CD20 antibody and its application in treating cancer
An antibody and CDR3 technology, applied in the field of biomedicine, can solve the problems of adverse reactions, restrictions on the long-term application of Rituximab, and a complete remission rate of only 10%, achieving the effect of enhancing anti-tumor activity
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Embodiment 1
[0034] Example 1. Anti-CD20 Antibody Preparation
[0035] BALB / C mice were immunized with NS / 0 transfected with human CD20. The cells were resuspended in PBS and mixed with complete Freund's adjuvant at a ratio of 1:1. The primary immunization method was intraperitoneal injection, and the immunization dose was 1X10 per mouse. 7 cells. Subsequent booster immunizations were followed by intraperitoneal injections with the same dose of unadjuvanted cells every 3 days. For the first three days of cell fusion, at 0.5X10 7 The cells were immunized by intravenous injection. After 3 days, the mice were sacrificed by cervical dislocation, and the spleen was removed to prepare a single-cell suspension in PBS. Splenocytes were washed 3 times with DMEM medium. The mouse myeloma cells SP2 / 0 in the logarithmic growth phase and the isolated mouse splenocytes were mixed at a ratio of 1:4 and washed twice with DMEM. Cell fusion was carried out by means of PEG fusion. Confluent cells were...
Embodiment 2
[0036] Example 2. Antibody Humanization
[0037] Take 5 × 10 hybridoma cells obtained in Example 1 7 Add 1ml Trizol, mix well and transfer to a 1.5mL centrifuge tube, let stand for 5 minutes at room temperature; add 0.2ml chloroform, shake for 15 seconds, stand for 2 minutes, centrifuge at 4°C, 12000g for 5 minutes, take the supernatant and transfer it to In a new 1.5ml centrifuge tube; add 0.5ml isopropanol, gently mix the liquid in the tube, stand at room temperature for 10 minutes, centrifuge at 12000g for 15 minutes at 4°C, discard the supernatant; add 1ml 75% ethanol, light Wash the precipitate lightly, centrifuge at 12000g at 4°C for 5 minutes, discard the supernatant, air dry the precipitate, add DEPC water to dissolve, and obtain total RNA. Take the total RNA, and then use a reverse transcription kit (PrimeScript RT Master Mix, purchased from Takara) to synthesize the first strand of cDNA; using cDNA as a template, use PCR to amplify the variable region of the antibod...
Embodiment 3
[0041] Example 3. Affinity of humanized CD20 antibody to human CD20
[0042] Referring to the instructions of the amino coupling kit and human anti-capture kit, the human antibody capture antibody (AHC) was immobilized on the surface of the S series sensor chip (Sereis S Sensor Chip CM5) by amino coupling. Equilibrate at room temperature for 20-30 min, and load the chip into the instrument. Dilute the antigen with equilibration buffer, dilute the antigen 10 nM starting dilution 5 concentration gradients, and set 2 zero concentrations (ie, equilibration buffer) and one repeat concentration (generally the lowest concentration repeat). The CD20 antibody h12H7-IgG1 was diluted to the experimental working concentration with equilibration buffer, and sealed at 2-8°C for later use. The antigen flow rate was set to 50 μL / min, and the binding time was set to 3 min. The flow rate of HBS-EP+ buffer was set to 50 μL / min, and the dissociation time was set to 10 min. Use 3MMgCl 2 As a r...
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