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A human antibody against human endothelin receptor type A that exhibits antitumor potency.

ABSTRACT: Endothelin receptor A (ET_A), a class A G-protein-coupled receptor (GPCR), is involved in the progression and metastasis of colorectal, breast, lung, ovarian, and prostate cancer. We overexpressed and purified human endothelin receptor type A in Escherichia coli and reconstituted it with lipid and membrane scaffold proteins to prepare an ET_A nanodisc as a functional antigen with a structure similar to that of native GPCR. By screening a human naive immune single-chain variable fragment phage library constructed in-house, we successfully isolated a human anti-ET_A antibody (AG8) exhibiting high specificity for ET_A in the β-arrestin Tango assay and effective inhibitory activity against the ET-1-induced signaling cascade via ET_A using either a CHO-K1 cell line stably expressing human ET_A or HT-29 colorectal cancer cells, in which AG8 exhibited IC₅₀ values of 56 and 51 nM, respectively. In addition, AG8 treatment repressed the transcription of inhibin βA and reduced the ET_A-induced phosphorylation of protein kinase B and extracellular regulated kinase. Furthermore, tumor growth was effectively inhibited by AG8 in a colorectal cancer mouse xenograft model. The human anti-ET_A antibody isolated in this study could be used as a potential therapeutic for cancers, including colorectal cancer.

SUBMITTER: Ju MS

PROVIDER: S-EPMC8492878 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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A human antibody against human endothelin receptor type A that exhibits antitumor potency.

Ju Man-Seok MS Ahn Hye-Mi HM Han Seong-Gu SG Ko Sanghwan S Na Jung-Hyun JH Jo Migyeong M Lim Chung Su CS Ko Byoung Joon BJ Yu Yeon Gyu YG Lee Won-Kyu WK Kim Youn-Jae YJ Jung Sang Taek ST

Experimental & molecular medicine 20210929 9

Endothelin receptor A (ET<sub>A</sub>), a class A G-protein-coupled receptor (GPCR), is involved in the progression and metastasis of colorectal, breast, lung, ovarian, and prostate cancer. We overexpressed and purified human endothelin receptor type A in Escherichia coli and reconstituted it with lipid and membrane scaffold proteins to prepare an ET<sub>A</sub> nanodisc as a functional antigen with a structure similar to that of native GPCR. By screening a human naive immune single-chain variab ...[more]

PMID: 34588605

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Project description:BackgroundIncreased titers of autoantibodies targeting the G-protein-coupled receptors angiotensin II type 1 receptor (AT1R) and endotelin-1 type A receptor (ETAR) are associated with severe coronavirus disease 2019 (COVID-19) infection. The aim of this study was to determine whether 1) these antibodies are specifically related to COVID-19 disease pathogenesis or increased during any severe respiratory illness, 2) if they are formed during illness, and 3) if they correlate with inflammatory markers or long-term symptoms.MethodsAntibodies against AT1R, ETAR, and antinuclear antibodies (ANAs) were measured in n=40 prospectively enrolled COVID-19 patients and n=207 COVID-19 patients included in a biobank. Clinical and laboratory findings were prospectively and retrospectively assessed in both cohorts, and results were combined for analysis. The presence of auto-antibodies against AT1R or ETAR in peripheral blood was compared between hospitalized patients with COVID-19 and controls (n=39). Additionally, AT1R and ETAR titers were compared between patients with an unfavorable disease course, defined as intensive care admission and/or death during hospital admission (n=121), to those with a favorable disease course (n=126). A subset of intubated patients with severe COVID-19 were compared to intubated patients with acute respiratory distress syndrome (ARDS) due to any other cause.ResultsSignificantly increased AT1R and ETAR antibody titers were found in COVID-19 patients compared to controls, while titers were equal between favorable and unfavorable COVID-19 disease course groups. On ICU, intubated patients with COVID-19 had significantly increased AT1R and ETAR titers compared to patients with ARDS due to any other cause. The titers did not correlate with baseline inflammatory markers during admission or with diffusion capacity, cognitive impairment, or fatigue measured at 3 months follow-up.ConclusionsIn patients hospitalized for COVID-19, antibodies against AT1R and ETAR are increased compared to controls and patients with ARDS due to other causes than COVID-19. The baseline antibody titers do not correlate with inflammatory markers or long-term symptoms in this study.

Project description:Study questionDoes endothelin-1 (ET-1) regulate matrix metalloproteinase (MMP) 14 and 15 production and invasion of human first trimester trophoblasts?Summary answerET-1 in pathophysiological concentrations down-regulates MMP14 and MMP15 expression via endothelin receptor (ETR) type B and decreases trophoblast migration and invasion.What is known alreadyMMP14 and MMP15 are involved in trophoblast invasion. Impairment of invasion has been linked to pregnancy complications such as pre-eclampsia (PE). ET-1 is up-regulated in PE.Study design, size, durationIn vitro study using primary human trophoblasts from 50 first trimester placentas (gestational week 7-12).Participants/materials, setting, methodsTrophoblasts were cultured in the absence or presence of 10-100 nM ET-1. MMP14 and MMP15 mRNA and protein were quantified by RT-qPCR and Western blotting, respectively. Selective antagonists for ETRA (BQ-123) or ETRB (BQ-788) were used to identify ETR subtypes involved. Functional ET-1 effects were tested in first trimester chorionic villous explants and transwell invasion assays. The roles of tumor necrosis factor (TNF)-α (25 ng/ml) and oxygen (1%) in ET-1 regulation of MMP14 and 15 expression were assessed by Western blotting.Main results and the role of chanceET-1 down-regulated MMP14 and MMP15 mRNA (-21% and -26%, respectively, P < 0.05) and protein levels (-18% and -22%, respectively, P < 0.05). This effect was mediated via ETRB. ET-1 decreased trophoblast outgrowth in placental explants (-24%, P < 0.05) and trophoblast invasion (-26%, P ≤ 0.01). TNF-α enhanced ET-1 mediated MMP15 down-regulation (by 10%, P < 0.05), whereas hypoxia abolished the effect of ET-1 on both MMPs.Large scale dataN/A.Limitations, reasons for cautionOnly primary trophoblasts were used in this study. Since trophoblast yield from first trimester placental material is limited, further aspects of MMP14 and 15 regulation could not be characterized. Other anti-invasive factors may be altered by ET-1 in trophoblasts and, thus, contribute to the reduced invasion, but have not been investigated. Oxygen levels similar to those found in the decidua (5-8% O2) were not analyzed in this study.Wider implications of the findingsET-1 modifies placental function already during the first trimester of pregnancy, the time-window when the placental changes implicated in PE occur. Thus, our results improve the understanding of the placental mechanisms underlying trophoblast invasion and PE.Study funding/competing interestsThe study was funded by the Oesterreichische Nationalbank (Anniversary Fund, project number: 14796) and the Herzfelder'sche Familienstiftung (to J.P.; number: 00685). AMM received funding from the Austrian Science Fund FWF (W1241) and the Medical University Graz through the PhD Program Molecular Fundamentals of Inflammation (DK-MOLIN). The authors have no conflict of interest.

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A human antibody against human endothelin receptor type A that exhibits antitumor potency.

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A human antibody against human endothelin receptor type A that exhibits antitumor potency.

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