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

Abstract

To provide compositions and methods relating to chimeric antigen receptor (CAR) polypeptides, and methods relating thereto.SOLUTION: In this embodiment, the present invention relates to engineered cells having chimeric antigen receptor polypeptides directed to at least two targets. In another embodiment, the present invention relates to engineered cells having chimeric antigen receptor polypeptides and an enhancer moiety.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application is an international PCT application claiming priority from U.S. Provisional Application No. 62 / 184,321, filed June 25, 2015, U.S. Patent Application No. 62 / 235,840, filed October 1, 2015, and U.S. Patent Application No. 62 / 244,435, filed October 21, 2015, the entire contents of which are incorporated herein by reference.

[0002] T cells, a type of lymphocyte, play a central role in cell-mediated immunity. Compared to other lymphocytes, B cells, and natural killer cells (NK cells), T cells have a T cell receptor (TCR) on their surface. Helper T cells (also called CD4+ T or CD4T cells) express the CD4 glycoprotein on their surface. Helper T cells are activated by peptide antigens presented by major histocompatibility complex (MHC) 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 by peptide antigens presented by MHC class I molecules. A subset of T cells, memory T cells, survive for a long time and respond to their cognate antigens, providing defense against the body by mounting an immune response based on memory of past infections and / or tumor cells.

[0003] T cells can be genetically engineered to produce special receptors on their surface called chimeric antigen receptors (CARs). CARs are proteins on T cells that recognize specific proteins (antigens) on tumor cells. These engineered CAR T cells are grown in the laboratory until they number in the billions, and then the grown CAR T cells are infused into patients. [Background technology]

[0004] To date, clinical trials using chimeric antigen receptor (CAR) T cells have shown great promise for the treatment of hematological malignancies resistant to standard chemotherapy. Most notably, CD19-specific CAR (CD19CAR) T cell therapy has shown remarkably favorable outcomes, including long-term remissions in B cell malignancies (see 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. 2013; Kochenderfer, Dudley et al. 2013; Lee, Shah et al. 2013; Park, Riviere et al. 2013; Maude, Frey et al. 2014).

[0005] 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), and in this respect CAR therapy has great clinical potential.

[0006] To date, current efforts have focused on demonstrating the efficacy of CAR T cells against various B-cell malignancies. Treatment of B-ALL with CD19CARs 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, and there are no general rules to validate or guide effective CAR design in this regard.

[0007] There are several obstacles that prevent the adoption of a more widespread approach in CAR therapy. One of the most common challenges is (1) the selection of antigen target and chimeric antigen receptor, (2) the design of the CAR, and (3) tumor heterogeneity, especially the variability in surface expression of tumor antigens. Targeting a single antigen carries the risk of immune escape, which can be overcome by targeting multiple desirable antigens.

[0008] Most CAR chimeric antigen receptors are scFvs derived from monoclonal antibodies, and some of these monoclonal antibodies have been used in clinical trials or treatment of diseases. However, they have limited efficacy, suggesting that alternative and more potent targeted approaches such as CARs are needed. 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 affect its function. In addition, the level of surface CAR expression on T cells or NK cells is influenced by the appropriate leader sequence and promoter. In addition, overexpressed CAR proteins can also be toxic to cells. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Provisional Application No. 62 / 184,321 [Patent Document 2] U.S. Patent Application No. 62 / 235,840 [Patent Document 3] U.S. Patent Application No. 62 / 244,435 [Non-patent literature]

[0010] [Non-Patent Document 1] Kochenderfer, Wilson et al. 2010 [Non-Patent Document 2] Kalos, Levine et al. 2011 [Non-Patent Document 3] Porter, Levine et al. 2011 [Non-Patent Document 4] Davila, Riviere et al. 2013 [Non-Patent Document 5] Grupp, Frey et al. 2013 [Non-Patent Document 6] Grupp, Kalos et al. 2013 [Non-Patent Document 7] Kalos, Nazimuddin et al. 2013 [Non-Patent Document 8] Kochenderfer, Dudley et al. 2013 [Non-Patent Document 9] Kochenderfer, Dudley et al. 2013 [Non-Patent Document 10] Lee, Shah et al. 2013 [Non-Patent Document 11] Park, Riviere et al. 2013 [Non-Patent Document 12] Maude, Frey et al. 2014 [Non-Patent Document 13] Abramson, Feldman et al. 2014 Summary of the Invention [Problem to be solved by the invention]

[0011] Thus, 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]

[0012] In this example, the disclosure provides details of 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 hinge region, 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, where the first antigen recognition domain is different from the second antigen recognition domain.

[0013] In another example, the disclosure provides a modified polypeptide comprising a chimeric antigen receptor and an enhancer.

[0014] In another example, the disclosure provides a modified polypeptide comprising a chimeric antigen receptor polypeptide and an enhancer.

[0015] In another embodiment, the disclosure provides 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, an engineered chimeric antigen receptor polypeptide, or a polynucleotide encoding the polypeptide.

[0016] In another embodiment, the disclosure provides details of engineered cells comprising the engineered polypeptides or polynucleotides described above.

[0017] In another embodiment, the disclosure provides a method of reducing the number of target cells, including, but not limited to, (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, the target cells comprising at least one cell surface antigen selected from the group consisting of interleukin 6 receptor, NY-ESO-1, alpha fetoprotein (AFP), glypican 3 (GPC3), BAFF-R, BCMA, TACI, LeY, CD5, CD13, CD14, CD15, CD19, CD20, CD22, CD33, CD41, CD45, CD61, CD64, CD68, CD117, CD123, CD138, CD267, CD269, CD38, Flt3 receptor, and CS1.

[0018] In another embodiment, the disclosure provides a method of treating B cell lymphoma, T cell lymphoma, multiple myeloma, chronic myelogenous leukemia, B cell acute lymphoblastic leukemia (B-ALL), and cell proliferative disorders comprising administering to a patient in need thereof any of the engineered cells described above. [Brief description of the drawings]

[0019] [Figure 1] Schematic diagram of cCAR configuration (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 is based on CARs composed of different components, each containing different or similar antigen recognition domains, hinge (H) regions, transmembrane (TM) domains, various co-stimulatory domains, and intracellular signaling domains. [Figure 2A]Schematic diagram of cCAR-T construct. The construct is a CAR with multiple modular units linked by P2A peptides, and expression is driven by the SFFV promoter. Upon cleavage of the linker, the cCARs split 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, 4-1BB in the area of ​​the CD33 CAR and CD28 sites on the CD123 CAR. [Figure 2B] Western blot shows the expression of CD33CD123 cCAR transduced into T cells. This figure shows the expression of two different CAR proteins, namely CD33 CAR and CD123 CAR. cCAR-T cells expressing both CD33 and CD123 CAR upon cleavage of the linker show two clear and consistently strong protein bands. Green fluorescent protein (GFP) was used as a negative control. [Figure 2C] Flow cytometry analysis represents the efficiency of transduction. 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 lentiviral vectors containing the CD33CD123cCAR construct and GFP-transduced cells as a control. The percentages indicated with yellow circles are a proxy for transduction efficiency. [Diagram 3] Schematic diagram showing how to generate compound CARs (cCARs). [Figure 4]Co-culture assay by incubation of the promyelocytic leukemia cell line HL60 with CD33CD123-2G CAR-T cells (cCAR). The potency of cCAR-T cells (lower panel) is compared with control GFP-transduced T cells (upper panel). Killing potency is measured by the population of CD33+ cells remaining after approximately 24 hours of incubation (circled in yellow). [Diagram 5] Co-culture assay by incubation of cCAR-T cells with myeloid leukemia cell line KG-1a, which expresses ~100% CD33 and ~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 ~24 h of incubation. [Figure 6] Co-culture assay by incubation of cCAR-T cells with an AML patient sample (herein referred to as AML-9). The patient cells are a mixed population of cells, e.g., leukemic cells, monocytes and other types of blast cells. CD33 and CD34 act as specific markers of leukemic cells and as indicators of CAR-T action. The CAR-T panel (right) is compared to control GFP-transduced T cells (middle). Killing efficacy is measured by the population of CD33+ / CD34+ cells remaining after at least 24 hours of incubation. [Figure 7] Co-culture assay by incubation of a B-ALL patient sample (herein referred to as Sp-BM-B6) with cCAR-T cells. The patient's cells are a mixed population of cells, e.g., leukemic cells, monocytes and other types of blast cells. CD34 acts as a specific marker for leukemic cells. The CAR-T panel (right) is compared to control GFP-transduced T cells (middle). 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 by incubation of cCAR NK-92 cells with HL-60. The performance of cCAR NK-92 cells 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 10] Co-culture assay by incubation of cCAR NK-92 cells with KG1a. Results of the cCAR NK cell panel are compared to those of 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 to HL-60 or KG1a by CD33CD123cCAR (CAR-CD33 / 123)NK-92 cells. Killing efficacy is measured by the population of CD33+ cells remaining after approximately 24 hours of incubation. [Figure 12] Comparison of the killing capacity of two populations of KG11 cells by CD33CD123 cCAR NK-92 cells with controls. Assays were performed at different cell ratios between CAR-CD33 / 123 (CD33CD123cCAR 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 13] Schematic representation of a cCAR. The construct contains an SFFV promoter that drives the expression of multiple modular units of the CAR linked by linkers. Upon cleavage of the linker, the cCAR splits 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 co-stimulatory domains, without limitation, 4-1BB (also called 4-BB) and / or CD28. [Figure 14A]Figure 14A-C. BCMA-CS1 cCAR construct diagram (BC1cCAR). (A) The construct consists of an SFFV promoter driving the expression of two modular units of CAR linked by a P2A peptide. Upon cleavage of this P2A peptide, the cCAR splits and engages targets expressing BCMA and / or CS1. The two unit CARs use the same co-stimulatory domain, 4-1BB. [Figure 14B] (B) Flow cytometry analysis of BC1cCAR expression shows that BC1cCAR (right, highlighted by a square) shows 15.3% positivity for F(Ab)2 compared to the isotype control (left). [Figure 14C] (C) Preliminary functional validation of BC1cCAR-T cells by co-culturing with K562 cells transduced with BCMA cDNA (BCMA-K562) (obtained from Kochenderfer, NIH). Bar graphs show solubility of BCMA-K562 cell line compared to control T cell activity as well as solubility of wild type K562 (wt-K562) compared to control. [Figure 14D] The BCMA-CS1-2G construct uses two different costimulatory domains, 4-1BB or CD28. The construct contains an SFFV promoter that drives the expression of two modular units of the CAR linked by a P2A peptide. Upon cleavage of this P2A peptide, the cCAR splits and engages targets expressing BCMA and / or CS1. The two unit CARs use different costimulatory domains, either 4-1BB or CD28. Flow cytometry analysis identified rare BC1cCAR expression on the T cell surface using F(Ab)2 (right, highlighted by a box). Gating was performed against an isotype control (left). [Figure 14E]Protein expression of BC1cCAR and BCMA-CS1-2G in HEK-293FT cells. HEK-293FT cells were transfected with lentiviral plasmids of GFP (lane 1), BC1cCAR (lane 2), and CD269-CS1-2G (lane 3). 48 hours after transfection, the supernatant was removed and the cells were harvested. The harvested cells were lysed and analyzed by Western blot with mouse anti-human CD3z antibody. [Figure 15] Co-culture with MM1S cell line. Co-culture was performed for less than 24 hours, and cells were harvested and analyzed by flow cytometry. Target MM1S cells (myeloma cells) were labeled with Cytotracker (CMTMR) dye to distinguish them from effector T cells. Populations were separated by anti-BCMA (CD269) and anti-CS1 (CD319) antibodies. Figure 15A, Co-culture results by flow cytometry. Figure 15B, Graphical summary of E:T ratio vs. solubility (right). [Figure 16] Co-culture with RPMI-8226 cell line. Co-cultures were performed for less than 24 hours, and cells were harvested and analyzed by flow cytometry. Target RPMI-8226 cells were labeled with Cytotracker (CMTMR) dye to distinguish effector T cells. Populations were distinguished by anti-BCMA (CD269) and anti-CS1 (CD319) antibodies. Figure 16A, Co-culture results by flow cytometry. Figure 16B, Graphical summary of E:T ratio and solubility. [Figure 17] Co-culture of U266 cell line. Co-culture was performed within 24 hours, cells were harvested and analyzed by flow cytometry. Target U266 cells were labeled with Cytotracker (CMTMR) dye to distinguish them from effector T cells. Populations were distinguished by anti-BCMA (CD269) and anti-CS1 (CD319) antibodies. (A) Flow cytometric results of co-culture. (B) Graphical summary of E:T ratio and lysis. [Figure 18A]Co-culture and specific lysis in MM10-G patient primary samples. Co-cultures were performed for less than 24 hours, and cells were collected and analyzed by flow cytometry. Target MM10-G cells were labeled with Cytotracker (CMTMR) dye to distinguish them from effector T cells. Populations were gated by anti-BCMA (CD269) and anti-CS1 (CD319) antibodies. Notably, gating shows that MM10-G presents distinct BCMA+ and CS1+ populations. Figure 18A, Co-culture results by flow cytometry. [Figure 18B] FIG. 18B, Graphical summary of E:T ratio and solubility. [Figure 19A] Co-culture and specific lysis in primary samples from MM7-G patients. Co-cultures were performed for less than 24 hours, and cells were harvested and analyzed by flow cytometry. Target MM7-G cells were labeled with Cytotracker (CMTMR) dye to distinguish them from effector T cells. Populations were gated by anti-BCMA (CD269) and anti-CS1 (CD319) antibodies. Figure 19A, Flow cytometric results of co-culture. [Figure 19B] FIG. 19B, Graphical summary of E:T ratio and solubility. [Figure 20A] Co-culture and specific lysis in primary samples from MM11-G patients. Co-cultures were performed for less than 24 hours, and cells were harvested and analyzed by flow cytometry. Target MM11-G cells were labeled with Cytotracker (CMTMR) dye to distinguish them from effector T cells. Populations were separated by anti-BCMA (CD269) and anti-CS1 (CD319) antibodies. Figure 20A, Co-culture results by flow cytometry. [Figure 20B] FIG. 20B, Graphical summary of E:T ratio and solubility. [Figure 21]CD269-CS1-BBCAR T cells exhibit anti-leukemic effects in vivo. Sublethally irradiated NSG mice were intravenously injected with luciferase-expressing MM.1S multiple myeloma cells the following day to induce measurable tumor formation. Three days later, mice were intravenously injected with 8x106 CD269-CS1-BBCAR NK cells or vector control NK cells. On days 3, 6, and 8, mice were subcutaneously injected with RediJect D-Luciferin and analyzed by IVIS imaging. The mean light intensity in mice injected with CD269-CS1-BBCAR NK and vector control T cells was compared. [Figure 22] Percent survival was determined and compared between the two groups based on the study in FIG. [Figure 23] 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 24] FIG. 1 is a schematic diagram providing an example of steps for generating CAR T or NK cells that target hematological malignancies. [Diagram 25] Generation of stable CD45 knockdown NK-92 cells using CRISPR / Cas9 lentiviral system and sorting of cells. Flow cytometry analysis showed the CD45 expression level on the surface of NK-92 cells (left panel). After transduction of sgCD45B CRISPR into NK-92 cells, the transduced cells were cultured in medium containing puromycin for several weeks. CD45 negative NK-92 cells were determined and sorted using CD45 antibody. The purity of stable NK45i-92 (CD45 knockdown) NK-92 cells was determined by flow cytometry analysis (right panel). This data indicates that NK45i-92 cells were successfully generated and obtained. [Figure 26]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 (▲) were counted 48 and 96 hours after seeding in 24-well plates. IL-2 was added at 48 hours. (n = 3 independent duplicate experiments). Data are mean + SD. These data showed that knockdown of the CD45 receptor on NK-92 cells showed similar cell proliferation curves compared to non-transduced or GFP-transduced NK-92 cells. [Figure 27A] Co-culture assay of CCRF-CEM (target:T) and GFP NK-92 cells or GFP NK45i-92 cells (effector:E) at a ratio of 5:1 (E:T). 16 h incubation. (A) Flow cytometric analysis of CCRF-CEM alone (blue dots in left panel), CCRF-CEM and control GFP-transduced NK-92 cells (middle 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 16 h and the effector T cell:target cell ratio was 5:1. All experiments were performed in duplicate. [Figure 27B] (B) Bar graph shows the percent of cell lysis by GFP-transduced NK45i-92 cells compared to control GFP-transduced NK92 cells in a co-culture assay with CCRF-CEM. These data suggest that knockdown of CD45 in NK-92 cells does not show significant differences in killing activity against CCRF-CEM cells compared to GFP-control NK-92 cells. Blue dots are in the upper left fraction. [Figure 28A]Co-culture assays with CCRF-CEM (target:T) and GFP NK-92, CD5CAR NK-92 or CD5CAR NK45i-92 cells (effector:E) at a ratio of 5:1 (E:T). Incubation for 16 hours. (A) Flow cytometry analysis of CCRF-CEM alone (left panel), CCRF-CEM and control GFP NK-92 cells (middle left panel), CD5CAR NK-92 cells (middle right panel), or CD5CAR 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 16 hours with a 5:1 effector T cell:target cell ratio. All experiments were performed in duplicate. [Figure 28B] (B) Bar graphs show the percent of cell lysis by CD5CAR NK-92 cells or CD5CAR NK45i-92 cells compared to control GFP NK92 cells in co-culture assays with CCRF-CEM. Data are mean + SD. Both CD5CAR NK cells and CD5CAR NK45i-92 cells show near 100% cell killing activity against CD5 positive CCRF-CEM compared to control GFP NK-92 cells. These data show that CD5CAR NK cells and CD5CAR NK45i-92 cells can effectively lyse CD5 expressing CCRF-CEM cells in in vitro co-culture assays compared to GFP control NK-92 cells and knockdown of CD45 has no effect on cell function on killing activity in NK-92 cells. The blue dots in the top left corner of the 4 fractions of the panels are seen in the first two panels from the left. [Figure 29A]The structure of CD45CAR and its expression configuration. (A) Schematic diagram of CD45CAR lentiviral vector. The CD45CAR configuration is a modularized signaling domain containing a leader sequence, anti-CD45scFv, hinge domain (H), transmembrane domain (TM), two costimulatory domains (CD28 and 4-1BB), defining it as a third generation CAR, and containing the intracellular signaling domain CD3 zeta. [Figure 29B] In (B), HEK-293FT cells were transfected with lentiviral plasmids for GFP (lane 1) and CD45CAR (lane 2). 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 antibody. [Figure 30A] Transduction of CD45CAR into NK45i-92 cells and sorting of CD45CAR-transduced cells. (A) After transduction of NK45i-92 cells with CD45CAR lentivirus, the expression level of CD45CAR on NK45i-92 (blue circled area in the right panel) was compared with NK45i-92 cells (left panel) by flow cytometry analysis. CD45CAR-expressing NK45i-92 cells were sorted, and the expression level of CD45 on the cell surface was measured by flow cytometry analysis. [Figure 30B] (B) Approximately 87% CD45CAR expression on the cell surface was detected by flow cytometry analysis. [Figure 31A]Co-culture assay with CCRF-CEM (target:T) and GFP NK-92 or CD45CAR NK45i-92 cells (effector:E). 5:1 (E:T) ratio. 16 hours of incubation. (A) Flow cytometry analysis of CCRF-CEM and co-culture with 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. All incubation times were 16 hours, with a 5:1 effector T cell:target cell ratio. All experiments were performed in duplicate. [Figure 31B] (B) Bar graph shows the percentage of cell lysis by CD45CAR NK45i-92 cells in a 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 an in vitro co-culture assay compared to GFP-control NK-92 cells. [Figure 32A] Co-culture assay with Jurkat cells (target:T) and GFP-control or CD45CAR NK45i-92 cells (effector:E) at 5:1 or 2:1 (E:T) ratios for 6 hours of incubation. (A) 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 cells (right panel). [Figure 32B](B) Flow cytometric analysis of co-culture assays using Jurkat cells (target:T) and control or CD45CAR NK45i-92 cells (effector:E). Co-culture assays were performed at ratios of 5:1 or 2:1 (E:T). The left panel shows the results of co-cultures with control GFP or CD45CAR NK45i-92 cells at a ratio of 5:1 (E:T), while the right panel shows the results of co-cultures with control GFP or CD45CAR NK45i-92 cells at a ratio of 2:1 (E:T). The blue dots in the panels indicate the remaining target Jurkat cells after the co-culture assay, and the red dots indicate the effector cells after the co-culture assay. All incubation times were 6 hours. All experiments were performed in duplicate. [Figure 32C] (C) Bar graphs show the percentage of cell lysis by CD45CAR NK45i-92 cells compared to control GFP NK92 cells at a 5:1 or 2:1 (E:T) ratio. Data are mean + SD. CD45CAR NK45i-92 cells show approximately 60% cell lysis rate 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 in an in vitro co-culture assay compared to GFP control NK-92 cells. [Figure 33A] Figure 33A-C. Co-culture assays with GFP-NK-92 cells (target:T) and non-transduced NK-92 cells or CD45CAR NK45i-92 cells (effector:E) at 5:1 or 2:1 (E:T) ratios. 6 hours of incubation. (A) Flow cytometry analysis with GFP-control NK-92 cells. These data demonstrate that approximately 99% of GFP-control NK-92 cells are GFP-positive cells (green dots). [Figure 33B](B) Flow cytometry analysis of co-culture assays with GFP control NK-92 cells (target:T) and non-transduced or CD45CAR NK45i-92 cells (effector:E). Co-culture assays were performed at a ratio of 5:1 or 2:1 (E:T). Left panels show co-cultures with non-transduced or CD45CAR NK45i-92 cells at a ratio of 5:1 (E:T), and right panels show co-cultures with non-transduced or CD45CAR NK45i-92 cells at a ratio of 2:1 (E:T). Green dots in the panels indicate target GFP NK-92 cells left by 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. [Figure 33C] (C) Bar graphs show the percentage of cell lysis of GFP NK-92 cells by CD45CAR NK45i-92 cells compared to non-transduced NK-92 cells at 5:1 or 2:1 (E:T) ratios. Data are mean + SD. CD45CAR NK45i-92 cells showed lysis of approximately 20% of GFP NK-92 cells at 2:1 (E:T) ratio and approximately 55% of GFP 92 cells at 5:1 (E:T) ratio compared to non-transduced 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 non-transduced NK-92 cells. Green dots are seen in the top right fraction of each panel. [Figure 33D] Transduction of CD45b-BB or CD45b-28 into NK45i-92 cells and sorting of CD45b-BB or CD45b-28 NK45i-92 transduced cells. (D) 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 (middle panel, blue boxed area) were examined by flow cytometry analysis compared to NK45i-92 cells (left panel). [Figure 33E](E) NK45i-92 cells expressing CD45b-BB or CD45b-28 CAR were sorted by flow cytometry analysis, which detected approximately 74% CD45b-BB CAR or approximately 82% CD45b-28 CAR expression on the cell surface. [Figure 33F] Figure 33F-G. Co-culture assay with REH cells (target:T) and GFP NK-92 cells, CD45CAR NK45i-92 cells, CD45b-BB NK45i-92 cells or CD45b-28 NK45i-92 cells (effector:E). 5:1 (E:T) ratio. 20 hour incubation. (F) Flow cytometric analysis of co-cultures with 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 left panel) or CD45b-28 NK45i-92 cells (right panel). Blue dots in all panels indicate remaining target REH cells after co-culture assay, and red dots indicate effector GFP or CARs-NK-92 cells after co-culture assay. REH is a B acute lymphoblastic cell line. All incubation times were 20 hours, and the effector NK cell:target cell ratio was 5:1. All experiments were performed in duplicate. [Figure 33G] (G) Bar graphs show 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 co-culture assays with REH cells. Data are mean + SD. Compared to control GFP NK-92 cells, CD45CAR NK45i-92 cells lyse approximately 76% of the cells, CD45b-BB NK45i-92 cells show approximately 79% cell lysis, and CD45b-28 NK45i-92 cells show 100% cell lysis against REH cells. These data suggest that all three CD45CARs effectively lyse REH cells. [Figure 34A]Schematic diagram to explain the structure and its expression in T or NK cells. (A) The combination of CAR (3rd generation) and sushi / IL-15 was constructed on an expression vector, and their expression was induced by the SFFV promoter. The CAR containing sushi / IL-15 is linked to a P2A cleavage sequence. The sushi / IL-15 part is composed of an IL-2 signal peptide linked to IL-5 via a 26 amino acid polyproline linker, fused to the sushi domain. [Figure 34B] (B) CAR and sushi / IL15 are present on T or NK cells. [Figure 35A] Expression of CD4IL15RA-CAR. (A) HEK-293FT cells were transfected with lentiviral plasmids for GFP (lane 1) and CD4IL15RA CAR (lane 2), and positive control CD4CAR (lane 3). 48 hours after transfection, the supernatant was removed and cells were harvested for Western blot with mouse anti-human CD3z antibody. [Figure 35B] (B) HEK-293 cells were transduced with either GFP (left) or CD4IL15RA-CAR (right) viral supernatants 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. [Diagram 36] Transduction of NK cells with CD4IL15RACAR. NK-92 cells were transduced with either GFP (left) or CD4IL15RACAR (right) viral supernatant from transfected HEK-293FT cells. The 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 on fresh medium containing 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 1:250 for 30 minutes. Cells were washed and stained with streptavidin-PE conjugate at 1:500 ratio, then washed, suspended in 2% formalin, and analyzed by flow cytometry. [Figure 37] Transduction of CD4IL15RACAR into T cells. Left: Western blot results. HEK-293FT cells were transfected with lentiviral plasmids for GFP (lane 1) and CD4IL15RA-CAR (lane 2). 48 hours after transfection, the supernatant was removed and cells were harvested for Western blot analysis using mouse anti-human CD3 zeta antibody. Right: CD4IL15RACAR expression. Activated T cells derived from umbilical cord blood buffy coats were transduced with either GFP (left) or CD4IL15RACAR (right) viral supernatants 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 were harvested and stained with goat anti-mouse F(Ab')2 antibody or isotype control for 30 minutes. Cells were transduced with either GFP (left) or CD4IL15RA (right), washed, stained with streptavidin-PE conjugate at a 1:250 ratio, washed, suspended in 2% formalin, and analyzed by flow cytometry. [Figure 38A] CD4CAR NK-92 cells and CD4IL15RA CAR NK-92 cells kill KARPAS 299 T leukemia cells in co-culture. (A) NK-92 cells transduced with either GFP control (top right), CD4CAR (bottom left), or CD4IL15RA (bottom right) lentiviral supernatant were incubated with KARPAS 299 cells at a 5:1 ratio. After 4 hours of co-culture, 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 labeling of only Karpas 299 cells. [Figure 38B] (B) The percentage of lysed target cells is shown in the graph. [Figure 39]CD4CAR NK-92 cells and CD4IL15RA CAR NK-92 cells kill MOLT4 T leukemia cells expressing CD4 in co-culture. NK-92 cells transduced with either GFP control (left), CD4CAR (middle) or CD4IL15RA (second from the right) lentiviral supernatants were incubated with MOLT4 cells at an effector:target ratio of 1:1 or 2:1. After overnight co-culture, cells were stained with mouse anti-human CD4 (APC) and CD56 (PerCp) antibodies and analyzed by flow cytometry (N=2). The top right panel shows only labeled MOLT4 cells. The percentage of lysed target cells is shown on the graph. [Diagram 40] CD4IL15RACAR T cells exhibit more potent anti-leukemia effects in vivo than CD4CAR. NSG mice were sublethally irradiated and injected intravenously (tail vein) with luciferase-expressing MOLM13 cells the following day to induce measurable tumor formation. Three days later, mice were injected intravenously with one therapeutic unit of 8x106 CD4CAR, or CD4IL15RACAR 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. [Diagram 41] Measurement and comparison of tumor shrinkage rates for three types of mice based on the study in Figure 40. The mean light intensity measured for mice injected with vector control T cells and mice injected with CD4CAR and CD4IL15RACAR T cells correlated with tumor shrinkage rates. For each of the two sets, the results for CD4CAR T are shown on the left and the results for CD4IL15RA CAR T are shown on the right. [Diagram 42] HEK293 cells were transduced with either EF1-GFP or SFFV-GFP viral supernatant at the indicated volumes in DMEM medium containing 10% FBS in 6-well tissue culture plates. Culture medium was changed the following morning. After 48 hours, GFP signals in transduced cells were visualized under an EVOS fluorescence microscope at 10x magnification. [Diagram 43]Based on the data in Figure 42, HEK293 cells transduced with either EF1-GFP or SFFV-GFP viral supernatant were harvested by trypsinization, suspended in formalin, and the percentage of GFP+ cells was identified by flow cytometry analysis using the FITC channel. [Diagram 44] Activated cord blood buffy coat T cells transduced with either low or high doses of EF1-GFP or SFFV-GFP viral supernatant were cultured and the percentage of GFP+ cells 7, 14, 21, and 28 days after transduction were analyzed by flow cytometry using the FITC channel after suspending the cells in formalin. (A) Percentage of GFP+ T cells for cells transduced with either low or high doses of viral supernatant. (B) Comparison of the percentage of GFP+ T cells transduced with high amount of EF1-GFP supernatant to the percentage of GFP+ cells in T cells transduced with low amount of SFFV-GFP supernatant. (50 μL of SFFV-GFP and 1 mL of EF1-GFP supernatant were used). (N=2). [Diagram 45] Ligand-receptor interactions in malignant plasma cells. APRIL ligand binds to TAC1 or BCMA. BAFF ligand binds to TAC1, BCMA, or BAFF-R. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present disclosure provides chimeric antigen receptor (CAR) constructs, methods of making and using same.

[0021] 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.

[0022] The first generation CAR contains CD3z as an intracellular signaling domain, while the second generation CAR contains at least one single costimulatory domain from various proteins. Examples of costimulatory domains include, but are not limited to, CD28, CD2, 4-1BB (CD137, also called "4-BB"), and OX-40 (CD124). The third generation CAR contains, but is not limited to, two costimulatory domains, such as CD28, 4-1BB, CD134 (OX-40), CD2 and / or CD137 (4-1BB).

[0023] As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably to 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 have. A polypeptide includes any peptide or protein having two or more amino acids linked together by peptide bonds. As used herein, it refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, commonly referred to in the art as proteins, of which there are many varieties. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0024] A "signal peptide" is a peptide sequence that directs transport and localization, and includes any peptide that is capable of binding to a cell organelle (such as the endoplasmic reticulum) and / or the cell surface.

[0025] A signal peptide, as used herein, is any secretory or transmembrane protein peptide or polypeptide that directs transport to the cell membrane and cell surface and provides correct localization. In particular, a signal peptide, as used herein, is a polypeptide, as used herein, that directs transport to the cell membrane and cell surface, and 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.

[0026] In this example, the signal peptide is a signal peptide that is cleaved after passing 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.

[0027] An "antigen recognition domain" includes a polypeptide that is selective for a target antigen, receptor, peptide ligand, or protein ligand or is selective for a targeting polypeptide.

[0028] The antigen recognition domain can be derived from any of a wide variety of extracellular domains or secreted proteins associated with 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 combined with 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 embodiment, the antigen recognition domain can be a receptor or a ligand. In a particular embodiment, 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 the compound CAR structure. In a particular embodiment, the chimeric receptor can be for any cancer for which a specific monoclonal or polyclonal antibody exists or can be made. 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.

[0029] The target-specific antigen recognition domain preferably comprises an antigen-binding domain capable of targeting an antibody against the target's antigen or a peptide that binds to the target's antigen, or a peptide or protein that binds to an antibody that binds to the target's 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 peptide or protein ligand that binds to the target (including but not limited to a growth factor receptor, cytokine receptor, or hormone receptor).

[0030] In this example, the antigen recognition domain comprises the binding portion or variable region of a monoclonal or polyclonal antibody directed against the target.

[0031] In another embodiment, the antigen recognition domain comprises a camelid single domain antibody or a portion thereof. In this embodiment, the camelid single domain antibody comprises a heavy chain antibody found in a camelid, or a VHH antibody. 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 isolated VHH antibodies from camelids, recombinant VHH antibodies derived from camelids, or camelid VHH synthetic antibodies.

[0032] In another embodiment, the antigen recognition domain comprises a ligand that engages their cognate receptor. As an example, APRIL is a ligand that binds to the TAC1 receptor or the BCMA receptor. According to 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. According to the invention disclosed herein, the antigen recognition domain comprises BAFF or a fragment thereof. In another embodiment, the antigen recognition domain is humanized.

[0033] It is understood that an antigen recognition domain can contain some variability in its sequence and still be selective for the target 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 polypeptide disclosed herein and still be considered as described herein and within the scope of this disclosure.

[0034] 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, These include CD30, EGFRvIII, immunoglobulins kappa and lambda, CD38, CD52, CD3, CD4, CD8, CD5, CD7, CD2, and CD138.

[0035] 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 / Any of the following proteins may be included: neu, 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.

[0036] 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 / These include exposed surface sites of neu, 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.

[0037] In another embodiment, 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.

[0038] In this example, the TACI antigen recognition domain comprises SEQ ID NO:24.

[0039] In this example, the BCMA antigen recognition domain comprises SEQ ID NO:25.

[0040] In this example, the CS1 antigen recognition domain comprises SEQ ID NO:26.

[0041] In this example, the BAFF-R antigen recognition domain comprises SEQ ID NO:27.

[0042] In this example, the CD33 antigen recognition domain comprises SEQ ID NO:28.

[0043] In this example, the CD123 antigen recognition domain comprises SEQ ID NO:29.

[0044] In this example, the CD19 antigen recognition domain comprises SEQ ID NO:30.

[0045] In this example, the CD20 antigen recognition domain comprises SEQ ID NO:31.

[0046] In another embodiment, the CD20 antigen recognition domain comprises SEQ ID NO:32.

[0047] In this example, the CD22 antigen recognition domain comprises SEQ ID NO:33.

[0048] In this example, the CD45 antigen recognition domain comprises SEQ ID NO:34.

[0049] For example, but not limited to, a hinge region is located between the sequences of the chimeric antigen receptor and at least one of the costimulatory domain and the signaling domain.

[0050] The hinge sequence can be obtained from any suitable sequence from any genus, including, for example, human or a portion thereof. Such hinge regions are known in the art. In this embodiment, the hinge region comprises a 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.

[0051] In this example, the hinge region comprises a CD8 hinge region.

[0052] In some examples, the hinge region includes, but is not limited to, those selected from immunoglobulins (eg, IgG1, IgG2, IgG3, IgG4, and IgD).

[0053] 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).

[0054] A transmembrane domain may be in the shape of an alpha-helical or beta-barrel, or a combination thereof. Transmembrane domains include polytopic proteins, which have many transmembrane segments, each alpha-helix, beta sheet, or a combination thereof.

[0055] In this embodiment, a transmembrane domain that is naturally associated with one of the domains of the CAR structure is used, in another embodiment, 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.

[0056] For example, transmembrane domains include the transmembrane domains of the T cell receptor alpha or beta chains, the CD3 zeta chain, CD28, CD3 epsilon, 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.

[0057] 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.

[0058] In this embodiment, the transmembrane domain is a CD8 transmembrane domain. In another embodiment, the transmembrane domain is a CD28 transmembrane domain. Such transmembrane domains are known in the art.

[0059] The signaling domain and the costimulatory domain comprise a polypeptide for activating an immune cell or activating at least some aspect of an immune cell signaling pathway.

[0060] In this example, the signaling domain includes polypeptides of functional signaling domains thereof, including CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIa, FcR beta (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.

[0061] In this embodiment, the CAR polypeptide comprises one or more costimulatory domains, which in this embodiment are functional signaling domains derived from proteins including ligands that bind at least one of OX40, CD27, CD28, CD30, CD40, PD-1, CD2, 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), active fragments thereof, functional derivatives thereof, and combinations thereof.

[0062] 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 may be collectively referred to as the extracellular domain.

[0063] The present disclosure further provides polynucleotides encoding the above-described chimeric antigen receptor polypeptides.

[0064] The term "polynucleotide," as used herein, is defined as a chain of nucleotides.

[0065] Polynucleotide includes DNA and RNA. Furthermore, nucleic acid is a polymer of nucleotides. Therefore, as used herein, nucleic acid and polynucleotide have the same meaning. Those skilled in the art have the general knowledge that nucleic acid is a polynucleotide, which can be hydrolyzed into monomeric "nucleotides". Monomeric nucleotides can be hydrolyzed into nucleosides. Polynucleotide as used herein includes, but is not limited to, any nucleic acid sequence obtained by any means available in the art, i.e., by conventional cloning techniques from recombinant libraries or cells and by synthetic means such as polymerase chain reaction (PCR).

[0066] A polynucleotide encoding a CAR can be easily prepared from the amino acid sequence of a particular CAR by any conventional method. The base 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, and the nucleic acid of the present invention can be prepared by standard molecular biology and / or chemical procedures. For example, a polynucleotide can be synthesized based on the base sequence, and the polynucleotide of the present invention can be prepared by combining DNA fragments obtained from a cDNA library using polymerase chain reaction (PCR).

[0067] In this example, the polynucleotides disclosed herein are part of a gene, an expression cassette or a cloning cassette.

[0068] The above polynucleotides can be cloned into vectors. A "vector" is a construct that contains an isolated polynucleotide and is used to deliver the isolated polynucleotide to the inside of 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. The term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like. In this example, vectors include cloning vectors, expression vectors, replication vectors, probe generation vectors, integration vectors, and sequencing vectors.

[0069] In this example, the vector is a viral vector. In this example, the viral vector is a retroviral vector or a lentiviral vector. In one embodiment, the engineered cells are transduced with a virus to express the polynucleotide sequence.

[0070] 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 the patient's cells by either in vivo or ex vivo means. Many retroviral systems are known in the art. In some examples, an adenoviral vector is used. Many adenoviral vectors are known in the art. In this example, a lentiviral vector is used.

[0071] Viral vector technology is well known in the art and 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, herpes viruses and lentiviruses.In general, suitable vectors include a replication origin that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0072] Lentiviral vectors are well known for their ability to transfer genes into human T cells with high efficiency, but expression of the vector-encoded genes depends on an internal promoter that drives their expression. Strong promoters are particularly important for the creation of third or fourth generation CARs, because the construct size of increased CARs with genes encoding additional costimulatory domains or proliferative cytokines does not guarantee equal expression. A variety of 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 body and maintain expression over a long period of time. The EF-1α promoter is a commonly chosen promoter for CAR expression.

[0073] The present invention relies on expression vectors with strong promoters for high-level gene expression in T cells or NK cells. In further embodiments, the inventors disclose strong promoters useful for high-level expression of CARs in T cells or NK cells. In a particular embodiment, a strong promoter associated with the SFFV promoter can be selectively introduced into an expression vector to obtain high levels of expression in T cells or NK cells and maintain expression for a long period of time. The expressed CARs, T cell costimulators and cytokines are used for immunotherapy.

[0074] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence, which is a strong constitutive promoter sequence capable of inducing high levels of expression in any manipulated polynucleotide sequence linked to it.

[0075] Another example of a suitable promoter is elongation growth factor-1a (EF-1a). However, other constitutive promoter sequences may also be used, including, but not limited to, 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 actin promoter, myosin promoter, hemoglobin promoter, creatine kinase promoter, etc. Furthermore, the present disclosure should not be limited to the use of constitutive promoters, and inducible promoters are also considered part of the present disclosure. The use of an inducible promoter provides a molecular switch that can turn on expression when expression of the linked polynucleotide sequence is desired or can turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0076] 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 11 alpha (EF) promoter, the CAG (chicken β-actin promoter with CMV enhancer) promoter, or the human elongation factor 1 alpha (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 element including all generations, preferably the third generation), inducible promoters (Clontech Laboratories, Mountain View, CA), or portions thereof, or combinations thereof.

[0077] In a preferred embodiment, the promoter is an SFFV promoter or a derivative thereof. Unexpectedly, it has been discovered in the present disclosure that the SFFV promoter shows stronger expression and higher persistence in transduced cells.

[0078] "Expression vector" refers to a vector that contains a recombinant polynucleotide that includes an expression control sequence operably linked to the 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 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. An expression vector can be a bicistronic or multicistronic expression vector. 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.

[0079] In this example, the disclosure provides engineered cells harboring at least one chimeric antigen receptor polypeptide or polynucleotide.

[0080] "Genetically engineered cells" refers to any cell of any organism that has been modified, transformed, or engineered by the addition or modification of a gene, DNA or RNA sequence, or a 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 the 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 the treatment of infectious diseases.

[0081] In this example, the engineered cells include immunoregulatory cells. Immunomodulatory cells include T cells, such as CD4 T cells (helper T cells), CD8 T cells (cytotoxic T cells, CTL), and memory T cells or T memory stem cells. In another example, the T cells include natural killer T cells (NK T cells).

[0082] In this embodiment, the engineered cells include natural killer cells. Natural killer cells are well known in the art. In this embodiment, 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.

[0083] NK cells provide antitumor effects without the risk of GvHD and are short-lived compared to T cells, therefore reducing the need for inducible suicide gene transfer in CAR configurations to clear modified cells, as NK cells are depleted soon after destroying cancer cells.

[0084] In accordance with the present disclosure, it has surprisingly been found that NK cells are cells that can be readily engineered to contain and express the chimeric antigen receptor polypeptides disclosed herein and then be readily accessible.

[0085] Because allogeneic or autologous NK cells induce rapid immune responses but have a limited life span and are cleared relatively quickly from the blood circulation, applicants have surprisingly discovered that there is less concern about persistent side effects when using CAR cell-based therapies.

[0086] This disclosure includes methods of making cCARs. In some embodiments, the cCARs are made using T cells. In other embodiments, the cCARs use primary NK cells or NK-92 cells isolated from peripheral or umbilical cord blood and are administered "off the shelf" to any mammal with disease or cancer.

[0087] According to one aspect of the invention, NK cells can be expanded and transfected with CAR polynucleotides 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 invention, NK-92 cells could be expanded and transfected with CAR. NK-92 is a continuously growing cell line that has 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 to be feasible. CAR-expressing NK-92 cells can be expanded in serum-free medium with or without co-culture with feeder cells. A pure population of NK-92 cells carrying the CAR of interest can be obtained by sorting.

[0088] In this example, the engineered cells include 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).

[0089] 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 altered to delete the native CD45 gene to prevent its expression and presence on the cell surface.

[0090] In some embodiments, the engineered cells carry an inducible suicide gene ("safety switch") or a combination of safety switches that can be incorporated on a vector, such as a retroviral vector, a lentiviral vector, an adenoviral vector, or a plasmid. The introduction of a "safety switch" greatly improves the safety profile of the compound CAR, limiting the target tumor or off-target tumor toxicity. "Safety switches" are inducible suicide genes, such as, but not limited to, the caspase 9 gene, thymidine kinase, cytosine deaminase (CD) or cytochrome P450. Other safety switches to eliminate undesirable modified T cells include the expression of CD20 or CD19 or truncated epidermal growth factor receptor in T cells. All possible safety switches are contemplated and are encompassed by the present invention.

[0091] In some embodiments, the suicide gene is integrated into the engineered cell genome.

[0092] In this example, the disclosure provides an engineered cell having 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.

[0093] Multiple CAR unit The present disclosure provides details of engineered cells that have at least two different CAR polypeptides.

[0094] As used herein, compound CAR (cCAR) or multiple CAR refers to an engineered cell with 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 signal transduction 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 signal transduction 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 that code for them as well.

[0095] 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.

[0096] In some embodiments, compound CARs target the same antigen. For example, cCARs target different epitopes or different 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 targets a different antigen specific to a side effect caused by a disease state.

[0097] In some embodiments, the compound CAR targets two different antigens.

[0098] It is very challenging to create compound CARs with various CAR units.

[0099] It is very challenging to create compound CARs with various CAR units.

[0100] (1) CAR-CAR interaction may have a detrimental effect, and appropriate CAR design is key to offset this adverse effect, (2) single-component compound CAR may increase the length of the expression cassette, which may result in a decrease in viral titer and protein expression level, (3) appropriate design is required to select a strategy, especially including various CAR body elements, for expressing multiple CARs in a single vector, (4) strong promoters are particularly important for compound CARs with additional units of CAR, (5) the hinge region of CAR should be designed such that the interaction of the hinge region between each CAR unit is preferably avoided, (6) two or more CAR units expressed in a cell may cause toxic effects (CAR and CAR interaction). Applicant provides novel and surprising CAR compositions and methods to overcome these hurdles.

[0101] In this embodiment, the present disclosure provides engineered cells with multiple CAR units, which allows a single engineered cell to target multiple antigens. Targeting multiple surface markers or antigens simultaneously with multiple CAR units prevents the selection of resistant clones and reduces tumor recurrence. Multiple CAR T cell immunotherapy with individual CAR configurations containing various domains and activation sites has not yet been deployed for any malignant tumor.

[0102] In one aspect of the invention, the cCAR comprises multiple CAR units. In some embodiments, the cCAR comprises at least two CAR units. In other embodiments, the cCAR comprises at least three CAR units. In other embodiments, the cCAR comprises at least four units.

[0103] In this example, the disclosure provides an engineered cell that has at least two different chimeric antigen receptor polypeptides, each having a different antigen recognition domain.

[0104] In a preferred embodiment, the engineered cells having 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.

[0105] 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, (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, where the first antigen recognition domain is different from the second antigen recognition domain.

[0106] In a preferred embodiment, each engineered CAR unit polynucleotide has a different nucleotide sequence to avoid homologous recombination.

[0107] In this example, the target of the first antigen recognition domain is interleukin 6 receptor, NY-ESO-1, alpha fetoprotein (AFP), glypican-3 (GPC3), BAFF-R, BCMA, TACI, LeY, CD5, CD13, CD14, CD15, CD19, CD20, CD22, CD33, CD41, CD61, CD64, CD68, CD117, CD123, CD138, CD267, CD269, CD38, Flt3 receptor, and and CS1, and a target of the second antigen recognition domain is selected from the group consisting of interleukin 6 receptor, NY-ESO-1, alpha-fetoprotein (AFP), glypican-3 (GPC3), BAFF-R, BCMA, TACI, LeY, CD5, CD13, CD14, CD15, CD19, CD20, CD22, CD33, CD41, CD61, CD64, CD68, CD117, CD123, CD138, CD267, CD269, CD38, Flt3 receptor, and combinations thereof.

[0108] 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 recognition domain. In this example, the engineered cell comprises the polypeptide of SEQ ID NO:3 and a polynucleotide corresponding to SEQ ID NO:4.

[0109] 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.

[0110] 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.

[0111] 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 a polypeptide of SEQ ID NO:9 and a polynucleotide corresponding to SEQ ID NO:10. In another example, the engineered cell comprises a polypeptide of SEQ ID NO:11 and a polynucleotide corresponding to SEQ ID NO:12.

[0112] In this example, the engineered cell contains 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.

[0113] 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 a polypeptide of SEQ ID NO: 19 and a polynucleotide corresponding to SEQ ID NO: 20. In this example, the engineered cell comprises a polypeptide of SEQ ID NO: 21 and a polynucleotide corresponding to SEQ ID NO: 22.

[0114] 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.

[0115] In this embodiment, each CAR unit comprises the same or a different hinge region. In another embodiment, each CAR unit comprises the same or a different transmembrane region. In another embodiment, each CAR unit comprises the same or a different intracellular domain.

[0116] In this example, each CAR unit contains a CD3 zeta chain signaling domain.

[0117] In this example, each separate CAR unit contains a different costimulatory domain to avoid interactions, e.g., a first chimeric antigen receptor polypeptide contains a 4-BB costimulatory domain and a second chimeric antigen receptor polypeptide contains a CD28 costimulatory domain.

[0118] In another embodiment, 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 embodiment, the hinge region is designed to eliminate or minimize hydrophobic residues to prevent undesired hydrophobic interactions.

[0119] Compound CAR can kill targets 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, hinge regions are selected to avoid interaction sites.

[0120] The compound CARs of the present invention can target the same or different tumor populations in T cells or NK cells. For example, a first CAR may target a bulk tumor population, and a next or second CAR may eradicate, for example, cancer or leukemia stem cells to avoid cancer recurrence.

[0121] According to the present invention, it has been surprisingly 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 causing downregulation of target antigens from the cancer. Surprisingly, it has also been found that this allows cancer cells to "hide" from CAR therapy due to "antigen escape" and tumor heterogeneity, with different tumor cells exhibiting different surface antigen expression profiles.

[0122] Engineered Cells with CAR Polypeptides and Enhancers In another example, the disclosure provides an engineered cell comprising at least one chimeric antigen receptor polypeptide and an enhancer.

[0123] In this example, the disclosure provides an engineered cell having at least two different chimeric antigen receptor polypeptides and an enhancer.

[0124] As used herein, enhancers include biological molecules that enhance or potentiate the activity of engineered cells having a chimeric antigen receptor polypeptide. Enhancers also include cytokines. In another embodiment, enhancers include IL-2, IL-7, IL-12, IL-15, IL-21, PD-1, PD-L1, CSF1R, CTAL-4, TIM-3, and TGFR-beta, its receptors, and functional fragments.

[0125] 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 of surface display and secretion are well known in the art. For example, the enhancer may be a fusion protein with a peptide that provides surface display or secretion into the extracellular space.

[0126] The effect of the 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.

[0127] In this example, the enhancer is IL-15. In this case, the additional factor is the IL-15 receptor and functional fragments thereof. Functional fragments include the IL-15 receptor, IL-15RA, and the sushi domain of IL-15RA. An example of a suitable sushi domain is contained in SEQ ID NO: 35. According to the present disclosure, any chimeric antigen receptor polypeptide disclosed herein comprises human interleukin 15 with human interleukin 2 signal peptide, SEQ ID NO: 36.

[0128] 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 are modified to express autocrine growth factors, including but not limited to IL-2, IL-7, IL21 or IL-15, to maintain survival after transfer in vivo. 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 expresses some or more than half of IL-15, IL15RA and IL-15 / IL-15R or IL15-RA / IL-15 or a combination thereof to enhance CAR T or NK survival or proliferation and improve memory CAR CD8+ T cell proliferation.

[0129] The present disclosure relates to engineered cells having any one or more of the CARs and IL-15, IL15RA and IL-15 / IL-15R or IL15-RA / IL-15, or portions or combinations described herein, that are used to treat cancer in patients by enhancing survival or persistence or proliferation of CAR T or NK.

[0130] In this example, the engineered cells include a CD4 chimeric antigen receptor polypeptide and IL-15RA (SEQ ID NO:1) and the corresponding polynucleotide (SEQ ID NO:2).

[0131] Methods for producing engineered cells Any of the polynucleotides disclosed herein can be introduced into engineered cells by any method known in the art.

[0132] In this example, the CAR polynucleotide is delivered to the engineered cell by any of the viral vectors disclosed herein.

[0133] In this example, any engineered cell disclosed herein may be constructed as a transient RNA-modified "biodegradable" version or derivative, or combination thereof, to achieve an improved safety profile or therapeutic index. The RNA-modified CAR of the present invention may be electroporated into T cells or NK cells. Expression of the compound CAR may be gradually decreased over several days.

[0134] 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.

[0135] Methods for producing engineered cells with multiple CAR units In another embodiment, the disclosure provides a method of producing an engineered cell having at least two CAR units.

[0136] In some embodiments, multiple units of the CAR are expressed in T cells or NK cells using bicistronic or multicistronic expression vectors. There are several strategies that can be used to construct bicistronic or multicistronic vectors, including but not limited to: (1) multiple promoters fused to the open reading frame of the CAR, (2) insertion of splicing signals between the units of the CAR, fusing the CAR whose expression is driven by a single promoter, (3) insertion of proteolytic cleavage sites (self-cleaving peptides) between the units of the CAR, and (iv) insertion of an internal ribosome entry site (IRES).

[0137] In a preferred embodiment, multiple CAR units are expressed in a single open reading frame (ORF), thereby generating a single polypeptide having multiple CAR units, in which an amino acid sequence or linker containing a high efficiency cleavage site is placed between each CAR unit.

[0138] 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.

[0139] Furthermore, in preferred embodiments, equal amounts of the truncations are present as shown by Western blot analysis.

[0140] 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 Flacheria Infection 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.

[0141] In the present embodiment, multiple CAR units are expressed under a single open reading frame (ORF), and expression is under the control of a strong promoter. Examples of strong promoters include the SFFV promoter and its derivatives.

[0142] The engineered cells have a CAR polypeptide and an enhancer In another example, the disclosure provides a method of generating an engineered cell that expresses at least one CAR unit and an enhancer.

[0143] 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. There are several strategies that can be used to construct bicistronic or multicistronic vectors, including but not limited to: (1) multiple promoters fused to the open reading frame of the CAR, (2) insertion of a splicing signal between the units of the CAR and fusing the CAR whose expression is driven by a single promoter, (3) insertion of a proteolytic cleavage site (self-cleaving peptide) between the units of the CAR, and (4) insertion of an internal ribosome entry site (IRES).

[0144] In a preferred embodiment, multiple CAR units are expressed in a single open reading frame (ORF), thereby producing a single polypeptide with 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.

[0145] Furthermore, in preferred embodiments, equal amounts of the truncations are present as shown by Western blot analysis.

[0146] Methods of Treatment Using the Compositions Disclosed Herein In another embodiment, the present invention provides a method of 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%-90% of acute lymphocytic and myeloid leukemias express CD45. CD45 is not found in non-hematopoietic origins. Furthermore, CD45 is expressed at high density with an average copy number of about 200,000 molecules per cell on malignant cells and leukocytes. CD45 is an ideal target for various hematologic malignancies. However, CAR T and NK cells also express CD45. Without inactivation of endogenous CD45, arming CAR T or NK cells to target CD45 may result in self-killing.

[0147] 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, namely, 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 called "antigen escape." The present disclosure preempts cancer antigen escape by recognizing a combination of two or more antigens to rapidly eliminate tumors.

[0148] The present invention provides a method for simultaneous targeting of multiple antigens using cCAR, improving tumor control by minimizing the possibility of tumor selection based on loss or downregulation of target antigens.

[0149] The disclosed invention includes compound (multiple or compound) cCARs on 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 that are linked by a linker and target the same or different antigens. For example, each CAR construct present in the compound CAR (cCAR) construct includes an antigen recognition domain, an extracellular domain, a transmembrane domain, and / or a cytoplasmic domain. The extracellular domain and the transmembrane domain can be from any such desired domain. The multiple CAR constructs are linked by a linker. The expression of the compound CAR construct is induced by a promoter. The linker may be a part of a peptide or protein that is self-cleaved after the protein or peptide is produced (also called a self-cleaving peptide).

[0150] In this example, the compound CAR of the present invention targets myelodysplastic syndromes and acute myeloid leukemia (AML). Myelodysplastic syndromes (MDS) are refractory hematopoietic stem cell malignancies that occur frequently 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. MDS and AML have been significantly studied, but no satisfactory treatments have been developed.

[0151] 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.

[0152] 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. Various effector cells include cytotoxic T cells, NK cells and NK-92 cells. The compositions and methods described in the present invention may be used in conjunction with other types of therapy for cancer, such as chemotherapy, surgery, radiation therapy, gene therapy, etc. The compositions and methods described in the present invention may also be used in other diseases that depend on immune response, such as inflammation, immune diseases and infectious diseases.

[0153] In some embodiments, the compound CARs of the invention may serve as a bridge to bone marrow transplantation by achieving complete remission for patients with minimal residual disease who are no longer responding to chemotherapy. In other embodiments, the compound CARs eliminate leukemic cells and support leukopenia with subsequent bone marrow stem cell rescue.

[0154] In some embodiments, the compound CARs of the present disclosure can combat an important mechanism by which cancer cells resist CAR activity by downregulating target antigens. In another example, the compound CARs of the present invention can also combat the heterogeneity of cancer cells, which is also a significant challenge in conventional CAR T / NK cell therapy. In a further example, the compound CARs disclosed are designed such that a first CAR targets a large tumor population and another CAR eradicates cancer or leukemia stem cells to avoid cancer recurrence.

[0155] In this embodiment, the disclosure provides a method of destroying a targeted cell by contacting a cell bearing the CD33 antigen or the CD123 antigen, or both, with an engineered cell having at least one chimeric antigen receptor polypeptide having a CD33 antigen recognition domain and a CD123 antigen recognition domain. The engineered cell can be a T cell or a NK cell.

[0156] Cells bearing at least one of the CD33 and CD123 antigens are acute myeloid leukemia, precursor acute lymphoblastic leukemia, chronic myeloproliferative neoplasms, chronic myelogenous leukemia, myelodysplastic syndromes, blastic plasmacytoma dendritic neoplasm (BPDCN), Hodgkin's lymphoma, mastocytosis, and hairy cell leukemia cells.

[0157] 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.

[0158] In a further embodiment, 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 bearing a CD33 unit and a CD123 unit are administered to a patient in need thereof.

[0159] In a further example, compound CARs in T or NK cells could be used to eradicate or kill CD34 + CD38 − leukemic stem cells or bulk leukemic cells expressing CD123 or CD33 or both.

[0160] 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. In further embodiments, the target antigen includes at least one of the group consisting of, but is not limited to, 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, EGFRvIII, immunoglobulin kappa and lambda, CD38, CD52, CD3, CD4, CD8, CD5, CD7, CD2, and CD138. The target antigen may also include viral or fungal antigens, such as E6 and E7 from human papillomavirus (HPV) or EBV (Epstein-Barr virus) antigens.

[0161] In some embodiments, the compound CAR targets cells expressing CD19 or CD123 antigens or both. The target cells are cancer cells, such as, but not limited to, B cell lymphoma or leukemia.

[0162] In a further embodiment, the compound CAR targets cells expressing CS1 and / or B cell maturation antigen (BCMA), or both. In another embodiment, the target cells are malignant plasma cells, such as, but not limited to, multiple myeloma.

[0163] In some examples, the compound CAR targets cells expressing multiple antigens, including but not limited to, CS1, BCMA, CD267, BAFF-R, CD38, CD138, CD52, CD19, CD20, interleukin 6 receptor, and NY-ESO-1 antigens. In another example, the target cell is a malignant plasma cell, such as, but not limited to, multiple myeloma.

[0164] In some embodiments, the compound CAR targets cells expressing multiple antigens, including, but not limited to, alphafetoprotein (AFP) and glypican-3 (GPC3). In other embodiments, the target cells include hepatocellular carcinoma, fibroblastoma, hepatoblastoma, undifferentiated embryonal sarcoma and mesenchymal tumor of the liver, lung squamous cell carcinoma, testicular undifferentiated germ cell tumor, liposarcoma, ovarian and teratoid yolk sac tumor, ovarian choriocarcinoma, ovarian clear cell carcinoma, and placental site trophoblastoma.

[0165] According to the present invention, T cells or NK cells containing compound CARs targeting different or the same antigens counteract tumor escape and allow simultaneous targeting of tumor cells.

[0166] T or NK host cells comprising the compound CARs disclosed herein are specifically depicted in this disclosure. The nucleotides and constructs of the compound CARs, sequences, host cells, vectors are considered part of this disclosure and are specifically depicted herein.

[0167] In some examples, the compound CAR is administered in combination with any chemotherapeutic agent currently in development or on the market. In some examples, the compound CAR is administered as a first-line treatment for diseases, including but not limited to hematological malignancies, cancer, non-hematological malignancies, inflammatory diseases, infectious diseases such as HIV and HTLV, and others. In this example, T cells expressing the compound CAR are co-administered with NK cells expressing the same or different compound CAR as an adaptive immune therapy. The compound CAR NK cells provide rapid, innate activity to target cells, while the compound T cells provide a relative sustained adaptive immune activity.

[0168] In this embodiment, cells expressing a compound CAR are administered as a bridge to bone marrow stem cell transplantation to a mammal, for example, a patient who is resistant to chemotherapy and ineligible for bone marrow stem cell transplantation.

[0169] In some examples, the compound CAR co-expresses a transgene and releases a transforming product, such as IL-12, in the targeted tumor lesion, further modulating the tumor microenvironment.

[0170] In this example, cells expressing a compound CAR are administered to a mammal for myeloablation of bone marrow as part of a treatment for a disease.

[0171] In certain embodiments, the compound CAR expressing cells can be T cells or NK cells and are administered to a mammal, e.g., a human. The disclosure provided includes methods of treating a mammal with a disorder or disease by administering the compound CAR. The target cells can be cancer cells or cells affected by any other disease state, such as infectious disease, inflammation, and autoimmune disease.

[0172] The present invention is intended to include the use of fragments, mutants or variants (e.g., modified forms) of compound CARs and antigens that retain the ability to induce stimulation and proliferation of T / NK cells. "Forms of a protein" are intended to mean proteins that share significant homology with at least one CAR or antigen and can achieve stimulation and proliferation of T / NK cells. As used herein, the term "biologically active" or "biologically active forms of a protein" is meant to include forms of a protein or variants that can result in anti-tumor activity of cells.

[0173] The compositions and methods of the present invention are used to generate populations of T cells or NK cells that deliver both primary and costimulatory signals in immunotherapy as a treatment for cancer, particularly lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, rhabdomyosarcoma, leukemia and lymphoma. The compositions and methods described in the present invention can be utilized in conjunction with other types of therapy for cancer, such as chemotherapy, surgery, radiation therapy, gene therapy, etc.

[0174] In some embodiments, the present invention discloses a method for reducing B cells, immature B cells, memory B cells, plasmablasts, long-lived plasma cells, or plasma cells in patients with autoimmune diseases by administering CAR or compound CAR T cells or NK cells to the patient. The target cells of the CAR are B or plasma cells expressing one or two or all of the antigens BCMA, TACI, and BAFF-R. Autoimmune diseases include systemic sclerosis, multiple sclerosis, psoriasis, dermatitis, inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), systemic lupus erythematosus, vasculitis, rheumatoid arthritis, Sjogren's syndrome, polymyositis, granulomatosis and vasculitis, Addison's disease, antigen-antibody complex-mediated disease, and anti-glomerular basement membrane disease.

[0175] Several extracellular markers have shown utility as tumor-associated antigens and are therefore currently being investigated as potential targets for CAR T / NK cell therapy. However, expression of these antigens on healthy tissues results in on-target, off-tumor toxicity, and off-target toxicity remains a major safety concern. Furthermore, a major limitation of CAR T / NK cell therapy is the potential for selection of antigen escape mutants when targeting molecules that are not essential for tumorigenesis. Thus, malignant cells that persist with little or no expression of the target antigen may evade CAR T / NK cells despite the high affinity action of their CARs.

[0176] According to the present invention, natural killer (NK) cells are an alternative cytotoxic effector for CAR killing.Unlike T cells, NK cells do not require preactivation and exhibit constitutive cytolytic function.Further expression of cCAR in NK cells allows NK cells to effectively kill cancer, especially cancer cells that are resistant to NK cell therapy.

[0177] Furthermore, NK cells are known to provide anti-cancer effects without the risk of inducing graft-versus-host disease (GvHD).

[0178] Studies have demonstrated an abnormal overexpression of CD123 on CD34 + CD38- AML cells, whereas their normal bone marrow counterparts CD34 + CD38- did not express CD123 (Jordan, Upchurch et al. 2000). This population of CD123 + , CD34 + CD38- is considered as LSCs (Chronic Myeloid Leukemia Stem Cells) because these cells can initiate and maintain the leukemic process in immunodeficient mice.

[0179] The number of CD34+ / CD38- / CD123+ LSCs can be used to predict the clinical outcome of AML patients. More than 15% CD34+ / CD38- / CD123+ cells in AML patients is associated with lack of complete remission and unfavorable cytogenetic profile. Furthermore, the presence of more than 1% CD34+ / CD38- / CD123+ cells may also have a negative impact on disease-free survival and overall survival.

[0180] Currently, treatments for MDS and AML focus on leukemic blasts, as they are highly abundant and clearly represent the most immediate problem for patients. Importantly, leukemic stem cells (LSCs) are quite distinct from the majority of other leukemic cells ("blast" cells), as they constitute a rare subpopulation. Killing blast cells provides short-term relief, but unless LSCs are destroyed, they constantly regrow and the patient's disease relapses. To achieve a permanent cure for MDS disease, it is essential to destroy LSCs. Unfortunately, standard drug regimens are not effective against LSCs in MDS or AML. It is therefore important to develop new therapies that can specifically target both the leukemic stem cell population and the bulk of the leukemic population. The compound CARs disclosed in the present invention target both of these populations and are specifically described herein.

[0181] According to the present invention, it has surprisingly been found that NK cells provide a ready-made product that can be used as an allogeneic product for healing.According to the present invention, therefore, cCAR cell therapy needs to be performed on a patient-specific basis, as required by the current state of the art.The applicant of the present invention has discovered a novel immunotherapy that does not require the isolation of the patient's lymphocytes or tumor-infiltrating lymphocytes to perform an effective CAR cell-based therapy.

[0182] Although allogeneic or autologous NK cells are expected to induce rapid immune responses, they have a limited life span and are relatively quickly eliminated from the blood circulation. Therefore, the applicants have surprisingly discovered that the use of cCAR cell-based therapy reduces the concern of persistent side effects.

[0183] According to one aspect of the present invention, NK cells can be expanded and transfected with cCAR according to 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 can be expanded and transfected with cCAR. NK-92 is a continuously growing cell line with the characteristics and features of natural killer (NK) cells. The NK-92 cell line is IL-2 dependent and has been proven to be a safe and suitable cell. cCAR-expressing NK-92 cells can be expanded in serum-free medium with or without co-culture with feeder cells. A pure population of interesting NK-92 carrying the cCAR of interest can be obtained by sorting.

[0184] Identification of appropriate surface target antigens is a prerequisite for developing CAR T / NK cells in adaptive immunotherapy.

[0185] In one aspect of the present invention, CD123 antigen is one of the targets of cCAR therapy. CD123 (alpha chain of interleukin 3 receptor) is overexpressed in various hematological malignancies, including acute myeloid leukemia (AML), B-cell acute lymphoblastic leukemia (B-ALL), hairy cell leukemia and blastic plasmacytoid dendritic tumor. CD123 is absent or minimally expressed on normal hematopoietic stem cells. More importantly, CD123 is expressed on a subset of leukemia cells related to leukemia stem cells (LSC), and its ablation is essential for preventing refractory disease and relapse.

[0186] In one aspect of the present invention, CD33 antigen is one of the targets of cCAR therapy.CD33 is a transmembrane receptor that is expressed on 90% of malignant cells in acute myeloid leukemia.Therefore, according to the present invention, the target antigens of CD123 and CD33 are particularly attractive from the viewpoint of safety.

[0187] According to the present invention, compound CD33CD123CARs can be highly effective in the therapeutic treatment of chronic myeloid leukemia (CML) population. In chronic myeloid leukemia (CML), there is a rare subset of cells that are CD34+CD38-. This population is believed to consist of LSCs. Increased numbers of LSCs are associated with disease progression. Small molecule Bcr-Abl tyrosine kinase inhibitors (TKIs) have been shown to significantly improve overall survival of CP-CML patients. However, LSCs are believed to be resistant to TKI therapy. There is an urgent need for novel therapies that target CML-resistant LSCs for the treatment of CML, and novel therapies are embodied in the compound CD33CD123CARs disclosed in the present invention. CD123 expression is high in the CD34+CD38- population. According to the present invention, compound CD33CD123CARs is highly effective in the therapeutic treatment of this population.

[0188] In this embodiment of the present invention, cCAR uses leukemia cells that express both CD123 and CD33 as therapeutic treatment.CD33 is expressed on the cells of myeloid lineage, myeloid leukemia blasts and mature monocytes, but not on normal pluripotent hematopoietic stem cells (Griffin, Linch et al., 1984).CD33 is widely expressed on leukemia cells in CML, myeloproliferative neoplasms and MDS.

[0189] Because a significant number of acute myeloid leukemia (AML) patients are refractory to standard chemotherapy regimens and experience disease relapse after treatment (Burnett 2012), the development of CAR T cell immunotherapy for AML has the potential to provide a much-needed better medical treatment. In the majority of these patients, leukemia cells express both CD123 and CD33, giving the compound CD33CD123 CARs disclosed herein broad clinical applicability. Thus, the present invention discloses novel multiple cCAR T / NK cell constructs that contain multiple CARs targeting multiple leukemia-associated antigens, thereby counteracting antigen escape mechanisms that target leukemia cells, including leukemia stem cells, through the synergistic effect of co-stimulatory domain activation, thereby providing a more potent, safer and more effective therapy.

[0190] The present invention further discloses a compound CAR structure that has enhanced antitumor activity against cells that co-express target antigens, and also retains sensitivity against tumor cells that express only one antigen. Furthermore, each CAR of the compound CAR has one or two costimulatory domains and has a strong killing ability in the presence of a specific target.

[0191] In preclinical studies of bispecific trans-signaling CARs targeting solid tumors, including breast and epithelial ovarian cancer, the CD3ζ intracellular signaling domain is separated from the costimulatory domain from the second generation CAR. In other words, one CAR contains a first generation CAR without the costimulatory domain, and another CAR lacks the CD3 zeta intracellular domain. Thus, the presence of both target antigens is required for T cell activation and potent killing. They were therefore proposed as a way to increase target specificity but at the expense of sensitivity and reduce extratumoral toxicity caused by expression in healthy tissues of one of the two target antigens. In this example, the compound CAR is a compound CD123CD19 CAR. It has been shown that more than 90% of a subset of the population of B-ALL expresses CD123. It is believed that, like AML and MDS, there is a rare LSC population in B-ALL. Thus, targeting both leukemic stem cells and large leukemic populations according to the present invention can be applied to B-ALLs. According to the present invention, CD123 and CD19 surface antigens expressed in B-ALL may be targets, since CD19 is widely expressed at different stages of the B-cell lymphocyte population.

[0192] Multiple myeloma (MM) is the second most common hematologic malignancy in the United States and originates from clonal plasma cells accumulated in bone marrow or extramedullary sites. MM is an incurable disease with a median survival of approximately 4.5 years (Kumar, Rajkumar et al. 2008). Anti-myeloma CARs in preclinical development have been developed, and CAR targets include CD38, CS1, B-cell maturation antigen (BCMA) and CD38. However, heterogeneity in surface antigen expression is common in malignant plasma cells (Ruiz-Arguelles and San Miguel 1994), making them a challenging target for CARs. Malignant plasma cells also express low levels of CD19. To date, myeloma stem cells have been shown to also express several B-cell markers, including CD19. Targeting this population may be effective in the treatment of myeloma in combination with standard therapies and other myeloma CAR therapies.

[0193] Multiple myeloma (MM) is a hematological malignancy involving clonal proliferation of plasma cells. Despite significant advances in treatment, myeloma remains an incurable disease. Therefore, novel therapeutic approaches are urgently needed.

[0194] CS1 (also called CD319 or SLAMF7) is a protein encoded by the SLAMF7 gene. The surface antigen CS1 is a robust marker of normal plasma cells and myeloma cells (malignant plasma cells).

[0195] Tumor necrosis factor receptor superfamily, member 17 (TNFRSF17), also known as B-cell maturation antigen (BCMA) or CD269, is expressed almost exclusively on plasma cells and late stages of malignant plasma cells. Its expression is absent in other tissues, indicating its potential as a target for CAR T or NK cells.

[0196] Malignant plasma cells show antigen heterogeneity with varying proportions for CD269 and CS1. A single CAR unit product targeting either CD269 or CS1 can target the majority of cells in the bulk tumor, resulting in an initial strong antitumor response. Subsequently, the remaining rare non-targeted cells proliferate and cause disease relapse. Although multiple myeloma is particularly heterogeneous, this phenomenon could certainly apply to other leukemias or tumors. A recent clinical trial from the NIH with BCMA CAR T cells showed promising results with complete responses in some patients with multiple myeloma. However, these patients relapsed after 17 weeks, which may be due to antigen escape. Antigen escape is also seen in treatment with CD19 CAR and NY-ESO1 CAR T cells. Therefore, more effective CAR T cell treatments are urgently needed to prevent relapse.

[0197] In one aspect of the invention, BCMA and CS1 are targets for BCMACS1 CAR therapy.

[0198] In some embodiments, the compound CAR targets cells expressing BCMA or CS1 antigens or both. The target cells may be cancer cells, such as, but not limited to, lymphoma, or leukemia or plasma cell neoplasms. In further embodiments, the plasma cell neoplasm is selected from plasma cell leukemia, multiple myeloma, plasmacytoma, heavy chain disease, amyloidosis, Waldestrom's macroglobulinoma, heavy chain disease, isolated osteoblastoma, monoclonal gammopathy of undetermined duration (MGUS), and smoldering multiple myeloma.

[0199] BAFF (B cell activating factor) and APRIL (proliferation-inducing ligand) are two TNF homologs that bind specifically with high affinity to TACI (also known as TNFRSF1 3B or CD267) and BCMA. BAFF (also known as BLyS) binds to BAFF-R and is functionally involved in enhancing late-stage survival and proliferation of B cells. BAFF has been shown to be associated with several autoimmune diseases. APRIL plays an important role in enhancing antibody class switching. Both BAFF and APRIL have been implicated as proliferation and persistence factors of malignant plasma cells.

[0200] The ligand-receptor interactions in malignant plasma cells are depicted in FIG.

[0201] In some embodiments, the compound CAR targets cells expressing TACI or CS1 antigens or both. In another embodiment, the compound CAR targets cells expressing TACI or CS1 antigens or both. The target cells may be cancer cells, such as, but not limited to, lymphomas, or leukemias or plasma cell neoplasms. In further embodiments, the plasma cell leukemia is selected from plasma cell leukemia, multiple myeloma, plasmacytoma, heavy chain disease, amyloidosis, Waldestrom's macroglobulinoma, heavy chain disease, isolated osteoblastoma, monoclonal gammopathy of undetermined type (MGUS), and smoldering multiple myeloma. The target cells may be B cells, immature B cells, naive B cells, centroblasts, centrocytes, memory B cells, plasmablasts, long-lived plasma cells, and one or two or more different cell types of plasma cells. These cells are involved in the development of immune-mediated diseases such as systemic sclerosis, multiple sclerosis, psoriasis, dermatitis, inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), systemic lupus erythematosus, vasculitis, rheumatoid arthritis, Sjögren's syndrome, polymyositis, granulomatous and vasculitis, Addison's disease, antigen-antibody complex-mediated diseases, and anti-glomerular basement membrane diseases.

[0202] In some embodiments, the compound CAR targets cells expressing BAFF-R or CS1 antigen, or both. In another embodiment, the compound CAR targets cells expressing BAFF-R or CS1 antigen, or both. The target cell may be a cancer cell, such as, but not limited to, lymphoma, or leukemia or plasma cell neoplasm. In further implementations, the plasma cell neoplasm is selected from plasma cell leukemia, multiple myeloma, plasmacytoma, heavy chain disease, amyloidosis, Waldestrom's macroglobulinoma, heavy chain disease, isolated osteoblastoma, monoclonal gammopathy of undetermined type (MGUS), and smoldering multiple myeloma.

[0203] In some embodiments, the compound CAR (cCAR) targets cells expressing one or two or all of the BAFF-R, BCMA, TACI and CS1 antigens.

[0204] In some embodiments, the CAR unit in the cCAR is 1) an scFv against any of BAFF-R, BCMA, TACI and CS1, 2) a hinge region, 3) a costimulatory domain, and an intracellular signaling domain.

[0205] In some embodiments, the CAR units in a cCAR include 1) a BCMA or TACI or BAFF-R binding domain or an APRIL binding domain, 2) a hinge region, 3) a costimulatory domain, and an intracellular signaling domain.

[0206] In further examples, the BCMA or TAC1 or BAFF-R binding domains may be part or all of the APRIL and BAFF molecules.

[0207] In some embodiments, the CAR units in a cCAR include 1) an scFv against BCMA or CS1, 2) a hinge region, 3) a costimulatory domain, and an intracellular signaling domain.

[0208] In further embodiments, a cCAR can comprise one or more CARs, each of which can have the same or different hinge region and costimulatory domain.

[0209] In further embodiments, the target antigen is selected from at least one of the group consisting of, but not limited to, 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, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, CD30, EGFRvIII, immunoglobulin kappa and lambda, CD38, CD52, CD3, CD4, CD8, CD5, CD7, CD2, and CD 138. Target antigens can also include viral or fungal antigens, such as E6 and E7 from human papillomavirus (HPV) or EBV (Epstein-Barr virus) antigens.

[0210] In some embodiments, the cCAR targets cells expressing CD19 or CD20 antigens, or both. In other embodiments, the cCAR targets cells expressing CD19 or CD22 antigens, or both. The target cells are cancer cells, such as B-cell lymphoma or leukemia.

[0211] Acute graft-versus-host disease (GVHD) remains the most important cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. In the effector phase of GVHD, the T cell receptor (TCR), a heterodimer of α and β chains, is expressed on the surface of T cells, where the TCR recognizes some antigens on HLA molecules on host cells, enhancing T cell proliferation and causing damage to host cells by releasing cytotoxins. Inactivation of the TCR gene is effective in preventing potential graft-versus-host reactions. Inactivation of the TCR may result in the prevention of TCR recognition in alloantigens and thus GVHD. The role of CD45 in NK cells is quite different from that of T cells. NK cells from CD45-deficient mice have normal cytotoxic activity against the prototypic tumor cell line Yac-1. Furthermore, CD45-deficient NK cells proliferate normally and respond to IL15 and IL-21. Thus, disruption or deletion of CD45 would not affect the killing and proliferation in NK cells. The present disclosure includes a method for permanent deletion of CD45 in T cells or NK cells, followed by stable introduction of a CD45-specific CAR. As a result, the engineered T cells exhibit the desired properties that are specifically redirected to CD45 without causing self-killing and response to antigen presentation. In further examples, the engineered T cells may have efficacy as a pre-existing treatment for treating malignant or other diseases. The present disclosure presents a method for engineering T cells to allow proliferation when TCR signaling is reduced or lost due to inactivation or deletion of endogenous CD45. Reduced or lost TCR signaling may result in the prevention of GVHD.

[0212] In a further example, T cells that reduce or lose TCR signaling by inactivating CD45 can be used as an "off the shelf" therapeutic product.

[0213] The present disclosure includes a method for modified T or NK cells, (a) modifying T or NK cells by inactivating CD45, (b) expanding these modified cells, (c) selecting modified T or NK cells that do not express CD45, and (d) introducing a CD45CAR.

[0214] In an embodiment, the chimeric antigen receptor (CAR) encoded by the CD45CAR gene comprises at least one of an antigen recognition domain, a hinge region, a transmembrane domain, and a T cell activation domain, and the antigen recognition domain is redirected to the CD45 surface antigen present on a cell. The antigen recognition domain comprises a monoclonal or polyclonal antibody against the CD45 antigen. The antigen recognition domain comprises the binding site or variable region of the monoclonal or polyclonal antibody.

[0215] In some embodiments, the modified T cells are obtained from an allogeneic donor and used "off the shelf."

[0216] Targeting CD45 with CAR T or NK cells may cause self-killing, since T and NK cells express this surface antigen. To overcome this drawback, the inventors propose to inactivate the CD45 gene using engineered CRISP / Cas9 systems, zinc finger nucleases (ZFNs) and TALE nucleases (TALENs) and meganucleases. CD45-deleted T or NK cells are then transduced with CARs that target neoplasms expressing CD45.

[0217] The present disclosure includes methods for eliminating or reducing abnormal or malignant cells in bone marrow, blood, and organs. In some embodiments, the malignant cells expressing CD45 include acute leukemia, chronic leukemia, B and T cell lymphoma, myeloid leukemia, acute lymphoblastic lymphoma or leukemia, primary effusion lymphoma, reticulohistiocytoma, transient myeloproliferative disorder of Down's syndrome, lymphocyte predominant Hodgkin's lymphoma, myeloid leukemia or sarcoma, dendritic cell tumor, histiocytic sarcoma, giant cell tumor of tendon sheath, interdigitating dendritic cell sarcoma, post-transplant lymphoproliferative disorder, and the like.

[0218] In some embodiments, CD45CAR cells can be used to create space in the bone marrow by removing hematopoietic cells for bone marrow stem cell transplantation while simultaneously removing leukemia / lymphoma cells or immune cells capable of graft rejection.

[0219] In a further example, CD45CAR cells could be used to pre-treat patients before they undergo a bone marrow transplant to receive stem cells. In a further example, CD45CAR could be used as a myeloblast conditioning regimen for hematopoietic stem cell transplantation.

[0220] In some embodiments, CD45CAR cells are utilized to treat or prevent residual disease following stem cell transplantation and / or chemotherapy.

[0221] In some embodiments, CD45CAR is part of an expressed gene or cassette. In a preferred embodiment, the expressed gene or cassette may include an accessory gene or tag or a portion thereof in addition to CD45CAR. The accessory gene may be an inducible suicide gene or a portion thereof, including, but not limited to, caspase 9 gene, thymidine kinase, cytosine deaminase (CD) or cytochrome P450. The ablation method with a "suicide gene" improves the safety of gene therapy and kills cells only when activated by a specific compound or molecule. In some embodiments, the suicide gene is inducible and is activated using a specific chemical derivative of dimerization (CID).

[0222] In some embodiments, the safety switch can include an accessory tag that is a c-myc tag, CD20, CD52 (Campath), a truncated EGFR gene (EGFRt) or a portion or combination thereof, etc. The accessory tag could be used as a non-immunogenic selection tool or as a tracking marker.

[0223] In some embodiments, the safety switch can include a 24 residue peptide corresponding to residues 254-277 of the RSV F glycoprotein of strain A2 (NSELLSLINDMPITNDQKKLMSNN).

[0224] In some embodiments, the safety switch may comprise the amino acid sequence of TNFα bound by a monoclonal anti-TNFα agent.

[0225] Administration of any of the engineered cells described herein may be supplemented with the co-administration of a CAR enhancing agent. For better therapeutic outcomes, examples of CAR enhancing agents include, but are not limited to, agents that target immune checkpoint pathways, immunomodulatory agents such as inhibitors of colony stimulating factor-1 receptor (CSF1R), which enhance CAR activity. Agents that target immune checkpoint pathways include small molecules, proteins, or antibodies that bind to the inhibitory immune receptors CTLA-4, PD-1, and PD-L1, resulting in CTLA-4 and PD-1 / PD-L1 blockade. As used herein, enhancing agents also include enhancers as described above.

[0226] A "patient" as used herein includes a mammal. The mammal referred to herein may be any mammal. The term "mammal" as used herein means any mammal, including but not limited to rodent mammals such as mice and hamsters, and mammals of Lagomorpha such as rabbits. The mammal may be from the order Carnivora, including Felines (cats) and Canines (dogs). The mammal may be from the order Artiodactyla, including Bovines (cattle) and Swines (pigs) or Equines (horses). Preferably, the mammal is a human. The patient includes a subject.

[0227] In certain embodiments, the patient is a human between 0-6 months, 6-12 months, 1-5 years, 5-10 years, 5-12 years, 10-15 years, 15-20 years, 13-19 years, 20-25 years, 25-30 years, 20-65 years, 30-35 years, 35-40 years, 40-45 years, 45-50 years, 50 years, 55-60 years, 60-65 years, 65-70 years, 70-75 years, 75-80 years, 80-85 years, 85-90 years, 90-95 years, or 95 to 100 years of age.

[0228] As used herein, the terms "effective amount" and "therapeutically effective amount" of engineered cells refer to an amount of engineered cells sufficient to provide a desired therapeutic or physiological or effect. Such effects or therapeutic effects include alleviation or amelioration of symptoms of a cell-mediated disease. Undesirable effects, e.g., side effects, sometimes occur along with the desired therapeutic effect. Thus, practitioners will balance potential benefits with potential risks to determine what is an appropriate "effective amount." The exact amount required will vary from patient to patient, depending on the subject's species, age, and general condition of the patient, mode of administration, etc. Thus, it may be impossible to specify an exact "effective amount." However, the appropriate "effective amount" term for any individual case may be determined using routine techniques by those in the art. In general, a particular engineered cell or group of engineered cells is provided in an amount and under conditions sufficient to reduce proliferation of the target cells.

[0229] Following administration in a delivery system for the treatment, inhibition or prevention of cancer, the effectiveness of the therapeutic engineered cells can be assessed using a variety of techniques well known to the skilled practitioner.

[0230] For example, one skilled in the art will understand that therapeutically engineered cells delivered with a chemotherapeutic adjuvant will be effective in treating or inhibiting a subject's cancer by observing that the cancer cell burden is reduced or prevented from further proliferation. Cancer cell burden can be measured by methods known in the art, for example, using a polymerase chain reaction assay to detect the presence of specific cancer cell nucleic acid, or by identifying specific cancer cell markers in blood, such as, but not limited to, measuring a sample from a subject or patient using antibodies, or by measuring the amount of circulating cancer cell antibody levels in a patient.

[0231] Throughout this specification, quantities are defined by ranges and lower and upper boundaries. Each lower boundary can define a combined range with each upper boundary. Each lower boundary and upper boundary should be considered as a separate entity. Deleted sentence from p67-18 to p69-5 because Janapnes words could not distinguish those english words by japanes

[0232] As used herein, a XXXX antigen recognition domain is a polypeptide that is selective for XXXX. Thus, XXXX is the target. For example, a CD38 antigen recognition domain is a polypeptide that is specific for CD38.

[0233] As used herein, CDXCAR refers to a chimeric antigen receptor having a CDX antigen recognition domain.

[0234] The present disclosure may be better understood with reference to the following illustrative examples, which are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed herein are made and evaluated, and are purely exemplary and are not intended to be limiting of the disclosure. EXAMPLES

[0235] Creation of compound CAR (cCAR) The structure of CD33CD123 cCAR follows the schematic diagram in Figure 1A. It contains an SFFV (spleen focus forming virus) promoter that induces the expression of a functional compound CAR (cCAR) with two different CAR units. The antigen receptor is headed by anti-CD33 and anti-CD123 scFv (single-chain variable fragment) nucleotide sequences. The picornavirus-derived P2A peptide is utilized due to the highly efficient mechanism of self-cleavage dynamics of the bicistronic gene structure. The self-cleaving P2A peptide serves to link together the two independent CAR units, CD33CAR and CD123CAR, during expression. The advantage of this approach over the internal ribosome entry site (IRES) commonly used in the literature is that the 2A peptide, with its small size and high cleavage efficiency, is sandwiched between the two upstream and downstream unit proteins. Furthermore, the use of the self-cleaving P2A peptide can avoid the problem of the difference in expression levels between the genes before and after the IRES when the IRES is applied.

[0236] The modular unit, CD33CAR, contains the CD33 scFv domain, CD8a hinge region, CD8a transmembrane domain, 4-BB costimulatory domain and the intracellular domain of the CD3 zeta chain. The second modular CAR, CD123CAR, has the same hinge, transmembrane and intracellular signaling domains as CD33CAR, but different scFv and costimulatory domains. CD33 CAR recognizes its corresponding antigen, and CD123 CAR binds its corresponding antigen. The hinge region was designed to avoid disulfide interactions. Different costimulatory domains, 4-BB and CD28, were used. CD33CD123 compound CARs were subcloned into lentiviral plasmids.

[0237] Creation of highly efficient compound CAR (cCAR) Compound CAR lentivirus was generated by transfection into HEK-293FT cells using Lipofectamine 2000 according to the manufacturer's instructions, except that for vectors containing large inserts as shown in Figure 2, twice the amount of vector DNA was used to increase titers. After 12 to 16 hours of incubation, the Lipofectamine-containing medium was removed and replaced with DMEM medium containing 10% FBS, 20 mM HEPES, 1 mM sodium pyruvate, and 1 mM sodium butyrate. After approximately 24 hours, the supernatant was harvested, refrigerated, and replaced with fresh medium. After another approximately 24 hours, it was harvested, combined with the previous supernatant, and filtered through a 0.45 μM filter disk. The supernatant was divided into aliquots, flash frozen in liquid nitrogen, and stored at -80°C. HEK-293 FT cells were harvested, cryopreserved, and lysed for subsequent electrophoresis and Western blotting.

[0238] PB (peripheral blood) or CB (human umbilical cord blood) buffy coat cells were activated with anti-CD3 antibody and IL-2 for 2 days. cCAR lentivirus supernatant was spinoculated onto retronectin-coated multiwell plates. Activated T cells were cultured at approximately 0.3 × 10 6 Cells were transduced in multiple wells containing lentiviral supernatant at a low concentration of cells / mL (Figure 2).

[0239] After the first overnight transduction, cells were not washed and were added directly to the next virus-coated plate for the second transduction unless the cells appeared abnormal. After the second overnight transduction, cells were washed, combined, and incubated in tissue culture-treated plates. CAR T cells were allowed to grow for up to 5 days before co-culture killing assays. After approximately 3 days of incubation, cells were incubated with goat anti-mouse F(Ab')2 or goat IgG (isotype) antibodies conjugated with biotin, washed, and subsequently incubated with streptavidin-PE and anti-human CD3 antibodies. After washing and suspension in 2% formalin, cells were analyzed by flow cytometry to determine transduction efficiency.

[0240] CD33CD123 cCAR characterization To confirm the construction of the compound CAR, transfected CD33CD123 cCAR HEK293T cells were analyzed by Western blot. Immunoblot with anti-CD3 zeta monoclonal antibody showed a band of the expected size of the compound CAR CD3 zeta fusion protein (Figure 1B). Importantly, two distinct band signals of similar intensity were observed on the blot, which is expected for the successful high cleavage action of the P2A peptide. As expected, no expression of CD3 zeta was observed for the GFP control vector. Surface expression of the scFv was also tested on HEK293 cells (Figure 1C) and primary T cells (Figure 1C).

[0241] To test the transduction efficiency of the compound CD33CD23CAR lentivirus in the HEK293 cell line, the cells were analyzed by flow cytometry (Beckman Coulter) (Figure 1C). Flow cytometry showed that approximately 67% of HEK cells expressed the CD33CD123 CAR. Human peripheral blood (PB) is often used for autologous T cell therapy. Human PB buffy coat cells were activated with anti-CD3 antibody and IL-2 and transduced with either the CD4CAR or control (GFP) lentivirus. After transduction, flow cytometry analysis showed that approximately 22% of T cells expressed the CD33CD123CAR (Figure 1C).

[0242] result Umbilical cord blood (UCB) and peripheral blood (PB) derived CD33CD123cCAR T cells specifically kill CD33-expressing tumor cells CD33CD123 cCAR T cells or GFP T cells (control) were incubated with target cells in ratios ranging from 0.5:1 to 50:1, preferably about 2:1, 5:1, 10:1, 20:1, 50:1 (about 100,000, 200,000, 500,000, about 1 million, or 2 million effector cells to about 50,000, 100,000, 200,000 target cells) in about 1-2 mL of T cell culture medium without IL-2 for about 24 hours. The target cells were leukemia cell lines and leukemia cells from leukemia patients. After about 24 hours of co-culture, the cells were stained with mouse anti-human CD33, CD123, CD34, and CD3 antibodies.

[0243] CD33CD123 cCAR T cells expressing CD33CAR and CD123 CAR were generated and tested for anti-leukemia function using HL60 and KG-1a cell lines. The HL60 cell line is a promyelocytic leukemia cell line that highly expresses CD33. Approximately 100% of its cell population is CD33+, and a small subset of it (<10%) is dim CD123+. In culture, this cell line was used to test to determine the efficacy of CD33CD123 CAR with an emphasis on targeting CD33-expressing leukemia cells. Furthermore, due to the strong expression of CD33 in HL60, the effect of CD33CD123cCAR may be quite strong. Indeed, under 24-hour co-culture conditions with various ratios of effector to target cells, CD33CD123cCAR showed significant killing effect on leukemia cells (Figure 3). CB-derived CD33CD123 CAR T cells were first tested for their ability to kill HL60 cells. By incubation for about 24 hours at low effector:target (E:T) ratios ranging from about 0.5:1 to 50:1, preferably 1:1 to about 5:1, more preferably about 2:1 to 4:1, CD33CD123 CAR cells eliminated about 55% of CD33-expressing HL60 cells compared to GFP controls. At a ratio of about 5:1, the killing capacity increased to about 82%.

[0244] CD33CD123 CAR derived from peripheral blood mononuclear cells (PBMC) was co-cultured with the myeloid leukemia cell line KG1a, which has approximately 100% but weak CD33 signal compared to HL60 and expresses 50-80% CD123. KG1a is therefore a relatively dual target cell population that is double positive for the antigen targeted by the CD33CD123 CAR. Low effector:target (E:T) ratios ranging from approximately 0.5:1 to 50:1 were examined with approximately 24 hours of incubation. At low E:T ratios of about 2:1, the CD33CD123 CAR showed moderate anti-leukemic activity of about 26%, and when the E:T ratio was increased to 10:1, the killing of KG1a was about 62% compared to the GFP control (Figure 4), indicating that the potency of the killing effect on HL60 cells utilizing more CAR activity than KG1a may be indicative of its intensity at the CD33 marker. These experiments provide evidence that the overall function of the CD33CD123 CAR is relevant to the antigen-presenting cell population.

[0245] An additional compound CAR was created, CD33CD123-BB cCAR. This compound CAR contains two independent CAR units, CD33 and CD123. The first CAR contains an scFv that binds to CD33, and the second CAR has a different scFv that recognizes CD123. Both CARs contain the same hinge region, transmembrane, costimulatory and intracellular domains. CD33CD123-BB cCAR lentiviruses were produced and their killing ability in KG-1a cells was tested. As shown in Figure 5, they were substantially killed at a ratio of about 10:1, but with lower efficacy than CD33CD123 cCAR.

[0246] CD33CD123 cCAR has activity against patient samples expressing CD33 and / or CD123 In addition to experiments with cell lines, studies were also performed on patient samples to test the functionality of individual CAR units. AML-9, an aggressive acute myeloid leukemia (AML), was used to test the efficacy of CD33CD123 cCAR. Due to the heterogeneity of the patient cell population, including multiple cell types in the AML-9 sample, leukemic blasts were gated on CD34 and CD33, which were the two positive markers. Depletion of this CD33+CD34+ population of leukemic cells was observed to be 48% with CAR T cells compared to the GFP control (Figure 6).

[0247] Leukemic cells that were CD123 positive and CD33 negative were also tested. For this purpose, a human B-cell acute lymphoblastic leukemia (B-ALL) sample, Sp-BM-B6, was selected. All leukemic blasts in this sample were CD34+CD33- and approximately 50% or more positive for CD123. The reduction of the CD34+ leukemic cell population by CD33CD123 cCAR T cells was approximately 86% compared to the GFP control (Figure 7). Based on cell line and human sample studies, our data strongly suggest that the compound CD33CD123 CAR can target leukemic cells expressing CD33 or CD123 or both.

[0248] CD33CD123 cCAR NK cells targeting leukemia cells expressing CD33 or CD23 or both Natural killer (NK) cells are CD56+ CD3- and can kill infected and tumor cells as efficiently as CD8+ T cells. Unlike CD8+ T cells, NK cells exert cytotoxicity against tumors without the need for activation to kill the cells. NK cells are safer effector cells because they avoid the potentially fatal complications of cytokine storm. However, the use of CD33 or CD123 or both CAR NK cells to kill leukemia is completely unexplored.

[0249] Generation of CD33CD123 cCAR NK cells NK-92 cells were transduced with CD33CD123 CAR lentiviral supernatant in two consecutive overnight transductions, with retronectin and virus-coated plates replaced at each transduction. Transduced cells were grown for 3 or 4 days, and then CAR expression was analyzed by flow cytometry. Cells were harvested and incubated with goat anti-mouse F(Ab')2 at a ratio of approximately 1:250 for approximately 30 minutes. Cells were washed, suspended, and stained with streptavidin-PE for approximately 30 minutes. Cells were washed, suspended in 2% formalin, and analyzed by flow cytometry. NK-92 cells expressing CD33CD123cCAR were then labeled as above, sorted on a FACSAria, and the top 0.2% of F(Ab')2-labeled cells were collected and cultured. Subsequent labeling of sorted and cultured cells showed that NK-92 cells were approximately 89% positive for anti-mouse F(Ab')2 (Figure 8).

[0250] CD33CD123 cCAR NK cells efficiently lyse or kill leukemia cells First, we tested the function of CD33CD123 cCAR NK-92 cells by measuring their ability to kill the HL-60 cancer cell line in co-culture. Virtually all HL-60 cells highly express CD33, whereas CD123 expression in this cell line is less than 10% (weak). Therefore, the killing ability of CD33CD123 cCAR is likely dependent on the ability of the cCAR to properly target CD33.

[0251] CD33CD123 cCAR NK-92 cells were co-cultured with HL-60 cells for approximately 24 hours in NK cell medium without IL-2. After incubation, CD33CD123 cCAR NK-92 cells were labeled and compared with non-CAR GFP NK-92 cells as a control. Dramatic killing of HL-60 cells by CD33CD123 cCAR NK-92 cells was observed compared to control GFP NK-92 cells. Furthermore, the killing ability of CD33CD123 cCAR NK-92 cells was dose-dependent and was nearly 100% at approximately a 10:1 cell ratio compared to the control (Figures 9 and 11).

[0252] Subsequent co-culture experiments with myeloid leukemia cell lines were performed with KG1a, which expresses CD33 in all cells, but at a moderate level compared to that of HL-60. The CD123 antigen is expressed on approximately 50-80% of KG1a cells. The experimental design was similar to the first HL-60 killing assay experiment described above, with the same incubation time, effector:cancer cell ratio, and GFP NK-92 cells as controls. The results showed a significant killing of KG1a cells by CD33CD123 cCAR NK-92 cells in a dose-dependent manner compared to the GFP NK-92 cell control. At an effector:target ratio of 10:1, the killing of KG1a cells by CD33CD123 cCAR NK-92 cells was approximately 85% compared to that of the GFP control (Figures 10 and 11).

[0253] Analysis of KG1a cells revealed that they were two distinct populations, CD33 + CD123- and CD33 + CD123-. Figure 11 showed that the killing of cells seen in both populations increased in a dose-dependent manner. Surprisingly, the double-positive population showed more efficient killing with increasing effector cell ratio, suggesting a possible synergistic effect of the two modular CARs, CD33 and CD123 (Figure 12).

[0254] Generation of CD19CD20, CD19CD22, CD19CD138 cCAR The three cCARs were generated using a method similar to the CD33CD123 cCAR described above (Figure 13).

[0255] Generation of cCARs, including BCMA CS1 cCAR and BCMA CD19 cCAR, for the treatment of multiple myeloma Preclinical studies of cCARs against target antigens including CD38, CS1, CD138, B-cell maturation antigen (BCMA) and CD38 have been developed. CD19 CARs have also shown some efficacy for the treatment of multiple myeloma in phase I clinical trials. However, since heterogeneity of surface antigen expression commonly occurs in malignant plasma cells (Ruiz-Arguelles and San Miguel 1994), it is unlikely that a single target will be sufficient to treat the disease. BCMA CS1 cCARs, BCMA CD19 cCARs, BCMA CD38 cCARs and BCMA CD138 cCARs were generated, and the experimental design was similar to that of the CD33CD123 cCAR described above.

[0256] Generation of cCARs, including BCMA CS1 cCAR (BC1cCAR), for the treatment of multiple myeloma Generation and characterization of BCMA CS1 cCAR (BC1cCAR) The modular design of BC1cCAR is designed to contain the CD8-derived hinge (H) and transmembrane (TM) regions fused to anti-CD269 (BCMA, B cell maturation antigen) single chain variable fragment (scFv) regions and anti-CD319 (CS1) scFv flanked by self-cleaving P2A peptides, and linked to the CD3ζ signaling domain and tandem 4-1BB coactivation domains (Figure 14A). The strong spleen focus forming virus promoter (SFFV) and CD8 leader sequence were used for efficient expression of the BC1cCAR CAR molecule on the T cell surface. The two unit CARs use the same co-stimulatory domain, 4-1BB. Transfected BC1cCAR HEK293T cells were subjected to Western blot analysis to confirm the compound structure. Immunoblots using anti-CD3 monoclonal antibodies showed a band of the predicted size for the compound CAR CD3 fusion protein (Figure 14E). Importantly, two distinct bands of signal of similar intensity were observed due to successful high cleavage of the P2A peptide, and as expected, no CD3 expression was observed with the GFP control vector.

[0257] Generation of BC1cCAR (cCAR) T-cells T cells isolated from umbilical cord blood (UCB) buffy coats were transduced with BC1cCAR lentivirus after 2 days of activation. The two CARs units used the same costimulatory domain 4-1BB. The transduction efficiency of BC1cCAR was determined to be approximately 15% by flow cytometry (Figure 14B). BC1cCAR T cells were first tested in CML (chronic myeloid leukemia) cell lines that are negative for myeloma markers BCMA and CS1. As expected, there was no lysis from either control or BC1cCAR T cells against wild-type K562 (Figure 14C). BCMA-K562 (Kochenderfer, NIH) are K562 cells transduced with cDNA expressing BCMA, and express BCMA in more than 80% of the cell population. BC1cCAR T cells were co-cultured with this cell line at E:T ratios of 2:1 and 5:1 and showed more than 30% lysis compared to controls (Figure 14C). These results are in agreement with those of other co-culture experiments performed with cell lines transduced with antigens against other CARs, such as CS1CAR T cells.

[0258] However, when different costimulatory domains, either 4-BB or CD28, were used for each unit of BCMA-CS1-2G (cCAR), low CAR surface expression was detected. This indicates that the appropriate selection of costimulatory domains is important to ensure surface CAR expression on T cells (Figure 14D). Although protein expression in HEK cells was detected by Western blotting (Figure 14E), no surface expression was detectable in activated T cells transduced with CD269-CS1-2G lentiviral supernatant. This could be due to an inability to transport the expressed protein to the cell membrane. In the future, the sequence of this structure may need to be optimized to allow better expression on the cell surface.

[0259] BC1cCAR T cells specifically lyse BCMA+ and CS1+ cell lines To evaluate the cytotoxic activity of BC1cCAR T-cells, we performed co-culture assays with myeloma cell lines MM1S (BMCA + CS1 +), RPMI-8226 (BCMA + CS1-) and U266 (BCMA + CS1dim). The ability of BC1cCAR T cells to lyse target cells was quantified by flow cytometry analysis, staining target cells with Cytotracker dye (CMTMR). In 24 h of co-culture, BC1cCAR showed virtually complete lysis of MM1S cells, with >90% reduction of target cells at an E:T ratio of 2:1 and >95% reduction at an E:T ratio of 5:1 (Figure 15). In RPMI-8226 cells, BC1cCAR lysed >70% of BCMA + target cells at an E:T ratio of 2:1 and >75% at an E:T ratio of 5:1 (Figure 16). In 24-h co-culture with U266 target cells, BC1cCAR inhibited BCMA + 80% of the U266 cells were lysed at an E:T ratio of 2:1, reaching saturation (Figure 17).

[0260] BC1cCAR T cells specifically target BCMA+ and CS1+ populations in primary patient myeloma samples Flow cytometry analysis of MM10-G patient samples reveals distinct and consistent BCMA+ and CS1+ population subsets (Figure 18). MM7-G samples display a fully BCMA+CS1+ phenotype, while MM11-G shows noisy BCMA+CS1+ phenotype likely due to the nature of being bone marrow aspirates. dim CS1 dim After 24 hours, BC1cCAR T cells showed >75% lysis at an E:T ratio of 5:1, increasing to >85% at 10:1 (Figure 19), indicating robust ablation of primary patient samples in MM7-G. For MM11-G (Figure 20), BC1cCAR T cells were able to lyse >45% of the BCMA+CS1+ population at E:T = 10:1.

[0261] BC1cCAR demonstrates targeted and specific lytic capacity by significantly ablating BCMA+CS1+ and BCMA-CS1+ subset populations in MM10-G co-cultures over 24 hours. At an E:T ratio of 2:1, BC1cCAR T cells ablate 60% of the BCMA+CS1+ population and 70% of the CS1+ only population. At an E:T ratio of 5:1, ablation of the CS1+ only population increases to 80% (Figure 18).

[0262] BC1cCAR T cells show significant control and reduction of tumors in vivo To evaluate the in vivo antitumor activity of C1cCAR T cells, we developed a xenogeneic mouse model in which luciferase-expressing MM.1S cells (a multiple myeloma cell line) were injected intravenously into sublethally irradiated NSG mice and were able to induce measurable leukemia formation. Three days after tumor cell injection, mice were injected with 8 × 10 6 A single dose of BC1cCAR T cells or vector control T cells was injected intravenously. On days 3, 6, and 8, mice were subcutaneously injected with RediJect D-Luciferin (Perkin Elmer) and tumor burden was measured by IVIS imaging (Figure 21). To compare the percentage of tumor cells after treatment with BC1cCAR T cells or vector control T cells, the average light intensity measured was compared for each mouse. Unpaired T-test (P = 0.0001) showed a highly significant difference between the two groups by day 8, with less light intensity and therefore less tumor burden in the BC1cCAR T cell injected group compared to the control group (p <0.0001). On day 1 and every other day thereafter, tumor size area was measured and the average tumor size between the two groups was compared (Figure 21). In summary, these in vivo data show that CD269-CS1-BBCAR T cells significantly reduce tumor burden in NSG mice injected with MM.1S when compared to vector control T cells.

[0263] CD45 CAR treatment Three pairs of sgRNAs are designed in CHOPCHOP to target genes of interest. The gene-specific sgRNAs are then cloned into a lentiviral vector (Lenti U6-sgRNA-SFFV-Cas9-puro-wpre) that expresses human Cas9 and a puromycin resistance gene linked with an E2A self-cleaving linker. The U6-sgRNA cassette precedes the Cas9 element. The expression of the sgRNA and Cas9puro is driven by the U6 and SFFV promoters, respectively (Figure 23).

[0264] The following gene-specific sgRNA sequences were used and constructed:

[0265] In a non-limiting example of the present invention, exemplary gene-specific sgRNAs are designed and constructed as follows: CD45 sgRNA construct: Lenti-U6-sgCD45a-SFFV-Cas9-puro GTGGTGTGAGTAGGTAA Lenti-U6-sgCD45b-SFFV-Cas9-puro GAGTTTTGCATTGGCGG Lenti-U6-sgCD45c-SFFV-Cas9-puro GAGGGTGGTTGTCAATG

[0266] Figure 24 shows the stages in the generation of CD45 CAR T or NK cells targeting hematological malignancies.

[0267] CRISPR / Cas nucleases targeting CD45 on NK cells NK-92 cells were transduced with gene-specific sgRNA carried by lentivirus. Loss of CD45 expression in NK-92 cells was determined by flow cytometry analysis. The CD45 negative population of NK-92 cells was sorted and expanded (Figure 25). The sorted and expanded CD45 negative NK-92 cells were used to generate CD45CAR NK cells. The resulting CD45CAR NK cells were used to test for their ability to kill CD45+ cells.

[0268] CD45-inactivated NK-92 cells after CRISPR / Cas nuclease targeting (NK 45i -92) Functional characterization We report that after CRISPR / Cas nuclease inactivation of CD45, NK 45i We demonstrated that cell proliferation of NK-92 cells was similar to that of wild-type NK-92 cells (Figure 26). Inactivation of CD45 did not significantly affect NK-92 cell proliferation. Furthermore, 45i The lytic potential of NK-92 cells was shown to be consistent with that of wild-type NK-92 when the cells were co-cultured with the leukemia cell line, CCRF (Figure 27).

[0269] To demonstrate that CD45-inactivated NK-92 are compatible with CAR lysis, NK 45i NK-92 cells and wild-type NK-92 were transduced with lentivirus expressing CD5CAR or GFP, respectively. The resulting CD5CAR NK 45i -92 cells and GFP NK 45i -92 were sorted by FACS and used to compare their ability to kill target cells. 45i When co-cultured with CD5-targeting leukemia cells, CCRF-CEM cells, CD5CAR NK-92 cells exhibited potent killing ability at the E:T ratios of 2:1 and 5:1. 45iWe showed that CD45-92 and CD5 CAR NK-92 cells had similar killing potency against CCRF-CEM cells (Figure 28), suggesting that loss of CD45 expression does not reduce the anti-tumor activity of CAR NK cells.

[0270] Generation of CD45CAR constructs We next 45i We investigated the response of CD45CAR in HEK293-92 cells to the CD45 antigen in leukemia cells. We generated CD45CAR, which consists of an anti-CD45 single-chain variable fragment (scFv) region linked to a CD3ζ signaling domain, CD8-derived hinge (H) and transmembrane (TM) regions, and tandem CD28 and 4-1BB coactivation domains (Figure 29A). We used a strong spleen focus forming virus promoter (SFFV) and CD8 leader sequence. CD45CAR protein was revealed by Western blot using HEK293-FT cells transfected with CD45CAR lentiviral plasmid compared to appropriate vector controls. Furthermore, immunoblotting with anti-CD3 zeta monoclonal antibody revealed a band of the expected size for CD45CAR protein in the samples of interest, whereas the vector control did not (Figure 29B).

[0271] CD45CAR NK 45i -92 NK cells CD45NK by fluorescence-activated cell sorting (FACS) 45i After collection of the areas enriched for CD45 NK-92 cells, the CD45CAR NK-92 transduction efficiency after sorting was determined to be 87% by flow cytometry (Figure 30). 45i Following FACS collection of -92 cells, CD45CAR expression levels remained consistently stable for at least 10 passages.

[0272] CD45CAR NK 45i -92 cells specifically lyse CD45+ leukemic cells. To evaluate the anti-leukemia activity of CD45CAR NK45i-92, we performed co-culture assays with T-ALL cell lines, CCRF-CEM and Jurkat, and NK cell lines and NK-92 cells, since these cells express CD45 (Figures 31, 32, 33). 45i We demonstrated that CD45CAR NK-92 cells consistently demonstrated potent lysis of leukemia cells. 45i -92 cells effectively lysed over 60% of CCRF-CEM cells after 6 hours of incubation at low potency against target cells (E:T ratio 5:1) (Figure 31). After 6 hours of co-culture, CD45CAR NK 45i -92 cells were also able to kill approximately 60% of Jurkat cells at E:T, 2:1 or 5:1 ratios (Figure 32). After 6 hours of co-culture, CD45CAR NK 45i NK-92 cells efficiently lysed 20% CD45 positive NK-92 cells at an E:T ratio of 2:1 and showed close to 60% lysis at E:T = 5:1 (Figures 33A-33C).

[0273] For further analysis of CD45-targeted hematologic malignancies, two additional CARs were developed using lentiviruses expressing either the CD45-28 or CD45-BB CARs to target NK 45i CD45-28 and CD45-BB CARs were generated by transducing NK-92 cells. CD45-28 and CD45-BB CARs contain a new anti-CD45 scFv that is distinct from the previously described CD45CAR. CD45-28 CAR uses the CD28 costimulatory domain, whereas CD45-BB has a 4-BB costimulatory domain. Both CARs use the hinge (H), transmembrane (TM) region and CD3ζ signaling domain from CD8. CD45CAR showed potent lysis in the B acute lymphoblastic cell line REH. CD45CAR NK 45i -92 cells lysed approximately 76% of REH cells. CD45b-BB CAR NK 45i -92 cells and CD45b-28 CAR NK 45iCD45b-28 CAR NK-92 cells showed approximately 79% and 100% lysis capacity of REH cells, respectively, compared to control GFP NK-92 cells (Figure 33D-G). 45i -92 cells showed the highest lytic potential for REH cells.

[0274] Augmentation of CAR T and NK cell function by IL15 and its receptor Recent studies have demonstrated that T cell persistence correlates well with the therapeutic efficacy of CAR T cells. Recent studies have demonstrated that injecting small numbers of high-quality CAR T cells engineered to have potent and durable high tumor activity is more important in contributing to antitumor activity than injecting volume.

[0275] Interleukin (IL)-15 is a cytokine that promotes lymphocyte development and hemostasis. Increased levels of IL-15 promote T cell proliferation and enhance T cell effector responses. Data from recent studies indicate that IL-15 is important for the generation and maintenance of memory CD8 T cells, one of the key factors associated with antitumor activity. IL-15 binds to the IL-15 receptor alpha chain (also called IL15RA or RA), which contributes to IL-15-mediated actions such as T cell survival, proliferation and generation of memory T cells.

[0276] IL-15RA binds to the βγ complex on the surface of T cells, and IL15 signals upon binding to this IL-15RA / βγ complex on the cell surface of T cells and other types of cells.

[0277] Recent data indicate that transfection of IL-15 alone does not significantly affect T cell function, whereas transfection of IL-15 / IL-15RA allows T cells to survive and proliferate spontaneously.

[0278] The efficacy of administered IL-15 alone may be limited by the availability of free IL-15RA and its short half-life. Administration of soluble IL-15 / RA complexes significantly enhanced the half-life and bioavailability of II-15 in vivo. Thus, treatment of mice with this complex rather than IL-15 alone results in robust proliferation and maintenance of memory CD8 T cells and NK cells. Recent studies have shown that a portion of the extracellular region of IL-15RA, also called the sushi domain, is required for IL15 binding (WEI et al., J. Immunol., vol.167(1), p:277-282,2001). IL-15 / RA or IL-15 / sushi fusion proteins containing linkers are more potent than IL-15 and soluble IL-15RA alone. The combination of IL-15 / RA or IL-15 / sushi can maximize IL-15 activity. However, because the length of the insert sequence may affect transfection efficiency and gene expression levels, it is unclear whether designs incorporating both CAR and Il-15 / RA or IL15 / sushi in the same construct would maintain the desired biological properties in T or NK cells.

[0279] The present disclosure provides details of engineered cells that have both CAR and IL15 / RA or IL15 / sushi in a single configuration. In some examples, the disclosure includes methods to generate higher titers of virus and use stronger promoters to induce both CAR and IL15 / RA or IL-15 / sushi.

[0280] In some embodiments, the disclosure provides engineered cells that contain (1) an antigen targeted by the CAR, such as, but not limited to, CD4, CD2, CD3, CD7, CD5, CD45, CD20, CD19, CD33, CD123, CS1, and B cell maturation antigen (BCMA), and (2) IL-15, (3) IL15RA (RA) or sushi. In further embodiments, the CAR contains one or more co-stimulatory endogenous domains, such as chimeric antigen receptor, CD28, CD2, 4-1BB, and OX40, the intracellular domain of CD3 zeta chain. In further embodiments, but not limited to, the strong promoter can be SFFV. CARs, IL-15 / RA or sushi, and an inducible suicide gene ("safety switch"), or combinations thereof, can be assembled on vectors, such as lentiviral, adenoviral, and retroviral vectors or plasmids. The introduction of a “safety switch” could significantly improve the safety profile and limit off-target or off-tumor toxicity of CARs.

[0281] Characterization of CD4IL15RA-CAR CD4IL15RA-CAR was generated, which contains a third generation CD4CAR linked to IL15RA (Figure 34). The CAR (third generation), sushi / IL-15 combination was assembled on an expression vector, and their expression was driven by the SFFV promoter (Figure 34). The CAR containing sushi / IL-15 is linked to a P2A cleavage sequence. The sushi / IL-15 part consists of the IL-2 signal peptide fused to the sushi domain, linked to IL-15 via a 26 amino acid poly-proline linker (Figure 34).

[0282] To verify the construct of CD4IL15RA, HEK293FT cells were transfected with either GFP (control) or CD4IL15RA lentiviral plasmids. Approximately 60 hours after transfection, both HEK-293FT cells and supernatants were harvested. Cells were lysed in RIPA buffer containing a protease inhibitor cocktail and electrophoresed. Gels were transferred to Immobilon FL blotting membranes, blocked, and probed with mouse anti-human CD3z antibody at a ratio of 1:500. After washing, the membrane was probed with goat anti-mouse HRP-conjugated antibody, washed, and exposed to film after treatment with HyGlo HRP substrate. CD4IL15RA-CAR was shown to be successfully expressed in HEK293 cells (lane 2, Figure 35a) next to lane 3 (arrow). CD4IL15RA-CAR lentiviral supernatant was used to further investigate by transduction of fresh HEK-293 cells. HEK-293 cells were transduced with either GFP or CD4IL15RA-CAR viral supernatant from HEK-293FT cells. Polybrene was added at 4 μL / mL. After 16 hours, the medium was changed and replaced with medium without viral supernatant or polybrene. Three days after transduction, cells were harvested and stained with goat anti-mouse F(Ab')2 antibody at a ratio of 1:250 for 30 minutes. Cells were washed and stained with streptavidin-PE conjugated antibody at a ratio of 1:500, washed, suspended in 2% formalin, and analyzed by flow cytometry. As shown in Figure 35b, HEK-293 cells transduced with CD4IL15RA-CAR lentivirus were 80% positive for F(Ab)2-PE (circled area in Figure 35b), while cells transduced with the GFP control lentivirus were only weakly positive for F(Ab)2-PE (35b left panel).

[0283] Generation of CD4IL15RA-CAR NK cells NK-92 cells were transduced with CD4IL15RA-CAR lentiviral supernatant. After 5 days of incubation, cells were harvested and incubated with goat anti-mouse F(Ab')2 at a ratio of 1:250 for 30 minutes. Cells were washed, suspended, and stained with streptavidin-PE for 30 minutes. Cells were washed, suspended in 2% formalin, and analyzed by flow cytometry, showing that nearly 70% of transduced cells expressed CD4IL15RA-CAR (circled area in Figure 36). Further experimental testing of CD4IL15RA-CAR includes in vitro and in vivo leukemia / lymphoma killing assays, as well as target killing ability and cell proliferation rate comparison with CD4CAR-transduced cells. The inventors also used the same method described above to generate CD19IL15RA-CAR.

[0284] Generation of CD4IL15RA-CAR T cells Human umbilical cord buffy coat cells were transduced with CD4IL15RA-CAR lentiviral supernatant. After 5 days of incubation, cells were harvested and incubated with goat anti-mouse F(Ab')2 at a ratio of 1:250 for 30 minutes. Cells were washed, suspended, and stained with streptavidin-PE antibody for 30 minutes. Cells were washed, suspended in 2% formalin, and analyzed by flow cytometry, which showed that 63% of the transduced cells expressed CD4IL15RA-CAR (circled area in Figure 37). Further experimental testing of CD4IL15RA-CAR includes in vitro and in vivo leukemia / lymphoma killing assays, as well as comparison of target killing ability and cell proliferation rate with CD4CAR-transduced cells.

[0285] CD4IL15RACAR NK cells were tested for anti-leukemic activity in vitro compared to CD4CAR NK cells by co-culture with the following CD4 positive cell lines, Karpas 299 and MOLT4. The Karpas 299 cell line is a patient-derived anaplastic large T-cell lymphoma. The CD4-expressing MOLT4 cell line is an acute lymphoblastic leukemia (T-ALL) established from peripheral blood of a 19-year-old patient. During a 4-hour co-culture experiment, at a 5:1 effector:target ratio, CD4IL15RA CAR NK cells demonstrated significant killing capacity (95%) of Karpas 299 cells at a higher rate than that of CD4CAR NK cells (82%, Figure 38). Similarly, when co-cultured 1:1 with MOLT4 cells, CD4IL15RA CAR NK cells lysed target cells at a higher rate (84%-65%) than CD4CAR NK cells in an overnight assay (Figure 39). These results indicate that CD4IL15 CAR NK cells can ablate tumor cells similar to CD4CAR NK cells.

[0286] CD4CAR and CD4IL15RA CAR T cells exhibit more potent antitumor activity in vivo than CD4CAR To evaluate the in vivo antitumor activity of CD4CAR and CD4IL15RACAR T cells and to test the possibility that CD4IL15RA CAR T cells may be more persistent relative to CD4CAR T cells, we developed a xenogeneic mouse model in which sublethally irradiated NSG mice were injected intravenously with luciferase-expressing MOLM13 cells, a 100% CD4 acute myeloid leukemia cell line (M5), allowing for measurable tumor formation. Three days after tumor cell injection, six mice were inoculated with 8 × 10 6Mice were injected intravenously with one course of CD4CAR, CD4IL15RACAR T cells or vector control T cells. On days 3, 6, 9 and 11, mice were subcutaneously injected with RediJect D-Luciferin (Perkin Elmer) and tumor burden was measured by IVIS imaging (Figure 40). On day 6, mice treated with CD4CAR T cells had a 52% lower tumor burden compared to the control, while mice treated with CD4IL15RA CAR T cells had a 74% lower tumor burden (Figure 41). On day 11, almost all tumor cells were lysed in both of these groups. Unpaired T test analysis showed that compared to the control, the CD4CAR and CD4IL15RACAR T cell treatment groups had lower light intensity and smaller tumor burden by day 9, a highly significant statistical difference (P = 0.0045).

[0287] Promoter testing using GFP reporter HEK293FT cells were transfected with lentiviral plasmids expressing GFP under the SFFV, EF1 or CAG promoter. Approximately 60 hours after transfection, supernatants were harvested from each. Relative viral titers were first determined by introducing supernatants from each of the three promoters into HEK293 cells. HEK-293 cells were transduced with each of the three GFP viral supernatants obtained from HEK-293FT cells. Polybrene was added to 4 μL / mL. The medium was changed 16 hours later and replaced with viral supernatant or medium without polybrene. Three days after transduction, cells were harvested, washed, suspended in 2% formalin, and the status of GFP expression (FITC) was analyzed by flow cytometry. GFP expression was seen in each sample, but was highest in cells transduced with viruses generated using the SFFV promoter.

[0288] Activated human umbilical cord buffy coat cells were transduced with GFP lentiviral supernatant from each promoter (amount based on HEK293 transduction efficiency results). After 5 days of incubation, cells were harvested, washed, suspended in 2% formalin, and analyzed for GFP expression by flow cytometry. 43% of cells expressed high levels (>10 3 ), whereas GFP expression in cells transduced with viruses using the promoters EF1 (15%) and CAG (3%) was considerably lower. After 5 days, cells analyzed in the same way showed almost the same percentages (46%, 15% and 3%, respectively, FIG. 23). These results indicate that the SFFV promoter results in stronger expression than the EF1 or CAG promoters and that expression remains high for at least 10 days after transduction. Further experimental tests include longer incubation times of the transduced cells beyond the 10 day period.

[0289] Methods are provided for generating a CAR gene that includes at least one of a T antigen recognition portion (at least one of CD4, CD8, CD3, CD5, CD7, and CD2, or a portion or combination thereof), a hinge region, and a T cell activation domain.

[0290] Methods for producing multiple CAR units (cCARs) are provided that include portions or combinations of CD33, CD123, CD19, CD20, CD22, CD269, CS1, CD38, CD52, ROR1, PSMA, CD138, and GPC3 or hinge regions and T cell activation domains. All references cited and / or disclosed herein are incorporated by reference in their entirety.

[0291] The methods provided include: 1) generating CAR T or NK cells that target CD45-expressing leukemias and lymphomas and avoid self-killing; and 2) generating "armored" CAR T or NK cells designed to overcome the inhibitory tumor microenvironment and exhibit enhanced anti-tumor activity and long-term persistence.

[0292] The present invention is not limited to the above described and illustrated embodiments, but can be modified and altered within the scope of the appended claims. Various publications, including patents, published applications, technical papers, and academic papers, are cited throughout this specification. Each cited publication is incorporated herein by reference in its entirety for all purposes. Various terms relating to aspects of the invention are used throughout the specification and claims. Such terms shall have their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms shall be interpreted consistent with the definitions provided herein.

[0293] Functional titer of viral vector particles in the supernatant (percentage (%) of GFP cells determined by flow cytometry) allows for a surrogate viral titer adjustment, as higher titer virus will infiltrate more cells, resulting in a cell population with a higher percentage of GFP expression. To determine the functional titer of viral vector particles in each of our supernatants, HEK293 cells were transduced with either EF1-GFP or SFFV-GFP viral supernatants in volumes of either 30 μL (low), 125 μL (medium), or 500 μL (high) per well of a 12-well tissue culture-treated plate. The following morning, medium was changed to DMEM containing 10% FBS.

[0294] The transduced cells were then trypsinized, washed, and suspended in formalin and analyzed by flow cytometry. The percentage of GFP+ cells in each condition was measured by flow cytometry using the FITC channel (Figure 43). In all cases, cells transduced with SFFV-GFP had a higher percentage of GFP+ cells than those transduced with the corresponding volume of EF1-GFP virus supernatant (50% and 18% GFP+ cells were observed when using low volume virus, 80% and 40% GFP+ cells were observed when using medium volume virus, and 82% and 70% GFP+ cells were observed when using high volume virus). Therefore, we determined that using the maximum amount of EF1 promoter virus was comparable to using the minimum volume of SFFV promoter virus in terms of titer, and the results of the following transduction experiments allow for a comparison of relative promoter strengths.

[0295] The transduced cells were also visualized with an EVOS fluorescence microscope using GFP at 20x magnification for each well and the same exposure conditions (Figure 42). Cells transduced with SFFV-GFP virus supernatant were dramatically brighter than cells transduced with EF1-GFP virus. Furthermore, comparing the images of the EF1 promoter under the use of high virus doses with the images of the SFFV promoter under the use of low virus doses shows similar fluorescence intensity. This indicates that the SFFV promoter is a stronger driver of gene expression.

[0296] Comparison of surface expression and persistence of different promoters in primary T cells (proportion of GFP cells in transduced T cells determined by flow cytometry shows expected differences in the GFP cell population as predicted from previous experiments in HEK293 cells) To determine promoter transduction efficiency and persistence of surface expression in primary T cells, activated cord blood buffy coat T cells were transduced with either 50 μL of SFFV-GFP or 1 mL of EF1-GFP viral supernatant in 12-well tissue culture plates precoated with RetroNectin (Clontech). After two overnight transductions, cells were cultured in T cell culture medium containing 300 IU / mL IL-2 (Peprotech) and incubated with 1.0–4.0 × 10 6 A cell density of 100 / mL was maintained. Cells were washed and suspended in formalin, and the percentage of GFP+ cells was determined by flow cytometry analysis using the FITC channel at 7, 14, 21, and 28 days after transduction. The percentage of GFP+ cells was consistently higher in T cells transduced with SFFV-GFP compared to EF1-GFP transduced T cells (Figure 44A). Further comparison showed that T cells transduced with a larger amount (1 mL) of EF1-GFP supernatant showed a greater decrease in the percentage of GFP+ cells between days 7 and 28 than cells transduced with a smaller amount (50 μL or 20-fold less) of SFFV-GFP supernatant (Figure 44B). This suggests that transduction with the SFFV promoter maintains better persistence of phenotypic expression in transduced cells.

[0297] BCMA or TACI or BAFF-R CAR NK cells or T cells target cells expressing at least one of the BCMA or TACI or BAFF-R CAR antigens To evaluate the cytotoxicity ability of CARs with at least one of BCMA or TACI or BAFF-R NK cells or T cells, co-culture assays are performed with cell lines or primary human cells expressing at least one of BCMA or TACI or BAFF-R. The ability of the aforementioned CAR NK cells or T cells to lyse target cells is quantified by flow cytometry analysis, and the target cells are stained with Cytotracker dye (CMTMR). Lysis is observed over a long culture time of 24 hours.

[0298] BAFF or APRIL CAR NK or T cells target cells expressing at least one of the BCMA or TACI or BAFF-R antigens The chimeric antigen receptor in the CAR is a ligand for BCMA or TACI or BAFF-R.

[0299] To evaluate the cytotoxicity potential of CARs with at least one of BCMA or TACI or BAFF-R NK or T cells, co-culture assays were performed with cell lines or human primary cells expressing at least one of BCMA or TACI or BAFF-R. The ability of the aforementioned CAR NK or T cells to lyse target cells was quantified by flow cytometry analysis, and the target cells were stained with Cytotracker dye (CMTMR). Lysis is observed in long-term cultures for 24 hours. [References] TIFF2024116259000001.tif209170TIFF2024116259000002.tif180169

Claims

1. 1. An engineered T or NK cell comprising an engineered chimeric antigen receptor polynucleotide to encode a chimeric antigen receptor polypeptide (CAR), the chimeric antigen receptor polypeptide comprising a signal peptide, an antigen binding domain, a hinge region, a transmembrane domain, at least one costimulatory domain, and a signaling domain, the antigen-binding domain is CD45; An engineered T or NK cell, wherein CD45 is disrupted or deleted in the engineered T or NK cell.

2. 2. The engineered T or NK cell of claim 1, wherein the CAR binds to CD45.

3. 2. The engineered T or NK cell of claim 1, wherein the gene encoding the cell surface CD45 antigen is knocked out or disrupted.

4. The engineered T or NK cell of claim 1, which is resistant to CAR T or NK cell self-killing.

5. The engineered T or NK cell of claim 1, wherein the CD45 antigen-binding domain comprises the binding portion or variable region of an antibody selective for CD45.

6. The engineered T or NK cell of claim 1, wherein the CD45 antigen recognition domain comprises a polypeptide selective for SEQ ID NO: 13, 15 or 17, or a polypeptide encoded by the corresponding polynucleotide sequence SEQ ID NO: 14, 16 or 18.

7. The engineered T or NK cell of claim 1, further comprising at least one enhancer selected from the group consisting of IL-15 or a functional fragment thereof, and an enhancer receptor comprising IL-15RA or a functional fragment thereof.

8. 10. Use of an engineered T or NK cell for the manufacture of a medicament for treating a cell proliferative disorder, said treatment comprising administering to a patient in need thereof the engineered cell of any one of claims 1 to 7, wherein the target of the antigen-binding domain is CD45.

9. 10. Use of engineered T or NK cells for the manufacture of a medicament for a method for creating space in bone marrow for bone marrow stem cell transplantation, comprising administering to a patient in need thereof the engineered cells of any one of claims 1 to 7, wherein the target of the antigen binding domain is CD45.

10. 10. Use of engineered T or NK cells for the manufacture of a medicament for a method for pre-treating a patient prior to undergoing a bone marrow transplant to receive stem cells, said pre-treatment comprising administering to a patient in need thereof the engineered cells of any one of claims 1 to 7, wherein the target of the antigen binding domain is CD45.

11. 10. Use of engineered T or NK cells for the manufacture of a medicament for a method for myeloablative conditioning for hematopoietic stem cell transplantation, comprising administering to a patient in need thereof the engineered cells of any one of claims 1 to 7, wherein the target of the antigen-binding domain is CD45.

12. 9. The use of engineered T or NK cells for the manufacture of a medicament for treating a cell proliferative disorder according to claim 8, wherein the cell proliferative disorder is selected from the group consisting of acute leukemia, chronic leukemia, B and T cell lymphoma, myeloid leukemia, acute lymphoblastic lymphoma or leukemia, primary effusion lymphoma, reticulohistiocytoma, transient myeloproliferative disorder of Down's syndrome, lymphocyte-predominant Hodgkin's lymphoma, myeloid leukemia or sarcoma, dendritic cell tumor, histiocytic sarcoma, giant cell tumor of tendon sheath, interdigitating dendritic cell sarcoma, and post-transplant lymphoproliferative disorder.

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