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Human Serum Albumin-Based Drug-Free Macromolecular Therapeutics: Apoptosis Induction by Coiled-Coil-Mediated Cross-Linking of CD20 Antigens on Lymphoma B Cell Surface.

ABSTRACT: A therapeutic platform-drug-free macromolecular therapeutics (DFMT)-that induces apoptosis in B cells by cross-linking of CD20 receptors, without the need for low molecular weight cytotoxic drug, is developed. In this report, a DFMT system is synthesized and evaluated based on human serum albumin (HSA) and two complementary coiled-coil forming peptides, CCE and CCK. Fab' fragment of anti-CD20 monoclonal antibody rituximab is attached to CCE (Fab'-CCE); multiple grafts of CCK are conjugated to HSA (HSA-(CCK)7 ). The colocalization of both nanoconjugates at the surface of non-Hodgkin's lymphoma (NHL) Raji cells is demonstrated by confocal fluorescence microscopy. The colocalization leads to coiled-coil formation, CD20 cross-linking, and apoptosis induction. The apoptotic levels are evaluated by Annexin V, Caspase 3, and terminal deoxynucleotidyl transferase dUTP nick end labeling assays. Selective surface binding of DFMT to CD20+ cells is validated in experiments on a coculture of CD20+ (Raji) and CD20-(DG-75) cells. It is found that DFMT can trigger calcium influx only in Raji cells, but not in DG-75 cells. A highly specific treatment for NHL and other B cell malignancies with considerable translational potential is presented by HSA-based DFMT system.

SUBMITTER: Zhang L

PROVIDER: S-EPMC6392022 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Human Serum Albumin-Based Drug-Free Macromolecular Therapeutics: Apoptosis Induction by Coiled-Coil-Mediated Cross-Linking of CD20 Antigens on Lymphoma B Cell Surface.

Zhang Libin L Fang Yixin Y Li Lian L Yang Jiyuan J Radford D Christopher DC Kopeček Jindřich J

Macromolecular bioscience 20180927 11

A therapeutic platform-drug-free macromolecular therapeutics (DFMT)-that induces apoptosis in B cells by cross-linking of CD20 receptors, without the need for low molecular weight cytotoxic drug, is developed. In this report, a DFMT system is synthesized and evaluated based on human serum albumin (HSA) and two complementary coiled-coil forming peptides, CCE and CCK. Fab' fragment of anti-CD20 monoclonal antibody rituximab is attached to CCE (Fab'-CCE); multiple grafts of CCK are conjugated to HS ...[more]

PMID: 30259654

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Project description:The Spike (S) glycoprotein of coronaviruses (CoV) mediates viral entry into host cells. It contains two hydrophobic heptad repeat (HR) regions, denoted HRN and HRC, which oligomerize the S glycoprotein into a trimer in the native state and when activated collapse into a six-helix bundle structure driving fusion of the host and viral membranes. Previous studies have shown that peptides of the HR regions can inhibit viral infectivity. These studies imply that the HR regions are accessible and that agents which can interact with them may prevent viral entry. In the present study, we have investigated an approach to generate antibodies that specifically recognize the HRN and HRC regions of the SARS-CoV spike (S) glycoprotein in order to evaluate whether these antibodies can inhibit viral infectivity and thus neutralize the SARS-CoV. In this regard, we incorporated HRN and HRC coiled-coil surface residues into a de novo designed two-stranded alpha-helical coiled-coil template for generating conformation-specific antibodies that recognize alpha-helices in proteins (Lu, S.M., Hodges, R.S., 2002. J. Biol. Chem. 277, 23515-23524). Eighteen surface residues from two regions of HRN and HRC were incorporated into the template and used to generate four anti-sera, HRN1, HRN2, HRC1, and HRC2. Our results show that all of the elicited anti-sera can specifically recognize HRN or HRC peptides and the native SARS-CoV S protein in an ELISA format. Flow cytometry (FACS) analysis, however, showed only HRC1 and HRC2 anti-sera could bind to native S protein expressed on the cell surface of Chinese hamster ovary cells, i.e., the cell surface structure of the S glycoprotein precluded the ability of the HRN1 or HRN2 anti-sera to see their respective epitope sites. In in vitro viral infectivity assays, no inhibition was observed for either HRN1 or HRN2 anti-serum, whereas both HRC1 and HRC2 anti-sera could inhibit SARS-CoV infection in a dose-dependent manner. Interestingly, the HRC1 anti-serum, which was a more effective inhibitor of viral infectivity compared to HRC2 anti-serum, could only bind the pre-fusogenic state of HRC, i.e., the HRC1 anti-serum did not recognize the six-helix bundle conformation (fusion state) whereas HRC2 anti-serum did. These results suggest that antibodies that are more specific for the pre-fusogenic state of HRC may be better neutralizing antibodies. Overall, these results clearly demonstrate that the two-stranded coiled-coil template acts as an excellent presentation system for eliciting helix-specific antibodies against highly conserved viral antigens and HRC1 and HRC2 peptides may represent potential candidates for use in a peptide vaccine against the SARS-CoV.

Dataset Information

Human Serum Albumin-Based Drug-Free Macromolecular Therapeutics: Apoptosis Induction by Coiled-Coil-Mediated Cross-Linking of CD20 Antigens on Lymphoma B Cell Surface.

Publications

Human Serum Albumin-Based Drug-Free Macromolecular Therapeutics: Apoptosis Induction by Coiled-Coil-Mediated Cross-Linking of CD20 Antigens on Lymphoma B Cell Surface.

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