RESUMO
Targeting the dimer interface for the epidermal growth factor receptor (EGFR) that is highly conserved in the structure and directly involved in dimerization may solve the resistance problem that plagues anti-EGFR therapy. Heavy chain single domain antibodies have promising prospects as therapeutic antibodies. A bispecific nanobody was constructed based on previously screened humanized nanobodies that target the ß-loop at the EGFR dimer interface, an anti-FcγRIIIa (CD16) of natural killer cells (NK) nanobodies and anti-human serum albumin (HSA) nanobodies. The target gene was effectively expressed and secreted while controlled by promoter GAP in Pichia pastoris X33, and the expressed product was purified with a cation exchange and nickel chelation chromatography. The bispecific nanobody specifically bound to the surfaces of EGFR-overexpressed human epidermal carcinoma A431 cells and effectively inhibited tumor cell growth both in vitro and in vivo. In the A431 cell nude mouse xenograft model, the growth inhibition effect from the bispecific nanobody was significantly increased with the assistance of peripheral blood mononuclear cells (PBMCs), which was consistent with the results obtained in vitro, suggesting that there was an antibody-dependent cell-mediated cytotoxicity (ADCC) effect. In addition, the intraperitoneal administration of bispecific nanobodies effectively reached tumor tissues in the shoulder dorsal region, but in significantly less distributed quantities than EGFR Dimer Nb77. To conclude, a bispecific nanobody targeting the EGFR dimer interface with ADCC effect was successfully constructed.
Assuntos
Anticorpos Biespecíficos/metabolismo , Antineoplásicos/farmacologia , Receptores ErbB/metabolismo , Multimerização Proteica , Anticorpos de Cadeia Única/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/isolamento & purificação , Membrana Celular/metabolismo , Proliferação de Células , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células NIH 3T3 , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Targeting the interaction interface is an effective strategy to obtain programmed death receptor 1 (PD-1)/PD-1 ligand 1 (PD-L1) nanobody blockers. To validate this strategy, the interaction interface between PD-1 and the PD-L1 extracellular domain were analyzed using Cn3D 4.1. The peptide PD-1125-136 located at the interface of PD-1 was selected as the antigen to screen nanobodies from a humanized nanobody phage display library. Six different nanobodies were screened, with molecular weights of 12 â¼ 13 kDa, excluding a single basic protein. The nanobody with the longest CDR3 region, termed PD-1-Nb-B20, was selected for further analysis. For mass production, the C-terminal His6-tagged nanobody coding sequence was optimized and cloned into pET-21b for over-expression under the T7 promoter in Escherichia coli BL21 (DE3). PD-1-Nb-B20 was expressed and pancreatic adenocarcinoma cells BxPC-3 over-expressing PD-L1 were selected for nanobody competitive inhibition assays. The purified nanobodies significantly inhibited PD-1 binding to the surface of target cells, indicating their ability to block the PD-1/PD-L1 interaction.
Assuntos
Antineoplásicos Imunológicos , Antígeno B7-H1/imunologia , Proteínas de Neoplasias/imunologia , Neoplasias Pancreáticas/imunologia , Receptor de Morte Celular Programada 1/imunologia , Anticorpos de Domínio Único , Células A549 , Antineoplásicos Imunológicos/química , Antineoplásicos Imunológicos/imunologia , Antineoplásicos Imunológicos/isolamento & purificação , Células HeLa , Humanos , Anticorpos de Domínio Único/química , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/isolamento & purificação , Neoplasias PancreáticasRESUMO
Objective To express, purify and identify the single-chain fragment variable (scFv) against human epidermal growth factor receptor 3 (HER3). Methods We searched NCBI for the light chain sequence and heavy chain sequence of anti-HER3 mAb LJM716 to construct the gene of scFv against HER3. The recombinant expression vector pGAPZαA-anit-HER3-scFv was constructed using the constitutive expression vector pGAPZαA and then electro-transformed into Pichia Pastoris X-33 to screen the strains with high expression of the protein of interest. After shaking flask fermentation, the supernatant was purified by hydrophobic chromatography and metal ion affinity chromatography. The purified product was identified by Western blotting and ELISA. Results The anti-HER3-scFv gene was successfully constructed and the strains with high expression of anti-HER3-scFv were obtained. The anti-HER3-scFv was purified to a purity of more than 95% by two-step chromatography, and the purified yield was 192 mg/L. Western blotting showed that the anti-HER3-scFv was correctly expressed and ELISA indicated that anti-HER3-scFv could specifically recognize HER3. Conclusion The anti-HER3-scFv has been successfully prepared.
Assuntos
Anticorpos Monoclonais , Receptor ErbB-3/imunologia , Anticorpos de Cadeia Única , Western Blotting , Ensaio de Imunoadsorção Enzimática , HumanosRESUMO
Epidermal growth factor receptor (EGFR) is an effective target for the treatment of many epithelial cancers. However, EGFR inhibitors have low clinical response rates and are prone to drug resistance arising from mutations and heterodimerization of EGFR. Therefore, targeting the highly conserved dimer interface of EGFR may be an effective strategy for improving the clinical response of anti-EGFR therapies. Nanobodies have significant advantages over conventional antibodies in terms of size, solubility, stability and cost-effectiveness. To investigate the feasibility of nanobodies targeting the dimer interface of EGFR as novel anticancer drugs, four nanobodies were screened from a commercial humanized nanobody phage antibody library using the EGFR237-267 peptide from the ß-hairpin loop of the dimer interface of EGFR as the antigen. A nanobody with an isoelectric point (pI) of 8.6, named EGFR dimer Nb77, was selected for further analysis of anticancer activities. EGFR dimer Nb77 was expressed in Escherichia coli Shuffle T7-B as a soluble (His)6-tagged protein and purified by a CM Sepharose column and a nickel-nitrilotriacetic acid (Ni-NTA) column. Purified EGFR dimer Nb77 could specifically bind to the surface of EGFR-overexpressing A431â¯cells in a dose-dependent and ligand-dependent manner, and this nanobody could effectively inhibit the growth of the tumour cells, with an inhibition rate similar to that of the monoclonal antibody EGFR dimer 5G9, which also targets the dimer interface of EGFR. This work is the first to prove that nanobodies targeting the dimer interface of EGFR have promising prospects as anticancer agents.