Engineered Antibody Fragment against the Urokinase Plasminogen Activator for Fast Delineation of Triple-Negative Breast Cancer by Positron Emission Tomography

Abstract

The urokinase plasminogen activator (uPA) and its cofactors are important regulators of tumor initiation and progression (including metastasis), and its overexpression is associated with unfavorable situations in cancer patients. We have previously used positron emission tomography (PET) imaging with a radiolabeled monoclonal antibody against the uPA (named ATN-291) to detect the uPA signaling activity in various cancer types; however, good tumor contrast can only be observed 24 h postinjection. To shorten the antibody circulation time and decrease interactions of ATN-291 with the mononuclear phagocyte system (MPS), our goal in this study is to develop an engineered antibody fragment (F­(ab′)2) from the parent antibody. By pepsin digestion and chromatography purification, ATN-291 F­(ab′)2 was obtained and characterized. Subsequently, it was conjugated with NOTA-Bn-NCS or fluorescein isothiocyanate (FITC) for PET imaging and fluorescence-mediated cellular analysis (i.e., flow cytometry or fluorescence microscopy). We confirmed that ATN-291 F­(ab′)2 still maintained a good targeting efficacy for the uPA in MDA-MB-231 cells (uPA+) and it had a faster blood clearance speed compared with ATN-291, while its interaction with MPS has been significantly decreased. In rodent tumor xenografts, radiolabeled ATN-291 F­(ab′)2 had a selective and persistent uptake in MDA-MB-231 tumors, with an early tumor-to-blood ratio of 1.3 ± 0.8 (n = 4) at 2 h postinjection from PET imaging. During our observation, radiolabeled ATN-291 F­(ab′)2 was excreted from both renal and hepatobiliary pathways. Radiolabeled ATN-291 F­(ab′)2 was also used for detecting uPA fluctuation during the tumor treatment in test animals. We concluded that radiolabeled ATN-291 F­(ab′)2 could be used as fast as PET cancer diagnostics with versatile applicability.

Therapeutic Strategies Targeting Urokinase and Its Receptor in Cancer

“…Subsequently, ATN-291 F(ab )2 was conjugated with NOTA-Bn-NCS for PET imaging. The monitoring of radiolabeled ATN-291 F(ab )2 permits to follow the changes of uPA levels all along the treatment [195].…”

Immuno-PET Imaging of TNF-α in Colitis Using 89Zr-DFO-infliximab

“…PET imaging has been employed in IBD diagnosis in the clinic using 18 F-FDG based on the increase of glucose metabolism in immune cells . Immuno-PET is a technology combining the sensitivity of PET and superior targeting specificity of monoclonal antibody (mAb), − which has also been used in IBD diagnosis. , Radiolabeled antibodies, 89 Zr-labeled GK1.5 cys-diabody, 89 Zr-labeled anti-CD11b, and anti-IL-1β mAbs, were used as PET probes in imaging of CD4 + T cells, CD11b-expressing cells, and innate immune cell-secreted IL-1β, respectively. , The immuno-PET imaging reveals the immune cells or pro-inflammatory cytokines activity during the colitis, which is a useful tool to monitor disease severity and predict treatment success.…”