Single-chain antigen-binding proteins capable of glycosylation, production and uses thereof

A polynucleotide encoding a single-chain antigen-binding polypeptide capable of glycosylation, comprising:

• (a) a first polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;
• (b) a second polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;
• and
• (c) a peptide linker linking said first and second polypeptides (a) and (b) into a single chain polypeptide having an antigen binding site,
• wherein said single-chain antigen-binding polypeptide has at least one tripeptide Asn-linked glycosylation sequence comprising Asn-Xaa-Yaa, wherein Xaa is an amino acid other than proline and Yaa is threonine or serine, said tripeptide glycosylation sequence capable of attaching a carbohydrate moiety at said Asn residue located at a position selected from the group consisting of
• (i) the amino acid position 11, 12, 13, 14 or 15 of said light chain variable region;
• (ii) the amino acid position 77, 78 or 79 of said light chain variable region;
• (iii) the amino acid position 11, 12, 13, 14 or 15 of said heavy chain variable region;
• (iv) the amino acid position 82B, 82C or 83 of said heavy chain variable region;
• (v) any amino acid position of said peptide linker;
• (vi) adjacent to the C-terminus of said second polypeptide (b);
• and
• (vii) combinations thereof, and
• wherein the glycosylated single-chain antigen-binding polypeptide is capable of binding an antigen.

A method of producing a single-chain antigen-binding polypeptide capable of glycosylation, comprising:

• (a) providing a first polynucleotide encoding a first polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;
• (b) providing a second polynucleotide encoding a second polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;
• and
• (c) linking said first and second polynucleotides (a) and (b) with a third polynucleotide encoding a peptide linker into a fourth polynucleotide encoding a single chain polypeptide having an antigen binding site,
• wherein said single-chain antigen-binding polypeptide has at least one tripeptide Asn-linked glycosylation sequence comprising Asn-Xaa-Yaa, wherein Xaa is an amino acid other than proline and Yaa is threonine or serine, said tripeptide glycosylation sequence capable of attaching a carbohydrate moiety at said Asn residue located at a position selected from the group consisting of
• (i) the amino acid position 11, 12, 13, 14 or 15 of said light chain variable region;
• (ii) the amino acid position 77, 78 or 79 of said light chain variable region;
• (iii) the amino acid position 11, 12, 13, 14 or 15 of said heavy chain variable region;
• (iv) the amino acid position 82B, 82C or 83 of said heavy chain variable region;
• (v) any amino acid position of said peptide linker;
• (vi) adjacent to the C-terminus of said second polypeptide (b);
• and
• (vii) combinations thereof, and
• wherein the glycosylated single-chain antigen-binding polypeptide is capable of binding an antigen;
• and
• (d) expressing said single-chain antigen-binding polypeptide of step (c) in a host cell, thereby producing a single-chain antigen-binding polypeptide capable of glycosylation.

A multivalent single-chain antigen-binding protein, comprising two or more single-chain antigen-binding polypeptides, each single-chain antigen-binding polypeptide comprising:

• (a) a first polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;
• (b) a second polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;
• and
• (c) a peptide linker linking said first and second polypeptides (a) and (b) into a single chain polypeptide having an antigen binding site,
• wherein at least one said single-chain antigen-binding polypeptide has at least one tripeptide Asn-linked glycosylation sequence comprising Asn-Xaa-Yaa, wherein Xaa is an amino acid other than proline and Yaa is threonine or serine, said tripeptide glycosylation sequence capable of attaching a carbohydrate moiety at said Asn residue located at a position selected from the group consisting of
• (i) the amino acid position 11, 12, 13, 14 or 15 of said light chain variable region;
• (ii) the amino acid position 77, 78 or 79 of said light chain variable region;
• (iii) the amino acid position 11, 12, 13, 14 or 15 of said heavy chain variable region;
• (iv) the amino acid position 82B, 82C or 83 of said heavy chain variable region;
• (v) any amino acid position of said peptide linker;
• (vi) adjacent to the C-terminus of said second polypeptide (b);
• and
• (vii) combinations thereof, and
• wherein the glycosylated single-chain antigen-binding polypeptide is capable of binding an antigen.

A method of producing a polypeptide having increased glycosylation, comprising:

• (a) providing to a polynucleotide encoding said polypeptide at least two tripeptide Asn-linked glycosylation sequences, wherein each said tripeptide glycosylation sequence comprises Asn-Xaa-Yaa, wherein Xaa is an amino acid other than proline and Yaa is threonine or serine, and wherein said tripeptide glycosylation sequences are in tandem such that said Asn residues are separated by two amino acid residues;
• (b) providing to said polynucleotide at least one set of two tripeptide Asn-linked glycosylation sequences, wherein said two tripeptide glycosylation sequences overlap such that said Asn residues are adjacent;
• and
• (c) expressing said polynucleotide in a host cell capable of attaching a carbohydrate moiety at said Asn residues.

A polypeptide having increased glycosylation produced by the process comprising:

• (a) providing to a polynucleotide encoding said polypeptide at least two tripeptide Asn-linked glycosylation sequences, wherein each said tripeptide glycosylation sequence comprises Asn-Xaa-Yaa, wherein Xaa is an amino acid other than proline and Yaa is threonine or serine, and wherein said tripeptide glycosylation sequences are in tandem such that said Asn residues are separated by two amino acid residues;
• (b) providing to said polynucleotide at least one set of two tripeptide Asn-linked glycosylation sequences, wherein said two tripeptide glycosylation sequences overlap such that said Asn residues are adjacent;
• and
• (c) expressing said polynucleotide in a host cell capable of attaching a carbohydrate moiety at said Asn residues.