Bivalent bispecific antibody hybrid protein expression and preparation methods

A method for expressing and preparing a bivalent bispecific antibody, the bivalent bispecific antibody comprising a first light chain and a first heavy chain of an antibody that specifically binds to a first antigen, and a second light chain and a second heavy chain of an antibody that specifically binds to a second antigen, the method comprising:

• S1:
• providing a first polynucleotide sequence and a second polynucleotide sequence, wherein the first polynucleotide sequence encodes a portion A antibody,
• wherein the second polynucleotide sequence encodes a portion B antibody, wherein the portion A antibody comprises the first light chain, the first heavy chain, an Fc chain of the second heavy chain, a part A of a hinge of the second heavy chain linked to the N-terminus of the Fc chain of the second heavy chain and a C-terminal fragment of a split intein (Ic) fused to the N terminus of then part A of the hinge of the second heavy chain, wherein the portion B antibody comprises the second light chain a VH+CH1 chain of the second heavy chain, a part B of the hinge of the second heavy chain linked to the C-terminus of the VH+CH1 chain of the second heavy chain, and a N-terminal fragment of the split intein (In) fused to the C terminus of the part B of the hinge of the second heavy chain, and
• S2:
• constructing a first mammalian cell expression vector and a second mammalian cell expression vector, wherein the first mammalian cell expression vector comprises the first polynucleotide sequence and is configured to express the portion A antibody, and the second mammalian cell expression vector comprises the second polynucleotide sequence and is configured to express the portion B antibody;
• S3:
• transfecting a first mammalian cell with the first mammalian expression vector, and inducing the first mammalian cell transfected with the first mammalian cell expression vector to express the portion A antibody;
• and transfecting a second mammalian cell with the second mammalian expression vector, and inducing the second mammalian cell transfected with the second mammalian cell expression vector to express the portion B antibody;
• and
• S4:
• purifying the expressed portion A antibody and the expressed portion B antibody respectively, and subjecting the Ic of the portion A antibody and the In of the portion B antibody to trans-splicing in vitro, to obtain the bivalent bispecific antibody,
• wherein
• the trans-splicing, in vitro, occurs at a temperature of 4-37° C., is continued for 5-20 min, and the concentration of the sulfhydryl compound is 0.05-2 mM and the split intein is Npu DNA E or Ssp DnaE,
• wherein the Fc chain of the second heavy chain in portion A antibody is fused to the VH+CH1 chain of the second heavy chain in portion B antibody by the Ic/In linkage of the part A of the hinge and the part B of the hinge of the second heavy chain, and
• wherein the part A of hinge is one portion of the hinge region of the antibody, the part B of hinge is another portion of said hinge region of the antibody, and part A and part B are fused to a whole of said hinge region after trans-splicing of the intein in S4.