Highly efficient hybridoma generation and screening strategy for anti-PD-1 monoclonal antibody development

Abstract

Programmed cell death protein 1 (PD-1) plays a significant role in suppressing antitumor immune responses. Cancer treatment with immune checkpoint inhibitors (ICIs) targeting PD-1 has been approved to treat numerous cancers and is the backbone of cancer immunotherapy. Anti-PD-1 molecule is necessary for next-generation cancer immunotherapy to further improve clinical efficacy and safety as well as integrate into novel treatment combinations or platforms. We developed a highly efficient hybridoma generation and screening strategy to generate high-potency chimeric anti-PD-1 molecules. Using this strategy, we successfully generated several mouse hybridoma and mouse/human chimeric clones that produced high-affinity antibodies against human PD-1 with high-quality in vitro PD-1/PD-L1 binding blockade and T cell activation activities. The lead chimeric prototypes exhibited overall in vitro performance comparable to commercially available anti-PD-1 antibodies and could be qualified as promising therapeutic candidates for further development toward immuno-oncology applications.

The Development of Robust Antibodies to Sarcospan, a Dystrophin- and Integrin-Associated Protein, for Basic and Translational Research

“…The isolated murine spleen cells were fused with SP2/0-Ag14 cell line (ATCC, CRL-1581TM) myeloma cells using polyethylene glycol PEG1500 (Roche Diagnostics, 10783641001, Rotkreuz, Switzerland). To enhance the cloning efficiency and prevent the loss of high-producing clones, a methylcellulose-based semisolid medium was chosen for the hybridoma selection and cloning [ 49 ] where the fused cell suspension was mixed with a medium containing methylcellulose (Sigma, M7027) and plated in 100 mm Petri dishes (Thermo Fisher Scientific, 172931). After 10 to 14 days, single colonies visible to the naked eye were individually picked from the semisolid media and transferred into single wells of 96-well culture plates containing 200 µL of ClonaCell-HY medium E (Stemcell, 03805, Vancouver, Canada).…”

Development and characterization of mouse anti-canine PD-L1 monoclonal antibodies and their expression in canine tumors by immunohistochemistry in vitro

“…Electrofusion was used for hybridoma generation, as previously described by Phakham et al. ( 2022 ). In brief, splenocytes were separated, filtered through a 100 µm cell strainer, and counted using a Countess 3 FL automated cell counter (Invitrogen, Waltham, MA, USA).…”

“…Cultured supernatants containing anti-cPD-L1 immunoglobulin were harvested by centrifugation (4500× g , at room temperature for 10 min) and then filtrated through a 0.45 µm PES syringe filter before being applied to the protein A FF column (Cytiva, South Logan, UT, USA), as described previously (Phakham et al. 2022 ). The purified antibody was quantified by spectrophotometry (A280) using a Cytation 5-cell imaging multi-mode reader (BioTek) and stored at 4 °C (short-term) or −80 °C (long-term).…”