A monoclonal antibody is an antibody that is highly uniform and unique to a particular epitope produced by a single B cell clone, and is referred to as a monoclonal antibody. Hybridoma technology is usually used to prepare hybridoma antibody technology. Based on cell fusion technology, sensitized B cells with the ability to secrete specific antibodies and myeloma cells with unlimited reproduction ability are fused to B cell hybridization tumor.
Types
Murine antibody
The basic principle of hybridoma monoclonal antibody preparation technology is to use polyethylene glycol as a cell fusion agent to fuse the spleen cells of the immunized mouse with the mouse myeloma cells with the ability to reproduce in vitro, in HAT selective medium. Under the action of the fusion, only the hybridoma cells with successful fusion are grown, and repeated immunological detection and single cell culture (cloning) are finally obtained to obtain a hybridoma cell line which can produce the desired monoclonal antibody and can continuously multiply. The cells are expanded and cultured, and inoculated into the peritoneal cavity of the mouse, and a high-priced monoclonal antibody can be obtained in the ascites produced. Since the application of monoclonal antibodies is in vitro development from in vitro diagnostics to in vivo tumor localization and treatment, murine monoclonal antibodies have been difficult to overcome, especially in vivo, when there are major histocompatibility antigens (MHC) and super Sensitive reaction problem. With the increasing use of murine monoclonal antibodies in clinical treatment, the need to reduce the immunogenicity of antibodies has become more and more urgent. To overcome this problem, scientists have used genetic engineering methods to humanize antibodies in mice. By constructing a human-mouse chimeric antibody, human anti-mouse antibodies were attenuated to some extent.
Human and mouse chimeric antibody
To overcome the problems of murine monoclonal antibodies, scientists have used genetic engineering methods to humanize antibodies in mice. By constructing a human-mouse chimeric antibody, human anti-mouse antibodies were attenuated to some extent. In 1984, Morrison et al. successfully constructed the first human murine monoclonal antibody, a chimeric antibody. A chimeric antibody refers to a monoclonal antibody produced by the constant region gene of a murine monoclonal antibody which is encoded by a genetic region recombination technique and which is encoded by a human antibody constant region gene and expressed in a suitable host cell.
Rationale: The specific recognition of antibody molecules, antigen binding is determined by the light chain and heavy chain variable regions, and the human anti-mouse antibody produced by the heterologous protein is mainly the antibody constant region. The mouse mAb constant region was replaced with a humanized constant region and spliced into a chimeric antibody, the variable regions of the heavy and light chains were derived from the mouse, and the constant region was derived from human. Briefly, chimeric antibodies have both antigen binding specificity and greatly reduce the heterogeneity of murine mAb. Chimeric antibody is an antibody originally developed by genetically engineered antibodies and has been widely used in tumor therapy and diagnostic methods. Although chimeric antibodies attenuate human anti-mouse antibody responses to some extent, there is still a small fraction of murine components. This directly leads to the rapid elimination of antibodies, thereby reducing the therapeutic effect.
Reshaped antibody
In 1986, Jones et al. successfully constructed the first modified antibody, also known as CDR-grafted antibody and humanized antibody, which refers to the replacement of the corresponding CDR sequence of the human antibody by the complementarity determining region (CDR) sequence in the murine monoclonal antibody variable region. Recombinant constitutes a CDR-grafted antibody that has both murine monoclonal antibody specificity and human antibody affinity. To date, more than 100 murine monoclonal antibodies have been humanized by CDR grafting. Rationale: The variable regions of the antibody heavy and light chains are composed primarily of CDRs and framework regions (FR). The six CDRs of the variable region are the regions responsible for the recognition and binding of antigens, and they are directly in contact with the antigen, which determines the specificity of the antibody. The framework region is other than the variable region, mainly plays a role in supporting CDRs, and their amino acid composition and arrangement are relatively difficult to change. Therefore, the CDR of the murine monoclonal antibody can be transplanted into the framework region of the human monoclonal antibody. It is possible to obtain the same antigen specificity as the murine mAb, and to minimize the heterogeneity of the murine mAb, thus obtaining a modified antibody. Compared with chimeric antibodies, the modified antibody further reduces the proportion of the murine fraction in the antibody and reduces the human anti-mouse antibody, but there are still antibodies that may lead to the production of anti-idiotypic antibodies and have some limitations, such as the relative construction method. Complex, time-consuming and labor-intensive; the crystal structure of the antibody and the micro-structure of the computer-simulated antibody have great problems; there are still many problems in reducing immunogenicity and maintaining antigen binding activity. Of course, it is imperative to find a simple and easy way to achieve both humanization and high immunological activity.
Surface amino acid residues humanized one-faced antibody
A method for reducing the immunogenicity of murine antibodies, which was completely different from CDR grafting, proposed by Padlan in 1991. [9] The theoretical basis is to analyze the surface exposure of a large number of murine monoclonal antibody variable regions and human monoclonal antibody variable region amino acid residues, and found that the position and number of these exposed amino acid residues are very conservative, not because of species Change with type. On the surface of the study, these exposed amino acid residues are the main source of immunogenicity in the murine variable region. By changing the amino acid residue corresponding to the human variable region in the amino acid residue of the framework region exposed by the variable region of the murine mAb to human, the surface of the variable region can be humanized, and the heterogeneity is eliminated without affecting The overall spatial conformation of the variable region.
Episode imprint selection
Epitope selection refers to a method of humanized antibody that binds to the highly efficient screening of phage antibody library technology, and can be obtained by digital screening to obtain fully human antibodies. Rationale: A heavy or light chain variable region gene of a murine mAb is paired with a heavy chain or light chain variable region gene library of a human antibody to obtain a heterozygous human mouse antibody library. With the efficient screening method of phage antibody library, the desired antibody can be obtained quickly, which is simpler than CDR transplantation and can obtain real human antibodies. Disadvantages: The screening workload is particularly large.
Small molecule antibody
Small molecule antibodies, as the name implies, are antibody fragments of smaller molecular weight, and the antigen binding site of the antibody molecule is limited to the variable regions of the heavy and light chains. Although the molecule is small, it retains the same specificity as the parental monoclonal antibody with the affinity of the parental monoclonal antibody. The categories mainly include: antigen binding sheet (Fab) antibody, Fv antibody, single chain antibody, single domain antibody and minimal recognition unit.
Fab antibody
The Fab antibody is a Fab-only molecule, and the Fab segment is formed by a disulfide bond between the intact light chain (constant region CL and variable region VLCL) and the heavy chain Fd segment (first constant region CH1 and variable region VH). The dimer, which is about one-third the size of the total antibody, contains only one antigen-binding site. The coding genes of the complete light and heavy chain Fd are ligated and expressed in E. coli to form a complete disulfide bond and a three-dimensional fold, which can preserve the function of Fab break. Often used in laboratory research tools.
Fv antibody
The Fv antibody consists only of the variable regions of the light and heavy chains by non-covalent linkages, and is the smallest functional fragment of the antibody that retains the intact antigen binding site. Since the fragment is connected by a non-covalent bond, the stability is not good and it is very easy to dissociate. An appropriate method is used to solve the problem of Fv fragment stability.
Single-chain antibody
The advent of single-chain antibodies solves the problem of stability of Fv antibodies. It is formed by linking the light chain and heavy chain variable regions by a suitable oligonucleotide sequence to form a single-stranded molecule, so it is called a single-chain antibody. The structure of a single chain greatly increases the stability of the Fv fragment. Single-chain antibodies have many advantages in clinical use as therapeutic agents relative to fully antibodies. But it also has shortcomings such as decreased affinity.
Single domain antibody
The single domain antibody contains only the heavy chain variable region and has a smaller structure than the subunit of Fv, which is a molecule having antigen binding activity. Single domain antibodies still have the same ability and stability to bind to antibodies as compared to intact antibodies.
Minimum identification unit
The smallest recognition unit smaller than the single domain antibody contains only a single CDR structure in the variable region, the molecular weight is very small, only about 1% of the complete antibody, and the affinity is also relatively low, so it is named as the smallest recognition unit. Although the minimal recognition unit has a small molecular weight and low affinity, it has the ability to bind to an antigen.
Human antibody
From the initial murine monoclonal antibody technology to humanized technology, monoclonal antibodies have made rapid progress in decades. The advent of humanization has made monoclonal antibodies basically solve the problem of human anti-rat origin. However, useful humanized antibody genes are derived from hybridoma cells. This process of hybridomas is complex and time consuming, and it is difficult to prepare autoantigen antibodies and fully human antibodies using hybridoma technology. These two major disadvantages have become a stumbling block in the application of genetically engineered antibodies. With the continuous advancement of science and technology, monoclonal antibodies have entered a new stage of development - human-derived monoclonal antibodies. It mainly includes phage antibody library technology, preparation of human-derived antibodies in transgenic mice, and ribosome display technology.
The development of monoclonal antibody drugs originated in 1975, and the advent of hybridoma technology has made it possible to prepare a large number of uniform murine monoclonal antibodies. In 1986, the first murine monoclonal antibody mumomonab-CD3 (OKT3) against post-transplant immune rejection was approved by the US Food and Drug Administration (FDA), but derived from murine lymphocyte hybridization. Tumor antibodies are recognized by the human immune system and cause severe human anti-mouse antibody (HAMA), which not only shortens the half-life of therapeutic monoclonal antibodies, but also reduces the efficacy and sometimes causes serious adverse reactions.
No comments:
Post a Comment