(357m) Bioprocess Development of Engineered Anti-CD276/CD47 Antibody-Drug Conjugates for Cancer Treatment

Targeted anti-cancer therapeutic agents possess superior efficacy for invasive and heterologous cancer treatment as compared to the conventional chemo therapies. This study aimed to establish a robust bioprocess for antibody-drug conjugates (ADCs) development to treat a highly metastatic and heterologous triple-negative breast cancers (TNBCs). First, two surface receptors, i.e. CD276 and CD47, were identified in TNBC patient samples using comparative membrane proteomics. Novel anti-CD276/CD47 monoclonal antibodies (mAbs) were developed using hybridoma technique and further engineered (i.e. chimeric re-construction and humanization in this study) to improve their translational potential such as high stability and low immunogenicity. Second, both transient expression using HEK 293F cell and fed-batch production using stable CHO DG44 cell line were developed and optimized to achieve high productivity, up to 2 g/L, of the engineered antibodies in 2 or 5-L stirred-tank bioreactors. The Protein A chromatography and ultrafiltration-based purification process was developed to achieve high purity of >99%. Third, an ELISA-based assay revealed nanomolar dissociation constant (K_d value) and high affinity to the targeted receptors in TNBCs. The flow cytometry analysis, confocal microscopy imaging and In Vivo Imaging System demonstrated the high cancer targeting specificity and drug delivery capability of the engineered anti-CD276/CD47 mAbs. Fourth, a tubulin inhibitor-mertansine was conjugated to the engineered mAb via sulfo-SMCC linker to construct ADCs. Finally, the in vitro cytotoxicity assay indicated high anti-cancer potency with IC₅₀ value of <5 nM. Both patient-derived xenograft and TNBC cell-derived xenograft mice models demonstrated the high tumor specificity and therapeutic efficacy of the constructed ADCs. This study established a robust bioprocess of CD276/CD47 mAb production, purification and conjugation for TNBC treatment.