Coiled coil and/or tether containing protein complexes and uses thereof

Abstract

The invention provides engineered protein complexes constructed using a coiled coil and/or a tether and methods for making, using, and purifying such complexes, such as multispecific antibodies or other multispecific Fc containing complexes.

1 . An antibody comprising:
 (a) a first polypeptide comprising a VH domain and a first coiled coil domain (CC), wherein the first CC comprises a heptad repeat of Formula I:   
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 29) 
                 
                     
                   (Xi X 2  X 3  X 4  X 5  X 6  X 7 ) n  (Formula I) 
                 
             
                
                
               
            
           
         
         Xi is a hydrophobic amino acid residue or Asparagine, 
         X 2 , X 3 , and X 6  are each any amino acid residue, 
         X 4  is a hydrophobic amino acid residue, and 
         X 5  and X 7  are each a charged amino acid residue; and 
         (b) a second polypeptide comprising a VH domain and a second coiled coil domain (CC), wherein the second CC comprises a heptad repeat of Formula II: 
       
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 30) 
                 
                     
                   X′, X′ 2  X′ 3  X′ 4  X′s X′ 6  X′ 7 ) n  (Formula II) 
                 
             
                
                
               
            
           
         
         X′ i is a hydrophobic amino acid residue or Asparagine, 
         X′ 2 , X′ 3 , and X′ 6  are each any amino acid residue, 
         X′ 4  is a hydrophobic amino acid residue, and 
         X′ 5  and X′ 7  are each a charged amino acid residue; 
         wherein n in Formula I and II is greater than or equal to 2; and 
         wherein, in each heptad repeat, the first CC comprises an X 5  residue that is opposite in charge to the X′ 7  residue in the second CC and the first CC comprises an X 7  residue that is opposite in charge to the X′ 5 residue in the second CC. 
       
     
     
         2 . The antibody of  claim 1 , wherein the first and second polypeptides each comprise a VH and a CH 1 domain. 
     
     
         3 . The antibody of  claim 2 , wherein the first and second polypeptides each further comprise a hinge domain. 
     
     
         4 . The antibody of  claim 1 , wherein said first and second polypeptides each further comprise a CH2 and a CH3 domain. 
     
     
         5 . The antibody of  claim 1 , wherein the first and second polypeptides each comprise VH, CHI, hinge, CH2, and CH3 domains positioned relative to each other in an N-terminal to C-terminal direction: VH-CH1-hinge-CH2-CH3. 
     
     
         6 . The antibody of  claim 1 , wherein said antibody further comprises a third and a fourth polypeptide, wherein said third polypeptide comprises a first VL domain and said fourth polypeptide comprises a second VL domain. 
     
     
         7 . The antibody of  claim 6 , wherein said VH domain of the first polypeptide is linked to the VL domain of the third polypeptide by a tether and the VH domain of the second polypeptide is linked to the VL domain of the fourth polypeptide by a tether. 
     
     
         8 . The antibody of  claim 6 , wherein the third polypeptide further comprises a first CL domain wherein said first VL and CL domains are positioned relative to each other within the third polypeptide in an N-terminal to C-terminal direction: VL-CL, and the fourth polypeptide further comprises a second CL domain, and wherein said second VL and CL domains are positioned relative to each other within the fourth polypeptide in an N-terminal to C-terminal direction: VL-CL. 
     
     
         9 . The antibody of  claim 1 , wherein the sequences of said first VL domain and said second VL domain are the same. 
     
     
         10 . The antibody of  claim 1 , wherein the N-terminus of the VH of at least one of said first or said second polypeptides is connected to the C-terminus of a CL with a tether. 
     
     
         11 . An antibody comprising:
 (a) a first polypeptide comprising a VH domain and a first coiled coil domain (CC), wherein the first CC comprises a heptad repeat of Formula I:   
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 29) 
                 
                     
                   (X, X 2  X 3  X 4  X 5  X 6  X 7 ) n  (Formula I) 
                 
             
                
                
               
            
           
         
         Xi is a hydrophobic amino acid residue or Asparagine, 
         X 2 , X 3 , and X are each any amino acid residue, 
         X 4  is a hydrophobic amino acid residue, and 
         X 5  and X 7  are each a charged amino acid residue; and 
         (b) a second polypeptide comprising a CH2 and CH3 domain and a second coiled coil (CC), wherein the second CC comprises a heptad repeat of Formula II: 
       
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 30) 
                 
                     
                   (X′, X′ 2  X′ 3  X′ 4  X′s X′ 6  X′ 7 ) n  (Formula II) 
                 
             
                
                
               
            
           
         
         X′ I is a hydrophobic amino acid residue or Asparagine, 
         X′ 2 , X′ 3 , and X′ 6  are each any amino acid residue, X′4 is a hydrophobic amino acid residue, and 
         X′ 5  and X′ 7  are each a charged amino acid residue; 
         wherein n in Formula I and II is greater than or equal to 2; and 
         wherein, in each heptad repeat, the first CC comprises an X 5  residue that is opposite in charge to the X′ 7  residue in the second CC and the first CC comprises an X 7  residue that is opposite in charge to the X′ 5  residue in the second CC. 
       
     
     
         12 . The antibody of  claim 11 , wherein the first polypeptide comprises a VH and CHI domain. 
     
     
         13 . The antibody of  claim 12 , wherein the first polypeptide further comprises a hinge domain. 
     
     
         14 . The antibody of  claim 12 , wherein the first polypeptide further comprises a CH2 and a CH3 domain. 
     
     
         15 . The antibody of  claim 11 , wherein the first polypeptide comprises VH, CH I, hinge, CH2, and CH3 domains positioned relative to each other in an N-terminal to C-terminal direction: VH-CH 1-hinge-CH2-CH3. 
     
     
         16 . The antibody of  claim 11 , wherein the antibody further comprises a third polypeptide, wherein the third polypeptide comprises a VL domain. 
     
     
         17 . The antibody of  claim 16 , wherein said third polypeptide further comprises a CL domain, and the VL and CL domains are positioned relative to each other in an N-terminal to C-terminal direction: VL-CL. 
     
     
         18 . The antibody of  claim 11 , wherein the N-terminus of the VH of said first polypeptide is connected to the C-terminus of a CL with a tether. 
     
     
         19 . The antibody of  claim 1 , wherein said hydrophobic amino acid residue in any of Xi, X′I, X4, and X′ 4  is selected from the group consisting of Alanine, Valine, Leucine, Isoleucine, Tryptophan, Phenylalanine, and Methionine. 
     
     
         20 . The antibody of  claim 1 , wherein said charged amino acid residue in any of X 5 , X′ 5)  X 7 , and X′ 7  is selected from the group consisting of Lysine, Arginine, Histidine, Aspartic Acid, and Glutamic Acid. 
     
     
         21 . The antibody of  claim 1 , wherein, in at least one heptad repeat of said first CC, Xi is Asparagine, and wherein the respective X′ 1 is Asparagine in at least one heptad repeat of said second CC. 
     
     
         22 . The antibody of  claim 1 , wherein
 (a) the first CC comprises a heptad repeat wherein   Xi is Leucine or Asparagine,   X 2  is Alanine or Glutamine,   X 3  is Alanine or Glutamine,   X 4  is Leucine,   X 5  is Glutamic Acid,   X is Lysine or Tryptophan, and   X 7  is Glutamic Acid; and   (b) the second CC comprises a heptad repeat wherein   X′ i is Leucine or Asparagine,   X′ 2  is Alanine or Glutamine,   X′ 3  is Alanine or Glutamine,   X′ 4  is Leucine,   X′ 5  is Lysine,   X′ 6  is Lysine or Tryptophan, and   X′ 7  is Lysine.   
     
     
         23 . The antibody of  claim 1 , wherein n is greater than or equal to 3. 
     
     
         24 . The antibody of  claim 23 , wherein n is greater than or equal to 4. 
     
     
         25 . The antibody of  claim 1 , wherein at least one of said first or said second CC is linked C-terminal to a constant domain of the antibody. 
     
     
         26 . The antibody of  claim 25 , wherein said constant domain is a CH3 domain and the first CC is linked C-terminal to a CH3 domain of the first polypeptide and the second CC is linked C-terminal to a CH3 domain of the second polypeptide. 
     
     
         27 . The antibody of  claim 25 , wherein linkage is by a cleavable linker sequence. 
     
     
         28 . The antibody of  claim 1 , wherein a Lys-C endopeptidase cleavage site is located N-terminal to at least one of said first or said second CC. 
     
     
         29 . An antibody comprising a first polypeptide comprising a VL, CL, tether, VH, CH1, CH2, and CH3 domain positioned relative to each other in an N-terminal to C-terminal direction: VL-CL-tether-VH-CH1-CH2-CH3 (Formula III). 
     
     
         30 . The antibody of  claim 29 , wherein said antibody further comprises a second polypeptide of Formula EL 
     
     
         31 . The antibody of  claim 1 , wherein the antibody is multispecific. 
     
     
         32 . The antibody of  claim 31 , wherein the antibody is capable of binding at least 2 antigens. 
     
     
         33 . The antibody of  claim 31 , wherein the antibody a capable of binding at least 2 epitopes on the same antigen. 
     
     
         34 . The antibody of  claim 1 , wherein said antibody is bispecific. 
     
     
         35 . The antibody of  claim 7 , wherein said tether comprises Glycine (G) and Serine (S) residues. 
     
     
         36 . The antibody of  claim 7 , wherein said tether is between 15 and 50 amino acids in length. 
     
     
         37 . The antibody of  claim 36 , wherein said tether is between 20 and 26 amino acids in length. 
     
     
         38 . The antibody of  claim 7 , wherein said tether comprises GGS repeats. 
     
     
         39 . The antibody of  claim 7 , wherein said tether is cleavable. 
     
     
         40 . The antibody of  claim 28 , wherein said antibody comprises a mutation that removes a Lys-C endopeptidase cleavage site. 
     
     
         41 . The antibody of  claim 40 , wherein said mutation that removes a Lys-C endopeptidase cleavage site is in a hinge domain. 
     
     
         42 . The antibody of  claim 41 , wherein said antibody has a K222A substitution (EU numbering system). 
     
     
         43 . The antibody of  claim 27 , wherein said tether or said linker is cleavable by one or more of the following endopeptidases: Furin, Thrombin, Genenase, Lys-C, Arg-C, Asp-N, Glu-C, Factor Xa, Tobacco Etch Virus Protease (TEV), Enterokinase, Human Rhinovirus C3 protease (HRV C3), and Kininogenase. 
     
     
         44 . The antibody of  claim 27 , wherein said tether or said linker comprises an Asparagine-Glycine peptide bond. 
     
     
         45 . The antibody of  claim 44 , wherein said Asparagine-Glycine peptide bond is cleavable by hydroxylamine. 
     
     
         46 . The antibody of  claim 1 , wherein said antibody comprises a constant region conjugated to a cytotoxic agent. 
     
     
         47 . The antibody of  claim 1 , wherein said antibody is expressed by a mammalian cell. 
     
     
         48 . The antibody of  claim 47 , wherein said mammalian cell is a CHO cell. 
     
     
         49 . The antibody of  claim 1 , wherein said antibody is expressed by a prokaryotic cell. 
     
     
         50 . The antibody of  claim 49 , wherein said prokaryotic cell is an  E. coli  cell. 
     
     
         51 . A method of producing an antibody, said method comprising the step of culturing a cell comprising a vector encoding the antibody of  claim 1  in a culture medium. 
     
     
         52 . The method of  claim 51 , wherein said method further comprises recovering said antibody from said cell or said culture medium. 
     
     
         53 . The method of  claim 52 , further comprising the steps of
 (a) capturing said antibody on a column comprising Protein A,   (b) eluting said antibody from said column, and   (c) diluting said eluted antibody into a solution containing a chaotropic agent or mild detergent.   
     
     
         54 . A method of maintaining a coiled coil containing antibody in solution, said method comprising maintaining said antibody in the presence of a chaotropic agent or mild detergent. 
     
     
         55 . The method of  claim 53 , wherein said chaotropic agent or mild detergent is Arginine, Guanidine-HCl, urea, lithium perchlorate, Histidine, Sodium Dodecyl Sulfate (SDS), Tween, Triton, or NP-40.