title: Drug-free macromolecular therapeutics: Impact of structure on induction of apoptosis in Raji B cells.
abstract: Recently, we developed a new paradigm in macromolecular therapeutics that avoids the use of low molecular weight drugs. The activity of the "drug-free macromolecular therapeutics" is based on the biorecognition of complementary motifs at cell surface resulting in receptor crosslinking and apoptosis induction. The system is composed of two nanoconjugates: (1) a single-stranded morpholino oligonucleotide (MORF1) attached to an anti-CD20 Fab' fragment (Fab'-MORF1); (2) multiple copies of complementary oligonucleotide MORF2 grafted to a linear polymer of N-(2-hydroxypropyl)methacrylamide (HPMA) - P-(MORF2)x. The two conjugates crosslink CD20 antigens via MORF1-MORF2 hybridization at the surface of CD20+malignant B-cells and induce apoptosis. Preclinical studies in a murine model of human non-Hodgkin's lymphoma showed cancer cells eradication and long-term survivors. The aim of this study was to determine the relationship between the detailed structure of the nanoconjugates and apoptosis induction in Raji cells to allow system optimization. The factors studied include the length of the MORF sequence, the valence of P-(MORF2)x(varying x), molecular weight of P-(MORF2)x, incorporation of a miniPEG spacer between Fab' and MORF1 and between polymer backbone and pendant MORF2, and comparison of two Fab' fragments, one from 1F5 antibody (Fab'1F5), the other from Rituximab (Fab'RTX). The results of apoptosis induction in human Burkitt's B-cell non-Hodgkin's lymphoma (NHL) Raji cells as determined using three apoptotic assays (Annexin V, Caspase 3, and TUNEL) indicated that: a) An improvement of apoptotic activity was observed for a 28 base pair MORF sequence when compared to MORFs composed of 20 and 25 base pairs. The differences depended on type of assay, concentration and exposure schedule (consecutive vs. premixed). b) The higher the valence of P-(MORF2)xthe higher the levels of apoptosis. c) Higher molecular weight of P-(MORF2)xinduced higher levels of apoptosis. d) A miniPEG8spacer was effective in enhancing apoptotic levels in contrast to a miniPEG2spacer. e) There was not a statistically significant difference when comparing Fab'1F5-MORF1 with Fab'RTX-MORF1.
authors.affiliation: Department of Pharmaceutics and Pharmaceutical Chemistry, CCCD, University of Utah, Salt Lake City, UT 84112, USA.
authors.forename: Libin
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Department of Pharmaceutics and Pharmaceutical Chemistry, CCCD, University of Utah, Salt Lake City, UT 84112, USA.
authors.forename: Yixin
authors.initials: Y
authors.lastname: Fang
authors.affiliation: Department of Pharmaceutics and Pharmaceutical Chemistry, CCCD, University of Utah, Salt Lake City, UT 84112, USA.
authors.forename: Jiyuan
authors.initials: J
authors.lastname: Yang
authors.affiliation: Department of Pharmaceutics and Pharmaceutical Chemistry, CCCD, University of Utah, Salt Lake City, UT 84112, USA; Department of Bioengineering, University of Utah, Salt Lake City, UT 84112, USA. Electronic address: jindrich.kopecek@utah.edu.
authors.forename: Jindřich
authors.initials: J
authors.lastname: Kopeček
mesh_terms: D018951:Antigens, CD20 / Q000008:administration & dosage / Q000737:chemistry; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000008:administration & dosage / Q000737:chemistry; D015394:Molecular Structure; D060172:Morpholinos / Q000008:administration & dosage / Q000737:chemistry
chemical_list: D018951:Antigens, CD20; D007140:Immunoglobulin Fab Fragments; D060172:Morpholinos
keywords: Apoptosis; B-cell lymphoma; Biorecognition; CD20; Drug-free macromolecular therapeutics; Morpholino oligonucleotide; N-(2-hydroxypropyl)methacrylamide (HPMA); Receptor crosslinking