# US 11,306,139 B2 · Glycosylated immunoglobulin single variable domains > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,306,139 B2 - Document ID: US11306139B2 - Status: Granted - Kind: B2 - Application number: 15/554,744 - Filed: 2016-03-18 - Published: 2018-02-15 - Granted: 2022-04-19 - Earliest claimed: 2015-03-20 - Assignees: Ablynx N.V. - Applicants: Ablynx N.V. - Inventors: Marie-Ange Buyse; Carlo Boutton - Web page: https://patentgrep.com/patent/US11306139B2 ## CPC - C07K 16/18: against material from animals or humans - C07K 2317/40: characterized by post-translational modification - C07K 2317/41: Glycosylation, sialylation, or fucosylation - C07K 2317/56: variable (Fv) region, i.e. VH and/or VL - C07K 2317/567: Framework region [FR] - C07K 2317/569: Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody® - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) ## 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 “ISF” 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. ## Claims ### Claim 1 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 complementary determining regions 1 to 3, wherein FR1 comprises the amino acid residues at positions 1-30, FR2 comprises the amino acid residues at positions 36-49, FR3 comprises the amino acid residues at positions 66-94, and FR4 comprises the amino acid residues at positions 103-113; and 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, 112, 113 or 114 is or can be glycosylated, wherein the numbering is according to Kabat numbering. ### Claim 2 (depends on 1) The heavy-chain immunoglobulin single variable domain according to claim 1, which contains an N-glycosylation site such that the amino acid residue at one of positions 10, 11, 12, 13, 14, 39, 40, 41, 42, 87, 89, 108, 112, 113 or 114 is an asparagine residue that is or can be N-glycosylated. ### Claim 3 (depends on 2) The heavy-chain immunoglobulin single variable domain according to claim 2 that contains an NXT or NXS motif, in which X can be any amino acid except proline, 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, 112, 113 or 114. ### Claim 4 (depends on 1) The heavy-chain immunoglobulin single variable domain according to claim 1, that is not glycosylated. ### Claim 5 (depends on 1) The heavy-chain immunoglobulin single variable domain according to claim 1, that is glycosylated at one of positions 10, 11, 12, 13, 14, 39, 40, 41, 42, 87, 89, 108, 112, 113 or 114. ### Claim 6 (depends on 1) The heavy-chain immunoglobulin single variable domain according to claim 1, which contains a glycosylation site such that the amino acid residue at one of positions 108, 112, 113 or 114 is or can be glycosylated. ### Claim 7 (depends on 6) The heavy-chain immunoglobulin single variable domain according to claim 6, which contains an N-glycosylation site such that the amino acid residue at one of positions 108, 112, 113 or 114 is an asparagine residue that is or can be N-glycosylated. ### Claim 8 (depends on 7) The heavy-chain immunoglobulin single variable domain according to claim 7 that contains an NXT or NXS motif, in which X can be any amino acid except proline, such that the asparagine (N) residue of the NXT/NXS motif is present at one of positions 108, 112, 113 or 114. ### Claim 9 (depends on 6) The heavy-chain immunoglobulin single variable domain according to claim 6, that is not glycosylated. ### Claim 10 (depends on 6) The heavy-chain immunoglobulin single variable domain according to claim 6, that is glycosylated at one of positions 108, 112, 113 or 114. ### Claim 11 (depends on 1) The heavy-chain immunoglobulin single variable domain according to claim 1, which 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). ### Claim 12 (depends on 1) A protein, polypeptide, construct or other chemical entity that comprises or consists essentially of at least one heavy-chain immunoglobulin single variable domain according to claim 1. ### Claim 13 (depends on 12) The protein or polypeptide of claim 12, wherein the at least one heavy-chain immunoglobulin single variable domain is at the C-terminal end of the protein or polypeptide. ### Claim 14 (depends on 1) A nucleotide sequence or nucleic acid encoding the heavy-chain immunoglobulin single variable domain according to claim 1 or a protein or polypeptide that comprises or consists essentially of at least one heavy-chain immunoglobulin single variable domain according to claim 1. ### Claim 15 (depends on 1) A composition comprising the heavy-chain immunoglobulin single variable domain according to claim 1. ### Claim 16 (depends on 15) The composition according to claim 15, which is a pharmaceutical composition. ### Claim 17 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 one of positions 10, 11, 12, 13, 14, 39, 40, 41, 42, 87, 89, 108, 112, 113 or 114 is or can be glycosylated, wherein the numbering is according to Kabat numbering. ### Claim 18 (depends on 17) A protein, polypeptide, construct or other chemical entity that comprises or consists essentially of at least one heavy-chain immunoglobulin single variable domain according to claim 17. ### Claim 19 (depends on 17) A nucleotide sequence or nucleic acid encoding the heavy-chain immunoglobulin single variable domain according to claim 17 or a protein or polypeptide that comprises or consists essentially of at least one heavy-chain immunoglobulin single variable domain according to claim 17. ### Claim 20 (depends on 17) A composition comprising the heavy-chain immunoglobulin single variable domain according to claim 17. ### Claim 21 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 position 11 is V or K; and which contains a glycosylation site such that the amino acid residue at one of positions 10, 12, 13, 14, 39, 40, 41, 42, 87, 89, 108, 110, 112, 113 or 114 is or can be glycosylated, wherein the numbering is according to Kabat numbering. ### Claim 22 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 position 14 is P; and which contains a glycosylation site such that the amino acid residue at one of positions 10, 11, 12, 13, 39, 40, 41, 42, 87, 89, 108, 112, 113 or 114 is or can be glycosylated, wherein the numbering is according to Kabat numbering. ### Claim 23 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 position 41 is A; and which contains a glycosylation site such that the amino acid residue at one of positions 10, 11, 12, 13, 14, 39, 40, 42, 87, 89, 108, 112, 113 or 114 is or can be glycosylated, wherein the numbering is according to Kabat numbering. ### Claim 24 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 position 89 is T or L; and 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, 108, 110, 112, 113 or 114 is or can be glycosylated, wherein the numbering is according to Kabat numbering.