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Dissection of ?4?7 integrin regulation by Rap1 using novel conformation-specific monoclonal anti-?7 antibodies.

ABSTRACT: Integrin activation is associated with conformational regulation. In this study, we developed a system to evaluate conformational changes in ?4?7 integrin. We first inserted the PA tag into the plexin-semaphorin-integrin (PSI) domain of ?7 chain, which reacted with an anti-PA tag antibody (NZ-1) in an Mn2+-dependent manner. The small GTPase Rap1 deficiency, as well as chemokine stimulation and the introduction of the active form of Rap1, Rap1V12, enhanced the binding of NZ-1 to the PA-tagged mutant integrin, and increased the binding affinity to mucosal addressing cell adhesion molecule-1 (MAdCAM-1). Furthermore, we generated two kinds of hybridomas producing monoclonal antibodies (mAbs) that recognized Mn2+-dependent epitopes of ?7. Both epitopes were exposed to bind to mAbs on the cells by the introduction of Rap1V12. Although one epitope in the PSI domain of ?7 was exposed on Rap1-deficienct cells, the other epitope in the hybrid domain of ?7 was not. These data indicate that the conversion of Rap1-GDP to GTP exerts two distinct effects stepwise on the conformation of ?4?7. The induction of colitis by Rap1-deficient CD4+ effector/memory T cells suggests that the removal of constraining effect by Rap1-GDP on ?4?7 is sufficient for homing of these pathogenic T cells into colon lamina propria (LP).

SUBMITTER: Sato T

PROVIDER: S-EPMC7413538 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Dissection of α4β7 integrin regulation by Rap1 using novel conformation-specific monoclonal anti-β7 antibodies.

Sato Tsuyoshi T Ishihara Sayaka S Marui Ryoya R Takagi Junichi J Katagiri Koko K

Scientific reports 20200806 1

Integrin activation is associated with conformational regulation. In this study, we developed a system to evaluate conformational changes in α4β7 integrin. We first inserted the PA tag into the plexin-semaphorin-integrin (PSI) domain of β7 chain, which reacted with an anti-PA tag antibody (NZ-1) in an Mn2+-dependent manner. The small GTPase Rap1 deficiency, as well as chemokine stimulation and the introduction of the active form of Rap1, Rap1V12, enhan ...[more]

PMID: 32764635

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Project description:BackgroundOne-third of breast cancers display amplifications of the ERBB2 gene encoding the HER2 kinase receptor. Trastuzumab, a humanized antibody directed against an epitope on subdomain IV of the extracellular domain of HER2 is used for therapy of HER2-overexpressing mammary tumors. However, many tumors are either natively resistant or acquire resistance against Trastuzumab. Antibodies directed to different epitopes on the extracellular domain of HER2 are promising candidates for replacement or combinatorial therapy. For example, Pertuzumab that binds to subdomain II of HER2 extracellular domain and inhibits receptor dimerization is under clinical trial. Alternative antibodies directed to novel HER2 epitopes may serve as additional tools for breast cancer therapy. Our aim was to generate novel anti-HER2 monoclonal antibodies inhibiting the growth of breast cancer cells, either alone or in combination with tumor necrosis factor-? (TNF-?).MethodsMice were immunized against SK-BR-3 cells and recombinant HER2 extracellular domain protein to produce monoclonal antibodies. Anti-HER2 antibodies were characterized with breast cancer cell lines using immunofluorescence, flow cytometry, immunoprecipitation, western blot techniques. Antibody epitopes were localized using plasmids encoding recombinant HER2 protein variants. Antibodies, either alone or in combination with TNF-?, were tested for their effects on breast cancer cell proliferation.ResultsWe produced five new anti-HER2 monoclonal antibodies, all directed against conformational epitope or epitopes restricted to the native form of the extracellular domain. When tested alone, some antibodies inhibited modestly but significantly the growth of SK-BR-3, BT-474 and MDA-MB-361 cells displaying ERBB2 amplification. They had no detectable effect on MCF-7 and T47D cells lacking ERBB2 amplification. When tested in combination with TNF-?, antibodies acted synergistically on SK-BR-3 cells, but antagonistically on BT-474 cells. A representative anti-HER2 antibody inhibited Akt and ERK1/2 phosphorylation leading to cyclin D1 accumulation and growth arrest in SK-BR-3 cells, independently from TNF-?.ConclusionsNovel antibodies against extracellular domain of HER2 may serve as potent anti-cancer bioactive molecules. Cell-dependent synergy and antagonism between anti-HER2 antibodies and TNF-? provide evidence for a complex interplay between HER2 and TNF-? signaling pathways. Such complexity may drastically affect the outcome of HER2-directed therapeutic interventions.

Dataset Information

Dissection of ?4?7 integrin regulation by Rap1 using novel conformation-specific monoclonal anti-?7 antibodies.

Publications

Dissection of α<sub>4</sub>β<sub>7</sub> integrin regulation by Rap1 using novel conformation-specific monoclonal anti-β<sub>7</sub> antibodies.

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