EXTH-29. MULTI-RECEPTOR TARGETING IN GBM. (5th November 2018)

• Record Type: Journal Article
• Authors: Sharma, Puja; Herpai, Denise; Rossmeisl, John; Tatter, Stephen; Debinski, Waldemar
• Is Part Of: Neuro-oncology. Volume 20(2018)Supplement 6
• Page Start: vi91
• Page End: vi91
• Publication Date: 2018-11-05
• Subjects: Brain Neoplasms -- Periodicals; Brain -- Tumors -- Periodicals; Brain -- Cancer -- Periodicals; Nervous system -- Cancer -- Periodicals; 616.99481
• DOI: 10.1093/neuonc/noy148.378
• Languages: English
• ISSNs: 1522-8517

Abstract

Abstract: Glioblastoma is a heterogeneous tumor for which all single-targeted agents failed. Being that a cytotoxic cocktail targeting IL-13RA2 and EphA2 receptors is effective in the treatment of spontaneous gliomas in dogs, we have been further pursuing molecular resection by targeting four tumor-specific/associated receptors at once: IL-13RA2, EphA2, EphA3 and EphB2. These receptors are over-expressed in most GBM tumor compartments responsible for tumor progression and recurrence. To this end, we have engineered a protein based on an immunoglobulin scaffold with a ligand targeting the three Eph receptors, ephrinA5 (eA5), at the N-terminal end and a ligand targeting IL-13RA2, IL-13.E13K, at the C-terminal end (QUAD). However, the protein produced in insect cells had frequently two bands on SDS-PAGE. We have therefore decided to change the order of the ligands and placed the ligand targeting IL-13RA2 at the N-terminal end of the protein and the other ligand and the C-terminus. We also started using High-Five cells for codon and yield optimization. The newer construct is produced as a single protein band on the SDS-PAGE and Western blot and can be purified to homogeneity. It bound all four receptors effectively in ELISA while the affinity toward the EphA3 was even better than in the original multivalent compound. We also obtained higher yields of the protein. To make our vector even more versatile, we have added a C-terminal cysteine providing a free thiol group for chemical conjugation with drugs. The QUAD-Cys was also obtained as a single band protein homodimer and the multi-targeted protein bound effectively to all targets of interest. Thus, we have optimized the production of a functional multivalent targeted vector protein for making drug conjugates. QUAD and QUAD-Cys based conjugates will be tested for their utility in glioma treatment using relevant clinical models like spontaneous brain tumors in dogs.