Engineered regulatory t cells and uses thereof

Abstract

The invention provides a lentiviral vector comprising a construct comprising from 5' to 3': (a) a first nucleotide sequence encoding FoxP3; (b) a second nucleotide sequence encoding a cleavable linker; and (c) a third nucleotide sequence encoding a CD19 CAR.

Description

[0001] ENGINEERED REGULATORY T CELLS AND USES THEREOF

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a lentiviral vector comprising a nucleotide sequence encoding FoxP3 and encoding a CD19 CAR, wherein the nucleotide sequence encodes from 5’ to 3’: FoxP3 - cleavable linker - CD19 CAR. The invention also relates to the use of the lentiviral vector for the production of a population of engineered CD19 CAR Tregs, a population of engineered CD19 CAR Tregs and the use thereof in the treatment or prevention of an autoimmune disease.

[0004] BACKGROUND TO THE INVENTION

[0005] Regulatory T cells (Tregs) represent a subgroup of CD4+ T lymphocytes endowed with immune suppressive capacities, which maintain immune homeostasis and immune tolerance, thereby preventing unwanted immune responses directed against self-antigens. Tregs have been largely studied in the context of autoimmune diseases and a reduction both in terms of numbers and function contributes to the pathogenesis of such diseases. Since their discovery, Tregs have represented attractive candidates for the treatment of autoimmune diseases due to their immunomodulatory properties. Several clinical trials in patients with autoimmune diseases have been conducted with polyclonal Tregs, which proved to be safe but also have limited efficacy.

[0006] The limited efficacy has been attributed to several factors, including imperfect selection processes for Tregs, poor persistence of infused Tregs (e,g. due to reprogramming of Tregs to Tconvs), insufficient numbers of disease-relevant antigen specific cells in the infused Treg products to achieve adequate immunosuppression, and the lack of antigen specificity to enable trafficking of the Tregs to the target tissue.

[0007] Several markers have been employed for the selection of Tregs, but none of them is specific for this subset. Indeed, Tregs are routinely selected using a combination of factors, which are partially shared by activated conventional T cells (Tconvs). Hence, current strategies for selecting Tregs during the manufacture of adoptive T cell therapies are imperfect, yielding mixed cell populations. Thus, there is a risk that a contaminant Tconv population in the final clinical Treg product could aggravate the underlying autoimmune disease by exacerbating the anti-self-antigen immune response.

[0008] It has also been demonstrated that, upon chronic inflammatory conditions, Tregs may be reprogrammed to Tconvs, further aggravating the inflammatory response in the context of autoimmune disease. Chimeric antigen receptors (CARs) are able to redirect Treg suppressive capacities in an antigen-specific manner and their efficacy in different autoimmune disease models has been reported. Efficacy of CAR-Tregs have been shown in preclinical models of type I diabetes and solid organ rejection. However, the lack of specific markers and intrinsic plasticity of T regs still limit the clinical use of CAR-Tregs.

[0009] Accordingly, there is a need in the art for improved Treg therapies.

[0010] SUMMARY OF THE INVENTION

[0011] The present inventors have developed a safe and efficacious Treg cellular product for the treatment of autoimmune diseases. The present inventors have designed a lentiviral vector which allows the co-expression of both an anti-CD19 CAR and the FoxP3 gene. The inventors have surprisingly found that the combination of both transgenes is able to redirect the Treg suppressive capacities in an antigen-specific manner, stabilise the Treg phenotype and reprogram Tconvs to suppressive cells, thus improving the safety and efficacy of the CAR- T reg-based cellular product. Hence, the lentiviral vector of the invention permits to simultaneously redirect and stabilize engineered Tregs that have been transduced with the vector.

[0012] The Treg product of the invention advantageously addresses several issues with current Treg products by:

[0013] 1) redirecting the antigen-specificity of regulatory T cells to increase their effectiveness;

[0014] 2) controlling the Tconv population that might contaminate the final cellular product due to imperfect selection processes with potential detrimental effects; and

[0015] 3) avoiding the reprogramming of Tregs to Tconvs in chronic inflammatory conditions following administration to the patient, thereby improving persistence of infused Tregs.

[0016] Accordingly, in a first aspect the invention provides a lentiviral vector comprising a construct encoding from 5’ to 3’:

[0017] (a) a first nucleotide sequence encoding FoxP3;

[0018] (b) a second nucleotide sequence encoding a cleavable linker; and

[0019] (c) a third nucleotide sequence encoding a CD19 CAR.

[0020] In some embodiments, the cleavable linker is T2A.

[0021] In some embodiments, the CD19 CAR comprises an antigen binding domain comprising a single-chain variable fragment (scFv). In some embodiments, the CAR comprises an antigen binding domain that is specific for CD19.

[0022] In some embodiments, the antigen-binding domain comprises: a) heavy chain variable region (VH) complementarity determining regions (CDRs) with the sequences: CDR1 - GVSLPDYGVS (SEQ ID NO: 5); CDR2 - VIWGSETTYYNSALKS (SEQ ID NO: 6); and CDR3 - HYYYGGSYAMDY (SEQ ID NO: 7); or variants thereof each having up to three amino acid substitutions, additions or deletions; and b) light chain variable region (VL) CDRs with the sequences: CDR1 - RASQDISKYLN (SEQ ID NO: 8); CDR2 - HTSRLHS (SEQ ID NO: 9); and CDR3 - QQGNTLPYT (SEQ ID NO: 10); or variants thereof each having up to three amino acid substitutions, additions or deletions.

[0023] In some embodiments, the antigen-binding domain comprises: a) heavy chain variable region (VH) complementarity determining regions (CDRs) with the sequences: CDR1 - SGVSLPDY (SEQ ID NO: 62); CDR2 - LEWLGVIWGSETTYYN (SEQ ID NO: 63); and CDR3 - HYYYGGSYAMD (SEQ ID NO: 64); or variants thereof each having up to three amino acid substitutions, additions or deletions; and b) light chain variable region (VL) CDRs with the sequences: CDR1 - QDISK (SEQ ID NO: 65); CDR2 - VKLLIYHTSRLH (SEQ ID NO: 66); and CDR3 - GNTLPY (SEQ ID NO: 67); or variants thereof each having up to three amino acid substitutions, additions or deletions.

[0024] In some embodiments, the antigen-binding domain comprises: a) heavy chain variable region (VH) complementarity determining regions (CDRs) with the sequences: CDR1 - SGVSLPDYGVS (SEQ ID NO: 68); CDR2 - LEWLGVIWGSETTYYNSALKS (SEQ ID NO: 69); and CDR3 - HYYYGGSYAMDY (SEQ ID NO: 70); or variants thereof each having up to three amino acid substitutions, additions or deletions; and b) light chain variable region (VL) CDRs with the sequences: CDR1 - RASQDISKYLN (SEQ ID NO: 71); CDR2 - VKLLIYHTSRLHS (SEQ ID NO: 72); and CDR3 - QQGNTLPYT (SEQ ID NO: 73); or variants thereof each having up to three amino acid substitutions, additions or deletions. In some embodiments, the antigen-binding domain comprises: a) heavy chain variable region (VH) complementarity determining regions (CDRs) comprising the sequences: CDR1 - GVSLPDY (SEQ ID NO: 74); CDR2 - VIWGSETTYYN (SEQ ID NO: 75); and CDR3 - HYYYGGSYAMD (SEQ ID NO: 76); or variants thereof each having up to three amino acid substitutions, additions or deletions; and b) light chain variable region (VL) CDRs comprising the sequences: CDR1 - QDISK (SEQ ID NO: 77); CDR2 - HTSRLH (SEQ ID NO: 78); and CDR3 - GNTLPY (SEQ ID NO: 79); or variants thereof each having up to three amino acid substitutions, additions or deletions.

[0025] In some embodiments, the antigen binding domain comprises: a) a VH domain comprising the sequence of SEQ ID NO: 13; and b) a VL domain comprising the sequence of SEQ ID NO: 14; or variants thereof, each having at least 75% sequence identity thereto.

[0026] In some embodiments, the CAR comprises the sequence as set forth in SEQ ID NO: 15 or a variant thereof having at least 75% sequence identity thereto.

[0027] In some embodiments, the antigen-binding domain comprises: a) heavy chain variable region (VH) complementarity determining regions (CDRs) with the sequences: CDR1 - GFDFSRYW(SEQ ID NO: 16); CDR2 - INLDSSTI (SEQ ID NO: 17); and CDR3 - ARRYDAMDY (SEQ ID NO: 18); or variants thereof each having up to three amino acid substitutions, additions or deletions; and b) light chain variable region (VL) CDRs with the sequences: CDR1 - ESVDDYGISF (SEQ ID NO: 19); CDR2 - AAP (SEQ ID NO: 20); and CDR3 - QQSKD (SEQ ID NO: 21); or variants thereof each having up to three amino acid substitutions, additions or deletions.

[0028] In some embodiments, the antigen binding domain comprises: a) a VH domain comprising the sequence of SEQ ID NO: 22; and b) a VL domain comprising the sequence of SEQ ID NO: 23; or variants thereof, each having at least 75% sequence identity thereto.

[0029] In some embodiments, the CAR comprises the sequence as set forth in SEQ ID NO: 26 or a variant thereof having at least 75% sequence identity thereto.

[0030] In some embodiments, the CD19 CAR comprises:

[0031] (a) a CD28, a CD8, and / or a CD4 transmembrane domain;

[0032] (b) an I gG 1 hinge, a CD8a spacer, LNGFR spacer or mCH2CH3 spacer; and / or

[0033] (c) a CD28 and / or a 4-1 BB co-stimulatory domain.

[0034] In some embodiments, the CD19 CAR comprises a CD28 transmembrane domain, an lgG1 hinge, a CD28 co-stimulatory domain, and a CD3 intracellular signalling domain.

[0035] In some embodiments, the lentiviral vector further comprises a hPGK promoter operably linked to the construct.

[0036] In some embodiments, the first nucleotide sequence comprises a sequence having at least 70% sequence identity to the sequence as set forth in SEQ ID NO: 1 .

[0037] In a further aspect, the invention provides a cell comprising a lentiviral vector of the invention. Suitably, the cell is in the form of a population of cells.

[0038] In a further aspect, the invention provides a population of cells comprising a lentiviral vector of the invention.

[0039] In some embodiments, the cell or population of cells has been transduced by the lentiviral vector.

[0040] In some embodiments, the cell has the phenotype CD3+CD4+CD25+CD127'FoxP3+.

[0041] In a further aspect, the invention provides a method of producing a population of CD19 CAR- Treg cells, comprising the steps of:

[0042] (a) providing a population of T cells;

[0043] (b) activating the population of T cells;

[0044] (c) transducing the population of T cells with a lentiviral vector according to the invention; and (d) culturing the population of T cells in the presence of IL-2 and rapamycin and under conditions suitable for the expression of the transgene.

[0045] In some embodiments, the population of T cells in step (a) comprises Tconv cells.

[0046] In some embodiments, the population of T cells in step (a) has the phenotype CD4+CD25+, CD4+CD25_or is a mixture comprising CD4+CD25+and CD4+CD25_cells. Suitably, the population of T cells in step (a) has the phenotype CD4+CD25+. Suitably, the population of T cells in step (a) is a mixture comprising CD4+CD25+and CD4+CD25_cells.

[0047] In some embodiments, step (b) comprises stimulating the population of T cells with anti-CD3 and anti-CD28 antibodies.

[0048] In some embodiments, step (d) comprises culturing the population of T cells in cell culture medium comprising rapamycin and adding IL-2 to the cell culture medium one or more time.

[0049] In some embodiments, step (d) comprises supplementing the cell culture medium with IL-2 on days 3, 6, 9 and 12 after activating the population of T cells in step (b).

[0050] In some embodiments, the population of CD19 CAR-Treg cells has the phenotype CD3+CD4+CD25+CD127-FoxP3+.

[0051] In a further aspect, the invention provides a population of CD19 CAR-Treg cells obtained or obtainable by the method according to the invention.

[0052] In a further aspect, the invention provides a pharmaceutical composition comprising a lentiviral vector, a cell, a population of cells or a population of CD19 CAR-Treg cells of the invention.

[0053] In a further aspect, the invention provides pharmaceutical composition, a cell or a population of CD19 CAR-Treg cells according to the invention for use in the treatment and / or prevention of an autoimmune disease.

[0054] In some embodiments, the autoimmune disease is selected from the group consisting of inflammatory arthritis (such as rheumatoid arthritis), type 1 diabetes mellitus, multiples sclerosis (MS), psoriasis, inflammatory bowel diseases, systemic lupus erythematosus (SLE), vasculitis, allergic inflammation (such as allergic asthma, atopic dermatitis, and contact hypersensitivity), Graves' disease, Hashimoto's thyroiditis, celiac disease, Crohn's disease, ulcerative colitis, Guillain-Barre syndrome, primary biliary sclerosis / cirrhosis, sclerosing cholangitis, autoimmune hepatitis, Raynaud's phenomenon, scleroderma, Sjogren's syndrome, Goodpasture's syndrome, Wegener's granulomatosis, polymyalgia rheumatica, temporal arteritis / giant cell arteritis, chronic fatigue syndrome (CFS), autoimmune Addison's Disease, ankylosing spondylitis, acute disseminated encephalomyelitis, antiphospholipid antibody syndrome, aplastic anemia, idiopathic thrombocytopenic purpura, Myasthenia gravis, opsoclonus myoclonus syndrome, optic neuritis, Ord's thyroiditis, pemphigus, pernicious anaemia, Reiter's syndrome, Takayasu's arteritis, warm autoimmune hemolytic anemia, Wegener's granulomatosis, fibromyalgia (FM), vitiligo, IPEX, graft versus host disease, undifferentiated connective tissue disease, mixed connective tissue disease, autoinflammatory syndromes (such as Familial mediterranean fever), still's disease, and adultonset still's disease.

[0055] In some embodiments, the autoimmune disease is systemic lupus erythematosus.

[0056] BRIEF DESCRIPTION OF THE FIGURES

[0057] Figure 1 - Fox19CAR-Treg in vitro generation and validation

[0058] A. Fox19CAR lentiviral vector (LV) and FoxP3 LV schematic representations. The FoxP3 gene and an anti-CD19 second-generation CAR are inserted in an unidirectional LV and under the control of a Phosphoglycerate Kinase (PGK) promoter. The CAR is composed by an anti- CD19 single-chain fragment variant (scFv), a hinge region as linker and the transmembrane and the intracellular portion of the human CD28 fused with the CD3 zeta-chain. The two genes are separated by a self-cleaving thosea-asigna virus (T2A) peptide. The vector encoding for the FoxP3 gene alone is a bi-directional LV encoding for the FoxP3 gene in sense (PGK promoter) and for the enhanced Green Fluorescent Protein (eGFP) in antisense (minimal CMV or mCMV promoter). Other LV components: LTR long terminal repeat, SD splice donor, SA splice acceptor, GA gag-pol element, RRE REV responsive element, cPPT central polypurine tract, pA polyadenilation signal, CTE constitutive transport element, WPRE woodchuck hepatitis virus post-transcriptional regulatory element.

[0059] B. Transduction efficiency of CAR19.28z LV, Fox19CAR LV and FoxP3 LV measured as percentage of either CAR+or GFP+cells among CD4+CD25+CD127_FoxP3+lymphocytes at day +14 after initial stimulation. Recombinant CD19 for UT-, 19CAR- and Fox19CAR-Tregs and GFP for FoxP3 LV respectively, were employed as transduction markers to estimate the percentage of CAR+cells. N=6 for UT-, Fox19CAR- and 19CAR-Tregs. N=3 for FoxP3-Tregs. One-way ANOVA with Tukey correction for multiple comparison. *** = p-value <0.001 .

[0060] C. Representative flow cytometry plot for co-localization of the CAR construct and FoxP3 in Fox19CAR-Tregs compared to untransduced (UT) cells. CAR expression was assessed using the human rCD19.

[0061] D. Expansion rate of UT-, 19CAR-, Fox19CAR- and FoxP3-Tregs, assessed at day +14 since the initial stimulation. The fold increase was calculated dividing the number of cells at day 14 with that at day 0. N=6 for UT-, Fox19CAR- and 19CAR-Tregs. N=3 for FoxP3-Tregs. Oneway ANOVA with Tukey correction for multiple comparison.

[0062] E. Polyclonal suppressive capacities of UT-, 19CAR- and Fox19CAR-Tregs. Regulatory T cells were co-cultured with autologous Peripheral Blood Mononuclear Cells (PBMCs), previously stained with a proliferation dye, in the presence of anti-CD3 / CD28 stimulation beads (T cell: beads = 10:1) at various effector-to-target ratios. Seven days after the stimulation their proliferation was assessed by flow cytometry. Results are expressed as Suppression Index, calculated as Suppression index = [1-(PBMCs’ proliferation with Tregs) / (PBMCs’ proliferation alone)] * 100. UT conventional T lymphocytes, cultured with anti- CD3 / CD28 stimulation beads and IL-7 and IL-15, has been included as negative control. N=4 for UT-, Fox19CAR- and 19CAR-Tregs. N=3 for UT conventional T cells. Two-way ANOVA with Tukey correction for multiple comparison.

[0063] F. Antigen-specific suppressive capacities of UT-, 19CAR-, Fox19CAR- and FoxP3-Tregs. Regulatory T cells were co-cultured with autologous B lymphocytes at various effector-to- target ratios. B cells were stimulated with a combination of anti-CD40L and anti-lg antibodies and were stained with a proliferation dye. After 3 days B cell proliferation was assessed by flow cytometry. Results are expressed as Suppression index = [1-(B cell proliferation with Tregs) / (B cell proliferation alone)] * 100. N=6 for UT-, Fox19CAR- and 19CAR-Tregs. N=3 for FoxP3-Tregs. Two-way ANOVA with Tukey correction for multiple comparison. * = p-value <0.05, ** = p-value <0.01 , **** = p-value <0.0001.

[0064] All the results are expressed as mean ± standard deviation.

[0065] Figure 2 - In vitro safety profile of Fox19CAR engineered CD4+CD25‘ T cells

[0066] A. Schematic representation of CD4+CD25_T cell engineering with different LV vectors and stimulation protocols. After the isolation by magnetic separation, CD4+CD25_T lymphocytes were stimulated with anti-CD3 / CD28 beads with two different cytokine cocktails: IL-7 / IL-15 (7 / 15) and IL-2 + rapamycin (2R). The first group was transduced with either CAR19.28z LV or Fox19CAR LV, whereas the second one received only the latter LV.

[0067] B. Schematic representation of Treg engineering with two different LV vectors. After the isolation by magnetic separation, CD4+CD25+T lymphocytes were stimulated with anti- CD3 / CD28 beads and maintained in IL-2 and rapamycin (2R) culture condition. Cells were transduced with either CAR19.28z LV or Fox19CAR LV.

[0068] C. Expansion rate of engineered and untransduced CD4+CD25_T cells in the presence of two different cytokine cocktails and transduced with two different LV vectors, assessed at day 14 since the initial stimulation. The fold increase was calculated dividing the number of cells at day 14 with that at day 0. N=9 for each group. One-way ANOVA with Tukey correction for multiple comparison. *** = p-value <0.001 , **** = p-value <0.0001.

[0069] D. Polyclonal suppressive capacities of either engineered or untransduced CD4+CD25_derived T cells. Cells were co-cultured with autologous Peripheral Blood Mononuclear Cells (PBMCs), previously stained with a proliferation dye, in the presence of anti-CD3 / CD28 stimulation beads (T cell: beads = 10:1) at various effector-to-target ratios. Seven days after the stimulation their proliferation was assessed by flow cytometry. The result is expressed as Suppression index, calculated as Suppression Index = [1-(PBMCs’ proliferation with Tregs) / (PBMCs’ proliferation alone)] * 100. N=4 for each group. Two-way ANOVA with Tukey correction for multiple comparison. * = p-value <0.05.

[0070] E. Heat map representing the relative abundance of each meta-cluster among different in vitro-expanded CD4+CD25+and, CD4+CD25_derived cellular products. Fifteen meta-clusters were identified using cytoChain for the unsupervised analysis of flow cytometry data. The darker the color, the higher the abundance of the cluster within the group. N=5.

[0071] F. Heat map reporting the intensity of the evaluated markers in each cluster. Engineered or untransduced Tregs and CD4+CD25_T lymphocyte phenotypes were analyzed by flow cytometry. Subsequently, samples were analyzed with cytoChain for an unsupervised scrutiny. After the removal of the noise due to the instrument instability, the expression of each marker was assessed by the software and 15 meta-clusters were identified according to the various combinations. The intensity of each marker was normalized, scaled to range from 0 (minimum) to 1 (maximum) and a color gradient was generated. For each cluster, the intensity of each marker is reported. Red and cyan boxes highlight the 4 most expressed meta-clusters as reported in figure 2E. N=5.

[0072] All the results are expressed as mean ± standard deviation.

[0073] Figure 3 - Efficacy of Fox19CAR-Tregs in an in vivo humanized mouse model of SLE

[0074] A. Generation of the humanized mouse model of SLE. 1 -day-old pups of NSG mice were irradiated and transplanted with 0.8-1 x105human cord-blood stem cells / mouse injected intraliver. The engraftment was monitored assessing the presence and the composition of human leukocytes on peripheral blood weekly by flow cytometry. After the establishment of a human immune system, mice were injected i.p. with pristane to induce a chronic inflammation. After 3 weeks, UT-, Fox19CAR-T regs or PBS were injected and their kinetic and the levels of human leukocytes in the peripheral blood were monitored weekly by flow cytometry. Mice were sacrificed 15 weeks after humanization.

[0075] B. Longitudinal assessment of human B lymphocytes in peripheral blood. Human B cells were identified as hCD45+CD19+lymphocytes. Absolute B cell counts were assessed by flow cytometry. Pristane injection is indicated with an arrow. N=38 mice. One-way ANOVA with Tukey correction for multiple comparison. * = p-value <0.05.

[0076] C. Longitudinal assessment of circulating Fox19CAR-Tregs in mouse peripheral blood after their injection. CAR+cells were identified with FITC-conjugated recombinant CD19 (rCD19). CAR-Treg absolute counts were assessed by flow cytometry. N=16 mice.

[0077] D. Absolute numbers of circulating human B lymphocytes in peripheral blood of humanized mice in the different groups of treatment after the injection of Fox19CAR-Tregs, UT-Tregs or PBS. Human B cells were identified as hCD45+CD19+lymphocytes. Absolute B cell counts were assessed by flow cytometry. N=34 (16 CAR-Tregs, 11 UT-Tregs, 7 PBS). Two-way ANOVA test with Tukey correction for multiple comparisons. * = p-value <0.05, ** = p-value <0.01.

[0078] All the results are expressed as mean ± standard deviation.

[0079] Mean IL-10 (E), IFN-g (F) and IL-6 (G) levels in peripheral blood before and 3 days after the injection of Fox19CAR-Tregs, UT-Tregs or PBS. As control, humanized mice not treated with pristane (NT) are included. Cytokine levels were assessed employing a bead-based immunoassay (Biolegend Legendplex 13-plex kit) according to the manufacturer’s instructions. Each dot represents a single mouse. N=37 (16 CAR-Tregs, 11 UT-Tregs, 7 PBS, 3 NT). Wilcoxon test for non-parametric paired data. * = p-value <0.05.

[0080] Figure 4 - Fox19CAR-Tregs immunomodulatory properties

[0081] A. Frequency of human anti-dsDNA auto-antibodies in mouse serum in the different groups of treatment. Auto-antibody presence was evaluated with immunofluorescence on HEK293T cells. Positivity was defined as the presence of signal with at a dilution of 1 :10 or higher. N=34 (16 CAR-Tregs, 11 UT-Tregs, 7 PBS). Chi-squared test. * = p-value <0.05

[0082] B. Percentage of regulatory T cells in the different organs at sacrifice in the different groups of treatment. Tregs were defined as CD3+CD4+CD25+CD127_FoxP3+lymphocytes and their frequency was assessed by flow cytometry. As control, humanized mice not treated with pristane (NT) are included. N=37 (16 CAR-Tregs, 11 UT-Tregs, 7 PBS, 3 NT). One-way ANOVA test with Tukey correction for multiple comparisons.

[0083] C. Percentage of total human CD45+cells and of the principal human T and B cell sub populations in the spleen in the different groups of treatment at sacrifice. The frequency of cells was assessed by flow cytometry. T cells were defined as CD3+cells. B cells were defined as: pre-B cells CD19+CD20’CD27’ cells, naive B cells CD19+CD20+CD27_cells, memory B cells CD19+CD20+CD27+cells, plasmablasts CD19+CD20’CD27+cells, plasma cells CD138+cells. The results are expressed as mean ± standard deviation. As control, humanized mice not treated with pristane (NT) are included. N=37 (16 CAR-Tregs, 11 UT-Tregs, 7 PBS, 3 NT). One-way ANOVA test with Tukey correction for multiple comparisons. * = p-value <0.05, ** = p-value <0.01 , *** = p-value <0.001.

[0084] D. Frequency and severity of the inflammatory lesions in the spleen at the sacrifice in the different groups of treatment. As control, humanized mice not treated with pristane (NT) are included. N=37 (16 CAR-Tregs, 11 UT-Tregs, 7 PBS, 3 NT). Chi-squared test.

[0085] E. Frequency and severity of the pulmonary inflammatory lesions at sacrifice in the different groups of treatment. As control, humanized mice not treated with pristane (NT) are included. N=37 (16 CAR-Tregs, 11 UT-Tregs, 7 PBS, 3 NT). Chi-squared test.

[0086] F. Representative pictures of the spleen in 4 different mice, one per group of treatment. Asterisk indicates the red pulp. Arrowhead indicates areas of granulomatous inflammation. Reference bar 100 pm.

[0087] G. Representative pictures of the lungs in 4 different mice, one per group of treatment. Asterisk indicates areas of granulomatous inflammation. Arrowhead indicates blood vessels. Reference bar 100 pm.

[0088] At euthanasia, tissues were stocked in 10% formalin. Specimens were prepared and stained with the hematoxylin-eosin coloration. Each organ was evaluated blindly by an expert pathologist and a specific score was assigned based on the extension of the lesions ranging from 0 (absent) to 5 (>80% of extension).

[0089] Figure 5 - Efficacy and safety of multiple Fox19CAR-Tregs in reshaping the B cell compartment

[0090] A. Generation of the humanized mouse model of SLE. Eight-weeks-old SGM-3 mice were irradiated and transplanted with 0.8-1 x 105human cord-blood stem cells / mouse intravenously injected. The engraftment was monitored assessing the presence and the composition of human leukocytes on peripheral blood weekly by flow cytometry. After the establishment of a human immune system, mice were injected i.p. with pristane to induce a chronic inflammation. After 3 weeks, Fox19CAR-Tregs, 19CAR-Tconvs or PBS were injected and their kinetic and the levels of human leukocytes in the peripheral blood were monitored weekly by flow cytometry. Mice were sacrificed 14 weeks after pristane administration.

[0091] B. Longitudinal assessment of circulating Fox19CAR-Tregs and 19CAR-Tconvs in mouse peripheral blood after their injection 3 weeks after pristane (early treatment). CAR+cells were identified with FITC-conjugated recombinant CD19 (rCD19). Absolute counts were assessed by flow cytometry. Black arrow indicates engineered cell injection. Green arrow denotes CAR- Treg second infusion. Results are expressed as mean ± standard deviation. N=11 (5 CAR- Tregs, 6 CAR-Tconvs). Two-way ANOVA test with Tukey correction for multiple comparisons.

[0092] C. Absolute numbers of circulating human B lymphocytes in peripheral blood of humanized mice in the different groups of early treatment (3 weeks after pristane) after the injection of Fox19CAR-Tregs, 19CAR-Tconvs or PBS. Human B cells were identified as hCD45+CD19+lymphocytes. Absolute B cell counts were assessed by flow cytometry. Black arrow indicates engineered cell injection. Green arrow denotes CAR-Treg second infusion. Results are expressed as mean ± standard deviation. N=17 (5 CAR-Tregs, 6 CAR-Tconvs, 6 PBS). Two- way ANOVA test with Tukey correction for multiple comparisons. * = p-value <0.05.

[0093] D. Heat map reporting the cytokine levels in peripheral blood of treated mice. Animals were single-injected with Fox19CAR-Tregs, 19CAR-Tconvs or PBS 3 weeks after pristane. Sera at baseline and 7 days after treatment were collected and analyzed with a bead-based assay (Biolegend Legendplex 13-plex kit) according to the manufacturer’s instructions. The amount of each cytokine was normalized across the various groups, scaled to range from 0 (minimum) to 1 (maximum), and a color gradient was generated. For each group, the relative abundance of each cytokine is reported. N=17 (5 CAR-Tregs, 6 CAR-Tconvs, 6 PBS).

[0094] E. Mean IL- 10 levels in peripheral blood before and 7 days after the injection of Fox19CAR- Tregs, 19CAR-Tconvs or PBS, 3 weeks after pristane. Cytokine levels were assessed employing a bead-based immunoassay (Biolegend Legendplex 13-plex kit) according to the manufacturer’s instructions. Results are expressed as mean ± standard deviation. N=17 (5 CAR-Tregs, 6 CAR-Tconvs, 6 PBS). Wilcoxon test for non-parametric paired data. * = p-value <0.05, **** = p-value <0.0001.

[0095] F. Percentage of CAR+cells in the bone marrow and the spleen in the different groups of treatment at sacrifice. Early treatment indicates animals treated 3 weeks after pristane, whereas late treatment denotes those injected 8 weeks after pristane. CAR+cells were identified with FITC-conjugated recombinant CD19 (rCD19) and their frequency was assessed by flow cytometry. The results are expressed as mean ± standard deviation. N=21 (5 early CAR-Tregs, 5 late CAR-Treg, 6 early CAR-Tconvs, 5 late CAR-Tconvs). One-way ANOVA test with Tukey correction for multiple comparisons. G. Percentage of total B cells and their sub-populations in the bone marrow in the different groups of treatment at sacrifice. Early treatment indicates animals treated 3 weeks after pristane, whereas late treatment denotes those injected 8 weeks after pristane. The frequency of cells was assessed by flow cytometry. B cells were identified as hCD45+CD19+lymphocytes. B cell sub-populations were defined as: pre-B cells CD19+CD20’CD27’ cells, naive B cells CD19+CD20+CD27_cells, memory B cells CD19+CD20+CD27+cells, plasmablasts CD19+CD20'CD27+cells, plasma cells CD138+cells. The results are expressed as mean ± standard deviation. N=27 (5 early CAR-Tregs, 5 late CAR-Tregs, 6 early CAR- Tconvs, 5 late CAR-Tconvs, 6 PBS). One-way ANOVA test with Tukey correction for multiple comparisons. * = p-value <0.05, ** = p-value <0.01.

[0096] H. Percentage of total B cells and their sub-populations in the spleen in the different groups of treatment at sacrifice. Early treatment indicates animals treated 3 weeks after pristane, whereas late treatment denotes those injected 8 weeks after pristane. The frequency of cells was assessed by flow cytometry. B cells were identified as hCD45+CD19+lymphocytes. B cell sub-populations were defined as: pre-B cells CD19+CD20'CD27' cells, naive B cells CD19+CD20+CD27' cells, memory B cells CD19+CD20+CD27+cells, plasmablasts CD19+CD20'CD27+cells, plasma cells CD138+cells. The results are expressed as mean ± standard deviation. N=27 (5 early CAR-Tregs, 5 late CAR-Tregs, 6 early CAR-Tconvs, 5 late CAR-Tconvs, 6 PBS). One-way ANOVA test with Tukey correction for multiple comparisons. * = p-value <0.05, ** = p-value <0.01.

[0097] I. Frequency and severity of the inflammatory lesions at the sacrifice in the different groups of early-administered treatment (3 weeks after pristane). At euthanasia, tissues were stocked in 10% formalin. Specimens were prepared and stained with the hematoxylin-eosin coloration. The spleen, the liver and the bone marrow were evaluated blindly by an expert pathologist a specific global score was assigned based on the extension of the lesions ranging from 0 (absent) to 5 (>80% of extension). N=17 (5 CAR-Tregs, 6 UT-Tregs, 6 PBS). Chi-squared test.

[0098] Figure 6

[0099] A. CAR19.28z bi-directional lentiviral vector (LV) schematic representation. The anti-CD19 second-generation CAR is under the control of a Phosphoglycerate Kinase (PGK) promoter. The CAR is composed by an anti-CD19 single-chain fragment variant (scFv), a linker peptide derived from the Nerve Growth Factor Receptor (NGFR) and the transmembrane and the intracellular portion of the human CD28 fused with the CD3 zeta-chain. In antisense, GFP is under the control of the minimal CMV promoter (mCMV). Other LV components: LTR long terminal repeat, SD splice donor, SA splice acceptor, GA gag-pol element, RRE REV responsive element, cPPT central polypurine tract, pA polyadenilation signal, CTE constitutive transport element, WPRE woodchuck hepatitis virus post-transcriptional regulatory element.

[0100] B. Schematic representation of the CAR-Treg generation protocol. CD4+CD25+T cells were isolated from peripheral lymphocytes of healthy donors with magnetic separation. The cells were activated in vitro with anti-CD3 / CD28 stimulation beads and expanded in the presence of IL-2 600 ILI / mL and rapamycin 100 nM. IL-2 was replenished every 2-3 days. After 2 days, cells were transduced with the lentiviral vector (LV) encoding for a second-generation anti- CD19 (CAR19.28z LV). Starting from day +14, functional assays were performed.

[0101] C. Efficiency of Treg transduction with CAR19.28z LV. The frequency of transduced cells was assessed by flow cytometry 14 and 21 days after stimulation, respectively. CAR+cells were identified according to the NGFR expression. Tregs were defined as CD3+CD4+CD25+CD127_FoxP3+cells. N=4. Paired Wilcoxon test for non-parametric variables.

[0102] D. Representative plot of 19CAR-Treg transduction. The histograms showed the differential GFP expression between 19CAR- and UT-Tregs.

[0103] E. Representative plot of CAR detection methods. The dot plot showed the CAR detection using 3 different methods: recombinant CD19 (rCD19) in PE, anti-NGFR (Nerve Growth Factor Receptor) antibody in PE-Cy7 and GFP (Green Fluorescent Protein). The rCD19 and anti- NGFR antibody signals are reported as dots, whereas the Mean Fluorescence Intensity of the GFP signal is reported as a color gradient, ranging from 0 (blue) to 262856 (red).

[0104] F. Expansion rate of UT and 19CAR-Tregs at day +14. The fold increase was calculated by dividing the number of cells at day +14 with that at day +0. N=4. Mann-Whitney test.

[0105] G. Percentage of regulatory T cells among T lymphocytes in either UT- or 19CAR-Tregs at day +14 and at day +21. Regulatory T cells were defined as CD3+CD4+CD25+CD127'FoxP3+cells and were identified by flow cytometry. N=4. Mann-Whitney test.

[0106] H. Engineered Treg suppressive capacities. Regulatory T cells were co-cultured with autologous Peripheral Blood Mononuclear Cells (PBMCs), previously stained with a proliferation dye, in the presence of anti-CD3 / CD28 stimulation beads (T cell: beads = 10:1) at an effector-to-target ratio of 1 :1. Seven days after the stimulation their proliferation was assessed by flow cytometry. Results are expressed as Suppression Index, calculated as Suppression index = [1 -(PBMCs’ proliferation with Tregs) / (PBMCs’ proliferation alone)] * 100. N=4. Student’s T-test.

[0107] I. Antigen-specific suppressive capacities of 19CAR-Tregs. UT- and 19CAR-Tregs were cocultured with autologous B lymphocytes at a Treg : B cell ratio of 1 :1 , in the presence of irradiated 3T3 cells, a murine fibroblast cell line, transduced with the CD40L to provide an exclusive stimulation signal to B cells. Prior to the culture, B lymphocytes were stained with a proliferation dye and 3 days after stimulation their proliferation was assessed by flow cytometry. Results are expressed as percentages of proliferating B lymphocytes among the groups. N = 5 for each group. One-way ANOVA with T ukey correction for multiple comparison. * = p-value <0.05.

[0108] J. Killing capacities of different lymphocyte subpopulations. 19CAR-Tregs, UT- and 19CAR- conventional T lymphocytes were co-cultured with CD19+ALL-CM tumor cells at an effector- to-target ratio of 1 :5. After 3 days, the number of viable tumor cells was evaluated by flow cytometry. Results are expressed as tumor fold increase, calculated as Tumor Fold Increase = (number of viable ALL-CM cells in the various conditions) / (number of viable ALL-CM alone). N=5 for each group. One-way ANOVA with Tukey correction for multiple comparison. * = p- value <0.05.

[0109] All the results are expressed as mean ± standard deviation.

[0110] Figure 7

[0111] A. Representative plot of CAR transduction. The contour plot showed the CAR expression in UT- (blue), 19CAR- (orange) and Fox19CAR-Tregs (green), detected with recombinant CD19 (rCD19) in PE.

[0112] B. Percentage of regulatory T cells among T lymphocytes in UT-, 19CAR-, Fox19CAR- or FoxP3-Tregs at day +21. Regulatory T cells were defined as CD3+CD4+CD25+CD127'FoxP3+cells and were identified by flow cytometry. N=5 for UT-, Fox19CAR- and 19CAR-Tregs. N=3 for FoxP3-Tregs. One-way ANOVA with Tukey correction for multiple comparison.

[0113] Figure 8

[0114] A. Schematic representation of naive- or CD4+CD25+-derived Treg engineering with two different LV vectors. After the isolation by magnetic separation, CD4+CD25+T lymphocytes were divided into two fractions. The first fraction was stimulated with anti-CD3 / CD28 beads, maintained in IL-2 and rapamycin (2R) culture condition and transduced with the Fox19CAR LV. The second fraction was further sorted to isolate CD127_CD45RA+cells using a cell sorter and subsequently stimulated with anti-CD3 / CD28 beads, maintained in IL-2 without rapamycin and transduced with the CAR19.28z LV.

[0115] B. Expansion rate of naive- or CD4+CD25+-derived Tregs transduced with CAR19.28z LV or Fox19CAR LV, respectively, or left untransduced, assessed at day +14 since the initial stimulation. The fold increase was calculated dividing the number of cells at day 14 with that at day 0. N=3 for each group. One-way ANOVA with Tukey correction for multiple comparison. = p-value <0.05.

[0116] C. Transduction efficiency of CAR19.28z LV and Fox19CAR LV on naive- or CD4+CD25+- derived Tregs measured as percentage of CAR+cells among CD4+CD25+CD127'FoxP3+lymphocytes at day +14 after initial stimulation. Recombinant CD19 (rCD19) was employed as transduction markers to estimate the percentage of CAR+cells. N=3 for each group. Oneway ANOVA with Tukey correction for multiple comparison.

[0117] D. Percentage of regulatory T cells among T lymphocytes in either naive- or CD4+CD25+- derived Tregs transduced with CAR19.28z LV or Fox19CAR LV, respectively, or left untransduced, assessed at day +21 since the initial stimulation. Regulatory T cells were defined as CD3+CD4+CD25+CD127_FoxP3+cells and were identified by flow cytometry. N=3. One-way ANOVA test with Tukey correction for multiple comparisons.

[0118] E. Antigen-specific suppressive capacities of naive- or CD4+CD25+-derived Tregs transduced with CAR19.28z LV or Fox19CAR LV, respectively, or left untransduced. Regulatory T cells were co-cultured with autologous B lymphocytes at various effector-to-target ratios. B cells were stimulated with a combination of anti-CD40L and anti-lg antibodies and were stained with a proliferation dye. After 3 days B cell proliferation was assessed by flow cytometry. Results are expressed as Suppression index = [1-(B cell proliferation with Tregs) / (B cell proliferation alone)] * 100. N=3 for each group. Two-way ANOVA with Tukey correction for multiple comparison. * = p-value <0.05.

[0119] F. Heat map reporting the cytokine profile secreted by engineered or untransduce dTregs upon antigen-specific stimulation. Naive- or CD4+CD25+-derived Tregs transduced with CAR19.28z LV or Fox19CAR LV, respectively, or left untransduced were cultured with autologous B cells. After 3 days, culture supernatants were collected and analyzed with a bead-based immunoassay (Biolegend Legendplex 13-plex kit) according to the manufacturer’s instructions. The amount of each cytokine was normalized across the various groups, scaled to range from 0 (minimum) to 1 (maximum), and a color gradient was generated. For each group, the relative abundance of each cytokine is reported. N=3 for each group. Kruskall- Wallis test.

[0120] All the results are expressed as mean ± standard deviation.

[0121] Figure 9

[0122] A. Transduction efficiency of CD4+CD25_engineered cells with either CAR19.28z LV or Fox19CAR LV upon culture in media supplemented with IL2 and rapamycin (2R) or IL7 and IL15 (7 / 15). The percentages of CAR+cells were assessed by flow cytometry using recombinant CD19 (rCD19) after 14 days of culture. N=9 for each group. One-Way ANOVA test with Tukey correction for multiple comparison.

[0123] B. Representative flow cytometry plot for co-localization of CAR and FoxP3 in 2R Fox19CAR and 7 / 15 UT cells obtained from CD4+CD25_sorted lymphocytes. CAR expression was assessed by flow cytometry at day +14 using the recombinant CD19 (rCD19).

[0124] C. Percentage of CD4+CD25+CD127'FoxP3+cells measured at day +21 in the different cellular products obtained upon manipulation of CD4+CD25_cells, culture in IL-2 and rapamycin (2R)- or IL-7 and IL-15 (7 / 15)- supplemented media, and transduced with CAR19.28z, Fox19CAR LV or left untreated. N=6 for each group. One-way ANOVA test with Tukey correction for multiple comparison.

[0125] D. Evaluation of the principal Treg-associated markers on the cellular products originated from sorted CD4+CD25+(Tregs) and CD4+CD25_cells. Marker expressions were assessed by flow cytometry after 21 days of culture and reported as Mean Fluorescent Index (MFI). N = 5 for each group. One-way ANOVA test with Tukey correction for multiple comparison. * = p-value <0.05, ** = p-value <0.01 , *** = p-value <0.001 , **** = p-value <0.0001.

[0126] E. Principal Component Analysis (PCA) showing the phenotype similarity of different samples derived from either engineered or untransduced T regs and CD4+CD25'-derived cells. Samples were analyzed by multi-parametric flow cytometry. Similarity was calculated employing cytoChain according to the similarity of expression of the evaluated markers. MDS = multidimensionality scaling. N=5 for each group.

[0127] All the results are expressed as mean ± standard deviation.

[0128] Figure 10

[0129] A. Longitudinal assessment of circulating human leukocytes in peripheral blood. Human leukocytes were identified as hCD45+cells. Absolute hCD45+cell counts were assessed by flow cytometry. Pristane injection is indicated with an arrow. N=38. One-way ANOVA with Tukey correction for multiple comparison.

[0130] B. Longitudinal assessment of human T lymphocytes in peripheral blood. Human T cells were identified as hCD45+CD3+lymphocytes. Absolute T cell counts were assessed by flow cytometry. Pristane injection is indicated with an arrow. N=38. One-way ANOVA with Tukey correction for multiple comparison. **** = p-value <0.0001.

[0131] C. Absolute numbers of circulating human leukocytes in peripheral blood of humanized mice in the different groups of treatment after the injection of Fox19CAR-Tregs, UT Tregs or PBS. Human leukocytes were identified as hCD45+cells. Absolute CD45+cell counts were assessed by flow cytometry. N=34 (16 CAR-Tregs, 11 UT-Tregs, 7 PBS). Two-way ANOVA test with Tukey correction for multiple comparisons.

[0132] D. Absolute numbers of circulating human T lymphocytes in peripheral blood of humanized mice in the different groups of treatment after the injection of Fox19CAR-Tregs, UT Tregs or PBS. Human T cells were identified as hCD45+CD3+lymphocytes. Absolute T cell counts were assessed by flow cytometry. N=34 (16 CAR-Tregs, 11 UT-Tregs, 7 PBS). Two-way ANOVA test with Tukey correction for multiple comparisons.

[0133] All the results are expressed as mean ± standard deviation.

[0134] Mean I L-1 b (E), IFN-a2 (F), TNF-a (G), CCL2 (H), IL-8 (I), IL-12p70 (J), IL-17A (K), IL-18 (L), IL-23 (M) and IL-33 (N) levels in peripheral blood before and 3 days after the injection of Fox19CAR-Treg, UT Tregs or PBS. As control, humanized mice not treated with pristane (NT) are included. Cytokine levels were assessed employing a bead-based immunoassay (Biolegend Legendplex 13-plex kit) according to the manufacturer’s instructions. Each dot represents a single mouse. N=37 (16 CAR-Tregs, 11 UT-Tregs, 7 PBS, 3 NT). Paired Wilcoxon test for non-parametric data.

[0135] Figure 11

[0136] A. Evaluation of the principal Treg-associated markers expressed by Tregs in the spleen, in the bone marrow and in kidneys in different groups of treatment. Tregs were identified as CD3+CD4+CD25+CD127'FoxP3+cells. Marker expressions were assessed by flow cytometry at sacrifice. Their expression is reported as Mean Fluorescent Index (MFI). LAP, GARP = Glycoprotein A Repetitions Predominant, CTLA-4 = Cytotoxic T Lymphocyte Antigen 4, ICOS = Inducible Costimulator, GITR = Glucocorticoid-induced TNFR-related, TIGIT = T cell Immunoreceptor with Ig and ITIM domains. As control, humanized mice not treated with pristane (NT) are included. N=37 (16 CAR-Tregs, 11 UT-Tregs, 7 PBS, 3 NT). One-way ANOVA test with Tukey correction for multiple comparisons. * = p-value <0.05.

[0137] B. Percentage of total human CD45+cells and of the principal human T and B cell subpopulations in the bone marrow in the different groups of treatment at sacrifice. The frequency of cells was assessed by flow cytometry. T cells were defined as CD3+cells. B cells were defined as: pre-B cells CD19+CD20'CD27' cells, naive B cells CD19+CD20+CD27_cells, memory B cells CD19+CD20+CD27+cells, plasmablasts CD19+CD20'CD27+cells, plasma cells CD138+cells. The results are expressed as mean ± standard deviation. As control, humanized mice not treated with pristane (NT) are included. N=37 (16 CAR-Tregs, 11 UT- Tregs, 7 PBS, 3 NT). One-way ANOVA test with Tukey correction for multiple comparisons. C. Percentage of total human CD45+cells and of the principal human T and B cell subpopulations in kidneys in the different groups of treatment at sacrifice. The frequency of cells was assessed by flow cytometry. T cells were defined as CD3+cells. B cells were defined as: pre-B cells CD19+CD20’CD27’ cells, naive B cells CD19+CD20+CD27_cells, memory B cells CD19+CD20+CD27+cells, plasmablasts CD19+CD20’CD27+cells, plasma cells CD138+cells. The results are expressed as mean ± standard deviation. As control, humanized mice not treated with pristane (NT) are included. N=37 (16 CAR-Tregs, 11 UT-Tregs, 7 PBS, 3 NT). One-way ANOVA test with Tukey correction for multiple comparisons. ** = p-value <0.01.

[0138] D. Frequency and grade of human reconstitution in the spleen at the sacrifice in the different groups of treatment, evaluated as representation of red and white pulp. N=37 (16 CAR-Tregs, 11 UT-Tregs, 7 PBS, 3 NT). Chi-squared test.

[0139] E. Frequency and severity of the tubular degeneration in kidneys at the sacrifice in the different groups of treatment. N=37 (16 CAR-Tregs, 11 UT-Tregs, 7 PBS, 3 NT). Chi-squared test.

[0140] F. Representative pictures of the kidneys in 4 different mice, one per group of treatment. Asterisk indicates areas of tubular degeneration and vacuolization. Reference bar 50 pm.

[0141] At euthanasia, tissues were stocked in 10% formalin. Specimens were prepared and stained with the hematoxylin-eosin coloration. Each organ was evaluated blindly by an expert pathologist and a specific score was assigned based on the extension of the lesions ranging from 0 (absent) to 5 (>80% of extension).

[0142] G. Immunohistochemistry staining for human CD3 on the spleen, one per group of treatment.

[0143] H. Immunohistochemistry staining for human CD20 on the spleen, one per group of treatment.

[0144] Figure 12

[0145] A. Absolute numbers of circulating human leukocytes in peripheral blood of humanized mice in the different groups of treatment after the injection of Fox19CAR-Tregs, 19CAR-Tconvs or PBS. Human leukocytes were identified as hCD45+cells. Absolute CD45+cell counts were assessed by flow cytometry. Black arrow indicates engineered cell injection. Green arrow denotes CAR-Treg second infusion. Results are expressed as mean ± standard deviation. N=17 (5 CAR-Tregs, 6 CAR-Tconvs, 6 PBS). Two-way ANOVA test with Tukey correction for multiple comparisons.

[0146] B. Absolute numbers of circulating human T lymphocytes in peripheral blood of humanized mice in the different groups of treatment after the injection of Fox19CAR-Tregs, 19CAR- Tconvs or PBS. Human T cells were identified as hCD45+CD3+lymphocytes. Absolute T cell counts were assessed by flow cytometry. Black arrow indicates engineered cell injection. Green arrow denotes CAR-Treg second infusion. Results are expressed as mean ± standard deviation. N=17 (5 CAR-Tregs, 6 CAR-Tconvs, 6 PBS). Two-way ANOVA test with Tukey correction for multiple comparisons.

[0147] C. Generation of the humanized mouse model of SLE. Eight-weeks-old SGM-3 mice were irradiated and transplanted with 0.8-1 x 105human cord-blood stem cells / mouse intravenously injected. The engraftment was monitored assessing the presence and the composition of human leukocytes on peripheral blood weekly by flow cytometry. After the establishment of a human immune system, mice were injected i.p. with pristane to induce a chronic inflammation. After 8 weeks, Fox19CAR-Tregs, 19CAR-Tconvs or PBS were injected and their kinetic and the levels of human leukocytes in the peripheral blood were monitored weekly by flow cytometry. Mice were sacrificed 14 weeks after pristane administration.

[0148] D. Longitudinal assessment of circulating Fox19CAR-Tregs and 19CAR-Tconvs in mouse peripheral blood after their injection 8 weeks after pristane (late treatment). CAR+cells were identified with FITC-conjugated recombinant CD19 (rCD19). Absolute counts were assessed by flow cytometry. Black arrow indicates engineered cell injection. Results are expressed as mean ± standard deviation. N=10 (5 CAR-Tregs, 5 CAR-Tconvs). Two-way ANOVA test with Tukey correction for multiple comparisons.

[0149] E. Absolute numbers of circulating human B lymphocytes in peripheral blood of humanized mice in the different groups of late treatment (8 weeks after pristane) after the injection of Fox19CAR-Tregs, 19CAR-Tconvs or PBS. Human B cells were identified as hCD45+CD19+lymphocytes. Absolute B cell counts were assessed by flow cytometry. Black arrow indicates engineered cell injection. Results are expressed as mean ± standard deviation. N=16 (5 CAR- Tregs, 5 CAR-Tconvs, 6 PBS). Two-way ANOVA test with Tukey correction for multiple comparisons.

[0150] F. Heat map reporting the cytokine levels in peripheral blood of treated mice. Animals were single-injected with Fox19CAR-Tregs, 19CAR-Tconvs or PBS 8 weeks after pristane administration or received two FoxCAR-Treg doses 3 and 8 weeks after pristane, respectively. Sera at baseline and 7 days after treatment of single-injected animals and of those that received the Fox19CAR-Treg re-administration 8 weeks after pristane were collected. Samples were analyzed with a bead-based assay (Biolegend Legendplex 13-plex kit) according to the manufacturer’s instructions. The amount of each cytokine was normalized across the various groups, scaled to range from 0 (minimum) to 1 (maximum), and a color gradient was generated. For each group, the relative abundance of each cytokine is reported. N=21 (5 late CAR-Tregs, 5 re-infused CAR-Tregs, 5 late CAR-Tconvs, 6 PBS). G. Mean IL-10 levels in peripheral blood before and 7 days after the single-injection of Fox19CAR-Tregs, 19CAR-Tconvs or PBS and Fox19CAR-Treg re-infusion, 8 weeks after pristane. Cytokine levels were assessed employing a bead-based immunoassay (Biolegend Legendplex 13-plex kit) according to the manufacturer’s instructions. Results are expressed as mean ± standard deviation. N=21 (5 late CAR-Tregs, 5 re-infused CAR-Tregs, 5 late CAR- Tconvs, 6 PBS). Wilcoxon test for non-parametric paired data. * = p-value <0.05, **** = p- value <0.0001.

[0151] H. Percentage of total human CD45+cells in the bone marrow and the spleen in the different groups of treatment at sacrifice. Early treatment indicates animals treated 3 weeks after pristane, whereas late treatment denotes those injected 8 weeks after pristane. The frequency of cells was assessed by flow cytometry. The results are expressed as mean ± standard deviation. N=27 (5 early CAR-Tregs, 5 late CAR-Treg, 6 early CAR-Tconvs, 5 late CAR- Tconvs, 6 PBS). One-way ANOVA test with Tukey correction for multiple comparisons.

[0152] I. Percentage of total T and CD4+ T cells in the bone marrow and the spleen in the different groups of treatment at sacrifice. Early treatment indicates animals treated 3 weeks after pristane, whereas late treatment denotes those injected 8 weeks after pristane. The frequency of cells was assessed by flow cytometry. T cells were identified as hCD45+CD3+lymphocytes. The results are expressed as mean ± standard deviation. N=27 (5 early CAR-Tregs, 5 late CAR-Tregs, 6 early CAR-Tconvs, 5 late CAR-Tconvs, 6 PBS). One-way ANOVA test with Tukey correction for multiple comparisons. * = p-value <0.05.

[0153] Figure 13 - In vitro expansion of Luc+Fox19CAR+Treg cells and Luc+ Treg.

[0154] A. Luc-GFP lentiviral vector. Bidirectional lentiviral vector that shows the GFP reporter gene under the murine cytomegalovirus (mCMV) promoter and Luciferase (Luc) under the human Phosphoglycerate kinase (PGK) promoter.

[0155] B. Fox19CAR lentiviral vector. Representation of the Fox19CAR construct with the FoxP3 transgene. The unidirectional lentiviral vector shows the FoxP3 gene under the human PGK promoter. The FoxP3 gene is separated from our second-generation CAR by a self-cleaving thosea-asigna virus (T2A) peptide. The second-generation CAR is composed by a single chain fragment variable (scFv) which confer the specificity against CD19. A hinge region link of the extracellular part with the intracellular portion of the human CD28 fused with the CD3 zetachain. In the picture, the other portions depicted in white represent components essential for the functionality of the viral vector. SD splicing donor, SA splicing acceptor, GA truncated gag sequence, RRE Rev responsive element, cPPT central polypurine tract.

[0156] C. Fold Increase of untransduced (UT, n=8) Tregs, Luc+ Tregs (n=5) and Luc+Fox19CAR- Tregs (n=8) assessed at day +14 of expansion.

[0157] D. Transduction efficiency of Luc-GFP (n=8) and Fox19CAR (n=11 ) measured as percentage of either CAR+ and / or GFP+ cells among cells in culture at day +14. Recombinant CD19 and GFP for Fox19CAR Tregs and GFP for Luc-GFP Tregs, were employed to assess the transduction percentage.

[0158] E. Purity of Luc+Fox19CAR+ cells was around 50% before enrichment.

[0159] F. Purity of Luc+Fox19CAR+ and single Luc+ Treg cells analyzed at flow cytometry pre and post cell sorting. Data are expressed as mean±SD.

[0160] Figure 14 - Humanized NSG mouse model. Generation of the humanized mouse model. 6 weeks-old adult NSG mice were irradiated and transplanted with 1 x 105human cord-blood stem cells / mouse injected intravenously. The engraftment of human stem cells was monitored assessing the presence of human leukocytes on peripheral blood weekly by flow cytometry. After 4 weeks of humanization, Luc+ Treg and Luc+ CAR-Treg cells were injected intravenously and their kinetic was monitored weekly in the peripheral blood by flow cytometry.

[0161] Figure 15 - Weight monitoring. Adult NSG mice have been weighted from week 4 after humanization until the sacrifice. Weights are expressed in grams. mean±SD, n=5.

[0162] Figure 16 - Human CD45+ (hCD45) cells in adult NSG humanized mice. (A-B) Absolute counts of human CD45+and CD14+ cells. n=5. (C-D) Longitudinal assessment of human B and T lymphocytes in peripheral blood. Human B cells were identified as hCD45+ CD19+. Human T lymphocytes were identified as hCD45+CD3+ cells. n=5. Results are expressed as mean ± standard deviation. Two-way ANOVA with Sidak's multiple comparisons test, upvalue 0.0001 **** P-value < 0.0001. The black arrow indicates Luc+CAR-Treg / Luc+ Treg infusion on day 0.

[0163] Figure 17- Injected Luc+ cells monitoring in vivo.

[0164] A. Absolute counts of Luc+human cells from the peripheral blood of the humanized NSG mice. Recombinant CD19 and GFP expression was used for CAR Tregs. GFP for Luc-GFP Tregs, were employed to assess the number of positive cells.

[0165] B. Total flux (in photons per seconds) measured for the Luc+ CART-Tregs injected mice was compared to that of Luc+Tregs injected mice control. Results are expressed as mean± standard deviation. Two-way ANOVA Sidak's multiple comparisons test. n=5.

[0166] Figure 18 - 1 VIS spectrum demonstrate low CAR-Treg persistence in vivo. Intra- ital Imaging System (IVIS) of mice injected with luciferase expressing Treg (Luc+Treg) and CAR-Tregs (Luc+CAR-Treg) at different timepoints. Average flux of IVIS imaging was measured and compared in the CAR-Treg treated mice (n=5) and the Treg treated mice (n=5) every 3 days. Min Radiance: 1.07e7, Max Radiance: 2.26e8.

[0167] Figure 19 - In vitro characterization of Fox19CAR-Tregs with 1 :3 Treg-to-beads stimulation and high IL2 doses.

[0168] A. CAR-Tregs were cultured with 500U / ml (green) and 1000 ll / rnl (pink) of IL2. Both conditions stimulated with 3:1 Treg-to-beads ratio. n=5.

[0169] B. The percentage of Fox19CAR LV transduction was assessed on day 14 of the culturing protocol using flow cytometry. Recombinant CD19 was used to stain CAR-Tregs. n=5.

[0170] C. CAR-Treg purity assessed by using flow cytometry. Based the expression of lineage markers CD3, CD4, CD25, CD127, FOXP3. n=5.

[0171] D. T memory subsets including T central memory (Tern, yellow), T stem cell memory (Tscm, red), T effector memory (Tern, light blue), and T effector memory CD45RA+ (TemRA, blue). n=5.

[0172] E. Exhaustion markers expressed in CAR-Treg cells cultured with varying IL-2 concentrations: 500 ll / rnl (green) and 1000 ll / rnl (pink). Data are represented as Mean±SD,

[0173] F. Polyclonal suppression assay. Results are expressed in terms of Suppression Index. Tukey's multiple comparisons test. n=3. **p-value<0.01. Tconv, conventional T cells.

[0174] Figure 20 - In vitro characterization of Fox19CAR-Tregs with 1:3 Treg-to-beads stimulation and low IL2 doses.

[0175] A. CAR-Tregs were cultured with 500U / ml (green) and 250 ll / rnl (yellow) of IL2. Both conditions stimulated with 3:1 Treg-to-beads ratio. n=5.

[0176] B. The percentage of Fox19CAR LV transduction was assessed on day 14 of the culturing protocol using flow cytometry. Recombinant CD19 was used to stain CAR-Tregs. n=5.

[0177] C. CAR-Treg purity assessed by using flow cytometry. Based the expression of lineage markers CD3, CD4, CD25, CD127, FOXP3. n=5.

[0178] D. T memory subsets including T central memory (Tern, yellow), T stem cell memory (Tscm, red), T effector memory (Tern, light blue), and T effector memory CD45RA+ (TemRA, blue). n=5. E. Exhaustion markers expressed in CAR-Treg cells cultured with varying IL-2 concentrations: 500 ll / ml (green, n=2) and 250 ll / ml (yellow, n=2). Data are represented as Mean±SD,

[0179] F. Polyclonal suppression assay. Results are expressed in terms of Suppression Index. Two- way ANOVA. Tukey's multiple comparisons test. *p-value 0.0102. Tconv, conventional T cells.

[0180] Figure 21 - In vitro characterization of Fox19CAR-Tregs with 1 :1 Treg-to beads stimulation and high IL2 doses.

[0181] A. CAR-Tregs were cultured with 500U / ml and 1 :3 Treg-beads ratio (green) or with 1 :1 Treg- beads ratio and 500U / ml IL2 (orange) or 1000U / ml IL2 (blue); n=4.

[0182] B. The percentage of Fox19CAR LV transduction was assessed on day 14 of the culturing protocol using flow cytometry. Recombinant CD19 was used to stain CAR-Tregs. n=4.

[0183] C. CAR-Treg purity assessed by using flow cytometry. Based the expression of lineage markers CD3, CD4, CD25, CD127, FOXP3. n=5.

[0184] D. T memory subsets including T central memory (Tern), T stem cell memory (Tscm), T effector memory (Tern), and T effector memory CD45RA+ (Teffector memory CD45RA+); n=4.

[0185] E. Exhaustion markers expressed in CAR-Treg cells cultured with varying IL-2 concentrations: 500U / ml (orange) and 1000U / ml (blue). n=2. Data are represented as Mean±SD,

[0186] F. Polyclonal suppression assay. Results are expressed in terms of Suppression Index. Tconv, conventional T cells.

[0187] Figure 22 - In vitro characterization of Fox19CAR-Tregs with restimulation and high IL2 doses.

[0188] A. CAR-Tregs were cultured with 500U / ml and 1 :3 Treg-beads ratio (green) or with 500U / ml and 1 :1 Treg-beads restimulation (blue) or 1000U / ml and 1 :1 Treg-beads restimulation (purple), n=5. One way ANOVA Dunnett's multiple comparisons test.

[0189] B. The percentage of Fox19CAR-LV transduction was assessed on day 14 of the culturing protocol using flow cytometry. n=5.

[0190] C. CAR-Treg purity assessed by using flow cytometry. Based the expression of lineage markers CD3, CD4, CD25, CD127, FOXP3. n=5.

[0191] D. T memory subsets including T central memory (Tern), T stem cell memory (Tscm), T effector memory (Tern), and T effector memory CD45RA+ (Teffector memory CD45RA+) n=5. E. Exhaustion markers expressed in CAR-Treg cells stimulated with 1 :1 Treg-to-beads ratio, restimulated and cultured with 500U / ml (blue) or 1000U / ml (purple) of IL-2. N=5. Data are represented as mean±SD. One-way ANOVA.

[0192] F. Polyclonal suppression assay. Results are expressed in terms of Suppression Index. Tconv, conventional T cells.

[0193] Figure 23 - In vitro characterization of Fox19CAR-Tregs with low IL2 doses and restimulation on day 9.

[0194] A. Control CAR-Tregs were stimulated with 1 :3 Treg-beads ratio and 500 ll / rnl (green, n=5) of IL2. CAR-Treg cells were also cultured with 1 :1 Treg-beads ratio and 250U / ml IL2 (pink, n=5) and restimulated (yellow, n=5).

[0195] B. The percentage of Fox19CAR-LV transduction was assessed on day 14 of the culturing protocol.

[0196] C. CAR-Treg purity assessed by using flow cytometry. Based the expression of lineage markers CD3, CD4, CD25, CD127, FOXP3. n=5.

[0197] D. T memory subsets including T central memory (Tern), T stem cell memory (Tscm), T effector memory (Tern), and T effector memory CD45RA+ (Teffector memory CD45RA+). n=5. Two-way ANOVA. *p-value < 0.05

[0198] E. Exhaustion markers expressed in CAR-Treg cells cultured with varying IL-2 concentrations. N=5. Data are represented as Mean±SD. One way ANOVA.

[0199] F. Polyclonal suppression assay. Results are expressed in terms of Suppression Index. Tconv, conventional T cells.

[0200] DETAILED DESCRIPTION OF THE INVENTION

[0201] Construct

[0202] The present inventors have developed constructs for co-expression of a CD19 CAR and FOXP3 in cells, particularly in immune cells for adoptive cell therapy, more particularly Treg cells or precursors therefor. As noted above, it is proposed to express FOXP3 alongside the CAR to redirect the Treg suppressive capacities in an antigen-specific manner, stabilise the Treg phenotype and reprogram Tconvs to suppressive cells, thus improving the safety and efficacy of the CAR-Treg-based cellular product.

[0203] The constructs are designed to encode the CD19 CAR and FOXP3 within a nucleic acid molecule (i.e. construct) in such a manner that the encoded polypeptides may be produced in the cell as discrete entities. This may be achieved by encoding a cleavable linker in the construct, in particular self-cleaving sequences, in between the nucleotide sequences encoding the respective components (i.e. encoding the CD19 CAR and FOXP3).

[0204] Accordingly, in one aspect, the invention provides a construct comprising from 5’ to 3’:

[0205] (a) a first nucleotide sequence encoding FoxP3;

[0206] (b) a second nucleotide sequence encoding a cleavable linker; and

[0207] (c) a third nucleotide sequence encoding a CD19 CAR.

[0208] Thus, the construct encodes from 5’ to 3’:

[0209] FoxP3 - cleavable linker - CD19 CAR.

[0210] In some embodiments, the construct further comprises a promoter operably linked to the first nucleotide sequence, the second nucleotide sequence and the third nucleotide sequence. Suitably, the promoter is a hPGK promoter.

[0211] In some embodiments, the construct consists of the first nucleotide sequence, the second nucleotide sequence and the third nucleotide sequence.

[0212] In some embodiments, the construct consists of the promoter, the first nucleotide sequence, the second nucleotide sequence and the third nucleotide sequence.

[0213] Preferably, the first nucleotide sequence and the third nucleotide sequence are separated only by the second nucleotide sequence.

[0214] FoxP3

[0215] FoxP3 is the master regulator of the regulatory pathway in the development and function of regulator T cells (Tregs).

[0216] An illustrative nucleotide sequence encoding FoxP3 is:

[0217] ATGCCCAACCCCAGGCCTGGCAAGCCCTCGGCCCCTTCCTTGGCCCTTGGCCCATCCCCAGG AGCCTCGCCCAGCTGGAGGGCTGCACCCAAAGCCTCAGACCTGCTGGGGGCCCGGGGCCCAG GGGGAACCTTCCAGGGCCGAGATCTTCGAGGCGGGGCCCATGCCTCCTCTTCTTCCTTGAAC CCCATGCCACCATCGCAGCTGCAGCTGCCCACACTGCCCCTAGTCATGGTGGCACCCTCCGG GGCACGGCTGGGCCCCTTGCCCCACTTACAGGCACTCCTCCAGGACAGGCCACATTTCATGC ACCAGCTCTCAACGGTGGATGCCCACGCCCGGACCCCTGTGCTGCAGGTGCACCCCCTGGAG AGCCCAGCCATGATCAGCCTCACACCACCCACCACCGCCACTGGGGTCTTCTCCCTCAAGGC CCGGCCTGGCCTCCCACCTGGGATCAACGTGGCCAGCCTGGAATGGGTGTCCAGGGAGCCGG CACTGCTCTGCACCTTCCCAAATCCCAGTGCACCCAGGAAGGACAGCACCCTTTCGGCTGTG CCCCAGAGCTCCTACCCACTGCTGGCAAATGGTGTCTGCAAGTGGCCCGGATGTGAGAAGGT CTTCGAAGAGCCAGAGGACTTCCTCAAGCACTGCCAGGCGGACCATCTTCTGGATGAGAAGG GCAGGGCACAATGTCTCC TCCAGAGAGAGATGGTACAGTC TC TGGAGCAGCAGC TGGTGC TG GAGAAGGAGAAGCTGAGTGCCATGCAGGCCCACCTGGCTGGGAAAATGGCACTGACCAAGGC TTCATCTGTGGCATCATCCGACAAGGGCTCCTGCTGCATCGTAGCTGCTGGCAGCCAAGGCC CTGTCGTCCCAGCCTGGTCTGGCCCCCGGGAGGCCCCTGACAGCCTGTTTGCTGTCCGGAGG CACCTGTGGGGTAGCCATGGAAACAGCACATTCCCAGAGTTCCTCCACAACATGGACTACTT CAAGTTCCACAACATGCGACCCCCTTTCACCTACGCCACGCTCATCCGCTGGGCCATCCTGG AGGCTCCAGAGAAGCAGCGGACACTCAATGAGATCTACCACTGGTTCACACGCATGTTTGCC TTCTTCAGAAACCATCCTGCCACCTGGAAGAACGCCATCCGCCACAACCTGAGTCTGCACAA GTGCTTTGTGCGGGTGGAGAGCGAGAAGGGGGCTGTGTGGACCGTGGATGAGCTGGAGTTCC GCAAGAAACGGAGCCAGAGGCCCAGCAGGTGTTCCAACCCTACACCTGGCCCC (SEQ ID NO : 1)

[0218] An illustrative FoxP3 amino acid sequence is:

[0219] MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLN PMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLE SPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAV PQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVL EKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPWPAWSGPREAPDSLFAVRR HLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFA FFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGP (SEQ ID NO : 2 )

[0220] In one embodiment, the first nucleotide sequence comprises or consists of SEQ ID NO: 1 or a sequence having at least 75% (suitably, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%) sequence identity thereto.

[0221] In one embodiment, the first nucleotide sequence comprises or consists of a sequence encoding SEQ ID NO: 2 or a sequence having at least 75% (suitably, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%) sequence identity thereto.

[0222] Cleavable linker

[0223] The cleavable linker may be a self cleaving sequence. Suitably, the self-cleaving sequence is a 2A self-cleaving peptide.

[0224] In some embodiments, the 2A self-cleaving peptide is selected from the group consisting of: P2A peptide, T2A peptide, E2A peptide, and F2A peptide. Preferably, the cleavable linker is a T2A peptide.

[0225] An illustrative nucleotide sequence encoding T2A is: 1 GAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCC (SEQ ID

[0226] NO : 3)

[0227] An illustrative T2A amino acid sequence is:

[0228] EGRGSLLTCGDVEENPGP (SEQ ID NO : 4)

[0229] In one embodiment, the second nucleotide sequence comprises or consists of SEQ ID NO: 3 or a sequence having at least 75% (suitably, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%) sequence identity thereto.

[0230] In one embodiment, the second nucleotide sequence comprises or consists of a sequence encoding SEQ ID NO: 4 or a sequence having at least 75% (suitably, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% at least 99%) sequence identity thereto.

[0231] Chimeric antigen receptor (CAR)

[0232] “Chimeric antigen receptor" or "CAR" or"CARs" as used herein refers to engineered receptors which can confer an antigen specificity onto cells (for example T cells such as naive T cells, central memory T cells, effector memory T cells or combinations thereof). CARs are also known as artificial T-cell receptors, chimeric T-cell receptors or chimeric immunoreceptors.

[0233] CARs are commonly classified into ‘generations’, by virtue of the composition of their intracellular signalling domain(s). All CARs typically comprise an extracellular antigen-binding domain, normally an scFv, joined to a membrane-anchoring transmembrane domain by a linker or spacer sequence. Whilst first generation CARs comprise a single intracellular signalling domain that is typically a single CD3 zeta chain, second and third generation CARs additionally comprise one or two further co-stimulatory domains (respectively), typically CD28, 4-1 BB, and / or QX-40. Fourth generation CARs are structurally similar to second generation CARs, however, are typically provided alongside an expression cassette (e.g., in a CAR T cell) that encodes an additional transgene such as a cytokine. Fifth generation CARs include additional co-stimulatory domains to selectively engage cellular pathways involved in cytokine signaling.

[0234] In one embodiment the CAR for use according to the invention is a first generation CAR.

[0235] In one embodiment the CAR for use according to the invention is a second generation CAR.

[0236] In one embodiment the CAR for use according to the invention is a third generation CAR.

[0237] In one embodiment the CAR for use according to the invention is a fourth generation CAR. In one embodiment the CAR for use according to the invention is a fifth generation CAR.

[0238] Preferably the CARs for use according to the invention comprise an antigen-specific targeting region, which may comprise an scFv, an extracellular domain such as a linker or hinge, a transmembrane domain, optionally one or more co-stimulatory domains, and an intracellular signaling domain.

[0239] Antigen-specific targeting domain

[0240] The antigen-specific targeting domain (or antigen-binding domain) provides the CAR with the ability to bind to the target antigen of interest. The antigen-specific targeting domain preferably targets an antigen of clinical interest against which it would be desirable to trigger an effector immune response that results in cell killing.

[0241] The antigen-specific targeting domain may be any protein or peptide that possesses the ability to specifically recognize and bind to a biological molecule (e.g., CD19). The antigen-specific targeting domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule of interest.

[0242] Illustrative antigen-specific targeting domains include antibodies or antibody fragments or derivatives, extracellular domains of receptors, ligands for cell surface molecules / receptors, or receptor binding domains thereof, and tumor binding proteins.

[0243] In a preferred embodiment, the antigen-specific targeting domain is, or is derived from, an antibody. An antibody-derived targeting domain can be a fragment of an antibody or a genetically engineered product of one or more fragments of the antibody, which fragment is involved in binding with the antigen. Examples include a variable region (Fv), a complementarity determining region (CDR), a Fab, a single chain variable fragment (scFv), a heavy chain variable region (VH), a light chain variable region (VL) and a camelid antibody (VHH).

[0244] In a preferred embodiment, the binding domain is a single chain variable fragment (scFv). The scFv may be, for example, a murine, human or humanized scFv.

[0245] "Complementarity determining region" or "CDR" with regard to an antibody or antigen-binding fragment thereof refers to a highly variable loop in the variable region of the heavy chain or the light chain of an antibody. CDRs can interact with the antigen conformation and largely determine binding to the antigen (although some framework regions are known to be involved in binding). The heavy chain variable region and the light chain variable region each contain 3 CDRs. "Heavy chain variable region" or "VH" refers to the fragment of the heavy chain of an antibody that contains three CDRs interposed between flanking stretches known as framework regions, which are more highly conserved than the CDRs and form a scaffold to support the CDRs.

[0246] "Light chain variable region" or "VL" refers to the fragment of the light chain of an antibody that contains three CDRs interposed between framework regions.

[0247] "Fv" refers to the smallest fragment of an antibody to bear the complete antigen binding site. An Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.

[0248] "Single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to one another directly or via a peptide linker sequence.

[0249] The target antigen of the present invention is CD19.

[0250] B-lymphocyte antigen CD19 (also known as B-lymphocyte surface antigen B4, T-cell surface antigen Leu-12, and CVID3) is expressed in all B-lineage cells in humans.

[0251] Preferably, the antigen-binding domain specifically binds to CD19.

[0252] As used herein, the term “specifically binds” means that the antigen-binding domain preferentially binds to a specific partner (i.e. to CD19) over a different binding partner. Suitably, the antigen-binding domain may bind to its specific partner (i.e. to CD19) with a dissociation constant of at least 10-6M.

[0253] In a preferred embodiment, the antigen-binding domain is a scFv.

[0254] In a preferred embodiment, the antigen-binding domain is a scFv with specificity for CD19.

[0255] In one embodiment, the antigen-binding domain comprises: a) heavy chain variable region (VH) complementarity determining regions (CDRs) with the sequences: CDR1 - GVSLPDYGVS (SEQ ID NO: 5); CDR2 - VIWGSETTYYNSALKS (SEQ ID NO: 6); and CDR3 - HYYYGGSYAMDY (SEQ ID NO: 7); or variants thereof each having up to three amino acid substitutions, additions or deletions; and b) light chain variable region (VL) CDRs with the sequences: CDR1 - RASQDISKYLN (SEQ ID NO: 8); CDR2 - VKLLIYHTSRLHS (SEQ ID NO: 9); and CDR3 - QQGNTLPYT (SEQ ID NO: 10); or variants thereof each having up to three amino acid substitutions, additions or deletions.

[0256] In one embodiment, the antigen-binding domain comprises: a) heavy chain variable region (VH) complementarity determining regions (CDRs) with the sequences: CDR1 - GVSLPDYGVS (SEQ ID NO: 5); CDR2 - VIWGSETTYYNSALKS (SEQ ID NO: 6); and CDR3 - HYYYGGSYAMDY (SEQ ID NO: 7); and b) light chain variable region (VL) CDRs with the sequences: CDR1 - RASQDISKYLN (SEQ ID NO: 8); CDR2 - HTSRLHS (SEQ ID NO: 9); and CDR3 - QQGNTLPYT (SEQ ID NO: 10).

[0257] In some embodiments, the antigen-binding domain comprises: a) heavy chain variable region (VH) complementarity determining regions (CDRs) with the sequences: CDR1 - SGVSLPDY (SEQ ID NO: 62); CDR2 - LEWLGVIWGSETTYYN (SEQ ID NO: 63); and CDR3 - HYYYGGSYAMD (SEQ ID NO: 64); or variants thereof each having up to three amino acid substitutions, additions or deletions; and b) light chain variable region (VL) CDRs with the sequences: CDR1 - QDISK (SEQ ID NO: 65); CDR2 - VKLLIYHTSRLH (SEQ ID NO: 66); and CDR3 - GNTLPY (SEQ ID NO: 67); or variants thereof each having up to three amino acid substitutions, additions or deletions.

[0258] In some embodiments, the antigen-binding domain comprises: a) heavy chain variable region (VH) complementarity determining regions (CDRs) with the sequences: CDR1 - SGVSLPDY (SEQ ID NO: 62); CDR2 - LEWLGVIWGSETTYYN (SEQ ID NO: 63); and CDR3 - HYYYGGSYAMD (SEQ ID NO: 64); and b) light chain variable region (VL) CDRs with the sequences: CDR1 - QDISK (SEQ ID NO: 65); CDR2 - VKLLIYHTSRLH (SEQ ID NO: 66); and CDR3 - GNTLPY (SEQ ID NO: 67).

[0259] In some embodiments, the antigen-binding domain comprises: a) heavy chain variable region (VH) complementarity determining regions (CDRs) with the sequences: CDR1 - SGVSLPDYGVS (SEQ ID NO: 68); CDR2 - LEWLGVIWGSETTYYNSALKS (SEQ ID NO: 69); and CDR3 - HYYYGGSYAMDY (SEQ ID NO: 70); or variants thereof each having up to three amino acid substitutions, additions or deletions; and b) light chain variable region (VL) CDRs with the sequences: CDR1 - RASQDISKYLN (SEQ ID NO: 71); CDR2 - VKLLIYHTSRLHS (SEQ ID NO: 72); and CDR3 - QQGNTLPYT (SEQ ID NO: 73); or variants thereof each having up to three amino acid substitutions, additions or deletions.

[0260] In some embodiments, the antigen-binding domain comprises: a) heavy chain variable region (VH) complementarity determining regions (CDRs) with the sequences: CDR1 - SGVSLPDYGVS (SEQ ID NO: 68); CDR2 - LEWLGVIWGSETTYYNSALKS (SEQ ID NO: 69); and CDR3 - HYYYGGSYAMDY (SEQ ID NO: 70); and b) light chain variable region (VL) CDRs with the sequences: CDR1 - RASQDISKYLN (SEQ ID NO: 71); CDR2 - VKLLIYHTSRLHS (SEQ ID NO: 72); and CDR3 - QQGNTLPYT (SEQ ID NO: 73).

[0261] In some embodiments, the antigen-binding domain comprises: a) heavy chain variable region (VH) complementarity determining regions (CDRs) comprising the sequences: CDR1 - GVSLPDY (SEQ ID NO: 74); CDR2 - VIWGSETTYYN (SEQ ID NO: 75); and CDR3 - HYYYGGSYAMD (SEQ ID NO: 76); or variants thereof each having up to three amino acid substitutions, additions or deletions; and b) light chain variable region (VL) CDRs comprising the sequences: CDR1 - QDISK (SEQ ID NO: 77); CDR2 - HTSRLH (SEQ ID NO: 78); and CDR3 - GNTLPY (SEQ ID NO: 79); or variants thereof each having up to three amino acid substitutions, additions or deletions.

[0262] In some embodiments, the antigen-binding domain comprises: a) heavy chain variable region (VH) complementarity determining regions (CDRs) comprising the sequences: CDR1 - GVSLPDY (SEQ ID NO: 74); CDR2 - VIWGSETTYYN (SEQ ID NO: 75); and CDR3 - HYYYGGSYAMD (SEQ ID NO: 76); and b) light chain variable region (VL) CDRs comprising the sequences: CDR1 - QDISK (SEQ ID NO: 77); CDR2 - HTSRLH (SEQ ID NO: 78); and CDR3 - GNTLPY (SEQ ID NO: 79).

[0263] In one embodiment, the antigen-binding domain comprises: a) heavy chain variable region (VH) complementarity determining regions (CDRs) with the sequences: CDR1 - GVSLPDYGVS (SEQ ID NO: 5); CDR2 - VIWGSETTYYNSALKS (SEQ ID NO: 6), or VIWGSETTYYSSSLKS (SEQ ID NO: 24), or VIWGSETTYYQSSLKS (SEQ ID NO: 25), or VIWGSETTYYNSSLKS (SEQ ID NO: 26); and CDR3 - HYYYGGSYAMDY (SEQ ID NO: 7); or variants thereof each having up to three amino acid substitutions, additions or deletions; and b) light chain variable region (VL) CDRs with the sequences: CDR1 - RASQDISKYLN (SEQ ID NO: 8); CDR2 - HTSRLHS (SEQ ID NO: 9); and CDR3 - QQGNTLPYT (SEQ ID NO: 10); or variants thereof each having up to three amino acid substitutions, additions or deletions.

[0264] An illustrative nucleotide sequence encoding a VH domain is:

[0265] GAAGTGCAGCTGCAGCAGAGCGGCCCTGGCCTGGTCGCCCCTAGCCAGAGCCTGTCCGTGAC CTGTACCGTGTCCGGCGTGTCCCTGCCCGACTACGGCGTGTCCTGGATCAGACAGCCCCCCA GAAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAGACAACCTACTACAACAGCGCC CTGAAGTCCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTGAAGATGAA CAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTACGGCGGCA GCTACGCTATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGTCCAGCTACGTGACAGTG TCTAGC (SEQ ID NO : 11)

[0266] An illustrative nucleotide sequence encoding a VL domain is:

[0267] TCTCTTCAGTTCCAGCTTGGTGCCTCCGCCGAAGGTGTAGGGCAGGGTGTTGCCTTGCTGAC AGAAGTAGGTAGCGATGTCTTCCTGTTCCAGGTTGGAGATGGTCAGGCTGTAGTCGGTGCCG CTGCCGCTGCCAGAAAATCTGCTGGGCACGCCGCTGTGCAGCCGGGAGGTGTGGTAGATCAG CAGCTTCACGGTGCCGTCGGGTTTCTGCTGATACCAGTTCAGGTACTTGCTGATGTCCTGGC TGGCCCGGCAGCTGATGGTCACTCTGTCGCCCAGGCTGGCGCTCAGGCTGCTGGTGGTCTGG GTCATCTGGATGTCCCGGGAGCACTGCACGCCCTTCAGGATGGCCACCAGGAACAGCCAGCT CAGGCCGAATTCCA (SEQ ID NO : 12)

[0268] In one embodiment, the third nucleotide sequence comprises SEQ ID NO: 11 or a sequence having at least 75% (suitably, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) sequence identity thereto. In one embodiment, the third nucleotide sequence comprises SEQ ID NO: 12 or a sequence having at least 75% (suitably, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%) sequence identity thereto.

[0269] An illustrative VH domain sequence is:

[0270] EVQLQQSGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSA LKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTTVTVSSYVTV SS (SEQ ID NO : 13)

[0271] An illustrative VL domain sequence is:

[0272] MEFGLSWLFLVAILKGVQCSRDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPD GTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTK LELK (SEQ ID NO : 14)

[0273] In one embodiment, the antigen-binding domain comprises or consists of: a) a VH domain comprising the sequence of SEQ ID NO: 13; and b) a VL domain comprising the sequence of SEQ ID NO: 14; or variants thereof, each having at least 75% (suitably, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto.

[0274] In one embodiment, the antigen-binding domain comprises a sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 13, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.

[0275] In one embodiment, the antigen-binding domain comprises a sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 14, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.

[0276] In one embodiment, the antigen-binding domain comprises or consists of: a) a VH domain comprising the sequence of SEQ ID NO: 13; and b) a VL domain comprising the sequence of SEQ ID NO: 14.

[0277] An exemplary anti-CD19 scFv sequence is: MEFGLSWLFLVAILKGVQCSRDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPD GTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTK LELKRGGGGSGGGGSGGGGSGGGGSEVQLQQSGPGLVAPSQSLSVTCTVSGVSLPDYGVSWI RQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHY YYGGSYAMDYWGQGTTVTVSSYVTVSSQ (SEQ ID NO : 15)

[0278] In one embodiment, the antigen-binding domain comprises or consists of a sequence having at least 75% sequence identity to the sequence of SEQ ID NO: 15, such as at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.

[0279] In one embodiment, the antigen-binding domain comprises or consists of a sequence comprising SEQ ID NOs: 5-10 and having at least 75% sequence identity to the sequence of SEQ ID NO: 15, such as at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.

[0280] In one embodiment, the antigen-binding domain comprises or consists of a sequence comprising SEQ ID NOs: 62-67 and having at least 75% sequence identity to the sequence of SEQ ID NO: 15, such as at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.

[0281] In one embodiment, the antigen-binding domain comprises or consists of a sequence comprising SEQ ID NOs: 68-73 and having at least 75% sequence identity to the sequence of SEQ ID NO: 15, such as at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.

[0282] In one embodiment, the antigen-binding domain comprises or consists of a sequence comprising SEQ ID NOs: 74-79 and having at least 75% sequence identity to the sequence of SEQ ID NO: 15, such as at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.

[0283] In one embodiment, the antigen-binding domain comprises a sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 15.

[0284] Nucleotide sequence encoding an exemplary scFv:

[0285] ATGGAATTCGGCCTGAGCTGGCTGTTCCTGGTGGCCATCCTGAAGGGCGTGCAGTGCTCCCG

[0286] GGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCA TCAGCTGCCGGGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGAC GGCACCGTGAAGCTGCTGATCTACCACACCTCCCGGCTGCACAGCGGCGTGCCCAGCAGATT TTCTGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGACA TCGCTACCTACTTCTGTCAGCAAGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACCAAG CTGGAACTGAAGAGAGGCGGCGGAGGCTCTGGTGGAGGCGGATCTGGCGGGGGAGGAAGTGG GGGCGGAGGATCTGAAGTGCAGCTGCAGCAGAGCGGCCCTGGCCTGGTCGCCCCTAGCCAGA GCCTGTCCGTGACCTGTACCGTGTCCGGCGTGTCCCTGCCCGACTACGGCGTGTCCTGGATC AGACAGCCCCCCAGAAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAGACAACCTA CTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGT TCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTAC TACTACGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGTCCAG CTACGTGACAGTGTCTAGCCAA (SEQ ID NO : 16)

[0287] In one embodiment, the third nucleotide sequence comprises SEQ ID NO: 16, or a sequence with at least 75% sequence identity, such as at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.

[0288] Co-stimulatory domain

[0289] The CAR for use according to the invention may also comprise one or more co-stimulatory domains. This domain may enhance cell proliferation, cell survival and development of memory cells.

[0290] In some embodiments, the co-stimulatory domains are fused together.

[0291] In some embodiments, the co-stimulatory domains are separated by linker sequences.

[0292] In some embodiments, the co-stimulatory domains are not separated by linker sequences.

[0293] Each co-stimulatory domain may comprise the co-stimulatory domain of any one or more of, for example, members of the TNFR super family, CD28, CD137 (4-1 BB), CD134 (0X40), DapIO, CD27, CD2, CD5, ICAM-1 , LFA-1 , Lek, TNFR-1 , TNFR-II, Fas, CD30, CD40 or combinations thereof. Co-stimulatory domains from other proteins may also be used with the CAR of the invention. Additional co-stimulatory domains will be apparent to those of skill in the art.

[0294] In some embodiments, the co-stimulatory domain is a 4-1 BB co-stimulatory domain.

[0295] In some embodiments, the co-stimulatory domain is a CD28 co-stimulatory domain.

[0296] In some embodiments, the CAR comprises two co-stimulatory domains.

[0297] In some embodiments, the one or more co-stimulatory domains comprise a CD28 co- stimulatory domain and a 4-1 BB co-stimulatory domain. In one embodiment, the CD28 co-stimulatory domain and 4-1 BB co-stimulatory domains are fused together.

[0298] In some embodiments, the co-stimulatory domain may comprise a composite co-stimulatory domain comprising one or more co-stimulatory domains according to the invention, or fragments thereof. As such, said domains may be fused in their entirety or smaller sections of said domains may be fused.

[0299] In one embodiment, the co-stimulatory domain is a composite co-stimulatory domain comprising a fragment or the entirety of a co-stimulatory domain selected from any two or more of: CD28, CD137 (4-1 BB), CD134 (0X40), DapIO, CD27, CD2, CD5, ICAM-1 , LFA-1 , Lek, TNFR-1 , TNFR-II, Fas, CD30, and CD40.

[0300] In one embodiment, the co-stimulatory domain comprises a sequence having at least 90% (such as at least 95%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO: 28.

[0301] Exemplary CD28 co-stimulatory domain:

[0302] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO : 28)

[0303] In one embodiment, the co-stimulatory domain comprises a sequence having at least 75% sequence identity to SEQ ID NO: 28, such as at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.

[0304] Intracellular signaling domain

[0305] The CAR for use according to the invention may also comprise an intracellular signaling domain. This domain may be cytoplasmic and may transduce the effector function signal and direct the cell to perform its specialized function.

[0306] Examples of intracellular signaling domains include, but are not limited to, chain of the T-cell receptor or any of its homologs (e.g., q chain, FcsRIy and chains, MB1 (Igo) chain, B29 (IgP) chain, etc.), CD3 polypeptides (A, 5 and E), syk family tyrosine kinases (Syk, ZAP 70, etc.), sre family tyrosine kinases (Lek, Fyn, Lyn, etc.) and other molecules involved in T-cell transduction, such as CD2, CD5 and CD28. The intracellular signaling domain may be, for example, human CD3 zeta chain, FcyRIII, FcsRI, cytoplasmic tails of Fc receptors, immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptors or combinations thereof. Additional intracellular signaling domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention.

[0307] In a preferred embodiment the intracellular signaling domain is a CD3- signalling domain.

[0308] In one embodiment, the intracellular signaling domain comprises a sequence having at least 90% sequence identity (such as at least 95%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity) to SEQ ID NO: 30.

[0309] Exemplary CD3- signalling domain:

[0310] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO : 30)

[0311] In one embodiment, the intracellular signaling domain is encoded by SEQ ID NO: 31 , or a sequence with at least 75% sequence identity (such as at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) thereto.

[0312] Nucleotide sequence encoding exemplary CD3- signalling domain:

[0313] TCAGCGAGGAGGCAGGGCCTGCATGTGAAGGGCGTCGTAGGTGTCCTTGGTGGCTGTACTGA GACCCTGGTAAAGGCCATCGTGCCCCTTGCCCCTCCGGCGCTCGCCTTTCATCCCAATCTCA CTGTAGGCCTCCGCCATCTTATCTTTCTGCAGTTCATTGTACAGGCCTTCCTGAGGGTTCTT CCTTCTCGGCTTTCCCCCCATCTCAGGGTCCCGGCCACGTCTCTTGTCCAAAACATCGTACT CCTCTCTTCGTCCTAGATTGAGCTCGTTATAGAGCTGGTTCTGGCCCTGCTGGTACGCGGGG GCGTCTGCGCTCCTGCTGAACTTCACTCT (SEQ ID NO : 31)

[0314] Transmembrane domain

[0315] The CAR for use according to the invention may also comprise a transmembrane domain. The transmembrane domain may comprise the transmembrane sequence from any protein which has a transmembrane domain, including any of the type I, type II or type III transmembrane proteins. The transmembrane domain of the CAR of the invention may also comprise an artificial hydrophobic sequence. The transmembrane domains of the CARs of the invention may be selected so as not to dimerize. Additional transmembrane domains will be apparent to those of skill in the art.

[0316] Examples of transmembrane (TM) regions used in CAR constructs are: 1) The CD28 TM region (Pule et al, Mol Ther, 2005, Nov;12(5):933-41 ; Brentjens et al, CCR, 2007, Sep 15; 13(18 Pt 1):5426-35; Casucci et al, Blood, 2013, Nov 14;122(20):3461-72.); 2) The 0X40 TM region (Pule et al, Mol Ther, 2005, Nov;12(5):933-41); 3) The 41 BB TM region (Brentjens et al, CCR, 2007, Sep 15; 13(18 Pt 1):5426-35); 4) The CD3 zeta TM region (Pule et al, Mol Ther, 2005, Nov;12(5):933-41 ; Savoldo B, Blood, 2009, Jun 18;113(25):6392-402.); 5) The CD8a TM region (Maher et al, Nat Biotechnol, 2002, Jan;20(1):70-5.; Imai C, Leukemia, 2004, Apr;18(4):676-84; Brentjens et al, CCR, 2007, Sep 15;13(18 Pt 1):5426-35; Milone et al, Mol Ther, 2009, Aug; 17(8): 1453-64.).

[0317] In some embodiments, the transmembrane domain is a CD28 transmembrane domain.

[0318] Exemplary CD28 transmembrane domain:

[0319] FWVLVWGGVLACYSLLVTVAFIIFWV (SEQ ID NO : 17)

[0320] In one embodiment, the transmembrane domain comprises a sequence having at least 75% sequence identity to SEQ ID NO: 17, such as at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.

[0321] In some embodiments, the transmembrane domain and intracellular co-stimulatory domain may be derived from the same molecule. Suitably, the transmembrane domain and intracellular co-stimulatory domain are derived from CD28.

[0322] Exemplary CD28 transmembrane domain and co-stimulatory domain:

[0323] FWVLVWGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRD FAAYRS (SEQ ID NO : 18)

[0324] In one embodiment, the transmembrane domain and co-stimulatory domain comprises a sequence having at least 75% sequence identity to SEQ ID NO: 18, such as at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto.

[0325] Exemplary nucleotide sequence encoding a CD28 transmembrane and co-stimulatory domain:

[0326] TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGC CTTTATTATTTTCTGGGTGGAGCGATAGGCTGCGAAGTCGCGTGGTGGGGCATAGGGCTGGT AATGCTTGCGGGTGGGCCCGGGGCGGCGGGGAGTCATGTTCATGTAGTCACTGTGCAGGAGC CTGCTCCTCTTACTCCT (SEQ ID NO : 29)

[0327] In one embodiment, the third nucleotide sequence comprises SEQ ID NO: 29, or a sequence with at least 75% sequence identity, such as at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity thereto. Spacer domain

[0328] The CAR for use according to the invention may comprise an extracellular spacer domain. The extracellular spacer domain may be attached to the antigen-specific targeting region and the transmembrane domain. The spacer domain may also be referred to as a hinge or linker.

[0329] In some embodiments, the spacer domain is an lgG1-derived hinge.

[0330] In one embodiment, the spacer domain comprises a sequence having at least 90% sequence identity (such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ ID NO: 32.

[0331] Exemplary lgG1 hinge:

[0332] IEPKSCDKTHTCPPCPP (SEQ ID NO : 32 )

[0333] In one embodiment, the spacer is encoded by SEQ ID NO: 33, or a sequence with at least 75% sequence identity (such as at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) thereto.

[0334] Nucleotide sequence encoding exemplary lgG1 hinge:

[0335] ATCGAGCCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCCTGCCCCCCC (SEQ ID NO : 33)

[0336] In one embodiment the spacer comprises a sequence having at least 90% (suitably, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 34.

[0337] Exemplary lgG4 hinge:

[0338] ATSSGESKYGPPCPPCP (SEQ ID NO : 34 )

[0339] The CAR of the present invention may comprise an extracellular spacer which comprises at least part of the extracellular domain of human low affinity nerve growth factor receptor (LNGFR) or a derivative thereof.

[0340] LNGFR is not expressed on the majority of human hematopoietic cells, thus allowing quantitative analysis of transduced gene expression by immunofluorescence, with single cell resolution. Thus, fluorescence activated cell sorter analysis of expression of LNGFR may be performed in transduced cells to study gene expression. Further details on analysis using LNGFR may be found in Mavilio (1994) Blood 83, 1988-1997. In one embodiment, the CAR of the invention comprises a truncated LNGFR (also known as ALNGFR). Preferably the LNGFR used in the present invention is truncated in its intracytoplasmic domain. Such a truncation is described in Mavilio (1994) Blood 83, 1988- 1997.

[0341] Thus, preferably the LNGFR spacer of the present invention comprises at least part of the extracellular domain or a derivative thereof but lacks the intracellular domain of LNGFR. The extracellular domain may comprise amino acids 29 - 250 of LNGFR or a derivative thereof.

[0342] Exemplary human LNGFR [UNIPROT accession P08138, TNR16_HUMAN]:

[0343] MGAGATGRAMDGPRLLLLLLLGVSLGGAKEACPTGLYTHSGECCKACNLGEGVAQPCGANQT VCE PCLDSVTFSDWS ATE PCKPC TE CVGLQSMS APCVEADDAVCRCAYGYYQDE TTGRCEA CRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADA ECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGWTTVMGSSQPWTRGTT DNLIPVYCSILAAVWGLVAYIAFKRWNSCKQNKQGANSRPVNQTPPPEGEKLHSDSGISVD SQSLHDQQPHTQTASGQALKGDGGLYSSLPPAKREEVEKLLNGSAGDTWRHLAGELGYQPEH IDSFTHEACPVRALLASWATQDSATLDALLAALRRIQRADLVESLCSESTATSPV (SEQ ID NO : 35)

[0344] Exemplary extracellular domain of the human LNGFR [UNIPROT accession P08138, TNR16_HUMAN, position 29 - 250]

[0345] KE AC PTGLYTHS GE CCKACNLGEGVAQPCGANQTVCE PCLDSVTFSDWS ATE PCKPC TE CV GLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDG TYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQE PEAPPEQDLIASTVAGWTTVMGSSQPWTRGTTDN (SEQ ID NO : 36)

[0346] Preferably the LNGFR lacks the signal peptide.

[0347] SEQ ID NO: 36 may also be referred to as the LNGFR wild type long spacer (NWL).

[0348] In one embodiment, the spacer comprises at least part of a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the extracellular domain of LNGFR (e.g., SEQ ID NO: 36). In one embodiment, the spacer comprises at least part of a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to amino acids 29-250 of the LNGFR protein (e.g., SEQ ID NO: 36).

[0349] In one embodiment, the spacer comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 36. In one embodiment, the spacer comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to amino acids 29-250 of SEQ ID NO: 36. In one embodiment, the spacer is encoded by SEQ ID NO: 37, or a sequence with at least 75% sequence identity thereto.

[0350] Nucleotide sequence encoding exemplary LNGFR spacer (NWL):

[0351] AAAGAGGCCTGCCCCACCGGCCTGTACACCCACAGCGGAGAGTGCTGCAAGGCCTGCAACCT GGGAGAGGGCGTGGCCCAGCCTTGCGGCGCCAATCAGACCGTGTGCGAGCCCTGCCTGGACA GCGTGACCTTCAGCGACGTGGTGTCCGCCACCGAGCCCTGCAAGCCTTGCACCGAGTGTGTG GGCCTGCAGAGCATGAGCGCCCCCTGCGTGGAAGCCGACGACGCCGTGTGTAGATGCGCCTA CGGCTACTACCAGGACGAGACAACCGGCAGATGCGAGGCCTGTAGAGTGTGCGAGGCCGGCA GCGGCCTGGTGTTCAGTTGTCAAGACAAGCAGAATACCGTGTGTGAAGAGTGCCCCGACGGC ACCTACAGCGACGAGGCCAACCACGTGGACCCCTGCCTGCCCTGCACTGTGTGCGAGGACAC CGAGCGGCAGCTGCGCGAGTGCACAAGATGGGCCGACGCCGAGTGCGAAGAGATCCCCGGCA GATGGATCACCAGAAGCACCCCCCCTGAGGGCAGCGACAGCACCGCCCCTAGCACCCAGGAA CCTGAGGCCCCTCCCGAGCAGGACCTGATCGCCTCTACAGTGGCCGGCGTGGTGACAACCGT GATGGGCAGCTCTCAGCCCGTGGTGACACGGGGCACCACCGACAAT (SEQ ID NO : 37)

[0352] In one embodiment, the spacer comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 38.

[0353] Exemplary LNGFR spacer:

[0354] KE AC PTGLYTHS GE CCKACNLGEGVAQPCGANQTVCE PCLDSVTFSDWS ATE PCKPC TE CV GLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDG TYSDEAARAADAECEE (SEQ ID NO : 38)

[0355] SEQ ID NO: 38 may also be referred to as the LNGFR mutated short spacer (NMS)

[0356] In one embodiment, the spacer is encoded by SEQ ID NO: 39, or a sequence with at least 75% (suitably, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity thereto.

[0357] Nucleotide sequence encoding exemplary LNGFR spacer:

[0358] AAAGAGGCCTGCCCCACCGGCCTGTACACCCACAGCGGAGAGTGCTGCAAGGCCTGCAACCT GGGAGAGGGCGTGGCCCAGCCTTGCGGCGCCAATCAGACCGTGTGCGAGCCCTGCCTGGACA GCGTGACCTTCAGCGACGTGGTGTCCGCCACCGAGCCCTGCAAGCCTTGCACCGAGTGTGTG GGCCTGCAGAGCATGAGCGCCCCCTGCGTGGAAGCCGACGACGCCGTGTGTAGATGCGCCTA CGGCTACTACCAGGACGAGACAACCGGCAGATGCGAGGCCTGTAGAGTGTGCGAGGCCGGCA GCGGCCTGGTGTTCAGTTGTCAGGACAAGCAGAACACCGTGTGTGAAGAGTGCCCCGACGGC ACCTACAGCGACGAGGCCGCCCGGGCCGCCGACGCCGAGTGCGAGGAA (SEQ ID NO : 39)

[0359] Further exemplary spacers are illustrated below.

[0360] Exemplary LNGFR spacer (LNGFR wild type short (NWS)): KEACPTGLYTHSGECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDWSATEPCKPCTECV GLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDG TYSDEANHVDPCLPCTVCEDTERQLRECTRWADAECEE (SEQ ID NO : 40)

[0361] Exemplary LNGFR spacer (LNGFR mutated long (NML))

[0362] KE AC PTGLYTHS GE CCKACNLGEGVAQPCGANQTVCE PCLDSVTFSDWS ATE PCKPC TE CV GLQSMSAPCVEADDAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDG TYSDEAARAADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGWTTVM GSSQPWTRGTTDN (SEQ ID NO : 41)

[0363] In one embodiment the spacer comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 36, 38, 40 or 41.

[0364] LNGFR comprises 4 TNFR-Cys domains (TNFR-Cys 1 , TNFR-Cys 2, TNFR-Cys 3 and TNFR- Cys 4). Sequences of the domains are exemplified below:

[0365] TNFR-Cys 1

[0366] ACPTGLYTHSGECCKACNLGEGVAQPCGANQTVC (SEQ ID NO : 42)

[0367] TNFR-Cys 2

[0368] PCLDSVTFSDWSATEPCKPCTECVGLQSMSAPCVEADDAVC (SEQ ID NO : 43)

[0369] TNFR-Cys 3

[0370] RCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVC (SEQ ID NO : 44 )

[0371] TNFR-Cys 4

[0372] ECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWADAEC (SEQ ID NO : 45)

[0373] In one embodiment, the spacer comprises TNFR-Cys 1 , 2 and 3 domains or fragments or derivatives thereof. In another embodiment, the spacer comprises the TNFR-Cys 1 , 2, 3 and 4 domains or fragments or derivatives thereof.

[0374] In one embodiment the spacer comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to TNFR-Cys 1 (SEQ ID NO: 42), a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to TNFR-Cys 2 (SEQ ID NO: 43), or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to TNFR-Cys 3 (SEQ ID NO: 44). The spacer may further comprise a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to TNFR-Cys 4 (SEQ ID NO: 45). Rather than comprise the full TNFR-Cys 4 domain, the spacer may comprise a TNFR-Cys 4 domain with the following amino acids deleted from said domain:

[0375] NHVDPCLPCTVCEDTERQLRECTRW (SEQ ID NO : 46)

[0376] In one embodiment, the NHVDPCLPCTVCEDTERQLRECTRW (SEQ ID NO: 46) amino acids are replaced with the following amino acids:

[0377] ARA

[0378] In one embodiment, the spacer lacks the LNGFR serine / threonine-rich stalk. In another embodiment, the spacer comprises the LNGFR serine / threonine-rich stalk.

[0379] The spacer may comprise or consist of a sequence of SEQ ID NO: 42 or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 42.

[0380] The spacer may comprise or consist of a sequence of SEQ ID NO: 43 or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 43.

[0381] The spacer may comprise or consist of a sequence of SEQ ID NO: 44 or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 44.

[0382] The spacer may comprise or consist of a sequence of SEQ ID NO: 45 or a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 45.

[0383] The spacer may comprise a mutated version of the lgG1 CH2CH3 spacer (mCH2CH3) that is unable to recognize the FcyRI (Hornbach et al., Gene Ther. 2000).

[0384] In one embodiment, the spacer comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 48.

[0385] Exemplary mCH2CH3 spacer:

[0386] EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVWDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO : 48)

[0387] In one embodiment, the spacer is encoded by SEQ ID NO: 49 or a sequence with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0388] Nucleotide sequence encoding exemplary mCH2CH3 spacer:

[0389] GAGCCCAAGAGCCCCGACAAGACCCACACCTGTCCCCCCTGTCCTGCCCCTCCAGTGGCCGG ACCTAGCGTGTTCCTGTTCCCCCCAAAGCCCAAGGACACCCTGATGATCGCCCGGACCCCCG AAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAAGTGAAGTTCAATTGGTAC GTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAACAGTACAACAGCAC CTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACA AGTGCAAGGTCTCCAACAAGGCCCTGCCTGCCCCCATCGAGAAAACCATCAGCAAGGCCAAG GGCCAGCCCCGCGAGCCCCAGGTGTACACACTGCCCCCCAGCCGGGACGAGCTGACCAAGAA CCAGGTGTCCCTGACCTGCCTCGTGAAAGGCTTCTACCCCAGCGATATCGCCGTGGAATGGG AGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGC TCATTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCCGGTGGCAGCAGGGCAACGTGTT CAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGA GCCCCGGCAAG (SEQ ID NO : 49)

[0390] The spacer may confer properties to the CAR such that it allows for immunoselection of cells, preferably Tregs, expressing said CAR.

[0391] The CAR of the present invention (e.g. comprising the spacer referred to herein) preferably enables T-cells expressing the CAR to mediate therapeutically significant effects.

[0392] The CAR of the present invention (e.g. comprising the spacer referred to herein) is preferably suitable for facilitating immunoselection of cells transduced with said CAR.

[0393] An exemplary CAR of the present invention comprising the LNGFR-based spacer may avoid activation of unwanted and potentially toxic off-target immune responses and may allow CAR- expressing T cells to persist in vivo without being prematurely cleared by the host immune system.

[0394] As described herein, the present invention also encompasses the use of variants, derivatives, homologues and fragments of the spacer elements described herein.

[0395] Exemplary CAR

[0396] The CAR is an anti-CD19 CAR.

[0397] In one embodiment, the CD19 CAR comprises: a) an anti-CD19 scFv; b) an lgG1 , CD8a, LNGFR or mCH2CH3 spacer domain; c) a CD28, a CD8, or a CD4 transmembrane domain; d) a CD28 and / or a 4-1 BB co-stimulatory domain; and / or e) a CD3- signalling domain.

[0398] In one embodiment, the CD19 CAR comprises a CD28 transmembrane domain. In one embodiment, the CD19 CAR comprises an lgG1 hinge.

[0399] In one embodiment, the CD19 CAR comprises a CD28 hinge.

[0400] In one embodiment, the CD19 CAR comprises a CD28a hinge.

[0401] In one embodiment, the CD19 CAR comprises a LNGFR spacer.

[0402] In one embodiment, the CD19 CAR comprises a CD28 co-stimulatory domain.

[0403] In one embodiment, the CD19 CAR comprises a 4-1 BB co-stimulatory domain.

[0404] In one embodiment, the CD19 CAR comprises a CD3 intracellular signalling domain.

[0405] In one embodiment, the CD19 CAR comprises an antigen-binding domain comprising an anti- CD19 scFv, lgG1 hinge, CD28 transmembrane and co-stimulatory domain, and a CD3- signalling domain.

[0406] In one embodiment, the CAR comprises an antigen-binding domain comprising an anti-CD19 scFv, CD28 hinge, CD28 transmembrane and co-stimulatory domain, and a CD3- signalling domain.

[0407] In one embodiment, the CAR comprises an antigen-binding domain comprising an anti-CD19 scFv, CD8a hinge, CD8a transmembrane domain, 4-1 BB co-stimulatory domain and a CD3- signalling domain.

[0408] In one embodiment, the CAR comprises an antigen-binding domain comprising an anti-CD19 scFv, lgG4 hinge, CD28 transmembrane domain, 4-1 BB co-stimulatory domain and a CD3- signalling domain.

[0409] In one embodiment, the CAR comprises SEQ ID NO: 15, SEQ ID NO: 32, SEQ ID NO: 18 and SEQ ID NO: 30 or sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0410] In one embodiment, the CAR comprises SEQ ID NO: 15, SEQ ID NO: 32, SEQ ID NO: 18 and SEQ ID NO: 30 or variants having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein the variant of SEQ ID NO: 15 comprises SEQ ID NOs: 5-10.

[0411] In one embodiment, the CAR comprises SEQ ID NO: 15, SEQ ID NO: 32, SEQ ID NO: 18 and SEQ ID NO: 30 or variants having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein the variant of SEQ ID NO: 15 comprises SEQ ID NOs: 62-67. In one embodiment, the CAR comprises SEQ ID NO: 15, SEQ ID NO: 32, SEQ ID NO: 18 and SEQ ID NO: 30 or variants having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein the variant of SEQ ID NO: 15 comprises SEQ ID NOs: 68-73.

[0412] In one embodiment, the CAR comprises SEQ ID NO: 15, SEQ ID NO: 32, SEQ ID NO: 18 and SEQ ID NO: 30 or variants having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein the variant of SEQ ID NO: 15 comprises SEQ ID NOs: 74-79.

[0413] In one embodiment, the CAR is encoded by SEQ ID NO: 16, SEQ ID NO: 33, SEQ ID NO: 29 and SEQ ID NO: 31 or sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0414] In one embodiment, the CAR comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 50.

[0415] Exemplary CD19 CAR:

[0416] MEFGLSWLFLVAILKGVQCSRDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPD GTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTK LELKRGGGGSGGGGSGGGGSGGGGSEVQLQQSGPGLVAPSQSLSVTCTVSGVSLPDYGVSWI RQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHY YYGGSYAMDYWGQGTTVTVSSYVYVSSQCIEPKSCDKTHTCPPCPPGDRFWVLVWGGVLAC YSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRS ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO : 50)

[0417] In one embodiment, the CAR comprises a protein that is encoded by a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 51.

[0418] Nucleotide sequence encoding exemplary CAR:

[0419] ATGGAATTCGGCCTGAGCTGGCTGTTCCTGGTGGCCATCCTGAAGGGCGTGCAGTGCTCCCG GGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCA TCAGCTGCCGGGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGAC GGCACCGTGAAGCTGCTGATCTACCACACCTCCCGGCTGCACAGCGGCGTGCCCAGCAGATT TTCTGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGACA

[0420] TCGCTACCTACTTCTGTCAGCAAGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACCAAG CTGGAACTGAAGAGAGGCGGCGGAGGCTCTGGTGGAGGCGGATCTGGCGGGGGAGGAAGTGG GGGCGGAGGATCTGAAGTGCAGCTGCAGCAGAGCGGCCCTGGCCTGGTCGCCCCTAGCCAGA GCCTGTCCGTGACCTGTACCGTGTCCGGCGTGTCCCTGCCCGACTACGGCGTGTCCTGGATC AGACAGCCCCCCAGAAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAGACAACCTA

[0421] CTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGT TCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTAC TACTACGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGTCCAG CTACGTGACAGTGTCTAGCCAATGCATCGAGCCCAAGAGCTGCGACAAGACCCACACCTGTC CCCCCTGCCCCCCCGGCGATCGCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGC TATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCT CCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACC AGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGC GCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACG AAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGC CGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAG GCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTA CCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTC CTCGCTAA (SEQ ID NO : 51)

[0422] In one embodiment, the CAR comprises an antigen-binding domain comprising an anti-CD19 scFv, LNGFR mutated short spacer (NWL), CD28 transmembrane and co-stimulatory domain and a CD3- signalling domain.

[0423] In one embodiment, the CAR comprises an antigen-binding domain comprising an anti-CD19 scFv, LNGFR mutated short spacer (NMS), CD28 transmembrane and co-stimulatory domain and a CD3- signalling domain.

[0424] In one embodiment, the CAR comprises SEQ ID NO: 15, SEQ ID NO: 36, SEQ ID NO: 18 and SEQ ID NO: 30 or sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0425] In one embodiment, the CAR comprises SEQ ID NO: 15, SEQ ID NO: 36, SEQ ID NO: 18 and SEQ ID NO: 30 or variants having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein the variant of SEQ ID NO: 15 comprises SEQ ID NOs: 5-10.

[0426] In one embodiment, the CAR comprises SEQ ID NO: 15, SEQ ID NO: 36, SEQ ID NO: 18 and SEQ ID NO: 30 or variants having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein the variant of SEQ ID NO: 15 comprises SEQ ID NOs: 62-67.

[0427] In one embodiment, the CAR comprises SEQ ID NO: 15, SEQ ID NO: 36, SEQ ID NO: 18 and SEQ ID NO: 30 or variants having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein the variant of SEQ ID NO: 15 comprises SEQ ID NOs: 68-73.

[0428] In one embodiment, the CAR comprises SEQ ID NO: 15, SEQ ID NO: 36, SEQ ID NO: 18 and SEQ ID NO: 30 or variants having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein the variant of SEQ ID NO: 15 comprises SEQ ID NOs: 74-79.

[0429] In one embodiment, the CAR is encoded by SEQ ID NO: 16, SEQ ID NO: 37, SEQ ID NO: 29 and SEQ ID NO: 31 or sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0430] In one embodiment, the CAR comprises SEQ ID NO: 15, SEQ ID NO: 38, SEQ ID NO: 18 and SEQ ID NO: 30 or sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0431] In one embodiment, the CAR comprises SEQ ID NO: 15, SEQ ID NO: 38, SEQ ID NO: 18 and SEQ ID NO: 30 or variants having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein the variant of SEQ ID NO: 15 comprises SEQ ID NOs: 5-10.

[0432] In one embodiment, the CAR comprises SEQ ID NO: 15, SEQ ID NO: 38, SEQ ID NO: 18 and SEQ ID NO: 30 or variants having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein the variant of SEQ ID NO: 15 comprises SEQ ID NOs: 62-67.

[0433] In one embodiment, the CAR comprises SEQ ID NO: 15, SEQ ID NO: 38, SEQ ID NO: 18 and SEQ ID NO: 30 or variants having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein the variant of SEQ ID NO: 15 comprises SEQ ID NOs: 68-73.

[0434] In one embodiment, the CAR comprises SEQ ID NO: 15, SEQ ID NO: 38, SEQ ID NO: 18 and SEQ ID NO: 30 or variants having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein the variant of SEQ ID NO: 15 comprises SEQ ID NOs: 74-79.

[0435] In one embodiment, the CAR is encoded by SEQ ID NO: 16, SEQ ID NO: 39, SEQ ID NO: 29 and SEQ ID NO: 31 or sequences having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto.

[0436] In one embodiment, the CAR comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 80.

[0437] Exemplary CD19 CAR:

[0438] MEFGLSWLFLVAILKGVQCSRDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPD GTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTK LELKRGGGGSGGGGSGGGGSGGGGSEVQLQQSGPGLVAPSQSLSVTCTVSGVSLPDYGVSWI RQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHY YYGGSYAMDYWGQGTTVTVSSYVTVSSQCIKEACPTGLYTHSGECCKACNLGEGVAQPCGAN QTVCE PCLDSVTFSDWS ATE PCKPC TE CVGLQSMS APCVEADDAVCRCAYGYYQDE TTGRC EACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCLPCTVCEDTERQLRECTRWA DAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIASTVAGWTTVMGSSQPWTRG TTDNRDRFWVLVWGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQ PYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPP R (SEQ ID NO : 80)

[0439] In one embodiment, the CAR comprises a protein that is encoded by a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 81.

[0440] Nucleotide sequence encoding exemplary CAR:

[0441] ATGGAATTCGGCCTGAGCTGGCTGTTCCTGGTGGCCATCCTGAAGGGCGTGCAGTGCTCCCG GGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCA TCAGCTGCCGGGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGAC GGCACCGTGAAGCTGCTGATCTACCACACCTCCCGGCTGCACAGCGGCGTGCCCAGCAGATT TTCTGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGACA TCGCTACCTACTTCTGTCAGCAAGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACCAAG CTGGAACTGAAGAGAGGCGGCGGAGGCTCTGGTGGAGGCGGATCTGGCGGGGGAGGAAGTGG GGGCGGAGGATCTGAAGTGCAGCTGCAGCAGAGCGGCCCTGGCCTGGTCGCCCCTAGCCAGA GCCTGTCCGTGACCTGTACCGTGTCCGGCGTGTCCCTGCCCGACTACGGCGTGTCCTGGATC AGACAGCCCCCCAGAAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAGACAACCTA CTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGT TCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTAC TACTACGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGTCCAG CTACGTGACAGTGTCTAGCCAATGCATAAAAGAGGCCTGCCCCACCGGCCTGTACACCCACA GCGGAGAGTGCTGCAAGGCCTGCAACCTGGGAGAGGGCGTGGCCCAGCCTTGCGGCGCCAAT CAGACCGTGTGCGAGCCCTGCCTGGACAGCGTGACCTTCAGCGACGTGGTGTCCGCCACCGA GCCCTGCAAGCCTTGCACCGAGTGTGTGGGCCTGCAGAGCATGAGCGCCCCCTGCGTGGAAG CCGACGACGCCGTGTGTAGATGCGCCTACGGCTACTACCAGGACGAGACAACCGGCAGATGC GAGGCCTGTAGAGTGTGCGAGGCCGGCAGCGGCCTGGTGTTCAGTTGTCAAGACAAGCAGAA TACCGTGTGTGAAGAGTGCCCCGACGGCACCTACAGCGACGAGGCCAACCACGTGGACCCCT GCCTGCCCTGCACTGTGTGCGAGGACACCGAGCGGCAGCTGCGCGAGTGCACAAGATGGGCC GACGCCGAGTGCGAAGAGATCCCCGGCAGATGGATCACCAGAAGCACCCCCCCTGAGGGCAG CGACAGCACCGCCCCTAGCACCCAGGAACCTGAGGCCCCTCCCGAGCAGGACCTGATCGCCT CTACAGTGGCCGGCGTGGTGACAACCGTGATGGGCAGCTCTCAGCCCGTGGTGACACGGGGC ACCACCGACAATCGCGATCGCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTA TAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCC TGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAG CCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGC AGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAA GAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCG AGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGC CTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACC AGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCT CGCTAA (SEQ ID NO : 81)

[0442] In one embodiment, the CAR comprises a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 82.

[0443] Exemplary CD19 CAR:

[0444] MEFGLSWLFLVAILKGVQCSRDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPD GTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTK LELKRGGGGSGGGGSGGGGSGGGGSEVQLQQSGPGLVAPSQSLSVTCTVSGVSLPDYGVSWI RQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHY YYGGSYAMDYWGQGTTVTVSSYVTVSSQCIKEACPTGLYTHSGECCKACNLGEGVAQPCGAN QTVCE PCLDSVTFSDWS ATE PCKPC TE CVGLQSMS APCVEADDAVCRCAYGYYQDE TTGRC EACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEAARAADAECEERDRFWVLVWGGVLAC YSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRS ADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO : 82 )

[0445] In one embodiment, the CAR comprises a protein that is encoded by a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 83.

[0446] Nucleotide sequence encoding exemplary CAR:

[0447] ATGGAATTCGGCCTGAGCTGGCTGTTCCTGGTGGCCATCCTGAAGGGCGTGCAGTGCTCCCG GGACATCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCA TCAGCTGCCGGGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGAC GGCACCGTGAAGCTGCTGATCTACCACACCTCCCGGCTGCACAGCGGCGTGCCCAGCAGATT TTCTGGCAGCGGCAGCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGACA TCGCTACCTACTTCTGTCAGCAAGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACCAAG

[0448] CTGGAACTGAAGAGAGGCGGCGGAGGCTCTGGTGGAGGCGGATCTGGCGGGGGAGGAAGTGG GGGCGGAGGATCTGAAGTGCAGCTGCAGCAGAGCGGCCCTGGCCTGGTCGCCCCTAGCCAGA GCCTGTCCGTGACCTGTACCGTGTCCGGCGTGTCCCTGCCCGACTACGGCGTGTCCTGGATC AGACAGCCCCCCAGAAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAGACAACCTA CTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGT TCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTAC

[0449] TACTACGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGTCCAG CTACGTGACAGTGTCTAGCCAATGCATAAAAGAGGCCTGCCCCACCGGCCTGTACACCCACA GCGGAGAGTGCTGCAAGGCCTGCAACCTGGGAGAGGGCGTGGCCCAGCCTTGCGGCGCCAAT CAGACCGTGTGCGAGCCCTGCCTGGACAGCGTGACCTTCAGCGACGTGGTGTCCGCCACCGA GCCCTGCAAGCCTTGCACCGAGTGTGTGGGCCTGCAGAGCATGAGCGCCCCCTGCGTGGAAG CCGACGACGCCGTGTGTAGATGCGCCTACGGCTACTACCAGGACGAGACAACCGGCAGATGC

[0450] GAGGCCTGTAGAGTGTGCGAGGCCGGCAGCGGCCTGGTGTTCAGTTGTCAGGACAAGCAGAA CACCGTGTGTGAAGAGTGCCCCGACGGCACCTACAGCGACGAGGCCGCCCGGGCCGCCGACG CCGAGTGCGAGGAACGCGATCGCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGC TATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCT CCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACC AGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGC GCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACG AAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGC CGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAG GCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTA CCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTC CTCGCTAA (SEQ ID NO : 83)

[0451] In one embodiment, the CAR comprises a sequence with at least 90% (suitably, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 50.

[0452] In one embodiment, the CAR consists of a sequence with at least 90% (suitably, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 50.

[0453] In one embodiment, the CAR comprises the sequence of SEQ ID NO: 50.

[0454] In one embodiment, the CAR comprises a sequence with at least 90% (suitably, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 50, wherein said sequence comprises the sequences according to SEQ ID NOs: 5-10.

[0455] In one embodiment, the CAR comprises a sequence with at least 90% (suitably, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 50, wherein said sequence comprises the sequences according to SEQ ID NOs: 62-67.

[0456] In one embodiment, the CAR comprises a sequence with at least 90% (suitably, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 50, wherein said sequence comprises the sequences according to SEQ ID NOs: 68-73.

[0457] In one embodiment, the CAR comprises a sequence with at least 90% (suitably, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 50, wherein said sequence comprises the sequences according to SEQ ID NOs: 74-79.

[0458] In one embodiment, the CAR comprises a sequence with at least 90% (suitably, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 80. In one embodiment, the CAR consists of a sequence with at least 90% (suitably, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 80.

[0459] In one embodiment, the CAR comprises the sequence of SEQ ID NO: 80.

[0460] In one embodiment, the CAR comprises a sequence with at least 90% (suitably, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 80, wherein said sequence comprises the sequences according to SEQ ID NOs: 5-10.

[0461] In one embodiment, the CAR comprises a sequence with at least 90% (suitably, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 80, wherein said sequence comprises the sequences according to SEQ ID NOs: 62-67.

[0462] In one embodiment, the CAR comprises a sequence with at least 90% (suitably, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 80, wherein said sequence comprises the sequences according to SEQ ID NOs: 68-73.

[0463] In one embodiment, the CAR comprises a sequence with at least 90% (suitably, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 80, wherein said sequence comprises the sequences according to SEQ ID NOs: 74-79.

[0464] In one embodiment, the CAR comprises a sequence with at least 90% (suitably, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 82.

[0465] In one embodiment, the CAR consists of a sequence with at least 90% (suitably, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 82.

[0466] In one embodiment, the CAR comprises the sequence of SEQ ID NO: 82.

[0467] In one embodiment, the CAR comprises a sequence with at least 90% (suitably, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 82, wherein said sequence comprises the sequences according to SEQ ID NOs: 5-10. In one embodiment, the CAR comprises a sequence with at least 90% (suitably, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 82, wherein said sequence comprises the sequences according to SEQ ID NOs: 62-67.

[0468] In one embodiment, the CAR comprises a sequence with at least 90% (suitably, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 82, wherein said sequence comprises the sequences according to SEQ ID NOs: 68-73.

[0469] In one embodiment, the CAR comprises a sequence with at least 90% (suitably, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 82, wherein said sequence comprises the sequences according to SEQ ID NOs: 74-79.

[0470] Exemplary constructs

[0471] In one embodiment, the vector comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 84.

[0472] ATGCCCAACCCCAGGCCTGGCAAGCCCTCGGCCCCTTCCTTGGCCCTTGGCCCATCCCCAGG AGCCTCGCCCAGCTGGAGGGCTGCACCCAAAGCCTCAGACCTGCTGGGGGCCCGGGGCCCAG GGGGAACCTTCCAGGGCCGAGATCTTCGAGGCGGGGCCCATGCCTCCTCTTCTTCCTTGAAC CCCATGCCACCATCGCAGCTGCAGCTGCCCACACTGCCCCTAGTCATGGTGGCACCCTCCGG GGCACGGCTGGGCCCCTTGCCCCACTTACAGGCACTCCTCCAGGACAGGCCACATTTCATGC ACCAGCTCTCAACGGTGGATGCCCACGCCCGGACCCCTGTGCTGCAGGTGCACCCCCTGGAG AGCCCAGCCATGATCAGCCTCACACCACCCACCACCGCCACTGGGGTCTTCTCCCTCAAGGC CCGGCCTGGCCTCCCACCTGGGATCAACGTGGCCAGCCTGGAATGGGTGTCCAGGGAGCCGG CACTGCTCTGCACCTTCCCAAATCCCAGTGCACCCAGGAAGGACAGCACCCTTTCGGCTGTG CCCCAGAGCTCCTACCCACTGCTGGCAAATGGTGTCTGCAAGTGGCCCGGATGTGAGAAGGT CTTCGAAGAGCCAGAGGACTTCCTCAAGCACTGCCAGGCGGACCATCTTCTGGATGAGAAGG GCAGGGCACAATGTCTCCTCCAGAGAGAGATGGTACAGTCTCTGGAGCAGCAGCTGGTGCTG GAGAAGGAGAAGCTGAGTGCCATGCAGGCCCACCTGGCTGGGAAAATGGCACTGACCAAGGC TTCATCTGTGGCATCATCCGACAAGGGCTCCTGCTGCATCGTAGCTGCTGGCAGCCAAGGCC CTGTCGTCCCAGCCTGGTCTGGCCCCCGGGAGGCCCCTGACAGCCTGTTTGCTGTCCGGAGG CACCTGTGGGGTAGCCATGGAAACAGCACATTCCCAGAGTTCCTCCACAACATGGACTACTT CAAGTTCCACAACATGCGACCCCCTTTCACCTACGCCACGCTCATCCGCTGGGCCATCCTGG AGGCTCCAGAGAAGCAGCGGACACTCAATGAGATCTACCACTGGTTCACACGCATGTTTGCC TTCTTCAGAAACCATCCTGCCACCTGGAAGAACGCCATCCGCCACAACCTGAGTCTGCACAA GTGCTTTGTGCGGGTGGAGAGCGAGAAGGGGGCTGTGTGGACCGTGGATGAGCTGGAGTTCC GCAAGAAACGGAGCCAGAGGCCCAGCAGGTGTTCCAACCCTACACCTGGCCCCGCTAGCAGA GCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGA ATTCGGCCTGAGCTGGCTGTTCCTGGTGGCCATCCTGAAGGGCGTGCAGTGCTCCCGGGACA TCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGC TGCCGGGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCAC CGTGAAGCTGCTGATCTACCACACCTCCCGGCTGCACAGCGGCGTGCCCAGCAGATTTTCTG GCAGCGGCAGCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGACATCGCT ACCTACTTCTGTCAGCAAGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACCAAGCTGGA ACTGAAGAGAGGCGGCGGAGGCTCTGGTGGAGGCGGATCTGGCGGGGGAGGAAGTGGGGGCG GAGGATCTGAAGTGCAGCTGCAGCAGAGCGGCCCTGGCCTGGTCGCCCCTAGCCAGAGCCTG TCCGTGACCTGTACCGTGTCCGGCGTGTCCCTGCCCGACTACGGCGTGTCCTGGATCAGACA GCCCCCCAGAAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAGACAACCTACTACA ACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTG AAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTA

[0473] CGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGTCCAGCTACG TGACAGTGTCTAGCCAATGCATAAAAGAGGCCTGCCCCACCGGCCTGTACACCCACAGCGGA GAGTGCTGCAAGGCCTGCAACCTGGGAGAGGGCGTGGCCCAGCCTTGCGGCGCCAATCAGAC CGTGTGCGAGCCCTGCCTGGACAGCGTGACCTTCAGCGACGTGGTGTCCGCCACCGAGCCCT GCAAGCCTTGCACCGAGTGTGTGGGCCTGCAGAGCATGAGCGCCCCCTGCGTGGAAGCCGAC GACGCCGTGTGTAGATGCGCCTACGGCTACTACCAGGACGAGACAACCGGCAGATGCGAGGC CTGTAGAGTGTGCGAGGCCGGCAGCGGCCTGGTGTTCAGTTGTCAAGACAAGCAGAATACCG TGTGTGAAGAGTGCCCCGACGGCACCTACAGCGACGAGGCCAACCACGTGGACCCCTGCCTG CCCTGCACTGTGTGCGAGGACACCGAGCGGCAGCTGCGCGAGTGCACAAGATGGGCCGACGC

[0474] CGAGTGCGAAGAGATCCCCGGCAGATGGATCACCAGAAGCACCCCCCCTGAGGGCAGCGACA GCACCGCCCCTAGCACCCAGGAACCTGAGGCCCCTCCCGAGCAGGACCTGATCGCCTCTACA GTGGCCGGCGTGGTGACAACCGTGATGGGCAGCTCTCAGCCCGTGGTGACACGGGGCACCAC CGACAATCGCGATCGCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCT TGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCAC AGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTA TGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGACG CCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAG GAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAG

[0475] GAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACA GTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGT CTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGCTA A (SEQ ID NO : 84 ; FoxP3-T2A-19CARNWL28z)

[0476] In one embodiment, the vector comprises a nucleotide sequence encoding a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 85.

[0477] MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLN PMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLE SPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAV PQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVL EKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPWPAWSGPREAPDSLFAVRR HLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFA FFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGPASR AEGRGSLLTCGDVEENPGPMEFGLSWLFLVAILKGVQCSRDIQMTQTTSSLSASLGDRVTIS CRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA TYFCQQGNTLPYTFGGGTKLELKRGGGGSGGGGSGGGGSGGGGSEVQLQQSGPGLVAPSQSL

[0478] SVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFL KMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTTVTVSSYVTVSSQCIKEACPTGLYTHSG ECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDWSATEPCKPCTECVGLQSMSAPCVEAD DAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEANHVDPCL PCTVCEDTERQLRECTRWADAECEEIPGRWITRSTPPEGSDSTAPSTQEPEAPPEQDLIAST VAGWTTVMGSSQPWTRGTTDNRDRFWVLVWGGVLACYSLLVTVAFIIFWVRSKRSRLLH SDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRRE EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQG LSTATKDTYDALHMQALPPR (SEQ ID NO : 85 ; FoxP3-T2A-19CARNWL28z)

[0479] In one embodiment, the vector comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 86.

[0480] ATGCCCAACCCCAGGCCTGGCAAGCCCTCGGCCCCTTCCTTGGCCCTTGGCCCATCCCCAGG AGCCTCGCCCAGCTGGAGGGCTGCACCCAAAGCCTCAGACCTGCTGGGGGCCCGGGGCCCAG GGGGAACCTTCCAGGGCCGAGATCTTCGAGGCGGGGCCCATGCCTCCTCTTCTTCCTTGAAC CCCATGCCACCATCGCAGCTGCAGCTGCCCACACTGCCCCTAGTCATGGTGGCACCCTCCGG GGCACGGCTGGGCCCCTTGCCCCACTTACAGGCACTCCTCCAGGACAGGCCACATTTCATGC ACCAGCTCTCAACGGTGGATGCCCACGCCCGGACCCCTGTGCTGCAGGTGCACCCCCTGGAG AGCCCAGCCATGATCAGCCTCACACCACCCACCACCGCCACTGGGGTCTTCTCCCTCAAGGC CCGGCCTGGCCTCCCACCTGGGATCAACGTGGCCAGCCTGGAATGGGTGTCCAGGGAGCCGG CACTGCTCTGCACCTTCCCAAATCCCAGTGCACCCAGGAAGGACAGCACCCTTTCGGCTGTG CCCCAGAGCTCCTACCCACTGCTGGCAAATGGTGTCTGCAAGTGGCCCGGATGTGAGAAGGT CTTCGAAGAGCCAGAGGACTTCCTCAAGCACTGCCAGGCGGACCATCTTCTGGATGAGAAGG GCAGGGCACAATGTCTCC TCCAGAGAGAGATGGTACAGTC TC TGGAGCAGCAGC TGGTGC TG GAGAAGGAGAAGCTGAGTGCCATGCAGGCCCACCTGGCTGGGAAAATGGCACTGACCAAGGC TTCATCTGTGGCATCATCCGACAAGGGCTCCTGCTGCATCGTAGCTGCTGGCAGCCAAGGCC CTGTCGTCCCAGCCTGGTCTGGCCCCCGGGAGGCCCCTGACAGCCTGTTTGCTGTCCGGAGG CACCTGTGGGGTAGCCATGGAAACAGCACATTCCCAGAGTTCCTCCACAACATGGACTACTT CAAGTTCCACAACATGCGACCCCCTTTCACCTACGCCACGCTCATCCGCTGGGCCATCCTGG AGGCTCCAGAGAAGCAGCGGACACTCAATGAGATCTACCACTGGTTCACACGCATGTTTGCC TTCTTCAGAAACCATCCTGCCACCTGGAAGAACGCCATCCGCCACAACCTGAGTCTGCACAA GTGCTTTGTGCGGGTGGAGAGCGAGAAGGGGGCTGTGTGGACCGTGGATGAGCTGGAGTTCC GCAAGAAACGGAGCCAGAGGCCCAGCAGGTGTTCCAACCCTACACCTGGCCCCGCTAGCAGA GCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGA ATTCGGCCTGAGCTGGCTGTTCCTGGTGGCCATCCTGAAGGGCGTGCAGTGCTCCCGGGACA TCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGC TGCCGGGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCAC CGTGAAGCTGCTGATCTACCACACCTCCCGGCTGCACAGCGGCGTGCCCAGCAGATTTTCTG GCAGCGGCAGCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGACATCGCT ACCTACTTCTGTCAGCAAGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACCAAGCTGGA ACTGAAGAGAGGCGGCGGAGGCTCTGGTGGAGGCGGATCTGGCGGGGGAGGAAGTGGGGGCG

[0481] GAGGATCTGAAGTGCAGCTGCAGCAGAGCGGCCCTGGCCTGGTCGCCCCTAGCCAGAGCCTG TCCGTGACCTGTACCGTGTCCGGCGTGTCCCTGCCCGACTACGGCGTGTCCTGGATCAGACA GCCCCCCAGAAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAGACAACCTACTACA ACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTG AAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTA CGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGTCCAGCTACG TGACAGTGTCTAGCCAATGCATAAAAGAGGCCTGCCCCACCGGCCTGTACACCCACAGCGGA GAGTGCTGCAAGGCCTGCAACCTGGGAGAGGGCGTGGCCCAGCCTTGCGGCGCCAATCAGAC CGTGTGCGAGCCCTGCCTGGACAGCGTGACCTTCAGCGACGTGGTGTCCGCCACCGAGCCCT GCAAGCCTTGCACCGAGTGTGTGGGCCTGCAGAGCATGAGCGCCCCCTGCGTGGAAGCCGAC GACGCCGTGTGTAGATGCGCCTACGGCTACTACCAGGACGAGACAACCGGCAGATGCGAGGC CTGTAGAGTGTGCGAGGCCGGCAGCGGCCTGGTGTTCAGTTGTCAGGACAAGCAGAACACCG TGTGTGAAGAGTGCCCCGACGGCACCTACAGCGACGAGGCCGCCCGGGCCGCCGACGCCGAG TGCGAGGAACGCGATCGCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAG CTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGC ACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCC TATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGA CGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAG AGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGA AGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTA CAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGG GTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGC TAA (SEQ ID NO : 86 ; FoxP3-T2A-19CARNMS28z)

[0482] In one embodiment, the vector comprises a nucleotide sequence encoding a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 87.

[0483] MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLN PMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLE SPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAV PQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVL EKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPWPAWSGPREAPDSLFAVRR HLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFA FFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGPASR AEGRGSLLTCGDVEENPGPMEFGLSWLFLVAILKGVQCSRDIQMTQTTSSLSASLGDRVTIS CRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA TYFCQQGNTLPYTFGGGTKLELKRGGGGSGGGGSGGGGSGGGGSEVQLQQSGPGLVAPSQSL SVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFL KMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTTVTVSSYVTVSSQCIKEACPTGLYTHSG ECCKACNLGEGVAQPCGANQTVCEPCLDSVTFSDWSATEPCKPCTECVGLQSMSAPCVEAD DAVCRCAYGYYQDETTGRCEACRVCEAGSGLVFSCQDKQNTVCEECPDGTYSDEAARAADAE CEERDRFWVLVWGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQP YAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSE I GMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPR (SEQ ID NO : 87 ; FoxP3-T2A-19CARNMS28z)

[0484] In one embodiment, the vector comprises a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 88.

[0485] ATGCCCAACCCCAGGCCTGGCAAGCCCTCGGCCCCTTCCTTGGCCCTTGGCCCATCCCCAGG AGCCTCGCCCAGCTGGAGGGCTGCACCCAAAGCCTCAGACCTGCTGGGGGCCCGGGGCCCAG GGGGAACCTTCCAGGGCCGAGATCTTCGAGGCGGGGCCCATGCCTCCTCTTCTTCCTTGAAC CCCATGCCACCATCGCAGCTGCAGCTGCCCACACTGCCCCTAGTCATGGTGGCACCCTCCGG GGCACGGCTGGGCCCCTTGCCCCACTTACAGGCACTCCTCCAGGACAGGCCACATTTCATGC ACCAGCTCTCAACGGTGGATGCCCACGCCCGGACCCCTGTGCTGCAGGTGCACCCCCTGGAG AGCCCAGCCATGATCAGCCTCACACCACCCACCACCGCCACTGGGGTCTTCTCCCTCAAGGC CCGGCCTGGCCTCCCACCTGGGATCAACGTGGCCAGCCTGGAATGGGTGTCCAGGGAGCCGG CACTGCTCTGCACCTTCCCAAATCCCAGTGCACCCAGGAAGGACAGCACCCTTTCGGCTGTG CCCCAGAGCTCCTACCCACTGCTGGCAAATGGTGTCTGCAAGTGGCCCGGATGTGAGAAGGT CTTCGAAGAGCCAGAGGACTTCCTCAAGCACTGCCAGGCGGACCATCTTCTGGATGAGAAGG GCAGGGCACAATGTCTCC TCCAGAGAGAGATGGTACAGTC TC TGGAGCAGCAGC TGGTGC TG GAGAAGGAGAAGCTGAGTGCCATGCAGGCCCACCTGGCTGGGAAAATGGCACTGACCAAGGC TTCATCTGTGGCATCATCCGACAAGGGCTCCTGCTGCATCGTAGCTGCTGGCAGCCAAGGCC CTGTCGTCCCAGCCTGGTCTGGCCCCCGGGAGGCCCCTGACAGCCTGTTTGCTGTCCGGAGG CACCTGTGGGGTAGCCATGGAAACAGCACATTCCCAGAGTTCCTCCACAACATGGACTACTT CAAGTTCCACAACATGCGACCCCCTTTCACCTACGCCACGCTCATCCGCTGGGCCATCCTGG AGGCTCCAGAGAAGCAGCGGACACTCAATGAGATCTACCACTGGTTCACACGCATGTTTGCC TTCTTCAGAAACCATCCTGCCACCTGGAAGAACGCCATCCGCCACAACCTGAGTCTGCACAA GTGCTTTGTGCGGGTGGAGAGCGAGAAGGGGGCTGTGTGGACCGTGGATGAGCTGGAGTTCC GCAAGAAACGGAGCCAGAGGCCCAGCAGGTGTTCCAACCCTACACCTGGCCCCGCTAGCAGA GCCGAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCCATGGA ATTCGGCCTGAGCTGGCTGTTCCTGGTGGCCATCCTGAAGGGCGTGCAGTGCTCCCGGGACA TCCAGATGACCCAGACCACCAGCAGCCTGAGCGCCAGCCTGGGCGACAGAGTGACCATCAGC TGCCGGGCCAGCCAGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCAC CGTGAAGCTGCTGATCTACCACACCTCCCGGCTGCACAGCGGCGTGCCCAGCAGATTTTCTG GCAGCGGCAGCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAGGAAGACATCGCT ACCTACTTCTGTCAGCAAGGCAACACCCTGCCCTACACCTTCGGCGGAGGCACCAAGCTGGA ACTGAAGAGAGGCGGCGGAGGCTCTGGTGGAGGCGGATCTGGCGGGGGAGGAAGTGGGGGCG GAGGATCTGAAGTGCAGCTGCAGCAGAGCGGCCCTGGCCTGGTCGCCCCTAGCCAGAGCCTG TCCGTGACCTGTACCGTGTCCGGCGTGTCCCTGCCCGACTACGGCGTGTCCTGGATCAGACA GCCCCCCAGAAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAGACAACCTACTACA ACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACAACAGCAAGAGCCAGGTGTTCCTG AAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGCGCCAAGCACTACTACTA CGGCGGCAGCTACGCTATGGACTACTGGGGCCAGGGCACCACCGTGACCGTGTCCAGCTACG TGACAGTGTCTAGCCAATGCATCGAGCCCAAGAGCTGCGACAAGACCCACACCTGTCCCCCC TGCCCCCCCGGCGATCGCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAG CTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGCTCCTGC ACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCC TATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCAGA CGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAG AGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGA AGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTA CAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGG GTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCTCCTCGC TAA (SEQ ID NO : 88 ; FoxP3-T2A-antiCD19CAR28z)

[0486] In one embodiment, the vector comprises a nucleotide sequence encoding a protein having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 89.

[0487] MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLN PMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLE SPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAV PQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKGRAQCLLQRE , VQSLEQQLVL EKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPWPAWSGPREAPDSLFAVRR HLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFA FFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGPASR AEGRGSLLTCGDVEENPGPMEFGLSWLFLVAILKGVQCSRDIQMTQTTSSLSASLGDRVTIS CRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA TYFCQQGNTLPYTFGGGTKLELKRGGGGSGGGGSGGGGSGGGGSEVQLQQSGPGLVAPSQSL SVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFL KMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTTVTVSSYVYVSSQCIEPKSCDKTHTCPP CPPGDRFWVLVWGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQP YAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSE I GMKGERRRGKGHDGLYQGLS TATKDTYDALHMQALPPR (SEQ ID NO : 89 ; FoxP3-T2A-antiCD19CAR28z)

[0488] Promoter

[0489] The vector of the present invention may comprise a promoter. Suitably, the protein-coding sequences (e.g. the first, second and third nucleotide sequences) are operably linked to a promoter.

[0490] A “promoter” may refer to a region of DNA that leads to initiation of transcription of a gene. Promoters are located near the transcription start sites of genes.

[0491] As used herein, a “tissue-specific promoter” may refer to a promoter which preferentially facilitates expression of a transgene in a specific type of cells or tissue. Suitably, a tissuespecific promoter may facilitate higher expression of a transgene in one cell type as compared to other cell types. Higher expression may be measured for example by measuring the expression of a transgene, for example green fluorescence protein (GFP), operably linked to the promoter, wherein expression of the transgene correlates with the ability of the promoter to facilitate expression of a gene. For example, a tissue-specific promoter may be a promoter which facilitates transgene expression levels at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 100% higher, at least 200% higher, at least 300% higher, at least 400% higher, at least 500% higher or at least 1000% higher in one cell type as compared to expression levels in other cell types.

[0492] In some embodiments, the promoter is a hPGK promoter, or a variant and / or fragment thereof. The variant retains the capacity to drive expression of a transgene which is operably coupled to the promoter. A skilled person will be able to arrive at such variants using methods known in the art. The variant may, for example, have at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the promoter.

[0493] A fragment of the promoter (or variants thereof) may be used, provided that the fragment retains the capacity to drive expression of a transgene which is operably coupled to the promoter. A skilled person will be able to arrive at such fragments using methods known in the art. The fragment may be, for example, at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides or at least 1000 nucleotides in length.

[0494] Exemplary hPGK promoter:

[0495] CCACGGGGTTGGGGTTGCGCCTTTTCCAAGGCAGCCCTGGGTTTGCGCAGGGACGCGGCTGC TCTGGGCGTGGTTCCGGGAAACGCAGCGGCGCCGACCCTGGGTCTCGCACATTCTTCACGTC CGTTCGCAGCGTCACCCGGATCTTCGCCGCTACCCTTGTGGGCCCCCCGGCGACGCTTCCTG CTCCGCCCCTAAGTCGGGAAGGTTCCTTGCGGTTCGCGGCGTGCCGGACGTGACAAACGGAA GCCGCACGTCTCACTAGTACCCTCGCAGACGGACAGCGCCAGGGAGCAATGGCAGCGCGCCG ACCGCGATGGGCTGTGGCCAATAGCGGCTGCTCAGCGGGGCGCGCCGAGAGCAGCGGCCGGG AAGGGGCGGTGCGGGAGGCGGGGTGTGGGGCGGTAGTGTGGGCCCTGTTCCTGCCCGCGCGG TGTTCCGCATTCTGCAAGCCTCCGGAGCGCACGTCGGCAGTCGGCTCCCTCGTTGACCGAAT CACCGACCTCTCTCCCCAGG

[0496] (SEQ ID NO : 54 )

[0497] In some embodiments, the promoter comprises the sequence as set forth in SEQ ID NO: 54, or a variant and / or fragment thereof.

[0498] The promoter may be a constitutive promoter. As used herein, a “constitutive promoter” is a promoter which is always active.

[0499] Alternatively, the promoter may be an inducible promoter. As used herein, an “inducible promoter” is a promoter which is only active under specific conditions. For example, expression of the transgene may be induced by a small molecule or drug (e.g. which binds to a promoter, regulatory sequence or to a transcriptional repressor or activator molecule) or by using an environmental trigger. Types of inducible promoter include chemically-inducible promoters (e.g. a Tet-on system); temperature-inducible promoters (e.g. Hsp70 or Hsp90- derived promoters); and light-inducible promoters. Suitably, the promoter is chemically- inducible. Any suitable method for engineering an inducible promoter may be used.

[0500] Lentiviral vector

[0501] In one aspect, the invention provides a lentiviral vector comprising a construct as described herein.

[0502] Lentiviral vector

[0503] A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. A lentiviral vector may be in the form of a lentiviral particle. In some embodiments, a lentiviral vector may comprise a lentiviral genome. As used herein, a lentiviral genome may refer to a genome that comprises at least one element derived or derivable from a lentivirus genome.

[0504] Lentivirus is a genus of retroviruses, which contain an RNA genome that is converted to DNA in the transduced cell by a reverse transcriptase. Lentiviral vectors can transduce a wide range of cell types and integrate into the host genome in both dividing and post-mitotic cells, resulting in long-term expression of the protein-coding sequence both in vitro and in vivo.

[0505] The basic genes required for lentivirus survival and function are the gag, pol, and env genes: gag encodes structural proteins; pol encodes enzymes required for reverse transcription and integration into the host cell genome; and env encodes the viral envelope glycoprotein. Lentiviruses may also have additional cis-acting elements, such as a rev response element (RRE), which enables the efficient export of RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of an infected target cell; a retroviral psi packaging element, which is involved in regulating the essential process of packaging the retroviral RNA genome into the viral capsid during replication; a primer binding site (PBS), where reverse transcription is initiated; the TAT activation region (TAR); splice donor and acceptor sites; and central and terminal polypurine tracts, which allow initiation of plus-strand synthesis.

[0506] In a lentivirus genome, the elements are typically flanked at both ends by regions called long terminal repeats (LTRs). The LTRs are responsible for integration and transcription. LTRs may also serve as enhancer-promoter sequences and can control the expression of the lentiviral genes. The LTRs themselves are identical or near-identical sequences that can typically be divided into three regions: U3, R and U5. LTRs may be naturally occurring or may be modified. For example, U3 and U5 modifications are described in Iwakuma et al. (1999) Virology 261 : 120-132.

[0507] The lentiviral vector of the present invention may comprise a minimal lentiviral genome. As used herein, a minimal lentiviral genome may mean that the lentiviral genome has been manipulated so as to remove the non-essential elements and to retain the essential elements in order to provide the required functionality to infect, transduce and deliver a nucleotide sequence of interest to a target host cell (see, for example, Kim et al. (1998) Journal of Virology 72: 811-816; Sertkaya et al. (2021) Scientific Reports 11 : 1-15).

[0508] A lentiviral genome may comprise from 5’ to 3’: a 5’ LTR, one or more lentiviral-derived cis- acting elements, and a 3’ LTR. Suitably, a lentiviral genome may comprise from 5’ to 3’: a 5’ LTR, a RRE, and a 3’ LTR. Suitably, a lentiviral genome may comprise from 5’ to 3’: a 5’ LTR, a retroviral psi packaging element, a RRE, and a 3’ LTR. Suitably, a lentiviral genome may comprise from 5’ to 3’: a 5’ LTR, a retroviral psi packaging element, a RRE, a cPPT, and a 3’ LTR. Suitably, a lentiviral genome may comprise from 5’ to 3’: a 5’ LTR, a PBS, a retroviral psi packaging element, a RRE, a cPPT, and a 3’ LTR.

[0509] A lentiviral genome may further comprise a protein-coding sequence (e.g. the construct) and, optionally, one or more regulatory elements (e.g. operably linked to the protein-coding sequence). Suitably, a lentiviral genome may comprise from 5’ to 3’: a 5’ LTR, a RRE, a protein-coding sequence, and a 3’ LTR. Suitably, a lentiviral genome may comprise from 5’ to 3’: a 5’ LTR, a retroviral psi packaging element, a RRE, a protein-coding sequence, and a 3’ LTR. Suitably, a lentiviral genome may comprise from 5’ to 3’: a 5’ LTR, a retroviral psi packaging element, a RRE, a cPPT, a protein-coding sequence, and a 3’ LTR. Suitably, lentiviral genome may comprise from 5’ to 3’: a 5’ LTR, a PBS, a retroviral psi packaging element, a RRE, a cPPT, a protein-coding sequence, and a 3’ LTR.

[0510] The lentiviral vector of the present invention may be replication-defective. Typically, at least part of one or more protein coding regions essential for replication may be removed from the lentiviral genome. This makes the lentivirus “replication-defective” or “replicationincompetent”. Suitably, one or more of gag, pol, rev, and env genes are deleted (at least partially) in a replication-defective lentivirus. Suitably, each of the gag, pol, rev, and env genes are deleted (at least partially) in a replication-defective lentivirus. Optionally, the lentivirus lacks a functional gag-pol and / or env gene and / or other genes essential for replication.

[0511] The lentiviral vector of the present invention may be derived from any lentivirus. As used herein “lentivirus-derived” or“lentivirus-based” may mean that the lentiviral genome comprises one or more elements from said lentivirus. For example, the coding regions of viral proteins may be deleted, but one or more cis-acting element may be retained from said lentivirus.

[0512] The lentiviral vector may be derived from a primate lentivirus. Examples of “primate” lentiviruses include, but are not limited to, human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). The lentiviral vector may be derived from a non-primate lentivirus (i.e. derived from a lentivirus which does not primarily infect primates, especially humans). Examples of “non-primate” lentiviruses include, but are not limited to, the prototype “slow virus” visna / maedi virus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV), feline immunodeficiency virus (FIV), and bovine immunodeficiency virus (BIV).

[0513] Suitably, the lentiviral vector of the present invention is a HIV-derived lentiviral vector. As used herein “HIV-derived” or “HIV-based” may mean that the lentiviral genome comprises one or more element from HIV. For example, the coding regions of HIV viral proteins may be deleted, and one or more HIV cis-acting element may retained in the lentiviral genome (see, for example, Johnson (2021) Molecular Therapy-Methods & Clinical Development 21 : 451-465). A HIV-derived lentiviral genome may comprise from 5’ to 3’: a 5’ LTR, one or more HIV-derived cis-acting elements (e.g. RRE and / or cPPT), and a 3’ LTR.

[0514] Suitably, the lentiviral vector of the present invention is a HIV-1-derived lentiviral vector. The prototype lentiviral vector system is based on HIV-1 (see, for example, Merten et al. (2016) Molecular Therapy-Methods & Clinical Development 3: 16017). It has been shown that sequences that extend into the gag open reading frame may be important for packaging of HIV-1. Therefore, HIV-1 vectors often contain the relevant portion of gag in which the translational initiation codon has been mutated. In addition, HIV-1 vectors often also contain a portion of the envgene that includes the RRE. Rev binds to RRE, which permits the transport of full-length or singly spliced mRNAs from the nucleus to the cytoplasm. In the absence of rev and / or a RRE, full-length HIV-1 RNAs may accumulate in the nucleus. Alternatively, a constitutive transport element from certain simple retroviruses such as Mason-Pfizer monkey virus can be used to relieve the requirement for rev and a RRE. A HIV-1-derived lentiviral genome may comprise from 5’ to 3’: a 5’ LTR, one or more HIV-1-derived cis-acting elements (e.g. a PBS, a retroviral psi packaging element, a RRE and / or a cPPT), and a 3’ LTR.

[0515] The lentiviral vector of the present invention may be a self-inactivating lentiviral vector. As used herein, “self-inactivating” or “SIN” lentiviral vector may comprise lentiviral genomes in which the lentiviral enhancer and promoter sequences have been deleted (see, for example, Zufferey (1998) Journal of Virology 72: 9873-9880; Miyoshi et al. (1998) Journal of Virology 72: 8150-8157). SIN lentiviral vectors can be generated and transduce non-dividing cells in vivo with an efficacy similar to that of wild-type vectors. The transcriptional inactivation of the long terminal repeat (LTR) in the SIN provirus can prevent mobilisation by replication- competent virus. This can also enable the regulated expression of genes from internal promoters by eliminating any cis-acting effects of the LTR.

[0516] The lentiviral vector vector of the present invention may be integration competent. As used herein, an “integration competent” lentiviral vector is capable of integrating into the genome of a host cell. In contrast to integration competent lentiviral vectors, integration defective lentiviral vectors (IDLVs) can be produced, for example by packaging the lentiviral vector with catalytically inactive integrase (such as an HIV integrase bearing the D64V mutation in the catalytic site) or by modifying or deleting essential att sequences from the lentiviral genome LTR, or by a combination of the above (see, for example. Wanisch et al. (2009) Molecular Therapy 17: 1316-1332). The lentiviral vector of the present invention may be replication-defective and integrating. The lentiviral vector of the present invention may be replication-defective, integrating, and selfinactivating. The lentiviral vector of the present invention may be replication-defective, integrating, self-inactivating, and HIV-derived.

[0517] A “lentiviral particle” may refer to an enveloped lentiviral genome. Lentiviral particles may be generated by co-transfection of a plasmid containing a lentiviral genome (e.g. a “transfer vector”) with helper plasmids (e.g. “packaging vectors” encoding gag-pol and / or rev, and “envelope vectors” encoding env) into host cells and harvesting of the lentivirus-containing supernatant afterwards.

[0518] The lentiviral vector of the present invention may be pseudotyped. Pseudotyping lentiviral vector with naturally occurring or engineered lentiviral envelopes can allow targeted transduction of specific cell types (see, for example, Joglekar et al. (2017) Human Gene Therapy Methods 28: 291-301).

[0519] Preferably, the lentiviral vector of the present invention is VSV-G pseudotyped. Vesicular stomatitis virus G protein (VSV-G) is a commonly used envelope protein for pseudotyping. VSV-G is a trimeric protein that binds phosphatidylserine and low-density lipoprotein receptors on a cell surface to endocytose into the cell.

[0520] The lentiviral vector of the present invention may be replication-defective, integrating, and VSV-G pseudotyped. The lentiviral vector of the present invention may be replicationdefective, integrating, self-inactivating, and VSV-G pseudotyped. The lentiviral vector of the present invention may be replication-defective, integrating self-inactivating, HIV-derived, and VSV-G pseudotyped.

[0521] Regulatory elements

[0522] The lentiviral vector of the present invention may further comprise one or more regulatory elements which may act pre- or post-transcriptional ly. Suitably, the protein-coding sequence (e.g. the construct) is operably linked to one or more regulatory elements which may act pre- or post-transcriptionally. The one or more regulatory elements may facilitate expression of the protein in liver cells (e.g. hepatocytes).

[0523] As used herein, a “regulatory element” may refer any nucleotide sequence that facilitates expression of a polypeptide, for example acts to increase expression of a transcript or to enhance mRNA stability. Suitable regulatory elements include for example promoters, enhancer elements, post-transcriptional regulatory elements, polyadenylation sites and Kozak sequences. Promoter

[0524] The lenti viral vector of the present invention may comprise a promoter as described herein. Suitably, the protein-coding sequence (e.g. the construct) is operably linked to a promoter as described herein.

[0525] Enhancer elements

[0526] The lentiviral vector of the present invention may comprise an enhancer. Suitably, the proteincoding sequence (e.g. the construct is operably linked to an enhancer.

[0527] An “enhancer” or “enhancer element” may refer a region of DNA that can be bound by proteins (activators) to increase the likelihood that transcription of a particular gene will occur. Enhancers are cis-acting. They can be located up to 1 Mbp (1 ,000,000 bp) away from the gene, upstream or downstream from the start site.

[0528] As used herein, a “tissue-specific enhancer” is an enhancer which preferentially facilitates expression of a gene in specific cells or tissues. Suitably, a tissue-specific enhancer may facilitate higher expression of a gene in specific cells types as compared to other cell types. Higher expression may be measured for example by measuring the expression of a transgene, for example green fluorescence protein (GFP), operably linked to the enhancer, wherein expression of the transgene correlates with the ability of the enhancer to facilitate expression of a gene. For example, a tissue-specific enhancer may be an enhancer which facilitates gene expression levels at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 100% higher, at least 200% higher, at least 300% higher, at least 400% higher, at least 500% higher or at least 1000% higher in a specific cell-type compared to expression levels in other cell types.

[0529] Suitable tissue-specific enhancers will be well known to the skilled person. Methods to identify the enhancer regions associated with genes will be well known to the skilled person.

[0530] Post-transcriptional regulatory elements

[0531] The lentiviral vector of the present invention may comprise one or more post-transcriptional regulatory elements. Suitably, the protein-coding sequence (e.g. the construct) is operably linked to one or more post-transcriptional regulatory elements. The post-transcriptional regulatory element may improve gene expression.

[0532] The lentiviral vector of the present invention may comprise a Woodchuck Hepatitis Virus Post- transcriptional Regulatory Element (WPRE). Suitably, the protein-coding sequence (e.g. the construct) is operably linked to a WPRE. Suitable WPRE sequences will be well known to those of skill in the art (see, for example, Zufferey et al. (1999) Journal of Virology 73: 2886-2892; Zanta-Boussif et al. (2009) Gene Therapy 16: 605-619). Suitably, the WPRE is a wild-type WPRE or is a mutant WPRE. For example, the WPRE may be mutated to abrogate translation of the woodchuck hepatitis virus X protein (WHX), for example by mutating the WHX ORF translation start codon.

[0533] In some embodiments, the WPRE comprises or consists of a nucleotide sequence that has at least 70% sequence to SEQ ID NO: 55 or a fragment thereof. Suitably, the WPRE comprises or consists of a nucleotide sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 55 or a fragment thereof.

[0534] In some embodiments, the WPRE comprises or consists of the nucleotide sequence SEQ ID NO: 55 or a fragment thereof.

[0535] AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCC TTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGG CTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCC GTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGG CATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGG CGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGAC AATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCAC CTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTC CTTCCCGC (SEQ ID NO : 55)

[0536] Polyadenylation sequence

[0537] The lentiviral vector of the present invention may comprise a polyadenylation sequence. Suitably, the protein-coding sequence (e.g. the construct) is operably linked to a polyadenylation sequence. A polyadenylation sequence may be inserted after the proteincoding sequence to improve transgene expression.

[0538] A polyadenylation sequence typically comprises a polyadenylation signal, a polyadenylation site and a downstream element: the polyadenylation signal comprises the sequence motif recognised by the RNA cleavage complex; the polyadenylation site is the site of cleavage at which a poly-A tails is added to the mRNA; the downstream element is a GT-rich region which usually lies just downstream of the polyadenylation site, which is important for efficient processing.

[0539] Suitable polyadenylation sequences will be well known to those of skill in the art (see, for example, Schambach et al. (2007) Molecular Therapy 15: 1167-1173; Choi et al. (2014) Molecular Brain 7: 1-10). Exemplary polyadenylation sequences include the bGH poly(A) signal sequence and SV40pA signal sequence. Kozak sequence

[0540] The lentiviral vector of the present invention may comprise a Kozak sequence. Suitably, the protein-coding sequence (e.g. the construct) is operably linked to a Kozak sequence. A Kozak sequence may be inserted before the start codon to improve the initiation of translation.

[0541] Suitable Kozak sequences will be well known to the skilled person (see, for example, Kozak (1987) Nucleic Acids Research 15: 8125-8148).

[0542] In some embodiments, the Kozak sequence comprises or consists of a nucleotide sequence that has at least 80% sequence identity to SEQ ID NO: 56 or a fragment thereof.

[0543] In some embodiments, the Kozak sequence comprises or consists of the nucleotide sequence SEQ ID NO: 56 or a fragment thereof.

[0544] GCCACC (SEQ ID NO : 56)

[0545] Other cis-acting elements

[0546] The lentiviral vector of the present invention may comprise any other suitable cis-acting elements, such as one or more of a rev response element (RRE); a retroviral psi packaging element; a primer binding site (PBS); a TAT activation region (TAR); splice donor and acceptor sites; and central and terminal polypurine tracts.

[0547] Long terminal repeats (LTRs)

[0548] The lentiviral vector of the present invention may comprise one or more long terminal repeat (LTR). LTRs are responsible for proviral integration and transcription. Typically, a naturally occurring LTR comprises U3, R, and U5 regions.

[0549] The lentiviral vector may comprise a 5’ LTR and / or a 3’ LTR. The lentiviral vector may comprise a 5’ LTR and a 3’ LTR. Suitably, a 5’ LTR comprises R and U5 regions, and optionally comprises a U3 region. Suitably, a 3’ LTR comprises U3, R and U5 regions.

[0550] Suitable LTR sequences will be well known to the skilled person (see, for example, Freeh et al. (1996) Virology 224: 256-267).

[0551] In some embodiments, a LTR comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 57 or a fragment thereof. Suitably, a LTR comprises or consists of a nucleotide sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 57 or a fragment thereof. In some embodiments, a LTR comprises or consists of the nucleotide sequence SEQ ID NO: 57 or a fragment thereof.

[0552] TGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTC TGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCC TCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTA ACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAG (SEQ ID NO : 57)

[0553] The lentiviral vector of the present invention may comprise one or more self-inactivating long terminal repeat (SIN-LTR). A “SIN-LTR” may comprise a deletion that abolishes transcription of the full-length virus after it has incorporated into a host cell. For example, a 3’ SIN-LTR may comprise a deletion in the U3 region removing the promoter / enhancer elements (see, for example, Zufferey et al. (1998) Journal of Virology 72: 9873-9880). This deletion is copied into the 5’ LTR after reverse transcription, thereby making the gene expression in target cells dependent on an internal promoter of choice.

[0554] Suitable SIN-LTR sequences will be well known to the skilled person (see, for example, Zufferey et al. (1998) Journal of Virology 72: 9873-9880; Miyoshi et al. (1998) Journal of Virology 72: 8150-8157).

[0555] In some embodiments, the 5’ LTR comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 58 or a fragment thereof. Suitably, the 5’ LTR comprises or consists of a nucleotide sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 58 or a fragment thereof.

[0556] In some embodiments, the 5’ LTR comprises or consists of the nucleotide sequence SEQ ID NO: 58 or a fragment thereof.

[0557] GGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACT GCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTG ACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAG (SEQ ID NO : 58)

[0558] In some embodiments, the 5’ LTR and / or the 3’ LTR comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 58 or a fragment thereof. Suitably, the 5’ LTR and / or the 3’ LTR comprises or consists of a nucleotide sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 58 or a fragment thereof.

[0559] In some embodiments, the 5’ LTR and / or the 3’ LTR comprises or consists of the nucleotide sequence SEQ ID NO: 58 or a fragment thereof. In some embodiments, the 5’ LTR and the 3’ LTR comprise or consist of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 58 or a fragment thereof. Suitably, the 5’ LTR and the 3’ LTR comprise or consist of a nucleotide sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 58 or a fragment thereof.

[0560] In some embodiments, the 5’ LTR and the 3’ LTR comprise or consist of the nucleotide sequence SEQ ID NO: 58 or a fragment thereof.

[0561] Primer binding site (PBS)

[0562] The lentiviral vector of the present invention may comprise a primer binding site (PBS). A PBS is a cis-acting element where a primer may bind to initiate reverse transcription of the RNA genome (see, for example, Lanchy et al. (1998) Journal of Biological Chemistry 273: 24425- 24432).

[0563] Suitable retroviral PBSs will be well known to the skilled person.

[0564] In some embodiments, a PBS comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 59 or a fragment thereof. Suitably, a PBS comprises or consists of a nucleotide sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 59 or a fragment thereof.

[0565] In some embodiments, a PBS comprises or consists of the nucleotide sequence SEQ ID NO: 59 or a fragment thereof.

[0566] TGGCGCCCGAACAGGGACTTGAAAGCGAAAGGGAAACCAGAGGAGCTCTCTCGACGCAGGAC TCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCGACTGGTGAGTACGCCAAAAA TTTTGACTAGCGGAGGCTAGAAGGAGAGAG (SEQ ID NO : 59)

[0567] Retroviral psi packaging element

[0568] The lentiviral vevtor of the present invention may comprise a retroviral psi packaging element. A retroviral psi packaging element is a cis-acting element which is involved in regulating the process of packaging the retroviral RNA genome into the viral capsid during replication (see, for example, McBride et al. (1997) Journal of Virology 71 : 4544-4554). A retroviral psi packaging element may form part of the 5’ region of the gag gene.

[0569] Suitable retroviral psi packaging elements will be well known to the skilled person.

[0570] In some embodiments, a retroviral psi packaging element comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 60 or a fragment thereof. Suitably, a retroviral psi packaging element comprises or consists of a nucleotide sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 60 or a fragment thereof.

[0571] In some embodiments, a retroviral psi packaging element comprises or consists of the nucleotide sequence SEQ ID NO: 60 or a fragment thereof.

[0572] ATGGGTGCGAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGG TTAAGGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCT AGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATACTGG GACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAATACAGTA GCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAGCTTTAGACAA GATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGCCGCTGAT ( SEQ ID NO : 60)

[0573] Rev response element (RRE)

[0574] The lentiviral vector of the present invention may comprise a rev response element (RRE). A RRE is a cis-acting element that enables the efficient export of RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of an infected target cell (see, for example, Pollard et al. (1998) Annual Review of Microbiology 52: 491-532).

[0575] Suitable RRE sequences will be well known to the skilled person.

[0576] In some embodiments, a RRE comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 61 or a fragment thereof. Suitably, a RRE comprises or consists of a nucleotide sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 61 or a fragment thereof.

[0577] In some embodiments, a RRE comprises or consists of the nucleotide sequence SEQ ID NO: 61 or a fragment thereof.

[0578] GGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGGGCGCAGCGTCAATGACGCTGAC GGTACAGGCCAGACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGG CGCAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCCTGGCTGTG GAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTT (SEQ ID NO : 61)

[0579] Central polypurine tract (cPPT)

[0580] The lentiviral vector of the present invention may comprise a central polypurine tract (cPPT). A cPPT may allow initiation of plus-strand synthesis (see, for example, Follenzi et al. (2000) Nature Genetics 25: 217-222). Suitable cPPT sequences will be well known to the skilled person.

[0581] In some embodiments, a cPPT comprises or consists of a nucleotide sequence that has at least 70% sequence identity to SEQ ID NO: 27 or a fragment thereof. Suitably, a cPPT comprises or consists of a nucleotide sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 27 or a fragment thereof.

[0582] In some embodiments, a cPPT comprises or consists of the nucleotide sequence SEQ ID NO: 27 or a fragment thereof.

[0583] AACTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACATAATAGCAA CAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTTTATC (SEQ ID NO : 27)

[0584] Cell

[0585] In a further aspect, the invention provides a cell comprising a lentiviral vector of the invention. Suitably, the cell is in the form of a population of cells.

[0586] In a further aspect, the invention provides a population of cells comprising a lentiviral vector of the invention.

[0587] In some embodiments, the cell or population of cells has been transduced by the lentiviral vector.

[0588] In some embodiments, the cell has the phenotype CD3+CD4+CD25+CD127’FoxP3+.

[0589] The cell may be a cell as described herein.

[0590] Method of producing engineered Tregs

[0591] In a further aspect, the invention provides a method of producing a population of CD19 CAR- Treg cells, comprising the steps of:

[0592] (a) providing a population of T cells;

[0593] (b) activating the population of T cells;

[0594] (c) transducing the population of T cells with a lentiviral vector according to the invention; and

[0595] (d) culturing the population of T cells in the presence of IL-2 and rapamycin and under conditions suitable for the expression of the transgene. In some embodiments, the population of T cells in step (a) comprises Treg cells.

[0596] In some embodiments, the population of T cells in step (a) comprises Tconv cells.

[0597] In some embodiments, the population of T cells in step (a) comprises a mixture of Tconv cells and Treg cells.

[0598] In some embodiments, the population of T cells in step (a) is CD25+, CD25' or is a mixture comprising CD25+and CD25' cells. Suitably, the population of T cells in step (a) is CD25+. Suitably, the population of T cells in step (a) is CD25-. Suitably, the population of T cells in step (a) is a mixture comprising CD4+CD25+and CD4+CD25_cells.

[0599] In some embodiments, the population of T cells in step (a) has the phenotype CD4+CD25+, CD4+CD25_or is a mixture comprising CD4+CD25+and CD4+CD25_cells. Suitably, the population of T cells in step (a) is CD4+CD25+. Suitably, the population of T cells in step (a) is CD4+CD25_. Suitably, the population of T cells in step (a) is a mixture comprising CD4+CD25+and CD4+CD25_cells.

[0600] In some embodiments, step (b) comprises stimulating the population of T cells with anti-CD3 and / or anti-CD28 antibodies.

[0601] In some embodiments, step (b) comprises stimulating the population of T cells with anti-CD3 and anti-CD28 antibodies.

[0602] In some embodiments, step (b) comprises stimulating the population of T cells with anti-CD3 and / or anti-CD28 antibody coated beads.

[0603] In some embodiments, the ratio of T cells to beads is about 1 :5, about 1 :3, about 1 :2, about 1 :1 , about 2:1 , about 3:1 , about 5:1. In some embodiments, the ratio of T cells to beads is about 1 :3. In some embodiments, the ratio of T cells to beads is about 1 :1.

[0604] Suitably, the transducing step (c) is performed after step (b). Thus, step (c) is carried out on the activated population of T cells produced in step (b).

[0605] In some embodiments, the method comprises a step of re-stimulating the population of T cells, such as the population of T cells produced in step (b) or step (c), with anti-CD3 and / or anti- CD28 antibodies.

[0606] For example, the T cells may be re-stimulated on day 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14 following step (b). In some embodiments, the T cells are re-stimulated on day 9 following step (b). Suitably, steps (c) and (d) may be performed in either order or simultaneously.

[0607] Suitably, step (b) may be performed in the presence of IL-2 and / or rapamycin. Suitably, step (b) is performed in the presence of rapamycin.

[0608] In one embodiment, the method comprises transducing the population of T cells with a lentiviral vector according to the invention followed by culturing the population of T cells in the presence of IL-2 and rapamycin and under conditions suitable for the expression of the transgene.

[0609] In one embodiment, the method comprises culturing the population of T cells in the presence of IL-2 and rapamycin and under conditions suitable for the expression of the transgene followed by transducing the population of T cells with a lentiviral vector according to the invention.

[0610] In one embodiment, the method comprises transducing the population of T cells with a lentiviral vector according to the invention in the presence of IL-2 and rapamycin and culturing the cells under conditions suitable for the expression of the transgene.

[0611] In some embodiments, step (d) comprises culturing the population of T cells in cell culture medium comprising rapamycin and adding IL-2 to the cell culture medium one or more time. Suitably, IL-2 is added to the cell culture medium once, twice, three times, four times or five times.

[0612] In some embodiments, step (d) comprises supplementing the cell culture medium with IL-2 on days 3, 6, 9 and 12 after activating the population of T cells in step (b).

[0613] Suitably, the cell culture medium in step (d) and / or step (b) comprises from about 20 pM to about 250 pM rapamycin. Suitably, the cell culture medium in step (d) and / or step (b) comprises from about 40 pM to about 200 pM, about 60 pM to about 150 pM, or about 80 pM to about 125 pM rapamycin. Suitably, the cell culture medium in step (d) and / or step (b) comprises about 20 pM, about 40 pM, about 60 pM, about 80 pM, about 100 pM, about 125 pM, about 150 pM, about 175 pM, about 200 pM, about 225 pM or about 250 pM rapamycin. Suitably, the cell culture medium in step (d) and / or step (b) comprises about 100 pM rapamycin.

[0614] Suitably, IL-2 is added to the cell culture medium in step (d) to a final concentration of from about 100 lU / ml to about 1000 lU / ml. Suitably, IL-2 is added to the cell culture medium in step (d) to a final concentration of from about 100 lU / ml to about 900 lU / ml, about 200 lU / ml to about 800 lU / ml, about 300 lU / ml to about 700 lU / ml, or about 400 lU / ml to about 600 lU / ml. Suitably, IL-2 is added to the cell culture medium in step (d) to a final concentration of about 100 lU / ml, about 200 lU / ml, about 300 lU / ml, about 400 lU / ml, about 500 lU / ml, about 600 lU / ml, about 700 lU / ml, about 800 lU / ml, about 900 lU / ml, about 1000 lU / ml. Suitably, IL-2 is added to the cell culture medium in step (d) to a final concentration of about 500 lU / ml.

[0615] In some embodiments, IL-2 is added to the cell culture medium in step (d) to a final concentration of about 100 lU / ml to about 500 lU / ml. Suitably, IL-2 is added to the cell culture medium in step (d) to a final concentration of from about 100 lU / ml to about 400 lU / ml, about 100 lU / ml to about 300 lU / ml, about 150 lU / ml to about 300 lU / ml, about 150 lU / ml to about 250 lU / ml. In some embodiments, IL-2 is added to the cell culture medium in step (d) to a final concentration of about 250 lU / ml.

[0616] Thus, in some embodiments, the method of producing a population of CD19 CAR-Treg cells, comprises the steps of:

[0617] (a) providing a population of T cells, optionally CD4+CD25+ cells;

[0618] (b) activating the population of T cells, optionally by stimulating the population of T cells with anti-CD3 and / or anti-CD28 antibodies, optionally wherein the anti-CD3 and / or anti- CD28 antibodies are coated on beads, optionally wherein the T cell to bead ratio is 1 :1 ;

[0619] (c) transducing the population of T cells with a lentiviral vector according to the invention; and

[0620] (d) culturing the population of T cells in the presence of IL-2 and rapamycin and under conditions suitable for the expression of the transgene, optionally wherein the IL-2 is added to the culture medium to a final concentration of 250 lU / ml; and

[0621] (e) optionally, re-stimulating the population of T cells with anti-CD3 and / or anti- CD28 antibodies.

[0622] In some embodiments, at least 70% (suitably, at least 75% or at least 80%) of the population of CD19 CAR-Treg cells produced in step (d) have the phenotype CD3+CD4+CD25+CD127_FoxP3+.

[0623] In some embodiments, the population of CD19 CAR-Treg cells produced in step (d) has the phenotype CD3+CD4+CD25+CD127'FoxP3+.

[0624] Suitably, the CD19 CAR+ cells produced in step (d) comprise at least 70% (suitably, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%) CD25+cells.

[0625] Engineered Regulatory T cells

[0626] In a further aspect, the invention provides a population of CD19 CAR-Treg cells obtained or obtainable by the method according to the invention.

[0627] In a further aspect, the invention provides a population of CD19 CAR-Treg cells obtained by the method according to the invention.

[0628] As used herein, the term "T cell" refers to lymphocytes (white blood cells) that function in cell- mediated immunity. The presence of a T cell receptor (TCR) on the cell surface distinguishes them from other lymphocytes. T cells do not present antigens and rely on other lymphocytes (natural killer cells, B cells, macrophages, dendritic cells) to aid in antigen presentation. Types of T cells include: T helper cells (TH cells), Memory T cells (Tern, Tern, or Temra), Regulatory T cells (Treg), Cytotoxic T cells (CTLs), Natural killer T cells (NKT cells), gamma delta T cells, and Mucosal associated invariant T cells (MAIT).

[0629] Treg cells play an important role for the maintenance of immunological tolerance by suppressing the action of autoreactive effector cells and have been shown to be critically involved in preventing the development of autoimmune reactions.

[0630] Accordingly, as described herein, a "regulatory T cell" or "Treg" refers to those T cells that have immunoregulatory properties and the ability to suppress the proliferation and / or effector function of other T cell populations. A number of cell surface molecules are used to characterize and define Treg cells as described below herein.

[0631] In some embodiments, a Treg cell can be a T cell expressing one or more markers selected from the group consisting of CTLA4; PDL1 ; LAP; GARP; CD25; and CD27. In some embodiments, a Treg cell can be a CD8-CD4+CD25+CD127+ cell. In some embodiments, a Treg cell can be a CD8-CD4dimCD25hiCD127low cell. In some embodiments, a Treg cell can be a CD8-CD4lowCD25hiCD127low cell.

[0632] One skilled in the art will be capable of assessing the molecules present on a cell using standard techniques, for example using immunofluorescence to detect commercially available antibodies bound to the marker molecules. The designators "hi" "high" "Io" "low" or "dim" are to indicate a cell with low or high expression of the marker as compared to a larger population of cells. Such designators are often used when sorting or identifying cells by FACS, in which gates can be established to divide cells based on the level of expression of the marker. In some embodiments, the cell population is a population of mammalian cells. In some embodiments, the cell population is a human cell population. In some embodiments, the cell population is autologous to a subject. In some embodiments, the cell is allogeneic to a subject. In some embodiments of any of the aspects, the cell is engineered to reduce expression of an endogenous T cell receptor and / or an endogenous MHC complex.

[0633] T cell, T cell precursor, stem cell, and / or iPSC can be obtained from the subject and engineered as described herein to provide an T cell which is autologous to the subject. The cell can be obtained from the subject immediately before engineering the cell, obtained from a culture of the subject's cells, and / or provided from a sample collected at an earlier date (e.g. a frozen sample).

[0634] In some embodiments, the T cell can be allogenic to the subject. To provide allogeneic cells, the T cell can be engineered, e.g., engineered to not express an endogenous T cell receptor and / or endogenous MHC complex. Such modifications are known in the art and can be engineered by, e.g., directed mutagenesis, directed deletion / insertions (e.g., via homologous recombination), CRISPR technology or the like. An endogenous T cell receptor and / or endogenous MHC complex gene can be engineered to, e.g., disable the promoter, provide a premature stop codon, or to delete the coding sequence of the gene. In some embodiments of any of the aspects, the cell can be engineered to comprise an inhibitory nucleic acid that inhibits the expression of the endogenous T cell receptor and / or one or more genes of the endogenous MHC complex. The engineering of the T cell can be performed in a mature T cell or performed in a T cell precursor cell (e.g., a stem cell or partially differentiated cell) and a T cell then differentiated from the precursor cell.

[0635] Methods of treatment

[0636] In a further aspect, the invention provides a pharmaceutical composition comprising a lentiviral vector, a cell, a population of cells or a population of CD19 CAR-Treg cells of the invention. Suitably, the pharmaceutical composition further comprises a pharmaceutically acceptable diluent, carrier or excipient.

[0637] In a further aspect, the invention provides a population of CD19 CAR-Treg cells according to the invention or a pharmaceutical composition according to the invention for use in the treatment and / or prevention of an autoimmune disease.

[0638] In a further aspect, the invention provides the use of a population of CD19 CAR-Treg cells according to the invention or of a pharmaceutical composition according to the invention for the manufacture of a medicament for the treatment and / or prevention of an autoimmune disease. In a further aspect, the invention provides a method of treating and / or preventing an autoimmune disease in a subject, the method comprising administering a population of CD19 CAR-Treg cells according to the invention or a pharmaceutical composition according to the invention to the subject.

[0639] As used herein, "autoimmune disease" refers to a class of diseases in which a subject's own antibodies react with host tissue or in which immune effector T cells are autoreactive to endogenous self-peptides and cause destruction of tissue. Thus an immune response is mounted against a subject's own antigens, referred to as self-antigens. A "self-antigen" as used herein refers to an antigen of a normal host tissue. Normal host tissue does not include cancer cells. An autoimmune condition, disease, or disorder is caused by the inability of one's immune system to distinguish between a foreign cell and a healthy cell. This results in one's immune system targeting one's healthy cells for programmed cell death. Non-limiting examples of an autoimmune disease include inflammatory arthritis (such as rheumatoid arthritis), type 1 diabetes mellitus, multiples sclerosis (MS), psoriasis, inflammatory bowel diseases, systemic lupus erythematosus (SLE), vasculitis, allergic inflammation (such as allergic asthma, atopic dermatitis, and contact hypersensitivity), Graves' disease, Hashimoto's thyroiditis, celiac disease, Crohn's disease, ulcerative colitis, Guillain-Barre syndrome, primary biliary sclerosis / cirrhosis, sclerosing cholangitis, autoimmune hepatitis, Raynaud's phenomenon, scleroderma, Sjogren's syndrome, Goodpasture's syndrome, Wegener's granulomatosis, polymyalgia rheumatica, temporal arteritis / giant cell arteritis, chronic fatigue syndrome (CFS), autoimmune Addison's Disease, ankylosing spondylitis, acute disseminated encephalomyelitis, antiphospholipid antibody syndrome, aplastic anemia, idiopathic thrombocytopenic purpura, Myasthenia gravis, opsoclonus myoclonus syndrome, optic neuritis, Ord's thyroiditis, pemphigus, pernicious anaemia, Reiter's syndrome, Takayasu's arteritis, warm autoimmune hemolytic anemia, Wegener's granulomatosis, fibromyalgia (FM), vitiligo, IPEX, graft versus host disease, undifferentiated connective tissue disease, mixed connective tissue disease, autoinflammatory syndromes (such as Familial mediterranean fever), still's disease, and adult-onset still's disease.

[0640] In some embodiments, the autoimmune disease is systemic lupus erythematosus.

[0641] Administration

[0642] The cells described herein can be administered to a subject having or diagnosed as having an autoimmune disease and / or at risk of having an autoimmune disease. In some embodiments, an effective amount of Treg cells as described herein is administered to a subject in order to alleviate a symptom of an autoimmune disease. As used herein, "alleviating a symptom" of a condition is ameliorating any condition or symptom associated with the condition. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the cells described herein to subjects are known to those of skill in the art.

[0643] In some embodiments, the CAR, cell, or pharmaceutical composition is administered to a subject locally.

[0644] Local administration may include administration to the tissue of interest.

[0645] In some embodiments, the CAR, cell, or pharmaceutical composition is administered to a subject systemically.

[0646] The term “systemic delivery” or “systemic administration” as used herein means that the agent of the invention is administered into the circulatory system, for example to achieve broad distribution of the agent. In contrast, topical or local administration restricts the delivery of the agent to a localised area.

[0647] Dosage

[0648] The skilled person can readily determine an appropriate dose of an agent of the invention to administer to a subject. Typically, a physician will determine the actual dosage that will be most suitable for an individual patient, which will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of the invention.

[0649] Subject

[0650] The term “subject” as used herein refers to either a human or non-human animal.

[0651] Examples of non-human animals include vertebrates, for example mammals, such as non- human primates (particularly higher primates), dogs, rodents (e.g. mice, rats or guinea pigs), pigs and cats. The non-human animal may be a companion animal.

[0652] Preferably, the subject is a human.

[0653] Proteins The term “protein” as used herein includes single-chain polypeptide molecules as well as multiple-polypeptide complexes where individual constituent polypeptides are linked by covalent or non-covalent means. The terms “polypeptide” and “peptide” as used herein refer to a polymer in which the monomers are amino acids and are joined together through peptide or disulfide bonds.

[0654] The proteins of the invention include any of the proteins disclosed herein with a methionine at the N-terminus.

[0655] Polynucleotides

[0656] Polynucleotides of the invention may, for example, comprise DNA or RNA. They may be single-stranded or double-stranded. It will be understood by a skilled person that numerous different polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that the skilled person may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the invention to reflect the codon usage of any particular host organism in which the polypeptides of the invention are to be expressed.

[0657] The polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or lifespan of the polynucleotides of the invention.

[0658] Polynucleotides such as DNA polynucleotides may be produced recombinantly, synthetically or by any means available to the skilled person. They may also be cloned by standard techniques.

[0659] Longer polynucleotides will generally be produced using recombinant means, for example using polymerase chain reaction (PCR) cloning techniques. This may involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking the target sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture with an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable vector.

[0660] Variants, derivatives, analogues and fragments

[0661] In addition to the specific polypeptides and polynucleotides mentioned herein, the invention also encompasses variants, derivatives and fragments thereof. In the context of the invention, a “variant” of any given sequence is a sequence in which the specific sequence of residues (whether amino acid or nucleic acid residues) has been modified in such a manner that the polypeptide or polynucleotide in question retains at least one or all of its endogenous functions. A variant sequence can be obtained by addition, deletion, substitution, modification, replacement and / or variation of at least one residue present in the naturally occurring polypeptide or polynucleotide.

[0662] The term “derivative” as used herein in relation to proteins or polypeptides of the invention includes any substitution of, variation of, modification of, replacement of, deletion of and / or addition of one (or more) amino acid residues from or to the sequence, providing that the resultant protein or polypeptide retains at least one or all of its endogenous functions.

[0663] Typically, amino acid substitutions may be made, for example from 1 , 2 or 3, to 10 or 20 substitutions, provided that the modified sequence retains the required activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogues.

[0664] Polypeptides used in the invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent polypeptide. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or the amphipathic nature of the residues as long as the endogenous function is retained. 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 having similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine.

[0665] Conservative substitutions may be made, for example according to the table below. Amino acids in the same block in the second column and in the same line in the third column may be substituted for each other:

[0666] The effect of additions, deletions, substitutions, modifications, replacements and / or variations may be predicted using any suitable prediction tool, for example SIFT (Vaser et al. (2016) Nature Protocols 11 : 1-9), PolyPhen-2 (Adzhubei et al. (2013) Current Protocols in Human Genetics 76: 7-20), CADD (Rentzsch et al. (2021) Genome Medicine 13: 1-12), REVEL (loannidis et al. (2016) The American Journal of Human Genetics 99: 877-885), MetaLR (Dong et al. (2015) Human Molecular Genetics 24: 2125-2137) and / or MutationAssessor (Reva et al. (2011) Nucleic Acids Research 39: e118-e118) or based on clinical data, for example ClinVar (Landrum et al. (2016) Nucleic Acids Research 44: D862-D868). Suitable additions, deletions, substitutions, modifications, replacements and / or variations may be considered tolerated, benign and / or likely benign.

[0667] Typically, a variant may have a certain sequence identity with the wild type amino acid sequence or the wild type nucleotide sequence.

[0668] In the present context, a variant sequence is taken to include an amino acid sequence which may be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical, suitably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the subject sequence. Although a variant can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties / functions), in the context of the present invention it is preferred to express in terms of sequence identity.

[0669] In the present context, a variant sequence is taken to include a nucleotide sequence which may be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical, suitably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the subject sequence. Although a variant can also be considered in terms of similarity, in the context of the present invention it is preferred to express it in terms of sequence identity.

[0670] Suitably, reference to a sequence which has a percent identity to any one of the SEQ ID NOs described herein refers to a sequence that has the stated percent identity over the entire length of the SEQ ID NO referred to.

[0671] Sequence identity 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 percent identity between two or more sequences.

[0672] Percent identity may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues. Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion in the amino acid or nucleotide sequence may cause the following residues or codons to be put out of alignment, thus potentially resulting in a large reduction in percent identity when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall identity score. This is achieved by inserting “gaps” in the sequence alignment to try to maximise local identity.

[0673] However, these more complex methods assign “gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids or nucleotides, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. “Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0674] Calculation of maximum percent identity therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (Devereux et al. (1984) Nucleic Acids Research 12: 387-395). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (Altschul, et al. (1990) Journal of Molecular Biology 215: 403-410), BLAST 2 (Tatusova et al. (1999) FEMS Microbiology Letters 174: 247-250), FASTA (Pearson et al. (1988) PNAS 85: 2444-2448), EMBOSS Needle (Madeira et al. (2019) Nucleic Acids Research 47: W636-W641) and the GENEWORKS suite of comparison tools. For some applications, it is preferred to use EMBOSS Needle.

[0675] Although the final percent identity can be measured, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix.

[0676] Once the software has produced an optimal alignment, it is possible to calculate percent sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. The percent sequence identity may be calculated as the number of identical residues as a percentage of the total residues in the SEQ ID NO referred to.

[0677] “Fragments” are also variants and the term typically refers to a selected region of the polypeptide or polynucleotide that is of interest either functionally or, for example, in an assay. “Fragment” thus refers to an amino acid or nucleic acid sequence that is a portion of a full- length polypeptide or polynucleotide.

[0678] Such variants, derivatives and fragments may be prepared using standard recombinant DNA techniques, such as site-directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5’ and 3’ flanking regions corresponding to the naturally-occurring sequence either side of the insertion site may be made. The flanking regions will contain convenient restriction sites corresponding to sites in the naturally- occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed in accordance with the invention to make the encoded polypeptide. These methods are only illustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used.

[0679] The polynucleotides used in the invention may be codon-optimised. Codon optimisation has previously been described in WO 1999 / 41397 and WO 2001 / 79518. Different cells differ in their usage of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type. By altering the codons in the sequence so that they are tailored to match with the relative abundance of corresponding tRNAs, it is possible to increase expression. It is also possible to decrease expression by deliberately choosing codons for which the corresponding tRNAs are known to be rare in the particular cell type. Thus, an additional degree of translational control is available.

[0680] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley & Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, J.M. and McGee, J.O’D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, M.J. (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, D.M. and Dahlberg, J.E. (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is herein incorporated by reference.

[0681] This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any 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, respectively.

[0682] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

[0683] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0684] The terms "comprising", "comprises" and "comprised of' as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms "comprising", "comprises" and "comprised of' also include the term "consisting of'.

[0685] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0686] The skilled person will understand that they can combine all features of the invention disclosed herein without departing from the scope of the invention as disclosed.

[0687] The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention. EXAMPLES

[0688] Materials & Methods

[0689] Cell culture

[0690] Cell lines

[0691] Acute lymphocytic leukemia cell line (ALL-CM) was grown in X-VIVO 15 (Lonza) supplemented with 10% FBS (fetal bovine serum, Euroclone), 1% penicillin / streptomycin (Lonza) and 1% glutamine (Lonza). HEK293T and CD40L+3T3 cells were grown in IMDM (Lonza) supplemented with 10% FBS (fetal bovine serum, Euroclone), 1% penicillin / streptomycin (Lonza) and 1% glutamine (Lonza). Cells were counted every 2-4 days by Trypan blue dye exclusion according to the cell line and plated at a concentration of 0.5-1 x 106cells / ml.

[0692] Primary T cell isolation and expansion

[0693] Peripheral blood mononuclear cells (PBMCs) were collected from healthy donors, after written informed consent, according to the San Raffaele Scientific Institutional Ethical Committee guidelines. PBMCs were isolated by Ficoll-Hypaque gradient separation (Lymphoprep; Fresenius) and were freshly used for the following protocols of specific T cell expansion. After the activation, cells were incubated at 37°C, 5% CO2, in a humidified cell culture incubator.

[0694] Primary regulatory T cell isolation

[0695] CD4+CD25+regulatory T cells were isolated by magnetic cell separation (Miltenyi), according to manufacturer’s instructions. Sorted CD4+CD25+were activated with cell-sized anti- CD3 / anti-CD28 magnetic beads (Dynabeads) in a 3:1 bead : T cell ratio and cultured in X- VIVO 15 (Lonza) supplemented with 10% human serum (Lonza), 1 % penicillin / streptomycin (Lonza) and glutamine (Lonza) with Ramapacyn (100nM). At day 2 after activation, IL-2 (500 Ul, Proleukin, Novartis) was added to cell culture and replenished every 2-3 days. After 14 days of cultures, beads were magnetically removed according to the manufacturer’s instructions. Starting from day 14, cells were employed for phenotypical evaluation and functional assays.

[0696] Naive Tregs were initially isolated from PBMCs by magnetic cell separation of CD4+CD25+cells. A subsequent selection of CD127'CD45RA+elements was done employing a Miltenyi Tyto cell sorter. Naive Tregs were subsequently activated with cell-sized anti-CD3 / anti-CD28 magnetic beads (Dynabeads) in a 1 :1 bead : T cell ratio and cultured in X- VIVO 15 (Lonza) supplemented with 10% human serum (Lonza), 1% penicillin / streptomycin (Lonza) and glutamine (Lonza) without Ramapacyn. At day 2 after activation, IL-2 (1000 Ul, Proleukin, Novartis) was added to cell culture and replenished every 2-3 days. After 9 days, cells were restimulated with anti-CD3 / anti-CD28 magnetic beads in a 1 :1 bead : T cell ratio. At day +14 after stimulation, beads were magnetically removed according to the manufacturer’s instructions. Starting from day 14, cells were employed for phenotypical evaluation and functional assays.

[0697] Primary conventional T cell isolation

[0698] Conventional T Lymphocytes were enriched from isolated PBMCs and stimulated with anti- CD3 / CD28 magnetic beads in a 3:1 bead : T cell ratio for 6 days. After 6 days of stimulation, beads were magnetically removed, and cells were maintained in X- VIVO 15 (Lonza) supplemented with 10% human serum (Lonza), 1% penicillin / streptomycin (Lonza) and glutamine (Lonza). T cells were cultured in the presence of IL-7 and IL-15 (5 ng / ml; PeproTech) to preserve early differentiated stem cell memory and central memory phenotype. Medium was replaced every 3-4 days and cells were counted by Trypan blue dye exclusion. Starting from day 14, cells were employed for phenotypical evaluation and functional assays.

[0699] CD4+CD25-T cells

[0700] CD4+CD25' T cells were collected from freshly isolated PBMCs after magnetic cell separation (Miltenyi) according to manufacturer’s instructions and were stimulated with cell-sized anti- CD3 / anti-CD28 magnetic beads in a 3:1 bead : T cell ratio. Cells were cultured with either IL- 7 / IL-15 (5ng / ml, Peprotech) or IL-2 (500 Ul, Proleukin, Novartis) and rapamycin (100nM) in X- VIVO 15 supplemented with 10% human serum (Lonza), 1 % penicillin / streptomycin (Lonza) and glutamine (Lonza). Beads were magnetically removed at day 14. Starting from day 14, cells were employed for phenotypical evaluation and functional assays. Medium was replaced every 3-4 days and cells were counted by Trypan blue dye exclusion.

[0701] Viral vectors

[0702] CAR constructs

[0703] CAR19.28z construct was generated by cloning the antigen-specific single chain fragment variable (scFv) into bi-directional lentiviral vector encoding for CAR backbone containing a NGFR-derived mutated-short spacer (NMS) (Casucci, M. et al., Front. Immunol. 9, 507 (2018)), a CD28 transmembrane and co-stimulatory domain and a CD3zeta endodomain under the control of a hPGK promoter. Green Fluorescent Protein (GFP) was cloned in antisense under the control of a mCMV promoter. Fox19CAR LV construct was generated by cloning in a unidirectional lentiviral vector both the human FoxP3 gene and an anti-CD19 second-generation CAR under the control of a hPGK promoter. Anti-CD19 CAR was composed by a CD19-specific scFv, a linker peptide derived from the mutated constant portion of lgG4, the transmembrane and intracellular portion of the CD28 and the CD3z chain. The two transgenes were linked by a thosea-asigna virus 2A (T2A) peptide. As control, we generated a unidirectional lentiviral vector encoding for the FoxP3 gene under the control of a hPGK promoter.

[0704] LV vector production

[0705] Third generation replication-defective and self-inactivating lentiviral vectors (LV) were produced by transient transfection of HEK-293T cells. Briefly, a solution containing the packaging plasmids pMDLg / pRRE (containing HIV-1 gag / pol genes), the plLVV01-Rev plasmid (encoding Rev protein), the envelope plasmid encoding the vesicular stomatitis virus glycoprotein (VSV-G), the pAdvantage (that enhances transient protein expression) and the plasmids carrying the transgene of interest was transfected in sub-confluent 293T cells using the calcium chloride precipitation method. Supernatants containing lentiviral particles were collected 48 hours later and filtered using a 0.22 pm filter, ultracentrifuged at 8000 g for 18 h at 4°C (Beckman Optima XL-100K Ultracentrifuge), aliquoted and cryopreserved.

[0706] T-cell transduction with LV vectors

[0707] At day +2 after stimulation, activated T cells (Treg, Tconv or CD4+CD25_lymphocytes) from healthy donors were collected and re-suspended at the concentration of 2,5x10A6 cells / ml in X-VIVO 15. LV vectors were added accordingly to viral titration to achieve an Multiplicity of Infection (MOI) of 10. T cells were kept 24 hours at 37°C and then fresh medium was added in each condition. Transduction efficiency was measured by flow cytometry at day +10 / +14. Recombinant CD19 reagent (Miltenyi) was used for CAR surface staining according to manufacturer’s instructions.

[0708] Functional in vitro assays

[0709] Polyclonal suppression assay

[0710] Autologous T lymphocytes (target cells) and engineered T cells (effector cells) were cocultured at decreasing Effector-to-Target ratio in the presence of anti-CD3 / anti-CD28 magnetic beads at 1 :10 bead : T cell ratio. To detect proliferating cells, target cells were stained with VioBlue proliferation dye (ThermoFisher), while engineered cells were stained with FarRed proliferation dye (ThermoFisher), according to manufacturer’s instruction. After 6 days, cells were analyzed by flow cytometry. Target and effector cells were discriminated according to the specific proliferation dye. Suppression index was calculated as follows: (1- (percentage of proliferating cells in the effector group) / (percentage of proliferating target cells alone)) x 100.

[0711] Antigen-specific suppression assay

[0712] CD19+B cells (target cells) were negatively sorted from cryopreserved autologous PBMCs using B cell isolation kit II (Miltenyi), according to manufacturer’s instruction. To detect proliferating cells, isolated B lymphocytes were labeled with VioBlue proliferation dye (ThermoFisher), according to manufacturer’s instruction, whereas engineered T cells (effector cells) were stained with FarRed proliferation dye (ThermoFisher), according to manufacturer’s instruction. Labeled target cells and effector cells were co-cultured either in a 1 :1 Effector-to- Target ratio in the presence of irradiated CD40L+3T3 cells (a fibroblast-derived murine cell line) or at different Effector-to-Target ratios and stimulated with anti-CD40 (0.5 ug / ml), anti- IgM / IgG (20 ug / ml) stimulating antibodies. Cells were analyzed by flow cytometry 3 days later. Suppression Index was calculated as follows: (1 -(percentage of proliferating cells in the effector group) / (percentage of proliferating target cells alone)) x 100.

[0713] Antigen-specific killing assay

[0714] For antigen-specific killing assay with tumor cell lines, CD19+ALL-CM cells were co-cultured for 3 days with either engineered or untransduced T cells at 1 :5 Effector-to-Target ratio. Cells were analyzed by flow cytometry. The number of residual ALL-CM cells and effector lymphocytes were counted according to the number of elements acquired with flow cytometry and to the total well volume. The anti-tumor activity was expressed as Elimination index as follows: 1 - (number of viable target cells in presence of redirected T cells / number of viable target cells in presence of untransduced T cells).

[0715] In vivo humanized models of SLE

[0716] Generation of the models

[0717] The experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC). At day 0, 1 -day-old NSG mouse (NSG, Charles-River Italia) pups were sub-lethally irradiated and infused intra-liver with 0.8-1x105cord-blood derived human CD34+hematopoietic stem cells. Mice were clinically monitored and weighed every week. Starting from week 5 after cord-blood infusion, the frequency of human cells in the peripheral blood was monitored every week. After the establishment of a full human immune system, chronic inflammation was induced in mice by intra-peritoneal injection of pristane (300pL, Sigma- Aldrich). A control group of humanized mice did not receive pristane to monitor the dynamics of the immune cells without a chronic inflammatory stimulus. Three weeks after pristane injection, mice were treated with 3.5 x106of anti-CD19 CAR T regs or UT T regs or PBS. Human chimerism and human cord-blood derived lymphoid compartment on peripheral blood were assessed weekly by flow cytometry. Mouse weight was monitored every week and, in the presence of weight loss greater than 20%, animals were euthanized. Spleen, bone marrow and kidneys were harvested for flow cytometry and pathology evaluations.

[0718] At day 0, 8 weeks-old SGM-3 mice (SGM-3, Charles-River Italia) were sub-lethally irradiated and intra-venously infused with 0.8-1x105cord-blood derived human CD34+hematopoietic stem cells. Mice were clinically monitored and weighed every week. Starting from week 5 after cord-blood infusion, the frequency of human cells in the peripheral blood was monitored every week. After the establishment of a full human immune system, chronic inflammation was induced in mice by intra-peritoneal injection of pristane (500pL, Sigma-Aldrich). Mice were subsequently randomized and divided either in the early or late treatment group. Early treatment group received three weeks after pristane injection 3.5 x106of anti-CD19 CAR- Tregs or anti-CD19 CAR-Tconvs or PBS. After 5 weeks (8 weeks after pristane), CAR-Treg treated mice received a second infusion of 3.5 x106of anti-CD19 CAR-Tregs. Late treatment group received 3.5 x106of anti-CD19 CAR-Tregs or anti-CD19 CAR-Tconvs 8 weeks after pristane injection.

[0719] Human chimerism and human cord-blood derived lymphoid compartment on peripheral blood were assessed weekly by flow cytometry. Mouse weight was monitored every week and, in the presence of weight loss greater than 20%, animals were euthanized. Spleen, liver, bone marrow ear pinnae and kidneys were harvested for flow cytometry and pathology evaluations.

[0720] Single-cell suspension from kidney was obtained by using multi-tissue dissociation kit II (Miltenyi) according to manufacturer’s instructions with GentleMACS apparatus. Single-cell suspension from spleen was obtained using 70 nm cell strainer. Cells from bone marrow were collected by flushing femurs with PBS. After red-blood cell lysis, cells were suspended in PBS supplemented with 5% FBS. Tregs phenotype and lymphoid immune cells were analyzed by flow cytometry.

[0721] Anti-dsDNA quantification

[0722] The presence of human anti-double-strand DNA (dsDNA) IgG auto-antibodies in mouse serum was assessed at euthanasia by immunofluorescence assay according to manufacturer’s instruction (QUANTA Lite dsDNA, Werfen Group). A dilution of 1 :10 was employed as a threshold for the positivity.

[0723] Pathological evaluation At euthanasia, mice were euthanized by cervical dislocation. Collected organs fixed in buffered 4% formalin and embedded in paraffin. Haematoxylin and eosin stained 3-pm paraffin sections were examined for histopathological analysis. The evaluation of the specimens was performed in double-blind by a pathologist specifically trained in mouse pathology. We assessed: the normal anatomy, the presence of lesions or fibrosis, the presence and the characteristics of immune cells. For each organ, we evaluated the presence of:

[0724] • Spleen: the extension of the red and the white pulp and their relative proportion, the presence and the composition of inflammatory cells and, eventually, of vasculitis and granulomas

[0725] • Liver: the anatomy of the liver and hepatocyte characteristics, the presence of fibrosis, the presence and the composition of the immune infiltrate

[0726] • Kidneys: the glomeruli amount and features, the presence of inflammatory cells and of tubular damages / degeneration

[0727] • Bone marrow: the presence and the composition of hematopoietic cells and their maturation, the presence of signs of hemophagocytic lympho-histiocytosis

[0728] • Ear pinnae: the presence and the composition of skin lesions, the organization and the thickness of the different layers and the presence of Graft-versus-Host Disease

[0729] Microscopic lesions were classified on a scale of 0 to 5 as minimal (1), mild (2), moderate (3), marked (4), or severe (5); minimal referred to the least extent discernible and severe the greatest extent possible.

[0730] Immunohistochemistry

[0731] Immunohistochemical analysis was performed on selected sections of spleen using rabbit anti-human CD3 (2GV6; Ventana) and mouse anti-human CD20 (L26; Ventana) in automated Ventana Discovery ULTRA system.

[0732] Multiparametric flow cytometry

[0733] For Treg phenotype, cells were labeled with titrated fluorescent monoclonal antibodies specific for CD3 (Biolegend), CD4 (Miltenyi), FoxP3 (eBioscience), CD45RA (BD Bioscience), CD62L (BD Bioscience), TIGIT (Biolegend), LAP (eBioscience), Helios (Biolegend), CD25 (Miltenyi), CD27 (BD Bioscience), CD137 (Biolegend), CD127 (Miltenyi), GARP (BD Bioscience), CTLA- 4 (Biolegend), ICOS (BD Bioscience), GITR (Biolegend). For 19CAR engineered cells, transduction efficiency was assessed with GFP or NGFR spacer (CD271 , BD Bioscience). Fox19CAR engineered cells were identified using biotinylated recombinant human CD19 reagent (Miltenyi). Biotin was detected using VioBright515-conjugated anti-biotin secondary antibody (REA, Miltenyi). For intra-nuclear staining, lymphocytes were stained with surface antibodies, washed, fixed and permeabilized with FoxP3 staining buffer set (Miltenyi), according to manufacturer’s instructions.

[0734] For in vivo experiments, whole blood was lysed with ACK (Ammonium-Chloride-Potassium) buffer for 10 minutes at room temperature to remove red blood cells. The reaction was then stopped with PBS supplemented with 5% FBS. Subsequently, samples were stained with recombinant CD19 reagent. After washing with PBS supplemented with 5% FBS, fluorochrome-conjugated monoclonal antibodies specific for mouse CD45 (Biolegend), human CD45 (Biolegend), CD3 (Biolegend), CD14 (Beckman Coulter), CD19 (Biolegend), CD56 (BeckmanCoulter) were added to samples. Human T cells were counted on peripheral blood using Flow Count fluorescent beads (Beckman Coulter), according to manufacturer’s instruction.

[0735] For Treg analysis in harvested organs, single-cell suspensions were labeled with fluorochrome-conjugated monoclonal antibodies specific for CD3 (Biolegend), CD4 (Miltenyi), FoxP3 (eBioscience), CD45RA (BD Bioscience), CD62L (BD Bioscience), TIGIT (Biolegend), LAP (eBioscience), Helios (Biolegend), CD25 (Miltenyi), CD27 (BD Bioscience), CD137 (Biolegend), CD127 (Miltenyi), GARP (BD Bioscience), CTLA-4 (Biolegend), ICOS (BD Bioscience), GITR (Biolegend). Recombinant CD19 reagent (Miltenyi) was used to detect CAR+T cell, as previously described. Prior to the staining, mouse FC blocking reagent was employed according to the manufacturer’s instructions (Miltenyi) to avoid specific binding and to reduce noise.

[0736] For immune cells infiltrating the harvested organs at sacrifice, single-cell suspensions were labeled with fluorochrome-conjugated monoclonal antibodies specific for CD3 (BD Bioscience), CD4 (Biolegend), CD19 (BD Bioscience), CD20 (Biolegend), CD138 (BeckmanCoulter), CD27 (Biolegend), CD14 (Biolegend), HLA-DR (BD Bioscience), PD-1 (Biolegend), CD16 (Beckman Coulter), CD206 (Biolegend).

[0737] For each experiment, dead cells were excluded by DAPI positive staining. Data were acquired using a BD FACS Canto II or a Symphony A5 (BD Biosciences) and analyzed with FlowJo version 10 software (TreeStar).

[0738] High dimensional flow cytometry analysis

[0739] Flow cytometry data were analyzed using cytoChain algorithm for unbiased high-dimensional analysis (Manfredi, F. et al. Flow cytometry data mining by cytoChain identifies determinants of exhaustion and sternness in TCR-engineered T cells. Eur. J. Immunol. 51 , 1992-2005 (2021)). Briefly, acquisition stability was evaluated using FlowJQC algorithm and all the channels’ fluorescence intensities transformed by the arcSin function. Successively, the optimized flowSet was analyzed by FlowSOM-based clustering algorithm: the resulting 50 clusters were collapsed into 20 meta-clusters by Consensus Cluster Plus. Marker expressions in each cluster were then organized in a heat map. The frequency of cluster composition in each group was then plotted and analyzed for statistically significant differences using Prism 9 (GraphPad Software)

[0740] Cytokine evaluation

[0741] Engineered cells were stimulated in an antigen-specific manner, according to the specific experiment. Culture supernatants were collected after 72 h of stimulation and analyzed with a bead-based assay (Biolegend Legendplex 13-plex kit) according to the manufacturer’s instruction by flow cytometry.

[0742] Mouse blood samples were collected at the baseline and 3 or 7 days after the cell injection according to the specific experiment. The samples were centrifugated and stored at -20°C. At the moment of the cytokine analysis, mouse sera were thawed and analyzed with a beadbased assay (Biolegend Legendplex 13-plex kit) according to the manufacturer’s instructions by flow cytometry.

[0743] Statistical analysis

[0744] Statistical analyses were performed with Prism 9 (GraphPad Software). Student t- test was used when comparing two independent groups. Two-way ANOVA was used when comparing three or more independent groups. For non -parametric variables, Mann-Whitney or Kruskall- Wallis tests were employed. P-value adjusted tests were employed to identify significant differences between groups. For categorical variables, chi-square test was used. For all comparisons, two-sided p-values were used, and p-value < 0.05 was considered statistically significant.

[0745] The detailed statistical analysis is reported in the description of each figure.

[0746] Example 1 - Engineered CD19 CAR-Tregs maintain immunomodulatory properties and acquire antigen specificity

[0747] Considering the relevant role of B cells, autoantibodies and immune complexes in lupus pathogenesis, we resolved to generate anti-CD19 CAR-Tregs to control self-reactive B lymphocytes. We developed a bi-directional lentiviral (LV) vector encoding for a second- generation anti-CD19 CAR constituted by a spacer region derived from the extracellular domain of the human low-affinity nerve growth factor receptor (LNGFR), the CD3 chain, and the intracellular portion of CD28 (CAR19.28z LV, Fig. 6a), the most active co-stimulatory domain for the CAR-Treg function. To generate anti-CD19 CAR-Tregs (19CAR-Tregs), we sorted CD4+CD25+cells from healthy donors’ (HD) peripheral blood mononuclear cells (PBMCs) and we expanded them in the presence of IL-2 and rapamycin (Battaglia, M. et al., J. Immunol. 177, 8338-8347 (2006)). We optimized the protocol for Treg expansion to include lentiviral transduction for CAR-Treg generation (Fig. 6b). Mean transduction efficiency, assessed by flow cytometry, was 39.4% ± 12.0%, with stable co-expression of the transgenes for up to 21 days of culture (Fig. 6c-e). 19CAR-Tregs and untransduced (UT) cells displayed a similar expansion rate (43.7 fold ± 11.1 for 19CAR-Tregs and 39.4 ± 14.7 for UT-Tregs) at days 14 and purity (median CD4+CD25+CD127-: 84.4% and 70,2% for 19CAR-Tregs and 89,7% and 67,6% for UT-Tregs) at days 14 and 21 after the initial stimulation (Fig. 6f-g), indicating that the lentiviral transduction impacts neither on Treg expansion nor on cell phenotype.

[0748] To verify the 19CAR-Treg suppressive capacities, we co-cultured autologous PBMCs with either 19CAR-Tregs or UT-Tregs in the presence of anti-CD3 / anti-CD28 stimulation beads. After 7 days, we measured the proliferation of PBMCs by flow cytometry and observed a comparable suppressive capacity in 19CAR-Tregs and UT-Tregs, with a suppression index of 83.0% ± 7.7% for UT-Tregs and 74.0% ± 19.3% for 19CAR-Tregs (Fig. 6h). These results indicate that the lentiviral transduction and the culture do not alter Treg function.

[0749] We then co-cultured autologous B lymphocytes with either 19CAR-Tregs or UT-Tregs. Briefly, we activated B cells, by challenging them with irradiated CD40L-transduced 3T3 cells. 19CAR- Tregs, but not UT-Tregs, suppressed the proliferation of autologous B lymphocytes (mean suppression indexes of 61 ,2%, and 0% respectively), highlighting the effectiveness with which CAR molecules redirect the suppressive activity in an antigen-specific manner (Fig. 6i).

[0750] In chronic inflammatory conditions, Tregs can be reprogrammed towards an effector phenotype. To exclude that the reduced B lymphocyte proliferation might be due to a CAR- Treg reprogramming and consequent B-cell killing, we co-cultured ALL-CM, a CD19+cell line, with either 19CAR-Tregs, 19CAR-Tconvs or UT-Tconvs. After 3 days, 19CAR-Tconvs killed target cells, while 19CAR-Tregs and UT-Tconvs equally spared CD19+targets (p-value <0.01) (Fig. 6j). Overall, the results indicate that CAR-Tregs can be generated and propagated with relative ease and that engineered Tregs retain their suppressive capacities, which unravel upon recognition of their specific antigens.

[0751] Example 2 - FoxP3-CD19-CAR-engineered Tregs maintain immunomodulatory properties and show superior antigen-specific suppressive functions.

[0752] The adoptive transfer of Tregs requires an extensive validation of their safety profile, since they might undergo reprogramming toward conventional Th 17 cells, and Tconvs might contaminate the final cellular product. Since FoxP3 over-expression redirects inflammatory phenotypes toward a suppressive function, we decided to stabilize the Tregs through the constitutive up-regulation of FoxP3, by developing a new LV encoding for the anti-CD19 CAR and FoxP3 genes separated by a thosea asigna virus 2A self-cleaving peptide (T2A) and under the control of a PGK promoter (Fox19CAR). As control, we employed a LV encoding for the FoxP3 gene alone, devoid of the CAR construct (FoxP3 LV) (Figure 1a).

[0753] We transduced sorted CD4+CD25+bona fide Treg cells with the new constructs according to the protocol described above. Transduction efficiency, measured at day 14, was higher for Fox19CAR and FoxP3 LV than for CAR19.28z LV (mean: 72.5% ± 12.3%; 91.1 % ± 1.5%; 46.1% ± 12,5% respectively; Fox19CAR and FoxP3 LV vs. CAR19.28z LV p-value <0.001) (Figure 1b). Fox19CAR LV transduction promoted robust co-localization of CAR and FoxP3, indicating that both transgenes were actively transcribed (Figure 1c). CAR expression was similar in 19CAR- and Fox19CAR-Tregs (Fig 7a). We did not find significant differences in terms of expansion between Fox19CAR-, FoxP3-, 19CAR- and UT-Tregs (mean Fold Expansion at day 14: 41.8 ± 5.2; 28.1 ± 8.0; 43.3 ± 5.2 and 41.0 ± 12.0 respectively Figure 1d). Finally, the Treg purity in the cellular products after 14 days of culture was identical in the four groups. (Fig. 7b).

[0754] In a 7-day polyclonal suppression assay we observed that Fox19CAR-Tregs suppressed the proliferation of activated PBMCs as efficiently as FoxP3-, 19CAR- and UT Tregs (Figure 1e) and when cultured together with autologous B lymphocytes, Fox19CAR engineered cells not only showed a potent antigen-specific suppression but also outperformed 19CAR-Tregs in controlling the B cell proliferation (Fox19CAR- vs FoxP3- and UT-Tregs p-value <0.0001 at 1 :1 and 1 :4 ratios; Fox19CAR- vs 19CAR-Tregs p-value <0.05 at 1 :1 and 1 :4 ratios) (Figure 1f). Overall, Fox19CAR-Tregs retain their suppressive capacities and display superior antigen-specific suppression than 19CAR-Tregs.

[0755] Naive-derived (CD4+CD25+CD127-CD45RA+ cells) Tregs have been recently employed to generate CAR-Tregs. Our protocol, based on the Fox19CAR LV and use of rapamycin in culture to transduce CD4+CD25+ sorted Tregs, could represent a valuable alternative approach. We thus compared Fox19CAR-Tregs with naive-derived anti-CD19 CAR-Tregs. We firstly isolated CD4+CD25+ cells from HD PBMCs with magnetic cell separation. Sorted cells were divided in two fractions: one was transduced with the Fox19CAR LV and cultured with rapamycin, whereas the second fraction was further sorted to isolate naive-Tregs and subsequently transduced with CD19.28z LV and kept in culture in the absence of rapamycin, following the protocol of Imura and coll. (Fig. 8a). After 14 days, naive-derived anti-CD19 CAR-Tregs showed a higher expansion rate compared to Fox19CAR-Tregs (mean fold increase: 436.9 ± 223.1 ; 19.3 ± 6.3, respectively; p-value <0.05) (Fig. 7b). No differences in transduction efficiency (mean: 64.6% ± 15.3% for naive-derived CAR-Tregs; 62.2% ± 15.7% for Fox19CAR-Tregs) or Treg purity (mean: 85.5% ± 6.4% for naive-derived CAR-Tregs; 80.7% ± 6.6% for Fox19CAR-Tregs) were detected at day +21 (Fig. 7c-d).

[0756] When cultured with autologous B lymphocytes, Fox19CAR-Tregs proved superior in suppressing the B cell proliferation than naive-derived CAR-Tregs, especially at lower effector-to-target ratios (p-value <0.05) (Fig. 7e). In terms of cytokine secretion, naive-derived and Fox19CAR-Tregs showed a similar profile upon antigen-specific stimulation (Fig. 7f).

[0757] Example 3 - FoxP3-CD19-CAR LV coupled with rapamycin-based culture efficiently promote a regulatory phenotype in Tconvs.

[0758] Contaminant Tconvs might affect the purity and ultimately the safety of Treg-based cellular products. On the other hand, by inducing a constitutive FoxP3 expression, the Fox19CAR construct could reprogram Tconvs contaminating the cellular product to a suppressive phenotype. To test this hypothesis, we transduced sorted CD4+CD25_Tconvs with Fox19CAR LV according to two distinct expansion protocols: the Treg protocol, based on IL-2 and rapamycin (IL2R) and the Tconv protocol, based on IL-7 and IL-15, in the absence of rapamycin (IL7 / 15). As negative control, we generated 19CAR-Tconvs starting from CD4+CD25_cells engineered with CAR19.28z LV, and expanded in the presence of IL-7 and IL-15 (Figure 2a and 2b).

[0759] Transduction efficiency was independent of the expansion protocol used and similar with the two LVs (IL2R Fox19CAR 90.7% ± 2.7%; IL7 / 15 Fox19CAR 84.2% ± 10.4%, IL7 / 15 19CAR 83.8% ± 9.0%) (Fig. 9a). Again, Fox19CAR transduced cells displayed a good co-localization of the two transgenes, independently of the culture conditions (Fig. 9b. No differences were observed in the expansion rate promoted by the two protocols or by the vector used (mean fold increase at day 14: IL2R Fox19CAR 20.7 ± 5.6; IL7 / 15 Fox19CAR 21.9 ± 10.0; IL7 / 15 19CAR 24.7 ± 7.8, Figure 2c) but we consistently observed a significantly higher expansion rate in engineered cells compared to the untransduced counterparts (19CAR- p-value <0.0001 and Fox19CAR-engineered cells p-value <0.001 both vs UT cells). A large fraction (40%) of transduced cells were characterized by a CD3+CD4+CD25+CD127_FoxP3+phenotype, independently of the culture conditions and the vector (Fig. 9c).

[0760] We then verified whether CD4+CD25_cells transduced with the Fox19CAR gained suppressive capacities by culturing autologous PBMCs with IL2R Fox19CAR-, IL7 / 15 Fox19CAR-, IL7 / 15 19CAR- or UT T cells in the presence of a polyclonal stimulus. Only IL2R Fox19CAR lymphocytes efficiently suppressed the proliferation of autologous T cells (mean suppression index 82.3% at 1 :1 ratio, Figure 2d). These results show that the Fox19CAR construct reprograms CD4+CD25_lymphocytes toward a suppressive phenotype and the effect depends on the presence of IL-2 and rapamycin.

[0761] Example 4 - Co-expression of Helios, FoxP3 and TIGIT identifies highly immune suppressive cellular products.

[0762] We then characterized in better details our Tconv (CD4+CD25_) and Treg (CD4+25+) derived engineered cellular products (Figure 2a and 2b) by a multi-parametric flow cytometry panel. CAR-Tconvs and UT counterparts were used as controls. Through a supervised gating strategy, we observed that UT Tregs express higher levels of Glycoprotein A Repetitions Predominant (GARP) than the other cellular products. We found a higher Cytotoxic T Lymphocyte Antigen 4 (CTLA-4) expression on both UT Tregs and Fox19CAR transduced CD4+CD25+and CD4+CD25_derived cellular products compared to 19CAR-Tconvs, and independently of the culture conditions. Conversely, Glucocorticoid-induced TNFR-related (GITR) protein expression was higher on CAR-Tconvs. Finally, cellular products with proved suppressive capacity displayed a higher expression of T cell Immunoreceptor with Ig and ITIM domains (TIGIT) compared to 7 / 15 Fox19CAR and 19CAR-Tconvs (Fig. 9d).

[0763] To capture the complexity of the dataset, we employed cytoChain, a software developed in our lab for the unbiased analysis and dimensionality reduction of flow cytometry data. Firstly, we performed a multi-dimensional scaling to identify potential similarities between samples, finding a good separation between regulatory cells and conventional CAR-T or untransduced lymphocytes. Interestingly, IL2R Fox19CAR CD4+CD25_engineered cells localized closer to Tregs than to Tconv cells, in line with the suppressive capacity observed with this Tconv- derived cellular product (Fig. 9e). Subsequently, we used the algorithm to classify the different cellular products and thus identified a total of 15 metaclusters. We analyzed more in details the cluster composition of each group and we found 4 differently expressed clusters: cluster 3 and 6 are composed by conventional T lymphocytes while cluster 5 and 12 are constituted by Tregs (Figure 2e-f).

[0764] 19CAR- and unmanipulated Tconvs were more represented in cluster 3 and 6. Cluster 3 was composed by CD4+CD25'CD127+FoxP3+cells, whereas cluster 6 by CD4+CD25’CD127’ FoxP3+ones. Both clusters were characterized by CD4+CD25_elements, indicating the presence of conventional pro-inflammatory T lymphocytes with a high FoxP3 expression due to their activation status. IL7 / 15 Fox19CAR-Tconvs were more represented in cluster 6, composed by activated Tconvs, thus suggesting their pro-inflammatory nature.

[0765] Engineered and unmanipulated Tregs were mainly represented in cluster 5 and 12, comprising CD4+CD25+CD127lowFoxP3+and CD4+CD25+CD127’FoxP3+elements, respectively, also characterized by the expression of Helios and TIGIT. These combinations of markers were compatible with a Treg signature. IL2R Fox19CAR-Tconv cells, the only Tconv-derived cellular product with suppressive capacity, were mainly represented in cluster 5, thus supporting their immunosuppressive nature. In addition, both clusters showed an intermediate CD45RA expression on Tregs compared to Tconvs in cluster 3 and 6, compatible with a mixed cell population with a high frequency of effector cells.

[0766] Collectively, the unsupervised analysis of flow cytometry data allowed to separate Treg and Tconv derived cellular products, with the exception of the IL2R Fox19CAR CD4+CD25_derived cells, that clusterized with Treg cells, in accordance with the suppressive activity. These findings confirm a Treg profile for suppressive T cells, and indicate a combination of Helios, Foxp3 and TIGIT as a signature associated to the suppressive capacity (Figure 2e).

[0767] Overall, the combination of a FoxP3 encoding vector with culture conditions designed for Tregs induces a suppressive phenotype also on Tconvs that might contaminate the initial cellular composition.

[0768] Example 5 - FoxP3-CD19-CAR-Tregs control autoimmunity in vivo.

[0769] We then turned to test in vivo the functionality and the safety profile of engineered Fox19CAR- Tregs. To do this, we employed a humanized mouse model of SLE that recapitulates chronic inflammatory tissue damage mediated by human immune cells. We transplanted human cord blood stem cells (0.8-1 x 10A5 CD34+cells / mouse) in irradiated 1 -day-old NSG mice. After having established a fully humanized immune system, we i.p. injected mice with pristane, a lipid moiety widely used for experimental SLE induction, since it leads to anti-DNA antibodies formation, lymphopenia and renal damage. Three weeks after, we administered Fox19CAR- Tregs or UT Tregs and analyzed the composition of the bone marrow, spleen, lung and kidney (Figure 3a).

[0770] Circulating human cells were detected 4-5 weeks after humanization and their concentration progressively increased (Figure 3b). After the appearance of T lymphocytes, at week 7-9, we induced experimental SLE by injection with pristane while maintaining a group of humanized control mice untreated. The treatment was effective, since the animals developed as expected autoantibodies, inflammatory involvement of the lung and the kidney and B cell lymphopenia (Figure 3b and Fig. 10a-b).

[0771] Three weeks after pristane administration, mice were randomized to receive either Fox19CAR-Tregs, UT-Tregs (3.5x106cells / mouse) or PBS. Fox19CAR-Treg blood concentration peaked 3 days after injection, then decreased at day +7 and remained stable up to day +10. After 14 days, the pool of engineered cells contracted, and virtually disappeared from the peripheral blood by day +21 (Figure 3c). After the injection of PBS or UT-Tregs, we found a progressive and significant reduction of B cells while huCD45+and CD3+T cell counts remained stable (Fig. 10c-d). Mice infused with Fox19CAR-Tregs had stable levels of circulating B cells for up to 10 days (Figure 3d).

[0772] We measured the serum levels of inflammatory and suppressive cytokines before and 3 days after cellular therapy. Fox19CAR-Treg treated mice showed a significant increase in levels of the immune-regulatory agent IL-10, whereas mice treated with UT-Tregs or PBS did not. Levels of inflammatory human cytokines, IFNgamma, IL6, TNFalpha, ILIbeta, CCL2, IL17, IL23, IL33 were not significantly increased (Figures 3e-g and Fig. 10e-n).

[0773] Collectively, these results indicate that Fox19CAR-Tregs can be safely administered and selectively stabilize B cell counts and delay progression of B cell lymphopenia. Indeed, the stable B cell levels obtained after CAR-Treg injection indicate a limited contaminant CAR- Tconvs if any, being B cell aplasia a hallmark of anti-CD19 CAR-T cell functionality both in patients and in mice.

[0774] Example 6 - Restored immune cell composition and reduced inflammation upon FoxP3- CD19-CAR-Treg infusion.

[0775] To investigate the effects of our cellular products on autoimmunity, we assessed autoantibodies and immune cells composition in the bone marrow and the spleen. Fox19CAR- Tregs significantly reduced the frequency of anti-dsDNA antibody development (80% vs 13% of mice treated with CAR-T regs), while UT Tregs was only partially effective (p-value <0.05) (Figure 4a). At sacrifice, no differences in terms of relative enrichment in human T regs or T reg phenotype were found between groups (Figure 4b and Fig. 11 a). The percentage of huCD45+leukocytes varied in different organs, but not between treated groups (Figure 4c and 11 b-c). In the spleen of PBS-injected animals, we observed increased frequencies of CD3+and CD4+T lymphocytes compared to non-pristane injected humanized mice (p-value <0.01 for CD3+and CD4+cells). Similar frequencies were observed also in UT Treg animals (p-value <0.001 for CD3+and CD4+cells). Conversely, CAR-Treg mice displayed lower levels of CD3+and CD4+T lymphocytes than the UT Treg (p-value <0.001 for CD3+and CD4+cells) and PBS groups (p-value <0.01 for CD3+and CD4+cells), comparable to those of non-pristane injected mice (Figure 4c). A similar situation was detectable for Naive B cells. The CAR-Treg group displayed a significantly higher frequency of naive B cells than the other two groups of pristane-treated mice, comparable to that observed in non-pristane injected animals (CAR- Treg group vs UT Treg one p-value <0.001). A similar restored homeostasis of the immune cell composition in CAR-Treg treated animals was observed in the bone marrow and kidney (Fig. 11b-c). Overall, these data indicate restoration of the immune composition of lymphoid organs upon CAR-Treg infusion.

[0776] Experimental SLE in pristane-injected humanized NSG mice was characterized by grossly altered spleen architecture, with multiple granulomatous lesions characterized by giant cells and signs of necrosis. The immune-mediated remodeling abated in Fox19CAR-Treg treated animals, with 60% of them with a normal or negative score (p <0.01). UT-Tregs did not exert significant effects on architecture or inflammatory response in the spleen, where lesions were more numerous than in humanized Pristane-treated NSG mice injected with PBS (Figure 4d and 4f), suggesting a detrimental role of Tconvs contaminating the cellular product. No significant differences were found in terms of spleen white and red pulp representation, indicating a similar humanization and supporting the flow cytometry data (Fig. 11 d).

[0777] Similarly, in the lungs of pristane-treated mice an inflammatory reaction characterized by a monocytic / macrophagic infiltrate and granulomatous-like lesions was present. Inflammatory cells localized mainly around blood vessels, also causing their occlusion forming inflammatory thrombi. Alveolar spaces were subverted with the complete loss of the normal architecture. UT-Tregs were ineffective in controlling the inflammatory reaction, even exacerbating it in some mice. Conversely, CAR-Treg mice showed a lower grade of inflammation, with 60% of animals with grade <1 lesions and preserved lung structure, normal alveoli and vessel permeability (p-value <0.05) (Figure 4e and 4g).

[0778] The kidney of pristane-injected animals underwent tubular degeneration and vacuolation, tinctorial changes and cellular sloughing, occasional interstitial and perivascular inflammatory cell infiltrates and sporadic granulomatous inflammation in all mice. Glomerular structures were mostly spared. The kidney remodeling was significantly reduced in Fox19CAR-Tregs treated animals, that displayed significantly less tubular lesions compared to both UT-Treg and PBS injected animals (p-value <0.01). Actually, the kidney of up to 40% of Fox19CAR- Tregs treated mice had no detectable lesions (Fig. 11 e-f).

[0779] Immunohistochemical analysis of the spleens revealed a prevalent human T cell infiltrate without a clear organization in pristane-treated mice injected with PBS, with only a minimal B cell component. UT Tregs treated animals displayed similar features with a greater B cell infiltrate, in the absence of a clear organization. Conversely, CAR-Treg injection completely restored the normal spleen organization with T and B cells almost exclusively represented inside the white pulp follicles, as in healthy humanized mice (Fig. 11 g-h). Collectively, these data further support the potent immunoregulatory effect of CAR-Tregs in this SLE model. Example 7 - Multiple FoxP3-CD19-CAR-Treg administrations are safe and effective in reshaping the B cell compartment in a prolonged humanized model of autoimmunity.

[0780] To explore the possibility of multiple infusions of Fox19CAR-Tregs and to verify their efficacy in a later stage of disease, we adapted the SLE model to SGM-3 mice, a strain characterized by a constitutive high expression of human IL-3 and GM-CSF, able to better promote human HSC engraftment, and characterized by a longer lifespan than humanized NGS mouse pups. We thus transplanted irradiated 8-weeks-old SGM-3 mice, with human cord blood stem cells (0.8-1 x10A5 CD34+ cells / mouse). After the establishment of a full human immune system, we injected the animals with pristane i.p. to induce the disease. Animals were randomized to receive Fox19CAR-Tregs (3.5 M of cells / mouse) or PBS at 3 and 8 weeks after pristane administration. Additionally, since the efficacy of anti-CD19 conventional CAR-T cells has been recently reported in refractory SLE in patients, in this model we also tested the effect 19CAR-Tconvs (3.5 M of cells / mouse), injected 3 weeks after pristane administration (early therapeutic regimen, Figure 5a).

[0781] Early administered CAR-Tconvs peaked in the peripheral blood at day +31 and at day +63, then contracted and disappeared by day +91 (Figure 5b). As expected, circulating human B cells disappeared in CAR-Tconvs treated mice and remained undetectable until day +77 (Figure 5c). Conversely, the administered CAR-Tregs, despite displaying a limited peak of expansion in the peripheral blood at day +28, induced a significant increase in circulating B cells at day +35 (p-value <0.05). Starting from day +49, we observed a gradual B cell lymphopenia also in CAR-Treg treated mice, with levels similar to those observed in PBS- injected animals. Of notice, the second infusion of the CAR-Tregs did not produce detectable CAR-Treg expansion nor did it further increase the B cell count (Fig. 5b-c).

[0782] Humanized animals showed a progressive contraction of the circulating hCD45+ and CD3+ cell pools, independently from the group of treatment (Fig. 12a-b). In the CAR-Treg mice, both administrations of engineered cells were associated with a transient increase of total hCD45+ cells. Conversely, CAR-Tconv treated animals showed a delayed transient hCD45+ cell expansion at day +56, with a peak at day +63, followed by a progressive reduction, reaching levels comparable to PBS-injected mice by the end of the experiment.

[0783] To evaluate the efficacy of engineered cells in a more advanced stage of disease, we compared the effects of Fox19CAR-Tregs or 19CAR-Tconvs injection at 8 weeks after pristane (late therapeutic regimen, Fig. 12c). Engineered CAR-Tregs and CAR-Tconvs showed a rapid expansion, followed by an immediate disappearance from the peripheral blood at day +28 and +31 after T cell infusion, respectively (Fig. 12d). CAR-Treg treated mice displayed a transient increase in circulating B cells at day +31 and +35 after T cell infusion, although not statistically significant (Fig. 12e).

[0784] To better elucidate the effects of engineered T cell infusion, we measured the levels of circulating cytokines before and 7 days after each cell administration, in both early and late therapeutic models. In the early therapeutic model, as expected, CAR-Tconv injection was associated with a relevant increase of perforin, Granzyme A and pro-inflammatory cytokines, like IFN-gamma, IL-6 and IL-10 (p-value <0.0001). On the contrary, CAR-Tregs produced high amounts of IL-10 (p-value <0.05), IL-2 (p-value 0.09) and IL-4, with negligible levels of other pro-inflammatory molecules (Figure 5d-e). CAR-Treg re-administration 5 weeks after the first injection showed a similar cytokine profile, with a significant increase of IL-10 (p-value <0.05) and a reduction of IL-17 and TNF-alpha (Fig. 12f-g). In the late therapeutic model, we observed a significant increase in IL-10 (p-value <0.0001) with negligible levels of IFN-gamma and IL-6 in CAR-Treg treated mice, whereas CAR-Tconv administration was associated with an increased IFN-gamma production (p-value <0.05) (Fig. 12f-g).

[0785] At sacrifice, engineered cells could be detected in the bone marrow with frequencies similar in all groups, while a higher proportion of 19CAR-Tconvs than CAR-Treg was detected in the spleen (Figure 5f). In bone marrow and spleen, animals injected with an early infusion of 19CAR-Tconv displayed the highest frequency of human CD45+ cells (p-value CD45+ cells early CAR-Tconvs vs. PBS 0.07; early CAR-Tconvs vs. late CAR-Tconvs 0.10; early CAR- Tconvs vs. early CAR-Tregs 0.12), mainly composed by T lymphocytes (%CD3+ cells in the BM early CAR-Tconvs vs. PBS p-value <0.05) (Fig. 12h-i).

[0786] In terms of B cells, early CAR-Treg mice showed a significantly higher frequency of CD19+ cells compared to CAR-Tconv and PBS-treated animals both in the bone marrow (early Fox19CAR-Tregs vs. PBS-injected mice p-value <0.05; early Fox19CAR-Tregs vs. late 19CAR-Tconv mice p-value <0.01) and in the spleen (early Fox19CAR-Tregs vs. PBS I early 19CAR-Tconv mice p-value <0.05; early Fox19CAR-Tregs vs. late 19CAR-Tconv I late Fox19CAR-Treg mice p-value <0.01), constituted by pre- / naive B cells and naive / memory B cells, respectively. B lymphocytes were still detectable in early 19CAR-Tconv mice, which presented an increased skew towards antibody-producing cells (% plasmablasts in early 19CAR-Tconvs vs. all other groups p-value <0.05 in the bone marrow and the spleen) (Figure 5g-h).

[0787] Pathological evaluation on the bone marrow, the spleen and the liver revealed the presence of an immune cell infiltrate composed mostly by monocytic / macrophagic and histiocytic elements and giant cells, a greatly compromised white pulp reconstitution and a skewing of the hematopoiesis towards myeloid elements with a great abundance of macrophages and eosinophils. Although present in all groups, CAR-Tconv treated animals showed a higher grade of the inflammatory lesions, whereas CAR-Treg and PBS injected mice appeared similar (p-value <0.05) (Figure 5i). Notably, one out of 5 CAR-Tconv treated mice developed cutaneous GvHD, which on the contrary was absent in all of those that received CAR-Tregs or PBS.

[0788] Collectively, these results confirm the efficacy of multiple CAR-Treg injections in the early phase of the disease in improving circulating B cells and in reshaping the human immune infiltrate in lymphoid organs. In addition, we confirm the favorable CAR-Treg safety profile, which do not display B cell killing or pro-inflammatory cytokine secretion.

[0789] Conclusions

[0790] In our study, we report that anti-CD19 CAR-Tregs acquire new antigen-specific suppressive capacities proving effective against B cells in vitro without detectable inflammatory activity. When employed in vivo in two different humanized mouse model of lupus, an early administration of engineered cells did not increase the overall inflammatory burden, reduced the extent of tissue damage, delayed the occurrence of B cell lymphopenia, restored the homeostasis of lymphoid organs while reducing the generation of autoantibodies.

[0791] These data confirm that CAR-Tregs exert a potent antigen-specific immunosuppressive activity both in vitro and in vivo. Their specificity for the CD19 B cell antigen did not endow them with the ability to kill B lymphocytes, but rather to exert their regulatory actions at the sites where B cells play their biological role, including the bone marrow, the spleen, and solid organs such as the lung and the kidney. Our results indicate that targeting Treg specificity by CAR to recognize B cells has dramatic effects on the disease natural history, quenching the pathogenic action of the small percentage of disease-causing clones while sparing the vast majority of bystander B cells, that remain able to play their homeostatic role in vivo. Moreover, the pathological evaluation and the flow cytometry analysis on the organs at sacrifice suggest an immunomodulatory activity that might extend beyond B lymphocytes, also involving T cells, probably due to a more extensive loco-regional effect, already described with other CAR-Treg products.

[0792] As already reported in clinical trials with polyclonal Tregs, CAR-Tregs showed a limited persistence. Modifications of the treatment protocol, such as co-administration of CAR-Tregs and low-dose IL-2 or prior lymphodepletion, might increase Treg survival and persistence. However, in light of the substantial effects observed in our models, we can speculate that an early administration of relatively short-lived CAR-Tregs could anyway play as a molecular switch, interrupting the feed forward cycle by which autoimmunity and tissue damage sustain each other in patients and allowing the restoration of immune homeostasis. This would be an ideal environment for cell therapy, as safety would increase if the desired biological effects could be achieved by transferring cells that do not normally survive for more than a few days.

[0793] Tregs don’t express specific markers and some of them are shared with conventional T lymphocytes, raising the possibility of contaminant Tconvs in the final cellular product. This aspect is particularly relevant with CAR-Tregs in autoimmunity, where CAR molecules specific for self-antigens are employed and unwanted Tconv transduction could lead to the production of potentially harmful self-reactive CAR-T cells.

[0794] Treg sorting strategies have been developed, achieving different grades of purity and cell yields. For a GMP-grade manufacturing, a compromise between feasibility, purity and cell yield is required. We sorted CD4+CD25+cells as bona fide Treg and used IL2 and rapamycin in the cell culture to promote Treg expansion without compromising purity, as described (Battaglia, M. et al., J. Immunol. 177, 8338-8347 (2006)). The results confirm that this approach is sustainable, as it meets the requirements for its application in the clinical setting.

[0795] Tregs may convert to pro-inflammatory Th17 T cells and lose the FoxP3 expression. We hypothesized that a constitutive overexpression of FoxP3 could stabilize the Treg phenotype and restrain contaminant Tconvs. Indeed, FoxP3 overexpression in conventional T lymphocytes converts Tconvs to a suppressive phenotype. We thus included the FoxP3 gene in the lentiviral vector (Fox19CAR LV). FoxP3 overexpression did not affect Treg expansion or functionality and in combination with IL-2 and rapamycin proved effective even in reprogramming CD4+CD25_T cells (bona fide Tconvs) to a suppressive phenotype. When infused in two different humanized mouse models, differently from reports with anti-CD19 CAR-Tconvs, Fox19CAR-Tregs did not cause B cell aplasia, Cytokine Release Syndrome (CRS) nor hemophagocytic lymphohystiocytosis (HLH), thus confirming the stability of their immunosuppressive phenotype. Interestingly, compared to CAR-Tconvs no signs of xenogenic GvHD were found in CAR-Treg treated mice, in line with the encouraging safety profile of these engineered cells.

[0796] The isolation of CD45RA+ naive Tregs, a purer and more undifferentiated subset, represents an alternative strategy to reduce contaminant Tconvs in Treg-based cellular products. It has been previously reported that naive-derived anti-CD19 CAR-Tregs are effective in suppressing B cell activation both in vitro and in vivo in a xenograft mouse model of GvHD. When directly compared, naive-derived CAR-Tregs display the highest expansion capacity but Fox19CAR-Tregs showed the highest suppressive activity. Our approach might represent an alternative to the prior art product, displaying an easier manufacturing and a greater flexibility, being employable with either bona fide Tregs or CD4+ Tconvs, potentially improving the scalability in clinical settings where autoimmune patients’ lymphopenia might be a limit. To properly evaluate the efficacy and the safety profile of CAR-Tregs and their interactions with the human immune system, we employed two humanized mouse models of pristane- induced SLE.

[0797] Elegant pre-clinical studies have suggested that conventional anti-CD19 CAR-T cells can be used to selectively deplete B lymphocytes, which play a role in the pathogenesis of SLE, improving the disease in genetic lupus-prone mice. More recently, the efficacy of anti-CD19 CAR-T cells in controlling the disease manifestations in 8 patients with refractory SLE up to 1 year after their injection has been shown. Conventional CAR-T cell use, however, might cause CRS or HLH, well-known CAR-T cell toxicities, and theoretically favor opportunistic infections due to the chronic depletion of B cells, even though this risk appears highly mitigated in recent studies. Further studies are required to evaluate the long-term efficacy and safety of such a therapy.

[0798] Compared to CAR-Tconvs, CAR-Tregs might display a better safety profile. In addition, by exerting an immune suppressive effect directly in lymphoid organs where antigen-presentation and antibody generation occur, anti-CD19 CAR-Tregs might interrupt the vicious cycle that sustains the disease and potentially restore immune tolerance.

[0799] Generally, compared to CAR-Tconvs, the CAR-Treg platform offers the unique opportunity of safely targeting self-antigens, potentially extending its applicability to other autoimmune diseases where B cell lymphodepletion is ineffective.

[0800] In conclusion, here we developed and validated a novel CAR-Treg cellular product with potent antigen-specific immune suppressive capacities both in vitro and in vivo. In addition, our engineered cells showed an optimal safety profile when employed in two humanized lupus mouse models, proving the stability of the suppressive phenotype. Collectively, these data provide a solid base for the future clinical translation of CAR-Tregs in autoimmunity.

[0801] Materials & Methods (Examples 8 and 9)

[0802] Cell lines

[0803] HEK293T cells were grown in IMDM (Lonza) supplemented with 10% FBS (fetal bovine serum, Euroclone), 1% penicillin / streptomycin (Lonza) and 1 % glutamine (Lonza).

[0804] Lentiviral vector production

[0805] Third generation replication-defective and self-inactivating lentiviral vectors (LV) were produced by transient transfection of HEK-293T cells. Briefly, a solution containing the packaging plasmids pMDLg / pRRE (containing HIV-1 gag / pol genes), the plLW01-Rev plasmid (encoding Rev protein), the envelope plasmid encoding the vesicular stomatitis virus glycoprotein (VSV-G), the pAdvantage (that enhances transient protein expression) and the plasmids carrying the transgene of interest was transfected in sub-confluent 293T cells using the calcium chloride precipitation method. Supernatants containing lentiviral particles were collected 48 hours later and filtered by 0.22-pm-filter. The supernatant of Fox19CAR lentiviral vectors was centrifugated at 7000 rpm, at 4° for 16h (Beckman Optima XL-100K Ultracentrifuge). Then it was aliquoted and cryopreserved.

[0806] Peripheral blood mononuclear cells isolation

[0807] Peripheral blood mononuclear cells (PBMCs) were collected from healthy donors, after written informed consent, according to the San Raffaele Scientific Institutional Ethical Committee guidelines. PBMCs were isolated by Ficoll density gradient separation (Lymphoprep) and were freshly used for the following protocols of specific T cell expansion.

[0808] Regulatory T cell isolation

[0809] Regulatory T cells defined as CD4+CD25+ cells were isolated from PBMCs derived from Healthy Donors by magnetic cell separation (Miltenyi, CD4+ CD25+ regulatory kit isolation kit human, 130-091-301), according to manufacturer’s instructions. Tregs were plated 0,1 M / ml in a 24 well-plate and stimulated with cell-sized anti-CD3 / anti-CD28 magnetic beads. They are cultured in X-VIVO15 (Lonza) supplemented with 10% human serum (Lonza), 1% penicillin / streptomycin (Lonza) and glutamine (Lonza) with Rapamycin (100nM / ml). IL-2 was continuously replenished every 2 days. At day 14, stimulation beads were magnetically removed, and Treg cells were counted and employed for phenotypic and functional studies.

[0810] Tree cell transduction

[0811] At day +2 after stimulation, activated Treg cells were transduced adding the virus to 1 ml of cells concentrated 0.2M / ml. The lentiviral vectors were added accordingly to the viral titration. Treg cells were kept 24 hours at 37°C and then fresh medium supplied with IL2 was added in each condition. Transduction efficiency was measured by flow cytometry at day 14 of expansion. Recombinant CD19 reagent (FMC63 PE, Miltenyi) was used for CAR surface staining according to manufacturer’s instructions.

[0812] To co-transduce the Luc-GFP and the Fox19CAR, activated Treg cells were transduced firstly with the Luc-GFP lentiviral vector as previously reported. On day 5, Luc+ Treg cells were centrifugated at 1500 rpm per 5 mins, re-suspended into fresh medium with rapamycin, plated and transduced with the Fox19CAR LV as previously discussed. Luc-GFP transduction was evaluated based on the GFP fluorescence. Treg culturing protocol

[0813] On day 0, Treg cells were stimulated using antiCD3 / CD28 magnetic beads at either a 1:3 or 1:1 ratio of Treg-beads. After transduction, IL2 (Proleukin, Novartis) was replenished every two days at concentrations of 250, 500, 1000 ll / rnl. The Treg cells were maintained at a concentration of approximately 2M / ml and split as necessary. Rapamycin was added to the fresh medium until day 14 of expansion, at which point the beads were removed using magnetic columns. Whether restimulation with 1:1 Treg-beads ratio was applied on day 9, the cells were counted and resuspended at a concentration of 0.15 M / ml.

[0814] Polyclonal suppression assay

[0815] PBMCs (target cells) and engineered T cells (effector cells) were co-cultured at decreasing Effector-to-Target ratio in the presence of anti-CD3 / anti-CD28 coated beads at 1 :10 beads to cell ratio. To detect proliferating cells, PBMCs were stained with FarRed proliferation dye, while engineered cells were stained with VioBlue proliferation dye, according to manufacturer’s instruction. After 6 days, cells were analyzed by flow cytometry. Suppression index was calculated as follows: %PBMCs’ proliferation when cultured with Tregs.

[0816] %PBMCs’ proliferation alone '

[0817] In vivo humanized mouse model

[0818] The experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC 1357). 6-7 weeks-old NOD-scid y mouse (NSG, Charles-River Italia) received sub- lethal total body irradiation (95”, 1.5 Gy) and infused intravenously with 0,08 M human cord blood-derived hematopoietic stem cells (CD34+ hHSC). Mice were clinically monitored and weighed every week. Starting from week 4 after cord-blood infusion, the frequency of human cells in the peripheral blood was monitored every week. Mice were intravenously injected with 200pL of Luc+ or Luc+Fox19CAR+ Tregs (3,5 M each).

[0819] Luc+ and Luc-Fox 19CAR+ Tree / cell sorting

[0820] Treg cells transduced with the Luc-GFP lentiviral vector and co-transduced with the Fox19CAR lentiviral vector were sorted using the MACSQuant Tyto. The staining of Fox19CAR-Treg cells was performed using FMC63 in PE. Luc+ Treg cells were sorted based on the GFP expression, while for Fox19CAR-Tregs we sorted cells based on the coexpression of the GPF and the Fox19CAR.

[0821] Bleedings For in vivo experiments, the blood harvested from the mice is centrifugated and the serum was collected and cryopreserved. 50 pL of whole blood was lysed with ACK (Ammonium- Chloride-Potassium) buffer for 10 minutes at room temperature to remove red blood cells and then stopped with PBS supplemented with 1 % FBS. Subsequently, samples were stained with recombinant CD19 reagent (Miltenyi, FMC63). Then, fluorochrome-conjugated monoclonal antibodies specific for mouse CD45 (Biolegend), human CD45 (Biolegend), hCD3 (Biolegend), hCD14 (Beckman Coulter), hCD19 (Biolegend), hCD56 (BeckmanCoulter) were added to samples. Flow Count fluorescent beads (Beckman Coulter) were used to assess cell count. cell n° of acquired events x qL beads

[0822] - = - - - — - - - — - - x beads concentration qL n of acquired beads x qL blood

[0823] Flow cytometry panels

[0824] For Treg phenotype, cells were labeled with titrated fluorescent monoclonal antibodies specific for antiCD19 CAR (FMC63, Miltenyi), CD3 (Biolegend), CD4 (Miltenyi), FoxP3 (eBioscience), CD45RA (BD Bioscience), CD62L (BD Bioscience), TIGIT (Biolegend), LAP (eBioscience), Helios (Biolegend), CD25 (Miltenyi), CD27 (BD Bioscience), CD137 (Biolegend), CD127 (Miltenyi), GARP (BD Bioscience), CTLA-4 (Biolegend), ICOS (BD Bioscience), GITR (Biolegend).

[0825] For Treg exhaustion phenotype, cells were labeled with titrated fluorescent monoclonal antibodies specific for LAG3 (Miltenyi), CD137 (BioLegend), CD25 (BioLegend), CD4 (BioLegend / lnvitrogen OKT4), HLA-DR (BD Horizon), CTLA4 (Biolegend), PD1 (BioLegend), KLRG1 (BioLegend), CD3 (BD Horizon), CD45RA (BD Bioscience), CD62L (BD Bioscience), TIM3 (Miltenyi), 2B4 (BioLegend) and TIGIT (Invitrogen).

[0826] For intra-nuclear staining, lymphocytes were first stained with surface antibodies, washed and fixed and permeabilized with FoxP3 staining buffer set (Miltenyi), according to manufacturer’s instructions. Intracellular staining was performed with anti-Helios and anti-FoxP3 antibodies.

[0827] Statistical Analysis

[0828] Statistical analyses were performed with Prism 8 (GraphPad Software). One way or two-way ANOVA was used when comparing three or more different subsets: p-value adjusted tests were employed to identify significant differences between groups. For all comparisons, two- sided p-values were used, and p-value < 0.05 was considered statistically significant.

[0829] Example 8 - In vivo persistence of Fox19CAR-Tregs

[0830] Example 8.1 - Generation of Luciferase+ CAR-Tregs To investigate and confirm the reduced CAR-Treg persistence in vivo, we decided to exploit the luciferase system to track the infused engineered cells. To do this, we modified the protocol for the CAR-Treg manufacturing to include a second transduction procedure with a lentiviral vector (LV) encoding for the luciferase and the Green Fluorescence Protein (GFP), as a transduction marker (Luc+CAR-Tregs) (Figure 13A). CD4+CD25+healthy donors’ Tregs were isolated from the peripheral blood, stimulated with anti-CD3 / CD28 beads and kept in culture with IL-2 and rapamycin. Cells were first transduced at day +2 with the Luc-GFP LV and at day +5 days with the Fox19CAR LV (Figure 13B). Polyclonal Tregs transduced only with the luciferase LV (Luc+Tregs) or unmanipulated (UT Tregs) were used as controls. After 14 days of culture, the regulatory T cells were counted, and their transduction was assessed by flow cytometry. Luc+CAR-Tregs and Luc+Tregs showed a similar expansion rate compared to UT Tregs (Figure 13C; mean±SD, UT 33.9±11.16; Luc+ 31.9±12.7; Luc-Fox19CAR 27.3±10.46). Luc-GFP expression was observed in 83.5±5.57% of cells in culture (mean±SD, n=5). In contrast, the co-transduction efficacy of Fox19CAR and GFP-LUC lentiviral vectors is 49.74±12.1 % (mean±SD, n=5, Figure 13D).

[0831] To increase the purity of the CAR-Treg product, Luc+CAR+Treg cells were sorted using the MACSQuant Tyto system. Luc+Treg cells were sorted based on GFP expression, while Luc+CAR-Treg cells were sorted based on the co-expression of GFP and the Fox19CAR molecule (Figure 13E). The final purity was 91.43±5% for Luc+Treg cells (mean±SD, n=3) and 88±2.6% for Luc+CAR-Treg cells (mean±SD, n=3, Figure 13F).

[0832] Altogether, these results validate the in vitro culture of Luc+Treg and CAR-Treg cells. Luc+CAR-Tregs exhibited similar expansion rates and phenotypes to UT cells, indicating that the co-transduction with Luc-GFP does not affect overall the quality of the cellular product.

[0833] Example 8.2 - In vivo CAR-Treg monitoring

[0834] In previous studies, we employed a 1-day-old NSG mouse model. The pups were irradiated and infused with human hematopoietic stem cells derived from heathy donors’ cord blood. Pristane, a lipidic moiety, was injected in humanized mice to cause an inflammation characterized by lymphopenia, damage to lymphoid organs, and autoantibody production, recapitulating some features of Systemic Lupus Erythematosus (SLE). However, pups showed short lifespan. To study the effects of multiple CAR-Treg injections and treatment at later disease stages, we employed adult SGM3 humanized mice. These mice are characterized by constitutive production of IL3 and GM-CSF to promote HSCs engraftment. Compared to pups, SGM3 humanized mice showed longer lifespan. However, this animal model was biased towards myeloid lineages in long-term hematopoiesis. Therefore, in this study, we employed an adult humanized NSG mouse model. We humanized a total of 10 sub-lethally irradiated 6 weeks-old adult NSG mice by injecting them intravenously with 0.08 M / mouse of human cord blood-derived hematopoietic stem cells (CD34+hHSC).

[0835] Starting 4 weeks after the humanization, we monitored the appearance of human immune cells in the peripheral blood by flow cytometry every week. At the appearance of the B cell in the periphery at week 4, we divided the animals into two groups, 5 mice each, and we injected them with 3,5 M of Luc+Tregs or Luc+CAR-Tregs, respectively (Figure 14). CAR-Treg injection was well tolerated. As shown in Figure 15, the mice did not experience weight loss or signs of Cytokine Release Syndrome (CRS), a well-known complication of conventional CAR-T cell administration.

[0836] We longitudinally monitored the level of humanization, and the human immune cells count. Following Treg infusion, both groups displayed an initial increase of the circulating hCD45+ cells, which peaked at day +11. At day +18, Luc+CAR-Treg treated animals showed a transient decrease in the human leukocyte count compared to Luc+Tregs. From day +32, both groups showed superimposable hCD45+cell counts till the end of the experiment, characterized by a progressive decline with the nadir at day +58 followed by an increase of the values until sacrifice (Figure 16A). Luc+Treg treated mice showed a human B cell kinetic like that observed with hCD45+cells, with a peak at day +11 , a gradual reduction reaching the nadir at day +58 and a new progressive increase of the values till day +78. Conversely, Luc+CAR-Treg mice displayed a transient mild reduction of the human B cells at day +5, which recovered at day +11. At day +18, circulating B lymphocytes showed a transient reduction, which gradually resolved in the next timepoints. Starting from day +32, the Luc+CAR-Treg group displayed human B cell counts comparable to that of the Luc+Treg one (Figure 16C).

[0837] Human monocyte counts were superimposable between the two groups, except for a transient increase of hCD14+cells in Luc+CAR-Treg mice at day +11 (Figure 16B). After the cell injection, circulating human T cells expanded in both groups, significantly more in Luc+CAR- Treg treated animals compared to Luc+Treg ones, reaching a peak at day +11 , followed by a gradual reduction till their disappearance at day +32. At day +65 human T cells started to increase in both groups till day +78 (Figure 16D).

[0838] We evaluated the dynamics of circulating engineered using a combination of flow cytometry and bioluminescence. After the injection, Luc+CAR-Tregs showed a significant greater expansion than GFP+Tregs (Figure 17A, mean±SD Luc+27.61 ±19.64; Luc+CAR+223.9±79.46; p-value < 0.0001) at day +5 and a subsequent contraction, disappearing from the peripheral blood after 32 days. Conversely, Luc+Tregs displayed a minimal expansion after their administration. These results were mirrored by the bioluminescence analysis that confirmed a significant Luc+CAR-Treg greater expansion than GFP+ones at day +5 and +7 (Figure 17B, mean±SD, day+5: Luc+Treg 7.8x108±4.4x108; Luc+CAR-Treg 1.23x1010±5.5x109, ****p-value < 0.0001 ; day+7: Luc+Treg 4.95x108±3.8x108; Luc+CAR-Tregs 5.41x109±5.7x109, ***p-value 0.0008). The analysis of the bioluminescence allowed us to assess the localization of the injected cells. Luc+CAR-Tregs showed a preferential accumulation in the lungs and in lymphoid organs like the spleen and femurs, already detectable 5 days after their injection (Figure 18).

[0839] Altogether, these results confirmed previous findings on CAR-Treg dynamics in the peripheral blood and further supported the observation of their low persistence after injection. Moreover, the employed humanized adult NSG mouse model has demonstrated a longer survival compared to humanized NSG pups for future studies, which allows the long-term evaluation of the CAR-Treg persistence.

[0840] Example 9 - Optimization of in-vitro culture of Fox19CAR-Treg cells

[0841] CAR-T cell manufacturing is one of the key factors that can influence their fitness and consequently their persistence in vivo. While this aspect has been extensively studied with conventional CAR-T cells (Watanabe, 2022, Front Immunol), no clear data are available about the optimal ex vivo Treg manufacturing. Battaglia et al. reported that a combination of anti- CD3 / CD28 stimulation beads, IL2 500 lll / ml and rapamycin represents a valuable approach for the Treg expansion (Battaglia & Roncarolo, 2006, J Immunol). However, several other Treg expansion protocols have been reported in literature, and no direct comparisons of the various strategies have been reported so far. We previously published that the use of rapamycin and IL2 is fundamental to improve the safety of CD4+CD25+derived CAR-Treg products generated using a LV to co-express both the FoxP3 gene and a second-generation CAR molecule (Figure 13B).

[0842] To improve the CAR-Treg persistence in vivo, we aimed at optimizing the manufacturing process trying to improve the cell fitness, contemporary maintaining the use of the rapamycin. Evaluating the expansion protocols available in literature, we identified 3 main variables: the amount of stimulation beads, the IL2 concentration and the number of stimulation steps. For this reason, we evaluated the efficacy of various combinations of these 3 elements in promoting the Treg expansion starting from CD4+CD25+cells, contemporary preserving their phenotype and suppressive functions. Table 1 summarizes the combinations employed for the CAR-Treg culture. Table 1

[0843] Example 9.1 - Impact of different combinations of IL-2 Concentration and anti-CD3 / CD28 bead amount on the Expansion, Phenotype and Suppressive Function of CAR-Tregs

[0844] The fundamental role of IL2 in Treg development and survival has been extensively demonstrated. To identify the optimal conditions supporting the in vitro culture of CAR-Tregs, we decided to start by culturing them with a higher IL-2 concentration compared to our standard protocol. Healthy Donors’ CD4+CD25+Tregs cells were isolated and stimulated with anti-CD3 / CD28 stimulation beads at a 1:3 Treg-to-beads ratio. CAR-Tregs were cultured in the presence of rapamycin and either 500 ll / rnl (3:1 500, standard protocol) or 1000 ll / rnl of IL2 (3:1 1000). On day +14, CAR-Tregs cultured with 500 or 1000 ll / rnl of IL2 showed a similar expansion rate (Figure 19A, mean Fl ± SD, standard 27.39±22.1; 1:3 1000 IL229.6+1Q.QQ).

[0845] The percentage of transduction was comparable between the two groups (Figure 19B, mean ±SD, Standard 66.2±6.82; 1:3 1000 IL2 65.73+7.03). The purity of the CAR-Treg products (assessed as percentage of CD4+CD25+CD127_FoxP3+cells) was high and comparable (Figure 19C, mean±SD; Standard 63.2±4.8; 1:3 1000 IL2 63.32±5.5). Based on the expression of CD45RA and CD62L, we evaluated the Treg memory phenotype of the two cellular products. As shown in Figure 19D, central memory T cells (Tern) represented the most abundant memory subset in both conditions (mean±SD; Standard 81 ,4±4.94%; 1:3 1000 IL2 80±6.1%). Stem memory and effector memory T cells were equally represented, each ill constituting approximately 10% of the population (mean±SD, Tscm Standard 9.2+2.4%; Tscm 1:3 1000 IL2 10.1±4.12%; Tem Standard 9±6.25%; 1:3 1000 IL29.3±5.92%).

[0846] Cellular exhaustion could be a contributing factor affecting the functionality and therapeutic efficacy of CAR-Tregs. While exhausted conventional T cells are characterized by a high expression of exhaustion markers, reduced proliferative potential, and diminished effector functions, the exhaustion of Tregs remains controversial. Indeed, regulatory T cells naturally express high levels of the several exhaustion markers, like CTLA-4 and TIGIT, to exert their suppressive functions, thus complicating the assessment of their exhaustion state. Lamarche at al. explored the CAR-Treg exhaustion state by chronically stimulating engineered cells with a constitutively active anti-GD2 CAR molecule, able to continuously provide activation signals. After 2 weeks of culture, anti-GD2 CAR-Tregs showed an impaired functionality with lower suppressive functions compared to unmanipulated Tregs and upregulated some inhibitor receptors, like Leukocyte-Activation Gene 3 (LAG-3), T-cell Immunoglobulin and Mucin domain 3 (TIMS), Programmed cell Death (PD-1), GITR and 4-1 BB (Lamarche et al., 2023, PNAS).

[0847] To characterize our Fox19CAR-Treg exhaustion profile at the end of the two different culture conditions, a new multiparametric flow cytometry panel was established. On day +21 , we analyzed the expression of several inhibitory receptors on CAR-Tregs, finding no differences between the two cellular products (Figure 19E). Once assessed the expansion rate and the phenotype of the two CAR-Treg groups cultured with the different IL2 concentrations, we wanted to assess their suppressive capacities upon a polyclonal stimulation. Only at 1 :1 Treg- Tconv ratio there is significant functional advantage of cells cultured with 500 U / ml compared to 1000 U / ml in suppressing PBMCs proliferation (Figure 19F, p-value < 0.05).

[0848] Therefore, we can conclude that CAR-Treg cells do not benefit from higher IL-2 concentrations. Although no difference in terms of phenotype was observed between the two groups, higher IL-2 doses interfered with the polyclonal suppressive activity.

[0849] Since Tregs have a high affinity for IL2 due to the expression of CD25, the high affinity alphachain of the IL2 receptor, at high levels and considering the detrimental effect of higher doses of IL2 on their suppressive capacities, we wanted to explore the CAR-Treg expansion and functionality when cultured with a lower IL2 amount.

[0850] Isolated CD4+CD25+Tregs were stimulated with a bead-to-Treg ratio of 3:1 in the presence of rapamycin and either 250 or 500 U / ml of IL2. After 14 days, the expansion rate was not significantly improved by a lower IL2 concentration (Figure 20A, mean±SD, Fold increase of 3:1 250 IL2 24.18±12.96; 3:1 500 IL2 25.01±8.88; n=5). The transduction (Figure 20B mean±SD; %CAR cells 1:3 250 IL2 68.8±20.53%; 1:3 500 IL2 66.68±19.44%; n=5) and the percentage of CD4+CD25+CD127’FoxP3+cells (mean±SD; purity of 3:1 250 IL2 69±13.22; 3:1 500 IL2 64±20.13; n=5) were consistently high and comparable between the two conditions (Figure 20C). Phenotypically, the two CAR-Treg products showed a similar composition of the memory compartment and a comparable expression of the exhaustion markers at day +21 (Figure 20D-E).

[0851] When functionally tested, preliminary data suggest that CAR-Treg cells cultured with 250 ll / rnl I L-2 exhibit a stronger suppression capability compared to those cultured with 500 ll / rnl. These findings suggest that while IL2 concentration does not significantly change the phenotype or expansion potential of CAR-Tregs, lower IL2 (250 ll / rnl) appears to enhance their suppressive functions. This further supports the idea that fine-tuning IL-2 concentration is critical for optimizing CAR-Treg in vitro culture.

[0852] Subsequently, we focused on discriminating the impact of different IL2 doses coupled with a lower amount of stimulatory anti-CD3 / CD28 beads. To do this, isolated CD4+CD25+Tregs were stimulated with a 1 :1 Treg-to-bead ratio in the presence of rapamycin and either 500 ll / rnl (500 IL2 1:1) or 1000 ll / rnl of IL2 (1000 IL2 1:1). No differences were detectable between the two groups in terms of fold increase (mean Fl ± SD, Fold increase of 3:1 500 !L220.28±.06; 1:1 500 !L2 21.8+4.66' 1:1 1000 IL2 16.99+6.22, n=4), efficiency of transduction (mean±SD; %CAR of 3:1 500 IL287.35±3.22%; 1:1 500 IL2 84.28±5.91 %; 1:1 1000 IL286.53±5%; n=4) and Treg purity (mean purity±SD; 3:1 500 IL258±10.48%; 1:1 500 IL258.78±25%; 1:1 1000 IL2 50.33±23.26%; n=4) (Figure 21A-C). The two CAR-Treg products showed a similar memory Treg phenotype, composed mainly by Tern (mean% Tcm±SD Standard 71.1±11.3%; 1:1 500 IL2 72+14.7%' 1 :1 1000 IL2 64.4±13.4%, n=4), whereas Tscm and Tern accounted for about 10% and 14.4% of the cells, respectively. At day +21 , we evaluated the expression of the inhibitory receptors without highlighting significant differences between the two conditions. We then evaluated the functional profile of engineered Tregs upon polyclonal stimulation. No differences were observed between the tested items in terms of suppression abilities against proliferating conventional T lymphocytes (Figure 21 F).

[0853] In conclusion, these data highlight the essential role covered by IL2 in culturing CAR-Treg cells and suggest that too high IL2 concentrations (e.g. 1000U / ml) might affect CAR-Treg functionality. By comparing different IL2 dosages we determined which conditions best preserve the regulatory properties of Fox19CAR-Treg cells. Additionally, we found that a lower Treg-to-bead ratio is sufficient to effectively stimulate Treg cells, challenging the standard use of a 1 :3 ratio. This reduced bead ratio offers cost-efficiency gain without compromising cell activation. Several Treg expansion protocols reported in literature encompass a second stimulation at day +9. To evaluate the impact of such a procedure together with different amounts of IL2, at day +0 we stimulated isolated CD4+CD25+Tregs with a bead-to-Treg ratio of 1 :1 in the presence of rapamycin and either 500 (500 IL2R) or 1000 ll / rnl of IL2 (1000 IL2R). These conditions were received a second round of stimulation at day +9 again with a Treg-to-bead ratio of 1 :1. As control, we employed a group of CAR-Tregs treated with our standard expansion protocol comprising a bead-to-Treg ratio of 3:1 , rapamycin and IL2 500 ll / rnl.

[0854] Compared to the standard control, restimulated CAR-Tregs culture with IL2 500 ll / rnl showed a greater expansion rate (Figure 22A; mean fold increase of 115.2±51.36; p-value 0.0021), whereas 1000 IL2 R CAR-Tregs showed a trend (mean fold increase of 87.42±20.77; p-value 0.0232). The transduction efficiency (Figure 22B, mean %CAR±SD; Standard 80.14±5.37%; 1: 1R 500 IL2 74.88±7.81 %; 1: 1R 1000 IL2 80.32±5.19%) and the Treg purity (Figure 22C, mean purity±SD; Standard 68.52±30.68%; 1: 1R 500 IL2 82.52±7.54%; 1: 1R 1000 IL2 85.2±3.82%) were comparable and consistently high across the conditions (Figure 22C). Moreover, the groups didn’t show major differences in the Treg memory phenotype (Figure 22D) and suppressive function (Figure 22F).

[0855] The analysis of the CAR-Treg exhaustion profile at day +21 revealed a significant Killer cell Lectin like Receptor G1 (KLRG1) decrease in the restimulated CAR-Treg groups compared to the standard (mean MFI±SD; Standard 274+37 vs. 1 :1 R 500IL2 188±48 p-value 0.016; Standard vs. 1 :1 R 1000IL2 197±45 p-value 0.028). Similar results were observed for CTLA4, whose expression is decreased in the restimulated CAR-Treg groups compared to the standard control (mean MFI±SD; Standard 3613±933 vs. 1 :1 R 500IL2 2406±327 p-value 0.0201 ; Standard vs. 1 :1 R 10001 L2 2470±482 p-value 0.0269).

[0856] Although not statistically significant, CAR-Tregs culture with the standard protocol showed trend toward a higher expression of Programmed cell Death 1 (PD1) compared to the restimulated conditions (vs. 1:1R 500IL2, p-value 0.07; vs. 1:1R 1000IL2 p-value 0.1027).

[0857] Considering the improved CAR-Treg functionality, we wanted to explore the potential benefits of combining together two stimulation steps with a lower amount of anti-CD3 / CD28 stimulation beads and a lower IL2 concentration. To evaluate the impact of this combination, at day +0 we stimulated isolated CD4+CD25+ Tregs with a 1 :1 bead-to-Treg ratio in the presence of rapamycin and 250 ll / rnl of IL2 (1 :1 R 250 IL2). Then, a subgroup of cells received a second round of stimulation with a 1 : 1 ratio at day +9. A group of CAR-T regs treated with our standard expansion protocol comprising a bead-to-Treg ratio of 3:1 , rapamycin and IL2 500 ll / rnl was employed as a control. Compared to the standard control, restimulated CAR-Tregs culture with 250 ll / rnl of IL2 showed a greater expansion rate despite the high variability between donors (Figure 23A; mean fold increase of 84.16±50.51 ; p-value 0.0346). Whereas 1 :1 250 IL2 CAR-Treg group showed a fold increase comparable to the standard control (mean fold increase of 31.79±13.92). The transduction efficiency (Figure 23B, mean %CAR±SD; Standard 83±5.66%; 1:1 250 IL2 79.94±7.28%; 1: 1R 250 IL2 79.3±6.27%) and the Treg purity (mean purity±SD; Standard 73.38±10%; 1:1 250 IL270±17.98%; 1: 1R 250 IL264.32±17.95%) were comparable and consistently high across the conditions (Figure 23C). The low ratio of Treg- beads associated with low IL2 concentration show reduced percentages of CAR-Treg cells with a central memory phenotype compared to the standard control (Figure 23D, mean Tcm±SD; Standard 66.82±8.3%; 1:1 250 IL2 51.58±9.19%; 1: 1R 250 IL2 71.18±9%). The groups didn’t show major differences in the exhaustion phenotypes (Figure 23E) and during functional evaluation (Figure 23F).

[0858] EMBODIMENTS

[0859] Various preferred features and embodiments of the present invention will now be described with reference to the following numbered paragraphs (paras).

[0860] 1. A lentiviral vector comprising a construct comprising from 5’ to 3’:

[0861] (a) a first nucleotide sequence encoding FoxP3;

[0862] (b) a second nucleotide sequence encoding a cleavable linker; and

[0863] (c) a third nucleotide sequence encoding a CD19 CAR.

[0864] 2. The lentiviral vector according to para 1 , wherein the cleavable linker is T2A.

[0865] 3. The lentiviral vector according to para 1 or para 2, wherein the CD19 CAR comprises an antigen binding domain comprising a single-chain variable fragment (scFv).

[0866] 4. The lentiviral vector according to any one of the preceding paras, wherein the CD19 CAR comprises:

[0867] (a) a CD28, a CD8, and / or a CD4 transmembrane domain;

[0868] (b) an I gG 1 hinge, CD8a spacer, LNGFR spacer or mCH2CH3 spacer; and / or

[0869] (c) a CD28 and / or a 4-1 BB co-stimulatory domain. 5. The lentiviral vector according to any one of the preceding paras, wherein the CD19 CAR comprises a CD28 transmembrane domain, an lgG1 hinge, a CD28 co-stimulatory domain and a CD3 intracellular signalling domain.

[0870] 6. The lentiviral vector according to any one of the preceding paras, wherein the lentiviral vector further comprises a hPGK promoter operably linked to the construct.

[0871] 7. The lentiviral vector according to any one of the preceding paras, wherein the first nucleotide sequence comprises a sequence having at least 70% sequence identity to the sequence as set forth in SEQ ID NO: 1.

[0872] 8. A cell comprising a lentiviral vector according to any one of the preceding paras.

[0873] 9. A population of cells comprising a lentiviral vector according to any one of paras 1-7.

[0874] 10. The cell according to para 8 or the population of cells according to para 9, wherein the cell or population of cells has been transduced by the lentiviral vector.

[0875] 11. A method of producing a population of CD19 CAR-Treg cells, comprising the steps of:

[0876] (a) providing a population of T cells;

[0877] (b) activating the population of T cells;

[0878] (c) transducing the population of T cells with a lentiviral vector according to any one of the preceding paras; and

[0879] (d) culturing the population of T cells in the presence of IL-2 and rapamycin and under conditions suitable for the expression of the transgene.

[0880] 12. The method according to para 11 , wherein the population of T cells in step (a) comprises Tconv cells.

[0881] 13. The method according to para 11 or para 12, wherein the population of T cells in step (a) has the phenotype CD4+CD25+, CD4+CD25_or is a mixture comprising CD4+CD25+and CD4+CD25- cells.

[0882] 14. The method according to any one of paras 11-13, wherein step (b) comprises stimulating the population of T cells with anti-CD3 and anti-CD28 antibodies.

[0883] 15. The method according to any one of paras 11-14, wherein step (d) comprises culturing the population of T cells in cell culture medium comprising rapamycin and adding IL-2 to the cell culture medium one or more time. 16. The method according to para 15, wherein step (d) comprises supplementing the cell culture medium with IL-2 on days 3, 6, 9 and 12 after activating the population of T cells in step (b).

[0884] 17. The method according to any one of paras 11-16, wherein the population of CD19 CAR-Treg cells has the phenotype CD3+CD4+CD25+CD127'FoxP3+.

[0885] 18. A population of CD19 CAR-Treg cells obtained or obtainable by the method according to any one of paras 11-17.

[0886] 19. The cell according to para 8, the population of cells according to para 9, or the population of CD19 CAR-Treg cells according to para 18 for use in the treatment and / or prevention of an autoimmune disease.

[0887] 20. The population of CD19 CAR-Treg cells for use according to para 19, wherein the autoimmune disease is selected from the group consisting of inflammatory arthritis (such as rheumatoid arthritis), type 1 diabetes mellitus, multiples sclerosis (MS), psoriasis, inflammatory bowel diseases, systemic lupus erythematosus (SLE), vasculitis, allergic inflammation (such as allergic asthma, atopic dermatitis, and contact hypersensitivity), Graves' disease, Hashimoto's thyroiditis, celiac disease, Crohn's disease, ulcerative colitis, Guillain- Barre syndrome, primary biliary sclerosis / cirrhosis, sclerosing cholangitis, autoimmune hepatitis, Raynaud's phenomenon, scleroderma, Sjogren's syndrome, Goodpasture's syndrome, Wegener's granulomatosis, polymyalgia rheumatica, temporal arteritis / giant cell arteritis, chronic fatigue syndrome (CFS), autoimmune Addison's Disease, ankylosing spondylitis, acute disseminated encephalomyelitis, antiphospholipid antibody syndrome, aplastic anemia, idiopathic thrombocytopenic purpura, Myasthenia gravis, opsoclonus myoclonus syndrome, optic neuritis, Ord's thyroiditis, pemphigus, pernicious anaemia, Reiter's syndrome, Takayasu's arteritis, warm autoimmune hemolytic anemia, Wegener's granulomatosis, fibromyalgia (FM), vitiligo, IPEX, graft versus host disease, undifferentiated connective tissue disease, mixed connective tissue disease, autoinflammatory syndromes (such as Familial mediterranean fever), still's disease, and adult-onset still's disease.

[0888] 21. The population of CD19 CAR-Treg cells for use according to para 19 or para 20, wherein the autoimmune disease is systemic lupus erythematosus.

[0889] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims

CLAIMS1. A lentivi ral vector comprising a construct comprising from 5’ to 3’:(a) a first nucleotide sequence encoding FoxP3;(b) a second nucleotide sequence encoding a cleavable linker; and(c) a third nucleotide sequence encoding a CD19 CAR.

2. The lentiviral vector according to claim 1 , wherein the cleavable linker is T2A.

3. The lentiviral vector according to claim 1 or 2, wherein the CD19 CAR comprises:(a) a CD28, a CD8, and / or a CD4 transmembrane domain;(b) an I gG 1 hinge, CD8a spacer, LNGFR spacer or mCH2CH3 spacer; and / or(c) a CD28 and / or a 4-1 BB co-stimulatory domain.

4. The lentiviral vector according to any one of the preceding claims, wherein the first nucleotide sequence comprises a sequence having at least 70% sequence identity to the sequence as set forth in SEQ ID NO: 1.

5. A cell comprising a lentiviral vector according to any one of the preceding claims.

6. A population of cells comprising a lentiviral vector according to any one of claims 1-4.

7. A method of producing a population of CD19 CAR-Treg cells, comprising the steps of:(a) providing a population of T cells;(b) activating the population of T cells;(c) transducing the population of T cells with a lentiviral vector according to any one of claims 1-4; and(d) culturing the population of T cells in the presence of IL-2 and rapamycin and under conditions suitable for the expression of the transgene.

8. The method according to claim 7, wherein the population of T cells in step (a) comprises Tconv cells.

9. The method according to claim 7 or claim 8, wherein the population of T cells in step (a) has the phenotype CD4+CD25+, CD4+CD25_or is a mixture comprising CD4+CD25+and CD4+CD25- cells.

10. The method according to any one of claims 7-9, wherein step (b) comprises stimulating the population of T cells with anti-CD3 and anti-CD28 antibodies.11 . The method according to any one of claims 7-10, wherein step (d) comprises culturing the population of T cells in cell culture medium comprising rapamycin and adding IL-2 to the cell culture medium one or more time.

12. The method according to claim 11 , wherein step (d) comprises supplementing the cell culture medium with IL-2 on days 3, 6, 9 and 12 after activating the population of T cells in step (b).

13. The method according to any one of claims 7-12, wherein the population of CD19 CAR- Treg cells has the phenotype CD3+CD4+CD25+CD127’FoxP3+.

14. A population of CD19 CAR-Treg cells obtained or obtainable by the method according to any one of claims 7-13.

15. The cell according to claim 5, the population of cells according to claim 6, or the population of CD19 CAR-Treg cells according to claim 14 for use in the treatment and / or prevention of an autoimmune disease, optionally systemic lupus erythematosus.

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