Vivaspin 20

The extracellular domain of LTβR was cloned into a modified pET28a vector with 6-His tag and HRV 3C Protease cleavage site at the N-terminal. The protein was refolded following standard protocol described elsewhere [23 (link)]. Briefly, the cells were sonicated in 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 2 M urea and 2% glycerol. The inclusion body was dissolved in 100 mM Tris-HCl, 8 M urea pH 8.0, then further purified using Ni-NTA column. The purified denatured protein sample was added dropwise into an agitating refolding buffer (100 mM Tris HCl, 2 mM EDTA, 400 mM L-arginine, 0.5 mM oxidized glutathione, 5 mM reduced glutathione, pH 8.0). The refolded protein was concentrated using Vivaspin 20 centrifugal concentrator (Cytiva, Marlborough, MA, USA) with a molecular weight cutoff at 10 kDa. The concentrated protein was buffer exchanged to 50 mM Tris-HCl pH 7.5, 100 mM NaCl and 2% glycerol by using Superdex 75 column (GE HealthCare, Chicago, IL, USA).

Cell pellets (2 g, wet weight) were thawed and resuspended in 50 mL lysis buffer (50 mM HEPES, pH 7.0 for MymA or 50 mM sodium phosphate, pH 7.5, for EthA, both supplemented with 300 mM NaCl, 10 mM imidazole, 8% glycerol). One milligram/milliliter lysozyme, 0,1% Triton X-100, 1 mM AEBSF [4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride], and 1 mM dithiothreitol were also added in this lysis buffer. The suspension was lysed on ice with an Emulsiflex C5 homogenizer (Avestin) using 100,000-kPa pulses, and clarified lysate was obtained after centrifugation at 22,000 × g for 30 min. The clarified lysate was applied to a 1-mL HisTrap HP column (Cytiva) equilibrated with lysis buffer supplemented with 20 mM imidazole. Nonspecific bound proteins were eluted from the beads with lysis buffer supplemented with 90 mM imidazole. Proteins of interest were eluted with elution buffer (lysis buffer supplemented with 300 mM imidazole).
Eluted fractions were 10-fold concentrated using Vivaspin 20 (30-kDa cutoff; Cytiva), and 500-μL fractions were loaded at 0.5 mL/min on a Superose 6 Increase 10/300 GL (Cytiva) equilibrated with buffer (50 mM HEPES, pH 7.0 or 50 mM sodium phosphate, pH 7.5, 300 mM NaCl for MymA and EthA, respectively). The collected fractions were concentrated, and total protein concentration was determined using the bicinchoninic acid (BCA) protein assay kit (Sigma-Aldrich).

Two ADCs were investigated within the experimental part of this study: ADC1 with two engineered cysteines for a site-directed DAR 2 conjugation and ADC2 for a cysteine-linked DAR 8 conjugation. For DAR 2, a functionalized mAb solution was generated through a full reduction with tris(2-carboxyethyl) phosphine hydrochloride (TCEP, EMD Millipore), followed by a buffer exchange using Vivaspin 20 (30 kDa MWCO, Cytiva) and a re-oxidation of the interchain disulfides with (L)-dehydroascorbic acid (DHAA, Sigma-Aldrich). For the ADC2 with a DAR of 8, a mild reduction of the interchain disulfides with TCEP was performed. In both cases, conjugation was carried out with a maleimide-functionalized payload that was dissolved in DMSO (Sigma-Aldrich). All other solutions were prepared with 20 mM sodium phosphate buffer (J.T. Baker), 1 mM EDTA (EMD Millipore), pH 7.0.