Anti-PD-1 nano antibody and application thereof
A nano-antibody, PD-1 technology, applied in the direction of antibodies, anti-tumor drugs, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve the problems of poor antibody uniformity and difficulty in reaching
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[0095] (1) Preparation and purification of nanobody samples:
[0096] a. Synthesize the gene sequence encoding the Nanobody sequence;
[0097] b. Construct the synthesized gene sequence on the pcDNA3.1 vector, transform it into Escherichia coli DH5α strain, shake and extract the pcDNA3.1 plasmid with the Omega plasmid extraction kit, and filter and sterilize it;
[0098] c. Cultivate CHO-S cells to 5×10 6 pcs / ml;
[0099] d. Put the pcDNA3.1 plasmid obtained in step b and the transfection reagent PEI in the ratio of 1:3 in the transfection medium ExpiCHO TM Mix well in Expression Medium and let stand for 20min, then add it to the CHO-S cells in step c, at 37℃, 6%CO 2 , 115rpm culture for 5 days;
[0100] e. The culture product is centrifuged and filtered with a 0.2 μm membrane to obtain the centrifugation supernatant;
[0101] f. Purify the centrifugal supernatant by ProA affinity chromatography, and use the Tris system buffer of pH 7.0 to wash off impurity proteins and ...
Embodiment 1
[0116] Example 1 Detection of known anti-PD-1 nanobodies
[0117] CN107814845A discloses a new anti-PD-1 nanobody and its application. The VHH sequence of the antibody is as follows:
[0118] SEQ ID NO.1 (CDR1 / CDR2 / CDR3: SEQ ID NO.2 / SEQ ID NO.3 / SEQ ID NO.4, underlined and bold):
[0119]
[0120] VHH-IgG4-Fc (PR antibody) was prepared according to the procedure described in "(1) Nanobody sample preparation" above, wherein the amino acid sequence of IgG4-Fc is shown in SEQ ID NO.5.
[0121] After purifying the VHH-IgG1-Fc expressed in HEK293 disclosed in CN107814845A (see Example 8 of CN107814845A) and VHH-IgG4-Fc, according to the above "(II) Appearance of Nanobody Samples and Detection of Visible Foreign Matter" The program was observed, and it was found that there were obvious floccules in the purified antibody samples. According to the procedure of "(3) CE-SDS detection of nanobody samples" above, it was found that split peaks were detected. The results are shown in ...
Embodiment 2
[0123] Example 2 Sequence Optimization Design and Preliminary Screening of Known Anti-PD-1 Nanobodies
[0124] In order to solve the defects of flocculent and poor homogeneity after the purification of PR antibody, the PR antibody was optimized, and the optimized design scheme is shown in Table 1.
[0125] Table 1. Optimization scheme for PR antibodies
[0126] optimized domain Number of optimizations (species) CDRs 2 FR 8 FR+CDR 12 FR+Hinge (hinge region) 1 FR+polypeptide (connecting peptide) 3 Configuration change (Fc+VHH, VHH attached to the C-terminus of Fc) 1
[0127]Note: The CDR in Table 1, the optimization number 2, refers to the design of two sequence optimization schemes for the CDR region. Similarly, 8 sequence optimization schemes were designed for the FR region; 8 sequence optimization schemes were designed for both FR and CDR regions; and 1 sequence optimization scheme was designed for both FR and Hinge (hing...
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