Multimeric protein-albumin conjugate, preparation method therefor, and use thereof

A method for manufacturing a tetramer protein-albumin conjugate, the method comprises:

• reacting an albumin and a linker,
• wherein the linker comprises a dienophile functional group, an anchor, and a thiol reactive moiety,
• wherein the dienophile functional group is a trans-cyclooctene or a derivative of trans-cyclooctene,
• wherein the thiol reactive moiety is selected from a maleimide or a derivative of maleimide, and a 3-arylpropiolonitriles or a derivative of 3-arylpropiolonitriles,
• wherein the thiol reactive moiety of the linker is bound with thiol moiety of albumin through reaction to form an albumin-linker conjugate;
• and
• reacting the albumin-linker conjugate and a tetramer protein variant,
• wherein, the tetramer protein variant is a tetramer that four of variant subunits are oligomerized,
• wherein each of the variant subunit comprises one 4-(1,2,3,4-tetrazine-3-yl) phenylalanines (frTet), whereby the tetramer protein comprises four frTet,
• wherein the tetrazine functional group of the frTet and the is bound with the dienophile functional group of the linker through Inverse Electron Demand Diels-Alder (IEDDA) reaction to form a tetramer protein-albumin conjugate,
• wherein the tetramer protein-albumin conjugate is characterized in that three or more albumins are conjugated to the tetramer protein variant through the linkers.

A method for manufacturing a tetramer protein-albumin conjugate, the method comprises:

• disrupting a cell,
• wherein, the cell comprises a tetramer protein variant,
• wherein the tetramer protein variant comprises at least one 4-(1,2,3,4-tetrazine-3-yl) phenylalanines (frTet);
• adding an albumin-linker conjugate to the cell disruption product,
• wherein, the albumin-linker conjugate comprises a trans-cyclooctene or a derivative of trans-cyclooctene as a dienophile functional group,
• wherein the tetrazine functional group of the frTet and the is bound with the dienophile functional group of the linker through Inverse Electron Demand Diels-Alder (IEDDA) reaction to form a multimeric protein-albumin conjugate;
• and
• obtaining the multimeric protein-albumin conjugate.