ADAMTS13 protein variants and uses thereof
The invention relates to ADAMTS13 protein variants comprising residues 1 to 685 of ADAMTS13 and wherein one or more N-linked glycosylation sites are added as compared to wild-type ADAMTS13 and/or one or more existing N-linked glycosylation sites are shifted as compared to wild-type ADAMTS13. The invention further relates to compositions comprising such ADAMTS13 variants, methods for their preparation and uses thereof, in particular as an antithrombotic agent, and in the treatment of thrombotic disease, thrombotic microangiopathy, thrombotic thrombocytopeniaurpura (TTP), hemolytic-uremic syndrome (HUS), ischemic stroke, systemic thrombosis, COVID19, antiphospholipid syndrome, pre-eclampsia/HELLP syndrome, sepsis and sickle cell disease.
1 . An ADAMTS13 protein variant having ADAMTS13 activity comprising amino acid residues corresponding to amino acid residues 1 to 685 of the amino acid sequence of SEQ ID NO: 1 and wherein one or more N-linked glycosylation sites are added as compared to a wild-type ADAMTS13 and/or one or more existing N-linked glycosylation sites are shifted as compared to a wild-type ADAMTS13 in a spacer domain comprising amino acid residues corresponding to amino acid residues S556 to A685 of the amino acid sequence of SEQ ID NO: 1.
2 . The ADAMTS13 protein variant of claim 1 comprising amino acid residues corresponding to amino acid residues 1 to 1427 of the amino acid sequence of SEQ ID NO: 1.
3 . The ADAMTS13 protein variant of claim 1 , wherein one or more existing N-linked glycosylation sites are shifted in part of the spacer domain comprising amino acid residues corresponding to amino acid residues R568 to R670 of the amino acid sequence of SEQ ID NO: 1.
4 . The ADAMTS13 protein variant of claim 1 , comprising an N-linked glycosylation site at an amino acid residue corresponding to amino acid residues selected from the group consisting of 568, 591, 608, 609, 636, 637, 665, 668, and combinations thereof of the amino acid sequence of SEQ ID NO: 1.
5 . The ADAMTS13 protein variant of claim 1 , comprising an N-linked glycosylation site at amino acid residue corresponding to amino acid residue 608 of the amino acid sequence of SEQ ID NO: 1.
6 . The ADAMTS13 protein variant of claim 1 , having proteolytic activity against Von Willebrand Factor (VWF) that is at least 10% of the proteolytic activity against VWF of wild-type ADAMTS13.
7 . The ADAMTS13 protein variant of claim 1 comprising an N-linked glycosylation site comprising an amino acid residue corresponding to amino acid residues selected from the group consisting of R568, L591, V604, V605, A606, G607, K608, M609, R636, L637, P638, R639, Y665, L668 and combinations thereof of the amino acid sequence of SEQ ID NO: 1.
8 . The ADAMTS13 protein variant of claim 1 , comprising a mutation selected from the group consisting of 568REY570 to 560NET570 (NGLY1), 591LFT593 to 591NFT593 (NGLY2), 608KMSI611 to 608NMSI611 (NGLY3), 608KMSI611 to 608KNST611 (NGLY4), 636RLPR639 to 636NLSR639 (NGLY5), 636RLPL639 to 636RNAS639 (NGLY6), 665YGNL668 to 665NVTL668 (NGLY7), 667NLTRP671 to 667LNVTA671 (NGLY8), and combinations thereof of the amino acid sequence of SEQ ID NO: 1.
9 . The ADAMTS13 protein variant of claim 8 , comprising the mutation 608KMSI611 to 608NMSI611 (NGLY3).
10 . The ADAMTS13 protein variant of claim 1 , further comprising a mutation at one or more amino acid residues.
11 . The ADAMTS13 protein variant of claim 10 , wherein said mutation at one or more amino acid residues is in the spacer domain comprising amino acid residues corresponding to amino acid residues S556 to A684 of the amino acid sequence of SEQ ID NO: 1.
12 . The ADAMTS13 protein variant of claim 1 , further comprising a mutation at an amino acid residue corresponding to amino acid residues selected from the group consisting of R568, L591, F592, R636, L637, L668, L591, F592, R636, L637, R660, Y661, Y665, L668 and combinations thereof of the amino acid sequence of SEQ ID NO: 1.
13 . The ADAMTS13 protein variant of claim 1 , comprising mutations R568A and Y665A or mutations L591A, R636A, L637A, and L668A.
14 . The ADAMTS13 protein variant of claim 1 , comprising an N-glycan at said one or more N-linked glycosylation sites that are added and/or wherein said one or more existing N-linked glycosylation sites that are shifted comprise an N-linked glycan.
15 . A pharmaceutical composition comprising the ADMATS13 protein variant of claim 1 and one or more pharmaceutically acceptable carriers, adjuvants, excipients and/or diluents.
16 . A method for treatment of a disorder characterized by aberrant Von Willebrand Factor (VWF) activity and/or VWF processing comprising administering to a subject in need thereof a therapeutically effective amount of the ADAMTS13 protein variant of claim 1 .
17 . The method of claim 16 , wherein said disorder is a thrombotic disease.
18 . The method of claim 17 , wherein said thrombotic disease is a thrombotic microangiopathy or a disorder selected from the group consisting of thrombotic thrombocytopenia purpura (TTP), including immune-mediated TTP (iTTP), hemolytic-uremic syndrome (HUS), ischemic stroke, systemic thrombosis, COVID19, antiphospholipid syndrome, preeclampsia/HELLP syndrome, sepsis, and sickle cell disease.
19 . The ADAMTS13 protein variant of claim 7 comprising a mutation selected from the group consisting of R568N, L591N, V604N, V605N, A606N, G607N, K608N, M609N, R636N, L637N, P638N, R639N, Y665N, L668N and combinations thereof.
20 . The ADAMTS13 protein variant of claim 12 , further comprising a mutation selected from the group consisting of R568K, R568A, R568N, L591A, F592Y, F592A, F592N, R636A, L637A, R660K, R660A, R660N, Y661F, Y661A, Y661N, Y665F, Y665A, Y665N, L668A, and combinations thereof.