Abstract
Antibodies are one of the most important groups of biomolecules for both clinical and basic research and have been developed as potential therapeutics. Affinity is the key feature for biological activity and clinical efficacy of an antibody, especially of therapeutic antibodies, and thus antibody affinity improvement is indispensable and still remains challenging. To address this issue, we developed the E. coli Assisted Speed affINity-maturation Evolution SyStem (EASINESS) for continuous directed evolution of Ag–Ab interactions. Two key components of EASINESS include a mutation system modified from error-prone DNA polymerase I (Pol I) that selectively mutates ColE1 plasmids in E. coli and a protein–protein interaction selection system from mDHFR split fragments. We designed a GCN4 variant which barely forms a homodimer, and during a single round of evolution, we reversed the homodimer formation activity from the GCN4 variant to verify the feasibility of EASINESS. We then selected a potential therapeutic antibody 18A4Hu and improved the affinity of the antibody (18A4Hu) to its target (ARG2) 12-fold in 7 days while requiring very limited hands-on time. Remarkably, these variants of 18A4Hu revealed a significant improved ability to inhibit melanoma pulmonary metastasis in a mouse model. These results indicate EASINESS could be as an attractive choice for antibody affinity maturation.
Highlights
As one of top groups of important biological molecules with the ability of specific targeting [1, 2], antibodies have been widely applied in basic research, clinical diagnostics, and therapeutics [3, 4]
An affinity maturation selection system is designed based on a proteinfragment complementation assay (PCA) system using murine dihydrofolate reductase, where E. coli relies on the interaction between antigen (Ag) and antibody (Ab) to form functional mDHFR for growth under the pressure of trimethoprim (TMP) (Figure 1A)
When Ag that binds to Ab, F1,2 is brought close to F3 to form the whole functional mDHFR (F1,2~F3), the activity of murine dihydrofolate reductase is proportional to the level of TMP [31]
Summary
As one of top groups of important biological molecules with the ability of specific targeting [1, 2], antibodies have been widely applied in basic research, clinical diagnostics, and therapeutics [3, 4]. Nearly 79 monoclonal antibody (mAb) drugs have been approved, and over 570 therapeutic antibodies are in the clinical development phase for treating various diseases, including cancer, inflammatory disease, and infectious disease [5,6,7]. Due to obvious advantages of small size, strong penetrability, and high specificity, scFvs are widely applied in targeted therapy, Continuous Antibody Affinity Maturation intracellular immunity diagnosis, and biological imaging detection [15,16,17]. High affinity is a key feature for the successful development of therapeutic antibodies. During development of therapeutic antibodies, antibody affinity improvement is a critical and essential standard procedure
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