Streamlined human antibody generation and optimization by exploiting designed immunoglobulin loci in a B cell line

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Abstract

Monoclonal antibodies (mAbs) are widely utilized as therapeutic drugs for various diseases, such as cancer, autoimmune diseases, and infectious diseases. Using the avian-derived B cell line DT40, we previously developed an antibody display technology, namely, the ADLib system, which rapidly generates antigen-specific mAbs. Here, we report the development of a human version of the ADLib system and showcase the streamlined generation and optimization of functional human mAbs. Tailored libraries were first constructed by replacing endogenous immunoglobulin genes with designed human counterparts. From these libraries, clones producing full-length human IgGs against distinct antigens can be isolated, as exemplified by the selection of antagonistic mAbs. Taking advantage of avian biology, effective affinity maturation was achieved in a straightforward manner by seamless diversification of the parental clones into secondary libraries followed by single-cell sorting, quickly affording mAbs with improved affinities and functionalities. Collectively, we demonstrate that the human ADLib system could serve as an integrative platform with unique diversity for rapid de novo generation and optimization of therapeutic or diagnostic antibody leads. Furthermore, our results suggest that libraries can be constructed by introducing exogenous genes into DT40 cells, indicating that the ADLib system has the potential to be applied for the rapid and effective directed evolution and optimization of proteins in various fields beyond biomedicine.

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Most cited references 44

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Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides.

J Wölle, Sarah Fischer, Andreas Plückthun … (2000)

By analyzing the human antibody repertoire in terms of structure, amino acid sequence diversity and germline usage, we found that seven V(H) and seven V(L) (four Vkappa and three Vlambda) germline families cover more than 95 % of the human antibody diversity used. A consensus sequence was derived for each family and optimized for expression in Escherichia coli. In order to make all six complementarity determining regions (CDRs) accessible for diversification, the synthetic genes were designed to be modular and mutually compatible by introducing unique restriction endonuclease sites flanking the CDRs. Molecular modeling verified that all canonical classes were present. We could show that all master genes are expressed as soluble proteins in the periplasm of E. coli. A first set of antibody phage display libraries totalling 2x10(9) members was created after cloning the genes in all 49 combinations into a phagemid vector, itself devoid of the restriction sites in question. Diversity was created by replacing the V(H) and V(L) CDR3 regions of the master genes by CDR3 library cassettes, generated from mixed trinucleotides and biased towards natural human antibody CDR3 sequences. The sequencing of 257 members of the unselected libraries indicated that the frequency of correct and thus potentially functional sequences was 61 %. Selection experiments against many antigens yielded a diverse set of binders with high affinities. Due to the modular design of all master genes, either single binders or even pools of binders can now be rapidly optimized without knowledge of the particular sequence, using pre-built CDR cassette libraries. The small number of 49 master genes will allow future improvements to be incorporated quickly, and the separation of the frameworks may help in analyzing why nature has evolved these distinct subfamilies of antibody germline genes. Copyright 2000 Academic Press.

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Mice with megabase humanization of their immunoglobulin genes generate antibodies as efficiently as normal mice.

A Murphy, L MacDonald, S. Stevens … (2014)

Mice genetically engineered to be humanized for their Ig genes allow for human antibody responses within a mouse background (HumAb mice), providing a valuable platform for the generation of fully human therapeutic antibodies. Unfortunately, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which their genetic humanization was carried out. Heretofore, HumAb mice have been generated by disrupting the endogenous mouse Ig genes and simultaneously introducing human Ig transgenes at a different and random location; KO-plus-transgenic humanization. As we describe in the companion paper, we attempted to make mice that more efficiently use human variable region segments in their humoral responses by precisely replacing 6 Mb of mouse Ig heavy and kappa light variable region germ-line gene segments with their human counterparts while leaving the mouse constant regions intact, using a unique in situ humanization approach. We reasoned the introduced human variable region gene segments would function indistinguishably in their new genetic location, whereas the retained mouse constant regions would allow for optimal interactions and selection of the resulting antibodies within the mouse environment. We show that these mice, termed VelocImmune mice because they were generated using VelociGene technology, efficiently produce human:mouse hybrid antibodies (that are rapidly convertible to fully human antibodies) and have fully functional humoral immune systems indistinguishable from those of WT mice. The efficiency of the VelocImmune approach is confirmed by the rapid progression of 10 different fully human antibodies into human clinical trials.

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Human antibodies from transgenic animals.

Nils Lonberg (2005)

Laboratory mice provide a ready source of diverse, high-affinity and high-specificity monoclonal antibodies (mAbs). However, development of rodent antibodies as therapeutic agents has been impaired by the inherent immunogenicity of these molecules. One technology that has been explored to generate low immunogenicity mAbs for in vivo therapy involves the use of transgenic mice expressing repertoires of human antibody gene sequences. This technology has now been exploited by over a dozen different pharmaceutical and biotechnology companies toward developing new therapeutic mAbs, and currently at least 33 different drugs in clinical testing--including several in pivotal trials--contain variable regions encoded by human sequences from transgenic mice. The emerging data from these trials provide an early glimpse of the safety and efficacy issues for these molecules. Nevertheless, actual product approval, the biggest challenge so far, is required to fully validate this technology as a drug discovery tool. In the future, it may be possible to extend this technology beyond rodents and use transgenic farm animals to directly generate and produce human sequence polyclonal sera.

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Author and article information

Contributors

Hidetaka Seo:

ORCID: http://orcid.org/0000-0002-8676-8955

hidetakaseo@bio.c.u-tokyo.ac.jp

Yukoh Nakazaki: antibody@chiome.co.jp

Journal

Journal ID (nlm-ta): Cell Mol Immunol

Journal ID (iso-abbrev): Cell Mol Immunol

Title: Cellular and Molecular Immunology

Publisher: Nature Publishing Group UK (London )

ISSN (Print): 1672-7681

ISSN (Electronic): 2042-0226

Publication date (Electronic): 26 May 2020

Publication date PMC-release: 26 May 2020

Publication date (Print): June 2021

Volume: 18

Issue: 6

Pages: 1545-1561

Affiliations

[1 ]GRID grid.26999.3d, ISNI 0000 0001 2151 536X, Department of Life Sciences, Graduate School of Arts and Sciences, , The University of Tokyo, ; Tokyo, Japan

[2 ]Research Laboratories, Chiome Bioscience Inc, Tokyo, Japan

Author information

Hidetaka Seo http://orcid.org/0000-0002-8676-8955

Atsushi Sawada http://orcid.org/0000-0002-6081-8643

Article

Publisher ID: 440

DOI: 10.1038/s41423-020-0440-9

PMC ID: 8166883

PubMed ID: 32457406

SO-VID: 8f9c2e87-9b3a-4392-b45a-4db0f9302798

License:

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

History

Date received : 13 February 2020

Date revision received : 6 April 2020

Date accepted : 7 April 2020

Funding

Funded by: FundRef https://doi.org/10.13039/501100001863, New Energy and Industrial Technology Development Organization (NEDO);

Custom metadata

ScienceOpen disciplines: Immunology

Keywords: immunoglobulin rearrangements,homologous recombination,antibody therapeutics,antibody engineering,drug discovery,cell biology,immunology

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ScienceOpen disciplines: Immunology

Keywords: immunoglobulin rearrangements, homologous recombination, antibody therapeutics, antibody engineering, drug discovery, cell biology, immunology

Streamlined human antibody generation and optimization by exploiting designed immunoglobulin loci in a B cell line

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Abstract

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Most cited references 44

Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides.

Mice with megabase humanization of their immunoglobulin genes generate antibodies as efficiently as normal mice.

Human antibodies from transgenic animals.

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