Non-human primate (NHP) antibody library construction using phage display is one of the most effective methods for identifying antibody candidates with high affinity and specificity. NHP antibody libraries using phage display are effective because NHPs (Old World Monkeys such as macaques) have a highly similar immune system to humans and produce antibodies that are highly similar in sequence and structure—particularly in the variable-domain framework regions—to human antibodies. Creative Biolabs delivers leading-edge NHP antibody library construction solutions using our unique phage display technology. We provide an end-to-end solution for identifying high-quality, therapeutic antibody candidates. Our competitive edge is the ability to take advantage of the close homology of NHP immunoglobulin genes to their human counterparts and the robust antibody responses in NHPs relative to humans, in order to identify high-affinity, highly specific antibodies to challenging targets that are refractory in other systems, and to combine this biological advantage with a streamlined process from immunization through to proprietary biopanning methods and germline humanization for a much smoother bench experience.
Monoclonal antibody library construction from NHPs involves several steps. The ultimate goal of the process is to produce an output library that has high affinity and specificity for the target antigen of interest. The first step is to immunize an NHP with a target antigen. The animal's immune system is primed to produce a diverse repertoire of high-quality antibodies. B cell populations from the animal are collected and used to make a library of antibody fragments, often in the scFv or Fab format. These genes are cloned into phage display vectors so that the antibody fragments are displayed on phage coat proteins, and a selection process called biopanning is then used to select the high-affinity binders. A key advantage of this approach is its ability to produce high affinity antibodies to human protein epitopes that have been challenging to access via rodent immunization or naïve/synthetic libraries. The resulting NHP antibodies are then germline humanized to increase similarity to human germline frameworks and to reduce potential immunogenicity; these research antibodies are not ready for clinical use and require extensive preclinical/clinical evaluation.
Our NHP antibody library construction service is a highly controlled process designed to give our clients the best chance of obtaining high quality antibody candidates:
The first step in the construction of an NHP monoclonal antibody library is to immunize the NHPs with the antigen of interest. This is typically done with a series of injections over a period of weeks or months, often with appropriate adjuvants, in order to elicit the strongest possible immune response from the animals. The resulting immune response produces a high diversity of antibodies that are specific for the antigen.
Once the NHP has mounted an immune response to the antigen, B cells are collected, typically from lymph nodes or bone marrow, which are then used to construct the phage display library. The genes encoding the variable regions of the antibodies are inserted into the bacteriophage genome, resulting in a library of millions of unique antibody fragments.
The next step is to screen the library in order to enrich binders with the desired specificity and affinity. This is typically done by exposing the library to the antigen and then selecting the phages displaying antibodies that bind to the antigen for further analysis, a process typically done in repeated rounds and is referred to as panning. The selected phages are then amplified, and the process is repeated in order to enrich for high-affinity antibodies.
Once the high-affinity antibodies have been selected, they can be further optimized. Germline humanization is a common approach used at this stage, which involves modifying the NHP antibodies in order to make them more similar to human antibodies while still retaining high affinity.
The final step in the process involves producing the full-size IgG antibodies. This is often accomplished by cloning the antibody fragment genes into mammalian expression vectors, which are then used to produce the antibodies in suitable host cells, such as CHO cells.
The ultimate goal of antibody discovery has been to identify potential drugs with the highest possible chance of being efficacious and safe for patients. The primary antibody discovery techniques for decades include rodent immunization, human/synthetic libraries, and more recently for specific use cases are NHP immune libraries. Each of these methods has advantages and disadvantages. Rodent-derived antibodies typically require humanization unless transgenic human-Ig mice are used; this process may require significant molecular engineering, which is time consuming, expensive and can sometimes compromise affinity. Synthetic or naïve human libraries avoid the need for humanization, but the antibodies often begin with modest affinities on average and benefit from in vitro affinity maturation before entering the clinic. This is where NHPs come in—especially Old World monkeys, such as cynomolgus and rhesus macaques. Their immune system and antibodies are highly similar to humans.
Fig.1 Human–rhesus IgG subclass structural & functional properties comparison.1
Published comparative analyses indicate that macaque immunoglobulin variable genes are about as close to human genes as human genes are to each other. This high degree of homology provides several advantages:
The NHP platform has been used in research to generate antibodies with value in a range of therapeutic areas:
While NHP antibodies are extremely similar to human antibodies, subtle differences in the framework regions of NHP antibodies may still be able to elicit an immune response in humans. To prevent this, scientists have developed a technique called germline humanization that aims to minimize framework deviations from human germline and thereby reduces or eliminates these small differences in order to make the final antibodies as indistinguishable from human antibodies as possible. This process is best viewed as complementary to traditional CDR-grafting/humanization strategies and involves aligning the antibody framework sequence to the closest human germline sequence, the antibody "blueprint" our own bodies naturally tolerate. Published data clearly illustrates the effectiveness of this approach. To quantitatively measure the similarity between a given antibody framework and the closest human germline sequence, researchers have developed a Germlinity Index (GI). Published data on human and NHP IgGs reveal that a typical human IgG has a GI of about 95% whereas NHP IgG's have a GI of ~90%. In one study, researchers took a NHP antibody that neutralizes anthrax toxin and, through germline humanization, increased its GI from 92% to 97.8% while maintaining the antibody's high affinity and neutralizing ability. The resulting modified antibody has a framework sequence that is even more human-like than a comparator fully human antibody in that dataset. With a data-driven approach, we increase human-likeness of the framework while preserving function.
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Q: Why use NHPs instead of transgenic mice that produce human antibodies?
A: While transgenic mice are a great tool, the NHP immune system is a closer match to our own. This can lead to antibodies with better properties and a wider range of epitope recognition. It's also a key advantage when the mouse version of a target protein is very different from the human one.
Q: What about the ethics of using Non-Human Primates?
A: We take ethics very seriously. All work with NHPs is done under strict ethical guidelines that put animal welfare first. We follow the 3Rs principles (Replacement, Reduction, and Refinement) to ensure we use the minimum number of animals necessary to get clear scientific results.
Q: What is the intellectual property (IP) situation for these antibodies?
A: The IP for antibody drugs can be complex, but the core technologies are well-established. Our clients receive full IP rights to the new antibody sequences discovered during their project. We always recommend consulting with an IP attorney for specific legal guidance.
Q: Can the final antibody be made in different formats?
A: Yes. Once we identify a lead scFv or Fab, we can easily convert its sequence into a full-length IgG, a bispecific antibody, or any other format needed for further development.
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Please kindly note that our services can only be used to support research purposes (Not for clinical use).
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