Anti-protease nexin-1 conformational single domain antibodies and uses thereof to control bleeding
Protease nexin-1 (PN-1) is a member of the serine protease inhibitor (Serpin)-family, with thrombin as its main target. Current polyclonal and monoclonal antibodies against PN-1 frequently cross-react with Plasminogen activator inhibitor-1 (PAI-1), a structurally and functionally homologous Serpin. Herein the inventors develop inhibitory single-domain antibodies (VHHs) showing specific binding to both human (hPN-1) and murine (mPN-1) PN-1 and which de not cross-react with PAI-1. Importantly, all VHHs could block PN-1 activity in plasma as well as PN-1 released from activated platelets, one of the main sources of PN-1 during hemostasis. Thus, the present invention relates to anti-protease nexin-1 (PN-1) conformational single domain antibodies and uses thereof in particular in the therapeutic field for the treatment of haemorrhagic diseases.
1 . A single domain antibody (sdAb) that targets protease Nexin-1 comprising formula (I), wherein the amino acid sequence of formula (I) has three complementary determining regions (CDR1, CDR2, and CDR3) and four framework regions (FR1 to FR4) from N-terminus to C-terminus:
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (I); wherein
CDR1 has the amino acid sequence set forth as SEQ ID NO: 1, CDR2 has the amino acid sequence set forth as SEQ ID NO:2 and CDR3 has the amino acid sequence set forth as SEQ ID NO:3.
2 . The single domain antibody according to claim 1 having at least 95% identity with the amino acid sequence set forth as SEQ ID NO:4.
3 . The single domain antibody according to claim 1 which comprises the amino acid sequence set forth as SEQ ID NO:4.
4 . A pharmaceutical composition comprising the single domain antibody of claim 1 and/or a polypeptide comprising
at least one copy of the single domain antibody and
a pharmaceutically acceptable carrier.
5 . A polypeptide comprising the single domain antibody according to claim 1 .
6 . The polypeptide according to claim 5 further comprising a second single domain antibody comprising an amino acid sequence of formula (I) comprising three complementary determining regions (CDR1a, CDR2b and CDR3c) and four framework regions (FR1 to FR4):
FR1-CDR1a-FR2-CDR2b-FR3-CDR3c-FR4 (I); wherein:
CDR1a has the amino acid sequence set forth as SEQ ID NO: 5 CDR2b has the amino acid sequence set forth as SEQ ID NO:6 and CDR3c has the amino acid sequence set forth as SEQ ID NO:7.
7 . The polypeptide according to claim 5 further comprising:
a second single domain antibody comprising an amino acid sequence of formula (I) comprising three complementary determining regions (CDR1d, CDR2e, and CDR3f) and four framework regions (FR1 to FR4):
FR1-CDR1d-FR2-CDR2e-FR3-CDR3f-FR4 (I); wherein
CDR1d has the amino acid sequence set forth as SEQ ID NO:9;
CDR2e has the amino acid sequence set forth as SEQ ID NO:10; and
CDR3f has the amino acid sequence set forth as SEQ ID NO:11.
8 . The polypeptide according to claim 5 which comprises the amino acid sequence set forth as SEQ ID NO:17.
9 . The polypeptide according to claim 6 which comprises the amino acid sequence set forth as SEQ ID NO:18.
10 . The polypeptide according to claim 7 which comprises the amino acid sequence set forth as SEQ ID NO:19.
11 . A pharmaceutical composition comprising the polypeptide according to claim 6 and a pharmaceutically acceptable carrier.
12 . A pharmaceutical composition comprising the polypeptide according to claim 7 and a pharmaceutically acceptable carrier.