An anti-EGFR × cotinine bispecific antibody complexed with cotinine-conjugated duocarmycin inhibits growth of EGFR-positive cancer cells with KRAS mutations

Abstract

Antibody-drug conjugates (ADCs) can selectively deliver cytotoxic agents to tumor cells and are frequently more potent than naked antibodies. However, optimization of the conjugation process between antibodies and cytotoxic agents and characterization of ADCs are laborious and time-consuming processes. Here, we describe a novel ADC platform using a tetravalent bispecific antibody that simultaneously binds to the tumor-associated antigen and a hapten conjugated to a cytotoxic agent. We selected cotinine as the hapten because it is not present in biological systems and is inert and nontoxic. We prepared an anti-epidermal growth factor receptor (EGFR) × cotinine bispecific antibody and mixed it with an equimolar amount of cotinine-conjugated duocarmycin to form the ADC. This ADC showed significant in vitro and in vivo antitumor activity against EGFR-positive, cetuximab-refractory lung adenocarcinoma cells with KRAS mutations.

Stereotypic neutralizing V H antibodies against SARS-CoV-2 spike protein receptor binding domain in patients with COVID-19 and healthy individuals

“…The genes encoding the selected scFv clones were cloned into a modified mammalian expression vector, containing the hFc or hCκ at the C terminus ( 44 , 47 ), before being transfected and purified by affinity chromatography, as described above. Genes encoding V H and V λ were amplified, cloned into a mammalian expression vector containing the CH1 and hinge regions of human IgG2 fused to the CH2 and CH3 regions of human IgG4 ( 48 , 49 ), and transfected into Expi293F cells (Invitrogen) as described previously ( 50 ). Then, IgG2/4 was purified by affinity chromatography using MabSelect columns with the AKTA pure chromatography system (GE Healthcare) following the manufacturer’s protocol.…”

Induction of Anti-Aquaporin 5 Autoantibody Production by Immunization with a Peptide Derived from the Aquaporin of Prevotella melaninogenica Leads to Reduced Salivary Flow in Mice

“…The variable genes encoding clone 44 were cloned into a modified expression vector containing the human IgG4 construct, and transfected into Expi293F cells (Invitrogen) as described previously ( 26 ). IgG was purified by affinity chromatography using MabSelect columns with the AKTA Pure chromatography system (GE Healthcare, Chicago, IL, USA) following the manufacturer's protocol.…”

Generation of a Nebulizable CDR-Modified MERS-CoV Neutralizing Human Antibody

“…For the expression of IgG 1 , genes encoding V H and V L were amplified from the phage clones, cloned into a mammalian expression vector, and transfected into HEK293F cells. Then, IgG 1 was purified by Protein A affinity chromatography as described previously [68]. Then the eluate containing IgG 1 was subjected to gel filtration chromatography.…”