Hybrid nanomaterials composed of synthetic and biological building blocks possess high potential for the design of nanomedicines. The use of self-assembling nanomaterials as “bio-mimics” may trigger cellular events and result in new therapeutic effects. Motivated by this rationale, we designed a therapeutic platform that mimics the mechanism of immune effector cells to crosslink surface receptors of target cells and induce apoptosis. This platform was tested against B-cell lymphomas that highly express the surface antigen CD20. Here, two nanoconjugates were synthesized: (1) an anti-CD20 Fab’ fragment covalently linked to a single-stranded morpholino oligonucleotide (MORF1), and (2) a linear polymer of N-(2-hydroxypropyl)methacrylamide (HPMA) grafted with multiple copies of the complementary oligonucleotide MORF2. We show that the two conjugates self-assemble via MORF1-MORF2 hybridization at the surface of CD20+ malignant B-cells, which crosslinks CD20 antigens and initiates apoptosis. When tested in a murine model of human non-Hodgkin’s lymphoma, the two conjugates, either administered consecutively or as a premixture, eradicated cancer cells and produced long-term survivors. The designed therapeutics contains no small-molecule cytotoxic compounds and is immune-independent, aiming to improve over chemotherapy, radiotherapy and immunotherapy. This therapeutic platform can be applied to crosslink any non-internalizing receptor and potentially treat other diseases.

The use of rituximab, an anti-CD20 mAb, in combination with chemotherapy is the current standard for the treatment of B-cell lymphomas. However, because of a significant number of treatment failures, there is a demand for new, improved therapeutics. Here, we designed a nanomedicine that crosslinks CD20 and directly induces apoptosis of B-cells without the need for toxins or immune effector functions. The therapeutic system comprises a pretargeting component (anti-CD20 Fab' conjugated with an oligonucleotide1) and a crosslinking component (N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer grafted with multiple complementary oligonucleotide2). Consecutive treatment with the two components resulted in CD20 clustering on the cell surface and effectively killed malignant B-cells in vivo. To enhance therapeutic efficacy, a two-step pretargeting approach was employed. We showed that the time lag between the two doses can be optimized based on pharmacokinetics and biodistribution of the Fab'-oligonucleotide1 conjugate. In a mouse model of human non-Hodgkin lymphoma (NHL), increasing the time lag from 1 h to 5 h resulted in dramatically improved tumor growth inhibition and animal survival. When the 5 h interval was used, the nanotherapy was more efficacious than rituximab and led to complete eradication of lymphoma cells with no signs of metastasis or disease recurrence. We further evaluated the nanomedicine using patient mantle cell lymphoma cells; the treatment demonstrated more potent apoptosis-inducing activity than rituximab hyper-crosslinked with secondary antibodies. In summary, our approach may constitute a novel treatment for NHL and other B-cell malignancies with significant advantages over conventional chemo-immunotherapy.

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.

This review highlights a unique research area in polymer-based nanomedicine designs. Drug-free macromolecular therapeutics induce apoptosis of malignant cells by the crosslinking of surface non-internalizing receptors. The receptor crosslinking is mediated by the biorecognition of high-fidelity natural binding motifs (such as antiparallel coiled-coil peptides or complementary oligonucleotides) that are grafted to the side chains of polymers or attached to targeting moieties against cell receptors. This approach features the absence of low-molecular-weight cytotoxic compounds. Here, we summarize the rationales, different designs, and advantages of drug-free macromolecular therapeutics. Recent developments of novel therapeutic systems for B-cell lymphomas are discussed, as well as relevant approaches for other diseases. We conclude by pointing out various potential future directions in this exciting new field.