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1.
Int J Mol Sci ; 22(12)2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34207911

RESUMO

Phage display technology is a widely used practical tool for isolating binding molecules against the desired targets in phage libraries. In the case of targeting the membrane protein with its natural conformation, conventional bio-panning has limitations on the efficient screening of the functionally relevant antibodies. To enrich the single-chain variable fragment (scFv) pools for recognizing the natural conformation of the membrane targets, the conventional bio-panning and screening process was modified to include the semi-automated cell panning protocol. Using FGFR3-overexpressing patient-derived cancer cells, biotin-X-DHPE was introduced and coupled to Streptavidin-coated magnetic beads for use in the solution-phage bio-panning procedure. The resulting clones of scFv were compared to the diversity of the binding region, especially on CDR-H3. The clones enriched further by cell-based panning procedure possessed a similar binding site and the CDR-H3 loop structure. The resulting antibodies inhibited cell growth and induced target degradation. This process may be a useful tool for screening biologically related antibodies that recognize natural conformational structure on cell membrane protein. Furthermore, cell-based panning has the potential to further expand to a high-throughput screening (HTS) system and automation process.


Assuntos
Antineoplásicos Imunológicos/química , Automação Laboratorial , Técnicas de Cultura de Células , Neoplasias/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Anticorpos de Cadeia Única/química , Humanos , Neoplasias/patologia , Células Tumorais Cultivadas
2.
ACS Omega ; 5(40): 25798-25809, 2020 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-33073104

RESUMO

c-Met, as a receptor expressed on the cell membrane, contributes to the growth and metastasis of tumors, as well as angiogenesis, mainly through the hepatocyte growth factor (HGF)/c-Met axis during tumor progression. Although several c-Met inhibitors, including small molecules and monoclonal antibody inhibitors, are currently being investigated, their clinical outcomes have not been promising. Development of an antibody-drug conjugate (ADC) against c-Met could be an attractive therapeutic strategy that would provide superior antitumor efficacy with broad-spectrum c-Met expression levels. In the present study, site-specific drug-conjugate technology was applied to develop an ADC using the human-mouse cross-reactive c-Met antibody and a prodrug pyrrolobenzodiazepine (PBD). The toxin payload was uniformly conjugated to the light-chain C-terminus of the native cIRCR201 antibody (drug-to-antibody ratio = 2), as confirmed using LC-MS. Using a high-throughput screening system, we found that cIRCR201-dPBD exhibited varying sensitivities depending on the expression levels of c-Met, and it induced receptor-mediated endocytosis and toxin-mediated apoptosis in 47 different cancer cell lines. cIRCR201-dPBD also showed significant antitumor activity on the MET-amplified cancer cells using in vivo xenograft models. Therefore, cIRCR201-dPBD could be a promising therapeutic strategy for tumors with c-Met expression.

3.
Genome Biol ; 21(1): 216, 2020 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-32847614

RESUMO

BACKGROUND: Glioblastoma (GBM) is a complex disease with extensive molecular and transcriptional heterogeneity. GBM can be subcategorized into four distinct subtypes; tumors that shift towards the mesenchymal phenotype upon recurrence are generally associated with treatment resistance, unfavorable prognosis, and the infiltration of pro-tumorigenic macrophages. RESULTS: We explore the transcriptional regulatory networks of mesenchymal-associated tumor-associated macrophages (MA-TAMs), which drive the malignant phenotypic state of GBM, and identify macrophage receptor with collagenous structure (MARCO) as the most highly differentially expressed gene. MARCOhigh TAMs induce a phenotypic shift towards mesenchymal cellular state of glioma stem cells, promoting both invasive and proliferative activities, as well as therapeutic resistance to irradiation. MARCOhigh TAMs also significantly accelerate tumor engraftment and growth in vivo. Moreover, both MA-TAM master regulators and their target genes are significantly correlated with poor clinical outcomes and are often associated with genomic aberrations in neurofibromin 1 (NF1) and phosphoinositide 3-kinases/mammalian target of rapamycin/Akt pathway (PI3K-mTOR-AKT)-related genes. We further demonstrate the origination of MA-TAMs from peripheral blood, as well as their potential association with tumor-induced polarization states and immunosuppressive environments. CONCLUSIONS: Collectively, our study characterizes the global transcriptional profile of TAMs driving mesenchymal GBM pathogenesis, providing potential therapeutic targets for improving the effectiveness of GBM immunotherapy.


Assuntos
Redes Reguladoras de Genes , Glioblastoma/genética , Macrófagos Associados a Tumor , Animais , Carcinogênese , Linhagem Celular Tumoral , Modelos Animais de Doenças , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Glioblastoma/patologia , Glioma/genética , Humanos , Imunoterapia , Macrófagos/metabolismo , Camundongos , Neurofibromina 1/genética , Fenótipo , Prognóstico , Células-Tronco , Transcriptoma , Microambiente Tumoral
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