Therapeutic T cell product

Abstract

Immune cells containing modifications that increase SERPINB9 expression or activity are disclosed. Also disclosed are compositions containing such cells, and methods of using such cells and compositions.

Description

[Technical Field]

[0001] This application claims priority to U.S. 63 / 330718, filed April 13, 2022, and U.S. 63 / 446520, filed February 17, 2023, the contents and elements of which are each incorporated herein by reference for all purposes.

[0002] The present disclosure relates to the field of molecular biology, more particularly cell therapy, and to methods of medical treatment and prophylaxis. [Background technology]

[0003] Adoptive cell therapy has emerged as a powerful and effective treatment for various cancers. For example, CD19 chimeric antigen receptor (CAR)-expressing T cells are highly effective against B-cell malignancies, and virus-specific T cells (VSTs) have shown great potential in treating patients with Epstein-Barr virus (EBV)-associated lymphoma and viral infections in hematopoietic stem cell transplant (HSCT) recipients.

[0004] To avoid graft-versus-host disease (GVHD) or graft rejection, most cell therapies are autologous. However, because such highly personalized therapies are produced from cells derived from patients with cancer or genetic diseases, there are many problems associated with the treatment. Furthermore, high costs and long "harvest-to-infusion" times limit the use of autologous cell therapies.

[0005] On the other hand, off-the-shelf third-party derived cells have served as a readily available therapeutic source for patients at the point of care, overcoming many of the problems associated with customized cell products. However, unwanted host immune responses to allogeneic third-party derived therapeutic cells limit their bioavailability and therapeutic efficacy. Efforts have been made to increase the persistence of therapeutic products and reduce their allogeneic clearance, for example, by expressing alloprotective receptors (ADRs) that target and kill alloreactive host T and NK cells (1, 2), by genetic knockout of HLA molecules (3) and CD52 (4, 5), and by cell encapsulation techniques that hide therapeutic agents from the host immune system (6). However, many of these approaches exert undesirable effects on the host, such as the elimination of alloreactive or pathogen-specific immune cells, which may lead to opportunistic infections. There remains a strong need for strategies to protect therapeutic cells without compromising host immunity.

[0006] Granzyme B (GzmB) is a serine proteinase that has been described as an important cytotoxic molecule utilized by T cells or natural killer (NK) cells for the elimination of allogeneic or pathogen-infected cells (7-9). Additionally, granzyme B produced within therapeutic cells after stimulation (i.e., intrinsic granzyme B) is associated with homeostatic cell death (Bird et al., Cell Death Differ. (2014) 21, 876-887). GzmB cleaves its target proteins, such as the proapoptotic Bcl-2 family member Bid or apoptotic caspase substrates (e.g., DNA-PK, PARP, and NuMA), causing mitochondrial instability and caspase activation, respectively, leading to apoptosis of the targeted cells (10, 11). Wild-type SERPINB9 (also referred to herein as SB9(WT)) inhibits GzmB by forming a stable covalent bond with GzmB and acting as a pseudosubstrate that prevents activation of apoptosis (12). A glutamic acid (340E) at the P1 position of the reactive center loop (RCL) of SB9 is important for its specificity for GzmB but limits GzmB's interaction with other caspases involved in Fas-mediated apoptosis (13). Substitution of the glutamic acid with an aspartic acid (E340D), forming a variant referred to herein as SB9(CAS), has been shown to broaden SB9 inhibition of both GzmB and Fas-mediated apoptosis (13). Additionally, SB9 wild-type was observed to be reactive oxygen species (ROS) sensitive, and the C341S and C342S conversions resulted in functional SB9, forming a variant herein designated SB9(ROS), which resists inactivation by ROS while maintaining GzmB inhibition ( 14 ). Summary of the Invention [Means for solving the problem]

[0007] In a first aspect, the present disclosure provides immune cells comprising a modification that increases SERPINB9 expression or activity. In particular, the present disclosure provides immune cells for use in methods of treatment or prevention by adoptive cell transfer, comprising a modification that increases SERPINB9 expression or activity.

[0008] In some embodiments, the immune cell comprises an exogenous nucleic acid encoding a SERPINB9 polypeptide. In some embodiments, the exogenous nucleic acid encoding a SERPINB9 polypeptide is or is contained in an expression vector, and optionally, the expression vector is a retroviral expression vector. In some embodiments, the SERPINB9 polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 1, 4, 5, 6, or 7, or a variant thereof having at least 85% amino acid sequence identity with SEQ ID NO: 1, 4, 5, 6, or 7.

[0009] In some embodiments, the immune cell is an effector immune cell, and optionally the effector immune cell is a T cell or a natural killer (NK) cell. In some embodiments, the immune cells comprise a nucleic acid encoding a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an antigen-binding domain that binds to a cancer-associated antigen selected from CD30, CD19, CD20, CD22, B7H3, c-Met, ROR1R, CD4, CD7, CD38, BCMA, mesothelin, EGFR, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY, and PSCA, and optionally, the CAR comprises an antigen-binding domain that binds to CD30.

[0010] In some embodiments, the immune cells are virus-specific T cells or activated T cells (ATCs). In some embodiments, the immune cells are virus-specific T cells. In some embodiments, the virus-specific T cells are specific for a virus selected from Epstein-Barr virus (EBV), adenovirus, cytomegalovirus (CMV), human papillomavirus (HPV), influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), herpes simplex virus (HSV), BK virus (BKV), or varicella-zoster virus (VZV). In some embodiments, the virus is EBV.

[0011] The present disclosure also provides a pharmaceutical composition comprising an immune cell described in this disclosure and a pharmaceutically acceptable carrier, diluent, excipient, or adjuvant. The present disclosure also provides an immune cell or pharmaceutical composition according to the present disclosure for use in a method of medical treatment or prevention.

[0012] The present disclosure also provides the use of an immune cell or pharmaceutical composition described in this disclosure in the manufacture of a medicament for use in a method of medical treatment or prophylaxis. The present disclosure also provides a method of treating or preventing a disease or condition in a subject, the method comprising administering to the subject a therapeutically or prophylactically effective amount of an immune cell or pharmaceutical composition described in the present disclosure.

[0013] The present disclosure also provides a method for reducing serine protease or caspase activity in a cell, the method comprising modifying the cell to increase SERPINB9 expression or activity.

[0014] The present disclosure also provides a method for increasing the resistance of a cell to the activity of a serine protease or caspase, the method comprising modifying the cell to increase the expression or activity of SERPINB9.

[0015] The present disclosure also provides a method for increasing the resistance of a cell to cell killing by granzyme B, the method comprising modifying the cell to increase the expression or activity of SERPINB9.

[0016] The present disclosure also provides a method for increasing the resistance of a cell to apoptosis mediated by a death receptor, the method comprising modifying the cell to increase the expression or activity of SERPINB9.

[0017] In some embodiments according to various aspects of the present disclosure, modifying a cell to increase SERPINB9 expression or activity comprises introducing a nucleic acid encoding a SERPINB9 polypeptide into the cell. In some embodiments, the nucleic acid encoding the SERPINB9 polypeptide is or is contained within an expression vector, and optionally the expression vector is a retroviral expression vector. In some embodiments, the SERPINB9 polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 1, 4, 5, 6, or 7, or a variant thereof having at least 85% amino acid sequence identity with SEQ ID NO: 1, 4, 5, 6, or 7.

[0018] In some embodiments, the cell is an effector immune cell, optionally the effector immune cell is a T cell or a natural killer (NK) cell. In some embodiments, the cells comprise a nucleic acid encoding a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an antigen-binding domain that binds to a cancer-associated antigen selected from CD30, CD19, CD20, CD22, B7H3, c-Met, ROR1R, CD4, CD7, CD38, BCMA, mesothelin, EGFR, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY, and PSCA, and optionally, the CAR comprises an antigen-binding domain that binds to CD30.

[0019] In some embodiments, the cells are virus-specific T cells. In some embodiments, the virus-specific T cells are specific for a virus selected from Epstein-Barr virus (EBV), adenovirus, cytomegalovirus (CMV), human papillomavirus (HPV), influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), herpes simplex virus (HSV), BK virus (BKV), or varicella-zoster virus (VZV). In some embodiments, the virus is EBV.

[0020] The present disclosure also provides an immune cell for use in treating or preventing cancer, comprising: a nucleic acid encoding a CAR comprising (i) an antigen-binding domain that binds to CD30 or CD19, (ii) a transmembrane domain, and (iii) a signaling domain that comprises an immunoreceptor tyrosine-based activation motif (ITAM), Immune cells are provided that contain modifications that increase the expression or activity of SERPINB9.

[0021] The present disclosure also provides the use of immune cells in the manufacture of a medicament for use in treating or preventing cancer, comprising: the immune cell comprises a nucleic acid encoding a CAR comprising (i) an antigen-binding domain that binds to CD30 or CD19, (ii) a transmembrane domain, and (iii) a signaling domain that comprises an immunoreceptor tyrosine-based activation motif (ITAM); The invention provides a use wherein the immune cell comprises a modification that increases the expression or activity of SERPINB9.

[0022] The present disclosure also provides a method of treating or preventing cancer in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of immune cells; the immune cell comprises a nucleic acid encoding a CAR comprising (i) an antigen-binding domain that binds to CD30 or CD19, (ii) a transmembrane domain, and (iii) a signaling domain that comprises an immunoreceptor tyrosine-based activation motif (ITAM); Methods are provided in which the immune cells comprise a modification that increases the expression or activity of SERPINB9.

[0023] In some embodiments, the immune cells are virus-specific T cells, and optionally, the immune cells are Epstein-Barr virus (EBV)-specific T cells. The present disclosure also provides an immune cell for use in treating or preventing cancer, comprising: virus-specific T cells, optionally Epstein-Barr virus (EBV)-specific T cells; Immune cells are provided that contain modifications that increase the expression or activity of SERPINB9.

[0024] The present disclosure also provides the use of immune cells in the manufacture of a medicament for use in treating or preventing cancer, comprising: the immune cells are virus-specific T cells, optionally the immune cells are Epstein-Barr virus (EBV)-specific T cells; The invention provides a use wherein the immune cell comprises a modification that increases the expression or activity of SERPINB9.

[0025] The present disclosure also provides a method of treating or preventing cancer in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of immune cells; the immune cells are virus-specific T cells, optionally the immune cells are Epstein-Barr virus (EBV)-specific T cells; Methods are provided in which the immune cells comprise a modification that increases the expression or activity of SERPINB9.

[0026] In some embodiments, the immune cell comprises a nucleic acid encoding a CAR comprising (i) an antigen-binding domain that binds to CD30 or CD19, (ii) a transmembrane domain, and (iii) a signaling domain that comprises an immunoreceptor tyrosine-based activation motif (ITAM).

[0027] In some embodiments according to various aspects of the present disclosure, the subject (i.e., the subject being treated / to be treated) is allogeneic with respect to the immune cells (i.e., the immune cells administered / to be administered pursuant to the treatment / prophylaxis).

[0028] In some embodiments according to various aspects of the present disclosure, the immune cell comprises an exogenous nucleic acid encoding a SERPINB9 polypeptide. In some embodiments, the exogenous nucleic acid encoding the SERPINB9 polypeptide is or is contained in an expression vector, and optionally the expression vector is a retroviral expression vector. In some embodiments, the SERPINB9 polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 1, 4, 5, 6, or 7, or a variant thereof having at least 85% amino acid sequence identity with SEQ ID NO: 1, 4, 5, 6, or 7.

[0029] BRIEF DESCRIPTION OF THE DRAWINGS Embodiments and experiments illustrating the principles of the present disclosure will now be described with reference to the accompanying drawings. [Brief explanation of the drawings]

[0030] [Figure 1] FIG. 1 is a schematic diagram of a process for producing activated, TCR knockout (KO) cells as therapeutic transplant cells. [Figure 2] FIG. 1 is a schematic diagram of a process for producing virus-specific cells (VSTs) as therapeutic transplant cells. [Figure 3] FIG. 1 is a schematic diagram of a process for producing CD30 knockout (KO), allogeneic T (allo-T) cells, which serves as an example of generating allo-T cells that do not express the CAR target antigen. [Figure 4A]Graphs and bar graphs show the expansion and expression of SERPINB9 (SB9) by T cells from two different donors transduced with either GFP or serpin-GFP. The fold change in T cell proliferation over the culture period from the day of transduction was determined from cell counts enumerated by trypan blue staining and a hemocytometer. Transduced T cells were harvested 5 days post-transduction, and GFP expression as a percentage of total CD3-positive cells and intracellular SB9 expression (median fluorescence intensity) of GFP-positive cells were determined by flow cytometry. (4A) Cumulative fold change in cell number over time is shown. (4B) GFP expression as a percentage of total CD3-positive cells is shown. (4C) Intracellular SB9 expression of GFP-positive cells is shown. [Figure 4B] Graphs and bar graphs show the expansion and expression of SERPINB9 (SB9) by T cells from two different donors transduced with either GFP or serpin-GFP. The fold change in T cell proliferation over the culture period from the day of transduction was determined from cell counts enumerated by trypan blue staining and a hemocytometer. Transduced T cells were harvested 5 days post-transduction, and GFP expression as a percentage of total CD3-positive cells and intracellular SB9 expression (median fluorescence intensity) of GFP-positive cells were determined by flow cytometry. (4A) Cumulative fold change in cell number over time is shown. (4B) GFP expression as a percentage of total CD3-positive cells is shown. (4C) Intracellular SB9 expression of GFP-positive cells is shown. [Figure 4C]Graphs and bar graphs show the expansion and expression of SERPINB9 (SB9) by T cells from two different donors transduced with either GFP or serpin-GFP. The fold change in T cell proliferation over the culture period from the day of transduction was determined from cell counts enumerated by trypan blue staining and a hemocytometer. Transduced T cells were harvested 5 days post-transduction, and GFP expression as a percentage of total CD3-positive cells and intracellular SB9 expression (median fluorescence intensity) of GFP-positive cells were determined by flow cytometry. (4A) Cumulative fold change in cell number over time is shown. (4B) GFP expression as a percentage of total CD3-positive cells is shown. (4C) Intracellular SB9 expression of GFP-positive cells is shown. [Figure 5A] Graphs and bar charts show the expansion, CD30.CAR (IgG1 spacer) expression, and SERPINB9 (SB9) expression of T cells from two different donors transduced with either GFP-CAR or serpin-CAR. The fold change in T cell proliferation over the culture period from the day of transduction was determined from cell counts enumerated by trypan blue staining and a hemocytometer. GFP-transduced T cells were used as a control. Transduced T cells were harvested 7 days post-transduction, and CD30.CAR expression as a percentage of total CD3-positive cells and intracellular SB9 expression (median fluorescence intensity) of CD30.CAR-positive cells were determined by flow cytometry. (5A) Cumulative fold change in cell number over time is shown. (5B) CD30.CAR expression as a percentage of total CD3-positive cells is shown. (5C) Intracellular SB9 expression of CD30.CAR-positive cells is shown. [Figure 5B]Graphs and bar charts show the expansion, CD30.CAR (IgG1 spacer) expression, and SERPINB9 (SB9) expression of T cells from two different donors transduced with either GFP-CAR or serpin-CAR. The fold change in T cell proliferation over the culture period from the day of transduction was determined from cell counts enumerated by trypan blue staining and a hemocytometer. GFP-transduced T cells were used as a control. Transduced T cells were harvested 7 days post-transduction, and CD30.CAR expression as a percentage of total CD3-positive cells and intracellular SB9 expression (median fluorescence intensity) of CD30.CAR-positive cells were determined by flow cytometry. (5A) Cumulative fold change in cell number over time is shown. (5B) CD30.CAR expression as a percentage of total CD3-positive cells is shown. (5C) Intracellular SB9 expression of CD30.CAR-positive cells is shown. [Figure 5C] Graphs and bar charts show the expansion, CD30.CAR (IgG1 spacer) expression, and SERPINB9 (SB9) expression of T cells from two different donors transduced with either GFP-CAR or serpin-CAR. The fold change in T cell proliferation over the culture period from the day of transduction was determined from cell counts enumerated by trypan blue staining and a hemocytometer. GFP-transduced T cells were used as a control. Transduced T cells were harvested 7 days post-transduction, and CD30.CAR expression as a percentage of total CD3-positive cells and intracellular SB9 expression (median fluorescence intensity) of CD30.CAR-positive cells were determined by flow cytometry. (5A) Cumulative fold change in cell number over time is shown. (5B) CD30.CAR expression as a percentage of total CD3-positive cells is shown. (5C) Intracellular SB9 expression of CD30.CAR-positive cells is shown. [Figure 6]This graph shows the cytolysis of KM-H2 cells by T cells transduced with GFP, CD30.CAR (IgG1 spacer), or SERPINB9 and CD30.CAR (IgG1 spacer) cells prepared from two different donors. CD30.CAR-positive T cells were normalized to 70% of the total cells in the culture (for GFP-CD30.CAR and serpin-CD30.CAR conditions) by the addition of GFP-transduced T cells. Transduced T cells (effector cells) were cocultured with CD30-expressing KM-H2 cells (target cells) at effector:target ratios of 1:1 and 2:1, and KM-H2 cell lysis was measured over 48 hours using xCELLigence Real-Time Cell Analysis software (Agilent). GFP-transduced T cells were used as a negative control. [Figure 7A] 7A and 7B are schematic diagrams and graphs showing that SERPINB9 expression protects transplanted T cells from immune rejection in vitro. (7A) A schematic diagram of the mixed lymphocyte reaction (MLR) assay and the constructs used is shown. GFP-transduced (negative control) or serpin-GFP-transduced TCR knockout (KO) transplanted T cells from two different donors were mixed with HLA-mismatched host PBMCs at a cell ratio of 1:10 (PBMC-mixed lymphocyte assay), and cell numbers were determined over 12 days. MLR assays were performed in the presence of IL-7 and IL-15, both at 10 ng / mL, and cultures were expanded if necessary. (7B) Cell counts of transplanted T cells cultured alone are shown. (7C) Cell counts of transplanted T cells cultured in a PBMC MLR are shown. (7D) Cell counts of host CD3-positive T cells are shown. (7E) Cell counts of host CD3-negative CD56-positive natural killer (NK) cells are shown. All data are shown as mean ± SD. [Figure 7B]7A and 7B are schematic diagrams and graphs showing that SERPINB9 expression protects transplanted T cells from immune rejection in vitro. (7A) A schematic diagram of the mixed lymphocyte reaction (MLR) assay and the constructs used is shown. GFP-transduced (negative control) or serpin-GFP-transduced TCR knockout (KO) transplanted T cells from two different donors were mixed with HLA-mismatched host PBMCs at a cell ratio of 1:10 (PBMC-mixed lymphocyte assay), and cell numbers were determined over 12 days. MLR assays were performed in the presence of IL-7 and IL-15, both at 10 ng / mL, and cultures were expanded if necessary. (7B) Cell counts of transplanted T cells cultured alone are shown. (7C) Cell counts of transplanted T cells cultured in a PBMC MLR are shown. (7D) Cell counts of host CD3-positive T cells are shown. (7E) Cell counts of host CD3-negative CD56-positive natural killer (NK) cells are shown. All data are shown as mean ± SD. [Figure 7C] 7A and 7B are schematic diagrams and graphs showing that SERPINB9 expression protects transplanted T cells from immune rejection in vitro. (7A) A schematic diagram of the mixed lymphocyte reaction (MLR) assay and the constructs used is shown. GFP-transduced (negative control) or serpin-GFP-transduced TCR knockout (KO) transplanted T cells from two different donors were mixed with HLA-mismatched host PBMCs at a cell ratio of 1:10 (PBMC-mixed lymphocyte assay), and cell numbers were determined over 12 days. MLR assays were performed in the presence of IL-7 and IL-15, both at 10 ng / mL, and cultures were expanded if necessary. (7B) Cell counts of transplanted T cells cultured alone are shown. (7C) Cell counts of transplanted T cells cultured in a PBMC MLR are shown. (7D) Cell counts of host CD3-positive T cells are shown. (7E) Cell counts of host CD3-negative CD56-positive natural killer (NK) cells are shown. All data are shown as mean ± SD. [Figure 7D]7A and 7B are schematic diagrams and graphs showing that SERPINB9 expression protects transplanted T cells from immune rejection in vitro. (7A) A schematic diagram of the mixed lymphocyte reaction (MLR) assay and the constructs used is shown. GFP-transduced (negative control) or serpin-GFP-transduced TCR knockout (KO) transplanted T cells from two different donors were mixed with HLA-mismatched host PBMCs at a cell ratio of 1:10 (PBMC-mixed lymphocyte assay), and cell numbers were determined over 12 days. MLR assays were performed in the presence of IL-7 and IL-15, both at 10 ng / mL, and cultures were expanded if necessary. (7B) Cell counts of transplanted T cells cultured alone are shown. (7C) Cell counts of transplanted T cells cultured in a PBMC MLR are shown. (7D) Cell counts of host CD3-positive T cells are shown. (7E) Cell counts of host CD3-negative CD56-positive natural killer (NK) cells are shown. All data are shown as mean ± SD. [Figure 7E] 7A and 7B are schematic diagrams and graphs showing that SERPINB9 expression protects transplanted T cells from immune rejection in vitro. (7A) A schematic diagram of the mixed lymphocyte reaction (MLR) assay and the constructs used is shown. GFP-transduced (negative control) or serpin-GFP-transduced TCR knockout (KO) transplanted T cells from two different donors were mixed with HLA-mismatched host PBMCs at a cell ratio of 1:10 (PBMC-mixed lymphocyte assay), and cell numbers were determined over 12 days. MLR assays were performed in the presence of IL-7 and IL-15, both at 10 ng / mL, and cultures were expanded if necessary. (7B) Cell counts of transplanted T cells cultured alone are shown. (7C) Cell counts of transplanted T cells cultured in a PBMC MLR are shown. (7D) Cell counts of host CD3-positive T cells are shown. (7E) Cell counts of host CD3-negative CD56-positive natural killer (NK) cells are shown. All data are shown as mean ± SD. [Figure 8A]Schematic, graph, and bar graph showing that SERPINB9 expression protects transplanted CD30.CAR (IgG1 spacer) T cells from T cell-mediated immune rejection in vitro. (8A) Schematic representation of the mixed lymphocyte reaction (MLR) assay and the constructs used. GFP-transduced (negative control) or serpin-GFP-transduced TCR knockout (KO) transplanted T cells from two different donors were mixed at a 1:1–2 cell ratio with CD30 KO alloreactive T cells from an HLA-mismatched host stimulated to recognize and kill the transplanted cells (CD30 KO alloreactive T cell mixed lymphocyte assay), and cell numbers were determined over a 3-day period. The assay was performed in the presence of both 10 ng / mL IL-7 and IL-15. (8B) Cell counts of transplanted T cells cultured alone are shown. (8C) Cell counts of transplanted T cells cultured in an MLR are shown. (8D) Cell counts of transplanted T cells after 3 days of culture in an MLR are shown. (8E) Cell counts of host CD3-positive T cells cultured in an MLR. All data represent the mean ± SD. [Figure 8B] Schematic, graph, and bar graph showing that SERPINB9 expression protects transplanted CD30.CAR (IgG1 spacer) T cells from T cell-mediated immune rejection in vitro. (8A) Schematic representation of the mixed lymphocyte reaction (MLR) assay and the constructs used. GFP-transduced (negative control) or serpin-GFP-transduced TCR knockout (KO) transplanted T cells from two different donors were mixed at a 1:1–2 cell ratio with CD30 KO alloreactive T cells from an HLA-mismatched host stimulated to recognize and kill the transplanted cells (CD30 KO alloreactive T cell mixed lymphocyte assay), and cell numbers were determined over a 3-day period. The assay was performed in the presence of both 10 ng / mL IL-7 and IL-15. (8B) Cell counts of transplanted T cells cultured alone are shown. (8C) Cell counts of transplanted T cells cultured in an MLR are shown. (8D) Cell counts of transplanted T cells after 3 days of culture in an MLR are shown. (8E) Cell counts of host CD3-positive T cells cultured in an MLR. All data represent the mean ± SD. [Figure 8C] Schematic, graph, and bar graph showing that SERPINB9 expression protects transplanted CD30.CAR (IgG1 spacer) T cells from T cell-mediated immune rejection in vitro. (8A) Schematic representation of the mixed lymphocyte reaction (MLR) assay and the constructs used. GFP-transduced (negative control) or serpin-GFP-transduced TCR knockout (KO) transplanted T cells from two different donors were mixed at a 1:1–2 cell ratio with CD30 KO alloreactive T cells from an HLA-mismatched host stimulated to recognize and kill the transplanted cells (CD30 KO alloreactive T cell mixed lymphocyte assay), and cell numbers were determined over a 3-day period. The assay was performed in the presence of both 10 ng / mL IL-7 and IL-15. (8B) Cell counts of transplanted T cells cultured alone are shown. (8C) Cell counts of transplanted T cells cultured in an MLR are shown. (8D) Cell counts of transplanted T cells after 3 days of culture in an MLR are shown. (8E) Cell counts of host CD3-positive T cells cultured in an MLR. All data represent the mean ± SD. [Figure 8D]Schematic, graph, and bar graph showing that SERPINB9 expression protects transplanted CD30.CAR (IgG1 spacer) T cells from T cell-mediated immune rejection in vitro. (8A) Schematic representation of the mixed lymphocyte reaction (MLR) assay and the constructs used. GFP-transduced (negative control) or serpin-GFP-transduced TCR knockout (KO) transplanted T cells from two different donors were mixed at a 1:1–2 cell ratio with CD30 KO alloreactive T cells from an HLA-mismatched host stimulated to recognize and kill the transplanted cells (CD30 KO alloreactive T cell mixed lymphocyte assay), and cell numbers were determined over a 3-day period. The assay was performed in the presence of both 10 ng / mL IL-7 and IL-15. (8B) Cell counts of transplanted T cells cultured alone are shown. (8C) Cell counts of transplanted T cells cultured in an MLR are shown. (8D) Cell counts of transplanted T cells after 3 days of culture in an MLR are shown. (8E) Cell counts of host CD3-positive T cells cultured in an MLR. All data represent the mean ± SD. [Figure 8E] Schematic, graph, and bar graph showing that SERPINB9 expression protects transplanted CD30.CAR (IgG1 spacer) T cells from T cell-mediated immune rejection in vitro. (8A) Schematic representation of the mixed lymphocyte reaction (MLR) assay and the constructs used. GFP-transduced (negative control) or serpin-GFP-transduced TCR knockout (KO) transplanted T cells from two different donors were mixed at a 1:1–2 cell ratio with CD30 KO alloreactive T cells from an HLA-mismatched host stimulated to recognize and kill the transplanted cells (CD30 KO alloreactive T cell mixed lymphocyte assay), and cell numbers were determined over a 3-day period. The assay was performed in the presence of both 10 ng / mL IL-7 and IL-15. (8B) Cell counts of transplanted T cells cultured alone are shown. (8C) Cell counts of transplanted T cells cultured in an MLR are shown. (8D) Cell counts of transplanted T cells after 3 days of culture in an MLR are shown. (8E) Cell counts of host CD3-positive T cells cultured in an MLR. All data represent the mean ± SD. [Figure 9A] Schematic, graph, and bar graph showing that overexpression of SB9 (CAS) in CD30.CAR-engrafted T cells does not affect CAR cytotoxic function and is superior in protecting engrafted cells from allogeneic rejection. (9A) Graph showing CD30.CAR(IgG1 spacer) ATC-mediated cell lysis of KM-H2 cells measured using the xCELLigence system. (9B) Schematic showing the in vitro coculture setup for 9C–9F. Engrafted TCRαβKO CD30.CAR(IgG1 spacer) ATCs were mixed at a 1:1 ratio with host CD30KO ATCs stimulated to recognize and kill engrafted cells. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (9C, 9D, and 9E) Graphs showing cell counts from representative graft-host pairs: (C) engrafted T cells expanding in monoculture, (D) engrafted T cells in coculture, and (E) host T cells in coculture. (9F)

[0031]

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[0032] (9G) Schematic diagram showing the in vitro co-culture setup for 9H-9K, showing the co-culture of transplanted CD30.CAR(4-1BB spacer)EBVST with host CD30 stimulated to recognize and kill transplanted cells. KOATC was mixed at a ratio of 1:1 to 4. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (9H, 9I, and 9J) Graphs showing cell counts from representative graft-host pairs: (H) transplanted T cells expanding in monoculture, (I) transplanted T cells in coculture, and (J) host T cells in coculture. (9K) Bar graph showing host-mediated graft killing. Each point represents a unique graft-host pair, and the bar represents the median (n = 4). The minimum graft:host ratio that resulted in 80% or greater host-mediated graft killing of CD30.CAR(4-1BB spacer) EBVST on day 4 was selected. P values ​​were determined by one-way ANOVA and Tukey's post-hoc test. *, P = 0.0150; **, P < 0.01. [Figure 9B] Schematic, graph, and bar graph showing that overexpression of SB9 (CAS) in CD30.CAR-engrafted T cells does not affect CAR cytotoxic function and is superior in protecting engrafted cells from allogeneic rejection. (9A) Graph showing CD30.CAR(IgG1 spacer) ATC-mediated cell lysis of KM-H2 cells measured using the xCELLigence system. (9B) Schematic showing the in vitro coculture setup for 9C–9F. Engrafted TCRαβKO CD30.CAR(IgG1 spacer) ATCs were mixed at a 1:1 ratio with host CD30KO ATCs stimulated to recognize and kill engrafted cells. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (9C, 9D, and 9E) Graphs showing cell counts from representative graft-host pairs: (C) engrafted T cells expanding in monoculture, (D) engrafted T cells in coculture, and (E) host T cells in coculture. (9F)

[0033]

number

[0034] (9G) Schematic diagram showing the in vitro co-culture setup for 9H-9K, showing the co-culture of transplanted CD30.CAR(4-1BB spacer)EBVST with host CD30 stimulated to recognize and kill transplanted cells. KO ATC was mixed at a ratio of 1:1 to 4. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (9H, 9I, and 9J) Graphs showing cell counts from representative graft-host pairs: (H) transplanted T cells expanding in monoculture, (I) transplanted T cells in coculture, and (J) host T cells in coculture. (9K) Bar graph showing host-mediated graft killing. Each point represents a unique graft-host pair, and the bar represents the median (n = 4). The minimum graft:host ratio that resulted in 80% or greater host-mediated graft killing of CD30.CAR(4-1BB spacer) EBVST on day 4 was selected. P values ​​were determined by one-way ANOVA and Tukey's post-hoc test. *, P = 0.0150; **, P < 0.01. [Figure 9C]Schematic, graph, and bar graph showing that overexpression of SB9 (CAS) in CD30.CAR-engrafted T cells does not affect CAR cytotoxic function and is superior in protecting engrafted cells from allogeneic rejection. (9A) Graph showing CD30.CAR(IgG1 spacer) ATC-mediated cell lysis of KM-H2 cells measured using the xCELLigence system. (9B) Schematic showing the in vitro coculture setup for 9C–9F. Engrafted TCRαβKO CD30.CAR(IgG1 spacer) ATCs were mixed at a 1:1 ratio with host CD30KO ATCs stimulated to recognize and kill engrafted cells. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (9C, 9D, and 9E) Graphs showing cell counts from representative graft-host pairs: (C) engrafted T cells expanding in monoculture, (D) engrafted T cells in coculture, and (E) host T cells in coculture. (9F)

[0035]

number

[0036] (9G) Schematic diagram showing the in vitro co-culture setup for 9H-9K, showing the co-culture of transplanted CD30.CAR(4-1BB spacer)EBVST with host CD30 stimulated to recognize and kill transplanted cells. KOATC was mixed at a ratio of 1:1 to 4. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (9H, 9I, and 9J) Graphs showing cell counts from representative graft-host pairs: (H) transplanted T cells expanding in monoculture, (I) transplanted T cells in coculture, and (J) host T cells in coculture. (9K) Bar graph showing host-mediated graft killing. Each point represents a unique graft-host pair, and the bar represents the median (n = 4). The minimum graft:host ratio that resulted in 80% or greater host-mediated graft killing of CD30.CAR(4-1BB spacer) EBVST on day 4 was selected. P values ​​were determined by one-way ANOVA and Tukey's post-hoc test. *, P = 0.0150; **, P < 0.01. [Figure 9D] Schematic, graph, and bar graph showing that overexpression of SB9 (CAS) in CD30.CAR-engrafted T cells does not affect CAR cytotoxic function and is superior in protecting engrafted cells from allogeneic rejection. (9A) Graph showing CD30.CAR(IgG1 spacer) ATC-mediated cell lysis of KM-H2 cells measured using the xCELLigence system. (9B) Schematic showing the in vitro coculture setup for 9C–9F. Engrafted TCRαβKO CD30.CAR(IgG1 spacer) ATCs were mixed at a 1:1 ratio with host CD30KO ATCs stimulated to recognize and kill engrafted cells. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (9C, 9D, and 9E) Graphs showing cell counts from representative graft-host pairs: (C) engrafted T cells expanding in monoculture, (D) engrafted T cells in coculture, and (E) host T cells in coculture. (9F)

[0037]

number

[0038] (9G) Schematic diagram showing the in vitro co-culture setup for 9H-9K, showing the co-culture of transplanted CD30.CAR(4-1BB spacer)EBVST with host CD30 stimulated to recognize and kill transplanted cells. KO ATC was mixed at a ratio of 1:1 to 4. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (9H, 9I, and 9J) Graphs showing cell counts from representative graft-host pairs: (H) transplanted T cells expanding in monoculture, (I) transplanted T cells in coculture, and (J) host T cells in coculture. (9K) Bar graph showing host-mediated graft killing. Each point represents a unique graft-host pair, and the bar represents the median (n = 4). The minimum graft:host ratio that resulted in 80% or greater host-mediated graft killing of CD30.CAR(4-1BB spacer) EBVST on day 4 was selected. P values ​​were determined by one-way ANOVA and Tukey's post-hoc test. *, P = 0.0150; **, P < 0.01. [Figure 9E]Schematic, graph, and bar graph showing that overexpression of SB9 (CAS) in CD30.CAR-engrafted T cells does not affect CAR cytotoxic function and is superior in protecting engrafted cells from allogeneic rejection. (9A) Graph showing CD30.CAR(IgG1 spacer) ATC-mediated cell lysis of KM-H2 cells measured using the xCELLigence system. (9B) Schematic showing the in vitro coculture setup for 9C–9F. Engrafted TCRαβKO CD30.CAR(IgG1 spacer) ATCs were mixed at a 1:1 ratio with host CD30KO ATCs stimulated to recognize and kill engrafted cells. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (9C, 9D, and 9E) Graphs showing cell counts from representative graft-host pairs: (C) engrafted T cells expanding in monoculture, (D) engrafted T cells in coculture, and (E) host T cells in coculture. (9F)

[0039]

number

[0040] (9G) Schematic diagram showing the in vitro co-culture setup for 9H-9K, showing the co-culture of transplanted CD30.CAR(4-1BB spacer)EBVST with host CD30 stimulated to recognize and kill transplanted cells. KOATC was mixed at a ratio of 1:1 to 4. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (9H, 9I, and 9J) Graphs showing cell counts from representative graft-host pairs: (H) transplanted T cells expanding in monoculture, (I) transplanted T cells in coculture, and (J) host T cells in coculture. (9K) Bar graph showing host-mediated graft killing. Each point represents a unique graft-host pair, and the bar represents the median (n = 4). The minimum graft:host ratio that resulted in 80% or greater host-mediated graft killing of CD30.CAR(4-1BB spacer) EBVST on day 4 was selected. P values ​​were determined by one-way ANOVA and Tukey's post-hoc test. *, P = 0.0150; **, P < 0.01. [Figure 9F] Schematic, graph, and bar graph showing that overexpression of SB9 (CAS) in CD30.CAR-engrafted T cells does not affect CAR cytotoxic function and is superior in protecting engrafted cells from allogeneic rejection. (9A) Graph showing CD30.CAR(IgG1 spacer) ATC-mediated cell lysis of KM-H2 cells measured using the xCELLigence system. (9B) Schematic showing the in vitro coculture setup for 9C–9F. Engrafted TCRαβKO CD30.CAR(IgG1 spacer) ATCs were mixed at a 1:1 ratio with host CD30KO ATCs stimulated to recognize and kill engrafted cells. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (9C, 9D, and 9E) Graphs showing cell counts from representative graft-host pairs: (C) engrafted T cells expanding in monoculture, (D) engrafted T cells in coculture, and (E) host T cells in coculture. (9F)

[0041]

number

[0042] (9G) Schematic diagram showing the in vitro co-culture setup for 9H-9K, showing the co-culture of transplanted CD30.CAR(4-1BB spacer)EBVST with host CD30 stimulated to recognize and kill transplanted cells. KO ATC was mixed at a ratio of 1:1 to 4. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (9H, 9I, and 9J) Graphs showing cell counts from representative graft-host pairs: (H) transplanted T cells expanding in monoculture, (I) transplanted T cells in coculture, and (J) host T cells in coculture. (9K) Bar graph showing host-mediated graft killing. Each point represents a unique graft-host pair, and the bar represents the median (n = 4). The minimum graft:host ratio that resulted in 80% or greater host-mediated graft killing of CD30.CAR(4-1BB spacer) EBVST on day 4 was selected. P values ​​were determined by one-way ANOVA and Tukey's post-hoc test. *, P = 0.0150; **, P < 0.01. [Figure 9G]Schematic, graph, and bar graph showing that overexpression of SB9 (CAS) in CD30.CAR-engrafted T cells does not affect CAR cytotoxic function and is superior in protecting engrafted cells from allogeneic rejection. (9A) Graph showing CD30.CAR(IgG1 spacer) ATC-mediated cell lysis of KM-H2 cells measured using the xCELLigence system. (9B) Schematic showing the in vitro coculture setup for 9C–9F. Engrafted TCRαβKO CD30.CAR(IgG1 spacer) ATCs were mixed at a 1:1 ratio with host CD30KO ATCs stimulated to recognize and kill engrafted cells. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (9C, 9D, and 9E) Graphs showing cell counts from representative graft-host pairs: (C) engrafted T cells expanding in monoculture, (D) engrafted T cells in coculture, and (E) host T cells in coculture. (9F)

[0043]

number

[0044] (9G) Schematic diagram showing the in vitro co-culture setup for 9H-9K, showing the co-culture of transplanted CD30.CAR(4-1BB spacer)EBVST with host CD30 stimulated to recognize and kill transplanted cells. KOATC was mixed at a ratio of 1:1 to 4. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (9H, 9I, and 9J) Graphs showing cell counts from representative graft-host pairs: (H) transplanted T cells expanding in monoculture, (I) transplanted T cells in coculture, and (J) host T cells in coculture. (9K) Bar graph showing host-mediated graft killing. Each point represents a unique graft-host pair, and the bar represents the median (n = 4). The minimum graft:host ratio that resulted in 80% or greater host-mediated graft killing of CD30.CAR(4-1BB spacer) EBVST on day 4 was selected. P values ​​were determined by one-way ANOVA and Tukey's post-hoc test. *, P = 0.0150; **, P < 0.01. [Figure 9H] Schematic, graph, and bar graph showing that overexpression of SB9 (CAS) in CD30.CAR-engrafted T cells does not affect CAR cytotoxic function and is superior in protecting engrafted cells from allogeneic rejection. (9A) Graph showing CD30.CAR(IgG1 spacer) ATC-mediated cell lysis of KM-H2 cells measured using the xCELLigence system. (9B) Schematic showing the in vitro coculture setup for 9C–9F. Engrafted TCRαβKO CD30.CAR(IgG1 spacer) ATCs were mixed at a 1:1 ratio with host CD30KO ATCs stimulated to recognize and kill engrafted cells. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (9C, 9D, and 9E) Graphs showing cell counts from representative graft-host pairs: (C) engrafted T cells expanding in monoculture, (D) engrafted T cells in coculture, and (E) host T cells in coculture. (9F)

[0045]

number

[0046] (9G) Schematic diagram showing the in vitro co-culture setup for 9H-9K, showing the co-culture of transplanted CD30.CAR(4-1BB spacer)EBVST with host CD30 stimulated to recognize and kill transplanted cells. KO ATC was mixed at a ratio of 1:1 to 4. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (9H, 9I, and 9J) Graphs showing cell counts from representative graft-host pairs: (H) transplanted T cells expanding in monoculture, (I) transplanted T cells in coculture, and (J) host T cells in coculture. (9K) Bar graph showing host-mediated graft killing. Each point represents a unique graft-host pair, and the bar represents the median (n = 4). The minimum graft:host ratio that resulted in 80% or greater host-mediated graft killing of CD30.CAR(4-1BB spacer) EBVST on day 4 was selected. P values ​​were determined by one-way ANOVA and Tukey's post-hoc test. *, P = 0.0150; **, P < 0.01. [Figure 9I]Schematic, graph, and bar graph showing that overexpression of SB9 (CAS) in CD30.CAR-engrafted T cells does not affect CAR cytotoxic function and is superior in protecting engrafted cells from allogeneic rejection. (9A) Graph showing CD30.CAR(IgG1 spacer) ATC-mediated cell lysis of KM-H2 cells measured using the xCELLigence system. (9B) Schematic showing the in vitro coculture setup for 9C–9F. Engrafted TCRαβKO CD30.CAR(IgG1 spacer) ATCs were mixed at a 1:1 ratio with host CD30KO ATCs stimulated to recognize and kill engrafted cells. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (9C, 9D, and 9E) Graphs showing cell counts from representative graft-host pairs: (C) engrafted T cells expanding in monoculture, (D) engrafted T cells in coculture, and (E) host T cells in coculture. (9F)

[0047]

number

[0048] (9G) Schematic diagram showing the in vitro co-culture setup for 9H-9K, showing the co-culture of transplanted CD30.CAR(4-1BB spacer)EBVST with host CD30 stimulated to recognize and kill transplanted cells. KOATC was mixed at a ratio of 1:1 to 4. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (9H, 9I, and 9J) Graphs showing cell counts from representative graft-host pairs: (H) transplanted T cells expanding in monoculture, (I) transplanted T cells in coculture, and (J) host T cells in coculture. (9K) Bar graph showing host-mediated graft killing. Each point represents a unique graft-host pair, and the bar represents the median (n = 4). The minimum graft:host ratio that resulted in 80% or greater host-mediated graft killing of CD30.CAR(4-1BB spacer) EBVST on day 4 was selected. P values ​​were determined by one-way ANOVA and Tukey's post-hoc test. *, P = 0.0150; **, P < 0.01. [Figure 9J] Schematic, graph, and bar graph showing that overexpression of SB9 (CAS) in CD30.CAR-engrafted T cells does not affect CAR cytotoxic function and is superior in protecting engrafted cells from allogeneic rejection. (9A) Graph showing CD30.CAR(IgG1 spacer) ATC-mediated cell lysis of KM-H2 cells measured using the xCELLigence system. (9B) Schematic showing the in vitro coculture setup for 9C–9F. Engrafted TCRαβKO CD30.CAR(IgG1 spacer) ATCs were mixed at a 1:1 ratio with host CD30KO ATCs stimulated to recognize and kill engrafted cells. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (9C, 9D, and 9E) Graphs showing cell counts from representative graft-host pairs: (C) engrafted T cells expanding in monoculture, (D) engrafted T cells in coculture, and (E) host T cells in coculture. (9F)

[0049]

number

[0050] (9G) Schematic diagram showing the in vitro co-culture setup for 9H-9K, showing the co-culture of transplanted CD30.CAR(4-1BB spacer)EBVST with host CD30 stimulated to recognize and kill transplanted cells. KO ATC was mixed at a ratio of 1:1 to 4. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (9H, 9I, and 9J) Graphs showing cell counts from representative graft-host pairs: (H) transplanted T cells expanding in monoculture, (I) transplanted T cells in coculture, and (J) host T cells in coculture. (9K) Bar graph showing host-mediated graft killing. Each point represents a unique graft-host pair, and the bar represents the median (n = 4). The minimum graft:host ratio that resulted in 80% or greater host-mediated graft killing of CD30.CAR(4-1BB spacer) EBVST on day 4 was selected. P values ​​were determined by one-way ANOVA and Tukey's post-hoc test. *, P = 0.0150; **, P < 0.01. [Figure 9K]Schematic, graph, and bar graph showing that overexpression of SB9 (CAS) in CD30.CAR-engrafted T cells does not affect CAR cytotoxic function and is superior in protecting engrafted cells from allogeneic rejection. (9A) Graph showing CD30.CAR(IgG1 spacer) ATC-mediated cell lysis of KM-H2 cells measured using the xCELLigence system. (9B) Schematic showing the in vitro coculture setup for 9C–9F. Engrafted TCRαβKO CD30.CAR(IgG1 spacer) ATCs were mixed at a 1:1 ratio with host CD30KO ATCs stimulated to recognize and kill engrafted cells. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (9C, 9D, and 9E) Graphs showing cell counts from representative graft-host pairs: (C) engrafted T cells expanding in monoculture, (D) engrafted T cells in coculture, and (E) host T cells in coculture. (9F)

[0051]

number

[0052] (9G) Schematic diagram showing the in vitro co-culture setup for 9H-9K, showing the co-culture of transplanted CD30.CAR(4-1BB spacer)EBVST with host CD30 stimulated to recognize and kill transplanted cells. KOATC was mixed at a ratio of 1:1 to 4. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (9H, 9I, and 9J) Graphs showing cell counts from representative graft-host pairs: (H) transplanted T cells expanding in monoculture, (I) transplanted T cells in coculture, and (J) host T cells in coculture. (9K) Bar graph showing host-mediated graft killing. Each point represents a unique graft-host pair, and the bar represents the median (n = 4). The minimum graft:host ratio that resulted in 80% or greater host-mediated graft killing of CD30.CAR(4-1BB spacer) EBVST on day 4 was selected. P values ​​were determined by one-way ANOVA and Tukey's post-hoc test. *, P = 0.0150; **, P < 0.01. [Figure 10A] 10A-10D are schematics, bar graphs, and graphs showing the production and characterization of TCRαβKOCD30.CAR (IgG1 spacer) ATCs expressing various forms of SB9. (10A) Schematic showing the production timeline for the generation of TCRαβKOCD30.CAR ATCs expressing various forms of SB9 when transduced with a retrovirus encoding a bicistronic construct (top panel) or retroviruses encoding CAR and SB9 separately (bottom panel). (10B) Bar graphs showing SB9 expression as measured by intracellular staining and subsequent flow cytometry analysis. (10C and 10D) Bar graphs showing CD30.CAR expression on CD3-positive T cells as (C) percentages and (D) geometric mean fluorescence intensity (MFI). (10E) Graphs showing TCRαβ expression on CD3 wild-type (WT) T cells or T cells after TCRαβKO gene modification using Crispr gene editing, as measured by flow cytometry analysis. [Figure 10B]10A-10D are schematics, bar graphs, and graphs showing the production and characterization of TCRαβKOCD30.CAR (IgG1 spacer) ATCs expressing various forms of SB9. (10A) Schematic showing the production timeline for the generation of TCRαβKOCD30.CAR ATCs expressing various forms of SB9 when transduced with a retrovirus encoding a bicistronic construct (top panel) or retroviruses encoding CAR and SB9 separately (bottom panel). (10B) Bar graphs showing SB9 expression as measured by intracellular staining and subsequent flow cytometry analysis. (10C and 10D) Bar graphs showing CD30.CAR expression on CD3-positive T cells as (C) percentages and (D) geometric mean fluorescence intensity (MFI). (10E) Graphs showing TCRαβ expression on CD3 wild-type (WT) T cells or T cells after TCRαβKO gene modification using Crispr gene editing, as measured by flow cytometry analysis. [Figure 10C] 10A-10D are schematics, bar graphs, and graphs showing the production and characterization of TCRαβKOCD30.CAR (IgG1 spacer) ATCs expressing various forms of SB9. (10A) Schematic showing the production timeline for the generation of TCRαβKOCD30.CAR ATCs expressing various forms of SB9 when transduced with a retrovirus encoding a bicistronic construct (top panel) or retroviruses encoding CAR and SB9 separately (bottom panel). (10B) Bar graphs showing SB9 expression as measured by intracellular staining and subsequent flow cytometry analysis. (10C and 10D) Bar graphs showing CD30.CAR expression on CD3-positive T cells as (C) percentages and (D) geometric mean fluorescence intensity (MFI). (10E) Graphs showing TCRαβ expression on CD3 wild-type (WT) T cells or T cells after TCRαβKO gene modification using Crispr gene editing, as measured by flow cytometry analysis. [Figure 10D]10A-10D are schematics, bar graphs, and graphs showing the production and characterization of TCRαβKOCD30.CAR (IgG1 spacer) ATCs expressing various forms of SB9. (10A) Schematic showing the production timeline for the generation of TCRαβKOCD30.CAR ATCs expressing various forms of SB9 when transduced with a retrovirus encoding a bicistronic construct (top panel) or retroviruses encoding CAR and SB9 separately (bottom panel). (10B) Bar graphs showing SB9 expression as measured by intracellular staining and subsequent flow cytometry analysis. (10C and 10D) Bar graphs showing CD30.CAR expression on CD3-positive T cells as (C) percentages and (D) geometric mean fluorescence intensity (MFI). (10E) Graphs showing TCRαβ expression on CD3 wild-type (WT) T cells or T cells after TCRαβKO gene modification using Crispr gene editing, as measured by flow cytometry analysis. [Figure 10E] 10A-10D are schematics, bar graphs, and graphs showing the production and characterization of TCRαβKOCD30.CAR (IgG1 spacer) ATCs expressing various forms of SB9. (10A) Schematic showing the production timeline for the generation of TCRαβKOCD30.CAR ATCs expressing various forms of SB9 when transduced with a retrovirus encoding a bicistronic construct (top panel) or retroviruses encoding CAR and SB9 separately (bottom panel). (10B) Bar graphs showing SB9 expression as measured by intracellular staining and subsequent flow cytometry analysis. (10C and 10D) Bar graphs showing CD30.CAR expression on CD3-positive T cells as (C) percentages and (D) geometric mean fluorescence intensity (MFI). (10E) Graphs showing TCRαβ expression on CD3 wild-type (WT) T cells or T cells after TCRαβKO gene modification using Crispr gene editing, as measured by flow cytometry analysis. [Figure 11A]11A-11B are schematics and graphs showing the production of alloreactive CD30KO host cells (allo-T) that recognize and kill transplanted cells. (11A) Schematic showing the production timeline for the generation of allo-T. (11B) Graph showing CD30 expression on allo-T cells as determined by staining with two different antibody clones, BY88 and BerH8, and flow cytometry analysis. [Figure 11B] 11A-11B are schematics and graphs showing the production of alloreactive CD30KO host cells (allo-T) that recognize and kill transplanted cells. (11A) Schematic showing the production timeline for the generation of allo-T. (11B) Graph showing CD30 expression on allo-T cells as determined by staining with two different antibody clones, BY88 and BerH8, and flow cytometry analysis. [Figure 12A]Schematic, SDS-PAGE, and bar graphs depicting the production and characterization of CD30.CAR (4-1BB spacer) EBVST expressing various forms of SB9. (12A) Schematic depicting the production timeline for the generation of CD30.CAR EBVST expressing various forms of SB9. (12B) SDS-PAGE analysis showing SB9 expression from a representative donor. The upper band (approximately 62 kDa) represents the preformed GzmB and SB9 complex in the cytoplasm, while the lower band (approximately 43 kDa) represents functional SB9 monomer. Transduction was performed with either low (10 ng / mL) or high (100 ng / mL) IL-15 concentrations, indicated by L and H, respectively. (12C) Bar graph depicting densitometric analysis of SB9 monomer normalized to the CD30.CAR EBVST control and calnexin as a loading control. (12D and 12E) Bar graphs showing CD30.CAR expression on CD3-positive T cells in (D) percentage and (E) geometric mean fluorescence intensity (MFI). n=4 donors. (12F) Bar graphs showing the percentage of CD3-positive T cells producing IFNγ and / or TNFα in response to EBV peptides, analyzed by intracellular cytokine staining and flow cytometry. n=4 donors. (12G, 12H, 12I, 12J, and 12K) Bar graphs showing flow cytometry analysis of (G) CD4 / CD8 subsets, (H) memory T cell subsets (CM: central memory, EM: effector memory, and TEMRA: T effector memory expressing CD45RA), (I) PD-1, Tim-3, Lag-3 expression, (J) CD39 expression, and (K) CD25 expression of CD30.CAR EBVST from a representative donor. [Figure 12B]Schematic, SDS-PAGE, and bar graphs depicting the production and characterization of CD30.CAR (4-1BB spacer) EBVST expressing various forms of SB9. (12A) Schematic depicting the production timeline for the generation of CD30.CAR EBVST expressing various forms of SB9. (12B) SDS-PAGE analysis showing SB9 expression from a representative donor. The upper band (approximately 62 kDa) represents the preformed GzmB and SB9 complex in the cytoplasm, while the lower band (approximately 43 kDa) represents functional SB9 monomer. Transduction was performed with either low (10 ng / mL) or high (100 ng / mL) IL-15 concentrations, indicated by L and H, respectively. (12C) Bar graph depicting densitometric analysis of SB9 monomer normalized to the CD30.CAR EBVST control and calnexin as a loading control. (12D and 12E) Bar graphs showing CD30.CAR expression on CD3-positive T cells in (D) percentage and (E) geometric mean fluorescence intensity (MFI). n=4 donors. (12F) Bar graphs showing the percentage of CD3-positive T cells producing IFNγ and / or TNFα in response to EBV peptides, analyzed by intracellular cytokine staining and flow cytometry. n=4 donors. (12G, 12H, 12I, 12J, and 12K) Bar graphs showing flow cytometry analysis of (G) CD4 / CD8 subsets, (H) memory T cell subsets (CM: central memory, EM: effector memory, and TEMRA: T effector memory expressing CD45RA), (I) PD-1, Tim-3, Lag-3 expression, (J) CD39 expression, and (K) CD25 expression of CD30.CAR EBVST from a representative donor. [Figure 12C]Schematic, SDS-PAGE, and bar graphs depicting the production and characterization of CD30.CAR (4-1BB spacer) EBVST expressing various forms of SB9. (12A) Schematic depicting the production timeline for the generation of CD30.CAR EBVST expressing various forms of SB9. (12B) SDS-PAGE analysis showing SB9 expression from a representative donor. The upper band (approximately 62 kDa) represents the preformed GzmB and SB9 complex in the cytoplasm, while the lower band (approximately 43 kDa) represents functional SB9 monomer. Transduction was performed with either low (10 ng / mL) or high (100 ng / mL) IL-15 concentrations, indicated by L and H, respectively. (12C) Bar graph depicting densitometric analysis of SB9 monomer normalized to the CD30.CAR EBVST control and calnexin as a loading control. (12D and 12E) Bar graphs showing CD30.CAR expression on CD3-positive T cells in (D) percentage and (E) geometric mean fluorescence intensity (MFI). n=4 donors. (12F) Bar graphs showing the percentage of CD3-positive T cells producing IFNγ and / or TNFα in response to EBV peptides, analyzed by intracellular cytokine staining and flow cytometry. n=4 donors. (12G, 12H, 12I, 12J, and 12K) Bar graphs showing flow cytometry analysis of (G) CD4 / CD8 subsets, (H) memory T cell subsets (CM: central memory, EM: effector memory, and TEMRA: T effector memory expressing CD45RA), (I) PD-1, Tim-3, Lag-3 expression, (J) CD39 expression, and (K) CD25 expression of CD30.CAR EBVST from a representative donor. [Figure 12D]Schematic, SDS-PAGE, and bar graphs depicting the production and characterization of CD30.CAR (4-1BB spacer) EBVST expressing various forms of SB9. (12A) Schematic depicting the production timeline for the generation of CD30.CAR EBVST expressing various forms of SB9. (12B) SDS-PAGE analysis showing SB9 expression from a representative donor. The upper band (approximately 62 kDa) represents the preformed GzmB and SB9 complex in the cytoplasm, while the lower band (approximately 43 kDa) represents functional SB9 monomer. Transduction was performed with either low (10 ng / mL) or high (100 ng / mL) IL-15 concentrations, indicated by L and H, respectively. (12C) Bar graph depicting densitometric analysis of SB9 monomer normalized to the CD30.CAR EBVST control and calnexin as a loading control. (12D and 12E) Bar graphs showing CD30.CAR expression on CD3-positive T cells in (D) percentage and (E) geometric mean fluorescence intensity (MFI). n=4 donors. (12F) Bar graphs showing the percentage of CD3-positive T cells producing IFNγ and / or TNFα in response to EBV peptides, analyzed by intracellular cytokine staining and flow cytometry. n=4 donors. (12G, 12H, 12I, 12J, and 12K) Bar graphs showing flow cytometry analysis of (G) CD4 / CD8 subsets, (H) memory T cell subsets (CM: central memory, EM: effector memory, and TEMRA: T effector memory expressing CD45RA), (I) PD-1, Tim-3, Lag-3 expression, (J) CD39 expression, and (K) CD25 expression of CD30.CAR EBVST from a representative donor. [Figure 12E]Schematic, SDS-PAGE, and bar graphs depicting the production and characterization of CD30.CAR (4-1BB spacer) EBVST expressing various forms of SB9. (12A) Schematic depicting the production timeline for the generation of CD30.CAR EBVST expressing various forms of SB9. (12B) SDS-PAGE analysis showing SB9 expression from a representative donor. The upper band (approximately 62 kDa) represents the preformed GzmB and SB9 complex in the cytoplasm, while the lower band (approximately 43 kDa) represents functional SB9 monomer. Transduction was performed with either low (10 ng / mL) or high (100 ng / mL) IL-15 concentrations, indicated by L and H, respectively. (12C) Bar graph depicting densitometric analysis of SB9 monomer normalized to the CD30.CAR EBVST control and calnexin as a loading control. (12D and 12E) Bar graphs showing CD30.CAR expression on CD3-positive T cells in (D) percentage and (E) geometric mean fluorescence intensity (MFI). n=4 donors. (12F) Bar graphs showing the percentage of CD3-positive T cells producing IFNγ and / or TNFα in response to EBV peptides, analyzed by intracellular cytokine staining and flow cytometry. n=4 donors. (12G, 12H, 12I, 12J, and 12K) Bar graphs showing flow cytometry analysis of (G) CD4 / CD8 subsets, (H) memory T cell subsets (CM: central memory, EM: effector memory, and TEMRA: T effector memory expressing CD45RA), (I) PD-1, Tim-3, Lag-3 expression, (J) CD39 expression, and (K) CD25 expression of CD30.CAR EBVST from a representative donor. [Figure 12F]Schematic, SDS-PAGE, and bar graphs depicting the production and characterization of CD30.CAR (4-1BB spacer) EBVST expressing various forms of SB9. (12A) Schematic depicting the production timeline for the generation of CD30.CAR EBVST expressing various forms of SB9. (12B) SDS-PAGE analysis showing SB9 expression from a representative donor. The upper band (approximately 62 kDa) represents the preformed GzmB and SB9 complex in the cytoplasm, while the lower band (approximately 43 kDa) represents functional SB9 monomer. Transduction was performed with either low (10 ng / mL) or high (100 ng / mL) IL-15 concentrations, indicated by L and H, respectively. (12C) Bar graph depicting densitometric analysis of SB9 monomer normalized to the CD30.CAR EBVST control and calnexin as a loading control. (12D and 12E) Bar graphs showing CD30.CAR expression on CD3-positive T cells in (D) percentage and (E) geometric mean fluorescence intensity (MFI). n=4 donors. (12F) Bar graphs showing the percentage of CD3-positive T cells producing IFNγ and / or TNFα in response to EBV peptides, analyzed by intracellular cytokine staining and flow cytometry. n=4 donors. (12G, 12H, 12I, 12J, and 12K) Bar graphs showing flow cytometry analysis of (G) CD4 / CD8 subsets, (H) memory T cell subsets (CM: central memory, EM: effector memory, and TEMRA: T effector memory expressing CD45RA), (I) PD-1, Tim-3, Lag-3 expression, (J) CD39 expression, and (K) CD25 expression of CD30.CAR EBVST from a representative donor. [Figure 12G]Schematic, SDS-PAGE, and bar graphs depicting the production and characterization of CD30.CAR (4-1BB spacer) EBVST expressing various forms of SB9. (12A) Schematic depicting the production timeline for the generation of CD30.CAR EBVST expressing various forms of SB9. (12B) SDS-PAGE analysis showing SB9 expression from a representative donor. The upper band (approximately 62 kDa) represents the preformed GzmB and SB9 complex in the cytoplasm, while the lower band (approximately 43 kDa) represents functional SB9 monomer. Transduction was performed with either low (10 ng / mL) or high (100 ng / mL) IL-15 concentrations, indicated by L and H, respectively. (12C) Bar graph depicting densitometric analysis of SB9 monomer normalized to the CD30.CAR EBVST control and calnexin as a loading control. (12D and 12E) Bar graphs showing CD30.CAR expression on CD3-positive T cells in (D) percentage and (E) geometric mean fluorescence intensity (MFI). n=4 donors. (12F) Bar graphs showing the percentage of CD3-positive T cells producing IFNγ and / or TNFα in response to EBV peptides, analyzed by intracellular cytokine staining and flow cytometry. n=4 donors. (12G, 12H, 12I, 12J, and 12K) Bar graphs showing flow cytometry analysis of (G) CD4 / CD8 subsets, (H) memory T cell subsets (CM: central memory, EM: effector memory, and TEMRA: T effector memory expressing CD45RA), (I) PD-1, Tim-3, Lag-3 expression, (J) CD39 expression, and (K) CD25 expression of CD30.CAR EBVST from a representative donor. [Figure 12H]Schematic, SDS-PAGE, and bar graphs depicting the production and characterization of CD30.CAR (4-1BB spacer) EBVST expressing various forms of SB9. (12A) Schematic depicting the production timeline for the generation of CD30.CAR EBVST expressing various forms of SB9. (12B) SDS-PAGE analysis showing SB9 expression from a representative donor. The upper band (approximately 62 kDa) represents the preformed GzmB and SB9 complex in the cytoplasm, while the lower band (approximately 43 kDa) represents functional SB9 monomer. Transduction was performed with either low (10 ng / mL) or high (100 ng / mL) IL-15 concentrations, indicated by L and H, respectively. (12C) Bar graph depicting densitometric analysis of SB9 monomer normalized to the CD30.CAR EBVST control and calnexin as a loading control. (12D and 12E) Bar graphs showing CD30.CAR expression on CD3-positive T cells in (D) percentage and (E) geometric mean fluorescence intensity (MFI). n=4 donors. (12F) Bar graphs showing the percentage of CD3-positive T cells producing IFNγ and / or TNFα in response to EBV peptides, analyzed by intracellular cytokine staining and flow cytometry. n=4 donors. (12G, 12H, 12I, 12J, and 12K) Bar graphs showing flow cytometry analysis of (G) CD4 / CD8 subsets, (H) memory T cell subsets (CM: central memory, EM: effector memory, and TEMRA: T effector memory expressing CD45RA), (I) PD-1, Tim-3, Lag-3 expression, (J) CD39 expression, and (K) CD25 expression of CD30.CAR EBVST from a representative donor. [Figure 12I]Schematic, SDS-PAGE, and bar graphs depicting the production and characterization of CD30.CAR (4-1BB spacer) EBVST expressing various forms of SB9. (12A) Schematic depicting the production timeline for the generation of CD30.CAR EBVST expressing various forms of SB9. (12B) SDS-PAGE analysis showing SB9 expression from a representative donor. The upper band (approximately 62 kDa) represents the preformed GzmB and SB9 complex in the cytoplasm, while the lower band (approximately 43 kDa) represents functional SB9 monomer. Transduction was performed with either low (10 ng / mL) or high (100 ng / mL) IL-15 concentrations, indicated by L and H, respectively. (12C) Bar graph depicting densitometric analysis of SB9 monomer normalized to the CD30.CAR EBVST control and calnexin as a loading control. (12D and 12E) Bar graphs showing CD30.CAR expression on CD3-positive T cells in (D) percentage and (E) geometric mean fluorescence intensity (MFI). n=4 donors. (12F) Bar graphs showing the percentage of CD3-positive T cells producing IFNγ and / or TNFα in response to EBV peptides, analyzed by intracellular cytokine staining and flow cytometry. n=4 donors. (12G, 12H, 12I, 12J, and 12K) Bar graphs showing flow cytometry analysis of (G) CD4 / CD8 subsets, (H) memory T cell subsets (CM: central memory, EM: effector memory, and TEMRA: T effector memory expressing CD45RA), (I) PD-1, Tim-3, Lag-3 expression, (J) CD39 expression, and (K) CD25 expression of CD30.CAR EBVST from a representative donor. [Figure 12J]Schematic, SDS-PAGE, and bar graphs depicting the production and characterization of CD30.CAR (4-1BB spacer) EBVST expressing various forms of SB9. (12A) Schematic depicting the production timeline for the generation of CD30.CAR EBVST expressing various forms of SB9. (12B) SDS-PAGE analysis showing SB9 expression from a representative donor. The upper band (approximately 62 kDa) represents the preformed GzmB and SB9 complex in the cytoplasm, while the lower band (approximately 43 kDa) represents functional SB9 monomer. Transduction was performed with either low (10 ng / mL) or high (100 ng / mL) IL-15 concentrations, indicated by L and H, respectively. (12C) Bar graph depicting densitometric analysis of SB9 monomer normalized to the CD30.CAR EBVST control and calnexin as a loading control. (12D and 12E) Bar graphs showing CD30.CAR expression on CD3-positive T cells in (D) percentage and (E) geometric mean fluorescence intensity (MFI). n=4 donors. (12F) Bar graphs showing the percentage of CD3-positive T cells producing IFNγ and / or TNFα in response to EBV peptides, analyzed by intracellular cytokine staining and flow cytometry. n=4 donors. (12G, 12H, 12I, 12J, and 12K) Bar graphs showing flow cytometry analysis of (G) CD4 / CD8 subsets, (H) memory T cell subsets (CM: central memory, EM: effector memory, and TEMRA: T effector memory expressing CD45RA), (I) PD-1, Tim-3, Lag-3 expression, (J) CD39 expression, and (K) CD25 expression of CD30.CAR EBVST from a representative donor. [Figure 12K]Schematic, SDS-PAGE, and bar graphs depicting the production and characterization of CD30.CAR (4-1BB spacer) EBVST expressing various forms of SB9. (12A) Schematic depicting the production timeline for the generation of CD30.CAR EBVST expressing various forms of SB9. (12B) SDS-PAGE analysis showing SB9 expression from a representative donor. The upper band (approximately 62 kDa) represents the preformed GzmB and SB9 complex in the cytoplasm, while the lower band (approximately 43 kDa) represents functional SB9 monomer. Transduction was performed with either low (10 ng / mL) or high (100 ng / mL) IL-15 concentrations, indicated by L and H, respectively. (12C) Bar graph depicting densitometric analysis of SB9 monomer normalized to the CD30.CAR EBVST control and calnexin as a loading control. (12D and 12E) Bar graphs showing CD30.CAR expression on CD3-positive T cells in (D) percentage and (E) geometric mean fluorescence intensity (MFI). n=4 donors. (12F) Bar graphs showing the percentage of CD3-positive T cells producing IFNγ and / or TNFα in response to EBV peptides, analyzed by intracellular cytokine staining and flow cytometry. n=4 donors. (12G, 12H, 12I, 12J, and 12K) Bar graphs showing flow cytometry analysis of (G) CD4 / CD8 subsets, (H) memory T cell subsets (CM: central memory, EM: effector memory, and TEMRA: T effector memory expressing CD45RA), (I) PD-1, Tim-3, Lag-3 expression, (J) CD39 expression, and (K) CD25 expression of CD30.CAR EBVST from a representative donor. [Figure 13A]Schematic, graph, and bar graph showing that overexpression of SB9 (CAS) in non-CAR-engrafted T cells (ATC and EBVST) is superior in protecting the engrafted cells from allogeneic rejection. (13A) Schematic showing the in vitro co-culture setup for 13B-13E. Engrafted TCRαβKO ATCs were mixed with HLA-mismatched host PBMCs at a ratio of 1:10-20. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (13B, 13C, and 13D) Graphs showing cell counts from representative graft-host pairs: (B) engrafted T cells expanding in monoculture, (C) engrafted T cells in coculture, and (D) host T and NK cells in coculture. (13E)

[0053]

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[0054] (13F) Schematic diagram showing the in vitro co-culture setup for 13G-13J, showing the relationship between transplanted EBVST and alloreactive CD30 cells stimulated to recognize and kill the transplanted cells. KO The host ATCs were mixed at a ratio of 1:1 to 4. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (13G, 13H, and 13I) Graphs showing cell counts from representative graft-host pairs: (G) transplanted T cells growing in monoculture, (H) transplanted T cells in coculture, and (I) host T cells in coculture. (13J)

[0055]

number

[0056] 1 is a bar graph showing host-mediated graft death calculated by (n=2). Each point represents a unique graft-host pair and the bar represents the median. Statistics were not performed because n<3. [Figure 13B] Schematic, graph, and bar graph showing that overexpression of SB9 (CAS) in non-CAR-engrafted T cells (ATC and EBVST) is superior in protecting the engrafted cells from allogeneic rejection. (13A) Schematic showing the in vitro co-culture setup for 13B-13E. Engrafted TCRαβKO ATCs were mixed with HLA-mismatched host PBMCs at a ratio of 1:10-20. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (13B, 13C, and 13D) Graphs showing cell counts from representative graft-host pairs: (B) engrafted T cells expanding in monoculture, (C) engrafted T cells in coculture, and (D) host T and NK cells in coculture. (13E)

[0057]

number

[0058] (13F) Schematic diagram showing the in vitro co-culture setup for 13G-13J, showing the relationship between transplanted EBVST and alloreactive CD30 cells stimulated to recognize and kill the transplanted cells. KO The host ATCs were mixed at a ratio of 1:1 to 4. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (13G, 13H, and 13I) Graphs showing cell counts from representative graft-host pairs: (G) transplanted T cells growing in monoculture, (H) transplanted T cells in coculture, and (I) host T cells in coculture. (13J)

[0059]

number

[0060] 1 is a bar graph showing host-mediated graft death calculated by (n=2). Each point represents a unique graft-host pair and the bar represents the median. Statistics were not performed because n<3. [Figure 13C] Schematic, graph, and bar graph showing that overexpression of SB9 (CAS) in non-CAR-engrafted T cells (ATC and EBVST) is superior in protecting the engrafted cells from allogeneic rejection. (13A) Schematic showing the in vitro co-culture setup for 13B-13E. Engrafted TCRαβKO ATCs were mixed with HLA-mismatched host PBMCs at a ratio of 1:10-20. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (13B, 13C, and 13D) Graphs showing cell counts from representative graft-host pairs: (B) engrafted T cells expanding in monoculture, (C) engrafted T cells in coculture, and (D) host T and NK cells in coculture. (13E)

[0061]

number

[0062] (13F) Schematic diagram showing the in vitro co-culture setup for 13G-13J, showing the relationship between transplanted EBVST and alloreactive CD30 cells stimulated to recognize and kill the transplanted cells. KOThe host ATCs were mixed at a ratio of 1:1 to 4. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (13G, 13H, and 13I) Graphs showing cell counts from representative graft-host pairs: (G) transplanted T cells growing in monoculture, (H) transplanted T cells in coculture, and (I) host T cells in coculture. (13J)

[0063]

number

[0064] 1 is a bar graph showing host-mediated graft death calculated by (n=2). Each point represents a unique graft-host pair and the bar represents the median. Statistics were not performed because n<3. [Figure 13D] Schematic, graph, and bar graph showing that overexpression of SB9 (CAS) in non-CAR-engrafted T cells (ATC and EBVST) is superior in protecting the engrafted cells from allogeneic rejection. (13A) Schematic showing the in vitro co-culture setup for 13B-13E. Engrafted TCRαβKO ATCs were mixed with HLA-mismatched host PBMCs at a ratio of 1:10-20. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (13B, 13C, and 13D) Graphs showing cell counts from representative graft-host pairs: (B) engrafted T cells expanding in monoculture, (C) engrafted T cells in coculture, and (D) host T and NK cells in coculture. (13E)

[0065]

number

[0066] (13F) Schematic diagram showing the in vitro co-culture setup for 13G-13J, showing the relationship between transplanted EBVST and alloreactive CD30 cells stimulated to recognize and kill the transplanted cells. KO The host ATCs were mixed at a ratio of 1:1 to 4. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (13G, 13H, and 13I) Graphs showing cell counts from representative graft-host pairs: (G) transplanted T cells growing in monoculture, (H) transplanted T cells in coculture, and (I) host T cells in coculture. (13J)

[0067]

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[0068] 1 is a bar graph showing host-mediated graft death calculated by (n=2). Each point represents a unique graft-host pair and the bar represents the median. Statistics were not performed because n<3. [Figure 13E] Schematic, graph, and bar graph showing that overexpression of SB9 (CAS) in non-CAR-engrafted T cells (ATC and EBVST) is superior in protecting the engrafted cells from allogeneic rejection. (13A) Schematic showing the in vitro co-culture setup for 13B-13E. Engrafted TCRαβKO ATCs were mixed with HLA-mismatched host PBMCs at a ratio of 1:10-20. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (13B, 13C, and 13D) Graphs showing cell counts from representative graft-host pairs: (B) engrafted T cells expanding in monoculture, (C) engrafted T cells in coculture, and (D) host T and NK cells in coculture. (13E)

[0069]

number

[0070] (13F) Schematic diagram showing the in vitro co-culture setup for 13G-13J, showing the relationship between transplanted EBVST and alloreactive CD30 cells stimulated to recognize and kill the transplanted cells. KO The host ATCs were mixed at a ratio of 1:1 to 4. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (13G, 13H, and 13I) Graphs showing cell counts from representative graft-host pairs: (G) transplanted T cells growing in monoculture, (H) transplanted T cells in coculture, and (I) host T cells in coculture. (13J)

[0071]

number

[0072] 1 is a bar graph showing host-mediated graft death calculated by (n=2). Each point represents a unique graft-host pair and the bar represents the median. Statistics were not performed because n<3. [Figure 13F]Schematic, graph, and bar graph showing that overexpression of SB9 (CAS) in non-CAR-engrafted T cells (ATC and EBVST) is superior in protecting the engrafted cells from allogeneic rejection. (13A) Schematic showing the in vitro co-culture setup for 13B-13E. Engrafted TCRαβKO ATCs were mixed with HLA-mismatched host PBMCs at a ratio of 1:10-20. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (13B, 13C, and 13D) Graphs showing cell counts from representative graft-host pairs: (B) engrafted T cells expanding in monoculture, (C) engrafted T cells in coculture, and (D) host T and NK cells in coculture. (13E)

[0073]

number

[0074] (13F) Schematic diagram showing the in vitro co-culture setup for 13G-13J, showing the relationship between transplanted EBVST and alloreactive CD30 cells stimulated to recognize and kill the transplanted cells. KO The host ATCs were mixed at a ratio of 1:1 to 4. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (13G, 13H, and 13I) Graphs showing cell counts from representative graft-host pairs: (G) transplanted T cells growing in monoculture, (H) transplanted T cells in coculture, and (I) host T cells in coculture. (13J)

[0075]

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[0076] 1 is a bar graph showing host-mediated graft death calculated by (n=2). Each point represents a unique graft-host pair and the bar represents the median. Statistics were not performed because n<3. [Figure 13G] Schematic, graph, and bar graph showing that overexpression of SB9 (CAS) in non-CAR-engrafted T cells (ATC and EBVST) is superior in protecting the engrafted cells from allogeneic rejection. (13A) Schematic showing the in vitro co-culture setup for 13B-13E. Engrafted TCRαβKO ATCs were mixed with HLA-mismatched host PBMCs at a ratio of 1:10-20. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (13B, 13C, and 13D) Graphs showing cell counts from representative graft-host pairs: (B) engrafted T cells expanding in monoculture, (C) engrafted T cells in coculture, and (D) host T and NK cells in coculture. (13E)

[0077]

number

[0078] (13F) Schematic diagram showing the in vitro co-culture setup for 13G-13J, showing the relationship between transplanted EBVST and alloreactive CD30 cells stimulated to recognize and kill the transplanted cells. KO The host ATCs were mixed at a ratio of 1:1 to 4. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (13G, 13H, and 13I) Graphs showing cell counts from representative graft-host pairs: (G) transplanted T cells growing in monoculture, (H) transplanted T cells in coculture, and (I) host T cells in coculture. (13J)

[0079]

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[0080] 1 is a bar graph showing host-mediated graft death calculated by (n=2). Each point represents a unique graft-host pair and the bar represents the median. Statistics were not performed because n<3. [Figure 13H] Schematic, graph, and bar graph showing that overexpression of SB9 (CAS) in non-CAR-engrafted T cells (ATC and EBVST) is superior in protecting the engrafted cells from allogeneic rejection. (13A) Schematic showing the in vitro co-culture setup for 13B-13E. Engrafted TCRαβKO ATCs were mixed with HLA-mismatched host PBMCs at a ratio of 1:10-20. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (13B, 13C, and 13D) Graphs showing cell counts from representative graft-host pairs: (B) engrafted T cells expanding in monoculture, (C) engrafted T cells in coculture, and (D) host T and NK cells in coculture. (13E)

[0081]

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[0082] (13F) Schematic diagram showing the in vitro co-culture setup for 13G-13J, showing the relationship between transplanted EBVST and alloreactive CD30 cells stimulated to recognize and kill the transplanted cells. KOThe host ATCs were mixed at a ratio of 1:1 to 4. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (13G, 13H, and 13I) Graphs showing cell counts from representative graft-host pairs: (G) transplanted T cells growing in monoculture, (H) transplanted T cells in coculture, and (I) host T cells in coculture. (13J)

[0083]

number

[0084] 1 is a bar graph showing host-mediated graft death calculated by (n=2). Each point represents a unique graft-host pair and the bar represents the median. Statistics were not performed because n<3. [Figure 13I] Schematic, graph, and bar graph showing that overexpression of SB9 (CAS) in non-CAR-engrafted T cells (ATC and EBVST) is superior in protecting the engrafted cells from allogeneic rejection. (13A) Schematic showing the in vitro co-culture setup for 13B-13E. Engrafted TCRαβKO ATCs were mixed with HLA-mismatched host PBMCs at a ratio of 1:10-20. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (13B, 13C, and 13D) Graphs showing cell counts from representative graft-host pairs: (B) engrafted T cells expanding in monoculture, (C) engrafted T cells in coculture, and (D) host T and NK cells in coculture. (13E)

[0085]

number

[0086] (13F) Schematic diagram showing the in vitro co-culture setup for 13G-13J, showing the relationship between transplanted EBVST and alloreactive CD30 cells stimulated to recognize and kill the transplanted cells. KO The host ATCs were mixed at a ratio of 1:1 to 4. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (13G, 13H, and 13I) Graphs showing cell counts from representative graft-host pairs: (G) transplanted T cells growing in monoculture, (H) transplanted T cells in coculture, and (I) host T cells in coculture. (13J)

[0087]

number

[0088] 1 is a bar graph showing host-mediated graft death calculated by (n=2). Each point represents a unique graft-host pair and the bar represents the median. Statistics were not performed because n<3. [Figure 13J] Schematic, graph, and bar graph showing that overexpression of SB9 (CAS) in non-CAR-engrafted T cells (ATC and EBVST) is superior in protecting the engrafted cells from allogeneic rejection. (13A) Schematic showing the in vitro co-culture setup for 13B-13E. Engrafted TCRαβKO ATCs were mixed with HLA-mismatched host PBMCs at a ratio of 1:10-20. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (13B, 13C, and 13D) Graphs showing cell counts from representative graft-host pairs: (B) engrafted T cells expanding in monoculture, (C) engrafted T cells in coculture, and (D) host T and NK cells in coculture. (13E)

[0089]

number

[0090] (13F) Schematic diagram showing the in vitro co-culture setup for 13G-13J, showing the relationship between transplanted EBVST and alloreactive CD30 cells stimulated to recognize and kill the transplanted cells. KO The host ATCs were mixed at a ratio of 1:1 to 4. Assays were performed in the presence of both 10 ng / mL IL-7 and IL-15. (13G, 13H, and 13I) Graphs showing cell counts from representative graft-host pairs: (G) transplanted T cells growing in monoculture, (H) transplanted T cells in coculture, and (I) host T cells in coculture. (13J)

[0091]

number

[0092] 1 is a bar graph showing host-mediated graft death calculated by (n=2). Each point represents a unique graft-host pair and the bar represents the median. Statistics were not performed because n<3. [Figure 14A]Schematics, images, bar graphs, and graphs showing that overexpression of SB9 (CAS) in CD30.CAR (4-1BB spacer) EBVST protects CD30.CAR EBVST from allogeneic rejection and improves antitumor efficacy both in vitro and in vivo. (14A) Schematic showing an in vitro triplicate culture setup for (B) and (C) in which engineered NALM6 (truncated CD30 positive and HLA I and II KO) and transplanted CD30.CAR EBVST were mixed with host CD30KO ATC stimulated to recognize and kill transplanted cells at a 1:1:1:4 ratio. Assays were performed in the absence of cytokines. (14B)

[0093]

number

[0094] (14C) A bar graph showing host-mediated graft death calculated by α = 0.01 (n = 5). Each point represents a unique graft-host pair, and the bar represents the median (n = 5). The lowest graft:host ratio that resulted in 60% or more host-mediated graft death of CD30.CAR EBVST at day 3 was selected. P values ​​were determined by one-way ANOVA and Holm-Sidak post-hoc test. *, P = 0.0193. (14C) A bar graph showing the percentage of round 2 tumor cells killed in triplicate cultures, normalized to tumor cells grown in monoculture. (14D) A schematic diagram showing the in vivo allorejection model for 14E-14F, showing the engineered NALM6 (a cleaved form of CD30 positive and HLA I and II receptors) KO )2.5×10 6 Individuals, NSG (MHC KO ) mice were intravenously injected. 18 days later, 5 × 10 eGFP-ffLuc-expressing CD30.CAR EBVST cells were transplanted. 6 and 5 × 10 alloreactive host T cells (allo-T). 6NALM6.eGFP-ffLuc 2.5×10 cells were co-infused intravenously. (14E) Bioluminescence image showing transplanted CD30.CAR EBVST levels captured by an IVIS Lumina S5 imaging system. (14F) Graph showing quantified bioluminescence signal from transplanted CD30.CAR EBVST over time. (14G) Schematic diagram showing the in vivo allorejection model for 14H-14K. Engineered NALM6.eGFP-ffLuc 2.5×10 cells were co-infused intravenously. 6 Individuals, NSG (MHC KO ) mice were intravenously injected, and 15 days later, transplanted with 5 x 10 CD30.CAR EBVST. 6 and 5 × 10 alloreactive host T cells (allo-T). 6 were co-infused intravenously. (14H and 14I) Flow cytometry analysis of blood samples at the indicated time points. Percentages of (H) engrafted CD30.CAR EBVST cells and (I) host allogeneic T cells in peripheral blood are shown. (14J) Bioluminescence images showing engineered NALM6.eGFP-ffLuc tumor growth captured by an IVIS Lumina S5 imaging system. (14K) Quantified bioluminescence signal from tumor cells over time, normalized to the level of disease at day 0. All graphs for in vivo data show mean + SD. P values ​​were determined using one-way ANOVA (K; day 11 after treatment) or two-way ANOVA (F and H: P values ​​are shown for comparisons between CD30.CAR+ alloT and SB9(CAS)-CD30.CAR+ alloT samples) with Dunnett's correction for multiple comparisons, comparing sample means to the mean of CD30.CAR+ alloT samples. [Figure 14B]Schematics, images, bar graphs, and graphs showing that overexpression of SB9 (CAS) in CD30.CAR (4-1BB spacer) EBVST protects CD30.CAR EBVST from allogeneic rejection and improves antitumor efficacy both in vitro and in vivo. (14A) Schematic showing an in vitro triplicate culture setup for (B) and (C) in which engineered NALM6 (truncated CD30 positive and HLA I and II KO) and transplanted CD30.CAR EBVST were mixed with host CD30KO ATC stimulated to recognize and kill transplanted cells at a 1:1:1:4 ratio. Assays were performed in the absence of cytokines. (14B)

[0095]

number

[0096] (14C) A bar graph showing host-mediated graft death calculated by α = 0.01 (n = 5). Each point represents a unique graft-host pair, and the bar represents the median (n = 5). The lowest graft:host ratio that resulted in 60% or more host-mediated graft death of CD30.CAR EBVST at day 3 was selected. P values ​​were determined by one-way ANOVA and Holm-Sidak post-hoc test. *, P = 0.0193. (14C) A bar graph showing the percentage of round 2 tumor cells killed in triplicate cultures, normalized to tumor cells grown in monoculture. (14D) A schematic diagram showing the in vivo allorejection model for 14E-14F, showing the engineered NALM6 (a cleaved form of CD30 positive and HLA I and II receptors) KO )2.5×10 6 Individuals, NSG (MHC KO ) mice were intravenously injected. 18 days later, 5 × 10 eGFP-ffLuc-expressing CD30.CAR EBVST cells were transplanted. 6 and 5 × 10 alloreactive host T cells (allo-T). 6NALM6.eGFP-ffLuc 2.5×10 cells were co-infused intravenously. (14E) Bioluminescence image showing transplanted CD30.CAR EBVST levels captured by an IVIS Lumina S5 imaging system. (14F) Graph showing quantified bioluminescence signal from transplanted CD30.CAR EBVST over time. (14G) Schematic diagram showing the in vivo allorejection model for 14H-14K. Engineered NALM6.eGFP-ffLuc 2.5×10 cells were co-infused intravenously. 6 Individuals, NSG (MHC KO ) mice were intravenously injected, and 15 days later, transplanted with 5 x 10 CD30.CAR EBVST. 6 and 5 × 10 alloreactive host T cells (allo-T). 6 were co-infused intravenously. (14H and 14I) Flow cytometry analysis of blood samples at the indicated time points. Percentages of (H) engrafted CD30.CAR EBVST cells and (I) host allogeneic T cells in peripheral blood are shown. (14J) Bioluminescence images showing engineered NALM6.eGFP-ffLuc tumor growth captured by an IVIS Lumina S5 imaging system. (14K) Quantified bioluminescence signal from tumor cells over time, normalized to the level of disease at day 0. All graphs for in vivo data show mean + SD. P values ​​were determined using one-way ANOVA (K; day 11 after treatment) or two-way ANOVA (F and H: P values ​​are shown for comparisons between CD30.CAR+ alloT and SB9(CAS)-CD30.CAR+ alloT samples) with Dunnett's correction for multiple comparisons, comparing sample means to the mean of CD30.CAR+ alloT samples. [Figure 14C]Schematics, images, bar graphs, and graphs showing that overexpression of SB9 (CAS) in CD30.CAR (4-1BB spacer) EBVST protects CD30.CAR EBVST from allogeneic rejection and improves antitumor efficacy both in vitro and in vivo. (14A) Schematic showing an in vitro triplicate culture setup for (B) and (C) in which engineered NALM6 (truncated CD30 positive and HLA I and II KO) and transplanted CD30.CAR EBVST were mixed with host CD30KO ATC stimulated to recognize and kill transplanted cells at a 1:1:1:4 ratio. Assays were performed in the absence of cytokines. (14B)

[0097]

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[0098] (14C) A bar graph showing host-mediated graft death calculated by α = 0.01 (n = 5). Each point represents a unique graft-host pair, and the bar represents the median (n = 5). The lowest graft:host ratio that resulted in 60% or more host-mediated graft death of CD30.CAR EBVST at day 3 was selected. P values ​​were determined by one-way ANOVA and Holm-Sidak post-hoc test. *, P = 0.0193. (14C) A bar graph showing the percentage of round 2 tumor cells killed in triplicate cultures, normalized to tumor cells grown in monoculture. (14D) A schematic diagram showing the in vivo allorejection model for 14E-14F, showing the engineered NALM6 (a cleaved form of CD30 positive and HLA I and II receptors) KO )2.5×10 6 Individuals, NSG (MHC KO ) mice were intravenously injected. 18 days later, 5 × 10 eGFP-ffLuc-expressing CD30.CAR EBVST cells were transplanted. 6 and 5 × 10 alloreactive host T cells (allo-T). 6NALM6.eGFP-ffLuc 2.5×10 cells were co-infused intravenously. (14E) Bioluminescence image showing transplanted CD30.CAR EBVST levels captured by an IVIS Lumina S5 imaging system. (14F) Graph showing quantified bioluminescence signal from transplanted CD30.CAR EBVST over time. (14G) Schematic diagram showing the in vivo allorejection model for 14H-14K. Engineered NALM6.eGFP-ffLuc 2.5×10 cells were co-infused intravenously. 6 Individuals, NSG (MHC KO ) mice were intravenously injected, and 15 days later, transplanted with 5 x 10 CD30.CAR EBVST. 6 and 5 × 10 alloreactive host T cells (allo-T). 6 were co-infused intravenously. (14H and 14I) Flow cytometry analysis of blood samples at the indicated time points. Percentages of (H) engrafted CD30.CAR EBVST cells and (I) host allogeneic T cells in peripheral blood are shown. (14J) Bioluminescence images showing engineered NALM6.eGFP-ffLuc tumor growth captured by an IVIS Lumina S5 imaging system. (14K) Quantified bioluminescence signal from tumor cells over time, normalized to the level of disease at day 0. All graphs for in vivo data show mean + SD. P values ​​were determined using one-way ANOVA (K; day 11 after treatment) or two-way ANOVA (F and H: P values ​​are shown for comparisons between CD30.CAR+ alloT and SB9(CAS)-CD30.CAR+ alloT samples) with Dunnett's correction for multiple comparisons, comparing sample means to the mean of CD30.CAR+ alloT samples. [Figure 14D]Schematics, images, bar graphs, and graphs showing that overexpression of SB9 (CAS) in CD30.CAR (4-1BB spacer) EBVST protects CD30.CAR EBVST from allogeneic rejection and improves antitumor efficacy both in vitro and in vivo. (14A) Schematic showing an in vitro triplicate culture setup for (B) and (C) in which engineered NALM6 (truncated CD30 positive and HLA I and II KO) and transplanted CD30.CAR EBVST were mixed with host CD30KO ATC stimulated to recognize and kill transplanted cells at a 1:1:1:4 ratio. Assays were performed in the absence of cytokines. (14B)

[0099]

number

[0100] (14C) A bar graph showing host-mediated graft death calculated by α = 0.01 (n = 5). Each point represents a unique graft-host pair, and the bar represents the median (n = 5). The lowest graft:host ratio that resulted in 60% or more host-mediated graft death of CD30.CAR EBVST at day 3 was selected. P values ​​were determined by one-way ANOVA and Holm-Sidak post-hoc test. *, P = 0.0193. (14C) A bar graph showing the percentage of round 2 tumor cells killed in triplicate cultures, normalized to tumor cells grown in monoculture. (14D) A schematic diagram showing the in vivo allorejection model for 14E-14F, showing the engineered NALM6 (a cleaved form of CD30 positive and HLA I and II receptors) KO )2.5×10 6 Individuals, NSG (MHC KO ) mice were intravenously injected. 18 days later, 5 × 10 eGFP-ffLuc-expressing CD30.CAR EBVST cells were transplanted. 6 and 5 × 10 alloreactive host T cells (allo-T). 6NALM6.eGFP-ffLuc 2.5×10 cells were co-infused intravenously. (14E) Bioluminescence image showing transplanted CD30.CAR EBVST levels captured by an IVIS Lumina S5 imaging system. (14F) Graph showing quantified bioluminescence signal from transplanted CD30.CAR EBVST over time. (14G) Schematic diagram showing the in vivo allorejection model for 14H-14K. Engineered NALM6.eGFP-ffLuc 2.5×10 cells were co-infused intravenously. 6 Individuals, NSG (MHC KO ) mice were intravenously injected, and 15 days later, transplanted with 5 x 10 CD30.CAR EBVST. 6 and 5 × 10 alloreactive host T cells (allo-T). 6 were co-infused intravenously. (14H and 14I) Flow cytometry analysis of blood samples at the indicated time points. Percentages of (H) engrafted CD30.CAR EBVST cells and (I) host allogeneic T cells in peripheral blood are shown. (14J) Bioluminescence images showing engineered NALM6.eGFP-ffLuc tumor growth captured by an IVIS Lumina S5 imaging system. (14K) Quantified bioluminescence signal from tumor cells over time, normalized to the level of disease at day 0. All graphs for in vivo data show mean + SD. P values ​​were determined using one-way ANOVA (K; day 11 after treatment) or two-way ANOVA (F and H: P values ​​are shown for comparisons between CD30.CAR+ alloT and SB9(CAS)-CD30.CAR+ alloT samples) with Dunnett's correction for multiple comparisons, comparing sample means to the mean of CD30.CAR+ alloT samples. [Figure 14E]Schematics, images, bar graphs, and graphs showing that overexpression of SB9 (CAS) in CD30.CAR (4-1BB spacer) EBVST protects CD30.CAR EBVST from allogeneic rejection and improves antitumor efficacy both in vitro and in vivo. (14A) Schematic showing an in vitro triplicate culture setup for (B) and (C) in which engineered NALM6 (truncated CD30 positive and HLA I and II KO) and transplanted CD30.CAR EBVST were mixed with host CD30KO ATC stimulated to recognize and kill transplanted cells at a 1:1:1:4 ratio. Assays were performed in the absence of cytokines. (14B)

[0101]

number

[0102] (14C) A bar graph showing host-mediated graft death calculated by α = 0.01 (n = 5). Each point represents a unique graft-host pair, and the bar represents the median (n = 5). The lowest graft:host ratio that resulted in 60% or more host-mediated graft death of CD30.CAR EBVST at day 3 was selected. P values ​​were determined by one-way ANOVA and Holm-Sidak post-hoc test. *, P = 0.0193. (14C) A bar graph showing the percentage of round 2 tumor cells killed in triplicate cultures, normalized to tumor cells grown in monoculture. (14D) A schematic diagram showing the in vivo allorejection model for 14E-14F, showing the engineered NALM6 (a cleaved form of CD30 positive and HLA I and II receptors) KO )2.5×10 6 Individuals, NSG (MHC KO ) mice were intravenously injected. 18 days later, 5 × 10 eGFP-ffLuc-expressing CD30.CAR EBVST cells were transplanted. 6 and 5 × 10 alloreactive host T cells (allo-T). 6NALM6.eGFP-ffLuc 2.5×10 cells were co-infused intravenously. (14E) Bioluminescence image showing transplanted CD30.CAR EBVST levels captured by an IVIS Lumina S5 imaging system. (14F) Graph showing quantified bioluminescence signal from transplanted CD30.CAR EBVST over time. (14G) Schematic diagram showing the in vivo allorejection model for 14H-14K. Engineered NALM6.eGFP-ffLuc 2.5×10 cells were co-infused intravenously. 6 Individuals, NSG (MHC KO ) mice were intravenously injected, and 15 days later, transplanted with 5 x 10 CD30.CAR EBVST. 6 and 5 × 10 alloreactive host T cells (allo-T). 6 were co-infused intravenously. (14H and 14I) Flow cytometry analysis of blood samples at the indicated time points. Percentages of (H) engrafted CD30.CAR EBVST cells and (I) host allogeneic T cells in peripheral blood are shown. (14J) Bioluminescence images showing engineered NALM6.eGFP-ffLuc tumor growth captured by an IVIS Lumina S5 imaging system. (14K) Quantified bioluminescence signal from tumor cells over time, normalized to the level of disease at day 0. All graphs for in vivo data show mean + SD. P values ​​were determined using one-way ANOVA (K; day 11 after treatment) or two-way ANOVA (F and H: P values ​​are shown for comparisons between CD30.CAR+ alloT and SB9(CAS)-CD30.CAR+ alloT samples) with Dunnett's correction for multiple comparisons, comparing sample means to the mean of CD30.CAR+ alloT samples. [Figure 14F]Schematics, images, bar graphs, and graphs showing that overexpression of SB9 (CAS) in CD30.CAR (4-1BB spacer) EBVST protects CD30.CAR EBVST from allogeneic rejection and improves antitumor efficacy both in vitro and in vivo. (14A) Schematic showing an in vitro triplicate culture setup for (B) and (C) in which engineered NALM6 (truncated CD30 positive and HLA I and II KO) and transplanted CD30.CAR EBVST were mixed with host CD30KO ATC stimulated to recognize and kill transplanted cells at a 1:1:1:4 ratio. Assays were performed in the absence of cytokines. (14B)

[0103]

number

[0104] (14C) A bar graph showing host-mediated graft death calculated by α = 0.01 (n = 5). Each point represents a unique graft-host pair, and the bar represents the median (n = 5). The lowest graft:host ratio that resulted in 60% or more host-mediated graft death of CD30.CAR EBVST at day 3 was selected. P values ​​were determined by one-way ANOVA and Holm-Sidak post-hoc test. *, P = 0.0193. (14C) A bar graph showing the percentage of round 2 tumor cells killed in triplicate cultures, normalized to tumor cells grown in monoculture. (14D) A schematic diagram showing the in vivo allorejection model for 14E-14F, showing the engineered NALM6 (a cleaved form of CD30 positive and HLA I and II receptors) KO )2.5×10 6 Individuals, NSG (MHC KO ) mice were intravenously injected. 18 days later, 5 × 10 eGFP-ffLuc-expressing CD30.CAR EBVST cells were transplanted. 6 and 5 × 10 alloreactive host T cells (allo-T). 6NALM6.eGFP-ffLuc 2.5×10 cells were co-infused intravenously. (14E) Bioluminescence image showing transplanted CD30.CAR EBVST levels captured by an IVIS Lumina S5 imaging system. (14F) Graph showing quantified bioluminescence signal from transplanted CD30.CAR EBVST over time. (14G) Schematic diagram showing the in vivo allorejection model for 14H-14K. Engineered NALM6.eGFP-ffLuc 2.5×10 cells were co-infused intravenously. 6 Individuals, NSG (MHC KO ) mice were intravenously injected, and 15 days later, transplanted with 5 x 10 CD30.CAR EBVST. 6 and 5 × 10 alloreactive host T cells (allo-T). 6 were co-infused intravenously. (14H and 14I) Flow cytometry analysis of blood samples at the indicated time points. Percentages of (H) engrafted CD30.CAR EBVST cells and (I) host allogeneic T cells in peripheral blood are shown. (14J) Bioluminescence images showing engineered NALM6.eGFP-ffLuc tumor growth captured by an IVIS Lumina S5 imaging system. (14K) Quantified bioluminescence signal from tumor cells over time, normalized to the level of disease at day 0. All graphs for in vivo data show mean + SD. P values ​​were determined using one-way ANOVA (K; day 11 after treatment) or two-way ANOVA (F and H: P values ​​are shown for comparisons between CD30.CAR+ alloT and SB9(CAS)-CD30.CAR+ alloT samples) with Dunnett's correction for multiple comparisons, comparing sample means to the mean of CD30.CAR+ alloT samples. [Figure 14G]Schematics, images, bar graphs, and graphs showing that overexpression of SB9 (CAS) in CD30.CAR (4-1BB spacer) EBVST protects CD30.CAR EBVST from allogeneic rejection and improves antitumor efficacy both in vitro and in vivo. (14A) Schematic showing an in vitro triplicate culture setup for (B) and (C) in which engineered NALM6 (truncated CD30 positive and HLA I and II KO) and transplanted CD30.CAR EBVST were mixed with host CD30KO ATC stimulated to recognize and kill transplanted cells at a 1:1:1:4 ratio. Assays were performed in the absence of cytokines. (14B)

[0105]

number

[0106] (14C) A bar graph showing host-mediated graft death calculated by α = 0.01 (n = 5). Each point represents a unique graft-host pair, and the bar represents the median (n = 5). The lowest graft:host ratio that resulted in 60% or more host-mediated graft death of CD30.CAR EBVST at day 3 was selected. P values ​​were determined by one-way ANOVA and Holm-Sidak post-hoc test. *, P = 0.0193. (14C) A bar graph showing the percentage of round 2 tumor cells killed in triplicate cultures, normalized to tumor cells grown in monoculture. (14D) A schematic diagram showing the in vivo allorejection model for 14E-14F, showing the engineered NALM6 (a cleaved form of CD30 positive and HLA I and II receptors) KO )2.5×10 6 Individuals, NSG (MHC KO ) mice were intravenously injected. 18 days later, 5 × 10 eGFP-ffLuc-expressing CD30.CAR EBVST cells were transplanted. 6 and 5 × 10 alloreactive host T cells (allo-T). 6NALM6.eGFP-ffLuc 2.5×10 cells were co-infused intravenously. (14E) Bioluminescence image showing transplanted CD30.CAR EBVST levels captured by an IVIS Lumina S5 imaging system. (14F) Graph showing quantified bioluminescence signal from transplanted CD30.CAR EBVST over time. (14G) Schematic diagram showing the in vivo allorejection model for 14H-14K. Engineered NALM6.eGFP-ffLuc 2.5×10 cells were co-infused intravenously. 6 Individuals, NSG (MHC KO ) mice were intravenously injected, and 15 days later, transplanted with 5 x 10 CD30.CAR EBVST. 6 and 5 × 10 alloreactive host T cells (allo-T). 6 were co-infused intravenously. (14H and 14I) Flow cytometry analysis of blood samples at the indicated time points. Percentages of (H) engrafted CD30.CAR EBVST cells and (I) host allogeneic T cells in peripheral blood are shown. (14J) Bioluminescence images showing engineered NALM6.eGFP-ffLuc tumor growth captured by an IVIS Lumina S5 imaging system. (14K) Quantified bioluminescence signal from tumor cells over time, normalized to the level of disease at day 0. All graphs for in vivo data show mean + SD. P values ​​were determined using one-way ANOVA (K; day 11 after treatment) or two-way ANOVA (F and H: P values ​​are shown for comparisons between CD30.CAR+ alloT and SB9(CAS)-CD30.CAR+ alloT samples) with Dunnett's correction for multiple comparisons, comparing sample means to the mean of CD30.CAR+ alloT samples. [Figure 14H]Schematics, images, bar graphs, and graphs showing that overexpression of SB9 (CAS) in CD30.CAR (4-1BB spacer) EBVST protects CD30.CAR EBVST from allogeneic rejection and improves antitumor efficacy both in vitro and in vivo. (14A) Schematic showing an in vitro triplicate culture setup for (B) and (C) in which engineered NALM6 (truncated CD30 positive and HLA I and II KO) and transplanted CD30.CAR EBVST were mixed with host CD30KO ATC stimulated to recognize and kill transplanted cells at a 1:1:1:4 ratio. Assays were performed in the absence of cytokines. (14B)

[0107]

number

[0108] (14C) A bar graph showing host-mediated graft death calculated by α = 0.01 (n = 5). Each point represents a unique graft-host pair, and the bar represents the median (n = 5). The lowest graft:host ratio that resulted in 60% or more host-mediated graft death of CD30.CAR EBVST at day 3 was selected. P values ​​were determined by one-way ANOVA and Holm-Sidak post-hoc test. *, P = 0.0193. (14C) A bar graph showing the percentage of round 2 tumor cells killed in triplicate cultures, normalized to tumor cells grown in monoculture. (14D) A schematic diagram showing the in vivo allorejection model for 14E-14F, showing the engineered NALM6 (a cleaved form of CD30 positive and HLA I and II receptors) KO )2.5×10 6 Individuals, NSG (MHC KO ) mice were intravenously injected. 18 days later, 5 × 10 eGFP-ffLuc-expressing CD30.CAR EBVST cells were transplanted. 6 and 5 × 10 alloreactive host T cells (allo-T). 6NALM6.eGFP-ffLuc 2.5×10 cells were co-infused intravenously. (14E) Bioluminescence image showing transplanted CD30.CAR EBVST levels captured by an IVIS Lumina S5 imaging system. (14F) Graph showing quantified bioluminescence signal from transplanted CD30.CAR EBVST over time. (14G) Schematic diagram showing the in vivo allorejection model for 14H-14K. Engineered NALM6.eGFP-ffLuc 2.5×10 cells were co-infused intravenously. 6 Individuals, NSG (MHC KO ) mice were intravenously injected, and 15 days later, transplanted with 5 x 10 CD30.CAR EBVST. 6 and 5 × 10 alloreactive host T cells (allo-T). 6 were co-infused intravenously. (14H and 14I) Flow cytometry analysis of blood samples at the indicated time points. Percentages of (H) engrafted CD30.CAR EBVST cells and (I) host allogeneic T cells in peripheral blood are shown. (14J) Bioluminescence images showing engineered NALM6.eGFP-ffLuc tumor growth captured by an IVIS Lumina S5 imaging system. (14K) Quantified bioluminescence signal from tumor cells over time, normalized to the level of disease at day 0. All graphs for in vivo data show mean + SD. P values ​​were determined using one-way ANOVA (K; day 11 after treatment) or two-way ANOVA (F and H: P values ​​are shown for comparisons between CD30.CAR+ alloT and SB9(CAS)-CD30.CAR+ alloT samples) with Dunnett's correction for multiple comparisons, comparing sample means to the mean of CD30.CAR+ alloT samples. [Figure 14I]Schematics, images, bar graphs, and graphs showing that overexpression of SB9 (CAS) in CD30.CAR (4-1BB spacer) EBVST protects CD30.CAR EBVST from allogeneic rejection and improves antitumor efficacy both in vitro and in vivo. (14A) Schematic showing an in vitro triplicate culture setup for (B) and (C) in which engineered NALM6 (truncated CD30 positive and HLA I and II KO) and transplanted CD30.CAR EBVST were mixed with host CD30KO ATC stimulated to recognize and kill transplanted cells at a 1:1:1:4 ratio. Assays were performed in the absence of cytokines. (14B)

[0109]

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[0110] (14C) A bar graph showing host-mediated graft death calculated by α = 0.01 (n = 5). Each point represents a unique graft-host pair, and the bar represents the median (n = 5). The lowest graft:host ratio that resulted in 60% or more host-mediated graft death of CD30.CAR EBVST at day 3 was selected. P values ​​were determined by one-way ANOVA and Holm-Sidak post-hoc test. *, P = 0.0193. (14C) A bar graph showing the percentage of round 2 tumor cells killed in triplicate cultures, normalized to tumor cells grown in monoculture. (14D) A schematic diagram showing the in vivo allorejection model for 14E-14F, showing the engineered NALM6 (a cleaved form of CD30 positive and HLA I and II receptors) KO )2.5×10 6 Individuals, NSG (MHC KO ) mice were intravenously injected. 18 days later, 5 × 10 eGFP-ffLuc-expressing CD30.CAR EBVST cells were transplanted. 6 and 5 × 10 alloreactive host T cells (allo-T). 6NALM6.eGFP-ffLuc 2.5×10 cells were co-infused intravenously. (14E) Bioluminescence image showing transplanted CD30.CAR EBVST levels captured by an IVIS Lumina S5 imaging system. (14F) Graph showing quantified bioluminescence signal from transplanted CD30.CAR EBVST over time. (14G) Schematic diagram showing the in vivo allorejection model for 14H-14K. Engineered NALM6.eGFP-ffLuc 2.5×10 cells were co-infused intravenously. 6 Individuals, NSG (MHC KO ) mice were intravenously injected, and 15 days later, transplanted with 5 x 10 CD30.CAR EBVST. 6 and 5 × 10 alloreactive host T cells (allo-T). 6 were co-infused intravenously. (14H and 14I) Flow cytometry analysis of blood samples at the indicated time points. Percentages of (H) engrafted CD30.CAR EBVST cells and (I) host allogeneic T cells in peripheral blood are shown. (14J) Bioluminescence images showing engineered NALM6.eGFP-ffLuc tumor growth captured by an IVIS Lumina S5 imaging system. (14K) Quantified bioluminescence signal from tumor cells over time, normalized to the level of disease at day 0. All graphs for in vivo data show mean + SD. P values ​​were determined using one-way ANOVA (K; day 11 after treatment) or two-way ANOVA (F and H: P values ​​are shown for comparisons between CD30.CAR+ alloT and SB9(CAS)-CD30.CAR+ alloT samples) with Dunnett's correction for multiple comparisons, comparing sample means to the mean of CD30.CAR+ alloT samples. [Figure 14J]Schematics, images, bar graphs, and graphs showing that overexpression of SB9 (CAS) in CD30.CAR (4-1BB spacer) EBVST protects CD30.CAR EBVST from allogeneic rejection and improves antitumor efficacy both in vitro and in vivo. (14A) Schematic showing an in vitro triplicate culture setup for (B) and (C) in which engineered NALM6 (truncated CD30 positive and HLA I and II KO) and transplanted CD30.CAR EBVST were mixed with host CD30KO ATC stimulated to recognize and kill transplanted cells at a 1:1:1:4 ratio. Assays were performed in the absence of cytokines. (14B)

[0111]

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[0112] (14C) A bar graph showing host-mediated graft death calculated by α = 0.01 (n = 5). Each point represents a unique graft-host pair, and the bar represents the median (n = 5). The lowest graft:host ratio that resulted in 60% or more host-mediated graft death of CD30.CAR EBVST at day 3 was selected. P values ​​were determined by one-way ANOVA and Holm-Sidak post-hoc test. *, P = 0.0193. (14C) A bar graph showing the percentage of round 2 tumor cells killed in triplicate cultures, normalized to tumor cells grown in monoculture. (14D) A schematic diagram showing the in vivo allorejection model for 14E-14F, showing the engineered NALM6 (a cleaved form of CD30 positive and HLA I and II receptors) KO )2.5×10 6 Individuals, NSG (MHC KO ) mice were intravenously injected. 18 days later, 5 × 10 eGFP-ffLuc-expressing CD30.CAR EBVST cells were transplanted. 6 and 5 × 10 alloreactive host T cells (allo-T). 6NALM6.eGFP-ffLuc 2.5×10 cells were co-infused intravenously. (14E) Bioluminescence image showing transplanted CD30.CAR EBVST levels captured by an IVIS Lumina S5 imaging system. (14F) Graph showing quantified bioluminescence signal from transplanted CD30.CAR EBVST over time. (14G) Schematic diagram showing the in vivo allorejection model for 14H-14K. Engineered NALM6.eGFP-ffLuc 2.5×10 cells were co-infused intravenously. 6 Individuals, NSG (MHC KO ) mice were intravenously injected, and 15 days later, transplanted with 5 x 10 CD30.CAR EBVST. 6 and 5 × 10 alloreactive host T cells (allo-T). 6 were co-infused intravenously. (14H and 14I) Flow cytometry analysis of blood samples at the indicated time points. Percentages of (H) engrafted CD30.CAR EBVST cells and (I) host allogeneic T cells in peripheral blood are shown. (14J) Bioluminescence images showing engineered NALM6.eGFP-ffLuc tumor growth captured by an IVIS Lumina S5 imaging system. (14K) Quantified bioluminescence signal from tumor cells over time, normalized to the level of disease at day 0. All graphs for in vivo data show mean + SD. P values ​​were determined using one-way ANOVA (K; day 11 after treatment) or two-way ANOVA (F and H: P values ​​are shown for comparisons between CD30.CAR+ alloT and SB9(CAS)-CD30.CAR+ alloT samples) with Dunnett's correction for multiple comparisons, comparing sample means to the mean of CD30.CAR+ alloT samples. [Figure 14K]Schematics, images, bar graphs, and graphs showing that overexpression of SB9 (CAS) in CD30.CAR (4-1BB spacer) EBVST protects CD30.CAR EBVST from allogeneic rejection and improves antitumor efficacy both in vitro and in vivo. (14A) Schematic showing an in vitro triplicate culture setup for (B) and (C) in which engineered NALM6 (truncated CD30 positive and HLA I and II KO) and transplanted CD30.CAR EBVST were mixed with host CD30KO ATC stimulated to recognize and kill transplanted cells at a 1:1:1:4 ratio. Assays were performed in the absence of cytokines. (14B)

[0113]

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[0114] (14C) A bar graph showing host-mediated graft death calculated by α = 0.01 (n = 5). Each point represents a unique graft-host pair, and the bar represents the median (n = 5). The lowest graft:host ratio that resulted in 60% or more host-mediated graft death of CD30.CAR EBVST at day 3 was selected. P values ​​were determined by one-way ANOVA and Holm-Sidak post-hoc test. *, P = 0.0193. (14C) A bar graph showing the percentage of round 2 tumor cells killed in triplicate cultures, normalized to tumor cells grown in monoculture. (14D) A schematic diagram showing the in vivo allorejection model for 14E-14F, showing the engineered NALM6 (a cleaved form of CD30 positive and HLA I and II receptors) KO )2.5×10 6 Individuals, NSG (MHC KO ) mice were intravenously injected. 18 days later, 5 × 10 eGFP-ffLuc-expressing CD30.CAR EBVST cells were transplanted. 6 and 5 × 10 alloreactive host T cells (allo-T). 6NALM6.eGFP-ffLuc 2.5×10 cells were co-infused intravenously. (14E) Bioluminescence image showing transplanted CD30.CAR EBVST levels captured by an IVIS Lumina S5 imaging system. (14F) Graph showing quantified bioluminescence signal from transplanted CD30.CAR EBVST over time. (14G) Schematic diagram showing the in vivo allorejection model for 14H-14K. Engineered NALM6.eGFP-ffLuc 2.5×10 cells were co-infused intravenously. 6 Individuals, NSG (MHC KO ) mice were intravenously injected, and 15 days later, transplanted with 5 x 10 CD30.CAR EBVST. 6 and 5 × 10 alloreactive host T cells (allo-T). 6 were co-infused intravenously. (14H and 14I) Flow cytometry analysis of blood samples at the indicated time points. Percentages of (H) engrafted CD30.CAR EBVST cells and (I) host allogeneic T cells in peripheral blood are shown. (14J) Bioluminescence images showing engineered NALM6.eGFP-ffLuc tumor growth captured by an IVIS Lumina S5 imaging system. (14K) Quantified bioluminescence signal from tumor cells over time, normalized to the level of disease at day 0. All graphs for in vivo data show mean + SD. P values ​​were determined using one-way ANOVA (K; day 11 after treatment) or two-way ANOVA (F and H: P values ​​are shown for comparisons between CD30.CAR+ alloT and SB9(CAS)-CD30.CAR+ alloT samples) with Dunnett's correction for multiple comparisons, comparing sample means to the mean of CD30.CAR+ alloT samples. [Figure 15A]Graph and bar graph showing that overexpression of SB9 (CAS) in CD19.CAR ATC and CD30.CAR (4-1BB spacer) ATC protects them from allogeneic rejection and improves anti-tumor efficacy in vitro. (15A) Graph showing flow cytometry analysis of engineered NALM6 cells overexpressing truncated CD30 (tCD30) and gene-edited for HLA class I and II knockout (HLADKO) sorted by FACS. (15B) Bar graph showing the percentage of tumor cells killed by host cells in a co-culture setting of engineered NALM6 tumor cells and host allogeneic T cells at a 1:1:1 ratio. (15C) Triplicate culture of CD19.CAR ATC-implanted cells, host ATC stimulated to recognize and kill the implanted cells, and engineered NALM6 at a 1:1:1 ratio.

[0115]

number

[0116] 15D is a graph showing host-mediated graft killing calculated by the ##EQU1## Assays were performed in the absence of cytokines. (15D) CD30.CAR ATC transplanted cells and host CD30 stimulated to recognize and kill the transplanted cells. KO 15E and 15F are graphs showing host-mediated graft killing in triplicate cultures of ATC and engineered NALM6 at a 1:4:1 ratio. Assays were performed in the absence of cytokines. (15E and 15F) Bar graphs showing tumor cell numbers counted by flow cytometry analysis on day 3 after triplicate culture with (E) CD19.CAR ATC or (F) CD30.CAR ATC. [Figure 15B]Graph and bar graph showing that overexpression of SB9 (CAS) in CD19.CAR ATC and CD30.CAR (4-1BB spacer) ATC protects them from allogeneic rejection and improves anti-tumor efficacy in vitro. (15A) Graph showing flow cytometry analysis of engineered NALM6 cells overexpressing truncated CD30 (tCD30) and gene-edited for HLA class I and II knockout (HLADKO) sorted by FACS. (15B) Bar graph showing the percentage of tumor cells killed by host cells in a co-culture setting of engineered NALM6 tumor cells and host allogeneic T cells at a 1:1:1 ratio. (15C) Triplicate culture of CD19.CAR ATC-implanted cells, host ATC stimulated to recognize and kill the implanted cells, and engineered NALM6 at a 1:1:1 ratio.

[0117]

number

[0118] 15D is a graph showing host-mediated graft killing calculated by the ##EQU1## Assays were performed in the absence of cytokines. (15D) CD30.CAR ATC transplanted cells and host CD30 stimulated to recognize and kill the transplanted cells. KO 15E and 15F are graphs showing host-mediated graft killing in triplicate cultures of ATC and engineered NALM6 at a 1:4:1 ratio. Assays were performed in the absence of cytokines. (15E and 15F) Bar graphs showing tumor cell numbers counted by flow cytometry analysis on day 3 after triplicate culture with (E) CD19.CAR ATC or (F) CD30.CAR ATC. [Figure 15C]Graph and bar graph showing that overexpression of SB9 (CAS) in CD19.CAR ATC and CD30.CAR (4-1BB spacer) ATC protects them from allogeneic rejection and improves anti-tumor efficacy in vitro. (15A) Graph showing flow cytometry analysis of engineered NALM6 cells overexpressing truncated CD30 (tCD30) and gene-edited for HLA class I and II knockout (HLADKO) sorted by FACS. (15B) Bar graph showing the percentage of tumor cells killed by host cells in a co-culture setting of engineered NALM6 tumor cells and host allogeneic T cells at a 1:1:1 ratio. (15C) Triplicate culture of CD19.CAR ATC-implanted cells, host ATC stimulated to recognize and kill the implanted cells, and engineered NALM6 at a 1:1:1 ratio.

[0119]

number

[0120] 15D is a graph showing host-mediated graft killing calculated by the ##EQU1## Assays were performed in the absence of cytokines. (15D) CD30.CAR ATC transplanted cells and host CD30 stimulated to recognize and kill the transplanted cells. KO 15E and 15F are graphs showing host-mediated graft killing in triplicate cultures of ATC and engineered NALM6 at a 1:4:1 ratio. Assays were performed in the absence of cytokines. (15E and 15F) Bar graphs showing tumor cell numbers counted by flow cytometry analysis on day 3 after triplicate culture with (E) CD19.CAR ATC or (F) CD30.CAR ATC. [Figure 15D]Graph and bar graph showing that overexpression of SB9 (CAS) in CD19.CAR ATC and CD30.CAR (4-1BB spacer) ATC protects them from allogeneic rejection and improves anti-tumor efficacy in vitro. (15A) Graph showing flow cytometry analysis of engineered NALM6 cells overexpressing truncated CD30 (tCD30) and gene-edited for HLA class I and II knockout (HLADKO) sorted by FACS. (15B) Bar graph showing the percentage of tumor cells killed by host cells in a co-culture setting of engineered NALM6 tumor cells and host allogeneic T cells at a 1:1:1 ratio. (15C) Triplicate culture of CD19.CAR ATC-implanted cells, host ATC stimulated to recognize and kill the implanted cells, and engineered NALM6 at a 1:1:1 ratio.

[0121]

number

[0122] 15D is a graph showing host-mediated graft killing calculated by the ##EQU1## Assays were performed in the absence of cytokines. (15D) CD30.CAR ATC transplanted cells and host CD30 stimulated to recognize and kill the transplanted cells. KO 15E and 15F are graphs showing host-mediated graft killing in triplicate cultures of ATC and engineered NALM6 at a 1:4:1 ratio. Assays were performed in the absence of cytokines. (15E and 15F) Bar graphs showing tumor cell numbers counted by flow cytometry analysis on day 3 after triplicate culture with (E) CD19.CAR ATC or (F) CD30.CAR ATC. [Figure 15E]Graph and bar graph showing that overexpression of SB9 (CAS) in CD19.CAR ATC and CD30.CAR (4-1BB spacer) ATC protects them from allogeneic rejection and improves anti-tumor efficacy in vitro. (15A) Graph showing flow cytometry analysis of engineered NALM6 cells overexpressing truncated CD30 (tCD30) and gene-edited for HLA class I and II knockout (HLADKO) sorted by FACS. (15B) Bar graph showing the percentage of tumor cells killed by host cells in a co-culture setting of engineered NALM6 tumor cells and host allogeneic T cells at a 1:1:1 ratio. (15C) Triplicate culture of CD19.CAR ATC-implanted cells, host ATC stimulated to recognize and kill the implanted cells, and engineered NALM6 at a 1:1:1 ratio.

[0123]

number

[0124] 15D is a graph showing host-mediated graft killing calculated by the ##EQU1## Assays were performed in the absence of cytokines. (15D) CD30.CAR ATC transplanted cells and host CD30 stimulated to recognize and kill the transplanted cells. KO 15E and 15F are graphs showing host-mediated graft killing in triplicate cultures of ATC and engineered NALM6 at a 1:4:1 ratio. Assays were performed in the absence of cytokines. (15E and 15F) Bar graphs showing tumor cell numbers counted by flow cytometry analysis on day 3 after triplicate culture with (E) CD19.CAR ATC or (F) CD30.CAR ATC. [Figure 15F]Graph and bar graph showing that overexpression of SB9 (CAS) in CD19.CAR ATC and CD30.CAR (4-1BB spacer) ATC protects them from allogeneic rejection and improves anti-tumor efficacy in vitro. (15A) Graph showing flow cytometry analysis of engineered NALM6 cells overexpressing truncated CD30 (tCD30) and gene-edited for HLA class I and II knockout (HLADKO) sorted by FACS. (15B) Bar graph showing the percentage of tumor cells killed by host cells in a co-culture setting of engineered NALM6 tumor cells and host allogeneic T cells at a 1:1:1 ratio. (15C) Triplicate culture of CD19.CAR ATC-implanted cells, host ATC stimulated to recognize and kill the implanted cells, and engineered NALM6 at a 1:1:1 ratio.

[0125]

number

[0126] 15D is a graph showing host-mediated graft killing calculated by the ##EQU1## Assays were performed in the absence of cytokines. (15D) CD30.CAR ATC transplanted cells and host CD30 stimulated to recognize and kill the transplanted cells. KO 15E and 15F are graphs showing host-mediated graft killing in triplicate cultures of ATC and engineered NALM6 at a 1:4:1 ratio. Assays were performed in the absence of cytokines. (15E and 15F) Bar graphs showing tumor cell numbers counted by flow cytometry analysis on day 3 after triplicate culture with (E) CD19.CAR ATC or (F) CD30.CAR ATC. [Figure 16A]Schematics, images, and graphs show that administration of alloreactive host T cells in vivo results in allogeneic rejection of transplanted T cells. (16A) Schematic of the in vivo allorejection model for 16B and 16C. 2.5 × 10 engineered NALM6 (truncated CD30 positive and HLA I and II KO) cells were intravenously injected into NSG (MHCKO) mice. 18 days later, 5 × 10 transplanted eGFP-ffLuc-expressing CD30.CAR (4-1BB spacer) EBVST cells were intravenously infused with or without 5 × 10 alloreactive host T cells (allo-T). (16B) Bioluminescence images showing transplanted CD30.CAR EBVST levels captured by an IVIS Lumina S5 imaging system. (16C) Graph showing quantified bioluminescence signal from transplanted CD30.CAR EBVST over time. (16D) Schematic diagram showing the in vivo allorejection model for 16E-16G. 2.5 x 10 engineered NALM6.eGFP-ffLuc cells were injected intravenously into NSG(MHCKO) mice. 15 days later, 5 x 10 transplanted CD30.CAR EBVST cells were infused intravenously with or without 5 x 10 alloreactive host T cells (allo-T). (16E) Graph showing flow cytometry analysis of blood samples at the indicated time points. The percentage of transplanted CD30.CAR EBVST cells in peripheral blood is shown. (16F) Bioluminescence images showing engineered NALM6.eGFP-ffLuc tumor growth captured by an IVIS Lumina S5 imaging system. (16G) Graph showing quantified bioluminescence signal from tumor cells over time, normalized to the level of disease at day 0. All graphs for in vivo data show mean + SD. P values ​​were determined using (E) two-way ANOVA and Sidak's correction for multiple comparisons or (C and G; day 11 after treatment) unpaired one-tailed t test. [Figure 16B]Schematics, images, and graphs show that administration of alloreactive host T cells in vivo results in allogeneic rejection of transplanted T cells. (16A) Schematic of the in vivo allorejection model for 16B and 16C. 2.5 × 10 engineered NALM6 (truncated CD30 positive and HLA I and II KO) cells were intravenously injected into NSG (MHCKO) mice. 18 days later, 5 × 10 transplanted eGFP-ffLuc-expressing CD30.CAR (4-1BB spacer) EBVST cells were intravenously infused with or without 5 × 10 alloreactive host T cells (allo-T). (16B) Bioluminescence images showing transplanted CD30.CAR EBVST levels captured by an IVIS Lumina S5 imaging system. (16C) Graph showing quantified bioluminescence signal from transplanted CD30.CAR EBVST over time. (16D) Schematic diagram showing the in vivo allorejection model for 16E-16G. 2.5 x 10 engineered NALM6.eGFP-ffLuc cells were injected intravenously into NSG(MHCKO) mice. 15 days later, 5 x 10 transplanted CD30.CAR EBVST cells were infused intravenously with or without 5 x 10 alloreactive host T cells (allo-T). (16E) Graph showing flow cytometry analysis of blood samples at the indicated time points. The percentage of transplanted CD30.CAR EBVST cells in peripheral blood is shown. (16F) Bioluminescence images showing engineered NALM6.eGFP-ffLuc tumor growth captured by an IVIS Lumina S5 imaging system. (16G) Graph showing quantified bioluminescence signal from tumor cells over time, normalized to the level of disease at day 0. All graphs for in vivo data show mean + SD. P values ​​were determined using (E) two-way ANOVA and Sidak's correction for multiple comparisons or (C and G; day 11 after treatment) unpaired one-tailed t test. [Figure 16C]Schematics, images, and graphs show that administration of alloreactive host T cells in vivo results in allogeneic rejection of transplanted T cells. (16A) Schematic of the in vivo allorejection model for 16B and 16C. 2.5 × 10 engineered NALM6 (truncated CD30 positive and HLA I and II KO) cells were intravenously injected into NSG (MHCKO) mice. 18 days later, 5 × 10 transplanted eGFP-ffLuc-expressing CD30.CAR (4-1BB spacer) EBVST cells were intravenously infused with or without 5 × 10 alloreactive host T cells (allo-T). (16B) Bioluminescence images showing transplanted CD30.CAR EBVST levels captured by an IVIS Lumina S5 imaging system. (16C) Graph showing quantified bioluminescence signal from transplanted CD30.CAR EBVST over time. (16D) Schematic diagram showing the in vivo allorejection model for 16E-16G. 2.5 x 10 engineered NALM6.eGFP-ffLuc cells were injected intravenously into NSG(MHCKO) mice. 15 days later, 5 x 10 transplanted CD30.CAR EBVST cells were infused intravenously with or without 5 x 10 alloreactive host T cells (allo-T). (16E) Graph showing flow cytometry analysis of blood samples at the indicated time points. The percentage of transplanted CD30.CAR EBVST cells in peripheral blood is shown. (16F) Bioluminescence images showing engineered NALM6.eGFP-ffLuc tumor growth captured by an IVIS Lumina S5 imaging system. (16G) Graph showing quantified bioluminescence signal from tumor cells over time, normalized to the level of disease at day 0. All graphs for in vivo data show mean + SD. P values ​​were determined using (E) two-way ANOVA and Sidak's correction for multiple comparisons or (C and G; day 11 after treatment) unpaired one-tailed t test. [Figure 16D]Schematics, images, and graphs show that administration of alloreactive host T cells in vivo results in allogeneic rejection of transplanted T cells. (16A) Schematic of the in vivo allorejection model for 16B and 16C. 2.5 × 10 engineered NALM6 (truncated CD30 positive and HLA I and II KO) cells were intravenously injected into NSG (MHCKO) mice. 18 days later, 5 × 10 transplanted eGFP-ffLuc-expressing CD30.CAR (4-1BB spacer) EBVST cells were intravenously infused with or without 5 × 10 alloreactive host T cells (allo-T). (16B) Bioluminescence images showing transplanted CD30.CAR EBVST levels captured by an IVIS Lumina S5 imaging system. (16C) Graph showing quantified bioluminescence signal from transplanted CD30.CAR EBVST over time. (16D) Schematic diagram showing the in vivo allorejection model for 16E-16G. 2.5 x 10 engineered NALM6.eGFP-ffLuc cells were injected intravenously into NSG(MHCKO) mice. 15 days later, 5 x 10 transplanted CD30.CAR EBVST cells were infused intravenously with or without 5 x 10 alloreactive host T cells (allo-T). (16E) Graph showing flow cytometry analysis of blood samples at the indicated time points. The percentage of transplanted CD30.CAR EBVST cells in peripheral blood is shown. (16F) Bioluminescence images showing engineered NALM6.eGFP-ffLuc tumor growth captured by an IVIS Lumina S5 imaging system. (16G) Graph showing quantified bioluminescence signal from tumor cells over time, normalized to the level of disease at day 0. All graphs for in vivo data show mean + SD. P values ​​were determined using (E) two-way ANOVA and Sidak's correction for multiple comparisons or (C and G; day 11 after treatment) unpaired one-tailed t test. [Figure 16E]Schematics, images, and graphs show that administration of alloreactive host T cells in vivo results in allogeneic rejection of transplanted T cells. (16A) Schematic of the in vivo allorejection model for 16B and 16C. 2.5 × 10 engineered NALM6 (truncated CD30 positive and HLA I and II KO) cells were intravenously injected into NSG (MHCKO) mice. 18 days later, 5 × 10 transplanted eGFP-ffLuc-expressing CD30.CAR (4-1BB spacer) EBVST cells were intravenously infused with or without 5 × 10 alloreactive host T cells (allo-T). (16B) Bioluminescence images showing transplanted CD30.CAR EBVST levels captured by an IVIS Lumina S5 imaging system. (16C) Graph showing quantified bioluminescence signal from transplanted CD30.CAR EBVST over time. (16D) Schematic diagram showing the in vivo allorejection model for 16E-16G. 2.5 x 10 engineered NALM6.eGFP-ffLuc cells were injected intravenously into NSG(MHCKO) mice. 15 days later, 5 x 10 transplanted CD30.CAR EBVST cells were infused intravenously with or without 5 x 10 alloreactive host T cells (allo-T). (16E) Graph showing flow cytometry analysis of blood samples at the indicated time points. The percentage of transplanted CD30.CAR EBVST cells in peripheral blood is shown. (16F) Bioluminescence images showing engineered NALM6.eGFP-ffLuc tumor growth captured by an IVIS Lumina S5 imaging system. (16G) Graph showing quantified bioluminescence signal from tumor cells over time, normalized to the level of disease at day 0. All graphs for in vivo data show mean + SD. P values ​​were determined using (E) two-way ANOVA and Sidak's correction for multiple comparisons or (C and G; day 11 after treatment) unpaired one-tailed t test. [Figure 16F]Schematics, images, and graphs show that administration of alloreactive host T cells in vivo results in allogeneic rejection of transplanted T cells. (16A) Schematic of the in vivo allorejection model for 16B and 16C. 2.5 × 10 engineered NALM6 (truncated CD30 positive and HLA I and II KO) cells were intravenously injected into NSG (MHCKO) mice. 18 days later, 5 × 10 transplanted eGFP-ffLuc-expressing CD30.CAR (4-1BB spacer) EBVST cells were intravenously infused with or without 5 × 10 alloreactive host T cells (allo-T). (16B) Bioluminescence images showing transplanted CD30.CAR EBVST levels captured by an IVIS Lumina S5 imaging system. (16C) Graph showing quantified bioluminescence signal from transplanted CD30.CAR EBVST over time. (16D) Schematic diagram showing the in vivo allorejection model for 16E-16G. 2.5 x 10 engineered NALM6.eGFP-ffLuc cells were injected intravenously into NSG(MHCKO) mice. 15 days later, 5 x 10 transplanted CD30.CAR EBVST cells were infused intravenously with or without 5 x 10 alloreactive host T cells (allo-T). (16E) Graph showing flow cytometry analysis of blood samples at the indicated time points. The percentage of transplanted CD30.CAR EBVST cells in peripheral blood is shown. (16F) Bioluminescence images showing engineered NALM6.eGFP-ffLuc tumor growth captured by an IVIS Lumina S5 imaging system. (16G) Graph showing quantified bioluminescence signal from tumor cells over time, normalized to the level of disease at day 0. All graphs for in vivo data show mean + SD. P values ​​were determined using (E) two-way ANOVA and Sidak's correction for multiple comparisons or (C and G; day 11 after treatment) unpaired one-tailed t test. [Figure 16G]Schematics, images, and graphs show that administration of alloreactive host T cells in vivo results in allogeneic rejection of transplanted T cells. (16A) Schematic of the in vivo allorejection model for 16B and 16C. 2.5 × 10 engineered NALM6 (truncated CD30 positive and HLA I and II KO) cells were intravenously injected into NSG (MHCKO) mice. 18 days later, 5 × 10 transplanted eGFP-ffLuc-expressing CD30.CAR (4-1BB spacer) EBVST cells were intravenously infused with or without 5 × 10 alloreactive host T cells (allo-T). (16B) Bioluminescence images showing transplanted CD30.CAR EBVST levels captured by an IVIS Lumina S5 imaging system. (16C) Graph showing quantified bioluminescence signal from transplanted CD30.CAR EBVST over time. (16D) Schematic diagram showing the in vivo allorejection model for 16E-16G. 2.5 x 10 engineered NALM6.eGFP-ffLuc cells were injected intravenously into NSG(MHCKO) mice. 15 days later, 5 x 10 transplanted CD30.CAR EBVST cells were infused intravenously with or without 5 x 10 alloreactive host T cells (allo-T). (16E) Graph showing flow cytometry analysis of blood samples at the indicated time points. The percentage of transplanted CD30.CAR EBVST cells in peripheral blood is shown. (16F) Bioluminescence images showing engineered NALM6.eGFP-ffLuc tumor growth captured by an IVIS Lumina S5 imaging system. (16G) Graph showing quantified bioluminescence signal from tumor cells over time, normalized to the level of disease at day 0. All graphs for in vivo data show mean + SD. P values ​​were determined using (E) two-way ANOVA and Sidak's correction for multiple comparisons or (C and G; day 11 after treatment) unpaired one-tailed t test. [Figure 17A]Schematic, graph, and bar graph showing that SB9 (CAS) overexpression improves CD30.CAR (4-1BB spacer) and CD19.CAR ATC expansion after continuous coculture with tumor cells. (17A) Schematic showing in vitro coculture of CAR ATC with engineered NALM6 tumor cells at a fixed ratio of 1:1 or 1:5 when CD30.CAR and CD19.CAR ATCs were studied, respectively. Every 2–3 days, cells were counted by flow cytometry and replated in coculture with fresh tumor cells at a fixed ratio. The process was repeated until the CAR T cells could no longer eliminate tumor cells. Experiments were performed in the absence of cytokines. (17B, 17C, 17F, and 17G) Graphs showing the cumulative proliferation fold change of CD30.CAR ATCs grown in (B and F) monoculture and (C and G) coculture. (17D and 17H) Bar graphs showing the number of tumor cells counted by flow cytometry on day 13. (17E and 17I) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD30.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 13). (17J, 17K, 17N, and 17O) Graphs showing the cumulative proliferation fold change of CD19.CAR ATCs grown in (J and N) monoculture and (K and O) co-culture. (17L and 17P) Bar graphs showing the number of tumor cells counted by flow cytometry on day 8. (17M and 17Q) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD19.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 8). [Figure 17B]Schematic, graph, and bar graph showing that SB9 (CAS) overexpression improves CD30.CAR (4-1BB spacer) and CD19.CAR ATC expansion after continuous coculture with tumor cells. (17A) Schematic showing in vitro coculture of CAR ATC with engineered NALM6 tumor cells at a fixed ratio of 1:1 or 1:5 when CD30.CAR and CD19.CAR ATCs were studied, respectively. Every 2–3 days, cells were counted by flow cytometry and replated in coculture with fresh tumor cells at a fixed ratio. The process was repeated until the CAR T cells could no longer eliminate tumor cells. Experiments were performed in the absence of cytokines. (17B, 17C, 17F, and 17G) Graphs showing the cumulative proliferation fold change of CD30.CAR ATCs grown in (B and F) monoculture and (C and G) coculture. (17D and 17H) Bar graphs showing the number of tumor cells counted by flow cytometry on day 13. (17E and 17I) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD30.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 13). (17J, 17K, 17N, and 17O) Graphs showing the cumulative proliferation fold change of CD19.CAR ATCs grown in (J and N) monoculture and (K and O) co-culture. (17L and 17P) Bar graphs showing the number of tumor cells counted by flow cytometry on day 8. (17M and 17Q) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD19.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 8). [Figure 17C]Schematic, graph, and bar graph showing that SB9 (CAS) overexpression improves CD30.CAR (4-1BB spacer) and CD19.CAR ATC expansion after continuous coculture with tumor cells. (17A) Schematic showing in vitro coculture of CAR ATC with engineered NALM6 tumor cells at a fixed ratio of 1:1 or 1:5 when CD30.CAR and CD19.CAR ATCs were studied, respectively. Every 2–3 days, cells were counted by flow cytometry and replated in coculture with fresh tumor cells at a fixed ratio. The process was repeated until the CAR T cells could no longer eliminate tumor cells. Experiments were performed in the absence of cytokines. (17B, 17C, 17F, and 17G) Graphs showing the cumulative proliferation fold change of CD30.CAR ATCs grown in (B and F) monoculture and (C and G) coculture. (17D and 17H) Bar graphs showing the number of tumor cells counted by flow cytometry on day 13. (17E and 17I) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD30.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 13). (17J, 17K, 17N, and 17O) Graphs showing the cumulative proliferation fold change of CD19.CAR ATCs grown in (J and N) monoculture and (K and O) co-culture. (17L and 17P) Bar graphs showing the number of tumor cells counted by flow cytometry on day 8. (17M and 17Q) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD19.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 8). [Figure 17D]Schematic, graph, and bar graph showing that SB9 (CAS) overexpression improves CD30.CAR (4-1BB spacer) and CD19.CAR ATC expansion after continuous coculture with tumor cells. (17A) Schematic showing in vitro coculture of CAR ATC with engineered NALM6 tumor cells at a fixed ratio of 1:1 or 1:5 when CD30.CAR and CD19.CAR ATCs were studied, respectively. Every 2–3 days, cells were counted by flow cytometry and replated in coculture with fresh tumor cells at a fixed ratio. The process was repeated until the CAR T cells could no longer eliminate tumor cells. Experiments were performed in the absence of cytokines. (17B, 17C, 17F, and 17G) Graphs showing the cumulative proliferation fold change of CD30.CAR ATCs grown in (B and F) monoculture and (C and G) coculture. (17D and 17H) Bar graphs showing the number of tumor cells counted by flow cytometry on day 13. (17E and 17I) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD30.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 13). (17J, 17K, 17N, and 17O) Graphs showing the cumulative proliferation fold change of CD19.CAR ATCs grown in (J and N) monoculture and (K and O) co-culture. (17L and 17P) Bar graphs showing the number of tumor cells counted by flow cytometry on day 8. (17M and 17Q) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD19.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 8). [Figure 17E]Schematic, graph, and bar graph showing that SB9 (CAS) overexpression improves CD30.CAR (4-1BB spacer) and CD19.CAR ATC expansion after continuous coculture with tumor cells. (17A) Schematic showing in vitro coculture of CAR ATC with engineered NALM6 tumor cells at a fixed ratio of 1:1 or 1:5 when CD30.CAR and CD19.CAR ATCs were studied, respectively. Every 2–3 days, cells were counted by flow cytometry and replated in coculture with fresh tumor cells at a fixed ratio. The process was repeated until the CAR T cells could no longer eliminate tumor cells. Experiments were performed in the absence of cytokines. (17B, 17C, 17F, and 17G) Graphs showing the cumulative proliferation fold change of CD30.CAR ATCs grown in (B and F) monoculture and (C and G) coculture. (17D and 17H) Bar graphs showing the number of tumor cells counted by flow cytometry on day 13. (17E and 17I) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD30.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 13). (17J, 17K, 17N, and 17O) Graphs showing the cumulative proliferation fold change of CD19.CAR ATCs grown in (J and N) monoculture and (K and O) co-culture. (17L and 17P) Bar graphs showing the number of tumor cells counted by flow cytometry on day 8. (17M and 17Q) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD19.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 8). [Figure 17F]Schematic, graph, and bar graph showing that SB9 (CAS) overexpression improves CD30.CAR (4-1BB spacer) and CD19.CAR ATC expansion after continuous coculture with tumor cells. (17A) Schematic showing in vitro coculture of CAR ATC with engineered NALM6 tumor cells at a fixed ratio of 1:1 or 1:5 when CD30.CAR and CD19.CAR ATCs were studied, respectively. Every 2–3 days, cells were counted by flow cytometry and replated in coculture with fresh tumor cells at a fixed ratio. The process was repeated until the CAR T cells could no longer eliminate tumor cells. Experiments were performed in the absence of cytokines. (17B, 17C, 17F, and 17G) Graphs showing the cumulative proliferation fold change of CD30.CAR ATCs grown in (B and F) monoculture and (C and G) coculture. (17D and 17H) Bar graphs showing the number of tumor cells counted by flow cytometry on day 13. (17E and 17I) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD30.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 13). (17J, 17K, 17N, and 17O) Graphs showing the cumulative proliferation fold change of CD19.CAR ATCs grown in (J and N) monoculture and (K and O) co-culture. (17L and 17P) Bar graphs showing the number of tumor cells counted by flow cytometry on day 8. (17M and 17Q) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD19.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 8). [Figure 17G]Schematic, graph, and bar graph showing that SB9 (CAS) overexpression improves CD30.CAR (4-1BB spacer) and CD19.CAR ATC expansion after continuous coculture with tumor cells. (17A) Schematic showing in vitro coculture of CAR ATC with engineered NALM6 tumor cells at a fixed ratio of 1:1 or 1:5 when CD30.CAR and CD19.CAR ATCs were studied, respectively. Every 2–3 days, cells were counted by flow cytometry and replated in coculture with fresh tumor cells at a fixed ratio. The process was repeated until the CAR T cells could no longer eliminate tumor cells. Experiments were performed in the absence of cytokines. (17B, 17C, 17F, and 17G) Graphs showing the cumulative proliferation fold change of CD30.CAR ATCs grown in (B and F) monoculture and (C and G) coculture. (17D and 17H) Bar graphs showing the number of tumor cells counted by flow cytometry on day 13. (17E and 17I) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD30.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 13). (17J, 17K, 17N, and 17O) Graphs showing the cumulative proliferation fold change of CD19.CAR ATCs grown in (J and N) monoculture and (K and O) co-culture. (17L and 17P) Bar graphs showing the number of tumor cells counted by flow cytometry on day 8. (17M and 17Q) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD19.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 8). [Figure 17H]Schematic, graph, and bar graph showing that SB9 (CAS) overexpression improves CD30.CAR (4-1BB spacer) and CD19.CAR ATC expansion after continuous coculture with tumor cells. (17A) Schematic showing in vitro coculture of CAR ATC with engineered NALM6 tumor cells at a fixed ratio of 1:1 or 1:5 when CD30.CAR and CD19.CAR ATCs were studied, respectively. Every 2–3 days, cells were counted by flow cytometry and replated in coculture with fresh tumor cells at a fixed ratio. The process was repeated until the CAR T cells could no longer eliminate tumor cells. Experiments were performed in the absence of cytokines. (17B, 17C, 17F, and 17G) Graphs showing the cumulative proliferation fold change of CD30.CAR ATCs grown in (B and F) monoculture and (C and G) coculture. (17D and 17H) Bar graphs showing the number of tumor cells counted by flow cytometry on day 13. (17E and 17I) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD30.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 13). (17J, 17K, 17N, and 17O) Graphs showing the cumulative proliferation fold change of CD19.CAR ATCs grown in (J and N) monoculture and (K and O) co-culture. (17L and 17P) Bar graphs showing the number of tumor cells counted by flow cytometry on day 8. (17M and 17Q) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD19.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 8). [Figure 17I]Schematic, graph, and bar graph showing that SB9 (CAS) overexpression improves CD30.CAR (4-1BB spacer) and CD19.CAR ATC expansion after continuous coculture with tumor cells. (17A) Schematic showing in vitro coculture of CAR ATC with engineered NALM6 tumor cells at a fixed ratio of 1:1 or 1:5 when CD30.CAR and CD19.CAR ATCs were studied, respectively. Every 2–3 days, cells were counted by flow cytometry and replated in coculture with fresh tumor cells at a fixed ratio. The process was repeated until the CAR T cells could no longer eliminate tumor cells. Experiments were performed in the absence of cytokines. (17B, 17C, 17F, and 17G) Graphs showing the cumulative proliferation fold change of CD30.CAR ATCs grown in (B and F) monoculture and (C and G) coculture. (17D and 17H) Bar graphs showing the number of tumor cells counted by flow cytometry on day 13. (17E and 17I) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD30.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 13). (17J, 17K, 17N, and 17O) Graphs showing the cumulative proliferation fold change of CD19.CAR ATCs grown in (J and N) monoculture and (K and O) co-culture. (17L and 17P) Bar graphs showing the number of tumor cells counted by flow cytometry on day 8. (17M and 17Q) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD19.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 8). [Figure 17J]Schematic, graph, and bar graph showing that SB9 (CAS) overexpression improves CD30.CAR (4-1BB spacer) and CD19.CAR ATC expansion after continuous coculture with tumor cells. (17A) Schematic showing in vitro coculture of CAR ATC with engineered NALM6 tumor cells at a fixed ratio of 1:1 or 1:5 when CD30.CAR and CD19.CAR ATCs were studied, respectively. Every 2–3 days, cells were counted by flow cytometry and replated in coculture with fresh tumor cells at a fixed ratio. The process was repeated until the CAR T cells could no longer eliminate tumor cells. Experiments were performed in the absence of cytokines. (17B, 17C, 17F, and 17G) Graphs showing the cumulative proliferation fold change of CD30.CAR ATCs grown in (B and F) monoculture and (C and G) coculture. (17D and 17H) Bar graphs showing the number of tumor cells counted by flow cytometry on day 13. (17E and 17I) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD30.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 13). (17J, 17K, 17N, and 17O) Graphs showing the cumulative proliferation fold change of CD19.CAR ATCs grown in (J and N) monoculture and (K and O) co-culture. (17L and 17P) Bar graphs showing the number of tumor cells counted by flow cytometry on day 8. (17M and 17Q) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD19.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 8). [Figure 17K]Schematic, graph, and bar graph showing that SB9 (CAS) overexpression improves CD30.CAR (4-1BB spacer) and CD19.CAR ATC expansion after continuous coculture with tumor cells. (17A) Schematic showing in vitro coculture of CAR ATC with engineered NALM6 tumor cells at a fixed ratio of 1:1 or 1:5 when CD30.CAR and CD19.CAR ATCs were studied, respectively. Every 2–3 days, cells were counted by flow cytometry and replated in coculture with fresh tumor cells at a fixed ratio. The process was repeated until the CAR T cells could no longer eliminate tumor cells. Experiments were performed in the absence of cytokines. (17B, 17C, 17F, and 17G) Graphs showing the cumulative proliferation fold change of CD30.CAR ATCs grown in (B and F) monoculture and (C and G) coculture. (17D and 17H) Bar graphs showing the number of tumor cells counted by flow cytometry on day 13. (17E and 17I) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD30.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 13). (17J, 17K, 17N, and 17O) Graphs showing the cumulative proliferation fold change of CD19.CAR ATCs grown in (J and N) monoculture and (K and O) co-culture. (17L and 17P) Bar graphs showing the number of tumor cells counted by flow cytometry on day 8. (17M and 17Q) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD19.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 8). [Figure 17L]Schematic, graph, and bar graph showing that SB9 (CAS) overexpression improves CD30.CAR (4-1BB spacer) and CD19.CAR ATC expansion after continuous coculture with tumor cells. (17A) Schematic showing in vitro coculture of CAR ATC with engineered NALM6 tumor cells at a fixed ratio of 1:1 or 1:5 when CD30.CAR and CD19.CAR ATCs were studied, respectively. Every 2–3 days, cells were counted by flow cytometry and replated in coculture with fresh tumor cells at a fixed ratio. The process was repeated until the CAR T cells could no longer eliminate tumor cells. Experiments were performed in the absence of cytokines. (17B, 17C, 17F, and 17G) Graphs showing the cumulative proliferation fold change of CD30.CAR ATCs grown in (B and F) monoculture and (C and G) coculture. (17D and 17H) Bar graphs showing the number of tumor cells counted by flow cytometry on day 13. (17E and 17I) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD30.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 13). (17J, 17K, 17N, and 17O) Graphs showing the cumulative proliferation fold change of CD19.CAR ATCs grown in (J and N) monoculture and (K and O) co-culture. (17L and 17P) Bar graphs showing the number of tumor cells counted by flow cytometry on day 8. (17M and 17Q) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD19.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 8). [Figure 17M]Schematic, graph, and bar graph showing that SB9 (CAS) overexpression improves CD30.CAR (4-1BB spacer) and CD19.CAR ATC expansion after continuous coculture with tumor cells. (17A) Schematic showing in vitro coculture of CAR ATC with engineered NALM6 tumor cells at a fixed ratio of 1:1 or 1:5 when CD30.CAR and CD19.CAR ATCs were studied, respectively. Every 2–3 days, cells were counted by flow cytometry and replated in coculture with fresh tumor cells at a fixed ratio. The process was repeated until the CAR T cells could no longer eliminate tumor cells. Experiments were performed in the absence of cytokines. (17B, 17C, 17F, and 17G) Graphs showing the cumulative proliferation fold change of CD30.CAR ATCs grown in (B and F) monoculture and (C and G) coculture. (17D and 17H) Bar graphs showing the number of tumor cells counted by flow cytometry on day 13. (17E and 17I) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD30.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 13). (17J, 17K, 17N, and 17O) Graphs showing the cumulative proliferation fold change of CD19.CAR ATCs grown in (J and N) monoculture and (K and O) co-culture. (17L and 17P) Bar graphs showing the number of tumor cells counted by flow cytometry on day 8. (17M and 17Q) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD19.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 8). [Figure 17N]Schematic, graph, and bar graph showing that SB9 (CAS) overexpression improves CD30.CAR (4-1BB spacer) and CD19.CAR ATC expansion after continuous coculture with tumor cells. (17A) Schematic showing in vitro coculture of CAR ATC with engineered NALM6 tumor cells at a fixed ratio of 1:1 or 1:5 when CD30.CAR and CD19.CAR ATCs were studied, respectively. Every 2–3 days, cells were counted by flow cytometry and replated in coculture with fresh tumor cells at a fixed ratio. The process was repeated until the CAR T cells could no longer eliminate tumor cells. Experiments were performed in the absence of cytokines. (17B, 17C, 17F, and 17G) Graphs showing the cumulative proliferation fold change of CD30.CAR ATCs grown in (B and F) monoculture and (C and G) coculture. (17D and 17H) Bar graphs showing the number of tumor cells counted by flow cytometry on day 13. (17E and 17I) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD30.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 13). (17J, 17K, 17N, and 17O) Graphs showing the cumulative proliferation fold change of CD19.CAR ATCs grown in (J and N) monoculture and (K and O) co-culture. (17L and 17P) Bar graphs showing the number of tumor cells counted by flow cytometry on day 8. (17M and 17Q) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD19.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 8). [Figure 17O]Schematic, graph, and bar graph showing that SB9 (CAS) overexpression improves CD30.CAR (4-1BB spacer) and CD19.CAR ATC expansion after continuous coculture with tumor cells. (17A) Schematic showing in vitro coculture of CAR ATC with engineered NALM6 tumor cells at a fixed ratio of 1:1 or 1:5 when CD30.CAR and CD19.CAR ATCs were studied, respectively. Every 2–3 days, cells were counted by flow cytometry and replated in coculture with fresh tumor cells at a fixed ratio. The process was repeated until the CAR T cells could no longer eliminate tumor cells. Experiments were performed in the absence of cytokines. (17B, 17C, 17F, and 17G) Graphs showing the cumulative proliferation fold change of CD30.CAR ATCs grown in (B and F) monoculture and (C and G) coculture. (17D and 17H) Bar graphs showing the number of tumor cells counted by flow cytometry on day 13. (17E and 17I) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD30.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 13). (17J, 17K, 17N, and 17O) Graphs showing the cumulative proliferation fold change of CD19.CAR ATCs grown in (J and N) monoculture and (K and O) co-culture. (17L and 17P) Bar graphs showing the number of tumor cells counted by flow cytometry on day 8. (17M and 17Q) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD19.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 8). [Figure 17P]Schematic, graph, and bar graph showing that SB9 (CAS) overexpression improves CD30.CAR (4-1BB spacer) and CD19.CAR ATC expansion after continuous coculture with tumor cells. (17A) Schematic showing in vitro coculture of CAR ATC with engineered NALM6 tumor cells at a fixed ratio of 1:1 or 1:5 when CD30.CAR and CD19.CAR ATCs were studied, respectively. Every 2–3 days, cells were counted by flow cytometry and replated in coculture with fresh tumor cells at a fixed ratio. The process was repeated until the CAR T cells could no longer eliminate tumor cells. Experiments were performed in the absence of cytokines. (17B, 17C, 17F, and 17G) Graphs showing the cumulative proliferation fold change of CD30.CAR ATCs grown in (B and F) monoculture and (C and G) coculture. (17D and 17H) Bar graphs showing the number of tumor cells counted by flow cytometry on day 13. (17E and 17I) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD30.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 13). (17J, 17K, 17N, and 17O) Graphs showing the cumulative proliferation fold change of CD19.CAR ATCs grown in (J and N) monoculture and (K and O) co-culture. (17L and 17P) Bar graphs showing the number of tumor cells counted by flow cytometry on day 8. (17M and 17Q) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD19.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 8). [Figure 17Q]Schematic, graph, and bar graph showing that SB9 (CAS) overexpression improves CD30.CAR (4-1BB spacer) and CD19.CAR ATC expansion after continuous coculture with tumor cells. (17A) Schematic showing in vitro coculture of CAR ATC with engineered NALM6 tumor cells at a fixed ratio of 1:1 or 1:5 when CD30.CAR and CD19.CAR ATCs were studied, respectively. Every 2–3 days, cells were counted by flow cytometry and replated in coculture with fresh tumor cells at a fixed ratio. The process was repeated until the CAR T cells could no longer eliminate tumor cells. Experiments were performed in the absence of cytokines. (17B, 17C, 17F, and 17G) Graphs showing the cumulative proliferation fold change of CD30.CAR ATCs grown in (B and F) monoculture and (C and G) coculture. (17D and 17H) Bar graphs showing the number of tumor cells counted by flow cytometry on day 13. (17E and 17I) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD30.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 13). (17J, 17K, 17N, and 17O) Graphs showing the cumulative proliferation fold change of CD19.CAR ATCs grown in (J and N) monoculture and (K and O) co-culture. (17L and 17P) Bar graphs showing the number of tumor cells counted by flow cytometry on day 8. (17M and 17Q) Bar graphs showing PD-1, Tim-3, and Lag-3 expression on CD19.CAR ATCs before the start of co-culture (day 0) and on the final day of co-culture (day 8). [Figure 18A]Schematic and bar graphs showing cytokine secretion profiles of CD30.CAR EBVST (4-1BB spacer) expressing various forms of SB9, with or without CAR-mediated stimulation. (18A) Schematic showing the in vitro coculture setup for 18B-18D. Effector CAR T cells were cocultured at a 1:1 ratio with either clonally selected engineered NALM6 (cleaved CD30 positive and HLA I and II KO) or KM-H2 tumor cells. Assays were performed in the absence of cytokines. (18B, 18C, and 18D) Bar graphs showing cytokine concentrations measured using a 13-plex-Immunology Multiplex Assay kit (Merck) from supernatants of cells cultured for 24 hours in (B) monoculture, (C) coculture with engineered NALM6 tumor cells, or (D) coculture with KM-H2 tumor cells. Bars represent mean values ​​+ SD. [Figure 18B] Schematic and bar graphs showing cytokine secretion profiles of CD30.CAR EBVST (4-1BB spacer) expressing various forms of SB9, with or without CAR-mediated stimulation. (18A) Schematic showing the in vitro coculture setup for 18B-18D. Effector CAR T cells were cocultured at a 1:1 ratio with either clonally selected engineered NALM6 (cleaved CD30 positive and HLA I and II KO) or KM-H2 tumor cells. Assays were performed in the absence of cytokines. (18B, 18C, and 18D) Bar graphs showing cytokine concentrations measured using a 13-plex-Immunology Multiplex Assay kit (Merck) from supernatants of cells cultured for 24 hours in (B) monoculture, (C) coculture with engineered NALM6 tumor cells, or (D) coculture with KM-H2 tumor cells. Bars represent mean values ​​+ SD. [Figure 18C]Schematic and bar graphs showing cytokine secretion profiles of CD30.CAR EBVST (4-1BB spacer) expressing various forms of SB9, with or without CAR-mediated stimulation. (18A) Schematic showing the in vitro coculture setup for 18B-18D. Effector CAR T cells were cocultured at a 1:1 ratio with either clonally selected engineered NALM6 (cleaved CD30 positive and HLA I and II KO) or KM-H2 tumor cells. Assays were performed in the absence of cytokines. (18B, 18C, and 18D) Bar graphs showing cytokine concentrations measured using a 13-plex-Immunology Multiplex Assay kit (Merck) from supernatants of cells cultured for 24 hours in (B) monoculture, (C) coculture with engineered NALM6 tumor cells, or (D) coculture with KM-H2 tumor cells. Bars represent mean values ​​+ SD. [Figure 18D] Schematic and bar graphs showing cytokine secretion profiles of CD30.CAR EBVST (4-1BB spacer) expressing various forms of SB9, with or without CAR-mediated stimulation. (18A) Schematic showing the in vitro coculture setup for 18B-18D. Effector CAR T cells were cocultured at a 1:1 ratio with either clonally selected engineered NALM6 (cleaved CD30 positive and HLA I and II KO) or KM-H2 tumor cells. Assays were performed in the absence of cytokines. (18B, 18C, and 18D) Bar graphs showing cytokine concentrations measured using a 13-plex-Immunology Multiplex Assay kit (Merck) from supernatants of cells cultured for 24 hours in (B) monoculture, (C) coculture with engineered NALM6 tumor cells, or (D) coculture with KM-H2 tumor cells. Bars represent mean values ​​+ SD. [Figure 19A]Schematic, graph, and bar graph showing that SB9 (CAS) overexpression improves the expansion and tumor-killing efficacy of CD30.CAR EBVST in vivo. (19A) Schematic showing the in vitro coculture of CD30.CAR (4-1BB spacer) EBVST with clonally selected, engineered NALM6 tumor cells (expressing truncated CD30 positive and HLA I and IIKO) at a fixed 1:1 ratio. Every 2–3 days, cells were counted by flow cytometry and replated in coculture with fresh tumor cells at a fixed ratio. The process was repeated until the CAR T cells could no longer eliminate tumor cells. Experiments were performed in the absence of cytokines. (19B and 19C) Graphs showing the cumulative proliferation fold change of CD30.CAR EBVST grown in (B) monoculture and (C) coculture. (19D) Bar graph showing the number of tumor cells counted by flow cytometry on day 8. (19E) Bar graph showing PD-1, Tim-3, and Lag-3 expression on CD30.CAR EBVST before the initiation of coculture (day 0) and on the final day of coculture (day 8). CD30.CAR EBVST produced from three donors were tested in vitro with similar findings. (19F) Schematic of the in vivo activation-induced cell death (AICD) model for 19G-19J. 2.5 × 106 clonally selected engineered NALM6 cells were injected intravenously into NSG (MHCKO) mice. 15 days later, 10 × 106 effector CD30.CAR (4-1BB spacer) EBVST were injected intravenously. (19G and 19H) (G) Images and graphs showing tumor bioluminescence captured by an IVIS Lumina S5 imaging system and (H) its quantification normalized to day 0. Quantified tumor burden (normalized to day 0) at 20 days after (19I) treatment is shown. Graphs show mean values ​​+ SD; P values ​​were determined using one-way analysis of variance and Dunnett's correction for multiple comparisons, comparing mean values ​​to the mean values ​​of SB9(CAS)-CD30.CAR samples.(19J) Flow cytometry analysis of blood samples collected from the facial vein at the indicated time points showing CD30.CAR EBVST levels in peripheral blood. Graphs show mean values ​​+ SD, and P values ​​(between CD30.CAR and SB9(CAS)-CD30.CAR samples) were determined using two-way analysis of variance with Dunnett's correction for multiple comparisons. [Figure 19B]Schematic, graph, and bar graph showing that SB9 (CAS) overexpression improves the expansion and tumor-killing efficacy of CD30.CAR EBVST in vivo. (19A) Schematic showing the in vitro coculture of CD30.CAR (4-1BB spacer) EBVST with clonally selected, engineered NALM6 tumor cells (expressing truncated CD30 positive and HLA I and IIKO) at a fixed 1:1 ratio. Every 2–3 days, cells were counted by flow cytometry and replated in coculture with fresh tumor cells at a fixed ratio. The process was repeated until the CAR T cells could no longer eliminate tumor cells. Experiments were performed in the absence of cytokines. (19B and 19C) Graphs showing the cumulative proliferation fold change of CD30.CAR EBVST grown in (B) monoculture and (C) coculture. (19D) Bar graph showing the number of tumor cells counted by flow cytometry on day 8. (19E) Bar graph showing PD-1, Tim-3, and Lag-3 expression on CD30.CAR EBVST before the initiation of coculture (day 0) and on the final day of coculture (day 8). CD30.CAR EBVST produced from three donors were tested in vitro with similar findings. (19F) Schematic of the in vivo activation-induced cell death (AICD) model for 19G-19J. 2.5 × 106 clonally selected engineered NALM6 cells were injected intravenously into NSG (MHCKO) mice. 15 days later, 10 × 106 effector CD30.CAR (4-1BB spacer) EBVST were injected intravenously. (19G and 19H) (G) Images and graphs showing tumor bioluminescence captured by an IVIS Lumina S5 imaging system and (H) its quantification normalized to day 0. Quantified tumor burden (normalized to day 0) at 20 days after (19I) treatment is shown. Graphs show mean values ​​+ SD; P values ​​were determined using one-way analysis of variance and Dunnett's correction for multiple comparisons, comparing mean values ​​to the mean values ​​of SB9(CAS)-CD30.CAR samples.(19J) Flow cytometry analysis of blood samples collected from the facial vein at the indicated time points showing CD30.CAR EBVST levels in peripheral blood. Graphs show mean values ​​+ SD, and P values ​​(between CD30.CAR and SB9(CAS)-CD30.CAR samples) were determined using two-way analysis of variance with Dunnett's correction for multiple comparisons. [Figure 19C]Schematic, graph, and bar graph showing that SB9 (CAS) overexpression improves the expansion and tumor-killing efficacy of CD30.CAR EBVST in vivo. (19A) Schematic showing the in vitro coculture of CD30.CAR (4-1BB spacer) EBVST with clonally selected, engineered NALM6 tumor cells (expressing truncated CD30 positive and HLA I and IIKO) at a fixed 1:1 ratio. Every 2–3 days, cells were counted by flow cytometry and replated in coculture with fresh tumor cells at a fixed ratio. The process was repeated until the CAR T cells could no longer eliminate tumor cells. Experiments were performed in the absence of cytokines. (19B and 19C) Graphs showing the cumulative proliferation fold change of CD30.CAR EBVST grown in (B) monoculture and (C) coculture. (19D) Bar graph showing the number of tumor cells counted by flow cytometry on day 8. (19E) Bar graph showing PD-1, Tim-3, and Lag-3 expression on CD30.CAR EBVST before the initiation of coculture (day 0) and on the final day of coculture (day 8). CD30.CAR EBVST produced from three donors were tested in vitro with similar findings. (19F) Schematic of the in vivo activation-induced cell death (AICD) model for 19G-19J. 2.5 × 106 clonally selected engineered NALM6 cells were injected intravenously into NSG (MHCKO) mice. 15 days later, 10 × 106 effector CD30.CAR (4-1BB spacer) EBVST were injected intravenously. (19G and 19H) (G) Images and graphs showing tumor bioluminescence captured by an IVIS Lumina S5 imaging system and (H) its quantification normalized to day 0. Quantified tumor burden (normalized to day 0) at 20 days after (19I) treatment is shown. Graphs show mean values ​​+ SD; P values ​​were determined using one-way analysis of variance and Dunnett's correction for multiple comparisons, comparing mean values ​​to the mean values ​​of SB9(CAS)-CD30.CAR samples.(19J) Flow cytometry analysis of blood samples collected from the facial vein at the indicated time points showing CD30.CAR EBVST levels in peripheral blood. Graphs show mean values ​​+ SD, and P values ​​(between CD30.CAR and SB9(CAS)-CD30.CAR samples) were determined using two-way analysis of variance with Dunnett's correction for multiple comparisons. [Figure 19D]Schematic, graph, and bar graph showing that SB9 (CAS) overexpression improves the expansion and tumor-killing efficacy of CD30.CAR EBVST in vivo. (19A) Schematic showing the in vitro coculture of CD30.CAR (4-1BB spacer) EBVST with clonally selected, engineered NALM6 tumor cells (expressing truncated CD30 positive and HLA I and IIKO) at a fixed 1:1 ratio. Every 2–3 days, cells were counted by flow cytometry and replated in coculture with fresh tumor cells at a fixed ratio. The process was repeated until the CAR T cells could no longer eliminate tumor cells. Experiments were performed in the absence of cytokines. (19B and 19C) Graphs showing the cumulative proliferation fold change of CD30.CAR EBVST grown in (B) monoculture and (C) coculture. (19D) Bar graph showing the number of tumor cells counted by flow cytometry on day 8. (19E) Bar graph showing PD-1, Tim-3, and Lag-3 expression on CD30.CAR EBVST before the initiation of coculture (day 0) and on the final day of coculture (day 8). CD30.CAR EBVST produced from three donors were tested in vitro with similar findings. (19F) Schematic of the in vivo activation-induced cell death (AICD) model for 19G-19J. 2.5 × 106 clonally selected engineered NALM6 cells were injected intravenously into NSG (MHCKO) mice. 15 days later, 10 × 106 effector CD30.CAR (4-1BB spacer) EBVST were injected intravenously. (19G and 19H) (G) Images and graphs showing tumor bioluminescence captured by an IVIS Lumina S5 imaging system and (H) its quantification normalized to day 0. Quantified tumor burden (normalized to day 0) at 20 days after (19I) treatment is shown. Graphs show mean values ​​+ SD; P values ​​were determined using one-way analysis of variance and Dunnett's correction for multiple comparisons, comparing mean values ​​to the mean values ​​of SB9(CAS)-CD30.CAR samples.(19J) Flow cytometry analysis of blood samples collected from the facial vein at the indicated time points showing CD30.CAR EBVST levels in peripheral blood. Graphs show mean values ​​+ SD, and P values ​​(between CD30.CAR and SB9(CAS)-CD30.CAR samples) were determined using two-way analysis of variance with Dunnett's correction for multiple comparisons. [Figure 19E]Schematic, graph, and bar graph showing that SB9 (CAS) overexpression improves the expansion and tumor-killing efficacy of CD30.CAR EBVST in vivo. (19A) Schematic showing the in vitro coculture of CD30.CAR (4-1BB spacer) EBVST with clonally selected, engineered NALM6 tumor cells (expressing truncated CD30 positive and HLA I and IIKO) at a fixed 1:1 ratio. Every 2–3 days, cells were counted by flow cytometry and replated in coculture with fresh tumor cells at a fixed ratio. The process was repeated until the CAR T cells could no longer eliminate tumor cells. Experiments were performed in the absence of cytokines. (19B and 19C) Graphs showing the cumulative proliferation fold change of CD30.CAR EBVST grown in (B) monoculture and (C) coculture. (19D) Bar graph showing the number of tumor cells counted by flow cytometry on day 8. (19E) Bar graph showing PD-1, Tim-3, and Lag-3 expression on CD30.CAR EBVST before the initiation of coculture (day 0) and on the final day of coculture (day 8). CD30.CAR EBVST produced from three donors were tested in vitro with similar findings. (19F) Schematic of the in vivo activation-induced cell death (AICD) model for 19G-19J. 2.5 × 106 clonally selected engineered NALM6 cells were injected intravenously into NSG (MHCKO) mice. 15 days later, 10 × 106 effector CD30.CAR (4-1BB spacer) EBVST were injected intravenously. (19G and 19H) (G) Images and graphs showing tumor bioluminescence captured by an IVIS Lumina S5 imaging system and (H) its quantification normalized to day 0. Quantified tumor burden (normalized to day 0) at 20 days after (19I) treatment is shown. Graphs show mean values ​​+ SD; P values ​​were determined using one-way analysis of variance and Dunnett's correction for multiple comparisons, comparing mean values ​​to the mean values ​​of SB9(CAS)-CD30.CAR samples.(19J) Flow cytometry analysis of blood samples collected from the facial vein at the indicated time points showing CD30.CAR EBVST levels in peripheral blood. Graphs show mean values ​​+ SD, and P values ​​(between CD30.CAR and SB9(CAS)-CD30.CAR samples) were determined using two-way analysis of variance with Dunnett's correction for multiple comparisons. [Figure 19F]Schematic, graph, and bar graph showing that SB9 (CAS) overexpression improves the expansion and tumor-killing efficacy of CD30.CAR EBVST in vivo. (19A) Schematic showing the in vitro coculture of CD30.CAR (4-1BB spacer) EBVST with clonally selected, engineered NALM6 tumor cells (expressing truncated CD30 positive and HLA I and IIKO) at a fixed 1:1 ratio. Every 2–3 days, cells were counted by flow cytometry and replated in coculture with fresh tumor cells at a fixed ratio. The process was repeated until the CAR T cells could no longer eliminate tumor cells. Experiments were performed in the absence of cytokines. (19B and 19C) Graphs showing the cumulative proliferation fold change of CD30.CAR EBVST grown in (B) monoculture and (C) coculture. (19D) Bar graph showing the number of tumor cells counted by flow cytometry on day 8. (19E) Bar graph showing PD-1, Tim-3, and Lag-3 expression on CD30.CAR EBVST before the initiation of coculture (day 0) and on the final day of coculture (day 8). CD30.CAR EBVST produced from three donors were tested in vitro with similar findings. (19F) Schematic of the in vivo activation-induced cell death (AICD) model for 19G-19J. 2.5 × 106 clonally selected engineered NALM6 cells were injected intravenously into NSG (MHCKO) mice. 15 days later, 10 × 106 effector CD30.CAR (4-1BB spacer) EBVST were injected intravenously. (19G and 19H) (G) Images and graphs showing tumor bioluminescence captured by an IVIS Lumina S5 imaging system and (H) its quantification normalized to day 0. Quantified tumor burden (normalized to day 0) at 20 days after (19I) treatment is shown. Graphs show mean values ​​+ SD; P values ​​were determined using one-way analysis of variance and Dunnett's correction for multiple comparisons, comparing mean values ​​to the mean values ​​of SB9(CAS)-CD30.CAR samples.(19J) Flow cytometry analysis of blood samples collected from the facial vein at the indicated time points showing CD30.CAR EBVST levels in peripheral blood. Graphs show mean values ​​+ SD, and P values ​​(between CD30.CAR and SB9(CAS)-CD30.CAR samples) were determined using two-way analysis of variance with Dunnett's correction for multiple comparisons. [Figure 19G]Schematic, graph, and bar graph showing that SB9 (CAS) overexpression improves the expansion and tumor-killing efficacy of CD30.CAR EBVST in vivo. (19A) Schematic showing the in vitro coculture of CD30.CAR (4-1BB spacer) EBVST with clonally selected, engineered NALM6 tumor cells (expressing truncated CD30 positive and HLA I and IIKO) at a fixed 1:1 ratio. Every 2–3 days, cells were counted by flow cytometry and replated in coculture with fresh tumor cells at a fixed ratio. The process was repeated until the CAR T cells could no longer eliminate tumor cells. Experiments were performed in the absence of cytokines. (19B and 19C) Graphs showing the cumulative proliferation fold change of CD30.CAR EBVST grown in (B) monoculture and (C) coculture. (19D) Bar graph showing the number of tumor cells counted by flow cytometry on day 8. (19E) Bar graph showing PD-1, Tim-3, and Lag-3 expression on CD30.CAR EBVST before the initiation of coculture (day 0) and on the final day of coculture (day 8). CD30.CAR EBVST produced from three donors were tested in vitro with similar findings. (19F) Schematic of the in vivo activation-induced cell death (AICD) model for 19G-19J. 2.5 × 106 clonally selected engineered NALM6 cells were injected intravenously into NSG (MHCKO) mice. 15 days later, 10 × 106 effector CD30.CAR (4-1BB spacer) EBVST were injected intravenously. (19G and 19H) (G) Images and graphs showing tumor bioluminescence captured by an IVIS Lumina S5 imaging system and (H) its quantification normalized to day 0. Quantified tumor burden (normalized to day 0) at 20 days after (19I) treatment is shown. Graphs show mean values ​​+ SD; P values ​​were determined using one-way analysis of variance and Dunnett's correction for multiple comparisons, comparing mean values ​​to the mean values ​​of SB9(CAS)-CD30.CAR samples.(19J) Flow cytometry analysis of blood samples collected from the facial vein at the indicated time points showing CD30.CAR EBVST levels in peripheral blood. Graphs show mean values ​​+ SD, and P values ​​(between CD30.CAR and SB9(CAS)-CD30.CAR samples) were determined using two-way analysis of variance with Dunnett's correction for multiple comparisons. [Figure 19H]Schematic, graph, and bar graph showing that SB9 (CAS) overexpression improves the expansion and tumor-killing efficacy of CD30.CAR EBVST in vivo. (19A) Schematic showing the in vitro coculture of CD30.CAR (4-1BB spacer) EBVST with clonally selected, engineered NALM6 tumor cells (expressing truncated CD30 positive and HLA I and IIKO) at a fixed 1:1 ratio. Every 2–3 days, cells were counted by flow cytometry and replated in coculture with fresh tumor cells at a fixed ratio. The process was repeated until the CAR T cells could no longer eliminate tumor cells. Experiments were performed in the absence of cytokines. (19B and 19C) Graphs showing the cumulative proliferation fold change of CD30.CAR EBVST grown in (B) monoculture and (C) coculture. (19D) Bar graph showing the number of tumor cells counted by flow cytometry on day 8. (19E) Bar graph showing PD-1, Tim-3, and Lag-3 expression on CD30.CAR EBVST before the initiation of coculture (day 0) and on the final day of coculture (day 8). CD30.CAR EBVST produced from three donors were tested in vitro with similar findings. (19F) Schematic of the in vivo activation-induced cell death (AICD) model for 19G-19J. 2.5 × 106 clonally selected engineered NALM6 cells were injected intravenously into NSG (MHCKO) mice. 15 days later, 10 × 106 effector CD30.CAR (4-1BB spacer) EBVST were injected intravenously. (19G and 19H) (G) Images and graphs showing tumor bioluminescence captured by an IVIS Lumina S5 imaging system and (H) its quantification normalized to day 0. Quantified tumor burden (normalized to day 0) at 20 days after (19I) treatment is shown. Graphs show mean values ​​+ SD; P values ​​were determined using one-way analysis of variance and Dunnett's correction for multiple comparisons, comparing mean values ​​to the mean values ​​of SB9(CAS)-CD30.CAR samples.(19J) Flow cytometry analysis of blood samples collected from the facial vein at the indicated time points showing CD30.CAR EBVST levels in peripheral blood. Graphs show mean values ​​+ SD, and P values ​​(between CD30.CAR and SB9(CAS)-CD30.CAR samples) were determined using two-way analysis of variance with Dunnett's correction for multiple comparisons. [Figure 19I]Schematic, graph, and bar graph showing that SB9 (CAS) overexpression improves the expansion and tumor-killing efficacy of CD30.CAR EBVST in vivo. (19A) Schematic showing the in vitro coculture of CD30.CAR (4-1BB spacer) EBVST with clonally selected, engineered NALM6 tumor cells (expressing truncated CD30 positive and HLA I and IIKO) at a fixed 1:1 ratio. Every 2–3 days, cells were counted by flow cytometry and replated in coculture with fresh tumor cells at a fixed ratio. The process was repeated until the CAR T cells could no longer eliminate tumor cells. Experiments were performed in the absence of cytokines. (19B and 19C) Graphs showing the cumulative proliferation fold change of CD30.CAR EBVST grown in (B) monoculture and (C) coculture. (19D) Bar graph showing the number of tumor cells counted by flow cytometry on day 8. (19E) Bar graph showing PD-1, Tim-3, and Lag-3 expression on CD30.CAR EBVST before the initiation of coculture (day 0) and on the final day of coculture (day 8). CD30.CAR EBVST produced from three donors were tested in vitro with similar findings. (19F) Schematic of the in vivo activation-induced cell death (AICD) model for 19G-19J. 2.5 × 106 clonally selected engineered NALM6 cells were injected intravenously into NSG (MHCKO) mice. 15 days later, 10 × 106 effector CD30.CAR (4-1BB spacer) EBVST were injected intravenously. (19G and 19H) (G) Images and graphs showing tumor bioluminescence captured by an IVIS Lumina S5 imaging system and (H) its quantification normalized to day 0. Quantified tumor burden (normalized to day 0) at 20 days after (19I) treatment is shown. Graphs show mean values ​​+ SD; P values ​​were determined using one-way analysis of variance and Dunnett's correction for multiple comparisons, comparing mean values ​​to the mean values ​​of SB9(CAS)-CD30.CAR samples.(19J) Flow cytometry analysis of blood samples collected from the facial vein at the indicated time points showing CD30.CAR EBVST levels in peripheral blood. Graphs show mean values ​​+ SD, and P values ​​(between CD30.CAR and SB9(CAS)-CD30.CAR samples) were determined using two-way analysis of variance with Dunnett's correction for multiple comparisons. [Figure 19J]Schematic, graph, and bar graph showing that SB9 (CAS) overexpression improves the expansion and tumor-killing efficacy of CD30.CAR EBVST in vivo. (19A) Schematic showing the in vitro coculture of CD30.CAR (4-1BB spacer) EBVST with clonally selected, engineered NALM6 tumor cells (expressing truncated CD30 positive and HLA I and IIKO) at a fixed 1:1 ratio. Every 2–3 days, cells were counted by flow cytometry and replated in coculture with fresh tumor cells at a fixed ratio. The process was repeated until the CAR T cells could no longer eliminate tumor cells. Experiments were performed in the absence of cytokines. (19B and 19C) Graphs showing the cumulative proliferation fold change of CD30.CAR EBVST grown in (B) monoculture and (C) coculture. (19D) Bar graph showing the number of tumor cells counted by flow cytometry on day 8. (19E) Bar graph showing PD-1, Tim-3, and Lag-3 expression on CD30.CAR EBVST before the initiation of coculture (day 0) and on the final day of coculture (day 8). CD30.CAR EBVST produced from three donors were tested in vitro with similar findings. (19F) Schematic of the in vivo activation-induced cell death (AICD) model for 19G-19J. 2.5 × 106 clonally selected engineered NALM6 cells were injected intravenously into NSG (MHCKO) mice. 15 days later, 10 × 106 effector CD30.CAR (4-1BB spacer) EBVST were injected intravenously. (19G and 19H) (G) Images and graphs showing tumor bioluminescence captured by an IVIS Lumina S5 imaging system and (H) its quantification normalized to day 0. Quantified tumor burden (normalized to day 0) at 20 days after (19I) treatment is shown. Graphs show mean values ​​+ SD; P values ​​were determined using one-way analysis of variance and Dunnett's correction for multiple comparisons, comparing mean values ​​to the mean values ​​of SB9(CAS)-CD30.CAR samples.(19J) Flow cytometry analysis of blood samples collected from the facial vein at the indicated time points showing CD30.CAR EBVST levels in peripheral blood. Graphs show mean values ​​+ SD, and P values ​​(between CD30.CAR and SB9(CAS)-CD30.CAR samples) were determined using two-way analysis of variance with Dunnett's correction for multiple comparisons. [Figure 20] Graphs showing CD30-positive B-cell acute lymphoblastic leukemia (B-ALL) tumor burden in two in vivo models used to test SB9-mediated protection of grafts against allorejection (allo-rejection model) and AICD (AICD model), respectively. Bioluminescence signals from engineered NALM6.eGFP-ffLuc prepared from either FACS sorting (allo-rejection model) or clonal selection (AICD model) were measured at baseline (day 0). Each point represents an individual mouse. Lines with error bars indicate mean values ​​+ SD. [Figure 21A]Schematic, bar graph, and graph showing that SB9 (CAS) overexpression, but not SB9 (WT) overexpression, protects CD30.CAR EBVST from Fas-mediated apoptosis. (21A) Schematic of the apoptotic pathways involved in allorejection and activation-induced cell death (AICD). (21B and 21C) Bar graphs showing luminescence measurements of (B) a CellTiter-Glo® assay taken 16 hours after seeding of CD30.CAR (4-1BB spacer) EBVST, or (C) a Caspase-Glo® 3 / 7 CellTiter-Glo® assay taken 45 minutes after seeding of CD30.CAR EBVST, with or without the presence of the pan-caspase inhibitor zVAD FMK (100 μM), on plates coated with PBS or anti-CD95 antibody. Each point within the bar graph represents a unique donor, and the bar represents the median (n = 3-4). P values ​​were determined by two-way ANOVA and Tukey's post-hoc test. **, P<0.01; ****, P<0.0001. (21D) Graph showing intracellular flow cytometry analysis of SB9(CAS)-His-CD30.CAR EBVST 16 hours after plating with SB9(CAS)-His-CD30.CAR EBVST, with or without the presence of the pan-caspase inhibitor zVAD FMK, on ​​plates coated with PBS or anti-CD95 antibody. SB9(CAS)-His-CD30.CAR EBVST produced from three donors were studied and found to have similar results. [Figure 21B]Schematic, bar graph, and graph showing that SB9 (CAS) overexpression, but not SB9 (WT) overexpression, protects CD30.CAR EBVST from Fas-mediated apoptosis. (21A) Schematic of the apoptotic pathways involved in allorejection and activation-induced cell death (AICD). (21B and 21C) Bar graphs showing luminescence measurements of (B) a CellTiter-Glo® assay taken 16 hours after seeding of CD30.CAR (4-1BB spacer) EBVST, or (C) a Caspase-Glo® 3 / 7 CellTiter-Glo® assay taken 45 minutes after seeding of CD30.CAR EBVST, with or without the presence of the pan-caspase inhibitor zVAD FMK (100 μM), on plates coated with PBS or anti-CD95 antibody. Each point within the bar graph represents a unique donor, and the bar represents the median (n = 3-4). P values ​​were determined by two-way ANOVA and Tukey's post-hoc test. **, P<0.01; ****, P<0.0001. (21D) Graph showing intracellular flow cytometry analysis of SB9(CAS)-His-CD30.CAR EBVST 16 hours after plating with SB9(CAS)-His-CD30.CAR EBVST, with or without the presence of the pan-caspase inhibitor zVAD FMK, on ​​plates coated with PBS or anti-CD95 antibody. SB9(CAS)-His-CD30.CAR EBVST produced from three donors were studied and found to have similar results. [Figure 21C]Schematic, bar graph, and graph showing that SB9 (CAS) overexpression, but not SB9 (WT) overexpression, protects CD30.CAR EBVST from Fas-mediated apoptosis. (21A) Schematic of the apoptotic pathways involved in allorejection and activation-induced cell death (AICD). (21B and 21C) Bar graphs showing luminescence measurements of (B) a CellTiter-Glo® assay taken 16 hours after seeding of CD30.CAR (4-1BB spacer) EBVST, or (C) a Caspase-Glo® 3 / 7 CellTiter-Glo® assay taken 45 minutes after seeding of CD30.CAR EBVST, with or without the presence of the pan-caspase inhibitor zVAD FMK (100 μM), on plates coated with PBS or anti-CD95 antibody. Each point within the bar graph represents a unique donor, and the bar represents the median (n = 3-4). P values ​​were determined by two-way ANOVA and Tukey's post-hoc test. **, P<0.01; ****, P<0.0001. (21D) Graph showing intracellular flow cytometry analysis of SB9(CAS)-His-CD30.CAR EBVST 16 hours after plating with SB9(CAS)-His-CD30.CAR EBVST, with or without the presence of the pan-caspase inhibitor zVAD FMK, on ​​plates coated with PBS or anti-CD95 antibody. SB9(CAS)-His-CD30.CAR EBVST produced from three donors were studied and found to have similar results. [Figure 21D]Schematic, bar graph, and graph showing that SB9 (CAS) overexpression, but not SB9 (WT) overexpression, protects CD30.CAR EBVST from Fas-mediated apoptosis. (21A) Schematic of the apoptotic pathways involved in allorejection and activation-induced cell death (AICD). (21B and 21C) Bar graphs showing luminescence measurements of (B) a CellTiter-Glo® assay taken 16 hours after seeding of CD30.CAR (4-1BB spacer) EBVST, or (C) a Caspase-Glo® 3 / 7 CellTiter-Glo® assay taken 45 minutes after seeding of CD30.CAR EBVST, with or without the presence of the pan-caspase inhibitor zVAD FMK (100 μM), on plates coated with PBS or anti-CD95 antibody. Each point within the bar graph represents a unique donor, and the bar represents the median (n = 3-4). P values ​​were determined by two-way ANOVA and Tukey's post-hoc test. **, P<0.01; ****, P<0.0001. (21D) Graph showing intracellular flow cytometry analysis of SB9(CAS)-His-CD30.CAR EBVST 16 hours after plating with SB9(CAS)-His-CD30.CAR EBVST, with or without the presence of the pan-caspase inhibitor zVAD FMK, on ​​plates coated with PBS or anti-CD95 antibody. SB9(CAS)-His-CD30.CAR EBVST produced from three donors were studied and found to have similar results. [Figure 22A]22A is a schematic, graph, and bar graph showing evaluation of SB9 (CAS)-mediated protection against Fas-mediated apoptosis in SB9 (CAS)-overexpressing NK cells. (22B) is a graph showing the manufacturing expansion of non-transduced (NT) and SB9 (CAS)-overexpressing NK cells. (22C) is a bar graph showing a CellTiter-Glo® assay taken 1 hour after plating NK cells with or without the presence of the pan-caspase inhibitor zVAD FMK (100 μM) on plates coated with PBS or anti-CD95 antibody. (19D) is a graph showing intracellular flow cytometry analysis of SB9 (CAS)-His tag expression 4 hours after plating SB9 (CAS)-His NK cells with or without the presence of the pan-caspase inhibitor zVAD FMK on plates coated with PBS or anti-CD95 antibody. SB9(CAS)-His NK cells produced from two donors were studied and found to have similar results. [Figure 22B] 22A is a schematic, graph, and bar graph showing evaluation of SB9 (CAS)-mediated protection against Fas-mediated apoptosis in SB9 (CAS)-overexpressing NK cells. (22B) is a graph showing the manufacturing expansion of non-transduced (NT) and SB9 (CAS)-overexpressing NK cells. (22C) is a bar graph showing a CellTiter-Glo® assay taken 1 hour after plating NK cells with or without the presence of the pan-caspase inhibitor zVAD FMK (100 μM) on plates coated with PBS or anti-CD95 antibody. (19D) is a graph showing intracellular flow cytometry analysis of SB9 (CAS)-His tag expression 4 hours after plating SB9 (CAS)-His NK cells with or without the presence of the pan-caspase inhibitor zVAD FMK on plates coated with PBS or anti-CD95 antibody. SB9(CAS)-His NK cells produced from two donors were studied and found to have similar results. [Figure 22C] 22A is a schematic, graph, and bar graph showing evaluation of SB9 (CAS)-mediated protection against Fas-mediated apoptosis in SB9 (CAS)-overexpressing NK cells. (22B) is a graph showing the manufacturing expansion of non-transduced (NT) and SB9 (CAS)-overexpressing NK cells. (22C) is a bar graph showing a CellTiter-Glo® assay taken 1 hour after plating NK cells with or without the presence of the pan-caspase inhibitor zVAD FMK (100 μM) on plates coated with PBS or anti-CD95 antibody. (19D) is a graph showing intracellular flow cytometry analysis of SB9 (CAS)-His tag expression 4 hours after plating SB9 (CAS)-His NK cells with or without the presence of the pan-caspase inhibitor zVAD FMK on plates coated with PBS or anti-CD95 antibody. SB9(CAS)-His NK cells produced from two donors were studied and found to have similar results. [Figure 22D]22A is a schematic, graph, and bar graph showing evaluation of SB9 (CAS)-mediated protection against Fas-mediated apoptosis in SB9 (CAS)-overexpressing NK cells. (22B) is a graph showing the manufacturing expansion of non-transduced (NT) and SB9 (CAS)-overexpressing NK cells. (22C) is a bar graph showing a CellTiter-Glo® assay taken 1 hour after plating NK cells with or without the presence of the pan-caspase inhibitor zVAD FMK (100 μM) on plates coated with PBS or anti-CD95 antibody. (19D) is a graph showing intracellular flow cytometry analysis of SB9 (CAS)-His tag expression 4 hours after plating SB9 (CAS)-His NK cells with or without the presence of the pan-caspase inhibitor zVAD FMK on plates coated with PBS or anti-CD95 antibody. SB9(CAS)-His NK cells produced from two donors were studied and found to have similar results. DETAILED DESCRIPTION OF THE INVENTION

[0127] The present disclosure is based on the present inventors' unexpected discovery that upregulating SERPINB9 expression / activity in immune cells increases their persistence / survival in the presence of allogeneic effector immune cells.This is believed to be the result of inhibiting the activity of granzyme B in such cells.Therefore, upregulating SERPINB9 expression / activity in cells used in methods for treating disease by adoptive cell transfer increases their persistence in recipient subjects, especially in the case of allogeneic transplantation.

[0128] It is shown herein that cells modified to upregulate SERPINB9 expression / activity grow / expand in vitro to a similar extent as comparable cells lacking such modifications, and when such cells are effector immune cells, they exhibit similar effector activity as comparable cells lacking modifications that upregulate SERPINB9 expression / activity. Also, cells modified in this manner have a comparable toxicity profile (i.e., to an allogeneic recipient subject) compared to comparable cells lacking such modifications. SERPINB9 SERPINB9 (also known as cytoplasmic antiproteinase 3 (CAP-3 / CAP3) or peptidase inhibitor 9 (PI-9)) is a protein identified by UniProt P50453. Human SERPINB9 has the amino acid sequence set forth in SEQ ID NO: 1. SERPINB9 is a member of the serpin family of protease inhibitors. SERPINB9 is an intracellular inhibitor of the cytotoxic lymphocytic serine protease granzyme B.

[0129] The structure and function of SERPINB9 are reviewed in, for example, Kaiserman et al., Cell Death Differ. (2010) 17(4):586-95; Bird et al., Mol Cell Biol. (1998), 18(11):6387-98; and Bird et al., Cell Death Differ. (2014) 21, 876-887, all of which are incorporated herein by reference in their entirety. SERPINB9 is present in the nucleus and cytoplasm of cells and is endogenously expressed in immune-privileged sites (e.g., placenta and lung), T cells, antigen-presenting cells, and hematopoietic stem cells. In T cells, SERPINB9 has been reported to prevent friendly killing by misdirected granzyme B at the immune synapse. SERPINB9 also inhibits the activity of serine proteases (e.g., granzyme B) against enzyme-producing cells. That is, SERPINB9 protects activated serine protease (e.g., granzyme B)-expressing effector immune cells from the action of the serine proteases they produce—i.e., SERPINB9 protects cells from autolysis by their own serine proteases (e.g., granzyme B).

[0130] Serpins present their exposed reactive center loop (RCL) to their target serine proteases, which then cleave the peptide bond between two residues, P1 and P1', of the serpin. This cleavage induces a conformational change in the serpin, irreversibly trapping the protease within a covalent complex. Residues surrounding P1 contribute to protease binding, and mutations of the serpin RCL have been shown to abolish inhibition and / or alter target specificity.

[0131] The RCL of human SERPINB9 is formed by positions 334 to 348 of SEQ ID NO: 1 and is shown in SEQ ID NO: 2. The P1 residue of human SERPINB9 is a glutamic acid residue at position 340 of SEQ ID NO: 1, and the P1' residue of human SERPINB9 is a cysteine ​​residue at position 341 of SEQ ID NO: 1.

[0132] The modifications T327R and E340A in human SERPINB9 have been shown to abolish the ability of SERPINB9 to inhibit granzyme B in vitro (see Bird et al., Mol Cell Biol. (1998), 18(11):6387-98). The E340A modification removes a critical glutamic acid residue in P1, and the T327R modification disrupts a conserved proximal hinge domain required for serpin loop mobility and inhibitory function.

[0133] Wild-type SERPINB9 has also been reported to inhibit caspase activity (see, e.g., Annand et al., Biochem. J. (1999) 342(Pt3):655-665). The modification E340D has been shown to reduce granzyme B inhibition by SERPINB9 but broaden its target specificity, increasing its inhibitory activity against caspases, and thus inhibiting Fas-mediated apoptosis (see, Bird et al., Mol Cell Biol. (1998), 18(11):6387-98).

[0134] The modifications C341S and C342S have been shown to reduce reactive oxygen species (ROS)-mediated inactivation of SERPINB9 while retaining serine protease inhibitory activity (see Mangan et al., J. Biol. Chem. (2016) 291(7):3626-38).

[0135] As used herein, "SERPINB9" refers to SERPINB9 from any species, including isoforms, fragments, variants or homologs from any species. "Fragment" generally refers to a small portion of a reference protein. A fragment of SERPINB9 may have a minimum length of 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, or 350 amino acids, and a maximum length of 20, 30, 40, 50, 100, 150, 200, 250, 300, or 350 amino acids. "Isoform" generally refers to a variant of a reference protein that is expressed by the same species as the species of the reference protein. "Homolog" generally refers to a variant of a reference protein that is produced by a species different from the species of the reference protein. Homologs include orthologs.

[0136] A "variant" generally refers to a protein having an amino acid sequence that contains one or more amino acid substitutions, insertions, deletions, or other modifications compared to the amino acid sequence of a reference protein, but that retains a significant degree of amino acid sequence identity (e.g., at least 70%) with the amino acid sequence of the reference protein.

[0137] In some embodiments, the SERPINB9 is from a mammal (e.g., a primate (rhesus monkey, cynomolgus monkey, or human) and / or a rodent (e.g., a rat or mouse)). SERPINB9 isoforms, fragments, variants, or homologs may optionally be characterized as having at least 70%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the amino acid sequence of an immature or mature SERPINB9 isoform from a given species, e.g., human.

[0138] In some embodiments, SERPINB9 is human SERPINB9. In some embodiments, SERPINB9 is cynomolgous macaque SERPINB9. In some embodiments, SERPINB9 is mouse SERPINB9 (also known as "SPI6").

[0139] The isoform, fragment, variant or homolog may optionally be a functional isoform, fragment, variant or homolog that has the functional activity of, for example, a reference SERPINB9 (e.g., human SERPINB9) as determined by analysis with a suitable assay for functional activity (e.g., protease inhibition (e.g., serine protease (e.g., granzyme B) and / or cysteine ​​protease (e.g., caspase) inhibition)).

[0140] In some embodiments, SERPINB9 comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1, for example, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% amino acid sequence identity.

[0141] Aspects and embodiments of the present disclosure relate to SERPINB9 variants. As referred to herein, a "SERPINB9 variant" refers to a SERPINB9 that includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) modifications compared to a reference SERPINB9.

[0142] "Modification" refers to a difference with respect to a reference amino acid sequence. The reference amino acid sequence may be the amino acid sequence encoded by the most common nucleotide sequence of a gene encoding a related protein. In this embodiment (and also more generally in the art), "modification" may also be referred to as "substitution" or "mutation."

[0143] In a preferred embodiment, the SERPINB9 variant described in the present disclosure comprises an amino acid sequence having at least 70% amino acid sequence identity with SEQ ID NO:1 and comprises one or more (e.g., 1 of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) modifications compared to SEQ ID NO:1.

[0144] The modification typically involves the replacement of an amino acid residue with a non-identical "replacement" amino acid residue. The replacement amino acid residue of the modification described herein may be a naturally occurring amino acid residue (i.e., encoded by the genetic code) selected from alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val), which is not identical to the amino acid residue at the relevant position in the amino acid sequence before modification. In some embodiments, the replacement amino acid residue of the modification may be a non-naturally occurring amino acid residue—i.e., an amino acid residue other than those listed in the preceding sentence. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, aib, and other amino acid residue analogs, such as those described in Ellman et al., Meth. Enzym. 202 (1991) 301-336.

[0145] The SERPINB9 variants described in this disclosure may contain modifications at specific positions in the amino acid sequence of SERPINB9. As used herein, when referring to a position in human wild-type SERPINB9 (i.e., having the amino acid sequence of SEQ ID NO: 1), the position in a homologous sequence corresponding to human wild-type SERPINB9 is also intended. Positions corresponding to positions identified in human wild-type SERPINB9 can be identified by sequence alignment, which can be performed using sequence alignment software such as ClustalOmega (Soding, J. 2005, Bioinformatics 21, 951-960).

[0146] In some embodiments, the SERPINB9 variants described herein comprise an alteration at one or more of the following positions (numbered relative to SEQ ID NO: 1): 340, 341, and 342. In some embodiments, the SERPINB9 variants comprise an alteration at position 340. In some embodiments, the SERPINB9 variants comprise an alteration at positions 341 and / or 342. In some embodiments, the SERPINB9 variants comprise an alteration at positions 340, 341, and / or 342.

[0147] In some embodiments, the SERPINB9 variant comprises 340D. In some embodiments, the SERPINB9 variant comprises 341S. In some embodiments, the SERPINB9 variant comprises 342S.

[0148] In some embodiments, a SERPINB9 variant comprises the amino acid sequence set forth in SEQ ID NO: 3, which amino acid sequence is not identical to SEQ ID NO: 2. In some embodiments, a SERPINB9 variant comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4, which amino acid sequence is not identical to SEQ ID NO: 1.

[0149] In some embodiments, SERPINB9 described in the present disclosure comprises or consists of an amino acid sequence having the amino acid sequence of SEQ ID NO: 1, 4, 5, 6 or 7, or an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 1, 4, 5, 6 or 7, for example, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more. In some embodiments, SERPINB9 described in the present disclosure comprises or consists of an amino acid sequence having the amino acid sequence of SEQ ID NO: 4, 5, 6 or 7, or an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 4, 5, 6 or 7, for example, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more. In some embodiments, SERPINB9 described in the present disclosure comprises or consists of an amino acid sequence having the amino acid sequence of SEQ ID NO: 4, 5 or 7, or an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 4, 5 or 7, for example, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more.

[0150] In some embodiments, SERPINB9 (or a SERPINB9 variant) comprises or consists of an amino acid sequence having the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 1, for example, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity.

[0151] In some embodiments, SERPINB9 (or a SERPINB9 variant) comprises or consists of an amino acid sequence having the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 4, for example, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity.

[0152] In some embodiments, SERPINB9 (or a SERPINB9 variant) comprises or consists of an amino acid sequence having the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 5, for example, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity.

[0153] In some embodiments, SERPINB9 (or a SERPINB9 variant) comprises or consists of an amino acid sequence having the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 6, for example, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity.

[0154] In some embodiments, SERPINB9 (or a SERPINB9 variant) comprises or consists of an amino acid sequence having the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 7, for example, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0155] In some embodiments, SERPINB9 (or a SERPINB9 variant) described herein does not comprise or consist of the amino acid sequence of SEQ ID NO: 1. In some embodiments, SERPINB9 comprises or consists of an amino acid sequence that is not identical to the amino acid sequence of SEQ ID NO: 1.

[0156] In some embodiments, SERPINB9 (or a SERPINB9 variant) described herein does not comprise or consist of the amino acid sequence of SEQ ID NO: 5. In some embodiments, SERPINB9 comprises or consists of an amino acid sequence that is not identical to the amino acid sequence of SEQ ID NO: 5.

[0157] In some embodiments, SERPINB9 (or a SERPINB9 variant) described herein does not comprise or consist of the amino acid sequence of SEQ ID NO: 6. In some embodiments, SERPINB9 comprises or consists of an amino acid sequence that is not identical to the amino acid sequence of SEQ ID NO: 6.

[0158] In some embodiments, SERPINB9 (or a SERPINB9 variant) described herein does not comprise or consist of the amino acid sequence of SEQ ID NO: 7. In some embodiments, SERPINB9 comprises or consists of an amino acid sequence that is not identical to the amino acid sequence of SEQ ID NO: 7.

[0159] In some embodiments, SERPINB9 (or a SERPINB9 variant) described herein does not comprise or consist of the amino acid sequence of SEQ ID NO: 47. In some embodiments, SERPINB9 comprises or consists of an amino acid sequence that is not identical to the amino acid sequence of SEQ ID NO: 47.

[0160] In some embodiments, one or more amino acids of an amino acid sequence referred to herein (e.g., SERPINB9 or a SERPINB9 variant) are substituted with another amino acid. A substitution includes replacing an amino acid residue with a non-identical "replacement" amino acid residue. The replacement amino acid residue of the substitution described in the present disclosure can be a naturally occurring amino acid residue (i.e., encoded by genetic code) selected from alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr) and valine (Val), which is not identical to the amino acid residue at the corresponding position of the equivalent unsubstituted amino acid sequence.In some embodiments, the replacement amino acid can be a non-naturally occurring amino acid residue—i.e., an amino acid residue other than the amino acid residues listed in the preceding sentence. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine, aib, and other amino acid residue analogs such as those described in Ellman et al., Meth. Enzym. (1991) 202:301-336.

[0161] In some embodiments, substitutions can be biochemically conservative. In some embodiments, when a substituted amino acid is shown in one of columns 1-5 of the table below, the replacement amino acid of the substitution is another, non-identical amino acid shown in the same column.

[0162] [Table 1]

[0163] To illustrate, in some embodiments where the substitution is for a Met residue, the replacement amino acid may be selected from Ala, Val, Leu, lie, Trp, Tyr, Phe, and norleucine.

[0164] In some embodiments, the replacement amino acid in the substitution may have the same side chain polarity as the amino acid residue it replaces. In some embodiments, the replacement amino acid in the substitution may have the same side chain charge (at pH 7.4) as the amino acid residue it replaces.

[0165] [Table 2]

[0166] That is, in some embodiments, a nonpolar amino acid is substituted with another non-identical nonpolar amino acid; in some embodiments, a polar amino acid is substituted with another non-identical polar amino acid; in some embodiments, an acidic polar amino acid is substituted with another non-identical acidic polar amino acid; in some embodiments, a basic polar amino acid is substituted with another non-identical basic polar amino acid; in some embodiments, a neutral amino acid is substituted with another non-identical neutral amino acid; in some embodiments, a positive amino acid is substituted with another non-identical positive amino acid; in some embodiments, a negative amino acid is substituted with another non-identical negative amino acid.

[0167] In some embodiments, substitutions may be functionally conservative, i.e., substitutions may not affect (or may not substantially affect) one or more functional properties (e.g., target binding) of an antigen-binding molecule containing the substitution, compared to a comparable unsubstituted molecule.

[0168] Aspects and embodiments of the present disclosure relate to SERPINB9 polypeptides. The SERPINB9 polypeptides described in the present disclosure can be SERPINB9 or SERPINB9 variants described in any of the embodiments described hereinabove.

[0169] The present disclosure also provides fusion polypeptides comprising a SERPINB9 polypeptide described herein and another polypeptide of interest. The amino acid sequence of the SERPINB9 polypeptide and the amino acid sequence of the other polypeptide of interest can be provided.

[0170] In accordance with such aspects and embodiments, the polypeptide of interest may be a molecule for directing the activity of immune cells to cells expressing a given target antigen. In some embodiments, the molecule for directing the activity of immune cells to cells expressing a given target antigen may be a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0171] According to such embodiments, the SERPINB9 polypeptide and other polypeptides of interest can be joined by a linker sequence. In some embodiments, the linker sequence can be a cleavable linker. That is, the linker sequence can include a sequence of amino acids capable of being cleaved. For example, the linker sequence can include a peptide bond—i.e., a sequence capable of acting as a substrate for an enzyme capable of cleaving the cleavage site. Many such cleavage sites are known to and can be used by those skilled in the art of molecular biology. In some embodiments, the cleavable linker can include a self-cleavage site. A self-cleavage site is automatically cleaved without the need for enzymatic treatment. A 2A cleavage site includes the canonical "NPGP" motif, which is cleaved at "G / P." 2A cleavage sites include P2A, T2A, E2A, and F2A self-cleavage sites. A linker sequence containing a 2A self-cleavage site is referred to herein as a 2A linker. Certain constructs described in the experimental examples use a P2A self-cleavage site (i.e., in a P2A linker). Certain constructs described in the experimental examples use a T2A self-cleavage site (ie, in the T2A linker).

[0172] In some embodiments, a fusion polypeptide comprises the structure N-terminus-[...]-[SERPINB9 polypeptide]-[other polypeptide of interest]-[...]-C-terminus. In some embodiments, a fusion polypeptide comprises the structure N-terminus-[...]-[SERPINB9 polypeptide]-[cleavable linker]-[other polypeptide of interest]-[...]-C-terminus. In some embodiments, a fusion polypeptide comprises the structure N-terminus-[...]-[SERPINB9 polypeptide]-[cleavable linker]-[CAR]-[...]-C-terminus. In some embodiments, a fusion polypeptide comprises the structure N-terminus-[...]-[SERPINB9 polypeptide]-[2A linker]-[CAR]-[...]-C-terminus. In some embodiments, a fusion polypeptide comprises the structure N-terminus-[...]-[SERPINB9 polypeptide]-[2A linker]-[CAR]-[2A linker]-[other polypeptide of interest]-[...]-C-terminus. In some embodiments, a fusion polypeptide comprises the structure N-terminus-[...]-[SERPINB9 polypeptide]-[2A linker]-[other polypeptide of interest]-[2A linker]-[CAR]-[...]-C-terminus. In some embodiments, a fusion polypeptide comprises the structure N-terminus-[...]-[other polypeptide of interest]-[SERPINB9 polypeptide]-[...]-C-terminus. In some embodiments, a fusion polypeptide comprises the structure N-terminus-[...]-[other polypeptide of interest]-[cleavable linker]-[SERPINB9 polypeptide]-[...]-C-terminus. In some embodiments, a fusion polypeptide comprises the structure N-terminus-[...]-[CAR]-[cleavable linker]-[SERPINB9 polypeptide]-[...]-C-terminus. In some embodiments, a fusion polypeptide comprises the structure N-terminus-[...]-[CAR]-[2A linker]-[SERPINB9 polypeptide]-[...]-C-terminus. In some embodiments, the fusion polypeptide comprises the structure N-terminus-[...]-[CAR]-[2A linker]-[SERPINB9 polypeptide]-[2A linker]-[other polypeptide of interest]-[...]-C-terminus.In some embodiments, a fusion polypeptide comprises the structure N-terminus-[...]-[CAR]-[2A linker]-[other polypeptide of interest]-[2A linker]-[SERPINB9 polypeptide]-[...]-C-terminus. In some embodiments, a fusion polypeptide comprises the structure N-terminus-[...]-[other polypeptide of interest]-[2A linker]-[SERPINB9 polypeptide]-[2A linker]-[CAR]-[...]-C-terminus. In some embodiments, a fusion polypeptide comprises the structure N-terminus-[...]-[other polypeptide of interest]-[2A linker]-[CAR]-[2A linker]-[SERPINB9 polypeptide]-[...]-C-terminus.

[0173] When used in depicting polypeptide structures herein, "[...]" indicates the optional presence of an additional polypeptide / protein domain / region of interest. For example, in the structures in the preceding section, an additional polypeptide / protein domain / region of interest may optionally be present upstream of the SERPINB9 polypeptide, before the N-terminus of the fusion polypeptide. Furthermore, when used in depicting polypeptide structures herein, "[-]" indicates an optional linker sequence. For example, in the last structure in the preceding section, a linker sequence may optionally be provided between the SERPINB9 polypeptide and the 2A linker.

[0174] In some embodiments, a polypeptide described in the present disclosure comprises or consists of an amino acid sequence having at least 70% amino acid sequence identity to SEQ ID NO: 43, 44, 45 or 46, e.g., 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more or 100% amino acid sequence identity. Agents for increasing SERPINB9 expression or activity Aspects and embodiments of the present disclosure relate to agents for increasing the expression or activity of SERPINB9. As used herein, "agents for increasing the expression or activity of SERPINB9" refers to agents for increasing the expression (i.e., gene and / or protein expression) or activity of SERPINB9. Agents that upregulate the expression and / or activity of SERPINB9 may alternatively be referred to as agents for increasing / enhancing the expression or activity of SERPINB9. Such agents may also be referred to as "SERPINB9 agonists" or "SERPINB9 enhancers."

[0175] As used herein, "expression" can refer to gene or protein expression. Gene expression encompasses transcription of DNA or RNA and can be measured by various means known to those skilled in the art, for example, by measuring mRNA levels by quantitative real-time PCR (qRT-PCR) or by reporter-based methods. Similarly, protein expression can be measured by various methods well known in the art, for example, by antibody-based methods, such as Western blot, immunohistochemistry, immunocytochemistry, flow cytometry, ELISA, ELISPOT, or reporter-based methods.

[0176] Upregulation of SERPINB9 expression can be characterized by an increase in the level of RNA encoding SERPINB9 and / or an increase in the level of SERPINB9 protein compared to the level in the absence of such upregulation.Similarly, upregulation of SERPINB9 activity can be characterized by an increase in the level of related activity compared to the level in the absence of such upregulation.

[0177] Thus, agents for increasing SERPINB9 expression or activity include agents that increase the level of SERPINB9 gene and / or protein expression, as well as agents that increase the level of SERPINB9 activity. It is understood that increase in the foregoing context refers to the level of SERPINB9 expression, or the level of associated SERPINB9 activity, observed in the absence of the agent.

[0178] As used herein, the "activity" of SERPINB9 may refer to the inhibition of proteases. The activity of SERPINB9 may be the inhibition of serine proteases (e.g., granzyme B) and / or cysteine ​​proteases (e.g., caspases). The inhibition of serine proteases (e.g., granzyme B) may include binding to and / or inactivating serine proteases.

[0179] In some embodiments, agents for increasing the expression or activity of SERPINB9 (i.e., SERPINB9 agonists) described herein have the following properties: Increasing the expression (e.g., gene and / or protein expression) of SERPINB9 Increasing the levels of RNA encoding SERPINB9 Increasing transcription of nucleic acid encoding SERPINB9 Reducing the degradation of RNA encoding SERPINB9 Increasing SERPINB9 protein levels reducing the degradation of SERPINB9 protein, or Increasing levels of correlates of SERPINB9 activity Indicates one or more of the following:

[0180] It is understood that a given SERPINB9 agonist can exhibit two or more of the properties listed in the preceding paragraph.A given SERPINB9 agonist can be evaluated for the properties listed in the preceding paragraph using suitable assay.For example, assay can be, for example, in vitro assay, optionally cell-based assay or cell-free assay.In some embodiments, assay can be, for example, in vivo assay, i.e., can be carried out in non-human animals.In some embodiments, assay can be, for example, ex vivo assay, i.e., can be carried out using cells / tissues / organs obtained from a subject.

[0181] When the assays are cell-based assays, they may involve treating cells with a given agent to determine whether the agent exhibits one or more of the listed properties. The assays may use chemical species labeled with a detectable entity to facilitate detection of the chemical species. The assays may involve treating cells separately with a range of amounts / concentrations of the given agent (e.g., serial dilutions) and then evaluating the listed properties. The cells used in such cell-based assays may express SERPINB9.

[0182] Agents for increasing the expression or activity of SERPINB9, which are capable of increasing the gene expression of SERPINB9, and / or increasing the level of the RNA encoding SERPINB9, and / or increasing the transcription of the nucleic acid encoding SERPINB9, and / or reducing the degradation of the RNA encoding SERPINB9, can be identified using an assay that includes detecting and / or quantifying the level of the RNA encoding SERPINB9. Such an assay can include quantifying the RNA encoding SERPINB9 by techniques well known to those skilled in the art, such as RT-qPCR, Northern blot, etc. This method can use primers and / or probes to detect and / or quantify the RNA encoding SERPINB9. Such an assay can include contacting cells in in vitro culture with a putative SERPINB9 agonist, and then (for example, after an appropriate period, i.e., a period sufficient to observe changes in the level of the RNA encoding SERPINB9) measuring the level of the RNA encoding SERPINB9. Such an assay may further comprise comparing the level of RNA encoding SERPINB9 in cells treated with a putative SERPINB9 agonist with the level of RNA encoding SERPINB9 detected under control conditions, in which the same type of cells are subjected to the same conditions except that, instead of being treated with the putative SERPINB9 agonist, the cells are not treated or are otherwise treated with a negative control agent known not to affect the level of RNA encoding SERPINB9.

[0183] Increased transcription of the nucleic acid encoding SERPINB9 can be the result of promoting the assembly and / or activity of factors required for transcription of DNA encoding SERPINB9. Decreased degradation of the RNA encoding SERPINB9 can be the result of reduced enzymatic degradation of the RNA encoding SERPINB9, for example, as a result of RNA interference (RNAi) and / or increased stability of the RNA encoding SERPINB9.

[0184] As used herein, "contacting" a cell with a given agent (e.g., a putative SERPINB9 agonist) can include applying the agent to the cell and / or mixing the agent with the cell. In some embodiments, the SERPINB9 agonist is provided to the cell in combination with one or more additional agents for promoting the introduction of the SERPINB9 agonist into the cell and / or promoting the uptake of the SERPINB9 agonist by the cell. For example, in embodiments where the SERPINB9 agonist is one or more nucleic acids or is encoded by one or more nucleic acids, the cell can be contacted with the nucleic acid and an agent for promoting the introduction of the nucleic acid into the cell, for example, by transfection or transduction.

[0185] Agents for increasing the expression or activity of SERPINB9, which can increase the level of SERPINB9 protein and / or reduce the degradation of SERPINB9 protein and / or increase the translation of mRNA encoding SERPINB9, can be identified using assays that include detecting the level of SERPINB9 protein, using techniques well known to those skilled in the art, such as antibody or reporter-based methods (Western blot, ELISA, immunohistochemistry / cytochemistry, etc.). This method can use an antibody specific to SERPINB9. Such an assay can include contacting cells in in vitro culture with a putative SERPINB9 agonist, and then (for example, after an appropriate period, i.e., a period sufficient for changes in the level of SERPINB9 protein to be observed) measuring the level of SERPINB9 protein. Such an assay may further comprise comparing the level of SERPINB9 protein in cells treated with a putative SERPINB9 agonist with the level of SERPINB9 protein detected under control conditions, in which the same type of cells are subjected to the same conditions except that instead of being treated with the putative SERPINB9 agonist, the cells are not treated or are otherwise treated with a negative control agent known not to affect the level of SERPINB9 protein.

[0186] An increase in the level of SERPINB9 protein can be the result of, for example, an increase in the level of RNA encoding SERPINB9, increased post-transcriptional processing of RNA encoding SERPINB9, or reduced degradation of SERPINB9 protein.

[0187] Agents for increasing SERPINB9 expression or activity that are capable of increasing the level of SERPINB9 function (e.g., a SERPINB9 function described hereinabove) can be identified using an assay that includes detecting the level of the relevant function. Detecting the level of a given function can include detecting and / or quantifying a correlate of the function. Such an assay can include contacting cells in in vitro culture with a putative SERPINB9 agonist and subsequently measuring the level of the relevant function and / or its correlate (e.g., after an appropriate period of time, i.e., a period sufficient to observe a change in the level of the relevant function and / or its correlate). Such an assay can further include comparing the level of SERPINB9 function in cells treated with the putative SERPINB9 agonist with the level of SERPINB9 function detected under control conditions, in which the same type of cells are subjected to the same conditions except that, instead of being treated with the putative SERPINB9 agonist, they are not treated or are otherwise treated with a negative control agent known not to affect the level of the relevant SERPINB9 function.

[0188] In some embodiments, a SERPINB9 agonist described in the present disclosure exhibits a SERPINB9 agonist activity in a given assay that is greater than 1-fold, e.g., 1.01-fold or greater, 1.02-fold or greater, 1.03-fold or greater, 1.04-fold or greater, 1.05-fold or greater, 1.1-fold or greater, 1.2-fold or greater, 1.3-fold or greater, 1.4-fold or greater, 1.5-fold or greater, 1.6-fold or greater, 1.7-fold or greater, 1.8-fold or greater, 1.9-fold or greater, 2.0-fold or greater, 2.1-fold or greater, 2.2-fold or greater, 2.3-fold or greater, 2.4-fold or greater, 2.5-fold or greater, 2.6-fold or greater, 2.7-fold or greater, 2.8-fold or greater, 2.9 ...1-fold or greater, 2.2-fold or greater, 2.3-fold or greater, 2.4-fold or greater, 2.5-fold or greater, 2.6-fold or greater, 2.7-fold or greater, 2.8-fold or greater, 2.9-fold or greater, 2.9-fold or greater, 2.1-fold or greater, 2.1-fold or greater, 2.2-fold or greater, 2.3-fold or greater, 2.4-fold or greater, 2.5-fold or greater, 2.6-fold or greater It is capable of increasing the expression (e.g., gene and / or protein expression) of SERPINB9 / increasing the level of RNA encoding SERPINB9 / increasing the transcription of nucleic acid encoding SERPINB9 / increasing the level of SERPINB9 protein / increasing the level of a correlate of SERPINB9 activity by one of the following: 0.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more.

[0189] In some embodiments, a SERPINB9 agonist described in the present disclosure is capable of reducing the degradation of SERPINB9 protein / reducing the degradation of RNA encoding SERPINB9 in a given assay by less than 1-fold, e.g., 0.99-fold or less, 0.95-fold or less, 0.9-fold or less, 0.85-fold or less, 0.8-fold or less, 0.75-fold or less, 0.7-fold or less, 0.65-fold or less, 0.6-fold or less, 0.55-fold or less, 0.5-fold or less, 0.45-fold or less, 0.4-fold or less, 0.35-fold or less, 0.3-fold or less, 0.25-fold or less, 0.2-fold or less, 0.15-fold or less, 0.1-fold or less, 0.05-fold or less, or 0.01-fold or less, of the level observed in the absence of the SERPINB9 agonist or in the presence of an equal amount of a control agent known not to possess such agonist activity.

[0190] In some embodiments, an agent for increasing the expression or activity of SERPINB9 according to the present disclosure is or comprises a nucleic acid encoding SERPINB9, eg, as described herein. Nucleic acids and vectors The present disclosure provides a nucleic acid or nucleic acids encoding a polypeptide of the present disclosure.For example, the present disclosure provides a nucleic acid or nucleic acids encoding a SERPINB9 polypeptide.Also provided is a nucleic acid or nucleic acids encoding an agent for increasing the expression or activity of SERPINB9.

[0191] In some embodiments, the nucleic acid comprises or consists of DNA and / or RNA. In some embodiments, the nucleic acid can be or be contained in a vector or vectors. That is, the nucleotide sequence of the nucleic acid can be contained in a vector. The agent for increasing the expression or activity of SERPINB9 / SERPINB9 can be produced intracellularly by transcription from a vector encoding the peptide / polypeptide, followed by translation of the transcribed RNA.

[0192] Thus, the present disclosure also provides a vector or vectors comprising a nucleic acid or nucleic acids described herein. The vector can facilitate delivery of a nucleic acid comprising / encoding a SERPINB9 polypeptide. The vector may be an expression vector containing the elements required to express a nucleic acid comprising / encoding a SERPINB9 polypeptide.

[0193] As used herein, a "vector" refers to a nucleic acid molecule used as a vehicle for transferring an exogenous nucleic acid into a cell. The vector may be a vector for expressing a nucleic acid in a cell. Such a vector may include a promoter sequence operably linked to a nucleotide sequence encoding the sequence to be expressed. The vector may also include a stop codon and an expression enhancer. Any suitable vector, promoter, enhancer, and stop codon known in the art may be used to express a peptide or polypeptide from the vector described in this disclosure.

[0194] The term "operably linked" can include the situation where a selected nucleic acid sequence and a regulatory nucleic acid sequence (e.g., a promoter and / or enhancer) are covalently linked in such a manner that expression of the nucleic acid sequence is under the influence or control of the regulatory sequence (thereby forming an expression cassette). Thus, a regulatory sequence is operably linked to a selected nucleic acid sequence if it is capable of affecting the transcription of the nucleic acid sequence. The resulting transcription product can then be translated into a desired peptide / polypeptide.

[0195] Suitable vectors include plasmids, binary vectors, DNA vectors, mRNA vectors, viral vectors (e.g., retroviral vectors, e.g., gamma retroviral vectors (e.g., murine leukemia virus (MLV)-derived vectors, e.g., SFG vectors), lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, vaccinia viral vectors, and herpes viral vectors), transposon-based vectors, and artificial chromosomes (e.g., yeast artificial chromosomes), as described, for example, in Maus et al., Annu Rev Immunol (2014) 32:189-225, or Morgan and Boyerinas, Biomedicines (2016) 4:9, both of which are incorporated herein by reference in their entireties.

[0196] In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector. In some embodiments, the vector is an MLV-derived vector. In some embodiments, the vector is an SFG vector.

[0197] In some embodiments, the vector may be a eukaryotic vector, for example, a vector containing elements necessary for the expression of nucleic acid from the vector in eukaryotic cells. In some embodiments, the vector may be a mammalian vector, for example, containing a cytomegalovirus (CMV) or SV40 promoter to drive expression. In some embodiments, the vector includes a cell- or tissue-specific promoter, for example, an immune cell-specific promoter.

[0198] In some embodiments, a vector is selected based on its tropism for the cell type / tissue / organ to which it is desired to deliver the nucleic acid. In some embodiments, a vector is selected based on its tropism for the cell type / tissue / organ to which it is desired to express SERPINB9. For example, it may be desired to deliver a nucleic acid encoding a SERPINB9 polypeptide to an immune cell (e.g., an effector immune cell, such as a T cell or an NK cell).

[0199] In some embodiments, the nucleic acids described herein include modifications to incorporate one or more moieties that facilitate delivery to and / or uptake by a cell type or tissue of interest (e.g., immune cells). In some embodiments, the nucleic acids described herein are linked (e.g., chemically conjugated) to one or more moieties that facilitate delivery to and / or uptake by a cell type or tissue of interest.

[0200] Modifications to and formulation of nucleic acids to facilitate targeted delivery to a cell type and / or tissue of interest are described, for example, in Lorenzer et al., J Control Release (2015) 203:1-15, which is incorporated herein by reference in its entirety. A moiety that facilitates delivery to and / or uptake by a cell type or tissue of interest may selectively bind to a target cell type / tissue of interest. The moiety may facilitate crossing of the cell membrane of cells of the target cell type and / or tissue of interest. The moiety may bind to a molecule expressed on the cell surface of the target cell type / tissue of interest. The moiety may facilitate internalization (e.g., by endocytosis) of the nucleic acid by the target cell type / tissue of interest.

[0201] In some embodiments, the nucleic acid further encodes another polypeptide of interest, e.g., a molecule for directing the activity of immune cells against cells expressing a given target antigen (e.g., a chimeric antigen receptor (CAR) or a T cell receptor (TCR)).

[0202] The nucleic acid may comprise a nucleotide sequence encoding a SERPINB9 polypeptide described herein and a nucleotide sequence encoding another polypeptide of interest. The nucleotide sequences encoding the SERPINB9 polypeptide and the other polypeptide of interest may be in the same reading frame and may not include a stop codon provided between the nucleotide sequences.

[0203] In some embodiments, the nucleic acids / vectors described herein are multicistronic (e.g., bicistronic, tricistronic, etc.), i.e., in some embodiments, the vector encodes an mRNA having multiple protein-coding regions. In some embodiments, the vector is bicistronic. In some embodiments, the vector contains a nucleic acid encoding an internal ribosome entry site (IRES). In some embodiments, the vector contains a nucleic acid that allows a SERPINB9 polypeptide and another polypeptide of interest (e.g., a CAR) to be translated separately from the same RNA transcript.

[0204] In some embodiments, the nucleic acid encodes a fusion polypeptide described herein, e.g., a fusion polypeptide comprising a SERPINB9 polypeptide described herein and a CAR described herein.

[0205] The nucleic acids and vectors described in this disclosure can be provided in a purified or isolated form, i.e., purified or isolated from other nucleic acids or naturally occurring biological material. SERPINB9 and application of drugs to increase SERPINB9 expression / activity The SERPINB9 polypeptides described in this disclosure, nucleic acids / vectors encoding the SERPINB9 polypeptides described in this disclosure, and agents for increasing SERPINB9 expression or activity described in this disclosure find use in a variety of applications.

[0206] Example 2.3 herein demonstrates that immune cells modified to upregulate SERPINB9 expression / activity are less susceptible to elimination by alloreactive immune cells compared to comparable cells lacking such modification. Increased SERPINB9 expression / activity appears to protect immune cells against the cytolytic activity of allogeneic effector immune cells, particularly granzyme B-mediated cytolysis.

[0207] The SERPINB9 polypeptides described in the present disclosure, the nucleic acids / vectors encoding the SERPINB9 polypeptides described in the present disclosure, and the agents for increasing the expression or activity of SERPINB9 described in the present disclosure include: Increase the level of SERPINB9 expression (i.e., gene or protein expression) in a cell; reducing the activity of serine proteases (e.g., granzyme B) in cells; reducing the activity of a caspase (e.g., caspase-1, -4, -5, -2, -3, -6, -7, -8, or -10) in a cell; Increase the resistance of cells (and / or decrease the susceptibility of cells) to cell killing by serine proteases (e.g., granzyme B); increasing the resistance of the cell (and / or decreasing the cell's susceptibility) to cell killing by cells expressing serine proteases (e.g., cells expressing granzyme B, e.g., effector immune cells); reducing the rate of cell killing by cells expressing serine proteases (e.g., cells expressing granzyme B, e.g., effector immune cells); Increase the resistance of cells to (and / or decrease the susceptibility of cells to) death receptor-mediated apoptosis (e.g., Fas-mediated apoptosis), Increase cell persistence / survival / proliferation in the presence of (e.g., when co-cultured with) allogeneic effector immune cells; Increase cell persistence / survival / proliferation in allogeneic subjects; reduce allograft rejection in a subject; Increase the anti-cancer activity of the cells in the presence of allogeneic effector immune cells (e.g., when co-cultured with allogeneic effector immune cells); Increase the anti-cancer activity of the cells in an allogeneic subject; or Increased cell persistence / survival / proliferation under conditions of chronic antigen exposure The present invention finds use in methods for and / or comprising the steps of

[0208] A "reduction" or "increase" according to the preceding paragraph can be, for example, a reduction or increase relative to the level of the relevant property normally exhibited by cells of that type in the absence of treatment with an article of the present disclosure (e.g., a reduction or increase compared to a baseline value for the level of the relevant property for cells of that type). The properties identified in the preceding paragraph can be assessed using suitable methods known to those of skill in the art.

[0209] The level of expression of SERPINB9 gene expression in the cells, and the level of SERPINB9 protein in the cells can be assessed, for example, as described herein above.

[0210] The level of activity of a given enzyme (e.g., a protease, a serine protease (e.g., granzyme B), a cysteine ​​protease (e.g., a caspase, e.g., caspase-1, -4, -5, -2, -3, -6, -7, -8, or -10)) in a cell can be assessed using an appropriate assay to detect and / or quantify the level of that activity. Such an assay can involve measuring the level of a substrate of the relevant enzyme and / or measuring the level of the product of the enzyme's activity over time. The method can include applying a substrate of the enzyme to the cell and detecting and / or quantifying the level of the substrate and / or the product of enzyme-mediated processing of the substrate after a given period of time. Such assays can use labeled chemical species for detection and / or quantification of the enzyme's substrate and / or the product of its activity, or can use reagents for detection and / or quantification of the enzyme's substrate and / or the product of its activity. Such assays can also use colorimetric substrates or reaction products.

[0211] For example, Granzyme B activity can be assessed using the Granzyme B Assay Kit (Catalog No. BML-AK711-0001) from Enzo Life Sciences, Inc., which uses the colorimetric substrate IEPD-pNA. Cleavage of the p-nitroanilide (pNA) group from IEPD-pNA increases absorbance at 405 nm, allowing Granzyme B activity to be detected and quantified. Other suitable assays for detecting and quantifying Granzyme B activity are well known in the art, including, for example, the Granzyme B Activity Assay Kit (Catalog No. MAK176) from Sigma-Aldrich. Granzyme B activity can also be assessed, for example, as described in Bird et al., Mol Cell Biol. (1998), 18(11):6387-98, hereinabove, which is incorporated herein by reference.

[0212] For further illustration, caspase activity may be assessed using, for example, the Caspase-Glo assay from Promega Corporation, which uses a luminescent caspase substrate and luciferase. Cleavage of the substrate liberates the luciferase substrate, aminoluciferin. Luciferase activity on aminoluciferin results in the production of light, allowing for the detection and quantification of caspase activity.

[0213] As used herein, a "serine protease" refers to an enzyme that cleaves peptide bonds within a polypeptide, where serine serves as a nucleophilic amino acid in the enzyme's active site. "Serine protease activity" refers to the catalysis of the cleavage of peptide bonds within a polypeptide by a serine protease. Serine proteases are reviewed, for example, in Patel, Allergol. Immunopathol. (Madr). (2017) 45(6):579-591, the entire contents of which are incorporated herein by reference.

[0214] In a preferred embodiment, the serine protease is granzyme.Granzyme is a serine protease contained in the cytoplasmic granules of effector immune cells such as cytotoxic T lymphocytes (CTL) and natural killer (NK) cells.Granzymes are reviewed, for example, in Chowdhury and Lieberman, Annu. Rev. Immunol. (2008) 26:389-420 (incorporated herein by reference in its entirety), and include granzymes A, B, H, K and M.In a preferred embodiment, the granzyme described in the present disclosure is granzyme B.Thus, in some embodiments, the serine protease is granzyme B.The biology of granzyme B is reviewed, for example, in Lord et al., Immunol. Rev. (2003) 193:31-38 and Trapani et al., Curr. Opin. Immunol. (2003) 15:533-43. Granzyme B induces apoptosis by cleaving polypeptides after aspartic acid residues and activating caspases (e.g., executioner caspase-3). Human granzyme B also directly cleaves bid and ICAD, activating the same mitochondrial and DNA damage pathways, respectively, to activate cell death.

[0215] As used herein, a "cysteine ​​protease" refers to an enzyme that cleaves peptide bonds within a polypeptide, where cysteine ​​serves as a nucleophilic amino acid in the enzyme's active site. "Cysteine ​​protease activity" refers to the catalysis of cleavage of peptide bonds within a polypeptide by a cysteine ​​protease. Cysteine ​​proteases are reviewed, for example, in Verma et al., Front. Pharmacol. (2016) 7:107, the entire contents of which are incorporated herein by reference.

[0216] In preferred embodiments, the cysteine ​​protease is a caspase. Caspases are cysteine-dependent, aspartate-directed proteases that play a role in programmed cell death, including apoptosis and pyroptosis. Caspases are reviewed, for example, in Julien and Wells, Cell Death & Differentiation (2017) 24:1380-1389 (incorporated herein by reference in its entirety), and include caspase-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -12, and -14. In some embodiments, the caspase described herein is a caspase involved in apoptosis, for example, selected from caspase-2, -8, -9, -10, -3, -6, and -7. In some embodiments, the caspase is an inducible caspase, for example, selected from caspase-2, -8, -9, and -10. In some embodiments, the caspase is an executioner caspase, e.g., selected from caspase-3, -6, and -7. In some embodiments, the caspase described herein is a caspase involved in pyroptosis, e.g., selected from caspase-1, -4, -5, and -12.

[0217] The resistance / susceptibility of cells to cell killing by serine proteases (e.g., granzyme B), the resistance / susceptibility of cells to cell killing by cells expressing serine proteases (e.g., granzyme B, e.g., effector immune cells), and the rate of cell killing by cells expressing serine proteases (e.g., granzyme B-expressing cells, e.g., effector immune cells) can be assessed using assays that involve detecting and / or quantifying cell lysis / cell killing.

[0218] In some embodiments, the serine protease (e.g., granzyme B) can be expressed by cells being evaluated for resistance / susceptibility to cell killing by a serine protease (e.g., granzyme B). In some embodiments, the serine protease (e.g., granzyme B) can be expressed by cells different from the cells being evaluated for resistance / susceptibility to cell killing by a serine protease (e.g., granzyme B). In some embodiments, the serine protease (e.g., granzyme B)-expressing cells are allogeneic with respect to the cells. That is, the granzyme B-expressing cells can be obtained / derived from a subject other than the subject from which the test cells are obtained / derived (e.g., a genetically non-identical subject). In some embodiments, the serine protease (e.g., granzyme B)-expressing cells can be autogeneic or autologous with respect to the cells. That is, the granzyme B-expressing cells can be obtained / derived from a genetically identical or the same subject as the subject from which the test cells are obtained / derived.

[0219] As used herein, "death receptor-mediated apoptosis" refers to programmed cell death (i.e., apoptosis) of cells expressing a death receptor, induced by death receptor activation. Death receptor-mediated apoptosis is reviewed, for example, in Green, Cold Spring Harb Perspect Biol. (2022) 14:a041053, the entire contents of which are incorporated herein by reference.

[0220] Death receptors belong to the tumor necrosis factor / nerve growth factor superfamily. They are type I transmembrane proteins with a conserved cytoplasmic death domain (DD). Death receptors are activated upon ligation with their cognate ligands. After activation, the DD promotes homotypic interaction with adaptor proteins via the death domain motif of the adaptor proteins, activating the caspase cascade and ultimately causing apoptosis. In some embodiments, the death receptor is selected from Fas (also known as CD95 and APO-1), tumor necrosis factor receptor-1 (TNFR1), TRAIL receptor-1 (also known as DR4), TRAIL receptor-2 (also known as DR5), death receptor 3 (DR3), death receptor 6 (DR6), nerve growth factor receptor (NGFR), and ectodysplasia-in-A receptor (EDAR). In some embodiments, the death receptor is Fas.

[0221] As used herein, "Fas-mediated apoptosis" refers to programmed cell death (i.e., apoptosis) of cells expressing the Fas receptor, induced by activation of the Fas receptor. Fas-mediated apoptosis is reviewed, for example, in Timmer et al., J. Pathol. (2002) 196(2):125-34, the entire contents of which are incorporated herein by reference. Fas-mediated apoptosis typically involves cross-linking of Fas by its ligand, FasL (e.g., expressed on the surface of effector immune cells), activating the caspase cascade and ultimately leading to apoptosis.

[0222] The resistance / susceptibility of cells to apoptosis mediated by a death receptor (e.g., Fas-mediated apoptosis) can be assessed using an assay that includes detecting and / or quantifying apoptosis mediated by a death receptor. Such an assay can include contacting cells with an agent for activating apoptosis mediated by a death receptor (e.g., a ligand for a given death receptor, or cells expressing a ligand for a given death receptor), and detecting and / or quantifying apoptosis of the cells. For example, such an assay can include contacting cells with an agent for activating Fas-mediated apoptosis (e.g., FasL or cells expressing FasL), and detecting and / or quantifying apoptosis of the cells. Detecting / quantifying apoptosis can include analyzing the expression or activity levels of one or more caspases, and / or detecting and / or quantifying live, dead, and / or apoptotic cells. Detecting / quantifying apoptosis may include detecting and / or quantifying one or markers of apoptosis (e.g., phosphatidylserine (PS) exposure, Bcl-2 family protein (e.g., Bax, Bak, Bid) activation, ROS production, caspase activation, mitochondrial membrane permeabilization, or DNA fragmentation).

[0223] Cell killing can be investigated using, for example, any of the methods outlined in Zaritskaya et al., Expert Rev Vaccines (2011), 9(6):601-616, which is incorporated herein by reference in its entirety. Examples of in vitro cytotoxicity / cell killing assays include release assays, e.g., 51These assays include the Cr release assay, lactate dehydrogenase (LDH) release assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) release assay, and calcein-acetoxymethyl (calcein-AM) release assay. These assays measure cell killing based on the detection of factors released from lysed cells. Cell killing of a given test cell type by a given effector immune cell type can be analyzed, for example, by co-culturing the test cells with the effector immune cells and measuring the number / percentage of viable / dead (e.g., lysed) test cells after a suitable period of time. Other suitable assays include the xCELLigence real-time cytolysis in vitro potency assay described in Cerignoli et al., PLoS One. (2018) 13(3):e0193498 (incorporated herein by reference in its entirety). An increase in resistance to cell killing by granzyme B-expressing cells (e.g., effector immune cells) and / or a decrease in susceptibility to cell killing by such cells compared to a baseline level of cell killing (e.g., for that cell type) can be determined by detecting a decrease in the number / percentage of dead (e.g., lysed) test cells and / or an increase in the number / percentage of live (e.g., viable, unlysed) test cells after a given period of time.

[0224] The rate of cell death of a given test cell type can be determined by analyzing cell lysis over time, for example, by determining the number / proportion of lysed and / or unlysed test cells at different time points. For example, such analysis can use the xCELLigence system described in Example 1.6 herein. A reduction in the rate of cell death compared to a baseline rate of cell death (e.g., for that cell type) can be determined by detecting a decrease in the number / proportion of dead (e.g., lysed) test cells and / or an increase in the number / proportion of live (e.g., viable, unlysed) test cells per time unit.

[0225] The persistence / survival / proliferation / expansion of a given cell / population thereof can be assessed in vitro by measuring or monitoring the number or proportion of such cells over time. Cell proliferation / expansion can be investigated by analyzing cell division or cell number over a period of time. Cell division can be analyzed, for example, by in vitro analysis of H-thymidine incorporation or by CFSE dilution assay, as described, for example, in Fulcher and Wong, Immunol Cell Biol (1999) 77(6):559-564, the entire contents of which are incorporated herein by reference. Proliferating cells can also be identified by analyzing 5-ethynyl-2'-deoxyuridine (EdU) incorporation using a suitable assay, as described, for example, in Buck et al., Biotechniques. 2008 June;44(7):927-9, and Sali and Mitchison, PNAS USA 2008 February 19;105(7):2415-2420, both of which are incorporated herein by reference in their entirety.

[0226] The level of cell proliferation or population expansion of a given cell type can also be assessed, for example, by counting the number of relevant cell types at one or more defined time points after culture in certain conditions.

[0227] An increase in persistence / survival can be determined by detection of a greater number / percentage of live (eg, viable, unlysed) cells after a given period of time. The cell killing assay described above can also be used to assess cell persistence / survival in the presence of allogeneic effector immune cells. For example, the persistence / survival of a given test cell type in the presence of allogeneic effector immune cells can be analyzed by co-culturing the test cells with the allogeneic effector immune cells and measuring the number / proportion of viable / dead (e.g., lysed) test cells after a suitable period of time. An increase in persistence / survival compared to a baseline level (e.g., for that cell type) can be determined by detecting a decrease in the number / proportion of dead (e.g., lysed) test cells and / or an increase in the number / proportion of live (e.g., viable, unlysed) test cells after a given period of time.

[0228] Suitable assays for assessing proliferation / expansion / persistence / survival in the presence of allogeneic effector immune cells include, for example, mixed lymphocyte reaction (MLR) assays, such as those described herein in Example 1.5.

[0229] According to the assays described herein, the given / defined period of time that the level of the relevant characteristic is subsequently evaluated can be any suitable period that provides a meaningful comparison between the level of the relevant characteristic between test cells and control cells in the relevant assay.In some embodiments, the level of the relevant characteristic is evaluated in or after a sufficient period of time that the maximum level of the relevant characteristic is obtained or has been obtained in the relevant assay.In some embodiments, the given / defined period of time is one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days.In some embodiments, the given / defined period of time is one of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days.

[0230] As used herein, when a given cell is referred to herein as being "allogeneic" with respect to a reference cell, it is obtained / derived from a subject other than the subject from which the reference cell is obtained / derived. Accordingly, the allogeneic effector immune cells referred to in the preceding paragraph are obtained / derived from a subject other than the subject from which the test cell is obtained / derived. In some embodiments, the allogeneic cell comprises MHC / HLA genes encoding MHC / HLA molecules (e.g., MHC class Iα and / or MHC class II molecules) that are not identical to the MHC / HLA molecules (e.g., MHC class Iα and / or MHC class II molecules) encoded by the MHC / HLA genes of the reference cell.

[0231] The survival / persistence of a given cell or a group of cells in a subject (e.g., an allogeneic subject) in vivo can be evaluated using a method, for example, by labeling cells with a detectable marker or reporter, introducing them into a subject, and monitoring their survival over time.Such a method includes, for example, labeling cells with firefly luciferase, and measuring the firefly luciferase activity at various time points by bioluminescence imaging, for example, after administering D-luciferin (for example, as described in Prescher and Contag, Curr. Opin. Chem. Biol. (2010) 14(1):80-9, the entirety of which is incorporated herein by reference).Compared to the baseline level (e.g., for that cell type), the increase in persistence / survival can be determined by detecting a greater number / proportion of test cells in the subject after a given period of time.

[0232] As used herein, when a subject is referred to herein as being "allogeneic" with respect to a reference cell, the subject is a subject other than the subject from which the reference cell is obtained / derived. Thus, the allogeneic subject referred to in the preceding paragraph is a subject other than the subject from which the test cell is obtained / derived. In some embodiments, the allogeneic subject comprises MHC / HLA genes encoding MHC / HLA molecules (e.g., MHC class Iα and / or MHC class II molecules) that are not identical to the MHC / HLA molecules (e.g., MHC class Iα and / or MHC class II molecules) encoded by the MHC / HLA genes of the reference cell.

[0233] A suitable assay for assessing the survival / persistence of a given cell or population of cells in an allogeneic subject (i.e., allogeneic to the test cells) in vivo can be performed in a surrogate allogeneic subject. A surrogate allogeneic subject can be established by injecting allogeneic immune cells (i.e., allogeneic to the test cells) into a non-allogeneic subject, for example, by injecting allogeneic effector immune cells into an MHC knockout mouse. Thus, a suitable assay for assessing the survival / persistence of a given cell or population of cells in an allogeneic subject in vivo can include co-injecting a given cell or population of cells with allogeneic immune cells (i.e., allogeneic to the test cells) into an MHC knockout mouse. In some embodiments, the survival / persistence of a given cell or population of cells in an allogeneic subject (e.g., a surrogate allogeneic subject) in vivo can be assessed essentially as described in Example 1.10 and Example 4.

[0234] Furthermore, the above-described method for assessing cell persistence / survival / proliferation in an allogeneic subject can be used to assess allograft rejection in the subject. Furthermore, the SERPINB9 polypeptides and other agents for increasing SERPINB9 expression or activity described in the present disclosure are useful for reducing / preventing graft rejection. Graft rejection refers to the destruction of transplanted cells / tissues / organs by the recipient's immune system after transplantation. When graft rejection is the rejection of an allograft, it can be referred to as allograft rejection. Increasing SERPINB9 expression or activity in cells confers resistance to serine protease (e.g., granzyme B)-mediated depletion by granzyme B-expressing cells (e.g., effector immune cells) in the recipient subject.

[0235] The anti-cancer activity of a given cell type or population of such cells can be assessed, for example, by analyzing cell killing of cancer cells by such cells, and / or correlates thereof.

[0236] Anti-cancer activity can be analyzed, for example, using an assay that detects and / or quantifies cell death of cancer cells in vitro. The cell death assays described hereinabove can be used to assess cell death of cancer cells.

[0237] For example, the anti-cancer activity of a given test cell type can be assessed by culturing the test cells with allogeneic effector immune cells and cancer cells in the presence of allogeneic effector immune cells and monitoring the number / proportion of live and / or dead (e.g., lysed) cancer cells over time. An increase in anti-cancer activity (e.g., compared to the level of anti-cancer activity exhibited by cells of that type in the absence of treatment with an article of the present disclosure) can be determined by detecting a decrease in the number / proportion of live cancer cells and / or an increase in the number / proportion of dead (e.g., lysed) cancer cells after a given period of time.

[0238] Suitable assays for assessing the anti-cancer activity of a cell or population of cells include, for example, a mixed lymphocyte reaction (MLR) assay. In some embodiments, the anti-cancer activity of a cell or population of cells can be assessed essentially as described in Example 1.5 herein.

[0239] Anti-cancer activity can also be analyzed, for example, using assays that detect and / or quantitate cell killing of cancer cells in a subject in vivo. For example, the anti-cancer activity of a given test cell type can be assessed using an assay in which the test cells are administered to a subject with cancer, and the number / proportion of cancer cells, cancer burden, and / or tumor volume are monitored over time. An increase in anti-cancer activity (e.g., compared to the level of anti-cancer activity exhibited by that type of cell in the absence of treatment with an article of the present disclosure) can be determined by detecting a decrease in the number / proportion of cancer cells, a decrease in cancer burden, and / or a decrease in tumor volume in the subject after a given period of time. In some embodiments, such assays can use cancer cells labeled with a detectable marker or reporter, and in vivo anti-cancer activity can be assessed by monitoring the number / proportion of cells over time. Such methods include, for example, labeling cancer cells with firefly luciferase and measuring firefly luciferase activity at various time points by bioluminescence imaging, e.g., after administration of D-luciferin (e.g., as described in Prescher and Contag, Curr. Opin. Chem. Biol. (2010) 14(1):80-9, which is incorporated herein by reference in its entirety).

[0240] In some embodiments, the anti-cancer activity of a cell or population of cells can be assessed essentially as described in Example 1.10 herein. The persistence / survival / proliferation of a cell or population of cells under conditions of chronic antigen exposure can be assessed in vitro by exposing the cells to continuous antigen challenge and measuring or monitoring the number or proportion of such cells over time or after a given period of time. For example, the persistence / survival of a given test cell type can be analyzed by serially co-culturing the test cells with cancer cells (e.g., multiple rounds of tumor challenge by reseeding the test cells with fresh cancer cells) and monitoring the number / proportion of live and / or dead (e.g., lysed) test cells after a suitable period of time. An increase in the persistence / survival of a given test cell type (e.g., compared to the level exhibited by cells of that type in the absence of treatment with an article of the present disclosure) can be determined by detecting a decrease in the number / proportion of dead (e.g., lysed) test cells and / or an increase in the number / proportion of live (e.g., viable, unlysed) test cells after a given period of time.

[0241] In some embodiments, "conditions of chronic antigen exposure" can refer to multiple rounds (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 rounds) of stimulation of immune cells with an antigen or cells containing / expressing an antigen, where the immune cells contain a specific receptor (e.g., a CAR having an antigen-binding domain that binds to the antigen, and / or a TCR that binds to an MHC:peptide complex containing a peptide of the antigen). In some embodiments, "conditions of chronic antigen exposure" can refer to multiple separate occasions (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 occasions) in which an antigen or cells containing / expressing an antigen, where the immune cells contain a specific receptor (e.g., a CAR having an antigen-binding domain that binds to the antigen, and / or a TCR that binds to an MHC:peptide complex containing a peptide of the antigen), is added to immune cells in culture. In some embodiments, the rounds of stimulation by antigen / antigen addition are performed at regular intervals, e.g., every 1, 2, 3, 4, or 5 days. In some embodiments, the rounds of stimulation by antigen / antigen addition are performed at regular intervals, e.g., every 2-3 days. In some embodiments, "conditions of antigen exposure" may refer to stimulation of immune cells in an in vivo model in which immune cells are implanted with tumors / tumor cells containing / expressing an antigen, wherein the immune cells contain specific receptors (e.g., a CAR having an antigen-binding domain that binds to the antigen, and / or a TCR that binds to an MHC:peptide complex containing a peptide of the antigen).

[0242] In some embodiments, the persistence / survival / proliferation of a cell or population of cells under conditions of chronic antigen exposure can be assessed essentially as described in Example 1.9 and / or Example 1.10, "Activation-Induced Cell Death (AICD) Model," herein.

[0243] In some embodiments, in the methods and uses described herein, the SERPINB9 polypeptide, nucleic acid, vector, or agent for increasing SERPINB9 expression or activity described herein increases the expression or activity of SERPINB9 in a cell by more than 1-fold, e.g., 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, 1.04-fold or more, 1.05-fold or more, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more, of the level of the relevant trait normally exhibited by cells of that type (e.g., the baseline value for the level of the relevant trait for cells of that type, i.e., in the absence of treatment with an article of the disclosure). increase the level of expression (i.e., gene or protein expression) of a cell; increase the resistance of a cell to cell killing by serine proteases (e.g., granzyme B); increase the resistance of a cell to cell killing by cells expressing serine proteases (e.g., granzyme B, e.g., effector immune cells); increase the resistance of a cell to apoptosis mediated by death receptors (e.g., Fas-mediated apoptosis); increase the persistence, survival, or proliferation of a cell in the presence of allogeneic effector immune cells; increase the persistence, survival, or proliferation of a cell in an allogeneic subject; increase the anti-cancer activity of a cell in the presence of allogeneic effector immune cells; increase the anti-cancer activity of a cell in an allogeneic subject; increase the persistence, survival, or proliferation of a cell under conditions of chronic antigen exposure.

[0244] In some embodiments, in the methods and uses described herein, the SERPINB9 polypeptide, nucleic acid, vector, or agent for increasing SERPINB9 expression or activity described herein increases the level of the relevant trait normally displayed by cells of that type (e.g., a baseline value for the level of the relevant trait for cells of that type) by less than 1 fold, for example, to one of 0.99 fold or less, 0.95 fold or less, 0.9 fold or less, 0.85 fold or less, 0.8 fold or less, 0.75 fold or less, 0.7 fold or less, 0.65 fold or less, 0.6 fold or less, 0.55 fold or less, 0.5 fold or less, 0.45 fold or less, 0.4 fold or less, 0.35 fold or less, 0.3 fold or less, 0.25 fold or less, 0.2 fold or less, 0.15 fold or less, 0.1 fold or less, 0.05 fold or less, or 0.01 fold or less. reduce the activity of serine proteases (e.g., granzyme B) in the cell; reduce the activity of caspases (e.g., caspase-1, -4, -5, -2, -3, -6, -7, -8, or -10) in the cell; reduce the susceptibility of the cell to cell killing by serine proteases (e.g., granzyme B); reduce the susceptibility of the cell to cell killing by cells expressing serine proteases (e.g., granzyme B, e.g., effector immune cells); reduce the susceptibility of the cell to apoptosis mediated by death receptors (e.g., Fas-mediated apoptosis); reduce the rate of cell killing by cells expressing serine proteases (e.g., granzyme B, e.g., effector immune cells); and reduce graft rejection in a subject. Cells modified to increase SERPINB9 expression or activity The present disclosure provides cells modified to increase SERPINB9 expression (i.e., gene and / or protein expression) or activity. When a cell is referred to herein in the singular (i.e., "a cell / the cell"), it is understood that a plurality / population of such cells is also contemplated.

[0245] Cells having increased SERPINB9 expression (i.e., gene and / or protein expression) or activity can be characterized by a level of SERPINB9 expression or a level of SERPINB9 activity that is greater than the level of expression / activity normally exhibited by that type of cell. Cells having increased SERPINB9 expression or activity can be characterized by a level of SERPINB9 expression or a level of SERPINB9 activity that is greater than the baseline value for the level of expression / activity for that type of cell.

[0246] Cells that have increased expression (ie, gene and / or protein expression) or activity of SERPINB9 may do so as a result of the treatments / modifications described herein.

[0247] In some embodiments, the cells are cells that have been treated / modified to increase the level or activity of SERPINB9 protein in the cells. In some embodiments, the cells are cells that have been treated / modified to increase the gene and / or protein expression of SERPINB9.

[0248] The present disclosure provides cells that contain or express the SERPINB9 polypeptides described herein.The present disclosure also provides cells that contain or express a nucleic acid (e.g., an exogenous nucleic acid) encoding the SERPINB9 polypeptides described herein.The present disclosure also provides cells that contain or express a vector encoding the SERPINB9 polypeptides described herein.

[0249] As used herein, "exogenous" nucleic acid refers to a nucleic acid that is not endogenous to the cell that contains the exogenous nucleic acid. An exogenous nucleic acid may not be encoded by the genome of the subject from which the cell is obtained / derived. A cell that contains an exogenous nucleic acid may contain it as a result of being modified to introduce, for example, a nucleic acid / vector encoding a SERPINB9 polypeptide into the cell, as described herein.

[0250] In some embodiments, the cells are cells into which a nucleic acid / vector encoding a SERPINB9 polypeptide described in the present disclosure has been introduced. Such cells can be characterized by a level of SERPINB9 expression or a level of SERPINB9 activity that is greater than the level of expression / activity exhibited by a comparable cell that has not been introduced with the nucleic acid / vector.

[0251] In some embodiments, the cells are cells treated with an agent for increasing SERPINB9 expression or activity, as described herein. Such cells can be characterized by a level of SERPINB9 expression or a level of SERPINB9 activity that is greater than the level of expression / activity exhibited by comparable cells that have not been treated with the agent.

[0252] The expression level of SERPINB9 can be measured by suitable means well known to those skilled in the art. The expression level of SERPINB9 gene can be analyzed using an assay that includes detecting and / or quantifying the level of RNA encoding SERPINB9. Such an assay can include quantifying the RNA encoding SERPINB9 by RT-qPCR, Northern blot, etc. This method can use primers and / or probes for detecting and / or quantifying the RNA encoding SERPINB9. The level of SERPINB9 protein can be analyzed using an assay that includes detecting and / or quantifying the level of SERPINB9 protein. Such an assay can include, for example, antibody / reporter-based methods (Western blot, ELISA, immunohistochemistry / cytochemistry, etc.), and can use, for example, an antibody specific to SERPINB9.

[0253] The level of SERPINB9 activity can be measured using a suitable assay for activity. Such assays include assays that analyze the inhibition of serine protease (e.g., granzyme B) activity. Such assays can include, for example, assessing the activity of serine protease, as described hereinabove. In some embodiments, SERPINB9 activity can be assessed, for example, as described in Bird et al., Mol Cell Biol. (1998), 18(11):6387-98, which is incorporated herein by reference.

[0254] In some embodiments, cells having increased SERPINB9 expression (i.e., gene and / or protein expression) or activity as described herein may exhibit a level of SERPINB9 expression or activity that is more than 1-fold, e.g., one of 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, 1.04-fold or more, 1.05-fold or more, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more, the level of expression or activity normally exhibited by cells of that type (e.g., a reference value for that level of expression or level of activity for that type of cell).

[0255] In some embodiments, cells containing or expressing a nucleic acid (e.g., an exogenous nucleic acid) or vector encoding a SERPINB9 polypeptide described in the present disclosure may exhibit a level of SERPINB9 expression or a level of SERPINB9 activity that is more than 1-fold, e.g., 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, 1.04-fold or more, 1.05-fold or more, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more, the level of expression or activity exhibited by a comparable cell that does not contain the nucleic acid / vector.

[0256] In some embodiments, cells treated with an agent for increasing SERPINB9 expression or activity as described herein may exhibit a level of SERPINB9 expression or a level of SERPINB9 activity that is more than 1-fold the level of expression or activity exhibited by comparable cells not treated with the agent, e.g., one of 1.01-fold or more, 1.02-fold or more, 1.03-fold or more, 1.04-fold or more, 1.05-fold or more, 1.1-fold or more, 1.2-fold or more, 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more. In some embodiments, cells treated with an agent for increasing SERPINB9 expression or activity as described herein may exhibit a level of SERPINB9 expression that is more than 1.3-fold, e.g., 1.3-fold or more, 1.4-fold or more, 1.5-fold or more, 1.6-fold or more, 1.7-fold or more, 1.8-fold or more, 1.9-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, or 10-fold or more, of the level of expression exhibited by comparable cells that do not contain the nucleic acid / vector (e.g., as determined by an analysis essentially as described in Example 1.7).

[0257] The cell can be a eukaryotic cell, for example, a mammalian cell. The mammal can be a primate (rhesus monkey, cynomolgus monkey, non-human primate or human) or a non-human mammal (for example, a rabbit, guinea pig, rat, mouse or other rodent (including any animal of the order Rodents), cat, dog, pig, sheep, goat, cattle (including cattle, such as dairy cows, or any animal of the order Bovidae), horse (including any animal of the order Equidae), donkey and non-human primate). In a preferred embodiment, the cell is a human cell.

[0258] In preferred embodiments, the cells are immune cells. The immune cells can be cells of hematopoietic origin, such as neutrophils, eosinophils, basophils, dendritic cells, lymphocytes, or monocytes. The lymphocytes can be, for example, T cells, B cells, NK cells, NKT cells, or innate lymphoid cells (ILCs) or their precursors (e.g., thymocytes or pre-B cells). The immune cells can express CD3 polypeptides (e.g., CD3γ, CD3ε, CD3ζ, or CD3δ), TCR polypeptides (TCRα or TCRβ), CD27, CD28, CD4, or CD8. In some embodiments, the immune cells are T cells, for example, CD3+ T cells. In some embodiments, the T cells are CD3+, CD4+ T cells. In some embodiments, the T cells are CD3+, CD8+ T cells. In some embodiments, the T cells are T helper cells (TH cells). In some embodiments, the T cell is a cytotoxic T cell (e.g., a cytotoxic T lymphocyte (CTL)). In some embodiments, the immune cell is a T cell or an NK cell.

[0259] In a preferred embodiment, the cell is an effector immune cell. As used herein, "effector immune cell" can be an immune cell that exhibits effector function. The effector immune cell can be a CD8+ T cell, a CD8+ cytotoxic T lymphocyte (CD8+ CTL), a CD4+ T cell, a CD4+ T helper cell, a NK cell, an IFNγ-producing cell, a memory T cell, a central memory T cell, an antigen-experienced T cell, or a CD45RO+ T cell. The effector immune cell can be characterized by one or more of the following characteristics: granzyme B expression, IFNγ expression, CD107a expression, IL-2 expression, TNFα expression, perforin expression, granulysin expression, and / or FAS ligand (FASL) expression. In some embodiments, the effector immune cell described in the present disclosure is a granzyme B-expressing cell.

[0260] The immune cells may be immune cells from any suitable source. The immune cells may be cells obtained / isolated from a subject, for example, a subject described herein. The immune cells may be suitable for administration to a subject following a therapeutic / prophylactic intervention described in the present disclosure. The cells may be cells obtained / isolated from a subject being treated according to a therapeutic / prophylactic intervention described in the present disclosure. The cells may be cells obtained / isolated from a subject other than a subject being treated according to a therapeutic / prophylactic intervention described in the present disclosure. The cells may be cells obtained / isolated from a healthy subject, for example, a subject not known to be suffering from a disease / condition.

[0261] The present disclosure also provides a method for producing a cell described herein, the method comprising introducing into the cell a nucleic acid or vector described herein, hi some embodiments, introducing into the cell a nucleic acid or vector described herein comprises transformation, transfection, electroporation, or transduction (e.g., retroviral transduction).

[0262] Transfection refers to the process of introducing nucleic acids into cells using means other than viral infection and is therefore a non-viral method. Transfection can be performed by physical / mechanical methods (including electroporation, sonoporation, magnetofection, gene microinjection, and laser irradiation) or chemical methods (liposome-based or non-liposome-based). Liposome-based transfection reagents are chemicals that allow the formation of positively charged lipid aggregates that can then merge with the phospholipid bilayer of cells to facilitate the entry of exogenous genetic material. Examples of liposome-based transfection reagents include, but are not limited to, Oligofectamine®, Lipofectamine®, and DharmaFECT®. Non-liposomal transfection reagents include, but are not limited to, calcium phosphate, nanoparticles, polymers, dendrimers, and non-liposomal lipids. One example of a non-liposomal transfection reagent is polyethyleneimine (PEI).

[0263] Electroporation can be performed, for example, as described in Koh et al., Molecular Therapy-Nucleic Acids (2013) 2, e114, which is incorporated herein by reference in its entirety.

[0264] Transduction is a process by which nucleic acids can be introduced into cells by a virus or viral vector. Thus, in some embodiments, the nucleic acid is contained in a viral vector, or the vector is a viral vector. Viral vector transduction of immune cells is described, for example, in Simmons and Alberola-Ila, Methods Mol Biol. (2016) 1323:99-108, the entire contents of which are incorporated herein by reference. Agents can be used in the methods of the present disclosure to improve transduction efficiency. Hexadimethrine bromide (polybrene) is a cationic polymer commonly used to improve transduction by neutralizing charge repulsion between virions and sialic acid residues expressed on the cell surface. Other agents commonly used to enhance transduction include, for example, poloxamer-based agents such as LentiBOOST (Sirion Biotech), Retronectin (Takara), Vectofusin (Miltenyi Biotech), and also SureENTRY (Qiagen) and ViraDuctin (Cell Biolabs).

[0265] In some embodiments, the method comprises centrifuging cells into which it is desired to introduce a nucleic acid described herein in the presence of cell culture medium containing a viral vector comprising the nucleic acid (referred to in the art as "spinfection").

[0266] In some embodiments, the method further comprises culturing the cells under conditions suitable for expression of the nucleic acid / vector by the cells. Methods for culturing (including generating and / or expanding) populations of immune cells in vitro / ex vivo are well known to those of skill in the art. Suitable culture conditions (i.e., cell culture medium, additives, stimuli, temperature, gas atmosphere), cell number, culture period, and method for introducing a nucleic acid encoding a polypeptide of interest into the cells can be determined by reference to, for example, Hombach et al., J Immunol (2001) 167:6123-6131; Ramos et al., J. Clin. Invest. (2017) 127(9):3462-3471; WO2021 / 245249 A1; WO2021 / 222927 A1; WO2021 / 222928 A1; WO2021 / 222929 A1; WO2015 / 028444 A1; or WO2016 / 008973 A1, all of which are incorporated herein by reference in their entireties.

[0267] Conveniently, cultures of cells described in the present disclosure can be maintained at 37° C. in a humidified atmosphere containing 5% CO. Cells in cell culture can be established and / or maintained at any suitable density, as can be readily determined by one of skill in the art.

[0268] Cell culture can be performed in any vessel suitable for the volume of culture, for example, a well of a cell culture plate, a cell culture flask, a bioreactor, etc. In some embodiments, cells are cultured in a bioreactor, for example, a bioreactor described in Somerville and Dudley, Oncoimmunology (2012) 1(8):1435-1437, the entire contents of which are incorporated herein by reference. In some embodiments, cells are cultured in a GRex cell culture vessel, for example, a GRex flask or a GRex 100 bioreactor.

[0269] In some embodiments, the methods are performed in vitro. The present disclosure also provides cells obtained or obtainable by the methods described herein, and of course populations of such cells.

[0270] In some embodiments, the cells are cells for use in a medical treatment or prevention method. Cells "for use in a medical treatment or prevention method" refer to cells that are suitable for use in a medical treatment or prevention method. Such cells may not contain certain drugs / contaminants that may make the cells unsuitable for such use.

[0271] In some embodiments, the cells are for use in methods of medical treatment or prevention by adoptive cell transfer (ACT). Adoptive cell transfer involves administering a cell / population of cells to a subject to treat / prevent a disease / condition. In particular, adoptive cell transfer typically involves administering immune cells (e.g., T cells) to a subject to provide the subject with a population of immune cells for treating / preventing a disease / condition, or to increase the number of such cells in the subject. In some embodiments, the adoptively transferred cells contain molecules for directing the activity of the immune cells against cells containing / expressing a given target antigen, e.g., a disease-associated antigen.

[0272] Adoptive cell transfer can involve isolating / obtaining cells (e.g., immune cells) from a subject, for example, by taking a blood sample from which the cells are isolated. The cells are then typically modified and / or expanded and then administered to either the same subject (in the case of adoptive transfer of autologous / autogeneic cells) or a different subject (in the case of adoptive transfer of allogeneic cells). Treatment typically aims to provide a population of cells with certain desired characteristics to a subject, or to increase the frequency of such cells with such characteristics in the subject.

[0273] Cells for use in methods of medical treatment or prevention by adoptive cell transfer may contain / express molecules to direct the activity of the cells towards cells expressing a given target antigen.

[0274] In some embodiments, the cells comprise a T cell receptor (TCR) to direct the activity of the cells to cells that present an MHC-peptide complex for which the TCR is specific. In some embodiments, the TCR is encoded by the genome of the subject from which the cells are obtained / derived. In some embodiments, the TCR is encoded by a nucleic acid introduced into the cells.

[0275] In some embodiments, the cells comprise a chimeric antigen receptor (CAR) to direct the activity of the cells to cells that express the antigen for which the CAR is specific. In some embodiments, the CAR is encoded by a nucleic acid introduced into the cell.

[0276] In some embodiments, the immune cells are immune cells engineered to express molecules for directing the activity of the immune cells against cells expressing a given target antigen (e.g., CAR-engineered immune cells or TCR-engineered immune cells) and / or immune cells specific for a disease-associated antigen (e.g., pathogen-specific immune cells, e.g., virus-specific immune cells). In some embodiments, the immune cells are disease-associated antigen-specific immune cells engineered to express molecules for directing the activity of the immune cells against cells containing / expressing the given target antigen. In some embodiments, the immune cells are virus-specific, CAR-engineered immune cells.

[0277] In some embodiments, the immune cells are engineered to express a chimeric antigen receptor (CAR) (i.e., the immune cells are CAR-engineered immune cells) or to express a T cell receptor (TCR) (i.e., the immune cells are TCR-engineered immune cells).

[0278] TCR-engineered immune cells are described, for example, in Zhao et al., Front. Immunol. (2021) 30;12:658753, which is incorporated herein by reference in its entirety. TCR-engineered immune cells can be modified to express a TCR specific for a given MHC-peptide complex, for example, through the introduction into the cells of nucleic acids encoding component polypeptides of the TCR.

[0279] In some embodiments, the immune cells contain / express a TCR encoded by a non-endogenous nucleic acid (i.e., a nucleic acid that is not encoded by the genome of the cell prior to introduction of a nucleic acid encoding a component polypeptide of the TCR into the cell).

[0280] In some embodiments, the methods described herein include introducing into immune cells (i) a nucleic acid encoding a molecule (e.g., a CAR or TCR) for directing the activity of immune cells toward cells containing / expressing a given target antigen, and (ii) a nucleic acid encoding a SERPINB9 polypeptide. According to such embodiments, the nucleic acids (i) and (ii) can be introduced into cells simultaneously or sequentially. When the nucleic acids (i) and (ii) are introduced simultaneously, they can be introduced in the form of a nucleic acid (e.g., a vector) containing both the nucleic acids (i) and (ii). Alternatively, the nucleic acids (i) and (ii) can be contained in separate nucleic acids (e.g., separate vectors). When the nucleic acids (i) and (ii) are introduced sequentially, the method may include (a) introducing the nucleic acid (i) or (ii) into an immune cell, and (b) subsequently (e.g., after a specified period of time, e.g., 12 hours to 14 days, e.g., after one of 1 to 7 days, 2 to 5 days, or 3 to 4 days), introducing another nucleic acid (i.e., a nucleic acid not introduced into the cell in (a)) into the immune cell.

[0281] The TCR-engineered immune cells can comprise a TCR specific for any MHC-peptide complex of interest. In some embodiments, the TCR of the TCR-engineered immune cells is specific for an MHC-peptide complex that includes a peptide of a disease-associated antigen.

[0282] Through engineering to express a TCR specific for a particular MHC-peptide complex, immune cells, T cells, can be directed to kill cells expressing the MHC-peptide complex. Binding of the TCR-engineered T cell to its cognate MHC-peptide complex triggers intracellular signaling, resulting in T cell activation. Activated TCR-engineered T cells are stimulated to divide and produce factors that lead to the killing of cells expressing the MHC-peptide complex.

[0283] In some embodiments, the cells described herein (i.e., cells comprising / expressing SERPINB9 as described herein, or cells comprising / expressing a nucleic acid / vector SERPINB9 as described herein) do not comprise a chimeric HLA accessory receptor (CHAR) to direct the activity of the cells to alloreactive T cells. CHARs and CHAR-expressing cells are described, for example, in US 2021 / 0238255 A1, the entire contents of which are incorporated herein by reference.

[0284] In some embodiments, the cells described herein (i.e., cells comprising / expressing SERPINB9 as described herein, or cells comprising / expressing a nucleic acid / vector SERPINB9 as described herein) have not been modified to comprise / express CHAR. In some embodiments, the cells do not comprise a nucleic acid (e.g., an exogenous nucleic acid) encoding CHAR. In some embodiments, the methods described herein do not comprise a step of introducing a nucleic acid (e.g., an exogenous nucleic acid) encoding CHAR into the cell.

[0285] In some aspects, the cells described in the present disclosure (i.e., cells containing / expressing SERPINB9 as described herein, or cells containing / expressing nucleic acid / vector SERPINB9 as described herein) have not been modified to express or overexpress cellular FLICE inhibitory protein (cFLIP) or a variant thereof.

[0286] Human cFLIP is a protein identified by UniProt O15519-1. Variants of cFLIP include polypeptides having at least 70% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) amino acid sequence identity with the amino acid sequence of human cFLIP. In some embodiments, variants of cFLIP may include the substitution K167R compared to UniProt O15519-1.

[0287] In some embodiments, the cells do not contain a nucleic acid (e.g., an exogenous nucleic acid) encoding cFLIP or a variant thereof. In some embodiments, the methods described herein do not include a step of introducing a nucleic acid (e.g., an exogenous nucleic acid) encoding cFLIP or a variant thereof into the cells.

[0288] As used herein, "disease-associated antigen" refers to an antigen whose presence indicates a given disease / disease state, or an antigen whose high levels positively correlate with a given disease / disease state. A disease-associated antigen may be an antigen whose expression is associated with the onset, progression, or severity of symptoms of a given disease. A disease-associated antigen may be associated with the cause or pathology of a disease, or may be an abnormality manifested as a result of a disease. A disease-associated antigen may be an antigen of an infectious agent or pathogen, a cancer-associated antigen, or an autoimmune disease-associated antigen.

[0289] In some embodiments, the disease-associated antigen is an antigen of a pathogen. The pathogen can be a prokaryote (bacteria), a eukaryote (e.g., a protozoan, a helminth, a fungus), a virus, or a prion. In some embodiments, the pathogen is an intracellular pathogen. In some embodiments, the pathogen is a virus, such as those described hereinabove. In some embodiments, the pathogen is a bacterium. The bacterium can be gram-positive or gram-negative. In particular, the present disclosure relates to the antigens of bacteria of the genera Bacillus, Bartonella, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia and Chlamydophila, Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacterium, and the like. Bacteria of the genera S. pylori, S. cerevisiae ... In particular, the present disclosure contemplates protozoa of the genera Entamoeba, Plasmodium, Giardia, Trypanosoma, Leishmania, Besnoitia, and Toxoplasma. In some embodiments, the pathogen is a fungus.In particular, the present disclosure contemplates fungi of the genera Candida, Aspergillus, Blastomyces, Coccidioides, Sporothrix, Cryptococcus, Histoplasma, Pneumocystis, Stachybotrys, Rhizopus, Mucor, Pneumocystis, Apophysomyces, Trichophyton, Microsporum, Epidermophyton, Fusarium, and Lichtheimia.

[0290] In some embodiments, the disease-associated antigen is a cancer-associated antigen. In some embodiments, the cancer-associated antigen is an antigen whose expression is associated with the onset, progression, or severity of symptoms of cancer. The cancer-associated antigen may be associated with cancer or cancer pathology, or may be an abnormality that appears as a result of cancer. I...

Claims

1. Immune cells containing modifications that increase the expression or activity of SERPINB9.

2. Containing exogenous nucleic acids encoding the SERPINB9 polypeptide, In some cases, the exogenous nucleic acid encoding the SERPINB9 polypeptide is an expression vector or is contained within an expression vector, and in some cases, the expression vector is a retroviral expression vector. Furthermore, depending on the circumstances, the SERPINB9 polypeptide may include or consist of the amino acid sequence of SEQ ID NOs: 1, 4, 5, 6, or 7, or variants thereof having at least 85% amino acid sequence identity with SEQ ID NOs: 1, 4, 5, 6, or 7. The immune cell according to claim 1.

3. The immune cell according to claim 1, wherein the immune cell is an effector immune cell, and optionally the effector immune cell is a T cell or a natural killer (NK) cell.

4. Nucleic acids that encode chimeric antigen receptors (CARs) Depending on the circumstances, the CAR may include an antigen-binding domain that binds to an autoimmune disease-related antigen, or to a cancer-related antigen selected from CD30, CD19, CD20, CD22, B7H3, c-Met, ROR1R, CD4, CD7, CD38, BCMA, mesothelin, EGFR, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY, and PSCA. The immune cell according to claim 1.

5. These are virus-specific T cells or activated T cells (ATCs). In some cases, the virus-specific T cells are specific to a virus selected from Epstein-Barr virus (EBV), adenovirus, cytomegalovirus (CMV), human papillomavirus (HPV), influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), herpes simplex virus (HSV), BK virus (BKV), or varicella-zoster virus (VZV), and in some cases, the virus is EBV. The immune cell according to claim 1.

6. A pharmaceutical composition comprising immune cells according to any one of claims 1 to 5 and a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.

7. The pharmaceutical composition according to claim 6 for use in a method of medical treatment or prevention.

8. Use of immune cells according to any one of claims 1 to 5 in the manufacture of a pharmaceutical product for use in a method of medical treatment or prevention.

9. A method for reducing the activity of a serine protease or caspase in a cell, comprising the step of modifying the cell to increase the expression or activity of SERPINB9.

10. A method for increasing the resistance of cells to the activity of serine proteases or caspases, comprising the step of modifying the cells to increase the expression or activity of SERPINB9.

11. A method for increasing the resistance of cells to cell death by granzyme B, comprising the step of modifying the cells to increase the expression or activity of SERPINB9.

12. A method for increasing a cell's resistance to apoptosis mediated by a cell death receptor, comprising the step of modifying the cell to increase the expression or activity of SERPINB9.

13. The method according to any one of claims 9 to 12, wherein the step of modifying the cells to increase the expression or activity of SERPINB9 includes the step of introducing a nucleic acid encoding a SERPINB9 polypeptide into the cells.

14. The method according to claim 13, wherein the nucleic acid encoding the SERPINB9 polypeptide is an expression vector or is contained therein, and optionally the expression vector is a retroviral expression vector.

15. The method according to claim 13, wherein the SERPINB9 polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 1, 4, 5, 6, or 7, or variants thereof having at least 85% amino acid sequence identity with SEQ ID NO: 1, 4, 5, 6, or 7.

16. The method according to claim 9, wherein the cells are effector immune cells, and optionally the effector immune cells are T cells or natural killer (NK) cells.

17. The aforementioned cells contain nucleic acids that encode chimeric antigen receptors (CARs), In some cases, the CAR includes an antigen-binding domain that binds to an autoimmune disease-related antigen or a cancer-related antigen selected from CD30, CD19, CD20, CD22, B7H3, c-Met, ROR1R, CD4, CD7, CD38, BCMA, mesothelin, EGFR, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY, and PSCA, and in some cases, the CAR includes an antigen-binding domain that binds to CD30. The method according to claim 9.

18. The aforementioned cells are virus-specific T cells. In some cases, the virus-specific T cells are specific to a virus selected from Epstein-Barr virus (EBV), adenovirus, cytomegalovirus (CMV), human papillomavirus (HPV), influenza virus, measles virus, hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), lymphocytic choriomeningitis virus (LCMV), herpes simplex virus (HSV), BK virus (BKV), or varicella-zoster virus (VZV), and in some cases, the virus is EBV. The method according to claim 9.

19. Immune cells for use in treating or preventing a disease or condition in a subject, These are virus-specific T cells, and in some cases, Epstein-Barr virus (EBV)-specific T cells. Modifications that increase the expression or activity of SERPINB9, The aforementioned immune cells.

20. The use of immune cells in the manufacture of a pharmaceutical product for use in treating or preventing a disease or condition in a subject, The immune cells are virus-specific T cells, and in some cases, the immune cells are Epstein-Barr virus (EBV)-specific T cells. The immune cells include modifications that increase the expression or activity of SERPINB9. The aforementioned use.

21. The immune cells or use for use according to claim 19 or claim 20, wherein the disease or condition is selected from autoimmune diseases, SLE, multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, type 1 diabetes, juvenile idiopathic arthritis, and celiac disease.

22. The immune cells or use according to claim 19 or 20, wherein the subject is homogeneous with respect to the immune cells.

23. The immune cells for use or use according to claim 19 or claim 20, wherein the immune cells comprise an exogenous nucleic acid encoding a SERPINB9 polypeptide.

24. The exogenous nucleic acid encoding the SERPINB9 polypeptide is an expression vector or contained therein, and optionally the expression vector is a retroviral expression vector, for use in immune cells or for use according to claim 19 or claim 20.

25. The SERPINB9 polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 1, 4, 5, 6, or 7, or variants thereof having at least 85% amino acid sequence identity with SEQ ID NO: 1, 4, 5, 6, or 7, according to claim 19 or claim 20, for use in immune cells or use.

26. The virus-specific T cells include an antigen-binding domain that binds to an autoimmune disease-related antigen, or to a cancer-related antigen selected from CD30, CD19, CD20, CD22, B7H3, c-Met, ROR1R, CD4, CD7, CD38, BCMA, mesothelin, EGFR, GPC3, MUC1, HER2, GD2, CEA, EpCAM, LeY, and PSCA, and optionally the CAR includes a nucleic acid encoding a chimeric antigen receptor (CAR) that includes an antigen-binding domain that binds to CD30. Immune cells or use for the purpose described in claim 19 or claim 20.

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