Affinity-controlled capture and release of engineered monoclonal antibodies by macroporous dextran hydrogels using coiled-coil interactions

Abstract

Long-term delivery is a successful strategy used to reduce the adverse effects of monoclonal antibody (mAb)-based treatments. Macroporous hydrogels and affinity-based strategies have shown promising results in sustained and localized delivery of the mAbs. Among the potential tools for affinity-based delivery systems, the de novo designed Ecoil and Kcoil peptides are engineered to form a high-affinity, heterodimeric coiled-coil complex under physiological conditions. In this study, we created a set of trastuzumab molecules tagged with various Ecoil peptides and evaluated their manufacturability and characteristics. Our data show that addition of an Ecoil tag at the C-termini of the antibody chains (light chains, heavy chains, or both) does not hinder the production of chimeric trastuzumab in CHO cells or affect antibody binding to its antigen. We also evaluated the influence of the number, length, and position of the Ecoil tags on the capture and release of Ecoil-tagged trastuzumab from macroporous dextran hydrogels functionalized with Kcoil peptide (the Ecoil peptide-binding partner). Notably, our data show that antibodies are released from the macroporous hydrogels in a biphasic manner; the first phase corresponding to the rapid release of residual, unbound trastuzumab from the macropores, followed by the affinity-controlled, slow-rate release of antibodies from the Kcoil-functionalized macropore surface.

Coiled-Coil-Based Biofunctionalization of 100 nm Gold Nanoparticles with the Trastuzumab Antibody for the Detection of HER2-Positive Cancer Cells

“…In addition, Baniahmad et al have previously shown that the manufacture of Ecoil-tagged TZM does not pose extra challenges compared to that of wild-type TZM. 65 From a translational and industrial point of view, the E/K coiled-coil-based biofunctionalization might furthermore help accelerate the production of many AuNP−protein conjugates for large-scale studies and/or applications. Indeed, developing different populations of Kcoil-decorated nanoparticles and producing Ecoil-tagged proteins can be performed independently, allowing for the easy and last-minute immobilization of any Ecoil-tagged protein with any type of Kcoil peptidedecorated nanoparticles.…”

“…However, once Ecoil tags are genetically encoded on TZM, the E/K coiled-coil biofunctionalization strategy shows many advantages, because it allows the easy production of AuNP-TZM bioconjugates while ensuring the integrity, orientation, and bioactivity of the immobilized antibodies. In addition, Baniahmad et al have previously shown that the manufacture of Ecoil-tagged TZM does not pose extra challenges compared to that of wild-type TZM . From a translational and industrial point of view, the E/K coiled-coil-based biofunctionalization might furthermore help accelerate the production of many AuNP–protein conjugates for large-scale studies and/or applications.…”

“…The wild-type trastuzumab antibody (TZM) was produced, purified, and characterized as described by Cambay et al The trastuzumab-(EVSALEK) 5 antibody (Ecoil-TZM), a recombinant TZM bearing a C-terminal (EVSALEK) 5 peptide (Ecoil) on each of its heavy chains, was produced, purified, and characterized as described by Baniahmad et al by the Human Health Therapeutics Centre of the National Research Council of Canada. Aliquots were prepared in phosphate-buffered saline (PBS) and were immediately stored at −80 °C until further use.…”

“…The wild-type trastuzumab antibody (TZM) was produced, purified, and characterized as described by Cambay et al 64 The trastuzumab-(EVSALEK) 5 antibody (Ecoil-TZM), a recombinant TZM bearing a C-terminal (EVSALEK) 5 peptide (Ecoil) on each of its heavy chains, was produced, purified, and characterized as described by Baniahmad et al 65 Sodium citrate (A12274.36) was purchased from Thermo Fisher Scientific and stored at room temperature (RT). 2,4,6-Trinitrobenzenesulfonic Acid (TNBS, BC86), 1% in methanol, was purchased from G-Biosciences (St. Louis, MO) and stored at −20 °C.…”

Cofunctionalization of Macroporous Dextran Hydrogels with Adhesive Peptides and Growth Factors Enables Vascular Spheroid Sprouting

“…36,37 These motifs are ubiquitous in nature and have been extensively studied, leading to the de novo design of synthetic coiled-coils, including the E/K heterodimer. 38 In previous studies, our research group has used this E/K coil affinity pair, composed of the two peptides Ecoil and Kcoil, for numerous applications, including (i) the development of bioassays, 39,40 (ii) the functionalization of substrates with Ecoil-tagged recombinant proteins, 17,41−45 and (iii) the controlled delivery of growth factors, 46 monoclonal antibodies, 47 and nanoparticles. 48 In this work, we propose the use of the E/K coiled-coil as a versatile approach to functionalize macroporous dextran hydrogels with growth factors for the implantation and sprouting of vascular spheroids in supplement-depleted medium.…”