Dynamis medium

Mouse anti-EGFR was purchased from BioXcell (Cat# BE-0279). Amino acid sequences of the heavy chain and light chain of hu-anti-EGFR (cetuximab) were retrieved from a database (32 (link)), cDNAs encoding both original cetuximab and the ACVC variant (A114C,V205C) were synthesized and cloned into the vector pGenHT1.0-DGV. Plasmids were transfected into CHO-K1SP cells to establish a stable cell line with the selection drug L-Methionine sulfoximine. To produce antibodies, stable cells with a starting density of 0.5 × 10⁶ were cultured in Dynamis™ medium (Gibco 26615-01) for 14 d. EfficientFeed™ B media supplemented with AGT™ (Gibco A25030-05) was added on day 3 (3% of initial volume), day 5 (4%), day 7 (5%), and day 9 (5%). The final culture supernatant was loaded onto a 5 mL protein A column (Cytiva); after washing with 10 column volumes of PBS, the antibody was eluted with 0.1 M Glycine (pH2.5) and further purified using a 5mL Hitrap® desalting column (Cytiva).

A fully human m276-SL expression plasmid containing S239C, L234A, L235A, and P329G (LALAPG) in the Fc domain of IgG1 was used to create a stable CHOK1 m276-SL expression cell line. For large scale production, stable cells were grown in Dynamis medium (Gibco, A2661501) with 25 μM MSX in fed-batch culture. The culture supernatant was collected on Day 12 or when the viability dropped below 60%, whichever occurred first. Antibody in the supernatant was purified using protein G affinity chromatography and dialyzed into PBS, pH 7.4. The purity of the antibody was monitored using SDS-PAGE and SEC-HPLC and antibodies with <5% aggregates were used for further ADC production. The DS-7300a ADC was generated by producing and purifying the B7-H3 humanized M30 antibody [Sequence 85 (HC) and 77 (LC) derived from US patent 9,371,395 B2] and randomly conjugating it to the exatecan derivative-based cytotoxic payload DXd (MedChemExpress, cat # HY-13631D) on endogenous cysteines via a cleavable tetrapeptide to create an average DAR of 4 as previously described.^{20 (link)} m276-Tesirine was generated by randomly conjugating the m276 parent antibody to the drug-linker Tesirine (MedChemExpress) to obtain an average DAR of 2 as previously described.^{67 (link)}

The complete pCHO vectors inserted with each mAb light and heavy chain were linearized by digestions with 15 µL of NruI enzyme for 50 µg of DNA. The digestion proceeded for 6 h at 37 °C. CHO-S cells (Gibco, Carlsbad, CA, USA) were transfected with the vectors for antibody expression. The procedure for stable transfection and the selection steps were performed with a Freedom ^TM CHO-S^TM Kit (Gibco, Carlsbad, CA, USA), following the manufacturer’s guidelines. At the end of each selection stage, aliquots of cells were frozen in liquid nitrogen. In four conditions, puromycin (Gibco, Carlsbad, CA, USA) and methotrexate (MTX) were used as selection agents. The pools were cultivated in a fed-batch for 14 days in Dynamis medium (Gibco, Carlsbad, CA, USA) to assess the specific productivity. The productivity of each stable pool was verified by glucose supplementation on days 3, 5, and 7. The mAbs were purified by affinity chromatography using protein-A Sepharose [17 (link)]. Purified protein was quantified by absorbance at 280 nm.