With the development of a series of major biotechnologies (such as PCR, antibody library, transgenic animal technology, etc.), antibody technology has gradually developed from chimeric antibody and modified antibody to humanized antibody today. Humanized antibodies have shown unique advantages and good application prospects in the treatment of tumors, autoimmune diseases and organ transplantation.
Construction Strategy of Humanized Antibody
Humanization of mouse antibody is to make it have a very similar profile with human antibody molecules through genetic modification, thus avoiding the recognition of human immune system and inducing HAMA reaction. Two principles should be followed in humanizing mouse-derived antibodies. First, the affinity and specificity of antibodies should be maintained, and secondly, the immunogenicity of antibodies should be reduced or eliminated.
Chimeric antibody
The first generation of humanized antibodies, simple chimeric antibodies, developed in the mid-1980s, replaced the constant region of murine monoclonal antibodies with human genes. The constructed chimeric antibody not only retains the specificity of antigen-antibody binding, but also greatly reduces the immunogenicity of mouse-derived monoclonal antibodies. Rituximab, as the first genetic engineering antibody for cancer therapy, is a chimeric antibody composed of murine variable region and human constant region. However, because the variable region (V) of chimeric antibody accounts for about 30% of the total antibody, the frame region (FR) of mouse-derived antibody V still has some immunogenicity, which can induce HAMA reaction. Primate-derived antibodies are also chimeric antibodies produced by immunizing macaques. Since the variable region of macaque antibody is almost the same as that of human, this kind of chimeric antibody does not need any change and does not cause antibody reaction.
F AB and F (ab') chimeric antibodies are prepared by recombining light and heavy chain variable region genes of functional antibodies with K chain and heavy chain CHl constant region genes of human antibodies, respectively, and cloning them into expression vectors to construct a mouse-to-human chimeric F AB gene expression vector, which is then transferred to host cells for expression. The structure of a hinged region between CHl and CH2 is a natural antibody molecule. Two of the Cys residues can form disulfide bonds, which covalently bind the two heavy chains together.
CDR transplantation antibody
In order to reduce the mouse-derived components, CDR transplantation antibody or modified antibody (Reshaped_Ab) appeared. This is a more complete humanized antibody, that is, the true humanization of antibodies. In addition to three complementary determinant regions (CDRs) of the antibodies, all of them are human structures. For example, "Taixinsheng", a new national class I cancer treatment drug, "Taixinsheng recombinant humanized anti-human epidermal growth factor receptor monoclonal antibody", uses advanced "CDR transplantation" technology, with a humanized degree of more than 95%, with higher safety and lower toxicity.
SDR transplantation antibody
Compared with chimeric antibodies, CDR transplanted antibodies can significantly reduce mouse-derived components, but sometimes humanized CDR antibodies with different genes may cause anti-individual genotype reactions. Specific determinant region (SDR) transfer antibody is a small number of residues, such as SDR, which are closely related to antigen binding of heterologous antibodies, transplanted to the corresponding position of human antibodies, further reducing the heterology. In this way, the potential immunogenicity of humanized antibodies is minimized.
Fully humaneantibody
Antibody library screening technology
Phage display technology
With the development of antibody engineering, bacteriophage display technology emerged as the times require. That is, a whole set of gene sequences encoding human antibodies were amplified from organisms by PCR technology, cloned into phage vectors, and expressed on the surface of bacteriophages in the form of fusion proteins, so that the specific binding of antigen and antibody can be conveniently used for screening and amplification. This technique can not only obtain monoclonal antibodies with human nature, but also screen the required genes directly from the library by using antigens without cell fusion or even through immune animals. The experimental period is short and the process is simple. This is a major breakthrough in the preparation of human antibodies.
Ribosome display
The technology links genotypes and phenotypes, transcribes and translates the DNA encoding proteins in vitro, due to the special processing and modification of DNA, such as removing the 3'terminal termination codon. When the ribosome is translated to the end of the RNA, due to the lack of termination codon, it stays at the 3'end of the RNA and does not detach, thus forming a protein-ribose-2 RNA trimer, solidifying the specific ligand of the target protein, then screening, decomposing the screened complex, releasing the RNA by RT-PCR, and the product of the PCR enters the next cycle. The labeled proteins and their coded gene sequences were enriched and separated. Specific and high affinity antibodies can be obtained by this technique.
Transgenic mice
In transgenic animals, there are several different ways to produce human antibodies. One way is to introduce lymphocytes from donors or cancer patients who have already produced certain immune responses into severe combined immunodeficiency mice (SCID) or Trimem mice. Hybridoma secreting human antibodies can be obtained by hybridizing mouse spleen cells with human myeloma cells. Another way to produce human antibodies is to inactivate the genes of mice themselves by gene knockout technology and introduce new genes to create transgenic mice carrying heavy and light chain gene clusters of human antibodies. The human DNA fragments carried by the transgenic mice with human antibody gene have complete functions, and can effectively carry out homotypic transformation and affinity maturation. Any target antigen can be used to immunize the mice to produce high affinity human antibodies.
Tomizuka et al. firstly used chromosomes as carriers to successfully cultivate transgenic mice, and prepared human antibodies with high affinity. Kirin Company of Japan used genetic engineering technology to make mice carry complete human chromosome 14, which contains all human antibody-producing genes. But up to now, no products produced by this technology have been published.
Clinical application of humanized antibody
In recent years, the emergence of humanized antibodies and human antibodies has brought new hope for clinical application. Chimeric antibodies and humanized antibodies account for more than 70% of the various antibodies currently in clinical research. At present, humanized antibodies are mainly used in the treatment of tumors, autoimmune diseases and cardiovascular diseases, as well as Anti-transplant rejection and anti-virus infection.
Application in cancer treatment
Monoclonal antibodies can effectively reduce the adverse reactions of traditional cancer drugs. These humanized monoclonal antibodies are mainly aimed at target molecules related to tumorigenesis and development, such as anti-CD20 chimeric antibody Ritu for non-Hodgkin lymphoma and anti-CD22 humanized antibody Epratuzumab, as well as humanized antibodies for tumor targets such as VEGF, EGFRL and CD33. These target molecules are expressed in normal tissues or cells, but over-expressed in many cancer cells, so they can be used as tumor-specific markers.
There are also humanized monoclonal antibodies that act by carrying anti-cancer drugs such as toxins, cytotoxic drugs, radionuclides, enzymes, chemotherapeutic drugs and other molecules. In this way, a "biological missile" is coupled to achieve specific killing effect on cancer cells.
Application in organ transplantation
How to avoid and minimize the rejection after transplantation in order to protect the function of transplanted organs is the key to the success of organ transplantation. In recent years, the use of antibody drugs as induction therapy for solid organ transplantation has been increasing. Anti-CD3 monoclonal antibodies, the first therapeutic antibodies approved to enter the U.S. market (1986), are used to reverse rejection of kidney, heart and liver transplantation.
Application in autoimmune diseases
In the treatment of autoimmune diseases, monoclonal antibodies inhibit excessive immunopathological reactions by eliminating activated cells, blocking their functions, or reducing elevated levels of pro-inflammatory cytokines to normal levels.
Studies have shown that antibodies are effective in treating rheumatoid arthritis, systemic lupus erythematosus, psoriasis, multiple sclerosis, scleroderma and ulcerative colitis. Infliximab is a chimeric anti-rINF-a monoclonal antibody developed jointly by Johnson and entoeor in the United States. It has a good curative effect on localized ileitis and rheumatoid arthritis, and patients have a good tolerance to it.
At present, the humanization method of antibody is still developing and improving. The goal is to improve the affinity of antibody, reduce the heterogenicity of antibody, and take into account the immunological activation of antibody. In addition, for different heterologous antibodies, choosing the appropriate humanized approach to construct more suitable humanized antibodies for clinical application will open up a new path for the development of new drugs and the treatment of some intractable diseases.
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