I. Introduction to Chimeric Antibodies

Human-mouse chimeric antibodies, that is, the variable region of the antibody comes from mouse monoclonal antibodies, while the constant region comes from human antibodies. It is produced by isolating the functional variable region gene from hybridoma cells, connecting it with the human Ig constant region gene, inserting it into an appropriate expression vector, and transfecting the host cell for expression. Chimeric antibodies not only retain the ability of the parent antibody to specifically bind to antigens, but also greatly reduce the immunogenicity of mouse-derived antibodies in the human body. Its half-life is significantly prolonged, and it is also significantly enhanced in mediating CDC and ADCC.

II. Preparation of Chimeric Antibodies (Key Technologies and Methods)

1. Variable Region Gene Cloning

1.1 RNA Extraction and Reverse Transcription

Take about 10^7 cells of the hybridoma cell line in the logarithmic growth phase, extract the total RNA of the cells according to the instructions of the Trizol RNA extraction kit, and then perform RT-PCR amplification, and recover and purify the amplified products for use.

1.2 Amplification of Light Chain and Heavy Chain V Region Genes

According to the primers designed by Orlandi et al. (1989) for amplifying mouse variable region genes, the variable region of the antibody gene was amplified using the above amplified products as templates. The heavy chain VH (about 360 bp) and light chain VL (about 330 bp) fragments were recovered and inserted into the Teasy vector, and 3 samples were sent for sequencing. The sequences obtained by sequencing were classified and homology analyzed in the GeneBank nucleic acid database.

Add restriction sites at both ends: Design primers containing restriction sites according to the above PCR product sequence, and amplify again so that the antibody variable region gene fragment has restriction sites that match the vector, which is convenient for insertion into the expression vector. Recover and purify the amplified product for later use.

2. Construction of Chimeric Antibody Expression Vector

2.1 Modification of monoclonal sites of human Ig heavy chain and light chain C region gene fragments containing signal peptides

Design upstream primer HLF (KOZAK+4G, i.e. GCCGCCATGG) containing the heavy chain signal peptide start site sequence and the restriction site Nhe I, and downstream primer HLR containing the Xho I site, using plasmid pAc-K-CH3 as a template to amplify the heavy chain signal peptide gene fragment; design upstream primer HCF containing XhoI and KpnI sites, and downstream primer HCR containing Furin (RKRR) sequence and restriction site Xba I, using plasmid pAc-K-CH3 as a template to amplify the heavy chain constant region gene fragment; connect the above fragments with Xho I to obtain a gene fragment containing the heavy chain signal peptide, MCS and heavy chain constant region.

Design the upstream primer LLF containing the restriction site Apa I, and the downstream primer LLR containing the EcoR I site, and use the plasmid pAc-K-CH3 as a template to amplify the light chain signal peptide gene fragment; design the upstream primer LCF containing the EcoR I and Hind I sites, and the downstream primer LCR containing Pme I, and use the plasmid pAc-K-CH3 as a template to amplify the light chain constant region gene fragment; connect the above fragments with EcoR I to obtain the gene fragment containing the light chain signal peptide, MCS and light chain constant region.

2.2 Construction of an empty expression vector containing signal peptide and human Ig heavy chain and light chain C region gene fragments

The above heavy and light chains were connected with the synthesized 2A sequence containing restriction sites (XbaI and ApaI) to obtain the gene fragment LigC (Leader + Ig Constant) containing signal peptides and human Ig heavy chain and light chain C region genes. Then, it was connected with the large fragment of pcDNA3.1-CA double-digested with Nhe I and Pme I to obtain the chimeric antibody eukaryotic dual expression vector pcDNA3. 1-CA (chimeric antibody, CA).

Figure 2 pcDNA3.1 map

2.3 Insertion of the V region genes containing the source Ig heavy chain and light chain

The double-digested expression vector pcDNA3.1-CA and purified VL were separated and purified by agarose gel electrophoresis, and then connected and transformed into Escherichia coli, and the pcDNA3.1-CA-VL positive clones were screened. After enrichment culture, the plasmid was extracted for enzyme digestion identification. Then, the double-digested expression vector pcDNA3.1-CA-VL and purified VH were separated and purified, and the corresponding enzyme-digested fragments were repeated to construct the recombinant expression vector pcDNA3.1-CA-XX (the first letter of the word antigen).

3. Transfection of Sp2/0 Cells

Inoculate an appropriate amount of Sp2/0 cells in a culture flask, culture for 12 hours, and transfect with purified vector pcDNA3.1-CA-XX DNA using Lipofectamine 2000 reagent according to the instructions of the kit. Set empty vector and cells without vector as controls. 72 hours after transfection, add selective medium containing MTX for screening. After 2 weeks, the positive clones grown were monocloned by limiting dilution method.

4. Identification and Screening of Positive Clones

Use antigen and enzyme-labeled goat anti-human IgG Fc fragment antibody to detect the specificity and recombinant nature of chimeric antibodies by indirect method. Coat the ELISA plate with an appropriate amount of antigen, add cell culture supernatant for reaction, and then add goat anti-human IgG Fc fragment-HRP enzyme-labeled antibody (adsorbed by mouse Ig) for incubation. After color development, measure the A490 absorbance.

5. Antibody Ascites Production

Transfected tumor cells were implanted in the peritoneal cavity, and ascites was extracted after 5-7 days. A mouse implanted with hybridoma cells can sometimes produce up to 10 ml of ascites. Wang Shuo et al. (2003) reported that the yield of chimeric antibodies obtained from ascites can reach 1~2 mg/ml.

III. Application of Chimeric Antibodies

Chimeric antibodies are currently mainly used in drug therapy. Compared with mouse antibodies, chimeric antibodies retain the high affinity of mouse-derived variables and have multiple immune killing functions of human Fc segments, thereby greatly reducing the incidence of human anti-mouse antibody reactions and improving clinical treatment effects. Compared with other small molecule genetic engineering antibodies, chimeric antibodies, as completed antibody molecules, have a longer half-life in the body and have multiple immune killing functions of human Fc segments.

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 high-affinity and low-immunogenicity chimeric antibody transformation services.