Covalent conjugation of a biologically stable
polymer to a therapeutic
protein, e.g., an antibody, holds many benefits such as prolonged plasma exposure of the
protein and improved
tumor uptake. Generation of defined conjugates is advantageous in many applications, and a range of site-selective conjugation methods have been reported. Many current coupling methods lead to dispersity in coupling efficiencies with subsequent conjugates of less-well-defined structure, which impacts reproducibility of manufacture and ultimately may impact successful translation to treat or image diseases. We explored designing stable, reactive groups for
polymer conjugation reactions that would lead to conjugates through the simplest and most abundant residue on most
proteins, the
lysine residue, yielding conjugates in high purity and demonstrating retention of mAb efficacy through surface plasmon resonance (SPR), cell targeting, and in vivo
tumor targeting. We utilized
squaric acid diesters as coupling agents for selective amidation of
lysine residues and were able to selectively conjugate one, or two, high-molecular-weight
polymers to a therapeutically relevant antibody, 528mAb, that subsequently retained full binding specificity. Water-soluble copolymers of N-(2-hydroxypropyl)
methacrylamide (
HPMA) and
N-isopropylacrylamide (NIPAM) were prepared by Reversible Addition-Fragmentation chain-Transfer (RAFT) polymerization and we demonstrated that a dual-
dye-labeled antibody-RAFT conjugate (528mAb-RAFT) exhibited effective
tumor targeting in model
breast cancer xenografts in mice. The combination of the precise and selective
squaric acid ester conjugation method, with the use of RAFT
polymers, leads to a promising strategic partnership for improved therapeutic
protein-
polymer conjugates having a very-well-defined structure.