Anti-PD-1 Monoclonal Antibody and Its Application
A monoclonal antibody, PD-1 technology, applied in the fields of immunology and antibody engineering, can solve the problems of short serum half-life, side effects that increase the cost of treatment, the frequency and amount of drug administration, and the speed of antibody clearance that affects the efficacy of treatment.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1H8
[0055] Protein expression of embodiment 1H8L2 mutant
[0056]On the basis of the humanized antibody H2L2 (anti-PD-1 IgG antibody), the 254th, 308th, and 434th amino acids in the FcRn binding site region of the heavy chain constant region of the antibody were mutated to threonine and proline, respectively. and alanine, named H8L2 (anti-PD-1 IgG class antibody variant).
[0057] That is, the target antibody H8L2 mutant, compared to the humanized antibody H2L2, the 254th, 308th, and 434th amino acids in the FcRn binding site region of the heavy chain constant region of the antibody are mutated to threonine, proline, and alanine, respectively. Acid, the sequence of other regions remains unchanged.
[0058] In actual operation, the nucleic acid sequence encoding the humanized antibody H8L2 is synthesized from the whole gene and constructed into an expression vector. The expression vector DNA was extracted and transfected into 293 mammalian cells. After cell transfection, antibod...
Embodiment 2H8
[0072] Example 2 H8L2 mutant recombinant humanized antibody ELISA experiment
[0073] For the H2L2 antibody and the H8L2 antibody prepared in Example 1, a comparative study of ELISA binding experiment and competition ELISA experiment was carried out, as follows:
[0074] 1. 18A10H8L2, 18A10H2L2 ELISA binding experiment
[0075] Specific steps are as follows:
[0076] 1) Coating antigen: PD-1-his antigen 0.25 μg / ml, 100 μl / well, coated overnight at 4°C;
[0077] 2) Block with 1% BSA (diluted in PBS) at 37°C for 2 hours, wash with 1×PBST (Tween-20, 1%) for 3 times, and pat dry;
[0078] 3) Primary antibody: 2 μg / ml, 1:3 gradient dilution with 7 gradient concentrations, the blank control group is PBS, and incubated at 37°C for 1 hour;
[0079] 4) Secondary antibody: Wash 3 times with PBST, pat dry gently, add 1:10000 diluted HRP enzyme-labeled goat anti-human IgG (H+L) secondary antibody to 100 μl per well, and incubate at 37°C for 1 hour;
[0080] 5) Color development: wash ...
Embodiment 3
[0121] Example 3 Determination of Kinetic Parameters of H8L2 and H2L2 Using Fortebio Molecular Interaction Instrument
[0122] Use the Fortebio molecular interaction instrument to measure and compare the kinetic parameters of H8L2 (prepared in Example 1) and H2L2, specifically as follows:
[0123] The biotin-labeled antigen PD-1 was immobilized on the surface of the SA sensor. After equilibrating in PBST, it was combined with the antibody H8L2. H8L2 was three-fold diluted with PBST, and the concentration was 200, 66.67, 22.22, 7.41, 2.47, 0.82, 0.27, 0 nM, Dissociated in PBST. The detection method of H2L2 is the same as that of H8L2. H8L2, H2L2 Kinetic Parameter Results Figure 4 . Depend on Figure 4 It can be seen that the mutation on FcRN has no effect on the kinetic parameters of the antibody.
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More