Anti-Tumor Necrosis Factor-Alpha Agents And Uses Thereof

Abstract

The present invention relates to polypeptides comprising one or more antibody single domains, or antigen-binding fragments thereof, directed against Tumor Necrosis Factor-alpha, in particular, two light chain variable domains in dimeric form, where the dimer has high solubility. It also relates to methods of using anti-Tumor Necrosis Factor-alpha polypeptides in treating inflammatory disorders, including rheumatoid arthritis. Compositions and methods for enhancing therapeutic potential of anti-Tumor Necrosis Factor-alpha polypeptides are provided, including linking the polypeptide to an albumin-binding domain and/or de-immunizing the polypeptide, to provide therapeutic agents with good solubility, enhanced serum half-life, and/or reduced immunogenicity, while substantially maintaining the specific binding properties of the anti-Tumor Necrosis Factor-alpha polypeptides.

1 . A polypeptide comprising an antibody molecule that binds tumor necrosis factor-alpha (TNF-alpha), said molecule comprising either
 (i) three complementary determining regions of the amino acid sequence SEQ ID NO:1, or a de-immunized variant thereof, said de-immunized variant formed by elimination of at least one T H  epitope in at least one of said three complementary determining regions of the amino acid sequence SEQ ID NO:1; or   (ii) three complementary determining regions of the amino acid sequence SEQ ID NO:2, or a de-immunized variant thereof, said de-immunized variant formed by elimination of at least one T H  epitope in at least one of said three complementary determining regions of the amino acid sequence SEQ ID NO:2.   
     
     
         2 . The polypeptide according to  claim 1  wherein said antibody molecule comprises SEQ ID NO:1 (VL18) or SEQ ID NO:2 (VL11). 
     
     
         3 . The polypeptide according to  claim 1  wherein said antibody molecule antagonizes binding of human TNF-alpha to a TNF-alpha receptor. 
     
     
         4 . The polypeptide according to  claim 3  wherein said antibody molecule further cross-reacts with at least one other mammalian TNF-alpha, wherein said mammal is not a primate;
 wherein said mammal is a rat or a mouse; or 
 wherein said antibody molecule cross-reacts with TNF-alpha of at least two other mammals, said at least two other mammals being a rodent and a non-rodent species. 
 
     
     
         5 - 7 . (canceled) 
     
     
         8 . The polypeptide according to  claim 1 , wherein said de-immunized variant comprises at least one amino acid substitution selected from the group consisting of A51V in conjunction with L54E or A51V in conjunction with L54R and T91A, said substitutions referring to amino acid positions in SEQ ID NO:1. 
     
     
         9 . The polypeptide according to  claim 1 , wherein said de-immunized variant comprises at least one amino acid substitution selected from the group consisting of R31S, A51V in conjunction with L54E or A51V in conjunction with L54R, T91A, and A100S, said substitutions referring to amino acid positions in SEQ ID NO:2. 
     
     
         10 . The polypeptide according to  claim 1 , wherein said de-immunized variant comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs:19-23 (five VL18 variants), SEQ ID NOs: 24-28 (five VL11 variants), and a TNF-alpha-binding fragment. 
     
     
         11 . The polypeptide according to  claim 1 , further comprising an albumin-binding domain linked thereto; or
 further comprising an albumin-binding domain linked thereto as a fusion; or   further comprising an albumin-binding domain linked thereto as a fusion via a linker; or   further comprising an albumin-binding domain linked thereto as a fusion via a peptide linker comprising the amino acid sequence SEQ ID NO:29 (3L).   
     
     
         12 .- 14 . (canceled) 
     
     
         15 . The polypeptide according to  claim 11 , wherein said albumin-binding domain further is characterized according to at least one of (i)-(iii):
 (i) said albumin-binding domain enhances the half-life of said polypeptide by five fold;   (ii) said albumin-binding domain corresponds to an albumin-binding domain isolated from  S. zooepidemicus  Z5; and   (iii) said albumin-binding domain comprises the amino acid sequence SEQ ID NO:30 (PEP), or an albumin-binding fragment or de-immunized derivative thereof, said de-immunized derivative formed by elimination of at least one T H  epitope in SEQ ID NO:30.   
     
     
         16 - 18 . (canceled) 
     
     
         19 . The polypeptide according to  claim 15 , wherein said de-immunized derivative of the albumin-binding domain of SEQ ID NO:30 comprises at least one amino acid substitution selected from the group consisting of E12D, T29H-K35D, and A45D, said substitutions referring to amino acid positions in SEQ ID NO:30; or
 wherein said de-immunized derivative of the albumin-binding domain of SEQ ID NO: 30 comprises the amino acid sequence SEQ ID NO:31.   
     
     
         20 . (canceled) 
     
     
         21 . A polypeptide comprising
 (i) a first light chain variable domain, said first light chain variable domain comprising three complementary determining regions of the amino acid sequence SEQ ID NO:1 (VL18), or a TNF-alpha-binding fragment or a first de-immunized variant thereof, said first de-immunized variant formed by elimination of at least one T H  epitope in said first light chain variable domain; and   (ii) a second light chain variable domain, said second light chain variable domain comprising three complementary determining regions (CDRs) of at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1-18, or a TNF-alpha-binding fragment or a second de-immunized variant thereof, said second de-immunized variant formed by elimination of at least one T H  epitope in said second light chain variable domain; and   wherein said first and second light chain variable domains together form a dimer that binds tumor necrosis factor (TNF-alpha).   
     
     
         22 . The polypeptide according to  claim 21 , wherein said second light chain variable domain comprises three complimentary determining regions (CDRs) of the amino acid sequence SEQ ID NO:2 (VL11), or said TNF-alpha-binding fragment or said second de-immunized variant thereof. 
     
     
         23 . The polypeptide according to  claim 22  wherein said dimer comprises the amino acid sequence SEQ ID NO:32 (VL18-3L-VL11), or a TNF-alpha-binding fragment thereof. 
     
     
         24 . The polypeptide according to  claim 22 , wherein said first de-immunized variant comprises at least one amino acid substitution selected from the group consisting of A51V in conjunction with L54R or A51V in conjunction with L54E and T91A, said substitutions referring to amino acid positions in SEQ ID NO:1; and/or
 wherein said second de-immunized variant comprises at least one amino acid substitution selected from the group consisting of A51V in conjunction with L54R or A51V in conjunction with L54E, T91S, and A100S, said substitutions referring to amino acid positions in SEQ ID NO:2.   
     
     
         25 . The polypeptide according to  claim 24 , wherein said polypeptide comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 35-39 (VL18-3L-VL11 variants), or said TNF-alpha-binding fragment thereof. 
     
     
         26 . The polypeptide according to  claim 21 , further comprising an albumin-binding domain linked to at least one of said first or second light chain variable domains; or
 further comprising an albumin-binding domain linked to at least one of said first or second light chain variable domains as a fusion; or   further comprising an albumin-binding domain linked to at least one of said first or second light chain variable domains as a fusion via a linker; or   further comprising an albumin-binding domain linked to at least one of said first or second light chain variable domains as a fusion via a peptide linker comprising the amino acid sequence SEQ ID NO:29 (3L).   
     
     
         27 . The polypeptide according to  claim 26  wherein said albumin-binding domain further is characterized according to at least one of (i) to (iii):
 (i) said albumin-binding domain enhances half-life of said polypeptide by five fold; 
 (ii) said albumin-binding domain corresponds to an albumin-binding domain isolated from  S. zooepidemicus  Z5; and 
 (iii) said albumin-binding domain comprises the amino acid sequence SEQ ID NO:30 (PEP), or an albumin-binding fragment or de-immunized derivative thereof. 
 
     
     
         28 . The polypeptide according to  claim 27 , wherein said polypeptide comprises the amino acid sequence SEQ ID NO: 33 (VL18-3L-VL11-PEP) or said TNF-alpha-binding fragment thereof. 
     
     
         29 . (canceled) 
     
     
         30 . The polypeptide according to  claim 27 , wherein said polypeptide comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 34 and 40-44 (VL18-3L-VL11-PEP variants), or said TNF-alpha-binding fragment thereof. 
     
     
         31 . A method for treating and/or delaying a TNF-alpha-related condition in a subject suffering therefrom and/or pre-disposed thereto, said method comprising:
 administering to said subject an effective amount of the polypeptide according to  claim 1  or  21 .   
     
     
         32 . The method of  claim 31  wherein said TNF-alpha-related condition is an inflammatory disorder or rheumatoid arthritis. 
     
     
         33 . (canceled) 
     
     
         34 . The method of  claim 31  wherein said polypeptide is administered no more than twice a month or no more than once a month. 
     
     
         35 . (canceled) 
     
     
         36 . A pharmaceutical composition comprising an effective amount of the polypeptide according to  claim 1  or  21  and a pharmaceutically acceptable carrier. 
     
     
         37 . A nucleic acid comprising a nucleotide sequence encoding the polypeptide according to  claim 1  or  21 . 
     
     
         38 . A vector comprising the nucleic acid according to  claim 37 . 
     
     
         39 . A host cell comprising the vector according to  claim 38 . 
     
     
         40 . A method of making the polypeptide according to  claim 1  or  21  comprising:
 (i) providing a host cell comprising a vector encoding said polypeptide; 
 (ii) culturing said cell under conditions allowing expression of said polypeptide; and 
 (iii) recovering said polypeptide from said culture.