Anti-CD20 multivalent HPMA copolymer-Fab′ conjugates for the direct induction of apoptosis

Abstract

A hybrid biomimetic system comprising high-molecular-weight, linear copolymer of N-(2-hydroxypropyl)methacrylamide (HPMA) grafted with multiple Fab′ fragments of anti-CD20 monoclonal antibody (mAb) was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization followed by attachment of Fab′ fragments via thioether bonds. Exposure of human non-Hodgkin’s lymphoma (NHL) Raji B cells to the multivalent conjugates resulted in crosslinking of CD20 receptors and commencement of apoptosis. Five conjugates with varying molecular weight and valence (amount of Fab′ per polymer chain) were synthesized. One of the copolymers contained enzyme degradable peptide sequences (GFLG) in the backbone. The multivalency led to higher avidity and apoptosis induction compared to unconjugated whole mAb. Time-dependent studies showed that the cytotoxicity of conjugates exhibited a slower onset at shorter exposure times than mAb hyper-crosslinked with a secondary Ab; however, at longer time intervals the HPMA copolymer conjugates achieved significantly higher biological efficacies. In addition, study of the relationship between the structure of conjugates and Raji B cell apoptosis revealed that both valency and polymer molecular weight influenced biological activities, while insertion of peptide sequences into the backbone was not a factor in vitro.

Immunogenicity of coiled-coil based drug-free macromolecular therapeutics

“…A copolymer of HPMA and APMA was synthesized using reversible addition-fragmentation chain transfer (RAFT) polymerization as previously described [28]. Briefly, the monomers HPMA and APMA, chain transfer agent CPDB, and V-501 were placed in an ampule and sealed after bubbling the solution with nitrogen.…”

Drug-free macromolecular therapeutics – a new paradigm in polymeric nanomedicines

“…Previously we attempted to increase the valence by using branched polymer backbones 113,114 or linear, high-MW copolymers synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. 115 Other researchers used biological polymers such as DNA or polypeptides as scaffolds to attach antibodies or the smaller size single-chain variable fragments (scFv). 17,116 These polyvalent constructs indeed achieved better efficacies than their monovalent counterparts.…”

“…Previously we have shown that, in addition to the valence, the MW of the polymer backbone also had a positive influence on the efficiency of CD20 crosslinking and apoptosis in vitro . 115 In the body, HPMA copolymers with larger MW tend to circulate longer in the blood. 64,65 This characteristic is favorable for targeting blood cancers such as lymphomas.…”

Antibody fragment-conjugated polymeric micelles incorporating platinum drugs for targeted therapy of pancreatic cancer

“…The hydrodynamic diameter of Mal-DACHPt/m remained close to 30 nm even after Fab' conjugation (Tables 1 and 2). This might be reasonable considering the diameter of Fab' as that of a volumeequivalent sphere of 4e6 nm [46] (Table 2). This relatively small size of anti-TF Fab'-DACHPt/m could be a significant advantage for effective extravasation and penetration in stroma-rich tumor tissues of pancreatic cancer [35].…”