Generation and diversification of recombinant monoclonal antibodies

  1. Keith F DeLuca
  2. Jeanne E Mick
  3. Amy Hodges Ide
  4. Wanessa C Lima
  5. Lori Sherman
  6. Kristin L Schaller
  7. Steven M Anderson
  8. Ning Zhao
  9. Timothy J Stasevich
  10. Dileep Varma
  11. Jakob Nilsson
  12. Jennifer G DeLuca  Is a corresponding author
  1. Colorado State University, United States
  2. University of Geneva, Switzerland
  3. University of Colorado Anschutz Medical Campus, United States
  4. Northwestern University, United States
  5. University of Copenhagen, Denmark

Abstract

Antibodies are indispensable tools used for a large number of applications in both foundational and translational bioscience research; however, there are drawbacks to using traditional antibodies generated in animals. These include a lack of standardization leading to problems with reproducibility, high costs of antibodies purchased from commercial sources, and ethical concerns regarding the large number of animals used to generate antibodies. To address these issues, we have developed practical methodologies and tools for generating low-cost, high-yield preparations of recombinant monoclonal antibodies and antibody fragments directed to protein epitopes from primary sequences. We describe these methods here, as well as approaches to diversify monoclonal antibodies, including customization of antibody species specificity, generation of genetically encoded small antibody fragments, and conversion of single chain antibody fragments (e.g. scFv) into full-length, bivalent antibodies. This study focuses on antibodies directed to epitopes important for mitosis and kinetochore function; however, the methods and reagents described here are applicable to antibodies and antibody fragments for use in any field.

Data availability

All data generated during this study are included in the manuscript. We will also deposit the plasmid text files and maps on our institutional repository and AddGene.

Article and author information

Author details

  1. Keith F DeLuca

    Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, United States
    Competing interests
    No competing interests declared.
  2. Jeanne E Mick

    Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, United States
    Competing interests
    No competing interests declared.
  3. Amy Hodges Ide

    Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, United States
    Competing interests
    No competing interests declared.
  4. Wanessa C Lima

    Geneva Antibody Facility, Faculty of Medicine, University of Geneva, Geneva, Switzerland
    Competing interests
    No competing interests declared.
  5. Lori Sherman

    CU Cancer Center Cell Technologies Shared Resource, University of Colorado Anschutz Medical Campus, Aurora, United States
    Competing interests
    No competing interests declared.
  6. Kristin L Schaller

    Department of Pediatric Hematology, Oncology and Bone Marrow Transplant, University of Colorado Anschutz Medical Campus, Aurora, United States
    Competing interests
    No competing interests declared.
  7. Steven M Anderson

    Department of Pathology, University of Colorado Anschutz Medical Campus, Aurora, United States
    Competing interests
    No competing interests declared.
  8. Ning Zhao

    Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, United States
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7092-6229
  9. Timothy J Stasevich

    Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, United States
    Competing interests
    No competing interests declared.
  10. Dileep Varma

    Department of Cell and Developmental Biology, Northwestern University, Chicago, United States
    Competing interests
    No competing interests declared.
  11. Jakob Nilsson

    The Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4100-1125
  12. Jennifer G DeLuca

    Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, United States
    For correspondence
    jdeluca@colostate.edu
    Competing interests
    Jennifer G DeLuca, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3598-1721

Funding

National Institute of General Medical Sciences (R35GM130365)

  • Jennifer G DeLuca

National Institute of General Medical Sciences (MIRA R35GM119728)

  • Timothy J Stasevich

National Institute of General Medical Sciences (K99GM141453)

  • Ning Zhao

National Institute of General Medical Sciences (R01GM135391)

  • Dileep Varma

National Science Foundation (MCB-1845761)

  • Timothy J Stasevich

National Cancer Institute (P30CA046934)

  • Lori Sherman
  • Steven M Anderson

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Silke Hauf, Virginia Tech, United States

Publication history

  1. Received: July 10, 2021
  2. Preprint posted: September 11, 2021 (view preprint)
  3. Accepted: December 20, 2021
  4. Accepted Manuscript published: December 31, 2021 (version 1)
  5. Accepted Manuscript updated: January 7, 2022 (version 2)
  6. Version of Record published: January 17, 2022 (version 3)

Copyright

© 2021, DeLuca et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,598
    Page views
  • 322
    Downloads
  • 1
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Keith F DeLuca
  2. Jeanne E Mick
  3. Amy Hodges Ide
  4. Wanessa C Lima
  5. Lori Sherman
  6. Kristin L Schaller
  7. Steven M Anderson
  8. Ning Zhao
  9. Timothy J Stasevich
  10. Dileep Varma
  11. Jakob Nilsson
  12. Jennifer G DeLuca
(2021)
Generation and diversification of recombinant monoclonal antibodies
eLife 10:e72093.
https://doi.org/10.7554/eLife.72093

Further reading

    1. Biochemistry and Chemical Biology
    Tiantian Wei et al.
    Research Article Updated

    The dual-specificity tyrosine phosphorylation-regulated kinase DYRK2 has emerged as a critical regulator of cellular processes. We took a chemical biology approach to gain further insights into its function. We developed C17, a potent small-molecule DYRK2 inhibitor, through multiple rounds of structure-based optimization guided by several co-crystallized structures. C17 displayed an effect on DYRK2 at a single-digit nanomolar IC50 and showed outstanding selectivity for the human kinome containing 467 other human kinases. Using C17 as a chemical probe, we further performed quantitative phosphoproteomic assays and identified several novel DYRK2 targets, including eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and stromal interaction molecule 1 (STIM1). DYRK2 phosphorylated 4E-BP1 at multiple sites, and the combined treatment of C17 with AKT and MEK inhibitors showed synergistic 4E-BP1 phosphorylation suppression. The phosphorylation of STIM1 by DYRK2 substantially increased the interaction of STIM1 with the ORAI1 channel, and C17 impeded the store-operated calcium entry process. These studies collectively further expand our understanding of DYRK2 and provide a valuable tool to pinpoint its biological function.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Lukas P Feilen et al.
    Research Article

    Cleavage of membrane proteins in the lipid bilayer by intramembrane proteases is crucial for health and disease. Although different lipid environments can potently modulate their activity, how this is linked to their structural dynamics is unclear. Here we show that the carboxy-peptidase-like activity of the archaeal intramembrane protease PSH, a homolog of the Alzheimer's disease-associated presenilin/γ-secretase is impaired in micelles and promoted in a lipid bilayer. Comparative molecular dynamics simulations revealed that important elements for substrate binding such as transmembrane domain 6a of PSH are more labile in micelles and stabilized in the lipid bilayer. Moreover, consistent with an enhanced interaction of PSH with a transition-state analog inhibitor, the bilayer promoted the formation of the enzyme´s catalytic active site geometry. Our data indicate that the lipid environment of an intramembrane protease plays a critical role in structural stabilization and active site arrangement of the enzyme-substrate complex thereby promoting intramembrane proteolysis.