Industry Insights with Amy Sheng on Nanobodies

This episode of Share Science features Amy Sheng, PhD, a technical account manager at Sino Biological. In this interview, Amy shares how nanobodies are emerging as important tools for tumor diagnosis and treatment, and how they are expected to revolutionize antibody-based drug therapies for a wide range of pathological conditions and especially cancer immunotherapy.

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Nanobodies are single-domain antibodies that were discovered nearly 30 years ago. They’re found in a lot of interesting animals such as alpacas, llamas, and cartilaginous fish like sharks and rays. Unlike traditional immunoglobulin-G antibodies from humans and mice that contain both heavy and light chains, nanobodies do not have light chains. Nanobodies bind to antigens through the single variable domain on a heavy chain (VHH). These single-domain antibodies with only VHH are currently the smallest naturally-derived antigen-binding fragments. That’s where the name “nanobody” comes from and I guess anything smaller than that would be “nobody.”

Their applications have been explored in a wide range of conditions such as neurological, infectious, and inflammatory diseases. I would also like to point out that they are widely used for tumor diagnosis and therapeutics. Besides treating human diseases, scientists also use nanobodies to detect and fight pathogens in agriculture. Moreover, some nanobodies are developed to detect small molecules for environmental monitoring.

There are many great advantages of nanobodies over the conventional ones which mainly come from their size and structure, as well as their development and production protocols. As we mentioned, the VHH is currently the smallest antigen-binding fragment. Due to their small size, nanobodies can penetrate tissues more efficiently and bind to antigens that are less accessible to conventional antibodies.

“Nanobodies are less immunogenic in humans and more soluble and stable [than conventional antibodies].”

Some nanobodies can bind to antigens at high temperatures, and some are functional in presence of ammonia, sunlight, or ethanol. These properties increase their shelf life and offer alternative delivery routes such as inhalation. From the production point of view, there is no need to sacrifice animals when developing nanobodies, and no need to pair variable fragment heavy and light chains since nanobodies contain only a single domain. Furthermore, since their folding and stability is less dependent on disulfide bond formation, many systems like Escherichia coli and plants can be used to express and produce nanobodies, which can greatly lower production costs.

“It all starts with animals.”

We know that those single-domain antibodies are found in sharks and rays, but they’re really hard to catch. Most of the experiments are done on camels; some are immunized and some are not. If we start with an immunized alpaca, blood is collected from the animal first, from which messenger RNA is extracted to construct complementary DNA (cDNA) by reverse transcription polymerase chain reaction. The cDNA is then amplified to isolate the VHH genes that are to be incorporated into plasmids.

Researchers construct the VHH library from bacteriophages and pan the library for a desired binder if they’re starting with a target protein. After good binders are obtained, we do sequencing and then reconstruct to express and purify them. The final nanobody products are, of course, validated in various assays to determine their stability, affinity, and specificity.

There are over 10 VHH antibodies in preclinical through phase III stages. Shortly before the pandemic, the first nanobody drug, caplacizumab, received EMA and FDA approval for treating rare blood clotting disorders and it entered the market this year. This drug consists of two identical humanized single variable domains connected by a short linker. Basically, it functions by blocking platelet aggregation. In phase III clinical trials, more than 50% of patients who received the drug reached normal platelet counts.

Another interesting nanobody drug is ozoralizumab, which is under review by Japan’s PMDA for the treatment of rheumatoid arthritis (RA). According to phase II and III results, ozoralizumab significantly reduced symptoms in patients with active RA. This improvement was seen as early three days following injection.

“There are many biotech companies and institutes investing in VHH development and we can definitely expect more emergent diagnostic or therapeutic toys from this field.”

Nanobody development is still quite a young field. More than 130 therapeutic monoclonal antibodies have been approved, compared to only one nanobody. A lot of studies are in phase I and II, and many researchers are still in very early development stages such as screening for good binders and increasing binding affinity.

Even though nanobodies have a lot of advantages, conventional antibody technology development has boomed so much more. It is truly unlikely that we will be able to have alpaca farms sitting close to big pharma, right? However, thanks to contract research organizations (CROs), it is getting easier and easier to immunize animals such as llamas or alpacas and obtain the initial set of libraries for screening, thereby generating more candidates for different applications.

Sino Biological has independently developed and established a nanobody platform with a large storage capacity. Through this platform, candidate nanobodies for the drug target PD-L1 were successfully obtained with competitive screening. In our experiments, we demonstrated that this nanobody’s affinity is comparable to that of positive control antibodies, while its blocking and cellular activities were superior to positive controls.

We also produce nanobodies through CROs for our customers. We only need an amino acid sequence to start, and we’ll perform all the steps from gene synthesis to construction, transformation, and purification. We have launched many high-throughput productions for screening purposes. For example, we can produce up to 1000 nanobody constructs per batch within three to four weeks.

“This way, scientists can perform screening on a large list of candidates and only pick ones with good yield, good stability, and good binding characteristics.”

Many of the constructs are bispecific or trispecific. Once the good candidates are selected, we can scale-up production for subsequent characterization experiments. To sum up, we can help researchers develop the best nanobodies for their applications by supplying reagents and offering screening and characterization services.