Anti-HER3/HER4 antigen-binding protein that binds her3 β-hairpin and HER4 β-hairpin
A technology for binding proteins, HVR-H2, applied in the field of anti-HER3/HER4 antigen-binding proteins, which can solve problems that have not been thoroughly characterized
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Embodiment 3b
[0424] f) the antibody exhibits a ratio of binding constants (Ka) in the presence of heregulin (Ka(+heregulin)) to that in the absence of heregulin (Ka(-heregulin)) (Ka(+hegulin)) of 4.0 or greater protein) / Ka(-heregulin)) binds to HER3-ECD (see Example 3b); and
[0425] The antibody exhibits a ratio of binding constants (Ka) in the presence of heregulin (Ka (+ heregulin)) to that in the absence of heregulin (Ka (- heregulin)) (Ka (+ heregulin)) of 20.0 or higher. / Ka(-heregulin)) binds HER4-ECD (see Example 3b);
[0426] and / or
[0427] g) the antibody binds at a ratio of molar ratio (MR) of 2.0 or greater in the presence of heregulin (MR(+hergulin)) and in the absence of heregulin (MR(- heregulin)) (MR(+ Heregulin) / MR(-Heregulin)) binds HER3-ECD (see Example 3b); and
[0428] The antibody binds at a molar ratio (MR) ratio of 5.0 or greater in the presence of heregulin (MR (+ heregulin)) and in the absence of heregulin (MR (- heregulin)) (MR (+ heregulin) ) / MR(-Heregulin)...
Embodiment approach
[0768] 1. A method for selecting an antigen-binding protein that binds to human HER3 and binds to human HER4, wherein the antigen-binding protein is within the amino acid sequence PQPLVYNKLTFQLEPNPHT (SEQ ID NO: 1) of human HER3 and within the amino acid sequence PQTFVYNPTTFQLEHNFNA of human HER4 ( within SEQ ID NO:2),
[0769] Which uses
[0770] a) at least one polypeptide comprising the amino acid sequence of SEQ ID NO: 1 selected from the group consisting of:
[0771] SEQ ID NO: 13 TtSlyD-FKBP-Her3,
[0772] SEQ ID NO: 17 TtSlyDcas-Her3,
[0773] SEQ ID NO: 18 TtSlyDcys-Her3,
[0774] SEQ ID NO: 19 TgSlyDser-Her3, and
[0775] SEQ ID NO:20 TgSlyDcys-Her3
[0776] with
[0777] b) at least one polypeptide comprising the amino acid sequence of SEQ ID NO:2 selected from the group consisting of:
[0778] SEQ ID NO: 21 TtSlyDcas-Her4,
[0779] SEQ ID NO: 22 TtSlyDcys-Her4,
[0780] SEQ ID NO:23 TgSlyDser-Her4, and
[0781] SEQ ID NO: 24 TgS...
Embodiment 1
[0929] 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
[0930] 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).
[0931] 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...
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