Glycosylated immunoglobulin single variable domains

Abstract

The present invention relates to glycosylated immunoglobulin variable domains, and in particular to glycosylated immunoglobulin single variable domains (the latter also being referred to herein by means of the abbreviation “ISV” or “ISVD”). The present invention relates to glycosylated immunoglobulin heavy-chain variable domains (also referred to herein as “VH domains”), and in particular to glycosylated immunoglobulin heavy-chain ISVD's. The invention in particular relates to immunoglobulin (single) variable domains that are glycosylated in such a way that the binding of said immunoglobulin (single) variable domains by so-called “pre-existing antibodies” is prevented and/or reduced (i.e. partially or essentially completely) compared to the same immunoglobulin (single) variable domain without the glycosylation of the invention being present. For example, the present invention relates to heavy-chain immunoglobulin variable domain, which contains a glycosylation site such that the amino acid residue at one of positions 10, 11, 12, 13, 14, 39, 40, 41, 42, 87, 89, 108, 110, 112, 113 or 114, and in particular one of positions 11, 13, 87, 89, 108, 110, 112, 113 or 114 (numbering according to Kabat) is or can be glycosylated.

1 .- 37 . (canceled) 
     
     
         38 . A method for generating, expressing, producing and/or manufacturing a heavy-chain immunoglobulin single variable domain or a polypeptide comprising the same; wherein the heavy-chain immunoglobulin single variable domain consists of formula 1: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4 and CDR1 to CDR3 refer to complementarity determining regions 1 to 3; wherein the heavy-chain immunoglobulin single variable domain forms a single functional antigen binding site; said method including the step of introducing one or more codons that encode a glycosylation site into those part(s) of the nucleotide sequence that encodes the heavy-chain immunoglobulin single variable domains such that the amino acid residue at one of positions 10, 11, 12, 13, 14, 39, 40, 41, 42, 87, 89, 108, 110, 112, 113 or 114 of the heavy-chain immunoglobulin single variable domain can be glycosylated, wherein the numbering is according to Kabat numbering; and optionally expressing the nucleic acid obtained in a suitable host cell or host organism. 
     
     
         39 . The method of  claim 38 , wherein an N-glycosylation site is introduced such that the amino acid residue at one of positions 10, 11, 12, 13, 14, 39, 40, 41, 42, 87, 89, 108, 110, 112, 113 or 114 is an asparagine residue that can be N-glycosylated. 
     
     
         40 . The method of  claim 39 , wherein an N-glycosylation site is introduced such that the heavy-chain immunoglobulin single variable domain contains an NXT or NXS motif, in which X can be any amino acid, such that the asparagine (N) residue of the NXT/NXS motif is present at one of positions 10, 11, 12, 13, 14, 39, 40, 41, 42, 87, 89, 108, 110, 112, 113 or 114. 
     
     
         41 . The method of  claim 38 , wherein the glycosylation site is introduced such that the amino acid residue at one of positions 108, 110, 112, 113 or 114 can be glycosylated. 
     
     
         42 . The method of  claim 41 , wherein an N-glycosylation site is introduced such that the amino acid residue at one of positions 108, 110, 112, 113 or 114 is an asparagine residue that can be N-glycosylated. 
     
     
         43 . The method of  claim 42 , wherein an N-glycosylation site is introduced such that the heavy-chain immunoglobulin single variable domain contains an NXT or NXS motif, in which X can be any amino acid, such that the asparagine (N) residue of the NXT/NXS motif is present at one of positions 108, 110, 112, 113 or 114. 
     
     
         44 . The method of  claim 38 , wherein the glycosylation site is introduced such that the amino acid residue at position 108 can be glycosylated. 
     
     
         45 . The method of  claim 44 , wherein an N-glycosylation site is introduced such that the amino acid residue at position 108 is an asparagine residue that can be N-glycosylated. 
     
     
         46 . The method of  claim 45 , wherein an N-glycosylation site is introduced such that the heavy-chain immunoglobulin single variable domain contains an NXT or NXS motif, in which X can be any amino acid, such that the asparagine (N) residue of the NXT/NXS motif is present at position 108. 
     
     
         47 . The method of  claim 38 , including the step of expressing the nucleic acid obtained in a host cell or host organism that is capable of glycosylating at least one of the glycosylation sites that is encoded by said nucleic acid. 
     
     
         48 . The method of  claim 38 , wherein the heavy-chain immunoglobulin single variable domain is or is derived from a variable domain of a camelid heavy chain antibody (VHH), a humanized variable domain of a camelid heavy chain antibody (humanized VHH), a variable domain of a heavy chain of a human antibody (human VH), or a camelized variable domain of the heavy chain of a human antibody (camelized human VH). 
     
     
         49 . A method for generating, expressing, producing and/or manufacturing a polypeptide according to  claim 38 , wherein the heavy-chain immunoglobulin single variable domain is at the C-terminal end of the polypeptide. 
     
     
         50 . A method for preventing or reducing the binding by pre-existing antibodies to a heavy-chain immunoglobulin single variable domain or a polypeptide comprising the same, wherein the heavy-chain immunoglobulin single variable domain consists of formula 1: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4 and CDR1 to CDR3 refer to complementarity determining regions 1 to 3; wherein the heavy-chain immunoglobulin single variable domain forms a single functional antigen binding site; said method including the step of introducing at least one glycosylation site into those part(s) of the nucleotide sequence that encodes the heavy-chain immunoglobulin single variable domains such that the amino acid residue at one of positions 10, 11, 12, 13, 14, 39, 40, 41, 42, 87, 89, 108, 110, 112, 113 or 114 of the heavy-chain immunoglobulin single variable domain can be glycosylated, wherein the numbering is according to Kabat numbering. 
     
     
         51 . The method of  claim 50 , wherein an N-glycosylation site is introduced such that the amino acid residue at one of positions 10, 11, 12, 13, 14, 39, 40, 41, 42, 87, 89, 108, 110, 112, 113 or 114 is an asparagine residue that can be N-glycosylated. 
     
     
         52 . The method of  claim 51 , wherein an N-glycosylation site is introduced such that the heavy-chain immunoglobulin single variable domain contains an NXT or NXS motif, in which X can be any amino acid, such that the asparagine (N) residue of the NXT/NXS motif is present at one of positions 10, 11, 12, 13, 14, 39, 40, 41, 42, 87, 89, 108, 110, 112, 113 or 114. 
     
     
         53 . The method of  claim 50 , wherein the glycosylation site is introduced such that the amino acid residue at one of positions 108, 110, 112, 113 or 114 can be glycosylated. 
     
     
         54 . The method of  claim 53 , wherein an N-glycosylation site is introduced such that the amino acid residue at one of positions 108, 110, 112, 113 or 114 is an asparagine residue that can be N-glycosylated. 
     
     
         55 . The method of  claim 54 , wherein an N-glycosylation site is introduced such that the heavy-chain immunoglobulin single variable domain contains an NXT or NXS motif, in which X can be any amino acid, such that the asparagine (N) residue of the NXT/NXS motif is present at one of positions 108, 110, 112, 113 or 114. 
     
     
         56 . The method of  claim 50 , wherein the glycosylation site is introduced such that the amino acid residue at position 108 can be glycosylated. 
     
     
         57 . The method of  claim 56 , wherein an N-glycosylation site is introduced such that the amino acid residue at position 108 is an asparagine residue that can be N-glycosylated. 
     
     
         58 . The method of  claim 57 , wherein an N-glycosylation site is introduced such that the heavy-chain immunoglobulin single variable domain contains an NXT or NXS motif, in which X can be any amino acid, such that the asparagine (N) residue of the NXT/NXS motif is present at position 108. 
     
     
         59 . The method of  claim 50 , wherein the heavy-chain immunoglobulin single variable domain is or is derived from a variable domain of a camelid heavy chain antibody (VHH), a humanized variable domain of a camelid heavy chain antibody (humanized VHH), a variable domain of a heavy chain of a human antibody (human VH), or a camelized variable domain of the heavy chain of a human antibody (camelized human VH). 
     
     
         60 . A method for preventing or reducing the binding by pre-existing antibodies to a polypeptide according to  claim 50 , wherein the heavy-chain immunoglobulin single variable domain is at the C-terminal end of the polypeptide. 
     
     
         61 . A heavy-chain immunoglobulin single variable domain that consists of formula 1: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein FR1 to FR4 refer to framework regions 1 to 4 and CDR1 to CDR3 refer to complementarity determining regions 1 to 3, wherein the heavy-chain immunoglobulin single variable domain forms a single functional antigen binding site; and which contains a glycosylation site such that the amino acid residue at positions 108 can be glycosylated, wherein the numbering is according to Kabat numbering. 
     
     
         62 . A nucleotide sequence or nucleic acid encoding: the heavy-chain immunoglobulin single variable domain according to  claim 61 ; or a protein or polypeptide that comprises or consists essentially of at least one heavy-chain immunoglobulin single variable domain according to  claim 61 . 
     
     
         63 . A host cell comprising an expression vector comprising the nucleotide sequence or the nucleic acid according to  claim 62 .