Drug-Free Macromolecular Therapeutics Induce Apoptosis in Cells Isolated from Patients with B Cell Malignancies with Enhanced Apoptosis Induction By Pretreatment with Gemcitabine

• Published in: Blood 2018-11, Vol.132 (Supplement 1), p.4426-4426
• Main Authors: Wang, Jiawei, Li, Lian, Yang, Jiyuan, Clair, Phillip M., Glenn, Martha, Stephens, Deborah M., Radford, D. Christopher, Kosak, Ken M., Deininger, Michael W., Shami, Paul J, Kopeček, Jindřich
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2018-99-112001

Background Anti-CD20 mAbs are an important element in the therapeutic armamentarium for B-cell malignancies such as chronic lymphocytic leukemia (CLL). Although new targeted drugs (Ibrutinib & Idelalisib) and recently developed anti-CD20 mAbs (obinutuzumab & ofatumumab) have significant activity, the clinical management is limited by toxicity or low sensitivity to anti-CD20 mAbs. Herein we propose Drug-Free Macromolecular Therapeutics (DFMT) as a novel class of therapeutics that amplifies CD20 crosslinking and triggers apoptosis. The unique features of DFMT include augmentation of multivalent CD20 crosslinking, immune effector independence and no cytotoxic agents. Its effectiveness has been demonstrated in vitro and in preclinical Non-Hodgkin Lymphoma (NHL) models including a CD20-deficient rituximab (RTX)-resistant model, in which DFMT combined with a long-circulating polymer-gemcitabine conjugate (2P-GEM), induced prolonged survival and tumor clearance from bone marrows.[Zhang R, et al. PNAS 2014; Li L, et al. ACS Nano 2018] Therefore, we conducted assessment of DFMT on patient samples. Methods Malignant B cells were isolated from patients diagnosed by the hematologic malignancies service at the Huntsman Cancer Institute. The majority of isolates were from CLL patients. CD20 expression of each sample was evaluated by flow cytometry. DFMT consisting of two nanoconjugates was used to treat the cells in consecutive administration: first, cells were incubated with Fab‘-MORF1 (bispecific engager, Fab’ fragment of RTX conjugated with a morpholino oligonucleotide); after 1 h, a crosslinking effector (CLE) containing multiple copies of complementary morpholino oligonucleotide (MORF2) was added and the cells were continually incubated for another 6-20 h (depending on assays). The CLE can be prepared by conjugation of MORF2 to either N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer precursor or human serum albumin. Crosslinking of CD20 receptors is initiated by the multiple hybridization of MORF1/MORF2 at the cell surface, and results in apoptosis (Fig. 1A). Annexin V and caspase 3 assays were used to quantify B-cell apoptosis. RTX followed by goat-anti-human (GAH) secondary antibody was used for comparison. GAH was added to imitate the function of Fcγ+ immune effector cells for crosslinking of RTX. When enough cells were available, a detailed evaluation of apoptosis induction was performed, i.e. plasma membrane rupture, genomic DNA fragmentation, mitochondrial