Multi-Specific Molecules

Abstract

The present disclosure relates to multi-specific molecules which are capable of simultaneously binding at least two different target antigens or epitopes. The molecules comprise at least one binding domain molecule (BDM) which binds to a first target antigen or epitope, the BDM being modified for selective binding to a heterologous target, coupled to a pharmacologically active protein or peptide which is an antibody or antigen-binding fragment thereof or a non-antibody protein or peptide which binds to a second target antigen or epitope, the BDMs being coupled to a C-terminus of a polypeptide present within the pharmacologically active protein or peptide.

1 .- 43 . (canceled) 
     
     
         44 . A multi-specific molecule capable of binding to two or more different target antigens or epitopes, the molecule comprising:
 at least one binding domain molecule (BDM) which binds to a first target antigen or epitope, the BDM comprising a V-like domain (VLD) scaffold which comprises an extracellular portion of human CTLA-4 in soluble monomeric form and having three exposed binding loops (BLs) contained within and wherein at least one of the three BLs are modified or replaced relative to their corresponding native sequence in the scaffold for selective binding to a heterologous target antigen or epitope; and   (ii) a pharmacologically active protein or peptide which is an antibody or antigen-biding fragment thereof or a non-antibody protein or peptide which binds to a second target antigen or epitope;   wherein at least one BDM is coupled to a C-terminus of each heavy and/or light chain of the antibody or antigen binding fragment thereof, or is coupled to a C-terminus of each polypeptide chain of the non-antibody protein or peptide.   
     
     
         45 . The molecule according to  claim 44 , wherein at least one BDM is coupled to a C-terminus of all antibody heavy and light chain polypeptides. 
     
     
         46 . The molecule according to  claim 44 , wherein the antibody is a full length antibody. 
     
     
         47 . The molecule according to  claim 44 , wherein the ratio of antibody chains to BDMs is 4:2 n  where n is a number between 1 and 5. 
     
     
         48 . The molecule according to  claim 44 , wherein the pharmacologically active protein is an antigen-binding fragment is selected from the group consisting of an Fab, Fab′, F(ab′) 2  or chemically linked F(ab′) 2 . 
     
     
         49 . The molecule according to  claim 48 , wherein the ratio of antigen-binding fragment chains to BDMs is 2:2 n  wherein n is a number between 0 and 5. 
     
     
         50 . The molecule according to  claim 44 , wherein at least one BDM is coupled to a C-terminus of the CH1, CH2 or CH3 domain of the heavy chain polypeptide. 
     
     
         51 . The molecule according to  claim 44 , wherein the pharmacologically active protein binds to its native target antigen or epitope. 
     
     
         52 . The molecule according to  claim 44 , wherein the first target antigen and the second target antigen are different. 
     
     
         53 . The molecule according to  claim 44 , wherein the first target epitope and the second target epitope are on the same or different antigens. 
     
     
         54 . The molecule according to  claim 44 , wherein the molecule is a bi-specific or a tri-specific. 
     
     
         55 . The molecule according to  claim 44 , wherein the molecule comprises a non-antibody protein or polypeptide and one, two, three, four, five or six BDMs. 
     
     
         56 . The molecule according to  claim 44 , wherein the molecule comprises an antibody or antigen-binding fragment thereof and one pair or two pairs of BDMs wherein the BDMs in the pair are identical. 
     
     
         57 . The molecule according to  claim 44 , wherein the at least one BDM is linked to one or more further BDMs. 
     
     
         58 . The molecule according to  claim 44 , wherein the molecule comprises least two BDMs, or at least one pair of BDMs, wherein each BDM (or BDM pair) binds to a different target antigen or epitope. 
     
     
         59 . The molecule according to  claim 44 , wherein each BDM binds to a target antigen or epitope that is different from the target antigen epitope to which the pharmacologically active protein or polypeptide binds. 
     
     
         60 . The molecule according to  claim 44 , wherein the non-antibody protein or peptide is selected from the group consisting of a blood clotting factor, an anticalin, a toxoid, a collagen binding protein, a human serum albumin (HSA), a TNF-alpha receptor binding protein, an integrin binding protein, a VEGF or mimetic thereof, an EPO or mimetic thereof, a C4 binding protein, a urokinase receptor antagonist, a lymphokine, a cytokine, an osteoprotegerin (OPG) or the extracellular domain of a protein selected from programmed cell death 1 protein (PD1), programmed death ligand 1 (PD-L1), NKG2D, MHC class I polypeptide related sequence A (MICA), MHC class I polypeptide related sequence B (MICB), UL1 6 binding protein (ULBP). 
     
     
         61 . The molecule according to  claim 44 , wherein the VLD scaffold consists of a framework sequence corresponding to residues 1 to 25, 34 to 54, 60 to 97 and 106 to 126 of SEQ ID NO:1 set forth in: 
       
         
           
                 
               
                   KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVC 
                 
                   AATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMY 
                 
                   PPPYYLGIGNGTQIYVIDPEPSPDSN. 
                 
             
                
                
                
               
            
           
         
       
     
     
         62 . The molecule according to  claim 61 , wherein the VLD scaffold comprises a sequence having at least about 70% sequence identity to residues 1 to 25, 34 to 54, 60 to 97 and 106 to 126 of SEQ ID NO:1 
     
     
         63 . The molecule according to  claim 61 , wherein the amino acid residues at positions 26 to 33, and/or positions 55 to 59 and/or positions 98 to 105 of SEQ ID NO:1 are modified or replaced with heterologous sequence. 
     
     
         64 . The molecule according to  claim 44 , wherein the BDM comprises or consists of the sequence set forth in: 
       
         
           
                 
               
                   (SEQ ID NO: 5) 
                 
                   KAMHVAQPAVVLASSRGIASFVCEY Xn1 VRVTVLRQADSQVTEVCAATY 
                 
                     Xn2 LTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKV Xn3 LGIGNGT 
                 
                   QIYVIDPEPSPDSN 
                 
             
                
                
                
                
               
            
           
         
         wherein X, X and X is any amino acid residue and n is a number between 5 and 15 and 1, 2, 3 indicate BLs 1, 2 and 3 respectively. 
       
     
     
         65 . The molecule according to  claim 64 , wherein Xn 1  is between 5 and 8 amino acids, Xn2 is between 5 and 8 amino acids, and Xn 3  is between 10 and 15 amino acids. 
     
     
         66 . The molecule according to  claim 64 , wherein:
 (i) Xn 1  is 8 amino acids;   (ii) Xn 2  is 5 amino acids; and   (iii) Xn 3  is between 10 and 15 amino acids.   
     
     
         67 . The molecule according to  claim 44 , wherein the at least one BDM is present as a monomer, dimer or a series of BDM monomers linked together. 
     
     
         68 . The molecule according to  claim 44 , wherein coupling of the at least one BDM and the pharmacologically active protein or peptide is by means of a linker, by direct fusion, by conjugation or by covalent or non-covalent bonding. 
     
     
         69 . The molecule according to  claim 68 , wherein the linker is a Gly-Ser peptide linker. 
     
     
         70 . The molecule according to  claim 44 , wherein the molecule binds selectively to cells that express two or more different target antigens or epitopes recognised by the individual BDM and pharmaceutically active protein moieties of the molecule but not to cells that express only one of the target antigens or epitopes. 
     
     
         71 . A polypeptide is selected from the group consisting of SEQ ID NOs: 19, 21, 22, 23, 25-28 or 29. 
     
     
         72 . A pharmaceutical composition comprising the molecule according to  claim 44 , together with a pharmaceutically acceptable carrier and/or excipient. 
     
     
         73 . The molecule according to  claim 44 , wherein the molecule is labelled with an agent to facilitate detection.