1050: Forgot Algebra
What is immunotoxin?
Malignant tumors are a kind of serious diseases that endanger human health. With the deterioration of human living environment and the increase of social pressure, the incidence and mortality of malignant tumors are gradually increasing. Surgery is the main treatment of tumors, supplemented by radiotherapy and chemotherapy, but surgery is not suitable for all patients and has certain damage to patients. Radiotherapy and chemotherapy lack specificity and have greater toxicity and side effects on normal tissues and organs. Targeted therapy has become the goal of many medical workers. The immunotoxin (IT) is a conjugate of cytotoxin and monoclonal antibody. This conjugate does not show activity in normal cells, especially in the circulatory system, but only releases effector molecules in tumor tissues. Therefore, the immunotoxin not only has the ability to specifically recognize tumor antigens, but also retains the toxicity of effector molecules to kill tumor cells.
Composition of immunotoxins
Selection of appropriate cytotoxins
1. Plant-derived cytotoxins: Plant-derived cytotoxins are ribosomal inactivating proteins (RIPs). RIPs widely exist in plants. According to their different structures, RIPs can be divided into two types:(1) Type I RIP (RIP-I) is a single chain protein with a relative molecular weight of about 30 kD and an isoelectric point of 8-10, which is a stable basic protein. (2) TypeⅡRIP (RIP-II) is a double-chain protein, in which proteins A and B are linked by a pair of disulfide bonds. The relative molecular weight of each chain is about 30kD. The function of the chain is similar to that of type I RIP, and its isoelectric point is generally 4.8-8, which is very acidic and unstable.RIPs have broadspectrum of anti-tumor activity, which may be due to the fact that there are many phytotoxin receptors on the surface of cancer cells or their receptors are easily endocytosed after binding to toxins.
2. Animal-derived cytotoxins: Cytotoxin (CTX) extracted from cobra is a protein with 60 amino acid residues and a relative molecular weight of 6000-7000. The CTX is soluble in water and resistant to strong acids, strongalkalis and hydrolase. It is extremely stable at room temperature. It has a selective, dose-dependent killing effect, which is characterized by direct action on the cell membrane and no inhibition on bone marrow and other germinal tissues.CTX, as a warhead for the construction of anti-tumor immunotoxin, has a relatively small molecular weight and can quickly infiltrate into the tumor foci and kill the tumor cells. CTX has no oligosaccharide side chain on the molecule, and is not easy to be removed by the liver because of its non-specific binding with the liver cells, thus shortening the half-life of drug action.And the strong membrane toxicity of CTX makes it impossible to perform endocytosis and kills cancer cells quickly. Therefore, the construction of immunotoxins by CTX is a promising approach for targeted therapy of tumors.
3. Microbial-derived cytotoxins: Microbial-derived cytotoxins include cytotoxins derived from bacterial and fungi.
Selection of appropriate carrier parts
(1). Monoclonal antibody vectors: As the carrier part of immunotoxic cords, antibodies have at least three functions:a. Like naked antibodies, they can effectively transport immunotoxins to the surface of targeted cells; b. They can retain the whole or part of naked antibodies, that is to say, the part of monoclonal antibodies is also an effective drug; c. They can induce cell phagocytosis of monoclonal antibodies into the pre-lysosome and lead to the effective release of effector molecules in cells.For antibodies targeting hematopoietic differentiation antigens, the internalization process sometimes requires the involvement of complements. At present, the commonly used anti-tumor monoclonal antibodies can theoretically be coupled with cytotoxins to prepare immunotoxins.
(2). Cytokine vectors: The binding of cytokine to its receptors on the cell surface can effectively mediate endocytosis of toxins. Cytokine receptor level is regulated by cell activation and differentiation. The expression levels of cytokine receptors in different cell subsets are different. Cytokines can select specific cell groups, so cytokines can be used as targeted carriers. There are a large number of cytokine receptors on the surface of many cancer cells.
Preparation of immunotoxin
1. Chemical cross-linking method: Chemical cross-linking method refers to linking antibodies or cytokines to cytotoxins through cross-linking agents.
2. Gene fusion method: This method is based on molecular biology technology. The targeted part is ligated to the toxic part bya linker, the target gene fragment is amplified by PCR, the target gene fragment and vector are digestedwith two restriction enzymes, then the target gene fragment is connected to the vector to prepare a recombinant vector. It is cloned into the expression plasmid and transferred into the expression bacteria such as Escherichia coli, which is induced by isopropyl-beta-D-thiogalactoside (IPTG), and the products generally exist in the form of insoluble inclusions.
Action mechanism of immunotoxin
The killing effect of immunotoxin on target cells usually includes the following steps. (1) Binding: binding to specific receptor antigens on the surface of target cells by antibody or other ligands; (2) Internalization: the entry of immunotoxin into cells is the prerequisite for the action of immunotoxin; (3) Killing the target cells: after internalization, immunotoxin induces apoptosis mainly by inhibiting the synthesis of cancer cell proteins or activating important apoptotic proteins.
Problems and prospects
At present, the main problem in the application of immunotoxin is that they are easily degraded after application in vivo, that is, the half-life is not long and unstable.And the half-life and stability of the immunotoxin depend on the size of the immunotoxin, the stability of the junction and its affinity with tumors, etc. Therefore, appropriate antibody and junction should be selected to prepare the immunotoxin.Most immunotoxins have non-specific toxicity, and therefore need to clone and modify the toxic and targeted parts of the immunotoxins to reduce their toxicity to non-target cells. Most immunotoxins have immunogenicity, which mainly depends on their toxic part. Therefore, it is necessary to modify the immunogenic parts of the toxins by location mutation or gene deletion or to reduce the immunogenicity by developing and applying humanized carriers and warheads. The immunotoxin obtained by chemical cross-linking method has a lowyield, and the immunotoxin obtained by the method is a mixture, which is difficult to separate by effective methods, so more effective methods for obtaining immunotoxinshould be found.In summary, although there are still some problems in targeted treatment of malignant tumors by immunotoxins, with the in-depth study of protein and gene engineering technology, immunotoxin has good application prospects in the targeted therapy for malignant tumors.
