These authors contributed equally to this work.Keywords: cell-penetrating antibody, cytosolic protein targeting, cellular internalization, endosomal release, IgG antibody, intracellular trafficking, next-generation antibodyAbbreviations: IgG, immunoglobulin G; VL, light chain variable domain; VH, heavy chain variable domain; LC, light chain; HC, heavy chain; CDR, complementarity-determining region.Full-length IgG antibodies cannot cross cell membranes of living cells; this limits their use for direct targeting of cytosolic proteins. Here, we describe a general strategy for the generation of intact, full-length IgG antibodies, herein called cytotransmabs, which internalize into living cells and localize in the cytosol. We first generated a humanized light chain variable domain (VL) that could penetrate into the cytosol of living cells and was engineered for association with various subtypes of human heavy chain variable domains (VHs). When light chains with humanized VL were coexpressed with 3 heavy chains (HCs), including 2 HCs of the clinically approved adalimumab (Humira Ò ) and bevacizumab (Avastin Ò ), all 3 purified IgG antibodies were internalized into the cytoplasm of living cells. Cytotransmabs primarily internalized into living cells by the clathrin-mediated endocytic pathway through interactions with heparin sulfate proteoglycan that was expressed on the cell surface. The cytotransmabs escaped into the cytosol from early endosomes without being further transported into other cellular compartments, like the lysosomes, endoplasmic reticulum, Golgi apparatus, and nucleus. Furthermore, we generated a cytotransmab that co-localized with the targeted cytosolic protein when it was incubated with living cells, demonstrating that the cytotransmab can directly target cytosolic proteins. Internalized cytotransmabs did not show any noticeable cytotoxicity and remained in the cytosol for more than 6 h before being degraded by proteosomes. These results suggest that cytotransmabs, which efficiently enter living cells and reach the cytosolic space, will find widespread uses as research, diagnostic, and therapeutic agents.

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In this study, the correlation between HAZ microstructure and low temperature impact toughness of bainitic steel plates was investigated. The Steel with high carbon content and finish cooling temperature has a higher volume fraction and large packet size of granular bainite compared to the steel with low carbon content and finish cooling temperature. The room temperature tensile properties of the two steels are similar, mostly because the microstructure is composed of acicular ferrite having fine grains. On the other hand, the low temperature Charpy absorbed energy of two steels showed a big difference. As the test temperature decreased, the Charpy absorbed energy of the steel with high carbon content and finish cooling temperature decreased more rapidly than the steel with low carbon content and finish cooling temperature. This is due to the high volume fraction of granular bainite and large packet size of granular bainite in the steel with high content and finish cooling temperature. HAZ specimens have a very complex microstructure with a mixture of acicular ferrite, granular bainite, and bainitic ferrite through the rapid cooling process. In the HAZ specimen with high carbon content, more volume fraction of granular bainite + bainitic ferrite was formed, and the packet size of granular bainite + bainitic ferrite was also coarse. Because of this result, the HAZ specimen with high carbon content has low Charpy absorbed energy compared to the HAZ specimen with low carbon content.