Prostate cancer (PCa) ranks as the second leading cause of cancer-related deaths among men in the United States. Prostate-specific membrane antigen (PSMA) represents a well-established biomarker of PCa, and its levels correlate positively with the disease progression, culminating at the stage of metastatic castration-resistant prostate cancer. Due to its tissue-specific expression and cell surface localization, PSMA shows superior potential for precise imaging and therapy of PCa. Antibody-based immunotherapy targeting PSMA offers the promise of selectively engaging the host immune system with minimal off-target effects. Here we report on the design, expression, purification, and characterization of a bispecific engager, termed 5D3-CP33, that efficiently recruits macrophages to the vicinity of PSMA-positive cancer cells mediating PCa death. The engager was engineered by fusing the anti-PSMA 5D3 antibody fragment to a cyclic peptide 33 (CP33), selectively binding the Fc gamma receptor I (FcγRI/CD64) on the surface of phagocytes. Functional parts of the 5D3-CP33 engager revealed a nanomolar affinity for PSMA and FcγRI/CD64 with dissociation constants of KD = 3 nM and KD = 140 nM, respectively. At a concentration as low as 0.3 nM, the engager was found to trigger the production of reactive oxygen species by U937 monocytic cells in the presence of PSMA-positive cells. Moreover, flow cytometry analysis demonstrated antibody-dependent cell-mediated phagocytosis of PSMA-positive cancer cells by U937 monocytes when exposed to 0.15 nM 5D3-CP33. Our findings illustrate that 5D3-CP33 effectively and specifically activates monocytes upon PSMA-positive target engagement, resulting in the elimination of tumor cells. The 5D3-CP33 engager can thus serve as a promising lead for developing new immunotherapy tools for the efficient treatment of PCa.

• MeSH
• antigeny povrchové * imunologie   metabolismus   MeSH
• glutamátkarboxypeptidasa II * imunologie   metabolismus   MeSH
• imunoterapie metody   MeSH
• lidé MeSH
• makrofágy imunologie   MeSH
• monocyty * imunologie   metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádory prostaty * imunologie   terapie   patologie   MeSH
• protilátky bispecifické * imunologie   farmakologie   MeSH
• receptory IgG metabolismus   imunologie   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny povrchové * MeSH
• FOLH1 protein, human MeSH Prohlížeč
• glutamátkarboxypeptidasa II * MeSH
• protilátky bispecifické * MeSH
• receptory IgG MeSH

Prostate cancer (PCa) tops the list of cancer-related deaths in men worldwide. Prostate-specific membrane antigen (PSMA) is currently the most prominent PCa biomarker, as its expression levels are robustly enhanced in advanced stages of PCa. As such, PSMA targeting is highly efficient in PCa imaging as well as therapy. For the latter, PSMA-positive tumors can be targeted directly by using small molecules or macromolecules with cytotoxic payloads or indirectly by engaging the immune system of the host. Here we describe the engineering, expression, purification, and biological characterization of bispecific T-cell engagers (BiTEs) that enable targeting PSMA-positive tumor cells by host T lymphocytes. To this end, we designed the 5D3-αCD3 BiTE as a fusion of single-chain fragments of PSMA-specific 5D3 and anti-CD3 antibodies. Detailed characterization of BiTE was performed by a combination of size-exclusion chromatography, differential scanning fluorimetry, and flow cytometry. Expressed in insect cells, BiTE was purified in monodisperse form and retained thermal stability of both functional parts and nanomolar affinity to respective antigens. 5D3-αCD3's efficiency and specificity were further evaluated in vitro using PCa-derived cell lines together with peripheral blood mononuclear cells isolated from human blood. Our data revealed that T-cells engaged via 5D3-αCD3 can efficiently eliminate tumor cells already at an 8 pM BiTE concentration in a highly specific manner. Overall, the data presented here demonstrate that the 5D3-αCD3 BiTE is a candidate molecule of high potential for further development of immunotherapeutic modalities for PCa treatment.

• Publikační typ
• časopisecké články MeSH