Customized Nanobody Immunogen (二)

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 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, most nanobody development is carried out through animal immunization of alpacas due to their ease of breeding and immunization, as well as high profitability. Phage display technology can be used to construct VHH antibody libraries, which are divided into native antibody libraries, immune antibody libraries, synthetic antibody libraries, and semi-synthetic antibody libraries.

Immunogens are classified into proteins, polysaccharides, nucleic acids (DNA and RNA), and cell lines based on their molecular weight and properties. Protein immunogens include enzymes, proteins, bacterial toxins, and other substances that have antigenicity and can trigger immune responses in the body. Nucleic acid immunogens include DNA and RNA. DNA immunogens express antigen proteins by introducing target genes into host cells, while RNA immunogens express antigen proteins in cells through mRNA. Cell line immunogens contain multiple antigenic epitopes that can stimulate complex immune responses in the body, and virus like particles (VLPs) are a commonly used cell line immunogen.

Fig. 1 Structure based immunogen design

(Figure source: Structure-based immunogen design — leading the way to the new age of precision vaccines - PMC (nih.gov))

Preparation of immunogens

When preparing DNA immunogens, it is necessary to choose a suitable vector system to ensure that the target DNA is correctly cloned into the vector, and that the DNA immunogen is effectively delivered to the host cell. The host cell should also be able to correctly express and process the generated antigens, and the transfection efficiency and expression level can be improved by optimizing the design and construction of the vector. The methods of immunogen delivery such as electroporation and liposome mediated transfection also need to be selected correctly to improve delivery efficiency. Due to the easy degradation of RNA, stabilizers should be added to ensure the integrity of RNA during the preparation of RNA immunogens. When storing RNA immunogens, temperature and pH conditions should also be strictly controlled. Optimizing the packaging system can improve the success rate and efficiency of RNA packaging. Cell line type immunogens should be protected from contamination during cell culture to ensure normal cell growth. When extracting immunogens from cells, appropriate and gentle extraction methods should be chosen to maintain the natural structure and activity of the protein.

Introducing specific epitopes onto antigens through genetic engineering can enhance the immunogenicity of the prepared immunogens. According to the expression properties of antigens, corresponding vector construction is carried out to improve the expression level and stability of antigens. The cultivation conditions of the expression system, such as temperature, pH value, and medium composition, can be optimized for antigen expression and folding after appropriate adjustments.

Key points of immunogen preparation

When preparing DNA immunogens, the first step is to construct plasmid DNA based on the antigen type and its function. The encoding gene of the target antigen is cloned into an appropriate plasmid vector, and promoters, enhancers, etc. are added to ensure efficient expression of the target antigen in the host cell. The vector can be modified, such as adding fluorescent tags, for subsequent tracking and detection. Amplify plasmid DNA through bacterial culture and extract plasmid DNA using alkaline lysis or plasmid extraction kit. The impurities were removed by gel filtration, ultracentrifugation and other purification methods to obtain high-purity plasmid DNA. The host's immune response is regularly monitored after immunization of animals with immunogens, and antibodies are collected and purified from the blood of immunized animals for subsequent use. When preparing RNA immunogens, cap structures and poly (A) tails can be added to RNA to enhance its stability and immunogenicity.

KMD Bioscience has been committed to VHH antibody library construction and nanobody library screening services for many years, with rich 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 services including antigen design vector construction and animal immunization. The main experimental steps include the construction and screening of the alpaca nanoantibody library, as well as the validation of its active functions. We also 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 large 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] Mullins JI, Nickle DC, Heath L, et al. Immunogen sequence: the fourth tier of AIDS vaccine design. Expert Rev Vaccines. 2004;3(4 Suppl):S151-9.

[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] Sesterhenn F, Bonet J, Correia BE. Structure-based immunogen design-leading the way to the new age of precision vaccines. Curr Opin Struct Biol. 2018;51:163-169.