In vitro display technologies are a group of methods used to display diverse antibody libraries on the surface of solid supports or particles like phage, yeast, ribosomes, and bacteria. They are powerful tools for the discovery and engineering of novel antibodies with desired binding properties and were first explored for antibody selection over 30 years ago.
Compared to immunization, in vitro display allows for precise selection conditions and immediate availability of the antibody sequence. They also have the adaptability for high-throughput processes, valuable in basic and applied research.1
The isolation of a desired candidate relies on the linkage of the phenotype to the genotype. This coupling allows for barcoding of billions of different protein variants and selection of binders through repeated high-throughput identification.2
High-Throughput Antibody Selection and Screening
A key strength of in vitro display technologies is their adaptability to high-throughput screening processes. Using automated systems, millions of antibody variants can be simultaneously displayed and tested against target antigens, allowing for the rapid selection of candidates with desired traits. High-throughput screening accelerates both basic and applied research, streamlining the process of antibody discovery and engineering.
In vitro display systems also provide immediate access to the genetic information of the selected antibody variant. This direct linkage between the antibody’s phenotype (its binding activity) and its genotype (the DNA or RNA sequence encoding it) is crucial for downstream applications, such as antibody optimization or humanization. This is in contrast to immunization-based methods, where obtaining the genetic sequence of an antibody often requires additional steps, such as hybridoma production and sequencing.
The process of screening relies heavily on iterative rounds of selection, often referred to as panning, where weak binders are eliminated, and strong binders are enriched. In some cases, multiple rounds of mutation and selection may be applied to further improve the binding properties of an antibody. The ability to cycle through these iterations in a controlled, high-throughput environment is one of the reasons why in vitro display technologies are so powerful.
Related: What Is Phage Display?
Phenotype-Genotype Linkage in Display Systems
A defining feature of in vitro display methods is the physical linkage between an antibody’s phenotype (its ability to bind to an antigen) and its genotype (the genetic information encoding the antibody). This linkage is essential for the identification and selection of antibodies from large, diverse libraries. The linkage varies depending on the system used:
Phage Display: In phage display, the genotype-phenotype link is achieved by displaying the antibody fragment on the surface of bacteriophage particles. The phage particle carries the DNA encoding the antibody within it, making it easy to recover and sequence the genetic information of successful candidates. Phage display is one of the most widely used display technologies due to its robustness and scalability.
Yeast Display: Yeast cells are engineered to express antibodies on their surface, with the corresponding genetic material present inside the cell. Yeast display offers the advantage of eukaryotic expression, which can be more relevant for the proper folding and glycosylation of complex proteins like antibodies. Additionally, yeast display allows for the use of flow cytometry, enabling precise quantification of antibody-antigen interactions and sorting of yeast cells displaying the strongest binders.
Ribosome Display: In ribosome display, the antibody is linked to the ribosome-mRNA complex during translation, ensuring that the genetic sequence is still physically associated with the displayed antibody. This method is entirely cell-free, making it possible to create very large libraries, and it allows for the direct selection of functional binders from diverse antibody repertoires.
Bacterial Display: Antibodies can also be displayed on the surface of bacteria such as Escherichia coli. This method can be valuable for the selection of antibody fragments like single-chain variable fragments (scFv). Bacterial display can be advantageous due to the ease of bacterial culture and the ability to perform selections in complex environments, such as under conditions of oxidative stress or in the presence of detergents.
Each of these systems offers specific advantages depending on the nature of the antibody being screened and the downstream application. The tight genotype-phenotype linkage in all these systems ensures that after selection, researchers can quickly and accurately retrieve the genetic sequence of the best-performing antibody, which is critical for further development and production.
Applications in Antibody Discovery and Engineering
In vitro display technologies are widely used in both basic research and applied industrial processes. In antibody discovery, these technologies have been pivotal in generating therapeutic antibodies for cancer, autoimmune diseases, and infectious diseases. Several blockbuster drugs, such as adalimumab (Humira), were developed using phage display technology. By allowing researchers to screen enormous libraries of antibody variants, in vitro display methods enable the discovery of antibodies with high specificity, low cross-reactivity, and superior binding kinetics.
In the context of antibody engineering, in vitro display systems are employed to fine-tune antibody properties, such as affinity, stability, and effector functions. For example, once an antibody with moderate affinity is identified, further rounds of selection can be performed to isolate variants with improved affinity through a process known as affinity maturation. Moreover, these methods can be adapted to select for other desirable properties, such as resistance to aggregation, enhanced solubility, or prolonged serum half-life, which are crucial factors in therapeutic antibody development.
Bradbury, A. R., Sidhu, S., Dübel, S., & McCafferty, J. (2011). Beyond natural antibodies: The power of in vitro display technologies. Nature Biotechnology, 29(3), 245-254. https://www.nature.com/articles/nbt.1791
Valldorf, B., Hinz, S., Russo, G., Pekar, L., Mohr, L., Klemm, J., Doerner, A., Krah, S., Hust, M. & Zielonka, S. (2022). Antibody display technologies: selecting the cream of the crop. Biological Chemistry, 403(5-6), 455-477. https://www.degruyter.com/document/doi/10.1515/hsz-2020-0377/html
Antibody specificity refers to an antibody's ability to selectively bind to a unique epitope on a target antigen while avoiding interactions with unrelated antigens. This property arises from the highly specialized antigen-binding site located in the variable region of the antibody, which determines its unique binding characteristics.
Antibody affinity refers to the strength of the binding interaction between a single antigen epitope and the paratope (binding site) of an antibody. This interaction is a fundamental measure of how well an antibody recognizes its specific antigen target.
Recombinant antibodies are produced using genetic engineering techniques, unlike traditional antibody production, where the immune system generates antibodies without direct control over their sequence. By introducing genes encoding antibody fragments into host cells, such as bacteria or mammalian cells, recombinant antibodies can be expressed, purified, and deployed for applications including research, diagnostics, and therapeutics.
Recombinant antibody expression is a biotechnological process that involves engineering and producing antibodies outside their natural context using recombinant DNA technology.
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