Proteins are large organic molecules formed through peptide bonds between amino acids. Natural proteins are typically unable to penetrate cell membranes and are easily degraded by protein-degrading enzymes, especially within cellular organelles such as lysosomes. This limitation has restricted effective intracellular protein targeting. In this study, a method was employed to construct a binding domain that binds to receptors expressed on the cell surface, allowing it to move into the cell without relying on other intracellular protein delivery platforms. After binding to the target receptor, this domain undergoes internalization and translocates to the endosome. Once located in the endosome, the constructed binding domain is released from the receptor, facilitated by a protein with endosomal escape capability, and then moves into the cytoplasm._x000D_
<br>Commonly, to induce affinity changes in an endosomal environment, histidine mutations are introduced into the antigen binding complementary determining region (CDR) regions associated with antigen binding. However, these improvements are limited due to the acidic environment of the tumor microenvironment (TME). Therefore, this study aims to enhance protein delivery efficiency based on an endosome reduction environment. The improved binding domain, designed to induce changes in protein affinity, is based on the monobody scaffold and is enhanced by introducing disulfide bonds into the loop region of the monobody. This modification enhances dissociation of the monobody from the receptor when the disulfide bond is reduced in the reduction environment, thereby improving efficiency._x000D_
<br>As a result, the affinity with EGFR, EpCAM, FAP, and Her2 receptors in the reduction environment decreased from 50-fold to a maximum of 77-fold compared to the non-reducing environment. The induced affinity changes in the endosomal environment enhance fusion protein delivery into the cytoplasm, improving intracellular delivery of cytotoxic fusion proteins, such as immunotoxins, thus expanding the therapeutic window for immunotoxins. In summary, this study provides a novel approach to enhance intracellular protein delivery by utilizing a reduction environment rather than the acidic environment of the endosome.
