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To address these challenges, antibody fragments are often multimerized 9, 10 and/or conjugated to larger proteins 11, which increases the size of antibody fragments, again reducing their ability to penetrate into the tumor. However, small antibody fragments have a low residence time in the body and often have a higher rate of dissociation ( k off) from the target compared with full-length antibodies, limiting their clinical utility 8. Small antibody fragments with low molecular weight can diffuse much deeper into tissues, presenting an excellent alternative to full-length antibodies. Some studies in patients with cancer estimate that only 0.01% of the injected antibodies accumulate per gram of solid tumor tissue 7. In addition, it has been shown that high-affinity antibodies bind to the periphery of the tumor tissues, forming a barrier and preventing their further penetration 6. Due to their large size, full-length antibodies are unable to diffuse deep into solid tumors 5. Although full-length antibodies have shown promise for treatment of several cancers, limited success has been demonstrated in eliminating solid tumors. These antibodies bind to cell surface receptors expressed at higher levels on cancer cells, addressing a major challenge of selective cell targeting in cancer therapy. Several antibodies and antibody fragments have been previously developed for the treatment of various diseases, including cancer 3, 4. Replacing these noncovalent interactions with a covalent bond while concurrently modulating the binding in response to an external stimulus can further expand the applications of antibodies. Finally, we demonstrated that in the absence of light, this pcY- and Bpa-containing mutant of 7D12 does not bind to EGFR, but irradiation with 365-nm light activates (1) specific binding and (2) covalent bond formation with EGFR.Īpplications of antibodies depend upon their specific binding to antigens, binding that is mediated by interactions, such as electrostatics, van der Waals, hydrophobic and hydrogen bonding, that are susceptible to changes in the microenvironment 1, 2. We then developed a method for site-specific incorporation of pcY and Bpa at two distinct sites in 7D12. Upon exposure to 365-nm light, this Bpa-containing 7D12 mutant forms a covalent bond with EGFR in an antigen-specific manner. We identified a position for installing Bpa in 7D12 that has minimal effect on 7D12–EGFR binding affinity in the absence of light. This was achieved by site-specific incorporation of photocaged tyrosine (pcY) for photoactivity and p-benzoyl-ʟ-phenylalanine (Bpa) for photoreactivity into 7D12. Here, we have introduced concurrent photoactivity and photoreactivity into an epidermal growth factor receptor (EGFR)-targeting antibody fragment, 7D12. Design of biomolecules that perform two or more distinct functions in response to light remains challenging.