POLYPEPTIDES COMPRISING IMMUNOGLOBULIN SINGLE VARIABLE DOMAINS TARGETING IL-13 AND TSLP
The present technology aims at providing a novel type of drug for treating a subject suffering from an inflammatory disease. Specifically, the present technology provides polypeptides comprising at least four immunoglobulin single variable domains (ISVDs), characterized in that at least two ISVDs bind to IL-13 and at least two ISVDs binds to TSLP. The present technology also provides nucleic acids, vectors and compositions.
1 .- 20 . (canceled)
21 . A method of producing a multivalent, multispecific polypeptide, comprising:
(a) selecting a first immunoglobulin single variable domain (ISVD), a second ISVD, a third ISVD, a fourth ISVD, and a fifth ISVD, wherein each of the first ISVD and the second ISVD specifically binds to IL-13, each of the third ISVD and the fourth ISVD specifically binds to TSLP, and the fifth ISVD specifically binds to human serum albumin;
(b) generating a nucleic acid molecule encoding a polypeptide that comprises a first segment and a second segment, wherein the first segment and the second segment are directly or indirectly linked, wherein:
(i) the first segment comprises, N-terminally to C-terminally, the first ISVD, a first linker, and the second ISVD, wherein the first linker consists of about 35 amino acids, wherein each of the about 35 amino acids is a glycine (G) or a serine(S); and
(ii) the second segment comprises, N-terminally to C-terminally, the third ISVD, a second linker, the fifth ISVD, a third linker, and the fourth ISVD, wherein each of the second linker and the third linker consists of about 9 amino acids, wherein each of the about 9 amino acids is a glycine (G) or a serine(S); and
(c) expressing the polypeptide from the nucleic acid molecule.
22 . The method of claim 21 , wherein:
the first linker consists of the amino acid sequence of SEQ ID NO: 83;
the second linker consists of the amino acid sequence of SEQ ID NO: 76; or
the third linker consists of the amino acid sequence of SEQ ID NO: 76.
23 . The method of claim 21 , wherein:
the first linker consists of the amino acid sequence of SEQ ID NO: 83;
the second linker consists of the amino acid sequence of SEQ ID NO: 76; and
the third linker consists of the amino acid sequence of SEQ ID NO: 76.
24 . The method of claim 21 , wherein the polypeptide further comprises a fourth linker, wherein the fourth linker consists of 9 amino acids, wherein each of the 9 amino acids is a glycine (G) or a serine(S).
25 . The method of claim 24 , wherein the fourth linker consists of the amino acid sequence of SEQ ID NO: 76.
26 . The method of claim 24 , wherein the polypeptide comprises, N-terminally to C-terminally, the structure of the first ISVD—the first linker—the second ISVD—the fourth linker—the third ISVD—the second linker—the fifth ISVD—the third linker—the fourth ISVD.
27 . The method of claim 24 , wherein the polypeptide comprises, N-terminally to C-terminally, the structure of the third ISVD—the second linker—the fifth ISVD—the third linker—the fourth ISVD—the fourth linker—the first ISVD—the first linker—the second ISVD.
28 . The method of claim 21 , wherein the first ISVD and the second ISVD consist of the same amino acid sequence.
29 . The method of claim 21 , wherein the third ISVD and the fourth ISVD consist of different amino acid sequences.
30 . A multivalent, multispecific polypeptide produced according to the method of claim 21 .
31 . A method of reducing binding of pre-existing antibodies to a multivalent polypeptide comprising:
(a) selecting a polypeptide comprising, N-terminally to C-terminally, a structure of a first immunoglobulin single variable domain (ISVD)—a first linker—a second ISVD, wherein the first linker has a length of greater than 9 amino acids;
(b) generating a modified polypeptide comprising, N-terminally to C-terminally, a structure of a third ISVD—a second linker—and a fourth ISVD, wherein the third ISVD is identical to the first ISVD or comprises one or more framework mutations relative to the first ISVD, wherein the fourth ISVD is identical to the second ISVD or comprises one or more framework mutations relative to the second ISVD, and wherein the second linker has a length of no greater than 9 amino acids; and
(c) measuring the binding of pre-existing antibodies to the polypeptide of (a) and the modified polypeptide of (b).
32 . The method of claim 31 , wherein each of the amino acids of the second linker is a glycine (G) or a serine(S).
33 . The method of claim 31 , wherein the second linker consists of the amino acid sequence of SEQ ID NO: 76.
34 . The method of claim 31 , wherein the modified polypeptide does not comprise a linker having a length of greater than 9 amino acids.
35 . The method of claim 31 , wherein the third ISVD or the fourth ISVD comprises a valine (V) at amino acid position 11, a leucine (L) at amino acid position 89, a lysine (K) or glutamine (Q) at amino acid position 110, a lysine (K) or glutamine (Q) at amino acid position 112, or a combination thereof, wherein amino acid positions are determined according to Kabat numbering.
36 . The method of claim 31 , wherein the third ISVD and the fourth ISVD comprises a valine (V) at amino acid position 11, a leucine (L) at amino acid position 89, a lysine (K) or glutamine (Q) at amino acid position 110, a lysine (K) or glutamine (Q) at amino acid position 112, or a combination thereof, wherein amino acid positions are determined according to Kabat numbering.
37 . A method of reducing binding of pre-existing antibodies to a multivalent polypeptide comprising:
(a) selecting a polypeptide comprising, N-terminally to C-terminally, a structure of a first immunoglobulin single variable domain (ISVD)—a first linker—a second ISVD—a second linker—a third ISVD—a third linker—a fourth ISVD—a fourth linker— and a fifth ISVD, wherein at least one of the first linker, the second linker, the third linker, and the fourth linker has a length of greater than 9 amino acids;
(b) generating a modified polypeptide, wherein the modified polypeptide does not comprise a linker having a length of greater than 9 amino acids but is otherwise essentially identical to the polypeptide of (a); and
(c) measuring the binding of pre-existing antibodies to the polypeptide of (a) and the modified polypeptide of (b).
38 . The method of claim 37 , wherein each of the linkers of the modified polypeptide consists of the same amino acid sequence.
39 . The method of claim 37 , wherein each of the linkers of the modified polypeptide is 9 amino acids in length.
40 . The method of claim 37 , wherein each of the linkers of the modified polypeptide consists of the amino acid sequence of SEQ ID NO: 76.