Selecting the Right Platform for Your Antibody Discovery Project
Contributed by John Charpentier, Ph.D. and Li Hui, M.D., Ph.D.
The variety of technology platforms available for therapeutic antibody discovery can be both exciting and daunting. Here we provide some general guidelines to help investigators understand their capabilities and applications.
Since the introduction of hybridoma technology in the 1970s, numerous antibody discovery platforms have been developed. Selecting the right one for your application requires an alignment of your project goals and an understanding of the capabilities and limitations of each.
Clarify your goals
The first priority is to clarify what you hope to achieve. Ask yourself the following:
- Is your goal to identify as many antigen-specific B cell clones as possible in one step?
- Do you want to source them in a human antibody transgenic mouse model to spare time-consuming humanization steps later?
- Do you want to develop a monoclonal antibody with single-target specificity or a multi-specific antibody?
- Do you plan to develop a candidate into an antibody-drug conjugate (ADC)?
- Is functional screening early in the discovery process a priority?
Answering questions like these will help you to select the platform technology best suited for your purposes.
Know what to expect
Biocytogen currently offers Beacon-based and FACS-based single B cell cloning discovery platforms. Regardless of which platform is used, many workflow steps are the same:
- When animals are immunized with your target antigen, antibody titers are measured to ensure desirable levels.
- Lymphoid tissues are collected from the animals and single cell suspensions are prepared.
- Antibody-producing B cells from these tissues are then enriched by magnetic separation before further analysis and characterization.
- After screening and export of desirable clones, cells of interest are lysed for RT-PCR and sequencing of their immunoglobulin variable domains. The sequences selected by the client for further validation are then cloned into expression vectors and transiently transfected into EXPi293 cells.
- Antibodies in supernatants collected from these transfection experiments are then validated and characterized by ex vivo methods, such as enzyme-linked immunosorbent assay (ELISA), flow cytometry, or surface plasmon resonance (SPR).
Hybridoma technology remains a low cost, popular means of discovering antibody clones of favorable affinity in mice or rabbits. However, newer single B cell screening platforms, including FACS- and Beacon®-based workflows, are quickly gaining popularity. Both allow for the identification of single, antigen-specific B cell clones in a high-throughput manner, in the former case by flow cytometry and in the latter by use of the Beacon® Optofluidic System.
Recognize each platform’s strengths and limitations
While hybridoma technology is the oldest and most well-known antibody discovery platform, it has some significant drawbacks. Screening is very time-consuming, and it can often take more than three months before specific hits are identified. Moreover, positive hybridoma clones are not stable and have the tendency to lose specificity against the antigen.
Beacon-based Screening Platform
By comparison, Beacon®-based single B cell cloning offers numerous advantages. First, it allows for much more rapid screening —as brief as one day—and since positive cells are captured so quickly, attrition rates are lower, and screening success rates are higher than those seen with hybridoma-based discovery. Screening with a single Beacon® chip (housing 11K to 20K B cells) can yield several hundred positive clones in one run, with the capacity to expand up to 4 chips. Second, there are multiple functional screening assays that the Beacon system can perform within one day: cross-reactivity binding assays, live cell binding assays and in-pen ligand competition assays. This means that by two weeks post-screening, you can obtain the sequences of antibodies that:
- recognize a specific antigen,
- bind to live cells that express the antigen on the cell surface, and
- competitively block the ligand of the antigen receptor.
Finally, since affinity maturation occurs in vivo prior to Beacon screening, the antigen- binding affinities of derived clones are higher as well.
After immunization, serum titer measurements (Left; gray curve represents a non-immunized control) confirm a successful immune response.
(Right) snapshot of antibody hits captured by the Beacon from single antibody-secreting B cells. Anywhere from 100-1000 antibody hits can be recovered, depending on the characteristics of your target.
FACS-based single B cell cloning technology also provides a very efficient and straightforward workflow. For targets that have soluble antigen available, positive B cells can be quickly sorted and identified. The FACS platform is normally not suitable for multi-pass transmembrane targets- however, the development of virus-like particles (VLP) makes it possible to screen and sort positive B cells for these difficult targets. Biocytogen has established a VLP workflow that allows for efficient immunization and antibody screening for challenging targets such as GPCRs and Ion channels.
Species Capabilities and Limitations
Single B cell cloning technology is a more universal and adaptable platform in that many species can be used, as long as the immunoglobulin genetic information is known and the screening reagents are well established. This is in contrast to hybridoma technology, which can only use mice, rats and rabbits as hosts.
If your aim is to develop antibodies from primary human cells, peripheral blood mononuclear cells (PBMCs) can be used to discover human antibodies targeting a specific antigen. Biocytogen has established both Beacon-based and FACS-based human PBMC antibody discovery. Please contact us for more details if you are interested.
RenMab™ and RenLite® -powered discovery
Immunoglobulin-humanized mice are a powerful adjunct for any thera
peutic antibody discovery project. Biocytogen’s industry-leading RenMab™ mice express fully contiguous, human heavy chain and kappa light chain variable domains, while RenLite® mice contain the full human heavy chain with a common light chain; both models were engineered to enable investigators to generate fully human therapeutic antibodies in vivo. These mice exhibit strong immune responses that are comparable to wild type mice, but also produce high affinity, high diversity antibodies with desirable developability. By sourcing B cell clones in immunoglobulin-humanized RenMab™ or RenLite® mice prior to screening, one can identify antibody hits from a humanized, affinity matured repertoire. Antibody discovery using RenMice spares costly and time-consuming humanization steps during later stages of therapeutic development, and allows for efficient assembly of fully human bispecific or multi-specific antibodies for therapeutic discovery.
Set up a meeting
Once you’ve clarified your project goals, determined which platform is most appropriate, and selected your preferred antibody source, it’s time to schedule a meeting with Biocytogen. Our antibody discovery team will evaluate your project, make recommendations, and together with our business managers share a price quote. They’ll also be able to answer any questions or concerns you may have about intellectual property, deliverables, and further development.