campath-1H, anti-CD2 antibodies, anti-CD3 antibodies, anti-CD20 antibodies, cyclophosphamide, fludarabine), mTOR inhibitors (e.g., sirolimus, everolimus), agents that inhibit costimulatory pathways (e.g., anti-CD40 / CD40L, CTAL4Ig), and / or agents that inhibit specific cytokines (IL-6, IL-17, TNFα, IL18).

[0287] Depending on the disease / condition and subject to be treated, and the route of administration, the cells, cell populations, or pharmaceutical compositions can be administered at various dosages (e.g., measured in cells / kg or cells / subject). In any case, the physician will determine the actual dosage most suitable for each subject, and the dosage will vary depending on the age, weight, and response of the particular subject. However, typically, for the cells herein, 5×10 7 ~3×10 9 cells / subject, or 10 8 ~2×10 9 It can be administered in cell / subject doses.

[0288] The cells may be suitably modified to make them suitable for use in a pharmaceutical composition, for example, the cells may be cryopreserved and thawed at an appropriate time before being infused into a subject.

[0289] The invention further includes the use of kits comprising the cells, cell populations, and / or pharmaceutical compositions described herein. Optionally, the kits are for use in the methods and uses described herein, e.g., the therapeutic methods described herein. Optionally, the kits include instructions for use of the kit components.

[0290] The cells, cell populations, and pharmaceutical compositions of the invention may be particularly useful in treating disorders associated with cells expressing ENTPD3 or in which ENTPD3 is localized at or near the disease site, particularly disorders that would benefit from the immunosuppressive or targeted killing activity of the cells of the invention.

[0291] The cells, cell populations, compositions, and vectors described herein may be used to treat, prevent, or reduce the risk of a disease or condition in a subject, particularly a disease or condition that can be treated by or using a CAR. The cells and compositions containing them are intended for adoptive cell therapy (ACT). A variety of conditions can be treated by administering cells expressing a CAR according to the present disclosure, including Treg cells in particular. As described above, these conditions may be responsive to immunosuppression, particularly to the immunosuppressive effects of Treg cells. Thus, the cells, cell populations, compositions, and vectors described herein may be used to induce or achieve immunosuppression in a subject. The administered Treg cells, or in vivo engineered Treg cells, may be targeted by expression of a CAR. Conditions suitable for such treatment include autoimmune or inflammatory diseases (e.g., type 1 diabetes), and more broadly, any condition associated with an undesired, unwanted, or harmful immune response. Furthermore, the cells, cell populations, compositions, and vectors described herein may be used to promote tissue repair and / or tissue regeneration.

[0292] The conditions to be treated or prevented include inflammation, or alternatively, conditions associated with or involving inflammation. The inflammation may be chronic or acute. Furthermore, the inflammation may be low-level inflammation or systemic inflammation.

[0293] The term "target cell" refers to any cell expressing ENTPD3 to which the cells of the present invention are directed to exert their therapeutic effect. In some embodiments, the target cell serves as a marker for the site of disease, i.e., to attract the cells of the present invention to exert their immunosuppressive effect. In some embodiments, the target cell is killed or neutralized by the cells of the present invention. As noted above, in some embodiments, the target cell is a pancreatic beta cell.

[0294] In particular, the disease or disorder to be treated may be type 1 diabetes. Other diseases or disorders that can be treated using the CARs described herein include, for example, autoimmune pancreatitis, type 2 diabetes, and insulinoma. In particular, CARs can be expressed in cells with immunosuppressive function (e.g., CD4+ or CD8+ T regulatory cells, tolerogenic NK cells or NKT cells, gamma delta cells, and immunoregulatory 1 cells (Tr1), as well as other cells that secrete immunomodulatory cytokines such as IL-10, TGFβ, IL-35, or amphiregulin) for the treatment of type 1 diabetes, autoimmune pancreatitis, or type 2 diabetes. CARs can be expressed in cells with effector function (e.g., T effector cells, NK cells, NKT cells) for the treatment of insulinoma.

[0295] Furthermore, the CARs described herein may be used to prevent rejection of transplanted cells expressing ENTPD3 (e.g., allogeneic islet transplants, xenogeneic islet transplants, and beta cell replacement therapies, including stem cell-derived beta cells). In particular, CARs can be expressed in cells with immunosuppressive function (e.g., CD4+ or CD8+ T regulatory cells, tolerogenic NK cells or NKT cells, gamma delta cells, and immunoregulatory 1 cells (Tr1), as well as other cells that secrete immunomodulatory cytokines such as IL-10, TGFβ, IL-35, or amphiregulin) to prevent rejection of transplanted cells expressing ENTPD3.

[0296] The engineered cells, e.g., Tregs, may be administered to a subject with a disease to alleviate, reduce, or ameliorate at least one symptom of the disease, e.g., hyperglycemia. The at least one symptom may be alleviated, reduced, or ameliorated by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%. Alternatively, the at least one symptom may be completely alleviated.

[0297] Engineered cells, such as Tregs, may be administered to a subject with a disease to slow, reduce, or prevent disease progression. The progression of the disease may be slowed, reduced, or prevented by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a subject not receiving the engineered cells. Alternatively, the progression of the disease may be halted entirely.

[0298] In particular, the disease to be treated may be type 1 diabetes. As described above, ENTPD3-specific CAR-Tregs may be able to slow the rate of pancreatic beta cell destruction by migrating to sites of ENTPD3 expression and controlling inflammation through a bystander effect.

[0299] Type 1 diabetes is a chronic autoimmune disease in which the immune system destroys pancreatic beta cells, which are responsible for producing insulin. This is caused by genetic and environmental factors. The destruction of beta cells reduces or eliminates insulin production in the body and causes inflammation in the pancreatic islets. Insulin is a hormone necessary for regulating glucose levels in the bloodstream, and before treatment, subjects with type 1 diabetes have excessively high blood sugar levels (hyperglycemia). Type 1 diabetes is a serious and lifelong condition. Currently, type 1 diabetes patients must carefully monitor their blood sugar levels and administer appropriate amounts of insulin (e.g., via injections or pumps). This treatment is not a cure and must be administered continuously. Over time, irregular blood sugar levels (e.g., wide fluctuations in blood sugar levels) can lead to long-term complications such as damage to the heart, eyes, feet, and kidneys, as well as reduced life expectancy. It has been noted that glycemic control is particularly poor in younger subjects, particularly those approximately 16 to 25 years of age; therefore, the treatments described herein may be particularly useful for this population.

[0300] Type 1 diabetes is a continuum of disease that progresses sequentially at variable but predictable rates through distinct, identifiable stages before symptoms appear. This is described in more detail in Insel et al, Diabetes Care. 2015; 38(10): 1964-1974. The ability to screen for risk and determine the stage of type 1 diabetes before symptomatic type 1 diabetes develops offers the opportunity for early intervention to delay and ultimately prevent the onset of clinical symptoms.

[0301] Individuals at high risk for developing type 1 diabetes can be identified through genetic screening. The HLA region on chromosome 6 accounts for approximately 30-50% of the genetic risk for type 1 diabetes, with the highest association with the HLA class II haplotypes RB1*0301-DQB1*0201 (DR3-DQ2) and DRB1*0401-DQB1*0302 (DR4-DQ8). The remaining genetic risk for type 1 diabetes is thought to be attributable to approximately 50 non-HLA genes or loci identified through candidate gene and genome-wide association study approaches. The largest contributors among non-HLA genes are the INS, PTPN22, CTLA4, and IL2RA genes. Individuals identified as at high risk for type 1 diabetes but who have not yet developed any signs of disease (i.e., before stage 1) can be prophylactically administered the CAR-Tregs described herein.

[0302] Stage 1 refers to individuals who have acquired two or more type 1 diabetes-associated islet autoantibodies (against insulin, GAD65, IA-2, and / or ZnT8) but have normal blood glucose levels.

[0303] Stage 2 refers to individuals who have acquired two or more of the type 1 diabetes-associated islet autoantibodies (anti-insulin, GAD65, IA-2, and / or ZnT8) but whose disease has progressed due to a loss of functional pancreatic beta-cell mass, resulting in impaired glucose tolerance or dysglycemia. Dysglycemia can be defined as fasting plasma glucose levels of 5.6 mmol / L or higher, or plasma glucose levels of 7.8 mmol / L or higher 2 hours after a 75-g oral glucose tolerance test (OGTT), elevated blood glucose levels at midpoint of the OGTT (30, 60, and 90-minute levels of 11.1 mmol / L or higher), and / or HbA1c levels of 5.7% (39 mmol / mol) or higher.

[0304] Stage 3 refers to an individual who exhibits typical clinical symptoms and signs of diabetes, including, for example, polyuria, polydipsia, weight loss, fatigue, and diabetic ketoacidosis (DKA).

[0305] The CAR-Tregs described herein may be used, for example, to treat subjects with stage 1, stage 2, and / or stage 3 disease. Alternatively, the CAR-Tregs described herein may be used to treat subjects at risk for T1D, i.e., pre-stage 1.

[0306] If a subject has stage 3 disease, CAR-Treg therapy should be initiated as early as possible to minimize pancreatic beta cell destruction and maximize preservation of remaining pancreatic beta cell function, which can be determined, for example, by measuring the subject's blood insulin or C-peptide levels. For example, a subject may be required to have a stimulated C-peptide level of at least 0.2 pmol / mL at the time of CAR-Treg administration, or a stimulated C-peptide level of at least 0.4 pmol / mL, e.g., obtained by a mixed meal challenge. Such a subject may be considered to have "recent-onset type 1 diabetes." For example, if a subject has stage 3 disease, CAR-Treg therapy can be initiated within about 24 weeks of symptom onset or diagnosis. For example, it may be initiated within about 20 weeks, about 16 weeks, about 15 weeks, about 14 weeks, about 12 weeks, about 8 weeks, or about 6 weeks of symptom onset or diagnosis. For example, CAR-Treg therapy may be initiated within 100 days of symptom onset or diagnosis.

[0307] The subject may be, for example, of any age. For example, the subject may be under 30, under 25, under 20, under 18, or under 16. In particular, the subject may be between 8 and 30 years old, particularly between 8 and 25 years old, between 8 and 16 years old, or between 16 and 25 years old.

[0308] Disease progression and pancreatic beta cell death, for example, can be monitored by several methods.

[0309] For example, pancreatic β cell imaging can be used to visualize the integrity of β cell.For example, CAR-Treg as described herein can maintain or increase the number of pancreatic β cells present in subject after administration.This can be observed, for example, when subject is administered with reduced dose of exogenous insulin or when subject is not administered with exogenous insulin.

[0310] For example, the subject's insulin concentration can be monitored. A normal fasting blood insulin concentration (blood insulin concentration after a subject has fasted (has eaten or drunk nothing but water) for at least 8 hours) is considered to be approximately 2-20 mIU / mL. For example, after administration to a subject, the CAR-Tregs described herein may maintain or increase the subject's fasting blood insulin concentration, e.g., within the normal range for fasting blood insulin. This may be observed, for example, when the subject is receiving a reduced dose of exogenous insulin or is not receiving exogenous insulin.

[0311] Insulin cell-free DNA (cfDNA) and unmethylated insulin can also be detected.

[0312] A subject's C-peptide concentration can be monitored. Pancreatic beta cells first produce a protein called "proinsulin." Each proinsulin is broken down into one molecule of insulin and one molecule of C-peptide. Both are released when blood glucose levels rise. Insulin and C-peptide are released in equal amounts, but they are broken down differently. Therefore, C-peptide can be used as a surrogate marker of beta-cell function. While the liver breaks down insulin at variable rates, the kidneys break down C-peptide at a fairly stable rate. Therefore, C-peptide may be a more reliable measure of insulin production and beta-cell function. Normal fasting (i.e., after at least 8 hours of fasting) C-peptide concentrations are thought to be approximately 0.8-3.85 ng / mL. For example, after administration to a subject, the CAR-Tregs described herein may maintain or increase the subject's fasting C-peptide concentration, e.g., within the normal fasting C-peptide range. This can be observed, for example, when the subject is receiving reduced doses of exogenous insulin or no exogenous insulin.

[0313] Additionally, the subject's blood glucose level can be monitored. Normal fasting blood glucose levels (i.e., after at least 8 hours of fasting) are considered to be approximately 3.9 to 6.9 mmol / L. Alternatively, or in addition, an oral glucose tolerance test can be performed to measure blood glucose levels (e.g., using 75 g of oral glucose). Normal blood glucose levels two hours after oral glucose administration are considered to be less than approximately 7.8 mmol / L. At intermediate time points before the two-hour mark (e.g., 30 minutes, 60 minutes, 90 minutes), normal blood glucose levels are considered to be less than approximately 11.1 mmol / L. For example, after administration to a subject, the CAR-Tregs described herein may maintain or increase the subject's fasting blood glucose level or OGTT blood glucose level, e.g., within the normal range for fasting blood glucose levels or OGTT blood glucose levels. This may be observed, for example, when the subject is receiving a reduced dose of exogenous insulin or is not receiving exogenous insulin.

[0314] A subject's hemoglobin (Hb) HbA1c level can be monitored. HbA1c is produced by the binding of glucose to hemoglobin and is a measure of average blood glucose levels over the past two to three months. A normal HbA1c level is considered to be approximately 4.0-5.6% (20-38 mmol / mol). For example, after administration to a subject, the CAR-Tregs described herein may maintain or increase the subject's HbA1c level, e.g., within the normal HbA1c range. This may be observed, for example, when the subject is receiving a reduced dose of exogenous insulin or is not receiving exogenous insulin.

[0315] Preferably, the subject is a mammal. Preferably, the subject is a human.

[0316] Suitably, the cells may be engineered Treg cells and the cell population may be a population of engineered Treg cells, which Treg cells have been engineered to express a CAR as described herein.

[0317] Suitably, the CAR may comprise an antigen-binding domain capable of specifically binding to ENTPD3, i.e., the antigen is ENTPD3.

[0318] Methods for treating diseases or conditions relate to therapeutic uses of the cells herein, and in this regard, the cells may be administered to a subject with an existing disease or condition to alleviate, reduce, or ameliorate at least one symptom associated with the disease or condition, and / or to slow, reduce, or prevent the progression of the disease.

[0319] Suitably, treating and / or preventing an autoimmune or inflammatory disease may refer to administering an effective amount of the cells (e.g., Tregs) such that a subject with the disease requires a reduced amount of an existing medication (e.g., exogenous insulin), or may be able to discontinue administration of the existing medication to the subject.

[0320] Preventing disease or condition refers to the prophylactic use of cells herein.In this regard, the cells can be administered to a subject who has not yet suffered from or developed the disease or condition and / or has not yet shown any symptoms of the disease or condition, in order to prevent the disease or condition, or to reduce or prevent the onset of at least one symptom associated with the disease or condition.The subject can be predisposed to the disease or condition, or can be considered to be at risk of developing the disease or condition (for example, type 1 diabetes before stage 1).

[0321] As used herein, the term "treatment" refers to a clinical intervention aimed at changing the natural course of a clinical pathological process in a treated individual. The desired effects of treatment include slowing the rate of progression, improving or alleviating the pathological state, and remission or improving the prognosis of a particular disease, disorder, or condition. An individual is considered to be effectively "treated" if, for example, one or more symptoms associated with a particular disease, disorder, or condition are alleviated or eliminated.

[0322] An "effective amount" refers to at least an amount that is effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. An effective amount can be provided in one or more administrations.

[0323] A "therapeutically effective amount" is at least the minimum concentration required to bring about a measurable improvement in a particular disease, disorder, or condition. As used herein, a therapeutically effective amount may vary depending on factors such as the patient's condition, age, sex, and weight, as well as the ability of the chimeric receptor to induce a desired response in an individual. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the cell, cell population, or pharmaceutical composition are outweighed by the therapeutically beneficial effects.

[0324] The terms "subject," "patient," and "individual" are used interchangeably herein and refer to a mammal, optionally a human. In particular, the terms subject, patient, and individual refer to a human having a disease or disorder as defined herein and in need of treatment.

[0325] In some embodiments of the invention, the patient may receive other therapies before, concurrently with, or after a treatment of the invention. For example, in some embodiments, the patient may be treated with other treatments for the treatment of symptoms associated with the disease or disorder.

[0326] The medical uses or methods herein may comprise the following steps: (i) isolating or providing a cell-containing sample; (ii) introducing a nucleic acid molecule, construct, or vector defined herein into a cell; and (iii) A step of administering the cells obtained in (ii) to a subject. The cells may be Tregs as defined herein.Before and / or after step (ii) of the method, an enriched Treg population may be isolated and / or generated from the cell-containing sample.For example, before and / or after step (ii), isolation and / or generation may be performed to isolate and / or generate an enriched Treg sample.Enrichment may be performed after step (ii) to enrich for cells and / or Tregs comprising the CAR, polynucleotide, and / or vector described herein. Preferably, the cells may be autologous cells. Preferably, the cells may be allogeneic cells. Preferably, the cells (e.g., engineered Tregs) may be administered in combination with one or more other therapeutic agents, such as lymphodepleting agents (e.g., those described above). The engineered cells, e.g., Tregs, may be administered simultaneously with the one or more other therapeutic agents or sequentially (i.e., before or after the one or more other therapeutic agents). The cells, e.g., Tregs, may be activated and / or expanded before or after introduction of the nucleic acid molecules described herein, e.g., by treatment with anti-CD3 monoclonal antibody, or with both anti-CD3 and anti-CD28 monoclonal antibodies. Expansion protocols are as described above. The cells, eg Tregs, may be washed after each step of the method, particularly after expansion. The population of engineered cells, eg, Treg cells, may be further enriched by any method known to one of skill in the art, eg, FACS or magnetic bead separation. Each step of the above production method may be carried out in a closed, sterile cell culture system.

[0327] The present invention may also provide a method for increasing the stability and / or suppressive function of cells, comprising introducing the nucleic acid molecule, expression construct, or vector provided herein into cells. The increase in suppressive 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 measuring the level of cytokines produced by the Tconv cells. The increase in suppressive function can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% compared to unmanipulated Tregs.

[0328] As defined herein, increased stability of a cell, e.g., a Treg, refers to an increase in the persistence or survival of the cell, or an increase in the proportion of cells that retain the Treg phenotype over a period of time (e.g., the proportion of cells that retain Treg markers such as FOXP3 and Helios), compared to unmanipulated Tregs. The increase in stability can be at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% increase in stability and can be measured by techniques known in the art, such as staining for Treg cell markers within the cell population and analysis by FACS.

[0329] The present invention also provides the use of CAR-Treg to reduce or prevent the cell death of pancreatic β cells, for example, in subjects with type 1 diabetes or subjects at risk of developing it, particularly subjects with recently developed type 1 diabetes.This can be determined, for example, by measuring the number of pancreatic β cells, blood insulin concentration, and / or blood C-peptide concentration in the subject.If the number of pancreatic β cells, blood insulin concentration, and / or blood C-peptide concentration are maintained or improved, it can be considered that the cell death rate of pancreatic β cells is reduced or cell death is prevented.This can be observed, for example, when the subject is administered with a reduced dose of exogenous insulin or when the subject is not administered with exogenous insulin.

[0330] Thus, the present invention also provides for the use of CAR-Tregs to maintain or increase fasting blood insulin and / or C-peptide levels, for example, in subjects with or at risk of developing type 1 diabetes, particularly subjects with recent-onset type 1 diabetes, which may be observed, for example, when the subject is receiving reduced doses of exogenous insulin or is not receiving exogenous insulin.

[0331] The present invention further provides the use of CAR-Treg to reduce or prevent hyperglycemia, for example, in subjects with type 1 diabetes or subjects at risk of developing it, particularly in subjects with recently developed type 1 diabetes.This can be determined, for example, by measuring the fasting blood glucose level and / or HbA1c level in the subject.If the blood glucose level and / or HbA1c level in the subject is maintained or improved, it can be considered that the hyperglycemia in the subject is reduced or prevented.This can be observed, for example, when the subject is administered with a reduced dose of exogenous insulin or when the subject is not administered with exogenous insulin.

[0332] Thus, the present invention also provides for the use of CAR-Tregs to maintain or reduce fasting blood glucose and / or HbA1c levels, for example, in subjects with or at risk of developing type 1 diabetes, particularly in subjects with recent-onset type 1 diabetes, which may be observed, for example, when the subject is receiving reduced doses of exogenous insulin or is not receiving exogenous insulin.

[0333] The CAR is a CAR of the present invention, i.e., the CAR comprises an antigen recognition domain that specifically binds to ENTPD3 (e.g., human ENTPD3), and can further have any of the characteristics of a CAR disclosed herein.

[0334] Those skilled in the art will understand that the scFv identified in the present invention can also be used in formats other than CAR structures. Thus, in a further aspect of the present invention, an antibody or antibody fragment that specifically binds to ENTPD3 is provided. The antibody or antibody fragment may, in particular, be an scFv. The antibody or antibody fragment, for example, an scFv, may comprise the CDR sequences, VH and VL sequences, or scFv sequences defined above for the antigen-binding domain of CAR. Thus, all sequences described above in relation to CDRs, VH and VL, and scFvs may also be included in the antibody or antibody fragment of this embodiment. In particular, the antibody or antibody fragment may comprise: (a) VH CDRs set forth in SEQ ID NOs: 1 to 3 and VL CDRs set forth in SEQ ID NOs: 4 to 6; (b) VH CDRs set forth in SEQ ID NOs: 7 to 9 and VL CDRs set forth in SEQ ID NOs: 10 to 12; (c) VH CDRs set forth in SEQ ID NOs: 13 to 15 and VL CDRs set forth in SEQ ID NOs: 16 to 18; (d) VH CDRs set forth in SEQ ID NOs: 19 to 21 and VL CDRs set forth in SEQ ID NOs: 22 to 24; (e) VH CDRs set forth in SEQ ID NOs: 25 to 27 and VL CDRs set forth in SEQ ID NOs: 28 to 30; (f) VH CDRs set forth in SEQ ID NOs: 31 to 33 and VL CDRs set forth in SEQ ID NOs: 34 to 36; (g) VH CDRs set forth in SEQ ID NOs: 37 to 39 and VL CDRs set forth in SEQ ID NOs: 40 to 42; (h) VH CDRs set forth in SEQ ID NOs: 43 to 45 and VL CDRs set forth in SEQ ID NOs: 46 to 48; (i) VH CDRs set forth in SEQ ID NOs: 49 to 51 and VL CDRs set forth in SEQ ID NOs: 52 to 54; (j) VH CDRs set forth in SEQ ID NOs: 55 to 57 and VL CDRs set forth in SEQ ID NOs: 58 to 60; (k) VH CDRs set forth in SEQ ID NOs: 61 to 63 and VL CDRs set forth in SEQ ID NOs: 64 to 66; (l) VH CDRs set forth in SEQ ID NOs: 67 to 69 and VL CDRs set forth in SEQ ID NOs: 70 to 72; (m) the VH CDRs set forth in SEQ ID NOs: 73 to 75 and the VL CDRs set forth in SEQ ID NOs: 76 to 78; or (n) VH CDRs set forth in SEQ ID NOs: 79 to 81 and VL CDRs set forth in SEQ ID NOs: 82 to 84; Alternatively, the CDRs may comprise one to three, more particularly one or two, amino acid sequence modifications in any of the above sequences.

[0335] 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., E. coli). Alternatively, antibodies or antibody fragments may be prepared by chemical synthesis using techniques well known in protein chemistry, such as solid-phase synthesis or synthesis in homogeneous solution.

[0336] N- or C-terminal fusion proteins may be made comprising the antibodies or antibody fragments defined herein, or alternatively, the antibodies may be conjugated to another molecule, such as a therapeutic or detectable molecule. Thus, the present invention further encompasses the antibodies or antibody fragments defined above conjugated to one or more additional molecules, such as immunoglobulins, hormones, growth factors, lectins, insulin, low-density lipoproteins, glucagon, endorphins, transferrin, tags, fluorescent dyes, radioisotopes, or therapeutic molecules (e.g., immunosuppressants).

[0337] Nucleic acid molecules comprising a nucleotide sequence encoding an antibody, antibody fragment, or fusion protein as defined herein are also provided, as are vectors and cells comprising the nucleic acid molecules.

[0338] These antibodies, antibody fragments, and fusion proteins / conjugates can be used to detect, identify, and / or image cells expressing ENTPD3 (e.g., imaging pancreatic islet masses or detecting insulinomas), and can also be used to deliver therapeutic molecules to target sites (e.g., cells expressing ENTPD3).

[0339] Therefore, in this regard, the present invention provides a method for detecting or imaging cells expressing ENTPD3, particularly pancreatic islet cells, comprising incubating the cells with an antibody, antibody fragment, or fusion protein / conjugate comprising the antibody or antibody fragment as defined herein, and determining whether the antibody, antibody fragment, or fusion protein / conjugate binds to the cells. From another perspective, the present invention provides a method for detecting or imaging cells expressing ENTPD3 in a subject, comprising administering to the subject an antibody, antibody fragment, or fusion protein / conjugate comprising the antibody or antibody fragment as defined herein, and determining or detecting the binding of the antibody, antibody fragment, or fusion protein / conjugate to the cells. In particular, detection may be performed by fluorescence, in which case the antibody, antibody fragment, or fusion protein / conjugate may be fluorescently labeled. Other detectable labels may also be used.

[0340] Finally, the present invention provides a method for delivering a therapeutic molecule to a target site where ENTPD3 is present or expressed, comprising administering to a subject a fusion protein or conjugate comprising an antibody or antibody fragment of the present invention and a therapeutic molecule.

[0341] The disclosure is not limited to the exemplary methods and materials disclosed herein, and methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise specified, nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation.

[0342] Where a range of values ​​is provided, each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range, unless the context clearly dictates otherwise, is also understood to be expressly disclosed. Each narrower range between any stated or intervening value in a stated range and any other stated or intervening value in that stated range is also encompassed within the disclosure. The upper and lower limits of these narrower ranges may each independently be included or excluded, and ranges where either, neither, or both limits are included in the narrower ranges are also encompassed within the disclosure. However, any explicitly excluded limit in a stated range is intended to be governed. When a stated range includes one or both limits, ranges excluding either or both included limits are also included within the disclosure.

[0343] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0344] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including," "includes," or "containing," and "contains," and are inclusive or open-ended and do not exclude additional, unrecited elements, elements, or method steps. The terms "comprising," "comprises," and "comprised of" also include the term "consisting of."

[0345] The publications referenced herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that such publications constitute prior art to the appended claims.

[0346] Further embodiments of the present invention are set forth in the following embodiments 1-49. 1. A chimeric antigen receptor (CAR) containing an antigen recognition domain that specifically binds to ENTPD3. 2. The CAR of embodiment 1, wherein the antigen recognition domain binds to human ENTPD3. 3.a. an exodomain comprising an antigen recognition domain; b. a transmembrane domain; c. An endodomain comprising an intracellular signaling domain. 4. The CAR of embodiment 3, further comprising a hinge domain and / or one or more costimulatory domains. 5. The CAR of embodiment 4, wherein the hinge domain is selected from CD28, CD8α, CD4, CD7, CH2CH3, an immunoglobulin hinge region, or a portion or variant thereof, and optionally, the CAR comprises a CD8α hinge region or a CH2CH3 hinge region. 6. The CAR of any one of embodiments 3 to 5, wherein the CAR comprises one or more transmembrane domains selected from the transmembrane domains of CD28, ICOS, CD8α, CD4, CD134 (OX40), CD137 (4-1BB), CD3ζ, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD154, CH2CH3, or portions or variants thereof; optionally, the CAR comprises a CD4 transmembrane domain, a CD28 transmembrane domain, a CD8α transmembrane domain, or a CH2CH3 transmembrane domain. 7. The CAR of any one of embodiments 4 to 6, wherein the costimulatory domain is selected from the intracellular domain of CD28, ICOS, CD134 (OX40), CD137 (4-1BB), CD27, or TNFRSF25, or a portion or variant thereof; and optionally, the CAR comprises a CD28 costimulatory domain. 8. The CAR of any one of embodiments 3 to 7, wherein the CAR comprises one or more intracellular signaling domains selected from the group consisting of a CD3 zeta signaling domain or any of its homologs, a CD3 polypeptide, a syk family tyrosine kinase, a src family tyrosine kinase, CD2, CD5, CD28, or a portion or variant thereof, optionally wherein the CAR comprises a CD3 zeta signaling domain. 9. The CAR of 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 costimulatory domain, and a CD3ζ signaling domain, wherein if the hinge domain is CD8α, the transmembrane domain is CD8α, and if the hinge domain is CH2CH3, the transmembrane domain is CD28 or CH2CH3. 10. The CAR of any one of embodiments 3 to 9, wherein the CAR comprises a signal peptide and / or a reporter peptide. 11. The CAR of any preceding embodiment, wherein the antigen recognition domain is an antibody, an antibody fragment, or derived from an antibody. 12. The CAR of any preceding embodiment, wherein the antigen recognition domain is a single chain antibody (scFv). 13. The antigen recognition domain is (i) VH CDR1 sequences, VH CDR2 sequences, and VH CDR3 sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and VL CDR1 sequences, VL CDR2 sequences, and VL CDR3 sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively; (ii) the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively; (iii) VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively, and VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively; (iv) VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively, and VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively; (v) VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 25, 26, and 27, respectively, and VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively; (vi) the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence set forth in SEQ ID NOs: 31, 32, and 33, respectively, and the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence set forth in SEQ ID NOs: 34, 35, and 36, respectively; (vii) the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence set forth in SEQ ID NOs: 37, 38, and 39, respectively, and the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence set forth in SEQ ID NOs: 40, 41, and 42, respectively; (viii) the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence set forth in SEQ ID NOs: 43, 44, and 45, respectively, and the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence set forth in SEQ ID NOs: 46, 47, and 48, respectively; (ix) VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively, and VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 52, 53, and 54, respectively; (x) VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 55, 56, and 57, respectively, and VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 58, 59, and 60, respectively; (xi) VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 61, 62, and 63, respectively, and VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 64, 65, and 66, respectively; (xii) the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence set forth in SEQ ID NOs: 67, 68, and 69, respectively, and the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence set forth in SEQ ID NOs: 70, 71, and 72, respectively; (xiii) the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 73, 74, and 75, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 76, 77, and 78, respectively; or (xiv) comprising the VH CDR1 sequence, the VH CDR2 sequence, and the VH CDR3 sequence set forth in SEQ ID NOs: 79, 80, and 81, respectively, and the VL CDR1 sequence, the VL CDR2 sequence, and the VL CDR3 sequence set forth in SEQ ID NOs: 82, 83, and 84, respectively; The CAR of any preceding embodiment, wherein one or more of the CDR sequences (i) to (xiv) above may optionally comprise one to three amino acid modifications relative to the CDR sequence, and in particular, one or more of the CDR sequences may optionally be modified by substitution, addition, or deletion of one to three amino acids. 14. The antigen recognition domain is (i) a VH domain comprising the sequence set forth in SEQ ID NO: 85, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 86, or a sequence having at least 70% sequence identity thereto; (ii) a VH domain comprising the sequence set forth in SEQ ID NO: 87, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 88, or a sequence having at least 70% sequence identity thereto; (iii) a VH domain comprising the sequence set forth in SEQ ID NO: 89, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 90, or a sequence having at least 70% sequence identity thereto; (iv) a VH domain comprising the sequence set forth in SEQ ID NO: 91, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 92, or a sequence having at least 70% sequence identity thereto; (v) a VH domain comprising the sequence set forth in SEQ ID NO: 93, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 94, or a sequence having at least 70% sequence identity thereto; (vi) a VH domain comprising the sequence set forth in SEQ ID NO: 95, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 96, or a sequence having at least 70% sequence identity thereto; (vii) a VH domain comprising the sequence set forth in SEQ ID NO: 97, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 98, or a sequence having at least 70% sequence identity thereto; (viii) a VH domain comprising the sequence set forth in SEQ ID NO: 99, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 100, or a sequence having at least 70% sequence identity thereto; (ix) a VH domain comprising the sequence set forth in SEQ ID NO: 101, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 102, or a sequence having at least 70% sequence identity thereto; (x) a VH domain comprising the sequence set forth in SEQ ID NO: 103, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 104, or a sequence having at least 70% sequence identity thereto; (xi) a VH domain comprising the sequence set forth in SEQ ID NO: 105, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 106, or a sequence having at least 70% sequence identity thereto; (xii) a VH domain comprising the sequence set forth in SEQ ID NO: 107, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 108, or a sequence having at least 70% sequence identity thereto; (xiii) a VH domain comprising the sequence set forth in SEQ ID NO: 109, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 110, or a sequence having at least 70% sequence identity thereto; or (xiv) The CAR of any preceding embodiment, comprising a VH domain comprising the sequence set forth in SEQ ID NO: 111, or a sequence with at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 112, or a sequence with at least 70% identity thereto. 15. The antigen recognition domain is (i) a VH domain comprising a sequence encoded by SEQ ID NO: 205, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 205, and a VL domain comprising a sequence encoded by SEQ ID NO: 206, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 206; (ii) a VH domain comprising a sequence encoded by SEQ ID NO: 207, or a sequence having at least 70% identity to 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 to the sequence encoded by SEQ ID NO: 208; (iii) a VH domain comprising a sequence encoded by SEQ ID NO: 209, or a sequence having at least 70% identity to the 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 to the sequence encoded by SEQ ID NO: 210; (iv) a VH domain comprising a sequence encoded by SEQ ID NO:211, or a sequence having at least 70% identity to the 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 to the sequence encoded by SEQ ID NO:212; (v) a VH domain comprising a sequence encoded by SEQ ID NO:213, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO:213, and a VL domain comprising a sequence encoded by SEQ ID NO:214, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO:214; (vi) a VH domain comprising a sequence encoded by SEQ ID NO: 215, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 215, and a VL domain comprising a sequence encoded by SEQ ID NO: 216, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 216; (vii) a VH domain comprising a sequence encoded by SEQ ID NO: 217, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 217, and a VL domain comprising a sequence encoded by SEQ ID NO: 218, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 218; (viii) a VH domain comprising a sequence encoded by SEQ ID NO: 219, or a sequence having at least 70% identity to 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 to the sequence encoded by SEQ ID NO: 220; (ix) a VH domain comprising a sequence encoded by SEQ ID NO: 221, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO: 221, and a VL domain comprising a sequence encoded by SEQ ID NO: 222, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO: 222; (x) a VH domain comprising a sequence encoded by SEQ ID NO: 223, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 223, and a VL domain comprising a sequence encoded by SEQ ID NO: 224, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 224; (xi) a VH domain comprising a sequence encoded by SEQ ID NO: 225, or a sequence having at least 70% identity to 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 to the sequence encoded by SEQ ID NO: 226; (xii) a VH domain comprising a sequence encoded by SEQ ID NO: 227, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 227, and a VL domain comprising a sequence encoded by SEQ ID NO: 228, or a sequence having at least 70% identity to the sequence encoded by SEQ ID NO: 228; (xiii) a VH domain comprising a sequence encoded by SEQ ID NO: 229, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO: 229, and a VL domain comprising a sequence encoded by SEQ ID NO: 230, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO: 230; or (xiv) The CAR of any preceding embodiment, comprising a VH domain comprising a sequence encoded by SEQ ID NO: 231, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO: 231, and a VL domain comprising a sequence encoded by SEQ ID NO: 232, or a sequence with at least 70% identity to the sequence encoded by SEQ ID NO: 232. 16. The antigen recognition domain is (i) the sequence set forth in SEQ ID NO: 113, or a sequence having at least 80% sequence identity thereto; (ii) the sequence set forth in SEQ ID NO: 114, or a sequence having at least 80% sequence identity thereto; (iii) the sequence set forth in SEQ ID NO: 115, or a sequence having at least 80% sequence identity thereto; (iv) the sequence set forth in SEQ ID NO: 116, or a sequence having at least 80% sequence identity thereto; (v) the sequence set forth in SEQ ID NO: 117, or a sequence having at least 80% sequence identity thereto; (vi) the sequence set forth in SEQ ID NO: 118, or a sequence having at least 80% sequence identity thereto; (vii) the sequence set forth in SEQ ID NO: 119, or a sequence having at least 80% sequence identity thereto; (viii) the sequence set forth in SEQ ID NO: 120, or a sequence having at least 80% sequence identity thereto; (ix) the sequence set forth in SEQ ID NO: 121, or a sequence having at least 80% sequence identity thereto; (x) the sequence set forth in SEQ ID NO: 122, or a sequence having at least 80% sequence identity thereto; (xi) the sequence set forth in SEQ ID NO: 123, or a sequence having at least 80% sequence identity thereto; (xii) the sequence set forth in SEQ ID NO: 124, or a sequence having at least 80% sequence identity thereto; (xiii) the sequence set forth in SEQ ID NO: 125, or a sequence having at least 80% sequence identity thereto; or (xiv) The CAR of any preceding embodiment, comprising or consisting of the sequence set forth in SEQ ID NO: 126, or a sequence having at least 80% sequence identity thereto. 17. The antigen recognition domain is (i) a sequence encoded by the sequence set forth in SEQ ID NO: 191 or a sequence having at least 80% identity to the sequence encoded by the sequence set forth in SEQ ID NO: 191; (ii) a sequence encoded by the sequence set forth in SEQ ID NO: 192 or a sequence having at least 80% identity to the sequence encoded by the sequence set forth in SEQ ID NO: 192; (iii) a sequence encoded by the sequence set forth in SEQ ID NO: 193 or a sequence having at least 80% identity to the sequence encoded by the sequence set forth in SEQ ID NO: 193; (iv) a sequence encoded by the sequence set forth in SEQ ID NO: 194 or a sequence having at least 80% identity to the sequence encoded by the sequence set forth in SEQ ID NO: 194; (v) a sequence encoded by the sequence set forth in SEQ ID NO: 195 or a sequence having at least 80% identity to the sequence encoded by the sequence set forth in SEQ ID NO: 195; (vi) a sequence encoded by the sequence set forth in SEQ ID NO: 196 or a sequence having at least 80% identity to the sequence encoded by the sequence set forth in SEQ ID NO: 196; (vii) a sequence encoded by the sequence set forth in SEQ ID NO: 197 or a sequence having at least 80% identity to the sequence encoded by the sequence set forth in SEQ ID NO: 197; (viii) a sequence encoded by the sequence set forth in SEQ ID NO: 198 or a sequence having at least 80% identity to the sequence encoded by the sequence set forth in SEQ ID NO: 198; (ix) a sequence encoded by the sequence set forth in SEQ ID NO: 199 or a sequence having at least 80% identity to the sequence encoded by the sequence set forth in SEQ ID NO: 199; (x) a sequence encoded by the sequence set forth in SEQ ID NO: 200 or a sequence having at least 80% identity to the sequence encoded by the sequence set forth in SEQ ID NO: 200; (xi) a sequence encoded by the sequence set forth in SEQ ID NO: 201 or a sequence having at least 80% identity to the sequence encoded by the sequence set forth in SEQ ID NO: 201; (xii) a sequence encoded by the sequence set forth in SEQ ID NO: 202 or a sequence having at least 80% identity to the sequence encoded by the sequence set forth in SEQ ID NO: 202; (xiii) a sequence encoded by the sequence set forth in SEQ ID NO: 203 or a sequence having at least 80% identity to the sequence encoded by the sequence set forth in SEQ ID NO: 203; or (xiv) The CAR of any preceding embodiment, comprising or consisting of a sequence encoded by, or having at least 80% identity to, the sequence set forth in SEQ ID NO: 204. 18. The CAR of any preceding embodiment, wherein the CAR comprises or consists of a sequence set forth in any one of SEQ ID NOs: 237-262, or a sequence having at least 80% identity thereto. 19. A nucleic acid molecule comprising a nucleotide sequence encoding a CAR of any preceding embodiment. 20. A vector comprising the nucleic acid molecule of embodiment 19. 21. The vector of embodiment 20, further comprising a nucleic acid molecule encoding a FOXP3 polypeptide. 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 embodiment 21. 23. The cell of embodiment 22, wherein the cell is a production host cell. 24. The cell of embodiment 22, wherein the cell is an immune cell, or a progenitor or precursor thereof, optionally a T cell or a precursor thereof, or a stem cell. 25. The cell of embodiment 22 or embodiment 24, wherein the cell is a regulatory T cell (Treg), or a precursor thereof, or an iPSC cell, and in particular, the cell further comprises an exogenous nucleic acid comprising a nucleotide sequence encoding a FOXP3 polypeptide. 26. A cell population comprising cells according to any one of embodiments 22, 24, or 25. 27. A cell population according to embodiment 26, comprising a plurality of cells according to any one of embodiment 22, embodiment 24, or embodiment 25, in particular a plurality of T cells according to embodiment 24, more particularly a plurality of Tregs according to embodiment 25. 28. The cell population of embodiment 27, wherein the clonality of the plurality of T cells, particularly Tregs, has not been modified ex vivo. 29. The cell population of embodiment 27 or embodiment 28, wherein the plurality of T cells, particularly Tregs, have polyclonal endogenous TCRs. 30. A pharmaceutical composition comprising a cell according to any one of embodiments 22, 24, or 25, a cell population according to any one of embodiments 26 to 29, or a vector according to embodiment 20 or embodiment 21. 31. A cell according to any one of embodiments 22, 24, or 25, a cell population according to any one of embodiments 26 to 29, or a pharmaceutical composition according to embodiment 30, for use in therapy. 32. The cell, cell population, or pharmaceutical composition for use according to embodiment 31, wherein said therapy is adoptive cell transfer therapy. 33. The cell of any one of embodiments 22, 24 or 25, the cell population of any one of embodiments 26 to 29, or the pharmaceutical composition of embodiment 30, for use in the treatment or prevention of an autoimmune or inflammatory disease, for use in inducing immunosuppression, or for use in promoting tissue repair and / or regeneration, in particular wherein the cell is a Treg. 34. The cell, cell population, or pharmaceutical composition for use according to embodiment 33, wherein said autoimmune or inflammatory disease is type 1 diabetes, for example recent type 1 diabetes. 35. A method for treating or preventing an autoimmune or inflammatory disease, such as type 1 diabetes, inducing immunosuppression, or promoting tissue repair and / or tissue regeneration, comprising administering a cell according to any one of embodiments 22, 24, or 25, in particular a Treg, a cell population according to any one of embodiments 26 to 29, or a pharmaceutical composition according to embodiment 30, in particular a pharmaceutical composition according to embodiment 30 comprising a Treg. 36. (i) isolating or providing a Treg-enriched cell sample from a subject; (ii) introducing into the Treg cells the nucleic acid molecule of embodiment 19 or the vector of embodiment 20 or embodiment 21; (iii) administering the Treg cells obtained in (ii) above to the subject. 37. Use of a cell according to any one of embodiments 22, 24, or 25, a cell population according to any one of embodiments 26 to 29, or a pharmaceutical composition according to embodiment 30, in the manufacture of a medicament for treating or preventing an autoimmune or inflammatory disease, such as type 1 diabetes, for inducing immunosuppression, or for promoting tissue repair and / or tissue regeneration in a subject, particularly wherein the cell is a Treg. 38. A method for producing a cell according to any one of embodiments 22, 24, or 25, comprising introducing into the cell (e.g., transducing or transfecting into the cell) a nucleic acid molecule according to embodiment 19 or a vector according to embodiment 20 or embodiment 21. 39. The method of embodiment 38, wherein the cells are Treg cells, and the method comprises isolating or providing a cell-containing sample comprising 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. 40. A cell obtained by the method according to embodiment 38 or embodiment 39. 41. A cell according to any one of embodiments 22, 24, or 25, a cell population according to any one of embodiments 26 to 29, or a pharmaceutical composition according to embodiment 30, for use in preventing cell death or reducing the rate of cell death of pancreatic beta cells in a subject. 42. A cell according to any one of embodiments 22, 24, or 25, a cell population according to any one of embodiments 26 to 29, or a pharmaceutical composition according to embodiment 30, for use in maintaining or increasing fasting blood insulin levels in a subject. 43. A cell according to any one of embodiments 22, 24, or 25, a cell population according to any one of embodiments 26 to 29, or a pharmaceutical composition according to embodiment 30, for use in maintaining or increasing fasting C-peptide levels in a subject. 44. A cell according to any one of embodiments 22, 24, or 25, a cell population according to any one of embodiments 26 to 29, or a pharmaceutical composition according to embodiment 30, for use in reducing or preventing hyperglycemia in a subject. 45. A cell according to any one of embodiments 22, 24, or 25, a cell population according to any one of embodiments 26 to 29, or a pharmaceutical composition according to embodiment 30, for use in maintaining or reducing fasting blood glucose levels in a subject. 46. ​​A cell according to any one of embodiments 22, 24, or 25, a cell population according to any one of embodiments 26 to 29, or a pharmaceutical composition according to embodiment 30, for use in maintaining or reducing HbA1c levels in a subject. 47. The cell of any one of embodiments 22, 24, or 25, the cell population of any one of embodiments 26 to 29, or the pharmaceutical composition of 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, the subject has recent-onset type 1 diabetes. 48. The cell of any one of embodiments 22, 24, or 25, the cell population of any one of embodiments 26 to 29, or the pharmaceutical composition of embodiment 30, for use according to any one of embodiments 41 to 47, wherein the subject is not receiving exogenous insulin. 49. The cell of any one of embodiments 22, 24, or 25, the cell population of any one of embodiments 26 to 29, or the pharmaceutical composition of 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 dose of insulin administered before administration of the CAR-Tregs.

[0347] The present invention will now be further described by way of examples. These examples are intended to assist those skilled in the art in practicing the present invention and are not intended to limit the scope of the present invention in any way.

[0348] Sequence Listing SEQ ID NO: 1 is the amino acid sequence of VH CDR1 of A8 GFTFDDYA SEQ ID NO: 2 is the amino acid sequence of VH CDR2 of A8 ISWNSGSI SEQ ID NO: 3 is the amino acid sequence of VH CDR3 of A8 AKGANYDILTGYRKDNWFDP SEQ ID NO: 4 is the amino acid sequence of VL CDR1 of A8 SSNIGSNY SEQ ID NO: 5 is the amino acid sequence of VL CDR2 of A8 SSN SEQ ID NO: 6 is the amino acid sequence of VL CDR3 of A8 AAWDDSLSGWV SEQ ID NO: 7 is the amino acid sequence of VH CDR1 of B2 GFTFSSYA SEQ ID NO: 8 is the amino acid sequence of VH CDR2 of B2 ISGSGGNT SEQ ID NO: 9 is the amino acid sequence of VH CDR3 of B2 AKGGSTSSGYRFDY SEQ ID NO: 10 is the amino acid sequence of VL CDR1 of B2 SGHSNYA SEQ ID NO: 11 is the amino acid sequence of VL CDR2 of B2 VNRDGSY SEQ ID NO: 12 is the amino acid sequence of VL CDR3 of B2 QTWGTGVQV SEQ ID NO: 13 is the amino acid sequence of VH CDR1 of B7 GYTFTSYA SEQ ID NO: 14 is the amino acid sequence of VH CDR2 of B7 INAGNGNT SEQ ID NO: 15 is the amino acid sequence of VH CDR3 of B7 ARDSGYDLFDY SEQ ID NO: 16 is the amino acid sequence of VL CDR1 of B7 SGSVSTTYY SEQ ID NO: 17 is the amino acid sequence of VL CDR2 of B7 KTN SEQ ID NO: 18 is the amino acid sequence of VL CDR3 of B7 LLYMGSGVWV SEQ ID NO: 19 is the amino acid sequence of VH CDR1 of C1 GYSFTSYW SEQ ID NO: 20 is the amino acid sequence of VH CDR2 of C1 IYPGDSGT SEQ ID NO: 21 is the amino acid sequence of VH CDR3 of C1 ARQQGAGAFDI SEQ ID NO: 22 is the amino acid sequence of VL CDR1 of C1 SRDVGGYNY SEQ ID NO: 23 is the amino acid sequence of VL CDR2 of C1 EVT SEQ ID NO: 24 is the amino acid sequence of the VL CDR3 of C1 SSYTSSSTVV SEQ ID NO: 25 is the amino acid sequence of VH CDR1 of C6 GFTFGDYA SEQ ID NO: 26 is the amino acid sequence of VH CDR2 of C6 IRSKAYGGTT SEQ ID NO: 27 is the amino acid sequence of VH CDR3 of C6 TRVPVVAGWYNWFDP SEQ ID NO: 28 is the amino acid sequence of VL CDR1 of C6 SSNIGSNY SEQ ID NO: 29 is the amino acid sequence of VL CDR2 of C6 RNN SEQ ID NO: 30 is the amino acid sequence of the VL CDR3 of C6 SSYAGSNNVV SEQ ID NO: 31 is the amino acid sequence of VH CDR1 of C8 GFTFDDYA SEQ ID NO: 32 is the amino acid sequence of VH CDR2 of C8 ISWNSGSI SEQ ID NO: 33 is the amino acid sequence of VH CDR3 of C8 AKGANYDILTGYRKDNWFDP SEQ ID NO: 34 is the amino acid sequence of VL CDR1 of C8 QTISNW SEQ ID NO: 35 is the amino acid sequence of VL CDR2 of C8 KAS SEQ ID NO: 36 is the amino acid sequence of the VL CDR3 of C8 QQYHSYSRT SEQ ID NO: 37 is the amino acid sequence of VH CDR1 of D3 GFTFDDYA SEQ ID NO: 38 is the amino acid sequence of VH CDR2 of D3 ISWNSGSI SEQ ID NO: 39 is the amino acid sequence of VH CDR3 of D3 AKGANYDILTGYEY SEQ ID NO: 40 is the amino acid sequence of VL CDR1 of D3 TGAVTSDHH SEQ ID NO: 41 is the amino acid sequence of VL CDR2 of D3 DTS SEQ ID NO: 42 is the amino acid sequence of the VL CDR3 of D3 FLYYSGTAI SEQ ID NO: 43 is the amino acid sequence of VH CDR1 of E11 GFTFSSYA SEQ ID NO: 44 is the amino acid sequence of VH CDR2 of E11 ISGSGGST SEQ ID NO: 45 is the amino acid sequence of VH CDR3 of E11 AKDDYDFWSGSLGNY SEQ ID NO: 46 is the amino acid sequence of VL CDR1 of E11 SSNIGSNY SEQ ID NO: 47 is the amino acid sequence of VL CDR2 of E11 ENN SEQ ID NO: 48 is the amino acid sequence of VL CDR3 of E11 AAWDDTLNAWV SEQ ID NO: 49 is the amino acid sequence of VH CDR1 of E4 GFTFDDYA SEQ ID NO: 50 is the amino acid sequence of VH CDR2 of E4 ISWNSGSI SEQ ID NO: 51 is the amino acid sequence of VH CDR3 of E4 AKGANYDILTGYRKDNWFDP SEQ ID NO: 52 is the amino acid sequence of VL CDR1 of E4 QSVGSS SEQ ID NO: 53 is the amino acid sequence of VL CDR2 of E4 DAS SEQ ID NO: 54 is the amino acid sequence of VL CDR3 of E4 QQRSNWPPYT SEQ ID NO: 55 is the amino acid sequence of VH CDR1 of F10 GFTFSSYG SEQ ID NO: 56 is the amino acid sequence of VH CDR2 of F10 ISYDGSNK SEQ ID NO: 57 is the amino acid sequence of VH CDR3 of F10 AKDHHPYGSSDSFDY SEQ ID NO: 58 is the amino acid sequence of VL CDR1 of F10 TGAVTRGHY SEQ ID NO: 59 is the amino acid sequence of VL CDR2 of F10 DTV SEQ ID NO: 60 is the amino acid sequence of VL CDR3 of F10 LLSFIDTRYPARYV SEQ ID NO: 61 is the amino acid sequence of VH CDR1 of A10 GYTFTSYG SEQ ID NO: 62 is the amino acid sequence of VH CDR2 of A10 ISAYNGNT SEQ ID NO: 63 is the amino acid sequence of VH CDR3 of A10 ARDDPWGSYRPRPFDY SEQ ID NO: 64 is the amino acid sequence of VL CDR1 of A10 SSNIGSNT SEQ ID NO: 65 is the amino acid sequence of VL CDR2 of A10 SNN SEQ ID NO: 66 is the amino acid sequence of VL CDR3 of A10 AAWDDSLNGWV SEQ ID NO: 67 is the amino acid sequence of VH CDR1 of B4 GGTFSSYA SEQ ID NO: 68 is the amino acid sequence of VH CDR2 of B4 IIPIFGTA SEQ ID NO: 69 is the amino acid sequence of VH CDR3 of B4 ARGASGYDWSLDY SEQ ID NO: 70 is the amino acid sequence of VL CDR1 of B4 QSLVYSDGNTY SEQ ID NO: 71 is the amino acid sequence of VL CDR2 of B4 KVS SEQ ID NO: 72 is the amino acid sequence of VL CDR3 of B4 MQGSRWPPT SEQ ID NO: 73 is the amino acid sequence of VH CDR1 of F8 GYSFISHW SEQ ID NO: 74 is the amino acid sequence of VH CDR2 of F8 IYPGDSGT SEQ ID NO: 75 is the amino acid sequence of VH CDR3 of F8 ARLADGGLQFDH SEQ ID NO: 76 is the amino acid sequence of VL CDR1 of F8 SSDVGGYNY SEQ ID NO: 77 is the amino acid sequence of VL CDR2 of F8 GVS SEQ ID NO: 78 is the amino acid sequence of the VL CDR3 of F8 NSYTSSSTYV SEQ ID NO: 79 is the amino acid sequence of VH CDR1 of E1 GFTFSSYW SEQ ID NO: 80 is the amino acid sequence of VH CDR2 of E1 IKQDGSEK SEQ ID NO: 81 is the amino acid sequence of VH CDR3 of E1 ARVPNYYDSSGTV SEQ ID NO: 82 is the amino acid sequence of VL CDR1 of E1 SGSIASNY SEQ ID NO: 83 is the amino acid sequence of VL CDR2 of E1 EDN SEQ ID NO: 84 is the amino acid sequence of VL CDR3 of E1 QSYDSTLLV SEQ ID NO: 85 is the amino acid sequence of the VH domain of A8 (CDRs are underlined) QVTLKESGGGLVQPGRSLRLSCAAS GFTFDDYA MHWVRQAPGKGLEWVSG ISWNSGSI GYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYC AKGANYDILTGYRKDNWFDP WGQGTLVTVSS SEQ ID NO: 86 is the amino acid sequence of the VL domain of A8 (CDRs are underlined) LPVLTQPPSASGTPGQKVTISCSGS SSNIGSNY VFWYEQLPGAAPKLLMY SSN QRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYC AAWDDSLSGWV FGGGTKLTVL SEQ ID NO: 87 is the amino acid sequence of the VH domain of B2 (CDRs are underlined) QVQLVQSGGGVVQPGRSLRLSCAAS GFTFSSYA MSWVRQAPGKGLEWVSA ISGSGGNT NYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC AKGGSTSSGYRFDY WGQGTLVTVSS SEQ ID NO: 88 is the amino acid sequence of the VL domain of B2 (CDRs are underlined) QLVLTQSPSASASLGASVKLTCTLS SGHSNYA IAWHQQQPEKGPRFLMN VNRDGSY TKGDGIPDRFSGSSSGAERYLTISSLQSDDEADYYC QTWGTGVQV FGGGTKLTVL SEQ ID NO: 89 is the amino acid sequence of the VH domain of B7 (CDRs are underlined) QVQLQQSGAEVKKPGASVKVSCKAS GYTFTSYA MHWVRQAPGQRLEWMGW INAGNGNT KYSQKLQGRVTITRDTSASTAYMELSSLRSEDTAVYYC ARDSGYDLFDY WGQGTLVTVSS SEQ ID NO: 90 is the amino acid sequence of the VL domain of B7 (CDRs are underlined) QAVVTQEPSFSVSPGGTVTLTCGLT SGSVSTTYY PSWYQQTPGQTPRTLIY KTN LRSPGVPDRFSGSILGNKAALTITGAQADDDSDYYC LLYMGSGVWV FGGGTRLTVL SEQ ID NO: 91 is the amino acid sequence of the VH domain of C1 (CDRs are underlined) QVQLVQSGAEVKKPGESLKISCKGS GYSFTSYW IGWVRQMPGKGLEWMGI IYPGDSGT RYSPSFQGQVTISADKSISTAYLQWSSLKASNTAMYYC ARQQGAGAFDI WGQGTMVTVSS SEQ ID NO: 92 is the amino acid sequence of the VL domain of C1 (CDRs are underlined) QSALTQPPSASGSPGQSVTISCTGT SRDVGGYNY VSWYQQHPGKAPKLILY EVT KRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYC SSYTSSSTVV FGGGTKLTVL SEQ ID NO: 93 is the amino acid sequence of the VH domain of C6 (CDRs are underlined) EVQLVQSGGGLVQPGRSLRLSCTAS GFTFGDYA MSWVRQAPGKGLEWVGF IRSKAYGGTT EYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYC TRVPVVAGWYNWFDP WGQGTLVTVSS SEQ ID NO: 94 is the amino acid sequence of the VL domain of C6 (CDRs are underlined) QSVLTQPPSASGTPGQRVTISCSGS SSNIGSNY VYWYQQLPGTAPKLLIY RNN QRPSGVPDRFSGSKSGNTASLTVSGLQAGDEADYYC SSYAGSNNVV FGGGTKLAVL SEQ ID NO: 95 is the amino acid sequence of the VH domain of C8 (CDRs are underlined) QVTLKESGGGLVQPGRSLRLSCAAS GFTFDDYA MHWVRQAPGKGLEWVSG ISWNSGSI GYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYC AKGANYDILTGYRKDNWFDP WGQGTLVTVSS SEQ ID NO: 96 is the amino acid sequence of the VL domain of C8 (CDRs are underlined) DIVMTQTPSTLSASVGDRVTITCRAS QTISNW LAWYQQKPGKAPKLLIY KAS TLESGVPSRFSGTGSGTEFTLTISSLQPDDFATYFC QQYHSYSRT FGQGTKVDV SEQ ID NO: 97 is the amino acid sequence of the VH domain of D3 (CDRs are underlined) QVQLVQSGGGLVQPGRSLRLSCAAS GFTFDDYA MHWVRQAPGKGLEWVSG ISWNSGSI GYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYC AKGANYDILTGYEY WGQGTLVTVSS SEQ ID NO: 98 is the amino acid sequence of the VL domain of D3 (CDRs are underlined) QAVVTQEPSLTVSPGETVTLTCGSS TGAVTSDHH PYWFQQKPGQVPRTLMH DTS TRYSWTPARFSGSIVGGKAALTLSGAQPEDEAEYHC FLYYSGTAI FGGGTKLTVL SEQ ID NO: 99 is the amino acid sequence of the VH domain of E11 (CDRs are underlined) EVQLVESGGGLVQPGGSLRLSCAAS GFTFSSYA MSWVRQAPGKGLEWVSA ISGSGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC AKDDYDFWSGSLGNY WGQGTLVTVSS SEQ ID NO: 100 is the amino acid sequence of the VL domain of E11 (CDRs are underlined) QSVLTQPPSASGTPGQRVTISCSGR SSNIGSNY VYWYQQPPGTAPKLLIY ENN HRPSGVPDRFSASKSGTSASLAISGLRSEDEADYYC AAWDDTLNAWV FGGGTKLTVL SEQ ID NO: 101 is the amino acid sequence of the VH domain of E4 (CDRs are underlined) QVTLKESGGGLVQPGRSLRLSCAAS GFTFDDYA MHWVRQAPGKGLEWVSG ISWNSGSI GYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYC AKGANYDILTGYRKDNWFDP WGQGTLVTVSS SEQ ID NO: 102 is the amino acid sequence of the VL domain of E4 (CDRs are underlined) DIVMTQSPATLSLSPGERATLSCRAS QSVGSS LAWFQQKPGQAPRLLIY DAS NRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC QQRSNWPPYT FGQGTKLEIK SEQ ID NO: 103 is the amino acid sequence of the VH domain of F10 (CDRs are underlined) QMQLVQSGGGVVQPGRSLRLSCAAS GFTFSSYG MHWVRQAPGKGLERVAV ISYDGSNK YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTALYYC AKDHHPYGSSDSFDY WGQGTLVTVSS SEQ ID NO: 104 is the amino acid sequence of the VL domain of F10 (CDRs are underlined) QAVVTQEPSLTVSPGGTVTLTCGSS TGAVTRGHY PYWLQQKPGQAPRTLIY DTV KKHSWTPARFSGSLVGGKAALTLSGAQPEDEADYYC LLSFIDTRYPARYV FGTGTKVTVL SEQ ID NO: 105 is the amino acid sequence of the VH domain of A10 (CDRs are underlined) QVQLVQSGAEVKKPGASVKVSCKAS GYTFTSYG ISWVRQAPGQGLEWMGW ISAYNGNT NYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYC ARDDPWGSYRPRPFDY WGQGTLVTVSS SEQ ID NO: 106 is the amino acid sequence of the VL domain of A10 (CDRs are underlined) QSVLTQPPSASGTPGQRVTISCSGS SSNIGSNT VNWYQQLPGTTPKLLIY SNN QRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYC AAWDDSLNGWV FGGGTKLTVL SEQ ID NO: 107 is the amino acid sequence of the VH domain of B4 (CDRs are underlined) EVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTA NYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYC ARGASGYDWSLDY WGQGTLVTVSS SEQ ID NO: 108 is the amino acid sequence of the VL domain of B4 (CDRs are underlined) KIVMTQSPLSLPVTLGQPASISCRSS QSLVYSDGNTY LNWFQQRPGQSPRRLIY KVS NRHSGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYFC MQGSRWPPT FGPGTKVEIK SEQ ID NO: 109 is the amino acid sequence of the VH domain of F8 (CDRs are underlined) QIQLVQSGTEVKKPGESLKISCKGS GYSFISHW IGWVRQMPGKGLEWMGI IYPGDSGT RYSPSFQGQATISADKSISTAYLQWSSLKASDTAMYYC ARLADGGLQFDH WGQGTLVTVSS SEQ ID NO: 110 is the amino acid sequence of the VL domain of F8 (CDRs are underlined) QSALTQPASVSGSPGQSITISCTGT SSDVGGYNY VSWYQQHPGKAPKLMIY GVS NRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYC NSYTSSSTYV FGTGTKVTVL SEQ ID NO: 111 is the amino acid sequence of the VH domain of E1 (CDRs are underlined) EVQLVQSGGGLVQPGGSLRLSCAAS GFTFSSYW MSWVRQAPGKGLEWVAN IKQDGSEK YYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC ARVPNYYDSSGTV WGQGTLVTVSS SEQ ID NO: 112 is the amino acid sequence of the VL domain of E1 (CDRs are underlined) NFMLTQPHSVSESPGKTVTISCTRS SGSIASNY VQWYQQRPGSAPTTVIY EDN QRPSGVPDRFSGSIDSSSNSASLTISGLEPEDEADYYC QSYDSTLLV FGGGTKLTVL SEQ ID NO: 113 is the amino acid sequence of the antigen recognition domain containing the VH and VL sequences of A8 QVTLKESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKGANYDILTGYRKDNWFDPWGQGTLVTVS SGGGGSGGGGSGGGGSLPVLTQPPSASGTPGQKVTISCSGSSSNIGSNYVFWYEQLPGAAPKLLMYSSNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTKLTVL SEQ ID NO: 114 is the amino acid sequence of the antigen recognition domain containing the VH and VL sequences of B2 QVQLVQSGGGVVQPGRSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGNTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGGSTSSGYRFDYWGQGTLVTVSSGG GGSGGGGSGGGGSQLVLTQSPSASASLGASVKLTCTLSSGHSNYAIAWHQQQPEKGPRFLMNVNRDGSYTKGDGIPDRFSGSSSGAERYLTISSLQSDDEADYYCQTWGTGVQVFGGGTKLTVL SEQ ID NO: 115 is the amino acid sequence of the antigen recognition domain of B7, including the VH and VL sequences. QVQLQQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGWINAGNGNTKYSQKLQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDSGYDLFDYWGQGTLVTVSSGGG GSGGGGSGGGGSQAVVTQEPSFSVSPGGTVTLTCGLTSGSVSTTYYPSWYQQTPGQTPRTLIYKTNLRSPGVPDRFSGSILGNKAALTITGAQADDDSDYYCLLYMGSGVWVFGGGTRLTVL SEQ ID NO: 116 is the amino acid sequence of the antigen recognition domain of C1, including the VH and VL sequences. QVQLVQSGAEVKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSGTRYSPSFQGQVTISADKSISTAYLQWSSLKASNTAMYYCARQQGAGAFDIWGQGTMVTVSSGGG GSGGGGSGGGGSQSALTQPPSASGSPGQSVTISCTGTSRDVGGYNYVSWYQQHPGKAPKLILYEVTKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTVVFGGGTKLTVL SEQ ID NO: 117 is the amino acid sequence of the antigen recognition domain containing the VH and VL sequences of C6 EVQLVQSGGGLVQPGRSLRLSCTASGFTFGDYAMSWVRQAPGKGLEWVGFIRSKAYGGTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRVPVVAGWYNWFDPWGQGTLVTVSS GGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGNTASLTVSGLQAGDEADYYCSSYAGSNNVVFGGGTKLAVL SEQ ID NO: 118 is the amino acid sequence of the antigen recognition domain containing the VH and VL sequences of C8 QVTLKESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKGANYDILTGYRKDNWFDPWGQGTLVT VSSGGGGSGGGGSGGGGSDIVMTQTPSTLSASVGDRVTITCRASQTISNWLAWYQQKPGKAPKLLIYKASTLESGVPSRFSGTGSGTEFTLTISSLQPDDFATYFCQQYHSYSRTFGQGTKVDV SEQ ID NO: 119 is the amino acid sequence of the antigen recognition domain containing the VH and VL sequences of D3 QVQLVQSGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKGANYDILTGYEYWGQGTLVTVSSG GGGSGGGGSGGGGSQAVVTQEPSLTVSPGETVTLTCGSSTGAVTSDHHPYWFQQKPGQVPRTLMHDTSTRYSWTPARFSGSIVGGKAALTLSGAQPEDEAEYHCFLYYSGTAIFGGGTKLTVL SEQ ID NO: 120 is the amino acid sequence of the antigen recognition domain of E11, including the VH and VL sequences. EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDDYDFWSGSLGNYWGQGTLVTVSSG GGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGRSSNIGSNYVYWYQQPPGTAPKLLIYENNHRPSGVPDRFSASKSGTSASLAISGLRSEDEADYYCAAWDDTLNAWVFGGGTKLTVL SEQ ID NO: 121 is the amino acid sequence of the antigen recognition domain of E4, including the VH and VL sequences. QVTLKESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSIGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKGANYDILTGYRKDNWFDPWGQGTLVTV SSGGGGSGGGGSGGGGSDIVMTQSPATLSLSPGERATLSCRASQSVGSSLAWFQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPYTFGQGTKLEIK SEQ ID NO: 122 is the amino acid sequence of the antigen recognition domain containing the VH and VL sequences of F10 QMQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLERVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTALYYCAKDHPYGSSDSFDYWGQGTLVTVSSGGG GSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCGSSTGAVTRGHYPYWLQQKPGQAPRTLIYDTVKKHSWTPARFSGSLVGGKAALTLSGAQPEDEADYYCLLSFIDTRYPARYVFGTGTKVTVL SEQ ID NO: 123 is the amino acid sequence of the antigen recognition domain containing the VH and VL sequences of A10 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDDPWGSYRPRPFDYWGQGTLVTVSSG GGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTTPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKLTVL SEQ ID NO: 124 is the amino acid sequence of the antigen recognition domain containing the VH and VL sequences of B4 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGASGYDWSLDYWGQGTLVTVSSGGG GSGGGGSGGGGSKIVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPGQSPRRLIYKVSNRHSGVPDRFSGSGSGTDFTLKISRVEAEEDVGVYFCMQGSRWPPTFGPGTKVEIK SEQ ID NO: 125 is the amino acid sequence of the antigen recognition domain containing the VH and VL sequences of F8 QIQLVQSGTEVKKPGESLKISCKGSGYSFISHWIGWVRQMPGKGLEWMGIIYPGDSGTRYSPSFQGQATISADKSISTAYLQWSSLKASDTAMYYCARLADGGLQFDHWGQGTLVTVSSGGG GSGGGGSGGGGSQSALTQPASVGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYGVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCNSYTSSSTYVFGTGTKVTVL SEQ ID NO: 126 is the amino acid sequence of the antigen recognition domain of E1, including the VH and VL sequences. EVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVPNYYDSSGTVWGQGTLVTVSSGG GGSGGGGSGGGGSNFMLTQPHSVSESPGKTVTISCTRSSGSIASNYVQWYQQRPGSAPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLEPEDEADYYCQSYDSTLLVFGGGTKLTVL SEQ ID NO: 127 is the amino acid sequence of human ENTPD3 (the two transmembrane domains are underlined, the extracellular domain is located between the two transmembrane domains, and one cytoplasmic domain is located at each end of the polypeptide) MFTVLTRQPCEQAGLKALYRTP TIIALVVLLVSIVVLVSITVI QIHKQEVLPPGLKYGIVLDAGSSRTTVYVYQWPAEKENNTGVVSQTFKCSVKGSGISSYGNNPQDVPRAFEECMQKVKGQVPSHLHGSTPIHLGATAGMRLLRLQNETAA NEVLESIQSYFKSQPFDFRGAQIISGQEEGVYGWITANYLMGNFLEKNLWHMWVHPHGVETTGALDLGGASTQISFVAGEKMDLNTSDIMQVSLYGYVYTLYTHSFQCYGR NEAEKKFLAMLLQNSPTKNHLTNPCYPRDYSISFTMGHVFDSLCTVDQRPESYNPNDVITFEGTGDPSLCKEKVASIFDFKACHDQETCSFDGVYQPKIKGPFVAFAGFY YTASALNLSGFSLDTFNSSTWNFCSQNWSQLPLLLPKFDEVYARSYCFSANYIYHLFVNGYKFTEETWPQIHFEKEVGNSSIAWSLGYMLSLTNQIPAESPLIRLPIEPP VFVGTLAFFTAAALLCLAFLA YLCSATRRKRHSEHAFDHAVDSD SEQ ID NO: 128 is the amino acid sequence of mouse ENTPD3 (the two transmembrane domains are underlined, the extracellular domain is located between the two transmembrane domains, and one cytoplasmic domain is located at each end of the polypeptide) MFTVMTRQPCEQAGFRALSRTP AIVTLVVLLVSIVVLVTLTLI QIRHPQVLPPGLKYGVVLDAGSSRTTVYVYQWPAEKENNTGVVSQTFRCSVKGSGISSYENNPQDAPKAFEDCILKVKEQVPEHLHGSTRIYLGATAGMRLLRLQNETAA REVLESIQSYFKSQPFDFRGAQIISGQEEGVYGWITANYIMGNFLEKNLWHMWVHPHGVDTTGALDLGGASTQISFVAGEKMEPNASDTVQVSLYGYTYTLYTHSFQCYGQ NEAEKKFLAMLLQSPSTEANISNPCYPQGYSTAFTLGHVFGSLCTEKQRPESYNSSKSVTFMGTGDPRLCREKVASVFDFNACQEQDACSFDGIYQPKVQGPFVAFAGFY YTASALNLSGFSLTSFNDSSWDFCRHTWSELPALLSRFDETYARSYCFSAHYIYHLLVNGYKFTEETWPQIRFEKEVGNSSIAWSLGYMLSLTNQIPAGSPLIHLPIQPP VFMGVLAFFTAIALLCLAFLL YLCSSFRTKERSENAFDQAVDSD SEQ ID NO: 129 is the amino acid sequence of the human CD8α transmembrane domain, representing amino acids 183 to 203 of human CD8α. IYIWAPLAGTCGVLLLSLVIT SEQ ID NO: 130 is the amino acid sequence of the CH2CH3 hinge domain PCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 131 is the amino acid sequence of a modified combination of the CD8α hinge domain and transmembrane domain (the transmembrane domain is underlined). FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFAD IYIWAPLAGTCGVLLLSLVIT LYCNHR SEQ ID NO: 132 is the amino acid sequence of a modified CD8α hinge domain FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFAD SEQ ID NO: 133 is the combined amino acid sequence of the hinge and transmembrane domains of wild-type CD8α (the transmembrane domain is underlined). FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD IYIWAPLAGTCGVLLLSLVIT LYCNHR SEQ ID NO: 134 is the amino acid sequence of the CH2CH3 hinge domain EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 135 is the amino acid sequence of the hinge and transmembrane domains of CD28 (the transmembrane domain is underlined) IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP FWVLVVVGGVLACYSLLVTVAFIIFWV SEQ ID NO: 136 is the amino acid sequence of the signal / leader sequence MALPVTALLLPLALLLHAAAP SEQ ID NO: 137 is the amino acid sequence of the wild-type CD8α leader sequence MALPVTALLLPLALLLHAARP SEQ ID NO: 138 is the amino acid sequence of the intracellular signaling domain of the CD3 zeta chain RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 139 is the combined amino acid sequence of the CD28 transmembrane domain and the CD28 intracellular signaling domain (the transmembrane domain is underlined). FWVLVVVGGVLACYSLLVTVAFIIFWV RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO: 140 is the amino acid sequence of the CD28 intracellular signaling domain WVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO: 141 is an exemplary amino acid sequence of the signaling domain of OX40 ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI SEQ ID NO: 142 is an exemplary amino acid sequence of the signaling domain of 4-1BB KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL SEQ ID NO: 143 is an exemplary amino acid sequence of the signaling domain of ICOS CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL SEQ ID NO: 144 is an exemplary amino acid sequence of the signaling domain of TNFRSF25 TYTYRHCWPHKPLVTADEAGMEALTPPPATHLSPLDSAHTLLAPPDSSEKICTVQLVGNSWTPGYPETQEALCPQVTWSWDQLPSRALGPAAAPTLSPESPAGSPAMMLQPGPQLYDVMDAVPARRWKEFVRTLGLREAEIEAVEVEIGRFRDQQYEMLKRWRQQQPAGLGAVYAALERMGLDGCVEDLRSRLQRGP SEQ ID NO: 145 is the amino acid sequence representing amino acids 266 to 551 of the human IL-2 receptor β chain. NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPL QPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV SEQ ID NO: 146 is the amino acid sequence representing a shortened and sequence-altered variant of SEQ ID NO: 145 (Y510) NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLPLNTDAYLSLQELQGQDPTHLV SEQ ID NO: 147 is an amino acid sequence representing a shortened and sequence-altered variant of SEQ ID NO: 145 (Y510 and Y392) NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV SEQ ID NO: 148 is the amino acid sequence of wild-type FOXP3 (UniProtKB Accession No. Q9BZS1) MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFL KHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPE FLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGP SEQ ID NO: 149 is the amino acid sequence of an N- and C-terminally truncated FOXP3 fragment described in WO 2019 / 241549 GGAHASSSSL NPMPPSQLQL PTLPLVMVAP SGARLGPLPH LQALLQDRPH FMHQLSTVDA HARTPVLQVH PLESPAMISL TPPTTATGVF SLKARPGLPP GINVASLEWV SREPALLCTF PNPSAPRKDS TLSAVPQSSY PLLANGVCKW PGCEKVFEEP EDFLKHCQAD HLLDEKGRAQ CLLQREMVQS LEQQLVLEKE KLSAMQAHLA GKMALTKASS VASSDKGSCC IVAAGSQGPV VPAWSGPREA PDSLFAVRRH LWGSHGNSTF PEFLHNMDYF KFHNMRPPFT YATLIRWAIL EAPEKQRTLN EIYHWFTRMF AFFRNHPATW KNAIRHNLSL HKCFVRVESE KGAVWTVDEL EF SEQ ID NO: 150 is the amino acid sequence of a FOXP3 variant with a mutation at amino acid 418 MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVF EEPEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHL WGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKR E QRPSRCSNPTPGP SEQ ID NO: 151 is the amino acid sequence of a FOXP3 variant with a mutation at amino acid 422 MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEE PEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGS HGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRP A RCSNPTPGP SEQ ID NO: 152 is the amino acid sequence of a FOXP3 variant with mutations at amino acids 418 and 422 MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVF EEPEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHL WGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKR E QRP A RCSNPTPGP SEQ ID NO: 153 is the amino acid sequence of an exemplary FOXP3 polypeptide MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVD AHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQA DHLLDEKGRAQCLLQREMVQSLEQVEELSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHN MRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGPEGRGSLLTCGDVEEN SEQ ID NO: 154 is a nucleotide sequence encoding an exemplary FOXP3 polypeptide SEQ ID NO: 155 is a nucleotide sequence encoding an exemplary FOXP3 polypeptide SEQ ID NO: 156 is the amino acid sequence of the P2A domain ATNFSLLKQAGDVEENPGP SEQ ID NO: 157 is the amino acid sequence of the T2A domain EGRGSLLTCGDVEENPGP SEQ ID NO: 158 is the amino acid sequence of the E2A domain QCTNYALLKLAGDVESNPGP SEQ ID NO: 159 is the amino acid sequence of the F2A domain VKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 160 is the amino acid sequence of the furin cleavage site RXXR SEQ ID NO: 161 is the amino acid sequence of the furin cleavage site RRKR SEQ ID NO: 162 is the nucleotide sequence of the SFFV promoter GTAACGCCATTTTGCAAGGCATGGAAAAATACCAAACCAAGAATAGAGAAGGTTCAGATCAAGGGCGGGTACATGAAAATAGCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTTTCGGCCCCGGCCCGGGGCCAAGAACAGATGGTCACCGCAGTTTCGGCCCCGGCCCGAGGCCAAGAACAGATGGTCCCCAGATA TGGCCCAACCCTCAGCAGTTTCCTTAAGACCCATCAGATGTTTCCAGGCTCCCCCAAGGACCTGAAATGACCCTGCGCCTTATTTGAATTAACCAATCAGCCTGCTTCTCGCTTCTGTTCGCGCCTCTGCTTCCCGAGCTCTATAAAAGAGCTCACAACCCCTCACTCGGCGCGCCAGTCCTCCGACAGACTGAGTCGGCCGG SEQ ID NO: 163 - Representative linker sequence GGGS SEQ ID NO: 164 - Representative linker sequence ETSGGGGSRL SEQ ID NO: 165 - Representative linker sequence SGGGGSGGGGSGGGGS SEQ ID NO: 166 - Representative linker sequence GGGGS SEQ ID NO: 167 - Representative linker sequence GGGGGS SEQ ID NO: 168 - Representative linker sequence GGGGGGS SEQ ID NO: 169 - Representative linker sequence GGGGSGGGGSGGGGS SEQ ID NO: 170 - Representative linker sequence GGGGG SEQ ID NO: 171 - Representative linker sequence GGGGSGGGGS SEQ ID NO: 172 - Representative linker sequence GGGGSGGGGSGGGGSGGGGS SEQ ID NO: 173 - Representative linker sequence GGGGGGG SEQ ID NO: 174 - Representative linker sequence G6 SEQ ID NO: 175 - Representative linker sequence G8 SEQ ID NO: 176 - Representative linker sequence KESGSVSSEQLAQFRSLD SEQ ID NO: 177 - Representative linker sequence EGKSSGSGSESKST SEQ ID NO: 178 - Representative linker sequence GSAGSAAGSGEF SEQ ID NO: 179 - Representative linker sequence SGGGGSAGSAAGSGEF SEQ ID NO: 180 - Representative linker sequence SGGGLLLLLLLLGGGS SEQ ID NO: 181 - Representative linker sequence SGGGAAAAAAAAGGGS SEQ ID NO: 182 - Representative linker sequence SGGGAAAAAAAAAAAAAAAAGGGS SEQ ID NO: 183 - Representative linker sequence SGALGGLALAGLLLAGLGLGAAGS SEQ ID NO: 184 - Representative linker sequence SLSLSPGGGGGPAR SEQ ID NO: 185 - Representative linker sequence SLSLSPGGGGGPARSLSLSPGGGGG SEQ ID NO: 186 - Representative linker sequence GSSGSS SEQ ID NO: 187 - Representative linker sequence GSSSSSS SEQ ID NO: 188 - Representative linker sequence GGSSSS SEQ ID NO: 189 - Representative linker sequence GSSSSS SEQ ID NO: 190 - Representative linker sequence SGGGGS SEQ ID NO: 191 is the nucleotide sequence encoding A8 and the signal sequence and tag (the sequence encoding the signal sequence and tag is underlined) ATGAAATACCTATTGCCTACGGCAGCCGCTGGCTTGCTGCTGCTGGCAGCTCAGCCGGCCATGGCC CAGGTCACCTTGAAGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTAGCATAGGCTATGCGGACTCTGTGAAGGGCCGATT CACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGGGGCTAATTACGATATTTTGACTGGTTATCGGAAGGATAACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTGCATCCGCCCCAAAGCTTG AAGAAGGTGAATTTTCAGAAGCACGCGTACTGCCTGTGCTGACTCAGCCCCTCAGCGTCTGGGACCCCCGGGCAGAAGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATTATGTATTCTGGTACGAGCAGCTCCCAGGAGCGGCCCCCAAGCTCCTCATGTATAGCAGTAATCAGAGGCCCTCAGGG GTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGACTATTATTGTGCAGCATGGGATGACAGCCTGAGTGGTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCACCGTCCTCT GCGGCCGCAGGTTCTGAACAAAAGCTGATCTCAGAAGAAGATCTATCCCATCATCACCATCATCAT SEQ ID NO: 192 is the nucleotide sequence encoding B2 and the signal sequence and tag (the sequence encoding the signal sequence and tag is underlined) ATGAAATACCTATTGCCTACGGCAGCCGCTGGCTTGCTGCTGCTGGCAGCTCAGCCGGCCATGGCC CAGGTCCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAACACAAACTACGCAGACTCCGTGAAGGGCC GGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGGGGAGTACTAGTAGTGGTTATCGTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTGCATCCGCCCCAAAGCTTGAAGAAGGTGAA TTTTTCAGAAGCACGCGTACAGCTTGTGCTGACTCAATCGCCCTCTGCCTCTGCCTCCCTGGGAGCCTCGGTCAAGCTCACCTGCACTCTGAGCAGTGGGCACAGCAACTACGCCATCGCATGGCATCAGCAGCAGCCAGAGAAGGGCCCTCGGTTCTTGATGAACGTTAATCGTGATGGCAGCTACACCAAGGGGGACG GGATCCTGATCGCTTCTCAGGCTCCAGCTCTGGGGCTGAGCGCTACCTCACCATCTCCAGCCTCCAGTCTGACGATGAGGCTGACTATTACTGTCAGACCTGGGGCACTGGCGTTCAAGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAAGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCT GCGGCCGCAGGTTCTGAACAAAAGCTGATCTCAGAAGAAGATCTATCCCATCATCACCATCATCAT SEQ ID NO: 193 is the nucleotide sequence encoding B7 and the signal sequence and tag (the sequence encoding the signal sequence and tag is underlined) ATGAAATACCTATTGCCTACGGCAGCCGCTGGCTTGCTGCTGCTGGCAGCTCAGCCGGCCATGGCC CAGGTACAGCTGCAGCAGTCAGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCTTCTGGATACACCTTCACTAGCTATGCTATGCATTGGGTGCGCCAGGCCCCGGAACAAAGGCTTGAGTGGATGGGATGGATCAACGCTGGCAATGGTAACACAAAATATTCACAGAAGCTCCAGG GCAGAGTCACCATTACCAGGGACACATCCGCGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAAGACACGGCTGTGTATTACTGTGCGAGAGATAGTGGCTACGACCTCTTTGACTACTGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTGCATCCGCCCCAAAGCTTGAAGAAGGTGAATTT TCAGAAGCACGCGTACAGGCTGTGGTGACTCAGGAGCCATCGTTCTCAGTGTCCCCTGGAGGGACAGTCACACTTACTTGTGGCTTGACCTCTGGCTCAGTCTCTACTACTTACTACCCCAGCTGGTACCAGCAGACCCCAGGCCAGACTCCACGCACACTCATCTACAAAACAAATCTTCGCTCTCCTGGGGTCC CTGATCGCTTCTCTGGCTCCATCCTTGGGAACAAAGCTGCCCTCACCATTACGGGGGCCCAGGCAGACGACGACTCTGATTACTACTGTTCTGCTGTATATGGGTAGTGGCGTTTGGGTGTTCGGCGGAGGGACCAGGTTGACCGTCCTGGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCT GCGGCCGCAGGTTCTGAACAAAAGCTGATCTCAGAAGAAGATCTATCCCATCATCACCATCATCAT SEQ ID NO: 194 is the nucleotide sequence encoding C1 and the signal sequence and tag (the sequence encoding the signal sequence and tag is underlined) ATGAAATACCTATTGCCTACGGCAGCCGCTGGCTTGCTGCTGCTGGCAGCTCAGCCGGCCATGGCC CAAGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCGGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGCTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGGTACCAGATACAGCCCGTCCTTCCAAG GCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGAACACCGCCATGTATTACTGTGCGAGACAACAGGGCGCGGTGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGGAGTGCATCCGCCCCAAAGCTTGAAGAAGGTGAATTT TCAGAAGCACGCGTACAGTCTGCCCTGACTCAGCCTCCCTCCGCGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACCAGCCGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAACACCCCGGCAAAGCCCCCAAACTCATACTTTATGAGGTCACTAAGCGGCCCTCAGGGGTCC CTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGCTCATATACAAGCAGCAGCACCGTGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCGTCCTCT GCGGCCGCAGGTTCTGAACAAAAGCTGATCTCAGAAGAAGATCTATCCCATCATCACCATCATCAT SEQ ID NO: 195 is the nucleotide sequence encoding C6 and the signal sequence and tag (the sequence encoding the signal sequence and tag is underlined) ATGAAATACCTATTGCCTACGGCAGCCGCTGGCTTGCTGCTGCTGGCAGCTCAGCCGGCCATGGCC GAAGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCAGGGCGGTCCCTGAGACTCTCCTGTACAGCTTCTGGATTCACCTTTGGTGATTATGCTATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTAGGTTTCATTAGAAGCAAAGCTTATGGTGGGACAACAGAATACGCCGCGTCTGTGAA AGGCAGATTCACCATCTCAAGAGATGATTCCAAAAGCATCGCCTATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTACTAGAGTCCCGGTGGTAGCTGGGTGGTACAACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTGCATCCGCCCCAAAGCTTGAAG AAGGTGAATTTTCAGAAGCACGCGTACAGTCTGTGCTGACGCAGCCGCCCTCAGCGTCTGGGACCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGGAGTAATTATGTATACTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGG GTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCTGACCGTCTCTGGGCTCCAGGCTGGGGATGAGGCCGATTATTACTGCAGCTCATATGCAGGCAGCAACAATGTGGTATTCGGCGGAGGGACCAAGCTGGCCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCACCGTCCTCT GCGGCCGCAGGTTCTGAACAAAAGCTGATCTCAGAAGAAGATCTATCCCATCATCACCATCATCAT SEQ ID NO: 196 is the nucleotide sequence encoding C8 and the signal sequence and tag (the sequence encoding the signal sequence and tag is underlined) ATGAAATACCTATTGCCTACGGCAGCCGCTGGCTTGCTGCTGCTGGCAGCTCAGCCGGCCATGGCC CAGGTTACCTTGAAGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTAGCATAGGCTATGCGGACTCTGTGAAG GGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGGGGCTAATTACGATATTTTGACTGGTTATCGGAAGGATAACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTGCA TCCGCCCCAAAGCTTGAAGAAGGTGAATTTTCAGAAGCACGCGTAGATATTGTGATGACTCAGACTCCTTCCACCCTGTCTGCATCTGTGGGAGACAGAGTCACCATCACTTGCCGGCCAGTCAGACTATTAGTAACTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTAATTTATAAG GCGTCTACTTTAGAAAGTGGGGTCCCATCAAGGTTCAGCGGCACTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTTTTGCCAACAATATCATAGTTATTCTCGGACGTTCGGCCAAGGGACCAAGGTCGATGTCGCACGAACTGTGGCTGCACCATCTGTC GCGGCCGCAGGTTCTGAACAAAAGCTGATCTCAGAAGAAGATCTATCCCATCATCACCATCATCAT SEQ ID NO: 197 is the nucleotide sequence encoding D3 and the signal sequence and tag (the sequence encoding the signal sequence and tag is underlined) ATGAAATACCTATTGCCTACGGCAGCCGCTGGCTTGCTGCTGCTGGCAGCTCAGCCGGCCATGGCC CAGGTTCAGCTGGTGCAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTAGCATAGGCTATGCGGACTCTGTGAAGGGC CGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGGGGCCAATTACGATATTTTGACTGGTTATGAGTACTGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTGCATCCGCCCCAAAGCTTGAAGAAGGT GAATTTTCAGAAGCACGCGTACAGGCTGTGGTGACTCAGGAGCCCTCACTGACTGTGTCCCCAGGAGAGACAGTCACTCTCACCTGTGGCTCCAGTACTGGAGCTGTCACCAGTGATCATCATCCCTACTGGTTCCAACAGAAGCCTGGCCAAGTCCCAGGACACTGATGCATGATACAAGCACCAGATACTCCTGG ACCCCTGCCCGGTTCTCAGGCTCCATCGTTGGGGGCAAAGCTGCCCTGACTCTTTCGGGTGCGCAGCCTGAGGATGAGGCTGAATATCACTGTTTCCTCTATTACAGTGGAACTGCGATATTCGGCGGAGGGACCAAGCTGACCGTCCTAAGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCGTCCTCT GCGGCCGCAGGTTCTGAACAAAAGCTGATCTCAGAAGAAGATCTATCCCATCATCACCATCATCAT SEQ ID NO: 198 is the nucleotide sequence encoding E11 and the signal sequence and tag (the sequence encoding the signal sequence and tag is underlined) ATGAAATACCTATTGCCTACGGCAGCCGCTGGCTTGCTGCTGCTGGCAGCTCAGCCGGCCATGGCCGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCC GGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGATGATTACGATTTTTGGAGTGGTTCCCTAGGGAACTACTGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTGCATCCGCCCCAAAGCTTGAAGAAGGT GAATTTTCAGAAGCACGCGTACAGTCTGTGCTGACGCAGCCGCCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGACGCAGCTCCAACATCGGAAGTAATTATGTATACTGGTACCAGCAGCCCCCAGGAACGGCCCCCAAACTCCTCATCTATGAGAATAATCACCGGCCCTCAGGGGTCC CTGACCGATTCTCTGCCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGACGACACCTTGAATGCTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCGTCCTCT GCGGCCGCAGGTTCTGAACAAAAGCTGATCTCAGAAGAAGATCTATCCCATCATCACCATCATCAT SEQ ID NO: 199 is the nucleotide sequence encoding E4 and the signal sequence and tag (the sequence encoding the signal sequence and tag is underlined) ATGAAATACCTATTGCCTACGGCAGCCGCTGGCTTGCTGCTGCTGGCAGCTCAGCCGGCCATGGCCCAGGTCACCTTGAAGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAGGTATTAGTTGGAATAGTGGTAGCATAGGCTATGCGGACTCTGTGAAG GGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGGGGCTAATTACGATATTTTGACTGGTTATCGGAAGGATAACTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTGCAT CCGCCCCAAAGCTTGAAGAAGGTGAATTTTCAGAAGCACGCGTAGATATTGTGATGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTGGCAGCTCCTTAGCCTGGTTCCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGC ATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCCCTACACTTTTGGCCAGGGGACCAAACTGGAGATCAAACGAACTGTGGCTGCACCATCTGTC GCGGCCGCAGGTTCTGAACAAAAGCTGATCTCAGAAGAAGATCTATCCCATCATCACCATCATCAT SEQ ID NO: 200 is the nucleotide sequence encoding F10 and the signal sequence and tag (the sequence encoding the signal sequence and tag is underlined) ATGAAATACCTATTGCCTACGGCAGCCGCTGGCTTGCTGCTGCTGGCAGCTCAGCCGGCCATGGCCCAGATGCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGCGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGAT TCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTCTGTATTACTGTGCGAAAGACCACCATCCGTATGGAAGTAGTGACTCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTGCATCCGCCCCAAAGCTTGAAGAAGGTGAATTT TCAGAAGCACGCGTACAGGCTGTGGTGACTCAGGAGCCCTCACTGACTGTGTCCCCAGGAGGGACAGTCACTCTCACCTGTGGCTCCAGCACTGGGGCTGTCACCAGGGGTCATTATCCCTATTGGTTGCAGCAGAAGCCTGGCCAAGCCCCCAGGACACTGATTTATGATACGGTCAAGAAACACTCCTGGACCCCTGCCC GGTTCTCTGGCTCCCTCGTTGGGGGCAAAGCTGCCCTGACCCTTTCGGGTGCGCAGCCTGAAGACGAGGCTGACTATTATTGCTTGCTCTCCTTTATTGATACTCGGTACCCTGCTCGGTATGTCTTCGGAACCGGGACCAAGGTCACCGTCCTAGGTCAGCCCAAGGCCAACCCCACTGTCACTCTGTTCCCGCCCTCCTCT GCGGCCGCAGGTTCTGAACAAAAGCTGATCTCAGAAGAAGATCTATCCCATCATCACCATCATCAT SEQ ID NO: 201 is the nucleotide sequence encoding A10 and the signal sequence and tag (the sequence encoding the signal sequence and tag is underlined) ATGAAATACCTATTGCCTACGGCAGCCGCTGGCTTGCTGCTGCTGGCAGCTCAGCCGGCCATGGCCCAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAG AGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGATGATCCGTGGGGTTCCTATCGGCCCCGTCCGTTTGACTACTGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTGCATCCGCCCCAAAGCTTGAAGAAG GTGAATTTTCAGAAGCACGCGTACAGTCTGTGCTGACGCAGCCGCCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATACTGTAAACTGGTACCAGCAGCTCCCAGGAACGACCCCCAAACTCCTCATCTATAGTAATAATCAGCGGCCCTCAGGGGTC CCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAATGGTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCGTCCTCT GCGGCCGCAGGTTCTGAACAAAAGCTGATCTCAGAAGAAGATCTATCCCATCATCACCATCATCAT SEQ ID NO: 202 is the nucleotide sequence encoding B4 and the signal sequence and tag (the sequence encoding the signal sequence and tag is underlined) ATGAAATACCTATTGCCTACGGCAGCCGCTGGCTTGCTGCTGCTGGCAGCTCAGCCGGCCATGGCCGAAGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCC AGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGAGCAAGTGGATATGACTGGTCCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTGCATCCGCCCCAAAGCTTGAA GAAGGTGAATTTTCAGAAGCACGCGTAAAAATTGTGATGACACAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAAAGCCTCGTATACAGTGATGAAACACCTACTTGAATTGGTTTCAGCAGAGGCC...

Claims

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

2. a. an exodomain comprising the antigen recognition domain; b. a transmembrane domain; c. an endodomain comprising an intracellular signaling domain; The CAR according to claim 1.

3. The CAR of claim 2, further comprising a hinge domain and / or one or more costimulatory domains.

4. the hinge domain is selected from CD28, CD8α, CD4, CD7, CH2CH3, or an immunoglobulin hinge region, or a portion or variant thereof, and optionally the CAR comprises a CD8α hinge region or a CH2CH3 hinge region; and / or the costimulatory domain is selected from the intracellular domain of CD28, ICOS, CD134 (OX40), CD137 (4-1BB), CD27, or TNFRSF25, or a portion or variant thereof; and optionally, the CAR comprises a CD28 costimulatory domain. The CAR according to claim 3.

5. the CAR comprises one or more transmembrane domains selected from the transmembrane domains of CD28, ICOS, CD8α, CD4, CD134 (OX40), CD137 (4-1BB), CD3ζ, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD154, or CH2CH3, or portions or variants thereof; optionally, the CAR comprises a CD4 transmembrane domain, a CD28 transmembrane domain, a CD8α transmembrane domain, or a CH2CH3 transmembrane domain; and / or the CAR comprises one or more intracellular signaling domains selected from the group consisting of a CD3ζ signaling domain or any of its homologs, a CD3 polypeptide, a Syk family tyrosine kinase, a Src family tyrosine kinase, CD2, CD5, or CD28, or a portion or variant thereof, and optionally the CAR comprises a CD3ζ signaling domain. The CAR according to any one of claims 2 to 4.

6. The CAR according to any one of claims 2 to 5, wherein the CAR comprises a CD8α or CH2CH3 hinge domain, a CD28, CD8α, or CH2CH3 transmembrane domain, a CD28 costimulatory domain, and a CD3ζ signaling 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. 10. The CAR of any one of the preceding claims, wherein the antigen recognition domain is an antibody, an antibody fragment, or derived from an antibody, optionally wherein the antigen recognition domain is a single chain antibody (scFv).

8. The antigen recognition domain is i. the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively; ii. VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively, and VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively; iii. The VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 73, 74, and 75, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 76, 77, and 78, respectively; iv. the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 25, 26, and 27, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively; v. the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 37, 38, and 39, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 40, 41, and 42, respectively; vi. the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively; vii. the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively; viii. the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively; ix. the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 43, 44, and 45, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 46, 47, and 48, respectively; x. the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 52, 53, and 54, respectively; xi. VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 55, 56, and 57, respectively, and VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 58, 59, and 60, respectively; xii. the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 61, 62, and 63, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 64, 65, and 66, respectively; xiii. the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 67, 68, and 69, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 70, 71, and 72, respectively; or xiv. comprising the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 79, 80, and 81, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 82, 83, and 84, respectively; One or more of the CDR sequences (i) to (xiv) may optionally contain one to three amino acid modifications relative to the CDR sequence, and in particular, one or more of the CDR sequences may optionally be modified by substitution, addition, or deletion of one to three amino acids; CAR according to any one of the preceding claims.

9. The antigen recognition domain is (i) a VH domain comprising the sequence set forth in SEQ ID NO: 87, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 88, or a sequence having at least 70% sequence identity thereto; (ii) a VH domain comprising the sequence set forth in SEQ ID NO: 91, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 92, or a sequence having at least 70% sequence identity thereto; (iii) a VH domain comprising the sequence set forth in SEQ ID NO: 109, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 110, or a sequence having at least 70% sequence identity thereto; (iv) a VH domain comprising the sequence set forth in SEQ ID NO: 93, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 94, or a sequence having at least 70% sequence identity thereto; (v) a VH domain comprising the sequence set forth in SEQ ID NO: 97, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 98, or a sequence having at least 70% sequence identity thereto; (vi) a VH domain comprising the sequence set forth in SEQ ID NO: 85, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 86, or a sequence having at least 70% sequence identity thereto; (vii) a VH domain comprising the sequence set forth in SEQ ID NO: 89, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 90, or a sequence having at least 70% sequence identity thereto; (viii) a VH domain comprising the sequence set forth in SEQ ID NO: 95, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 96, or a sequence having at least 70% sequence identity thereto; (ix) a VH domain comprising the sequence set forth in SEQ ID NO: 99, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 100, or a sequence having at least 70% sequence identity thereto; (x) a VH domain comprising the sequence set forth in SEQ ID NO: 101, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 102, or a sequence having at least 70% sequence identity thereto; (xi) a VH domain comprising the sequence set forth in SEQ ID NO: 103, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 104, or a sequence having at least 70% sequence identity thereto; (xii) a VH domain comprising the sequence set forth in SEQ ID NO: 105, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 106, or a sequence having at least 70% sequence identity thereto; (xiii) a VH domain comprising the sequence set forth in SEQ ID NO: 107, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 108, or a sequence having at least 70% sequence identity thereto; or (xiv) a VH domain comprising the sequence set forth in SEQ ID NO: 111, or a sequence having at least 70% sequence identity thereto; and a VL domain comprising the sequence set forth in SEQ ID NO: 112, or a sequence having at least 70% sequence identity thereto; CAR according to any one of the preceding claims.

10. The antigen recognition domain is (i) the sequence set forth in SEQ ID NO: 114, or a sequence having at least 80% sequence identity thereto; (ii) the sequence set forth in SEQ ID NO: 116, or a sequence having at least 80% sequence identity thereto; (iii) the sequence set forth in SEQ ID NO: 125, or a sequence having at least 80% sequence identity thereto; (iv) the sequence set forth in SEQ ID NO: 117, or a sequence having at least 80% sequence identity thereto; (v) the sequence set forth in SEQ ID NO: 119, or a sequence having at least 80% sequence identity thereto; (vi) the sequence set forth in SEQ ID NO: 113, or a sequence having at least 80% sequence identity thereto; (vii) the sequence set forth in SEQ ID NO: 115, or a sequence having at least 80% sequence identity thereto; (viii) the sequence set forth in SEQ ID NO: 118, or a sequence having at least 80% sequence identity thereto; (ix) the sequence set forth in SEQ ID NO: 120, or a sequence having at least 80% sequence identity thereto; (x) a sequence set forth in SEQ ID NO: 121, or a sequence having at least 80% sequence identity thereto; (xi) the sequence set forth in SEQ ID NO: 122, or a sequence having at least 80% sequence identity thereto; (xii) the sequence set forth in SEQ ID NO: 123, or a sequence having at least 80% sequence identity thereto; (xiii) the sequence set forth in SEQ ID NO: 124, or a sequence having at least 80% sequence identity thereto; or (xiv) comprising or consisting of the sequence set forth in SEQ ID NO: 126, or a sequence having at least 80% sequence identity thereto; CAR according to any one of the preceding claims.

11. 10. The CAR of any one of the preceding claims, wherein the CAR comprises or consists of a sequence according to any one of SEQ ID NOs: 237 to 262, in particular the CAR comprises or consists of a sequence according to SEQ ID NOs: 238, 240, 249, 241 or 243.

12. 10. A nucleic acid molecule comprising a nucleotide sequence encoding a CAR according to any one of the preceding claims.

13. 13. A vector comprising the nucleic acid molecule of claim 12, and optionally further comprising a nucleic acid molecule encoding a FOXP3 polypeptide.

14. A cell comprising the CAR of any one of claims 1 to 11, the nucleic acid molecule of claim 12, or the vector of claim 13.

15. 15. The cell of claim 14, wherein the cell is a regulatory T cell (Treg), or a precursor thereof, or an iPSC cell, and particularly wherein the cell further comprises an exogenous nucleic acid comprising a nucleotide sequence encoding a FOXP3 polypeptide.

16. A cell population comprising the cells of claim 14 or claim 15.

17. 17. The cell population of claim 16, wherein the plurality of cells is a plurality of T cells, particularly Tregs, and the plurality of T cells has a polyclonal endogenous TCR.

18. A pharmaceutical composition comprising a cell according to claim 14 or claim 15, a cell population according to claim 16 or claim 17, or a vector according to claim 13.

19. 19. A cell according to any one of claims 14 or 15, a cell population according to claim 16 or 17, or a pharmaceutical composition according to claim 18, for use in therapy.

20. 20. The cell, cell population, or pharmaceutical composition for use according to claim 19, wherein said therapy is adoptive cell transfer therapy.

21. 19. A cell according to claim 14 or claim 15, a cell population according to claim 16 or claim 17, or a pharmaceutical composition according to claim 18, for use in the treatment or prevention of an autoimmune or inflammatory disease, for use in inducing immunosuppression, or for use in promoting tissue repair and / or tissue regeneration, in particular wherein the cell is a Treg.

22. 22. The cell, cell population, or pharmaceutical composition for use according to claim 21, wherein the autoimmune or inflammatory disease is type 1 diabetes, such as recent onset type 1 diabetes.

23. A method for producing the cell described in claim 14 or claim 15, comprising the step of introducing the nucleic acid molecule described in claim 12 or the vector described in claim 13 into a cell.

24. Cells obtained by the method of claim 23.

25. an antibody or antibody fragment, optionally a single chain antibody (scFv), that specifically binds to ENTPD3, (i) VH CDR1 sequences, VH CDR2 sequences, and VH CDR3 sequences set forth in SEQ ID NOs: 7, 8, and 9, respectively, and VL CDR1 sequences, VL CDR2 sequences, and VL CDR3 sequences set forth in SEQ ID NOs: 10, 11, and 12, respectively; (ii) VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 19, 20, and 21, respectively, and VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 22, 23, and 24, respectively; (iii) the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 73, 74, and 75, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 76, 77, and 78, respectively; (iv) the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 25, 26, and 27, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 28, 29, and 30, respectively; (v) the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 37, 38, and 39, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 40, 41, and 42, respectively; (vi) VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 1, 2, and 3, respectively, and VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively; (vii) the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 13, 14, and 15, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively; (viii) the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 31, 32, and 33, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 34, 35, and 36, respectively; (ix) the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 43, 44, and 45, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 46, 47, and 48, respectively; (x) the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 49, 50, and 51, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 52, 53, and 54, respectively; (xi) the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 55, 56, and 57, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 58, 59, and 60, respectively; (xii) the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 61, 62, and 63, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 64, 65, and 66, respectively; (xiii) the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 67, 68, and 69, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 70, 71, and 72, respectively; or (xiv) comprising the VH CDR1, VH CDR2, and VH CDR3 sequences set forth in SEQ ID NOs: 79, 80, and 81, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences set forth in SEQ ID NOs: 82, 83, and 84, respectively; One or more of the CDR sequences (i) to (xiv) may optionally contain one to three amino acid modifications relative to the CDR sequence, and in particular, one or more of the CDR sequences may optionally be modified by substitution, addition, or deletion of one to three amino acids; An antibody or antibody fragment.

26. The antibody or antibody fragment (i) a VH domain comprising the sequence set forth in SEQ ID NO: 87, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 88, or a sequence having at least 70% sequence identity thereto; (ii) a VH domain comprising the sequence set forth in SEQ ID NO: 91, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 92, or a sequence having at least 70% sequence identity thereto; (iii) a VH domain comprising the sequence set forth in SEQ ID NO: 109, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 110, or a sequence having at least 70% sequence identity thereto; (iv) a VH domain comprising the sequence set forth in SEQ ID NO: 93, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 94, or a sequence having at least 70% sequence identity thereto; (v) a VH domain comprising the sequence set forth in SEQ ID NO: 97, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 98, or a sequence having at least 70% sequence identity thereto; (vi) a VH domain comprising the sequence set forth in SEQ ID NO: 85, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 86, or a sequence having at least 70% sequence identity thereto; (vii) a VH domain comprising the sequence set forth in SEQ ID NO: 89, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 90, or a sequence having at least 70% sequence identity thereto; (viii) a VH domain comprising the sequence set forth in SEQ ID NO: 95, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 96, or a sequence having at least 70% sequence identity thereto; (ix) a VH domain comprising the sequence set forth in SEQ ID NO: 99, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 100, or a sequence having at least 70% sequence identity thereto; (x) a VH domain comprising the sequence set forth in SEQ ID NO: 101, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 102, or a sequence having at least 70% sequence identity thereto; (xi) a VH domain comprising the sequence set forth in SEQ ID NO: 103, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 104, or a sequence having at least 70% sequence identity thereto; (xii) a VH domain comprising the sequence set forth in SEQ ID NO: 105, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 106, or a sequence having at least 70% sequence identity thereto; (xiii) a VH domain comprising the sequence set forth in SEQ ID NO: 107, or a sequence having at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 108, or a sequence having at least 70% sequence identity thereto; or (xiv) The antibody or antibody fragment of claim 25, comprising a VH domain comprising the sequence set forth in SEQ ID NO: 111, or a sequence with at least 70% sequence identity thereto, and a VL domain comprising the sequence set forth in SEQ ID NO: 112, or a sequence with at least 70% identity thereto.

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