Dual specificity polypeptide molecule

Abstract

The present invention relates to a bispecific polypeptide molecule comprising a first polypeptide chain and a second polypeptide chain providing a binding region derived from a T cell receptor (TCR) being specific for a major histocompatibility complex (MHC)-associated viral peptide epitope, and a binding region derived from an antibody capable of recruiting human immune effector cells by specifically binding to a surface antigen of said cells, as well as methods of making the bispecific polypeptide molecule, and uses thereof.

1 . A dual specificity polypeptide molecule selected from the group of molecules comprising a first polypeptide chain and a second polypeptide chain, wherein:
 the first polypeptide chain comprises a first binding region of a variable domain (VD1) of an antibody specifically binding to a cell surface antigen of a human immune effector cell, and   a first binding region of a variable domain (VR1) of a TCR specifically binding to an MHC-associated peptide epitope, and   a first linker (LINK1) connecting said domains;   the second polypeptide chain comprises a second binding region of a variable domain (VR2) of a TCR specifically binding to an MHC-associated peptide epitope, and   a second binding region of a variable domain (VD2) of an antibody specifically binding to a cell surface antigen of a human immune effector cell, and   a second linker (LINK2) connecting said domains;   wherein said first binding region (VD1) and said second binding region (VD2) associate to form a first binding site (VD1)(VD2) that binds a cell surface antigen of a human immune effector cell;   said first binding region (VR1) and said second binding region (VR2) associate to form a second binding site (VR1)(VR2) that binds said MHC-associated peptide epitope;   wherein said two polypeptide chains are fused to human IgG hinge domains and/or human IgG Fc domains or dimerizing portions thereof; and   wherein the said two polypeptide chains are connected by covalent and/or non-covalent bonds between said hinge domains and/or Fc-domains; and   wherein said dual specificity polypeptide molecule is capable of simultaneously binding the cell surface molecule and the MHC-associated peptide epitope, and dual specificity polypeptide molecules, wherein the order of the binding regions in the two polypeptide chains is selected from VD1-VR1 and VR2-VD2 or VD1-VR2 and VR1-VD2, or VD2-VR1 and VR2-VD1 or VD2-VR2 and VR1-VD1, and wherein the domains are either connected by LINK1 or LINK2.   
     
     
         2 . The dual specificity polypeptide molecule according to  claim 1 , wherein the order of the binding regions in the polypeptide chains is selected from VD1-VR1 and VD2-VR2; and wherein the domains are connected by LINK1 or LINK2, respectively. 
     
     
         3 . The dual specificity polypeptide molecule according to  claim 1 , wherein the linker-sequences LINK1 and/or LINK2 contain at least one sequence motif selected from GGGS, GGGGS, TVLRT, TVSSAS, and TVLSSAS. 
     
     
         4 . The dual specificity polypeptide molecule according to  claim 1 , wherein said first and second polypeptide chains further comprise at least a hinge domain and an Fc domain or portions thereof derived from human IgG1, IgG2 or IgG4. 
     
     
         5 . The dual specificity polypeptide molecule according to  claim 4 , wherein said Fc domain comprises at least one effector function silencing mutation at a residue selected from positions 233, 234, 235, 236, 297 and 331, optionally wherein said effector function silencing mutation is generated by replacing at least one residue in position 233, 234, 235, 236, and 331 with the corresponding residue derived from IgG2 or IgG4. 
     
     
         6 . The dual specificity polypeptide molecule according to  claim 3 , wherein said Fc domain comprises a CH3 domain comprising at least one mutation that facilitates the formation of heterodimers. 
     
     
         7 . The dual specificity polypeptide molecule according to  claim 6 , wherein said mutations are located at any position selected from 366, 368, 405, and 407, optionally, wherein said mutations comprise T366W and T366'S, L368A′ and Y407′V as knob-into-hole mutations. 
     
     
         8 . The dual specificity polypeptide molecule according to  claim 3 , wherein said Fc domain comprises CH2 and CH3 domain(s) comprising at least two additional cysteine residues, for example S354C and Y349C or L242C and K334C. 
     
     
         9 . The dual specificity polypeptide molecules according to  claim 1 , wherein said antibody-derived domains VD1 and VD2 display an engineered disulfide bridge introducing a covalent bond between VD1 and VD2 and where said cysteines are introduced into framework region (FR) 4 in case of VL and framework region 2 in case of VH. 
     
     
         10 . The dual specificity polypeptide molecule according to  claim 1 , wherein said cell surface molecule is known to induce the activation of immune cells, or is at least one selected from the group consisting of immune response-related molecules, CD3, such as the CD3γ, CD3δ, and CD3ε chains, CD4, CD7, CD8, CD10, CD11 b, CD11c, CD14, CD16, CD18, CD22, CD25, CD28, CD32a, CD32b, CD33, CD41, CD41b, CD42a, CD42b, CD44, CD45RA, CD49, CD55, CD56, CD61, CD64, CD68, CD94, CD90, CD117, CD123, CD125, CD134, CD137, CD152, CD163, CD193, CD203c, CD235a, CD278, CD279, CD287, Nkp46, NKG2D, GITR, FcεRI, TCRα/β and TCRγ/β, HLA-DR. 
     
     
         11 . The dual specificity polypeptide molecule according to  claim 1 , wherein the regions in the first polypeptide chain comprise SEQ ID NO: 28 for VD1, SEQ ID NO: 29 for VR1, SEQ ID NO: 30 for LINK1; and the regions in the second polypeptide chain comprise SEQ ID NO: 31 for VD2, SEQ ID NO: 32 for VR2, and SEQ ID NO: 30 for LINK2. 
     
     
         12 . The dual specificity polypeptide molecule according to  claim 3 , wherein the FC region in the first polypeptide chain comprises SEQ ID NO: 26 or SEQ ID NO: 47 (Fc1), and the FC region in the second polypeptide chain comprises SEQ ID NO: 27 or SEQ ID NO: 48 (Fc2). 
     
     
         13 . A dual specificity polypeptide molecule comprising a first polypeptide chain comprising SEQ ID NO: 16 or SEQ ID NO: 43 or SEQ ID NO: 45 or SEQ ID NO: 51, 53, 55, or 57, and a second polypeptide chain comprising SEQ ID NO: 17 or SEQ ID 44 or SEQ ID NO: 46 or SEQ ID NO: 52, 54, 56, or 58. 
     
     
         14 . The dual specificity polypeptide molecule according to  claim 1 , wherein said molecule carries a detectable label. 
     
     
         15 . The dual specificity polypeptide molecule according to  claim 1 , wherein said first binding site (VD1)(VD2) that binds the cell surface antigen of said immune cells is humanized; and/or said second binding site (VR1)(VR2) that binds said MHC-associated peptide epitope is maturated to achieve higher affinity and/or stability. 
     
     
         16 . A nucleic acid encoding for the first polypeptide chain and/or the second polypeptide chain according to  claim 1 , or an expression vector comprising at least one of said nucleic acids. 
     
     
         17 . A host cell comprising and optionally expressing a vector as defined in  claim 16 . 
     
     
         18 . A pharmaceutical composition comprising the dual specificity polypeptide molecule according to  claim 1  together with one or more pharmaceutically acceptable carriers or excipients. 
     
     
         19 . A method for treating a disease or disorder comprising administering a therapeutically effective amount of the dual specificity polypeptide molecule according to  claim 1  to a patient in need thereof. 
     
     
         20 . The method of  claim 19 , wherein the dual specificity polypeptide molecule comprising a first polypeptide chain comprising SEQ ID NO: 16 or SEQ ID NO: 43 or SEQ ID NO: 45 or SEQ ID NO: 51, 53, 55, or 57, and a second polypeptide chain comprising SEQ ID NO: 17 or SEQ ID 44 or SEQ ID NO: 46 or SEQ ID NO: 52, 54, 56, or 58.