The tetramer technology enables specific, efficient and direct quantification of antigen-specific CTI-activity assays, and can be applied to immunological research and detection, specific immunotherapy, and vaccine efficacy monitoring.
MHC class I molecules serve as antigen presentation and recognition in the CD8+ cellular immune system. Due to their specificity, MHC-peptide complexes play an important role in immunotherapy. Although MHC-polypeptide complexes are specifically recognized by TCR, most natural TCR receptors are not only less stable but also have lower affinity for MHC-polypeptide complexes.
In order to solve the above two deficiencies, targeting tumor epitopes, the preparation of TCR-like antibodies with high affinity and solubility is the best alternative to wild-type TCR receptors. As a new immune molecule, this type of TCR-like antibody plays a role in recognizing and killing tumor cells.
Immunotherapy targeting T cell epitopes
Cells and cancer cells in the autoimmune system disorder express specific peptides that bind to cell surface MHC molecules. Tumor cells expressing specific short peptides in cancer patients can be recognized by CTLs. Despite this recognition mechanism, tumor cell apoptosis escape is common in tumor tissues.
In recent years, with the gradual deepening of research on tumor-associated antigens, people are also designing and validating relevant immunotherapy programs for tumor-specific antigens, and have achieved certain results in the specific treatment of cancer patients. There are many researches on tumor-associated antigens expressed by tumor cells, which can be roughly divided into three categories:
1) Tumor-specific antigens caused by protein mutations, such as B-catenin mutations in colon cancer.
2) Antigen expressed by certain defined cell lines
3) derived from high expression or amplification of genes, such as HER-2/neu
As for immunotherapy programs, most researchers have focused on how to enhance acquired immunity to tumor cells. These include acquired immune T cells, cancer cell short peptide vaccines, autologous cancer vaccines, and DC vaccines. These different methods have made great progress in the clinic.
Cell-based research efforts face challenges such as the mechanism of apoptosis, although these challenges limit the anti-tumor activity of CTL. However, the use of antibodies, through the simulation method can better solve this problem. Specific tumor-associated MHC I-polypeptide complexes play a specific immunomodulatory role. Using this property, an MHC I-polypeptide complex containing a specific short peptide can be used as a specific antigen on the surface of a tumor cell.
Such an antigen not only serves as a targeting molecule for cell therapy, but also serves to distinguish between cancer cells and normal cells. Moreover, a series of short peptides that are well-recognized by TCR are selected. These short peptides and MHC form an MHC-short peptide complex and can recognize most common tumors.
The mechanism by which TCR-like antibodies specifically recognize tumor cells is identical to that of MHC-short peptide complexes recognized by T cells. Based on the cell therapy program, it is easily restricted by many factors such as the state of the cell itself. Compared with cell therapy, soluble antibodies have higher yield; tumor infiltration ability is stronger than T cells, and it is superior to T cells as a treatment.
TCR-like antibodies have two major advantages in the immune system: from a biological point of view, TCR-like antibodies have better antigen specificity. As a drug, its stability is stronger than that of T cells, and T cells are more affected by the outside world. At the same time, tumor cells are affected by the tumor microenvironment, and the expression of MHC molecules on the surface of most tumor cells is down-regulated. Based on this understanding, high-affinity TCR-like antibodies are particularly suitable for low-density MHC-expressing antigens.
T cell receptor-like antibody
MHC-restricted antigen-specific T cell antibodies are few and difficult to prepare. TCR recognizes linear short peptides that bind to MHC molecules. This mechanism differs from the B cell recognition mechanism in that B cells recognize the three-dimensional structure of soluble or cell membrane antibodies and are not restricted by any MHC. At this point, T and B cells exhibit recognition mechanisms for two different functions in the immune system.
T cells recognize antigenic peptides presented by MHC class I or class II molecules on the cell membrane surface to recognize cells. B cells do not have a selective recognition mechanism. In recent years, research and development work on TCR-like antibodies has made great progress. Many research teams have the ability to make this antibody, and many TCR-like antibodies against tumor and viral T cell epitopes have been developed.
It is possible to obtain a THC-like antibody preparation that is recognized by T cells and naturally folds the MHC-short peptide complex after purification. The MHC-short peptide complex can be obtained by E. coli expression. Escherichia coli expresses MHC heavy chain and β-globulin, respectively, and after folding in vitro, the MHC-short peptide complex is obtained by affinity with a short peptide. The folded MHC-short peptide complex is required to be high, and the prepared complex is required to be very pure and folded correctly.
The prepared MHC-short peptide complex is biotinylated to screen for specific TCR-like antibodies in vitro. Many laboratories try to obtain epitopes that can be recognized by T cells, but many laboratories have difficulty preparing them. In 1996, Andersen et al. demonstrated the use of phage display technology as a means to obtain specific antibodies.
After they fuse the antigenic protein on the surface of the phage particle, they display the corresponding antibody by display screening. There are different genes in the phagemid that express different sequence proteins on the surface of the phage. After several rounds of screening and bacterial culture, the desired phage is eluted. Phage display exhibits a stronger positive selectivity than hybridoma technology.
Many research teams have built phage peptide libraries to screen for appropriate antibodies. The phage display technology was first able to isolate mouse MHC class I antibodies with short peptide specificity and MHC restriction. Thereafter, human HLA-related disease antibodies were also prepared.
TCR-like antibody screening
The specificity and affinity of a TCR-like antibody determines whether the antibody is good or bad. Specific T cell antibodies with MHC restriction are more difficult to prepare. However, in recent years, through the efforts of many teams, the use of tumor or viral T cell epitopes to prepare TCR sample antibodies has been greatly developed.
A number of TCR-like antibodies are obtained by combining phage display in vitro screening methods and developing appropriate immunization procedures. Phage display screening, high flux, screening pressure. Before screening, a large number of gene banks can be constructed, and the gene bank can be introduced into the phage, and the optimal antibody can be screened as much as possible.
3.1 phage display library
Phage display technology is mainly to fuse foreign genes to the surface of phage particles. Each phage particle displays an antibody. A display library is constructed and the library sequence is constructed into phage by genetic means. Each phage is shown to display a unique antibody gene. The larger the library, the more information is covered, and the more useful antibodies are available through display technology.
3.1.1 original library
Phage display screens for affinity TCR antibodies and is often not effective for therapeutic purposes. Selection of affinity TCR antibody genes, both random mutations and target mutations can increase TCR-like antibody affinity. Chames et al (2000) reported that after screening for TCR samples with affinity for tumor soluble T cell receptor (scTCR) epitopes, random mutations were made in Fab, and their affinity was increased by 18-fold by in vitro screening.
Renner et al. (2003) analyzed the TCR-like Fab 3M4E5 protein structure of HLA-A2/NY-ESO-1, identified some key amino acids that determine the affinity, and made site-directed mutagenesis at key amino acid positions without changing the helical structure of the MHC recognition site. In this case, the TCR-like Fab affinity is increased by a factor of 20.
TCR-like antibodies derived from phage display are presented as monovalent antibodies. At the same time, such antibodies can be purified in the field with higher stability in bacteria. Monovalent fragments can be used to conduct studies such as antigen screening and structural analysis. In addition, monovalent fragments can be used as targets for transposable proteins.
However, the antibody that appears in the form of monovalent has a weak affinity, especially in the research of low-density epitopes of tumors. To compensate for this deficiency, Fab or scFV is prepared in a tetrameric form, and the labeling of fluorescent molecules to the tetramer can be used to analyze the affinity for the MHC-short peptide complex.
3.1.2 immune library
Phage display immunoblot construction, this step complements phage display screening. The recombinant MHC-short peptide complex was used to immunize mice, and then mouse spleen cells were used to construct a phage display system. The mice used should use mice with their own MHC deletion and stably expressing the human HLA gene. This type of mouse is immunized with an MHC-polypeptide complex. This type of mouse can increase the rate of TCR-like antibody acquisition.
Phage screening yields a series of affinity antibody Fab fragments. The affinity Fab fragment gene information is thus obtained. Such gene sequences are cloned into a vector to prepare an antibody. What is the affinity of such antibodies? Further evaluation is needed. The ELISA method for evaluating antibody affinity is generally accepted by researchers.
TCR-like antibodies were evaluated using the MHC-peptide complex and coated by ELISA. Bhavna Verma et al (2010) used breast cancer as a model to select hCGB Ag antigen. The HLA-short peptide form was prepared by the short peptide GVLPALPQV on the Ag antigen and HLA-A2. The mice were immunized to obtain antibodies.
In evaluating the antibody, the HLA-short peptide complex was prepared as an MHC tetramer, and the tetramer was coated on the enzyme-linked plate. Different candidate TCR-like antibodies were prepared, and ELISA values were determined to evaluate different candidate TCR-like antibodies.
The MHC tetramers are cross-linking of four MHC-short peptide complexes on streptavidin. The MHC tetramer is coated on the enzyme-linked plate, and the affinity of the antibody is determined by ELISA to achieve high-throughput detection.
Anna Sergeeva et al. (2011) used mouse HLA-A2-PR1 peptide complex to immunize mice and isolate mouse spleen cells in the study of TCR-like antibodies in human acute myeloid leukemia. Monoclonal cells are cultured to prepare antibodies. The MHC-PR1 polypeptide complex was coated on an enzyme-linked plate and subjected to the same quantitative ELISA screening. Of the 2850 clones, one (8F4) affinity antibody was screened.
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