US20170362297A1 - Chimeric antigen receptors and methods of use thereof - Google Patents

Chimeric antigen receptors and methods of use thereof Download PDF

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US20170362297A1
US20170362297A1 US15/537,779 US201515537779A US2017362297A1 US 20170362297 A1 US20170362297 A1 US 20170362297A1 US 201515537779 A US201515537779 A US 201515537779A US 2017362297 A1 US2017362297 A1 US 2017362297A1
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Wayne A. Marasco
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Dana Farber Cancer Institute Inc
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • the present invention relates generally to chimeric antigen receptor cells for and methods of using same for treatment cancer and other disorders.
  • T lymphocytes recognize specific antigens through interaction of the T cell receptor (TCR) with short peptides presented by major histocompatibility complex (MHC) class I or II molecules.
  • TCR T cell receptor
  • MHC major histocompatibility complex
  • APCs professional antigen-presenting cells
  • TCR activation in the absence of co-stimulation can result in unresponsiveness and clonal anergy.
  • APCs professional antigen-presenting cells
  • Chimeric antigen receptors have been constructed that consist of binding domains derived from natural ligands or antibodies specific for cell-surface components of the TCR-associated CD3 complex. Upon antigen binding, such chimeric antigen receptors link to endogenous signaling pathways in the effector cell and generate activating signals similar to those initiated by the TCR complex. Since the first reports on chimeric antigen receptors, this concept has steadily been refined and the molecular design of chimeric receptors has been optimized. Aided by advances in recombinant antibody technology, chimeric antigen receptors targeted to a wide variety of antigens on the surface of cancer cells and of cells infected by human immunodeficiency virus (HIV) have been generated.
  • HIV human immunodeficiency virus
  • the invention provides a chimeric antigen receptor (CAR) having an intracellular signaling domain, a transmembrane domain and an extracellular domain.
  • CAR chimeric antigen receptor
  • the transmembrane domain further includes a stalk region positioned between the extracellular domain and the transmembrane domain.
  • the transmembrane domain includes CD28.
  • the CAR further includes one or more additional costimulatory molecules positioned between the transmembrane domain and the intracellular signaling domain.
  • the costimulatory molecules is CD28, 4-1BB, ICOS, or OX40.
  • the intracellular signaling domain is for example a CD3 zeta chain.
  • the extracellular domain is an antibody such as a Fab or a scFV. Preferably the antibody is specific for BCMA, CA-9, CD138, CCR4, or the influenza virus.
  • nucleic acids encoding the CAR of the invention further including a nucleic acid encoding a polypeptide positioned after the intracellular signaling domain.
  • the polypeptide is an antibody such as scFV.
  • the antibody is specific for CCR4, PD-1, PDL-1, PD-L2, CXCR4, or GITR.
  • the invention provides a genetically engineered cell which express and bear on the cell surface membrane the chimeric antigen receptor of the invention.
  • the cell is a T-cell or an NK cell.
  • the T cell is CD4 + or CD8 + .
  • the cell is a mixed population of CD4 + and CD8 cells + .
  • the cell is further engineered to express and secrete a polypeptide such as for example an antibody.
  • FIG. 1 ADCC of CAIX-specific Abs. 1 ⁇ g/ml CAIX-specific scFv-Fc minibodies were added to the target tumor cells in the presence of human PBMC (E:T 25:1). Similar results were obtained in 2 experiments. Irrelevant anti-SARS scFv-Fc (11A) and anti-CCR4 scFv-Fc (48) minibodies were used as negative controls. A, CAIX+ sk-rc-09 cells; B, CAIX+ sk-rc-52 cells; C, CAIX ⁇ sk-rc-59 cells.
  • FIG. 2 Construction and expression of CAIX-specific CARs.
  • the 1 st generation CAR, scFv-CD8-TCR (CD8 CAR) is composed of a specific anti-CAIX scFv that is coupled to truncated human CD8 ⁇ extracellular domain, hinge (H), transmembrane (TM) and intracellular regions, then to the signaling domain of human TCR ⁇ .
  • the 2 nd generation CAR, scFv-CD28-TCR ⁇ (CD28 CAR), contains anti-CAIX scFv fused with human CD28 extracellular, TM and intracellular signaling domain to TCR ⁇ .
  • Both anti-CAIX CARs were cloned into a bicistronic self-inactivating (SIN) lentiviral vector with expression driven by an internal eF1- ⁇ promoter.
  • the CAR control construct contains an irrelevant anti-HIV CCRS specific A8 scFv substitution.
  • anti-CAIX scFv CARs were stained with CAIX-Fc fusion protein and C9-tag (TETSQVAPA) was stained with 1D4 antibody.
  • LAK only were served as unstained cell control (i) or stained with 2 nd antibody (ii. PE-anti-human IgG and iii. APC-anti-mouse IgG) were used as staining controls.
  • FIG. 3 Effector functions of CAIX-specific CARTs.
  • A Cytokine secretion. Anti-CAIX CART, irrelevant CART or activated control T cells (LAK) were cocultivated overnight with kidney cancer cell lines sk-rc-52 (CAIX+) and sk-rc-59 (CAIX ⁇ ) for cytokine production. One representative out of 2-3 results is shown.
  • B ELISPOT. G36 CART or control A8 CART cells were added to tumor cells overnight. IFN- ⁇ or granzyme B secreting T cells detected by ELISPOT. Similar results were obtained in 2-3 experiments.
  • C Cytokine secretion. Anti-CAIX CART, irrelevant CART or activated control T cells (LAK) were cocultivated overnight with kidney cancer cell lines sk-rc-52 (CAIX+) and sk-rc-59
  • FIG. 4 Clonal expansion of CART cells after tumor contact.
  • B Clonal enrichment. In tumor stimulation experiments, cultures from CART- and LAK cells were assayed on one week and two weeks by flow cytometry for expression of CART and T-cell subset. One representative of two results is shown.
  • FIG. 5 Regression of established human RCC xenografts by CART cells.
  • Athymic null mice were inoculated subcutaneously with 7.5 ⁇ 10 6 sk-rc-52 and 5 ⁇ 10 6 sk-rc-59 RCC tumor cells at left and right flank respectively.
  • mice were injected i.v. with 50 ⁇ 10 6 G36 CD28 CART cells, A8 CD28 CART cells ( ⁇ 20% CAR+), LAK, or PBS alone.
  • FIG. 6 In vivo anti-tumor activity of CAR+ T-cells.
  • A. Expression of ZsGreen by CART cells is shown in upper panel. CART cells were pre-stained with Far Red dye, cytospun and examined by fluorescent microscopy (lower panel).
  • the higher magnification view shows the locations of granzyme B+ T cells (shown by arrows) and the corresponding H&E slide shows the tumor necrosis (shown by n).
  • Granzyme B+ T cells are distributed at the edge of tumor (middle panel) and inside the tumor (lower panel).
  • FIG. 7 CAIX ⁇ sk-rc-52 tumors treated with control LAK cells showed negative granzyme B staining (left) (upper panel) and the corresponding histology was shown in H&E (right).
  • FIG. 8 Low background staining of granzyme B in CAIX ⁇ sk-rc-59 tumors treated with G36 CD28z CART cells
  • FIG. 9 Low background staining of granzyme B in CAIX ⁇ sk-rc-59 tumors treated with LAK cells.
  • FIG. 10 Positive control of granzyme B staining was performed on sk-rc-52 tumors which was local injected with G36 CD28z CART cells (left) and tumor morphology was shown in H&E (right).
  • FIG. 11 Expression of ⁇ CAIX CAR and ⁇ PD-L1 scFv-Fc transiently transfected 293T cells.
  • 10 6 of 293T cells were transfected with or without lentiviral pHAGE-EF1 ⁇ G36-C9tag-CD28-CD3zeta-IRES-anti-PDL1 scFv-Fc (IgG4)-WPRE or the control pHAGE-EF1 ⁇ -A716-C9tag-CD28-CD3zeta-IRES-ZsGreen-WPRE plasmids.
  • Cells and supernatant were harvested at 24 and 48 hours post transfection.
  • FIG. 12 (A) Total PBMCs from 4 donors were stimulated with 50 ng/ml SEB and treated with 10 ug/ml anti-PDL1 (#42) sIgG4 or anti-influenza sIgG4. Restoration of IFN ⁇ production (upper) or TNFa production (lower) was measured. (B) Parental anti-CCR4 antibody, c1567IgG, inhibited chemotaxis of Tregs to CCL22. (C) CD4 + CD25 ⁇ T cells were CFSE-labeled, incubated with Tregs at 10:1 ratio, and then stimulated with anti-CCR4 mAb and control mAb in the presence of anti-CD3/28 co-stimulation.
  • the CFSE-labeled Teffs were harvested and CFSE intensity was analyzed by flow cytometry. The proliferation of Teffs were only observed when the coculture incubated with anti-GITR mAb. Right, IFN ⁇ production in the same cultures was further measured by MSD.
  • CFSE-labeled Teffs (5 ⁇ 10 4 ) and unlabeled Tregs (5 ⁇ 10 3 ) were co-incubated with 20 ⁇ g/ml PHA in 96-well plates for 5 days.
  • the CFSE-labeled Teffs were harvested and CFSE intensity was analyzed by flow cytometry. Teffs were proliferated after 5-day incubation with PHA, but not in the Teff/Treg coculture. Left, IFN ⁇ production in the same cultures was measured by MSD.
  • FIG. 13 is an illustration showing different CART configurations according to the invention.
  • FIG. 14 is an illustration showing different armed CART configurations according to the invention.
  • FIG. 15 is a further illustration showing different armed CART configurations according to the invention.
  • FIG. 16 is a series of illustrations and graphs indicating chimeric antigen receptors (CAR) constructs for CD8+ T cell transduction.
  • CAR chimeric antigen receptors
  • the second cassette after the Internal Ribosome Entry Site (IRES) sequence, is responsible by coding the soluble Anti-PD-L1 IgG1 or IgG4 isotypes or the Anti-Severe Acute respiratory syndrome (SARS) coronavirus IgG1.
  • IRS Internal Ribosome Entry Site
  • SARS Anti-Severe Acute respiratory syndrome coronavirus IgG1.
  • the pHAGE vectors were transfected together with the packaging plasmids (Gag, Rev, Tat and VSVG) into 293T LentiX cells using Polyethyleneimine. The viruses were harvested two days after transfection, purified and concentrated using LentiX Concentrator (ClontechTM), according to the manufacturer instructions.
  • T cells were transduced with the lentiviruses to generate Anti-CAIX CART Cells, which are able to recognize CAIX positive RCC, and also release Anti-PD-L1 IgG1 or IgG4 in the tumor microenvironment to block PD-1/PD-L1-induced T cell exhaustion.
  • C Percentage of CART cells 14 days after transduction, representing the stable long-term expression of CAR by the integrated lentiviruses in CD8+ T cells.
  • the CD8+ T cells were selected using DynabeadsTM CD8 Positive isolation Kit (Life Technologies) and activated with DynabeadsTM Human T Cell Activator CD3/CD28 (Life Technologies) in the presence of IL-21 50 U/mL. IL-21 was added to the medium every 2 days. After 14 days, the CART cells were incubated with human CAIX-Fc or BCMA-Fc, followed by incubation with an APC conjugated anti-human Fc IgG and analyzed by FACS.
  • the CD8+ CART cells were previously cultivated for five days after transduction with Anti CAIX CAR able to express anti-PD-L1 IgG1 (Anti-CAIX/Anti-PD-L1 IgG1), IgG4 (Anti-CAIX/Anti-PD-L1 IgG4) or a unspecific Anti-SARS Ab (Anti-CAIX/Anti SARS IgG1) or Anti-BCMA CAR (negative control) able to express an unspecific anti-SARS Ab (Anti-BCMA/Anti SARS IgG1) and activated with Dynabeads Human T Activator CD3/CD28 (Life Technologies) in the presence of IL-21 50 U/mL.
  • the beads were removed and the CART cells were cultured with skrc52 CAIX+PD-L1 ⁇ and IL-21 (50 U/mL), which was added to the medium every 2 days for 21 days.
  • the results represent the average ⁇ SD of three donors in duplicate.
  • FIG. 17 is a series of graphs that illustrate CART cell effector function.
  • A Viability of Skrc59 CAIX+/PD-L1+ cells or
  • B Skrc52 CAIX ⁇ /PD-L1 ⁇ cells incubated overnight (O.N.) with CART cells Anti-CAIX/Anti-PD-L1 IgG1, Anti-CAIX/Anti-PD-L1 IgG4, Anti-CAIX/Anti SARS IgG1 or Anti-BCMA/Anti SARS IgG1.
  • CART cells were used 4 days after lentiviral transduction. The cell viability was evaluated by MTT (Molecular ProbesTM).
  • the IFN ⁇ secretion was evaluated using the Human IFN ⁇ ELISA Ready-SET-Go Kit (eBiosciencesTM). *P ⁇ 0.05 for all CART cells compared to Anti-BCMA/Anti-SARS IgG1.
  • G Ab-dependent cell-mediated cytotoxicity of skrc59 CAIX+/PD-L1+ or
  • H skrc52 CAIX ⁇ /PD-L1 ⁇ after incubation with the supernatant (SN) of CART cells containing 500 ng/mL of the Anti-PD-L1 IgG1, Anti-PD-L1 IgG4 or the Anti-SARS IgG1.
  • CART cells were incubated for six days with Dynabeads Human T Activator CD3/CD28 (Life Technologies) in the presence of IL-21 50 U/mL. After 7 days, the medium containing the mAbs was harvested NK cells were purified using an EasySepTM Human NK Cell Enrichment Kit (StemCell Technologies). RCC cell lines Skrc59 CAIX+PD-L1+ and Skrc52 CAIX ⁇ PD-L1 ⁇ were used as target cells and plated at 1.5 ⁇ 10 3 /well in a 96-well plate.
  • RCC cells were incubated for 1 hour, 37° C., with 50 ⁇ L of the CART cells supernatant adjusted for 500 ng/mL of the respective Ab Anti-PD-L1 IgG1, Anti-PD-L1 IgG4 or Anti-SARS IgG1. After the incubation, the cells were washed with medium and incubated with 12.5:1, 25:1 or 50:1 NK cells for 4 h, 37° C. Culture supernatants were harvested by centrifugation and LDH measured in the supernatant by CytoTox 96® Non-Radioactive Cytotoxicity Assay (PromegaTM) at 490 nm. These results represent the average ⁇ SD of three donors in duplicate.
  • FIG. 18 is a series of graphs that depict CART cells expression of exhaustion markers.
  • the CD8+ CART cells were selected using DynabeadsTM CD8 Positive isolation Kit (Life Technologies), activated with DynabeadsTM Human T Activator CD3/CD28 (Life Technologies) and transduced with the following CARs: Anti-CAIX/Anti-PD-L1 IgG1, Anti-CAIX/Anti-PD-L1 IgG4, Anti-CAIX/Anti SARS IgG1 or Anti-BCMA/Anti SARS IgG1. These cells were cultured in the presence of IL-21 50 U/mL and Dynabeads Human T Activator CD3/CD28 for five days. After this period the CART cells were co-cultured with Skrc-59 CAIX+PD-L1+ for 2 days in order to stimulate exhaustion.
  • CART cells were stained with FITC-conjugated anti-human PD-1, PE-conjugated anti-human Tim3 and PerCP/Cy5.5 anti-human Lag3 and analyzed by FACS.
  • D Viability of Skrc59 CAIX positive/PD-L1 positive cells after incubation with exhausted CART cells. The cell viability was evaluated by MTT (Molecular Probes). *P ⁇ 0.05 compared to both Anti-CAIX/Anti-SARS IgG1 and Anti-BCMA/Anti-SARS IgG1. **P ⁇ 0.05 compared to Anti-CAIX/Anti-PD-L1 IgG1. These results represent the average ⁇ SD of three donors.
  • FIG. 19 is a series of images and graphs that depict the effect that CART cells have in an orthotopic model of human RCC.
  • the tumor bioluminescence was quantified after 5 minutes of luciferin IP injection using IVIS.
  • C Tumor growth curve.
  • FIG. 20 is a series of graphs and histological images that depict antitumor activity from CART cells.
  • TIL tumor infiltrating lymphocytes
  • the kidney tumors from all mice were divided in two parts and one of them was fragmented in small pieces and digested with collagenase and DNAse to extraction of TIL.
  • the CART cells were analyzed for the exhaustion markers PD-1, Tim-3 and Lag3. *P ⁇ 0.05 compared with untransduced, Anti-BCMA/Anti SARS IgG1 CAR and Anti-CAIX/Anti-SARS IgG1.
  • B Detection of Ki67 as a tumor cell proliferation marker and granzyme B to analyze CART cells activity in the tissue.
  • the scale bars represent the magnification of the images of each column [500 ⁇ m (40 ⁇ ), 100 ⁇ m (200 ⁇ ) or 50 ⁇ m (400 ⁇ )].
  • C A series of graphs present percentage quantification of TIL staining positive for Granzyme B, PD-L1-IHC, and Ki67. Also presented in (C) is a Ki67-DAB Pixel count of TILs.
  • FIG. 21 is a series of graphs that depict the evaluation of IL-2 versus IL-21 to CD8+ CART cells proliferation.
  • a and B Proliferation of CART transduced cells in the presence of IL-2 or IL-21 evaluated 48 h, 72 h and 120 h after transduction with lentiviruses.
  • A Anti-CAIX CART cells or
  • B Unspecific Anti-BCMA CART cells (both with ZsGreen in the second cassette).
  • the CD8+ T cells were selected using DynabeadsTM CD8 Positive isolation Kit (Life Technologies) and activated with DynabeadsTM Human T Activator CD3/CD28 (Life Technologies) in the presence of IL2 or IL-21 50 U/mL (PeprotechTM)
  • the CART cells transduction was evaluated by ZsGreen expression using FACS. The data represents the average ⁇ SD of two donors. *p ⁇ 0.05 comparing IL-21 with non treated control (Ctr); **p ⁇ 0.05 comparing IL21 with IL-2.
  • C and D Viability of RCC cells treated with CD8+ CART cells cultivated in the presence of IL-2 or IL-21.
  • the viability was evaluated by MTT after an overnight incubation of CART cells Anti-BCMA, Anti-CAIX or Untransduced T cells with (C) Skrc-59 CAIX+/PD-L1+ and (F) Skrc-52 CAIX ⁇ /PD-L1 ⁇ RCC cells.
  • the CART cells were previously cultivated in the presence of IL2 or IL-21 50 U/mL for 120 hours. These results represent the average ⁇ SD of two donors in triplicate. *P ⁇ 0.05 comparing Anti-CAIX CAR with Anti-BCMA CAR or untransduced T cells.
  • FIG. 22 is a series of flow cytometry graphs that depict the expression of PD-1 and CAIX in the renal cell carcinoma (RCC) lines.
  • A Negative control
  • B Skrc52 CAIX-PD-L1 ⁇
  • C Skrc52 CAIX+PD-L1 ⁇
  • D Skrc59 CAIX+PD-L1+.
  • the cells were stained with Anti-human CAIX antibody developed with APC-Anti-human Fc IgG and Biotinylated Anti human PD-L1 antibody developed with PE-Avidin. The analysis was performed by FACS.
  • FIG. 23 is a series of graphs that depict the characterization of CART cells.
  • A Proliferation of total CD8+ T cells two or four days after transduction Anti CAIX CAR/Anti-PD-L1 IgG1), (Anti-CAIX CAR/Anti-PD-L1 IgG4), Anti-CAIX CAR/Anti SARS IgG1) or Anti-BCMA CAR/Anti SARS IgG1).
  • the CD8+ T cells were selected using DynabeadsTM CD8 Positive isolation Kit (Life Technologies) and activated with DynabeadsTM Human T Activator CD3/CD28 (Life Technologies) in the presence of IL-21 50 U/mL. IL-21 was added to the medium every 2 days.
  • the proliferation was evaluated by FACS using Counting Beads (Molecular Probes).
  • B Concentration of CAR-transduced T cells two and four days after transduction. The CART cells were incubated with human CAIX-Fc or BCMA-Fc, followed by incubation with an APC conjugated anti-human Fc IgG and analyzed by FACS.
  • C Percentage of CART cells 2 and 4 days after transduction. The results represent the average ⁇ SD of three donors in duplicate.
  • FIG. 24 is a series of graphs that depict the effect that CART cells have in an orthotopic model of human RCC.
  • the tumor bioluminescence was quantified after 5 minutes of luciferin IP injection using IVIS. In the Day 17, more 2.5 ⁇ 106 CART cells were injected.
  • **P ⁇ 0.05 compared to untransduced T cells ***P ⁇ 0.05 compared to Anti-CAIX/Anti-SARS IgG1.
  • TIL Total tumor infiltrating lymphocytes
  • FIGS. 25A and 25B are a series of graphs and histological images that depict Human natural killer (NK) cells in the tumors treated with Anti-CAIX CART cells releasing anti-PD-L1 IgG1 Ab.
  • A Percentage of CD56+ cells (NK marker) in the tumors. Two mice of each group were injected with 4.5 ⁇ 106 NK cells one day before the euthanasia. The kidney tumors from all mice were divided in two parts and one of them was fragmented in small pieces and digested with collagenase and DNAse to extraction of NK. NK cells present in the tumor were stained with APC-Anti-CD56 Ab and analyzed by FACS. *P ⁇ 0.05.
  • the slides were developed using 3,3′-diaminobenzidine (DAB) and counterstained with hematoxylin.
  • DAB 3,3′-diaminobenzidine
  • the images were obtained in an Olympus BX51 microscopy using a DP71 digital camera (Olympus) and analyzed in the DP Controller Software (Olympus).
  • the quantification was performed using the IHC Profiler Plugin of ImageJ Software (23).
  • the scale bars represent the magnification of the images (400 ⁇ ). *P ⁇ 0.05 compared with untransduced, **P ⁇ 0.05 compared with untransduced and Anti-BCMA/Anti-SARS IgG1.
  • the present invention relates to a chimeric antigen receptor (CAR) particularly adapted to immune cells used in immunotherapy.
  • CAR chimeric antigen receptor
  • a double immunotherapeutic strategy is described based on the blocking of T cell exhaustion using anti-PD-L1 antibodies secreted by targeted anti-CAIX CAR T cells combined in a single lentivirus construct for improving cancer treatment.
  • An emerging mechanism associated with the progression of RCC and other tumors is the immune checkpoint pathway, which consists in cellular interactions that prevent excessive activation of T cells under normal conditions, allowing T cell function in a self-limited manner.
  • immune checkpoint pathway As an evasion mechanism, many tumors are able to stimulate the expression of immune checkpoint molecules, resulting in an anergic phenotype of T cells that cannot restrain tumor progression.
  • Emerging clinical data highlight the importance of one inhibitory ligand and receptor pair as an immune checkpoint: the programmed death-ligand 1 (PD-L1; B7-H1 and CD274) and programmed death receptor-1 (PD-1; CD279), in preventing killing of cancer cells by cytotoxic T-lymphocytes.
  • PD1 receptor is expressed by many cell types like T cells, B cells, Natural Killer cells (NK) and host tissues. Tumors and Antigen-presenting cells (APC) expressing PD-L1 can block T cell receptor (TCR) signaling of cytotoxic T-lymphocytes through binding to receptor PD-1, decreasing the production of cytokines and T cell proliferation.
  • TCR T cell receptor
  • PD-L1 overexpression can be found in many tumor types and may also mediate an immunosuppressive function through its interaction with other proteins, including CD80 (B7.1), blocking its ability to activate T cells through binding to CD28.
  • CAR tumor-directed chimeric antigen receptors
  • CARTS lymphocytes
  • a single CAR targets two or more antigens.
  • the CARTS are further modified to express and secrete one or more polypeptides, such as for example an antibody or a cytokine.
  • Such CARTS are referred to herein as armed CARTS. Armed CARTS allow for simultaneous secretion of the polypeptide locally at the targeted site (i.e., tumor site).
  • CAR chimeric antigen receptor
  • scFv single chain variable antibody fragment
  • CART cells antigen-presenting cells
  • the lymphocytes include a receptor that is chimeric, non-natural and engineered at least in part by the hand of man.
  • the engineered chimeric antigen receptor has one, two, three, four, or more components, and in some embodiments the one or more components facilitate targeting or binding of the lymphocyte to one or more tumor antigen-comprising cancer cells.
  • the CAR according to the invention generally comprises at least one transmembrane polypeptide comprising at least one extracellular ligand-biding domain and; one transmembrane polypeptide comprising at least one intracellular signaling domain; such that the polypeptides assemble together to form a Chimeric Antigen Receptor.
  • extracellular ligand-binding domain is defined as an oligo- or polypeptide that is capable of binding a ligand.
  • the domain will be capable of interacting with a cell surface molecule.
  • the extracellular ligand-binding domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state.
  • the extracellular ligand-binding domain can comprise an antigen binding domain derived from an antibody against an antigen of the target.
  • the antigen of the target can be a tumor-associated surface antigen, such as ErbB2 (HER2/neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, .beta.-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostase, prostase specific antigen (PSA), P
  • the CAR is specific for BMCA, CAIX, CCR4, PD-L1, PD-L2, PD1, Glucocorticoid-Induced Tumor Necrosis Factor Receptors (GITR), Severe acute respiratory syndrome (SARS), influenza, flavivirus or Middle East Respiratory Syndrome (MERS).
  • GMCA Tumor Necrosis Factor Receptors
  • SARS Severe acute respiratory syndrome
  • influenza influenza
  • flavivirus or Middle East Respiratory Syndrome MERS.
  • said extracellular ligand-binding domain is a single chain antibody fragment (scFv) comprising the light (V L ) and the heavy (V H ) variable fragment of a target antigen specific monoclonal antibody joined by a flexible linker.
  • scFv single chain antibody fragment
  • said scFv antibody is specific for BMCA, CAIX, CCR4, PD-L1, PD-L2, PD1, GITR, SARS, influenza, flavivirus or MERS.
  • Exemplary antibodies useful in constructing the CAR according to the invention includes antibodies disclosed in for example: WO/2005/060520, WO/2006/089141, WO/2007/065027, WO/2009/086514, WO/2009/079259, WO/2011/153380, WO/2014/055897, WO 2015/143194, WO 2015/164865, WO 2013/166500, and WO 2014/144061; PCT/US2015/054202, PCT/US2015/054010 and 62/144,729 the contents of which are hereby incorporated by reference in their entireties.
  • Exemplary anti-PDL1 antibodies include antibodies having containing a VH nucleotide sequence having SEQ ID NO: 1485 and a VL nucleotide sequence having SEQ ID NO: 1487; a VH nucleotide sequence having SEQ ID NO: 1485 and a VL nucleotide sequence having SEQ ID NO: 1487; a VH nucleotide sequence having SEQ ID NO: 1489 and a VL nucleotide sequence having SEQ ID NO: 1491; a VH nucleotide sequence having SEQ ID NO: 1493 and a VL nucleotide sequence having SEQ ID NO: 1495; a VH nucleotide sequence having SEQ ID NO: 1497 and a VL nucleotide sequence having SEQ ID NO: 1499; a VH nucleotide sequence having SEQ ID NO: 1501 and a VL nucleotide sequence having SEQ ID NO: 1503; a VH nucleotide sequence having SEQ
  • Exemplary anti-PDL1 antibodies include antibodies having containing a VH amino acid sequence having SEQ ID NO: 970 and a VL amino acid sequence having SEQ ID NO: 971; a VH amino acid having SEQ ID NO: 1486 and a VL polypeptide sequence having SEQ ID NO: 1488 a VH amino acid having SEQ ID NO: 1490 and a VL polypeptide sequence having SEQ ID NO: 1492 a VH amino acid having SEQ ID NO: 1494 and a VL polypeptide sequence having SEQ ID NO: 1496 a VH amino acid having SEQ ID NO: 1498 and a VL polypeptide sequence having SEQ ID NO: 1500 a VH amino acid having SEQ ID NO: 1502 and a VL polypeptide sequence having SEQ ID NO: 1504 a VH amino acid having SEQ ID NO: 1506 and a VL polypeptide sequence having SEQ ID NO: 1508 a VH amino acid having SEQ ID NO: 1510 and a VL polypeptide sequence having
  • the anti-PDL1 antibodies have a heavy chain with three CDRs including the amino acid sequences SEQ ID NO: 1541, 1554, 1569 respectively and a light chain with three CDRs including the amino acid sequences 1584, 1599, 1610 respectively; or a heavy chain with three CDRs comprising the amino acid sequences 1543, 1556, 1571 and a light chain with three CDRs comprising the amino acid sequences 1586, 1600, 1612; or a heavy chain with three CDRs comprising the amino acid sequences 1544, 1557, 1572 and a light chain with three CDRs comprising the amino acid sequences 1587, 1601, 1613; or a heavy chain with three CDRs comprising the amino acid sequences 1545, 1558, 1573 and a light chain with three CDRs comprising the amino acid sequences 1588, 1602, 1614; or a heavy chain with three CDRs comprising the amino acid sequences 1546, 1559, 1574 and a light chain with three CDRs comprising the
  • Exemplary SARS neutralizing antibodies include antibodies having containing a VH nucleotide sequence having SEQ ID NO: 1626 and a VL nucleotide sequence having SEQ ID NO: 1628; a VH nucleotide sequence having SEQ ID NO: 1630 and a VL nucleotide sequence having SEQ ID NO: 1639; a VH nucleotide sequence having SEQ ID NO: 1634 and a VL nucleotide sequence having SEQ ID NO: 1640; a VH nucleotide sequence having SEQ ID NO: 1632 and a VL nucleotide sequence having SEQ ID NO: 1641; a VH nucleotide sequence having SEQ ID NO: 1633 and a VL nucleotide sequence having SEQ ID NO: 1642; a VH nucleotide sequence having SEQ ID NO: 1634 and a VL nucleotide sequence having SEQ ID NO: 1643; a VH nucleotide sequence having SEQ ID
  • Exemplary anti-CXCR4 antibody include antibodies having a VH amino acid sequence having SEQ ID NO: 771 and a VL amino acid sequence having SEQ ID NO: 779; a VH amino acid sequence having SEQ ID NO: 772 and a VL amino acid sequence having SEQ ID NO: 780; a VH amino acid sequence having SEQ ID NO: 773 and a VL amino acid sequence having SEQ ID NO: 781; a VH amino acid sequence having SEQ ID NO: 774 and a VL amino acid sequence having SEQ ID NO: 782; a VH amino acid sequence having SEQ ID NO: 775 and a VL amino acid sequence having SEQ ID NO: 783; a VH amino acid sequence having SEQ ID NO: 776 and a VL amino acid sequence having SEQ ID NO: 784; a VH amino acid sequence having SEQ ID NO: 777 and a VL amino acid sequence having SEQ ID NO: 785; or a VH amino acid sequence having SEQ ID NO: 778 and
  • the anti-CXCR4 antibodies have a heavy chain with three CDRs including the amino acid sequences SEQ ID NO: 803, 804, 805 respectively and a light chain with three CDRs including the amino acid sequences 806, 807, 808 respectively; or a heavy chain with three CDRs comprising the amino acid sequences 809, 810, 811, respectively and a light chain with three CDRs comprising the amino acid sequences 812, 813, 814, respectively; or a heavy chain with three CDRs comprising the amino acid sequences 815, 816, 817 respectively and a light chain with three CDRs comprising the amino acid sequences 818, 819, 820 respectively; or a heavy chain with three CDRs comprising the amino acid sequences 827, 828, 829 respectively and a light chain with three CDRs comprising the amino acid sequences 830, 831, 832 respectively; or a heavy chain with three CDRs comprising the amino acid sequences 833, 834, 835, respectively and
  • Exemplary anti-CA IX antibodies include antibodies having containing a VH amino acid sequence having SEQ ID NO: 845 and a VL amino acid sequence having SEQ ID NO: 846; a VH amino acid sequence having SEQ ID NO: 847 and a VL amino acid sequence having SEQ ID NO: 868; a VH amino acid sequence having SEQ ID NO: 848 and a VL amino acid sequence having SEQ ID NO: 869; a VH amino acid sequence having SEQ ID NO: 849 and a VL amino acid sequence having SEQ ID NO: 870; a VH amino acid sequence having SEQ ID NO: 850 and a VL amino acid sequence having SEQ ID NO: 871; a VH amino acid sequence having SEQ ID NO: 851 and a VL amino acid sequence having SEQ ID NO: 872; a VH amino acid sequence having SEQ ID NO: 852 and a VL amino acid sequence having SEQ ID NO: 873; a VH amino acid sequence having SEQ ID NO: 853 and
  • the anti-CA IX antibodies have a heavy chain with three CDRs including the amino acid sequences SEQ ID NO: 803, 804, 805 respectively and a light chain with three CDRs including the amino acid sequences 806, 807, 808 respectively; or a heavy chain with three CDRs comprising the amino acid sequences 899, 915, 909 and a light chain with three CDRs comprising the amino acid sequences 905, 906, 952 or a heavy chain with three CDRs comprising the amino acid sequences 899, 915, 909 and a light chain with three CDRs comprising the amino acid sequences 935, 943, 953 or a heavy chain with three CDRs comprising the amino acid sequences 899, 915, 909 and a light chain with three CDRs comprising the amino acid sequences 935, 906, 954 or a heavy chain with three CDRs comprising the amino acid sequences 910, 916, 923 and a light chain with three CDRs comprising the amino acid sequences 936
  • Exemplary CC-chemokine receptor 4 (CCR4) antibodies include antibodies having containing a VH nucleotide sequence having SEQ ID NO: 969 and a VL nucleotide sequence having SEQ ID NO: 971; a VH nucleotide sequence having SEQ ID NO: 969 and a V L nucleotide sequence having SEQ ID NO: 972.
  • Exemplary CCR4 antibodies include antibodies having containing a VH amino acid sequence having SEQ ID NO: 970 and a V L amino acid sequence having SEQ ID NO: 971.
  • the CCR4 antibodies have a heavy chain with three CDRs including the amino acid sequences SEQ ID NO: 973, 974, 975 respectively and a light chain with three CDRs including the amino acid sequences 976, 977, 978 respectively.
  • MERS-CoV Middle East Respiratory Syndrome Coronavirus
  • Exemplary anti-Middle East Respiratory Syndrome coronavirus (MERS-CoV) antibody include antibodies having a VH nucleotide sequence having SEQ ID NO: 677and a VL nucleotide sequence having SEQ ID NO:679; a VH nucleotide sequence having SEQ ID NO: 681and a VL nucleotide sequence having SEQ ID NO:683; a VH nucleotide sequence having SEQ ID NO: 685and a VL nucleotide sequence having SEQ ID NO:687; a VH nucleotide sequence having SEQ ID NO: 689and a VL nucleotide sequence having SEQ ID NO:692; a VH nucleotide sequence having SEQ ID NO: 693and a VL nucleotide sequence having SEQ ID NO:695; a VH nucleotide sequence having SEQ ID NO: 697and a VL nucleotide sequence having SEQ ID NO:699
  • Exemplary anti-Middle East Respiratory Syndrome coronavirus (MERS-CoV) antibody include antibodies having a VH amino acid sequence SEQ ID NO: 678 and a VL amino acid sequence having SEQ ID NO: 680; a VH amino acid sequence SEQ ID NO: 682 and a VL amino acid sequence having SEQ ID NO: 684; a VH amino acid sequence SEQ ID NO: 686 and a VL amino acid sequence having SEQ ID NO: 688; a VH amino acid sequence SEQ ID NO: 690 and a VL amino acid sequence having SEQ ID NO: 692; a VH amino acid sequence SEQ ID NO: 694 and a VL amino acid sequence having SEQ ID NO: 696; a VH amino acid sequence SEQ ID NO: 698 and a VL amino acid sequence having SEQ ID NO: 700; and a VH amino acid sequence SEQ ID NO: 702 and a VL amino acid sequence having SEQ ID NO: 704.
  • the anti-Middle East Respiratory Syndrome coronavirus (MERS-CoV) antibody has a heavy chain with three CDRs including the amino acid sequences of 705, 706, and 707 and a light chain with three CDRs including the amino acid sequences 722, 723, and 724; a heavy chain with three CDRs including the amino acid sequences of 708, 709, and 710 and a light chain with three CDRs including the amino acid sequences 725, 726, and 727; a heavy chain with three CDRs including the amino acid sequences of 711, 712, and 713 and a light chain with three CDRs including the amino acid sequences 728, 729, and 730; a heavy chain with three CDRs including the amino acid sequences of 711, 735, and 715 and a light chain with three CDRs including the amino acid sequences 731, 732, and 733; a heavy chain with three CDRs including the amino acid sequences of 711, 735, and 716 and a
  • Exemplary anti-human GITR antibody include antibodies having a VH nucleotide sequence having SEQ ID NO: 1361 and a VL nucleotide sequence having SEQ ID NO: 1363; a VH nucleotide sequence having SEQ ID NO: 1365 and a VL nucleotide sequence having SEQ ID NO:1367; a VH nucleotide sequence having SEQ ID NO: 1369 and a VL nucleotide sequence having SEQ ID NO: 1371; a VH nucleotide sequence having SEQ ID NO: 1381 and a VL nucleotide sequence having SEQ ID NO: 1375; a VH nucleotide sequence having SEQ ID NO: 1377 and a VL nucleotide sequence having SEQ ID NO: 1379; a VH nucleotide sequence having SEQ ID NO: 1381 and a VL nucleotide sequence having SEQ ID NO: 1383; a VH nucleotide sequence having SEQ ID
  • Exemplary anti-human GITR antibody include antibodies having a VH amino acid sequence having SEQ ID NO: 1362 and a VL amino acid sequence having SEQ ID NO: 1364; a VH amino acid having SEQ ID NO: 1366 and a VL polypeptide sequence having SEQ ID NO:1368; a VH amino acid sequence having SEQ ID NO: 1371 and a VL amino acid sequence having SEQ ID NO: 1372; a VH amino acid sequence having SEQ ID NO: 1382 and a VL amino acid sequence having SEQ ID NO: 1376; a VH nucleotide sequence having SEQ ID NO: 1378 and a VL nucleotide sequence having SEQ ID NO: 1380; a VH amino acid having SEQ ID NO: 1382 and a VL polypeptide sequence having SEQ ID NO: 1384; a VH amino acid sequence having SEQ ID NO: 1386 and a VL amino acid sequence having SEQ ID NO: 1388; a VH amino acid sequence having SEQ ID NO
  • the anti-human GITR antibody has a heavy chain with three CDRs including the amino acid sequences 1405, 1406, and 1407 and a light chain with three CDRs including the amino acid sequences1408, 1409, and 1410 respectively; a heavy chain with three CDRs including the amino acid sequences 1411, 1412, and 1413 and a light chain with three CDRs including the amino acid sequences1414, 1415, and 1416 respectively; a heavy chain with three CDRs including the amino acid sequences 1417, 1418, and 1419 and a light chain with three CDRs including the amino acid sequences1420, 1421, and 1422 respectively; a heavy chain with three CDRs including the amino acid sequences 1423, 1424, and 1425 and a light chain with three CDRs including the amino acid sequences1426, 1427, and 1428 respectively; a heavy chain with three CDRs including the amino acid sequences 1429, 1430, and 1431 and a light chain with three CDRs including the amino acid sequences143
  • Exemplary anti-West Nile virus envelope protein E (WINE) antibody include antibodies having a VH nucleotide sequence having a VH amino acid sequence having SEQ ID NO: 1224 and a VL amino acid sequence having SEQ ID NO: 1226.
  • Exemplary anti-West Nile virus envelope protein E (WINE) antibody include antibodies having a VH nucleotide sequence having SEQ ID NO: 1225 and a VL nucleotide sequence having SEQ ID NO: 1227.
  • the anti-West Nile virus envelope protein E (WNE) antibody has a heavy chain with three CDRs including the amino acid sequences 1244, 1245, and 1246 and a light chain with three CDRs including the amino acid sequences 1247, 1248, and 1249 respectively.
  • Exemplary anti-CC-chemokine receptor 4 (CCR4) antibody include antibodies having a VH nucleotide sequence having SEQ ID NO: 1329 and a VL nucleotide sequence having SEQ ID NO: 1331; a VH nucleotide sequence having SEQ ID NO: 1333 and a V L nucleotide sequence having SEQ ID NO:1335; a VH nucleotide sequence having SEQ ID NO: 1337 and a V L nucleotide sequence having SEQ ID NO: 1192; a VH nucleotide sequence having SEQ ID NO: 1341 and a V L nucleotide sequence having SEQ ID NO: 1343; or a VH nucleotide sequence having SEQ ID NO: 1357 and a V L nucleotide sequence having SEQ ID NO:1359.
  • Exemplary anti-CC-chemokine receptor 4 (CCR4) antibody include antibodies having a V H amino acid sequence having SEQ ID NO: 1330 and a V L amino acid sequence having SEQ ID NO: 1332; a V H amino acid sequence having SEQ ID NO: 1334 and a V L amino acid sequence having SEQ ID NO: 1336; a V H amino acid sequence having SEQ ID NO: 1338 and a V L amino acid sequence having SEQ ID NO: 1340; a V H amino acid sequence having SEQ ID NO: 1342 and a V L amino acid sequence having SEQ ID NO: 1344; or a V H amino acid sequence having SEQ ID NO: 1358 and a V L amino acid sequence having SEQ ID NO: 1360.
  • the anti-CC-chemokine receptor 4 (CCR4) antibody has a heavy chain with three CDRs including the amino acid sequences 1203, 1208, and 1211 and a light chain with three CDRs including the amino acid sequences 1207, 1209, and 1216 respectively; or a heavy chain with three CDRs including the amino acid sequences 1204, 1208, and 1212 and a light chain with three CDRs including the amino acid sequences 1207, 1209, and 1217 respectively; or a heavy chain with three CDRs including the amino acid sequences 1204, 1208, and 1213 and a light chain with three CDRs including the amino acid sequences 1207, 1209, and 1217 respectively; or a heavy chain with three CDRs including the amino acid sequences 1205, 1208, and 1214 and a light chain with three CDRs including the amino acid sequences 1207, 1209, and 1218 respectively; or a heavy chain with three CDRs including the amino acid sequences 1206, 1208, and 1210 and a light chain with three CDRs including the amino acid sequences
  • Exemplary anti-human immunoglobulin heavy chain variable region germline gene VH1-69 antibody include antibodies having a VH nucleotide sequence having SEQ ID NO: 1153 and a VL nucleotide sequence having SEQ ID NO: 1155; or a VH nucleotide sequence having SEQ ID NO: 1163 and a VL nucleotide sequence having SEQ ID NO:1155.
  • Exemplary anti-human immunoglobulin heavy chain variable region germline gene VH1-69 antibody include antibodies having a V H amino acid sequence having SEQ ID NO: 1154 and a V L amino acid sequence having SEQ ID NO: 1156; or a V H amino acid sequence having SEQ ID NO: 1164 and a V L amino acid sequence having SEQ ID NO: 1156.
  • the anti-human immunoglobulin heavy chain variable region germline gene VH1-69 antibody has a heavy chain with three CDRs including the amino acid sequences 1157, 1158, and 1159 and a light chain with three CDRs including the amino acid sequences 1160, 1161, and 1162 respectively.
  • Exemplary anti-influenza antibody include antibodies having a VH nucleotide sequence having SEQ ID NO: 981 and a VL nucleotide sequence having SEQ ID NO: 983; a VH nucleotide sequence having SEQ ID NO: 985 and a VL nucleotide sequence having SEQ ID NO: 989; a VH nucleotide sequence having SEQ ID NO: 987 and a VL nucleotide sequence having SEQ ID NO: 991; a VH nucleotide sequence having SEQ ID NO: 993 and a VL nucleotide sequence having SEQ ID NO: 997; a VH nucleotide sequence having SEQ ID NO: 995 and a VK nucleotide sequence having SEQ ID NO: 999; a VH nucleotide sequence having SEQ ID NO: 1001 and a VL nucleotide sequence having SEQ ID NO: 1005; a VH nucleotide sequence having SEQ ID
  • Exemplary anti-influenza antibody include antibodies having a VH amino acid sequence having SEQ ID NO: 982 and a VL amino acid sequence having SEQ ID NO: 984; a VH amino acid sequence having SEQ ID NO: 986 and a VL amino acid sequence having SEQ ID NO: 988; a VH amino acid sequence having SEQ ID NO: 986 and a VL amino acid sequence having SEQ ID NO: 990; a VH amino acid sequence having SEQ ID NO: 992 and a VL amino acid sequence having SEQ ID NO: 994; a VH amino acid sequence having SEQ ID NO: 992 and a VK amino acid sequence having SEQ ID NO: 996; a VH amino acid sequence having SEQ ID NO: 998 and a VL amino acid sequence having SEQ ID NO: 1000; a VH amino acid sequence having SEQ ID NO: 998 and a VL amino acid sequence having SEQ ID NO: 1002; a VH amino acid sequence having SEQ ID NO: 1004 and a V
  • the anti-influenza antibody has a heavy chain with three CDRs including the amino acid sequences of 1023, 1031, and 1039 and a light chain with three CDRs including the amino acid sequences 1047, 1059, and 1071; a heavy chain with three CDRs including the amino acid sequences of 1023, 1032, and 1040 and a light chain with three CDRs including the amino acid sequences 1048, 1060, and 1072; a heavy chain with three CDRs including the amino acid sequences of 1025, 1032, and 1040 and a light chain with three CDRs including the amino acid sequences 1057, 1069, and 1081; a heavy chain with three CDRs including the amino acid sequences of 1026, 1033, and 1041 and a light chain with three CDRs including the amino acid sequences 1049, 1061, and 1073; a heavy chain with three CDRs including the amino acid sequences of 1026, 1033, and 1041 and a light chain with three CDRs including the amino acid sequences
  • Exemplary anti-influenza antibodies include antibodies having containing a VH nucleotide sequence having SEQ ID NO: 397 and a nucleotide sequence having SEQ ID NO: 398; a VH nucleotide sequence having SEQ ID NO: 399 and a V L nucleotide sequence having SEQ ID NO:400; a VH nucleotide sequence having SEQ ID NO: 401 and a V L nucleotide sequence having SEQ ID NO: 402; a VH nucleotide sequence having SEQ ID NO: 403 and a VL nucleotide sequence having SEQ ID NO: 404; or a VH nucleotide sequence having SEQ ID NO: 405 and a VL nucleotide sequence having SEQ ID NO:406; or a VH nucleotide sequence having SEQ ID NO: 407 and a VL nucleotide sequence having SEQ ID NO:408; or a VH nucleotide sequence having S
  • Exemplary anti-influenza antibodies antibody include antibodies having containing a VH amino acid sequence having SEQ ID NO: 469 and a VL amino acid sequence having SEQ ID NO: 470; a VH amino acid having SEQ ID NO: 471 and a V L polypeptide sequence having SEQ ID NO:472; a VH amino acid sequence having SEQ ID NO: 473 and a V L amino acid sequence having SEQ ID NO: 474; a VH amino acid sequence having SEQ ID NO: 475 and a VL amino acid sequence having SEQ ID NO: 476; or a VH nucleotide sequence having SEQ ID NO: 477 and a VL nucleotide sequence having SEQ ID NO:478; a VH amino acid sequence having SEQ ID NO: 479 and a VL amino acid sequence having SEQ ID NO: 480; a VH amino acid sequence having SEQ ID NO: 481 and a VL amino acid sequence having SEQ ID NO: 482; a VH amino acid sequence having
  • the anti-influenza antibodies antibody has a heavy chain with three CDRs including the amino acid sequences SEQ ID NO: 1, 37, 73 respectively and a light chain with three CDRs including the amino acid sequences 109, 145, 181 respectively; or a heavy chain with three CDRs comprising the amino acid sequences 2, 38, 74 respectively and a light chain with three CDRs comprising the amino acid sequences 110, 146, 182, respectively; or a heavy chain with three CDRs comprising the amino acid sequences 3, 39, 75 respectively and a light chain with three CDRs comprising the amino acid sequences 111, 147, 183, respectively; or a heavy chain with three CDRs comprising the amino acid sequences 4, 40, 76 respectively and a light chain with three CDRs comprising the amino acid sequences 112, 148, 184, respectively; or a heavy chain with three CDRs comprising the amino acid sequences 5, 41, 77 respectively and a light chain with three CDRs comprising the amino acid sequences comprising the
  • anti-influenza antibodies include those having the amino acid or nucleic acid sequences shown in the below Table 1.
  • HCDR1 GFTFSNYG SEQ ID NO: 1671
  • HCDR2 ISFDGSKK SEQ ID NO: 1672
  • HCDR3 CAKLPSPYYFDSRFVWVA SEQ ID NO: 1673
  • ASAFHFW LCDR1 SSNIGGNT SEQ ID NO: 1674
  • LCDR2 TNS SEQ ID NO: 1675
  • LCDR3 CAAWDDSLNGQVF SEQ ID NO: 1676
  • 3I14V L D94N CAAWDNSLNGQVF SEQ ID NO: 1677
  • binding domain than scFv can also be used for predefined targeting of lymphocytes, such as camelid single-domain antibody fragments or receptor ligands, antibody binding domains, antibody hypervariable loops or CDRs as non limiting examples.
  • said transmembrane domain further comprises a stalk region between said extracellular ligand-binding domain and said transmembrane domain.
  • the term “stalk region” used herein generally means any oligo- or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain. In particular, stalk region are used to provide more flexibility and accessibility for the extracellular ligand-binding domain.
  • a stalk region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.
  • Stalk region may be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4 or CD28, or from all or part of an antibody constant region.
  • the stalk region may be a synthetic sequence that corresponds to a naturally occurring stalk sequence, or may be an entirely synthetic stalk sequence.
  • said stalk region is a part of human CD8 alpha chain
  • the signal transducing domain or intracellular signaling domain of the CAR of the invention is responsible for intracellular signaling following the binding of extracellular ligand binding domain to the target resulting in the activation of the immune cell and immune response.
  • the signal transducing domain is responsible for the activation of at least one of the normal effector functions of the immune cell in which the CAR is expressed.
  • the effector function of a T cell can be a cytolytic activity or helper activity including the secretion of cytokines.
  • the term “signal transducing domain” refers to the portion of a protein which transduces the effector signal function signal and directs the cell to perform a specialized function.
  • Signal transduction domain comprises two distinct classes of cytoplasmic signaling sequence, those that initiate antigen-dependent primary activation, and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal.
  • Primary cytoplasmic signaling sequence can comprise signaling motifs which are known as immunoreceptor tyrosine-based activation motifs of ITAMs.
  • ITAMs are well defined signaling motifs found in the intracytoplasmic tail of a variety of receptors that serve as binding sites for syk/zap70 class tyrosine kinases.
  • ITAM used in the invention can include as non limiting examples those derived from TCR zeta, FcR gamma, FcR beta, FcR epsilon, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b and CD66d.
  • the signaling transducing domain of the CAR can comprise the CD3 zeta signaling domain, or the intracytoplasmic domain of the Fc epsilon RI beta or gamma chains.
  • the signaling is provided by CD3 zeta together with co-stimulation provided by CD28 and a tumor necrosis factor receptor (TNFr), such as 4-1BB or OX40), for example.
  • TNFr tumor necrosis factor receptor
  • the intracellular signaling domain of the CAR of the present invention comprises a co-stimulatory signal molecule.
  • the intracellular signaling domain contains 2, 3, 4 or more co-stimulatory molecules in tandem.
  • a co-stimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient immune response.
  • Co-stimulatory ligand refers to a molecule on an antigen presenting cell that specifically binds a cognate co-stimulatory molecule on a T-cell, thereby providing a signal which, in addition to the primary signal provided by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, mediates a T cell response, including, but not limited to, proliferation activation, differentiation and the like.
  • a co-stimulatory ligand can include but is not limited to CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, M1CB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds with B7-H3.
  • a co-stimulatory ligand also encompasses, inter alia, an antibody that specifically binds with a co-stimulatory molecule present on a T cell, such as but not limited to, CD27, CD28, 4-IBB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83.
  • an antibody that specifically binds with a co-stimulatory molecule present on a T cell such as but not limited to, CD27, CD28, 4-IBB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LTGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83.
  • a “co-stimulatory molecule” refers to the cognate binding partner on a T-cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the cell, such as, but not limited to proliferation.
  • Co-stimulatory molecules include, but are not limited to an MHC class 1 molecule, BTLA and Toll ligand receptor.
  • costimulatory molecules include CD27, CD28, CD8, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and a ligand that specifically binds with CD83 and the like.
  • said signal transducing domain is a TNFR-associated Factor 2 (TRAF2) binding motifs, intracytoplasmic tail of costimulatory TNFR member family.
  • Cytoplasmic tail of costimulatory TNFR family member contains TRAF2 binding motifs consisting of the major conserved motif (P/S/A)X(Q/E)E) or the minor motif (PXQXXD), wherein X is any amino acid.
  • TRAF proteins are recruited to the intracellular tails of many TNFRs in response to receptor trimerization.
  • transmembrane polypeptides comprise the ability to be expressed at the surface of an immune cell, in particular lymphocyte cells or Natural killer (NK) cells, and to interact together for directing cellular response of immune cell against a predefined target cell.
  • the different transmembrane polypeptides of the CAR of the present invention comprising an extracellular ligand-biding domain and/or a signal transducing domain interact together to take part in signal transduction following the binding with a target ligand and induce an immune response.
  • the transmembrane domain can be derived either from a natural or from a synthetic source.
  • the transmembrane domain can be derived from any membrane-bound or transmembrane protein.
  • amino acid sequence functional variants of the polypeptide can be prepared by mutations in the DNA which encodes the polypeptide.
  • Such variants or functional variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence. Any combination of deletion, insertion, and substitution may also be made to arrive at the final construct, provided that the final construct possesses the desired activity, especially to exhibit a specific anti-target cellular immune activity.
  • the functionality of the CAR of the invention within a host cell is detectable in an assay suitable for demonstrating the signaling potential of said CAR upon binding of a particular target.
  • this assay allows the detection of a signaling pathway, triggered upon binding of the target, such as an assay involving measurement of the increase of calcium ion release, intracellular tyrosine phosphorylation, inositol phosphate turnover, or interleukin (IL) 2, interferon .gamma., GM-CSF, IL-3, IL-4 production thus effected.
  • IL interleukin
  • Embodiments of the invention include cells that express a CAR (i.e, CARTS).
  • the cell may be of any kind, including an immune cell capable of expressing the CAR for cancer therapy or a cell, such as a bacterial cell, that harbors an expression vector that encodes the CAR.
  • the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations.
  • host cell refers to a eukaryotic cell that is capable of replicating a vector and/or expressing a heterologous gene encoded by a vector.
  • a host cell can, and has been, used as a recipient for vectors.
  • a host cell may be “transfected” or “transformed,” which refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a transformed cell includes the primary subject cell and its progeny.
  • engineered and “recombinant” cells or host cells are intended to refer to a cell into which an exogenous nucleic acid sequence, such as, for example, a vector, has been introduced.
  • a host cell is a T cell, including a cytotoxic T cell (also known as TC, Cytotoxic T Lymphocyte, CTL, T-Killer cell, cytolytic T cell, CD8+ T-cells or killer T cell); NK cells and NKT cells are also encompassed in the invention.
  • cytotoxic T cell also known as TC, Cytotoxic T Lymphocyte, CTL, T-Killer cell, cytolytic T cell, CD8+ T-cells or killer T cell
  • NK cells and NKT cells are also encompassed in the invention.
  • Some vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells.
  • control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells.
  • One of skill in the art would further understand the conditions under which to incubate all of the above described host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.
  • the cells can be autologous cells, syngeneic cells, allogenic cells and even in some cases, xenogeneic cells.
  • the cells become neoplastic, in research where the absence of the cells after their presence is of interest, or other event.
  • the invention further includes CARTS that are modified to secrete one or more polypeptides.
  • the polypeptide can be for example an antibody or cytokine.
  • the antibody is specific for CAIX, GITR, PD-L1, PD-L2.
  • Armed CART can be constructed by including a nucleic acid encoding the polypeptide of interest after the intracellular signaling domain.
  • a nucleic acid encoding the polypeptide of interest after the intracellular signaling domain.
  • IRES internal ribosome entry site
  • the methods and compositions presented herein provide a target-specific Anti-CAIX CAR T cell of second generation armed with the power to secrete anti-PD-L1 IgGs in the RCC milieu to combat T cell exhaustion.
  • the human Anti-CAIX CAR containing the CD28 co-stimulatory domain was chosen based in its killing activity and low immunogenicity in human cRCC xenografts in mice.
  • the Anti-CAIX CART cells-secreting Anti-PD-L1 IgG1 or IgG4 was extensively compared with an unrelated Anti-BCMA CAR or with an Anti-CAIX CAR, both secreting an irrelevant Anti-SARS IgG1.
  • Anti-CAIX CART cells have capacity to undergo clonal expansion when in contact with CAIX+ RCC cells, and were also activated, releasing high levels of IFN ⁇ and IL-2, which could improve tumor suppression. These Anti-CAIX CART cells are also able to secrete high levels of Anti-PD-L1 IgG1 or IgG4 and these antibodies can interact specifically with PD-L1, inducing downregulation of the exhaustion markers PD-1, Tim-3 and Lag-3 in vitro and in vivo.
  • Anti-PD-L1 antibodies secreted in the tumor microenvironment are able to revert T cell exhaustion, facilitating the Anti-CAIX CART cells antitumor activity.
  • These Anti-CAIX CART cells mainly the ones secreting anti-PD-L1, are also able to diminish the proliferation of CAIX+ RCC cells, resulting in a slow tumor growth and small tumor size and weight in an orthotopic NSG mice model of RCC.
  • Anti-CAIX CART cell secreting the IgG1 isotype of Anti-PD-L1 was also able to induce ADCC in vitro and increased the number of human NK cells infiltrating the tumor site in vivo.
  • the human NK cells were injected only in 2 mice of each group, two days before their euthanasia to determine the NK cells capacity to recognize the IgG1 isotype of the Anti-PD-L1 in vivo, which was attested.
  • the injection of human NK cells was not made in the beginning of the treatment once our previous experience with this mice model showed that these cells last only for a few days in their blood
  • the injection of CART cells into mice was performed without the addition of interleukins to avoid their influence in the therapeutic effect of CART cells alone.
  • CART cells can be maintained with the use of cytokines such as, for example, IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21.
  • cytokines such as, for example, IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21.
  • Cytokines sharing the ⁇ c receptor like IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 are important for the development and maintenance of memory T cells.
  • IL-21 promote a less differentiated phenotype, associated with an enrichment of tumor-specific CD8 T cells, with increased anti-tumor effect in a mouse melanoma model when compared to IL-2 or IL-15.
  • CART cells are maintained with IL-21.
  • CAIX is a consistent marker for development of cancer targeted systemic therapies due to it overexpression in many tumors, remarkably in cRCC, but it is also expressed physiologically in a few tissues.
  • the anti-CAIX scFv in the CAR recognizes the catalytic domain of CAIX, located in the central portion of the protein, which could increase its specificity to sites of higher expression of CAIX.
  • CD28 is replaced by 41BB in the CAR constructs.
  • T cell exhaustion is common in cancer and these T cells present low capacities of proliferation and cytokine production associated with high apoptosis rate and expression of inhibitory receptors like PD-1, Tim-3 and Lag3.
  • New strategies for preventing T cell exhaustion include of PD-1/PD-L1 axis.
  • the methods and compositions presented herein provide Anti-CAIX CART cells-secreting Anti-PD-L1 IgG1 or IgG4 that can diminish T cell exhaustion, improving CART cell efficiency in the cRCC treatment in vitro and in vivo.
  • Expression vectors that encode the CARs can be introduced as one or more DNA molecules or constructs, where there may be at least one marker that will allow for selection of host cells that contain the construct(s).
  • the constructs can be prepared in conventional ways, where the genes and regulatory regions may be isolated, as appropriate, ligated, cloned in an appropriate cloning host, analyzed by restriction or sequencing, or other convenient means. Particularly, using PCR, individual fragments including all or portions of a functional unit may be isolated, where one or more mutations may be introduced using “primer repair”, ligation, in vitro mutagenesis, etc., as appropriate. The construct(s) once completed and demonstrated to have the appropriate sequences may then be introduced into the CTL by any convenient means.
  • the constructs may be integrated and packaged into non-replicating, defective viral genomes like Adenovirus, Adeno-associated virus (AAV), or Herpes simplex virus (HSV) or others, including retroviral vectors or lentiviral vectors, for infection or transduction into cells.
  • the constructs may include viral sequences for transfection, if desired.
  • the construct may be introduced by fusion, electroporation, biolistics, transfection, lipofection, or the like.
  • the host cells may be grown and expanded in culture before introduction of the construct(s), followed by the appropriate treatment for introduction of the construct(s) and integration of the construct(s). The cells are then expanded and screened by virtue of a marker present in the construct.
  • markers that may be used successfully include hprt, neomycin resistance, thymidine kinase, hygromycin resistance, etc.
  • homologous recombination one may use either .OMEGA. or O-vectors. See, for example, Thomas and Capecchi, Cell (1987) 51, 503-512; Mansour, et al., Nature (1988) 336, 348-352; and Joyner, et al., Nature (1989) 338, 153-156.
  • the constructs may be introduced as a single DNA molecule encoding at least the CAR and optionally another gene, or different DNA molecules having one or more genes.
  • Other genes include genes that encode therapeutic molecules or suicide genes, for example.
  • the constructs may be introduced simultaneously or consecutively, each with the same or different markers.
  • Vectors containing useful elements such as bacterial or yeast origins of replication, selectable and/or amplifiable markers, promoter/enhancer elements for expression in prokaryotes or eukaryotes, etc. that may be used to prepare stocks of construct DNAs and for carrying out transfections are well known in the art, and many are commercially available.
  • the cells according to the invention can be used for treating cancer, viral infections or autoimmune disorders in a patient in need thereof.
  • said isolated cell according to the invention can be used in the manufacture of a medicament for treatment of a cancer, viral infections of autoimmune disorders, in a patient in need thereof.
  • the present invention relies on methods for treating patients in need thereof, said method comprising at least one of the following steps: (a) providing a chimeric antigen receptor cells according to the invention and (b) administrating the cells to said patient.
  • Said treatment can be ameliorating, curative or prophylactic. It may be either part of an autologous immunotherapy or part of an allogenic immunotherapy treatment.
  • autologous it is meant that cells, cell line or population of cells used for treating patients are originating from said patient or from a Human Leucocyte Antigen (HLA) compatible donor.
  • HLA Human Leucocyte Antigen
  • allogeneic is meant that the cells or population of cells used for treating patients are not originating from said patient but from a donor.
  • the invention is particularly suited for allogenic immunotherapy, insofar as it enables the transformation of T-cells, typically obtained from donors, into non-alloreactive cells. This may be done under standard protocols and reproduced as many times as needed.
  • the resulted modified T cells may be pooled and administrated to one or several patients, being made available as an “off the shelf” therapeutic product.
  • Cancers that can be used with the disclosed methods are described in the previous section. Said treatment can be used to treat patients diagnosed with cancer, viral infection, autoimmune disorders or Graft versus Host Disease (GvHD). Cancers that may be treated include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors.
  • the cancers may comprise nonsolid tumors (such as hematological tumors, for example, leukemias and lymphomas) or may comprise solid tumors.
  • Types of cancers to be treated with the CARs of the invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas.
  • carcinoma a malignant neoplasm originating from a malignant neoplasm originating from tumors.
  • sarcomas e.g., sarcomas, carcinomas, and melanomas.
  • adult tumors/cancers and pediatric tumors/cancers are also included.
  • It can be a treatment in combination with one or more therapies against cancer selected from the group of antibodies therapy, chemotherapy, cytokines therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy and radiation therapy.
  • therapies against cancer selected from the group of antibodies therapy, chemotherapy, cytokines therapy, dendritic cell therapy, gene therapy, hormone therapy, laser light therapy and radiation therapy.
  • said treatment can be administrated into patients undergoing an immunosuppressive treatment.
  • the present invention preferably relies on cells or population of cells, which have been made resistant to at least one immunosuppressive agent due to the inactivation of a gene encoding a receptor for such immunosuppressive agent.
  • the immunosuppressive treatment should help the selection and expansion of the T-cells according to the invention within the patient.
  • the cell compositions of the present invention are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAM PATH.
  • chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAM PATH.
  • the cell compositions of the present invention are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rittman.
  • subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation.
  • subjects receive an infusion of the expanded immune cells of the present invention.
  • expanded cells are administered before or following surgery.
  • Said modified cells obtained by any one of the methods described here can be used in a particular aspect of the invention for treating patients in need thereof against Host versus Graft (HvG) rejection and Graft versus Host Disease (GvHD); therefore in the scope of the present invention is a method of treating patients in need thereof against Host versus Graft (HvG) rejection and Graft versus Host Disease (GvHD) comprising treating said patient by administering to said patient an effective amount of modified cells comprising inactivated TCR alpha and/or TCR beta genes.
  • the invention is particularly suited for allogenic immunotherapy, insofar as it enables the transformation of T-cells, typically obtained from donors, into non-alloreactive cells. This may be done under standard protocols and reproduced as many times as needed.
  • the resulted modified T cells may be pooled and administrated to one or several patients, being made available as an “off the shelf” therapeutic product.
  • the cells may be introduced into a host organism, e.g. a mammal, in a wide variety of ways.
  • the cells may be introduced at the site of the tumor, in specific embodiments, although in alternative embodiments the cells hone to the cancer or are modified to hone to the cancer.
  • the number of cells that are employed will depend upon a number of circumstances, the purpose for the introduction, the lifetime of the cells, the protocol to be used, for example, the number of administrations, the ability of the cells to multiply, the stability of the recombinant construct, and the like.
  • the cells may be applied as a dispersion, generally being injected at or near the site of interest.
  • the cells may be in a physiologically-acceptable medium.
  • the cells are encapsulated to inhibit immune recognition and placed at the site of the tumor.
  • the cells may be administered as desired. Depending upon the response desired, the manner of administration, the life of the cells, the number of cells present, various protocols may be employed. The number of administrations will depend upon the factors described above at least in part.
  • the administration of the cells or population of cells according to the present invention may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation.
  • the compositions described herein may be administered to a patient subcutaneously, intradermaly, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally.
  • the cell compositions of the present invention are preferably administered by intravenous injection.
  • the administration of the cells or population of cells can consist of the administration of 10 4 -10 9 cells per kg body weight, preferably 10 5 to 10 6 cells/kg body weight including all integer values of cell numbers within those ranges.
  • the cells or population of cells can be administrated in one or more doses.
  • said effective amount of cells are administrated as a single dose.
  • said effective amount of cells are administrated as more than one dose over a period time. Timing of administration is within the judgment of managing physician and depends on the clinical condition of the patient.
  • the cells or population of cells may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determination of optimal ranges of effective amounts of a given cell type for a particular disease or conditions within the skill of the art.
  • An effective amount means an amount which provides a therapeutic or prophylactic benefit.
  • the dosage administrated will be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired.
  • the system is subject to many variables, such as the cellular response to the ligand, the efficiency of expression and, as appropriate, the level of secretion, the activity of the expression product, the particular need of the patient, which may vary with time and circumstances, the rate of loss of the cellular activity as a result of loss of cells or expression activity of individual cells, and the like. Therefore, it is expected that for each individual patient, even if there were universal cells which could be administered to the population at large, each patient would be monitored for the proper dosage for the individual, and such practices of monitoring a patient are routine in the art.
  • the CARs of the present invention may be expressed from an expression vector. Recombinant techniques to generate such expression vectors are well known in the art.
  • vector is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated.
  • a nucleic acid sequence can be “exogenous,” which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found.
  • Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs).
  • YACs artificial chromosomes
  • expression vector refers to any type of genetic construct comprising a nucleic acid coding for a RNA capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes.
  • Expression vectors can contain a variety of “control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra.
  • a “promoter” is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled. It may contain genetic elements at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors, to initiate the specific transcription a nucleic acid sequence.
  • the phrases “operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence.
  • a promoter generally comprises a sequence that functions to position the start site for RNA synthesis.
  • the best known example of this is the TATA box, but in some promoters lacking a TATA box, such as, for example, the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation. Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30 110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well.
  • a coding sequence “under the control of” a promoter one positions the 5′ end of the transcription initiation site of the transcriptional reading frame “downstream” of (i.e., 3′ of) the chosen promoter.
  • the “upstream” promoter stimulates transcription of the DNA and promotes expression of the encoded RNA.
  • promoter elements frequently are flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
  • the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
  • individual elements can function either cooperatively or independently to activate transcription.
  • a promoter may or may not be used in conjunction with an “enhancer,” which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.
  • a promoter may be one naturally associated with a nucleic acid sequence, as may be obtained by isolating the 5 prime′ non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as “endogenous.”
  • an enhancer may be one naturally associated with a nucleic acid sequence, located either downstream or upstream of that sequence.
  • certain advantages will be gained by positioning the coding nucleic acid segment under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment.
  • a recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment.
  • promoters or enhancers may include promoters or enhancers of other genes, and promoters or enhancers isolated from any other virus, or prokaryotic or eukaryotic cell, and promoters or enhancers not “naturally occurring,” i.e., containing different elements of different transcriptional regulatory regions, and/or mutations that alter expression.
  • promoters that are most commonly used in recombinant DNA construction include the lactamase (penicillinase), lactose and tryptophan (trp) promoter systems.
  • sequences may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCRTM, in connection with the compositions disclosed herein (see U.S. Pat. Nos. 4,683,202 and 5,928,906, each incorporated herein by reference).
  • control sequences that direct transcription and/or expression of sequences within non-nuclear organelles such as mitochondria, chloroplasts, and the like, can be employed as well.
  • promoter and/or enhancer that effectively directs the expression of the DNA segment in the organelle, cell type, tissue, organ, or organism chosen for expression.
  • Those of skill in the art of molecular biology generally know the use of promoters, enhancers, and cell type combinations for protein expression, (see, for example Sambrook et al. 1989, incorporated herein by reference).
  • the promoters employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins and/or peptides.
  • the promoter may be heterologous or endogenous.
  • any promoter/enhancer combination could also be used to drive expression.
  • Use of a T3, T7 or SP6 cytoplasmic expression system is another possible embodiment.
  • Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.
  • tissue-specific promoters or elements as well as assays to characterize their activity, is well known to those of skill in the art.
  • a specific initiation signal also may be required for efficient translation of coding sequences. These signals include the ATG initiation codon or adjacent sequences. Exogenous translational control signals, including the ATG initiation codon, may need to be provided. One of ordinary skill in the art would readily be capable of determining this and providing the necessary signals
  • IVS internal ribosome entry sites
  • Vectors can include a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector.
  • MCS multiple cloning site
  • Restriction enzyme digestion refers to catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in a nucleic acid molecule. Many of these restriction enzymes are commercially available. Use of such enzymes is widely understood by those of skill in the art. Frequently, a vector is linearized or fragmented using a restriction enzyme that cuts within the MCS to enable exogenous sequences to be ligated to the vector.
  • “Ligation” refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions are well known to those of skill in the art of recombinant technology.
  • Splicing sites termination signals, origins of replication, and selectable markers may also be employed.
  • a plasmid vector is contemplated for use to transform a host cell.
  • plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts.
  • the vector ordinarily carries a replication site, as well as marking sequences which are capable of providing phenotypic selection in transformed cells.
  • E. coli is often transformed using derivatives of pBR322, a plasmid derived from an E. coli species.
  • pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.
  • the pBR plasmid, or other microbial plasmid or phage must also contain, or be modified to contain, for example, promoters which can be used by the microbial organism for expression of its own proteins.
  • phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts.
  • the phage lambda GEMTM 11 may be utilized in making a recombinant phage vector which can be used to transform host cells, such as, for example, E. coli LE392.
  • plasmid vectors include pIN vectors (Inouye et al., 1985); and pGEX vectors, for use in generating glutathione S transferase (GST) soluble fusion proteins for later purification and separation or cleavage.
  • GST glutathione S transferase
  • Other suitable fusion proteins are those with galactosidase, ubiquitin, and the like.
  • Bacterial host cells for example, E. coli , comprising the expression vector, are grown in any of a number of suitable media, for example, LB.
  • suitable media for example, LB.
  • the expression of the recombinant protein in certain vectors may be induced, as would be understood by those of skill in the art, by contacting a host cell with an agent specific for certain promoters, e.g., by adding IPTG to the media or by switching incubation to a higher temperature. After culturing the bacteria for a further period, generally of between 2 and 24 h, the cells are collected by centrifugation and washed to remove residual media.
  • Components of the present invention may be a viral vector that encodes one or more CARs of the invention.
  • Non-limiting examples of virus vectors that may be used to deliver a nucleic acid of the present invention are described below.
  • a particular method for delivery of the nucleic acid involves the use of an adenovirus expression vector.
  • adenovirus vectors are known to have a low capacity for integration into genomic DNA, this feature is counterbalanced by the high efficiency of gene transfer afforded by these vectors.
  • “Adenovirus expression vector” is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to ultimately express a tissue or cell specific construct that has been cloned therein.
  • Knowledge of the genetic organization or adenovirus, a 36 kb, linear, double stranded DNA virus allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus and Horwitz, 1992).
  • the nucleic acid may be introduced into the cell using adenovirus assisted transfection. Increased transfection efficiencies have been reported in cell systems using adenovirus coupled systems (Kelleher and Vos, 1994; Cotten et al., 1992; Curiel, 1994).
  • Adeno associated virus (AAV) is an attractive vector system for use in the cells of the present invention as it has a high frequency of integration and it can infect nondividing cells, thus making it useful for delivery of genes into mammalian cells, for example, in tissue culture (Muzyczka, 1992) or in vivo.
  • AAV has a broad host range for infectivity (Tratschin et al., 1984; Laughlin et al., 1986; Lebkowski et al., 1988; McLaughlin et al., 1988). Details concerning the generation and use of rAAV vectors are described in U.S. Pat. Nos. 5,139,941 and 4,797,368, each incorporated herein by reference.
  • Retroviruses are useful as delivery vectors because of their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and of being packaged in special cell lines (Miller, 1992).
  • a nucleic acid e.g., one encoding the desired sequence
  • a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al., 1983).
  • Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al., 1975).
  • Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. Lentiviral vectors are well known in the art (see, for example, Naldini et al., 1996; Zufferey et al., 1997; Blomer et al., 1997; U.S. Pat. Nos. 6,013,516 and 5,994,136). Some examples of lentivirus include the Human Immunodeficiency Viruses: HIV-1, HIV-2 and the Simian Immunodeficiency Virus: SIV. Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe.
  • Recombinant lentiviral vectors are capable of infecting non-dividing cells and can be used for both in vivo and ex vivo gene transfer and expression of nucleic acid sequences.
  • recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Pat. No. 5,994,136, incorporated herein by reference.
  • One may target the recombinant virus by linkage of the envelope protein with an antibody or a particular ligand for targeting to a receptor of a particular cell-type.
  • a sequence (including a regulatory region) of interest into the viral vector, along with another gene which encodes the ligand for a receptor on a specific target cell, for example, the vector is now target-specific.
  • viral vectors may be employed as vaccine constructs in the present invention.
  • Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988), Sindbis virus, cytomegalovirus and herpes simplex virus may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).
  • a nucleic acid to be delivered may be housed within an infective virus that has been engineered to express a specific binding ligand.
  • the virus particle will thus bind specifically to the cognate receptors of the target cell and deliver the contents to the cell.
  • a novel approach designed to allow specific targeting of retrovirus vectors was developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification can permit the specific infection of hepatocytes via sialoglycoprotein receptors.
  • Suitable methods for nucleic acid delivery for transfection or transformation of cells are known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by ex vivo transfection, by injection, and so forth. Through the application of techniques known in the art, cells may be stably or transiently transformed.
  • eukaryotic cells and tissues removed from an organism in an ex vivo setting are known to those of skill in the art.
  • cells or tissues may be removed and transfected ex vivo using nucleic acids of the present invention.
  • the transplanted cells or tissues may be placed into an organism.
  • a nucleic acid is expressed in the transplanted cells.
  • compositions described herein may be comprised in a kit.
  • one or more cells for use in cell therapy and/or the reagents to generate one or more cells for use in cell therapy that harbors recombinant expression vectors may be comprised in a kit.
  • the kit components are provided in suitable container means.
  • kits may be packaged either in aqueous media or in lyophilized form.
  • the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial.
  • the kits of the present invention also will typically include a means for containing the components in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.
  • the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly ueful.
  • the container means may itself be a syringe, pipette, and/or other such like apparatus, from which the formulation may be applied to an infected area of the body, injected into an animal, and/or even applied to and/or mixed with the other components of the kit.
  • kits may be provided as dried powder(s).
  • the powder can be reconstituted by the addition of a suitable solvent.
  • the solvent may also be provided in another container means.
  • kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent.
  • kits that are to be used for cell therapy are provided in a kit, and in some cases the cells are essentially the sole component of the kit.
  • the kit may comprise reagents and materials to make the desired cell.
  • the reagents and materials include primers for amplifying desired sequences, nucleotides, suitable buffers or buffer reagents, salt, and so forth, and in some cases the reagents include vectors and/or DNA that encodes a CAR as described herein and/or regulatory elements therefor.
  • the kit suitable for extracting one or more samples from an individual.
  • the apparatus may be a syringe, scalpel, and so forth.
  • the kit in addition to cell therapy embodiments, also includes a second cancer therapy, such as chemotherapy, hormone therapy, and/or immunotherapy, for example.
  • a second cancer therapy such as chemotherapy, hormone therapy, and/or immunotherapy, for example.
  • the kit(s) may be tailored to a particular cancer for an individual and comprise respective second cancer therapies for the individual.
  • methods of the present invention for clinical aspects are combined with other agents effective in the treatment of hyperproliferative disease, such as anti-cancer agents.
  • An “anti-cancer” agent is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer. More generally, these other compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell.
  • This process may involve contacting the cancer cells with the expression construct and the agent(s) or multiple factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the expression construct and the other includes the second agent(s).
  • Tumor cell resistance to chemotherapy and radiotherapy agents represents a major problem in clinical oncology.
  • One goal of current cancer research is to find ways to improve the efficacy of chemo- and radiotherapy by combining it with other therapies.
  • cell therapy could be used similarly in conjunction with chemotherapeutic, radiotherapeutic, or immunotherapeutic intervention, as well as pro-apoptotic or cell cycle regulating agents.
  • the present inventive therapy may precede or follow the other agent treatment by intervals ranging from minutes to weeks.
  • the other agent and present invention are applied separately to the individual, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and inventive therapy would still be able to exert an advantageously combined effect on the cell.
  • Cancer therapies also include a variety of combination therapies with both chemical and radiation based treatments.
  • Combination chemotherapies include, for example, abraxane, altretamine, docetaxel, herceptin, methotrexate, novantrone, zoladex, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding agents, taxol, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin and methotre
  • chemotherapy for the individual is employed in conjunction with the invention, for example before, during and/or after administration of the invention
  • Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
  • Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
  • contacted and “exposed,” when applied to a cell are used herein to describe the process by which a therapeutic construct and a chemotherapeutic or radiotherapeutic agent are delivered to a target cell or are placed in direct juxtaposition with the target cell.
  • both agents are delivered to a cell in a combined amount effective to kill the cell or prevent it from dividing.
  • Immunotherapeutics generally rely on the use of immune effector cells and molecules to target and destroy cancer cells.
  • the immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell.
  • the antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing.
  • the antibody also may be conjugated to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent.
  • 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 and NK cells.
  • Immunotherapy other than the inventive therapy described herein could thus be used as part of a combined therapy, in conjunction with the present cell therapy.
  • the general approach for combined therapy is discussed below.
  • the tumor cell must bear some marker that is amenable to targeting, i.e., is not present on the majority of other cells. Many tumor markers exist and any of these may be suitable for targeting in the context of the present invention.
  • Common tumor markers include PD-1, PD-L1, CTLA4, carcinoembryonic antigen, prostate specific antigen, urinary tumor associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and p155.
  • the secondary treatment is a gene therapy in which a therapeutic polynucleotide is administered before, after, or at the same time as the present invention clinical embodiments.
  • a variety of expression products are encompassed within the invention, including inducers of cellular proliferation, inhibitors of cellular proliferation, or regulators of programmed cell death.
  • Curative surgery is a cancer treatment that may be used in conjunction with other therapies, such as the treatment of the present invention, chemotherapy, radiotherapy, hormonal therapy, gene therapy, immunotherapy and/or alternative therapies.
  • Curative surgery includes resection in which all or part of cancerous tissue is physically removed, excised, and/or destroyed.
  • Tumor resection refers to physical removal of at least part of a tumor.
  • treatment by surgery includes laser surgery, cryosurgery, electrosurgery, and miscopically controlled surgery (Mohs' surgery). It is further contemplated that the present invention may be used in conjunction with removal of superficial cancers, precancers, or incidental amounts of normal tissue.
  • a cavity may be formed in the body.
  • Treatment may be accomplished by perfusion, direct injection or local application of the area with an additional anti-cancer therapy.
  • Such treatment may be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months.
  • These treatments may be of varying dosages as well.
  • agents may be used in combination with the present invention to improve the therapeutic efficacy of treatment.
  • additional agents include immunomodulatory agents, agents that affect the upregulation of cell surface receptors and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion, or agents that increase the sensitivity of the hyperproliferative cells to apoptotic inducers.
  • Immunomodulatory agents include tumor necrosis factor; interferon alpha, beta, and gamma; IL-2 and other cytokines; F42K and other cytokine analogs; or MIP-1, MIP-1beta, MCP-1, RANTES, and other chemokines.
  • cell surface receptors or their ligands such as Fas/Fas ligand, DR4 or DR5/TRAIL would potentiate the apoptotic inducing abililties of the present invention by establishment of an autocrine or paracrine effect on hyperproliferative cells. Increases intercellular signaling by elevating the number of GAP junctions would increase the anti-hyperproliferative effects on the neighboring hyperproliferative cell population.
  • cytostatic or differentiation agents can be used in combination with the present invention to improve the anti-hyerproliferative efficacy of the treatments.
  • Inhibitors of cell adhesion are contemplated to improve the efficacy of the present invention.
  • cell adhesion inhibitors are focal adhesion kinase (FAKs) inhibitors and Lovastatin. It is further contemplated that other agents that increase the sensitivity of a hyperproliferative cell to apoptosis, such as the antibody c225, could be used in combination with the present invention to improve the treatment efficacy.
  • FAKs focal adhesion kinase
  • Lovastatin Lovastatin
  • Human CAIX+ renal cell carcinoma cell lines sk-rc-52, sk-rc-09 and CAIX ⁇ sk-rc-59 were obtained from Dr. Gerd Ritter, Memorial Sloan-Kettering Cancer Center, New York. They were cultured at 37° C. with 5% CO 2 in R-10 complete medium containing RPMI 1640 medium (Life Technologies) supplemented with 10% FCS, 2 mmol/L L-glutamine, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin (Sigma).
  • Primary human T cells were maintained in R-10 with 10% human serum and 100 IU/ml recombinant human interleukin 2 (IL-2) (Chiron).
  • IL-2 human interleukin 2
  • Human embryonic kidney cell line 293T (ATCC) and mouse fibroblast NIH3T3 cells (ATCC) were grown in D-10 complete medium (Life Technologies) containing DMEM medium with 10% FCS, 100 U/ml penicillin, and 100 ⁇ g/ml streptomycin (Sigma).
  • Leukopacks obtained from the blood bank of the Children's Hospital Boston were collected from healthy volunteers with written informed consent.
  • Human ccRCC cell lines were obtained from Dr. Gerd Ritter (Memorial Sloan-Kettering Cancer Center, New York). These cells were cultivated in RPMI 1640 Medium (Life Technologies) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS, Gibco), 100 IU/ml penicillin and 100 ⁇ g/ml streptomycin.
  • 293T CRL-11268, ATCC
  • Lenti-X 293T Clontech cells were grown in DMEM Medium (Life Technologies) supplemented with 10% FBS, 100 IU/ml penicillin and 100 ⁇ g/ml streptomycin. All cell lines used in this project were transduced with luciferase through lentiviral transduction and maintained at 37° C. with 5% CO2.
  • the Skrc52 cells were selected for CAIX ⁇ /PD-L1 ⁇ and CAIX+/PD-L1 ⁇ cell populations by Fluorescence activated cell sorting (FACS) sorting. Skrc59 cells were engineered to express high levels of human CAIX and CAIX+/PD-L1+ were selected by FACS sorting.
  • the anti-PD-L1 antibody (Ab) used to construct the CART cells was previously selected using a 27-billion-member human scFv phage display library against a full length PD-L1 in the form of paramagnetic proteoliposomes (manuscript in preparation).
  • the DNA sequences that encoded the anti-PD-L1 scFv (Clone 42)-Fc IgG1 or IgG4 were codon optimized and synthesized (Genewiz) containing the restriction sites for 5′ NdeI and 3′ ClaI to allow further scFv-Fc cloning to replace ZsGreen in the lentiviral vector pHAGE-eIF ⁇ signal-scFvG36 (anti-CAIX)-C9TAG-linker-CD28-CD3 ⁇ -IRES-ZsGreen (e.g. CD28z).
  • This anti-CAIX original vector was previously constructed and published (21).
  • the DNA sequence that coded for the anti-SARS scFv was amplified by PCR from the plasmid pHAGE CMV-Anti-SARS (11A) scFv-Fc-CD28-gp41-IRES-ZsGreen to insert the restriction sites for 5′ MluI and 3′ XbaI for further cloning using the MluI forward primer 5′ TCG ACG CGT GAG GTG CAG CTG GTG CAG T 3′ and XbaI reverse primer 5′ TCC TCT AGA CAG GAC GGT GAC CTT GGT CC 3′.
  • CAIX-specific scFv antibodies were isolated from a non-immune human scFv phage library as previously reported and submitted to GenBank with accession numbers of GQ903548-GQ903561 23 .
  • scFv-coding DNA fragments from the pFarber phagemid were digested with SfiI/NotI sites and subcloned into the mammalian expression vector pcDNA3.1-F105L-hinge-stuffer which has a human IgG1 F105 leader sequence and the human IgG1 hinge-CH2-CH3 Fc portion to express scFv-Fc antibodies.
  • Plasmids of scFv-Fc were transiently transfected into 293T cells by lipofectamine 2000 (Invitrogen), and expressed antibodies were purified using Sepharose protein A beads (Amersham Bioscience). Specific binding to CAIX was tested by staining with phage scFv antibodies or scFv converted into scFv-Fc format antibodies by incubation with CAIX-expressing 293T and sk-rc-52 cell lines, and with CAIX negative 293T and sk-rc-59 cell lines. In these experiments, irrelevant anti-HIV CCRS antibody (clone A8) 25 or anti-SARS antibody (11A) 24 and fluorescently conjugated secondary antibodies alone were used as negative controls.
  • Pz1, scFv-CD8-TCR ⁇ , and P28z, scFv-CD28-TCR ⁇ , DNA constructs in phagemid vector pSL1180 were obtained from Dr. Michel Sadelain, Memorial Sloan-Kettering Cancer Center, New York.
  • Pz1 the scFv and TCR ⁇ intracellular domain are appended to N- and C-terminus of human CD8a chain, respectively.
  • P28z the scFv and TCR ⁇ sequences are appended to the N- and C-terminus of human CD28, respectively.
  • the amino acid sequence of human CD8a is 71 residues in length, consisting of 47 (aa 137-183), 23 (aa 184-206), and 2 (aa 207-208) residues of the CD8a extracellular and hinge, transmembrane, and cytoplasmic domains, respectively.
  • the CD28 sequence in P28z is 107 residues in length, consisting of 40 (aa 114-153), 23 (aa 154-176), and 44 (aa 177-220) residues of the CD28 extracellular, transmembrane, and cytoplasmic domains respectively.
  • the human CD3 ⁇ intracellular domain common to both CARs consists of 112 amino acids (aa 52-163).
  • the nucleic acid sequence encoding an internal C9-tag (a nine-amino acid peptide of human rhodopsin, TETSQVAPA) with a GGGGS linker was amplified by PCR and was fused upstream with CD8-TCR ⁇ and CD28-TCR ⁇ sequences with 5′ NotI site and 3′ PacI sites.
  • the primers used for cloning chimeric TCR ⁇ constructs are
  • the chimeric TCR constructs tagged with internal C9 peptide were cloned into the pcDNA3.1-F105L-hinge stuffer vector containing anti-CXCR4 scFv-Fc, clone 48, using NotI and PacI restriction sites. This design allowed us to insert chimeric TCR receptor constructs to replace Fc portion fragment. Later, anti-CAIX scFv (clone G36) and anti-CCRS scFv (clone A8, as irrelevant scFv control) antibody fragments were cloned to replace anti-CXCR4 scFv at SfiI/NotI sites to create CAIX-specific chimeric TCR constructs.
  • the lentivirus vector pHAGE-CMV-DsRed-IRES-ZsGreen, and four HIV helper plasmids pHDM-Hgpm2 (HIV gag-pol), pMD-tat, pRC/CMV-rev, and an Env VSV-G pseudotype were obtained from Dr. Richard Mulligan, of the Virus Production Core at The Harvard Gene Therapy Initiative in Boston.
  • the CMV promoter in pHAGE-CMV-IRES-ZsGreen was replaced by an EF1 ⁇ promoter derived from the pSIN lentivirus vector at SpeI/NotI sites.
  • Lentivirus was produced by five plasmid transient transfection into 293T cells using lipofectamine 2000 as per the manufacturer's instructions (Invitrogen). Cells were prepared for 80% confluence in 15 cm Petri dishes (Nalge Nunc) and transfected with 30 ⁇ g of total plasmid DNA. The ratio of vector plasmids (pHDM-Hgpm2 (HIV gag-pol): pMD-tat: pRC/CMV-rev: Env VSV-G pseudotype) was 20:1:1:1:2.
  • virus supernatant was harvested on day 3, filtrated through a 0.45 ⁇ m filter, and concentrated by ultracentrifugation (Beckman Coulter, Fullerton, Calif.) for 90 minutes at 16,500 rpm (48,960 ⁇ g, Beckman SW28 rotor) and 4° C.
  • the virus pellets were resuspended in R-10 medium and kept frozen at ⁇ 80° C.
  • Lentiviruses were produced by transient transfection of five plasmids into 293T cells using Polyethyleneimine (PEI). Briefly, each 80% confluent 293T cells in 15 cm plate (Nalge Nunc) was transfected with 30 ⁇ g of total five plasmids, being 5 ⁇ g of each structural plasmid pHDH-Hgpm2 (HIV gag-pol), pMD-tat; pRC/CMV-rev and Env VSV-G, and 10 ⁇ g of the main plasmid codifying the CAR (anti-CAIX/anti-PD-L1 IgG1, anti-CAIX/anti-PD-L1 IgG4, anti-CAIX/anti SARS IgG1 or anti-BCMA/anti SARS IgG1). The virus supernatant was concentrated using Lenti-X Concentrator (Clontech), following the manufacturer instructions, and kept frozen at ⁇ 80° C.
  • Lenti-X Concentrator (Clontech),
  • Human PBMCs were isolated by ficoll density gradient separation and were activated with 2 ⁇ g/ml PHA (Sigma) plus 100 IU/ml human IL-2 for 4 days.
  • the cells were infected with two or three rounds of lentivirus transduction at multiplicity of infection ( M OI) of 10-20 in the presence of 10 ⁇ g/ml DEAE.
  • M OI multiplicity of infection
  • transduced T cells were collected for phenotypic and functional analyses in vitro, or were expanded for in vivo experiments.
  • PBMCs peripheral blood mononuclear cells
  • the assays performed to determine if IL-2 or IL-21 were the best cytokine to induce anti-CAIX CART cells proliferation were performed with 50 IU/mL of each cytokine.
  • the CD8+ T cells were activated with Dynabeads Human T-Activator CD3/CD28 (Life Technologies) using a ratio of 1:1.
  • the cells were transduced with the Lentiviruses at a multiplicity of infection of 20 and 10 ⁇ g/mL of Diethylaminoethyl. All the assays were performed in triplicate and using T cells from three different healthy donors.
  • Transduction efficiency of human primary T cells was assessed by expression of a reporter gene (ZsGreen).
  • the CAIX-Fc protein was expressed from a pcDNA3.1 plasmid that encoded amino acids 38-397 of CAIX followed by human IgG1 hinge, CH2 and CH3 domains, the CAIX signal peptide (aa 1-37) was replaced with Ig leader sequence.
  • Expression of scFv(G250) on transduced T cells was tested by staining the cells with 1 ⁇ g CAIX-Fc protein, and then APC-conjugated mouse anti-human IgG antibody (Jackson ImmunoResearch).
  • TETSQVAPA internal rhodopsin nonapeptide C9 tag of the scFv domain of TCR constructs on transduced T cells was detected by staining with 5 ⁇ g mouse 1D4 antibody followed by APC-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch).
  • the subsets of human cells in culture during clonal expansion experiment were stained with fluorescence conjugated mouse anti-human antibodies (Invitrogen) against CD3 (clone S4.1), CD4 (clone S3.5) or CD8 (clone 3B5). In all cell staining, five hundred thousand cells were stained with antibodies at recommended concentration according to company's instruction. The matched isotype control antibodies for each sample were used and the cells were analyzed using a FACSCalibur cytometer (Becton-Dickinson).
  • transduction of 293T cells or CD8+ T cells was confirmed by FACS analysis of the anti-CAIX or anti-BCMA expression.
  • the cells were stained with 10 ⁇ g/mL of human CAIX-Fc produced in our lab or human BCMA-mouse-Fc (AB Bioscience) and then developed with 1:250 APC-conjugated mouse anti-human IgG Ab (Southern Biotech) or goat-anti mouse IgG Ab (Biolegend), respectively.
  • CountBrightTM Absolute Counting Beads was used for the proliferation and clonal expansion assays.
  • TIL Tumor-infiltrating Lymphocytes
  • CAIX and PD-L1 were used 10 ⁇ g/mL of the anti-human CAIX mAb (Clone G36), produced in our laboratory, and 10 ⁇ g/mL of the biotinylated mouse anti-human PD-L1 (Biolegend).
  • the primary antibodies were detected using 1:250 APC-conjugated anti-human Ab and PE-conjugated avidin, respectively, and analyzed by FACS.
  • Cytotoxicity assays were performed using the DELFIA EuTDA Cytotoxicity kit (Perkin Elmer, Boston, Mass.) in accordance with the manufacturer's instructions. Briefly, target tumor cells were labeled with a fluorescent ligand (BATDA) for 30 minutes at 37° C. and 1 ⁇ 10 4 labeled cells were loaded per well in 96-well U-bottom plate. For antibody-dependent cellular cytotoxicity (ADCC) assay, a panel of anti-CAIX scFv-Fc antibodies or irrevelant scFv-Fc antibody at a concentration of 1 ⁇ g/ml or 5 ⁇ g/ml was added separately.
  • DCC antibody-dependent cellular cytotoxicity
  • the assay was set up with ratios of effector cells (human PBMC) to target cells (E:T) at 50:1, 25:1 and 12.5:1.
  • T cell cytoxicity assay different ratios of effector cells (nontransduced or transduced T cells) to target cells (E:T) were prepared (100:1, 50:1 and 25:1).
  • the culture was incubated for 4 hours in humidified 5% CO 2 at 37° C. After the plate was spun for 5 minutes at 500 ⁇ g, 20 ⁇ l of supernatant was transferred to a flat-bottom plate. 200 ⁇ l of Europium solution was added and the fluorescence released from the cells was read by fluorometer (VictorTM, PerkinElmer).
  • the control for spontaneous release was prepared by culturing the labeling cells only and the control for maximum release was made by adding lysis buffer (kit provided) to the labeling cells.
  • RCC cell lines sk-rc-52 (CAIX+) or sk-rc-59 (CAIX ⁇ ) were seeded overnight at 1 ⁇ 10 6 per well in a 24-well plate, followed by 1 ⁇ 10 6 untransduced or transduced T cells.
  • T cells were washed with PBS twice to remove human IL-2. After overnight incubation, the supernatant was harvested and analyzed for IL-2 and IFN- ⁇ by ELISA (e-Bioscience).
  • ELISA e-Bioscience
  • a membrane was developed using AEC substrate solution and the number of spots was counted by ELISPOT plate reader (C.T.L. Cellular Technology).
  • the total level of IgG secreted to the medium of transduced cells was detected using Human IgG ELISA Quantitation Set (Bethyl Laboratories).
  • the Anti-PD-L1 Abs secreted by transduced CD8+ CART Cells were purified with Protein A sepharose beads (GE Healthcare) and biotinylated using the EZ-Link Sulfo-NHS-LC-Biotin (Thermo Scientific). These antibodies were incubated with 5 ⁇ g/mL of human PD-L1 produced in the lab, which was pre-immobilized in MaxiSorp plates (Nunc) by 2 hours, RT.
  • Tumor cells were irradiated (3,000 rads) and seeded at 2.5 ⁇ 10 5 per well. T cells were added at 1 ⁇ 10 6 in culture medium containing R-10 plus 100 IU/ml human IL-2 for a week culture. T cells were split to maintain suitable density and re-stimulated with tumor cells weekly. The number of T cells was counted every 3 or 4 days for 2 weeks. The percentage expression of ZsGreen by transduced T cells and T cell subsets were determined weekly by fluorescence-activated cell sorting (FACS). For cytokine secretion studies after tumor cell contact, T cells that were in contact with irradiated tumor cells for one or two weeks were washed, incubated with fresh tumor cells overnight and culture supernatants were collected after 24 hrs for analysis.
  • FACS fluorescence-activated cell sorting
  • Skrc52 CAIX+/PD-L1 ⁇ and Skrc52 CAIX ⁇ /PD-L1 ⁇ cells were irradiated with 3,000 rads and seeded at 2.5 ⁇ 10 5 per well. 1 ⁇ 10 6 T cells were added at the culture medium containing 50 IU/ml human IL-21 every two days. T cells were split to maintain suitable density and re-stimulated with tumor cells weekly. T cell number was counted once a week for 3 weeks by FACS.
  • LDH Lactate dehydrogenase
  • the tumor BLI was quantified after 7, 14, 23 and 30 days of CART cells injection. A second injection of 2.5 ⁇ 10 6 CAR or untransduced T cells was made on day 17.
  • mice were sacrificed at 30 days post tumor engraftment by standard CO2 inhalation, and tumors were harvested and weighted.
  • the kidney tumors from all mice were divided in two equal parts and one of them was fragmented in small pieces and digested with collagenase 0.5 U/mL and DNAse 1.0 mg/mL to TIL extraction, which were analyzed for the expression of the exhaustion markers and the percentage of CART cells by FACS.
  • the other part was fixed in 10% buffered formaldehyde, and submitted to immunohistochemistry for different markers.
  • Two mice of each group were injected with 4.5 ⁇ 106 NK cells 2 days before the euthanasia. NK cells present in the tumor were stained with APC-Anti-CD56 Ab and analyzed by FACS. Animal experiments were performed in accordance with the guidelines of the DFCI Animal Care Committee.
  • sk-rc-52 due to immune-rejection of sk-rc-52 in 6-8 week-old female BALB/c nude mice and to accelerate in vivo growth properties, five million cells were subcutaneously inoculated into the mice, harvested, and expanded in vitro. The cell line was then passaged two more times in nude mice and the passaged cells were expanded for further experiments (subclone 4-1). For the therapeutic experiments, 5 million sk-rc-59 and 7.5 million passaged sk-rc-52 cells were subcutaneously inoculated on opposing flanks into nude mice to yield comparable tumor growth rates. After 7 days, tumors grew to the size of ⁇ 6 mm, and 50 million nontransduced or transduced T cells were injected intravenously.
  • mice were also treated with 20,000 IU human IL-2 by peritoneal injection every two days. Tumor size was measured by caliper in two dimensions and the mean of two tumor diameter was reported here. Animal experiments were performed in accordance with the guidelines of the Dana Farber Cancer Institute Animal Care Committee. Mice were sacrificed when tumors reached 15-mm diameter or 2,000 mm 3 and tumors were harvested.
  • the cultured T cells were washed twice using PBS and resupended in 2 ⁇ M Far Red DDAO-SE CellTrace dye (Molecular Probe) in PBS for 15 minutes at 37° C. Then the cells were washed with culture medium twice and cytospun on the glass slide. Far red pre-stained CART cells with ZsGreen coexpression were visualized using confocal microscopy (Zeiss) at the Optical Imaging Core facility, Harvard NeuroDiscovery Center.
  • Far Red DDAO-SE CellTrace dye Molecular Probe
  • tumors were prepared for frozen sections for ApopTag Peroxidase In Situ Apoptosis Detection kit (Millipore). Cryosections were incubated with TdT enzyme (Millipore) for 1 hour. Rabbit anti-DIG (Dako) was added and incubated for 30 minutes and then Cy3-conjugated anti-rabbit antibody (Invitrogen) was added and incubated for 30 minutes. Sections were mounted with DAPI antifade mounting medium and fluorescent images were examined using confocal microscopy.
  • Xenograft tumors and mouse spleens were harvested, fixed in 10% formalin/PBS solution, and submitted to the Harvard Medical School, Rodent Histopathology Core Facility. Paraffin-embedded sections were dewaxed with xylene and rehydrated through graded alcohols before staining. Immunohistochemistry staining was performed by incubating with anti-human granzyme B antibody (Dako, clone GrB-7 (1:200)) as a primary antibody for 1 hour followed by secondary anti-rabbit antibody (Pierce) or anti-mouse antibody (Dako) for 30 minutes. Sections were developed using DAB substrate and counterstained with hematoxylin.
  • the fixed tumors were paraffin-embedded, sectioned at four-micrometer, placed on slides and prepared for IHQ.
  • the tissues were stained with the anti human: Ki67 (Vector, VP-K451), PD-L1 (Clone 405.9A11, produced in Dr. Gordon Freeman's lab), granzyme B (Abcam, ab4059) or NCAM (CD56) (Abcam, ab133345) antibodies, followed by secondary HRP conjugated anti-rabbit Ab or HRP-Avidin.
  • Ki67 Vector, VP-K451
  • PD-L1 Cell 405.9A11
  • granzyme B Abcam, ab4059
  • NCAM CD56
  • the images were obtained in an Olympus BX51 microscopy using a DP71 digital camera (Olympus) and analyzed in the DP Controller Software (Olympus).
  • the image quantification was performed using the IHC Profiler Plugin of ImageJ Software as described in Varghese F, Bukhari A B, Malhotra R, De A.
  • IHC Profiler an open source plugin for the quantitative evaluation and automated scoring of immunohistochemistry images of human tissue samples. PloS one. 2014; 9:e96801.
  • FIGS. 16-23 are representative of at least three experiments unless otherwise noted.
  • the statistical significance of the data was evaluated using ANOVA and Tukey posttest. P ⁇ 0.05 was considered significant.
  • the statistical analysis was performed using the IBM SPSS Statistics software version 20.
  • 2 nd generation CD28 CAR was generated, consisting of scFvG36 fused to truncated extracellular, transmembrane and intracellular domains of CD28 plus signaling domain of TCR ⁇ (G36-CD28z) ( FIG. 2A ).
  • Irrelevant 2 nd generation CD28 CAR was made by using anti-HIV CCRS (clone A8) scFv instead 25 .
  • PHA mitogen was used to stimulate peripheral blood lymphocytes for 3 days.
  • Concentrated lentivirus supernatants were used to infect human primary T cells in the presence of cationic reagent DEAE as it increased the transduction rate of 1.5-2 ⁇ fold as compared with polybrene (data not shown).
  • the transduction rate of primary T cells ranged from 17% to 45% by ZsGreen expression in FACS analysis.
  • a representative experiment showing ZsGreen expression in circa 25% by primary CART cells following lentivirus transduction is shown in FIG. 2B , left column CAIX-Fc fusion protein can bind to the G36-CD8z and -CD28z CART cells but not to control A8-CD28z CART cells ( FIG. 2B , middle column).
  • both 1 st and 2 nd generation G36 expressing CART cells showed elevated levels of cytokine secretion with 2 nd generation G36-CD28z CART cells secreting higher amounts of type I cytokines which reflects their higher activation status compared to 1 st generation G36-CD8z CART cells.
  • G36-CD28z CART cells secreted 6.5 ⁇ , 2.3 ⁇ and 4 ⁇ more IL-2, IFN ⁇ and IL-17, respectively than G36-CD8z CART cells.
  • Specificity of cytokine secretion induction by the two G36 CART cells is seen by their minimal stimulation with CAIX ⁇ sk-rc-59 cells.
  • G36-CD28z CART cells became high capacity IFN- ⁇ producing cells ( FIG. 3B ).
  • G36-CD28z CART cells produced 6 times more spots than seen for G36-CD8z CART cells upon interaction with CAIX+ sk-rc 52 tumor cells and 12 times more spots than seen after interaction with CAIX ⁇ sk-rc-59 tumor cells.
  • G36-CD28z CART cells had a higher amount of granzyme B-secreting spots after contact with CAIX+ tumors as compared with G36-CD8z CART cells and control T cells.
  • G36-CD28z CART cells An in vitro cytotoxicity assay was established to further evaluate the killing activity of the different G36 CART cells.
  • G36-CD28z CART cells and its' twice in vivo passaged subclone 4-1 exhibited the highest amount of cytolysis of CAIX+ tumor sk-rc-52 ( FIG. 3C ).
  • G36-CD28z CART cells showed 2-3 fold higher cytotoxicity than G36-CD8z CART cells and with low ratio of 5:1, G36-CD28z CART cells showed 8-9 fold higher lysis than G36-CD8z CART cells.
  • G36-CD8z CART cells still exhibited good cytotoxicity with up to more than 60% tumor lysis using 100:1 of E:T ratio.
  • Irrelevant A8-CD28z CART cells and control T cell LAK showed the background non-specific tumor lysis with around 20% lysis when using the highest 100:1 of E:T ratio.
  • transduced and untransduced T cells showed background lysis.
  • Proliferating T cells were also harvested to examine their enrichment on CAIX+ tumor cell contact.
  • CAIX ⁇ tumor contact there was no change in the percentage of any CART cells within the population.
  • CAIX+ sk-rc-52 tumor cells there was enrichment in both populations of G36 CART cells.
  • G36-CD28z CART cells the positive population was enriched from 18% on day 0 to 52% on day 8 to 88% on day 16.
  • Expression of G36-CD8z CART cells was enriched from 19% on day 0 (same levels at T cells only) to 32% on day 8, and to 72% on day 16.
  • No expansion of A8-CD28z CART cells was seen over the two week study ( FIG. 4B ).
  • the percentage of CD8 cells remained constant throughout the 16 day study under all conditions ( FIG. 4C ).
  • Transduced T cells that were in contact with irradiated tumor cells for one or two weeks were also tested for cytokine secretion after 24 hours of contact with fresh non-irradiated tumor cells.
  • G36-CD28z and G36-CD8z CART cells Upon contact with CAIX+ tumor (sk-rc-52) for one or two weeks, G36-CD28z and G36-CD8z CART cells showed similar IFN- ⁇ secretion levels although costimulatory signaling through G36-CD28z CAR yielding 2 ⁇ to 2.5 ⁇ more IFN- ⁇ secretion than seen for G36-CD8z CAR (Table 1).
  • IL-2 secretion two weeks of tumor contact for G36-CD28z and G36-CD8z CART cells exhibited more IL-2 secretion than one week of contact.
  • G36-CD28z CART cells yielding 5 ⁇ more IL2 than G36-CD8z CART cell on one week of contact and 2.5 ⁇ more on contact for two weeks.
  • G36-CD28z CART cells in contact with tumor cells for two weeks secreted 3.3 ⁇ more IL-2 than one time tumor contact whereas G36-CD8z CART gave 6.8 ⁇ more IL-2 secretion after two weeks compared to after one week of tumor contact.
  • treated and untreated CAIX ⁇ sk-rc-59 tumors had average size of 6.09 ⁇ 0.02 mm on day 4 and 9.29 ⁇ 0.12 mm on day 25 (within four tested groups). They exhibited the same tumor growth rate in control groups and T-cell treated groups. Untreated CAIX+ tumors that received no T cells showed similar tumor size as CAIX ⁇ tumors, with an average size of 6.09 ⁇ 0.13 mm on day 4 and 9.15 ⁇ 0.11 mm on day 25. However, the tumor size of G36-CD28z CART cell treated mice showed statistically significant reduction in size compared to no T-cell treated mice at every time point that was examined over the 25 day study ( FIG. 5 ).
  • G36-CD28z CART treatment also led to a greater reduction in tumor size than seen with A8-CD28z CART cell and LAK treated mice on day 7 (p ⁇ 0.05) and on day 25 (p ⁇ 0.001), as calculated by two-tailed t test.
  • tumor size of G36-CD28z CART cell treated mice was significant smaller than that of no T-cell treated mice through the 29 day experiment.
  • G36-CD28z CART cell treated mice also had smaller tumors than were seen with A8 CD28z CART cell and LAK treated mice on day 8 to day 26 with p ⁇ 0.01 and on day 29 with p ⁇ 0.001 ( FIG. 5 ).
  • Partial regression of CAIX+ tumor was considered when the tumor size was smaller than 30% volume of control CAIX ⁇ tumor in a same mouse receiving the same T-cell. Partial tumor regression was observed in a high percentage of cases using G36-CD28z CART cells (10 out of 15, (67%)), but only infrequently in irrelevant target A8-CD28z CART cells (1 out of 15, (7%)) and in activated T cell LAKs (2 out of 15, (13%)) (Table 2). Frequency of partial regression response was found to be statistically significant for mice treated with G36-CD28z CART cells versus control A8-CD28z CART cells and LAKs at p ⁇ 0.001 and p ⁇ 0.005, respectively by Fisher test.
  • Example 9 Anti-Carbonic Anhydrase IX Chimeric Antigen Receptor T Cells Releasing Anti-PD-L1 Antibodies Revert T Cell Exhaustion and Regress Renal Cell Carcinoma in a Humanized Mouse Model
  • FIG. 23 The CART cell functionality is demonstrated in FIG. 23 , where CD8 T cells transduced with all CARS were able to proliferate in the presence of IL-21 and anti-CD8/CD28 beads ( FIGS. 23A and 23B ), achieving transduction levels of 65-90% after four days ( FIG. 23C ).
  • FIG. 16C Fourteen days after transduction, we evaluated the stable long-term expression of CAR by the integrated lentiviruses ( FIG. 16C ), which was maintained around 25-50% for all CARs.
  • Total IgG levels secreted by CD8 T cells was also determined, ranging around 300-650 ng/mL after 4 days ( FIG. 16D ).
  • the binding specificity of the anti-PD-L1 IgG1 and IgG4 antibodies for human PD-L1 was also confirmed ( FIG. 16E ).
  • the levels of biotinylated anti-PDL1 IgG1 and 4 were significantly lower than total IgG, which could be explained by the fact that a portion of the IgG was wasted during the purification process.
  • the ability of anti-CAIX CART cells to undergo clonal expansion in the presence of CAIX+ RCC cells was established ( FIGS. 16F and 16G ). Anti-CAIX CART cells cannot expand significantly in the presence of CAIX ⁇ RCC cells.
  • Anti-CAIX CART Cells-Secreting Anti-PD-L1 Antibodies can Diminish T Cell Exhaustion In Vitro.
  • FIGS. 18A, 18B and 18C A circa 50% decrease in the exhaustion markers Lag-3, Tim3 and PD-1 was found in the anti-PD-L1 IgG1 and IgG4 anti-CAIX CART groups ( FIGS. 18A, 18B and 18C , respectively) after induction of exhaustion compared to the parental anti-CAIX or irrelevant anti-BCMA CART cells. At this point, the killing activity of anti-CAIX CART cells without anti-PD-L1 was retested over Skrc59 CAIX+/PD-L1+ cells. As can be seen in FIG.
  • the anti-CAIX CART cells had lost their killing activity against CAIX+/PD-L1+ RCC in vitro to a level that was similar to the irrelavent CAR group, establishing that the anti-CAIX CART cells became anergic.
  • the RCC viability diminished 25-50% for the anti-CAIX CAR anti-PD-L1 IgG1 and IgG4 CART groups, thereby providing evidence that the checkpoint blockade elicited by the presence of secreted anti-PD-L1 IgGs can lead to diminished T cell exhaustion.
  • Anti-CAIX CART Cells Secreting Anti-PD-L1 Antibodies can Further Decrease Tumor Growth in an Orthotopic Mouse Model of Human RCC.
  • NSG mice were used to establish an orthotopic RCC model by injecting Skrc-59 CAIX+PD-L1+luciferase+ positive RCC cells under the kidney capsule followed by i.v. injection of 1.0 ⁇ 107 CART or untransduced T cells (Day 0) and repeat treatment on Day 17 with a lower dose (2.5 ⁇ 10 6 ) of the same cells.
  • FIGS. 19A-19C demonstrate that all three anti-CAIX CART cell groups showed decreased RCC growth compared to irrelevant anti-BCMA CART cells or untransduced cells over the course of the experiment with the marked anti-tumor effects of the anti-CAEX CART cells secreting anti-PD-L1 IgG1 or IgG4 becoming evident at day 23 and 30 ( FIGS. 19A and 19B ).
  • FIGS. 19A and 19B show that the tumors were 2-3 times smaller in the anti-PD-L1-secreting CART cells when compared with parental anti-CAIX CART cells and the two control groups.
  • FIG. 24A We also analyzed CD45+ T cell survival in the mouse blood to gauge their survival in this passive transfer model.
  • the effector activity of CART cells and their influence over RCC proliferation in vivo was evaluated by the immunohistochemical detection of granzyme B in the TIL, the tumor proliferation marker Ki67 and the tumor immunosuppressive protein PD-L1 ( FIGS. 20B and 20C ).
  • the granzyme B staining showed the effector activity of CD8+ cells, especially in the RCC tumors treated with the anti-CAIX CART cells secreting anti-PD-L1 IgG4, which presented an augmented percentage of high positive stained cells ( FIGS. 20B and 20C ).
  • PD-L1 expression decreased dramatically in the tumors treated with Antianti-CAIX CART cells, being more expressive in the Anti-CAIX/Anti-PD-L1 IgG-secreting groups ( FIGS. 20B and 20C ).
  • Ki67 expression decreased significantly in the Anti-CAIX/Anti-PD-L1 IgG-secreting groups, as we can visualize in the total DAB pixel count graph, however when the intensity among the positive nuclei staining (Ki67 IHC quantification graph) was evaluated, we can note that the Anti-CAIX/Anti-PD-L1 IgG4 presented the lowest intensity of Ki67 expression ( FIGS. 20B and 20C ).
  • Anti-CAIX CART Cells Secreting Anti-PD-L1 IgG1 Antibodies can Recruit NK Cells to the Tumor.
  • the tumors of the mice treated with Anti-CAIX/Anti-PD-L1 IgG1 that received an injection of NK cells 2 days before the euthanasia showed the presence of 40% more NK cells inside the tumor, when compared with Anti-BCMA/Anti-SARS IgG1, as detected by Anti-CD56 staining by FACS ( FIG. 25A ).
  • the increase in NK cells in the Anti-CAIX/Anti-PD-L1 IgG1 group was also detected and quantified by IHC ( FIG. 25B ). Very few NK cells were also detected in the groups treated with CART cells secreting an unspecific IgG1 Ab.

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AU2022200257A1 (en) 2022-02-10
KR20170090506A (ko) 2017-08-07
AU2020204373A1 (en) 2020-07-23
IL252295A0 (en) 2017-07-31
WO2016100985A2 (en) 2016-06-23
IL252295B2 (en) 2023-10-01
AU2022200257B2 (en) 2023-11-16
AU2020204373B2 (en) 2021-10-21
CA2968412A1 (en) 2016-06-23
WO2016100985A3 (en) 2016-08-18
EP3233900A2 (en) 2017-10-25
CN108064252A (zh) 2018-05-22
BR112017013176A2 (pt) 2018-05-15
AU2015364245B2 (en) 2020-04-02
IL302660A (en) 2023-07-01
JP2022064952A (ja) 2022-04-26
US20210061877A1 (en) 2021-03-04
AU2024200592A1 (en) 2024-02-22
RU2017125531A (ru) 2019-01-21
IL252295B1 (en) 2023-06-01

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