bispecific fusion protein
A technology of fusion protein and binding specificity, which is applied in the field of pharmaceutical composition containing this fusion protein and bispecific fusion protein
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Embodiment 1
[0451] Example 1. Preparation of Representative Bispecific Fusion Proteins
[0452] A bispecific fusion protein is made in which the targeting polypeptide domain binds DNA and the activation domain is NRG1. The two domains are connected by a modified human serum albumin (HSA) linker. NRG1 was recombinantly fused amino-terminally to an HSA linker incorporating a short linker polypeptide and the anti-DNA scFv was recombinantly fused carboxy-terminally to a modified HSA linker incorporating an additional short linker polypeptide. The modified HSA linker contains 2 amino acid substitutions. The cysteine residue at position 34 of native HSA was mutated to serine to reduce potential protein heterogeneity due to oxidation at this site. Pharmacological half-life is reduced by asparagine at position 503 of native HSA, which may be sensitive to deamidation, which is mutated to glutamine. The modified HSA linker confers an extended circulating half-life to the bispecific fusion prot...
Embodiment 2
[0453] Example 2. In vitro activity of bispecific fusion proteins
[0454] The activities of the two components of the representative bispecific fusion protein prepared in Example 1 (wherein the targeting polypeptide domain binds DNA and the activation domain is NRG1) were tested by ELISA, and the ELISA was designed so that only when the Activity occurs when the 2 arms of the bispecific fusion protein simultaneously bind their substrate. ELISA was performed essentially as described by Stokes et al., J. Clin. Pathol. 35(5):566-573 (1982) and Gripenberg et al., Scand. J. Immunol. 1:151-157 (1978). More specifically, a PBS solution of 1-50 ng / ml bispecific fusion protein was added to the plate wells of pre-adsorbed DNA (anti-DS-DNA antibody ELISA kit (Alpha Diagnostic International International), AutogenBioclear, UK (Dist by AutogenBioclear, UK))) and incubated and washed according to the manufacturer's guidelines until the step of adding detection antibody. At this stage, 100...
Embodiment 3
[0456] Example 3. In vivo activity of bispecific fusion proteins
[0457] The in vivo activity of the representative bispecific fusion protein prepared in Example 1 was determined by detecting the signal change in the module regulated by the activation domain of the fusion protein. For the activation domain of this fusion protein NRG1, activity was assessed by detecting an increase in phosphorylated ErbB-3 in heart cells treated with the bispecific fusion compared to untreated or mock-treated hearts. Myocardial infarction was generated in C57BL / 6 mice by ligation of the left coronary artery (LCA) after tracheal intubation, ventilation and thoracotomy. Coronary occlusion was confirmed by acute examination of left ventricular wall discoloration, ST evaluation of ECG before chest closure. Sham-operated mice underwent the same surgical procedure without LCA ligation.
[0458] Hearts from normal mice or mice after induction of myocardial infarction, mice treated with control and ...
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