Cryopreservation method for surgical Tregs

Abstract

The present invention relates to a method for preserving CD62L expression in a regulatory T cell (Treg) population that has undergone cryopreservation, the method comprising introducing a polynucleotide encoding a FOXP3 polypeptide into the Treg population prior to cryopreservation. The present invention also relates to a method for preserving CD62L in a Treg population after cryopreservation, the method comprising introducing a polynucleotide encoding a FOXP3 polypeptide into the Treg population and cryopreserving the Treg population. Furthermore, the present invention relates to the use of an exogenous polynucleotide encoding FOXP3 for preserving CD62L expression in a Treg population after cryopreservation, as well as to cryopreserved engineered Tregs, pharmaceutical compositions comprising the cryopreserved engineered Tregs, and therapeutic uses thereof.

Description

Technical Field

[0001] The present invention relates to a method for cryopreserving regulatory T cells (Tregs). In particular, the present invention relates to a method for increasing FOXP3 expression in Tregs before cryopreservation in order to preserve the suppressive function of Tregs after cryopreservation. The present invention also relates to cryopreserved engineered Tregs, pharmaceutical compositions comprising cryopreserved engineered Tregs, and their therapeutic uses.

Background Art

[0002] In recent years, there has been increasing interest in the use of regulatory T cells (Tregs) in clinical settings for adoptive cell therapy (ACT) for treating various different pathologies. Tregs have been proposed for use in the control of unwanted immune responses based on their immunosuppressive function. For example, Tregs have been used in the treatment of autoimmune or allergic diseases, immunomodulation in transplantation, and the improvement and / or prevention of immune-mediated organ damage in inflammatory diseases. In addition, genetic engineering of Tregs has been performed to express a T cell receptor (TCR) or chimeric antigen receptor (CAR) with new specificities, thereby providing the advantage of targeted immunosuppression. The safety and efficacy of Treg therapy have been demonstrated by various Phase I trials, and several Phase II trials are currently underway.

[0003] The primary sources of Tregs for therapeutic use are the patient's own blood (either directly collected from blood vessels or as a leukocyte apheresis product) or umbilical cord blood. While the number of Tregs obtained from these sources is small, a significant number is needed to effectively suppress the immune system. Therefore, ex-vivo expansion is necessary to obtain a sufficient number of Tregs for patient injection. A typical isolation and proliferation protocol following Good Manufacturing Practice (GMP) guidelines takes approximately 9 to 21 days. Processing and freezing these cells in sufficient quantities, making them readily available for patient use when needed, would be highly advantageous, as it would avoid prolonged processing that could negatively impact Treg quality. This approach would allow for greater flexibility in therapy planning and injection timing, and would enable cell harvesting prior to subsequent Treg injections and pharmacological therapy. Therefore, the ability to cryopreserve Tregs is crucial.

[0004] The effects of freeze-thaw cycles on Treg cell populations are not clearly defined. Several research groups have reported that cryopreservation may have detrimental effects on Tregs, potentially reducing Treg viability, causing abnormal cytokine secretion, and altering the expression of surface markers essential for proper Treg suppression and migration.

[0005] In particular, Florek et al, PLoS One, 2015, DOI: 10.137 1A study (journal.pone.0145763) revealed that freezing and thawing of Treg cells leads to a loss of CD62L (L-selectin) expression, impairing their protective ability against graft-versus-host disease (GvHD). In thawed Treg cells, the ability to bind to MADCAM1, the binding partner of CD62L, decreased, and homing to secondary lymphoid organs was impaired. Other studies have also shown that the expression of CD62L (L-selectin) and CCR5 on T cells is lost after cryopreservation (De Boer et al, Bone Marrow Transplant, 1998, 22:1103-10 and Hattori et al, Exp Hematol, 2001, 29:114-22). Treg cells lacking CD62L expression showed reduced transport capacity and localization, resulting in decreased compartmentalization of the Treg-regulated immune response (Sakaguchi et al, Cell, 2008, 133:775-87). Loss of CD62L expression after thawing can be restored by culturing overnight (De Boer et al., 1998 and Hattori et al., 2001). However, resting cells in this way may not be practical in clinical applications because thawing is usually performed bedside, making further culturing impossible.

[0006] Golab et al (Oncotarget, 2018, vol.9, (No.11), pp:9728-9740) proposed two cell banking strategies for Treg therapy: cryopreservation of CD4+ cells for subsequent isolation / proliferation of Tregs, and ex vivo proliferation of Tregs (CD4+). + CD25 hi CD127 lo / - We evaluated the cryopreservation of cells. Ex vivo proliferated Treg cells were more sensitive to the cryopreservation process than CD4+ cells, and their cell viability after thawing was significantly reduced, along with a decrease in Treg marker expression (i.e., the phenotype was CD4+). + CD25 hi CD127 - and CD4+ FoxP3 + It was found that the frequency of cells decreased. The low survival rate and phenotypic impairment of Tregs after thawing were overcome by restimulating the Tregs during a subsequent 13-day ex vivo proliferation. Similarly, Peters et al (PLoS One, 2008, 3:e3161) showed that the inhibitory ability of Tregs after thawing is reversible by 10 days of stimulation and proliferation.

[0007] However, as mentioned above, these additional culture and proliferation steps are not feasible in clinical settings, and are time-consuming and involve additional risks, thus negating the advantages of using cryopreserved Treg cells compared to newly isolated cells. Therefore, there is still a need for improved cryopreservation methods that would allow cryopreserved Treg cells to be administered to patients immediately after thawing, without the lengthy post-thawing rescue process currently required in the laboratory. [Overview of the Initiative]

[0008] In this regard, the inventors have surprisingly found that the expression of exogenous FOXP3 provides a protective effect against the Treg phenotype after cryopreservation. The inventors have confirmed that Tregs transduced with FOXP3 maintain the expression of CD62L (i.e., L-selectin) even after freeze-thawing. CD62L expression is essential for the migration and homing functions of Tregs and is therefore important for the immunosuppressive function of Tregs. For this reason, transducing Tregs with FOXP3 prior to cryopreservation (for example, as part of the processing of necessary cells) maintains the immunomodulatory and suppressive functions of Tregs after thawing, making the cells immediately available for use in patients. The inventors have thus provided a solution to the problems associated with Treg cryopreservation, avoiding further culture of the cells, and further providing a pathway to Treg therapies that can be effectively used in clinical settings.

[0009] Therefore, the present invention provides a modified Treg that maintains its immunosuppressive properties even after cryopreservation, thereby enhancing its clinical efficacy and safety. Cryopreserved Tregs can be used for a variety of therapeutic applications, such as inducing tolerance to grafts in subjects, or treating and / or preventing graft rejection, graft-versus-host disease, inflammation, autoimmune diseases, allergic diseases, neuroinflammatory diseases such as amyotrophic lateral sclerosis (ALS), metabolic diseases such as type 1 diabetes, or suppressing immune responses.

[0010] In a first embodiment, the present invention provides a method for preserving CD62L expression in a cryopreserved population of regulatory T cells (Tregs) or Tregs, comprising introducing a polynucleotide encoding a FOXP3 polypeptide into the Treg population or Tregs before cryopreservation.

[0011] The Treg population or Treg may have higher CD62L expression after cryopreservation than the corresponding unmodified Treg population or Treg after cryopreservation (i.e., a Treg population or Treg that has not been transduced with the polynucleotide encoding FOXP3).

[0012] The method described above may further include the step of cryopreserving the Treg population or Tregs. Thus, the present invention may further provide a method for preserving CD62L expression in a regulatory T cell (Treg) population or Tregs for cryopreservation, comprising the steps of (a) introducing a polynucleotide encoding a FOXP3 polypeptide into a Treg population or Tregs, and (b) cryopreserving the Treg population or Tregs.

[0013] Alternatively, the present invention may provide a method for cryopreserving a Treg (or population of Tregs), comprising (a) introducing a polynucleotide encoding FOXP3 into a Treg or population of Tregs, and (b) cryopreserving the Treg or population of Tregs, wherein the cryopreserved Treg or population of Tregs has a higher CD62L expression level compared to the corresponding unoperated Treg or population of Tregs.

[0014] In another embodiment, the present invention provides a method for producing a cryopreserved Treg or population of Tregs having a CD62L level equivalent to that of a corresponding non-cryopreserved Treg or population of Tregs, comprising: (a) introducing a polynucleotide encoding FOXP3 into a Treg or population of Tregs; and (b) cryopreserving the Treg or population of Tregs.

[0015] Those skilled in the art will understand that the steps of the methods described herein are typically arranged in an order in which they operate, for example, that step (a) should be performed prior to step (b).

[0016] The method of the present invention may further comprise one or more of the following additional steps: isolating a Treg or Treg population from a sample before introducing a polynucleotide encoding FOXP3 into the Treg or Treg population; thawing the Treg or Treg population; and / or administering the thawed Treg or Treg population. The method may further comprise a step of growing the Treg population or Treg prior to cryopreservation. Alternatively, the Treg or Treg population of the present invention may not be cryopreserved until grown. The cryopreserved Treg or Treg population of the present invention may be immediately used as an off-the-shelf therapeutic product without requiring further growth, modification, or alternative additional culture steps.

[0017] In this regard, the present invention further provides a method for preserving CD62L expression in a Treg or Treg population thawed from a cryopreserved state, comprising introducing a polynucleotide encoding a FOXP3 polypeptide into the Treg or Treg population prior to cryopreservation and thawing.

[0018] Alternatively, the present invention provides a method for preserving CD62L expression in a Treg or Treg population after thawing from a cryopreserved state, comprising: (a) introducing a polynucleotide encoding FOXP3 into the Treg or Treg population; (b) cryopreserving the Treg or Treg population; and (c) thawing the Treg or Treg population. The Treg or Treg population can be used immediately after / during thawing without additional growth. The method may further comprise (i) enriching a Treg from a sample presumed to contain a Treg to produce a Treg-enriched sample prior to step (a); and (ii) growing the Treg or Treg population from the Treg-enriched sample after step (a) and before step (b) to produce a manipulated-growth Treg or Treg population. Step (i) may further comprise depleting CD8+ cells.

[0019] In another embodiment, the present invention provides a method for producing a thawed Treg or Treg population having a CD62L level equivalent to that of a corresponding unfreezed Treg or Treg population, comprising: (a) introducing a polynucleotide encoding FOXP3 into the Treg or Treg population; (b) cryopreserving the Treg or Treg population; and (c) thawing the Treg or Treg population. The Treg or Treg population can be used immediately after / during thawing without additional growth. The method may further comprise (i) enriching a Treg from a starting cell sample presumed to contain Tregs before step (a) to produce a Treg-enriched sample; and (ii) growing the Treg or Treg population from the Treg-enriched sample after step (a) and before step (b) to produce a manipulated-growth Treg or Treg population. Step (i) may further comprise depleting CD8+ cells.

[0020] The sample (also known as a Treg cell-containing sample or simply a cell-containing sample) may contain, or consist of, whole blood, umbilical cord blood, leukocyte cones, blood cones, peripheral blood mononuclear cells (PBMCs), or leucopacks. Typically, the sample may contain, or consist of, leukocyte cones or one or more leucopacks. Typically, the sample is derived from a human donor. The donor may be healthy, have a disease such as a neurodegenerative disease (e.g., ALS) or an autoimmune disease (e.g., type 1 diabetes), or be a transplant patient.

[0021] The cryopreservation of the Treg population (or Treg) may include the following steps: (bi) suspending the Treg population in a cryopreservation medium; (bii) freezing the Treg population from step (bi); and (biii) storing the Treg population from step (bii) at a temperature lower than -130°C. A suitable cryopreservation medium may be any cryopreservation medium known in the art, as detailed below. The cryopreservation medium preferably contains one or more cryoprotective agents, suitable cryoprotective agents will be described later. The cryopreservation medium and the cryoprotective agent(s)(one or more) preferably comply with Good Manufacturing Practice (GMP) standards.

[0022] The method of the present invention may further include one or more of the following steps: pre-cooling the Treg population (or Treg) and / or one or more (preferably all) reagents or devices used in any step of cryopreservation (step (b)); freezing the Treg population (or Treg) at a controlled freezing rate of approximately -1°C / min (in step (b)); and / or storing the Treg population (or Treg) at -80°C for up to 24 hours prior to step (biii). “Reagents” may include, for example, a cryopreservation medium, and “devices” may include, for example, any freezing device that maintains a controlled freezing rate. Suitable reagents and devices may be any reagents or devices known in the art that are used for cryopreservation. Such reagents and devices are preferably compliant with GMP standards.

[0023] The above step (biii) may include storing the Treg group in liquid nitrogen. The temperature of the liquid nitrogen may be approximately -196°C.

[0024] The step of thawing the Treg population (or Tregs) includes raising the temperature of the Treg composition from a temperature lower than -130°C to a temperature between approximately 0°C and 10°C, and optionally, the temperature increase of the Treg population includes placing the Treg composition in a water bath maintained at approximately 37°C.

[0025] The cryopreservation step and / or the thawing step may be performed in a closed system or in a grade A environment according to GMP standards.

[0026] The Treg population is CD4 + CD25 + CD127 - cells and / or CD4 s + s] CD25 + CD127 low cells, or by selecting CD4 + CD25 hi CD127 - cells and / or CD4 + CD25 + CD127 low cells, may be isolated from a sample. The Treg population is CD45RA + cells, preferably CD4 + CD25 + CD127 low CD45RA + cells, may be isolated by selection. Thus, the cryopreserved Treg population of the present invention is CD4 + CD25 + CD127 - Treg cells, CD4 + CD25 + CD127 low Treg cells, and / or CD4 + CD25 + CD127 low CD45RA + Treg cells, may contain at least 70%, at least 80%, at least 90%, or at least 95%.

[0027] Furthermore, the cryopreserved Treg population may contain less than 20% CD8+ cells, less than 10% CD8+ cells, preferably less than 5% CD8+ cells, and more preferably less than 2% CD8+ cells. In this regard, the method of the present invention may include a step of depleting CD8+ cells before the transduction step (i.e., before step (a) of the method described above, which includes introducing a polynucleotide encoding FOXP3 into the Treg or Treg population).

[0028] In a second embodiment, the present invention provides the use of an exogenous polynucleotide encoding FOXP3 for preserving CD62L expression in a cryopreserved Treg population or Treg, or in a Treg population or Treg for cryopreservation. In particular, as described above, the exogenous polynucleotide is introduced into a Treg population or Treg, and its expression provides a protective effect on the CD62L expression level in the Treg population or Treg.

[0029] A third aspect of the present invention provides a cryopreserved manipulated Treg population or Treg comprising an exogenous polynucleotide encoding a FOXP3 polypeptide, wherein the manipulated Treg population or Treg has a higher CD62L expression level after cryopreservation than the corresponding cryopreserved unmanipulated Treg population.

[0030] The present invention further provides a Treg population or Treg (e.g., cryopreserved or thawed cells) that can be obtained or obtained by the method of the present invention. Alternatively, the present invention provides a thawed Treg or Treg population comprising an exogenous polynucleotide encoding a FOXP3 polypeptide, wherein the manipulated Treg or Treg population has a higher CD62L expression level after thawing than the corresponding unmanipulated Treg or Treg population after thawing.

[0031] The FOXP3 polypeptide may contain an amino acid sequence or functional fragment that is at least 80% identical to SEQ ID NO: 1. The polynucleotide encoding FOXP3 may be present in the expression vector.

[0032] The method may further include introducing a polynucleotide encoding an exogenous T cell receptor (TCR) or a polynucleotide encoding a chimeric antigen receptor (CAR) into the Treg population or Treg, or the cryopreserved manipulated Treg population or Treg may contain a polynucleotide encoding an exogenous T cell receptor (TCR) or a polynucleotide encoding a chimeric antigen receptor (CAR). The polynucleotide encoding the FOXP3 polypeptide and the polynucleotide encoding the exogenous TCR or CAR may be provided by a single expression vector.

[0033] The TCR or CAR may be directed to any desired target molecule, particularly a target molecule expressed on a target cell. Suitable TCRs and CARs will be described later. In one embodiment, the CAR is directed to an HLA antigen, for example, HLA-A2.

[0034] The vector may include a first polynucleotide encoding the FOXP3 polypeptide and a second polynucleotide encoding the exogenous TCR or CAR, wherein the first and second polynucleotides are operably linked to the same promoter, and the first polynucleotide is upstream of the second polynucleotide. An internal self-cleaving sequence may be present between the polynucleotide encoding FOXP3 and the polynucleotide encoding the exogenous TCR or CAR. Suitable promoters and self-cleaving sequences will be described later.

[0035] The method of the present invention may include introducing a polynucleotide encoding a safety switch containing a suicide moiety into the Treg population or Treg, or the cryopreserved operational Treg population or operational Treg of the present invention may contain a safety switch containing a suicide moiety. Thus, in one embodiment, a polynucleotide / nucleic acid molecule encoding FOXP3 and a polynucleotide / nucleic acid molecule optionally encoding a CAR / TCR may further encode a safety switch containing a suicide moiety. In particular, nucleic acid molecules and constructs may be designed to encode distinct components (e.g., three components (e.g., FOXP3, exogenous TCR or CAR, and a safety switch)) within a single nucleic acid molecule or construct, so that the components may be produced in the cell as individual components, i.e., separate entities (i.e., not linked to each other and physically distinct). Thus, the components encoded by the nucleic acid molecule may, after expression, be located separately inside or on the cell as distinct, different, or separate functional polypeptides. This is achieved by encoding cleavage sequences, particularly self-cleavage sequences, within the nucleic acid molecule between the nucleotide sequences encoding each component.

[0036] The polynucleotides disclosed herein (e.g., those encoding FOXP3 and / or exogenous TCRs or CARs and / or the safety switches) may be introduced into a Treg population or Tregs by viral transduction, preferably retroviral transduction or lentiviral transduction. In a fourth aspect, the present invention provides a pharmaceutical composition comprising a cryopreserved operational Treg population or Treg, or a thawed Treg population, as described herein.

[0037] In a fifth aspect, the present invention provides cryopreserved operational Treg populations or Tregs, thawed Tregs or Treg populations, or pharmaceutical compositions described herein, for use in the prevention and / or treatment of diseases.

[0038] In a sixth aspect, the present invention provides the use of cryopreserved operated Treg populations or Tregs, thawed Tregs or Treg populations, or pharmaceutical compositions described herein in the manufacture of agents for the prevention and / or treatment of diseases.

[0039] In a seventh aspect, the present invention provides a method for preventing and / or treating a disease, comprising administering to a cryopreserved operational Treg population or Treg, a thawed Treg or Treg population, or a pharmaceutical composition described herein.

[0040] The aforementioned diseases may include autoimmune diseases, allergic diseases, transplant rejection, graft-versus-host disease, inflammation, neuroinflammatory diseases such as amyotrophic lateral sclerosis (ALS), or metabolic diseases such as diabetes (e.g., type 1 diabetes). The present invention also relates to the cryopreserved manipulated Treg population or Treg described herein for use in suppressing immune responses, or the pharmaceutical composition described herein for use in suppressing immune responses, or the use of the cryopreserved manipulated Treg population or Treg or pharmaceutical composition described herein in suppressing immune responses.

[0041] In one embodiment, a method is provided for improving the homing ability of a cryopreserved Treg or Treg population to secondary lymphoid organs compared to a corresponding unmanipulated Treg or Treg population after cryopreservation, the method comprising the step of introducing a polynucleotide encoding a FOXP3 polypeptide into the Treg or Treg population before cryopreservation.

[0042] In further embodiments, a method is provided for removing a population of cryopreserved Tregs or Tregs as defined herein, comprising a nucleic acid molecule / polynucleotide encoding FOXP3, an expression construct, or a vector, and a safety switch, the method comprising the step of exposing the cryopreserved Tregs or Treg population to a separate cell removal agent (such as an antibody) that recognizes a suicide moiety in the safety switch. The cell removal agent can target the cells to be removed by binding to the suicide moiety. The method may be an in vitro method.

[0043] The present invention also provides a method for enhancing the stability and / or inhibitory function of Treg or Treg populations after cryopreservation, comprising the step of introducing a nucleic acid molecule / polynucleotide, expression construct, or vector provided herein into the cells before cryopreservation.

[0044] The present invention provides ex vivo grown and cryopreserved therapeutic Treg cells or Treg populations that maintain high viability, purity, and potency, as seen in the grown Treg cells or Treg populations before cryopreservation, without further proliferation after thawing. Thus, the cells of the present invention can be used as off-the-shelf therapeutic agents.

[0045] The protection required by this invention is as defined in the claims. [Brief explanation of the drawing]

[0046] [Figure 1] -Survival Rate- Figure 1 shows the survival rates of fresh Tregs (i.e., non-frozen Tregs) and frozen Tregs transduced with a construct expressing FOXP3 (C1), compared to the survival rates of fresh and frozen Tregs that were not transduced, i.e., non-GMO. Approximately 75% of cells remained viable after freezing in both transduced and non-transduced Tregs. [Figure 2]-Transduction- Figure 2 shows that both fresh and frozen Tregs transduced with C1 express FOXP3. [Figure 3] -Cell Surface Expression of CD62L- Figure 3 shows the cell surface expression of CD62L in fresh Tregs (i.e., non-frozen Tregs) and frozen Tregs transduced with a construct expressing FOXP3 (C1), and in non-transduced (non-GMO) fresh Tregs and frozen Tregs. The percentage of CD62L expression is significantly increased in frozen transduced Tregs compared to frozen non-transduced Tregs. [Figure 4] -Survival Rates in Different Cryopreservation Solutions- Figure 4 shows the survival rates of Tregs transduced with or without a construct expressing FOXP3 (C1), non-transduced (Non-GMO) (non-transduced Tregs do not express exogenous FOXP3), fresh Tregs (i.e., unfrozen Tregs) ("Before Freezing" column), and frozen Tregs. Frozen Tregs were frozen in three different cryopreservation solutions: Solution 1, Solution 2, and Solution 3, at five different cell densities for each solution. Under all conditions, the survival rate was close to 100%. [Figure 5] -Transduction in Different Cryopreservation Solutions- Figure 5 shows transduction in fresh Tregs ("Before Freezing" column) transduced or not transduced with a construct expressing FOXP3 (C1), as well as transduction in Tregs transduced or not transduced with C1 (Non-GMO) and then frozen in cryopreservation solutions 1, 2, or 3 at five different cell densities for each solution. In both fresh and frozen states, Tregs transduced with C1 expressed high levels of FOXP3. This high expression level was maintained in frozen Tregs across all three different cryopreservation solutions 1, 2, and 3 and all five different cell densities. [Figure 6]-Cell Surface Expression of CD62L in Different Cryopreservation Solutions- Figure 6 shows the cell surface expression of CD62L in fresh Tregs transduced or untransduced (Non-GMO) with a construct expressing FOXP3 (C1) ("Before Freezing" column), as well as in Tregs transduced or untransduced (Non-GMO) with C1, then frozen in cryopreservation solutions 1, 2, or 3 at five different cell densities for each solution. The percentage of CD62L expression was increased in Tregs transformed with C1 compared to untransformed Tregs and was comparable across all three cryopreservation solutions and all five different cell densities.

[0047] [Detailed explanation] The inventors have surprisingly found that transduction of exogenous FOXP3 into regulatory cells preserves CD62L expression at a level that maintains regulatory function even after cryopreservation. Therefore, the present invention provides a method for preserving CD62L expression in a cryopreserved regulatory T cell (Treg) population (or Treg), comprising introducing a polynucleotide encoding the FOXP3 polypeptide into the Treg population before cryopreservation. Alternatively, the present invention provides a method for producing a cryopreserved Treg or Treg population having a level of CD62L equivalent to that of a corresponding non-cryopreserved Treg or Treg population, comprising (a) introducing a polynucleotide encoding FOXP3 into the Treg or Treg population, and (b) cryopreserving the Treg or Treg population. The present invention also provides a cryopreserved pup of manipulated Tregs (or manipulated Tregs) comprising an exogenous polynucleotide encoding a FOXP3 polypeptide, characterized in that the manipulated Treg pup has higher CD62L expression after cryopreservation than the corresponding unmanipulated Treg pup after cryopreservation (or CD62L expression at a level equivalent to that of the corresponding unmanipulated Treg or Treg pup after cryopreservation).

[0048] [Regulatory T cells] Regulatory T cells (Tregs) or T regulatory cells are immune cells that have immunosuppressive functions that control cytotoxic immune responses and are essential for maintaining immune tolerance. As used herein, the term Treg refers to T cells with immunosuppressive functions. As used herein, T cells refer to lymphocytes including any type of T cell, such as αβT cells (e.g., CD8 or CD4+), γδT cells, memory T cells, or Treg cells.

[0049] Preferably, the terms immunosuppressive function and “immune response” may refer to the ability of Treg cells to reduce or inhibit one or more of the numerous physiological and cellular effects promoted by the immune system in response to stimuli such as pathogens, alloantigens, or autoantigens. Examples of such effects include enhanced proliferation of conventional T cells (Tconv) and secretion of inflammatory cytokines. Both of these effects can be used as indicators of the strength of the immune response. A relative weakening of the immune response by Tconv in the presence of Treg cells is considered to indicate the immunosuppressive ability of Treg cells. For example, a relative decrease in cytokine secretion is considered to indicate a weakening of the immune response and thus indicates the immunosuppressive ability of Treg cells. Treg cells can also suppress the immune response by regulating the expression of costimulatory molecules on antigen-presenting cells (APCs) such as B cells, dendritic cells, and macrophages. The expression levels of CD80 and CD86 can be used to evaluate the in vitro suppressive efficacy after co-culturing activated Treg cells.

[0050] Assays for measuring the strength of the immune response and thereby assessing the inhibitory ability of Tregs are known in the art. Specifically, antigen-specific Tconv cells are co-cultured with Tregs, and the corresponding antigen peptide is added to the co-culture to stimulate the response from the Tconv cells. The degree of Tconv cell proliferation and / or the amount of cytokine IL-2 secreted by Tconv cells in response to the addition of the peptide can be used as an indicator of the inhibitory ability of the co-cultured Tregs.

[0051] Antigen-specific Tconv cells co-cultured with a Treg (or Treg population) disclosed herein may proliferate 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 95%, or 99% less than Tconv cells cultured in the absence of the Treg. For example, antigen-specific Tconv cells co-cultured with the cryopreserved Treg of the present invention may proliferate 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% less than Tconv cells cultured in the presence of an unmanipulated cryopreserved Treg.

[0052] Antigen-specific Tconv cells co-cultured with the Treg described herein may express at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% less effector cytokines compared to corresponding Tconv cells cultured in the absence of the Treg (e.g., in the presence of an unprocessed cryopreserved Treg). The effector cytokines may be selected from IL-2, IL-17, TNFα, GM-CSF, IFN-γ, IL-4, IL-5, IL-9, IL-10, and IL-13. Preferably, the effector cytokines may be selected from IL-2, IL-17, TNFα, GM-CSF, and IFN-γ.

[0053] Several different subpopulations of Tregs have been identified, and these may express different specific markers or different levels of specific markers. Tregs are generally T cells that express the markers CD4, CD25, and FOXP3 (CD4 + CD25 + FOXP3 + Treg cells may also express CTLA-4 (cytotoxic T lymphocyte-associated molecule-4) or GITR (glucocorticoid-inducible TNF receptor).

[0054] Treg cells are present in peripheral blood, lymph nodes, and tissues, and the Tregs for use as described herein include thymic-derived native Treg (nTreg) cells, peripherally produced Tregs, and inducible Treg (iTreg) cells.

[0055] Tregs can be identified by using the cell surface markers CD4 and CD25 in the absence of the surface protein CD127, or in combination with low levels of CD127 expression (CD4 + CD25 + / hi CD127 - or CD4 + CD25 + CD127 low The use of such markers to identify Tregs is well known in the art and is described, for example, in Liu et al. (JEM; 2006; 203; 7(10); 1701-1711).

[0056] Treg is CD4 + CD25 + FOXP3 + T cells, CD4 + CD25 + CD127 - T cells, or CD4 + CD25 + FOXP3 + CD127 - / low T cell @.

[0057] Preferably, the Treg may be a natural Treg (nTreg). As used herein, the term “natural Treg” means a thymus-derived Treg. Natural Tregs are CD4 + CD25 + FOXP3 + Helios + Neuropilin1 +Compared to iTregs, nTregs exhibit high expression of PD-1 (programmed cell death-1, pdcd1), Neuropilin1 (Nrp1), Helios (Ikzf2), and CD73. nTregs can be distinguished from iTregs based on the individual expression of Helios protein or Neuropilin1 (Nrp1).

[0058] Tregs may have demethylated Treg-specific demethylation regions (TSDRs). TSDRs are important methylation-sensitive factors that regulate Foxp3 expression (Polansky, JK, et al, 2008. European Journal of Immunology, 38(6), pp.1654-1663).

[0059] Even more suitable Treg cells include Tr1 cells (which do not express Foxp3 and have high IL-10 production) and CD8 + FOXP3 + T cells and γδFOXP3 + T cells are an example, but are not limited to them.

[0060] Treg includes naive Treg (CD45RA) + FoxP3 low ), Effector / Memory Treg (CD45RA - FoxP3 high ), and cytokine-producing Tregs (CD45RA - FoxP3 low It is known that there are different subpopulations, including ). "Memory Treg" is a Treg that expresses CD45RO, and CD45RO +It is thought that these cells have increased levels of CD45RO compared to naive Tregs (e.g., CD45RO is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% higher), and preferably do not express CD45RA (mRNA and / or protein) or have low levels of it compared to naive Tregs (e.g., CD45RA is at least 80%, 90%, or 95% lower compared to naive Tregs). A "cytokine-producing Treg" is a Treg that, compared to a naive Treg, does not express CD45RA (mRNA and / or protein) or expresses it at a very low level (e.g., at least 80%, 90%, or 95% less CD45RA compared to a naive Treg), and has a low level of FOXP3 compared to a memory Treg (e.g., FOXP3 is less than 50%, 60%, 70%, 80%, or 90% compared to a memory Treg). A cytokine-producing Treg may also be a Treg capable of producing interferon-gamma and having lower in vitro repressive power compared to a naive Treg (e.g., less than 50%, 60%, 70%, 80%, or 90% repressive power compared to a naive Treg).

[0061] In this specification, expression level may refer to mRNA or protein expression. In particular, with respect to cell surface markers such as CD45RA, CD25, CD4, CD45RO, and CD62L, expression may refer to cell surface expression, i.e., the amount or relative amount of the marker protein expressed on the cell surface. Expression levels can be determined by methods known in the art. For example, mRNA expression levels may be determined by Northern blotting / array analysis, and protein expression may be determined by Western blotting, or preferably by FACS using cell surface expression antibody staining.

[0062] In particular, Tregs may be naive Tregs. In this specification, "naive regulatory T cells, naive T regulatory cells, or naive Tregs" as used synonymously refers to Treg cells that express CD45RA (in particular, those that express CD45RA on their cell surface). Therefore, naive Tregs are CD45RA + It is stated that naive Tregs generally represent Tregs that are not activated via the endogenous TCR by peptides / MHC, while effector / memory Tregs relate to Tregs that are activated via the endogenous TCR by stimulation. Typically, naive Tregs may express at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more CD45RA than non-naive Treg cells (e.g., memory Treg cells). Alternatively, naive Treg cells may express at least 2, 3, 4, 5, 10, 50, or 100 times more CD45RA than non-naive Treg cells (e.g., memory Treg cells). The level of CD45RA expression can be readily determined by methods of the art, such as flow cytometry using commercially available antibodies. Typically, non-naive Treg cells either do not express CD45RA or express it at low levels.

[0063] In particular, naive Tregs do not need to express CD45RO, and CD45RO - It may be considered that naive Tregs may express CD45RO at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less than memory Tregs, or alternatively, express CD45RO at least 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 10, 1 / 50, or 1 / 100 compared to memory Treg cells.

[0064] As mentioned above, naive Tregs express CD25, but depending on the origin of the naive Treg, the expression level of CD25 may be lower than that of memory Tregs. For example, in naive Tregs isolated from peripheral blood, the expression level of CD25 may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or even 90% lower than that of memory Tregs. Such naive Tregs may be considered to express moderate to low levels of CD25. However, those skilled in the art will understand that such differences may not be observed in naive Tregs isolated from umbilical cord blood.

[0065] A naive Treg as defined herein is typically a CD4 + CD25 + FOXP3 + CD127 low CD45RA + That's fine.

[0066] As used herein, low CD127 expression refers to CD4 from the same subject or donor. + This refers to a lower level of CD127 expression compared to unregulated cells or Tcon cells. More specifically, naive Tregs express CD4 from the same subject or donor. + Compared to unregulated or Tcon cells, CD127 expression may be 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or less than 10%. CD127 levels can be assessed by methods standard in the art, including flow cytometry of cells stained with an anti-CD127 antibody.

[0067] Typically, naive Tregs do not express CCR4, HLA-DR, CXCR3, and / or CCR6, or express them at low levels. More specifically, naive Tregs may express CCR4, HLA-DR, CXCR3, and CCR6 at lower levels compared to memory Tregs, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower levels.

[0068] Naive Treg also has CCR7 + and CD31 + Further markers, including those mentioned above, may be expressed. Isolated naive Tregs can be identified by methods known in the art, such as determining the presence or absence of one or more of the aforementioned markers on the cell surface of isolated cells. For example, CD45RA, CD4, CD25, and CD127low can be used to determine whether a cell is a naive Treg. Methods for determining whether isolated cells are naive Tregs or have a desired phenotype can be carried out as described below in relation to any additional steps that may be performed, and methods for determining the presence and / or expression levels of cell markers are well known in the art, such as flow cytometry using commercially available antibodies.

[0069] Preferably, the Treg or Treg population is isolated from a sample, the sample comprising or consisting of whole blood, umbilical cord blood, leukocyte cone, blood cone, peripheral blood mononuclear cells (PBMCs), or one or more leucopacks obtained from a subject. The Treg may be isolated from PBMCs obtained from a subject. The Treg may be isolated from leukocyte cone obtained from a subject. The Treg may be isolated from one or more leucopacks obtained from a subject. Preferably, the subject from which the sample is obtained is a mammal, preferably a human. Typically, the sample may be obtained by blood collection or apheresis.

[0070] Preferably, the Treg cells are either matched (e.g., HLA-matched) or autologous to the target to which the manipulated Treg cells are administered. Preferably, the target to be treated (i.e., the target to which the manipulated cells are administered) is a mammal, preferably a human. Allogeneic Treg cells can be used, for example, when the cells have been subjected to gene editing technology to prevent rejection or GvHD. Thus, the Treg cells may be generated ex vivo from the patient's own peripheral blood (first party), or, in the case of hematopoietic stem cell transplantation, from the donor's peripheral blood (second party), or from an unrelated donor (third party).

[0071] Preferably, Tregs are part of a population of cells. Preferably, the Treg population contains at least 60% Tregs, for example, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% Tregs. In one embodiment, the Treg population may contain only Tregs. Such a population may be referred to as a “Treg population” or “enriched Treg population.” The terms “Treg population” and “population of Tregs” are used synonymously herein. Methods for obtaining or isolating a Treg population from a sample are described below. Those skilled in the art will understand that the Treg population may also contain other cell types that do not have a regulatory phenotype. Furthermore, the introduction of a polynucleotide encoding FOXP3 may increase the proportion of Tregs in the Treg population during the implementation of the method of the present invention. The Treg population may contain different subpopulations of Tregs, or it may contain a single subpopulation of Tregs, for example, naive Tregs.

[0072] In some embodiments, the isolation / enrichment step in the method of the present invention includes depleting CD8+ cells in order to provide a high-purity Treg population. Depletion of CD8-expressing cells relates to reducing the proportion or number of such cells in the population, but does not necessarily result in the complete removal of all CD8-expressing cells. In some embodiments, it is sufficient for at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of CD8 cells to be depleted from the Treg population. CD8 depletion can be achieved by using CD8 beads in accordance with a standard protocol.

[0073] In some embodiments, the Tregs may be derived from ex vivo differentiation of inducible progenitor cells (e.g., iPSCs) or embryonic progenitor cells into Tregs (e.g., direct differentiation into Tregs, or indirect differentiation via differentiation into Tcon cells or conversion into Tregs, as described below). The nucleic acid molecules or vectors described herein may be introduced into inducible progenitor cells or embryonic progenitor cells before or after differentiation into Tregs. Preferred methods of differentiation are known in the art and disclosed in Haque et al, J Vis Exp., 2016, 117, 54720 (incorporated herein by reference).

[0074] As used herein, “conventional T cell” or the terms Tcon or Tconv (as used herein synonymously) mean a T lymphocyte cell that expresses the αβ T cell receptor (TCR) and a coreceptor which may be differentiation antigen group 4 (CD4) or differentiation antigen group 8 (CD8), and which does not have immunosuppressive function. Conventional T cells are found in peripheral blood, lymph nodes, and tissues. Preferably, the modified Treg may be generated from Tcon by introducing a nucleic acid containing a sequence encoding FOXP3. Alternatively, the modified Treg may be generated from Tcon by in vitro culture of CD4+CD25-FOXP3- cells in the presence of IL-2 and TGF-β.

[0075] The present invention provides Treg cells, or Treg populations, as defined or described herein. As used herein, “Treg” may also be referred to as “cell” or “Treg cell,” and as used herein, “Treg population” may also be referred to as “cell population” or “Treg cell population.” It will be understood that a Treg population may include both Treg cells of the present invention, which include nucleic acid molecules / polynucleotide molecules, polypeptide molecules, expression constructs, or vectors as defined herein, and Treg cells that do not include nucleic acid molecules / polynucleotide molecules, polypeptide molecules, expression constructs, or vectors described herein, for example, cells that have not been transfected or transfected. In a preferred embodiment, all Treg cells in the population may contain the nucleic acid molecule / polynucleotide molecule, polypeptide molecule, expression construct, or vector described herein, but a cell population is provided having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of cells containing the nucleic acid molecule / polynucleotide molecule, polypeptide molecule, expression construct, or vector of the present invention.

[0076] Natural killer (NK) cells are immune cells that have been reported to have regulatory effects. In particular, NK cells that are CD56 bright may exhibit regulatory properties. In this regard, the present invention provides the use of NK cells or NK cell populations in any method of the present invention (i.e., replacing Treg cells or Treg populations with NK cells or NK populations). NK cells or NK populations containing exogenous polynucleotides encoding the FOXP3 polypeptide are also provided, said NK cells or NK populations having higher CD62L expression after cryopreservation compared to the corresponding unoperated NK cells or NK populations after cryopreservation.

[0077] [Forkheadbox P3 Protein (FOXP3)] In this invention, the expression of FOXP3 in Treg cells is increased by introducing a polynucleotide encoding the FOXP3 polypeptide (sometimes referred to as the first polynucleotide herein) into the cells.

[0078] "FOXP3" is an abbreviation for the forkhead box P3 protein. FOXP3 is a member of the FOX protein family of transcription factors and functions as a major regulator of regulatory pathways in the development and function of regulatory T cells. As used herein, "FOXP3" encompasses variants, isoforms, and functional fragments of FOXP3.

[0079] The levels of FOXP3 mRNA and / or FOXP3 protein in cells (or populations of cells) may be increased compared to the corresponding unmodified cells (or populations of cells). For example, the levels of FOXP3 mRNA and / or FOXP3 protein in cells (or populations of such cells) modified according to the present invention may be increased by at least 1.5 times, at least 2 times, at least 5 times, at least 10 times, at least 50 times, at least 100 times, or at least 150 times compared to the corresponding unmodified cells (or populations of such cells). The cells are Tregs, or the populations of cells are populations of Tregs.

[0080] Preferably, the levels of FOXP3 mRNA and / or FOXP3 protein in cells (or populations of such cells) modified according to the present invention may be increased by at least 1.5, 2, or 5 times compared to the levels in the corresponding cells (or populations of such cells) that have not been modified according to the present invention. The cells are Tregs, or the population of cells is a population of Tregs.

[0081] Techniques for measuring specific mRNA and protein levels are well known in the art. mRNA levels in cell populations such as Treg cells may be measured by techniques such as Affymetrix eBioscience PrimeFlow RNA assay, Northern blotting, gene expression linkage analysis (SAGE), or quantitative polymerase chain reaction (qPCR). Protein levels in cell populations may be measured by techniques such as flow cytometry, high-performance liquid chromatography (HPLC), liquid chromatography / mass spectrometry (LC / MS), Western blotting, or enzyme-linked immunosorbent assay (ELISA).

[0082] A "FOXP3 polypeptide" is a polypeptide possessing FOXP3 activity, i.e., a polypeptide capable of functioning as a transcription factor that binds to FOXP3 target DNA and modulates the development and function of Tregs. In particular, a FOXP3 polypeptide may have the same or similar activity as wild-type FOXP3 (SEQ ID NO: 1), for example, at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, or 150% of the activity of wild-type FOXP3 polypeptide. Techniques for measuring transcription factor activity are well known in the art. For example, transcription factor DNA binding activity may be measured by ChIP. The transcriptional regulatory activity of a transcription factor may be measured by quantifying the expression level of the gene it regulates. Gene expression may be quantified by measuring the levels of mRNA and / or protein produced from the gene using techniques such as Northern blotting, SAGE, qPCR, HPLC, LC / MS, Western blotting, or ELISA. Genes regulated by FOXP3 include cytokines such as IL-2, IL-4, and IFN-γ (Siegler et al. Annu. Rev. Immunol. 2006, 24: 209-26, incorporated herein by reference). As detailed below, FOXP3 or the FOXP3 polypeptide includes its functional fragments, variants, and isoforms, including, for example, the functional fragment, variants, and isoforms of SEQ ID NO: 1.

[0083] A “functional fragment of FOXP3” may refer to a portion or region of the FOXP3 polypeptide, or a portion or region of a polynucleotide encoding the FOXP3 polypeptide, having the same or similar activity as the full-length FOXP3 polypeptide or polynucleotide. The functional fragment may have at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the activity of the full-length FOXP3 polypeptide or polynucleotide. Those skilled in the art will be able to generate functional fragments based on the known structural and functional characteristics of FOXP3. These are described, for example, in Song, X., et al., 2012. Cell reports, 1(6), pp.665-675; Lopes, JE, et al., 2006. The Journal of Immunology, 177(5), pp.3133-3142; and Lozano, T., et al., 2013. Frontiers in oncology, 3, p.294. Furthermore, FOXP3 fragments with shortened N-terminuses and C-terminuses are described in International Publication No. 2019 / 241549 (incorporated herein by reference), for example, having the sequence of Sequence ID No. 5, described below.

[0084] A “FOXP3 variant” may contain an amino acid sequence or nucleotide sequence having at least 50%, at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identity with the FOXP3 polypeptide or the polynucleotide encoding the FOXP3 polypeptide, preferably an amino acid sequence or nucleotide sequence having at least 95%, at least 97%, or at least 99% identity. A FOXP3 variant may have the same or similar activity as the wild-type FOXP3 polypeptide or polynucleotide, for example, at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, or 150% of the activity of the wild-type FOXP3 polypeptide or polynucleotide. Those skilled in the art will be able to generate FOXP3 variants based on the known structural and functional characteristics of FOXP3 and / or using conserved substitutions. FOXP3 mutants may have a turnover time (or degradation rate) in Treg cells similar to or the same as wild-type FOXP3, for example, at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the turnover time (or degradation rate) of wild-type FOXP3 in Tregs. Some FOXP3 mutants may have a reduced turnover time (or degradation rate) compared to wild-type FOXP3, for example, FOXP3 mutants having amino acid substitutions at amino acid positions 418 and / or 422 of SEQ ID NO: 1, e.g., S418E and / or S422A, described in International Publication No. 2019 / 241549 (incorporated herein by reference), as described in SEQ ID NOs: 2-4. These SEQ ID NOs: 2-4 represent the aa418 mutant, the aa422 mutant, and the aa418 and aa422 mutants, respectively.

[0085] Preferably, the FOXP3 polypeptide encoded by the nucleic acid molecules, constructs, or vectors described herein may include a human FOXP3 polypeptide sequence such as UniProtKB accession Q9BZS1 (SEQ ID NO: 1), or a functional fragment or variant thereof.

[0086] In some embodiments of the present invention, the FOXP3 polypeptide comprises an amino acid sequence or a functional fragment thereof having at least 70% identity with SEQ ID NO: 1. Preferably, the FOXP3 polypeptide comprises an amino acid sequence or a functional fragment thereof having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO: 1. In some embodiments, the FOXP3 polypeptide comprises SEQ ID NO: 1 or a functional fragment thereof, or comprises SEQ ID NO: 1 or a functional fragment thereof.

[0087] In some embodiments, as described above, the FOXP3 polypeptide may include mutations in residues 418 and / or 422 of SEQ ID NO: 1, as described in SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0088] In some embodiments of the present invention, the FOXP3 polypeptide may be shortened at the N-terminus and / or C-terminus to obtain a functional fragment. In particular, the functional fragment of FOXP3 with shortened N-terminus and C-terminus may include the amino acid sequence of SEQ ID NO: 5 or a functional variant thereof having at least 80%, 85%, 90%, 95%, or 99% identity to said amino acid sequence, or may consist of such amino acids or their functional variants.

[0089] Preferably, the FOXP3 polypeptide may be a variant of SEQ ID NO: 1, for example, a native variant. Preferably, the FOXP3 polypeptide is an isoform of SEQ ID NO: 1. For example, the FOXP3 polypeptide may contain a deletion at amino acid positions 72 to 106 compared to SEQ ID NO: 1. Alternatively, the FOXP3 polypeptide may contain a deletion at amino acid positions 246 to 272 compared to SEQ ID NO: 1.

[0090] Preferably, the FOXP3 polypeptide comprises SEQ ID NO: 6 or a functional fragment thereof. SEQ ID NO: 6 represents an exemplary FOXP3 polypeptide.

[0091] Preferably, the FOXP3 polypeptide comprises or consists of an amino acid sequence or functional fragment thereof that is at least 70% identical to SEQ ID NO: 6. Preferably, the FOXP3 polypeptide comprises or consists of an amino acid sequence or functional fragment thereof that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 6. In some embodiments, the FOXP3 polypeptide comprises or consists of SEQ ID NO: 6 or a functional fragment thereof.

[0092] Preferably, the FOXP3 polypeptide may be a variant of SEQ ID NO: 6, for example, a native variant. Preferably, the FOXP3 polypeptide may be an isoform of SEQ ID NO: 6 or a functional fragment thereof. For example, the FOXP3 polypeptide may contain a deletion at amino acid positions 72 to 106 compared to SEQ ID NO: 6. Alternatively, the FOXP3 polypeptide may contain a deletion at amino acid positions 246 to 272 compared to SEQ ID NO: 6.

[0093] Preferably, the polynucleotide encoding the FOXP3 polypeptide includes or consists of the polynucleotide sequence described in SEQ ID NO: 7. SEQ ID NO: 7 represents an exemplary FOXP3 nucleotide sequence.

[0094] In some embodiments of the present invention, the polynucleotide encoding the FOXP3 polypeptide or variant comprises a nucleotide sequence that is at least 70% identical to SEQ ID NO: 7, or a fragment thereof that encodes a functional FOXP3 polypeptide. Preferably, the polynucleotide encoding the FOXP3 polypeptide or variant comprises a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 7, or a functional fragment thereof. In some embodiments of the present invention, the polynucleotide encoding the FOXP3 polypeptide or variant comprises SEQ ID NO: 7 or a fragment thereof that encodes a functional FOXP3 polypeptide, or consists of SEQ ID NO: 7 or a fragment thereof that encodes a functional FOXP3 polypeptide.

[0095] Preferably, the polynucleotide encoding the FOXP3 polypeptide includes or consists of the polynucleotide sequence described in SEQ ID NO: 8. SEQ ID NO: 8 represents another exemplary FOXP3 nucleotide.

[0096] In some embodiments of the present invention, the polynucleotide encoding the FOXP3 polypeptide or variant comprises a polynucleotide sequence that is at least 70% identical to SEQ ID NO: 8, or a fragment thereof encoding a functional FOXP3 polypeptide. Preferably, the polynucleotide encoding the FOXP3 polypeptide or variant comprises a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to SEQ ID NO: 8, or a fragment thereof encoding a functional FOXP3 polypeptide. In some embodiments of the present invention, the polynucleotide encoding the FOXP3 polypeptide or variant comprises SEQ ID NO: 8 or a fragment thereof encoding a functional FOXP3 polypeptide, or consists of SEQ ID NO: 8 or a fragment thereof encoding a functional FOXP3 polypeptide.

[0097] Preferably, the polynucleotide encoding the FOXP3 polypeptide or a functional fragment or variant thereof may be codon-optimized. Preferably, the polynucleotide encoding the FOXP3 polypeptide or a functional fragment or variant thereof may be codon-optimized for expression in human cells.

[0098] As used herein, the term “mutant” includes any substitution, mutation, modification, replacement, deletion, and / or addition of one or more amino acid residues to or from a sequence, provided that the resulting protein or polypeptide retains the desired function. Alternatively, as used herein, a mutant or derivative is a functional mutant or functional derivative.

[0099] Typically, amino acid substitutions may range from one, two, or three substitutions to ten or twenty substitutions, provided that the modified sequence retains the desired activity or capability. Amino acid substitutions may include the use of analogues that do not exist in nature.

[0100] Proteins or peptides may also have deletions, insertions, or substitutions of amino acid residues that undergo silent changes, resulting in functionally equivalent proteins. Intentional amino acid substitutions may be made based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues, as long as endogenous function is preserved. For example, aspartic acid and glutamic acid are negatively charged amino acids, lysine and arginine are positively charged amino acids, and asparagine, glutamine, serine, threonine, and tyrosine are amino acids with similar hydrophilic values ​​and uncharged head groups.

[0101] Conservative substitutions may be performed, for example, according to Table 1 below. [Table 1]

[0102] The comparison of sequences can be performed visually, or more commonly, using readily available sequence comparison programs. These commercially available computer programs can calculate the percentage of identical sequences.

[0103] Sequence identity can also be calculated over consecutive sequences. That is, one sequence is aligned with the other, and each amino acid in one sequence is directly compared residue by residue with the corresponding amino acid in the other sequence. This is called "ungapped" alignment. Typically, such ungapped alignment is performed only for relatively short sequences of residues.

[0104] While this is a very simple and consistent method, it fails to account for the fact that, for example, in a pair of otherwise identical sequences, a single insertion or deletion in the nucleotide sequence can cause the subsequent codons to become misaligned, thus potentially significantly reducing the homology score when global alignment is performed. Therefore, most sequence comparison methods are designed to create an optimal alignment that takes possible insertions and deletions into account without excessively disadvantageing the overall homology score. This is achieved by inserting "gaps" into the sequence alignment to maximize local homology.

[0105] However, these more complex methods assign a "gap penalty" to each gap in the alignment, thereby ensuring that the sequence alignment with the fewest possible gaps achieves a higher score than the sequence alignment with the most gaps, reflecting a higher correlation between the two sequences being compared for the same number of identical amino acids. Typically, an "affine gap cost" is used, which places a relatively high cost on the presence of gaps and a lower cost on each subsequent residue within those gaps. This is the most commonly used gap scoring system. Naturally, a higher gap penalty results in a more optimized alignment with fewer gaps. Most alignment programs allow you to change the gap penalty. However, when using such software for sequence comparison, it is preferable to use the default values. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for gaps and -4 for each extension.

[0106] Therefore, calculating the maximum homology / maximum sequence identity requires first creating an optimal alignment that takes gap penalties into account. One suitable computer program for performing such alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Res. 12: 387). Other software capable of sequence comparison includes, but is not limited to, the BLAST package (Ausubel et al. (1999), see Chapter 18), FASTA (Atschul et al. (1990) J.Mol.Biol. 403-410), and the GENEWORKS comparison tools suite. Both BLAST and FASTA are available for offline and online searches (see Ausubel et al. (1999), pp. 7-58 to 7-60). However, depending on the application, the GCG Bestfit program is preferable. Another tool called BLAST 2 Sequences is also available for comparing protein and nucleotide sequences (see FEMS Microbiol. Lett. (1999) 174: 247-50; FEMS Microbiol. Lett. (1999) 177: 187-8).

[0107] While the final homology rate can be measured in terms of identity, the alignment process itself is not typically based on all-or-nothing pair comparisons. Instead, a scaled similarity score matrix is ​​commonly used, which assigns a score to each pair comparison based on chemical similarity or evolutionary distance. An example of such a commonly used matrix is ​​the BLOSUM62 matrix, which is the default matrix for a set of BLAST programs. GCG Wisconsin programs generally use either publicly available default values ​​or custom symbol comparison tables, if provided (see user manual for details). Depending on the application, it may be preferable to use the publicly available default values ​​for the GCG package, or, in the case of other software, to use a default matrix such as BLOSUM62. Preferably, the identity rate is determined across the entire reference sequence and / or query sequence. Once the software has created the optimal alignment, it becomes possible to calculate the homology rate, preferably the sequence identity rate. The software typically does this as part of the sequence comparison and generates a numerical result.

[0108] Mutants and fragments can be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. For insertions, synthetic DNA encoding the insert (insertion portion) may be prepared along with 5' and 3' flanking regions corresponding to naturally occurring sequences on either side of the insertion site. Each flanking region contains a convenient restriction site corresponding to a location in the naturally occurring sequence, thereby allowing the sequence to be cleaved by an appropriate enzyme(s) and the synthetic DNA to ligate the cleavage site. The DNA is then expressed according to the present invention to produce the encoded protein. These methods are merely examples of numerous standard techniques known in the art for DNA sequencing, and other known techniques may be used.

[0109] Those skilled in the art will understand that FOXP3 expression within a Treg can be indirectly increased by introducing polynucleotides into the cell that encode proteins that increase FOXP3 transcription and / or translation, or that increase the half-life of FOXP3 (e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%), or that enhance the function of FOXP3 (e.g., function determined by the repressive ability of the transduced Treg, as measured above). For example, the introduction of polynucleotides that increase endogenous FOXP3 transcription into a Treg can be done by interacting them with non-coding sequences (CNS, e.g., CNS1, CNS2, or CNS3) found upstream of the endogenous FOXP3 promoter or coding region. In particular, FOXP3 expression within a Treg may be increased by introducing polynucleotides that encode one or more of c-Rel, p65, Smad3, or CREB.

[0110] In this embodiment, the present invention further provides a method for preserving CD62L expression in a cryopreserved regulatory T cell (Treg) population, comprising introducing a polynucleotide that encodes a protein that increases the transcription and / or translation of FOXP3, or a protein that increases the half-life or function of FOXP3.

[0111] Alternatively, the present invention provides a method for preserving a population of regulatory T cells (Tregs) or CD62L expression in Tregs for cryopreservation, further comprising the steps of (a) introducing a polynucleotide encoding a protein that increases the transcription and / or translation of FOXP3, or a protein that increases the half-life or function of FOXP3, into a population of Tregs or Tregs, and (b) cryopreserving the population of Tregs or Tregs.

[0112] Alternatively, the present invention may provide a method for cryopreserving a Treg (or population of Tregs) comprising (a) introducing a polynucleotide encoding a protein that increases the transcription and / or translation of FOXP3, or a protein that increases the half-life or function of FOXP3, into a Treg or population of Tregs, and (b) cryopreserving the Treg or population of Tregs, wherein the cryopreserved Treg or population of Tregs has a higher CD62L expression level compared to the corresponding unmanipulated Treg or population of Tregs.

[0113] In another embodiment, the present invention provides a method for producing cryopreserved Tregs or populations of Tregs having CD62L levels equivalent to those of non-cryopreserved Tregs or populations of Tregs, comprising: (a) introducing a polynucleotide encoding a protein that increases the transcription and / or translation of FOXP3, or a protein that increases the half-life or function of FOXP3, into a Treg or population of Tregs; and (b) cryopreserving the Tregs or populations of Tregs.

[0114] Those skilled in the art will also understand that by introducing a polynucleotide directly encoding CD62L into cells described herein (e.g., Tregs), function (e.g., repressive function) or stability can be preserved after cryopreservation. In this embodiment, the present invention further provides a method for preserving the function or stability of Tregs or Treg populations (or other cells, e.g., NK cells) during / after cryopreservation, comprising the step of introducing a polynucleotide encoding CD62L into the cells or cell population prior to cryopreservation. The present invention further provides cryopreserved cells (in particular, Tregs) containing a polynucleotide encoding exogenous CD62L, the cells having equivalent function or stability to the corresponding non-cryopreserved cells. The polynucleotide may encode CD62L comprising the amino acid sequence of SEQ ID NO: 9 or a functional fragment or functional variant thereof (i.e., a fragment or variant that retains at least 50%, 60%, 70%, 80%, 90%, or 95% of the function of CD62L in SEQ ID NO: 9). The variant may have at least 60%, 70%, 80%, 90%, or 95% identity with respect to sequence number 9.

[0115] [Expression of phenotypic markers] CD62L, also known as L-selectin, is a homing receptor or cell surface adhesion molecule found on the extracellular surface of certain T lymphocytes (e.g., Tregs) and other immune cells such as neutrophils. CD62L belongs to the selectin family of proteins that recognize sialylated carbohydrate groups. CD62L is cleaved by ADAM17.

[0116] CD62L plays a crucial role in the interaction between lymphocytes and endothelial cells, and CD62L expression is associated with a more repressive population obtained after Treg proliferation. CD62L acts as a "homing receptor" for Tregs to enter secondary lymphoid tissues via high endothelial venules. Ligands present on endothelial cells bind to CD62L-expressing Tregs, slowing the rate of Treg transport through the bloodstream and thus facilitating their entry into secondary lymphoid organs.

[0117] As used herein, the terms “homing” or “home” mean moving to a target, such as a site or location containing a binding target. When referring to Treg cells expressing CD62L, this may mean moving to a site where MADCAM1, the binding partner of CD62L, is expressed.

[0118] CD62L is a cell surface marker, and among the many markers, the presence of CD62L in particular indicates that proliferating cells retain the Treg phenotype. Cells that retain the Treg phenotype are also likely to retain their normal repressive function.

[0119] CD62L typically has the following amino acid sequence. MIFPWKCQSTQRDLWNIFKLWGWTMLCCDFLAHHGTDCWT YHYSEKPMNW QRARRFCRDNYTDLVAIQNKAEIEYLEKTLPFSRSYYWIGIRKIGGIWTW VGTNKSLTEE AENWGDGEPNNKKNKEDCVEIYIKRNKDAGKWNDDACHKLKAALCYTASCQPWSCSGHGECVEIINNYTCNCDVGYYGPQCQFVIQCEPLEAPELGTMDCTHPLGNFSFSSQCAFSCSEG TNLTGIEETTCGPGFNWSSPEPTCQVIQCE PLSAPDLGIM NCSHPLASFS FTSACTFICS EGTELIGKKKTICESSGIWS NPSPICQKLD KSFSMIKEGD YNPLFIPVAV MVTAFSGLAF IIWLARRLKK GKKSKRSMND PY(sequence number 9)

[0120] The cryopreserved operated Tregs (or Treg populations) described herein may preserve the expression of cell surface markers, particularly the cell surface expression of CD62L, compared to cryopreserved Tregs that do not contain exogenous FOXP3. As used herein, the terms “preserved,” “preserving,” or “preservation” mean that the expression level of the cell surface marker (e.g., CD62L) remains at or equivalent to the expression level of the cell surface marker on the corresponding fresh (i.e., non-cryopreserved or non-frozen) Treg. That is, the expression of the cell surface marker only needs to be the same as (i.e., not significantly different from) the expression level of the cell surface marker on the fresh Treg, or it may be as little as 1% less than the expression on the fresh Treg, or at most 30% less. Preferably, the expression of the cell surface marker is 5% or less, 10% or less, 15% or less, 20% or less, or 25% or less less. Typically, cryopreserved Treg cells and fresh Treg cells used to compare the expression levels of a cell surface marker (typically CD62L) will be understood to be of the same species or derived from the same species. For example, the level of CD62L in frozen naive CD45RA+ Tregs should be compared to the level of CD62L in fresh or unfrozen naive CD56RA+ Tregs. Typically, the corresponding non-cryopreserved cells (Tregs) will have been processed or treated in the same manner as the cells of the present invention or the cells used in the present invention, and the same polynucleotide sequence will have been introduced into the corresponding non-cryopreserved cells, except that the cryopreservation process (and any thawing process) is not performed. Thus, the corresponding non-cryopreserved cells are typically the corresponding manipulated non-cryopreserved cells.

[0121] Alternatively, as used herein, “preserved,” “preserving,” or “preservation” means that the amount of cell surface marker on the cryopreserved operated Treg described herein is higher (in particular, significantly higher) than the amount of cell surface marker on the corresponding untransduced cryopreserved Treg. Preferably, the expression level of the cell surface marker is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 80% higher than the expression level of the cell surface marker on the untransduced cryopreserved Treg. As used herein, the terms “preserved” or “preserving” are synonymous with the terms “maintained” or “maintaining.”

[0122] As used herein, the term “higher CD62L expression” means that the cryopreserved manipulated Tregs described herein exhibit increased (or elevated) CD62L expression levels compared to cryopreserved Tregs without exogenous FOXP3. Therefore, where the corresponding unmanipulated cells or Tregs are referred herein, they typically refer to cells or Tregs that have undergone, or are scheduled to undergo, the same cryopreservation process (and any thawing process) as the cells or Tregs of the present invention or those used herein, but which have not been introduced with the exogenous polynucleotide encoding FOXP3.

[0123] A cryopreserved Treg or Treg population comprising a polynucleotide, polypeptide, expression construct, or vector as defined herein (and thus having a preserved CD62L expression level as defined herein, and / or a higher CD62L expression level) may exhibit increased repressive activity (e.g., increased repressive activity by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) compared to an unmanipulated cryopreserved Treg or Treg population as defined herein.

[0124] CD62L expression can be determined by any method known in the art, including flow cytometry. Antibodies for detecting CD62L and determining its expression level are commercially available, such as Thermofisher CD62L (L-selectin) monoclonal antibodies (MEL-14 or DREG-56).

[0125] As described herein, the polynucleotide encoding the FOXP3 polypeptide is introduced into the Treg prior to cryopreservation. The term “prior to cryopreservation” means before the Treg is cryopreserved (i.e., before the Treg is frozen). Therefore, the Treg described herein is not subjected to the cryopreservation process before the introduction of the polynucleotide encoding the FOXP3 polypeptide. Typically, the polynucleotide is introduced into the Treg after isolation and before growth (both isolation and growth can be performed before cryopreservation).

[0126] [Crozen preservation] As used herein, the terms “cryopreservation” or “cryopreserving” mean freezing a Treg or population of Tregs under conditions that the cells remain viable (e.g., during freezing and after any subsequent thawing steps). Cell viability can be measured by any method well known in the art, e.g., flow cytometry using live / dead staining (e.g., LIVE / DEAD® Fixable Near-IR-Dead Cell Stain (Thermofisher)). Typically, at least 50%, 60%, 70%, 80%, 90%, or 95% of cells remain viable during and after cryopreservation. Thus, viability refers to live cells. Those skilled in the art will understand that cryopreservation can keep cells viable by applying one or more conditions (e.g., specific temperature, freeze / thaw rate, and / or use of cryopreservatives) that effectively prevent cell death and maintain the cell structure. These conditions are publicly known in the relevant technical field and are described below.

[0127] "Cryopreserved Tregs" or "cryopreserved Treg populations" refer to Tregs or Treg populations that have been subjected to and are cryopreserved (e.g., in a cryopreserved or frozen state) under conditions in which the cells remain viable as defined above. As previously mentioned, "cryopreserved Tregs" include exogenous FOXP3. "Cryopreserved Tregs" may also be referred to as "cryopreserved therapeutic Tregs or Treg populations" or "cryopreserved operational Tregs or Treg populations."

[0128] "Treg or Treg population that has undergone cryopreservation" refers to Treg or Treg population that has undergone cryopreservation as described above, and which is either still cryopreserved (e.g., in a cryopreserved state) or may no longer be cryopreserved, that is, which includes Treg or Treg populations that are thawed (i.e., were previously cryopreserved).

[0129] As used herein, “after cryopreservation” means that the cryopreservation process has been carried out. A cryopreserved Treg or Treg population may be in a cryopreserved state (e.g., in storage), or it may be thawed or in the process of thawing.

[0130] "Non-freezed Treg" or "non-freezed Treg population" refers to Treg cells or Treg populations that have not undergone cryopreservation, that is, have never been cryopreserved or frozen. Such cells are sometimes referred to as "fresh" as an alternative term.

[0131] "Freezing," "frozen," or "frozen state" have their usual meanings in the art and refer to the process or result of a liquid changing into a solid. To freeze a Treg or a group of Tregs means to lower the temperature of the Treg or group of Tregs to a temperature at which the particles can no longer maintain enough energy to overcome the attractive forces between them.

[0132] Frozen-preserved operational Tregs or Treg populations are typically stored at -196°C. However, cryopreserved operational Tregs or Treg populations may be stored at any temperature below the freezing point (i.e., any temperature below 0°C) in which the cells (or some cells) remain viable, as described above. For example, they may be stored at any temperature between -50°C and -196°C, between -80°C and -196°C, or below -55°C, below -60°C, below -65°C, below -70°C, below -75°C, below -80°C, below -85°C, below -90°C, below -95°C, below -100°C, below -105°C, below -110°C, below -115°C, below -120°C, below -125°C, below -130°C, below -135°C, below -140°C, below -145°C, below -150°C, below -155°C, below -160°C, below -165°C, or below -170°C. Cryopreserved Tregs or Treg populations may be stored at any temperature from below the freezing point to -196°C, for example, below -175°C, below -180°C, below -185°C, or below -190°C. Preferably, cryopreserved Tregs or Treg populations may be stored at -80°C, below -130°C, or in the gas phase of liquid nitrogen at approximately -196°C. More preferably, cryopreserved Tregs or Treg populations may be stored in liquid nitrogen.

[0133] In the cryopreservation process of the present invention, the Treg or Treg population, one or more (preferably all) of the reagents used (e.g., cryopreservation media) and devices (e.g., control rate devices and freezers, many of which are known in the art, e.g., ThermoFisher's CryoMed) may be pre-cooled (or pre-chilled) at the start of the cryopreservation process. As used herein, “pre-chilled” or “pre-chilling” means lowering the temperature to between approximately 4°C and 8°C. This ensures that the temperature shock to the cells is minimized. The pre-chilling step may be performed for, for example, approximately 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 30 minutes.

[0134] During cryopreservation, Treg populations are typically protected by contact with a cryopreservation medium (also known as a cryopreservation solution) containing one or more cryoprotective agents described herein. This is usually done before lowering the temperature of the Treg or Treg population to the cryopreservation temperature, i.e., the temperature at which the Treg or Treg population is stored. Before lowering the temperature, the Treg population may be centrifuged to form a cell pellet and resuspended in the cryopreservation medium. The cryopreservation medium typically contains a complete growth medium together with a cryoprotective agent as described above, and the complete growth medium may be specifically optimized to provide viable cells upon thawing. Many different types of cryopreservation media are commercially available, for example, ImmunoCult-XF T cell medium (StemCell). Preferably, the cryopreservation medium may be any medium that conforms to GMP standards. The cryoprotective agent is a substance used to protect living tissue from freeze damage caused by the formation of ice crystals. Cryoprotective agents are classified into two general categories: permeable cryoprotective agents that can pass through cell membranes and impermeable cryoprotective agents, and either type of cryoprotective agent can be used according to the cryopreservation step of the method of the present invention. Examples of permeable cryoprotective agents include, but are not limited to, dimethyl sulfoxide (DMSO), glycerol, sucrose, and 1,2-propanediol. Examples of impermeable cryoprotective agents include, but are not limited to, hydroxyethyl starch, albumin, and polyvinylpyrrolidone. The most commonly used permeable cryoprotective agent is DMSO, which is often used in combination with an impermeable substance such as autologous plasma, serum albumin, and / or hydroxyethyl starch. Any cryoprotective agent known in the art may be used in the method of the present invention. Preferably, the cryoprotective agent used in the method of the present invention is a cryoprotective agent that conforms to Good Manufacturing Process (GMP) standards.

[0135] Those skilled in the art will understand that the number of Tregs that may be resuspended in cryopreservation medium (i.e., medium containing one or more cryoprotective agents) may depend on the dose provided to the target, and that such doses may vary between Treg products and depending on the disease or condition being treated. The Treg population can be cryopreserved at any desired cell density.

[0136] The optimal freezing rate for a Treg or Treg population can be determined by several factors, including the permeability of the Treg cell membrane to water, the ratio of cell surface to volume, and the type and concentration of cryoprotectant used. The Treg or Treg population may be continuously cooled in the range of about 4°C to -80°C, and more specifically, a controlled freezing rate (e.g., about -1°C / min) may be used. Alternatively, the controlled freezing rate may be, for example, about -2°C / min, -3°C / min, -4°C / min, or -5°C / min. Any suitable controlled-rate freezing device known in the art may be used to achieve freezing at a controlled rate. Those skilled in the art will understand that ice nucleation (i.e., induction of freezing of the population by ice crystal formation in the extracellular fluid) can be initiated at about -5°C by any suitable means. Once the temperature reaches about -80°C, the Treg or population can be directly transferred to liquid nitrogen (-196°C) for storage.

[0137] Tregs or Treg populations may be stored at -80°C for any desired period of time, for example, from approximately 4 to 48 hours, preferably from 4 to 16 hours, or up to 24 hours, after which they may be transferred to liquid nitrogen storage below -130°C. Alternatively, once the Tregs or Treg populations reach -80°C, they may be immediately transferred to liquid nitrogen storage below -130°C. Storage in liquid nitrogen may be carried out for any desired period of time. Storage may be carried out for any number of days or weeks. Typically, storage may be carried out for a long period of time, i.e., any number of months, one year, or any number of years, for example, two years, five years, ten years, twenty years, fifty years, or one hundred years.

[0138] Alternatively, Tregs or Treg clusters may be cryopreserved by vitrification. Vitrification is a very rapid cryopreservation method that allows Tregs or Treg clusters to be cooled from approximately 37°C to -196°C in a short time (typically less than 1 second), resulting in extremely rapid cooling.

[0139] Tregs or Treg populations may be thawed after cryopreservation. “Thawing,” “thawed,” or “thawed state” have their usual meanings in the art and refer to the process or result of a solid changing into a liquid. Thawing a Treg or Treg population means raising the temperature of the Treg or population to a temperature at which its particles have enough energy to overcome the interparticle attractive forces. Therefore, heat is usually applied to thaw a Treg or Treg population. Typically, thawed cells are capable of proliferation and / or function as Tregs (e.g., exerting their immunosuppressive function). Thus, a thawed Treg or Treg population refers to a Treg or population subjected to both cryopreservation and the thawing process. Those skilled in the art will understand that a thawed Treg population or the thawing process does not necessarily result in the complete thawing of the population, and some cells within the population may remain frozen. For example, a thawed Treg population may contain at least 10%, 20%, 30%, 40%, or 50% of frozen or cryopreserved cells. Alternatively, a thawed Treg population may contain 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less of frozen or cryopreserved cells.

[0140] The optimal thawing rate varies depending on the cryopreservation conditions used. Generally, rapid heating may be applied to cryopreserved Treg cells, which can be achieved by placing the composition in a water bath at a temperature of approximately 37°C or by agitating (e.g., shaking) it in the water bath. The Tregs or Treg population may be warmed to a cooling temperature (approximately 0°C to 10°C) until small ice crystals remain, or they may be warmed to, for example, human body temperature of approximately 37°C for direct administration to a patient.

[0141] In one embodiment, the cryopreserved Treg or Treg population may be warmed to a temperature at which it changes from solid to liquid and administered directly to the patient.

[0142] The Treg or Treg population described herein may be administered parenterally, for example, by intravenous administration or infusion techniques. Parenteral administration as used herein means administration methods other than enteral and topical administration, and is usually by injection, including intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intra-articular, transtracheal, intradermal, intraperitoneal, subcutaneous, subcapsular, subarachnoid, intravertebral, and intracisional injections and infusions.

[0143] [T cell receptors (TCRs) and chimeric antigen receptors (CARs)] Preferably, the Treg or Treg population of the present invention may further contain an exogenous TCR or CAR.

[0144] TCRs are cell surface molecules that, as part of directing the immune response, bind to fragments of antigens bound to major histocompatibility complex (MHC) molecules on antigen-presenting cells. Preferably, TCRs may be recombinant proteins, in other words, TCRs may be exogenous proteins that are not spontaneously expressed by the Treg of the present invention.

[0145] As used herein, the terms “TCR” and “CAR (chimeric antigen receptor)” refer to engineered receptors that can confer antigen specificity to Treg cells (e.g., Treg).

[0146] CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors. A CAR typically comprises an extracellular domain containing an antigen-specific targeting region, referred herein to as an antigen-binding domain; a transmembrane domain; an endodomain optionally containing one or more costimulatory domains; and an intracellular signaling domain. The antigen-binding domain is typically bound to the transmembrane domain by a hinge domain. The design of CARs and the various domains they may contain is well known in the art.

[0147] The antigen-binding domain of a CAR may be derived from, or obtained from, any protein or polypeptide that binds to (i.e., has affinity for) a desired target antigen, or more generally, a desired target molecule. This may be, for example, a ligand or receptor, or a physiologically binding protein or part thereof to the target molecule, or a synthetic protein or derivative protein. The target molecule may generally be expressed on the surface of a cell, for example, a target cell or a cell near a target cell (for bystander effect), but this is not necessarily required. Depending on the nature and specificity of the antigen-binding domain, the CAR may also recognize soluble molecules, for example, if the antigen-binding domain is based on or derived from a cell receptor.

[0148] The antigen-binding domain most commonly originates from the antibody variable chain (for example, generally in the form of scFv), but may also be generated from other molecules, such as the T cell receptor variable domain, or, as mentioned above, a receptor for a ligand or other binding molecule.

[0149] CARs are typically expressed as polypeptides containing a signal sequence (also known as a leader sequence), and more specifically, as polypeptides containing a signal sequence that directs the CAR towards the cell's plasma membrane. This is generally located upstream of the antigen-binding domain, adjacent to or near the antigen-binding domain. Thus, the extracellular domain of the CAR, or ectodomain, may contain both the signal sequence and the antigen-binding domain.

[0150] The antigen-binding domain gives the CAR the ability to bind to a specific antigen of interest. Preferably, the antigen-binding domain targets a clinically relevant antigen or an antigen at the disease site.

[0151] As described above, the antigen-binding domain may be any protein or peptide having the ability to specifically recognize and bind to a biomolecule (e.g., a cell surface receptor or its components). The antigen-binding domain may include any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for the biomolecule of interest. Exemplary antigen-specific targeting domains include antibodies or antibody fragments or antibody derivatives, extracellular domains of receptors, ligands or their receptor-binding domains for cell surface molecules / cell surface receptors, and tumor-binding proteins. As will be discussed later, the antigen-specific targeting domain may preferably be an antibody or antibody-derived, and may also include other antigen-specific targeting domains, such as antigen-specific targeting domains formed from combinations of antigen-specific peptides with MHC or HLA that can bind to the TCR of active Tcon cells at the transplantation site, inflammation site, or disease site.

[0152] The antigen-binding domain may be an antibody or derived from an antibody. An antibody-derived binding domain may be a fragment of an antibody, or a genetically modified product of one or more fragments of an antibody, the fragments being involved in binding to the antigen. Examples include a variable region (Fv), a complementation-determining region (CDR), Fab or F(ab')2, or in a single chain (e.g., as ScFv) in either direction (e.g., V). L -V H or V H -V L Examples include light chain variable regions and heavy chain variable regions that can be bonded. L and / or V H The sequences may be modified. In particular, the framework region may be modified (for example, it may be substituted, for example, to humanize the antigen-binding domain). Other examples include heavy chain variable regions (VH), light chain variable regions (VL), camel antibodies (VHH), and single-domain antibodies (sAb).

[0153] Typically, the binding domain is a single-chain antibody (scFv). The scFv may be a mouse scFv, a human scFv, or a humanized scFv.

[0154] With respect to an antibody or its antigen-binding fragment, the "complementarity-determining region" or "CDR" refers to a highly variable loop within the variable region of the antibody's heavy or light chain. CDRs can interact with the higher-order structure (conformation) of the antigen and largely determine binding to the antigen (although several framework regions are known to be involved in binding). The heavy chain variable region and the light chain variable region each contain three CDRs. The "heavy chain variable region" or "VH" refers to a fragment of the antibody's heavy chain containing three CDRs inserted between flanking stretches known as framework regions. Framework regions are more conserved than CDRs and form a scaffold supporting the CDRs. The "light chain variable region" or "VL" refers to a fragment of the antibody's light chain containing three CDRs inserted between framework regions. The "Fv" refers to the smallest fragment of the antibody that has a complete antigen-binding site. The Fv fragment consists of a single light chain variable region bound to a single heavy chain variable region. A "single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light-chain variable region and a heavy-chain variable region linked to each other either directly or via a peptide linker sequence. Antibodies that specifically bind to a given antigen can be prepared using methods well known in the art. Such methods include phage display, methods for generating human antibodies or humanized antibodies, or methods using transgenic animals or plants engineered to produce human antibodies. Phage display libraries of partially or fully synthetic antibodies are available and can be screened for antibodies or fragments thereof that can bind to a target molecule. Phage display libraries of human antibodies are also available. After identification, the amino acid sequence or polynucleotide sequence encoding the antibody can be isolated and / or determined.

[0155] A CAR may be directed to any desired target antigen or target molecule. This antigen or molecule may be selected depending on the intended therapy and the condition to be treated. For example, it may be an antigen or molecule associated with a particular condition, or an antigen or molecule associated with cells that are to be targeted in order to treat that condition. Typically, the antigen or molecule is a cell surface antigen or cell surface molecule.

[0156] The term "directed against" is synonymous with "specific for" or "anti." In other words, a CAR recognizes a target molecule. This means that the CAR can specifically bind to a specified antigen or a given antigen, i.e., a target. In particular, the antigen-binding domain of the CAR can specifically bind to the target molecule or target antigen (more specifically, when the CAR is expressed on the surface of a cell, especially on the surface of an immunoeffector cell). Specific binding can be distinguished from nonspecific binding to a non-target molecule or non-target antigen. Thus, a cell expressing a CAR is directed or redirected to specifically bind to a target cell expressing the target molecule or target antigen, in particular, to a target cell expressing the target antigen or target molecule on its cell surface.

[0157] Antigens that can be targeted by CARs include, but are not limited to, antigens expressed on cells associated with transplanted organs, autoimmune diseases, allergic diseases, metabolic diseases (e.g., diabetes), and inflammatory diseases (e.g., neurodegenerative diseases).

[0158] Antigens associated with organ transplantation, and / or cells associated with transplanted organs, include, but are not limited to, HLA antigens present in transplanted organs but absent in patients, or antigens whose expression increases during transplant rejection, such as CCL19, MMP9, SLC1A3, MMP7, HMMR, TOP2A, GPNMB, PLA2G7, CXCL9, FABP5, GBP2, CD74, CXCL10, UBD, CD27, CD48, and CXCL11.

[0159] In one embodiment, the CAR is directed towards HLA antigens, particularly HLA-A2 antigens.

[0160] Antibodies against such antigens are publicly known in the art, and conveniently, scFv can be obtained or generated based on known or available antibodies. In this regard, VH and VL sequences, as well as CDR sequences, have been made public to assist in the preparation of such antibody-binding domains, for example, in International Publication No. 2020 / 044055. The disclosures of this document are incorporated herein by reference. Antigen-binding domains disclosed in International Publication No. 2020 / 044055, or any of the CDR sequence, VH sequence, and / or VL sequence, can be used.

[0161] In one embodiment, the antigen-binding domain includes the VH CDR1, VH CDR2, and VH CDR3 sequences described in SEQ ID NOs. 10, 11, and 12, respectively, and the VL CDR1, VL CDR2, and VL CDR3 sequences described in SEQ ID NOs. 13, 14, and 15, respectively.

[0162] If a CDR includes an amino acid sequence modification, this may be a deletion, addition, or substitution of amino acid residues in the CDR sequence described above. More specifically, the modification may be an amino acid substitution, such as a conservative amino acid substitution, as described above. The longer the CDR, the more amino acid residue modifications are permitted. For a CDR with a length of 5 or 7 amino acid residues, one or two residues, for example, one residue, may be subject to modification. In general, zero, one, two, or three modifications may be made to any particular CDR sequence. Furthermore, in one embodiment, CDR1 and CDR2 may be modified, while CDR3 remains unchanged. In another embodiment, all three CDRs may be modified. In yet another embodiment, none of the CDRs are modified.

[0163] The antigen-binding domain may be in the form of an scFV containing the above-mentioned VH domain sequence and VL domain sequence in either order, for example, VH-VL. The VH sequence and VL sequence may be linked by a linker sequence.

[0164] A suitable linker can be easily selected and can be any suitable length from 1 amino acid (e.g., Gly) to 30 amino acids. For example, it may be any length from 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids to any length from 12, 15, 18, 20, 21, 25, or 30 amino acids, for example, 5-30, 5-25, 6-25, 10-15, 12-25, 15-25, etc.

[0165] Preferably, the CAR also includes a hinge domain for keeping the extracellular domain, particularly the antigen-binding domain, away from the cell surface, and a transmembrane domain. The hinge domain and transmembrane domain may include hinge sequences and transmembrane sequences derived from any protein having a hinge domain and / or transmembrane domain, including any type I, type II, or type III transmembrane protein.

[0166] The transmembrane domain of CAR may also include artificial hydrophobic sequences. The transmembrane domain of CAR may be selected to avoid dimerization. Further transmembrane domains will be apparent to those skilled in the art. Examples of transmembrane (TM) regions used in CAR constructs include: 1) CD28 TM region (Pule et al, Mol Ther, 2005, Nov;12(5):933-41; Brentjens et al, CCR, 2007, Sep 15;13(18 Pt 1):5426-35; Casucci et al, Blood, 2013, Nov 14;122(20):3461-72.), 2) OX40 TM region (Pule et al, Mol Ther, 2005, Nov;12(5):933-41), 3) 41BB TM region (Brentjens et al, CCR, 2007, Sep 15;13(18 Pt 1):5426-35), 4) CD3 zeta TM region (Pule et al, Mol Ther, 2005, Nov;12(5):933-41; Di Stasi et al., Blood, 2009, Jun 18;113(25):6392-402.), 5) CD8a™ region (Maher et al, Nat Biotechnol, 2002, Jan;20(1):70-5.; Imai C, Leukemia, 2004, Apr;18(4):676-84; Brentjens et al, CCR, 2007, Sep 15;13(18 Pt 1):5426-35; Milone et al, Mol Ther, 2009, Aug;17(8):1453-64.). Other transmembrane domains that can be used include those derived from CD4, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, or CD154.

[0167] The hinge domain can be conveniently derived from the same protein as the transmembrane domain, but this is not essential.

[0168] Alternatively, the CAR may contain a domain derived from the CD8α transmembrane domain.

[0169] The endodomains of CARs described herein contain motifs necessary to transmit effector function signals upon antigen binding and to instruct cells expressing the CAR to perform its specific function. In particular, the endodomain may contain one or more (e.g., two or three) immune receptor tyrosine activation motifs (ITAMs), which typically contain the amino acid sequence YXXL / I (where X is any amino acid). Examples of intracellular signaling domains include, but are not limited to, any of the ζ-chain endodomains of T cell receptors or their homologs (e.g., η-chain, FcεR1γ and β-chain, MB1(Igα)-chain, B29(Igβ)-chain, etc.), CD3 polypeptide domains (Δ, δ, and ε), syk family tyrosine kinases (Syk, ZAP70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.), and other molecules involved in T cell transduction such as CD2, CD5, and CD28. The intracellular signaling domain may include the human CD3 zeta chain endodomain, FcyRIII, FcsRI, the cytoplasmic tail of the Fc receptor, an immunoreceptor tyrosine activation motif (ITAM) with a cytoplasmic receptor, or a combination thereof.

[0170] Other signaling domains that may be used include the signaling domains of CD28 or CD27 or their variants. Further intracellular signaling domains will be apparent to those skilled in the art and may be used in connection with alternative embodiments of the present invention.

[0171] A CAR may include a compound endodomain, for example, a fusion of the intracellular portion of a T cell costimulatory molecule with the intracellular portion of CD3ζ. Such a compound endodomain may be referred to as a second-generation CAR, capable of simultaneously transmitting activation and costimulatory signals after antigen recognition. The most commonly used costimulatory domain is the CD28 costimulatory domain, which delivers the most potent costimulatory signal, i.e., immunological signal 2, that induces T cell proliferation. The CAR endodomain may also include one or more TNF receptor family signaling domains, such as the signaling domains of OX40, 4-1BB, ICOS, or TNFRSF25.

[0172] [Safety switch] The safety switch polypeptide provides a suicide moiety within or on the cells expressing the safety switch polypeptide. This is useful as a safety mechanism that allows cryopreserved cells administered to a subject to be removed if necessary, or more generally, if desired or required, for example, after the cells have exerted or worn off their therapeutic effect. As described above, the Treg of the present invention or the Treg used in the present invention may additionally contain a safety switch polypeptide.

[0173] A suicide moiety has the ability to induce cell death, or more generally, the disappearance or removal of a cell. An example of a suicide moiety is a suicide protein encoded by a suicide gene, which may be expressed in or on a Treg as described herein. In this specification, a suicide moiety refers to a suicide polypeptide, which is a polypeptide that can remove a cell under tolerable conditions, i.e., under induced or on conditions.

[0174] The suicide portion may be a polypeptide or amino acid sequence that can be activated by an activator administered to the target and exert cell-removal activity, or a polypeptide or amino acid sequence that has the activity to exert cell-removal activity in the presence of a substrate that can be administered to the target. In certain embodiments, the suicide portion may be a target of a cell-removal agent administered separately to the target. The cell-removal agent can target cells to be removed by binding to the suicide portion. In particular, the suicide portion may be recognizable by an antibody, and when a safety switch polypeptide is expressed on the surface of a cell, the antibody binds to the safety switch polypeptide, causing the cell to disappear or be removed.

[0175] The suicide moiety may be HSV-TK or iCasp9. However, it is preferable that the suicide moiety is an epitope recognized by a cell removal antibody or other binding molecule capable of inducing cell removal, or contains such an epitope. In such embodiments, the safety switch polypeptide is expressed on the surface of the cell.

[0176] In particular, the suicide portion may be a CD20 epitope recognized by the antibody rituximab. Therefore, in the safety switch polypeptide, the suicide portion may include a minimal epitope based on a CD20-derived epitope recognized by the antibody rituximab. Cryopreserved cells expressing a safety switch polypeptide containing this sequence can be selectively killed using the antibody rituximab or an antibody having binding specificity for rituximab. When the safety switch polypeptide is expressed on the cell surface, and the expressed polypeptide is exposed to or comes into contact with rituximab or an antibody having the same binding specificity, cell death occurs as a result.

[0177] For example, the suicide constructs in International Publication No. 2013 / 153391 (incorporated herein by reference) may be used in the Treg or Treg population described herein.

[0178] [Polynucleotides] Nucleic acid molecules and polynucleotides / nucleic acid sequences as defined and used synonymously herein may include DNA or RNA. They may be single-stranded or double-stranded. Those skilled in the art will understand that, as a result of genetic coding degeneracy, many different nucleic acid molecules / polynucleotides may encode the same polypeptide.

[0179] The nucleic acid molecule / polynucleotide can be modified using any method available in the art. Such modifications may be made to improve the in vivo activity or lifetime of the nucleic acid molecule / polynucleotide as defined herein.

[0180] Nucleic acid molecules / polynucleotides / nucleotide sequences, such as DNA nucleic acid molecules / polynucleotides / sequences, can be prepared by recombination, synthesis, or any means available to those skilled in the art. They can also be cloned using standard techniques.

[0181] Longer nucleic acid molecules / polynucleotides / nucleotide sequences are generally prepared using recombinant methods, such as polymerase chain reaction (PCR) cloning techniques. This involves creating a primer pair (e.g., about 15-30 nucleotides) flanking the target sequence to be cloned, contacting the primers with mRNA or cDNA obtained from animal or human cells, performing a polymerase chain reaction under conditions that induce amplification of the desired region, isolating the amplified fragment (e.g., by purifying the reaction mixture on an agarose gel), and recovering the amplified DNA. The primers may be designed to contain appropriate restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable vector.

[0182] The nucleic acid molecules / polynucleotides described herein may further include nucleic acid sequences encoding a selectable marker. Suitable selectable markers are well known in the art and include, but are not limited to, fluorescent proteins such as GFP. Preferably, the selectable marker may be a fluorescent protein, e.g., GFP, YFP, RFP, tdTomato, dsRed, or a variant thereof. In some embodiments, the fluorescent protein is GFP or a GFP variant. The nucleic acid sequences encoding the selectable marker may be provided in combination with the nucleic acid molecules herein in the form of nucleic acid constructs. Such nucleic acid constructs may be provided in vectors.

[0183] Preferably, the selected marker / reporter domain may be a luciferase-based reporter, a PET reporter (e.g., sodium-iodine cotransporter (NIS)), or a membrane protein (e.g., CD34, low affinity nerve growth factor receptor (LNGFR)).

[0184] Nucleic acid sequences encoding FOXP3 and one or more other polypeptides (e.g., CARs, safety switches, and / or selection markers) may be separated from each other by one or more co-expression sites, when contained within a single construct, allowing each polypeptide to be expressed as a separate entity. Preferred co-expression sites are known in the art and include, for example, intra-sequence ribosome entry sites (IRESs) and autocleavage sites contained in the nucleic acid molecule, including those defined below. In some embodiments, this may be a 2A cleavage site, as described below.

[0185] Suitable self-cleavage domains include P2A sequences, T2A sequences, E2A sequences, and F2A sequences.

[0186] The use of selection markers is advantageous because they allow cells (e.g., Tregs) that have been successfully introduced (to express the encoded FOXP3 polypeptide and, optionally, CAR and safety switch polypeptides) to be selected and isolated from a starting cell population using common methods, such as flow cytometry.

[0187] The expression of polynucleotides described herein, such as the polynucleotide encoding FOXP3, will typically be controlled by a promoter. A “promoter” is a region of DNA that initiates the transcription of a gene. Promoters are located upstream of the DNA (near the 5' region of the sense strand) and near the transcription start site of the gene. Any suitable promoter can be used, and its selection can be easily made by those skilled in the art. Promoters may come from any source and may be viral promoters or eukaryotic promoters, including mammalian or human promoters (i.e., physiological promoters). In some embodiments, the promoter is a viral promoter. Specific promoters include LTR promoters, EFS (or its functional truncations), SFFV, PGK, and CMV. In some embodiments, the promoter is SFFV or a viral LTR promoter. "Operatably ligated to the same promoter" means that the transcription of each of the polynucleotide sequences may begin from the same promoter (for example, the transcription of the first, second, and third polynucleotide sequences may begin from the same promoter), and that each of the nucleotide sequences is positioned and oriented so that transcription begins from that promoter. Polynucleotides operably ligated to a promoter are under the transcriptional regulation of that promoter.

[0188] [vector] In some embodiments of the present invention, the polynucleotide is contained within an expression vector. As used herein, the term “expression vector” means a construct that enables the expression of the FOXP3 polypeptide (or another polypeptide that yields a conserved level of CD62L).

[0189] A vector is a tool that enables or facilitates the transfer of an entity from one environment to another. As used herein, and as an example, some vectors used in recombinant nucleic acid technology enable the transfer of entities such as nucleic acid segments (e.g., heterologous DNA segments such as heterologous cDNA segments) into target cells.

[0190] The vector may be a non-viral vector or a viral vector. Examples of vectors used in recombinant nucleic acid technology include, but are not limited to, plasmids, mRNA molecules (e.g., in vitro transcribed mRNA), chromosomes, artificial chromosomes, and viruses. The vector may also be, for example, naked nucleic acid (e.g., DNA). In its simplest form, the vector itself may be the nucleotide of interest.

[0191] The vectors used herein may be, for example, plasmids, mRNA, or viral vectors, and may include a promoter for the expression of a nucleic acid molecule / polynucleotide (as described below) and optionally, regulatory factors of said promoter. The term “nucleic acid molecule” as used herein includes polynucleotides.

[0192] In one embodiment, the vector is a viral vector, such as a retroviral vector, or, for example, a lentiviral vector or a gamma-retroviral vector.

[0193] The vector may further include additional promoters, for example, in one embodiment, the promoter may be an LTR, such as a retroviral LTR or a lentiviral LTR. Long terminal repeats (LTRs) are identical sequences of DNA repeated hundreds or thousands of times, found at both ends of a retrotransposon or proviral DNA formed by the reverse transcription of retroviral RNA. LTRs are used by viruses to insert genetic material into the host genome. LTRs contain signals for gene expression, such as enhancers, promoters (which may have both transcriptional enhancers and regulatory elements), transcription initiation (e.g., capping), transcriptional terminators, and polyadenylation signals. Preferably, the vector may include 5'LTRs and 3'LTRs.

[0194] The vector may include one or more additional regulatory sequences that can act pre-transcriptionally or post-transcriptionally. A “regulatory sequence” is any sequence that promotes the expression of the polypeptide, for example, by increasing the expression of the transcript or enhancing mRNA stability. Suitable regulatory sequences include, for example, enhancer elements, post-transcriptional regulatory elements, and polyadenylation sites. Preferably, the additional regulatory sequences may be located in one or more LTRs. Preferably, the vector may include, for example, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) operably ligated to the promoter.

[0195] Vectors containing nucleic acid molecules / polynucleotides described herein can be introduced into cells using various techniques known in the art, such as transformation and transduction. Several techniques are known in the art, including, for example, infection of recombinant viral vectors such as retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, baculovirus vectors, and herpes simplex virus vectors, direct injection of nucleic acids, and transformation by bioristic methods.

[0196] Nonviral delivery systems include, but are not limited to, DNA transfection methods. Here, transfection involves a process of delivering a gene to a target cell using a nonviral vector. Nonviral delivery systems may include liposome-mediated cell membrane-permeable peptides or amphipathic cell membrane-permeable peptides, preferably complexed with nucleic acid molecules or constructs.

[0197] Typical transfection methods include electroporation, DNA bioristic transfection, lipid-mediated transfection, compressed DNA-mediated transfection, liposome transfection, immunoliposome transfection, lipofectin transfection, cationic drug-mediated transfection, cationic facial amphiphile transfection (CFA) (Nat. Biotechnol. (1996) 14: 556), and combinations thereof.

[0198] Modified Treg cells may be generated by introducing nucleic acid molecules, constructs, or vectors as defined herein by one of many means, including transduction with a viral vector or transfection using DNA or RNA.

[0199] Treg cells as defined herein may be prepared by introducing nucleic acid molecules / polynucleotides, constructs, or vectors as defined herein into cells (e.g., by transduction or transfection).

[0200] Suitable cells have been described in detail above, but these cells may be derived from a sample isolated from the subject. The subject may be a donor subject or a subject of treatment (i.e., the cells may be autologous cells or donor cells for introduction into another recipient, such as allogeneic cells).

[0201] The cells can be produced by a method comprising the following steps: (i) A step of isolating a sample containing Treg cells from a subject, or a step of preparing a sample containing cells; and (ii) A step of introducing a nucleic acid molecule, construct, or vector as defined herein into the Treg cell-containing sample (for example, by transduction or transfection) to obtain a population of manipulated Treg cells.

[0202] A sample enriched with target Treg cells may be isolated, enriched, and / or generated from the cell-containing sample before and / or after step (ii) of the method. For example, Treg (or other target cells) may be isolated, enriched, and / or generated before and / or after step (ii) to isolate, enrich, or generate a Treg-enriched sample. Isolation and / or enrichment from the cell-containing sample may be performed after step (ii) to enrich Treg (or other target cells) containing nucleic acid molecules / polynucleotides, constructs, and / or vectors encoding FOXP3 as described herein.

[0203] Treg-enriched samples can be isolated or enriched by any method known to those skilled in the art, for example, by FACS and / or magnetic bead sorting. Treg-enriched samples may also be generated from cell-containing samples by any method known to those skilled in the art, for example, from Tcon cells by introducing DNA or RNA encoding FOXP3, and / or from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells. Other methods for isolating and / or enriching target cells are known in the art.

[0204] Preferably, the manipulated target cells can be generated by a method comprising the following steps: (i) a step of isolating a sample enriched with target cells, or a step of preparing a sample enriched with target cells; and (ii) A step of introducing a nucleic acid, construct, or vector as defined herein into a sample enriched with target cells (for example, by transduction or transfection) to obtain a population of engineered target cells.

[0205] The target cells may be Treg cells, or their precursors or progenitor cells. Isolation of Treg cells may include the following steps: (i) the step of isolating CD4+ T cells; and (ii) A step of isolating Treg cells from the CD4+ T cells.

[0206] The isolation may include selecting the T cells or Treg cells using immunomagnetic beads or fluorescence-activated cell sorting (FACS).

[0207] "Engineered cells," "engineered Tregs," or "engineered Treg populations" mean cells that contain or have been modified to express polynucleotides that are not naturally present in the cell. Those skilled in the art will understand that while Tregs express FOXP3, the engineered cells described herein will contain polynucleotide sequences that are not naturally present and encode FOXP3 (e.g., including promoters, regulatory sequences, CARs, TCRs, etc., that are not naturally present). Methods for manipulating cells are known in the art and include, but are not limited to, transduction methods such as retroviral or lentiviral transduction, transfection methods including lipofection (such as DNA or RNA-based transient transfection), polyethylene glycol methods, calcium phosphate methods, and electroporation methods for genetic modification of cells, as described above. Nucleic acid sequences can be introduced into cells using any suitable method. Nucleic acids may also be introduced using non-viral techniques such as amphipathic cell membrane permeable peptides.

[0208] Unmanipulated Tregs or cells do not contain the exogenous polynucleotide / nucleic acid molecule encoding FOXP3. Therefore, unmanipulated Tregs or cells may contain other exogenous polynucleotides (i.e., those other than FOXP3). In one embodiment, the unmanipulated Tregs or cells do not contain any exogenous polynucleotide / nucleic acid molecule.

[0209] Preferably, the manipulated cells are modified cells, for example, cells modified by transduction or transfection. Preferably, the manipulated cells are cells that have been modified or whose genomes have been altered, for example, by transduction or transfection. Preferably, the manipulated cells are cells that have been modified or whose genomes have been altered by retroviral transduction. Preferably, the manipulated cells are cells that have been modified or whose genomes have been altered by lentiviral transduction.

[0210] As used herein, the term “introduced” refers to a method for inserting foreign nucleic acids, such as DNA or RNA, into cells. As used herein, the term “introduced” includes both transduction and transfection methods. Transfection is the process of introducing nucleic acids into cells by a non-viral method. Transduction is the process of introducing foreign DNA or RNA into cells via a viral vector. Manipulated cells can be produced by introducing the nucleic acids described herein by one of many means, including transduction by a viral vector or transfection using DNA or RNA.

[0211] Cells may be activated and / or proliferated before or after the introduction of nucleic acids / polynucleotides as described herein, for example, by treatment with an anti-CD3 monoclonal antibody or with both an anti-CD3 monoclonal antibody and an anti-CD28 monoclonal antibody. The cells may also be proliferated in the presence of an anti-CD3 monoclonal antibody and an anti-CD28 monoclonal antibody in combination with IL-2. Preferably, IL-2 may be replaced with IL-15. Other components that may be used in cell (e.g., Treg) proliferation protocols include, but are not limited to, rapamycin, total trans retinoic acid (ATRA), and TGFβ. As used herein, the term “activated” means that cells have been stimulated to undergo proliferation. As used herein, the term “expanded” means that cells or a population of cells have been induced to proliferate. The proliferation of a population of cells can be measured, for example, by counting the number of cells present in the population. The phenotype of a cell can be determined by methods known in the art, such as flow cytometry.

[0212] [Composition] A pharmaceutical composition is a composition comprising a therapeutically effective amount of a pharmaceutically active agent, i.e., the Treg or Treg cell population, or such an agent. The pharmaceutical composition preferably contains a pharmaceutically acceptable carrier, diluent, or excipient (including combinations thereof). Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical field and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (ARGennaro, ed., 1985). The choice of pharmaceutically acceptable carrier, excipient, or diluent can be made considering the intended route of administration and standard pharmaceutical practices. The pharmaceutical composition may also contain, as a carrier, excipient, or diluent, or in addition thereof, any suitable binder, lubricant, suspending agent, coating agent, or solubilizer. It will be understood that the pharmaceutical composition of the present invention may be a cryopreserved pharmaceutical composition.

[0213] "Pharmacologically acceptable" includes the fact that the formulation is sterile and pyrogen-free (free of pyrogens). The carrier, diluent, and / or excipient must be "acceptable" in the sense that they are compatible with the cells or vector and are not harmful to the recipient. Typically, the carrier, diluent, and excipient are sterile and pyrogen-free saline or infusion media, but other acceptable carriers, diluents, and excipients may be used.

[0214] Examples of pharmaceutically acceptable carriers include, for example, water, saline solutions, alcohol, silicone, wax, petrolatum, vegetable oil, polyethylene glycol, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, fragrance oils, fatty acid monoglycerides, fatty acid diglycerides, petroethral fatty acid esters, hydroxymethylcellulose, and polyvinylpyrrolidone.

[0215] The Tregs, Treg populations, or pharmaceutical compositions described herein may be administered in a manner suitable for treating and / or preventing a desired disease or condition, and appropriate routes of administration are described herein. Dosage and frequency of administration are determined by various factors, including the condition and the type and severity of the disease or condition, although appropriate doses may be determined by clinical trials. Pharmaceutical compositions may be formulated accordingly.

[0216] The Tregs, Treg populations, or pharmaceutical compositions described herein may be administered parenterally, for example, intravenously, or by infusion techniques. The Tregs, Treg populations, or pharmaceutical compositions may also be administered in the form of a sterile aqueous solution, which may contain other substances, such as salts or glucose sufficient to make the solution isotonic with blood. The aqueous solution may be appropriately buffered (preferably to pH 3-9). The pharmaceutical compositions can be formulated accordingly. Preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

[0217] The pharmaceutical composition may contain cells in an infusion medium, for example, in a sterile isotonic solution. The pharmaceutical composition may be sealed in glass or plastic ampoules, disposable syringes, or multi-dose vials.

[0218] The Treg, Treg population, or pharmaceutical composition may be administered as a single dose or as multiple doses. In particular, the Treg, Treg population, or pharmaceutical composition may be administered as a single, one-time dose. The pharmaceutical composition may be formulated accordingly.

[0219] The pharmaceutical composition may further comprise one or more active agents. The pharmaceutical composition may further comprise a lymphocyte depleting agent (e.g., thymoglobulin, Campath-1H, anti-CD2 antibody, anti-CD3 antibody, anti-CD20 antibody, cyclophosphamide, fludarabine), an inhibitor of mTOR (e.g., sirolimus, everolimus), an agent that inhibits a co-stimulatory pathway (e.g., anti-CD40 / CD40L, CTAL4Ig), and / or an agent that inhibits a specific cytokine (IL-6, IL-17, TNFalpha, IL18), and the like, one or more other therapeutic agents.

[0220] Depending on the disease / condition to be treated and the subject, as well as the route of administration, the Tregs, Treg population, or pharmaceutical composition can be administered at various dosages (e.g., measured as number of cells / kg or number of cells / subject). In any case, the physician determines the actual dosage most suitable for any individual subject, and that dosage will vary depending on the age, weight, and response of that particular subject. However, typically, for the cells herein, 5×10 7 ~3×10 9 cells per subject, or 1×10 8 ~2×10 9 cells can be administered.

[0221] The Treg cells, Treg populations, and pharmaceutical compositions described herein may be used for the treatment or prevention of diseases or conditions. The cells and compositions containing them are for adoptive cell therapy (ACT). Various conditions can be treated by administration of Treg cells, for example, Treg cells expressing TCRs or CARs. As described above, these may be immunosuppressive conditions, particularly those responding to the immunosuppressive effects of Treg cells. Therefore, the cells, cell populations, and pharmaceutical compositions described herein may be used to induce or achieve immunosuppression in a subject. Administered Treg cells, or Treg cells modified in vivo, may be targeted by the expression of TCRs or CARs. Conditions suitable for such treatment include infections, neurodegenerative diseases, inflammatory diseases, or metabolic diseases, or more broadly, conditions associated with any undesirable, unnecessary, or harmful immune response.

[0222] In particular, the Treg cells, Treg populations, and compositions provide means for inducing tolerance to grafts, for treating and / or preventing cellular and / or humoral graft rejection, for treating and / or preventing graft-versus-host disease (GvHD), autoimmune or allergic diseases, neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), and metabolic diseases such as type 1 diabetes; or for promoting tissue repair and / or tissue regeneration; or for relieving inflammation and / or suppressing immune responses. The cells, cell populations, and compositions may be used in methods comprising the step of administering the cells, cell populations, or compositions described herein to a target.

[0223] In this specification, “inducing tolerance to a graft” means inducing tolerance in a recipient to the transplanted organ. In other words, inducing tolerance to a graft means reducing the level of the recipient’s immune response to the donor’s transplanted organ. Inducing tolerance to a transplanted organ may reduce the amount of immunosuppressant drugs required by the transplant recipient, or may allow for the discontinuation of immunosuppressant drugs. In this regard, the present invention further provides a method for inducing tolerance to a transplanted organ in a subject, comprising administering to the subject a Treg, population, or composition described herein.

[0224] For example, the Treg may be administered to a subject with a disease to alleviate, reduce, or improve at least one symptom of the disease, such as jaundice, dark urine, itching, abdominal distension, abdominal tenderness, fatigue, nausea, vomiting, and / or loss of appetite. The at least one symptom may be alleviated, reduced, or improved by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%, or the at least one symptom may be completely relieved.

[0225] The Treg cells may be administered to a subject with the disease to delay, reduce, or halt the progression of the disease. The progression of the disease may be delayed, reduced, or halted by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a subject not administered the manipulated cells, or the progression of the disease may be completely halted.

[0226] In one embodiment, the subject is a transplant recipient undergoing immunosuppressive therapy.

[0227] Preferably, the subject is a mammal. Preferably, the subject is a human.

[0228] Transplants can be selected from liver transplants, kidney transplants, heart transplants, lung transplants, pancreas transplants, intestinal transplants, stomach transplants, bone marrow transplants, vascularized composite tissue grafts, and skin transplants.

[0229] The Treg, Treg population, or pharmaceutical composition of the present invention can be administered to a patient immediately after or during thawing without further proliferation. The Treg, Treg population, or pharmaceutical composition may be administered to a patient within approximately 5, 10, 15, or 30 minutes after thawing, or within approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours.

[0230] A method for treating a disease or condition relates to the therapeutic use of cells as described herein. In this regard, cells may be administered to a subject having a pre-existing disease or condition in order to alleviate, reduce or improve at least one symptom associated with that disease or condition, and / or to delay, reduce or halt the progression of the disease. Preferably, treating and / or preventing cellular and / or humoral transplant rejection may mean administering an effective amount of cells (e.g., Tregs) such that the amount of immunosuppressant drugs required by the transplant recipient is reduced, or it may mean enabling the discontinuation of immunosuppressant drugs.

[0231] The prevention of disease or condition relates to the prophylactic use of cells as described herein. In this regard, cells may be administered to subjects who have not yet contracted or developed a disease or condition and / or who do not exhibit any symptoms of such disease or condition, in order to prevent such disease or condition, or to reduce or prevent the development of at least one symptom associated with such disease or condition. Such subjects may be predisposed to the disease or condition, or are considered to be at risk of developing such disease or condition.

[0232] Autoimmune or allergic diseases may be selected from inflammatory skin diseases including psoriasis and dermatitis (e.g., atopic dermatitis); responses associated with inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis); dermatitis; allergic symptoms such as food allergies, eczema, and asthma; rheumatoid arthritis; systemic lupus erythematosus (SLE) (including lupus nephritis and cutaneous lupus); diabetes mellitus (e.g., type 1 diabetes or insulin-dependent diabetes mellitus); multiple sclerosis; neurodegenerative diseases, e.g., amyotrophic lateral sclerosis (ALS); chronic inflammatory demyelinating polyneuropathy (CIPD); and juvenile diabetes mellitus.

[0233] In some embodiments, the disease or disorder to be treated is a neurological / neurodegenerative disease or disorder / condition. In some embodiments, the neurological / neurodegenerative disease or disorder is associated with inflammation. Thus, in some embodiments, the present invention may find usefulness in treating or preventing (e.g., reducing the risk thereof) neuroinflammation or related diseases or disorders. Neuroinflammation may be chronic or acute, preferably chronic. Neuroinflammation may be central nervous system or peripheral nervous system neuroinflammation, preferably central nervous system neuroinflammation.

[0234] In some embodiments, the neurological disease, neurological disorder, or neurological injury is selected from amyotrophic lateral sclerosis (ALS), dementia, frontotemporal dementia, Alzheimer's disease, vascular dementia, mixed dementia, Creutzfeldt-Jakob disease, chronic inflammatory demyelinating polyneuropathy (CIDP), Huntington's disease, tauopathic diseases, Nasu-Hakola disease, central nervous system lupus, Parkinson's disease, Lewy body dementia, multiple system atrophy (Shy-Drager syndrome), progressive supranuclear palsy, cortical basal ganglionic degeneration, acute disseminated encephalomyelitis, seizures, spinal cord injury, traumatic brain injury (e.g., ischemic and traumatic brain injury), depression, autism spectrum disorder, and multiple sclerosis. In some embodiments, the neurological disease, neurological disorder, or neurological injury is amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, multiple sclerosis, ischemic and traumatic brain injury, depression, and autism spectrum disorder.

[0235] Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease or Lou Gehrig's disease, is a debilitating disease of various etiologies characterized by rapidly progressing weakness, muscle atrophy and fasciculations, muscle spasms, difficulty speaking (dysarthria), difficulty swallowing (dysphagia), and difficulty breathing (dyspnea).

[0236] Therefore, the present invention provides Treg, Treg populations, or pharmaceutical compositions (for example, cryopreserved, thawed, or Treg populations obtained by the methods of the present invention) for use in therapy.

[0237] Preferably, the Treg may be self-derived. Preferably, the Treg may be of the same type but different in origin.

[0238] Preferably, the Treg (e.g., the manipulated Treg) may be administered in combination with one or more other therapeutic agents, such as lymphocyte depletion agents (e.g., those described above).

[0239] The Treg may be administered simultaneously with or sequentially (for example, before or after the one or more other therapeutic agents) to the one or more other therapeutic agents. Activation and / or proliferation of the Treg may be performed before or after the introduction of the nucleic acid molecules described herein, for example, by treatment with an anti-CD3 monoclonal antibody, or by treatment with both an anti-CD3 monoclonal antibody and an anti-CD28 monoclonal antibody. The proliferation protocol is as described above. The activation and / or proliferation process is preferably performed before cryopreservation. It is preferable that further activation and / or proliferation is not required after thawing.

[0240] The Treg may be washed after each step of the method, especially after proliferation.

[0241] The population of Treg cells may be further enriched by any method known to those skilled in the art, for example, by FACS or magnetic bead sorting.

[0242] Each step in this manufacturing method and cryopreservation method may be carried out in a closed, sterile cell culture system.

[0243] As previously stated, the present invention further provides a method for improving the homing ability of a cryopreserved Treg or Treg population to secondary lymphoid organs compared to a corresponding unmanipulated Treg or Treg population after cryopreservation, the method comprising the step of introducing a polynucleotide encoding a FOXP3 polypeptide into the Treg or Treg population before cryopreservation.

[0244] To “enhance the homing ability” of a Treg or Treg population means an increased ability to home to or locate in secondary lymphoid organs after in vivo administration compared to a Treg or Treg population that does not contain the exogenous polynucleotide encoding FOXP3 (e.g., an increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%). Homing ability can be measured by any known imaging technique.

[0245] Furthermore, the present invention provides a method for enhancing the stability and / or repressive function of a Treg or Treg population after cryopreservation, comprising the step of introducing a nucleic acid molecule / polynucleotide, expression construct, or vector provided herein into cells before cryopreservation. Enhanced stability means an enhancement of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of stability (e.g., compared to a corresponding cryopreserved unmanipulated Treg). Stability can be determined by measuring the number of Tregs present over time using methods known in the art. Enhanced repressive function of a Treg means an enhancement of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of repressive function (e.g., compared to a corresponding cryopreserved unmanipulated Treg). Repressive function can be determined by any well-known assay, including those described herein. Alternatively, the present invention provides a method for preserving the stability and / or repressive function of a Treg or Treg population after cryopreservation, comprising the step of introducing a nucleic acid molecule / polynucleotide, expression construct, or vector provided herein into cells before cryopreservation, wherein the stability and / or repressive function is equivalent to that of the corresponding non-cryopreserved operational Treg. Equivalent function / stability means that the function or stability may be equivalent to or identical to that of the corresponding non-cryopreserved operational Treg, for example, having at least 70%, 80%, or 90% of the stability or function.

[0246] This disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of embodiments of this disclosure. Numerical ranges include the number defining the range. Unless otherwise specified, all nucleic acid sequences are written from left to right in the 5' to 3' direction, and amino acid sequences are written from left to right in the amino to carboxy direction. The exact nucleic acid and amino acid sequences were obtained directly from the inventors.

[0247] Where a range of values ​​is provided, each intervening value between the upper and lower limits of that range, up to one-tenth of the lower limit unit unless otherwise clearly indicated in the context, is understood to be specifically disclosed. Any smaller range between any stated value or intervening value within the stated range and any other stated value or intervening value within the stated range is each included in this disclosure. The upper and lower limits of these smaller ranges may independently be included in or excluded from such ranges, and each range where either the upper or lower limit is included in the smaller range, where neither the upper or lower limit is included in the smaller range, or where both the upper and lower limits are included in the smaller range, is also included in this disclosure, except for any specifically excluded upper or lower limits within the stated range. If the stated range includes one or both of the upper and lower limits, the range excluding one or both of the upper and lower limits that they include is also included in this disclosure.

[0248] Please note that the singular forms "a," "an," and "the" used herein and in the appended claims refer to multiple subjects unless otherwise clearly indicated by the context.

[0249] As used herein, the terms “comprising,” “comprises,” and “comprised of” are synonymous with “including,” “includes,” or “containing,” and are comprehensive or open-ended, and do not exclude any additional components, elements, or process steps not described herein. The terms “comprising,” “comprises,” and “comprised of” also include the term “consisting of.”

[0250] The publications described in this specification merely show the disclosure content before the filing date of the present application and are incorporated herein by reference. Any content in this specification should not be construed as admitting that such publications constitute prior art against the appended claims.

[0251] Various modifications and changes to the disclosed methods, cells, compositions, and uses of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been disclosed in connection with specific preferred embodiments, it should be understood that the invention claimed in the claims should not be unduly limited to such specific embodiments. Indeed, various modifications to the disclosed forms for carrying out the present invention that are apparent to those skilled in the art are also intended to be within the scope of the following claims.

[0252] Here, the present invention will be further described by way of examples. These examples are intended to assist those of ordinary skill in the art in practicing the present invention and are not intended to limit the scope of the present invention in any way.

Examples

[0253] <Materials and Methods> [Cloning] Construct 1 (C1) was designed in-house, the entire sequence was codon-optimized for expression in human cells, and manufactured. The construct was cloned into the pMP71 backbone, D5a high-efficiency bacteria were transformed with the plasmid, and grown with the selective agent ampicillin. DNA was extracted using a Miniprep Kit (Qiagen). The insert was transferred into the lentiviral backbone by PCR cloning. Construct 1 (C1) represents the construct of the present disclosure described in the claims.

[0254] [PBMC Recovery] Leukocyte cones were supplied by NHS Blood and Transplant and leukopacks were obtained from BioIVT. PBMCs were isolated from the cones using a density centrifugation protocol. Briefly, blood was diluted 1:1 with 1×PBS and overlaid on Ficoll-Paque (GE Healthcare). The samples were centrifuged and the leukocyte layer was removed and washed with PBS.

[0255] [Treg Isolation Protocol] Blood cones and leukopacks were subjected to CD4 enrichment by negative selection using RosetteSep™ Human CD4+ T Cell Enrichment Cocktail. Subsequently, CD4+ cells were isolated using density centrifugation. Then, CD4+CD25+ T cells were isolated by positive selection using CD25 MicroBeads II (Miltenyi). The CD4+CD25+ fraction was stained with flow cytometry antibodies CD4 FITC (OKT4, Biolegend), CD25 PE-Cy7 (BC96, Biolegend), CD127 BV421 (A019D5, Biolegend), CD45RA BV510 (HI100, Biolegend), and LIVE / DEAD™ Fixable Near-IR Dead Cell Stain (Thermofisher) and then FACS sorting was performed. CD4+CD25+CD127low CD45RA+ (CD45RA+ Tregs) were sorted and used.

[0256] [Treg Medium and Expansion] Human regulatory T cells were activated with Human T-Activator CD3 / CD28 Dynabeads™ (Gibco) and cultured in XVIVO (Lonza) supplemented with IL-₂ and 5% human serum (Sigma-Aldrich). The Treg medium was replenished to the cells every 2 - 3 days. A second stimulation with Dynabeads™ was performed to promote further expansion of Treg cells.

[0257] [Transfection and production of virus particles] (Lentivirus) HEK293T cells were seeded in DMEM (Dulbecco's Modified Eagle's Medium) + 10% fetal bovine serum (FBS) and cultured for 24 hours. Transfection reagents were allowed to return to room temperature and mixed with the target DNA construct / plasmid, packaging plasmid (pD8.91), and viral envelope (pVSV-G). Diluted DNA was mixed with PEI and added to HEK293T cells. The supernatant was collected 48 hours after transfection, filtered, and the virus was concentrated.

[0258] [Transduction of T cells] Treg cells were activated with anti-CD3 and anti-CD28 Dynabeads (Gibco) and resuspended in T cell medium. Non-tissue culture-treated 24-well plates were coated with RetroNectin (Takara Bio, Otsu, Japan) and the cell suspension was added along with the lentiviral supernatant. The cells were incubated, and the medium was changed every other day. Twelve days after transduction, the cells were used in experiments.

[0259] [Flow cytometry staining to measure transduction and FOXP3 expression] Treg cells were isolated from the culture, washed, and then stained first with LIVE / DEAD® Fixable Near-IR Dead Cell Stain (Thermofisher) in PBS, followed by staining 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, permeabilized, and stained with anti-Foxp3 PE (150D / E4, Thermofisher) antibody. Cells were analyzed using an Attune NxT flow cytometer.

[0260] [Phenotyping of transduced cells by flow cytometry] Treg cells were isolated from the culture and stained using dextramer and LIVE / DEAD® Fixable Near-IR Dead Cell Stain as described above. Cell surface staining was performed using anti-CD62L BV650 (Biolegend). Cells were permeabilized and stained with anti-Foxp3 PE (150D / E4, Thermofisher).

[0261] [Cryopreservation and thawing of human Treg cells] After 14 days of in vitro proliferation, Dynabeads were removed from the Treg cells using magnetism, and the cells were rested in cell culture medium containing 1% human serum at a density of 1 × 10^6 / mL. The following day, half of the cells were cryopreserved, and the other half were used fresh for phenotypic analysis. For cryopreservation, the rested Treg cells were resuspended in cryopreservation medium and frozen at a density of 0.5–10 × 10^6 / mL. 6 The procedure was carried out by obtaining a cell suspension at a concentration of cells / mL. Next, the cells were transferred to a cryovial using a sterile Pasteur pipette, and the cryovial was frozen at -80°C for 4 to 6 hours in a pre-cooled freezer container. Finally, the cryovial was transferred to a liquid nitrogen tank. Thawing of the Treg cells was performed by immersing the cryovial in a 37°C water bath for 2 to 3 minutes. The thawed cell suspension was gently poured into a conical tube containing cell culture medium with 20% fetal bovine serum (FBS), and the cells were centrifuged at 300×g for 10 minutes. After centrifugation, the cells were counted and used for phenotypic analysis.

[0262] [Testing methods for different cryopreservation solutions and different cell densities] Construction 1(C1), a proprietary construct, was designed and manufactured using a standard protocol. This construct represents the construct of the present disclosure as described in the claims. Naive Treg cells were isolated from LeucoPak and sorted according to a standard protocol. The isolated cells were activated with CD3 / CD28 beads and cultured in XVIVO(Lonza) supplemented with IL-2 and human serum. The cells were replenished with culture medium every 2-3 days. A second stimulation with CD3 / CD28 beads was performed during culture to promote further proliferation of Treg cells. Treg cells were either transduced without transduction or transduced with C1 during culture using a lentiviral vector (the lentiviral vector was manufactured according to a standard protocol). The vector and IL-2 were added directly to the Treg cells in culture. After proliferation, a portion of the cells were collected for counting and flow cytometry analysis ("Pre-freeze"). The remaining cells were aliquoted into five different cell volumes. The cells were spun down, and the cell pellets were resuspended in 1 ml of cryopreservation solution 1, 2, or 3 to obtain five different cell densities for each cryopreservation solution (ranging from 1 × original cell density to 00 × original cell density). The cells were then aliquoted into cryovials and incubated at -80°C for 24 hours, after which they were transferred to liquid nitrogen. Thawing of Treg cells was performed by placing the cryovials in a 37°C water bath until small ice crystals remained. Subsequently, cells were analyzed by flow cytometry to evaluate transduction, FOXP3 expression, and phenotype. This was done using our proprietary "health" flow cytometry panel and our proprietary "maturation" flow cytometry panel. Briefly, the health panel measures viability / death, CD4 / CD25, FOXP3, RQR8 (using anti-CD34 antibody to indicate transduction), and apoptosis markers. The maturation panel measures viability / death, CD4, CD45RA (naive / SCM), CD45RA-CCR7+ (central memory), CD45RA-CCR7- (effector memory), and CD62L. The results are shown in Figures 4 to 6.

[0263] [Data Analysis] Flow cytometry data were analyzed using the Flow Cytometry Analysis software FlowJo (Flowjo, LLC). All statistical analyses were performed using Graphpad Prism v.8 (Graphpad, Software).

[0264] (Example 1 - Survival Rate Evaluation) As described above, fresh and frozen non-transduced Tregs, i.e., non-GMO Tregs that do not contain exogenous FOXP3, and fresh and frozen Tregs transduced with a FOXP3-expressing construct (C1 (Construction 1)) were collected and stained with a fixable viability-determining dye for flow cytometry analysis. Cells from eight different donors were evaluated. Construction 1 (C1) is a construct containing FOXP3, a safety switch, and a CD34 selection marker. Transduced cells can be identified by detecting the CD34 selection marker using an anti-CD34 antibody (e.g., QBEND). C1 represents the construct of this disclosure as described in the claims. Figure 1 shows that the survival rate of frozen transduction Tregs is approximately 75%, which is equivalent to the survival rate of non-transduction Tregs.

[0265] (Example 2 - Transduction and FOXP3 Expression) Transduction efficiency in fresh and frozen Treg cells transduced with a FOXP3-expressing construct (C1) was evaluated as described above. Transduced cells were selected using an anti-CD34 antibody. Cells from six different donors were evaluated. Figure 2 shows that FOXP3 was expressed in both fresh and frozen Tregs transduced with C1.

[0266] (Example 3 - Phenotypic evaluation (Cell surface expression of CD62L)) Fresh and frozen non-GMO transducible Tregs (i.e., Tregs without exogenous FOXP3), and fresh and frozen transducible Tregs expressing an experimental construct (C1 (construct 1)) expressing FOXP3 were collected and stained with anti-CD62L antibody conjugated to fluorophores, as described above, for flow cytometry analysis. Cells from eight different donors were evaluated. Figure 2 shows that in FOXP3-transduced and untransduced Treg cells, CD62L expression levels remain close to 100% when these cells are not frozen. CD62L expression levels decrease after cryopreservation in both transduced and untransduced cells. However, most importantly, FOXP3-transduced Treg cells maintain significantly higher levels of CD62L expression after freeze-thawing compared to untransduced Treg cells.

[0267] (Example 4 - Survival rate evaluation with different cryopreservation solutions) Treg cells were either transduced with a construct expressing FOXP3 (C1 (construct 1)) or not transduced, i.e., non-GMO Treg cells that did not contain exogenous FOXP3. Some of these Treg cells were used for viability analysis in a fresh state (the "Before Freezing" column in Figure 4), while the remainder were frozen in three different cryopreservation solutions: Solution 1, Solution 2, and Solution 3, at five different cell densities for each solution. As described above, the fresh and frozen Treg cells were harvested and stained with viability-determining dyes for flow cytometry analysis. Cells transduced with C1 were selected using an anti-CD34 antibody. Cells from five different donors were evaluated. Figure 4 shows that the survival percentages of frozen transduced and frozen non-transduced Treg cells were nearly 100%, comparable to those of fresh transduced and fresh non-transduced Treg cells, and that there was little change in the survival percentages across all three different cryopreservation solutions 1, 2, and 3, as well as all five cell densities, for both transduced and non-transduced cells.

[0268] (Example 5 - Transduction and FOXP3 Expression in Different Cryopreservation Solutions) The transduction efficiency was evaluated in fresh Tregs (see the "Before freezing" column in Figure 5) that were either transduced with the construct (C1) expressing FOXP3 or not transduced (i.e., non-genetically modified Tregs (Non-GMO) without exogenous FOXP3), and also in cryopreserved Tregs frozen at five different cell densities for each solution in cryopreservation solutions 1, 2, or 3, which were either transduced with C1 or not transduced. Cells transduced with C1 were selected using an anti-CD34 antibody. Cells from five different donors were evaluated. Figure 5 shows that FOXP3 was expressed at high levels in both fresh and cryopreserved Tregs transduced with C1, and the percentage of FOXP3 expression was constant across all three cryopreservation solutions and all five cell densities.

[0269] (Example 6 - Phenotypic Evaluation (Cell Surface Expression of CD62L) in Different Cryopreservation Solutions) Tregs were either transduced with the construct (C1 (Construct 1)) expressing FOXP3 or not transduced, i.e., non-genetically modified Tregs (Non-GMO) without exogenous FOXP3. Some of these Tregs were used for phenotypic analysis in the fresh state (the "Before freezing" column in Figure 6), and the rest were frozen at five different cell densities for each of the three different cryopreservation solutions, solution 1, solution 2, or solution 3. As described above, fresh and cryopreserved Tregs were recovered and stained with an anti-CD62L antibody conjugated to a fluorophore for flow cytometry analysis. Cells from five different donors were evaluated. Figure 6 shows that in Tregs transduced with FOXP3 (C1), the expression level of CD62L was maintained at nearly 90% across all three cryopreservation solutions 1, 2, and 3 and all five cell densities, whereas in non-transduced Tregs, CD62L expression was significantly lower under all conditions.

Claims

1. A method for preparing a composition comprising a Treg population for immediate administration to a subject, (a) Steps to isolate the Treg population from the sample, (b) A step of introducing a polynucleotide encoding a FOXP3 polypeptide into the Treg population, (c) A step of cryopreserving the Treg group, and (d) A step of melting the group, The method is one which does not include any further culture or growth steps after step (d).

2. The method according to claim 1, further comprising the step of growing the Treg population before step (c).

3. The method according to claim 1 or 2, wherein the sample comprises whole blood, umbilical cord blood, leukocyte cones, blood cones, peripheral blood mononuclear cells (PBMCs), or one or more leucopacks.

4. The method according to any one of claims 1 to 3, wherein step (c) includes the following steps: (ci) A step of suspending the Treg population in a culture medium for cryopreservation; (cii) a step of freezing the Treg group of step (ci); and (Ciii) A step of storing the Treg group from step (Ciii) at a temperature lower than -130°C.

5. The method according to claim 4, further comprising the following steps: Prior to step (c), a step of pre-cooling the Treg population and / or one or more reagents and devices used in the cryopreservation step; Step (c) is to freeze and store the Treg group at a controlled freezing rate of approximately -1°C / min; and / or Prior to the above step (ciii), the step of storing the Treg group at -80°C for a maximum of 24 hours.

6. The method according to any one of claims 1 to 5, wherein the melting of the Treg group includes raising the temperature of the Treg group from a temperature lower than -130°C to a temperature between approximately 0°C and 10°C.

7. The method according to any one of claims 1 to 6, wherein the Treg population is isolated by selecting (i) or (ii) below: (i) CD4 + CD25 + CD127 - Cells and / or CD4 + CD25 + CD127 low cell; or (ii) CD4 + CD25 hi CD127 - cells and / or CD4 + CD25 + CD127 low cells.

8. The aforementioned Treg group is CD45RA + The method according to any one of claims 1 to 7, wherein cells are isolated by selection.

9. The method according to any one of claims 1 to 8, wherein the FOXP3 polypeptide comprises an amino acid sequence or a functional fragment thereof that is at least 90% identical to SEQ ID NO:

1.

10. The method according to any one of claims 1 to 9, wherein the polynucleotide encoding FOXP3 is present in the expression vector.

11. The method according to any one of claims 1 to 10, further comprising introducing a polynucleotide encoding an exogenous T cell receptor (TCR) or a polynucleotide encoding a chimeric antigen receptor (CAR) into the Treg population.

12. The method according to claim 11, wherein the polynucleotide encoding the FOXP3 polypeptide and the polynucleotide encoding the exogenous TCR or the CAR are provided by a single expression vector.

13. The method according to claim 12, wherein the vector comprises a first polynucleotide encoding the FOXP3 polypeptide and a second polynucleotide encoding the exogenous TCR or CAR, the first polynucleotide and the second polynucleotide being operably linked to the same promoter, and the first polynucleotide being upstream of the second polynucleotide.

14. The method according to any one of claims 11 to 13, wherein an internal self-cleavage sequence exists between the polynucleotide encoding FOXP3 and the polynucleotide encoding the exogenous TCR or CAR.

15. A composition comprising a Treg population for use in the prevention and / or treatment of a disease, wherein the Treg population is obtained by the method of any one of claims 1 to 14, and the Treg population is to be administered to a subject immediately after thawing.

16. Use of a Treg population in the manufacture of a drug for the prevention and / or treatment of a disease, wherein the Treg population is obtained by the method of any one of claims 1 to 14, and the Treg population is intended to be administered to a subject immediately after thawing.

17. The composition according to claim 15, wherein the disease is an autoimmune disease or an allergic disease.

18. The composition according to claim 15, wherein the disease is transplant rejection or graft-versus-host disease.

19. The composition according to claim 15, used for suppressing the immune response.

20. The composition according to claim 15, wherein the disease is a neurodegenerative disease.

21. The composition according to claim 15, wherein the disease is type 1 diabetes.

22. Use of the Treg population according to claim 16, wherein the disease is an autoimmune disease or an allergic disease.

23. Use of the Treg population according to claim 16, wherein the disease is transplant rejection or graft-versus-host disease.

24. Use of the Treg population according to claim 16 in suppressing the immune response.

25. Use of the Treg population according to claim 16, wherein the disease is a neurodegenerative disease.

26. Use of the Treg population according to claim 16, wherein the disease is type 1 diabetes.

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