Ligands specific for the pcpe-1 glycoprotein and uses thereof

Abstract

The present invention relates to a ligand specific for the PCPE-1 glycoprotein, characterized by being a nanobody. It also relates to a ligand specific for the PCPE-1 glycoprotein, characterized in that it inhibits its activity.

Description

[0001] PCPE-1 glycoprotein-specific ligands and their uses

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to ligands specific for the PCPE-1 (Procollagen C-proteinase enhancer-1) protein. It also relates to their use in medical imaging and in diagnostic and treatment methods. More particularly, it relates to two types of ligands: some capable of binding to PCPE-1 for use in imaging, and other so-called antagonist ligands capable of binding to PCPE-1 and inhibiting its activity, for use in therapy. In one embodiment of the invention, these antagonist ligands are used for the diagnosis and treatment of fibrosis and cancer.

[0004] BACKGROUND OF THE INVENTION

[0005] Fibrillar collagens are the most abundant proteins in the human body and major components of extracellular matrices. They shape organs and tissues and play a crucial role in maintaining their homeostasis or repairing them after injury. Long considered to have a structural role in tissues, collagens are now recognized as integral players in numerous cellular processes such as cell adhesion, proliferation, migration, and differentiation.

[0006] However, these collagens are also associated with numerous pathological processes and are prime targets for the development of new diagnostic tools or new treatments.

[0007] Thus, fibrosis, which is characterized by excessive deposition of extracellular matrix composed mainly of collagen fibers, is a major common denominator of many pathologies that strongly affects disease progression, efficacy and delivery of therapies (Henderson et al. 2020). Furthermore, the production of abnormal collagen by tumor cells or by the microenvironment plays a major role in the immune escape of these cells and strongly contributes to their metastatic capacity as well as their dormancy and resistance to treatments (Shi et al. 2022).

[0008] In fact, targeting collagen and more specifically all the key steps enabling its biosynthesis, secretion, maturation or macrostructuring are all targets of interest to limit the excessive production of collagen associated with these processes. Examples include LARP6, the P4H complex, HSP47, SHMT2, LOX, etc. (Shi et al. 2022). Among the targets mentioned, those involved in the proteolytic maturation of the N- and C-terminal regions seem particularly interesting. Thus, BTPs (bone morphogenetic protein- 1 (BMP1) / tolloid-like proteinases) are the main proteases responsible for the removal of C-propeptides from fibrillar procollagens, a process generally considered to be the rate-limiting step in the formation of type 1 collagen fibrils.However, although BTPs are prime targets, they possess, in addition to collagens, activity directed against many other substrates involved in several pathways (activation of growth factors, angiogenesis, mineralization, tumor progression, etc.), which means that inhibiting their activity could lead to undesirable side effects.

[0009] Another key regulator of collagen fibrillogenesis is PCPE-1 (Procollagen C-Proteinase Enhancer-1), a secreted glycoprotein that specifically stimulates the C-terminal cleavage of fibrillar procollagen by BTPs. PCPE-1 is composed of two CUB (Complement-Uegf-BMP-1) domains and a C-terminal NTR (Netrin-like) domain, separated by a long linker. The CUB domains are necessary and sufficient for PCPE-1 activity (Kronenberg 2009; Vadon 2011) and this is due to their direct and close interaction with the C-propeptide of procollagens. This interaction is thought to allow the local destructuring of the procollagen trimer that facilitates cleavage by BTPs, and explains why PCPE-1 has no effect on other BMP-1 substrates. Furthermore, data have described the overexpression of PCPE-1 in different fibrotic contexts (Lagoutte et al. Matrix Biol Plus 2021), designating it as a promising target for monitoring and / or treating fibrosis.In particular, a correlation between the expression level of PCPE-1 and fibrosis, especially cardiac fibrosis, has been established (Lagoutte et al., BioRxiv, 2021). Direct detection of PCPE-1 in biological fluids by immunoassay has also been proposed as a diagnostic method (US2012270246 for bone formation, W02017065206À1 for NASH).

[0010] However, there is currently no robust method to detect PCPE-1 non-invasively in tissues, and thus monitor the accumulation of fibrotic collagen. Furthermore, the lack of effective tools to inhibit the action of PCPE-1 has until now prevented the evaluation of a pharmacological strategy targeting this protein.

[0011] International application WO 2019 / 080284 teaches that the PCPE-1 protein is a therapeutic target for combating fibrosis-type diseases, including skin fibrosis and difficult healing. The strategy considered in this document is the inhibition of PCPE-1 expression, via the injection of siRNAs intended to block the translation of the gene into protein.

[0012] However, in some therapeutic cases, it is preferable to inhibit the activity of a protein rather than limit its expression. However, to date, no drug capable of blocking the interaction between the PCPE-1 protein and the C-propeptides of procollagen has been reported. In the diagnosis of cardiac fibrosis, histological analysis of cardiac biopsies remains the traditional technique, despite its invasive nature and the fact that this technique only allows analysis of a small portion of the cardiac tissue, not necessarily representative of the whole. The use of MRI associated with the injection of gadolinium now allows imaging of large fibrotic areas but is not completely specific for fibrosis, and does not detect interstitial fibrosis.

[0013] One of the characteristics of fibrosing pathologies and cancers is their progressive characteristic, which implies that even if, particularly in the case of fibrosis, it is the result of stress or injury, the excessive production of collagen is continuous and only leads to pathological and functional effects after a sometimes very long period. Hence a significant need to be able to detect early and monitor the evolution of this excessive production of collagen over time. As such, molecular imaging, coupled with increasingly sensitive detection systems (isotopic imaging, fluorescence, etc.), now appears to be an essential tool for the longitudinal monitoring of these pathologies as well as for monitoring the therapeutic effect of new treatments.

[0014] To date, the main approach used for direct molecular imaging of collagen, particularly in fibrotic contexts, is based on the use of probes directly targeting collagen, mainly by hybridization. Examples include the peptides collagenlin, EP-3533 or CBP8 (Désogère et al., 2019).

[0015] Although the results are promising, other indirect routes, such as the one described in the present invention, would have certain advantages by possibly limiting the signal / background noise ratio. This is particularly the case for probes targeting integrin collagen receptors.

[0016] The use of immunoglobulins for targeting proteins of interest has been widely proven, both for therapeutic and molecular imaging purposes, and they now represent the majority of marketed biomedicines. In recent years, immunoglobulin scaffolds, lacking the undesirable properties of conventional immunoglobulins (large molecular weight, heterotetrameric composition, disulfide bonds), have emerged as alternatives to conventional antibodies. Among them, nanobodies, also called VHH (Variable Heavy-chain domains of Heavy-chain antibodies), derived from the heavy chain of camelid immunoglobulins, discovered in the early 1990s, have demonstrated their interest in both therapeutic and diagnostic applications (Muyldermans, 2021).Nanobodies are the smallest antibody fragments (approximately 15 kDa), highly soluble and stable, and can be easily produced in large quantities in prokaryotic systems. Furthermore, their convex paratope is well suited to bind to hard-to-target cavities on the antigen surface. Three types of approaches can be used to select antigen-specific nanobodies: from immune libraries, naive libraries, or synthetic libraries. An immune library is generated by immunizing a camelid with the target. These are the most commonly used nanobody libraries, with a limitation for some targets that are not immunogenic or too toxic for animal immunization. Due to these limitations, several synthetic libraries have been developed. Selection can be made among these libraries to identify nanobodies specific to one or more target proteins.To date, more than 1400 VHHs have been successfully selected and are used as research tools, in biotechnological or diagnostic applications.

[0017] BRIEF DESCRIPTION OF THE INVENTION

[0018] The present invention relates to a specific ligand of the PCPE-1 glycoprotein, characterized in that it is a nanobody, also designated by the abbreviation VHH (Variable domain of the Heavy chain of Heavy chain-only antibodies).

[0019] The present invention also relates to a specific ligand of the PCPE-1 glycoprotein, characterized in that it inhibits its activity. This specific ligand is also called a PCPE-1 antagonist ligand. In particular, it is a PCPE-1 antagonist nanobody.

[0020] Another object of the invention is a specific ligand of the PCPE-1 glycoprotein consisting of a combination of two or three nanobodies as described above.

[0021] The present invention also relates to a nucleic acid encoding a nanobody or a combination of two or three nanobodies, as described in the present application.

[0022] The present invention also relates to a specific ligand antagonist of the PCPE-1 glycoprotein for its use as a medicament, and more specifically for its medical use in the treatment of cancer or fibrosis, in particular cardiac fibrosis.

[0023] The present invention also relates to a specific ligand of the PCPE-1 glycoprotein as described in the present application, characterized in that it is coupled to a detectable marker, in particular a marker used in medical imaging.

[0024] Another object of the invention is the use of such a specific ligand of the PCPE-1 glycoprotein coupled to a detectable marker, for the in vivo monitoring of a pathological state by medical imaging.

[0025] Another subject of the invention is a specific ligand of the PCPE-1 glycoprotein as described above, for its theranostic use for the treatment and in vivo monitoring of a pathological condition by medical imaging. Finally, the present application relates to a kit for determining the activity and / or the quantity of the PCPE-1 glycoprotein in a biological sample in vitro or ex vivo, comprising:

[0026] - a specific ligand for the PCPE-1 glycoprotein as described above, and

[0027] - detection reagents.

[0028] FIGURE CAPTIONS

[0029] Figure 1: SPR characterization of the PCPE-1 / VHHs interaction

[0030] Figure 1A: Injection of 200 nM of VHHs selected by immunization on immobilized PCPE-1 (645 RU i.e. Resonance Units).

[0031] Figure 1 B: Injection of 200 nM of VHHs selected from a synthetic library onto immobilized PCPE-1 (645 RU).

[0032] Figure 2: Specificity of VHHs

[0033] Figure 2A: % inhibition of the interaction between VHHs (50 nM) and immobilized PCPE-1 when the CUB1 or CUB2 domains (250 nM) are co-injected.

[0034] Figure 2B: Injection of 250 nM VHH-I5 followed by an injection of the same concentration of VHH-I5 in combination with 250 nM of a second VHH.

[0035] Figure 2C: Injection of 250 nM VHH-H4 followed by an injection of the same concentration of VHH-H4 in combination with 250 nM of a second VHH.

[0036] Figure 2D: VHHs I5 is biotinylated and immobilized on a streptavidin chip (813 RU), then increasing concentrations of PCPE-1 (in black) or PCPE-2 (in gray) are injected.

[0037] Figure 2E: VHH H4 is biotinylated and immobilized on a streptavidin chip (491 RU), then increasing concentrations of PCPE-1 or PCPE-2 are injected.

[0038] D, E: Interactions are modeled (dotted line) using the Langmuir model (Biacore T200 Evaluation software, Cytiva)

[0039] Figure 3: Inhibition of PCPE-1 activity by VHHs

[0040] Figure 3A: % inhibition of PCPE-1 / mini-procollagen III interaction in the presence of VHHs: the interaction of PCPE-1 (5 nM) with immobilized mini-procollagen III is measured and then compared to the interaction obtained when PCPE-1 (5 nM) is co-injected with 250 nM of VHHs. Mean ± SD, n = 3.

[0041] Figure 3B: Effect of VHHs on PCPE-1 activity: CPU I -Long is incubated with BMP-1 and 75 nM PCPE-1 in the absence or presence of 2 pM VHHs (1 h, 37°C). Analysis by SDS-PAGE (4-20% gel, in the presence of DTT, Coomassie blue staining). The % inhibition is calculated by comparison with the PCPE-1 condition alone (gel representative of n=3 experiments). Figure 3C: Immunodetection of procollagen I and its C-terminal cleavage products in the culture medium of rat heart fibroblasts, after 48 h of culture in the absence or presence of PCPE-1 (5 pg / ml) and VHHs (15 pg / ml). Analysis by western blot using an anti-procollagen I C-propeptide antibody (LF41).

[0042] Figure 3D: Relative quantification of the released C-propeptide, under the conditions of Fig. 3C, by densitometry (mean ± SD, n = 3).

[0043] Figure 4: Characterization of diabody D1 (H4-GSs-l5) of sequence SEQ ID NO. 21

[0044] Figure 4A: SPR analysis of the interaction of D1 with immobilized PCPE-1. Sequential injections of increasing concentrations of D1 (0-250 nM) and modeling of the interaction (dotted line) using the Langmuir model (Biacore T200 Evaluation software, Cytiva).

[0045] Figure 4B: Inhibition of PCPE-1 / mini-procollagen III interaction in the presence of D1 diabody: the interaction of PCPE-1 (5 nM) with immobilized mini-procollagen III is measured and then compared to the interaction obtained when PCPE-1 (5 nM) is co-injected with increasing amounts (0-200 nM) of D1. The % residual PCPE-1 binding is plotted as a function of D1 concentration.

[0046] Figure 4C: D1 inhibits PCPE-1 activity on procollagen III: CPU I -Long is incubated with BMP-1 and 75 nM PCPE-1 in the absence or presence of 2 pM monovalent or bivalent nanobody (1 h, 37°C). Analysis by SDS-PAGE (4-20% gel, Coomassie blue staining). The % inhibition is calculated by comparison with the PCPE-1 alone condition.

[0047] Figure 4D: D1 inhibits PCPE-1 activity on procollagens I and II: Mini I or Mini II are incubated with BMP-1 and 75 nM PCPE-1, in the absence or presence of 2 pM D1 (1 h, 37°C). Analysis is performed by SDS-PAGE (8% gel, Coomassie blue staining). The % inhibition is calculated by comparison with the PCPE-1 alone condition.

[0048] Figure 4E: Immunodetection of procollagen I and its C-terminal cleavage products in the culture medium of rat heart fibroblasts, after 48 hours of culture in the absence or presence of PCPE-1 (5 pg / ml) and diabody D1 (15 pg / ml). Western blot analysis using an anti-procollagen I C-propeptide antibody (LF41).

[0049] Figure 4F: Relative quantification of the released C-propeptide, under the conditions of Fig. 4E, by densitometry (mean ± SD, n = 3).

[0050] Figure 4G: Immunodetection of procollagen I and its C-terminal cleavage products in the culture medium of human skin fibroblasts, after 72 h of culture in the absence or presence of PCPE-1 (5 pg / ml), TGF-beta (5 ng / ml) and diabody D1 (15 pg / ml). Western blot analysis using an anti-procollagen I C-propeptide antibody (LF41).

[0051] Figure 4H: Relative quantification of the released C-propeptide, under the conditions of Fig. 4G, by densitometry (mean ± SD, n = 3). Figure 5: Use of a nanobody coupled to a detectable label for the detection of PCPE-1

[0052] Figure 5A: SPR characterization of the PCPE-1 / biotinylated VHH-H4 interaction. Biotinylation of VHH-H4 allows its oriented capture on a streptavidin chip. The presence of PCPE-1 in the injected sample is detected by its interaction with VHH-H4.

[0053] Figure 5B: Detection by ELISA: Calibration curve obtained by adding increasing amounts of purified human PCPE-1. Concentration of PCPE-1 (ng / ml) in human plasma samples, mean ± SD of 3 determinations, each in duplicate.

[0054] Figure 6: Characteristics of the VHH-H4-Ga nanobody 68 for use in imaging

[0055] Figure 6A: Pharmacokinetics of VHH-H4-Ga 68. Measurement of half-life time in blood; dosage during the 2.5 h following injection, expressed as % of the injected dose (%ID, mean ± SD, gamma count)

[0056] Figure 6B: Biodistribution of VHH-H4-Ga 68 determined ex-vivo 2.5 hours after injection, expressed as a % of the injected dose (%ID, mean ± SD) for each organ (gamma counting)

[0057] Figure 6C: Detection range of VHH-H4-Ga 68 on rat heart section. Autoradiography (phosphorimager) after one hour of incubation and one hour of exposure.

[0058] DETAILED DESCRIPTION OF THE INVENTION

[0059] The present invention relates to a specific ligand of the PCPE-1 glycoprotein, characterized in that it is a nanobody (also called VHH).

[0060] The glycoprotein PCPE-1 (Procollagen C-proteinase enhancer-1) is a key regulator of collagen fibrillogenesis. The human protein is referenced in the UniProt database under reference Q15113, where its polypeptide sequence is described.

[0061] Collagens are synthesized as soluble precursors called procollagens. These undergo proteolytic maturation before they can assemble into collagen fibers. This rate-limiting step is regulated by the glycoprotein PCPE-1, which specifically stimulates the C-terminal cleavage of fibrillar procollagen by BTP proteases, which are responsible for the removal of C-propeptides from fibrillar procollagens. For this purpose, PCPE-1 interacts closely with the collagen C-propeptide via its two CUB (Complement-Uegf-BMP-1) domains.

[0062] The term "specific ligand" refers to a compound that interacts with a protein in a non-covalent, reversible, and specific manner.

[0063] A ligand is said to be "specific" when, on the one hand, it binds preferentially to its target protein among a multitude of target proteins with similar structures; and on the other hand, it exhibits an affinity, that is to say a force of interaction between said ligand and the target protein, judged to be sufficiently strong. The affinity is quantitatively measured by the association / dissociation equilibrium constant, also called affinity constant or equilibrium dissociation constant, abbreviated "KD". The lower the KD value, the higher the binding affinity between the ligand and its target protein.

[0064] For the purposes of the present invention, a ligand is considered to bind specifically to PCPE-1 if its affinity constant KD is less than 100 nM, or less than 80 nM, or less than 50 nM, and more preferably if it is less than 30 nM. In view of these numerical indications, the person skilled in the art will be able to determine which are the specific ligands of the invention.

[0065] A nanobody refers to a single-domain antibody, which is an antibody fragment composed of a single monomeric variable antibody domain, which corresponds to the variable domain of the single heavy chain of antibodies of the type found in camelids, which naturally lack light chains. A nanobody is capable of selectively binding to a specific antigen. It has the advantage of having a molecular weight of only 12 to 15 kDa, which is about 10 times less than common antibodies, which have a molecular weight of 150 to 160 kDa. A nanobody is also much less bulky than a conventional antibody. In addition, the synthesis process, particularly in bacterial cells, is facilitated due to this simplified structure. The first single-domain antibodies were designed from heavy-chain antibodies found in camelids; these are designated VH H fragments.

[0066] In the present application, the terms nanobody and VHH are used interchangeably and both refer to a single-domain antibody.

[0067] The nanobodies each have three CDRs, designated CDR1, CDR2, and CDR3 respectively.

[0068] The nanobodies according to the invention may in particular be llama nanobodies or synthetic nanobodies.

[0069] A combination of two nanobodies, covalently coupled, with or without a bonding agent, is referred to as a "diabody" or "diacorps" in French, or even as a "bivalent nanobody".

[0070] A combination of three nanobodies is referred to as a "tribody" or "trivalent nanobody" in French.

[0071] Other specific ligands of the PCPE-1 glycoprotein are also included in the invention, in particular any protein structure equivalent to a nanobody, excluding antibodies. Examples of such protein structures ("protein scaffold") capable of specifically binding to target epitopes are presented in the review by (Gebauer & Skerra, 2019) The invention also relates to a specific ligand of the PCPE-1 glycoprotein, characterized in that it inhibits its interaction with the C-terminal domain of procollagens, which has the effect of inhibiting the activity of this PCPE-1 glycoprotein.

[0072] Some PCPE-1-specific ligands have, in addition to the ability to bind to this glycoprotein, the ability to inhibit the activity of said PCPE-1 glycoprotein. They are then called "PCPE-1 antagonist ligands".

[0073] The term "antagonist" refers to a compound that interacts with a target protein exhibiting physiological activity, reducing or even eliminating the physiological activity of said protein.

[0074] As shown in Example 3, significant inhibition of PCPE-1 activity corresponds to:

[0075] - either an inhibition of its interaction with the C-terminal domains of procollagen;

[0076] - either an inhibitory action on the cleavage of procollagen, which is normally stimulated by the action of PCPE-1.

[0077] More specifically, this inhibition of activity is characterized by an inhibition of the interaction of PCPE-1 with the C-terminal domains of procollagen.

[0078] This inhibition of PCPE-1 activity is considered significant when it is greater than 40%, or even at least 50%, and preferably greater than 60%.

[0079] These PCPE-1 antagonist ligands may be of any nature, and in particular be synthetic chemical compounds, nucleic acids, or even protein structures, and in particular a polypeptide chain.

[0080] According to a preferred embodiment, the PCPE-1 antagonist ligand is a polypeptide chain, and in particular is a nanobody (VHH). It is understood that any polypeptide structure equivalent to a nanobody is included in the invention.

[0081] Specific nanobodies

[0082] The inventors identified two families of nanobodies possessing either PCPE-1 binding activity for use in medical imaging and diagnosis, or PCPE-1 binding and inhibitory activity for use as a drug.

[0083] These nanobodies were selected from a synthetic nanobody library or after immunization of a llama and possess PCPE-1 binding activity measured by surface plasmon resonance as presented in Figure 1. They were selected to possess a KD of less than 100 nM and more precisely less than 30 nM (see Table 2 in the examples).

[0084] These nanobodies were sequenced, along with their CDRs; the sequences are shown in Table 1 below. Table 1. Sequences

[0085] As is well known to those skilled in the art, the combination of CDR1, CDR2 and CDR3 is sufficient to define an antigen binding site.

[0086] For the purposes of the invention, the term "CDR" for "complementarity determining region" refers to the amino acid sequences, which together define the binding affinity and specificity of the natural Fv region of a native binding site of an immunoglobulin.

[0087] According to a specific implementation of the invention, the PCPE-1 specific ligand is a nanobody whose polypeptide sequence includes three complementarity determining regions (CDRs) with PCPE-1, characterized in that among these three CDRs, at least one CDR has at least 80% identity with one of the sequences SEQ ID NO. 1 to SEQ ID NO. 15.

[0088] According to another specific implementation, among these three CDRs called CDR1, CDR2 and CDR3, each CDR is defined as follows:

[0089] 1, CDR1 has at least 80% identity with one of the following sequences: SEQ ID NO. 1, 4, 7, 10 or 13; ii. CDR2 has at least 80% identity with one of the following sequences: SEQ ID NO.

[0090] 2, 5, 8, 1 1 or 14; and iii. CDR3 has at least 80% identity with one of the following sequences: SEQ ID NO.

[0091] 3, 6, 9, 12 or 15. According to another particular implementation:

[0092] - the CDR1 of the nanobody has at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with at least one of the CDR1 sequences mentioned in (i) above, and

[0093] - the CDR2 of the nanobody has at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with at least one of the CDR2 sequences mentioned in (ii) above, and

[0094] - the CDR3 of the nanobody has at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with at least one of the CDR3 sequences mentioned in (iii) above.

[0095] The identity percentages referred to in the disclosure of the present invention are determined after optimal alignment of the sequences to be compared, which may therefore include one or more additions, deletions, truncations and / or substitutions.

[0096] This percentage of identity can be calculated by any sequence analysis method well known to those skilled in the art.

[0097] The percentage of identity can be determined after global alignment of the sequences to be compared taken in their entirety, over their entire length. In addition to manually, it is possible to determine the global alignment of sequences using the algorithm of Needleman and Wunsch (1970).

[0098] For nucleotide sequences, sequence comparison can be performed using any software well known to those skilled in the art, such as Needle software. Parameters used may include: "Gap Open" equal to 10.0, "Gap Extend" equal to 0.5, and the EDNÀFULL matrix (EMBOSS version of NCBI NUC4.4).

[0099] For amino acid sequences, the sequence comparison can be performed using any software well known to those skilled in the art, such as Needle software. The parameters used may include, in particular: “Gap Open” equal to 10.0, “Gap Extend” equal to 0.5 and the BLOSUM62 matrix.

[0100] Preferably, the percentage of identity defined within the framework of the present invention is determined by means of a global alignment of the sequences to be compared over their entire length.

[0101] According to another embodiment, the PCPE-1 glycoprotein-specific ligand is a nanobody which comprises three complementarity determining regions (CDRs) with PCPE-1, said three CDRs having at least 80% sequence identity with the following sequence combinations: a) CDR1: SEQ ID NO. 1, CDR2: SEQ ID NO. 2 and CDR3: SEQ ID NO. 3; or b) CDR1: SEQ ID NO. 4, CDR2: SEQ ID NO. 5 and CDR3: SEQ ID NO. 6; or c) CDR1: SEQ ID NO. 7, CDR2: SEQ ID NO. 8 and CDR3: SEQ ID NO. 9; or d) CDR1: SEQ ID NO. 10, CDR2: SEQ ID NO. 11 and CDR3: SEQ ID NO. 12; or e) CDR1: SEQ ID NO. 13, CDR2: SEQ ID NO. 14 and CDR3: SEQ ID NO. 15.

[0102] According to a particular implementation:

[0103] - the three CDRs of the nanobody have at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the CDR sequences mentioned in (a) above, or

[0104] - the three CDRs of the nanobody have at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the CDR sequences mentioned in (b) above, or

[0105] - the three CDRs of the nanobody have at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the CDR sequences mentioned in (c) above, or

[0106] - the three CDRs of the nanobody have at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the CDR sequences mentioned in (d) above, or

[0107] - the three CDRs of the nanobody have at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the CDR sequences mentioned in (e) above.

[0108] According to another embodiment, the PCPE-1 glycoprotein-specific ligand is a nanobody which comprises three complementarity determining regions (CDRs) with PCPE-1, said three CDRs having the following sequences: a) CDR1: SEQ ID NO. 1, CDR2: SEQ ID NO. 2 and CDR3: SEQ ID NO. 3; or b) CDR1: SEQ ID NO. 4, CDR2: SEQ ID NO. 5 and CDR3: SEQ ID NO. 6; or c) CDR1: SEQ ID NO. 7, CDR2: SEQ ID NO. 8 and CDR3: SEQ ID NO. 9; or d) CDR1: SEQ ID NO. 10, CDR2: SEQ ID NO. 11 and CDR3: SEQ ID NO. 12; or e) CDR1: SEQ ID NO. 13, CDR2: SEQ ID NO. 14 and CDR3: SEQ ID NO. 15.

[0109] According to another implementation of the invention, the specific ligand of the PCPE-1 glycoprotein is a nanobody whose polypeptide sequence has at least 80% identity with one of the sequences SEQ ID NO. 16 to SEQ ID NO. 20.

[0110] In particular, this nanobody has a polypeptide sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the sequences SEQ ID NO. 16 to SEQ ID NO. 20.

[0111] Preferably, the substitutions observed in the polypeptide sequences will be placed in the domains outside the CDRS, called the “framework” regions of the nanobody.

[0112] More preferably, the CDRs are conserved and have 100% identity with the sequences mentioned above in (a) for SEQ ID NO. 16; in (b) for SEQ ID NO. 17; in (c) for SEQ ID NO. 18; in (d) for SEQ ID NO. 19; and in (e) for SEQ ID NO. 20.

[0113] According to a preferred embodiment, the nanobody of the invention has a polypeptide sequence consisting of the sequence SEQ ID NO. 16; or SEQ ID NO. 17; or SEQ ID NO.

[0114] 18; or SEQ ID NO. 19; or SEQ ID NO. 20. Combination of nanobodies

[0115] In another embodiment, the present invention describes the association of these nanobodies within the same diabody or tribody type structure. This association is preferably achieved via the addition of a linking agent between the protein sequences of each nanobody. Those skilled in the art will be able to select the most suitable linking agent, in particular among those described in the review by Kwon, 2019. According to a preferred implementation, the GSs sequence (GSGSGSGSGSGSGSGS, SEQ ID NO. 38) is used as a linking agent.

[0116] According to one implementation of the invention, the specific ligand of the PCPE-1 glycoprotein consists of a combination of two or three nanobodies as described above, i.e. a diabody or a tribody, optionally comprising one or two binding agents, in particular one or two binding peptides (one for a diabody, two for a tribody).

[0117] According to a particular implementation of the invention, a diabody is made up of the association of a nanobody having a binding activity to CUB1 and another having a binding activity to CUB2, it being understood that each nanobody can be positioned either upstream or downstream of the binding peptide.

[0118] A diabody according to the invention may in particular consist of any combination of two polypeptide sequences each having at least 80% or 90% sequence identity with one of the sequences SEQ ID NO. 16, 17, 18, 19 or 20, optionally further comprising a binding agent.

[0119] More specifically, a diabody will consist of any combination of two polypeptide sequences each having a sequence chosen from the sequences SEQ ID NO. 16 to SEQ ID NO. 20, optionally linked by a linking agent, in particular by a linking peptide.

[0120] This combination of two nanobodies (diabodies) may in particular have a polypeptide sequence having at least 80% or 90% identity with the sequence SEQ ID NO. 21, and in particular at least 95%, 98%, 99% or 100% identity with the sequence SEQ ID NO. 21.

[0121] More preferably, in this diabody, the CDRs of the nanobodies are conserved and present 100% identity with the sequences mentioned above in (a) for SEQ ID NO. 16 (H4) and in (c) for SEQ ID NO. 18 (I5).

[0122] According to the present invention, this type of diabody may correspond to I5-Iinker-H4 or H4-Iinker-I5. An example of an amino acid sequence for the H4-Iinker-I5 diabody corresponds to the sequence SEQ ID NO. 21. According to a preferred implementation, a diabody according to the invention has a polypeptide sequence consisting of the sequence SEQ ID NO. 21.

[0123] Another subject of the invention relates to a nucleic acid comprising a nucleic sequence coding for a nanobody or a combination of two or three nanobodies according to the present invention.

[0124] In a particular embodiment, the nucleic acid according to the invention comprises or consists of a nucleic sequence encoding a nanobody defined by one of the amino acid sequences SEQ ID NO. 16 to 20 or encoding a diabody defined by the sequence SEQ ID NO. 21.

[0125] This nucleic acid is a DNA or RNA molecule, which can be included in any suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.

[0126] The term "vector" refers to the vehicle by which the DNA or RNA sequence can be introduced into a host cell, so as to transform the host and promote the expression (e.g. transcription and translation) of the introduced nucleic acid sequence. Such a vector advantageously comprises regulatory elements, such as a promoter, an enhancer, a terminator, etc., to induce the expression of the polypeptide.

[0127] Therefore, another subject of the invention relates to a vector comprising a nucleic acid according to the invention.

[0128] Another subject of the invention is a host cell having integrated a vector as described above, and thus capable of expressing the nanobody, diabody or tribody according to the invention.

[0129] Uses of the PCPE-1 specific ligands according to the invention

[0130] The present invention also relates to the PCPE-1 specific ligands as defined above for use as a contrast agent in non-invasive medical imaging, for use in diagnostic methods and for use as a medicament.

[0131] Furthermore, the ligands described in the present invention, possessing both binding activity and inhibitory activity of PCPE-1, are ideal candidates for a so-called theranostic approach.

[0132] Finally, it relates to a pharmaceutical composition comprising one of these ligands in association with a pharmaceutically acceptable vehicle.

[0133] The compounds according to the invention can be used in immunoassays (ELISA, Westernblot, immunofluorescence, immunohistochemistry) to detect PCPE-1 in biological fluids or tissues, in vitro or ex vivo. In particular, the present invention relates to an antagonist ligand of the PCPE-1 glycoprotein as described above, for its use as a medicament.

[0134] The present invention also relates to an antagonist ligand of the PCPE-1 glycoprotein as described above, for its therapeutic use in the treatment of cancer or fibrosis, in particular cardiac fibrosis.

[0135] Fibrosis, also known as sclerosis, occurs as a result of substantial tissue destruction or when inflammation occurs in a location where tissues do not regenerate. Fibrosis is a condition characterized by excessive synthesis and deposition of extracellular matrix (ECM), leading to pathological scarring and stiffening that can lead to death when vital organs are affected (heart, liver, lung, etc.). There are several types of fibrosis: cardiac fibrosis, pulmonary fibrosis, hepatic fibrosis, renal fibrosis, muscular fibrosis, etc.

[0136] Cancer is also a disease where collagen plays an important role, although this role is complex and dependent on the tumor type (Shi et al., 2021).

[0137] The present invention also relates to a method of treating cancer or fibrosis, in particular cardiac fibrosis, comprising administering to a patient suffering from cancer or fibrosis, a PCPE-1 antagonist ligand, inhibiting its activity, as described above.

[0138] In the context of the invention, a "patient" means a human or non-human mammal, such as a rodent (rat, mouse, rabbit), a primate (chimpanzee), a feline (cat), or a canine (dog). Preferably, the patient is a human being, in particular suffering from cancer or fibrosis, and in particular cardiac fibrosis.

[0139] Ligands coupled to detectable markers

[0140] The present invention also relates to a specific ligand of the PCPE-1 glycoprotein as defined above, characterized in that it is coupled to a marker detectable in medical imaging.

[0141] By "PCPE-1-specific ligand coupled to a detectable label" is meant herein that the detectable label is linked, directly or indirectly, to the ligand, or is incorporated into the ligand. The detectable label may in particular be linked to the ligand by substitution, by complexation or by chelation.

[0142] For the purposes of the present invention, a "detectable marker" means a compound that produces a detectable signal. When combined with a tracer, it makes it possible to monitor the fate of the tracer in the body. A detectable marker here refers in particular to a marker detectable in medical imaging. The detectable marker used may in particular be an MRI contrast agent, a scintigraphy contrast agent, an X-ray imaging contrast agent, an ultrasound contrast agent, or an optical imaging contrast agent.

[0143] Examples of detectable markers include radionuclides, fluorophores such as fluorescein, Alexa, cyanine; chemiluminescent compounds such as luminol; bioluminescent compounds such as luciferase or alkaline phosphatase; contrast agents such as nanoparticles or gadolinium; and quantum dots.

[0144] The choice of the appropriate detectable marker, which depends on the detection system used, is within the reach of those skilled in the art.

[0145] Labeling can be directed, via the introduction of a terminal cysteine, or a recognition sequence by sortase or another ligase. Labeling can also be achieved by direct coupling after activation of the lysines.

[0146] The detectable marker is notably a fluorophore, a chromophore or luminescent compound or a label detectable by an antibody. It can also be a chelator, such as NODÀGÀ, which is then coupled to a radioisotope, such as Ga68.

[0147] Advantageously, the ligand thus modified retains its ability to bind to PCPE-1.

[0148] The PCPE-1-specific ligand coupled to a detectable label can be used to detect PCPE-1 protein in an immunoassay or in cell culture, as illustrated in Example 5.

[0149] The present invention also relates to the use of a specific PCPE-1 ligand coupled to a detectable marker as defined above, for the in vivo monitoring of a pathological state by medical imaging.

[0150] In particular, the invention relates to the use of said PCPE-1 specific ligand coupled to a detectable marker as a contrast agent in medical imaging, in particular non-invasive in vivo medical imaging.

[0151] For the purposes of the invention, a contrast agent designates a substance which, when administered into the body, makes it possible to detectably mark organs or structures (tissue, cell, receptor) which, without a contrast agent, are barely or not at all visible in medical imaging.

[0152] Advantageously, the PCPE-1-specific ligand coupled to the marker has pharmacokinetic characteristics compatible with its use as a contrast agent (rapid clearance but sufficient persistence to allow imaging, renal elimination, no accumulation in healthy animals). This is notably illustrated in Example 6. These properties can, if necessary, be modulated by PEGylation, or other modification of the charge or lipophilicity of the molecules. The present invention also relates to a method of medical imaging, in particular non-invasive in vivo medical imaging, in which a PCPE-1-specific ligand coupled to a detectable marker as defined above is administered to a patient, then the patient is subjected to a medical imaging protocol.

[0153] It also relates to the use of a PCPE-1 specific ligand coupled to a detectable marker as defined above, for the manufacture of a contrast agent useful for medical imaging, in particular non-invasive in vivo medical imaging.

[0154] Theranostic use

[0155] The term theranostics is a neologism constructed from the terms therapy and diagnosis, which corresponds to a medical approach aimed at favoring the simultaneous development of diagnostic and therapeutic aspects. In particular, it involves using compounds that allow both the in vivo visualization of a patient's clinical situation and the treatment of the condition concerned with the same compound.

[0156] The PCPE-1-specific ligands according to the invention, in particular those exhibiting an inhibitory action on PCPE-1 activity and coupled to a detectable marker, are entirely suitable for theranostic use.

[0157] Thus, the present invention relates to a PCPE-1 specific ligand coupled to a detectable marker, for its theranostic use for the treatment and in vivo monitoring of a pathological condition by medical imaging. Preferably, this ligand is a PCPE-1 antagonist.

[0158] Detection kit

[0159] The present invention also relates to a kit for determining the activity and / or quantity of the PCPE-1 glycoprotein in a biological sample in vitro or ex vivo, comprising:

[0160] - a specific ligand of the PCPE-1 glycoprotein according to the invention, and

[0161] - detection reagents.

[0162] This PCPE-1 specific ligand will in particular be a ligand coupled to a detectable marker.

[0163] A biological sample means any type of sample from a living body, including the body of a patient, and includes, but is not limited to, blood, serum, plasma, urine, cerebrospinal fluid, and tears. EXAMPLES

[0164] MATERIALS AND METHODS

[0165] Protein production and purification

[0166] PCPE-1 (native human or with a C-terminal 8his tag), Miniprocollagen I (Mini I, Twinstrep tag at the N-terminal of the α2 chain), Mini-procollagen II (Mini II, 6His tag at the N-terminal), Mini-procollagen III (Mini III, C-myc tag at the N-terminal), and BMP-1 (C-terminal flag tag) proteins were produced in HEK 293-EBNA or 293-F cells and purified as described. The CUB domains of PCPE-1 were prepared by limited proteolysis of CUB1 NTR and CUB2NTR as described by Kronenberg et al. CPI 11 - Long and PCPE-2 (with an N-terminal 6-his tag) were produced by transient transfection of HEK 293T cells and purified as described.

[0167] The antigen used for nanobody selection is the CUB1 CUB2 region of the PCPE-1 protein (corresponding to amino acids 1 to 279). It was cloned into the pHLsec vector, fused with a 6-His tag and an N-terminal HRV-3C protease cleavage sequence. It was then produced by transient transfection of 293-F cells, grown at 37°C, 125 rpm, 8% CO2 in Freestyle™ 293 medium (Gibco), according to the procedure described by Pulido et al. CUB1 CUB2 was purified on a Ni-excel column (5 ml; Cytiva), then treated with HRV-3C protease at 4°C overnight to remove the histidine tag. After gel filtration with a HiLoad Superdex 75 16 / 600 SEC column (size exclusion chromatography, Cytiva) equilibrated in 20 mM HEPES pH 7.4, 0.3 M NaCl, the protein was stored at -80°C in the same buffer. Protein concentration was determined using a Nanodrop 2000 (Thermo Scientific).

[0168] A biotinylated form of CUB1 CUB2 was prepared for biopanning and phage ELISA. For this, the coding sequence for the CUB1 CUB2 region was cloned into the pHL-Avitag3 vector between the EcoRI and KpnI sites and the protein was expressed in 293-T as above. After purification on Cobalt resin, 60 pM of CUB1 CUB2avi was biotinylated using 1 pM GST-BirA (Biotin-protein ligase) in 50 mM bicine buffer, 300 mM potassium glutamate pH 8.3 in the presence of 10 mM ATP and 50 pM d-biotin for 5 h at 30 °C. GST-BirA (cloned into the pGex vector, a gift from Y. Zhao, STRUBI, Oxford, UK) was produced in E. coli BL21(DE3)pLysS bacteria. Purification on cobalt resin followed by gel filtration with a Superdex S75 column (20 mM HEPES buffer pH 7.4, 0.3 M NaCl) allowed the removal of excess BirA and biotin and the production of a pure and 95% biotinylated protein.

[0169] Selection of nanobodies in llamas

[0170] Llama nanobodies were generated by the Architecture and Function of Biological Macromolecules Laboratory platform (Marseille, France). Briefly, a llama was immunized with five successive injections of 1 mg of CUB1 CUB2 at one-week intervals. After 39 days, blood was collected, and the nanobody library was generated as previously described. Two biopanning steps on 50 nM biotinylated CUB1 CUB2 enriched phages expressing nanobodies specifically recognizing CUB1 CUB2. 48 clones (phagemid) were selected and their affinity for human PCPE-1 determined by phage ELISA. Positive clones were sequenced and their sequences aligned and analyzed using ESPript 3.0. Eight nanobodies were selected for further characterization: 11, I2, I3, I4, I5, I7, 110 and 111.

[0171] Generation of synthetic nanobodies

[0172] The synthetic nanobodies were selected by Hybrigenics Services SÀS. After three biopanning steps on biotinylated CUB1CUB2 using the hsd2ab nanobody library (Moutel et al., 2016), 90 clones were tested by phage ELISA for their ability to bind to PCPE1. After sequencing, a library of 24 unique positive clones was obtained. Ten of these were selected for further characterization: H1 to H10.

[0173] Production and purification of nanobodies

[0174] Synthetic nanobodies were cloned into the pET29b(+) vector, fused with the pelB signal sequence and a C-terminal 6-His tag. Llama nanobodies were cloned into the pHEN6 vector following the pelB sequence and fused with a C-terminal 6-His tag.

[0175] To construct the diabody, the VHH-15 nanobody was amplified by PCR with the addition of a NotI restriction site and a GSs sequence (GSGSGSGSGSGSGSGS, SEQ ID NO. 38) at the N-terminus. The GSs-VHH-15 construct was then cloned following VHH-H4 into pET29b(+) by NotI / XhoI digestion to yield the bivalent nanobody VHH-H4-GSs-VHH-15 (D1).

[0176] The resulting plasmids were then transformed into E. coli T7 express bacteria (NEB). When the bacterial density reached OD600 = 0.8, protein expression was triggered by treatment with 0.1 mM IPTG (BD-1 -thiogalactopyranoside) and the cells were incubated overnight at 28°C in Terrify Broth medium containing 0.1% glucose. The cells were then harvested by centrifugation at 4000 rpm for 15 min at 4°C and resuspended in TES buffer (200 mM Tris HCl pH 8.0, 500 mM Sucrose, 50 nM EDTA). The periplasmic fraction is prepared by osmotic shock and the nanobodies are purified by affinity chromatography on nickel resin (Qiagen) and gel filtration (Superdex 75 16 / 600; Cytiva) in 20 mM HEPES, 0.3 M NaCl pH 7.4 buffer.

[0177] Some nanobodies were also cloned into the pHEN vector containing the LPETG sequence (recognition site for Sortase, SEQ ID NO. 39) and a C-terminal 6-His tag (gift from Dr. Leo Hanke, Karolinska Institut, Stockholm, Sweden), and produced and purified as described in (Hanke et al., 2020). In parallel, Sortase A 5M cloned into the pET30b vector (Àddgene #51140), with a C-terminal 6-His tag, was produced and purified according to the supplier's instructions. The nanobodies were biotinylated on their C-terminus using 50 pM Sortase 5M and 200 pM GGGK-biotin (Covalab) in 50 mM HEPES buffer pH 7.5, 150 mM NaCl, 10 mM CaCl, 2 h at 25 °C. Sortase 5M and excess nanobodies were removed by passage on Ni-NTA resin (Qiagen; 2 ml), then excess biotin by passage on Zeba spin (7K MWCO, Thermo Fisher Scientific).

[0178] SPR analyses

[0179] Surface plasmon resonance (SPR) experiments were performed on a Biacore T200 instrument (Cytiva). Ligand proteins, dissolved in 10 mM HEPES pH 7.4 buffer for PCPE-1 or 10 mM sodium acetate pH 4.5 for mini-procollagen III and streptavidin, were immobilized on CM5 chip by amine coupling. Analytes were diluted in running buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 5 mM CaCl, 0.05% P2O) and injected at a flow rate of 30 or 50 pL / min. Competition, epitope binning and kinetics experiments were all performed at 25°C. Regeneration was achieved by successive injection of 2M guanidine chloride and 0.5 M EDTA for 30 sec. Sensorgrams were analyzed using Biacore T200 Evaluation Software v3.2.1 (Cytiva), using best-fit models. IC50 was determined by nonlinear regression using GraphPad Prism (v8.2.1).

[0180] Activity tests

[0181] To characterize the effects of nanobodies on PCPE-1 activity, 400 nM CPIII-Long (or Mini I or Mini II) was incubated for 1 h at 37 °C with 2.5 nM BMP-1 and 75 nM PCPE-1 (when present) in the presence or absence of 2 pM nanobodies in the following buffer: 20 mM HEPES pH 7.4, 0.15 M NaCl, 5 mM CaCl2, 0.02% n-octyl-BD-glucopyranoside. Samples were analyzed by SDS-PÀGE on 4-20% gradient gels (Criterion; Biorad) and Instant Blue staining (Euromedex). The level of activation by PCPE-1 was assessed as the ratio of the intensity of the C-propeptide band to the intensity of all long CPI II bands (cleaved and uncleaved), after normalization with the BMP-1 alone condition, using ImageQuantTL software (Cytiva).

[0182] Effect of nanobodies on C-terminal maturation of fibrillar collagens in vitro

[0183] Rat heart fibroblasts were cultured in DMEM, 10% FCS (Eurobio), 1% AAS (Antibiotic-antimycotic solution; Thermo Fisher) at 37 °C, 5% CO2. At 70% cell confluence, serum was removed and replaced with serum-free medium containing PCPE-1 (5 pg-mL' 1 ) or / and 15 pg-mL' 1 nanobodies. After 48 h of culture, the supernatants are harvested and the procollagen is analyzed by western blotting using an anti-C-propeptide antibody (LF41, gift from Dr. Larry W. Fisher, Bethesda, USA). Quantification of the bands is performed using ImageQuantTL software (Cytiva).

[0184] Human skin fibroblasts (donated from the tissue and cell bank of the Edouard Herriot Hospital, Lyon) were cultured in the same way.

[0185] ELISA analyses

[0186] 100 ng of an anti-PCPE-1 antibody in a carbonate buffer pH 9.6 is coated on the bottom of a microplate overnight at 4°C. After blocking with a 1% BSA solution, increasing amounts of PCPE-1 (purified human protein) are added and left in contact for 1 h 30 min at room temperature. PCPE-1 is detected by adding a biotinylated VHH, for example VHH-I5 (100 ng) for 1 h at room temperature, followed by the addition of extravidin-HRP and detection using TMB solution. For detection of PCPE-1 in blood, human plasma samples are diluted 50-fold and analyzed as above.

[0187] Marking of VHH-H4 and study of its characteristics

[0188] VHH-H4 is bioconjugated to NODAGA and then labeled with galium 68 according to the procedure described in (Renard et al, 2020). Its biodistribution is evaluated in rats after iv injection of 25 pg (12 MBq). For this, 9 blood samples (150-200 pL) were taken during the 2.5 hours following the injection, after which the animal was euthanized. The tissues collected were analyzed by gamma counting.

[0189] Statistical analyses

[0190] Unless otherwise stated, data represent the mean ± standard deviation of at least three independent experiments, calculated using Graphpad Prism 8.2 software.

[0191] RESULTS

[0192] Example 1. Selection and characterization of nanobodies directed against PCPE-1

[0193] Figure 1 presents the results of the selection of PCPE-1-binding nanobodies from the llama nanobody library or a synthetic library.

[0194] Since the CUB1 and CUB2 domains of PCPE-1 are necessary and sufficient for its activity, we chose to use as an antigen a protein containing these two domains, but lacking the NTR domain. The tags used for purification were removed to avoid the selection of nonspecific ligands. Two strategies were carried out in parallel: an in vivo selection of llama nanobodies by immunization of the animal and an in vitro selection from a library of synthetic nanobodies.

[0195] For the immune library, nanobodies were generated by five successive injections of recombinant CUB1 CUB2 into a llama, followed by two biopanning steps. 48 clones were analyzed by phage-ELISA, resulting in 11 unique clones after sequencing. These 11 clones can be classified into six families based on their CDR3. The first family is the most represented with 5 clones while the others have only one or two variants. 3 members of family 1 (VHH-11, VHH-15 and VHH-17) and one member of each of the other families were selected, produced in the periplasm of E. coli and purified. Their affinity for PCPE-1 was measured by surface plasmon resonance (SPR) (Fig. 1A). All, except VHH-110, bind to PCPE-1 (Table 2). It is noted that nanobodies belonging to the same family have very different behaviors.In family 1, the interaction of VHH-11 with PCPE-1 appears less stable than those of VHH-I5 and -I7, leading to a significantly higher dissociation constant (Table 2). Among the other llama nanobodies, only VHH-I3 is a potent ligand of PCPE-1, with dissociation constants in the nanomolar range.

[0196] Synthetic nanobody selection was performed using three rounds of phage display, and then 90 clones were randomly selected and tested by phage ELISA for their ability to bind to PCPE-1. 32% of the clones gave a positive signal, corresponding to 24 unique clones after sequencing. 7 of these 24 clones also gave a positive signal when tested against immobilized CUB1 NTR. 10 clones (including these 7) were finally produced and purified, and their affinity for PCPE-1 was measured by SPR (Fig. 1 B). Of the 10, only half gave a clear interaction with PCPE-1 when injected at 200 nM, the best results being obtained with VHH-H4 and VHH-H10 (Fig. 1 B) whose KDS are in the nanomolar range (Table 2).

[0197] Measurement of the KD affinity constant of the different nanobodies studied

[0198] Nanobodies from the synthetic nanobody bank are designated by names beginning with the letter H; nanobodies from the serum of an immunized llama are designated by names beginning with I.

[0199] Figure 1A shows the PCPE-1 binding activity of the eight llama serum nanobodies: 11, 12, 13, 14, 15, 17, 110 and 111.

[0200] Figure 1 B shows the PCPE-1 binding activity of the following ten synthetic nanobodies: H1 to H10.

[0201] Table 2 below shows the KD affinity constants of seven llama-derived nanobodies (as noted above, nanobody 110 does not bind at all), and the ten synthetic nanobodies. Table 2 Affinity constants below 100 nM are representative of strong binding; this is the case for the following nanobodies: I3, I5, I7, H4 and H10.

[0202] Example 2. Determination of regions recognized by nanobodies

[0203] Figure 2 shows the results of the determination of the regions of PCPE-1 recognized by the antibodies. Competition experiments by co-injection of the nanobodies with the CUB1 or CUB2 domain of PCPE-1 were performed (Fig. 2A). When mixed with CUB2, nanobodies I3, I5 and I7 were found to be unable to bind to PCPE-1, whereas co-injection with CUB1 had almost no effect, indicating that these nanobodies interact mainly with the CUB2 domain of PCPE-1. In contrast, the interaction of synthetic nanobodies H4 and H10 with PCPE-1 was prevented by co-injection with CUB1 and not with CUB2, indicating that they interact with the CUB1 domain.

[0204] For the best ligands in each library (I5 and H4), we then determined whether the other nanobodies possessed the same epitope or were able to bind simultaneously to PCPE-1. To this end, the PCPE-1 chip was first saturated with the I5 or H4 nanobody. While no change in signal was observed when VHH-I5 was subsequently co-injected with the other llama nanobodies, co-injection of VHH-I5 and VHH-H4 or H10 led to a strong increase in signal (Fig. 2B), indicating that they bind simultaneously to distinct sites on PCPE-1. Similarly, when the surface was first saturated with VHH-H4 (Fig. 2C), the llama nanobodies were still able to interact with PCPE-1 while VHH-H10 could no longer bind. These results confirm that the two libraries have different epitopes, but that within a library, the epitopes are rather conserved.

[0205] PCPE-2 is a protein from the same family as PCPE-1, with a 43% identical amino acid sequence. It shares some activities with PCPE-1, but also has very different specific functions. The specificity of the nanobody interaction for PCPE-1 compared to PCPE-2 was analyzed by SPR. For this, VHH-I5 and VHH-H4 were biotinylated on their C-terminus using sortase, then immobilized on a streptavidin surface. In both cases, the interaction with PCPE-2 is almost undetectable (KD > 1 pM) while PCPE-1 binds very strongly (KD = 1.5 nM (VHH-I5) and KD = 3.9 nM (VHH-H4), Fig. 2D-E and Table 3 below). The affinity of VHH-I5 and -H4 for PCPE-1 is therefore at least 250 times stronger than for PCPE-2.

[0206] Table 3

[0207] Example 3: Inhibitory antagonist effects on PCPE-1 of the nanobodies according to the invention

[0208] Figure 3 shows the results of the inhibitory effect of the nanobodies according to the invention on the activity of PCPE-1. The activity of PCPE-1 is mediated by its direct interaction with the C-TJ propeptides of procollagens. We therefore observed whether the nanobodies were able to prevent the binding of PCPE-1 to a mini-procollagen III (Mini III, composed of the C-terminal domains of procollagen III: end of the triple helix, C-telopeptide and C-propeptide) using SPR competition experiments.

[0209] The results show that VHH-I3, -I5, -I7, -H4 and -H10 inhibit the binding of PCPE-1 to Mini III while VHH-11, -I2, -I4 and -111 have no effect (Fig. 3A).

[0210] VHH-H4, -H10 and VHH-I5 are the most effective, with 50-75% inhibition at 250 nM.

[0211] Moreover, their antagonistic effects are additive, and co-injection of a mixture of VHH-I5 and VHH-H4 with PCPE-1 virtually eliminates its interaction with Mini III. This inhibitory effect increases with concentration, with IC50s estimated at approximately 45 nM (VHH-H4), 24 nM (VHH-I5), and 20 nM (mix of VHH-I5 and VHH-H4) in the presence of 5 nM PCPE-1.

[0212] However, while complete inhibition is achieved at high concentrations for VHH-H4, the addition of VHH-I5 alone does not completely block the PCPE-1 / Mini III interaction, whereas inhibition is complete when VHH-H4 and VHH-I5 are combined.

[0213] Since the nanobodies prevent PCPE-1 binding to procollagens, the next step was to see if they also inhibit PCPE-1 activity in vitro. CPH l-Long (a model substrate of procollagen III) is cleaved by incubation with the protease BMP-1 and this cleavage is increased in the presence of PCPE-1 (Fig. 3B, 2.4-fold increase under these conditions). The addition of VHH-11, VHH-I2, VHH-I4 or VHH-111 has very little effect on PCPE-1 activity. In contrast, the addition of VHH-I3, VHH-I5, VHH-I7, VHH-H4 or VHH-H10 leads to a 25-45% decrease in activation, and this is completely inhibited by the simultaneous use of VHH-I5 and VHH-H4. It should also be noted that the basal activity of BMP-1 does not appear to be modified by the presence of VHHs, despite the fact that BMP-1 also contains three CUB domains.

[0214] Finally, the nanobodies were evaluated in a cell-based assay for their ability to modulate procollagen I cleavage.

[0215] To mimic fibrotic conditions, rat heart fibroblasts were treated with recombinant human PCPE-1 protein (Fig. 3C,D). Addition of PCPE-1 to the medium increased the release of C-propeptide from procollagen I by an average factor of about 2.5. The presence of VHH-I5 and VHH-H4 inhibited the effect of PCPE-1 (by about 40%), as shown by the decrease in C-propeptide (Fig. 3C). Clearly, co-treatment with VHH-I5 and VHH-H4 completely blocked the effect of exogenous PCPE-1, with a return to the basal amount of C-propeptide release. Furthermore, treatment of cells with VHHs alone (in the absence of added PCPE-1) had no effect on C-propeptide release. All these results suggest that the selected ligands are potent antagonists of PCPE-1, capable of blocking the stimulation of C-terminal maturation of procollagens by PCPE-1.

[0216] Example 4: Construction and characterization of a diabody (divalent nanobody)

[0217] Previous results suggest that combining I5 and H4 nanobodies in a single polypeptide chain could further enhance their affinity for PCPE-1 due to site cooperativity.

[0218] Figure 4 shows the results obtained for the diab-D1 diabody of SEQ ID NO. 21, produced in bacteria.

[0219] The interaction of diab-D1 with PCPE-1 was assessed by SPR, by injecting increasing concentrations of diab-D1 onto immobilized PCPE-1. The benefit of VHH fusion is clearly visible with the avidity it provides, which is manifested by an improved dissociation constant (KD = 0.32 nM) and a much slower dissociation (Fig. 4A).

[0220] On the other hand, competition experiments were performed to determine whether diab-D1 had a more potent antagonistic potency than the nanobodies used individually (Fig. 4B). The calculated IC50 for diab-D1 is estimated at 3.5 nM, which is significantly lower than that of VHH-I5 and VHH-H4 alone or co-injected (5- to 13-fold) under the same conditions. Similarly, the addition of diab-D1 completely blocks the stimulation of CPH I-Long cleavage by PCPE-1 (Fig. 4C).

[0221] Effect of diab-D1 on the cleavage of procollagens I and II

[0222] The diab-D1 diabody is also able to block the activity of PCPE-1 on other fibrillar collagens. Indeed, the addition of diab-D1 inhibits the activation of Miniprocollagen I (Mini I) and II (Mini II) cleavage by PCPE-1 as illustrated in Figure 4D.

[0223] Modulation of collagen maturation by diab-D1

[0224] In cell culture (Fig. 4E, 4F), diab-D1 is able to completely block the effect of PCPE-1 addition on the cleavage of endogenous procollagen I by rat heart fibroblasts. On the other hand, and unlike individual VHHs, the diabody also inhibits C-propeptide cleavage in the absence of added PCPE-1.

[0225] Diabody also inhibits the cleavage of procollagen I produced by human skin fibroblasts, either in the basal state or under “fibrotic” conditions mimicked by TGF-beta stimulation, or in the presence of added exogenous PCPE-1.

[0226] This is illustrated in Figures 4G and 4H, which show immunodetection of procollagen I and its C-terminal cleavage products in the culture medium of human skin fibroblasts, after 72 h of culture in the absence or presence of PCPE-1, TGF-beta and diab-D1. Further measurements were performed by immunofluorescence, on human fibroblasts in culture (results not shown). It was found that the matrix deposited by the cells is also affected, since the addition of diab-D1 leads to a decrease in the collagen deposition detected by immunofluorescence.

[0227] Example 5: Nanobody coupled to a detectable marker

[0228] Nanobodies containing the LPETG sequence (SEQ ID NO. 39) were coupled to biotin using Sortase as described above. The coupled nanobodies can be attached to streptavidin, allowing the detection of PCPE1 in a complex mixture, by SPR, ELISA or Western Blot.

[0229] Figure 5A represents the SPR study of the interaction of PCPE-1 with the biotinylated H4 nanobody, captured on a chip coupled to streptavidin.

[0230] Figure 5B shows the detection of PCPE-1 using a sandwich ELISA. The sample containing PCPE-1 is captured by an anti-PCPE-1 antibody. The addition of biotinylated VHH-I5 allows its detection, via the use of streptavidin coupled to HRP or a fluorophore such as Alexafluor488. The method is very sensitive and can easily detect 0-1 ng of PCPE-1. This allows the amount of PCPE-1 present in human plasma to be determined, which is approximately 510 ± 70 ng / ml.

[0231] Alternatively, PCPE-1 can be detected by sandwich ELISA between H4 and I5.

[0232] The nanobodies were also coupled to a radioactive label, NODAGA-Ga 68 .

[0233] For this, VHH-H4 was first bioconjugated to NODAGA, then labeled with galium 68 according to the procedure described in (Renard, 2020).

[0234] The biodistribution and pharmacokinetics of the nanobody were then evaluated in rats after intravenous injection of 25 pg (12 MBq) of H4-Ga 68 , in order to determine the elimination pathways and areas of accumulation of the molecule.

[0235] The results are presented in Figure 6A (pharmacokinetics) and 6B (biodistribution in the main organs: heart, lungs, blood, liver, spleen, kidneys and bladder).

[0236] The H4-Ga nanobody 68 can also be used to detect PCPE-1 on tissue sections and phosphorimager detection: see Figure 6C. BIBLIOGRAPHY

[0237] US20120270246

[0238] W02017065206

[0239] W02019080284

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[0241] Shi R, Zhang Z, Zhu A, Xiong X, Zhang J, Xu J, Sy MS, Li C. Targeting type I collagen for cancer treatment. Int J Cancer. 2022 Feb 28. doi: 10.1002 / ijc.33985

[0242] Kronenberg, D., Vadon-Le Goff, S., Bourhis, J. M., Font, B., Eichenberger, D., Hulmes, D. J. & Moali, C. (2009). Strong cooperativity and loose geometry between CUB domains are the basis for procollagen C-proteinase enhancer activity. J Biol Chem. 284, 33437-33446

[0243] Vadon-Le Goff, S., Kronenberg, D., Bourhis, J. M., Bijakowski, C., Raynal, N., Ruggiero, F., Farndale, R. W., Stocker, W., Hulmes, D. J. a Moali, C. Procollagen C-proteinase enhancer stimulates procollagen processing by binding to the C-propeptide region only. J. Biol. Chem. 286, 38932-38938 (2011 ).

[0244] Lagoutte P, Bettier E, Vadon-Le Goff S, Moali C. Procollagen C-proteinase enhancer-1 (PCPE-1 ), a potential biomarker and therapeutic target for fibrosis. Matrix Biol Plus. 2021 Apr 20; 11 :100062. doi: 10.1016 / j.mbplus.2O21 .100062.

[0245] Lagoutte, P., Oudot, A., Dussoyer, M., Goncalves, V., Bouchot, O., Vandroux, D., Bellaye, P. S., Moali, C. a Vadon-Le Goff, S. Procollagen C-Proteinase Enhancer 1 (PCPE-1 ) is a marker of myocardial fibrosis and impaired cardiac function in a murine model of pressure overload. bioRxiv 2021 ; doi: https: / / doi.org / 10.1101 / 2021.03.05.434071

[0246] Désogère P, Montesi SB, Caravan P. Molecular Probes for Imaging Fibrosis and Fibrogenesis. Chemistry. 2019 Jan 24;25(5):1128-1141 . doi:10.1002 / chem.201801578.

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[0250] Kwon NY, Kim Y, Lee JO. Structural diversity and flexibility of diabodies. Methods. 2019 Feb 1;154:136-142. doi: 10.1016 / j.ymeth.2018.09.005. Epub 2018 Sep 25. PMID: 30261312. Pulido D, Sharma U, Vadon-Le Goff S, Hussain S, Cordes S, Mariano N, Bettier E, Moali C, Aghajari N, Hohenester E, Hulmes DJS. Structural Basis for the Acceleration of Procollagen Processing by Procollagen C-Proteinase Enhancer-1. Structure. 2018 Oct 2;26(10):1384-1392.e3. doi:10.1016 / j.str.2O18.06.011 Moutel S, Bery N, Bernard V, Keller L, Lemesre E, de Marco A, Ligat L, Rain JC, Favre G, Olichon A, Perez F. NaLi-H1 : A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies. Elife. 2016 Jul 19;5:e16228. doi: 10.7554 / eLife.16228.

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Claims

DEMANDS 1. Specific ligand of the glycoprotein PCPE-1 characterized in that it is a nanobody.

2. Specific ligand of the glycoprotein PCPE-1 characterized in that it inhibits its interaction with the C-terminal domain of procollagens.

3. Specific ligand of the PCPE-1 glycoprotein according to claim 2, characterized in that it is a nanobody.

4. Specific ligand of the PCPE-1 glycoprotein according to any one of claims 1 or 3, characterized in that said nanobody comprises three complementarity-determining regions (CDRs) with PCPE-1, and that among these three CDRs, at least one CDR exhibits at least 80% identity with one of the sequences SEQ ID NO. 1 to SEQ ID NO.

15.

5. Specific ligand of the glycoprotein PCPE-1 according to claim 4, characterized in that each of the three CDRs designated CDR1, CDR2 and CDR3 is defined as follows: i. CDR1 has at least 80% identity with one of the following sequences: SEQ ID NO. 1, 4, 7, 10 or 13; ii. CDR2 has at least 80% identity with one of the following sequences: SEQ ID NO. 2, 5, 8, 11 or 14; and iii. CDR3 has at least 80% identity with one of the following sequences: SEQ ID NO. 3, 6, 9, 12 or 15.

6. Specific ligand of the glycoprotein PCPE-1 according to any one of claims 1 or 3 to 5, the peptide sequence of which has at least 80% identity with one of the sequences SEQ ID NO. 16 to SEQ ID NO.

20.

7. Specific ligand of the PCPE-1 glycoprotein consisting of a combination of two or three nanobodies according to any one of claims 1, 3, 4, 5 or 6, in particular of a combination of two polypeptide sequences each having at least 80% identity with one of the sequences SEQ ID NO. 16 to SEQ ID NO. 20, optionally comprising one or two additional linkers.

8. Specific ligand of the glycoprotein PCPE-1 according to claim 7, the polypeptide sequence of which has at least 80% identity with the SEQ ID NO. 21 sequence.

9. Nucleic acid encoding a nanobody according to any one of claims 1 or 3 to 6, or encoding a combination of two or three nanobodies according to any one of claims 7 or 8.

10. Specific ligand of the glycoprotein PCPE-1 according to any one of claims 2 to 8, for its use as a medicinal product.

11. Specific ligand of the glycoprotein PCPE-1 according to any one of claims 2 to 8, for its medical use in the treatment of cancer or fibrosis, in particular cardiac fibrosis.

12. Specific ligand of the PCPE-1 glycoprotein according to any one of claims 1 to 8, characterized in that it is coupled to a detectable marker.

13. Use of a specific ligand of the PCPE-1 glycoprotein according to claim 12, for the in vivo monitoring of a pathological state by medical imaging.

14. Specific ligand of the glycoprotein PCPE-1 according to claim 12, for its theranostic use for the treatment and in vivo monitoring of a pathological condition by medical imaging.

15. Kit for determining the activity and / or quantity of PCPE-1 glycoprotein in an in vitro or ex vivo biological sample, comprising: - a specific ligand of the PCPE-1 glycoprotein according to any one of claims 1 to 8 or 12, and - detection reagents.

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