ABSTRACT
Small-cell lung cancer (SCLC) is an aggressive cancer for which immune checkpoint inhibitors (ICI) have had only limited success. Bispecific T-cell engagers are promising therapeutic alternatives for ICI-resistant tumors, but not all patients with SCLC are responsive. Herein, to integrate CD137 costimulatory function into a T-cell engager format and thereby augment therapeutic efficacy, we generated a CD3/CD137 dual-specific Fab and engineered a DLL3-targeted trispecific antibody (DLL3 trispecific). The CD3/CD137 dual-specific Fab was generated to competitively bind to CD3 and CD137 to prevent DLL3-independent cross-linking of CD3 and CD137, which could lead to systemic T-cell activation. We demonstrated that DLL3 trispecific induced better tumor growth control and a marked increase in the number of intratumoral T cells compared with a conventional DLL3-targeted bispecific T-cell engager. These findings suggest that DLL3 trispecific can exert potent efficacy by inducing concurrent CD137 costimulation and provide a promising therapeutic option for SCLC.
Subject(s)
CD3 Complex , Intracellular Signaling Peptides and Proteins , Lung Neoplasms , Membrane Proteins , Small Cell Lung Carcinoma , T-Lymphocytes , Tumor Necrosis Factor Receptor Superfamily, Member 9 , Small Cell Lung Carcinoma/immunology , Small Cell Lung Carcinoma/pathology , Small Cell Lung Carcinoma/drug therapy , Small Cell Lung Carcinoma/therapy , Small Cell Lung Carcinoma/metabolism , Humans , Lung Neoplasms/immunology , Lung Neoplasms/pathology , Lung Neoplasms/drug therapy , Tumor Necrosis Factor Receptor Superfamily, Member 9/metabolism , CD3 Complex/immunology , Animals , Mice , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Membrane Proteins/immunology , Antibodies, Bispecific/pharmacology , Antibodies, Bispecific/therapeutic use , Cell Line, Tumor , Lymphocyte Activation/immunology , Female , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/metabolism , Xenograft Model Antitumor AssaysABSTRACT
We previously reported that monoclonal antibodies (mAbs) with a high isoelectric point (pI) value tended to exhibit fast plasma clearance (CL) and large steady-state volume of distribution (Vdss) in mice. However, the positive correlation between pI, CL, and Vdss cannot be described by the reported physiologically based pharmacokinetic (PBPK) models, in which FcRn-mediated transcytosis of mAbs is set to be minimal compared to convection-mediated transport. To address this issue, physiological parameters (lymph flow rate, reflection coefficient, endothelial uptake clearance, and FcRn concentration) were optimized based on the pharmacokinetic profiles of mAbs with various pI values in wild type and FcRn-deficient (beta-2-microglobulin knockout [KO]) mice. Simulations using the PBPK model developed in this study showed a positive correlation between pI, CL and Vdss observed in wild-type mice. Therefore, this model successfully characterized our hypothetical mechanism that an electrostatic positive interaction between mAbs and the endothelial membrane enhances FcRn-mediated transcytosis of mAbs, resulting in large Vdss. We sought to determine the right contribution of the two pathways of antibody distribution to the interstitial space and established a new model that could effectively capture the effect of pI on FcRn-mediated distribution of mAbs in the body.
Subject(s)
Antibodies, Monoclonal , Models, Biological , Mice , Animals , Antibodies, Monoclonal/pharmacokinetics , Biological Transport , Kinetics , Mice, Knockout , Receptors, Fc/genetics , Receptors, Fc/metabolism , Histocompatibility Antigens Class I/metabolismABSTRACT
Adenosine triphosphatase phospholipid transporting 8B1 (ATP8B1) deficiency, an ultrarare autosomal recessive liver disease, includes severe and mild clinical forms, referred to as progressive familial intrahepatic cholestasis type 1 (PFIC1) and benign recurrent intrahepatic cholestasis type 1 (BRIC1), respectively. There is currently no practical method for determining PFIC1 or BRIC1 at an early disease course phase. Herein, we assessed the feasibility of developing a diagnostic method for PFIC1 and BRIC1. A nationwide Japanese survey conducted since 2015 identified 25 patients with cholestasis with ATP8B1 mutations, 15 of whom agreed to participate in the study. Patients were divided for analysis into PFIC1 (n = 10) or BRIC1 (n = 5) based on their disease course. An in vitro mutagenesis assay to evaluate pathogenicity of ATP8B1 mutations suggested that residual ATP8B1 function in the patients could be used to identify clinical course. To assess their ATP8B1 function more simply, human peripheral blood monocyte-derived macrophages (HMDMs) were prepared from each patient and elicited into a subset of alternatively activated macrophages (M2c) by interleukin-10 (IL-10). This was based on our previous finding that ATP8B1 contributes to polarization of HMDMs into M2c. Flow cytometric analysis showed that expression of M2c-related surface markers cluster of differentiation (CD)14 and CD163 were 2.3-fold and 2.1-fold lower (95% confidence interval, 2.0-2.5 for CD14 and 1.7-2.4 for CD163), respectively, in patients with IL-10-treated HMDMs from PFIC1 compared with BRIC1. Conclusion: CD14 and CD163 expression levels in IL-10-treated HMDMs may facilitate diagnosis of PFIC1 or BRIC1 in patients with ATP8B1 deficiency.
Subject(s)
Adenosine Triphosphatases/deficiency , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Cholestasis/metabolism , Lipopolysaccharide Receptors/metabolism , Macrophages/metabolism , Receptors, Cell Surface/metabolism , Adenosine Triphosphatases/metabolism , Adolescent , Adult , Child , Child, Preschool , Cholestasis/diagnosis , Cholestasis/pathology , Female , Humans , Interleukin-10/pharmacology , Liver/metabolism , Liver/pathology , Macrophages/pathology , Male , Mutagenesis/genetics , Mutation , Young AdultABSTRACT
Agonistic antibodies targeting CD137 have been clinically unsuccessful due to systemic toxicity. Because conferring tumor selectivity through tumor-associated antigen limits its clinical use to cancers that highly express such antigens, we exploited extracellular adenosine triphosphate (exATP), which is a hallmark of the tumor microenvironment and highly elevated in solid tumors, as a broadly tumor-selective switch. We generated a novel anti-CD137 switch antibody, STA551, which exerts agonistic activity only in the presence of exATP. STA551 demonstrated potent and broad antitumor efficacy against all mouse and human tumors tested and a wide therapeutic window without systemic immune activation in mice. STA551 was well tolerated even at 150 mg/kg/week in cynomolgus monkeys. These results provide a strong rationale for the clinical testing of STA551 against a broad variety of cancers regardless of antigen expression, and for the further application of this novel platform to other targets in cancer therapy. SIGNIFICANCE: Reported CD137 agonists suffer from either systemic toxicity or limited efficacy against antigen-specific cancers. STA551, an antibody designed to agonize CD137 only in the presence of extracellular ATP, inhibited tumor growth in a broad variety of cancer models without any systemic toxicity or dependence on antigen expression.See related commentary by Keenan and Fong, p. 20.This article is highlighted in the In This Issue feature, p. 1.
Subject(s)
Adenosine Triphosphate , Neoplasms , Animals , Antibodies, Monoclonal/pharmacology , Antigens, Neoplasm , Immunotherapy , Mice , Neoplasms/drug therapy , Tumor Microenvironment , Tumor Necrosis Factor Receptor Superfamily, Member 9ABSTRACT
Progressive familial intrahepatic cholestasis type 1 (PFIC1), a rare inherited recessive disease resulting from a genetic deficiency in ATP8B1, progresses to liver failure. Because of the difficulty of discriminating PFIC1 from other subtypes of PFIC based on its clinical and histological features and genome sequencing, an alternative method for diagnosing PFIC1 is desirable. Herein, we analyzed human peripheral blood monocyte-derived macrophages (HMDM) and found predominant expression of ATP8B1 in interleukin-10 (IL-10)-induced M2c, a subset of alternatively activated macrophages. SiRNA-mediated depletion of ATP8B1 in IL-10-treated HMDM markedly suppressed the expression of M2c-related surface markers and increased the side scatter (SSC) of M2c, likely via impairment of the IL-10/STAT3 signal transduction pathway. These phenotypic features were confirmed in IL-10-treated HMDM from four PFIC1 patients with disease-causing mutations in both alleles, but not in those from four patients with other subtypes of PFIC. This method identified three PFIC1 patients in a group of PFIC patients undiagnosed by genome sequencing, an identical diagnostic outcome to that achieved by analysis of liver specimens and in vitro mutagenesis studies. In conclusion, ATP8B1 deficiency caused incomplete polarization of HMDM into M2c. Phenotypic analysis of M2c helps to identify PFIC1 patients with no apparent disease-causing mutations in ATP8B1.
Subject(s)
Adenosine Triphosphatases/deficiency , Cholestasis/blood , Cholestasis/metabolism , Macrophages/metabolism , Monocytes/pathology , Adenosine Triphosphatases/metabolism , Adolescent , Biomarkers/metabolism , Child , Child, Preschool , Cholestasis/diagnosis , Cholestasis/pathology , Female , Humans , Interleukin-10/metabolism , Liver/metabolism , Liver/pathology , Macrophages/pathology , Male , Mutagenesis/genetics , Phenotype , STAT3 Transcription Factor/metabolism , Signal Transduction , gamma-Glutamyltransferase/metabolismABSTRACT
AIM: Benign recurrent intrahepatic cholestasis type 2 (BRIC2) is caused by mutations in ABCB11, a gene encoding the bile salt export pump (BSEP) that mediates biliary bile salt secretion, and presents with repeated intermittent cholestasis with refractory itching. Currently, no effective medical therapy has been established. We previously provided experimental and clinical evidence suggesting the therapeutic potential of 4-phenylbutyrate (4PB) for the cholestatic attacks of BRIC2. METHODS: After examining the potential therapeutic use of 4PB treatment by in vitro studies, a patient with BRIC2 was treated p.o. with 4PB at gradually increasing doses (200, 350, and 500 mg/kg per day) for 4 months. Biochemical, histological and clinical data were collected. RESULTS: The patient was diagnosed with BRIC2 because he had non-synonymous mutations (c.1211A>G [p.D404G] and 1331T>C [p.V444A]) in ABCB11, reduced hepatocanalicular expression of BSEP and low biliary bile salt concentrations. In vitro analysis showed that 4PB treatment partially restored the decreased expression of BSEP caused by p.D404G mutation. During the first 2 months of 4PB therapy at 200 and 350 mg/kg per day, the patient had no relief from his symptoms. No beneficial effect was observed after additional treatment with bilirubin absorption and endoscopic nasobiliary drainage. However, after starting treatment at a dose of 500 mg/kg per day, the patient's liver function tests and intractable itching were markedly improved. No apparent side-effects were observed during or after 4PB therapy. The symptoms relapsed within 1.5 months after cessation of 4PB therapy. CONCLUSION: 4PB therapy would have a therapeutic effect on the cholestatic attacks of BRIC2.
ABSTRACT
BACKGROUND: Progressive familial intrahepatic cholestasis type 1 (PFIC1), an inherited liver disease caused by mutations in ATP8B1, progresses to severe cholestasis with a sustained intractable itch. Currently, no effective therapy has been established for PFIC1. Decreased function of the bile salt export pump (BSEP) in hepatocytes is suggested to be responsible for the severe cholestasis observed in PFIC1. We found a previously unidentified pharmacological effect of 4-phenylbutyrate (4PB) that increases the expression and function of BSEP. Here, we tested 4PB therapy in three patients with PFIC1. METHODS: The therapeutic potency of 4PB in these patients was tested by oral administration of this drug with gradually increasing dosage (200, 350, and 500 mg/kg/day) for 6 months. Biochemical, histological, and clinical data were collected. RESULTS: 4PB therapy had no beneficial effect on the patients' liver functions, as assessed by biochemical and histological analyses, despite an increase in hepatic BSEP expression. However, therapy with 4PB at a dosage of 350 or 500 mg/kg/day significantly relieved the intractable itch. Serum levels of potential pruritogens in cholestasis were much higher than the reference ranges during the 4PB therapy. CONCLUSIONS: 4PB therapy may be a new medication for patients with intractable cholestatic pruritus and may improve quality of life for patients and their families.
Subject(s)
Cholestasis, Intrahepatic/complications , Genetic Diseases, Inborn/complications , Phenylbutyrates/therapeutic use , Pruritus/drug therapy , Administration, Oral , Adolescent , Child , Child, Preschool , Cholestasis, Intrahepatic/genetics , Cholestasis, Intrahepatic/physiopathology , Disease Progression , Dose-Response Relationship, Drug , Genetic Diseases, Inborn/genetics , Genetic Diseases, Inborn/physiopathology , Humans , Phenylbutyrates/administration & dosage , Pruritus/etiologyABSTRACT
To examine the effects of 4-phenylbutyrate (4PB) therapy in a patient with progressive familial intrahepatic cholestasis type 2. A homozygous c.3692G>A (p.R1231Q) mutation was identified in ABCB11. In vitro studies showed that this mutation decreased the cell-surface expression of bile salt export pump (BSEP), but not its transport activity, and that 4PB treatment partially restored the decreased expression of BSEP. Therapy with 4PB had no beneficial effect for 1 month at 200 mg/kg/day and the next month at 350 mg/kg/day but partially restored BSEP expression at the canalicular membrane and significantly improved liver tests and pruritus at a dosage of 500 mg/kg/day. We conclude that 4PB therapy would have a therapeutic effect in patients with progressive familial intrahepatic cholestasis type 2 who retain transport activity of BSEP per se.
Subject(s)
Cholestasis, Intrahepatic/drug therapy , Gastrointestinal Agents/therapeutic use , Phenylbutyrates/therapeutic use , Pruritus/drug therapy , ATP Binding Cassette Transporter, Subfamily B, Member 11 , ATP-Binding Cassette Transporters/genetics , Cholestasis, Intrahepatic/complications , Cholestasis, Intrahepatic/genetics , Female , Genetic Markers , Homozygote , Humans , Infant , Liver Function Tests , Point Mutation , Pruritus/etiologyABSTRACT
PURPOSE: The expression of a multispecific organic anion transporter, OATP1B3/SLCO1B3, is associated with clinical prognosis and survival of cancer cells. The aims of present study were to investigate the involvement of epigenetic regulation in mRNA expression of a cancer-type variant of OATP1B3 (Ct-OATP1B3) in cancer cell lines. METHODS: The membrane localization and transport functions of Ct-OATP1B3 were investigated in HEK293 cells transiently expressing Ct-OATP1B3. DNA methylation profiles around the transcriptional start site of Ct-OATP1B3 in cancer cell lines were determined. The effects of a DNA methyltransferase inhibitor and siRNA knockdown of methyl-DNA binding proteins (MBDs) on the expression of Ct-OATP1B3 mRNA were investigated. RESULTS: 5'-RACE identified the TSS of Ct-OATP1B3 in PK-8 cells. Ct-OATP1B3 was localized on the plasma membrane, and showed the transport activities of E217ßG, fluvastatin, rifampicin, and Gd-EOB-DTPA. The CpG dinucleotides were hypomethylated in Ct-OATP1B3-positive cell lines (DLD-1, TFK-1, PK-8, and PK-45P) but were hypermethylated in Ct-OATP1B3-negative cell lines (HepG2 and Caco-2). Treatment with a DNA methyltransferase inhibitor and siRNA knockdown of MBD2 significantly increased the expression of Ct-OATP1B3 mRNA in HepG2 and Caco-2. CONCLUSIONS: Ct-OATP1B3 is capable of transporting its substrates into cancer cells. Its mRNA expression is regulated by DNA methylation-dependent gene silencing involving MBD2.
Subject(s)
Gene Expression Regulation, Neoplastic , Neoplasms/genetics , Organic Anion Transporters, Sodium-Independent/genetics , Cell Line , Cell Line, Tumor , DNA Methylation , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Epigenesis, Genetic , Humans , Neoplasms/metabolism , Organic Anion Transporters, Sodium-Independent/metabolism , RNA Interference , RNA, Messenger/genetics , Solute Carrier Organic Anion Transporter Family Member 1B3ABSTRACT
Multidrug resistance-associated protein 4 (MRP4/ABCC4) makes a vital contribution to the bodily distribution of drugs and endogenous compounds because of its cellular efflux abilities. However, little is known about the mechanism regulating its cell surface expression. MRP4 has a PDZ-binding motif, which is a potential sequence that modulates the membrane expression of MRP4 via interaction with PDZ adaptor proteins. To investigate this possible relationship, we performed GST pull-down assays and subsequent analysis with matrix-assisted laser desorption/ionization-time of flight mass spectrometry. This method identified sorting nexin 27 (SNX27) as the interacting PDZ adaptor protein with a PDZ-binding motif of MRP4. Its interaction was confirmed by a coimmunoprecipitation study using HEK293 cells. Knockdown of SNX27 by siRNA in HEK293 cells raised MRP4 expression on the plasma membrane, increased the extrusion of 6-[(14)C]mercaptopurine, an MRP4 substrate, and conferred resistance against 6-[(14)C]mercaptopurine. Cell surface biotinylation studies indicated that the inhibition of MRP4 internalization was responsible for these results. Immunocytochemistry and cell surface biotinylation studies using COS-1 cells showed that SNX27 localized to both the early endosome and the plasma membrane. These data suggest that SNX27 interacts with MRP4 near the plasma membrane and promotes endocytosis of MRP4 and thereby negatively regulates its cell surface expression and transport function.
Subject(s)
Cell Membrane/metabolism , Endosomes/metabolism , Gene Expression Regulation/physiology , Multidrug Resistance-Associated Proteins/biosynthesis , Sorting Nexins/metabolism , Amino Acid Motifs , Animals , Biological Transport, Active/physiology , COS Cells , Cell Membrane/genetics , Chlorocebus aethiops , Endosomes/genetics , Gene Knockdown Techniques , HEK293 Cells , HeLa Cells , Hep G2 Cells , Humans , Multidrug Resistance-Associated Proteins/genetics , PDZ Domains , Protein Binding , RNA, Small Interfering , Sorting Nexins/geneticsABSTRACT
UNLABELLED: The bile salt export pump (BSEP) mediates the biliary excretion of bile salts and its dysfunction induces intrahepatic cholestasis. Reduced canalicular expression of BSEP resulting from the promotion of its internalization is one of the causes of this disease state. However, the molecular mechanism underlying BSEP internalization from the canalicular membrane (CM) remains unknown. We have shown previously that 4-phenylbutyrate (4PBA), a drug used for ornithine transcarbamylase deficiency (OTCD), inhibited internalization and subsequent degradation of cell-surface-resident BSEP. The current study found that 4PBA treatment decreased significantly the expression of α- and µ2-adaptin, both of which are subunits of the AP2 adaptor complex (AP2) that mediates clathrin-dependent endocytosis, in liver specimens from rats and patients with OTCD, and that BSEP has potential AP2 recognition motifs in its cytosolic region. Based on this, the role of AP2 in BSEP internalization was explored further. In vitro analysis with 3×FLAG-human BSEP-expressing HeLa cells and human sandwich-culture hepatocytes indicates that the impairment of AP2 function by RNA interference targeting of α-adaptin inhibits BSEP internalization from the plasma membrane and increases its cell-surface expression and transport function. Studies using immunostaining, coimmunoprecipitation, glutathione S-transferase pulldown assay, and time-lapse imaging show that AP2 interacts with BSEP at the CM through a tyrosine motif at the carboxyl terminus of BSEP and mediates BSEP internalization from the CM of hepatocytes. CONCLUSION: AP2 mediates the internalization and subsequent degradation of CM-resident BSEP through direct interaction with BSEP and thereby modulates the canalicular expression and transport function of BSEP. This information should be useful for understanding the pathogenesis of severe liver diseases associated with intrahepatic cholestasis.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Adaptor Protein Complex 2/physiology , Bile Canaliculi/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 11 , Adaptor Protein Complex alpha Subunits/genetics , Adaptor Protein Complex alpha Subunits/physiology , Animals , Biological Transport , Cell Polarity , HeLa Cells , Humans , Male , Phenylbutyrates/pharmacology , Rats , Rats, Sprague-Dawley , UbiquitinationABSTRACT
UNLABELLED: ATP-binding cassette transporter A1 (ABCA1) plays an essential role in the biogenesis of high-density lipoprotein in liver and in the prevention of foam cell formation in macrophages by mediating the efflux of cellular cholesterol and phospholipids to apolipoprotein A-I (apoA-I). Our current study investigated the mechanism of degradation of cell surface-resident ABCA1, focusing on ubiquitination. A coimmunoprecipitation study indicated the presence of ubiquitinated ABCA1 in the plasma membrane of the human hepatoma cell line, HuH-7, of cells from mouse liver, and of macrophages differentiated from the human acute monocytic leukemia cell line, THP-1 (THP-1 macrophages). In HuH-7 cells, degradation of cell surface-resident ABCA1 was inhibited by the overexpression of a dominant-negative form of ubiquitin. Moreover, the disruption of the endosomal sorting complex required for transport (ESCRT) pathway, a dominant mechanism for ubiquitination-mediated lysosomal degradation, by the knockdown of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), significantly delayed the degradation of cell surface-resident ABCA1. This was accompanied by an increase in ABCA1 expression as well as in apoA-I-mediated [3H]-cholesterol efflux function. The effect of HRS knockdown was also observed after calpain inhibitor treatment, which is reported to retard ABCA1 degradation. The induction of ABCA1 by HRS knockdown was confirmed in THP-1 macrophages. CONCLUSION: Together with the fact that lysosomal inhibitor treatments increased ABCA1 expression in HuH-7 and THP-1 macrophages, these results suggest that ubiquitination mediates the lysosomal degradation of cell surface-resident ABCA1 through the ESCRT pathway, and thereby controls the expression and cholesterol efflux function of ABCA1. This mechanism seems to mediate ABCA1 degradation independently of the calpain-involving pathway. The modulation of ABCA1 ubiquitination could thus be a potential new therapeutic target for antiatherogenic drugs.