WO2019060174A1 - Récepteurs antigéniques chimériques à signalisation de nfkb améliorée - Google Patents

Récepteurs antigéniques chimériques à signalisation de nfkb améliorée Download PDF

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WO2019060174A1
WO2019060174A1 PCT/US2018/050417 US2018050417W WO2019060174A1 WO 2019060174 A1 WO2019060174 A1 WO 2019060174A1 US 2018050417 W US2018050417 W US 2018050417W WO 2019060174 A1 WO2019060174 A1 WO 2019060174A1
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car
cells
cell
taa
domain
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PCT/US2018/050417
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Marco L. DAVILLA
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H. Lee Moffitt Cancer Center And Research Institute, Inc.
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Priority to AU2018335266A priority Critical patent/AU2018335266A1/en
Priority to EP18859613.4A priority patent/EP3684424A4/fr
Priority to CA3070861A priority patent/CA3070861A1/fr
Priority to US16/632,091 priority patent/US11434290B2/en
Priority to JP2020516722A priority patent/JP2021500859A/ja
Publication of WO2019060174A1 publication Critical patent/WO2019060174A1/fr
Priority to US17/929,457 priority patent/US20230265184A1/en

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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
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    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
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    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
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    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
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    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • 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
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C12N5/0638—Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]
    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • 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)
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    • 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
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    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
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    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2510/00—Genetically modified cells
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • Immunotherapy sometimes called biological therapy, biotherapy, or biological response modifier therapy
  • the human immune system is an untapped resource for cancer therapy and that effective treatment can be developed once the components of the immune system are properly harnessed.
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • CAR chimeric antigen receptor
  • the disclosed CARs comprise a costimulatory signaling region with one or more mutations in the cytoplasmic domains of CD28 and/or 41 BB that enhance signaling that CAR-T cell function.
  • the mutated costimulatory signaling region reduces CAR-T cell exhaustion.
  • the CD28 domain includes 3 intracellular subdomains (YMNM, PRRP, and PYAP) that regulate signaling pathways post TCR-stimulation.
  • the disclosed CAR comprises mutation or deletion of one or more of these subdomains that enhances CAR-T cell function, e.g. reducing CAR-T cell exhaustion.
  • N FKB nuclear factor kappaB
  • CARs chimeric antigen receptors
  • CARs chimeric antigen receptors
  • the co-stimulatory protein 41 BB CD137
  • TRAF proteins can independently regulate CAR
  • each TRAF has a different impact on CAR T cell function.
  • CARs comprising one or more 41 BB domains with mutations that enhance binding to specific TRAF proteins, such as TRAF1 , TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, or any combination thereof.
  • the 41 BB mutation enhances TRAF1- and/or TRAF2-dependent proliferation and survival of the T-cell, e.g. through NF-kB.
  • the 41 BB mutation enhances TRAF3-dependent antitumor efficacy, e.g. through ⁇ 7/ ⁇ .
  • TRAF proteins can in some cases enhance CAR T cell function independent of N FKB and 41 BB.
  • TRAF proteins can in some cases enhance CD28 co-stimuation in T cells. Therefore, also disclosed herein are immune effector cells co-expressing CARs with one or more TRAF proteins, such as TRAF1 , TRAF2, TRAF3, TRAF4, TRAF5, TRAF6, or any combination thereof.
  • the CAR is any CAR that targets a tumor antigen.
  • first-generation CARs typically had the intracellular domain from the ⁇ 3 ⁇ chain
  • second-generation CARs added intracellular signaling domains from various costimulatory protein receptors (e.g., CD28, 41 BB, ICOS) to the endodomain of the CAR to provide additional signals to the T cell.
  • the CAR is the disclosed CAR with enhanced 41 BB activation.
  • the CAR polypeptides further comprise one or more deletions or mutations in CD3zeta and/or 41 BB that enhance CAR T cell function.
  • the disclosed CAR polypeptides contain in an
  • the disclosed polypeptides can also contain a transmembrane domain and an endodomain capable of activating an immune effector cell.
  • the endodomain can contain an intracellular signaling domain and optionally one or more co-stimulatory signaling regions.
  • the anti-TAA binding agent is in some embodiments an antibody fragment that specifically binds a TAA.
  • the antigen binding domain can be a Fab or a single-chain variable fragment (scFv) of an antibody that specifically binds a TAA.
  • the anti-TAA binding agent is in some embodiments an aptamer that specifically binds the TAA.
  • the anti-TAA binding agent can be a peptide aptamer selected from a random sequence pool based on its ability to bind TAA.
  • the anti-TAA binding agent can also be a natural ligand of TAA, or a variant and/or fragment thereof capable of binding the TAA.
  • the intracellular signaling domain is a CD3 zeta ( ⁇ 3 ⁇ ) signaling domain.
  • the costimulatory signaling region contains 1 , 2, 3, or 4 cytoplasmic domains of one or more intracellular signaling molecules.
  • the cell can be an immune effector cell selected from the group consisting of an alpha-beta T cells, a gamma-delta T cell, a Natural Killer (NK) cells, a Natural Killer T (NKT) cell, a B cell, an innate lymphoid cell (I LC), a cytokine induced killer (CIK) cell, a cytotoxic T lymphocyte (CTL), a lymphokine activated killer (LAK) cell, and a regulatory T cell.
  • an immune effector cell selected from the group consisting of an alpha-beta T cells, a gamma-delta T cell, a Natural Killer (NK) cells, a Natural Killer T (NKT) cell, a B cell, an innate lymphoid cell (I LC), a cytokine induced killer (CIK) cell, a cytotoxic T lymphocyte (CTL), a lymphokine activated killer (LAK) cell, and a regulatory T cell.
  • the cell exhibits an anti-tumor immunity when the antigen binding domain of the CAR binds to the TAA on a tumor. Also disclosed is a method of providing an anti-tumor immunity in a subject with a TAA-expressing cancer that involves administering to the subject an effective amount of an immune effector cell genetically modified with a disclosed TAA-specific CAR.
  • FIG. 1 is a comparison of mouse T cells with mCD19 targeted CARs having different intracellular domains.
  • A Cytotoxicity assay. CAR T cells were co-cultured with EL4-mCD19 cells at indicated E:T ratios. Cytotoxicity was evaluated with a Chromium release assay. Data are representative of three independent experiments in triplicate.
  • B Cytokine production. CAR T cells were co-cultured with 3T3-mCD19 cells for 24 hr. Supernatant were collected for Luminex assay. Data are
  • FIG. 2 shows at a stress test dose mCD19-targeted CAR T cells containing a human 4-1 BB endodomain (m19-humBBz) display comparable in vivo function to m1928z CAR T cells.
  • A Amino acid sequence alignment of mouse (SEQ ID NO:25) and human (SEQ ID NO: 1) 4-1 BB endodomains. Identical amino acids are indicated with an asterisk.
  • B Intracellular IFNv and TNFa in CAR T cells upon mCD19 antigen stimulation. One million transduced T cells were co-cultured with 1 ⁇ 10 5 irradiated 3T3-mCD19 for 4 hr in the presence of protein transport inhibitor. Cells were subjected to flow cytometry. Data are representative of two independent experiments performed in triplicate.
  • C Cytotoxicity assay. CAR T cells were co-cultured with 3T3- mCD19 at a E:T ratio of 10: 1 and target cell killing was monitored on an
  • FIG. 3 shows persistence of m19-humBBz CAR T cells is required for optimal function in vivo.
  • CAR T CD3+CAR+
  • Donor T CD3+Thy 1.1+
  • FIG. 4 shows m19-humBBz CAR T cells have higher anti-apoptotic protein expression than m1928z CAR T cells.
  • C BCL2 and BCL-XL expression in CAR T cells by flow cytometry. Day 4 CAR T cells were intracellularly stained with anti-BCL2 and anti-BCL-XL antibodies and subjected to flow cytometry.
  • FIG. 5 shows N F-KB signaling regulates the viability and proliferation of 4- 1 BB-based CAR T cells.
  • A CAR expression (mCherry) after transduction (left) and N F-KB up-regulation (right) in N F-KB/293/GFP-LUC reporter cells. Reporter cells were transduced with mouse CD19-targeted CARs and N F- ⁇ signaling was measured by flow cytometry for GFR Data are representative of two independent experiments.
  • (B) m19-humBBz CAR T cells have greater N F- ⁇ signaling than m1928z CAR T cells after antigen stimulation.
  • C Three million CAR T cells derived from N F-KB-RE-IUC transgenic mice were co-cultured with 3T3-mCD19 cells at a 10:1 ratio for 4 hr. Cell lysates were evaluated using a luciferase assay. Data are representative of 3 independent experiments in triplicate.
  • C Amino acid sequences of human wild type (SEQ ID NO: 1) and mutated (SEQ ID NOs:2-5) 4-1 BB endodomains evaluated in hCD19-targeted CARs. Amino acid numbers of the 4-1 BB endodomain are shown.
  • D N F- ⁇ signaling of hCD19 CAR transduced reporter cells.
  • N F-KB/293/GFP-LUC reporter cells were retrovirally transduced with hCD19 targeted CARs. Percentages of GFP+ cells were measured by flow cytometry, which reflect NF- ⁇ signaling. Data are from one experiment and done in triplicate.
  • E Viability on day 16 and
  • F proliferation of hCD19 targeted CAR T cells cultured in vitro. Human T cells were isolated from healthy donor PBMC at day 0. CAR T cells were harvested, beads removed and co-cultured with 3T3-hCD19 cells at 5: 1 ratio for 2 weeks. Cell numbers were measured at indicated timepoints.
  • G Cytotoxicity of hCD19 targeted CAR T cells.
  • CAR T cells were co-cultured with 3T3-hCD19 cells at 10: 1 ratio.
  • Target cell killing was monitored by RTCA.
  • E E
  • F F
  • G data are from one single experiment in triplicate. Data represent mean ⁇ SD for Figure B-F. Cytotoxicity curves show mean only. Cell expansion curves, two-way ANOVA; all other data, unpaired t test. *p ⁇ 0.05; **p ⁇ 0.01 ; ***p ⁇ 0.001 ; ****p ⁇ 0.0001 ; ns, not significant.
  • FIG. 6 shows TRAF1 inhibition negatively impacts N F- ⁇ signaling and m19- humBBz CAR T cell function.
  • A Effect of TRAF dominant negative (DN) proteins on m19-humBBz-induced N F- ⁇ signaling in N F-KB/293/GFP-LUC reporter cells.
  • Reporter cells were retrovirally transduced with cerulean-tagged TRAF1 DN, TRAF2 DN or TRAF3 DN constructs followed by transduction with m19-humBBz CAR. Cells were subjected to flow cytometry for NF- ⁇ signaling, shown as GFP+ cells. Data are representative of two independent experiments.
  • C Viability and proliferation of wild type and Traf1 "A mCD19-targeted CAR T cells.
  • CAR T cells were produced from wild type B6 mice or Traf1 "A mice and proliferation was evaluated by fold change from the initial cell number to final cell yield at Day 4. Cell viability was measured by trypan blue staining on an automated cell counter (BIO-RAD). Data are from a single experiment in triplicate.
  • FIG. 7 shows TRAF2 over-expression modulates 4-1 BB based human CAR T function.
  • A N F- ⁇ signaling in human CD19 CAR (h19BBz) transduced N F-KB293 reporter cells by increasing N F- ⁇ . Cells were transduced with h19BBz CAR with or without TRAFs. N F- ⁇ was measured by GFP. Data are from one experiment in triplicate.
  • B Viability and (C) cell expansion of h19BBz CAR T cells with TRAF over- expression upon antigen stimulation. CAR T cells were co-cultured with 3T3-hCD19 at a 10: 1 ratio and cell numbers and viability were measured daily for 3 days.
  • CAR T cells were co-cultured with CHO-hCD33 at a 10: 1 ratio. Target cell killing was monitored by RTCA.
  • F Fold change of h33BBz T cell production with or without TRAF2 co-transduction. CAR T cells were produced and proliferation was evaluated by fold change from the initial cell number to final cell yield.
  • G h33BBz CAR T cell expansion in vitro upon antigen stimulation. CAR T cells were stained with
  • FIG. 8 shows immune phenotype of mCD19 targeted CAR T and dose titration of in vivo efficacy.
  • A Immune phenotype of transduced T cells used in 5x106 dose in vivo study ( Figure 1C&D). Cells were pre-gated on single live cells.
  • FIG. 9 shows gene expression of fluorescent-protein tagged CAR T cells.
  • A Schematic of genetic constructs for mCD19 targeted CARs. Shown are the long terminal repeats (LTR), packaging signal ⁇ , splice donor (SD), splice acceptor (SA), VH and VL regions of the scFv (single-chain variable fragment), the extracellular hinge (EC), transmembrane (TM), and intracellular regions of the retroviral construct.
  • LTR long terminal repeats
  • SD splice donor
  • SA splice acceptor
  • VH and VL regions of the scFv single-chain variable fragment
  • EC extracellular hinge
  • TM transmembrane
  • TM transmembrane
  • B Comparison of fluorescence protein and Protein L as a method to evaluate CAR expression.
  • C Principal component analysis
  • E A heatmap of the 205 differentially expressed genes. The list of 205 genes is included in Supplemental Tables 1-4.
  • CAR T cells with the m19z, m1928z, or m19-musBBz CAR tagged to the fluorescent protein GFP were incubated with 3T3-mCD19 AAPC at 10: 1 E:T ratio overnight, FACS-sorted, and lysed to isolate RNA. Each group of CAR T cells was transduced, stimulated, and sorted independently in triplicates.
  • FIG. 10 shows fluorescent protein tagged CAR T cells function similarly to non-tagged counterparts.
  • A Cytokines released by fluorescent protein tagged CAR T cells upon antigen stimulation. Day 4 CAR T cells were co-cultured with 3T3- mCD19 at 10: 1 ratio for 24 hr. Supernatant was subjected to luminex assay for IFNv and TNFa.
  • B Immune phenotype of CAR T cells with a fluorescent protein tag. Day 4 CAR T cells were harvested, beads removed and subjected to flow cytometry. Cells were pre-gated on single live cells (top) or CD3+CAR+ cells (middle & bottom).
  • mice were i.p. injected with CTX at 250 mg/kg and then one day later i.v.
  • FIG. 11 shows transduction efficiency and immune phenotype of mCD19 targeted CAR T cells for survival study (Figure 2D). Data are representative of four independent productions used to generate CAR T cells for the survival experiments of Figure 2D. For transduction efficiency (top panel), cells were pre-gated on single live cells. For immune phenotype (middle and bottom panels), cells were pre-gated on single live CAR T (CD3+CAR+) cells.
  • FIG. 12 shows transduction efficiency and immune phenotype of CAR T cells used in irradiated CAR T study (Figure 3B-3C).
  • Day 4 transduced cells were harvested, beads removed, stained with antibodies and subjected to flow cytometry.
  • For transduction efficiency (Top panels), cells were pre-gated on single live cells.
  • For immune phenotype (middle and bottom panels), cells were pre-gated on CD3+CAR+ cells.
  • FIG. 13 shows differential gene expression of CD4+ m19-humBBz CAR T cells.
  • T cells with the m19z, m1928z, or m19-humBBz CAR were incubated with 3T3- mCD19 AAPC at 10: 1 E:T ratio, FACS-sorted, and lysed to isolate RNA. Each group of CAR T cells was transduced, stimulated, and sorted independently in biologic triplicates.
  • A PCA of mouse CD19-targeted CAR T cells stimulated with antigen.
  • B Venn Diagram demonstrating the number of genes differentially expressed in m19- humBBz CAR T cells compared to m19z and m1928z CAR T cells.
  • C GSEA demonstrates gene sets correlating to NF- ⁇ B regulatory pathways are differentially expressed in m19-humBBz CAR T cells versus m19z or m1928z CAR T cells.
  • FIG. 14 shows CAR expression and CD4/CD8 subsets of human CD19 targeted CAR T cells for Figure 5E-G.
  • CAR expression top
  • immune phenotype bottom
  • cells were pre-gated on CD3+CAR+ cells.
  • FIG. 15 shows transduction efficiency and immune phenotype of mCD19 targeted wild type (WT) and Trail-'- CAR T cells used for in vivo study ( Figure 6D).
  • Day 4 transduced cells were harvested, beads removed, stained with antibodies and subjected to flow cytometry.
  • For transduction efficiency (top panel), cells were pre- gated on single live cells.
  • For CD4/CD8 subsets (middle panel) and memory subsets (bottom panel) cells were pre-gated on single live CAR T (CD3+CAR+) cells.
  • FIG. 16 shows mutated m19-musBBz CAR T cells have increased NF-kB signaling, improved cytokine production, anti-apoptosis, and in vivo function.
  • A NF- kB signaling in mCD19 targeted CAR T cells.
  • CAR T cells derived from NF-k B-RE- luc transgenic mice were co-cultured with 3T3-mCD19 for 4 hr. Cell lysates were prepared and subjected to a luciferase assay. Bioluminescence was measured and correlates to NF-kB signaling. Data are representative of three independent experiments.
  • B Intracellular IFNv and
  • C BCL-XL expression in CD8+ CAR T cells stimulated with 3T3-mCD19. Data are representative of two independent
  • FIG. 17 shows TRAF and CAR co-expression in human CD19-targeted CAR T cells.
  • A CAR and TRAF expression in hCD19 targeted CAR T cells before antigen stimulation for viability, proliferation, and cytotoxicity assays (Figure 7B-D). Cells were pre-gated on single live cells. Data are one representative of 3 healthy donors.
  • B CAR and TRAF expression in hCD19 CAR T cells 3 days after stimulation with 3T3-hCD19. Cells were pre-gated on single live cells. Data are from one experiment in triplicate. Numbers indicate percentages of gated cells.
  • C Cytokine production.
  • CAR T cells were activated on 3T3-hCD19 at 10: 1 ratio. After 24 hr supernatant were harvested and cytokines were measured by ELLA. Bar graphs shown as mean ⁇ SD. Data are one representative of 3 different healthy donors in triplicate. All data, unpaired t test, ns, not significant.
  • FIG. 18 shows TRAF2 over-expressed h19BBz CAR T cells show similar in vivo efficacy to h19BBz CAR T cells in an aggressive leukemia model.
  • NSG mice were implanted with leukemia by i.v. injecting 5x10 5 NALM6-GL cells. Four days later, mice were i.v. injected with 3x10 5 -1x10 6 CAR T cells. Blood samples were collected weekly. Leukemia burden was evaluated weekly using bioluminescence imaging. Survival was monitored.
  • CAR chimeric antigen receptor
  • CD28 and/or 4-1 BB that enhance signaling that CAR-T cell function.
  • immune effector cells such as T cells or Natural Killer (NK) cells, that are engineered to express these CARs. Therefore, also disclosed are methods for providing an anti-tumor immunity in a subject with TAA-expressing cancers that involves adoptive transfer of the disclosed immune effector cells engineered to express the disclosed CARs.
  • the mutated costimulatory signaling region reduces CAR-T cell exhaustion.
  • the CD28 domain includes 3 intracellular subdomains
  • the disclosed CAR comprises mutation or deletion of one or more of these subdomains that enhances CAR-T cell function, e.g. reducing CAR-T cell exhaustion.
  • the disclosed CARs comprises altered phosphorylation at Y206 and/or Y218.
  • the disclosed CAR comprises an attenuating mutation at Y206, which will reduce the activity of the CAR.
  • the disclosed CAR comprises an attenuating mutation at Y218, which will reduce expression of the CAR.
  • any amino acid residue such as alanine or phenylalanine, can be substituted for the tyrosine to achieve attenuation.
  • the tyrosine at Y206 and/or Y218 is substituted with a phosphomimetic residue.
  • the disclosed CAR substitution of Y206 with a phosphomimetic residue which will increase the activity of the CAR.
  • the disclosed CAR comprises substitution of Y218 with a phosphomimetic residue, which will increase expression of the CAR.
  • the phosphomimetic residue can be phosphotyrosine.
  • a CAR may contain a combination of phosphomimetic amino acids and substitution(s) with non-phosphorylatable amino acids in different residues of the same CAR.
  • a CAR may contain an alanine or phenylalanine substitution in Y209 and/or Y191 PLUS a phosphomimetic substitution in Y206 and/or Y218.
  • N FKB nuclear factor kappaB
  • CARs chimeric antigen receptors
  • CARs chimeric antigen receptors
  • the co-stimulatory protein 41 BB CD137
  • TRAF tumor necrosis factor receptor-associated factor
  • the disclosed CARs comprise two or more copies of 41 BB.
  • the disclosed CARs comprise one or more 41 BB domains with mutations that modulate binding to TRAF proteins, such as TRAF1 , TRAF2, TRAF3, or any combination thereof.
  • TRAF proteins can have both positive and/or negative regulatory effects on NFKB. These bind directly to 41 BB or bind to other proteins that are bound to 41 BB.
  • the disclosed mutations enhance association of TRAFs that potentiate NFKB and reduce association of TRAFs the attenuate NFKB signaling.
  • the cytoplasmic domain of 41 BB is responsible for binding to TRAF proteins. Therefore, in some embodiments, the disclosed CAR comprises two or more copies of the cytoplasmic domain of 41 BB. Moreover, in order to provide finer control over TRAF activity, the cytoplasmic domain of 41 BB can contain mutations that regulate TRAF association.
  • the cytoplasmic domain of 41 BB comprises the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:1). As disclosed herein, the regions of this domain responsible for TRAF binding are underlined in SEQ ID NO:1. Therefore, the disclosed CARs can comprise cytoplasmic domain(s) of 41 BB having at least one mutation in these underligned sequences that enhance TRAF-binding and/or enhance N FKB signaling.
  • the cytoplasmic domain of 41 BB comprises the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTAAAAGCSCRFPEEEEGGCEL (SEQ ID NO:2, Mut01).
  • the cytoplasmic domain of 41 BB comprises the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPAAAAGGCEL (SEQ ID NO:3, Mut02). In some cases, the cytoplasmic domain of 41 BB comprises the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTAAAAGCSCRFPAAAAGGCEL (SEQ ID NO:4, Mut03).
  • the cytoplasmic domain of 41 BB comprises the amino acid sequence
  • the cytoplasmic domain of 41 BB comprises the amino acid sequence
  • the cytoplasmic domain of 41 BB comprises the amino acid sequence
  • the cytoplasmic domain of 41 BB comprises the amino acid sequence
  • the cytoplasmic domain of 41 BB comprises the amino acid sequence
  • the cytoplasmic domain of 41 BB comprises the amino acid sequence
  • the cytoplasmic domain of 41 BB comprises the amino acid sequence
  • the cytoplasmic domain of 41 BB comprises the amino acid sequence
  • the cytoplasmic domain of 41 BB comprises the amino acid sequence
  • the cytoplasmic domain of 41 BB comprises the amino acid sequence
  • the cytoplasmic domain of 41 BB comprises the amino acid sequence
  • the cytoplasmic domain of 41 BB comprises the amino acid sequence
  • the cytoplasmic domain of 41 BB comprises the amino acid sequence
  • the cytoplasmic domain of 41 BB comprises the amino acid sequence
  • the cytoplasmic domain of 41 BB comprises the amino acid sequence
  • the cytoplasmic domain of 41 BB comprises the amino acid sequence
  • the cytoplasmic domain of 41 BB comprises the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO:1 , but has at least 1 , 2, 3, 4, 5, 6, or 7 amino acid substitutions of an underlined amino acid).
  • the amino acid substitution is a conservative substitution.
  • the amino acid is substituted for a non-acidic residue.
  • the cytoplasmic domain of 41 BB comprises the amino acid sequence KRGRKKLLYI FKQPFMRPVQTTX1X2X3X4GCSCRFPEEEEGGCEL (SEQ ID NO:21 , where Xi is not Gin, wherein X2 is not Glu, X3 is not Glu, where X 4 is not Asp, or any combination thereof).
  • the cytoplasmic domain of 41 BB comprises the amino acid sequence KRGRKKLLYI FKQPFMRPVQTTQEEDGCSCRFPX5X6X7X8GGCEL (SEQ ID NO:22, wherein X5 is not Glu, wherein ⁇ is not Glu, wherein X 7 is not Glu, wherein Xs is not Glu, or any combination thereof).
  • the cytoplasmic domain of 41 BB comprises the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTXj) ⁇ 2) ⁇ GCSCRFPX ⁇ ) ⁇ 7 X8GGCEL (SEQ ID NO:23, where Xi is not Gin, wherein X2 is not Glu, X3 is not Glu, where X* is not Asp, wherein X5 is not Glu, wherein 3 is not Glu, wherein X 7 is not Glu, wherein Xs is not Glu, or any combination thereof).
  • the cytoplasmic domain of 41 BB comprises the amino acid sequence
  • CARs generally incorporate an antigen recognition domain from the single- chain variable fragments (scFv) of a monoclonal antibody (mAb) with transmembrane signaling motifs involved in lymphocyte activation (Sadelain M, et al. Nat Rev Cancer 2003 3:35-45).
  • scFv single- chain variable fragments
  • mAb monoclonal antibody
  • CAR chimeric antigen receptor
  • the disclosed CAR is generally made up of three domains: an ectodomain, a transmembrane domain, and an endodomain.
  • the ectodomain comprises the TAA- binding region and is responsible for antigen recognition. It also optionally contains a signal peptide (SP) so that the CAR can be glycosylated and anchored in the cell membrane of the immune effector cell.
  • SP signal peptide
  • the transmembrane domain (TD) is as its name suggests, connects the ectodomain to the endodomain and resides within the cell membrane when expressed by a cell.
  • the endodomain is the business end of the CAR that transmits an activation signal to the immune effector cell after antigen recognition.
  • the endodomain can contain an intracellular signaling domain (ISD) and a co-stimulatory signaling region (CSR).
  • ISD intracellular signaling domain
  • CSR co-stimulatory signaling region
  • the disclosed CARs have a CSR comprising a mutated form of 41 BB that enhances N FKB signaling.
  • the disclosed CAR is defined by the formula:
  • SP represents an optional signal peptide
  • TAA represents a TAA-binding region
  • HG represents an optional hinge domain
  • TM represents a transmembrane domain
  • CSR represents the co-stimulatory signaling region
  • ISD represents an intracellular signaling domain
  • the CAR can be a TRUCK, Universal CAR, Self-driving CAR, Armored CAR, Self-destruct CAR, Conditional CAR, Marked CAR, TenCAR, Dual CAR, or sCAR.
  • TRUCKs T cells redirected for universal cytokine killing co-express a chimeric antigen receptor (CAR) and an antitumor cytokine.
  • Cytokine expression may be constitutive or induced by T cell activation.
  • CAR specificity targeted by CAR specificity, localized production of pro-inflammatory cytokines recruits endogenous immune cells to tumor sites and may potentiate an antitumor response.
  • Universal, allogeneic CAR T cells are engineered to no longer express endogenous T cell receptor (TCR) and/or major histocompatibility complex (MHC) molecules, thereby preventing graft-versus-host disease (GVHD) or rejection, respectively.
  • TCR T cell receptor
  • MHC major histocompatibility complex
  • CAR T cells engineered to be resistant to immunosuppression may be genetically modified to no longer express various immune checkpoint molecules (for example, cytotoxic T lymphocyte-associated antigen 4 (CTLA4) or programmed cell death protein 1 (PD1 )), with an immune checkpoint switch receptor, or may be administered with a monoclonal antibody that blocks immune checkpoint signaling.
  • CTL4 cytotoxic T lymphocyte-associated antigen 4
  • PD1 programmed cell death protein 1
  • a self-destruct CAR may be designed using RNA delivered by electroporation to encode the CAR.
  • inducible apoptosis of the T cell may be achieved based on ganciclovir binding to thymidine kinase in gene-modified lymphocytes or the more recently described system of activation of human caspase 9 by a small- molecule dimerizer.
  • a conditional CAR T cell is by default unresponsive, or switched Off', until the addition of a small molecule to complete the circuit, enabling full transduction of both signal 1 and signal 2, thereby activating the CAR T cell.
  • T cells may be engineered to express an adaptor-specific receptor with affinity for subsequently administered secondary antibodies directed at target antigen.
  • Marked CAR T cells express a CAR plus a tumor epitope to which an existing monoclonal antibody agent binds. In the setting of intolerable adverse effects, administration of the monoclonal antibody clears the CAR T cells and alleviates symptoms with no additional off-tumor effects.
  • TanCAR T cell expresses a single CAR consisting of two linked single-chain variable fragments (scFvs) that have different affinities fused to intracellular co-stimulatory domain(s) and a ⁇ 3 ⁇ domain. TanCAR T cell activation is achieved only when target cells co-express both targets.
  • scFvs linked single-chain variable fragments
  • a dual CAR T cell expresses two separate CARs with different ligand binding targets; one CAR includes only the ⁇ 3 ⁇ domain and the other CAR includes only the co-stimulatory domain(s). Dual CAR T cell activation requires co-expression of both targets on the tumor.
  • a safety CAR (sCAR) consists of an extracellular scFv fused to an
  • sCAR T cells co-expressing a standard CAR become activated only when encountering target cells that possess the standard CAR target but lack the sCAR target.
  • the antigen recognition domain of the disclosed CAR is usually an scFv.
  • An antigen recognition domain from native T- cell receptor (TCR) alpha and beta single chains have been described, as have simple ectodomains (e.g. CD4 ectodomain to recognize HIV infected cells) and more exotic recognition components such as a linked cytokine (which leads to recognition of cells bearing the cytokine receptor).
  • TCR T- cell receptor
  • the endodomain is the business end of the CAR that after antigen recognition transmits a signal to the immune effector cell, activating at least one of the normal effector functions of the immune effector cell.
  • Effector function of a T cell may be cytolytic activity or helper activity including the secretion of cytokines. Therefore, the endodomain may comprise the "intracellular signaling domain" of a T cell receptor (TCR) and optional co-receptors. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal.
  • TCR T cell receptor
  • Cytoplasmic signaling sequences that regulate primary activation of the TCR complex that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (ITAMs).
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • Examples of ITAM containing cytoplasmic signaling sequences include those derived from CD8, ⁇ 3 ⁇ , CD35, CD3y, CD3e, CD32 (Fc gamma Rl la), DAP10, DAP12, CD79a, CD79b, FcyRlY, FCYRI I IY, FceR ⁇ (FCERI B), and FCERIY (FCERIG).
  • the intracellular signaling domain is derived from
  • CD3 zeta ⁇ CD3Q (TCR zeta, GenBank accno. BAG36664.1).
  • T-cell surface glycoprotein CD3 zeta ( ⁇ 3 ⁇ ) chain also known as T-cell receptor T3 zeta chain or CD247 (Cluster of Differentiation 247), is a protein that in humans is encoded by the CD247 gene.
  • First-generation CARs typically had the intracellular domain from the ⁇ 3 ⁇ chain, which is the primary transmitter of signals from endogenous TCRs.
  • Second- generation CARs add intracellular signaling domains from various costimulatory protein receptors (e.g. , CD28, 41 BB, ICOS) to the endodomain of the CAR to provide additional signals to the T cell.
  • costimulatory protein receptors e.g. , CD28, 41 BB, ICOS
  • Preclinical studies have indicated that the second generation of CAR designs improves the antitumor activity of T cells.
  • third-generation CARs combine multiple signaling domains to further augment potency. T cells grafted with these CARs have demonstrated improved expansion, activation, persistence, and tumor-eradicating efficiency independent of costimulatory receptor/ligand interaction (Imai C, et al.
  • the endodomain of the CAR can be designed to comprise the ⁇ 3 ⁇ signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR of the invention.
  • the cytoplasmic domain of the CAR can comprise a ⁇ 3 ⁇ chain portion and a
  • the costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule.
  • a costimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen.
  • Examples of such molecules include CD27, CD28, 41 BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, and NKG2D.
  • CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, and NKG2D CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, and NKG2D.
  • the CAR comprises a hinge sequence.
  • a hinge sequence is a short sequence of amino acids that facilitates antibody flexibility (see, e.g., Woof et al. , Nat. Rev. Immunol. , 4(2): 89-99 (2004)).
  • the hinge sequence may be positioned between the antigen recognition moiety and the transmembrane domain.
  • the hinge sequence can be any suitable sequence derived or obtained from any suitable molecule. In some embodiments, for example, the hinge sequence is derived from a CD8a molecule or a CD28 molecule.
  • the transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.
  • the transmembrane region may be derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8 (e.g. , CD8 alpha, CD8 beta), CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154, KI RDS2, OX40, CD2, CD27, LFA-1
  • CD11 a, CD18 CD11 , CD18
  • ICOS CD278
  • 4-1 BB CD137
  • GITR CD40, BAFFR
  • HVEM LIGHTR
  • SLAM F7, NKp80 KLRF1
  • CD160 CD19, I L2R beta, IL2R gamma, I L7R a, ITGA1 , VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11 d, ITGAE, CD103, ITGAL, CD11 a, LFA-1 , ITGAM , CD11 b, ITGAX, CD11 c, ITGB1 , CD29, ITGB2, CD18, LFA-1 , ITGB7, TNFR2, DNAM 1 (CD226) , SLAMF4 (CD244, 2B4) , CD84, CD96 (Tactile) , CEACAM 1 , CRTAM, Ly9 (CD
  • the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In some cases, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.
  • a short oligo- or polypeptide linker such as between 2 and 10 amino acids in length, may form the linkage between the transmembrane domain and the endoplasmic domain of the CAR.
  • the CAR has more than one transmembrane domain, which can be a repeat of the same transmembrane domain, or can be different transmembrane domains.
  • the CAR is a multi-chain CAR, as described in WO2015/039523, which is incorporated by reference for this teaching.
  • a multi-chain CAR can comprise separate extracellular ligand binding and signaling domains in different transmembrane polypeptides.
  • the signaling domains can be designed to assemble in juxtamembrane position, which forms flexible architecture closer to natural receptors, that confers optimal signal transduction.
  • the multichain CAR can comprise a part of an FCERI alpha chain and a part of an FCERI beta chain such that the FCERI chains spontaneously dimerize together to form a CAR.
  • Tables 1 and 2 below provide some example combinations of TAA-binding region, co-stimulatory signaling regions, and intracellular signaling domain that can occur in the disclosed CARs.
  • 41 BB/CD28* mutated 41 BB alone or mutated 41 BB in combination with mutated
  • the anti-TAA binding agent is single chain variable fragment (scFv) antibody.
  • the affinity/specificity of an anti-TAA scFv is driven in large part by specific sequences within complementarity determining regions (CDRs) in the heavy (VH) and light (VL) chain. Each VH and VL sequence will have three CDRs (CDR1 , CDR2, CDR3).
  • the anti-TAA binding agent is an affinity maturated scFv. In some cases, the anti-TAA has a dissociation constant (K D ) for the TAA that is less than 50 nM, 40 nM, 30 nM, 25 nM, 20 nM, 15 nM, or 10 nM.
  • K D dissociation constant
  • the anti-TAA binding agent is derived from natural antibodies, such as monoclonal antibodies.
  • the antibody is human.
  • the antibody has undergone an alteration to render it less immunogenic when administered to humans.
  • the alteration comprises one or more techniques selected from the group consisting of chimerization, humanization, CDR- grafting, deimmunization, and mutation of framework amino acids to correspond to the closest human germline sequence.
  • Tumor antigens are proteins that are produced by tumor cells that elicit an immune response, particularly T-cell mediated immune responses.
  • the additional antigen binding domain can be an antibody or a natural ligand of the tumor antigen. The selection of the additional antigen binding domain will depend on the particular type of cancer to be treated.
  • the tumor antigen is selected from the group CD19, TAG-72, CD99, CLEC12A, TIM3, CD83, CD123, TIM3, CD33, and any combination thereof.
  • tumor antigens include the following: Differentiation antigens such as tyrosinase, TRP-1 , TRP-2 and tumor-specific multilineage antigens such as MAGE-1 , MAGE-3, BAGE, GAGE-1 , GAGE-2, pi 5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor- suppressor genes such as p53, Ras, HER-2/neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7.
  • Differentiation antigens such as tyrosinase, TRP-1 , TRP-2 and tumor-specific multilineage antigens such as MAGE-1 , MAGE-3, B
  • Tumor antigens include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), EGFRvlll, IL-IIRa, IL-13Ra, EGFR, FAP, B7H3, Kit, CA LX, CS-1 , MUC1 , BCMA, bcr-abl, HER2, ⁇ -human chorionic gonadotropin, alphafetoprotein (AFP), ALK, CD19, cyclin Bl, lectin-reactive AFP, Fos-related antigen 1 , ADRB3, thyroglobulin, EphA2, RAGE-1 , RUI, RU2, SSX2, AKAP-4, LCK, OY-TESI, PAX5, SART3, CLL-1 , fucosyl GM 1 , GloboH, MN-CA IX, EPCAM, EVT6- AML, TGS5, human telomerase reverse transcriptase, plysialic acid,
  • polynucleotides and polynucleotide vectors encoding the disclosed CARs that allow expression of the CARs in the disclosed immune effector cells.
  • Nucleic acid sequences encoding the disclosed CARs, and regions thereof can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques.
  • the gene of interest can be produced synthetically, rather than cloned.
  • nucleic acids encoding CARs is typically achieved by operably linking a nucleic acid encoding the CAR polypeptide to a promoter, and incorporating the construct into an expression vector.
  • Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.
  • the disclosed nucleic acid can be cloned into a number of types of vectors.
  • the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid.
  • Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • the expression vector may be provided to a cell in the form of a viral vector.
  • Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001 , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals.
  • Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers.
  • the polynucleotide vectors are lentiviral or retroviral vectors.
  • retroviruses provide a convenient platform for gene delivery systems.
  • a selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
  • CMV immediate early cytomegalovirus
  • EF-1 a Elongation Growth Facto a
  • constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, MND (myeloproliferative sarcoma virus) promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter.
  • the promoter can alternatively be an inducible promoter. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a
  • progesterone promoter and a tetracycline promoter.
  • promoter elements e.g., enhancers
  • promoters regulate the frequency of transcriptional initiation.
  • these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
  • the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
  • the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes.
  • Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
  • Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene. Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.
  • the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.
  • the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art.
  • the expression vector can be transferred into a host cell by physical, chemical, or biological means.
  • Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like.
  • Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al.
  • Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
  • Viral vectors, and especially retroviral vectors have become the most widely used method for inserting genes into mammalian, e.g., human cells.
  • Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).
  • an exemplary delivery vehicle is a liposome.
  • the nucleic acid may be associated with a lipid.
  • the nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a "collapsed" structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape.
  • Lipids are fatty substances which may be naturally occurring or synthetic lipids.
  • lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Lipids suitable for use can be obtained from commercial sources.
  • dimyristyl phosphatidylcholine can be obtained from Sigma, St. Louis, Mo.
  • dicetyl phosphate can be obtained from K & K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc, (Birmingham, Ala.).
  • immune effector cells that are engineered to express the disclosed CARs (also referred to herein as "CAR-T cells.” These cells are preferably obtained from the subject to be treated (i.e. are autologous). However, in some embodiments, immune effector cell lines or donor effector cells (allogeneic) are used. Immune effector cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. Immune effector cells can be obtained from blood collected from a subject using any number of techniques known to the skilled artisan, such as
  • immune effector cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM gradient or by counterflow centrifugal elutriation.
  • a specific subpopulation of immune effector cells can be further isolated by positive or negative selection techniques.
  • immune effector cells can be isolated using a combination of antibodies directed to surface markers unique to the positively selected cells, e.g., by incubation with antibody-conjugated beads for a time period sufficient for positive selection of the desired immune effector cells.
  • enrichment of immune effector cells population can be accomplished by negative selection using a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • the immune effector cells comprise any leukocyte involved in defending the body against infectious disease and foreign materials.
  • the immune effector cells can comprise lymphocytes, monocytes, macrophages, dentritic cells, mast cells, neutrophils, basophils, eosinophils, or any combinations thereof.
  • the immune effector cells can comprise T lymphocytes.
  • T cells or T lymphocytes can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T-cell receptor
  • TCR tumor necrosis factor receptor
  • T cells on the cell surface. They are called T cells because they mature in the thymus (although some also mature in the tonsils). There are several subsets of T cells, each with a distinct function.
  • T helper cells assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells become activated when they are presented with peptide antigens by MHC class II molecules, which are expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or assist in the active immune response. These cells can differentiate into one of several subtypes, including TH1 , TH2, TH3, TH17, TH9, or TFH, which secrete different cytokines to facilitate a different type of immune response.
  • APCs antigen-presenting cells
  • Cytotoxic T cells destroy virally infected cells and tumor cells, and are also implicated in transplant rejection. These cells are also known as CD8 + T cells since they express the CD8 glycoprotein at their surface. These cells recognize their targets by binding to antigen associated with MHC class I molecules, which are present on the surface of all nucleated cells. Through IL-10, adenosine and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated to an anergic state, which prevents autoimmune diseases.
  • Memory T cells are a subset of antigen-specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen, thus providing the immune system with "memory” against past infections.
  • Memory cells may be either CD4 + or CD8 + .
  • Memory T cells typically express the cell surface protein CD45RO.
  • Regulatory T cells (T reg cells), formerly known as suppressor T cells, are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell-mediated immunity toward the end of an immune reaction and to suppress auto-reactive T cells that escaped the process of negative selection in the thymus. Two major classes of CD4 + T reg cells have been described— naturally occurring T reg cells and adaptive T reg cells.
  • Natural killer T (NKT) cells (not to be confused with natural killer (NK) cells) bridge the adaptive immune system with the innate immune system. Unlike conventional T cells that recognize peptide antigens presented by major
  • NKT cells recognize glycolipid antigen presented by a molecule called CD1 d.
  • the T cells comprise a mixture of CD4+ cells. In other embodiments, the T cells are enriched for one or more subsets based on cell surface expression. For example, in some cases, the T comprise are cytotoxic CD8 + T lymphocytes. In some embodiments, the T cells comprise ⁇ T cells, which possess a distinct T-cell receptor (TCR) having one ⁇ chain and one ⁇ chain instead of a and ⁇ chains.
  • TCR T-cell receptor
  • NK cells are CD56 + CD3- large granular lymphocytes that can kill virally infected and transformed cells, and constitute a critical cellular subset of the innate immune system (Godfrey J, et al. Leuk Lymphoma 2012 53:1666-1676). Unlike cytotoxic CD8 + T lymphocytes, NK cells launch cytotoxicity against tumor cells without the requirement for prior sensitization, and can also eradicate MHC-I- negative cells (Narni-Mancinelli E, et al. Int Immunol 2011 23:427-431). NK cells are safer effector cells, as they may avoid the potentially lethal complications of cytokine storms (Morgan RA, et al. Mol Ther 2010 18:843-851), tumor lysis syndrome (Porter DL, et al. N Engl J Med 2011 365:725-733), and on-target, off-tumor effects.
  • NK cells have a well-known role as killers of cancer cells, and NK cell impairment has been extensively documented as crucial for progression of MM (Godfrey J, et al. Leuk Lymphoma 2012 53: 1666-1676; Fauriat C, et al. Leukemia 2006 20:732-733), the means by which one might enhance NK cell-mediated anti- MM activity has been largely unexplored prior to the disclosed CARs.
  • Immune effector cells expressing the disclosed CARs can elicit an anti-tumor immune response against TAA-expressing cancer cells.
  • the anti-tumor immune response elicited by the disclosed CAR-modified immune effector cells may be an active or a passive immune response.
  • the CAR-mediated immune response may be part of an adoptive immunotherapy approach in which CAR- modified immune effector cells induce an immune response specific to TAA.
  • the cells may be genetically engineered to express the disclosed CARs, then infused back into the patient.
  • compositions may comprise a target cell population as described herein, in combination with one or more pharmaceutically or
  • compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol;
  • proteins proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and
  • compositions for use in the disclosed methods are in some
  • compositions may be administered in any manner appropriate treat MM.
  • the quantity and frequency of administration will be determined by such factors as the condition of the patient, and the severity of the patient's disease, although appropriate dosages may be determined by clinical trials.
  • an immunologically effective amount When “an immunologically effective amount”, “an anti-tumor effective amount”, “an tumor-inhibiting effective amount”, or “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the T cells described herein may be administered at a dosage of 10 4 to 10 9 cells/kg body weight, such as 10 5 to 10 6 cells/kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages.
  • the cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988).
  • the optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.
  • T cells can be activated from blood draws of from 10 cc to 400 cc. In certain embodiments, T cells are activated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc. Using this multiple blood draw/multiple reinfusion protocol may serve to select out certain populations of T cells.
  • compositions described herein may be administered to a patient subcutaneously, intradermal ⁇ , intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
  • the disclosed compositions are administered to a patient by intradermal or subcutaneous injection.
  • the disclosed compositions are administered by i.v. injection.
  • the compositions may also be injected directly into a tumor, lymph node, or site of infection.
  • the disclosed CAR-modified immune effector cells are administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities, including but not limited to thalidomide, dexamethasone, bortezomib, and lenalidomide.
  • the CAR-modified immune effector cells may be used in combination with
  • immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies
  • immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation.
  • the CAR- modified immune effector cells 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
  • the cell compositions of the present invention are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan.
  • B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan.
  • 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.
  • the cancer of the disclosed methods can be any TAA-expressing cell in a subject undergoing unregulated growth, invasion, or metastasis.
  • the cancer can be any neoplasm or tumor for which radiotherapy is currently used.
  • the cancer can be a neoplasm or tumor that is not sufficiently sensitive to radiotherapy using standard methods.
  • the cancer can be a sarcoma, lymphoma, leukemia, carcinoma, blastoma, or germ cell tumor.
  • a representative but non-limiting list of cancers that the disclosed compositions can be used to treat include lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, kidney cancer, lung cancers such as small cell lung cancer and non-small cell lung cancer, neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, endometrial cancer, cervical cancer, cervical carcinoma, breast cancer, epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer,
  • hematopoietic cancers hematopoietic cancers
  • testicular cancer colon and rectal cancers
  • prostatic cancer pancreatic cancer
  • the disclosed CARs can be used in combination with any compound, moiety or group which has a cytotoxic or cytostatic effect.
  • Drug moieties include
  • chemotherapeutic agents which may function as microtubulin inhibitors, mitosis inhibitors, topoisomerase inhibitors, or DNA intercalators, and particularly those which are used for cancer therapy.
  • the disclosed CARs can be used in combination with a checkpoint inhibitor.
  • the two known inhibitory checkpoint pathways involve signaling through the cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed-death 1 (PD-1) receptors.
  • CTLA-4 cytotoxic T-lymphocyte antigen-4
  • PD-1 receptor also known as CD279
  • CD279 is expressed on the surface of activated T cells. Its ligands, PD-L1 (B7-H1 ; CD274) and PD-L2 (B7-DC; CD273), are expressed on the surface of APCs such as dendritic cells or macrophages.
  • PD-L1 is the predominant ligand, while PD- L2 has a much more restricted expression pattern.
  • an inhibitory signal is transmitted into the T cell, which reduces cytokine production and suppresses T-cell proliferation.
  • Checkpoint inhibitors include, but are not limited to antibodies that block PD-1 (Nivolumab (BMS-936558 or MDX1106), CT-011 , MK- 3475), PD-L1 (MDX-1 105 (BMS-936559), MPDL3280A, MSB0010718C), PD-L2 (rHlgM12B7), CTLA-4 (Ipilimumab (MDX-010), Tremelimumab (CP-675,206)), IDO, B7-H3 (MGA271), B7-H4, TIM3, LAG-3 (BMS-986016).
  • PD-1 Nonvolumab (BMS-936558 or MDX1106)
  • CT-011 PDX-1 105
  • MPDL3280A MSB0010718C
  • PD-L2 rHlgM12B7
  • CTLA-4 Ipilimumab (MDX-010), Tremelimumab (CP-675,206)
  • IDO B
  • Anti-PD-L1 antibodies and uses therefor are described in U.S. Patent No. 8,552, 154, which is incorporated by reference for these antibodies.
  • Anticancer agent comprising anti-PD-1 antibody or anti-PD-L1 antibody are described in U.S. Patent No. 8,617,546, which is
  • the PDL1 inhibitor comprises an antibody that specifically binds PDL1 , such as BMS-936559 (Bristol-Myers Squibb) or MPDL3280A (Roche).
  • the PD1 inhibitor comprises an antibody that specifically binds PD1 , such as lambrolizumab (Merck), nivolumab (Bristol-Myers Squibb), or MEDI4736 (AstraZeneca).
  • Human monoclonal antibodies to PD-1 and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutics are described in U.S. Patent No. 8,008,449, which is incorporated by reference for these antibodies.
  • Anti-PD-L1 antibodies and uses therefor are described in U.S. Patent No. 8,552, 154, which is incorporated by reference for these antibodies.
  • Anticancer agent comprising anti-PD-1 antibody or anti-PD-L1 antibody are described in U.S. Patent No. 8,617,546, which is
  • the disclosed CARs can be used in combination with other cancer immunotherapies.
  • immunotherapy uses components of the immune system to direct targeted cytotoxic activity against cancer cells, without necessarily initiating an immune response in the patient, while active immunotherapy actively triggers an endogenous immune response.
  • Passive strategies include the use of the monoclonal antibodies (mAbs) produced by B cells in response to a specific antigen.
  • mAbs monoclonal antibodies
  • mAbs have been the biggest success story for immunotherapy; the top three best-selling anticancer drugs in 2012 were mAbs.
  • rituximab (Rituxan, Genentech), which binds to the CD20 protein that is highly expressed on the surface of B cell malignancies such as non- Hodgkin's lymphoma (NHL).
  • NHL non- Hodgkin's lymphoma
  • CLL chronic lymphocytic leukemia
  • trastuzumab Herceptin; Genentech
  • HER2 human epidermal growth factor receptor 2
  • T cell receptor activation plus co-stimulation, which can be provided through ligation of tumor necrosis factor receptor family members, including OX40 (CD134) and 4-1 BB
  • OX40 is of particular interest as treatment with an activating (agonist) anti- OX40 mAb augments T cell differentiation and cytolytic function leading to enhanced anti-tumor immunity against a variety of tumors.
  • such an additional therapeutic agent may be selected from an antimetabolite, such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine or cladribine.
  • an antimetabolite such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine or cladribine.
  • such an additional therapeutic agent may be selected from an alkylating agent, such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives, such as carboplatin .
  • an alkylating agent such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives, such as carboplatin .
  • such an additional therapeutic agent may be selected from an anti-mitotic agent, such as taxanes, for instance docetaxel, and paclitaxel, and vinca alkaloids, for instance vindesine, vincristine, vinblastine, and vinorelbine.
  • an anti-mitotic agent such as taxanes, for instance docetaxel, and paclitaxel
  • vinca alkaloids for instance vindesine, vincristine, vinblastine, and vinorelbine.
  • such an additional therapeutic agent may be selected from a topoisomerase inhibitor, such as topotecan or irinotecan, or a cytostatic drug, such as etoposide and teniposide.
  • a topoisomerase inhibitor such as topotecan or irinotecan
  • a cytostatic drug such as etoposide and teniposide.
  • such an additional therapeutic agent may be selected from a growth factor inhibitor, such as an inhibitor of ErbBI (EGFR) (such as an EGFR antibody, e.g. zalutumumab, cetuximab, panitumumab or nimotuzumab or other EGFR inhibitors, such as gefitinib or erlotinib), another inhibitor of ErbB2 (HER2/neu) (such as a HER2 antibody, e.g. trastuzumab, trastuzumab-DM I or pertuzumab) or an inhibitor of both EGFR and HER2, such as lapatinib).
  • a growth factor inhibitor such as an inhibitor of ErbBI (EGFR) (such as an EGFR antibody, e.g. zalutumumab, cetuximab, panitumumab or nimotuzumab or other EGFR inhibitors, such as gefitinib or erlotinib), another
  • a disclosed antibody is used in combination with ofatumumab, zanolimumab, daratumumab, ranibizumab, nimotuzumab, panitumumab, hu806, daclizumab (Zenapax), basiliximab (Simulect), infliximab (Remicade), adalimumab (Humira), natalizumab (Tysabri), omalizumab (Xolair), efalizumab (Raptiva), and/or rituximab.
  • a therapeutic agent for use in combination with a CARs for treating the disorders as described above may be an anti-cancer cytokine, chemokine, or combination thereof.
  • suitable cytokines and growth factors include IFNy, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL- 24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa (e.g., INFa2b), IFN , GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim, and TNFa.
  • Suitable chemokines may include Glu-Leu-Arg (ELR)- negative chemokines such as IP-10, MCP-3, MIG, and SDF-la from the human CXC and C-C chemokine families.
  • Suitable cytokines include cytokine derivatives, cytokine variants, cytokine fragments, and cytokine fusion proteins.
  • a therapeutic agent for use in combination with a CARs for treating the disorders as described above may be a cell cycle
  • a cell cycle control/apoptosis regulator may include molecules that target and modulate cell cycle control/apoptosis regulators such as (i) cdc-25 (such as NSC 663284), (ii) cyclin-dependent kinases that overstimulate the cell cycle (such as flavopiridol (L868275, HMR1275), 7- hydroxystaurosporine (UCN-01 , KW-2401), and roscovitine (R-roscovitine,
  • telomerase modulators such as BIBR1532, SOT-095, GRN163 and compositions described in for instance US 6,440,735 and US 6,713,055
  • telomerase modulators such as BIBR1532, SOT-095, GRN163 and compositions described in for instance US 6,440,735 and US 6,713,055
  • Non- limiting examples of molecules that interfere with apoptotic pathways include TNF- related apoptosis-inducing ligand (TRAIL)/apoptosis-2 ligand (Apo-2L), antibodies that activate TRAIL receptors, IFNs, and anti-sense Bcl-2.
  • a therapeutic agent for use in combination with a CARs for treating the disorders as described above may be a hormonal regulating agent, such as agents useful for anti-androgen and anti-estrogen therapy.
  • hormonal regulating agents are tamoxifen, idoxifene, fulvestrant, droloxifene, toremifene, raloxifene, diethylstilbestrol, ethinyl estradiol/estinyl, an antiandrogene (such as flutaminde/eulexin), a progestin (such as such as hydroxyprogesterone caproate, medroxy- progesterone/provera, megestrol acepate/megace), an adrenocorticosteroid (such as hydrocortisone, prednisone), luteinizing hormone- releasing hormone (and analogs thereof and other LHRH agonists such as buserelin and goserelin),
  • aminoglutethimide/cytraden, exemestane aminoglutethimide/cytraden, exemestane
  • a hormone inhibitor such as octreotide/sandostatin
  • a therapeutic agent for use in combination with an CARs for treating the disorders as described above may be an anti-cancer nucleic acid or an anti-cancer inhibitory RNA molecule.
  • Combined administration may be simultaneous, separate, or sequential.
  • the agents may be administered as one composition or as separate compositions, as appropriate.
  • Radiotherapy may comprise radiation or associated administration of radiopharmaceuticals to a patient is provided.
  • the source of radiation may be either external or internal to the patient being treated (radiation treatment may, for example, be in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)).
  • Radioactive elements that may be used in practicing such methods include, e.g., radium, cesium-137, iridium-192, americium-241 , gold-198, cobalt-57, copper- 67, technetium-99, iodide-123, iodide-131 , and indium-1 11.
  • the disclosed CARs is administered in combination with surgery.
  • CAR-T cells may be designed in several ways that enhance tumor cytotoxicity and specificity, evade tumor immunosuppression, avoid host rejection, and prolong their therapeutic half-life.
  • TRUCK T-cells Redirected for Universal Cytokine Killing
  • TRUCK T-cells Redirected for Universal Cytokine Killing
  • cytokines such as IL-12 that promote tumor killing. Because these cells are designed to release a molecular payload upon activation of the CAR once localized to the tumor environment, these CAR-T cells are sometimes also referred to as 'armored CARs'.
  • cytokines as cancer therapies are being investigated both pre-clinically and clinically, and may also prove useful when similarly incorporated into a TRUCK form of CAR-T therapy.
  • IL-2 IL-3.
  • IL-4 IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, M-CSF, GM-CSF, IFN-a, IFN- ⁇ , TNF-a, TRAIL, FLT3 ligand, Lymphotactin, and TGF- ⁇ (Dranoff 2004).
  • "Self-driving" or "homing" CAR-T cells are engineered to express a chemokine receptor in addition to their CAR.
  • chemokines can be upregulated in tumors
  • incorporation of a chemokine receptor aids in tumor trafficking to and infiltration by the adoptive T-cell, thereby enhancing both specificity and functionality of the CAR-T (Moon 2011).
  • Universal CAR-T cells also possess a CAR, but are engineered such that they do not express endogenous TCR (T-cell receptor) or MHC (major histocompatibility complex) proteins. Removal of these two proteins from the signaling repertoire of the adoptive T-cell therapy prevents graft-versus-host-disease and rejection, respectively.
  • Armored CAR-T cells are additionally so named for their ability to evade tumor immunosuppression and tumor-induced CAR-T hypofunction.
  • CAR-Ts possess a CAR, and may be engineered to not express checkpoint inhibitors.
  • these CAR-Ts can be co-administered with a monoclonal antibody (mAb) that blocks checkpoint signaling.
  • mAb monoclonal antibody
  • Administration of an anti-PDL1 antibody significantly restored the killing ability of CAR TILs (tumor infiltrating lymphocytes).
  • PD1-PDL1 and CTLA-4- CD80/CD86 signaling pathways have been investigated, it is possible to target other immune checkpoint signaling molecules in the design of an armored CAR-T including LAG-3, Tim-3, IDO-1 , 2B4, and KIR.
  • TILs intracellular inhibitors of TILs include phosphatases (SHP1), ubiquitin-ligases (i.e., cbl-b), and kinases (i.e., diacylglycerol kinase) .
  • Armored CAR-Ts may also be engineered to express proteins or receptors that protect them against or make them resistant to the effects of tumor-secreted cytokines.
  • CTLs cytotoxic T lymphocytes
  • transduced cells showed notably increased antitumor activity in vivo when compared to their control counterparts.
  • Tandem and dual CAR-T cells are unique in that they possess two distinct antigen binding domains.
  • a tandem CAR contains two sequential antigen binding domains facing the extracellular environment connected to the intracellular costimulatory and stimulatory domains.
  • a dual CAR is engineered such that one extracellular antigen binding domain is connected to the intracellular costimulatory domain and a second, distinct extracellular antigen binding domain is connected to the intracellular stimulatory domain. Because the stimulatory and costimulatory domains are split between two separate antigen binding domains, dual CARs are also referred to as "split CARs". In both tandem and dual CAR designs, binding of both antigen binding domains is necessary to allow signaling of the CAR circuit in the T-cell. Because these two CAR designs have binding affinities for different, distinct antigens, they are also referred to as "bi-specific" CARs.
  • CAR-T cells are a form of "living therapeutic" as a form of "living therapeutic".
  • off-switches safety mechanisms
  • conditional control mechanisms Both self-destruct and marked/tagged CAR-T cells for example, are engineered to have an "off-switch” that promotes clearance of the CAR-expressing T-cell.
  • a self-destruct CAR-T contains a CAR, but is also engineered to express a pro-apoptotic suicide gene or "elimination gene” inducible upon administration of an exogenous molecule.
  • HSV-TK herpes simplex virus thymidine kinase
  • Fas iCasp9
  • CD20 MYC tag
  • truncated EGFR endothelial growth factor receptor
  • HSK will convert the prodrug ganciclovir (GCV) into GCV-triphosphate that incorporates itself into replicating DNA, ultimately leading to cell death.
  • GCV prodrug ganciclovir
  • iCasp9 is a chimeric protein containing components of FK506-binding protein that binds the small molecule AP1903, leading to caspase 9 dimerization and apoptosis.
  • a marked/tagged CAR-T cell is one that possesses a CAR but also is engineered to express a selection marker.
  • Truncated EGFR is one such targetable antigen by the anti-EGFR mAb, and administration of cetuximab works to promotes elimination of the CAR-T cell.
  • CARs created to have these features are also referred to as sCARs for 'switchable CARs', and RCARs for 'regulatable CARs'.
  • a "safety CAR” also known as an
  • inhibitory CAR is engineered to express two antigen binding domains. One of these extracellular domains is directed against a tumor related antigen and bound to an intracellular costimulatory and stimulatory domain. The second extracellular antigen binding domain however is specific for normal tissue and bound to an intracellular checkpoint domain such as CTLA4, PD1 , or CD45. Incorporation of multiple intracellular inhibitory domains to the iCAR is also possible. Some inhibitory molecules that may provide these inhibitory domains include B7-H1 , B7-1 , CD160, PIH, 2B4, CEACAM (CEACAM-1. CEACAM-3, and/or CEACAM-5), LAG-3, TIGIT, BTLA, LAIR1 , and ⁇ .
  • iCARs are also a form of bi-specific CAR-T cells.
  • the safety CAR-T engineering enhances specificity of the CAR-T cell for tumor tissue, and is advantageous in situations where certain normal tissues may express very low levels of a tumor associated antigen that would lead to off target effects with a standard CAR (Morgan 2010).
  • a conditional CAR-T cell expresses an
  • extracellular antigen binding domain connected to an intracellular costimulatory domain and a separate, intracellular costimulator.
  • the costimulatory and stimulatory domain sequences are engineered in such a way that upon administration of an exogenous molecule the resultant proteins will come together intracellular ⁇ to complete the CAR circuit. In this way, CAR-T activation can be modulated, and possibly even 'fine-tuned' or personalized to a specific patient. Similar to a dual CAR design, the stimulatory and costimulatory domains are physically separated when inactive in the conditional CAR; for this reason these too are also referred to as a "split CAR".
  • two or more of these engineered features may be combined to create an enhanced, multifunctional CAR-T.
  • a CAR-T cell with either dual- or conditional- CAR design that also releases cytokines like a TRUCK.
  • a dual-conditional CAR-T cell could be made such that it expresses two CARs with two separate antigen binding domains against two distinct cancer antigens, each bound to their respective costimulatory domains. The costimulatory domain would only become functional with the stimulatory domain after the activating molecule is administered.
  • the cancer must express both cancer antigens and the activating molecule must be administered to the patient; this design thereby incorporating features of both dual and conditional CAR-T cells.
  • CAR-T cells are created using ⁇ - ⁇ T cells, however ⁇ - ⁇ T cells may also be used.
  • the described CAR constructs, domains, and engineered features used to generate CAR-T cells could similarly be employed in the generation of other types of CAR-expressing immune cells including NK (natural killer) cells, B cells, mast cells, myeloid-derived phagocytes, and NKT cells.
  • a CAR-expressing cell may be created to have properties of both T-cell and NK cells.
  • the transduced with CARs may be autologous or allogeneic.
  • CAR expression may be used including retroviral transduction (including ⁇ -retroviral), lentiviral transduction,
  • transposon/transposases Small Beauty and PiggyBac systems
  • messenger RNA transfer-mediated gene expression Gene editing (gene insertion or gene deletion/disruption) has become of increasing importance with respect to the possibility for engineering CAR-T cells as well.
  • CRISPR-Cas9, ZFN (zinc finger nuclease), and TALEN (transcription activator like effector nuclease) systems are three potential methods through which CAR-T cells may be generated. Definitions
  • amino acid sequence refers to a list of abbreviations, letters, characters or words representing amino acid residues.
  • the amino acid abbreviations used herein are conventional one letter codes for the amino acids and are expressed as follows: A, alanine; B, asparagine or aspartic acid; C, cysteine; D aspartic acid; E, glutamate, glutamic acid; F, phenylalanine; G, glycine; H histidine; I isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; Z, glutamine or glutamic acid.
  • antibody refers to an immunoglobulin, derivatives thereof which maintain specific binding ability, and proteins having a binding domain which is homologous or largely homologous to an immunoglobulin binding domain. These proteins may be derived from natural sources, or partly or wholly synthetically produced.
  • An antibody may be monoclonal or polyclonal.
  • the antibody may be a member of any immunoglobulin class from any species, including any of the human classes: IgG, IgM, IgA, IgD, and IgE.
  • antibodies used with the methods and compositions described herein are derivatives of the IgG class.
  • antibodies are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules that selectively bind the target antigen.
  • aptamer refers to oligonucleic acid or peptide molecules that bind to a specific target molecule. These molecules are generally selected from a random sequence pool. The selected aptamers are capable of adapting unique tertiary structures and recognizing target molecules with high affinity and specificity.
  • a "nucleic acid aptamer” is a DNA or RNA oligonucleic acid that binds to a target molecule via its conformation, and thereby inhibits or suppresses functions of such molecule.
  • a nucleic acid aptamer may be constituted by DNA, RNA, or a combination thereof.
  • a "peptide aptamer” is a combinatorial protein molecule with a variable peptide sequence inserted within a constant scaffold protein. Identification of peptide aptamers is typically performed under stringent yeast dihybrid conditions, which enhances the probability for the selected peptide aptamers to be stably expressed and correctly folded in an intracellular context.
  • carrier means a compound, composition, substance, or structure that, when in combination with a compound or composition, aids or facilitates preparation, storage, administration, delivery, effectiveness, selectivity, or any other feature of the compound or composition for its intended use or purpose.
  • a carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.
  • chimeric molecule refers to a single molecule created by joining two or more molecules that exist separately in their native state.
  • the single, chimeric molecule has the desired functionality of all of its constituent molecules.
  • One type of chimeric molecules is a fusion protein.
  • fusion protein refers to a polypeptide formed by the joining of two or more polypeptides through a peptide bond formed between the amino terminus of one polypeptide and the carboxyl terminus of another polypeptide.
  • the fusion protein can be formed by the chemical coupling of the constituent polypeptides or it can be expressed as a single polypeptide from nucleic acid sequence encoding the single contiguous fusion protein.
  • a single chain fusion protein is a fusion protein having a single contiguous polypeptide backbone. Fusion proteins can be prepared using conventional techniques in molecular biology to join the two genes in frame into a single nucleic acid, and then expressing the nucleic acid in an appropriate host cell under conditions in which the fusion protein is produced.
  • identity refers to sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base, then the molecules are identical at that position. A degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences.
  • Various alignment algorithms and/or programs may be used to calculate the identity between two sequences, including FASTA, or BLAST which are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default setting.
  • polypeptides having at least 70%, 85%, 90%, 95%, 98% or 99% identity to specific polypeptides described herein and preferably exhibiting substantially the same functions, as well as polynucleotide encoding such polypeptides are contemplated.
  • a similarity score will be based on use of BLOSUM62.
  • BLASTP is used, the percent similarity is based on the BLASTP positives score and the percent sequence identity is based on the BLASTP identities score.
  • BLASTP "Identities" shows the number and fraction of total residues in the high scoring sequence pairs which are identical; and BLASTP “Positives” shows the number and fraction of residues for which the alignment scores have positive values and which are similar to each other.
  • amino acid sequences having these degrees of identity or similarity or any intermediate degree of identity of similarity to the amino acid sequences disclosed herein are contemplated and encompassed by this disclosure.
  • the polynucleotide sequences of similar polypeptides are deduced using the genetic code and may be obtained by conventional means, in particular by reverse translating its amino acid sequence using the genetic code.
  • nucleic acid refers to a natural or synthetic molecule comprising a single nucleotide or two or more nucleotides linked by a phosphate group at the 3' position of one nucleotide to the 5' end of another nucleotide.
  • the nucleic acid is not limited by length, and thus the nucleic acid can include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • operably linked to refers to the functional relationship of a nucleic acid with another nucleic acid sequence. Promoters, enhancers, transcriptional and translational stop sites, and other signal sequences are examples of nucleic acid sequences operably linked to other sequences.
  • operable linkage of DNA to a transcriptional control element refers to the physical and functional relationship between the DNA and promoter such that the transcription of such DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes the DNA.
  • peptide protein
  • polypeptide are used interchangeably to refer to a natural or synthetic molecule comprising two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another.
  • pharmaceutically acceptable refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio.
  • protein domain refers to a portion of a protein, portions of a protein, or an entire protein showing structural integrity; this determination may be based on amino acid composition of a portion of a protein, portions of a protein, or the entire protein.
  • a "spacer” as used herein refers to a peptide that joins the proteins comprising a fusion protein. Generally a spacer has no specific biological activity other than to join the proteins or to preserve some minimum distance or other spatial relationship between them. However, the constituent amino acids of a spacer may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity of the molecule.
  • a specified ligand or antibody when referring to a polypeptide (including antibodies) or receptor, refers to a binding reaction which is determinative of the presence of the protein or polypeptide or receptor in a heterogeneous population of proteins and other biologies.
  • a specified ligand or antibody under designated conditions (e.g. immunoassay conditions in the case of an antibody), a specified ligand or antibody "specifically binds" to its particular "target” (e.g. an antibody specifically binds to an endothelial antigen) when it does not bind in a significant amount to other proteins present in the sample or to other proteins to which the ligand or antibody may come in contact in an organism.
  • a first molecule that "specifically binds" a second molecule has an affinity constant (Ka) greater than about 10 5 M _ (e.g., 10 6 M- ⁇ 10 7 M- ⁇ 10 8 M- ⁇ 10 9 M- ⁇ 10 10 M -1 , 10 11 M -1 , and 10 12 M _ or more) with that second molecule.
  • Ka affinity constant
  • subject refers to any individual who is the target of administration or treatment.
  • the subject can be a vertebrate, for example, a mammal.
  • the subject can be a human or veterinary patient.
  • patient refers to a subject under the treatment of a clinician, e.g., physician.
  • terapéuticaally effective refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.
  • transformation and “transfection” mean the introduction of a nucleic acid, e.g., an expression vector, into a recipient cell including introduction of a nucleic acid to the chromosomal DNA of said cell.
  • treatment refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder.
  • This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • variant refers to an amino acid or peptide sequence having conservative amino acid substitutions, non-conservative amino acid subsitutions (i.e. a degenerate variant), substitutions within the wobble position of each codon (i.e. DNA and RNA) encoding an amino acid, amino acids added to the C-terminus of a peptide, or a peptide having 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to a reference sequence.
  • vector refers to a nucleic acid sequence capable of transporting into a cell another nucleic acid to which the vector sequence has been linked.
  • expression vector includes any vector, (e.g., a plasmid, cosmid or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to a transcriptional control element).
  • Example 1 4-1 BB enhancement of CAR T function requires NF- ⁇ and TRAFs
  • mice C57BL/6, Thy1.1 (B6.PL-Thy1a/CyJ), and Rag1-/- (B6.129S7- Rag1tm1 Mom/J) mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and N F-KB-RE-IUC (BALB/c-Tg(Rela-luc)31Xen) transgenic mice were purchased from Taconic (Hudson, NY). Trail-'- mice were gifts from Dr. Tania Watts of University of Toronto and were maintained and bred in the animal facility of Moffitt. NSG mice (NOD.Cg-Prkdcscid ll2rgtm1Wjl/SzJ) were purchased from Jackson Laboratory and bred in the animal facility of Moffitt.
  • mice Female and/or male mice at 8-12 weeks of age were used for the study.
  • mice were injected i.v. with ⁇ -ALL (1 * 10 6 cells/mouse, day 0), followed by i.p. cyclophosphamide (250-300 mg/kg, day 6-7) and mCD19-targeted CAR T cells (0.15-5*10 6 CAR T cells/mouse, day 7-10).
  • Mice were monitored for illness and sacrificed when there was evidence of leukemia progression, such as decreased activity, hunched posture, and ruffled coat.
  • blood and/or bone marrow were collected for analyses.
  • mice were i.v. injected with 1 ⁇ 10 6 mCD19-targeted CAR T cells. Blood and BM were collected for flow cytometry.
  • the ⁇ -ALL cell line has been described (Davila ML, et al. PLoS One. 2013 8(4):e61338).
  • the cells were cultured with irradiated (30 Gy) NIH/3T3 fibroblasts as feeders.
  • the culture medium consists of equal volume of 1) IMDM supplemented with 2 mM L-glutamine, 55 ⁇ ⁇ -Mercaptoethanol, 100 U/ml Penicillin, 100 ⁇ g/ml Streptomycin and 10% FBS and 2) DM EM supplemented with 2 mM L-glutamine, 100 U/ml Penicillin, 100 g/ml Streptomycin and 10% calf serum.
  • 3T3-mCD19 and 3T3-hCD19 cells are NIH/3T3 cells retrovirally transduced with mouse or human CD19 and were used as target cells.
  • CHO-hCD33 cells are Chinese hamster ovary (CHO) cells retrovirally transduced with human CD33 and were used as target cells for human CD33 targeted CAR T cells.
  • NIH/3T3 and CHO cells were purchased from ATCC (Manassas, VA).
  • Mouse T cell complete medium consists of RPMI1640 medium, 10% FBS, 1 mM sodium pyruvate, 1 *NEAA (Non-essential Amino Acids), 10 mM HEPES, 55 ⁇ ⁇ -Mercaptoethanol, 2 mM L- glutamine, 100 U/ml Penicillin and 100 ⁇ g/ml Streptomycin.
  • Human PBMCs from healthy donors were purchased from ReachBio (Seattle, WA).
  • Human T cell complete medium consists of RPMI 1640 medium, 10% FBS, 2 mM L-glutamine, 100 U/ml Penicillin and 100 ⁇ g/ml Streptomycin. All medium and supplements were from ThermoFisher Scientific (Waltham, MA).
  • N F-KB/293/GFP-LUCTM Transcriptional Reporter Cells were purchased from System Biosciences (Palo Alto, CA), maintained and used according to the manufacturer's instructions.
  • FFLuc-GFP NALM6 (NALM6- GL) cells have been described (Zhao Z, et al. Cancer Cell. 2015 28(4):415-28).
  • TRAF and TRAF DN dominant negative constructs include the coding sequences, glycine serine linker, cerulean, and stop codon, which were synthesized and subcloned into the SFG retroviral vector. TRAF DN coding sequences have been described (Duckett CS, et al. Mol Cell Biol. 1997 17(3): 1535-42).
  • TRAF1 DN (184- 417aa) consists only of the TRAF domain, TRAF2 DN (87-501 aa) lacks the ring finger domain, and TRAF3 DN (382-568aa) also lacks the ring finger domain. All SFG constructs were calcium phosphate transfected into H29 cells. Retroviral supernatants of transfected H29 cells were harvested and used to transduce Phoenix E cells for mouse T cell transduction or RD114 cells for human T cell transduction. Retroviral supernatant of Phoenix E or RD114 producer cells were harvested, 0.45 ⁇ filtered and used to transduce mouse or human T cells as described (Davila ML, et al. PLoS One.
  • TRAF overexpressed CAR T cells T cells were co-transduced with retrovirus containing CAR or TRAF at day 1 and day 2. At day 3 or day 4 CAR T cells were collected, beads removed, and subjected to counting and viability evaluation before downstream experimental use. Viability was measured by staining cells with trypan blue and enumerated on an automated cell counter (Bio-Rad, Hercules, CA).
  • Transduction efficiency was estimated as percentage of GFP+ or Cherry+ live cells as detected by flow cytometry.
  • CAR expression was evaluated by staining T cells with 1 ⁇ g Biotin-Protein L (GenScript, Piscataway, NJ) followed by fluorochrome-conjugated streptavidin (eBioscience) and flow cytometry as described (Zheng Z, et al. Journal of translational medicine. 2012 10:29).
  • CAR T cell doses were normalized based on CAR gene-transfer but not sorted to exclude CAR-negative T cells so the total T cell dose varied.
  • CAR T cells were irradiated at 10 Gy. Development of the CD33- targeted CARs are described in Supplementary Methods.
  • anti-mouse or anti-human antibodies with clones listed were obtained from eBioscience (San Diego, CA): anti-mCD16/CD32 (93), anti-mB220 (RA3-6B2), anti-mCD19 (eBio1 D3), anti-mCD3 (145-2C1 1), anti-mCD4 (GK1.5), anti-mCD8 (53- 6.7), anti-mThy1.1 (HIS51), anti-mCD44 (1 M7), antimCD62L (MEL-14), anti- mTER119 (TER-119), anti-mCD11 b (M1/70), anti-mGrl (RB6-8C5), antimNKH (PK136), anti-mlFNY (XMG1.2), anti-mTNFa (MP6-XT22), and anti-mBcl2 (10C4).
  • Anti-mCD3 17A2
  • anti-mCD4 RM4- 5
  • anti-mCD8 53-6.7
  • Anti-BCL-XL 54H6 was from Cell Signaling Technology (Danvers, MA).
  • T cells were harvested, fixed and permeabilized with Intracellular Fixation and Permeabilization Buffer Set (eBioscience) followed by antibody staining. The manufacturer's instruction was followed. Peripheral blood samples were stained with antibodies and lysed afterwards using BD FACS lysing solution (Davila ML, et al. PLoS One. 2013 8(4):e61338). For some experiments, Countbright beads (Thermo Fisher Scientific, Waltham, MA) were used for cell quantitation. All samples were analyzed with a 5-laser BD LSRII (BD Biosciences) and data were analyzed using FlowJo software (Tree Star, Ashland, OR).
  • mice CAR T cells were co-cultured with 1 x 105 3T3-mCD19 cells for 24 hr.
  • Supernatants were harvested and analyzed using a mouse luminex kit (R&D Systems, Minneapolis, MN). Data were collected on a Luminex 100 system (Luminex, Austin, TX). The manufacturer's instructions were followed.
  • CAR T cells were co-cultured with 3T3-hCD19 cells at 10: 1 for 24 hr.
  • Supernatants were harvested and analyzed using a Simple Plex Assay Kit (R&D systems) on an Ella machine (ProteinSimple, San Jose, CA). Manufacturer's instructions were followed.
  • a 4-hour chromium release assay was performed with EL4-mCD19 as target cells and mouse CD19-targeted CAR T cells as effectors. Our methods have been described (Davila ML, et al. PLoS One. 2013 8(4):e61338). Cytotoxicity assays were also run on an xCELLigence RTCA (real time cell analysis) instrument (ACEA)
  • 3T3-mCD19 or 3T3-hCD19 cells were seeded at 10,000 cells per well in an E-Plate 96.
  • mouse or human CAR T cells were resuspended in fresh complete medium without IL2 and added onto target cells at different E:T ratios and cell growth was monitored.
  • CAR T cells were stimulated with 3T3-mCD19 at a 10: 1 ratio for 4 hr.
  • Cell lysates were prepared using 240 ⁇ of cell lysis buffer (Cell Signaling Technology, Danvers, MA) for 6x 106 CAR T cells.
  • 30 ⁇ of reduced and denatured cell lysates were electrophoresed through a 10% Mini-PROTEAN TGX Precast gel (Bio-Rad, Hercules, California), transferred to nitrocellulose blot membranes, blocked, and the membranes were cut based on molecular weight to probe different proteins. The membranes were incubated with primary antibody at 1 : 1000 overnight at 4 degrees.
  • Blots were washed and incubated with HRP-linked anti-rabbit IgG (Cell Signaling Technology) at 1 : 10,000 for 1 hr at room temperature. Blots were washed again and incubated with SuperSignal west femto maximum sensitivity substrate
  • IP immunoprecipitation
  • N F-KB/293/GFP-LUC cells (System Biosciences, Palo Alto, CA) were retrovirally transduced with TRAF or TRAF-DN constructs and CD19-targeted CARs.
  • N F-KB signaling was evaluated by measuring GFP expression with flow cytometry.
  • CAR T cells were generated from N F-KB-RE-IUC transgenic splenocytes. CAR T cells were co-cultured with irradiated (30 Gy) 3T3-mCD19 cells in 6-well plates for 4 hr. For each group, T cells, normalized to 3x 106 mCD19-targeted CAR T cells per well, were incubated with 3x 105 3T3-mCD19 cells per well. After stimulation, cell lysates were prepared using Cell Culture Lysis Reagent (Promega, Madison, Wl). Luciferase assay was performed using a luciferase assay kit (Promega) according to the manufacturer's instructions.
  • Normalized numbers (1 or 2* 10 6 ) of human CAR T cells were co-cultured with 2x 105 3T3-hCD19 AAPC per well in non-tissue culture treated 6-well plates in triplicate.
  • Cells were grown in human T cell complete medium supplemented with 60 lU/ml IL2 and split every 2-3 days or whenever the medium turned yellow.
  • Cell viability and total cell numbers in each well were measured daily or every 2-4 days (T isolation as day 0) on a cell counter (Bio-Rad) with trypan blue staining.
  • CAR T cells were stained with eFluor670 proliferation dye (eBioscience) and then co-cultured with target cells at 5: 1 ratio for 4 days.
  • Cytotoxicity curves were compared using Kolmogorov-Smirnov test. Human CAR T cell in vitro proliferation were compared using two-way ANOVA. Survival was compared using logrank test. Statistical analyses were conducted using GraphPad Prism software 7 (Graphpad, La Jolla, CA) and the R software package. *P ⁇ 0.05 is considered significant. **P ⁇ 0.01 ; ***P ⁇ 0.001 ; ****P ⁇ 0.0001 ; ns, not significant.
  • RNA-SEQ. For m19z, m1928z and m19-humBBz comparison, three million CAR T cells were incubated with 1 * 10 6 3T3-mCD19 cells for 48 hr. Live CD4+ CAR T cells were sorted into Trizol. RNA was isolated according to manufacturer's instructions and evaluated for quality. The Genomic Core performed mRNA enrichment and cDNA library preparation using the lllumina Tru-seq stranded mRNA sample prep kit. Final RNA-seq libraries were reviewed for size and quality on the Agilent TapeStation, followed by quantitative PCR-based quantitation with the Kapa Library Quantification Kit.
  • Anti-CD33 antibodies were developed at the Vanderbilt Antibody and Protein Resource using standard methods (Markham NO, et al. Hybridoma (Larchmt). 2012 31 (4): 246-54). Briefly, after completing a series of immunizations splenocytes of immunized mice were isolated and fused to a non-lg secreting myeloma cell line and grown in a semi-solid plate. Antibody-secreting clusters were identified in semi-solid plates and selected for clonal expansion in 96 well plates. During expansion supernatant was collected and assayed for CD33 binding by ELISA as well as flow cytometry.
  • hybridomas were selected for expansion and isolation of RNA, which was used to amplify IgH and IgL rearrangements.
  • scFv were designed and cloned into the Ncol/Notl sites of our human CD19-targeted CAR in the SFG retroviral cassette. This allowed replacement of the anti-human CD19 scFv with anti-human CD33 scFv. These constructs were then used to produce gammaretroviral supernatant as described in Methods.
  • NALM6 leukemia mouse model has been described (Zhao Z, et al. Cancer Cell. 2015 28(4):415-28). Briefly, NALM6-GL cells were i.v. injected to NSG mice at 5x10 5 dose. Four days later, mice were treated with 3x10 5 -1x10 6 human CD19 targeted CAR T cells. Human CD19 targeted CAR T cells with excess TRAF2 were made by CAR and mouse TRAF2 co-transduction or transduction with a bicistronic construct combining CAR and human TRAF2. Blood samples were collected weekly for flow cytometry. Leukemia burden was evaluated weekly using bioluminescence imaging on an MS system. Survival was monitored. Mice were sacrificed when they develop signs of progressive leukemia.
  • CD19 targeted CAR T cells with a mouse 4-1 BB endodomain (m19-musBBz) eradicate leukemia less efficaciously than T cells with a CAR containing a CD28 endodomain (m1928z).
  • mCD19-targeted CARs which are all murine-derived with the same extracellular rat-origin anti-mCD19 scFv paired to mouse CD8a hinge and transmembrane domains. They differ only in their intracellular activation and co- stimulatory domains by including no domains ( ⁇ 19 ⁇ ), ⁇ 3 ⁇ alone (m19z), or CD3z paired with the CD28 (m1928z) or the 4-1 BB co-stimulatory domain (m19-musBBz).
  • mCD19 mouse CD19 targeted CAR T cells in an immune competent mouse model (Davila ML, et al. PLoS One.
  • m1928z CAR T cells provided superior protection against leukemia compared to m19-musBBz or m19z CAR T cells ( Figure 1 E). Also, m1928z CAR T cells had enhanced in vivo B cell aplasia and donor T cell persistence compared to m19-musBBz ( Figure 1 F).
  • Mouse CD19 targeted CAR T cells with a fluorescent protein tag showed reproducible patterns of CAR expression (Figure 9B).
  • Figures 9C-9E There is a collection of 205 probesets differentially expressed by m19-musBBz CAR T cells compared to m19z and m1928z CAR T cells ( Figures 9C-9E). This includes the upregulation of effector genes (Gzmf, Ifng, Prf1), as well as exhaustion genes or transcription factors (Havcr2, CD244, Klrgl , Eomes) in m19z and m1928z CAR T cells (Tables 1-4).
  • CD19-targeted CAR T cells After 4 hr stimulation with 3T3-mCD19 AAPC, intracellular flow cytometry demonstrated m1928z CD8+ CAR T cells were 8.4% positive for IFNv, which is significantly greater than m19-humBBz (1.4%) or m19- musBBz CD8+ CAR T cells (average 0.3%) ( Figure 2B). In addition, m1928z CAR T cells produced the greatest amount of TNFa. There was also enhancement
  • m19-humBBz CAR T cells rely on persistence to enhance function in vivo
  • CAR T cells required persistence for optimal function in vivo
  • CAR T cells (10 Gy) prior to injection.
  • C57BL/6 mice were i.p. injected with cyclophosphamide followed by CAR T cells (Figure 12) one day later.
  • CAR T cell persistence and B cell killing in peripheral blood and BM were evaluated one week after CAR T cell transfer.
  • irradiation significantly reduced persistence of m19-humBBz but not m1928z CAR T cells
  • GSEA GSEA Enrichment Analysis
  • mCD19-targeted T cells with a CAR containing a 4- 1 BB domain have enhanced proliferation and NF- ⁇ signaling.
  • the 41 BB endodomain mutants were located in previously identified (Jang IK, et al. Biochem Biophys Res Commun. 1998 242(3):613-20; Arch RH, et al.
  • TRAFI/NF-KB is required for optimal m19-humBBz CAR T cell function in vivo
  • NF- ⁇ signaling in T cells is mediated, at least in part, through the binding of TRAF1-3 to the intracellular domain of 4-1 BB
  • TRAF2 is critical for enhanced function mediated by 4-1 BB co-stimulation in CAR T cells but no direct evidence exists (Zhao Z, et al. Cancer Cell. 2015 28(4):415-28; Gomes-Silva D, et al. Cell Rep. 2017 21 (1): 17-26).
  • TRAF dominant negative (DN) proteins into NF-KB/293/GFP- Luc reporter cells followed by m19-humBBz CAR transduction.
  • Gene-transfer for CAR and TRAF DN proteins was confirmed by flow cytometry ( Figure 6A).
  • NF- ⁇ signaling with TRAF1 DN decreased ( Figure 6A, GFP%: 45.5% vs. 13.4%; GFP MFI: 10929 vs. 1688).
  • the TRAF3 DN group also displayed decreased NF- ⁇ ( Figure 6A, GFP%: 45.5% vs. 30.8%; GFP MFI: 10929 vs. 6056), although not to the same extent as the TRAF1 DN group.
  • N F- ⁇ signaling in the TRAF2 DN group was greater than the m19-humBBz control ( Figure 6A, GFP+%: 63.5% vs. 45.5%; GFP MFI: 14309 vs. 10929).
  • TRAF1 could be identified binding to the CAR.
  • N F-KB/293/GFP-LUC reporter cells After transduction of N F-KB/293/GFP-LUC reporter cells with the m19-humBBz CAR we isolated the CAR by Protein L binding and assayed for retention of TRAF1.
  • Both h19BBz CAR T cell groups enhanced leukemia killing and survival compared to untransduced T cells, but neither group appeared superior to each other. This may be due to the nature of the aggressiveness of the NALM6/NSG mouse model (Zhao Z, et al. Cancer Cell. 2015 28(4):415-28), which requires rapid leukemia killing so that even 2nd generation CAR T cells do not prevent death.
  • N F-KB signaling drives different metabolic or signaling pathways in CAR T cells, which is a hypothesis that we are evaluating.
  • NF- ⁇ signaling is required for maintaining memory T cells and can also increase mitochondrial respiration (Knudson KM, et al. Proc Natl Acad Sci U S A. 2017 114(9):E1659-E67; Mauro C, et al. Nat Cell Biol. 201 1 13(10): 1272-9).
  • TRAF proteins regulate T cell function by linking extracellular activation receptors, including 4-1 BB, and intracellular signaling pathways thereby impacting T cell differentiation, proliferation, survival and cytokine production (Watts TH. Annu Rev Immunol. 2005 23:23-68; So T, et al. Tohoku J Exp Med. 2015 236(2): 139-54). TRAF proteins have been speculated as having multiple roles in transducing 4-1 BB co-stimulation in CARs but to date, none have been able to confirm or define their specific roles (Zhao Z, et al. Cancer Cell.
  • TRAF1 and TRAF3 are required for optimal NF- ⁇ activation by 4- 1 BB co-stimulation, which we confirmed for TRAF1 in primary mouse CAR T cells (Figure 6).
  • a TRAF2-DN inhibitor increased NF- ⁇ signaling of m19-humBBz CARs, which may be due to its role in degrading the NF-KB-inducing Kinase (NIK) since it serves as a negative regulator of the alternative NF- ⁇ signaling pathway (Zarnegar BJ, et al. Nat Immunol. 2008 9(12): 1371-8).
  • TRAF2 or TRAF3 in primary human h19BBz CAR T cells enhanced viability, proliferation and cytotoxicity (Figure 7A-7D).
  • T cells with h19BBz and excess TRAF2 dramatically increased NF- ⁇ compared to h19BBz CAR T cells (7.7-fold) further confirming that increased NF- ⁇ enhances CAR T cell function ( Figure 7A).
  • TRAFs and CARs into T cells may allow potentiation of both

Abstract

L'invention concerne des polypeptides de récepteurs antigéniques chimériques (CAR), qui peuvent être utilisés avec un transfert adoptif de cellules pour cibler et éliminer des cancers, comprenant une région de signalisation costimulatrice ayant une forme mutée d'un domaine cytoplasmique de CD28 qui améliore la fonction des lymphocytes T à CAR (CAR-T), une forme mutée d'un domaine cytoplasmique de 41BB qui améliore une signalisation de facteur nucléaire kappaB (NFκB) ou une combinaison de ces dernières. L'invention concerne également des cellules effectrices immunitaires, telles que des lymphocytes T ou des cellules tueuses naturelles (NK), qui sont modifiées pour exprimer ces CAR. L'invention concerne également des cellules effectrices immunitaires co-exprimant un CAR et une ou plusieurs protéines TRAF. Par conséquent, l'invention concerne également des méthodes consistant à conférer une immunité anti-tumorale à un sujet atteint d'une tumeur associée à un cancer exprimant des antigènes qui implique un transfert adoptif des cellules effectrices immunitaires décrites.
PCT/US2018/050417 2017-06-08 2018-09-11 Récepteurs antigéniques chimériques à signalisation de nfkb améliorée WO2019060174A1 (fr)

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WO2019165116A1 (fr) * 2018-02-26 2019-08-29 Fred Hutchinson Cancer Research Center Compositions et méthodes pour immunothérapie cellulaire
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CN113203781A (zh) * 2021-05-13 2021-08-03 桂林电子科技大学 一种基于RGO-CS-Hemin@Pt NPs纳米材料和适配体检测GPC3的方法
WO2021202801A1 (fr) * 2020-03-31 2021-10-07 Fred Hutchinson Cancer Research Center Augmentation de la mort cellulaire négative pour l'antigène dans des immunothérapies ciblées par un antigène
WO2021244654A1 (fr) * 2020-06-05 2021-12-09 Nanjing Legend Biotech Co., Ltd. Production de cytokine induite par activation dans des cellules immunitaires
CN114729319A (zh) * 2019-09-06 2022-07-08 新加坡国立大学 用含有单个itam信号传导基序的合成受体修饰的t细胞
WO2022173703A1 (fr) * 2021-02-10 2022-08-18 The Regents Of The University Of California Récepteurs immunitaires avec des domaines co-stimulateurs synthétiques
JP2023513819A (ja) * 2020-02-13 2023-04-03 ベイジン イムノチャイナ ファーマシューティカルズ カンパニー リミテッド キメラ抗原受容体の最適化
US11851491B2 (en) 2016-11-22 2023-12-26 TCR2 Therapeutics Inc. Compositions and methods for TCR reprogramming using fusion proteins
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CA3070861A1 (fr) 2019-03-28

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