Il-1beta inhibitor composition and use thereof
A kind of technology of composition, purposes, be used in the field of compositions derived from heterodimeric IL-1R1/IL-1RAcP-
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Embodiment 1
[0070] Example 1 : Construction of a plasmid for expression of the polypeptide of the present invention.
[0071] An optimized gene sequence comprising a sequence encoding residues 1 to 333 of amino acid residues of IL-1R1, or a sequence of residues 1 to 358 of IL-1RAcP was chemically synthesized. The resulting fragment was cloned in frame into an acceptor intermediate vector containing sequences encoding Fc-V or Fc-II via Hindlll and BspEI restriction sites. The obtained positive clones were verified by DNA sequencing. The final constructs were called PKN001'-IL-1R-FcV and PKN001'-RAcP-FcII, respectively.
[0072] The gene encoding IL-1R1-Fc-V was then cloned into a second intermediate vector called PKN002 via Hindlll and EcoRI restriction sites. Positive clones were screened by double restriction digestion, and the correct plasmid insert sequence was verified by DNA sequencing. The final construct was called PKN002-IL-1R-FcV.
[0073] Finally, the expression cassette f...
Embodiment 2
[0075] Example 2 : Generation of stable cell lines for expressing polypeptides of the invention.
[0076]Stable clones of CHO-K1 cells co-expressing the hIL1-R1-hlgG1-Fc polypeptide of SEQ ID NO.1 and the hIL-1RAcP-hlgG1-Fc polypeptide of SEQ ID NO.2 have been generated by standard cell biology protocols. The expression plasmid PKN012-IL1R-FcV-RAcP-FcII described in Example 1 was used to generate a stable cell line for high-efficiency expression of the polypeptide. The expression level of the polypeptide in multiple clones was about or exceeded 100 mg / L in 7-day batch culture. One clonal cell line showed expression levels of about or over 300 mg / L in shake flask batch culture.
[0077] Material
[0078] Chinese hamster ovary cells (CHO-K1) were obtained from ATCC (CCL-61 TM ) of frozen material. Cells were adapted in-house to CDCHO medium. Media and reagents were obtained from commercial sources. Based on complete adaptation, cells are grown to high density for several...
Embodiment 3
[0092] Example 3 : Purification of the polypeptide of the present invention.
[0093] The hIL1-Rl-hlgG1-Fc polypeptide of SEQ ID NO.1 and the hIL-1RAcP-hlgG1-Fc polypeptide of SEQ ID NO.2 were essentially co-expressed as CHO-K1, as described in Example 2 above. Cells were harvested and lysed using well-established protocols. After clarification of the cell lysate, the supernatant containing the expressed polypeptide of hlL1-R1-hlgG1-Fc / hIL-1RAcP-hlgG1-Fc at a protein concentration of about 0.4 mg / ml was applied to a Protein A affinity column (ProteinAffinitycolumn ). The affinity purification step was performed according to the procedure outlined in Table 2. Protein A eluates containing hIL1-Rl-hlgG1-Fc / hIL-1RAcP-hlgG1-Fc at a pH of approximately 3.5-3.7 were incubated for 45-60 minutes to potentially inactivate the presence of contaminating material.
[0094] After approximately 45 minutes of incubation of the material at room temperature, pH 3.6, its pH was adjusted to ...
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