Effect of Chain‐End Chemistries on the Efficiency of Coupling Antibodies to Polymers Using Unnatural Amino Acids

Abstract

Novel conjugates that incorporate strategies for increasing the therapeutic payload, such as targeted polymeric delivery vehicles, have great potential in overcoming limitations of conventional antibody therapies that often exhibit immunogenicity and limited drug loading. Click chemistry has significantly expanded the toolbox of effective strategies for developing hybrid polymer‐biomolecule conjugates, however, effective systems require orthogonality between the polymer and biomolecule chemistries to achieve efficient coupling. Here, three cycloaddition‐based strategies for antibody conjugation to polymeric carriers are explored and show that a purely radical‐based method for polymer synthesis and subsequent biomolecule attachment has a trade‐off between coupling efficiency of the antibody and the ability to synthesize polymers with controlled chemical properties. It is shown that careful consideration of both coupling chemistries as well as the potential effect of how this modulates the chemical properties of the polymer nanocarrier should be considered during the development of such systems. The strategies described offer insight into improving conjugate development for therapeutic and theranostic applications. In this system, polymerization using conventional and established reversible addition fragmentation chain transfer (RAFT) agents, followed by multiple post‐modification steps, always leads to systems with more defined chemical architectures compared to strategies that utilize alkyne‐functional RAFT agents.

Stimuli-Responsive Sulfoxide Polymer–Protein Conjugates with Improved Pharmacokinetics and Tumor Delivery

“…The disappearance of the SDS-PAGE band for Tf-OMSEA and the appearance of long tailing bands toward the high-molecular-weight region also verified the successful synthesis of the Tf-polymer conjugates (Figure d). This observed streaking effect for protein–polymer conjugates is a well-known phenomenon in PAGE and is attributed to molar mass dispersity, shape of the materials, and the hindrance from the polymeric component when moving through the gel. , …”

“…This observed streaking effect for protein−polymer conjugates is a well-known phenomenon in PAGE and is attributed to molar mass dispersity, shape of the materials, and the hindrance from the polymeric component when moving through the gel. 59,60 Following successful synthesis, the Tf-PMSEA and Tf-POEGA conjugates were fluorescently labeled with two fluorophores by reacting the azide-containing monomer in the polymer chain with Cy5 DBCO and Tf protein with Cy7.5 NHS ester to enable the separate visualization of the polymer and protein in subsequent in vivo investigations. A sample of unconjugated Tf was also separately labeled with the Cy7.5 NHS ester as a control for comparison with the Tf-polymer conjugates (Figure 3e).…”

Click‐on Antibody Fragments for Customisable Targeted Nanomedicines – Site‐specific Tetrazine and Azide Functionalisation through Non‐canonical Amino Acid incorporation

“…In this recently published study, the nanobody‐conjugated particles showed increased cell association in spheroid models through confocal microscopy [20] . Likewise, ScJ591 has also been expressed with azide functionality and conjugated to a polymer showing high conjugate cell association through flow cytometry [9a] . Interestingly, lower cell associations were observed for 2 K mPEG conjugates compared to all other tested formats (Figure 3A), which is most likely due to partial masking of the protein by linear PEG under the conditions of the experiment.…”

“…Hence position 42, a protruding loop structurally far from the CDRs of JVZ007 [15] (a prostate‐specific membrane antigen (PSMA) binding nanobody) was selected as the mutation site for incorporating tetrazine functionality (Figure 1, right). However, it was anticipated that an N‐terminal positioning for ScJ591 (scFv derived from PSMA binding monoclonal antibody J591 [16] ) would not significantly interfere with binding, based on structural analysis and previous work in our labs [9a] . In both cases, the C‐terminal appeared to be a far better positioning for conjugation purposes.…”

Enhancement of Sensitivity for Retroreflection‐Based Biosensor by Controlling Polymer Brush on Janus Particles

“…The RJPs described above were modified with a dibenzocyclooctyne (DBCO)‐oligonucleotide (DNA2), and the glass surface was functionalized with the azide group and was conjugated with another DBCO‐oligonucleotide (DNA1). [ 46,51–53 ] As shown in Figure 5c, in the presence of the target DNA, the RJPs were aligned on the surface in the same orientation, resulting in enhanced retroreflective signals compared to the background.…”