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1.
Leukemia ; 32(2): 402-412, 2018 02.
Article in English | MEDLINE | ID: mdl-28951562

ABSTRACT

Current clinical outcomes using chimeric-antigen receptors (CARs) against multiple myeloma show promise in the eradication of bulk disease. However, these anti-BCMA (CD269) CARs observe relapse as a common phenomenon after treatment due to the reemergence of either antigen-positive or -negative cells. Hence, the development of improvements in CAR design to target antigen loss and increase effector cell persistency represents a critical need. Here, we report on the anti-tumor activity of a CAR T-cell possessing two complete and independent CAR receptors against the multiple myeloma antigens BCMA and CS1. We determined that the resulting compound CAR (cCAR) T-cell possesses consistent, potent and directed cytotoxicity against each target antigen population. Using multiple mouse models of myeloma and mixed cell populations, we are further able to show superior in vivo survival by directed cytotoxicity against multiple populations compared to a single-expressing CAR T-cell. These findings indicate that compound targeting of BCMA and CS1 on myeloma cells can potentially be an effective strategy for augmenting the response against myeloma bulk disease and for initiation of broader coverage CAR therapy.


Subject(s)
Multiple Myeloma/immunology , Receptors, Chimeric Antigen/immunology , Animals , B-Cell Maturation Antigen/immunology , Cell Line, Tumor , Cytotoxicity, Immunologic/immunology , Humans , K562 Cells , Male , Mice , Mice, Inbred NOD , Neoplasm Recurrence, Local/immunology , Receptors, Antigen, T-Cell/immunology , Signaling Lymphocytic Activation Molecule Family/immunology , T-Lymphocytes/immunology , Xenograft Model Antitumor Assays/methods
2.
Leukemia ; 31(10): 2151-2160, 2017 10.
Article in English | MEDLINE | ID: mdl-28074066

ABSTRACT

The outlook for T-cell malignancies remain poor due to the lack of effective therapeutic options. Chimeric antigen receptor (CAR) immunotherapy has recently shown promise in clinical trials for B-cell malignancies, however, designing CARs for T-cell based disease remain a challenge due to the shared surface antigen pool between normal and malignant T-cells. Normal T-cells express CD5 but NK (natural killer) cells do not, positioning NK cells as attractive cytotoxicity cells for CD5CAR design. Additionally, CD5 is highly expressed in T-cell acute lymphoblastic leukemia (T-ALL) and peripheral T-cell lymphomas (PTCLs). Here, we report a robust anti-CD5 CAR (CD5CAR) transduced into a human NK cell line NK-92 that can undergo stable expansion ex vivo. We found that CD5CAR NK-92 cells possessed consistent, specific, and potent anti-tumor activity against a variety of T-cell leukemia and lymphoma cell lines as well as primary tumor cells. Furthermore, we were able to demonstrate significant inhibition and control of disease progression in xenograft mouse models of T-ALL. The data suggest that CAR redirected targeting for T-cell malignancies using NK cells may be a viable method for new and complementary therapeutic approaches that could improve the current outcome for patients.


Subject(s)
Antigens, Neoplasm/immunology , CD5 Antigens/immunology , Immunotherapy, Adoptive/methods , Killer Cells, Natural/immunology , Lymphoma, T-Cell, Peripheral/therapy , Molecular Targeted Therapy , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/therapy , Recombinant Fusion Proteins/immunology , 4-1BB Ligand/genetics , 4-1BB Ligand/immunology , Animals , CD28 Antigens/immunology , CD3 Complex/genetics , CD3 Complex/immunology , CD8 Antigens/immunology , Cell Line, Tumor , Coculture Techniques , Cytotoxicity, Immunologic , Humans , Killer Cells, Natural/transplantation , Lymphoma, T-Cell, Peripheral/pathology , Mice , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Salvage Therapy , Single-Chain Antibodies/genetics , Single-Chain Antibodies/immunology , Transduction, Genetic , Tumor Necrosis Factor Receptor Superfamily, Member 9/immunology , Xenograft Model Antitumor Assays
3.
Leukemia ; 30(3): 701-7, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26526988

ABSTRACT

Peripheral T-cell lymphomas (PTCLs) are aggressive lymphomas with no effective upfront standard treatment and ineffective options in relapsed disease, resulting in poorer clinical outcomes as compared with B-cell lymphomas. The adoptive transfer of T cells engineered to express chimeric antigen receptors (CARs) is a promising new approach for treatment of hematological malignancies. However, preclinical reports of targeting T-cell lymphoma with CARs are almost non-existent. Here we have designed a CAR, CD4CAR, which redirects the antigen specificity of CD8+ cytotoxic T cells to CD4-expressing cells. CD4CAR T cells derived from human peripheral blood mononuclear cells and cord blood effectively redirected T-cell specificity against CD4+ cells in vitro. CD4CAR T cells efficiently eliminated a CD4+ leukemic cell line and primary CD4+ PTCL patient samples in co-culture assays. Notably, CD4CAR T cells maintained a central memory stem cell-like phenotype (CD8+CD45RO+CD62L+) under standard culture conditions. Furthermore, in aggressive orthotropic T-cell lymphoma models, CD4CAR T cells efficiently suppressed the growth of lymphoma cells while also significantly prolonging mouse survival. Combined, these studies demonstrate that CD4CAR-expressing CD8+ T cells are efficacious in ablating malignant CD4+ populations, with potential use as a bridge to transplant or stand-alone therapy for the treatment of PTCLs.


Subject(s)
Adoptive Transfer , CD8-Positive T-Lymphocytes/immunology , Lymphoma, T-Cell, Peripheral/therapy , Mutant Chimeric Proteins/genetics , Receptors, Artificial/genetics , Animals , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/pathology , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/transplantation , Cell Engineering , Cell Line, Tumor , Coculture Techniques , Cytotoxicity, Immunologic , Gene Expression , Humans , Leukemia/genetics , Leukemia/immunology , Leukemia/pathology , Lymphoma, T-Cell, Peripheral/genetics , Lymphoma, T-Cell, Peripheral/immunology , Lymphoma, T-Cell, Peripheral/mortality , Male , Mice , Mice, Inbred NOD , Mutant Chimeric Proteins/immunology , Neoplasm Transplantation , Primary Cell Culture , Receptors, Artificial/immunology , Survival Analysis
4.
Article in English | MEDLINE | ID: mdl-11554302

ABSTRACT

A number of laboratories have shown that those types of DNA damage that are generally reparable by base excision repair are efficiently repaired in mtDNA. In contrast, most types of damage that require other sorts of repair machinery are not effectively repaired in mtDNA. We have shown that a set of highly purified mitochondrial proteins, including AP endonuclease (APE), DNA polymerase gamma, and mtDNA ligase, is capable of efficiently repairing abasic (AP) sites in mtDNA. These three enzymes appear to conduct all four steps in a conventional BER mechanism: incision, removal of the 5'-deoxyribosephosphate by dRP lyase, polymerization, and ligation. Both DNA polymerase gamma and mtDNA ligase possess some dRP lyase activity. DNA polymerase gamma is a member of the family A of DNA polymerases, with clear homology to DNA pol I of E. coli, while mtDNA ligase is an alternatively expressed form of DNA ligase III. The dRP lyase activities discovered in these mitochondrial enzymes are not unique, but are found in all representatives tested of the family-A DNA polymerases and of the ATP-dependent DNA ligases. These dRP lyase activities have low turnover rates that may have important implications for the overall process of BER. All proteins involved in maintenance of mtDNA are encoded in the nuclear genome and must be directed to mitochondria in order to act on mtDNA. Thus, it is evident that the scope of DNA repair activities undertaken within mitochondria is determined by the set of nucleus-encoded DNA repair enzymes that are capable of being imported into the organelle. A review of DNA repair proteins that may be imported into mitochondria in various organisms will be presented.


Subject(s)
DNA Ligases/genetics , DNA Repair , DNA, Mitochondrial/genetics , Animals , Bacterial Proteins/genetics , Carbon-Oxygen Lyases/genetics , Carbon-Oxygen Lyases/metabolism , Cell Nucleus/enzymology , DNA Damage , DNA Ligase ATP , DNA Ligases/metabolism , DNA Polymerase gamma , DNA, Mitochondrial/metabolism , DNA-(Apurinic or Apyrimidinic Site) Lyase , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolism , Deoxyribonuclease IV (Phage T4-Induced) , Fungal Proteins/genetics , Genes , Humans , Mice , Oxidative Stress , Poly-ADP-Ribose Binding Proteins , Sequence Homology, Amino Acid , Xenopus Proteins , Xenopus laevis/metabolism
5.
J Biol Chem ; 275(17): 12509-14, 2000 Apr 28.
Article in English | MEDLINE | ID: mdl-10777538

ABSTRACT

Mitochondrial DNA polymerase gamma (pol gamma) is active in base excision repair of AP (apurinic/apyrimidinic) sites in DNA. Usually AP site repair involves cleavage on the 5' side of the deoxyribose phosphate by AP endonuclease. Previous experiments suggested that DNA pol gamma acts to catalyze the removal of a 5'-deoxyribose phosphate (dRP) group in addition to playing the conventional role of a DNA polymerase. We confirm that DNA pol gamma is an active dRP lyase and show that other members of the family A of DNA polymerases including Escherichia coli DNA pol I also possess this activity. The dRP lyase reaction proceeds by formation of a covalent enzyme-DNA intermediate that is converted to an enzyme-dRP intermediate following elimination of the DNA. Both intermediates can be cross-linked with NaBH(4). For both DNA pol gamma and the Klenow fragment of pol I, the enzyme-dRP intermediate is extremely stable. This limits the overall catalytic rate of the dRP lyase, so that family A DNA polymerases, unlike pol beta, may only be able to act as dRP lyases in repair of AP sites when they occur at low frequency in DNA.


Subject(s)
Carbon-Oxygen Lyases/metabolism , DNA-Directed DNA Polymerase/metabolism , Escherichia coli Proteins , Base Sequence , Catalysis , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , DNA Polymerase I/metabolism , DNA Polymerase gamma , DNA Repair , DNA-(Apurinic or Apyrimidinic Site) Lyase , Deoxyribonuclease IV (Phage T4-Induced) , Mitochondria/enzymology , Models, Chemical , Molecular Sequence Data , Moloney murine leukemia virus/enzymology , RNA-Directed DNA Polymerase/metabolism , Temperature , Time Factors
6.
J Biol Chem ; 273(14): 7888-93, 1998 Apr 03.
Article in English | MEDLINE | ID: mdl-9525883

ABSTRACT

Apurinic/apyrimidinic (AP) sites occur frequently in DNA as a result of spontaneous base loss or following removal of a damaged base by a DNA glycosylase. The action of many AP endonuclease enzymes at abasic sites in DNA leaves a 5'-deoxyribose phosphate (dRP) residue that must be removed during the base excision repair process. This 5'-dRP group may be removed by AP lyase enzymes that employ a beta-elimination mechanism. This beta-elimination reaction typically involves a transient Schiff base intermediate that can react with sodium borohydride to trap the DNA-enzyme complex. With the use of this assay as well as direct 5'-dRP group release assays, we show that T4 DNA ligase, a representative ATP-dependent DNA ligase, contains AP lyase activity. The AP lyase activity of T4 DNA ligase is inhibited in the presence of ATP, suggesting that the adenylated lysine residue is part of the active site for both the ligase and lyase activities. A model is proposed whereby the AP lyase activity of DNA ligase may contribute to the repair of abasic sites in DNA.


Subject(s)
DNA Ligases/metabolism , DNA Repair , DNA/metabolism , Animals , Base Sequence , Binding Sites , DNA/genetics , Female , Molecular Sequence Data , Substrate Specificity , Xenopus
7.
Mol Cell Biol ; 18(3): 1257-65, 1998 Mar.
Article in English | MEDLINE | ID: mdl-9488440

ABSTRACT

Mutations in mitochondrial DNA (mtDNA) cause a variety of relatively rare human diseases and may contribute to the pathogenesis of other, more common degenerative diseases. This stimulates interest in the capacity of mitochondria to repair damage to mtDNA. Several recent studies have shown that some types of damage to mtDNA may be repaired, particularly if the lesions can be processed through a base excision mechanism that employs an abasic site as a common intermediate. In this paper, we demonstrate that a combination of enzymes purified from Xenopus laevis mitochondria efficiently repairs abasic sites in DNA. This repair pathway employs a mitochondrial class II apurinic/apyrimidinic (AP) endonuclease to cleave the DNA backbone on the 5' side of an abasic site. A deoxyribophosphodiesterase acts to remove the 5' sugar-phosphate residue left by AP endonuclease. mtDNA polymerase gamma fills the resulting 1-nucleotide gap. The remaining nick is sealed by an mtDNA ligase. We report the first extensive purification of mtDNA ligase as a 100-kDa enzyme that functions with an enzyme-adenylate intermediate and is capable of ligating oligo(dT) strands annealed to poly(rA). These properties together with preliminary immunological evidence suggest that mtDNA may be related to nuclear DNA ligase III.


Subject(s)
Carbon-Oxygen Lyases/metabolism , DNA Ligases/metabolism , DNA Repair , DNA, Mitochondrial , DNA-Directed DNA Polymerase/metabolism , Mitochondria/enzymology , Phosphoric Diester Hydrolases/metabolism , Amino Acid Sequence , Animals , Carbon-Oxygen Lyases/classification , Cell Fractionation , DNA/biosynthesis , DNA Polymerase gamma , DNA-(Apurinic or Apyrimidinic Site) Lyase , Deoxyribonuclease IV (Phage T4-Induced) , HeLa Cells , Humans , Molecular Sequence Data , Templates, Genetic , Xenopus laevis
8.
J Biol Chem ; 270(16): 9202-6, 1995 Apr 21.
Article in English | MEDLINE | ID: mdl-7721837

ABSTRACT

Mitochondrial DNA is subject to oxidative damage generating 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) residues and to spontaneous or induced base loss generating abasic sites. Synthetic oligonucleotides containing these lesions were prepared and used as templates to determine their effects on the action of Xenopus laevis DNA polymerase gamma. An analogue of an abasic site in DNA, tetrahydrofuran, was found to inhibit elongation by DNA polymerase gamma. When the DNA polymerase was able to complete translesional synthesis, a dA residue was incorporated opposite the abasic site. In contrast, elongation by DNA polymerase gamma was not inhibited by an 8-oxo-dG residue in the template strand. The polymerase inserted dA opposite 8-oxo-dG in approximately 27% of the extended products. The effects of these lesions on the 3'-->5' exonuclease proofreading activity of DNA polymerase gamma were also investigated. The 3'-->5' exonuclease activity excised any of the four normal bases positioned opposite either a tetrahydrofuran residue or 8-oxo-dG, suggesting that proofreading may not play a major role in avoiding misincorporation at abasic sites or 8-oxo-dG residues in the template. Thus, both of these lesions have the prospect of causing high rates of mutation during mtDNA replication.


Subject(s)
DNA Damage , DNA Polymerase III/physiology , DNA, Mitochondrial/metabolism , Mitochondria/enzymology , 8-Hydroxy-2'-Deoxyguanosine , Animals , Base Sequence , DNA Replication , Deoxyguanosine/analogs & derivatives , Deoxyguanosine/pharmacology , Female , Molecular Sequence Data , Xenopus laevis
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