(175u) Engineering an ?-P-Selectin Antibody for Quantifying Drug Delivery in Glioblastoma

Glioblastoma Multiforme (GBM), the most common and aggressive form of primary brain cancer, has a 5-year survival rate of ~5%, creating a clear medical need for more effective therapies. A major obstacle is the migration of neoplastic cells from the main bulk of the tumor, where the blood-brain barrier (BBB) may be compromised, to regions in which intact BBB prevents accumulation of anti-neoplastic agents. Many strategies have been proposed to improve drug delivery to GBM, including local injection (e.g., convection enhanced delivery) and receptor mediated transcytosis of protein or nanoparticle delivery systems. Recently, P-selectin, an inducible endothelial marker associated with tumors and sites of inflammation, has been identified as a potential target for drug delivery to GBM. Specifically, both fucoidan and dendritic polyglycerol sulfate nanocarriers, which have inherent affinity for P-selectin, have been shown to target brain endothelium and even potentially cross tumor endothelial cells via receptor-mediated transcytosis. While these approaches may have translational potential, the relative delivery of P-selectin targeted therapeutics to normal vs. tumor endothelium has yet to be quantified. The goal of our work was to create a recombinant antibody specific for murine P-selectin to enable quantification of affinity ligand and drug conjugate delivery to GBM-associated vs. normal brain endothelium. Lysate from the RMP-1 hybridoma, which produces a mouse and rat-P-selectin cross-reactive antibody (PMID 9219035), was obtained from Dr. Andrew Issekutz. Following isolation of total cellular RNA, sequences of the variable regions of the heavy and light chain were obtained and inserted into a pTT5 expression vector. CDRs obtained from Dr. Stephen Tomlinson were used to generate alternative α-P-selectin mAbs Psel2.3 and Psel2.12, which are cross-reactive between human and mouse P-selectin. HEK293 cells were transiently co-transfected with plasmids containing the heavy and light chain of the antibodies, and the protein was purified using protein A agarose beads. The antibodies were characterized using HPLC-SEC and SDS-PAGE under reduced and non-reduced conditions. All mAbs had yields of greater than 14 mg/L, and the purities were determined to be >99% after characterization. Cells expressing mouse P-selectin were made for flow cytometry-based cellular binding and compared to a non-expressing control. The binding affinity of RMP-1 was found using both a secondary antibody and conjugating AF-647 to RMP-1, giving a K_D of 1.12 ± 0.29 nM and 1.40 ± 0.30 nM, respectively, with insignificant binding to control cells. Psel2.3 and Psel2.12, however, had lower K_D values of 11.87 ± 3.18 and 11.65 ± 1.35, respectively. Based on these results, we successfully engineered multiple recombinant mouse P-selectin antibodies that can be expressed with good yield, high purity, and high specificity towards its target. We used quantitative radiolabeling by conjugating radioisotope I-125 to the P-selectin antibodies to track localization of the antibody in vivo. Comparison of these antibodies with other relevant antibodies such as inflammation targeting α-VCAM-1, BBB targeting α-CD98hc, and IgG isotype control in wild-type mice will be used to determine base pharmacokinetics (PK) and tissue distribution of the antibodies, while further study in a glioblastoma model can determine PK changes due to inflammation and variations in receptor expression. This project will assess the efficacy of an α-P-selectin antibody in improving targeted localization in GMB-associated endothelium, providing a novel path for the development of enhanced protein therapeutics for the treatment of glioblastoma.