HLA-specific chimeric antigen receptor

Abstract

1. A vector comprising a first polynucleotide encoding a FOXP3 polypeptide and a second polynucleotide encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen recognition domain that specifically binds to a human leukocyte antigen (HLA), wherein the first polynucleotide and the second polynucleotide are operably linked to the same promoter, and the first polynucleotide is upstream of the second polynucleotide.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to a vector encoding a FOXP3- and HLA-specific chimeric antigen receptor (CAR). The present invention also relates to modified regulatory T cells comprising said vector, and their therapeutic use.

[0002] Background of the Invention Allotransplantation of foreign organs or tissues has the potential to save lives, but can also lead to serious and complex complications. After solid organ transplantation, immune-mediated rejection necessitates the use of long-term and comprehensive immunosuppression, limiting the lifespan of the transplanted allograft tissue (Perkey, E. and Maillard, I., 2018. Annual Review of Pathology: Mechanisms of Disease, 13, pp.219-245).

[0003] For example, acute cellular rejection occurs in 15-25% of liver transplant recipients using tacrolimus as the basis of immunosuppressive therapy (Choudhary, NS, et al., 2017. Journal of clinical and experimental hepatology, 7(4), pp.358-366). Acute immune-mediated rejection occurs in 30-40% of kidney transplant recipients (Roberts, DM, et al., 2012. Transplantation, 94(8), pp.775-783).

[0004] Although acute transplant rejection usually responds well to treatment, chronic rejection can present a challenging situation. For example, a significant proportion of liver transplant patients do not respond to increased immunosuppression. Chronic rejection often leads to retransplantation or death (Choudhary, NS, et al., 2017. Journal of clinical and experimental hepatology, 7(4), pp.358-366).

[0005] Antigens present in the transplanted organ but absent in the patient are the primary cause of immune-mediated rejection. In particular, human leukocyte antigens (HLA) present in the transplanted organ but absent in the patient are important causes of transplant rejection. HLA-A, HLA-B, and HLA-DR are the major transplant antigens, and recent clinical data indicate that HLA matching also influences the outcome of HSCT clinical trials. Acute rejection is primarily the result of a T cell-mediated response, while chronic rejection may also be due to an antibody-mediated response (Choo, SY, 2007. Yonsei medical journal, 48(1), pp.11-23).

[0006] HLA typing can be used to match patients with transplant donors and reduce the risk of transplant rejection. HLA matching has had a major clinical impact, for example, in kidney and bone marrow transplants. However, in heart, liver, and lung transplants, allocation is primarily based on medical urgency, donor availability, and waiting time (Sheldon, S. and Poulton, K., 2006. In Transplantation immunology, pp. 157-174, Humana Press; and Choo, SY, 2007. Yonsei medical journal, 48(1), pp. 11-23).

[0007] Furthermore, HLA matching by Sanger Sequencing-Based Typing (SBT), the current standard of care, has significant limitations. SBT typically only sequences a subset of HLA gene regions, precluding the identification of potential functional differences outside of those regions. Furthermore, DNA sequences generated by SBT have phase ambiguity, which affects 53% of tested samples and represents a significant source of potential error. HLA genotype ambiguity often necessitates extensive additional testing to ensure accurate matching between patients and potential donors (Allen, ES, et al., 2018. Human Immunology, 79(12), pp. 848-854). As a result, there remains a need for improved methods for down-regulating immune responses in transplant patients, particularly when the transplanted organ has antigens (eg, HLA) that are absent in the patient.

[0008] Summary of the Invention Regulatory T cells (Tregs) are a type of T cell that regulates the activity of the immune system. Generally, Tregs are immunosuppressive, down-regulating immune responses to stimuli. In particular, Tregs suppress the activation and proliferation of normal T cells, whereas some types of normal T cells are directly involved in the immune response (e.g., cytotoxic T cells).

[0009] The suppressive effect of Tregs can be directed to specific target cells by expression of a chimeric antigen receptor (CAR) that recognizes an antigen expressed on the surface of the target cell. The present invention uses Tregs containing a CAR against an HLA antigen (e.g., HLA-A2) to induce tolerance to transplanted tissue in a subject or to treat and / or prevent transplant rejection.

[0010] However, there is a risk that over time, the engineered Tregs may lose their ability to suppress immune responses, thereby reducing the efficacy of any Treg immunotherapy and / or necessitating multiple infusions of the engineered Tregs. There is also a risk that the engineered Tregs (i.e., Tregs expressing a CAR) may acquire an effector phenotype over time. Furthermore, for example, if T effector cells are present in the starting cell population, engineered T effector cells may be generated as a by-product of generating the engineered Tregs.

[0011] This is problematic because, contrary to the immunosuppressive effects of Tregs, modified T effector cells may augment or promote immune-mediated damage of target cells.

[0012] We surprisingly found that exogenous FOXP3 in regulatory T cells (Tregs) (which already express endogenous FOXP3) can enhance regulatory function. We also surprisingly found that expression of exogenous FOXP3 in Tregs (which already express endogenous FOXP3) can reduce the risk of Tregs acquiring an effector phenotype and reduces the risk of generating modified T effector cells during the generation of modified Tregs.

[0013] Furthermore, the inventors have determined that the configuration in which the polynucleotide encoding FOXP3 precedes the polynucleotide encoding the CAR in a 5' to 3' direction ensures that CAR expression can only occur when FOXP3 is expressed, and that CAR expression does not occur in the absence of FOXP3. In the context of engineered Tregs, this is particularly advantageous, as it greatly reduces the risk of Tregs acquiring an effector phenotype and / or reduces the risks associated with introducing the CAR into T effector cells in the starting population.

[0014] Thus, the present invention provides HLA-specific Tregs with enhanced efficacy and safety, which may be used to induce tolerance to transplanted tissue in a subject or to treat and / or prevent transplant rejection.

[0015] In a first aspect, the present invention provides a vector comprising a first polynucleotide encoding a FOXP3 polypeptide and a second polynucleotide encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen recognition domain that specifically binds to a human leukocyte antigen (HLA), wherein the first polynucleotide and the second polynucleotide are operably linked to the same promoter, and the first polynucleotide is upstream of the second polynucleotide. Preferably, the antigen recognition domain specifically binds to HLA-A2.

[0016] The vector may comprise a polynucleotide encoding a cleavage site between the first polynucleotide and the second polynucleotide, and / or an internal ribosome entry site (IRES) between the first polynucleotide and the second polynucleotide. Preferably, the vector may comprise a self-cleaving sequence between the first polynucleotide and the second polynucleotide, and preferably, the self-cleaving sequence is a polynucleotide sequence encoding a 2A self-cleaving peptide. The 2A self-cleaving peptide may be selected from the group consisting of a P2A peptide, a T2A peptide, an E2A peptide, and an F2A peptide.

[0017] The antigen-recognition domain is an antibody, an antibody fragment, or is derived from an antibody. Preferably, the antigen-recognition domain is an antigen-binding fragment (Fab), a single-chain antibody (scFv), or a single-domain antibody (sdAb). Preferably, the antigen-recognition domain is a single-chain antibody (scFv).

[0018] The CAR comprises a transmembrane (TM) domain and an intracellular signaling domain. The CAR may also comprise a hinge domain and / or one or more costimulatory domains. Preferably, the CAR comprises a CD8 hinge domain, a CD8 TM domain, a CD28 signaling domain, and a CD3 zeta signaling domain.

[0019] The vector may be a viral vector, preferably a retroviral vector or a lentiviral vector.

[0020] In another aspect, the present invention provides a modified T cell comprising a vector according to the present invention.

[0021] In another aspect, the present invention provides modified regulatory T cells (Tregs) comprising a vector according to the present invention.

[0022] In another aspect, the invention provides a polynucleotide encoding a FOXP3 polypeptide, or a vector, according to the invention for use in enhancing the ability of engineered HLA-specific Tregs to suppress an immune response, preferably an immune response against cells expressing HLA.

[0023] The present invention also provides a method for enhancing the ability of engineered HLA-specific Tregs to suppress an immune response, the method comprising introducing into said Tregs a polynucleotide encoding a FOXP3 polypeptide as described herein, or introducing into said Tregs a vector of the present invention, preferably wherein said immune response is directed against cells expressing HLA.

[0024] The present invention also provides use of a polynucleotide encoding a FOXP3 polypeptide as described herein, or a vector as described herein, for enhancing the ability of engineered HLA-specific Tregs to suppress an immune response, said use comprising introducing the polynucleotide encoding the FOXP3 polypeptide into said Tregs, or introducing the vector into said Tregs, preferably wherein said immune response is directed against cells expressing HLA.

[0025] In another related aspect, the present invention provides a method for enhancing the ability of modified HLA-specific Tregs to suppress an immune response, the method comprising introducing into said Tregs a first polynucleotide encoding a FOXP3 polypeptide and a second polynucleotide encoding an HLA-specific CAR.

[0026] In another related aspect, the invention provides a method for enhancing the ability of engineered HLA-specific Tregs to suppress an immune response, the method comprising introducing into a cell-containing sample a first polynucleotide encoding a FOXP3 polypeptide and a second polynucleotide encoding an HLA-specific CAR, the method comprising: (a) the cell-containing sample comprises or consists of Tregs, and / or (b) the cell-containing sample comprises or consists of peripheral blood mononuclear cells (PBMCs), and Tregs are enriched from the cell-containing sample before or after introducing the first polynucleotide and / or the second polynucleotide; and / or (c) the cell-containing sample comprises or consists of PBMCs, and Tregs are generated from the cell-containing sample before or after introducing the first polynucleotide and / or the second polynucleotide; and / or (d) the cell-containing sample comprises or consists of pluripotent stem cells (PSCs) (e.g., induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs)), and Tregs are differentiated from the PSCs before or after introducing the first polynucleotide and / or the second polynucleotide. Preferably, the cell-containing sample is isolated from the body.

[0027] In another aspect, the invention provides a polynucleotide encoding a FOXP3 polypeptide, or a vector according to the invention, for use in reducing the risk of modified HLA-specific Tregs acquiring an effector phenotype. The present invention also provides a method for reducing the risk that modified HLA-specific Tregs will acquire an effector phenotype, said method comprising introducing a polynucleotide encoding a FOXP3 polypeptide into said Tregs, or introducing a vector of the present invention into said Tregs.

[0028] The present invention also provides the use of a polynucleotide encoding a FOXP3 polypeptide, or a vector as described herein, to reduce the risk that modified HLA-specific Tregs will acquire an effector phenotype, said use comprising introducing a polynucleotide encoding a FOXP3 polypeptide into said Tregs, or introducing said vector into said Tregs.

[0029] In another related aspect, the invention provides a method of reducing the risk of modified HLA-specific Tregs acquiring an effector phenotype, said method comprising introducing into said Tregs a first polynucleotide encoding a FOXP3 polypeptide and a second polynucleotide encoding an HLA-specific CAR.

[0030] In another related aspect, the invention provides a method of reducing the risk that modified HLA-specific Tregs will acquire an effector phenotype, said method comprising introducing into a cell-containing sample a first polynucleotide encoding a FOXP3 polypeptide and a second polynucleotide encoding an HLA-specific CAR, said method comprising: (a) the cell-containing sample comprises or consists of Tregs, and / or (b) the cell-containing sample comprises or consists of PBMCs, and Tregs are enriched from the cell-containing sample before or after introducing the first polynucleotide and / or the second polynucleotide; and / or (c) the cell-containing sample comprises or consists of PBMCs, and Tregs are generated from the cell-containing sample before or after introducing the first polynucleotide and / or the second polynucleotide; and / or (d) the cell-containing sample comprises or consists of pluripotent stem cells (PSCs) (e.g., induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs)), and Tregs are differentiated from the PSCs before or after introducing the first polynucleotide and / or the second polynucleotide. Preferably, the cell-containing sample is isolated from the body.

[0031] In another aspect, the invention provides a FOXP3 polypeptide or vector according to the invention for use in reducing the risk of generating modified HLA-specific T effector cells during the generation of modified HLA-specific Tregs.

[0032] The present invention also provides a method for reducing the risk of generating modified HLA-specific T effector cells during the generation of modified HLA-specific Tregs, said method comprising introducing into said Tregs a polynucleotide encoding a FOXP3 polypeptide as described herein, or introducing into said Tregs a vector as described herein.

[0033] The present invention also provides the use of a polynucleotide encoding a FOXP3 polypeptide as described herein, or a vector as described herein, to reduce the risk of generating modified HLA-specific T effector cells during the generation of modified HLA-specific Tregs, said use comprising introducing the polynucleotide encoding said FOXP3 polypeptide into said Tregs, or introducing the vector into said Tregs.

[0034] In another related aspect, the invention provides a method of reducing the risk of generating modified HLA-specific T effector cells during the generation of modified HLA-specific Tregs, the method comprising introducing into said Tregs a first polynucleotide encoding a FOXP3 polypeptide and a second polynucleotide encoding an HLA-specific CAR.

[0035] In another related aspect, the invention provides a method for reducing the risk of generating modified HLA-specific T effector cells during the generation of modified HLA-specific Tregs, the method comprising introducing into a cell-containing sample a first polynucleotide encoding a FOXP3 polypeptide and a second polynucleotide encoding an HLA-specific CAR, the method comprising: (a) the cell-containing sample contains Tregs and / or T effector cells; and / or (b) the cell-containing sample comprises or consists of PBMCs, and Tregs are enriched from the cell-containing sample before or after introducing the first polynucleotide and / or the second polynucleotide; and / or (c) the cell-containing sample comprises or consists of PBMCs, and Tregs are generated from the cell-containing sample before or after introducing the first polynucleotide and / or the second polynucleotide; and / or (d) the cell-containing sample comprises or consists of pluripotent stem cells (PSCs) (e.g., induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs)), and Tregs are differentiated from the PSCs before or after introducing the first polynucleotide and / or the second polynucleotide. Preferably, the cell-containing sample is isolated from the body.

[0036] According to the present invention, the HLA described herein is preferably HLA-A2, for example, preferably, the HLA-specific CAR is an HLA-A2-specific CAR, the modified HLA-specific Treg is a modified HLA-A2-specific Treg, and the modified HLA-specific T effector cell is a modified HLA-A2-specific T effector cell.

[0037] In these embodiments, the first polynucleotide and / or the second polynucleotide may be introduced by viral transduction, such as retroviral or lentiviral transduction. Preferably, the first polynucleotide and the second polynucleotide are introduced into a single vector, and optionally the first polynucleotide and the second polynucleotide are operably linked to the same promoter. More preferably, the single vector is the vector of the present invention.

[0038] In another aspect, the present invention provides modified Tregs obtainable or obtained by the methods according to the present invention.

[0039] In another aspect, the present invention provides a pharmaceutical composition comprising a vector, modified T cell, or modified Treg according to the present invention.

[0040] In another aspect, the invention provides a vector, modified T cells or Tregs, or pharmaceutical composition according to the invention for use in inducing tolerance to transplanted tissue in a subject, or for use in the treatment and / or prevention of transplant rejection or graft-versus-host disease (GvHD) in a subject, or for use in the treatment and / or prevention of an autoimmune or allergic disease in a subject, or for use in tissue repair and / or tissue regeneration in a subject, or for use in ameliorating chronic inflammation in a subject, preferably said subject is a human. In another related aspect, the present invention provides a method for inducing tolerance to transplanted tissue in a subject, or for treating and / or preventing transplant rejection or GvHD in a subject, said method comprising administering to said subject a vector, modified T cells or Tregs, or pharmaceutical composition of the present invention, preferably said subject is human.

[0041] The method comprises: (i) isolating or providing a cell-containing sample (e.g., from said subject), said cell-containing sample comprising or consisting of PBMCs; (ii) introducing the vector of the present invention into the cell-containing sample, The cell-containing sample comprises or consists of Tregs, and Tregs are enriched from the cell-containing sample before or after introducing the vector; and / or Tregs are generated from the cell-containing sample before or after introducing the vector; and / or the cell-containing sample comprises or consists of pluripotent stem cells (PSCs) (such as induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs)) and Tregs are differentiated from the PSCs (such as iPSCs or hESCs) before or after introducing the vector. [Brief explanation of the drawings]

[0042] [Figure 1] Figure 1 - Proliferation of Tconv cells (donor 1) Figure 1 shows the proliferation of Tconv cells transduced with the TCR construct with and without peptide (indicated by *), as well as proliferation of the same cells in the presence of mock-transduced Tregs (white bars), TCR construct-transduced Tregs (black bars), or TCR construct-transduced Tregs with FOXP3 (grey bars) at various Treg:Tconv ratios. [Figure 2] Figure 2 - IL-2 production of Tconv cells (donor 1) Figure 2 shows the IL-2 production of Tconv cells transduced with the TCR construct with and without peptide (indicated by *), as well as the IL-2 production of the same cells in the presence of mock-transduced Tregs (white bars), TCR construct-transduced Tregs (black bars), or TCR construct-transduced Tregs with FOXP3 (grey bars) at various Treg:Tconv ratios. [Figure 3]Figure 3 - Proliferation of Tconv cells (donor 2) Figure 3 shows the proliferation of Tconv cells (obtained from a different donor than that in Figure 1) transduced with the TCR construct with and without peptide, as well as proliferation of the same cells in the presence of mock-transduced Tregs (white bars), TCR construct-transduced Tregs (black bars), or TCR construct-transduced Tregs with FOXP3 (grey bars) at various Treg:Tconv ratios. [Figure 4] Figure 4 - IL-2 production of Tconv cells (donor 2) Figure 4 shows IL-2 production (indicated by *) of Tconv cells (obtained from a different donor than that in Figure 2) transduced with the TCR construct with and without peptide, as well as proliferation of the same cells in the presence of mock-transduced Tregs (white bars), TCR construct-transduced Tregs (black bars), or TCR construct-transduced Tregs with FOXP3 (grey bars) at various Treg:Tconv ratios. [Figure 5] Figure 5 - Mean Fluorescence Intensity (MFI) of Treg Markers. Figure 5 shows the mean fluorescence intensity (MFI) of Treg markers (FOXP3, CD25, and CTLA-4) in mock-transduced Tregs, or TCR- or TCR+FOXP3-transduced Tregs analyzed by flow cytometry 7–10 days after transduction. Points represent individual experiments. One-way ANOVA was used for statistical analysis. p<0.05*, p<0.005**. [Figure 6] Figure 6 - MFI of FOXP3, CD25, and CTLA-4 on transduced Tregs Figure 6 shows the MFI of FOXP3, CD25, and CTLA-4 on transduced Tregs. Each line represents a single experiment showing the MFI of the marker on the same Tregs transduced with TCR or TCR+FOXP3. [Figure 7]Figure 7 - Proliferation of Tconv cells (donor 3) Figure 7 shows the proliferation of Tconv cells (obtained from a different donor than those in Figures 1 and 3) transduced with the TCR construct with and without peptide, and in the presence of mock-transduced Tregs (white bars), TCR construct-transduced Tregs (black bars), TCR construct-transduced Tregs with FOXP3 (grey bars), or Tconv cells transduced with the TCR construct and FOXP3, i.e., inducible Tregs (red bars, right bar of each data set), at various Treg:Tconv ratios. [Figure 8] Figure 8 - IL-2 production by Tconv cells (donor 3) Figure 8 shows the level of IL-2 production by Tconv cells (from the same donor as in Figure 7) transduced with the TCR construct with and without peptide, as well as their IL-2 production in the presence of mock-transduced Tregs (white bars), TCR construct-transduced Tregs (black bars), TCR construct-transduced Tregs with FOXP3 (grey bars), or Tconv-transduced with the TCR construct with FOXP3, i.e., inducible Tregs (red bars, right bar of each data set), at various Treg:Tconv ratios. [Figure 9-1]Figure 9 - TCR-transduced regulatory T cells can engraft in irradiated hosts but require exogenous FOXP3 expression to prevent the accumulation of TCR+FOXP3- cells. Thy1.1+CD4+CD25+ Tregs were isolated by bead separation from lymph nodes or splenocytes of HLA-DRB*0401 transgenic mice. Tregs were transduced with TCR, TCR+ mouse FOXP3, or cultured with virus-free supernatant (mock). One day after transduction, TCR- or TCR+FOXP3-transduced cells were infused into HLA-DRB*0401 transgenic hosts conditioned with 4 Gy irradiation. Seven weeks later, engraftment of transduced Tregs was measured using flow cytometry. A. One day after transduction, transduction efficiency was measured by expression of human variable 2.1 and mouse Foxp3. [Figure 9-2] B. Splenocytes from mice receiving TCR or TCR+FOXP3 transduced Tregs were stained with Thy1.1 to identify transferred cells (upper panel) as well as FOXP3+TCR (lower panel). [Figure 9-3] C. Cumulative data (n=3) showing the fold change in transduction efficiency (left panel) and the fold change in absolute number of transduced cells (right panel) for TCR- or TCR+FOXP3-transduced Tregs compared to the day of infusion. Error bars represent the standard error of the mean. Statistical analysis by unpaired t-test. D. Representative expression of FOXP3 in transduced cells 7 weeks after transfer. Graphs show the cumulative percentage of FOXP3+ cells within the transduced population at week 7 (left) and the fold change in FOXP3+ cells compared to the day of infusion (n=3). Error bars represent the standard error of the mean. *p=>0.05, **p=>0.01 determined by unpaired t-test. [Figure 10A]Figure 10 - Tregs expressing exogenous FOXP3 maintain Treg function after 7 weeks in vivo, whereas Tregs not expressing exogenous FOXP3 acquire the ability to produce effector cytokines. A. Splenocytes were cultured for 4 hours with CD86+HLA-DR4+CHO cells pulsed with an irrelevant peptide or 10 uM MBP. IL-2 and IFNg production was measured by flow cytometry. FACS plots show CD45.1 cells containing TCR-only expressing Tregs (top panel) and Thy1.1 cells containing TCR+FOXP3-expressing Tregs. [Figure 10B] The graph in B shows cumulative IL-2 and IFNg production by TCR-expressing Tregs (dark gray) and TCR+FOXP3-expressing Tregs (light gray). Error bars indicate standard deviation of the mean (n=3). [Figure 11A] Figure 11A - Example of HLA-A2-specific CAR construct design. Schematic diagram of an example vector encoding an HLA-A2-specific CAR (designated A2 CAR): Construct FC: Represents a construct encoding 5'-FOXP3-P2A-A2 CAR-3'. Construct RC: Represents a construct encoding 5'-R-P2A-A2 CAR-3', where R represents other genes. Construct C: Represents a construct encoding only A2 CAR. Construct CR: Represents a construct encoding 5'-A2 CAR-P2A-R-3', where R represents other genes. [Figure 11B]Figure 11B - Generation of FOXP3 / HLA-A2 CAR-Tregs. Schematic diagram showing the generation and expansion of FOXP3 / HLA-A2 CAR-Tregs. Phoenix-GP (P.gp) cells, a retroviral packaging cell line stably expressing gag pol, were seeded at 1x106 cells / 10mm cell culture dish. The following day, CD4+CD25hiCD127low cells were isolated and activated with anti-CD3 / CD28 beads in the absence of IL-2. On the same day, P.gp cells were transfected with constructs encoding the envelope and FOXP3 / HLA.A2-CAR using Fugene transfection reagent. Two days after activation, Tregs were transduced with the g-retrovirus and IL-2 was added. Cells were refed with medium and IL-2 every two days. Transduction efficacy was verified on day 6 using HLA.A2 dextramers. Tregs were further expanded using new anti-CD3 / CD28 beads. [Figure 12A] Figure 12 - Expression of HLA-A2-specific CAR and FOXP3 in transduced Tregs. Figure 12 shows the expression levels of HLA-A2-specific CAR (A2 CAR), FOXP3, and other gene R, measured by flow cytometry, in Tregs transduced with constructs FC, RC, C, and CR, compared to mock controls. Figure 12A shows FACS plots gated on CD3+CD4+ for dextramer (HLA) versus SSC-A for mock Tregs and Tregs transduced with constructs FC, RC, C, and CR. Tregs transduced with each construct expressed the HLA-A2-specific CAR. [Figure 12B]Figure 12B shows a CD3+CD4+dextramer+-gated FACS plot of FOXP3 expression versus the expression of the other gene R for mock Tregs and Tregs transduced with constructs FC, RC, C, and CR. The gene R was expressed at high levels in Tregs transduced with constructs RC and CR. FOXP3 expression was observed in all Tregs, but was significantly higher in construct FC, especially compared to the expression of the HLA-A2-specific CAR alone (construct C). [Figure 13A] Figure 13 - Expression of HLA-A2-specific CAR and FOXP3 in transduced and expanded Tregs. Figure 13 shows the expression levels of HLA-A2-specific CAR (A2 CAR), FOXP3, and other gene R, measured by flow cytometry, in Tregs transduced and expanded with constructs FC, RC, C, and CR, compared to mock controls. Figure 13A shows FACS plots gated on CD3+CD4+ for dextramer (HLA) versus SSC-A for expanded mock Tregs and Tregs transduced and expanded with constructs FC, RC, C, and CR. Tregs transduced with each construct continued to express HLA-A2-specific CARs after expansion. [Figure 13B]Figure 13B shows a CD3+CD4+dextramer+-gated FACS plot of FOXP3 expression versus expression of other gene R for expanded mock Tregs and Tregs expanded after transduction with constructs FC, RC, C, and CR. After expansion, FOXP3 expression decreased in Tregs transduced with constructs RC, C, and CR. In contrast, FOXP3 expression did not decrease in Tregs transduced with construct FC after expansion. Consequently, FOXP3 expression was significantly higher in construct FC after expansion, especially compared with that of the HLA-A2-specific CAR alone (construct C). [Figure 14-1] Figure 14 - Phenotypic lineage of Tregs transduced with constructs encoding FOXP3 and an HLA-A2-specific CAR. Figure 14 shows the expression of phenotypic lineage markers in Tregs transduced with constructs FC, RC, C, and CR. Tregs transduced with construct FC maintained the Treg phenotypic lineage while exhibiting enhanced FOXP3 expression. [Figure 14-2] Figure 14 - Phenotypic lineage of Tregs transduced with constructs encoding FOXP3 and an HLA-A2-specific CAR. Figure 14 shows the expression of phenotypic lineage markers in Tregs transduced with constructs RC, C, and CR. Tregs transduced with construct FC maintained the Treg phenotypic lineage while exhibiting enhanced FOXP3 expression.

[0043] Detailed Description The suppressive properties of Tregs can be used therapeutically to ameliorate and / or prevent immune-mediated organ damage in transplants. The suppressive effects of Tregs can be directed to specific target cells by expression of chimeric antigen receptors (CARs) that recognize antigens expressed on the surface of target cells. In transplant rejection, the CAR can be directed against an HLA antigen (e.g., HLA-A2) present in the graft donor but absent in the graft recipient. In GvHD, the CAR can be directed against an HLA antigen (e.g., HLA-A2) present in the recipient but absent in the graft donor. Surprisingly, the present inventors found that expression of exogenous FOXP3 in regulatory T cells (Tregs) (which already express endogenous FOXP3) can enhance regulatory function. Surprisingly, the inventors have found that expression of exogenous FOXP3 in Tregs (which already express endogenous FOXP3) can reduce the risk of Tregs acquiring an effector phenotype and reduces the risk of generating modified T effector cells during the generation of modified Tregs. In particular, the configuration in which FOXP3 precedes the CAR in a 5' to 3' orientation ensures that CAR expression can occur only when FOXP3 is expressed, and that CAR expression does not occur in the absence of FOXP3.

[0044] Thus, the present invention provides HLA-specific Tregs (particularly HLA-A2-specific Tregs) with enhanced efficacy and safety, which may be used to treat and / or prevent transplant rejection or graft-versus-host disease.

[0045] Various preferred features and embodiments of the present invention will now be described by way of non-limiting example.

[0046] Please note that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0047] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including," "includes," or "containing," and "contains," and are inclusive or open-ended and do not exclude additional, unrecited elements, components, or method steps. The terms "comprising," "comprises," and "comprised of" also include the term "consisting of."

[0048] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application, and nothing herein should be construed as an admission that such publications constitute prior art to the appended claims.

[0049] This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise specified, any nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation.

[0050] Forkhead box P3 protein (FOXP3) In the present invention, expression of FOXP3 is increased in a cell (e.g., Treg) by introducing a polynucleotide encoding a FOXP3 polypeptide (sometimes referred to herein as a first polynucleotide) into the cell.

[0051] "FOXP3" is the abbreviation for forkhead box P3 protein. FOXP3 is a member of the FOX protein family of transcription factors and functions as a master regulator of regulatory pathways in regulatory T cell development and function.

[0052] "Increasing FOXP3 expression" means increasing the level of FOXP3 mRNA and / or protein in a cell (or population of cells) compared to a corresponding unmodified cell (or population of cells). For example, the level of FOXP3 mRNA and / or protein in a cell (or population of such cells) modified according to the present invention may be increased by at least 1.5-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, or at least 150-fold over the level in a corresponding cell (or population of such cells) that has not been modified according to the present invention. Preferably, the cells are Tregs and the population of cells is a population of Tregs.

[0053] Suitably, the level of FOXP3 mRNA and / or protein in a cell (or population of such cells) modified according to the invention may be increased by at least 1.5-fold, at least 2-fold, or at least 5-fold over the level in a corresponding cell (or population of such cells) that has not been modified according to the invention. Preferably, the cells are Tregs and the population of cells is a population of Tregs.

[0054] The technology of measuring the level of specific mRNA and protein is well known in the art.The level of mRNA in a population of cells such as Treg can be measured by techniques such as Affymetrix eBioscience PrimeFlow RNA assay, Northern blotting, serial analysis of gene expression (SAGE) or quantitative polymerase chain reaction (qPCR).The level of protein in a population of cells can be measured by techniques such as flow cytometry, high performance liquid chromatography (HPLC), liquid chromatography / mass spectrometry (LC / MS), Western blotting or enzyme-linked immunosorbent assay (ELISA).

[0055] A "FOXP3 polypeptide" is a polypeptide having FOXP3 activity, i.e., a polypeptide capable of binding to FOXP3 target DNA and functioning as a transcription factor regulating the development and function of Tregs. In particular, a FOXP3 polypeptide may have the same or similar activity as wild-type FOXP3 (SEQ ID NO: 1), e.g., 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. 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 level of mRNA and / or protein produced from the gene using techniques such as Northern blotting, SAGE, qPCR, HPLC, LC / MS, Western blotting, or ELISA. Genes regulated by FOXP3 include cytokines such as IL-2, IL-4, and IFN-γ (Siegler et al. Annu. Rev. Immunol. 2006, 24: 209-26, incorporated herein by reference).

[0056] A "functional fragment of FOXP3" may refer to a portion or region of a FOXP3 polypeptide or a portion or region of a polynucleotide encoding a FOXP3 polypeptide that has the same or similar activity as a full-length FOXP3 polypeptide or polynucleotide. The functional fragment may have at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the activity of a full-length FOXP3 polypeptide or polynucleotide. One skilled in the art would be able to generate functional fragments based on the known structural and functional characteristics of FOXP3. For example, see Song, X., et al., 2012, Cell Reports, 1(6), pp.665-675; Lopes, JE, et al., 2006, The Journal of Immunology, 177(5), pp.3133-3142; and Lozano, T., et al., 2013, Frontiers in Oncology, 3, p.294.

[0057] A "FOXP3 variant" may comprise an amino acid or nucleotide sequence that may be at least 50%, at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, preferably at least 95%, at least 97%, or at least 99% identical to a FOXP3 polypeptide or a polynucleotide encoding a FOXP3 polypeptide. A FOXP3 variant may have the same or similar activity as a 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 a wild-type FOXP3 polypeptide or polynucleotide. One skilled in the art would be able to generate FOXP3 variants based on known structural and functional characteristics of FOXP3 and / or using conservative substitutions.

[0058] FOXP3 polypeptide sequence Suitably, the FOXP3 polypeptide may comprise the polypeptide sequence of human FOXP3, such as UniProtKB Accession No. Q9BZS1 (SEQ ID NO: 1), or a functional fragment thereof. FOXP3, UniProtKB Accession Number Q9BZS1 (SEQ ID NO: 1): MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFL KHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPE FLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGP

[0059] In some embodiments of the invention, the FOXP3 polypeptide comprises an amino acid sequence, or a functional fragment thereof, that is at least 70% identical to SEQ ID NO: 1. Suitably, the FOXP3 polypeptide comprises an amino acid sequence, or a 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: 1. In some embodiments, the FOXP3 polypeptide comprises SEQ ID NO: 1 or a functional fragment thereof, or consists of SEQ ID NO: 1 or a functional fragment thereof.

[0060] Preferably, the FOXP3 polypeptide may be a variant of SEQ ID NO: 1, such as a natural variant. Preferably, the FOXP3 polypeptide is an isoform of SEQ ID NO: 1. For example, the FOXP3 polypeptide may contain a deletion of amino acids 72 to 106 compared to SEQ ID NO: 1. Alternatively, the FOXP3 polypeptide may contain a deletion of amino acids 246 to 272 compared to SEQ ID NO: 1.

[0061] Suitably, the FOXP3 polypeptide comprises SEQ ID NO: 2 or a functional fragment thereof. Example of a FOXP3 Polypeptide (SEQ ID NO: 2) MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVD AHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQA DHLLDEKGRAQCLLQREMVQSLEQVEELSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHN MRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGPEGRGSLLTCGDVEEN

[0062] Suitably, the FOXP3 polypeptide comprises an amino acid sequence, or a functional fragment thereof, that is at least 70% identical to SEQ ID NO: 2. Suitably, the FOXP3 polypeptide comprises an amino acid sequence, or a 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: 2. In some embodiments, the FOXP3 polypeptide comprises SEQ ID NO: 2, or a functional fragment thereof, or consists of SEQ ID NO: 2, or a functional fragment thereof.

[0063] Preferably, the FOXP3 polypeptide may be a variant of SEQ ID NO: 2, such as a natural variant. Preferably, the FOXP3 polypeptide is an isoform of SEQ ID NO: 2 or an isoform of a functional fragment thereof. For example, the FOXP3 polypeptide may contain a deletion of amino acids 72 to 106 compared to SEQ ID NO: 2. Alternatively, the FOXP3 polypeptide may contain a deletion of amino acids 246 to 272 compared to SEQ ID NO: 2.

[0064] FOXP3 polynucleotide sequence Suitably, said polynucleotide encoding a FOXP3 polypeptide comprises or consists of the polynucleotide sequence set out in SEQ ID NO:3. Example of a FOXP3 Polynucleotide (SEQ ID NO: 3)

[0065] In some embodiments of the invention, the polynucleotide encoding the FOXP3 polypeptide or variant comprises a polynucleotide sequence that is at least 70% identical to SEQ ID NO: 3, or a functional fragment thereof. Suitably, 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: 3, or a functional fragment thereof. In some embodiments of the invention, the polynucleotide encoding the FOXP3 polypeptide or variant comprises SEQ ID NO: 3, or a functional fragment thereof, or consists of SEQ ID NO: 3, or a functional fragment thereof.

[0066] Suitably, said polynucleotide encoding a FOXP3 polypeptide comprises or consists of the polynucleotide sequence set out in SEQ ID NO:4. Example of a FOXP3 Polynucleotide (SEQ ID NO: 4)

[0067] In some embodiments of the invention, the polynucleotide encoding the FOXP3 polypeptide or variant comprises a polynucleotide sequence that is at least 70% identical to SEQ ID NO: 4, or a functional fragment thereof. Suitably, 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: 4, or a functional fragment thereof. In some embodiments of the invention, the polynucleotide encoding the FOXP3 polypeptide or variant comprises SEQ ID NO: 4, or a functional fragment thereof, or consists of SEQ ID NO: 4, or a functional fragment thereof.

[0068] Preferably, the polynucleotide encoding the FOXP3 polypeptide or functional fragment or variant thereof may be codon-optimized. Preferably, the polynucleotide encoding the FOXP3 polypeptide or functional fragment or variant thereof may be codon-optimized for expression in human cells.

[0069] HLA-specific chimeric antigen receptor In the present invention, HLA-specific cells are generated by introducing into cells a polynucleotide (sometimes referred to herein as a second polynucleotide) encoding an HLA-specific chimeric antigen receptor (CAR).

[0070] As used herein, "chimeric antigen receptor" or "CAR" or "CARs" refers to a genetically engineered receptor that can confer antigen specificity to cells, such as Tregs. CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors. CARs of the present invention comprise a binding domain specific for HLA, preferably HLA-A2, and optionally include a hinge domain, a transmembrane domain, and an endodomain (including an intracellular signaling domain and, optionally, one or more costimulatory domains).

[0071] A polynucleotide encoding a CAR can be transferred into cells, for example, using a retroviral vector. In this way, a large number of antigen-specific T cells can be generated by adoptive cell transfer. When a CAR binds to a target antigen, an activation signal is transmitted to cells expressing the CAR (e.g., Treg). In this way, the CAR directs the modified Treg to cells expressing the target antigen, thereby suppressing the immune response to the antigen or cells containing the antigen.

[0072] antigen recognition domain The CAR of the present invention comprises an antigen recognition domain. As used herein, " antigen recognition domain " refers to the extracellular portion of CAR, which determines the antigen binding ability of CAR.In some embodiments of the invention, antigen recognition domain provides CAR with the ability to bind to HLA.In this way, antigen recognition domain targets HLA.

[0073] The human leukocyte antigen (HLA) system or complex is a complex of genes that encodes the major histocompatibility complex (MHC) proteins in humans. HLA is responsible for regulating the human immune system. Preferably, the HLA is selected from the group consisting of HLA-A2, HLA-A1, HLA-C0701, HLA-A3, HLA-A11, and HLA-A2402. Preferably, the HLA is HLA-A2. "HLA-A2" may also be referred to as HLA-A*02, HLA-A02, and HLA-A*2. HLA-A*02 is a specific group of class I major histocompatibility complex (MHC) alleles at the HLA-A locus.

[0074] Preferably, the antigen-binding domain may comprise an antigen-binding domain capable of binding to HLA (preferably HLA-A2) present in the graft / transplant donor but absent in the graft / trasplant recipient, or vice versa. For example, if the transplant is an organ transplant, the HLA (preferably HLA-A2) may be present in the transplanted organ but absent in the patient. If the transplant is HSCT (e.g., bone marrow transplant), the HLA (preferably HLA-A2) may be present in the patient but absent in the transplanted organ.

[0075] The antigen recognition domain may bind to, and preferably specifically bind to, one or more regions or epitopes within HLA (preferably HLA-A2). An epitope, also known as an antigenic determinant, is the part of an antigen that is recognized by an antigen recognition domain (e.g., an antibody). In other words, an epitope is the specific part of an antigen to which an antibody binds. Suitably, the antigen recognition domain binds to, and preferably specifically binds to, one region or epitope within HLA (preferably HLA-A2). It will be appreciated by those skilled in the art that specific binding may occur to more than one region within HLA (preferably HLA-A2), for example due to protein / polypeptide folding.

[0076] The antigen recognition domain used in the present invention may selectively or specifically bind to HLA (preferably HLA-A2), and therefore, its binding affinity to HLA (preferably HLA-A2) may be greater than its binding affinity to other proteins / molecules. Suitably, as used herein, "specifically binds" means that the antigen recognition domain does not bind to other proteins, or binds with significantly lower affinity (e.g., at least 10-, 50-, 100-, 500-, 1000-, or 10,000-fold lower than its affinity to HLA (preferably HLA-A2)) than its binding affinity to other proteins. Thus, the antigen recognition domain referred to herein may bind to HLA (preferably HLA-A2) with an affinity that is at least 10-, 50-, 100-, 500-, 1000-, or 10,000-fold higher than its affinity to other proteins. For example, the binding affinity of the antigen recognition domain may be measured using methods well known in the art, such as using the Lineweaver-Burk plot method, or by using commercially available binding model software, such as the 1:1 binding model in BIAcore 1000 Evaluation software. Preferably, an HBS-P buffer system (0.01 M Hepes, pH 7.4, 0.15 M NaCl, 0.05% Surfactant P20) is used.

[0077] The antigen recognition domain (also known as an antigen-specific targeting domain) may be any protein or peptide capable of specifically recognizing and binding to HLA (preferably HLA-A2). The antigen recognition domain includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for HLA (preferably HLA-A2). Examples of antigen recognition domains include antibodies or antibody fragments or derivatives, extracellular domains of receptors, ligands for cell surface molecules / receptors or their receptor-binding domains, and tumor-binding proteins.

[0078] Preferably, the antigen-recognition domain is an antibody (Ab) or is derived from an antibody (Ab). The antibody-derived antigen-recognition domain may be an antibody fragment or a genetically engineered product of one or more of the antibody fragments, which are involved in binding to the antigen. Examples include camelid antibodies (VHH), antigen-binding fragments (Fab), variable regions (Fv), single-chain antibodies (scFv), single-domain antibodies (sdAb), heavy chain variable regions (VH), light chain variable regions (VL), and complementarity-determining regions (CDR). In a preferred embodiment, the antigen recognition domain is a single chain antibody (scFv).

[0079] Antibodies recognize antigens through the fragment antigen-binding (Fab) variable region. Antibodies are glycoproteins that belong to the immunoglobulin superfamily. They make up the majority of the gamma globulin fraction of blood proteins. They typically consist of two large heavy chains and two small light chains. Camelid antibodies (VHH) lack light chains and consist of two heavy chains attached to variable domains.

[0080] The term "antigen-binding fragment" (Fab) refers to the region of an antibody that binds to an antigen, and each of the heavy and light chains comprises one constant region and one variable region.

[0081] "Fv" is the smallest antibody fragment containing a complete antigen-binding site. The Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain.

[0082] A "single-chain antibody" (scFv) is an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to each other directly or via a peptide linker sequence. Suitable linkers can be readily selected and can have any suitable length, such as from 1 amino acid (e.g., Gly) to 30 amino acids, including any of 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, to any of 12, 15, 18, 20, 21, 25, and 30 amino acids, such as 5-30 amino acids, 5-25 amino acids, 6-25 amino acids, 10-15 amino acids, 12-25 amino acids, and 15-25 amino acids. The peptide linker sequence is typically about 10-25 amino acids in length, rich in glycines for flexibility, and rich in serine or threonine for solubility. Examples of flexible linkers include glycine polymers (Gly). n (n is an integer greater than or equal to 1), glycine-serine polymer, glycine-alanine polymer, alanine-serine polymer, and other flexible linkers known in the art. The linker may contain one or more "GS" domains. Linkers may have different characteristics, such as flexible, rigid, or cleavable. The peptide linker sequence can connect the N-terminus of the heavy chain variable region to the C-terminus of the light chain variable region, or vice versa. The heavy chain variable region and the light chain variable region may be linked via a linker sequence (X)n, where X is any amino acid and n is an integer between 1 and 30. The linker sequence may be any linker sequence known in the art.

[0083] "Single domain antibodies" (sdAbs), also known as nanobodies, are antibody fragments consisting of a single monomeric variable antibody domain. Thus, an sdAb may be a heavy chain variable region (VH) or a light chain variable region (VL).

[0084] A "heavy chain variable region" or "VH" refers to a fragment of an antibody heavy chain containing three CDRs flanked by flanking stretches known as framework regions, which are more highly conserved than the CDRs and form the scaffold for CDR retention. A "light chain variable region" or "VL" refers to a fragment of an antibody light chain containing three CDRs flanked by framework regions.

[0085] A "complementarity-determining region" or "CDR" of an antibody or antigen-binding fragment thereof refers to the hypervariable loops in the variable region of the heavy or light chain of the antibody. CDRs can interact with the antigen structure and primarily determine antigen binding (although some framework regions have also been shown to be involved in binding). The heavy and light chain variable regions each contain three CDRs (heavy chain CDRs 1, 2, and 3 and light chain CDRs 1, 2, and 3, numbered from the amino terminus to the carboxy terminus).

[0086] The CDRs of the heavy and light chain variable regions of an antibody can be predicted from the antibody heavy and light chain variable region sequences using prediction software available in the art, such as the Abysis algorithm, or using IMGT / V-QUEST software, e.g., the IMGT algorithm (ImMunoGeneTics) available at www.IMGT.org (see, e.g., Lefranc et al., 2009 NAR 37:D1006-D1012 and Lefranc 2003, Leukemia 17: 260-266). CDR regions identified by either algorithm are considered equally suitable for use in the present invention. CDRs can vary in length depending on the antibody from which they are predicted and between the heavy and light chains. Thus, the three heavy chain CDRs of an intact antibody can be of different lengths (or the same length), and the three light chain CDRs of an intact antibody can be of different lengths (or the same length). For example, the CDRs may range in length from 2 or 3 amino acids to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. In particular, the CDRs may be 3 to 14 amino acids in length, for example at least 3 amino acids and less than 15 amino acids.

[0087] It should be noted that, where necessary, Kabat nomenclature is followed herein to define the positions of CDRs (Kabat et al., 1991, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 647-669).

[0088] Antibodies, derivatives, and fragments thereof that specifically bind to HLA (preferably HLA-A2) can be prepared using methods well known to those skilled in the art. Such methods include phage display, methods for generating human or humanized antibodies, or methods using transgenic animals or plants engineered to make human antibodies. Phage display libraries of partially or fully synthetic antibodies are available, and the libraries can be screened for antibodies or fragments thereof that can bind to HLA (preferably HLA-A2). Phage display libraries of human antibodies are also available. Once the amino acid or polynucleotide sequence encoding the antibody (or its derivative or fragment) is identified, it can be isolated and / or measured. The sequence of the antibody can be used to design suitable derivatives or fragments thereof.

[0089] Examples of antibodies, derivatives and fragments thereof that can be used in the present invention are further described below.

[0090] The antigen recognition domain may comprise at least one CDR (e.g., CDR3), which can be predicted from an antibody (or antibody fragment) that binds to HLA (preferably HLA-A2), or from an antibody that binds to a variant of such a predicted CDR (e.g., a variant with one, two, or three amino acid substitutions). It will be understood that a molecule containing three or fewer CDR regions (e.g., a single CDR, or a portion thereof) will retain the antigen-binding activity of the antibody from which the CDRs are derived. It has been described in the art that molecules containing two CDR regions can bind to target antigens, for example, in the form of minibodies (Vaughan and Sollazzo, 2001, Combinational Chemistry & High Throughput Screening, 4, 417-430). It has also been described that molecules containing a single CDR can exhibit strong binding activity to targets (Nicaise et al., 2004, Protein Science, 13: 1882-91).

[0091] In this regard, the antigen-recognition domain may comprise one or more variable heavy chain CDRs, for example, one, two, or three variable heavy chain CDRs. Alternatively / additionally, the antigen-recognition domain may comprise one or more variable light chain CDRs, for example, one, two, or three variable light chain CDRs. The antigen-recognition domain may comprise three heavy chain CDRs and / or three light chain CDRs (particularly a heavy chain variable region comprising three CDRs and / or a light chain variable region comprising three CDRs), where at least one CDR, and preferably all CDRs, may be derived from an antibody that binds to HLA (preferably HLA-A2) and may be selected from one of the CDR sequences shown below.

[0092] The antigen-recognition domain may comprise any combination of variable heavy chain CDRs and variable light chain CDRs, for example, a combination of one variable heavy chain CDR and one variable light chain CDR, a combination of two variable heavy chain CDRs and one variable light chain CDR, a combination of two variable heavy chain CDRs and two variable light chain CDRs, a combination of three variable heavy chain CDRs and one or two variable light chain CDRs, a combination of one variable heavy chain CDR and two or three variable light chain CDRs, or a combination of three variable heavy chain CDRs and three variable light chain CDRs. Preferably, the antigen-recognition domain may comprise a combination of three variable heavy chain CDRs (CDR1, CDR2, and CDR3) and / or three variable light chain CDRs (CDR1, CDR2, and CDR3).

[0093] The one or more CDRs present in an antigen-recognition domain do not all need to be derived from the same antibody, so long as the domain has the above-mentioned binding activity. Thus, some CDRs may be predicted from the heavy or light chain of an antibody that binds to HLA (preferably HLA-A2), while other CDRs may be predicted from a different antibody that binds to HLA (preferably HLA-A2). In this case, it would be preferable for CDR3 to be predicted from an antibody that binds to HLA (preferably HLA-A2). However, particularly when more than one CDR is present in the antigen-recognition domain, it is preferable that the CDRs be predicted from antibodies that bind to HLA (preferably HLA-A2), and particularly preferably from antibodies that bind to the same region or epitope of said HLA. A combination of CDRs may be used from different antibodies, particularly antibodies that bind to the same region or epitope.

[0094] In a particularly preferred embodiment, the antigen recognition domain comprises three CDRs predicted from the variable heavy chain sequence of an antibody (or antibody fragment) that binds to HLA (preferably HLA-A2) and / or three CDRs predicted from the variable light chain sequence of an antibody (or antibody fragment) (preferably the same antibody or antibody fragment) that binds to HLA (preferably HLA-A2).

[0095] In some embodiments, the antigen-recognition domain is an antibody or is derived from an antibody (e.g., a Fab, scFv, or sdAb), and the antibody comprises one or more CDR regions selected from SEQ ID NOs: 5 to 133, or a derivative thereof (e.g., a derivative comprising one, two, or three substitutions, preferably one substitution). In other words, in some embodiments, the antigen-recognition domain comprises one or more CDR regions selected from SEQ ID NOs: 5 to 133, or a derivative thereof (e.g., a derivative comprising one, two, or three substitutions, preferably one substitution). Suitably, the antigen-recognition domain comprises three CDR regions selected from SEQ ID NOs: 5 to 133, or a derivative thereof.

[0096] [Table 1-1] [Table 1-2]

[0097] Preferably, the antigen-binding domain comprises CDRs (CDR1, CDR2, and CDR3) or derivatives thereof selected from the same variable chain. For example, the antigen-binding domain may comprise CDRs selected from SEQ ID NOs: 5 to 7, 8 to 10, 11 to 13, 14 to 16, 17 to 19, 20 to 22, 23 to 25, 26 to 28, 29 to 31, 32 to 34, 35 to 37, 38 to 40, 41 to 43, 44 to 46, 47 to 49, 50 to 52, 53 to 55, 56 to 58, 59 to 61, 62 to 64, 65 to 67, 68 to 70, 71 to 73, or the like. The fragments may comprise SEQ ID NOs: 74 to 76, SEQ ID NOs: 77 to 79, SEQ ID NOs: 80 to 82, SEQ ID NOs: 83 to 85, SEQ ID NOs: 86 to 88, SEQ ID NOs: 89 to 91, SEQ ID NOs: 92 to 94, SEQ ID NOs: 95 to 97, SEQ ID NOs: 98 to 100, SEQ ID NOs: 101 to 103, SEQ ID NOs: 104 to 106, SEQ ID NOs: 107 to 109, SEQ ID NOs: 110 to 112, SEQ ID NOs: 113 to 115, SEQ ID NOs: 116 to 118, SEQ ID NOs: 119 to 121, SEQ ID NOs: 122 to 124, SEQ ID NOs: 125 to 127, SEQ ID NOs: 128 to 130, and / or SEQ ID NOs: 131 to 133, or derivatives thereof.

[0098] In a preferred embodiment, the antigen recognition domain comprises a combination of variable heavy and variable light CDRs as follows: (i) SEQ ID NOs: 5 to 7, and SEQ ID NOs: 17 to 19, or derivatives thereof; (ii) SEQ ID NOs: 8 to 10, and SEQ ID NOs: 20 to 22, or derivatives thereof; (iii) SEQ ID NOs: 11 to 13, and SEQ ID NOs: 23 to 25, or derivatives thereof; (iv) SEQ ID NOs: 14 to 16, and SEQ ID NOs: 26 to 28, or derivatives thereof; (v) SEQ ID NOs: 14 to 16, and SEQ ID NOs: 29 to 31, or derivatives thereof; (vi) SEQ ID NOs: 14 to 16, and SEQ ID NOs: 32 to 34, or derivatives thereof; (vii) SEQ ID NOs: 14 to 16, and SEQ ID NOs: 35 to 37, or derivatives thereof; (viii) SEQ ID NOs: 14 to 16, and SEQ ID NOs: 38 to 40, or derivatives thereof; (ix) SEQ ID NOs: 14 to 16, and SEQ ID NOs: 41 to 43, or derivatives thereof; (x) SEQ ID NOs: 14 to 16, and SEQ ID NOs: 44 to 46, or derivatives thereof; (xi) SEQ ID NOs: 14 to 16, and SEQ ID NOs: 47 to 49, or derivatives thereof; (xii) SEQ ID NOs: 14 to 16, and SEQ ID NOs: 50 to 52, or derivatives thereof; (xiii) SEQ ID NOs: 14 to 16, and SEQ ID NOs: 53 to 55, or derivatives thereof; (xiv) SEQ ID NOs: 14 to 16, and SEQ ID NOs: 56 to 58, or derivatives thereof; (xv) SEQ ID NOs: 14 to 16, and SEQ ID NOs: 59 to 61, or derivatives thereof; (xvi) SEQ ID NOs: 62 to 64, and SEQ ID NOs: 98 to 100, or derivatives thereof; (xvii) SEQ ID NOs: 65 to 67, and SEQ ID NOs: 101 to 103, or derivatives thereof; (xviii) SEQ ID NOs: 68 to 70, and SEQ ID NOs: 104 to 106, or derivatives thereof; (xix) SEQ ID NOs: 71 to 73, and 107 to 109, or derivatives thereof; (xx) SEQ ID NOs: 74 to 76, and SEQ ID NOs: 110 to 112, or derivatives thereof; (xxi) SEQ ID NOs: 77 to 79, and SEQ ID NOs: 113 to 115, or derivatives thereof; (xxii) SEQ ID NOs: 80 to 82, and SEQ ID NOs: 116 to 118, or derivatives thereof; (xxiii) SEQ ID NOs: 83 to 85, and SEQ ID NOs: 119 to 121, or derivatives thereof; (xxiv) SEQ ID NOs: 86 to 88, and SEQ ID NOs: 122 to 124, or derivatives thereof; (xxv) SEQ ID NOs: 89 to 91, and SEQ ID NOs: 125 to 127, or derivatives thereof; (xxvi) SEQ ID NOs: 92 to 94, and SEQ ID NOs: 128 to 130, or derivatives thereof; (xxvii) SEQ ID NOs: 95 to 97, and SEQ ID NOs: 131 to 133, or derivatives thereof.

[0099] The antigen-binding domain may comprise or consist of a variable heavy domain selected from SEQ ID NOs: 134 to 149, or a variant that is at least 80% identical to one or more of SEQ ID NOs: 134 to 149. The variant comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to one or more of SEQ ID NOs: 134 to 149, or a functional fragment thereof.

[0100] [Table 2]

[0101] The antigen-binding domain may comprise or consist of a variable light domain selected from SEQ ID NOs: 150 to 176, or a variant that is at least 80% identical to one or more of SEQ ID NOs: 150 to 176. The variant comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to one or more of SEQ ID NOs: 150 to 176, or a functional fragment thereof.

[0102] [Table 3-1] [Table 3-2]

[0103] Suitably, the antigen recognition domain comprises a combination of a variable heavy domain and a variable light domain. Preferably, the antigen recognition domain comprises a combination of a variable heavy domain and a variable light domain selected from: (i) an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 134 and an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 150; (ii) an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 135 and an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 151; (iii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 136 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 152; (iv) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 137 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 153; (v) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 137 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 154; (vi) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 137 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 155; (vii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 137 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 156; (viii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 137 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 157; (ix) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 137 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 158; (x) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 137 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 159; (xi) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 137 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 160; (xii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 137 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 161; (xiii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 137 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 162; (xiv) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 137 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 163; (xv) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 137 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 164; (xvi) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 138 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 165; (xvii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 139 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 166; (xviii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 140 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 167; (xix) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 141 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 168; (xx) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 142 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 169; (xxi) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 143 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 170; (xxii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 144 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 171; (xxiii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 145 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 172; (xxiv) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 146 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 173; (xxv) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 147 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 174; (xxvi) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 148 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 175; (xxvii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 149 and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 176;

[0104] The antigen-binding domain may comprise or consist of an amino acid sequence selected from SEQ ID NOs: 177 to 203, or a variant having at least 80% identity to one or more of SEQ ID NOs: 177 to 203. The variant comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to one or more of SEQ ID NOs: 177 to 203, or a functional fragment thereof. The antigen-binding domain may comprise a linker sequence designated (X)n, where X is any amino acid and n is an integer between 1 and 30. The linker sequence may be any linker sequence known in the art. Antigen-binding domain example 1 (SEQ ID NO: 177) QVQLVQSGGGVVQPGGSLRVSCAASGVTLSDYGMHWVRQAPGKGLEWVAFIRNDGSDKYYADSVKGRFTISRDNSEKTVSLQMSSLRAEDTAVYYCAKNGESGPLDYWYLDLW G-(X)n-DVVMTQSPSSLSASVGDRVTITCQSSLDISHYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTHFTFTISSLQPEDFATYYCQQYDNLPLTFGGGTKLEIK Antigen-binding domain example 2 (SEQ ID NO: 178) QVQLVQSGGGVVQPGGSLRVSCAASGVTLSDYGMHWVRQAPGKGLEWMAFIRNDGSDKYYADSVKGRFTISRDNSKKTVSLQMSSLRAEDTAVYYCAKNGESGPLDYWYFDLWGR GT-(X)n-DVVMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPPTFGGGGTKLTVLG Antigen-Binding Domain Example 3 (SEQ ID NO: 179) QVQLVQSGGGVVQPGGSMRVSCAASGVTLSDYGMHWVRQAPGKGLEWVAFIRNDGSDKYYADSVRGRFTISRDNSKKTVFLQMNSLRAEDTAVYYCAKNGESGPLDYWYFDLWGR GT-(X)n-DIVLMQSPSFLSASVGDRVTITCRASHGINNYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQYDSYPPTFGRTKVEIKR Antigen Binding Domain Example 4 (SEQ ID NO: 180) QVQLVQSGGGVVQPGGSLRVSCAASGVTLSDYGMHWVRQAPGKGLEWMAFIRNDGSDKYYADSVKGRFTISRDNSKKTVSLQMSSLRAEDTAVYYCAKNGESGPLDYWYFDLWGR GT-(X)n-DVVMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDFATYYCQQYSSFPLTFGGGTKVDIK Antigen-Binding Domain Example 5 (SEQ ID NO: 181) QVQLVQSGGGVVQPGGSLRVSCAASGVTLSDYGMHWVRQAPGKGLEWMAFIRNDGSDKYYADSVKGRFTISRDNSKKTVSLQMSSLRAEDTAVYYCAKNGESGPLDYWYFDLWGR GT-(X)n-DVVMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQEPGKAPKLLIYDETHLDSGVPSRFTGSRSGTDFTLTISSLQPEDFATYYCQQYDSLPPTFGGGGTKVDIK Antigen-Binding Domain Example 6 (SEQ ID NO: 182) QVQLVQSGGGVVQPGGSLRVSCAASGVTLSDYGMHWVRQAPGKGLEWMAFIRNDGSDKYYADSVKGRFTISRDNSKKTVSLQMSSLRAEDTAVYYCAKNGESGPLDYWYFDLWGR GT-(X)n-DVVMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPITFGGGTKVDIK Antigen-Binding Domain Example 7 (SEQ ID NO: 183) QVQLVQSGGGVVQPGGSLRVSCAASGVTLSDYGMHWVRQAPGKGLEWMAFIRNDGSDKYYADSVKGRFTISRDNSKKTVSLQMSSLRAEDTAVYYCAKNGESGPLDYWYFDLWGR GT-(X)n-DVVMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPSTFGGGTKVDIK Antigen-Binding Domain Example 8 (SEQ ID NO: 184) QVQLVQSGGGVVQPGGSLRVSCAASGVTLSDYGMHWVRQAPGKGLEWMAFIRNDGSDKYYADSVKGRFTISRDNSKKTVSLQMSSLRAEDTAVYYCAKNGESGPLDYWYFDLWGR GT-(X)n-DVVMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDFGTYYCQQYNTYPLTFGGGTKVDIK Antigen Binding Domain Example 9 (SEQ ID NO: 185) QVQLVQSGGGVVQPGGSLRVSCAASGVTLSDYGMHWVRQAPGKGLEWMAFIRNDGSDKYYADSVKGRFTISRDNSKKTVSLQMSSLRAEDTAVYYCAKNGESGPLDYWYFDLWGR GT-(X)n-DVVMTQSPSSLTASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTFTLSIDSLQPEDFATYYCQQYHTYPLTFGGGTKVDIK Antigen-Binding Domain Example 10 (SEQ ID NO: 186) QVQLVQSGGGVVQPGGSLRVSCAASGVTLSDYGMHWVRQAPGKGLEWMAFIRNDGSDKYYADSVKGRFTISRDNSKKTVSLQMSSLRAEDTAVYYCAKNGESGPLDYWYFDLWGR GT-(X)n-DVVMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKVDIK Antigen-Binding Domain Example 11 (SEQ ID NO: 187) QVQLVQSGGGVVQPGGSLRVSCAASGVTLSDYGMHWVRQAPGKGLEWMAFIRNDGSDKYYADSVKGRFTISRDNSKKTVSLQMSSLRAEDTAVYYCAKNGESGPLDYWYFDLWGR GT-(X)n-DVVMTQSPSSLSASVGDRVTITCRTSQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNNYPLTFGGGTKVDIK Antigen Binding Domain Example 12 (SEQ ID NO: 188) QVQLVQSGGGVVQPGGSLRVSCAASGVTLSDYGMHWVRQAPGKGLEWMAFIRNDGSDKYYADSVKGRFTISRDNSKKTVSLQMSSLRAEDTAVYYCAKNGESGPLDYWYFDLWGR GT-(X)n-DVVMTQSPSSLSASVGDRVTITCQASQDISNYLAWYQQKPGRAPTLLIFAASNLQSGVPSRFSGSGSGTEFTLTISGLQPEDFATYYCLQDSSYPPTFGGGTKVDIK Antigen Binding Domain Example 13 (SEQ ID NO: 189) QVQLVQSGGGVVQPGGSLRVSCAASGVTLSDYGMHWVRQAPGKGLEWMAFIRNDGSDKYYADSVKGRFTISRDNSKKTVSLQMSSLRAEDTAVYYCAKNGESGPLDYWYFDLWGR GT-(X)n-DVVMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGRAPTLLIYKASNLQSGVPSRFSGSGSGTEFTLTISSLQPDDFASYYCQQYSNYPLTFGGGTKVDIK Antigen Binding Domain Example 14 (SEQ ID NO: 190) QVQLVQSGGGVVQPGGSLRVSCAASGVTLSDYGMHWVRQAPGKGLEWMAFIRNDGSDKYYADSVKGRFTISRDNSKKTVSLQMSSLRAEDTAVYYCAKNGESGPLDYWYFDLWGR GT-(X)n-DVVMTQSPSFLSASVGDRVTITCRASHGISNYFAWYQQKPGKAPKLLIYATSTLQSGVPSRFSGSGSGTEFTLTISGLQPEDFATYYCQQYSSYPLTFGGGTKVDIK Antigen Binding Domain Example 15 (SEQ ID NO: 191) QVQLVQSGGGVVQPGGSLRVSCAASGVTLSDYGMHWVRQAPGKGLEWMAFIRNDGSDKYYADSVKGRFTISRDNSKKTVSLQMSSLRAEDTAVYYCAKNGESGPLDYWYFDLWGR GT-(X)n-DVVMTQSPSTLSAYVGDRITITCRASRGISNYLAWYQQKPGKAPKLLIYATSTLQSGVPLRFSGSGSGTEFTLTISGLQPEDFATYYCQQYDSYPPTFGGGTKVDIK Antigen Binding Domain Example 16 (SEQ ID NO: 192) EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGYDSRGSYYYMDVWGKGTTV TVSS-(X)n-QSVLTQPPSTSGTPGQRVTISSCSGSSSNIGGNAVNWYQHFPGTAPTLLIYSNNQRPSGVPERFSGSKSGTSASLTVSGLQAEDEADYYCTAWDDSLRGYLFGTGTKVTVL Antigen Binding Domain Example 17 (SEQ ID NO: 193) QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDREELLALFGGMDVWGQGTT VTVSS-(X)n-QPVLTQPSSVSVAPGQTARITCGGNNIGSKSVHWYQQKPGQAPVLVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEARDEADYYCHVWDAKTNHQVFGGGTRLTVQ Antigen Binding Domain Example 18 (SEQ ID NO: 194) EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARPQSRWLQSGDAFDIWGQGTMV TVSS-(X)n-QPVLTQPRSVSGSPGQSVTISCTGTSSDVGGYNRVSWYQQTPGTAPKLMIYEVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTVVFGGGTKLTVL Antigen Binding Domain Example 19 (SEQ ID NO: 195) QVQLQQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGRINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARDLTGTLLFDYWGQGTLVTV SS-(X)n-QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNGVKWYQQLPGTAPKLVIYRDYQRPSGVPDRFSGSKSGTSASLAISGLQSEDEAKYYCAAWDDSLNVVFGGGTQLTVL Antigen Binding Domain Example 20 (SEQ ID NO: 196) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRATITADESTSTAYMELSSLRSEDTAVYYCARRAERWLHLSGAFDIWGQGTMV TVSS-(X)n-QPVLTQSSSASGTPGQRVAISCSGSSSNVGSNTVNWYQQSPGTAPKLLISSNHQRPSGVPDRFSGSKFGTSASLAISGLQSEDEADYYCGAWDDSLNGYVFGSGTKVTVL Antigen Binding Domain Example 21 (SEQ ID NO: 197) QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTMSRDNAKNSLYLQMNSLRVEDSAVYYCATGHYGDYVWGQGALVTVSS -(X)n-QAGLTQPPSASGTPGQRVTISSCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGPVFGGGTKLTVL Antigen Binding Domain Example 22 (SEQ ID NO: 198) QVQLQQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMGWINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCARVSGGAFDIWGQGTVVTVS S-(X)n-QSALTQPASVSGSPGQSITISCTGTGSDVGGYKYVSWYQHHPGKAPRLIIYDVNYWPSGVSHRFSGSKSGNTASLTISGLQSEDEADYYCSSYRTGDTWVFGGGTKLTVL Antigen Binding Domain Example 23 (SEQ ID NO: 199) QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYGFSWVRQAPGQGLEWMGEIIPMFGTANYAQKLQGRVTITAETSTSTVYMELSSLRSEDTATYYCARVPRSSSGYNYGMDVWGQGTT VTVSS-(X)n-DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSAPRTFGQGTKVEIK Antigen Binding Domain Example 24 (SEQ ID NO: 200) QVQLQQSGPGLLKPSQTLSLTCAVSGDSVSTNSGAWSWIRQSPSRGLEWLGRTYYRSKWSTDYALSLQSRVTIKSDRSKNQFSLQLDSVTPEDTAIYYCARENWNSGGFDYWGQGTLVT VPS-(X)n-QPVLTQSSSASGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYEVSKRPSGVPDRFSGSKSGSTASLTVSGLQAEDEAEYYCSSYAGSNNYVFGTGTKVTVL Antigen Binding Domain Example 25 (SEQ ID NO: 201) EVQLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARAASRWEPGDAFDIWGQGTMV TVSS-(X)n-QPVLTQSSSVSVAPKTARVTCGGDNIGKSVHWYQQRAGQAPVLVISHDTDRPSGIPERFSGSKSGTSASLAISGLRSEDEADYYCAVWDASLGGSWLFGGGTKLTVL Antigen Binding Domain Example 26 (SEQ ID NO: 202) QVQLVQSGAEVKKPGASVKVSCKTSGYTFTSYDISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRRLRSDDTAVYYCARGGRWLRSASSFDYWGQGTLVTV SS-(X)n-QAGLTQPPSVSGAPGQRVTISCTGSSSNIGAAYDVHWYQQLPGAAPKLLIFGDSNRPSGVPDRFSGSKSDTSASLAITGLQAEDEADYYCQSFDSSLSGSRVFGGGTKLTVL Antigen Binding Domain Example 27 (SEQ ID NO: 203) EVQLVESGGGLVKPGGSLRLSCAASGFTFSDHYMSWVRQAPGKGLEWVSYITSGGSSIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGLDSSAYQGRAFDIWGQGTMV TVSS-(X)n-LPVLTQPPSASGTPGQRVTISCSGSSSNIGSNPVHWYQQLPGTAPKLLVYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDVSLSGVVFGGGTKLTVL

[0105] The variants of the antigen recognition domain described herein retain antigen-binding ability. For example, the variants may bind to HLA-A2 at a level that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the binding level of the corresponding reference amino acid sequence. The variants may bind to HLA-A2 at a level close to or the same as the corresponding reference amino acid sequence, or may bind to HLA-A2 at a higher level than the corresponding reference amino acid sequence (e.g., at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% higher). Thus, the antigen recognition domain may comprise or consist of an amino acid sequence containing one or more (e.g., one, two, three, four, five, or six) CDRs of SEQ ID NOS: 5-133 (the underlined portions of SEQ ID NOS: 134-176, shown above). The framework regions may be substituted, mutated, modified, exchanged, deleted and / or added to one (or more) amino acid residues.

[0106] Thus, in some embodiments, the antigen recognition domain comprises or consists of the following sequence: (i) an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 134, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 5 to 7, respectively, and / or an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 150, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 17 to 19, respectively; (ii) an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 135, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 8 to 10, respectively, and / or an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 151, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 20 to 22, respectively; (iii) an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 136, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 11 to 13, respectively, and / or an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 152, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 23 to 25, respectively; (iv) an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 137, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 14 to 16, respectively, and / or an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 153, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 26 to 28, respectively; (v) an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 137, which comprises the CDR1, CDR2, and CDR3 regions of SEQ ID NOs: 14 to 16, respectively, and / or an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 154, which comprises the CDR1, CDR2, and CDR3 regions of SEQ ID NOs: 29 to 31, respectively; (vi) an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 137, the amino acid sequence comprising the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 14 to 16, respectively, and / or an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 155, the amino acid sequence comprising the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 32 to 34, respectively; (vii) an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 137, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 14 to 16, respectively, and / or an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 156, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 35 to 37, respectively; (viii) an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 137, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 14 to 16, respectively, and / or an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 157, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 38 to 40, respectively; (ix) an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 137, the amino acid sequence comprising the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 14 to 16, respectively, and / or an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 158, the amino acid sequence comprising the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 41 to 43, respectively; (x) an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 137, and that includes the CDR1, CDR2, and CDR3 regions of SEQ ID NOs: 14 to 16, respectively, and / or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 159, and that includes the CDR1, CDR2, and CDR3 regions of SEQ ID NOs: 44 to 46, respectively; (xi) an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 137, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 14 to 16, respectively, and / or an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 160, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 47 to 49, respectively; (xii) an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 137, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 14 to 16, respectively, and / or an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 161, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 50 to 52, respectively; (xiii) an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 137, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 14 to 16, respectively, and / or an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 162, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 53 to 55, respectively; (xiv) an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 137, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 14 to 16, respectively, and / or an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 163, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 56 to 58, respectively; (xv) an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 137, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 14 to 16, respectively, and / or an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 164, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 59 to 61, respectively; (xvi) an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 138, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 62 to 64, respectively, and / or an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 165, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 98 to 100, respectively; (xvii) an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 139, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 65 to 67, respectively, and / or an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 166, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 101 to 103, respectively; (xviii) an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 140, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 68 to 70, respectively, and / or an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 167, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 104 to 106, respectively; (xix) an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 141, the amino acid sequence comprising the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 71 to 73, respectively, and / or an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 168, the amino acid sequence comprising the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 107 to 109, respectively; (xx) an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 142, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 74 to 76, respectively, and / or an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 169, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 110 to 112, respectively; (xxi) an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 143, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 77 to 79, respectively, and / or an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 170, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 113 to 115, respectively; (xxii) an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 144, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 80 to 82, respectively, and / or an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 171, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 116 to 118, respectively; (xxiii) an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 145, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 83 to 85, respectively, and / or an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 172, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 119 to 121, respectively; (xxiv) an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 146, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 86 to 88, respectively, and / or an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 173, which comprises the CDR1 region, CDR2 region, and CDR3 region of SEQ ID NOs: 122 to 124, respectively; (xxv) an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 147, which comprises the CDR1, CDR2, and CDR3 regions of SEQ ID NOs: 89 to 91, respectively, and / or an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 174, which comprises the CDR1, CDR2, and CDR3 regions of SEQ ID NOs: 125 to 127, respectively; (xxvi) an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 148, which comprises the CDR1, CDR2, and CDR3 regions of SEQ ID NOs: 92 to 94, respectively, and / or an amino acid sequence having an identity of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% to SEQ ID NO: 175, which comprises the CDR1, CDR2, and CDR3 regions of SEQ ID NOs: 128 to 130, respectively; or (xxvii) An amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 149, and that includes CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 95 to 97, respectively, and / or an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 176, and that includes CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 131 to 133, respectively.

[0107] In some embodiments, the antigen recognition domain comprises or consists of the following sequence: (i) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 177, wherein the variable heavy domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 5 to 7, respectively, and the variable light domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 17 to 19, respectively; (ii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 178, wherein the variable heavy domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 8 to 10, and the variable light domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 20 to 22, respectively; (iii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 179, wherein the variable heavy domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 11 to 13, respectively, and the variable light domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 23 to 25, respectively; (iv) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 180, wherein the variable heavy domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 14 to 16, respectively, and the variable light domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 26 to 28, respectively; (v) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 181, wherein the variable heavy domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 14 to 16, respectively, and the variable light domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 29 to 31, respectively; (vi) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 182, wherein the variable heavy domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 14 to 16, respectively, and the variable light domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 32 to 34, respectively; (vii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 183, wherein the variable heavy domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 14 to 16, respectively, and the variable light domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 35 to 37, respectively; (viii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 184, wherein the variable heavy domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 14 to 16, respectively, and the variable light domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 38 to 40, respectively; (ix) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 185, wherein the variable heavy domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 14 to 16, respectively, and the variable light domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 41 to 43, respectively; (x) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 186, wherein the variable heavy domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 14 to 16, respectively, and the variable light domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 44 to 46, respectively; (xi) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 187, wherein the variable heavy domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 14 to 16, respectively, and the variable light domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 47 to 49, respectively; (xii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 188, wherein the variable heavy domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 14 to 16, respectively, and the variable light domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 50 to 52, respectively; (xiii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 189, wherein the variable heavy domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 14 to 16, respectively, and the variable light domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 53 to 55, respectively; (xiv) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 190, wherein the variable heavy domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 14 to 16, respectively, and the variable light domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 56 to 58, respectively; (xv) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 191, wherein the variable heavy domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 14 to 16, respectively, and the variable light domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 59 to 61, respectively; (xvi) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 192, wherein the variable heavy domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 62 to 64, respectively, and the variable light domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 98 to 100, respectively; (xvii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 193, wherein the variable heavy domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 65 to 67, respectively, and the variable light domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 101 to 103, respectively; (xvii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 194, wherein the variable heavy domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 68 to 70, respectively, and the variable light domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 104 to 106, respectively; (xix) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 195, wherein the variable heavy domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 71 to 73, respectively, and the variable light domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 107 to 109, respectively; (xx) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 196, wherein the variable heavy domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 74 to 76, respectively, and the variable light domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 110 to 112, respectively; (xxi) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 197, wherein the variable heavy domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 77 to 79, respectively, and the variable light domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 113 to 115, respectively; (xxii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 198, wherein the variable heavy domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 80 to 82, respectively, and the variable light domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 116 to 118, respectively; (xxiii) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 199, wherein the variable heavy domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 83 to 85, respectively, and the variable light domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 119 to 121, respectively; (xxiv) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 200, wherein the variable heavy domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 86 to 88, respectively, and the variable light domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 122 to 124, respectively; (xxv) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 201, wherein the variable heavy domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 89 to 91, respectively, and the variable light domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 125 to 127, respectively; (xxvi) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 202, wherein the variable heavy domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 92 to 94, respectively, and the variable light domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 128 to 130, respectively; or (xxvii) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 203, wherein the variable heavy domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 95 to 97, respectively, and the variable light domain comprises CDR1, CDR2, and CDR3 regions consisting of SEQ ID NOs: 131 to 133, respectively.

[0108] Hinge domain The CAR may comprise a hinge domain. As used herein, the "hinge domain," also referred to as the "spacer domain," refers to the extracellular portion of the CAR that separates the antigen-binding domain from the transmembrane domain. The hinge may provide flexibility for accessing the target antigen. For example, a long spacer provides extra flexibility to the CAR, making it easier to access membrane-proximal epitopes.

[0109] Suitable hinge domains will be apparent to those skilled in the art (e.g., Guedan, S., et al., 2018. Molecular Therapy-Methods & Clinical Development, 12, 145-156). Suitable hinge domains include, but are not limited to, CD28 hinge domain, CD8 hinge domain, IgG hinge domain, and IgD hinge domain. Preferably, the hinge domain is a CD8 or CD28 hinge domain.

[0110] Most preferably, the hinge domain is a CD8 hinge domain. Suitably, the hinge domain may comprise the amino acid sequence set forth as SEQ ID NO: 222, or a variant having at least 80% identity to SEQ ID NO: 222. Example of a CD8 hinge domain (SEQ ID NO: 222): TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD Suitably, the variant may have at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:222.

[0111] Suitably, said hinge domain is a CD28 hinge domain. Suitably, said hinge domain may comprise the amino acid sequence set forth as SEQ ID NO: 221, or a variant having at least 80% identity to SEQ ID NO: 221. Example of a CD28 hinge domain (SEQ ID NO: 221): IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP Suitably, the variant may have at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:221.

[0112] Suitably, the CAR may encode a tag, such as a c-Myc tag (EQKLISEEDL - SEQ ID NO: 223). Suitably, the tag may be incorporated into the extracellular domain of the CAR, such as into the hinge domain of the extracellular domain. An example of a CD28 hinge domain incorporating a c-Myc tag is shown below. Suitably, the hinge domain may comprise the amino acid sequence shown as SEQ ID NO: 224, or a variant having at least 80% identity to SEQ ID NO: 224. Example of a CD28 hinge domain with an integrated c-Myc tag (SEQ ID NO: 224): IEVEQKLISEEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP Suitably, the variant may have at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:224.

[0113] Transmembrane domain The CAR may comprise a transmembrane domain. As used herein, a "transmembrane domain" refers to a portion of a CAR that anchors the CAR within the Treg cell membrane. Thus, the transmembrane domain can span or reside within the Treg cell membrane. A transmembrane domain may be derived from a protein that includes extracellular and / or intracellular portions, and thus, as used herein, a transmembrane domain may be associated with not only the portion that is within or spans the cell membrane, but also the extracellular and / or intracellular residues derived from the protein from which it was derived. For example, a transmembrane domain may be associated with a hinge domain derived from its protein from which it was derived, e.g., a transmembrane domain derived from CD8 may be associated with a hinge domain derived from CD8. Also, a transmembrane domain derived from CD8 or CD28 may be associated with a CD8 or CD28 costimulatory domain, for example. Those skilled in the art will appreciate that the transmembrane domain may also be synthetic, e.g., de novo designed, and may not be derived from a protein with a transmembrane domain. The presence of a transmembrane domain within the cell membrane can be determined using any suitable method known in the art, such as fluorescent labeling using a fluorescent microscope.

[0114] Suitable transmembrane domains will be clear to those skilled in the art.The transmembrane domain may comprise the transmembrane sequence from any protein that has a transmembrane domain, such as type I transmembrane protein, type II transmembrane protein, or type III transmembrane protein.The transmembrane domain of CAR may also comprise an artificial hydrophobic sequence.The transmembrane domain may be selected so that it does not dimerize.

[0115] Examples of transmembrane (TM) domains used in CAR constructs are shown below. 1)CD28TM domain (Pule et al, Mol Ther, 2005, Nov;12(5):933-41; Brentjens et al, CCR, 2007, Sep15;13(18Pt1):5426-35; Casucci et al, Blood, 2013, Nov14;122(20):3461-72); 2) OX40 TM domain (Pule et al, Mol Ther, 2005, Nov;12(5):933-41); 3) 41BB TM domain (Brentjens et al, CCR, 2007, Sep15;13(18Pt1):5426-35); 4) CD3 zeta TM domain (Pule et al, Mol Ther, 2005, Nov;12(5):933-41; Savoldo B, Blood, 2009, Jun18;113(25):6392-402); 5)CD8 alpha TM domain (Maher et al, Nat Biotechnol, 2002, Jan;20(1):70-5.; Imai C, Leukemia, 2004, Apr;18(4):676-84; Brentjens et al, CCR, 2007, Sep15;13(18Pt1):5426-35; Milone et al. al, Mol Ther, 2009, Aug;17(8):1453-64); 6) ICOS TM domain; 7) CD4 TM domain.

[0116] Most preferably, the CAR may comprise a CD8 transmembrane domain. Suitably, the transmembrane domain may comprise the amino acid sequence set forth as SEQ ID NO: 225, or a variant having at least 80% identity to SEQ ID NO: 225. An example of a CD8™ domain (AA183-203) (SEQ ID NO: 225): IYIWAPLAGTCGVLLLSLVIT Suitably, the variant may have at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:225.

[0117] Most preferably, the CAR may comprise a CD8 hinge domain and a CD8 transmembrane domain. Suitably, the hinge and transmembrane domain may comprise the amino acid sequence set forth as SEQ ID NO: 226, or a variant having at least 80% identity to SEQ ID NO: 226. Example of a CD8 hinge domain and CD8 transmembrane domain (SEQ ID NO: 226): TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVIT Suitably, the variant may be at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:226.

[0118] Suitably, the CAR may comprise a CD28 hinge domain and a CD8 transmembrane domain. Suitably, the hinge and transmembrane domain may comprise the amino acid sequence set forth as SEQ ID NO: 227, or a variant having at least 80% identity to SEQ ID NO: 227. Example of a CD28 hinge domain and CD8 transmembrane domain (SEQ ID NO: 227): IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPIYIWAPLAGTCGVLLLSLVIT Suitably, the variant may be at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:227.

[0119] Suitably, the CAR may comprise a CD28 transmembrane domain. Suitably, the transmembrane domain may comprise the amino acid sequence set forth as SEQ ID NO: 228, or a variant having at least 80% identity to SEQ ID NO: 228. An example of a CD28™ domain (AA153-179) (SEQ ID NO: 228): FWVLVVVGGVLACYSLLVTVAFIIFWV Suitably, the variant may have at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:228.

[0120] Those skilled in the art will appreciate that a transmembrane domain variant must be capable of spanning or spanning the cell membrane, i.e., be capable of being a transmembrane domain.

[0121] End Domain A CAR may comprise an endodomain comprising one or more intracellular signaling domains and, optionally, one or more costimulatory domains. The CAR may comprise one or more intracellular signaling domains.

[0122] As used herein, an "intracellular signaling domain" refers to the intracellular portion of a CAR that is involved in transmitting the message of an effective CAR that binds to HLA (preferably HLA-A2) inside Tregs, thereby inducing Treg functions, such as immunosuppressive functions.

[0123] Suitable intracellular signaling domains will be apparent to those skilled in the art. The intracellular signaling domain is necessary to transmit effector function signals and direct Tregs to exert their specialized functions upon antigen binding. Examples of intracellular signaling domains include, but are not limited to, the T cell receptor zeta chain or its homologs (e.g., eta chain, FcεR1γ and β chains, 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, or signaling domains thereof. The intracellular signaling domain may be the human CD3 zeta signaling domain, FcyRIII, FcsRI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine-based activation motif (ITAM), or a combination thereof.

[0124] Most preferably, the intracellular signaling domain may comprise the intracellular signaling domain of human CD3 zeta signaling domain. Suitably, the intracellular signaling domain may comprise the amino acid sequence set forth as SEQ ID NO:229, or a variant having at least 80% identity to SEQ ID NO:229. Example of a CD3 zeta signaling domain (SEQ ID NO: 229): RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR Suitably, the variant may have at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:229.

[0125] The intracellular signaling domain of the CAR may comprise a CD28 signaling domain. Suitably, the intracellular signaling domain may comprise the amino acid sequence set forth as SEQ ID NO: 230, or a variant having at least 80% identity to SEQ ID NO: 230. Example of a CD28 signaling domain (SEQ ID NO: 230): RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS Suitably, the variant may have at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity to SEQ ID NO:230.

[0126] The intracellular signaling domain of the CAR may comprise a CD27 signaling domain. Suitably, the intracellular signaling domain may comprise the amino acid sequence set forth as SEQ ID NO: 231, or a variant having at least 80% identity to SEQ ID NO: 231. Example of a CD27 signaling domain (SEQ ID NO: 231): QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP In one embodiment, the intracellular signaling domain comprises a signaling motif that is at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:231.

[0127] Still other intracellular signaling domains will be apparent to those skilled in the art and may be used in connection with alternative embodiments of the present invention.

[0128] The CAR may also comprise one or more costimulatory domains.

[0129] As used herein, a "co-stimulatory domain" refers to the intracellular portion of a CAR that can promote Treg function (e.g., immunosuppressive function), proliferation, and / or persistence.

[0130] Thus, a CAR may comprise a compound endodomain comprising one or more of the above-described costimulatory domains fused to an intracellular signaling domain, such as CD3ζ. Such compound endodomains may be referred to as second-generation CARs, which can simultaneously transmit activation and costimulatory signals after antigen recognition. The most commonly used costimulatory domain is the CD28 costimulatory domain, which provides the most potent costimulatory signal, i.e., immunological signal 2, that induces Treg proliferation. Suitable costimulatory domains will be apparent to those skilled in the art.

[0131] Thus, the CAR may preferably comprise a CD28 costimulatory domain. Suitably, the one or more costimulatory domains may comprise the amino acid sequence set forth as SEQ ID NO: 230, or a variant having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO: 230.

[0132] Preferably, the one or more costimulatory domains may comprise the amino acid sequence set forth as SEQ ID NO:231, or a variant having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%) to SEQ ID NO:231.

[0133] Suitably, the one or more costimulatory domains may comprise one or more signalling domains of the TNF receptor family, such as the signalling domains of OX40, 4-1BB, ICOS, or TNFRSF25.

[0134] Exemplary sequences of the signaling domains of OX40, 4-1BB, ICOS, or TNFRSF25 are shown below. The one or more costimulatory domains may comprise one or more of SEQ ID NOs: 232-235, or a variant having at least 80% identity to one or more of SEQ ID NOs: 232-235. Example of an OX40 signaling domain (SEQ ID NO: 232): ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI Example of a 41BB signaling domain (SEQ ID NO: 233) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL Example of an ICOS signaling domain (SEQ ID NO: 234) CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL Example of a TNFRSF25 signaling domain (SEQ ID NO: 235): TYTYRHCWPHKPLVTADEAGMEALTPPPATHLSPLDSAHTLLAPPDSSEKICTVQLVGNSWTPGYPETQEALCPQVTWSWDQLPSRALGPAAAPTLSPESPAGSPAMMLQPGPQLYDVMDAVPARRWKEFVRTLGLREAEIEAVEVEIGRFRDQQYEMLKRWRQQQPAGLGAVYAALERMGLDGCVEDLRSRLQRGP The one or more costimulatory domains may comprise variants of one or more of the OX40, 4-1BB, ICOS, and TNFRSF25 signaling domains that are at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 232-235.

[0135] The endodomain of the CAR may comprise a CD28 signaling domain and a CD3 zeta signaling domain. Suitably, the endodomain may comprise the amino acid sequence set forth as SEQ ID NO: 236, or a variant having at least 80% identity to SEQ ID NO: 236. Examples of CD28 signaling domains and CD3 zeta signaling domains (SEQ ID NO: 236): RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR The endodomain may comprise a variant that is at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:236.

[0136] A variant of an intracellular signaling domain and / or costimulatory domain may have the same or a similar function as the corresponding wild-type intracellular signaling domain and / or costimulatory domain, e.g., at least 40, 50, 60, 70, 80, 90, 95, 100, 110, 120, 130, 140, or 150% of the function of the wild-type domain (e.g., the signaling ability of the wild-type domain).

[0137] Other domains In some embodiments, the CAR comprises one or more signal peptides.

[0138] The CAR may contain a leader sequence that directs the CAR to the endoplasmic reticulum pathway for expression on the cell surface. An example of a leader sequence is the CD8 leader sequence. Exemplary leader sequences are shown below as SEQ ID NOs: 237 and 241. Example of a CD8 leader (SEQ ID NO: 237) MALPVTALLLPLALLLHAARP Example of a leader sequence (SEQ ID NO: 241) MALPVTALLLPLALLLHAAAP

[0139] The leader sequence may comprise or consist of a variant having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 237 or 241.

[0140] In some embodiments, the CAR comprises one or more reporter domains, optionally in combination with a self-cleaving or cleavage domain.

[0141] Suitable reporter domains are well known in the art and include, but are not limited to, fluorescent proteins such as GFP. The use of a selectable marker is advantageous because the reporter domain allows Tregs successfully transfected with the polynucleotide or vector of the invention (Tregs transfected to express the encoded CAR) to be selected and isolated from the starting cell population using common methods, such as flow cytometry. Preferably, the reporter domain may be a luciferase-based reporter, a PET reporter (e.g., sodium iodide symporter (NIS)), or a membrane protein (e.g., CD34, low-affinity nerve growth factor receptor (LNGFR)).

[0142] The nucleic acid sequence encoding the CAR and the nucleic acid sequence encoding the reporter domain may be separated by a co-expression site that allows each polypeptide to be expressed as an individual entity. Suitable co-expression sites are known in the art, such as internal ribosome entry sites (IRES) and self-cleaving peptides. Suitable self-cleaving or cleavage domains include, but are not limited to, P2A peptide, T2A peptide, E2A peptide, F2A peptide, and furin site.

[0143] Example CAR construct Examples of CARs for use in the present invention are provided below. The CAR may comprise an amino acid sequence that is at least 80% identical (e.g., at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) to one of SEQ ID NOs: 209 or 210. Preferably, any such variant retains at least part of the function compared to SEQ ID NOs: 209 or 210. For example, the variant may have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the function of the amino acid sequence set forth as one of SEQ ID NOs: 209 or 210. The variant or derivative may have a level of function that is close to or the same as that of one of SEQ ID NOs: 209 or 210, or may have a higher level of function (e.g., at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% higher) than the amino acid sequence set forth as one of SEQ ID NOs: 209 or 210. CD8 hinge-CD8TM domain-CD28 signaling domain-CD3 zeta signaling domain (SEQ ID NO: 209): TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRV KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD28 hinge-CD8TM domain-CD28 signaling domain-CD3 zeta signaling domain (SEQ ID NO: 210): IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPIYIWAPLAGTCGVLLLSLVITRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFS RSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0144] In particular, SEQ ID NO: 209 or a variant thereof may be used in combination with an antigen-binding domain comprising the following (i), (ii), or (iii): (i) SEQ ID NOs: 8 to 10 and SEQ ID NOs: 20 to 22, or derivatives thereof; (ii) SEQ ID NOs: 11 to 13 and SEQ ID NOs: 23 to 25, or derivatives thereof; or (iii) SEQ ID NOs: 14 to 16 and SEQ ID NOs: 26 to 28, or derivatives thereof.

[0145] The vector of the present invention may in particular comprise a CAR comprising domains as shown in the table below. [Table 4]

[0146] Examples of polynucleotide sequences encoding SEQ ID NO:209 and SEQ ID NO:210 are provided below. A polynucleotide encoding an HLA-specific CAR may comprise a sequence that is at least 70% identical (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) to SEQ ID NO:211 or 212. Preferably, any such variant encodes an HLA-specific CAR that has at least partial function compared to SEQ ID NO:209 or 210. For example, the HLA-specific CAR encoded by said variant may have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the function of the amino acid sequence set forth as SEQ ID NO:209 or 210. The HLA-specific CAR encoded by the variant may have a level of function close to or the same as that of one of SEQ ID NOs: 209 or 210, or may have a higher level of function (e.g., at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% higher) than the amino acid sequence set forth as one of SEQ ID NOs: 209 or 210. An example of a polynucleotide sequence encoding SEQ ID NO:209 (SEQ ID NO:211): ACCACCACCCCCGCCCCCCGCCCCCCCACCCCCGCCCCCACCATCGCCAGCCAGCCCCTGAGCCTGCGCCCCGAGGCCTGCCGCCCCGCCGCCGGCGGCGCCGTGCACACCCGCGGCCTGGACTTCGCCTGCGACATCTACATCTGGGCCCCCCTGGCCGGCACCTGCGGCGTGCTGCTGCTGAGCCTGGTGATCACCCGCAGCAAGCGCAGCCGCCTGCTGCACAGCGACTACATGAACATGACCCCCCGCCGCCCCGGCCCCACCCGCAAGCACTACCAGCCCTACGCCCCCCCCCGCGACTTCGCCGCCTACCGCAGCCGCGTGAAGTTCAGCCGCAGCGCCGACGCCCCCGCCTACCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCCGCCGCGAGGAGTACGACGTGCTGGACAAGCGCCGCGGCCGCGACCCCGAGATGGGCGGCAAGCCCCGCCGCAAGAACCCCCAGGAGGGCCTGTACAACGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGCCGCCGCGGCAAGGGCCACGACGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCCCGC Example of a polynucleotide sequence encoding SEQ ID NO: 210 (SEQ ID NO: 212): ATCGAGGTGATGTACCCCCCCCCTACCTGGACAACGAGAAGAGCAACGGCACCATCATCCACGTGAAGGGCAAGCACCTGTGCCCCAGCCCCCTGTTCCCCGGCCCAGCAAGCCCATCTACATCTGGGCCCCCCTGGCCGGCACCTGCGGCGTGCTG CTGCTGAGCCTGGTGATCACCCGCAGCAAGCGCAGCCGCCTGCTGCACAGCGACTACATGAACATGACCCCCCGCCGCCCCGGCCCCACCCGCAAGCACTACCAGCCCTACGCCCCCCCGCGACTTCGCCGCCTACCGCAGCCGCGTGAAGTTCAGCC GCAGCGCCGACGCCCCCGCCTACCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCCGCCGCGAGGAGTACGACGTGCTGGACAAGCGCCGCGGCCGCGACCCCGAGATGGGCGGCAAGCCCCGCCGCAAGAACCCCCAGGAGGGCCTGTA CAACGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGCCGCCGCGGCAAGGGCCACGACGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCCCGC

[0147] Relative Position of the First and Second Polynucleotides In a preferred embodiment, the first polynucleotide encoding FOXP3 is upstream of the second polynucleotide encoding the HLA-specific CAR. Thus, in a preferred embodiment, the first polynucleotide and the second polynucleotide are operably linked to the same promoter, and the first polynucleotide is upstream of the second polynucleotide.

[0148] When the term "upstream" is used herein in reference to a polynucleotide, the "upstream" polynucleotide refers to the 5' side of the "downstream" polynucleotide. In other words, in a preferred embodiment, the vector may have a 5' FOXP3-HLA-specific CAR 3' orientation. In a more preferred embodiment, the vector may have a 5' promoter-FOXP3-HLA-specific CAR 3' structure. Herein, FOXP3 expression is directly driven by the promoter for optimal expression.

[0149] Importantly, the 5' to 3' configuration of FOXP3 preceding the CAR ensures that CAR expression can only occur when (exogenous) FOXP3 is expressed, and that CAR expression does not occur in the absence of FOXP3. In the context of engineered Tregs, this is particularly advantageous, as it reduces the risk of Tregs acquiring an effector phenotype and / or reduces the risks associated with introducing the CAR into T effector cells present in the starting population.

[0150] Cleavage sites and internal ribosome entry sites The polynucleotide encoding FOXP3 may be separated from the polynucleotide encoding the HLA-specific CAR by a nucleic acid sequence that allows both the nucleic acid sequence encoding FOXP3 and the nucleic acid sequence encoding the HLA-specific CAR to be expressed from the same mRNA transcript.

[0151] For example, the vector may contain an internal ribosome entry site (IRES) between (i) the nucleic acid sequence encoding FOXP3 and (ii) the nucleic acid sequence encoding the HLA-specific CAR. The IRES is a nucleotide sequence that allows translation initiation in the middle of an mRNA sequence. Preferably, the vector may have a structure of 5' promoter-FOXP3-IRES-HLA-specific CAR 3'.

[0152] Preferably, the vector may comprise (i) a nucleic acid sequence encoding FOXP3 and (ii) a nucleic acid sequence encoding an HLA-specific CAR linked by a cleavage domain. Such sequences may be self-cleaving during protein production or may be cleaved by common enzymes present in cells. Preferably, the cleavage domain may be self-cleaving. Thus, by including a cleavage domain in the polypeptide sequence, the first and second polypeptides can be expressed as a single polypeptide, which is then cleaved into individual functional polypeptides. Preferably, the vector may have the following structure: 5' promoter-FOXP3-cleavage domain-HLA-specific CAR 3'. A suitable cleavage domain may comprise a furin site (e.g., SEQ ID NO: 238 or 239). Furin site - cleavage domain: RXXR (SEQ ID NO: 238) (preferentially RRKR (SEQ ID NO: 239)

[0153] Preferably, the vector may comprise (i) a nucleic acid sequence encoding FOXP3 and (ii) a nucleic acid sequence encoding an HLA-specific CAR linked by a self-cleaving sequence. Such sequences self-cleave during protein production. Suitably, the vector may have a structure of 5' promoter-FOXP3-self-cleaving sequence-HLA-specific CAR 3'.

[0154] Preferably, the self-cleaving sequence is a polynucleotide sequence encoding a 2A self-cleaving peptide. Suitably, the vector may have a structure of 5' promoter-FOXP3-2A self-cleaving peptide-HLA-specific CAR 3'.

[0155] Overall, compared to other methods for co-expression of multiple genes, 2A peptides result in relatively high levels of downstream protein expression, and their small size reduces the risk of interfering with the function of co-expressed genes (Liu, Z., et al., 2017. Scientific Reports, 7(1), p.2193).

[0156] Furthermore, the mechanism of 2A-mediated "self-cleavage" is that the ribosome skips the formation of a glycyl-prolyl peptide bond at the C-terminus of 2A. A highly conserved sequence, GDVEXNPGP (SEQ ID NO: 240), is shared by different 2As at the C-terminus and is essential for steric hindrance and ribosome skipping. 2A-mediated skipping can occur in three ways: (1) skipping occurs, and translation resumes, resulting in two "cleaved" proteins: the protein upstream of 2A is attached with the complete 2A peptide except for the C-terminal proline, and the protein downstream of 2A is attached with a single N-terminal proline; (2) skipping occurs, but the ribosome is dropped off, and translation does not continue, resulting in only the protein upstream of 2A; (3) skipping does not occur, and translation continues, resulting in a fusion protein. Because of the risk of (2), the configuration in which FOXP3 precedes CAR in the 5' to 3' direction ensures that CAR expression can occur only when FOXP3 is expressed, and that CAR expression does not occur in the absence of FOXP3.

[0157] Suitable self-cleaving peptides include P2A peptides, T2A peptides, E2A peptides, and F2A peptides.

[0158] Suitably, the vector may have the following structure: (i) 5′ promoter-FOXP3-P2A-HLA-specific CAR 3′; (ii) 5′ promoter-FOXP3-T2A-HLA-specific CAR 3′; (iii) 5' promoter-FOXP3-E2A-HLA-specific CAR 3'; or (iv) 5' promoter-FOXP3-F2A-HLA-specific CAR 3'.

[0159] Preferably, the vector may have the following structure: 5' promoter-FOXP3-P2A-HLA-specific CAR 3'.

[0160] Exemplary sequences of P2A, T2A, E2A, and F2A peptides are shown below. The self-cleaving sequence may comprise or consist of a polynucleotide sequence encoding any of SEQ ID NOs: 213, 215, 217, 219, 242, 244, 246, or 248, or a variant having at least 80% identity to any of SEQ ID NOs: 213, 215, 217, 219, 242, 244, 246, or 248. Example of a P2A peptide cleavage domain (SEQ ID NO: 213): GSGATNFSLLKQAGDVEENPGP Example of a T2A peptide cleavage domain (SEQ ID NO: 215): GSGEGRGSLLTCGDVEENPGP Example of an E2A peptide cleavage domain (SEQ ID NO: 217): GSGQCTNYALLKLAGDVESNPGP Example of an F2A peptide cleavage domain (SEQ ID NO: 219): GSGVKQTLNFDLLKLAGDVESNPGP An example of a P2A peptide cleavage domain (SEQ ID NO: 242): ATNFSLLKQAGDVEENPGP Example of a T2A peptide cleavage domain (SEQ ID NO: 244): EGRGSLLTCGDVEENPGP Example of an E2A peptide cleavage domain (SEQ ID NO: 246): QCTNYALLKLAGDVESNPGP Example of an F2A peptide cleavage domain (SEQ ID NO: 248): VKQTLNFDLLKLAGDVESNPGP The self-cleaving sequence may comprise or consist of a polynucleotide sequence encoding a variant that is at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 213, 215, 217, 219, 242, 244, 246, or 248.

[0161] Examples of polynucleotide sequences encoding the P2A, T2A, E2A, and F2A peptides are shown below. The self-cleaving sequence may comprise or consist of a polynucleotide selected from any of SEQ ID NOs: 214, 216, 218, 220, 243, 245, 247, or 249, or a variant having at least 80% identity to any of SEQ ID NOs: 214, 216, 218, 220, 243, 245, 247, or 249. Example of a P2A peptide cleavage domain (SEQ ID NO: 214): GGCAGCGGCGCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCC Example of a T2A peptide cleavage domain (SEQ ID NO: 216): GGCAGCGGCGAGGGCCGCGGCAGCCTGCTGACCTGCGGCGACGTGGAGGAGAACCCCGGCCCC Example of an E2A peptide cleavage domain (SEQ ID NO: 218): GGCAGCGGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC Example of an F2A peptide cleavage domain (SEQ ID NO: 220): GGCAGCGGCGTGAAGCAGACCCTGAACTTCGACCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC An example of a P2A peptide cleavage domain (SEQ ID NO: 243): GCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCCGGCCCC Example of a T2A peptide cleavage domain (SEQ ID NO: 245): GAGGGCCGCGGCAGCCTGCTGACCTGCGGCGACGTGGAGGAGAACCCCGGCCCC Example of an E2A peptide cleavage domain (SEQ ID NO: 247): CAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC Example of an F2A peptide cleavage domain (SEQ ID NO: 249): GTGAAGCAGACCCTGAACTTCGACCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC The self-cleaving sequence may comprise or consist of a variant that has at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 214, 216, 218, 220, 243, 245, 247, or 249.

[0162] Polynucleotides and Polypeptides In the present invention, HLA-specific cells are generated by introducing into cells a polynucleotide encoding a FOXP3 polypeptide (sometimes referred to herein as a first polynucleotide) and a polynucleotide encoding an HLA-specific chimeric antigen receptor (CAR) (sometimes referred to herein as a second polynucleotide).

[0163] The terms "polynucleotide" and "nucleic acid" are intended to be synonymous with each other. A polynucleotide may be any suitable type of nucleotide sequence, for example, a synthetic RNA / DNA sequence, a cDNA sequence, or a partial genomic DNA sequence.

[0164] The term "polypeptide" is synonymous with "protein" and refers to a series of residues, typically L-amino acids, joined together by peptide bonds between the alpha amino and carboxyl groups of adjacent amino acids.

[0165] Many different polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code, and one skilled in the art can make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotide to reflect the codon usage of any particular host organism in which the polypeptide is expressed.

[0166] The polynucleotide may comprise DNA or RNA, may be single-stranded or double-stranded, and may contain synthetic or modified nucleotides. Several different types of modifications to oligonucleotides are known in the art. These modifications include methylphosphonate and phosphorothioate backbones, and the addition of acridine or polylysine chains to the 3' and / or 5' ends of the molecule. Polynucleotides may be modified by any method known in the art. Such modifications may improve the in vivo activity or lifespan of the polynucleotide.

[0167] The polynucleotide may be in isolated or recombinant form. The polynucleotide may be incorporated into a vector, and the vector may be incorporated into a host cell.

[0168] The polynucleotide may be codon-optimized. Different cells have different frequencies of use of certain codons. This codon bias corresponds to a bias in the relative abundance of certain tRNAs in cell types. By adjusting the codons in the sequence to match the relative abundance of the corresponding tRNAs, expression can be increased. Preferably, the polynucleotide may be codon-optimized for expression in a mouse disease model. Preferably, the polynucleotide may be codon-optimized for expression in a human subject.

[0169] Many viruses, such as HIV and other lentiviruses, utilize numerous rare codons, and by changing these to correspond to commonly used mammalian codons, increased expression of the packaging components in mammalian producer cells can be achieved. Codon usage tables are well known in the art for mammalian cells, as well as for a variety of other organisms. Codon optimization may also involve the removal of mRNA instability motifs and cryptic splice sites.

[0170] Variants, derivatives, and fragments In addition to the specific polypeptides and polynucleotides referred to herein, the use of derivatives, variants, and fragments thereof is also encompassed by the present invention.

[0171] The term "derivative" as used herein in relation to a protein or polypeptide of the invention includes substitution, mutation, modification, substitution, deletion, and / or addition of one (or more) amino acid residues from or to the sequence, provided that the resulting polypeptide retains a desired function (e.g., if the derivative or variant is an antigen-binding domain, the desired function may be the ability of the antigen-binding domain to bind to a target antigen, or if the derivative or variant is a signaling domain, the desired function may be the ability of the domain to signal (e.g., activate or inactivate a downstream molecule)). The variant or derivative may have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the function of the corresponding reference sequence, or may have a similar or identical level of function of the corresponding reference sequence, or may have a higher level of function than the corresponding reference sequence, for example at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% higher than the unaltered sequence.

[0172] Typically, amino acid substitutions may be made, for example, from 1, 2, or 3 to 10 or 20 substitutions, provided that the modified sequence retains the required activity or potency, e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the activity compared to the corresponding reference sequence, or a similar or identical level of activity compared to the corresponding reference sequence, or a higher level of activity than the corresponding reference sequence, e.g., at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% higher than the unmodified sequence. Non-naturally occurring analogs may be used for amino acid substitutions.

[0173] Proteins or peptides used in the present invention may also have deletions, insertions, or substitutions of amino acid residues that produce a silent change and result in a functionally equivalent protein. Deliberate amino acid substitutions may be made based on the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues, so long as the inherent function is maintained. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with uncharged polar head groups with similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0174] Conservative substitutions may be made, for example, according to the following table: Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other. [Table 5]

[0175] Said derivatives may be homologues or variants. As used herein, the terms "homologue" or "variant" refer to entities that have a certain homology with the wild-type amino acid sequence and the wild-type nucleotide sequence. The term "homology" can be equated with "identity".

[0176] A homologous or variant sequence may comprise an amino acid or nucleotide sequence that may be at least 50%, at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, preferably at least 95%, at least 97%, or at least 99% identical to its reference sequence. Typically, the homolog will have similar chemical properties / functions, e.g., contain the same binding site as the reference amino acid sequence or the amino acid sequence encoded by the reference nucleotide sequence. Although homology can also be considered in terms of similarity (i.e., amino acid residues with similar chemical properties / functions), in the context of the present invention, it is preferred to express homology in terms of sequence identity.

[0177] Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percent homology or percent identity between two or more sequences. Percent homology may also be calculated for consecutive sequences, i.e., one sequence is aligned with the other and each amino acid in one sequence is directly compared, residue by residue, to the corresponding amino acid in the other sequence. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

[0178] While this is a very simple and consistent method, it does not take into account that in a pair of sequences that are identical except for, for example, a single insertion or deletion, that insertion or deletion in the nucleotide sequence may cause the following codon to be moved out of alignment, potentially resulting in a significant decrease in percent homology when a global alignment is performed. As a result, most sequence comparison methods are designed to produce optimal alignments that take into account possible insertions and deletions without unduly penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to maximize local homology.

[0179] However, these more complex methods assign a "gap penalty" to each gap that occurs during alignment, such that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible (reflecting greater similarity between the two compared sequences) achieves a higher score than one with many gaps. An "affine gap cost" is typically used, which imposes a relatively high cost for the presence of a gap and a smaller penalty for each subsequent residue within the gap. This is the most commonly used gap scoring system. Higher gap penalties naturally produce optimized alignments with fewer gaps. Most alignment programs allow for the modification of gap penalties. However, it is preferable to use the default values ​​when using such software for sequence comparison. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0180] Calculating maximum percentage homology therefore first requires the creation of an optimal alignment, taking into account gap penalties. A suitable computer program for performing such alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Res. 12: 387). Examples of other software capable of performing sequence comparison include, but are not limited to, the BLAST package (see Ausubel et al. (1999) ibid-Ch. 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410) and the GENEWORKS comparison tool suite. Both BLAST and FASTA are available for offline and online searches (see Ausubel et al. (1999) ibid, pages 7-58 to 7-60). However, for some applications, it is preferable to use the GCG Bestfit program. Another tool called BLAST2Sequences is also available for comparing protein and nucleotide sequences (see FEMS Microbiol. Lett. (1999) 174:247-50; FEMS Microbiol. Lett. (1999) 177:187-8).

[0181] Although the final percentage homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pairwise comparison. Instead, a scaled similarity score matrix is ​​commonly used, which assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. One example of such a matrix commonly used is the BLOSUM62 matrix (the default matrix for the BLAST suite of programs). GCG Wisconsin programs typically use either the public default values ​​or a custom symbol comparison table, if supplied (see user manual for further details). However, for some applications, it is preferred to use the default values ​​in the GCG package, and for other software, it is preferred to use a default matrix such as BLOSUM62. Preferably, percent identity is measured across the entire reference and / or query sequence.

[0182] Once the software has produced an optimal alignment, it is possible to calculate percent homology, preferably percent sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

[0183] A "fragment" typically refers to a selected region of a polypeptide or polynucleotide of interest in terms of function. Thus, a "fragment" refers to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide, respectively.

[0184] Such derivatives, variants, and fragments may be prepared using standard recombinant DNA techniques, such as site-directed mutagenesis. Where an insert is made, synthetic DNA encoding the insert may be made, with 5' and 3' flanking regions corresponding to the naturally occurring sequence on either side of the insertion site. The flanking regions contain appropriate restriction enzyme sites corresponding to sites in the naturally occurring sequence, so that the sequence can be cleaved with the appropriate enzyme(s) and the synthetic DNA ligated to the cleavage sites. The DNA is then expressed in accordance with the invention to produce the encoded protein. These methods are merely illustrative of the many standard techniques well known in the art for manipulating DNA sequences; other known techniques may also be used.

[0185] vector In some embodiments of the invention, the polynucleotide encoding FOXP3 (sometimes referred to herein as the first polynucleotide) and / or the polynucleotide encoding the HLA-specific CAR (sometimes referred to herein as the second polynucleotide) are contiguous portions of a vector.

[0186] In a preferred embodiment, the first polynucleotide encoding FOXP3 and the second polynucleotide encoding the HLA-specific CAR are present in a single vector.

[0187] A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. According to the present invention, by way of example, some vectors used in recombinant nucleic acid technology are capable of transferring an entity, such as a segment of nucleic acid (e.g., a heterologous DNA segment, such as a heterologous cDNA segment), into a target cell.

[0188] Vectors may be non-viral or viral. Examples of vectors used in recombinant nucleic acid technology include, but are not limited to, plasmids, mRNA molecules (e.g., in vitro transcribed mRNA), chromosomes, artificial chromosomes, and viruses. Vectors may also be, for example, naked nucleic acids (e.g., DNA). In the simplest form, the vector itself may be the nucleotide of interest. Preferably, the vector can be continuously expressed at a high level in the host cell.

[0189] Suitably, the vector used in the present invention may be, for example, a plasmid, mRNA or viral vector, hi a preferred embodiment, the vector is a viral vector.

[0190] Many viral systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Using techniques well known in the art, a selected gene can be inserted into a vector and packaged into retroviral particles. The recombinant virus can then be isolated and delivered to cells of interest either in vivo or ex vivo. The vector may comprise a promoter for expression of the polynucleotide, and optionally one or more regulators of that promoter. In a preferred embodiment, the first polynucleotide encoding FOXP3 and the second polynucleotide encoding the HLA-specific CAR are present in a single vector, and the first polynucleotide and the second polynucleotide are optionally operably linked to the same promoter (e.g., LTR).

[0191] The vectors of the present invention may be introduced into cells using various techniques known in the art, such as transformation or transduction. Several techniques are known in the art, such as infection with recombinant viral vectors, such as retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, baculoviral vectors, and herpes simplex viral vectors; direct injection of nucleic acids; and biolistic transformation. Many vectors can be used for transduction / gene transfer.

[0192] Non-viral delivery systems include, but are not limited to, DNA transfer methods. Gene transfer includes the process of using a non-viral vector to deliver a gene to a target cell. Non-viral delivery systems can include liposomes or amphiphilic cell-penetrating peptides, preferably complexed with a polynucleotide of the present invention.

[0193] Typical gene transfer methods include electroporation, DNA gene guns, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofection, cationic drug-mediated transfection, cationic facial amphiphiles (CFAs) (Nat. Biotechnol. (1996) 14: 556), and combinations thereof.

[0194] viral vectors The vector of the present invention may be a viral vector. The viral vector may be any viral vector known to those skilled in the art. In particular, many viral-based systems have been developed for gene transfer into mammalian cells. Preferably, the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector, a poxvirus vector, or a vaccinia virus vector. Preferably, the viral vector is a retroviral vector (e.g., a gamma retroviral vector) or a lentiviral vector. More preferably, the viral vector is a lentiviral vector.

[0195] Preferably, the vector used in the present invention is a retroviral vector that has been genetically modified so that the virus cannot replicate or produce infectious progeny virus particles once it enters target cells. There are many retroviruses that are widely used to deliver genes in both tissue culture conditions and live organisms. Examples include, but are not limited to, murine leukemia virus (MLV), human immunodeficiency virus (HIV-1), equine infectious anemia virus (EIAV), mouse mammary tumor virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myelocytomatosis virus-29 (MC29), and avian erythroblastosis virus (AEV), as well as all other members of the retroviridae family, including lentiviruses. A detailed list of retroviruses may be found in Coffin et al., 1997, "retroviruses", Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763.

[0196] The basic structure of a retroviral genome is that located between or within the 5'LTR and 3'LTR are a packaging signal that enables packaging of the genome, a primer binding site, an integration site that enables integration into the host cell genome, and the gag, pol, and env genes that encode packaging components (these are polypeptides necessary for the assembly of viral particles).More complex retroviruses have additional features that enable efficient transport of the integrated proviral RNA transcript from the cell nucleus to the cytoplasm of infected target cells, such as the rev and RRE sequences in HIV.

[0197] In the provirus, these genes are flanked at both ends by regions called long terminal repeats (LTRs). The LTRs are responsible for proviral integration and transcription. LTRs also function as enhancer-promoter sequences, controlling viral gene expression. Encapsidation of retroviral RNA is mediated by the psi sequence located at the 5' end of the viral genome.

[0198] The LTR itself is an identical sequence that can be divided into three elements, designated U3, R, and U5. U3 is derived from a sequence unique to the 3' end of the RNA. R is derived from a sequence repeated at both ends of the RNA, and U5 is derived from a sequence unique to the 5' end of the RNA. The sizes of the three elements can vary considerably between different retroviruses.

[0199] In the retroviral vector genome of the present invention, gag, pol, and env may be absent or may not function. The R regions at both ends of the RNA are repeated sequences. U5 and U3 represent unique sequences at the 5' and 3' ends of the RNA genome, respectively.

[0200] Preferably, the envelope allows for the transduction of human cells, preferably T cells, most preferably Tregs. Examples of suitable env genes include, but are not limited to, VSV-G, MLV amphotropic env such as 4070A env, RD114 feline leukemia virus env, or hemagglutinin (HA) from influenza virus. The Env protein may be capable of binding to receptors on a limited number of human cell types or may be a genetically engineered envelope containing a targeting moiety. The Env and gag-pol coding sequences are transcribed from a promoter and, optionally, an enhancer active in the selected packaging cell line, with the transcription unit terminated by a polyadenylation signal. For example, if the packaging cell is a human cell, a suitable promoter-enhancer combination may be that from the human cytomegalovirus major-immediate-early (hCMV-MIE) gene and a polyadenylation signal from the SV40 virus. Other suitable promoters and polyadenylation signals are known in the art.

[0201] In a preferred embodiment, the vector of the present invention is a lentiviral vector. Lentiviral vectors are part of a larger group of retroviral vectors. A detailed list of lentiviruses can be found in Coffin et al. "Retroviruses" 1997 Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763. Briefly, lentiviruses are divided into primate and non-primate groups. Examples of primate lentiviruses include, but are not limited to, human immunodeficiency virus (HIV), the causative agent of human acquired immunodeficiency syndrome (AIDS), and simian immunodeficiency virus (SIV). The non-primate lentivirus group includes the prototype "slow virus" Visna / Maedi virus (VMV), as well as the related Caprine Arthritis-Encephalitis Virus (CAEV), Equine Infectious Anemia Virus (EIAV), and the more recently described Feline Immunodeficiency Virus (FIV), and Bovine Immunodeficiency Virus (BIV).

[0202] What distinguishes the lentivirus family from other types of retroviruses is that lentiviruses can infect both dividing and non-dividing cells. In contrast, other retroviruses are unable to infect non-dividing or slowly dividing cells that make up, for example, muscle, brain, lung, and liver tissues. Because lentiviruses can transduce well-differentiated / primary cells, the use of lentiviral screening strategies allows for library selection in primary target non-dividing or slowly dividing host cells.

[0203] The vector of the present invention may be packaged into a viral particle. Methods for packaging viral particles are well known to those skilled in the art. For example, a method for producing and packaging a retroviral vector is described in Merten, OW, 2004. The Journal of Gene Medicine: A cross-disciplinary journal for research on the science of gene transfer and its clinical applications, 6(S1), pp.S105-S124. For example, a method for producing and packaging a lentiviral particle is described in Merten, OW, et al., 2016. Molecular Therapy-Methods & Clinical Development, 3, p.16017 and Zufferey, R., 2002. Production of lentiviral vectors. In Lentiviral Vectors (pp. 107-121). Springer, Berlin, Heidelberg.

[0204] Example vector constructs Examples of vectors for use in the present invention are provided below. Suitably, the vector may comprise (5' to 3'): (i) a polynucleotide sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) to SEQ ID NO:3, or a functional fragment thereof of or comprising said polynucleotide sequence or a functional fragment thereof mosquito A first polynucleotide comprising: and , cleavage domain, and / or IRES and,a second polynucleotide comprising a polynucleotide sequence that is at least 70% identical (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 211; (ii) a polynucleotide sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) to SEQ ID NO:3, or a functional fragment thereof of or comprising said polynucleotide sequence or a functional fragment thereof mosquito A first polynucleotide comprising: and , cleavage domain, and / or IRES and, a second polynucleotide comprising a polynucleotide sequence that is at least 70% identical (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 212; (iii) a polynucleotide sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) to SEQ ID NO:4, or a functional fragment thereof of or comprising said polynucleotide sequence or a functional fragment thereof mosquito A first polynucleotide comprising: and , cleavage domain, and / or IRES and, a second polynucleotide comprising a polynucleotide sequence that is at least 70% identical (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 211; or (iv) a polynucleotide sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) to SEQ ID NO:4, or a functional fragment thereof of or comprising said polynucleotide sequence or a functional fragment thereof mosquito A first polynucleotide comprising: and , cleavage domain, and / or IRES and, A second polynucleotide comprising a polynucleotide sequence that is at least 70% identical (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 212.

[0205] The first polynucleotide and the second polynucleotide may be operably linked to the same promoter. Suitably, the vector is a viral vector, preferably a retroviral vector or a lentiviral vector.

[0206] Suitably, the vector may comprise (5' to 3'): (i) a polynucleotide sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) to SEQ ID NO:3, or a functional fragment thereof of or comprising said polynucleotide sequence or a functional fragment thereof mosquito A first polynucleotide comprising: and a self-cleaving sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 214; a second polynucleotide comprising a polynucleotide sequence that is at least 70% identical (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 211; (ii) a polynucleotide sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) to SEQ ID NO:3, or a functional fragment thereof of or comprising said polynucleotide sequence or a functional fragment thereof mosquito A first polynucleotide comprising: and a self-cleaving sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 214; a second polynucleotide comprising a polynucleotide sequence that is at least 70% identical (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 212; (iii) a polynucleotide sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) to SEQ ID NO:4 of or comprising said polynucleotide sequence or a functional fragment thereof mosquito A first polynucleotide comprising: and a self-cleaving sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 214; a second polynucleotide comprising a polynucleotide sequence that is at least 70% identical (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 211; or (iv) a polynucleotide sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) to SEQ ID NO:4, or a functional fragment thereof of or comprising said polynucleotide sequence or a functional fragment thereof mosquito A first polynucleotide comprising: and a self-cleaving sequence having at least 70% identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 214;A second polynucleotide comprising a polynucleotide sequence that is at least 70% identical (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 212.

[0207] The first polynucleotide and the second polynucleotide may be operably linked to the same promoter. Suitably, the vector is a viral vector, preferably a retroviral vector or a lentiviral vector.

[0208] Suitably, the vector may comprise (5' to 3'): (i) a polynucleotide sequence having at least 95% identity to SEQ ID NO: 3 or a functional fragment thereof of a polynucleotide sequence comprising or having at least 95% identity to SEQ ID NO: 3, or a functional fragment thereof mosquito A first polynucleotide comprising: and a self-cleaving sequence having at least 95% identity to SEQ ID NO: 214; a second polynucleotide comprising a polynucleotide sequence that is at least 70% identical (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 211; (ii) a polynucleotide sequence having at least 95% identity to SEQ ID NO: 3, or a functional fragment thereof of a polynucleotide sequence comprising or having at least 95% identity to SEQ ID NO: 3, or a functional fragment thereof mosquito A first polynucleotide comprising: and a self-cleaving sequence having at least 95% identity to SEQ ID NO: 214; a second polynucleotide comprising a polynucleotide sequence that is at least 70% identical (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 212; (iii) a polynucleotide sequence having at least 95% identity to SEQ ID NO:4 of a polynucleotide sequence comprising or having at least 95% identity to SEQ ID NO:4 mosquito A first polynucleotide comprising: and a self-cleaving sequence having at least 95% identity to SEQ ID NO: 214; a second polynucleotide comprising a polynucleotide sequence that is at least 70% identical (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 211; or (ii) a polynucleotide sequence having at least 95% identity to SEQ ID NO: 4, or a functional fragment thereof of a polynucleotide sequence comprising or having at least 95% identity to SEQ ID NO: 4, or a functional fragment thereof mosquito A first polynucleotide comprising: and a self-cleaving sequence having at least 95% identity to SEQ ID NO: 214; A second polynucleotide comprising a polynucleotide sequence that is at least 70% identical (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) to SEQ ID NO: 212.

[0209] The first polynucleotide and the second polynucleotide may be operably linked to the same promoter. Suitably, the vector is a viral vector, preferably a retroviral vector or a lentiviral vector.

[0210] Regulators of gene expression The vector of the present invention may comprise a promoter for expression of a polynucleotide(s). When the first polynucleotide encoding FOXP3 and the second polynucleotide encoding an HLA-specific CAR are present in the same vector, the first polynucleotide and the second polynucleotide may be operably linked to the same promoter.

[0211] A "promoter" is a region of DNA that initiates the initiation of transcription of a gene. A promoter is located upstream of the DNA (toward the 5' region of the signal strand), near the transcription start site of the gene. Any suitable promoter may be used, the selection of which would be readily apparent to one of skill in the art.

[0212] In one embodiment, the promoter may be an LTR, for example an LTR of a vector (eg, a retroviral or lentiviral LTR).

[0213] Long terminal repeats (LTRs) are hundreds or thousands of identical sequences of DNA found at either end of retrotransposons or proviral DNA formed by reverse transcription of retroviral RNA. They are used by viruses to insert genetic material into the host genome. Signals for gene expression are found within the LTRs and include enhancers, promoters (which can have both transcription enhancers or regulatory elements), transcription initiation (including capping), transcription terminators, and polyadenylation signals.

[0214] Preferably, the vector of the present invention may comprise a 5'LTR and a 3'LTR. When the first polynucleotide encoding FOXP3 and the second polynucleotide encoding an HLA-specific CAR are present in the same vector, the first polynucleotide and the second polynucleotide may be operably linked to the same LTR.

[0215] The vector of the present invention may contain one or more additional regulatory sequences that can act pre- or post-transcriptionally. A "regulatory sequence" is any sequence that promotes the expression of a polypeptide, for example, any sequence that acts to increase the expression of a transcript or enhance mRNA stability. Suitable regulatory sequences include, for example, enhancer elements, post-transcriptional regulatory elements, and polyadenylation sites. Preferably, the additional regulatory sequence may be present in the LTR(s).

[0216] Suitably, the vector may comprise a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), e.g., operably linked to the promoter. Suitably, the vector may comprise the nucleotide sequence set forth as SEQ ID NO:250, or a variant having at least 80% identity to SEQ ID NO:250. Suitably, the variant may have at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:250. Example of WPRE (SEQ ID NO: 250) AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTCCTCCTTGTAT AAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCC CTCCCTATTGCCAGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTATGTTGCCACCTGGATT CTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGC

[0217] cell In one embodiment, the present invention provides a cell comprising the vector of the present invention. Suitably, the cell is a T cell or a T cell precursor.

[0218] T cells are a type of lymphocyte that develop in the thymus gland and play a central role in the immune response. T cells can be distinguished from other lymphocytes by the presence of T cell receptors on their cell surface. These immune cells arise as precursor cells derived from the bone marrow and differentiate into several distinct types upon migration to the thymus. T cell differentiation may continue after leaving the thymus. T cells are grouped into a series of subsets based on their function. CD4 T cells and CD8 T cells are selected in the thymus, but further differentiate at the periphery into specialized cells with distinct functions. T cell subsets are initially defined by function but also have related gene or protein expression patterns. Conventional and adaptive T cells include helper CD4+ T cells, cytotoxic CD8+ T cells, memory T cells, and regulatory CD4+ T cells. Innate T cells include natural killer T cells, mucosal-associated invariant T cells, and gamma delta T cells.

[0219] Regulatory T cells (Treg) Regulatory T cells (Tregs) are immune cells with suppressive functions that control cytopathic immune responses and are essential for maintaining immune tolerance. In one embodiment, the present invention provides a Treg comprising the vector of the present invention. In other words, the present invention provides a modified Treg.

[0220] As used herein, "modified Tregs" refers to Tregs that have been modified to contain or express a polynucleotide not naturally encoded by Tregs, particularly a polynucleotide encoding a FOXP3 polypeptide and / or a polynucleotide encoding an HLA-specific CAR, as described herein. Methods for modifying Tregs are known in the art, including, but not limited to, genetic modification of Tregs, for example, by transduction, such as retroviral or lentiviral transduction, gene transfer (e.g., transient DNA or RNA transfection), lipofection, polyethylene glycol transduction, calcium phosphate transduction, and electroporation. Any suitable method can be used to introduce a nucleic acid sequence into Tregs.

[0221] As used herein, the term "Treg" refers to T cells with immunosuppressive function.

[0222] Preferably, "immunosuppressive function" may refer to the ability of Tregs to reduce or inhibit one or more of the numerous physiological cellular effects (effects) promoted by the immune system in response to stimuli such as pathogens, antigens, e.g., alloantigens, or autoantigens. Examples of such effects include increased proliferation of conventional T cells (Tconv) and secretion of pro-inflammatory cytokines. Any such effect may be used as an indicator of the strength of the immune response. A relatively weaker immune response by Tconvs in the presence of Tregs may indicate the ability of Tregs to suppress the immune response. For example, a relative decrease in cytokine secretion indicates a weakened immune response and, therefore, the ability of Tregs to suppress the immune response. Tregs can also suppress immune responses by regulating the expression of costimulatory molecules on antigen-presenting cells (APCs), such as B cells, dendritic cells, and macrophages. The expression levels of CD80 and CD86 can be used to investigate the suppressive ability of activated Tregs in vitro after coculture.

[0223] Assays for measuring the strength of immune responses and thereby the suppressive ability of Tregs are known in the art. In particular, antigen-specific Tconv cells may be co-cultured with Tregs, and peptides of the corresponding antigen may be added to the co-culture to stimulate responses from Tconv cells. The degree of proliferation of Tconv cells and / or the amount of cytokine IL-2 secreted by Tconv cells in response to the addition of the peptide may be used as an indicator of the suppressive ability of the co-cultured Tregs.

[0224] Antigen-specific Tconv cells co-cultured with Tregs of the invention may proliferate 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 95% or 99% less compared to the proliferation of the same Tconv cells cultured in the absence of the Tregs of the invention. Antigen-specific Tconv cells co-cultured with Tregs of the invention may express at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% less effector cytokines compared to corresponding Tconv cells cultured in the absence of Tregs of the invention.

[0225] The effector cytokine may be selected from IL-2, IL-17, TNFα, GM-CSF, IFN-γ, IL-4, IL-5, IL-9, IL-10, and IL-13. Suitably, the effector cytokine may be selected from IL-2, IL-17, TNFα, GM-CSF, and IFN-γ.

[0226] Preferably, Tregs express the markers CD4, CD25, and FOXP3 (CD4 + CD25 + FOXP3 + )-expressing T cells.

[0227] Marker levels can be measured by any method known to one of skill in the art, for example, by flow cytometry.

[0228] Tregs may also express CTLA-4 (cytotoxic T lymphocyte-associated molecule 4) and / or GITR (glucocorticoid-induced TNF receptor).Treg cells are present in peripheral blood, lymph nodes, and tissues. Preferably, by using the cell surface markers CD4 and CD25 in the absence or combination with the surface protein CD127, which is expressed at low levels (CD4 + CD25 + CD127 - , or CD4 + CD25 + CD127 low , or CD4 + CD25 hi CD127 - , or CD4 + CD25 hi CD127 low ), Tregs may be identified. Such markers for use in identifying Tregs are known in the art and are described, for example, in Liu et al. (JEM;2006;203;7(10);1701-1711).

[0229] The Tregs are CD4 + CD25 + FOXP3 + T cells, or CD4 + CD25 hi FOXP3 + It may be a T cell. The Tregs are CD4 + CD25 + CD127 - T cells, or CD4 + CD25 hi CD127 - It may be a T cell. The Tregs are CD4 + CD25 + FOXP3 + CD127 -T cells, or CD4 + CD25 hi FOXP3 + CD127 - It may be a T cell.

[0230] The Treg may have a demethylated Treg-specific demethylation region (TSDR), which is a key methylation-sensitive element that controls the expression of FOXP3 (Polansky, JK, et al., 2008. European Journal of Immunology, 38(6), pp.1654-1663).

[0231] The Tregs may be natural Tregs or thymus-derived Tregs, adaptive Tregs or peripherally-derived Tregs, or in vitro-induced Tregs (Abbas, AK, et al., 2013. Nature immunology, 14(4), p.307-308). Preferably, the Tregs are CD4 + CD25 + FOXP3 + Helios + Neuropilin 1 + Preferably, the Tregs are natural Tregs.

[0232] Further preferred examples of Treg include Tr1 cells, CD8 + FOXP3 + T cells; and γδFOXP3 + These include, but are not limited to, T cells.

[0233] Suitably, the Tregs are isolated from peripheral blood mononuclear cells (PBMCs) obtained from a subject. Suitably, the subject is a mammal, preferably a human.

[0234] Preferably, the Tregs are matched (e.g., HLA-matched) to the subject to whom the modified Tregs are administered, or are autologous to the subject. Preferably, the subject to whom the modified Tregs are administered is a mammal, preferably a human. The Tregs may be generated ex vivo (ex vivo), either from the patient's own peripheral blood (first party), or in the setting of a hematopoietic stem cell transplant from donor peripheral blood (second party), or from peripheral blood from an unrelated donor (third party). Preferably, the Tregs are autologous to the subject to whom the modified Tregs are administered.

[0235] In a preferred embodiment, the Tregs are isolated and matched (e.g., HLA-matched) from peripheral blood mononuclear cells (PBMCs) obtained from the subject, or are autologous to the subject to whom the modified Tregs are administered.

[0236] Preferably, the Tregs are part of a population of Tregs. Preferably, the population of Tregs comprises at least 70% Tregs, such as at least 75%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% Tregs. Such a population may be referred to as an "enriched Treg population" or an "enriched Treg sample."

[0237] As used herein, "conventional T cells" or Tcon refers to T lymphocyte cells that express the αβ T cell receptor (TCR) and a non-immunosuppressive co-receptor, which can be cluster of differentiation 4 (CD4) or cluster of differentiation 8 (CD8). Conventional T cells are present in peripheral blood, lymph nodes, and tissues. Engineered Tregs express CD4 in the presence of IL-2 and TGF-β. + CD25 - FOXP3 - It may be produced from Tcon by in vitro culture of cells.

[0238] The Tregs of the present invention may be derived from stem cells. In particular, the Tregs of the present invention may be derived from in vitro stem cells. The Tregs may be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells into Tregs. The polynucleotides or vectors of the present invention may be introduced into the inducible progenitor cells or embryonic progenitor cells before or after differentiation into Tregs.

[0239] As used herein, the term "stem cell" refers to an undifferentiated cell that can indefinitely give rise to more stem cells of the same type, and from which other specialized cells can be differentiated. Stem cells are pluripotent. Stem cells may be, for example, embryonic stem cells or adult stem cells.

[0240] As used herein, the term "progenitor cell" refers to a cell that can differentiate to form one or more cell types, but has limited self-renewal in vitro. Preferably, the cells can be differentiated into T cells, such as Tregs. Preferably, the cells may be embryonic stem cells (ESCs). Preferably, the cells are hematopoietic stem cells or hematopoietic progenitor cells. Preferably, the cells are induced pluripotent stem cells (iPSCs). Preferably, the cells may be obtained from umbilical cord blood. Preferably, the cells may be obtained from adult peripheral blood.

[0241] In some embodiments, hematopoietic stem and progenitor cells (HSPCs) may be obtained from umbilical cord blood, which can be collected according to techniques well known in the art (e.g., U.S. Patent Nos. 7,147,626 and 7,131,958, which are incorporated herein by reference). In one embodiment, HSPCs may be obtained from pluripotent stem cell sources, such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs).

[0242] As used herein, the term "hematopoietic stem and progenitor cells" or "HSPCs" refers to cells that express the antigen marker CD34 (CD34+), and populations of such cells. In certain embodiments, the term "HSPCs" refers to cells identified by the presence of the antigen marker CD34 (CD34+) and the absence of lineage (lin) markers. Populations of cells that contain cells that are CD34+ and / or Lin(-) include hematopoietic stem cells and hematopoietic progenitor cells.

[0243] HSPCs can be obtained or isolated from adult bone marrow, such as the femur, pelvis, ribs, sternum, or other bones. Bone marrow aspirate containing HSPCs can be obtained or isolated directly from the pelvis using a needle and syringe. Other sources of HSPCs include umbilical cord blood, placental blood, mobilized peripheral blood, Wharton's jelly, placenta, fetal blood, fetal liver, or fetal spleen. In certain embodiments, harvesting a sufficient amount of HSPCs for therapeutic use may require mobilization of stem and progenitor cells in a subject.

[0244] As used herein, the term "induced pluripotent stem cells" or "iPSCs" refers to non-pluripotent cells that have been reprogrammed to a pluripotent state. Once a subject's cells have been reprogrammed to a pluripotent state, they can then be programmed into a desired cell type, such as a hematopoietic stem or progenitor cell (HSC or HPC, respectively).

[0245] As used herein, the term "reprogramming" refers to a method of increasing the potency of a cell towards a less differentiated state.

[0246] As used herein, the term "programming" refers to a method of reducing the potency of a cell or causing a cell to differentiate into a more differentiated state.

[0247] The present invention also provides modified Tregs that have higher FOXP3 expression than unmodified Tregs, and modified Tregs that have higher FOXP3 expression than corresponding unmodified Tregs.

[0248] By "higher FOXP3 expression" is meant that the levels of FOXP3 mRNA or protein are higher in the modified Tregs than before the Tregs were engineered by artificial intervention that alters gene expression. "Higher FOXP3 expression" may be defined and measured as described herein.

[0249] Suitably, the levels of FOXP3 mRNA and / or protein in modified Tregs (or populations of such Tregs) according to the present invention may be increased by at least 1.5-fold, at least 2-fold, or at least 5-fold over the levels in corresponding unmodified Tregs (or populations of such Tregs).

[0250] Suitably, the levels of CD25 mRNA and / or protein in the modified Tregs (or populations of such Tregs) of the present invention may be increased by at least 1.5-fold, at least 2-fold, or at least 5-fold over the levels in corresponding unmodified Tregs (or populations of such Tregs). Suitably, the levels of CTLA-4 mRNA and / or protein in the modified Tregs (or populations of such Tregs) of the present invention may be increased by at least 1.5-fold, at least 2-fold, or at least 5-fold over the levels in corresponding unmodified Tregs (or populations of such Tregs).

[0251] The modified Tregs of the present invention may comprise an exogenous polynucleotide encoding a FOXP3 polypeptide. An "exogenous polynucleotide" is a polynucleotide that originates outside of a Treg.

[0252] T effector cells The present invention may reduce the risk of generating modified T effector cells, for example, the risk of generating modified T effector cells during the generation of modified Tregs.

[0253] T effector cells are relatively short-lived activated cells that defend the body in immune responses. T effector cells include cytotoxic T cells and helper T cells that perform cell-mediated responses. Therefore, T effector cells can be cytotoxic T cells or helper T cells. T effector cells can express low levels of FOXP3, and FOXP3 low Alternatively, they may be FOXP3-. Preferably, the T effector cells do not have immunosuppressive function.

[0254] Most cytotoxic T cells express a subset of surface markers, such as CD8, CD45, and CD54. + FOXP3 low cells or CD8 + FOXP3 - It may also be a cell. Preferably, the cytotoxic T cells are CD8 + CD45 + FOXP3 low cells or CD8 + CD45 + FOXP3 - It may also be a cell. Preferably, the cytotoxic T cells are CD8 + CD54 + FOXP3 low cells or CD8 + CD54 + FOXP3 - It may also be a cell. Preferably, the cytotoxic T cells are CD8 + CD45 + CD54 + FOXP3 low cells or CD8 + CD45 + CD54 + FOXP3 - It may also be a cell.

[0255] Helper T cells (also known as T helper cells, Th cells, or CD4+ cells) are a type of T cell that plays a key role in the immune system, particularly the adaptive immune system. They assist the activity of other immune cells by releasing cellular cytokines. They are essential for antibody class switching of B cells, activation and proliferation of cytotoxic T cells, and maximizing the bactericidal capacity of phagocytes such as macrophages. T helper subtypes include Th1 cells, Th2 cells, Th9 cells, Th17 cells, Th22 cells, and Tfh cells. Preferably, helper T cells are CD4+ cells without immunosuppressive function. Advantageously, helper T cells are CD4 + FOXP3 low cells or CD4 + FOXP3 - It may also be a cell.

[0256] Cell creation method The modified Tregs of the present invention may be generated by introducing a polynucleotide encoding a FOXP3 polypeptide (sometimes referred to herein as a first polynucleotide) and / or a polynucleotide encoding an HLA-specific chimeric antigen receptor (CAR) (sometimes referred to herein as a second polynucleotide), as described herein.

[0257] As used herein, the term "introduce" refers to methods for inserting foreign DNA into cells, including both gene transfer and transduction methods. Gene transfer is the process of introducing nucleic acid into cells by non-viral methods. Transduction is the process of introducing foreign DNA into cells via a viral vector.

[0258] The modified Tregs of the present invention may be generated by introducing (eg, by transduction or gene transfer) the polynucleotide(s) or vectors defined herein into Tregs. Suitably, the Tregs may be derived from a sample isolated from a subject, and the Tregs may be further separated from the sample by any suitable method, for example by magnetic separation.

[0259] The modified Tregs of the present invention may be generated by a method comprising the following steps. (i) isolating or obtaining a cell-containing sample (e.g., from a subject), and (ii) transducing or transducing the cell-containing sample with a polynucleotide encoding a FOXP3 polypeptide and / or a polynucleotide encoding an HLA-specific CAR, as described herein, or a vector as described herein (e.g., a vector encoding a 5' FOXP3-HLA-specific CAR 3'), to obtain a population of modified cells. Preferably, the cell-containing sample comprises or consists of PBMCs.

[0260] Suitably, a Treg-enriched sample may be isolated, enriched, and / or generated from the cell-containing sample before and / or after step (ii) of the method. For example, isolation, enrichment, and / or generation of Tregs may be performed before and / or after step (ii) to isolate, enrich, and / or generate a Treg-enriched sample. Isolation and / or enrichment may be performed after step (ii) to enrich for cells and / or Tregs comprising the CAR, polynucleotide(s), and / or vector of the invention. The Treg-enriched sample may be isolated or enriched by any method known to those of skill in the art, for example, by FACS and / or magnetic bead separation. Preferably, the cells are Tregs as defined herein.

[0261] Preferably, the modified Tregs of the present invention may be generated by a method comprising the following steps: (i) isolating or obtaining a Treg-enriched sample (e.g., from a subject); and (ii) transducing or transducing the Treg-enriched sample with a first polynucleotide encoding a FOXP3 polypeptide and / or a second polynucleotide encoding an HLA-specific CAR as described herein, or a vector as described herein (e.g., a vector encoding a 5' FOXP3-HLA-specific CAR 3') to obtain a population of modified Treg cells of the invention.

[0262] Before or after introduction of the polynucleotide(s) or vector, the cells and / or Tregs may be activated and / or expanded, 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. Tregs may also be expanded in the presence of anti-CD3 and anti-CD28 monoclonal antibodies in combination with IL-2. Preferably, IL-2 may be replaced with IL-15. Additional components that may be used in Treg expansion protocols include, but are not limited to, rapamycin, all-trans retinoic acid (ATRA), and TGFβ.

[0263] As used herein, "activated" means that a cell or population of cells is stimulated to cause proliferation of the cell(s). As used herein, "expanded" means that proliferation of a cell or population of cells is induced. The proliferation of a population of cells may be measured, for example, by counting the number of cells present in the population. The phenotype of a cell may be measured by methods known in the art, such as flow cytometry.

[0264] The Tregs may be washed after each step of the method, particularly after expansion.

[0265] The modified Treg population may be further enriched by any method known to one of skill in the art, for example, by FACS and / or magnetic bead separation.

[0266] The steps of the manufacturing method may be carried out in a closed, sterile cell culture system.

[0267] Enhance engineered Treg immunosuppression A polynucleotide encoding a FOXP3 polypeptide (eg in a vector according to the invention) may be introduced into Tregs to increase the expression of FOXP3 and thus enhance the ability of said Tregs to suppress an immune response. Thus, the present invention provides a polynucleotide encoding a FOXP3 polypeptide as described herein for use in enhancing the ability of HLA-specific engineered Tregs to suppress an immune response, preferably wherein the engineered HLA-specific Tregs are HLA-A2-specific Tregs.

[0268] The present invention provides use of a polynucleotide encoding a FOXP3 polypeptide as described herein for enhancing the ability of modified HLA-specific Tregs to suppress immune responses, preferably modified HLA-A2-specific Tregs.

[0269] The present invention provides a first polynucleotide encoding a FOXP3 polypeptide as described herein and a second polynucleotide encoding a chimeric antigen receptor (CAR) as described herein for use in enhancing the ability of engineered HLA-specific Tregs to suppress immune responses, preferably immune responses against cells expressing HLA. The CAR may comprise a single-chain antibody (scFv) antigen recognition domain as described herein that specifically binds to a human leukocyte antigen (HLA). The first polynucleotide and the second polynucleotide may be operably linked to the same promoter. The first polynucleotide may be upstream of the second polynucleotide. The scFv antigen recognition domain may specifically bind to HLA-A2.

[0270] The present invention provides a vector as described herein for use in enhancing the ability of modified HLA-specific Tregs to suppress immune responses, preferably modified HLA-A2-specific Tregs.

[0271] The present invention provides a vector as described herein for use in enhancing the ability of engineered HLA-specific Tregs to suppress an immune response, the vector comprising a first polynucleotide encoding a FOXP3 polypeptide as described herein and a second polynucleotide encoding a chimeric antigen receptor (CAR) as described herein, the CAR comprising a single-chain antibody (scFv) antigen recognition domain that specifically binds to a human leukocyte antigen (HLA), the first polynucleotide and the second polynucleotide being operably linked to the same promoter, and the first polynucleotide being upstream of the second polynucleotide. The antigen recognition domain may specifically bind to HLA-A2.

[0272] The present invention provides a vector as described herein for use in enhancing the ability of engineered HLA-specific Tregs to suppress an immune response, the vector comprising a first polynucleotide encoding a FOXP3 polypeptide as described herein and a second polynucleotide encoding a chimeric antigen receptor (CAR) as described herein, the CAR comprising an antigen recognition domain that specifically binds to a human leukocyte antigen (HLA), the first polynucleotide and the second polynucleotide being operably linked to the same promoter, the first polynucleotide being upstream of the second polynucleotide, and the vector further comprising a polynucleotide encoding a cleavage site as described herein between the first and second polynucleotides and / or an internal ribosome entry site (IRES) as described herein between the first and second polynucleotides. The antigen recognition domain may specifically bind to HLA-A2.

[0273] The present invention provides use of a vector as described herein to enhance the immune response suppression ability of modified HLA-specific Tregs, preferably modified HLA-A2-specific Tregs.

[0274] The present invention provides a method for enhancing the ability of modified HLA-specific Tregs to suppress immune responses, comprising introducing a vector as described herein into Tregs.

[0275] The vector may comprise a first polynucleotide encoding a FOXP3 polypeptide as described herein and a second polynucleotide encoding a chimeric antigen receptor (CAR) as described herein, wherein the CAR comprises a single-chain antibody (scFv) antigen recognition domain that specifically binds to a human leukocyte antigen (HLA), the first polynucleotide and the second polynucleotide being operably linked to the same promoter, and the first polynucleotide being upstream of the second polynucleotide. The antigen recognition domain may specifically bind to HLA-A2.

[0276] The vector may comprise a first polynucleotide encoding a FOXP3 polypeptide as described herein and a second polynucleotide encoding a chimeric antigen receptor (CAR) as described herein, wherein the CAR comprises an antigen recognition domain that specifically binds to a human leukocyte antigen (HLA), the first polynucleotide and the second polynucleotide being operably linked to the same promoter, the first polynucleotide being upstream of the second polynucleotide, and the vector further comprising a polynucleotide encoding a cleavage site as described herein between the first and second polynucleotides, and / or an internal ribosome entry site (IRES) as described herein between the first and second polynucleotides. The antigen recognition domain may specifically bind to HLA-A2.

[0277] The present invention provides a method for enhancing the immune response suppression ability of engineered Tregs, the method comprising introducing into the Tregs a first polynucleotide encoding a FOXP3 polypeptide as described herein and a second polynucleotide encoding an HLA-specific CAR as described herein. Preferably, the HLA-specific CAR is an HLA-A2-specific CAR. The HLA-specific CAR may comprise a single-chain antibody (scFv) antigen-recognition domain.

[0278] The present invention provides a method for enhancing the ability of engineered HLA-specific Tregs to suppress an immune response, the method comprising introducing into a cell-containing sample a first polynucleotide encoding a FOXP3 polypeptide as described herein and a second polynucleotide encoding an HLA-specific CAR as described herein; (a) the cell-containing sample comprises or consists of Tregs, and / or (b) the cell-containing sample comprises or consists of peripheral blood mononuclear cells (PBMCs), and Tregs are enriched from the cell-containing sample before or after introducing the first polynucleotide and / or the second polynucleotide; and / or (c) the cell-containing sample comprises or consists of PBMCs, and Tregs are generated from the cell-containing sample before or after introducing the first polynucleotide and / or the second polynucleotide; and / or (d) the cell-containing sample comprises or consists of pluripotent stem cells (PSCs) (e.g., induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs)), and Tregs are differentiated from PSCs before or after introducing the first polynucleotide and / or second polynucleotide.

[0279] Preferably, the modified HLA-specific Treg is a modified HLA-A2-specific Treg, and the HLA-specific CAR is an HLA-A2-specific CAR. The HLA-specific CAR may comprise a single-chain antibody (scFv) antigen-recognition domain.

[0280] Suitably, said first polynucleotide and / or said second polynucleotide are introduced by viral transduction, preferably by retroviral or lentiviral transduction.

[0281] Preferably, the first polynucleotide and the second polynucleotide are introduced into a single vector, and the first polynucleotide and the second polynucleotide may be operably linked to the same promoter. The vector may be the vector of the present invention.

[0282] In other words, the present invention provides a method for enhancing the immune response suppression ability of modified HLA-specific Tregs (preferably HLA-A2-specific Tregs), comprising introducing a vector as described herein into a cell-containing sample; (a) the cell-containing sample comprises or consists of Tregs, and / or (b) the cell-containing sample comprises or consists of peripheral blood mononuclear cells (PBMCs), and Tregs are enriched from the cell-containing sample before or after introducing the vector; and / or (c) the cell-containing sample comprises or consists of PBMCs, and Tregs are generated from the cell-containing sample before or after introducing the vector; and / or (d) the cell-containing sample comprises or consists of pluripotent stem cells (PSCs) (e.g., induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs)), and Tregs are differentiated from PSCs before or after introducing the first polynucleotide and / or second polynucleotide.

[0283] Preferably, the vector comprises an HLA-specific CAR. The HLA-specific CAR may be an HLA-A2-specific CAR. The HLA-specific CAR may comprise a single-chain antibody (scFv) antigen-recognition domain.

[0284] The phrase "enhancing the ability to suppress an immune response" means increasing the suppressive effect of Tregs (or a population of such Tregs) on an immune response compared to the suppressive effect of corresponding Tregs (or a population of such Tregs) that have not been modified by introducing a first polynucleotide encoding a FOXP3 polypeptide as described herein and a second polynucleotide encoding an HLA-specific CAR as described herein, and / or by introducing a vector as described herein. Preferably, the immune response is an immune response against cells expressing HLA, more preferably an immune response against cells expressing HLA-A2. The increase in suppressive effect may be at least 10, 20, 30, 40, 50, 60, 70, 80 or 90% and may be measured by various means, such as measuring a decrease in the production of IL-2 by T effector cells (e.g., at least a 10, 20, 30, 40, 50, 60, 70, 80 or 90% decrease), or an increase in Treg-associated cytokines such as IL10 (e.g., at least a 10, 20, 30, 40, 50, 60, 70, 80 or 90% increase).

[0285] The term "immune response" refers to a number of physiological cellular events promoted by the immune system in response to stimuli such as pathogens or autoantigens. Examples of such events include proliferation of Tconv cells and increased cytokine secretion. Any such event may be used as an indicator of the strength of the immune response. A relatively weaker Tconv-mediated immune response in the presence of engineered Tregs compared to unmodified Tregs indicates a relatively enhanced ability of the engineered Tregs to suppress the immune response. For example, a relatively reduced cytokine secretion is an indicator of a weakened immune response, and thus indicates an enhanced ability of Tregs to suppress the immune response.

[0286] Assays for measuring the strength of immune responses and thereby the suppressive ability of Tregs are known in the art. In particular, antigen-specific Tconv cells may be co-cultured with Tregs, and peptides of the corresponding antigen may be added to the co-culture to stimulate responses from Tconv cells. The degree of proliferation of Tconv cells and / or the amount of cytokine IL-2 secreted by Tconv cells in response to the addition of the peptide may be used as an indicator of the suppressive ability of the co-cultured Tregs.

[0287] The proliferation of antigen-specific Tconv cells co-cultured with the modified Tregs of the present invention (i.e., with increased FOXP3 expression) may be 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% less than the same Tconv cells co-cultured with corresponding unmodified Tregs (i.e., without increased FOXP3 expression). Preferably, the Tconv cells are HLA-specific Tconv cells. More preferably, the Tconv cells are HLA-A2-specific Tconv cells.

[0288] Antigen-specific Tconv cells co-cultured with the modified Tregs of the present invention (i.e., with increased FOXP3 expression) may exhibit at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, or at least a 60% greater reduction in effector cytokines than corresponding Tconv cells co-cultured with corresponding unmodified Tregs (i.e., without increased FOXP3 expression). Preferably, the Tconv cells are HLA-specific Tconv cells. More preferably, the Tconv cells are HLA-A2-specific Tconv cells.

[0289] The production of antigen-specific Tconv cells co-cultured with the modified Tregs of the present invention (i.e., with increased FOXP3 expression) may produce 10% or less, 20% or less, 30% or less, 40% or less, 50% or less, or 60% or less of effector cytokines than the same Tconv cells co-cultured with corresponding unmodified Tregs (i.e., without increased FOXP3 expression). Preferably, the Tconv cells are HLA-specific Tconv cells. More preferably, the Tconv cells are HLA-A2-specific Tconv cells.

[0290] The effector cytokine may be selected from IL-2, IL-17, TNFα, GM-CSF, IFN-γ, IL-4, IL-5, IL-9, IL-10, and IL-13. Suitably, the effector cytokine may be selected from IL-2, IL-17, TNFα, GM-CSF, and IFN-γ.

[0291] Antigen-specific Tconv cells co-cultured with the modified Tregs of the present invention (i.e., with increased FOXP3 expression) may suppress IL-2 production to 2-fold, 4-fold, 8-fold, 10-fold, or 20-fold lower than the number of corresponding unmodified Tregs (i.e., without increased FOXP3 expression). Preferably, the Tconv cells are HLA-specific Tconv cells. More preferably, the Tconv cells are HLA-A2-specific Tconv cells.

[0292] Reduce the risk that engineered Tregs will acquire an effector phenotype Introducing a polynucleotide encoding a FOXP3 polypeptide (e.g., in a vector of the present invention) into Tregs may increase FOXP3 expression, thereby reducing the risk of the modified Tregs acquiring an effector phenotype. Furthermore, a vector of the present invention in which FOXP3 precedes the HLA-specific CAR in a 5' to 3' orientation ensures that CAR expression can only occur when FOXP3 is expressed, and that CAR expression does not occur in the absence of FOXP3. This may further reduce the risk of the modified HLA-specific Tregs acquiring an effector phenotype.

[0293] Accordingly, the present invention provides a polynucleotide encoding a FOXP3 polypeptide as described herein for use in reducing the risk of modified HLA-specific Tregs acquiring an effector phenotype, preferably wherein said modified HLA-specific Tregs are modified HLA-A2-specific Tregs.

[0294] The present invention provides use of a polynucleotide encoding a FOXP3 polypeptide as described herein to reduce the risk of modified HLA-specific Tregs acquiring an effector phenotype, preferably wherein the modified HLA-specific Tregs are modified HLA-A2-specific Tregs.

[0295] The present invention provides use of a first polynucleotide encoding a FOXP3 polypeptide as described herein and a second polynucleotide encoding a chimeric antigen receptor (CAR) as described herein to reduce the risk of engineered HLA-specific Tregs acquiring an effector phenotype. The CAR may comprise a single-chain antibody (scFv) antigen recognition domain as described herein that specifically binds to a human leukocyte antigen (HLA). The first polynucleotide and the second polynucleotide may be operably linked to the same promoter. The first polynucleotide may be upstream of the second polynucleotide. The scFv antigen recognition domain may specifically bind to HLA-A2.

[0296] The present invention provides a vector as described herein for use in reducing the risk of modified HLA-specific Tregs acquiring an effector phenotype, preferably wherein the modified HLA-specific Tregs are modified HLA-A2-specific Tregs.

[0297] The present invention provides the use of a vector as described herein to reduce the risk of modified HLA-specific Tregs acquiring an effector phenotype, preferably wherein the modified HLA-specific Tregs are modified HLA-A2-specific Tregs.

[0298] The present invention provides a method for reducing the risk that modified HLA-specific Tregs will acquire an effector phenotype, said method comprising introducing into said Tregs a vector as described herein.

[0299] The vector may comprise a first polynucleotide encoding a FOXP3 polypeptide as described herein and a second polynucleotide encoding a chimeric antigen receptor (CAR) as described herein, wherein the CAR comprises a single-chain antibody (scFv) antigen recognition domain that specifically binds to a human leukocyte antigen (HLA), the first polynucleotide and the second polynucleotide being operably linked to the same promoter, and the first polynucleotide being upstream of the second polynucleotide. The antigen recognition domain may specifically bind to HLA-A2.

[0300] The vector may comprise a first polynucleotide encoding a FOXP3 polypeptide as described herein and a second polynucleotide encoding a chimeric antigen receptor (CAR) as described herein, wherein the CAR comprises an antigen recognition domain that specifically binds to a human leukocyte antigen (HLA), the first polynucleotide and the second polynucleotide being operably linked to the same promoter, the first polynucleotide being upstream of the second polynucleotide, and the vector further comprising a polynucleotide encoding a cleavage site as described herein between the first and second polynucleotides, and / or an internal ribosome entry site (IRES) as described herein between the first and second polynucleotides as described herein. The antigen recognition domain may specifically bind to HLA-A2.

[0301] The present invention provides a method for reducing the risk that modified HLA-specific Tregs acquire an effector phenotype, the method comprising introducing into the Tregs a first polynucleotide encoding a FOXP3 polypeptide as described herein and a second polynucleotide encoding an HLA-specific CAR as described herein. Preferably, the modified HLA-specific Tregs are modified HLA-A2-specific Tregs, and the HLA-specific CAR is an HLA-A2-specific CAR. The HLA-specific CAR may comprise a single-chain antibody (scFv) antigen-recognition domain.

[0302] The present invention provides a method for reducing the risk that modified HLA-specific Tregs will acquire an effector phenotype, said method comprising introducing into a cell-containing sample a first polynucleotide encoding a FOXP3 polypeptide as described herein and a second polynucleotide encoding an HLA-specific CAR as described herein; (a) the cell-containing sample comprises or consists of Tregs, and / or (b) the cell-containing sample comprises or consists of peripheral blood mononuclear cells (PBMCs), and Tregs are enriched from the cell-containing sample before or after introducing the first polynucleotide and / or the second polynucleotide; and / or (c) the cell-containing sample comprises or consists of PBMCs, and Tregs are generated from the cell-containing sample before or after introducing the first polynucleotide and / or the second polynucleotide; and / or (d) the cell-containing sample comprises or consists of pluripotent stem cells (PSCs) (e.g., induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs)), and Tregs are differentiated from PSCs before or after introducing the first polynucleotide and / or second polynucleotide.

[0303] Preferably, the modified HLA-specific Treg is a modified HLA-A2-specific Treg, and the HLA-specific CAR is an HLA-A2-specific CAR. The HLA-specific CAR may comprise a single-chain antibody (scFv) antigen-recognition domain.

[0304] Suitably, said first polynucleotide and / or said second polynucleotide are introduced by viral transduction, preferably by retroviral or lentiviral transduction.

[0305] Preferably, the first polynucleotide and the second polynucleotide are introduced into a single vector, and the first polynucleotide and the second polynucleotide may be operably linked to the same promoter. The vector may be the vector of the present invention.

[0306] Therefore, the present invention provides a method for reducing the risk that modified HLA-specific Tregs acquire an effector phenotype, the method comprising introducing the vector of the present invention into the Tregs. Preferably, the modified HLA-specific Tregs are modified HLA-A2-specific Tregs.

[0307] The present invention provides a method for reducing the risk that modified HLA-specific Tregs (e.g., HLA-A2-specific Tregs) acquire an effector phenotype, the method comprising introducing a vector according to the present invention into a cell-containing sample, (a) the cell-containing sample comprises or consists of Tregs, and / or (b) the cell-containing sample comprises or consists of PBMCs, and Tregs are enriched from the cell-containing sample before or after introducing the vector; and / or (c) the cell-containing sample comprises or consists of PBMCs, and Tregs are generated from the cell-containing sample before or after introducing the vector; and / or (d) the cell-containing sample comprises or consists of pluripotent stem cells (PSCs) (e.g., induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs)), and Tregs are differentiated from PSCs before or after introducing the first polynucleotide and / or second polynucleotide.

[0308] Preferably, the vector comprises an HLA-specific CAR. The HLA-specific CAR may be an HLA-A2-specific CAR. The HLA-specific CAR may comprise a single-chain antibody (scFv) antigen-recognition domain.

[0309] The phrase "reducing the risk that modified Tregs will acquire an effector phenotype" may refer to reducing the likelihood or rate at which Tregs (or a population of such Tregs) acquire an effector phenotype compared to the likelihood or rate of corresponding Tregs that have not been modified by introducing a polynucleotide encoding a FOXP3 polypeptide as described herein and / or a polynucleotide encoding an HLA-specific CAR (e.g., an HLA-A2-specific CAR) as described herein, and / or a vector as described herein. Preferably, the Tregs are HLA-specific Tregs, more preferably HLA-A2-specific Tregs. Suitably, the likelihood is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100%. Suitably, the rate is at least 1.5-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold slower.

[0310] The phrase "acquire an effector phenotype" refers to Tregs acquiring a phenotype associated with T effector cells and / or losing a phenotype associated with Tregs.

[0311] Suitably, T cells that have acquired an effector phenotype may have reduced levels of FOXP3, CD25 and / or CTLA-4, preferably reduced levels of FOXP3. The methods described herein may be used to measure levels of FOXP3, CD25 and / or CTLA-4 mRNA and / or protein.

[0312] Suitably, T cells that have acquired an effector phenotype have reduced levels of FOXP3 after 1 week or more, 2 weeks or more, 3 weeks or more, 4 weeks or more, 5 weeks or more, 6 weeks or more, 7 weeks or more, or 8 weeks or more, preferably after 7 weeks or more. Suitably, T cells that have acquired an effector phenotype may have reduced levels of FOXP3 mRNA and / or protein by at least 1.5-fold, at least 2-fold, or at least 5-fold or more.

[0313] Suitably, T cells that have acquired an effector phenotype have reduced levels of CD25 after 1 week or more, 2 weeks or more, 3 weeks or more, 4 weeks or more, 5 weeks or more, 6 weeks or more, 7 weeks or more, or 8 weeks or more, preferably after 7 weeks or more. Suitably, T cells that have acquired an effector phenotype may have reduced levels of CD25 mRNA and / or protein by at least 1.5-fold, at least 2-fold, or at least 5-fold or more.

[0314] Suitably, T cells that have acquired an effector phenotype have reduced levels of CTLA-4 after 1 week or more, 2 weeks or more, 3 weeks or more, 4 weeks or more, 5 weeks or more, 6 weeks or more, 7 weeks or more, or 8 weeks or more, preferably after 7 weeks or more. Suitably, T cells that have acquired an effector phenotype may have reduced levels of CTLA-4 mRNA and / or protein by at least 1.5-fold, at least 2-fold, or at least 5-fold or more.

[0315] Reduce the risk of generating modified T effector cells Introducing a polynucleotide encoding a FOXP3 polypeptide (e.g., in a vector of the invention) into T effector cells increases FOXP3 expression, thereby reducing the risk of generating modified T effector cells, for example, during the generation of modified Tregs. Furthermore, a vector of the invention in which FOXP3 precedes the HLA-specific CAR in a 5' to 3' orientation ensures that CAR expression can only occur when FOXP3 is expressed, and that CAR expression does not occur in the absence of FOXP3. This may further reduce the risk of generating modified HLA-specific T effector cells during the generation of modified Tregs.

[0316] Thus, the present invention provides polynucleotides encoding FOXP3 polypeptides as described herein for use in reducing the risk of generating modified T effector cells, e.g., modified HLA-specific T effector cells, and preferably for reducing the risk during the generation of modified Tregs, preferably for use in reducing the risk during the generation of modified HLA-specific Tregs. More preferably, said modified HLA-specific T effector cells are modified HLA-A2-specific T effector cells, and said modified HLA-specific Tregs are modified HLA-A2-specific Tregs.

[0317] The present invention provides the use of a polynucleotide encoding a FOXP3 polypeptide as described herein for reducing the risk of generating modified T effector cells, e.g., modified HLA-specific T effector cells, preferably for reducing the risk of generation during the generation of modified Tregs, preferably for reducing the risk of generation during the generation of modified HLA-specific Tregs. More preferably, said modified HLA-specific T effector cells are modified HLA-A2-specific T effector cells, and said modified HLA-specific Tregs are modified HLA-A2-specific Tregs.

[0318] The present invention provides the use of a first polynucleotide encoding a FOXP3 polypeptide as described herein and a second polynucleotide encoding an HLA-specific chimeric antigen receptor (CAR) as described herein to generate engineered T effector cells, e.g., engineered HLA-specific T effector cells, preferably to reduce the risk of Tregs being generated during the generation of engineered Tregs. More preferably, the engineered HLA-specific T effector cells are engineered HLA-A2-specific T effector cells, and the engineered HLA-specific Tregs are engineered HLA-A2-specific Tregs. The CAR may comprise a single-chain antibody (scFv) antigen recognition domain as described herein that specifically binds to a human leukocyte antigen (HLA). The first polynucleotide and the second polynucleotide may be operably linked to the same promoter. The first polynucleotide may be upstream of the second polynucleotide. The scFv antigen recognition domain may specifically bind to HLA-A2.

[0319] Thus, the present invention provides a vector as described herein for use in reducing the risk of generating modified T effector cells, preferably for use in reducing the risk of generating modified Tregs during generation. Preferably, the modified T effector cells are modified HLA-specific T effector cells and the modified Tregs are modified HLA-specific Tregs. More preferably, the modified T effector cells are modified HLA-A2-specific T effector cells and the modified HLA-specific Tregs are modified HLA-A2-specific Tregs.

[0320] The present invention provides the use of a vector as described herein for reducing the risk of generating modified T effector cells, preferably for reducing the risk of generating modified Tregs during generation. Preferably, the modified T effector cells are modified HLA-specific T effector cells, and the modified Tregs are modified HLA-specific Tregs. More preferably, the modified T effector cells are modified HLA-A2-specific T effector cells, and the modified HLA-specific Tregs are modified HLA-A2-specific Tregs.

[0321] The present invention provides a method for reducing the risk of generating modified T effector cells, preferably during the generation of modified Tregs, the method comprising introducing a vector as described herein into the T effector cells. Preferably, the modified T effector cells are modified HLA-specific T effector cells, and the modified Tregs are modified HLA-specific Tregs. More preferably, the modified T effector cells are modified HLA-A2-specific T effector cells, and the modified HLA-specific Tregs are modified HLA-A2-specific Tregs.

[0322] The vector may comprise a first polynucleotide encoding a FOXP3 polypeptide as described herein and a second polynucleotide encoding a chimeric antigen receptor (CAR) as described herein, wherein the CAR comprises a single-chain antibody (scFv) antigen recognition domain that specifically binds to a human leukocyte antigen (HLA), the first polynucleotide and the second polynucleotide being operably linked to the same promoter, and the first polynucleotide being upstream of the second polynucleotide. The antigen recognition domain may specifically bind to HLA-A2.

[0323] The vector may comprise a first polynucleotide encoding a FOXP3 polypeptide as described herein and a second polynucleotide encoding a chimeric antigen receptor (CAR) as described herein, wherein the CAR comprises an antigen recognition domain that specifically binds to a human leukocyte antigen (HLA), the first polynucleotide and the second polynucleotide are operably linked to the same promoter, the first polynucleotide is upstream of the second polynucleotide, and the vector further comprises a polynucleotide encoding a cleavage site as described herein between the first and second polynucleotides, and / or an internal ribosome entry site (IRES) as described herein between the first and second polynucleotides. The antigen recognition domain may specifically bind to HLA-A2.

[0324] The present invention provides a method for reducing the risk of generating modified HLA-specific T effector cells, preferably during the generation of modified HLA-specific Tregs, comprising introducing into the T effector cells a first polynucleotide encoding a FOXP3 polypeptide as described herein and a second polynucleotide encoding an HLA-specific CAR as described herein. Preferably, the modified HLA-specific T effector cells are modified HLA-A2-specific T effector cells, the modified HLA-specific Tregs are modified HLA-A2-specific Tregs, and the HLA-specific CAR is an HLA-A2-specific CAR. The HLA-specific CAR may comprise a single-chain antibody (scFv) antigen-recognition domain.

[0325] The present invention provides a method for reducing the risk of generating modified HLA-specific T effector cells, preferably during the generation of modified HLA-specific Tregs, said method comprising introducing into a cell-containing sample a first polynucleotide encoding a FOXP3 polypeptide as described herein and a second polynucleotide encoding an HLA-specific CAR as described herein; (a) the cell-containing sample contains T effector cells and / or Tregs; and / or (b) the cell-containing sample comprises or consists of peripheral blood mononuclear cells (PBMCs), and Tregs are enriched from the cell-containing sample before or after introducing the first polynucleotide and / or the second polynucleotide; and / or (c) the cell-containing sample comprises or consists of PBMCs, and Tregs are generated from the cell-containing sample before or after introducing the first polynucleotide and / or the second polynucleotide; and / or (d) the cell-containing sample comprises or consists of pluripotent stem cells (PSCs) (e.g., induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs)), and Tregs are differentiated from PSCs before or after introducing the first polynucleotide and / or second polynucleotide.

[0326] Preferably, the modified HLA-specific T effector cells are modified HLA-A2-specific T effector cells, the modified HLA-specific Tregs are modified HLA-A2-specific Tregs, and the HLA-specific CARs are HLA-A2-specific CARs. The HLA-specific CARs may comprise a single-chain antibody (scFv) antigen-recognition domain.

[0327] Suitably, said first polynucleotide and / or said second polynucleotide are introduced by viral transduction, preferably by retroviral or lentiviral transduction.

[0328] Preferably, the first polynucleotide and the second polynucleotide are introduced into a single vector, and the first polynucleotide and the second polynucleotide may be operably linked to the same promoter. The vector may be the vector of the present invention.

[0329] Therefore, the present invention provides a method for reducing the risk of generating modified HLA-specific T effector cells, preferably during the generation of modified HLA-specific Tregs, said method comprising introducing a vector according to the present invention into said T effector cells. Preferably, said modified HLA-specific T effector cells are modified HLA-A2-specific T effector cells, and said modified HLA-specific Tregs are modified HLA-A2-specific Tregs.

[0330] The present invention provides a method for reducing the risk of generating modified HLA-specific T effector cells (e.g. modified HLA-A2-specific T effector cells), preferably a method for reducing the risk of generating modified HLA-specific Tregs (e.g. modified HLA-A2-specific Tregs) during their generation, said method comprising introducing a vector according to the present invention into a cell-containing sample, (a) the cell-containing sample comprises or consists of T effector cells and / or Tregs, and / or (b) the cell-containing sample comprises or consists of PBMCs, and Tregs are enriched from the cell-containing sample before or after introducing the vector; and / or (c) the cell-containing sample comprises or consists of PBMCs, and Tregs are generated from the cell-containing sample before or after introducing the vector; and / or (d) the cell-containing sample comprises or consists of pluripotent stem cells (PSCs) (e.g., induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs)), and Tregs are differentiated from PSCs before or after introducing the first polynucleotide and / or second polynucleotide.

[0331] Preferably, the vector comprises an HLA-specific CAR. The HLA-specific CAR may be an HLA-A2-specific CAR. The HLA-specific CAR may comprise a single-chain antibody (scFv) antigen-recognition domain.

[0332] The phrase "reducing the risk of generating modified T effector cells" can mean reducing the likelihood or rate of generating modified T effector cells (e.g., HLA-specific T effector cells or HLA-A2-specific T effector cells) compared to the likelihood or rate of corresponding unmodified T effector cells (e.g., HLA-specific T effector cells or HLA-A2-specific T effector cells) by introducing a polynucleotide encoding a FOXP3 polypeptide as described herein and / or a vector as described herein. Preferably, the likelihood or rate is reduced during generation of modified Tregs (e.g., HLA-specific Tregs or HLA-A2-specific Tregs). Suitably, the likelihood is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, or 100%. Preferably, the rate is at least 1.5 times, at least 2 times, at least 5 times, at least 10 times, at least 20 times, at least 50 times, or at least 100 times slower.

[0333] Suitably, when a polynucleotide or vector encoding a FOXP3 polypeptide as described herein is introduced, the number of modified T effector cells (e.g., HLA-specific T effector cells or HLA-A2-specific T effector cells) generated during the generation of modified Tregs (e.g., HLA-specific Tregs or HLA-A2-specific Tregs) may be reduced compared to the number of corresponding modified T effector cells (e.g., HLA-specific T effector cells or HLA-A2-specific T effector cells) generated during the generation of the corresponding modified Tregs (e.g., HLA-specific Tregs or HLA-A2-specific Tregs) when only a polynucleotide encoding a CAR (e.g., an HLA-specific CAR or HLA-A2-specific CAR) is used, but not a polynucleotide or vector encoding FOXP3 of the invention. Preferably, the number of modified T effector cells (e.g., HLA-specific T effector cells or HLA-A2-specific T effector cells) generated during the generation of modified Tregs (e.g., HLA-specific Tregs or HLA-A2-specific Tregs) is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%, or 100%.

[0334] Pharmaceutical Composition Pharmaceutical compositions comprising the cells of the invention (eg, modified Tregs of the invention), or the vectors of the invention are also provided. A pharmaceutical composition is a composition comprising or consisting of a therapeutically effective amount of a pharmaceutically active agent, i.e., vector and / or Treg. Preferably, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent, or excipient (including combinations thereof). "Pharmaceutically acceptable" includes the meaning that the formulation is sterile and pyrogen-free. The carrier, diluent, and / or excipient must be "acceptable" in the sense of being compatible with the Tregs or vector and not deleterious to the recipient. Typically, the carrier, diluent, and / or excipient will be a sterile, pyrogen-free physiological or infusion medium, although other acceptable carriers, diluents, and excipients may be used.

[0335] Acceptable carriers, diluents, and excipients for therapeutic use are well known in the pharmaceutical arts. The choice of pharmaceutical carrier, excipient, or diluent can be selected taking into account the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may include, as or in addition to the carrier, excipient, or diluent, any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), or solubilizing agent(s).

[0336] Examples of pharmaceutically acceptable carriers include water, saline, alcohol, silicone, wax, petrolatum, vegetable oils, polyethylene glycol, propylene glycol, liposomes, sugar, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oils, fatty acid monoglycerides, fatty acid diglycerides, petroleum esters of fatty acids, hydroxymethylcellulose, polyvinylpyrrolidone, and the like.

[0337] The Tregs or pharmaceutical compositions of the present invention may be administered in a manner suitable for treating and / or preventing the diseases described herein. The dosage and frequency of administration are determined by factors such as the condition of the subject and the type and severity of the subject's disease, and appropriate dosages may be determined through clinical trials. The pharmaceutical compositions may be appropriately formulated.

[0338] The Tregs or pharmaceutical compositions described herein may be administered parenterally, e.g., intravenously, or by infusion techniques. The Tregs or pharmaceutical compositions may be administered in the form of a sterile aqueous solution containing other substances, e.g., sufficient salts or glucose, to make the solution isotonic with blood. The aqueous solution may be suitably buffered (preferably pH 3-9). The pharmaceutical composition may be suitably formulated. The preparation of suitable parenteral formulations under sterile conditions may be readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

[0339] The pharmaceutical composition may comprise the Tregs of the invention in an infusion vehicle, such as a sterile isotonic solution, and may be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0340] The Tregs or pharmaceutical compositions may be administered in a single dose or multiple doses. In particular, the Tregs or pharmaceutical compositions may be administered in a single, disposable dose. The pharmaceutical compositions may be appropriately formulated.

[0341] The Tregs or pharmaceutical compositions may be administered at various dosages (e.g., measured as cells / kg, cells / subject, etc.) depending on the disease and subject being treated and the route of administration. In any event, a physician will determine the actual dosage most suitable for any individual subject, which will vary depending on the age, weight, and response of the subject. Typically, however, for the Tregs of the present invention, a dosage of 5x10 cells / subject is administered. 7 ~3x10 9 cells, or 10 8 ~2x10 9 The cells may be administered.

[0342] The Tregs may be suitably modified for use in a pharmaceutical composition, for example, the Tregs may be cryopreserved and thawed at an appropriate time before being infused into a subject.

[0343] The pharmaceutical composition may further comprise one or more other therapeutic agents, such as lymphodepleting agents (e.g., thymoglobulin, campath-1H, anti-CD2 antibodies, anti-CD3 antibodies, anti-CD20 antibodies, cyclophosphamide, fludarabine), mTOR inhibitors (e.g., sirolimus, everolimus), agents that inhibit costimulatory pathways (e.g., anti-CD40 / CD40L, CTAL4Ig), and / or specific cytokines (IL-6, IL-17, TNFalpha, IL18).

[0344] Further included in the present invention is the use of kits comprising the Tregs, polynucleotides, vectors, and / or pharmaceutical compositions of the present invention. Preferably, the kits are for use in the methods and uses described herein, e.g., the therapeutic methods described herein. Preferably, the kits include instructions for use of the kit components.

[0345] Treatment and / or prevention of disease organ transplantation The present invention provides a method for inducing tolerance to transplantation, comprising administering the modified Tregs or pharmaceutical compositions of the present invention to a subject, suitably the subject is a mammal, preferably a human. Induction of transplant tolerance reduces the level of the recipient's immune response to the donor transplant tissue.

[0346] Thus, the present invention provides methods for treating and / or preventing transplant rejection, comprising the step of administering to a subject the modified Tregs or pharmaceutical compositions of the present invention.

[0347] The subject may be a transplant recipient and the transplant may be selected from liver, kidney, heart, lung, pancreas, small intestine, stomach, bone marrow, vascularized composite tissue transplant, and skin transplant. Preferably, the transplant is a liver transplant.

[0348] The modified Tregs may be administered to a subject not experiencing transplant rejection to prevent or reduce the likelihood of transplant rejection, reducing the likelihood of transplant rejection by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a subject not receiving the modified Tregs, or preventing said transplant rejection entirely.

[0349] The modified Tregs may be administered to a subject not experiencing symptoms of transplant rejection to reduce the likelihood of the onset of one symptom of transplant rejection, such as pain or tenderness at the transplant site, flu-like symptoms, fever, weight changes (e.g., weight gain), and fatigue, and the at least one symptom may be reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a subject not receiving the modified Tregs, or the at least one symptom may be prevented entirely.

[0350] The engineered Tregs or pharmaceutical compositions may be administered to a subject experiencing transplant rejection to counteract or slow the progression of transplant rejection, or to reduce, alleviate, or ameliorate at least one symptom of transplant rejection, such as pain or tenderness at the transplant site, flu-like symptoms, fever, weight changes (e.g., weight gain), and fatigue. At least one symptom may be reduced, alleviated, or ameliorated by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%, or at least one symptom may be completely cured.

[0351] The subject may be a transplant recipient undergoing immunosuppressive therapy. Advantageously, the present invention may reduce the amount of immunosuppressive medication required by the transplant recipient or allow for discontinuation of immunosuppressive medication.

[0352] Graft-versus-host disease The present invention provides a method for treating and / or preventing graft-versus-host disease (GvHD), comprising administering the modified Treg or pharmaceutical composition of the present invention to a subject, suitably a mammal, preferably a human.

[0353] Preferably, the subject is a transplant recipient. The subject may be a transplant recipient, and the transplanted tissue may be selected from liver, kidney, heart, lung, pancreas, small intestine, stomach, bone marrow, vascularized composite tissue transplant, and skin transplant. Preferably, the transplant is a bone marrow transplant. GvHD is a common complication that occurs after receiving transplanted tissue from a genetically different individual. GvHD is commonly associated with stem cell transplants, such as those that occur with bone marrow transplants. GvHD also occurs with other forms of transplanted tissue, such as liver transplants. White blood cells from the donor's immune system remaining in the tissue from the donor (the graft) recognize the recipient (the host) as foreign (non-self). The white blood cells present in the transplanted tissue then attack the recipient's own body cells, resulting in GvHD. GvHD can be acute or chronic. In general terms, acute graft-versus-host disease is characterized by selective damage to the liver, skin, mucous membranes, and gastrointestinal tract.

[0354] Preferably, the HLA-specific CAR may comprise an antigen-binding domain capable of specifically binding to an HLA present in the recipient but not present in the graft / trasplant donor.

[0355] The subject may be undergoing immunosuppressive therapy.

[0356] The engineered Tregs may be administered to a subject with GvHD to slow, reduce, or stop disease progression by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a subject not administered the engineered Tregs, or may completely stop the progression of the disease.

[0357] The engineered Tregs or pharmaceutical compositions may be administered to a subject with GvHD to reduce, alleviate, or ameliorate at least one symptom of GvHD, such as rash or itchy skin, jaundice, nausea, vomiting, diarrhea, abdominal cramps, dry or irritated eyes, dry mouth, shortness of breath, difficulty swallowing, weight loss, fatigue, and muscle weakness or pain. At least one symptom may be reduced, alleviated, or ameliorated by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%, or at least one symptom may be completely cured.

[0358] The modified Tregs may be administered to a subject who is not affected and / or does not exhibit any symptoms of GvHD to prevent or reduce the likelihood of GvHD. The modified Tregs reduce the likelihood or prevent at least one symptom of GvHD, such as rash or itchy skin, jaundice, nausea, vomiting, diarrhea, abdominal cramps, dry or irritated eyes, dry mouth, shortness of breath, difficulty swallowing, weight loss, fatigue, and muscle weakness or pain. The at least one symptom may be reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a subject not administered the modified Tregs, or at least one symptom may be completely prevented.

[0359] Preferably, the therapeutic method of the present invention may comprise a step of administering to a subject modified Tregs according to the present invention or modified Tregs obtainable (e.g. obtained) by a method according to the present invention.

[0360] Suitably, the method for treating and / or preventing a disease may comprise administering to a subject the modified Tregs of the present invention (e.g., in a pharmaceutical composition as described herein).

[0361] The method may include the following steps: (i) isolating or providing a cell-containing sample; (ii) introducing into said cells a polynucleotide(s) or vector defined herein; and (iii) administering to a subject the cells from (ii). Preferably, the cells are Tregs as defined herein.

[0362] Suitably, an enriched Treg population may be isolated and / or generated from the cell-containing sample prior to and / or after step (ii) of the method. For example, isolation and / or generation may be performed prior to and / or after step (ii) to isolate and / or generate an enriched Treg sample. Enrichment may be performed after step (ii) to enrich for cells and / or Tregs comprising the CAR, polynucleotide(s), and / or vector of the invention.

[0363] Suitably, said polynucleotide(s) or vector may be introduced by transduction and / or gene transfer.

[0364] Suitably, the cells may be autologous and / or allogeneic cells.

[0365] Preferably, the engineered Tregs may be administered in combination with one or more other therapeutic agents, such as lymphodepleting agents (e.g., thymoglobulin, Cambus-1H, anti-CD2 antibodies, anti-CD3 antibodies, anti-CD20 antibodies, cyclophosphamide, fludarabine), mTOR inhibitors (e.g., sirolimus, everolimus), agents that inhibit costimulatory pathways (e.g., anti-CD40 / CD40L, CTAL4Ig), and / or specific cytokines (IL-6, IL-17, TNFalpha, IL18). The engineered Tregs may be administered simultaneously with or sequentially (i.e., before or after) the one or more other therapeutic agents.

[0366] Example The present invention is further illustrated by examples which are intended to aid those skilled in the art in practicing the invention and are not intended to limit the scope of the invention.

[0367] Example 1A - Isolation of natural Tregs CD4+ T cells were isolated using a CD4+ positive selection kit. Cells were then stained with flow cytometry antibodies CD4, CD25, and CD127 before FACS sorting using a BD ARIA. CD4+CD25hiCD127- Tregs and CD4+CD25-CD127+ Tconvs were collected into polypropylene tubes. Cell sorting purity was determined by adding FOXP3 PE antibody. Purity of CD4+CD25+CD127-FOXP3+ cells was typically >70%.

[0368] Example 1B - Transduction of natural Tregs with FOXP3 On day 0, FACS-sorted Tregs and Tconvs were activated separately for 48 hours by culturing 1:1 with anti-CD3 and anti-CD28 beads. On day 2, cells were counted and cultured at 1x10 in complete RPMI (Tconvs) or Texmacs medium (Tregs). 6 Cells were resuspended at 1000 μg / mL. Non-tissue culture-treated 24-well plates were pre-treated by coating with retronectin, followed by blocking with 2% bovine serum albumin in PBS and washing twice with PBS. The final concentration of IL-2 was 300 μg / mL for Tconv and 1000 μg / mL for Treg. After incubating the cells overnight at 37°C, the supernatant was removed and fresh complete medium and IL-2 were added. The medium was changed every other day. Tconv cells were grown in RPMI-1640 (Gibco) supplemented with 10% heat-inactivated fetal bovine serum, 100 units / mL penicillin, 100 μg / mL streptomycin, and 2 mM L-glutamine. Regulatory T cells were cultured in Texmacs medium (Miltenyi) supplemented with 100 units / mL penicillin and 100 μg / mL streptomycin. On days 7-10, flow cytometry analysis was performed to analyze the level of transduction through the expression of mouse TCR constant regions and FOXP3.

[0369] Example 1: Proliferation and IL-2 production of stimulated Tconv cells in the presence of C-FOXP3-transduced natural Tregs Chinese hamster ovary (CHO) cells transduced with human HLA-DR4 and CD80 or CD86 were loaded with MBP111-129 (LSRFSWGAEGQRPGFGYGG) (10 μM / ml) on day 10. The suspension was incubated for 2 hours under standard tissue culture conditions, then irradiated, washed, and resuspended at the appropriate concentration. Transduced responder T cells were stained with CFSE cell tracker dye in warmed PBS at 37°C for 3 minutes, followed by the addition of an equal volume of warmed FBS and an additional 3 minutes of incubation. Cells were washed with 5 volumes of complete RPMI medium, then counted and transduced cells were counted at 1x10 6 Regulatory T cells were removed from culture, washed, and transduced cells were resuspended at 1x10 cells / ml. 6 Cells were resuspended in complete RPMI at 1 / ml. Cells were plated at various ratios of 1 Treg:0.1 CHO cells:Tconv for 4 days. On day 4, cells were stained with a viability dye and analyzed by flow cytometry. Proliferation rates were measured by gating on "live" cells and then gating on the population of cells with low CFSE fluorescence compared to cells cultured without peptide.

[0370] Figure 1 shows the proliferation of TCR-transduced Tconv cells with and without peptide (blue bars), as well as the proliferation of the same cells in the presence of mock Tregs (white bars), TCR-transduced Tregs, or TCR+FOXP3-transduced Tregs. On day 4, supernatants were collected and analyzed by ELISA for IL-2 production. Figure 2 shows IL-2 production by TCR-transduced Tconv cells with and without peptide (blue bars), and proliferation of the same cells in the presence of mock Tregs (white bars), TCR-transduced Tregs, or TCR+FOXP3-transduced Tregs.

[0371] Example 2 - T cells from different donors The experiment described in Example 1 was repeated using T cells from a different donor. FIG. 3 shows the proliferation rate of TCR-transduced T cells. FIG. 4 shows the concentration of IL-2 in the supernatants collected from the co-culture experiments.

[0372] Example 3 - Expression of Treg markers in transduced natural Tregs On days 7-10, mock-transduced or TCR- or TCR+FOXP3-transduced Tregs were analyzed by flow cytometry for the expression of Treg markers (FOXP3, CD25, and CTLA-4). Figure 5 shows the mean fluorescence intensity (MFI) for each marker. Points represent individual experiments. One-way ANOVA was used for statistical analysis; p<0.05*, p<0.005**. Figure 6 shows the same data presented in a different format: each line represents one experiment showing the MFI of the marker on the same Tregs transduced with TCR or TCR+FOXP3.

[0373] Example 4 - Transduced natural Tregs compared to inducible Tregs As described in Example 1C, CD80 + CD86 + DR4 + CHO cells were allowed to incorporate the peptide, irradiated, and then 0.1x10 6Transduced responder T cells were stained with CFSE cell tracking dye in warmed PBS at 37°C for 3 minutes, followed by addition of an equal volume of warmed FBS and incubation for an additional 3 minutes. The cells were washed with 5 volumes of complete medium, then counted and transduced cells were counted at 1x10 6 The transduction efficiency of Tconv and Treg cells was measured by flow cytometry. Treg cells were removed from the culture medium, washed, and transduced cells were resuspended at 1x10 cells / ml. 6 Cells were resuspended in complete RPMI at 1 Treg:0.1 CHO cells:Tconv at various ratios. Proliferation was measured by analyzing the dilution of Tconv stained with carboxyfluorescein succinimidyl ester (CFSE). The data in Figure 7 show that TCR+FOXP3-transduced natural Tregs suppress proliferation more effectively than TCR+FOXP3-transduced Tconv cells (i.e., inducible Tregs). Supernatants were collected from the culture medium and analyzed for IL-2 by ELISA. The data presented in Figure 8 show that TCR+FOXP3-transduced natural Tregs suppress IL-2 production more effectively than TCR+FOXP3-transduced Tconv cells (i.e., inducible Tregs).

[0374] Example 5A - Tregs expressing exogenous FOXP3 engraft, persist, and retain FoxP3, CD25, and TCR expression Thy1.1+CD4+CD25+ Tregs or CD45.1+CD4+CD25+ Tregs were isolated from lymph nodes and splenocytes of HLA-DRB*0401 transgenic mice by bead separation. CD45.1+ Tregs were transduced with TCR, and Thy1.1+ Tregs were transduced with TCR+ murine FOXP3. One day after transduction, TCR- or TCR+FOXP3-transduced cells were infused at a 1:1 ratio into HLA-DRB*0401 transgenic hosts conditioned with 4 Gy irradiation. FACS plots show the CD45.1:Thy1.1 ratio of the infused cells and their respective FOXP3 expression. After 7 weeks, engrafted cells were identified by TCR staining using flow cytometry, the CD45.1:Thy1.1 ratio within the TCR+ population was measured, and the phenotype of engrafted CD45.1 (TCR-transduced Tregs) or Thy1.1 (TCR+FOXP3-transduced Tregs) cells was examined by staining for FOXP3 and CD25. Thy1.1+CD4+CD25+ Tregs were isolated by bead separation from lymph nodes or splenocytes of HLA-DRB*0401 transgenic mice. Tregs were transduced with TCR, TCR+ mouse FOXP3, or cultured with virus-free supernatant (mock). One day after transduction, TCR- or TCR+FOXP3-transduced cells were infused into HLA-DRB*0401 transgenic hosts conditioned with 4 Gy irradiation. Seven weeks later, engraftment of transduced Tregs was measured using flow cytometry. Figure 9A shows transduction efficiency, measured by expression of human variable 2.1 and mouse Foxp3, one day after transduction. Figure 9B shows splenocytes from mice administered TCR- or TCR+FOXP3-transduced Tregs stained with Thy1.1 to identify transduced cells (top panel) and FOXP3 and TCR (bottom panel). Figure 9C shows cumulative data for TCR- or TCR+FOXP3-transduced Tregs, showing the fold change in transduction efficiency (left panel) and the fold change in absolute number of transduced cells (right panel) compared to the day of infusion. Figure 9D shows representative expression of FOXP3 in transduced cells 7 weeks after infusion. Graphs show the cumulative percentage of FOXP3+ cells in the transduced population at week 7 (left) and the fold change in FOXP3+ cells compared to the day of infusion.

[0375] Example 5B - Tregs expressing exogenous FOXP3 retain Treg function after 7 weeks in vivo, whereas Tregs not expressing exogenous FOXP3 acquire the ability to produce effector cytokines Splenocytes were cultured for 4 hours with CD86+HLA-DR4+ CHO cells pulsed with an irrelevant peptide or 10 μM MBP. Tregs expressing exogenous FOXP3 retained Treg function in vivo after 7 weeks, as indicated by the lack of effector cytokine production, whereas Tregs not expressing exogenous FOXP3 acquired the ability to produce effector cytokines (Figure 10). Although Examples 1-5 are exemplified using TCRs, the results are broadly applicable, for example, to other TCR constructs and CAR constructs, such as those containing antigen-binding domains that target HLA-A2.

[0376] Example 6A - Tregs transduced with constructs encoding FOXP3 and an HLA-A2-specific CAR express both genes and express significantly higher levels of FOXP3 compared to Tregs with only endogenous FOXP3 Tregs were isolated from PBMCs by CD4+ and CD25+ enrichment, and the enriched cells were stained for CD4, CD25, CD127, and CD45RA and sorted by FACS. Enriched Tregs were transduced with one of four constructs. Figure 11A shows a schematic diagram of the constructs used. Construct FC: Represents a construct encoding 5'-FOXP3-P2A-A2 CAR-3'. Construct RC: Represents a construct encoding 5'-R-P2A-A2 CAR-3', where R represents other genes. Construct C: Represents a construct encoding only A2 CAR. Construct CR: Represents a construct encoding 5'-A2 CAR-P2AR-3', where R represents other genes. FIG. 11B shows an outline of the transduction method. The enriched transduced cells were expanded using the following protocol. Day 0, Gibco TM Dynabeads TM Together with Human T-Activator CD3 / CD28, TexMACS TM 0.25x10 in 6 / ml On day 2, TexMACS was performed on Retronectin-coated plates with IL-2 and virus. TM 0.1x10 in 6 / ml Days 4-9: T25 flasks / 6-well plates containing TexMACS IL-2TM 0.25x10 in 6 Resuspend to / ml Day 15: TexMACS containing IL-2 in a T25 flask TM 0.25x10 in 6 Resuspend to / ml Figure 12 shows the expression levels of HLA-A2-specific CAR (A2 CAR), FOXP3, and other genes R, measured by flow cytometry, in Tregs transduced with constructs FC, RC, C, and CR, compared to mock controls. Figure 12A shows that Tregs transduced with each construct expressed the HLA-A2-specific CAR, both in the downstream (construct RC) and upstream (construct CR) constructs. Figure 12B shows that FOXP3 was expressed in all Tregs, but was significantly higher in construct FC, especially compared to expression in the HLA-A2-specific CAR alone (construct C).

[0377] Example 6B - Tregs transduced with constructs encoding FOXP3 and HLA-A2-specific CAR maintain FOXP3 expression Figure 13 shows the expression levels of HLA-A2-specific CAR (A2 CAR), FOXP3, and other genes R, measured by flow cytometry, in Tregs transduced with constructs FC, RC, C, and CR and expanded, compared to mock controls. FIG. 13A shows that Tregs transduced with each construct still expressed HLA-A2-specific CARs after further expansion. Figure 13B shows that FOXP3 expression decreased in Tregs transduced with constructs RC, C, and CR after further expansion. In contrast, FOXP3 expression did not decrease in Tregs transduced with construct FC after further expansion. Consequently, FOXP3 expression was significantly higher in construct FC after further expansion, especially when compared to that of the HLA-A2-specific CAR alone (construct C).

[0378] Example 7 - Tregs transduced with constructs encoding FOXP3 and HLA-A2-specific CAR maintain the Treg phenotype lineage while enhancing FOXP3 expression The media used in the growth protocol were TM X-VIVO-15 containing 5% AB serum (rather than TM and the cell concentration on day 0 was changed to (0.25x10 6 / ml) 0.2x10 6 / ml, except for CD4 + CD25 + CD127 - Tregs were FACS sorted, activated, transduced and expanded as described in Example 6A. T cells were removed from the culture medium and treated with dextramer and LIVE / DEAD TMFixable Near-IR was used to stain live cells with HLA-A2-specific CAR. Surface staining of cells was performed in Brilliant Stain Buffer (BD) containing anti-CD25 PE-Cy7, anti-CD62L PE-CF594, anti-TIGIT BV605, anti-CD45RO BUV395, and anti-CD223 BV711 for 20–30 min at 4°C in the dark. Cells were washed with FACS buffer and resuspended in fixation / permeabilization solution. The cells were incubated for 30 min at 4°C in the dark. Permeabilized cells were washed with 1x permeabilization buffer and resuspended in 50 μL of 1x permeabilization buffer containing anti-CTLA-4 BV421 and anti-Foxp3 PE for 30 min at 4°C in the dark. Cells were then washed with 1x permeabilization buffer, resuspended in FACS buffer, and analyzed by flow cytometry. In each sample, transduced (TD) cells were identified as Dextramer+, and other cells were considered non-transduced (NTD); mean fluorescence intensity (MFI) was measured for each phenotypic lineage marker in TD and NTD cells. 0 represents no change, and 2 1 indicates a two-fold increase in expression. Figure 14 shows the expression of phenotypic lineage markers in Tregs transduced with constructs FC, RC, C, and CR. Tregs transduced with construct FC maintained the Treg phenotypic lineage while exhibiting enhanced FOXP3 expression.

[0379] All publications mentioned herein are incorporated herein by reference. Various modifications and variations of the methods, cells, compositions, and uses of the present invention disclosed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been disclosed in connection with certain preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Various modifications of the disclosed modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the following claims.

Claims

1. 1. A vector comprising a first polynucleotide encoding a FOXP3 polypeptide and a second polynucleotide encoding a chimeric antigen receptor (CAR), the CAR comprises an antigen recognition domain that specifically binds to a human leukocyte antigen (HLA); the first polynucleotide and the second polynucleotide are operably linked to the same promoter; A vector, wherein the first polynucleotide is upstream of the second polynucleotide.

2. The vector according to claim 1, wherein the antigen recognition domain specifically binds to HLA-A2.

3. The vector according to claim 1 or 2, which satisfies at least one selected from the group consisting of the following (a) to (g): (a) the vector comprises a polynucleotide encoding a cleavage site between the first polynucleotide and the second polynucleotide, and / or a polynucleotide encoding an internal ribosome entry site (IRES) between the first polynucleotide and the second polynucleotide; (b) the vector comprises a self-cleaving sequence between the first polynucleotide and the second polynucleotide; (c) the vector comprises a polynucleotide sequence encoding a 2A self-cleaving sequence between the first polynucleotide and the second polynucleotide; (d) the FOXP3 polypeptide is comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1 or 2; or consisting of an amino acid sequence having at least 90% identity to SEQ ID NO: 1 or 2; (e) the first polynucleotide is comprises a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 3 or 4; or consisting of a polynucleotide sequence having at least 90% identity to SEQ ID NO: 3 or 4; (f) the second nucleotide sequence comprises a polynucleotide sequence that is at least 90% identical to SEQ ID NO: 211 or 212; (g) the promoter is a long terminal repeat (LTR); (h) The vector is a viral vector, a retroviral vector, or a lentiviral vector.

4. The vector according to any one of claims 1 to 3, which satisfies at least one selected from the group consisting of the following (a) to (e): (a) the antigen recognition domain is an antibody, an antibody fragment, or an antibody-derived domain; (b) the antigen recognition domain is an antigen-binding fragment (Fab), a single-chain antibody (scFv), or a single-domain antibody (sdAb); (c) The antigen recognition domain comprises any one of the following (i) to (xxvii): (i) a variable heavy domain comprising CDR1, CDR2 and CDR3 regions comprising or consisting of SEQ ID NOs: 5-7, respectively, and a variable light domain comprising CDR1, CDR2 and CDR3 regions comprising or consisting of SEQ ID NOs: 17-19, respectively; (ii) a variable heavy domain comprising CDR1, CDR2 and CDR3 regions comprising or consisting of SEQ ID NOs: 8-10, respectively, and a variable light domain comprising CDR1, CDR2 and CDR3 regions comprising or consisting of SEQ ID NOs: 20-22, respectively; (iii) a variable heavy domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs: 11-13, respectively, and a variable light domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs: 23-25, respectively; (iv) a variable heavy domain comprising CDR1, CDR2 and CDR3 regions comprising or consisting of SEQ ID NOs: 14-16, respectively, and a variable light domain comprising CDR1, CDR2 and CDR3 regions comprising or consisting of SEQ ID NOs: 26-28, respectively; (v) a variable heavy domain comprising CDR1, CDR2 and CDR3 regions comprising or consisting of SEQ ID NOs: 14-16, respectively, and a variable light domain comprising CDR1, CDR2 and CDR3 regions comprising or consisting of SEQ ID NOs: 29-31, respectively; (vi) a variable heavy domain comprising CDR1, CDR2 and CDR3 regions comprising or consisting of SEQ ID NOs: 14-16, respectively, and a variable light domain comprising CDR1, CDR2 and CDR3 regions comprising or consisting of SEQ ID NOs: 32-34, respectively; (vii) a variable heavy domain comprising CDR1, CDR2 and CDR3 regions comprising or consisting of SEQ ID NOs: 14-16, respectively, and a variable light domain comprising CDR1, CDR2 and CDR3 regions comprising or consisting of SEQ ID NOs: 35-37, respectively; (viii) a variable heavy domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs: 14-16, respectively, and a variable light domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs: 38-40, respectively; (ix) a variable heavy domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs: 14-16, respectively, and a variable light domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs: 41-43, respectively; (x) a variable heavy domain comprising CDR1, CDR2 and CDR3 regions comprising or consisting of SEQ ID NOs: 14-16, respectively, and a variable light domain comprising CDR1, CDR2 and CDR3 regions comprising or consisting of SEQ ID NOs: 44-46, respectively; (xi) a variable heavy domain comprising CDR1, CDR2 and CDR3 regions comprising or consisting of SEQ ID NOs: 14-16, respectively, and a variable light domain comprising CDR1, CDR2 and CDR3 regions comprising or consisting of SEQ ID NOs: 47-49, respectively; (xii) a variable heavy domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs: 14-16, respectively, and a variable light domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs: 50-52, respectively; (xiii) a variable heavy domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs: 14-16, respectively, and a variable light domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs: 53-55, respectively; (xiv) a variable heavy domain comprising CDR1, CDR2 and CDR3 regions comprising or consisting of SEQ ID NOs: 14-16, respectively, and a variable light domain comprising CDR1, CDR2 and CDR3 regions comprising or consisting of SEQ ID NOs: 56-58, respectively; (xv) a variable heavy domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs: 14-16, respectively, and a variable light domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs: 59-61, respectively; (xvi) a variable heavy domain comprising CDR1, CDR2 and CDR3 regions comprising or consisting of SEQ ID NOs: 62-64, respectively, and a variable light domain comprising CDR1, CDR2 and CDR3 regions comprising or consisting of SEQ ID NOs: 98-100, respectively; (xvii) a variable heavy domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs: 65-67, respectively, and a variable light domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs: 101-103, respectively; (xviii) a variable heavy domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs: 68-70, respectively, and a variable light domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs: 104-106, respectively; (xix) a variable heavy domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs:71-73, respectively, and a variable light domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs:107-109, respectively; (xx) a variable heavy domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs:74-76, respectively, and a variable light domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs:110-112, respectively; (xxi) a variable heavy domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs:77-79, respectively, and a variable light domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs:113-115, respectively; (xxii) a variable heavy domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs: 80-82, respectively, and a variable light domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs: 116-118, respectively; (xxiii) a variable heavy domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs: 83-85, respectively, and a variable light domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs: 119-121, respectively; (xxiv) a variable heavy domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs: 86-88, respectively, and a variable light domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs: 122-124, respectively; (xxv) a variable heavy domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs: 89-91, respectively, and a variable light domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs: 125-127, respectively; (xxvi) a variable heavy domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs: 92-94, respectively, and a variable light domain comprising CDR1, CDR2, and CDR3 regions comprising or consisting of SEQ ID NOs: 128-130, respectively; or (xxvii) a variable heavy domain comprising CDR1, CDR2 and CDR3 regions comprising or consisting of SEQ ID NOs: 95-97, respectively, and a variable light domain comprising CDR1, CDR2 and CDR3 regions comprising or consisting of SEQ ID NOs: 131-133, respectively; (d) the antigen recognition domain satisfies any one of (i) to (xxvii) defined in (c) above, and comprises a variable heavy domain having at least 90% identity to one or more of SEQ ID NOs: 134 to 149, and a variable light domain having at least 90% identity to one or more of SEQ ID NOs: 150 to 176; (e) the antigen recognition domain comprises an amino acid sequence that is at least 90% identical to one or more of SEQ ID NOs: 177-203, or consists of an amino acid sequence that is at least 90% identical to one or more of SEQ ID NOs: 177-203.

5. The vector according to any one of claims 1 to 4, which satisfies at least one selected from the group consisting of the following (a) to (k): (a) the CAR comprises a transmembrane (TM) domain and an intracellular signaling domain; (b) the CAR comprises a transmembrane (TM) domain and an intracellular signaling domain, as well as a hinge domain and / or one or more costimulatory domains; (c) the CAR comprises one or more hinge domains selected from the group consisting of a CD28 hinge domain, a CD8 hinge domain, an IgG hinge domain, and an IgD hinge domain. (d) the CAR comprises one or more TM domains selected from the group consisting of a CD28 TM domain, an ICOS TM domain, a CD8 TM domain, a CD4 TM domain, an OX40 TM domain, a 4-1BB TM domain, and a CD3 zeta TM domain; (e) the CAR comprises a CD8 hinge domain and / or a CD8 TM domain. (f) the CAR comprises one or more costimulatory domains selected from the group consisting of a CD28 signaling domain, an ICOS signaling domain, an OX40 signaling domain, a 4-1BB signaling domain, a CD27 signaling domain, or a TNFRSF25 signaling domain. (g) the CAR comprises one or more intracellular signaling domains selected from the group consisting of a CD3 zeta signaling domain, a CD3 polypeptide, a syk family tyrosine kinase, a src family tyrosine kinase, a CD2 signaling domain, a CD5 signaling domain, and a CD8 signaling domain. (h) the CAR comprises a CD3 zeta signaling domain and / or a CD28 signaling domain. (i) the CAR comprises a CD8 hinge domain, a CD8 TM domain, a CD28 signaling domain, and a CD3 zeta signaling domain; (j) the CAR comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 209 or SEQ ID NO:

210. (k) The vector comprises: (i), (ii), (iii), or (iv): (i) a first polynucleotide comprising, or consisting of, a polynucleotide sequence that is at least 90% identical to SEQ ID NO:3, and a second polynucleotide comprising a self-cleaving sequence that is at least 90% identical to SEQ ID NO:214 and a polynucleotide sequence that is at least 90% identical to SEQ ID NO:211; (ii) a first polynucleotide comprising a polynucleotide sequence at least 90% identical to SEQ ID NO:3 or consisting of a polynucleotide sequence at least 90% identical to SEQ ID NO:3, and a second polynucleotide comprising a self-cleaving sequence at least 90% identical to SEQ ID NO:214 and a polynucleotide sequence at least 90% identical to SEQ ID NO:212; (iii) a first polynucleotide comprising a polynucleotide sequence at least 90% identical to SEQ ID NO:4 or consisting of a polynucleotide sequence at least 90% identical to SEQ ID NO:4, a self-cleaving sequence at least 90% identical to SEQ ID NO:214, and a second polynucleotide comprising a polynucleotide sequence at least 90% identical to SEQ ID NO:211; or (iv) a first polynucleotide comprising a polynucleotide sequence at least 90% identical to SEQ ID NO:4, or consisting of a polynucleotide sequence at least 90% identical to SEQ ID NO:4, and a second polynucleotide comprising a self-cleaving sequence at least 90% identical to SEQ ID NO:214, and a polynucleotide sequence at least 90% identical to SEQ ID NO:

212.

6. 6. A modified T cell comprising the vector of any one of claims 1 to 5, wherein the modified T cell is a modified regulatory T cell (Treg).

7. 6. The vector of claim 1 for use in enhancing the ability of modified HLA-specific Tregs to suppress an immune response in a patient, wherein the immune response is an immune response against cells expressing HLA.

8. 10. A method for enhancing the ability of modified HLA-specific Tregs to suppress an immune response, comprising introducing a vector according to any one of claims 1 to 5 into (i) the Tregs or (ii) a cell-containing sample, (a) the cell-containing sample comprises or consists of Tregs; and / or (b) the cell-containing sample comprises or consists of peripheral blood mononuclear cells (PBMCs), and Tregs are enriched from the cell-containing sample before or after introducing the vector; and / or (c) the cell-containing sample comprises or consists of PBMCs, and Tregs are generated from the cell-containing sample before or after introducing the vector. method.

9. The method of claim 8, wherein the HLA is HLA-A2.

10. The method of claim 8 or 9, wherein the vector is introduced by retroviral or lentiviral transduction.

11. 6. The vector of any one of claims 1 to 5 for use in reducing the risk of modified HLA-specific Tregs acquiring an effector phenotype in a patient.

12. 10. A method for reducing the risk of modified HLA-specific Tregs acquiring an effector phenotype, comprising introducing a vector according to any one of claims 1 to 5 into said Tregs.

13. 1. A method for reducing (i) the risk of modified HLA-specific Tregs acquiring an effector phenotype, and / or (ii) the risk of generating modified HLA-specific T effector cells during generation of modified HLA-specific Tregs, comprising:

6. The method of claim 1, further comprising introducing a vector according to claim 1 into a cell-containing sample. (a) the cell-containing sample comprises or consists of Treg and / or effector cells; and / or (b) the cell-containing sample comprises or consists of PBMCs, and Tregs are enriched from the cell-containing sample before or after introducing the vector; and / or (c) The method, wherein the cell-containing sample comprises or consists of PBMCs, and Tregs are generated from the cell-containing sample before or after introducing the vector.

14. 6. Use of a vector according to any one of claims 1 to 5 to reduce the risk of generating modified HLA-specific T effector cells during the generation of modified HLA-specific Tregs.

15. The vector according to claim 11, wherein the HLA is HLA-A2.

16. Use of the vector according to claim 14 or the method according to claim 12 or 13, wherein the HLA is HLA-A2.

17. Modified Treg obtainable or obtained by the method of any one of claims 8 to 10.

18. A pharmaceutical composition comprising the modified T cells of claim 6 or the modified Tregs of claim 17.

19. 19. A pharmaceutical composition according to claim 18 for use in inducing tolerance to transplanted tissue in a subject or for use in treating and / or preventing transplant rejection or graft-versus-host disease (GvHD) in a subject, comprising: The pharmaceutical composition, wherein the subject is a human.

20. 19. A pharmaceutical composition according to claim 18 for use in a method for inducing tolerance to transplanted tissue in a subject or for treating and / or preventing transplant rejection or GvHD in a subject, comprising: The method comprises the following steps (i) and (ii): (i) isolating or providing a cell-containing sample from said subject, said cell-containing sample comprising or consisting of PBMCs; (ii) introducing the vector of any one of claims 1 to 5 into the cell-containing sample, the cell-containing sample comprises Tregs or consists of PBMCs; and / or wherein Tregs are enriched from said cell-containing sample before or after introducing a vector according to any one of claims 1 to 5 and / or are generated from said cell-containing sample before or after introducing a vector according to any one of claims 1 to 5; The pharmaceutical composition, wherein the subject is a human.

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