Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Dec 3:12:747267.
doi: 10.3389/fimmu.2021.747267. eCollection 2021.

EASINESS: E. coli Assisted Speedy affINity-maturation Evolution SyStem

Hai-Nan Zhang  1 , Jun-Biao Xue  1 , Aru Ze-Ling Wang  2 , He-Wei Jiang  1 , Siva Bhararth Merugu  2 , Da-Wei Li  2 , Sheng-Ce Tao  1
Affiliations

EASINESS: E. coli Assisted Speedy affINity-maturation Evolution SyStem

Hai-Nan Zhang et al. Front Immunol. .

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.

Keywords: 18A4Hu; antibody affinity improvement; directed evolution; error-prone DNA polymerase I; protein-fragment complementation assay.

PubMed Disclaimer

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Schematic and workflow of the EASINESS. (A) Anatomy of the key cell in EASINESS, i.e., E. coli JS200-ΔthyaΔfola. E. coli JS200-ΔthyaΔfola carries two plasmids: the mutation plasmid (MP), which mainly enables mutagenesis, and the target plasmid (TP), which encodes an evolving antibody part (Ab-F3). The antigen part (Ag-F1,2) was expressed in MP. The reconstitution survives on selecting media with trimethoprim (TMP). The resistance to TMP is proportional to the binding affinity between Ag and Ab. The red star indicates E. coli JS200 with the gene thya and fola knocked out. (B) The workflow of EASINESS. On day 1, the E. coli JS200-ΔthyaΔfola with MP and TP was grown in LB medium with thymine at 30°C overnight and then was shifted to 37°C in 2× YT medium with thymine to mutagenesis. On days 2–8, mutation and screening at 37°C were conducted. Strains were plated to select positive clones with reconstituted mDHFR activity through Ag-Ab binding. To select Ab with higher affinity to the designated target protein, the positive clones were iterated on plates with higher concentrations of TMP until the concentration reached 1,000 ng/μl. The chemical structural formula of TMP was drawn in InDraw web version (http://www.integle.com/static/indraw).
Figure 2
Figure 2
Recovery of the homodimer formation activity in mutated GCN4 by EASINESS. (A) The structure of the GCN4 leucine zipper dimer (PDB: 1CE9). (B, C) Native and SDS-polyacrylamide gel electrophoresis (PAGE) analysis of the dimer formation of GCN4WT and GCN43mut (L12E, L19A, and N16A). (D, E) Reversing the mutation of GCN43mut using EASINESS. Strain-4 carrying the mutation plasmid pEP (PolI3mut) was grown in the presence of 10 μg/ml TMP, and three mutation sites were reversed from GCN43mut to GCN4WT according to Sanger sequencing. Strain-1: GCN4WT-F1,2 and GCN4WT-F3 with pWT (PolIWT), Strain-2: GCN4WT-F1,2 and GCN4WT-F3 with pEP (PolI3mut), Strain-3: GCN4WT-F1,2 and GCN43mut-F3 with pWT, Strain-4: GCN4WT-F1,2 and GCN43mut-F3 with pEP. An additional mutation (AAA) at the end of GCN43mut was applied as a marker to differentiate the reversed mutant from wild-type GCN4.
Figure 3
Figure 3
Improved binding affinity of 18A4HuscFv to its target protein by EASINESS. (A) Plausible evolution trajectories of 18A4HuscFv by EASINESS in gradient concentrations of TMP from 0 to 1,000 (μg/ml). (B) These mutated sites of 18A4HuscFv located in the signal peptide or antibody backbone. The red star indicates stop codon. (C) The location of mutated residues from five variants in different domains of 18A4HuscFv. (D–I) Biolayer interferometry (BLI) assays for measuring the binding affinity to AGR2 of select 18A4HuscFv mutants.
Figure 4
Figure 4
18A4Hu variants inhibit tumor metastasis in the lungs of BALB/c nude mice. (A) Three scFv mutants (S102G, E131K, or D226N) were placed back to the human IgG backbone. (B) Schematic illustration of the treatment design. BALB/c nude mice were implanted with 5 × 105 per mouse B16F10 tumor cells. Day 2 after injection, mice were treated with the antibody 18A4Hu and its variants by intraperitoneal (i.p.) injection (100 mg per kg in 200 μl 1 × PBS) one for every three days for five times. (C, D) Representative macroscopic images (C) of lungs from 18A4Hu- and variant-treated animals to quantify lung metastases (D) after i.v. engraftment of B16F10 cells. (E, F) Representative macroscopic images of H&E staining in lung (E) and quantification of metastatic pulmonary metastasis nodules (F) from 18A4Hu- and variant-treated animals. Data are represented as the mean ± s.e.m. of n = 3/4 mice (D), n = 5 (F). p values calculated with unpaired Student’s t-test (D, F). Scale bar, 2 mm (F), or 1 mm (F).
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Uhlen M, Fagerberg L, Hallstrom BM, Lindskog C, Oksvold P, Mardinoglu A, et al. . Tissue-Based Map of the Human Proteome. Science (2015) 347:1260419. doi: 10.1126/science.1260419 - DOI - PubMed
    1. Thul PJ, Akesson L, Wiking M, Mahdessian D, Geladaki A, Blal HA, et al. . A Subcellular Map of the Human Proteome. Science (2017) 356:eaal3321. doi: 10.1126/science.aal3321 - DOI - PubMed
    1. Borrebaeck CA. Antibodies in Diagnostics - From Immunoassays to Protein Chips. Immunol Today (2000) 21:379–82. doi: 10.1016/S0167-5699(00)01683-2 - DOI - PubMed
    1. Reader RH, Workman RG, Maddison BC, Gough KC. Advances in the Production and Batch Reformatting of Phage Antibody Libraries. Mol Biotechnol (2019) 61:801–15. doi: 10.1007/s12033-019-00207-0 - DOI - PMC - PubMed
    1. Lu RM, Hwang YC, Liu IJ, Lee CC, Tsai HZ, Li HJ, et al. . Development of Therapeutic Antibodies for the Treatment of Diseases. J BioMed Sci (2020) 27:1. doi: 10.1186/s12929-019-0592-z - DOI - PMC - PubMed

Publication types

-