Polymeric nanoparticles displaying tumor necrosis factor on their surface (TNF nanocytes) are useful carrier systems capable of mimicking the bioactivity of membrane-bound TNF. Thus, TNF nanocytes are potent activators of TNF receptor 1 and 2 leading to a striking enhancement of apoptosis. However, in vivo applications are hampered by potential systemic toxicity. Here, using TNF nanocytes as a model system, we developed a procedure to generate targeted lipid-coated particles (TLP) in which TNF activity is shielded. The TLPs generated here are composed of an inner single-chain TNF (scTNF)-functionalized, polymeric nanoparticle core surrounded by a lipid coat endowed with polyethylene glycol (PEG) for sterical stabilization and a single-chain Fv (scFv) fragment for targeting. Using a scFv directed against the tumor stroma marker fibroblast activation protein (FAP) we show that TLP and scTNF-TLP specifically bind to FAP-expressing, but not to FAP-negative cells. Lipid coating strongly reduced nonspecific binding of particles and scTNF-mediated cytotoxicity towards FAP-negative cells. In contrast, an increased cytotoxicity of TLP was observed for FAP-positive cells. Thus, through liposome encapsulation, nanoparticles carrying bioactive molecules, which are subject to nonselective uptake and activity towards various cells and tissues, can be converted into target cell-specific composite particles exhibiting a selective activity towards antigen-positive target cells. Besides safe and targeted delivery of death ligands such as TNF, TLP should be suitable for various diagnostic and therapeutic applications, which benefit from a targeted delivery of reagents embedded into the particle core or displayed on the core particle surface.
The family of tumor necrosis factor receptors (TNFRs) and their ligands form a regulatory signaling network that controls immune responses. Various members of this receptor family respond differently to the soluble and membrane-bound forms of their respective ligands. However, the determining factors and underlying molecular mechanisms of this diversity are not yet understood. Using an established system of chimeric TNFRs and novel ligand variants mimicking the bioactivity of membranebound TNF (mTNF), we demonstrate that the membrane-proximal extracellular stalk regions of TNFR1 and TNFR2 are crucial in controlling responsiveness to soluble TNF (sTNF). We show that the stalk region of TNFR2, in contrast to the corresponding part of TNFR1, efficiently inhibits both the receptor's enrichment/clustering in particular cell membrane regions and ligandindependent homotypic receptor preassembly, thereby preventing sTNF-induced, but not mTNF-induced, signaling. Thus, the stalk regions of the two TNFRs not only have implications for additional TNFR family members, but also provide potential targets for therapeutic intervention.
Immunoliposomes generated by coupling of antibodies to the liposomal surface allow for an active targeting of entrapped compounds to diseased areas. Single-chain Fv fragments (scFv) represent the smallest part of an antibody containing the entire antigen-binding site. They can be coupled in a defined and site-directed manner through genetically engineered cysteine residues, for example, those added at the C-terminus. Here, we have performed a comparative analysis of various scFv' variants with cysteine residues present at the end of a C-terminal extension of varying length and composition (HC variants) or introduced in the linker sequence connecting the variable heavy and light chain domain (LC variants). Using a scFv fragment directed against fibroblast activation protein (FAP) as a model antibody, we could show that all variants can be employed for the generation of active immunoliposomes, although the presence of three additional cysteine residues in one scFv' molecule resulted in decreased binding of immunoliposomes compared to that of immunoliposomes generated with scFv' molecules containing only one additional cysteine residue. In order to further improve the scFv' format by reducing the number of additional amino acid residues, we also generated molecules with the hexahistidyl-tag incorporated into the linker sequence together with a cysteine residue either at position 1 or 3 of the linker sequence (LCH variants). These newly designed scFv' molecules may be particularly suitable for the generation of immunoliposomes and other antibody conjugates, limiting the number of additional residues in these antibody molecules to a minimum.
Dimeric assembly of antibody fragments and other therapeutic molecules can result in increased binding and improved bioactivity. Here, we investigated the use of the IgM heavy chain domain 2 (MHD2) as covalently linked homodimerization module. Fusion of single-chain fragment variable (scFv) molecules directed against epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 to the N- and/or C-terminus of the MHD2, respectively, resulted in molecules with single or dual specificity for tumor cells. Bispecific tetravalent molecules were further generated by fusing a bispecific single-chain diabody directed against EGFR and epithelial cell adhesion molecule to the N-terminus of the MHD2. By combining an anti-EGFR scFv with a single-chain derivative of tumor necrosis factor, a tetravalent bifunctional fusion protein was produced. This fusion protein exhibited improved TNF activity, also mimicking the membrane-bound form of TNF, as shown by the activation of TNFR2-mediated cell killing. Furthermore, the scFv moiety allowed for an antigen-dependent delivery of TNF to EGFR-positive cells and an improved stimulatory TNF action on these cells. Thus, we established the MHD2 as a versatile module for the generation of bispecific and bifunctional fusion proteins.
Immunoliposomes generated by coupling of antibodies to the liposomal surface allow for an active tissue targeting, e.g., through binding to tumor cell-specific receptors. Instead of whole antibodies, single-chain Fv fragments (scFv), which represent the smallest part of an antibody containing the entire antigen-binding site, find increasing usage as targeting moiety. Here we provide protocols for the preparation of type II scFv immunoliposomes by the conventional coupling method as well as the post-insertion method. Furthermore protocols to analyze binding of these immunoliposomes to antigen-expressing cells as well as internalization through receptor-mediated endocytosis are included.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.