Humanized monoclonal antibodies have basically solved the immunogenicity problem of mouse antibodies, but the humanization process is still complicated and expensive, requiring extensive computer model design and a large number of experiments to determine the harmful effects of various amino acids on target selectivity and binding affinity. In order to completely eliminate the adverse effects of heterologous antibodies, people applied phage display technology to the expression and cloning of antibodies, resulting in phage antibody library technology. As a result, antibody engineering technology has entered a new stage of development, and the production and application of fully humanized antibodies have gradually matured.

I.  Fully Humanized Antibodies

Fully humanized antibodies refer to the transfer of all human antibody-encoding genes to genetically engineered antibody gene-deficient animals through transgenic or transchromosome technology, so that the animals express human antibodies and achieve the purpose of fully humanized antibodies.

II. Preparation Methods of Fully Humanized Antibodies

1. Display Technology

(1) Phage display technology: This technology uses phage surface display technology. First, the full set of variable region genes of antibodies are amplified by PCR and cloned into phage display vectors. In this way, the antibody genes are expressed and displayed on the phage surface. Then, after multiple rounds of "adsorption-elution-amplification" process, specific antibodies are screened and enriched. This technology links phenotype and genotype together, combining antigen recognition ability and re-amplification. It is an extremely efficient expression and screening system.

(2) Ribosome antibody library technology uses the basic principle of ribosome display. First, the antibody variable region gene library amplified by PCR is constructed into the ribosome display system, and then transcribed and translated in the non-cellular translation system. Due to the particularity of this system, the antibody will eventually form an "antibody fragment-ribosome-mRNA" complex, that is, a ribosome-displayed antibody complex. Then the ribosome-displayed antibody library is affinity screened with the corresponding antigen, and the screened complex is used to obtain the target antibody gene DNA through RT-PCR. The obtained DNA can enter the next round of display screening or be sequenced and analyzed.

(3) Yeast antibody library technology usually fuses antibody fragments with a or a-lectin respectively and displays them on the surface of yeast cells. At present, a-lectin display technology is widely used. Yeast antibody library display technology inherits the characteristics of phage display and can screen target genes according to the characteristics of the encoded protein. The yeast display system can not only be screened by traditional biological panning methods, but also by flow cytometry sorting, which greatly reduces the screening workload and improves the screening efficiency.

2. Transgenic Mice

There are several transgenic mice for preparing fully human antibodies, including human peripheral blood lymphocyte-severe combined immunodeficiency mice, human immunoglobulin genome mice and human chromosome mice. The first method is widely used in immunology research, but there are few reports on the preparation of fully human antibodies; the latter two methods began in the late 20th century and are technically difficult, but they have attracted much attention because they can prepare complete high-affinity human antibody molecules.

3. Single-cell PCR

Single-cell PCR is a technology for constructing fully human monoclonal antibodies. It directly obtains the antibody light and heavy chain variable region genes in a single target B cell through single-cell RCR technology, and then recombines them into an expression vector containing the light and heavy chain constant regions of human IgG, and then expresses them through transfection of mammalian cells to prepare fully human monoclonal antibodies against specific antigens.

The basic process of single-cell PCR to construct fully human monoclonal antibodies is as follows: (1) Collect peripheral blood or bone marrow mononuclear cells from vaccine-immunized or immune-responsive individuals. (2) Through B cell surface-specific molecular markers, antigen-specific plasma cells or memory B lymphocytes are obtained by flow cytometry, immunomagnetic bead sorting and other methods. (3) Amplify the antibody light and heavy chain variable region genes in the cell by single-cell RT-PCR and Nested PCR methods, sequence and identify them. (4) Clone the antibody light and heavy chain variable region genes into a eukaryotic expression vector containing human antibody light and heavy chain constant region genes, and transfect 293T cells for antibody expression. (5) Identify the expression product. The specific binding activity of the antibody to the corresponding antigen can be detected by Western blot, indirect ELISA and other methods; the biological function of the antibody can be detected by cell tests or animal tests.

III. Application of Humanized Antibodies

1. Application in Autoimmune Diseases

In the treatment of autoimmune diseases, humanized monoclonal antibodies inhibit excessive immunopathological reactions by clearing activated cells, blocking their functions, or reducing elevated pro-inflammatory cytokine levels to normal levels. Existing studies have shown that antibodies have good therapeutic effects on systemic lupus erythematosus, multiple sclerosis, rheumatoid arthritis and ulcerative colitis, and will be applied to the treatment of more diseases as technology develops.

2. Application in Tumor Treatment

The development of some humanized monoclonal antibodies is mainly aimed at target molecules related to tumor occurrence and development, such as the chimeric antibody Ritu against CD20 for the treatment of non-Hodgkin's lymphoma and humanized antibodies developed for tumor targets such as VEGF and CD33. These target molecules are expressed in normal cells, but are highly/lowly expressed in some tumor cells, so they can be used as tumor-specific markers.

3. Application in Organ Transplantation

The application of some developed humanized monoclonal antibodies in organ transplantation can reduce the rejection reaction after transplantation to protect the function of transplanted organs, such as the therapeutic antibody anti-CD3. Monoclonal antibodies are used to reverse the rejection of heart, kidney and liver transplants.

TekBiotech has been committed to antibody research for many years. Antibody humanization is an important part of experimental research on the production and preparation of recombinant antibodies. Obtaining humanized antibodies with high specificity and affinity plays an important role in effective antibody treatment of many diseases. We have rich experience in antibody engineering construction. Based on the antibody phage display library platform, we can provide chimeric antibody transformation services with high affinity and low immunogenicity.

TekBiotech has mature antibody humanization technology, which can humanize antibody sequences and ensure that the affinity of the modified humanized antibodies remains at the same order of magnitude. We provide guaranteed humanization services to facilitate your experiments. We are committed to building a complete one-stop technical service platform, which can provide a series of services from bioinformatics analysis, humanized antibody design, antibody expression and purification, to affinity determination, antibody affinity maturation, etc. to meet the needs of different customers.