Method for enhancing the inhibitory properties of Treg cells

Abstract

To provide a method for enhancing the ability of regulatory T cells (Tregs) to suppress immune responses.SOLUTION: Increase in FOXP3 expression in Tregs which already express endogenous FOXP3 (e.g., by introducing exogenous FOXP3) enhances the regulatory function of the Tregs to a greater degree than the regulatory function provided by expressing exogenous FOXP3 in conventional T cells which do not express endogenous FOXP3.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to methods for enhancing the ability of regulatory T cells (Tregs) to suppress immune responses. In particular, the present invention relates to methods for increasing FOXP3 expression in Tregs. The present invention further relates to genetically modified Tregs provided by the methods of the present invention, as well as methods and uses of such genetically modified Tregs. [Background technology]

[0002] Regulatory T cells (Tregs) are a type of T cell that modulates the activity of the immune system. Generally, Tregs are immunosuppressive and downregulate the immune response to stimuli. In particular, Tregs suppress the induction and proliferation of normal T cells (some types of which are directly involved in the immune response, e.g., cytotoxic T cells). The suppressive effect of Tregs can be directed to specific antigens by expressing recombinant T cell receptor (TCR) constructs that recognize peptides that match epitopes found in those specific antigens. Similarly, the suppressive effect of Tregs can be directed to specific targets by expressing chimeric antigen receptors (CARs) that recognize antigens expressed on the surface of target cells. Normal T cells can be differentiated into a regulatory phenotype ex vivo by expressing FOXP3 in them.

[0003] In autoimmune and inflammatory central nervous system (CNS) diseases, the immune system attacks its own antigens. For example, in multiple sclerosis (MS), the most common neurological disorder in young adults, the immune system attacks the myelin sheath of neurons in the central nervous system.

[0004] Current treatments for autoimmune and inflammatory CNS diseases generally involve suppressing the immune system. For example, some treatments include bone marrow transplantation in combination with the administration of cell proliferation inhibitors and immunosuppressants. Autologous hematopoietic stem cell transplantation can show sustained beneficial effects in some patients, but the procedure requires aggressive myeloablative conditioning, which carries considerable toxicity and risk.

[0005] While some disease-modifying therapies (DMTs) have been shown to reduce the frequency of clinical relapses, most patients continue to clinically worsen under current treatment schedules. Neither DMTs nor stem cell transplantation can mediate CNS-specific suppression of the immunopathology of autoimmune and inflammatory CNS diseases.

[0006] Currently, there are no effective treatments for autoimmune and inflammatory CNS diseases. Treatment typically focuses only on alleviating symptoms by suppressing the immune system as a whole. Therapies that specifically target the local immune responses associated with the onset and progression of CNS diseases are needed. [Overview of the Initiative]

[0007] To our surprise, the inventors discovered that exogenous FOXP3 expression in regulatory T cells (Tregs) (which already express endogenous FOXP3) enhances their regulatory function.

[0008] Therefore, the present invention provides a method for enhancing the ability of regulatory T cells (Tregs) to suppress the immune response.

[0009] The Tregs of the present invention are either inducible Tregs or innate Tregs that originate from normal T cells. For example, the Tregs of the present invention are either inducible Tregs or innate Tregs that originate from normal T cells in vivo. Suitable Treg cells include innate Treg (nTreg) cells derived from the thymus and inducible Treg (iTreg) cells that are generated in the periphery. In other words, the Tregs of the present invention express endogenous FOXP3. Surprisingly, the inventors found that increasing FOXP3 expression in Tregs that already express endogenous FOXP3 (for example, by introducing exogenous FOXP3) enhances the regulatory function of Tregs more significantly than the regulatory function brought about by expressing exogenous FOXP3 in normal T cells that do not express endogenous FOXP3.

[0010] The inventors have further found that increasing FOXP3 expression in Tregs already expressing endogenous FOXP3 allows for improved retention of the Treg functional profile in vivo after administration to the subject. For example, congenital Tregs that do not express exogenous FOXP3 may lose their Treg profile after administration to the subject. For instance, it has been found that congenital Tregs that do not express exogenous FOXP may have decreased levels of FOXP3 expression and the ability to produce pro-inflammatory effector cytokines after a period following administration to the subject. The Tregs provided by the present invention may retain FOXP3 expression and exhibit a reduced ability to produce pro-inflammatory effector cytokines after a period following administration to the subject.

[0011] In a preferred embodiment, the Treg of the present invention is an innate Treg.

[0012] In one embodiment, the present invention provides a method for generating a population of regulatory T cells (Tregs), comprising providing a first population of Tregs and generating a second population of Tregs by increasing FOXP3 expression in the first population of Tregs.

[0013] The present invention provides a method for enhancing the ability of a Treg to suppress the immune response, which includes increasing FOXP3 expression in the Treg.

[0014] In some embodiments of the present invention, FOXP3 expression is increased by introducing a polynucleotide encoding the FOXP3 protein into the Treg.

[0015] In some embodiments of the present invention, the method for enhancing the ability of regulatory T cells (Tregs) to suppress the immune response is as follows (a) and (b), namely (a) Isolating Treg from a cell population, and (b) Increase FOXP3 expression in Tregs. Includes.

[0016] Depending on the context, the term "Treg" may refer to a group of Tregs (i.e., multiple Tregs).

[0017] The present invention also provides genetically modified Tregs that can be obtained or obtained by the method of the present invention.

[0018] The present invention also provides genetically modified Tregs that exhibit higher FOXP3 expression than non-genetically modified Tregs.

[0019] In some embodiments, the genetically modified Treg includes an exogenous polynucleotide encoding the FOXP3 protein.

[0020] The present invention also provides a pharmaceutical composition containing the genetically modified Treg of the present invention.

[0021] The present invention also provides genetically modified Tregs or pharmaceutical compositions of the present invention for use in the prevention and / or treatment of diseases.

[0022] The present invention also provides the use of the genetically modified Treg in the manufacture of pharmaceuticals.

[0023] The present invention also provides a method for the prevention and / or treatment of a disease, comprising administering the genetically modified Treg or composition of the present invention to a subject. [Brief explanation of the drawing]

[0024] [Figure 1] Figure 1 shows the proliferation of Tconv cells transduced with and without peptides (indicated by "*"), as well as the proliferation of the same cells in the presence of mock transduced Tregs with different Treg:Tconv ratios (white bars), Tregs transduced with TCR constructs (black bars), or Tregs transduced with both TCR constructs and FOXP3 (gray bars). [Figure 2]Figure 2 shows IL-2 production (indicated by "*") in Tconv cells transduced with or without peptides, as well as IL-2 production in the presence of pseudotransduced Tregs with different Treg:Tconv ratios (white bars), Tregs transduced with TCR constructs (black bars), or Tregs transduced with TCR constructs and FOXP3 (gray bars). [Figure 3] Figure 3 shows the proliferation (indicated by "*") of Tconv cells transduced with a TCR construct (obtained from a different donor than in Figure 1), with and without the use of peptides, as well as the proliferation of the same cells in the presence of pseudotransduced Tregs with different Treg:Tconv ratios (white bars), Tregs transduced with a TCR construct (black bars), or Tregs transduced with both a TCR construct and FOXP3 (gray bars). [Figure 4] Figure 4 shows IL-2 production (indicated by "*") of Tconv cells transduced with or without peptide-containing TCR constructs (obtained from a different donor than in Figure 2), as well as proliferation of the same cells in the presence of pseudotransduced Tregs with different Treg:Tconv ratios (white bars), Tregs transduced with TCR constructs (black bars), or Tregs transduced with TCR constructs and FOXP3 (gray bars). [Figure 5] Figure 5 shows the mean fluorescence intensity (MFI) of Treg markers (FOXP3, CD25, and CTLA-4) in pseudotransduction Tregs, TCRs, or TCR+FOXP3-transduced Tregs, analyzed by flow cytometry 7–10 days after transduction. The dots represent individual experiments. One-way ANOVA was used for statistical analysis (p<0.05*, p<0.005**). [Figure 6] Figure 6 shows the multiple factor infiltration (MFI) of FOXP3, CD25, and CTLA-4 in transduced Tregs. Each line represents a single experiment showing MFI of a marker on the same Treg transduced with TCR or TCR+FOXP3. [Figure 7]Figure 7 shows the proliferation of Tconv cells (obtained from different donors than those in Figures 1 and 3) with and without peptide use, as well as the proliferation of the same cells in the presence of pseudotransduced Tregs with different Treg:Tconv ratios (white bars), Tregs with TCR constructs (black bars), Tregs with TCR constructs and FOXP3 (gray bars), or Tconv cells with TCR constructs and FOXP3, i.e., inducible Tregs (red bars, bars on the right in each dataset). [Figure 8] Figure 8 shows the levels of IL-2 production by transduced Tconv cells (obtained from the same donor as in Figure 7), with or without peptide use, as well as the levels of IL-2 production by the same cells in the presence of pseudotransduced Tregs with different Treg:Tconv ratios (white bars), transduced Tregs with TCR constructs (black bars), transduced Tregs with TCR constructs and FOXP3 (gray bars), or transduced Tconv cells with TCR constructs and FOXP3, i.e., inducible Tregs (red bars, bars on the right in each dataset). [Figure 9-1] Figure 9 shows that regulatory T cells transduced with TCR can engraft in irradiated hosts, but exogenous FOXP3 expression is required to prevent the accumulation of TCR+FOXP3- cells. Thy1.1+CD4+CD25+Treg cells were isolated from lymph node and splenocytes of HLA-DRB*0401 transgenic mice by bead sorting. Treg cells were transduced with TCR or TCR+mouse FOXP3, or Treg cells were cultured with virus-free supernatant (sham). One day after transduction, TCR or TCR+FOXP3 transduced cells were injected into HLA-DRB*0401 transgenic hosts conditioned with 4 Gy irradiation. After 7 weeks, engraftment of transduced Treg cells was measured using flow cytometry. A. Transduction efficiency was measured one day after transduction through the expression of human variable 2.1 and mouse Foxp3. [Figure 9-2]Figure 9 shows that regulatory T cells transduced with TCR can engraft in irradiated hosts, but exogenous FOXP3 expression is required to prevent the accumulation of TCR+FOXP3- cells. Thy1.1+CD4+CD25+Treg cells were isolated from lymph node and spleen cells of HLA-DRB*0401 transgenic mice by bead sorting. Treg cells were transduced with TCR or TCR+mouse FOXP3, or Treg cells were cultured with virus-free supernatant (sham). One day after transduction, TCR or TCR+FOXP3 transduced cells were injected into HLA-DRB*0401 transgenic hosts conditioned with 4 Gy irradiation. After 7 weeks, engraftment of transduced Treg cells was measured using flow cytometry. Splenocytes obtained from mice administered with transduced B.TCR or TCR+FOXP3-containing Treg cells were stained with Thy1.1 to identify the transferred cells (upper panel) as well as FOXP3 and TCR (lower panel). [Figure 9-3]Figure 9 shows that regulatory T cells transduced with TCR can engraft in irradiated hosts, but exogenous FOXP3 expression is required to prevent the accumulation of TCR+FOXP3- cells. Thy1.1+CD4+CD25+Treg cells were isolated from lymph node and spleen cells of HLA-DRB*0401 transgenic mice by bead sorting. Treg cells were transduced with TCR or TCR+mouse FOXP3, or Treg cells were cultured with virus-free supernatant (sham). One day after transduction, TCR or TCR+FOXP3 transduced cells were injected into HLA-DRB*0401 transgenic hosts conditioned with 4 Gy irradiation. After 7 weeks, engraftment of transduced Treg cells was measured using flow cytometry. C. Cumulative data (n=3) showing the doubling of transduction efficiency (left panel) and the doubling of the absolute number of transduced cells (right panel) compared to the day of injection for Treg cells transduced with TCR or TCR+FOXP3. Error bars indicate the standard error of the mean. Statistical analysis by independent t-test. D. Typical expression of FOXP3 in transduced cells 7 weeks after transplantation. The graph shows the cumulative percentage of FOXP3+ cells in the transduced population at week 7 (left) and the doubling of FOXP3+ cells compared to the day of injection (n=3). Error bars indicate the standard error of the mean. *p=>0.05, **p=>0.01 were measured by independent t-test. [Figure 10A] Figure 10 shows that Tregs expressing exogenous FOXP3 retain Treg functionality after 7 weeks in vivo, while Tregs not expressing exogenous FOXP3 acquire the ability to produce effector cytokines. In A, splenocytes were cultured for 4 hours with CD86+HLA-DR4+CHO cells pulsed with an unrelated peptide or 10 μM MBP. IL-2 and IFNg production was measured by flow cytometry. The FACS plot shows CD45.1 cells containing Tregs expressing only TCR (upper panel) and Thy1.1 cells containing Tregs expressing TCR+FOXP3. [Figure 10B]Figure 10 shows that Tregs expressing exogenous FOXP3 retain Treg functionality even after 7 weeks in vivo, while Tregs not expressing exogenous FOXP3 acquire the ability to produce effector cytokines. The graph in B shows cumulative IL-2 and IFNγ production by TCR-expressing (dark gray) and TCR + FOXP3-expressing (light gray) Tregs. Error bars indicate the standard deviation of the mean (n = 3). [Figure 11] Figure 11 shows schematic diagrams of exemplary retroviral vectors encoding (A) TCR α and β chains and (B) FOXP3 + TCR α and β chains. **BEST MODE FOR CARRYING OUT THE INVENTION**

[0025] The present invention provides a method for enhancing the ability of regulatory T cells (Tregs) to suppress an immune response, which includes increasing FOXP3 expression in the Tregs.

[0026] Regulatory T cells The term "regulatory T cells" (Tregs) refers to T cells that express the markers CD4, CD25, and FOXP3 (CD4 + CD25 + FOXP3 + ). Tregs may be identified using the cell surface markers CD4 and CD25, in the absence or in combination with low-level expression of the surface protein CD127 (CD4 + CD25 + CD127 - or CD4 + CD25 + CD127 low ). Tregs may also express high levels of CTLA-4 (cytotoxic T lymphocyte-associated molecule-4) or GITR (glucocorticoid-induced TNF receptor) on the cell surface. Unlike normal T cells, Tregs do not produce IL-2 and are therefore anergic at baseline.

[0027] The term "natural Treg" refers to Treg cells originating from the thymus. Natural Treg cells are CD4+CD25+FOXP3+Helios+Neuropilin 1+. The term "natural Treg" distinguishes thymic Treg cells from "induced Treg cells" that originate from normal T cells outside the thymus. Compared to induced Treg cells, natural Treg cells show higher expression of PD-1 (programmed cell death-1, pdcd1), Neuropilin 1 (Nrp1), Helios (Ikzf2), and CD73. Natural Treg cells may be further distinguished from induced Treg cells based on individual expression of Helios protein or Neuropilin 1 (Nrp1).

[0028] As used herein, the term "inducible regulatory T cell" (iTreg) refers to mature extrathymic CD4 cells. + CD4 cells originate from normal T cells. + CD25 + FOXP3 + Helios - Neuropilin 1 - This refers to T cells. For example, iTreg cells are CD4 cells in the presence of IL-2 and TGF-β. + CD25 - FOXP3 - It can be induced in vitro from cells.

[0029] As appropriate, the Treg cells express FOXP3 from the endogenous FoxP3 gene of their cells.

[0030] As appropriate, the Treg is CD4 + CD25 + FOXP3 + Treg may also be acceptable.

[0031] As appropriate, the Treg is CD4 + CD25 + CD127 - Treg may also be acceptable.

[0032] As appropriate, the Treg is CD4+ CD25 + CD127 low Treg may also be acceptable.

[0033] As appropriate, the Treg is CD4 + CD25 + CD127 - CD45RA + Treg may also be acceptable.

[0034] As appropriate, the Treg is CD4 + CD25 + CD127 low CD45RA + Treg may also be acceptable.

[0035] As appropriate, the Treg is CD4 + CD25 + FOXP3 + CD127 - Treg may also be acceptable.

[0036] As appropriate, the Treg is CD4 + CD25 + FOXP3 + CD127 low Treg may also be acceptable.

[0037] As appropriate, the Treg is CD4 + CD25 + FOXP3 + Helios + It is Treg.

[0038] As appropriate, the Treg is CD4 + CD25 + FOXP3 + Neuropilin 1 + It is Treg.

[0039] As appropriate, the Treg is CD4 + CD25 + FOXP3 + Helios + Neuropilin 1 + It is Treg.

[0040] Where appropriate, the Treg is a human Treg. Where appropriate, the Treg is a human Treg and the FOXP3 is a human FOXP3.

[0041] The term "Tconv cells" refers to normal T cells, specifically T cells that are not Treg cells.

[0042] In some embodiments, the Treg of the present invention may be derived from stem cells. In particular, the Treg of the present invention may be induced from stem cells in vitro.

[0043] In another embodiment, the cells are progenitor cells.

[0044] As used herein, the term "stem cell" means an undifferentiated cell capable of infinitely generating many more stem cells of the same type, from which other specialized cells may arise through differentiation. Stem cells are pluripotent. Stem cells may be, for example, embryonic stem cells or adult stem cells.

[0045] As used herein, the term “progenitor cell” means a cell that can differentiate to form one or more types of cells, but has limited self-renewal capabilities in vitro.

[0046] The aforementioned cells can be differentiated into T cells such as Tregs as appropriate.

[0047] Depending on the circumstances, the cells may have the ability to differentiate into T cells such as Tregs that express FOXP3.

[0048] The cells may, as appropriate, be embryonic stem cells (ESCs). The cells may, as appropriate, be hematopoietic stem cells or hematopoietic progenitor cells. The cells may, as appropriate, be induced pluripotent stem cells (iPSCs). The cells may, as appropriate, be obtained from umbilical cord blood. The cells may, as appropriate, be obtained from adult peripheral blood.

[0049] In some embodiments, hematopoietic stem and progenitor cells (HSPCs) may be obtained from umbilical cord blood. Umbilical cord blood can be collected according to techniques known in the art (e.g., U.S. Patents 7,147,626 and 7,131,958, which are incorporated herein by reference).

[0050] In some embodiments, HSPCs may be obtained from pluripotent stem cell sources, such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs).

[0051] As used herein, the terms “hematopoietic stem and progenitor cells” or “HSPC” refer to cells that express the antigen marker CD34 (CD34 + ) refers to cells and populations of such cells. In certain embodiments, the term "HSPC" refers to the presence of the antigen marker CD34 (CD34 + This refers to cells identified by the absence of ) and cell lineage (lin) markers. CD34 + and / or Lin( - Examples of cell populations that include ) cells include hematopoietic stem cells and hematopoietic progenitor cells.

[0052] HSPCs can be obtained or isolated from adult bone marrow, including the femur, hip, ribs, sternum, and other bones. Bone marrow aspirates containing HSPCs can be obtained or isolated directly from the hip using needles and syringes. Other sources of HSPCs include umbilical cord blood, placental blood, mobilized peripheral blood, Wharton's gelatin, placenta, fetal blood, fetal liver, or fetal spleen. In certain embodiments, mobilization of stem and progenitor cells in the subject may be required to obtain a sufficient amount of HSPCs for therapeutic use.

[0053] As used herein, the terms “induced pluripotent stem cells” or “iPSCs” refer to non-pluripotent cells that have been reprogrammed to a pluripotent state. Once the non-pluripotent cells of interest have been reprogrammed to a pluripotent state, they can then be programmed into a desired cell type, such as hematopoietic stem cells or hematopoietic progenitor cells (HSCs and HPCs, respectively).

[0054] As used herein, the term “reprogramming” refers to a method of improving the capabilities of cells to a more undifferentiated state.

[0055] As used herein, the term “programming” refers to a method of reducing the capabilities of a cell or differentiating a cell into a more differentiated state.

[0056] Immune response The expression "enhances the ability to suppress the immune response" means increasing the inhibitory effect of a Treg (or population of such Tregs) on the immune response compared to the inhibitory effect of a corresponding Treg (or population of such Tregs) that has not been modified by the method of the present invention.

[0057] The term "immune response" refers to the numerous physiological and cellular effects promoted by the immune system in response to stimuli such as pathogens or autoantigens. Specific examples of such effects include increased proliferation of Tconv cells and cytokine secretion. Any such effect may be used as an indicator of the strength of the immune response. A relatively weaker immune response by Tconv in the presence of modified Tregs compared to unmodified Tregs indicates a relative enhancement of the modified Tregs in suppressing the immune response. For example, a relative decrease in cytokine secretion would be an indicator of a weaker immune response, and thus an indicator of an enhanced ability of Tregs to suppress the immune response.

[0058] Assays for measuring indicators of immune response intensity and thereby measuring the inhibitory ability of Tregs are known in this field. Specifically, antigen-specific Tconv cells may be co-cultured with Tregs, and the response from the Tconv cells may be stimulated by adding the peptide of the corresponding antigen to the co-culture. The degree of proliferation of the Tconv cells and / or the amount of cytokine IL-2 they secrete in response to the addition of the peptide may be used as an indicator of the inhibitory ability of the co-cultured Tregs.

[0059] Antigen-specific Tconv cells co-cultured with the Treg of the present invention exhibiting increased FOXP3 expression may proliferate 5, 10, 15, 20, 25, 30, 35, or 40% less than the same Tconv cells co-cultured with a corresponding Treg that does not exhibit increased FOXP3 expression.

[0060] Antigen-specific Tconv cells co-cultured with the Treg of the present invention exhibiting increased FOXP3 expression may show at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% greater reductions in effector cytokines compared to corresponding Tconv cells co-cultured with corresponding Tregs that do not exhibit increased FOXP3 expression.

[0061] Antigen-specific Tconv cells co-cultured with the Treg of the present invention exhibiting increased FOXP3 expression may produce 10%, 20%, 30%, 40%, 50%, 60%, or less effector cytokines than corresponding Tconv cells co-cultured with a corresponding Treg that does not exhibit increased FOXP3 expression.

[0062] 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.

[0063] The effector cytokine may be selected from IL-2, IL-17, TNFα, GM-CSF, and IFN-γ as appropriate.

[0064] Antigen-specific Tconv cells co-cultured with the Treg cells of the present invention exhibiting increased FOXP3 expression may achieve suppression of IL-2 production at 1 / 2, 1 / 4, 1 / 8, 1 / 10, or 1 / 20 the cell number of the corresponding Treg cells that do not exhibit increased FOXP3 expression.

[0065] FOXP3 "FOXP3" is an abbreviation for the forkhead box P3 protein. FOXP3 is a member of the FOX protein family of transcription factors and functions as a major regulator of regulatory pathways in the development and function of regulatory T cells.

[0066] "Increasing FOXP3 expression" means increasing the level of FOXP3 mRNA and / or protein in a Treg (or population of such Tregs) compared to a corresponding Treg (or population of such Tregs) that has not been modified by the method of the present invention. For example, the level of FOXP3 mRNA and / or protein in a Treg (or population of such Tregs) modified by the method of the present invention can be increased by at least 1.5 times, at least 2 times, at least 5 times, at least 10 times, at least 50 times, at least 100 times, or at least 150 times compared to the level in a corresponding Treg (or population of such Tregs) that has not been modified by the method of the present invention.

[0067] Depending on the circumstances, the levels of FOXP3 mRNA and / or protein in a Treg (or population of such Tregs) modified by the method of the present invention can be increased by at least 1.5 times compared to the levels in the corresponding Treg (or population of such Tregs) that have not been modified by the method of the present invention.

[0068] Depending on the circumstances, the levels of FOXP3 mRNA and / or protein in a Treg (or population of such Tregs) modified by the method of the present invention can be increased by at least twice as much as the levels in the corresponding Treg (or population of such Tregs) that have not been modified by the method of the present invention.

[0069] Depending on the circumstances, the levels of FOXP3 mRNA and / or protein in a Treg (or population of such Tregs) modified by the method of the present invention can be increased by at least five times compared to the levels in the corresponding Treg (or population of such Tregs) that have not been modified by the method of the present invention.

[0070] Techniques for measuring specific mRNA and protein levels are well known in this field. mRNA levels in cell populations such as Treg may be measured by techniques such as Affymetrix eBioscience Prime Flow RNA Assay, Northern blotting, sequential gene expression analysis (SAGE), or quantitative polymerase chain reaction (qPCR). Protein levels in cell populations may be measured by techniques such as flow cytometry, high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC / MS), Western blotting, or enzyme-linked immunosorbent assay (ELISA).

[0071] In some embodiments of the present invention, FOXP3 expression is increased by introducing a polynucleotide encoding the FOXP3 polypeptide into isolated Treg cells.

[0072] The term "introduction" refers to methods for inserting foreign DNA into cells, including both transfection and transduction. Transfection is the process of introducing nucleic acids into cells by non-viral methods. Transduction is the process of introducing foreign DNA into cells via a viral vector.

[0073] A "FOXP3 polypeptide" is a polypeptide possessing FOXP3 activity, i.e., a polypeptide capable of functioning as a transcription factor that binds to FOXP3 target DNA and regulates the development and function of Tregs. Techniques for measuring transcription factor activity are well known in this field. 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 level of expression of the gene regulated by the transcription factor. Gene expression may be quantified by measuring the level 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).

[0074] Polynucleotides and polypeptides The terms “polynucleotide” and “nucleic acid” are intended to be synonymous. A polynucleotide may be any suitable type of nucleotide sequence, such as a synthetic RNA / DNA sequence, a cDNA sequence, or a partial genomic DNA sequence.

[0075] The term "polypeptide" is synonymous with "protein" and typically refers to a series of residues, usually L-amino acids, linked to one another by peptide bonds between the α-amino and carboxyl groups of adjacent amino acids.

[0076] Numerous different polynucleotides may encode the same polypeptide as a result of the degeneracy of the genetic code. Those skilled in the art can reflect the codon usage frequency of any particular host organism in which the polypeptide is expressed by making nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotide.

[0077] The polynucleotide may contain DNA or RNA, may be single-stranded or double-stranded, and may even contain synthetic or modified nucleotides. Several different types of modifications to oligonucleotides are known in the art. These modifications include methylphosphonate and phosphorothioate backbones, and the addition of acridine or polylysine chains to the 3' and / or 5' ends of the molecule. The polynucleotide may be modified by any method in the art. Such modifications may improve the in vivo activity or lifetime of the polynucleotide.

[0078] The polynucleotide may be in an isolated or recombinant form. The polynucleotide may be incorporated into a vector, or the vector may be incorporated into a host cell.

[0079] The polynucleotide may have optimized codons. Different cells use different codons. This codon bias corresponds to a bias in the relative abundance of a particular tRNA in a cell type. Expression can be increased by modifying the codons in the sequence to match the relative abundance of the corresponding tRNA. The polynucleotide may, as appropriate, have optimized codons for expression in a mouse model of disease. The polynucleotide may, as appropriate, have optimized codons for expression in human subjects.

[0080] Many viruses, including HIV and other lentiviruses, utilize numerous rare codons. By modifying these to correspond to commonly used mammalian codons, increased expression of packaging components in mammalian producer cells can be achieved. Codon frequency tables for mammalian cells and various other organisms are publicly known in this field. Codon optimization may also involve the removal of mRNA unstable motifs and latent splice sites.

[0081] FOXP3 polypeptide sequence Depending on the context, the FOXP3 polypeptide may be a polypeptide sequence of human FOXP3, for example, UniProtKB accession number Q9BZS1, or a functional fragment thereof, i.e. It may include TIFF0007839774000001.tif38164.

[0082] In some embodiments of the present invention, the FOXP3 polypeptide comprises an amino acid sequence or a functional fragment thereof that is at least 80% identical to SEQ ID NO: 3. Optionally, the FOXP3 polypeptide comprises an amino acid sequence or a functional fragment thereof that is at least 85, 90, 95, 98, or 99% identical to SEQ ID NO: 3. In some embodiments, the FOXP3 polypeptide comprises SEQ ID NO: 3 or a functional fragment thereof.

[0083] The FOXP3 polypeptide may optionally be a variant of SEQ ID NO: 3, for example, a natural variant. The FOXP3 polypeptide may optionally be an isoform of SEQ ID NO: 3. For example, the FOXP3 polypeptide may contain deletions at amino acid positions 72-106 with respect to SEQ ID NO: 3. Alternatively, the FOXP3 polypeptide may contain deletions at amino acid positions 246-272 with respect to SEQ ID NO: 3.

[0084] Depending on the circumstances, the FOXP3 polypeptide may be represented by Sequence ID No. 4 or its functional fragment, i.e. Includes TIFF0007839774000002.tif39163.

[0085] The FOXP3 polypeptide may optionally include an amino acid sequence or functional fragment thereof that is at least 80% identical to SEQ ID NO: 4. The polypeptide may optionally include an amino acid sequence or functional fragment thereof that is 85, 90, 95, 98, or 99% identical to SEQ ID NO: 4.

[0086] The FOXP3 polypeptide may optionally be a variant of SEQ ID NO: 4, for example, a natural variant. The FOXP3 polypeptide may optionally be an isoform or functional fragment of SEQ ID NO: 4. For example, the FOXP3 polypeptide may contain deletions at amino acid positions 72-106 with respect to SEQ ID NO: 4. Alternatively, the FOXP3 polypeptide may contain deletions at amino acid positions 246-272 with respect to SEQ ID NO: 4.

[0087] FOXP3 polynucleotide sequence As appropriate, the FOXP3 polypeptide is represented by Sequence ID No. 1, i.e. It is encoded by the polynucleotide sequence represented by TIFF0007839774000003.tif62163TIFF0007839774000004.tif48164.

[0088] In some embodiments of the present invention, the polynucleotide encoding the FOXP3 polypeptide or variant comprises a polynucleotide sequence or a functional fragment thereof that is at least 80% identical to SEQ ID NO: 1. Optionally, the polynucleotide encoding the FOXP3 polypeptide or variant comprises a polynucleotide sequence or a functional fragment thereof that is at least 85, 90, 95, 98, or 99% identical to SEQ ID NO: 1. In some embodiments of the present invention, the polynucleotide encoding the FOXP3 polypeptide or variant comprises SEQ ID NO: 1 or a functional fragment thereof.

[0089] As appropriate, the FOXP3 polypeptide is represented by Sequence ID No. 2, i.e. Encoded by the polynucleotide sequence shown in TIFF0007839774000005.tif111163.

[0090] In some embodiments of the present invention, the polynucleotide encoding the FOXP3 polypeptide or variant comprises a polynucleotide sequence or a functional fragment thereof that is at least 80% identical to SEQ ID NO: 2. Optionally, the polynucleotide encoding the FOXP3 polypeptide or variant comprises a polynucleotide sequence or a functional fragment thereof that is at least 85, 90, 95, 98, or 99% identical to SEQ ID NO: 2. In some embodiments of the present invention, the polynucleotide encoding the FOXP3 polypeptide or variant comprises SEQ ID NO: 2 or a functional fragment thereof.

[0091] The polynucleotide encoding the FOXP3 polypeptide or its variant may have its codons optimized as appropriate. The polynucleotide encoding the FOXP3 polypeptide or its variant may have its codons optimized for expression in human cells as appropriate.

[0092] Sequence comparison Sequence comparison can be performed visually or, more commonly, using readily available sequence comparison programs. These publicly and commercially available computer programs can calculate sequence identity between two or more sequences.

[0093] Sequence identity may be calculated across multiple consecutive sequences; that is, one sequence is aligned with another sequence, and then each amino acid in one sequence is directly compared with its corresponding amino acid in the other sequence, one residue at a time. This is called a "gapless" alignment. Typically, such gapless alignment is performed only over a relatively small number of residues (e.g., fewer than 50 consecutive amino acids).

[0094] While this method is very simple and consistent, it does not take into account, for example, that a single insertion or deletion in an otherwise identical sequence pair may exclude subsequent amino acid residues from the alignment, potentially leading to a significant decrease in homology percentage when performing a global alignment. Therefore, most sequence comparison methods are designed to produce an optimal alignment that takes into account all possible insertions and deletions without excessively penalizing the overall homology score. This is achieved by attempting to maximize local homology by inserting "gaps" into the sequence alignment.

[0095] However, these more complex methods assign a "gap penalty" to each gap in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible (reflecting a higher relevance between the two comparison sequences) scores higher than one with many gaps. Typically, an "affine gap cost" is used, which imposes a relatively high cost on the presence of gaps and a smaller penalty on each subsequent residue within those gaps. This is the most commonly used gap scoring system. A high gap penalty naturally produces an optimized alignment with fewer gaps. Most alignment programs allow you to change the gap penalty. However, when using such software for sequence comparison, it is preferable to use the default values. For example, when using the GCG Wisconsin Bestfit package (see below), the default gap penalty for amino acid sequences is -12 for one gap and -4 for each extension.

[0096] Therefore, calculating the maximum percentage of sequence identity first requires generating an optimal alignment that takes gap penalties into account. A suitable computer program for performing such alignment is the GCG Wisconsin Bestfit package (incorporated herein by reference, University of Wisconsin, USA; Devereux et al., 1984, Nucleic Acids Research 12:387). Other software capable of performing sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid - Chapter 18), FASTA (incorporated herein by reference, Atschul et al., 1990, J. Mol. Biol., 403-410), and the GENEWORKS comparison tools suite. Both BLAST and FASTA are available for offline and online searches (incorporated herein by reference, Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However, it is preferable to use the GCG Bestfit program.

[0097] The sequence identity may be determined over the entire sequence as appropriate. The sequence identity may also be determined over the entire range of candidate sequences to be compared with the sequences listed herein as appropriate.

[0098] While final sequence identity can be measured in terms of identity, the alignment process itself is generally not based on all-or-nothing pair comparisons. Instead, a scaled similarity score matrix is ​​typically used, which assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. A commonly used example of such a matrix is ​​the BLOSUM62 matrix (the default matrix for the BLAST program suite). The GCG Wisconsin program generally uses either public default values ​​or custom symbol comparison tables, if provided (see the user manual for further details). Preferably, use public default values ​​for the GCG package, or the default matrix, such as BLOSUM62, for other software.

[0099] Once the software generates the optimal alignment, the sequence identity percentage can be calculated. Typically, the software performs this as part of a sequence comparison and generates a numerical result.

[0100] vector In some embodiments of the present invention, the polynucleotide encoding FOXP3 is a contiguous portion of the expression vector.

[0101] The term "expression vector" refers to a construct that enables the expression of the FOXP3 polypeptide. Where appropriate, the expression vector is a cloning vector.

[0102] Suitable vectors include, but are not limited to, plasmids, viral vectors, transposons, or nucleic acids that are complexed with polypeptides or immobilized on solid-phase particles.

[0103] Preferably, the expression vector has the ability to maintain high levels of expression in host cells.

[0104] The expression vector may be a retroviral vector. The expression vector may be based on the MP71 vector skeleton, or may be derivable from the MP71 vector skeleton. The expression vector may lack a full-length or truncated woodchuck hepatitis response element (WPRE).

[0105] In some embodiments of the present invention, the vector also encodes a T cell receptor (TCR).

[0106] TCRs are cell surface molecules that bind to fragments of antigens bound to major histocompatibility complex (MHC) molecules on antigen-presenting cells as part of directing the immune response. The TCR may, as appropriate, be a recombinant protein; in other words, the TCR may be an exogenous protein that is not spontaneously expressed by the Treg of this invention.

[0107] In some embodiments of the present invention, the vector also encodes a chimeric antigen receptor (CAR).

[0108] CARs are recombinant cell surface molecules expressed by genetically modified T cells that bind to antigens expressed on the surface of other cells as part of directing the immune response. More specifically, CARs are proteins that transfer the specificity of antigen conjugates, such as monoclonal antibodies (mAbs), to the effector function of T cells. Their typical form is a type I transmembrane domain protein with an antigen-recognizing amino terminus, a spacer, and a transmembrane domain, all linked to a compound endodomain that transmits T cell survival and activation signals.

[0109] If the vector includes a polynucleotide encoding TCR or CAR in addition to the polynucleotide encoding FOXP3, the vector may have a 5' FOXP3-TCR / CAR 3' orientation. Therefore, the polynucleotide encoding FOXP3 may be on the 5' side of the polynucleotide encoding CAR or TCR.

[0110] The polynucleotide encoding FOXP3 may be separated from the polynucleotide encoding TCR or CAR by a nucleic acid sequence that allows both the nucleic acid sequence encoding FOXP3 and the nucleic acid sequence encoding TCR or CAR to be expressed from the same mRNA transcript.

[0111] For example, the polynucleotide may include an internal ribosome entry site (IRES) between (i) FOXP3 and (ii) a nucleic acid sequence encoding a TCR or CAR. The IRES is a nucleotide sequence that enables translation initiation in the middle of the mRNA sequence.

[0112] The polynucleotide may include (i) a nucleic acid sequence encoding a FOXP3 and (ii) a TCR or CAR, linked by an internal self-cleavage sequence.

[0113] The vector may optionally have the following structure: 5' strong promoter (e.g., LTR)-FoxP3-2A-CAR / TCR-3'LTR. In this case, FOXP3 expression is directly driven by the strong LTR promoter for optimal expression. The 2A sequence is present before the CAR / TCR, so CAR / TCR expression depends on both LTR promoter activity and 2A cleavage activity. Importantly, the arrangement in which FOXP3 precedes the CAR / TCR in the 5'→3' direction ensures that CAR / TCR expression can only occur if FOXP3 is expressed, and that CAR / TCR expression does not occur without FOXP3. This is particularly advantageous in the context of genetically engineered Tregs to reduce the risk of the genetically engineered Tregs acquiring an effector phenotype and / or to reduce the risks associated with introducing CAR or TCR into T effector cells present in the initial population.

[0114] The internal self-cleaving sequence may be any sequence that enables the separation of the polypeptide comprising (i) FOXP3 and (ii) TCR or CAR.

[0115] The cleavage site may be a self-cleavage site such that, once the polypeptide is produced, the polypeptide is immediately cleaved into individual peptides without requiring any external cleavage activity.

[0116] The term "cleavage" is used herein for convenience, but the cleavage site may separate the peptide into individual substances by mechanisms other than classical cleavage. For example, in the case of the foot-and-mouth disease virus (FMDV) 2A autocleavage peptide, various models have been proposed to explain the "cleavage" activity, i.e., proteolysis, autoproteolysis, or translation by host cell proteinases (Donnelly et al. (2001) J. Gen. Virol. 82:1027-1041, incorporated herein by reference). The precise mechanism of such "cleavage" is not important to the purposes of this invention, as long as the cleavage site is located between protein-coding nucleic acid sequences and causes the protein to be expressed as a separate substance.

[0117] The self-cleaving peptide may be a 2A self-cleaving peptide obtained from an aphthous virus or cardiovirus.

[0118] Variants can be considered in terms of similarity (i.e., amino acid residues with similar chemical properties / functions), and preferably, variants are expressed in terms of sequence identity.

[0119] Sequence comparison can be performed visually or, more commonly, using readily available sequence comparison programs. These publicly and commercially available computer programs can calculate sequence identity between two or more sequences.

[0120] The FOXP3 polypeptide expressed from this vector may be located at the N-terminus of a self-cleaving peptide, such as a 2A self-cleaving peptide. Such a FOXP3-2A polypeptide may contain the sequence shown in SEQ ID NO: 5 or 6, or a variant of SEQ ID NO: 5 or 6 that is at least 80% identical to said sequence. The variant may be at least 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 5 or 6.

[0121] Sequence ID 5 TIFF0007839774000006.tif33164 Sequence ID 6 TIFF0007839774000007.tif32163

[0122] Virus-induced transduction In some embodiments of the present invention, the polynucleotide encoding FOXP3 is introduced into isolated Treg cells by viral transduction.

[0123] Examples of viral delivery systems include, but are not limited to, adenovirus vectors, adeno-associated virus (AAV) vectors, herpesvirus vectors, retrovirus vectors, lentivirus vectors, and baculovirus vectors.

[0124] In some embodiments, the polynucleotide encoding FOXP3 is introduced into isolated Treg cells by retroviral transduction.

[0125] Retroviruses are RNA viruses that have a different life cycle than lytic viruses. In this respect, retroviruses are infectious agents that replicate via DNA intermediates. When a retrovirus infects a cell, its genome is converted into DNA by an enzyme called reverse transcriptase. This DNA copy then acts as a template for producing a new RNA genome and the viral coding proteins necessary for assembling infectious viral particles.

[0126] Many retroviruses exist, such as mouse leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), mouse mammary cancer virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney's mouse leukemia virus (Mo-MLV), FBR mouse osteosarcoma virus (FBR MSV), Moloney's mouse sarcoma virus (Mo-MSV), Abelson's mouse leukemia virus (A-MLV), avian myelocytosis virus-29 (MC29), and avian erythroblastosis virus (AEV), as well as all other retroviral viruses, including lentiviruses.

[0127] A detailed list of retroviruses can be found in Coffin et al. (incorporated herein by reference, “Retroviruses” 1997 Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763).

[0128] Lentiviruses also belong to the retrovirus family, but they can infect both dividing and non-dividing cells (Lewis et al. 1992 EMBO J. 3053-3058, incorporated herein by reference).

[0129] For efficient infection of human cells, viral particles may be packaged with a bispecies-targeting envelope or a gibbon leukemia virus envelope.

[0130] Isolation of Treg In some embodiments, the method according to the present invention is as follows (a) and (b), namely (a) Isolating Treg from a cell population, and (b) Increase FOXP3 expression in the Treg. Includes.

[0131] The phrase "isolate Tregs from a cell population" means separating Tregs from a heterogeneous mixture of multiple different cell types. A suitable cell population would be one obtained from a sample of a human subject.

[0132] If appropriate, the Treg cells are isolated as a group of Treg cells.

[0133] As appropriate, the Treg population may include at least 70% Tregs, for example, 75%, 85%, 90%, or 95% Tregs.

[0134] In some embodiments of the present invention, the cell population includes peripheral blood mononuclear cells (PBMCs) or consists of PMBCs.

[0135] PBMCs are any blood cells with a round nucleus that are found in the circulating pool of blood rather than being trapped in the bone marrow, liver, spleen, or lymphatic system. PBMCs consist of monocytes and lymphocytes (T cells, B cells, and NK cells). Techniques for isolating PBMCs from whole blood are well known in this field. For example, PBMCs can be separated from blood samples by adding a density gradient medium such as Ficoll (GE Healthcare) followed by centrifugation. Different types of cells in the blood separate into various layers, including a layer containing PBMCs.

[0136] In some embodiments of the present invention, the isolation of the Treg is performed by CD4 + This includes isolating T cells. In some embodiments, the isolation of Treg cells is performed using CD4 + To isolate T cells, and thereafter the CD4 + This includes isolating the Treg cells from T cells.

[0137] CD4 (cluster of differentiation 4) is a co-receptor of T cell receptors expressed by various types of T cells. +Cell isolation involves separating T cells containing Tregs from an initial cell population. Subsequently, the Tregs may be isolated from this T cell-rich population.

[0138] Techniques for isolating specific cell types from heterogeneous cell populations are well known in this field. Specific examples include the use of immunomagnetic beads and fluorescence-activated cell sorting.

[0139] In some embodiments of the present invention, the isolation of the Treg population includes the use of immunomagnetic beads. Various companies (e.g., Miltenyi Biotec, Stem Cell Technologies, ThermoFisher Scientific) offer kits containing immunomagnetic beads for the isolation of specific types of T cells (see, for example, Fallarino et al. (2003) Modulation of tryptophan catabolism by regulatory T cells. Nat. Immunol. 4: 1206-1212, incorporated herein by reference). These isolation kits utilize antibodies widely available in the art against T cell surface proteins such as CD8, CD25, CD49b, and others. For example, CD4 + Cells, cell populations, non-CD4 + The cells may first be subjected to negative selection by incubation with a biotin-conjugated antibody against a cell marker (e.g., CD8) and then removal using anti-biotin magnetic beads. Subsequently, the Treg cells may be subjected to positive selection by incubation with anti-CD25 labeled beads.

[0140] In some embodiments of the present invention, the isolation of the Treg population includes fluorescence-activated cell sorting (FACS). In some embodiments, the Treg is isolated on its CD4 + CD25 hi CD127 - Select according to phenotype.

[0141] Innate Tregs may be selected from inducible Tregs based on the expression of helios protein or neuropilin 1. In some embodiments of the present invention, the innate Tregs are selected based on their CD4 + CD25 + FOXP3 + Helios + Neuropilin 1 + You may also select based on phenotype.

[0142] FACS is a form of flow cytometry well known in this field. In FACS, cells are suspended in a liquid and passed through a detection system that analyzes various properties. Cells can be sorted according to their properties using this method. In particular, FACS labels molecules using fluorescent antibodies and sorts cells according to the degree of fluorescence, which indicates the expression level of specific molecules (see Adan et al., Flow cytometry: basic principles and applications, Crit. Rev. Biotechnol. 2017 Mar;37(2):163-176, incorporated herein by reference).

[0143] Genetically modified Treg The present invention also provides genetically modified Tregs, such as those produced by the method of the present invention.

[0144] The term "genetically modified Treg" refers to a Treg gene that has been manipulated through human intervention to alter its gene expression.

[0145] The present invention also provides genetically modified Tregs that exhibit higher FOXP3 expression than non-genetically modified Tregs.

[0146] The present invention also provides a Treg that exhibits higher FOXP3 expression than the corresponding non-genetically modified Treg.

[0147] "High FOXP3 expression" means that the level of FOXP3 mRNA or protein in a genetically engineered Treg is higher than the level before the Treg was manipulated by human intervention to alter its gene expression.

[0148] "High FOXP3 expression" can be defined and measured as described herein.

[0149] Depending on the circumstances, the levels of CD25 mRNA and / or protein in a Treg (or population of such Tregs) modified by the method of the present invention can be increased by at least 1.5 times compared to the levels in the corresponding Treg (or population of such Tregs) that have not been modified by the method of the present invention.

[0150] Depending on the circumstances, the levels of CD25 mRNA and / or protein in a Treg (or population of such Tregs) modified by the method of the present invention can be increased by at least twice as much as the levels in the corresponding Treg (or population of such Tregs) that have not been modified by the method of the present invention.

[0151] Depending on the circumstances, the levels of CD25 mRNA and / or protein in a Treg (or population of such Tregs) modified by the method of the present invention can be increased by at least five times compared to the levels in the corresponding Treg (or population of such Tregs) that have not been modified by the method of the present invention.

[0152] Depending on the circumstances, the levels of CTLA-4 mRNA and / or protein in a Treg (or population of such Tregs) modified by the method of the present invention can be increased by at least 1.5 times compared to the levels in the corresponding Treg (or population of such Tregs) that have not been modified by the method of the present invention.

[0153] Depending on the circumstances, the levels of CTLA-4 mRNA and / or protein in a Treg (or population of such Tregs) modified by the method of the present invention can be increased by at least twice as much as the levels in the corresponding Treg (or population of such Tregs) that have not been modified by the method of the present invention.

[0154] Depending on the circumstances, the levels of CTLA-4 mRNA and / or protein in a Treg (or population of such Tregs) modified by the method of the present invention can be increased by at least five times compared to the levels in the corresponding Treg (or population of such Tregs) that have not been modified by the method of the present invention.

[0155] In some embodiments of the present invention, the genetically modified Treg comprises an exogenous polynucleotide encoding a FOXP3 polypeptide.

[0156] An "exogenous polynucleotide" is a polynucleotide derived from outside the Treg. This exogenous polynucleotide may be introduced into the Treg as part of an expression vector. Therefore, this exogenous polynucleotide may be contiguous with expression vector elements such as a promoter.

[0157] In some embodiments of the present invention, the FOXP3 polypeptide comprises an amino acid sequence or a functional fragment thereof that is at least 80% identical to SEQ ID NO: 3 or 4. Optionally, the FOXP3 polypeptide comprises an amino acid sequence or a functional fragment thereof that is at least 85%, 90%, 95%, 98%, or 99% identical to SEQ ID NO: 3 or 4. In some embodiments, the FOXP3 polypeptide comprises SEQ ID NO: 3 or 4 or a functional fragment thereof.

[0158] In some embodiments of the present invention, the exogenous polynucleotide encoding FOXP3 comprises a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 1 or 2. In some embodiments of the present invention, the polynucleotide encoding FOXP3 is identical to SEQ ID NO: 1 or 2.

[0159] In some embodiments of the present invention, the exogenous polynucleotide encoding FOXP3 is a contiguous portion of the vector.

[0160] In some embodiments of the present invention, the vector also encodes a T cell receptor (TCR).

[0161] In some embodiments of the present invention, the vector comprises a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 5.

[0162] composition The present invention also provides a pharmaceutical composition containing the genetically modified Treg of the present invention.

[0163] Such pharmaceutical compositions may contain pharmaceutically acceptable carriers, diluents, excipients, or adjuvants. The choice of pharmaceutically acceptable carriers, excipients, or diluents can be made in relation to the intended route of administration and standard pharmaceutical practices. The pharmaceutical compositions may contain, as carriers, excipients, or diluents (or in addition thereto), any suitable binder(s), lubricants(s), suspending agents(s), coating agents(s), solubilizers(s), and other carrier substances.

[0164] The pharmaceutical compositions described above should typically be sterile and stable under manufacturing and storage conditions. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous media, pastes, and embedded sustained-release or biodegradable formulations. Sterile injection formulations may be prepared using non-toxic, parenterally acceptable diluents or solvents. The pharmaceutical compositions of the present invention may contain pharmaceutically acceptable dispersants, wetting agents, suspending agents, isotonic agents, coatings, antimicrobial and antifungal agents, carriers, excipients, salts, or stabilizers that are not toxic to the target at the dosage and concentration used. Preferably, such compositions may further contain pharmaceutically acceptable carriers or excipients for use in the treatment of a disease, adapted to a given method and / or site of administration, for example, parenteral (e.g., subcutaneous, intradermal, or intravenous injection) or intrathecal administration.

[0165] The above composition may be manufactured using current Good Manufacturing Practices (cGMP).

[0166] The pharmaceutical composition containing the genetically modified Treg may optionally contain organic solvents, such as, but not limited to, methyl acetate, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), dimethoxyethane (DME), and dimethylacetamide (including mixtures or combinations thereof).

[0167] The pharmaceutical composition may, as appropriate, not contain endotoxins.

[0168] Prevention and / or treatment of disease The present invention also provides genetically modified Tregs or pharmaceutical compositions of the present invention for use in the prevention and / or treatment of diseases.

[0169] The present invention also provides the use of the genetically modified Treg in the manufacture of agents for the prevention and / or treatment of diseases.

[0170] The present invention also provides a method for preventing and / or treating a disease, comprising administering the genetically modified Treg or composition of the present invention to a subject.

[0171] Preferably, the method for preventing and / or treating the disease includes administering the pharmaceutical composition of the present invention to a subject.

[0172] The term “to treat / treat / treat (to administer / treat / treat)” refers to administering the genetically modified Treg or pharmaceutical composition of the present invention to a subject having a pre-existing disease or condition in order to alleviate, reduce or improve at least one symptom associated with the disease, and / or to slow, suppress or prevent the progression of the disease.

[0173] "Prevention" or "the act of preventing" (or "prophylaxis") refers to delaying or preventing the onset of symptoms of a disease. Prevention may be absolute (that the disease does not occur) or it may be effective only for some individuals or for a limited period of time.

[0174] In some embodiments of the present invention, the subject of the method of the present invention is a mammal, preferably a cat, dog, horse, donkey, sheep, pig, goat, cattle, mouse, rat, rabbit, or guinea pig. Preferably, the subject is a human.

[0175] The administration of the pharmaceutical composition of the present invention can be achieved by utilizing any of the various routes that make the active ingredient bioavailable. For example, genetically modified Treg or the pharmaceutical composition can be administered intravenously, intrathecally, orally and parenterally, intranasally, intraperitoneally, subcutaneously, percutaneously, or intramuscularly.

[0176] The genetically modified Treg or pharmaceutical composition of the present invention may be administered intravenously as appropriate. The genetically modified Treg or pharmaceutical composition of the present invention may be administered intrathecally as appropriate.

[0177] Typically, a physician determines the most appropriate dosage for each individual patient, and this dosage varies depending on the patient's age, weight, and response. The dosage is sufficient to reduce and / or prevent the symptoms of the disease.

[0178] Those skilled in the art understand, for example, that the route of delivery (e.g., oral vs. intravenous vs. subcutaneous) may affect the required dosage (and vice versa). For example, concentrated delivery may be preferable when a particularly high concentration of the drug is desired within a specific site or location. Other factors to consider when optimizing the route and / or administration schedule for a given treatment regimen include, for example, the disease being treated (e.g., type or stage), the clinical condition of the subject (e.g., age, overall health), the presence or absence of concomitant therapies, and other factors known to healthcare professionals.

[0179] The amount of medication prescribed is sufficient to stabilize or improve the symptoms of the disease.

[0180] The present invention also provides a method for treating and / or preventing a disease, comprising the step of administering a pharmaceutical composition containing cells, such as T cells according to the present invention, to a target.

[0181] Depending on the circumstances, the methods for preventing and / or treating the aforementioned diseases are as follows: (i) to (iii), namely (i) Isolation of Treg from the subject, (ii) Introducing a polynucleotide sequence encoding the FOXP3 polypeptide into the Treg (i.e., genetically modifying the Treg), (iii) Administering genetically modified Treg to the subject It may include.

[0182] Tregs may be isolated from a patient by taking a blood sample and isolating the Tregs from the blood sample using techniques known in the art, such as those described herein under the heading "Isolation of Tregs."

[0183] The polynucleotide encoding the FOXP3 polypeptide may be introduced into Treg using techniques known in the art, such as those described herein under the heading "Viral Transduction."

[0184] If appropriate, the genetically modified Treg cells may be grown in vitro before administration to the target organism. The Treg cells may also be grown in vitro by culturing them in TexMACX® medium.

[0185] disease The diseases that can be treated and / or prevented by the methods and use of the present invention may be any diseases involving a pathological immune response.

[0186] The aforementioned disease may be, for example, cancer, an infectious disease, or an autoimmune disease.

[0187] In some embodiments of the present invention, the disease is an autoimmune disease.

[0188] The aforementioned diseases may involve the central nervous system (CNS) in systemic autoimmune and inflammatory diseases such as Behçet's disease, sarcoidosis, systemic lupus erythematosus, juvenile idiopathic arthritis, scleroderma, and Sjögren's syndrome.

[0189] The aforementioned disease may be any disease in which MBP is the antigen, for example, MBP is the autoantigen.

[0190] Depending on the context, the aforementioned disease may be an autoimmune or inflammatory central nervous system disease (e.g., a chronic neurodegenerative condition).

[0191] Depending on the context, the aforementioned disease may also be a chronic neurodegenerative condition such as multiple sclerosis (MS), Alzheimer's disease, Parkinson's disease, neurotropic viral infection, stroke, paraneoplastic disorders, and traumatic brain injury.

[0192] In some embodiments of the present invention, the disease is multiple sclerosis. Optionally, the disease is chronic progressive multiple sclerosis. Optionally, the disease is relapsing / remittent multiple sclerosis.

[0193] Depending on the circumstances, the aforementioned disease may be HLA-DRB1 * 0401 positive case is observed. Appropriately, the disease is multiple sclerosis and the subject is HLA-DRB1 * 0401 positive. Appropriately, the disease is chronic progressive multiple sclerosis, and the subject is HLA-DRB1 * The test is positive for 0401. The disease is appropriately classified as relapsing / remitting multiple sclerosis, and the subject is HLA-DRB1. * 0401 is positive.

[0194] Multiple sclerosis Multiple sclerosis (MS) is the most common neurological disorder in young adults in Europe and the United States. MS is characterized as a demyelinating disease, a chronic degenerative disease of the central nervous system in which there is a gradual destruction of myelin in and around the brain and / or spinal cord, interfering with neural connections and causing muscle weakness, loss of coordination and speech, and visual impairment.

[0195] Several types or patterns of MS progression have been identified, including clinically isolated syndrome (CIS), relapsing-remitting MS (RRMS), primary progressive MS (PPMS), and secondary progressive MS (SPMS). In some individuals, the increase or progression of disability is very slow, while in others it may occur more rapidly. However, generally, recovery from seizures becomes increasingly incomplete, symptoms tend to worsen, and disability progresses.

[0196] While some disease-modifying therapies (DMTs) have been shown to reduce the frequency of clinical relapses, most patients continue to clinically worsen under current treatment schedules. Autologous hematopoietic stem cell transplantation may have a sustained beneficial effect on patients, but the procedure requires potent myeloablative conditioning with considerable toxicity. Neither DMT nor stem cell transplantation can mediate antigen-specific suppression of MS immunopathology. While we do not wish to be bound by theory, in the future, a single dose of the genetically modified Treg of this invention may result in sustained suppression of MS immunopathology without systemic side effects. This would have a significant impact on disease progression in people with MS.

[0197] Depending on the circumstances, the genetically modified Treg or pharmaceutical composition of the present invention may reduce or improve one or more symptoms of MS, including decreased or lost vision, stumbling and irregular gait, slurred speech, frequent urination and urinary incontinence, mood swings and depression, muscle spasms and paralysis.

[0198] The present invention further provides methods for inducing immune tolerance to grafts, methods for treating and / or preventing cellular and / or humoral graft rejection, and methods for treating and / or preventing graft-versus-host disease (GvHD), the methods comprising the step of administering the genetically modified Treg or pharmaceutical composition of the present invention to a subject.

[0199] As used herein, "inducing immune tolerance to a graft" means inducing immune tolerance in the recipient to the transplanted organ. In other words, inducing immune tolerance to a graft means reducing the level of the recipient's immune response to the transplanted organ from the donor. Inducing immune tolerance to the transplanted organ may reduce the amount of immunosuppressant medication required by the graft recipient, or may allow for the discontinuation of immunosuppressant medication.

[0200] In one embodiment, the subject is a graft recipient undergoing immunosuppressive therapy.

[0201] The graft may be selected from the liver, kidney, heart, lung, pancreas, intestine, stomach, bone marrow, angiogenic composite tissue graft, and skin graft.

[0202] This disclosure is not limited to the exemplary methods and materials disclosed herein, and any similar or equivalent methods and materials described herein may be used in the practice or study of embodiments of this disclosure. Numerical ranges include the numerical values ​​that define the range. Unless otherwise indicated, all nucleic acid sequences are written from left to right in the 5' to 3' direction, and all amino acid sequences are written from left to right in the amino-terminus to carboxy-terminus direction.

[0203] Where a range of values ​​is provided, each intervening value between the upper and lower limits of that range, up to one-tenth of the lower limit unit unless otherwise explicitly indicated in the context, is also specifically disclosed. Any narrower range between any stated value or intervening value within the stated range and any other stated value or intervening value within that stated range is each included in this disclosure. The upper and lower limits of these narrower ranges may be included in or excluded from such ranges independently, and each range that includes one of the limit values, does not include either, or includes both is also included in this disclosure, provided that any limit value is specifically excluded within that stated range. If a stated range includes one or both of its limit values, any range that excludes one or both of those included limit values ​​is also included in this disclosure.

[0204] The singular forms "a," "an," and "the" refer to multiple objects unless explicitly indicated otherwise in the context.

[0205] The terms “comprising,” “comprises,” and “comprised of,” as used herein, are synonymous with “including,” “includes,” or “containing,” and are inclusive or open-ended, not excluding additional, undescribed members, elements, or method steps. The terms “comprising,” “comprises,” and “comprised of” also include the term “consisting of.”

[0206] Embodiments of the present invention may be combined.

[0207] The publications discussed herein are provided solely for disclosure prior to the filing date of this application. Nothing in this specification should be construed as an acknowledgment that such publications constitute prior art with respect to the claims attached herein.

[0208] The present invention will be further described here through examples, which are intended to assist those skilled in the art in carrying out the invention and are not intended in any way to limit the scope of the invention. (Examples) [Example 1A]

[0209] Example 1A - Isolation of Innate Treg CD4 + T cells are CD4 + Cells were isolated using a positive selection kit. The cells were then stained with flow cytometry antibodies CD4, CD25, and CD127 using BD ARIA before FACS sorting. + CD25 hi CD127 - Treg and CD4 + CD25 - CD127 +Tconv was collected in a polypropylene tube. The purity of cell sorting was measured by the addition of FOXP3 PE antibody. CD4 + CD25 + CD127 - FOXP3 + The purity of the cells was usually >70%. [Example 1B]

[0210] Example 1B - Transduction of native Tregs using FOXP3 On day 0, Tregs and Tconvs sorted by FACS were separately activated for 48 hours by culturing them 1:1 with anti-CD3 and anti-CD28 beads. On day 2, the cells were counted and resuspended at 1×10 6 / mL in complete RPMI (Tconv) or Texmacs medium (Treg). Non-tissue culture-treated 24-well plates were pre-prepared by coating with Retronectin and then blocking with 2% bovine serum albumin in PBS and further washing twice with PBS. The final concentration of IL-2 was 300 μ / mL for Tconv and 1000 μ / mL for Treg. After incubating the cells overnight at 37°C, the supernatant was removed and fresh complete medium and IL-2 were replenished. The medium was changed every other day.

[0211] Tconv cells were grown in RPMI-1640 (Gibco) supplemented with 10% heat-inactivated fetal bovine serum, 100 units / mL of penicillin, 100 μg / mL of streptomycin, and 2 mM of L-glutamine. Regulatory T cells were cultured in Texmacs medium (Miltenyi) supplemented with 100 units / mL of penicillin and 100 μg / mL of streptomycin.

[0212] Flow cytometry analysis was performed on days 7 - 10 to evaluate the level of transduction through the expression of mouse TCR constant region and FOXP3. [Example 1C]

[0213] Example 1: Proliferation and IL-2 production of stimulated Tconv cells in the presence of transduced C-FOXP3 innate Treg cells. On day 10, Chinese hamster ovary (CHO) cells transduced with human HLA-DR4 and CD80 or CD86 were introduced with MBP111-129 (LSRFSWGAEGQRPGFGYGG) (10 μM / mL). The suspension was incubated under standard tissue culture conditions for 2 hours, then irradiated, washed, and resuspended at an appropriate concentration.

[0214] Transduced responder T cells were stained with CFSE cell tracking dye in warm PBS at 37°C for 3 minutes, then an equal volume of warm FBS was added and incubated for another 3 minutes. After washing the cells with 5 × volume of complete RPMI medium, they were counted and then 1 × 10⁶ cells were obtained. 6 The transduced cells were resuspended in 10¹ cells / mL. Regulatory T cells were removed from the culture, washed, and then resuspended in complete RPMI at a rate of 1 × 10¹⁶ cells. 6 The cells were resuspended in 1 transduced cell / mL. The cells were plated for 4 days in a ratio of 1 Treg:0.1 CHO cells:various Tconv.

[0215] On day 4, cells were stained with a viability-determinating dye and analyzed by flow cytometry. Proliferation rates were measured by gateding "live" cells, and then gateding a population of cells that showed lower CFSE fluorescence compared to cells cultured without the peptide.

[0216] Figure 1 shows the proliferation of TCR-transduced Tconv cells with and without peptide use (blue bars), as well as the proliferation of the same cells in the presence of pseudoTregs (white bars), TCR-transduced Tregs, or TCR+FOXP3-transduced Tregs.

[0217] On the fourth day, the supernatant was collected and analyzed for IL-2 production by ELISA.

[0218] Figure 2 shows the IL-2 production (blue bars) of TCR-transduced Tconv cells with and without peptide, as well as the proliferation of the same cells in the presence of mock Treg (white bars), TCR-transduced Treg or TCR+FOXP3-transduced Treg. [Example 2]

[0219] Example 2 - T cells obtained from different donors The experiments described in Example 1 were repeated using T cells obtained from different donors. Figure 3 shows the proliferation rate of TCR-transduced T cells. Figure 4 shows the concentration of IL-2 in the supernatant collected in the co-culture experiment. [Example 3]

[0220] Example 3 - Expression of Treg markers in transduced naïve Treg Mock-transduced Treg or Treg transduced with TCR or TCR+FOXP3 were analyzed by flow cytometry for the expression of Treg markers (FOXP3, CD25 and CTLA-4) on days 7 - 10.

[0221] Figure 5 shows the mean fluorescence intensity (MFI) of each marker. The dots represent individual experiments. 1-way ANOVA was used for statistical analysis (p<0.05 * , p<0.005 ** ).

[0222] Figure 6 represents the same data in a different form. Each line represents a single experiment showing the MFI of markers on the same Treg transduced with TCR or TCR+FOXP3. [Example 4]

[0223] Example 4 - Transduced naïve Treg compared with induced Treg As described in Example 1C, CD80 + CD86 + DR4 + After incorporating the peptide into CHO cells and irradiating them, 0.1×106 The cells were resuspended at a concentration of 100 cells / mL. Transduced responder T cells were stained with CFSE cell tracking dye in warm PBS at 37°C for 3 minutes, then incubated for another 3 minutes with an equal volume of warm FBS.

[0224] After washing the cells with 5 × volume of complete medium, they were counted and then 1 × 10⁻⁶ cells were added. 6 Transduced cells were resuspended at 1 × 10¹⁶ cells / mL. Transduction efficiency of Tconv and Treg was measured by flow cytometry. Treg cells were removed from the culture, washed, and then resuspended in complete RPMI at a rate of 1 × 10¹⁶ cells / mL. 6 Transduced cells were resuspended at 1 cell / mL. Cells were cultured on plates in a ratio of 1 Treg:0.1 CHO cells:various Tconv. Growth was measured by analyzing the dilution of Tconv stained with carboxyfluorescein succinimidyl ester (CFSE).

[0225] The data in Figure 7 shows that innate Treg cells transduced with TCR+FOXP3 suppress proliferation more efficiently than Tconv cells transduced with TCR+FOXP3 (i.e., inducible Treg cells).

[0226] The supernatant was collected from the culture medium and analyzed for IL-2 by ELISA. The data shown in Figure 8 demonstrate that innate Treg cells transduced with TCR+FOXP3 suppress IL-2 production more efficiently than Tconv cells transduced with TCR+FOXP3 (i.e., inducible Treg cells). [Example 6A]

[0227] Example 6A - Treg expressing exogenous FOXP3 engrafts, persists, and retains FoxP3, CD25, and TCR expression. Thy1.1 + CD4 + CD25 + Or CD45.1 + CD4 + CD25 + Treg is sorted by bead by HLA-DRB *Isolated from lymph node and spleen cells of 0401 transgenic mice. CD45.1 + TCR was transduced into Treg, and Thy1.1 + Treg cells were transduced with TCR+ mouse FOXP3. One day after transduction, TCR or TCR+FOXP3 transduced cells were conditioned with 4 Gy irradiation in HLA-DRB cells. * The cells were injected into 0401 transgenic hosts in a 1:1 ratio. FACS plots show the CD45.1:Thy1.1 ratio of the injected cells and their respective FOXP3 expression.

[0228] After 7 weeks, engrafted cells were identified by staining for TCR using flow cytometry. + The ratio of CD45.1 to Thy1.1 within the population was measured, and the phenotype of engrafted CD45.1 (Treg cells transduced with TCR) or Thy1.1 (Treg cells transduced with TCR + FOXP3) cells was examined by staining for FOXP3 and CD25.

[0229] Thy1.1 + CD4 + CD25 + Treg is HLA-DRB by bead sorting. * Treg cells were isolated from lymph nodes and spleen cells of 0401 transgenic mice. Treg cells were transduced with TCR or TCR+ mouse FOXP3, or Treg cells were cultured with virus-free supernatant (sham). One day after transduction, TCR or TCR+FOXP3 transduced cells were conditioned with 4 Gy irradiation in HLA-DRB. *Injected into transgenic hosts. Seven weeks later, the engraftment of transduced Tregs was measured by flow cytometry. Figure 9A shows the transduction efficiency measured through the expression of human variable 2.1 and mouse Foxp3 one day after transduction. Figure 9B shows splenocytes obtained from mice administered with Tregs transduced with TCR or TCR+FOXP3, stained with Thy1.1 to identify the transferred cells (upper panel) and FOXP3 and TCR (lower panel). Figure 9C shows cumulative data indicating the fold change in transduction efficiency (left panel) and the fold change in the absolute number of transduced cells (right panel) for Tregs transduced with TCR or TCR+FOXP3, relative to the day of injection. Figure 9D shows the typical expression of FOXP3 in transduced cells seven weeks after transfer. The graph shows the cumulative value of the percentage of FOXP3 + cells in the transduced population at week 7 (left), and the fold change in FOXP3 + cells relative to the day of injection. [Example 6B]

[0230] Example 6B - Tregs expressing exogenous FOXP3 retain Treg functionality even after 7 weeks in vivo, while Tregs not expressing exogenous FOXP3 acquire the ability to produce effector cytokines Splenocytes were cultured with CD86 + HLA-DR4<00,00121>CHO cells pulsed with an irrelevant peptide or 10 uM MBP for 4 hours. Tregs expressing exogenous FOXP3 retained Treg functionality even after 7 weeks in vivo, as indicated by the lack of effector cytokine production, while Tregs not expressing exogenous FOXP3 acquired the ability to produce effector cytokines (Figure 10).

[0231] All publications mentioned in the above specification are hereby incorporated by reference into this specification. Various modifications and changes to the described methods and systems of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed in the claims should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art of molecular biology, cellular immunology or related fields are intended to be within the scope of the appended claims. This disclosure includes the following embodiments. [1] A method for enhancing the ability of regulatory T cells (Tregs) to suppress an immune response, comprising introducing a polynucleotide encoding a FOXP3 polypeptide into the Tregs. [2] The following (i) or (ii), i.e. (i) The FOXP3 polypeptide contains an amino acid sequence or functional fragment that is at least 80% identical to SEQ ID NO: 3 or 4, or (ii) The polynucleotide encoding the FOXP3 polypeptide includes a polynucleotide sequence or a functional fragment thereof that is at least 80% identical to SEQ ID NO: 1 or 2, The method described in Embodiment 1. [3] The method according to Embodiment 1 or 2, wherein the polynucleotide encoding FOXP3 is a contiguous portion of the expression vector. [4] The method according to any one of embodiments 1 to 3, further comprising introducing a polynucleotide encoding an exogenous T cell receptor (TCR) or a polynucleotide encoding a chimeric antigen receptor (CAR) into the Treg. [5] The method according to Embodiment 4, wherein the polynucleotide encoding the FOXP3 polypeptide and the polynucleotide encoding the exogenous TCR or the CAR are provided by a single expression vector. [6] The method according to Embodiment 5, wherein the vector comprises a nucleic acid having a 5' FOXP3-X-TCR / CAR orientation, where X is an internal self-cleavage sequence. [7] The method according to any one of Embodiments 1 to 6, wherein the polynucleotide encoding FOXP3 is introduced into an isolated Treg by viral transduction, and optionally the polynucleotide encoding FOXP3 is introduced into an isolated Treg by retroviral transduction. [8] The following (a) and (b), namely (a) Isolating Treg from a cell population, and (b) Increase FOXP3 expression in the Treg. The method according to any one of embodiments 1 to 7, including the method described above. [9] The method according to Embodiment 8, wherein the cell population includes peripheral blood mononuclear cells (PBMCs) or consists of PMBCs.

[10] Isolating the Treg is To isolate CD4+ T cells, and To isolate the Treg from the CD4+ T cells. The method according to embodiment 8 or 9, including the method described in embodiment 8 or 9.

[11] The method according to any one of embodiments 8 to 10, wherein the isolation of the Treg includes selection using immunomagnetic beads or fluorescence-activated cell sorting (FACS).

[12] The method according to any one of embodiments 8 to 11, wherein the Treg is isolated by selecting (i) CD4+CD25+CD127- and / or CD4+CD25+CD127low cells, and (ii) CD4+CD25hiCD127- and / or CD4+CD25+CD127low cells.

[13] The method according to any one of embodiments 8 to 12, wherein the Treg is isolated by selection from FOXP3+ cells, preferably by selection from CD4+CD25+FOXP3+Helios+Neuropilin1+ cells.

[14] Genetically modified Tregs that can be obtained or obtained by the method described in any of Embodiments 1 to 13.

[15] A genetically engineered Treg containing an exogenous polynucleotide encoding the FOXP3 polypeptide and exhibiting higher FOXP3 expression than the corresponding non-genetically engineered Treg.

[16] The genetically modified Treg according to Embodiment 15, wherein the FOXP3 polypeptide comprises (i) an amino acid sequence or functional fragment thereof that is at least 80% identical to SEQ ID NO: 3 or 4, or (ii) a polynucleotide sequence or functional fragment thereof that is at least 80% identical to SEQ ID NO: 1 or 2.

[17] The genetically modified Treg according to Embodiment 16, wherein the exogenous polynucleotide encoding FOXP3 is a contiguous portion of the expression vector.

[18] A genetically modified Treg according to any one of embodiments 14 to 17, further comprising a polynucleotide encoding an exogenous T cell receptor (TCR) or a polynucleotide encoding a chimeric antigen receptor (CAR).

[19] The genetically modified Treg according to Embodiment 18, wherein the polynucleotide encoding the FOXP3 polypeptide and the polynucleotide encoding the exogenous TCR or the CAR are provided by single expression.

[20] The genetically engineered Treg according to Embodiment 19, wherein the vector comprises a nucleic acid having a 5' FOXP3-X-TCR / CAR orientation, where X is an internal self-cleavage sequence.

[21] A pharmaceutical composition comprising a genetically modified Treg according to any of Embodiments 14 to 20.

[22] A genetically modified Treg according to any of Embodiments 14 to 20, or a pharmaceutical composition according to Embodiment 21, for use in the prevention and / or treatment of a disease.

[23] Use of genetically modified Tregs according to any of Embodiments 14-20 in the manufacture of agents for the prevention and / or treatment of disease.

[24] A method for preventing and / or treating a disease, comprising administering to a subject a genetically modified Treg or pharmaceutical composition according to any of Embodiments 14 to 21.

[25] The genetically modified Treg for use according to Embodiment 22, or a pharmaceutical composition for use, the use of the genetically modified Treg according to Embodiment 23, or the method according to Embodiment 24, wherein the disease is an autoimmune disease, preferably multiple sclerosis.

[26] A genetically modified Treg according to any of embodiments 13 to 17 for use in the prevention and / or treatment of a disease in which the disease is graft rejection or graft-versus-host disease.

[27] Use of polynucleotides encoding the FOXP3 polypeptide to enhance the ability of regulatory T cells (Tregs) to suppress the immune response.

[0232] This disclosure includes the following sequence information. SEQUENCE LISTING <110> UCL Business LTD <120> METHOD FOR ENHANCING THE SUPPRESSIVE PROPERTIES OF TREG CELLS <130> PA23-581 <140> <141> 2019-04-17 <150> GB 1806331.3 <151> 2018-04-18 <150> GB 1806330.5 <151> 2018-04-18 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 1296 <212> DNA <213> Artificial Sequence <220> <223> FOXP3 polynucleotide sequence <400> 1 atgcccaacc ccaggcctgg caagccctcg gccccttcct tggcccttgg cccatcccca 60 ggagcctcgc ccagctggag ggctgcaccc aaagcctcag acctgctggg ggcccggggc 120 ccagggggaa ccttccaggg ccgagatctt cgaggcgggg cccatgcctc ctcttcttcc 180 ttgaacccca tgccaccatc gcagctgcag ctgcccacac tgcccctagt catggtggca 240 ccctccgggg cacggctggg ccccttgccc cacttacagg cactcctcca ggacaggcca 300 catttcatgc accagctctc aacggtggat gcccacgccc ggacccctgt gctgcaggtg 360 caccccctgg agagcccagc catgatcagc ctcacaccac ccaccaccgc cactggggtc 420 ttctccctca aggcccggcc tggcctccca cctgggatca acgtggccag cctggaatgg 480 gtgtccaggg agccggcact gctctgcacc ttcccaaatc ccagtgcacc caggaaggac 540 agcacccttt cggctgtgcc ccagagctcc tacccactgc tggcaaatgg tgtctgcaag 600 tggcccggat gtgagaaggt cttcgaagag ccagaggact tcctcaagca ctgccaggcg 660 gaccatcttc tggatgagaa gggcagggca caatgtctcc tccagagaga gatggtacag 720 tctctggagc agcagctggt gctggagaag gagaagctga gtgccatgca ggcccacctg 780 gctgggaaaa tggcactgac caaggcttca tctgtggcat catccgacaa gggctcctgc 840 tgcatcgtag ctgctggcag ccaaggccct gtcgtcccag cctggtctgg cccccgggag 900 gcccctgaca gcctgtttgc tgtccggagg cacctgtggg gtagccatgg aaacagcaca 960 ttcccagagt tcctccacaa catggactac ttcaagttcc acaacatgcg accccctttc 1020 acctacgcca cgctcatccg ctgggccatc ctggaggctc cagagaagca gcggacactc 1080 aatgagatct accactggtt cacacgcatg tttgccttct tcagaaacca tcctgccacc 1140 tggaagaacg ccatccgcca caacctgagt ctgcacaagt gctttgtgcg ggtggagagc 1200 gagaaggggg ctgtgtggac cgtggatgag ctggagttcc gcaagaaacg gagccagagg 1260 cccagcaggt gttccaaccc tacacctggc ccctga 1296 <210> 2 <211> 1352 <212> DNA <213> Artificial Sequence <220> <223> FOXP3 polynucleotide sequence <400> 2 gaattcgtcg acatgcccaa ccccagaccc ggcaagcctt ctgccccttc tctggccctg 60 ggaccatctc ctggcgcctc cccatcttgg agagccgccc ctaaagccag cgatctgctg 120 ggagctagag gccctggcgg cacattccag ggcagagatc tgagaggcgg agcccacgcc 180 tctagcagca gcctgaatcc catgccccct agccagctgc agctgcctac actgcctctc 240 gtgatggtgg cccctagcgg agctagactg ggccctctgc ctcatctgca ggctctgctg 300 caggaccggc cccactttat gcaccagctg agcaccgtgg acgcccacgc cagaacacct 360 gtgctgcagg tgcaccccct ggaaagccct gccatgatca gcctgacccc tccaaccaca 420 gccaccggcg tgttcagcct gaaggccaga cctggactgc cccctggcat caatgtggcc 480 agcctggaat gggtgtcccg cgaacctgcc ctgctgtgca ccttccccaa tcctagcgcc 540 cccagaaagg acagcacact gtctgccgtg ccccagagca gctatcccct gctggctaac 600 ggcgtgtgca agtggcctgg ctgcgagaag gtgttcgagg aacccgagga cttcctgaag 660 cactgccagg ccgaccatct gctggacgag aaaggcagag cccagtgcct gctgcagcgc 720 gagatggtgc agtccctgga acagcagctg gtgctggaaa aagaaaagct gagcgccatg 780 caggcccacc tggccggaaa gatggccctg acaaaagcca gcagcgtggc cagctccgac 840 aagggcagct gttgtatcgt ggccgctggc agccagggac ctgtggtgcc tgcttggagc 900 ggacctagag aggcccccga tagcctgttt gccgtgcgga gacacctgtg gggcagccac 960 ggcaactcta ccttccccga gttcctgcac aacatggact acttcaagtt ccacaacatg 1020 aggccccct tcacctacgc caccctgatc agatgggcca ttctggaagc ccccgagaag 1080 cagcggaccc tgaacgagat ctaccactgg tttacccgga tgttcgcctt cttccggaac 1140 caccccgcca cctggaagaa cgccatccgg cacaatctga gcctgcacaa gtgcttcgtg 1200 cgggtggaaa gcgagaaggg cgccgtgtgg acagtggacg agctggaatt tcggaagaag 1260 cggtcccaga ggcccagccg gtgtagcaat cctacacctg gccctgaggg cagaggaagt 1320 ctgctaacat gcggtgacgt cgaggagaat cc 1352 <210> 3 <211> 431 <212> PRT <213> Artificial Sequence <220> <223> FOXP3 polypeptide sequence - UniProtKB accession Q9BZS1 <400> 3 Put Pro Donkey Pro Arg Pro Gly Lys Pro Ser Ala Pro Ser Leu Ala Leu 1 5 10 15 Gly Pro Ser Pro Gly Ala Ser Pro Ser Trp Arg Ala Ala Pro Lys Ala 20 25 30 Ser Asp Leu Leu Gly Ala Arg Gly Pro Gly Gly Thr Phe Gln Gly Arg 35 40 45 Asp Leu Arg Gly Gly Ala His Ala Ser Ser Ser Ser Leu Asn Pro Met 50 55 60 Pro Pro Ser Gln Leu Gln Leu Pro Thr Leu Pro Leu Val Met Val Ala 65 70 75 80 Pro Ser Gly Ala Arg Leu Gly Pro Leu Pro His Leu Gln Ala Leu Leu 85 90 95 Gln Asp Arg Pro His Phe Met His Gln Leu Ser Thr Val Asp Ala His 100 105 110 Ala Arg Thr Pro Val Leu Gln Val His Pro Leu Glu Ser Pro Ala Met 115 120 125 Ile Ser Leu Thr Pro Pro Thr Thr Ala Thr Gly Val Phe Ser Leu Lys 130 135 140 Ala Arg Pro Gly Leu Pro Pro Gly Ile Asn Val Ala Ser Leu Glu Trp 145 150 155 160 Val Ser Arg Glu Pro Ala Leu Leu Cys Thr Phe Pro Asn Pro Ser Ala 165 170 175 Pro Arg Lys Asp Ser Thr Leu Ser Ala Val Pro Gln Ser Ser Tyr Pro 180 185 190 Leu Leu Ala Asn Gly Val Cys Lys Trp Pro Gly Cys Glu Lys Val Phe 195 200 205 Glu Glu Pro Glu Asp Phe Leu Lys His Cys Gln Ala Asp His Leu Leu 210 215 220 Asp Glu Lys Gly Arg Ala Gln Cys Leu Leu Gln Arg Glu Met Val Gln 225 230 235 240 Ser Leu Glu Gln Gln Leu Val Leu Glu Lys Glu Lys Leu Ser Ala Met 245 250 255 Gln Ala His Leu Ala Gly Lys Met Ala Leu Thr Lys Ala Ser Ser Val 260 265 270 Ala Ser Ser Asp Lys Gly Ser Cys Cys Ile Val Ala Ala Gly Ser Gln 275 280 285 Gly Pro Val Val Pro Ala Trp Ser Gly Pro Arg Glu Ala Pro Asp Ser 290 295 300 Leu Phe Ala Val Arg Arg His Leu Trp Gly Ser His Gly Asn Ser Thr 305 310 315 320 Phe Pro Glu Phe Leu His Asn Met Asp Tyr Phe Lys Phe His Asn Met 325 330 335 Arg Pro Pro Phe Thr Tyr Ala Thr Leu Ile Arg Trp Ala Ile Leu Glu 340 345 350 Ala Pro Glu Lys Gln Arg Thr Leu Asn Glu Ile Tyr His Trp Phe Thr 355 360 365 Arg Met Phe Ala Phe Phe Arg Asn His Pro Ala Thr Trp Lys Asn Ala 370 375 380 Ile Arg His Asn Leu Ser Leu His Lys Cys Phe Val Arg Val Glu Ser 385 390 395 400 Glu Lys Gly Ala Val Trp Thr Val Asp Glu Leu Glu Phe Arg Lys Lys 405 410 415 Arg Ser Gln Arg Pro Ser Arg Cys Ser Asn Pro Thr Pro Gly Pro 420 425 430 <210> 4 <211> 441 <212> PRT <213> Artificial Sequence <220> <223> FOXP3 polypeptide sequence <400> 4 Met Pro Asn Pro Arg Pro Gly Lys Pro Ser Ala Pro Ser Leu Ala Leu 1 5 10 15 Gly Pro Ser Pro Gly Ala Ser Pro Ser Trp Arg Ala Ala Pro Lys Ala 20 25 30 Ser Asp Leu Leu Gly Ala Arg Gly Pro Gly Gly Thr Phe Gln Gly Arg 35 40 45 Asp Leu Arg Gly Gly Ala His Ala Ser Ser Ser Ser Leu Asn Pro Met 50 55 60 Pro Pro Ser Gln Leu Gln Leu Pro Thr Leu Pro Leu Val Met Val Ala 65 70 75 80 Pro Ser Gly Ala Arg Leu Gly Pro Leu Pro His Leu Gln Ala Leu Leu 85 90 95 Gln Asp Arg Pro His Phe Met His Gln Leu Ser Thr Val Asp Ala His 100 105 110 Ala Arg Thr Pro Val Leu Gln Val His Pro Leu Glu Ser Pro Ala Met 115 120 125 Ile Ser Leu Thr Pro Pro Thr Thr Ala Thr Gly Val Phe Ser Leu Lys 130 135 140 Ala Arg Pro Gly Leu Pro Pro Gly Ile Asn Val Ala Ser Leu Glu Trp 145 150 155 160 Val Ser Arg Glu Pro Ala Leu Leu Cys Thr Phe Pro Asn Pro Ser Ala 165 170 175 Pro Arg Lys Asp Ser Thr Leu Ser Ala Val Pro Gln Ser Ser Tyr Pro 180 185 190 Leu Leu Ala Asn Gly Val Cys Lys Trp Pro Gly Cys Glu Lys Val Phe 195 200 205 Glu Glu Pro Glu Asp Phe Leu Lys His Cys Gln Ala Asp His Leu Leu 210 215 220 Asp Glu Lys Gly Arg Ala Gln Cys Leu Leu Gln Arg Glu Met Val Gln 225 230 235 240 Ser Leu Glu Gln Val Glu Glu Leu Ser Ala Met Gln Ala His Leu Ala 245 250 255 Gly Lys Met Ala Leu Thr Lys Ala Ser Ser Val Ala Ser Ser Asp Lys 260 265 270 Gly Ser Cys Cys Ile Val Ala Ala Gly Ser Gln Gly Pro Val Val Pro 275 280 285 Ala Trp Ser Gly Pro Arg Glu Ala Pro Asp Ser Leu Phe Ala Val Arg 290 295 300 Arg His Leu Trp Gly Ser His Gly Asn Ser Thr Phe Pro Glu Phe Leu 305 310 315 320 His Asn Met Asp Tyr Phe Lys Phe His Asn Met Arg Pro Pro Phe Thr 325 330 335 Tyr Ala Thr Leu Ile Arg Trp Ala Ile Leu Glu Ala Pro Glu Lys Gln 340 345 350 Arg Thr Leu Asn Glu Ile Tyr His Trp Phe Thr Arg Met Phe Ala Phe 355 360 365 Phe Arg Asn His Pro Ala Thr Trp Lys Asn Ala Ile Arg His Asn Leu 370 375 380 Ser Leu His Lys Cys Phe Val Arg Val Glu Ser Glu Lys Gly Ala Val 385 390 395 400 Trp Thr Val Asp Glu Leu Glu Phe Arg Lys Lys Arg Ser Gln Arg Pro 405 410 415 Ser Arg Cys Ser Asn Pro Thr Pro Gly Pro Glu Gly Arg Gly Ser Leu 420 425 430 Leu Thr Cys Gly Asp Val Glu Glu Asn 435,440 <210> 5 <211> 452 <212> PRT <213> Artificial Sequence <220> <223> FOXP3-2A polypeptide sequence <400> 5 Met Pro Asn Pro Arg Pro Gly Lys Pro Ser Ala Pro Ser Leu Ala Leu 1 5 10 15 Gly Pro Ser Pro Gly Ala Ser Pro Ser Trp Arg Ala Ala Pro Lys Ala 20 25 30 Ser Asp Leu Leu Gly Ala Arg Gly Pro Gly Gly Thr Phe Gln Gly Arg 35 40 45 Asp Leu Arg Gly Gly Ala His Ala Ser Ser Ser Ser Leu Asn Pro Met 50 55 60 Pro Pro Ser Gln Leu Gln Leu Pro Thr Leu Pro Leu Val Met Val Ala 65 70 75 80 Pro Ser Gly Ala Arg Leu Gly Pro Leu Pro His Leu Gln Ala Leu Leu 85 90 95 Gln Asp Arg Pro His Phe Met His Gln Leu Ser Thr Val Asp Ala His 100 105 110 Ala Arg Thr Pro Val Leu Gln Val His Pro Leu Glu Ser Pro Ala Met 115 120 125 Ile Ser Leu Thr Pro Pro Thr Thr Ala Thr Gly Val Phe Ser Leu Lys 130 135 140 Ala Arg Pro Gly Leu Pro Pro Gly Ile Asn Val Ala Ser Leu Glu Trp 145 150 155 160 Val Ser Arg Glu Pro Ala Leu Leu Cys Thr Phe Pro Asn Pro Ser Ala 165 170 175 Pro Arg Lys Asp Ser Thr Leu Ser Ala Val Pro Gln Ser Ser Tyr Pro 180 185 190 Leu Leu Ala Asn Gly Val Cys Lys Trp Pro Gly Cys Glu Lys Val Phe 195 200 205 Glu Glu Pro Glu Asp Phe Leu Lys His Cys Gln Ala Asp His Leu Leu 210 215 220 Asp Glu Lys Gly Arg Ala Gln Cys Leu Leu Gln Arg Glu Met Val Gln 225 230 235 240 Ser Leu Glu Gln Gln Leu Val Leu Glu Lys Glu Lys Leu Ser Ala Met 245 250 255 Gln Ala His Leu Ala Gly Lys Met Ala Leu Thr Lys Ala Ser Ser Val 260 265 270 Ala Ser Ser Asp Lys Gly Ser Cys Cys Ile Val Ala Ala Gly Ser Gln 275 280 285 Gly Pro Val Val Pro Ala Trp Ser Gly Pro Arg Glu Ala Pro Asp Ser 290 295 300 Leu Phe Ala Val Arg Arg His Leu Trp Gly Ser His Gly Asn Ser Thr 305 310 315 320 Phe Pro Glu Phe Leu His Asn Met Asp Tyr Phe Lys Phe His Asn Met 325 330 335 Arg Pro Pro Phe Thr Tyr Ala Thr Leu Ile Arg Trp Ala Ile Leu Glu 340 345 350 Ala Pro Glu Lys Gln Arg Thr Leu Asn Glu Ile Tyr His Trp Phe Thr 355 360 365 Arg Met Phe Ala Phe Phe Arg Asn His Pro Ala Thr Trp Lys Asn Ala 370 375 380 Ile Arg His Asn Leu Ser Leu His Lys Cys Phe Val Arg Val Glu Ser 385 390 395 400 Glu Lys Gly Ala Val Trp Thr Val Asp Glu Leu Glu Phe Arg Lys Lys 405 410 415 Arg Ser Gln Arg Pro Ser Arg Cys Ser Asn Pro Thr Pro Gly Pro Gly 420 425 430 Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn 435 440 445 Pro Gly Pro Ser 450 <210> 6 <211> 462 <212> PRT <213> Artificial Sequence <220> <223> FOXP3-2A polypeptide sequence <400> 6 Met Pro Asn Pro Arg Pro Gly Lys Pro Ser Ala Pro Ser Leu Ala Leu 1 5 10 15 Gly Pro Ser Pro Gly Ala Ser Pro Ser Trp Arg Ala Ala Pro Lys Ala 20 25 30 Ser Asp Leu Leu Gly Ala Arg Gly Pro Gly Gly Thr Phe Gln Gly Arg 35 40 45 Asp Leu Arg Gly Gly Ala His Ala Ser Ser Ser Ser Leu Asn Pro Met 50 55 60 Pro Pro Ser Gln Leu Gln Leu Pro Thr Leu Pro Leu Val Met Val Ala 65 70 75 80 Pro Ser Gly Ala Arg Leu Gly Pro Leu Pro His Leu Gln Ala Leu Leu 85 90 95 Gln Asp Arg Pro His Phe Met His Gln Leu Ser Thr Val Asp Ala His 100 105 110 Ala Arg Thr Pro Val Leu Gln Val His Pro Leu Glu Ser Pro Ala Met 115 120 125 Ile Ser Leu Thr Pro Pro Thr Thr Ala Thr Gly Val Phe Ser Leu Lys 130 135 140 Ala Arg Pro Gly Leu Pro Pro Gly Ile Asn Val Ala Ser Leu Glu Trp 145 150 155 160 Val Ser Arg Glu Pro Ala Leu Leu Cys Thr Phe Pro Asn Pro Ser Ala 165 170 175 Pro Arg Lys Asp Ser Thr Leu Ser Ala Val Pro Gln Ser Ser Tyr Pro 180 185 190 Leu Leu Ala Asn Gly Val Cys Lys Trp Pro Gly Cys Glu Lys Val Phe 195 200 205 Glu Glu Pro Glu Asp Phe Leu Lys His Cys Gln Ala Asp His Leu Leu 210 215 220 Asp Glu Lys Gly Arg Ala Gln Cys Leu Leu Gln Arg Glu Met Val Gln 225 230 235 240 Ser Leu Glu Gln Val Glu Glu Leu Ser Ala Met Gln Ala His Leu Ala 245 250 255 Gly Lys Met Ala Leu Thr Lys Ala Ser Ser Val Ala Ser Ser Asp Lys 260 265 270 Gly Ser Cys Cys Ile Val Ala Ala Gly Ser Gln Gly Pro Val Val Pro 275 280 285 Ala Trp Ser Gly Pro Arg Glu Ala Pro Asp Ser Leu Phe Ala Val Arg 290 295 300 Arg His Leu Trp Gly Ser His Gly Asn Ser Thr Phe Pro Glu Phe Leu 305 310 315 320 His Asn Met Asp Tyr Phe Lys Phe His Asn Met Arg Pro Pro Phe Thr 325 330 335 Tyr Ala Thr Leu Ile Arg Trp Ala Ile Leu Glu Ala Pro Glu Lys Gln 340 345 350 Arg Thr Leu Asn Glu Ile Tyr His Trp Phe Thr Arg Met Phe Ala Phe 355 360 365 Phe Arg Asn His Pro Ala Thr Trp Lys Asn Ala Ile Arg His Asn Leu 370 375 380 Ser Leu His Lys Cys Phe Val Arg Val Glu Ser Glu Lys Gly Ala Val 385 390 395 400 Trp Thr Val Asp Glu Leu Glu Phe Arg Lys Lys Arg Ser Gln Arg Pro 405 410 415 Ser Arg Cys Ser Asn Pro Thr Pro Gly Pro Glu Gly Arg Gly Ser Leu 420 425 430 Leu Thr Cys Gly Asp Val Glu Glu Asn Gly Ala Thr Asn Phe Ser Leu 435 440 445 Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Ser 450 455 460 <210> 7 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> MBP111-129 <400> 7 Leu Ser Arg Phe Ser Trp Gly Ala Glu Gly Gln Arg Pro Gly Phe Gly 1 5 10 15 Tyr Gly Gly

Claims

1. A method for enhancing the ability of regulatory T cells (Tregs) to suppress the immune response, The above method involves, (i) A polynucleotide encoding the FOXP3 polypeptide, and (ii) Polynucleotides encoding chimeric antigen receptors (CARs), This includes introducing an expression vector containing nucleic acids, The nucleic acid has the orientation 5' FOXP3 - CAR 3', and The polynucleotide encoding the FOXP3 polypeptide and the polynucleotide encoding the CAR are separated from each other by a nucleic acid sequence that allows both FOXP3 and CAR to be expressed from the same mRNA transcript, thereby enhancing the ability of the Treg to suppress the immune response. The aforementioned method.

2. (i) or (ii) below, that is (i) The FOXP3 polypeptide contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 3 or 4, or (ii) The polynucleotide encoding the FOXP3 polypeptide includes a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 1 or 2, The method according to claim 1.

3. The method according to claim 1 or 2, wherein the polynucleotide encoding FOXP3 is a contiguous portion of an expression vector.

4. The method according to any one of claims 1 to 3, wherein the polynucleotide encoding FOXP3 is introduced into an isolated Treg by viral transduction.

5. The method according to claim 4, wherein the polynucleotide encoding FOXP3 is introduced into an isolated Treg by retroviral transduction.

6. (a) and (b) below, namely (a) Isolating Treg from a cell population, and (b) Increase FOXP3 expression in the Treg. The method according to any one of claims 1 to 5, including the method described in any one of claims 1 to 5.

7. The method according to claim 6, wherein the cell population includes peripheral blood mononuclear cells (PBMCs) or consists of PMBCs.

8. Isolating the aforementioned Treg CD4 ＋ To isolate T cells, and CD4 ＋ Isolating the Treg from T cells. The method according to claim 6 or 7, including the method described in claim 6 or 7.

9. The method according to any one of claims 6 to 8, wherein the isolation of the Treg includes selection using immunomagnetic beads or fluorescence-activated cell sorting (FACS).

10. The Tregs are selected from (i) CD4 ＋ CD25 ＋ CD127 － and / or CD4 ＋ CD25 ＋ CD127 low cells, (ii) CD4 ＋ CD25 hi CD127 － and / or CD4 ＋ CD25 ＋ CD127 low cells and isolated by selection, the method according to any one of claims 6 to 9.

11. The aforementioned Treg is FOXP3 ＋ The method according to any one of claims 6 to 10, wherein cells are isolated by selection.

12. The aforementioned Treg to CD4 ＋ CD25 ＋ FOXP3 ＋ Helios ＋ Neuropilin 1 ＋ The method according to claim 11, wherein cells are isolated by selection.

13. The method according to any one of claims 1 to 12, wherein the nucleic acid has the orientation 5' FOXP3 - X - CAR, and X is an internal self-cleavage sequence.

14. A genetically modified Treg obtained by the method described in any one of claims 1 to 13.

15. A genetically engineered Treg comprising an exogenous polynucleotide encoding a FOXP3 polypeptide and a polynucleotide encoding a chimeric antigen receptor (CAR), and exhibiting higher FOXP3 expression than the corresponding non-genetically engineered Treg, The polynucleotide encoding the FOXP3 polypeptide and the polynucleotide encoding the CAR are delivered by a single expression vector. The vector contains nucleic acid having the orientation 5' FOXP3 - CAR 3', The genetically engineered Treg, wherein the polynucleotide encoding the FOXP3 polypeptide and the polynucleotide encoding CAR are separated from each other by a nucleic acid sequence that allows both FOXP3 and CAR to be expressed from the same mRNA transcript.

16. The genetically engineered Treg according to claim 15, wherein the FOXP3 polypeptide comprises (i) an amino acid sequence that is at least 90% identical to SEQ ID NO: 3 or 4, or (ii) a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 1 or 2.

17. The genetically engineered Treg according to any one of claims 14 to 16, wherein the nucleic acid has a 5' FOXP3-X-CAR orientation and X is an internally self-cleaved sequence.

18. A pharmaceutical composition comprising a genetically modified Treg according to any one of claims 14 to 17.

19. A genetically modified Treg according to any one of claims 14 to 17, or a pharmaceutical composition according to claim 18, for use in the prevention and / or treatment of a disease.

20. Genetically modified Treg for use in the prevention and / or treatment of diseases involving a pathological immune response, wherein the prevention and / or treatment of the disease involving a pathological immune response is achieved by suppressing the response, The aforementioned Treg is, (i) A polynucleotide encoding the FOXP3 polypeptide, and (ii) Polynucleotides encoding chimeric antigen receptors (CARs), It is genetically engineered by introducing an expression vector containing nucleic acids into Treg cells. The nucleic acid has the orientation 5' FOXP3 - CAR 3', The polynucleotide encoding the FOXP3 polypeptide and the polynucleotide encoding the CAR are separated from each other by a nucleic acid sequence that allows both FOXP3 and CAR to be expressed from the same mRNA transcript, and the Treg has the ability to suppress an enhanced immune response. Genetically engineered Treg for the aforementioned use.

21. A genetically engineered Treg for use according to claim 20, wherein the genetically engineered Treg is a genetically engineered Treg according to any one of claims 14 to 17.

22. A genetically engineered Treg for use or a pharmaceutical composition for use according to any one of claims 19 to 21, wherein the disease is an autoimmune disease.

23. The genetically modified Treg for use or pharmaceutical composition for use according to claim 22, wherein the disease is multiple sclerosis.

24. A genetically modified Treg according to any one of claims 14 to 17, for use in the prevention and / or treatment of graft rejection or graft-versus-host disease.

25. The in vitro use of a polynucleotide encoding a FOXP3 polypeptide to enhance the ability of regulatory T cells (Tregs) to suppress the immune response, wherein the use is: (i) A polynucleotide encoding the FOXP3 polypeptide, and (ii) Polynucleotides encoding chimeric antigen receptors (CARs), This includes introducing an expression vector containing nucleic acids into the Treg, The nucleic acid has the orientation 5' FOXP3 - CAR 3', The in vitro use wherein the polynucleotide encoding the FOXP3 polypeptide and the polynucleotide encoding CAR are separated from each other by a nucleic acid sequence that allows both FOXP3 and CAR to be expressed from the same mRNA transcript.

26. The polynucleotide encoding FOXP3, or the FOXP3 polypeptide, The use according to claim 25, which is as described in any one of claims 2 to 5.

27. The aforementioned use, (a) Isolating Treg cells from a cell population, and (b) Increase FOXP3 expression in the Treg, The in vitro use according to claim 25 or 26, including the use described in claim 25 or 26.

28. (i) The cell population includes peripheral blood mononuclear cells (PBMCs) or consists of PMBCs, (ii) Isolating Treg is CD4 ＋ To isolate T cells, and the CD4 ＋ Including the isolation of Treg cells from T cells, (iii) Isolation of Tregs, including selection using immunomagnetic beads or fluorescence-activated cell sorting (FACS), (iv) The Treg is (a) CD4 + CD25 + CD127 - and / or CD4 + CD25 + CD127 low (b) cells, or CD4 + CD25 hi CD127 - and / or CD4 + CD25 + CD127 low Cells are isolated by selection. (v) The Treg is FOXP3 + Cells are isolated by selection, and in some cases the Treg is CD4 + CD25 + FOXP3 + Helios ＋ Neuropilin 1 ＋ Cells are isolated by selection. In vitro use as described in any one of claims 25 to 27.

29. The in vitro use according to any one of claims 25 to 28, wherein the nucleic acid has the orientation 5' FOXP3 - X - CAR, and X is an internally self-cleaving sequence.

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