Table of Contents
Antibody discovery technology plays a vital role in drug development, mainly discovering antibodies with specific binding activity by screening antibody libraries or antibodies obtained from animal immunization. This can help researchers accelerate the drug development process. It can also help researchers better understand diseases, including disease pathogenesis and discovering new therapeutic targets.
Using animals for research, development, and manufacturing has been controversial in many fields for decades, including antibody research, where most antibody-based reagents and therapeutics have historically been produced using animal immunization.
There is an ongoing discussion about the pros and cons of using animal immunization for antibody discovery versus using display technology for in vitro selection from recombinant libraries of non-animal origin. While it has been argued that recombinant display libraries could reduce animal consumption, some scientists believe that the number of animals used in immunization campaigns is dwarfed by the number sacrificed in preclinical studies. Therefore, improved quality control of antibodies before in vivo studies will significantly impact animal consumption.
Source of Antibodies
Antibodies can be purified directly from the plasma of immunized animals, hybridoma cell cultures (also derived from immunized animals), and recombinant cell lines (such as the Chinese hamster ovary cell line). Each production method is still used to produce antibodies for therapeutic, diagnostic, and research reagents, including affinity chromatography, and can produce antibodies with varying clonality.
Isolation of antibodies from animal plasma requires the continuous use of animals for discovery and manufacturing. This fundamentally differs from using hybridoma technology and B-cell screening, where animal immunization is a one-time investment. Therefore, some disadvantages will undoubtedly be, including batch-to-batch variability, high animal consumption, and undefined clonal composition for plasma-derived antibodies.
Likewise, hybridoma cell lines used for “monoclonal” antibody production sometimes contain two or more generative antibody genes, affecting product clonality and downstream applications that may require monospecificity. In contrast, antibodies produced using recombinant expression techniques can be made into products with a more uniform monoclonal composition, thereby improving reproducibility.
Benefits and Drawbacks of Animal-Derived Antibody Discovery
Immunization of animals has been used for over 120 years to raise antibodies for research, diagnostic and therapeutic purposes. In addition to being a proven and technically straightforward method, the use of animal immunizations has the following benefits that are effective for both (recombinant) monoclonal and polyclonal antibodies:
- Generate antibodies that have undergone an in vivo affinity maturation process, resulting in high affinity and stability in vivo.
- Gain valuable information on antibody lineage information and paratope patterns using high-throughput sequencing.
- Does not require exposure to the antigen to produce antibodies (e.g., by using DNA/RNA immunization)
The Medicilon antibody development platform has been deeply involved in antibody-drug research and development for many years. Its technical services include ADA antibody development technical services and druggable antibody development technical services, ADA rabbit polyclonal antibody development, ADA mouse hybridoma monoclonal antibody development, phage display rabbit monoclonal antibody development services, mouse monoclonal antibody development technical services, mouse/rabbit single B cell antibody development technical services and other projects.
Since the establishment of the platform, it has accumulated many well-known domestic and foreign pharmaceutical companies and scientific research institution customers. It has successfully helped the antibody projects of pharmaceutical companies such as Jemincare and Bio-Thera to be approved for clinical trials. It has achieved continuous cooperation for many years, gaining the constant trust of customers.
The production of antibodies in the body is a complex process that has undergone natural evolution and optimization over hundreds of millions of years. Multiple IGHV, IGLV, and IGKV germline genes, each encoding two antigen-binding loops, are highly variable in sequence and structure and can be used for antibody production. These genes can recombine with the IGHD and IGHJ genes or the IGLJ and IGKJ genes, forming the third and most variable antigen-binding loop. During this recombination process, imprecise ligation, nucleotide deletions, and non-templated nucleotide insertions will generate primary antibody repertoires with many paratopes, forming cavities, grooves, planes, or overhanging structures.
It has been argued that one of the disadvantages of animal-derived antibodies is that they may be immunogenic because they are not of human origin. However, introducing humanization techniques and transgenic animals for antibody production has made this argument less relevant. Although similar to access to in vitro methods, their use may be limited by high cost and the need for more expertise.
However, legitimate concerns about antibodies of animal origin remain: Immunization is usually quite time-consuming (repeat injections are generally required within 2-3 months), the antigen needs to be immunogenic or needs to be further modified to increase immunogenicity When combined with denatured adjuvants, antigens may lose significant 3D structural motifs and provide limited control over antibody responses during immunization, because of this little control, bias in antibody responses to certain immunodominant antigenic regions limits sequence and epitope diversity.
Benefits and Drawbacks of Non-animal-Derived Antibody Discovery
Antibodies of non-animal origin are usually discovered from recombinant antibody libraries using in vitro display methods. A vital advantage of these methods is overcoming many disadvantages associated with animal-derived antibody discovery. For example, recombinant antibody libraries and display selection technology offer the benefit of slightly reducing the use of animals in antibody discovery, as animals are only required to validate antibodies discovered in vitro, and animals are no longer needed to find them out.
Combining recombinant libraries with in vitro display technology allows for identifying affinity reagents, including antibody fragments, for highly toxic and non-immunogenic targets. Thus, the explorable target space of recombinant display libraries exceeds that of animal-based antibody discovery.
Furthermore, complete experimental control and the ability to manipulate different steps in the discovery process allow targeted and rational antibody discovery against defined antigen conformations or epitopes with custom properties. These properties may include the ability to bind antigens only under one specific set of conditions and release it under another set of conditions, such as non-physiological pH, which may improve the circulation of these antibodies, thereby significantly extending their half-life and therapeutic efficacy.
Conclusion
In summary, neither animal immunization nor the combined use of recombinant antibody libraries and display technologies is superior to antibody discovery. Both animal immunization and recombinant libraries have unique advantages in finding antibodies suitable for help, and in vitro, display technologies have many benefits. However, the limited availability and high cost of high-quality (synthetic or natural) antibody libraries have severely hindered the utilization of in vitro technologies.
When antibodies are discovered using recombinant library display techniques, other benefits of using animal immunization are lost, including antibody lineage information and the possibility to produce antibodies without exposure to the antigen itself (e.g., by using DNA/RNA immunization techniques).
To overcome these shortcomings, animal immunization can be combined with display technology, thereby retaining the advantages of in vivo affinity maturation and high-throughput selection under experimental conditions. Notably, while the combination of technologies can already improve the properties of the resulting antibody repertoire, it can also provide a large dataset that can be exploited using machine learning. This presents a golden opportunity to harness the potential of a third compelling method in our arsenal.
References:
[1] Andreas H Laustsen, Victor Greiff, et al. Animal Immunization, in Vitro Display Technologies, and Machine Learning for Antibody Discovery. Trends Biotechnol. 2021 Dec;39(12):1263-1273. doi: 10.1016/j.tibtech.2021.03.003. Epub 2021 Mar 25.
[2] Alison Gray, Andrew R M Bradbury, et al. Animal-free alternatives and the antibody iceberg. Nat Biotechnol. 2020 Nov;38(11):1234-1239. doi: 10.1038/s41587-020-0687-9.
[3] Manuela B Pucca, Felipe A Cerni, et al. History of Envenoming Therapy and Current Perspectives. Front Immunol. 2019 Jul 10;10:1598. doi: 10.3389/fimmu.2019.01598. eCollection 2019.
