Compositions of chimeric antigen receptors (CARS) and methods for use thereof

Abstract

To provide improved chimeric antigen receptor-based therapies that allow for more effective, safe, and efficient targeting of T-cell associated malignancies.SOLUTION: The present disclosure provides chimeric antigen receptors, compositions, and methods thereof. In one embodiment, the present disclosure provides a method of treating autoimmune diseases and asthma, and preventing or mediating organ rejection in a subject.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of prior U.S. Provisional Application No. 62 / 523,147, filed June 21, 2017, and U.S. Continuing Application No. 15 / 538,620, filed June 21, 2017, all of which are incorporated herein by reference in their entireties. [Background technology]

[0002] T cells, a type of lymphocyte, play a central role in cell-mediated immunity. They are distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of T cell receptors (TCRs) on their cell surface. Helper T cells, also known as CD4+ T or CD4T cells, express the CD4 glycoprotein on their surface. Helper T cells are activated upon exposure to peptide antigens presented by MHC (major histocompatibility complex) class II molecules. Once activated, these cells rapidly proliferate and secrete cytokines that regulate the immune response. Cytotoxic T cells, also known as CD8+ T cells or CD8 T cells, express the CD8 glycoprotein on their surface. CD8+ T cells are activated upon exposure to peptide antigens presented by MHC class I molecules. A subset of T cells, memory T cells, survive for long periods and respond to their cognate antigens by providing the immune system with a "memory" of past infections and / or tumor cells.

[0003] T cells can be genetically engineered to produce special receptors called chimeric antigen receptors (CARs) on their surface. CARs are proteins on T cells that can recognize specific proteins (antigens) on tumor cells. These engineered CAR T cells are grown in the laboratory until they number in the billions. The expanded population of CAR T cells is then infused into the patient.

[0004] To date, clinical trials using chimeric antigen receptor (CAR) T cells have shown great promise as a treatment for hematologic malignancies resistant to standard chemotherapy. Most notably, CD19-specific CAR (CD19CAR) T-cell therapy has shown remarkably favorable results, including long-term remissions, in B-cell malignancies. (References: Kochenderfer, Wilson et al. 2010, Kalos, Levine et al. 2011, Porter, Levine et al. 2011, Davila, Riviere et al. 2013, Grupp, Frey et al. 2013, Grupp, Kalos et al. 2013, Kalos, Nazimuddin et al. 2013, Kochenderfer, Dudley et al. al. 2013, Kochenderfer, Dudley et al. 2013, Park, Riviere et al. 2014). [Prior art documents] [Non-patent literature]

[0005] [Patent Document 1] WO 01 / 96584 [Patent Document 2] WO 01 / 29058 [Patent Document 3] U.S. Patent No. 6,326,193 Summary of the Invention [Problem to be solved by the invention]

[0006] Despite the success of CAR therapy in B-cell leukemia and lymphoma, CAR therapy for T-cell malignancies has not yet been fully established. T-cell malignancies have a poorer prognosis compared to B-cell malignancies (Abramson, Feldman et al. 2014), potentially addressing an even greater clinical need.

[0007] To date, current efforts have focused on demonstrating the efficacy of CAR T cells against various B-cell malignancies. Treatment of B-ALL with CD19 CARs has an initial remission rate of approximately 90%, but most of these relapse within one year. Relapse is due, at least in part, to antigen escape. Therefore, more effective CAR T-cell therapies to prevent relapse are urgently needed. Target discovery and selection are the first steps, as no general rules guarantee or guide effective CAR design in this regard.

[0008] There are several obstacles preventing the adoption of a broader approach in CAR therapy. The most common challenges are: (1) selection of antigen target and chimeric antigen receptor; (2) CAR design; (3) tumor heterogeneity, particularly variability in surface expression of tumor antigens. Targeting a single antigen carries the risk of immune escape, which can be overcome by targeting multiple desired antigens; and (4) an immunosuppressive microenvironment. CAR T cells are suppressed and may even be inactivated upon reaching the tumor site.

[0009] Most CAR chimeric antigen receptors are scFvs derived from monoclonal antibodies, and some of these monoclonal antibodies have been used in clinical trials or for the treatment of diseases. However, their efficacy has been limited, suggesting the need for alternative and more potent targeting approaches such as CARs. scFvs are the most commonly used chimeric antigen receptors for CARs. However, CAR affinity binding and the location of the recognized epitope on the antigen can potentially affect its function. Furthermore, the level of surface CAR expression on T cells or NK cells is affected by the appropriate leader sequence and promoter. Furthermore, overexpressed CAR proteins can be toxic to cells.

[0010] Therefore, there remains a need for improved chimeric antigen receptor-based therapies that are more effective, safer, and more efficiently acting for T-cell-related malignancies. [Means for solving the problem]

[0011] In one embodiment, the disclosure provides an engineered cell comprising a chimeric antigen receptor polypeptide comprising a signal peptide, an antigen recognition domain, a hinge region, a transmembrane domain, a signaling domain, and a costimulatory domain, wherein the antigen recognition domain comprises one of FcER1A, FcER1, IgE, CD19, BCMA, or CD45.

[0012] In another embodiment, the disclosure provides an engineered polypeptide comprising a chimeric antigen receptor polypeptide comprising a signal peptide, an antigen recognition domain, a hinge region, a transmembrane domain, a signaling domain, and a costimulatory domain, wherein the antigen recognition domain comprises one of FcER1A, CD19, BCMA, or CD45, and a high-efficiency cleavage site is disposed between the chimeric antigen receptor polypeptide and at least one enhancer.

[0013] In another embodiment, the present disclosure provides an engineered cell comprising a first chimeric antigen receptor polypeptide comprising a first signal peptide, a first antigen recognition domain, a first hinge region, a first transmembrane domain, a first signaling domain, and a first costimulatory domain, and a second chimeric antigen receptor polypeptide comprising a second signal peptide, a second antigen recognition domain, a second hinge region, a second transmembrane domain, a second signaling domain, and a second costimulatory domain, wherein the first antigen recognition domain and the second antigen recognition domain are different, and the first antigen recognition domain and the second antigen rejection domain are selected from the group consisting of CD4, CD19, CD33, CD123, CLL-1, BAFFR, BCMA, and CS-1.

[0014] In another embodiment, the present disclosure provides an engineered polypeptide comprising a first chimeric antigen receptor polypeptide comprising a first signal peptide, a first antigen recognition domain, a first hinge region, a first transmembrane domain, a first signaling domain, and a first costimulatory domain, and a second chimeric antigen receptor polypeptide comprising a second signal peptide, a second antigen recognition domain, a second hinge region, a second transmembrane domain, a second signaling domain, and a second costimulatory domain, wherein the first antigen recognition domain and the second antigen recognition domain are different, and the first antigen recognition domain and the second antigen rejection domain are selected from the group consisting of CD4, CD19, CD33, CD123, BAFFR, CLL-1, BCMA, and CS-1.

[0015] In another embodiment, the present disclosure provides a method of treating an autoimmune disease, comprising administering the engineered cells described above to a patient in need thereof, the autoimmune disease comprising lupus erythematosus (SLE), including systemic lupus, multiple sclerosis (MS), inflammatory bowel disease (IBD), rheumatoid arthritis, Sjogren's syndrome, dermatomyososis, autoimmune hemolytic anemia, neuromyelitis optica (NMO), NMO spectrum disorder (NMOSD), idiopathic thrombocytopenic purpura (ITP), antineutrophil cytoplasmic autoantibodies (ANCA) associated with systemic autoimmune small vessel vasculitis syndrome or microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA, Wegener's granulomatosis), or eosinophilic granulomatosis with polyangiitis (EGPA, Churg-Strauss syndrome).

[0016] In another embodiment, the present disclosure provides a method of treating asthma, the method comprising administering the engineered cells described above.

[0017] In another embodiment, the present disclosure provides a method of treating organ rejection, the method comprising administering the engineered cells described above.

[0018] In one embodiment, the present disclosure provides an engineered cell having a first chimeric antigen receptor polypeptide comprising a first antigen recognition domain, a first signal peptide, a first hinge region, a first transmembrane domain, a first costimulatory domain, and a first signal domain, and a second chimeric antigen receptor polypeptide comprising a second antigen recognition domain, a second signal peptide, a second hinge region, a second transmembrane domain, a second costimulatory domain, and a second signaling domain, wherein the first antigen recognition domain is different from the second antigen recognition domain.

[0019] In another embodiment, the disclosure provides a modified polypeptide comprising a chimeric antigen receptor and an enhancer.

[0020] In another embodiment, the disclosure provides a modified polypeptide comprising a chimeric antigen receptor polypeptide and an enhancer.

[0021] In another embodiment, the present disclosure provides an engineered chimeric antigen receptor polypeptide, a polypeptide comprising a signal peptide, a CD45 antigen recognition domain, a hinge region, a transmembrane domain, at least one costimulatory domain, and a signaling domain, or a polynucleotide encoding the aforementioned polypeptide.

[0022] In another embodiment, the present disclosure provides an engineered cell harboring the engineered polypeptide or polynucleotide described above.

[0023] In another embodiment, the present disclosure provides methods for reducing the number of target cells, including methods comprising: (i) contacting the target cells with an effective amount of engineered cells having at least one chimeric antigen receptor polypeptide, the engineered cells having different chimeric antigen receptor polypeptides, each chimeric antigen receptor polypeptide being independent; and (ii) optionally measuring the reduction in the number of said target cells. Target cells include interleukin 6 receptor, ROR1, PSMA, PSCA, MAGE A3, glycolipids, glypican 3, F77, GD-2, WT1, CEA, HER-2 / neu, IL13Rα2, Met, mesothelin, EGFR, EGFRvIII, MUC16, NKG2D ligands, thyroglobulin, MAGE -3, MAGE-4, MAGE-5, MAGE-6, alpha-fetoprotein, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, MUC1, MUC2, and MUC3. , MUC4, MUC5, CD30, EGFRvIII, CD33, CD123, CLL-1, immunoglobulin kappa and lambda, CD38, CD52, CD19, CD20, CD22, CD38, CD45, BCMA, CS1, BAFF receptor, TACI, CD3, CD4, CD8, CD5, CD7, CD2, CD45, CD70, and CD138.

[0024] In another embodiment, the present disclosure provides a method of treating B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, multiple myeloma, acute myeloid leukemia, chronic myelogenous leukemia, chronic myeloproliferative neoplasms, myelodysplastic syndromes, granulocytic sarcoma, histiocytic sarcoma, B-cell acute lymphoblastic leukemia (B-ALL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic myelomonocytic leukemia, and cell proliferative disorders, comprising administering to a patient in need thereof any of the engineered cells described above.

[0025] In some embodiments, the disclosed inventions include methods and compositions for controlling the proliferation of T cells, such as CAR T cells or therapeutic T cells, using, for example, CAMPATH. The methods further relate to compositions and methods for purging CAR T cells using CAMPATH after tumor depletion or in emergency situations, such as unexpected side effects caused by CAR therapy. In further embodiments, CD52 can be incorporated into CD5CAR-engineered cells or any CAR-engineered cells and used as a "safety switch" for CAR therapy. [Brief explanation of the drawings]

[0026] [Figure 1] Schematic diagram of a cCAR construct (hereafter referred to as "multiple CAR or compound CAR"). Multiple or compound CARs target multiple antigens (e.g., cell type 1 or cell type 2 or the same cell type). Multiple or cCAR T cell immunotherapy contains component CARs that contain different or the same antigen recognition domains, hinge regions, transmembrane domains, various costimulatory domains, and intracellular signaling domains. [Figure 2A] Schematic diagram of the cCAR-T construct. The construct contains an SFFV promoter that drives the expression of multiple modular units of the CAR linked by P2A peptides. Upon cleavage of the linker, the cCARs separate and engage targets expressing CD33 and / or CD123, respectively. As a novel cCAR construct, the activation domains of the construct include, but are not limited to, the 4-IBB region on the CD33 CAR segment and the CD28 region on the CD123 CAR. [Figure 2B]Western blot showing the expression of transduced CD33CD123 cCAR-T cells. This figure shows the expression of two different CAR proteins: CD33 CAR and CD123 CAR. cCAR-T cells expressing both CD33 and CD123 CAR upon linker cleavage show two distinct and consistently strong protein bands. Green fluorescent protein (GFP) was used as a negative control. [Figure 2C] Flow cytometry showing transduction efficiency. The top panel shows lentiviral titers of CD33CD123cCAR (also called CD33CD123-2G-CAR) tested in 293FT HEK (human embryonic kidney) cells to measure maximum transduction efficiency before use in UCB (umbilical cord blood) and PB (peripheral blood). The bottom panel shows CD33CD123cCAR (also called CD33CD123-2G-CAR) T cells transduced with a lentiviral vector containing the CD33CD123cCAR construct and GFP-transduced cells as a control. The percentage indicated by the yellow circle is a proxy for transduction efficiency. [Figure 3] Schematic diagram showing the method for producing compound CARs (cCARs). HEK-293-FT cells were transfected with compound CAR plasmid DNA and Lipofectamine 2000. Viral supernatants were harvested, filtered, and stored at -80°C at approximately 36 and 60 hours. T cells were activated with anti-mouse CD3 antibody and IL-2 for at least two days. Activated T cells were transduced at least once with thawed lentivirus on retronectin-coated plates at a cell density of 0.3 x 106 T cells / mL for approximately two days. After at least one overnight transduction, the T cell number was reduced to increase transduction efficiency. After transduction, cells were washed and expanded. Flow analysis (F(Ab')2 labeling) was performed on day 3 to confirm CAR efficiency, and the cells were expanded for a total of 5-7 days. cCAR T cells were co-cultured with target cells in vitro, and their killing effect on cancer cells was evaluated in vivo (in mice). [Figure 4]Co-culture assay showing incubation of the promyelocytic leukemia cell line HL60 with CD33CD123-2G CAR-T cells (cCAR). The potency of the cCAR-T cells (lower panel) is compared with a GFP-transduced T cell control (upper panel). Killing efficacy is measured by the population of CD33+ cells remaining after approximately 24 hours of incubation (circled in yellow). [Figure 5] Co-culture assay of cCAR-T cells incubated with the myeloid leukemia cell line KG-1a, which expresses approximately 100% CD33 and approximately 50-80% CD123. cCAR-T cells (lower panel) are compared to control GFP-transduced T cells (upper panel). Killing efficacy is measured by the population of CD33+ cells remaining after approximately 24 hours of incubation. [Figure 6] CD33CD123 cCAR-T cells are co-cultured with AML-9 at a 5:1 ratio. The co-culture assay demonstrates the incubation of cCAR-T cells with an AML patient sample (referred to here as AML-9). The patient cells are a mixed population of cells, e.g., leukemia cells, monocytes, and other types of blast cells. CD33 and CD34 act as specific markers for leukemia cells and as indicators of CAR-T activity. The CAR-T panel (right) is compared with control GFP-transduced T cells (center). Killing efficacy is measured by the population of CD33+ / CD34+ cells remaining after at least 24 hours of incubation. [Figure 7] CD33CD123 cCAR-T cells are cocultured with Sp-BM-B6 at a 5:1 ratio. The coculture assay demonstrates the incubation of cCAR-T cells with a B-ALL patient sample (referred to here as Sp-BM-B6). The patient's cells represent a mixed population of cells, e.g., leukemia cells, monocytes, and other types of blast cells. CD34 acts as a specific marker for leukemia cells. The CAR-T panel (right) is compared with control GFP-transduced T cells (center). Killing efficacy is measured by the population of CD34+ cells remaining after at least 24 hours of incubation. [Figure 8] Expression of CD33CD123 cCAR on NK-92 cells. Expression of CD33CD123 cCAR is detected using a goat anti-mouse F(ab)2 antibody. [Figure 9] Co-culture assay showing incubation of CD33CD123 cCAR NK-92 cells with HL-60. cCAR NK-92 cells are compared to GFP-transduced NK-92 cells. Killing efficacy is measured by the population of CD33+ cells remaining after approximately 24 hours of incubation. [Figure 10] Co-culture assay showing incubation of cCAR NK-92 cells with KG1a. The cCAR NK cell panel is compared to GFP-transduced NK-92 cells. Killing efficacy is measured by the population of CD33+ cells remaining after approximately 24 hours of incubation. [Figure 11] Dose-dependent response of CD33CD123cCAR (CAR-CD33 / 123) NK-92 cells to HL-60 or KG1a. Killing efficacy is measured by the population of CD33+ cells remaining after approximately 24 hours of incubation. [Figure 12] Comparison of the killing ability of two populations of KG11 cells by CD33CD123 cCAR NK-92 cells with control. Assays were performed at different cell ratios between CAR-CD33 / 123 (CD33CD123 cCAR NK-92 cells) and target cells kG1a. Killing efficacy was measured by the population of CD33+CD123+ or CD33+CD123- cells remaining after approximately 24 hours of incubation. [Figure 13A]This diagram, linked by P2A and T2A, shows the inclusion of both cCAR-T and 4-1BBL in a single construct. The construct consists of two modular units: the CAR A peptide and an enhancer, the SFFV promoter, which drives expression of 4-1BBL. Cleavage of the linker releases the cCAR and 4-1BBL, which then engage targets expressing CD33 and / or CD123 and 4-1BBL. Compound CAR, CD33CD123 CAR T cells are costimulated not only by CD28 but also by the 4-1BB ligand (4-1BBL or CD137L). The CD3-zeta signaling domain completes the assembly of this CAR-T. [Figure 13B] Expression of the CD33CD123-41BBL-2G construct in T cells. Peripheral blood-derived T cells from healthy donors were transduced with the CD33CD123-4-1BBL-2G construct by incubating them with 2 ml of viral supernatant in a 6-well plate. CAR expression was assayed by F(ab)' labeling of the CAR protein surface, followed by FACS analysis. Transduced cells were compared with control T cells by simultaneous labeling. Expression was quantified on day 1 after the end of the transduction period; the transduced population is circled. [Figure 14] This diagram, linked by P2A and T2A, shows the inclusion of both cCAR-T and IL-15 / IL-15sushi in a single construct. The construct consists of two modular units, the CARs and an enhancer, the SFFV promoter, which drives expression of IL-15 / IL-15sushi. Cleavage of the linker releases the cCAR and IL-15 / IL-15sushi, allowing them to engage targets expressing CD33 and / or CD123. The CD3-zeta signaling domain completes the assembly of this CAR-T. Enhancers include, but are not limited to, the IL-15 / IL-15sushi on the cCAR. [Figure 15]Schematic representation of a cCAR. The construct contains an SFFV promoter driving expression of multiple modular units of the CAR, connected by linkers. Upon cleavage of the linker, the cCAR cleaves and engages targets expressing various target antigens, combinations of CD19 and / or CD20, and / or CD22 and / or CD138. Multiple cCARs utilize the same or different costimulatory domains, including, but not limited to, 4-1BB (also referred to as 4-BB) and / or CD28. [Figure 16] Activated T cells were transduced to generate CD19CD20-2G and CD19CD22-2G CAR T cells (all L8). (16A) Design of compound CARs. (16B) Western blot. HEK-293T cells were transfected with lentiviral plasmids for control vector (lane 1), CD19CD20-2G (lane 2), and CD19CD22-2G (lane 3). 48 hours after transfection, the supernatant was removed and cells were harvested. Cells were lysed for Western blot and probed with mouse anti-human CD3z primary antibody and goat anti-mouse HRP secondary antibody. (16C) PMBC buffy coat T cells were activated with anti-CD3 antibody for 3 days. Cells were transduced with either control vector (left), CD19CD20-2G (middle), or CD19CD22-2G (right) lentiviral supernatant. After 3 days of incubation, cells were harvested and incubated with biotin-conjugated goat anti-mouse Fab2 or goat IgG antibody for 30 minutes. Cells were washed, suspended, and stained with streptavidin-PE and mouse anti-human CD3-PerCp for 30 minutes. Cells were washed, suspended in 2% formalin, and analyzed by flow cytometry to measure CAR efficiency. (N=2) [Figure 17]Expression of compound CD19CD22CAR T cells using different leader sequences. PMBC buffy coat T cells were activated with anti-CD3 antibody for 3 days. Cells were transduced with either control vector (left), L8-CD19CD22-2GCAR (center left), L45-CD19CD22-2GCAR (center right), or CSF-CD19CD22-2GCAR (right) lentiviral supernatant. Supernatants were each concentrated 3x. After 3 days of incubation, cells were harvested and incubated with biotin-conjugated goat anti-mouse Fab2 or goat IgG antibody for 30 minutes. Cells were washed, suspended, and stained with streptavidin-PE and mouse anti-human CD3-PerCp for 30 minutes. Cells were washed, suspended in 2% formalin, and analyzed by flow cytometry to measure CAR efficiency. (N=2). [Figure 18] Comparison of transduction efficiency using concentrated and non-concentrated L8-CD19CD22-2G or L8-CD19CD20-2G lentiviral supernatants. A. PMBC buffy coat T cells were activated with anti-CD3 antibody for 3 days. Cells were transduced with either control vector (left), non-concentrated (center) L8-CD19CD22-2GCAR, or 3x-concentrated L8-CD19CD22-2GCAR (right) lentiviral supernatant. After 3 days of incubation, cells were harvested and incubated with biotin-conjugated goat anti-mouse Fab2 or goat IgG antibody for 30 minutes. Cells were washed, suspended, and stained with streptavidin-PE and mouse anti-human CD3-PerCp for 30 minutes. Cells were washed, suspended in 2% formalin, and analyzed by flow cytometry to measure CAR efficiency. (N=2). B. The same experiment was performed with non-concentrated or 2.5-fold concentrated lentiviral vector of the L8-CD19CD20-2G construct. [Figure 19]L8-CD19CD22-2G CAR T cells lyse SP53 tumor cells in overnight coculture. Activated PMBC T cells transduced with either control (top) or L8-CD19CD22-2G (bottom) lentiviral supernatant were incubated at effector:target cell ratios of 1:1 (left), 2:1 (middle), and 5:1 (right). After 24 hours of incubation at 37°C, samples were washed, stained with anti-human CD3-PerCp and anti-human CD19-APC, washed, and analyzed by flow cytometry. Only SP53 cells are shown in the upper right, and a summary of lysis rates at each ratio is shown in the lower right. (N=2). [Figure 20] L8-CD19CD22-2G CAR T cells lyse JeKo-1 tumor cells in overnight coculture. Activated PMBC T cells transduced with either control (left) or 3x-enriched L8-CD19CD22-2G lentiviral supernatant (middle) were incubated with JeKo-1 cells at effector:target cell ratios of 2:1 (top) and 5:1 (bottom). After 24 h of incubation at 37°C, samples were washed, stained with anti-human CD3-PerCp and anti-human CD19-APC, washed, and analyzed by flow cytometry. A summary of JeKo-1 cell lysis and cell lysis is shown on the right. (N=2). [Figure 21] L8-CD19CD22-2G CAR T cells lyse AML patient cells in overnight coculture. Activated PMBC T cells transduced with either control (left) or 3x-enriched L8-CD19CD22-2G lentiviral supernatant (middle) were incubated with CMTMR-stained cells from a patient diagnosed with AML (PT1) at effector:target cell ratios of 2:1 (top) and 5:1 (bottom). After 24 hours of incubation at 37°C, samples were washed, stained with anti-human CD3-PerCp and anti-human CD19-APC, washed, and analyzed by flow cytometry. A summary of patient cells alone and cell lysis is shown on the right. (N=2). [Figure 22A]L8-CD19CD22-2G CAR T cells deplete CD19+ B-ALL patient cells. Activated PMBC T cells transduced with either control (left) or L8-CD19CD22-2G (center) lentiviral supernatant were incubated at a 1:1 ratio with CMTMR-stained cells from a patient with B-ALL (PT2) for 4 days in the presence of 2.5% FBS and IL-2. After this incubation at 37°C, samples were washed, stained with anti-human CD3-PerCp and anti-human CD19-APC, washed, and analyzed by flow cytometry. Stained patient cells cultured alone for 4 days are shown on the right. [Figure 22B] L-8-CD19CD22-2G cCAR T cells demonstrate efficacy against CD22+ K562 cells. A CD22-expressing artificial K562 cell line (K562xp22) was generated via transduction of wild-type K562 cells. The antitumor activity of the CD19CD22cCAR, which targets the minor CD22+ population of K562 cells, was then tested. Coculture experiments at a 1:1 ratio (effector:target) demonstrated a moderately significant cytotoxic effect against the CD22-expressing K562 population compared with controls. Cocultures were stained for CD3, CD19, and CD22, and effector and target populations were separated by flow cytometry. The results were graphed. The cytotoxicity results are consistent with other reported values ​​for antitumor activity against artificial antigen-presenting cell lines. [Figure 23] Various transduction schemes for BC1cCAR lentivirus. (A) Method 1 consists of two transductions of 24 hours each and is the baseline transduction scheme. The scheme proceeds as shown. (B) Method 2 has the same methodology as Method 1, but the second transduction is replaced by continuous incubation. (C) The revised Method 2 directly incubates cells with viral supernatant for 48 hours. [Figure 24A]Diagram of CAR construct and comparison of transduction methodologies. (24A) The modular design of BC1cCAR consists of an anti-CD269 (BCMA) single-chain variable fragment (scFv) domain, a CD319 (CS1) scFv fused to a self-cleaving P2A peptide, a CD8-derived hinge (H) and transmembrane (TM) region, and tandem CD28 and 4-1BB coactivation domains linked to a CD3ζ signaling domain. For efficient expression of the CD3CAR molecule on the T cell surface, the strong spleen focus-forming virus promoter (SFFV) and CD8 leader sequence were used. (24B) Expression of BC1cCAR was measured by flow cytometry against isotype controls. The boxed population indicates transduced CAR cells. [Figure 24B] Diagram of CAR construct and comparison of transduction methodologies. (24A) The modular design of BC1cCAR consists of an anti-CD269 (BCMA) single-chain variable fragment (scFv) domain, a CD319 (CS1) scFv fused to a self-cleaving P2A peptide, a CD8-derived hinge (H) and transmembrane (TM) region, and tandem CD28 and 4-1BB coactivation domains linked to a CD3ζ signaling domain. For efficient expression of the CD3CAR molecule on the T cell surface, the strong spleen focus-forming virus promoter (SFFV) and CD8 leader sequence were used. (24B) Expression of BC1cCAR was measured by flow cytometry against isotype controls. The boxed population indicates transduced CAR cells. [Figure 24C]Diagram of CAR construct and comparison of transduction methodologies. (24A) The modular design of BC1cCAR consists of an anti-CD269 (BCMA) single-chain variable fragment (scFv) domain, a CD319 (CS1) scFv fused to a self-cleaving P2A peptide, a CD8-derived hinge (H) and transmembrane (TM) region, and tandem CD28 and 4-1BB coactivation domains linked to a CD3ζ signaling domain. For efficient expression of the CD3CAR molecule on the T cell surface, the strong spleen focus-forming virus promoter (SFFV) and CD8 leader sequence were used. (24B) Expression of BC1cCAR was measured by flow cytometry against isotype controls. The boxed population indicates transduced CAR cells. [Figure 24D] Protein expression of BC1cCAR and BCMA-CS1-2G in HEK-293FT cells. HEK-293FT cells were transfected with lentiviral plasmids for GFP (lane 1) and BC1cCAR (lane 2). After 48 hours, the supernatant was removed and the cells were discarded. Cells were lysed for Western blot and probed with mouse anti-human CD3z antibody. Optimization of the method improves transduction efficiency. [Figure 25A]In vitro evaluation of BC1cCAR T cells against myeloma cell lines. (25A) BC1cCAR and control T cells were cultured with BCMA-highly positive MM1S and RPMI-8226 cells at E:T ratios of 2:1 and 5:1 for 24 hours. Target MM1S and RPMI-8226 cells were stained with Cytotracker dye (CMTMR) to distinguish them from effector T cells. Populations were gated with anti-BCMA (CD269) and anti-CS1 (CD319 antibody) along with CMTMR-PE. Target U266 cells were labeled with Cytotracker dye (CMTMR) to distinguish them from effector T cells. (25B) Reduction of U266 targets. BC1cCAR and control T cells were incubated with U266 cells expressing subsets of BCMA and CS1. Target tumor cells were stained as described above, and similar gating conditions were applied. The tumor population is boxed. (25C) Summary of BC1cCAR T activity against human myeloma cell lines in vitro. Graphical summary of in vitro cytotoxicity of BC1cCAR T cells against various myeloma cell lines at E:T ratios of 2:1 and 5:1. [Figure 25B]In vitro evaluation of BC1cCAR T cells against myeloma cell lines. (25A) BC1cCAR and control T cells were cultured with BCMA-highly positive MM1S and RPMI-8226 cells at E:T ratios of 2:1 and 5:1 for 24 hours. Target MM1S and RPMI-8226 cells were stained with Cytotracker dye (CMTMR) to distinguish them from effector T cells. Populations were gated with anti-BCMA (CD269) and anti-CS1 (CD319 antibody) along with CMTMR-PE. Target U266 cells were labeled with Cytotracker dye (CMTMR) to distinguish them from effector T cells. (25B) Reduction of U266 targets. BC1cCAR and control T cells were incubated with U266 cells expressing subsets of BCMA and CS1. Target tumor cells were stained as described above, and similar gating conditions were applied. The tumor population is boxed. (25C) Summary of BC1cCAR T activity against human myeloma cell lines in vitro. Graphical summary of in vitro cytotoxicity of BC1cCAR T cells against various myeloma cell lines at E:T ratios of 2:1 and 5:1. [Figure 25C]In vitro evaluation of BC1cCAR T cells against myeloma cell lines. (25A) BC1cCAR and control T cells were cultured with BCMA-highly positive MM1S and RPMI-8226 cells at E:T ratios of 2:1 and 5:1 for 24 hours. Target MM1S and RPMI-8226 cells were stained with Cytotracker dye (CMTMR) to distinguish them from effector T cells. Populations were gated with anti-BCMA (CD269) and anti-CS1 (CD319 antibody) along with CMTMR-PE. Target U266 cells were labeled with Cytotracker dye (CMTMR) to distinguish them from effector T cells. (25B) Reduction of U266 targets. BC1cCAR and control T cells were incubated with U266 cells expressing subsets of BCMA and CS1. Target tumor cells were stained as described above, and similar gating conditions were applied. The tumor population is boxed. (25C) Summary of BC1cCAR T activity against human myeloma cell lines in vitro. Graphical summary of in vitro cytotoxicity of BC1cCAR T cells against various myeloma cell lines at E:T ratios of 2:1 and 5:1. [Figure 26A]Characterization of the antitumor activity of BC1cCAR T cells against primary myeloma tumor cells. (26A) Dose-dependent effect against MM7-G primary biphenotypic tumors. BC1cCAR and control T cells were cultured against BCMA + CS1 + primary myeloma cells MM7-G for 24 hours. Target cells were pre-stained with CMTMR, and cultures were performed at E:T ratios of 2:1, 5:1, and 10:1. Populations were gated by BCMA and CS1 along with CMTMR. Flow cytometry plots showing circled populations represent target tumor populations (left). For clarity, a bar graph summarizing in vitro cytotoxicity is used (right). (26B) Specific reduction of populations in MM10-G. Co-culture with MM10-G primary tumor cells was performed under similar conditions. MM10-G reveals distinct populations when stained with anti-CS1 and anti-BCMA antibodies. The BCMA + CS1 + double positive population is colored purple, while only the CS1 + population is colored dark blue. The cytotoxicity of BC1cCAR T cells against each population is summarized in the bar graph below. (26C) Dose-dependent effect of CS1dim against BCMAneg. MM11-G primary tumors. A third experiment using BCMAdimCS1dim primary cells (MM11-G) summarizes the cytotoxic effect of BC1cCAR cells across a range of E:T dosages. (26D) Summary panel graph showing cytotoxicity of BC1cCAR T cells against myeloma cell lines and primary tumor cells with various BCMA and CS1 hybrids. [Figure 26B]Characterization of the antitumor activity of BC1cCAR T cells against primary myeloma tumor cells. (26A) Dose-dependent effect against MM7-G primary biphenotypic tumors. BC1cCAR and control T cells were cultured against BCMA + CS1 + primary myeloma cells MM7-G for 24 hours. Target cells were pre-stained with CMTMR, and cultures were performed at E:T ratios of 2:1, 5:1, and 10:1. Populations were gated by BCMA and CS1 along with CMTMR. Flow cytometry plots showing circled populations represent target tumor populations (left). For clarity, a bar graph summarizing in vitro cytotoxicity is used (right). (26B) Specific reduction of populations in MM10-G. Co-culture with MM10-G primary tumor cells was performed under similar conditions. MM10-G reveals distinct populations when stained with anti-CS1 and anti-BCMA antibodies. The BCMA + CS1 + double positive population is colored purple, while only the CS1 + population is colored dark blue. The cytotoxicity of BC1cCAR T cells against each population is summarized in the bar graph below. (26C) Dose-dependent effect of CS1dim against BCMAneg. MM11-G primary tumors. A third experiment using BCMAdimCS1dim primary cells (MM11-G) summarizes the cytotoxic effect of BC1cCAR cells across a range of E:T dosages. (26D) Summary panel graph showing cytotoxicity of BC1cCAR T cells against myeloma cell lines and primary tumor cells with various BCMA and CS1 hybrids. [Figure 26C]Characterization of the antitumor activity of BC1cCAR T cells against primary myeloma tumor cells. (26A) Dose-dependent effect against MM7-G primary biphenotypic tumors. BC1cCAR and control T cells were cultured against BCMA + CS1 + primary myeloma cells MM7-G for 24 hours. Target cells were pre-stained with CMTMR, and cultures were performed at E:T ratios of 2:1, 5:1, and 10:1. Populations were gated by BCMA and CS1 along with CMTMR. Flow cytometry plots showing circled populations represent target tumor populations (left). For clarity, a bar graph summarizing in vitro cytotoxicity is used (right). (26B) Specific reduction of populations in MM10-G. Co-culture with MM10-G primary tumor cells was performed under similar conditions. MM10-G reveals distinct populations when stained with anti-CS1 and anti-BCMA antibodies. The BCMA + CS1 + double positive population is colored purple, while only the CS1 + population is colored dark blue. The cytotoxicity of BC1cCAR T cells against each population is summarized in the bar graph below. (26C) Dose-dependent effect of CS1dim against BCMAneg. MM11-G primary tumors. A third experiment using BCMAdimCS1dim primary cells (MM11-G) summarizes the cytotoxic effect of BC1cCAR cells across a range of E:T dosages. (26D) Summary panel graph showing cytotoxicity of BC1cCAR T cells against myeloma cell lines and primary tumor cells with various BCMA and CS1 hybrids. [Figure 26D]Characterization of the antitumor activity of BC1cCAR T cells against primary myeloma tumor cells. (26A) Dose-dependent effect against MM7-G primary biphenotypic tumors. BC1cCAR and control T cells were cultured against BCMA + CS1 + primary myeloma cells MM7-G for 24 hours. Target cells were pre-stained with CMTMR, and cultures were performed at E:T ratios of 2:1, 5:1, and 10:1. Populations were gated by BCMA and CS1 along with CMTMR. Flow cytometry plots showing circled populations represent target tumor populations (left). For clarity, a bar graph summarizing in vitro cytotoxicity is used (right). (26B) Specific reduction of populations in MM10-G. Co-culture with MM10-G primary tumor cells was performed under similar conditions. MM10-G reveals distinct populations when stained with anti-CS1 and anti-BCMA antibodies. The BCMA + CS1 + double positive population is colored purple, while only the CS1 + population is colored dark blue. The cytotoxicity of BC1cCAR T cells against each population is summarized in the bar graph below. (26C) Dose-dependent effect of CS1dim against BCMAneg. MM11-G primary tumors. A third experiment using BCMAdimCS1dim primary cells (MM11-G) summarizes the cytotoxic effect of BC1cCAR cells across a range of E:T dosages. (26D) Summary panel graph showing cytotoxicity of BC1cCAR T cells against myeloma cell lines and primary tumor cells with various BCMA and CS1 hybrids. [Figure 27A]Functional validation of BC1cCAR antigen specificity. (27A) We engineered the CML cell line K562 to independently express either BCMA or CS1. Wild-type K562 is shown as a negative peak, while BCMA-expressing K562 (BCMAxpK562) and CS1-expressing K562 (CS1xpK562) show population shifts depending on the proportion of each antigen expressed. (27B) Short-term (4-12 h) incubation of BC1cCAR T cells with either BCMAxpK562 or CS1xpK562 demonstrates antigen-specific cytotoxicity that correlates with increasing E:T doses. Experiments with wild-type K562 were performed as a negative control. To compare the efficacy of BC1cCAR against CS1xpK562 cells, we generated a single CS1-specific CAR, depicted by the red bar in each plot. Specific anti-CS1 activity was also observed against CS1dim NK-92 cells after 24 hours of culture. (27C) Comparison of single-antigen CAR and BC1cCAR T cells in a mixed cell assay. Long-term culture was performed for 48 hours using BCMAxpK562 cells mixed with CS1xpK562 cells at a 5:1 ratio. BC1cCAR, CS1-CAR, BCMA-CAR, and control T cells were added to each well at a 5:1 E:T ratio and analyzed by flow cytometry. Histogram plots showing the residual population of BCMA or CS1 cells are shown for each experimental condition, with red lines demarcating the boundaries of the T cell or target tumor population. Numbers in the histogram plots represent the percentage of the residual population of target tumor cells. (27D) Activity of BC1cCAR T cells against the CS1 subset in primary bone marrow aspirates. Further coculture experiments were performed using bone marrow aspirate samples as a CS1-expressing minority subset. BC1cCAR or control T cells were added at a 2:1 (left panel), 5:1 (middle panel), or 10:1 (right panel) ratio, and the circled populations represent CS1-expressing populations. Results are analyzed by flow cytometry (top). Summary graphs of anti-CS1 activity against myeloid subsets are shown (below). [Figure 27B]Functional validation of BC1cCAR antigen specificity. (27A) We engineered the CML cell line K562 to independently express either BCMA or CS1. Wild-type K562 is shown as a negative peak, while BCMA-expressing K562 (BCMAxpK562) and CS1-expressing K562 (CS1xpK562) show population shifts depending on the proportion of each antigen expressed. (27B) Short-term (4-12 h) incubation of BC1cCAR T cells with either BCMAxpK562 or CS1xpK562 demonstrates antigen-specific cytotoxicity that correlates with increasing E:T doses. Experiments with wild-type K562 were performed as a negative control. To compare the efficacy of BC1cCAR against CS1xpK562 cells, we generated a single CS1-specific CAR, depicted by the red bar in each plot. Specific anti-CS1 activity was also observed against CS1dim NK-92 cells after 24 hours of culture. (27C) Comparison of single-antigen CAR and BC1cCAR T cells in a mixed cell assay. Long-term culture was performed for 48 hours using BCMAxpK562 cells mixed with CS1xpK562 cells at a 5:1 ratio. BC1cCAR, CS1-CAR, BCMA-CAR, and control T cells were added to each well at a 5:1 E:T ratio and analyzed by flow cytometry. Histogram plots showing the residual population of BCMA or CS1 cells are shown for each experimental condition, with red lines demarcating the boundaries of the T cell or target tumor population. Numbers in the histogram plots represent the percentage of the residual population of target tumor cells. (27D) Activity of BC1cCAR T cells against the CS1 subset in primary bone marrow aspirates. Further coculture experiments were performed using bone marrow aspirate samples as a CS1-expressing minority subset. BC1cCAR or control T cells were added at a 2:1 (left panel), 5:1 (middle panel), or 10:1 (right panel) ratio, and the circled populations represent CS1-expressing populations. Results are analyzed by flow cytometry (top). Summary graphs of anti-CS1 activity against myeloid subsets are shown (below). [Figure 27C]Functional validation of BC1cCAR antigen specificity. (27A) We engineered the CML cell line K562 to independently express either BCMA or CS1. Wild-type K562 is shown as a negative peak, while BCMA-expressing K562 (BCMAxpK562) and CS1-expressing K562 (CS1xpK562) show population shifts depending on the proportion of each antigen expressed. (27B) Short-term (4-12 h) incubation of BC1cCAR T cells with either BCMAxpK562 or CS1xpK562 demonstrates antigen-specific cytotoxicity that correlates with increasing E:T doses. Experiments with wild-type K562 were performed as a negative control. To compare the efficacy of BC1cCAR against CS1xpK562 cells, we generated a single CS1-specific CAR, depicted by the red bar in each plot. Specific anti-CS1 activity was also observed against CS1dim NK-92 cells after 24 hours of culture. (27C) Comparison of single-antigen CAR and BC1cCAR T cells in a mixed cell assay. Long-term culture was performed for 48 hours using BCMAxpK562 cells mixed with CS1xpK562 cells at a 5:1 ratio. BC1cCAR, CS1-CAR, BCMA-CAR, and control T cells were added to each well at a 5:1 E:T ratio and analyzed by flow cytometry. Histogram plots showing the residual population of BCMA or CS1 cells are shown for each experimental condition, with red lines demarcating the boundaries of the T cell or target tumor population. Numbers in the histogram plots represent the percentage of the residual population of target tumor cells. (27D) Activity of BC1cCAR T cells against the CS1 subset in primary bone marrow aspirates. Further coculture experiments were performed using bone marrow aspirate samples as a CS1-expressing minority subset. BC1cCAR or control T cells were added at a 2:1 (left panel), 5:1 (middle panel), or 10:1 (right panel) ratio, and the circled populations represent CS1-expressing populations. Results are analyzed by flow cytometry (top). Summary graphs of anti-CS1 activity against myeloid subsets are shown (below). [Figure 27D]Functional validation of BC1cCAR antigen specificity. (27A) We engineered the CML cell line K562 to independently express either BCMA or CS1. Wild-type K562 is shown as a negative peak, while BCMA-expressing K562 (BCMAxpK562) and CS1-expressing K562 (CS1xpK562) show population shifts depending on the proportion of each antigen expressed. (27B) Short-term (4-12 h) incubation of BC1cCAR T cells with either BCMAxpK562 or CS1xpK562 demonstrates antigen-specific cytotoxicity that correlates with increasing E:T doses. Experiments with wild-type K562 were performed as a negative control. To compare the efficacy of BC1cCAR against CS1xpK562 cells, we generated a single CS1-specific CAR, depicted by the red bar in each plot. Specific anti-CS1 activity was also observed against CS1dim NK-92 cells after 24 hours of culture. (27C) Comparison of single-antigen CAR and BC1cCAR T cells in a mixed cell assay. Long-term culture was performed for 48 hours using BCMAxpK562 cells mixed with CS1xpK562 cells at a 5:1 ratio. BC1cCAR, CS1-CAR, BCMA-CAR, and control T cells were added to each well at a 5:1 E:T ratio and analyzed by flow cytometry. Histogram plots showing the residual population of BCMA or CS1 cells are shown for each experimental condition, with red lines demarcating the boundaries of the T cell or target tumor population. Numbers in the histogram plots represent the percentage of the residual population of target tumor cells. (27D) Activity of BC1cCAR T cells against the CS1 subset in primary bone marrow aspirates. Further coculture experiments were performed using bone marrow aspirate samples as a CS1-expressing minority subset. BC1cCAR or control T cells were added at a 2:1 (left panel), 5:1 (middle panel), or 10:1 (right panel) ratio, and the circled populations represent CS1-expressing populations. Results are analyzed by flow cytometry (top). Summary graphs of anti-CS1 activity against myeloid subsets are shown (below). [Figure 28A]Long-term continuous killing assay and tumor rechallenge. (28A) Scheme for constructing the long-term continuous killing assay. Initial cultures were set up with CAR or control cells at a 1:1 E:T ratio with MM1S tumor cells and assayed for 168 hours without exogenous cytokines. After 48 hours, a small sample was collected and analyzed by flow cytometry, and MM1S cells were added back to each treatment well. This process was repeated until the 168 hour time point. (28B) T cell proliferation and response after 48 hours. Images were taken on the day of flow cytometry analysis, and cells were stained with anti-BCMA, anti-CS1, and anti-CD3 antibodies. MM1S cells express high levels of BCMA + with a large proportion of CS1 + cells. The blue circled population represents the presence of MM1S tumors. (28C) CAR cell proliferation and antigen decline after 108 hours. Similar imaging and flow cytometry analysis were performed at 108 hours. [Figure 28B] Long-term continuous killing assay and tumor rechallenge. (28A) Scheme for constructing the long-term continuous killing assay. Initial cultures were set up with CAR or control cells at a 1:1 E:T ratio with MM1S tumor cells and assayed for 168 hours without exogenous cytokines. After 48 hours, a small sample was collected and analyzed by flow cytometry, and MM1S cells were added back to each treatment well. This process was repeated until the 168 hour time point. (28B) T cell proliferation and response after 48 hours. Images were taken on the day of flow cytometry analysis, and cells were stained with anti-BCMA, anti-CS1, and anti-CD3 antibodies. MM1S cells express high levels of BCMA + with a large proportion of CS1 + cells. The blue circled population represents the presence of MM1S tumors. (28C) CAR cell proliferation and antigen decline after 108 hours. Similar imaging and flow cytometry analysis were performed at 108 hours. [Figure 28C]Long-term continuous killing assay and tumor rechallenge. (28A) Scheme for constructing the long-term continuous killing assay. Initial cultures were set up with CAR or control cells at a 1:1 E:T ratio with MM1S tumor cells and assayed for 168 hours without exogenous cytokines. After 48 hours, a small sample was collected and analyzed by flow cytometry, and MM1S cells were added back to each treatment well. This process was repeated until the 168 hour time point. (28B) T cell proliferation and response after 48 hours. Images were taken on the day of flow cytometry analysis, and cells were stained with anti-BCMA, anti-CS1, and anti-CD3 antibodies. MM1S cells express high levels of BCMA + with a large proportion of CS1 + cells. The blue circled population represents the presence of MM1S tumors. (28C) CAR cell proliferation and antigen decline after 108 hours. Similar imaging and flow cytometry analysis were performed at 108 hours. [Figure 29A] BC1cCAR T cells exhibit anti-leukemia effects in vivo. (29A) IVIS imaging of a mouse model injected with MM1S Luc+. NSG mice were sublethally irradiated and intravenously injected with luciferase-expressing MM1S multiple myeloma cells to induce measurable tumor formation. Three days later, mice were intravenously injected with 5x106 BC1cCAR T cells or control GFP T cells. On days 3, 6, 8, and 11, mice were subcutaneously injected with RediJect D-Luciferin and analyzed by IVIS imaging. (29B) BC1cCAR T cells control MM1S tumor growth. The mean light intensity measured in mice injected with BC1cCAR T cells was compared to the mean light intensity in mice injected with GFP control T cells. (29C) BC1cCAR T cells improve mouse survival. The survival rate of mice was measured and compared between the two groups, and a log-rank Mantel-Cox test was performed to show that the survival rate was significantly improved. [Figure 29B]BC1cCAR T cells exhibit anti-leukemia effects in vivo. (29A) IVIS imaging of a mouse model injected with MM1S Luc+. NSG mice were sublethally irradiated and intravenously injected with luciferase-expressing MM1S multiple myeloma cells to induce measurable tumor formation. Three days later, mice were intravenously injected with 5x106 BC1cCAR T cells or control GFP T cells. On days 3, 6, 8, and 11, mice were subcutaneously injected with RediJect D-Luciferin and analyzed by IVIS imaging. (29B) BC1cCAR T cells control MM1S tumor growth. The mean light intensity measured in mice injected with BC1cCAR T cells was compared to the mean light intensity in mice injected with GFP control T cells. (29C) BC1cCAR T cells improve mouse survival. The survival rate of mice was measured and compared between the two groups, and a log-rank Mantel-Cox test was performed to show that the survival rate was significantly improved. [Figure 29C] BC1cCAR T cells exhibit anti-leukemia effects in vivo. (29A) IVIS imaging of a mouse model injected with MM1S Luc+. NSG mice were sublethally irradiated and intravenously injected with luciferase-expressing MM1S multiple myeloma cells to induce measurable tumor formation. Three days later, mice were intravenously injected with 5x106 BC1cCAR T cells or control GFP T cells. On days 3, 6, 8, and 11, mice were subcutaneously injected with RediJect D-Luciferin and analyzed by IVIS imaging. (29B) BC1cCAR T cells control MM1S tumor growth. The mean light intensity measured in mice injected with BC1cCAR T cells was compared to the mean light intensity in mice injected with GFP control T cells. (29C) BC1cCAR T cells improve mouse survival. The survival rate of mice was measured and compared between the two groups, and a log-rank Mantel-Cox test was performed to show that the survival rate was significantly improved. [Figure 29D]BCMA-CAR and BC1 cCAR T cells showed significant anti-leukemia effects in a xenograft mouse model in a mixture of BCMA and CS1-expressing K562 cells. BCMA-expressing luciferase-positive K562 cells were mixed with CS1-expressing luciferase-positive K562 cells at a 4:1 ratio. Then, 24 hours after sublethal irradiation, the mixed K562 cells (0.5 x 106 cells) were injected intravenously (day 1). Three days later, BCMA CAR T cells, BC1 cCAR T cells, or control T cells were injected intravenously into each mouse (n = 5 per group). Dorsal tumor burden was measured using an IVIS imaging system on days 3, 7, 10, and 12. On day 7, BCMA mouse #3 had a large tumor. On day 10, dorsal comparison with the control group showed BCMA = 47.7% less tumor burden, cCAR = 53.8% less tumor burden. On day 12 (ventral view only), dorsal comparison showed BCMA vs. control = 43.8% less tumor burden, cCAR vs. control = 60.7% less tumor burden. [Figure 29E] Significant reduction in tumor burden in vivo with BCMA and BC1 cCAR T cells. Percent reduction over time compared to control in mice treated with BCMA CAR T cells or cCAR (BC1 cCAR). [Figure 30A] BC1cCAR transduction into NK-92 cells. (30A) The modular design of BC1cCAR was constructed as previously described. (30B) CAR expression on the surface of NK-92 cells. NK-92 cells were transduced with this construct by incubating with viral supernatant for 48 hours, and surface expression of the CAR protein was detected by labeling with a F(ab)' antibody. The transduced population is circled and compared to control NK-92 cells. [Figure 30B]BC1cCAR transduction into NK-92 cells. (30A) The modular design of BC1cCAR was constructed as previously described. (30B) CAR expression on the surface of NK-92 cells. NK-92 cells were transduced with this construct by incubating with viral supernatant for 48 hours, and surface expression of the CAR protein was detected by labeling with a F(ab)' antibody. The transduced population is circled and compared to control NK-92 cells. [Figure 31A] Characterization of BC1cCAR NK-92 antitumor properties. (31A) BC1cCAR NK cells lyse myeloma cell lines and primary cells. BC1cCAR NK-92 cells were incubated with primary MM7-G tumor cells as well as U266, RPMI-8226, and MM1S myeloma cell lines. Co-culture was performed at an E:T ratio of 5:1 for 2 hours and labeled with anti-CS1 and anti-BCMA antibodies to separate the populations. The tumor population is circled. MM7-G primary tumor cells were stained with cell cytotracker dye (CMTMR) to distinguish them from NK-92 cells and are circled. A summary of BC1cCAR NK-92 cytotoxic activity is presented in a bar graph (31B). (31C) The antigen-specific activity of BC1cCAR NK-92 cells was tested with artificially BCMA-expressing K562 (BCMAxpK562) and CS1-expressing K562 (CS1xpK562) cells. Co-culture was performed at an E:T ratio of 5:1 for 4 hours. The K562 population was pre-stained with CMTMR and circled on the flow cytometry plot. The bar graph visualizes a summary of anti-tumor activity. [Figure 31B]Characterization of BC1cCAR NK-92 antitumor properties. (31A) BC1cCAR NK cells lyse myeloma cell lines and primary cells. BC1cCAR NK-92 cells were incubated with primary MM7-G tumor cells as well as U266, RPMI-8226, and MM1S myeloma cell lines. Co-culture was performed at an E:T ratio of 5:1 for 2 hours and labeled with anti-CS1 and anti-BCMA antibodies to separate the populations. The tumor population is circled. MM7-G primary tumor cells were stained with cell cytotracker dye (CMTMR) to distinguish them from NK-92 cells and are circled. A summary of BC1cCAR NK-92 cytotoxic activity is presented in a bar graph (31B). (31C) The antigen-specific activity of BC1cCAR NK-92 cells was tested with artificially BCMA-expressing K562 (BCMAxpK562) and CS1-expressing K562 (CS1xpK562) cells. Co-culture was performed at an E:T ratio of 5:1 for 4 hours. The K562 population was pre-stained with CMTMR and circled on the flow cytometry plot. The bar graph visualizes a summary of anti-tumor activity. [Figure 31C]Characterization of BC1cCAR NK-92 antitumor properties. (31A) BC1cCAR NK cells lyse myeloma cell lines and primary cells. BC1cCAR NK-92 cells were incubated with primary MM7-G tumor cells as well as U266, RPMI-8226, and MM1S myeloma cell lines. Co-culture was performed at an E:T ratio of 5:1 for 2 hours and labeled with anti-CS1 and anti-BCMA antibodies to separate the populations. The tumor population is circled. MM7-G primary tumor cells were stained with cell cytotracker dye (CMTMR) to distinguish them from NK-92 cells and are circled. A summary of BC1cCAR NK-92 cytotoxic activity is presented in a bar graph (31B). (31C) The antigen-specific activity of BC1cCAR NK-92 cells was tested with artificially BCMA-expressing K562 (BCMAxpK562) and CS1-expressing K562 (CS1xpK562) cells. Co-culture was performed at an E:T ratio of 5:1 for 4 hours. The K562 population was pre-stained with CMTMR and circled on the flow cytometry plot. The bar graph visualizes a summary of anti-tumor activity. [Figure 32A]Generation and characterization of different BAFF-CAR constructs. (32A) L45-BAFF-28 CAR expression on the surface of T cells. T cells were transduced with the L45-BAFF-28 CAR, and surface expression was assessed using a F(ab)' antibody. Gating was compared to control. (32B) Dependence of CAR expression on the leader sequence. BAFF-CAR constructs with different leader sequences were tested to determine whether transduction efficiency could be improved. Detection on the cell surface was assessed using a F(ab)' antibody, and the transduced population is circled. (32C) Dependence of CAR expression on structural design. Different leader sequences and structural designs (additional units) included on additional BAFF-CAR constructs were tested and used to determine whether CAR transduction could be improved. The transduced population is boxed and gated compared to control T cells. CSF-BAFF-28 41BBL is a BAFF CAR co-expressing 4-1BBL (41BBL) with a CSF leader sequence, and CSF-BAFF-28 IL-15RA is a BAFF CAR co-expressing IL-15 / IL-15SUS (IL-15RA) with a CSF leader sequence. [Figure 32B]Generation and characterization of different BAFF-CAR constructs. (32A) L45-BAFF-28 CAR expression on the surface of T cells. T cells were transduced with the L45-BAFF-28 CAR, and surface expression was assessed using a F(ab)' antibody. Gating was compared to control. (32B) Dependence of CAR expression on the leader sequence. BAFF-CAR constructs with different leader sequences were tested to determine whether transduction efficiency could be improved. Detection on the cell surface was assessed using a F(ab)' antibody, and the transduced population is circled. (32C) Dependence of CAR expression on structural design. Different leader sequences and structural designs (additional units) included on additional BAFF-CAR constructs were tested and used to determine whether CAR transduction could be improved. The transduced population is boxed and gated compared to control T cells. CSF-BAFF-28 41BBL is a BAFF CAR co-expressing 4-1BBL (41BBL) with a CSF leader sequence, and CSF-BAFF-28 IL-15RA is a BAFF CAR co-expressing IL-15 / IL-15SUS (IL-15RA) with a CSF leader sequence. [Figure 32C]Generation and characterization of different BAFF-CAR constructs. (32A) L45-BAFF-28 CAR expression on the surface of T cells. T cells were transduced with the L45-BAFF-28 CAR, and surface expression was assessed using a F(ab)' antibody. Gating was compared to control. (32B) Dependence of CAR expression on the leader sequence. BAFF-CAR constructs with different leader sequences were tested to determine whether transduction efficiency could be improved. Detection on the cell surface was assessed using a F(ab)' antibody, and the transduced population is circled. (32C) Dependence of CAR expression on structural design. Different leader sequences and structural designs (additional units) included on additional BAFF-CAR constructs were tested and used to determine whether CAR transduction could be improved. The transduced population is boxed and gated compared to control T cells. CSF-BAFF-28 41BBL is a BAFF CAR co-expressing 4-1BBL (41BBL) with a CSF leader sequence, and CSF-BAFF-28 IL-15RA is a BAFF CAR co-expressing IL-15 / IL-15SUS (IL-15RA) with a CSF leader sequence. [Figure 33] Characterization of the antitumor properties of L45-BAFF-28 CAR T. L45-BAFF-28 CAR T cells have antitumor activity against MM1S tumor cells. L45-BAFF-28 CAR T cells were cultured with MM1S myeloma cells at an E:T ratio of 3:1 for 48 hours. Replicate samples are shown. Cytotoxic activity is summarized in a bar graph. [Figure 34A]Characterization of antitumor activity with different BAFF-CAR constructs and potentiation. (34A) BAFF-CAR constructs on MM1S cells. L8-BAFF-28IL-15 / IL-15sushi and L8-BAFF-28-41BBL CARs were cultured on MM1S tumor cells at an E:T ratio of 5:1 for 24 hours. The tumor population is outlined. (34B) BAFF-CAR constructs on SP53 cells. Both CAR and L45-BAFF-28 CAR were cultured on Sp53 tumor cells (B lineage) at an E:T ratio of 5:1 for 24 hours. (34C) Summary bar graph of cytotoxic activity. [Figure 34B] Characterization of antitumor activity with different BAFF-CAR constructs and potentiation. (34A) BAFF-CAR constructs on MM1S cells. L8-BAFF-28IL-15 / IL-15sushi and L8-BAFF-28-41BBL CARs were cultured on MM1S tumor cells at an E:T ratio of 5:1 for 24 hours. The tumor population is outlined. (34B) BAFF-CAR constructs on SP53 cells. Both CAR and L45-BAFF-28 CAR were cultured on Sp53 tumor cells (B lineage) at an E:T ratio of 5:1 for 24 hours. (34C) Summary bar graph of cytotoxic activity. [Figure 34C] Characterization of antitumor activity with different BAFF-CAR constructs and potentiation. (34A) BAFF-CAR constructs on MM1S cells. L8-BAFF-28IL-15 / IL-15sushi and L8-BAFF-28-41BBL CARs were cultured on MM1S tumor cells at an E:T ratio of 5:1 for 24 hours. The tumor population is outlined. (34B) BAFF-CAR constructs on SP53 cells. Both CAR and L45-BAFF-28 CAR were cultured on Sp53 tumor cells (B lineage) at an E:T ratio of 5:1 for 24 hours. (34C) Summary bar graph of cytotoxic activity. [Figure 35]Schematic diagram showing the cCAR construct. The construct consists of an SFFV promoter driving the expression of two modular units of the CAR linked by a P2A peptide. Upon cleavage of this P2A peptide, the cCARs separate and engage targets expressing BCMA and / or CD19. The two CAR units use different or the same costimulatory domains. The costimulatory domain can be, but is not limited to, 4-1BB or CD28. [Figure 36A] Characterization of BCMA CAR units. (36A) BCMA CARs effectively deplete BCMA+ MM1S cells. BCMA CARs were transduced into T cells and co-cultured with MM1S tumor cells. CS1 CARs were also generated and used for their potency. MM1S cells are significantly double-positive for both BCMA and CS1. Co-culture was performed for 48 hours, and BCMA and CS1 antibodies were used to identify the tumor center. The boxed population represents residual MM1S tumor cells after culture. (36B) BCMA CARs effectively lyse BCMA+ primary tumor cells. (36B) BCMA CARs and CS1 CARs were also evaluated for their antitumor properties against primary MM7-G myeloma patient cells. The MM7-G population is predominantly BCMA+CS1+, with a minor but significant CS1+ population present. Both BCMA CAR and CS1 CAR were used in tandem to differentiate tumor populations from CAR cells by BCMA and cell tracker (CMTMR) to assess cytotoxicity. [Figure 36B]Characterization of BCMA CAR units. (36A) BCMA CARs effectively deplete BCMA+ MM1S cells. BCMA CARs were transduced into T cells and co-cultured with MM1S tumor cells. CS1 CARs were also generated and used for their potency. MM1S cells are significantly double-positive for both BCMA and CS1. Co-culture was performed for 48 hours, and BCMA and CS1 antibodies were used to identify the tumor center. The boxed population represents residual MM1S tumor cells after culture. (36B) BCMA CARs effectively lyse BCMA+ primary tumor cells. (36B) BCMA CARs and CS1 CARs were also evaluated for their antitumor properties against primary MM7-G myeloma patient cells. The MM7-G population is predominantly BCMA+CS1+, with a minor but significant CS1+ population present. Both BCMA CAR and CS1 CAR were used in tandem to differentiate tumor populations from CAR cells by BCMA and cell tracker (CMTMR) to assess cytotoxicity. [Figure 37A] Characterization of CD19CAR. (37A) Design of the CD19CAR unit. (37B) Western blot. HEK-293T cells were transfected with the control vector (lane 1) and CD19-2G (lane 2) lentiviral plasmids. 48 hours after transfection, the supernatant was removed and the cells were harvested. Cells were lysed for Western blot and probed with mouse anti-human CD3z primary antibody and goat anti-mouse HRP secondary antibody. C. PMBC buffy coat T cells were activated with anti-CD3 antibody for 3 days. Cells were transduced with either the control vector (left) or L8-CD19-2G (right) lentiviral supernatant. After 3 days of incubation, cells were harvested and incubated with biotin-conjugated goat anti-mouse Fab2 or goat IgG antibody for 30 minutes. Cells were washed, suspended, and stained with streptavidin-PE and mouse anti-human CD3-PerCp for 30 minutes. The cells were washed, suspended in 2% formalin, and analyzed by flow cytometry to measure CAR efficiency (N=2). [Figure 37B]Characterization of CD19CAR. (37A) Design of the CD19CAR unit. (37B) Western blot. HEK-293T cells were transfected with the control vector (lane 1) and CD19-2G (lane 2) lentiviral plasmids. 48 hours after transfection, the supernatant was removed and the cells were harvested. Cells were lysed for Western blot and probed with mouse anti-human CD3z primary antibody and goat anti-mouse HRP secondary antibody. C. PMBC buffy coat T cells were activated with anti-CD3 antibody for 3 days. Cells were transduced with either the control vector (left) or L8-CD19-2G (right) lentiviral supernatant. After 3 days of incubation, cells were harvested and incubated with biotin-conjugated goat anti-mouse Fab2 or goat IgG antibody for 30 minutes. Cells were washed, suspended, and stained with streptavidin-PE and mouse anti-human CD3-PerCp for 30 minutes. The cells were washed, suspended in 2% formalin, and analyzed by flow cytometry to measure CAR efficiency (N=2). [Figure 37C] Characterization of CD19CAR. (37A) Design of the CD19CAR unit. (37B) Western blot. HEK-293T cells were transfected with the control vector (lane 1) and CD19-2G (lane 2) lentiviral plasmids. 48 hours after transfection, the supernatant was removed and the cells were harvested. Cells were lysed for Western blot and probed with mouse anti-human CD3z primary antibody and goat anti-mouse HRP secondary antibody. C. PMBC buffy coat T cells were activated with anti-CD3 antibody for 3 days. Cells were transduced with either the control vector (left) or L8-CD19-2G (right) lentiviral supernatant. After 3 days of incubation, cells were harvested and incubated with biotin-conjugated goat anti-mouse Fab2 or goat IgG antibody for 30 minutes. Cells were washed, suspended, and stained with streptavidin-PE and mouse anti-human CD3-PerCp for 30 minutes. The cells were washed, suspended in 2% formalin, and analyzed by flow cytometry to measure CAR efficiency (N=2). [Figure 38A]Expression of compound CD19CAR T cells using different leader sequences. (38A) CAR constructs were designed to express fusion proteins with different leader sequences. (38B) PMBC buffy coat T cells were activated with anti-CD3 antibody for 3 days. Cells were transduced with lentiviral supernatants containing either a control vector (left), HA-CD19-2G (top center), IL2-CD19-2G (top right), L8-CD19-2G (bottom center left), L45-CD19-2G (middle right), or CSF-CD19-2GCAR (bottom right). After 3 days of incubation, cells were harvested and incubated with biotin-conjugated goat anti-mouse Fab2 or goat IgG antibody for 30 minutes. Cells were washed, suspended, and stained with streptavidin-PE and mouse anti-human CD3-PerCp for 30 minutes. Cells were washed, suspended in 2% formalin, and analyzed by flow cytometry to measure CAR efficiency. (N=2) [Figure 38B] Expression of compound CD19CAR T cells using different leader sequences. (38A) CAR constructs were designed to express fusion proteins with different leader sequences. (38B) PMBC buffy coat T cells were activated with anti-CD3 antibody for 3 days. Cells were transduced with lentiviral supernatants containing either a control vector (left), HA-CD19-2G (top center), IL2-CD19-2G (top right), L8-CD19-2G (bottom center left), L45-CD19-2G (middle right), or CSF-CD19-2GCAR (bottom right). After 3 days of incubation, cells were harvested and incubated with biotin-conjugated goat anti-mouse Fab2 or goat IgG antibody for 30 minutes. Cells were washed, suspended, and stained with streptavidin-PE and mouse anti-human CD3-PerCp for 30 minutes. Cells were washed, suspended in 2% formalin, and analyzed by flow cytometry to measure CAR efficiency. (N=2) [Figure 39]Expression of CD19CAR on T cells using different CD19 scFv sequences. (39A) CAR constructs were designed to express fusion proteins with different scFv sequences. (39B) PMBC buffy coat T cells were activated with anti-CD3 antibody for 3 days. Cells were transduced with either a control vector (left), L8-CD19-2G (center), IL2-CD19-2G (top right), or L8-CD19b-BB-2G (right) lentiviral supernatant. After 3 days of incubation, cells were harvested and incubated with biotin-conjugated goat anti-mouse Fab2 or goat IgG antibody for 30 minutes. Cells were washed, suspended, and stained with streptavidin-PE and mouse anti-human CD3-PerCp for 30 minutes. Cells were washed, suspended in 2% formalin, and analyzed by flow cytometry to measure CAR efficiency. (N=2) [Figure 40] L8-CD19-2G and CD19b-BB CAR T cells lyse SP53 tumor cells in overnight coculture. Activated PMBC T cells transduced with either control (left), L8-CD19-2G (center), or L8-CD19b-BB-2G (right) lentiviral supernatant were incubated with SP53 cells at effector:target cell ratios of 2:1 (top) and 5:1 (bottom). After 24 hours of incubation at 37°C, samples were washed, stained with anti-human CD3-PerCp and anti-human CD19-APC, washed, and analyzed by flow cytometry. A summary of SP53 cell lysis and cell lysis is shown on the far right. (N=2) [Figure 41]L8-CD19-2G and CD19b-BB CAR T cells lyse JeKo-1 tumor cells in overnight coculture. Activated PMBC T cells transduced with either control (left), L8-CD19-2G (center), or L8-CD19b-BB-2G (right) lentiviral supernatant were incubated with JeKo-1 cells at effector:target cell ratios of 2:1 (top) and 5:1 (bottom). After 24 h of incubation at 37°C, samples were washed, stained with anti-human CD3-PerCp and anti-human CD19-APC, washed, and analyzed by flow cytometry. A summary of JeKo-1 cell lysis alone and cytolysis is shown on the far right. (N=2). [Figure 42] L8-CD19-2G and L8-CD19b-BB-2G CAR T cells lyse AML patient cells in overnight coculture. Activated PMBC T cells transduced with either control (left), L8-CD19-2G (center), or L8-CD19b-BB-2G (right) lentiviral supernatant were incubated with CMTMR-stained AML patient cells at effector:target cell ratios of 2:1 (top) and 5:1 (bottom). After 24 hours of incubation at 37°C, samples were washed, stained with anti-human CD3-PerCp and anti-human CD19-APC, washed, and analyzed by flow cytometry. A summary of cell lysis with pre-stained patient cells only is shown on the far right. (N=2). [Figure 43]L8-CD19-2G and L8-CD19b-BB-2G CAR T cells deplete CD19+ patient cells. Activated PMBC T cells transduced with either control (left), L8-CD19-2G (center), or L8-CD19b-BB-2G (right) lentiviral supernatant were incubated with CMTMR-stained cells from a patient with B-ALL. L8-CD19b-BB-2G T cells were incubated with patient cells at a 1:1 ratio overnight (top), and L8-CD19b-BB-2GT cells were incubated with patient cells at a 5:1 ratio for 40 hours (bottom). After this incubation at 37°C, samples were washed, stained with anti-human CD3-PerCp and anti-human CD19-APC, washed, and analyzed by flow cytometry. Only pre-stained patient cells are shown on the far right. (N=2). [Figure 44] Schematic diagram showing the cCAR construct. The construct consists of an SFFV promoter driving the expression of two modular units of the CAR linked by a P2A peptide. Upon cleavage of this P2A peptide, the cCAR separates and engages targets expressing BCMA and / or CD19b. The two units of the CAR use different or the same costimulatory domains. The costimulatory domain can be 4-1BB or CD28. [Figure 45A]Generation and characterization of different BAFF-CAR constructs. (45A) L45-BAFF-28 CAR was transduced into T cells, and surface expression was assessed using a F(ab)' antibody. Gating was compared to control. (45B) BAFF-CAR constructs with different leader sequences were tested to determine whether they could improve transduction efficiency. Surface detection was assessed using a F(ab)' antibody, and the transduced population is outlined. (45C) Additional BAFF-CAR constructs, including different leader sequences and construct designs (additional units), were tested and used to determine whether CAR transduction rates could be improved. The transduced population is outlined and gated compared to control T cells. CSF-BAFF-28 41BBL is a BAFF CAR co-expressing 4-1BBL (41BBL) with a CSF leader sequence. CSF-BAFF-28IL-15 / IL-15sushi is a BAFF CAR that co-expresses IL-15 / IL-15sushi with the CSF leader sequence. [Figure 45B]Generation and characterization of different BAFF-CAR constructs. (45A) L45-BAFF-28 CAR was transduced into T cells, and surface expression was assessed using a F(ab)' antibody. Gating was compared to control. (45B) BAFF-CAR constructs with different leader sequences were tested to determine whether they could improve transduction efficiency. Surface detection was assessed using a F(ab)' antibody, and the transduced population is outlined. (45C) Additional BAFF-CAR constructs, including different leader sequences and construct designs (additional units), were tested and used to determine whether CAR transduction rates could be improved. The transduced population is outlined and gated compared to control T cells. CSF-BAFF-28 41BBL is a BAFF CAR co-expressing 4-1BBL (41BBL) with a CSF leader sequence. CSF-BAFF-28IL-15 / IL-15sushi is a BAFF CAR that co-expresses IL-15 / IL-15sushi with the CSF leader sequence. [Figure 45C]Generation and characterization of different BAFF-CAR constructs. (45A) L45-BAFF-28 CAR was transduced into T cells, and surface expression was assessed using a F(ab)' antibody. Gating was compared to control. (45B) BAFF-CAR constructs with different leader sequences were tested to determine whether they could improve transduction efficiency. Surface detection was assessed using a F(ab)' antibody, and the transduced population is outlined. (45C) Additional BAFF-CAR constructs, including different leader sequences and construct designs (additional units), were tested and used to determine whether CAR transduction rates could be improved. The transduced population is outlined and gated compared to control T cells. CSF-BAFF-28 41BBL is a BAFF CAR co-expressing 4-1BBL (41BBL) with a CSF leader sequence. CSF-BAFF-28IL-15 / IL-15sushi is a BAFF CAR that co-expresses IL-15 / IL-15sushi with the CSF leader sequence. [Figure 46A] L45-BAFF-28 CAR T cells have antitumor activity against MM1S tumor cells. Characterization of the antitumor properties of L45-BAFF-28 CAR T. (46A) In vitro BAFF CAR cytotoxic activity summarized from (46B). (46B) L45-BAFF-28 CAR T cells have antitumor activity against MM1S tumor cells. L45-BAFF-28 CAR T cells were cultured with MM1S myeloma cells at an E:T ratio of 3:1 for 48 hours. Duplicate samples are shown. [Figure 46B]L45-BAFF-28 CAR T cells have antitumor activity against MM1S tumor cells. Characterization of the antitumor properties of L45-BAFF-28 CAR T. (46A) In vitro BAFF CAR cytotoxic activity summarized from (46B). (46B) L45-BAFF-28 CAR T cells have antitumor activity against MM1S tumor cells. L45-BAFF-28 CAR T cells were cultured with MM1S myeloma cells at an E:T ratio of 3:1 for 48 hours. Duplicate samples are shown. [Figure 47] Characterization of antitumor activity using different BAFF-CAR constructs and enhancements. (47A) L8-BAFF-28IL-15 / IL-15sushi and L8-BAFF-28 4-1BBL CARs were cultured with MM1S tumor cells at an E:T ratio of 5:1 for 24 hours. The tumor population is outlined. (47B) Both CARs and the L45-BAFF-28 CAR were cultured with Sp53 tumor cells (B lineage) at an E:T ratio of 5:1 for 24 hours. [Figure 48] CRISPR / Cas9 interference system. Expression of sgRNA and Cas9-puromycin is driven by the U6 and SFFV promoters, respectively. Cas9 is linked to the puromycin resistance gene by an E2A self-cleaving sequence. [Figure 49A] Steps for generating CAR T or NK cells to target hematologic malignancies. [Figure 49B]We generated stable CD45-knockdown NK-92 cells using the CRISPR / Cas9 lentiviral system and sorted the cells. Flow cytometry analysis showed the CD45 expression level on the surface of NK-92 cells (left panel). After transducing NK-92 cells with sgCD45B CRISPR, the transduced cells were cultured in medium containing puromycin for several weeks. CD45-negative NK-92 cells were identified and sorted using a CD45 antibody. The purity of stable NK45i-92 (CD45-knockdown) NK-92 cells was determined by flow cytometry analysis (right panel). This data demonstrates that we successfully generated and obtained NK45i-92 cells. [Figure 50] Cell proliferation curves of wild-type, GFP-transduced NK-92, or NK45i-92 NK cells. To evaluate the effect of CD45 knockdown (KD) on cell proliferation in NK-92 cells, NK-92 (●), GFP-transduced NK-92 (■), and NK45i-92 (▲) cells were counted 48 and 96 hours after seeding in 24-well plates. IL-2 was added at 48 hours. (n = 3 independent replicate experiments). Data are mean + SD. These data demonstrate that knockdown of the CD45 receptor on NK-92 cells results in similar cell proliferation curves compared to untransduced or GFP-transduced NK-92 cells. 24-well plate, n = 3 independent replicate experiments, IL-2 was added at 48 hours. [Figure 51]Co-culture assay using CCRF-CEM (target:T) and GFP NK-92 cells or GFP NK45i-92 cells (effector:E) at a 5:1 (E:T) ratio for 16 hours. (51A) Flow cytometry analysis of CCRF-CEM alone (blue dots in the left panel), CCRF-CEM co-cultured with control GFP-transduced NK-92 cells (center panel), or GFP NK45i-92 cells (right panel). Blue dots in all panels indicate remaining target CCRF-CEM cells, and red dots indicate effector cells in the co-culture assay. All incubation times were 6 hours, and the effector T cell:target cell ratio was 5:1. All experiments were performed in duplicate. (51B) Bar graph showing the percent cell lysis by GFP-transduced NK45i-92 cells compared to control GFP-transduced NK92 cells in a coculture assay with CCRF-CEM. These data suggest that knockdown of CD45 in NK-92 cells does not significantly affect the killing activity of CCRF-CEM cells compared to GFP-control NK-92 cells. [Figure 52]Co-culture assay of CCRF-CEM (target:T) with GFP NK-92, CD5CAR NK-92, or CD5CAR NK45i-92 cells (effector:E) at a 5:1 (E:T) ratio and 16 hours of incubation. (52A) From right to left, co-culture assays of CCRF-CEM alone (left panel) and CCRF-CEM with control GFP NK-92 cells (left center panel), CD5CAR NK-92 cells (right center panel), and CD5CAR NK45i. Blue dots in all panels indicate remaining target CCRF-CEM cells, and red dots indicate effector cells in the co-culture assay. The incubation time was 16 hours, and the effector T cell:target cell ratio was 5:1. All experiments were performed in duplicate. (52B) Bar graph shows the percentage of cell lysis by CD5CAR NK-92 cells or CD5CAR NK45i-92 cells compared to control GFP NK92 cells in a co-culture assay with CCRF-CEM. Data are mean + SD. Both CD5CAR NK cells and CD5CAR NK45i-92 cells exhibit nearly 100% cell killing activity against CD5-positive CCRF-CEM compared to control GFP NK-92 cells. These data indicate that CD5CAR NK cells and CD5CAR NK45i-92 cells can effectively lyse CD5-expressing CCRF-CEM cells in an in vitro co-culture assay compared to GFP control NK-92 cells, and that knockdown of CD45 does not affect NK-92 cell killing function. [Figure 53]Construction and expression of the CD45CAR construct. (53A) Schematic diagram of the CD45CAR lentiviral vector. The CD45CAR construct is a modular signaling domain containing a leader sequence, anti-CD45 scFv, hinge domain (H), transmembrane domain (TM), and two costimulatory domains (CD28 and 4-1BB). It is defined as a third-generation CAR and contains the intracellular signaling domain CD3 zeta. (53B) HEK-293FT cells were transfected with lentiviral plasmids encoding GFP (lane 1) and CD45CAR (lane 2). 48 hours after transfection, the supernatant was removed and the cells were harvested. Cells were lysed for Western blot and probed with mouse anti-human CD3z antibody. [Figure 54] Transduction of CD45CAR into NK45i-92 cells and sorting of CD45CAR-transduced cells. After transduction of NK45i-92 cells with CD45CAR lentivirus, the expression level of CD45CAR on NK45i-92 (area circled in blue in the right panel) was compared with that of NK45i-92 cells (left panel) by flow cytometry analysis. CD45CAR-expressing NK45i-92 cells were sorted, and the CD45 expression level on the cell surface was determined by flow cytometry analysis. Approximately 87% of CD45CAR was expressed on the cell surface by flow cytometry analysis. [Figure 55]Co-culture assay of CCRF-CEM (target:T) and GFP NK-92 or CD45CAR NK45i-92 cells (effector:E) at a 5:1 (E:T) ratio for 16 hours. (55A) Flow cytometry analysis of co-cultures of CCRF-CEM and control GFP-transduced NK-92 cells (left panel) or CD45CAR NK45i-92 cells (right panel). Blue dots in all panels indicate remaining target CCRF-CEM cells, and red dots indicate effector NK-92 cells in the co-culture assay. The incubation time was 16 hours, and the effector T cell:target cell ratio was 5:1. All experiments were performed in duplicate. (55B) Bar graphs show the cytolysis rate by CD45CAR NK45i-92 cells in the co-culture assay with CCRF-CEM compared to control GFP NK92 cells. Data are mean + SD. CD45CAR NK45i-92 cells show approximately 70% cell lysis against CCRF-CEM cells compared to control GFP NK-92 cells. These data suggest that CD45CAR NK45i-92 cells effectively lyse CD45-expressing CCRF-CEM cells in in vitro co-culture assays compared to GFP-control NK-92 cells. [Figure 56A]Co-culture assay of Jurkat cells (target:T) and GFP-control or CD45CAR NK45i-92 cells (effector:E) at a 5:1 or 2:1 (E:T) ratio for 6 hours. (56A) Jurkat cells were stained with CMTMR cell tracker dye followed by flow cytometry analysis. These data show that Jurkat cells are CD45 positive (left panel) and mostly CD56 negative (right panel). (56B and 56C) Flow cytometry analysis of co-culture assay of Jurkat cells (target:T) and control or CD45CAR NK45i-92 cells (effector:E). Co-culture assays were performed at a ratio of 5:1 (56B) or 2:1 (56C) (E:T). The left panel shows the results of co-culture with control GFP or CD45CAR NK45i-92 cells at a 5:1 (E:T) ratio, while the right panel shows the results of co-culture with control GFP or CD45CAR NK45i-92 cells at a 2:1 (E:T) ratio. Blue dots in the panels indicate the remaining target Jurkat cells after the co-culture assay, and red dots indicate effector cells in the co-culture assay. Incubation time was 6 hours. All experiments were performed in duplicate. (56C) Bar graphs show the percentage of cytolysis by CD45CAR NK45i-92 cells compared to control GFP NK92 cells at a 5:1 or 2:1 (E:T) ratio. Data are means + SD. CD45CAR NK45i-92 cells demonstrated approximately 60% cytolysis against Jurkat cells compared to control GFP NK-92 cells in both conditions. This data suggests that CD45CAR NK45i-92 cells effectively lyse Jurkat cells expressing CD45 on the cell surface compared to GFP control NK-92 cells in an in vitro co-culture assay. [Figure 56B]Co-culture assay of Jurkat cells (target:T) and GFP-control or CD45CAR NK45i-92 cells (effector:E) at a 5:1 or 2:1 (E:T) ratio for 6 hours. (56A) Jurkat cells were stained with CMTMR cell tracker dye followed by flow cytometry analysis. These data show that Jurkat cells are CD45 positive (left panel) and mostly CD56 negative (right panel). (56B and 56C) Flow cytometry analysis of co-culture assay of Jurkat cells (target:T) and control or CD45CAR NK45i-92 cells (effector:E). Co-culture assays were performed at a ratio of 5:1 (56B) or 2:1 (56C) (E:T). The left panel shows the results of co-culture with control GFP or CD45CAR NK45i-92 cells at a 5:1 (E:T) ratio, while the right panel shows the results of co-culture with control GFP or CD45CAR NK45i-92 cells at a 2:1 (E:T) ratio. Blue dots in the panels indicate the remaining target Jurkat cells after the co-culture assay, and red dots indicate effector cells in the co-culture assay. Incubation time was 6 hours. All experiments were performed in duplicate. (56C) Bar graphs show the percentage of cytolysis by CD45CAR NK45i-92 cells compared to control GFP NK92 cells at a 5:1 or 2:1 (E:T) ratio. Data are means + SD. CD45CAR NK45i-92 cells demonstrated approximately 60% cytolysis against Jurkat cells compared to control GFP NK-92 cells in both conditions. This data suggests that CD45CAR NK45i-92 cells effectively lyse Jurkat cells expressing CD45 on the cell surface compared to GFP control NK-92 cells in an in vitro co-culture assay. [Figure 56C]Co-culture assay of Jurkat cells (target:T) and GFP-control or CD45CAR NK45i-92 cells (effector:E) at a 5:1 or 2:1 (E:T) ratio for 6 hours. (56A) Jurkat cells were stained with CMTMR cell tracker dye followed by flow cytometry analysis. These data show that Jurkat cells are CD45 positive (left panel) and mostly CD56 negative (right panel). (56B and 56C) Flow cytometry analysis of co-culture assay of Jurkat cells (target:T) and control or CD45CAR NK45i-92 cells (effector:E). Co-culture assays were performed at a ratio of 5:1 (56B) or 2:1 (56C) (E:T). The left panel shows the results of co-culture with control GFP or CD45CAR NK45i-92 cells at a 5:1 (E:T) ratio, while the right panel shows the results of co-culture with control GFP or CD45CAR NK45i-92 cells at a 2:1 (E:T) ratio. Blue dots in the panels indicate the remaining target Jurkat cells after the co-culture assay, and red dots indicate effector cells in the co-culture assay. Incubation time was 6 hours. All experiments were performed in duplicate. (56C) Bar graphs show the percentage of cytolysis by CD45CAR NK45i-92 cells compared to control GFP NK92 cells at a 5:1 or 2:1 (E:T) ratio. Data are means + SD. CD45CAR NK45i-92 cells demonstrated approximately 60% cytolysis against Jurkat cells compared to control GFP NK-92 cells in both conditions. This data suggests that CD45CAR NK45i-92 cells effectively lyse Jurkat cells expressing CD45 on the cell surface compared to GFP control NK-92 cells in an in vitro co-culture assay. [Figure 57A]Co-culture assay with GFP-NK-92 cells (target:T) and untransduced NK-92 cells or CD45CAR NK45i-92 cells (effector:E) at a 5:1 or 2:1 (E:T) ratio for 6 hours. (57A) Flow cytometry analysis using GFP-control NK-92 cells. These data demonstrate that approximately 99% of GFP-control NK-92 cells were GFP-positive (green dots). (57B) Flow cytometry analysis of co-culture assay with GFP-control NK-92 cells (target:T) and untransduced or CD45CAR NK45i-92 cells (effector:E). Co-culture assays were performed at a ratio of 5:1 (57A) or 2:1 (E:T) (57C). The left panel shows co-cultures with untransduced or CD45CAR NK45i-92 cells at a 5:1 (E:T) ratio, while the right panel shows co-cultures with untransduced or CD45CAR NK45i-92 cells at a 2:1 (E:T) ratio. Green dots in the panels indicate target GFP NK-92 cells remaining from the co-culture assay, and red dots indicate effector cells after the co-culture assay. Incubation time was 6 hours. All experiments were performed in duplicate. (57C) Bar graphs show the percentage of cytolysis of GFP NK-92 cells by CD45CAR NK45i-92 cells compared to untransduced NK-92 cells at a 5:1 or 2:1 (E:T) ratio. Data are mean + SD. CD45CAR NK45i-92 cells demonstrated approximately 20% lysis of GFP NK-92 cells at a 2:1 (E:T) ratio and approximately 55% lysis of GFP NK-92 cells at a 5:1 (E:T) ratio compared to untransduced NK-92 cells. This data suggests that CD45CAR NK45i-92 cells effectively lyse GFP NK-92 cells expressing CD45 on the cell surface in in vitro co-culture assays compared to untransduced NK-92 cells. [Figure 57B]Co-culture assay with GFP-NK-92 cells (target:T) and untransduced NK-92 cells or CD45CAR NK45i-92 cells (effector:E) at a 5:1 or 2:1 (E:T) ratio for 6 hours. (57A) Flow cytometry analysis using GFP-control NK-92 cells. These data demonstrate that approximately 99% of GFP-control NK-92 cells were GFP-positive (green dots). (57B) Flow cytometry analysis of co-culture assay with GFP-control NK-92 cells (target:T) and untransduced or CD45CAR NK45i-92 cells (effector:E). Co-culture assays were performed at a ratio of 5:1 (57A) or 2:1 (E:T) (57C). The left panel shows co-cultures with untransduced or CD45CAR NK45i-92 cells at a 5:1 (E:T) ratio, while the right panel shows co-cultures with untransduced or CD45CAR NK45i-92 cells at a 2:1 (E:T) ratio. Green dots in the panels indicate target GFP NK-92 cells remaining from the co-culture assay, and red dots indicate effector cells after the co-culture assay. Incubation time was 6 hours. All experiments were performed in duplicate. (57C) Bar graphs show the percentage of cytolysis of GFP NK-92 cells by CD45CAR NK45i-92 cells compared to untransduced NK-92 cells at a 5:1 or 2:1 (E:T) ratio. Data are mean + SD. CD45CAR NK45i-92 cells demonstrated approximately 20% lysis of GFP NK-92 cells at a 2:1 (E:T) ratio and approximately 55% lysis of GFP NK-92 cells at a 5:1 (E:T) ratio compared to untransduced NK-92 cells. This data suggests that CD45CAR NK45i-92 cells effectively lyse GFP NK-92 cells expressing CD45 on the cell surface in in vitro co-culture assays compared to untransduced NK-92 cells. [Figure 57C]Co-culture assay with GFP-NK-92 cells (target:T) and untransduced NK-92 cells or CD45CAR NK45i-92 cells (effector:E) at a 5:1 or 2:1 (E:T) ratio for 6 hours. (57A) Flow cytometry analysis using GFP-control NK-92 cells. These data demonstrate that approximately 99% of GFP-control NK-92 cells were GFP-positive (green dots). (57B) Flow cytometry analysis of co-culture assay with GFP-control NK-92 cells (target:T) and untransduced or CD45CAR NK45i-92 cells (effector:E). Co-culture assays were performed at a ratio of 5:1 (57A) or 2:1 (E:T) (57C). The left panel shows co-cultures with untransduced or CD45CAR NK45i-92 cells at a 5:1 (E:T) ratio, while the right panel shows co-cultures with untransduced or CD45CAR NK45i-92 cells at a 2:1 (E:T) ratio. Green dots in the panels indicate target GFP NK-92 cells remaining from the co-culture assay, and red dots indicate effector cells after the co-culture assay. Incubation time was 6 hours. All experiments were performed in duplicate. (57C) Bar graphs show the percentage of cytolysis of GFP NK-92 cells by CD45CAR NK45i-92 cells compared to untransduced NK-92 cells at a 5:1 or 2:1 (E:T) ratio. Data are mean + SD. CD45CAR NK45i-92 cells demonstrated approximately 20% lysis of GFP NK-92 cells at a 2:1 (E:T) ratio and approximately 55% lysis of GFP NK-92 cells at a 5:1 (E:T) ratio compared to untransduced NK-92 cells. This data suggests that CD45CAR NK45i-92 cells effectively lyse GFP NK-92 cells expressing CD45 on the cell surface in in vitro co-culture assays compared to untransduced NK-92 cells. [Figure 57D]Transduction of CD45b-BB or CD45b-28 into NK45i-92 cells and sorting of CD45b-BB or CD45b-28 NK45i-92 transduced cells. The costimulatory domain of CD45b-BB is 4-1BB, and the costimulatory domain of CD45b-28 is CD28. After transduction of NK45i-92 cells with CD45b-BB or CD45b-28 lentivirus, the expression levels of CD45b-BB CAR or CD45b-28 CAR on NK45i-92 (blue boxed area in the middle panel) were determined by flow cytometry analysis compared with NK45i-92 cells (left panel). NK45i-92 cells expressing CD45b-BB or CD45b-28 CAR were sorted, and the expression levels of CD45b-BB or CD45b-28 on the cells were determined by flow cytometry analysis. Flow cytometry analysis detected approximately 74% CD45b-BB CAR or approximately 82% CD45b-28 CAR expression on the cell surface. [Figure 57E]Coculture assay of REH cells (target:T) with GFP NK-92 cells, CD45CAR NK45i-92 cells, CD45b-BB NK45i-92 cells, or CD45b-28 NK45i-92 cells (effector:E) at a 5:1 (E:T) ratio for 20 hours. The top row shows flow cytometry analysis of cocultures of REH cells alone (left panel), REH cells and control GFP-transduced NK-92 cells (second left panel), CD45CAR NK45i-92 cells (middle panel), CD45b-BB NK45i-92 cells (fourth panel from the left), or CD45b-28 NK45i-92 cells (right panel). Blue dots in all panels indicate remaining target REH cells after the coculture assay, and red dots indicate effector GFP or CARs-NK-92 cells after the coculture assay. All incubation times were 20 hours, and the effector NK cell:target cell ratio was 5:1. All experiments were performed in duplicate. The bar graph below shows the percentage of cell lysis by CD45CAR NK45i-92 cells, CD45b-BB NK45i-92 cells, or CD45b-28 NK45i-92 cells compared to control GFP NK92 cells in a co-culture assay with REH cells. Data are the mean + SD. Compared to control GFP NK-92 cells, CD45CAR NK45i-92 cells lysed approximately 76% of cells, CD45b-BB NK45i-92 cells showed approximately 79% cytolysis, and CD45b-28 NK45i-92 cells showed 100% cytolysis against REH cells. These data suggest that all three CD45CARs effectively lyse REH cells, considered CD45-expressing B cells, in in vitro co-culture assays compared to GFP control NK92 cells. [Figure 57F]Co-culture assay of U937 cells (target:T) with GFP NK-92 cells or CD45b-28 NK45i-92 cells at a 2:1 (E:T) ratio for 20 hours. Flow cytometry analysis of co-cultures of U937 cells (monocytic leukemia cell line) alone (left panel), U937 cells with control GFP-transduced NK-92 cells (middle panel), or CD45b-28 NK45i-92 cells (right panel). Blue dots in all panels indicate remaining target U937 cells, and red dots indicate effector GFP or CD45b-28 NK45i-92 cells in the co-culture assay. The incubation time was 6 hours, and the effector NK cell:target cell ratio was 2:1. F-B, Bar graphs show the percent cytolysis by CD45b-28 NK45i-92 cells compared to control GFP NK92 cells in a coculture assay with U937 cells. CD45b-28 NK45i-92 show approximately 81% cytolysis of U937 cells compared to control GFP NK-92 cells. [Figure 57G] Co-culture assay of MOLM-13 cells (target:T) with GFP NK-92 cells or CD45b-28 NK45i-92 cells at a 5:1 (E:T) ratio for 20 hours. Flow cytometry analysis of GA and co-cultures of MOLM13 cells (monocytic leukemia cell line) alone (left panel), MOLM13 cells with control GFP-transduced NK-92 cells (center panel), or CD45b-28 NK45i-92 cells (right panel). Blue dots in all panels indicate remaining target MOLM13 cells, and red dots indicate effector GFP or CD45b-28 NK45i-92 cells from the co-culture assay. The incubation time was 20 hours, and the effector NK cell:target cell ratio was 5:1. GB, Bar graph shows the percent of cytolysis by CD45b-28 NK45i-92 cells compared to control GFP NK92 cells in a co-culture assay with MOLM13 cells. CD45b-28 NK45i-92 show approximately 91.6% cytolysis against MOLM13 cells compared to control GFP NK-92 cells. [Figure 57H] Co-culture assay of Jeko-1 cells (target:T) with GFP NK-92 cells or CD45b-28 NK45i-92 cells at a 2:1 (E:T) ratio for 6 hours. Flow cytometry analysis of co-cultures with HA, Jeko-1 cells (mantle cell lymphoma) alone (left panel), Jeko-1 cells and control GFP-transduced NK-92 cells (center panel), or CD45b-28 NK45i-92 cells (right panel). Blue dots in all panels indicate remaining target Jeko-1 cells, and red dots indicate effector GFP or CD45b-28 NK45i-92 cells in the co-culture assay. The incubation time was 6 hours, and the effector NK cell:target cell ratio was 2:1. H-B, Bar graph shows percent cytolysis by CD45b-28 NK45i-92 cells compared to control GFP NK92 cells in a coculture assay with Jeko-1 cells. CD45b-28 NK45i-92 show approximately 44.6% cytolysis against Jeko-1 cells compared to control GFP NK-92 cells. [Figure 57I] Coculture assay in which SP53 cells (target:T) and GFP NK-92 cells or CD45b-28 NK45i-92 cells were incubated at a 2:1 (E:T) ratio for 6 hours. IA, Flow cytometry analysis of SP53 cells (mantle cell lymphoma cell line) alone (left panel), cocultured with Jeko-1 cells and control GFP-transduced NK-92 cells (center panel), or CD45b-28 NK45i-92 cells. Blue dots in all panels indicate remaining target SP53 cells, and red dots indicate effector GFP or CD45b-28 NK45i-92 cells in the coculture assay. The incubation time was 6 hours, and the effector NK cell:target cell ratio was 2:1. IB, Bar graphs show the percent cytolysis by CD45b-28 NK45i-92 cells in the coculture assay with SP53 cells compared to control GFP NK92 cells. CD45b-28 NK45i-92 show approximately 45% cytolysis against SP53 cells compared to control GFP NK-92 cells. [Figure 57J] Depletion of CD34(+) cord blood stem cells in 48-hour co-culture. CD34(+) stem cells derived from human umbilical cord blood were co-cultured with either control or CD45b-28 CAR NK cells for 48 hours before labeling at a low 2:1 ratio (effector:target). Approximately 96% of CD34(+) cells were deleted compared to controls. [Figure 58A] The diagram, linked by P2A, shows that both the cCAR-T and 4-1BBL or IL-15 / IL-15sushi are a single construct. The construct consists of an SFFV promoter driving expression of the CAR and an enhancer, 4-1BBL. Upon cleavage of the linker, the CD45 CAR (or CD45b CAR) and 4-1BBL or IL-15 / IL-15sushi split and engage targets expressing CD45. The CD45 CAR T cells are co-stimulated not only by CD28 but also by 4-1BB ligand (4-1BBL or CD137L) or IL-15 / IL-15sushi. The CD3-zeta signaling domain completes the assembly of this CAR-T. [Figure 58B] Flow cytometry analysis of surface CD45b CAR expression levels on CD45b-28-2G-4-1BBL CAR-transduced NK45i-92 cells. The left panel (NK92 cells) and center panel (GFP-NK92) are negative controls, and the right panel shows surface expression of CD45b CAR labeled with goat anti-mouse F(AB')2-PE against the ScFv region (encircled by the blue curve). Transduced cells expressed 86.99% CD45b-CAR on the cell surface. [Figure 58C]Flow cytometry analysis of surface CD45b CAR expression levels on NK45i-92 cells transduced with CD45b-28-2G-IL15 / IL-15sushi CAR. The left panel (NK92 cells) and center panel (GFP-NK92) show the negative control, while the right and left panels show the surface expression of CD45b CAR labeled with goat anti-mouse F(AB')2-PE against the ScFv region (encircled by the blue curve). CD45b-28-2GIL15RA CD45b-28-2G-IL-15 / IL-15sushi virally transduced cells expressed 55.96% more CD45b-CAR on the cell surface than the negative control. [Figure 59] Schematic diagram illustrating the construct and its expression in T or NK cells. (59A) The combination of a CAR (3rd generation) and the IL-15 / sushi domain of the IL-15 alpha receptor is on an expression vector, and their expression is driven by the SFFV promoter. The CAR containing IL-15 / sushi is linked to the P2A self-cleaving sequence. The IL-15 / sushi portion consists of the IL-2 signal peptide fused to IL-15 and linked to the sushi domain via a 26-amino acid poly-proline linker. (59B) The CAR and IL-15 / sushi are present on T or NK cells. [Figure 60] Expression of CD4IL-15 / IL-15sushi. (60A) HEK-293FT cells were transfected with lentiviral plasmids for GFP (lane 1) and CD4IL-15 / IL-15sushiCAR (lane 2). 48 hours after transfection, the supernatant was removed and cells were harvested for Western blotting using a mouse anti-human CD3z antibody. (60B) HEK-293 cells were transduced with either GFP (left) or CD4IL-15 / IL-15sushiCAR (right) viral supernatant from transfected HEK-293FT cells. After 3 days of incubation, cells were harvested, stained with goat anti-mouse F(Ab')2, and analyzed by flow cytometry. [Figure 61] Transduction of NK cells with CD4 IL-15 / IL-15sus CAR. NK-92 cells were transduced with either GFP (left) or CD4 IL-15 / IL-15sushi CAR (right) viral supernatant from transfected HEK-293FT cells. The second transduction was performed 24 hours after the first transduction. At 24 hours after the second transduction, cells were harvested, washed, and transferred to tissue culture plates containing fresh medium and IL-2. After 3 days of incubation, cells were harvested and stained with goat anti-mouse F(Ab')2 antibody or goat IgG (control) at a 1:250 dilution for 30 minutes. Cells were washed, stained with streptavidin-PE conjugate at a 1:500 dilution, washed, suspended in 2% formalin, and analyzed by flow cytometry. [Figure 62] Transduction of CD4IL15RACAR (CD4IL-15 / IL-15sushi) into T cells. Left: Western blot results. HEK-293FT cells were transfected with GFP (lane 1) and CD4IL15RA-CAR (lane 2) lentiviral plasmids. 48 hours after transfection, the supernatant was removed, and cells were harvested for Western blot analysis using a mouse anti-human CD3 zeta antibody. Right: Expression of CD4IL15RACAR. Activated T cells derived from umbilical cord blood buffy coats were transduced with either GFP (left) or concentrated CD4IL15RACAR (right) viral supernatant from transfected HEK-293FT cells. A second transduction was performed 24 hours after the first transduction. 24 hours after the second transduction, cells were harvested, washed, and transferred to tissue culture plates containing fresh medium and IL-2. After 3 days of incubation, cells transduced with either GFP (left) or CD4IL15RA CAR (right) were harvested and stained with goat anti-mouse F (Ab'). Cells were washed, stained with streptavidin-PE conjugate at a dilution of 1:500, washed, suspended in 2% formalin, and analyzed by flow cytometry. [Figure 63A]CD4CAR NK-92 cells and CD4IL-15 / IL-15sushi CAR NK-92 cells kill KARPAS 299 T leukemia cells in coculture. (63A) NK-92 cells transduced with either GFP control (top right), CD4CAR (bottom left), or CD4IL-15 / IL-15sushi (bottom right) lentiviral supernatant were incubated with KARPAS 299 cells at a 5:1 ratio. After 4 hours of coculture, cells were stained with mouse anti-human CD4 (APC) and CD3 (PerCp) antibodies and analyzed by flow cytometry (N = 2). The top left panel shows only labeled Karpas 299 cells. The percentage of lysed target cells is shown in the graph (63B). [Figure 63B] CD4CAR NK-92 cells and CD4IL-15 / IL-15sushi CAR NK-92 cells kill KARPAS 299 T leukemia cells in coculture. (63A) NK-92 cells transduced with either GFP control (top right), CD4CAR (bottom left), or CD4IL-15 / IL-15sushi (bottom right) lentiviral supernatant were incubated with KARPAS 299 cells at a 5:1 ratio. After 4 hours of coculture, cells were stained with mouse anti-human CD4 (APC) and CD3 (PerCp) antibodies and analyzed by flow cytometry (N = 2). The top left panel shows only labeled Karpas 299 cells. The percentage of lysed target cells is shown in the graph (63B). [Figure 64]CD4CAR NK-92 cells and CD4IL-15 / IL-15sushi CAR NK-92 cells kill MOLT4 T leukemia cells in coculture. NK-92 cells transduced with either GFP control (left), CD4CAR (center), or CD4IL-15 / IL-15sushi (second from right) lentiviral supernatant were incubated with MOLT4 cells at an effector:target ratio of 1:1 or 2:1. After overnight coculture, cells were stained with mouse anti-human CD4 (APC) and CD56 (PerCp) antibodies and analyzed by flow cytometry (N = 2). The upper right panel shows only labeled MOLT4 cells. The graph shows the percentage of target cell lysis. [Figure 65A] CD4CAR and CD4IL-15 / IL-15sushi CAR T cells demonstrate anti-leukemia effects in vivo. NSG mice were sublethally irradiated and injected intravenously (tail vein) with luciferase-expressing MOLM13 cells the following day to induce measurable tumor formation (65A). MOLM-13 cells are nearly 100% CD4+. Three days later, mice were intravenously injected with a course of 8x106 CD4CAR, CD4IL-15 / IL-15sushi CAR T cells, or vector control T cells. On days 3, 6, 9, and 11, mice were injected subcutaneously with RediJect D-Luciferin and analyzed by IVIS imaging (65B). [Figure 65B] CD4CAR and CD4IL-15 / IL-15sushi CAR T cells demonstrate anti-leukemia effects in vivo. NSG mice were sublethally irradiated and injected intravenously (tail vein) with luciferase-expressing MOLM13 cells the following day to induce measurable tumor formation (65A). MOLM-13 cells are nearly 100% CD4+. Three days later, mice were intravenously injected with a course of 8x106 CD4CAR, CD4IL-15 / IL-15sushi CAR T cells, or vector control T cells. On days 3, 6, 9, and 11, mice were injected subcutaneously with RediJect D-Luciferin and analyzed by IVIS imaging (65B). [Figure 65C] (65C) The mean light intensity measured for mice injected with vector control T cells was compared to that for mice injected with CD4CAR and CD4IL-15 / IL-15sushi T cells to determine the lysis rate in correlation with the remaining tumor burden. (65D) Mouse survival was measured and compared among the three groups. [Figure 65D] (65C) The mean light intensity measured for mice injected with vector control T cells was compared to that for mice injected with CD4CAR and CD4IL-15 / IL-15sushi T cells to determine the lysis rate in correlation with the remaining tumor burden. (65D) Mouse survival was measured and compared among the three groups. [Figure 66A] CD4IL15 / IL-15sushiCAR NK cells demonstrate potent anti-leukemia activity under stressful conditions in vivo. NSG mice were sublethally irradiated, and the following day, luciferase-expressing Jurkat cells were injected intravenously (tail vein) to induce measurable tumor formation (66A). Jurkat cells were <60% CD4+. Three days later, mice were intravenously injected with 8x106 CD4CAR, CD4IL-15 / IL-15sushiCAR NK cells, or vector control NK cells for one course. On days 3, 7, 10, and 14, mice were subcutaneously injected with RediJect D-Luciferin and underwent IVIS imaging (66B). [Figure 66B]CD4IL15 / IL-15sushiCAR NK cells demonstrate potent anti-leukemia activity under stressful conditions in vivo. NSG mice were sublethally irradiated, and the following day, luciferase-expressing Jurkat cells were injected intravenously (tail vein) to induce measurable tumor formation (66A). Jurkat cells were <60% CD4+. Three days later, mice were intravenously injected with 8x106 CD4CAR, CD4IL-15 / IL-15sushiCAR NK cells, or vector control NK cells for one course. On days 3, 7, 10, and 14, mice were subcutaneously injected with RediJect D-Luciferin and underwent IVIS imaging (66B). [Figure 66C] Lysis rates were quantified in correlation with residual tumor burden by comparing the mean light intensity in vector control NK-injected mice with that in CD4CAR and CD4IL-15 / IL-15sushi NK-injected mice. [Figure 66D] Lysis rates were quantified in correlation with residual tumor burden by comparing the mean light intensity in vector control NK-injected mice with that in CD4CAR and CD4IL-15 / IL-15sushi NK-injected mice. [Figure 67A] Repeat in vivo experiment demonstrating potent lysis of Jurkat tumor cells by CD4I-15 / IL-15sushi CAR NK cells showing results similar to those described in Figure 66. [Figure 67B] Repeat in vivo experiment demonstrating potent lysis of Jurkat tumor cells by CD4I-15 / IL-15sushi CAR NK cells showing results similar to those described in Figure 66. [Figure 68A]Effect of secreted IL-15 / IL-15sushi on CAR and adjacent non-transduced cells. NK-92 cells stably expressing either CD4CAR or CD4IL15RA (CD4IL-15 / IL-15sushi) were mixed with NK-92 cells stably expressing GFP at a 50:50 ratio. These cells were co-cultured with or without IL-2. (68A) Photographs taken with a fluorescence microscope at 20x magnification on days 0 (initiation of co-culture) and 7 without IL-2 addition. (68B) Total cell number calculated throughout the experiment (up to day 14) for NK-92 cells co-cultured with or without IL-2. [Figure 68B] Effect of secreted IL-15 / IL-15sushi on CAR and adjacent non-transduced cells. NK-92 cells stably expressing either CD4CAR or CD4IL15RA (CD4IL-15 / IL-15sushi) were mixed with NK-92 cells stably expressing GFP at a 50:50 ratio. These cells were co-cultured with or without IL-2. (68A) Photographs taken with a fluorescence microscope at 20x magnification on days 0 (initiation of co-culture) and 7 without IL-2 addition. (68B) Total cell number calculated throughout the experiment (up to day 14) for NK-92 cells co-cultured with or without IL-2. [Figure 69]Comparison of the effects of secreted IL-15 and IL-15 / IL-15sushi on NK-92 cell proliferation. CD4IL-15 / IL-15sushi, CD4IL-15, and control-transduced NK-92 cells were cultured in standard NK cell medium at 250,000 cells per day in the absence of IL-2 for 6 days. Although both transduced cells had 10% surface CAR expression, CD4IL15-IL15sushi-transduced NK-92 cells were able to proliferate approximately three-fold higher than CD4IL-15-transduced NK-92 cells by day 6. On day 4, proliferation of CD4IL-15-transduced NK-92 cells was slightly higher than control but significantly lower than that of CD4IL-15 / IL15sushi-transduced NK-92 cells. This study identifies the importance of the IL-15 / IL-15sushi co-expressed functional complex in promoting NK-92 cell proliferation. [Figure 70] Schematic diagram showing a Treg CAR T construct targeting Tregs. The construct consists of an SFFV promoter driving the expression of two units of a chimeric antigen receptor linked by a P2A peptide. Each unit contains a CD45 leader peptide sequence (signal peptide). Upon cleavage of the linker, the two peptide units are separated and engage targets expressing CD4 and CD25. The CD4 chimeric antigen receptor polypeptide unit contains a signal peptide, CD4 antigen recognition domain, hinge region, transmembrane domain, and CD3 zeta chain. The CD25 chimeric antigen receptor polypeptide unit contains a signal peptide, CD25 antigen recognition domain, hinge region, transmembrane domain, and costimulatory domain(s). Treg CARs can enhance the lytic activity of cells co-expressing CD4 and CD25 while minimizing the expression of cells bearing CD4 or CD25 antigens. [Figure 71A]Characterization of CD4 zeta CD25 CAR. (71A) CD4 zeta CD25 CAR was transduced into T cells via 48-hour viral incubation and stained with F(ab)' antibody to assay CAR surface expression. The boxed population represents transduced cells. (71B) C4-25z CAR (CD4 zeta CD25 CAR, Treg CAR) was characterized using CD4 and CD25 antibodies to validate structure-function. The two most relevant populations, CD4 + CD25 + and CD4 - CD25 +, are boxed. The disappearance of double-positive populations and other phenotypic groups is summarized in the adjacent bar graph. [Figure 71B] Characterization of CD4 zeta CD25 CAR. (71A) CD4 zeta CD25 CAR was transduced into T cells via 48-hour viral incubation and stained with F(ab)' antibody to assay CAR surface expression. The boxed population represents transduced cells. (71B) C4-25z CAR (CD4 zeta CD25 CAR, Treg CAR) was characterized using CD4 and CD25 antibodies to validate structure-function. The two most relevant populations, CD4 + CD25 + and CD4 - CD25 +, are boxed. The disappearance of double-positive populations and other phenotypic groups is summarized in the adjacent bar graph. [Figure 72] CD4zetaCD25 CAR T cells primarily target cells co-expressing CD4 and CD25. Three days after activation, PMBC buffy coat T cells were transduced with either control vector (left), CD4CAR (center), or CD4zetaCD25 (right) lentiviral supernatant. T cells were then harvested and incubated with mouse anti-human CD25-PE and mouse anti-human CD4-APC for 30 minutes. Cells were washed, suspended in 2% formalin, and analyzed by flow cytometry. [Figure 73A]Schematic diagram showing the CD5CAR-52 construct. The construct consists of an SFFV promoter driving co-expression of CD5CAR and the CD52 surface antigen. Cleavage of the P2A linker results in the CD5 chimeric antigen receptor polypeptide unit, which consists of a signal peptide, CD5 antigen recognition domain, hinge region, transmembrane domain, and costimulatory domains of CD28 and CD3 zeta chain. The CD5 peptide consists of a signal peptide, CD52 antigen recognition domain (anti-CD52 scFv), hinge region, and transmembrane domain (derived from CD28). [Figure 73B] Experimental design for determining the loss of CD5CAR-52 T cells in the blood. NSG mice were sublethally irradiated and CD5CAR-52 T cells (5 × 106 cells) were injected intravenously into each mouse. Approximately 24 hours later, PBS or 0.1 mg / kg CAMPATH was administered intraperitoneally (IP). N = 3. Six and 24 hours later, peripheral blood was collected from each mouse and labeled with CD3 and CD45 antibodies to determine the loss of CAR-T cells as an acute response to CAMPATH treatment. Five days later, whole blood was collected from each mouse and labeled with CD3 and CD45 antibodies to determine CAR-T cell persistence. CAR-T cells were quantified using flow cytometry analysis. [Figure 73C]Disappearance of CD5CAR-52 T cells in peripheral blood after 6 and 24 hours with and without CAMPATH treatment. Flow cytometry analysis shows the persistence of CD5CAR-52 T cells (blue dots) in the peripheral blood of mice with and without CAMPATH treatment. Blood samples were labeled with CD3 and CD45 antibodies to detect CD5CAR-52 T cells. Blood samples from mice not injected with CAR-T cells (left panel) showed no CD3- or CD45-positive cells (negative control). Mice injected with 0.1 mg / kg CAMPATH showed a loss of CD5CAR-52 T cells from the blood after 6 hours (center panel) and 24 hours (right panel) compared to mice not treated with CAMPATH after 6 hours (second panel from the left) and 24 hours (second panel from the right). N = 3. These results suggest that CAMPATH treatment can quickly remove CAR-T cells from the blood. [Figure 73D] Loss of CD5CAR-52 T cells in whole blood after 5 days with and without CAMPATH treatment. Flow cytometry analysis shows the persistence of CD5CAR-52 T cells (blue dots) in whole blood samples from mice with and without CAMPATH treatment. Blood samples were labeled with CD3 and CD45 antibodies to detect the persistence of CD5CAR-52 T cells. Blood samples from mice not injected with CAR-T cells (left panel) showed no CD3- or CD45-positive cells (negative control). Mice treated with 0.1 mg / kg CAMPATH lost CD5CAR-52 T cells in whole blood samples after 5 days (right panel) compared to mice not injected with CAMPATH (center panel). These results also suggest that CAMPATH treatment can clear CAR-T cells from the blood. [Figure 74] HEK293 cells were transduced with either EF1-GFP or SFFV-GFP viral supernatant in 6-well tissue culture plates in DMEM containing 10% FBS, using the indicated volumes. The medium was changed the following morning. 48 hours after transduction, cells were visualized with GFP at 10x magnification using an EVOS fluorescence microscope. [Figure 75]Using the volumes shown in the previous figure, HEK293 cells transduced with either EF1-GFP or SFFV-GFP viral supernatant were trypsinized, harvested, suspended in formalin, and the percentage of GFP+ cells was determined by flow cytometry analysis using the FITC channel. [Figure 76] Activated cord blood buffy coat T cells transduced with either low or high doses of EF1-GFP or SFFV-GFP viral supernatant were trypsinized, suspended in formalin, and analyzed by flow cytometry using the FITC channel to measure the percentage of GFP + cells 7, 14, 21, and 28 days after transduction. (76A) Percentage of GFP + T cells for cells transduced with either low or high doses of viral supernatant. (76B) Comparison of the percentage of GFP + T cells transduced with high doses of EF1-GFP supernatant with the percentage of GFP + cells in T cells transduced with low amounts of SFFV-GFP supernatant. (50 μL of SFFV-GFP and 1 mL of EF1-GFP supernatant were used.) (N=2). [Figure 77] Ligand-receptor interactions in malignant plasma cells. APRIL ligand binds to TAC1 or BCMA. BAFF ligand binds to TAC1, BCMA, or BAFF-R. [Figure 78]Steps in tumor elimination using CARs co-expressing secreted IL-15 / IL-15sushi. 78A, Tumor and its microenvironment. In the tumor microenvironment, macrophages, T cells, dendritic cells, and NK cells are immune response cells against tumors. They secrete low levels of unstable endogenous IL-15, which forms a complex with the soluble extracellular domain of IL-15RA. This complex forms a more stable molecule, greatly promoting the survival and proliferation of immune cells. In the tumor microenvironment, cancer cells express programmed cell death ligand-1 (PD-L1), a transmembrane protein thought to play a major role in suppressing the immune system during certain events, including cancer. PD-L1 binds to its receptor PD-1, found on activated T cells, B cells, and myeloid cells, and suppresses the immune activity of these cells. 78B, CAR T or NK cells can be carriers for delivering enhancers to the tumor microenvironment. CAR T or NK cells are engineered to co-express a secreted fusion protein, IL-15 / IL-15sushi fusion. In 78C, the engineered CAR T or NK cells bind to target tumor cells (either a subset or all cells). In 78D, CAR T or NK cells target cancer cells in the tumor microenvironment, bind to the CAR target antigen, and induce tumor cell lysis and the secretion of large amounts of soluble IL-15 / IL-15sushi fusion from the expanded CAR T or NK cells. The soluble IL-15 / IL-15sushi fusion is stable and functions as an unexpectedly potent immunomodulatory agent for CAR T / NK cells and their neighboring tumor immune response cells. The secreted IL-15 / IL-15sushi protein is involved in the transport of other T cells, dendritic cells, macrophages, and NK cells into the tumor microenvironment, which lyses tumor cells by compensating for the inability of CAR T or NK cells to eliminate non-targeted cancer cells. 2) Enhancement of CAR T / NK cell persistence and antitumor activity. Overexpression of IL-15 / IL-15sushi overwhelms the ability of PD-L1 to suppress immune responses.Preferably, this CAR therapy can be used synergistically with the administration of checkpoint inhibitors, including but not limited to PD-L1 and CTLA-4 inhibitors, to increase efficacy. [Figure 79] Surface markers during B cell to plasma cell development are shown. Both BAFF and APRIL bind to receptors, BCMA and TACI. BAFF also binds to the BAFF-R receptor. [Figure 80] Protein sequence alignment of IL-2 signal peptides across different species. [Figure 81] Protein sequence alignment of the BAFF extracellular domain across different species. [Figure 82A] A model of IgE production and allergic inflammation. IgE antibodies are initially produced by activated B cells, which differentiate into IgE plasma cells. IgE is released from plasma cells and binds to the FceR1 receptor complex present on mast cells, basophils, or eosinophils, triggering the release of allergic mediators. CARs can be designed to target or eliminate IgE-producing plasma cells and the basophils or eosinophils responsible for the release of allergic mediators. [Figure 82B] Composition of FcER1A CAR construct: The FcER1A CAR construct contains a leader sequence, the extracellular domain of FcER1A, a hinge domain (H), a transmembrane domain (TM), a costimulatory domain (including but not limited to CD28 or 4-1BB), and an intracellular signaling domain CD3 zeta. [Figure 82C] Protein sequence alignment of the extracellular domain of FcER1A across different species. The target may include a portion of the surface-exposed region of FcER1A. [Figure 82D]FcER1A CAR targets and lyses IgE-producing U266 plasma cells. Control and FcER1A CAR T cells were incubated with a myeloma cell line, U266, strictly positive for BCMA (pre-stained with Celltracker CMTMR) at a 5:1 E:T ratio. Co-cultures were set up at 48 hours of incubation with flow cytometry acquisition using CD3 and BCMA antibodies for analysis. The blue population represents BCMA + U266 cells. [Figure 82E] Configuration of anti-FcER1A or FcER1 complex CAR construct. The FcER1A or FcER1 complex CAR construct comprises a leader sequence, an scFv against FcER1A or FcER1 complex, a hinge domain (H), a transmembrane domain (TM), a costimulatory domain (including, but not limited to, CD28 or 4-1BB), and an intracellular signaling domain CD3 zeta. [Figure 83A] Schematic diagram of a cCAR construct. The construct contains an SFFV promoter driving the expression of multiple modular units of the CAR linked by P2A cleavage peptides. Upon cleavage of the P2A linker, the cCAR separates and binds to targets expressing CD19 and / or CD123. Each unit of the CAR possesses an scFv against the antigen, a hinge domain (H), a transmembrane domain (TM), a costimulatory domain (including, but not limited to, CD28 or 4-1BB), and the intracellular signaling domain CD3 zeta chain. For novel cCAR constructs, the activation domains of the construct may include, but are not limited to, 4-1BB on the CD19 CAR segment and the CD28 region on the CD123 CAR. [Figure 83B]CAR expression: CD19b-123-2G. Peripheral blood mononuclear cells were transduced with either a control vector (center) or a CD19b CD123-2G (cCAR) CAR lentiviral vector (right). After 48 hours of recovery, cells were labeled with an anti-mouse F(Ab')2-biotin antibody to detect the CAR phenotype. Non-transduced activated T cells are shown on the left. [Figure 83C] Coculture: CD19bCD123 CAR T vs. KG1-a, 16 / 48 hours. CD19bCD123-2G cCAR can eradicate the CD123-expressing KG1- tumor cell line in coculture assays. Coculture experiments were performed at an effector-to-target ratio of 5:1 for 16 and 48 hours and analyzed directly by flow cytometry with mouse anti-human CD3pPerCp and mouse anti-human CD123-APC. Each assay included target cells (KG1-a) vs. control (left), cCAR (center), T cells, or target cells alone (right). N = 2. [Figure 83D] Coculture: CD19bCD123 CAR T vs. K562-CD19xp, 16 / 48 hours. CD19bCD123-2G cCAR can eradicate CD19-expressing K562 tumor cell lines in coculture assays. Coculture experiments were performed at an effector-to-target ratio of 5:1 for 16 and 48 hours and analyzed directly by flow cytometry with mouse anti-human CD3pPerCp and mouse anti-human CD19-PE. Each assay included target cells (K562 tumor cells artificially expressing the CD19 antigen) versus control (left), cCAR (center), T cells, or target cells alone (right). N = 2. [Figure 83E]Coculture: CD19bCD123 CAR T vs. SP53 for 16 hours. CD19bCD123-2G cCAR can eliminate the CD19-expressing SP53 mantle cell lymphoma cell line in a coculture assay. Coculture experiments were performed at an effector-to-target ratio of 5:1 for 16 hours and analyzed directly by flow cytometry with mouse anti-human CD3pPerCp and mouse anti-human CD19-PE. Each assay included target cells (SP53) vs. control (left), or cCAR (center), T cells, or target cells alone (right). N = 2. [Figure 84A] CD19b-CD123 cCAR mice can effectively control tumor growth in an AML tumor model. NSG mice were sublethally irradiated and 24 hours later, 1 x 106 luciferase-expressing MOLM-13 cells were intravenously injected (day 1) to induce measurable tumor formation. Three days after tumor injection, mice were injected with 10 x 106 control or CD19b-CD123 CAR T cells. On days 6, 8, and 11, mice were subcutaneously injected with RediJect D-luciferin and subjected to IVIS imaging. Tumor intensity was quantified as luciferin signal (photons / second). [Figure 84B]CD19bCD123 CAR T cells lyse MOLM13 tumor cells in an in vivo mouse model survival curve. NSG mice injected with MOLM13 tumor cells survived significantly longer when treated with CD19b-CD123 CAR T cells. Ten sublethally irradiated NSG mice were intravenously injected with MOLM13 cells to induce measurable tumor formation. Three days later, half were intravenously injected with CD19b-CD123 CAR T cells, while the other half were injected with vector control T cells. Following the IVIS imaging experiment described above, mice were observed daily for symptoms of severe illness and were euthanized when movement became significantly impaired. All control mice died by day 18, while CD19b-CD123 CAR T-treated mice survived up to 15 days longer than control mice (Figure 84B). This difference between groups was shown to be significant by the Mantel-Cox test (0.0031) and the Gehan-Breslow-Wilcoxon test (P = 0.0043). [Figure 84C] CD19bCD123 CAR T cells lyse REH tumor cells in an in vivo mouse model. CD19bCD123 CAR T cells demonstrate long-lasting antitumor efficacy in vivo. NSG mice were sublethally irradiated and intravenously injected with 1.0 x 106 luciferase-expressing REH cells (day 0) to induce measurable tumor formation. Starting 3 days after tumor cell injection, mice received a course of intravenous injection of 10 x 106 CD19bCD123 CAR T cells or vector control T cells. Mice were injected subcutaneously with RediJect D-luciferin on day 16 and underwent IVIS imaging. [Figure 85A]Schematic diagram showing the cCAR-T construct. The construct contains an SFFV strong promoter that drives the expression of multiple modular units of the CAR linked by a P2A self-cleaving peptide. Upon cleavage of the linker, the cCAR separates and binds to targets expressing BCMA (CD269) and / or CD19b. For novel cCAR constructs, the activation domains of the construct may include, but are not limited to, the 4-1BB region on the BCMA CAR unit and the CD28 region on the CD19b CAR unit. [Figure 85B] Transduction efficiency of T cells transduced with cCAR, CD19b-BB-2G, and CD269-2G lentiviral vectors. Expression of CAR lentivirus-transduced peripheral blood-derived T cells. Peripheral blood T cells were transduced with either a control vector, CD269CD19b-2G (cCAR), CD269-2G, or CD19b-BB-2G CAR lentiviral vector. The lentiviral vector CD269CD19b-2G contains two units of CAR, CD269-2G and CD19b-BB-2G, which target both the CD269 and CD19 antigens. After 48 hours of recovery, cells were labeled with an anti-mouse F(Ab')2-biotin antibody to detect the CAR phenotype. Panel 1 (left) contains untransduced cells. Panel 2 (second from left) shows CD269-CD19b-2G cCAR T cells, and panels 3 (second from right) and 4 (far right) show expression data for CD269-2G CAR and CD19b-BB 2G CAR T cells. [Figure 85C]BCMA-CD19b cCAR T cells lyse CD19-expressing K562 cells. Coculture experiments were performed for 16 hours at an effector-to-target ratio of 5:1 and analyzed directly by flow cytometry with mouse anti-human CD3pPerCp and mouse anti-human CD19-PE. Each assay consisted of cocultures containing either control T cells (far left), cCAR (second from left), BCMA-2G CAR (second from right), or CD19b-BB-2G CAR (far right) T cells with target cells (K562 tumor cells (K19) artificially expressing the CD19 antigen). Target cells alone (K-19xp) are shown at the bottom. N = 2. [Figure 85D] BCMA-CD19b cCAR T cells lyse BCMA-expressing K562 cells. Coculture experiments were performed for 16 hours at an effector-to-target ratio of 5:1 and analyzed directly by flow cytometry with mouse anti-human CD3pPerCp and mouse anti-human BCMA-APC. Each assay included target cells (K562 tumor cells artificially expressing the BCMA antigen (k-BCMA)) and either control (second from left), cCAR (second from left), CD269-2G CAR (second from right), or CD19b-BB-2G CAR (far right) T cells. Target cells only, BCMA-K (bottom), N = 2. [Figure 85E] CAR specifically lyses only its own target epitope. Figure showing that BCMA-CD19b cCAR T cells do not lyse wild-type K562 cells. Coculture experiments were performed for 16 hours at an effector-to-target ratio of 5:1 and analyzed directly by flow cytometry with mouse anti-human CD3pPerCp and mouse anti-human BCMA-APC. Each assay included target cells (K562 wild-type tumor cells that do not express the CD19 antigen (A), CD269 antigen (B), or wt (C)) versus control (far left), cCAR (second from left), CD269-2G (center), or CD19b-BB-2G CAR (second from right) T cells. Target cells alone (K562 wild-type, far right). N = 2. [Figure 86A]BCMA-CD19b cCAR cells were lysed with a mixture of K562 cells expressing the artificial antigens CD19 and BCMA at an E:T ratio of ~5:2. Control and BCMA-CD19b cCAR cells were incubated with K562 cells expressing either CD19 (K-19) or BCMA (K-BCMA). K-19 and K-BCMA cells were mixed at a 1:1 ratio (10:10 cells), and then control or T cells were added to a final E:T ratio of 5:2. Cultures were incubated for 24 and 48 hours, and residual target antigen populations in the cultures were quantified by flow cytometry using CD3, BCMA, and CD19 antibodies. The purple population represents BCMA-CD19+CD3- K-19 cells, while the blue-green population represents BCMA+CD19-CD3- K-BCMA cells. N = 2. [Figure 86B] BCMA-CD19b cCAR T cells can lyse the BCMA+ multiple myeloma cell line MM1S. Control and BCMA-CD19b CAR cells were incubated with the myeloma cell line MM1S, which is strictly positive for BCMA, at a 5:1 E:T ratio. Co-cultures were set up at 24 and 48 hours of incubation and analyzed by flow cytometry acquisition using CD3 and BCMA antibodies. Purple populations represent BCMA+ MM1S cells. N = 2 [Figure 86C] BCMA-CD19b cCAR lyses MM1S cells with high efficiency. Control, CD19b, and BCMA-CD19b CAR cells were incubated with the myeloma cell line MM1S (pre-stained with Celltracker (CMTMR) and strictly positive for BCMA at E:T ratios of 2:1 and 5:1). Co-cultures were set up at 48 hours of incubation and analyzed by flow cytometry using CD3 and BCMA antibodies. The blue population represents BCMA + MM1S cells. [Figure 86D]BCMA-CD19b cCAR lyses RPMI-8226 multiple myeloma cells with high efficiency. Control, CD19b, and BCMA-CD19b CAR cells were incubated with the strictly BCMA-positive myeloma cell line RPMI-8226 (pre-stained with Celltracker CMTMR) at E:T ratios of 2:1 and 5:1. Co-cultures were set up for 48 hours and analyzed by flow cytometry using CD3 and BCMA antibodies. The blue population represents BCMA+ RPMI-8226 cells. [Figure 86E] BCMA-CD19b cCAR lyses U266 with high efficiency. Control, CD19b, and BCMA-CD19b CAR cells were incubated with the strictly BCMA-positive myeloma cell line U266 (pre-stained with Celltracker CMTMR) at E:T ratios of 2:1 and 5:1. Co-cultures were set up with a 48-hour incubation time and analyzed by flow cytometry acquisition using CD3 and BCMA antibodies. The blue population represents BCMA+ U266 cells. [Figure 87A] BCMA-CD19b cCAR T cells can lyse mixed tumor populations, myeloma cells, and B-ALL cells. Control and BCMA-CD19b CAR cells were incubated with a mixture of CD19 + REHREH cells and BCMA + RPMI-8226 cells. Co-cultures were set up with increasing E:T ratios and a 24-hour incubation time and analyzed by flow cytometry acquisition using CD3, CD19, and BCMA antibodies. Purple populations represent BCMA + RPMI-8226 cells, and blue populations represent REHREH cells. N = 2. BCMA-CD19b cCAR T cells can be used to clear plasma cell and B cell populations associated with autoimmune disease. [Figure 87B]BCMA-CD19b cCAR T cells can lyse mixed tumor populations, myeloma cells, and B-ALL cells. Control and BCMA-CD19b CAR cells were incubated with a mixture of CD19+ REH cells and a small number of BCMA+ RPMI-8226 cells. Co-cultures were set up at increasing E:T ratios with a 48-hour incubation time and analyzed by flow cytometry acquisition using CD3, CD19, and BCMA antibodies. Purple populations represent BCMA+ RPMI-8226 cells, and blue populations represent REH cells. N = 2. REH (REH) is a CD19+ B-ALL cell line, and RPMI-8226 is a BCMA-expressing myeloma cell line. [Figure 88A] CD269-CD19b cCAR T cells can target primary myeloma cells MM7-G. BCMA-CD19b cCAR lyses primary myeloma cells MM7-G. Control and BCMA-CD19b CAR cells were incubated with the BCMA + MM7-G primary myeloma cell subset. MM7-G cells were pre-stained with CMTMR Celltracker dye. Cocultures were analyzed by flow cytometry acquisition using CD3 and BCMA antibodies at 2:1 and 5:1 E:T ratios with a 24-hour incubation time. The circled population represents BCMA + MM7-G cells. Comparison with CD19b and BCMA single CARs is presented. [Figure 88B]BCMA-CD19b cCAR mice can control the growth of mixed tumor populations. BCMA-CD19b cCAR lyses mixed antigen-specific tumor cell populations in vivo. NSG mice were sublethally irradiated, and 24 hours later, they were intravenously injected with a 1:1 mixture of 1x106 luciferase-expressing CD19+ REH and BCMA+ MM1S cells (day 1) to induce measurable tumor formation. Three days after tumor injection, mice were injected with 10x106 control or BCMA-CD19b cCAR T cells. On days 6, 8, and 11, mice were subcutaneously injected with RediJect D-luciferin and imaged via IVIS. Tumor intensity was quantified as luciferin signal (photons / second). [Figure 88C] BCMA-CD19b cCAR mice can control the growth of mixed tumor populations (ventral view). BCMA-CD19b cCAR lyses mixed antigen tumor cell populations in vivo. NSG mice were sublethally irradiated and 24 hours later, 1 x 106 luciferase-expressing CD19+ REH and BCMA+ MM1S cells were intravenously injected (day 1) in a 1:1 mixture to induce measurable tumor formation. Three days after tumor injection, mice were injected with 10 x 106 control, CD269-CD19b cCAR, or T cells. On days 6, 8, and 11, mice were subcutaneously injected with RediJect D-luciferin and imaged via IVIS. Tumor intensity was quantified as luciferin signal (photons / second). [Figure 89A]Schematic diagram of a cCAR-T construct with two CAR units, BCMA and CS1 (BC1cCAR). The construct contains an SFFV promoter that drives the expression of multiple modular CAR units linked by a P2A peptide. Upon cleavage of the linker, the cCAR separates and binds to targets expressing BCMA (CD269) and / or CS1 (CD319 or Slamf7). For novel cCAR constructs, the activation domain of the construct can include, but is not limited to, both the BCMA and CS1 CAR segments and 4-1BB. The BCMA CAR unit of the cCAR can be selected from either the BCMA-A7D-28-2G CAR or the BCMA-C11D-28-2G CAR. The cCAR's CS1 CAR unit can be selected from one of the following groups: CS1-mu34-28-2G CAR, CS1-mu90-28-2G CAR, and CS1-hu63-28-2G CAR. [Figure 89B]Analysis of BCMA-CS1 cCAR T cell persistence and tumor reduction. A compound CAR (BCMA-CS1 cCAR) was generated and its function characterized above (Figures 24 to 29). To model potential antigen escape or multiple antigen tumor populations, we designed a xenogeneic mouse model in which sublethally irradiated NSG mice were intravenously injected with luciferase-expressing K562 cells expressing either BCMA or CS1. BCMA- and CS1-expressing K562 cells (BCMA-K562 and CS1-K562) were further selected for expression after puromycin selection and established as a stable, homogenous single-antigen population. BCMA- and CS1-expressing K562 cells were mixed at a 4:1 ratio before injection to model potential antigen escape. BCMA- and CS1-expressing K562 cells were then used to screen for tumor and CAR T cell persistence. Whole blood and liver tissue samples were collected from mice in the CS1-K562 experimental group at the time of sacrifice and screened for tumor and CAR T cell persistence using CD3, CD45, and CS1 antibodies. Two representative flow diagrams are shown. All control and cCAR mice showed the same trend for each mouse in each treatment group (n = 19). Compared to cCAR-treated mice, which had abundant T cell populations and no tumor populations (Figure 89B), control mice showed very low or no T cell populations, with few remaining T cells (blue), and apparent CS1-K562 tumor populations (purple). Similar experimental conditions and harvesting were performed for the BCMA-K562 experimental group, and a similar trend of tumor clearance and T cell persistence was observed in cCAR-treated mice (Figure 89C). [Figure 89C]Analysis of BCMA-CS1 cCAR T cell persistence and tumor reduction. A compound CAR (BCMA-CS1 cCAR) was generated and its function characterized above (Figures 24 to 29). To model potential antigen escape or multiple antigen tumor populations, we designed a xenogeneic mouse model in which sublethally irradiated NSG mice were intravenously injected with luciferase-expressing K562 cells expressing either BCMA or CS1. BCMA- and CS1-expressing K562 cells (BCMA-K562 and CS1-K562) were further selected for expression after puromycin selection and established as a stable, homogenous single-antigen population. BCMA- and CS1-expressing K562 cells were mixed at a 4:1 ratio before injection to model potential antigen escape. BCMA- and CS1-expressing K562 cells were then used to screen for tumor and CAR T cell persistence. Whole blood and liver tissue samples were collected from mice in the CS1-K562 experimental group at the time of sacrifice and screened for tumor and CAR T cell persistence using CD3, CD45, and CS1 antibodies. Two representative flow diagrams are shown. All control and cCAR mice showed the same trend for each mouse in each treatment group (n = 19). Compared to cCAR-treated mice, which had abundant T cell populations and no tumor populations (Figure 89B), control mice showed very low or no T cell populations, with few remaining T cells (blue), and apparent CS1-K562 tumor populations (purple). Similar experimental conditions and harvesting were performed for the BCMA-K562 experimental group, and a similar trend of tumor clearance and T cell persistence was observed in cCAR-treated mice (Figure 89C). [Figure 90]Expression of peripheral blood-derived T cells transduced with CAR lentivirus. Peripheral blood T cells were transduced with either a control vector (bottom right), BCMA-A7D-28-2G, BCMA-C11D-28-2G, CS1-mu34-28-2G, CS1-mu90-28-2G, or CS1-hu63-28-2G CAR lentiviral vector. After 48 hours of recovery, cells were labeled with an anti-mouse F(Ab')2-biotin antibody to detect the CAR phenotype. The upper left panel contains untransduced cells. The upper center and far right panels show BCMA-A7D-2G and BCMA-C11D-2G CAR T cells, respectively, while the second right and far right panels show expression data for CS1-mu34, CS1-mu90, and CS1-hu63 CAR T cells, respectively. [Figure 91A] Schematic diagram of the CD123b-CD33b cCAR, a cCAR-T construct that targets CD123, CD33, or both antigens. The construct contains an SFFV promoter that drives the expression of multiple modular CAR units linked by P2A peptides. Upon cleavage of the linker, the cCAR separates and binds to targets expressing CD123 and / or CD33. For novel cCAR constructs, the activation domains of the construct may include, but are not limited to, 4-1BB on the CD123 CAR unit and CD28 on the 33b CAR unit. [Figure 91B] Expression of CD123bCD33b CAR T cells. PMBC buffy coat T cells were activated with anti-CD3 antibody for 3 days. Cells were transduced with either control vector (center) or CD123bCD33b CAR (right) lentiviral supernatant. After 3 days of incubation, cells were harvested and labeled for flow cytometry. Pre-activated PMBC (left) were also labeled with the same antibodies and procedures as the transduced cells. [Figure 91C]CD123bCD33b CAR T cells can eliminate CD33-expressing MOLM13 tumor cell lines in coculture assays. Coculture experiments were performed for 16 hours at an effector-to-target ratio of 2:1 or 5:1 and analyzed directly by flow cytometry for CD33 and CD3. Each assay included MOLM13 target cells and control (left), CD123bCD33b CAR T cells (center), and target cells alone (right). The plots on the bottom right show the CD33 and CD123 phenotypes of MOLM13 cells. [Figure 91D] CD123bCD33b CAR T cells can eliminate the CD33-expressing U937 tumor cell line in coculture assays. CD123bCD33b-2G CAR T cells eliminate CD33+ / CD123- U937 cells. Coculture experiments were performed for 16 hours at an effector-to-target ratio of 2:1 or 5:1 and analyzed directly by flow cytometry for CD33 and CD3. Each assay included U937 target cells and control (left), CD123bCD33b CAR T cells (center), and target cells alone (right). The plots on the bottom right show the CD33 and CD123 phenotypes of U937 cells. [Figure 91E] CD123bCD33b CAR T cells can eliminate AML patient cells (PT-1) expressing CD33 and CD123 in coculture assays. Coculture experiments were performed for 24 hours at effector-to-target ratios of 2:1 and 5:1 and analyzed directly by flow cytometry for CD3 and CD33. Assays included AML patient cells versus control (left), CD123CD33 CAR T cells (center), and target cells alone (far right). [Figure 91F]CD123bCD33b CAR T cells are able to eliminate B-ALL patient cells (PT-2) expressing CD123 and CD19 in coculture assays. B-ALL14-BM (PT2). Coculture experiments were performed for 24 hours at an effector-to-target ratio of 2:1 and analyzed directly by flow cytometry for CD3 and CD123. Assays included B-ALL patient cells versus control (left), CD123CD33 CAR T cells (center), and target cells alone (far right). [Figure 91G] CD123-CD33-28-2G cCAR (cCAR) T cells can selectively and potently lyse CD33-expressing target cells. CD123b-CD33b-28-2G eliminates the CD33-specific population. (A) cCAR T cells were incubated with the T-ALL cell line Jurkat transduced to express CD33. Although CD33-expressing Jurkat (Jurkat xp33) cells comprise a small fraction of the overall Jurkat population, compared to controls, cCAR T cells were able to completely eliminate CD33-expressing target cells, but not CD33-negative Jurkat cells, after 24 hours of culture, even at an E:T ratio of 2:1. Flow plot showing depletion of CD33+ Jurkat cells (purple). Jurkat cells were pre-labeled with a cytotracker (CMTMR-PE). (B) Histogram visualization of the depletion of the CD33+ cell population after cCAR treatment (pink) versus control (gray). (C) Graphical summary of CD33 + Jurkat cell lysis by cCAR after coculture. [Figure 91H] CD123b-CD33b-2G cCAR T cells eliminate CD34+ AML leukemic blasts. Leukemic blasts were gated for CD34 expression, and the population was then expressed as a percentage of %gated. AML-18-G cells (human AML sample) were almost exclusively CD34+ leukemic blasts. Elimination of primary AML CD34+ bulk disease was analyzed via FACS. N = 2 [Figure 91I]CD123b-CD33b-2G cCAR T cells eradicate human leukemia stem cells (AML-18-G primary cells). Leukemia stem cells were first gated for CD34 expression, and then the population was isolated and analyzed for CD34 and CD38 expression. Gating revealed that both types of CD34-positive populations, CD34+CD38- and CD34+CD38+, were essentially eradicated by cCAR T cells, as analyzed by the total % of remaining cells. CD34+ / CD38- leukemia stem cells were specifically eradicated. [Figure 91J] CD123b-CD33b-2G cCAR T cells can target and eliminate primary human B-ALL cells expressing CD123. Depletion of CD34+ / CD123+ leukemia cells in cases of CD19CAR relapse. To test the elimination of primary B-ALL cells, cocultures were performed using the CD123b-CD33b-2G cCAR construct. Cocultures were performed at various E:T ratios (2:1, 5:1, and 10:1, as shown here) and stained with antibody combinations for population analysis. CD34, CD38, CD33, CD123, and CD19 markers were analyzed. Potential B-ALL relapse cells after CD19CAR treatment were isolated and shown by flow cytometry as a mixture of CD19+ and CD19- cells (circled in blue) that were also CD34+ / CD123+. Depletion of this population by cCAR T cells is shown in the figure. The pink population represents CD34+ cells, but are simultaneously negative for CD123, CD19, and CD33. N = 2 [Figure 91K] Significant efficacy of CD123b-CD33b-2G cCAR T cells in eliminating tumor cells. Co-cultures were established with increasing E:T ratios from 0.25:1 effector:target cells to a 10:E:T ratio. Co-cultures were incubated overnight and pre-labeled with a cytotracker (CMTMR dye) to isolate the tumor population from the effector cells. Flow cytometry analysis was performed to measure the elimination of target tumor cells. [Figure 91L]CD123b-CD33b CAR T cells demonstrate in vivo antitumor activity against cell lines expressing the CD33 antigen. NSG mice were sublethally irradiated and intravenously injected with 1.0 x 106 luciferase-expressing U937 cells (day 0) to induce measurable tumor formation. Starting 3 days after tumor cell injection, mice were intravenously injected with a course of 10 x 106 CD123b-CD33b CAR T cells or vector control T cells. On days 3 and 6, mice were subcutaneously injected with RediJect D-luciferin and imaged via IVIS. (91L) Dorsal view; (91M) Ventral view. [Figure 91M] CD123b-CD33b CAR T cells demonstrate in vivo antitumor activity against cell lines expressing the CD33 antigen. NSG mice were sublethally irradiated and intravenously injected with 1.0 x 106 luciferase-expressing U937 cells (day 0) to induce measurable tumor formation. Starting 3 days after tumor cell injection, mice were intravenously injected with a course of 10 x 106 CD123b-CD33b CAR T cells or vector control T cells. On days 3 and 6, mice were subcutaneously injected with RediJect D-luciferin and imaged via IVIS. (91L) Dorsal view; (91M) Ventral view. [Figure 91N] CD123b-CD33b CAR T cells demonstrate in vivo antitumor activity against cell lines expressing both CD33 and CD123 antigens. NSG mice were sublethally irradiated and intravenously injected with 1.0 x 106 luciferase-expressing U937 cells (day 0) to induce measurable tumor formation. Beginning three days after tumor cell injection, mice were intravenously injected with a course of 10 x 106 CD123bCD33b CAR T cells or vector control T cells. On days 3 and 6, mice were subcutaneously injected with RediJect D-luciferin and imaged via IVIS. (91N) Dorsal view. (91O) Ventral view. [Figure 91O]CD123b-CD33b CAR T cells demonstrate in vivo antitumor activity against cell lines expressing both CD33 and CD123 antigens. NSG mice were sublethally irradiated and intravenously injected with 1.0 x 106 luciferase-expressing U937 cells (day 0) to induce measurable tumor formation. Beginning three days after tumor cell injection, mice were intravenously injected with a course of 10 x 106 CD123bCD33b CAR T cells or vector control T cells. On days 3 and 6, mice were subcutaneously injected with RediJect D-luciferin and imaged via IVIS. (91N) Dorsal view. (91O) Ventral view. [Figure 92] Schematic diagram of a cCAR-T construct. The construct contains an SFFV promoter that drives the expression of multiple modular CAR units linked by P2A peptides. Upon cleavage of the linker, the cCAR separates and binds to targets expressing CD33 and / or CLL-1. For novel cCAR constructs, the activation domains of the construct may include, but are not limited to, the 4-1BB region on the CD33 CAR segment and the CD28 region on the CLL-1 CAR segment. [Figure 93] Schematic diagram of the cCAR-T construct. The construct contains an SFFV promoter that drives the expression of multiple modular CAR units linked by P2A peptides. Upon cleavage of the linker, the cCAR splits and binds to targets expressing CD4 and / or CD123. For novel cCAR constructs, the activation domains of the construct may include, but are not limited to, the 4-1BB region on the CD4 segment and the CD28 region on the CD123 CAR segment. [Figure 94]Schematic diagram illustrating the construct and its expression in T or NK cells. A) A combination of a CAR (including but not limited to third-generation CARs) and the sushi domain of the IL-15α receptor (referred to as IL-15sushi) is assembled on an expression vector, and its expression is driven by an SFFV promoter. The CAR containing the IL-15 / IL-15 sushi is linked to a P2A self-cleaving sequence. The IL-15 / sushi portion consists of the IL-2 signal peptide fused to IL-15, linked to the IL-15α receptor domain via a 26-amino acid poly-proline linker. B) The CAR and IL-15 / IL-15 sushi are presented on T or NK cells. [Figure 95] Schematic diagram showing a CAR with an IL-15 / IL_15 sushi anchor. A) The construct contains an SFFV promoter driving expression of the CAR and the IL-15 / IL-15 sushi anchor (also referred to as the anchor), linked by a P2A peptide. Upon cleavage of this P2A peptide, the IL-15 / IL-15 anchor CAR splits into the CAR and the IL-15 / IL-15 sushi anchor. The IL-15 / sushi portion of the anchor consists of the IL-2 signal peptide fused to the IL-15 alpha receptor sushi domain via a 26-amino acid poly-proline linker. Both the CAR and anchor contain a hinge (H) region and a transmembrane (TM) domain. The CAR also contains an scFv, a costimulatory domain (including, but not limited to, CD28 or 4-1BB), and an intracellular signaling domain, CD3 zeta chain, while the anchor lacks these components. B) IL-15 / IL-15sushi is immobilized on the surface of T or NK cells. [Figure 96A]Schematic diagram showing the CAR enhancer construct. The construct contains an SFFV promoter driving expression of the CAR and enhancer 4-1BBL (CD137L) linked by a P2A peptide. Upon cleavage of this P2A peptide, the CAR construct containing 4-1BBL is split into the CAR polypeptide and the full-length 4-1BBL protein. The CAR contains a leader sequence, scFv, hinge (H) region, and transmembrane (TM) domain. CARs also contain costimulatory domains (including, but not limited to, CD28 or 4-1BB) and intracellular signaling domains, such as the CD3 zeta chain; however, 4-1BBL lacks these components. 4-1BBL provides synergistic effects with CD28 or 4-1BB on T cell activation or antitumor activity, including, but not limited to, T cell activation. [Figure 96B] Schematic diagram showing the CAR-enhancer construct. The construct contains an SFFV promoter driving the expression of CAR and an enhancer IL-15 linked by a P2A peptide. Upon cleavage of this P2A peptide, the IL-15-containing CAR construct splits into the CAR polypeptide and the full-length IL-15 protein. The CAR contains a leader sequence, scFv, hinge (H) region, and transmembrane domain (TM). CARs also contain costimulatory domains (including, but not limited to, CD28 or 4-1BB) and intracellular signaling domains, such as the CD3 zeta chain, whereas IL-15 lacks these components. IL-15 synergizes with CD28 or 4-1BB for T cell activation, proliferation, or antitumor activity. The IL-15 signal peptide of IL-15 is replaced with the IL-2 signal peptide (leader sequence), a strong signal peptide that results in highly efficient IL-15 secretion. [Figure 97A]Generation of CD4-3G-IL-15 / IL-15suhi- and CD4-3G-IL-15 / IL-15sushi-anchors and their expression on NK92 cells by FACS analysis. NK92 cells were transduced with CD4-3G, CD4-3G-IL-15 / IL-15sushi-, and CD4-3G-IL-15 / IL-15 anchor (CD4-3G-IL-15 / IL-15sushi-anchor) CAR lentiviruses, and their surface CAR expression (boxed in blue in the upper panel) was determined by flow cytometry analysis using F(Ab')2 surface staining and CD56 antibody staining, compared with untransduced or GFP-transduced NK92 cells (negative control). Transduced NK92 cells showed CAR expression levels of CD4-3G- (78.3%), CD4-3G-IL-15 / IL-15sushi (97.1%), or CD4-3G-IL-15 / IL-15anchor (93.4%) compared to untransduced NK92 cells. [Figure 97B]Coculture assays compared the effects of IL-15 / IL-15sushi and IL-15 / IL-15sushi anchor secretion on CAR and non-transduced neighboring cells. Selected NK92 cells stably transduced with CD4-3G-IL-15 / IL-15sushi or CD4-3G-IL15 / IL-15 anchor CAR (see Figure 97A) proliferated at similar rates in the absence of IL-2 compared with NK92 cells stably transduced with CD4-3G CAR or GFP lentivirus, which were unable to proliferate in the absence of IL-2 (data not shown). This study points to the importance of co-expression of the functional complex IL-15 / IL-15sushi or IL-15 / IL-15 anchor in promoting CAR-transduced NK-92 cell proliferation. Figure 97B: Coculture cell proliferation analysis using GFP-NK92 cells and CD4CARs-NK92 cells without the addition of IL-2 to the cell culture medium. Next, we tested the effect of IL-15 / IL-15sushi and IL-15 / IL-15sushi anchor secretion on untransduced neighboring cells. Sorted NK-92 cells stably expressing CD4-3G, CD4-3G-IL-15 / IL-15sushi, or CD4CAR-3G-IL-15 / IL-15sushi anchored CAR were mixed with GFP+ NK-92 cells at a 50:50 ratio. These cells were co-cultured with or without IL-2. Total cell numbers were calculated for the entire experiment (up to day 10) for NK-92 cells co-cultured with or without IL-2. Co-cultured CD4-3G or CD4-3G-IL-15 / IL-15sushi anchored CAR-transduced cells or GFP-NK92 cells proliferated less compared to when co-cultured with CD4-3G-IL-15 / IL-15sushi CAR-NK92. Figure 97C, FACS analysis of the percentage of GFP-NK92 cells co-cultured with CD4CAR-3G-IL-15 / IL-15sushi transduced or CD4CAR-3G-IL-15 / IL-15sushi anchored NK92 cells.Flow cytometry analysis was used to compare the effects of IL-15 / IL-15sushi and IL-15 / IL-15sushi anchor secretion on CAR and non-transduced neighboring cells. The percentage of GFP+ NK cells was significantly reduced to background levels (3.27%), while the percentage of GFP+ NK cells remained at a high level (25.87%) when co-cultured with CD4-3G-IL-15 / IL-15sushi. Figure 97D, Day-dependent percentage of GFP-NK92 cells co-cultured with CD4CAR-3G-IL-15 / IL-15sushi-transduced or CD4CAR-3G-IL-15 / IL-15sushi anchor NK92 cells. Summary of the effects of IL-15 / IL-15sushi and IL-15 / IL-15sushi anchor secretion on CAR and non-transduced neighboring NK92 cells by flow cytometry analysis. GFP+ NK92 cells exhibited significantly longer survival in coculture with CD4-3G-IL-15 / IL-15sushi CAR-transduced NK-92 compared with CD4-3G-IL-15 / IL-15sushi-anchored CAR-NK92 in the absence of IL-2. These studies demonstrate that secretion of IL-15 / IL-15sushi complexes significantly impacts CAR cells and their neighboring non-CAR cells. In contrast, IL-15 / IL-15sushi-anchored CAR cells had a similar effect to IL-15 / IL-15sushi secretion on CAR cells, but only a limited effect on neighboring non-CAR cells. [Figure 97C]Coculture assays compared the effects of IL-15 / IL-15sushi and IL-15 / IL-15sushi anchor secretion on CAR and non-transduced neighboring cells. Selected NK92 cells stably transduced with CD4-3G-IL-15 / IL-15sushi or CD4-3G-IL15 / IL-15 anchor CAR (see Figure 97A) proliferated at similar rates in the absence of IL-2 compared with NK92 cells stably transduced with CD4-3G CAR or GFP lentivirus, which were unable to proliferate in the absence of IL-2 (data not shown). This study points to the importance of co-expression of the functional complex IL-15 / IL-15sushi or IL-15 / IL-15 anchor in promoting CAR-transduced NK-92 cell proliferation. Figure 97B: Coculture cell proliferation analysis using GFP-NK92 cells and CD4CARs-NK92 cells without the addition of IL-2 to the cell culture medium. Next, we tested the effect of IL-15 / IL-15sushi and IL-15 / IL-15sushi anchor secretion on untransduced neighboring cells. Sorted NK-92 cells stably expressing CD4-3G, CD4-3G-IL-15 / IL-15sushi, or CD4CAR-3G-IL-15 / IL-15sushi anchored CAR were mixed with GFP+ NK-92 cells at a 50:50 ratio. These cells were co-cultured with or without IL-2. Total cell numbers were calculated for the entire experiment (up to day 10) for NK-92 cells co-cultured with or without IL-2. Co-cultured CD4-3G or CD4-3G-IL-15 / IL-15sushi anchored CAR-transduced cells or GFP-NK92 cells proliferated less compared to when co-cultured with CD4-3G-IL-15 / IL-15sushi CAR-NK92. Figure 97C, FACS analysis of the percentage of GFP-NK92 cells co-cultured with CD4CAR-3G-IL-15 / IL-15sushi transduced or CD4CAR-3G-IL-15 / IL-15sushi anchored NK92 cells.Flow cytometry analysis was used to compare the effects of IL-15 / IL-15sushi and IL-15 / IL-15sushi anchor secretion on CAR and non-transduced neighboring cells. The percentage of GFP+ NK cells was significantly reduced to background levels (3.27%), while the percentage of GFP+ NK cells remained at a high level (25.87%) when co-cultured with CD4-3G-IL-15 / IL-15sushi. Figure 97D, Day-dependent percentage of GFP-NK92 cells co-cultured with CD4CAR-3G-IL-15 / IL-15sushi-transduced or CD4CAR-3G-IL-15 / IL-15sushi anchor NK92 cells. Summary of the effects of IL-15 / IL-15sushi and IL-15 / IL-15sushi anchor secretion on CAR and non-transduced neighboring NK92 cells by flow cytometry analysis. GFP+ NK92 cells exhibited significantly longer survival in coculture with CD4-3G-IL-15 / IL-15sushi CAR-transduced NK-92 compared with CD4-3G-IL-15 / IL-15sushi-anchored CAR-NK92 in the absence of IL-2. These studies demonstrate that secretion of IL-15 / IL-15sushi complexes significantly impacts CAR cells and their neighboring non-CAR cells. In contrast, IL-15 / IL-15sushi-anchored CAR cells had a similar effect to IL-15 / IL-15sushi secretion on CAR cells, but only a limited effect on neighboring non-CAR cells. [Figure 97D]Coculture assays compared the effects of IL-15 / IL-15sushi and IL-15 / IL-15sushi anchor secretion on CAR and non-transduced neighboring cells. Selected NK92 cells stably transduced with CD4-3G-IL-15 / IL-15sushi or CD4-3G-IL15 / IL-15 anchor CAR (see Figure 97A) proliferated at similar rates in the absence of IL-2 compared with NK92 cells stably transduced with CD4-3G CAR or GFP lentivirus, which were unable to proliferate in the absence of IL-2 (data not shown). This study points to the importance of co-expression of the functional complex IL-15 / IL-15sushi or IL-15 / IL-15 anchor in promoting CAR-transduced NK-92 cell proliferation. Figure 97B: Coculture cell proliferation analysis using GFP-NK92 cells and CD4CARs-NK92 cells without the addition of IL-2 to the cell culture medium. Next, we tested the effect of IL-15 / IL-15sushi and IL-15 / IL-15sushi anchor secretion on untransduced neighboring cells. Sorted NK-92 cells stably expressing CD4-3G, CD4-3G-IL-15 / IL-15sushi, or CD4CAR-3G-IL-15 / IL-15sushi anchored CAR were mixed with GFP+ NK-92 cells at a 50:50 ratio. These cells were co-cultured with or without IL-2. Total cell numbers were calculated for the entire experiment (up to day 10) for NK-92 cells co-cultured with or without IL-2. Co-cultured CD4-3G or CD4-3G-IL-15 / IL-15sushi anchored CAR-transduced cells or GFP-NK92 cells proliferated less compared to when co-cultured with CD4-3G-IL-15 / IL-15sushi CAR-NK92. Figure 97C, FACS analysis of the percentage of GFP-NK92 cells co-cultured with CD4CAR-3G-IL-15 / IL-15sushi transduced or CD4CAR-3G-IL-15 / IL-15sushi anchored NK92 cells.Flow cytometry analysis was used to compare the effects of IL-15 / IL-15sushi and IL-15 / IL-15sushi anchor secretion on CAR and non-transduced neighboring cells. The percentage of GFP+ NK cells was significantly reduced to background levels (3.27%), while the percentage of GFP+ NK cells remained at a high level (25.87%) when co-cultured with CD4-3G-IL-15 / IL-15sushi. Figure 97D, Day-dependent percentage of GFP-NK92 cells co-cultured with CD4CAR-3G-IL-15 / IL-15sushi-transduced or CD4CAR-3G-IL-15 / IL-15sushi anchor NK92 cells. Summary of the effects of IL-15 / IL-15sushi and IL-15 / IL-15sushi anchor secretion on CAR and non-transduced neighboring NK92 cells by flow cytometry analysis. GFP+ NK92 cells exhibited significantly longer survival in coculture with CD4-3G-IL-15 / IL-15sushi CAR-transduced NK-92 compared with CD4-3G-IL-15 / IL-15sushi-anchored CAR-NK92 in the absence of IL-2. These studies demonstrate that secretion of IL-15 / IL-15sushi complexes significantly impacts CAR cells and their neighboring non-CAR cells. In contrast, IL-15 / IL-15sushi-anchored CAR cells had a similar effect to IL-15 / IL-15sushi secretion on CAR cells, but only a limited effect on neighboring non-CAR cells. [Figure 97E]Cell proliferation analysis of GFP-NK92 cells cocultured with CD4CARs-NK92 cells using a transwell coculture assay without IL-2 in the cell culture medium. A chamber (transwell) culture assay was used to compare the effects of IL-15 / IL-15sushi secretion and IL-15 / IL-15sushi anchoring on CAR and non-transduced neighboring cells. To further determine whether this effect was due solely to secreted proteins or to interactions between cocultured cells, we designed an experiment in which GFP-NK92 cells were cultured in a chamber below CD4-3G or CD4-3G-IL-15 / IL-15sush or CD4-3G-IL-15 / IL-15sush-anchored CAR NK92 cells, or non-transduced NK92 cells. Under these conditions, only proteins, not cells, can pass through the membrane separating the two cultures. The number of GFP-NK92 cells in the absence of IL-2 was counted from days 2 to 10. In contrast, GFP NK92 cells in the upper chamber of NK-92 cells or NK-92 cells transduced with CD4-3G or CD4-30-IL-15 / IL-15sushi anchored CARs died by day 10, whereas GFP NK92 cells on NK92 cells transduced with CD4-IL-15 / IL-15sushi CARs survived and proliferated by day 10, indicating that IL-15 / IL-15sushi proteins secreted by CD4-3G-IL-15 / IL-15sushi NK92 cells, rather than direct cell-to-cell contact, kept the cells alive. In this model, the upper chamber represents the tumor microenvironment, and IL-15 / IL-15sushi secretion from CD4-3G-IL-15 / IL-15sushi NK cells improves T cell or NK cell survival. The IL-15 / IL-15 anchor has a major effect on the growth of transduced NK92 cells and a lesser extent on direct cell-cell interactions with neighboring untransduced cells. In other words, the IL-15 / IL-15 anchor has only a limited effect on neighboring untransduced cells. [Figure 97F]Comparison of the effects of IL-15 / IL-15sushi secretion, IL-15 / IL-15sushi anchor, and IL-15 secretion on in vivo CAR efficacy. Luciferase-expressing Jurkate cells (1x106 cells) were intravenously injected 24 hours after sublethal irradiation (day 1). Approximately 50% of Jurkate cells expressed CD4. On days 6 and 9, 5x106 control GFP-, CD4-3G-, CD4-3G-IL15 / IL-15sush, IL-15 / IL-15sushi anchor, and secreting IL-15 (containing the IL-2 signal peptide) CAR-NK92 cells were intravenously injected into each mouse (n = 2 per group). One CD4-3G-IL-15 / IL-15sushi NK92-treated mouse died as a result of the injection (NK92 cell mass). Based on IVIS analysis, all CD4-3G CARs equipped with IL-15 / IL-15 sushi, CD4-3G-IL-15 sushi anchor, and IL-15 (containing the IL-2 signal peptide) demonstrated stronger anti-leukemia effects than GFP or CD4-3G controls. Among these CARs, CD4-3G CARs equipped with IL-15 / IL-15 sushi demonstrated superior efficacy compared to other versions of CD4-3G CARs based on IVIS analysis. Interestingly, tumor burden gradually decreased in CD4-3G-IL-15 / IL-15 sushi anchor NK92-treated mice. Percentages indicate the percentage of tumor reduction compared to the GFP control. [Figure 98A]Generation of CD45b-28-4-1BBL-NK92 and CD45b-28-IL-15 / IL-15sushi-NK92 cells. As described above (Figures 57D and 57E), this CAR, CD45b-28 CAR, was generated with 4-1BBL (CD45b-28-4-1BBL) and IL-15 / IL-15sushi (CD45b-28-IL-15 / IL-15sushi). NK92 cells were transduced with CD45b-28, CD4-3G-4-1BBL, and CD45b-28-IL-15 / IL-15sushi-CAR lentivirus, and their surface CAR expression (boxed in blue on the top panel) was determined compared to untransduced or GFP-transduced NK92 cells (negative control) by sorting and flow cytometry analysis using F(Ab')2 surface staining and CD56 antibody staining. [Figure 98B] Analysis of cell proliferation of CD45b-28-IL-15 / IL-15suhi-NK92 cells in the presence or absence of exogenous IL2 in cell culture medium. Comparison of the effects of secreting the IL-15 / IL-15 / IL-15sushi complex or co-expressing 4-1BBL on NK-92 cell proliferation. CD45b-28 CARs are equipped with IL-15 / IL-15sushi (CD45b-28-IL-15 / IL-15sush) or 4-1BBL (CD45b-28-4-1BBL). We compared cell proliferation curves of untransduced, GFP-transduced, sorted CD45b-28-, sorted CD45b-28-IL-15 / IL-15sushi, or sorted CD45b-28-4-1BBL-transduced NK92 cells in the absence or presence of IL-2. There was no significant difference in cell proliferation between sorted NK92 cells stably transduced with CD45b-28-IL-15 / IL-15sushi in the absence or presence of IL-2. However, sorted NK92 cells stably transduced with CARs containing 4-1BBL, such as CD45b-28-4-1BBL, were unable to proliferate in the absence of IL-2. This study highlights the important issue that coexpression of 4-1BBL does not support the proliferation of CAR-transduced NK-92 cells. [Figure 98C]Comparison of the effects of secreting IL-15 / IL-15 / IL-15sushi complexes or coexpressing 4-1BBL on antitumor activity. Luciferase-expressing MOLM-13 cells (1x106 cells) were intravenously injected 24 hours after sublethal irradiation (day 1). On days 4 and 5, 5x106 control GFP-, CD45b-28-, CD45b-CAR-28-4-1BBL-, or CD45b-28-IL-15 / IL-15sushi CAR-NK92 cells were intravenously injected into each mouse (n = 2 per group). One control NK92-treated mouse and one CD45b-28-IL-15 / IL-15sushi NK92-treated mouse died during the injection process. Figure 98C. CD45b-28-NK92 cells failed to demonstrate significant anti-leukemic effects against the MOLM-13 (human acute monocytic leukemia) cell line in an in vivo xenograft mouse model. Dorsal tumor burden was measured using an IVIS imaging system on days 3, 7, and 9. No differences in tumor burden were observed between control NK92 cell-treated mice and CD45b-28 CAR NK92-treated mice by IVIS imaging analysis. However, CD45b-28 CARs with 4-1BBL (CD45b-28-4-1BBL) or IL-15 / IL-15sushi (CD45b-28-IL-15 / IL-15sushi) demonstrated robust and sustained anti-tumor activity in vivo (Figure 98D). Dorsal tumor burden was measured using an IVIS imaging system on days 3, 7, and 9. No differences in tumor burden were observed by IVIS image analysis in both control NK92 cell-treated and CD45b-CAR-28-NK92-treated mice. Figure 98E, Percent inhibition of tumor burden (MOLM-13 cells) in mice treated with CD45b-28-4-1BBL- or CD45b-CAR-28-IL-15 / IL-15sushi-NK92 cells compared to control on days 7 and 9.Unlike secreting IL-15 / IL-15sushi, 4-1BBL failed to induce the survival or proliferation of NK-92 cells in vitro (Figure 98B), but 4-1BBL was shown to be a potent enhancer of CAR antitumor activity in vivo (Figures 98D and 98E). [Figure 98D]Comparison of the effects of secreting IL-15 / IL-15 / IL-15sushi complexes or coexpressing 4-1BBL on antitumor activity. Luciferase-expressing MOLM-13 cells (1x106 cells) were intravenously injected 24 hours after sublethal irradiation (day 1). On days 4 and 5, 5x106 control GFP-, CD45b-28-, CD45b-CAR-28-4-1BBL-, or CD45b-28-IL-15 / IL-15sushi CAR-NK92 cells were intravenously injected into each mouse (n = 2 per group). One control NK92-treated mouse and one CD45b-28-IL-15 / IL-15sushi NK92-treated mouse died during the injection process. Figure 98C. CD45b-28-NK92 cells failed to demonstrate significant anti-leukemic effects against the MOLM-13 (human acute monocytic leukemia) cell line in an in vivo xenograft mouse model. Dorsal tumor burden was measured using an IVIS imaging system on days 3, 7, and 9. No differences in tumor burden were observed between control NK92 cell-treated mice and CD45b-28 CAR NK92-treated mice by IVIS imaging analysis. However, CD45b-28 CARs with 4-1BBL (CD45b-28-4-1BBL) or IL-15 / IL-15sushi (CD45b-28-IL-15 / IL-15sushi) demonstrated robust and sustained anti-tumor activity in vivo (Figure 98D). Dorsal tumor burden was measured using an IVIS imaging system on days 3, 7, and 9. No differences in tumor burden were observed by IVIS image analysis in both control NK92 cell-treated and CD45b-CAR-28-NK92-treated mice. Figure 98E, Percent inhibition of tumor burden (MOLM-13 cells) in mice treated with CD45b-28-4-1BBL- or CD45b-CAR-28-IL-15 / IL-15sushi-NK92 cells compared to control on days 7 and 9.Unlike secreting IL-15 / IL-15sushi, 4-1BBL failed to induce the survival or proliferation of NK-92 cells in vitro (Figure 98B), but 4-1BBL was shown to be a potent enhancer of CAR antitumor activity in vivo (Figures 98D and 98E). [Figure 98E]Comparison of the effects of secreting IL-15 / IL-15 / IL-15sushi complexes or coexpressing 4-1BBL on antitumor activity. Luciferase-expressing MOLM-13 cells (1x106 cells) were intravenously injected 24 hours after sublethal irradiation (day 1). On days 4 and 5, 5x106 control GFP-, CD45b-28-, CD45b-CAR-28-4-1BBL-, or CD45b-28-IL-15 / IL-15sushi CAR-NK92 cells were intravenously injected into each mouse (n = 2 per group). One control NK92-treated mouse and one CD45b-28-IL-15 / IL-15sushi NK92-treated mouse died during the injection process. Figure 98C. CD45b-28-NK92 cells failed to demonstrate significant anti-leukemic effects against the MOLM-13 (human acute monocytic leukemia) cell line in an in vivo xenograft mouse model. Dorsal tumor burden was measured using an IVIS imaging system on days 3, 7, and 9. No differences in tumor burden were observed between control NK92 cell-treated mice and CD45b-28 CAR NK92-treated mice by IVIS imaging analysis. However, CD45b-28 CARs with 4-1BBL (CD45b-28-4-1BBL) or IL-15 / IL-15sushi (CD45b-28-IL-15 / IL-15sushi) demonstrated robust and sustained anti-tumor activity in vivo (Figure 98D). Dorsal tumor burden was measured using an IVIS imaging system on days 3, 7, and 9. No differences in tumor burden were observed by IVIS image analysis in both control NK92 cell-treated and CD45b-CAR-28-NK92-treated mice. Figure 98E, Percent inhibition of tumor burden (MOLM-13 cells) in mice treated with CD45b-28-4-1BBL- or CD45b-CAR-28-IL-15 / IL-15sushi-NK92 cells compared to control on days 7 and 9.Unlike secreting IL-15 / IL-15sushi, 4-1BBL failed to induce the survival or proliferation of NK-92 cells in vitro (Figure 98B), but 4-1BBL was shown to be a potent enhancer of CAR antitumor activity in vivo (Figures 98D and 98E). [Figure 98F] This diagram shows a CAR with 4-1BBL and IL-15 / IL-15sushi in a single construct, linked by P2A and T2A to create a super CAR. The construct contains an SFFV promoter driving the expression of three segments: CAR, 4-1BBL, and IL-15 / IL-15sushi. Upon cleavage of the linker (P2A and T2A), the CAR, 4-1BBL, and IL-15 / IL-15sushi are released and engage the target. The CAR contains an scFv, hinge region, transmembrane domain, costimulatory domain (including, but not limited to, CD28 or 4-1BB), and intracellular signaling, CD3 zeta chain. 4-1BBL, IL-15 / IL-sushi, or both, can activate T or NK cells and synergize with CD28 or 4-1BB for sustained or antitumor activity. [Figure 98G] This diagram shows a CAR with 4-1BBL and IL-15 / IL-15sushi in a single construct, linked by P2A and T2A to create a super CAR. The construct contains an SFFV promoter driving the expression of three segments: CAR, 4-1BBL, and IL-15 / IL-15sushi. Upon cleavage of the linker (P2A and T2A), the CAR, 4-1BBL, and IL-15 / IL-15sushi are released and engage the target. The CAR contains an scFv, hinge region, transmembrane domain, costimulatory domain (including, but not limited to, CD28 or 4-1BB), and intracellular signaling, CD3 zeta chain. 4-1BBL, IL-15 / IL-sushi, or both, can activate T or NK cells and synergize with CD28 or 4-1BB for sustained or antitumor activity. [Figure 99A] CD45b-28-IL-15 / IL-15sushi CAR-NK92 cells exhibited significant anti-leukemic effects against Jurkat (human acute T-cell leukemia) cell lines in an in vivo xenograft mouse model. Luciferase-expressing Jurkat cells (1x106 cells) were intravenously injected 24 hours after sublethal irradiation (day 1). On days 4 and 7, 5x106 control GFP-CD45b-28 or CD45b-28-IL-15 / IL-15sushi CAR-NK92 cells were intravenously injected into each mouse (n = 2 per group). Dorsal and ventral tumor burden was measured on days 3, 6, 9, 11, 14, and 20 using an IVIS imaging system. Compared to mice treated with control NK92 cells or CD45b-28 CAR NK92 cells, mice treated with CD45b-28-IL-15 / IL-15sushi CAR NK92 cells had much smaller tumor burdens. Figure 99B, Comparison of total flux values ​​(photons / second) of the anti-leukemic effect between mice treated with control, CD45b-28-, or CD45b-28-IL-15 / IL-15sushi CAR NK92 cells against Jurkat (human acute T-cell leukemia) cell lines in an in vivo xenograft mouse model. Total flux levels in both the dorsal and ventral sides increased in a time-dependent manner in mice treated with control NK92 cells (black line in graph) and CD45b-28-NK92 cells (red line in graph). Meanwhile, mice treated with CD45b-28-IL-15 / IL-15sushi CAR NK92 cells (blue line in the graph) showed significant inhibition of tumor progression compared to control and CD45b-CAR-28-NK92 cell-injected mice. Figure 9C, Tumor (Jurkat cell) inhibition rate in mice treated with CD45b-28 CAR or CD45b-28-IL-15 / IL-15sushi CAR NK92 cells compared to control on days 9, 11, 14, and 20. [Figure 99B]CD45b-28-IL-15 / IL-15sushi CAR-NK92 cells exhibited significant anti-leukemic effects against Jurkat (human acute T-cell leukemia) cell lines in an in vivo xenograft mouse model. Luciferase-expressing Jurkat cells (1x106 cells) were intravenously injected 24 hours after sublethal irradiation (day 1). On days 4 and 7, 5x106 control GFP-CD45b-28 or CD45b-28-IL-15 / IL-15sushi CAR-NK92 cells were intravenously injected into each mouse (n = 2 per group). Dorsal and ventral tumor burden was measured on days 3, 6, 9, 11, 14, and 20 using an IVIS imaging system. Compared to mice treated with control NK92 cells or CD45b-28 CAR NK92 cells, mice treated with CD45b-28-IL-15 / IL-15sushi CAR NK92 cells had much smaller tumor burdens. Figure 99B, Comparison of total flux values ​​(photons / second) of the anti-leukemic effect between mice treated with control, CD45b-28-, or CD45b-28-IL-15 / IL-15sushi CAR NK92 cells against Jurkat (human acute T-cell leukemia) cell lines in an in vivo xenograft mouse model. Total flux levels in both the dorsal and ventral sides increased in a time-dependent manner in mice treated with control NK92 cells (black line in graph) and CD45b-28-NK92 cells (red line in graph). Meanwhile, mice treated with CD45b-28-IL-15 / IL-15sushi CAR NK92 cells (blue line in the graph) showed significant inhibition of tumor progression compared to control and CD45b-CAR-28-NK92 cell-injected mice. Figure 9C, Tumor (Jurkat cell) inhibition rate in mice treated with CD45b-28 CAR or CD45b-28-IL-15 / IL-15sushi CAR NK92 cells compared to control on days 9, 11, 14, and 20. [Figure 99C]CD45b-28-IL-15 / IL-15sushi CAR-NK92 cells exhibited significant anti-leukemic effects against Jurkat (human acute T-cell leukemia) cell lines in an in vivo xenograft mouse model. Luciferase-expressing Jurkat cells (1x106 cells) were intravenously injected 24 hours after sublethal irradiation (day 1). On days 4 and 7, 5x106 control GFP-CD45b-28 or CD45b-28-IL-15 / IL-15sushi CAR-NK92 cells were intravenously injected into each mouse (n = 2 per group). Dorsal and ventral tumor burden was measured on days 3, 6, 9, 11, 14, and 20 using an IVIS imaging system. Compared to mice treated with control NK92 cells or CD45b-28 CAR NK92 cells, mice treated with CD45b-28-IL-15 / IL-15sushi CAR NK92 cells had much smaller tumor burdens. Figure 99B, Comparison of total flux values ​​(photons / second) of the anti-leukemic effect between mice treated with control, CD45b-28-, or CD45b-28-IL-15 / IL-15sushi CAR NK92 cells against Jurkat (human acute T-cell leukemia) cell lines in an in vivo xenograft mouse model. Total flux levels in both the dorsal and ventral sides increased in a time-dependent manner in mice treated with control NK92 cells (black line in graph) and CD45b-28-NK92 cells (red line in graph). Meanwhile, mice treated with CD45b-28-IL-15 / IL-15sushi CAR NK92 cells (blue line in the graph) showed significant inhibition of tumor progression compared to control and CD45b-CAR-28-NK92 cell-injected mice. Figure 9C, Tumor (Jurkat cell) inhibition rate in mice treated with CD45b-28 CAR or CD45b-28-IL-15 / IL-15sushi CAR NK92 cells compared to control on days 9, 11, 14, and 20. [Figure 100A]Expression of CD19b-IL15 / IL15sushi CAR T cells. The diagram above shows the CD19b-IL-15 / IL-15sushi construct. T cells isolated from peripheral blood (PB) were transduced with lentivirus expressing either control or CD19b-IL15 / IL15sushi constructs. Flow cytometry using CD3 and F(ab)' antibodies was performed to assay the CAR transduction rate; the transduced population is shown in blue. N = 2. [Figure 100B] CD19b-IL15 / IL15sushi CAR T cells efficiently deplete CD19+ Sp53 cells. CD19b-IL15 / IL15sushi achieves potent lysis of Sp53 target cells. Coculture experiments were performed for 24 hours at effector-to-target ratios ranging from 1:1 to 5:1 and analyzed directly by flow cytometry using mouse anti-human CD3pPerCp and mouse anti-human CD19-PE. Each assay included target cells (Sp53, all CD19+) incubated with either control or CAR T cells. N = 2. [Figure 100C] Bar graph summarizing the cytotoxic activity from the results in Figure 100B. [Figure 100D] IVIS analysis was performed on both the dorsal ( Figure 100 D) and ventral ( Figure 100 E) fields. CD19b-IL15 / IL15sushi CAR-treated mice showed a significant reduction in tumor burden of up to 10% per luminescence dose, consistent with our expectation that CARs equipped with IL-15 / IL-15sushi would have more potent antitumor activity ( Figures 100 D and 100E ). [Figure 100E] IVIS analysis was performed on both the dorsal ( Figure 100 D) and ventral ( Figure 100 E) fields. CD19b-IL15 / IL15sushi CAR-treated mice showed a significant reduction in tumor burden of up to 10% per luminescence dose, consistent with our expectation that CARs equipped with IL-15 / IL-15sushi would have more potent antitumor activity ( Figures 100 D and 100E ). [Figure 101A] CD4CAR T cell loss with or without CAMPATH treatment. Experimental design (101A) and determination of CD4CAR T cell loss in peripheral blood (101B and 101C). CD4CAR T cells (10x106 cells) were intravenously injected into sublethally irradiated NSG mice (6 mice total). The following day, PBS or 0.1 mg / kg CAMPATH was administered intraperitoneally (IP) to three mice (N = 3). After 6 and 24 hours, peripheral blood was collected from each mouse and labeled with CD3 and CD45 antibodies to measure CD4CAR T cell loss due to CAMPATH treatment as an acute phase response. [Figure 101B] CD4CAR T cell loss with or without CAMPATH treatment. Experimental design (101A) and determination of CD4CAR T cell loss in peripheral blood (101B and 101C). CD4CAR T cells (10x106 cells) were intravenously injected into sublethally irradiated NSG mice (6 mice total). The following day, PBS or 0.1 mg / kg CAMPATH was administered intraperitoneally (IP) to three mice (N = 3). After 6 and 24 hours, peripheral blood was collected from each mouse and labeled with CD3 and CD45 antibodies to measure CD4CAR T cell loss due to CAMPATH treatment as an acute phase response. [Figure 101C] CD4CAR T cell loss with or without CAMPATH treatment. Experimental design (101A) and determination of CD4CAR T cell loss in peripheral blood (101B and 101C). CD4CAR T cells (10x106 cells) were intravenously injected into sublethally irradiated NSG mice (6 mice total). The following day, PBS or 0.1 mg / kg CAMPATH was administered intraperitoneally (IP) to three mice (N = 3). After 6 and 24 hours, peripheral blood was collected from each mouse and labeled with CD3 and CD45 antibodies to measure CD4CAR T cell loss due to CAMPATH treatment as an acute phase response. [Figure 102]Summary of the effect of CAMPATH treatment as a "safety switch" for engineered CD4CAR T cells. A, CD4CAR T cell loss in peripheral blood 6 and 48 hours after CAMPATH injection. B, CD4CAR T cell loss in mouse whole blood and liver 5 days after CAMPATH infusion. C, Analysis of engineered CAR T cell loss in various tissues. [Figure 103] The BCMA CAR was constructed as a modular signaling domain containing a leader sequence, an scFv against the BCMA antigen, a hinge domain (H), a transmembrane domain (TM), a costimulatory domain (4-1BB), and the intracellular signaling domain CD3 zeta (see above). BCMA-IL-15 / IL-15sushi is a BCMA CAR equipped with IL-15 / IL-15sushi (Figure 103). [Figure 104] Expression of BCMA-IL15 / IL15sushi CAR T cells. T cells isolated from peripheral blood (PB) were transduced with lentivirus expressing either vector control or the BCMA-IL15 / IL15sushi construct. Flow cytometry using CD3 and F(ab)' antibodies was performed to assay CAR transduction rates; the transduced population is shown in blue. N = 2. [Figure 105A] BCMA-IL15 / IL15sushi CAR T cells show evidence of low-level antitumor activity against CD19+ cells. BCMA-IL15 / IL15sushi targets some CD19+ SP53 cells. Coculture experiments were performed for 24 hours at effector-to-target ratios ranging from 1:1 to 5:1 and analyzed directly by flow cytometry using mouse anti-human CD3pPerCp and mouse anti-human CD19-PE. Each assay included target cells (Sp53, all CD19+) incubated with either vector control or CAR T cells. N = 2. [Figure 105B] A bar graph summarizing the cytotoxic activity from the results in Figure 105B. [Figure 106A]Composition of the GD3 CAR construct. The GD2 CAR construct is a modular signaling domain containing a leader sequence, GD2 scFv, hinge domain (H), transmembrane domain (TM), costimulatory domain (CD28 or 4-1BB), and intracellular signaling domain CD3 zeta. [Figure 106B] Composition of the GD3 CAR construct. The GD3 CAR construct is a modular signaling domain containing a leader sequence, GD3 scFv, a hinge domain (H), a transmembrane domain (TM), a costimulatory domain (CD28 or 4-1BB), and the intracellular signaling domain CD3 zeta. [Figure 107A] GD2 CAR surface expression in human T cells. Activated peripheral blood mononuclear cells were transduced with either vector control (left) or GD2CAR (right) lentiviral vectors. After 48 hours of recovery, cells were labeled with anti-mouse F(Ab')2-biotin antibody to detect the CAR phenotype. [Figure 107B] When Y79 cells were labeled with mouse anti-human GD2 and CD56, GD2 expression was observed in almost all retinal lipoma Y79 cells (N = 2). [Figure 107C] Y79 retinoblastoma cells were co-cultured with GD2CAR T cells for 24 hours. GD2CAR T cells can efficiently lyse the GD2-expressing Y79 retinoblastoma cell line in 24-hour co-culture assays. Co-culture experiments were performed for 24 hours at effector-to-target ratios ranging from 1:2 to 2:1 and analyzed directly by flow cytometry for CD56 and GD2. Each assay consisted of Y79 target cells and control T cells (top row), and GD2CAR T cells (bottom row). [Figure 107D]Y79 retinoblastoma cells were co-cultured with GD2CAR T cells for 24 hours. GD2CAR T cells can eliminate GD2-expressing Y79 retinoblastoma cell lines in co-culture assays. Co-culture experiments were performed for 24 hours at effector-to-target ratios ranging from 5:1 to 20:1 and analyzed directly by flow cytometry for CD56 and GD2 expression. Each assay consisted of Y79 target cells and control T cells (top row), and GD2CAR T cells (bottom row). All data were analyzed in duplicate. [Figure 107E] Y79 retinoblastoma cells were co-cultured with GD2CAR T cells for 72 hours. GD2CAR T cells can erase the GD2-expressing Y79 retinoblastoma cell line in 72-hour co-culture assays. Co-culture experiments were performed for 72 hours at effector-to-target ratios ranging from 1:2 to 2:1 and analyzed directly by flow cytometry for CD56 and GD2. Each assay consisted of Y79 target cells and control T cells (top row), and GD2CAR T cells (bottom row). [Figure 107F] GD2CAR T cells can eradicate the GD2-expressing Y79 retinoblastoma cell line in coculture assays. Coculture experiments were performed for 72 hours at effector-to-target ratios ranging from 5:1 to 20:1 and analyzed directly by flow cytometry for CD56 and GD2 expression. Each assay consisted of Y79 target cells and control T cells (top row), and GD2CAR T cells (bottom row). All data were replicated in duplicate. [Figure 107G] Summary of the percentage of Y79 tumor cells lysed by GD2CAR T cells after 24 and 72 hours of co-culture at ratios ranging from 1:2 to 20:1. (N = 2) [Figure 108A]Transduction and sorting of GD2CAR NK-92 cells. (A) NK-92 cells were transduced with control (left) or GD2CAR (right) lentiviral supernatant. After recovery, cells were labeled with goat anti-mouse F(Ab')2-antibody to detect the CAR phenotype. (B) GD2CAR + NK cells were sorted on a FACS Aria. After 10 days of recovery and expansion, vector control (left) or sorted GD2CAR (right) NK cells were labeled with anti-mouse F(Ab')2-antibody to detect the CAR phenotype. [Figure 108B] Anti-GD2 CAR NK-92 cells against the Y79 neuroblastoma cell line. GD2 CAR NK-92 cells demonstrate anti-tumor activity against the GD2+ neuroblastoma cell line, Y79. Y79 neuroblastoma cells were pre-labeled with a cytotracker (CMTMR) dye and cultured for 24 hours with control NK-92 or anti-GD2 CAR NK-92 cells at various E:T ratios. The GD2-positive Y79 cell population is purple, indicating a dual CMTMR+ GD2+ phenotype. Lysis rates are summarized in a bar graph (right). [Figure 108C] Increasing doses correlate with increased cytotoxicity. Summary of the percentage of Y79 tumor cells lysed by GD2CAR NK-92 cells at ratios of 2:1 to 10:1 shows that increasing doses correlate with increased cytotoxicity. [Figure 109] Schematic diagram of the GD2-GD3 cCAR construct. This construct contains an SFFV promoter driving the expression of multiple modular CAR units linked by P2A cleavage peptides. Upon cleavage of the P2A linker, the cCAR cleaves and engages targets expressing GD2 and / or GD3. Each CAR unit contains an scFv against the antigen, a hinge domain (H), a transmembrane domain (TM), a costimulatory domain (including, but not limited to, CD28 or 4-1BB), and an intracellular signaling domain, CD3 zeta chain. As a novel cCAR construct, the activation domains of the construct include, but are not limited to, the 4-1BB domain on the GD2 CAR segment and the CD28 region on the GD3 CAR. DETAILED DESCRIPTION OF THE INVENTION

[0027] The present disclosure provides chimeric antigen receptor (CAR) constructs, methods for making and using same.

[0028] A chimeric antigen receptor (CAR) polypeptide comprises a signal peptide, an antigen recognition domain, a hinge region, a transmembrane domain, at least one costimulatory domain, and a signaling domain.

[0029] First-generation CARs contain CD3z as an intracellular signaling domain, while second-generation CARs contain at least one single costimulatory domain derived from various proteins. Examples of costimulatory domains include, but are not limited to, CD28, CD2, 4-1BB (CD137, also known as "4-BB"), and OX-40 (CD124). Third-generation CARs contain two costimulatory domains, such as, but not limited to, CD28, 4-1BB, CD134 (OX-40), CD2, and / or CD137 (4-1BB).

[0030] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds having amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that a protein or peptide sequence can contain. Polypeptides include any peptide or protein having two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and long chains, commonly referred to in the art as proteins, of which there are many varieties. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins, among others. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0031] A "signal peptide" is a peptide sequence that directs transport and localization, and includes any peptide that can bind to a cell organelle (such as the endoplasmic reticulum) and / or the cell surface. As used herein, the terms "signal peptide" and "leader sequence" are used interchangeably.

[0032] A signal peptide, as used herein, is any secreted or transmembrane protein peptide or polypeptide that directs transport to the cell membrane and cell surface and provides for correct localization. In particular, a signal peptide of the present disclosure is a polypeptide of the present disclosure that directs transport to the cell membrane, in which the extracellular portion of the polypeptide is displayed on the cell surface, the transmembrane portion spans the plasma membrane, and the active domain is present in the cytoplasmic portion, or interior of the cell.

[0033] In this example, the signal peptide is cleaved after passage through the endoplasmic reticulum (ER), i.e., a cleavable signal peptide. In this example, the signal peptide is a human protein type I, II, III, or IV. In this example, the signal peptide comprises an immunoglobulin heavy chain signal peptide.

[0034] In this example, the signal peptide comprises a signal peptide derived from human CD45 (UniProtKB / Swiss-Prot accession number P08575). The CD45 signal peptide is 23 amino acids long (MYLWLKLLAFGFAFLDTEVFVTG). In some examples, the signal peptide may be a functional fragment of the CD45 signal peptide. A functional fragment comprises at least a 10-amino acid fragment of the CD45 signal peptide that targets the attached polypeptide to the cell membrane and cell surface. Examples of fragments of the human CD45 signal peptide include MYLWLKLLAFG, FAFLDTEVFVTG, and LKLLAFGFAFLDTE.

[0035] Functional equivalents of the human CD45 signal peptide are also contemplated. As used herein, "functional equivalent" is understood as a mutant that exhibits an amino acid substitution other than those specifically mentioned at least one of the aforementioned sequence positions, but which results in a mutation that still exhibits the same or similar properties with respect to the wild-type CD45 signal peptide. Functional equivalents include polypeptides having at least 80%, at least 85%, at least 90%, or at least 95% identity to the human CD45 signal peptide, a functional fragment thereof, or a functional equivalent thereof.

[0036] Functional equivalents also include CD45 signal peptides from homologous proteins from other species. Examples of these signal peptides include the signal peptide from mouse CD45 (MGLWLKLLAFGFALLDTEVFVTG), rat CD45 (MYLWLKLLAFSLALLGPEVFVTG), sheep CD45 (MTMYLWLKLLAFGFAFLDTAVSVAG), chimpanzee CD45 (MYLWLKLLAFGFAFLDTEVFVTG), and monkey CD45 (MTMYLWLKLLAFGFAFLDTEVFVAG).

[0037] In another embodiment, the signal peptide comprises the sequence: MX 1 LWLKLLAF X 2 X 3 AX 4 LX 5 X 6 X 7 VX 8 VX 9 G, those are X 1 , X 2 , X3, X 4 , X 5 , X 6 , X 7 , X 8 , and X 9 are each independently Y, G, S, F, L, D, P, T, E, or A. In this example, X 1 is Y or G, X 2 is G, S, or X 3 and X 4 are independently F or L, X 5 is D or G, X 6 is P or T, X 7 is E or A, X 8 is F or S, and X 9 is A or T.

[0038] In this example, the signal peptide comprises the signal peptide from human CD8a (MALPVTALLLPLALLLHAARP). In some examples, the signal peptide could be a functional fragment of the CD8a signal peptide. A functional fragment comprises at least a 10 amino acid fragment of the CD8a signal peptide that targets the attached polypeptide to the cell membrane and cell surface. Examples of fragments of the human CD8a signal peptide include MALPVTALLLALALHAHAA, MALPVTALLLP, PVTALLLPLALL, and LLLPLALLLHAARP.

[0039] In another example, the signal peptide comprises the signal peptide from human CD8b (MRPRLWLLLAAQLTVLHGNSV). In some examples, the signal peptide may be a functional fragment of the CD8b signal peptide. A functional fragment comprises at least a 10 amino acid fragment of the CD8b signal peptide that targets the attached polypeptide to the cell membrane and cell surface. Examples of fragments of the human CD8b signal peptide include MRPRLWLLLAAQ, RLWLLLAAQLTVLHG, and LWLLLAAQLTVLHGNSV.

[0040] Functional equivalents of the human CD8a or CD8b signal peptide are also contemplated. As used herein, "functional equivalent" is understood as a mutant that exhibits an amino acid substitution other than those specifically mentioned at at least one of the aforementioned sequence positions, but which results in a mutation that still exhibits the same or similar properties with respect to the wild-type CD8a or CD8b signal peptide. Functional equivalents include polypeptides having at least 80%, at least 85%, at least 90%, or at least 95% identity to the human CD8 signal peptide, a functional fragment thereof, or a functional equivalent thereof.

[0041] Functional equivalents also include the CD8a and CD8b signal peptides from homologous proteins from other species.

[0042] In this example, the signal peptide comprises a signal peptide derived from human IL-2. The IL-2 signal peptide is 23 amino acids long (MYRMQLLSCIALSLALVTNS). In some examples, the signal peptide may be a functional fragment of the IL-2 signal peptide. Functional fragments include at least a 10 amino acid fragment of the IL-2 signal peptide that directs the attached polypeptide to the cell membrane and cell surface. Examples of fragments of the human IL-2 signal peptide include MYRMQLLSCIAL, QLLSCIALSLAL, and SCIALSLALVTNS.

[0043] Functional equivalents of the human IL-2 signal peptide are also contemplated. As used herein, "functional equivalent" is understood as a mutant that exhibits an amino acid substitution other than those specifically mentioned at least one of the aforementioned sequence positions, but which results in a mutation that still exhibits the same or similar properties as the wild-type IL-2 signal peptide. Functional equivalents include polypeptides having at least 80%, at least 85%, at least 90%, or at least 95% identity to the human IL-2 signal peptide, a functional fragment thereof, or a functional equivalent thereof.

[0044] Functional equivalents also include IL-2 signal peptides from homologous proteins from other species. See, for example, Figure 80.

[0045] In another embodiment, the signal peptide comprises the sequence: MYX 1 X 2 QLX 3 SCX 4 X 5 LX 6 LX 7 LX 8 X 9 X 10 X 11 , they are X 1 , X 2 , X3, X 4 , X 5 , X 6 , X7 , X 8 , X 9 , X 10 , and X 11 are each independently R, K, S, M, I, V, L, A, I, T, N, S, or G. In this example, X 1 is R, K or S, X 2 is M, I or V, X 3 is L or A, X 4 and X 5 are independently I, A, V or T, X 6 is S or T, X 7 is A or V, X 8 , X 9 , X 10 And X 11 are independently V, L, T, A, N, S, or G.

[0046] An "antigen recognition domain" includes a polypeptide that is selective for a target antigen, receptor, peptide ligand, or protein ligand or for a targeting polypeptide.

[0047] The antigen recognition domain can be derived from any of a wide variety of extracellular domains or secreted proteins involved in ligand binding and / or signal transduction. The antigen recognition domain can be configured as a single-chain fragment variable (scFv) that includes a portion of an Ig heavy chain linked to a portion of an Ig light chain and specifically binds to a target antigen. The antibody can be a monoclonal or polyclonal antibody, or any type that specifically binds to a target antigen. In another example, the antigen recognition domain can be a receptor or a ligand. In certain examples, the target antigen is specific to a particular disease state, which may be any type of cell surface antigen that can be recognized by at least one chimeric receptor structure on a compound CAR structure. In certain examples, the chimeric receptor can be related to any cancer for which a specific monoclonal or polyclonal antibody exists or can be produced. In particular, cancers such as neuroblastoma, small cell lung cancer, melanoma, ovarian cancer, renal cell carcinoma, colon cancer, Hodgkin's lymphoma, and childhood acute lymphoblastic leukemia have antigens specific for the chimeric receptor.

[0048] In some examples, the antigen recognition domain can be a non-antibody protein scaffold that can be engineered to bind to various specific targets with high affinity, such as, but not limited to, centirin, a scaffold protein based on the human consensus tenascin FN3 domain and typically smaller than the scFv molecule of a CAR molecule.

[0049] The target-specific antigen recognition domain preferably comprises an antigen-binding domain capable of targeting an antibody against the target antigen or a peptide that binds to the target antigen, or a peptide or protein that binds to an antibody that binds to the target antigen, or a peptide or protein ligand that binds to a receptor on the target (including, but not limited to, a growth factor, cytokine, or hormone), or a domain derived from a receptor that binds to a peptide or protein ligand that binds to a target (including, but not limited to, a growth factor receptor, cytokine receptor, or hormone receptor).

[0050] In this example, the antigen recognition domain comprises the binding portion or variable region of a monoclonal or polyclonal antibody directed against (selective for) the target.

[0051] In another example, the antigen recognition domain comprises a camelid single domain antibody or portion thereof. In this example, the camelid single domain antibody comprises a heavy chain antibody or VHH antibody found in camelids. Camelid VHH antibodies (e.g., camel, dromedary, llama, and alpaca) refer to variable fragments of camelid single chain antibodies (see Nguyen et al., 2001; Muyldermans, 2001), and include camelid, recombinant camelid-derived VHH antibodies, or VHH antibodies isolated from camelid VHH synthetic antibodies.

[0052] In another example, the antigen-recognition domain comprises a single-chain fragment variable (scFv), which is the binding variable region of a monoclonal antibody. An scFv comprises one light chain and one heavy chain antibody. In a specific embodiment, the antigen-recognition domain comprises two different heavy chain domains (VHH). Each heavy chain domain binds to a different epitope of the same antigen or different antigens. In one embodiment, the antigen-recognition domain comprises a single heavy chain domain.

[0053] In another embodiment, the antigen recognition domain comprises a ligand that binds to their cognate receptor. As an example, APRIL is a ligand that binds to the TAC1 receptor or the BCMA receptor. In accordance with the invention disclosed herein, the antigen recognition domain comprises APRIL or a fragment thereof. As a further example, BAFF is a ligand that binds to the BAFF-R receptor or the BCMA receptor. In accordance with the invention disclosed herein, the antigen recognition domain comprises BAFF or a fragment thereof. In another embodiment, the antigen recognition domain is humanized.

[0054] It is understood that an antigen recognition domain can contain some variability in its sequence and still be selective for the targets disclosed herein. Thus, an antigen recognition domain polypeptide that is at least 95%, at least 90%, at least 80%, or at least 70% identical to the antigen recognition domain polypeptides disclosed herein and still be considered as described herein and within the scope of the present disclosure.

[0055] Targets include interleukin 6 receptor, NY-ESO-1, alpha-fetoprotein (AFP), glypican-3 (GPC3), BCMA, BAFF-R, TACI, LeY, CD5, CD13, CD14, CD15, CD19, CD20, CD22, CD33, CD41, CD61, CD64, CD68, CD117, CD123, CD138, CD267, CD269, CD38, Flt3 receptor, CS1, CD45, ROR1, PSMA, MAGE A3, glycolipids, glypican-3, F77, GD-2, WT1, CEA, HER-2 / neu, MAGE-3, MAGE-4, MAGE-5, MAGE-6, alpha-fetoprotein, CA19-9, CA72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, CD30, These include EGFRvIII, immunoglobulins kappa and lambda, CD38, CD52, CD3, CD4, CD8, CD5, CD7, CD2, and CD138.

[0056] In another embodiment, the target is any portion of interleukin 6 receptor, NY-ESO-1, alpha fetoprotein (AFP), glypican-3 (GPC3), BCMA, BAFF-R, TACI, LeY, CD5, CD13, CD14, CD15, CD19, CD20, CD22, CD33, CD41, CD61, CD64, CD68, CD117, CD123, CD138, CD267, CD269, CD38, Flt3 receptor, CS1, CD45, TACI, ROR1, PSMA, MAGE A3, glycolipids, glypican 3, F77, GD-2, WT1, CEA, HER-2 / neu, Includes any of the following proteins: MAGE-3, MAGE-4, MAGE-5, MAGE-6, alpha-fetoprotein, CA19-9, CA72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, CD30, EGFRvIII, immunoglobulins kappa and lambda, CD38, CD52, CD3, CD4, CD8, CD5, CD7, CD2, and CD138.

[0057] In this example, the targets are interleukin 6 receptor, NY-ESO-1, alpha fetoprotein (AFP), glypican-3 (GPC3), BCMA, BAFF-R, TACI, LeY, CD5, CD13, CD14, CD15, CD19, CD20, CD22, CD33, CD41, CD61, CD64, CD68, CD117, CD123, CD138, CD267, CD269, CD38, Flt3 receptor, CS1, CD45, TACI, ROR1, PSMA, MAGE A3, glycolipids, glypican 3, F77, GD-2, WT1, CEA, HER-2 / neu, These include exposed surface sites of MAGE-3, MAGE-4, MAGE-5, MAGE-6, alpha-fetoprotein, CA19-9, CA72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, CD30, EGFRvIII, immunoglobulins kappa and lambda, CD38, CD52, CD3, CD4, CD8, CD5, CD7, CD2, and CD138 polypeptides.

[0058] For example, as shown in Figure 81, the target includes a surface-exposed region of BAFF. The target can include a portion of a surface-exposed region of BAFF. For example, the portion of BAFF includes residues 1-200, 1-100, 50-150, or 100-200 of human BAFF.

[0059] In another example, the target antigen comprises a viral or fungal antigen, such as a portion thereof or a surface-exposed region thereof, such as E6 and E7 from human papillomavirus (HPV) or EBV (Epstein-Barr virus) antigens.

[0060] In another example, the target antigen comprises FcER1A, FCER1, and IgE. FCER1 is a high-affinity IgE receptor comprising an alpha chain (FcER1A), a beta chain, and two gamma chains. The target may be present on a cell surface. Examples of cells include plasma cells, mast cells, basophils, or eosinophils.

[0061] In one embodiment, the target comprises the extracellular domain of FcER1A. The target also includes a fragment or portion of the extracellular domain of FcER1A. For example, the target includes residues 1-178, 1-100, 50-150, or 100-178 of human FcER1A. In another embodiment, the target includes the extracellular domain of the FCER1 receptor. In this example, the TACI antigen recognition domain comprises SEQ ID NO:24. In this example, the BCMA antigen recognition domain comprises SEQ ID NO:25. In this example, the CS1 antigen recognition domain comprises SEQ ID NO:26. In this example, the BAFF-R antigen recognition domain comprises SEQ ID NO:27. In this example, the CD33 antigen recognition domain comprises SEQ ID NO:28. In this example, the CD123 antigen recognition domain comprises SEQ ID NO:29. In this example, the CD19 antigen recognition domain comprises SEQ ID NO:30. In this example, the CD20 antigen recognition domain comprises SEQ ID NO:31. In another embodiment, the CD20 antigen recognition domain comprises SEQ ID NO:32. In this example, the CD22 antigen recognition domain comprises SEQ ID NO:33. In this example, the CD45 antigen recognition domain comprises SEQ ID NO:34. In this example, the CD4 antigen recognition domain comprises SEQ ID NO:35. In this example, the CD25 antigen recognition domain comprises SEQ ID NO:36.

[0062] The hinge region is located between sequences including, but not limited to, a chimeric antigen receptor and at least one costimulatory domain and signaling domain. The hinge sequence can be derived from any suitable sequence from any genus, including, for example, humans or portions thereof. Such hinge regions are known in the art. In this example, the hinge region includes the hinge region of a human protein, including CD-8 alpha, CD28, 4-1BB, OX40, CD3-zeta, T cell receptor alpha or beta chain, CD3 zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, and combinations thereof.

[0063] In this example, the hinge region comprises a CD8 hinge region.

[0064] In some examples, the hinge region comprises, but is not limited to, an immunoglobulin (eg, IgG1, IgG2, IgG3, IgG4, and IgD).

[0065] A transmembrane domain comprises a hydrophobic polypeptide that spans the cell membrane. In particular, a transmembrane domain spans from one side of the cell membrane (extracellular) to the other side of the cell membrane (intracellular or cytoplasmic).

[0066] Transmembrane domains can be in the form of alpha helices or beta barrels, or a combination thereof. Transmembrane domains include polytopic proteins, which have many transmembrane segments, each alpha-helix, beta sheet, or a combination thereof.

[0067] In this example, a transmembrane domain naturally associated with one of the domains of the CAR structure is used, hi another example, the transmembrane domain is modified by selection or amino acid substitution to avoid binding of such domain with transmembrane domains of the same or different surface membrane proteins or to minimize interactions with other members of the receptor complex.

[0068] For example, transmembrane domains include the transmembrane domains of the T cell receptor α or β chain, the CD3 zeta chain, CD28, CD3ε, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD68, CD134, CD137, ICOS, CD41, CD154, functional derivatives thereof, and combinations thereof.

[0069] In this example, a transmembrane domain is artificially designed such that more than 25%, more than 50%, or more than 75% of the amino acid residues in the domain are hydrophobic residues such as leucine and valine. In this example, a triplet of phenylalanine, tryptophan, and valine is found at each end of the synthetic transmembrane domain.

[0070] In this example, the transmembrane domain is a CD8 transmembrane domain. In another example, the transmembrane domain is a CD28 transmembrane domain. Such transmembrane domains are known in the art.

[0071] The signaling domain and the costimulatory domain comprise a polypeptide for activating an immune cell or activating at least some aspects of an immune cell signaling pathway.

[0072] In this example, the signaling domain includes polypeptides of functional signaling domains thereof, including CD3 zeta, common FcRγ (FCER1G), FcγRIIa, FcRβ (Fc epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DNAX activation protein 10 (DAP10), DNAX activation protein 12 (DAP12), active fragments thereof, functional derivatives thereof, and combinations thereof. Such signaling domains are known in the art.

[0073] In this embodiment, the CAR polypeptide comprises one or more costimulatory domains, such as IL-15 receptor alpha, IL-15 receptor alpha cytoplasmic domain, B7-1 / CD80, CD28; B7-2 / CD86, CTLA-4, B7-H1 / PD-L1, ICOS, B7-H2, PD-1, B7-H3, PD-L2, B7-H4, PDCD6, BTLA, 4-1BB / TNFRSF9 / CD137, CD40 Ligand / TNFSF5, 4-1BB Ligand / TNFSF9, GITR / TNFRSF18, BAFF / BLyS / TNFSF13B, GITR Ligand / TNFSF18, BAFF R / TNFRSF13C, HVEM / TNFRSF14, CD27 / TNFRSF7, LIGHT / TNFSF14, and CD27. Ligand / TNFSF7, OX40 / TNFRSF4, CD30 / TNFRSF8, OX40 Ligand / TNFSF4, CD30 Ligand / TNFSF8, TACI / TNFRSF13B, CD40 / TNFRSF5, B4 / CD244 / SLAMF4, CD84 / SLAMF5, BLAME / SLAMF8, CD229 / SLAMF3, CD2, CD27, CRACC / SLAMF7, CD2F-10 / SLAMF9, NTB-A / SLAMF6, CD48 / SLAMF2, SLAM / CD150, CD58 / LFA-3, Ikaros, CD53, Integrin alpha 4 / CD49d, CD82 / Kai-1, Integrin alpha 4 beta 1, CD90 / Thy1, ntegrin alpha 4 beta 7 / LPAM-1, CD96;LAG-3, CD160, LMIR1 / CD300A, CRTAM, TCL1A, DAP12, TIM-1 / KIM-1 / HAVCR, Dectin-1 / CLEC7A, TIM-4, DPPIV / CD26, TSLP, EphB6, TSLP R, and ligands that bind to at least one of HLA-DR, OX40, CD30, CD40, PD-1, CD7, CD258, natural killer group 2 member C (NKG2C), natural killer group 2 member D (NKG2D), B7-H3 or CD83, ICAM-1, LFA-1 (CD11a / CD18), ICOS, and 4-1BB (CD137), CDS, ICAM-1, LFA-1 (CD1a / CD18), CD40, CD27, CD7, B7-H3, NKG2C, Functional signaling domains derived from proteins including PD-1, ICOS, active fragments thereof, functional derivatives thereof, and combinations thereof;

[0074] As used herein, the at least one costimulatory domain and the signaling domain may be collectively referred to as the intracellular domain. As used herein, the hinge region and the antigen recognition domain may be collectively referred to as the extracellular domain.

[0075] The present disclosure further provides polynucleotides encoding the above-described chimeric antigen receptor polypeptides.

[0076] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Polynucleotides include DNA and RNA. Furthermore, nucleic acids are polymers of nucleotides. Therefore, as used herein, nucleic acids and polynucleotides have the same meaning. Those skilled in the art generally know that nucleic acids are polynucleotides, which can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, any nucleic acid sequence obtained by any means available in the art, for example, conventional cloning techniques from recombinant libraries or cells, and cloning by synthetic means such as polymerase chain reaction (PCR).

[0077] Polynucleotides encoding CARs can be easily prepared from the amino acid sequence of a specific CAR by any conventional method. The nucleotide sequence encoding the amino acid sequence can be obtained from the aforementioned NCBI RefSeq ID or the amino acid sequence of each domain can be obtained from the GenBenk accession number. The nucleic acids of the present invention can be prepared by standard molecular biology and / or chemical procedures. For example, polynucleotides can be synthesized based on the nucleotide sequence, or polynucleotides of the present invention can be prepared by combining DNA fragments obtained from a cDNA library using polymerase chain reaction (PCR).

[0078] In this example, the polynucleotides disclosed herein are part of a gene, an expression cassette, or a cloning cassette.

[0079] The polynucleotides described above can be cloned into vectors. A "vector" is a construct that contains an isolated polynucleotide and is used to deliver the isolated polynucleotide into a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, phagemids, cosmids, and viruses. Viruses include phages and phage derivatives. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and lentiviral vectors. In this example, vectors include cloning vectors, expression vectors, replication vectors, probe generation vectors, integration vectors, and sequencing vectors.

[0080] In this example, the vector is a viral vector. In this example, the viral vector is a retroviral or lentiviral vector. In one embodiment, the engineered cells are transduced with a virus to express the polynucleotide sequence.

[0081] Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene is inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to patient cells by either in vivo or ex vivo means. Many retroviral systems are known in the art. In some embodiments, an adenoviral vector is used. Many adenoviral vectors are known in the art. In this embodiment, a lentiviral vector is used.

[0082] Viral vector technology is well known in the art, and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses and lentiviruses.Generally, suitable vectors comprise a replication origin that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (for example, WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0083] Lentiviral vectors are well known for their ability to efficiently transfer genes into human T cells; however, expression of the vector-encoded gene depends on the internal promoter that drives its expression. Strong promoters are particularly important for the generation of third- or fourth-generation CARs, as the increased construct size of CARs carrying genes encoding additional costimulatory domains or proliferative cytokines does not guarantee equal expression. Various promoters exist with different strengths and cell-type specificities. Gene therapy using CAR T cells relies on the ability of T cells to express sufficient CAR molecules and maintain expression over long periods of time. The EF1α promoter is commonly chosen for CAR expression.

[0084] The present disclosure provides expression vectors comprising strong promoters for high-level gene expression in T cells or NK cells. In further embodiments, the present disclosure provides strong promoters useful for high-level expression of CARs in T cells or NK cells. In certain embodiments, a strong promoter associated with an SFFV promoter can be selectively introduced into an expression vector to achieve high-level expression in T cells or NK cells and maintain expression over a long period of time. The expression of CARs, T cell costimulators, and cytokines can be used in immunotherapy.

[0085] One example of a suitable promoter is the immediate-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of inducing high-level expression of any manipulated polynucleotide sequence linked to it. Another example of a suitable promoter is the elongation growth factor-1a (EF-1a) promoter. However, other constitutive promoter sequences can also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate-early promoter, Rous sarcoma virus promoter, and human gene promoters such as the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present disclosure should not be limited to the use of constitutive promoters; inducible promoters are also considered part of this disclosure. The use of an inducible promoter provides a molecular switch that can turn on expression of the linked polynucleotide sequence when expression is desired or turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, a metallothionein promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0086] Expression of the chimeric antigen receptor polynucleotide may be achieved, for example, using, but not limited to, the SFFV (spleen focus-forming virus) (e.g., SEQ ID NO: 23) or human elongation factor 11α (EF) promoter, the CAG (chicken β-actin promoter with a CMV enhancer) promoter, or the human elongation factor 1α (EF) promoter. Examples of low-strength / low-expression promoters include, but are not limited to, the simian virus 40 (SV40) early promoter, the cytomegalovirus (CMV) immediate-early promoter, the ubiquitin C (UBC) promoter, and the phosphoglycerate kinase 1 (PGK) promoter, or portions thereof. Inducible expression of the chimeric antigen receptor may be achieved, for example, using, but not limited to, tetracycline-responsive promoters, including, but not limited to, TRE3GV (Tet-responsive elements, including all generations, preferably the third generation), inducible promoters (Clontech Laboratories, Mountain View, CA), or portions thereof, or combinations thereof.

[0087] In a preferred embodiment, the promoter is an SFFV promoter or a derivative thereof. Unexpectedly, the SFFV promoter has been discovered in the present disclosure to exhibit stronger expression and higher persistence in transduced cells.

[0088] An "expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression. Other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide. Expression vectors can be bicistronic or multicistronic expression vectors. Bicistronic or multicistronic expression vectors include (1) multiple promoters fused to each open reading frame, (2) insertion of splicing signals between genes, fusing genes whose expression is driven by a single promoter, (3) insertion of proteolytic cleavage sites (self-cleaving peptides) between genes, and (iv) insertion of internal ribosome entry sites (IRES) between genes.

[0089] In this example, the disclosure provides engineered cells harboring at least one chimeric antigen receptor polypeptide or polynucleotide.

[0090] "Genetically engineered cells" refers to any cell of any organism that has been modified, transformed, or manipulated by the addition or modification of a gene, DNA or RNA sequence, or protein or polypeptide. The isolated cells, host cells, and genetically engineered cells of the present disclosure include isolated immune cells, such as NK cells and T cells, that contain DNA or RNA sequences encoding a chimeric antigen receptor or chimeric antigen receptor complex and express the chimeric receptor on their cell surface. For example, isolated host cells and engineered cells that activate NK cells or activate T lymphocytes can be used for the treatment of cancer and infectious diseases.

[0091] In this example, the engineered cells include immunoregulatory cells, including T cells such as CD4 T cells (helper T cells), CD8 T cells (cytotoxic T cells, CTLs), and memory T cells or T memory stem cells. In another example, the T cells include natural killer T cells (NK T cells).

[0092] In this example, the engineered cells include natural killer cells. Natural killer cells are well known in the art. In this example, the natural killer cells include cell lines such as NK-92 cells. Further examples of NK cell lines include NKG, YT, NK-YS, HANK-1, YTS cells, and NKL cells.

[0093] NK cells provide antitumor effects without the risk of GvHD and are short-lived compared to T cells. Therefore, NK cells are depleted immediately after destroying cancer cells, reducing the need for inducible suicide gene transfer in CAR constructs to clear engineered cells.

[0094] In accordance with the present disclosure, it has surprisingly been found that NK cells are readily accessible cells that can then be engineered to contain and express the chimeric antigen receptor polypeptides disclosed herein.

[0095] Because allogeneic or autologous NK cells induce rapid immune responses but have a limited lifespan and are cleared relatively quickly from the circulation, applicants have surprisingly discovered that there is less concern about persistent side effects when using CAR cell-based therapies.

[0096] According to one aspect of the present invention, NK cells can be expanded and transfected with a CAR polynucleotide in accordance with the present invention. NK cells can be obtained from umbilical cord blood, peripheral blood, iPS cells, and embryonic stem cells. According to one aspect of the present invention, NK-92 cells may be expanded and transfected with a CAR. NK-92 is a continuously growing cell line that possesses the characteristics and features of natural killer (NK) cells (Arai, Meagher et al., 2008). The NK-92 cell line is IL-2 dependent, safe (Arai, Meagher et al., 2008), and has been proven feasible. CAR-expressing NK-92 cells can be grown in serum-free medium with or without co-culture with feeder cells. A pure population of NK-92 cells carrying a desired CAR can be obtained by sorting.

[0097] In this example, the engineered cells comprise allogeneic T cells obtained from a donor that have been modified to inactivate components of the TCR (T cell receptor) involved in MHC recognition, such that the TCR-deficient T cells will not cause graft-versus-host disease (GVHD).

[0098] In some embodiments, the engineered cells may be modified to prevent expression of a cell surface antigen, for example, the engineered cells may be genetically modified to delete the native CD45 gene to prevent its expression and presence on the cell surface.

[0099] In some embodiments, the engineered cells carry an inducible suicide gene ("safety switch") or a combination of safety switches that can be incorporated onto a vector, such as a retroviral vector, a lentiviral vector, an adenoviral vector, or a plasmid. The introduction of a "safety switch" significantly improves the safety profile of compound CARs, limiting on-target or off-target tumor toxicity. "Safety switches" include, but are not limited to, inducible suicide genes such as caspase 9, thymidine kinase, cytosine deaminase (CD), or cytochrome P450. Other safety switches for eliminating unwanted modified T cells include the expression of CD20, CD52, CD19, or truncated epidermal growth factor receptor (EGFR) on T cells. All possible safety switches are contemplated and encompassed by the present invention.

[0100] In some embodiments, the suicide gene is integrated into the engineered cell genome.

[0101] In this example, the disclosure provides engineered cells comprising a CD45-chimeric antigen receptor polynucleotide. In this example, the CD45 CAR polypeptide comprises SEQ ID NO: 13 and corresponds to the polynucleotide sequence SEQ ID NO: 14. In another example, the CD45 CAR polypeptide comprises SEQ ID NO: 15 and corresponds to the polynucleotide sequence SEQ ID NO: 16. In another example, the CD45 CAR polypeptide comprises SEQ ID NO: 17 and corresponds to the polynucleotide sequence SEQ ID NO: 18.

[0102] In a specific example, the engineered cells comprise a CD45 CAR linked to IL15 / IL-15sushi via a P2A cleavage sequence. The polypeptide provided in this example comprises SEQ ID NO 43 and the corresponding polynucleotide sequence SEQ ID NO 44.

[0103] In a particular example, the engineered cells comprise a CD45 CAR linked to 4-1BBL (CD137L) via a P2A cleavage sequence. The polypeptide provided in this example comprises SEQ ID NO 42 and the corresponding polynucleotide sequence SEQ ID NO 41.

[0104] In one embodiment, the engineered cell comprises a CD22 CAR comprised of a chimeric antigen receptor polypeptide having a CD22 antigen recognition domain. In one embodiment, the engineered cell comprises the polypeptide of SEQ ID NO: 130 and a polynucleotide corresponding to SEQ ID NO: 131.

[0105] Multiple CAR unit In this example, the present disclosure provides an engineered cell having at least two different or separate CAR units. The two CAR units may be complete or incomplete CAR units. As used herein, "different CAR polypeptides" and "different CAR polypeptide units" are used interchangeably.

[0106] The present disclosure provides chimeric antigen receptor polypeptides having a signal peptide, an antigen recognition domain, a hinge region, a transmembrane domain, a signaling domain, and at least one costimulatory domain, which are defined as CAR units or complete CAR units. As used herein, incomplete CAR units include polypeptides having a signal peptide, an antigen recognition domain, a hinge region, a transmembrane domain, and a signaling domain or at least one costimulatory domain. Incomplete CAR units do not include the signaling domain and at least one costimulatory domain, but do not include one or the other.

[0107] In this example, the disclosure provides an engineered cell having a first chimeric antigen receptor polypeptide having a first antigen recognition domain and a costimulatory domain (first incomplete CAR unit), and a second chimeric antigen receptor polypeptide having a second antigen recognition domain and a signaling domain (second incomplete CAR unit), wherein the first antigen recognition domain is different from the second antigen recognition domain.

[0108] Thus, engineered cells with two incomplete CAR units are only fully activated when both target antigens bind to the antigen recognition domains. This strategy provides added specificity in that the engineered cells are not fully activated until the target binds to the antigen recognition domain of each incomplete CAR unit.

[0109] In a further embodiment, the engineered cells have two incomplete CAR units, and one of the antigen recognition domains may be specific for and capable of binding to biotin, HIS, MYC, HA, agarose, V5, maltose, GST, GFP, CD52, CD20, 4-1BB, or CD28.

[0110] As used herein, compound CAR (cCAR) or multiple CAR refers to an engineered cell having at least two different chimeric antigen receptor polypeptides. As used herein, "different chimeric antigen receptor polypeptides" have unique antigen-recognition domains, signal peptides, hinge regions, transmembrane domains, at least one costimulatory domain, and signaling domains. Thus, two unique chimeric antigen receptor polypeptides will have different antigen-recognition domains. The signal peptides, hinge regions, transmembrane domains, at least one costimulatory domain, and signaling domains may be the same or different between two different chimeric antigen receptor polypeptides. As used herein, chimeric antigen receptor (CAR) units refer to different chimeric antigen receptor polypeptides or polynucleotides encoding them.

[0111] As used herein, a unique antigen recognition domain is also one that is target-specific or targets a single target or a single epitope of a target.

[0112] In some embodiments, compound CARs target the same antigen. For example, cCARs target different epitopes or portions of a single antigen. In some embodiments, each CAR unit present in a compound CAR targets a different antigen in the same or different disease state, or a different antigen specific to a side effect caused by a disease state.

[0113] In some embodiments, the compound CAR targets two different antigens.

[0114] The creation of compound CARs with various CAR units is very challenging: (1) CAR-CAR interactions may have deleterious effects, and appropriate CAR design is key to offsetting these adverse effects; (2) a single-component compound CAR may increase the length of the expression cassette, which may result in reduced viral titers and protein expression levels; (3) to select a strategy for expressing multiple CARs in a single vector, appropriate design including various CAR body elements is necessary; (4) a strong promoter is particularly important for compound CARs with additional CAR units; (5) the hinge region of the CAR needs to be designed so that interactions between the hinge regions of each CAR unit are preferably avoided; and (6) expressing two or more CAR units in a cell may cause toxic effects (CAR-CAR interactions). The applicant provides novel and surprising CAR compositions and methods that overcome these hurdles.

[0115] In this example, the present disclosure provides engineered cells with multiple CAR units, allowing a single engineered cell to target multiple antigens. Simultaneous targeting of multiple surface markers or antigens with multiple CAR units prevents the selection of resistant clones and reduces tumor recurrence. Multiple CAR T cell immunotherapy using individual component CARs containing different domains and activation sites has not yet been deployed for any malignant tumor.

[0116] In one aspect of the invention, a cCAR comprises multiple CAR units. In some embodiments, a cCAR comprises at least two CAR units. In other embodiments, a cCAR comprises at least three CAR units. In other embodiments, a cCAR comprises at least four units.

[0117] In one embodiment, the present disclosure provides an engineered cell having at least two distinct chimeric antigen receptor polypeptides, each having a different antigen recognition domain.

[0118] In this example, the engineered cells bearing at least two different chimeric antigen receptor polypeptides are T cells. The T cells can be engineered not to express a cell surface antigen. For example, the T cells can be engineered not to express the CD45 cell surface antigen.

[0119] In a preferred embodiment, the engineered cells comprising at least two different chimeric antigen receptor polypeptides are primary NK cells isolated from peripheral blood or umbilical cord blood, and are NK-92 cells, and are administered "off the shelf" to any mammal with disease or cancer.

[0120] In this example, the engineered cell comprises (i) a first chimeric antigen receptor polypeptide comprising a first antigen recognition domain, a first signal peptide, a first hinge region, a first transmembrane domain, a first costimulatory domain, and a first signaling domain, and (ii) a second chimeric antigen receptor polypeptide comprising a second antigen recognition domain, a second signal peptide, a second hinge region, a second transmembrane domain, a second costimulatory domain, and a second signaling domain, wherein the first antigen recognition domain is different from the second antigen recognition domain.

[0121] In a preferred embodiment, each engineered CAR unit polynucleotide has a different nucleotide sequence to avoid homologous recombination.

[0122] In this example, the target of the first antigen recognition domain is ROR1, PSMA, PSCA (prostate stem cell antigen), MAGE A3, glycolipids, glypican 3, F77, GD-2, WT1, CEA, HER-2 / neu, IL13Rα2, Met, mesothelin, EGFR, EGFRvIII, MUC16, NKG2D ligand, thyroglobulin, MAGE-3, MAGE-4, MAGE-5, MAGE-6, alpha-fetoprotein, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART- 1, MUC1, MUC2, MUC3, MUC4, MUC5, CD30, EGFRvIII, CD33, CD123, CLL-1, immunoglobulin kappa and lambda, CD38, CD52, CD19, CD20, CD22, CD38, BCMA, CS1, BAFF receptor, TACI, CD3, CD4, CD8, CD5, CD7, CD2, CD45, CD70, CD138, interleukin-6 receptor, NY-ESO-1, alpha-fetoprotein (AFP), glypican-3 (GPC3), BAFF-R, BCMA, TACI, LeY, CD4, CD5, CD13, CD14, CD15 The target of the second recognition domain is selected from the group consisting of CD19, CD20, CD22, CD33, CD41, CD61, CD64, CD68, CD117, CD123, CD138, CD267, CD269, CD38, Flt3 receptor, CLL-1, and CS1, and the target of the second recognition domain is selected from the group consisting of ROR1, PSMA, PSCA (prostate stem cell antigen), MAGE A3, glycolipids, glypican 3, F77, GD-2, WT1, CEA, HER-2 / neu, IL13Rα2, Met, mesothelin, EGFR, EGFRvIII, MUC16, NKG2D ligand, thyroglobulin, MAGE-3, MAGE-4, MAGE-5, MAGE-6, alpha-fetoprotein, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, MUC1, MUC2, MUC3, MUC4, MUC5, CD30, EGFRvIII, CD33, CD123, CLL-1, immunoglobulin kappa and lambda, CD38, CD52, CD19, CD20, CD22, CD38, BCMA, CS1, BAFF receptor, TACI, CD3, CD4, CD8, CD5, CD7, CD2, CD45, CD70 and CD138.

[0123] In this example, the engineered cell contains a first chimeric antigen receptor polypeptide having a CD19 antigen recognition domain and a second chimeric antigen receptor polypeptide having a CD20 recognition domain. In this example, the engineered cell contains the polypeptide of SEQ ID NO:3 and a polynucleotide corresponding to SEQ ID NO:4.

[0124] In this example, the engineered cell comprises a first chimeric antigen receptor polypeptide having a CD19 antigen recognition domain and a second chimeric antigen receptor polypeptide having a CD22 antigen recognition domain. In this example, the engineered cell comprises the polypeptide of SEQ ID NO:5 and a polynucleotide corresponding to SEQ ID NO:6.

[0125] In this example, the engineered cell comprises a first chimeric antigen receptor polypeptide having a CD19 antigen recognition domain and a second chimeric antigen receptor polypeptide having a CD123 antigen recognition domain. In this example, the engineered cell comprises the polypeptide of SEQ ID NO:7 and a polynucleotide corresponding to SEQ ID NO:8.

[0126] In one embodiment, the engineered cell comprises a first chimeric antigen receptor polypeptide having a BCMA antigen recognition domain and a second chimeric antigen receptor polypeptide having a CD19 antigen recognition domain.

[0127] In one embodiment, the engineered cell comprises a first chimeric antigen receptor polypeptide having a BAFFR antigen recognition domain and a second chimeric antigen receptor polypeptide having a CD19 antigen recognition domain.

[0128] In one embodiment, the engineered cell comprises a first chimeric antigen receptor polypeptide having a BCMA antigen recognition domain and a second chimeric antigen receptor polypeptide having a CS1 antigen recognition domain.

[0129] In one embodiment, the engineered cell comprises a first chimeric antigen receptor polypeptide having a CD33 antigen recognition domain and a second chimeric antigen receptor polypeptide having a CLL-1 antigen recognition domain.

[0130] In one embodiment, the engineered cell comprises a first chimeric antigen receptor polypeptide having a CD4 antigen recognition domain and a second chimeric antigen receptor polypeptide having a CLL-1 antigen recognition domain.

[0131] In one embodiment, the engineered cell comprises a first chimeric antigen receptor polypeptide having a CD4 antigen recognition domain and a second chimeric antigen receptor polypeptide having a CD123 antigen recognition domain.

[0132] In one embodiment, the engineered cell comprises a first chimeric antigen receptor polypeptide having a CD19 antigen recognition domain and a second chimeric antigen receptor polypeptide having a CS-1 antigen recognition domain.

[0133] In this example, the engineered cell comprises a first chimeric antigen receptor polypeptide having a CD33 antigen recognition domain and a second chimeric antigen receptor polypeptide having a CD123 antigen recognition domain. In this example, the engineered cell comprises the polypeptide of SEQ ID NO:9 and a polynucleotide corresponding to SEQ ID NO:10. In another example, the engineered cell comprises the polypeptide of SEQ ID NO:11 and a polynucleotide corresponding to SEQ ID NO:12.

[0134] In this example, the engineered cells contain a first chimeric antigen receptor polypeptide having a BAFF-R antigen recognition domain and a second chimeric antigen receptor polypeptide having a CS1 antigen recognition domain.

[0135] In this example, the engineered cell comprises a first chimeric antigen receptor polypeptide having a CD269 antigen recognition domain and a second chimeric antigen receptor polypeptide having a CS1 antigen recognition domain. In this example, the engineered cell comprises the polypeptide of SEQ ID NO: 19 and a polynucleotide corresponding to SEQ ID NO: 20. In this example, the engineered cell comprises the polypeptide of SEQ ID NO: 21 and a polynucleotide corresponding to SEQ ID NO: 22.

[0136] In this example, the engineered cells comprise a first chimeric antigen receptor polypeptide having a CD33 antigen recognition domain and a second chimeric antigen receptor polypeptide having a CD123 antigen recognition domain.

[0137] In this example, each CAR unit comprises the same or a different hinge region. In another example, each CAR unit comprises the same or a different transmembrane region. In another example, each CAR unit comprises the same or a different intracellular domain.

[0138] In this example, each CAR unit contains a CD3 zeta chain signaling domain

[0139] In this example, each separate CAR unit contains a different costimulatory domain to avoid interactions, e.g., the first chimeric antigen receptor polypeptide contains a 4-BB costimulatory domain and the second chimeric antigen receptor polypeptide contains a CD28 costimulatory domain.

[0140] In another example, the hinge region is designed to eliminate amino acids that may cause undesired intra- or intermolecular interactions. For example, the hinge region is designed to eliminate or minimize cysteine ​​residues to prevent disulfide bond formation. In another example, the hinge region is designed to eliminate or minimize hydrophobic residues to prevent undesired hydrophobic interactions.

[0141] Compound CARs can kill targets either alone or in combination. Multiple or compound CARs contain the same or different hinge regions, the same or different transmembrane domains, the same or different costimulatory domains, and the same or different intracellular domains. Preferably, the hinge regions are selected to avoid interaction sites.

[0142] The compound CARs of the present invention can target the same or different tumor populations in T cells or NK cells. For example, one CAR may target a bulk tumor population, and a subsequent or second CAR may eradicate cancer or leukemia stem cells, for example, to prevent cancer recurrence.

[0143] According to the present invention, it has surprisingly been found that compound CARs in T or NK cells targeting different or the same tumor populations combat tumor factors that cause cancer cells to be resistant to CAR killing activity, thereby downregulating target antigens from the cancer cell surface. Surprisingly, it has also been found that this allows cancer cells to "hide" from CAR therapy due to "antigen escape" and tumor heterogeneity, where different tumor cells exhibit different surface antigen expression profiles.

[0144] Engineered cells with a CAR polypeptide and an enhancer In another example, the present disclosure provides an engineered cell comprising at least one chimeric antigen receptor polypeptide and an enhancer.

[0145] In this example, the disclosure provides engineered cells having at least two different chimeric antigen receptor polypeptides and an enhancer.

[0146] As used herein, an enhancer includes a biological molecule that promotes or enhances the activity of engineered cells harboring a chimeric antigen receptor polypeptide. Enhancers also include cytokines. In another example, enhancers include IL-2, IL-7, IL-10, IL-12, IL-15, IL-18, IL-21, PD-1, PD-L1, CSF1R, CTAL-4, TIM-3, and TGFR-beta, its receptors, and functional fragments.

[0147] The enhancer can be expressed by the engineered cells described herein and displayed on the surface of the engineered cells, or the enhancer can be secreted by the engineered cells into the surrounding extracellular space. Methods for surface display and secretion are well known in the art. For example, the enhancer can be a fusion protein with a peptide that provides surface display or secretion into the extracellular space.

[0148] The effect of an enhancer may be complemented by additional factors, such as enhancer receptors and functional fragments thereof, which may be co-expressed with the enhancer as a fusion protein or expressed as separate peptides and secreted into the extracellular space.

[0149] Enhancers are cytokines secreted by engineered CAR cells and are designed to be co-expressed with the CAR polypeptide. Mass release occurs upon CAR engagement with the cognate antigen. Inflammatory cells surrounding tumor cells have a significant correlation with cancer cell progression and metastasis. Inflammatory cells include innate immune response cells such as T cells, NK cells, macrophages, and dendritic cells, whose proliferation and antitumor activity are regulated by cytokines. CAR cells, such as CAR T cells or NK cells, bind to target cancer cells and induce the massive secretion of enhancers from the proliferating CAR T / NK cells. The secreted enhancers efficiently promote the survival, differentiation, and activation of immune response cells against cancer cells. Co-expression of enhancers with CARs can compensate for the inability of CAR T or NK cells to eliminate non-target cancer cells (Figure 78).

[0150] CAR cells are cytokine carriers, and cytokines can be delivered to target cancer sites by CAR cells to reduce systemic toxicity caused by high doses of exogenous cytokines (Figure 78).

[0151] To improve the sustained survival or long-term persistence of CAR cells, a membrane-bound enhancer can be co-expressed with the CAR to improve CAR persistence.

[0152] In this example, the enhancer is IL-15. In this case, the additional factor is the IL-15 receptor and its functional fragments. Functional fragments include the IL-15 receptor, IL-15RA, and the sushi domain of IL-15RA (IL-15sushi). Soluble IL-15RA or IL15sushi significantly enhances IL-15 functional activity by preventing IL-15 degradation. Soluble IL-15 / IL-15RA or IL-15 / IL-15sushi complexes are more stable and much more stimulatory than IL-15 alone in vivo.

[0153] In this example, IL-15 is co-expressed as a fusion protein with at least one of the IL-15 receptor, IL-15RA, and the sushi domain of IL-15RA (IL-15sushi). In this example, the IL-15 receptor, IL-15RA, or IL-15RA sushi domain (IL-15sushi) is at the N-terminus of IL-15. In another example, the IL-15 receptor, IL-15RA, or IL-15RA sushi domain (IL-15sushi) is at the C-terminus of IL-15. As used herein, IL-15 / IL-15sushi indicates that IL-15sushi is at the C-terminus of IL-15 in the fusion protein, and IL-15sushi / il-15 indicates that IL-15sushi is at the N-terminus of IL-15 in the fusion protein.

[0154] In some embodiments, IL-15 can be fused to the soluble domain of IL-15Rα(sushi) to form a heterodimeric complex (IL-15 / IL-15sushi) that is stable in solution and exhibits increased biological activity compared to the uncomplexed IL-15.

[0155] In some embodiments, IL-15 is the IL-15N72D mutant, which is fused to the soluble domain of IL-15Rα(sushi) to form a stable complex in solution, which has increased biological activity compared to uncomplexed IL-15. The mutant IL-15N72D can enhance the biological activity of IL-15 (US20120177595 A1).

[0156] In some embodiments, the IL-15 and IL-15 receptor or functional fragment thereof polypeptides are on a single polypeptide molecule and are separated by a peptide linker, the peptide linker being 1-25 amino acid residues in length, 25-100 amino acid residues in length, or 50-200 amino acid residues in length, which linker may include a high-efficiency cleavage site as described herein.

[0157] An example of a suitable sushi domain includes the CAR construct, SEQ ID NO: 1. In accordance with the present disclosure, any of the chimeric antigen receptor polypeptides disclosed herein can be co-expressed with human interleukin-15, SEQ ID NO: 2, with the human interleukin-2 signal peptide.

[0158] Interleukin (IL)-15 and its specific receptor chain, IL-15Rα (IL-15-RA), play important functional roles in various effector cells, including NK and CD8+ T cells. CD8+ T cells can be engineered to express autocrine growth factors, including, but not limited to, IL-2, IL-7, IL-21, or IL-15, to maintain survival after in vivo transfer. Without wishing to be bound by theory, it is believed that IL-15 can overcome CD4 deficiency to induce primary and memory CD8+ T cells. Overexpression of IL-15-RA or IL-15 IL-RA fusions on CD8+ T cells significantly enhances their survival and proliferation in vitro and in vivo. In some embodiments, the CD4 CAR or any CAR co-expresses at least one of IL-15, IL15RA, and IL-15 / IL-15R or IL15-RA / IL-15 or IL-15 / IL-15 sushi, or a portion thereof, or a combination thereof, to enhance CAR T or NK survival or proliferation and improve memory CAR CD8+ T cell proliferation.

[0159] The present disclosure provides CAR polypeptides as described herein and engineered cells having at least one of IL-15, IL-15RA, IL-15sushi, IL-15 / IL-15RA, IL15-RA / IL-15, IL-15 / IL-15sushi, IL15sushi / IL-15, fragments thereof, combinations thereof that enhance survival or persistence or proliferation of CAR T or NK to treat cancer in a patient.

[0160] In another example, the disclosure provides a method for the treatment of IL-15, IL-15RA, IL-15sushi, IL-15 / IL-15RA, IL15-RA / IL-15, IL-15 / IL-15sushi, IL15sushi / IL-15, functional fragments thereof, and combinations thereof, and at least one separate CAR polypeptide, wherein the antigen recognition domain is selected from the group consisting of NY-ESO-1, alpha fetoprotein (AFP), glypican-3 (GPC3), BCMA, BAFF-R, BCMA, TACI, LeY, CD5, CD7, CD2, CD3, CD14, CD15, CD19, CD20, CD22, CD33, CD41, CD61, CD64, CD68, CD117, CD123, CD138, CD267, CD269, CD38, Flt3 receptor, ROR1, PSMA, MAGE A3, Engineered cells are provided that contain at least one of glycolipids, F77, GD-2, WT1, CEA, HER-2 / neu, MAGE-3, MAGE-4, MAGE-5, MAGE-6, CA19-9, CA72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, CD30, EGFRvIII, immunoglobulin kappa and lambda, CD38, and CS1. Target antigens can also include viral or fungal antigens, such as E6 and E7 from human papillomavirus (HPV) or EBV (Epstein-Barr virus) antigens. In further embodiments, antigen-recognizing polypeptides (scFvs) and polynucleotides corresponding to CD2, CD3, CD5, CD7, and CD52 are described in PCT Application No. PCT / US2016 / 39306, the contents of which are incorporated herein by reference.

[0161] Enhancer of CAR function IL-15 / IL15sushi enhancer In one embodiment, a CAR construct containing an IL-15 / IL15sushi enhancer is shown in Figure 94. The CAR contains a secreted IL-15 / IL-15sushi complex. The IL-15 / IL-15sushi-containing CAR is linked to a P2A self-cleaving sequence. The IL-15 / IL-15sushi portion consists of an IL-2 signal peptide fused to IL-15 and linked to the sushi domain of the IL-15 alpha receptor via an amino acid linker. The linker length can vary. In one embodiment, the linker is 1-20 amino acids in length, and in another embodiment, the linker is 20-40 amino acids in length. For example, the linker can be a 26-amino acid polyproline linker. The CAR contains an scFv, a costimulatory domain (including, but not limited to, CD28 or 4-1BB), and an intracellular signaling domain, CD3 zeta chain. The IL-15 signal peptide in IL-15 is replaced with the IL-2 signal peptide (leader sequence), a strong signal peptide that provides high efficiency of IL-15 / IL-15 sucrose secretion.

[0162] The IL-15 may be a variant of IL-15N72D, as described, among others, in US8507222, the contents of which are incorporated herein by reference.

[0163] Potential drawbacks to using NK cells in CAR therapy include a lack of persistence, which may reduce long-term efficacy.

[0164] Finding matching donor T cells to generate CAR T cells is challenging because mismatched T cells can cause graft-versus-host disease (GVHD) if they attach to the recipient's tissues.

[0165] In one embodiment, the present disclosure includes a method for generating chimeric antigen receptor (CAR)-modified NK cells with long lifespan or long-term persistence in vivo for treating disease. Surprisingly, it has been found that CAR NK cells co-expressing IL-15 / IL-15sushi can extend long-term survival.

[0166] Surprisingly, we found that CAR NK cells co-expressing IL-15 / IL-15sushi could extend survival by at least 4 weeks, at least 2 months, or at least 3 months.

[0167] In a further embodiment, extended CAR NK cell survival can be achieved by co-expressing IL-15 / IL-15.

[0168] In some embodiments, IL-15 / IL-15sushi co-expressing CAR NK cells can be scaled up and used off-the-shelf. In one embodiment, IL-15 / IL-15sushi co-expressing CAR NK cells can continue to provide supportive cytokine signaling, which is important for survival after infusion in patients.

[0169] In certain embodiments, NK cells may be derived from human peripheral blood mononuclear cells (PBMCs), leukopheresis fraction (PBSCs), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, or umbilical cord blood.

[0170] In one embodiment, the present disclosure provides a related method in which NK cells targeting tumor cells can act as a carrier for delivering an enhancer, IL-15 / IL-15 sushi, to the tumor microenvironment. The CAR NK cells are engineered to co-express secreted IL-15 / IL-15 sushi. The engineered CAR NK cells in the tumor microenvironment target tumor cells, bind to the CAR target antigen, and induce tumor cell lysis and the proliferation of CAR NK cells, resulting in the secretion of large amounts of soluble IL-15 / IL-15 sushi. The secretion of IL-15 / IL-15 sushi can further extend the survival of the CAR NK cells.

[0171] In certain embodiments, tumor removal can be achieved by a combination of at least one or more of the following steps: (1) Binding of the CAR-engineered NK cells disclosed herein to a portion of tumor cells by targeting the CAR antigen. (2) The proliferation of CAR NK cells co-expressing this molecule triggers the massive secretion of IL-15 / IL-15 sushi. (3) Recruitment and stimulation of various innate and adaptive immune cells against tumors. (4) secreting IL-15 / IL-15 extends the survival or persistence of CAR NK cells for at least 4 weeks, at least 2 months, or at least 3 months or 1 year. (5) Administration of checkpoint blockade, such as PD-L1 and CTLA-4 inhibitors, reduces tumor suppressors present in tumors.

[0172] IL-15 / IL15sushi anchor enhancer In one embodiment, a CAR construct with an IL-15 / IL-15sushi anchor is shown in Figure 95. The CAR / IL-15 / IL-15sushi anchor construct consists of an SFFV promoter driving expression of a CAR and an IL-15 / IL-15sushi anchor (also referred to as the anchor) linked by a P2A peptide. Upon cleavage of this P2A peptide, the IL-15 / IL-15 anchor CAR separates into the CAR and the IL-15 / IL-15sushi anchor. The IL-15 / IL-15sushi portion of the anchor consists of an IL-2 signal peptide fused to IL-15 and linked to the sushi domain of the IL-15α receptor via an amino acid linker. The length of the linker can vary. In one embodiment, the linker is 1-20 amino acids in length; in another embodiment, the linker is 20-40 amino acids in length. For example, the linker can be a 26-amino acid polyproline linker. Both the CAR and the anchor comprise a hinge (H) region and a transmembrane (TM) domain. The CAR also contains an scFv, a costimulatory domain (including, but not limited to, CD28 or 4-1BB), and an intracellular signaling domain, CD3 zeta chain, while the anchor lacks these components. The IL-15 / IL-15 sushi anchor provides synergistic effects with CD28 or 4-1BB for T cell activation or antitumor activity. The inclusion of the IL-15 / IL-15 sushi anchor makes the CAR more potent.

[0173] The IL-15 may be a variant of IL-15N72D, as described, among others, in US8507222, the contents of which are incorporated herein by reference.

[0174] In one embodiment, the present disclosure includes a method for generating chimeric antigen receptor (CAR)-modified NK cells with long lifespan or long-term persistence in vivo for treating disease. Surprisingly, it has been found that CAR NK cells co-expressing the IL-15 / IL-15sushi anchor can extend long-term survival.

[0175] Surprisingly, we found that CAR NK cells co-expressing the IL-15 / IL-15sushi anchor could extend survival by at least 4 weeks, at least 2 months, or at least 3 months.

[0176] In a further embodiment, extended survival of CAR NK cells can be achieved by co-expressing an IL-15 / IL-15 anchor.

[0177] In some embodiments, CAR NK cells co-expressing the IL-15 / IL-15sushi anchor can be scaled up and used off-the-shelf.

[0178] In one embodiment, CAR NK cells co-expressing the IL-15 / IL-15sushi anchor are able to maintain supportive cytokine signaling, which is important for survival after infusion in patients.

[0179] In certain embodiments, NK cells may be derived from human peripheral blood mononuclear cells (PBMCs), leukopheresis fraction (PBSCs), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, or umbilical cord blood.

[0180] 4-1BBL enhancer In another embodiment, a CAR construct with a 4-1BBL enhancer is shown in Figure 96A. The CAR 4-1BBL construct consists of a CAR, an enhancer linked by a P2A peptide, and an SFFV promoter driving the expression of 4-1BBL (CD137L). When the P2A peptide is cleaved, the CAR construct containing 4-1BBL separates into the CAR polypeptide and the full-length 4-1BBL protein. The CAR contains a leader sequence, scFv, hinge (H) region, and transmembrane domain (TM). CARs also contain costimulatory domains (including, but not limited to, CD28 or 4-1BB) and intracellular signaling domains, such as the CD3 zeta chain, whereas 4-1BBL lacks these components. 4-1BBL provides synergistic effects with CD28 or 4-1BB on T cell activation or antitumor activity. Loading 4-1BBL makes the CAR more potent.

[0181] IL-15 enhancer CAR function can be enhanced by incorporating the secretion enhancer IL-15 shown in Figure 96B. The CAR 4-IL-15 construct consists of an SFFV promoter driving the expression of the CAR and enhancer IL-15 linked by a P2A peptide. Upon cleavage of this P2A peptide, the IL-15-containing CAR construct separates into the CAR polypeptide and the full-length IL-15 protein. The CAR contains a leader sequence, scFv, hinge (H) region, and transmembrane domain (TM). CARs also contain costimulatory domains (including, but not limited to, CD28 or 4-1BB) and intracellular signaling domains, such as the CD3 zeta chain, whereas IL-15 lacks these components. IL-15 secretion synergizes with CD28 or 4-1BB for T cell activation or antitumor activity. CARs are more potent when they secrete IL-15. The IL-15 signal peptide of IL-15 was replaced with the IL-2 signal peptide (leader sequence), a strong signal peptide that provides high efficiency of IL-15 secretion.

[0182] In some embodiments, the engineered cells comprise at least one enhancer. In such embodiments, the CAR polypeptide and enhancer are expressed on a single polypeptide molecule with two high-efficiency cleavage sites. In one embodiment, the two high-efficiency cleavage sites are different. In another embodiment, the high-efficiency cleavage sites are the same. In one embodiment, the CAR polypeptide is expressed with the 4-1BBL and IL-15 / IL-sushi enhancers on a single polypeptide molecule, and P2A and T2A high-efficiency cleavage sites are used. An example of such an embodiment is shown in Figure 98F.

[0183] In one embodiment, the present disclosure includes a method for generating chimeric antigen receptor (CAR)-modified NK cells with long life or long-term persistence in vivo for treating disease. Surprisingly, it has been found that CAR NK cells that co-express IL-15 can extend long-term survival.

[0184] Surprisingly, we found that CAR NK cells co-expressing IL-15 could extend survival by 4 weeks, 2 months, or 3 months.

[0185] In a further embodiment, extended CAR NK cell survival can be achieved by co-expressing IL-15.

[0186] In some embodiments, IL-15 co-expressing CAR NK cells can be scaled up and used off-the-shelf.

[0187] In certain embodiments, NK cells may be derived from human peripheral blood mononuclear cells (PBMCs), leukopheresis fraction (PBSCs), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, or umbilical cord blood.

[0188] Without wishing to be bound by theory, it is believed that IL-15 / IL-15sushi and other types of IL-15 or IL-15RA proteins or protein fragments thereof may provide a synergistic effect of CAR polypeptides when combined with checkpoint inhibitors or modulators (e.g., anti-PD-1).

[0189] In one example, the disclosure provides CD4 CAR-engineered cells comprising IL-15 / IL-15sushi (SEQ ID NO: 1) and a corresponding polynucleotide (SEQ ID NO: 2). In one example, the disclosure provides a method of providing long-term durable remission in a cancer patient by administering CD4 CAR-engineered cells comprising IL-15 / IL-15sushi to a patient in need thereof. Without being bound by theory, it is believed that co-expression of IL-15 / IL-15sushi with a CD4 CAR polypeptide results in long-term durable remission in the patient by increasing the sensitivity of target cancer cells to CAR recognition or by recruiting innate immune cells against the cancer cells.

[0190] In this example, the disclosure provides engineered cells having a CD45 chimeric antigen receptor polypeptide and IL-15 / IL-15sushi (SEQ ID NO: 44) and the corresponding nucleotide sequence (SEQ ID NO: 43).

[0191] In this example, the present disclosure provides a method for providing long-term durable remission in cancer patients by administering CD45 CAR-engineered cells containing IL-15 / IL-15sushi to a patient in need thereof. Without being bound by theory, it is believed that co-expression of IL-15 / IL-15sushi and a CD45 CAR polypeptide results in long-term durable remission in patients by increasing the sensitivity of target cancer cells to CAR recognition or by recruiting innate immune cells against the cancer cells.

[0192] In this example, the engineered cells contain a CD45 chimeric antigen receptor polypeptide and 4-1BBL (SEQ ID NO: 74) and the corresponding nucleotide sequence (SEQ ID NO: 73).

[0193] In this example, the present disclosure provides a method of providing long-term durable remission to a patient suffering from cancer by administering to a patient in need thereof CD45 CAR-engineered cells that co-express 4-1BBL. Without being bound by theory, it is believed that co-expression of 4-1BBL and CD45CAR results in long-term durable remission in the patient by increasing the persistence of the CAR-engineered cells.

[0194] In this example, the engineered cells comprise a CD19 chimeric antigen receptor polypeptide and IL-15 / IL-15sushi (SEQ ID NO: 59) and the corresponding polynucleotide (SEQ ID NO: 60). In this example, the present disclosure provides a method of providing long-term durable remission in cancer patients by administering CD19 CAR-engineered cells containing IL-15 / IL-15sushi to a patient in need thereof. Without being bound by theory, it is believed that co-expression of IL-15 / IL-15sushi with a CD19 CAR results in long-term durable remission in patients by either increasing the sensitivity of target cancer cells to CAR recognition or by recruiting innate immune cells against the cancer cells.

[0195] In this example, the engineered cells comprise a CD20 chimeric antigen receptor polypeptide and IL-15 / IL-15sushi (SEQ ID NO: 58) and the corresponding polynucleotide (SEQ ID NO: 57). In this example, the present disclosure provides a method of providing long-term durable remission in cancer patients by administering CD20 CAR-engineered cells containing IL-15 / IL-15sushi to a patient in need thereof. Without being bound by theory, it is believed that co-expression of IL-15 / IL-15sushi with a CD20 CAR results in long-term durable remission in patients by sensitizing target cancer cells to CAR recognition or by recruiting innate immune cells against the cancer cells.

[0196] In this example, the engineered cells comprise a CD22 chimeric antigen receptor polypeptide and IL-15 / IL-15sushi (SEQ ID NO: 62) and the corresponding polynucleotide (SEQ ID NO: 61). In this example, the present disclosure provides a method of providing long-term durable remission in cancer patients by administering CD22 CAR-engineered cells containing IL-15 / IL-15sushi to a patient in need thereof. Without being bound by theory, it is believed that co-expression of IL-15 / IL-15sushi with a CD22 CAR results in long-term durable remission in patients by either increasing the sensitivity of target cancer cells to CAR recognition or by recruiting innate immune cells against the cancer cells.

[0197] In this embodiment, the engineered cells comprise a CD269 chimeric antigen receptor polypeptide and IL-15 / IL-15sushi (SEQ ID NO: 44) and corresponding polynucleotide (SEQ ID NO: 45). In this example, the disclosure provides a method of providing long-term durable remission in cancer patients by administering CD269 CAR-engineered cells containing IL-15 / IL-15sushi to a patient in need thereof. Without being bound by theory, it is believed that co-expression of IL-15 / IL-15sushi with a CD269 CAR results in long-term durable remission in patients by sensitizing target cancer cells for CAR recognition or by recruiting innate immune cells against cancer cells, such as CD269(BCMA)dim-positive plasma cells or myeloma cells.

[0198] In this example, the engineered cells contain the CD4 polypeptide of SEQ ID NO:90 and the corresponding polynucleotide of SEQ ID NO:89.

[0199] In this example, the engineered cells comprise a CD4 chimeric antigen receptor polypeptide and IL-15 / IL-15sushi (SEQ ID NO: 96) and the corresponding polynucleotide (SEQ ID NO: 95). In this example, the present disclosure provides a method for providing long-term durable remission in cancer patients by administering CD4 CAR-engineered cells containing IL-15 / IL-15sushi to a patient in need thereof. Without being bound by theory, it is believed that co-expression of IL-15 / IL-15sushi with a CD4 CAR results in long-term durable remission in patients by either increasing the sensitivity of target cancer cells to CAR recognition or by recruiting innate immune cells against the cancer cells.

[0200] In this example, the engineered cells comprise a CD4 chimeric antigen receptor polypeptide and IL-15 / IL-15RA (membrane-bound) (SEQ ID NO: 98) and the corresponding polynucleotide (SEQ ID NO: 97). In this example, the present disclosure provides a method for providing long-term durable remission in cancer patients by administering CD4 CAR-engineered cells containing IL-15 / IL-15RA to a patient in need thereof. A single construct contains both a CD4CAR and an IL-15 / IL-15 sushi anchor linked by a P2A cleavage peptide. The IL-15 / IL-15 sushi anchor has the IL-2 signal peptide IL-15 bound to a hinge domain (CD8a) and a transmembrane domain (IL-15RA) following the sushi domain. Without wishing to be bound by theory, it is believed that co-expression of IL-15 / IL-15RA (membrane-bound) with a CD4 CAR results in long-term durable remission in patients by increasing the persistence of the CAR-engineered cells.

[0201] In this example, the engineered cells comprise a compound CAR, a CD33CD123 polypeptide, and IL-15 / IL-15sushi (SEQ ID NO: 40) and a corresponding polynucleotide (SEQ ID NO: 39). In this example, the disclosure provides a method of providing long-term durable remission in cancer patients by administering CD33CD123 compound CAR-engineered cells containing IL-15 / IL-15sushi to a patient in need thereof. Without being bound by theory, it is believed that co-expression of IL-15 / IL-15sushi with a CD33CD123 CAR results in long-term durable remission in patients by either sensitizing the target cancer cells to CAR recognition or by recruiting innate immune cells against the cancer cells.

[0202] In this example, the engineered cells comprise a compound CAR, a CD33CD123 polypeptide, and 4-1BBL (SEQ ID NO: 38) and a corresponding polynucleotide (SEQ ID NO: 37). In this example, the disclosure provides a method of providing long-term durable remission in cancer patients by administering CD33CD123 compound CAR-modified cells that co-express 4-1BBL to a patient in need thereof. Without wishing to be bound by theory, it is believed that co-expression of 4-1BBL with CD33CD123cCAR increases the persistence of cCAR-engineered cells, thereby resulting in long-term sustainable remission in patients.

[0203] In this example, the engineered cells contain a BAFF CAR polypeptide having a CD45 leader sequence (SEQ ID NO: 78) and the corresponding polynucleotide sequence (SEQ ID NO: 77).

[0204] In this example, the engineered cells contain a BAFF CAR polypeptide having a CD8a leader sequence (comprising SEQ ID NO:80) and the corresponding polynucleotide sequence (SEQ ID NO:79).

[0205] In this example, the engineered cells contain a BAFF CAR polypeptide and IL-15 / IL-15sushi (SEQ ID NO: 84) and the corresponding polynucleotide (SEQ ID NO: 83).

[0206] In this example, the present disclosure provides a method for providing long-term durable remission in cancer patients by administering BAFF CAR-engineered cells containing IL-15 / IL-15sushi to a patient in need thereof. Without being bound by theory, it is believed that co-expression of IL-15 / IL-15sushi with BAFF CAR results in long-term durable remission in patients by either increasing the sensitivity of target cancer cells to CAR recognition or by recruiting innate immune cells against cancer cells, such as plasma cells or myeloma cells that weakly express the BAFF receptor, CD269 (BCMA).

[0207] In this example, the engineered cells comprise a BAFF CAR polypeptide and 4-1BBL (SEQ ID NO: 82) and the corresponding polynucleotide (SEQ ID NO: 81). In this example, the disclosure provides a method of providing long-term durable remission in a cancer patient by administering BAFF CAR-engineered cells co-expressing 4-1BBL to a patient in need thereof. Without wishing to be bound by theory, it is believed that co-expression of 4-1BBL with BAFF CAR can provide long-term durable remission in patients by increasing the persistence of the CAR-engineered cells.

[0208] In this example, the engineered cells contain the compound CAR, the BAFF CD19b polypeptide of SEQ ID NO:86 and the corresponding polynucleotide of SEQ ID NO:85.

[0209] In this example, the present disclosure provides a method of treating an autoimmune disorder in a patient by administering BAFF CD19b compound CAR-engineered cells to a patient in need thereof. Without wishing to be bound by theory, it is believed that BAFF CD19b compound CAR-engineered cells provide better therapeutic outcomes for B cell and plasma cell depletion associated with autoimmune disease.

[0210] In this example, the engineered cells comprise the APRIL CD19b compound CAR polypeptide of SEQ ID NO:88 and the corresponding polynucleotide of SEQ ID NO:77.

[0211] In this example, the present disclosure provides a method for depleting B cells and plasma cells in a patient in need thereof by administering APRIL CD19b compound CAR-engineered cells to the patient in need thereof. Without wishing to be bound by theory, it is believed that APRIL CD19b compound CAR-engineered cells can provide better therapeutic outcomes by depleting B cells and plasma cells associated with autoimmune diseases.

[0212] In this example, the engineered cells comprise a compound CAR, a CD269 CS1 polypeptide of SEQ ID NO: 48, and a corresponding polynucleotide of SEQ ID NO: 47. In this example, the disclosure provides a method of treating myeloma in a patient in need thereof by administering CD269CS1 compound CAR engineered cells to the patient.

[0213] Without wishing to be bound by theory, it is believed that CD269 CS1 compound CAR-modified cells provide better treatment outcomes for myeloma patients and prevent antigen escape or disease relapse.

[0214] In this example, the engineered cells comprise the compound CAR, the CD269 CD19b polypeptide of SEQ ID NO:50 and the corresponding polynucleotide of SEQ ID NO:49.

[0215] In this example, the present disclosure provides a method for depleting B cells and plasma cells in a patient in need thereof by administering CD269 CD19b compound CAR-engineered cells to the patient. Without wishing to be bound by theory, it is believed that CD269 CD19b compound CAR-engineered cells result in better therapeutic outcomes for patients suffering from autoimmune diseases due to the depletion of B cells and plasma cells associated with the autoimmune disease.

[0216] In this example, the engineered cells comprise another compound CAR, the CD269 CS1 polypeptide of SEQ ID NO: 52 and the corresponding polynucleotide of SEQ ID NO: 51. In this example, the disclosure provides a method for depleting B cells and plasma cells in a patient in need thereof by administering CD269 CD19 compound CAR-engineered cells to the patient. Without being bound by theory, it is believed that CD269 CD19 compound CAR-engineered cells result in better therapeutic outcomes in patients suffering from autoimmune diseases due to the depletion of B cells and plasma cells associated with the autoimmune disease.

[0217] In this example, the disclosure provides an engineered cell comprising a CD19 chimeric antigen receptor polynucleotide. In this example, the CD19 CAR polypeptide comprises SEQ ID NO: 54 and the corresponding polynucleotide sequence of SEQ ID NO: 53. In another example, the CD19 CAR polypeptide comprises SEQ ID NO: 56 and the corresponding polynucleotide sequence of SEQ ID NO: 55.

[0218] In this example, the engineered cells contain a CD30 CAR polypeptide and an IL-15 / IL-15sushi polypeptide (SEQ ID NO: 100) and corresponding polynucleotide (SEQ ID NO: 99). The target disease is malignant Hodgkin's lymphoma, which involves cancer cells expressing CD30.

[0219] In this example, the present disclosure provides a method for reactivating T cells and innate immune cells in a patient's tumor microenvironment by administering CD30CAR-engineered cells that secrete IL-15 / IL-15 complexes to a patient in need thereof. Without being bound by theory, the IL-15 / IL-15 complexes (e.g., IL-15 / IL-15sushi complexes) secreted from the engineered cells reactivate T cells and innate immune cells in the tumor microenvironment, and subsequently restore or enhance anti-tumor immune responses against Hodgkin's lymphoma or anaplastic large cell lymphoma.

[0220] In this example, the present disclosure provides a method for restoring or enhancing T cell or innate immune cell activation or proliferation involving co-expression of a CAR polypeptide disclosed herein with IL-15 / IL-15sushi.

[0221] In another example, the present disclosure provides a chimeric antigen receptor polypeptide having an antigen recognition domain specific for the CD30 antigen.

[0222] In this example, the CD30CAR comprises at least one costimulatory domain. In another embodiment, the CD30CAR comprises at least two costimulatory domains.

[0223] In some embodiments, the present disclosure includes methods of co-expressing IL-15 / IL-15sushi with CD30CAR. In further embodiments, high-volume secretion of stable, functional IL-15 / IL-15sushi complexes occurs upon binding of the CAR to target cells.

[0224] In another example, the present disclosure provides a method of treating a patient suffering from Hodgkin's lymphoma or a cancer associated with malignant cells expressing the CD30 antigen by administering CD30 CAR-engineered cells to a patient in need thereof. Examples of malignant cells expressing CD30 include anaplastic large cell lymphoma.

[0225] Malignant Hodgkin lymphomas possess CD30+ Reed-Sternberg or Reed-Sternberg-like cells, which are surrounded by an overwhelming number of T cells and innate immune cells. These T or innate immune cells are immunologically tolerant because they cannot eliminate cancer cells. Therefore, one of the key aspects of treating Hodgkin lymphoma is to reactivate T cells and innate immune cells in the tumor microenvironment and then restore or enhance their antitumor immune responses.

[0226] In some embodiments, the disclosure includes a method for co-expression of IL-15 / IL-15sushi and CD30CAR, in which engineered CD30CAR T or NK cells bind to target cancer cells and induce the proliferation of CD30CAR T or NK cells to secrete large amounts of IL-15 / IL-15sushi, thereby effectively restoring or enhancing T or innate immune cells against cancer cells to overcome the immunosuppressive tumor microenvironment.

[0227] In this example, the present disclosure provides a method for providing long-term, durable remission in cancer patients by administering CD30 CAR-engineered cells co-expressing IL-15 / IL-15sushi to patients in need thereof. Without being bound by theory, it is believed that co-expression of IL-15 / IL-15sushi with CD30CAR results in long-term, durable remission in patients by overcoming the immunosuppressive tumor microenvironment, thereby increasing the susceptibility of target cancer cells to CAR recognition or by recruiting innate immune cells.

[0228] In some examples, the present disclosure provides engineered cells that co-express IL-15 / IL-15sushi and CD30CAR polypeptides. Without being bound by theory, it is believed that the combination of CD30CAR engineered cells and co-expression of IL-15 / IL-15sushi provides a synergistic effect when combined with a checkpoint inhibitor or modulator (e.g., anti-PD-1).

[0229] In some examples, the present disclosure provides a method of treating Hodgkin's lymphoma in a patient in need thereof by administering to the patient CD30 CAR-engineered cells co-expressing IL-15 / IL-15sushi. Without being bound by theory, co-expression of a CD30CAR polypeptide with IL-15 / IL-15sushi provides better results for the treatment of Hodgkin's lymphoma or anaplastic large cell lymphoma than CD30CAR alone, because CD30 is not expressed on all cancer cells.

[0230] In some embodiments, the present disclosure provides methods for providing long-term, durable remission to cancer patients by administering APRIL CAR-engineered cells co-expressing IL-15 / IL-15sushi to patients in need thereof. Without being bound by theory, it is believed that co-expression of IL-15 / IL-15sushi and APRIL CAR polypeptides results in long-term remission in patients by either increasing the sensitivity of CAR recognition by target cancer cells or by recruiting natural cells to cancer cells. The APRIL receptor, CD269 (BCMA), is weakly expressed on plasma cells and myeloma cells.

[0231] Steps for tumor elimination by CAR co-expressing secreted IL-15 / IL-15sushi (Figure 78) In certain embodiments, the present disclosure provides a method for the elimination of tumor cells, comprising contacting the tumor cells with CAR-engineered cells that co-express IL-2 to destroy the tumor cells.

[0232] IL-15 was originally thought to be an interleukin-2 (IL-2)-like factor for T and NK cells. Unlike IL-2, IL-15 is a survival factor for memory T cells.

[0233] In certain embodiments, tumor removal can be achieved by a combination of at least one or more of the following steps: (1) binding of the CAR-engineered T cells or NK cells disclosed herein to a portion of tumor cells by targeting the CAR or NK antigen; (2) The proliferation of CAR T / NK cells co-expressing this molecule leads to the secretion of large amounts of IL-15 / IL-15sushi or IL-2, with an extended half-life. (3) Recruitment and stimulation of various innate and adaptive immune cells against tumors. (4) Administration of checkpoint blockade, such as PD-L1 and CTLA-4 inhibitors, reduces tumor suppressors present in tumors.

[0234] Without wishing to be bound by theory, it is believed that the combination of the above steps provides a potent anti-tumor effect through a coordinated innate and adaptive immune response.

[0235] The engineered cells and methods described herein (Figure 78) are suitable for the treatment of any cancer with specific monoclonal or polyclonal antibodies that exist or can be produced according to the current state of the art. In particular, the following cancers are contemplated and considered within the scope of this disclosure: neuroblastoma, lung cancer, melanoma, ovarian cancer, renal cell carcinoma, colon cancer, brain cancer, Hodgkin's lymphoma, B-cell lymphoma / leukemia and T-cell lymphoma / leukemia, hepatocellular carcinoma, fibrolamellar carcinoma, hepatoblastoma, undifferentiated embryonal sarcoma and mesenchymal hamartoma of the liver, squamous cell carcinoma of the lung, testicular nonseminomatous germ cell tumor, liposarcoma, ovarian and extragonadal yolk sac tumor, ovarian choriocarcinoma, teratoma, ovarian clear cell carcinoma, and trophoblastic tumor of the placental site. All of these cancers have cell surface antigens that can be targeted by the chimeric antigen receptor polypeptides and methods disclosed herein.

[0236] In another embodiment, the target cells are hepatocellular carcinoma, fibrolamellar carcinoma, hepatoblastoma, undifferentiated embryonal sarcoma and mesenchymal hamartoma of the liver, lung squamous cell carcinoma, testicular nonseminomatous germ cell tumor, liposarcoma, ovarian and extragonadal yolk sac tumor, ovarian choriocarcinoma, teratoma ova, clear cell carcinoma, and trophoblastic tumor of the placental site.

[0237] Many tumors escape specific CAR T / NK killing due to loss of target antigen or CAR T or NK depletion. The present disclosure provides a method for preventing tumor escape by administering CAR-engineered cells containing the enhancers or cytokines disclosed herein, particularly IL-15 or IL-2, to the tumor site. This is believed to directly stimulate innate and adaptive immune responses. Furthermore, IL-15 and / or IL-2 secretion from CAR-engineered cells is believed to promote proliferation of infused CAR T or CAR NK cells and infiltration of immune cells into the tumor site, which may lead to tumor destruction.

[0238] In some embodiments, the half-life and therapeutic activity can be increased by the presence of an Fc domain, such as IL-15Fc or IL-2Fc. In the case of the IL-15 cytokine, IL-15 / IL-15sushi or IL-15 / IL-15sushiFc is preferred. The Fc domain is referred to as an IgG Fc domain fused to various effector molecules (so-called Fc fusion proteins).

[0239] Single-antigen-specific CAR immunotherapies, such as, but not limited to, CD19, CD20, CD22, CD2, CD3, CD4, CD5, CD7, CD33, CD30, CD123, CD45, BCMA, CS1, BAFF, TACI, and APRIL CARs, are at risk of remission in patients due to complete loss of the target antigen or changes in target antigen expression. Based on this, the present disclosure provides methods for providing long-term durable remission in patients by administering engineered cells having co-expression of the CAR polypeptides disclosed herein and IL-15 / IL-15sushi, thereby increasing the sensitivity of target cancer cells to CAR recognition or recruiting innate immune cells to cancer cells.

[0240] Some solid tumors or lymphomas are difficult to eradicate entirely with CAR T cells. Furthermore, the immunosuppressive microenvironment must be overcome, as CAR T cells are simply inactivated or suppressed upon contact with the tumor.

[0241] In some examples, the present disclosure provides methods for co-expressing secreted IL-15 / IL-15sushi and a chimeric antigen receptor polypeptide in engineered cells.

[0242] In some examples, the present disclosure provides methods for increasing the in vivo half-life of CAR-engineered cells by co-expressing secreted IL-15 / IL-15sushi in the engineered cells. Without being bound by theory, it is believed that the secreted IL-15 / IL-15sushi complex is functionally stable and effectively promotes survival of CAR-containing engineered cells.

[0243] In some examples, the present disclosure provides methods for delivering IL-15 / IL-15sushi to targeted cancer sites using CARs as carriers to promote proliferation of innate immune response cells against cancer cells, prevent tumor microenvironment suppression, and reduce systemic toxicity associated with high doses of exogenous cytokines.

[0244] In some examples, the present disclosure provides methods for delivering IL-15 / IL-15sushi to targeted cancer sites using CARs as carriers to recruit other effector immune cells to the site and help them kill cancer cells.

[0245] In some examples, the present disclosure provides methods for delivering IL-15 / IL-15sushi to targeted cancer sites using CARs as carriers to activate bystander immunity to eradicate cancer cells that lose antigens to CAR T / NK cells.

[0246] Methods for producing engineered cells Any of the polynucleotides disclosed herein can be introduced into engineered cells by any method known in the art.

[0247] In this example, the CAR polynucleotide is delivered to the engineered cell by any of the viral vectors disclosed herein.

[0248] In this example, to achieve an improved safety profile or therapeutic index, any engineered cell disclosed herein may be constructed as a transient RNA-modified "biodegradable" version or derivative, or a combination thereof. The RNA-modified CAR of the present invention may be electroporated into T cells or NK cells. Expression of the compound CAR may gradually decrease over several days.

[0249] In some embodiments of the present invention, any of the engineered cells disclosed herein may be constructed with a transposon system (also called "Sleeping Beauty") that integrates the CAR DNA into the host genome without viral DNA.

[0250] Methods for generating engineered cells with multiple CAR units In another example, the present disclosure provides a method for producing an engineered cell having at least two CAR units.

[0251] In some embodiments, multiple units of the CAR are expressed in T cells or NK cells using bicistronic or multicistronic expression vectors. Several strategies can be used to construct bicistronic or multicistronic vectors, including, but not limited to, (1) multiple promoters fused to the CAR open reading frame, (2) insertion of splicing signals between CAR units, fusing the CAR so that its expression is driven by a single promoter, (3) insertion of proteolytic cleavage sites (self-cleaving peptides) between CAR units, and (iv) insertion of an internal ribosome entry site (IRES).

[0252] In a preferred embodiment, multiple CAR units are expressed in a single open reading frame (ORF), thereby creating a single polypeptide with multiple CAR units, in which an amino acid sequence or linker containing a high-efficiency cleavage site is located between each CAR unit.

[0253] As used herein, high cleavage efficiency is defined as cleavage of 50% or more, 70% or more, 80% or more, or 90% or more of the translated protein. Cleavage efficiency may be assessed by Western blot analysis as described by Kim 2011.

[0254] Furthermore, in preferred embodiments, there are equal amounts of cleavage products as shown by Western blot analysis.

[0255] Examples of high-efficiency cleavage sites include, but are not limited to, porcine teschovirus-1 2A (P2A), FMDV 2A (abbreviated as F2A herein), equine rhinitis A virus (ERAV) 2A (E2A), Thosea asigna virus (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A), and flacherie virus 2A (BmIFV2A), or combinations thereof. In a preferred embodiment, the high-efficiency cleavage site is P2A. The high-efficiency cleavage site is used herein by reference to the description in Kim JH, Lee SR, Li LH, Park HJ, Park JH, Lee KY, et al. (2011) High Cleavage Efficiency of a 2A Peptide Derived from Porcine Teschovirus-1 in Human Cell Lines, Zebrafish, and Mice. PLoS ONE 6(4): e18556.

[0256] In the present example, multiple CAR units are expressed under the control of a single open reading frame (ORF), and expression is under the control of a strong promoter, such as the SFFV promoter and its derivatives.

[0257] The engineered cells have the CAR polypeptide and the enhancer In another example, the present disclosure provides a method of generating an engineered cell that expresses at least one CAR unit and an enhancer.

[0258] In some embodiments, at least one CAR unit and enhancer are expressed in T cells or NK cells using a bicistronic or multicistronic expression vector. Several strategies can be used to construct bicistronic or multicistronic vectors, including but not limited to: (1) multiple promoters fused to the CAR open reading frame; (2) inserting a splicing signal between the CAR units and fusing the CAR so that expression is driven by a single promoter; (3) inserting a proteolytic cleavage site (self-cleaving peptide) between the CAR units; and (4) inserting an internal ribosome entry site (IRES).

[0259] In a preferred embodiment, at least one CAR unit and an enhancer are expressed in a single open reading frame (ORF), thereby producing a single polypeptide having at least one CAR unit and an enhancer. In this embodiment, an amino acid sequence or linker containing a high-efficiency cleavage site is placed between each CAR unit and between the CAR unit and the enhancer. In this embodiment, the ORF is under the control of a strong promoter. Examples of strong promoters include the SFFV promoter and its derivatives.

[0260] Furthermore, in preferred embodiments, there are equal amounts of cleavage products as shown by Western blot analysis.

[0261] Methods of Treatment Using the Compositions Disclosed Herein In another embodiment, the present invention provides a method for targeting CD45 for conditioning prior to allogeneic transplantation in cancer therapy. CD45, also known as leukocyte common antigen (LCA), is a tyrosine phosphatase expressed on virtually all cells of hematopoietic origin except for erythrocytes and platelets. Most hematologic malignancies express CD45. For example, 85% to 90% of acute lymphocytic leukemias and myeloid leukemias express CD45. CD45 is not found in non-hematopoietic origins. Furthermore, CD45 is expressed at high density on malignant cells and leukocytes, with an average copy number of approximately 200,000 molecules per cell. CD45 is an ideal target for various hematologic malignancies. However, CAR T and NK cells also express CD45. Without inactivation of endogenous CD45, CAR T or NK cells equipped with CD45-targeted CARs may result in self-killing.

[0262] The association of CD45 with the TCR complex is essential for regulating T cell activation in response to antigen. The inability of CD45-deficient T cells to present antigen is due to reduced signaling through T cell receptors (TCRs). TCRs are cell surface receptors that play an essential role in T cell activation in response to antigen presentation. TCRs are generally made up of two chains, α and β, associated with the transducing subunit, CD3, to form the T cell receptor complex present on the cell surface.

[0263] Surprisingly, the multiple CARs (Compound CARs, cCARs) of the present invention have been found to combat an important mechanism by which cancer cells resist CAR activity: downregulation or heterologous expression of target antigens from the cancer cell surface. This mechanism allows cancer cells to "hide" from CAR therapy, a phenomenon also known as "antigen escape." The present disclosure preempts cancer antigen escape by recognizing a combination of two or more antigens to rapidly eliminate tumors.

[0264] The present disclosure provides a method for simultaneous targeting of multiple antigens using cCARs to improve tumor control by minimizing the possibility of tumor selection based on loss or downregulation of target antigens.

[0265] The disclosed invention includes compound (multiple or compound) cCARs in T cells or NK cells that target different or the same surface antigens present in tumor cells. The compound chimeric antigen receptor of the present invention includes at least multiple chimeric receptor constructs linked by a linker and targeting the same or different antigens. For example, each CAR construct in a compound CAR (cCAR) construct includes an antigen recognition domain, an extracellular domain, a transmembrane domain, and / or a cytoplasmic domain. The extracellular domain and transmembrane domain can be derived from any desired domain. Multiple CAR constructs are linked by a linker. Expression of the compound CAR construct is driven by a promoter. The linker may be a portion of a peptide or protein that self-cleaves after the protein or peptide is produced (also called a self-cleaving peptide).

[0266] In this example, the compound CAR of the present invention targets myelodysplastic syndrome and acute myeloid leukemia (AML). Myelodysplastic syndrome (MDS) is a frequently occurring, incurable hematopoietic stem cell malignancy among the elderly, with approximately 14,000 new cases occurring in the United States each year. Approximately 30-40% of MDS cases progress to AML. As the population ages, the incidence of MDS continues to increase. Although MDS and AML have been extensively studied, satisfactory treatments have not yet been developed.

[0267] The compositions and methods of the present invention are used to generate populations of T lymphocytes or NK cells that deliver both primary and costimulatory signals in immunotherapy for the treatment of cancer, including the treatment of lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, brain cancer, sarcoma, leukemia, and lymphoma, among others.

[0268] Immunotherapeutics generally rely on immune effector cells and molecules to target and destroy cancer cells. Effectors may be lymphocytes with surface molecules that directly or indirectly interact with tumor cell targets. Various effector cells include cytotoxic T cells, NK cells, and NK-92 cells. The compositions and methods described herein may be used in conjunction with other types of cancer therapy, such as chemotherapy, surgery, radiation therapy, and gene therapy. The compositions and methods described herein may also be used in other diseases that depend on the immune response, such as inflammation, immune disorders, and infectious diseases.

[0269] In some embodiments, the compound CARs of the present disclosure may serve as a bridge to bone marrow transplant by achieving complete remission for patients with minimal residual disease who are no longer responding to chemotherapy. In other embodiments, the compound CARs eliminate leukemia cells and support leukopenia with subsequent bone marrow stem cell rescue.

[0270] In some embodiments, the disclosed compound CARs can combat an important mechanism by which cancer cells resist CAR activity: downregulation of target antigens. In another example, the disclosed compound CARs can also combat cancer cell heterogeneity, a significant challenge in conventional CAR T / NK cell therapy. In a further example, the disclosed compound CARs are designed such that one CAR targets a large tumor population and another CAR eradicates cancer or leukemia stem cells to avoid cancer recurrence.

[0271] In this embodiment, the present disclosure provides a method for destroying targeted cells by contacting cells bearing the CD33 antigen, the CD123 antigen, or both, with engineered cells having at least one chimeric antigen receptor polypeptide having the CD33 antigen recognition domain and the CD123 antigen recognition domain. The engineered cells can be T cells or NK cells.

[0272] Cells bearing at least one of the CD33 antigen and the CD123 antigen are acute myeloid leukemia, precursor acute lymphoblastic leukemia, chronic myeloproliferative neoplasm, chronic myeloid leukemia, myelodysplastic syndrome, blastic plasmacytoma dendritic neoplasm (BPDCN), Hodgkin's lymphoma, mastocytosis, and hairy cell leukemia cells.

[0273] In another embodiment, the present disclosure provides a method for providing a myeloablative conditioning regimen for hematopoietic stem cell transplantation, in which T or NK engineered cells having a CD33 unit and a CD123 unit are administered to a patient in need thereof.

[0274] In a further example, the present disclosure provides a method of eradicating or killing leukemic stem cells (LSCs) or bulk leukemic cells, or both, that express CD123 or CD33, in which T or NK engineered cells having a CD33 unit and a CD123 unit are administered to a patient in need thereof.

[0275] In a further example, compound CARs in T or NK cells could be used to eradicate or kill CD34 + CD38 − leukemia stem cells or bulk leukemia cells expressing CD123 or CD33 or both.

[0276] In some embodiments, the compound CAR targets cells expressing CD19 or CD20 antigens, or both. In another example, the compound CAR targets cells expressing CD19 or CD22 antigens, or both. The target cells may be cancer cells, such as, but not limited to, B-cell lymphoma or leukemia cells. In further embodiments, the target antigen includes, but is not limited to, at least one from the group consisting of ROR1, PSMA, MAGE A3, glycolipids, glypican 3, F77, GD-2, WT1, CEA, HER-2 / neu, MAGE-3, MAGE-4, MAGE-5, MAGE-6, alpha-fetoprotein, CA19-9, CA72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, CD30, MUC1, MUC2, MUC3, MUC4, MUC5, EGFRvIII, immunoglobulin kappa and lambda, CD38, CD52, CD3, CD4, CD8, CD5, CD7, CD2, CD45, and CD138. Target antigens can also include viral or fungal antigens, such as E6 and E7 from human papillomavirus (HPV) or EBV (Epstein-Barr virus) antigens.

[0277] In some embodiments, the compound CAR-engineered cells target cells bearing the cell surface CD19 antigen, the cell surface CD123 antigen, or both, and the target cells are cancer cells, such as, but not limited to, B-cell lymphoma or leukemia cells.

[0278] Clinical trials of CD19 CAR T-cell therapy have shown that although 80-94% of patients with B-ALL achieve complete remission, a significant proportion of patients ultimately relapse. The high prevalence of CD123 expression in B-ALL suggests that B-ALL can be used as a CAR target.

[0279] In some embodiments, the compound CAR targets cells that express the CD19 or CD123 antigen, or both. Without being bound by theory, it is believed that CD19 and / or CD123 compound CAR engineered cells counteract tumor escape by eliminating the CD19 or CD123 antigen or preventing relapse of B-ALL or other types of B-cell lymphoma / leukemia.

[0280] In some embodiments, the compound CAR targets cells that express the CD19 or BAFFR antigen, or both. Without wishing to be bound by theory, it is believed that cells engineered with CD19 and / or BAFFR compound CARs counteract tumor escape due to loss of the CD19 or BAFFR antigen or prevent relapse of B-ALL or other types of B-cell lymphoma / leukemia.

[0281] In a further example, the CD19 and / or CD20 compound CAR engineered cells target cells bearing the cell surface CD19 antigen and / or CD20 cell surface antigen. In another example, the target cells are aggressive B-cell lymphomas / leukemias, including but not limited to B-ALL, high-grade B-cell lymphoma, low-grade B-cell lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, mantle cell lymphoma, CLL, marginal zone B-cell lymphoma, and follicular lymphoma.

[0282] Without wishing to be bound by theory, it is believed that CD19 and / or CD20 CAR-engineered cells provide an effective means of protection against antigen escape in adoptive T / NK cell therapy of B-cell malignancies, preventing disease relapse.

[0283] The CAR target cells are selected from the group consisting of congenital acute lymphoblastic leukemia, B-cell lymphoma / leukemia, chronic lymphocytic leukemia / lymphoma, mantle lymphoma, follicular lymphoma, marginal zone B-cell lymphoma, diffuse large B-cell lymphoma, Burkitt lymphoma, blastic plasmacytoma dendritic neoplasm (BPDCN), Hodgkin lymphoma, and hairy cell leukemia cells bearing at least one of the antigens CD19, CD20, CD22, BAFF, and CD123.

[0284] In this example, the engineered cell comprises a first chimeric antigen receptor polypeptide having a CD19 antigen recognition domain and a second chimeric antigen receptor polypeptide having a CD22 antigen recognition domain. In this example, the engineered cell comprises the polypeptide of SEQ ID NO:64 and a polynucleotide corresponding to SEQ ID NO:63.

[0285] In this example, the engineered cell comprises a first chimeric antigen receptor polypeptide having a CD19 antigen recognition domain and a second chimeric antigen receptor polypeptide having a CD20 antigen recognition domain. In this example, the engineered cell comprises the polypeptide of SEQ ID NO:66 and the corresponding polynucleotide of SEQ ID NO:65.

[0286] In this example, the engineered cell comprises a first chimeric antigen receptor polypeptide having a CD19 antigen recognition domain and a second chimeric antigen receptor polypeptide having a CD123 antigen recognition domain. In this example, the engineered cell comprises the polypeptide of SEQ ID NO:68 and the corresponding polynucleotide of SEQ ID NO:67.

[0287] Multiple myeloma is an incurable disease characterized by uncontrolled proliferation of plasma cells in the bone marrow. CS1 and BCMA are widely expressed on myeloma cells but not on hematopoietic stem / progenitor cells. Therefore, BCMA and CS1 are ideal targets for CAR T / NK cell therapy.

[0288] In a further example, the present disclosure provides compound CAR-engineered cells having a CS1 (SLAM7) antigen recognition domain and / or an antigen recognition domain targeting B-cell maturation antigen (BCMA). In another example, the target cell is a malignant plasma cell, such as, but not limited to, multiple myeloma.

[0289] Without wishing to be bound by theory, it is believed that compound CAR engineered cells bearing at least one of the CS1 and BCMA antigen recognition domains have enhanced functionality against multiple myeloma and counteracting antigen escape.

[0290] In some embodiments, the CAR targets cells expressing multiple antigens, including but not limited to, CS1, BCMA, CD267, BAFF-R, CD38, CD138, CD52, CD19, TACI, CD20, interleukin 6 receptor, and NY-ESO-1 antigen. In another embodiment, the target cell is a plasma cell, a B cell, or a malignant plasma cell, such as but not limited to multiple myeloma.

[0291] In some embodiments, the compound CAR targets cells expressing multiple antigens, including but not limited to, CS1, BCMA, CD267, BAFF-R, CD38, CD138, CD52, CD19, TACI, CD20, interleukin-6 receptor, and NY-ESO-1 antigen. In other embodiments, the target cells are plasma cells, B cells, malignant plasma cells, such as, but not limited to, multiple myeloma.

[0292] In some embodiments, the compound CAR targets cells that express multiple antigens, including, but not limited to, alpha-fetoprotein (AFP) and glypican-3 (GPC3). In other...

Claims

1. An engineered cell comprising a chimeric antigen receptor polypeptide comprising a signal peptide, an antigen recognition domain, a hinge region, a transmembrane domain, a signaling domain, and a costimulatory domain, and the antigen recognition domain comprises one of FcER1A, FcER1, Ig E, CD19, BCMA, or CD45.

2. The engineered cell of claim 1, wherein the costimulatory domain is CD28 or 4-1 BB and the signaling domain is CD3 zeta.

3. 2. The engineered cell of claim 1, further comprising one or more enhancers selected from the group consisting of IL-15 / IL-15 sushi, IL-15 / 1L-15 sushi anchor, 4-1BBL, and IL-15.

4. An engineered polypeptide comprising a chimeric antigen receptor polypeptide comprising a signal peptide, an antigen recognition domain, a hinge region, a transmembrane domain, a signaling domain, and a costimulatory domain, wherein the antigen recognition domain comprises one of FcER1A, CD19, BCMA, or CD45, and at least one enhancer, and a high efficiency cleavage site disposed between the chimeric antigen receptor polypeptide and the enhancer.

5. The engineered polypeptide of claim 4, wherein the modified polypeptide comprises two enhancers and two high efficiency cleavage sites.

6. 5. The engineered polypeptide of claim 4, wherein the high efficiency cleavage site is selected from the group consisting of P2A, T2A, E2A, and F2A.

7. An engineered polynucleotide encoding the polypeptide of claim 4.

8. a first chimeric antigen receptor polypeptide comprising a first signal peptide, a first antigen recognition domain, a first hinge region, a first transmembrane domain, a first signaling domain, and a first costimulatory domain; and a second chimeric antigen receptor polypeptide comprising a second signal peptide, a second antigen recognition domain, a second hinge region, a second transmembrane domain, a second signaling domain, and a second costimulatory domain, the domain and the second antigen recognition domain being different; and the first antigen recognition domain and the second antigen rejection domain are selected from the group consisting of CD4, CD19, CD33, CD123, CLL-1, BAFFR, BCMA, and CS-1.

9. The engineered cell of claim 8, wherein the first antigen recognition domain and the second antigen recognition domain are selected from the group including CD19 and CD123, CD19 and BAFFR, BCMA and CD19, BCMA and CS1, CD123 and CD33, CD33 and CLL-1, CD4 and CD123, CD19 and CS-1, and CD4 and CLL-1.

10. 1. An engineered polypeptide comprising: a first chimeric antigen receptor polypeptide comprising a first signal peptide, a first antigen recognition domain, a first hinge region, a first transmembrane domain, a first signaling domain, and a first costimulatory domain; and a second chimeric antigen receptor polypeptide comprising a second signal peptide, a second antigen recognition domain, a second hinge region, a second transmembrane domain, a second signaling domain, and a second costimulatory domain, wherein the first antigen recognition domain and the second antigen recognition domain are different, and the first antigen recognition domain and the second antigen rejection domain are selected from the group consisting of CD4, CD19, CD33, CD123, BAFFR, CLL-1, BCMA, and CS-1.

11. 11. The engineered polypeptide of claim 10, wherein the first antigen recognition domain and the second antigen recognition domain are selected from the group including CD19 and CD123, BCMA and CD19, CD19 and BAFFR, BCMA and CS1, CD123 and CD33, CD33 and CLL-1, CD4 and CD123, CD19 and CS-1, and CD4 and CLL-1.

12. 11. The engineered polypeptide of claim 10, wherein the first costimulatory domain and the second costimulatory domain are different.

13. 11. The engineered polypeptide of claim 10, wherein the high efficiency cleavage site is located between the first antigen recognition domain and the second antigen recognition domain.

14. 10. An engineered polynucleotide encoding the polypeptide of claim 9.

15. A method for treating asthma comprising administering to a patient in need thereof a composition comprising the engineered cells of claim 1, wherein the chimeric antigen receptor comprises FcER1A or FcER1.

16. A method for lysing a target cell bearing an FcER1A FcER1 or Ig E cell surface antigen, comprising contacting the cell with an engineered cell according to claim 1.

17. 16. The method of claim 15, wherein the target cells comprise plasma cells, mast cells, or basophil cells.

18. 11. A method of treating an autoimmune disease comprising administering the engineered cells of claim 1 to a patient in need thereof, wherein the autoimmune disease comprises systemic lupus erythematosus (SLE), multiple sclerosis (MS), inflammatory bowel disease (IBD), rheumatoid arthritis, Sjogren's syndrome, dermatomyositis, autoimmune hemolytic anemia, neuromyelitis optica (NMOD), NMO spectrum disorder (NMOSD), idiopathic thrombocytopenic purpura (ITP), antineutrophil cytoplasmic autoantibodies (ANCA) associated with systemic autoimmune small vessel vasculitis syndrome or microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA, Wegener's granulomatosis, granulomatosis, granulomatosis, EG) or eosinophilic granulomatosis with polyangiitis (EGPA, Churg-Strauss syndrome).

19. 10. A method of treating an autoimmune disease comprising administering the engineered cells of claim 7 to a patient in need thereof, wherein the autoimmune disease comprises systemic lupus erythematosus (SLE), multiple sclerosis (MS), inflammatory bowel disease (IBD), rheumatoid arthritis, Sjogren's syndrome, dermatomyositis, autoimmune hemolytic anemia, neuromyelitis optica (NMOD), NMO spectrum disorder (NMOSD), idiopathic thrombocytopenic purpura (ITP), antineutrophil cytoplasmic autoantibodies (ANCA) associated with systemic autoimmune small vessel vasculitis syndrome or microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA, Wegener's granulomatosis, granulomatosis, granulomatosis, EG) or eosinophilic granulomatosis with polyangiitis (EGPA, Churg-Strauss syndrome).

20. 4. A method for depleting a target cell comprising contacting the target cell with the engineered cell of claim 3, wherein the target cell has one or more of the following cell surface antigens: FcER1A, FcERi, IgE, CD19, BCMA, or CD45.

21. 10. A method of depleting a target cell comprising contacting the target cell with the engineered cell of claim 9, wherein the target cell has one or more of the following cell surface antigens CD4, CD19, CD33, CD123, BAFFR, CLL-1, BCMA, and CS-1.

22. 13. A method of treating asthma in a patient, comprising administering to the patient an effective amount of the cells of claim 1.

23. 10. A method of preventing or mediating organ rejection in a patient, comprising administering to said patient an effective amount of the cells of claim 7.

24. an engineered cell comprising a first chimeric antigen receptor polypeptide comprising a first signal peptide, a first antigen recognition domain, a first hinge region, a first transmembrane domain, a first signaling domain, and a first costimulatory domain, and optionally a second chimeric antigen receptor polypeptide comprising one or more of a second signal peptide, a second antigen recognition domain, a second hinge region, a second transmembrane domain, a second signaling domain, and a second costimulatory domain, wherein the first antigen recognition domain and the second antigen recognition domain are different, and the first antigen recognition domain and the second antigen rejection domain are selected from the group consisting of CD4, CD19, CD33, CD123, CLL-1, BAFFR, BCMA, and CS-1, or one or more enhancers selected from the group consisting of IL-15 / IL-15 sushi, IL-15 / 1L-15 sushi anchor, 4-1BBL, and IL-15.

25. Hereinafter, a chimeric antigen receptor polypeptide comprising a signal peptide, an antigen recognition domain, a hinge region, a transmembrane domain, a signal transduction domain, and a costimulatory domain, and an engineered cell, wherein the antigen recognition domain comprises one of GD2 and GD3.

26. 26. The engineered cell of claim 25, wherein the costimulatory domain is CD28 or 4-1 BB and the signaling domain is CD3 zeta.

27. 26. The engineered cell of claim 25, further comprising one or more enhancers selected from the group consisting of IL-15 / IL-15 sushi, IL-15 / IL-15 sushi anchor, 4-1BBL, and IL-15.

28. An engineered polypeptide comprising a chimeric antigen receptor polypeptide comprising a signal peptide, an antigen recognition domain, a hinge region, a transmembrane domain, a signaling domain, and a costimulatory domain, wherein said antigen recognition domain comprises one of GD2 and GD3, and optionally at least one enhancer, wherein a high efficiency cleavage site is disposed between the chimeric antigen receptor polypeptide and the enhancer.

29. 30. The engineered polypeptide of claim 28, wherein the engineered polypeptide comprises two enhancers and two high efficiency cleavage sites.

30. 30. The engineered polypeptide of claim 29, wherein the high efficiency cleavage site is selected from the group consisting of P2A, T2A, E2A, and F2A.

31. 26. An engineered polynucleotide encoding the engineered polypeptide of claim 25.

32. Hereinafter, an engineered cell comprising a first chimeric antigen receptor polypeptide comprising a first signal peptide, a GD2 antigen recognition domain, a first hinge region, a first transmembrane domain, a first signaling domain, and a first costimulatory domain, and a second chimeric antigen receptor polypeptide comprising a second signal peptide, a GD3 antigen recognition domain, a second hinge region, a second transmembrane domain, a second signaling domain, and a second costimulatory domain.

33. Hereinafter, an engineered polypeptide comprising a first chimeric antigen receptor polypeptide comprising a first signal peptide, a GD2 antigen recognition domain, a first hinge region, a first transmembrane domain, a first signaling domain, and a first costimulatory domain, and a second chimeric antigen receptor polypeptide comprising a second signal peptide, a GD3 antigen recognition domain, a second hinge region, a second transmembrane domain, a second signaling domain, and a second costimulatory domain.

34. 34. The engineered polypeptide of claim 33, wherein the first costimulatory domain and the second costimulatory domain are different.

35. 34. The engineered polypeptide of claim 33, wherein the high efficiency cleavage site is located between the first antigen recognition domain and the second antigen recognition domain.

36. 34. An engineered polynucleotide encoding the engineered polypeptide of claim 33.

37. A method for increasing the persistence of CAR engineered cells in a patient, the method comprising administering to the patient engineered cells that co-express one or more enhancers with a GD2 or GD3 engineered CAR polypeptide.

38. A method for lysing a target cell bearing a GD2 or GD3 cell surface antigen, comprising contacting the target cell with an engineered cell described in claim 26.

39. 26. A method for depleting a target cell comprising contacting the target cell with the engineered cell of claim 25, wherein the target cell has one or more of the following cell surface antigens: GD2 and GD3.

40. 26. A method of treating a cell proliferative disorder comprising administering the engineered cell of claim 25 to a patient in need thereof.

41. 41. The method of claim 40, wherein the cell proliferative disorder is selected from the group consisting of medulloblastoma / primary neuroectodermal tumors of the central nervous system (CNS), malignant glioma, neuroblastoma, retinoblastoma, ependymoma, sarcoma, melanoma, breast cancer, ovarian cancer, glioblastoma, Ewing's sarcoma, and small cell lung cancer.

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