Macrocyclization of Interferon–Poly(α-amino acid) Conjugates Significantly Improves the Tumor Retention, Penetration, and Antitumor Efficacy

Abstract

Cyclization and polymer conjugation are two commonly used approaches for enhancing the pharmacological properties of protein drugs. However, cyclization of parental proteins often only affords a modest improvement in biochemical or cell-based in vitro assays. Moreover, very few studies have included a systematic pharmacological evaluation of cyclized protein-based therapeutics in live animals. On the other hand, polymer-conjugated proteins have longer circulation half-lives but usually show poor tumor penetration and suboptimal pharmacodynamics due to increased steric hindrance. We herein report the generation of a head-to-tail interferon-poly(α-amino acid) macrocycle conjugate circ-P(EGGlu)-IFN by combining the aforementioned two approaches. We then compared the antitumor pharmacological activity of this macrocycle conjugate against its linear counterparts, N-P(EGGlu)-IFN, C-IFN-P(EGGlu), and C-IFN-PEG. Our results found circ-P(EGGlu)-IFN to show considerably greater stability, binding affinity, and in vitro antiproliferative activity toward OVCAR3 cells than the three linear conjugates. More importantly, circ-P(EGGlu)-IFN exhibited longer circulation half-life, remarkably higher tumor retention, and deeper tumor penetration in vivo. As a result, administration of the macrocyclic conjugate could effectively inhibit tumor progression and extend survival in mice bearing established xenograft human OVCAR3 or SKOV3 tumors without causing severe paraneoplastic syndromes. Taken together, our study provided until now the most relevant experimental evidence in strong support of the in vivo benefit of macrocyclization of protein-polymer conjugates and for its application in next-generation therapeutics.

Broadening the scope of sortagging

“…In their subsequent work, it turned out that the cyclization of such an protein-poly(amino acid) conjugate yields particularly interesting protein-based therapeutics. 202 A G 3 -poly(amino acid)-SPh was conjugated to a cystein at the N-terminus of Cys-IFN-LPETG and this conjugate cyclized by SML. The macrocycle combines the advantages of polymer conjugation and cyclization of therapeutic proteins which was evident by greater stability, longer circulation half-life and higher tumor retention compared to the linear counterparts.…”

Therapeutic Protein PEPylation: The Helix of Nonfouling Synthetic Polypeptides Minimizes Antidrug Antibody Generation

“…26−32 We envisage that the chemical modification of proteins by synthetic polypeptides, which we call PEPylation, could open up enormous possibilities. 33−35 Particularly, the chemical diversity of synthetic polypeptides has been greatly expanded by incorporating noncanonical amino acids via ring-opening polymerization (ROP) of α-amino acid N -carboxyanhydrides (NCA) and utilizing d -amino acids. 20 Notably, during the preparation of this manuscript, Jiang et al reported the nonspecific grafting of zwitterionic polypeptides to uricase, 36 which showed extraordinarily low immunogenicity and outstanding safety profile in vivo.…”

Polypeptides–Drug Conjugates for Anticancer Therapy

“…[73,74] They also found that head-to-tail macrocyclization of protein-P(OEG3-Glu)n conjugates enhanced the tumor retention, penetration, and antitumor efficacy of protein therapeutics. [71] Recent, our group synthesized an amphiphilic triblock copolymer methoxy poly(ethylene glycol)-block-poly(l-glutamic acid)block-poly-( -propargyl-L-glutamate) (mPEG-b-PLGA-b-PPLG). The PLGA block of mPEG-b-PLGA-b-PPLG was modified with dopamines containing catechol groups by amidation reaction, and the PPLG block was modified with a small tertiary-amine molecule by click reaction, generating a novel triblock copolymer, mPEG-b-PGCA-b-PGTA.…”