Development of A Prostate 3D Spheroid Model for Assessing Tumor Uptake of Different Antibody Designs

Desmond Lewis

doi:10.17918/00001584

Targeted radionuclide therapy has shown improved overall survival in patients with metastatic castration resistant prostate cancer (mCRPC). Most recently, Pluvicto (177Lu-PSMA-617), a small molecule PSMA ligand conjugated to the beta emitter 177Lu was approved for treatment of mCRPC. Although, initial responses have been encouraging, further improvements are being explored outside of small molecules and beta emitters. Novel large molecule radionuclide carriers are being developed, including monoclonal antibodies and fragment-based designs that may offer unique advantages to small molecule carriers. As radionuclides are often more difficult to work with and pose a health risk if not properly trained, pre-clinical testing with cytotoxic payloads can often serve as a surrogate for radionuclide payload uptake and assessment. Conventionally, efficacy of these competing antibodies has been assessed through measuring cell viability after treatment in 2D culture conditions. The use of 2D culture allows antibodies maximal access to cells and does not measure tumor penetration, a delineating factor in vitro. As a result, full length and fragment antibody drug conjugates may appear similar in binding, internalization, and cytotoxicity. 3D spheroid culture can assess antibody penetration and recapitulate features of solid tumors more closely than 2D culture conditions. Here a C42B prostate cancer spheroid was developed to obtain a model suitable for high-throughput screening using live-cell imaging. C42B Spheroids composed of 1000 cells were found to be reproducible, having steady growth and consistent shape. The spheroids were characterized and observed to decrease in both prostate specific membrane antigen (PSMA) expression and individual cell size following the development of hypoxia, quantified by expression of HIF-1[alpha] at day seven of spheroid growth. This suggest that C42B spheroids are less sensitive to drug treatment than 2D culture due to the decrease in PSMA expression and the added penetration required in 3D culture. C42B spheroids were also observed to develop oxygen gradients resulting in hypoxia and necrosis at the core. This further validated the ability of spheroids to reproduce features of tumors not observed in 2D culture. The onset of necrosis also determined the optimal time to measure penetration of competing antibody designs. A monoclonal antibody and its digested fragment were labeled with a fluorescent dye and penetration was assessed using live-cell imaging. Spheroids treated with the competing antibodies showed a significant increase in fragment penetration compared to the full-length monoclonal antibody (P < 0.0001) over a period of 63 hours. These findings indicate that spheroids can differentiate between competing antibodies using penetration, a feature of tumors not measured using 2D culture. Spheroids allow competing antibodies to be assessed in an environment more relevant than current 2D culture conditions.

Development of A Prostate 3D Spheroid Model for Assessing Tumor Uptake of Different Antibody Designs

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Development of A Prostate 3D Spheroid Model for Assessing Tumor Uptake of Different Antibody Designs

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