US20200283534A1 - CHIMERIC ANTIGEN RECEPTORS (CARs), COMPOSITIONS AND METHODS THEREOF - Google Patents

CHIMERIC ANTIGEN RECEPTORS (CARs), COMPOSITIONS AND METHODS THEREOF Download PDF

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US20200283534A1
US20200283534A1 US16/312,769 US201616312769A US2020283534A1 US 20200283534 A1 US20200283534 A1 US 20200283534A1 US 201616312769 A US201616312769 A US 201616312769A US 2020283534 A1 US2020283534 A1 US 2020283534A1
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cells
car
cell
domain
antigen recognition
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Yupo Ma
Kevin PINZ
Xun JIANG
Masayuki Wada
Kevin Chen
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Icell Gene Therapeutics LLC
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Icell Gene Therapeutics LLC
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Priority claimed from PCT/US2016/039306 external-priority patent/WO2016210293A1/fr
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Priority to US16/312,769 priority Critical patent/US20200283534A1/en
Assigned to ICELL GENE THERAPEUTICS LLC reassignment ICELL GENE THERAPEUTICS LLC ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: WADA, MASAYUKI, CHEN, KEVIN, JIANG, Xun, MA, YUPO, PINZ, Kevin
Publication of US20200283534A1 publication Critical patent/US20200283534A1/en
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Definitions

  • T cells a type of lymphocyte, play a central role in cell-mediated immunity. They are distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface.
  • T helper cells also called CD4+ T or CD4 T cells, express CD4 glycoprotein on their surface. Helper T cells are activated when exposed to peptide antigens presented by MHC (major histocompatibility complex) class II molecules. Once activated, these cells proliferate rapidly and secrete cytokines that regulate immune response. Cytotoxic T cells, also known as CD8+ T cells or CD8 T cells, express CD8 glycoprotein on the cell surface.
  • the CD8+ T cells are activated when exposed to peptide antigens presented by MHC class I molecules.
  • Memory T cells a subset of T cells, persist long term and respond to their cognate antigen, thus providing the immune system with “memory” against past infections and/or tumor cells.
  • CARs chimeric antigen receptors
  • CD19-specific CAR (CD19CAR) T-cell therapies have had remarkable results including long-term remissions in B-cell malignancies (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, Vietnamesemuddin 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).
  • CAR therapy in this respect has the potential to further address a great clinical need.
  • CAR T cells may be suppressed and de-activated on arrival at the tumor site.
  • CAR chimeric antigen receptors are scFvs derived from monoclonal antibodies and some of these monoclonal antibodies have been used in the clinical trials or treatment for diseases. However, they have limited efficacy, which suggests that alternative and more potent targeting approaches, such as CARs are required.
  • scFvs are the most commonly used chimeric antigen receptor for CARs.
  • CAR affinity binding and locations of the recognized epitope on the antigen could affect the function. Additionally the level of the surface CAR expression on the T cells or NK cells is affected by an appropriate leader sequence and promoter. Furthermore, overexpressed CAR proteins can be toxic to cells.
  • the present disclosure provides an engineered cell having a first chimeric antigen receptor polypeptide including a first antigen recognition domain, a first signal peptide, a first hinge region, a first transmembrane domain, a first co-stimulatory domain, and a first signaling domain; and a second chimeric antigen receptor polypeptide including a second antigen recognition domain, a second signal peptide, a second hinge region, a second transmembrane domain, a second co-stimulatory domain, and a second signaling domain; wherein the first antigen recognition domain is different than the second antigen recognition domain.
  • the present disclosure provides an engineered polypeptide including a chimeric antigen receptor and an enhancer.
  • the present disclosure provides an engineered polypeptide including a chimeric antigen receptor polypeptide and an enhancer.
  • the present disclosure provides an engineered cell having the engineered polypeptide or polynucleotide described above.
  • the present disclosure provides a method of reducing the number of target cells including the steps of (i.) contacting said target cells with an effective amount of an engineered cell having at least one chimeric antigen receptor polypeptide, for engineered cells having multiple chimeric antigen receptor polypeptides, each chimeric antigen receptor polypeptide is independent; and (ii.) optionally, assaying for the reduction in the number of said cells.
  • the target cells include 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, BAFF, APRIL, 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.
  • the present disclosure provides methods for treating B-cell lymphoma, T-cell lymphoma, multiple myeloma, chronic myeloid leukemia, B-cell acute lymphoblastic leukemia (B-ALL), and cell proliferative diseases by administering any of the engineered cells described above to a patient in need thereof.
  • B-ALL B-cell acute lymphoblastic leukemia
  • FIG. 1 A schematic representation of cCAR construct (hereinafter, “multiple CAR or compound CAR”). Multiple or compound CAR targets multiple antigens (e.g. cell type 1 or cell type 2 or the same cell type). Multiple or cCAR T cell immunotherapies comprises individual component CAR comprising a different or same antigen recognition domain, a hinge region, a transmembrane domain, various co-stimulatory domain(s) and an intracellular signaling domain.
  • multiple CAR or compound CAR targets multiple antigens (e.g. cell type 1 or cell type 2 or the same cell type).
  • Multiple or cCAR T cell immunotherapies comprises individual component CAR comprising a different or same antigen recognition domain, a hinge region, a transmembrane domain, various co-stimulatory domain(s) and an intracellular signaling domain.
  • FIG. 2A A schematic representation of cCAR-T construct.
  • the construct comprises a SFFV promoter driving the expression of-multiple modular units of CARs linked by a P2A peptide.
  • the cCARs split and engage upon targets expressing CD33 and/or CD123.
  • the activation domains of the construct may include, but is not limited to, 4-1BB on the CD33 CAR segment and a CD28 region on the CD123 CAR.
  • FIG. 2B A Western blot depicting the expression of transduced CD33CD123 cCAR-T cells.
  • the figure depicts expression of two different CAR proteins, i.e., CD33 CAR and CD123 CARs.
  • the cCAR-T cells expressing both CD33 and CD123 CARs upon cleavage of the linker generate two distinct and consistently intense protein bands.
  • Green Fluroscent Protein (GFP) is included as negative control.
  • FIG. 2C Flow cytometry representing the efficiency of transduction.
  • Upper panel shows the lentiviral titer for CD33CD123 cCARs (also referred to as CD33CD123-2G-CAR) tested on 293FT HEK (human embryonic kidney) cells to gauge maximum transduction efficiency before usage on UCB (umbilical cord blood) and PB (peripheral blood) T-cells.
  • Lower panel shows CD33CD123 cCAR (also referred to as CD33CD123-2G-CAR) T-cells transduced with lentiviral vectors comprising CD33CD123 cCAR construct and GFP-transduced cells as control Percentages indicated by yellow circles are proxies for transduction efficiency.
  • FIG. 3 Schematic showing a method of generating a high-efficiency compound CAR (cCAR).
  • HEK-293-FT cells are transfected with compound CAR plasmid DNA and lipofectamine 2000; viral supernatant collected at about 36 hr and at about 60 h; filtered and stored at ⁇ 80° C.
  • T cells are activated with anti-mouse CD3 antibody and IL-2 for at least 2 days.
  • Activated T cells are transduced at least once with thawed lentivirus on retronectin-coated plates; After at least one overnight transductions at 0.3 ⁇ 106 T cells/mL for about 2 days, the number of T cells was reduced in order to increase transduction efficiency.
  • cCAR T cells are co-cultured with target cells in vitro and cCAR T cells killing efficacy of cancer cells is assessed in vivo (mice).
  • FIG. 4 A co-culture assay representing the incubation of CD33CD123-2G CAR-T cells (cCAR) with the promyelocytic leukemia cell line HL60. cCAR-T cell (lower panel) is compared to control GFP transduced T-cell (upper panel). The efficacy of the killing is measured by the population of CD33+ cells that is left over after incubation for about 24 hours (enclosed in yellow circles).
  • FIG. 5 A co-culture assay representing incubation of cCAR-T cells with the myelogenous leukemia cell line KG-1a, which expresses about 100% CD33 and about 50-80% CD123.
  • cCAR-T cell lower panel
  • control GFP transduced T-cell upper panel
  • the efficacy of the killing is measured by the population of CD33+ cells that is left over after incubation for about 24 hours.
  • FIG. 6 CD33CD123 cCAR-T cells co-cultures with AML-9 at 5:1.
  • a co-culture assay representing incubation of cCAR-T cells with AML patient samples (here referred to as AML-9).
  • the patient cells include mixed populations of cells, such as for example, leukemia cells, monocytes, and other types of blasts.
  • CD33 acts as a marker for CAR-T action as well as CD34, a specific marker for leukemia cells.
  • the CAR-T panel (right) is compared to control GFP transduced T-cells (middle).
  • the efficacy of the killing is measured by the population of CD33+/CD34+ cells that is left over after incubation for at least 24 hours.
  • FIG. 7 CD33CD123 cCAR-T cells co-cultures with Sp-BM-B6 at 5:1.
  • a co-culture assay representing incubation of cCAR-T cells with B-ALL patient samples here referred to as Sp-BM-B6.
  • the patient cells include mixed populations of cells, such as, for example, leukemia cells, monocytes, and other types of blasts.
  • CD34 acts as a specific marker for leukemia cells.
  • the CAR-T panel (right) is compared to control GFP transduced T-cells (middle). The efficacy of the killing is measured by the population of CD34+ cells left over after incubation for at least 24 hours.
  • FIG. 8 CD33CD123 cCAR expression in NK-92 cells.
  • the CD33CD123 cCAR expression are detected using goat-anti-mouse antibody, F(ab)2.
  • FIG. 9 A co-culture assay representing incubation of CD33CD123 cCAR NK-92 cells with HL-60.
  • the cCAR NK-92 cells are compared with GFP transduced NK-92 cells.
  • the efficacy of the killing is measured by the population of CD33+ cells left over after incubation for about 24 hours.
  • FIG. 10 A co-culture assay representing incubation of cCAR NK-92 cells with KG1a.
  • the cCAR NK cell panel is compared with GFP transduced NK-92 cells.
  • the efficacy of the killing is measured by the population of CD33+ cells left over after incubation for about 24 hours.
  • FIG. 11 Dose response of CD33CD123 cCAR (CAR-CD33/123) NK-92 cells with HL-60 or KG1a. The efficacy of the killing is measured by the population of CD33+ cells left over after incubation for about 24 hours.
  • FIG. 12 A comparison of CD33CD123 cCAR NK-92 cell killing ability with control in two populations of KG11 cells. Assays were performed at different ratios of CAR-CD33/123 (CD33CD123 cCAR NK-92 cells) and target cells, kG1a. The efficacy of the killing is measured by the population of CD33+CD123+ or CD33+CD123-cells left over after incubation for about 24 hours.
  • FIG. 13A Links by P2A and T2A schematic showing both cCAR-T and 4-1BBL in a single construct.
  • the construct consists of a SFFV promoter driving the expression of two modular units of CARs A peptide and an enhancer, 4-1BBL.
  • the cCARs and 4-1BBL split and engage upon targets expressing CD33 and/or CD123 and 4-1BBL.
  • Compound CAR, CD33CD123 CAR T cells received not only costimulation through the CD28 but also 4-1BB ligand (4-1BBL or CD137L).
  • the CD3-zeta signaling domain complete the assembly of this CAR-T.
  • FIG. 13B Expression the CD33CD123-41BBL-2G construct in T-cells.
  • T-cells derived from peripheral blood from healthy donors were transduced with the CD33CD123-4-1BBL-2G construct in 6-well plates incubated with 2 ml of virus supernatant.
  • CAR expression was assayed with F(ab)′ labeling for surface expression of the CAR protein and subsequently underwent FACS analysis.
  • Transduced cells were compared to control T-cells labeled at the same time. Expression was determined and transduced population encircled on plot 1 day after end of transduction period.
  • FIG. 14 Links by P2A and T2A schematic showing both cCAR-T and IL-15/IL-15sushi in a single construct.
  • the construct consists of a SFFV promoter driving the expression of two modular units of CARs and an enhancer, IL-15/IL-15sushi.
  • the cCARs and IL-15/IL-15sushi split and engage upon targets expressing CD33 and/or CD123.
  • the CD3-zeta signaling domain completes the assembly of this CAR-T.
  • the enhancers include, but not limited to, IL-15/IL-15sushi on cCAR.
  • FIG. 15 A schematic representation of cCAR.
  • the construct comprises a SFFV promoter driving the expression of multiple modular units of CARs linked by a linker. Upon cleavage of the linker, the cCARs split and engage upon targets expressing combinations of various target antigens: CD19 and/or CD20, and/or CD22 and/or 138. Multiple cCARs utilize the same or different co-stimulatory domains, such as, without limiting 4-1BB (also labeled as 4-BB) and/or CD28.
  • 4-1BB also labeled as 4-BB
  • FIG. 16 Activated T cells transduced to make CD19CD20-2G, CD19CD22-2G CAR T cells (all are L8).
  • 16 A Design of compound CARs.
  • 16 B Western blot. HEK-293T cells were transfected with lentiviral plasmids for control vector (lane 1), CD19CD20-2G (lane 2), and CD19CD22-2G (lane 3). 48 hours after transfection, supernatant was removed, and cells were also harvested. Cells were lysed for Western blot and probed with mouse anti-human CD3z primary antibody, and goat anti-mouse HRP secondary antibody.
  • 16 C PMBC buffy coat T cells were activated 3 days with anti-CD3 antibody.
  • FIG. 17 Expression of compound CD19CD22CAR T cells using different leader sequences.
  • PMBC buffy coat T cells were activated 3 days with anti-CD3 antibody.
  • Cells were transduced with either control vector (left), L8-CD19CD22-2GCAR (middle left), L45-CD19CD22-2GCAR, (middle right) or CSF-CD19CD22-2GCAR (right) lentiviral supernatant. The supernatants were each 3 ⁇ concentrated. After 3 days of incubation, cells were harvested and incubated with goat anti-mouse Fab2 or goat IgG antibodies conjugated with biotin for 30 minutes.
  • FIG. 18 Comparison of transduction efficiency using concentrated vs unconcentrated L8-CD19CD22-2G or L8-CD19CD20-2G lentiviral supernatant.
  • FIG. 19 L8-CD19CD22-2G CAR T cells lyse SP53 tumor cells in overnight co-culture.
  • Activated PMBC T cells transduced with either control (top row), L8-CD19CD22-2G, or (bottom row) lentiviral supernatant were incubated with SP53 cells at the ratios of 1:1 (left) 2:1 (middle) and 5:1 (right), effector:target cells.
  • samples were washed and stained with anti-human CD3-PerCp and anti-human CD19-APC, washed, and analyzed by flow cytometry.
  • FIG. 20 L8-CD19CD22-2G CAR T cells lyse JeKo-1 tumor cells in overnight co-culture.
  • Activated PMBC T cells transduced with either control (left), or L8-CD19CD22-2G, (middle) 3 ⁇ concentrated lentiviral supernatant were incubated with JeKo-1 cells at the ratios of 2:1 (top) and 5:1 (bottom), effector:target cells.
  • FIG. 21 L8-CD19CD22-2G CAR T cells lyse AML patient cells in overnight co-culture.
  • FIG. 22A L8-CD19CD22-2G CAR T cells deplete CD19+B-ALL patient cells.
  • Activated PMBC T cells transduced with either control (left), or L8-CD19CD22-2G, (middle) lentiviral supernatant were incubated with CMTMR-stained cells from a patient with B-ALL (PT2) at a 1:1 ratio for 4 days in the presence of 2.5% FBS and IL-2. Following this incubation at 37° C., samples were washed and stained with anti-human CD3-PerCp and anti-human CD19-APC, washed, and analyzed by flow cytometry. Prestained patient cells cultured alone for 4 days are shown on the right.
  • FIG. 22B L-8-CD19CD22-2G cCAR T-cells show effect on CD22 + K562 cells.
  • a co-culture experiment at 1:1 ratio (effective:target) show a modest significant cytotoxic effect on K562 expressing CD22 population compared to the control.
  • Co-cultures were stained with CD3, CD19 and CD22 to separate effector and target populations by flow cytometry. The result was graphed. Cytotoxicity results remain consistent with other numbers reported for anti-tumor activity against artificial antigen presenting cell lines.
  • FIG. 23 Various transduction schemes for BC1cCAR lentivirus.
  • A Method 1 consisting of a 2 ⁇ transduction for 24 hours each time is a baseline transduction scheme. Scheme proceeds according to the figure.
  • B Method 2 possesses the same methodology as Method 1, however, the second transduction is replaced by continued incubation.
  • C Method 2 revised uses viral supernatant incubated with cells directly for 48 hours.
  • FIGS. 24A-24C CAR construct scheme and comparison of transduction methodologies.
  • BC1cCAR's modular design consists of an anti-CD269 (BCMA) single-chain variable fragment (scFv) region fused to an anti-CD319 (CS1) scFv by a self-cleaving P2A peptide, CD8-derived hinge (H) and transmembrane (TM) regions, and tandem CD28 and 4-1BB co-activation domains linked to the CD3 ⁇ signaling domain.
  • a strong spleen focus forming virus promoter (SFFV) and a CD8 leader sequence were used for efficient expression of the CD3CAR molecule on the T-cell surface.
  • SFFV spleen focus forming virus promoter
  • CD8 leader sequence were used for efficient expression of the CD3CAR molecule on the T-cell surface.
  • BC1cCAR's expression was measured via flow cytometry against an isotype control. Population encircled represents transduced CAR cells.
  • FIG. 24D Protein expression of BC1cCAR and BCMA-CS1-2G in HEK-293FT cells.
  • HEK-293FT cells were transfected with lentiviral plasmids for GFP (lane 1), BC1cCAR (lane 2) 48 hours after transfection, supernatant was removed, and cells were also removed. Cells were lysed for Western blot and probe with mouse anti-human CD3z antibody.
  • C Transduction efficiency is improved by optimal methods.
  • FIGS. 25A-25C in vitro evaluation of BC1cCAR T-cells against myeloma cell lines.
  • CMTMR-PE Cytotracker
  • Target U266 cells were labeled with Cytotracker (CMTMR) dye to distinguish it from effector T-cells. Encircled populations represent tumor cells.
  • FIGS. 26A-26D Characterization of BC1cCAR T-cell anti-tumor activity against primary myeloma tumor cells.
  • 26 A Dose dependent effect on MM7-G primary double phenotype tumors. BC1cCAR and control T-cells were cultured against BCMA + CS1 + primary myeloma cells MM7-G for 24 hours. Target cells were pre-stained with CMTMR and cultures were carried out in E:T ratios of 2:1, 5:1, and 10:1. Populations were gated by BCMA and CS1, along with CMTMR, and flow cytometry plots with populations encircled represent target tumor populations (left). Bar graph summarizing in vitro cytotoxicity is shown for clarity (right).
  • FIGS. 27A-27D Functional validation of BC1cCAR antigenic specificity.
  • 27 A We engineered a CML cell line, K562, to express either BCMA or CS1 independently. Wild-type K562 shows as a negative peak, while BCMA expressing K562 (BCMAxpK562) and CS1 expressing K562 (CS xpK562) show population shifts in their respective antigen expression ranges.
  • 27 B Short term (4 hour-12 hour) cultures of BC1cCAR T-cells against either BCMAxpK562 or CS1 xpK562 show antigen specific cytotoxicity correlating with E:T dosage increase. Experiments against wild-type K562 were performed as a negative control.
  • a CS1-specific single CAR was generated to compare efficacy with BC1cCAR against CS1xpK562 cells and are delineated with red bars in the respective plot. Anti-CS1 specific activity was also seen against CS1 dim NK-92 cells after 24 hours of culture.
  • FIGS. 28A-28C Long-term sequential killing assay and tumor re-challenge.
  • 28 A Scheme for construction of long-term sequential killing assay. Assay was conducted over a period of 168 hours with no exogenous cytokines where the initial culture was set-up with a 1:1 E:T ratio of CAR cells or control cells mixed with MM1S tumor cells. After 48 hours, flow cytometry analysis was acquired for a small sample collection and MM1S cells re-introduced into each treatment well. Repeated until the 168 hour time-point.
  • 28 B T-cell proliferation and response after 48 hours.
  • FIG. 29A-29C BC1cCAR T-cells demonstrate anti-leukemic effects in vivo.
  • 29 A IVIS imaging of MM1S Luc+ injected mouse model. NSG mice were sublethally irradiated and intravenously injected with luciferase-expressing MM1S multiple myeloma cells to induce measurable tumor formation. After 3 days, the mice were intravenously injected with 5 ⁇ 10 6 BC1cCAR T-cells or control GFP T-cells. On days 3, 6, 8 and 11, mice were injected subcutaneously with RediJect D-Luciferin and subjected to IVIS imaging.
  • 29 B BC1cCAR T-cells control MM1S tumor growth.
  • FIG. 29D BCMA-CAR and BC1 cCAR T-cells demonstrate a profound anti-leukemic effect on a mixture of K562 cells expressing BCMA and CS1 in xenograft mouse model.
  • Luciferase positive K562 cells expressing BCMA are mixed with luciferase positive K562 cells expressing CS1 at a ratio of 4:1 BCMA to CS1 K562 cells.
  • the mixed K562 cells (0.5 ⁇ 10 6 cells) were then injected intravenously (day 1) at 24 h later after sub-lethal irradiation.
  • FIG. 29E BCMA and BC1 cCAR T-cells in vivo significant reduction of tumor burden. Percent reduction relative to control in mice treated with BCMA CAR T-cells or cCAR (BC1 cCAR) relative to control over time.
  • FIGS. 30A-30B BC1cCAR transduction into NK-92 cells.
  • FIGS. 31A-31B Characterization of BC1cCAR NK-92 anti-tumor properties.
  • MM7-G primary tumor cells were stained with cell cytotracker dye (CMTMR) to distinguish from NK-92 cells and are encircled.
  • CTMR cell cytotracker dye
  • Summary bar graph of BC1cCAR NK-92 cytotoxic activity is presented as a visualization aid.
  • BC1cCAR NK-92 cells were tested for antigen specific activity using artificially generated BCMA expressing K562 (BCMAxpK562) and CS1 expressing K562 (CS1xpK562) cells. Co-cultures were carried out over 4 hours at an E:T ratio of 5:1. K562 populations were previously stained with CMTMR and encircled in the flow cytometry plots. Bar graph summarizing anti-tumor activity to visualize.
  • FIGS. 32A-32C Generation and characterization of different BAFF-CAR constructs.
  • CSF-BAFF-28 41BBL is a BAFF CAR co-expressing 4-1BBL (41BBL) with a CSF leader sequence.
  • CSF-BAFF-28 IL-15RA is a BAFF CAR co-expressing IL-15/IL-15sushi (IL-15RA) with a CSF leader.
  • FIG. 33 Characterization of L45-BAFF-28 CAR T anti-tumor properties.
  • FIGS. 34A-34B Characterization of anti-tumor activity using different BAFF-CAR constructs and enhancements.
  • 34 A BAFF-CAR constructs against MM1S cells. L8-BAFF-28IL-15/IL-15sushi and L8-BAFF-28-41BBL CARs were cultured for 24 hours against MM1S tumor cells at an E:T ratio of 5:1. Tumor populations are encircled.
  • 34 B BAFF-CAR constructs against SP53 cells. Both CARs and L45-BAFF-28 CAR were cultured against Sp53 tumor cells (B-lineage) at an E:T ratio of 5:1 for 24 hours.
  • 34 C Summary bar graph of cytotoxic activity.
  • FIG. 35 A schematic showing cCAR construct.
  • the construct consists a SFFV promoter driving the expression of two modular units of CAR linked by a P2A peptide. Upon cleavage of this P2A peptide, the cCARs split and engage upon targets expressing BCMA and/or CD19. Two unit CARs use different or same co-stimulatory domain.
  • a co-stimulatory domain could be, but limited to, 4-1BB or CD28.
  • FIGS. 36A-36B Characterization of the BCMA CAR unit.
  • the BCMA CAR and CS1 CAR were also evaluated for its anti-tumor properties against primary MM7-G myeloma patient cells.
  • the MM7-G population is a majority BCMA + CS1 population with minority but significant CS1 + only populations as well.
  • Both BCMA CAR and CS1 CAR were used in tandem to evaluate cytotoxicity with BCMA and cytotracker (CMTMR) used to differentiate tumor populations from CAR cells.
  • CTMR cytotracker
  • FIGS. 37A-37C Characterization of CD19 CARs.
  • 37 A Design of CD19CAR unit.
  • 37 B Western blot. HEK-293T cells were transfected with lentiviral plasmids for control vector (lane 1) and CD19-2G (lane 2). 48 hours after transfection, supernatant was removed, and cells were also harvested. Cells were lysed for Western blot and probed with mouse anti-human CD3z primary antibody, and goat anti-mouse HRP secondary antibody.
  • C PMBC buffy coat T cells were activated 3 days with anti-CD3 antibody.
  • FIGS. 38A-38B Expression of compound CD19CAR T cells using different leader sequences.
  • 38 A CAR constructs were designed to express the fusion protein with different leader sequences.
  • 38 B PMBC buffy coat T cells were activated 3 days with anti-CD3 antibody. Cells were transduced with either control vector (left), HA-CD19-2G (top middle), IL2-CD19-2G (top right), L8-CD19-2G (lower middle left), L45-CD19-2G, (lower middle right) or CSF-CD19-2GCAR (lower right) lentiviral supernatant.
  • FIGS. 39A-39B Expression of CD19CAR on T cells using different CD19 scFv sequences.
  • 39 A CAR constructs were designed to express the fusion protein with different scFv sequences.
  • 39 B PMBC buffy coat T cells were activated 3 days with anti-CD3 antibody. Cells were transduced with either control vector (left), L8-CD19-2G (middle), IL2-CD19-2G (top right), or L8-CD19b-BB-2G (right) lentiviral supernatant. After 3 days of incubation, cells were harvested and incubated with goat anti-mouse Fab2 or goat IgG antibodies conjugated with biotin for 30 minutes.
  • FIG. 40 L8-CD19-2G and CD19b-BB CAR T cells lyse SP53 tumor cells in overnight co-culture.
  • FIG. 41 L8-CD19-2G and CD19b-BB CAR T cells lyse JeKo-1 tumor cells in overnight co-culture.
  • FIG. 42 L8-CD19-2G and L8-CD19b-BB-2G CAR T cells lyse AML patient cells in overnight co-culture.
  • FIG. 43 L8-CD19-2G and L8-CD19b-BB-2G CAR T cells deplete CD19+ patient cells.
  • Activated PMBC T cells transduced with either control (left), L8-CD19-2G, (middle) or L8-CD19b-BB-2G (right) lentiviral supernatant were incubated with CMTMR-stained cells from a patient with B-ALL.
  • L8-CD19-2G T cells were incubated with patient cells at a 1:1 ratio for overnight (top), while L8-CD19b-BB-2G T cells were incubated with patient cells at a 5:1 ratio for 40 hours (bottom).
  • FIG. 44 A schematic showing cCAR construct.
  • the construct consists a SFFV promoter driving the expression of two modular units of CAR linked by a P2A peptide. Upon cleavage of this P2A peptide, the cCARs split and engage upon targets expressing BCMA and/or CD19b. Two unit CARs use different or same co-stimulatory domain. A co-stimulatory domain could be 4-1BB or CD28.
  • FIGS. 45A-45C Generation and characterization of different BAFF-CAR constructs.
  • 45 C Additional BAFF-CAR constructs containing different leader sequences and construct designs (additional units) were validated and used to determine if CAR transduction could be improved. Transduced populations are encircled and gating compared to control T-cells.
  • CSF-BAFF-28 41BBL is a BAFF CAR co-expressing 4-1BBL (41BBL) with a CSF leader sequence.
  • CSF-BAFF-28IL-15/IL-15sushi is a BAFF CAR co-expressing IL-15/IL-15sushi with a CSF leader.
  • FIGS. 46A-46B L45-BAFF-28 CAR T-cells possess anti-tumor activity against MM1S tumor cells. Characterization of L45-BAFF-28 CAR T anti-tumor properties.
  • L45-BAFF-28 CAR T-cells were cultured for 48 hours at an E:T ratio of 3:1 against MM1S myeloma cells. Duplicate samples are shown.
  • FIGS. 47A-47B Characterization of anti-tumor activity using different BAFF-CAR constructs and enhancements.
  • 47 A L8-BAFF-28IL-15/IL-15sushi and L8-BAFF-28 4-1BBL CARs were cultured for 24 hours against MM1S tumor cells at an E:T ratio of 5:1. Tumor populations are encircled.
  • 47 B Both CARs and L45-BAFF-28 CAR were cultured against Sp53 tumor cells (B-lineage) at an E:T ratio of 5:1 for 24 hours.
  • FIG. 48 CRISPR/Cas9 interference system.
  • the expression of sgRNA and Cas9 puromycin is driven by the U6 and SFFV promoters, respectively.
  • the Cas9 is linked with puromycin resistant gene by E2A self-cleaving sequences.
  • FIG. 49A Steps of generation of CAR T or NK cell targeting hematologic malignancies.
  • FIG. 49B Generation and cell sorting of stable CD45 knockdown NK-92 cells using CRISPR/Cas9 lentivirus system.
  • Flow cytometry analysis indicated the CD45 expression levels on NK-92 cell surface (left panels).
  • transduced sgCD45B CRISPR into NK-92 cells transduced cells were cultured in medium containing puromycin for a few weeks.
  • CD45 negative NK-92 cells were determined using CD45 antibody and were sorted.
  • the purity of stable NK 45i -92 (CD45 knockdown) NK-92 cells were determined by Flow cytometry analysis (right panel). This data showed that we we successfully generated and obtained NK 45i -92 cells.
  • FIG. 50 Cell growth curve of wild type, GFP transduced NK-92 or NK 45i -92NK cells.
  • KD CD45-knockdown
  • FIGS. 51A-51B Co-culture assay with CCRF-CEM (target: T) and GFP NK-92 or GFP NK 45i -92 cells (effector: E) at 5:1 (E:T) ratio and 16 hour incubation.
  • 51 A Flow cytometry analysis of CCRF-CEM only (blue dot in left panel), in co-culture with CCRF-CEM and control GFP transduced NK-92 cells (middle panel) or GFP NK 45i -92 cells (right panel). Blue dots in all of panels indicates the leftover target CCRF-CEM cells and red dots shows effector cells by co-culture assay. The majority of the blue dots are in the upper left square of each experiment.
  • FIGS. 52A-52B Co-culture assay with CCRF-CEM (target: T) and GFP NK-92, CD5CAR NK-92 or CD5CAR NK 45i -92 cells (effector: E) at 5:1 (E:T) ratio and 16 hour incubation.
  • ( 52 A) Flow cytometry analysis of CCRF-CEM only (left panel), in co-culture with CCRF-CEM and control GFP NK-92 cells (middle left panel), CD5CAR NK-92 cells (middle right panel), CD5CAR NK 45i -92 cells (right panel) from right to left.
  • CD5CAR NK-cells and CD5CAR NK 45i -92 cells can effectively lyse CCRF-CEM cells that express CD5 compared to GFP-control NK-92 cells in vitro co-culture assay and prof that knockdown of CD45 does not affect cell function for killing activity in NK-92 cells.
  • FIGS. 53A-53B Organization of the CD45CAR construct and its expression.
  • 53 A Schematic representation of the CD45CAR lentiviral vector.
  • the CD45CAR construct is a modularized signaling domain containing: a leader sequence, an anti-CD45scFv, a hinge domain (H), a transmembrane domain (TM), two co-stimulatory domains (CD28 and 4-1BB) that define the construct as a 3 rd generation CAR, and the intracellular signaling domain CD3 zeta.
  • 53 B HEK-293FT cells were transfected with lentiviral plasmids for GFP (lane 1) and CD45CAR (lane 2). 48 hours after transfection, supernatant was removed, and cells were also removed. Cells were lysed for Western blot and probe with mouse anti-human CD3z antibody.
  • FIG. 54 Transduction of CD45CAR into NK 45i -92 cells and cell sorting of CD45CAR transduced cells.
  • the expression levels of CD45CAR on NK 45i -92 were determined by flow cytometry analysis (circled in blue at middle panel) compared to NK 45i -92 cells (left panel) after CD45CAR lentviruses were transduced into NK 45i -92 cells.
  • CD45CAR expressed NK 45i -92 cells were sorted and CD45 expression levels on cell surface were determined by Flow cytometry analysis (right panel). About 87% of CD45CAR expression on cell surface was detected by flow cytometry analysis.
  • FIGS. 55A-55B Co-culture assay with CCRF-CEM (target: T) and GFP NK-92 or CD45CAR NK 45i -92 cells (effector: E) at 5:1 (E:T) ratio and 16 hour incubation.
  • ( 55 A) Flow cytometry analysis of in co-culture with CCRF-CEM and control GFP transduced NK-92 cells (left panel) or CD45CAR NK 45i -92 cells (right panel). Blue dots in all of panels indicates the leftover target CCRF-CEM cells and red dots shows effector NK-92 cells by co-culture assay. All of incubation time were 16 h and the ratio of effector T-cells:target cell is 5:1.
  • FIGS. 56A-56C Co-culture assay with Jurkat cells (target: T) and GFP-control or CD45CAR NK 45i -92 cells (effector: E) at 5:1 or 2:1 (E:T) ratio and 6 hour incubation.
  • ( 56 A) Flow cytometry analysis was carried out after Jurkat cells were stained by CMTMR cell tracker dye. These data show that Jurkat cells are CD45 positive (left panels) and mostly CD56 negative cells (right panel).
  • 56 B Flow cytometry analysis of co-culture assay with Jurkat cells (target: T) and control or CD45CAR NK 45i -92 cells (effector: E). The ratio of co-culture assay was performed in 5:1 or 2:1 (E:T).
  • CD45CAR NK 45i -92 cells shows about 60% cell lysis against Jurkat cells compared to control GFP NK-92 cells in both conditions. This data suggests that CD45CAR NK 45i -92 cells effectively lyse Jurkat cells that express CD45 on cell surface compared to GFP-control NK-92 cells in vitro co-culture assay.
  • FIG. 57A-57C Co-culture assay with GFP-NK-92 cells (target: T) and non-transduced NK-92 cells or CD45CAR NK 45i -92 cells (effector: E) at 5:1 or 2:1 (E:T) ratio, 6 hour incubation.
  • 57 A Flow cytometry analysis was carried out using GFP control NK-92 cells. These data proof that GFP control NK-92 cells are about 99% GFP positive cells (green dots).
  • ( 57 B) Flow cytometry analysis of co-culture assay with GFP control NK-92 cells (target: T) and non-transduced or CD45CAR NK 45i -92 cells (effector: E).
  • the ratio of co-culture assay was performed in 5:1 ( 57 A) or 2:1 (E:T) ( 57 C). Left panels showed that in co-culture with non-transduced or CD45CAR NK 45i -92 cells in 5:1 (E:T) ratio and right panels indicated that in co-culture with non-transduced or CD45CAR NK 45i -92 cells in 2:1 (E:T) ratio. Green dots in panels indicate the leftover target GFP NK-92 cells and red dots represent effector cells by co-culture assay. All of incubation times were 6 h. All experiments were performed in duplicate.
  • FIG. 57D Transduction of CD45b-BB or CD45b-28 into NK 45i -92 cells and cell sorting of CD45b-BB or CD45b-28 transduced NK 45i -92 cells.
  • the co-stimulatory domain for CDb-BB is 4-1BB while co-stimulatory domain for CD45b-28 is CD28.
  • the expression levels of CD45b-BB or CD45b-28 on NK 45i -92 were determined by flow cytometry analysis (circled in blue at middle panel) compared to NK 45i -92 cells (left panel) after CD45b-BB or CD45b-28 on lentviruses were transduced into NK 45i -92 cells.
  • CD45b-BB or CD45b-28 on expressed NK 45i -92 cells were sorted and CD45b-BB or CD45b-28 on expression levels on cell surface were determined by Flow cytometry analysis (right panel). About 74% of CD45b-BB or 82% of CD45b-28 on expression on cell surface was detected by flow cytometry analysis.
  • FIG. 57E Co-culture assay with REH cells (target: T) and GFP NK-92 cells, CD45CAR NK 45i -92 cells, CD45b-BB NK 45i -92 cells or CD45b-28 NK 45i -92 cells at 5:1 (E:T) ratio and 20 hour incubation.
  • Upper Flow cytometry analysis of CREH cells only (left panel), in co-culture with with REH cells and control GFP transduced NK-92 cells (2 nd left panel), CD45CAR NK 45i -92 cells (middle panel), CD45b-BB NK 45i -92 cells (4 th from left panel) or CD45b-28 NK 45i -92 cells (right panel).
  • CD45CAR NK 45i -92 cells shows about 76% cell lysis
  • CD45b-BB NK 45i -92 cells shows about 79% cell lysis
  • CD45b-28 NK 45i -92 shows 100% cell lysis against REH cells compared to control GFP NK-92 cells.
  • FIGS. 57FA-57FI Co-culture assay with U937 cells (target: T) and GFP NK-92 cells or CD45b-28 NK 45i -92 cells. at 2:1 (E:T) ratio for 20 hours.
  • FA Flow cytometry analysis of U937 cells (monocytic leukemia cell line) only (left panel), in co-culture with U937 cells and control GFP transduced NK-92 cells (middle panel) or CD45b-28 NK 45i -92 cells (right panel). Blue dots in all of panels indicates the leftover target U937 cells and red dots shows effector GFP or CD45b-28 NK 45i -92 cells by co-culture assay.
  • FB Bar graph indicates the percent of cell lysis by CD45b-28 NK 45i -92 cells compared to the control GFP NK92 cells in co-culture assay with U937 cells.
  • CD45b-28 NK 45i -92 shows about 81% cell lysis against U937 cells compared to control GFP NK-92 cells.
  • FIGS. 57GA-57GB Co-culture assay with MOLM-13 cells (target: T) and GFP NK-92 cells or CD45b-28 NK 45i -92 cells at 5:1 (E:T) ratio for 20 hours.
  • GA Flow cytometry analysis of MOLM13 cells (monocytic leukemic cell line) only (left panel), in co-culture with Molm13 cells and control GFP transduced NK-92 cells (middle panel) or CD45b-28 NK 45i -92 cells (right panel). Blue dots in all of panels indicates the leftover target MOLM13 cells and red dots shows effector GFP or CD45b-28 NK 45i -92 cells by co-culture assay.
  • GB Bar graph indicates the percent of cell lysis by CD45b-28 NK 45i -92 cells compared to the control GFP NK92 cells in co-culture assay with MOLM13 cells.
  • CD45b-28 NK 45i -92 shows about 91.6% cell lysis against Molm13 cells compared to control GFP NK-92 cells.
  • FIGS. 57HA-57HB Co-culture assay with Jeko-1 cells (target: T) and GFP NK-92 cells or CD45b-28 NK 45i -92 cells at 2:1 (E:T) ratio for 6 hours.
  • HA Flow cytometry analysis of Jeko-1 cells (T cell acute lymphoblastic cell line) only (left panel), in co-culture with Jeko-1 cells and control GFP transduced NK-92 cells (middle panel) or CD45b-28 NK 45i -92 cells (right panel). Blue dots in all of panels indicates the leftover target Jeko-1 cells and red dots shows effector GFP or CD45b-28 NK 45i -92 cells by co-culture assay.
  • FIGS. 57IA-57IB Co-culture assay with SP53 cells (target: T) and GFP NK-92 cells or CD45b-28 NK 45i -92 cells at 2:1 (E:T) ratio for 6 hour incubation.
  • IA Flow cytometry analysis of SP53 cells (mantle cell lymphoma cell line) only (left panel), in co-culture with Jeko-1 cells and control GFP transduced NK-92 cells (middle panel) or CD45b-28 NK 45i -92 cells (right panel). Blue dots in all of panels indicates the leftover target SP53 cells and red dots shows effector GFP or CD45b-28 NK 45i -92 cells by co-culture assay.
  • FIG. 57J Elimination of CD34(+) umbilical chord blood stem cells in 48 hr co-culture.
  • CD34(+) stem cells derived from human umbilical cord blood were co-cultured with either Control or CD45b-28 CAR NK cells for 48 hr prior to labeling at a low ratio of 2:1 (effective:target). About 96% of CD34(+) cells were eliminated comparing to the control.
  • FIG. 58A A Link by P2A schematic showing both cCAR-T and 4-1BBL or IL-15/IL-15sushi in a single construct.
  • the construct consists of a SFFV promoter driving the expression of CAR and an enhancer, 4-1BBL.
  • the CD45 CAR (or CD45b CAR) and 4-1BBL or IL-15/IL-15sush split and engage upon targets expressing CD45.
  • CD45 CAR T cells received not only costimulation through the CD28 but also 4-1BB ligand (4-1BBL or CD137L) or IL-15/IL-15sushi.
  • the CD3-zeta signaling domain completes the assembly of this CAR-T.
  • FIG. 58B Surface CD45b CAR expression levels on CD45b-28-2G-4-1BBL CAR transduced NK 45i -92 cells were determined using flow cytometry analysis. Left panel(NK92 cells) and middle panel (GFP-NK92) indicated negative control and right panel showed the surface expression of CD45b CAR which was labeled using goat anti-mouse F(AB′)2-PE against ScFv region (circled in blue). Transduced cells expressed 86.99% of CD45b-CAR on the cell surface.
  • FIG. 58C Surface CD45b CAR expression levels on CD45b-28-2G-IL15/IL-15sushi CAR transduced NK45i-92 cells were determined using flow cytometry analysis. Left panel(NK92 cells) and middle panel (GFP-NK92) indicated negative control and right and right panel showed the surface expression of CD45b CAR which was labeled using goat anti-mouse F(AB′)2-PE against ScFv region (circled in blue). CD45b-28-2G IL15RA virus transduced cells expressed 55.96% of CD45b-CAR on cells surface compared to negative control cells.
  • FIGS. 59A-59B Schematic diagram to elucidate the construct and its expression in T or NK cells.
  • 59 A a combination of CAR, (third generation), and IL-15/sushi domain of the IL-15 alpha receptor, is assembled on an expression vector and their expression is driven by the SFFV promoter.
  • CAR with IL-15/sushi is linked with the P2A self-cleaving sequence.
  • the IL-15/sushi portion is composed of IL-2 signal peptide fused to IL-15 and linked to sushi domain via a 26-amino acid poly-proline linker.
  • 59 B CAR and IL-15/sushi are present on the T or NK cells.
  • FIG. 59C Surface CD45b CAR expression levels on CD45b-28-2G-IL-15/IL-15sushi CAR transduced NK 45i -92 cells were determined using flow cytometry analysis. Left panel (NK92 cells) and middle panel (GFP-NK92) indicated negative control and right and right panel showed the surface expression of CD45b CAR which was labeled using goat anti-mouse F(AB′)2-PE against ScFv region (circled in blue). CD45b-28-2G IL-15/IL-15sushi virus transduced cells expressed 55.96% of CD45b-CAR on cells surface compared to negative control cells. CD45b-28-2G-IL-15/IL-15sushi NK cells showed a robust functional activity.
  • FIGS. 60A-60B CD4IL-15/IL-15sushi expression.
  • 60 A HEK-293FT cells were transfected with lentiviral plasmids for GFP (lane 1) and CD4IL-15/IL-15sushi CAR (lane 2). 48 hours after transfection, supernatant was removed, and cells were also removed for a Western blot with mouse anti-human CD3z antibody.
  • 60 B HEK-293 cells were transduced with either GFP (left) or CD4IL-15/IL-15sushi-CAR(right) viral supernatant from transfected HEK-293FT cells. After 3 days incubation, cells were harvested, stained with goat-anti-mouse F(Ab′)2 and analyzed by flow cytometry.
  • FIG. 61 Transduction of NK cells with CD4IL-15/IL-15sushi CAR.
  • NK-92 cells were transduced with either GFP (left) or CD4 IL-15/IL-15sushi CAR (right) viral supernatant from transfected HEK-293FT cells.
  • a second transduction was performed 24 hours after the first. 24 hours after the second transduction, cells were harvested, washed and moved to tissue culture plates with fresh media and IL-2. After 3 days 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, washed, suspended in 2% formalin, and analyzed by flow cytometry.
  • FIG. 62 Transduction of T cells with CD4IL15RACAR.
  • Left is the Western blot.
  • HEK-293FT cells were transfected with lentiviral plasmids for GFP (lane 1) and CD4IL15RA-CAR (lane 2). 48 hours after transfection, supernatant was removed, and cells were also collected for a Western blot with mouse anti-human CD3zeta antibody.
  • Right is CD4IL15RACAR expression.
  • Activated T cells from cord blood buffy coat were transduced with either GFP (left) or concentrated CD4IL15RACAR (right) viral supernatant from transfected HEK-293FT cells. A second transduction was performed 24 hours after the first.
  • FIGS. 63A-63B CD4CAR NK-92 cells and CD4IL-15/IL-15sushi CAR NK-92 cells eliminate KARPAS 299 T leukemic cells in co-culture.
  • FIG. 64 CD4CAR NK-92 cells and CD4IL-15/IL-15sushi CAR NK-92 cells eliminate MOLT4 T leukemic cells in co-culture.
  • the upper right panel shows labeled MOLT4 cells alone. The percentage of target cells lysed is shown in the graph.
  • FIGS. 65A and 65B CD4CAR and CD4IL-15/IL-15sushi CAR T cells demonstrate anti-leukemic effects in vivo.
  • NSG mice were sublethally irradiated and intravenously (tail vein) injected the following day with luciferase-expressing MOLM13 cells to induce measurable tumor formation ( 65 A).
  • MOLM-13 cells are nearly 100% CD4+.
  • the mice were intravenously injected with one course of 8 ⁇ 10 6 CD4CAR, or CD4IL-15/IL-15sushi CAR T cells, or vector control T control cells.
  • mice were injected subcutaneously with RediJect D-Luciferin and subjected to IVIS imaging ( 65 B).
  • FIGS. 65C and 65D Average light intensity measured for the CD4CAR and CD4IL-15/IL-15sushi CAR T injected mice was compared to that of vector control T injected mice, and correlated with remaining tumor burden to determine a percent lysis.
  • 65 D Percent survival of mice was measured and compared between the three groups.
  • FIGS. 66A and 66B CD4IL15/IL-15sushi CAR NK cells demonstrate robust anti-leukemic activity under stressful condition in vivo.
  • NSG mice were sublethally irradiated and intravenously (tail vein) injected the following day with luciferase-expressing Jurkat cells to induce measurable tumor formation ( 66 A).
  • Jurkat cells are less than 60% CD4+.
  • the mice were intravenously injected with one course of 8 ⁇ 10 6 CD4CAR, or CD4IL-15/IL-15sushi CAR NK cells, or vector control NK cells.
  • mice On days 3, 7, 10, and 14, mice were injected subcutaneously with RediJect D-Luciferin and subjected to IVIS imaging ( 66 B).
  • FIG. 66C Average light intensity measured for the CD4CAR and CD4IL-15/IL-15sushi NK injected mice was compared to that of vector control NK injected mice, and correlated with remaining tumor burden to determine a percent lysis.
  • FIG. 67 Repeat of the in vivo experiment demonstrating robust lysis of Jurkat tumor cells by CD4I-15/IL-15sushi CAR NK cells showing similar results to those described in FIG. 66 .
  • FIGS. 68A-68B Effect of secreted IL-15/IL-15sushi on CAR and non-transduced neighboring cells.
  • NK-92 cells stably expressing either CD4CAR or CD4IL15RA were mixed in a 50:50 ratio with NK-92 cells stably expressing GFP. These cells were co-cultured either with IL-2 added or no IL-2.
  • 68 A Photographs taken on a fluorescent microscope at 20 ⁇ on Day 0 (start of co-culture) and Day 7, without the addition of IL-2.
  • FIG. 69 Comparing the effect of secreted IL-15 and IL-15sushi on NK-92 cell growth.
  • CD4IL-15/IL-15sushi, CD4 IL-15, and control transduced NK-92 cells were cultured from 250,000 cells in regular NK cell media but in the absence of IL-2 for up to 6 days. Both transduced cells had 10% surface CAR expression, while CD4IL15-IL15sushi transduced NK-92 cells were able to expand at a rate approximately 3-fold higher than the CD4 IL-15 transduced NK-92 cells on day 6.
  • FIG. 70 A schematic showing the Treg CAR T construct targeting Tregs.
  • the construct consists of a SFFV promoter driving the expression of two units of chimeric antigen receptors linked by a P2A peptide. Each unit contains a CD45 leader peptide sequence (signal peptide). Upon cleavage of the linker, two units of peptide are divided and engage upon targets expressing CD4 and CD25.
  • the CD4 chimeric antigen receptor polypeptide unit comprises a signal peptide, a CD4 antigen recognition domain, a hinge region, a transmembrane domain and CD3 zeta chain;
  • CD25 chimeric antigen receptor polypeptide unit comprises a signal peptide, a CD25 antigen recognition domain, a hinge region, a transmembrane domain, a co-stimulatory domain (s).
  • the Treg CAR can potentiate the lysis activity of a cell co-expressing CD4 and CD25 while minimizing a cell bearing CD4 or CD25 antigen.
  • FIGS. 71A-71B Characterization of the CD4 zeta CD25 CAR.
  • 71 A The CD4 zeta CD25 CAR was transduced into T-cells via viral incubation for 48 hours and stained with F(ab)′ antibody to assay CAR surface expression. Encircled populations represent transduced cells.
  • 71 B The C4-25z CAR was characterized using CD4 and CD25 antibodies to validate the construct function. Two most relevant populations are encircled: CD4 + CD25 + and CD4 ⁇ CD25 + . The depletion of the double positive population and other phenotype groups are summarized in the bar graph adjacent.
  • FIG. 72 CD4zetaCD25 CAR T cells target cells mainly co-expressing CD4 and CD25. 3 days after activation, PMBC buffy coat T cells transduced with either control vector (left), CD4CAR (middle) or CD4zetaCD25(right) lentiviral supernatant were harvested and incubated with mouse anti-human CD25-PE and mouse anti-human CD4-APC for 30 minutes. Cells were washed and suspended in 2% formalin, and analyzed by flow cytometry.
  • FIG. 73A A schematic showing the CD52-52 construct.
  • the construct consists of a SFFV promoter driving the co-expression of CD5CAR and CD52 surface antigen.
  • the CD5 chimeric antigen receptor polypeptide unit comprises a signal peptide, a CD5 antigen recognition domain, a hinge region, a transmembrane domain and CD3 zeta chain;
  • CD5 peptide comprises a signal peptide, a CD52 antigen recognition domain, a hinge region, a transmembrane domain (derived from CD28).
  • FIG. 73B Experimental design to determine depletion of CD5CAR-52 T cells in blood.
  • peripheral blood was collected from each mouse and labeled using CD3 and CD45 antibodies to determine the depletion of CAR-T cells as acute phase response by CAMPATH treatment.
  • whole blood was collected from each mouse and labeled using CD3 and CD45 antibodies to determine the persistency of CAR-T cells as well.
  • CAR-T-cells were determined using Flow cytometry analysis.
  • FIG. 73C Depletion of CD5CAR-52 T in peripheral blood after 6 h and 24 h later with or without CAMPATH treatment.
  • Flow cytometry analysis shows persistence of CD5CAR-52 T-cells (Blue dots) in peripheral blood of mouse with or without CAMPATH treatment.
  • Blood samples were labeled with CD3 and CD45 antibodies to detect CD5CAR-52 T-cells.
  • Blood samples from un-infused CAR-T cells did not show CD3 and CD45 positive cells (negative control).
  • FIG. 73D Depletion of CD5CAR-52 T in whole blood after 5 days later with or without CAMPATH treatment.
  • Flow cytometry analysis shows persistence of CD5CAR-52 T-cells (Blue dots) in whole blood samples from mouse with or without CAMPATH treatment.
  • Blood samples were labeled with CD3 and CD45 antibodies to detect CD5CAR-52 T-cells persistence.
  • Blood samples from uninfused CAR-T cells did not show CD3 and CD45 positive cells (negative control).
  • 0.1 mg/kg of CAMPATH treated mice eliminate CD5CAR-52 T-cells (right panels) compared to CAMPATH uninjected mouse (middle panels) after 5 days later in whole blood samples.
  • FIG. 74 HEK 293 cells were transduced with either EF1-GFP or SFFV-GFP viral supernatant, using the volumes indicated, in DMEM with 10% FBS in a 6 well tissue culture plate. Culture media was changed the following morning. Forty-eight hours later, transduced cells were visualized on an EVOS fluorescent microscope using GFP at 10 ⁇ .
  • FIG. 75 HEK 293 cells transduced with either EF1-GFP or SFFV-GFP viral supernatant, using the volumes from the previous figure, were trypsinized, suspended in formalin, and subjected to flow cytometry analysis, using the FITC channel to determine the percentage of GFP+ cells.
  • FIGS. 76A-76B Activated cord blood buffy coat T cells transduced with either EF1-GFP or SFFV-GFP viral supernatant, with either low or high amounts of viral supernatant, were trypsinized, suspended in formalin, and subjected to flow cytometry analysis, using the FITC channel to determine the percentage of GFP+ cells, 7, 14, 21 and 28 days after transduction.
  • FIG. 77 Ligand receptor interactions in malignant plasma cells.
  • the APRIL ligand binds TAC1 or BCMA.
  • the BAFF ligand binds TAC1, BCMA, or BAFF-R.
  • FIG. 78 Steps for elimination of tumor by CAR co-expressing secretory IL-15/IL-15sushi.
  • I tumor and its microenviroment.
  • Macrophages, T cells, dendritic cells and NK cells are immune response cells against tumor in the tumor microenvironment and they secrete a low level of endogenous IL-15, which is unstable, which complexes with the soluble extracellular domain of IL-15RA. The complex forms a more stable molecule, which greatly enhances immune cell survival and expansion.
  • cancer cells express programmed death ligand 1 (PD-L1) as a transmembrane protein that has been considered to play a major role in suppressing the immune system during particular events including cancer.
  • PD-L1 programmed death ligand 1
  • PD-L1 binds to its receptor, PD-1, found on activated T cells, B cells, and myeloid cells, to suppress these cell immune activities.
  • CAR T or NK cells targeting tumor cells could be a carrier to deliver an enhancer to the tumor microenvironment.
  • CAR T or NK cells are engineered to co-express a secretory fusion protein, IL-15/IL-15sushi fusion.
  • Engineered CAR T or NK cells bind to targeted tumor cells (either subset or all cells).
  • the secreted IL-15/IL-15sushi protein would be involved in trafficking of other T cells, dendritic cells, macrophages and NK cells to the tumor microenvironment, which then also: 1) lyse the tumor cells by supplementing the defect that CAR T or NK cells are unable to eliminate non-targeting cancer cells; 2) enhance CAR T/NK cell persistency and anti-tumor activity.
  • the overexpression of IL-15/IL-15sushi overwhelms the PD-L1 ability to suppress the immune response.
  • this CAR therapy could be used synergistically with administration of a checkpoint blockage including, but not limited to PD-L1, CTLA-4 inhibitor for even greater efficacy.
  • FIG. 79 Surface markers during B cell and plasma cell development are shown. Both BAFF and APRIL binds to receptors, BCMA and TACI. BAFF also binds to BAFF-R receptor.
  • FIG. 80 Protein sequence alignment of IL-2 signal peptide among different species.
  • FIG. 81 Protein sequence alignment of BAFF extracellular domain among different species.
  • the disclosure provides chimeric antigen receptor (CAR) compositions, methods of making and using thereof.
  • CAR chimeric antigen receptor
  • a chimeric antigen receptor (CAR) polypeptide includes a signal peptide, an antigen recognition domain, a hinge region, a transmembrane domain, at least one co-stimulatory domain, and a signaling domain.
  • First-generation CARs include CD3z as an intracellular signaling domain, whereas second-generation CARs include at least one single co-stimulatory domain derived from various proteins.
  • co-stimulatory domains include, but are not limited to, CD28, CD2, 4-1BB (CD137, also referred to as “4-BB”), and OX-40 (CD124).
  • Third generation CARs include two co-stimulatory domains, such as, but not limited to, CD28, 4-1BB, CD134 (OX-40), CD2, and/or CD137 (4-1BB).
  • peptide As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound having amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can be included in a protein's or peptide's sequence.
  • Polypeptides include any peptide or protein having two or more amino acids joined to each other by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides, and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins, among others.
  • the polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
  • a “signal peptide” includes a peptide sequence that directs the transport and localization of the peptide and any attached polypeptide within a cell, e.g. to a certain cell organelle (such as the endoplasmic reticulum) and/or the cell surface.
  • signal peptide and “leader sequence” are used interchangeably.
  • the signal peptide is a peptide of any secreted or transmembrane protein that directs the transport of the polypeptide of the disclosure to the cell membrane and cell surface, and provides correct localization of the polypeptide of the present disclosure.
  • the signal peptide of the present disclosure directs the polypeptide of the present disclosure to the cellular membrane, wherein the extracellular portion of the polypeptide is displayed on the cell surface, the transmembrane portion spans the plasma membrane, and the active domain is in the cytoplasmic portion, or interior of the cell.
  • the signal peptide is cleaved after passage through the endoplasmic reticulum (ER), i.e. is a cleavable signal peptide.
  • the signal peptide is human protein of type I, II, III, or IV.
  • the signal peptide includes an immunoglobulin heavy chain signal peptide.
  • the signal peptide includes the signal peptide from human CD45. (UniProtKB/Swiss-Prot Accession Number P08575).
  • the CD45 signal peptide is 23 amino acids in length (MYLWLKLLAFGFAFLDTEVFVTG).
  • the signal peptide may be a functional fragment of the CD45 signal peptide.
  • a functional fragment includes a fragment of at least 10 amino acids of the CD45 signal peptide that directs the appended polypeptide to the cell membrane and cell surface. Examples of fragments of the human CD45 signal peptide include: MYLWLKLLAFG, FAFLDTEVFVTG, and LKLLAFGFAFLDTE.
  • Functional equivalents of the human CD45 signal peptide have also been contemplated.
  • “functional equivalents” are to be understood as mutants that exhibit, in at least one of the abovementioned sequence positions, an amino acid substitution other than the one mentioned specifically, but still lead to a mutant which show the same or similar properties with respect to the wild-type CD45 signal peptide.
  • Functional equivalents include polypeptides having at least 80%, at least 85%, at least 90%, or at least 95% identity to the human CD45 signal peptide, functional fragments thereof, or functional equivalents thereof.
  • Functional equivalents also include CD45 signal peptides from homologous proteins from other species.
  • Examples of these signal peptides include signal peptide from mouse CD45 (MGLWLKLLAFGFALLDTEVFVTG); signal peptide from rat CD45 (MYLWLKLLAFSLALLGPEVFVTG); signal peptide from sheep CD45 (MTMYLWLKLLAFGFAFLDTAVSVAG); signal peptide from chimpanzee CD45 (MYLWLKLLAFGFAFLDTEVFVTG); and signal peptide from monkey CD45 (MTMYLWLKLLAFGFAFLDTEVFVAG.
  • the signal peptide includes the following sequence: MX 1 LWLKLLAF X 2 X 3 AX 4 LX 5 X 6 X 7 VX 8 VX 9 G; wherein X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , and X 9 are independently Y, G, S, F, L, D, P, T, E, or A.
  • the signal peptide includes the signal peptide from human CD8a (MALPVTALLLPLALLLHAARP).
  • the signal peptide may be a functional fragment of the CD8a signal peptide.
  • a functional fragment includes a fragment of at least 10 amino acids of the CD8a signal peptide that directs the appended polypeptide to the cell membrane and cell surface. Examples of fragments of the human CD8a signal peptide include: MALPVTALLLPLALLLHAA, MALPVTALLLP, PVTALLLPLALL, and LLLPLALLLHAARP.
  • the signal peptide includes the signal peptide from human CD8b (MRPRLWLLLAAQLTVLHGNSV).
  • the signal peptide may be a functional fragment of the CD8b signal peptide.
  • a functional fragment includes a fragment of at least 10 amino acids of the CD8b signal peptide that directs the appended polypeptide to the cell membrane and cell surface. Examples of fragments of the human CD8b signal peptide include: MRPRLWLLLAAQ, RLWLLLAAQLTVLHG, and LWLLLAAQLTVLHGNSV.
  • Functional equivalents of the human CD8a or CD8b signal peptide have also been contemplated.
  • “functional equivalents” are to be understood as mutants which exhibit, in at least one of the abovementioned sequence positions, an amino acid substitution other than the one mentioned specifically, but still lead to a mutant which show the same or similar properties with respect to the wild-type CD8a or CD8b signal peptide.
  • Functional equivalents include polypeptides having at least 80%, at least 85%, at least 90%, or at least 95% identity to the human CD8 signal peptide, functional fragments thereof, or functional equivalents thereof.
  • Functional equivalents also include CD8a and CD8b signal peptides from homologous proteins from other species.
  • the signal peptide includes the signal peptide from human IL-2.
  • the IL-2 signal peptide is 23 amino acids in length (MYRMQLLSCIALSLALVTNS).
  • the signal peptide may be a functional fragment of the IL-2 signal peptide.
  • a functional fragment includes a fragment of at least 10 amino acids of the IL-2 signal peptide that directs the appended polypeptide to the cell membrane and cell surface. Examples of fragments of the human IL-2 signal peptide include: MYRMQLLSCIAL, QLLSCIALSLAL, and SCIALSLALVTNS.
  • Functional equivalents of the human IL-2 signal peptide have also been contemplated.
  • “functional equivalents” are to be understood as mutants which exhibit, in at least one of the abovementioned sequence positions, an amino acid substitution other than the one mentioned specifically, but still lead to a mutant which show the same or similar properties with respect to the wild-type IL-2 signal peptide.
  • Functional equivalents include polypeptides having at least 80%, at least 85%, at least 90%, or at least 95% identity to the human IL-2 signal peptide, functional fragments thereof, or functional equivalents thereof.
  • Functional equivalents also include IL-2 signal peptides from homologous proteins from other species. See for example FIG. 80 .
  • the signal peptide includes the following sequence: MYX 1 X 2 QLX 3 SCX 4 XLX 6 LX 7 LXX 9 X 10 X 11 ; wherein X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , and X 11 are independently R, K, S, M, I, V, L, A, I, T, N, S, or G.
  • X 1 is R, K, or S
  • X 2 is M, I, or V
  • X 3 is L or A
  • X 4 and X 5 are independently I, A, V, or T
  • X 6 is S or T
  • X 7 is A or V
  • X 8 , X 9 , X 10 , and X 11 are independently V, L, T, A, N, S, or G.
  • the “antigen recognition domain” includes a polypeptide that is selective for or targets an antigen, receptor, peptide ligand, or protein ligand of the target; or a polypeptide of the target.
  • the antigen recognition domain may be obtained from any of the wide variety of extracellular domains or secreted proteins associated with ligand binding and/or signal transduction.
  • the antigen recognition domain may include a portion of Ig heavy chain linked with a portion of Ig light chain, constituting a single chain fragment variable (scFv) that binds specifically to a target antigen.
  • the antibody may be monoclonal or polyclonal antibody or may be of any type that binds specifically to the target antigen.
  • the antigen recognition domain can be a receptor or ligand.
  • the target antigen is specific for a specific disease condition and the disease condition may be of any kind as long as it has a cell surface antigen, which may be recognized by at least one of the chimeric receptor construct present in the compound CAR architecture.
  • the chimeric receptor may be for any cancer for which a specific monoclonal or polyclonal antibody exists or is capable of being generated.
  • 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 receptors.
  • antigen recognition domain can be non-antibody protein scaffolds, such as but not limited to, centyrins, non-antibody protein scaffolds that can be engineered to bind a variety of specific targets with high affinity.
  • Centyrins are scaffold proteins based on human consensus tenascin FN3 domain, are usually smaller than scFv molecules CAR molecules.
  • the target specific antigen recognition domain preferably includes an antigen binding domain derived from an antibody against an antigen of the target, or a peptide binding an antigen of the target, or a peptide or protein binding an antibody that binds an antigen of the target, or a peptide or protein ligand (including but not limited to a growth factor, a cytokine, or a hormone) binding a receptor on the target, or a domain derived from a receptor (including but not limited to a growth factor receptor, a cytokine receptor or a hormone receptor) binding a peptide or protein ligand on the target.
  • an antigen binding domain derived from an antibody against an antigen of the target, or a peptide binding an antigen of the target, or a peptide or protein binding an antibody that binds an antigen of the target, or a peptide or protein ligand (including but not limited to a growth factor, a cytokine, or a hormone) binding a receptor on the target,
  • the antigen recognition domain includes the binding portion or variable region of a monoclonal or polyclonal antibody directed against (selective for) the target.
  • the antigen recognition domain includes Camelid single domain antibody, or portions thereof.
  • Camelid single-domain antibodies include heavy-chain antibodies found in camelids, or VHH antibody.
  • a VHH antibody of camelid (for example camel, dromedary, llama, and alpaca) refers to a variable fragment of a camelid single-chain antibody (See Nguyen et al, 2001; Muyldermans, 2001), and also includes an isolated VHH antibody of camelid, a recombinant VHH antibody of camelid, or a synthetic VHH antibody of camelid.
  • the antigen recognition domain includes ligands that engage their cognate receptor.
  • APRIL is a ligand that binds the TAC1 receptor or the BCMA receptor.
  • the antigen recognition domain includes APRIL, or a fragment thereof.
  • BAFF is a ligand that binds the BAFF-R receptor or the BCMA receptor.
  • the antigen recognition domain includes BAFF, or a fragment thereof.
  • the antigen recognition domain is humanized.
  • the antigen recognition domain may include some variability within its sequence and still be selective for the targets disclosed herein. Therefore, it is contemplated that the polypeptide of the antigen recognition domain may be 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 selective for the targets described herein and be within the scope of the disclosure.
  • the target includes interleukin 6 receptor, NY-ESO-1, alpha fetoprotein (AFP), glypican-3 (GPC3), BCMA, BAFF-R, TACI, LeY, 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, Glycolipid, 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, immunoglobin kappa and lambda, CD38, CD52, CD3, CD4, CD8, CD5, CD7, CD2, and CD138
  • the target includes any portion interleukin 6 receptor, NY-ESO-1, alpha fetoprotein (AFP), glypican-3 (GPC3), BCMA, BAFF-R, TACI, LeY, 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, Glycolipid, 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, immunoglobin kappa and lambda, CD38, CD52, CD3, CD4, CD8, CD5, CD7
  • the target includes surface exposed portions of interleukin 6 receptor, NY-ESO-1, alpha fetoprotein (AFP), glypican-3 (GPC3), BCMA, BAFF-R, TACI, LeY, 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, Glycolipid, 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, immunoglobin kappa and lambda, CD38, CD52, CD3, CD4, CD8, CD5,
  • the target includes the surface exposed regions of BAFF, as shown in FIG. 81 .
  • the target may include a portion of the surface exposed regions of BAFF.
  • portions of BAFF include residues 1-200, 1-100, 50-150, or 100-200 of human BAFF.
  • the target antigens include viral or fungal antigens, such as E6 and E7 from the human papillomavirus (HPV) or EBV (Epstein Barr virus) antigens; portions thereof; or surface exposed regions thereof.
  • viral or fungal antigens such as E6 and E7 from the human papillomavirus (HPV) or EBV (Epstein Barr virus) antigens; portions thereof; or surface exposed regions thereof.
  • the TACI antigen recognition domain includes SEQ ID NO. 24.
  • the BCMA antigen recognition domain includes SEQ ID NO. 25.
  • the CS1 antigen recognition domain includes SEQ ID NO. 26.
  • the BAFF-R antigen recognition domain includes SEQ ID NO. 27.
  • the CD33 antigen recognition domain includes SEQ ID NO. 28.
  • the CD123 antigen recognition domain includes SEQ ID NO. 29.
  • the CD19 antigen recognition domain includes SEQ ID NO. 30.
  • the CD20 antigen recognition domain includes SEQ ID NO. 31. In another embodiment, the CD20 antigen recognition domain includes SEQ ID NO. 32.
  • the CD22 antigen recognition domain includes SEQ ID NO. 33.
  • the CD45 antigen recognition domain includes SEQ ID NO. 34.
  • the CD4 antigen recognition domain includes SEQ ID NO. 35
  • the CD25 antigen recognition domain includes SEQ ID NO. 36
  • the hinge region is a sequence positioned between for example, including, but not limited to, the chimeric antigen receptor, and at least one co-stimulatory domain and a signaling domain.
  • the hinge sequence may be obtained including, for example, from any suitable sequence from any genus, including human or a part thereof. Such hinge regions are known in the art.
  • the hinge region includes the hinge region of a human protein including CD-8 alpha, CD28, 4-1BB, OX40, CD3-zeta, T cell receptor a or 3 chain, a CD3 zeta chain, CD28, CD3c, CD45, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, and combinations thereof.
  • a human protein including CD-8 alpha, CD28, 4-1BB, OX40, CD3-zeta, T cell receptor a or 3 chain, a CD3 zeta chain, CD28, CD3c, CD45, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, and combinations thereof.
  • the hinge region includes the CD8 a hinge region.
  • the hinge region includes one selected from, but not limited to, immunoglobulin (e.g. IgG1, IgG2, IgG3, IgG4, and IgD).
  • immunoglobulin e.g. IgG1, IgG2, IgG3, IgG4, and IgD.
  • the transmembrane domain includes a hydrophobic polypeptide that spans the cellular membrane.
  • the transmembrane domain spans from one side of a cell membrane (extracellular) through to the other side of the cell membrane (intracellular or cytoplasmic).
  • the transmembrane domain may be in the form of an alpha helix or a beta barrel, or combinations thereof.
  • the transmembrane domain may include a polytopic protein, which has many transmembrane segments, each alpha-helical, beta sheets, or combinations thereof.
  • the transmembrane domain that is naturally associated with one of the domains in the CAR is used.
  • the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
  • a transmembrane domain includes a transmembrane domain of a T-cell receptor a or ⁇ chain, a CD3 zeta chain, CD28, CD3c, 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.
  • the transmembrane domain is artificially designed so that more than 25%, more than 50% or more than 75% of the amino acid residues of the domain are hydrophobic residues such as leucine and valine.
  • a triplet of phenylalanine, tryptophan and valine is found at each end of the synthetic transmembrane domain.
  • the transmembrane domain is the CD8 transmembrane domain. In another embodiment, the transmembrane domain is the CD28 transmembrane domain. Such transmembrane domains are known in the art.
  • the signaling domain and co-stimulatory domain include polypeptides that provide activation of an immune cell to stimulate or activate at least some aspect of the immune cell signaling pathway.
  • the signaling domain includes the polypeptide of a functional signaling domain of CD3 zeta, common FcR gamma (FCER1G), Fc gamma R11a, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DNAX-activating protein 10 (DAP10), DNAX-activating protein 12 (DAP12), active fragments thereof, functional derivatives thereof, and combinations thereof.
  • FCER1G common FcR gamma
  • FcR beta Fc Epsilon Rib
  • CD3 gamma CD3 delta
  • CD3 epsilon CD79a
  • CD79b CD79b
  • DAP10 DNAX-activating protein 10
  • DAP12 DNAX-activating protein 12
  • the CAR polypeptide further includes one or more co-stimulatory domains.
  • the co-stimulatory domain is a functional signaling domain from a protein including 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; a ligand that binds to at least one of CD83, ICAM-1, LFA-1 (CD11a/CD18), ICOS, and 4-1BB (CD137); CDS; ICAM-1; LFA-1 (CD1a/CD18); CD40; CD27; CD7; B7-H3; NKG2C; PD-1; ICOS; active fragments thereof; functional derivatives thereof; and combinations thereof.
  • the at least one co-stimulatory domain and signaling domain may be collectively referred to as the intracellular domain.
  • the hinge region and the antigen recognition domain may be collectively referred to as the extracellular domain.
  • the present disclosure further provides a polynucleotide encoding the chimeric antigen receptor polypeptide described above.
  • polynucleotide as used herein is defined as a chain of nucleotides. Polynucleotide includes DNA and RNA. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable.
  • nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides.
  • polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and polymerase chain reaction (PCR), and the like, and by synthetic means.
  • recombinant means i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and polymerase chain reaction (PCR), and the like, and by synthetic means.
  • the polynucleotide encoding the CAR is easily prepared from an amino acid sequence of the specified CAR by any conventional method.
  • a base sequence encoding an amino acid sequence can be obtained from the aforementioned NCBI RefSeq IDs or accession numbers of GenBenk for an amino acid sequence of each domain, and the nucleic acid of the present disclosure can be prepared using a standard molecular biological and/or chemical procedure.
  • a polynucleotide can be synthesized, and the polynucleotide of the present disclosure can be prepared by combining DNA fragments which are obtained from a cDNA library using a polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the polynucleotide disclosed herein is part of a gene, or an expression or cloning cassette.
  • a “vector” is a composition of matter which includes an isolated polynucleotide and which can be used to deliver the isolated polynucleotide to the interior of a cell.
  • vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, phagemid, cosmid, and viruses.
  • Viruses include phages, phage derivatives.
  • the term “vector” includes an autonomously replicating plasmid or a virus.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
  • vectors include cloning vectors, expression vectors, replication vectors, probe generation vectors, integration vectors, and sequencing vectors.
  • the vector is a viral vector.
  • the viral vector is a retroviral vector or a lentiviral vector.
  • the engineered cell is virally transduced to express the polynucleotide sequence.
  • retroviruses provide a convenient platform for gene delivery systems.
  • a selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to cells of the patient either in vivo or ex vivo.
  • retroviral systems are known in the art.
  • adenovirus vectors are used.
  • a number of adenovirus vectors are known in the art.
  • lentivirus vectors are used.
  • Viruses which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient and unique restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).
  • Lentiviral vectors have been well known for their capability of transferring genes into human T cells with high efficiency but expression of the vector-encoded genes is dependent on the internal promoter that drives their expression.
  • a strong promoter is particularly important for the third or fourth generation of CARs that bear additional co-stimulatory domains or genes encoding proliferative cytokines as increased CAR body size does not guarantee equal levels of expression.
  • Gene therapies using CAR T cells rely on the ability of T cells to express adequate CAR body and maintain expression over a long period of time.
  • the EF-1 ⁇ promoter has been commonly selected for the CAR expression.
  • the present disclosure provides an expression vector containing a strong promoter for high level gene expression in T cells or NK cells.
  • the inventor discloses a strong promoter useful for high level expression of CARs in T cells or NK cells.
  • a strong promoter relates to the SFFV promoter, which is selectively introduced in an expression vector to obtain high levels of expression and maintain expression over a long period of time in T cells or NK cells. Expressed genes prefer CARs, T cell co-stimulatory factors and cytokines used for immunotherapy.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
  • CMV immediate early cytomegalovirus
  • EF-1 a Elongation Growth Factor-1 a
  • constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter.
  • SV40 simian virus 40
  • MMTV mouse mammary tumor virus
  • HSV human immunodeficiency virus
  • LTR long terminal repeat
  • MoMuLV promoter MoMuLV promoter
  • an avian leukemia virus promoter an Epstein-Barr virus immediate early promoter
  • Rous sarcoma virus promoter as well as human gene promoters such as
  • inducible promoters are also contemplated as part of the disclosure.
  • the use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence, which is operatively linked when such expression is desired, or turning off the expression when expression is not desired.
  • inducible promoters include, but are not limited to a metalothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
  • chimeric antigen receptor polynucleotide may be achieved using, for example, expression vectors including, but not limited to, at least one of a SFFV (spleen-focus forming virus) (for example, SEQ ID NO. 23) or human elongation factor 11 ⁇ (EF) promoter, CAG (chicken beta-actin promoter with CMV enhancer) promoter human elongation factor 1 ⁇ (EF) promoter.
  • SFFV single-focus forming virus
  • EF human elongation factor 11 ⁇
  • CAG chicken beta-actin promoter with CMV enhancer
  • Examples of less-strong/lower-expressing promoters utilized may include, but is not limited to, the simian virus 40 (SV40) early promoter, cytomegalovirus (CMV) immediate-early promoter, Ubiquitin C (UBC) promoter, and the phosphoglycerate kinase 1 (PGK) promoter, or a part thereof.
  • Inducible expression of chimeric antigen receptor may be achieved using, for example, a tetracycline responsive promoter, including, but not limited to, TRE3GV (Tet-response element, including all generations and preferably, the 3rd generation), inducible promoter (Clontech Laboratories, Mountain View, Calif.) or a part or a combination thereof.
  • the promoter is an SFFV promoter or a derivative thereof. It has been unexpectedly discovered that SFFV promoter provides stronger expression and greater persistence in the transduced cells in accordance with the present disclosure.
  • “Expression vector” refers to a vector including a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector includes sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
  • the expression vector may be a bicistronic or multicistronic expression vector.
  • Bicistronic or multicistronic expression vectors may include (1) multiple promoters fused to each of the open reading frames; (2) insertion of splicing signals between genes; fusion of genes whose expressions are driven by a single promoter; (3) insertion of proteolytic cleavage sites between genes (self-cleavage peptide); and (iv) insertion of internal ribosomal entry sites (IRESs) between genes.
  • the disclosure provides an engineered cell having at least one chimeric antigen receptor polypeptide or polynucleotide.
  • An “engineered cell” means any cell of any organism that is modified, transformed, or manipulated by addition or modification of a gene, a DNA or RNA sequence, or protein or polypeptide.
  • 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 the DNA or RNA sequences encoding a chimeric antigen receptor or chimeric antigen receptor complex and express the chimeric receptor on the cell surface.
  • Isolated host cells and engineered cells may be used, for example, for enhancing an NK cell activity or a T lymphocyte activity, treatment of cancer, and treatment of infectious diseases.
  • the engineered cell includes immunoregulatory cells.
  • Immunoregulatory cells include T-cells, such as CD4 T-cells (Helper T-cells), CD8 T-cells (Cytotoxic T-cells, CTLs), and memory T cells or memory stem cell T cells.
  • T-cells include Natural Killer T-cells (NK T-cells).
  • the engineered cell includes Natural Killer cells. Natural killer cells are well known in the art. In one embodiment, 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.
  • NK cells mediate anti-tumor effects without the risk of GvHD and are short-lived relative to T-cells. Accordingly, NK cells would be exhausted shortly after destroying cancer cells, decreasing the need for an inducible suicide gene on CAR constructs that would ablate the modified cells.
  • NK cells provide a readily available cell to be engineered to contain and express the chimeric antigen receptor polypeptides disclosed herein.
  • NK cells can be expanded and transfected with CAR polynucleotides in accordance to the present disclosure.
  • NK cells can be derived from cord blood, peripheral blood, iPS cells and embryonic stem cells.
  • NK-92 cells may be expanded and transfected with CAR.
  • NK-92 is a continuously growing cell line that has features and characteristics of natural killer (NK) cells (Arai, Meagher et al. 2008).
  • NK-92 cell line is IL-2 dependent and has been proven to be safe (Arai, Meagher et al. 2008) and feasible.
  • CAR expressing NK-92 cells can be expanded in the serum free-medium with or without co-culturing with feeder cells. A pure population of NK-92 carrying the CAR of interest may be obtained by sorting.
  • engineered cells include allogeneic T cells obtained from donors that are modified to inactivate components of TCR (T cell receptor) involved in MHC recognition. As a result, TCR deficient T cells would not cause graft versus host disease (GVHD).
  • TCR T cell receptor
  • the engineered cell may be modified to prevent expression of cell surface antigens.
  • an engineered cell may be genetically modified to delete the native CD45 gene to prevent expression and cell surface display thereof.
  • the engineered cell includes an inducible suicide gene (“safety switch”) or a combination of safety switches, which may be assembled on a vector, such as, without limiting, a retroviral vector, lentiviral vector, adenoviral vector or plasmid.
  • a “safety switch” greatly increases safety profile and limits on-target or off-tumor toxicities of the compound CARs.
  • the “safety switch” may be an inducible suicide gene, such as, without limiting, caspase 9 gene, thymidine kinase, cytosine deaminase (CD) or cytochrome P450.
  • Other safety switches for elimination of unwanted modified T cells involve expression of CD20 or CD52 or CD19 or truncated epidermal growth factor receptor in T cells. All possible safety switches have been contemplated and are embodied in the present disclosure.
  • the suicide gene is integrated into the engineered cell genome.
  • the present disclosure provides an engineered cell having a CD45 chimeric antigen receptor polynucleotide.
  • the CD45 CAR polypeptide includes SEQ ID NO. 13 and corresponding polynucleotide sequence SEQ ID NO. 14.
  • the CD45 CAR polypeptide includes SEQ ID NO. 15, and corresponding polynucleotide sequence SEQ ID NO. 16.
  • the CD45 CAR polypeptide includes SEQ ID NO. 17, and corresponding polynucleotide sequence SEQ ID NO. 18.
  • the engineered cell includes CD45 CAR linked to IL15/IL-15sushi via the P2A cleavage sequence.
  • a polypeptide providing this embodiment includes SEQ ID No. 43 and corresponding polynucleotide sequence SEQ ID No. 44.
  • the engineered cell includes CD45 CAR linked to 4-1BBL (CD137L) via the P2A cleavage sequence.
  • a polypeptide providing this embodiment includes SEQ ID No. 41 and corresponding polynucleotide sequence SEQ ID No. 42.
  • the present disclosure provides an engineered cell having at least two distinct or separate CAR units.
  • the two CAR units may be complete CAR units or incomplete CAR units.
  • “distinct CAR polypeptide” and “distinct CAR polypeptide unit” are used interchangeably.
  • the present disclosure provides chimeric antigen receptor polypeptides having a signal peptide, antigen recognition domain, a hinge region, a transmembrane domain, a signaling domain, and at least one co-stimulatory domain, defining a CAR unit or a complete CAR unit.
  • an incomplete CAR unit includes a polypeptide having a signal peptide, antigen recognition domain, a hinge region, a transmembrane domain, and a signaling domain or at least one co-stimulatory domain.
  • An incomplete CAR unit will not contain a signaling domain and at least one co-stimulatory domain, but one or the other.
  • the present disclosure provides an engineered cell having a first chimeric antigen receptor polypeptide having a first antigen recognition domain and a co-stimulatory domain (first incomplete CAR unit); and a second chimeric antigen receptor polypeptide having a second antigen recognition domain and a signaling domain (second incomplete CAR unit); wherein the first antigen recognition domain is different than the second antigen recognition domain.
  • an engineered cell having two incomplete CAR units will only be fully activated when both target antigens are bound to the antigen recognition domain.
  • This strategy provides added specificity in that the engineered cells are not fully activated until targets are bound at the antigen recognition domain of each incomplete CAR unit.
  • one of the antigen recognition domains may be specific for and bind streptavidin, biotin, HIS, MYC, HA, agarose, V5, Maltose, GST, GFP, CD52, CD20, 4-1BB, or CD28.
  • compound CAR or multiple CAR refers to an engineered cell having at least two complete and distinct chimeric antigen receptor polypeptides.
  • a “distinct chimeric antigen receptor polypeptide” has a unique antigen recognition domain, a signal peptide, a hinge region, a transmembrane domain, at least one costimulatory domain, and a signaling domain. Therefore, two unique chimeric antigen receptor polypeptides will have different antigen recognition domains.
  • the signal peptide, hinge region, transmembrane domain, at least one costimulatory domain, and signaling domain may be the same or different between the two distinct chimeric antigen receptor polypeptides.
  • a chimeric antigen receptor (CAR) unit refers to a distinct chimeric antigen receptor polypeptide, or a polynucleotide encoding for the same.
  • a unique antigen recognition domain is one that is specific for or targets a single target, or a single epitope of a target.
  • the compound CAR targets the same antigen.
  • cCAR targets different epitopes or parts of a single antigen.
  • each of the CAR units present in the compound CAR targets different antigen specific to the same or different disease condition or side effects caused by a disease condition.
  • the compound CAR targets two different antigens.
  • the present disclosure provides an engineered cell having multiple CAR units. This allows a single engineered cell to target multiple antigens. Targeting multiple surface markers or antigens simultaneously with a multiple CAR unit prevents selection of resistant clones and reduces tumor recurrence. Multiple CAR T cell immunotherapies, with each individual component CAR comprising various domains and activation sites has not yet been developed for any malignancies.
  • cCAR includes multiple CAR units. In some embodiments, cCAR includes at least two CAR units. In another embodiment, the cCAR includes at least three CAR units. In another embodiment, the cCAR includes at least four units.
  • the present disclosure provides an engineered cell having at least two distinct chimeric antigen receptor polypeptides, each having a different antigen recognition domain.
  • the engineered cell having at least two distinct chimeric antigen receptor polypeptides is a T-cell.
  • the T-cell may be engineered so that it does not express a cell surface antigen.
  • a T-cell may be engineered so that it does not express a CD45 cell surface antigen.
  • the engineered cell having at least two distinct chimeric antigen receptor polypeptides is a primary NK cell isolated from the peripheral blood or cord blood and NK-92 cells, such that it is administered “off-the-shelf” to any mammal with a disease or cancer.
  • the engineered cell includes (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 co-stimulatory domain, and a first signaling domain; and (ii.) a second chimeric antigen receptor polypeptide comprising a second antigen recognition domain, a second signal peptide, a second hinge region, a second transmembrane domain, a second co-stimulatory domain, and a second signaling domain.
  • the first antigen recognition domain is different from the second antigen recognition domain.
  • each engineered CAR unit polynucleotide have different nucleotide sequences in order to avoid homologous recombination.
  • the target of the first 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 CS1; and the target of the second 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, Fl
  • the engineered cell includes a first chimeric antigen receptor polypeptide having a CD19 antigen recognition domain and second chimeric antigen receptor polypeptide having a CD20 recognition domain. In one embodiment, this engineered cell includes a polypeptide of SEQ ID NO. 3 and corresponding polynucleotide of SEQ ID NO. 4.
  • the engineered cell includes a first chimeric antigen receptor polypeptide having a CD19 antigen recognition domain and second chimeric antigen receptor polypeptide having a CD22 antigen recognition domain. In one embodiment, this engineered cell includes a polypeptide of SEQ ID NO. 5 and corresponding polynucleotide of SEQ ID NO. 6.
  • the engineered cell includes a first chimeric antigen receptor polypeptide having a CD19 antigen recognition domain and second chimeric antigen receptor polypeptide having a CD123 antigen recognition domain. In one embodiment, this engineered cell includes a polypeptide of SEQ ID NO. 7 and corresponding polynucleotide of SEQ ID NO. 8.
  • the engineered cell includes a first chimeric antigen receptor polypeptide having a CD33 antigen recognition domain and second chimeric antigen receptor polypeptide having a CD123 antigen recognition domain.
  • this engineered cell includes a polypeptide of SEQ ID NO. 9 and corresponding polynucleotide of SEQ ID NO. 10.
  • this engineered cell includes a polypeptide of SEQ ID NO. 11 and corresponding polynucleotide of SEQ ID NO. 12.
  • the engineered cell includes a first chimeric antigen receptor polypeptide having a BAFF-R antigen recognition domain and second chimeric antigen receptor polypeptide having a CS lantigen recognition domain.
  • the engineered cell includes a first chimeric antigen receptor polypeptide having a CD269 antigen recognition domain and second chimeric antigen receptor polypeptide having a CS1 antigen recognition domain.
  • the engineered cell includes a polypeptide including SEQ ID NO. 19 and corresponding polynucleotide SEQ ID NO. 20.
  • the engineered cell includes a polypeptide including SEQ ID NO. 21 and corresponding polynucleotide SEQ ID NO. 22.
  • the engineered cell includes a first chimeric antigen receptor polypeptide having a CD33 antigen recognition domain and second chimeric antigen receptor polypeptide having a CD123 antigen recognition domain.
  • each CAR unit includes the same or different hinge region. In another embodiment, each CAR unit includes the same or different transmembrane region. In another embodiment, each CAR unit includes the same or different intracellular domain.
  • each CAR unit includes the CD3 zeta chain signaling domain.
  • each distinct CAR unit includes different co-stimulatory domains to avoid interaction.
  • the first chimeric antigen receptor polypeptide includes a 4-BB co-stimulatory domain; and the second chimeric antigen receptor polypeptide includes a CD28 co-stimulatory domain.
  • the hinge region is designed to exclude amino acids that may cause undesired intra- or intermolecular interactions.
  • the hinge region may be designed to exclude or minimize cysteine residues to prevent formation of disulfide bonds.
  • the hinge region may be designed to exclude or minimize hydrophobic residues to prevent unwanted hydrophobic interactions.
  • Compound CAR can perform killing independently or in combination.
  • Multiple or compound CAR comprises same or different hinge region, same or different transmembrane, same or different co-stimulatory and same or different intracellular domains.
  • the hinge region is selected to avoid the interaction site.
  • the compound CAR of the present disclosure may target same or different tumor populations in T or NK cells.
  • the first CAR for example, may target the bulky tumor population and the next or the second CAR, for example, may eradicate cancer or leukemic stem cells, to avoid cancer relapses.
  • the compound CAR in a T or NK cells targeting different or same tumor populations combat tumor factors causing cancer cells resistant to the CAR killing activity, thereby producing down regulation of the target antigen from the cancer cell surface. It was also surprisingly found that this enables the cancer cell to “hide” from the CAR therapy referred to as “antigen escape” and tumor heterogeneity, by which different tumor cells can exhibit distinct surface antigen expression profiles.
  • the present disclosure provides an engineered cell having at least one chimeric antigen receptor polypeptide and an enhancer.
  • the present disclosure provides an engineered cell having at least two distinct chimeric antigen receptor polypeptides and an enhancer.
  • an enhancer includes a biological molecule that promotes or enhances the activity of the engineered cell having the chimeric antigen receptor polypeptide.
  • Enhancers include cytokines.
  • enhancers include IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, PD-1, PD-L1, CSF1R, CTAL-4, TIM-3, and TGFR beta, receptors for the same, and functional fragments thereof.
  • Enhancers may be expressed by the engineered cell described herein and displayed on the surface of the engineered cell or the enhancer may be secreted into the surrounding extracellular space by the engineered cell. Methods of surface display and secretion are well known in the art.
  • the enhancer may be a fusion protein with a peptide that provides surface display or secretion into the extracellular space.
  • the effect of the enhancer may be complemented by additional factors such as enhancer receptors and functional fragments thereof.
  • additional factors may be co-expressed with the enhancer as a fusion protein, or expressed as a separate polypeptide and secreted into the extracellular space.
  • Enhancers can be cytokines secreted from engineered CAR cells and are designed to co-express with the CAR polypeptide. A massive release occurs upon CAR engagement of cognate antigen. Inflammatory cells surrounding tumor cells have a significant correlation with cancer cell progression and metastasis. Inflammatory cells could include T cells and innate immune response cells, such as NK cells, macrophages, and dendritic cells and their proliferation and anti-tumor activity are regulated by cytokines. CAR cells such as CAR T or NK cells bind to targeted cancer cells and trigger massive secretion of enhancers from the expansion of CAR T/NK cells. The secreted enhancers efficiently promote survival, differentiation and activation of immune response cells against cancer cells. The co-expression of an enhancer(s) with CAR can supplement the defect that CAR T or NK cells are unable to eliminate non-targeting cancer cells ( FIG. 78 ).
  • CAR cells can be a carrier of cytokines, and cytokines can be delivered to targeted cancer sites by CAR cells to reduce systemic toxicity with high-dose exogenous cytokines ( FIG. 78 ).
  • a membrane bound enhancer can be co-expressed with CAR to improve CAR persistency.
  • the enhancer is IL-15.
  • the additional factor described above 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 (IL-15sushi). Soluble IL-15RA or IL15sushi profoundly potentiates IL-15 functional activity by prevention of IL-15 degradation. Soluble IL-15/IL-15RA or IL-15/IL-15sushi complexes are stable and much more stimulatory than IL-15 alone in vivo.
  • IL-15 is co-expressed as a fusion protein with at least one of IL-15 receptor, IL-15RA, and the sushi domain of IL-15RA (IL-15sushi).
  • the IL-15 receptor, IL-15RA, or the sushi domain of IL-15RA (IL-15sushi) is at the N-terminus of IL-15.
  • the IL-15 receptor, IL-15RA, or the sushi domain of IL-15RA (IL-15sushi) is at the C-terminus of IL-15.
  • IL-15/IL-15 sushi denotes that IL-15 sushi is at the C-terminus of IL-15 in a fusion protein
  • IL-15sushi/il-15 denotes that IL-15 sushi is at the N-terminus of IL-15 in a fusion protein.
  • IL-15 and the IL-15 receptor or functional fragments thereof polypeptide is on a single polypeptide molecule and is separated by a peptide linker, the peptide linker may be 1-25 amino acid residues in length, 25-100 amino acid residues in length, or 50-200 amino acid residues in length.
  • This linker may include a high efficiency cleavage site described herein.
  • An example of a suitable sushi domain includes a CAR construct, SEQ ID NO. 1.
  • any chimeric antigen receptor polypeptide disclosed herein may be co-expressed with the Human Interleukin 15 with human interleukin 2 signal peptide SEQ ID NO. 2.
  • CD8+ T cells can be modified to express autocrine growth factors including, but not limited to, IL-2, 11-7, IL21 or IL-15, to sustain survival following transfer in vivo.
  • IL-15 overcomes the CD4 deficiency to induce primary and recall memory CD8T cells.
  • Overexpression of IL-15-RA or an IL-15 IL-RA fusion on CD8 T cells significantly enhances its survival and proliferation in-vitro and in-vivo.
  • CD4CAR or any CAR is co-expressed with at least one of IL-15, IL15RA and IL-15/IL-15R or IL15-RA/IL-15, or a part or a combination thereof, to enhance survival or proliferation of CAR T or NK, and to improve expansion of memory CAR CD8+ T cells.
  • the present disclosure provides an engineered cell having a CAR polypeptide as described herein and at least one of IL-15, IL-15RA, IL-15sushi, IL-15/IL-15RA, IL15-RA/IL-15, IL-15/IL-15sushi, IL15sushi/IL-15, fragment thereof, a combination thereof, to enhance survival or persistence or proliferation of CAR T or NK for treating cancer in a patient.
  • the present disclosure provides an engineered cell having at least one of recombinant IL-15, IL-15RA, IL-15sushi, IL-15/IL-15RA, IL15-RA/IL-15, IL-15/IL-15sushi, IL15sushi/IL-15, functional fragment thereof, and combination thereof; and at least one distinct CAR polypeptide wherein the antigen recognition domain includes NY-ESO-1, alpha fetoprotein (AFP), glypican-3 (GPC3), BCMA, BAFF-R, BCMA, TACI, LeY, CD5, CD7, CD2, CD3, CD4, CD45, CD13, CD14, CD15, CD19, CD20, CD22, CD33, CD41, CD61, CD64, CD68, CD117, CD123, CD138, CD267, CD269, CD38, Flt3 receptor, ROR1, PSMA, MAGE A3, Glycolipid, F77, GD-2, WT1, CEA, HER-2/neu, MAGE-3
  • the target antigens can also include viral or fungal antigens, such as E6 and E7 from the human papillomavirus (HPV) or EBV (Epstein Barr virus) antigens.
  • viral or fungal antigens such as E6 and E7 from the human papillomavirus (HPV) or EBV (Epstein Barr virus) antigens.
  • E6 and E7 from the human papillomavirus (HPV) or EBV (Epstein Barr virus) antigens.
  • HPV human papillomavirus
  • EBV Epstein Barr virus
  • IL-15/IL-15sushi and other types of IL-15 or IL-15RA proteins or protein fragments thereof provide synergistic efficacy of a CAR polypeptide when combined with checkpoint inhibitors or modulators (e.g. anti-PD-1).
  • the disclosure provides a CD4 CAR engineered cell that includes IL-15/IL-15sushi (SEQ ID NO. 1), and corresponding polynucleotide (SEQ ID NO. 2).
  • the present disclosure provides a method of providing long-term durable remission in cancer patients by administering a CD4 CAR engineered cell that includes IL-15/IL-15sushi to a patient in need thereof.
  • co-expression of IL-15/IL-15sushi with a CD4 CAR polypeptide provides long-term durable remission in patients by increasing the sensitivity of CAR recognition of target cancer cells or recruiting innate immune cells to cancer cells.
  • the present disclosure provides engineered cell having a CD45 chimeric antigen receptor polypeptide and IL-15/IL-15sushi (SEQ ID NO. 44), and corresponding nucleotides (SEQ ID NO. 43).
  • the present disclosure provides a method of providing long-term durable remission in cancer patients by administering a CD45 CAR engineered cell that includes IL-15/IL-15sushi to a patient in need thereof.
  • a CD45 CAR engineered cell that includes IL-15/IL-15sushi to a patient in need thereof.
  • co-expression of IL-15/IL-15sushi with a CD45 CAR polypeptide provides long-term durable remission in patients by increasing the sensitivity of CAR recognition of target cancer cells or recruiting innate immune cells to cancer cells.
  • the engineered cell includes a CD45 chimeric antigen receptor polypeptide and 4-1BBL (SEQ ID NO. 74), and corresponding nucleotides (SEQ ID NO. 73).
  • the present disclosure provides a method of providing long-term durable remission in patients suffering from cancer by administering a CD45 CAR engineered cell that co-expresses 4-1BBL to a patient in need thereof.
  • a CD45 CAR engineered cell that co-expresses 4-1BBL to a patient in need thereof.
  • co-expression of 4-1BBL with a CD45 CAR provides long-term durable remission in patients by increasing the persistence of CAR engineered cells.
  • the engineered cell includes a CD19 chimeric antigen receptor polypeptide and IL-15/IL-15sushi (SEQ ID NO. 59), and corresponding polynucleotide (SEQ ID NO. 60).
  • the present disclosure provides a method of providing long-term durable remission in cancer patients by administering a CD19 CAR engineered cell that includes IL-15/IL-15sushi to a patient in need thereof.
  • co-expression of IL-15/IL-15sushi with a CD19 CAR provides long-term durable remission in patients by increasing the sensitivity of CAR recognition of target cancer cells or recruiting innate immune cells to cancer cells.
  • the engineered cell includes a CD20 chimeric antigen receptor polypeptide and IL-15/IL-15sushi (SEQ ID NO. 58), and corresponding polynucleotide (SEQ ID NO. 57).
  • the present disclosure provides a method of providing long-term durable remission in cancer patients by administering a CD20 CAR engineered cell that includes IL-15/IL-15sushi to a patient in need thereof.
  • co-expression of IL-15/IL-15sushi with a CD20 CAR provides long-term durable remission in patients by increasing the sensitivity of CAR recognition of target cancer cells or recruiting innate immune cells to cancer cells.
  • the engineered cell includes a CD22 chimeric antigen receptor polypeptide and IL-15/IL-15sushi (SEQ ID NO. 62), and corresponding polynucleotide (SEQ ID NO. 61).
  • the present disclosure provides a method of providing long-term durable remission in cancer patients by administering a CD22 CAR engineered cell that includes IL-15/IL-15sushi to a patient in need thereof.
  • co-expression of IL-15/IL-15sushi with a CD22 CAR provides long-term durable remission in patients by increasing the sensitivity of CAR recognition of target cancer cells or recruiting innate immune cells to cancer cells.
  • the engineered cell includes a CD269 chimeric antigen receptor polypeptide and IL-15/IL-15sushi (SEQ ID NO. 44), and corresponding polynucleotide (SEQ ID NO. 45).
  • the present disclosure provides a method of providing long-term durable remission in cancer patients by administering a CD269 CAR engineered cell that includes IL-15/IL-15sushi to a patient in need thereof.
  • IL-15/IL-15sushi with a CD269 CAR provides long-term durable remission in patients by increasing the sensitivity of CAR recognition of target cancer cells or recruiting innate immune cells to cancer cells as plasma cells or myeloma cells are usually dim CD269 (BCMA) positive.
  • the engineered cell includes a CAR, CD4 polypeptide of SEQ ID NO. 90, and corresponding polynucleotide of SEQ ID NO. 89.
  • the engineered cell includes a CD4 chimeric antigen receptor polypeptide and IL-15/IL-15sushi (SEQ ID NO. 96), and corresponding polynucleotide (SEQ ID NO. 95).
  • the present disclosure provides a method of providing long-term durable remission in cancer patients by administering a CD4 CAR engineered cell that includes IL-15/IL-15sushi to a patient in need thereof.
  • co-expression of IL-15/IL-15sushi with a CD4 CAR provides long-term durable remission in patients by increasing the sensitivity of CAR recognition of target cancer cells or recruiting innate immune cells to cancer cells.
  • the engineered cell includes a CD4 chimeric antigen receptor polypeptide and IL-15/IL-15RA (membrane bound) (SEQ ID NO. 98), and corresponding polynucleotide (SEQ ID NO. 97).
  • the present disclosure provides a method of providing long-term durable remission in cancer patients by administering a CD4 CAR engineered cell that includes IL-15/IL-15RA to a patient in need thereof.
  • co-expression of IL-15/IL-15RA (membrane bound) with a CD4 CAR provides long-term durable remission in patients by increasing the persistence of CAR engineered cells.
  • the engineered cell includes a compound CAR, CD33CD123 polypeptide and IL-15/IL-15sushi (SEQ ID NO. 40), and corresponding polynucleotide (SEQ ID NO. 39).
  • the present disclosure provides a method of providing long-term durable remission in cancer patients by administering a CD33CD123 compound CAR engineered cell that includes IL-15/IL-15sushi to a patient in need thereof.
  • co-expression of IL-15/IL-15sushi with a CD33CD123 CAR provides long-term durable remission in patients by increasing the sensitivity of CAR recognition of target cancer cells or recruiting innate immune cells to cancer cells.
  • the engineered cell includes a compound CAR, CD33CD123 polypeptide and 4-1BBL(SEQ ID NO. 38), and corresponding polynucleotide (SEQ ID NO. 37).
  • the present disclosure provides a method of providing long-term durable remission in cancer patients by administering a CD33CD123 compound CAR engineered cell that co-expresses 4-1BBL to a patient in need thereof.
  • co-expression of 4-1BBL with a CD33CD123 cCAR provides long-term durable remission in patients by increasing the persistency of cCAR engineered cells.
  • the engineered cell includes a BAFF CAR polypeptide with a CD45 leader sequence (SEQ ID NO. 78) and corresponding polynucleotide sequence (SEQ ID NO. 77).
  • the engineered cell includes BAFF CAR polypeptide with a CD8a leader sequence (includes SEQ ID NO. 80) and corresponding polynucleotide sequence (SEQ ID NO. 79).
  • the engineered cell includes a BAFF CAR polypeptide and IL-15/IL-15sushi (SEQ ID NO. 84), and corresponding polynucleotide (SEQ ID NO. 83).
  • the present disclosure provides a method of providing long-term durable remission in cancer patients by administering a BAFF CAR engineered cell that includes IL-15/IL-15sushi to a patient in need thereof.
  • a BAFF CAR engineered cell that includes IL-15/IL-15sushi to a patient in need thereof.
  • co-expression of IL-15/IL-15sushi with a BAFF CAR provides long-term durable remission in patients by increasing the sensitivity of CAR recognition of target cancer cells or recruiting innate immune cells to cancer cells as BAFF receptor, CD269 (BCMA) is weakly expressed in plasma cells and myeloma cells.
  • the engineered cell includes a BAFF CAR polypeptide and 4-1BBL (SEQ ID NO. 82), and corresponding polynucleotide (SEQ ID NO. 81).
  • the present disclosure provides a method of providing long-term durable remission in cancer patients by administering a BAFF CAR engineered cell co-expresses 4-1BBL to a patient in need thereof. Without wishing to be bound by theory, it is believed that co-expression of 4-1BBL with a BAFF CAR can provide long-term durable remission in patients by increasing the persistence of CAR engineered cells.
  • the engineered cell includes a compound CAR, BAFF CD19b polypeptide of SEQ ID NO. 86 and corresponding polynucleotide of SEQ ID NO. 85.
  • the present disclosure provides a method of treating an autoimmune disorder in a patients by administering a BAFF CD19b compound CAR engineered cell to a patient in need thereof.
  • a BAFF CD19b compound CAR engineered cell provides a better therapeutic outcome for depletion of B-cells and plasma cells associated with autoimmune disorders.
  • the engineered cell includes a APRIL CD19b compound CAR polypeptide of SEQ ID NO. 88 and corresponding polynucleotide of SEQ ID NO. 77.
  • the present disclosure provides a method of depleting B-cells and plasma cells in a patient in need thereof by administering a APRIL CD19b compound CAR engineered cell to a patient in need thereof.
  • a APRIL CD19b compound CAR engineered cell can provide a better therapeutic outcome for depletion of B-cells and plasma cells associated with autoimmune disorders.
  • the engineered cell includes a compound CAR, CD269 CS1 polypeptide of SEQ ID NO. 48 and corresponding polynucleotide of SEQ ID NO. 47.
  • the present disclosure provides a method of treating myeloma in a patient by administering a CD269CS1 compound CAR engineered cell to a patient in need thereof.
  • CD269 CS1 compound CAR engineered cells provide a better therapeutic outcome for patients with myeloma, and prevent antigen escape or disease relapse.
  • the engineered cell includes a compound CAR, CD269 CD19b polypeptide of SEQ ID NO. 50 and corresponding polynucleotide of SEQ ID NO. 49.
  • the present disclosure provides a method of depleting B-cells and plasma cells in patients by administering a CD269 CD19b compound CAR engineered cell to a patient in need thereof.
  • CD269 CD19b compound CAR engineered cells provide a better therapeutic outcomes for patients suffering from an autoimmune disorder by depletion of B-cells and plasma cells associated with autoimmune disorders.
  • the engineered cell includes another compound CAR, CD269 CD19 polypeptide of SEQ ID NO. 52 and corresponding polynucleotide of SEQ ID NO. 51.
  • the present disclosure provides a method of depleting B-cells and plasma cells in patients by administering a CD269 CD19 compound CAR engineered cell to a patient in need thereof. Without wishing to be bound by theory, it is believed that CD269 CD19 compound CAR engineered cells provide a better therapeutic outcomes in patients suffering from an autoimmune disorder by depletion of B-cells and plasma cells associated with autoimmune disorders.
  • the present disclosure provides an engineered cell having a CD19 chimeric antigen receptor polynucleotide.
  • the CD19 CAR polypeptide includes SEQ ID NO. 54 and corresponding polynucleotide sequence SEQ ID NO. 53.
  • the CD19 CAR polypeptide includes SEQ ID NO. 56, and corresponding polynucleotide sequence SEQ ID NO. 55
  • the engineered cell includes a CD30 CAR polypeptide, and IL-15/IL-15sushi polypeptide (SEQ ID NO. 100), and corresponding polynucleotide (SEQ ID NO. 99).
  • the targeted disease is malignant Hodgkin lymphoma with cancer cells expressing CD30.
  • the present disclosure provides a method of re-activating T-cell and innate immune cells in the tumor microenvironment patients by administering a CD30CAR engineered cell that secretes IL-15/IL-15 complexes to a patient in need thereof.
  • IL-15/IL-15 complexes e.g. IL-15/IL-15sushi complexes
  • engineered cells can re-activate T-cell and innate immune cells in the tumor microenvironment and then restore or augment their anti-tumor immune responses for Hodgkin lymphoma or anaplastic large cell lymphoma.
  • the present disclosure provides a method of restoring or augmenting T-cell or innate immune cell activation or expansion including coexpression of IL-15/IL-15sushi with a CAR polypeptide disclosed herein.
  • the disclosure provides a chimeric antigen receptor polypeptide having an antigen recognition domain specific for a CD30 antigen.
  • the CD30CAR includes at least one-costimulatory domain. In another embodiment, the CD30CAR includes at least two co-stimulatory domains.
  • the disclosure includes a method of co-expressing IL-15/IL-15sushi with CD30CAR.
  • massive secretion of stable, functional IL-15/IL-15sushi complexes occurs upon binding of CAR to target cells.
  • the present disclosure provides a method of treating a patient suffering from Hodgkin's lymphoma or a cancer associated with a malignant cell expressing CD30 antigen by administering a CD30 CAR engineered cell to a patient in need thereof.
  • a malignant cells expressing CD30 includes anaplastic large cell lymphoma.
  • T cells and innate immune cells Malignant Hodgkin lymphoma bears CD30+ Reed-Sternberg or Reed-Sternberg like cells, which are surrounded by an overwhelming numbers of T cells and innate immune cells. These T or innate immune cells are immunologically tolerant as they fail to eliminate cancer cells. Therefore, one of critical aspects for treating Hodgkin lymphoma is to re-activate T-cell and innate immune cells in the tumor microenvironment and then restore or augment their anti-tumor immune responses.
  • the present disclosure comprises a method of co-expression of IL-15/IL-15sushi with a CD30CAR.
  • Engineered CD30CAR T or NK cells bind to targeted cancer cells, trigger massive secretion of IL-15/IL-15sushi from the expansion of CD30CAR T or NK cells, whereby secreting IL-15/IL-15sushi efficiently restore or augment T or innate immune cells against cancer cells to overcome immunosuppressive tumor microenvironment.
  • the present disclosure provides a method of providing long-term durable remission in a cancer patient by administering a CD30 CAR engineered cell that co-express IL-15/IL-15sushi to a patient in need thereof.
  • a CD30 CAR engineered cell that co-express IL-15/IL-15sushi to a patient in need thereof.
  • co-expression of IL-15/IL-15sushi with a CD30CAR provides long-term durable remission in patients by increasing the sensitivity of CAR recognition of target cancer cells or recruiting innate immune cells against target cancer cells to overcome immunosuppressive tumor microenvironment.
  • the present disclosure provides an engineered cell that co-expresses IL-15/IL-15sushi and a CD30CAR polypeptide.
  • IL-15/IL-15sushi co-expresses IL-15/IL-15sushi and a CD30CAR polypeptide.
  • CD30CAR engineered cell with co-expression of IL-15/IL-15sushi provides synergistic efficacy when combined with checkpoint inhibitors or modulators (e.g. anti-PD-1).
  • the present disclosure provides a method of treating Hodgkin's lymphoma in a patient by administering a CD30 CAR engineered cell that co-expresses IL-15/IL-15sushi to a patient in need thereof.
  • a CD30 CAR engineered cell that co-expresses IL-15/IL-15sushi to a patient in need thereof.
  • co-expression of CD30CAR polypeptide and IL-15/IL-15sushi provides better outcomes for treatment of Hodgkin's lymphoma or anaplastic large cells than CD30CAR alone as CD30 is not expressed in all cancer cells.
  • the present disclosure provides a method of provide long-term durable remission in a cancer patient by administering a APRIL CAR engineered cell that co-expresses IL-15/IL-15sushi to a patient in need thereof.
  • a APRIL CAR engineered cell that co-expresses IL-15/IL-15sushi to a patient in need thereof.
  • co-expression of IL-15/IL-15sushi with a APRIL CAR polypeptide provides long-term durable remissions in patients by increasing the sensitivity of CAR recognition of target cancer cells or recruiting innate cells to cancer cells.
  • APRIL receptor, CD269 (BCMA) is weakly expressed in plasma cells and myeloma cells.
  • the present disclosure provides a method for elimination of tumor cells including contacting said tumor cell with a CAR engineered cell that co-expresses IL-2 to destroy said tumor cell.
  • IL-15 was originally considered as an interleukin-2 (IL-2)-like factor for T and NK cells. Unlike IL-2, IL-15 is a survival factor for memory T cells.
  • IL-2 interleukin-2
  • IL-15 is a survival factor for memory T cells.
  • elimination of tumor can be achieved by combination of at least one or more of the following steps:
  • the engineered cells and methods described herein are suitable for the treatment of any cancer wherein specific monoclonal or polyclonal antibodies exist or are capable of being generated in accordance with the current state of the art.
  • cancers have been contemplated and are considered within the scope of the present disclosure, neuroblastoma, lung cancer, melanoma, ovarian cancer, renal cell carcinoma, colon cancer, brain cancer, Hodgkin's lymphoma, B cell lymphoma/leukemia and T cell lymphomalleukemia. All of which have cell surface antigens that may be targeted by the chimeric antigen receptor polypeptides and methods disclosed herein.
  • the present disclosure provides a method to overcome this escape. Without wishing to be bound by theory, the present disclosure provides a method of preventing tumor escape by administering a CAR engineered cell having an enhancer or cytokine as disclosed herein, in particular IL-15 or IL-2 to a tumor site by CAR engineered cell. It is believed that this directly stimulates innate and adaptive immune responses. Furthermore, it is believed that IL-15 and/or IL-2 secretion from CAR engineered cells promote the expansion of infused CAR T cells or CAR NK cells and infiltration of immune cells to the tumor site, which is believed to result in tumor destruction.
  • a CAR engineered cell having an enhancer or cytokine as disclosed herein, in particular IL-15 or IL-2 to a tumor site by CAR engineered cell. It is believed that this directly stimulates innate and adaptive immune responses.
  • IL-15 and/or IL-2 secretion from CAR engineered cells promote the expansion of infused CAR T cells or CAR NK cells and infiltration of immune cells
  • Fc domain such IL-15Fc or IL-2Fc.
  • Fc domain is referred to as the IgG Fc-domain fused to various effector molecules (so-called Fc-fusion proteins).
  • Single antigen-directed CAR immunotherapy bears a risk of remission in patients due to the complete loss of target antigen or changes of target antigen expression.
  • the present disclosure provides a method of providing long-term durable remission in patients by administering an engineered cell having a CAR polypeptide disclosed herein and co-expression of IL-15/IL-15sushi to increase the sensitivity of CAR recognition of target cancer cells or recruiting innate immune cells to cancer cells.
  • CAR T cells may end up simply being inactivated or suppressed when contacting tumor.
  • the present disclosure provides a method of co-expressing secretory IL-15/IL-15sushi and a chimeric antigen receptor polypeptide in an engineered cell.
  • the present disclosure provides a method of increasing CAR engineered cell in vivo half life by co expressing secretory IL-15/IL-5sushi in said engineered cell.
  • secretory IL-15/IL-5sushi in said engineered cell.
  • the present disclosure provides a method of delivering IL-15/IL-15sushi to targeted cancer sites using CAR as a carrier to promote the proliferation of innate immune response cells against cancer cells, prevent the tumor microenvironment suppression, and reduce systemic toxicity with high-dose exogenous cytokines.
  • the present disclosure provides a method of delivering IL-15/IL-15sushi to targeted cancer sites using CAR as a carrier to recruit other effector immune cells to the site and help them to kill cancer cells.
  • the present disclosure provides a method of delivering IL-15/IL-15sushi to targeted cancer sites using CAR as a carrier to activate bystander immunity to eradicate cancer cells that lose the antigen for CAR T/NK cells.
  • polynucleotides disclosed herein may be introduced into an engineered cell by any method known in the art.
  • CAR polynucleotides are delivered to the engineered cell by any viral vector as disclosed herein.
  • the any of the engineered cells disclosed herein be constructed as a transient RNA-modified “biodegradable” version or derivatives, or a combination thereof.
  • the RNA-modified CARs of the present disclosure may be electroporated into T cells or NK cells.
  • the expression of the compound CAR may be gradually diminished over few days.
  • any of the engineered cells disclosed herein may be constructed in a transponson system (also called a “Sleeping Beauty”), which integrates the CAR DNA into the host genome without a viral vector.
  • a transponson system also called a “Sleeping Beauty”
  • the present disclosure provides a method making an engineered cell having at least two CAR units.
  • multiple units of CAR are expressed in a T or NK cell using bicistronic or multicistronic expression vectors.
  • bicistronic or multicistronic vectors There are several strategies which can be employed to construct bicistronic or multicistronic vectors including, but not limited to, (1) multiple promoters fused to the CARs' open reading frames; (2) insertion of splicing signals between units of CAR; fusion of CARs whose expressions are driven by a single promoter; (3) insertion of proteolytic cleavage sites between units of CAR (self-cleavage peptide); and (iv) insertion of internal ribosomal entry sites (IRESs).
  • IVSs internal ribosomal entry sites
  • multiple CAR units are expressed in a single open reading frame (ORF), thereby creating a single polypeptide having multiple CAR units.
  • ORF open reading frame
  • an amino acid sequence or linker containing a high efficiency cleavage site is disposed between each CAR unit.
  • high cleavage efficiency is defined as more than 50%, more than 70%, more than 80%, or more than 90% of the translated protein is cleaved. Cleavage efficiency may be measured by Western Blot analysis, as described by Kim 2011.
  • cleavage product there are equal amounts of cleavage product, as shown on a Western Blot analysis.
  • high efficiency cleavage sites examples include porcine teschovirus-1 2A (P2A), FMDV 2A (abbreviated herein as F2A); equine rhinitis A virus (ERAV) 2A (E2A); and Thoseaasigna virus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A) and flacherie Virus 2A (BmIFV2A), or a combination thereof.
  • the high efficiency cleavage site is P2A.
  • High efficiency cleavage sites are described in Kim J H, Lee S-R, Li L-H, Park H-J, Park J-H, Lee K Y, et al.
  • expression is under the control of a strong promoter.
  • strong promoters include the SFFV promoter, and derivatives thereof.
  • the present disclosure provides a method making an engineered cell that expresses at least one CAR unit and an enhancer.
  • At least one CAR unit and enhancer is expressed in a T or NK cell using bicistronic or multicistronic expression vectors.
  • bicistronic or multicistronic vectors There are several strategies which can be employed to construct bicistronic or multicistronic vectors including, but not limited to, (1) multiple promoters fused to the CARs' open reading frames; (2) insertion of splicing signals between units of CAR; fusion of CARs whose expressions are driven by a single promoter; (3) insertion of proteolytic cleavage sites between units of CAR (self-cleavage peptide); and (4) insertion of internal ribosomal entry sites (IRESs).
  • IVSs internal ribosomal entry sites
  • At least one CAR unit and an enhancer are expressed in a single open reading frame (ORF), thereby creating a single polypeptide having at least one CAR unit and an enhancer.
  • ORF open reading frame
  • an amino acid sequence or linker containing a high efficiency cleavage site is disposed between each CAR unit and between a CAR unit and enhancer.
  • the ORF is under the control of a strong promoter. Examples of strong promoters include the SFFV promoter, and derivatives thereof.
  • cleavage product there are equal amounts of cleavage product, as shown on a Western Blot analysis.
  • CD45 is also known as leukocyte common antigen (LCA) and is a tyrosine phosphatase expressed on virtually all cells of hematopoietic origin except erythrocytes and platelets. Most hematologic malignancies express CD45. For instance, 85% to 90% acute lymphoid and myeloid leukernias express CD45. (1CD45 is not found in non-hematopoietic origin. In addition, CD45 is expressed at a high density of an average copy number of approximately 200,000 molecules per cells on malignant cells and leukocytes. CD45 presents an ideal target for a variety of hematologic malignancies. H-lowever, CAR T and NK cells also express CD45. Without inactivation of endogenous CD45, CAR T or NK cells armed with CARs targeting CD45 may result in self-killing.
  • LCA leukocyte common antigen
  • TCRs T cell receptors
  • TCRs are cell surface receptors that play an essential role in the activation of T cells in response to the presentation of antigen.
  • the TCR is generally made from two chains, alpha and beta, which are associated with the transducing subunits, the CD3, to form the T-cell receptor complex present on the cell surface.
  • CARs Compound CARs, cCAR
  • cCAR compound CARs
  • This mechanism allows the cancer cell to “hide” from the CAR therapy, a phenomenon referred to as ‘antigen escape’.
  • the present disclosure pre-empts cancer antigen escape by recognizing a combination of two or more antigens to rapidly eliminate the tumor.
  • the disclosure provides a method of simultaneous targeting of multi-antigens using a cCAR resulting in improved tumor control by minimizing the possibility of tumor selection on the basis of target antigen loss or down-regulation.
  • the disclosed disclosure includes compound (multiple or compound) cCAR in a T or NK cell targeting different or same surface antigens present in tumor cells.
  • the compound chimeric antigen receptors of the present disclosure comprise at least multiple chimeric receptor constructs linked by a linker and target of the same or different antigens.
  • each of the 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 transmembrane domain can be derived from any desired source for such domains.
  • the multiple CAR constructs are linked by a linker.
  • the expression of the compound CAR construct is driven by a promoter.
  • the linker may be a peptide or a part of a protein, which is self-cleaved after a protein or peptide is generated (also called as a self-cleaving peptide).
  • the compound CARs of the present disclosure target Myelodysplastic Syndrome and acute myeloid leukemia (AML) populations.
  • Myelodysplastic Syndrome (MDS) remains an incurable hematopoietic stem cell malignancy that occurs most frequently among the elderly, with about 14,000 new cases each year in the USA. About 30-40% of MDS cases progress to AML. The incidence of MDS continues to increase as our population ages. Although MDS and AML have been studied intensely, no satisfactory treatments have been developed.
  • compositions and methods of this disclosure can be used to generate a population of T lymphocyte or NK cells that deliver both primary and co-stimulatory signals for use in immunotherapy in the treatment of cancer, in particular, the treatment of lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, brain cancer, sarcoma, leukemia and lymphoma.
  • Immunotherapeutics generally rely on the use of immune effector cells and molecules to target and destroy cancer cells.
  • the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target.
  • Various effector cells include cytotoxic T cells, NK cells, and NK-92 cells.
  • the compositions and methods described in the present disclosure may be utilized in conjunction with other types of therapy for cancer, such as chemotherapy, surgery, radiation, gene therapy, and so forth.
  • the compositions and methods described in the present disclosure may be utilized in other disease conditions that rely on immune responses such as inflammation, immune diseases, and infectious diseases.
  • the compound CAR of the present disclosure may act as a bridge to bone marrow transplant, by achieving complete remission for patients who have minimal residual disease and are no longer responding to chemotherapy.
  • the compound CAR eliminates leukemic cells followed by bone marrow stem cell rescue to support leukopenia.
  • the compound CAR of the present disclosure can combat a key mechanism by which cancer cells resist CAR activity by the down-regulation of the target antigen.
  • the invented compound CAR can also combat the heterogeneity of cancer cells, which creates significant challenges in a regular CAR T/NK cell therapy.
  • the disclosed compound CAR is designed that the first CAR targets the bulky tumor population and another eradicates cancer or leukemic stem cells to avoid cancer relapses.
  • the present disclosure provides a method of destroying cells having a CD33 antigen or a CD123 antigen, or both by contacting said cells with an engineered cell having at least one of chimeric antigen receptor polypeptide having a CD33 antigen recognition domain and chimeric antigen receptor polypeptide having a CD23 antigen recognition domain.
  • the engineered cell may be a T or NK cell.
  • Cells having at least one of the CD33 antigen and the CD123 antigen include acute myeloid leukemia, precursor acute lymphoblastic leukemia, chronic myeloproliferative neoplasms, chronic myeloid leukemia, myelodysplasia syndromes, blastic plasmocytoid dendritic neoplasms (BPDCN), Hodgkin's lymphoma, mastocytosis, and hairy cell leukemia cells.
  • BPDCN blastic plasmocytoid dendritic neoplasms
  • the present disclosure provides a method of providing myeloblative conditioning regimens for hematopoietic stem cell transplantation.
  • a T or NK engineered cell having a CD33 unit and a CD123 unit is administered to a patient in need thereof.
  • the present disclosure provides a method of eradicating or killing leukemic stem cells (LSCs) or bulk leukemic cells expressing CD123 or CD33, or both.
  • LSCs leukemic stem cells
  • a T or NK engineered cell having a CD33 unit and a CD123 unit is administered to a patient in need thereof.
  • the compound CAR in a T or NK cell may be used to eradicate or kill CD34+CD38 ⁇ leukemic stem cells or bulk leukemic cells expressing CD123 or CD33 or both.
  • a compound CAR targets cells expressing CD19 or CD20 antigens or both. In another embodiment, a compound CAR targets cells expressing CD19 or CD22 antigens or both.
  • the targeted cells may be cancer cells, such as, without limiting, B-cell lymphomas or leukemias.
  • the target antigens can include at least one of this group, but not limited to, ROR1, PSMA, MAGE A3, Glycolipid, 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, immunoglobin kappa and lambda, CD38, CD52, CD3, CD4, CD8, CD5, CD7, CD2, and CD138.
  • the target antigens can also include viral or fungal antigens, such as E6 and E7 from the human papillomavirus (HPV) or EBV (Epstein Barr virus) antigens.
  • the compound CAR engineered cells target cells having cell surface CD19 antigen or cell surface CD123 antigen or both.
  • the targeted cells are cancer cells, such as, without limiting, B-cell lymphomas or leukemias.
  • the compound CAR targets cells expressing CD19 or CD123 antigen or both. Without wishing to be bound by theory, it is believed that CD19 and/or CD123 compound CAR engineered cells offset tumor escape due to the loss of CD19 or CD123 antigen or prevent B-ALL or other type B-cell lymphoma/leukemia relapse.
  • the CD19 and/or CD20 compound CAR engineered cells target cells having cell surface CD19 antigens and/or CD20 cell surface antigens.
  • the targeted cells are malignant B cell lymphoma/leukemia such as, without limiting, B-ALL, high grade B cell lymphoma, low grade B-cell lymphoma, diffuse large B cell lymphoma, Burkett lymphoma, mantle cell lymphoma, CLL, marginal zone B cell lymphoma and follicular lymphoma.
  • CD19 and/or CD20 CAR engineered cells provide an effective safeguard against antigen escape and prevent disease relapse in adoptive T/NK-cell therapy for B-cell malignancies.
  • CAR target cells having at least one of the antigens CD19, CD20, CD22, and CD123 include precursor acute lymphoblastic leukemia, B-cell lymphoma/leukemia, chronic lymphocytic leukemia/lymphoma, mantle lymphoma, follicular lymphoma, marginal zone B cell lymphoma, diffuse large B cell lymphoma, Burkett lymphoma, blastic plasmocytoid dendritic neoplasms (BPDCN), Hodgkin's lymphoma, and hairy cell leukemia cells.
  • precursor acute lymphoblastic leukemia include precursor acute lymphoblastic leukemia, B-cell lymphoma/leukemia, chronic lymphocytic leukemia/lymphoma, mantle lymphoma, follicular lymphoma, marginal zone B cell lymphoma, diffuse large B cell lymphoma, Burkett lymphoma, blastic plasmocytoid dendritic ne
  • the engineered cell includes a first chimeric antigen receptor polypeptide having a CD19 antigen recognition domain and second chimeric antigen receptor polypeptide having a CD22 antigen recognition domain. In one embodiment, this engineered cell includes a polypeptide of SEQ ID NO. 64 and corresponding polynucleotide of SEQ ID NO. 63.
  • the engineered cell includes a first chimeric antigen receptor polypeptide having a CD19 antigen recognition domain and second chimeric antigen receptor polypeptide having a CD20 antigen recognition domain. In one embodiment, this engineered cell includes a polypeptide of SEQ ID NO. 66 and corresponding polynucleotide of SEQ ID NO. 65.
  • the engineered cell includes a first chimeric antigen receptor polypeptide having a CD19 antigen recognition domain and second chimeric antigen receptor polypeptide having a CD123 antigen recognition domain. In one embodiment, this engineered cell includes a polypeptide of SEQ ID NO. 68 and corresponding polynucleotide of SEQ ID NO. 67.
  • CS1 and BCMA are widely expressed on myeloma cells, but is not expressed in hematopoietic stem/progenitor cells. Therefore, BCMA and CS1 are ideal targets for CAR T/NK cell therapy.
  • the present disclosure provides compound CAR engineered cell having a CS1 (SLAM7) antigen recognition domain and/or an antigen recognition domain that targets B-cell maturation antigens (BCMA).
  • the targeted cells are malignant plasma cells, such as, but not limited to, multiple myeloma.
  • a compound CAR engineered cell having at least one of CS1 and BCMA antigen recognition domain enhances functionality against multiple myeloma and offset antigen escape.
  • a CAR targets cells expressing multiple antigens including, but not limited to, CS1, BCMA, CD267, BAFF-R, CD38, CD138, CD52, CD19, TACI, CD20, interleukin 6 receptor, and NY-ESO-1 antigens.
  • the targeted cells are plasma cells, B-cells, malignant plasma cells such as, without limiting, multiple myeloma.
  • the compound CAR targets cells expressing multiple antigens including, but not limited to, CS1, BCMA, CD267, BAFF-R, CD38, CD138, CD52, CD19, TACI, CD20, interleukin 6 receptor, and NY-ESO-1 antigens.
  • the targeted cells are malignant plasma cells such as, without limiting, multiple myeloma.
  • the compound CAR targets cells expressing multiple antigens including but not limited to, alpha fetoprotein (AFP) and Glypican-3 (GPC3).
  • the targeting cells are hepatocellular carcinoma, fibrolamellar carcinoma, hepatoblastoma, undifferentiated embryonal sarcoma and mesenchymal hamartoma of liver, lung-squamous cell carcinoma, testicular nonseminomatous germ cell tumors, liposarcoma, ovarian and extragonadal yolk sac tumors, ovarian choriocarcinoma, teratomas, ovarian clear cell carcinoma, and placental site trophoblastic tumor.
  • AFP alpha fetoprotein
  • GPC3 Glypican-3
  • the present disclosure provides compound CAR engineered T cells or NK cells that target different or the same antigens offset tumor escape and provides simultaneous targeting of tumor cells.
  • T or NK host cells comprising compound CAR disclosed herein is embodied in the present disclosure.
  • the nucleotide and polypeptide constructs, sequences, host cells, and vectors of the compound CAR are considered to be part of the present disclosure and is embodied herein.
  • the compound CAR engineered cell is administrated in combination with any chemotherapy agents currently being developed or available in the market.
  • the compound CAR engineered cell is administrated as a first line treatment for diseases including, but not limited to, hematologic malignancies, cancers, non-hematologic malignances, inflammatory diseases, infectious diseases such as HIV and HTLV and others.
  • T cells expressing the compound CAR engineered cells are co-administrated with NK cells expressing the same or different compound CAR as an adaptive immunotherapy.
  • Compound CAR NK cells provide rapid, innate activity targeting cells while compound T cells provide relative long-lasting adaptive immune activity.
  • the compound CAR engineered cells are administrated as a bridge to bone marrow stem transplantation for mammals, e.g. patients who are resistant to chemotherapies and are not qualified for bone marrow stem cell transplantation.
  • the compound CAR co-expresses a transgene and releases a transgenic product, such as IL-12 in the targeted tumor lesion and further modulates the tumor microenvironment.
  • compound CAR engineered cells are administrated to a mammal for bone marrow myeloid ablation as a part of the treatment to a disease.
  • the cells expressing a compound CAR can be T cells or NK cells, administrated to a mammal, e.g. human.
  • the presented disclosure includes a method of treating a mammal having a disorder or disease by administration of a compound CAR.
  • the targeted cells may be cancer cells such as, or cells affected by any other disease condition, such as infectious diseases, inflammation, and autoimmune disorders.
  • the present disclosure is intended to include the use of fragments, mutants, or variants (e.g., modified forms) of the compound CAR or antigens that retain the ability to induce stimulation and proliferation of TINK cells.
  • a “form of the protein” is intended to mean a protein that shares a significant homology with at least one CAR or antigen and is capable of effecting stimulation and proliferation of T/NK cells.
  • biologically active or “biologically active form of the protein,” as used herein, are meant to include forms of the proteins or variants that are capable of effecting anti-tumor activity of the cells.
  • compositions and methods of this disclosure can be used to generate a population of TINK cells that deliver both primary and co-stimulatory signals for use in immunotherapy in the treatment of cancer, in particular the treatment of lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, rhabdornyosarcoma, leukemia and lymphoma.
  • the compositions and methods described in the present disclosure may be utilized in conjunction with other types of therapy for cancer, such as chemotherapy, surgery, radiation, gene therapy, and so forth.
  • CAR T/NK cell therapy Multiple extracellular cell markers are now being studied for value as tumor-associated antigens and thus potential targets for CAR T/NK cell therapy.
  • expression of these antigens on healthy tissue leading to on-target, off-tumor adverse events remains a major safety concern in addition to off-target toxicities.
  • a major limitation of CAR T/NK cell therapy is in the possibility of selecting for antigen escape variants when targeting molecules non-essential to tumorigenesis.
  • malignant cells that persist with little or no expression of the target antigens may evade CAR T/NK cells, despite their high-affinity action.
  • NK cells represent alternative cytotoxic effectors for CAR driven killing. Unlike T-cells, NK cells do not need pre-activation and constitutively exhibit cytolytic functions. Further expression of cCARs in NK cells allow NK cells to effectively kill cancers, particularly cancer cells that are resistant to NK cell treatment.
  • NK cells are known to mediate anti-cancer effects without the risk of inducing graft-versus-host disease (GvHD).
  • CD123+, CD34+CD38 ⁇ has been considered as LSCs as these cells are able to initiate and maintain the leukemic process in immunodeficient mice.
  • the number of CD34+/CD38 ⁇ /CD123+ LSCs can be used to predict the clinical outcome for AML patients.
  • the CD34+/CD38 ⁇ /CD123+ cells greater than 15% in AML patients, are associated with a lack of complete remission and unfavorable cytogenetic profiles.
  • the presence of more than 1% of CD34+/CD38 ⁇ /CD123+ cells could also have a negative impact on disease-free survival and overall survival.
  • LSCs leukemic stem cells
  • NK cells provide an off-the-shelf product that may be used as an allogeneic product for treatment.
  • cCAR cell therapy needs to be performed on a patient-specific basis as required by the current state of art.
  • the applicants of the present disclosure have discovered a novel immunotherapy, where the patient's lymphocytes or tumor infiltrated lymphocytes need not be isolated for an effective CAR cell based therapy.
  • NK cells Allogeneic or autologous NK cells are expected to induce a rapid immune response but disappear relatively rapidly from the circulation due to their limited lifespan. Thus, applicants surprisingly discovered that there is reduced concern of persisting side effects using cCAR cell based therapy.
  • NK cells can be expanded and transfected with cCAR in accordance to the present disclosure.
  • NK cells can be derived from cord blood, peripheral blood, iPS cells and embryonic stem cells.
  • NK-92 cells may be expanded and transfected with cCAR.
  • NK-92 is a continuously growing cell line that has features and characteristics of natural killer (NK) cells.
  • NK-92 cell line is IL-2 dependent and has been proven to be safe and feasible.
  • cCAR expressing NK-92 cells can be expanded in the serum free-medium with or without co-culturing with feeder cells. A pure population of NK-92 carrying the cCAR of interest may be obtained by sorting.
  • CD123 antigen is one of the targets for cCAR therapy.
  • CD123 the alpha chain of the interleukin 3 receptor
  • CD123 is overexpressed on a variety of hematologic malignancies, including acute myeloid leukemia (AML), B-cell acute lymphoblastic leukemia (B-ALL), hairy cell leukemia, and blastic plasmocytoid dendritic neoplasms.
  • AML acute myeloid leukemia
  • B-ALL B-cell acute lymphoblastic leukemia
  • hairy cell leukemia and blastic plasmocytoid dendritic neoplasms.
  • CD123 is absent or minimally expressed on normal hematopoietic stem cells. More importantly, CD123 is expressed on a subset of leukemic cells related to leukemic stem cells (LSCs), the ablation of which is essential in preventing disease refractoriness and relapse.
  • LSCs leukemic stem cells
  • CD33 antigen is one of the targets for cCAR therapy.
  • CD33 is a transmembrane receptor expressed on 90% of malignant cells in acute myeloid leukemia.
  • CD123 and CD33 target antigens are particularly attractive from a safety standpoint.
  • the compound CD33CD123 CARs may be highly effective for therapeutic treatment of chronic myeloid leukemia (CML) population.
  • CML chronic myeloid leukemia
  • CML chronic myeloid leukemia
  • LSCs lymphosar cells
  • TKI Bcr-Abl tyrosine kinase inhibitor
  • a novel therapy targeting CML resistant LSCs is urgently needed for treatment of CML and the novel therapy is embodied in the compound CD33CD123 CAR disclosed in the present disclosure.
  • CD123 expression is high in the CD34+CD38-population.
  • the compound CD33CD123 CARs is highly effective for therapeutic treatment of this population.
  • leukemic cells expressing both CD123 and CD33 in the cCAR is used as a therapeutic treatment.
  • CD33 is expressed on cells of myeloid lineage, myeloid leukemic blasts, and mature monocytes but not normal pluripotent hematopoietic stem cells. CD33 is widely expressed in leukemic cells in CML, myeloproliferative neoplasms, and MDS.
  • the present disclosure discloses a novel multiple cCAR T/NK cell construct comprising multiple CARs targeting multiple leukemia-associated antigens, thereby offsetting antigen escape mechanism, targeting leukemia cells, including leukemic stem cells, by synergistic effects of co-stimulatory domain activation, thereby providing a more potent, safe and effective therapy.
  • the present disclosure further discloses a compound CAR construct with enhanced potency of anti-tumor activity against cells co-expressing target antigens, and yet retains sensitivity to tumor cells only expressing one antigen.
  • each CAR of the compound CAR includes one or two co-stimulatory domains and exhibits potent killing capability in the presence of the specific target.
  • the compound CAR is a compound CD123CD19 CAR.
  • B-ALLs express CD123 in a subset of population. Like AML and MDS, it has been considered that a rare LSC population exists in B-ALL. Therefore, targeting both leukemic stem cell and bulky leukemic populations in accordance to the present disclosure, can be applied to B-ALLs.
  • CD123 and CD19 surface antigens expressed in the B-ALLs may be targets as CD19 is widely expressed in different stages of B-cell lymphoid populations, in accordance with the present disclosure.
  • MM Multiple myeloma
  • CAR targets include CD38, CS1, and B cell maturation Antigen (BCMA).
  • BCMA B cell maturation Antigen
  • heterogeneity of surface antigen expression commonly occurs in malignant plasma cells, which makes it a difficult target for CARs.
  • Malignant plasma cells also express low levels of CD19.
  • myeloma stem cells also express some B-cell markers including CD19. Targeting this population could be effective in the treatment of myeloma in conjunction with standard and other myeloma CAR therapies.
  • MM Multiple myeloma
  • myeloma is a haematological malignancy with a clonal expansion of plasma cells.
  • myeloma remains an incurable disease; thus novel therapeutic approaches are urgently needed.
  • CS1 (also called as CD319 or SLAMF7) is a protein encoded by the SLAMF7 gene.
  • the surface antigen CS1 is a robust marker for normal plasma cells and myeloma cells (malignant plasma cells).
  • Tumour necrosis factor receptor superfamily, member 17 also referred to as B-cell maturation antigen (BCMA) or CD269 is almost exclusively expressed at the terminal stages of plasma cells and malignant plasma cells. Its expression is absent other tissues, indicating the potential as a target for CAR T or NK cells.
  • BCMA B-cell maturation antigen
  • Malignant plasma cells display variable degrees of antigenic heterogeneity for CD269 and CS1.
  • a single CAR unit product targeting either CD269 or CS1 could target the majority of the cells in a bulk tumor resulting in an initial robust anti-tumor response. Subsequently residual rare non-targeted cells are expanded and cause a disease relapse. While multiple myeloma is particularly heterogeneous, this phenomena could certainty apply to other leukemias or tumors.
  • a recent clinical trial at NIH using BCMA CAR T cells showed a promising result with a complete response in some patients with multiple myeloma. However, these patients relapsed after 17 weeks, which may be due to the antigen escape. The antigen escape is also seen in CD19 CAR and NY-ESO1 CAR T cell treatments. Thus, there is an urgent need for more effective CAR T cell treatment in order to prevent the relapse.
  • BCMA and CS1 are the targets for BCMACS1 CAR therapy.
  • a compound CAR targets cells expressing BCMA or CS1 antigens or both.
  • the targeted cells may be cancer cells, such as, without limiting, lymphomas, or leukemias or plasma cell neoplasms.
  • plasma cell neoplasms is selected from plasma cell leukemia, multiple myeloma, plasmacytoma, heavy chain diseases, amyloidosis, waldestrom's macroglobulinema, heavy chain diseases, solitary bone plasmacytoma, monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma.
  • MGUS monoclonal gammopathy of undetermined significance
  • the present disclosure provides a compound CAR polypeptide engineered cell that targets cells expressing BCMA or CD19 antigens or both.
  • the targeted cells may be cancer cells, such as, but not limited to, lymphomas, or leukemias or plasma cell neoplasms.
  • plasma cell neoplasms are selected from plasma cell leukemia, multiple myeloma, plasmacytoma, heavy chain diseases, amyloidosis, waldestrom's macroglobulinema, heavy chain diseases, solitary bone plamacytoma, monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma.
  • MGUS monoclonal gammopathy of undetermined significance
  • BAFF B-cell-activation factor
  • APRIL a proliferation-induced ligand
  • BAFF B-cell-activation factor
  • APRIL a proliferation-induced ligand
  • BAFF-R TNF homologs that bind specifically TACI (also called as TNFRSF1 3B or CD267) and BCMA with high affinity.
  • BAFF also known as BLyS
  • BAFF-R binds BAFF-R and functionally involves in the enhancement of survival and proliferation of later stage of B cells.
  • BAFF has been shown to involve some autoimmune disorders.
  • APRIL plays an important role in the enhancement of antibody class switching. Both BAFF and APRIL have been implicated as growth and survival factors for malignant plasma cells.
  • the present disclosure provides a compound CAR engineered cell that targets cells expressing TACI or CS1 antigens or both.
  • the targeted cells may be cancer cells, such as, without limiting, lymphomas, or leukemias or plasma cell neoplasms.
  • plasma cell neoplasms is selected from plasma cell leukemia, multiple myeloma, plasmacytoma, heavy chain diseases, amyloidosis, waldestrom's macroglobulinema, heavy chain diseases, solitary bone plamacytoma, monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma.
  • the target cells may also be one or two or multiple different cell types of B cells, immature B cells, na ⁇ ve B cells, centroblasts, centrocytes, memory B cells, plasmablasts, long lived plasma cells, plasma cells.
  • autoimmune diseases include systemic scleroderma, multiple sclerosis, psoriasis, dermatitis, inflammatory bowel diseases (such as Crohn's disease and ulcerative colitis), systemic lupus erythematosus, vasculitis, rheumatoid arthritis, Sjorgen's syndrome, polymyositis, granulomatosis and vasculitis, Addison's disease, antigen-antibody complex mediated diseases, and anti-glomerular basement membrane disease.
  • autoimmune diseases include systemic scleroderma, multiple sclerosis, psoriasis, dermatitis, inflammatory bowel diseases (such as Crohn's disease and ulcerative colitis), systemic lupus erythematosus, vasculitis, rheumatoid arthritis, Sjorgen's syndrome, polymyositis, granulomatosis and vasculitis, Addison's disease, antigen-antibody complex mediated diseases
  • the present disclosure provides a compound CAR engineered cell that targets cells expressing BAFF-R or CS1 antigens or both.
  • a compound CAR engineered cell that targets cells expressing BAFF-R or CS1 antigens or both may be cancer cells, such as, without limiting, lymphomas, or leukemias or plasma cell neoplasms.
  • plasma cell neoplasms is selected from plasma cell leukemia, multiple myeloma, plasmacytoma, heavy chain diseases, amyloidosis, waldestrom's macroglobulinema, heavy chain diseases, solitary bone plamacytoma, monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma.
  • B and plasma cells play a critical role in the pathogenesis of autoimmune disorders as they are the sources of autoantibody production. B and plasma cells may contribute to disease progression and relapse through antigen presentation, cytokine secretion, or antibody production. Deletion of B cells or plasma cells or both using CAR T/NK cell approaches can be a beneficial therapy.
  • An organ transplant represents a new life for a person and organs that can be transplanted could include the kidneys, heart, lungs, pancreas and intestine.
  • organs that can be transplanted could include the kidneys, heart, lungs, pancreas and intestine.
  • HLA human leukocyte antigens
  • compositions and methods relating to CAR engineered cells that target cells expressing CD19 or CD20 that result in the deletion of B cells but spare long-lived plasma cells in patients with antibody mediated organ rejection or autoimmune disorders including, but not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and pemphigus vulgaris and multiple sclerosis (MS).
  • SLE systemic lupus erythematosus
  • RA rheumatoid arthritis
  • MS pemphigus vulgaris and multiple sclerosis
  • the present disclosure also provides compositions and methods relating to CAR engineered cells that target cells expressing BCMA or BAFF-R, TACI which results in the deletion of plasma cells in patients with antibody mediated organ rejection or autoimmune disorders including, but not limited to, systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and pemphigus vulgaris and multiple sclerosis (MS).
  • SLE systemic lupus erythematosus
  • RA rheumatoid arthritis
  • MS pemphigus vulgaris and multiple sclerosis
  • compositions and methods relating to CAR engineered cells for CARs depleting mature, memory B cells, and short, long lived plasma cells for treatment of autoimmune disorders and organ antibody-mediated organ rejection In one embodiment, the present disclosure provides a method for depleting mature, memory B cells, and short, long lived plasma cells using one or more of the following strategies:
  • a compound CAR targets cells expressing one or two or all of BAFF-R, BCMA, TACI and CS1 antigens.
  • a unit of CAR in a cCAR can comprise: 1) a scFv against either BAFF-R, BCMA, TACI and CS1; 2) a hinge region; 3) co-stimulatory domain (s) and intracellular signaling domain.
  • BAFF CAR can be a unit of CAR in a cCAR comprises: 1) BCMA or TACI or BAFF-R binding domain; 2) a hinge region; 3) co-stimulatory domain (s) and intracellular signaling domain.
  • APRIL CAR can be a unit of CAR in a cCAR comprises: 1) BCMA or TACI binding domain; 2) a hinge region; 3) co-stimulatory domain (s) and intracellular signaling domain.
  • BCMA or TAC1 or BAFF-R binding domain can be a part of or entire APRIL and BAFF molecules.
  • a unit of CAR in a cCAR can comprise: 1) a scFv against BCMA or CS1; 2) a hinge region; 3) co-stimulatory domain (s) and intracellular signaling domain.
  • a unit of CAR in a cCAR can comprise: 1) a scFv against BCMA or CD19; 2) a hinge region; 3) co-stimulatory domain (s) and intracellular signaling domain.
  • a unit of CAR in a cCAR can comprise: 1) a scFv against BCMA or CD20; 2) a hinge region; 3) co-stimulatory domain (s) and intracellular signaling domain.
  • a unit of CAR in a cCAR can comprise: 1) BAFF-R binding domain or a scFv against BCMA; 2) a hinge region; 3) co-stimulatory domain (s) and intracellular signaling domain.
  • a unit of CAR in a cCAR can comprise: 1) BAFF-R binding domain or a scFv against CD19; 2) a hinge region; 3) co-stimulatory domain (s) and intracellular signaling domain.
  • a unit of CAR in a cCAR can comprise: 1) BAFF-R binding domain or a scFv against CD20; 2) a hinge region; 3) co-stimulatory domain (s) and intracellular signaling domain.
  • TACI, BCMA and BAFF-R are receptors for BAFF. It is also unexpected that some myeloma cells express TACI or BAFF-R over BCMA.
  • the disclosure provides a method of comprising a BAFF CAR targeting a cell expressing at least one of receptors, BAFF-R, TACI and BCMA to improve therapeutic efficacy and reduce the risk of antigen disease escape.
  • the affinity for BAFF binding to BCMA is within the micromolar range, which is much lower than that of BAFF-R or TACI in the nanomolar range.
  • the disclosure provides a method of generating a compound cCAR comprising BAFF and BCMA CARs to complement some of myeloma cells that cannot be eliminated by a BAFF CAR.
  • cCAR can comprise one or two or multiple units of CAR. Each unit CAR could bear same or different hinge region and co-stimulatory domain.
  • cCAR polypeptide in further embodiments, cCAR polypeptides two or more CAR polypeptide units. Each unit CAR could bear a different polynucleotide sequence to avoid a homologous recombination.
  • pooled CAR engineered cells which are generated by at least two separate CAR T or NK cells.
  • pooled CAR engineered cells include a population of engineered cells having more than one distinct CAR polypeptide unit.
  • pooled engineered cells include a population of engineered cells with a distinct CAR polypeptide and a population of engineered cells with a different and distinct CAR polypeptide.
  • the pooled CAR engineered cells include engineered cells having cCAR polypeptides.
  • the pooled CAR T or NK cells can be completed by the following steps:
  • the different engineered cells may be individual expanded and independently or sequentially administered.
  • the target antigens can include at least one of this group, but not limited to, ROR1, PSMA, MAGE A3, Glycolipid, 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, immunoglobin kappa and lambda, CD38, CD52, CD3, CD4, CD8, CD5, CD7, CD2, and CD138.
  • the target antigens can also include viral or fungal antigens, such as E6 and E7 from the human papillomavirus (HPV) or EBV (Epstein Barr virus) antigens.
  • a cCAR engineered cell targets a cell expressing either CD19 or CD20 antigens or both of them.
  • a cCAR engineered cells target a cell expressing either CD19 or CD22 antigens or both of them.
  • the targeted cells are normal B cells associated with autoimmune disorders or cancer cells such as B-cell lymphomas or leukemias.
  • Acute graft-versus-host disease remains the most important cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation.
  • TCR T cell receptor
  • TCR T cell receptor
  • a heterodimer of alpha and beta chains is expressed on the surface of T cells, TCR recognizes some antigens on the HLA molecule on host cells, enhances T cell proliferation, and releases cytotoxic agents that cause the damage on host cells.
  • TCR gene inactivation is efficient at preventing potential graft-versus-host reaction. The inactivation of TCRs can result in the prevention of the TCR recognition of alloantigen and thus GVHD.
  • the role of CD45 on NK cells is quite different from that of T cells.
  • NK cells from CD45-defficient mice have normal cytotoxic activity against the prototypic tumor cell line, Yac-1.
  • CD45-deficient NK cells proliferate normally and respond to IL-15 and IL-21. Therefore, CD45 disruption or deletion would not affect the NK cell killing and proliferation.
  • the present disclosure includes methods of permanent deletion of CD45 in a T or NK cell with subsequent stable introduction of CD45-specific CARs.
  • the engineered T cells display the desired properties of redirected specificity for CD45 without causing self-killing and response to presentation of antigen.
  • the engineered T cells may have efficacy as an off-the-shelf therapy for treating malignancies or other diseases.
  • the present disclosure relates to a method where T-cells are engineered to allow proliferation when TCR signaling is reduced or lost through the inactivation or deletion of endogenous CD45.
  • T cells reducing or losing the TCR signaling by the inactivation of CD45 could be used as an “off the shelf” therapeutic product.
  • the present disclosure includes methods of modified T or NK cells, which comprises: (a) modifying T or NK cells by inactivating CD45; (b) expanding these modified cells; (c) sorting modified T or NK cells, which do not express CD45; (d) introducing CD45CAR.
  • the CD45CAR gene encodes a chimeric antigen receptor (CAR), wherein the CAR comprises at least one of an antigen recognition domain, a hinge region, a transmembrane domain, and T cell activation domains, and the antigen recognition domain is redirected against CD45 surface antigen present on a cell.
  • the antigen recognition domain includes a monoclonal antibody or a polyclonal antibody directed against CD45 antigen.
  • the antigen recognition domain includes the binding portion or a variable region of a monoclonal or a polyclonal antibody.
  • engineered CD45CAR T/NK cells comprise secretory IL-15/IL-15sushi complexes, which can promote expansion of specific CAR T/NK cells, and promote infiltrate of innate immune cells to the tumor sites resulting in tumor destruction.
  • the present disclosure provides an alternative strategy in which engineered CD45 CAR T cells receive not only costimulation through the CD28 pathway but also co-express the 4-1BB ligand (CD137L), which provide high therapeutic efficacy.
  • the modified T cells are obtained from allogeneic donors and used as an “off-the-shelf product”.
  • Targeting CD45 using CAR T or NK cells may cause self-killing as T and NK cells express this surface antigen.
  • the present disclosure provides engineered cells that are deficient in CD45.
  • an engineered cell is deficient for a particular gene when expression of the gene is reduced or eliminated. Reduction or elimination of expression can be accomplished by any genetic method known in the art.
  • the CD45 gene may be inactivated by using engineered CRISPR/Cas9 system, zinc finger nuclease (ZFNs) and TALE nucleases (TALENs) and meganucleases. The loss of CD45 in T or NK cells is further transduced with CARs targeting neoplasms expressing CD45.
  • malignant cells expressing CD45 are present in patients with acute leukemia, chronic leukemia T cell lymphomas, 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, dendrocytoma, histiocytic sarcoma, Giant cell tumor of tendon sheath, interdigitating dendritic cell sarcoma, post-transplant lymphoproliferative disorders, etc.
  • Hematopoietic stem cell transplantation has been widely used for the treatment of hematologic malignancies or non-hematologic diseases.
  • Non-hematologic diseases include genetic disorders and immunodeficiencies and autoimmune disorders.
  • Genetic disorders include, not limited to, sickle cell disease, thalassemia and lysosomal storage diseases.
  • patients are required to undergo a conditional therapy to deplete hematopoietic stem/progenitor cells in the bone marrow niches to promote the donor stem cell engraftment and proliferation.
  • conditional therapies which cause severe toxicity and long-term complications particularly in non-hematopoietic organs such as severe intestinal mucositis.
  • conventional conditional therapies could destruct bone marrow niches resulting hematopoietic cell recovery.
  • the safety concerns represent a major obstacle in effort to expand HSCT to a variety of non-hematologic diseases.
  • CD45 is expressed only on hematopoietic cells and targeting CD45 should minimize the toxicity to non-hematopoietic organs.
  • CD45CAR cells are used to make space in the bone marrow for bone marrow stem cell transplant by removing hematopoietic cells, at the same time removing leukemic/lymphoma cells or immunologic cells capable of graft rejection.
  • CD45CAR engineered cells are used to deplete hematopoietic stem/progenitor cells while the architecture and vasculature of the bone marrow are preserved, in contrast to the disruptive effects of total body irradiation on these tissues.
  • Preservation of the architecture and vasculature of the bone marrow allows faster hematopoietic recovery after transient CD45CAR treatment.
  • CD45CAR cells may be used for pre-treatment of patients before their undergoing a bone marrow transplant to receive stem cells.
  • CD45CAR can be used as myeloblative conditioning regimens for hematopoietic stem cell transplantation.
  • CD45CAR engineered cell therapy is transient for allowing faster recovery of bone marrow and peripheral hematopoietic cells.
  • Transient therapy may be accomplished by using short lived engineered cells or providing an engineered cell having the suicide system as described herein.
  • the present disclosure comprises a method of selectively depleting or ablating an endogenous hematopoietic stem/progenitor population, where the endogenous hematopoietic stem/progenitor cells expressing CD45, by contacting said cells with CD45CAR engineered cell that specifically targets CD45 expressing hematopoietic stem/progenitor cells.
  • CD45CAR cells are utilized for treating or preventing a residual disease after stem cell transplant and/or chemotherapy.
  • the CD45CAR is part of an expressing gene or a cassette.
  • the expressing gene or the cassette includes an accessory gene or a tag or a part thereof, in addition to the CD45CAR.
  • the accessory gene may be an inducible suicide gene or a part thereof, including, but not limited to, caspase 9 gene, thymidine kinase, cytosine deaminase (CD) or cytochrome P450.
  • the “suicide gene” ablation approach improves safety of the gene therapy and kills cells only when activated by a specific compound or a molecule.
  • the suicide gene is inducible and is activated using a specific chemical inducer of dimerization (CID).
  • the safety switch can include the accessory tags are a c-myc tag, CD20, CD52 (Campath), truncated EGFR gene (EGFRt) or a part or a combination thereof.
  • the accessory tag may be used as a nonimmunogenic selection tool or for tracking markers.
  • safety switch can include a 24-residue peptide that corresponds to residues 254-277 of the RSV F glycoprotein A2 strain (NSELLSLINDMPITNDQKKLMSNN).
  • a safety switch can include the amino acid sequence of TNF a bound by monoclonal anti-TNF a drugs.
  • Interleukin (IL)-15 is a cytokine that promotes the development and hemostasis of lymphocytes. Increased levels of IL-15 promote T-cell proliferation and enhance T cell effector response. Data from recent studies have shown that IL-15 is crucial for the generation and maintenance of memory CD8 T-cells, one of the key factors associated with anti-tumor activity. IL-15 binds the IL-15 receptor alpha chain (also called IL-15RA or RA) contributing to IL-15-mediated effects such as T-cell survival, proliferation and memory T cell generation.
  • IL-15 receptor alpha chain also called IL-15RA or RA
  • IL-15RA binds the ⁇ y complex in the surface of T cells and IL-15 signals by binding with this IL-15RA/ ⁇ y complex on the cell surface of T cells and other types of cells.
  • Transfection of IL-15 alone does not significantly influence T-cell function, transfection of IL-15/IL-15RA allows T cells to survive and proliferate autonomously.
  • 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 greatly enhanced II-15 half-life and bioavailability in vivo. Therefore, treatment of mice with this complex, but not with IL-15 alone results in robust proliferation and maintenance of memory CD8 T cells and NK cells.
  • a portion of the extracellular region of IL-15RA called sushi domain (IL-15sushi) is required for its binding of IL-15 (WEI et al., J. Immunol., vol. 167(1), p:277-282, 2001).
  • the IL-15/sushi fusion protein is also called IL-15/IL-15sushi containing the linker is more potent than IL-15 and soluble IL-15RA (IL-15sushi) alone.
  • the combination of IL-15/RA (membrane bound form) or IL-15/sushi (soluble form) can maximize IL-15 activity.
  • the membrane bound form, IL-15/RA would not release of free IL-15 by keeping IL-15 bound to IL-15RA on the surface of transduced cells.
  • the soluble IL-15/IL-15sushi released from transduced cells are able to promote the expansion of transduced cells and their neighbor cells, and further stimulate them against tumor.
  • the present disclosure provides an engineered cell having both CAR and IL-15/IL-15sushi or IL-15/RA in a single construct.
  • the disclosure includes methods to generate higher virus titer and use a stronger promoter to drive both CAR and IL-15/RA or IL-15/IL-15sushi.
  • the present disclosure provides an engineered cell having: (1) a CAR targeting an antigen including, but not limited to, CD4, CD2, CD3, CD7, CD5, CD45, CD20, CD19, CD33, CD123, CS1, and B-cell mature antigen (BCMA); and (2) IL-15; (3) IL-15RA (membrane bound) or sushi (IL-15/IL-15sushi.
  • a CAR targeting an antigen including, but not limited to, CD4, CD2, CD3, CD7, CD5, CD45, CD20, CD19, CD33, CD123, CS1, and B-cell mature antigen (BCMA); and (2) IL-15; (3) IL-15RA (membrane bound) or sushi (IL-15/IL-15sushi.
  • CAR comprises chimeric antigen receptor, one or more of co-stimulatory endodomains including, but not limited to, CD28, CD2, 4-1BB, 4-1BBL (CD137L), B7-2/CD86, CTLA-4, B7-H1/PD-L1, ICOS, B7-H2, PD-1, B7-H3, PD-L2, B7-H4, CD40 Ligand/TNFSF5, DPPIV/CD26, DAP12 and OX40, and intracellular domain of CD3 zeta chain.
  • a strong promoter can be, but is not limited to, SFFV.
  • CARs, IL-15/RA or sushi and inducible suicide gene (“safety switch”), or a combination can be assembled on a vector, such as a lentiviral vector, adenoviral vector and retroviral vector or a plasmid.
  • a vector such as a lentiviral vector, adenoviral vector and retroviral vector or a plasmid.
  • safety switch could significantly increase safety profile, and limit on-target or off-tumor toxicities of CARs.
  • the engineered cell includes a CD2 chimeric antigen receptor polypeptide and IL-15/IL-15sushi (SEQ ID NO. 102), and corresponding polynucleotide (SEQ ID NO. 101).
  • a CD2 chimeric antigen receptor polypeptide and IL-15/IL-15sushi SEQ ID NO. 102
  • corresponding polynucleotide SEQ ID NO. 101
  • the engineered cell includes a CD3 chimeric antigen receptor polypeptide and IL-15/IL-15sushi (SEQ ID NO. 104), and corresponding polynucleotide (SEQ ID NO. 103).
  • a CD3 chimeric antigen receptor polypeptide and IL-15/IL-15sushi SEQ ID NO. 104
  • corresponding polynucleotide SEQ ID NO. 103
  • the engineered cell includes a CD7 chimeric antigen receptor polypeptide and IL-15/IL-15sushi (SEQ ID NO. 106), and corresponding polynucleotide (SEQ ID NO. 105).
  • the present disclosure provides a method of providing long-term durable remission in a cancer patient by administering a CD7 CAR engineered cell that co-expresses IL-15/IL-15sushi to a patient in need thereof.
  • co-expression of IL-15/IL-15sushi with a CD7 CAR provides long-term durable remissions in patients by increasing the sensitivity of CAR recognition of target cancer cells or recruiting innate immune cells to cancer cells.
  • the engineered cell includes a CD5 chimeric antigen receptor polypeptide and IL-15/IL-15sushi (SEQ ID NO. 107), and corresponding polynucleotide (SEQ ID NO. 108).
  • the present disclosure provides a method of providing long-term durable remission in a cancer patient by administering a CD5 CAR engineered cell that co-expresses IL-15/IL-15sushi to a patient in need thereof.
  • co-expression of IL-15/IL-15sushi with a CD5 CAR provides long-term durable remissions in patients by increasing the sensitivity of CAR recognition of target cancer cells or recruiting innate immune cells to cancer cells.
  • Tregs Regulatory T cells
  • Tregs are a sub-population of T cells which regulate the immune system and maintain tolerance of self-antigens.
  • Tregs constitute about 1-5% of total CD4+ T cells in blood with diverse clinical applications in transplantation, allergy, asthma, infectious diseases, graft versus host disease (GVHD), and autoimmunity.
  • the biomarkers for Tregs are CD4, Foxp3 and CD25. Tregs are considered to be derived from Na ⁇ ve CD4 cells.
  • Tregs play an important role in suppressing tumor immunity and hindering the body's innate ability to control the growth of cancerous cells.
  • Tregs expand in patients with cancer and are often enriched in the tumor microenvironment. Tregs cab infiltrate tumors and limit antitumor immunity as well. Depletion of Treg can render mice capable of rejecting tumors that normally grow progressively.
  • Tregs are very difficult to effectively discern from effector T cells and NK cells, making them difficult to study.
  • the engineered cell includes a first chimeric antigen receptor polypeptide having a CD4 antigen recognition domain and second chimeric antigen receptor polypeptide having a CD25 antigen recognition domain.
  • this engineered cell includes a polypeptide of SEQ ID NO. 92 with a CD45 leader sequence and corresponding polynucleotide of SEQ ID NO. 91.
  • the engineered cell includes a first chimeric antigen receptor polypeptide having a CD4 antigen recognition domain and second chimeric antigen receptor polypeptide having a CD25 antigen recognition domain.
  • this engineered cell includes a polypeptide of SEQ ID NO. 94 with a CD8a leader sequence and corresponding polynucleotide of SEQ ID NO. 93.
  • T lymphocytes are a subtype of white blood cells that play a key role in cell-mediated immunity. T cells are divided into CD4 and CD8 cells. Natural killer cells (NK cells) are a type of cytotoxic cells critical to the innate immunity.
  • Tregs are a type of CD4+ cells mediating immune tolerance and suppression and are distinguished from helper T cells. Tregs express CD4, CD25 and Foxp3 (CD4CD25+ regulatory T cells).
  • Tregs are enriched in the tumor microenvironment and considered to be important for hindering antitumor immune responses and promoting cancer cell tolerance. Increased numbers of infiltrating Tregs in tumors have been associated poor survival in a variety of cancers including hematologic malignancies and solid tumors.
  • Tregs appear to be preferentially trafficked to the tumor microenvironment and play a critical role of immunosuppression (Ethan M. Shevach et al, Annual Review of Immunology, Vol. 18: 423-449, 2000).
  • Tregs for cancer treatments by targeting CD25, resulting in a partial reduction of Tregs.
  • CD25 is also expressed in activated CD4, CD8 and NK cells.
  • CD25 expression can be seen in activated B cells, macrophages, osteoblasts, some thymocytes, some myeloid precursors and some oligodendocytes.
  • a very high-level of Treg depletion is required for efficacy (Xingrui Li et al, Eur. J. Immunol. 2010. 40: 3325-3335).
  • the CAR design is to redirect patient or donor immune cells against a specific “target” antigen in a major-histocompatibility complex (MHC)—independent manner.
  • the CAR protein construct usually includes a number of modular components or regions integral to function.
  • the module for “target” recognition is the extracellular single-chain variable fragment (scFv). This component is derived from a monoclonal antibody with specific direction against a carefully selected target antigen.
  • a hinge region of appropriate length tandem to the scFv conveys mobility of the scFv region to allow for optimal binding to the target protein.
  • the transmembrane domain region serves as a conduit between the extracellular binding regions and co-activation domains' such as CD28 and/or 4-1BB.
  • the final module includes the CD3 zeta intracellular signaling domain.
  • the present disclosure provides a method for a novel Treg CAR (also called CD4zetaCD25CAR or C4-25z CAR) construct targeting a cell co-expressing CD4 and CD25.
  • the Treg CAR depletes Tregs specifically while sparing most of cells that do not co-express CD4 and CD25.
  • T cell activation could be achieved upon simultaneous engagement of two scFv molecules against CD4 and CD25 in a Treg CAR.
  • both T cell activation and co-stimulation signals are provided using two distinct/separate chimeric antigen receptor polypeptides.
  • a TregCAR includes (1) a first chimeric antigen receptor polypeptide unit comprising a first signal peptide, a first antigen recognition domain, a first hinge region, a first transmembrane domain, and an intracellular signaling domain; and (2) a second chimeric antigen receptor polypeptide unit comprising a second signal peptide, a second antigen recognition domain, a second hinge region, a second transmembrane domain, and a co-stimulatory domain (s).
  • Both the first chimeric antigen receptor polypeptide unit and the second chimeric engineered polypeptide unit are expressed on a single polypeptide molecule, and wherein an amino acid sequence comprising a high efficiency cleavage site is disposed between the first chimeric antigen receptor polypeptide unit and the second chimeric antigen receptor polypeptide unit.
  • the Treg CAR potentiates the lysis activity of a cell co-expressing CD4 and CD25 while minimizing a cell bearing only CD4 or CD25 antigen.
  • the nucleotide sequence of the first chimeric antigen receptor polypeptide unit is different from the second chimeric engineered polypeptide unit in order to avoid a homologous recombination
  • the high efficiency cleavage site in Treg CAR is P2A.
  • the target of the first antigen recognition domain is either CD4 or CD25 and the target of the second antigen recognition domain is either CD4 or CD25; wherein the first antigen recognition domain is different than the second antigen recognition domain.
  • the antigen recognition domain includes the binding portion or variable region of a monoclonal or polyclonal antibody directed against (selective for) the target.
  • the target antigen is CD4 or CD25.
  • the T or NK cell comprising Treg CARs targeting different or same antigens.
  • the T or NK cell comprises Treg CARs targeting Tregs expressing CD4 and CD25 while sparing most of cells, which do not co-express CD4 and CD25.
  • the T or NK cell comprises Treg CARs depleting Tegs.
  • the present disclosure provides a method of generation of Treg CAR useful for treating or preventing a CD4+CD25+ Foxp3+ T regulatory cell (Treg) related disease in a subject is provided.
  • the diseases treated with Treg CAR include, but not limiting to, cancers.
  • the present disclosure provides a method of generation of Treg CAR useful for treating or preventing a CD4+CD25+ Foxp3+ T regulatory cell (Treg) related Cancers including, but not limited, hepatocellular carcinoma, fibrolamellar carcinoma, hepatoblastoma, undifferentiated embryonal sarcoma and mesenchymal hamartoma of liver, lung-squamous cell carcinoma, testicular nonseminomatous germ cell tumors, liposarcoma, ovarian and extragonadal yolk sac tumors, ovarian choriocarcinoma, teratomas, ovarian clear cell carcinoma, placental site trophoblastic tumor, lymphoma and leukemia.
  • Treg T regulatory cell
  • the present disclosure provides a method of generation of Treg CAR useful for inhibiting the growth of a tumor in a subject is provided.
  • the Treg CAR is administrated in combination with any chemotherapy agents currently being developed or available in the market. In some embodiments, the Treg CAR is administrated as a first line treatment for diseases including, but not limited to, hematologic malignancies and cancers.
  • the cells expressing a Treg CAR are co-administrated with immunomodulatory drugs, such as, but not limited to, CTLA-4 and PD-1/PD-L1 blockades, or cytokines, such as IL-2, IL-15 or IL-15/IL-15sushi or IL-15/RA, and IL-12 or inhibitors of colony stimulating factor-1 receptor (CSF1R) for better therapeutic outcomes.
  • immunomodulatory drugs such as, but not limited to, CTLA-4 and PD-1/PD-L1 blockades, or cytokines, such as IL-2, IL-15 or IL-15/IL-15sushi or IL-15/RA, and IL-12 or inhibitors of colony stimulating factor-1 receptor (CSF1R) for better therapeutic outcomes.
  • the cells expressing a Treg CAR are co-administrated with other immunomodulatory drugs or CAR-expressing cells to provide synergistic effects in a subject.
  • the cells expressing a Treg CAR can be T cells or NK cells, administrated to a mammal, e.g. human.
  • the Treg CAR is used in immunotherapy in the treatment of cancers.
  • the cancers may be selected from the group consisting of lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, cervical cancer, head or neck cancer, stomach cancer, liver cancer, neuroblastoma, rhabdomyosarcoma, leukemia and lymphoma.
  • the compositions and methods described in the present disclosure may be utilized in conjunction with other types of therapy for cancer, such as chemotherapy, surgery, radiation, gene therapy, and so forth.
  • the Treg CAR of the present disclosure may be constructed as a transient RNA-modified “biodegradable” version or derivatives, or a combination thereof.
  • the RNA-modified CARs of the present disclosure may be electroporated into T cells or NK cells.
  • the expression of the Treg CAR may be gradually diminished over few days.
  • a method for treating cancers using Treg CAR in a subject is embodied in the present disclosure.
  • the method comprises:
  • T/NK cells from a subject or donor(s).
  • Culturing the lymphocytes/T cells or NK cells (3) Introducing an Treg CAR construct into the cultured T cells or NK cells.
  • Expanding Treg CAR T cells or NK cells Treating the subject by administering a therapeutically effective amount thereto.
  • TILs tumor-infiltrating lymphocytes
  • TILs can be obtained from a tumor tissue sample and expanding the number of TILs.
  • Treg CAR T or NK cells were used to co-culture with TILs to deplete Treg population to enhance TIL responses to cancers, which is valuable to the disease therapies.
  • CD4CAR bear a set of CAR body including an antigen recognition domain, a hinge region, a co-stimulatory domain (s) and an intracellular domain (CD3 zeta chain).
  • CD4CAR depletes Tregs, and then enhances T-cell responses to cancer cells and improves therapeutic outcomes of anti-tumor activity.
  • CD25CAR bear a set of CAR body including an antigen recognition domain, a hinge region, a co-stimulatory domain (s) and an intracellular domain (CD3 zeta chain).
  • CD25CAR depletes Tregs, and then enhances T-cell responses to cancer cells and improves therapeutic outcomes of anti-tumor activity.
  • CAR enhancing agents include immunomodulatory drugs that enhance CAR activities, such as, but not limited to agents that target immune-checkpoint pathways, inhibitors of colony stimulating factor-1 receptor (CSFIR) for better therapeutic outcomes.
  • Agents that target immune-checkpoint pathways include small molecules, proteins, or antibodies that bind inhibitory immune receptors CTLA-4, PD-1, and PD-L1, and result in CTLA-4 and PD-1/PD-L1 blockades.
  • enhancing agent includes enhancer as described above.
  • patient includes mammals.
  • the mammal referred to herein can be any mammal.
  • the term “mammal” refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits.
  • the mammals may be from the order Carnivora, including Felines (cats) and Canines (dogs).
  • the mammals may be from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses).
  • the mammals may be of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes).
  • the mammal is a human.
  • a patient includes subject.
  • the patient is a human 0 to 6 months old, 6 to 12 months old, I to 5 years old, 5 to 10 years old, 5 to 12 years old, 10 to 15 years old, 15 to 20 years old, 13 to 19 years old, 20 to 25 years old, 25 to 30 years old, 20 to 65 years old, 30 to 35 years old, 35 to 40 years old, 40 to 45 years old, 45 to 50 years old, 50 to 55 years old, 55 to 60 years old, 60 to 65 years old, 65 to 70 years old, 70 to 75 years old, 75 to 80 years old, 80 to 85 years old, 85 to 90 years old, 90 to 95 years old or 95 to 100 years old.
  • an effective amount and “therapeutically effective amount” of an engineered cell as used herein mean a sufficient amount of the engineered cell to provide the desired therapeutic or physiological or effect or outcome. Such, an effect or outcome includes reduction or amelioration of the symptoms of cellular disease. Undesirable effects, e.g. side effects, are sometimes manifested along with the desired therapeutic effect; hence, a practitioner balances the potential benefits against the potential risks in determining what an appropriate “effective amount” is.
  • the exact amount required will vary from patient to patient, depending on the species, age and general condition of the patient, mode of administration and the like. Thus, it may not be possible to specify an exact “effective amount”. However, an appropriate “effective amount” in any individual case may be determined by one of ordinary skill in the art using only routine experimentation. Generally, the engineered cell or engineered cells is/are given in an amount and under conditions sufficient to reduce proliferation of target cells.
  • the efficacy of the therapeutic engineered cell can be assessed in various ways well known to the skilled practitioner. For instance, one of ordinary skill in the art will understand that a therapeutic engineered cell delivered in conjunction with the chemo-adjuvant is efficacious in treating or inhibiting a cancer in a patient by observing that the therapeutic engineered cell reduces the cancer cell load or prevents a further increase in cancer cell load.
  • Cancer cell loads can be measured by methods that are known in the art, for example, using polymerase chain reaction assays to detect the presence of certain cancer cell nucleic acids or identification of certain cancer cell markers in the blood using, for example, an antibody assay to detect the presence of the markers in a sample (e.g., but not limited to, blood) from a subject or patient, or by measuring the level of circulating cancer cell antibody levels in the patient.
  • polymerase chain reaction assays to detect the presence of certain cancer cell nucleic acids or identification of certain cancer cell markers in the blood using, for example, an antibody assay to detect the presence of the markers in a sample (e.g., but not limited to, blood) from a subject or patient, or by measuring the level of circulating cancer cell antibody levels in the patient.
  • the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” or any other variation thereof, are intended to cover a non-exclusive inclusion.
  • a process, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such process, article, or apparatus.
  • “or” refers to an inclusive “or” and not to an exclusive “or”. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).
  • any examples or illustrations given herein are not to be regarded in any way as restrictions on, limits to, or express definitions of any term or terms with which they are utilized. Instead, these examples or illustrations are to be regarded as being described with respect to one particular embodiment and as being illustrative only. Those of ordinary skill in the art will appreciate that any term or terms with which these examples or illustrations are utilized will encompass other embodiments which may or may not be given therewith or elsewhere in the specification and all such embodiments are intended to be included within the scope of that term or terms. Language designating such nonlimiting examples and illustrations includes, but is not limited to: “for example,” “for instance,” “e.g.,” and “in one embodiment.”
  • each member may be combined with any one or more of the other members to make additional sub-groups.
  • additional sub-groups specifically contemplated include any one, two, three, or four of the members, e.g., a and c; a, d, and e; b, c, d, and e; etc.
  • a XXXX antigen recognition domain is a polypeptide that is selective for XXXX.
  • XXXX denotes the target as discussed herein and above.
  • a CD38 antigen recognition domain is a polypeptide that is specific for CD38.
  • CDXCAR refers to a chimeric antigen receptor having a CDX antigen recognition domain.
  • a CAR engineered cell is an engineered cell as described herein that includes a chimeric antigen receptor polypeptide.
  • a CD45 engineered cell is an engineered cell having a CD45 chimeric antigen receptor polypeptide as disclosed herein.
  • a compound CAR (cCAR) engineered cell is an engineered cell as described herein that includes at least two distinct chimeric antigen receptor polypeptides.
  • a CD19CD22 compound CAR engineered cell is an engineered cell as described herein that includes 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.
  • the construction of the CD33CD123 cCAR follows the schematic in FIGS. 1 and 2A . It includes SFFV (spleen focus-forming virus) promoter that drives the expression of the functional compound CAR (cCAR) bearing two different units of CARs.
  • SFFV single focus-forming virus
  • the antigen receptor head a scFv (single-chain variable fragment) nucleotide sequence of the anti-CD33 and anti-CD123.
  • a P2A peptide derived from picornavirus is utilized due to the highly efficient mechanism of its self-cleaving dynamics for bicistronic genetic constructs.
  • the self-cleaving P2A peptide serves to link the two independent units of CARs, CD33CAR, and CD123CAR together during expression.
  • IRES internal ribosomal entry site
  • the modular unit, CD33CAR includes the CD33 scFv domain, a CD8a hinge region, a CD8a transmembrane domain, 4-BB co-stimulatory domain and an intracellular domain of CD3 zeta chain.
  • the second modular CAR, CD123CAR bears the same hinge, transmembrane and intracellular signaling domains as CD33CAR but different scFv, and co-stimulatory domains.
  • the CD33 CAR recognizes its corresponding antigen and the CD123 CAR binds to its corresponding antigen.
  • the hinge region was designed such that sequences where disulfide interactions are avoided. Different co-stimulatory domains, 4-BB and CD28 were used.
  • the CD33CD123 compound CAR was subcloned into a lentiviral plasmid.
  • Compound CAR lentivirus was generated by transfection of HEK-293 FT cells with Lipofectamine 2000 according to manufacturer's directions, except with 2 ⁇ the vector DNA due to a large size of insert, in order to increase titer as shown in FIG. 2 .
  • media containing Lipofectamine was removed and replaced with DMEM containing 10% FBS, 20 mM HEPES, 1 mM sodium pyruvate and 1 mM sodium butyrate.
  • the supernatant was harvested and refrigerated, and replaced with fresh media. After about another 24 hours, this was collected and combined with the previous supernatant, and filtered through a 0.45 ⁇ M filter disc. Supernatant was split into aliquots, flash frozen with liquid nitrogen and stored at ⁇ 80° C.
  • HEK-293 FT cells were harvested, stored frozen, and lysed for subsequent electrophoresis and Western blotting ( FIG. 2B ).
  • PB peripheral blood
  • CB human umbilical cord blood buffy coat cells
  • cCAR lentiviral supernatant was spinoculated onto retronectin-coated multiwell plates.
  • Activated T cells were transduced in multiple wells with lentiviral supernatant at a low concentration of about 0.3 ⁇ 10 6 cells/mL to increase transduction efficiency ( FIG. 3 ).
  • CAR T cells were allowed to expand for up to about 5 days prior to co-culture killing assays. After about 3 days of incubation, cells were incubated with goat anti-mouse F(Ab′)2 or goat IgG (isotype) antibodies conjugated with biotin, washed and followed by incubation with streptavidin-PE and conjugated anti-human CD3. After washing and suspension in 2% formalin, cells were then analyzed by flow cytometry to determine percent transduction efficiency.
  • Transfected CD33CD123 cCAR HEK293T cells were subjected to Western blot analysis in order to confirm the compound construct.
  • Immunoblot with an anti-CD3 ⁇ monoclonal antibody showed bands of predicted size for the compoundCAR CD3 ⁇ fusion protein ( FIG. 2B ).
  • two distinct bands of similar intensity were observed on the blot signaling the successful high cleavage action of the P2A peptide as expected.
  • No CD3 ⁇ expression was seen for the GFP control vector as expected.
  • the surface expression of scFv was also tested on HEK 293 cells ( FIG. 2C ) and primary T cells ( FIG. 2C ).
  • the compound CD33CD23CAR lentivirus was tested for transduction efficiency in the HEK293 cell line and analyzed by flow cytometry (Beckman Coulter) ( FIG. 2C ). Flow cytometry showed that about 67% of HEK cells expressed CD33CD123 CARs.
  • 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 CD4CAR or control (GFP) lentiviruses. After transduction, flow cytometry analysis showed that about 22% of T-cells expressed the CD33CD123CAR ( FIG. 2C ).
  • CD33CD123 cCAR T cells or GFP T cells were incubated with target cells at ratios ranging from 0.5:1 from 50:1, preferably, at about 2:1, 5:1, 10:1, 20:1, 50:1, at 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, respectively) in about 1-2 mL T cell culture media, without IL-2 for about 24h.
  • Target cells were leukemic cell lines and leukemia cells from a patient with leukemia. After about 24 hours of co-culture, cells were stained with mouse anti-human CD33, CD123, CD34 and CD3 antibodies.
  • CD33CD123 cCAR T cells expressing the CD33CAR and CD123 CAR were generated and tested for anti-leukemic functions using the HL60 and KG-1a cell lines.
  • the HL60 cell line is a promyelocytic leukemia cell line highly enriched for CD33. About 100% of its cell population is CD33+ with a small subset ( ⁇ 10%) of it being dim CD123+. In culture, this cell line was tested to determine the effectiveness of the CD33CD123 CAR with an emphasis on targeting CD33-expressing leukemic cells. Additionally, due to the strong expression of CD33 in HL60, it is CD33CD123 cCAR action may be profound.
  • CD33CD123 cCAR exhibited significant leukemic cell killing properties ( FIG. 4 ).
  • CB-derived CD33CD123 CAR T-cells were first tested for their ability to kill HL60 cells.
  • E:T effector:target
  • CD33CD123 CAR cells eliminated about 55% of the CD33 expressing HL60 cells when compared to GFP control.
  • the killing action rose to about 82%.
  • CD33CD123 CAR derived from peripheral blood mononuclear cells (PBMCs) were co-cultured with the myelogenous leukemia cell line KG1a, which also expresses about 100% CD33 at moderate levels compared to HL60 and 50-80% CD123.
  • KG1a is, therefore, a relatively dual target cell population that is double positive for the antigens targeted by the CD33CD123 CAR.
  • E:T effector:target
  • CD33CD123-BB cCAR has been generated (data not shown).
  • This compound CAR comprises two independent units of CARs, CD33 and CD123.
  • the first CAR comprises scFv binding to CD33 and the second CAR bears a different scFv recognizing CD123.
  • Both CARs contain the same hinge region, transmembrane, co-stimulatory and intracellular domains.
  • CD33CD123-BB cCAR lentiviruses were produced and their killing ability was tested in KG-1a cells. As shown in FIG. 5 , there was substantial killing at a ratio of about 10:1 but it is less potent than that of CD33CD123 cCAR.
  • CD33CD123 cCAR Possesses Activity against Patient Samples Expressing CD33 and/or CD123
  • CD33CD123 cCAR T cells Leukemic cells that were CD123 positive and CD33 negative were also tested.
  • B-ALL human B cell acute lympoblastic leukemia
  • Sp-BM-B6 Sp-BM-B6 was chosen. All leukemic blasts in this sample were CD34+CD33 ⁇ , and more than about 50% positive for CD123. Depletion of the CD34+ leukemic cell population by CD33CD123 cCAR T cells was about 86% as compared to that of the GFP control ( FIG. 7 ). Based on the cell line and human sample studies, our data strongly suggest that the compound CD33CD123 CAR is able to target leukemic cells expressing CD33 or CD123 or both.
  • Natural killer (NK) cells are CD56+CD3- and can efficiently kill infected and tumor cells like CD8+ T cells. Unlike CD8+ T cells, NK cells launch cytotoxicity against tumors without the requirement of activation to kill cells. NK cells are safer effector cells, as they may avoid the potentially lethal complications of cytokine storms. However, the use of either CD33 or CD123 or both CAR NK cells in killing leukemias is entirely unexplored.
  • NK-92 cells were transduced with CD33CD123 CAR lentiviral supernatant in two consecutive overnight transductions with a change of retronectin- and virus-coated plates in between.
  • the transduced cells were expanded for 3 or 4 days and then analyzed by flow cytometry for CAR expression.
  • Cells were harvested and incubated with goat anti-mouse F(Ab′)2 at about 1:250 for about 30 minutes.
  • Cells were washed, suspended and stained with streptavidin-PE for about 30 minutes. Cells were washed and suspended in 2% formalin, and analyzed by flow cytometry.
  • NK-92 cells expressing CD33CD123 cCAR were then labeled as above and sorted on FACSAria, with the top 0.2% of F(Ab′)2-expressing cells collected and cultured. Subsequent labeling of sorted, expanded cells showed about 89% of NK-92 cell positive for anti-mouse F(Ab′)2 ( FIG. 8 ).
  • CD33CD123 cCAR NK-92 cells were tested by assessing their ability to kill a HL-60 cancerous cell line in co-culture. Virtually all HL-60 cells highly express CD33 but CD123 expression in this cell line is only less than 10% (weak). Therefore, it is likely that the killing ability of CD33CD123cCAR is dependent on the ability for cCAR to properly targeting CD33.
  • CD33CD123 cCAR NK-92 cells were co-cultured with the HL-60 cells for about 24 hours in NK cell media without IL-2. After the incubation, the CD33CD123 cCAR NK-92 cells were labeled and compared to a control of non-CAR, GFP NK-92 cells. Dramatic killing of HL-60 cells by CD33CD123 cCAR NK-92 cells was observed as compared to the control, GFP NK-92 cells. Moreover, the killing ability of CD33CD123 cCAR NK-92 cells was dose-dependent, with a about 10 to 1 ratio of about 100% compared to the control ( FIGS. 9 and 11 ).
  • a second co-culture experiment using the myeloid leukemia cell line was performed using KG1a, which expresses CD33 in all cells but at a moderate level compared to that of HL-60.
  • the CD123 antigen is expressed in about 50-80% of KG1a cells.
  • the experimental design was similar to the first experiment of the HL-60 killing assay described above, with the same incubation time, effector:cancer cell ratios and GFP NK-92 cell controls. Results show a remarkable killing of KG1a cells by CD33CD123 cCAR NK-92 cells in a dose-dependent manner as compared to the GFP NK-92 cell control. At a ratio of effector:target of 10:1, killing of KG1a cells by CD33CD123 cCAR NK-92 cells was about 85% as compared to that of GFP control ( FIGS. 10 and 11 ).
  • FIG. 11 showed a dose dependent increase in cell killing seen in both populations. Surprisingly, the double positive population showed a higher efficient killing for each increased ratio, suggesting a possible synergistic effect of two modular CARs of CD33 and CD123 ( FIG. 12 ).
  • CD33CD123 CAR T cells received not only costimulation through the CD28 but also co-express the 4-1BB ligand (4-1BBL or CD137L) in a single construct, which provide the better therapeutic efficacy ( FIG. 13A ).
  • T-cells derived from peripheral blood from healthy donors were transduced with the CD33CD123-4-1BBL-2G construct in 6-well plates incubated with 2 ml of virus supernatant.
  • CAR expression was assayed with F(ab)′ labeling for surface expression of the CAR protein and subsequently underwent FACS analysis. Transduced cells were compared to control T-cells labeled at the same time.
  • CD33CD123-41BBL-2G CAR expression on T cells was approximately 60% ( FIG. 13B ).
  • CD33CD123 CAR improves functional activates when 4-1BBL was included in the construct.
  • IL-15/IL-15sushi was also included in CD33CD123 CAR construct as an alternative approach. Both compound CAR, CD33CD123 and IL-15/IL-15sushi were in a single construct ( FIG. 14 ). IL-15/IL-15sushi is able to promote the expansion of CAR T/NK cells, and infiltrate of innate immune cells to the tumor site, which could result in better tumor destruction
  • the three cCARs have been generated ( FIG. 15 ) using the similar strategy to that of the CD33CD123 cCAR described above.
  • the construction of the compound CAR follows the schematic in FIG. 16A . It comprises of SFFV (spleen focus-forming virus) promoter that drives the expression of the functional cCAR bearing two different units of CARs.
  • the first CAR is the complete L8-CD19-2G CAR (using human CD8a leader sequence, called L-8), linked to the complete second CAR (either CD20-2G or CD22-2G) by a high efficient P2A self-cleaving peptide, derived from picornavirus.
  • L-8 human CD8a leader sequence, called L-8
  • P2A self-cleaving peptide derived from picornavirus.
  • the entire sequence is in frame as to result in initially one large fusion protein which is cleaved in half prior to cell surface expression. This method ensures equal expression levels of both CARs.
  • the cCAR DNA molecules were subsequently sub-cloned into the same lentiviral plasmid as above.
  • Lentiviral vector supernatant was generated from HEK293T cells transfected with either CD19CD20-2G or CD19CD22-2G vector construct or control vector. After collection of lentiviral supernatant was collected, cells were harvested, lysed, and electrophoresed prior to Western blot transfer. Incubation of blot membrane with anti-human CD3zeta antibody resulted in two distinct bands representing each CAR unit after cleavage; the CD19CAR is slightly larger than the CD20 or CD22 CAR units ( FIG. 18B ).
  • peripheral blood mononuclear buffy coat cells were activated for three days and transduced with concentrated CD19CD20-2G, CD19CD22-2G or control vector lentiviral supernatant on non-tissue culture plates coated with retronectin.
  • the transduction procedure was repeated 24 hours after the first transduction.
  • CAR expression on the T-cell surface was demonstrated three days after transduction by staining transduced T cells with goat anti-mouse F(Ab′)2 antibody and mouse anti-human CD3.
  • FIG. 16 shows that 26.9% of cells transduced with concentrated L8-CD19CD20-2G lentiviral supernatant and 35.6% of T cells transduced with concentrated CD19CD22-2G lentiviral supernatant were positive for both F(Ab′)2 and CD3 as determined by flow cytometry, when compared to the control transduction.
  • leader sequence that would result in the highest level of cell surface expression of cCAR, three constructs were made that incorporated leader sequences for human CD8a (L8), CD45 (L45), and colony stimulating factor (CSF) ( FIG. 17 ). Following transduction of human peripheral blood T cells with lentiviral supernatant generated from each of these vectors, transduction efficiency for the T cells was determined using F(Ab′)2 antibody as above. The L8 leader sequence again led to the highest transduction efficiency (43.8%), followed by L45 (9.8%) and CSF (1.3%). ( FIG. 17 ). This shows that the optimal design of a compound CAR, like a single CAR, depends in part on the leader sequence for surface CAR expression.
  • lentiviral supernatant for CD19CD20-2G and CD19CD22-2G was centrifuged at 3,880 ⁇ g for 24 hours. The resulting viral pellets were suspended in media at one third their original volume, making them 3 ⁇ concentrated. This concentrate was used to transduce activated T cells in the same volume as non-concentrated virus.
  • FIG. 10 a shows that CAR efficiency for T cells transduced with 3 ⁇ concentrated CD19CD22-2G lentiviral supernatant nearly tripled, while CAR efficiency for T cells transduced with 2.5 ⁇ concentrated CD19CD20-2G lentiviral supernatant increased nearly 10-fold ( FIG. 18 ). This illustrates the importance of concentrating lentiviral vector for the longer cCAR constructs.
  • T cell co-culture killing assays were performed to determine the ability of L8-CD19CD22-2G and L8-CD19CD20-2G CAR T cells to effectively lyse the CD19+ cell lines, SP53 and JeKo-1 (both mantle cell lymphoma lines). Briefly, each target cell line was pre-labeled with CMTMR membrane dye, and then co-cultured with either vector control, L8-CD19CD22-2G or L8-CD19CD20-2G CAR T cells at ratios of 2:1 and 5:1 effector:target cells (200,000 or 500,000 effector cells to 100,000 target cells, in 1 mL T cell media without serum or IL-2).
  • the L8-CD19CD22-2G CAR T cells demonstrated robust lysis of tumor cells ( FIG. 19 ), lysing 53.4% and 93% of the SP53 cells at 2:1 and 5:1 ratios, respectively. At the same ratios, the L8-CD19CD22-2G CAR T cells were able to lyse 69% and 97.3% of the JeKo-1 cells ( FIG. 20 ).
  • L8-CD19CD22-2G CAR T cells lysed 54.3% and 77% of the AML patient cells with aberrant expression of CD19 at 2:1 and 5:1 ratios, respectively, in an overnight co-culture, and lysed 84.3% of the B-ALL tumor cells at a 1:1 ratio in a four day co-culture with 2.5% FBS and IL-2 added to the media ( FIGS. 21, 22A ).
  • FIGS. 21, 22A As these AML patient cells only comprised 65% blasts and 75% of them expressed CD19, it was likely that L8-CD19CD22-2G CAR T cells were able to eliminate the entire CD19 positive blast population.
  • CD19 CAR has also demonstrated some efficacy for the treatment of multiple myeloma in a phase I clinical trial.
  • BCMA CS1 cCAR, BCMA CD19 cCAR, BCMA CD38 cCAR and BCMA CD138 cCAR were generated and the experimental design was similar to that of CD33CD123 cCAR as described above.
  • BCMA CS1 cCAR BCMA CS1 cCAR
  • BCMA-CS1 cCAR BC1cCAR
  • transduction of compound CAR constructs in general lack high efficiency gene transfer rates compared to single antigen CARs. Whether due to construct size or metabolic stress on effector cells or other factors, optimization of a transduction schema for compound CARs remain necessary.
  • Method 1 was a 2 ⁇ transduction for 24 hours each time and uses retronectin coated plates incubated with virus first, aspirated, then incubated with T-cells to a final concentration of 0.5 ⁇ 10 6 cells/ml.
  • Method 2 used the same viral retronectin procedure, however, it exchanged the 2 nd transduction period for continued incubation to a total of 48 hours of incubation with a final cell density of 0.3 ⁇ 10 6 cells/ml.
  • Method 2 revised uses an incubation scheme where viral supernatant was directly incubated with cells for 48 hours on a retronectin coated plate at a cell density of 0.3 ⁇ 10 6 cells/ml ( FIG. 23 ).
  • BC1cCAR's modular design consists of an anti-CD269 (BCMA) single-chain variable fragment (scFv) region fused to an anti-CD319 (CS1) scFv by a self-cleaving P2A peptide, CD8-derived hinge (H) and transmembrane (TM) regions, and 4-1BB co-activation domains linked to the CD3 ⁇ signaling domain ( FIG. 24A ).
  • a strong spleen focus forming virus promoter (SFFV) and a CD8 leader sequence were used for efficient expression of the BC1cCAR molecule on the T-cell surface.
  • T-cells isolated from human peripheral blood buffy coats were transduced with BC1cCAR lentivirus after 2 days of activation.
  • Transfected BC1cCAR HEK293T cells were subjected to Western blot analysis in order to confirm the compound construct.
  • Immunoblot with an anti-CD3 ⁇ monoclonal antibody showed bands of predicted size for the compound CAR CD3 ⁇ fusion protein ( FIG. 24D ). Importantly, two distinct bands of similar intensity were observed on the blot signaling the successful high cleavage action of the P2A peptide as expected. No CD3 ⁇ expression was seen for the GFP control vector as expected.
  • BC1cCAR T-Cells Specifically Lyse BCMA + and CS1 + Myeloma Cell Lines
  • BC1cCAR T-cells To assess the cytotoxicity ability of BC1cCAR T-cells, we conducted co-culture assays against myeloma cell lines: MM1S (BMCA + CS1 + ), RPMI-8226 (BCMA + CS1 ⁇ ), and U266 (BCMA + CS1 dim ). The ability of the BC1cCAR T-cells to lyse the target cells was quantified by flow cytometry analysis, and target cells were stained with Cytotracker dye (CMTMR). In 24 hour co-cultures, the BC1cCAR exhibited virtually complete lysis of MM1S cells, with over 90% depletion of target cells at an E:T ratio of 2:1 and over 95% depletion at an E:T of 5:1 ( FIGS.
  • CTMR Cytotracker dye
  • BC1cCAR T-Cells Specifically Target BCMA + and CS1 + Populations in Primary Patient Myeloma Samples
  • BC1cCAR T cells show robust dose-dependent ablation of the MM7-G primary patient sample, with over 75% lysis at an E:T ratio of 5:1, increasing to over 85% at 10:1 ( FIG. 26A ).
  • BC1cCAR also show targeted and specific lysis ability, by significantly ablating both the BCMA + CS1 + and the BCMA-CS1 + population subsets in MM10-G co-cultures.
  • BC1cCAR T-cells ablate over 60% of the BCMA + CS1 + population, and 70% of the CS1 + only population ( FIG. 26B ).
  • E:T ratio of 5:1 the ablation of CS1 + only population increases to 80% ( FIG. 26B ).
  • BC1cCAR T-cells were also able to demonstrate cytotoxic activity in a dose-dependent manner as well ( FIG. 26C ).
  • BC1cCAR T cells exhibit robust anti-tumor activity against both myeloma cell lines and primary tumor cells presenting different combinations of BCMA and CS1 ( FIG. 26D )
  • BC1cCAR T-cells In short-term cultures ( ⁇ 24 hrs), BC1cCAR T-cells exhibited cytotoxic activity against BCMAxpK562 cells while showing no effect against wild-type K562 cells ( FIG. 27B ). Next, short-term cultures against CS1xpK562 cells show similar responses against CS1 expressing target cells. Furthermore, BC1cCAR T-cells appeared to have a stronger cytotoxic effect than a CS1-specific CAR against CS1xpK562 cells ( FIG. 27B ). Further validation of the anti-CS1 activity was performed on CS1 dim expressing NK-92 cells where cytotoxicity exhibited as a dose-dependent effect ( FIG. 27B ).
  • BC1cCAR T-Cells Exhibit Persistency and Sequential Killing Ability Even with Tumor Re-Challenge
  • control T-cells show no response and proliferation yielding a tumor population that has now expanded by twice its initial number.
  • all CARs still retain a high degree of cytotoxicity even without exogenous cytokines.
  • the newly inputted MM1S cells have been virtually depleted by both BCMA-CAR and the BC1cCAR with significant cytotoxicity still observed from the CS1-CAR.
  • flow cytometry show a diminished CS1-CAR population and a relative growth in the tumor antigen population to ⁇ 17% ( FIG. 28C ), suggesting that the CS1-CAR T-cells may be faltering.
  • BC1cCAR T-Cells Exhibit Significant Control and Reduction of Tumor In Vivo
  • mice were intravenously injected with 5 ⁇ 10 6 BC1cCAR T-cells or control GFP cells in a single dose.
  • mice were injected subcutaneously with RediJect D-Luciferin (Perkin Elmer) and subjected to IVIS imaging to measure tumor burden ( FIG. 29A ).
  • BC1cCAR T-cells exhibit improved cytotoxic effect in a mixed antigen xenogeneic mouse model.
  • mice were sublethally irradiated and intravenously injected with luciferase-expressing K562 cells expressing either stably transduced BCMA or CS1.
  • BCMA and CS1 expressing K562 cells were further sorted for expression following puromycin selection and established as stable homogenous single antigen populations.
  • BCMA and CS1 expressing K562 cells were then mixed at a 4:1 ratio respectively before injection to model potential antigen escape.
  • mice were intravenously injected with a course 15 ⁇ 10 6 control T-cells, BCMA-specific CAR, or BC1cCAR T-cells.
  • mice Two control mice died as a result of injection procedure as a result of technical issues during T-cell infusion and cell aggregation.
  • mice were injected subcutaneously with RediJect D-Luciferin (Perkin Elmer) and subjected to IVIS imaging to visualize tumor burden ( FIG. 19D ).
  • Average light intensity (signifying tumor burden) measured for the BC1cCAR T-cell injected mice was compared to that of a BCMA-specific CAR and GFP control injected mice in order to determine the control of tumor growth by treatment ( FIG. 29D ).
  • day 10 both the BCMA-specific CAR and BC1cCAR T-cells exhibited over 47% tumor reduction compared to control.
  • BC1cCAR construct into a model NK cell line, NK-92.
  • the construct was successfully able to be transduced via lentiviral incubation for 48 hours into NK-92 cells and resulted in a surface expression profile of 62.1% after gene-transfer ( FIGS. 30A and 30B ).
  • NK-92 cells maintained at densities of 0.3-0.5 ⁇ 10 6 cells/ml resulted in stable populations.
  • BC1cCAR anti-tumor activity in vitro we conducted co-cultures against myeloma cell lines and a primary patient sample.
  • the BC1cCAR approached 80% lysis against MM1S, U266, and RPMI-8226 cell lines at E:T ratios of 5:1 in culture. It also successfully lysed over 60% of the primary MM7-G tumor ( FIGS. 31A and 31B ). These results are similar in terms of comparability with BC1cCAR T-cells.
  • BCMAxpK562 BCMAxpK562
  • CS1xpK562 cells BC1cCAR NK cells
  • BC1cCAR's modular design consists of an anti-CD269 (BCMA) single-chain variable fragment (scFv) region fused to an anti-CD319 (CS1) scFv by a self-cleaving P2A peptide, CD8-derived hinge (H) and transmembrane (TM) regions, and 4-1BB co-activation domains linked to the CD3 ⁇ signaling domain ( FIG. 35 ).
  • BCMA anti-CD269
  • CS1 scFv single-chain variable fragment
  • BCMA CAR unit was able to potently lyse any BCMA + population.
  • the single-chain variable fragment (scFv) nucleotide sequences of the anti-CD19 molecule was used for two different constructs, CD19-2G and CD19b-BB CAR.
  • the CD19CAR was designed with 4-1BB co-activation domain fused to the CD3zeta signaling domain, making it a second generation CAR ( FIG. 37A ).
  • CD19-targeting second generation CAR T-cells have previously been used in clinical trials.
  • SFFV spleen focus-forming virus promoter
  • CD19CAR constructs using the leader sequences of CD45, CSF, human albumin (HA) or IL-2 were also made.
  • the anti-CD19 scFv was separated from the intracellular signaling domains by CD-8 derived hinge (H) and transmembrane (TM) regions ( FIG. 37 ).
  • the CD19CAR DNA molecules, with different leader sequences or different scFv sequences, were also subsequently sub-cloned into a lentiviral plasmid.
  • Lentiviral vector supernatant was generated from HEK293T cells transfected with CD19-2G vector construct and control vector. After collection of lentiviral supernatant was collected, cells were harvested, lysed, and electrophoresed prior to Western blot transfer. Incubation of blot membrane with anti-human CD3zeta antibody resulted in a ⁇ 56 kDa band in the lane containing lysate from cells transfected with CD19-2G, the predicted size for the expressed fusion protein ( FIG. 37B ).
  • peripheral blood mononuclear buffy coat cells were activated for three days and transduced with L8-CD19-2G, or control vector lentiviral supernatant on non-tissue culture plates coated with retronectin.
  • the transduction procedure was repeated 24 hours after the first transduction.
  • CAR expression on the T-cell surface was demonstrated three days after transduction by staining transduced T cells with goat anti-mouse Fab antibody and mouse anti-human CD3.
  • FIG. 37C shows that 19.8% of cells transduced with the L8-CD19-2G virus were positive for both F(Ab′)2 and CD3 as determined by flow cytometry, when compared to the control transduction.
  • leader sequences for human CD8a (L8), CD45 (L45), colony stimulating factor (CSF), human albumin (HA), and IL2 FIG. 38A .
  • CD8a human CD8a
  • L45 CD45
  • CSF colony stimulating factor
  • HA human albumin
  • IL2 IL2
  • CD19-2G CAR To determine the scFv sequence of CD19 that would result in the highest level of cell surface expression of CD19-2G CAR, two different sequences were used in the design of CD19-2G CAR ( FIG. 39A ), CD19 and CD19b. Both used the L8 leader sequence. Following transduction of human peripheral blood T cells with lentiviral supernatant generated from each of these vectors under the same condition, transduction efficiency for the T cells was determined using F(Ab′)2 antibody as above. The CD19-2G construct resulted in 18.2% CAR cells, but the CD19b-BB-2G construct resulted in 54.7% CAR efficiency ( FIG. 39 b ). This shows that the optimal design of CD19-2G CAR also depends in part on the sequence of the scFv used.
  • T cell co-culture killing assays were performed to determine the ability of CD19-2G and CD19b-BB-2G CAR T cells to effectively lyse the CD19+ cell lines, SP53 and JeKo-1 (both mantle cell lymphoma lines). Briefly, each target cell line was pre-labeled with CMTMR membrane dye, and then co-cultured with either vector control, L8-CD19-2G or L8-CD19b-BB-2G CAR T cells at ratios of 2:1 and 5:1 effector:target cells (200,000 or 500,000 effector cells to 100,000 target cells, in 1 mL T cell media without serum or IL-2).
  • CD19-2G and CD19b 2G CAR T cells displayed robust lysis of B cell lines, SP53 and Jeko-1 ( FIG. 41 ).
  • CD19-2G and CD19b-BB-2G CAR T Cells Eliminate CD19+ Cells from AML and B-ALL Patient Samples
  • Viral titers generally decrease as the size of insert increases and the sequence of CD19b scFv provided a higher titer for CD19b CAR ( FIG. 39 ). Therefore, CD19b scFv was used to generate the compound BCMA CD19b CAR ( FIG. 44 ). BCMA CD19b CAR.
  • sgRNA Three pairs of sgRNA are designed with CHOPCHOP to target the gene of interest. Gene-specific sgRNAs are then cloned into the lentiviral vector (Lenti U6-sgRNA-SFFV-Cas9-puro-wpre) expressing a human Cas9 and puromycin resistance genes linked with an E2A self-cleaving linker.
  • the U6-sgRNA cassette is in front of the Cas9 element.
  • the expression of sgRNA and Cas9puro is driven by the U6 promoter and SFFV promoter, respectively ( FIG. 48 ).
  • exemplary gene-specific sgRNAs have been designed and constructed as set forth below:
  • CD45 sgRNA construct
  • FIG. 49A shows steps of generation of CD45 CAR T or NK cell targeting hematologic malignancies.
  • Lentiviruses carried gene-specific sgRNAs were used to transduce NK-92 cells.
  • the loss of CD45 expression on NK-92 cells was determined by flow cytometry analysis.
  • the CD45 negative population of NK-92 cells was sorted and expanded ( FIG. 49B ).
  • 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 their ability of killing CD45+ cells.
  • NK 45i -92 cells were similar to that of the wild NK-92 cells ( FIG. 50 ). Inactivation of CD45 did not significantly affect the cell proliferation of NK-92. In addition, we showed that the lysis ability of NK 45i -92 cells was compatible to that of wild type, NK-92 when cells were co-cultured with leukemic cells, CCRF ( FIG. 51 ).
  • NK 45i -92 cells and their wild type, NK-92 were transduced with lentiviruses expressing CD5CAR or GFP.
  • the resulting CD5CAR NK 45i -92 cells and GFP NK 45i -92 were sorted by FACS, and used to compare their ability of killing targeted cells.
  • CD5CAR NK 45i -92 cells displayed the ability of robustly killing CD5 target leukemic cells at ratios (E:T), 2:1 and 5:1 when they were co-cultured with CCRF-CEM cells.
  • CD45CAR in NK 45i -92 cells response to the CD45 antigen in leukemic cells.
  • CD45CAR consists of an anti-CD45 single-chain variable fragment (scFv) region, CD8-derived hinge (H) and transmembrane (TM) regions, and tandem CD28 and 4-1BB co-activation domains linked to the CD3 ⁇ signaling domain ( FIG. 53A ).
  • SFFV spleen focus forming virus promoter
  • CD45CAR protein was characterized by Western blot of HEK293-FT cells transfected with CD45CAR lentiviral plasmid with appropriate vector control. Additionally, anti-CD3zeta monoclonal antibody immunoblots revealed bands of predicted size for the CD45CAR protein with no bands observed in vector control ( FIG. 53B ).
  • CD45CAR NK-92 transduction efficiency was determined to be 87%, as determined by flow cytometry ( FIG. 54 ) after sorting. After FACS collection of NK 45i -92 cells, CD45CAR expression levels remained consistently stable for at least 10 passages.
  • CD45CAR NK 45i -92 anti-leukemic activity we conducted co-culture assays using T-ALL cell lines, CCRF-CEM and Jurkat, and NK cell line and NK-92 cells since they all express CD45 ( FIGS. 55, 56 and 57 ).
  • CD45CAR NK 45i -92 cells consistently displayed robust lysis of leukemic cells.
  • E:T ratio 5:1 CD45CAR NK 45i -92 cells effectively lysed more than 60% of CCRF-CEMcells ( FIG. 55 ).
  • CD45CAR NK 45i -92 cells were also able to eliminate about 60% of Jurkat cells at a ratio of E:T, 2:1 or 5:1 ( FIG. 56 ).
  • CD45CAR NK 45i -92 cells efficiently lysed 20% CD45 positive NK-92 cells at an E:T ratio of 2:1, with close to 60% lysis at an E:T of 5:1 ( FIGS. 57A-57C ).
  • CD45-28 and CD45-BB CARs contain a new anti-CD45 scFv, which is different from that of CD45CAR described above.
  • CD45-28 CAR uses a CD28 co-stimulatory domain while the CD45-BB bears a 4-BB co-stimulatory domain. Both CARs use the CD8-derived hinge (H), transmembrane (TM) regions and CD3 ⁇ signaling domain.
  • CD45CARs displayed robust lysis of B acute lymphoblastic cell line, REH.
  • CD45CAR NK45i-92 cells lysed about 76% REH cells.
  • CD45b-BB CAR NK45i-92 cells and CD45b-28 CAR NK45i-92 cells showed about 79% and 100% lysis of REH cells, respectively compared to control GFP NK-92 cells ( FIG. 57D-57E ).
  • CD45b-28 CAR NK45i-92 cells exhibited the highest ability of lysis of REH cells (B-ALL cells).
  • CD4b-28CAR CD45b-28 CAR NK45i-92 cells could lyse other types of leukemic cells.
  • co-culture assay was performed with U937 cells (target: T) and GFP NK-92 cells or CD45b-28 NK 45i -92 cells (effector: E) at 2:1 (E:T) ratio for 20 hours, CD45b-28 NK 45i -92 cells exhibited a robust anti-leukemic activity with about 81% cell lysis against U937 cells compared to control GFP NK-92 cells.
  • U937 is an acute myeloid leukemia cell line.
  • MOLM-13 cells target: T
  • GFP NK-92 cells or CD45b-28 NK45i-92 cells effector: E
  • E 5:1
  • FIG. 57G MOLM-13 cells are derived from a patient with aggressive acute monocytic leukemia.
  • the anti-leukemic activities were also examined in two mantle cell lines, SP53 and Jeko ( FIGS. 57H and I).
  • CD45b-28 NK 45i -92 cells could lyse CD34+ hematopoietic stem/progenitor cells.
  • CD34(+) stem cells derived from human umbilical cord blood were co-cultured with either control or CD45b-28 CAR NK cells for 48 hr at a low ratio of 2:1 (effective:target).
  • CD45b-28 NK 45i -92 cells nearly eliminate CD34+ hematopoietic precursor cells ( FIG. 57J ) compared to the control.
  • CD45 CAR cells received not only costimulation through the CD28 but also co-express the 4-1BB ligand (4-1BBL or CD137L) in a single construct, which provide the better therapeutic efficacy ( FIG. 58A ) and their example is described below:
  • CD45b-28-2G-4-1BBL was generated and the generated CD45b CAR cells could receive both co-stimulatory pathways, CD28 and 4-1BB.
  • CD45b-28-2G-4-1BBL viruses were concentrated by 4 fold and used to transduce NK 45i -92 cells. Its CAR surface expression was about 87% ( FIG. 58B ).
  • CD45b-28-2G-4-1BBL viruses were concentrated by 4 fold and used for transduction. Anti-tumor activity of CD45b-2G CAR cells was significantly improved when 4-1BBL was included in the construct.
  • CD45 CAR construct An enhancer, IL-15/IL-15sushi was also included in CD45 CAR construct as an alternative approach to enhance CD45 CAR anti-tumor activity. Both CD45 CAR and IL-15/IL-15sushi were in a single construct ( FIG. 58 ). Anti-tumor activity of CD45b-2G CAR cells is significantly improved when IL-15/IL-15sushi is included in the construct.
  • CD45b-28-2G-IL-15/IL-15sushi NK cells was generated. Surface CD45b CAR expression were about 60%. ( FIG. 58C ). Anti-tumor activity of CD45b-2G CAR cells was significantly improved when IL-15/IL-15sushi was included in the construct.
  • the CD4IL-15/IL-15sushi-CAR has been generated and it contains the third generation of CD4CAR linked to IL-15/IL-15sushi ( FIG. 59 ).
  • a combination of CAR, (third generation), sushi/IL-15 is assembled on an expression vector and their expression is driven by the SFFV promoter ( FIG. 59 ).
  • CAR with IL-15/IL-15sushi is linked with the P2A cleaving sequence.
  • the IL-15/IL-15sushi portion is composed of IL-2 signal peptide fused to IL-15 linked to IL-15susi via a 26-amino acid poly-proline linker ( FIG. 59 ).
  • the IL-2 signal peptide provide a better secreting signal.
  • the stable, functional complexes of IL-15/IL-15sushi can be secreted from the transduced cells and the secretion is directed by IL-2 signal peptide.
  • HEK293FT cells were transfected with lentiviral plasmids for either GFP (control) or. CD4IL-15/IL-15sushi. Approximately 60 hours after transfection, both HEK-293FT cells and supernatant were collected. Cells were lysed in RIPA buffer containing protease inhibitor cocktail and electrophoresed. The gel was transferred to Immobilon FL blotting membrane, blocked, and probed with mouse anti-human CD3z antibody at 1:500. After washes, membrane was probed with goat anti-mouse HRP conjugate, washed, and exposed to film following treatment with HyGlo HRP substrate.
  • the CD4IL-15/IL-15sushi was successfully expressed in HEK 293 cells (Lane 2, FIG. 60 a ).
  • the CD4IL-15/IL-15sushi lentiviral supernatant was further examined by the transduction of fresh HEK-293 cells ( FIG. 60A ).
  • HEK-293 cells were transduced with either GFP or CD4IL-15/IL-15sushi CAR viral supernatant from transfected HEK-293FT cells.
  • Polybrene was added to 4 ⁇ L/mL. Media was changed after 16 hours and replaced with media containing no viral supernatant or polybrene.
  • Three days after transduction, cells were harvested and stained with goat-anti-mouse F(Ab′)2 antibody at 1:250 for 30 minutes.
  • FIG. 60 b shows that HEK-293 cells that were transduced with the CD4IL-15/IL-15sushi CAR lentivirus were 80% positive for F(Ab)2-PE (circled, FIG. 60B ), while transduction with GFP control lentivirus was minimal for F(Ab)2-PE ( FIG. 60 ).
  • NK-92 cells were transduced with concentrated CD4IL-15/IL-15sushi-CAR lentiviral supernatant. After 5 days incubation, cells were harvested and incubated with goat anti-mouse F(Ab′)2 at 1:250 for 30 minutes. Cells were washed, suspended and stained with streptavidin-PE for 30 minutes. Cells were washed and suspended in 2% formalin, and analyzed by flow cytometry, resulting in nearly 70% of the transduced cells expressing CD4IL-15/IL-15sushi-CAR (circled, FIG. 61 .
  • CD4IL-15/IL-15sushi-CAR Further experimental tests for CD4IL-15/IL-15sushi-CAR included leukemia/lymphoma killing assays in vitro and vivo, and comparison of target killing and proliferation rates with cells transduced with CD4CAR. The inventor also used the same strategy described above to generate CD19IL-15/IL-15sush CAR, CD20IL-15/IL-15sush CAR and CD22IL-15/IL-15sush CAR.
  • CD4IL-15/IL-15sushi-CAR Human umbilical cord buffy coat cells were transduced with concentrated CD4IL-15/IL-15sushi-CAR lentiviral supernatant. After 5 days incubation, cells were harvested and incubated with goat anti-mouse F(Ab′)2 at 1:250 for 30 minutes. Cells were washed, suspended and stained with streptavidin-PE for 30 minutes. Cells were washed and suspended in 2% formalin, and analyzed by flow cytometry, resulting in 63% of the transduced cells expressing CD4IL-15/IL-15sushi-CAR(circled, FIG. 62 ). Further experimental tests for CD4IL-15/IL-15sushi-CAR will include leukemia/lymphoma killing assays in vitro and vivo, and comparison of target killing and proliferation rates with cells transduced with CD4CAR.
  • CD4IL-15/IL-15Sushi CAR NK Cells were Tested for Anti-Leukemic Activity Relative to CD4CAR NK Cells In Vitro by Co-Culturing them with the Following CD4 Positive Cell Lines: Karpas 299 and MOLT4.
  • the Karpas 299 cell line was derived from a patient with anaplastic large T cell lymphoma.
  • the MOLT4 cell line expressing CD4 was established from the peripheral blood of a 19-year-old patient with acute lymphoblastic leukemia (T-ALL).
  • T-ALL acute lymphoblastic leukemia
  • CD4IL-15/IL-15sushi CAR NK cells showed profound killing (95%) of Karpas 299 cells at a 5:1 ratio of effector:target, at an even higher rate than that of CD4CAR NK cells (82%; FIG. 63 ).
  • CD4IL-15/IL-15sushi CAR NK cells lysed target cells at a higher rate (84% to 65%) than CD4CAR NK cells in an overnight assay ( FIG. 64 ).
  • mice were injected subcutaneously with RediJect D-Luciferin (Perkin Elmer) and subjected to IVIS imaging to measure tumor burden ( FIG. 65B ).
  • Average light intensity measured for the CD4CAR and CD4IL-15/IL-15sushi CAR T cell injected mice was compared to that of vector control T cell injected mice in order to determine the percentage of tumor cells in treated versus control mice ( FIG. 65C ).
  • CD4CAR T cell-treated mice had a 52% lower tumor burden relative to control on Day 6, whereas CD4IL-15/IL-15sushi CAR T cell-treated mice had a 74% lower tumor burden.
  • nearly all tumor cells had been lysed in both of these groups.
  • CD4IL-15/IL-15Sushi CAR NK Cells Exhibit Robust and Persistent Anti-Tumor Activity in Vivo
  • NK CAR cells bear a short half-life property and leukemic Jurkat cells show less than 60% CD4+ phenotype ( FIG. 66A ).
  • FIG. 66A it allows us to investigate how secretory soluble IL-15sushi affects the CAR functions in terms of its persistence and killing capability.
  • mice In contrast with MOLM-13 cells, Jurkat cells show less than 60% CD4+ phenotype (FIG. 66 A 5 ).
  • Three days following Jurkat cell injection mice were intravenously injected with a course of 10 ⁇ 10 6 either CD4CAR, CD4IL-15/IL-15sushi, or vector control NK cells. On day 3 (the day before treatment), 7, 10, and 14, mice were subjected to IVIS imaging to measure tumor burden ( FIG. 66B ). Average light intensity measured for the CD4CAR and CD4IL-15/IL-15sushi NK injected mice was compared to that of vector control NK injected mice to determine percent lysis of Jurkat cells ( FIG. 66C ).
  • NK-92 cells stably transduced with either CD4CAR or CD4IL-15/IL-15sushi were cultured in the presence or absence of IL-2 to determine if IL-15/IL-15sushi secretion alone could lead to survival and expansion.
  • CD4CAR-expressing NK cells cultured without IL-2 died by Day 7, while CD4IL-15/IL-15sushi-expressing NK cells cultured without IL-2 expanded at approximately the same rate as either CD4CAR or CD4IL-15/IL-15sushi cells cultured with IL-2 ( FIG. 68B ), showing that secreted IL-15/IL-15sushi could substitute for IL-2.
  • NK cells secreting IL-15/IL-15sushi could aid in the survival and expansion of non-transduced NK-92 cells in a co-culture.
  • an equal ratio of NK GFP-expressing cells were cultured with either CD4CAR- or CD4IL-15/IL-15sushi-expressing NK cells, in the presence or absence of IL-2. Cells were counted every 2-3 days ( FIG. 8A ). By Day 7, CD4CAR NK cells given no IL-2 had died, but CD4IL-15/IL-15sushi NK cells without IL-2 had survived and expanded at approximately the same rate as either CD4CAR or CD4IL-15/IL-15sushi cells cultured with IL-2.
  • FIG. 69 GFP NK and either control vector NK or CD4IL-15/IL-15sushi NK cells were mixed at a 10 to 1 ratio, with no IL-2. By Day 6, cells co-cultured with control cells had all died, but survival of cells cultured with NK cells secreting IL-15/IL-15sushi survived until Day 10.
  • the upper chamber represents the tumor microenvironment, in which the survival of T cells or NK cells is improved by the secretion of IL-15/IL-15sushi from the CD4IL-15/IL-15sushi NK cells.
  • CD4CAR and CD4IL-15/IL-15sushi transduced T cells were compared in the presence or absence of IL-2. Total cell counts calculated throughout the experiment (up to Day 17) for transduced cells with or without IL-2. CD4IL-15/IL-15sushi transduced T cells appeared to be more tolerant to the absence of IL-2 than that of CD4CAR transduced T cells.
  • Treg CAR also called CD4zetaCD25CAR or C4-25z
  • SFFV single focus-forming virus
  • the CD4 chimeric antigen receptor polypeptide unit comprises a CD45 signal peptide, a CD4 antigen recognition domain, a hinge region (derived CD8a), a transmembrane domain (CD8a) and CD3 zeta chain;
  • CD25 chimeric antigen receptor polypeptide unit comprises a CD45 signal peptide, a CD25 antigen recognition domain, a hinge region (CD8a), a transmembrane domain (CD8a), a co-stimulatory domain (s), CD28.
  • the Treg CAR can potentiate the lysis activity of a cell co-expressing CD4 and CD25 while minimizing a cell bearing CD4 or CD25 antigen alone.
  • CD4zetaCD25CAR C4-25z
  • Treg CAR The CD4zetaCD25CAR (C4-25z) (Treg CAR) was transduced in an assay. Compared to control T-cells, CD4zetaCD25CAR cells show ⁇ 15% surface expression and this was sufficient to observe the following phenotype validation of construct function ( FIG. 70A ). CD4zetaCD25 CAR cells and control T-cells were both assayed with CD4 and CD25 antibody to look for logic gated behavior using flow cytometry analysis. Due to the construct design, the CD4zetaCD25CAR cell would potentiate the lysis activity for cells co-expressing both CD4 and CD25 antigens.
  • CD4zetaCD25 CAR We further characterized CD4zetaCD25 CAR by comparing it with CD4 CAR.
  • CD4CAR T cells had a profound lysis ability of cells expressing CD4 only while CD4zetaCD25CAR T cells had a limited killing ability on this population ( FIG. 71 ).
  • CD4zetaCD25CAR T cells also showed virtually complete depletion of cells expressing both CD4 and CD25 antigens ( FIG. 71 ).
  • the disclosed disclosure also comprises methods of improving the CD4zetaCD25CAR therapeutic activity.
  • the example is described below.
  • An engineered CD4zetaCD25CAR cell was prepared in accordance with the present disclosure.
  • CD4zetaCD25CAR Targeted Cells killing by CD4zetaCD25CAR is improved when co-expressed with 4-1BBL or IL-15/IL-15sushi or IL-15/IL-15RA.
  • safety switch greatly increases safety profile and the “safety switch” may be an inducible suicide gene, such as, without limiting, caspase 9 gene, thymidine kinase, cytosine deaminase (CD) or cytochrome P450.
  • Other safety switches for elimination of unwanted modified T cells involve co-expression of CD20 or CD52 or CD19 or truncated epidermal growth factor receptor in T cells.
  • T-cells and B-cells express CD52 on the cell surface and a CD52 specific antibody, CAMPATH (alemtuzumab), can eliminate CD52+ cells from circulation.
  • CAMPATH an antigen specific antibody
  • FIG. 72A This additional CD52 construct mechanically separates the signaling from native CD52. The aim was to preempt the possibility of native CD52 antigen escape on CAR T-cell surface after CAMPATH treatment.
  • CD5CAR-CD52 lentiviral protein and expression were confirmed via western blot and flow cytometry analysis using CD52 antibody on transduced HEK293 cells. We also found that co-expressing CD52 would affect the CAR T cell functions.
  • FIGS. 72B and 72C To assess the effect of CAR elimination by CAMPATH (alemtuzumab) treatment, we conducted in vivo procedures as described ( FIGS. 72B and 72C ). We intravenously injected 5 ⁇ 10 6 CD5CAR-52 T-cells into irradiated mice. Next day, we added 0.1 mg/kg of CAMPATH or PBS via IP injection for 3 mice of each group. After 6 and 24 hours following CAMPATH treatment, we collected peripheral blood from the mouse tail and determined presence of CD5CAR-52 T-cells by FACS analysis. CAMPATH injection virtually completely deplete CD5CAR-CD52 T-cells in blood at both 6 h and 24 h ( FIG. 72C ).
  • CD5CAR-CD52 cells were also completely depleted in both the bone marrow and spleen ( FIG. 72D ). These findings support the use of CAMPATH as a useful strategy in acting as a safety trigger to deplete CAR-T cells from circulation and lymphoid organs.
  • the engineered cell includes a CD5 chimeric antigen receptor polypeptide and an anchor CD52 (SEQ ID NO. 70), and corresponding polynucleotide (SEQ ID NO. 69).
  • CD52 is incorporated into CD5 CAR engineered cell or any CAR engineered cell and can be used as a “safety switch” for CAR therapy.
  • HEK293FT cells were transfected with lentiviral plasmids expressing GFP under the SFFV, EF1 or CAG promoters. Approximately 60 hours after transfection, supernatant was collected from each. Relative viral titer was determined by first transducing HEK293 cells with supernatant from each of the 3 promoters. HEK-293 cells were transduced with GFP viral supernatant from each of the 3 transfected HEK-293FT cells. Polybrene was added to 4 ⁇ L/mL. Media was changed after 16 hours and replaced with media containing no viral supernatant or polybrene.
  • GFP expression was seen in each sample, but was highest for the cells transduced with virus made using the SFFV promoter.
  • Activated human umbilical cord buffy coat cells were transduced with GFP lentiviral supernatant (amount based on the results of the HEK293 transduction efficiency) from each of the promoters. After 5 days incubation, cells were harvested, washed and suspended in 2% formalin, and analyzed by flow cytometry for GFP expression. 43% of cells expressed GFP at high levels (>10 3 ) while GFP-expression for cells transduced with virus using promoters EF1 (15%) and CAG (3%) were considerably lower. Five days later, cells analyzed the same way showed nearly the same percentages for each (46%, 15% and 3%, respectively). These results indicate that SFFV promoter leads to stronger expression than EF1 or CAG promoters, and that the expression remains high for at least 10 days post-transduction. Further experimental tests will include longer incubation times for transduced cells beyond the 10-day window.
  • HEK 293 cells were transduced with either EF1-GFP or SFFV-GFP viral supernatant, with either 30 ⁇ L (low), 125 ⁇ L (medium), or 500 ⁇ L (high) per well of a 12 well tissue-culture treated plate. Culture media was changed the following morning to DMEM plus 10% FBS ( FIG. 73 ).
  • Transduced cells were then trypsinized, washed, and suspended in formalin and subjected to flow analysis.
  • the percentage of GFP+ cells in each of the conditions was determined by flow cytometry using the FITC channel ( FIG. 74 ).
  • the percentage of GFP+ was higher in cells transduced with SFFV-GFP than the cells transduced with the corresponding volume of EF1-GFP viral supernatant (50% to 18% for low, 80% to 40% for medium, and 82% to 70% for high). From this, we determined that using the highest volume of EF1-promoter virus was comparable to using the lowest volume of SFFV-promoter virus in terms of titer, and would allow for comparison of relative promoter strengths for the following transduction experiments
  • Transduced cells were also visualized on an EVOS fluorescent microscope using GFP at 20 ⁇ at the same exposure conditions for each well ( FIG. 73 ).
  • Cells transduced with SFFV-GFP viral supernatant were dramatically brighter than cells transduced with EF1-GFP.
  • comparing the image of the EF1-promoter under high viral volume loads with the image of the SFFV-promoter using low viral volume loads show similar fluorescent intensity. This suggests that the SFFV promoter is a stronger driver of gene expression.
  • activated cord blood buffy coat T cells were transduced with either 50 ⁇ L of SFFV-GFP or 1 mL of EF1-GFP EF1-GFP viral supernatant, in 12-well tissue culture-treated plates pre-coated with retronectin (Clontech). Following two overnight transductions, cells were cultured on T cell media with 300 IU/mL IL-2 (Peprotech) and maintained at 1.0-4.0 ⁇ 10 6 /mL. Cells were washed, suspended in formalin, and subjected to flow cytometry analysis, using the FITC channel to determine the percentage of GFP+ cells, on 7, 14, 21 and 28 days after transduction.
  • the percentage of GFP+ cells was consistently higher for T cells transduced with SFFV-GFP compared to EF1-GFP-transduced T cells ( FIG. 75A ), even as the percentage of total GFP+ cells decreased over this period.
  • a further comparison showed that T cells transduced with the higher (1 mL) amount of EF1-GFP supernatant actually decreased in percentage relative to the percent of GFP+ cells transduced with the lower amount (50 ⁇ L, or 20-fold less) of SFFV-GFP, between Day 7 and Day 28, from over 60% to under 40% ( FIG. 75B ). This suggests that transduction using the SFFV promoter led to greater persistence of transduced cells.
  • Methods of generating the CAR gene including at least one of a T antigen recognition moiety (at least one of CD4, CD8, CD3, CD5, CD7, and CD2, or a part or a combination thereof), a hinge region and T-cell activation domains is provided.
  • Methods of generating multiple units of CARs (cCAR) targeting antigen (s) including at least one of CD33, CD123, CD19, CD20, CD22, CD269, CS1, CD38, CD52, ROR1, PSMA, BAFF, TACI, CD138, and GPC3, or a part or a combination of a hinge region and T-cell activation domains is provided.
  • the provided methods also include: 1) generating of the CAR T or NK cells targeting leukemias and lymphomas expressing CD45 and avoiding self-killing; 2) generation of “armored” CAR T or NK cells designed to both overcome the inhibitory tumor microenvironment and exhibit enhanced anti-tumor activity and long-term persistence.
  • Sequence Listing is disclosed on a computer-readable ASCII text file titled, “sequence_listing.txt”, created on Dec. 22, 2016.
  • the sequence-listing.txt file is 508 KB in size.

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US20240141041A1 (en) 2024-05-02
CA3029197A1 (fr) 2017-12-28
JP2019522973A (ja) 2019-08-22
EP3474867A1 (fr) 2019-05-01

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