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.

Fig. 1

Synthesis of multivalent HPMA copolymer-Fab′ conjugates targeted to B cell antigen CD20. (A) Schemes for the synthesis of polymer precursors (P-NH₂, P-mal) and multivalent conjugates (P-Fab′). (B) Schematics of polymer conjugate architectures.

Fig. 2

(Top panel) SEC profiles of harvested 1F5 mAb, digested F(ab′)₂ and reduced Fab′ fragments by ÄKTA FPLC (Superdex 200 HR10/30 column, PBS). Purity of all products from each step were >95%. (Bottom panel) SEC profiles of representative P-NH₂ (P1) and P-Fab′ (P1-Fab′) by ÄKTA FPLC (Superpose 6 HR10/30 column, acetate buffer + 30% acetonitrile v/v).

Fig. 3

Confocal fluorescence microscopic images of Raji B cells exposed to (A) PBS, (B) 1F5 mAb labeled with FITC, (C) F(ab′)₂ antibody fragment labeled with rhodamine, and (D) P-Fab′ conjugate labeled with rhodamine. Raji cells (2.5 × 10⁵) were stained with varying concentrations of each compound for 2 h prior to analysis.

Fig. 4

Apoptosis induction of Raji B cell analyzed by (A) caspase-3 activity, (B) Annexin V binding, and (C) TUNEL assays. Quantification was performed by flow cytometry. All experiments were carried out in at least triplicate (data shown as mean ± SD). Statistics was performed by comparing each group with the untreated (*: p < 0.05).

Fig. 5

Time-dependent cell viability study assessed by propidium iodide (PI) binding. Treatment conditions were identical to apoptosis assays. Quantification was performed by flow cytometry. Experiments were carried out in triplicate (data shown as mean ± SD). (■) Untreated; (●) mAb+2°Ab; (▲) P1-Fab′; (▽) P2-Fab′; (×) P2a-Fab′; (○) P2b-Fab′; (□) P3-Fab′.

Fig. 6

Cell apoptosis evaluated by Annexin V binding in (A) time-dependent, and (B) concentration-dependent assays. All experiments were carried out in triplicate (data shown as mean ± SD). Statistical analyses (unless otherwise indicated) were performed by comparing each group with the corresponding shortest time interval or lowest concentration (*: p < 0.05, by Student’s t test).