Identification of a moderate affinity CD22 binding peptide and in vitro optimization of peptide-targeted nanoparticles for selective uptake by CD22+ B-cell malignancies

Abstract

B cell malignancies, such as B cell leukemia and lymphoma, have CD22 overexpression with ∼7% of patients. A short CD22 binding peptide (PV3) with a moderate affinity (K_d ∼ 9 μM) was identified by screening multiple peptide candidates determined through analysis of CD22-epratuzumab complex crystal structure. PV3 binding specificity was confirmed via competitive binding inhibition, then was used as the targeting moiety on CD22-targeted liposomal nanoparticle (TNP^PV3) formulations. To maximize the potential therapeutic outcome of TNP^PV3 formulation, nanoparticle design parameters, such as peptide hydrophilicity, ethylene glycol linker length, valency, and particle size were optimized for maximum selective cellular uptake by CD22+ malignant cancer cells. The effects of altering design parameters one at a time on TNP uptake were evaluated using flow cytometry, and the optimal parameters for TNP^PV3 were determined to be 8% peptide density, EG18 linker, and 3 lysines of 100 nm nanoparticles. This optimally designed TNP^PV3 achieved ∼4 and 40-fold enhancement of cellular uptake by CD22+ Raji cells over CD22− Jurkat and MOLT-4 cells, respectively, demonstrating selectivity for malignant cells with CD22 overexpression. Overall, this study establishes PV3 to be CD22 binding peptide with proven effectiveness as a targeting element. In future, the optimal TNP^PV3 formulation will potentially achieve maximal in vivo therapeutic outcomes by efficiently targeting CD22+ blood cancer cells in vivo.