Trifunctional molecule and application thereof

A trifunctional molecule, selected from any one of the following:

• (1) the trifunctional molecule comprising a first functional domain capable of binding to CD19, a second functional domain capable of binding to and activating CD3 molecule on a surface of T cell, and a third functional domain capable of binding to and activating a T cell positive costimulatory molecule, wherein the first functional domain is an anti-CD19 scFv, the second functional domain is an anti-CD3 scFv, and the third functional domain is an scFv against T cell positive costimulatory molecule or the extracellular domain of a ligand of the T cell positive costimulatory molecule;
• (2) the trifunctional molecule comprising a first functional domain capable of binding to CD19, a second functional domain capable of binding to and activating CD3 molecule on the surface of T cell, and a third functional domain capable of binding to and blocking a T cell negative costimulatory molecule, wherein the first functional domain is an anti-CD19 scFv, the second functional domain is an anti-CD3 scFv, and the third functional domain is an scFv against T cell negative costimulatory molecule,
• wherein each of the scFvs comprises a heavy chain variable region and a light chain variable region;
• wherein the first functional domain and the second functional domain are connected by a first linker, and the second functional domain and the third functional domain are connected by a second linker;
• wherein the first linker is a linker fragment consisting of G4S units, and the amino acid sequence of the linker fragment consisting of the G4S units comprises any one of SEQ ID NO. 23, SEQ ID NO. 25 or SEQ ID NO. 27;
• and
• wherein the second linker is a hinge domain fragment of immunoglobulin IgD, and the amino acid sequence of the hinge domain fragment of immunoglobulin IgD comprises SEQ ID NO. 29.