Pushing the Barriers in Next-Gen Antibody Development

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BioPharm International, BioPharm International, June 2023, Volume 36, Issue 06
Pages: 15–17

New fields are being explored to bring novel antibody candidates to the pipeline.

Continued development of new and next-generation antibodies is spurred by the increasing demand for more effective and custom-tailored biologic therapies. While the bio/pharma industry is seeing a shift toward personalized medicines, ongoing work in research and drug discovery is generating new biologic modalities, such as next-generation antibodies.

In particular, bispecific antibodies are next in line after the decades of success that monoclonal antibodies (mAbs) have had in the industry. Bispecifics were mainly developed for cancer treatment, notes Luca Varani, PhD, Structural Biology group leader at Switzerland’s Institute for Research in Biomedicine, and founder of CLBiotech, a 2022 start-up company focused on nanobody engineering.

Bispecifics (and beyond that, multispecific antibodies) can be simply viewed as a cocktail in a single molecule, or rather, the concept is that of a single molecule that has the property of a cocktail drug. However, Varani emphasizes, bispecifics can do things that a conventional cocktail drug cannot, and that is, bispecifics act synergistically. This is considered a novel mechanism of action, he notes.

“Bispecifics can achieve molecular functions that are not achievable by monoclonal antibodies,” Varani says. “If you think strategically, you could design a bispecific that acts like a cocktail.”

Ideally, Varani, says, a developer would want to look for bispecifics that do something that monoclonals cannot do, such as, for instance, having a bispecific that bridges two molecules on different cells. Or, another example could be using bispecifics to bridge different viruses, which would cause aggregation with the end result being that the virus is neutralized. Yet again, bispecifics can be used to bridge immune cells, such as T cells, to cancer cells.

“You attach the cancer cell on one end, and you bring the T cell immune system against the cancer with the other,” Varani demonstrates. “You cannot do that with a cocktail.”

Challenges in purification

Bispecific antibodies present challenges in downstream processing due to “shuffling.” To demonstrate this phenomenon, Varani says to imagine the antibodies having hands. In the case of a mAb, there are two hands. In the case of a bispecific or multispecific antibody, there are four or more hands. Shuffling occurs when the “left hand” of antibody 1, for example, binds with the left hand or the right hand of antibody 2. “This is a mess,” Varani asserts. “This complicates production purification, and you thus have to remove the forms that you do not want.”

“On COVID, for instance, I worked on bispecific [production]. It’s a human format. It’s identical to a monoclonal, but it is bispecific [in function]. Bispecific production yield was very low. Although the cost of production of bispecifics has gone down [significantly], there are still regulatory costs,” Varani says.


“Do you really have a bispecific? Are you producing a molecule that hits both its targets simultaneously? Does it remain a bispecific or does it open up once inside the body, how stable is it? These are all questions for which molecule characterization must be done,” Varani adds.

It is much more difficult to remove unwanted forms due to shuffling in bispecific antibody production compared with mAb production. Because shuffling must be avoided, product yield consequently tends to be lower, Varani says.

Although the purification process has since been improved, Varani notes that some trace amounts of unwanted bispecific antibody forms may be left behind. To that end, the more effective solution to minimizing shuffling and the formation of unwanted product is in the upstream, with molecular engineering, or rather, antibody engineering.

There are a number of solutions that can be derived through engineering. For example, one can engineer what’s known as “single chains” wherein a bispecific antibody can be forced into a single-chain format (rather than a folded molecule, the natural state of an antibody). “This is something very elegant,” Varani says. “You change the interface between heavy and light chains between the two hands [of the antibody] so that they are complementary to each other.”

Beyond bispecifics: VHH antibodies

Looking beyond bispecific antibodies, a new subcategory of antibodies is rising through the ranks: single variable domain on a heavy chain (VHH) antibodies, otherwise known as nanobodies (1), which are derived from camelids (e.g., llamas, alpacas, camels, etc.). One advantage that VHH antibodies hold is that they are single-chain antibodies, making them less structurally complex and smaller in size than human double-chain antibodies. Being single chain, VHH antibodies are not subject to shuffling, making their production much easier, according to Varani. Their small size and simpler structure also make them extremely stable.

In discussing the potential of VHH antibodies to act as immunotherapeutic agents, Varani says: “Being smaller, they have a much higher penetration in tumors. They get in much better than [conventional] antibodies. They can pass biological barriers as well, such as the blood-brain barrier.” Passing through the blood-brain barrier is a task that conventional antibodies cannot do. So, Varani explains, if conventional antibodies are injected into the blood, less than 1% of them will reach the brain, making them ineffective in treating neurological disorders, for instance. The supposition, then, is that VHH antibodies may have the ability to reach targets that conventional antibodies cannot reach.

However, with the pros come the cons. One major disadvantage that VHH antibodies have is that they have a much shorter half-life in vivo, Varani points out. Therefore, duration in the patient’s body may not be sufficient to confer full therapeutic benefit. “They have some advantages, but they also have some problems,” Varani states.

Yet, in the search for next-generation antibodies, VHH antibodies still hold strong potential. According to Varani, development work for VHH antibodies is currently booming after seeing flat activity prior to 2020, and clinical trial programs are in the pipeline. Importantly, the first VHH antibody product, Nanozora (anti-tumor necrosis factor alpha Nanobody), was approved in Japan in September 2022 for treating rheumatoid arthritis (2). Taisho Pharmaceutical developed Nanozora in Japan under a license agreement with Ablynx, a Sanofi company (2).


1. Bever C. S.; Dong J. X.; Vasylieva N.; et al. VHH Antibodies: Emerging Reagents for the Analysis of Environmental Chemicals. Anal Bioanal Chem. 2016 Sep, 408 (22), 5985–6002. DOI: 10.1007/s00216-016-9585-x
2. Taisho Pharmaceutical, Notification of Approval to Manufacture and Market Nanozora 30mg Syringes for S.C. Injection, a Therapy for Rheumatoid Arthritis, in Japan Japan’s First Nanobody Therapeutic. Press Release, Sept. 26, 2022.

About the author

Feliza Mirasol is science editor for BioPharm International.

Article Details

BioPharm International
Volume 36, No. 6
June 2023
Pages: 15–17


When referring to this article, please cite it as Mirasol, F. Pushing the Barriers in Next-Gen Antibody Development. BioPharm International 2023, 36 (6), 15–17.