18F-labeled anti-human CD20 cys-diabody for same-day immunoPET in a model of aggressive B cell lymphoma in human CD20 transgenic mice

Abstract

Purpose Metabolic imaging using [ 18 F]FDG is the current standard for clinical PET; however, some malignancies (e.g., indolent lymphomas) show low avidity for FDG. The majority of B cell lymphomas express CD20, making it a valuable target both for antibody-based therapy and imaging. We previously developed PET tracers based on the humanised anti-CD20 antibody obinutuzumab (GA101). Preclinical studies showed that the smallest bivalent fragment, the cys-diabody (GAcDb, 54.5 kDa) with a peak uptake at 1-2 h post-injection and a biological half-life of 2-5 h, is compatible with short-lived positron emitters such as fluorine-18 (18 F, t 1/2 110 min), enabling same-day imaging. Methods GAcDb was radiolabeled using amine-reactive N-succinimidyl 4-[ 18 F]-fluorobenzoate ([ 18 F]SFB), or thiol-reactive N-[2-(4-[ 18 F]-fluorobenzamido)ethyl]maleimide ([ 18 F]FBEM) for site-specific conjugation to C-terminal cysteine residues. Both tracers were used for immunoPET imaging of the B cell compartment in human CD20 transgenic mice (hCD20TM). [ 18 F]FB-GAcDb immunoPET was further evaluated in a disseminated lymphoma (A20-hCD20) syngeneic for hCD20TM and compared to [ 18 F]FDG PET. Tracer uptake was confirmed by ex vivo biodistribution. Results The GAcDb was successfully 18 F-radiolabeled using two different conjugation methods resulting in similar specific activities and without impairing immunoreactivity. Both tracers ([ 18 F]FB-GAcDb and [ 18 F]FBEM-GAcDb) specifically target human CD20-expressing B cells in transgenic mice. Fast blood clearance results in high contrast PET images as early as 1 h post injection enabling same-day imaging. [ 18 F]FB-GAcDb immunoPET detects disseminated lymphoma disease in the context of normal tissue expression of hCD20, with comparable sensitivity as [ 18 F]FDG PET but with added specificity for the therapeutic target. Conclusions [ 18 F]FB-GAcDb and [ 18 F]FBEM-GAcDb could monitor normal B cells and B cell malignancies non-invasively and quantitatively in vivo. In contrast to [ 18 F]FDG PET, immunoPET provides not only information about the extent of disease but also about presence and localisation of the therapeutic target.

A Dual-Modality Linker Enables Site-Specific Conjugation of Antibody Fragments for 18F-Immuno-PET and Fluorescence Imaging

“…The analysis suggested that sameday imaging would be best accomplished with the diabody format (3-to 5-h interval after injection) and that if the imaging figure of merit was calculated for 18 F, only the diabody was competitive, as the larger-sized fragments have half-lives that are too long and the smaller monovalent scFv showed much shorter tumor retention. The in vivo results using 18 F-DMLsCy5-A2cDb presented in this work indeed show that same-day immuno-PET imaging is feasible and further corroborate previous studies with 18 F-labeled diabodies in several models (human epidermal growth factor receptor 2, carcinoembryonic antigen, CD20) (29)(30)(31).…”

Distinguishing Between Monomeric scFv and Diabody in Solution Using Light and Small Angle X-ray Scattering

“…In a broad range of scattering angles and concentrations the experimental data showed the expected linear behavior and consequently the Zimm formalism can be applied to these linear regions. In Figure 8 the results with a linear fit and the extrapolation according to the Equations (13) and (14). The intercept of both extrapolated lines with the y-axis is at 2.0666 × 10 −5 mol·g −1 .…”

“…Antibodies feature highly specific binding to a wide range of antigens. Therefore, antibodies are being increasingly used for therapeutical purposes, such as passive immunization [5,6], immunomodulation (e.g., as therapy for multiple sclerosis) [7,8], cancer therapy (e.g., Trastuzumab [9][10][11] or monomethyl auristatin E [12]) or for medical imaging (e.g., immunoscintigraphy [13,14]). Besides the use of whole antibodies, the application of antibody fragments is widely spread and provides further advantages [15,16].…”

A promising cancer diagnosis and treatment strategy: targeted cancer therapy and imaging based on antibody fragment

“…Based on the comprehension of antibody structure and the development of immunology, molecular biology, diversified antibody fragments (as illustrated in Figure 1) are constructed through antibody engineering. All antibody fragments retain antigen-specific [26]. Meanwhile, current antibody fragment preparation techniques are relatively complete and there are many methods to choose from, such as phage display technology, yeast display technology [27][28][29].…”