Prostate-Specific Membrane Antigen (PSMA) is an established biomarker for the imaging and experimental therapy of prostate cancer (PCa), as it is strongly upregulated in high-grade primary, androgen-independent, and metastatic lesions. Here, we report on the development and functional characterization of recombinant single-chain Fv (scFv) and Fab fragments derived from the 5D3 PSMA-specific monoclonal antibody (mAb). These fragments were engineered, heterologously expressed in insect S2 cells, and purified to homogeneity with yields up to 20 mg/L. In vitro assays including ELISA, immunofluorescence and flow cytometry, revealed that the fragments retain the nanomolar affinity and single target specificity of the parent 5D3 antibody. Importantly, using a murine xenograft model of PCa, we verified the suitability of fluorescently labeled fragments for in vivo imaging of PSMA-positive tumors and compared their pharmacokinetics and tissue distribution to the parent mAb. Collectively, our data provide an experimental basis for the further development of 5D3 recombinant fragments for future clinical use.

Laboratory of Structural Biology Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Prumyslova 595 252 50 Vestec Czech Republic

Mass Spectrometry Group Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences IOCB and Gilead Research Center Flemingovo náměstí 542 2 160 00 Prague Czech Republic

The Russell H Morgan Department of Radiology and Radiological Science Johns Hopkins Medical Institutions 1550 Orleans St Baltimore MD 21231 USA

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Targeting Prostate Cancer Using Bispecific T-Cell Engagers against Prostate-Specific Membrane Antigen