Methods for maintaining suppressive activity of regulatory T cells

Abstract

The present invention provides a method for maintaining the ability of regulatory T cells (Tregs) to suppress immune responses under pro-inflammatory conditions, the method comprising introducing a polynucleotide encoding a FOXP3 polypeptide into Tregs.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a method for maintaining the ability of regulatory T cells (Tregs), particularly CAR Tregs, to suppress immune responses under proinflammatory conditions. In particular, the present invention relates to a method for increasing FOXP3 expression in Tregs. The present invention further relates to the engineered Tregs provided by the method of the present invention, as well as methods and uses of such engineered Tregs.

[0002] 2. Background of the Invention In autoimmune and inflammatory diseases of the central nervous system (CNS), the immune system attacks self-antigens, such as in multiple sclerosis (MS), the most common neurological disease in young adults, where the immune system attacks the myelin sheath of neurons in the CNS. Current treatments for autoimmune and inflammatory diseases of the CNS generally involve suppression of the immune system, for example by bone marrow transplantation combined with cytostatic and immunosuppressive drugs. Although autologous hematopoietic stem cell transplantation can provide durable beneficial effects in some subjects, this approach requires aggressive myeloablative conditioning, which is associated with significant toxicities and risks. Although several disease-modifying therapies (DMTs) have been approved to reduce the frequency of clinical relapse, most patients continue to deteriorate clinically with current treatment schedules. Neither DMTs nor stem cell transplantation can mediate CNS-specific suppression of the immunopathology of CNS autoimmune and inflammatory diseases.

[0003] Currently, there are no effective treatments for autoimmune and inflammatory diseases of the CNS. Treatments focus solely on alleviating symptoms, usually through general suppression of the immune system. There is a need for therapies that specifically target local immune responses involved in the development and progression of CNS diseases.

[0004] Regulatory T cells (Tregs) are a type of T cell that regulates the activity of the immune system. In general, Tregs are immunosuppressive, downregulating immune responses to stimuli. In particular, Tregs suppress the induction and proliferation of conventional T cells, some of which are directly involved in the immune response (e.g., cytotoxic T cells). The suppressive action of Tregs can be directed to a specific antigen by expressing recombinant T cell receptor (TCR) constructs that recognize peptides that match epitopes present on the antigen in question. Similarly, the suppressive action of Tregs can be directed to a specific target by expressing chimeric antigen receptors (CARs) that recognize antigens expressed on the surface of target cells. Conventional T cells can be differentiated ex vivo into a regulatory phenotype by expressing FOXP3 in the cells.

[0005] Regulatory T cells (Tregs) can modulate immune responses through multiple mechanisms, and thus therapeutic use of Tregs has been proposed to induce immune tolerance in transplantation, autoimmune diseases, and chronic inflammatory conditions. FOXP3 is considered the master transcription factor for Tregs, driving the expression of CD25, CTLA-4, and the suppression of proinflammatory cytokines such as IL-2. It has been shown that some cells with all the hallmarks of Tregs can, under certain conditions, lose FOXP3 expression and acquire pathogenic features, such as the expression of effector cytokines. This result is particularly problematic for antigen-specific Treg therapy, raising significant safety concerns.

[0006] Summary of the Invention The inventors surprisingly found that expressing exogenous FOXP3 in regulatory T cells (Tregs), which already express endogenous FOXP3, can maintain their regulatory function when exposed to pro-inflammatory conditions. Thus, the present invention provides methods for maintaining the ability of regulatory T cells (Tregs) to suppress immune responses when exposed to pro-inflammatory conditions.

[0007] The Treg of the present invention is a natural Treg or an inducible Treg generated from a conventional T cell. For example, the Treg of the present invention is a natural Treg or an inducible Treg generated from a conventional T cell in vivo. Suitable Treg cells include natural Treg (nTreg) cells derived from the thymus and inducible Treg (iTreg) cells generated in the periphery. In other words, the Treg of the present invention expresses endogenous FOXP3. Surprisingly, the present inventors confirmed that by increasing FOXP3 expression (e.g., by introducing exogenous FOXP3) in Tregs that already express endogenous FOXP3, the regulatory function of Tregs when exposed to proinflammatory conditions is maintained at a similar level to that of corresponding Tregs that are not exposed to proinflammatory conditions (e.g., cultured under non-proinflammatory conditions). In contrast, Tregs with endogenous levels of FOXP3 (e.g., Tregs without exogenous FOXP3) have been shown to lose their suppressive ability under proinflammatory conditions. Thus, the presence of exogenous FOXP3 in Tregs that already express endogenous FOXP3 appears to confer functional stability to Tregs compared to cells that only express endogenous FOXP3 (particularly native or wild-type endogenous FOXP3), which would be prone to functional decline under proinflammatory conditions. Therefore, from another perspective, the present invention further provides a method for maintaining the functional stability of Tregs under proinflammatory conditions, comprising increasing FOXP3 expression in Tregs (e.g., expressing exogenous FOXP3), or a method for maintaining suppressive function in Tregs under proinflammatory conditions, comprising increasing FOXP3 expression in Tregs (e.g., expressing exogenous FOXP3).

[0008] In a preferred embodiment, the Tregs of the present invention are naturally occurring Tregs.

[0009] The present invention provides a method for maintaining the ability of Tregs to suppress immune responses under pro-inflammatory conditions, comprising increasing FOXP3 expression in Tregs.

[0010] In some embodiments of the invention, FOXP3 expression is increased by introducing a polynucleotide encoding the FOXP3 protein into Tregs.

[0011] In some embodiments of the invention, the method for maintaining the ability of regulatory T cells (Tregs) to suppress an immune response under proinflammatory conditions comprises: (a) isolating Tregs from a cell population; (b) increasing FOXP3 expression in the Tregs.

[0012] Suitably, Treg may refer to a population of Tregs (ie, a plurality of Tregs).

[0013] Each method of the invention may comprise the additional step of exposing Treg cells to pro-inflammatory conditions.

[0014] The present invention also provides genetically engineered Tregs obtainable or obtained by the methods of the present invention.

[0015] The present invention also provides pharmaceutical compositions comprising the engineered Tregs of the present invention.

[0016] The present invention also provides the genetically engineered Treg of the present invention or the pharmaceutical composition of the present invention for use in the prevention and / or treatment of a disease.

[0017] The present invention also provides the use of the engineered Tregs of the present invention in the manufacture of a medicament.

[0018] The present invention also provides methods for the prevention and / or treatment of diseases comprising administering the engineered Tregs or compositions of the present invention to a subject.

[0019] The present invention also provides the use of a polynucleotide encoding a FOXP3 polypeptide to maintain the ability of regulatory T cells (Tregs) to suppress immune responses under pro-inflammatory conditions.

[0020] The present invention further provides the use of a nucleic acid molecule comprising a nucleotide sequence encoding FOXP3 to maintain the suppressive function or functional stability of Tregs when exposed to pro-inflammatory conditions, wherein said Tregs comprise said nucleic acid molecule. [Brief description of the drawings]

[0021] Description of the drawings [Figure 1] Figure 1 shows an outline of the instability assay method used to measure the functional stability of Tregs. Cells are incubated either under control conditions or in the presence of three proinflammatory cytokines. [Diagram 2] Figure 2 shows the construct design used in the instability assay. Two constructs were used: construct VIII encoding only CAR (HLA-A2 CAR) and construct I encoding FOXP3 and CAR (encoding polynucleotide sequences separated by a 2A self-cleaving peptide). Figure 2 further illustrates the instability assay. [Diagram 3] Figure 3 shows the results of an instability assay performed with a Treg population transduced with a polynucleotide encoding construct I and exposed to proinflammatory conditions. FOXP3 expression was measured in transduced and non-transduced cells, and these cells were subjected to a suppression assay. The transduced cells had a higher percentage of FOXP3 compared to non-transduced cells, and were able to suppress even after exposure to proinflammatory conditions compared to non-transduced cells, which were unable to suppress after exposure to proinflammatory conditions. [Figure 4]FIG. 4 shows the suppressive capacity of Tregs transduced with either Construct I or Construct VIII when exposed to proinflammatory conditions. Tregs expressing Construct I (and therefore expressing exogenous FOXP3 and CAR) maintain suppressive function after exposure to proinflammatory cytokines. Clear circles indicate Construct I containing cells exposed to control conditions, while grey circles indicate Construct I containing cells after exposure to proinflammatory conditions, confirming the maintenance of suppressive function. In contrast, Tregs expressing Construct VIII (no exogenous FOXP3, only CAR) fail to suppress after exposure to proinflammatory conditions, showing a similar low level of suppression that is comparable to non-transduced cells.

[0022] Detailed Description The present invention provides a method for maintaining the ability of regulatory T cells (Tregs) to suppress immune responses when exposed to pro-inflammatory conditions, the method comprising increasing FOXP3 expression in Tregs (e.g., by expressing exogenous FOXP3).

[0023] regulatory T cells The term "regulatory T cells" (Treg) refers to T cells that express the markers CD4, CD25, and FOXP3 (CD4 + CD25 + FOXP3 + Tregs are characterized by the cell surface markers CD4 and CD25 in the absence or in combination with low expression of the surface protein CD127 (CD4 + CD25 + CD127 - or CD4 + CD25 + CD127 low ) can be identified. Tregs can also express high levels of CTLA-4 (cytotoxic T lymphocyte-associated molecule-4) or GITR (glucocorticoid-inducible TNF receptor) on their cell surface. Unlike conventional T cells, Tregs do not produce IL-2 and are therefore anergic at baseline.

[0024] The term "natural Tregs" refers to thymus-derived Tregs. Natural Tregs are CD4 + CD25 + FOXP3 + Helios + Neuropilin 1 + The term "natural Treg" distinguishes thymus-derived Treg from "inducible Treg" which arise from extrathymic conventional T cells. Compared to inducible Treg, natural Treg highly expresses PD-1 (programmed cell death-1, pdcd1), neuropilin 1 (Nrp1), Helios (Ikzf2), and CD73. Natural Treg can be distinguished from inducible Treg based on the individual expression of Helios protein or neuropilin 1 (Nrp1).

[0025] As used herein, the term "induced regulatory T cells" (iTregs) refers to CD4 T cells that arise from extrathymic mature CD4+ conventional T cells. + CD25 + FOXP3 + Helios - Neuropilin 1 - It refers to T cells. For example, iTregs can be induced in vitro from CD4+CD25-FOXP3- cells in the presence of IL-2 and TGF-β.

[0026] Suitably, Tregs express FOXP3 from the cell's endogenous FoxP3 gene. Preferably, Tregs are CD4 + CD25 + FOXP3 + It may also be Treg. Preferably, Tregs are CD4 + CD25 + CD127 - It may also be Treg. Preferably, Tregs are CD4 + CD25 + CD127 low It may also be Treg. Preferably, Tregs are CD4+ CD25 + CD127 - CD45RA + It may also be Treg. Preferably, Tregs are CD4 + CD25 + CD127 low CD45RA + It may also be Treg. Preferably, Tregs are CD4 + CD25 + FOXP3 + CD127 - It may also be Treg. Preferably, Tregs are CD4 + CD25 + FOXP3 + CD127 low It may also be Treg. Preferably, Tregs are CD4 + CD25 + FOXP3 + Helios + It may also be Treg. Preferably, Tregs are CD4 + CD25 + FOXP3 + Neuropilin 1 + It may also be Treg. Preferably, Tregs are CD4 + CD25 + FOXP3 + Helios + Neuropilin 1 + It may also be Treg.

[0027] Preferably, the Tregs are human Tregs. Preferably, the Tregs are human Tregs and the FOXP3 is human FOXP3.

[0028] Typically, as used herein, a "corresponding Treg" has the same or similar phenotype as the target or comparative Treg, but may differ in either the genetic modification performed (typically, as used herein, an increase in FOXP3 levels) or the conditions to which the Treg is exposed (typically, as used herein, proinflammatory or non-proinflammatory conditions). The same or similar phenotype means that the Tregs may express the same or similar markers (e.g., may have at least 80%, 90%, or 95% of the marker molecules in common, in the same or similar amounts, e.g., at least 70%, 80%, or 90% of the respective levels).

[0029] In one embodiment, the methods of the present invention can be used in particular to stabilize the function, maintain suppressive action, or prevent loss of suppressive function of Treg or Treg populations with unstable phenotype under proinflammatory conditions as described herein. In particular, expressing exogenous FOXP3 in such unstable Tregs can have a disproportionately positive effect on maintaining (and thus preventing loss of) function as described herein. Thus, in particular, Tregs used in the present invention can have reduced expression levels of Helios and / or have more than 50% methylated TSDRs, for example more than 60%, more than 70%, or more than 80% methylated TSDRs. Demethylated TSDRs are associated with endogenous FOXP3 expression, and thus methylation of TSDRs is associated with reduced endogenous FOXP3 expression and reduced Treg stability. In another view, an unstable or less stable Treg population as used herein may be a population in which more than 30%, more than 40%, or more than 50% of the Treg cells in the population have methylated TSDR. The methylation status of TSDR can be determined by any method known in the art, including bisulfite sequencing.

[0030] Alternatively, Tregs or populations of Tregs having a reduced stability or unstable phenotype may be characterized by expression of IL2. Since Tregs with a stable phenotype typically do not express IL2, expression of IL2 by Tregs may signify an unstable phenotype. Thus, as used herein, Tregs or populations of Tregs may express IL2, e.g., 10%, 20%, 30%, 40%, or 50% more IL2 than Treg cells with a stable phenotype. It will be understood by those skilled in the art that, while typically at least 10%, 20%, 30%, 40%, or 50% of Tregs in a Treg population may express IL2, not all Treg cells in a Treg population may express IL2 for the Treg population to be considered to have an unstable phenotype or to be unstable (e.g., reduced stability). As described above, expression of exogenous FOXP3 in such cells can result in a stabilized phenotype (e.g., reduced expression of IL2 or reversion to a cell that does not express IL2), resulting in a cell or cell population that is able to maintain suppressive function under pro-inflammatory conditions.

[0031] Treg or Treg population as used herein can be obtained from patient, typically human patient.The Treg or Treg population from such patient can have a reduced or unstable phenotype compared with the Treg cell that can be isolated from healthy donor or subject.In particular, said Treg or Treg population can be obtained from the patient who has undergone transplantation, for example, liver, pancreatic islet or kidney transplantation, or the patient who has a condition associated with undesired inflammation, for example, an autoimmune condition such as type I diabetes or IBD.

[0032] The term "Tconv cells" refers to conventional T cells and refers to T cells that are not Treg.

[0033] In one embodiment, the Tregs of the present invention may be derived from stem cells, in particular, the Tregs of the present invention may be obtained in vitro from stem cells. In another embodiment, the cell is a progenitor cell.

[0034] As used herein, the term "stem cell" refers to an undifferentiated cell that can give rise indefinitely to more stem cells of the same type, from which other specialized cells may arise by differentiation. Stem cells are multipotent. Stem cells may be, for example, embryonic stem cells or adult stem cells.

[0035] As used herein, the term "progenitor cell" refers to a cell that can differentiate to form one or more cell types, but has limited self-renewal in vitro.

[0036] Suitably, the cells are capable of differentiating into T cells, such as Tregs. Advantageously, the cells have the capacity to differentiate into T cells that express FOXP3, such as Tregs. Suitably, the cells may be embryonic stem cells (ESCs). Suitably, the cells are hematopoietic stem cells or hematopoietic progenitor cells. Suitably, the cells are induced pluripotent stem cells (iPSCs). Suitably, the cells may be obtained from umbilical cord blood. Suitably, the cells may be obtained from adult peripheral blood.

[0037] In some embodiments, hematopoietic stem and progenitor cells (HSPCs) may be obtained from umbilical cord blood, which can be collected according to techniques known in the art (e.g., U.S. Pat. Nos. 7,147,626 and 7,131,958, which are incorporated herein by reference).

[0038] In one embodiment, HSPCs may be obtained from a pluripotent stem cell source, such as induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs).

[0039] As used herein, the term "hematopoietic stem and progenitor cells" or "HSPCs" refers to cells that express the antigenic marker CD34 (CD34+) and populations of such cells. In certain embodiments, the term "HSPCs" refers to cells identified by the presence of the antigenic marker CD34 (CD34+) and the absence of lineage (lin) markers. Cell populations that contain CD34+ and / or Lin(-) cells include hematopoietic stem cells and hematopoietic progenitor cells.

[0040] HSPCs can be harvested or isolated from adult bone marrow (including femur, hip bone, rib, sternum, and other bones). Bone marrow aspirate containing HSPCs can be harvested or isolated directly from the hip bone using a needle and syringe. Other sources of HSPCs include umbilical cord blood, placental blood, mobilized peripheral blood, Wharton's jelly, placenta, fetal blood, fetal liver, or fetal spleen. In certain embodiments, to harvest sufficient amounts of HSPCs for therapeutic use, it may be necessary to mobilize stem and progenitor cells in a subject.

[0041] As used herein, the term "induced pluripotent stem cell" or "iPSC" refers to non-pluripotent cells that are reprogrammed into a pluripotent state. Once cells from a subject are reprogrammed into a pluripotent state, they can be programmed into a desired cell type, such as hematopoietic stem cells or hematopoietic progenitor cells (HSC and HPC, respectively).

[0042] As used herein, the term "reprogramming" refers to a method of increasing the potency of a cell to a less differentiated state.

[0043] As used herein, the term "programming" refers to a method of reducing the differentiation potential of a cell or causing a cell to differentiate into a more differentiated state.

[0044] Immune response The expression "maintaining the ability to suppress immune responses" means maintaining the suppressive effect of Tregs (or a population of such Tregs) on immune responses under proinflammatory conditions (e.g., after or during exposure to proinflammatory conditions) at a level similar to that of corresponding Tregs (particularly Tregs (or a population of such Tregs) modified in the same manner or containing the same modification, e.g., to increase the expression level of FOXP3 (e.g., from an exogenous nucleic acid molecule or polynucleotide)) but not exposed to proinflammatory conditions (e.g., cultured under non-proinflammatory conditions, e.g., in the absence of IFNγ, IL1β, and / or IL6). In this specification, "similar level" refers to a difference (reduction or increase) of the above-mentioned suppressive function from the suppressive function of the corresponding Treg when not exposed to proinflammatory conditions within 10%, within 20%, within 30%, within 40%, or within 50%. In particular, the similar level may be the same level. The phrase "maintaining the ability to suppress an immune response" is used interchangeably herein with the phrases "maintaining functional stability" and "maintaining suppressive function." Maintaining suppressive function / functional stability / ability to suppress an immune response typically refers to when Tregs are / have been exposed to or under proinflammatory conditions.

[0045] Alternatively, Tregs with increased levels of FOXP3 (e.g., with exogenous polynucleotides encoding FOXP3) may be more capable of suppressing immune responses when under or exposed to proinflammatory conditions, compared to Tregs without increased levels of FOXP3, particularly Tregs without exogenous polynucleotides encoding FOXP3, e.g., untransduced Tregs or Tregs transduced with constructs that do not encode FOXP3. As described further below, the present inventors have shown that cells without exogenous FOXP3 lose their suppressive ability when exposed to proinflammatory conditions, whereas Tregs with exogenous FOXP3 can exert suppressive function under proinflammatory conditions at a level similar to that of Tregs cultured under non-proinflammatory conditions (e.g., in a medium containing IL2). The increased ability of Tregs to suppress an immune response as described herein may be at least a 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% increase compared to Tregs that do not have increased levels of FOXP3.

[0046] According to this alternative embodiment, the present invention also provides a method for enhancing the ability of regulatory T cells (Tregs) to suppress immune responses under pro-inflammatory conditions compared to Tregs that have not had their levels of FOXP3 increased (e.g. compared to Tregs that have endogenous levels of FOXP3 or compared to Tregs that have not been modified to have increased levels of FOXP3), comprising introducing a polynucleotide encoding a FOXP3 polypeptide into Tregs.

[0047] In another aspect, the present invention provides a method for preventing the loss of suppressive function in Treg cells when exposed to proinflammatory conditions, comprising expressing increased levels of FOXP3 in Treg cells (particularly from an exogenous nucleic acid molecule comprising a polynucleotide sequence encoding FOXP3). The method (and each method of the present invention described herein) may further comprise a step of incubating Treg cells under proinflammatory conditions, for example with any one of IL-6, IFNγ, and / or IL1β. In particular, as described herein, Tregs may be genetically engineered to comprise a nucleic acid molecule expressing FOXP3 and a CAR, in particular such that the nucleotide sequence encoding FOXP3 is 5' to the nucleotide sequence encoding the CAR. Furthermore, Tregs may be CD45RA+, in particular CD4+CD25+CD127loCD45RA+.

[0048] Loss of suppressive function (also referred to as loss of functional stability) in this specification refers to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% loss of suppressive function. Typically, such loss of suppressive function may occur when Treg cells are not genetically engineered to increase the expression of FOXP3 (particularly, are not genetically engineered to have an exogenous nucleic acid encoding FOXP3), as described above. Preventing loss of suppressive function may result in engineered Tregs having similar or the same suppressive function (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% similar suppressive function) under proinflammatory conditions as the same engineered cells under non-proinflammatory conditions.

[0049] "Exogenous" as defined herein means that a protein is expressed in a cell of the invention from a polynucleotide sequence that has been introduced into the cell (i.e., not from a polynucleotide sequence that is naturally present in the cell, i.e., an endogenous polynucleotide sequence).

[0050] The term "immune response" refers to several physiological and cellular actions that the immune system promotes in response to stimuli such as pathogens or self-antigens. Such actions include, for example, increased proliferation of Tconv cells and secretion of cytokines. Any such action can 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 would indicate a relatively enhanced ability of modified Tregs to suppress immune responses. For example, a relatively decreased cytokine secretion would indicate a weakened immune response and thus an enhanced ability of Tregs to suppress immune responses. Alternatively, a similar immune response by Tconv in the presence of modified Tregs exposed to or under proinflammatory conditions compared to modified Tregs not exposed to proinflammatory conditions (or only exposed to non-proinflammatory conditions) would indicate a maintained ability of modified Tregs to suppress immune responses. As described above, in this specification, "similar" means that the above functions are within 10%, 20%, 30%, 40%, or 50% difference (decrease or increase) from the function of the corresponding Treg when not exposed to proinflammatory conditions (or when exposed only to non-proinflammatory conditions).

[0051] The assay for measuring the index of the strength of immune response and thus the suppressive ability of Treg is known in the art.In particular, antigen-specific Tconv cells can be co-cultured with Treg, and the peptide of the corresponding antigen can be added to the co-culture system to stimulate the response from Tconv cells.The degree of proliferation of Tconv cells and / or the amount of cytokine IL-2 secreted by Tconv cells in response to the addition of the peptide can be used as an index of the suppressive ability of co-cultured Treg.

[0052] Antigen-specific Tconv cells co-cultured with Tregs as described herein that have increased FOXP3 expression may show a 5%, 10%, 15%, 20%, 25%, 30%, 35% or 40% reduction in proliferation after exposure of the Tregs to proinflammatory conditions, compared to the same Tconv cells co-cultured with corresponding Tregs that do not have increased FOXP3 expression. Alternatively, from another perspective, antigen-specific Tconv cells co-cultured with Tregs as described herein that have increased FOXP3 expression and that have been exposed to proinflammatory conditions may show similar levels of proliferation compared to antigen-specific Tconv cells co-cultured with corresponding Tregs that have increased FOXP3 expression but have not been exposed to proinflammatory conditions (or that have only been exposed to non-proinflammatory conditions).

[0053] Antigen-specific Tconv cells co-cultured with Tregs as described herein that have increased FOXP3 expression may exhibit at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% greater reduction in effector cytokines after exposure of Tregs to proinflammatory conditions than corresponding Tconv cells co-cultured with corresponding Tregs that do not have increased FOXP3 expression. Alternatively, looking at it another way, antigen-specific Tconv cells co-cultured with Tregs that have increased FOXP3 expression exposed to proinflammatory conditions may exhibit similar levels of effector cytokines compared to corresponding Tconv cells co-cultured with Tregs that have increased FOXP3 expression that have not been exposed to proinflammatory conditions (or that have only been exposed to non-proinflammatory conditions).

[0054] Antigen-specific Tconv cells co-cultured with Tregs as described herein that have increased FOXP3 expression may produce 10% or less, 20% or less, 30% or less, 40% or less, 50% or less, 60% or less of effector cytokines compared to corresponding Tconv cells co-cultured with corresponding Tregs that do not have increased FOXP3 expression after exposure of the Tregs to proinflammatory conditions. Alternatively, from another perspective, antigen-specific Tconv cells co-cultured with Tregs as described herein that have increased FOXP3 expression and are exposed to proinflammatory conditions may produce similar levels of effector cytokines compared to corresponding Tregs that have increased FOXP3 expression and are not exposed to proinflammatory conditions (or are only exposed to non-proinflammatory conditions).

[0055] 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. Suitably, the effector cytokine may be selected from IL-2, IL-17, TNFα, GM-CSF, and IFN-γ.

[0056] Antigen-specific Tconv cells co-cultured with Tregs of the present invention that have increased FOXP3 expression can achieve suppression of IL-2 production at 1 / 2, 1 / 4, 1 / 8, 1 / 10, or 1 / 20 the cell number of corresponding Tregs that do not have increased FOXP3 expression.

[0057] Pro-inflammatory conditions As used herein, the term "proinflammatory condition" refers to a condition capable of inducing, causing, promoting, or prolonging inflammation in a subject. A proinflammatory condition can be identified by the presence of one or more proinflammatory cytokines.

[0058] The exposure of Treg cells described herein to proinflammatory conditions, typically represented by the presence of one or more proinflammatory cytokines, may be for any period of time. The exposure of Tregs to proinflammatory conditions may occur in vitro (e.g., by culturing Tregs in the presence of one or more proinflammatory cytokines (which may be exogenously added to the culture system or may be produced by the cells during culture, particularly during co-culture)) or in vivo (e.g., when Tregs reach a proinflammatory environment). The one or more proinflammatory cytokines may be present in any amount, but are typically present in an amount that can affect non-transduced Tregs (e.g., at least 1 pg / ml, 5 pg / ml, 10 pg / ml, 50 pg / ml, or 100 pg / ml). Thus, each method of the invention may be in vitro, in vivo (e.g., following administration of Tregs to a patient, e.g., a human patient, with inflammation), or may include both in vitro and in vivo steps (e.g., genetic manipulation of Tregs and / or culture under or exposure to pro-inflammatory conditions may be performed in vitro (or ex vivo) and / or in vivo).

[0059] As mentioned above, the exposure of Treg cells to proinflammatory conditions can be any period, but typically is long-term, particularly at least 2 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, or 72 hours, more particularly at least 4 days, 5 days, 6 days, or 7 days.Typically, in vivo, proinflammatory conditions increase over time, and the level of proinflammatory cytokines can increase over a long period.Therefore, particularly in an in vivo situation, if Tregs are present at the site where proinflammatory cytokines are present for a long period, for example, more than 1 day, more than 2 days, more than 3 days, more than 4 days, more than 5 days, more than 6 days, or more than 7 days, they can be considered to be exposed to proinflammatory conditions. Tregs (or populations of Tregs) as described herein may exhibit sustained suppressive activity after exposure to proinflammatory conditions (e.g., at least 1 hour, 2 hours, 6 hours, 12 hours, or 24 hours) or during exposure to proinflammatory conditions (e.g., while the Tregs are under or exposed to proinflammatory conditions). "Exposed to" proinflammatory conditions includes prior exposure and ongoing exposure.

[0060] Proinflammatory cytokines are molecules that can be secreted from immune cells such as helper T cells and / or macrophages and promote inflammation in a subject or host. Proinflammatory cytokines include IL1β, IL6, IL12, IL18, IFNγ, GM-CSF, and TNFα. In particular, proinflammatory conditions include the presence of one or more proinflammatory cytokines (e.g., at least one, two, or three proinflammatory cytokines), more particularly, the presence of any one or more of IL1β, IL6, and TNFα, e.g., having the sequence of SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10, respectively, or functional variants thereof that typically have at least 80%, 90%, or 95% sequence identity thereto. IL1β (SEQ ID NO:8) [ka] IL6 (SEQ ID NO: 9) [ka] TNFα (SEQ ID NO: 10) [ka]

[0061] Typically, Tregs exposed to pro-inflammatory conditions as defined herein are exposed to one or more of IL1B, TNFα, and IL6 for at least 3 days, and in particular are exposed to all of IL1B, TNFα, and IL6 for at least 3 days.

[0062] Tregs that are not exposed to proinflammatory conditions may be those that have not been cultured in vitro and those that have not been exposed in vivo to one or more proinflammatory cytokines for a long period of time, for example, more than 2 hours, more than 6 hours, more than 12 hours, more than 24 hours, more than 48 hours, or more than 72 hours. Such Tregs may also be referred to as being exposed only to non-proinflammatory conditions. Typically, Tregs that are not exposed to proinflammatory conditions or that are exposed only to non-proinflammatory conditions are those that have not been incubated or cultured with any one or more of proinflammatory cytokines, such as IL6, TNFα, and / or IL1B, typically any one of IL6, TNFα, and IL1B.

[0063] FOXP3 "FOXP3" is the abbreviation for forkhead box P3 protein. FOXP3 is a member of the FOX protein family of transcription factors and functions as a master regulator of regulatory pathways in the development and function of regulatory T cells.

[0064] "Increasing FOXP3 expression" refers to increasing the levels of FOXP3 mRNA and / or protein in Tregs (or a population of Tregs) compared to corresponding unmodified Tregs (or a population of such Tregs). For example, the levels of FOXP3 mRNA and / or protein in Tregs (or a population of such Tregs) modified as described herein may be increased at least 1.5-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 150-fold compared to the levels in corresponding unmodified Tregs (or a population of such Tregs).

[0065] Suitably, the levels of FOXP3 mRNA and / or protein in Tregs (or a population of such Tregs) modified as described herein may be increased by at least 1.5-fold over the levels in corresponding Tregs (or a population of such Tregs) that have not been modified as described herein. Suitably, the levels of FOXP3 mRNA and / or protein in Tregs (or a population of such Tregs) modified as described herein may be increased by at least two-fold compared to the levels in corresponding Tregs (or a population of such Tregs) that have not been modified as described herein. Suitably, the levels of FOXP3 mRNA and / or protein in Tregs (or a population of such Tregs) modified as described herein may be increased by at least 5-fold over the levels in corresponding Tregs (or a population of such Tregs) that have not been modified as described herein.

[0066] The techniques for measuring the level of specific mRNA and protein are well known in the art.The mRNA level in a population of cells, such as Treg, can be measured by techniques such as Affymetrix's eBioscience Prime Flow RNA assay, Northern blotting, serial analysis of gene expression (SAGE), or quantitative polymerase chain reaction (qPCR).The protein level in a population of cells can be measured by techniques such as flow cytometry, high performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LC / MS), Western blotting, or enzyme-linked immunosorbent assay (ELISA).

[0067] In some embodiments of the invention, FOXP3 expression is increased by introducing a polynucleotide encoding a FOXP3 polypeptide into isolated Tregs.

[0068] The term "introduce" refers to a method for inserting foreign DNA into a cell, including both transfection and transduction methods. Transfection is the process of introducing nucleic acid into a cell by non-viral methods. Transduction is the process of introducing foreign DNA into a cell via a viral vector. In particular, introduction is performed before Tregs are exposed to proinflammatory conditions.

[0069] A "FOXP3 polypeptide" is a polypeptide having FOXP3 activity, i.e., a polypeptide that can bind to FOXP3 target DNA and function as a transcription factor that regulates the development and function of Tregs. Techniques for measuring transcription factor activity are well known in the art. For example, transcription factor DNA binding activity can be measured by ChIP. The transcriptional regulatory activity of a transcription factor can be measured by quantifying the expression level of the gene it regulates. Gene expression can be quantified by measuring the 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).

[0070] Polynucleotides and Polypeptides The terms "polynucleotide" and "nucleic acid" are intended to be synonymous with each other. 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.

[0071] The term "polypeptide" is synonymous with "protein" and refers to a series of residues, typically L-amino acids, joined one to the other by peptide bonds typically between the alpha-amino and carboxyl groups of adjacent amino acids. As a result of the degeneracy of the genetic code, many different polynucleotides can encode the same polypeptide. One skilled in the art can make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotide to reflect the codon usage of the particular host organism in which the polypeptide is expressed.

[0072] Polynucleotides may comprise DNA or RNA, may be single-stranded or double-stranded, and may comprise synthetic or modified nucleotides. Several different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, and the addition of acridine or polylysine chains to the 3' and / or 5' ends of the molecule. Polynucleotides can be modified by any method in the art. Such modifications may improve the in vivo activity or life span of the polynucleotide.

[0073] The polynucleotide may be in isolated or recombinant form. The polynucleotide may be incorporated into a vector and the vector may be incorporated into a host cell. The polynucleotide may be codon-optimized. Different cells use certain codons differently. This codon bias corresponds to the bias in the relative abundance of certain tRNAs in a cell type. It is possible to increase expression by changing the codons in the sequence to match the relative abundance of the corresponding tRNA. Preferably, the polynucleotide may be codon-optimized for expression in a mouse disease model. Preferably, the polynucleotide may be codon-optimized for expression in a human subject.

[0074] Many viruses, including HIV and other lentiviruses, use a large number of rare codons. Changing these codons to correspond to commonly used mammalian codons can increase expression of the packaging components in mammalian producer cells. Codon usage tables are known in the art for mammalian cells as well as a variety of other organisms. Codon optimization can also include removal of mRNA instability motifs and cryptic splice sites.

[0075] FOXP3 Polypeptide Sequence Suitably, the FOXP3 polypeptide may comprise the polypeptide sequence of human FOXP3, such as UniProtKB Accession No: Q9BZS1, or a functional fragment thereof. MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGP (SEQ ID NO: 3)

[0076] In some embodiments of the invention, the FOXP3 polypeptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 3, or a functional fragment thereof. Suitably, the FOXP3 polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 3, or a functional fragment thereof. In some embodiments, the FOXP3 polypeptide comprises SEQ ID NO: 3, or a functional fragment thereof.

[0077] Suitably, the FOXP3 polypeptide may be a variant of SEQ ID NO: 3, such as a naturally occurring variant. Suitably, the FOXP3 polypeptide is an isoform of SEQ ID NO: 3. For example, the FOXP3 polypeptide may comprise a deletion of amino acids at positions 72 to 106 relative to SEQ ID NO: 3. Alternatively, the FOXP3 polypeptide may comprise a deletion of amino acids at positions 246 to 272 relative to SEQ ID NO: 3.

[0078] Suitably, the FOXP3 polypeptide comprises SEQ ID NO:4 or a functional fragment thereof. MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAH ARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLL DEKGRAQCLLQREMVQSLEQVEELSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFT YATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGPEGRGSLLTCGDVEEN (SEQ ID NO: 4)

[0079] Suitably, the FOXP3 polypeptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 4, or a functional fragment thereof. Suitably, the polypeptide comprises an amino acid sequence having 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 4, or a functional fragment thereof.

[0080] Suitably, the FOXP3 polypeptide may be a variant of SEQ ID NO: 4, such as a naturally occurring variant. Suitably, the FOXP3 polypeptide is an isoform of SEQ ID NO: 4 or a functional fragment thereof. For example, the FOXP3 polypeptide may comprise a deletion of amino acids at positions 72 to 106 relative to SEQ ID NO: 4. Alternatively, the FOXP3 polypeptide may comprise a deletion of amino acids at positions 246 to 272 relative to SEQ ID NO: 4.

[0081] FOXP3 Polypeptide Sequence Suitably, the FOXP3 polypeptide is encoded by the polynucleotide sequence set forth in SEQ ID NO:1.

[0082] In some embodiments of the invention, the polynucleotide encoding a FOXP3 polypeptide or variant comprises a polynucleotide sequence having at least 80% identity to SEQ ID NO: 1, or a functional fragment thereof. Suitably, the polynucleotide encoding a FOXP3 polypeptide or variant comprises a polynucleotide sequence having at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 1, or a functional fragment thereof. In some embodiments of the invention, the polynucleotide encoding a FOXP3 polypeptide or variant comprises SEQ ID NO: 1, or a functional fragment thereof.

[0083] Suitably, the FOXP3 polypeptide is encoded by the polynucleotide sequence set forth in SEQ ID NO:2.

[0084] In some embodiments of the invention, the polynucleotide encoding a FOXP3 polypeptide or variant comprises a polynucleotide sequence having at least 80% identity to SEQ ID NO: 2, or a functional fragment thereof. Suitably, the polynucleotide encoding a FOXP3 polypeptide or variant comprises a polynucleotide sequence having at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO: 2, or a functional fragment thereof. In some embodiments of the invention, the polynucleotide encoding a FOXP3 polypeptide or variant comprises SEQ ID NO: 2, or a functional fragment thereof.

[0085] Suitably, the polynucleotide encoding the FOXP3 polypeptide or variant thereof may be codon-optimised. Suitably, the polynucleotide encoding the FOXP3 polypeptide or variant thereof may be codon-optimised for expression in a human cell.

[0086] Sequence comparison Sequence comparisons can be performed by eye, or more usually, with the aid of readily available sequence comparison programs. Such publicly and commercially available computer programs can calculate the sequence identity between two or more sequences. Sequence identity may be calculated over a contiguous sequence, i.e., one sequence is aligned with the other and each amino acid in one sequence is directly compared, residue by residue, to the corresponding amino acid in the other sequence. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues (e.g., less than 50 contiguous amino acids). This is a very simple and consistent method, but it does not take into account that in a pair of sequences that are identical except for, for example, one insertion or deletion, the insertion or deletion may cause subsequent amino acid residues to fall out of alignment, resulting in a significant decrease in percent homology when a global alignment is performed. Most sequence comparison methods are therefore designed to produce optimal alignments that take into account possible insertions and deletions without unduly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to try to maximize local homology.

[0087] However, these more complex methods assign a "gap penalty" to each gap that occurs during the alignment, so that for the same number of identical amino acids, a sequence alignment with as few gaps as possible (reflecting a higher relatedness between the two sequences being compared) will achieve a higher score than an alignment with more gaps. An "affine gap cost" is usually used, which imposes a relatively high cost for the presence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. Higher gap penalties will naturally produce an optimized alignment with fewer gaps. Most alignment programs allow the gap penalty to be modified. 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 a gap and -4 for each extension.

[0088] Therefore, the calculation of maximum % sequence identity first requires the creation of an optimal alignment, taking into account gap partitions. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al., 1984, Nucleic Acids Research 12:387, incorporated herein by reference). Other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999, ibid., Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410, incorporated herein by reference), and the GENEWORKS comparison tool suite. Both BLAST and FASTA are available for offline and online searches (see Ausubel et al., 1999, ibid., pp. 7-58-7-60, incorporated herein by reference). However, it is preferred to use the GCG Bestfit program.

[0089] Suitably, sequence identity may be determined over the entire sequence. Suitably, sequence identity may be determined over the entire candidate sequence which is compared to the sequences described herein.

[0090] Although the final sequence identity can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pairwise comparison. Instead, a scaled similarity score matrix is ​​commonly used that assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. One example of such a matrix commonly used is the BLOSUM62 matrix (the default matrix for the BLAST suite of programs). GCG Wisconsin programs generally use either the public default values ​​or a custom symbol comparison table if supplied (see user manual for further details). Preferably, the GCG package uses the public default values, while for other software, a default matrix such as BLOSUM62 is used.

[0091] Once the software has produced an optimal alignment, it is possible to calculate % sequence identity - the software typically does this as part of the sequence comparison and generates a numerical result.

[0092] vector In some embodiments of the invention, the polynucleotide encoding FOXP3 is a contiguous part of an expression vector.

[0093] The term "expression vector" refers to a construct that allows for the expression of a FOXP3 polypeptide. Preferably, the expression vector is a cloning vector. Suitable vectors include, but are not limited to, plasmids, viral vectors, transposons, nucleic acids complexed with a polypeptide, or nucleic acids immobilized on solid particles. Preferably, the expression vector is capable of continuous high level expression in the host cell. The expression vector may be a retroviral vector. The expression vector may be based on or derived from the MP71 vector backbone. The expression vector may lack a full-length or truncated version of the Woodchuck Hepatitis Response Element (WPRE).

[0094] In some embodiments of the invention, the vector also encodes a T cell receptor (TCR). TCR is a cell surface molecule that binds to fragments of antigens bound to major histocompatibility complex (MHC) molecules on antigen-presenting cells as part of inducing an immune response. Advantageously, TCR may be a recombinant protein. In other words, TCR may be an exogenous protein that is not naturally expressed by the Treg of the present invention.

[0095] In some embodiments of the invention, the vector also encodes a chimeric antigen receptor (CAR). CARs are recombinant cell surface molecules expressed by genetically engineered T cells that bind to antigens expressed on the surface of other cells as part of inducing an immune response. More specifically, CARs are proteins that transfer the specificity of antigen binders, such as monoclonal antibodies (mAbs), to the effector functions of T cells. CARs usually take the form of a type I transmembrane domain protein with an amino terminus that recognizes the antigen, a spacer, and a transmembrane domain, all connected to a composite endodomain that transmits survival and activation signals for the T cell.

[0096] If the vector comprises a polynucleotide encoding a TCR or CAR (e.g., an anti-HLA-A2 CAR) in addition to a polynucleotide encoding FOXP3, the vector may have an orientation of 5' FOXP3-TCR / CAR 3'. Thus, the polynucleotide encoding FOXP3 may be 5' to the polynucleotide encoding the CAR or TCR. Suitably, the polynucleotide encoding FOXP3 may be separated from the polynucleotide encoding the TCR or CAR by a nucleic acid sequence that allows both the nucleic acid sequence encoding FOXP3 and the nucleic acid sequence encoding the TCR or CAR to be expressed from the same mRNA transcript.

[0097] For example, a polynucleotide may contain an internal ribosome entry site (IRES) between (i) the nucleic acid sequence encoding FOXP3 and (ii) the nucleic acid sequence encoding a TCR or CAR. An IRES is a nucleotide sequence that allows translation initiation in the middle of an mRNA sequence.

[0098] The polynucleotide may comprise a nucleic acid sequence encoding (i) FOXP3 and (ii) a TCR or CAR linked by an internal self-cleaving sequence.

[0099] Advantageously, the vector may have the structure 5' strong promoter (e.g. LTR)-FoxP3-2A-CAR / TCR-3'LTR, where FOXP3 expression is directly driven by the strong LTR promoter for optimal expression. Due to the presence of a 2A sequence in front of the CAR / TCR, expression of the CAR / TCR depends on both LTR promoter activity and 2A cleavage activity. Importantly, the arrangement of FOXP3 preceding the CAR / TCR in a 5' to 3' direction allows CAR / TCR expression only when FOXP3 is expressed, and without FOXP3, CAR / TCR expression will not occur. This is particularly advantageous in the present context of engineered Tregs, as it reduces the risk of the engineered Tregs acquiring an effector phenotype and / or reduces the risks associated with the introduction of CAR or TCR into T effector cells present in the starting population.

[0100] The internal self-cleaving sequence may be any sequence that allows for separation of a polypeptide comprising (i) FOXP3 and (ii) a TCR or CAR.

[0101] The cleavage site may be autocleaving such that once the polypeptide is produced it is immediately cleaved into individual peptides without the need for external cleavage activity.

[0102] Although the term "cleavage" is used herein for convenience, the cleavage site may separate the peptide into individual entities by mechanisms other than classical cleavage. For example, for the foot and mouth disease virus (FMDV) 2A autocleaving peptide, various models have been proposed to explain the "cleavage" activity, such as proteolysis by host cell proteinases, autoproteolysis, or translational action (Donnelly et al (2001) J. Gen. Virol. 82:1027-1041, incorporated herein by reference). The exact mechanism of such "cleavage" is not important for the purposes of the present invention, so long as the cleavage site, when located between nucleic acid sequences encoding proteins, allows the proteins to be expressed as separate entities.

[0103] The auto-cleaving peptide may be a 2A auto-cleaving peptide from an aphthovirus or a cardiovirus.

[0104] Variants can be considered in terms of similarity (ie, amino acid residues having similar chemical properties / functions), but are preferably expressed in terms of sequence identity.

[0105] Sequence comparisons can be performed by eye, or more usually, with the aid of readily available sequence comparison programs. Such publicly and commercially available computer programs can calculate the sequence identity between two or more sequences.

[0106] Suitably, the FOXP3 polypeptide expressed from the vector may be located N-terminal to a self-cleaving peptide, such as a 2A self-cleaving peptide. Such a FOXP3-2A polypeptide may comprise a sequence as shown in SEQ ID NO:5 or SEQ ID NO:6, or a variant of SEQ ID NO:5 or SEQ ID NO:6 having at least 80% identity to SEQ ID NO:5 or SEQ ID NO:6. Suitably, said variant may have at least 85%, 90%, 95%, 97%, 98% or 99% identity to SEQ ID NO:5 or SEQ ID NO:6. SEQ ID NO:5 MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHA RTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDE KGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPP FTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGPGATNFSLLKQAGDVEENPGPS SEQ ID NO:6 MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHART PVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKGRAQ CLLQREMVQSLEQVEELSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAIL EAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGPEGRGSLLTCGDVEENGATNFSLLKQAGDVEENPGPS

[0107] Viral transduction In some embodiments of the invention, a polynucleotide encoding FOXP3 is introduced into isolated Tregs by viral transduction.

[0108] Viral delivery systems include, but are not limited to, adenoviral vectors, adeno-associated viral (AAV) vectors, herpes viral vectors, retroviral vectors, lentiviral vectors, and baculoviral vectors.

[0109] In some embodiments, a polynucleotide encoding FOXP3 is introduced into isolated Tregs by retroviral transduction.

[0110] Retroviruses are RNA viruses with a life cycle that is distinct from that of lytic viruses. In this regard, retroviruses are infectious agents that replicate through a DNA intermediate. When a retrovirus infects a cell, its genome is converted to a DNA form by the enzyme reverse transcriptase. This DNA copy serves as a template for the production of new RNA genomes and the virus-encoded proteins required for the assembly of infectious viral particles.

[0111] Many retroviruses exist, such as murine leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myelocytomatosis virus-29 (MC29), and avian erythroblastosis virus (AEV), as well as all other Retroviridae viruses, including lentiviruses.

[0112] A detailed list of retroviruses is provided in Coffin et al. ("Retroviruses" 1997 Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763, incorporated herein by reference).

[0113] Lentiviruses also belong to the Retroviridae family, but they are able to infect both dividing and non-dividing cells (Lewis et al. 1992 EMBO J. 3053-3058, incorporated herein by reference).

[0114] For efficient infection of human cells, viral particles may be packaged with an amphotropic or gibbon ape leukemia virus envelope.

[0115] Isolation of Tregs In some embodiments, the method of the present invention further comprises: (a) isolating Tregs from a cell population; (b) increasing the expression of FOXP3 in the Treg. Suitably, exogenous FOXP3 expression in Tregs is increased by introducing an exogenous polynucleotide encoding a FOXP3 polypeptide.

[0116] The term "isolating Tregs from a cell population" refers to separating Tregs from a heterogeneous mixture of multiple different types of cells. Preferably, the cell population is derived from a sample obtained from a human subject.

[0117] Suitably, said Tregs are isolated as a population of Tregs. Suitably, the population of Tregs comprises at least 70% Tregs, such as 75%, 85%, 90% or 95% Tregs.

[0118] In some embodiments of the invention, the cell population comprises or consists of peripheral blood mononuclear cells (PBMCs).

[0119] PBMCs are blood cells with round nuclei that are present in the circulating blood, rather than being sequestered in the bone marrow, liver, spleen, or lymphatic system. PBMCs consist of monocytes and lymphocytes (T cells, B cells, NK cells). Techniques for isolating PBMCs from whole blood are known in the art. For example, PBMCs can be separated from a blood sample by adding a density gradient medium, such as Ficoll (GE Healthcare), followed by centrifugation. Different types of cells in the blood separate into different layers, including a layer containing PBMCs.

[0120] In some embodiments of the invention, isolating Tregs comprises isolating CD4 +In some embodiments, isolating T cells includes isolating CD4 + Isolation of T cells followed by CD4 + This involves isolating Tregs from T cells.

[0121] CD4 (cluster of differentiation 4) is a co-receptor for the T cell receptor expressed by many types of T cells. + Cell isolation separates T cells, including Tregs, from the initial cell population, and Tregs can then be isolated from this T cell-enriched population.

[0122] Techniques for isolating specific cell types from a heterogeneous cell population are known in the art, for example, the use of immunomagnetic beads and fluorescence activated cell sorting.

[0123] In some embodiments of the invention, isolating a population of Tregs comprises using immunomagnetic beads. Various companies (e.g., Miltenyi Biotec, Stem Cell Technologies, ThermoFisher Scientific) provide kits that contain immunomagnetic beads for isolating specific types of T cells (e.g., 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 against T cell surface proteins, such as CD8, CD25, CD49b, etc., that are widely available in the art. For example, cell populations can be isolated from non-CD4 + CD4 by incubating with a biotin-conjugated antibody against a marker for CD4+ cells (e.g., CD8) and removing these cells using anti-biotin magnetic beads. +Cells can first be negatively selected, and then Tregs can be positively selected by incubation with anti-CD25 labeled beads.

[0124] In some embodiments of the invention, isolating the population of Tregs comprises fluorescence-activated cell sorting (FACS). + CD25 hi CD127 - Select according to phenotype.

[0125] Natural Tregs may be selected from inducible Tregs based on their expression of Helios protein or neuropilin 1. In some embodiments of the invention, natural Tregs are selected based on their expression of CD4 + CD25 + FOXP3 + Helios + Neuropilin 1 + Selection may also be according to phenotype.

[0126] FACS is a form of flow cytometry and is well known in the art. During FACS, cells are suspended in a fluid and passed through a detection system that analyzes various characteristics. Using this method, cells can be sorted according to their properties. In particular, in FACS, fluorescent antibodies are used to label molecules and cells are sorted according to their fluorescence, which indicates the level of expression of a particular molecule (see Adan et al. Flow cytometry: basic principles and applications Crit. Rev. Biotechnol. 2017 Mar; 37(2): 163-176, incorporated herein by reference).

[0127] Genetically engineered Tregs Each method of the invention provides Tregs that exhibit higher FOXP3 expression relative to corresponding non-genetically engineered Tregs that are capable of maintaining suppressive function when exposed to pro-inflammatory conditions.

[0128] By "higher FOXP3 expression" is meant that the levels of FOXP3 mRNA or protein in genetically engineered Tregs are higher than the levels before the Tregs were artificially engineered to alter their gene expression. "Higher FOXP3 expression" may be defined and determined as described herein.

[0129] Suitably, the levels of CD25 mRNA and / or protein in Tregs (or a population of such Tregs) modified as described herein may be increased by at least 1.5-fold over the levels in corresponding Tregs (or a population of such Tregs) that have not been modified as described herein. Suitably, the levels of CD25 mRNA and / or protein in Tregs (or a population of such Tregs) modified as described herein may be increased by at least two-fold compared to the levels in corresponding Tregs (or a population of such Tregs) that have not been modified as described herein. Suitably, the levels of CD25 mRNA and / or protein in Tregs (or a population of such Tregs) modified as described herein may be increased by at least 5-fold over the levels in corresponding Tregs (or a population of such Tregs) that have not been modified as described herein.

[0130] Suitably, the levels of CTLA-4 mRNA and / or protein in Tregs (or a population of such Tregs) modified as described herein may be increased by at least 1.5-fold over the levels in corresponding Tregs (or a population of such Tregs) that have not been modified as described herein. Suitably, the levels of CTLA-4 mRNA and / or protein in Tregs (or a population of such Tregs) modified as described herein may be increased by at least two-fold compared to the levels in corresponding Tregs (or a population of such Tregs) that have not been modified as described herein. Suitably, the levels of CTLA-4 mRNA and / or protein in Tregs (or a population of such Tregs) modified as described herein may be increased by at least 5-fold over the levels in corresponding Tregs (or a population of such Tregs) that have not been modified as described herein.

[0131] In some embodiments of the present invention, the genetically engineered Treg comprises an exogenous polynucleotide encoding a FOXP3 polypeptide.

[0132] An "exogenous polynucleotide" is a polynucleotide that originates outside of a Treg. An exogenous polynucleotide may be introduced into a Treg as part of an expression vector. Thus, an exogenous polynucleotide may be contiguous with an expression vector element such as a promoter.

[0133] In some embodiments of the invention, the FOXP3 polypeptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO:3 or SEQ ID NO:4, or a functional fragment thereof. Suitably, the FOXP3 polypeptide comprises an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO:3 or SEQ ID NO:4, or a functional fragment thereof. In some embodiments, the FOXP3 polypeptide comprises SEQ ID NO:3 or SEQ ID NO:4, or a functional fragment thereof.

[0134] In some embodiments of the invention, the exogenous polynucleotide encoding FOXP3 comprises a polynucleotide sequence having at least 80% identity to SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments of the invention, the polynucleotide encoding FOXP3 is identical to SEQ ID NO: 1 or SEQ ID NO:2.

[0135] In some embodiments of the invention, the exogenous polynucleotide encoding FOXP3 is a contiguous part of a vector.

[0136] In some embodiments of the invention, the vector also encodes a T cell receptor (TCR) or CAR, in particular an anti-HLA A2 CAR.

[0137] In some embodiments of the invention, the vector comprises a polynucleotide sequence having at least 80% identity to SEQ ID NO: 5. In some embodiments of the invention, the vector comprises a polynucleotide sequence identical to SEQ ID NO:5.

[0138] Therefore, the present invention provides genetically engineered Tregs expressing exogenous FOXP3, obtainable or obtainable by the methods of the present invention, which are capable of maintaining their suppressive function when exposed to pro-inflammatory conditions.

[0139] The genetically engineered Tregs are (i) isolating Tregs from a cell population; (ii) introducing an exogenous FOXP3 polypeptide-encoding polynucleotide into the isolated Treg to maintain the ability of the Treg to suppress immune responses under proinflammatory conditions.

[0140] The Treg may be a CD4+CD25+CD127- / lowCD45RA+ Treg.

[0141] The invention also provides methods of making engineered Tregs as disclosed herein, comprising the step of introducing into a regulatory T cell or a pluripotent or multipotent cell one or more polynucleotide sequences encoding exogenous FOXP3, CAR, or exogenous TCR, with the subsequent step, in the case of introducing into a pluripotent or multipotent cell, of differentiating the cell into a regulatory T cell.

[0142] Said regulatory T cells may be isolated from a subject, in particular by apheresis followed by enrichment. Alternatively, said regulatory T cells may be obtained by differentiation of pluripotent cells, in particular iPSCs.

[0143] composition The present invention also provides pharmaceutical compositions comprising engineered Tregs obtainable or obtainable by the methods of the present invention.

[0144] Such pharmaceutical compositions may include a pharma- ceutically acceptable carrier, diluent, excipient, or adjuvant. The choice of pharmaceutical carrier, excipient, or diluent can be selected having regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may include as (or in addition to) the carrier, excipient, or diluent, suitable binders, lubricants, suspending agents, coating agents, solubilizing agents, and other carrier agents.

[0145] Pharmaceutical compositions typically need to be sterile and stable under the conditions of manufacture and storage. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Sterile injectable formulations may be prepared using non-toxic parenterally acceptable diluents or solvents. Pharmaceutical compositions of the present invention include pharma-ceutically acceptable dispersing agents, wetting agents, suspending agents, isotonic agents, coating agents, antibacterial and antifungal agents, carriers, excipients, salts, or stabilizers that are non-toxic to subjects at the dosages and concentrations employed. Preferably, such compositions may further comprise pharma-ceutically acceptable carriers or excipients for use in the treatment of disease, compatible with a given method and / or site of administration, for example, for parenteral administration (e.g., subcutaneous, intradermal, or intravenous injection) or intrathecal administration.

[0146] The compositions may be manufactured using current good manufacturing practices (cGMP).

[0147] Suitably, pharmaceutical compositions comprising engineered Tregs may contain an organic solvent, such as, but not limited to, methyl acetate, dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), dimethoxyethane (DME), and dimethylacetamide, including mixtures or combinations thereof. Preferably, the pharmaceutical composition is endotoxin-free. Advantageously, the present invention relates to a pharmaceutical composition comprising genetically engineered T cells (Tregs) capable of suppressing an immune response under pro-inflammatory conditions, comprising: (iii) isolating Tregs from the cell population; and (vi) introducing a polynucleotide encoding an exogenous FOXP3 polypeptide into the isolated Treg to maintain the ability of the Treg to suppress immune responses under proinflammatory conditions.

[0148] The Treg may be a CD4+CD25+CD127- / lowCD45RA+ Treg.

[0149] Prevention and / or treatment of disease The present invention also provides a genetically engineered Treg obtainable or obtained by the method of the invention, or a pharmaceutical composition of the invention, for use in the prevention and / or treatment of a disease.

[0150] The present invention also provides the use of engineered Tregs obtainable or obtained by the method of the invention in the manufacture of a medicament for use in the prevention and / or treatment of disease.

[0151] The present invention also provides a method for the prevention and / or treatment of disease comprising administering to a subject the engineered Tregs or compositions of the present invention.

[0152] The method for preventing and / or treating the above diseases preferably comprises administering the pharmaceutical composition of the present invention to a subject.

[0153] The term "treat / treatment / treating" refers to the administration of the engineered Tregs or pharmaceutical compositions of the invention to a subject having an existing disease or condition to relieve, reduce, or ameliorate at least one symptom associated with the disease and / or to slow, inhibit, or prevent the progression of the disease.

[0154] "Prevention" / "preventing" (or prophylaxis) refers to delaying or preventing the onset of disease symptoms. Prevention may be absolute (such that the disease does not develop) or may be effective only in some individuals or for a limited period of time.

[0155] In some embodiments of the invention, the subject of the method of the invention is a mammal, preferably a cat, dog, horse, donkey, sheep, pig, goat, cow, mouse, rat, rabbit, or guinea pig. Preferably, the subject is a human.

[0156] Administration of the pharmaceutical compositions of the present invention can be accomplished using any of a variety of routes that render the active ingredient bioavailable. For example, the engineered Tregs or pharmaceutical compositions can be administered intravenously, intrathecally, by oral and parenteral routes, intranasally, intraperitoneally, subcutaneously, transdermally, or intramuscularly.

[0157] Preferably, the genetically engineered Tregs or pharmaceutical compositions of the invention are administered intravenously. Preferably, the genetically engineered Tregs or pharmaceutical compositions of the invention are administered intrathecally.

[0158] Typically, a physician will determine the dosage that will be most suitable for an individual subject, which will vary with the age, weight, and response of the particular subject. The dosage will be that amount that is sufficient to reduce and / or prevent symptoms of the disease.

[0159] Those skilled in the art will understand that, for example, the delivery route (e.g., oral, intravenous, subcutaneous, etc.) can affect the required dosage (and the required dosage can affect the delivery route). For example, if a particularly high concentration of the drug is desired in a particular area or location, focused delivery may be preferred. Other factors to consider when optimizing the route and / or administration schedule for a given treatment regimen can include, for example, the disease being treated (e.g., type or stage, etc.), the clinical condition of the subject (e.g., age, overall health, etc.), the presence or absence of concomitant therapy, and other factors known to medical practitioners.

[0160] The amount administered is such that is sufficient to stabilize or ameliorate symptoms of the disease. The present invention also provides a method for treating and / or preventing a disease, the method comprising the step of administering to a subject a pharmaceutical composition comprising a cell, such as a T cell, according to the present invention. Preferably, the method for preventing and / or treating the above diseases comprises: (i) isolating Tregs from a subject; (ii) introducing a polynucleotide sequence encoding a FOXP3 polypeptide into said Tregs (i.e., genetically engineering said Tregs); (iii) administering the engineered Treg to the subject.

[0161] Tregs may be isolated from a patient by taking a blood sample and isolating Tregs from the blood sample using techniques known in the art, such as those described herein under the heading "Isolation of Tregs."

[0162] A polynucleotide encoding a FOXP3 polypeptide may be introduced into Tregs using techniques known in the art, such as those described herein under the heading "Viral Transduction."

[0163] Suitably, the engineered Tregs may be expanded in vitro prior to administration to a subject. Tregs may be expanded in vitro by culturing in TexMACX® medium.

[0164] disease The disease to be treated and / or prevented by the methods and uses of the present invention may be any disease associated with a pathological immune response. The disease may be, for example, cancer, an infectious disease, or an autoimmune disease.

[0165] In some embodiments of the invention, the disease is an autoimmune disease.

[0166] The above diseases may be accompanied by central nervous system (CNS) involvement in systemic autoimmune and inflammatory diseases such as Behçet's disease, sarcoidosis, systemic lupus erythematosus, juvenile idiopathic arthritis, scleroderma, and Sjögren's syndrome. The disease may be any disease in which MBP is an antigen, for example in which MBP is an autoantigen.

[0167] Suitably, the disease may be an autoimmune or inflammatory disease of the central or peripheral nervous system (eg, a chronic neurodegenerative disease). Suitably, the disease may be a chronic neurodegenerative condition such as multiple sclerosis (MS), Alzheimer's disease, Parkinson's disease, neurotropic viral infections, stroke, paraneoplastic disorders, and traumatic brain injury.

[0168] The present invention further provides methods for inducing tolerance to transplantation, treating and / or preventing cellular and / or humoral transplant rejection, and treating and / or preventing graft-versus-host disease (GvHD), comprising administering to a subject a genetically engineered Treg or pharmaceutical composition of the present invention. As used herein, "inducing tolerance to transplantation" refers to inducing tolerance to a transplanted organ in a recipient. In other words, inducing tolerance to transplantation means reducing the level of the recipient's immune response to the donor transplanted organ. Inducing tolerance to a transplanted organ allows the transplant recipient to reduce the amount of immunosuppressant required or to discontinue the immunosuppressant.

[0169] In one embodiment, the subject is a transplant recipient undergoing immunosuppressive therapy. The transplant may be selected from a liver transplant, a kidney transplant, a heart transplant, a lung transplant, a pancreas transplant, an intestine transplant, a stomach transplant, a bone marrow transplant, a vascularized composite tissue transplant, and a skin transplant.

[0170] use The present invention further provides the use of a polynucleotide encoding a FOXP3 polypeptide to maintain the ability of regulatory T cells (Tregs) to suppress immune responses under proinflammatory conditions.As mentioned above, the maintenance of the suppressive function of the Tregs of the present invention that are exposed or have been exposed to proinflammatory conditions is provided by increasing FOXP3 expression by introducing a polynucleotide encoding a FOXP3 polypeptide into Tregs.Here, the maintenance of suppressive function is compared to the corresponding Tregs that contain a polynucleotide encoding a FOXP3 polypeptide but have not been exposed or have not been exposed to proinflammatory conditions (i.e., only exposed to non-proinflammatory conditions).Therefore, the polynucleotide encoding FOXP3 is used to maintain the suppressive function of Tregs under proinflammatory conditions.

[0171] Viewed another way, the present invention provides the use of a polynucleotide encoding a FOXP3 polypeptide to enhance the ability of regulatory T cells (Tregs) to suppress immune responses under pro-inflammatory conditions, compared to Tregs that do not contain an exogenous FOXP3 polypeptide-encoding polynucleotide. Viewed another way, the present invention provides the use of a polynucleotide encoding a FOXP3 polypeptide to prevent the loss of suppressive function of Treg cells under pro-inflammatory conditions.

[0172] Furthermore, the present invention may provide a method for increasing the proportion or amount of Tregs comprising an exogenous polynucleotide encoding FOXP3 within a population of Tregs, the method comprising exposing the population of Tregs to pro-inflammatory conditions. In particular, the increase in the percentage or amount of Tregs comprising an exogenous polynucleotide encoding FOXP3 in the population may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% and may be measured directly by flow cytometry using an antibody that binds to FOXP3 (after cell permeabilization) or by any of the methods described above. Typically, Tregs that do not contain exogenous FOXP3 have lower FOXP3 levels and less survival under proinflammatory conditions than transduced Tregs. Thus, exposure of a mixed population of Tregs to proinflammatory conditions can be used to purify Tregs comprising an exogenous polynucleotide encoding FOXP3 or to increase the number or percentage of those cells present in the population. As described above, this can be achieved by exposure to any one or more proinflammatory cytokines, but in particular by exposure to IL1B, IL6, and / or TNFα.

[0173] The disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, nucleic acid sequences are written left to right in 5' to 3' orientation and amino acid sequences are written left to right in amino to carboxy orientation.

[0174] Where a range of values ​​is provided, each intervening value between the upper and lower limits of that range, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, is also specifically disclosed. Each narrower range between any stated or intervening value in a stated range and any other stated or intervening value in that stated range is also encompassed in the disclosure. The upper and lower limits of these narrower ranges may independently be included or excluded in the range, and each range in which either, neither or both limits are included in the narrower range is also encompassed in the disclosure, unless a specifically excluded limit is within the stated range. When a stated range includes one or both limits, ranges excluding either or both of the included limits are also included in the disclosure.

[0175] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0176] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including," "includes," or "containing," and are inclusive or open-ended and do not exclude additional, unrecited elements, components, or method steps. The terms "comprising," "comprises," and "comprised of" also include the term "consisting of."

[0177] The embodiments of the present invention may be combined.

[0178] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the appended claims. The present invention will now be further described by way of examples, which are intended to assist those skilled in the art in practicing the present invention and are not intended to limit the scope of the present invention.

[0179] Working Example material and method Cloning: Two constructs (Figure 2) were designed in-house and produced with all sequences codon-optimized for expression in human cells. Constructs were cloned and transformed into D5a high efficiency bacteria with the plasmids and grown with the selection agent ampicillin. DNA was extracted using a Miniprep Kit (Qiagen). Inserts were transferred into the lentiviral backbone by PCR cloning.

[0180] Collection of PBMCs: Leukocyte cones were provided by NHS Blood and Transplant. PBMCs were isolated using a density centrifugation protocol. Briefly, blood was diluted 1:1 with 1x PBS and layered onto Ficoll-Paque (GE Healthcare). Samples were centrifuged and the leukocyte layer was removed and washed with PBS.

[0181] Treg and Tconv Isolation Protocol: HLA-A * Treg populations were derived using blood cones from 02 negative donors. Blood cones were CD4 enriched by negative selection using RosetteSep™ Human CD4+ T Cell Enrichment Cocktail. CD4+ cells were then isolated using density centrifugation. CD4+CD25+ T cells were then isolated by positive selection using CD25 MicroBeads II (Miltenyi). The CD4+CD25+ fraction was stained with flow cytometry antibodies CD4 FITC (OKT4, Biolegend), CD25 PE-Cy7 (BC96, Biolegend), CD127 BV421 (A019D5, Biolegend), CD45RA BV510 (HI100, Biolegend), and LIVE / DEAD™ Fixable Near-IR-Dead Cell Stain (Thermo Fisher) followed by FACS sorting.

[0182] Treg culture medium and expansion: Human regulatory T cells were cultured in Texmacs medium (Miltenyi) supplemented with IL-2 and activated with Human T-Activator CD3 / CD28 Dynabeads™ (Gibco). Cells were re-fed with Treg culture medium supplemented with IL-2 every 2-3 days. A second stimulation with Dynabeads™ was performed to promote further expansion of Treg cells.

[0183] Transfection and generation of viral particles Lentivirus: HEK293T cells were seeded in DMEM (Dulbecco's Modified Eagle Medium) + 10% fetal bovine serum (FBS) and cultured for 24 hours. The transfection reagent was brought to room temperature and mixed with the DNA construct / plasmid of interest, packaging plasmid (pD8.91), and viral envelope (pVSV-G). The diluted DNA was mixed with PEI and added to HEK293T. 48 hours after transfection, the supernatant was collected and filtered to concentrate the virus.

[0184] Flow cytometry staining to measure CAR and RQR8 expression T cells were removed from the culture system, washed with FACS buffer, and soaked in FACS buffer for HLA-A * 02 HLA-A staining using specific dextramer (WB2720-APC, Immudex) * 02-specific CARs were identified. Cells were then stained first with LIVE / DEAD™ Fixable Near-IR-Dead Cell Stain (Thermo Fisher) in PBS, followed by anti-CD4 AF700 (RPA-T4, BD), anti-CD34 FITC (QBEND / 10, Thermo Fisher), and anti-CD3 PE-Cy7 in FACS staining buffer. For intracellular staining of FOXP3, cells were fixed, permeabilized, and stained with anti-Foxp3 PE (150D / E4, Thermo Fisher) antibody. Cells were analyzed on a BD LSRII flow cytometer.

[0185] Instability assay Frozen Tregs transduced as described above were thawed and incubated under two different conditions. In the first condition, Tregs were cultured under standard conditions as described above. In the second condition, Tregs were cultured in the presence of additional IL6 (4 ng / ml), IL1β (10 ng / ml), and TNFα (10 ng / ml). Cells were cultured for 7 days, after which suppression assays were performed.

[0186] Suppression assay To evaluate the ability of Tregs to suppress effector T cell activation, Teff cells were labeled with CFSE dye. Teff cells were co-cultured with different concentrations of Treg cells (Treg:Teff ratios of 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64). For activation, CD3 / CD28 beads (1:100) were added. For CAR-dependent activation, HLA A2 expressing cells are used. Cells are harvested 72 hours after activation and analyzed by flow cytometry. CFSE dilution is used as a surrogate marker for Teff cell proliferation.

[0187] result Figure 3 shows the expression levels of FOXP3 in transduced and non-transduced cells for construct I (i.e., construct expressing additional exogenous FOXP3). It can be seen that transduced Tregs have higher levels of FOXP3 compared to non-transduced cells in the population. Thus, transduction with construct I increases the levels of FOXP3 in cells. Furthermore, Figure 3 shows a comparison of the suppressive function of transduced and non-transduced cells under proinflammatory conditions: cells expressing exogenous additional FOXP3 are able to maintain their suppressive function even after exposure to proinflammatory cytokines. This maintenance of suppressive activity is further demonstrated in FIG. 4, which shows a comparison of suppressive function or capacity of cells transduced with construct I (with exogenous FOXP3 and CAR expression) and construct VIII (expressing the same CAR as construct I, but without exogenous FOXP3). Cells transduced with construct I are able to maintain suppressive function compared to cells transduced with construct VIII, which are unable to suppress after exposure to proinflammatory conditions. Cells transduced with construct I exposed to proinflammatory conditions only exhibit a slight decrease in suppressive function compared to the same cells cultured under standard conditions (no proinflammatory cytokines), demonstrating how effective the presence of exogenous FOXP3 is in maintaining suppressive function under proinflammatory conditions. Considering that Treg products need to function under proinflammatory conditions in vivo, the presence of exogenous FOXP3 is important to provide a product that can retain functional capacity.

[0188] All publications mentioned herein are incorporated herein by reference.Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art that do not depart from the scope and spirit of the invention.Although the invention has been described in connection with certain preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments.Indeed, various modifications of the above-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 following claims.

Claims

1. 1. A method for maintaining the ability of regulatory T cells (Tregs) to suppress an immune response under proinflammatory conditions, the method comprising introducing into said Tregs a polynucleotide encoding a FOXP3 polypeptide.

2. The method of claim 1, further comprising incubating the Tregs under pro-inflammatory conditions, in particular with any one or more of IL-6, IFNγ, and / or IL1β.

3. (i) the FOXP3 polypeptide comprises an amino acid sequence having at least 80% identity to SEQ ID NO:3 or SEQ ID NO:4, or a functional fragment thereof; or (ii) the polynucleotide encoding the FOXP3 polypeptide comprises a polynucleotide sequence having at least 80% identity to SEQ ID NO:1 or SEQ ID NO:2, or a functional fragment thereof.

4. 10. The method of any one of the preceding claims, wherein the polynucleotide encoding FOXP3 is a contiguous part of an expression vector.

5. 2. The method of any one of the preceding claims, further comprising introducing into said Tregs a polynucleotide encoding an exogenous T cell receptor (TCR) or a polynucleotide encoding a chimeric antigen receptor (CAR).

6. 6. The method of claim 5, wherein the polynucleotide encoding the FOXP3 polypeptide and the polynucleotide encoding the exogenous TCR or CAR are provided by a single expression vector.

7. The method of claim 6, wherein the vector comprises a nucleic acid having a 5' FOXP3-TCR / CAR 3' orientation.

8. 13. The method of any one of the preceding claims, wherein the polynucleotide encoding FOXP3 is introduced into the isolated Tregs by viral transduction, optionally wherein the polynucleotide encoding FOXP3 is introduced into the isolated Tregs by retroviral transduction.

9. (a) isolating Tregs from a cell population; 2. The method of any one of the preceding claims, comprising: (b) increasing FOXP3 expression in said Tregs.

10. 10. The method of claim 9, wherein the cell population comprises or consists of peripheral blood mononuclear cells (PBMCs).

11. isolating the Tregs CD4 + Isolating T cells; The CD4 + and isolating Tregs from the T cells.

12. 12. The method of any one of claims 9 to 11, wherein the isolation of Tregs comprises immunomagnetic bead sorting or fluorescence activated cell sorting (FACS).

13. The Treg is (i) CD4 + CD25 + CD127 - and / or CD4 + CD25 + CD127 low cells; (ii) CD4 + CD25 hi CD127 - and / or CD4 + CD25 + CD127 low 13. The method of any one of claims 9 to 12, wherein the cells are isolated by selecting the cells.

14. The Treg is FOXP3 + and preferably, said Tregs are isolated by selecting CD4 + CD25 + FOXP3 + Helios + Neuropilin 1 + 14. The method of any one of claims 9 to 13, wherein the cells are isolated by selecting the cells.

15. 15. Genetically engineered Tregs obtainable or obtained by the method of any one of claims 1 to 14.

16. A pharmaceutical composition comprising the genetically engineered Treg of claim 15.

17. 17. The genetically engineered Treg of claim 15 or the pharmaceutical composition of claim 16 for use in the prevention and / or treatment of a disease.

18. 16. Use of the genetically engineered Treg of claim 15 in the manufacture of a medicament for the prevention and / or treatment of a disease.

19. 20. A method for the prevention and / or treatment of a disease comprising administering to a subject a genetically engineered Treg or a pharmaceutical composition according to any one of claims 15 to 18.

20. 20. The genetically engineered Treg for use according to claim 17 or the pharmaceutical composition for use according to claim 17, the use of the genetically engineered Treg according to claim 18 or the method according to claim 19, wherein the disease is an autoimmune disease, preferably wherein the disease is multiple sclerosis.

21. The genetically engineered Treg of claim 15 for use in the prevention and / or treatment of a disease which is transplant rejection or graft-versus-host disease.

22. Use of a polynucleotide encoding a FOXP3 polypeptide to maintain the ability of regulatory T cells (Tregs) to suppress immune responses under pro-inflammatory conditions.

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