Bispecific antibodies and uses thereof

A method of making bispecific antibody or antigen-binding fragment thereof, the method comprising:

• (a) selecting a first antigen and a second antigen, and identifying a first antibody or antigen-binding fragment thereof that binds to the first antigen and a second antibody or antigen-binding fragment thereof that binds to the second antigen, wherein the first antibody or antigen-binding fragment thereof comprises a first heavy chain variable region (VHa) and a first light chain variable region (VLa), and the second antibody or antigen-binding fragment thereof comprises a second heavy chain variable region (VHb) and a second light chain variable region (VLb);
• (b) determining the amino acid sequence of VHa, VLa, VHb, and VLb;
• (c) aligning the amino acid sequences of VLa and VLb and determining that the sequence homology between VLa and VLb is greater than 80%;
• (d) designing a common light chain variable region (VLc), wherein the VLc, when it associates with VHa, maintains the affinity to the first antigen;
• (e) redesigning the VHa and VHb sequences, thereby obtaining VHa′ and VHb′ to increase the difference of biochemical or biophysical characteristics between a first protein comprising two polypeptides each comprising VHa′ and two polypeptides each comprising VLc, and a second protein comprising two polypeptides each comprising VHb′ and two polypeptides each comprising VLc;
• and
• (f) producing a bispecific antibody or antigen-binding fragment thereof that has two light chain variable regions and two heavy chain variable regions, wherein the two light variable regions each comprises VLc, and the two heavy chain variable regions comprise VHa′ and VHb′ respectively.

A method of making bispecific antibody or antigen-binding fragment thereof, the method comprising:

• (a) selecting a first antigen and a second antigen, and identifying a first antibody or antigen-binding fragment thereof that binds to the first antigen and a second antibody or antigen-binding fragment thereof that binds to the second antigen, wherein the first antibody or antigen-binding fragment thereof comprises a first heavy chain variable region (VHa) and a first light chain variable region (VLa), and the second antibody or antigen-binding fragment thereof comprises a second heavy chain variable region (VHb) and a second light chain variable region (VLb);
• (b) determining the amino acid sequence of VHa, VLa, and VLb;
• (c) aligning the amino acid sequences of VLa and VLb and determining the sequence homology between VLa and VLb is less than 80%;
• (d) replacing all light chain variable regions in a phage display antibody library with the VLa, and panning against the second antigen to obtain a third heavy chain variable region (VHc);
• (e) redesigning the VHa and VHc sequences, thereby obtaining VHa′ and VHc′ to increase the difference of biochemical or biophysical characteristics between a first protein comprising two polypeptides each comprising VHa′ and two polypeptides each comprising VLa, and a second protein comprising two polypeptides each comprising VHc′ and two polypeptides each comprising VLa;
• and
• (f) producing a bispecific antibody or antigen-binding fragment thereof that has two light chain variable regions and two heavy chain variable regions, wherein the two light variable regions each comprises VLa, and the two heavy chain variable regions comprise VHa′ and VHc′ respectively.