Development and Validation of an Immuno-PET Tracer as a Companion Diagnostic Agent for Antibody-Drug Conjugate Therapy to Target the CA6 Epitope

Abstract

Purpose:To develop and compare three copper 64 ( 64 Cu)-labeled antibody fragments derived from a CA6-targeting antibody (huDS6) as immuno-positron emission tomography (immuno-PET)-based companion diagnostic agents for an antibody-drug conjugate by using huDS6. Materials and Methods:Three antibody fragments derived from huDS6 were produced, purified, conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and evaluated in the following ways: (a) the affinity of the fragments and the DOTA conjugates was measured via flow cytometry, (b) the stability of the labeled fragments was determined ex vivo in human serum over 24 hours, and (c) comparison of the in vivo imaging potential of the fragments was evaluated in mice bearing subcutaneous CA6-positive and CA6-negative xenografts by using serial PET imaging and biodistribution. Isotype controls with antilysozyme and anti-DM4 B-Fabs and blocking experiments with an excess of either B-Fab or huDS6 were used to determine the extent of the antibody fragment 64 Cu-DOTA-B-Fab binding specificity. Immunoreactivity and tracer kinetics were evaluated by using cellular uptake and 48-hour imaging experiments, respectively. Statistical analyses were performed by using t tests, one-way analysis of variance, and Wilcoxon and Mann-Whitney tests. Results:The antibody fragment 64Cu-DOTA-B-Fab was more than 95% stable after 24 hours in human serum, had an immunoreactivity of more than 70%, and allowed differentiation between CA6-positive and CA6-negative tumors in vivo as early as 6 hours after injection, with a 1.7-fold uptake ratio between tumors. Isotype and blocking studies experiments showed tracer-specific uptake in antigen-positive tumors, despite some nonspecific uptake in both tumor models. Conclusion:Three antibody fragments were produced and examined as potential companion diagnostic agents. 64 Cu-DOTA-BFab is a stable and effective immuno-PET tracer for CA6 imaging in vivo.q RSNA, 2015

Human umbilical cord plasma proteins revitalize hippocampal function in aged mice

“…Conjugation of recombinant mouse TIMP2 (R&D) and BSA (Sigma) with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was performed according to standard published procedures using metal-free buffers 46, 47 . Briefly, a solution of DOTA-NHS ester (Macrocyclics) in dimethyl sulfoxide (25 mmol l −1 ; 9–12 μl) was added to 200 μl of HEPES buffer (0.1 mol l −1 , pH 8.8) containing 100 μg of TIMP2 or BSA, and the reaction mixture was incubated at room temperature for 1 h. Excess DOTA-NHS was removed by Zeba Spin Desalting Columns (0.5 ml, 7K molecular weight cut-off, ThermoFisher Scientific), and the resulting solution was buffer-exchanged into ammonium acetate buffer (0.1 M, pH 5.5) for 64 Cu labelling.…”

“…Radiolabelling of DOTA–TIMP2 and DOTA–BSA with 64 Cu (half-life = 12.7 h) was performed using standard methods with some modifications 46, 47, 48 . In brief, DOTA–TIMP2 (25 μg) or DOTA–BSA (25 μg) in 50 μl of 0.25 mol l −1 ammonium acetate buffer (0.1 M, pH 5.5) was mixed with pH-balanced [ 64 Cu]Cl 2 solution (44–74 MBq, pH 5–5.5, University of Wisconsin, Madison) at 37 °C with gentle shaking at 300 r.p.m.…”

An antibody drug conjugate targeting a GSTA glycosite-signature epitope of mucin1 expressed by non-small cell lung cancer

“…28 However, the exact glycopeptide sequences in every individual cancer patients are highly variable and diverse, due to the "assembly line" nature of glycosylation pathway. 29 Big data on the glycopeptide epitopes caused by such abnormal glycosylation on specific glycoproteins in cancer population is unavailable. We previously predicted the glycopeptide sequences in lung cancer by MATLAB software.…”

Theranostics Using Antibodies and Antibody-Related Therapeutics

“…Naked antibody uptake is assumed to reflect ADC uptake and thus may predict whether a patient will respond to ADC therapy. This approach was used in 3 preclinical trials in mice and 1 study in both mice and nonhuman primates (37)(38)(39)(40). Organ biodistribution and tracer tumor uptake were assessed.…”