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Engineering reversible cell-cell interactions using enzymatically lipidated chemically self-assembled nanorings.

ABSTRACT: Multicellular biology is dependent on the control of cell-cell interactions. These concepts have begun to be exploited for engineering of cell-based therapies. Herein, we detail the use of a multivalent lipidated scaffold for the rapid and reversible manipulation of cell-cell interactions. Chemically self-assembled nanorings (CSANs) are formed via the oligomerization of bivalent dihydrofolate reductase (DHFR²) fusion proteins using a chemical dimerizer, bis-methotrexate. With targeting proteins fused onto the DHFR² monomers, the CSANs can target specific cellular antigens. Here, anti-EGFR or anti-EpCAM fibronectin-DHFR² monomers incorporating a CAAX-box sequence were enzymatically prenylated, then assembled into the corresponding CSANs. Both farnesylated and geranylgeranylated CSANs efficiently modified the cell surface of lymphocytes and remained bound to the cell surface with a half-life of >3 days. Co-localization studies revealed a preference for the prenylated nanorings to associate with lipid rafts. The presence of antigen targeting elements in these bifunctional constructs enabled them to specifically interact with target cells while treatment with trimethoprim resulted in rapid CSAN disassembly and termination of the cell-cell interactions. Hence, we were able to determine that activated PBMCs modified with the prenylated CSANs caused irreversible selective cytotoxicity toward EGFR-expressing cells within 2 hours without direct engagement of CD3. The ability to disassemble these nanostructures in a temporally controlled manner provides a unique platform for studying cell-cell interactions and T cell-mediated cytotoxicity. Overall, antigen-targeted prenylated CSANs provide a general approach for the regulation of specific cell-cell interactions and will be valuable for a plethora of fundamental and therapeutic applications.

SUBMITTER: Wang Y

PROVIDER: S-EPMC8179657 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:A number of clever recombinant methodologies have been developed that recapitulate the valencies of IgG's (bivalent) and IgA's (tetravalent). Although higher synthetic valencies have been achieved by conjugation of either monoclonal antibodies or single-chain antibodies to nanoparticles and liposomes, a method for the preparation of recombinant antibodies with valencies similar to IgM's (decavalent) but considerably less than what is generally found after antibody particle conjugation has yet to be devised. Recently, we have developed a methodology for the design of bivalent Chemically Self-Assembled Antibody Nanorings (CSANs). We now report the crystal structure of the nanoring subunit composed of the E. coli DHFR dimer and a methotrexate dimerizer (MTX2-C9) containing a visible nine methylene linker and a protocol for the preparation of CSANs from this subunit with valencies similar to IgM's, ranging from 8-10 single chain antibodies (scFvs). The multivalent CSANs were reversibly assembled from a fusion protein dihydrofolate reductase (DHFR)-DHFR-antiCD3 scFv containing a single glycine linker between the two DHFR scaffolding proteins. We also demonstrate that, similar to the parental bivalent anti-CD3 monoclonal antibody (mAB), anti-CD3 CSANs selectively bind to CD3+ leukemia cells and undergo rapid internalization through a caveolin-independent pathway that requires cholesterol, actin polymerization, and protein tyrosine kinase activation. While treatment with the monoclonal antibody leads to T-cell activation and nearly complete loss (i.e., 90%) of the surface displayed T-cell receptor (TCR), only 25-30% of the TCR down regulate and no significant T-cell proliferation is observed after treatment of peripheral blood mononuclear cells (PBMCs) with anti-CD3 CSANs. Consistent with the proliferation findings, 15-25% less CD25 (IL-2 receptor) was found on the surface of PBMCs treated with either the polyvalent or bivalent anti-CD3 CSANs, respectively, than on PBMCs treated with the parental mAB. Comparative experiments with F(ab')2 derived from the mAB confirm that the activation of the T-cells by the mAB is dependent on the Fc domain, and thus interactions of the PBMC T-cells with accessory cells, such as macrophages. Taken together, our results demonstrate that anti-CD3 CSANs with valencies ranging from 2 to 8 could be employed for radionuclide, drug, or potentially oligonucleotide delivery to T-cells without, as has been observed for other antibody conjugated nanoparticles, the deleterious effects of activation observed for mAB. Further the CSAN construct may be adapted for the preparation of other multivalent scFvs.

Project description:Synthetic nucleic acids have shown great potential in the treatment of various diseases. Nevertheless, the selective delivery to a target tissue has proved challenging. The coupling of nucleic acids to targeting peptides, proteins, and antibodies has been explored as an approach for their selective tissue delivery. Nevertheless, the preparation of covalently coupled peptides and proteins that can also undergo intracellular release as well as deliver more than one copy of the nucleic acid has proved challenging. Recently, we have developed a novel method for the rapid noncovalent conjugation of nucleic acids to targeting single chain antibodies (scFv) using chemically self-assembled nanostructures (CSANs). CSANs have been prepared by the self-assembly of two dihydrofolate reductase molecules (DHFR(2)) and a targeting scFv in the presence of bis-methotrexate (bis-MTX). The valency of the nanorings can be tuned from one to eight subunits, depending on the length and composition of the linker between the dihydrofolate reductase molecules. To explore their potential for the therapeutic delivery of nucleic acids as well as the ability to expand the capabilities of CSANs by incorporating smaller cyclic targeting peptides, we prepared DHFR(2) proteins fused through a flexible peptide linker to cyclic-RGD, which targets ?v?3 integrins, and a bis-MTX chemical dimerizer linked to an antisense oligonucleotide (bis-MTX-ASO) that has been shown to silence expression of eukaryotic translation initiation factor 4E (eIF4E). Monomeric and multimeric cRGD-CSANs were then prepared with bis-MTX-ASO and shown to undergo endocytosis in the breast cancer cell line, MDA-MB-231, which overexpresses ?v?3. The bis-MTX-ASO was shown to undergo endosomal escape resulting in the knock down of eIF4E with at least the same efficiency as ASO delivered by oligofectamine. The modularity, flexibility, and common method of conjugation may prove to be a useful general approach for the targeted delivery of ASOs, as well as other nucleic acids to cells.

Project description:Chemically self-assembled nanorings (CSANs) are made of dihydrofolate reductase (DHFR) fusion proteins and have been successfully used in vitro for cellular cargo delivery and cell surface engineering applications. However, CSANs have yet to be evaluated for their in vivo stability, circulation, and tissue distribution. In an effort to evaluate CSANs in vivo, we engineered a site-specifically PEGylated epidermal growth factor receptor (EGFR) targeting DHFR molecules, characterized their self-assembly into CSANs with bivalent methotrexates (bis-MTX), visualized their in vivo tissue localization by microPET/CT imaging, and determined their ex vivo organ biodistribution by tissue-based gamma counting. A dimeric DHFR (DHFR(2)) molecule fused with a C-terminal EGFR targeting peptide (LARLLT) was engineered to incorporate a site-specific ketone functionality using unnatural amino acid mutagenesis. Aminooxy-PEG, of differing chain lengths, was successfully conjugated to the protein using oxime chemistry. These proteins were self-assembled into CSANs with bis-MTX DHFR dimerizers and characterized by size exclusion chromatography and dynamic light scattering. In vitro binding studies were performed with fluorescent CSANs assembled using bis-MTX-FITC, while in vivo microPET/CT imaging was performed with radiolabeled CSANs assembled using bis-MTX-DOTA[(64)Cu]. PEGylation reduced the uptake of anti-EGFR CSANs by mouse macrophages (RAW 264.7) up to 40% without altering the CSAN's binding affinity toward U-87 MG glioblastoma cells in vitro. A significant time dependent tumor accumulation of (64)Cu labeled anti-EGFR-CSANs was observed by microPET/CT imaging and biodistribution studies in mice bearing U-87 MG xenografts. PEGylated CSANs demonstrated a reduced uptake by the liver, kidneys, and spleen resulting in high contrast tumor imaging within an hour of intravenous injection (9.6% ID/g), and continued to increase up to 24 h (11.7% ID/g) while the background signal diminished. CSANs displayed an in vivo profile between those of rapidly clearing small molecules and slow clearing antibodies. Thus, CSANs offer a modular, programmable, and stable protein based platform that can be used for in vivo drug delivery and imaging applications.

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Engineering reversible cell-cell interactions using enzymatically lipidated chemically self-assembled nanorings.

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