mRNA Immune Antibody Preparation Service

In recent years, with the development of mRNA synthesis, chemical modification of mRNA and delivery technology, mRNA stability and translation efficiency have been greatly improved, and immunogenicity is gradually controllable, which is widely used in the field of tumor immunotherapy as well as vaccines. After many years of animal immunization, Tek Biotech has formed a unique set of nucleic acid immunization technology, including mRNA immunization and DNA immunization, of which the mRNA immunization technology tends to be mature, and it is applicable to the development of neutralizing antibodies for GPCR receptor proteins, ion channel proteins, and other multiple transmembrane proteins, as well as the development of neutralizing antibodies for receptor proteins in the form of complex, such as the development of neutralizing antibodies for CD3 complex proteins. Neutralizing antibodies for complex proteins.

Fig1 mRNA immunization

Tek Biotech is able to provide mRNA immunoantibody preparation services for a wide range of antigens (including but not limited to PTM proteins, intracellular proteins, and secreted proteins). In addition, Tek Biotech has one-stop technical services ranging from gene synthesis and modification, AAV vector construction, animal immunization, fusion and screening of hybridoma cells, and antibody production. In addition, we have many downstream services such as antigen-antibody binding assay, antigen-antibody affinity determination, antibody cell line validation, etc., which provide strong support for our customers' R&D and production of antibody drugs.

█ Immunogenicity of mRNA

Purified mRNA contains only single-stranded RNA (ssRNA) molecules, which are encapsulated by the delivery system and endocytosed into the nuclear endosome. Upon entry into the nuclear endosome, the mRNA is released from the delivery system and recognized by the membrane-localized Toll-like receptor (TLR)7and TLR-8, leading to the activation of the downstream myeloid differentiation marker 88 (MyD88), causing the activation of interferon (IF) type I, which is a marker of myeloid differentiation, causing the activation of myeloid differentiation marker 88 (MyD88). The release from the delivery system and recognition by the Toll-like receptor (TLR)7 and TLR-8 localized on the membrane leads to the downstream activation of myeloid differentiation marker 88 (MyD88), which causes the activation of interferon (IFN) and the secretion of inflammatory factors.

█ Preparation of IVT mRNA Vaccine

1. Preparation of IVT mRNA: antigen selection, linear DNA template synthesis, in vitro transcription and mRNA purification. When the target antigen is selected after sequencing the genome of the pathogen, it is inserted into the plasmid DNA template. The production DNA template should contain the antigen sequence, the 5′ and 3′ UTRs, and the T7 promoter upstream of the 5′ UTR. The in vitro transcription process requires a DNA template, modified or unmodified nucleosides, and T7 RNA polymerase.

2. mRNA entry into the cytoplasm requires the use of vectors. mRNA vaccine delivery vectors include viral vectors and non-viral vectors: viral vectors are mainly lentiviruses, adenovirus-associated viruses, and Sendai viruses, etc. Viral vectors may cause immune responses while delivering nucleic acids, thus affecting the effect of the vaccine. Non-viral vectors mainly include liposomes, dendritic cells (DC), inorganic nanoparticles, and cationic cell-penetrating peptides.

█ mRNA Delivery System

1. Naked mRNA direct injection method: directly injected into the tissue, usually using buffers such as sucrose, alginate solution and other buffers to dilute the mRNA to some extent. The advantages are low cost, convenience and quickness. The disadvantages are that mRNA is easily degraded by RNA enzymes and difficult to cross the cell membrane.

2. In vitro DC loading: mRNA is loaded or electroporated into dendritic cells (DCs) in vitro, and in vitro loading of dendritic cells facilitates the effective targeting of antigen-presenting cells.

3. Cationic nanoemulsions: an oil-in-water based delivery method consisting of squalene and surfactants such as tween and tocopherol, etc. MF59 and AS03 are 2 oil-in-water emulsion adjuvants that were used in the 2009 influenza vaccine study.

4. Cationic peptides and polymers: Curevac has developed m RNA vaccine delivery for rabies and influenza A using fish protein, which is rich in basic amino acid residues such as arginine and lysine, and is able to stabilize m RNA by complexing with negatively charged m RNA.

5. LNP: LNP contains four components: cationic ionizable lipids, polyethylene glycol (PEG) lipids, phospholipids, and cholesterol. Cationic ionizable lipid is one of the most important components of LNP, and its ionization coefficient is basically the same as that of intracellular endosomes, which not only promotes the smooth packaging of m RNA into LNP, but also participates in the intracellular release of mRNA from endosomes; PEG lipids provide colloidal stability and prevent protein from binding to nanoparticles, which reduces the clearance of LNP by the reticuloendothelial system, and increases the length of the somatic circulation; neutral phospholipids such as distearoyl-sn-glycero-phospho-cho-line (DSPC) and dipalmitoyl-phosphatidylcholine (Dipalmitoyl (DSPC) and dipalmitoyl-phosphatidylcholine (DPPC), which provide a bilayer phospholipid stabilizing structure, and cholesterol, which provides rigidity, improves the stiffness of the LNP and prevents leakage of LNP components.

█ mRNA Drug Advantages

(1) It is easy to produce and purify in vitro, removing the complex process of protein drug and viral vector preparation, and at the same time avoiding host protein and viral source contamination;

(2) The production process of IVT mRNA is highly versatile and can be quickly applied to the production of different target proteins, saving drug development time and improving efficiency;

(3) mRNA only needs to enter the cytoplasm to be translated into proteins, without entering the nucleus, so there is no gene insertion and integration, improving drug safety;

(4) The half-life can be changed by adjusting the sequence modification and delivery carrier;

(5) Clinical trials have found that although the protein expression of mRNA is transient, it is effective for immunotherapeutic applications for tumors and facilitates pharmacokinetic and dosage control.

█ mRNA Immunization Service Process (nano-antibody as an example)

█ Service Advantages

-- No need for recombinant proteins, hassle-free full-length membrane protein immunization, low cost (compared to VLP)

-- Ion channel proteins, multiple transmembrane protein immunizations

-- Generation of antibodies with high affinity and specificity;

-- High specificity of target immunization, high purity of mRNA immunizing antigen (compared to cellular immunization)