Affinity maturation of antibodies by combinatorial codon mutagenesis versus error-prone PCR
In vitro affinity maturation of therapeutic monoclonal antibodies is commonly applied to achieve desired properties, such as improved binding kinetics and affinity. Currently there are no universally accepted protocols for generation of variegated antibody libraries or selection thereof. Here, we pe...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2020-01-01
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| Series: | mAbs |
| Subjects: | |
| Online Access: | https://www.tandfonline.com/doi/10.1080/19420862.2020.1803646 |
| _version_ | 1818481357959987200 |
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| author | Jan Fredrik Simons Yoong Wearn Lim Kyle P. Carter Ellen K. Wagner Nicholas Wayham Adam S. Adler David S. Johnson |
| author_facet | Jan Fredrik Simons Yoong Wearn Lim Kyle P. Carter Ellen K. Wagner Nicholas Wayham Adam S. Adler David S. Johnson |
| author_sort | Jan Fredrik Simons |
| collection | DOAJ |
| description | In vitro affinity maturation of therapeutic monoclonal antibodies is commonly applied to achieve desired properties, such as improved binding kinetics and affinity. Currently there are no universally accepted protocols for generation of variegated antibody libraries or selection thereof. Here, we performed affinity maturation using a yeast-based single-chain variable fragment (scFv) expression system to compare two mutagenesis methods: random mutagenesis across the entire V(D)J region by error-prone PCR, and a novel combinatorial mutagenesis process limited to the complementarity-determining regions (CDRs). We applied both methods of mutagenesis to four human antibodies against well-known immuno-oncology target proteins. Detailed sequence analysis showed an even mutational distribution across the entire length of the scFv for the error-prone PCR method and an almost exclusive targeting of the CDRs for the combinatorial method. Though there were distinct mutagenesis profiles for each target antibody and mutagenesis method, we found that both methods improved scFv affinity with similar efficiency. When a subset of the affinity-matured antibodies was expressed as full-length immunoglobulin, the measured affinity constants were mostly comparable to those of the respective scFv, but the full-length antibodies were inferior to their scFv counterparts for one of the targets. Furthermore, we found that improved affinity for the full-length antibody did not always translate into enhanced binding to cell-surface expressed antigen or improved immune checkpoint blocking ability, suggesting that screening with full-length antibody or antigen-binding fragment formats might be advantageous and the subject of a future study. |
| first_indexed | 2024-12-10T11:33:49Z |
| format | Article |
| id | doaj.art-ac6112ed99b44416933c0b907771131b |
| institution | Directory Open Access Journal |
| issn | 1942-0862 1942-0870 |
| language | English |
| last_indexed | 2024-12-10T11:33:49Z |
| publishDate | 2020-01-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | mAbs |
| spelling | doaj.art-ac6112ed99b44416933c0b907771131b2022-12-22T01:50:29ZengTaylor & Francis GroupmAbs1942-08621942-08702020-01-0112110.1080/19420862.2020.1803646Affinity maturation of antibodies by combinatorial codon mutagenesis versus error-prone PCRJan Fredrik Simons0Yoong Wearn Lim1Kyle P. Carter2Ellen K. Wagner3Nicholas Wayham4Adam S. Adler5David S. Johnson6GigaGen, Inc., South San Francisco, CA, USAGigaGen, Inc., South San Francisco, CA, USAGigaGen, Inc., South San Francisco, CA, USAGigaGen, Inc., South San Francisco, CA, USAGigaGen, Inc., South San Francisco, CA, USAGigaGen, Inc., South San Francisco, CA, USAGigaGen, Inc., South San Francisco, CA, USAIn vitro affinity maturation of therapeutic monoclonal antibodies is commonly applied to achieve desired properties, such as improved binding kinetics and affinity. Currently there are no universally accepted protocols for generation of variegated antibody libraries or selection thereof. Here, we performed affinity maturation using a yeast-based single-chain variable fragment (scFv) expression system to compare two mutagenesis methods: random mutagenesis across the entire V(D)J region by error-prone PCR, and a novel combinatorial mutagenesis process limited to the complementarity-determining regions (CDRs). We applied both methods of mutagenesis to four human antibodies against well-known immuno-oncology target proteins. Detailed sequence analysis showed an even mutational distribution across the entire length of the scFv for the error-prone PCR method and an almost exclusive targeting of the CDRs for the combinatorial method. Though there were distinct mutagenesis profiles for each target antibody and mutagenesis method, we found that both methods improved scFv affinity with similar efficiency. When a subset of the affinity-matured antibodies was expressed as full-length immunoglobulin, the measured affinity constants were mostly comparable to those of the respective scFv, but the full-length antibodies were inferior to their scFv counterparts for one of the targets. Furthermore, we found that improved affinity for the full-length antibody did not always translate into enhanced binding to cell-surface expressed antigen or improved immune checkpoint blocking ability, suggesting that screening with full-length antibody or antigen-binding fragment formats might be advantageous and the subject of a future study.https://www.tandfonline.com/doi/10.1080/19420862.2020.1803646Affinity maturationmutagenesis libraryyeast scFv display |
| spellingShingle | Jan Fredrik Simons Yoong Wearn Lim Kyle P. Carter Ellen K. Wagner Nicholas Wayham Adam S. Adler David S. Johnson Affinity maturation of antibodies by combinatorial codon mutagenesis versus error-prone PCR mAbs Affinity maturation mutagenesis library yeast scFv display |
| title | Affinity maturation of antibodies by combinatorial codon mutagenesis versus error-prone PCR |
| title_full | Affinity maturation of antibodies by combinatorial codon mutagenesis versus error-prone PCR |
| title_fullStr | Affinity maturation of antibodies by combinatorial codon mutagenesis versus error-prone PCR |
| title_full_unstemmed | Affinity maturation of antibodies by combinatorial codon mutagenesis versus error-prone PCR |
| title_short | Affinity maturation of antibodies by combinatorial codon mutagenesis versus error-prone PCR |
| title_sort | affinity maturation of antibodies by combinatorial codon mutagenesis versus error prone pcr |
| topic | Affinity maturation mutagenesis library yeast scFv display |
| url | https://www.tandfonline.com/doi/10.1080/19420862.2020.1803646 |
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