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

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.

• Klíčová slova
• NAALADase, antibody fragment, glutamate carboxypeptidase II, in vivo imaging, monoclonal antibody, prostate cancer, prostate-specific membrane antigen,
• MeSH
• antigeny povrchové imunologie   MeSH
• buněčné linie MeSH
• buňky PC-3 MeSH
• fluorescence MeSH
• glutamátkarboxypeptidasa II imunologie   MeSH
• hmyz MeSH
• jednořetězcové protilátky imunologie   MeSH
• lidé MeSH
• monoklonální protilátky imunologie   MeSH
• myši nahé MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory prostaty imunologie   MeSH
• rekombinantní proteiny imunologie   MeSH
• xenogenní modely - testy antitumorózní aktivity metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny povrchové MeSH
• FOLH1 protein, human MeSH Prohlížeč
• glutamátkarboxypeptidasa II MeSH
• jednořetězcové protilátky MeSH
• monoklonální protilátky MeSH
• rekombinantní proteiny MeSH

Prostate cancer (PC) is a potentially high-risk disease and the most common cancer in American men. It is a leading cause of cancer-related deaths in men in the US, second only to lung and bronchus cancer. Advanced and metastatic PC is initially treated with androgen deprivation therapy (ADT), but nearly all cases eventually progress to castrate-resistant prostate cancer (CRPC). CRPC is incurable in the metastatic stage but can be slowed by some conventional chemotherapeutics and second-generation ADT, such as enzalutamide and abiraterone. Therefore, novel therapeutic strategies are urgently needed. Prostate-specific membrane antigen (PSMA) is overexpressed in almost all aggressive PCs. PSMA is widely used as a target for PC imaging and drug delivery. Anti-PSMA monoclonal antibodies (mAbs) have been developed as bioligands for diagnostic imaging and targeted PC therapy. However, these mAbs are successfully used in PC imaging and only a few have gone beyond phase-I for targeted therapy. The 5D3 mAb is a novel, high-affinity, and fast-internalizing anti-PSMA antibody. Importantly, 5D3 mAb demonstrates a unique pattern of cellular localization to the centrosome after internalization in PSMA(+) PC3-PIP cells. These characteristics make 5D3 mAb an ideal bioligand to deliver tubulin inhibitors, such as mertansine, to the cell centrosome, leading to mitotic arrest and elimination of dividing PC cells. We have successfully developed a 5D3 mAb- and mertansine (DM1)-based antibody-drug conjugate (ADC) and evaluated it in vitro for binding affinity, internalization, and cytotoxicity. The in vivo therapeutic efficacy of 5D3-DM1 ADC was evaluated in PSMA(+) PC3-PIP and PSMA(-) PC3-Flu mouse models of human PC. This therapeutic study has revealed that this new anti-PSMA ADC can successfully control the growth of PSMA(+) tumors without inducing systemic toxicity.

• Klíčová slova
• 5D3 antibody, MCC linker, anti-PSMA antibody, antibody-drug conjugates (ADC), drug delivery, mertansine (DM1), prostate cancer, prostate-specific membrane antigen (PSMA), targeted therapy,
• MeSH
• androsteny farmakologie   MeSH
• antagonisté androgenů farmakologie   MeSH
• antigeny povrchové metabolismus   MeSH
• benzamidy farmakologie   MeSH
• buňky PC-3 MeSH
• centrozom metabolismus   MeSH
• fenylthiohydantoin farmakologie   MeSH
• glutamátkarboxypeptidasa II metabolismus   MeSH
• imunokonjugáty farmakologie   MeSH
• lidé MeSH
• modulátory tubulinu farmakologie   MeSH
• monoklonální protilátky farmakologie   MeSH
• myši nahé MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory prostaty rezistentní na kastraci farmakoterapie   metabolismus   MeSH
• nitrily farmakologie   MeSH
• xenogenní modely - testy antitumorózní aktivity MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• abiraterone MeSH Prohlížeč
• androsteny MeSH
• antagonisté androgenů MeSH
• antigeny povrchové MeSH
• benzamidy MeSH
• enzalutamide MeSH Prohlížeč
• fenylthiohydantoin MeSH
• FOLH1 protein, human MeSH Prohlížeč
• glutamátkarboxypeptidasa II MeSH
• imunokonjugáty MeSH
• modulátory tubulinu MeSH
• monoklonální protilátky MeSH
• nitrily MeSH

Prostate cancer is primarily fatal after it becomes metastatic and castration-resistant despite novel combined hormonal and chemotherapeutic regimens. Hence, new therapeutic concepts and drug delivery strategies are urgently needed for the eradication of this devastating disease. Here we report the highly specific, in situ click chemistry driven pretargeted delivery of cytotoxic drug carriers to PSMA(+) prostate cancer cells. Anti-PSMA 5D3 mAb and its F(ab')2 fragments were functionalized with trans-cyclooctene (TCO), labeled with a fluorophore, and used as pretargeting components. Human serum albumin (ALB) was loaded with the DM1 antitubulin agent, functionalized with PEGylated tetrazine (PEG4-Tz), labeled with a fluorophore, and used as the drug delivery component. The internalization kinetics of components and the therapeutic efficacy of the pretargeted click therapy were studied in PSMA(+) PC3-PIP and PSMA(-) PC3-Flu control cells. The F(ab')2 fragments were internalized faster than 5D3 mAb in PSMA(+) PC3-PIP cells. In the two-component pretargeted imaging study, both components were colocalized in a perinuclear location of the cytoplasm of PC3-PIP cells. Better colocalization was achieved when 5D3 mAb was used as the pretargeting component. Consecutively, the in vitro cell viability study shows a significantly higher therapeutic effect of click therapy in PC3-PIP cells when 5D3 mAb was used for pretargeting, compared to its F(ab')2 derivative. 5D3 mAb has a longer lifetime on the cell surface, when compared to its F(ab')2 analogue, enabling efficient cross-linking with the drug delivery component and increased efficacy. Pretargeting and drug delivery components were cross-linked via multiple bioorthogonal click chemistry reactions on the surface of PSMA(+) PC cells forming nanoclusters, which undergo fast cellular internalization and intracellular transport to perinuclear locations.

• Klíčová slova
• PSMA(+) prostate cancer, bioorthogonal click chemistry, drug delivery, nanomedicine, pretargeted therapy,
• MeSH
• albuminy MeSH
• antigeny povrchové imunologie   MeSH
• antitumorózní látky fytogenní terapeutické užití   MeSH
• cyklooktany chemie   MeSH
• fluorbenzeny chemie   MeSH
• glutamátkarboxypeptidasa II imunologie   metabolismus   MeSH
• imunoglobuliny - Fab fragmenty chemie   metabolismus   terapeutické užití   MeSH
• lidé MeSH
• maytansin terapeutické užití   MeSH
• modulátory tubulinu terapeutické užití   MeSH
• monoklonální protilátky chemie   metabolismus   terapeutické užití   MeSH
• nádorové buněčné linie MeSH
• nádory prostaty farmakoterapie   enzymologie   metabolismus   MeSH
• nanomedicína MeSH
• syntetická chemie okamžité shody metody   MeSH
• systémy cílené aplikace léků metody   MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• albuminy MeSH
• AlexaFluor 488 carboxylic acid tetrafluorophenyl ester MeSH Prohlížeč
• antigeny povrchové MeSH
• antitumorózní látky fytogenní MeSH
• cyklooktany MeSH
• fluorbenzeny MeSH
• FOLH1 protein, human MeSH Prohlížeč
• glutamátkarboxypeptidasa II MeSH
• imunoglobuliny - Fab fragmenty MeSH
• maytansin MeSH
• modulátory tubulinu MeSH
• monoklonální protilátky MeSH