Novel Anti-egfr antibody polypeptide
An antibody and heavy chain technology, applied in the direction of antibodies, peptides, specific peptides, etc.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0212] Example 1: Generation of Antigens, Antibodies and Cell Lines
[0213] 1. Preparation of human EGFR, human EGFRvIII, cynomolgus monkey EGFR and mouse EGFR-ECD proteins
[0214] The gene encoding human EGFR or EGFRvIII extracellular domain (ECD) was inserted into the expression vector pcDNA3.3 with 6-histidine (6xHis) tag or human Fc (hFc) and fused to the C-terminus of EGFR ECD. The gene encoding cynomolgus monkey EGFR (monkey EGFR) ECD was inserted into the expression vector pcDNA3.3 with human Fc (hFc) and fused to the C-terminus of EGFR ECD. The gene encoding mouse EGFR ECD was inserted into the expression vector pcDNA3.3 with human Fc (hFc) and fused to the C-terminus of EGFR ECD. The plasmid was transfected into HEK293 cells with PlasFect (Bioline). 6xHis-tagged proteins were purified from the collected supernatant using a Ni column followed by an ion exchange column. hFc fusion protein was purified with protein A column.
[0215] 2. Preparation of reference ant...
Embodiment 2
[0223] Example 2: Generation of VHH and VHH-Fc
[0224] 1. Immunity
[0225] In order to induce the humoral immune response of camelids to EGFR, the recombinant hFc-labeled human EGFR ECD protein is subcutaneously injected every 1 to 3 weeks, and the dose of each injection is 50ug to 200ug, a total of 6-8 doses.
[0226] 2. Serum titer detection
[0227] Using the EGFR-expressing CHO-K1 cell line and control CHO-K1 cells, the presence or absence of anti-EGFR antibodies in serum was detected by flow cytometry. The titers of anti-human epidermal growth factor receptor (EGFR) and anti-mouse EGFR were detected with W562-CHO-K1.hPro1.E9 and W562-CHO-K1.mPro1.F3 cell lines, respectively. cells at 1 x 10 5 Cells / well were plated in 96-well U-bottom plates (BD, Franklin Lakes, NJ, USA), incubated with serial dilutions of pre-immune or immune sera for 1 hour at 4°C, and then treated with FITC-conjugated goat antibody. Llama IgG antibody (Novas Bio, Littleton, CO, USA) was incubated...
Embodiment 3
[0243] Example 3: Identification in vitro
[0244] 1. Targeted FACS Binding Analysis
[0245] Cells transfected with EGFR and A431 cell line overexpressing EGFR were used to detect the binding of antibody to cell surface EGFR by FACS. Take 1x 10 5 Cells / well were plated in a 96-well plate and centrifuged at 210 g for 5 min at 4°C. After removing the supernatant, the cell pellet was resuspended with test purified antibody serially diluted in 1% BSA-PBS and incubated at 4°C for 1 hour, followed by immunoassay with FITC-conjugated goat anti-human IgG (Jackson Immunoassay). ) at 4°C for 1 hour. Rinse twice with 200 μl / well of 1% BSA-PBS between each step. Finally, cells were resuspended in 100 μl 1% BSA-PBS for flow cytometry analysis (Intellicyt). As shown in Figure 1, W5626-2C2-z22 and W5626-2C10-z5 interacted with cell surface human ( Figure 1A ),monkey( Figure 1B ) and mice ( Figure 1C ) EGFR binding. W5626-2C10.VHH and W5626-2C2.VHH (c-Myc and his-tagged VHH fragme...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More