Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
The unique qualities of antibodies—high affinity and specificity for their targets—make them one of the most powerful and indispensable tools in biology. Commonly used immunoassays such as ELISAs, Westerns, flow cytometry and immunoprecipitation are constantly evolving as the variety and diversity of antibodies available continue to improve. Antibodies used as therapeutics are also emerging as successful clinical tools. Today researchers can tweak antibodies to generate “made-to-order” products selected and optimized for specific applications—also known as antibody engineering. Here are some current tools being used to better engineer and screen for antibodies that can be used for novel detection and discoveries.
Monitor binding kinetics
The study of antibodies and how they interact with and bind to target molecules is benefiting greatly from the use of surface plasmon resonance (SPR). SPR uses the detection of reflected light at different angles to measure the interactions of molecules without the use of labels or tags. GE Healthcare Life Sciences offers several SPR-based instruments. According to Jason Schuman, senior Biacore product specialist for GE Healthcare Life Sciences business, the Biacore T200 and 4000 systems are most commonly used for antibody screening and characterization. They are also used for determining antibody affinity, kinetics and active concentrations
One of the biggest challenges facing researchers who design antibody therapeutics, says Schuman, is discriminating between small differences in candidate antibodies. “When companies are developing many candidates, and they have to choose which lead candidates to spend money developing, it is very important and also hard to resolve fine differences between them,” he says. That’s where technology like SPR becomes so valuable. It enables researchers to measure binding kinetics, with parameters such as off rates. “If you’re looking at something with a mechanism of action like a receptor blockade, then the off rate is critically important,” he says, “because there is a strong correlation between off rate and therapeutic efficacy.”
According to Schuman, the biggest recent development in antibody engineering is the use of alternate scaffolds, such as antibody drug conjugates (ADCs) and bispecifics. In the latter case, two antibody domains are linked together such that the structure binds two targets instead of just one. Biacore instruments are often used for characterizing such alternate scaffolds, says Schuman, including developing validated assays for regulatory agency approval.
ForteBio’s Octet instruments utilize biolayer interferometry (BLI), an optical analytical technology that monitors distinct changes in the interference pattern of white light reflected from two surfaces: a layer of immobilized protein on a biosensor tip and an internal reference layer. This method uses biosensors and label-free quantitation for rapid and reliable comprehensive kinetic analyis in real time.
According to Dominic Andrada, senior product manager at ForteBio (a division of Pall Life Sciences), “Antibody engineering is a very iterative process.” Andrada shares that researchers are often sensitive to time lines. “Label-free methods have to be totally reliable, because reliable kinetics data comes from optimization and confirmation.” If they can shorten a 1.5-hour procedure into 15 minutes, he says, then researchers can feel free to do that extra experiment to confirm previous findings. Reproducibility and confirmation are key factors in identifying and selecting antibody clones.
Bio-Rad Laboratories also offers a label-free SPR-based system to measure antibody affinity, specificity and kinetics in real time. The ProteOn™ XPR36 protein interaction array system can analyze up to 36 interactions simultaneously in a 6x6 array.
Needle in a haystack
Screening antibody-producing cell lines to identify and select the best clonal candidates can be a time-consuming and laborious process. Sometimes this challenge is easier to tackle with automation. Molecular Devices offers the ClonePix2™ system, which can screen thousands of cell colonies and automatically rank them by antibody expression level, which is measured by cellular-fluorescence intensity. High secreting clones are picked and screened for the rate of growth and clonality verification by CloneSelect™ imager. Their Qpix™ 400 Systems also screen phage libraries used to choose candidates for anti-cancer and anti-inflammatory therapeutic antibodies. These systems “are referred to as ‘Ferraris’ by our customers, as they are ultrafast and can screen 3,000 clones per hour in white light or fluorescence,” says Natalia Lysaya, global products manager in the biotherapeutics business at Molecular Devices. Clone-screening and imaging systems like these help researchers rise to the challenge of “finding a needle in a haystack,” says Lysaya. “In other words, finding a unique, high-value clone that expresses desired levels of protein or antibody of interest, exhibits stability and is monoclonal.”
Do it yourself
Antibody-engineering tools are emerging not only from companies but also from academic researchers such as Andrew Beavil, senior lecturer in molecular biophysics at King's College London, who studies IgE antibodies and receptors. Beavil’s colleagues were trying to use IgE isotype antibodies for cancer immunotherapy, but the options then available were either too expensive (commercial vector-based methods) or too labor intensive (transient expression-based methods). “They needed a fast method to clone and express antibodies and the ability to switch isotype and potentially species,” says Beavil. “We decided to develop our own vectors based on the bi-cistronic pVITRO1 mammalian vectors from Invivogen” [1].
The resulting system uses ligation-independent cloning and a set of vectors that cover all human-antibody isotypes. “The cloning is fast, and a stable cell line can be obtained in just a few weeks, which is then easy to maintain by people [familiar with] regular cell culture,” says Beavil. The vectors are available from nonprofit plasmid distributor Addgene. Beavil’s group has used the vectors for studies in allergy and allergy/oncology applications [2,3]. “We are hopeful that this free toolkit will make life much easier for academic labs to investigate the role of different antibody isotypes and generally speed things up,” he says.
An antibody-related alternative
Avacta Life Sciences provides another antibody-related alternative with its Affimers, small proteins (about 120 amino acids in size). “The original scaffold was engineered to lack significant binding to human proteins, [so] there is lower background when Affimers are used in ligand-binding assays, or more effective targeting when using Affimers as therapeutic molecules,” says Avacta’s CSO, Paul Ko Ferrigno. Affimers are suitable for many types of applications—expressed in cultured cells, used to detect biomolecules in vitro, or immobilized in microarrays or biosensors.
Affimers may shine in one application where antibodies struggle, says Ferrigno—studies of cell signaling pathways and drug development. “Biology is mostly mediated by protein-protein interactions,” he says. “If an Affimer binds to a protein-interaction surface and competes with a natural partner, you may be able to create an observable phenotype.” Using Affimers to show a link between protein inhibition and desired phenotype, he says, may be more cost-effective when validating possible drug candidates.
Fire the engineering up!
Methods for engineering and analyzing antibodies are growing in many areas—including screening for antibody-producing clones, analyzing antibody kinetics, generating customized do-it-yourself antibodies and venturing into Affimers. With evolving production and screening technologies available, chances are good that you can find recent innovations in antibody engineering to move your research forward.
References
[1] Dodev, TS, et al., “A tool kit for rapid cloning and expression of recombinant antibodies,” Sci Rep, 4:5885, 2014. [PMID: 25073855]
[2] Dodev, TS, et al., “Inhibition of allergen-dependent IgE activity by antibodies of the same specificity but different class,” Allergy, 70(6):720-724, 2015. [PMID: 25758595]
[3] Karagiannis, SN, et al., “Recombinant IgE antibodies for passive immunotherapy of solid tumours: from concept towards clinical application,” Cancer Immunol Immunother, 61(9):1547-1564, 2012. [PMID: 22139135]
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