Year-end Sale % OFF 
Abstract
Monoclonal antibodies (mAbs) have enabled numerous basic research discoveries and therapeutic approaches for many protein classes. However, there still exist a number of target classes, such as multi-pass membrane proteins, for which antibody discovery is difficult, due in part to lack of high quality, recombinant protein. Here we describe the impact of several parameters on antigen expression and the development of mAbs against human claudin 4 (CLDN4), a potential multi-indication cancer target. Using gene gun-based DNA delivery and bioluminescence imaging, we optimize promoter type by comparing expression profiles of four robust in vivo promoters. In addition, we observe that most vectors rapidly lose expression, ultimately reaching almost background levels by three days post-delivery. Recognizing this limitation, we next explored skin pretreatment strategies as an orthogonal method to further boost the efficiency of mAb generation. We show that SDS pretreatment can boost antigen expression, but fails to significantly increase mAb discovery efficiency. In contrast, we find that sandpaper pretreatment yields 5-fold more FACS+ anti-CLDN4 hybridomas, without impacting antigen expression. Our findings coupled with other strategies to improve DNA immunizations should improve the success of mAb discovery against other challenging targets and enable the generation of critical research tools and therapeutic candidates.
Highlights
Monoclonal antibodies bind their targets with high affinity and specificity, making them critical research tools and therapeutic agents
We demonstrate that pretreatment of the skin prior to gene gun delivery can increase antigen expression level as well as polyclonal Abs (pAb) titers against claudin 4 (CLDN4)
Antigen expression during the first 2–3 days has shown to be critical to driving immune responses to gene gun-based immunizations [20, 21]
Summary
Monoclonal antibodies (mAbs) bind their targets with high affinity and specificity, making them critical research tools and therapeutic agents. A wide variety of both in vitro selection technologies, such as phage or yeast display, and in vivo immunization methods exist for antibody discovery. For targets in which high quality, recombinant protein can be obtained, both avenues have proven to robustly deliver diverse panels of mAbs [1,2,3]. When recombinant protein is limiting, which is often the case for multi-spanning membrane proteins (MPs), existing antibody discovery strategies can fail to generate large panels of mAbs [4, 5].
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
