Method for maintaining inhibitory activity of regulatory t cells

Abstract

The present invention provides a method of maintaining the ability of a regulatory T cell (Treg) to inhibit an immune response under pro-inflammatory conditions, the method comprising introducing a polynucleotide encoding a FOXP3 polypeptide into the Treg.

Description

Technical Field

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

[0002] In autoimmune and inflammatory central nervous system (CNS) diseases, the immune system attacks self-antigens. For example, in multiple sclerosis (MS), the most common neurological disorder in young people, the immune system attacks the myelin sheath of CNS neurons.

[0003] Current treatments for autoimmune and inflammatory CNS diseases typically suppress the immune system. For example, one treatment involves bone marrow transplantation, along with administration of cytostatic and immunosuppressive drugs. Autologous hematopoietic stem cell transplantation can have lasting beneficial effects in some subjects, but the procedure requires aggressive myeloablative conditioning, which carries significant toxicity and risk.

[0004] Although several disease-modifying therapies (DMTs) have been approved to reduce the frequency of clinical relapses, most patients continue to experience clinical deterioration under current treatment regimens. Neither DMTs nor stem cell transplantation mediate CNS-specific suppression of the immunopathology of autoimmune and inflammatory CNS diseases.

[0005] Currently, there are no effective treatments for autoimmune and inflammatory CNS diseases. Treatment focuses solely on alleviating their symptoms, usually through global suppression of the immune system. A therapy is needed that specifically targets the local immune response associated with the development and progression of CNS diseases.

[0006] Regulatory T cells (Treg) are T cells that regulate the activity of the immune system. Generally, Treg has an immunosuppressive effect and downregulates the immune response to stimulation. In particular, Treg inhibits the induction and proliferation of conventional T cells, and some types of conventional T cells are directly involved in immune responses (such as cytotoxic T cells). The inhibitory effect of Treg can be directed to specific antigens by expressing recombinant T cell receptor (TCR) constructs, which recognize peptides that match the epitopes found in the antigen in question. Similarly, the inhibitory effect of Treg can be directed to specific targets by expressing a chimeric antigen receptor (CAR) that recognizes antigens expressed on the surface of target cells. Conventional T cells can be differentiated into a regulatory phenotype in vitro by expressing FOXP3 in the cells.

[0007] Regulatory T cells (Tregs) can modulate immune responses through a variety of mechanisms; therefore, their therapeutic use has been proposed for inducing immune tolerance in transplantation, autoimmune diseases, and chronic inflammatory conditions. FOXP3 is considered to be the master transcription factor for Tregs, driving the expression of CD25, CTLA-4, and suppressing proinflammatory cytokines such as IL-2. It has been shown that some cells with all the characteristics of Tregs lose FOXP3 expression and acquire pathogenic features, such as expression of effector cytokines, under certain conditions. This result is particularly problematic in the context of antigen-specific Treg therapies—posing significant safety concerns. Summary of the invention

[0008] The inventors surprisingly found that exogenous FOXP3 expression in regulatory T cells (Tregs), which already express endogenous FOXP3, allows to maintain their regulatory function when exposed to pro-inflammatory conditions.

[0009] Thus, 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.

[0010] The Treg of the present invention is a natural Treg or an induced Treg developed from a conventional T cell. For example, the Treg of the present invention is a natural Treg or an induced Treg developed from a conventional T cell in vivo. Suitable Treg cells include thymus-derived natural Treg (nTreg) cells and peripherally produced induced Treg (iTreg) cells. In other words, the Treg of the present invention expresses endogenous FOXP3. Surprisingly, the inventors have determined that increasing FOXP3 expression (e.g., by introducing exogenous FOXP3) in Tregs that have already expressed endogenous FOXP3 maintains the regulatory function of Tregs when exposed to proinflammatory conditions at a level similar to that of the corresponding Tregs (e.g., Tregs cultured under non-inflammatory conditions) that are not exposed to proinflammatory conditions. In contrast, Tregs with endogenous FOXP3 levels (e.g., Tregs without exogenous FOXP3) have been shown to lose their inhibitory ability under proinflammatory conditions. Therefore, the presence of exogenous FOXP3 in Tregs that already express endogenous FOXP3 appears to provide functional stability to Tregs compared to cells that only have endogenous FOXP3 expression (particularly natural or wild-type endogenous FOXP3 expression), while cells that only have endogenous FOXP3 expression appear to be susceptible 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 a step of increasing FOXP3 expression in Tregs (e.g., expressing exogenous FOXP3), or provides a method for maintaining the suppressive function of Tregs under proinflammatory conditions, comprising a step of increasing FOXP3 expression in Tregs (e.g., expressing exogenous FOXP3).

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

[0012] The present invention provides a method for maintaining the ability of Treg to suppress immune response under pro-inflammatory conditions, comprising increasing FOXP3 expression in Treg.

[0013] In some embodiments of the present invention, FOXP3 expression is increased by introducing a polynucleotide encoding FOXP3 protein into Tregs.

[0014] In some embodiments of the invention, the method of maintaining the ability of regulatory T cells (Tregs) to suppress immune responses under pro-inflammatory conditions comprises:

[0015] (a) isolating Tregs from a cell population; and

[0016] (b) increasing the expression of FOXP3 in the Tregs.

[0017] Suitably, said Treg refers to a population of Tregs (ie a plurality of Tregs).

[0018] The method of the invention may comprise the additional step of exposing said Treg cells to pro-inflammatory conditions.

[0019] The present invention also provides engineered Tregs obtainable or obtained by the methods of the present invention.

[0020] The present invention also provides a pharmaceutical composition comprising the engineered Treg of the present invention.

[0021] The present invention also provides the engineered Treg of the present invention or the pharmaceutical composition of the present invention for use in preventing and / or treating a disease.

[0022] The present invention also provides use of the engineered Treg of the present invention in manufacturing a drug.

[0023] The present invention also provides a method for preventing and / or treating a disease, comprising administering the engineered Treg or the composition of the present invention to a subject.

[0024] The present invention also provides the use of a polynucleotide encoding a FOXP3 polypeptide in maintaining the ability of regulatory T cells (Treg) to suppress immune responses under pro-inflammatory conditions.

[0025] The present invention further provides a use of a nucleic acid molecule comprising a nucleotide sequence encoding FOXP3 for maintaining the suppressive function or functional stability of Treg when exposed to proinflammatory conditions, wherein the Treg comprises the nucleic acid molecule. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Shown is the outline method of the instability assay used to determine the functional stability of Treg cells incubated under control conditions or in the presence of three pro-inflammatory cytokines.

[0027] Figure 2 The construct design used in the instability assay is shown. Two constructs were used, namely construct VIII encoding only CAR (HLA-A2 CAR) and construct I encoding FOXP3 and CAR (the encoding polynucleotide sequence was separated by a 2A self-cleaving peptide). Figure 2 Further shown is the instability assay.

[0028] Figure 3Results of instability assays using a population of Tregs transduced with construct I encoding polynucleotides exposed to proinflammatory conditions are shown. FOXP3 expression was determined in transduced and non-transduced cells and used in suppression assays. Transduced cells had a higher % 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.

[0029] Figure 4 The ability of Tregs transduced with construct I or construct VIII to suppress when exposed to proinflammatory conditions is shown. Tregs expressing construct I (thus expressing exogenous FOXP3 and CAR) maintain their inhibitory effects after exposure to proinflammatory cytokines. Transparent circles show cells containing construct I when exposed to control conditions, and gray circles show cells containing construct I after exposure to proinflammatory conditions, where the maintenance of inhibitory function can be seen. In contrast, Tregs expressing construct VIII (no exogenous FOXP3, only CAR) cannot suppress after exposure to proinflammatory conditions, and have similar low levels of suppression compared to untransduced cells. DETAILED DESCRIPTION

[0030] The present invention provides a method for maintaining the ability of regulatory T cells (Tregs) to suppress immune responses when exposed to proinflammatory conditions, comprising increasing FOXP3 expression in Tregs (eg, by expressing exogenous FOXP3).

[0031] Regulatory T cells

[0032] The term "regulatory T cells" (Treg) refers to cells that express the markers CD4, CD25, and FOXP3 (CD4 + CD25 + FOXP3 + ) T cells. In the absence of surface protein CD127, cell surface markers CD4 and CD25 can be used to identify Tregs, or cell surface markers CD4 and CD25 can be used in combination with low-level expression of surface protein CD127 to identify Tregs (CD4 + CD25 + CD127 - or CD4 + CD25 + CD127 低 ). Tregs may also express high levels of CTLA-4 (cytotoxic T lymphocyte-associated molecule-4) or GITR (glucocorticoid-induced TNF receptor) on the cell surface. Unlike conventional T cells, Tregs do not produce IL-2 and are therefore inactive at baseline.

[0033] The term "natural Treg" refers to thymus-derived Treg. Natural Treg is CD4 + CD25 + FOXP3 + Helios + Neuropilin 1 + . The term "natural Treg" distinguishes thymus-derived Treg from "induced Treg", which develops from conventional T cells outside the thymus. Natural Treg have higher expression of PD-1 (programmed cell death-1, pdcd1), neuropilin 1 (Nrp1), Helios (Ikzf2), and CD73 compared to induced Treg. Natural Treg can be distinguished from induced Treg based on the expression of Helios protein or neuropilin 1 (Nrp1), respectively.

[0034] As used herein, the term "induced regulatory T cells" (iTreg) refers to CD4+ conventional T cells that mature outside the thymus. + CD25 + FOXP3 + Helios - Neuropilin 1 - For example, iTregs can be induced in vitro from CD4+CD25-FOXP3- cells in the presence of IL-2 and TGF-β.

[0035] Suitably, Tregs express FOXP3 from the cell's endogenous FoxP3 gene.

[0036] Suitably, Tregs may be CD4 + CD25 + FOXP3 + Treg.

[0037] Suitably, Tregs may be CD4 + CD25 + CD127 - Treg.

[0038] Suitably, Tregs may be CD4 + CD25 + CD127 低 Treg.

[0039] Suitably, Tregs may be CD4 + CD25 + CD127 - CD45RA + Treg.

[0040] Suitably, Tregs may be CD4 + CD25 + CD127 低 CD45RA + Treg

[0041] Suitably, Tregs may be CD4 + CD25 + FOXP3 + CD127 - Treg.

[0042] Suitably, Tregs may be CD4 + CD25 + FOXP3 + CD127 低 Treg.

[0043] Suitably, Tregs are CD4 + CD25 + FOXP3 + Helios + Treg.

[0044] Suitably, Tregs are CD4 + CD25 + FOXP3 + Neuropilin 1 + Treg.

[0045] Suitably, Tregs are CD4 + CD25 + FOXP3 + Helios + Neuropilin 1 + Treg.

[0046] Suitably, the Treg is a human Treg. Suitably, the Treg is a human Treg and FOXP3 is human FOXP3.

[0047] Typically, "corresponding Tregs" as used herein have the same or similar phenotype as the Tregs of interest or comparison, but may differ in the genetic modification performed (generally increased FOXP3 levels herein) or the conditions to which the Tregs are exposed (generally pro-inflammatory vs. non-inflammatory conditions herein). The same or similar phenotype means that the Tregs can express the same or similar markers (e.g., at least 80%, 90%, or 95% of their shared marker molecules can have the same or similar amounts, such as at least 70%, 80%, or 90% of the levels).

[0048] In one embodiment, the method of the present invention can be particularly used for Treg or Treg groups with unstable phenotypes to stabilize function, maintain inhibitory effects or prevent the loss of inhibitory function under proinflammatory conditions as described herein. In particular, the expression of exogenous FOXP3 in such unstable Tregs can have a disproportionate positive effect on the maintenance of function as described herein (thereby preventing loss of function). Therefore, in particular, the Treg used in the present invention can express reduced levels of Helios and / or can have more than 50% methylated TSDR, such as more than 60%, 70% or 80% methylated. Demethylated TSDR is associated with endogenous FOXP3 expression, so methylation of TSDR is associated with the reduction of endogenous FOXP3 expression and the deterioration of Treg stability. From another perspective, the unstable or less stable Treg group that may be used herein may be a group in which more than 30%, 40% or 50% of Treg cells in the group may have methylated TSDR. The methylation state of TSDR can be determined by any method known in the art, including bisulfite sequencing.

[0049] Alternatively, Treg or Treg groups with reduced stability or unstable phenotype can be characterized by IL2 expression. Treg with a stable phenotype does not usually express IL2, so Treg expressing any IL2 may indicate that the phenotype is unstable. Therefore, Treg or Treg groups used herein can express IL2, for example, 10%, 20%, 30%, 40% or 50% IL2 can be expressed more than Treg cells with a stable phenotype. Technicians will understand that although Treg groups are considered to have unstable phenotypes or instability (e.g., reduced stability), not all Treg cells in the group can express IL2, although generally, at least 10%, 20%, 30%, 40% or 50% of Treg in Treg groups can express IL2. As described above, the expression of exogenous FOXP3 in such cells can lead to phenotypic stability (e.g., IL2 expression reduction or recovery to cells that do not express IL2) and cells or cell groups that can maintain inhibitory function under proinflammatory conditions.

[0050] Treg or Treg groups used herein can be obtained from patients (typically human patients). Compared with Treg cells that can be separated from healthy donors or subjects, such patient Treg or Treg groups may have a reduced or unstable phenotype. In particular, Treg or Treg groups can be obtained from patients receiving transplantation (such as liver, islet or kidney transplantation), or from patients suffering from disorders (such as autoimmune disorders, such as type I diabetes or IBD) associated with adverse inflammation.

[0051] The term "Tconv cell" means conventional T cell, which refers to T cells other than Treg.

[0052] In one aspect, the Tregs of the present invention can be derived from stem cells. In particular, the Tregs of the present invention can be derived from stem cells in vitro.

[0053] In another aspect, the cell is a progenitor cell.

[0054] As used herein, the term "stem cell" refers to an undifferentiated cell that is capable of indefinitely producing more stem cells of the same type, and can produce other specialized cells by differentiation. Stem cells are multipotent. Stem cells can be, for example, embryonic stem cells or adult stem cells.

[0055] As used herein, the term "progenitor cell" refers to a cell that is capable of differentiating to form one or more types of cells but has limited self-renewal in vitro.

[0056] Suitably, the cell is capable of differentiating into a T cell, such as a Treg.

[0057] Suitably, the cell has the ability to differentiate into a T cell (eg, a Treg) expressing FOXP3.

[0058] Suitably, the cell may be an embryonic stem cell (ESC). Suitably, the cell is a hematopoietic stem cell or hematopoietic progenitor cell. Suitably, the cell is an induced pluripotent stem cell (iPSC). Suitably, the cell may be obtained from umbilical cord blood. Suitably, the cell may be obtained from adult peripheral blood.

[0059] In certain aspects, hematopoietic stem and progenitor cells (HSPCs) can be obtained from umbilical cord blood.Umbilical cord blood 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).

[0060] In one aspect, HSPCs can be obtained from pluripotent stem cell sources, such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs).

[0061] As used herein, the term "hematopoietic stem and progenitor cells" or "HSPC" refers to cells expressing the antigen marker CD34 (CD34+) and populations of such cells. In a specific embodiment, the term "HSPC" refers to cells identified by the presence of the antigen marker CD34 (CD34+) and the absence of lineage (lin) markers. Cell populations comprising CD34+ and / or Lin(-) cells include hematopoietic stem cells and hematopoietic progenitor cells.

[0062] HSPC can be obtained or separated from adult bone marrow (including femur, hip bone, rib, sternum and other bones). A bone marrow aspirate containing HSPC can be obtained or separated directly from the hip bone using a needle and syringe. Other sources of HSPC include cord blood, placental blood, mobilized peripheral blood, Wharton's jelly, placenta, fetal blood, fetal liver or fetal spleen. In a specific embodiment, collecting a sufficient number of HSPC for therapeutic applications may require mobilization of stem cells and progenitor cells in the subject.

[0063] As used herein, the term "induced pluripotent stem cell" or "iPSC" refers to a non-pluripotent cell that has been reprogrammed to a pluripotent state. Once a subject's cells are reprogrammed to a pluripotent state, the cells can be programmed into a desired cell type, such as hematopoietic stem cells or progenitor cells (HSCs and HPCs, respectively).

[0064] As used herein, the term "reprogramming" refers to a process of increasing the potency of a cell to a less differentiated state.

[0065] As used herein, the term "programming" refers to a process of reducing the potency of a cell or differentiating a cell to a more differentiated state.

[0066] Immune response

[0067] The expression "maintaining the ability to suppress immune response" refers to maintaining the suppressive effect of Treg (or the Treg group) on immune response under proinflammatory conditions (e.g., after or during exposure to proinflammatory conditions) at a level similar to that of the corresponding Treg (particularly, Treg (or the Treg group) modified in the same manner or containing the same modification, for example, to express increased FOXP3 levels (particularly FOXP3 from exogenous nucleic acid molecules or polynucleotides)) that is not exposed to proinflammatory conditions (e.g., cultured under non-inflammatory conditions, such as cultured under conditions in the absence of IFNγ, IL1β and / or IL6). "Similar levels" used herein refer to suppressive functions that differ (decreased or increased) by no more than 10%, 20%, 30%, 40% or 50% from the suppressive functions of the corresponding Treg when not exposed to proinflammatory conditions. In particular, similar levels can be the same level. The expression "maintaining the ability to suppress immune response" is used interchangeably herein with the expressions "maintaining functional stability" and "maintaining suppressive function". Maintenance of suppressive function / functional stability / ability to suppress immune responses is typically when Tregs are / have been exposed to or are under pro-inflammatory conditions.

[0068] From another perspective, when under proinflammatory conditions or exposed to proinflammatory conditions, Tregs containing increased FOXP3 levels (e.g., containing exogenous polynucleotides encoding FOXP3) may have an increased ability to suppress immune responses compared to Tregs that do not have increased FOXP3 levels, particularly compared to Tregs that do not contain exogenous polynucleotides encoding FOXP3 (e.g., untransduced Tregs or Tregs transduced with constructs that do not encode FOXP3). As discussed further below, the inventors have demonstrated that cells that do not contain exogenous FOXP3 lose their suppressive ability when exposed to proinflammatory conditions, while Tregs containing exogenous FOXP3 can have suppressive function at a similar level to Tregs cultured under non-inflammatory conditions (e.g., in a medium containing IL2) under proinflammatory conditions. The increased ability of Tregs described herein to suppress immune responses may be increased by at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% compared to Tregs that do not have increased FOXP3 levels.

[0069] According to this alternative embodiment, the present invention also provides a method for increasing the ability of regulatory T cells (Tregs) to suppress immune responses under proinflammatory conditions, compared to Tregs that do not have increased FOXP3 levels (e.g., compared to Tregs that have endogenous FOXP3 levels, or compared to Tregs that have not been modified to have increased FOXP3 levels), comprising introducing a polynucleotide encoding a FOXP3 polypeptide into the Tregs.

[0070] From another point of view, the present invention provides a method for preventing Treg cells from losing their inhibitory function when exposed to proinflammatory conditions, including expressing increased FOXP3 levels in Treg cells (particularly from exogenous nucleic acid molecules containing polynucleotide sequences encoding FOXP3). The method (and the method of the present invention as described herein) may further include a step of incubating Treg cells under proinflammatory conditions, for example, incubating with any of IL-6, IFNγ and / or IL1β. In particular, as described herein, Treg can be engineered to contain nucleic acid molecules expressing FOXP3 and CAR, particularly wherein the nucleotide sequence encoding FOXP3 is located at 5' of the nucleotide sequence encoding CAR. Further, Treg cells can be CD45RA+, particularly CD4+CD25+CD127 low CD45RA+.

[0071] The loss of inhibitory function (also referred to as loss of functional stability) used herein refers to the loss of inhibitory function by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%. Generally, as described above, when Treg cells are not engineered to have increased FOXP3 expression (particularly not engineered to have exogenous nucleic acids encoding FOXP3), the loss of such inhibitory function may occur. Preventing the loss of inhibitory function can cause engineered Treg to have similar or identical inhibitory function (e.g., inhibitory function at least 50%, 60%, 70%, 80%, 90%, 95% or 99% similar) under proinflammatory conditions with the same engineered cells under non-inflammatory conditions.

[0072] "Exogenous" as defined herein means that the intracellular protein of the invention is expressed from a polynucleotide sequence that has been introduced into the cell (ie, not from a polynucleotide sequence originally present in the cell, ie, an endogenous polynucleotide sequence).

[0073] The term "immune response" refers to a series of physiological and cellular effects promoted by the immune system in response to stimuli (such as pathogens or self-antigens). Examples of such effects include increased Tconv cell proliferation and increased cytokine secretion. Any such effect can be used as an indicator of the strength of the immune response. Compared with unmodified Treg, the relatively weak immune response of Tconv in the presence of modified Treg indicates that the modified Treg has a relatively enhanced suppression of the immune response. For example, a relative reduction in cytokine secretion will indicate that the immune response is weaker, so the ability of Treg to suppress the immune response is enhanced. Alternatively, compared with modified Treg that is not exposed to proinflammatory conditions (or only exposed to non-inflammatory conditions), the immune response of Tconv is similar in the presence of modified Treg exposed to proinflammatory conditions or under proinflammatory conditions, indicating that the modified Treg maintains the ability to suppress the immune response. As described above, "similar" as used herein refers to the functional difference (reduction or increase) of Treg corresponding to when not exposed to proinflammatory conditions (or only exposed to non-inflammatory conditions) not exceeding 10%, 20%, 30%, 40% or 50%.

[0074] Assays for measuring immune response strength indicators and thus measuring the suppressive capacity of Tregs are known in the art. In particular, antigen-specific Tconv cells can be co-cultured with Tregs, and peptides of the corresponding antigens are added to the co-culture to stimulate the response of the Tconv cells. The extent of proliferation of Tconv cells in response to the addition of peptides and / or the amount of cytokine IL-2 they secrete can be used as an indicator of the suppressive capacity of co-cultured Tregs.

[0075] After the Tregs are exposed to proinflammatory conditions, the level of proliferation of antigen-specific Tconv cells co-cultured with Tregs having increased FOXP3 expression as described herein can be 5%, 10%, 15%, 20%, 25%, 30%, 35% or 40% lower than the same Tconv cells co-cultured with corresponding Tregs without increased FOXP3 expression; or from another perspective, compared with antigen-specific Tconv cells co-cultured with corresponding Tregs with increased FOXP3 expression that have not been exposed to proinflammatory conditions (or have only been exposed to non-inflammatory conditions), antigen-specific Tconv cells co-cultured with Tregs having increased FOXP3 expression as described herein and exposed to proinflammatory conditions can proliferate at similar levels.

[0076] After the Tregs are exposed to proinflammatory conditions, antigen-specific Tconv cells co-cultured with Tregs with increased FOXP3 expression as described herein can show a reduction in effector cytokines that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or at least 60% higher than the corresponding Tconv cells co-cultured with the corresponding Tregs without increased FOXP3 expression; or from another perspective, compared to the antigen-specific Tconv cells co-cultured with the corresponding Tregs with increased FOXP3 expression that are not exposed to proinflammatory conditions (or are only exposed to non-inflammatory conditions), the antigen-specific Tconv cells co-cultured with Tregs with increased FOXP3 expression and exposed to proinflammatory conditions can show similar levels of effector cytokines.

[0077] After the Tregs are exposed to proinflammatory conditions, antigen-specific Tconv cells co-cultured with Tregs having increased FOXP3 expression as described herein can produce 10%, 20%, 30%, 40%, 50%, 60% or less of the effector cytokines of the corresponding Tconv cells co-cultured with the corresponding Tregs without increased FOXP3 expression; or from another perspective, compared with the antigen-specific Tconv cells co-cultured with the corresponding Tregs with increased FOXP3 expression that are not exposed to proinflammatory conditions (or are only exposed to non-inflammatory conditions), antigen-specific Tconv cells co-cultured with Tregs having increased FOXP3 expression as described herein and exposed to proinflammatory conditions can produce similar levels of effector cytokines.

[0078] 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.

[0079] Suitably, the effector cytokine may be selected from IL-2, IL-17, TNFα, GM-CSF and IFN-γ.

[0080] Antigen-specific Tconv cells co-cultured with the Tregs having increased FOXP3 expression of the present invention can achieve inhibition of IL-2 production at 1 / 2, 1 / 4, 1 / 8, 1 / 10 or 1 / 20 of the number of corresponding Treg cells without increased FOXP3 expression.

[0081] Pro-inflammatory conditions

[0082] As used herein, "proinflammatory conditions" refers to conditions that are capable of inducing, causing, promoting or prolonging inflammation in a subject. Proinflammatory conditions can be identified by the presence of one or more proinflammatory cytokines.

[0083] As described herein, Treg cells are exposed to proinflammatory conditions (generally represented by the presence of one or more proinflammatory cytokines) and can be any time period. Treg exposure to proinflammatory conditions can occur in vitro (for example, by culturing Treg in the presence of one or more proinflammatory cytokines, the proinflammatory cytokines can be exogenously added to the culture or can be produced by cells during culture, particularly during co-culture) or in vivo (for example, when Treg arrives at a proinflammatory environment). One or more proinflammatory cytokines can exist in any amount, but generally exist in an amount that can act on non-transduced Treg (for example, at least 1pg / ml, 5pg / ml, 10pg / ml, 50pg / ml or 100pg / ml). Therefore, the method of the present invention can be in vitro, in vivo (for example, after Treg is administered to a patient (such as a human patient) suffering from inflammation) or can include in vitro and in vivo steps (for example, under proinflammatory conditions or exposed to proinflammatory conditions, the engineering of Treg and / or any culture can occur in vitro (or ex vivo) and / or in vivo).

[0084] As described above, Treg cells are exposed to proinflammatory conditions for any period of time, but are generally for a long period of time, particularly at least 2, 6, 12, 24, 48 or 72 hours, more particularly at least 4, 5, 6 or 7 days. Typically, in vivo, proinflammatory conditions may increase over time, and the level of proinflammatory cytokines may increase over a long period of time. Therefore, particularly in an in vivo environment, if Treg exists for a long period of time at the site where proinflammatory cytokines are present, for example, more than 1, 2, 3, 4, 5, 6 or 7 days, it can be considered that Treg is exposed to proinflammatory conditions. As described herein, Treg (or Treg population) can be exposed to proinflammatory conditions after (e.g., at least 1, 2, 6, 12 or 24 hours later) or during (e.g., when Treg is under or exposed to proinflammatory conditions) the maintenance of the inhibitory effect is shown. "When exposed" to proinflammatory conditions includes previous exposure and current exposure.

[0085] Proinflammatory cytokines are molecules that may be secreted by immune cells (e.g., helper T cells and / or macrophages) and may 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 1, 2, or 3 proinflammatory cytokines), more particularly, any one or more of IL1β, IL6, and TNFα, for example, sequences having SEQ ID NO.8, SEQ ID NO.9, or SEQ ID NO.10, respectively, or functional variants thereof, which typically have at least 80%, 90%, or 95% sequence identity thereto.

[0086] IL1β (SEQ ID NO.8)

[0087]

[0088] IL6 (SEQ ID NO.9)

[0089]

[0090] TNFα (SEQ ID NO.10)

[0091]

[0092] Typically, Tregs exposed to pro-inflammatory conditions as defined herein are exposed to any one or more of IL1B, TNFα and IL6 for at least 3 days, in particular to all of IL1B, TNFα and IL6 for at least 3 days.

[0093] Tregs not exposed to proinflammatory conditions may not be cultured in vitro or exposed to one or more proinflammatory cytokines in vivo for a long period of time, for example, more than 2, 6, 12, 24, 48 or 72 hours. The Tregs may be referred to as being exposed only to non-inflammatory conditions. Typically, Tregs not exposed to proinflammatory conditions or only exposed to non-inflammatory conditions will not be incubated or cultured with proinflammatory cytokines, for example, incubated or cultured with any one or more of IL6, TNFα and / or IL1B (typically incubated or cultured with any one of IL6, TNFα and IL1B).

[0094] FOXP3

[0095] "FOXP3" is the abbreviation for forkhead box P3 protein. FOXP3 is a member of the FOX protein family of transcription factors and plays a role as a master regulator of regulatory pathways in the development and function of regulatory T cells.

[0096] "Increasing FOXP3 expression" refers to increasing the level of FOXP3 mRNA and / or protein in Treg (or the Treg group) compared to the unmodified corresponding Treg (or the Treg group). For example, the level of FOXP3 mRNA and / or protein in the Treg (or the Treg group) modified as described herein can be increased to at least 1.5 times, at least 2 times, at least 5 times, at least 10 times, at least 50 times, at least 100 times, at least 150 times higher than the level in the unmodified corresponding Treg (or the Treg group).

[0097] Suitably, the level of FOXP3 mRNA and / or protein in a Treg (or population of such Tregs) modified as described herein may be increased to at least 1.5 times higher than the level in a corresponding Treg (or population of such Tregs) not modified as described herein.

[0098] Suitably, the level of FOXP3 mRNA and / or protein in a Treg (or such a population of Tregs) modified as described herein may be increased to at least 2-fold higher than the level in a corresponding Treg (or such a population of Tregs) not modified as described herein.

[0099] Suitably, the level of FOXP3 mRNA and / or protein in a Treg (or such a population of Tregs) modified as described herein may be increased to at least 5-fold higher than the level in a corresponding Treg (or such a population of Tregs) not modified as described herein.

[0100] Techniques for measuring specific mRNA and protein levels are well known in the art. The mRNA levels in a cell (such as Treg) group can be measured by techniques such as Affymetrix ebioscience prime flow RNA assay, Northern blot, serial analysis of gene expression (SAGE) or quantitative polymerase chain reaction (qPCR). The protein levels in a cell group can be measured by techniques such as flow cytometry, high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC / MS), Western blot or enzyme-linked immunosorbent assay (ELISA).

[0101] In some embodiments of the present invention, FOXP3 expression is increased by introducing a polynucleotide encoding a FOXP3 polypeptide into isolated Tregs.

[0102] The term "introduction" refers to a method for inserting foreign DNA into cells, including transfection and transduction methods. Transfection is the process of introducing nucleic acid into cells by non-viral methods. Transduction is the process of introducing foreign DNA into cells by viral vectors. In particular, the introduction is performed before Tregs are exposed to pro-inflammatory conditions.

[0103] "FOXP3 polypeptide" is a polypeptide with FOXP3 activity, i.e., a polypeptide that can bind to FOXP3 target DNA and act as a transcription factor that regulates Treg development and function. The 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 by the gene using techniques such as Northern blotting, SAGE, qPCR, HPLC, LC / MS, Western blotting or ELISA. Genes regulated by FOXP3 include cytokines such as IL-2, IL-4 and IFN-γ (Siegler et al., Annu. Rev. Immunol. 2006, 24: 209-26, incorporated herein by reference).

[0104] Polynucleotides and Peptides

[0105] 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.

[0106] The term "polypeptide" is synonymous with "protein" and refers to a series of residues, usually L-amino acids, connected to each other typically by peptide bonds between the α-amino and carboxyl groups of adjacent amino acids.

[0107] Due to the degeneracy of the genetic code, a variety of different polynucleotides can encode the same polypeptide. A skilled person can make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotide to reflect the codon usage of any specific host organism in which the polypeptide will be expressed.

[0108] Polynucleotides can comprise DNA or RNA, can be single-stranded or double-stranded, and can include synthetic or modified nucleotides. A variety of different types of oligonucleotide modifications are known in the art. These include methylphosphonate and phosphorothioate backbones, adding acridine or polylysine chains at the 3' and / or 5' ends of the molecule. Polynucleotides can be modified by any method in the art. Such modifications can enhance the in vivo activity or lifespan of the polynucleotides.

[0109] The polynucleotide may be in isolated or recombinant form. It may be incorporated into a vector, which may be incorporated into a host cell.

[0110] The polynucleotides can be codon optimized. Different cells use different specific codons. This codon preference corresponds to the relative abundance preference of a specific tRNA in a cell type. By changing the codons in the sequence to match the relative abundance of the corresponding tRNA, expression can be increased. Suitably, the polynucleotides can be codon optimized to be expressed in a mouse disease model. Suitably, the polynucleotides can be codon optimized to be expressed in a human subject.

[0111] Many viruses, including HIV and other lentiviruses, use a large number of rare codons, and by changing these codons to correspond to commonly used mammalian codons, increased expression of packaging components in mammalian production cells can be achieved. Codon usage tables for mammalian cells and various other organisms are known in the art. Codon optimization can also involve the removal of mRNA instability motifs and cryptic splice sites.

[0112] FOXP3 polypeptide sequence

[0113] Suitably, the FOXP3 polypeptide may comprise the polypeptide sequence of human FOXP3, such as UniProtKB accession number Q9BZS1, or a functional fragment thereof:

[0114] MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKH CQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLH NMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGP(SEQ ID NO:3)

[0115] In some embodiments of the invention, the FOXP3 polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 3 or a functional fragment thereof. Suitably, the FOXP3 polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 98% or 99% identical 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.

[0116] Suitably, the FOXP3 polypeptide may be a variant, such as a natural variant, of SEQ ID NO: 3. Suitably, the FOXP3 polypeptide is an isoform of SEQ ID NO: 3. For example, the FOXP3 polypeptide may comprise a deletion of amino acids 72-106 relative to SEQ ID NO: 3. Alternatively, the FOXP3 polypeptide may comprise a deletion of amino acids 246-272 relative to SEQ ID NO: 3.

[0117] Suitably, the FOXP3 polypeptide comprises SEQ ID NO:4 or a functional fragment thereof:

[0118] MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADH LLDEKGRAQCLLQREMVQSLEQVEELSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRP PFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGPEGRGSLLTCGDVEEN(SEQ IDNO:4).

[0119] Suitably, the FOXP3 polypeptide comprises an amino acid sequence at least 80% identical to SEQ ID NO: 4 or a functional fragment thereof Suitably, the polypeptide comprises an amino acid sequence 85%, 90%, 95%, 98% or 99% identical to SEQ ID NO: 4 or a functional fragment thereof.

[0120] Suitably, the FOXP3 polypeptide may be a variant, such as a natural variant, of SEQ ID NO: 4. 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 72-106 relative to SEQ ID NO: 4. Alternatively, the FOXP3 polypeptide may comprise a deletion of amino acids 246-272 relative to SEQ ID NO: 4.

[0121] FOXP3 polynucleotide sequence

[0122] Suitably, the FOXP3 polypeptide is encoded by the polynucleotide sequence shown in SEQ ID NO: 1:

[0123]

[0124] In some embodiments of the present invention, the polynucleotide encoding the FOXP3 polypeptide or variant comprises a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 1 or a functional fragment thereof. Suitably, the polynucleotide encoding the FOXP3 polypeptide or variant comprises a polynucleotide sequence that is at least 85%, 90%, 95%, 98% or 99% identical to SEQ ID NO: 1 or a functional fragment thereof. In some embodiments of the present invention, the polynucleotide encoding the FOXP3 polypeptide or variant comprises SEQ ID NO: 1 or a functional fragment thereof.

[0125] Suitably, the FOXP3 polypeptide is encoded by the polynucleotide sequence shown in SEQ ID NO:2:

[0126]

[0127] In some embodiments of the present invention, the polynucleotide encoding the FOXP3 polypeptide or variant comprises a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 2 or a functional fragment thereof. Suitably, the polynucleotide encoding the FOXP3 polypeptide or variant comprises a polynucleotide sequence that is at least 85%, 90%, 95%, 98% or 99% identical to SEQ ID NO: 2 or a functional fragment thereof. In some embodiments of the present invention, the polynucleotide encoding the FOXP3 polypeptide or variant comprises SEQ ID NO: 2 or a functional fragment thereof.

[0128] Suitably, the polynucleotide encoding the FOXP3 polypeptide or variant thereof may be codon-optimized. Suitably, the polynucleotide encoding the FOXP3 polypeptide or variant thereof may be codon-optimized for expression in human cells.

[0129] Sequence comparison

[0130] Sequence comparisons can be performed by eye, or more usually with the aid of readily available sequence comparison programs. These publicly and commercially available computer programs can calculate the sequence identity between two or more sequences.

[0131] Sequence identity can be calculated over contiguous sequences, i.e., one sequence is aligned with the other sequence and each amino acid in one sequence is compared directly to the corresponding amino acid in the other sequence, one residue at a time. This is called a "non-gap" alignment. Typically, such non-gap alignments are performed only over a relatively short number of residues (e.g., less than 50 contiguous amino acids).

[0132] While this is a very simple and consistent approach, it does not take into account that, for example, in a pair of otherwise identical sequences, one insertion or deletion will cause the following amino acid residues to be misplaced and therefore may result in a large reduction in % homology when a global alignment is performed. Therefore, most sequence comparison methods are designed to produce an optimal alignment that takes into account possible insertions and deletions without overly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment in an attempt to maximize local homology.

[0133] In some embodiments, the present invention relates to the method for comparing the sequence of amino acids in the present invention. The method comprises the following steps: (1) the alignment of amino acids in the present invention and the alignment of amino acids in the present invention. The method comprises the following steps: (1) the alignment of amino acids in the present invention and the alignment of amino acids in the present invention. The method comprises the following steps: (1) the alignment of amino acids in the present invention and the alignment of amino acids in the present invention. The method comprises the following steps: (2) the alignment of amino acids in the present invention and the alignment of amino acids in the present invention. The method comprises the following steps: (1) the alignment of amino acids in the present invention and the alignment of amino acids in the present invention. The method comprises the following steps: (1) the alignment of amino acids in the present invention and the alignment of amino acids in the present invention. The method comprises the following steps: (2) the alignment of amino acids in the present invention and the alignment of amino acids in the present invention. The method comprises the following steps: (1) the alignment of amino acids in the present invention and the alignment of amino acids in the present invention.

[0134] Therefore, calculating maximum % sequence identity first needs to generate optimal comparison, and consider gap penalty.The suitable computer program for performing such comparison is GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux etc., 1984, Nucleic Acids Research 12:387, incorporated herein by reference).The example of other software that can carry out sequence comparison includes but is not limited to BLAST package (referring to Ausubel etc., 1999, the same-the 18th chapter), FASTA (Atschul etc., 1990, J.Mol.Biol., 403-410, incorporated herein by reference) and GENEWORKS comparison tool suite.BLAST and FASTA can be used for offline and online search (referring to Ausubel etc., 1999, the same, 7-58 to 7-60 pages, incorporated herein by reference).However, preferably use GCG Bestfit program.

[0135] Suitably, sequence identity may be determined over the entire sequence.Suitably, sequence identity may be determined over the entire candidate sequence compared to a sequence described herein.

[0136] Although the final sequence identity can be measured in terms of identity, the alignment process itself is not usually based on an all-or-nothing pairwise comparison. Instead, a scaled similarity score matrix is ​​usually used, which assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. A common example of such a matrix is ​​the BLOSUM62 matrix (the default matrix for the BLAST suite of programs). The GCG Wisconsin program typically uses public default values ​​or a custom symbol comparison table (if provided) (see the user manual for more details). Preferably, the public default values ​​of the GCG package are used, or in the case of other software, the default matrix, such as BLOSUM62, is used.

[0137] Once the software has produced an optimal alignment, the % sequence identity can be calculated. The software typically performs this as part of the sequence comparison and generates a numerical result.

[0138] Carrier

[0139] In some embodiments of the present invention, the polynucleotide encoding FOXP3 is a continuous part of an expression vector.

[0140] The term "expression vector" refers to a construct capable of expressing a FOXP3 polypeptide. Suitably, the expression vector is a cloning vector.

[0141] Suitable vectors may include, but are not limited to, plasmids, viral vectors, transposons, or nucleic acids complexed with the polypeptide or immobilized on solid particles.

[0142] Preferably, the expression vector is capable of sustained high-level expression in host cells.

[0143] The expression vector may be a retroviral vector. The expression vector may be based on or derivable from an MP71 vector backbone. The expression vector may lack a full-length or truncated version of the woodchuck hepatitis response element (WPRE).

[0144] In some embodiments of the invention, the vector also encodes a T cell receptor (TCR).

[0145] A TCR is a cell surface molecule that binds to a fragment of an antigen bound by a major histocompatibility complex (MHC) molecule on an antigen presenting cell as part of directing an immune response. Suitably, the TCR may be a recombinant protein, in other words, the TCR may be an exogenous protein that is not naturally expressed by the Treg of the invention.

[0146] In some embodiments of the invention, the vector further encodes a chimeric antigen receptor (CAR).

[0147] CAR is a recombinant cell surface molecule expressed by engineered T cells, which binds to antigens expressed on other cell surfaces as part of directing immune responses. More specifically, CAR is a protein that transplants the specificity of an antigen binding agent (e.g., a monoclonal antibody (mAb)) to the effector function of T cells. Their common form is a type I transmembrane domain protein with an antigen recognition amino terminal, a spacer, a transmembrane domain, all of which are connected to a composite internal domain that transmits T cell survival and activation signals.

[0148] Wherein, in addition to the polynucleotide encoding FOXP3, the vector also comprises a polynucleotide encoding TCR or CAR (e.g., anti-HLA-A2 CAR); the vector may have the following orientation: 5'FOXP3-TCR / CAR 3'. Therefore, the polynucleotide encoding FOXP3 may be 5' of the polynucleotide encoding CAR or TCR.

[0149] Suitably, the polynucleotide encoding FOXP3 may be separated from the polynucleotide encoding TCR or CAR by a nucleic acid sequence that enables both the nucleic acid sequence encoding FOXP3 and the nucleic acid sequence encoding TCR or CAR to be expressed from the same mRNA transcript.

[0150] For example, the polynucleotide may comprise an internal ribosome entry site (IRES) between the nucleic acid sequences encoding (i) FOXP3 and (ii) TCR or CAR. IRES is a nucleotide sequence that allows translation to be initiated in the middle of an mRNA sequence.

[0151] The polynucleotide may comprise nucleic acid sequences encoding (i) FOXP3 and (ii) TCR or CAR connected by an internal self-cleavage sequence.

[0152] Suitably, the vector may have the following structure: 5' strong promoter (eg, LTR) -FoxP3-2A-CAR / TCR-3'LTR. Here, FOXP3 expression is driven directly by a strong LTR promoter to achieve optimal expression. There is a 2A sequence in front of CAR / TCR, so the expression of CAR / TCR depends on LTR promoter activity and 2A cleavage activity. Importantly, the configuration of FOXP3 in front of CAR / TCR in the 5' to 3' direction ensures that CAR / TCR expression only occurs when FOXP3 is expressed, and CAR / TCR expression without FOXP3 does not occur. This is a special advantage of the engineered Treg herein because it reduces the risk of engineered Treg acquiring an effector phenotype and / or reduces the risk of introducing CAR or TCR into T effector cells present in the starting population.

[0153] The internal self-cleavage sequence may be any sequence capable of separating the polypeptides comprising (i) FOXP3 and (ii) TCR or CAR.

[0154] The cleavage site may be self-cleaving, such that when the polypeptide is produced it is immediately cleaved into individual peptides without the need for any external cleavage activity.

[0155] The term "cleavage" is used herein for convenience, but the cleavage site can separate the peptide into individual entities by mechanisms other than traditional cleavage. For example, for the foot-and-mouth disease virus (FMDV) 2A self-cleavage peptide, various models have been proposed to explain the "cleavage" activity: proteolysis by host cell proteases, autoproteolysis, or translation effects (Donnelly et al. (2001) J. Gen. Virol. 82: 1027-1041, incorporated herein by reference). The exact mechanism of this "cleavage" is not important for the purposes of the present invention, as long as the cleavage site, when located between nucleic acid sequences encoding the protein, results in the expression of the protein as a separate entity.

[0156] The self-cleaving peptide may be a 2A self-cleaving peptide from foot-and-mouth disease virus or cardiovirus.

[0157] Variants may be considered based on similarity (ie, amino acid residues having similar chemical properties / functions), preferably variants are represented based on sequence identity.

[0158] Sequence comparisons can be performed by eye, or more usually with the aid of readily available sequence comparison programs. These publicly and commercially available computer programs can calculate the sequence identity between two or more sequences.

[0159] Suitably, the FOXP3 polypeptide expressed from the vector of the invention may be located at the N-terminus of a self-cleaving peptide (e.g., a 2A self-cleaving peptide). Such a FOXP3-2A polypeptide may comprise a sequence as shown in SEQ ID NO: 5 or 6; or a variant that is at least 80% identical to SEQ ID NO: 5 or 6. Suitably, the variant may be at least 85%, 90%, 95%, 98% or 99% identical to SEQ ID NO: 5 or 6.

[0160] SEQ ID NO:5

[0161] MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRLWGSHGNSTFPPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGPGATNFSLLKQAGDVEENPGPS

[0162] SEQ ID NO:6

[0163] MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQVEELSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGPEGRGSLTCGDVEENGATNFSLLKQAGDVEENPGPS

[0164] Viral transduction

[0165] In some embodiments of the present invention, the polynucleotide encoding FOXP3 is introduced into the isolated Tregs by viral transduction.

[0166] Viral delivery systems include, but are not limited to, adenoviral vectors, adeno-associated viral (AAV) vectors, herpes viral vectors, retroviral vectors, lentiviral vectors, baculoviral vectors.

[0167] In some embodiments, the polynucleotide encoding FOXP3 is introduced into the isolated Tregs by retroviral transduction.

[0168] Retroviruses are RNA viruses that have a different life cycle than lytic viruses. In this regard, a retrovirus is an infectious entity that replicates via a DNA intermediate. When a retrovirus infects a cell, its genome is converted into a DNA form by the enzyme reverse transcriptase. The DNA copy serves as a template for the production of a new RNA genome and virally encoded proteins necessary for the assembly of infectious viral particles.

[0169] There are many types of retroviruses, 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 myelocytoma virus-29 (MC29) and avian erythroblastosis virus (AEV) as well as all other retroviruses including lentiviruses.

[0170] A detailed list of retroviruses can be found 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).

[0171] Lentiviruses also belong to the Retroviridae family, but they can infect both dividing and non-dividing cells (Lewis et al., 1992 EMBO J. 3053-3058, incorporated herein by reference).

[0172] To efficiently infect human cells, viral particles can be packaged with an amphotropic envelope or the Gibbon Ape Leukemia Virus envelope.

[0173] Isolation of Tregs

[0174] In some embodiments, the method according to the present invention comprises:

[0175] (a) isolating Tregs from a cell population; and

[0176] (b) increasing the expression of FOXP3 in the Tregs.

[0177] Suitably, exogenous FOXP3 expression in Tregs is increased by introducing an exogenous polynucleotide encoding a FOXP3 polypeptide.

[0178] The expression "isolating Tregs from a cell population" means isolating Tregs from a heterogeneous mixture of multiple different types of cells. Suitably, the cell population is from a sample from a human subject.

[0179] Suitably, Tregs are isolated as Treg populations.

[0180] Suitably, the Treg population comprises at least 70% Tregs, such as 75%, 85%, 90% or 95% Tregs.

[0181] In some embodiments of the invention, the cell population comprises or consists of peripheral blood mononuclear cells (PBMCs).

[0182] PBMC is any blood cell with a round nucleus found in the blood circulation pool, rather than being sequestered in the bone marrow, liver, spleen or lymphatic system. PBMC consists of monocytes and lymphocytes (T cells, B cells and NK cells). Techniques for separating PBMC from whole blood are known in the art. For example, PBMC 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 are separated into different layers, including a layer containing PBMC.

[0183] In some embodiments of the present invention, isolating Tregs comprises isolating CD4 + In some embodiments, isolating Tregs comprises isolating CD4 + T cells, subsequently from CD4 + Isolation of Tregs from T cells.

[0184] CD4 (Cluster of Differentiation 4) is a co-receptor for the T cell receptor expressed by various types of T cells. + The separation of cells separates T cells, including Treg cells, from the initial cell population. Tregs can then be isolated from this T cell enriched population.

[0185] Techniques for isolating specific cell types from a heterogeneous cell population are known in the art. Examples include the use of immunomagnetic beads and fluorescence activated cell sorting.

[0186] In some embodiments of the invention, separation of Treg populations includes the use of immunomagnetic beads. Various companies (e.g., Miltenyi Biotec, Stem Cell Technologies, ThermoFisher Scientific) provide kits containing immunomagnetic beads for separating specific types of T cells (see, e.g., Fallarino et al. (2003) Modulation of tryptophan catabolism by regulatory T cells. Nat. Immunol. 4: 1206-1212, incorporated herein by reference). These separation kits utilize antibodies against T cell surface proteins (e.g., CD8, CD25, CD49b, etc.) widely available in the art. For example, the cell population can be firstly separated from the Treg population by a PCR reaction with antibodies against non-CD4 + CD4 + The cells are negatively selected. Tregs can then be positively selected by incubating with anti-CD25 labeled beads.

[0187] In some embodiments of the invention, isolating a Treg population comprises fluorescence activated cell sorting (FACS). In some embodiments, Tregs are sorted based on their CD4 + CD25 高 CD127 - Phenotype sorting.

[0188] Natural Tregs can be sorted from induced Tregs based on the expression of Helios protein or neuropilin 1. In some embodiments of the present invention, natural Tregs can be sorted based on their CD4 + CD25 + FOXP3 + Helios + Neuropilin 1 + Natural Tregs were sorted by phenotype.

[0189] FACS is a kind of flow cytometry known in the art. During FACS, cells are suspended in a liquid and flow through a detection system that analyzes various characteristics. The method can be used to sort cells according to the characteristics of the cells. In particular, in FACS, fluorescent antibodies are used to label molecules, and cells are sorted according to their fluorescence levels, which indicate the expression level of specific molecules (see Adan et al. Flow cytometry: basic principles and applications, Crit. Rev. Biotechnol. March 2017; 37 (2): 163-176, incorporated herein by reference).

[0190] Engineered Treg

[0191] The method of the present invention provides a Treg having higher expression of FOXP3 compared to corresponding non-engineered Treg, which is able to maintain its suppressive function when exposed to pro-inflammatory conditions.

[0192] "Higher FOXP3 expression" means that the level of FOXP3 mRNA or protein in the engineered Treg is higher than that before the Treg gene expression is altered by artificial intervention.

[0193] "Higher FOXP3 expression" can be defined and determined as described herein.

[0194] Suitably, the level of CD25 mRNA and / or protein in a Treg (or such a population of Tregs) modified as described herein may be increased to at least 1.5 times higher than the level in a corresponding Treg (or such a population of Tregs) not modified as described herein.

[0195] Suitably, the level of CD25 mRNA and / or protein in a Treg (or such a Treg population) modified as described herein may be increased to at least 2-fold higher than the level in a corresponding Treg (or such a Treg population) not modified as described herein.

[0196] Suitably, the level of CD25 mRNA and / or protein in a Treg (or such a Treg population) modified as described herein may be increased to at least 5-fold higher than the level in a corresponding Treg (or such a Treg population) not modified as described herein.

[0197] Suitably, the level of CTLA-4 mRNA and / or protein in a Treg (or such a Treg population) modified as described herein may be increased to at least 1.5 times higher than the level in a corresponding Treg (or such a Treg population) not modified as described herein.

[0198] Suitably, the level of CTLA-4 mRNA and / or protein in a Treg (or such a population of Tregs) modified as described herein may be increased to at least 2-fold higher than the level in a corresponding Treg (or such a population of Tregs) not modified as described herein.

[0199] Suitably, the level of CTLA-4 mRNA and / or protein in a Treg (or such a population of Tregs) modified as described herein may be increased to at least 5-fold higher than the level in a corresponding Treg (or such a population of Tregs) not modified as described herein.

[0200] In some embodiments of the present invention, the engineered Treg comprises an exogenous polynucleotide encoding a FOXP3 polypeptide.

[0201] An "exogenous polynucleotide" is a polynucleotide derived from outside of a Treg. An exogenous polynucleotide can be introduced into a Treg as part of an expression vector. Thus, an exogenous polynucleotide can be linked to an expression vector element (eg, a promoter).

[0202] In some embodiments of the present invention, the FOXP3 polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 3 or 4 or a functional fragment thereof. Suitably, the FOXP3 polypeptide comprises an amino acid sequence that is at least 85%, 90%, 95%, 98% or 99% identical to SEQ ID NO: 3 or 4 or a functional fragment thereof. In some embodiments, the FOXP3 polypeptide comprises SEQ ID NO: 3 or 4 or a functional fragment thereof.

[0203] In some embodiments of the present invention, the exogenous polynucleotide encoding FOXP3 comprises a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 1 or 2. In some embodiments of the present invention, the exogenous polynucleotide encoding FOXP3 is identical to SEQ ID NO: 1 or 2.

[0204] In some embodiments of the present invention, the exogenous polynucleotide encoding FOXP3 is a continuous part of the vector.

[0205] In some embodiments of the present invention, the vector also encodes a T cell receptor (TCR) or CAR, particularly an anti-HLA A2 CAR.

[0206] In some embodiments of the invention, the vector comprises a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 5. In some embodiments of the invention, the vector comprises a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 5.

[0207] Thus, the present invention provides an engineered Treg expressing exogenous FOXP3 obtainable or obtained by the method of the present invention, wherein the Treg can maintain its suppressive function when exposed to pro-inflammatory conditions.

[0208] Engineered Tregs can be obtained by methods including:

[0209] (i) isolating Tregs from the cell population; and

[0210] (ii) introducing a polynucleotide encoding an exogenous FOXP3 polypeptide into the isolated Treg to maintain the ability of Treg to suppress immune response under pro-inflammatory conditions.

[0211] Tregs can be CD4+CD25+CD127- / low CD45RA+ Tregs.

[0212] In addition, the present invention provides a method for preparing an engineered Treg as disclosed herein, comprising the step of introducing one or more polynucleotide sequences encoding exogenous FOXP3, CAR or exogenous TCR into T regulatory cells or multipotent or pluripotent cells, wherein when one or more polynucleotide sequences are introduced into multipotent or pluripotent cells, the method comprises a subsequent step of differentiating the cells into T regulatory cells.

[0213] T regulatory cells can be isolated from a subject, in particular by blood separation and subsequent enrichment; or T regulatory cells can be obtained by differentiation of pluripotent cells, in particular iPSCs.

[0214] Composition

[0215] The present invention also provides a pharmaceutical composition comprising the engineered Tregs obtainable or obtained by the method of the present invention.

[0216] The pharmaceutical composition may include a pharmaceutically acceptable carrier, diluent, excipient or adjuvant. Pharmaceutical carriers, excipients or diluents may be selected according to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition may include any suitable one or more adhesives, one or more lubricants, one or more suspending agents, one or more coating agents, one or more solubilizing agents and other carriers as carriers, excipients or diluents (or except carriers, excipients or diluents).

[0217] Pharmaceutical compositions should generally be sterile and stable under manufacturing and storage conditions. Preparations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous carriers, pastes, and implantable sustained release or biodegradable preparations. Sterile injection preparations can be prepared using nontoxic parenteral acceptable diluents or solvents. Pharmaceutical compositions of the present invention can include pharmaceutically acceptable dispersants, wetting agents, suspending agents, isotonic agents, coatings, antibacterial and antifungal agents, carriers, excipients, salts or stabilizers, which are nontoxic to the subject at the dosage and concentration employed. Preferably, this composition can further include a pharmaceutically acceptable carrier or excipient for the treatment of a disease, which is compatible with a given method of administration and / or site of administration, for example, for parenteral (e.g., subcutaneous, intradermal or intravenous injection) or intrathecal administration.

[0218] The compositions can be manufactured using current good manufacturing practices (cGMP).

[0219] Suitably, the pharmaceutical composition comprising engineered Tregs may comprise an organic solvent such as, but not limited to, methyl acetate, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), dimethoxyethane (DME) and dimethylacetamide, including mixtures or combinations thereof.

[0220] Suitably, the pharmaceutical composition is free of endotoxin.

[0221] Suitably, the present invention provides a pharmaceutical composition comprising engineered T cells (Tregs) having the ability to suppress immune responses under pro-inflammatory conditions, the engineered T cells being obtained by a process comprising:

[0222] (ii) isolating Tregs from the cell population; and

[0223] (ii) introducing a polynucleotide encoding an exogenous FOXP3 polypeptide into the isolated Treg to maintain the ability of Treg to suppress immune response under pro-inflammatory conditions.

[0224] Tregs can be CD4+CD25+CD127- / low CD45RA+ Tregs.

[0225] Prevent and / or treat disease

[0226] The present invention also provides engineered Tregs obtainable or obtained by the method of the present invention, or a pharmaceutical composition of the present invention, for use in preventing and / or treating a disease.

[0227] The present invention also provides use of the engineered Treg obtainable or obtained by the method of the present invention in the manufacture of a medicament for preventing and / or treating a disease.

[0228] The present invention also provides a method for preventing and / or treating a disease, comprising administering the engineered Treg or the composition of the present invention to a subject.

[0229] Preferably, the method for preventing and / or treating a disease comprises administering the pharmaceutical composition of the present invention to a subject.

[0230] The term "treatment" refers to administering the engineered Tregs or pharmaceutical compositions of the present invention to a subject with an existing disease or condition to alleviate, reduce or improve at least one symptom associated with the disease and / or slow, reduce or prevent the progression of the disease.

[0231] "Prevention" (or prophylaxis) means delaying or preventing the onset of disease symptoms. Prevention may be absolute (so that the disease never occurs) or may be effective only in certain people or for a limited time.

[0232] 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.

[0233] Administration of the pharmaceutical compositions of the invention can be accomplished using any of a variety of routes that make the active ingredients bioavailable. For example, engineered Tregs or pharmaceutical compositions can be administered intravenously, intrathecally, by oral and parenteral routes, intranasally, intraperitoneally, subcutaneously, transdermally, or intramuscularly.

[0234] Suitably, the engineered Treg or pharmaceutical composition of the invention is administered intravenously. Suitably, the engineered Treg or pharmaceutical composition of the invention is administered intrathecally.

[0235] Typically, a physician will determine the dosage most suitable for an individual subject, and the dosage will vary with the age, weight and response of the particular subject. The dosage is sufficient to reduce and / or prevent symptoms of the disease.

[0236] For example, it will be appreciated by those skilled in the art that the route of delivery (e.g., oral vs. intravenous vs. subcutaneous, etc.) may affect the desired dose (and vice versa). For example, if a particularly high concentration of an agent needs to be achieved within a particular site or location, then concentrated delivery may be preferred. Other factors to be considered when optimizing the route and / or dosing schedule for a given treatment regimen may 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 a combination therapy, and other factors known to medical practitioners.

[0237] The dosage is sufficient to stabilize or ameliorate symptoms of the disease.

[0238] The present invention also provides a method for treating and / or preventing a disease, comprising the step of administering a pharmaceutical composition comprising the cells (eg, T cells) according to the present invention to a subject.

[0239] Suitably, the method of preventing and / or treating a disease may comprise:

[0240] (i) isolating Tregs from a subject;

[0241] (ii) introducing (ie, engineering) a polynucleotide sequence encoding a FOXP3 polypeptide into said Treg; and

[0242] (iii) administering the engineered Tregs to a subject.

[0243] Tregs can be isolated from a patient by collecting a blood sample and isolating Tregs from the blood sample using techniques known in the art, such as those described under the heading "Isolating Tregs" in this specification.

[0244] A polynucleotide encoding a FOXP3 polypeptide can be introduced into Tregs using techniques known in the art (eg, those described under the heading "viral transduction" in this specification).

[0245] Suitably, the engineered Tregs may be expanded in vitro prior to administration to a subject. Tregs are cultured in culture medium to expand Tregs in vitro.

[0246] disease

[0247] 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.

[0248] The disease can be, for example, cancer, an infectious disease, or an autoimmune disease.

[0249] In some embodiments of the invention, the disease is an autoimmune disease.

[0250] The disease may be a systemic autoimmune and inflammatory disease associated with the central nervous system (CNS), such as Behcet's disease, sarcoidosis, systemic lupus erythematosus, juvenile idiopathic arthritis, scleroderma and Sjögren's syndrome.

[0251] The disease may be any disease in which MBP is an antigen, for example, MBP is a self-antigen.

[0252] Suitably, the disease may be an autoimmune disease and an inflammatory central nervous system disease or a peripheral nervous system disease (eg a chronic neurodegenerative disease).

[0253] Suitably, the disease may be a chronic neurodegenerative disease such as multiple sclerosis (MS), Alzheimer's disease, Parkinson's disease, neurotropic viral infection, stroke, paraneoplastic disease and traumatic brain injury.

[0254] The present invention further provides a method for inducing transplant tolerance, treating and / or preventing cellular and / or humoral transplant rejection, treating and / or preventing graft-versus-host disease (GvHD), comprising the step of administering the engineered Treg or pharmaceutical composition of the present invention to a subject.

[0255] As used herein, "inducing transplant tolerance" refers to inducing tolerance to a transplanted organ in a recipient. In other words, inducing transplant tolerance refers to reducing the level of the recipient's immune response to the donor transplanted organ. Inducing transplant tolerance can reduce the amount of immunosuppressive drugs required by the transplant recipient, or can allow immunosuppressive drugs to be discontinued.

[0256] In one embodiment, the subject is a transplant recipient undergoing immunosuppressive therapy.

[0257] The transplant may be selected from the group consisting of liver, kidney, heart, lung, pancreas, intestine, stomach, bone marrow, vascularized composite tissue transplants and skin transplants.

[0258] use

[0259] The present invention further provides the use of polynucleotides encoding FOXP3 polypeptides in maintaining the ability of regulatory T cells (Tregs) to suppress immune responses under proinflammatory conditions. As previously described, the maintenance of the suppressive function of Tregs of the present invention that have been or are being exposed to proinflammatory conditions is provided by increasing FOXP3 expression by introducing a polynucleotide encoding a FOXP3 polypeptide into Tregs, wherein the maintenance of the suppressive function is compared to a corresponding Treg that contains a polynucleotide encoding a FOXP3 polypeptide but has not been or is not being exposed to proinflammatory conditions (i.e., only exposed to non-inflammatory conditions). Therefore, FOXP3 encoding polynucleotides are used to maintain the suppressive function of Tregs under proinflammatory conditions.

[0260] From another perspective, the present invention provides the use of a polynucleotide encoding a FOXP3 polypeptide to increase the ability of regulatory T cells (Treg) to suppress immune responses under proinflammatory conditions compared to Tregs that do not contain exogenous FOXP3 polypeptide encoding polynucleotides.

[0261] From another perspective, the present invention provides the use of a polynucleotide encoding a FOXP3 polypeptide to prevent Treg cells from losing their suppressive function under pro-inflammatory conditions.

[0262] In addition, the present invention may provide a method for increasing the percentage or number of Tregs comprising an exogenous polynucleotide encoding FOXP3 in a Treg population, comprising exposing the Treg population to pro-inflammatory conditions.

[0263] In particular, the increase in the percentage or number of Tregs comprising an exogenous polynucleotide encoding FOXP3 in the population can be an increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, and can be directly measured by flow cytometry using an antibody that binds to FOXP3 (after cell permeabilization), or directly measured by any of the methods discussed above. Typically, Tregs that do not contain exogenous FOXP3 have lower FOXP3 levels than transduced Tregs, and lower activity under proinflammatory conditions. Therefore, the mixed Treg population is exposed to proinflammatory conditions for the purpose of purifying Tregs comprising exogenous polynucleotides encoding FOXP3, or increasing the number or percentage of these cells present in the population. As previously described, this can be achieved by exposure to any one or more proinflammatory cytokines, but in particular can be achieved by exposure to IL1B, IL6 and / or TNFα.

[0264] The present 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 the embodiments of the present disclosure. Numerical ranges include the numbers defining the ranges. Unless otherwise indicated, any nucleic acid sequence is written from left to right in a 5' to 3' direction, and any amino acid sequence is written from left to right in an amino to carboxyl terminal direction.

[0265] Where a numerical range is provided, each intermediate value between the upper and lower limits of the range is also specifically disclosed, up to one tenth of the unit of the lower limit, unless the context clearly dictates otherwise. Each smaller range between any specified value or intermediate value in the specified range and any other specified value or intermediate value in the specified range is encompassed in the present disclosure. The upper and lower limits of these smaller ranges may be independently included or excluded in the range, and each range in which the smaller range includes either, neither, or both limits is also encompassed in the present disclosure, subject to any explicitly excluded limits in the range. If the range includes one or two limits, the range excluding one or both of the limits is also encompassed in the present disclosure.

[0266] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0267] As used herein, the terms "comprising," "including," and "consisting of are synonymous with "comprising," "including," or "containing," "including," and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps. The terms "comprising," "including," and "consisting of also include the term "consisting of."

[0268] The embodiments of the present invention may be combined.

[0269] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that such publications constitute prior art to the appended claims.

[0270] The present invention will now be further described by way of examples, which are intended to help technicians implement the present invention and are not intended to limit the scope of the present invention in any way.

[0271] Example

[0272] Materials and methods

[0273] clone:

[0274] 2 constructs ( Figure 2 ) was designed in-house and the entire sequence was codon optimized for expression in human cells and manufactured. The construct was cloned and D5a efficient bacteria were transformed with the plasmid and grown with the selection agent ampicillin. DNA was extracted using the MiniprepKit (Qiagen). The insert was transferred into the lentiviral backbone by PCR cloning.

[0275] Collection of PBMCs:

[0276] Leukocyte cones were provided by NHS Blood and Transplant. PBMCs were isolated using a density centrifugation protocol. Briefly, blood was diluted 1:1 with 1× PBS and layered on Ficoll-Paque (GE Healthcare). Samples were centrifuged and the buffy coat was removed and washed in PBS.

[0277] Treg and Tconv isolation protocol:

[0278] Blood cones from HLA-A*02 negative donors were used to obtain Treg populations. TMHuman CD4+ T cell enrichment mixture was enriched for CD4 by negative selection of blood cones. Subsequently, CD4+ cells were isolated using density centrifugation. CD4+CD25+ T cells were then isolated by positive selection using CD25 microbeads II (Miltenyi). Prior to FACS sorting, flow cytometry antibodies CD4 FITC (OKT4, Biolegend), CD25 PE-Cy7 (BC96, Biolegend), CD127BV421 (A019D5, Biolegend), CD45RA BV510 (HI100, Biolegend), and LIVE / DEAD were used. TM The CD4+CD25+ fraction was stained with a fixable near infrared-dead cell stain (Thermofisher).

[0279] Treg Culture Media and Expansion:

[0280] Human regulatory T cells were cultured in Texmacs medium (Miltenyi) supplemented with IL-2 and stained with human T activator CD3 / CD28 Dynabeads. TM (Gibco). The cells were re-fed with Treg medium supplemented with IL-2 every 2 to 3 days. Dynabeads were used TM A second round of stimulation was performed to promote further expansion of Treg cells.

[0281] Transfection and production of viral particles

[0282] Lentivirus:

[0283] HEK293T cells were inoculated in DMEM (Dulbecco's modified Eagle's medium) + 10% fetal bovine serum (FBS) and cultured for 24 hours. The transfection reagent was placed at room temperature and mixed with the DNA construct / plasmid of interest, packaging plasmid (pD8.91) and viral envelope (pVSV-G). PEI was added to the diluted DNA and mixed, and added to HEK293T. The supernatant was collected 48 hours after transfection, filtered and the virus was concentrated.

[0284] Flow cytometric staining to determine CAR and RQR8 expression

[0285] T cells were removed from the culture and washed in FACS buffer and stained for HLA-A*02-specific CAR using HLA-A*02-specific Dextramer (WB2720-APC, Immudex) in FACS buffer. TMCells were stained with fixable near infrared-dead cell stain (Thermofisher) and then stained with anti-CD4 AF700 (RPA-T4, BD), anti-CD34 FITC (QBEND / 10, Thermofisher) and anti-CD3 PE-Cy7 in FACS staining buffer. For intracellular staining of FOXP3, cells were fixed and permeabilized and then stained with anti-Foxp3 PE (150D / E4, Thermofisher) antibody. Cells were analyzed on a BD LSRII flow cytometer.

[0286] Instability assay

[0287] Frozen Tregs transduced as described above were thawed and incubated under two different conditions. The first condition was to culture Tregs under the standard conditions described above. The second condition was to culture Tregs in the additional presence of IL6 (4 ng / ml), IL1β (10 ng / ml) and TNFα (10 ng / ml). The cells were cultured for 7 days and then subjected to suppression assays.

[0288] Inhibition assay

[0289] In order to evaluate the ability of Treg to inhibit the activation of effector T cells, Teff cells were labeled with CFSE dye. Teff cells were co-cultured with Treg cells at different concentrations (Treg: Teff ratios were 1: 1, 1: 2, 1: 4, 1: 8, 1: 16, 1: 32, and 1: 64). CD3 / CD28 beads (1: 100) were added for activation. For CAR-dependent activation, HLA A2 expressing cells were used. After 72 hours of activation, cells were collected and analyzed by flow cytometry. CFSE dilution was used as a surrogate marker for Teff cell proliferation.

[0290] result

[0291] Figure 3 The expression level of FOXP3 in transduced cells vs non-transduced cells of construct I (i.e., a construct expressing additional exogenous FOXP3) is shown. It can be seen that the level of FOXP3 in transduced Tregs is higher than that in non-transduced cells in the population. Therefore, transduction with construct I increases the level of FOXP3 in cells.

[0292] also, Figure 3 We show that under pro-inflammatory conditions, transduced cells have suppressive function compared to non-transduced cells. Cells expressing exogenous extra FOXP3 maintain their suppressive function even after exposure to pro-inflammatory cytokines.

[0293] This inhibitory effect is maintained Figure 4 This was further confirmed in Figure 4 Comparison of inhibitory function or ability between cells transduced with construct I (with exogenous FOXP3 expression and CAR expression) and construct VIII (expressing the same CAR as construct I but without exogenous FOXP3 expression) is shown. After exposure to proinflammatory conditions, cells transduced with construct I were able to maintain their inhibitory function, while cells transduced with construct VIII could not be inhibited. Cells transduced with construct I exposed to proinflammatory conditions showed only a slight decrease in inhibitory function compared to the same cells cultured under standard conditions (without proinflammatory cytokines), which shows how effective the presence of exogenous FOXP3 is for maintaining inhibitory function under proinflammatory conditions. Given that Treg products will need to function under proinflammatory conditions in vivo, the presence of exogenous FOXP3 is very important for providing products that can retain their functional capabilities.

[0294] All publications mentioned in the above description are incorporated herein by reference. It will be apparent to those skilled in the art that various modifications and variations of the methods and systems of the present invention will be apparent without departing from the scope and spirit of the present invention. Although the present invention has been described in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be overly limited to these specific embodiments. In fact, various modifications apparent to those skilled in the art of molecular biology, cellular immunology or related fields for implementing the described methods of the present invention are intended to fall within the scope of the following claims.

Claims

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

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

3. The method according to claim 1 or 2, wherein: (i) the FOXP3 polypeptide comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 3 or 4 or a functional fragment thereof; or (ii) The polynucleotide encoding the FOXP3 polypeptide comprises a polynucleotide sequence that is at least 80% identical to SEQ ID NO: 1 or 2 or a functional fragment thereof.

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

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

6. The method according to 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 the following orientation: 5' FOXP3-TCR / CAR 3'.

8. The method according to any one of the preceding claims, wherein the polynucleotide encoding FOXP3 is introduced into the isolated Treg by viral transduction; alternatively, wherein the polynucleotide encoding FOXP3 is introduced into the isolated Treg by retroviral transduction.

9. A method according to any one of the preceding claims, comprising: (a) Isolation of Tregs from cell populations; and (b) increasing the expression of FOXP3 in the Tregs.

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

11. The method according to claim 9 or 10, wherein isolating Tregs comprises Isolation of CD4 + T cells; and From the CD4 + The Tregs are isolated from T cells.

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

13. The method according to any one of claims 9 to 12, wherein the Tregs are selected by (i) CD4 + CD25 + CD127 - and / or CD4 + CD25 + CD127 低 cells; (ii) CD4 + CD25 高 CD127 - and / or CD4 + CD25 + CD127 低 Cells to separate.

14. The method according to any one of claims 9 to 13, wherein the Treg is selected by FOXP3 + cells to be isolated; preferably, wherein the Tregs are isolated by selecting CD4 + CD25 + FOXP3 + Helios + Neuropilin 1 + Cells to separate.

15. Engineered Tregs obtainable or obtained by the method of any one of claims 1-14.

16. A pharmaceutical composition comprising the engineered Treg according to claim 15.

17. The engineered Treg according to claim 15 or the pharmaceutical composition according to claim 16, for use in preventing and / or treating a disease.

18. Use of the engineered Treg according to claim 15 in the manufacture of a medicament for preventing and / or treating a disease.

19. A method for preventing and / or treating a disease, comprising administering the engineered Treg or the pharmaceutical composition according to any one of claims 15-18 to a subject.

20. The engineered Treg for use according to claim 17 or the pharmaceutical composition for use, the use of 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 engineered Treg according to claim 15, for use in preventing and / or treating a disease, wherein the disease 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 (Treg) to suppress immune responses under proinflammatory conditions.

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