ABSTRACT
Glutamine is a major nutrient for cancer cells during rapid proliferation. Alanine-serine-cysteine (ASC) transporter 2 (ASCT2; SLC1A5) mediates glutamine uptake in a variety of cancer cells. We previously reported that KM8094, a novel anti-ASCT2 humanized monoclonal antibody, possesses anti-tumor efficacy in gastric cancer patient-derived xenografts. The aim of this study was to investigate the molecular mechanism underlying the effect of KM8094 and to further substantiate the preclinical feasibility of using KM8094 as a potential therapeutic agent against gastric cancer. First, ASCT2 was found to be highly expressed in cancer tissues derived from gastric cancer patients by an immunohistochemical analysis. Next, we performed in vitro studies using multiple gastric cancer cell lines and observed that several gastric cancer cells expressing ASCT2 showed glutamine-dependent cell growth, which was repressed by KM8094. We found that KM8094 inhibited the glutamine uptake, leading to the reduction of glutathione (GSH) level and the elevation of oxidative stress. KM8094 suppressed the cell cycle progression and increased the apoptosis. Furthermore, KM8094 exerted antibody dependent cellular cytotoxicity (ADCC) against human gastric cancer cells in vitro. Finally, in vivo studies revealed that KM8094 suppressed tumor growth in several gastric cancer xenografts. This effect was enhanced by docetaxel, one of the agents commonly used in gastric cancer therapy. Thus, our findings suggest that KM8094 is a potential new therapeutic agent for gastric cancer expressing ASCT2, which blocks the cellular glutamine metabolism and possesses ADCC activity.
ABSTRACT
ASC amino acid transporter 2 (ASCT2), also known as solute linked carrier family 1 member A5 (SLC1A5) is a Na+-dependent glutamine/neutral amino acid transporter. ASCT2 acts as a high-affinity transporter of L-glutamine (Gln) and has been reported to be up-regulated in a variety of cancerous tissues including stomach, liver, and kidney. In this study, we evaluated anti-tumor efficacy of a novel anti-ASCT2 humanized monoclonal antibody, KM8094, which has a neutralizing activity against glutamine uptake, as a therapeutic antibody against gastric cancer and explored clinical predictive biomarker candidates by utilizing patient-derived xenograft (PDX) mouse models. Anti-tumor efficacy studies revealed that some of the PDX models used were responsive to KM8094 and the others were not. Interestingly, we observed a correlation between anti-tumor efficacy and low antigen expression as well as low basal levels of glutamine uptake, suggesting ASCT2 expression level could be a potential predictive biomarker for KM8094. We then further explored predictive biomarker candidates by multi-omics analysis on gastric cancer PDX mouse models. As a result, a few potential candidates such as TFF2, MUC13, and ANG were selected by gene expression and DNA methylation array analyses. In addition, metabolomics analysis revealed clear differences in intracellular energy status and redox status between responsive and non-responsive PDX models.
ABSTRACT
Many ovarian cancer patients often show peritoneal metastasis with malignant ascites. However, unmet medical needs remain regarding controlling these symptoms after tumors become resistant to chemotherapies. We developed KHK2805, a novel anti-folate receptor α (FOLR1) humanized antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The primary aim of the present study was to evaluate whether the anti-tumor activity of KHK2805 was sufficient for therapeutic application against peritoneal dissemination and malignant ascites of platinum-resistant ovarian cancer in preclinical models. Here, both the ADCC and CDC of KHK2805 were evaluated in ovarian cancer cell lines and patient-derived samples. The anti-tumor activity of KHK2805 was evaluated in a SCID mouse model of platinum-resistant peritoneal dissemination. As results, KHK2805 showed specific binding to FOLR1 with high affinity at a novel epitope. KHK2805 exerted potent ADCC and CDC against ovarian cancer cell lines. Furthermore, primary platinum-resistant malignant ascites cells were susceptible to autologous ADCC with KHK2805. Patient-derived sera and malignant ascites induced CDC of KHK2805. KHK2805 significantly reduced the total tumor burden and amount of ascites in SCID mice with peritoneal dissemination and significantly prolonged their survival. In addition, the parental rat antibody strongly stained serous and clear cell-type ovarian tumors by immunohistochemistry. Overall, KHK2805 showed cytotoxicity against both ovarian cancer cell lines and patient-derived cells. These translational study findings suggest that KHK2805 may be promising as a novel therapeutic agent for platinum-resistant ovarian cancer with peritoneal dissemination and malignant ascites.
ABSTRACT
PURPOSE: Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family and is an important therapeutic target in some types of human cancers. KHK2866 is a humanized anti-HB-EGF monoclonal antibody IgG that neutralizes HB-EGF activity by inhibiting the binding of HB-EGF to its receptors. The phase I study of KHK2866 was discontinued because of neuropsychiatric toxicity. In this study, the pharmacokinetics of KHK2866 was evaluated by (89)Zr-immuno-PET study and the determination of drug concentrations in serum and cerebrospinal fluid using cynomolgus monkeys was performed in order to predict neurotoxicity in a reverse-translational manner. METHODS: KHK2866 was radiolabeled with (89)Zr for preclinical evaluations in normal cynomolgus monkeys and its distribution was analyzed. Furthermore, as a separate study, KHK2866 concentrations in serum and cerebrospinal fluid were determined after administration of a single dose. RESULTS: PET studies with monkeys revealed (89)Zr-KHK2866 accumulation in the liver, spleen and joints of multiple parts, but not in brain. In addition, the pharmacokinetic analyses in serum and CSF demonstrated a low penetration of KHK2866 into the brain. CONCLUSIONS: These studies indicate the difficulty of prediction for neuropsychiatric toxicity of monoclonal antibodies in human by means of pharmacokinetic evaluations using cynomolgus monkeys.
Subject(s)
Antibodies, Monoclonal, Humanized/immunology , Antibodies, Monoclonal, Humanized/pharmacokinetics , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacokinetics , Heparin-binding EGF-like Growth Factor/immunology , Animals , Antibodies, Monoclonal/blood , Antibodies, Monoclonal/cerebrospinal fluid , Antibodies, Monoclonal, Humanized/blood , Antibodies, Monoclonal, Humanized/cerebrospinal fluid , Female , Humans , Macaca fascicularis , Positron-Emission Tomography/methods , Zirconium/blood , Zirconium/cerebrospinal fluid , Zirconium/pharmacokineticsABSTRACT
Antibody-dependent cellular cytotoxicity (ADCC) is dependent on the fucose content of oligosaccharides bound to monoclonal antibodies (MAbs). As MAbs with a low fucose content exhibit high ADCC activity, it is important to control the defucosylation levels (deFuc%) of MAbs and to analyze the factors that affect deFuc%. In this study, we observed that the deFuc% was inversely related to culture medium osmolality for MAbs produced in the rat hybridoma cell line YB2/0, with r (2) values as high as 0.92. Moreover, deFuc% exhibited the same correlation irrespective of the type of compound used for regulating osmolality (NaCl, KCl, fucose, fructose, creatine, or mannitol) at a culture scale ranging from 1 to 400 L. We succeeded in controlling MAb deFuc% by maintaining a constant medium osmolality in both perfusion and fed-batch cultures. In agreement with these observations, reverse transcription PCR analyses revealed decreased transcription of genes involved in glycolysis, GDP-fucose supply, and fucose transfer under hypoosmotic conditions.
ABSTRACT
Chinese hamster ovary (CHO) cells are the most common host cells and are widely used in the manufacture of approved recombinant therapeutics. They represent a major new class of universal hosts in biopharmaceutical production. However, there remains room for improvement to create more ideal host cells that can add greater value to therapeutic recombinant proteins at reduced production cost. A promising approach to this goal is biallelic gene knockout in CHO cells, as it is the most reliable and effective means to permanent phenotypic change, owing to the complete removal of gene function. In this chapter, we describe a biallelic gene knockout process in CHO cells, as exemplified by the successful targeted disruption of both FUT8 alleles encoding alpha-1,6-fucosyltransferase gene in CHO/DG44 cells. Wild-type alleles are sequentially disrupted by homologous recombination using two targeting vectors to generate homozygous disruptants, and the drug-resistance gene cassettes remaining on the alleles are removed by a Cre/loxP recombination system so as not to leave the extraphenotype except for the functional loss of the gene of interest.
Subject(s)
CHO Cells/enzymology , Gene Targeting/methods , Alleles , Animals , Base Sequence , Blotting, Southern , CHO Cells/drug effects , Cricetinae , Cricetulus , DNA Primers/genetics , Drug Resistance/genetics , Fucosyltransferases/genetics , Gene Deletion , Genetic Vectors , Phenotype , Polymerase Chain Reaction , Recombination, Genetic , TransfectionABSTRACT
To investigate the functional expression of adenosine A3 receptor (A3AR) in mammalian living tissues, we generated an apoaequorin-transgenic mouse that expresses jellyfish apoaequorin throughout its body. The expression of apoaequorin under the control of a strong CAG promoter was detected in various tissues, including the abdominal skin, adipose, ear, brain, esophagus, heart, inferior vena cava vessel, kidney, lens, liver, lung, pancreas, skeletal muscle, spleen, tail, testis, and thymus. The transgene was mapped to the C1-2 region of chromosome 16 by Fluorescence in situ hybridization analysis. Among these transgenic mouse tissues, we succeeded in detecting elevated responses of intracellular Ca2+ as a light emission of aequorin induced by the A3AR agonist in the pancreas, brain, and testis, the last two of which are known to be main tissues abundantly expressing A3AR. The A3AR agonist led to the phosphorylation of both extracellular signal-regulated kinase 1/2 and protein kinase B in mouse pancreas, and all the intracellular responses via A3AR were antagonized by the A3AR-specific antagonist. In addition, the mRNA expression of A3AR and the A3AR-induced intracellular responses were also found in the rat pancreatic acinar cell line AR42J. These results suggest that pancreas is one of the main tissues functionally expressing A3AR in mammalians in vivo, and that the present approach using transgenic mice that express apoaequorin throughout their bodies will facilitate the functional analysis of proteins of interest.
Subject(s)
Aequorin/metabolism , Apoproteins/metabolism , Calcium/metabolism , Mice, Transgenic/genetics , Pancreas/metabolism , Receptor, Adenosine A3/physiology , Scyphozoa/chemistry , Animals , Calcium Signaling , Mice , Mice, Inbred C57BL , Mice, Transgenic/metabolism , Receptor, Adenosine A3/drug effects , Recombinant Proteins/metabolismABSTRACT
Several methods have been described to enhance antibody-dependent cellular cytotoxicity (ADCC) using different host cells that produce antibody with reduced levels of fucose on their carbohydrates. We compared the suitability of these methods for the serum-free fed-batch production of antibody for clinical trials and commercial uses. Recombinant anti-human CD20 chimeric IgG1-producing clones were established from host-cells that have been shown to produce more than 90% fucose-negative antibody. The cell lines were a FUT8 (alpha-1,6-fucosyltransferase) knockout Chinese hamster ovary (CHO) cell line, Ms704, and two Lens culinaris agglutinin (LCA)-resistant cell lines, one derived from a variant CHO line, Lec13 and the other from a rat hybridoma cell line, YB2/0. The amount of fucose-negative antibody produced by Lec13 and YB2/0 significantly decreased with the culture. The increase in fucosylation was due to remaining synthesis of GDP-fucose via de novo pathway for the CHO line and the elevation of FUT8 expression by the YB2/0 cells. In contrast, Ms704 cells stably produced fucose-negative antibody with a consistent carbohydrate structure until the end of the culture. The productivity of the Ms704 cells reached 1.76 g/L with a specific production rate (SPR) of 29 pg/cell/day for 17 days in serum-free fed-batch culture using a 1 L spinner bioreactor. Our results demonstrate that FUT8 knockout has the essential characteristics of host cells for robust manufacture of fucose-negative therapeutic antibodies with enhanced ADCC.
Subject(s)
Antibody Formation , Antibody-Dependent Cell Cytotoxicity , Fucose/immunology , Animals , Bioreactors , CHO Cells/immunology , Cell Culture Techniques/methods , Cell Line , Cricetinae , Culture Media, Serum-Free , DNA, Complementary/genetics , Fucosyltransferases/genetics , Fucosyltransferases/metabolism , Gene Deletion , Monosaccharides/chemistry , Monosaccharides/isolation & purification , Oligosaccharides/chemistry , Oligosaccharides/immunology , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Although the adenosine A(3) receptor (A3AR), which is a G(i/o) protein-coupled receptor, has attracted considerable interest as a potential target for drugs against asthma or inflammation, the in vivo evaluation of the antagonists using rodents in the first step of drug development has been hampered by the lack of highly potent antagonists for the rodent A3AR. To evaluate the pharmacological effects of human A3AR antagonists in mice, we previously generated A3AR-humanized mice, in which the mouse A3AR gene was replaced by its human counterpart. However, the human A3AR did not lead to the phosphoinositide 3-kinase (PI3K) gamma-signaling pathway such as IgE/antigen-dependent mast cell degranulation, probably due to the uncoupling of the mouse G(i/o) protein(s). To overcome the uncoupling, we here generated A3AR functionally humanized mice by replacing the mouse A3AR gene with a human/mouse chimeric A3AR sequence in which whole intracellular regions of the human A3AR were substituted for the corresponding regions of the mouse A3AR. The chimeric A3AR led to intracellular Ca(2+) elevation and activation of the PI3Kgamma-signaling pathway, which are equivalent to the actions induced by A3AR in wild-type mice. The human A3AR antagonist had the same binding affinities for the chimeric A3AR as the human A3AR and completely antagonized this potentiation. This is the first direct evidence that the uncoupling of mouse G protein(s) to the human A3AR is due to a sequence difference in the intracellular regions of A3AR. The A3AR functionally humanized mice can be widely employed for pharmacological evaluations of the human A3AR antagonists.
Subject(s)
Adenosine A3 Receptor Antagonists , Calcium/metabolism , Cell Degranulation/drug effects , Chimera/metabolism , Mast Cells/metabolism , Models, Animal , Adenosine/analogs & derivatives , Adenosine/pharmacology , Amino Acid Sequence , Animals , Blotting, Western , Cell Degranulation/immunology , Cell Membrane/drug effects , Cell Membrane/metabolism , Chimera/genetics , Class Ib Phosphatidylinositol 3-Kinase , Humans , Isoenzymes/metabolism , Mast Cells/drug effects , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , Molecular Sequence Data , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Protein Binding , Receptor, Adenosine A3/genetics , Receptor, Adenosine A3/metabolism , Receptors, G-Protein-Coupled/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Sequence Homology, Amino Acid , Species SpecificityABSTRACT
Selective antagonists for the adenosine A(3) receptor (A3AR), a member of the G protein-coupled receptors, have been indicated as potential drugs for anti-asthma or anti-inflammation. However, potent antagonists for the rodent A3AR have not been identified. To evaluate the pharmacological effects of human A3AR antagonists in mice, we here generated A3AR-humanized mice, in which the mouse A3AR gene was replaced by its human counterpart. The expression levels of human A3AR in the A3AR-humanized mice were equivalent to those of mouse A3AR in wild-type mice. Elevation of the intracellular Ca(2+) concentration induced by an A3AR agonist was observed in bone marrow-derived mast cells from the A3AR-humanized mice and this Ca(2+) mobilization was completely antagonized by a human A3AR antagonist. However, antigen-dependent degranulation was not potentiated by the A3AR agonist in the mast cells from A3AR-humanized mice. The agonist-stimulated human A3AR did not lead to the phosphorylation of either extracellular signal-regulated kinase 1/2 or protein kinase B in A3AR-humanized mice. The rate of human A3AR internalization in the mast cells was also markedly decreased compared with that of mouse A3AR in the mast cells. These results demonstrate that the human A3AR is insufficient to activate phosphoinositide 3-kinase gamma-dependent signaling pathways in mice, probably due to the uncoupling of member(s) of the G proteins, which are capable of activating phosphoinositide 3-kinase gamma, to the human A3AR, despite the mouse G protein(s) responsible for the Ca(2+) elevation are coupled with the human A3AR.
Subject(s)
Calcium Signaling/physiology , Calcium/metabolism , Phosphatidylinositol 3-Kinases/physiology , Receptor, Adenosine A3/physiology , Adenosine/analogs & derivatives , Adenosine/pharmacology , Adenosine A3 Receptor Antagonists , Animals , Bone Marrow Cells/drug effects , Bone Marrow Cells/pathology , Calcium/antagonists & inhibitors , Cell Degranulation/immunology , Chimera , Class Ib Phosphatidylinositol 3-Kinase , Gene Expression/drug effects , Humans , Immunoglobulin E/immunology , Iodine Radioisotopes , Isoenzymes/chemistry , Isoenzymes/physiology , Male , Mast Cells/drug effects , Mice , Mice, Inbred C57BL/genetics , Mice, Inbred ICR/genetics , Mice, Knockout/genetics , Mice, Knockout/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Phenotype , Phosphatidylinositol 3-Kinases/chemistry , Phosphorylation , Protein Kinases/metabolism , Purines/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptor, Adenosine A3/drug effects , Receptors, Leukotriene B4/genetics , Receptors, Leukotriene B4/metabolism , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methodsABSTRACT
We explored the possibility of converting established antibody-producing cells to cells producing high antibody-dependent cellular cytotoxicity (ADCC) antibodies. The conversion was made by constitutive expression of small interfering RNA (siRNA) against alpha1,6 fucosyltransferase (FUT8). We found two effective siRNAs, which reduce FUT8 mRNA expression to 20% when introduced into Chinese hamster ovary (CHO)/DG44 cells. Selection for Lens culinaris agglutinin (LCA)-resistant clones after introduction of the FUT8 siRNA expression plasmids yields clones producing highly defucosylated (approximately 60%) antibody with over 100-fold higher ADCC compared to antibody produced by the parental cells (approximately 10% defucosylated). Moreover, the selected clones remain stable, producing defucosylated antibody even in serum-free fed-batch culture. Our results demonstrate that constitutive FUT8 siRNA expression can control the oligosaccharide structure of recombinant antibody produced by CHO cells to yield antibodies with dramatically enhanced ADCC.