Nanoantibody (Nb) refers to the antigen-binding domain of pure heavy chain antibody (HCAb) circulating naturally in camels and some shark species, which is called VHH in camels and VNAR in sharks. The nanoantibody is composed of a single immunoglobulin domain with a molecular weight of about 15KDa. The nanoantibody only retains 10% of the original antigenic specificity of the normal IgG size.

Compared with normal antibodies, nanoantibodies have a series of unique advantages, such as stronger physical and chemical properties such as solubility and stability, and lower production costs.

The earliest report on nanoantibodies was in 1993.

In 1993, the team of Professor Raymond Hamers of VUB in Brussels, Belgium, reported for the first time the small antibodies from the blood of camels in Nature. This antibody naturally lacks the light chain and heavy chain CH1 constant region, which is composed of heavy chain variable region, hinge region and CH2 and CH3 constant region. The antibody active fragments obtained by cloning its heavy chain variable region are called nanoantibody, VHH or single domain antibody (sdAb).

In 2000, VUB transferred a series of patents on VHH to the Vlaams Institute of Biotechnology (VIB) in Belgium. In 2002, VIB incubated the world's leading nanoantibody research company, Ablynx.

Nanobody is the registered trademark of Ablynx Company, and is now used to refer to single domain antibody (sdAb).

On January 29, 2018, Sanofi successfully acquired Ablynx for $4.8 billion. In September of the same year, EMA approved the nano-antibody drug Cablivi (Caplacizumab) of Sanofi for the treatment of adult acquired thrombocytopenic purpura (aTTP), becoming the first nano-antibody drug on the market in the world. Cablivi is a member of the Ablynx product pipeline.

With the launch of the world's first nanoantibody drug Cablivi and the unique advantages of nanoantibody in scientific research and clinical application, the development prospect of nanoantibody has attracted more and more attention. At present, many pharmaceutical companies have invested in the research and development of nanoantibodies.

Among them, it includes Loki Biology, a pioneer in the field of nanoantibody in China. Loki Biologics is a clinical stage company dedicated to the research and development of innovative nano antibody drugs. It is also the only company in China with the ability to develop nano antibody drugs in the whole process.

Where is the relationship between Loki organisms and nanoantibodies? What are the key points in the development of nanoantibodies? What are the potential of nanoantibodies that can be exploited?

With many questions and taking the opportunity of the 4th Bio-ONE in 2022, the Baijiayan team had the honor to interview Dr. Wan Yakun, the founder of Rocky Biology, and asked him to chat about "those things" about nanoantibodies.

Baiaogu&Biopharmaceutical Editorial Team has set up a special column of "Baijiayan", focusing on the upstream and downstream of the bio-pharmaceutical industry, business and technology, humanities and society, and your attention. Welcome your recommendation and suggestions. In the 39th issue, you are presented with a wonderful interview by Wan Yakun.

Introduction to "Baijia" in this issue:

Wan Yakun: Dr. Wan Yakun is the founder of Shanghai Luoqi Bio-pharmaceutical Technology Co., Ltd. and the current CEO of the company. Dr. Wan has been committed to the research of nano antibodies for 16 years, and has enjoyed a high reputation and popularity in the field of nano antibodies. He was selected into the "Hundred Talents Plan" of the Chinese Academy of Sciences Shanghai Institute of Materia Medica, won the six talent summit plans of Jiangsu Province, the "High level Creative Talents Plan" of the Ministry of Education, "New Century Excellent Talents", "Outstanding Innovation and Entrepreneurship Talents in Zhangjiang National Independent Innovation Demonstration Zone in 2021" and other honorary titles. The enterprise "Luoqi Biology" founded by it was selected as one of the top 50 most innovative pharmaceutical enterprises in China in 2020 and the top 100 pharmaceutical innovative seed enterprises in China in 2022.

In the field of scientific research, Dr. Wan has published more than 60 scientific research papers in recent years, including more than 50 SCI academic papers as the first author and corresponding author. A total of more than 80 domestic and foreign invention patents have been applied, of which 61 have been published and 30 have been authorized.

In the field of new drug research and development, Dr. Wan has established China's first nanoantibody research and development team, and has successfully cooperated with many well-known pharmaceutical companies at home and abroad. With a complete R&D, pilot test and GMP production system, Loki Biotech is the only company in China that has the ability to develop the whole process of nanoantibody. In addition, the production of Loqi Biological 500L Pichia pastoris GMP pilot production workshop fills the industry gap in this field, which is extremely scarce and competitive. The company's LQ036 project is positioned as the world's first inhaled nanoantibody asthma treatment drug, and is carrying out clinical phase I in Australia and China. LQ043, an innovative drug for respiratory diseases, also entered the IND declaration in 2022. Loki Biology will ensure that 1-2 projects are promoted to the clinical research stage every year.

Loki biological independent platform shows the differentiation advantage of nanoantibody

Since the first therapeutic antibody entered the clinic in 1986, the research of antibody has been developing rapidly. Up to now, nearly 100 antibody drugs have been approved worldwide, and antibody drugs have become the backbone of modern biological macromolecular drugs. With the development of technology, a variety of new antibody technologies emerge in an endless stream, and nanoantibodies are unique due to their unique differences. As early as 2004, Dr. Wan was exposed to nanoantibodies when he was a postdoctoral fellow at the American Institute of Systems Biology. When he returned to China in 2011, Dr. Wan set up the first research and development team of nanoantibody in China, and set up the first nanoantibody engineering center in China at Southeast University, officially opening a new chapter in the research of nanoantibody in China.

At first, nanoantibodies were first found in camels, and then in camels and cartilaginous fish.

"I am a native of Xinjiang, and I have lived in a variety of camels since childhood. I think this is a kind of fate." Dr. Wan said, "Luoqi Biology has built the largest camel breeding base in China in Jining, Shandong Province. At present, it has successfully immunized more than 300 camels, involving more than 400 research targets. It must be the largest base in the world that uses camels as nanoantibodies."

The large camel breeding base has solved the problem of animal resources, which is the source of nanoantibody research and development. The strong molecular discovery antibody resource base is an important part of industrialization. Through full-time veterinarians and full-time breeding personnel, the efficient and professional immune blood collection process can be realized, which can make the operation more standardized, the process more controllable, and the cost can be greatly reduced. Dr. Wan told us: "The name of the company is Luoqi Biology. The word" Luo "is homophonic with the word" Luo ". Luoqi is also the company's vision - to start a healthy future with camel antibody."

Looking back on the development of nanoantibodies and the heavyweight transactions in the field of nanoantibodies in recent years, it can be said that the curtain of the era of nanoantibodies is beginning. The world's first nanoantibody drug Cablivi (caplacizumab), which was approved by EMA in 2018 and FDA in 2019, was also updated in the third quarter of this year. In 2020, Merck and Novartis reached a blockbuster deal of 450 million euros in the development of monodomain antibody drugs (M6495) for osteoarthritis. In addition, the domestic leading Corning Jerry's Envolizumab (KN035) was approved for marketing in China in November 2021. KN035 is also an Fc fusion anti-PD-L1 nanoantibody. In February of this year, the BCMA CAR-T product (Carvykti) approved by the FDA of Nanjing Legend Biology and Johnson&Johnson also used nanoantibody technology.

这些信息表明，纳米抗体通过自身的优势在国内外生物医药领域有着较活跃的表现，这也代表了整个创新药的趋势，市场上都在找差异化，找创新突破点。从纳米抗体这个落脚点来看，因为它只含有一个VH片段，不需要FC，所以可以做到尺寸很小，也不完全依赖哺乳细胞系统生产表达。把它的 VH片段表达出来，就能够发挥它的优势。从洛启生物身上我们也能看到纳米抗体具体的优势体现。

The first advantage is the production system. Nanoantibodies can be expressed by microorganisms without FC. However, we know that the cost of microbial expression systems and conventional mammalian expression systems will differ by an order of magnitude. And the fermentation cycle and technological process will also be more convenient.

The second is the diversity of drug delivery methods. The stability of nanoantibodies is very strong. For example, the candidate pipeline for aerosol inhalation of nanoantibodies developed by Loki Biological for respiratory diseases (asthma) can be directly administered to the focus area to achieve therapeutic effect. Because of the molecular weight, stability and other factors, conventional antibody is difficult to develop atomized products.

Dr. Wan said: "Our company's platform has given full play to these advantages of nanoantibodies." Unlike conventional antibodies, the production of nanoantibodies does not have a process-based platform reference, so it has a relatively high requirement for CMC level and project experience. Dr. Wan continued, "As the first company in China to use Pichia pastoris to express nanoantibodies, Rocky Biology has established its own perfect CMC platform that meets the drug reporting standards. At the same time, each project also needs a strong molecular discovery platform to provide enough candidate molecules for the analysis and screening of drug resistance."

The difference in the production of nanoantibodies is significant, unlike the expression of conventional monoclonal antibodies in a range. The expression output of nano-antibody products of Loqi Biology can reach the level of 20g/L, so there must be a powerful nano-antibody discovery platform to support the later screening of proprietary drugs.

"Rocky Biological has independently established five core platforms, namely, nanoantibody rapid screening platform, Pichia pastoris CMC process development platform, inhaled macromolecular drug research and development platform, nanoantibody long-acting platform and nanoantibody dual-antibody platform. Among them, the nanoantibody rapid screening platform can efficiently generate candidate molecules with a large base in a short period of time, providing a good candidate molecular reserve basis for the evaluation of the CMC link For the development of nanoantibody technology, Loki Biology has a complete platform system, which can effectively connect and coordinate the efficient screening of nanoantibody, Pichia pastoris expression and drug analysis. " Dr. Wan said.

Loqi Biology has a platform from the construction of highly expressed and stable yeast cell lines to the development of mature nanoantibody CMC, which runs through the whole process of nanoantibody drug development, including the validation of drug formation, preparation development, nanoantibody quality standards, as well as the registration and application strategy, and clinical program design, and has gained enough project experience and accumulation.

"The accumulation of these experiences has helped Rocky Biology become the only enterprise in China that covers the whole industrial chain from the discovery of nanoantibodies to the production of Pichia pastoris, and has promoted the candidate pipeline to the clinical stage. I think this is the characteristic and differentiation advantage of Rocky Biology or nanoantibodies," said Dr. Wan.

In addition, like conventional antibodies, nanoantibodies should also pay attention to two issues in the development process and clinical stage, one is safety, the other is effectiveness.

How to develop the patient enrollment criteria? How to design the dosage? How to conduct a comprehensive drug toxicity study? Such issues need careful consideration. A good clinical trial plan needs constant discussion and repeated adjustment according to the data.

"It is very important to complete clinical trials with high quality. From the experience of LQ036 project at the clinical stage, how to select the appropriate clinical PI, as well as the management of the company's internal projects and the screening of clinical CROs, all of which can play a key role in the success of clinical projects," said Dr. Wan.

The label of inhaled drug treatment for respiratory diseases drives the multiple application potential of nanoantibodies

After years of technical reserves and research and development, nanoantibodies have gradually shown great application potential in new antibodies.

First, based on the advantages of small size, nanoantibodies have unique advantages in bispecific and multi-specific aspects;

Second, in the field of ADC, nano-antibody ADC has a high penetration rate of solid tumors, and is also suitable for dual-target ADC products;

Third, in immunocell therapy, if ordinary antibody is used to make ScFv in CAR part, it will bring about the problems of decreased affinity and steric hindrance, which can be avoided by using nanoantibody;

Fourth, in the field of nuclear medicine, nanoantibodies can be used for treatment in addition to diagnosis by adding isotopes. Nano antibody has high affinity and specificity, and has the advantage of short half-life as a diagnosis;

Fifth, in the field of gene therapy, because nanoantibodies have only a single chain and can be continuously expressed, their expression and activity have certain advantages.

In terms of drug delivery methods, nanoantibodies also have many different applications. For example, in ophthalmic diseases, nanoantibodies can be made into local eye drops to solve the problem of vitreous injection, and in respiratory diseases, atomization treatment can be used to improve patients' drug compliance. In addition, it is also a future direction to develop related products with the characteristics that nanoantibodies can cross the blood-brain barrier.

"Nanoantibody has a bigger advantage, that is, it can solve the problem of neck blockage in China in terms of affinity filler. These have expanded the development space of nanoantibody at a deeper level," Dr. Wan added.

As a platform-type company, Rocky Biology has accumulated many potential targets through the immunization of more than 300 camels. In combination with the differentiated application direction of nanoantibodies, the company's pipeline is mainly targeted at autoimmune diseases, focusing on the layout of respiratory diseases, and cooperating with other companies in ophthalmic diseases or cell therapy. "Our first pipeline LQ036 has many firsts in China: the first zero-issue supplement approved biomacromolecule for respiratory administration, the first nanoantibody expressed by microorganisms, and the first inhaled biomacromolecule therapy in the treatment of asthma, filling many gaps in China."

Luoqi Biological hopes to establish the label of inhalation drug treatment of respiratory diseases to establish the leading position of macromolecular respiratory disease treatment industry, and then quickly drive other projects. In addition, taking advantage of the differentiation of nano-antibodies, Loki Biologics is also laying out some innovative projects that cross the blood-brain barrier.

"In general, Loki Biotech is based on the field of nanoantibodies, taking respiratory diseases as the core, tamping the breadth and depth of the pipeline, and hopes to quickly grow into the second Ablynx, or surpass the Ablynx enterprise." Mr. Wan said with a smile.

The growth of enterprises comes from the transformation of all aspects of individuals

From a scientific research worker to the head of an enterprise that has been established for five years, it seems that Dr. Wan has achieved both the practical spirit of scientists and the management ability of entrepreneurs.

"I think this change is a change of thinking, involving the change of management mode, and also reflecting the process of personal growth," said Dr. Wan.

Scientific research achievements are the most initial link in the development environment, and scientific researchers mainly focus on academic articles. In the process of becoming an entrepreneur, the way of thinking needs to be changed. We should always remember the mission of the enterprise, develop competitive products, and produce certain economic value and social benefits. Therefore, for entrepreneurs, the steering direction of the company needs careful investigation and careful project establishment, not only to answer some scientific questions, but also to meet the needs of the market.

"Don't be shy about talking about benefits. Only when talking about benefits can enterprises survive for a long time. In this process, I think entrepreneurs must do some comprehensive training, such as how to control the project budget nodes, the matching degree of capital promotion, and the degree of financing." Dr. Wan concluded.

With regard to scientists as enterprises, Mr. Wan continued to share: "In addition, an open mind is also very important. It is necessary to rapidly expand from the depth of scientific and focused technology to the breadth of issues such as commercialization, sensitivity to the capital market, and team building." From the work at the beginning of the company's establishment, to the elaboration of personnel recruitment, corporate logo, corporate vision, and the formulation of the entire company's mission, In the early stage, the founders need to do it by themselves, which puts forward high requirements for the founders of enterprises, and an open mind helps to maintain good learning ability.

"The last point is also the point that I want to emphasize most," Dr. Wan continued, "Scientific research workers and entrepreneurs have some differences in their work thinking patterns. For example, scientific research workers are more focused on taking root in the subdivided fields, and more focused on the innovation results in a certain field of scientific research; while entrepreneurs need to consider more 'big and comprehensive'. Specifically, as scientific workers, they are more concerned about how to express creative ideas in a certain field with experimental data; and entrepreneurs need to consider the production of The effectiveness of the product also needs to consider how to effectively use the limited funds to enable the development of the team and how to stimulate the potential of the whole team. In addition to the differences, the two modes of thinking complement each other. For example, every step in the business battlefield also requires the thinking of scientists - cautious and cautious. From a scientist to an entrepreneur, it is an all-round transformation of identity. How to transition the two ways of thinking and how to make the two ways of thinking complement each other is a particular concern of scientists in entrepreneurship. "

Note: Coincidence

"In fact, at the beginning of the establishment of the project, the company went to the outside to find the microorganism CMO or CDMO to make nanoantibodies. But at that time, most of the CMO and CDMO in China were mainly CHO, so we decided to do it ourselves," said Dr. Wan.

At present, the GMP plant built by Loki Biological has been completed, which can provide the products of clinical phase I and phase II microbial expression, including peptides, cytokines and vaccines. "I think the platform construction mentioned above is very valuable. It can not only meet our own needs, but also generate some cooperation with other companies."

It has to be said that opportunities sometimes arise from self-solving the unmet needs of the market.

Bio-ONE 2022 Fourth Annual Bio-Process Industry Summit

As one of the industry benchmarking conferences of biological technology, the fourth Bio-ONE 2022 Annual Bio-Process Industry Summit will be held in Shanghai on November 17-18. The theme of this conference is "technology innovation, leaping and changing", and four thematic forums will be set up, namely, antibody upstream process development, antibody downstream and preparation process development, cell gene drug process development, commercial production and "smart" manufacturing trend. Dr. Wan Yakun, CEO of Shanghai Qiluo Biologics, will attend the conference and give a speech on "Innovative downstream process development strategy of nanoantibody drugs"! Take you on the journey of exploring technological innovation and promote the development process of the next generation of biopharmaceutical production process!