Customized Nanobody Immunogen (三)

In 1993, a native antibody lacking a light chain (VL) was discovered in the serum of camelids. Its structure is simple, consisting only of two heavy chains (VH), and its volume is very small, about 15 kDa, hence it is called a nanobody or single domain antibody. Later, this antibody was also found in animals such as alpacas and sharks. Based on its unique structure and broad application prospects, the preparation of nanobodies has a broad application market, especially in the field of drug development. Due to the strong penetration of nanobodies, it has become an ideal choice for treating tumor diseases, which helps to improve the efficacy of drugs and reduce side effects. Although antibodies with similar structures also exist in shark animals, due to the ease of breeding and immunization of animals such as alpacas, as well as their high profitability, most nanobody development is carried out through alpaca immunization. Phage display technology can be used to construct nanobody libraries, which are divided into natural antibody libraries, immune antibody libraries, synthetic antibody libraries, and semi synthetic antibody libraries.

There are various types of immunogens used in animal immunization, and viral immunogens, as a commonly used type of immunogen, include whole virus inactivated vaccines, subunit vaccines, viral vector immunity, and mRNA vaccines. By physically or chemically inactivating the virus, its antigenicity is preserved and pathogenicity is removed. Whole virus inactivated vaccines can stimulate the body to produce an immune response, but the virus needs to be completely inactivated, and the preparation process is relatively complex. The subunit vaccine only uses virus surface proteins as antigens, so this vaccine has high safety, but adjuvants need to be added to enhance the immune effect. Adenoviruses, lentiviruses, and other viruses are genetically modified and used as vectors. The target antigen gene is inserted into the viral genome to induce immune responses through viral replication and expression. The immune effect of viral vector immunity is efficient and long-lasting, but its preparation process is complex and requires strict control of biological safety. The mRNA encoding viral antigens is directly introduced into cells to express the antigens and stimulate immune responses, enabling the rapid preparation of mRNA vaccines. However, it is difficult to ensure the stability and delivery efficiency of mRNA. Virus type immunogens have become a hot topic in the field of infectious disease prevention and control, and efficient and safe virus vaccines have also been applied in various disease fields.

Fig. 1 Antibody response to polysaccharide pores in HIV-1 Env and Ebola GP. (Figure source: Influence of glycosylation on the immunogenicity and antigenicity of viral immunogens - ScienceDirect)

Precautions for preparing viral immunogens

The selection of inactivation method is crucial in the preparation of whole virus inactivated vaccines, and subsequent verification of whether the virus has been completely inactivated can be achieved through cytotoxicity testing and infectivity testing. When preparing subunit vaccines and viral vector vaccines, it is necessary to ensure that the antigen is correctly expressed in host cells and highly purified. Therefore, appropriate expression systems and purification techniques are important. When using vaccines, appropriate doses of adjuvants can be added to enhance the immune response of the vaccine.

When preparing immunogens, it is necessary to ensure that the virus titer reaches a level that can provide sufficient antigen stimulation in order to induce effective immune responses. But if the virus titer is too high, it may lead to a strong immune response and even cause diseases. Therefore, it is necessary to control the virus titer within a safe range while ensuring immune efficacy. Correspondingly, the amount of immunogen should not be too high, as long as it can induce an immune response. In addition, using high-purity immunogens can reduce non-specific reactions and improve the specificity of immune responses. The neutralizing effect of antibodies prepared by using viral immunogens to immunize animals is strong, which can bind to antigens on the surface of viral particles, prevent the virus from binding to host cells, and thus block the virus from entering cells. And antibodies can also exert antiviral effects through other mechanisms, such as mediating immune cell phagocytosis and clearance of virus particles, interfering with the virus replication cycle, etc. These mechanisms work together to effectively resist viral infections.

KMD Bioscience has been dedicated to the construction and screening of nanobody libraries for many years, with extensive experience in nanobody development. Based on our mature antibody discovery service platform, hundreds of alpaca nanoantibody library construction services are successfully delivered every year. KMD Bioscience has established a comprehensive and mature nanobody preparation service platform. Based on phage display technology, we can provide main experimental steps including antigen design, alpaca immunization, alpaca nanoantibody library construction and screening, and activity function verification, and provide customers with high specificity and affinity alpaca nanoantibody library construction services. And we will conduct comprehensive analysis of the nano sequence information and verify it through various experiments, such as EC50 determination, affinity analysis, flow cytometry verification, etc. We have multiple phage antibody library construction platforms including M13, T4, T7, and λ phages, which can meet the different needs of customers and provide personalized single domain antibody development services. KMD Bioscience specializes in building different types of phage display libraries, such as immune libraries, natural libraries, semi synthetic libraries, synthetic libraries, etc. The nanobody library we have constructed has a large capacity and can produce high affinity nano antibodies. We can provide customers with various bacteriophage vectors including pMECS, pComb3X, and pCANTAB 5E. We have strains such as TG1 Escherichia coli, XL1 Blue, and ER2738, which can be used for phage infection after expanded cultivation. The antibody library we have built has a huge capacity of up to 10 ^ 9, with a high insertion rate of target fragments, which is beneficial for screening nanobodies that satisfy customers. We can also express and purify the selected nanobodies according to customer needs. In addition to prokaryotic expression systems, we also have various eukaryotic expression systems for antibody proteins, such as mammalian cells, yeast cells, plant and insect cell expression systems, etc., which can produce high-quality nanobodies for customers.

Reference

[1] Newby ML, Allen JD, Crispin M. Influence of glycosylation on the immunogenicity and antigenicity of viral immunogens. Biotechnol Adv. 2024;70:108283.

[2] De Groot AS, Khan S, Mattei AE, et al. Does human homology reduce the potential immunogenicity of non-antibody scaffolds? Front Immunol. 2023;14:1215939.

[3] Doneva N, Dimitrov I. Viral Immunogenicity Prediction by Machine Learning Methods. Int J Mol Sci. 2024;25(5):2949.