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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

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posted on 2023-03-31, 20:11 authored by Kirstin A. Zettlitz, Richard Tavaré, Scott M. Knowles, Kristopher K. Steward, John M. Timmerman, Anna M. Wu

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.

Funding

NIH

Ralph and Marjorie Crump to the Crump Institute

UCLA Jonsson Comprehensive Cancer Center

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ARTICLE ABSTRACT

Purpose: The B-cell antigen CD20 provides a target for antibody-based positron emission tomography (immunoPET). We engineered antibody fragments targeting human CD20 and studied their potential as immunoPET tracers in transgenic mice (huCD20TM) and in a murine lymphoma model expressing human CD20.Experimental Design: Anti-CD20 cys-diabody (cDb) and cys-minibody (cMb) based on rituximab and obinutuzumab (GA101) were radioiodinated and used for immunoPET imaging of a murine lymphoma model. Pairwise comparison of obinutuzumab-based antibody fragments labeled with residualizing (89Zr) versus non-residualizing (124I) radionuclides by region of interest analysis of serial PET images was conducted both in the murine lymphoma model and in huCD20TM to assess antigen modulation in vivo.Results:124I-GAcDb and 124I-GAcMb produced high-contrast immunoPET images of B-cell lymphoma and outperformed the respective rituximab-based tracers. ImmunoPET imaging of huCD20TM showed specific uptake in lymphoid tissues. The use of the radiometal 89Zr as alternative label for GAcDb and GAcMb yielded greater target-specific uptake and retention compared with 124I-labeled tracers. Pairwise comparison of 89Zr- and 124I-labeled GAcDb and GAcMb allowed assessment of in vivo internalization of CD20/antibody complexes and revealed that CD20 internalization differs between malignant and endogenous B cells.Conclusions: These obinutuzumab-based PET tracers have the ability to noninvasively and quantitatively monitor CD20-expression and have revealed insights into CD20 internalization upon antibody binding in vivo. Because they are based on a humanized mAb they have the potential for direct clinical translation and could improve patient selection for targeted therapy, dosimetry prior to radioimmunotherapy, and prediction of response to therapy. Clin Cancer Res; 23(23); 7242–52. ©2017 AACR.

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