Effective Optimization of Antibody Affinity by Phage Display Integrated with High-Throughput DNA Synthesis and Sequencing Technologies
Open accessAbstract
Phage display technology has been widely used for antibody affinity maturation for decades. The limited library sequence diversity together with excessive redundancy and labour-consuming procedure for candidate identification are two major obstacles to widespread adoption of this technology. We hereby describe a novel library generation and screening approach to address the problems. The approach started with the targeted diversification of multiple complementarity determining regions (CDRs) of a humanized anti-ErbB2 antibody, HuA21, with a small perturbation mutagenesis strategy. A combination of three degenerate codons, NWG, NWC, and NSG, were chosen for amino acid saturation mutagenesis without introducing cysteine and stop residues. In total, 7,749 degenerate oligonucleotides were synthesized on two microchips and released to construct five single-chain antibody fragment (scFv) gene libraries with 4 x 10(6) DNA sequences. Deep sequencing of the unselected and selected phage libraries using the Illumina platform allowed for an in-depth evaluation of the enrichment landscapes in CDR sequences and amino acid substitutions. Potent candidates were identified according to their high frequencies using NGS analysis, by-passing the need for the primary screening of target-binding clones. Furthermore, a subsequent library by recombination of the 10 most abundant variants from four CDRs was constructed and screened, and a mutant with 158-fold increased affinity (Kd = 25.5 pM) was obtained. These results suggest the potential application of the developed methodology for optimizing the binding properties of other antibodies and biomolecules.
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Conventional in vitro affinity maturation usually takes a long time with poor efficiency due to its small library content, incompatible optimization for multiple goals, and other technical obstacles.
“The conventional in vitro affinity maturation usually takes a long time with poor efficiency due to its small library content, incompatible optimization for multiple goals, and other technical obstacles (32, 49).”
From Deep learning guided optimization of human antibody against SARS-CoV-2 variants with broad neutralization (2022) · cites this paper as [49]
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Cites
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BackgroundMethodSelf-citation
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“The paired-end reads, in part, compensate for the limited length of reads, allowing the sequencing of at least an entire variable domain (VH or VL), and improving the global error rate by reading the CDRs twice (Figure 2) [57,61,62].”
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“In another study, the introduction of diversity into the sequence of anti ErbB2 Ab only at CDRH3 did not result in affinity enhanced variant, while beneficial mutants could be obtained by engineering one of the other contacting CDRs (CDRH1,H2,L1 or L3).(48) This emphasizes that the importance of CDRH3 differ between A…”
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ResultNannini, F., Senicar, L., Parekh, F., Kong, K. J., Kinna, A., Bughda, R., Sillibourne, J., Hu, X. S., Ma, B., Bai, Y., et al. (2020) · mAbs · doi:10.1080/19420862.2020.1864084 · Cited by 24 · see citing works
“NGS technologies have been used in combination with phage display in a number of applications.(24,25) While NGS allows in-depth analysis of phage display libraries, its widespread use has been hampered by limitations in read length(26,27) and signal integrity(16) of several sequencing technologies.”
MethodSimultaneous affinity maturation and developability enhancement using natural liability-free CDRs
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High-throughput retrieval of physical DNA for NGS-identifiable clones in phage display library
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“However the leap has not yet made a dramatic impact on in vivo panning [5, 38, 87, 118, 120].”
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“Inspired by previous pioneering research in this area (D’Angelo et al., 2014; Glanville et al., 2015; Hu et al., 2015), we analyzed the HC and LC of the unselected and selected libraries using Illumina Hiseq3000 platform.”
MethodKnödler, M., Frank, K., Kerpen, L., & Buyel, J. (2023) · Bioengineered · doi:10.1080/21655979.2023.2244235 · Cited by 13 · see citing works
“Accordingly, we will improve the targeting of our RIT by both maturing the affinity of the antibody (fragment) toward CD64, for example by multisitedirected mutagenesis [78] or phage display [79], and by replacing the scFv domain with an antibody heavy or light chain that will be co-expressed with the complementary ch…”
MethodAdvances in next-generation sequencing (NGS) applications in drug discovery and development
Wang, H., Huang, J., Fang, X., Liu, M., Fan, X., & Li, Y. (2025) · Expert Opinion on Drug Discovery · doi:10.1080/17460441.2025.2481262 · Cited by 14 · see citing works
“In a notable study, this approach was used to improve the affinity of a scFv fragment targeting ErbB2 [45].”
Lu, S., Li, Y., Wang, F., Nan, X., & Zhang, S. (2021) · IEEE Transactions on Computational Biology and Bioinformatics · doi:10.1109/tcbb.2021.3083001 · Cited by 14 · see citing works
Perween, R., Ahmed, S., Shrivastava, T., Parray, H. A., Singh, B., Pindari, K. S., Sharma, C., Shukla, S., Sinha, S., Panchal, A. K., et al. (2021) · Biotechnology Progress · doi:10.1002/btpr.3136 · Cited by 13 · see citing works
“The codon usage for mutagenesis in CDRs of these libraries (synthetic or semi-synthetic) are often NNN, NNK, NWG, NWC, and NSG codons, which increase the frequency and chance of incorporation of amber codon into CDRs.(9,10) According to the binomial distribution of codons there are chances that 25% of phage ELISA posi…”
BackgroundStutz, C. C., Georgieva, J. V., & Shusta, E. V. (2018) · AIChE Journal · doi:10.1002/aic.16360 · Cited by 13 · see citing works
Dormeshkin, D., Shapira, M., Dubovik, S., Kavaleuski, A., Katsin, M., Migas, A., Meleshko, A., & Semyonov, S. (2022) · Frontiers in Immunology · doi:10.3389/fimmu.2022.965446 · Cited by 14 · see citing works
“Nevertheless, affinity improvement of H7 was quite low in comparison with successful cases of affinity increasing by more than 2 orders of magnitude (44).”
BackgroundGallo, E. (2024) · Journal of Biomedical Science · doi:10.1186/s12929-024-01018-5 · Cited by 13 · see citing works
“[ 60 ] T h e d ee p s e q u e n c i n g o f a ffi n i t y m a t u r a t i o n A b l i b r a r i e s a ll o w e d f o r a n i n - d e p t h e v a l u a - t i o n o f t h e e n r i c h m e n t l a n d s c a p e s i n C D R s e q u e n c e s a n d a m i n o a c i d s u b s t i t u t i o n s .”
BackgroundInfluentialSeidel-Greven, M., Addai‐Mensah, O., Spiegel, H., Dipah, G. N. C., Schmitz, S., Breuer, G., Frempong, M., Reimann, A., Klockenbring, T., Fischer, R., et al. (2021) · Malaria Journal · doi:10.1186/s12936-020-03548-3 · Cited by 11 · see citing works
“Several methods have been described, including random mutagenesis of CDRH3 and chain shuffling, resulting in the generation of affinity-matured variants [43, 64].”
Smith, M. D., Case, M., Makowski, E. K., & Tessier, P. M. (2023) · Bioinformatics · doi:10.1093/bioinformatics/btad446 · Cited by 10 · see citing works
“…or the abundance of an antibody sequence found after selection, which assumes that variants with the highest affinities become most prevalent during the sorting process (Ravn et al. 2010, 2013, D’Angelo et al. 2014, Hu et al. 2015, Lopez et al. 2017, Barreto et al. 2019, Ferrara et al. 2020).”
BackgroundZhuang, X., Watts, N. R. M., Palmer, I. W., Kaufman, J. D., Dearborn, A. D., Trenbeath, J. L., Eren, E., Steven, A. C., Rader, C. M., & Wingfield, P. T. (2017) · Journal of Biological Chemistry · doi:10.1074/jbc.m117.802272 · Cited by 10 · see citing works
Wang, Z., Cheng, L., Guo, G., Cheng, B., Hu, S., Zhang, H., Zhu, Z., & Niu, L. (2019) · Acta Crystallographica Section D Structural Biology · doi:10.1107/s2059798319006995 · Cited by 10 · see citing works
“The humanization level of HuA21 was greater than 95% (Hu et al., 2015; Xu et al., 2015).”
BackgroundSelf-citationBacterial cell surface characterization by phage display coupled to high-throughput sequencing
Grun, C. N., Jain, R., Schniederberend, M., Shoemaker, C. B., Nelson, B., & Kazmierczak, B. I. (2024) · Nature Communications · doi:10.1038/s41467-024-51912-7 · Cited by 9 · see citing works
“We corrected typographical errors in the references and supplemental tables and made minor stylistic edits to comply with the editorial requirements checklist.”
Matsunaga, R., & Tsumoto, K. (2025) · Protein Science · doi:10.1002/pro.70065 · Cited by 9 · see citing works
Lee, D. C. P., Raman, R., Ghafar, N. A., & Budigi, Y. (2021) · Antiviral Research · doi:10.1016/j.antiviral.2021.105105 · Cited by 8 · see citing works
Tumor-Targeting Domains for Chimeric Antigen Receptor T Cells
Bezverbnaya, K., Mathews, A., Sidhu, J., Helsen, C. W., & Bramson, J. L. (2016) · Immunotherapy · doi:10.2217/imt-2016-0103 · Cited by 9 · see citing works
Ribeiro, R., Moreira, J. N., & Gonçalves, J. (2024) · Scientific Reports · doi:10.1038/s41598-024-61230-z · Cited by 7 · see citing works
“…that only one round of panning may not be sufficient to decrease the percentage of non-functional sequences, such as sequences with stop codons 68 , it is suggested that such value is due to the quality of the constructed library, sequencing errors 69 , or bias during bacteria infection 49 .”
BackgroundMethodResultInfluentialThe Use of Somatic Hypermutation for the Affinity Maturation of Therapeutic Antibodies
Bowers, P. M., Boyle, W. J., & Damoiseaux, R. D. (2018) · Methods in molecular biology · doi:10.1007/978-1-4939-8648-4_24 · Cited by 8 · see citing works
MBE: model-based enrichment estimation and prediction for differential sequencing data
Busia, A., & Listgarten, J. (2023) · Genome biology · doi:10.1186/s13059-023-03058-w · Cited by 7 · see citing works
“…5, 10, 12, 25], and functional enrichment analysis [26], and have wide-ranging biologically significant applications, including antibody design [27, 28]; profiling pathogen proteomes for epitopes and major histocompatibility complex binding [17, 18]; improving thermostability [23]; assessing…”
BackgroundLi, R., Hu, S., Chang, Y., Zhang, Z., Zha, Z., Huang, H., Shen, G., Liu, J., Song, L., & Wei, W. (2016) · International Journal of Molecular Sciences · doi:10.3390/ijms17040563 · Cited by 8 · see citing works
“HuA21 was developed by phage display and antibody affinity maturation technologies on the basis of chA21 with a higher affinity, and the degree of humanization was more than 95% [21].”
Self-citationCohen-Khait, R., & Schreiber, G. (2018) · Biochemistry · doi:10.1021/acs.biochem.8b00172 · Cited by 7 · see citing works
Wang, Z., ZHOU, L., & WU, X. (2024) · Oncology Research Featuring Preclinical and Clinical Cancer Therapeutics · doi:10.32604/or.2024.048564 · Cited by 6 · see citing works
“Among the selection screening methods, phage display is widely recognized as the most prevalent technique [164].”
Brockmann, E., Pyykkö, M., Hannula, H., Khan, K., Lamminmäki, U., & Huovinen, T. (2020) · New Biotechnology · doi:10.1016/j.nbt.2020.09.002 · Cited by 6 · see citing works
de Souza, L. R., Scott, B. M., Bhakta, V., Donkor, D. A., Perruzza, D. L., & Sheffield, W. P. (2018) · Methods in molecular biology · doi:10.1007/978-1-4939-8645-3_3 · Cited by 6 · see citing works
Harwardt, J., Geyer, F. K., Schoenfeld, K., Baumstark, D., Molkenthin, V., & Kolmar, H. (2024) · Antibodies · doi:10.3390/antib13020036 · Cited by 5 · see citing works
“Hu et al. utilized the targeted diversification of multiple CDR regions of a humanized anti-receptor tyrosine kinase 2 (ErbB2) antibody together with phage display to identify a mutant with a 158-fold-increased affinity [21].”
BackgroundTherapeutic Antibodies for Infectious Diseases: Recent Past, Present, and Future
Subramaniam, S., Saville, J. W., Feng, F., & Freiburger, L. A. (2025) · Biochemistry · doi:10.1021/acs.biochem.5c00192 · Cited by 5 · see citing works
Hu, F. J., Lundqvist, M., Uhlén, M., & Rockberg, J. (2019) · Nucleic Acids Research · doi:10.1093/nar/gkz050 · Cited by 4 · see citing works
“The structures of antibodyantigen complexes indicate that the majority, if not all of the six CDRs, may contribute to antigen-binding in most cases (17,18).”
BackgroundMethods to Produce Monoclonal Antibodies for the Prevention and Treatment of Viral Infections
Merkuleva, I. A., Щербаков, Д. Н., & Ilyichev, A. A. (2022) · Russian Journal of Bioorganic Chemistry · doi:10.1134/s1068162022020169 · Cited by 5 · see citing works
Qudsia, S., Merugu, S. B., Mangukiya, H. B., Negi, H., Wu, Z., & Li, D. (2018) · Biochemical and Biophysical Research Communications · doi:10.1016/j.bbrc.2018.03.131 · Cited by 5 · see citing works
Gunaratne, G. S., Gallant, J. P., Ott, K. L., Broome, P. L., Celada, S., West, J. L., Mixdorf, J. C., Aluicio‐Sarduy, E., Engle, J. W., Boros, E., et al. (2025) · Communications Biology · doi:10.1038/s42003-025-08610-x · Cited by 4 · see citing works
Lu, S., Li, Y., Nan, X., & Zhang, S. (2021) · Lecture notes in computer science · doi:10.1007/978-3-030-91415-8_16 · Cited by 4 · see citing works
Fakih, T. M., Wijaya, S., & Priani, S. E. (2021) · Jurnal Sains Farmasi & Klinis · doi:10.25077/jsfk.8.3.316-322.2021 · Cited by 4 · see citing works
Learning what not to select for in antibody drug discovery
Gao, B., Han, J., & Reddy, S. T. (2022) · Cell Reports Methods · doi:10.1016/j.crmeth.2022.100258 · Cited by 3 · see citing works
“In recent years, deep sequencing has become a valuable tool for antibody screening and discovery (Parola et al., 2018), including the sequencing of display libraries to augment selection of candidates with high affinity to target antigens (Hu et al., 2015).”
BackgroundReview on Monoclonal Antibodies (mAbs) as a Therapeutic Approach for Type 1 Diabetes
Agarwal, G., & Patel, M. (2023) · Current Diabetes Reviews · doi:10.2174/1573399820666230831153249 · Cited by 3 · see citing works
Phage display screening of TIGIT-specific antibody for antitumor immunotherapy
Li, D., Hu, S., Qinglin, F., Bao, W., Zhou, W., Xu, T., Ye, T., Liu, H., & Song, L. (2019) · Bioscience Biotechnology and Biochemistry · doi:10.1080/09168451.2019.1617107 · Cited by 3 · see citing works
“ture gene (NCBI Reference Sequence: NM_006505) was synthesized and digested with BamHI and XhoI enzymes, then cloned into pCD-Fc vector to construct pCD-Fc-CD155-FL plasmid [21].”
MethodSelf-citationStructure-Based Antibody Paratope Prediction with 3D Zernike Descriptors and SVM
Daberdaku, S. (2020) · Lecture notes in computer science · doi:10.1007/978-3-030-34585-3_4 · Cited by 3 · see citing works
Nur, A., Lai, J. Y., Ch’ng, A. C. W., Choong, Y. S., Isa, W. Y. H. W., & Lim, T. S. (2024) · International Journal of Biological Macromolecules · doi:10.1016/j.ijbiomac.2024.134217 · Cited by 2 · see citing works
Optimizing Yeast Homologous Recombination for Splicing Large Coronavirus Genome Fragments
Xiong, G., Huang, X., Hu, A., Meng, Z., Cui, J., Feng, Y., Chen, Z., Lu, Y., Yang, Q., & Liu, G. (2024) · International Journal of Molecular Sciences · doi:10.3390/ijms252413742 · Cited by 2 · see citing works
Gunaratne, G. S., Gallant, J. P., Ott, K. L., Broome, P. L., Celada, S., West, J. L., Mixdorf, J. C., Aluicio‐Sarduy, E., Engle, J. W., Boros, E., et al. (2025) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2025.01.13.632555 · Cited by 2 · see citing works
Cui, J., Hu, A., Xiong, X.-H., Wang, Q., Chen, Z., Chen, Z., Lu, Y., Xia, X., Chen, H., & Liu, G. (2024) · International Journal of Molecular Sciences · doi:10.3390/ijms252111514 · Cited by 1 · see citing works
Natali, E. N., Hersch, J., Freiberg, C., & Steigele, S. (2025) · mAbs · doi:10.1080/19420862.2025.2555346 · Cited by 1 · see citing works
“…or recombinant library generated by molecular biology techniques), the library type ( e.g ., random mutagenesis libraries, positional or combinatorial mutation libraries for affinity maturation), 40,41 and the genealogy of pools ( e.g ., exposure to different antigens/targets and off-targets).”
High Affinity Maturated Human Antibodies from Naïve and Synthetic Antibody Repertoires
Lim, C. C., Choong, Y. S., & Lim, T. S. (2018) · InTech eBooks · doi:10.5772/intechopen.71664 · Cited by 1 · see citing works
“Recently, this approach was applied to construct few scFv gene libraries with degenerate oligonucleotides synthesized on two DNA microchips in parallel [115].”
BackgroundMethodApplications of Machine Learning for Predicting Selection Outcomes in Antibody Phage Display
Hogan, D. W. (2016) · University Library - University of Saskatchewan (University of Saskatchewan)
Generation and use of new tools for the characterisation of gut hormone receptors
Biggs, E. K. (2018) · Apollo (University of Cambridge) · doi:10.17863/cam.37487
Beyond Single Clones: High-Throughput Sequencing in Antibody DiscoveryPreprint
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Seidel-Greven, M., Addai‐Mensah, O., Spiegel, H., Chiegoua Dipah, G. N., Schmitz, S., Breuer, G., Frempong, M., Reimann, A., Klockenbring, T., Fischer, R., et al. (2021) · Figshare · doi:10.6084/m9.figshare.13558840
Hoffstedt, M., Wowra, J., Wätzig, H., & Baumann, K. (2026) · mAbs · doi:10.1080/19420862.2026.2644655
“23 In such settings, extrapolation beyond the observed sequence space is neither expected nor desirable, as predictions far from measured variants are inherently more unreliable.”
Wei, M., Bai, S., Chen, W., Wu, J., Pan, Y., Zhao, W., Qian, W., Cao, J., Pan, G., Bai, X., et al. (2026) · Phenomics · doi:10.1007/s43657-025-00295-w
“The maximum library capacities of phage and yeast display are almost 10 10 –10 11 and 10 6 –10 7 , respectively, yet these are insufficient to access the enormous sequence space of CDR regions, which is estimated to be as large as 10 60 (Hu et al. 2015).”
Self-citationSzataniak, I., & Packi, K. (2026) · Antibodies · doi:10.3390/antib15050081
“Unlike conventional colony screening and Sanger sequencing, NGS identified both dominant and low-frequency variants, allowing antibody candidates to be selected based on enrichment patterns across successive rounds of biopanning rather than random clone selection [37,38].”
InfluentialTemplate-Choice Bias in Deep Sequencing of Antibody Phage Display Libraries
Yang, H., Lee, H. K., Ryu, T., Yoo, D. K., & Chung, J. (2026) · Biomolecules · doi:10.3390/biom16101379
“In fact, however, two distinct implications are conflated in this procedure: first, bio-panning provides affinity-based enrichment [4,5]; and second, it enriches for “good expressors”, clones that tend to both propagate preferentially in the E. coli host and package efficiently into bacteriophage…”
- Authors
- Hu, D., Hu, S., Wan, W., Xu, M., Du, R., Zhao, W., Gao, X., Liu, J., Liu, H., & Hong, J.
- Affiliations
- University of Science and Technology of China (CN)
- Corresponding author
- Liu, Haiyan
- Hong, Jiong
- Venue
- PLOS ONE
- Publisher
- Public Library of Science (PLoS)
- Published
- 2015-06-05
- Volume
- 10
- Issue
- 6
- Pages
- e0129125
- ISSN
- 1932-6203 (ISSN-L)
- PMC
- PMC4457833
- OpenAlex
- W2128673241
- Cited by
- 86
- References
- 35
- Citations by year
- 2026: 4 · 2025: 6 · 2024: 11 · 2023: 6 · 2022: 6 · 2021: 15 · 2020: 7 · 2019: 9 · 2018: 13 · 2017: 5 · 2016: 3 · 2015: 1
- FWCI
- 5.94
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- PLoS ONE (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Figshare (submittedVersion, cc-by)
- Funders
- National Natural Science Foundation of China
- University of Science and Technology of China
- National Key Research and Development Program of China
- Topics
- Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Monoclonal and Polyclonal Antibodies Research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chemical Synthesis and Analysis
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › vaccines and immunoinformatics approaches
- Keywords
- affinity maturation, phage display, saturation mutagenesis, degenerate oligonucleotides, single-chain antibody fragments
- MeSH terms
- Antibody Affinity, Base Sequence, Humans, Gene Library, Mutagenesis, Sequence Analysis, DNA, Erb-b2 Receptor Tyrosine Kinases, Peptide Library, Complementarity Determining Regions, Single-Chain Antibodies, High-Throughput Nucleotide Sequencing, Antibodies, Monoclonal, Humanized
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