Antigen-binding fragment of a humanized T-cell activating V-domain immunosuppressive factor
A technology combining fragments and immunosuppression, applied in the direction of anti-animal/human immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, immunoglobulin, etc. Insufficient efficacy and other problems to achieve good pharmacokinetic properties, inhibition of tumor growth, and strong specificity
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Embodiment 1
[0043] 6-8 week-old female BALB / c mice (purchased from Shanghai Jiesijie Experimental Animal Co., Ltd.) were used as experimental animals for mouse immunization experiments. For the first immunization, 50 μg of human VISTA protein (purchased from Beijing Yiqiao Shenzhou) was mixed with complete Freund’s adjuvant to form an emulsion, and injected into the abdominal cavity of mice at an injection volume of 1 ml / mouse, and 25 μg of human VISTA protein was mixed with incomplete Freund’s adjuvant every 2 weeks. The adjuvant was fully mixed to form an emulsion for booster immunization. Booster immunization was performed three times. One week after the last immunization, the venous blood of the mice was collected and serum was separated, and the antibody titer was measured by ELISA method, and the mouse cells with high titer were selected to prepare hybridoma preparation cells. Splenocyte suspension.
[0044] Collect logarithmically grown myeloma cells (SP2 / 0) to prepare immune splee...
Embodiment 2
[0046] Screening of hybridoma culture supernatants for anti-human VISTA antibodies. Specifically, human VISTA (purchased from Beijing Yiqiao Shenzhou) was coated with a buffer solution on a 96-well high-adsorption enzyme plate, with a coating volume of 100 μL per well, and then washed with a buffer solution for 3 times; The buffer was blocked and incubated at 25°C for 1 h, the blocking volume was 280 μL / well, after the incubation was completed, the buffer was washed 3 times, and 75 μL of supernatant samples (S1-S85) and positive serum ( Control, CK1-5), incubated at 25°C for 1 hour, washed with buffer 5 times; added 100 μL of anti-mouse IgG antibody diluted in 1% BSA buffer at a ratio of 1 / 10000 to each well, the anti-mouse IgG The antibody was labeled with horseradish peroxidase, incubated at 25°C for 1 hour, and washed 5 times with buffer; 100 μL of colorimetric substrate 3,3',5,5'-tetramethylbenzidine (TMB) was added to each well, 30 After 10 minutes of color development a...
Embodiment 3
[0048] The screened clones having both antigen-binding activity and antigen-neutralizing activity were determined for the DNA sequence of the antibody. First, RNA prep Pure kit (Tiangen) was used to extract cellular mRNA according to the instructions. Then the cDNA first strand was synthesized using Quant Script RT kit (Tiangen). The first strand of cDNA produced by reverse transcription was used in the subsequent PCR reaction, and the target band obtained by PCR amplification was cloned into the pGEM-T vector, and the single clone was picked and sequenced by Nanjing GenScript Biotechnology Co., Ltd.
[0049] The antibody light chain variable region and the antibody heavy chain variable region are amplified by PCR, and the complementarity determining region sequences can be obtained after excluding the framework region sequence; the amino acid sequences of the three complementarity determining regions CDR-L1 of the light chain are shown in SEQ ID NO: 1; the amino acid sequenc...
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