Anti-HER3 antibody that binds to her3β-hairpin
An antibody and immunoconjugate technology, applied in the direction of antibodies, antibody medical components, anti-blood group antigen immunoglobulin, etc., can solve problems such as inaccessibility
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[0427] 1. An isolated antibody binding to human HER3,
[0428] where the antibody contains
[0429] a) Variable heavy domain VH (VH-A) having the amino acid sequence of SEQ ID NO:33.
[0430] 2. An isolated antibody that binds to human HER3,
[0431] where the antibody contains
[0432] a) a variable heavy chain domain VH (VH-A) having the amino acid sequence of SEQ ID NO: 33; and
[0433] b) a variable light domain VL (VL-D) having the amino acid sequence of SEQ ID NO:41, a variable light domain VL (VL-B) having the amino acid sequence of SEQ ID NO:39, or having the amino acid sequence of SEQ ID NO :42 variable light domain VL (VL-E).
[0434] 3. An isolated antibody that binds to human HER3,
[0435] where the antibody contains
[0436] a) a variable heavy chain domain VH (VH-A) having the amino acid sequence of SEQ ID NO: 33; and
[0437] b) Variable light domain VL (VL-D) having the amino acid sequence of SEQ ID NO:41.
[0438] 4. An isolate...
Embodiment 1
[0515] Antigen and Screening Protein Preparation - Generation of functional β-hairpin HER3 and β-hairpin HER4 constructs for selection of antibodies that bind HER3 β-hairpin and HER4 β-hairpin
[0516] To generate functional β-hairpin HER3 and HER4 constructs, the amino acid sequences of the β-hairpins of HER3 (SEQ ID NO: 1 ) and HER4 (SEQ ID NO: 2) were grafted into the SlyD polypeptide framework comprising the FKBP domain. In such constructs, the grafted β-hairpin is freely accessible, in contrast to the hidden structure in the native unactivated conformation of HER3 or HER4 (in the absence of a ligand such as HRG) (see figure 1 c and 1d, where the β-hairpin of HER3 is hidden).
[0517] All fusion SlyD polypeptides can be purified and refolded using almost the same protocol. Escherichia coli BL21(DE3) cells transformed with specific expression plasmids were grown at 37°C in LB medium containing corresponding antibiotics for selective growth (kanamycin 30 μg / ml, or am...
Embodiment 2
[0528] a) Immunization and selection of HER3 antibody
[0529] To generate antibodies against HER3 and HER4 β-hairpins, Balb / C, NMRI or SJL mice were immunized with different antigens. The following proteins were used as antigens: full-length HER3 ECD, or epitope scaffold proteins TtSlyD-FKBP12-Her3, TtSlyDcys-Her3, TtSlyDcas-Her3, TgSlyDcys-Her3 and TgSlyDser-Her3. The TtSlyD-FKBP12-Her3 variant represents the first epitope scaffold for generating HER3 dimerization domain-specific antibodies. Although the general rationale for using SlyD variants as epitope scaffolds could have been demonstrated earlier using the first generation SlyD-FKBP12 scaffolds, improved scaffold variants with higher stability were developed. These SlyD variants were derived from Thermus thermophilus and Thermus gamma-resistant.
[0530] All mice were immunized three times at time points 0, 6 and 10 weeks after the start of the immunization campaign. At each time point each mouse was immunized ...
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