Abstract
Antibody-drug conjugates (ADCs) used nowadays in clinical practice are mixtures of antibody molecules linked to a varying number of toxins at different positions. Preclinical studies have shown that the therapeutic index of these traditional ADCs can be improved by the site-specific linkage of toxins. However, current approaches to produce homogeneous ADCs have several limitations, such as low protein expression and slow reaction kinetics. In this protocol we describe how to set up an expression system to incorporate a cyclopropene derivative of lysine (CypK) into antibodies using genetic code expansion. This minimal bioorthogonal handle allows rapid conjugation of tetrazine derivatives through an inverse-demand Diels-Alder cycloaddition. The expression system here reported enables the facile production and purification of trastuzumab bearing CypK in each of the heavy chains. We explain how to link the antibody to the toxin monomethyl auristatin E and characterize the immunoconjugate by hydrophobic interaction chromatography and mass spectrometry. Finally, we describe assays to assess the stability in human serum of the dihydropyridazine linkage resulting from the conjugation and to test the selective cytotoxicity of the ADC for breast cancer cells with high levels of HER2 receptor.
Highlights
Antibody-drug conjugates (ADCs) combine the selectivity of biotherapeutics and the potency of small cytotoxic molecules
Firstgeneration ADCs approved by the Food and Drug Administration (FDA) rely on the modification of lysines and cysteines, which generates mixtures of molecules modified at different positions with decreased pharmacokinetic properties[2]
The cycloaddition is completed within 20 h using 2 equivalents of tetrazine-vcMMAE without acetonitrile (Figure 2c)
Summary
Antibody-drug conjugates (ADCs) combine the selectivity of biotherapeutics and the potency of small cytotoxic molecules. We selected the first position of the CH1 domain on the heavy chain to encode the ncAA (HC-118TAG) This is the most commonly modified site in ADCs23 and is known as HC-118 (EU numbering) but has been referred to as HC-121 (sequence position) and HC-114 (Kabat numbering)[24]. The selectivity and potency of trastuzumab(MMAE)[2] is assessed by comparing the cytoxicity of the ADC across cell lines expressing different levels of HER2. This assay provides a functional proof of the ADC stability when performed after incubating the immunoconjugate in human serum
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call