Engineered regulatory T cells

Abstract

The present invention provides modified regulatory T cells that can recognize liver cells, treat and / or prevent transplant rejection or immune-mediated damage, and promote liver cell regeneration. [Solution] The present invention provides modified regulatory T cells (Tregs) comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen-recognition domain that specifically binds to the asialoglycoprotein receptor (ASGR). The present invention also provides a method for promoting liver tissue repair and / or regeneration in a subject, the method comprising administering to the subject modified Tregs comprising a CAR or a pharmaceutical composition comprising the modified Tregs, wherein the CAR comprises a liver-specific antigen-recognition domain.

Description

[Technical Field]

[0001] The present invention relates to modified regulatory T cells and the therapeutic use of such cells in immune-mediated liver damage, particularly modified regulatory T cells that can recognize liver cells, treat and / or prevent transplant rejection or immune-mediated damage, and promote liver cell regeneration. [Background technology]

[0002] The liver is a vital organ that supports nearly all other organs in the body. Its strategic location and critical function make it susceptible to disease, particularly immune-mediated damage. Globally, there are approximately 2 million deaths due to liver disease each year, with 1 million due to complications of cirrhosis and 1 million due to viral hepatitis and hepatocellular carcinoma. Currently, cirrhosis is the 11th leading cause of death worldwide. It is among the top 20 causes of disability-adjusted life years and years of life lost due to premature death, accounting for 1.6% and 2.1% of the global burden, respectively (Asrani, SK, et al., 2018. Journal of Hepatology, 70, pp. 151-171).

[0003] Common autoimmune liver diseases, including primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), damage the liver and lead to cirrhosis. The disease burden in PBC is often manifested as reduced quality of life, poor health, and significant symptoms such as fatigue, itching, and depression. PSC is a risk factor for cholangiocarcinoma (CTC), gallbladder cancer, and colorectal cancer, and may contribute to premature mortality. Patients with PSC have a fourfold increased risk of death and a 398-fold increased risk of developing CTC compared with the general population (Asrani, SK, et al., 2018. Journal of Hepatology, 70, pp. 151-171).

[0004] Cirrhosis can also result from inflammatory liver disorders, such as viral hepatitis or steatohepatitis. In 2010, there were 300,000 deaths due to viral hepatitis, a 46% increase from 1990. Viral hepatitis was the 10th leading cause of death in 1990, but increased to become the 7th leading cause by 2013. In 2015, there were 1.34 million deaths due to viral hepatitis-related illnesses, a number comparable to tuberculosis (1.37 million) and exceeding HIV (1.06 million) and malaria (0.44 million) (Asrani, SK, et al., 2018. Journal of Hepatology, 70, pp. 151-171).

[0005] Transplant tolerance and survival are also important issues during liver transplantation. Acute cellular rejection occurs in 15–25% of liver transplant recipients receiving tacrolimus-based immunosuppressive therapy and typically improves with the use of steroids. While acute rejection usually responds well to treatment, chronic rejection presents a challenging situation, and a significant proportion of patients do not respond to increased immunosuppression. Chronic rejection often leads to retransplantation or death (Choudhary, NS, et al., 2017. Journal of Clinical and Experimental Hepatology, 7(4), pp.358–366). Five-year mortality rates are lowest in patients undergoing liver transplantation for primary biliary cholangitis. Recurrent diseases include autoimmune hepatitis, primary biliary cirrhosis, and primary sclerosing cholangitis (Hirschfield, GM, et al., 2009. BMJ, 338, p.b1670).

[0006] Liver transplants can also cause immune-mediated damage caused by graft-versus-host disease, which is reported to occur in 0.1% to 2% of cases and has a mortality rate of greater than 75% (Akbulut, S., et al., 2012. World journal of gastroenterology, 18(37), p.5240).

[0007] The liver possesses unique regenerative capabilities that can help alleviate immune-mediated injury. However, in the setting of chronic immune-mediated injury, liver cells undergo senescence and their regenerative capacity declines, contributing to the development of liver failure and increasing patient morbidity and mortality (Aravinthan, AD and Alexander, GJ, 2016. Journal of hepatology, 65(4), pp.825-834). Furthermore, liver regeneration in response to immune-mediated injury can be detrimental, potentially leading to cirrhosis and liver cancer (Michalopoulos, GK, 2017. Hepatology, 65(4), pp.1384-1392).

[0008] Therefore, a therapy that can treat and / or prevent immune-mediated damage and promote liver regeneration would have great potential for the treatment of liver disease.Therefore, there is a need for a therapy that can treat and / or prevent immune-mediated damage and promote liver regeneration. Summary of the Invention

[0009] In a first aspect, the present invention provides an engineered regulatory T cell (Treg) comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen recognition domain that specifically binds to the asialoglycoprotein receptor (ASGR).

[0010] In some embodiments, the CAR comprises an endodomain comprising a STAT5 association motif and a JAK1 and / or JAK2 binding motif, hi some other embodiments, the endodomain comprises a JAK3 binding motif and / or does not comprise a STAT3 association motif.

[0011] Suitably, the CAR endodomain does not comprise the amino acid sequence YXXQ (SEQ ID NO: 133). Suitably, the IL2Rβ portion of the CAR endodomain does not comprise the amino acid sequence YXXQ (SEQ ID NO: 133).

[0012] The modified Tregs of the invention with STAT5 signaling may be particularly effective in providing a survival advantage to the modified CAR-Tregs after antigen recognition relative to the subject's general T cell population. In particular, in situations where the use of immunosuppressive agents reduces IL-2 acquisition, such as in transplantation, STAT5 signaling in the CAR-Tregs provides an additional survival and functional advantage to the Tregs of the invention in an otherwise hostile microenvironment.

[0013] In another aspect, the present invention provides pharmaceutical compositions comprising the modified Tregs of the present invention.

[0014] In another aspect, the invention provides an engineered Treg or a pharmaceutical composition according to the invention for use in inducing tolerance to liver transplant tissue in a subject, or for use in treating and / or preventing liver transplant rejection, hepatic graft-versus-host disease (GvHD), autoimmune liver disease, or inflammatory liver damage in a subject. In another aspect, the present invention provides a method for inducing tolerance to liver transplant tissue in a subject, or for treating and / or preventing liver transplant rejection, hepatic graft-versus-host disease (GvHD), autoimmune liver disease, or inflammatory liver damage in a subject, said method comprising the step of administering to said subject an engineered Treg or pharmaceutical composition of the present invention.

[0015] In another aspect, the present invention relates to the use of modified Tregs of the present invention for the manufacture of a medicament for inducing tolerance to a liver transplant in a subject, or for the manufacture of a medicament for treating and / or preventing liver transplant rejection, hepatic graft-versus-host disease (GvHD), autoimmune liver disease, or inflammatory liver damage in a subject. In another aspect, the present invention provides an engineered regulatory T cell (Treg) comprising a chimeric antigen receptor (CAR), or a pharmaceutical composition comprising said engineered Treg, for use in repairing and / or regenerating liver tissue in a subject, wherein said CAR comprises a liver-specific antigen recognition domain.

[0016] In another aspect, the present invention provides a method of promoting liver tissue repair and / or regeneration in a subject, the method comprising administering to the subject engineered regulatory T cells (Tregs) comprising a chimeric antigen receptor (CAR), or a pharmaceutical composition comprising said engineered Tregs, wherein said CAR comprises a liver-specific antigen recognition domain.

[0017] In another aspect, the present invention provides use of modified Tregs comprising a chimeric antigen receptor (CAR), wherein the CAR comprises a liver-specific antigen recognition domain, in the manufacture of a medicament for promoting repair and / or regeneration of liver tissue in a subject.

[0018] In another aspect, the present invention provides modified regulatory T cells (Tregs) comprising a chimeric antigen receptor (CAR), or a pharmaceutical composition comprising said modified Tregs, for use in the treatment and / or prevention of cirrhosis, acute liver failure, or acute-onset chronic liver failure in a subject, wherein said CAR comprises a liver-specific antigen recognition domain.

[0019] In another aspect, the present invention provides a method for treating and / or preventing cirrhosis, acute liver failure, or acute-onset chronic liver failure in a subject, the method comprising administering to the subject engineered regulatory T cells (Tregs) comprising a chimeric antigen receptor (CAR), or a pharmaceutical composition comprising the engineered Tregs, wherein the CAR comprises a liver-specific antigen recognition domain.

[0020] In another aspect, the present invention provides use of modified Tregs comprising a chimeric antigen receptor (CAR), wherein the CAR comprises a liver-specific antigen recognition domain, in the manufacture of a medicament for treating and / or preventing cirrhosis, acute liver failure, or acute-onset chronic liver failure in a subject.

[0021] The present invention also provides chimeric antigen receptors (CARs), which comprise an antigen-recognition domain that specifically binds to the asialoglycoprotein receptor (ASGR) and further comprises a CD3 zeta signaling domain. In some embodiments, the CAR comprises an endodomain comprising a STAT5-associated motif and a JAK1 and / or JAK2-binding motif. In some other embodiments, the endodomain comprises a JAK3-binding motif and / or does not comprise a STAT3-associated motif. Thus, in one embodiment, the present invention provides a CAR, which comprises an antigen-recognition domain that specifically binds to ASGR and comprises a STAT5-associated motif and a JAK1 and / or JAK2-binding motif, preferably a JAK3-binding motif but does not comprise a STAT3-associated motif.

[0022] The present invention also provides a polynucleotide, nucleic acid, or vector encoding a CAR of the present invention.

[0023] The present invention further provides a method of producing modified Tregs according to the present invention, said method comprising: (i) isolating or obtaining a cell-containing sample from a subject; and (ii) transducing or transfecting the cell-containing sample with a polynucleotide, nucleic acid, or vector encoding a CAR to obtain a population of modified cells; The cell-containing sample contains Tregs and / or Tregs are enriched and / or generated from the cell-containing sample before or after step (ii). [Brief explanation of the drawings]

[0024] [Figure 1]Figure 1 - Example design of anti-ASGR1 CAR construct. Schematic diagram of an example anti-ASGR1 CAR construct. (A) An anti-ASGR1 CAR construct comprising an ASGR1 VH antigen recognition domain, a CD8α hinge domain, CD28TM, a CD28 signaling domain, a CD3z signaling domain, a P2A cleavage domain, and eGFP. (B) A schematic diagram of an example anti-ASGR1 CAR construct comprising an ASGR1 VH antigen recognition domain, a CD28 hinge domain, CD28TM, a CD28 signaling domain, a CD3z signaling domain, a P2A cleavage domain, and eGFP. [Figure 2] Figure 2 - Generation of anti-ASGR1 CAR-Tregs. Histograms showing the generation of anti-ASGR1 CAR-Tregs by FACS analysis. (A) Untransduced Tregs. (B) Transduced Tregs. CD4+CD25hiCD127 cells were isolated and activated with anti-CD3 / CD28 beads. Two days after activation, Tregs were transduced with a lentivirus containing the ASGR1-CAR and a GFP reporter gene. Transduced and untransduced Tregs were cultured for 10 days, and the efficacy of transduction was assessed by measuring GFP. [Figure 3] Figure 3 - Confirmation of ASGR1 expression in HepG2 cell line. Histogram showing ASGR1 levels in K562 and HepG2 cells by FACS analysis. (A) K562 cells (B) HepG2 cells [Figure 4A]Figure 4 - Assessment of antigen specificity of anti-ASGR1 CAR-Tregs. FACS histograms showing CD69 expression in response to culture with (i) medium alone, (ii) HepG2 cells, and (iii) anti-CD3 / CD28 beads. (A) Transduced Tregs (GFP+). (B) Transduced Tregs (GFP-). (C) Untransduced Tregs (GFP-). In the presence of medium alone, no upregulation of CD69 is observed. Following culture with HepG2 cells, only GFP+ transduced Tregs (but not GFP-negative transduced or untransduced cells) upregulate CD69. In contrast, when cultured with nonspecific anti-CD3 / CD28 bead stimulation, all cells upregulate CD69. [Figure 4B] (B) Transduced Tregs (GFP-). [Figure 4C] (C) Untransduced Tregs (GFP-). [Figure 5] Figure 5 - Evaluation of antigen-specific suppressive function of anti-ASGR1 CAR-Treg (1:1 Treg:Teff). Histogram showing proliferation of activated effector CD4+CD25- T cells by FACS analysis. Suppression assay shows the effect of anti-ASGR1 CAR-Treg on the proliferative capacity of effector T cells stained with proliferation dye and pre-activated with anti-CD3 / CD28 beads. Anti-ASGR1 CAR Treg could effectively suppress proliferation of conventional T cells in response to HepG2 stimulation. [Figure 6] Figure 6 - Evaluation of antigen-specific suppressive function of anti-ASGR1 CAR-Tregs (1:1, 1:2, and 1:5 Treg:Teff). Suppression assay results for Treg:Teff ratios of 1:1, 1:2, and 1:5. Suppression assay shows the effect of anti-ASGR1 CAR-Tregs on the proliferative capacity of effector T cells stained with proliferation dye and pre-activated with anti-CD3 / CD28 beads. Anti-ASGR1 CAR Tregs were able to effectively suppress the proliferation of normal T cells in response to HepG2 stimulation, but at a lower rate than untransduced Tregs. [Figure 7] Figure 7 - Example designs of anti-HLA.A2 IL2R CAR constructs. Schematic diagram of example anti-HLA.A2 CAR constructs containing various combinations of IL2R endodomains. (A) dCAR construct: HLA.A2scFv antigen recognition domain; CD28 hinge domain; CD28TM and eGFP. (B) CD28z construct: HLA.A2scFv antigen recognition domain; CD28 hinge domain; CD28TM; CD28 signaling domain; CD3z signaling domain and eGFP. (C) IL2R construct 1: HLA.A2scFv antigen recognition domain; CD28 hinge domain; CD28TM; CD28 signaling domain; truncated IL2RB endodomain; CD3z signaling domain and eGFP. (D) IL2R construct 1: HLA.A2scFv antigen recognition domain; CD28 hinge domain; CD28TM; CD28 signaling domain; truncated IL2RG; truncated IL2RB endodomain; CD3z signaling domain and eGFP. (E) IL2R construct 1: HLA.A2scFv antigen recognition domain; CD28 hinge domain; CD28TM; CD28 signaling domain; truncated IL2RB endodomain; CD3z signaling domain; FP2A cleavage domain and eGFP. [Figure 8] Figure 8 - Generation of anti-HLA.A2 IL2R CAR-Tregs. Schematic diagram showing the generation and expansion of anti-HLA.A2IL2RCAR-Tregs. (A) Isolated CD4+CD25hiCD127low cells were isolated and activated with anti-CD3 / CD28 beads. Three days after activation, Tregs were transduced with a lentivirus containing an HLA.A2-CAR and a GFP reporter gene. Fresh medium and 1000 IU / ml IL-2 were added every two days. Transduced and untransduced Tregs were cultured for 10 days, and GFP was measured to determine the effect of transduction. Tregs were further expanded using fresh anti-CD3 / CD28 beads. (B) Fold change expansion of untransduced Tregs or Tregs transduced with various CAR constructs at 10 days post-activation. [Figure 9] Figure 9 - Quantification of the effect of transduction of anti-HLA.A2 IL2R constructs over time. GFP expression was analyzed at different time points after cell activation for Tregs not transduced with the CAR construct and for Tregs transduced with the CAR construct. (A) Representative contour plot of GFP expression from HLA-A2 IL2R CAR Tregs 7 days after transduction. (B) Quantification of GFP+ CAR Tregs among viable CD4+ cells 7 days after transduction. (C) Quantification of GFP expression from HLA-A2 IL2R CAR Tregs over time. [Figure 10] Figure 10 - Quantification of cell surface expression of anti-HLA.A2 IL2R CAR construct on transduced Tregs. Membrane expression of the CAR construct on non-transduced and transduced Tregs was analyzed by PE-conjugated HLA-A*0201 / CINGVCWTV dextramer (Immudex, Copenhagen, Denmark). (A) Representative contour plot of GFP+Dextramer+CAR Tregs 7 days post-transduction. (B) Quantification of Dextramer+ cells among GFP+Tregs 7 days post-transduction. [Figure 11] Figure 11 - Phenotypic characterization of CAR Tregs after polyclonal cell expansion. Tregs were cultured and expanded for 15 days in the presence of anti-CD3 / CD28 activation beads and IL-2. Treg-associated markers FOXP3, HELIOS, CTLA4, and TIGIT were analyzed by FACS on untransduced and transduced Tregs to examine phenotypic lineage stability at day 15 of culture. [Figure 12A]Figure 12 - Evaluation of antigen specificity of anti-HLA.A2IL2RCAR Tregs. Untransduced and transduced Tregs were cultured for 18 hours in the presence of different stimuli. CD69 and CD137 activation markers were analyzed to examine specific versus nonspecific cell activation. (A) Representative contour plot showing CD69 expression in response to culture with K562 cells transduced with HLA.A1 or HLA.A2 molecules. GFP signal was used to select transduced Tregs. [Figure 12B] (B) Quantification of CD69 and CD137 expression on Tregs after 18 h of culture with medium alone (no stimulation), with anti-CD3 / CD28 beads (nonspecific stimulation), and with K562-HLA.A1 and K562-HLA.A2 cells. [Figure 12C] (C) Representative histogram showing CD69 expression on Tregs after 18 hours of culture with HLA.A1 and HLA.A2B cell lines using different cell-to-cell ratios. [Figure 13] Figure 13 - STAT5 phosphorylation analysis as an indicator of IL2R CAR signaling. Transduced CAR Tregs were placed in IL2-free medium overnight. STAT5 phosphorylation in Tregs was examined by FACS analysis after 10 and 120 minutes of culture in medium alone, with 1000 IU / ml IL-2, and in the presence of HLA.A2-Ig-based artificial APCs (generated according to the protocol described in DOI:10.3791 / 2801). (A) Representative contour plots showing GFP and phosphoSTAT5 expression in transduced CAR-Tregs after 10 minutes of culture in medium alone, with a 1:1 ratio of HLA.A2 beads, and with 1000 IU / ml IL-2. Histogram showing STAT5 phosphorylation in Tregs cultured for 120 minutes with a 1:1 ratio of HLA.A2 beads or with medium alone (unstimulated). [Figure 14]Figure 14-(i) Evaluation of Treg survival after non-specific activation and HLA.A2-specific activation with SEQ ID NOs: 11, 12, and 13, respectively, or their derivatives, in the absence of IL-2. CAR-transduced Tregs of different constructs were cultured with anti-CD3 / 28 activation beads and K562.A2-expressing cells in the absence of IL-2. Cell survival was examined 7 days after activation by FACS analysis. (A) Representative histogram of CAR-Tregs showing cell viability of GFP+ cells based on viability dye staining 7 days after activation without IL-2. (B) Percentage of surviving GFP+ Tregs after 7 days of culture with anti-CD3 / 28 beads and K562-HLA.A2 cells in the absence of IL-2 (*p<0.05, ANOVA analysis with Tukey's post hoc correction). [Figure 15] Figure 15 - Treg suppressive ability assay: Assessment of the immunomodulatory function of Tregs by analysis of modulation of costimulatory molecules on B cells. After co-culture with Tregs, B cells were analyzed for CD80 and CD86 expression to assess the ability of Tregs to reduce the expression of costimulatory molecules on antigen-presenting cells. A fixed number of live A2-expressing B cells (20K / well) were co-cultured overnight with titrated numbers of Treg products (A2-negative donors) (200, 100, 50, 25, 12.5K). FACS analysis of CD86 and CD80 costimulatory markers on B cells. [Figure 16] Figure 16 - Evaluation of the effect of preactivated, untransduced Tregs on in vitro albumin production by primary hepatocytes. Primary hepatocytes were cultured for 7 days alone or in the presence of preactivated regulatory T cells. Addition of regulatory T cells resulted in improved albumin secretion that could be detected in the supernatant. A similar effect was observed when hepatocytes were cultured in the presence of conditioned medium from cultures containing activated regulatory T cells, but in the absence of regulatory T cells. This figure shows that the addition of preactivated, untransduced Tregs increases albumin production by primary hepatocytes in vitro. [Figure 17]Figure 17 - Evaluation of the effect of preactivated, untransduced Tregs and effector T cells on in vitro albumin production by primary hepatocytes. Primary hepatocytes were cultured for 3 days alone, in the presence of preactivated regulatory T cells, preactivated effector T cells, or both. The addition of regulatory T cells resulted in improved albumin secretion, detectable in the supernatant. This was not observed when hepatocytes were cultured with effector T cells, which tended to decrease albumin levels. The beneficial effect of regulatory T cells on albumin levels was also observed when regulatory T cells were combined with preactivated effector T cells. This figure shows that the addition of preactivated, untransduced Tregs, but not effector T cells, increased albumin production by primary hepatocytes in vitro. [Figure 18] Figure 18 - Assessing the effect of anti-ASGPR CAR Tregs on in vitro albumin production by primary hepatocytes. Primary hepatocytes were cultured alone, in the presence of resting naive regulatory T cells, or in the presence of resting regulatory T cells lentivirally transduced to express anti-ASGPR CAR. Only Tregs bearing anti-ASGPR and thus capable of antigen-specific activation in response to hepatocyte-expressed ASGPR significantly affected albumin levels in the supernatant. This figure shows that the addition of preactivated anti-ASGPR CAR-expressing Tregs increased albumin production by primary hepatocytes in vitro, exceeding the levels achieved using untransduced Tregs.

[0025] Detailed Description The present invention provides modified Tregs comprising a CAR, which provides an activator signal to Tregs only when the CAR binds to a liver-specific antigen (e.g., ASGR), thus improving the retention, function, and survival of the modified Tregs, particularly in the liver microenvironment. The modified Tregs of the present invention increase or improve liver regeneration and tissue repair upon binding to a liver-specific antigen (e.g., ASGR).

[0026] Various preferred features and embodiments of the present invention will now be described by way of non-limiting example. It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including," "includes," or "containing," and "contains" and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps. The terms "comprising," "comprises," and "comprised of" also include the term "consisting of."

[0027] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application, and nothing herein should be construed as an admission that such publications constitute prior art to the appended claims.

[0028] This disclosure is not limited by the exemplary methods and materials disclosed herein, and methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise specified, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation.

[0029] Engineered regulatory T cells (Tregs) Regulatory T cells (Tregs) are immune cells with suppressive functions that control cytopathic immune responses and are essential for maintaining immune tolerance.

[0030] As used herein, the term Treg refers to T cells with immunosuppressive function. Preferably, "immunosuppressive function" refers to the ability of Treg to reduce or inhibit one or more of the numerous physiological cellular events promoted by the immune system in response to stimuli such as pathogens, antigens (e.g., alloantigens, or autoantigens). Examples of such events include proliferation of normal T cells (Tconv) and secretion of pro-inflammatory cytokines. Any of these events may be used as an indicator of the strength of the immune response. A relatively weaker immune response mediated by Tconv in the presence of Tregs may indicate the ability of Tregs to suppress the immune response. For example, a relative decrease in cytokine secretion indicates a weakened immune response and, therefore, the ability of Tregs to suppress the immune response. Tregs can also suppress immune responses by regulating the expression of costimulatory molecules on antigen-presenting cells (APCs), such as B cells, dendritic cells, and macrophages. The expression levels of CD80 and CD86 can be used to examine the suppressive ability of activated Tregs in vitro after coculture.

[0031] Assays for measuring the strength of immune responses and thereby the suppressive ability of Tregs are well known in the art. In particular, antigen-specific Tconv cells may be co-cultured with Tregs, and peptides of the corresponding antigen may be added to the co-culture to stimulate responses from Tconv cells. The degree of proliferation of Tconv cells and / or the amount of cytokine IL-2 secreted by Tconv cells in response to the addition of the peptide may be used as an indicator of the suppressive ability of the co-cultured Tregs.

[0032] The proliferation of antigen-specific Tconv cells co-cultured with Tregs of the invention may be 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 95%, or 99% less compared to the proliferation of the same Tconv cells cultured in the absence of Tregs of the invention.

[0033] Antigen-specific Tconv cells co-cultured with Tregs of the invention may express at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% less effector cytokines compared to corresponding Tconv cells cultured in the absence of Tregs of the invention.

[0034] The effector cytokine may be selected from IL-2, IL-17, TNFα, GM-CSF, IFN-γ, IL-4, IL-5, IL-9, IL-10, and IL-13.

[0035] Suitably, the effector cytokine may be selected from IL-2, IL-17, TNFα, GM-CSF, and IFN-γ.

[0036] Preferably, Tregs express the markers CD4, CD25, and FOXP3 (CD4 + CD25 + FOXP3 + "FOXP3" is an abbreviation for forkhead box P3 protein. FOXP3 is a member of the FOX protein family of transcription factors and functions as a master regulator of regulatory pathways in the development and function of regulatory T cells.

[0037] Marker levels may be determined by any method known to those skilled in the art, for example, by flow cytometry. Tregs also express CTLA-4 (cytotoxic T lymphocyte-associated molecule 4) and / or GITR (glucocorticoid-induced TNF receptor). Treg cells are present in peripheral blood, lymph nodes, and tissues.

[0038] Preferably, by using the cell surface markers CD4 and CD25 in the absence or combination of the surface protein CD127, which is expressed at low levels (CD4 + CD25 + CD127 - , or CD4 + CD25 + CD127 low , or CD4 + CD25 hi CD127 - , or CD4 + CD25 hi CD127 low ), Tregs may be identified. The use of such markers for the identification of Tregs is well known in the art and is described, for example, by Liu et al. (JEM; 2006; 203; 7(10); 1701-1711).

[0039] The Tregs are CD4 + CD25 + FOXP3 + T cells, or CD4 + CD25 hi FOXP3 + It may be a T cell.

[0040] The Tregs are CD4 + CD25 + CD127 - T cells, or CD4 + CD25 hi CD127 - The Treg may be a CD4 T cell. + CD25 + FOXP3 + CD127 - T cells, or CD4 + CD25 hi FOXP3 + CD127 - It may be a T cell.

[0041] The Treg may have a Treg-specific demethylation region (TSDR). The TSDR is an important methylation-sensitive element that regulates the expression of FOXP3 (Polansky, JK, et al., 2008. European journal of immunology, 38(6), pp.1654-1663). The Treg may be a natural Treg or a thymus-derived Treg, an adaptive Treg or a peripherally-derived Treg, or an in vitro-induced Treg (Abbas, AK, et al., 2013. Nature immunology, 14(4), pp.307-308). Preferably, the Treg is a CD4 + CD25 + FOXP3 + Helios + Neuropilin 1 + Further preferred examples of Treg include Tr1 cells (which do not express Foxp3 and produce a high level of IL-10); + FOXP3 + T cells; and γδFOXP3 + These include, but are not limited to, T cells.

[0042] Preferably, the Tregs are isolated from peripheral blood mononuclear cells (PBMCs) obtained from the subject. Preferably, the subject is a mammal, preferably a human. Preferably, the Tregs are matched (e.g., HLA-matched) to the subject to whom the modified Tregs are administered, or are autologous to the subject. Preferably, the subject to whom the modified Tregs are administered is a mammal, preferably a human. The Tregs may be generated ex vivo (ex vivo), either from the patient's own peripheral blood (first party), or in the setting of a hematopoietic stem cell transplant from donor peripheral blood (second party), or from peripheral blood from an unrelated donor (third party). Preferably, the Tregs are autologous to the subject to whom the modified Tregs are administered. In a preferred embodiment, the Tregs are isolated and matched (e.g., HLA-matched) from peripheral blood mononuclear cells (PBMCs) obtained from the subject, or are autologous to the subject to whom the modified Tregs are administered. Preferably, the Tregs are part of a population of Tregs. Suitably, the population of Tregs is at least 70% Tregs, for example at least 75%, 85%, 90%, 95%, 97%, 98%, or 99% Tregs. Such a population may be referred to as an "enriched Treg population" or "enriched Treg sample."

[0043] In some embodiments, Tregs may be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells into Tregs. As used herein, "normal T cells" or Tcon refers to T lymphocyte cells that express the αβ T cell receptor (TCR) and a co-receptor, which may be cluster of differentiation 4 (CD4) or cluster of differentiation 8 (CD8), and lack immunosuppressive function. Normal T cells reside in peripheral blood, lymph nodes, and tissues. Preferably, modified Tregs may be generated by introducing DNA or RNA encoding FOXP3 in addition to DNA or RNA encoding a CAR described herein by one of a number of means, including transduction using a viral vector or gene transfer using DNA or RNA on the same or a different vector. Alternatively, the modified Tregs may be generated by transducing CD4 in the presence of IL-2 and TGF-β.+ CD25 - FOXP3 - It may be produced from Tcon by in vitro culture of cells.

[0044] The Tregs may be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells into Tregs. The polynucleotide or vector of the invention may be introduced into the inducible progenitor cells or embryonic progenitor cells before or after differentiation into Tregs.

[0045] As used herein, "modified Treg" refers to a Treg that has been modified to contain or express a polynucleotide that is not naturally encoded by Treg, particularly the above-mentioned CAR. Methods for modifying Treg are well known in the art, including, but not limited to, genetic modification of Treg, such as transduction, e.g., retroviral or lentiviral transduction, gene transfer (e.g., transient transfection of DNA or RNA), e.g., lipofection, polyethylene glycol, calcium phosphate, and electroporation. Any suitable method may be used to introduce a nucleic acid sequence into cells.

[0046] Chimeric Antigen Receptor As used herein, a "chimeric antigen receptor" or "CAR" or "CARs" refers to an engineered receptor that confers antigen specificity to cells, in this case, Tregs. CARs are also known as artificial T cell receptors, chimeric T-cell receptors, or chimeric immunoreceptors. The CARs of the present invention comprise a binding domain specific to a liver antigen (e.g., ASGR), optionally a hinge domain, a transmembrane domain, and an endodomain (comprising an intracellular signaling domain and optionally one or more costimulatory domains). Typically, the CARs of the present invention may be expressed or present as a single polypeptide chain, for example, a single polypeptide chain comprising a binding domain specific to a liver antigen (e.g., ASGR), optionally a hinge domain, a transmembrane domain, and an endodomain (comprising an intracellular signaling domain and optionally one or more costimulatory domains). In particular, typically, the intracellular signaling domain and optionally one or more costimulatory domains may be present together on a single polypeptide chain. Thus, a CAR of the invention can comprise more than one polypeptide chain, e.g., a dual polypeptide or CAR system, although in certain embodiments, a CAR of the invention is not a dual CAR.

[0047] The polynucleotide encoding CAR can be introduced into Treg, for example, by using a retroviral vector.In this way, a large number of antigen-specific T cells can be generated by adoptive cell transfer.When CAR binds to target antigen, it transmits an activation signal to the Treg that expresses CAR.In this way, CAR directs the specificity of modified Treg to cells that express target antigen.

[0048] The Tregs of the present invention may comprise one or more different CARs of the present invention and may further comprise exogenous polynucleotides encoding other polypeptides.

[0049] antigen recognition domain The CAR of the present invention comprises an antigen recognition domain. As used herein, "antigen recognition domain" refers to the extracellular portion of the CAR, which determines the antigen binding ability of the CAR. In one embodiment of the present invention, the antigen recognition domain provides the CAR with the ability to bind to a liver-specific antigen, preferably the asialoglycoprotein receptor (ASGR). Thus, the antigen recognition domain targets a liver-specific antigen, preferably ASGR.

[0050] A liver-specific antigen is one that is preferentially expressed in liver tissue, such as hepatocytes, parenchymal cells, Kupffer cells, stellate cells, and / or liver sinusoidal endothelial cells. Preferably, a liver-specific antigen is an antigen that has a higher expression level in liver tissue (e.g., at least one of hepatocytes, parenchymal cells, Kupffer cells, stellate cells, and liver sinusoidal endothelial cells) compared to other tissues. For example, a liver-specific antigen may be an antigen that has an expression level that is 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 than the expression level in other tissues. Preferably, a liver-specific antigen is expressed exclusively in liver tissue, i.e., is not detectably expressed in other tissues.

[0051] The liver-specific antigen may be accessible to Treg cells (although it should be understood that the liver-specific antigen need not be accessible to all liver cell types that express the antigen). For example, the liver-specific antigen may be expressed on the cell surface. Examples of suitable liver-specific antigens include asialoglycoprotein receptor (ASGR), sodium / taurocholate cotransporting polypeptide (NTCP), mannose-6-phosphate receptor, type VI collagen receptor, PDGF receptor, scavenger receptor class A, scavenger receptor class B type I, heparan sulfate, glycyrrhizin receptor, HDL receptor, LDL receptor, transferrin receptor, insulin receptor, alpha-2 macroglobulin receptor, ferritin receptor, uroplasminogen receptor, and thrombin receptor.

[0052] Preferably, the antigen recognition domain specifically binds to the asialoglycoprotein receptor (ASGR). Most preferably, the antigen recognition domain binds to human ASGR. In some embodiments, the antigen recognition domain binds to both human ASGR and ASGR from other animals, such as mouse ASGR. Suitably, the antigen recognition domain may specifically bind to human ASGR.

[0053] The asialoglycoprotein receptor (ASGR or ASGPR) is a C-type lectin primarily expressed on the sinusoidal surface of hepatocytes. ASGR is composed of a large 48 kDa subunit (ASGR1) and a small 40 kDa subunit (ASGR2). The primary role of ASGR is to bind, internalize, and subsequently remove glycoproteins containing terminal galactose or N-acetylgalactosamine residues (asialoglycoproteins) from the circulation (Roggenbuck, D., et al., 2012. Autoimmunity Highlights, 3(3), p.119). In normal hepatocytes, ASGR is polarized on the sinusoidal and basolateral surfaces of the plasma hepatocyte membrane. However, during liver inflammation, ASGR expression shifts toward the bile canalicular membrane. In end-stage liver disease (cirrhosis), ASGR is overexpressed and serum levels of asialoglycoprotein increase (Roggenbuck, D., et al., 2012. Autoimmunity Highlights, 3(3), p.119).

[0054] The antigen recognition domain may bind to ASGR1 and / or ASGR2, preferably ASGR1. The antigen recognition domain may specifically bind to ASGR1 and / or ASGR2, preferably ASGR1.

[0055] Human ASGR1 (UniProt entry P07306) is encoded by the ASGR1 gene. Splice translation variants encoding multiple isoforms have been observed for this gene (Harris, RL, et al., 2012. Molecular biology international, 2012, Article ID 283974, 10 pages). The longer transcript contains all eight exons and is much larger, encoding the full-length ASGR1 (isoform a, 291 amino acids). The shorter transcript has an in-frame deletion of exon 3, resulting in a deletion of 39 residues (isoform b, 252 amino acids). Isoform b lacks the transmembrane domain and is secreted as a soluble protein. Exemplary sequences for human ASGR1 isoforms a and b are shown below (SEQ ID NOs: 1 and 2, respectively).

[0056] SEQ ID NO:1 - Human ASGR1 isoform a (NP_001662.1) MTKEYQDLQHLDNEESDHHQLRKGPPPPQPLLQRLCSGPRLLLLSLGLSLLLLVVVCVIGSQNSQLQEELRGLRETFSNFTASTEAQVKGLSTQGGNVGRKMKSLESQLEKQQKDLSEDHSSLLLHVKQFVSDLRSLSCQMAALQ GNGSERTCCPVNWVEHERSCYWFSRSGKAWADADNYCRLEDAHLVVVTSWEEQKFVQHHIGPVNTWMGLHDQNGPWKWVDGTDYETGFKNWRPEQPDDWYGHGLGGGEDCAHFTDDGRWNDDVCQRPYRWVCETELDKASQEPPLL SEQ ID NO:2 - Human ASGR1 isoform b (NP_001184145.1) MTKEYQDLQHLDNEESDHHQLRKDSQLQEELRGLRETFSNFTASTEAQVKGLSTQGGNVGRKMKSLESQLEKQQKDLSEDHSSLLLHVKQFVSDLRSLSCQMAALQGNGSERTCCPVNWVEHERSCYWFSRSGKAWADADNYCRLEDAHLVVVTSWEEQKFVQHHIGPVNTWMGLHDQNGPWKWVDGTDYETGFKNWRPEQPDDWYGHGLGGGEDCAHFTDDGRWNDDVCQRPYRWVCETELDKASQEPPLL

[0057] The antigen recognition domain may bind, preferably specifically bind, to human ASGR1 isoform a and / or human ASGR1 isoform b, preferably human ASGR1 isoform a. The antigen recognition domain may bind, preferably specifically bind, to one or more polypeptides having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 1 and / or SEQ ID NO: 2. Preferably, the antigen recognition domain may bind, preferably specifically bind, to polypeptides having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO: 1. Human ASGR2 (UniProt entry P07307) is encoded by the ASGR2 gene. Alternatively, splice translation variants encoding multiple isoforms have been observed for this gene (Harris, RL, et al., 2012. Molecular biology international, 2012, Article ID 283974, 10 pages). ASGR2 generates five transcripts (TH2', T1, T2, T3, and T4) encoding four isoforms (a-d) containing different in-frame deletions resulting from alternative exon splicing events. Isoforms a-c contain five amino acids that act as a proteolytic cleavage signal near the junction between the transmembrane domain and the CRD. Isoforms b and d lack this signal and therefore are not proteolytically cleaved, remain membrane-bound, and oligomerize with ASGR1 isoform a to form native ASGR at the cell surface. Exemplary sequences for ASGR2 isoforms a-d are shown below (SEQ ID NOs: 3-6, respectively).

[0058] SEQ ID NO:3 - Human ASGR2 isoform a (NP_001172.1 and NP_550434.1) MAKDFQDIQQLSSEENDHPFHQGEGPGTRRLNPRRGNPFLKGPPPAQPLAQRLCSMVCFSLLALSFNILLLVVICVTGSQSEGHRGAQLQAELRSLKEAFSNFSSSTLTEVQAISTHGGSVGDKITSLGAKLEKQQQDLKADHDALLFHLKHFPV DLRFVACQMELLHSNGSQRTCCPVNWVEHQGSCYWFSHSGKAWAEAEKYCQLENAHLVVINSWEEQKFIVQHTNPFNTWIGLTDSDGSWKWVDGTDYRHNYKNWAVTQPDNWHGHELGGSEDCVEVQPDGRWNDDFCLQVYRWVCEKRRNATGEVA SEQ ID NO:4 - Human ASGR2 isoform b (NP_550435.1) MAKDFQDIQQLSSEENDHPFHQGPPPAQPLAQRLCSMVCFSLLALSFNILLLVVICVTGSQSAQLQAELRSLKEAFSNFSSSTLTEVQAISTHGGSVGDKITSLGAKLEKQQQDLKADHDALLFHLKHFPVDLRFVACQMELL HSNGSQRTCCPVNWVEHQGSCYWFSHSGKAWAEAEKYCQLENAHLVVINSWEEQKFIVQHTNPFNTWIGLTDSDGSWKWVDGTDYRHNYKNWAVTQPDNWHGHELGGSEDCVEVQPDGRWNDDFCLQVYRWVCEKRRNATGEVA SEQ ID NO:5 - Human ASGR2 isoform c (NP_550436.1) MAKDFQDIQQLSSEENDHPFHQGPPPAQPLAQRLCSMVCFSLLALSFNILLLVVICVTGSQSEGHRGAQLQAELRSLKEAFSNFSSSTLTEVQAISTHGGSVGDKITSLGAKLEKQQQDLKADHDALLFHLKHFPVDLRFVACQME LLHSNGSQRTCCPVNWVEHQGSCYWFSHSGKAWAEAEKYCQLENAHLVVINSWEEQKFIVQHTNPFNTWIGLTDSDGSWKWVDGTDYRHNYKNWAVTQPDNWHGHELGGSEDCVEVQPDGRWNDDFCLQVYRWVCEKRRNATGEVA SEQ ID NO: 6 - Human ASGR2 isoform d (NP_001188281.1) MAKDFQDIQQLSSEENDHPFHQGEGPGTRRLNPRRGNPFLKGPPPAQPLAQRLCSMVCFSLLALSFNILLLVVICVTGSQSAQLQAELRSLKEAFSNFSSSTLTEVQAISTHGGSVGDKITSLGAKLEKQQQDLKADHDALLFHLKHFPVDLR FVACQMELLHSNGSQRTCCPVNWVEHQGSCYWFSHSGKAWAEAEKYCQLENAHLVVINSWEEQKFIVQHTNPFNTWIGLTDSDGSWKWVDGTDYRHNYKNWAVTQPDNWHGHELGGSEDCVEVQPDGRWNDDFCLQVYRWVCEKRRNATGEVA

[0059] The antigen recognition domain may bind, preferably specifically bind, to human ASGR2 isoforms a, b, c, and / or d, preferably human ASGR2 isoforms b and / or d. The antigen recognition domain may bind, preferably specifically bind, to one or more polypeptides that are at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 3 to 6. Preferably, the antigen recognition domain binds, preferably specifically binds, to a polypeptide that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 4 or at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 6.

[0060] The antigen recognition domain may bind, preferably specifically bind, to (a) human ASGR, including human ASGR1 isoform a and human ASGR2 isoform b, and / or (b) human ASGR, including human ASGR1 isoform a and human ASGR2 isoform d. The antigen recognition domain may bind, and preferably specifically match, (a)(i) one or more polypeptides having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO:1, and (ii) one or more polypeptides having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO:4, and / or (b)(i) one or more polypeptides having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO:1, and (ii) one or more polypeptides having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to SEQ ID NO:6. In some embodiments, the antigen recognition domain binds to both human ASGR1 and / or ASGR2 and ASGR1 and / or ASGR2 from other animals, such as mouse ASGR1 and / or mouse ASGR2. Thus, in some embodiments, the antigen recognition domain exhibits cross-reactivity between human ASGR and mouse ASGR. For example, if the antigen recognition domain binds to human ASGR1 isoform a, in some embodiments, it also binds to mouse ASGR1 isoform a. Examples of mouse ASGR1 isoforms a and b and mouse ASGR2 isoforms a and b are shown below (SEQ ID NOs: 7-10).

[0061] SEQ ID NO:7 - Mouse ASGR1 isoform a (NP_033844.1 and NP_001278060.1) MTKDYQDFQHLDNDNDHHQLRRGPPPTPRLLQRLCSGSRLLLLSSSLSILLLVVVCVITSQNSQLREDLLALRQNFSNLTVSTEDQVKALSTQGSSVGRKMKLVESKLEKQQKDLTEDHSSLLLHVKQLVSDVRSLSCQMAA FRGNGSERTCCPINWVEYEGSCYWFSSSVRPWTEADKYCQLENAHLVVVTSRDEQNFLQRHMGPLNTWIGLTDQNGPWKWVDGTDYETGFQNWRPEQPDNWYGHGLGGGEDCAHFTTDGRWNDDVCRRPYRWVCETKLDKAN SEQ ID NO:8 - Mouse ASGR1 isoform b (NP_001278061.1) MTKDYQDFQHLDNDNDHHQLRRGPPPTPRLLQRLCSGSRLLLLSSSLSILLLVVVCVITSQNSQLREDLLALRQNFSNLTVSTEDQVKALSTQGSSVGRKMKLVESKLEKQQKDLTEGSERTCCPIN WVEYEGSCYWFSSSVRPWTEADKYCQLENAHLVVTSRDEQNFLQRHMGPLNTWIGLTDQNGPWKWVDGTDYETGFQNWRPEQPDNWYGHGLGGGEDCAHFTTDGRWNDDVCRRPYRWVCETKLDKAN SEQ ID NO:9—Mouse ASGR2 isoform a (NP_031519.1, NP_001300854.1, and NP_001300855.1) MEKDCQDIQQLDSEENDHQLSGDDEHGSHVQDPRIENPHWKGQPLSRPFPQRLCSTFRLSLLALAFNILLLVVICVVSSQSIQLQEEFRTLKETFSNFSSSTLMEFGALDTLGGSTNAILTSWLAQLEEKQQQLKADHSTLLFHLKHFPM DLRTLTCQLAYFQSNGTECCPVNWVEFGGSCYWFSRDGLTWAEADQYCQLENAHLLVINSREEQDFVVKHRSQFHIWIGLTDRDGSWKWVDGTDYRSNYRNWAFTQPDNWQGHEQGGGEDCAEILSDGHWNDNFCQQVNRWVCEKRRNITH SEQ ID NO: 10 - Mouse ASGR2 isoform b (NP_001300856.1) MEFGALDTLGGSTNAILTSWLAQLEEKQQQLKADHSTLLFHLKHFPMDLRTLTCQLAYFQSNGTECCPVNWVEFGGSCYWFSRDGLTWAEADQYCQLEN AHLLVINSREEQDFVVKHRSQFHIWIGLTDRDGSWKWVDGTDYRSNYRNWAFTQPDNWQGHEQGGGEDCAILSDGHWNDNFCQQVNRWVCEKRRNITH

[0062] The antigen recognition domain may specifically bind to mouse ASGR1 isoform a, mouse ASGR1 isoform b, mouse ASGR2 isoform a, and / or mouse ASGR2 isoform b, preferably specifically bind to mouse ASGR1 isoform a. The antigen recognition domain may bind to, preferably specifically match, the equivalent human protein. The antigen recognition domain may bind to, preferably specifically bind to, one or more polypeptides that are at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 7-10. Preferably, the antigen recognition domain binds to, preferably specifically binds to, a polypeptide that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 1 and at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 7.

[0063] The antigen recognition domain may bind to, and preferably specifically match, one or more regions or epitopes within a liver-specific antigen, for example, within ASGR. An epitope, also known as an antigenic determinant, is a portion of an antigen that is recognized by an antigen recognition domain (e.g., an antibody). That is, an epitope is a specific portion of an antigen to which an antigen recognition domain binds. Preferably, the antigen recognition domain binds to, and preferably specifically matches, a region or epitope within a liver-specific antigen, for example, within ASGR.

[0064] The antigen recognition domain used in the present invention may selectively or specifically bind to a liver-specific antigen, preferably ASGR, and thus may have a higher binding affinity to the liver-specific antigen, preferably ASGR, than to other proteins / molecules. Preferably, "specifically bind" herein means that the antigen recognition domain does not bind to other proteins or binds with a significantly lower affinity (e.g., 10, 50, 100, 500, 1000, or 10,000 times lower than the affinity to the antigen it specifically binds) compared to its binding affinity to the antigen it specifically binds to. Thus, the antigen recognition domain referred to herein may bind to its antigen, e.g., ASGR, with an affinity that is 10, 50, 100, 500, 1000, or 10,000 times lower than its binding affinity to other proteins. The binding affinity of the antigen recognition domain can be determined using methods well known in the art, such as a method using a Biacore system. The antigen recognition domain may have a high binding affinity to a liver-specific antigen, e.g., ASGR, i.e., 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, or 10 -12The antigen recognition domain may have a binding affinity for a liver-specific antigen, such as ASGR, corresponding to a Kd of less than 100 nM, 50 nM, 20 nM, or 10 nM, more preferably less than 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, or 1 nM, and most preferably less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 nM. The antigen recognition domain may have a binding affinity for a liver-specific antigen, such as ASGR, corresponding to a Kd of about 1 pM to about 100 nM, about 1 pM to about 10 nM, or about 1 pM to about 1 nM.

[0065] Any suitable method may be used to determine the Kd. However, where convenient, the Kd may be determined using a surface plasmon resonance (SPR) assay or system (e.g., a Biacore assay or system). For example, the Kd may be determined by establishing a saturation curve by testing various concentrations of the antigen-recognition domain against various concentrations of antigen in vitro, e.g., using a Lineweaver-Burk plot, or by using commercially available binding model software, such as the 1:1 binding model in the BIAcore 1000 Evaluation software. Preferably, an HBS-P buffer system (0.01 M Hepes, pH 7.4, 0.15 M NaCl, 0.05% surfactant P20) is used. In particular, if the antigen-recognition domain is an antibody or antibody fragment or is derived from an antibody, the Kd may be appropriate if the antigen-recognition domain is in any format, such as an scFv or sdAb format, or another format described elsewhere herein.

[0066] The antigen recognition domain has a melting temperature of 50°C, 55°C, 60°C, 65°C, or higher, preferably 55°C or higher, for example, 55°C to 75°C. The melting temperature may be determined by any method known to those skilled in the art. Preferably, the melting temperature is determined by differential scanning calorimetry. Preferably, the antigen recognition domain is heated at 180°C / hour in 1 mg / mL PBS, and the detectable heat change is measured. The midpoint of the transition is the apparent melting temperature.

[0067] The antigen recognition domain (also known as an antigen-specific targeting domain) may be a protein or peptide capable of specifically recognizing and binding to a liver-specific antigen, preferably the asialoglycoprotein receptor (ASGR). The antigen recognition domain includes naturally occurring, synthetic, semisynthetic, or recombinantly produced binding partners of a liver-specific antigen, preferably the asialoglycoprotein receptor (ASGR). Examples of antigen recognition domains include antibodies or antibody fragments or derivatives, extracellular domains of receptors, ligands for cell surface molecules / receptors or their receptor-binding domains, and tumor-binding proteins. For example, suitable ligands for ASGR include beta-D-galactose and N-acetylgalactosamine (GalNAc). Preferably, the antigen-recognition domain is an antibody (Ab) or is derived from an antibody (Ab). The antibody-derived antigen-recognition domain may be an antibody fragment or a genetically engineered product of one or more of the antibody fragments, which are involved in binding to the antigen. Examples include camelid antibody (VHH), antigen-binding fragment (Fab), variable region (Fv), single-chain antibody (scFv), single-domain antibody (sdAb), heavy chain variable region (VH), light chain variable region (VL), and complementarity-determining region (CDR). In a preferred embodiment, the antigen-recognition domain is a single-chain antibody (scFv) or a single-domain antibody (sdAb). Most preferably, the antigen-recognition domain is a single-domain antibody (sdAb).

[0068] Antibodies recognize antigens via their fragment antigen-binding (Fab) variable regions. Antibodies are glycoproteins belonging to the immunoglobulin superfamily, making up the majority of the gamma globulin fraction of blood proteins. They typically consist of two long heavy chains and two small light chains, each consisting of a basic structural unit. Camelid antibodies (VHHs) lack light chains and consist of two heavy chains attached to variable domains.

[0069] The term "antigen-binding fragment" (Fab) refers to the region of an antibody that binds to an antigen, and each of the heavy and light chains comprises one constant region and one variable region.

[0070] "Fv" refers to the smallest antibody fragment containing an intact antigen-binding site. An Fv fragment consists of one light chain variable region linked to one heavy chain variable region. A "single-chain antibody" (scFv) is a modified antibody consisting of a light chain variable region and a heavy chain variable region connected to each other directly or via a peptide linker sequence. The peptide linker sequence is typically about 10 to 25 amino acids long and is rich in glycine for flexibility and serine or threonine for solubility. The peptide linker sequence can connect the N-terminus of the heavy chain variable region to the C-terminus of the light chain variable region, or vice versa.

[0071] A "single domain antibody" (sdAb), also known as a nanobody, is an antibody fragment consisting of a single monomeric variable antibody domain. Thus, an sdAb may be a heavy chain variable region (VH) or a light chain variable region (VL). A "heavy chain variable region" or "VH" refers to a fragment of an antibody heavy chain comprising three CDRs flanked by flanking regions known as framework regions, which are more highly conserved than the CDRs and form a scaffold for CDR retention. A "light chain variable region" or "VL" refers to a fragment of an antibody light chain comprising three CDRs flanked by framework regions.

[0072] A "complementarity-determining region" or "CDR" of an antibody or antigen-binding fragment thereof refers to the hypervariable loops in the variable region of the heavy or light chain of the antibody. CDRs can interact with the antigen structure and primarily determine antigen binding (although some framework regions have been shown to be involved in binding). The heavy and light chain variable regions each contain three CDRs (heavy chain CDR1, 2, and 3 and light chain CDR1, 2, and 3, numbered from the amino terminus to the carboxy terminus). The CDRs of the heavy and light chain variable regions of an antibody can be predicted from the antibody heavy and light chain variable region sequences using prediction software available in the art, such as the Abysis algorithm, or IMGT / V-QUEST software, e.g., the IMGT algorithm (ImMunoGeneTics) available at www.IMGT.org (see, e.g., Lefranc et al., 2009 NAR 37:D1006-D1012 and Lefranc 2003, Leukemia 17: 260-266). CDR regions identified by either algorithm are considered equally suitable for use in the present invention. CDRs can vary in length depending on the antibody from which they are predicted and between the heavy and light chains. Thus, the three heavy chain CDRs of an intact antibody may be of different lengths (or the same length), and the three light chain CDRs of an intact antibody may be of different lengths (or the same length). For example, CDRs may range from 2 or 3 amino acids in length to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length. In particular, CDRs may be 3 to 14 amino acids in length, e.g., at least 3 amino acids and less than 15 amino acids.

[0073] It should be noted that, where necessary, Kabat nomenclature is followed herein to define the positions of CDRs (Kabat et al., 1991, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 647-669).

[0074] Antibodies, derivatives, and fragments thereof that specifically bind to a liver-specific antigen, preferably the asialoglycoprotein receptor (ASGR), can be prepared using methods well known to those skilled in the art. Such methods include phage display, methods for generating human or humanized antibodies, or methods using transgenic animals or plants engineered to produce human antibodies. Phage display libraries of partially or fully synthetic antibodies are available, and such libraries can be screened for antibodies or fragments thereof that can bind to target molecules. Phage display libraries of human antibodies are also available. Once the amino acid or polynucleotide sequence encoding the antibody (or its derivative or fragment) is identified, it can be isolated and / or characterized. The antibody sequence can be used to design suitable derivatives or fragments thereof. Suitable antibodies, derivatives, and fragments thereof that specifically bind to a liver-specific antigen, preferably the asialoglycoprotein receptor (ASGR), are well known. For example, anti-NTCP antibodies are described in Stieger, B., et al., 1994, Gastroenterology, 107(6), pp.1781-1787. Anti-mannose-6-phosphate receptor antibodies are described in von Figura, K., et al., 1984, The EMBO journal, 3(6), pp.1281-1286. Anti-PDGF receptor antibodies are described in Ogawa, S., et al., 2010, Hepatology Research, 40(11), pp.1128-1141. Anti-scavenger receptor class A antibodies are described in Tomokiyo, RI, et al., 2002, Atherosclerosis, 161(1), pp.123-132. Meuleman, P., et al., 2012. Hepatology, 55(2), pp. 364-372, describes an anti-scavenger receptor class B type I antibody. Gao, W., et al., 2014. Hepatology, 60(2), pp. 576-587, describes an anti-heparan sulfate antibody.Antibodies, derivatives and fragments thereof that specifically bind to liver-specific antigens are commercially available. The sequences of the known antibodies can be used to design suitable derivatives or fragments thereof.

[0075] Examples of antibodies, derivatives and fragments thereof that can be used in the present invention are further described below.

[0076] The antigen recognition domain may comprise at least one CDR (e.g., CDR3), which can be deduced from an antibody that binds to a liver-specific antigen, preferably an antibody that binds to the asialoglycoprotein receptor (ASGR) (or a variant of such a deduced CDR, e.g., a variant with one, two, or three amino acid substitutions). It will be understood that a molecule comprising three or fewer CDR regions (e.g., a single CDR, or a portion thereof) will retain the antigen-binding activity of the antibody from which the CDRs are derived. It has been described in the art that molecules comprising two CDR regions can bind to target antigens, for example, in the form of minibodies (Vaughan and Sollazzo, 2001, Combinational Chemistry & High Throughput Screening, 4, 417-430). It has also been described that molecules comprising one CDR can exhibit strong binding activity to targets (Nicaise et al., 2004, Protein Science, 13: 1882-91). In this regard, the antigen-recognition domain may comprise one or more variable heavy chain CDRs, for example one, two or three variable heavy chain CDRs. Alternatively / in addition, the antigen-recognition domain may comprise one or more variable light chain CDRs, for example one, two or three variable light chain CDRs. The antigen-recognition domain may comprise three heavy chain CDRs and / or three light chain CDRs (particularly a heavy chain variable region comprising three CDRs and / or a light chain variable region comprising three CDRs), in which at least one CDR, and preferably all CDRs, may be derived from an antibody that binds to a liver-specific antigen, preferably ASGR, and may be selected from one of the CDR sequences shown below.The antigen-recognition domain may comprise a combination of variable heavy chain CDRs and variable light chain CDRs, for example, a combination of one variable heavy chain CDR and one variable light chain CDR, a combination of two variable heavy chain CDRs and one variable light chain CDR, a combination of two variable heavy chain CDRs and two variable light chain CDRs, a combination of three variable heavy chain CDRs and one or two variable light chain CDRs, a combination of one variable heavy chain CDR and two or three variable light chain CDRs, or a combination of three variable heavy chain CDRs and three variable light chain CDRs. Preferably, the antigen-recognition domain comprises a combination of three variable heavy chain CDRs (CDR1, CDR2, and CDR3) or three variable light chain CDRs (CDR1, CDR2, and CDR3), i.e., three CDRs. Alternatively, the antigen-recognition domain can comprise a combination of three variable heavy chain CDRs (CDR1, CDR2, and CDR3) and three variable light chain CDRs (CDR1, CDR2, and CDR3), or alternatively, six CDRs. As long as the antigen-recognition domain has the above-mentioned binding activity, one or more CDRs present in the domain do not all need to be derived from the same antibody. Thus, one CDR may be deduced from the heavy or light chain of an antibody that binds to a liver-specific antigen, such as ASGR, while another CDR may be deduced from another antibody that binds to the same liver-specific antigen (e.g., ASGR). In this case, it would be preferable for CDR3 to be deduced from an antibody that binds to a liver-specific antigen, such as ASGR. However, particularly when more than one CDR is present in the antigen-recognition domain, it is preferable for these CDRs to be deduced from an antibody that binds to a liver-specific antigen, such as ASGR. A combination of CDRs from different antibodies, particularly antibodies that bind to the same desired region or epitope, may also be used. Exemplary and preferred CDR sequences are provided elsewhere herein.

[0077] In a particularly preferred embodiment, the antigen recognition domain comprises three CDRs deduced from the variable heavy chain sequence of an antibody that binds to a liver-specific antigen, e.g., ASGR, and / or three CDRs deduced from the variable light chain sequence of an antibody (preferably the same antibody) that binds to a liver-specific antigen, e.g., ASGR. Exemplary and preferred CDR sequences are described elsewhere herein.

[0078] In some embodiments, the antigen-recognition domain is an antibody or is derived from an antibody (e.g., a Fab, scFv, or sdAb), and the antibody comprises one or more CDR regions selected from SEQ ID NOs: 11 to 73, or derivatives thereof. In other words, in some embodiments, the antigen-recognition domain comprises one or more CDR regions selected from SEQ ID NOs: 11 to 73, or derivatives thereof. Preferably, the antigen-recognition domain comprises three CDR regions selected from SEQ ID NOs: 11 to 73, or derivatives thereof. [Table 1]

[0079] Preferably, the antigen-binding domain comprises CDRs (CDR1, CDR2, and CDR3) selected from the same variable chain, or derivatives thereof. For example, the antigen-binding domain may comprise SEQ ID NOs: 11 to 13, 14 to 16, 17 to 19, 20 to 22, 23 to 25, 26 to 28, 29 to 31, 32 to 34, 35 to 37, 38 to 40, 41 to 43, 44 to 46, 47 to 49, 50 to 52, 53 to 55, 56 to 58, 59 to 61, 62 to 64, 65 to 67, 68 to 70, and / or 71 to 73, or derivatives thereof.

[0080] Preferably, the antigen-binding domain may comprise SEQ ID NOs: 11 to 13, 14 to 16, 17 to 19, 20 to 22, 23 to 25, 26 to 28, and / or 29 to 31, or a derivative thereof. Preferably, the antigen-binding domain may comprise SEQ ID NOs: 11 to 13, 14 to 16, 17 to 19, 23 to 25, 26 to 28, and / or 29 to 31, or a derivative thereof.

[0081] Preferably, the antigen-binding domain may comprise SEQ ID NOs: 11 to 13, 14 to 16, or 17 to 19, and / or SEQ ID NOs: 23 to 25, 26 to 28, or 29 to 31, or a derivative thereof.

[0082] In a preferred embodiment, the antigen recognition domain comprises one or more CDR regions selected from SEQ ID NOs: 11 to 31. Suitably, the antigen recognition domain comprises three CDR regions selected from SEQ ID NOs: 11 to 31, or derivatives thereof.

[0083] In a preferred embodiment, the antigen recognition domain comprises the following sequence: (i) CDR1 sequence EKYAMA (SEQ ID NO: 11), CDR2 sequence RISARG VT (SEQ ID NO: 12), and CDR3 sequence HKRHEHTRFDS (SEQ ID NO: 13), or derivatives thereof; (ii) CDR1 sequence RYTMG (SEQ ID NO: 14), CDR2 sequence AIGPPG SNTYYADSVKG (SEQ ID NO: 15), and CDR3 sequence WVMLRGRF DY (SEQ ID NO: 16), or derivatives thereof; (iii) CDR1 sequence DYGMG (SEQ ID NO: 17), CDR2 sequence AIGRN GSQTYYADSVKG (SEQ ID NO: 18), and CDR3 sequence LRRGRGL NTFTLDY (SEQ ID NO: 19), or derivatives thereof; (iv) CDR1 sequence AAGMG (SEQ ID NO: 20), CDR2 sequence AIGRNG SQTYYADSVKG (SEQ ID NO: 21), and CDR3 sequence LRRGRGLN TFTLDY (SEQ ID NO: 22), or derivatives thereof; (v) CDR1 sequence RASQAIGRWLL (SEQ ID NO: 23), CDR2 sequence PGSRLRS (SEQ ID NO: 24), and CDR3 sequence QQAYAWPPT (SEQ ID NO: 25), or derivatives thereof; (vi) the CDR1 sequence RASQAIGRWLL (SEQ ID NO: 26), the CDR2 sequence PGSRLQS (SEQ ID NO: 27), and the CDR3 sequence QQAYQLPVT (SEQ ID NO: 28), or derivatives thereof; and / or (vii) CDR1 sequence RASQAIGRWLL (SEQ ID NO: 29), CDR2 sequence PGSRLQS (SEQ ID NO: 30), and CDR3 sequence QQAYSLPPT (SEQ ID NO: 31), or derivatives thereof.

[0084] Most preferably, the antigen recognition domain comprises the CDR1 sequence EKYAMA (SEQ ID NO: 11), the CDR2 sequence RISARGVT (SEQ ID NO: 12), and the CDR3 sequence HKRHEHTRFDS (SEQ ID NO: 13), or a derivative thereof. Preferably, the antigen recognition domain is an sdAb comprising the CDR1 sequence EKYAMA (SEQ ID NO: 11), the CDR2 sequence RISARGVT (SEQ ID NO: 12), and the CDR3 sequence HKRHEHTRFDS (SEQ ID NO: 13), or a derivative thereof.

[0085] In other embodiments, the antigen recognition domain comprises CDR1, CDR2, and CDR3 regions, wherein the CDR1, CDR2, and CDR3 regions comprise the following: (i), (ii), or (iii): (i) SEQ ID NOs: 11, 12, and 13, respectively, or derivatives thereof, and SEQ ID NOs: 23, 24, and 25, respectively, or derivatives thereof, or SEQ ID NOs: 26, 27, and 28, respectively, or derivatives thereof, or SEQ ID NOs: 29, 30, and 31, respectively, or derivatives thereof, preferably wherein the antigen recognition domain comprises SEQ ID NOs: 11, 12, and 13, respectively, and comprises CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: 23, 24, and 25, respectively: (ii) SEQ ID NOs: 14, 15, and 16, respectively, or derivatives thereof, and SEQ ID NOs: 23, 24, and 25, respectively, or derivatives thereof, or SEQ ID NOs: 26, 27, and 28, respectively, or derivatives thereof, or SEQ ID NOs: 29, 30, and 31, respectively, or derivatives thereof, preferably wherein the antigen recognition domain comprises SEQ ID NOs: 14, 15, and 16, respectively, and comprises CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: 26, 27, and 28, respectively: (iii) SEQ ID NOs: 17, 18, and 19, respectively, or derivatives thereof, and SEQ ID NOs: 23, 24, and 25, respectively, or derivatives thereof, or SEQ ID NOs: 26, 27, and 28, respectively, or derivatives thereof, or SEQ ID NOs: 29, 30, and 31, respectively, or derivatives thereof, preferably wherein the antigen recognition domain comprises SEQ ID NOs: 17, 18, and 19, respectively, and comprises CDR1, CDR2, and CDR3 regions comprising SEQ ID NOs: 26, 27, and 28, respectively.

[0086] Thus, in such embodiments, the antigen recognition domain comprises CDR1, CDR2, and CDR3 regions, wherein the CDR1, CDR2, and CDR3 regions comprise SEQ ID NOs: 11, 12, and 13, respectively, or derivatives thereof; and SEQ ID NOs: 23, 24, and 25, respectively, or derivatives thereof.

[0087] The antigen recognition domain comprises CDR1, CDR2, and CDR3 regions, and the CDR1, CDR2, and CDR3 regions comprise SEQ ID NOs: 11, 12, and 13, respectively, or derivatives thereof; and SEQ ID NOs: 26, 27, and 28, respectively, or derivatives thereof.

[0088] The antigen recognition domain comprises CDR1, CDR2, and CDR3 regions, and the CDR1, CDR2, and CDR3 regions comprise SEQ ID NOs: 11, 12, and 13, respectively, or derivatives thereof; and SEQ ID NOs: 29, 30, and 31, respectively, or derivatives thereof.

[0089] The antigen recognition domain comprises CDR1, CDR2, and CDR3 regions, and the CDR1, CDR2, and CDR3 regions comprise SEQ ID NOs: 14, 15, and 16, respectively, or derivatives thereof; and SEQ ID NOs: 23, 24, and 25, respectively, or derivatives thereof.

[0090] The antigen recognition domain comprises CDR1, CDR2, and CDR3 regions, and the CDR1, CDR2, and CDR3 regions comprise SEQ ID NOs: 14, 15, and 16, respectively, or derivatives thereof; and SEQ ID NOs: 26, 27, and 28, respectively, or derivatives thereof.

[0091] The antigen recognition domain comprises CDR1, CDR2, and CDR3 regions, wherein the CDR1, CDR2, and CDR3 regions comprise SEQ ID NOs: 14, 15, and 16, respectively, or derivatives thereof; and SEQ ID NOs: 29, 30, and 31, respectively, or derivatives thereof.

[0092] The antigen recognition domain comprises CDR1, CDR2, and CDR3 regions, and the CDR1, CDR2, and CDR3 regions comprise SEQ ID NOs: 17, 18, and 19, respectively, or derivatives thereof; and SEQ ID NOs: 23, 24, and 25, respectively, or derivatives thereof.

[0093] The antigen recognition domain comprises CDR1, CDR2, and CDR3 regions, and the CDR1, CDR2, and CDR3 regions comprise SEQ ID NOs: 17, 18, and 19, respectively, or derivatives thereof; and SEQ ID NOs: 26, 27, and 28, respectively, or derivatives thereof.

[0094] The antigen recognition domain comprises CDR1, CDR2, and CDR3 regions, and the CDR1, CDR2, and CDR3 regions comprise SEQ ID NOs: 17, 18, and 19, respectively, or derivatives thereof; and SEQ ID NOs: 29, 30, and 31, respectively, or derivatives thereof.

[0095] CARs comprising such antigen recognition domains also constitute a further aspect of the present invention.

[0096] Preferably, the antigen recognition domain comprising six CDRs is an scFv.

[0097] The present invention includes "derivatives" of the CDR regions (as well as VH and VL (VK) regions and CDR regions therein) described above and elsewhere herein. The term "derivative" as used herein is defined in the "Variants, Derivatives, and Fragments" section below. It will be understood that one or more amino acid substitutions may be made within a CDR while retaining antigen-binding ability. For example, a CDR derivative may contain up to three amino acid substitutions, e.g., three amino acid substitutions, two amino acid substitutions, or one amino acid substitution. In particular, in some embodiments, the CDR derivative contains one amino acid substitution and retains antigen-binding ability. The derivative may also be a variant, e.g., a variant having at least 80% or 90% identity to the CDR.

[0098] In some embodiments, the antigen recognition domain comprises or consists of an amino acid sequence that is at least about 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 74-94. [Table 2]

[0099] In a preferred embodiment, the antigen recognition domain comprises or consists of an amino acid sequence that is at least about 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 74-80.

[0100] Most preferably, the antigen-recognition domain comprises or consists of an amino acid sequence that is at least about 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to ASGR1 VH1 (SEQ ID NO: 74). In some embodiments, the antigen-recognition domain comprises or consists of the amino acid sequence of SEQ ID NO: 74, 75, or 76, or a derivative thereof, and / or the amino acid sequence of SEQ ID NO: 78, 79, or 80, or a derivative thereof. In preferred embodiments, the antigen-recognition domain comprises or consists of the amino acid sequences of SEQ ID NOs: 74 and 78, or a derivative thereof; SEQ ID NOs: 75 and 79, or a derivative thereof; or SEQ ID NOs: 76 and 80, or a derivative thereof.

[0101] Thus, in such embodiments, the antigen recognition domain comprises or consists of SEQ ID NOs: 74 and 78, or a derivative thereof. The antigen recognition domain comprises or consists of SEQ ID NOs: 74 and 79, or a derivative thereof. The antigen recognition domain comprises or consists of SEQ ID NOs: 74 and 80, or a derivative thereof.

[0102] The antigen recognition domain comprises or consists of SEQ ID NOs: 75 and 78, or a derivative thereof. The antigen recognition domain comprises or consists of SEQ ID NOs: 75 and 79, or a derivative thereof. The antigen recognition domain comprises or consists of SEQ ID NOs: 75 and 80, or a derivative thereof.

[0103] The antigen recognition domain comprises or consists of SEQ ID NOs: 76 and 78, or a derivative thereof. The antigen recognition domain comprises or consists of SEQ ID NOs: 76 and 79, or a derivative thereof. The antigen recognition domain comprises or consists of SEQ ID NOs: 76 and 80, or a derivative thereof.

[0104] Preferably, the antigen-recognition domain comprising the VH and VL (VK) domains is an scFv, which may comprise a linker between the VH and VL (VK) domains.

[0105] The antigen recognition domain may further comprise SEQ ID NO: 74 or a derivative thereof, and any one of SEQ ID NOs: 81, 82, or 88 to 94, or a derivative thereof.

[0106] The antigen recognition domain may further comprise SEQ ID NO: 75 or a derivative thereof, and any one of SEQ ID NOs: 81, 82, or 88 to 94, or a derivative thereof.

[0107] The antigen recognition domain may further comprise SEQ ID NO: 76 or a derivative thereof, and any one of SEQ ID NOs: 81, 82, or 88 to 94, or a derivative thereof.

[0108] The antigen recognition domain may further comprise SEQ ID NO: 77 or a derivative thereof and any one of SEQ ID NOs: 78 to 82 or 88 to 94 or a derivative thereof.

[0109] The antigen recognition domain may further comprise SEQ ID NO: 83 or a derivative thereof and any one of SEQ ID NOs: 78 to 82 or 88 to 94 or a derivative thereof.

[0110] The antigen recognition domain may further comprise SEQ ID NO: 84 or a derivative thereof and any one of SEQ ID NOs: 78 to 82 or 88 to 94 or a derivative thereof.

[0111] The antigen recognition domain may further comprise SEQ ID NO: 85 or a derivative thereof and any one of SEQ ID NOs: 78 to 82 or 88 to 94 or a derivative thereof.

[0112] The antigen recognition domain may further comprise SEQ ID NO: 86 or a derivative thereof and any one of SEQ ID NOs: 78 to 82 or 88 to 94 or a derivative thereof.

[0113] The antigen recognition domain may further comprise SEQ ID NO: 87 or a derivative thereof and any one of SEQ ID NOs: 78 to 82 or 88 to 94 or a derivative thereof.

[0114] Some examples of scFvs (scFv fragments) are outlined below. EVQLLESGGGLVQPGGSLRLSCAASGFTFEKYAMAWVRQAPGKGLEWVSRISARGVTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHKRHEHTRFDSWGQGTLVTVSS LVTVSSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCRASQAIGRWLLWYQQKPGKAPKLLIGPGSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFVTYYCQQAYAWPPTFGQGTKVEIKR (SEQ ID NO: 173) ELQLLEFGGGLVQPGGSLRLSCTTSGFTFSRYTMGWVRQAPGKGLEWVSAIGPPGSNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWVMLRRGRFDYWGQGTLVTVSS LVTVSSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCRASQAIGRWLLWYQQKPGKAPKHLIGPGSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYQLPVTFGQGTKVEIKR (SEQ ID NO: 174) EVQLLESGGGLVQPGGSLRLSCAASGFTFEDYGMGWVRQAPGKGLEWVSAIGRNGSQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLRRGRGLNTFTLDYWGQGTLVTVSS LVTVSSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCRASQAIGRWLLWYQQKPGKAPKLLIGPGSRLQSGVPSRFSGSGSGTDFTLTIGSLQPEDFATYYCQQAYSLPPTFGQGTKVEIKR (SEQ ID NO: 175)

[0115] CARs comprising such antigen recognition domains also constitute a further aspect of the present invention.

[0116] The variants of the antigen-recognition domains described herein retain antigen-binding ability. For example, the variants may bind to ASGR at a level that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the binding level of the corresponding reference amino acid sequence. The variants or derivatives may bind to ASGR at a level similar to or identical to the binding level of the corresponding reference amino acid sequence, or may bind to ASGR at a level that is higher than the corresponding reference amino acid sequence (e.g., at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% higher). Thus, the antigen-recognition domain may comprise or consist of an amino acid sequence containing ASGR1 VH1-4 (SEQ ID NOS: 11-22) and / or ASGR1 VK1-3 (SEQ ID NOS: 23-31) (the underlined portions of SEQ ID NOS: 74-77 and 78-80, respectively). The antigen recognition domain may comprise or consist of an amino acid sequence including the ASGR1 VH1-3 (SEQ ID NOS: 11-19) and / or ASGR1 VK1-3 (SEQ ID NOS: 23-31) (the underlined portions of SEQ ID NOS: 74-76 and 78-80, respectively). One (or more) amino acid residues may be substituted, mutated, modified, replaced, deleted, and / or added in the framework regions.

[0117] Thus, in some embodiments, the antigen recognition domain may comprise or consist of the following sequence: (i) an amino acid sequence having at least about 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to ASGR1 VH1 (SEQ ID NO: 74), the amino acid sequence comprising the CDR1 sequence EKYAMA (SEQ ID NO: 11), the CDR2 sequence RISARGVT (SEQ ID NO: 12), and the CDR3 sequence HKRHEHTRFDS (SEQ ID NO: 13); (ii) an amino acid sequence having at least about 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to ASGR1 VH2 (SEQ ID NO: 75), the amino acid sequence comprising the CDR1 sequence RYTMG (SEQ ID NO: 14), the CDR2 sequence AIGPPGSNTYYADSVKG (SEQ ID NO: 15), and the CDR3 sequence WV MLRGRFDY (SEQ ID NO: 16); (iii) an amino acid sequence having at least about 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to ASGR1 VH3 (SEQ ID NO: 76), the amino acid sequence comprising the CDR1 sequence DYGMG (SEQ ID NO: 17), the CDR2 sequence AIGRNGSQTYYADSVKG (SEQ ID NO: 18), and the CDR3 sequence LRRGRGLNTFTLDY (SEQ ID NO: 19); (iv) an amino acid sequence having at least about 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to ASGR1 VH4 (SEQ ID NO: 77), the amino acid sequence comprising CDR1 sequence AAGMG (SEQ ID NO: 20), CDR2 sequence AIGRNGSQTYYADSVKG (SEQ ID NO: 21), and CDR3 sequence LR RGRGLNTFTLDY (SEQ ID NO: 22); (v) an amino acid sequence having at least about 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to ASGR1 VK1 (SEQ ID NO: 78), the amino acid sequence comprising the CDR1 sequence RASQAIGRWLL (SEQ ID NO: 23), the CDR2 sequence PGSRLRS (SEQ ID NO: 24), and the CDR3 sequence QQAYAWP PT (SEQ ID NO: 25); (vi) an amino acid sequence having at least about 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to ASGR1 VK2 (SEQ ID NO: 79), the amino acid sequence comprising the CDR1 sequence RASQAIGRWLL (SEQ ID NO: 26), the CDR2 sequence PGSRLQS (SEQ ID NO: 27), and the CDR3 sequence QQAYQLPVT (SEQ ID NO: 28); or (vii) An amino acid sequence having at least about 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity to ASGR1 VK3 (SEQ ID NO: 80), comprising the CDR1 sequence RASQAIGRWLL (SEQ ID NO: 29), the CDR2 sequence PGSRLQS (SEQ ID NO: 30), and the CDR3 sequence QQAYS LPPT (SEQ ID NO: 31).

[0118] In a preferred embodiment, the antigen recognition domain comprises or consists of an amino acid sequence that is at least about 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to ASGR1 VH1 (SEQ ID NO: 74), and the amino acid sequence comprises the CDR1 sequence EKYAMA (SEQ ID NO: 11), the CDR2 sequence RISARGVT (SEQ ID NO: 12), and the CDR3 sequence HKRHEHTRFDS (SEQ ID NO: 13), or derivatives thereof.

[0119] Although the CARs of the invention may comprise more than one antigen recognition domain, as defined above, i.e., may bind more than one liver-specific antigen, or more than one epitope within a liver-specific antigen, for example as part of a dual or two polypeptide or CAR system in certain embodiments, the CARs of the invention may comprise only one or a single antigen recognition domain.

[0120] Hinge domain CAR may also comprise a hinge domain. In this specification, the "hinge domain", also referred to as "spacer domain", is the extracellular part of the CAR that separates the antigen-binding domain from the transmembrane domain. The hinge may provide flexibility for accessing the target antigen. For example, a long spacer provides the CAR with extra flexibility, making it easier to access epitopes proximal to the membrane.

[0121] Suitable hinge domains will be apparent to those skilled in the art (e.g., Guedan, S., et al., 2018. Molecular Therapy-Methods & Clinical Development, 12, 145-156). Suitable hinge domains include, but are not limited to, CD28 hinge domain, CD8α hinge domain, IgG hinge domain, and IgD hinge domain. Preferably, the hinge domain is a CD8α or CD28 hinge domain.

[0122] Suitably, the hinge domain may comprise the amino acid sequence shown as SEQ ID NO:95 or a variant having at least 80% identity to SEQ ID NO:95. An example of a CD28 hinge domain (SEQ ID NO: 95): IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP

[0123] Suitably, the variant may be at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO:95.

[0124] Suitably, the hinge domain may comprise the amino acid sequence shown as SEQ ID NO:96, or a variant having at least 80% identity to SEQ ID NO:96. Example of a CD8 alpha hinge domain (SEQ ID NO: 96): TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0125] Suitably, the variant may be at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO:96.

[0126] Suitably, the CAR may encode a tag, such as a c-Myc tag (EQKLISEEDL - SEQ ID NO: 97). Suitably, said tag may be incorporated into the extracellular domain of the CAR, such as the hinge domain of the extracellular domain. An example of a CD28 hinge domain with an integrated c-Myc tag is shown below: Example of a CD28 hinge domain with an integrated c-Myc tag (SEQ ID NO: 98): IEVEQKLISEEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP

[0127] Suitably, the variant may be at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO:98.

[0128] Transmembrane domain The CAR may comprise a transmembrane domain. The "transmembrane domain" herein refers to a portion of the CAR that anchors the CAR in the cell membrane of Treg. Thus, the transmembrane domain can span or be present within the cell membrane of Treg. The transmembrane domain may be derived from a protein that includes an extracellular and / or intracellular portion, and thus the transmembrane domain herein may be attached not only to the portion that is within or spans the cell membrane, but also to the extracellular and / or intracellular residues derived from the protein from which it was derived. For example, the transmembrane domain may be attached to the hinge domain derived from its protein from which it was derived, for example, a transmembrane domain derived from CD8α may be attached to the hinge domain derived from CD8α. The presence of the transmembrane domain in the cell membrane can be determined using any suitable method known in the art, such as fluorescent labeling using a fluorescence microscope.

[0129] Suitable transmembrane domains will be clear to those skilled in the art.The transmembrane domain may comprise the transmembrane sequence from any protein with a transmembrane domain, such as type I transmembrane protein, type II transmembrane protein, or type III transmembrane protein.The transmembrane domain of CAR may also comprise an artificial hydrophobic sequence.The transmembrane domain may be selected so as not to dimerize.

[0130] Examples of transmembrane (TM) domains used in CAR construction are shown below. 1)CD28 TM domain (Pule et al, Mol Ther, 2005, Nov;12(5):933-41; Brentjens et al, CCR, 2007, Sep 15;13(18 Pt 1):5426-35; Casucci et al, Blood, 2013, Nov 14;122(20):3461-72.); 2)OX40 TM domain (Pule et al, Mol Ther, 2005, Nov;12(5):933-41); 3)41BB TM domain (Brentjens et al, CCR, 2007, Sep 15;13(18 Pt 1):5426-35); 4)CD3 zeta TM domain (Pule et al, Mol Ther, 2005, Nov;12(5):933-41; Savoldo B, Blood, 2009, Jun 18;113(25):6392-402.); 5) CD8 TM domain (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.); 6) ICOS TM domain; 7) CD4 TM domain.

[0131] Suitably, the CAR may comprise a CD28 transmembrane domain. Suitably, the transmembrane domain may comprise the amino acid sequence shown as SEQ ID NO: 99, or a variant having at least 80% identity to SEQ ID NO: 99. Example of CD28 TM domain (AA 153-179) (SEQ ID NO: 99): FWVLVVVGGVLACYSLLVTVAFIIFWV

[0132] Suitably, the variant may be at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO:99.

[0133] Suitably, the CAR may comprise a CD8α transmembrane domain. Suitably, the transmembrane domain may comprise the amino acid sequence set forth as SEQ ID NO: 165, or a variant having at least 80% identity to SEQ ID NO: 165. Example of CD8α TM domain (AA 183-203) (SEQ ID NO: 165): IYIWAPLAGTCGVLLLSLVIT

[0134] Suitably, the variant may be at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO:165.

[0135] Suitably, the CAR may comprise a CD28 hinge and transmembrane domain (optionally with a c-Myc tag). Suitably, said hinge and transmembrane domain may comprise the amino acid sequence set forth as SEQ ID NO: 100 or SEQ ID NO: 101, or a variant having at least 80% identity to SEQ ID NO: 100 or SEQ ID NO: 101. Example of a CD28 hinge domain and transmembrane domain (SEQ ID NO: 100): IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV Example of CD28 hinge domain and transmembrane domain with c-Myc tag (SEQ ID NO: 101): IEVEQKLISEEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV

[0136] Suitably, the variant may be at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO:100 or SEQ ID NO:101.

[0137] Suitably, the CAR may comprise a CD8α hinge domain and a CD28 transmembrane domain. Suitably, said hinge and transmembrane domain may comprise the amino acid sequence set forth as SEQ ID NO: 102, or a variant having at least 80% identity to SEQ ID NO: 102. Example of a CD8α hinge domain and CD28 transmembrane domain (SEQ ID NO: 102): TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWV

[0138] Suitably, the variant may be at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO:102.

[0139] Suitably, the CAR may comprise a CD28 hinge domain and a CD8α transmembrane domain. Suitably, said hinge and transmembrane domain may comprise the amino acid sequence set forth as SEQ ID NO: 166, or a variant having at least 80% identity to SEQ ID NO: 166. Example of a CD28 hinge domain and CD8α transmembrane domain (SEQ ID NO: 166): IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPIYIWAPLAGTCGVLLLSLVIT

[0140] Suitably, the variant may be at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO:166.

[0141] End Domain A CAR may comprise an endodomain comprising one or more intracellular signaling domains and, optionally, one or more costimulatory domains.

[0142] A CAR may comprise one or more intracellular signaling domains.

[0143] As used herein, the term "intracellular signaling domain" refers to the intracellular portion of a CAR that is involved in transmitting the message of an effective CAR that binds to a liver-specific antigen (e.g., ASGR) into Tregs to induce Treg functions, such as immunosuppressive function. Suitable intracellular signaling domains will be apparent to those skilled in the art. The intracellular signaling domain is necessary to transmit effector function signals and instruct Tregs to exert their specialized functions upon antigen binding. Examples of intracellular signaling domains include, but are not limited to, the zeta chain endodomain of a T cell receptor or its homologues (e.g., the eta chain, FcεR1γ and β chains, MB1 (Igα) chain, B29 (Igβ) chain, etc.), CD3 polypeptide domains (Δ, δ, and ε), syk family tyrosine kinases (e.g., Syk, ZAP 70, etc.), src family tyrosine kinases (e.g., Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transduction, such as CD2, CD5, and CD28. The intracellular signaling domain may be a human CD3 zeta signaling domain, FcyRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine-based activation motif (ITAM), or a combination thereof. Preferably, the intracellular signaling domain comprises a human CD3 zeta signaling domain. Preferably, the CD3 zeta signaling domain is located on the same polypeptide chain as an antigen recognition domain that binds to or specifically binds to ASGR. Preferably, the intracellular signaling domain comprises the amino acid sequence set forth in SEQ ID NO: 103, or a variant having at least 80% identity to SEQ ID NO: 103. Example of a CD3 zeta signaling domain (SEQ ID NO: 103): RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0144] Suitably, the variant may be at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO:103.

[0145] The intracellular signaling domain of the CAR may comprise a CD28 signaling domain. Suitably, the intracellular signaling domain may comprise the amino acid sequence set forth as SEQ ID NO: 104, or a variant having at least 80% identity to SEQ ID NO: 104. Example of a CD28 signaling domain (SEQ ID NO: 104): RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0146] Suitably, the variant may be at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO:104.

[0147] The intracellular signaling domain of the CAR may comprise a CD27 signaling domain. Suitably, the intracellular signaling domain may comprise the amino acid sequence set forth as SEQ ID NO: 105, or a variant having at least 80% identity to SEQ ID NO: 105. An example of a CD27 signaling domain (SEQ ID NO: 105): QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP

[0148] In one embodiment, the intracellular signaling domain comprises a signaling motif that is at least 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO:105.

[0149] Still other intracellular signaling domains will be apparent to those skilled in the art and may be used in connection with alternative embodiments of the present invention.

[0150] CARs may also contain one or more costimulatory domains. As used herein, a "costimulatory domain" refers to an intracellular portion of a CAR that promotes Treg function (e.g., immunosuppressive function), proliferation, and / or persistence. Thus, a CAR may contain a compound endodomain comprising one or more costimulatory domains fused to an intracellular signaling domain, such as CD3ζ. Such compound endodomains may be referred to as second-generation CARs that can simultaneously transmit activation and costimulatory signals after antigen recognition. The most commonly used costimulatory domain is the costimulatory domain of CD28, which provides the most potent costimulatory signal, i.e., immunological signal 2, that induces Treg proliferation. Suitable costimulatory domains will be apparent to those skilled in the art.

[0151] Suitably, the one or more costimulatory domains may comprise the amino acid sequence set forth as SEQ ID NO: 104 or 105, or a variant having at least 80% (e.g., 85, 90, 95, 97, 98 or 99%) identity to SEQ ID NO: 104 or 105. Suitably, the one or more costimulatory domains may comprise one or more signalling domains of the TNF receptor family, such as the signalling domains of OX40, 4-1BB, ICOS, or TNFRSF25.

[0152] Examples of signaling domains of OX40, 4-1BB, ICOS, or TNFRSF25 are shown below: The one or more costimulatory domains may comprise one or more of SEQ ID NOs: 106-109, or a variant having at least 80% identity to one or more of SEQ ID NOs: 106-109. An example of an OX40 signaling domain (SEQ ID NO: 106): ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI Example of a 41BB signaling domain (SEQ ID NO: 107) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL Example of an ICOS signaling domain (SEQ ID NO: 108) CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL Example of a TNFRSF25 signaling domain (SEQ ID NO: 109): TYTYRHCWPHKPLVTADEAGMEALTPPPATHLSPLDSAHTLLAPPDSSEKICTVQLVGNSWTPGYPETQEALCPQVTWSWDQLPSRALGPAAAPTLSPESPAGSPAMMLQPGPQLYDVMDAVPARRWKEFVRTLGLREAEIEAVEVEIGRFRDQQYEMLKRWRQQQPAGLGAVYAALERMGLDGCVEDLRSRLQRGP

[0153] The one or more costimulatory domains may comprise variants of one or more of the OX40, 4-1BB, ICOS and TNFRSF25 signaling domains that are at least 85, 90, 95, 97, 98 or 99% identical to any one of SEQ ID NOs: 106-109.

[0154] The endodomain of the CAR may comprise a CD28 signaling domain and a CD3 zeta signaling domain. Suitably, the endodomain may comprise the amino acid sequence set forth as SEQ ID NO: 110, or a variant having at least 80% identity to SEQ ID NO: 110. Examples of CD28 signaling domains and CD3 zeta signaling domains (SEQ ID NO: 110): RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0155] Suitably, the variant may be at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO:110.

[0156] In some embodiments, the CAR comprises an endodomain comprising a STAT5 association motif and a JAK1 and / or JAK2 binding motif.

[0157] Signal Transducer and Activator of Transcription 5 (STAT5) is a transcription factor involved in the IL-2 signaling pathway and plays a key role in the function, stability, and survival of Tregs by promoting the expression of genes such as FOXP3, IL2RA, and BCLXL. STAT5 requires phosphorylation for function and nuclear translocation. IL-2 ligation results in STAT5 phosphorylation by activating Jak1 and Jak3 kinases via specific signaling domains present in the IL-2Rβ and IL-2Rγ chains, respectively. Jak1 (or Jak2) can phosphorylate STAT5 without requiring Jak3, but transphosphorylation of both Jak1 and Jak3 can increase STAT5 activity, stabilizing its activity. As used herein, a "STAT5-associated motif" refers to an amino acid motif that contains tyrosine and can bind to the STAT5 polypeptide. The ability of an associated motif to bind to STAT5 can be determined using methods well known in the art for determining protein-protein interactions. For example, co-immunoprecipitation followed by Western blotting can be performed.

[0158] The present invention therefore provides engineered Tregs comprising a CAR capable of binding to a liver-specific antigen, preferably the asialoglycoprotein receptor, and the CAR also provides a STAT5-mediated pro-survival signal to Tregs only upon antigen binding. In particular, after antigen recognition, the CAR clusters and transmits a signal to the engineered Tregs via its intracellular signaling domain (endodomain). When the CAR contains an endodomain containing a STAT5-associated motif and a JAK1- and / or JAK2-binding motif, CAR clustering recruits and activates STAT5 and JAK1 and / or JAK2, providing a signal that enhances the function and survival of the engineered Tregs in an antigen-specific manner, independent of IL-2 acquisition in the microenvironment.

[0159] The engineered Tregs of the invention equipped with STAT5 signaling may be particularly effective in providing a survival advantage to the engineered CAR-Tregs after antigen recognition relative to the subject's general T cell population. Particularly in situations where IL-2 acquisition is reduced using immunosuppressants, such as in transplantation, STAT5 signaling of the CAR-Tregs provides an additional survival and functional advantage to the cells of the invention in an otherwise hostile microenvironment.

[0160] Preferably, the CAR endodomain may comprise two or more STAT5-associated motifs as defined herein.For example, the CAR endodomain may comprise two, three, four, five or more STAT5-associated motifs as defined herein.Preferably, the CAR endodomain may comprise two or three STAT5-associated motifs as defined herein.

[0161] Preferably, the STAT5 association motif is located in the cytoplasmic domain of a transmembrane protein, for example, the STAT5 association motif may be derived from an interleukin receptor (IL) receptor endodomain or a hormone receptor.

[0162] The CAR endodomain may comprise an amino acid sequence selected from any of the chains of interleukin receptors of which STAT5 is a downstream component, such as amino acids 266 to 551 of the IL-2 receptor β chain (National Center for Biotechnology Information (NCBI) Reference Sequence (REFSEQ): NP_000869.1, SEQ ID NO: 111), amino acids 265 to 459 of the IL-7R α chain (NCBI REFSEQ: NP_002176.2, SEQ ID NO: 112), IL-7RA 2Y chain truncated (SEQ ID NO: 113), amino acids 292 to 521 of the IL-9R chain (NCBI REFSEQ: NP_002177.2, SEQ ID NO: 114), amino acids 257 to 825 of the IL-4R α chain (NCBI REFSEQ: NPJD00409.1, SEQ ID NO: 115), amino acids 461 to 897 of the IL-3R β chain (NCBI Alternatively, the cytoplasmic domain comprising amino acids 314 to 502 of the IL-17Rβ chain (NCBI REFSEQ:NP_000386.1, SEQ ID NO: 116), or amino acids 314 to 502 of the IL-17Rβ chain (NCBI REFSEQ:NP_061195.2, SEQ ID NO: 117), may be used. The entire region of the cytoplasmic domain of an interleukin receptor chain may also be used. SEQ ID NO: 111-IL7RB (amino acid numbers (AA) 265 to 551 of NP_000869.1) NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPL QPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV SEQ ID NO: 112-IL7RA (AA 265-459 of NP_002176.2) KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSSNQEEAYVTMSSFYQNQ SEQ ID NO: 113-IL7RA 2Y truncated KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQPILTSLGSNQEEAYVTMSSFYQNQ SEQ ID NO: 114-IL9R (AA 292-521 of NP_002177.2) KLSPRVKRIFYQNVPSPAMFFQPLYSVHNGNFQTWMGAHGAGVLLSQDCAGTPQGALEPCVQEATALLTCGPARPWKSVALEEEQEGPGTRLPGNLSSEDVLPAGCTEWRVQTLA YLPQEDWAPTSLTRPAPPDSEGSRSSSSSSSSNNNNYCALGCYGGWHLSALPGNTQSSGPIPALACGSCDHQGLETQQGVAWVLAGHCQRPGLHEDLQGMLLPSVLSKARSWTF SEQ ID NO: 115-IL4RA (AA 257-825 of NPJD00409.1) KIKKEWWDQIPNPARRSRLVAIIIQDAQGSQWEKRSRGQEPAKCPHWKNCLTKLLPCFLEHNMKRDEDPHKAAKEMPFQGSGKSAWCPVEISKTVLWPESISVVRCVELFEAPVECEEEEEVEEEKGSFCASPESSRDDFQEG REGIVARLTESLFLDLLGEENGGFCQQDMGESCLLPPSGSTSAHMPWDEFPSAGPKEAPPWGKEQPLHLEPSPASPTQSPDNLTCTETPLVIAGNPAYRSFSNSLSQSPCPRELGPDPLLARHLEEVEPEMPCVPQLSEPT TVPQPEPETWEQILRRNVLQHGAAAAPVSAPTSGYQEFVHAVEQGGTQASAVVGLGPPGEAGYKAFSSLLASSAVSPEKCGFGASSGEEGYKPFQDLIPGCPGDPAPVPVPLFTFGLDREPPRSPQSSHLPSSSPEHLGLEP GEKVEDMPKPPLPQEQATDPLVDSLGSGIVYSALTCHLCGHLKQCHGQEDGGQTPVMASPCCGCCCGDRSSPPTTPLRAPDPSPGGVPLEASLCPASLAPSGISEKSKSSSSFHPAPGNAQSSSQTPKIVNFVSVGPTYMRVS SEQ ID NO: 116-IL3RB (AA 461-897 of NP_000386.1) RFCGIYGYRLRRKWEEKIPNPSKSHLFQNGSAELWPPGSMSAFTSGSPPHQGPWGSRFPELEGVFPVGFGDSEVSPLTIEDPKHVCDPPSGPDTTPAASDLPTEQPPSP QPGPPAASHTPEKQASSFDFNGPYLGPPHSRSLPDILGQPEPPQEGGSQKSPPPGSLEYLCLPAGGQVQLVPLAQAMGPGQAVEVERRPSQGAAGSPSLESGGGPAPPA LGPRVGGQDQKDSPVAIPMSSGDTEDPGVASGYVSSADLVFTPNSGASSVSLVPSLGLPSDQTPSLCPGLASGPPGAPGPVKSGFEGYVELPPIEGRSPRRSPRNNPVPP EAKSPVLNPGERPADVSPTSPQPEGLLVLQQVGDYCFLPGLGPGPLSLRSKPSSPGPGPEIKNLDQAFQVKKPPGQAVPQVPVIQLFKALKQQDYLSLPPWEVNKPGEVC SEQ ID NO: 117-IL17RB (AA 314-502 of NP_061195.2) RHERIKKTSFSTTTLLPPIKVLVVYPSEICFHHTICYFTEFLQNHCRSEVILEKWQKKKIAEMGPVQWLATQKKAADKVVFLLSNDVNSVCDGTCGKSEGSPSENSQDLFPLAFNLFCSDLRSQIHLHKYVVVYFREIDTKDDYNALSVCPKYHLMKDATAFCAELLHVKQQVSAGKRSQACHDGCCSL

[0163] The CAR endodomain may comprise a STAT5-associated motif, including an amino acid sequence set forth as one of SEQ ID NOs: 111-117, or a variant thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to one of SEQ ID NOs: 111-117. For example, the variant may bind to STAT5 at a level that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the binding level of the amino acid sequence set forth as one of SEQ ID NOs: 111-117. The variant or derivative may bind to STAT5 at a level similar to or identical to that of one of SEQ ID NOs: 111-117, or may bind to STAT5 at a higher level (e.g., at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% higher) than the amino acid sequence set forth as one of SEQ ID NOs: 111-117.

[0164] For example, the STAT5-associated motif may be derived from IL2Rβ, IL7Rα, IL-3Rβ (CSF2RB), IL-9R, IL-4Rα, IL-17Rβ, erythropoietin receptor, thrombopoietin receptor, growth hormone receptor, and prolactin receptor.

[0165] The STAT5 association motif may comprise the amino acid motif YXXF / L (SEQ ID NO: 118), where X is any amino acid.

[0166] Suitably, the STAT5 association motif may comprise the amino acid motif YCTF (SEQ ID NO: 119), YFFF (SEQ ID NO: 120), YLSL (SEQ ID NO: 121), or YLSLQ (SEQ ID NO: 122).

[0167] Suitably, the STAT5 association motif may comprise the amino acid motif YLSLQ (SEQ ID NO: 122).

[0168] The CAR endodomain may comprise one or more of the STAT5-associated motifs, including the amino acid motifs YCTF (SEQ ID NO: 119), YFFF (SEQ ID NO: 120), YLSL (SEQ ID NO: 121), and / or YLSLQ (SEQ ID NO: 122).

[0169] The CAR endodomain may comprise a first STAT5-associated motif comprising the amino acid motif YLSLQ (SEQ ID NO: 122) and a second STAT5-associated motif comprising the amino acid motif YCTF (SEQ ID NO: 119) or YFFF (SEQ ID NO: 120).

[0170] The CAR endodomain may contain the STAT5-associated motifs YLSLQ (SEQ ID NO: 122), YCTF (SEQ ID NO: 119), and YFFF (SEQ ID NO: 120).

[0171] As used herein, a "JAK1 and / or JAK2 binding motif" refers to a BOX motif that confers association with the tyrosine kinases JAK1 and / or JAK2. Suitable JAK1 and JAK2 binding motifs are described, for example, by Ferrao & Lupardus (Frontiers in Endocrinology; 2017; 8(71); incorporated herein by reference).

[0172] Methods well known in the art for determining protein-protein interactions may be used to determine whether the motif is capable of binding to JAK1 and / or JAK2, for example, co-immunoprecipitation followed by Western blotting.

[0173] The JAK1 and / or JAK2 binding motif may occur inherently within the cytoplasmic domain of a transmembrane protein, for example, the JAK1 and / or JAK2 binding motif may be derived from IFNLR1, IFNAR, IFNGR1, IL10RA, IL20RA, IL22RA, IFNGR2, or IL10RB.

[0174] The JAK1-binding motif may comprise an amino acid motif set forth as SEQ ID NOs: 123-129 or a variant thereof that is capable of binding to JAK1. For example, the variant may bind to JAK1 at a level that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the binding level of the amino acid sequence set forth as one of SEQ ID NOs: 123-129. The variant or derivative may be capable of binding to JAK1 at a level similar to or identical to that of one of SEQ ID NOs: 123-129, or may be capable of binding to JAK1 at a higher level (e.g., at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% higher) than the amino acid sequence set forth as one of SEQ ID NOs: 123-129. [Table 3]

[0175] A variant may contain one, two, or three amino acid differences compared to any of SEQ ID NOs: 123-129 and retain the ability to bind JAK1. The variant may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 123-129. In a preferred embodiment, the JAK1 binding domain may comprise SEQ ID NO: 123 or a variant thereof that is capable of binding to JAK1.

[0176] The JAK2-binding motif may comprise an amino acid motif set forth as SEQ ID NOs: 130-132 or a variant thereof capable of binding to JAK2. For example, the variant may bind to JAK2 at a level that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the binding level of the amino acid sequence set forth as one of SEQ ID NOs: 130-132. The variant or derivative may be capable of binding to JAK2 at a level similar to or identical to that of one of SEQ ID NOs: 130-132, or may be capable of binding to JAK2 at a higher level (e.g., at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% higher) than the amino acid sequence set forth as one of SEQ ID NOs: 130-132. [Table 4]

[0177] A variant may contain one, two, or three amino acid differences compared to any of SEQ ID NOs: 130-132 and retain the ability to bind JAK2.

[0178] The variant may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 130-132.

[0179] Methods well known in the art for determining protein-protein interactions can be used to determine whether a JAK1-binding motif or a JAK2-binding motif can bind to JAK1 or JAK2, for example, co-immunoprecipitation followed by Western blotting.

[0180] Preferably, the endodomain of the CAR described herein may be free of a "Signal Transducer and Activator of Transcription 3" (STAT3)-associated motif. STAT3 has been described as a deleterious signal for Treg stability and function. For example, STAT3 signaling promotes the expression of pro-inflammatory genes such as IL17, IL21, and IL22. Thus, the use of a CAR that does not contain a STAT3-associated motif provides particular advantages in the modified Tregs of the present invention.

[0181] The STAT3-associated motif may comprise the amino acid sequence YXXQ (SEQ ID NO: 133) (wherein X is any amino acid), which can bind to STAT3. Methods well known in the art for determining protein-protein interactions may be used to determine whether the STAT3-associated motif can bind to STAT3. For example, co-immunoprecipitation followed by Western blotting may be performed. Preferably, the CAR endodomain does not comprise the amino acid sequence YXXQ (SEQ ID NO: 133) (wherein X is any amino acid).

[0182] A "STAT3-associated motif" may refer to an amino acid motif that contains tyrosine and is capable of functionally binding to a STAT3 polypeptide (i.e., resulting in activation of the STAT3 polypeptide) when present in a Treg. Preferably, the CAR endodomain does not contain an amino acid motif that contains tyrosine and is capable of binding to a STAT3 polypeptide. For example, preferably, the CAR endodomain does not contain an amino acid motif that contains tyrosine and is capable of functionally binding to a STAT3 polypeptide (i.e., resulting in activation of the STAT3 polypeptide) when present in a Treg.

[0183] Preferably, the CAR endodomain is incapable of inducing productive STAT3 and / or STAT1 signaling when expressed in Tregs. In other words, when expressed in Tregs, the CAR may not be able to functionally bind to STAT3 and / or STAT1 and / or induce phosphorylation and activation of STAT3 and / or STAT1. Preferably, the CAR is incapable of inducing STAT3- and / or STAT1-dependent transcriptional activation when expressed in Tregs. Suitably, the IL2Rβ endodomain portion of the CAR may not contain a STAT3 association motif as defined herein.

[0184] Suitably, the CAR endodomain may comprise an IL2Rβ endodomain. Suitably, the CAR endodomain may comprise the amino acid sequence set forth as SEQ ID NO: 134, or a variant having at least 80% sequence identity to SEQ ID NO: 134. Example of an IL2Rβ endodomain (SEQ ID NO: 134) NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPL QPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV

[0185] The variant may be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:134.

[0186] Suitably, the CAR endodomain may comprise a truncated IL2Rβ endodomain. Suitably, the CAR endodomain may comprise the amino acid sequence set forth as SEQ ID NO: 135 or SEQ ID NO: 136, or a variant of SEQ ID NO: 135 or SEQ ID NO: 136 having at least 80% sequence identity thereto. IL2RB truncated - Example of Y510 (SEQ ID NO: 135) NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLPLNTDAYLSLQELQGQDPTHLV IL2RB truncated - Example of Y510 & Y392 (SEQ ID NO: 136) NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV

[0187] The variant may be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:135 or SEQ ID NO:136.

[0188] Transphosphorylation of both Jak1 / 2 and Jak3 can increase STAT5 activity, thereby stabilizing its activity. Preferably, the CAR endodomain described herein may further comprise a JAK3-binding motif. The term "JAK3-binding motif" as used herein refers to a BOX motif that binds to the tyrosine kinase JAK3. Suitable JAK3-binding motifs are described, for example, by Ferrao & Lupardus (Frontiers in Endocrinology; 2017; 8(71); incorporated herein by reference).

[0189] Methods well known in the art for determining protein-protein interactions may be used to determine whether the motif is capable of binding to JAK3, for example, co-immunoprecipitation followed by Western blotting.

[0190] The JAK3-binding motif may occur endogenously within the cytoplasmic domain of a transmembrane protein, for example, the JAK3-binding motif may be derived from the IL-2Rγ polypeptide.

[0191] The JAK3 binding motif may comprise the amino acid motif set forth as SEQ ID NO: 137 or 138 or a variant thereof that is capable of binding to JAK3. JAK3 binding motif 1 (SEQ ID NO: 137) ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEI JAK3 binding motif 2 (SEQ ID NO: 138) ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET

[0192] The variant may be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 137 or 138. In some embodiments, the CAR comprises an endodomain comprising a STAT5 association motif, a JAK1 and / or JAK2 binding motif, and a JAK3 binding motif.

[0193] In a preferred embodiment, the CAR endodomain comprises one or more JAK1 binding domains and at least one JAK3 binding domain. Suitably, the CAR endodomain may comprise SEQ ID NO: 139 or a variant having at least 85%, 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 139. SEQ ID NO: 139 (Example of an endodomain sequence containing CD28, IL2RG-T52, IL2RB-Y510, and CD3 zeta signaling domains) RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPE ISPLEVLERDKVTQLLPLNTDAYLSLQELQGQDPTHLVRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0194] Suitably, the CAR endodomain may comprise SEQ ID NO: 140 or a variant having at least 85%, 90%, 95%, 97%, 98%, or 99% identity to SEQ ID NO: 140. SEQ ID NO: 140 (an example of an endodomain sequence containing CD28, IL2RG-T52, IL7RA-2Y, and CD3 zeta signaling domains) RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIKKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFP QQPILTSLGSNQEEAYVTMSSFYQNQRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPG

[0195] Other domains In some embodiments, the CAR comprises one or more signal peptides. The CAR may comprise a leader sequence that directs the CAR to the endoplasmic reticulum pathway for expression on the cell surface. An example of a leader sequence is the CD8 leader (SEQ ID NO: 141). CD8 leader (SEQ ID NO: 141) MALPVTALLLPLALLLHAARP

[0196] In some embodiments, the CAR comprises one or more reporter domains, optionally in combination with a self-cleaving or cleavage domain.

[0197] Suitable reporter domains are well known in the art and include, but are not limited to, fluorescent proteins such as GFP. The use of a selectable marker is advantageous because the reporter domain allows Tregs into which a polynucleotide or vector of the invention has been introduced (such that the encoded CAR is expressed) to be selected and isolated from a starting cell population using common methods, such as, for example, flow cytometry.

[0198] Preferably, the reporter domain is a fluorescent protein, such as GFP, YFP, RFP, tdTomato, dsRed, or a variant thereof. In some embodiments, the fluorescent protein is GFP or a GFP variant. Preferably, the GFP variant comprises the amino acid sequence set forth as SEQ ID NO: 142, or a variant having at least 80% identity to SEQ ID NO: 142. Example of eGFP (SEQ ID NO: 142) MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDT LVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0199] Suitably, the variant may be at least 85%, 90%, 95%, 97%, 98% or 99% identical to SEQ ID NO:142.

[0200] Suitably, the reporter domain may be a luciferase-based reporter, a PET reporter (e.g., sodium iodide symporter (NIS)), or a membrane protein (e.g., CD34, low affinity nerve growth factor receptor (LNGFR)).

[0201] The nucleic acid sequence encoding the CAR and the reporter domain may be separated by a co-expression site, allowing each polypeptide to be expressed as an individual entity. Suitable co-expression sites are well known in the art, and include, for example, an internal ribosome entry site (IRES) or a self-cleaving peptide.

[0202] Thus, the CAR may further comprise a self-cleavage domain or cleavage domain. Such a sequence may self-cleave during protein synthesis or may be cleaved by common enzymes present in cells. Thus, including such a self-cleavage domain or cleavage domain in a polypeptide sequence allows the first and second polypeptides to be expressed as a single polypeptide that is subsequently cleaved into individual functional polypeptides. Suitable self-cleavage domains or cleavage domains include, but are not limited to, the P2A peptide, T2A peptide, E2A peptide, F2A peptide, and furin site (SEQ ID NOs: 143-148). P2A peptide-cleavage domain: GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 143) T2A peptide-cleavage domain: GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 144) E2A peptide-cleavage domain: GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 145) F2A peptide-cleavage domain: GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 146) Furin site-cleavage domain RXXR (SEQ ID NO: 147) (preferentially RRKR (SEQ ID NO: 148))

[0203] CAR example Examples of CARs for use in the present invention are provided below. A CAR may have a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to one or more of SEQ ID NOs: 149-152 or 167-169. Preferably, any such variants have at least partial functionality compared to SEQ ID NOs: 149-152 or 167-169. For example, the variants may have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the function of the amino acid sequence set forth as one of SEQ ID NOs: 149-152 or 167-169. The variant or derivative may have a level of function similar to or identical to that of one of SEQ ID NOs: 149-152 or 167-169, or may have a level of function that is higher (e.g., at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% higher) than the amino acid sequence set forth as one of SEQ ID NOs: 149-152 or 167-169. SEQ ID NO: 149 - Example of a CAR1 CAR comprising CD8 leader, ASGR1 VH, CD8α hinge, CD28 transmembrane, CD28 cytoplasmic, and CD3z cytoplasmic MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFTFEKYAMAWVRQAPGKGLEWVSRISARGVTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHKRHEHTRFDSWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF ACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 150 - Example of CAR2 CAR comprising CD8 leader, ASGR1 VH, c-Myc tagged CD28 hinge, CD28 transmembrane, CD28 cytoplasmic, and CD3z cytoplasmic MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFTFEKYAMAWVRQAPGKGLEWVSRISARGVTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHKRHEHTRFDSWGQGTLVTVSSAAAIEVEQKLISEEDLLDNEKSNGTIIHVKGKHLCPSPLFPG PSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 151 - Example of a CAR3 CAR comprising CD8 leader, ASGR1 VH, CD8α hinge, CD28 transmembrane, CD28 cytoplasmic, CD3z cytoplasmic, FP2A domain, and GFP MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFTFEKYAMAWVRQAPGKGLEWVSRISARGVTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHKRHEHTRFDSWGQGTLVTVSSTTTPAPRPPTPAPTIASQ PLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRRKRGSGATNFSLLKQAGDVEENPGPTRGGGATMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHM KQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK SEQ ID NO: 152 - Example of a CAR4 CAR comprising CD8 leader, ASGR1 VH, CD8α hinge, CD28 transmembrane, CD28 cytoplasmic, CD3z cytoplasmic, FP2A domain, and GFP MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFTFEKYAMAWVRQAPGKGLEWVSRISARGVTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHKRHEHTRFDSWGQGTLVTVSSAAAIEVEQKLISEEDLLD NEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRRKRGSGATNFSLLKQAGDVEENPGPTRGGGATMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHM KQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK SEQ ID NO: 167 - Example of a CAR5 CAR comprising a CD8 leader, ASGR1 VH (VH1, SEQ ID NO: 74), linker, ASGR1 VL (VK1, SEQ ID NO: 78), c-Myc tagged CD28 hinge, CD28 transmembrane, CD28 cytoplasmic, and CD3z cytoplasmic MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFTFEKYAMAWVRQAPGKGLEWVSRISARGVTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHKRHEHTRFDSWGQGTLVTVSS LVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQAIGRWLLWYQQKPGKAPKLLIGPGSRLRSGVPSRFSGSGSGTDFTLTISSLQPEDFVTYYCQQAYAWPPTFGQGTKVEIKRAAAIEVEQKLISEEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLAC YSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 168 - Example of CAR6 CAR comprising CD8 leader, ASGR1 VH (VH2, SEQ ID NO: 75), linker, ASGR1 VL (VK2, SEQ ID NO: 79), c-Myc tagged CD28 hinge, CD28 transmembrane, CD28 cytoplasmic, and CD3z cytoplasmic MALPVTALLLPLALLLHAARPELQLLEFGGGLVQPGGSLRLSCTTSGFTFSRYTMGWVRQAPGKGLEWVSAIGPPGSNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWVMLRGRFDYWGQGTLVTVSS LVTVSSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCRASQAIGRWLLWYQQKPGKAPKHLIPGSRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAYQLPVTFGQGTKVEIKRAAAIEVEQKLISEEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLAC YSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 169 - Example of a CAR7 CAR comprising a CD8 leader, ASGR1 VH (VH3, SEQ ID NO: 76), linker, ASGR1 VL (VK3, SEQ ID NO: 80), c-Myc tagged CD28 hinge, CD28 transmembrane, CD28 cytoplasmic, and CD3z cytoplasmic MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFTFEDYGMGWVRQAPGKGLEWVSAIGRNGSQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKLRRGRGLNTFTLDYWGQGTLVTVSS LVTVSSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCRASQAIGRWLLWYQQKPGKAPKLLIPGGSRLQSGVPSRFSGSGSGTDFTLTIGSLQPEDFATYYCQQAYSLPPTFGQGTKVEIKRAAAIEVEQKLISEEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLAC YSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0204] Variants, derivatives, and fragments In addition to the specific proteins, peptides, and nucleotides mentioned herein, the present invention also encompasses the use of derivatives, variants, and fragments thereof. The term "derivative" herein in relation to a protein or polypeptide of the present invention includes the substitution, mutation, modification, replacement, deletion, and / or addition of one (or more) amino acid residues from or to the sequence, provided that the resulting protein or polypeptide retains a desired function (e.g., the ability of the antigen-binding domain to bind to a target antigen, if the derivative or variant is an antigen-binding domain, or the ability of the domain to signal (e.g., activate or inactivate a downstream molecule)). The variant or derivative may have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the function compared to the corresponding reference sequence; a similar level of function or the same level of function compared to the corresponding reference sequence; or a higher level of function than the corresponding reference sequence, for example at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% increased function compared to the unmodified sequence.

[0205] Typically, amino acid substitutions may consist of, for example, one, two, or three to ten or twenty substitutions, provided that the resulting sequence retains the desired activity or ability, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the activity compared to the corresponding reference sequence, or at a similar or the same level of activity compared to the corresponding reference sequence, or at a higher level of activity than the corresponding reference sequence, e.g., at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% higher than the unmodified sequence. Amino acid substitutions may also include the use of non-naturally occurring analogs.

[0206] Proteins or peptides used in the present invention may also have deletions, insertions, or substitutions of amino acid residues, which may result in silent changes and result in functionally equivalent proteins. Deliberate amino acid substitutions may be made based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues, so long as the essential function is maintained. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with uncharged polar head groups with similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0207] Conservative substitutions may be made, for example, according to the following table: Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other. [Table 5]

[0208] Said derivatives may be homologues or variants. The terms "homologue" or "variant" as used herein refer to entities that have a certain homology with the wild-type amino acid sequence and the wild-type nucleotide sequence. The term "homology" can be equated with "identity".

[0209] A homologous or variant sequence may comprise an amino acid sequence or a nucleotide sequence that may be at least 50%, 55%, 65%, 75%, 85%, or 90% identical to the subject sequence, preferably at least 95%, 97%, or 99% identical. Typically, the homolog will have similar chemical properties / functions, e.g., contain the same binding site as the subject amino acid sequence or the amino acid sequence encoded by the subject nucleotide sequence. Although homology can also be considered in terms of similarity (i.e., amino acid residues with similar chemical properties / functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.

[0210] Homology comparisons can be performed visually, or more commonly, using readily available sequence comparison programs. These commercially available computer programs can calculate the percentage homology or identity between two or more sequences. Percent homology may also be calculated for consecutive sequences; that is, one sequence is aligned with the other, and each amino acid in one sequence is directly compared, residue by residue, with the corresponding amino acid in the other sequence. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

[0211] While this is a very simple and consistent method, it does not take into account that in a pair of sequences that are identical except for, for example, a single insertion or deletion, that insertion or deletion in the nucleotide sequence may cause the following codon to be moved out of alignment, potentially resulting in a significant decrease in percent homology when a global alignment is performed. As a result, most sequence comparison methods are designed to produce optimal alignments that take into account possible insertions and deletions without unduly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to maximize local homology.

[0212] However, these more complex methods assign a "gap penalty" to each gap that occurs during alignment, such that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible (reflecting a higher similarity between the two compared sequences) achieves a higher score than one with many gaps. An "affine gap cost" is typically used, which imposes a relatively high cost for the presence of a gap and a smaller penalty for each subsequent residue within the gap. This is the most commonly used gap scoring system. Higher gap penalties naturally produce optimized alignments with fewer gaps. Most alignment programs allow the gap penalty to be modified. However, it is preferable to use the default values ​​when using such software for sequence comparison. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0213] Calculating maximum percentage homology therefore first requires the creation of an optimal alignment, taking into account gap penalties. A suitable computer program for performing such alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Res. 12: 387). Examples of other software capable of performing sequence comparison include, but are not limited to, the BLAST package (see Ausubel et al. (1999) ibid - Ch. 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410) and the GENEWORKS comparison tool suite. Both BLAST and FASTA are available for offline and online searches (see Ausubel et al. (1999) ibid, pages 7-58 to 7-60). However, for some applications, it is preferable to use the GCG Bestfit program. Another tool called BLAST2 Sequences is also available for comparing protein and nucleotide sequences (see FEMS Microbiol. Lett. (1999) 174:247-50; FEMS Microbiol. Lett. (1999) 177:187-8).

[0214] Although the final percentage homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pairwise comparison. Instead, a scaled similarity score matrix is ​​commonly used, which assigns scores to pairwise comparisons based on chemical similarity or evolutionary distance. One example of such a matrix commonly used is the BLOSUM62 matrix (the default matrix for the BLAST suite of programs). GCG Wisconsin programs typically use either the public default values ​​or a custom symbol comparison table, if supplied (see user manual for further details). However, for some applications, it is preferred to use the default values ​​in the GCG package, and for other software, it is preferred to use a default matrix such as BLOSUM62. Preferably, percent identity is determined across the entire reference and / or query sequence.

[0215] Once the software has produced an optimal alignment, it is possible to calculate percent homology, preferably percent sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

[0216] A "fragment" typically refers to a selected region of a polypeptide or polynucleotide of interest in terms of function. Thus, a "fragment" refers to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide.

[0217] Such derivatives, variants, and fragments may be prepared using standard recombinant DNA techniques, such as site-directed mutagenesis. When an insertion is made, a sequence encoding the inserted portion is provided, along with 5' and 3' flanking regions corresponding to the naturally occurring sequence on either side of the insertion position. Synthetic DNA may be made that encodes the naturally occurring protein. The flanking regions contain appropriate restriction enzyme sites corresponding to sites in the naturally occurring sequence, so that the sequence can be cleaved with the appropriate enzyme(s) and the synthetic DNA ligated at the cleavage site. The DNA is then expressed in accordance with the invention to produce the encoded protein. These methods are merely illustrative of the many standard techniques well known in the art for manipulating DNA sequences; other known techniques may also be used.

[0218] Pharmaceutical Composition Pharmaceutical compositions comprising the engineered Tregs or CARs of the present invention are also provided. Pharmaceutical compositions are compositions comprising or consisting of a therapeutically effective amount of a pharmaceutically active agent, i.e., the Tregs. Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent, or excipient (including combinations thereof). "Pharmaceutically acceptable" includes the meaning that the formulation is sterile and pyrogen-free. The carrier, diluent, and / or excipient must be "acceptable" in the sense of having affinity for Tregs and not harmful to the recipient. Typically, the carrier, diluent, and / or excipient is a sterile, pyrogen-free physiological or infusion medium, although other acceptable carriers, diluents, and excipients may be used.

[0219] Acceptable carriers, diluents, and excipients for therapeutic use are well known in the pharmaceutical arts. The choice of pharmaceutical carrier, excipient, or diluent can be selected taking into account the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient, or diluent a suitable binder, lubricant, suspending agent, coating agent, or solubilizing agent.

[0220] Examples of pharmaceutically acceptable carriers include, for example, water, saline, alcohol, silicone, wax, petrolatum, vegetable oil, polyethylene glycol, propylene glycol, liposomes, sugar, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oils, fatty acid monoglycerides, fatty acid diglycerides, petroleum fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

[0221] The Tregs or pharmaceutical compositions of the present invention may be administered in a manner suitable for treating and / or preventing the diseases described herein. The dosage and frequency of administration are determined by factors such as the condition of the subject and the type and severity of the subject's disease, and appropriate dosages may be determined through clinical trials. The pharmaceutical compositions may be appropriately formulated. The Tregs or pharmaceutical compositions described herein may be administered parenterally, e.g., intravenously, or by infusion techniques. The Tregs or pharmaceutical compositions may be administered in the form of a sterile aqueous solution containing other substances, e.g., sufficient salts or glucose, to make the solution isotonic with blood. The aqueous solution may be suitably buffered (preferably pH 3-9). The pharmaceutical composition may be suitably formulated. The preparation of suitable parenteral formulations under sterile conditions may be readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

[0222] The pharmaceutical composition may comprise the Tregs of the invention in an infusion vehicle, such as a sterile isotonic solution, and may be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0223] The Tregs or pharmaceutical compositions may be administered in a single dose or multiple doses. In particular, the Tregs or pharmaceutical compositions may be administered in a single, disposable dose. The pharmaceutical compositions may be appropriately formulated.

[0224] The Tregs or pharmaceutical compositions may be administered at various dosages (e.g., measured as cells / kg, cells / subject, etc.) depending on the disease and subject being treated and the route of administration. In any event, a physician will determine the actual dosage most appropriate for an individual subject, which will vary depending on the subject's age, weight, and response. Typically, however, for the Tregs of the present invention, a dosage of 5x10 per subject is used. 7 ~3x10 9 cells, or 10 8 ~2x10 9 The Tregs may be administered in a single dose. The Tregs may be appropriately modified for use in a pharmaceutical composition. For example, the Tregs may be cryopreserved and thawed at an appropriate time before being infused into a subject.

[0225] The pharmaceutical composition may further comprise one or more other therapeutic agents, such as lymphodepleting agents (e.g., thymoglobulin, Camptus-1H, anti-CD2 antibodies, anti-CD3 antibodies, anti-CD20 antibodies, cyclophosphamide, fludarabine), mTOR inhibitors (e.g., sirolimus, everolimus), agents that inhibit costimulatory pathways (e.g., anti-CD40 / CD40L, CTAL4Ig), and / or specific cytokines (IL-6, IL-17, TNFalpha, IL18).

[0226] Further included in the present invention is the use of kits comprising the Tregs and / or pharmaceutical compositions of the present invention. Preferably, the kits are for use in the methods and uses described herein, e.g., the therapeutic methods described herein. Preferably, the kits include instructions for use of the components of the kit.

[0227] Methods for treating and / or preventing diseases The modified Tregs may be administered to a subject with an existing disease or condition to lessen, alleviate, or ameliorate at least one symptom associated with the disease and / or to slow, reduce, or stop the progression of the disease.

[0228] For example, engineered Tregs may be administered to a subject with a liver disease (e.g., liver transplant rejection, liver GvHD, autoimmune liver disease, liver inflammation, liver failure, etc.) to reduce, alleviate, or ameliorate at least one symptom of the disease, such as jaundice, dark urine, itching, abdominal distension, tenderness, fatigue, nausea or vomiting, and / or loss of appetite. At least one symptom may be reduced, alleviated, or ameliorated by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%, or at least one symptom may be completely cured.

[0229] Engineered Tregs may be administered to subjects with liver disease (e.g., liver transplant rejection, liver GvHD, autoimmune liver disease, liver inflammation, liver failure, etc.) to slow, reduce, or stop the progression of the disease by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to subjects not administered the engineered Tregs, or may completely stop the progression of the disease. Alternatively, the engineered Tregs may be administered to a subject who is not yet afflicted and / or does not exhibit symptoms of the disease to prevent the disease or to alleviate or prevent at least one symptom associated with the disease, although the subject may be predisposed to the disease or may be at risk of developing the disease.

[0230] For example, modified Tregs may be administered to a subject not already suffering from or not showing symptoms of liver disease (e.g., liver transplant rejection, liver GvHD, autoimmune liver disease, liver inflammation, liver failure, etc.) to alleviate or prevent at least one symptom of liver disease, such as jaundice, dark urine, itching, abdominal distension, tenderness, fatigue, nausea or vomiting, and / or loss of appetite. The at least one symptom may be alleviated by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a subject not administered modified Tregs, or the at least one symptom may be prevented entirely.

[0231] The modified Tregs may be administered to a subject who does not already have and / or exhibits symptoms of liver disease (e.g., liver transplant rejection, liver GvHD, autoimmune liver disease, liver inflammation, liver failure, etc.) to prevent liver disease. Liver disease may be reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a subject not administered the modified Tregs, or liver disease may be prevented entirely.

[0232] Liver diseases include conditions such as liver transplant rejection, liver GvHD, autoimmune liver disease, liver inflammation, and liver failure, and in particular liver diseases associated with an undesirable increase in immune responses in a subject, which can lead to liver damage or destruction. In certain embodiments, liver diseases may not include, for example, liver cancer, and more particularly, hepatocellular carcinoma (HCC). Thus, preferably, liver diseases include conditions that may be treated or prevented by reducing immune responses, particularly in or near the liver (e.g., by at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% reduction). Preferably, the therapeutic methods of the present invention may comprise administering to a subject an engineered Treg according to the present invention, or obtainable (e.g., obtained) by a method according to the present invention, or a polynucleotide or vector defined herein (e.g., in a pharmaceutical composition described herein).

[0233] Suitably, the method for treating and / or preventing a disease may comprise administering to a subject the modified Tregs of the present invention (e.g., in a pharmaceutical composition as described herein).

[0234] The method may include the following steps: (i) isolating or providing a cell-containing sample; (ii) introducing a polynucleotide or vector as defined herein into said cells; and (iii) administering to a subject the cells from (ii).

[0235] Suitably, said cells are Tregs as defined herein.

[0236] Suitably, an enriched Treg population may be isolated and / or generated from the cell-containing sample prior to and / or after step (ii) of the method. For example, isolation and / or generation may be performed prior to and / or after step (ii) to isolate and / or generate an enriched Treg sample. Enrichment may be performed after step (ii) to enrich for cells and / or Tregs comprising the CAR, polynucleotide, and / or vector of the invention.

[0237] Suitably, the polynucleotide or vector may be introduced by transduction and / or gene transfer.

[0238] Preferably, the cells are autologous and / or allogeneic cells.

[0239] Preferably, the engineered Tregs may be administered in combination with one or more other therapeutic agents, such as lymphodepleting agents (e.g., thymoglobulin, campath-1H, anti-CD2 antibodies, anti-CD3 antibodies, anti-CD20 antibodies, cyclophosphamide, fludarabine), mTOR inhibitors (e.g., sirolimus, everolimus), agents that inhibit costimulatory pathways (e.g., anti-CD40 / CD40L, CTAL4Ig), and / or specific cytokines (IL-6, IL-17, TNFalpha, IL-18). The engineered Tregs may be administered simultaneously with or sequentially (i.e., before or after) the one or more other therapeutic agents.

[0240] Liver transplantation Liver transplantation is currently the only available treatment option for patients with end-stage liver disease. Despite recent advances in immunosuppressants, acute allograft rejection remains a common complication of liver transplantation, with an incidence of 20%–40% of liver transplants. In most cases, rejection occurs within one month after liver transplantation. Early rejection episodes do not significantly compromise long-term transplant success or patient outcomes. In contrast, late allograft rejection (more than 3–6 months after liver transplantation) is associated with poor graft survival (Dogan, N., et al., 2018. Journal of International Medical Research, 46(9), pp.3979–3990).

[0241] The present invention provides a method for inducing tolerance to a liver transplant, comprising administering an engineered Treg or pharmaceutical composition of the present invention to a subject, preferably a mammal, preferably a human.

[0242] As used herein, "inducing tolerance to a liver transplant" refers to inducing tolerance to the transplanted liver in a recipient. In other words, inducing tolerance to a liver transplant means reducing the level of the recipient's immune response to the donor transplant organ. Inducing tolerance to the transplanted liver may reduce the incidence of rejection, reduce the amount of immunosuppressant required by the transplant patient, or even allow the immunosuppressant to be discontinued.

[0243] The present invention also provides a method for treating and / or preventing liver transplant rejection, the method comprising administering the modified Treg or pharmaceutical composition of the present invention to a subject. Preferably, the subject is a mammal, preferably a human. Preferably, treating and / or preventing liver transplant rejection may mean reducing the amount of immunosuppressant required by the liver transplant recipient or allowing the immunosuppressant to be discontinued.

[0244] In one embodiment, the subject is a liver transplant recipient undergoing immunosuppressive therapy. In one embodiment, the present invention promotes liver tissue repair and / or liver regeneration, preferably in addition to inducing tolerance to the liver transplant tissue or treating and / or preventing liver transplant rejection.

[0245] Graft-versus-host disease The present invention provides a method for treating and / or preventing hepatic graft-versus-host disease, the method comprising administering the engineered Treg or pharmaceutical composition of the present invention to a subject. The subject may be a liver transplant recipient. Suitably, the subject is a mammal, preferably a human.

[0246] GvHD is a complication that commonly occurs after receiving a tissue transplant from a genetically different individual. GvHD is commonly associated with stem cell transplants, such as those that occur with bone marrow transplants. GvHD also applies to other forms of transplanted tissue, such as liver transplants. White blood cells from the donor's immune system that remain in the donated tissue (transplant) recognize the recipient (host) as foreign (non-self). The white blood cells in the transplanted tissue then attack the recipient's own body cells, causing GvHD. Acute graft-versus-host disease is typically characterized by selective damage to the liver, skin, mucous membranes, and digestive tract. Therefore, the subject may have liver damage, i.e., liver GvHD.

[0247] In some embodiments, the subject is a transplant recipient, and the transplant is selected from a liver, kidney, heart, lung, pancreas, small intestine, stomach, bone marrow, vascularized composite tissue transplant, and skin transplant. Preferably, the subject is a liver transplant recipient.

[0248] In one embodiment, the subject is a liver transplant recipient undergoing immunosuppressive therapy. In one embodiment, in addition to treating and / or preventing liver GvHD, the present invention promotes liver tissue repair and / or liver regeneration.

[0249] Autoimmune Liver Disease The present invention provides a method for treating and / or preventing autoimmune liver disease, comprising administering an engineered Treg or pharmaceutical composition of the present invention to a subject. Suitably, the subject is a mammal, preferably a human.

[0250] Preferably, the autoimmune liver disease is a chronic autoimmune liver disease.

[0251] The autoimmune liver disease may be selected from one or more of autoimmune hepatitis, primary biliary cholangitis, and / or (primary) sclerosing cholangitis. Autoimmune liver diseases are chronic, slowly progressing inflammatory liver diseases that may have overlapping characteristics (Decock, S., McGee, P. and Hirschfield, GM, 2009. Bmj, 339, p.b3305).

[0252] Autoimmune hepatitis is usually a relapsing immune-mediated liver disease. Clinically, patients present with joint pain and fatigue, and one-third of patients develop cirrhosis. Early laboratory abnormalities are characterized by elevated liver enzymes (transaminases) (Decock, S., McGee, P. and Hirschfield, GM, 2009. Bmj, 339, p.b3305). Autoimmune hepatitis can also present as an acute illness that, if untreated, can lead to liver failure and death.

[0253] Primary biliary cholangitis (PBC) is a slowly progressive, chronic, cholestatic disease characterized by small duct granulomatous cholangitis and biochemical cholestasis (elevated alkaline phosphatase). Currently, 60% of patients diagnosed with PBC are asymptomatic, and most have noncirrhotic disease. When symptoms are present, fatigue, itching, and discomfort in the right upper quadrant are common but do not indicate the severity of the disease. However, even in the absence of symptoms, PBC patients have significantly reduced long-term survival compared to the general population (Decock, S., McGee, P., and Hirschfield, GM, 2009. Bmj, 339, p.b3305).

[0254] Primary sclerosing cholangitis (PSC) is a chronic cholestatic liver disease characterized by fibroinflammatory destruction of intrahepatic and / or extrahepatic bile ducts. There is currently no cure for PSC. Once symptomatic, there is an approximately 50% chance of needing transplantation and a 10% risk of cholangiocarcinoma over 10 years (Decock, S., McGee, P. and Hirschfield, GM, 2009. Bmj, 339, p.b3305).

[0255] In one embodiment, the present invention promotes liver tissue repair and / or liver regeneration in addition to treating and / or preventing autoimmune liver disease.

[0256] Liver inflammation The present invention provides a method for treating and / or preventing inflammatory liver damage, comprising administering the modified Treg or pharmaceutical composition of the present invention to a subject. Suitably, the subject is a mammal, preferably a human.

[0257] Inflammation of liver tissue is also known as hepatitis. Hepatitis can be acute or chronic. Acute hepatitis may resolve spontaneously, but it can progress to chronic hepatitis or, rarely, acute liver failure. Over time, the chronic condition can lead to scarring of the liver (cirrhosis), chronic liver failure, and / or liver cancer. Signs and symptoms of cirrhosis include jaundice, ascites (accumulation of fluid in the abdominal cavity), fatigue, and hepatic encephalopathy (brain dysfunction caused by liver failure).

[0258] Causes of hepatitis can be broadly divided into infectious, metabolic, alcoholic, ischemic, autoimmune, and genetic categories. Thus, in some embodiments, hepatitis is selected from infectious, metabolic, alcoholic, ischemic, autoimmune, and genetic hepatitis. Infectious hepatitis includes viral, parasitic, and bacterial hepatitis. Viral hepatitis is inflammation of the liver caused by a viral infection. It can present as an acute condition with a relatively rapid onset, such as a new infection, or as a chronic condition. The most common causes of viral hepatitis are five unrelated hepatotropic viruses: hepatitis types A, B, C, D, and E. Other viruses, such as cytomegalovirus, Epstein-Barr virus, and yellow fever, can also cause liver inflammation. Dozens of cases of herpes simplex virus causing viral hepatitis have also been reported.

[0259] Parasitic hepatitis is inflammation of the liver caused by parasitic infection. Among protozoans, Trypanosoma cruzi, Leishmania, and Plasmodium can all cause liver inflammation. Among helminths, the tapeworm Tapeworm Diplocarpon gonorrhoeae infects the liver, causing the characteristic hepatic echinococcosis. Liver flukes, such as Fasciola hepatica and Clonorchis sinensis, infect the bile duct and cause advanced hepatitis and liver fibrosis. Bacterial hepatitis is inflammation of the liver caused by bacterial infection. Acute hepatitis is caused by Neisseria meningitidis, Neisseria gonorrhoeae, Clostridium henselae, Borrelia burgdorferi, Salmonella, Brucella, and Campylobacter. Chronic or granulomatous hepatitis is seen in infections with Mycobacteria, Tropheryma whippurii, Treponema pallidum, Coxiella, and Rickettsia. Alcoholic hepatitis is inflammation of the liver due to excessive alcohol consumption. Alcoholic hepatitis can have a chronic course that can lead to cirrhosis, liver failure, and / or cancer, or it can present as an acute condition. Severe cases of acute alcoholic hepatitis have a 50% three-month mortality rate. Many chemical agents, including pharmaceuticals, industrial toxins, and herbal and dietary supplements, can also cause toxic hepatitis.

[0260] Nonalcoholic steatohepatitis, the most common form of metabolic hepatitis, is within the spectrum of nonalcoholic fatty liver disease (NAFLD). Nonalcoholic fatty liver disease occurs in people who rarely or never use alcohol but instead have a high association with metabolic syndrome, obesity, insulin resistance and diabetes, and hypertriglyceridemia. Nonalcoholic fatty liver disease can result in nonalcoholic steatohepatitis. Steatohepatitis is a type of fatty liver disease characterized by liver inflammation accompanied by hepatic fat accumulation, which can lead to cirrhosis, liver failure, and / or liver cancer. Ischemic hepatitis, also known as ischemic liver injury or shock liver, is a condition defined as acute liver damage caused by inadequate blood flow (and resulting in inadequate oxygen delivery) to the liver. Genetic causes of hepatitis include alpha-1 antitrypsin deficiency, hemochromatosis, and Wilson's disease.

[0261] In some embodiments, the liver inflammation has no identifiable cause.

[0262] In one embodiment, the present invention promotes liver tissue repair and / or liver regeneration in addition to treating and / or preventing inflammatory liver damage.

[0263] Liver repair or regeneration The present invention provides a method for promoting liver tissue repair and / or liver regeneration, comprising administering the modified Tregs or pharmaceutical compositions of the present invention to a subject. Suitably, the subject is a mammal, preferably a human. The subject may have cirrhosis, acute liver failure, or acutely exacerbated chronic liver failure.

[0264] As used herein, "liver regeneration" may refer to the intact reproduction of liver structure and function after injury (Cordero-Espinoza, L. and Huch, M., 2018. The Journal of Clinical Investigation, 128(1), pp. 85-96). For example, liver regeneration may result in the recovery of at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the original liver mass. The methods of the present invention may also reduce the time it takes to achieve maximal liver mass, for example, by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a subject not administered the engineered Tregs or pharmaceutical composition.

[0265] As used herein, "liver tissue repair" may refer to the restoration of liver structure and function, along with scarring (i.e., fibrosis), following injury (e.g., chronic injury) (Cordero-Espinoza, L. and Huch, M., 2018. The Journal of Clinical Investigation, 128(1), pp.85-96). This may be characterized by the replacement of functional parenchyma with a reticular extracellular matrix (ECM). The structure of the liver may be altered, and optimal function may be hindered. For example, liver repair may result in the restoration of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% of the original liver function. The methods of the present invention may reduce the time it takes to reach maximal liver function, for example, by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a subject not administered the engineered Tregs or pharmaceutical composition.

[0266] The modified Tregs of the present invention may express genes involved in liver regeneration and tissue repair to promote robust liver repair and / or regeneration.

[0267] Primary adult hepatocytes are long-lived and do not normally divide, but they maintain the ability to proliferate in response to inflammatory injury or after partial hepatectomy. This ability is most clearly demonstrated in rodents by the two-thirds partial hepatectomy model. In this model, two-thirds of the liver is surgically removed, and the remaining liver expands until the original liver mass is restored—approximately one week after surgery—after which the regenerative process ceases. In humans, liver regeneration occurs most frequently after liver injury due to ischemia or hepatitis (Taub, R., 2004. Nature Reviews Molecular Cell Biology, 5(10), pp. 836–847). Similar phenomena occur in humans undergoing partial hepatectomy for the treatment of liver tumors or during living donor liver transplantation. Furthermore, liver regeneration occurs in humans after hepatocyte injury due to ischemia, intoxication, and acute or chronic hepatitis.

[0268] Preferably, the liver may be injured and / or damaged by hepatitis. In some embodiments, in addition to promoting liver tissue repair and / or liver regeneration, the modified Tregs of the present invention also treat hepatitis. The hepatitis may be selected from infectious hepatitis, metabolic hepatitis, alcoholic hepatitis, ischemic hepatitis, autoimmune hepatitis, and hereditary hepatitis. In one embodiment, the hepatitis may not be fulminant or chronic hepatitis.

[0269] Liver regeneration has made it possible to use liver segments from living donors for transplantation, thereby increasing the number of organs available for transplantation. The increase in the number of liver transplants has been achieved by using tissue from living related donors and small-sized transplant organs, but for this, at least some liver regeneration or repair is necessary for the transplant to be successful (Taub, R., 2004. Nature reviews Molecular cell biology, 5(10), p.836-847).

[0270] Thus, the subject may be a liver transplant recipient or a patient undergoing partial hepatectomy (e.g., as a living donor for a liver transplant or because of a liver tumor requiring surgical resection). The liver may be a transplanted liver. Humans with any liver condition, including cirrhosis (liver fibrosis), steatosis (fatty liver), and even conditions associated with aging, have impaired liver regeneration and, as a result, increased morbidity and mortality in response to liver injury or damage (Taub, R., 2004. Nature reviews Molecular cell biology, 5(10), p.836-847).

[0271] Thus, the subject may have impaired liver regeneration, preferably due to one or more of acute liver failure, cirrhosis, acutely exacerbated chronic liver failure, hepatitis, steatosis, steatohepatitis, and aging.

[0272] In one embodiment, the present invention promotes liver tissue repair and / or liver regeneration, and also treats and / or prevents immune-mediated damage. For example, the present invention may induce tolerance to liver transplant tissue in a subject, or treat and / or prevent liver transplant rejection, hepatic graft-versus-host disease (GvHD), autoimmune liver disease, or inflammatory liver damage in a subject.

[0273] Preferred CARs for use in these embodiments for repair and / or regeneration (and other embodiments involving repair and / or regeneration described herein) are capable of increasing or stimulating albumin production, e.g., increasing albumin concentration or levels and / or stimulating albumin production in the liver, e.g., in liver cells or liver tissue, e.g., hepatocytes.

[0274] Thus, yet another aspect of the present invention provides modified regulatory T cells (Tregs) comprising a chimeric antigen receptor (CAR), or a pharmaceutical composition comprising said modified Tregs, for use in increasing or stimulating albumin production, or for use in increasing albumin concentrations or levels and / or stimulating albumin production, in liver tissue or cells of a subject, wherein said CAR comprises a liver-specific antigen recognition domain.

[0275] In another aspect, this aspect of the invention provides a method of increasing or stimulating albumin production or increasing albumin levels in liver tissue or cells of a subject, the method comprising administering to the subject engineered regulatory T cells (Tregs) comprising a chimeric antigen receptor (CAR), or a pharmaceutical composition comprising the engineered Tregs, wherein the CAR comprises a liver-specific antigen recognition domain.

[0276] In another aspect, this aspect of the invention provides the use of an engineered regulatory T cell (Treg) comprising a chimeric antigen receptor (CAR), or a pharmaceutical composition comprising said engineered Treg, in the manufacture of a medicament or composition for use in increasing or stimulating albumin production, or for use in increasing albumin concentration or levels and / or stimulating albumin production, in liver tissue or cells of a subject, wherein said CAR comprises a liver-specific antigen recognition domain. Suitable and preferred liver-specific antigen recognition domains, such as antigen recognition domains that specifically bind to ASGR, are described elsewhere herein. Other preferred embodiments for this aspect are also described elsewhere herein.

[0277] Hepatocytes typically comprise the majority of liver mass, and albumin levels or albumin concentrations in hepatocytes, or in the supernatants obtained when hepatocyte or liver tissue samples are cultured in vitro or in a patient's blood, are markers of differentiation, function, and viability of liver cells, including hepatocytes. Thus, determining the effect of Tregs comprising a chimeric antigen receptor (CAR) on albumin levels in such cells provides a measure or indicator of the ability of CAR Tregs to mediate trophic effects (e.g., trophic signals that promote hepatocyte function and / or viability) and / or cytoprotective effects on liver cells or liver tissue in response to antigenic stimulation.

[0278] Tregs comprising a chimeric antigen receptor (CAR) capable of increasing or stimulating albumin production or increasing albumin levels or concentrations can be identified using suitable assays. Suitable assays are well known in the art. For example, routine in vitro assays can be readily employed to measure albumin levels in appropriate liver cells or in supernatants from these cells (e.g., to investigate the level of secreted albumin). Levels can be conveniently measured and compared in the presence and absence of appropriate CAR Tregs. Hepatocytes, particularly human hepatocytes, such as primary human hepatocytes, are particularly suitable. Example 14 provides an exemplary assay. As described above, upon antigen stimulation, such CARs can in turn mediate trophic and / or cytoprotective effects on liver cells, making them particularly suitable for use in aspects of the present invention involving liver repair and / or regeneration. Albumin levels or concentrations in patients following treatment with the modified Tregs described herein can be measured via blood samples using routine procedures, as described above.

[0279] As used herein, reference to an increase or stimulation (or equivalent thereof) of albumin production or albumin level or concentration in liver cells or liver tissue includes any measurable increase or augmentation or improvement when compared to a suitable control. Preferably, such an increase or the like is a significant increase, preferably a clinically significant and statistically significant increase, e.g., with a probability value of <0.05 when compared to the level or value of a suitable control.

[0280] Suitable controls will be readily identified by one skilled in the art and would include, for example, the production, levels or concentration of albumin observed in the presence of a CAR Treg of the invention (e.g., a transduced CAR Treg of the invention) in comparison to the absence of said CAR Treg (e.g., in comparison to an untreated sample, such as in comparison to an appropriate control medium alone), or, for example, the production, levels or concentration of albumin observed in the presence of a CAR Treg of the invention (e.g., a transduced CAR Treg of the invention) in comparison to the presence of untransduced Tregs (e.g., activated or non-activated / resting untransduced Tregs).

[0281] In embodiments where increased albumin production, levels, or concentrations in cells of the liver are observed with the CAR Tregs of the invention (e.g., the transduced CAR Tregs of the invention), it is preferred that an improvement or increase in albumin levels or concentrations of at least (or up to) 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 7-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, or 50-fold is observed. For example, an improvement or increase in albumin levels or concentrations of at least (or up to) 5-fold, 7-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, or 50-fold may be observed with preferred CAR Tregs of the invention in comparison to the levels or concentrations observed in the absence of the CAR Tregs of the invention, e.g., compared to an untreated sample or appropriate control medium alone. For example, in comparison to the levels observed in non-transduced or non-gene-transduced Tregs, an improvement or increase in albumin levels or concentrations of at least (or up to) 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 7-fold, 10-fold may be observed in preferred CAR Tregs of the present invention when compared to, for example, non-transduced activated or non-activated / resting Tregs.

[0282] Liver fibrosis, cirrhosis, acute liver failure, and acute-onset chronic liver failure The present invention provides methods for treating and / or preventing liver fibrosis, cirrhosis, acute liver failure, or acutely exacerbated chronic liver failure.

[0283] Amelioration of liver fibrosis, cirrhosis, acute liver failure, or acutely exacerbated chronic liver failure typically requires treating and / or preventing immune-mediated damage to the liver and promoting liver regeneration.

[0284] Liver fibrosis is a major cause of death because it alters the structure of the organ and prevents its normal function. Liver fibrosis occurs as a result of the wound healing process caused by chronic liver diseases, such as viral hepatitis, alcoholic liver disease, nonalcoholic fatty liver disease, and other liver disorders. Excessive accumulation of extracellular matrix (ECM), rich in fibrillar collagen, is a typical manifestation of liver fibrosis. Excessive accumulation of ECM alters the normal structure of the liver, resulting in pathophysiological damage to the organ (Suk, KT and Kim, DJ, 2015. World Journal of Hepatology, 7(3), p.607).

[0285] Liver cirrhosis is defined as an advanced stage of liver fibrosis, accompanied by the destruction of the hepatic vasculature and liver structure. Histologically, cirrhosis occurs when small nodules of fibrous tissue are formed in response to chronic injury. As a result, liver structure collapses due to liver fibrosis and / or cirrhosis, hemodynamic instability occurs, and portal hypertension develops (Suk, KT and Kim, DJ, 2015. World Journal of Hepatology, 7(3), p.607).

[0286] Acute liver failure is defined herein as the sudden development of hepatocellular dysfunction, particularly coagulopathy and encephalopathy, in patients without pre-existing liver disease. For example, "acute liver failure" may be defined as the onset of encephalopathy within 26 weeks of the onset of liver symptoms. This may be further classified as "fulminant liver failure," which requires the onset of encephalopathy within 8 weeks, and "subfulminant," which specifies the onset of encephalopathy between 8 and 26 weeks. Alternatively, "hyperacute" is defined as onset within 7 days, "acute" as onset between 7 and 28 days, and "subacute" as onset between 28 and 24 weeks.

[0287] Acute-onset chronic liver failure is characterized by acute decompensation of chronic liver disease, which is associated with organ damage and high short-term mortality. Alcoholic and chronic viral hepatitis are the most common underlying liver diseases (Hernaez, R., et al., 2017. Gut, 66(3), pp.541-553).

[0288] Polynucleotides The present invention further provides a polynucleotide encoding a CAR of the present invention.

[0289] Polynucleotides of the present invention may comprise DNA or RNA. They may be single-stranded or double-stranded. Those skilled in the art will understand that many different polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code. Furthermore, it will be understood that those skilled in the art may use routine techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by a polynucleotide of the present invention to reflect the codon usage of the particular host organism in which the polypeptide of the present invention will be expressed.

[0290] The polynucleotides may be modified by any method available in the art in order to enhance the in vivo activity or lifespan of the polynucleotides of the invention.

[0291] Polynucleotides, such as DNA polynucleotides, may be produced recombinantly, synthetically, or by any means available to those of skill in the art. Polynucleotides may be cloned using standard techniques.

[0292] Longer polynucleotides will generally be generated using recombinant DNA techniques, for example, using polymerase chain reaction (PCR) cloning techniques. This will involve creating a pair of primers (e.g., about 15-30 nucleotides) that flank the target sequence to be cloned, contacting the primers with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions that result in amplification of the desired region, isolating the amplified fragment (e.g., by purifying the reaction mixture on an agarose gel), and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable vector.

[0293] The polynucleotides used in the present invention may be codon-optimized. Codon optimization has been previously described in WO1999 / 41397 and WO2001 / 79518. Different cells use different codons. This codon bias corresponds to a bias in the relative abundance of certain tRNAs in a cell type. By changing the codons in the sequence to match the relative abundance of the corresponding tRNA, expression can be increased. Similarly, expression can be decreased by deliberately selecting codons whose corresponding tRNAs are known to be scarce in a particular cell type. In this way, the degree of translational control can be further increased.

[0294] Examples of nucleotide sequences encoding exemplary CARs of the invention (SEQ ID NOS: 149-152 or 167-169) are shown below: Polynucleotides encoding CARs of the invention may have a sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to one or more of SEQ ID NOS: 153-156 or 170-172. SEQ ID NO: 153 - Example nucleotide sequence encoding an example CAR1 CAR comprising CD8 leader, ASGR1 VH, CD8α hinge, CD28 transmembrane, CD28 cytoplasmic, and CD3z cytoplasmic SEQ ID NO: 154 - Example of a nucleotide sequence encoding an example of a CAR2 CAR comprising a CD8 leader, ASGR1 VH, c-Myc tagged CD28 hinge, CD28 transmembrane, CD28 cytoplasmic, and CD3z cytoplasmic SEQ ID NO: 155 - Example nucleotide sequence encoding an example CAR3 CAR comprising CD8 leader, ASGR1 VH, CD8α hinge, CD28 transmembrane, CD28 cytoplasmic, CD3z cytoplasmic, FP2A domain, and GFP SEQ ID NO: 156 - Example nucleotide sequence encoding an example of a CAR4 CAR comprising a CD8 leader, ASGR1 VH, CD8α hinge, CD28 transmembrane, CD28 cytoplasmic, CD3z cytoplasmic, FP2A domain, and GFP SEQ ID NO: 170 - Example of a nucleotide sequence encoding an example of a CAR5 CAR comprising a CD8 leader, ASGR1 VH (VH1, SEQ ID NO: 74), a linker, ASGR1 VL (VK1, SEQ ID NO: 78), a c-Myc tagged CD28 hinge, CD28 transmembrane, CD28 cytoplasmic, and CD3z cytoplasmic SEQ ID NO: 171 - Example of a nucleotide sequence encoding an example of a CAR6 CAR comprising a CD8 leader, ASGR1 VH (VH2, SEQ ID NO: 75), a linker, ASGR1 VL (VK2, SEQ ID NO: 79), a c-Myc tagged CD28 hinge, CD28 transmembrane, CD28 cytoplasmic, and CD3z cytoplasmic SEQ ID NO: 172 - Example of a nucleotide sequence encoding an example of a CAR7 CAR comprising a CD8 leader, ASGR1 VH (VH3, SEQ ID NO: 76), a linker, ASGR1 VL (VK3, SEQ ID NO: 80), a c-Myc tagged CD28 hinge, CD28 transmembrane, CD28 cytoplasmic, and CD3z cytoplasmic

[0295] As discussed above, the polynucleotides of the invention may also encode polypeptides in addition to the CAR defined herein, such as reporter or marker polypeptides or proteins. In particular, the polynucleotides of the invention may further encode a FOXP3 polypeptide.

[0296] In one embodiment, the Tregs of the present invention may comprise one or more polynucleotides encoding the CAR and FOXP3 polypeptides of the present invention (exogenous FOXP3 polypeptides), and thus the Tregs of the present invention may be generated by introducing polynucleotides encoding the CAR and FOXP3 polypeptides of the present invention into cells. The CAR and FOXP3 polypeptides of the present invention may be encoded by a single polynucleotide sequence or by different polynucleotides.

[0297] "FOXP3" is an abbreviation for forkhead box P3 protein. FOXP3 is a member of the FOX protein family of transcription factors and functions as a master regulator of regulatory pathways in regulatory T cell development and function. As used herein, "FOXP3" includes variants, isoforms, and functional fragments of FOXP3.

[0298] A "FOXP3 polypeptide" is a polypeptide having FOXP3 activity, i.e., a polypeptide capable of binding to FOXP3 target DNA and functioning as a transcription factor regulating the development and function of Tregs. In particular, a FOXP3 polypeptide may have the same or similar activity as wild-type FOXP3 (SEQ ID NO: 157), e.g., at least 40, 50, 60, 70, 80, 90, 95, 100, 110, 120, 130, 140, or 150% of the activity of wild-type FOXP3 polypeptide. Thus, a FOXP3 polypeptide encoded by a nucleotide sequence described herein may have increased or decreased activity compared to wild-type FOXP3. Techniques for measuring transcription factor activity are well known in the art. For example, transcription factor DNA binding activity may be measured by ChIP. The transcriptional regulatory activity of a transcription factor may be measured by quantifying the expression level of the gene it regulates. Gene expression may be quantified by measuring the levels of mRNA and / or protein produced from the gene using techniques such as Northern blotting, SAGE, qPCR, HPLC, LC / MS, Western blotting, or ELISA. Genes regulated by FOXP3 include cytokines such as IL-2, IL-4, and IFN-γ (Siegler et al. Annu. Rev. Immunol. 2006, 24: 209-26, incorporated herein by reference). As described in detail below and above, FOXP3 or FOXP3 polypeptides include functional fragments, variants, and isoforms thereof, e.g., SEQ ID NO: 157.

[0299] A "functional fragment of FOXP3" refers to a portion or region of a FOXP3 polypeptide or a polynucleotide (i.e., a nucleotide sequence) that encodes a FOXP3 polypeptide having the same or similar activity as a full-length FOXP3 polypeptide or polynucleotide. The functional fragment may have at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the activity of the full-length FOXP3 polypeptide or polynucleotide. Those skilled in the art will be able to generate functional fragments based on the well-known structural and functional characteristics of FOXP3. For example, see Song, X., et al., 2012. Cell Reports, 1(6), pp.665-675; Lopes, JE, et al., 2006. The Journal of Immunology, 177(5), pp.3133-3142; and Lozano, T., et al, 2013. Frontiers in oncology, 3, p.294. N- and C-terminal truncated FOXP3 fragments are also described in WO2019 / 241549 (incorporated herein by reference), e.g., having SEQ ID NO: 157, as shown below.

[0300] A "FOXP3 variant" may comprise an amino acid or nucleotide sequence that may be at least 50%, at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, preferably at least 95%, at least 97%, or at least 99% identical to a FOXP3 polypeptide or a polynucleotide encoding a FOXP3 polypeptide, e.g., SEQ ID NO: 157. A FOXP3 variant may have the same or similar activity as wild-type FOXP3, e.g., at least 40, 50, 60, 70, 80, 90, 95, 100, 110, 120, 130, 140, or 150% of the activity of a wild-type FOXP3 polypeptide or polynucleotide. One skilled in the art would be able to generate FOXP3 variants based on the known structural and functional characteristics of FOXP3 and / or using conservative substitutions. Compared to wild-type FOXP3, FOXP3 variants may have a similar or the same metabolic time (or degradation rate) in Treg cells, e.g., at least 40, 50, 60, 70, 80, 90, 95, 99, or 100% of the metabolic time of wild-type FOXP3 in Treg cells. Some FOXP3 variants may have a reduced metabolic time (or degradation rate) compared to wild-type FOXP3, e.g., FOXP3 variants having amino acid substitutions, e.g., S418E and / or S422A, at amino acids 418 and / or 422 of SEQ ID NO: 157, as described in WO2019 / 241549 (incorporated herein by reference), and are set forth in SEQ ID NOs: 158-160, which represent the aa418 mutant, the aa422 mutant, and the aa418 and aa422 mutants, respectively.

[0301] Suitably, the FOXP3 polypeptide encoded by the nucleic acid molecule, construct or vector described herein may comprise or consist of the polypeptide sequence of human FOXP3, such as UniProtKB Accession No. Q9BZS1 (SEQ ID NO: 157) or a functional fragment or variant thereof.

[0302] In some embodiments of the invention, the FOXP3 polypeptide comprises or consists of an amino acid sequence that is at least 70% identical to SEQ ID NO: 157, or a functional fragment thereof. Suitably, the FOXP3 polypeptide comprises or consists of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 157, or a functional fragment thereof. In some embodiments, the FOXP3 polypeptide comprises or consists of SEQ ID NO: 157, or a functional fragment thereof.

[0303] In some embodiments, as described above, the FOXP3 polypeptide may include a mutation at residues 418 and / or 422 of SEQ ID NO: 157, as set forth in SEQ ID NO: 158, SEQ ID NO: 159, or SEQ ID NO: 160.

[0304] In some embodiments of the present invention, a FOXP3 polypeptide may be truncated at the N- and / or C-terminus to produce a functional fragment. In particular, an N- and C-terminally truncated functional fragment of FOXP3 may comprise or consist of the amino acid sequence of SEQ ID NO: 161, or a functional variant thereof having at least 80, 85, 90, 95, or 99% identity thereto.

[0305] Preferably, the FOXP3 polypeptide may be a variant of SEQ ID NO: 157, such as a natural variant. Preferably, the FOXP3 polypeptide may be an isoform of SEQ ID NO: 157. For example, the FOXP3 polypeptide may be deleted from amino acids 72 to 106 compared to SEQ ID NO: 157. Alternatively, the FOXP3 polypeptide may be deleted from amino acids 246 to 272 compared to SEQ ID NO: 157.

[0306] Suitably, the FOXP3 polypeptide comprises SEQ ID NO: 162 or a functional fragment thereof. SEQ ID NO: 162 represents an example of a FOXP3 polypeptide.

[0307] Suitably, the FOXP3 polypeptide comprises or consists of an amino acid sequence that is at least 70% identical to SEQ ID NO: 162, or a functional fragment thereof. Suitably, the FOXP3 polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 162, or a functional fragment thereof. In some embodiments, the FOXP3 polypeptide comprises or consists of SEQ ID NO: 162, or a functional fragment thereof.

[0308] Preferably, the FOXP3 polypeptide may be a variant of SEQ ID NO: 162, such as a natural variant. Preferably, the FOXP3 polypeptide is an isoform of SEQ ID NO: 162 or a functional fragment thereof. For example, the FOXP3 polypeptide may be deleted from amino acids 72 to 106 compared to SEQ ID NO: 162. Alternatively, the FOXP3 polypeptide may be deleted from amino acids 246 to 272 compared to SEQ ID NO: 162.

[0309] Suitably, said polynucleotide encoding a FOXP3 polypeptide may comprise or consist of the nucleotide sequence shown in SEQ ID NO: 163, which nucleotide sequence represents an example of a FOXP3 nucleotide sequence.

[0310] In some embodiments of the invention, the polynucleotide encoding the FOXP3 polypeptide or variant comprises a nucleotide sequence at least 70% identical to SEQ ID NO: 163, or a functional fragment thereof that encodes a functional FOXP3 polypeptide. Suitably, the polynucleotide encoding the FOXP3 polypeptide or variant comprises a polynucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 163, or a functional fragment thereof that encodes a functional FOXP3 polypeptide. In some embodiments of the invention, the polynucleotide encoding the FOXP3 polypeptide or variant comprises or consists of SEQ ID NO: 163, or a functional fragment thereof that encodes a functional FOXP3 polypeptide.

[0311] Suitably, said polynucleotide encoding a FOXP3 polypeptide may comprise or consist of the polynucleotide sequence set forth in SEQ ID NO: 164, which represents another example of a FOXP3 nucleotide sequence.

[0312] In some embodiments of the invention, the polynucleotide encoding the FOXP3 polypeptide or variant comprises a nucleotide sequence at least 70% identical to SEQ ID NO: 164, or a fragment thereof that encodes a functional FOXP3 polypeptide. Suitably, the polynucleotide encoding the FOXP3 polypeptide or variant comprises a polynucleotide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 164, or a functional fragment thereof that encodes a functional FOXP3 polypeptide. In some embodiments of the invention, the polynucleotide encoding the FOXP3 polypeptide or variant comprises or consists of SEQ ID NO: 164, or a functional fragment thereof that encodes a functional FOXP3 polypeptide.

[0313] Preferably, the polynucleotide encoding the FOXP3 polypeptide or functional fragment or variant thereof may be codon-optimized. Preferably, the polynucleotide encoding the FOXP3 polypeptide or functional fragment or variant thereof may be codon-optimized for expression in human cells. SEQ ID NO: 157 FOXP3, UniProtKB accession number Q9BZS1: MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFL KHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPE FLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGP SEQ ID NO: 158 FOXP3, aa418 variant: MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFL KHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPE FLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKREQRPSRCSNPTPGP SEQ ID NO: 159 FOXP3, aa422 variant: MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFL KHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPE FLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPARCSNPTPGP SEQ ID NO: 160 FOXP3, aa418 and 422 variants: MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFL KHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPE FLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKREQRPARCSNPTPGP SEQ ID NO: 161 FOXP3, truncated mutant: GGAHASSSSL NPMPPSQLQL PTLPLVMVAP SGARLGPLPH LQALLQDRPH FMHQLSTVDA HARTPVLQVH PLESPAMISL TPPTTATGVF SLKARPGLPP GINVASLEWV SREPALLCTF PNPSAPRKDS TLSAVPQSSY PLLANGVCKW PGCEKVFEEP EDFLKHCQAD HLLDEKGRAQ CLLQREMVQS LEQQLVLEKE KLSAMQAHLA GKMALTKASS VASSDKGSCC IVAAGSQGPV VPAWSGPREA PDSLFAVRRH LWGSHGNSTF PEFLHNMDYF KFHNMRPPFT YATLIRWAIL EAPEKQRTLN EIYHWFTRMF AFFRNHPATW KNAIRHNLSL HKCFVRVESE KGAVWTVDEL EF SEQ ID NO: 162 FOXP3, mutant examples: MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVD AHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQA DHLLDEKGRAQCLLQREMVQSLEQVEELSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHN MRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGPEGRGSLLTCGDVEEN SEQ ID NO: 163 FOXP3, Examples of FOXP3 Polynucleotides SEQ ID NO: 164 FOXP3, examples of FOXP3 polynucleotides:

[0314] vector The invention further provides a vector encoding a CAR of the invention, which vector may comprise a polynucleotide of the invention, e.g., encoding a CAR of the invention, and optionally encoding another polypeptide, e.g., a FOXP3 polypeptide.

[0315] A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. According to the present invention, for example, some vectors used in recombinant nucleic acid technology can transfer an entity, such as a segment of nucleic acid (e.g., a heterologous DNA segment, such as a heterologous cDNA segment), into a target cell. The vector can be non-viral or viral. Examples of vectors used in recombinant nucleic acid technology include, but are not limited to, plasmids, mRNA molecules (e.g., in vitro transcribed mRNA), chromosomes, artificial chromosomes, and viruses. A vector can also be, for example, a naked nucleic acid (e.g., DNA). In its simplest form, the vector itself can be the nucleotide of interest.

[0316] The vectors used in the present invention may be, for example, plasmid, mRNA or viral vectors and may comprise a promoter for the expression of the polynucleotide and, optionally, a regulator of that promoter.

[0317] Vectors containing the polynucleotides of the present invention may be introduced into cells using various techniques known in the art, such as transformation and transduction. Several techniques are known in the art, including infection with recombinant viral vectors such as retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, baculoviral vectors, and herpes simplex viral vectors; direct injection of nucleic acids; and biolistic transformation. Non-viral delivery systems include, but are not limited to, DNA transfer methods. Gene transfer includes the process of using non-viral vectors to deliver genes to target cells. Non-viral delivery systems can include liposomes or amphiphilic cell-penetrating peptides, preferably complexed with the polynucleotides of the present invention.

[0318] Typical gene transfer methods include electroporation, DNA gene guns, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofection, cationic drug-mediated transfection, cationic facial amphiphiles (CFAs) (Nat. Biotechnol. (1996) 14: 556), and combinations thereof.

[0319] Multiple vectors could be used for transduction / transfection, for example vectors encoding different CARs of the invention, or vectors encoding a CAR of the invention and an additional polypeptide.

[0320] Cell creation method The modified Tregs of the present invention may be generated by introducing DNA or RNA encoding a CAR as defined herein by one of a number of means, such as transduction with a viral vector or gene transfer with DNA or RNA. The modified Tregs of the present invention are generated by introducing a polynucleotide or vector defined herein into a Treg (e.g., by transduction or gene transfer). Alternatively, as described further below, the modified Tregs of the present invention may be generated by introducing a polynucleotide or vector defined herein into a cell that is not a Treg and converting (e.g., differentiating or reprogramming) the cell to have a Treg phenotype. The polynucleotide or vector may be introduced before or after conversion.

[0321] Suitably, the Tregs may be derived from a sample isolated from a subject, from which they may be further separated by any suitable method, for example by magnetic separation.

[0322] The modified Tregs of the present invention may be generated by a method comprising the following steps. (i) isolating or obtaining a cell-containing sample from a subject; and (ii) transducing or transfecting said cell-containing sample with a polynucleotide, nucleic acid, or vector encoding a CAR of the present invention to obtain a population of modified cells.

[0323] Suitably, a Treg-enriched sample may be isolated, enriched, and / or generated from the cell-containing sample before and / or after step (ii) of the method. For example, isolation, enrichment, and / or generation of Tregs may be performed before and / or after step (ii) to isolate, enrich, and / or generate the Treg-enriched sample. To enrich for cells and / or Tregs comprising the CAR, polynucleotide, and / or vector of the invention, isolation and / or enrichment may be performed after step (ii).

[0324] The Treg-enriched sample may be isolated or enriched by any method known to those of skill in the art, for example, by FACS and / or magnetic bead separation. The Treg-enriched sample may be generated from a cell-containing sample by any method known to those of skill in the art, for example, from Tcon cells by introducing DNA or RNA encoding FOXP3 and / or from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells.

[0325] Suitably, said cells are Tregs as defined herein.

[0326] Preferably, the modified Tregs of the present invention may be generated by a method comprising the following steps: (i) isolating or obtaining a Treg-enriched sample from a subject; and (ii) transducing or transducing the Treg-enriched sample with a polynucleotide, nucleic acid, or vector encoding a CAR of the invention to provide a population of modified Treg cells of the invention.

[0327] The cells and / or Tregs may be activated and / or expanded before or after introduction of a polynucleotide encoding a CAR described herein, for example, by treatment with an anti-CD3 monoclonal antibody, or by treatment with anti-CD3 and anti-CD28 monoclonal antibodies.

[0328] The cells and / or Tregs may also be expanded in the presence of anti-CD3 and anti-CD28 monoclonal antibodies in combination with IL-2. Preferably, IL-2 may be replaced with IL-15. Additional components that may be used in Treg expansion protocols include, but are not limited to, rapamycin, all-trans retinoic acid (ATRA), and TGFβ.

[0329] As used herein, "activated" means that a cell or population of cells is stimulated to cause cell proliferation. As used herein, "proliferated" means that proliferation of a cell or population of cells is induced. The proliferation of a population of cells may be measured, for example, by counting the number of cells present in the population. The phenotype of a cell may be determined by methods well known in the art, such as flow cytometry.

[0330] The cells and / or Tregs may be washed after each step of the method, particularly after expansion.

[0331] The modified cell or Treg population may be further enriched by any method known to those skilled in the art, for example, by FACS and / or magnetic bead separation. The steps of the production method may be carried out in a closed, sterile cell culture system. [Example]

[0332] The present invention is further illustrated by examples which are intended to aid those skilled in the art in practicing the invention and are not intended to limit the scope of the invention.

[0333] Example 1 - Design of anti-ASGR1 CAR construct Chimeric antigen receptors (CARs) were designed to contain an antigen recognition domain derived from a single-domain antibody (sdAb) known to specifically bind to ASGR, a transmembrane domain (TM) derived from CD28 (aa 153-179), and an intracellular signaling domain containing the signaling domains of CD3ζ and CD28. An example construct is shown below and in Figure 1. Anti-ASGR1 CAR construct 1 (SEQ ID NO: 151) comprising a CD8 leader, an ASGR1 VH antigen recognition domain, a CD8α hinge domain, a CD28 transmembrane domain, a CD28 cytoplasmic signaling domain, a CD3z cytoplasmic signaling domain, an FP2A domain, and GFP. MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFTFEKYAMAWVRQAPGKGLEWVSRISARGVTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHKRHEHTRFDSWGQGTLVTVSSTTTPAPRPPTPAPTIASQ PLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRRKRGSGATNFSLLKQAGDVEENPGPTRGGGATMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHM KQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK Anti-ASGR1 CAR construct 2 (SEQ ID NO: 152) comprising a CD8 leader, an ASGR1 VH antigen recognition domain, a c-Myc-tagged CD28 hinge domain, a CD28 transmembrane domain, a CD28 cytoplasmic signaling domain, a CD3z cytoplasmic signaling domain, an FP2A domain, and GFP. MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFTFEKYAMAWVRQAPGKGLEWVSRISARGVTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHKRHEHTRFDSWGQGTLVTVSSAAAIEVEQKLISEEDLLD NEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQK DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRRRKRGSGATNFSLLKQAGDVEENPGPTRGGGATMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHM KQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0334] Example 2 - Generation of anti-ASGR1 CAR-Tregs Anti-ASGR1 CAR-Tregs were generated. CD4+CD25hiCD127- Tregs were isolated and activated with anti-CD3 / CD28 beads. Two days after activation, Tregs were transduced with a lentivirus containing an anti-ASGR1 CAR and a GFP reporter gene. Transduced and untransduced Tregs were cultured for 10 days, and GFP was measured to assess the efficacy of transduction. Figure 2 shows that 67.3% of transduced Tregs expressed GFP (i.e., contained the anti-ASGR1 CAR construct).

[0335] Example 3 - Confirmation of ASGR1 expression in HepG2 cell line HepG2 cells are a suitable in vitro model system for studying polarized human hepatocytes. ASGR1 expression in HepG2 was confirmed by FACS and compared with that in K562 cells, an immortalized human myeloid leukemia cell line. Figure 3 shows that ASGR1 was highly expressed in HepG2 cells. HepG2 cells were used as CAR-Treg target cells in the following experiments.

[0336] Example 4 - Evaluation of antigen specificity of anti-ASGR1 CAR-Tregs After culturing the ASGR1-expressing cell line HepG2, we examined Treg activation and proliferation and confirmed that only Tregs expressing anti-ASGR1 CAR upregulated CD69 in response to ASGR1 stimulation. Figure 4 shows that CD69 upregulation is not observed in the presence of medium alone. Following culture with HepG2 cells, only GFP+ transduced Tregs (but not GFP-negative transduced or untransduced cells) upregulate CD69. In contrast, when cultured with nonspecific anti-CD3 / CD28 bead stimulation, all cells upregulate CD69.

[0337] Example 5 - Evaluation of antigen-specific suppressive function of anti-ASGR1 CAR-Tregs To examine the ability of anti-ASGR1 CAR Tregs to antigen-specifically suppress the proliferation of activated effector CD4+CD25- T cells, preactivated CD4+CD25- effector T cells were cocultured with anti-ASGR1 CAR Tregs or with non-transduced Tregs in the presence of HepG2 cells. Teff preactivation was performed under the following conditions: CD4+CD25- T cells were activated with CD3 / CD28 beads 1:10 for 14 hours. Suppression assay conditions were as follows: 3-day culture; 100,000 Teff; 30,000 HepG2; comparison of non-transduced Tregs and anti-ASGR1 CAR-Tregs. The effect of anti-ASGR1 CAR-Tregs on the proliferation capacity of effector T cells stained with proliferation dye and preactivated with anti-CD3 / CD28 beads was demonstrated by suppression assay (Figures 5 and 6). Compared with untransduced Tregs, anti-ASGR1 CAR Tregs were able to effectively suppress the proliferation of normal T cells in response to HepG2 stimulation at a low Treg-to-T effector ratio.

[0338] Example 6 - Generation of anti-HLA.A2 IL2RCAR-Tregs The effect of endodomains comprising a STAT5-associated motif and a JAK1 and / or JAK2-binding motif (optionally comprising a JAK3-binding motif and / or no STAT3-associated motif) is demonstrated in Examples 6-13 using HLA.A2-CAR constructs. While the exemplified antigen-binding domains target HLA.A2, the endodomains are believed to be broadly applicable and may be applicable to CAR constructs comprising antigen-binding domains targeting liver-specific antigens, such as ASGR.

[0339] CD4+CD25hiCD127low cells were isolated and activated with anti-CD3 / CD28 beads. Three days after activation, Tregs were transduced with a lentivirus containing an HLA.A2-CAR construct and a GFP reporter gene (Figure 7). Cell proliferation of total Tregs after polyclonal activation showed no significant difference between transduced and non-transduced Tregs (Figure 8).

[0340] Example 7 - Quantification of the efficacy of transduction of anti-HLA.A2 IL2R constructs over time GFP expression was analyzed at different time points after cell activation in Tregs that were not transduced with CAR constructs and in Tregs that were transduced with CAR constructs. The frequency of GFP+ cells was analyzed to assess the efficacy of transduction of various constructs and the persistence of expression over the Treg expansion period. Tregs containing dCAR, CD28z, constructs 1, 2, and 3 showed similar expression frequencies after transduction. The percentage of GFP+ cells in all Tregs was maintained throughout polyclonal cell expansion (Figure 9).

[0341] Example 8 - Quantification of cell surface expression of anti-HLA.A2 IL2R CAR constructs on transduced Tregs The membrane expression of the CAR construct on untransduced and transduced Tregs was analyzed by PE-conjugated HLA-A*0201 / CINGVCWTV dextramer (Immudex, Copenhagen, Denmark). The CAR protein on the cell surface (HLA-A2 dextramer) + The frequency of Tregs expressing ) was similar across all constructs (Figure 10).

[0342] Example 9 - Phenotypic characterization of CAR Tregs after polyclonal cell expansion Tregs were cultured and expanded for 15 days in the presence of anti-CD3 / CD28 activation beads and IL-2. Treg-associated markers FOXP3, HELIOS, CTLA4, and TIGIT were analyzed by FACS on untransduced and transduced Tregs to examine phenotypic lineage stability at day 15 of culture. Untransduced and CAR-transduced mice had similar levels of Treg lineage-associated protein expression and function after polyclonal expansion (Figure 11).

[0343] Example 10 - Evaluation of antigen specificity of anti-HLA.A2 IL2RCAR Tregs Untransduced and transduced Tregs were cultured for 18 hours in the presence of different stimuli. CD69 and CD137 activation markers were analyzed to examine specific and nonspecific cell activation. Tregs transduced with CD28z, Construct 1 CAR, Construct 2 CAR, and Construct 3 CAR showed similar specificity for HLA-A2 molecules based on the expression of T cell activation markers. CD69 and CD137 expression did not increase on inactivated cells or after culture with HLA-A1-expressing cells. The dCAR constructs, lacking the signaling endodomain, did not demonstrate activation (Figure 12).

[0344] Example 11 - STAT5 phosphorylation analysis as an indicator of IL2R CAR signaling Transduced CAR Tregs were placed in IL2-free medium overnight. STAT5 phosphorylation in Tregs was assessed by FACS analysis after 10 and 120 minutes of culture in medium alone, with 1000 IU / ml IL-2, or in the presence of HLA.A2-Ig-based artificial APCs (generated according to the protocol described in DOI:10.3791 / 2801). Introduction of the IL2R endodomain into the CAR construct demonstrated efficient STAT5 phosphorylation after CAR activation with HLA-A2 molecules. No significant increase in pSTAT5 was detected on CAR Tregs lacking the IL2R endodomain after culture with HLA-A2 beads (Figure 13).

[0345] Example 12 - Evaluation of Treg survival after non-specific activation and HLA.A2-specific activation in the absence of IL-2 CAR-transduced Tregs with different constructs were cultured with anti-CD3 / 28 activation beads and K562.A2-expressing cells in the absence of IL-2. Cell survival was examined 7 days after activation by FACS analysis. After cell culture with HLA-A2-expressing cells, Tregs expressing CAR constructs containing the IL2R endodomain showed increased cell survival compared to standard CD28z. This difference was not observed after polyclonal activation of Tregs, indicating that the effect was dependent on CAR signaling (Figure 14).

[0346] Example 13 - Treg suppression assay: Evaluation of the immunoregulatory function of Tregs by analyzing the modulation of costimulatory molecules on B cells After co-culture with Tregs, B cells were analyzed for CD80 and CD86 expression to assess the ability of Tregs to reduce the expression of costimulatory molecules on antigen-presenting cells. Tregs expressing CD28z, Construct 1 CAR, and Construct 2 CAR showed greater suppressive function compared to untransduced and dCAR-expressing Tregs. CD80 and CD86 expression on B cells was downregulated only after culture with Tregs signaling via CAR molecules (Figure 15).

[0347] Example 14 - Evaluation of the effect of CAR Tregs on albumin production in liver cells method: The cells were cultured at 225 K / cm on type I collagen (from rat tail)-coated plates in Williams' E medium (Gibco) supplemented with 10% FBS, 2 mM L-glutamine, 1% ITS, 10 nM Hepes, and 100 U / L penicillin / streptomycin, with or without 50 IU / mL human recombinant interleukin-2 (Aldesleukin, Novartis). 2Cryopreserved adult human primary hepatocytes (Lonza) were seeded at a cell density of 36K / well in half-area 96-well plates or transwell plates. Once hepatocytes attached after 24 hours, CD4+CD25+Foxp3+ regulatory T cells were added to the culture medium in the presence or absence of CD4+CD25- effector T cells. The supernatant was replaced every 48 hours and cryopreserved for analysis. Indirect coculture was performed using HTS transwell membranes (0.4 μm pore size; CORNING Inc.). For some experiments, regulatory T cells and effector T cells were preactivated in the presence of anti-CD3 and anti-CD28 beads. For other experiments, non-preactivated regulatory T cells were lentivirally transduced with an anti-ASGPR1 CAR (Construct 1 described in Example 1) before being added to the culture plate. In some experiments, hepatocytes were cultured with culture supernatant obtained from the culture medium containing expanded regulatory T cells. Albumin concentrations, a marker of hepatocyte function and viability, were measured in the media using an enzyme-linked immunosorbent assay for human albumin (ELISA - Bethyl Laboratories). For statistical analysis, a Kruskal-Wallis nonparametric test with Dunn's inter-group post-tests was performed. Asterisks indicate p<0.05. Pairwise comparisons of hepatocytes only and p<0.05 are indicated. Plots shown are representative of three independent experiments.

[0348] Results: To determine the ability of anti-ASGPR CAR Tregs to mediate trophic and cytoprotective effects on liver cells in response to antigenic stimulation, we set up coculture experiments in which primary human hepatocytes were cultured with various combinations of Tregs and / or effector T cells for up to 7 days. To investigate the effects on hepatocyte phenotype and function, we quantified supernatant production of albumin, which correlates with hepatocyte synthetic function, viability, and lineage stability. Addition of Tregs preactivated in the presence of anti-CD3 / anti-CD28 beads to cultured primary hepatocytes resulted in increased albumin levels. This effect was significant after 3 days of coculture but was no longer significant by day 7, reflecting the need for repeated antigenic stimulation of Tregs to exert their trophic effects on hepatocytes (Figure 16). The beneficial effects of Tregs do not require cell-to-cell contact, as they were replicated by culturing hepatocytes with conditioned medium obtained from activated Tregs (Figure 17). Notably, the conditioned medium was repeatedly added to the culture plates every 48 hours, eliciting a more durable beneficial effect than pre-activated Tregs, which typically require weekly reactivation. In contrast to Tregs, effector T cells were incapable of increasing albumin levels (Figure 17). When resting (non-preactivated) Tregs were used, the effect was weaker and not significant (Figure 18). A significant effect was only obtained when resting Tregs expressed an anti-ASGPR CAR that could recognize ASGPR in cultured hepatocytes and trigger Treg activation (Figure 18).

[0349] These results indicate that regulatory T cells (Tregs) can provide trophic and cytoprotective effects to liver cells. This requires Treg activation and can occur in the absence of cell-to-cell contact. Treg activation can be achieved by stimulating Tregs with conventional T cell stimuli, such as anti-CD3 / anti-CD28 beads, or by transducing Tregs with CARs that can induce T cell activation after recognizing hepatocyte-specific antigens, such as ASGPR.

[0350] All publications mentioned herein are incorporated herein by reference. Various modifications and variations of the methods, cells, compositions, and uses of the present invention disclosed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been disclosed in connection with certain preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Various modifications of the disclosed modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the following claims.

Claims

1. A modified regulatory T cell (Treg) containing a chimeric antigen receptor (CAR), The CAR includes an antigen recognition domain that specifically binds to the asial glycoprotein receptor (ASGR). The CAR is a modified regulatory T cell (Treg) comprising a transmembrane (TM) domain, a CD3 zeta intracellular signaling domain, and a CD28 costimulatory signaling domain.

2. The antigen recognition domain specifically binds to ASGR1 and / or ASGR2, preferably ASGR1, and / or The antigen recognition domain binds to human ASGR, and / or The antigen-recognition domain binds to one or more polypeptides selected from human ASGR1 isoform a, human ASGR1 isoform b, human ASGR2 isoform a, human ASGR2 isoform b, human ASGR2 isoform c, and human ASGR2 isoform d, preferably the antigen-recognition domain binds to human ASGR1 isoform a, and / or The antigen recognition domain binds to ASGR with an affinity of 1 pM to 100 nM, 1 pM to 10 nM, or 1 pM to 1 nM. A modified Treg according to claim 1.

3. The antigen recognition domain is either an antibody, an antibody fragment, or derived from an antibody. Preferably, the antigen recognition domain is an antigen-binding fragment (Fab), a single-chain antibody (scFv), or a single-domain antibody (sdAb), and preferably a single-domain antibody (sdAb). A modified Treg according to claim 1 or 2.

4. The antigen recognition domain includes a CDR1 region, a CDR2 region, and a CDR3 region, The aforementioned CDR1 region, CDR2 region, and CDR3 region are, Includes sequence numbers 11, 12, and 13, respectively; Includes sequence numbers 14, 15, and 16, respectively; Includes sequence numbers 17, 18, and 19, respectively; Includes sequence numbers 20, 21, and 22, respectively; Includes sequence numbers 23, 24, and 25, respectively; Each includes sequence numbers 26, 27, and 28; and / or, Each includes sequence numbers 29, 30, and 31, Preferably, the antigen recognition domain includes a CDR1 region, a CDR2 region, and a CDR3 region containing SEQ ID NOs. 11, 12, and 13, respectively, and / or The antigen recognition domain includes a CDR1 region, a CDR2 region, and a CDR3 region, and the CDR1 region, CDR2 region, and CDR3 region include (i), (ii), or (iii) below: (i) Each of the sequence numbers 11, 12, and 13 and each of the sequence numbers 23, 24, and 25, or each of the sequence numbers 26, 27, and 28, or each of the sequence numbers 29, 30, and 31, preferably the antigen recognition domain comprises CDR1, CDR2, and CDR3 regions each comprising sequence numbers 11, 12, and 13 and each of the sequence numbers 23, 24, and 25: (ii) Each of the sequence numbers 14, 15, and 16, and each of the sequence numbers 23, 24, and 25, or each of the sequence numbers 26, 27, and 28, or each of the sequence numbers 29, 30, and 31, preferably the antigen recognition domain comprises CDR1, CDR2, and CDR3 regions, each comprising sequence numbers 14, 15, and 16, and each comprising sequence numbers 26, 27, and 28: (iii) Each of the following: SEQ ID NOs: 17, 18, and 19, and each of the following: SEQ ID NOs: 23, 24, and 25, or each of the following: SEQ ID NOs: 26, 27, and 28, or each of the following: SEQ ID NOs: 29, 30, and 31, preferably the antigen recognition domain includes SEQ ID NOs: 17, 18, and 19, and each of the following: CDR1, CDR2, and CDR3 regions including SEQ ID NOs: 26, 27, and 28, and / or The antigen recognition domain contains or consists of one amino acid sequence from SEQ ID NOs: 74 to 80, preferably the antigen recognition domain contains or consists of the amino acid sequence of SEQ ID NO:

74. A modified Treg as described in claim 3.

5. The CAR is (i) comprising one or more hinge domains selected from the group consisting of CD28 hinge domains, CD8α hinge domains, IgG hinge domains, and IgD hinge domains, preferably the CAR comprising a CD8α or CD28 hinge domain, and / or (ii) A modified Treg according to any one of claims 1 to 4, comprising one or more transmembrane domains selected from the group consisting of a CD28 transmembrane domain, an ICOS transmembrane domain, a CD8α transmembrane domain, a CD4 transmembrane domain, an OX40 transmembrane domain, a 4-1BB transmembrane domain, and a CD3 zeta transmembrane domain, preferably wherein the CAR comprises a CD28 transmembrane domain.

6. The CAR comprises a CD8α or CD28 hinge domain, a CD28 transmembrane domain, the CD28 costimulatory signaling domain, and the CD3 zeta intracellular signaling domain, and / or The CAR comprises a signal peptide and / or reporter peptide linked by a self-cleaving domain or a cleavage domain, and / or The CAR contains or consists of one amino acid sequence from sequence numbers 149-152 or 167-169. A modified Treg according to any one of claims 1 to 5.

7. The CAR includes an end domain comprising a STAT5-related motif and a JAK1 and / or JAK2 binding motif, Preferably, The end domain contains a JAK3 binding motif, and / or the end domain does not contain a STAT3-related motif, A modified Treg according to any one of claims 1 to 6, wherein the end domain does not contain the amino acid sequence YXXQ (SEQ ID NO: 133).

8. A modified Treg according to any one of claims 1 to 6, wherein the CAR does not include an end domain containing a STAT5-related motif, a JAK1 binding motif, and / or a JAK2 binding motif, preferably the end domain does not include a STAT5-related motif.

9. The aforementioned Treg is CD4 + CD25 + CD127 - T cells, and / or CD4 + CD25 + FOXP3 + It is a T cell, Preferably, the Treg comprises an exogenous polynucleotide encoding a FOXP3 polypeptide. A modified Treg according to any one of claims 1 to 8.

10. The CD3 zeta intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 103 or a variant having at least 80% identity with SEQ ID NO: 103, and / or The CD28 costimulatory signaling domain includes the amino acid sequence of SEQ ID NO: 104 or a variant that is at least 80% identical to SEQ ID NO:

104. A modified Treg according to any one of claims 1 to 9.

11. A pharmaceutical composition comprising a modified regulatory T cell (Treg) according to any one of claims 1 to 10.

12. For use in inducing tolerance to liver transplant tissue in subjects, or For use in the treatment and / or prevention of liver transplant rejection, liver graft-versus-host disease (GvHD), autoimmune liver disease, or inflammatory liver injury in the subject, A pharmaceutical composition according to claim 11, Preferably, The autoimmune liver disease is primary biliary cholangitis and / or primary sclerosing cholangitis, or The inflammatory liver disorder is cirrhosis, acute liver failure, or acutely exacerbated chronic liver failure, and / or the inflammatory liver disorder is due to alcohol, viral hepatitis, fatty liver disease, ischemia, or drug toxicity, or the cause of the inflammatory liver disorder is unidentifiable. The pharmaceutical composition according to claim 11.

13. A pharmaceutical composition comprising a modified regulatory T cell (Treg) according to any one of claims 1 to 10 for use in the repair and / or regeneration of target liver tissue, Preferably, the liver in question is injured and / or damaged due to ischemia, partial hepatectomy, or inflammation. More preferably, the liver of the subject is injured and / or damaged by inflammation, and the inflammation is cirrhosis, acute liver failure, or acutely exacerbated chronic liver failure, and / or the inflammation is due to alcohol, viral hepatitis, fatty liver disease, ischemia, or drug toxicity, or the cause of the inflammation is unidentifiable. Pharmaceutical composition.

14. The pharmaceutical composition according to claim 13, wherein the subject is in a state where liver regeneration is weakened, preferably due to one or more of the following: acute liver failure, cirrhosis, acutely exacerbated chronic liver failure, hepatitis, steatohepatitis, and aging. Preferably, the subject has cirrhosis, acute liver failure, or acutely exacerbated chronic liver failure, A pharmaceutical composition for use according to claim 13.

15. A pharmaceutical composition comprising a modified regulatory T cell (Treg) according to any one of claims 1 to 10, for use in increasing or stimulating albumin production in target liver tissue.

16. The CD3 zeta intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 103 or a variant having at least 80% identity with SEQ ID NO: 103, and / or The CD28 costimulatory signaling domain includes the amino acid sequence of SEQ ID NO: 104 or a variant that is at least 80% identical to SEQ ID NO:

104. A pharmaceutical composition according to any one of claims 13 to 15.

17. A pharmaceutical composition for use according to any one of claims 13 to 16, The subject is a mammal, preferably a human. Pharmaceutical composition.

18. A method for producing modified regulatory T cells (Treg) according to any one of claims 1 to 10, The process includes the step of transducing or genetically modifying a cell-containing sample with a polynucleotide, nucleic acid, or vector encoding the CAR, thereby providing a population of modified cells. A method wherein the cell-containing sample contains Treg, and / or Treg is enriched before or after the transduction or gene transfer, and / or is generated from the cell-containing sample.

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