Nanobodies against tumor necrosis factor-alpha

Abstract

The present invention relates to improved Nanobodies™ against Tumor Necrosis Factor-alpha (TNF-alpha), as well as to polypeptides comprising or essentially consisting of one or more of such Nanobodies. The invention also relates to nucleic acids encoding such Nanobodies and polypeptides; to methods for preparing such Nanobodies and polypeptides; to host cells expressing or capable of expressing such Nanobodies or polypeptides; to compositions comprising such Nanobodies, polypeptides, nucleic acids or host cells; and to uses of such Nanobodies, such polypeptides, such nucleic acids, such host cells or such compositions, in particular for prophylactic, therapeutic or diagnostic purposes, such as the prophylactic, therapeutic or diagnostic purposes.

1 .- 111 . (canceled) 
     
     
         112 . A TNF-alpha epitope that comprises the following amino acid residues of a trimer of TNF-alpha: Gln at position 88 of monomer A; Lys at position 90 of monomer A and Glu at position 146 of monomer B, and
 optionally at least one, preferably two or more, more preferably 5 or more of the following amino acid residues: Gly at position 24 of monomer A, Gln at position 25 of monomer A, Thr at position 72 of monomer A, His at position 73 of monomer A, Val at position 74 of monomer A, Leu at position 75 of monomer A, Thr at position 77 of monomer A, Thr at position 79 of monomer A, Ile at position 83 of monomer A, Thr at position 89 of monomer A, Val at position 91 of monomer A, Asn at position 92 of monomer A, Ile at position 97 of monomer A, Arg at position 131 of monomer A, Glu at position 135 of monomer A, Ile at position 136 of monomer A, Asn at position 137 of monomer A, Arg at position 138 of monomer A, Pro at position 139 of monomer A, Asp at position 140 of monomer A, Pro at position 20 of monomer B, Arg at position 32 of monomer B, Lys at position 65 of monomer B, Lys at position 112 of monomer B, Tyr at position 115 of monomer B, Ala at position 145 of monomer B, and Ser at position 147 of monomer B.   
     
     
         113 . An isolated immunoglobulin molecule that binds an epitope on the trimer of TNF-alpha that comprises the following amino acid residues: Gln at position 88 of monomer A; Lys at position 90 of monomer A and Glu at position 146 of monomer B, and
 optionally at least one, preferably two or more, more preferably 5 or more of the following amino acid residues: Gly at position 24 of monomer A, Gln at position 25 of monomer A, Thr at position 72 of monomer A, His at position 73 of monomer A, Val at position 74 of monomer A, Leu at position 75 of monomer A, Thr at position 77 of monomer A, Thr at position 79 of monomer A, Ile at position 83 of monomer A, Thr at position 89 of monomer A, Val at position 91 of monomer A, Asn at position 92 of monomer A, Ile at position 97 of monomer A, Arg at position 131 of monomer A, Glu at position 135 of monomer A, Ile at position 136 of monomer A, Asn at position 137 of monomer A, Arg at position 138 of monomer A, Pro at position 139 of monomer A, Asp at position 140 of monomer A, Pro at position 20 of monomer B, Arg at position 32 of monomer B, Lys at position 65 of monomer B, Lys at position 112 of monomer B, Tyr at position 115 of monomer B, Ala at position 145 of monomer B, and Ser at position 147 of monomer B.   
     
     
         114 . The isolated immunoglobulin molecule according to  claim 113 , wherein the sequence of the immunoglobulin molecule is SEQ ID NO: 52 or SEQ ID NO: 96. 
     
     
         115 . A protein or polypeptide comprising two or more immunoglobulin molecules according to  claim 113 . 
     
     
         116 . The protein or polypeptide according to  claim 115 , wherein the sequence of the protein or polypeptide is SEQ ID NO: 417, SEQ ID NO: 419 or SEQ ID NO: 420. 
     
     
         117 . The isolated immunoglobulin molecule according to  claim 113  wherein the immunoglobulin molecule comprises three complementarity determining regions (CDR1 to CDR3 respectively), in which:
 a. CDR1 comprises the amino acid sequence DYWMY (SEQ ID NO: 164); or an amino acid sequence that has only 1 amino acid difference with the amino acid sequence DYWMY (SEQ ID NO: 164); 
 b. CDR2 comprises the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 232); or an amino acid sequence that has 2 or only 1 amino acid difference(s) with the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 232); and 
 c. CDR3 comprises the amino acid sequence SPSGFN (SEQ ID NO: 300); or an amino acid sequence that has only 1 amino acid difference with the amino acid sequence SPSGFN (SEQ ID NO: 300). 
 
     
     
         118 . The isolated immunoglobulin molecule according to  claim 117 , wherein CDR1 comprises the amino acid sequence DYWMY (SEQ ID NO: 164), CDR2 comprises the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 232), and CDR3 comprises the amino acid sequence SPSGFN (SEQ ID NO: 300). 
     
     
         119 . The isolated immunoglobulin molecule according to  claim 117 , wherein CDR1 consists of the amino acid sequence DYWMY (SEQ ID NO: 164), CDR2 consists of the amino acid sequence EINTNGLITKYPDSVKG (SEQ ID NO: 232), and CDR3 consists of the amino acid sequence SPSGFN (SEQ ID NO: 300). 
     
     
         120 . The isolated immunoglobulin molecule according to  claim 117 , further comprising 4 framework regions (FR1 to FR4). 
     
     
         121 . The protein or polypeptide according to  claim 115 , wherein each immunoglobulin molecule binds an epitope on TNF-alpha (and in particular of the TNF-alpha trimer) that lies in and/or forms part of the receptor binding site(s) of the TNF trimer, such that said polypeptide, upon binding to a TNF trimer, is capable of inhibiting or reducing the TNF receptor crosslinking that is mediated by said TNF trimer and/or the signal transduction that is mediated by such receptor crosslinking. 
     
     
         122 . The protein or polypeptide according to  claim 115 , wherein each immunoglobulin molecule binds an epitope on TNF-alpha (and in particular of the TNF-alpha trimer) that lies in and/or forms part of the receptor binding site(s) of the TNF trimer, such that said polypeptide is capable of intramolecular binding to at least two TNF receptor binding sites on a TNF trimer. 
     
     
         123 . The isolated immunoglobulin sequence according to  claim 113 , wherein said immunoglobulin molecule is chosen from the group consisting of heavy chain antibodies, conventional 4-chain antibodies, Nanobodies, VHH domains, VH/VL domains, Fab, scFv, F(ab′) 2 , VH domains, VL domains, single domain antibodies, diabodies, immunoglobulin variable domains, and parts, domains and fragments thereof. 
     
     
         124 . The protein or polypeptide according to  claim 115 , wherein the two immunoglobulin molecules are directly linked to each other. 
     
     
         125 . The protein of polypeptide according to  claim 115 , wherein the two immunoglobulin molecules are linked to each other via a linker. 
     
     
         126 . The protein or polypeptide according to  claim 125 , wherein the linker is an amino acid sequence. 
     
     
         127 . The protein or polypeptide according to  claim 126 , wherein the linker is an amino acid sequence that comprises at least 14 amino acids, more preferably at least 17 amino acids, such as about 20-40 amino acids. 
     
     
         128 . The protein or polypeptide according to  claim 126 , wherein the linker comprises glycine and serine residues. 
     
     
         129 . The protein or polypeptide according to  claim 115 , wherein the protein or polypeptide is pegylated. 
     
     
         130 . The protein or polypeptide according to  claim 115 , which further comprises at least one immunoglobulin molecule that binds human serum albumin. 
     
     
         131 . The protein or polypeptide according to  claim 130 , wherein the at least one immunoglobulin molecule that binds human serum albumin has the CDR sequences present in ALB 8 (SEQ ID NO: 102). 
     
     
         132 . An isolated nucleic acid comprising a nucleotide sequence that encodes an immunoglobulin molecule according to  claim 113 . 
     
     
         133 . An isolated host cell, comprising a nucleic acid according to  claim 132 . 
     
     
         134 . A pharmaceutical composition comprising at least one immunoglobulin molecule according to  claim 113 , and optionally at least one pharmaceutically acceptable carrier. 
     
     
         135 . A method of treating at least one condition mediated by TNF-alpha, selected from inflammation, rheumatoid arthritis, COPD, asthma, Crohn's disease, ulcerative colitis, inflammatory bowel syndrome, multiple sclerosis, Addison's disease, Autoimmune hepatitis, Autoimmune parotitis, Diabetes Type I, Epididymitis, Glomerulonephritis, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, Hemolytic anemia, Systemic lupus erythematosus, Male infertility, Multiple sclerosis, Myasthenia Gravis, Pemphigus, Psoriasis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Scleroderma, Sjogren's syndrome, Spondyloarthropathies, Thyroiditis, and Vasculitis; comprising administering an immunoglobulin molecule according to  claim 113  to a patient in need of such treatment.