Protein a affinity chromatography

The extracellular domains of human CD33 (positions 18–256) and human EGFR (positions 25–642) were cloned with a C-terminal TEV protease cleavage site, 8× polyhistidine, and AviTag^TM (Avidity, LLC). For the scFvs, the heavy and light-chain variable domains were paired using a (GGGGS)₄ linker and fused to a C-terminal 8× polyhistidine tag. All constructs were expressed using the Expi293 system (Thermo Fisher Scientific) and purified by Ni Sepharose Excel column (GE Healthcare Life Sciences) followed by size-exclusion chromatography over a Superdex 75 or Superdex 200 column (GE Healthcare Life Sciences) equilibrated in 1× PBS (Corning). Site-specific biotinylation of CD33 through the AviTag^TM was performed according to the manufacturer’s protocols. scFv-Fc constructs reformatted from scFv libraries were purified by Protein A affinity chromatography (GE Healthcare Life Sciences).

NCI-N87 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and OE-19 cells were obtained from the European Collection of Cell Culture (ECACC, Porton Down, UK). The cell culture medium was RPMI-1640 supplemented with 10% fetal bovine serum (FBS), and antibiotics and cells were cultured at 37°C under 5% CO₂. Trastuzumab and palivizumab was produced by Genentech (South San Francisco, CA, USA) and MedImmune, LLC (Gaithersburg, MD, USA), respectively. ChromPure human IgG (Jackson ImmunoResearch, West Grove, PA, USA) was used as human IgG control antibody for in vitro assays. IgG antibodies were produced using the Freestyle 293 system (Invitrogen, Carlsbad, CA, USA) and purified using protein-A affinity chromatography (GE Healthcare, Piscataway, NJ, USA). Endotoxin was removed with an Endotoxin Removal Kit (GenScript, Piscataway, NJ, USA), and endotoxin levels were determined using an Endotoxin Detection Kit (GenScript). Recombinant proteins were produced as secreted proteins using the Freestyle 293 system and purified using protein-A or Ni-NTA chromatography (Qiagen, Valencia, CA, USA) for Fc-tagged or His-tagged proteins, respectively.

The anti-LUNX antibody was introduced in our previous work [42 (link)] and humanized to replace the Fc region with the human IgG1 Fc region. Briefly, S-35-8 (IgG2a-k) LUNX hybridoma was raised against a His-tagged LUNX protein and screened by ELISA. The hybridoma was cultured in RPMI-1640 medium. The S-35-8 anti-LUNX antibody was purified by protein A affinity chromatography (GE Healthcare Bio-Sciences AB).

The PRLR-DbsAb was generated by BAPTS system, which was previously described in detail [27 (link),28 (link)]. CD3 antibody-fusion protein (Fragment A) was expressed in a stably transfected cell line CHO while PRLR antibody-fusion protein (Fragment B) was expressed in 293E cells using transient gene expression (TGE) technology. Both Fragment A and Fragment B were purified by Protein L affinity chromatography (GE Healthcare). The monoclonal antibodies used in this study including PRLR antibody (PRLR mAb), CD3 antibody (CD3 mAb) and PD-1 antibody (PD-1 mAb) were expressed by 293E cells and purified by Protein A affinity chromatography (GE Healthcare). The affinity of CD3 mAb and PD-1 mAb to their antigens was previously confirmed [27 (link),29 (link)]. The amino acid sequence used for the PRLR mAb is identical to that of the PRLR monoclonal antibody LFA102 [5 (link)]. All recombinant antibodies were dialyzed overnight to phosphate buffer saline (PBS) and sterilized by filtration using a 0.22 μm filter.