RAFT Polymer-Antibody Conjugation: Squaramide Ester Chemistry Leads to Conjugates with a Therapeutic Anti-EGFR Antibody with Full Retention of Activity and Increased Tumor Uptake In Vivo.

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.

Investigation of a Dual siRNA/Chemotherapy Delivery System for Breast Cancer Therapy.

Multidrug resistance (MDR) is a problem that is often associated with a poor clinical outcome in chemotherapeutic cancer treatment. MDR may potentially be overcome by utilizing synergistic approaches, such as combining siRNA gene therapy and chemotherapy to target different mechanisms of apoptosis. In this study, a strategy is presented for developing multicomponent nanomedicines using orthogonal and compatible chemistries that lead to effective nanotherapeutics. Hyperbranched polymers were used as drug carriers that contained doxorubicin (DOX), attached via a pH-sensitive hydrazone linkage, and ataxia-telangiectasia mutated (ATM) siRNA, attached via a redox-sensitive disulfide group. This nanomedicine also contained cyanine 5 (Cy5) as a diagnostic tracer as well as in-house developed bispecific antibodies that allowed targeting of the epidermal growth factor receptor (EGFR) present on tumor tissue. Highly efficient coupling of siRNA was achieved with 80% of thiol end-groups on the hyperbranched polymer coupling with siRNA. This attachment was reversible, with the majority of siRNA released in vitro under reducing conditions as desired. In cellular studies, the nanomedicine exhibited increased DNA damage and cancer cell inhibition compared to the individual treatments. Moreover, the nanomedicine has great potential to suppress the metabolism of cancer cells including both mitochondrial respiration and glycolytic activity, with enhanced efficacy observed when targeted to the cell surface protein EGFR. Our findings indicated that co-delivery of ATM siRNA and DOX serves as a more efficient therapeutic avenue in cancer treatment than delivery of the single species and offers a potential route for synergistically enhanced gene therapy.

Poly(HPMA-co-NIPAM) copolymer as an alternative to polyethylene glycol-based pharmacokinetic modulation of therapeutic proteins.

PEGylation is the standard approach for prolonging the plasma exposure of protein therapeutics but has limitations. We explored whether polymers prepared by Reversible Addition-Fragmentation chain-Transfer (RAFT) may provide better alternatives to polyethylene glycol (PEG). Four RAFT polymers were synthesised with varying compositions, molar mass (Mn), and structures, including a homopolymer of N-(2-hydroxypropyl)methacrylamide, (pHPMA) and statistical copolymers of HPMA with poly(ethylene glycol methyl ether acrylate) p(HPMA-co-PEGA); HPMA and N-acryloylmorpholine, p(HPMA-co-NAM); and HPMA and N-isopropylacrylamide, p(HPMA-co-NIPAM). The intravenous pharmacokinetics of the polymers were then evaluated in rats. The in vitro activity and in vivo pharmacokinetics of p(HPMA-co-NIPAM)-conjugated trastuzumab Fab' and full length mAb were then evaluated. p(HPMA-co-NIPAM) prolonged plasma exposure more avidly compared to the other p(HPMA) polymers or PEG, irrespective of molecular weight. When conjugated to trastuzumab-Fab', p(HPMA-co-NIPAM) prolonged plasma exposure of the Fab' similar to PEG-Fab'. The generation of anti-PEG IgM in rats 7 days after intravenous and subcutaneous dosing of p(HPMA-co-NIPAM) conjugated trastuzumab mAb was also examined and was shown to exhibit lower immunogenicity than the PEGylated construct. These data suggest that p(HPMA-co-NIPAM) has potential as a promising copolymer for use as an alternative conjugation strategy to PEG, to prolong the plasma exposure of therapeutic proteins.

Surface potentials in Langmuir monolayers of unidirectionally oriented alpha-helical diblock copolypeptides.

The surface potentials and effective dipole moments of alpha-helical amphiphilic diblock copolypeptides during monolayer compression at the air-water interface are reported. Amphiphilic diblock copolypeptides (PLGA-b-PMLGSLGs) of poly(alpha-L-glutamic acid) (PLGA) and poly(gamma-methyl-L-glutamate-ran-gamma-stearyl-L-glutamate) with 30 mol % of stearyl substituents (PMLGSLG) of various block lengths were studied during the double-brush formation process at the water surface. Upon monolayer spreading of PLGA-b-PMLGSLGs, surface potentials of hundreds of millivolts were recorded, attributed to the dipole moments of water molecules reorienting due to interactions with the monolayers. Upon compression, the effective dipole moments derived from the surface potentials of the PLGA-b-PMLGSLG monolayers decrease gradually, most likely as a result of the immersion of the hydrophilic block in water and cancellation of the interactions between the hydrophobic block and the underlying water molecules. The polypeptide macrodipole moment immersed in water was apparently effectively screened out. The remaining effective dipole moment of the monolayer contributes mainly to the hydrophobic block, and upon tilting away from the water surface toward the surface normal, it was found to increase with the hydrophobic block length, indicating the gradual formation of unidirectional aligned polypeptide molecules in the double-brush monolayer.