Engineered Bifunctional Proteins for Targeted Cancer Therapy: Prospects and Challenges

Abstract

Bifunctional proteins (BFPs) are a class of therapeutic agents produced through genetic engineering and protein engineering, and are increasingly used to treat various human diseases, including cancer. These proteins usually have two or more biological functions—specifically recognizing different molecular targets to regulate the related signaling pathways, or mediating effector molecules/cells to kill tumor cells. Unlike conventional small‐molecule or single‐target drugs, BFPs possess stronger biological activity but lower systemic toxicity. Hence, BFPs are considered to offer many benefits for the treatment of heterogeneous tumors. In this review, the authors briefly describe the unique structural feature of BFP molecules and innovatively divide them into bispecific antibodies, cytokine‐based BFPs (immunocytokines), and protein toxin‐based BFPs (immunotoxins) according to their mode of action. In addition, the latest advances in the development of BFPs are discussed and the potential limitations or problems in clinical applications are outlined. Taken together, future studies need to be centered on understanding the characteristics of BFPs for optimizing and designing more effective such drugs.

De Novo Evolution of an Antibody‐Mimicking Multivalent Aptamer via a DNA Framework

“…However, due to the lack of an Fc region, the flexible linker makes non‐IgG‐like bispecific antibodies have a shorter serum half‐life, weaker stability, and less biological activity compared to the IgG‐like bispecific antibodies (contains Fab, Fab’, and Fc region). [ 30 ] Similarly, a flexible linker applied for the assembly of a bivalent aptamer allows two aptamers to move randomly, resulting in a large entropy barrier (loss in rotational and translational entropy) when the bivalent aptamer binds to target molecules. So, the benefit of multivalent interactions is not maximized due to the flexibility of scaffold linkers.…”

“…As shown in Scheme 1, a Y-shaped bispecific antibody contains a constant region (Fc) and two variable antigen-binding fragment arms (Fab and Fab'), which can recognize and bind to two different antigens or two different epitopes of one antigen. [30,31] Like Fab and Fab' arms, two different nonoverlapping DNA monovalent aptamers can bind to two distinct epitopes on one target. More design details are presented in Figure S4, Supporting Information.…”

Targeted Mutation of a Non-catalytic Gating Residue Increases the Rate of Pseudomonas aeruginosa d-Arginine Dehydrogenase Catalytic Turnover

“…The success of the E 246 mutation in increasing the rate of Pa DADH turnover with d -arginine is a prime example of how deconstructing catalytic processes and targeting specific steps in the catalytic cycle of an enzyme can be explored for the bioengineering of biocatalysts. An important implication of this study is that we have engineered a d -amino acid oxidizing enzyme that can detect and consume its substrate faster at 500 s –1 , providing a highly efficient system for applications in the food and l -amino acid industries. − With the surging interest in gating residues as targets for protein engineering toward therapeutic development, it is vital to identify the properties of gating residues that alter enzyme activity and specificity. ,− ,,− Given that gates control the flux of substances in and out of the active site, ,,− and because most gating residues are distal from the active site, their mutations tend to alter substrate selectivity and not enzyme catalysis. ,,,, This study demonstrates that the proximity of a gating residue to the active site and interactions with ligands are two important factors to consider during protein engineering. Residues like E 246 of Pa DADH, which is close to the active site and interacts with a substrate or product, could be prime targets for engineering faster biological catalysts.…”

“…Scientists do not fully understand the effects of enzyme motions on enzyme catalysis, such as loop dynamics. Hence, engineered proteins often exhibit poor catalytic abilities compared to their native proteins. − Most enzymes undergo natural evolutionary processes that fashion them for better functionality in an organism, including mutations into more active forms that result in improved enzyme flexibility for catalysis. − , For protein engineering for therapeutics, such as in treating cardiovascular diseases and cancer therapy, − and for applications in the food industry, studies on flexible enzyme dynamics are also necessary to generate better biocatalysts.…”

Optimized Heterologous Expression and Efficient Purification of a New TRAIL-Based Antitumor Fusion Protein SRH–DR5-B with Dual VEGFR2 and DR5 Receptor Specificity

“…Modern bioengineering approaches make it possible to create bifunctional proteins (BFPs) of various types for targeted cancer therapy, which simultaneously activate or block two signaling pathways [ 39 ]. TRAIL represents one of the most promising cancer treatments due to its specificity, safety and encouraging results in vitro and in vivo.…”