Steric single domain antibody against protease nexin-1 and its use

Abstract

Protease nexin-1 (PN-1) is a member of the serine protease inhibitor (serpin) family whose primary target is thrombin. 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, we developed inhibitory single-domain antibodies (VHHs) that specifically bind to both human (hPN-1) and mouse (mPN-1) PN-1 and do not cross-react with PAI-1. Importantly, all VHHs were able to block PN-1 activity in plasma as well as PN-1 released from activated platelets, one of the major sources of PN-1 during hemostasis. Thus, the present invention relates to conformational single-domain antibodies against PN-1 and their use in therapeutic applications, particularly for the treatment of bleeding disorders.

Description

Technical Field

[0001] Field of the Invention: The present invention relates to a structurally single-domain antibody against protease nexin-1 (PN-1), and in particular to its use in the therapeutic field.

[0002] Background of the Invention: Current treatments for controlling bleeding episodes in hemophilia patients and more generally in patients with chronic bleeding disorders have many drawbacks. The treatment of these patients with inhibitors is limited to factor VIII or factor IX bypassing agents such as recombinant factor VIIa or plasma-derived activated prothrombin complex. However, these products are expensive and a significant number of patients do not respond to these drugs. Therefore, due to the limitations of current treatments that largely rely on intravenous injection of recombinant or plasma-derived factor VIII or factor IX, new strategies aimed at targeting natural anticoagulant proteins have emerged.

[0003] The inventors have previously demonstrated that platelet PN-1 is a negative regulator of both thrombin activity and generation, and that PN-1 deficiency promotes coagulation in vivo (Boulaftali et al. 2010). The inventors have shown that targeting PN-1 can treat factor VIII or factor IX deficiency, and have demonstrated that blockade of PN-1 plays a role in the treatment of bleeding disorders.

[0004] In this specification, the inventors develop inhibitory single domain antibodies (VHHs or nanobodies) against protease nexin-1 (PN-1), a natural and potent thrombin inhibitor, which is a platelet protease. The inventors emphasize the difficulty of generating specific antibodies against PN-1. Most of the commercially available antibodies against PN-1 tested by the inventors were found to be non-specific. The nanobodies of the present invention appear to be the first specific biological tools capable of blocking the antithrombin activity of both mouse and human PN-1. Thus, they are good candidates for blocking PN-1 in platelets and restoring the hemostatic balance in hemophilia patients.

[0005] Summary of the Invention: Protease nexin-1 (PN-1), also known as serpin 2, is a member of the serpin family of serine protease inhibitors, an important regulator in many biological events. PN-1 is a serpin that is hardly detectable in plasma but is found in many organs and is produced by most cell types, including monocytes, platelets, and vascular cells. PN-1 is a 45-50 kDa glycoprotein encoded by the serpin 2 gene on human chromosome 2q33-q35. PN-1 is a single chain of 378 amino acid residues that contains three cysteine residues that do not form disulfide bonds within the protein core of the molecule (Bouton et al., 2012 and Mc Grogan et al 1988 Boulaftali et al., 2010). The inventors have previously demonstrated that targeting PN-1 improves thrombin generation in patients with mild and moderate hemophilia. These findings establish the requirements for inhibition of PN-1 as a specific anticoagulant in platelets and demonstrate that blockade of PN-1 plays a role in the treatment of bleeding disorders.

[0006] In the present application, the inventors describe the successful selection and characterization of a structurally single-domain antibody against protease nexin-1 (PN-1). The sequence of the antibody is shown in Table 1. Phage display experiments were performed using a llama-derived or synthetic nanobody library to screen for VHHs that recognize both human and mouse PN-1. Using the llama-derived library, a total of seven rounds of panning and 940 tested VHHs yielded 52 candidates that recognize PN-1. However, careful characterization revealed that all of these VHHs also recognize plasminogen activator inhibitor-1 (PAI-1). This lack of specificity prompted the inventors to screen a synthetic library, resulting in the isolation of seven positive nanobodies that efficiently bind to both human and mouse PN-1 without cross-reacting with other serpins. Four were found to be able to inhibit the antithrombin activity of human and mouse PN-1. In particular, the inventors isolated three distinct specific anti-PN-1 clones that bind to three separate epitopes. Interestingly, all three single-domain antibodies were found to be specific for PN-1 without cross-reacting with other serpins or plasminogen activator inhibitor-1 (PAI-1). Thus, these antibodies appear to be the first specific biological tools to block antithrombin and may be useful for restoring the hemostatic balance in hemophilia patients.

[0007] Accordingly, the present invention relates to single-domain antibodies against protease nexin-1 (PN-1), and particularly to their use in the therapeutic field. In particular, the present invention is defined by the claims.

[0008] Detailed description of the invention: The inventors developed single-domain antibodies (sdAbs) containing three specific CDRs that enable selective binding to protease nexin-1 (PN-1). These sdAbs constitute an implementation tool as a therapeutic agent.

[0009] Definition As used herein, "protease nexin-1" or "PN-1" has its general meaning in the art and refers to protease nexin-1, also known as serpin 2. PN-1 refers to a member of the serine protease inhibitors, called serpins, which has its general meaning in the art and is an important regulator in many biological events. PN-1 is a serpin that is hardly detectable in plasma but is found in many organs and is produced by most cell types (including monocytes, platelets, and vascular cells). PN-1 is a glycoprotein of 45 to 50 kDa encoded by the serpin 2 gene on human chromosome 2q33-q35. PN-1 is a single chain of 378 amino acid residues that contains three cysteine residues that do not form disulfide bonds within the protein core of the molecule (Bouton et al., 2012 and Mc Grogan et al 1988 Boulaftali et al., 2010).

[0010] As used herein, the term "single domain antibody" has its general meaning in the art and refers to the single-chain variable domain of a type of antibody that can be found in camelid mammals that are naturally lacking in light chains. Such single domain antibodies are also referred to as VHH or "nanobodies (registered trademark)". For a general description of (single) domain antibodies, reference is also made to the prior art listed above, as well as European Patent No. 0368684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21(11):484-490; and International Publication Nos. 06 / 030220, 06 / 003388. Nanobodies have a molecular weight approximately one-tenth that of the human IgG molecule, and the protein has a physical diameter of only a few nanometers. One result of the small size is that camelid nanobodies can bind to antigenic sites that may not appear functionally accessible to larger antibody proteins, i.e., camelid nanobodies are useful as reagents for detecting antigens that cannot be found using classical immunological techniques and as candidates for therapeutic agents. Thus, yet another result of the small size is that nanobodies can inhibit as a result of binding to specific sites within the grooves or narrow gaps of target proteins, and thus can be useful in terms of the ability to approximate the function of classical small molecule drugs more than classical antibodies. Further, due to their low molecular weight and compact size, nanobodies are extremely heat-stable, stable to extreme pH, stable to digestion by proteolysis, and have weak antigenicity. Another result is that nanobodies can readily move from the circulatory system into tissues, even cross the blood-brain barrier, and treat disorders affecting neural tissue. Nanobodies can further facilitate drug transport across the blood-brain barrier. See U.S. Patent Application Publication No. 20040161738, published Aug. 19, 2004. These features, combined with low antigenicity in humans, indicate excellent therapeutic potential.The amino acid sequence and structure of a single-domain antibody can be considered to be composed of four framework regions, namely "Framework Region 1" or "FR1", "Framework Region 2" or "FR2", "Framework Region 3" or "FR3", and "Framework Region 4" or "FR4", which are referred to as "FR" in the art and in this specification, respectively; the framework regions are each interrupted by three complementarity-determining regions, namely "Complementarity-Determining Region 1" or "CDR1", "Complementarity-Determining Region 2" or "CDR2", and "Complementarity-Determining Region 3" or "CDR3", which are referred to as "CDR" in the art. Therefore, a single-domain antibody can be defined as an amino acid sequence having the general structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity-determining regions 1 to 3. In the context of the present invention, the amino acid residues of a single-domain antibody are numbered according to the general numbering for the VH domain as indicated by the amino acid numbering by the International ImMunoGeneTics Information System (http: / / imgt.cines.fr / ).

[0011] As used herein, the term "amino acid sequence" has its ordinary meaning and is the amino acid sequence that imparts its primary structure to a protein. According to the present invention, an amino acid sequence may be modified using one, two, or three conservative amino acid substitutions without a significant decrease in the interactive binding ability. By "conservative amino acid substitution" is meant that an amino acid may be replaced with another amino acid having a similar side chain. Amino acid families having similar side chains are defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., glycine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0012] According to the present invention, a first amino acid sequence having at least 70% identity to a second amino acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; or 99% identity to the second amino acid sequence. The identity of amino acid sequences is typically determined using a suitable sequence alignment algorithm such as BLASTP (Karlin and Altschul, 1990) and default parameters.

[0013] According to the meaning of the present invention, "identity" is calculated by comparing two aligned arrays in a comparison window. Sequence alignment makes it possible to determine the number of common positions (nucleotides or amino acids) for two sequences in a comparison window. Therefore, identity is obtained by dividing the number of common positions by the total number of positions in the comparison window and multiplying by 100. The determination of sequence identity may be performed manually or by well-known computer programs.

[0014] As used herein, the terms "purified" and "isolated" with respect to the single domain antibodies of the present invention mean that the single domain antibodies are present in the absence of substantially other biological macromolecules of the same type. The term "purified" as used herein preferably means that there is at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and more preferably at least 98% by weight of the antibody, compared to the total weight of the macromolecule.

[0015] As used herein, the term "nucleic acid molecule" has its general meaning in the art and refers to a DNA molecule or an RNA molecule.

[0016] Single domain antibodies and polypeptides The sequences of interest in this application are shown in Table 1 below:

[0017]

Table 1

[0018] The first object of the present invention is three complementarity determining regions (CDR1 to CDR3) and four framework regions (FR1 to FR4): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4(I) (wherein, CDR1 is the following sequence: X1 -T-W-X 4 -X 5 -E-I (where X 1 is S or D, and X 4 is F or R, and X 5 is R or L)) and has; and CDR2 has the following sequence: S-X2-X3-X4-W-H-A (where X2 is D or E, X3 is P or D, and X4 is T or G); and CDR3 has the sequence shown as SEQ ID NO: 3 or SEQ ID NO: 7) relates to an isolated single-domain antibody (sdAb) comprising the amino acid sequence of formula (I) shown by

[0019] In some embodiments, the isolated single-domain antibody described in the present invention comprises CDR1 having the sequence shown as SEQ ID NO: 1, CDR2 having the sequence shown as SEQ ID NO: 2, and CDR3 having the sequence shown as SEQ ID NO: 3 (the "B11 derivative").

[0020] In some embodiments, the isolated single-domain antibody described in the present invention comprises CDR1 having the sequence shown as SEQ ID NO: 5, CDR2 having the sequence shown as SEQ ID NO: 6, and CDR3 having the sequence shown as SEQ ID NO: 7 (the "F06 derivative").

[0021] Another object of the present invention is formula (I) consisting of three complementarity-determining regions (CDR1 to CDR3) and four framework regions (FR1 to FR4): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (I) (wherein CDR1 has the sequence shown as SEQ ID NO: 9; and CDR2 has the sequence shown as SEQ ID NO: 10; and CDR3 has the sequence shown as SEQ ID NO: 11)) relates to an isolated single-domain antibody comprising the amino acid sequence shown by (the "A08 derivative").

[0022] In other words, the present invention relates to an amino acid sequence represented by Formula (I) consisting of three complementarity determining regions (CDR1 to CDR3) and four framework regions (FR1 to FR4): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (I) (wherein CDR1 has a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 5, and SEQ ID NO: 9; and CDR2 has a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 6, and SEQ ID NO: 10; and CDR3 has a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 7, and SEQ ID NO: 11) and relates to an isolated single domain antibody (sdAb) comprising the amino acid sequence shown.

[0023] In some embodiments, FR1 has at least 70% identity to the amino acid sequence of SEQ ID NO: 13, FR2 has at least 70% identity to the amino acid sequence of SEQ ID NO: 14, FR3 has at least 70% identity to the amino acid sequence of SEQ ID NO: 15, and FR4 has at least 70% identity to the amino acid sequence of SEQ ID NO: 16.

[0024] In some embodiments, the isolated single domain antibody described in the present invention has at least 70% identity to the sequence shown as SEQ ID NO: 4 (the "B11 derivative").

[0025] In some embodiments, the isolated single domain antibody described in the present invention has at least 70% identity to the sequence shown as SEQ ID NO: 4 and comprises the sequences of CDR1, CDR2, and CDR3 shown as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[0026] In some embodiments, the isolated single domain antibody described in the present invention comprises the sequence shown as SEQ ID NO: 4 (the "B11").

[0027] In some embodiments, the isolated single domain antibody described in the present invention has the sequence shown as SEQ ID NO: 4.

[0028] In some embodiments, the isolated single domain antibody described in the present invention has at least 70% identity to the sequence shown as SEQ ID NO: 8 (the "F06 derivative").

[0029] In some embodiments, the isolated single domain antibody described in the present invention has at least 70% identity to the sequence shown as SEQ ID NO: 8 and comprises the sequences of CDR1, CDR2, and CDR3 shown as SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.

[0030] In some embodiments, the isolated single domain antibody described in the present invention comprises the sequence shown as SEQ ID NO: 8 (the "F06").

[0031] In some embodiments, the isolated single domain antibody described in the present invention has the sequence shown as SEQ ID NO: 8.

[0032] In some embodiments, the isolated single domain antibody described in the present invention has at least 70% identity to the sequence shown as SEQ ID NO: 12.

[0033] In some embodiments, the isolated single domain antibody described in the present invention has at least 70% identity to the sequence shown as SEQ ID NO: 12 (the "A08 derivative") and comprises the sequences of CDR1, CDR2, and CDR3 shown as SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11 (the "F06 derivative").

[0034] In some embodiments, the isolated single domain antibody described in the present invention comprises the sequence shown as SEQ ID NO: 12 (the "A08").

[0035] In some embodiments, the isolated single domain antibody described in the present invention has the sequence shown as SEQ ID NO: 12.

[0036] In some embodiments, the isolated single domain antibody is a "humanized" single domain antibody.

[0037] As used herein, the term "humanized" refers to a single domain antibody of the invention in which the amino acid sequence corresponding to the amino acid sequence of the native VHH domain has been "humanized", i.e., one or more amino acid residues within the amino acid sequence of the native VHH sequence (and particularly within the framework sequence) have been replaced by one or more amino acid residues present at the corresponding position(s) within the VH domain derived from a conventional human chain antibody. Methods for humanizing single domain antibodies are well known in the art. Typically, the humanizing substitutions should be selected such that the resulting humanized single domain antibody still retains the desirable properties of the single domain antibody of the invention. One of ordinary skill in the art can determine and select appropriate humanizing substitutions or combinations of appropriate humanizing substitutions.

[0038] A further aspect of the invention refers to a cross-competing single domain antibody that cross-competes with respect to the binding of the single domain antibody of the invention to PN-1.

[0039] In some embodiments, the cross-competing single domain antibody of the invention consists of three complementarity determining regions (CDR1 to CDR3) and four framework regions (FR1 to FR4), of formula (I): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (I) (wherein, CDR1 has the following sequence: X 1 -T-W-X 4 -X 5 -E-I (where X 1 is S or D, X 4 is F or R, X 5 is R or L); and CDR2 has the following sequence: S-X2-X3-X4-W-H-A (where X2 is D or E, X3 is P or D, and X4 is T or G); and CDR3 has a sequence shown as SEQ ID NO: 3 or SEQ ID NO: 7). A single-domain antibody comprising the amino acid sequence shown competes crosswise with respect to binding to PN-1.

[0040] In some embodiments, the cross-competing single-domain antibody of the present invention competes crosswise with respect to binding to PN-1 with a single-domain antibody comprising CDR1 having the sequence shown as SEQ ID NO: 1, CDR2 having the sequence shown as SEQ ID NO: 2, and CDR3 having the sequence shown as SEQ ID NO: 3.

[0041] In some embodiments, the cross-competing single-domain antibody of the present invention competes crosswise with respect to binding to PN-1 with a single-domain antibody comprising CDR1 having the sequence shown as SEQ ID NO: 5, CDR2 having the sequence shown as SEQ ID NO: 6, and CDR3 having the sequence shown as SEQ ID NO: 7.

[0042] In some embodiments, the cross-competing single-domain antibody of the present invention competes crosswise with respect to binding to PN-1 with a single-domain antibody comprising CDR1 having the sequence shown as SEQ ID NO: 9, CDR2 having the sequence shown as SEQ ID NO: 10, and CDR3 having the sequence shown as SEQ ID NO: 11.

[0043] In some embodiments, the cross-competing single-domain antibody of the present invention competes crosswise with respect to binding to PN-1 with a single-domain antibody comprising an amino acid selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, and SEQ ID NO: 12.

[0044] As used herein, the term "cross-competing" refers to single-domain antibodies that share the ability to bind to a particular region of an antigen. In the present disclosure, "cross-competing" single-domain antibodies have the ability to interfere with the binding of another single-domain antibody to an antigen in a standard competitive binding assay. Such single-domain antibodies may, according to non-limiting theory, bind to the same or related or proximal (e.g., structurally similar or spatially proximal) epitopes as the single-domain antibodies with which they compete. Cross-competition exists when single-domain antibody A reduces the binding of single-domain antibody B by at least 60%, particularly at least 70%, and more specifically at least 80% compared to a positive control lacking one of said single-domain antibodies, and vice versa. Those skilled in the art understand that competition can be evaluated in various assay settings. One suitable assay involves the use of Biacore technology (e.g., by using a Biacore 3000 instrument (Biacore, Uppsala, Sweden)), which can measure the range of interaction using surface plasmon resonance technology. Another assay for measuring cross-competition uses an ELISA-based approach. Additionally, high-throughput processes for "binning" antibodies based on their cross-competition are described in International Patent Application Publication No. WO 2003 / 48731.

[0045] According to the present invention, cross-competing antibodies as described above retain the activity of a single antibody and comprise CDR1 having the sequence shown as SEQ ID NO: 1, CDR2 having the sequence shown as SEQ ID NO: 2, and CDR3 having the sequence shown as SEQ ID NO: 3.

[0046] According to the present invention, cross-competing antibodies as described above retain the activity of a single antibody and comprise CDR1 having the sequence shown as SEQ ID NO: 5, CDR2 having the sequence shown as SEQ ID NO: 6, and CDR3 having the sequence shown as SEQ ID NO: 7.

[0047] According to the present invention, the cross-competing antibodies as described above retain the activity of a single antibody and include CDR1 having the sequence shown as SEQ ID NO: 9, CDR2 having the sequence shown as SEQ ID NO: 10, and CDR3 having the sequence shown as SEQ ID NO: 11.

[0048] According to the present invention, the cross-competing antibodies as described above retain the activity of a single antibody and include amino acids selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 8, and SEQ ID NO: 12.

[0049] A further aspect of the present invention refers to a polypeptide comprising at least one single-domain antibody of the present invention.

[0050] Typically, the polypeptide of the present invention is fused to at least one further amino acid sequence at its N-terminus, at its C-terminus, or at both its N-terminus and its C-terminus, i.e., it comprises a single-domain antibody of the present invention such that a fusion protein is provided. According to the present invention, a polypeptide comprising only one single-domain antibody is herein referred to as a "monovalent" polypeptide. A polypeptide comprising or consisting essentially of two or more single-domain antibodies according to the present invention is herein referred to as a "multivalent" polypeptide.

[0051] In some embodiments, the polypeptide comprises at least one single domain antibody of the invention and at least one other binding unit (i.e., directed against another epitope, antigen, target, protein, or polypeptide), which is also typically a single domain antibody. Such polypeptides are herein referred to as "multispecific" polypeptides, as opposed to polypeptides that contain the same single domain antibody ("monospecific" polypeptides). Thus, in some embodiments, the polypeptides of the invention may also provide at least one additional binding site directed against any desired protein, polypeptide, antigen, epitope, or antigenic determinant. The binding site may be directed against the same protein, polypeptide, antigen, epitope, or antigenic determinant to which the single domain antibody of the invention is directed, or it may be directed against a different protein, polypeptide, antigen, epitope, or antigenic determinant than the single domain antibody of the invention.

[0052] A "bispecific" polypeptide of the invention is a polypeptide comprising at least one single domain antibody directed against a first antigen (i.e., protease nexin-1, PN-1) and at least one additional binding site directed against a second antigen (i.e., different from PN-1), while a "trispecific" polypeptide of the invention is a polypeptide comprising at least one single domain antibody directed against a first antigen (i.e., PN-1), at least one additional binding site directed against a second antigen (i.e., different from PN-1), and at least one additional binding site directed against a third antigen (i.e., different from both antigens, i.e., the first and second antigens).

[0053] In some embodiments, the additional binding site is directed against a serum protein, thus extending the half-life of the single domain antibody. Typically, the serum protein is albumin.

[0054] Typically, one or more additional binding sites may comprise one or more portions, fragments, or domains of conventional chain antibodies (especially human antibodies) and / or heavy chain antibodies. For example, the single domain antibodies of the present invention may be linked to conventional (typically human) VH or VL, optionally via a linker sequence.

[0055] In some embodiments, the polypeptide comprises a single-domain antibody of the invention linked to an immunoglobulin domain. For example, the polypeptide comprises a single-domain antibody of the invention linked to an Fc portion (e.g., human Fc). The Fc portion can be useful for increasing the half-life of the single-domain antibody of the invention and even its production. For example, the Fc portion can bind to serum proteins, thus extending the half-life of the single-domain antibody. In some embodiments, at least one single-domain antibody may also be linked to one or more (typically human) CH1 domains, and / or CH2 domains, and / or CH3 domains, optionally via a linker sequence. For example, a single-domain antibody linked to a suitable CH1 domain can, for example, when used together with a suitable light chain, give rise to an antibody fragment / structural analog of a conventional Fab fragment or F(ab')2 fragment (however, here, one or both of the conventional VH domains or, in the case of an F(ab')2 fragment, one or both are replaced by the single-domain antibody of the invention). In some embodiments, one or more single-domain antibodies of the invention are linked to one or more constant domains (e.g., two or three constant domains that can be used as part of an Fc portion / to form an Fc portion), to an Fc portion, and / or to one or more antibody moieties, fragments, or domains that can confer one or more effector functions on the polypeptide of the invention and / or binding ability to one or more Fc receptors. For example, for this purpose, without being limited thereto, one or more additional amino acid sequences may include, for example, one or more CH2 domains and / or CH3 domains of an antibody derived from a heavy chain antibody and more typically a conventional human chain antibody; and / or may form an Fc region derived from, for example, IgG (e.g., derived from IgG1, IgG2, IgG3, or IgG4), derived from IgE, or derived from another human Ig, such as IgA, IgD, or IgM.For example, International Publication No. 94 / 04678 describes a heavy chain antibody (i.e., a single domain antibody) comprising a VHH domain of Camelidae or a humanized derivative thereof, wherein the CH2 domain and / or CH3 domain of Camelidae are replaced by the human CH2 domain and CH3 domain to obtain an immunoglobulin consisting of two heavy chains, each comprising a single domain antibody and the human CH2 domain and CH3 domain (but not containing any CH1 domain), and the immunoglobulin has effector functions conferred by the CH2 domain and CH3 domain, and the immunoglobulin can function without any light chain present.

[0056] In some embodiments, the polypeptide is as described in International Publication No. 2006 / 064136. In particular, the polypeptide can consist of i) a first fusion protein, wherein the CL constant domain of an antibody is fused at its N-terminus to the C-terminus of a single domain antibody (i.e., a single antibody directed against PN-1) described in the present invention, and ii) a second fusion protein, wherein the CH1 constant domain of an antibody is fused at its N-terminus to the C-terminus of a single domain antibody directed against an antigen different from PN-1. In another specific embodiment, the polypeptide consists of a first fusion protein (wherein the CH1 constant domain of the antibody is fused at its N-terminus to the C-terminus of a single domain antibody directed against an activation trigger molecule (e.g., CD16) on effector cells) and a second fusion protein (wherein the CL constant domain of the antibody is fused at its N-terminus to the C-terminus of the single domain antibody (i.e., PN-1) of the present invention).

[0057] In some embodiments, the polypeptide of the present invention - the following sequences: X 1 - T - W - X 4 - X 5 - E - I (wherein X 1 is S or D, X 4 is F or R, X 5 is R or L) having CDR1; and - A CDR2 having the following sequence: S-X2-X3-X4-W-H-A (where X2 is D or E, X3 is P or D, and X4 is T or G); and - A CDR3 having the sequence shown as SEQ ID NO: 3 or SEQ ID NO: 7 comprising at least one single-domain antibody.

[0058] In some embodiments, the polypeptide is a monovalent paratopic polypeptide. As used herein, the term "monovalent paratopic" polypeptide means a polypeptide comprising a single-domain antibody of the invention linked to a second single-domain antibody as defined herein, wherein these two single-domain antibodies are directed against the same epitope of one antigen.

[0059] In some embodiments, the monovalent paratopic antibody of the invention comprises at least two single-domain antibodies comprising a CDR1 having the sequence shown as SEQ ID NO: 1, a CDR2 having the sequence shown as SEQ ID NO: 2, and a CDR3 having the sequence shown as SEQ ID NO: 3.

[0060] In some embodiments, the monovalent paratopic polypeptide of the invention comprises at least two single-domain antibodies having at least 70% identity to the sequence shown as SEQ ID NO: 4.

[0061] In some embodiments, the monovalent paratopic polypeptide of the invention has at least 70% identity to the sequence shown as SEQ ID NO: 4 and comprises at least two single-domain antibodies comprising CDR1, CDR2, and CDR3 shown as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

[0062] In some embodiments, the monovalent paratopic polypeptide of the invention comprises at least two single-domain antibodies having the sequence shown as SEQ ID NO: 4.

[0063] In some embodiments, the monovalent paratope polypeptide of the present invention comprises the sequence shown as SEQ ID NO: 17.

[0064] In some embodiments, the monovalent paratope antibody of the present invention comprises at least two single-domain antibodies comprising a CDR1 having the sequence shown as SEQ ID NO: 5, a CDR2 having the sequence shown as SEQ ID NO: 6, and a CDR3 having the sequence shown as SEQ ID NO: 7.

[0065] In some embodiments, the monovalent paratope polypeptide of the present invention comprises at least two single-domain antibodies having at least 70% identity to the sequence shown as SEQ ID NO: 8.

[0066] In some embodiments, the monovalent paratope polypeptide of the present invention has at least 70% identity to the sequence shown as SEQ ID NO: 8 and comprises at least two single-domain antibodies comprising CDR1, CDR2, and CDR3 shown as SEQ ID NOs: 5, 6, and 7.

[0067] In some embodiments, the monovalent paratope polypeptide of the present invention comprises at least two single-domain antibodies having the sequence shown as SEQ ID NO: 8.

[0068] In some embodiments, the monovalent paratope polypeptide of the present invention comprises at least two single-domain antibodies comprising a CDR1 having the sequence shown as SEQ ID NO: 9, a CDR2 having the sequence shown as SEQ ID NO: 10, and a CDR3 having the sequence shown as SEQ ID NO: 11.

[0069] In some embodiments, the monovalent paratope polypeptide of the present invention comprises at least two single-domain antibodies having at least 70% identity to the sequence shown as SEQ ID NO: 12.

[0070] In some embodiments, the monovalent paratope polypeptide of the present invention has at least 70% identity to the sequence shown as SEQ ID NO: 12 and comprises at least two single-domain antibodies comprising CDR1, CDR2, and CDR3 shown as SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively.

[0071] In some embodiments, the monovalent paratope polypeptide of the present invention comprises at least two single-domain antibodies having the sequence shown as SEQ ID NO: 12.

[0072] In some embodiments, the polypeptide is a bivalent paratope polypeptide. As used herein, the term "bivalent paratope" polypeptide means a polypeptide comprising a single-domain antibody as defined herein and a second single-domain antibody, wherein the two single-domain antibodies can bind to two different epitopes of one antigen (e.g., PN-1), and the epitopes generally do not bind simultaneously to one single-specific immunoglobulin, e.g., a conventional antibody or one single-domain antibody. The bivalent paratope polypeptides described in the present invention are composed of single-domain antibodies having different epitope specificities and do not contain pairs of complementary variable domains that bind to the same epitope. Therefore, they do not compete with each other for binding to PN-1.

[0073] In some embodiments, the bivalent paratope polypeptide of the present invention comprises a B11 derivative as defined above and an F06 derivative as defined above.

[0074] In some embodiments, the bivalent paratopic antibody of the present invention comprises: i) a first single-domain antibody comprising a CDR1 having the sequence shown as SEQ ID NO: 1, a CDR2 having the sequence shown as SEQ ID NO: 2, and a CDR3 having the sequence shown as SEQ ID NO: 3; and ii) a second single-domain antibody comprising a CDR1 having the sequence shown as SEQ ID NO: 5, a CDR2 having the sequence shown as SEQ ID NO: 6, and a CDR3 having the sequence shown as SEQ ID NO: 7.

[0075] In some embodiments, the bivalent paratopic polypeptide of the present invention comprises: i) a first single-domain antibody having at least 70% identity to the sequence shown as SEQ ID NO: 4; and ii) a second single-domain antibody having at least 70% identity to the sequence shown as SEQ ID NO: 8.

[0076] In some embodiments, the bivalent paratopic polypeptide of the present invention comprises: i) a first single-domain antibody having at least 70% identity to the sequence shown as SEQ ID NO: 4 and comprising CDR1, CDR2, and CDR3 shown as SEQ ID NOs: 1, 2, and 3; and ii) a second single-domain antibody having at least 70% identity to the sequence shown as SEQ ID NO: 8 and comprising CDR1, CDR2, and CDR3 shown as SEQ ID NOs: 5, 6, and 7.

[0077] In some embodiments, the bivalent paratopic polypeptide of the present invention comprises: i) a first single-domain antibody having the sequence shown as SEQ ID NO: 4; and ii) a second single-domain antibody having the sequence shown as SEQ ID NO: 8.

[0078] In some embodiments, the bivalent paratopic polypeptide has the sequence shown as SEQ ID NO: 18.

[0079] In some embodiments, the bivalent paratopic polypeptide of the present invention comprises a B11 derivative as defined above and an A08 derivative as defined above.

[0080] In some embodiments, the bivalent paratopic antibody of the present invention comprises: i) a first single-domain antibody comprising a CDR1 having the sequence shown as SEQ ID NO: 1, a CDR2 having the sequence shown as SEQ ID NO: 2, and a CDR3 having the sequence shown as SEQ ID NO: 3; and ii) a second single-domain antibody comprising a CDR1 having the sequence shown as SEQ ID NO: 9, a CDR2 having the sequence shown as SEQ ID NO: 10, and a CDR3 having the sequence shown as SEQ ID NO: 11.

[0081] In some embodiments, the bivalent paratopic polypeptide of the present invention comprises: i) a first single-domain antibody having at least 70% identity to the sequence shown as SEQ ID NO: 4; and ii) a second single-domain antibody having at least 70% identity to the sequence shown as SEQ ID NO: 12.

[0082] In some embodiments, the bivalent paratopic polypeptide of the present invention comprises: i) a first single-domain antibody having at least 70% identity to the sequence shown as SEQ ID NO: 4 and comprising CDR1, CDR2, and CDR3 shown as SEQ ID NOs: 1, 2, and 3; and ii) a second single-domain antibody having at least 70% identity to the sequence shown as SEQ ID NO: 12 and comprising CDR1, CDR2, and CDR3 shown as SEQ ID NOs: 9, 10, and 11.

[0083] In some embodiments, the bivalent paratopic polypeptide of the present invention comprises: i) a first single-domain antibody having the sequence shown as SEQ ID NO: 4; and ii) a second single-domain antibody having the sequence shown as SEQ ID NO: 12.

[0084] In some embodiments, the bivalent paratopic polypeptide has the sequence shown as SEQ ID NO: 19.

[0085] In some embodiments, the bivalent paratopic polypeptide of the invention comprises an F06 derivative as defined above and an A08 derivative as defined above.

[0086] In some embodiments, the bivalent paratopic antibody of the invention comprises: i) a first single-domain antibody comprising a CDR1 having the sequence shown as SEQ ID NO: 5, a CDR2 having the sequence shown as SEQ ID NO: 6, and a CDR3 having the sequence shown as SEQ ID NO: 7; and ii) a second single-domain antibody comprising a CDR1 having the sequence shown as SEQ ID NO: 9, a CDR2 having the sequence shown as SEQ ID NO: 10, and a CDR3 having the sequence shown as SEQ ID NO: 11.

[0087] In some embodiments, the bivalent paratopic polypeptide of the invention comprises: i) a first single-domain antibody having at least 70% identity to the sequence shown as SEQ ID NO: 8; and ii) a second single-domain antibody having at least 70% identity to the sequence shown as SEQ ID NO: 12.

[0088] In some embodiments, the bivalent paratopic polypeptide of the invention comprises: i) a first single-domain antibody having at least 70% identity to the sequence shown as SEQ ID NO: 8 and comprising CDR1, CDR2, and CDR3 shown as SEQ ID NOs: 5, 6, and 7; and ii) a second single-domain antibody having at least 70% identity to the sequence shown as SEQ ID NO: 12 and comprising CDR1, CDR2, and CDR3 shown as SEQ ID NOs: 9, 10, and 11.

[0089] In some embodiments, the bivalent paratopic polypeptide of the invention comprises: i) a first single-domain antibody having the sequence shown as SEQ ID NO: 8; and ii) a second single-domain antibody having the sequence shown as SEQ ID NO: 12.

[0090] In some embodiments, the two single-domain antibodies of the bivalent paratopic polypeptide or monovalent paratopic polypeptide of the invention may be directly linked to each other (i.e., without using a linker) or linked via a linker. The linker is typically a linker peptide and would be selected according to the invention to enable binding of the two single-domain antibodies to each of at least two different epitopes of PN-1. Suitable linkers will depend inter alia on the epitopes, and in particular on the distance between the epitopes on PN-1 to which the single-domain antibodies bind, and will become apparent to those skilled in the art based on the disclosure herein after some limited routine experimentation. Also, the two single-domain antibodies that bind to PN-1 may be linked to each other via a third single-domain antibody (wherein the two single-domain antibodies may be directly linked to the third domain antibody or may be linked via a suitable linker). Such a third single-domain antibody may be, for example, a single-domain antibody that provides an extended half-life. For example, the latter single-domain antibody may be a single-domain antibody capable of binding to a (human) serum protein such as (human) serum albumin or (human) transferrin as further described herein. In some embodiments, two or more single-domain antibodies that bind to PN-1 are linked continuously (either directly or via a suitable linker), and a third (one) single-domain antibody (which, as described above, may provide an extended half-life) is connected directly or via a linker to one of these two or more of the foregoing single-domain antibodies. Suitable linkers are described herein in relation to the particular polypeptides of the invention and may include, for example but not by way of limitation, an amino acid sequence that preferably has a length of 9 or more amino acids, more preferably at least 17 amino acids in length, for example about 20 to 40 amino acids in length. However, the upper limit is not critical and is selected, for example, for reasons of convenience in the production of biopharmaceuticals of such polypeptides. The linker sequence may be a natural sequence or a non-natural sequence.When used for therapeutic purposes, the linker is preferably non-immunogenic in the subject to whom the anti-EGFR (epidermal growth factor receptor) polypeptide of the present invention is administered. One useful group of linker sequences are linkers derived from the hinge region of heavy chain antibodies as described in WO 96 / 34103 and WO 94 / 04678. Another example is a polyalanine linker sequence, such as Ala-Ala-Ala. Even more preferred examples of linker sequences are Gly / Ser linkers of various lengths, such as (gly4ser)3, (gly4ser)4, (gly4ser), (gly3ser), gly3, and (gly3ser2)3.

[0091] In some embodiments, it is contemplated that the polypeptide of the present invention used in the treatment methods of the present invention may be modified to improve its therapeutic efficacy. Such modifications of therapeutic compounds can be used to reduce toxicity, extend circulation time, or modify biodistribution. For example, the toxicity of an important therapeutic compound can be significantly reduced by combination with a wide variety of drug carrier vehicles that modify biodistribution.

[0092] In some embodiments, the polypeptide comprises a single domain antibody of the present invention linked to an immunoglobulin domain. For example, the polypeptide comprises a single domain antibody of the present invention linked to an Fc portion (e.g., human Fc). The Fc portion can be useful for increasing even the half-life and production of the single domain antibody of the present invention. For example, the Fc portion can bind to serum proteins, thus extending the half-life of the single domain antibody.

[0093] A strategy for improving the lifespan of drugs is the use of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the form of uptake into cells, change permeability through physiological barriers; and modify the rate of disappearance from the body. Water-soluble polymers containing a drug moiety as a terminal group, as part of the backbone, or as a pendant group on the polymer chain have been synthesized to achieve either a targeting effect or a sustained release effect.

[0094] Polyethylene glycol (PEG) is widely used as a drug carrier because of its high biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and reduce toxicity. PEG can be conjugated to active substances through the hydroxyl groups at the ends of the chains and via other chemical methods. However, PEG itself is limited to a maximum of two active substances per molecule. In a different approach, copolymers of PEG and amino acids have been explored as novel biomaterials that retain the biocompatible properties of PEG but have the advantage of having multiple attachment points per molecule (allowing more drugs to be loaded) and can be synthetically designed to fit various applications. Those skilled in the art are aware of PEGylation techniques for the effective modification of drugs. For example, drug delivery polymers consisting of alternating polymers of PEG and trifunctional monomers (such as lysine) are being used by VectraMed (Plainsboro, NJ). The PEG chains (typically below 2000 daltons) are linked to the a- and e-amino groups of lysine through stable urethane bonds. Such copolymers provide reactive pendant groups (the carboxylic acid groups of lysine) at precisely controlled and predetermined intervals along the polymer chain while retaining the desired properties of PEG. The reactive pendant groups can be used for derivatization, crosslinking, or conjugation with other molecules. These polymers are useful for generating stable, long-circulating prodrugs by varying the molecular weight of the polymer, the molecular weight of the PEG segment, and the cleavable bond between the drug and the polymer. The molecular weight of the PEG segment affects the spacing of the drug / linker complex and the amount of drug per molecular weight of the conjugate (larger drugs are loaded onto smaller PEG segments). Generally, increasing the total molecular weight of the block copolymer conjugate will extend the circulation half-life of the conjugate. Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the glomerular filtration threshold (e.g., less than 45 kDa).Furthermore, by using a linker on the polymer backbone that is important for maintaining the circulation half-life and in vivo distribution, the therapeutic agent can be maintained in prodrug form until it is released from the backbone polymer by a specific trigger, typically enzymatic activity within the target tissue. For example, this type of tissue-activated drug delivery is particularly useful when delivery to a specific site of in vivo distribution is required and the therapeutic agent is released at or near the diseased site. The linker libraries used for activated drug delivery are known to those of skill in the art and can be based on enzyme kinetics, the ubiquity of the activating enzyme, and the cleavage specificity of the selected disease-specific enzyme (see, e.g., the technology established by VectraMed, Plainsboro, NJ). Such linkers can be used to modify the polypeptides of the invention described herein for therapeutic agent delivery.

[0095] According to the present invention, the single domain antibodies and polypeptides of the invention can be produced by conventional automated peptide synthesis methods or by recombinant expression. The general principles for designing and making proteins are well known to those of skill in the art.

[0096] The single domain antibodies and polypeptides of the invention can be synthesized in solution or on a solid support according to conventional techniques. A variety of automated synthesizers are commercially available and can be used according to known protocols such as those described in Stewart and Young; Tam et al., 1983; Merrifield, 1986 and Barany and Merrifield, Gross and Meienhofer, 1979. The single domain antibodies and polypeptides of the invention may also be synthesized by solid phase techniques using an exemplary peptide synthesizer such as the Model 433A from Applied Biosystems. The purity of any given protein made through automated peptide synthesis or through recombinant methods can be determined using reverse phase HPLC analysis. The chemical authenticity of each peptide can be established by any method well known to those of skill in the art.

[0097] Nucleic Acids, Vectors, Recombinant Host Cells, and Their Use As an alternative to automated peptide synthesis, recombinant DNA technology can be used, where the nucleotide sequence encoding the selected protein is inserted into an expression vector, transformed or transfected into a suitable host cell, and cultured under conditions suitable for expression as described hereinbelow. The recombinant method is particularly preferred for producing longer polypeptides.

[0098] A variety of expression vector / host systems can be used to contain and express sequences encoding peptides or proteins. These include bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors (Giga-Hama et al., 1999); insect cell lines infected with viral expression vectors (e.g., baculovirus, see Ghosh et al., 2002); plant cell lines transfected with viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid; see, for example, Babe et al., 2000); or animal cell lines, but are not limited thereto. Those skilled in the art know various techniques for optimizing protein expression in mammals. For example, Kaufman, 2000; Colosimo et al., 200. Mammalian cells useful for the production of recombinant proteins include, but are not limited to, VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (e.g., COS-7), W138 cells, BHK cells, HepG2 cells, 3T3 cells, RIN cells, MDCK cells, A549 cells, PC12 cells, K562 cells, and 293 cells. Exemplary protocols for the recombinant expression of peptide substrates or fusion polypeptides in bacteria, yeast, and other invertebrates are known to those skilled in the art and are briefly described herein below. Mammalian host systems for the expression of recombinant proteins are also well known to those skilled in the art. The host cell line can be selected for specific capabilities useful for processing the expressed protein or for generating specific post-translational modifications that may be useful in conferring activity to the protein. Such modifications of polypeptides include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing that cleaves the "prepro" form of the protein can also be important for correct insertion, folding, and / or function.A variety of host cells, such as CHO cells, HeLa cells, MDCK cells, 293 cells, WI38 cells, etc., have specific cellular machinery and characteristic mechanisms for such post-translational activity and can be selected to ensure correct modification and processing of the introduced foreign protein.

[0099] In the recombinant production of the single-domain antibodies and polypeptides of the present invention, it will be necessary to use a vector containing a polynucleotide molecule for encoding the single-domain antibodies and polypeptides of the present invention. Methods for preparing such vectors and methods for producing host cells transformed with such vectors are well known to those skilled in the art.

[0100] Accordingly, a further object of the present invention relates to a nucleic acid molecule encoding the single-domain antibody and / or polypeptide described in the present invention.

[0101] Typically, said nucleic acid is a DNA molecule or an RNA molecule, which may be contained in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage, or viral vector. The terms "vector", "cloning vector" and "expression vector" as used herein mean a vehicle by which a DNA sequence or an RNA sequence (e.g., a foreign gene) can be introduced into a host cell, thus transforming the host and facilitating the expression (e.g., transcription and translation) of the introduced sequence. The terms "expression vector", "expression construct" or "expression cassette" are used synonymously throughout this specification and mean any kind of gene construct containing a nucleic acid encoding a gene product, a part or all of which nucleic acid coding sequence can be transcribed.

[0102] Accordingly, a further aspect of the present invention relates to a vector comprising the nucleic acid of the present invention. Such a vector may include regulatory sequences, such as promoters, enhancers, terminators, etc., which, when administered to a subject, cause or direct the expression of the antibody. Examples of promoters and enhancers used in expression vectors for animal cells include the early promoter and enhancer of SV40 (Mizukami T. et al. 1987), the LTR promoter and enhancer of Moloney murine leukemia virus (Kuwana Y et al. 1987), the promoter of immunoglobulin H chain (Mason JO et al. 1985) and enhancer (Gillies SD et al. 1983), etc. Any expression vector for animal cells can be used as long as it can insert and express the gene encoding the human antibody constant region. Examples of suitable vectors include pAG107 (Miyaji H et al. 1990), pAGE103 (Mizukami T et al. 1987), pHSG274 (Brady G et al. 1984), pKCR (O'Hare K et al. 1981), pSG1βd2-4- (Miyaji H et al. 1990), etc. Other examples of plasmids include replicating plasmids containing an origin of replication, or integrating plasmids, such as pUC, pcDNA, pBR, etc. Other examples of viral vectors include adenoviral vectors, retroviral vectors, herpes viral vectors, and adeno-associated viral vectors. Such recombinant viruses may be produced by techniques known in the art, for example, by transfecting packaging cells, or by transient transfection using helper plasmids or viruses. Typical examples of viral packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc.Detailed protocols for producing such replication-defective recombinant viruses can be found, for example, in WO 95 / 14785, WO 96 / 22378, U.S. Patent No. 5,882,877, U.S. Patent No. 6,013,516, U.S. Patent No. 4,861,719, U.S. Patent No. 5,278,056, and WO 94 / 19478.

[0103] The selection of an expression vector suitable for the expression of the peptide or polypeptide of the present invention, of course, depends on the specific host cell that is intended to be used, which is within the skill of the art.

[0104] For expression, the vector is provided with appropriate signals such as enhancers / promoters, which are derived from both viral and mammalian sources and can be used to drive the expression of the nucleic acid of interest in the host cell. Usually, the nucleic acid to be expressed is under the transcriptional control of a promoter. A "promoter" refers to a DNA sequence that is recognized by the synthetic machinery of the cell or introduced synthetic machinery and is required to initiate the specific transcription of a gene. The nucleotide sequence is operably linked when the regulatory sequence is functionally related to the DNA encoding the protein of interest (e.g., a single domain antibody). Thus, a promoter nucleotide sequence is operably linked to a given DNA sequence when the promoter nucleotide sequence directs the transcription of the sequence.

[0105] A further aspect of the present invention relates to host cells that have been transfected, infected, or transformed with the nucleic acids and / or vectors described in the present invention.

[0106] The term "transformation" means the introduction of an "exogenous" (i.e., exogenous or extracellular) gene, DNA sequence, or RNA sequence into a host cell, whereby the host cell will express the introduced gene or sequence to produce the desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. A host cell that receives and expresses the introduced DNA or RNA is "transformed".

[0107] Using the nucleic acids of the present invention, the antibodies of the present invention can be produced in a suitable expression system. The term "expression system" means, for example, a host cell and a compatible vector under suitable conditions for the expression of a protein encoded by foreign DNA, which is carried by a vector and introduced into a host cell. General expression systems include E. coli host cells and plasmid vectors, insect host cells, and baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria) and eukaryotic cells (e.g., yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include E. coli, Kluyveromyces, or Saccharomyces yeast, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.), and primary or established mammalian cell cultures (e.g., produced from lymphocytes, fibroblasts, embryonic cells, epithelial cells, nerve cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cells (ATCC CRL1581), mouse P3X63-Ag8.653 cells (ATCC CRL1580), CHO cells deficient in dihydrofolate reductase (hereinafter referred to as the "DHFR gene" in this specification) (Urlaub G et al; 1980), rat YB2 / 3HL.P2.G11.16Ag.20 cells (ATCC CRL1662, hereinafter referred to as "YB2 / 0 cells" in this specification), and the like. The present invention also relates to a method for producing a recombinant host cell expressing the antibody according to the present invention, the method comprising: (i) introducing the recombinant nucleic acid or vector as described above into a competent host cell in vitro or ex vivo; (ii) culturing the obtained recombinant host cell in vitro or ex vivo; and (iii) optionally, selecting cells that express and / or secrete the antibody. Such recombinant host cells can be used for the production of the antibodies of the present invention.

[0108] The antibodies of the present invention are appropriately isolated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0109] Therapeutic Methods and Uses The single-domain antibodies and polypeptides of the present invention are used as inhibitors of protease nexin-1 (PN-1).

[0110] Accordingly, the single-domain antibodies and polypeptides of the present invention are particularly suitable for the prevention or treatment of hemorrhagic diseases in subjects in need thereof.

[0111] The present invention also relates to a method for preventing or treating a hemorrhagic disease in a subject in need thereof, comprising the step of administering to the subject an effective amount of the single-domain antibody and / or polypeptide of the present invention.

[0112] As used herein, the term "subject" refers to a mammal. In a preferred embodiment of the present invention, the subject described in the present invention refers to any subject (preferably a human) suffering from or prone to suffering from a hemorrhagic disease. In another preferred embodiment, the subject described in the present invention refers to any subject (preferably a human) suffering from or prone to suffering from a hemorrhagic disease associated with a deficiency of factor V, factor VIII, factor IX, and / or factor XI.

[0113] As used herein, the term "bleeding disorder" has its ordinary meaning in the art and refers to bleeding disorders as revised in the World Health Organization's classification D65-D69. The term "bleeding disorder" also refers to permanent bleeding disorders, rare bleeding disorders, bleeding disorders associated with deficiencies of factor V, factor VIII, factor IX and / or factor XI. The term "bleeding disorder" also refers to hemophilia, hereditary factor VIII deficiency (unspecified hemophilia, hemophilia A, classical hemophilia); hereditary factor IX deficiency (Christmas disease, factor IX deficiency with functional abnormality, deficiency of plasma thromboplastin component [PTC], hemophilia B); coagulation abnormalities such as von Willebrand disease, vascular hemophilia, factor VIII deficiency associated with vascular abnormalities, vascular hemophilia; hereditary factor XI deficiency (hemophilia C, deficiency of plasma thromboplastin antecedent [PTA]); hereditary deficiencies of other coagulation factors (congenital afibrinogenemia, deficiency of accelerin, proaccelerin, factor I [fibrinogen], factor II [prothrombin], factor V [unstable], factor VII [stable], factor X [Stuart-Prower factor], factor XII [Hageman factor], and factor XIII [fibrin stabilizing factor], abnormal fibrinogenemia (congenital), hypoproconvertinemia, Owren disease); bleeding disorders due to circulating anticoagulants (bleeding during long-term use of anticoagulants, hyperheparinaemia, increased antithrombin, anti-VIIIa factor antibody, anti-IXa factor antibody, anti-Xa antibody, and anti-XIa factor antibody, Coding-Hint); acquired coagulation factor deficiencies (coagulation factor deficiencies due to liver disease and vitamin K deficiency); primary thrombophilia (activated protein C resistance [factor V Leiden mutation], deficiencies of antithrombin, protein C, and protein S, mutation of the prothrombin gene); other thrombophilias (presence of anticardiolipin syndrome, antiphospholipid syndrome, lupus anticoagulant); bleeding disorders such as purpura, allergic purpura, qualitative platelet abnormalities, thrombocytopenia, capillary fragility (hereditary) and vascular pseudohemophilia. The term "bleeding disorder" also refers to bleeding episodes in bleeding disorders such as hemophilia and other rare bleeding disorders.

[0114] In some embodiments, the bleeding disorder is a bleeding disorder associated with a deficiency of factor V, factor VIII, factor IX, and / or factor XI.

[0115] In some embodiments, the bleeding disorder is hemophilia.

[0116] In some embodiments, the bleeding disorder is hemophilia A or hemophilia B.

[0117] In some embodiments, the bleeding disorder is mild or moderate hemophilia A.

[0118] In some embodiments, the bleeding disorder is mild or moderate hemophilia B.

[0119] The term "mild hemophilia A" has its ordinary meaning in the art and refers to a bleeding disorder defined by 5-40% factor VIII in plasma.

[0120] The term "moderate hemophilia A" has its ordinary meaning in the art and refers to a bleeding disorder defined by 1-5% factor VIII in plasma.

[0121] The term "severe hemophilia A" has its ordinary meaning in the art and refers to a bleeding disorder defined by less than 1% factor VIII in plasma.

[0122] The term "mild hemophilia B" has its ordinary meaning in the art and refers to a bleeding disorder defined by 5-40% factor IX in plasma.

[0123] The term "moderate hemophilia B" has its ordinary meaning in the art and refers to a bleeding disorder defined by 1-5% factor IX in plasma.

[0124] The term "severe hemophilia B" has its ordinary meaning in the art and refers to a bleeding disorder defined by less than 1% factor IX in plasma.

[0125] Accordingly, the invention also relates to the single domain antibodies and polypeptides of the invention for use in preventing the development of resistance to clotting factors.

[0126] In some embodiments, the single domain antibody or polypeptide of the invention is used in combination with classical treatments of bleeding disorders.

[0127] Accordingly, the invention refers to a method for preventing or treating a bleeding disorder in a subject in need thereof, comprising administering to the subject i) an effective amount of the single domain antibody and / or polypeptide of the invention, and ii) a classical treatment of the bleeding disorder.

[0128] As used herein, the term "classical treatment of bleeding disorder" refers to any natural or synthetic compound used for the treatment of bleeding disorders.

[0129] According to the invention, the compounds used for the treatment of bleeding disorders can be selected from the group consisting of clotting factors; desmopressin, antifibrinolytics such as tranexamic acid and epsilon - aminocaproic acid; fibrin sealants; antibodies that mimic the function of clotting factors such as emicizumab.

[0130] As used herein, the term "antibody that mimics the function of clotting factor" refers to an antibody that exhibits the activity of a clotting factor that is deficient in hemophilia. An antibody that mimics the function of factor VIII, such as emicizumab, can bind to both activated factor IX and factor X and mediate the activation of factor X.

[0131] In some embodiments, the single domain antibody or polypeptide of the invention is used in combination with an antibody that mimics factor VIII.

[0132] In some embodiments, the single domain antibodies or polypeptides of the invention are used in combination with emicizumab.

[0133] Accordingly, in a further aspect, the invention relates to the single domain antibodies and polypeptides of the invention in combination with emicizumab for use in the prevention or treatment of bleeding disorders in a subject in need thereof.

[0134] As used herein, the term "coagulation factor" has its ordinary meaning in the art and refers to factor VIII (FVIII), factor IX (FIX), factor VIIa (FVIIa), plasma-derived activated prothrombin complex, and fibrinogen. The term "coagulation factor" also relates to recombinant or purified coagulation factors.

[0135] In some embodiments, the single domain antibodies or polypeptides of the invention are used in combination with one or more coagulation factors.

[0136] Accordingly, in a further aspect, the invention relates to the single domain antibodies and polypeptides of the invention in combination with one or more coagulation factors for use in the prevention or treatment of bleeding disorders in a subject in need thereof.

[0137] Typically, the single domain antibodies and polypeptides of the invention and classical treatments for bleeding disorders as described above are administered to a subject in a therapeutically effective amount.

[0138] The "therapeutically effective amount" of the single domain antibodies and polypeptides of the present invention as described above means a sufficient amount of the inhibitor. However, it will be understood that the total daily dosage of the inhibitors and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dosage level in any particular subject will depend on the disorder being treated, and the severity of the disorder; the activity of the specific inhibitor being used; the specific composition being used, the age, weight, general health, sex, and diet of the subject; the time of administration, the route of administration, and the rate of excretion of the specific inhibitor being used; the duration of the treatment; drugs used in combination with or simultaneously with the specific inhibitor being used; and various factors including similar factors well known in the medical arts. For example, it is well within the skill of the art to start with a dosage of the single domain antibodies and polypeptides of the present invention at a level lower than that required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the product may vary over a wide range of 0.01 to 1,000 mg per adult per day. Typically, the composition contains 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250, and 500 mg of the inhibitor of the present invention for adjustment of the dosage according to the symptoms in the subject to be treated. Pharmaceutical products typically contain from about 0.01 mg to about 500 mg of the single domain antibodies and polypeptides of the present invention, preferably from 1 mg to about 100 mg of the single domain antibodies and polypeptides of the present invention. The effective amount of the drug is usually supplied at dosage levels of from 0.0002 mg / kg to about 20 mg / kg (body weight) per day, particularly from about 0.001 mg / kg to 7 mg / kg (body weight) per day.

[0139] In certain embodiments, the single domain antibodies and polypeptides described in the present invention can be used at concentrations from 0.01 μM to 20 μM, and in particular, the inhibitors of the present invention can be used at concentrations of 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 20.0 μM.

[0140] The therapeutically effective amount of the coagulation factor of the present invention is well known in the art. Typically, the therapeutically effective amount of the coagulation factor relates to from about 10 IU to 300 IU / kg (body weight), particularly from about 10 IU to 100 IU / kg (body weight) of the coagulation factor. In particular, the therapeutically effective amount of the coagulation factor relates to amounts of the coagulation factor of 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0 IU / kg (body weight).

[0141] In certain embodiments, the coagulation factors described in the present invention may be used at low doses to avoid the development of resistance to the coagulation factor. Typically, the term "low dose" refers to from about 5 IU to 40 IU / kg (body weight) of the coagulation factor (e.g., factor VIII). In particular, the term "low dose" refers to amounts of the coagulation factor of about 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0 IU / kg (body weight).

[0142] According to the present invention, the single domain antibodies and polypeptides of the present invention are administered sequentially or simultaneously with one or more coagulation factors.

[0143] Pharmaceutical Compositions and Kits of the Present Invention Typically, the single domain antibodies and polypeptides of the present invention can form pharmaceutical compositions in combination with pharmaceutically acceptable excipients and optionally a sustained release matrix, such as a biodegradable polymer. Accordingly, the single domain antibodies and polypeptides of the present invention are administered to a subject in the form of a pharmaceutical composition.

[0144] "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when appropriately administered to a mammal, particularly a human. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type.

[0145] In the pharmaceutical composition of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical, or rectal administration, the active ingredient can be administered to animals and humans, alone or in combination with another active ingredient, in a unit dosage form, as a mixture with a conventional pharmaceutical support. Suitable unit dosage forms include oral route dosage forms, such as tablets, gel capsules, powders, granules, and oral suspensions or solutions, sublingual and buccal administration dosage forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal, and intranasal administration dosage forms, as well as rectal administration dosage forms.

[0146] Preferably, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for injectable formulations. These can be, in particular, isotonic and sterile aqueous saline solutions (such as monosodium phosphate or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc., or mixtures of such salts), or, optionally, dried, especially lyophilized, compositions that allow the reconstitution of the injection solution when sterile water or physiological saline is added.

[0147] Pharmaceutical dosage forms suitable for injection use include sterile aqueous solutions or dispersions; formulations containing sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the immediate preparation of sterile injection solutions or dispersions. In all cases, the dosage form must be sterile and must have a fluidity such that it can be easily handled with a syringe. It must be stable under the conditions of manufacture and storage and must be preserved from the contaminating action of microorganisms such as bacteria and fungi.

[0148] Solutions containing the inhibitor of the present invention as a free base or a pharmaceutically acceptable salt can be prepared in water appropriately mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof and in oils. Under normal storage and use conditions, these preparations contain preservatives that prevent the growth of microorganisms.

[0149] The inhibitor of the present invention can be formulated into a composition in neutral form or in the form of a salt. Pharmaceutically acceptable salts include acid addition salts (formed using the free amino groups of the protein), which are formed using inorganic acids such as hydrochloric acid or phosphoric acid, or such organic acids as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed using free carboxyl groups can also be derived from inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, or iron hydroxide, and such organic bases as isopropylamine, trimethylamine, histidine, procaine, etc.

[0150] The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Suitable fluidity can be maintained, for example, by the use of coating agents such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Protection against the action of microorganisms can be brought about by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents such as sugars or sodium chloride. Prolongation of the absorption of injectable compositions can be brought about by using substances that delay absorption in the composition, such as aluminum monostearate and gelatin.

[0151] Sterile injectable solutions are prepared by incorporating the required amount of the active compound in a suitable solvent, together with, if necessary, several of the other ingredients enumerated above, and then filtering sterilizing. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques, by which powders of the active ingredient and any additional desired ingredients are obtained from their previously sterile-filtered solutions.

[0152] Upon formulation, the liquid formulation will be administered in a manner compatible with the dosage formulation and in therapeutically effective amounts. The formulations are easily administered in various dosage forms, such as injection solutions of the above types, but drug release capsules and the like can also be used.

[0153] For parenteral administration in an aqueous solution, for example, the liquid formulation should be appropriately buffered if necessary, and the liquid diluent should first be made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this regard, the sterile aqueous media that can be used will be known to those skilled in the art in view of the present disclosure. Some variation in dosage will necessarily be made depending on the condition of the subject being treated. The person responsible for administration will determine the appropriate dosage for each individual subject in any event.

[0154] In addition to the inhibitors of the present invention formulated for parenteral administration such as intravenous or intramuscular injection, other pharmaceutically acceptable dosage forms include, for example, tablets or other solid forms for oral administration; liposome formulations; sustained release capsule formulations; and any other dosage forms currently in use.

[0155] The pharmaceutical composition of the present invention may contain any further other agents used for the prevention or treatment of hemorrhagic diseases.

[0156] In one embodiment, the additional active agent may be included in the same composition or administered separately.

[0157] In another embodiment, the pharmaceutical composition of the present invention relates to a combined preparation for the simultaneous, separate, or sequential use for the prevention and treatment of hemorrhagic diseases.

[0158] Finally, the present invention also provides a kit comprising at least one single domain antibody or polypeptide of the present invention. Kits containing single domain antibodies or polypeptides against PN-1 of the present invention find use in therapeutic methods.

[0159] The present invention will be further described by the following drawings and examples. However, these examples and drawings should not be construed as limiting the scope of the present invention in any way.

Brief Description of the Drawings

[0160]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

[0161] Example 1: Materials and methods The inventors determined the inhibition of the antithrombin catalytic activity of human and mouse PN-1 using a chromogenic substrate for thrombin S-2238 (Chromogenix, Montpellier, France). S-2238 is a short peptide specific for thrombin, covalently linked to pNA (4-nitroaniline). When S-2238 is cleaved by thrombin, free pNA is released, which can be detected at 405 nm by a spectrophotometer. Human or mouse PN-1 (20 nM) was incubated at 37 °C for 5 min in Hepes buffer (20 mM Hepes, 0.15 M NaCl, pH 7.5, 0.1% human serum albumin) with or without a diabody (10 or 1 μM of B11-Bv, B11-F06, or B11-A08), then incubated with thrombin (1 nM) at 37 °C for 10 min, and then the chromogenic substrate S-2238 (2 mM) was added. The change in absorbance at 405 nm was recorded for 90 min. Residual thrombin activity was expressed as the ratio of the activity measured in the presence of PN-1 incubated with or without the diabody to the activity of thrombin alone.

[0162] The inventors confirmed the specificity of the diabody for PN-1 by measuring the effect of the diabody on the inhibition of uPA by PAI-1 using the chromogenic substrate PNAPEP-1344 (Chromogenix, Montpellier, France). Human PAI (10 nM) was incubated at 37 °C for 5 min in PBS buffer containing 0.1% human serum albumin with or without a diabody (10 or 1 μM of B11-Bv, B11-F06, or B11-A08), then incubated with uPA (1.3 nM) at 37 °C for 10 min, and then the chromogenic substrate S-1344 (0.4 mM) was added to initiate the reaction. The change in absorbance at 405 nm was recorded for 120 min. Residual uPA activity was expressed as the ratio of the activity measured in the presence of PN-1 incubated with or without the diabody to the activity of uPA alone.

[0163] The inventors determined the IC50 of the bivalent hybrid against human PN-1 using a chromogenic substrate for thrombin S-2238 (Chromogenix, Montpellier, France). Human or mouse PN-1 (20 nM) was incubated with increasing concentrations of the bivalent hybrid (10 or 1 μM of B11-Bv, B11-F06, or B11-A08) in Hepes buffer (20 mM Hepes, 0.15 M NaCl, pH 7.5, 0.1% human serum albumin) at 37 °C for 5 min, then incubated with thrombin (1 nM) at 37 °C for 10 min, and then the chromogenic substrate S-2238 (2 mM) was added. The change in absorbance at 405 nm was recorded for 90 min. The % inhibition in the graph was equal to the % of PN-1 activity.

[0164] Results Inhibition of antithrombin activity of human and mouse PN-1 Using a llama-derived library, 52 candidates that recognize PN-1 were obtained by a total of 7 rounds of panning and 940 tested VHHs. Three nanobodies (B11, F06, and A08) were found to be able to inhibit the antithrombin activity of human and mouse PN-1. Combinations of dimers were tested that showed better affinity and inhibitory activity. Indeed, the hybrids B11-Bv, B11-F06, and B11-A08 were able to inhibit the antithrombin activity of human and mouse PN-1 (Figs. 2E - F).

[0165] The first specific anti-PN-1 antibody Careful characterization of candidates recognizing the llama-derived library led to 52 candidates that also recognize plasminogen activator inhibitor-1 (PAI-1) by all these VHHs. Furthermore, all commercially available anti-PN-1 antibodies tested by the inventors were found to be non-specific. Herein, the inventors found that the single domain antibodies B11, F06 and A08, as well as the bivalent B11-Bv; B11-F06, B11-A08 of the hybrids, efficiently bind to both human and mouse PN-1 without cross-reaction with PAI-1, the most phylogenetically close serpins homolog of PN-1 (Figure 2E-G).

[0166] The inventors previously showed that PN-1 inhibitors, such as anti-PN-1 antibodies, improve thrombin generation in patients with mild and moderate hemophilia. The inventors establish that blockade of PN-1 plays a role in the treatment of bleeding disorders. Herein, the inventors describe the first specific single domain antibodies that can block the antithrombin activity of both mouse and human PN-1 without cross-reacting with other serpins. These single domain antibodies can be a powerful tool, particularly in the treatment of bleeding disorders in hemophilia patients.

[0167] Example 2: Materials and Methods Materials Isopropyl β-D-1-thiogalactopyranoside (IPTG), human serum albumin (HSA), bovine serum albumin (BSA) were from Sigma-Aldrich (Saint-Quentin Fallavier, France). Polyclonal rabbit anti-c-Myc tag antibody labeled with peroxidase, polyclonal rabbit anti-6x histidine tag antibody were from Abcam (Paris, France). Human and mouse PN-1 and human α-thrombin were prepared as described 20、21The reagents for prothrombin time and activated prothrombin time were manufactured by Stago Diagnostica (Ané rue sur Seine, France). The chromogenic substrates PNAPEP-1344 and PNAPEP-0238 were manufactured by Cryopep (Montpellier, France). Dynabeads M-450 epoxy beads, Escherichia coli (E. coli) TG1 cells, terrific broth (TB) medium, and lysogeny broth (LB) medium were manufactured by Thermo Fisher Scientific (Villebon sur Yvette, France). Plasminogen activator inhibitor-1 (PAI-1) was manufactured by Stago BNL (Reuil, France). AEBSF was manufactured by vWR (Fontenay sous Bois, France).

[0168] Construction of anti-PN-1 VHH from a lymphocyte library by phage display The immunization of one llama (Llama glama) was outsourced to the Cancer Research Center (University of Aix-Marseille, Marseille, France). 22 Briefly, the llama was immunized with an equal ratio of Freund's incomplete adjuvant and 100 μg of a mixture of human or mouse PN-1. After blood collection, mRNA was extracted from lymphocytes, and a VHH library was constructed as described. 22~24 The library contained a DNA fragment encoding VHH, which was cloned into the pHEN6-phagemid vector and transformed in 25 electrocompetent E. coli TG1 cells (Thermo Fisher Scientific) to generate a library of 0.9×10 8 clones. The TG1 VHH library was infected with M13K07 helper phage to enable the expression of VHH on the surface.

[0169] Negative and positive selection of VHH To reduce the number of VHHS that cross-react with PAI-1, phage particles were first incubated with beads coated with 100 μg of PAI-1 (for 1 hour at room temperature in PBS-3% bovine serum albumin). Unbound phage was passed over beads coated with a mixture of mouse PN-1 and human PN-1 (50 μg each). The captured phage was eluted with 0.5 mg / ml trypsin. Three consecutive rounds of enrichment were performed.

[0170] Selection of anti-PN1 specific VHHS After infecting TG1 bacteria with the isolated phage, the cells were grown for 4 hours in TB medium with stirring, then 1 mM IPTG was added to induce the expression of VHHS at 30 °C. Eighteen hours after induction, the cells were centrifuged and the periplasmic extract was lysed in TES buffer (200 mM Tris HCl, pH 8, 0.5 mM EDTA, and 500 mM sucrose) at 4 °C for 1 hour, then collected after lysing for 30 minutes in TES buffer diluted 4-fold with PBS. The released soluble protein was tested for binding to PN-1, PAI-1, or bovine serum albumin. Protein in PBS (1 μg / well) was coated onto Nunc maxisorp plates (Thermo Fisher Scientific) overnight at 4 °C. After saturation (PBS-bovine serum albumin 3%, 1 hour at 37 °C), the bound VHHS were probed using a polyclonal anti-6x histidine tag antibody conjugated to alkaline phosphatase, detected via hydrolysis of 3,3’,5,5’-tetramethylbenzidine (TMB), and the absorbance at 450 nm was decoded.

[0171] Isolation of anti-PN-1 VHHS from a synthetic library In a second approach, a synthetic VHHS library was used to isolate anti-PN-1 VHHS (Hybrigenics Services SAS, Paris, France). 3 × 10 9This hs2dAb (humanized synthetic single domain antibody) phage display library containing individual VHHs was first incubated with beads coated with human PAI-1 to reduce non-specific binding substances. Subsequently, the unbound VHHs were incubated with beads coated with mouse or human PN-1. A total of three phage displays were performed, and the removal process was repeated during each. After three times, 90 E. coli clones were randomly picked up and analyzed for binding to human and mouse PN-1 as well as PAI-1. Phage clones showing a signal increased by more than 5-fold to PN-1 compared to PAI-1 were judged as specific binding substances to PN-1 (mouse, human, or both). By sequencing the positive clones, the presence of 18 different positive VHHs was found. Four of these recognized both mouse PN-1 and human PN-1 and were subcloned into the pHEN2 vector for further characterization.

[0172] Subcloning, Expression, and Purification of VHH Anti-PN-1 VHHs were directly transformed into E. coli WK6 cells. Each clone was first grown overnight at 37°C with gentle stirring in 10 mL of LB medium / 100 μg / mL ampicillin / 2% glucose / 1 mM MgCl 2 and then 3 mL of this pre-culture was used to inoculate 330 mL of TB medium / 100 μg / mL ampicillin / 0.1% glucose / 1 mM MgCl 2It was inoculated. The culture solution was stirred until OD600 reached 0.8 - 1 (170 rpm, 37 °C). Thereafter, the expression of VHH was induced by 1 mM IPTG and the medium was left to grow overnight (170 rpm, 28 °C). The periplasmic protein was extracted by sonication for 30 minutes using a cycle of 10 seconds on / 10 seconds off (Fisher Scientific, Illkirch, France). The lysis rate was confirmed by (absorbance after lysis / absorbance before lysis) × 100. Next, the histidine-tagged VHH was purified via Co2+ affinity chromatography as instructed (vWR). Minor contaminants were removed via size exclusion chromatography using 20 mM Hepes (pH 7.4) / 0.1 M NaCl as the equilibration buffer. The purified VHH showed a homogeneity of over 95% as evaluated via SDS-Page and Coomassie staining. For the bivalent VHH, two copies of the monovalent sdAb were separated by a linker consisting of [GGGs]3 and a C-terminal 6x histidine tag, enabling purification via Co2+ affinity chromatography.

[0173] ELISA for human PN-1 and mouse PN-1 Microtiter half-well plates (Greiner Bio-One, Courtaboeuf, France) were coated with 0.5 μg / well of mouse PN-1 or human PN-1 at 4 °C overnight, and then incubated with various concentrations of VHH (0 - 20 μg / mL) in PBS / 0.1% bovine serum albumin 0.1% Tween-20 at 37 °C for 2 hours. The bound anti-PN-1 VHH was probed in the same buffer using a peroxidase-labeled polyclonal anti-cMyc tag antibody for monovalent VHH or an anti-6x histidine tag antibody for bivalent VHH, detected via hydrolysis of TMB, and the absorbance at 450 nm was decoded.

[0174] Inhibition of thrombin activity by PN-1 In a 96-well plate (Greiner Bio-One), various concentrations of anti-PN-1 VHH were incubated with 10 nM of PN-1 for 15 minutes at room temperature in 20 mM Hepes / 0.15 M NaCl / 0.1% human serum albumin. Then, 1 nM of purified thrombin was added to the wells and incubated for 30 minutes at room temperature. Thrombin activity was quantified by measuring the rate of release of p-nitroaniline from the chromogenic substrate H-D-Phe-Pip-Arg-pNA (PNAPEP-0238) (0.2 mM) at 37 °C and decoded at 405 nm using a multi-well plate reader. A slightly modified protocol was used to calculate the IC50 for each VHH: anti-PN-1 VHH was used at concentrations from 0 to 2 μM, the incubation of VHH was carried out for 5 minutes, and then it was incubated with 1 nM of thrombin for 10 minutes. The PNAPEP-0238 substrate was added at a concentration of 0.2 mM and incubated at 37 °C for 2 hours. Quantification of thrombin activity was performed as described above.

[0175] Inhibition of thrombin activity by endogenous PN-1 Blood samples were collected in tubes with the anticoagulant ACD-A solution. The blood was centrifuged at 120 g for 15 minutes to recover platelet-rich plasma (PRP). Then, 2 μL / mL of apyrase (5 mg / mL) and 1 μL / mL of PGE1 (10 mM) were added, and the platelet-rich plasma was centrifuged at 1200 g at 20 °C for 12 minutes. The pellet was gently resuspended in wash buffer (3.6 mM citrate, 0.5 mM glucose, 0.5 mM KCl, 10.3 mM NaCl, 2 mM CaCl2, pH 6.5, 0.30% bovine serum albumin, 2 μL / mL of apyrase (5 mg / mL) and 1 μL / mL of PGE1 (10 mM)) and centrifuged at 1200 g at 20 °C for 12 minutes. Platelets were resuspended in reaction buffer (0.03 mM NaH 2 PO 4 , 0.5 mM Hepes, 0.55 mM glucose, 0.2 mM MgCl 2 , 0.1 mM KCl, 113.7 mM NaCl, 12 mM NaHCO 3 , 2 mM CaCl2 in pH 7.3 and 0.3% bovine serum albumin, adjusted to a concentration of 5×10 8 cells / mL. For the state of "activated platelets", 50 μM of TRAP (thrombin receptor activating peptide) was added to the platelets at 37 °C for 30 minutes. The platelets were centrifuged at 1200 g for 10 minutes at room temperature, and the supernatant was retained. In a 96-well plate (Greiner Bio-One), 1 μM of VHH was added to 80 μL of the supernatant of activated or non-activated platelets and incubated at room temperature for 15 minutes. Then, 0.1 nM of thrombin was added to each well and incubated at room temperature for 30 minutes. The PNAPEP-0238 substrate was added at a concentration of 0.2 mM and incubated at 37 °C for 2 hours. The quantification of thrombin activity was performed as described above.

[0176] Inhibition of uPA (urokinase plasminogen activator) by human PAI-1 In a 96-well plate (Greiner Bio-One), 10 μM of anti-PN-1 VHH was incubated with 10 nM of PAI-1 at room temperature for 15 minutes in 20 mM Hepes pH 7.5, 150 mM NaCl, 0.1% human serum albumin, pH 7.5. Subsequently, 1.3 nM of uPA was added. After incubation at room temperature for 10 minutes, the wells were quantified for uPA activity by measuring the release rate of p-nitroaniline from the chromogenic substrate Glu-Gly-Arg-pNA, PNAPEP-1344 (0.2 mM) at 37 °C and read at 405 nm by a multi-well plate reader.

[0177] Testing the specificity of VHH by biolayer interferometry (BLI) analysis Equilibrium binding was performed via BLI analysis using an OctetQK instrument (ForteBio, Reading, UK). Monovalent VHH (25 μg / mL) diluted in 100 mM MES (pH 5.0) was immobilized onto an amine-reactive biosensor. After quenching with ethanolamine, the VHH-coated sensor was incubated with human or mouse PN-1 or human PAI-1 (1 μM) in PBS-Tween 0.1% buffer for 15 min to allow association. Subsequently, the biosensor was incubated in PBS-Tween 0.1% buffer for 10 min to initiate dissociation. In an alternative experiment, the reactive center loop of biotinylated PN-1 at 50 μg / mL in PBS-Tween 0.1% buffer was immobilized onto a streptavidin sensor. Prior to association, 5 U of thrombin or PBS-Tween buffer was incubated with the sensor at 37 °C for 30 min, followed by association of divalent VHH (100 μg / mL) at 37 °C for 15 min. Subsequently, the biosensor was placed in PBS-tween 0.1% buffer for 10 min to initiate dissociation. Data were analyzed using Octet software version 4.0.

[0178] Modified prothrombin time (PT) 50 μl of normal pooled mouse plasma (MNPP) was diluted 8-fold with Owren-Koller buffer and 50 μL of factor II-deficient (prothrombin-deficient) lyophilized plasma (Stago Diagnostica) was added. The mixture was incubated with 62.5 nM of mouse PN-1 in the presence or absence of 288 nM of divalent VHH at 37 °C for 15 min. The prothrombin time was initiated by adding 100 μL of Neoplastin CI (Stago Diagnostica) to the mixture. Clot formation was measured by a coagulometer STArt (Stago Diagnostica). The assay was performed 3 times with the same experiment repeated 2 times at 37 °C.

[0179] Modified activated partial thromboplastin time (aPTT) 50 μl of factor VIII-deficient mouse plasma was diluted 8-fold with Owren-Koller buffer, to which 50 μL of factor VIII-deficient lyophilized plasma and 50 μL of PTT reagent (Stago Diagnostica) were added. After incubation for 100 seconds, 625 nM of VHH and 500 nM of mouse PN-1 were added. After incubation at 37 °C for 240 seconds, the activated partial thromboplastin time test was initiated by the addition of 100 μL of 0.025 M CaCl 2 The addition of which. Clot formation was measured by a coagulometer. The assay was performed 3 times at 37 °C with the same experiment being repeated 2 times.

[0180] Statistical analysis Data were presented as mean values and standard error (SD) and analyzed using Prism software. One-way analysis of variance was used for comparison of numerical values with the control. P-values less than 0.05 or less than 0.0001 were judged to be significant and highly significant, respectively.

[0181] Results: Development of anti-PN-1 VHHs from a synthetic VHH library To obtain specific and potent inhibitory anti-PN-1 VHHs, the inventors used a synthetic VHH library. The VHHs were selected using the same protocol as that used for classical VHHs. Four of the strongest binders were selected for further characterization: H12, B11, F06, and A08. ELISAs of unadsorbed phage against human PN-1, mouse PN-1, and PAI-1 presented in Figure 1 highlight that all four VHHs individually recognize human PN-1 and mouse PN-1, and that they do not recognize human PAI-1.

[0182] Characteristics of anti-PN-1 VHHs obtained from the synthetic library The purified VHHs were analyzed for their interactions with PAI-1 and both human and mouse PN-1. Consistent with the initial screening, none of the four selected VHHs showed relevant binding to human or mouse PAI-1 (Figs. 2A–B). In contrast, VHHs H12, B11, F06, and A08 all showed dose-dependent binding to both human and mouse PN-1 (Figs. 2C–D). To evaluate the inhibitory activity of the various VHHs against PN-1, the inventors next performed an assay measuring thrombin activity in the presence of PN-1 and the various VHHs. Although most efficient in binding to both human and mouse PN-1, VHH H12 only partially interfered with the inhibition of thrombin activity mediated by PN-1, restoring thrombin activity to 74 ± 5% and 53 ± 17% for human and mouse PN-1, respectively (Figs. 2E–F). VHHs B11, F06, and A08 were found to be more efficient in enhancing the inhibition of thrombin by human PN-1, with the restoration of thrombin activity being 100 ± 7% for B11, 100 ± 5% for F06, and 100 ± 11% for A08 (Fig. 2E). Similarly strong inhibitory ability was also observed against mouse PN-1: 92 ± 10% for B11, 96 ± 9% for F06, and 94 ± 14% for A08 (Fig. 2F). None of these VHHs affected the inhibition of u-PA by PAI-1 (Fig. 2G). In conclusion, this second selection strategy yielded VHHs that are selective and inhibitory against both human and mouse PN-1.

[0183] Determination of IC50 for VHHs B11, F06, A08 To determine the 50% inhibitory concentration of B11, F06, and A08 against human PN-1 and mouse PN-1, the inventors incubated various concentrations of VHH with 10 nM of PN-1 and 1 nM of thrombin. The degradation of the PNAPEP-0238 substrate was observed over a 2-hour period. The calculated IC50 values for the tested VHH against human PN-1 were 0.05 ± 0.01 μM for B11, 0.29 ± 0.03 μM for F06, and 0.96 ± 0.39 μM for A08 (Figure 3A). Using mouse PN-1, the calculated IC50 values were 1.58 ± 0.24 μM for B11, 0.56 ± 0.13 μM for F06, and 2.88 ± 0.77 μM for A08 (Figure 3A). To particularly increase the inhibitory effect of the VHH against mouse PN-1, the inventors next generated three different bivalent constructs, namely B11bv, B11F06, and B11A08 (Figure 3B). After this engineering step, the inventors confirmed whether the purified bivalent VHH retained the ability to bind to human PN-1 and mouse PN-1 (Figures 4A - B). The inventors also confirmed that they still did not recognize PAI-1 (not shown).

[0184] The IC50 of the bivalent VHH was significantly improved compared to the monovalent VHH, with values of 41 ± 7 nM for B11bv, 62 ± 6 nM for B11F06, and 142 ± 28 nM for B11A08 against human PN-1. The IC50 for mouse PN-1 was also improved, with values of 825 ± 9 nM, 414 ± 141 nM, and 524 ± 174 nM for B11bv, B11F06, and B11A08, respectively (Figure 4C).

[0185] In an attempt to identify the binding region of the VHHs on PN-1, the inventors performed biolayer interferometry analysis to test the interaction between peptides covering the reactive center loop region of PN-1 (either intact or after cleavage by thrombin) and bivalent VHHs. All three bivalent sdAbs efficiently bound to the peptides of the reactive center loop, demonstrating that they are directed against this region of the protein (Figure 4D). The binding to this peptide strongly decreased upon cleavage of the reactive center loop by thrombin in each of the bivalent constructs. These data are not inconsistent with the binding of VHHB11 to the C-terminal part of the reactive center loop (released from the sensor after cleavage by thrombin) or to an epitope overlapping the P1-P’1 cleavage site. None of the VHHs appears to recognize the N-terminal part of the reactive center loop (which remains bound to the sensor after cleavage by thrombin). The data do not discriminate between the binding of F06 and the binding of A08 to the C-terminal part of the reactive center loop or to an epitope outside the reactive center loop.

[0186] Effect of bivalent VHHs on in vitro coagulation assays To test the effect of bivalent VHHs on coagulation, the inventors performed coagulation assays of modified prothrombin time and activated prothrombin time using mouse plasma with mouse PN-1.

[0187] First, the prothrombin time was developed using wild-type plasma incubated with prothrombin-deficient lyophilized plasma to enable the use of only a small amount of mouse PN-1. Using this improved assay, the prothrombin time was 69.0 ± 2.4 seconds and was extended to 123.8 ± 6.6 seconds after the addition of 62.5 nM of mouse PN-1 (Figure 5A). At a concentration of 288 nM, all three bivalent VHHs were able to restore the prothrombin time but had no effect on the prothrombin time in the absence of added mouse PN-1 (83.5 ± 1.1 seconds for B11bv; 83.3 ± 1.5 seconds for B11F06; 93.2 ± 4.4 seconds for B11A08). In this assay, B11A08 was slightly less efficient than the other two VHHs.

[0188] The inventors also tested the effect of bivalent VHHs on the improved activated prothrombin time (Figure 5B). Similar to the prothrombin time, the inventors designed special conditions where the activated prothrombin time was extended by using factor VIII-deficient mouse plasma to make the test more sensitive to PN-1. The activated prothrombin time in factor VIII-deficient mouse plasma was 97.0 ± 2.7 seconds and increased 1.6-fold to 148.4 ± 7.9 seconds when mouse PN-1 was added. The three VHHs significantly shortened the activated prothrombin time, and B11F06 was the most efficient (112.5 ± 7.0 seconds; Figure 5B).

[0189] Next, the inventors investigated whether their bivalent VHHs could also inhibit endogenous PN-1 present within the α-granules of platelets. For this experiment, supernatants derived from non-activated or TRAP (thrombin receptor agonist peptide)-activated platelets were used as the PN-1 source. The efficacy of secreted PN-1 in the inhibition of thrombin activity was thus examined. As expected, when supernatants derived from non-activated platelets were incubated with thrombin, no inhibition was measured as PN-1 was not released from the α-granules of platelets (Figure 5C). In contrast, residual thrombin activity decreased to 18 ± 15% in the presence of supernatants derived from TRAP-activated platelets (Figure 5D). The bivalent VHHs abolished the inhibition of thrombin activity by secreted platelet PN-1, and thrombin activity recovered to 97 ± 23% with B11bv, 88 ± 21% with B11F06, and 100 ± 15% with B11A08.

[0190] The inventors had previously shown that platelets release PN-1 upon their activation and that this PN-1 impairs thrombin generation. Indeed, by using supernatants from non-activated and TRAP-activated platelets, the inventors were able to confirm the release of thrombin-inhibitory molecules (Figures 5C–D). Interestingly, this inhibitory effect could be neutralized by the addition of VHHs, demonstrating that they can interact with and inhibit PN-1 released from platelets.

[0191] Collectively, the inventors developed a strategy for the development of inhibitory VHHs that target PN-1 and do not cross-react with PAI-1. These VHHs can be used as research tools to better understand the role of PN-1 in physiological and pathological processes. Furthermore, they can be explored for their potential therapeutic applications, for example, in the treatment of hemophilia.

[0192] References: Throughout this application, various references describe the state of the art in the technical field to which the present invention pertains. The disclosures of these references are hereby incorporated herein by reference.

[0193] [Table 2]

Claims

**Claim 1** An isolated single-domain antibody (sdAb) that binds to protease nexin-1, comprising an amino acid sequence represented by formula (I): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (I) comprising i) CDR1 having the sequence shown as SEQ ID NO: 1, CDR2 having the sequence shown as SEQ ID NO: 2, and CDR3 having the sequence shown as SEQ ID NO: 3; or ii) CDR1 having the sequence shown as SEQ ID NO: 5, CDR2 having the sequence shown as SEQ ID NO: 6, and CDR3 having the sequence shown as SEQ ID NO: 7 and consisting of three complementarity-determining regions (CDR1-CDR3) and four framework regions (FR1-FR4). **Claim 2** The isolated single-domain antibody according to claim 1, having at least 90% identity to the sequence shown as SEQ ID NO: 4 or SEQ ID NO:

8. **Claim 3** The isolated single-domain antibody according to claim 2, comprising the sequence shown as SEQ ID NO: 4 or SEQ ID NO:

8. **Claim 4** An isolated single-domain antibody that binds to protease nexin-1, comprising an amino acid sequence represented by formula (I): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (I) (wherein CDR1 has the sequence shown as SEQ ID NO: 9; and CDR2 has the sequence shown as SEQ ID NO: 10; and CDR3 has the sequence shown as SEQ ID NO: 11) and consisting of three complementarity-determining regions (CDR1-CDR3) and four framework regions (FR1-FR4). **Claim 5** The isolated single-domain antibody according to claim 4, having at least 90% identity to the sequence shown as SEQ ID NO:

12. **Claim 6** The isolated single-domain antibody according to claim 5, comprising the sequence shown as SEQ ID NO:

12. **Claim 7** a) Two single-domain antibodies that bind to protease nexin-1, comprising CDR1 having the sequence shown as SEQ ID NO: 1, CDR2 having the sequence shown as SEQ ID NO: 2, and CDR3 having the sequence shown as SEQ ID NO: 3; b) A first single-domain antibody that binds to protease nexin-1, comprising a CDR1 having the sequence shown as SEQ ID NO: 1, a CDR2 having the sequence shown as SEQ ID NO: 2, and a CDR3 having the sequence shown as SEQ ID NO: 3, and a second single-domain antibody that binds to protease nexin-1, comprising a CDR1 having the sequence shown as SEQ ID NO: 5, a CDR2 having the sequence shown as SEQ ID NO: 6, and a CDR3 having the sequence shown as SEQ ID NO: 7; or c) A first single-domain antibody that binds to protease nexin-1, comprising a CDR1 having the sequence shown as SEQ ID NO: 1, a CDR2 having the sequence shown as SEQ ID NO: 2, and a CDR3 having the sequence shown as SEQ ID NO: 3, and a second single-domain antibody that binds to protease nexin-1, comprising a CDR1 having the sequence shown as SEQ ID NO: 9, a CDR2 having the sequence shown as SEQ ID NO: 10, and a CDR3 having the sequence shown as SEQ ID NO: 11 A polypeptide that binds to protease nexin-1, comprising the same.

8. The polypeptide according to claim 7, comprising a sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO:

19.

9. A nucleic acid molecule encoding the single-domain antibody according to claim 1 or 4 and / or the polypeptide according to claim 7.

10. A vector comprising the nucleic acid according to claim 9.

11. A host cell transfected, infected, or transformed by the nucleic acid according to claim 9 and / or the vector according to claim 10.

12. A pharmaceutical composition for preventing or treating a bleeding disorder in a subject in need thereof, containing an effective amount of the single-domain antibody according to claim 1 or 4 and / or the polypeptide according to claim 7.

13. The pharmaceutical composition according to claim 12, wherein the bleeding disorder is hemophilia.

14. A pharmaceutical composition comprising the single-domain antibody according to claim 1 or 4 and / or the polypeptide according to claim 7.

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