Multimodal Imaging Technology Effectively Monitors HER2 Expression in Tumors Using Trastuzumab-Coupled Organic Nanoparticles in Patient-Derived Xenograft Mice Models

Trastuzumab is a monoclonal antibody targeting human epidermal growth factor 2 (HER2), which has been successfully used in the treatment of patients with breast cancer and gastric cancer; however, problems concerning its cardiotoxicity, drug resistance, and unpredictable efficacy still remain. Herein, we constructed novel organic dopamine–melanin nanoparticles (dMNs) as a carrier and then surface-loaded them with trastuzumab to construct a multifunctional nanoprobe named Her-PEG-dMNPs. We used micro-PET/CT and PET/MRI multimodality imaging to evaluate the retention effect of the nanoprobe in HER2 expression in gastric cancer patient-derived xenograft (PDX) mice models after labeling of the radionuclides 64Cu or 124I and MRI contrast agent Mn2+. The nanoprobes can specifically target the HER2-expressing SKOV-3 cells in vitro (3.61 ± 0.74 vs. 1.24 ± 0.43 for 2 h, P = 0.002). In vivo, micro-PET/CT and PET/MRI showed that the 124I-labeled nanoprobe had greater contrast and retention effect in PDX models than unloaded dMNPs as carrier (1.63 ± 0.07 vs. 0.90 ± 0.04 at 24 h, P = 0.002), a similarity found in 64Cu-labeled Her-PEG-dMNPs. Because 124I has a longer half-life and matches the pharmacokinetics of the nanoparticles, we focused on the further evaluation of 124I-Her-PEG-dMNPs. Furthermore, immunohistochemistry staining confirmed the overexpression of HER2 in the animal model. This study developed and validated novel HER2-specific multimodality imaging nanoprobes for quantifying HER2 expression in mice. Through the strong retention effect of the tumor site, it can be used for the promotion of monoclonal antibody treatment effect and process monitoring.

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EB10, an Anti-FLT3 Monoclonal Antibody, Selectively Targets ALL Cell Lines and Primary ALL Blasts without Interfering with Normal Hematopoiesis.

Blood ◽

10.1182/blood.v104.11.2744.2744 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2744-2744 ◽

Cited By ~ 1

Author(s):

Obdulio Piloto ◽

Patrick Brown ◽

Li Li ◽

Bao Nguyen ◽

Kyu-Tae Kim ◽

...

Keyword(s):

Monoclonal Antibody ◽

Cell Lines ◽

Scid Mice ◽

Cytotoxic Agents ◽

Radioactive Isotopes ◽

Leukemic Cells ◽

Class Iii ◽

Antibody Treatment

Abstract The class III receptor tyrosine kinase, FLT3, is expressed by >90% of B-lineage acute lymphoblastic leukemias (ALL) blasts. In addition, it is expressed at extremely high levels in ALL patients with MLL-rearrangements or hyperdiploidy and sometimes mutated in these same patients. In this report, we investigated the effects of EB10, an anti-human FLT3 monoclonal antibody capable of preventing binding of FLT3 ligand (FL), on ALL cell lines and primary cells. In vitro studies, examining the ability of EB10 to inhibit FLT3 activation and downstream signaling in ALL cell lines and primary blasts, yielded variable results. In some cell lines FLT3 phosphorylation was inhibited and with it, downstream activation of pathways involving MAPK, AKT, and STAT5 phosphorylation. However, several cell lines actually exhibited FLT3 activation upon antibody treatment, possibly because of antibody-mediated receptor dimerization, and subsequent activation of downstream pathways. Nevertheless, through antibody-mediated cellular cytotoxicity (ADCC) such an antibody could still prove efficacious against leukemia cells in vivo. In fact, EB10 treatment significantly prolongs survival and/or reduces engraftment of ALL cell lines and primary ALL blasts in NOD/SCID mice. This effect might be even more pronounced in a host that was less immune compromised than are NOD/SCID mice. The leukemic cells surviving EB10 treatment in the mice were characterized by FACS analysis and found to express low levels or no FLT3. In contrast to the reduction in engraftment of human ALL primary blasts, EB10 treatment of NOD/SCID mice did not reduce engraftment of human hematopoietic CD34+ cells. Taken together, these data demonstrate that EB10 is selectively cytotoxic to ALL blasts while having little effect on normal hematopoiesis. Such an antibody, either naked or conjugated to radioactive isotopes or cytotoxic agents, may prove useful in the therapy of infant ALL as well as childhood and adult ALL patients whose blasts typically express FLT3.

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Defucosylated Mouse–Dog Chimeric Anti-EGFR Antibody Exerts Antitumor Activities in Mouse Xenograft Models of Canine Tumors

Cells ◽

10.3390/cells10123599 ◽

2021 ◽

Vol 10 (12) ◽

pp. 3599

Author(s):

Guanjie Li ◽

Tomokazu Ohishi ◽

Mika K. Kaneko ◽

Junko Takei ◽

Takuya Mizuno ◽

...

Keyword(s):

Monoclonal Antibody ◽

Cell Line ◽

Growth Factor Receptor ◽

High Sensitivity ◽

Osteosarcoma Cell ◽

Antibody Treatment ◽

Antitumor Effects ◽

Vitro Analysis

The epidermal growth factor receptor (EGFR) contributes to tumor malignancy via gene amplification and protein overexpression. Previously, we developed an anti-human EGFR (hEGFR) monoclonal antibody, namely EMab-134, which detects hEGFR and dog EGFR (dEGFR) with high sensitivity and specificity. In this study, we produced a defucosylated mouse–dog chimeric anti-EGFR monoclonal antibody, namely E134Bf. In vitro analysis revealed that E134Bf highly exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against a canine osteosarcoma cell line (D-17) and a canine fibroblastic cell line (A-72), both of which express endogenous dEGFR. Moreover, in vivo administration of E134Bf significantly suppressed the development of D-17 and A-72 compared with the control dog IgG in mouse xenografts. These results indicate that E134Bf exerts antitumor effects against dEGFR-expressing canine cancers and could be valuable as part of an antibody treatment regimen for dogs.

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68Ga-Labeled GX1 Dimer: A Novel Probe for PET/Cerenkov Imaging Targeting Gastric Cancer

Frontiers in Oncology ◽

10.3389/fonc.2021.750376 ◽

2021 ◽

Vol 11 ◽

Author(s):

Jipeng Yin ◽

Bo Xin ◽

Mingru Zhang ◽

Xiaoli Hui ◽

Na Chai ◽

...

Keyword(s):

Gastric Cancer ◽

Nude Mice ◽

Competitive Inhibition ◽

Standardized Uptake Value ◽

Cell Uptake ◽

Tumor Site ◽

Pet Ct ◽

Better Than

PurposeTo synthesize the dimer of GX1 and identify whether its affinity and targeting are better than those of GX1. To prepare 68Ga-DOTA-KEK-(GX1)2 and to apply it to PET and Cerenkov imaging of gastric cancer.Methods68Ga-DOTA-KEK-(GX1)2 was prepared, and the labeling yield and stability were determined. Its specificity and affinity were verified using an in vitro cell binding assay and competitive inhibition test, cell immunofluorescence, and cell uptake and efflux study. Its tumor-targeting ability was determined by nano PET/CT and Cerenkov imaging, standardized uptake value (SUV), signal-to-background ratio (SBR) quantification, and a biodistribution study in tumor-bearing nude mice.Results68Ga-DOTA-KEK-(GX1)2 was successfully prepared, and the labeling yield was more than 97%. It existed stably for 90 min in serum. The binding of 68Ga-DOTA-KEK-(GX1)2 to cocultured HUVECs (Co-HUVECs) was higher than that to human umbilical vein endothelial cells (HUVECs), BGC823 cells, and GES cells. It was also higher than that of 68Ga-DOTA-GX1, indicating that the dimer did improve the specificity and affinity of GX1. The binding of KEK-(GX1)2 to Co-HUVECs was significantly higher than that of GX1. Additionally, the uptake of 68Ga-DOTA-KEK-(GX1)2 by Co-HUVECs was higher than that of 68Ga-DOTA-GX1 and reached a maximum at 60 min. Nano PET/CT and Cerenkov imaging showed that the tumor imaging of the nude mice injected with 68Ga-DOTA-KEK-(GX1)2 was clear, and the SUV and SBR value of the tumor sites were significantly higher than those of the nude mice injected with 68Ga-DOTA-GX1, indicating that the probe had better targeting in vivo. Finally, the biodistribution showed quantitatively that when organs such as the kidney and liver metabolized rapidly, the radioactivity of the tumor site of the nude mice injected with 68Ga-DOTA-KEK-(GX1)2 decreased relatively slowly. At the same time, the percentage of injected dose per gram (%ID/g) of the tumor site was higher than that of other normal organs except the liver and kidney at 60 min, which indicated that the tumor had good absorption of the probe.ConclusionGX1 was modified successfully, and the in vivo and in vitro properties of the GX1 dimer were significantly better than those of GX1. The imaging probe, 68Ga-DOTA-KEK-(GX1)2, was successfully prepared, which provides a candidate probe for PET and Cerenkov diagnosis of gastric cancer.

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Genomic ERBB2/ERBB3 mutations promote PD-L1-mediated immune escape in gallbladder cancer: a whole-exome sequencing analysis

Gut ◽

10.1136/gutjnl-2018-316039 ◽

2018 ◽

Vol 68 (6) ◽

pp. 1024-1033 ◽

Cited By ~ 23

Author(s):

Maolan Li ◽

Fatao Liu ◽

Fei Zhang ◽

Weiping Zhou ◽

Xiaoqing Jiang ◽

...

Keyword(s):

Monoclonal Antibody ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Ectopic Expression ◽

Sequencing Analysis ◽

Antibody Treatment ◽

Whole Exome ◽

Recurrent Mutations

ObjectivesPatients with gallbladder carcinoma (GBC) lack effective treatment methods largely due to the inadequacy of both molecular characterisation and potential therapeutic targets. We previously uncovered a spectrum of genomic alterations and identified recurrent mutations in the ErbB pathway in GBC. Here, we aimed to study recurrent mutations of genes and pathways in a larger cohort of patients with GBC and investigate the potential mechanisms and clinical significance of these mutations.DesignWe performed whole-exome sequencing (WES) in 157 patients with GBC. Functional experiments were applied in GBC cell lines to explore the oncogenic roles of ERBB2/ERBB3 hotspot mutations, their correlation with PD-L1 expression and the underlying mechanisms. ERBB inhibitors and a PD-L1 blocker were used to evaluate the anticancer activities in co-culture systems in vitro and in vivo.ResultsWES identified ERBB2 and ERBB3 mutations at a frequency of 7%–8% in the expanded cohort, and patients with ERBB2/ERBB3 mutations exhibited poorer prognoses. A set of in vitro and in vivo experiments revealed increased proliferation/migration on ERBB2/ERBB3 mutation. Ectopic expression of ERBB2/ERBB3 mutants upregulated PD-L1 expression in GBC cells, effectively suppressed normal T-cell-mediated cytotoxicity in vitro through activation of the PI3K/Akt signalling pathway and contributed to the growth and progression of GBC in vivo. Treatment with an ERBB2/ERBB3 inhibitor or a PD-L1 monoclonal antibody reversed these immunosuppressive effects, and combined therapy revealed promising therapeutic activities.ConclusionsERBB2/ERBB3 mutations may serve as useful biomarkers in identifying patients who are sensitive to ERBB2/ERBB3 inhibitors and PD-L1 monoclonal antibody treatment.Trial registration numberNCT02442414;Pre-results.

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From AVATAR Mice to Patients: RC48-ADC Exerted Promising Efficacy in Advanced Gastric Cancer With HER2 Expression

Frontiers in Pharmacology ◽

10.3389/fphar.2021.757994 ◽

2022 ◽

Vol 12 ◽

Author(s):

Zuhua Chen ◽

Jiajia Yuan ◽

Yingying Xu ◽

Cheng Zhang ◽

Zhongwu Li ◽

...

Keyword(s):

Gastric Cancer ◽

Antitumor Activity ◽

Antitumor Effect ◽

Dependent Manner ◽

Her2 Expression ◽

Preclinical Research ◽

Her2 Positive ◽

Pdx Models

RC48-ADC is a novel humanized antibody specific for human epidermal growth factor receptor 2 (HER2)in conjugation with a microtubule inhibitor via a cleavable linker. This study was to evaluate the antitumor activity and mechanism of RC48-ADC in gastric cancer (GC) and explore the population that may benefit from RC48-ADC treatment. Four human GC cell lines and nine patient-derived xenograft (PDX) models were exploited to evaluate the antitumor effect of RC48-ADC or trastuzumab treatment in vitro and in vivo. The expression and phosphorylation of HER2 were assessed by immunohistochemistry (IHC) staining. Critical molecules of downstream PI3K/AKT and cell cycle and apoptosis signaling pathways were detected and quantified by immunoblotting. Combined with preliminary results of preclinical research, three patients with IHC3+, IHC2+/FISH+, and IHC2+/FISH- of HER2 were enrolled to verify the efficacy of RC48-ADC treatment in advanced GC. In vitro, RC48-ADC had superior antiproliferative effects in a dose-dependent manner on GC cells, especially on HER2-positive cells. In vivo, RC48-ADC exceeded trastuzumab in GC PDX models with HER2 expression, even in models with moderate to low expression of HER2. Further exploration of mechanism showed that RC48-ADC exerted the antitumor effect by inhibiting phosphorylation of HER2, inducing G2/M phase arrest and cell apoptosis in HER2-expressed PDX models. In clinical practice, RC48-ADC had satisfactory efficacy in HER2-positive and HER2 moderately expressed GC patients and demonstrated promising efficacy in HER2-positive patients who have progressed after anti-HER2 therapy. In conclusion, RC48-ADC exerted promising antitumor activity in HER2-positive as well as score of 2+ in IHC and ISH-negative AGC patients after progression of systematic treatment.

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The Anticoagulant Properties of a Modified Form of Protein S

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647048 ◽

1988 ◽

Vol 60 (02) ◽

pp. 298-304 ◽

Cited By ~ 17

Author(s):

C A Mitchell ◽

S M Kelemen ◽

H H Salem

Keyword(s):

Monoclonal Antibody ◽

Anticoagulant Activity ◽

Activated Protein C ◽

Factor Xa ◽

Protein S ◽

Human Neutrophil Elastase ◽

Factor X ◽

Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

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Interrogation of gender disparity uncovers androgen receptor as the transcriptional activator for oncogenic miR-125b in gastric cancer

Cell Death and Disease ◽

10.1038/s41419-021-03727-3 ◽

2021 ◽

Vol 12 (5) ◽

Author(s):

Ben Liu ◽

Meng Zhou ◽

Xiangchun Li ◽

Xining Zhang ◽

Qinghua Wang ◽

...

Keyword(s):

Gastric Cancer ◽

Androgen Receptor ◽

Signaling Pathway ◽

Gender Bias ◽

Gender Disparity ◽

Gene Set Enrichment Analysis ◽

Cancer Type ◽

Female Patients

AbstractThere is a male preponderance in gastric cancer (GC), which suggests a role of androgen and androgen receptor (AR). However, the mechanism of AR signaling in GC especially in female patients remains obscure. We sought to identify the AR signaling pathway that might be related to prognosis and examine the potential clinical utility of the AR antagonist for treatment. Deep learning and gene set enrichment analysis was used to identify potential critical factors associated with gender bias in GC (n = 1390). Gene expression profile analysis was performed to screen differentially expressed genes associated with AR expression in the Tianjin discovery set (n = 90) and TCGA validation set (n = 341). Predictors of survival were identified via lasso regression analyses and validated in the expanded Tianjin cohort (n = 373). In vitro and in vivo experiments were established to determine the drug effect. The GC gender bias was attributable to sex chromosome abnormalities and AR signaling dysregulation. The candidates for AR-related gene sets were screened, and AR combined with miR-125b was associated with poor prognosis, particularly among female patients. AR was confirmed to directly regulate miR-125b expression. AR-miR-125b signaling pathway inhibited apoptosis and promoted proliferation. AR antagonist, bicalutamide, exerted anti-tumor activities and induced apoptosis both in vitro and in vivo, using GC cell lines and female patient-derived xenograft (PDX) model. We have shed light on gender differences by revealing a hormone-regulated oncogenic signaling pathway in GC. Our preclinical studies suggest that AR is a potential therapeutic target for this deadly cancer type, especially in female patients.

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LncRNA UCA1 promotes development of gastric cancer via the miR-145/MYO6 axis

Cellular & Molecular Biology Letters ◽

10.1186/s11658-021-00275-8 ◽

2021 ◽

Vol 26 (1) ◽

Author(s):

An Yang ◽

Xin Liu ◽

Ping Liu ◽

Yunzhang Feng ◽

Hongbo Liu ◽

...

Keyword(s):

Gastric Cancer ◽

Cell Proliferation ◽

Cancer Cells ◽

Cell Apoptosis ◽

Gastric Cancer Cell ◽

Gastric Cancer Cells ◽

Gastric Cancer Cell Lines ◽

Development Of Gastric Cancer

Abstract Background Long noncoding RNA (lncRNA), urothelial carcinoma-associated 1 (UCA1) is aberrantly expressed in multiple cancers and has been verified as an oncogene. However, the underlying mechanism of UCA1 in the development of gastric cancer is not fully understood. In the present study, we aimed to identify how UCA1 promotes gastric cancer development. Methods The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) data were used to analyze UCA1 and myosin VI (MYO6) expression in gastric cancer. Western blot and quantitative real-time PCR (QPCR) were performed to test the expression level of the UCA1/miR-145/MYO6 axis in gastric cancer cell lines and tissues. The roles of the UCA1/miR-145/MYO6 axis in gastric cancer in vitro and in vivo were investigated by CCK-8 assay, flow cytometry, siRNAs, immunohistochemistry, and a mouse xenograft model. The targeted relationship among UCA1, miR-145, and MYO6 was predicted using LncBase Predicted v.2 and TargetScan online software, and then verified by luciferase activity assay and RNA immunoprecipitation. Results UCA1 expression was higher but miR-145 expression was lower in gastric cancer cell lines or tissues, compared to the adjacent normal cell line or normal tissues. Function analysis verified that UCA1 promoted cell proliferation and inhibited cell apoptosis in the gastric cancer cells in vitro and in vivo. Mechanistically, UCA1 could bind directly to miR-145, and MYO6 was found to be a downstream target gene of miR-145. miR-145 mimics or MYO6 siRNAs could partly reverse the effect of UCA1 on gastric cancer cells. Conclusions UCA1 accelerated cell proliferation and inhibited cell apoptosis through sponging miR-145 to upregulate MYO6 expression in gastric cancer, indicating that the UCA1/miR-145/MYO6 axis may serve as a potential therapeutic target for gastric cancer.

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A Novel Mouse Monoclonal Antibody C42 against C-Terminal Peptide of Alpha-1-Antitrypsin

International Journal of Molecular Sciences ◽

10.3390/ijms22042141 ◽

2021 ◽

Vol 22 (4) ◽

pp. 2141

Author(s):

Srinu Tumpara ◽

Elena Korenbaum ◽

Mark Kühnel ◽

Danny Jonigk ◽

Beata Olejnicka ◽

...

Keyword(s):

Monoclonal Antibody ◽

Flow Cytometry ◽

Western Blotting ◽

Complex Mixture ◽

Immunoglobulin M ◽

Confocal Laser ◽

Enzyme Linked Immunosorbent Assay ◽

Dot Blot

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359–394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1–0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.

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Activation of MAT2A-RIP1 signaling axis reprograms monocytes in gastric cancer

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-001364 ◽

2021 ◽

Vol 9 (2) ◽

pp. e001364

Author(s):

Yan Zhang ◽

Hui Yang ◽

Jun Zhao ◽

Ping Wan ◽

Ye Hu ◽

...

Keyword(s):

Gastric Cancer ◽

Cell Metabolism ◽

Vital Role ◽

Methionine Metabolism ◽

Promoter Regions ◽

Normal Tissues ◽

Methionine Cycle

BackgroundThe activation of tumor-associated macrophages (TAMs) facilitates the progression of gastric cancer (GC). Cell metabolism reprogramming has been shown to play a vital role in the polarization of TAMs. However, the role of methionine metabolism in function of TAMs remains to be explored.MethodsMonocytes/macrophages were isolated from peripheral blood, tumor tissues or normal tissues from healthy donors or patients with GC. The role of methionine metabolism in the activation of TAMs was evaluated with both in vivo analyses and in vitro experiments. Pharmacological inhibition of the methionine cycle and modulation of key metabolic genes was employed, where molecular and biological analyses were performed.ResultsTAMs have increased methionine cycle activity that are mainly attributed to elevated methionine adenosyltransferase II alpha (MAT2A) levels. MAT2A modulates the activation and maintenance of the phenotype of TAMs and mediates the upregulation of RIP1 by increasing the histone H3K4 methylation (H3K4me3) at its promoter regions.ConclusionsOur data cast light on a novel mechanism by which methionine metabolism regulates the anti-inflammatory functions of monocytes in GC. MAT2A might be a potential therapeutic target for cancer cells as well as TAMs in GC.

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