Covalent multi-specific antibody

Abstract

Disclosed herein are novel bispecific and tri-specific antibodies with increased stability and use thereof for therapy.

1 . An engineered antibody, comprising:
 (i) a first polypeptide that from N-terminal to C-terminal comprises a second light chain variable domain (VL2) binding a second target and a first heavy chain variable domain (VH1) binding a first target, wherein the VL2 is linked to the VH1 via a linker;   (ii) a second polypeptide that from N-terminal to C-terminal comprises a first light chain variable domain (VL1) binding a first target and a second heavy chain variable domain (VH2) binding a second target, as well as a cysteine-containing hinge domain and a CH2—CH3 domain of IgG, wherein the VL1 is linked to the VH2 via a linker;   (iii) a third polypeptide that from N-terminal to C-terminal comprises a cysteine-containing hinge domain and a CH2—CH3 domain of IgG; wherein:
 VL1 and VH1 associate to form a domain capable of binding the first target which is CD3; 
 VL2 and VH2 associate to form a domain capable of binding the second target which is CD19; 
 VL2 is covalently linked to VH2 via a disulfide bond and VL2 and VH2 independently comprise one or more substitutions that introduce charged amino acids, which are electrostatically unfavorable to homodimer formation; 
 the cysteine-containing hinge domains of the second and the third polypeptides are covalently linked via a disulfide bond. 
   
     
     
         2 . The antibody of  claim 1 , wherein the amino acid sequence of VL1 is SEQ ID NO.:1, the amino acid sequence of VH1 is SEQ ID NO.:2, the amino acid sequence of VL2 is SEQ ID NO.:3 and the amino acid sequence of VH2 is SEQ ID NO.:4. 
     
     
         3 . The antibody of  claim 1 , wherein the amino acid sequence of the first polypeptide is SEQ ID NO.:5, the amino acid sequence of the second polypeptide is SEQ ID NO.:6 and the amino acid sequence of the third polypeptide is SEQ ID NO.:7. 
     
     
         4 . An engineered antibody, comprising:
 (i) a first polypeptide that from N-terminal to C-terminal comprises a second light chain variable domain (VL2) binding a second target and a first heavy chain variable domain (VH1) binding a first target, wherein the VL2 is linked to the VH1 via a linker;   (ii) a second polypeptide that from N-terminal to C-terminal comprises a first light chain variable domain (VL1) binding a first target and a second heavy chain variable domain (VH2) binding a second target, as well as a cysteine-containing hinge domain and a CH2—CH3 domain of IgG, wherein the VL1 is linked to the VH2 via a linker;   (iii) a third polypeptide that from N-terminal to C-terminal comprises a third heavy chain variable domain (VH3) binding a third target, CH1 domain of IgG, a cysteine-containing hinge domain and a CH2—CH3 domain of IgG, wherein the VH3 is linked to CH1 via a linker; and   (iv) a fourth polypeptide that from N-terminal to C-terminal comprises a third light chain variable domain (VL3) binding the third target, a cysteine-containing light chain constant domain (CL), wherein the VL3 is linked to CL via a linker; wherein:
 VL1 and VH1 associate to form a domain capable of binding the first target; 
 VL2 and VH2 associate to form a domain capable of binding the second target; 
 VL3 and VH3 associate to form a domain capable of binding the third target; 
 VL2 is covalently linked to VH2 via a disulfide bond and VL2 and VH2 independently comprise one or more substitutions that introduce charged amino acids, which are electrostatically unfavorable to homodimer formation; 
 CH1 is covalently linked to CL via a disulfide bond; 
 the cysteine-containing hinge domains of the second and the third polypeptides are covalently linked via a disulfide bond. 
   
     
     
         5 . The antibody of  claim 4 , wherein the first target is CD3, the second target is CD19 and the third target is CD20. 
     
     
         6 . The antibody of  claim 4 , wherein the first target is CD20, the second target is CD19 and the third target is CD3. 
     
     
         7 . The antibody of  claim 5 , wherein the amino acid sequence of VL1 is SEQ ID NO.:1, the amino acid sequence of VH1 is SEQ ID NO.:2, the amino acid sequence of VL2 is SEQ ID NO.:3, the amino acid sequence of VH2 is SEQ ID NO.:4, the amino acid sequence of VL3 is SEQ ID NO.:8, and the amino acid sequence of VH3 is SEQ ID NO.:9. 
     
     
         8 . The antibody of  claim 6 , wherein the amino acid sequence of VL1 is SEQ ID NO.:8, the amino acid sequence of VH1 is SEQ ID NO.:9, the amino acid sequence of VL2 is SEQ ID NO.:3, the amino acid sequence of VH2 is SEQ ID NO.:4, the amino acid sequence of VL3 is SEQ ID NO.:1, and the amino acid sequence of VH3 is SEQ ID NO.:2. 
     
     
         9 . The antibody of  claim 5 , wherein the amino acid sequence of the first polypeptide is SEQ ID NO.: 10, the amino acid sequence of the second polypeptide is SEQ ID NO.: 11, the amino acid sequence of the third polypeptide is SEQ ID NO.: 12 and the amino acid sequence of the fourth polypeptide is SEQ ID NO.: 13. 
     
     
         10 . The antibody of  claim 6 , wherein the amino acid sequence of the first polypeptide is SEQ ID NO.: 14, the amino acid sequence of the second polypeptide is SEQ ID NO.: 15, the amino acid sequence of the third polypeptide is SEQ ID NO.: 16 and the amino acid sequence of the fourth polypeptide is SEQ ID NO.: 17.