Mutant antibodies and conjugation thereof

Abstract

The present invention relates to a polypeptide comprising 7 β-strands A, B, C, D, E, F, and G sequentially connected together by connecting chains of amino acids, and a first α-helix sequentially located on the EF chain between β-strands E and F, wherein the β-strands are arranged so as to form a first β-sheet comprising β-strands A, B, D, and E, and a second β-sheet comprising β-strands C, F and G, said first and second β-sheets being covalently bonded together so as to form a first Ig domain; wherein the EF chain between β-strands E and F comprises the sequence X 1 -X 2 -X 3 -X 4 -K 5 H 6 (SEQ ID NO:98), and X 1 , X 3 and X 4 are each independently any amino acid residue, characterized in that X 2 is selected from the group consisting of A, G, I, V, L, R, S, T, Q, P, N, M, H, W, and pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, and prodrugs thereof

1 . A polypeptide comprising an immunoglobulin domain comprising 7 β-strands A, B, C, D, E, F, and G sequentially connected together by chains of connecting amino acids,
 wherein the β-strands are arranged so as to form a first β-sheet comprising β-strands A, B, D, and E, and a second β-sheet comprising β-strands C, F and G, said first and second β-sheets being covalently bonded together; 
 wherein β-strands E and F are connected together by an EF chain, and said EF chain comprises the sequence, 
 X 1 -X 2 -X 3 -X 4 -K 5− H 6  (SEQ ID NO:98), and wherein X 1 , X 3  and X 4  are each independently any amino acid residue, and 
 characterized in that X 2  is selected from the group consisting of A, G, I, V, L, R, S, T, Q, P, N, M, H, W, and 
 pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, and prodrugs thereof. 
 
     
     
         2 . The polypeptide as claimed in  claim 1 , wherein the EF chain comprises a sequence selected from the group consisting of SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, and SEQ ID NO:224. 
     
     
         3 . The polypeptide as claimed in  claim 1 , wherein the EF chain is between 6 and 12 residues long. 
     
     
         4 . The polypeptide as claimed in  claim 1 , wherein the EF chain comprises an α-helix. 
     
     
         5 . The polypeptide as claimed in  claim 4 , wherein the α-helix on the EF chain comprises residues X 1  and X 2 . 
     
     
         6 . The polypeptide as claimed in  claim 5 , wherein β-strands C and D are connected together by a CD chain, comprising a CD motif selected from the group consisting of SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:248, SEQ ID NO:249, SEQ ID NO:250, SEQ ID NO:251, SEQ ID NO:252, and SEQ ID NO:253, said CD motif beginning at the first or second residue of said CD chain. 
     
     
         7 . The polypeptide as claimed in  claim 6 , wherein the CD motif is SEQ ID NO:251. 
     
     
         8 . The polypeptide as claimed in  claim 6 , wherein the CD motif does not form part of an α-helix. 
     
     
         9 . A polypeptide comprising an antibody constant domain comprising the residues K and H at positions corresponding to positions 80 and 81 of SEQ ID NO:6 when said antibody constant domain is aligned with the sequence of SEQ ID NO:6, and characterized in that the antibody constant domain further comprises a residue selected from the group consisting of A, G, I, V, L, R, S, T, Q, P, N, M, H, W at a position corresponding to position 77 of SEQ ID NO:6, pharmaceutically acceptable salts, stereoisomers, tautomers, solvates, and prodrugs thereof. 
     
     
         10 . A polypeptide as claimed in  claim 9 , further comprising a residue selected from the group consisting of V, I and L at a position corresponding to residue 42 of SEQ ID NO:6. 
     
     
         11 . A polypeptide as claimed in  claim 9 , further comprising a residue selected from the group consisting of D, E, N, and Q at a position corresponding to residue 43 of SEQ ID NO:6. 
     
     
         12 . A polypeptide as claimed in  claim 9 , wherein the residues at positions corresponding to residues 42 and 43 of SEQ ID NO:6 are not in α-helical formation. 
     
     
         13 . The polypeptide as claimed in  claim 1 , wherein the polypeptide is a mammalian antibody constant domain. 
     
     
         14 . The polypeptide as claimed in  claim 13 , wherein the constant domain is connected to an antibody variable domain. 
     
     
         15 . The polypeptide as claimed in  claim 1 , wherein the ε-amino group of the side chain of K 5  of SEQ ID NO:98 is covalently attached to a linker. 
     
     
         16 . The polypeptide as claimed in  claim 15 , wherein the linker comprises a formula selected from the group consisting of X 1 —Y—Z 1 , X 1 -Φ-Y 1 —Z 1 , and X 1 —Y 1 -Φ-Z, wherein Φ is a cleavable group, X 1  is a group covalently connectable to at least one Effector Moiety, Y 1  is a linear or branched connecting chain, and Z is a group covalently connected to the ε-amino group of the side chain of K 5  of SEQ ID NO:98. 
     
     
         17 . The polypeptide as claimed in  claim 16 , wherein the cleavable group Φ is present, and is of the formula 
       
         
           
           
               
               
           
         
       
       wherein the wavy line and parallel line each indicate a point of attachment to either the X 1 , Y 1  or Z 1  group as appropriate. 
     
     
         18 . The polypeptide as claimed in  claim 16 , wherein the linker is selected from the group consisting of: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         
           
           
               
               
           
         
         wherein m, n, j and k are each independently a range whose lower limits are selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and whose upper limit is selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30, and wherein the overall length of the linker does not exceed 200 atoms. 
       
     
     
         19 . The polypeptide as claimed in  claim 16 , wherein the Effector Moiety is a therapeutic agent, protein, peptide, nucleic acid, aptamer, small molecule, protein agonist, protein antagonist, metabolic regulator, hormone, toxin, growth factor, or diagnostic agent. 
     
     
         20 . The polypeptide as claimed in  claim 19 , wherein the Effector Moiety is a toxin, and comprises the formula: 
       
         
           
           
               
               
           
         
         or a pharmaceutically acceptable salt or solvate thereof, wherein, independently for each occurrence, 
       
       
         
           
           
               
               
           
         
       
       R 11  is 
       
         
           
           
               
               
           
         
       
       Y 2  is —C 2 -C 20  alkylene-, —C 2 -C 20  heteroalkylene-; —C 3 -C 8  carbocyclo-, -arylene-, —C 3 -C 8 heterocyclo-, —C 1 -C 10 alkylene-arylene-, -arylene-C 1 -C 10 alkylene-, —C 1 -C 10 alkylene-(C 3 -C 8  carbocyclo)-, -(C 3 -C 8 carbocyclo)-C 1 -C 10 alkylene-, —C 1 -C 10 alkylene-(C 3 -C 8 heterocyclo)- or —(C 3 -C 8  heterocyclo)-C 1 -C 10 alkylene-; 
       Z 2  is 
       
         
           
           
               
               
           
         
       
       R 12  is hydrogen, C 1 -C 8  alkyl or C 1 -C 8  haloalkyl; 
       R 13A  and R 13B  are either of the following:
 (iii) R 13A  is hydrogen, C 1 -C 8  alkyl, C 1 -C 8  haloalkyl, C 3 -C 8  carbocyclyl, C 1 -C 10  heterocyclyl, aryl, heteroaralkyl, aralkyl or halogen; and
 R 13B  is C 1 -C 8  alkyl, C 1 -C 8  haloalkyl, C 3 -C 8  carbocyclyl, C 1 -C 10  heterocyclyl, aryl, heteroaralkyl or aralkyl or halogen; or 
 
 (iv) R 13A  and R 13B  taken together are C 2 -C 8  alkylene or C 1 -C 8  heteroalkylene; R 14A  and R 14B  are either of the following: 
 (iii) R 14A  is hydrogen, C 1 -C 8  alkyl, C 1 -C 8  haloalkyl, C 3 -C 8  carbocyclyl, C 1 -C 10  heterocyclyl, aryl, heteroaralkyl or aralkyl; and
 R 14B  is hydrogen, C 1 -C 8  alkyl, C 1 -C 8  haloalkyl, C 3 -C 8  carbocyclyl, C 1 -C 10  heterocyclyl, aryl, heteroaralkyl or aralkyl; or 
 
 (iv) R 14A  and R 14B  taken together are C 2 -C 8  alkylene or C 1 -C 8  heteroalkylene; 
 
       R 15  is 
       
         
           
           
               
               
           
         
       
       C 1 -C 10  heterocyclyl, C 3 -C 8  carbocycly and C 6 -C 14  aryl optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of —C 1 -C 8  alkyl, —C 1 -C 8  alkyl-N(R′) 2 , —C 1 -C 8  alkyl-C(O)R′, —C 1 -C 8  alkyl-C(O)OR′—O—(C 1 -C 8  alkyl), —C(O)R′, —OC(O)R′, —C(O)OR′, —C(O)N(R′) 2 , —NHC(O)R′, —S(O) 2 R′, —S(O)R′, —OH, halogen, —N 3 , —N(R′) 2 , —CN, —NHC(═NH)NH 2 , —NHCONH 2 , —S(═O) 2 R′ and —SR′, wherein each R′ is independently selected from the group consisting of hydrogen, C 1 -C 8  alkyl and unsubstituted aryl, or two R′ can, together with the nitrogen to which they are attached, form a C 1 -C 10  heterocyclyl; 
       or R 15  is 
       
         
           
           
               
               
           
         
       
       optionally substituted with 1, 2, 3, 4 or 5 groups independently selected from the group consisting of C 1 -C 8  alkyl, —C 1 -C 8  alkyl-N(R′) 2 , —C 1 -C 8  alkyl-C(O)R′, —C 1 -C 8  alkyl-C(O)OR′, —O—(C 1 -C 8  alkyl), —C(O)R, —OC(O)R, —C(O)OR, —C(O)N(R′) 2 , —NHC(O)R, —S(O) 2 R′, —S(O)R′, —OH, halogen, —N 3 , —N(R′) 2 , —CN, —NHC(═NH)NH 2 , —NHCONH 2 , —S(═O) 2 R′, —SR′ and arylene-R′, wherein each R′ is independently selected from the group consisting of hydrogen, C 1 -C 8  alkyl, C 1 -C 8 heterocyclyl, C 1 -C 10 alkylene-C 3 -C 8 heterocyclyl and aryl, or two R′ can, together with the nitrogen to which they are attached, form a C 1 -C 10  heterocyclyl; 
       R 16  is hydrogen, —C 1 -C 8  alkyl, —C 2 -C 8  alkenyl, —C 2 -C 8  alkynyl or —C 1 -C 8  haloalkyl; 
       R 22  is hydrogen, C 1 -C 4  alkyl, C 1 -C 10  heterocyclyl or C 6 -C 14  aryl; 
       R 23  is C 1 -C 10  heterocyclyl; and 
       R 17  is independently selected for each occurrence from the group consisting of F, Cl, I and Br; 
       R 20  is -aryl, —C 1 -C 10 alkylene-aryl, where aryl on R 10  comprising aryl is substituted with [R 17 ] h ;
 h is 5; and 
 X is O or S; 
 provided that when R 13A  is hydrogen X is S. 
 
     
     
         21 . The polypeptide as claimed in  claim 19 , wherein the Effector Moiety is a toxin, and is selected from the group consisting of 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         22 . The polypeptide as claimed in  claim 1 , wherein the polypeptide comprises an antibody constant light (CL) domain. 
     
     
         23 . The polypeptide as claimed in  claim 22 , wherein the CL domain is a kappa domain (CLκ). 
     
     
         24 . The polypeptide as claimed in  claim 23 , wherein the CLκ comprises a sequence selected from the group consisting of SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, and SEQ ID NO:122. 
     
     
         25 . The polypeptide as claimed in  claim 23 , wherein the CLκ comprises an N-terminal portion defined by SEQ ID NO:225 and a C′ terminal portion defined by SEQ ID NO:226 contiguously connected together by an intermediate sequence selected from the group consisting of SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, and SEQ ID NO:224. 
     
     
         26 . The polypeptide as claimed in  claim 22 , wherein the CL domain is a lambda domain (CLλ), and comprises a sequence selected from the group consisting of SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:141, SEQ ID NO:144, SEQ ID NO:143, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, SEQ ID NO:242, SEQ ID NO:243, and SEQ ID NO:244. 
     
     
         27 . The polypeptide as claimed in  claim 22 , wherein the CL domain is a lambda domain (CLλ), and wherein the CLλ comprises an N′ terminal portion defined by one of SEQ ID NO:227, SEQ ID NO:228, SEQ ID NO:229, SEQ ID NO:230, SEQ ID NO:231, SEQ ID NO:232, or SEQ ID NO:233 contiguously connected together by an intermediate sequence to a C′ terminal portion defined by either of SEQ ID NO:234 or SEQ ID NO:235, the intermediate sequence being selected from the group consisting of SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:222, SEQ ID NO:223, and SEQ ID NO:224. 
     
     
         28 . The polypeptide as claimed in  claim 1 , comprising a sequence selected from the group consisting of SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:169, SEQ ID NO:236, SEQ ID NO:237, SEQ ID NO:238, SEQ ID NO:239, SEQ ID NO:240, SEQ ID NO:241, and SEQ ID NO:254, or a polypeptide at least about 85% identical thereof. 
     
     
         29 . A composition comprising the polypeptide as claimed in  claim 15 , wherein at least about 70% of the Linker in the composition or sample is conjugated to the ε-amino group of the side chain of K 5  of SEQ ID NO:98. 
     
     
         30 . A composition comprising multiples of the polypeptide as claimed in  claim 15 , wherein at least about 70% of the polypeptides comprises the Linker covalently attached to the ε-amino group of the side chain of K 5  of SEQ ID NO:98. 
     
     
         31 . An antibody, or antigen binding portion thereof, wherein the antibody comprises the polypeptide as claimed in  claim 1 . 
     
     
         32 . The antibody as claimed in  claim 31 , wherein the antibody comprises VH and VL domains from an antibody selected from the group consisting of h38C2, rituximab, cetuximab, infliximab, adalimumab, natalizumab, omalizumab, ranibizumab, trastuzumab and palivizumab. 
     
     
         33 . A pharmaceutical composition comprising the polypeptide as claimed in  claim 1 , and further comprising an acceptable carrier. 
     
     
         34 . A nucleic acid encoding the polypeptide as claimed in  claim 1 . 
     
     
         35 . A host cell comprising the nucleic acid as claimed in  claim 34 . 
     
     
         36 . A vector comprising the nucleic acid as claimed in  claim 35 . 
     
     
         37 . A method for preparing a multifunctional antibody conjugate (MAC) comprising a polypeptide as claimed in  claim 1 , comprising covalently attaching the Linker to a leaving group Z* of the formula: 
       
         
           
           
               
               
           
         
         where R 1  is F or Cl, h=2, 3, 4 or 5 and reacting the Linker-Z* group with the polypeptide.