Engineered Antibody Constructs for Targeting Protein Aggregates

The four bivalent monoclonal IgG
antibodies and proteins used in the experiments were designed, expressed,
and purified according to earlier published work.^{6 (link),21 (link)} The
RmAb158 monoclonal antibodies selectively bind to Aβ protofibrils,^{22 (link)} whereas the RmAb2G7 monoclonal antibodies selectively
bind to high-mobility group box 1 (HGMB1) proteins.^{23 (link)} In short, the heavy and light chain scFv8D3 transferrin
receptor transporter variable region sequence^{20 (link)} was connected to the C-terminus of the RmAb2G7 or RmAb158 light
chain with in-house designed linkers (APGSYTGSAPG or APGSGTGSAPG,
respectively). Figure 2 shows the cartoon representations of the antibody design, showing
the location of conjugated scFv8D3 in the modified antibodies.
The four recombinant antibodies were expressed
using Expi293 cells
(Thermo Fisher) transiently transfected with pcDNA3.4 vectors using
polyethyleneimine (PEI) as the transfection reagent. All antibodies
were purified on a protein G column (Cytiva) and eluted with an increasing
gradient of 0.7% acetic acid. The buffer was exchanged for phosphate-buffered
saline (PBS) (Gibco) immediately after elution, and the protein concentration
was determined at A280.

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Morrison J.I., Petrovic A., Metzendorf N.G., Rofo F., Yilmaz C.U., Stenler S., Laudon H, & Hultqvist G. (2023). Standardized Preclinical In Vitro Blood–Brain Barrier Mouse Assay Validates Endocytosis-Dependent Antibody Transcytosis Using Transferrin-Receptor-Mediated Pathways. Molecular Pharmaceutics, 20(3), 1564-1576.

Publication 2023

Expi293 cells protein G column phosphate-bufferedsaline (PBS)

A protein Acetic acid Antibodies Antibody Buffer Cells High mobility group box 1 proteins Light Monoclonal antibodies Phosphate Protein Protein g Transfection Transporter Vectors

Corresponding Organization : Uppsala University

Other organizations : BioArctic Neuroscience (Sweden)

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Variable analysis

independent variables

Four bivalent monoclonal IgG antibodies and proteins designed, expressed, and purified

dependent variables

Selective binding of RmAb158 monoclonal antibodies to Aβ protofibrils
Selective binding of RmAb2G7 monoclonal antibodies to high-mobility group box 1 (HGMB1) proteins

control variables

The heavy and light chain scFv8D3 transferrin receptor transporter variable region sequence was connected to the C-terminus of the RmAb2G7 or RmAb158 light chain with in-house designed linkers (APGSYTGSAPG or APGSGTGSAPG, respectively)

controls

No positive or negative controls were explicitly mentioned in the provided information.

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