Serum albumin binding nanobody compositions and methods for using the same
Disclosed herein are compositions and methods for increasing the stability of recombinant nanobodies. Also disclosed herein are recombinant nanobodies comprising an IL-2 polypeptide that bind serum albumin and uses thereof for treating cancers.
1 . A recombinant nanobody comprising a human serum albumin (HSA) binding polypeptide and an interleukin-2 (IL-2) polypeptide, wherein the HSA binding polypeptide specifically binds to an HSA epitope selected from the group consisting of epitope 1, epitope 2, epitope 3, and epitope 4,
wherein epitope 1 comprises, as determined starting from amino acid residue 25 of SEQ ID NO: 1, the amino acid residues 298-307, 311, 332-341, and 371-386 of SEQ ID NO: 1,
wherein epitope 2 comprises, as determined starting from amino acid residue 25 of SEQ ID NO: 1, the amino acid residues 5-13, 62-67, 93-99, and 228-266 of SEQ ID NO: 1,
wherein epitope 3 comprises, as determined starting from amino acid residue 25 of SEQ ID NO: 1, the amino acid residues 226-230 and 298-337 of SEQ ID NO: 1, and
wherein epitope 4 comprises, as determined starting from amino acid residue 25 of SEQ ID NO: 1, the amino acid residues 33-38 and 111-145 of SEQ ID NO: 1; and
wherein the HSA binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.
2 . The recombinant nanobody of claim 1 , wherein the HSA binding polypeptide specifically binds to a human HSA and a mouse serum albumin.
3 . The recombinant nanobody of claim 1 , wherein the IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 103, or a functional fragment thereof.
4 . The recombinant nanobody of claim 1 , wherein the HSA binding polypeptide is linked to the IL-2 polypeptide via a linker.
5 . The recombinant nanobody of claim 4 , wherein the linker comprises the amino acid sequence of SEQ ID NO: 6.
6 . The recombinant nanobody of claim 1 , wherein the recombinant nanobody is less than about 50 kDa.
7 . The recombinant nanobody of claim 1 , wherein the HSA binding polypeptide does not decrease an affinity between the IL-2 polypeptide and an IL-2 receptor thereof.
8 . The recombinant nanobody of claim 1 , wherein the recombinant nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.
9 . A method of treating a cancer responsive to interleukin-2 (IL-2) in a subject in need thereof, comprising administering a therapeutically effective amount of a recombinant nanobody comprising a human serum albumin (HSA) binding polypeptide and an IL-2 polypeptide, wherein the HSA binding polypeptide specifically binds to an HSA epitope selected from the group consisting of epitope 1, epitope 2, epitope 3, and epitope 4,
wherein epitope 1 comprises, as determined starting from amino acid residue 25 of SEQ ID NO: 1, the amino acid residues 298-307, 311, 332-341, and 371-386 of SEQ ID NO: 1,
wherein epitope 2 comprises, as determined starting from amino acid residue 25 of SEQ ID NO: 1, the amino acid residues 5-13, 62-67, 93-99, and 228-266 of SEQ ID NO: 1,
wherein epitope 3 comprises, as determined starting from amino acid residue 25 of SEQ ID NO: 1, the amino acid residues 226-230 and 298-337 of SEQ ID NO: 1, and
wherein epitope 4 comprises, as determined starting from amino acid residue 25 of SEQ ID NO: 1, the amino acid residues 33-38 and 111-145 of SEQ ID NO: 1; and
wherein the HSA binding polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.
10 . The method of claim 9 , wherein the HSA binding polypeptide specifically binds to a human HSA and a mouse serum albumin.
11 . The method of claim 9 , wherein the IL-2 polypeptide comprises the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 103, or a functional fragment thereof.
12 . The method of claim 9 , wherein the HSA binding polypeptide is linked to the IL-2 polypeptide via a linker.
13 . The method of claim 12 , wherein the linker comprises the amino acid sequence of SEQ ID NO: 6.
14 . The method of claim 9 , wherein the recombinant nanobody is less than about 50 kDa.
15 . The method of claim 9 , wherein the HSA binding polypeptide does not decrease an affinity between the IL-2 polypeptide and an IL-2 receptor thereof.
16 . The method of claim 9 , wherein the recombinant nanobody comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, SEQ ID NO: 8, and SEQ ID NO: 9.
17 . The method of claim 9 , wherein the recombinant nanobody has an in vivo half-life about 100 times higher than a natural IL-2 polypeptide.
18 . The method of claim 9 , wherein the cancer is selected from the group consisting of melanoma and renal carcinoma.
19 . The recombinant nanobody of claim 1 , wherein the HSA binding polypeptide comprises the amino acid sequence of SEQ ID NO: 2.
20 . The recombinant nanobody of claim 1 , wherein the HSA binding polypeptide comprises the amino acid sequence of SEQ ID NO: 3.
21 . The recombinant nanobody of claim 1 , wherein the HSA binding polypeptide comprises the amino acid sequence of SEQ ID NO: 4.
22 . The recombinant nanobody of claim 1 , wherein the recombinant nanobody comprises the amino acid sequence of SEQ ID NO: 7.
23 . The recombinant nanobody of claim 1 , wherein the recombinant nanobody comprises the amino acid sequence of SEQ ID NO: 8.
24 . The recombinant nanobody of claim 1 , wherein the recombinant nanobody comprises the amino acid sequence of SEQ ID NO: 9.