Engineering of Surface Proteins in Extracellular Vesicles for Tissue-Specific Targeting

Extracellular vesicles (EVs) have in the recent decades gained an important stand as vehicles enabling cell-to-cell transport and communication. With the advanced development towards their clinical use and increasing versatility of potential applications, improving their tissue-specific targeting in order to enhance their functionality in drug delivery opened as a challenging engineering field. In the past, the question of specific intercellular contact has been addressed by decoration of the EV surface with agents able of specific target recognition. An attractive possibility here is the modification of strongly overexpressed EV surface marker proteins towards recognition of target cells. As these proteins are involved in a plethora of biological functions in EV biogenesis, cargo targeting and intercellular transfer, a minimal impact on protein architecture upon modifications is desirable, which would also increase the stability of the exosomal preparation intended for therapeutic use. This chapter focuses on the possibilities of engineering of the EV marker proteins towards antigen-recognition units broadly applicable to endow EVs with tissue-targeting functionality

Superior SARS-CoV-2 RBD antigen designs for highly specific, quantitative serotests

Quantitative high-quality SARS-CoV-2 serotests that are easy-to-implement have been gaining great importance as means to characterize and monitor the magnitude of infection- or vaccine-induced immunity over time and are of particular interest for academic laboratories doing COVID-19 research or small diagnostic laboratories with basic equipment Please click Download on the upper right corner to see the full abstract

constant domain exchanged fab enables specific light chain pairing in heterodimeric bispecific seed antibodies

Abstract Background Bispecific antibodies promise to broadly expand the clinical utility of monoclonal antibody technology. Several approaches for heterodimerization of heavy chains have been established to produce antibodies with two different Fab arms, but promiscuous pairing of heavy and light chains remains a challenge for their manufacturing. Methods We have designed a solution in which the CH1 and CL domain pair in one of the Fab fragments is replaced with a CH3-domain pair and heterodimerized to facilitate correct modified Fab-chain pairing in bispecific heterodimeric antibodies based on a strand-exchange engineered domain (SEED) scaffold with specificity for epithelial growth factor receptor and either CD3 or CD16 (FcγRIII). Results Bispecific antibodies retained binding to their target antigens and redirected primary T cells or NK cells to induce potent killing of target cells. All antibodies were expressed at a high yield in Expi293F cells, were detected as single sharp symmetrical peaks in size exclusion chromatography and retained high thermostability. Mass spectrometric analysis revealed specific heavy-to-light chain pairing for the bispecific SEED antibodies as well as for one-armed SEED antibodies co-expressed with two different competing light chains. Conclusion Incorporation of a constant domain-exchanged Fab fragment into a SEED antibody yields functional molecules with favorable biophysical properties. General significance Our results show that the novel engineered bispecific SEED antibody scaffold with an incorporated Fab fragment with CH3-exchanged constant domains is a promising tool for the generation of complete heterodimeric bispecific antibodies with correct light chain pairing

Fab antibody fragment-functionalized liposomes for specific targeting of antigen-positive cells

Liposomes functionalized with monoclonal antibodies or their antigen-binding fragments have attracted much attention as specific drug delivery devices for treatment of various diseases including cancer. The conjugation of antibodies to liposomes is usually achieved by covalent coupling using cross-linkers in a reaction that might adversely affect the characteristics of the final product. Here we present an alternative strategy for liposome functionalization: we created a recombinant Fab antibody fragment genetically fused on its C-terminus to the hydrophobic peptide derived from pulmonary surfactant protein D, which became inserted into the liposomal bilayer during liposomal preparation and anchored the Fab onto the liposome surface. The Fab-conjugated liposomes specifically recognized antigen-positive cells and efficiently delivered their cargo, the Alexa Fluor 647 dye, into target cells in vitro and in vivo. In conclusion, our approach offers the potential for straightforward development of nanomedicines functionalized with an antibody of choice without the need of harmful cross-linkers.This work has received funding from the European Union's Seventh Framework Program (FP7/2007-2013; grant agreement NMP4-LA-2009-228827 NANOFOL) and Horizon 2020 Research and Innovation Program (grant agreement No 683356 - FOLSMART), further from the Portuguese Foundation for Science and Technology under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020.info:eu-repo/semantics/publishedVersio

Stabilisation of the Fc Fragment of Human IgG1 by Engineered Intradomain Disulfide Bonds

We report the stabilization of the human IgG1 Fc fragment by engineered intradomain disulfide bonds. One of these bonds, which connects the N-terminus of the CH3 domain with the F-strand, led to an increase of the melting temperature of this domain by 10°C as compared to the CH3 domain in the context of the wild-type Fc region. Another engineered disulfide bond, which connects the BC loop of the CH3 domain with the D-strand, resulted in an increase of Tm of 5°C. Combined in one molecule, both intradomain disulfide bonds led to an increase of the Tm of about 15°C. All of these mutations had no impact on the thermal stability of the CH2 domain. Importantly, the binding of neonatal Fc receptor was also not influenced by the mutations. Overall, the stabilized CH3 domains described in this report provide an excellent basic scaffold for the engineering of Fc fragments for antigen-binding or other desired additional or improved properties. Additionally, we have introduced the intradomain disulfide bonds into an IgG Fc fragment engineered in C-terminal loops of the CH3 domain for binding to Her2/neu, and observed an increase of the Tm of the CH3 domain for 7.5°C for CysP4, 15.5°C for CysP2 and 19°C for the CysP2 and CysP4 disulfide bonds combined in one molecule

Graphical presentation of secondary and tertiary structure of the CH3 domain and of an Fc fragment, and location of engineered disulfide bonds.

<p>A: 2D-presentation (“Collier de perles” <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030083#pone.0030083-Ruiz1" target="_blank">[32]</a>) of the secondary structure of the CH3 domain. The residues that were mutated to Cysteine to form the engineered disulfide bonds are indicated in green (CysP2) and red (CysP4). Native disulfide bond residues are indicated in yellow. B: 3D cartoon presentation (created using the PyMOL Molecular Graphics System, Version 1.3, Schrödinger, LLC) of the human IgG1 Fc fragment (Protein Data Bank (<a href="http://www.pdb.org/" target="_blank">http://www.pdb.org/</a>) accession 1OQO) with the positions that were mutated to create the engineered disulfide bonds indicated in green and red as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030083#pone-0030083-g001" target="_blank">Figure 1A</a>. The native disulfide bonds are shown in yellow.</p

Thermograms of wild-type and disulfide bond stabilized Fc mutants obtained with DSC measurements.

<p>A: wild-type Fc, B: FcCysP2, C: FcCysP4, D: FcCysP24, E: overlay of all 5 proteins. For each protein the point of reversibility of melting of CH2 domain was determined which is shown as a dashed line in the inset graphs.</p

Thermograms of Her2/neu binding Fcab H10-03-6 and its disulfide bond stabilized derivatives obtained with DSC measurements.

<p>Thermograms of Her2/neu binding Fcab H10-03-6 and its disulfide bond stabilized derivatives obtained with DSC measurements.</p

Spectroscopic characterization of disulfide bond stabilized Fc-mutants: (A) far-UV CD spectra and (B) near-UV CD spectra of stabilized Fc-mutants.

<p>The measurements were carried out at a protein concentration of 570 µg/ml in PBS at 25°C.</p

BLI analyses of FcRn interaction with wild-type Fc and with disulfide bond stabilized Fc-mutants.

<p>Biotinylated FcRn was immobilized to streptavidin tips. Association of the Fc fragment was observed at pH 6.0 and a sharp dissociation was induced by shifting the pH to 7.4.</p