89Zr- and 124I-anti-CD20 immunoPET SI from ImmunoPET of Malignant and Normal B Cells with ⁸⁹Zr- and ¹²⁴I-Labeled Obinutuzumab Antibody Fragments Reveals Differential CD20 Internalization In Vivo

Supplementary data Table S1. Ex vivo biodistribution (24 h p.i.) of antibody fragments in SCID mice bearing 38C13-huCD20, blocked or CD20-negative 38C13 subcutaneous allografts. Data are presented as mean (%ID/g) with standard deviation ({plus minus} SEM). Table S2. Ex vivo biodistribution (24 h p.i.) of GA101 cys-diabody and cys-minibody fragments in human CD20 transgenic mice (huCD20TM). Data are presented as mean (%ID/g) with standard deviation ({plus minus} SEM). Figure S1 Biochemical characterization. (A) SDS-PAGE analysis of purified rituximab-based cys-diabody (RxcDb, 52 kDa) and cys-minibody (RxcMb, 82 kDa) shows correctly assembled dimers under non-reducing conditions (NR) and monomeric fragments under reducing conditions. (B) Size exclusion chromatography of engineered antibody fragments in comparison to full-length rituximab IgG. Rx cMb eluted as a single peak corresponding to the expected molecular weight. Rx cDb eluted slightly later, corresponding to the 52 kDa dimer but also showed a smaller second peak of higher molecular weight. Figure S2. (A) Schematic representation of site-specific conjugation to the C-terminal cys-tag of cys-diabody and cys-minibody antibody fragments, respectively. (B) Reduction and conjugation of Alexa Fluor 488 Maleimide (using the example of the cys-diabody) results in monomeric fragments migrating under non-reducing conditions in SDS-PAGE shown as Coomassie stain (left panel) and fluorescence (right panel). (C) Size exclusion chromatography analysis comparing unconjugated (black) and mal-DFo conjugated (grey) antibody fragments (using Superdex 75 column for cys-diabodies and Superdex 200 column for cys-minibodies. Figure S3. Obinutuzumab (GA101)-based antibody fragments retain type II binding characteristics. Figure S4. Immunoreactive fractions of radiolabeled antibody fragments were determined by cell binding studies using CD20 positive Ramos cells and CD20 negative Bc-1 cells. Shown is one representative plot for 124I-GAcDb. Immunoreactive fractions for obinutuzumab (GA1010)-based fragments ranged from 65-85 %. Figure S5 89Zr-GAcMb immunoPET imaging of huCD20TM. Figure S6 Immunohistochemical staining of human CD20 expression in murine 38C13 B-cell lymphoma allografts.