Ligands Specific to PCPE-1 Glycoprotein and Their Use

Abstract

This application relates to a ligand specific for the PCPE-1 glycoprotein, which is characterized by being a nanobody. This application also relates to a ligand specific for the PCPE-1 glycoprotein, which is characterized by inhibiting its activity.

Description

Technical Field

[0001] The present invention relates to ligands specific for the PCPE-1 (procollagen C-proteinase enhancer-1) protein. The present invention also relates to its use in medical imaging and diagnostic and treatment methods. More specifically, the present invention focuses on two types of ligands: a ligand that binds to PCPE-1 and can be used for imaging, and another so-called antagonist ligand that binds to PCPE-1, inhibits its activity, and can be used for treatment. In one embodiment of the present invention, these antagonist ligands are used for the diagnosis and treatment of fibrosis and cancer.

Background Art

[0002] Fibrillar collagens are the most abundant proteins in the human body and are the main components of the extracellular matrix. They shape organs and tissues and play important roles in maintaining their homeostasis or repairing them after injury. Collagen has long been thought to have a structural role in tissues, but is now recognized as playing an essential role in many cellular processes such as cell adhesion, proliferation, migration, and differentiation.

[0003] However, these collagens are also associated with a number of pathological processes and are major targets for the development of new diagnostic tools or treatments.

[0004] Therefore, fibrosis, which is characterized by the excessive deposition of the extracellular matrix mainly composed of collagen fibers, is a major common factor in many pathologies and strongly affects the progression of diseases and the effectiveness and implementation of treatments (Henderson et al., 2020). Furthermore, the production of abnormal collagen by tumor cells or the microenvironment plays a major role in the immune evasion of these cells and strongly contributes to their metastatic ability and their dormancy and resistance to treatment (Shi et al., 2022).

[0005] The targeting of collagen, more specifically all the key stages involved in its biosynthesis, secretion, maturation or macro-structuration, are all targets of interest for limiting the excessive collagen production associated with these processes. These include LARP6, 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, BTP (bone morphogenetic protein-1 (BMP1) / Tolloid-like proteinase) is the main protease involved in the removal of the C-propeptide from procollagen fibrils, a process generally considered to be the rate-limiting step in the formation of type I collagen fibrils. However, while BTP is a major target, in addition to collagen, they have activity against a number of other substrates involved in several pathways (such as growth factor activation, angiogenesis, calcification, tumor progression, etc.), which means that inhibiting their activity can lead to undesirable side effects.

[0006] Another important regulator of collagen fibril formation is PCPE-1 (procollagen C-proteinase enhancer-1), a secreted glycoprotein that specifically stimulates the C-terminal cleavage of procollagen fibrils by BTP. 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 domain is necessary and sufficient for PCPE-1 activity because of its direct and close interaction with the C-propeptide of procollagen (Kronenberg 2009, Vadon 2011). This interaction is thought to allow for the local disruption of procollagen trimers that promotes cleavage by BTP, and PCPE Explain whether -1 has any effect on other BMP-1 substrates. Additionally, data has shown the overexpression of PCPE-1 in different fibrosis situations (Lagoutte et al., Matrix Biol Plus 2021), targeting it as a promising target for the monitoring and / or treatment of fibrosis. In particular, a correlation has been established between PCPE-1 expression levels and fibrosis, especially myocardial fibrosis (Lagoutte et al., BioRxiv, 2021). The direct detection of PCPE-1 in biological fluids by immunoassay has also been proposed as a diagnostic method (U.S. Patent Application Publication No. 2012270246 for bone formation, International Publication No. 2017065206A1 for NASH).

[0007] However, currently, there is no robust method to non-invasively detect PCPE-1 in tissues and thus monitor fibrotic collagen accumulation. Furthermore, the lack of an effective tool to inhibit the action of PCPE-1 has so far hindered the evaluation of pharmacological strategies targeting this protein.

[0008] International Publication No. 2019 / 080284 teaches that the PCPE-1 protein is a therapeutic target for combating fibrotic diseases, including dermal fibrosis and difficult scarring. The strategy considered in this paper is the inhibition of PCPE-1 expression by the injection of siRNAs designed to block the translation of the gene into protein.

[0009] Nevertheless, in certain treatments, it is preferable to inhibit the activity of the protein rather than restricting its expression. To date, no agent has been reported to block the interaction between the PCPE-1 protein and procollagen C-propeptide.

[0010] In the diagnosis of myocardial fibrosis, histological analysis of endomyocardial biopsy is still used as a traditional method, despite the fact that it is invasive, can only analyze a very small part of the heart tissue, and does not necessarily represent the whole. The combination of MRI and gadolinium injection now enables imaging of large fibrotic areas, but it is not completely specific for fibrosis and does not detect interstitial fibrosis.

[0011] One of the characteristics of fibrotic lesions and cancer is that they are progressive. This means that, especially in the case of fibrosis, even if it is the result of stress or injury, the overproduction of collagen continues, and sometimes pathological and functional effects only occur after a very long period. Therefore, there is a great need to be able to detect this excessive collagen production at an early stage and monitor its progression over time. Molecular imaging, combined with increasingly sensitive detection systems (isotope imaging, fluorescence, etc.), is now an essential tool for long-term monitoring of these conditions and for monitoring the therapeutic effects of new treatments.

[0012] To date, especially in the context of fibrosis, the main approach employed for direct molecular imaging of collagen has been based mainly on the use of probes that directly target collagen by hybridization. Examples include collagen peptides, EP-3533, or CBP8 (Desogere et al., 2019).

[0013] The results are promising, but other indirect pathways, such as those disclosed in the present invention, will offer distinct advantages by potentially limiting the background signal-to-noise ratio. This is particularly true for probes that target collagen integrin receptors.

[0014] The use of immunoglobulins to target proteins of interest has been widely demonstrated for both therapeutic and molecular imaging purposes, and they are currently biopharmaceuticals on the market It occupies most of the products. In recent years, an immunoglobulin scaffold lacking the undesirable properties of conventional immunoglobulins (high molecular weight, heterotetrameric composition, disulfide bonds) has emerged as an alternative to conventional antibodies. Among them, nanobodies, also known as VHH (Variable heavy chain domain of heavy chain antibodies), derived from the heavy chain of camelid immunoglobulins discovered in the early 1990s, have demonstrated interest in both therapy and diagnosis (Muyldermans, 2021). Nanobodies are the smallest antibody fragments (about 15 kDa), highly soluble and stable, and can be easily produced in large quantities in prokaryotic systems. In addition, their convex paratopes are well-suited for binding to target-difficult cavities on the antigen surface.

[0015] To select antigen-specific nanobodies, three approaches can be used: immune libraries, naive libraries, and synthetic libraries. Immune libraries are created by immunizing camelid animals with a target. These are the most widely used nanobody libraries, but some targets are not immunogenic or are too toxic for animal immunization. Due to these limitations, several synthetic libraries have been developed. Selection can be performed from these libraries to identify nanobodies specific to one or more target proteins. To date, more than 1400 VHHs have been selected and successfully used as research tools in biotechnology or diagnostic applications.

Summary of the Invention

[0016] The present invention relates to a ligand specific for the PCPE-1 glycoprotein, which is a nanobody also represented by the abbreviation VHH (Variable domain of the Heavy chain of Heavy chain-only antibodies, Variable domain of the heavy chain of heavy chain-only antibodies). It is characterized by a ligand that is a nanobody also represented by the variable domain of the heavy chain of heavy chain-only antibodies.

[0017] The present invention also relates to a ligand specific for the PCPE-1 glycoprotein, which is characterized by inhibiting its activity. This specific ligand is also known as a PCPE-1 antagonist ligand. In particular, this is a PCPE-1 antagonist nanobody.

[0018] Another object of the present invention is a ligand specific for the PCPE-1 glycoprotein, which consists of a combination of two or three of the above-mentioned nanobodies.

[0019] The present invention also relates to a nucleic acid encoding the nanobody or a combination of two or three nanobodies described in the present application.

[0020] The present invention also relates to a specific antagonist ligand for the PCPE-1 glycoprotein for use as a medicament, more specifically for medical use in the treatment of cancer or fibrosis, particularly myocardial fibrosis.

[0021] The present invention also relates to a ligand specific for the glycoprotein PCPE-1 described in the present application, which is characterized by binding to a detectable marker, particularly a marker used in medical imaging.

[0022] Another object of the present invention is the use of such a specific ligand for the PCPE-1 glycoprotein, which is bound to a detectable marker, for in vivo monitoring of pathological conditions by medical imaging.

[0023] Another object of the present invention is a specific ligand for the PCPE-1 glycoprotein as described above for its theranostic use in the treatment and in vivo monitoring of pathological conditions by medical imaging.

[0024] Finally, the present application is a kit for determining the activity and / or amount of PCPE-1 glycoprotein in a biological sample in vitro or ex vivo, - a ligand specific for the above-mentioned PCPE-1 glycoprotein, and - a detection reagent, comprises.

Brief Description of Drawings

[0025]

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Mode for Carrying Out the Invention

[0026] The present invention relates to a ligand specific for the PCPE-1 glycoprotein, which is characterized by being a nanobody (also known as VHH).

[0027] PCPE-1 (procollagen C-proteinase enhancer-1) glycoprotein is an important regulator of collagen fiber formation. The human protein is listed in the UniProt database under the reference number Q15113, and its polypeptide sequence is described.

[0028] Collagen is synthesized in the form of a soluble precursor known as procollagen. These undergo proteolytic maturation before they can assemble into collagen fibers. This limiting step is regulated by the PCPE-1 glycoprotein, which specifically stimulates the C-terminal cleavage of fibrillar procollagen by the BTP protease and is responsible for the removal of the C-propeptide from fibrillar procollagen. To achieve this, PCPE-1 interacts closely with the collagen C-propeptide via its two CUB (complement-Uegf-BMP-1) domains.

[0029] The term "specific ligand" refers to a compound that interacts with a protein in a non-covalent, reversible and specific manner.

[0030] A ligand is said to be "specific" if, on the one hand, it binds preferentially to its target protein among a number of target proteins with similar structures, and on the other hand, it exhibits an affinity that is considered to be sufficiently strong, i.e., the strength of the interaction between the ligand and the target protein. The affinity is expressed as an affinity constant or an equilibrium dissociation constant, or abbreviated as "K" DIt is quantitatively measured by the association / dissociation equilibrium constant, also known as "K". D The lower the K value, the higher the binding affinity between the ligand and its target protein.

[0031] Within the scope of the present invention, a ligand is considered to specifically bind to PCPE-1 when its affinity constant K D is less than 100 nM, or less than 80 nM, or less than 50 nM, and more preferably less than 30 nM. Regarding these figures, those skilled in the art will be able to determine which ligands are specific to the present invention.

[0032] Nanobodies are single-domain antibodies, corresponding to antibody fragments composed of a single monomeric variable antibody domain, which correspond to the variable domains of the heavy chain of the type of antibody found in camelids and originally lack a light chain. Nanobodies can selectively bind to specific antigens. This has the advantage that it has a molecular weight of only 12 - 15 kDa, which is about one-tenth of that of a general antibody with a molecular weight of 150 - 160 kDa. Nanobodies also occupy much less space than conventional antibodies. Furthermore, this simplified structure makes the synthesis process easier, especially in bacterial cells. The first single-domain antibodies were developed from heavy-chain antibodies found in camelids (these are known as VHH fragments).

[0033] In the present application, the terms "nanobody" and "VHH" are used interchangeably and both refer to single-domain antibodies.

[0034] Each nanobody has three CDRs, designated CDR1, CDR2, and CDR3.

[0035] The nanobodies according to the present invention can in particular be llama nanobodies or synthetic nanobodies.

[0036] Regardless of the presence or absence of a binder, a combination of two covalently linked nanobodies is a "di It is called a "body" or a "bivalent nanobody".

[0037] A combination of three nanobodies is known as a "tribody" or a "trivalent nanobody".

[0038] Also included in the present invention are other ligands specific for the PCPE-1 glycoprotein, in particular any nanobody-equivalent protein structures other than antibodies. Examples of such protein structures ("protein scaffolds") that can specifically bind to a target epitope are presented in the review by (Gebauer & Skerra, 2019).

[0039] The present invention also relates to a ligand specific for the PCPE-1 glycoprotein, which is characterized in that it inhibits its interaction with the C-terminal domain of procollagen, thereby inhibiting the activity of this PCPE-1 glycoprotein.

[0040] In addition to the ability to bind to this glycoprotein, certain PCPE-1-specific ligands have the ability to inhibit the activity of the PCPE-1 glycoprotein. These are then called "PCPE-1 antagonist ligands".

[0041] The term "antagonist" refers to a compound that interacts with a physiologically active target protein and reduces or further suppresses the physiological activity of the protein.

[0042] As shown in Example 3, significant inhibition of PCPE-1 activity is - inhibition of the interaction with the C-terminal domain of procollagen, - or, inhibition of procollagen cleavage stimulated by the action of PCPE-1 under normal conditions, corresponding to.

[0043] More specifically, this inhibition of activity is characterized by inhibition of the interaction between PCPE-1 and the C-terminal domain of procollagen.

[0044] This inhibition of PCPE-1 activity is considered significant if it is more than 40%, or even at least 50%, preferably more than 60%.

[0045] These PCPE-1 antagonist ligands can be of any type including synthetic chemical compounds, nucleic acids or protein structures, especially those containing polypeptide chains.

[0046] According to a preferred embodiment, the PCPE-1 antagonist ligand is a polypeptide chain, especially a nanobody (VHH). It is understood that any polypeptide structure equivalent to a nanobody is included in the present invention.

[0047] Specific nanobody The inventors have identified two families of nanobodies having either PCPE-1 binding activity for use in medical imaging and diagnosis, or PCPE-1 binding and inhibitory activity for use as a drug.

[0048] These nanobodies are selected from a synthetic nanobody library or after llama immunization and have PCPE-1 binding activity as measured by surface plasmon resonance as shown in Figure 1. They were selected to have a K D of less than 100 nM, more specifically less than 30 nM (see, for example, Table 2).

[0049] These nanobodies were sequenced together with their CDRs. The results are summarized in Table 1 below. summarized.

[0050]

Table 1-1

[0051]

Table 1-2

Table 1-3

[0052] As is well known to those skilled in the art, the combination of CDR1, CDR2 and CDR3 is sufficient to define the antigen-binding site.

[0053] For the purposes of the present invention, the term "CDR" (complementarity determining region) refers to an amino acid sequence that together defines the binding affinity and specificity of the natural Fv region of the natural immunoglobulin binding site.

[0054] According to a particular embodiment of the present invention, the PCPE-1 specific ligand is a nanobody, the polypeptide sequence of which comprises three complementarity determining regions (CDRs) with PCPE-1, and at least one of these three CDRs has at least 80% identity with one of the sequences of SEQ ID NO: 1 to SEQ ID NO: 15.

[0055] According to another particular embodiment, of these three CDRs designated as CDR1, CDR2 and CDR3, respectively i. CDR1 has at least 80% identity with one of SEQ ID NO: 1, 4, 7, 10 or 13, ii. CDR2 has at least 80% identity with one of SEQ ID NO: 2, 5, 8, 11 or 14, iii. CDR3 is defined as having at least 80% identity with one of SEQ ID NO: 3, 6, 9, 12 or 15.

[0056] According to another particular embodiment, - 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, - 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 described in (ii) above, - The CDR3 of the nanobody is defined as having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with at least one of the CDR3 sequences described in (iii) above.

[0057] The percentage of identity referred to in the present invention is determined after the optimal alignment of the sequences being compared and thus it may include one or more additions, deletions, truncations and / or substitutions.

[0058] This percentage of identity can be calculated by any sequence analysis method well known to those skilled in the art.

[0059] The percentage of identity can be determined over their entire length after global alignment of the sequences being compared. Similar to manual alignment, global sequence alignment can be determined using the algorithm of Needleman and Wunsch (1970). using.

[0060] For nucleotide sequences, sequence comparison can be carried out using any software program well known to those skilled in the art such as Needle. The parameters used can include, in particular, "Gap Open" equal to 10.0, "Gap Extend" equal to 0.5 and the EDNAFULL matrix (EMBOSS version of NCBI NUC4.4).

[0061] For amino acid sequences, sequence comparison can be carried out using any software program well known to those skilled in the art such as Needle. The parameters used can include, in particular, "Gap Open" equal to 10.0, "Gap Extend" equal to 0.5 and the BLOSUM62 matrix.

[0062] Preferably, the percentage of identity defined within the scope of the present invention is determined by global alignment of the sequences being compared over their entire length.

[0063] According to another embodiment, the ligand specific for the PCPE-1 glycoprotein is a nanobody comprising three complementarity determining regions (CDRs) with PCPE-1, and the three CDRs exhibit 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.

[0064] According to a particular embodiment, - the three CDRs of the nanobody exhibit at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the CDR sequences described in (a) above, or - the three nanobody CDRs have at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the CDR sequences described in (b) above, or - the three CDRs of the nanobody have at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the CDR sequences described in (c) above, or - the three CDRs of the nanobody have at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the CDR sequences described in (d) above, or - the three CDRs of the nanobody have at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the CDR sequences described in (e) above.

[0065] According to another embodiment, a ligand specific for the PCPE-1 glycoprotein is a nanobody comprising three complementarity determining regions (CDRs) with PCPE-1, and the three CDRs have 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.

[0066] According to another embodiment of the present invention, a ligand specific for the PCPE-1 glycoprotein is a nanobody whose polypeptide sequence has at least 80 % identity with one of the sequences of SEQ ID NOs: 16 to 20.

[0067] In particular, this nanobody has a polypeptide sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the sequences of SEQ ID NOs: 16 to 20.

[0068] Preferably, the substitutions observed in the polypeptide sequence are located in domains outside the CDRs called the "framework" regions of the nanobody.

[0069] More preferably, the CDRs are conserved and show 100% identity with the sequences described above in (a) of SEQ ID NO: 16, (b) of SEQ ID NO: 17, (c) of SEQ ID NO: 18, (d) of SEQ ID NO: 19, and (e) of SEQ ID NO: 20.

[0070] According to a preferred embodiment, the nanobody of the present invention has a polypeptide sequence consisting of the sequence of SEQ ID NO: 16, or SEQ ID NO: 17, or SEQ ID NO: 18, or SEQ ID NO: 19, or SEQ ID NO: 20.

[0071] Nanobody combination In another embodiment, the present invention discloses the association of these nanobodies within a single diabody or tribody structure. This association is preferably achieved by adding a binder between the protein sequences of each nanobody. Those skilled in the art will know how to select the most appropriate binder, especially from those disclosed in the review by Kwon, 2019. According to a preferred embodiment, GS 8 The sequence (GSGSGSGSGSGSGSGS, SEQ ID NO: 38) is used as the binder.

[0072] According to one embodiment of the present invention, a specific ligand of PCPE-1 glycoprotein consists of a combination of two or three nanobodies as described above, optionally including one or two binders, especially one or two binding peptides (one for a diabody and two for a tribody), i.e., consisting of a diabody or a tribody.

[0073] According to a particular embodiment of the present invention, it is understood that the diabody consists of a combination of a nanobody having CUB1 binding activity and another nanobody having CUB2 binding activity, and each nanobody can be located either upstream or downstream of the binding peptide.

[0074] In particular, the diabody according to the present invention can consist of any combination of two polypeptide sequences each having at least 80% or 90% sequence identity with one of the sequences of SEQ ID NOs: 16, 17, 18, 19 or 20, and optionally further includes a binder.

[0075] More specifically, the diabody consists of any combination of two polypeptide sequences having sequences selected from the sequences of SEQ ID NOs: 16 to 20 and optionally linked by a binder, especially a binding peptide.

[0076] In particular, this combination of two nanobodies (diabodies) may have a polypeptide sequence that is at least 80% or 90% identical to the sequence of SEQ ID NO: 21, in particular at least 95%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 21.

[0077] More preferably, in this diabody, the CDRs of the nanobodies are conserved and show 100% identity with the sequences described above for (a) with respect to SEQ ID NO: 16 (H4) and (c) with respect to SEQ ID NO: 18 (I5).

[0078] According to the present invention, this type of diabody may correspond to I5-linker-H4 or H4-linker-I5. An example of the amino acid sequence of the H4-linker-I5 diabody corresponds to the sequence of SEQ ID NO: 21.

[0079] According to a preferred embodiment, the diabody according to the present invention has a polypeptide sequence consisting of the sequence of SEQ ID NO: 21.

[0080] A further object of the present invention relates to a nucleic acid comprising a nucleic acid sequence encoding a nanobody according to the present invention or a combination of two or three nanobodies.

[0081] In certain embodiments, the nucleic acid according to the present invention encodes a nanobody defined by one of the amino acid sequences of SEQ ID NOs: 16-20, or comprises or consists of a nucleic acid sequence encoding a diabody defined by the sequence of SEQ ID NO: 21.

[0082] This nucleic acid is a DNA or RNA molecule that can be contained in any suitable vector such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.

[0083] The term "vector" refers to an agent capable of introducing a DNA or RNA sequence 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 vectors advantageously contain regulatory elements (e.g., promoters, activators, terminators, etc.) for inducing polypeptide expression.

[0084] As a result, another object of the present invention relates to a vector containing a nucleic acid according to the present invention.

[0085] Another object of the present invention is a host cell incorporating the above vector and thus capable of expressing a nanobody, diabody, or tribody according to the present invention.

[0086] Use of a PCPE-1 specific ligand according to the present invention The present invention also relates to a PCPE-1 specific ligand 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.

[0087] Furthermore, the ligands disclosed in the present invention having both PCPE-1 binding and inhibitory activity are ideal candidates for so-called seranostic approaches.

[0088] Finally, the present invention relates to a pharmaceutical composition containing one of these ligands together with a pharmaceutically acceptable vehicle.

[0089] The compounds according to the present invention can be used in immunoassays (ELISA, Western blot, immunofluorescence, immunohistochemistry) for detecting PCPE-1 in biological fluids or tissues in vitro or ex vivo.

[0090] In particular, the present invention relates to the above PCPE-1 glycoprotein antagonist ligand for use as a medicament.

[0091] The present invention also relates to the above-mentioned PCPE-1 glycoprotein antagonist ligand for its therapeutic use in the treatment of cancer or fibrosis, particularly myocardial fibrosis.

[0092] Fibrosis, also known as sclerosis, occurs when tissue is substantially destroyed or when inflammation occurs in areas where tissue cannot regenerate. Fibrosis is a pathological condition characterized by the excessive synthesis and deposition of the extracellular matrix (ECM), leading to pathological scarring and sclerosis, and can be fatal if vital organs (such as the heart, liver, and lungs) are affected. There are several types of fibrosis (such as myocardial fibrosis, pulmonary fibrosis, hepatic fibrosis, renal fibrosis, muscular fibrosis, etc.). Cancer is also a disease in which collagen plays an important role, but this role is complex and depends on the tumor type (Shi et al., 2021).

[0093] The present invention also relates to a method for treating cancer or fibrosis, particularly myocardial fibrosis, comprising administering a PCPE-1 antagonist ligand to a patient suffering from cancer or fibrosis to inhibit its activity as described above.

[0094] In the context of the present invention, "patient" refers to 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, particularly a human suffering from cancer or fibrosis, particularly myocardial fibrosis.

[0095] A ligand bound to a detectable marker

[0096] The present invention also relates to a ligand specific for the PCPE-1 glycoprotein as defined above, characterized in that it is bound to a marker detectable in a medical image.

[0097] ​"A PCPE-1 specific ligand conjugated to a detectable marker" as used herein means that the detectable marker is directly or indirectly linked to or incorporated into the ligand. In particular, the detectable marker can be linked to the ligand by substitution, complex formation or chelation.

[0098] In the context of the present invention, a "detectable marker" refers to a compound that generates a detectable signal. When combined with a tracer, it can be used to monitor the results of the tracer in the body. Here, the detectable marker particularly refers to a marker detectable in medical imaging.

[0099] The detectable marker used can 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.

[0100] Examples of detectable markers include radioactive elements, 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.

[0101] The selection of a suitable detectable marker depends on the detection system used and is a matter for those skilled in the art.

[0102] Marking can be oriented via introduction of a terminal cysteine or a recognition sequence by sortase or another ligase. Marking can also be achieved by direct binding after lysine activation.

[0103] The detectable marker is particularly a fluorophore, a chromophore or a luminescent compound or an antibody-detectable label. It can also be a chelating agent (e.g., NODAGA), which is then bound to a radioisotope (e.g., Ga68).

[0104] Advantageously, the ligand thus modified retains its ability to bind to PCPE-1.

[0105] A PCPE-1 specific ligand conjugated to a detectable marker can be used to detect PCPE-1 protein in immunoassays or cell cultures, as shown in Example 5.

[0106] The present invention also relates to the use of a PCPE-1 specific ligand conjugated to a detectable marker as defined above for the in vivo monitoring of pathological conditions by medical imaging.

[0107] In particular, the present invention relates to the use of a PCPE-1 specific ligand conjugated to a detectable marker as a contrast agent in medical imaging, particularly non-invasive in vivo medical imaging.

[0108] For the purposes of the present invention, a contrast agent is defined as a substance that, when administered to the body, enables an organ or structure (tissue, cell, receptor) to be labeled in a detectable manner, which would be difficult or impossible to see in a medical image without the contrast agent.

[0109] Advantageously, the PCPE-1 specific ligand conjugated to the marker has pharmacokinetic properties that are suitable for its use as a contrast agent (rapid clearance, but sufficient persistence to enable imaging, renal clearance, no accumulation in healthy animals). This is shown in particular in Example 6. These properties can be adjusted, if necessary, by PEGylation or other modifications of the charge or lipophilicity of the molecule.

[0110] The present invention also relates to a method for medical imaging, particularly non-invasive in vivo medical imaging, comprising administering to a patient a PCPE-1 specific ligand conjugated to a detectable marker as defined above, and then subjecting the patient to a medical imaging protocol.

[0111] The present invention also relates to the use of a PCPE-1 specific ligand conjugated to a detectable marker as defined above for the manufacture of a contrast agent useful for medical images, in particular non-invasive in vivo medical images.

[0112] Cerasnostic applications Cerasnostics is a neologism derived from the terms "therapy" and "diagnosis" and corresponds to a medical approach that emphasizes the simultaneous progression of the diagnostic and therapeutic aspects. In particular, the aim is to use compounds that can be used both for visualizing the in vivo clinical situation of a patient and for treating the condition in question with the same compound.

[0113] The PCPE-1 specific ligand according to the present invention, in particular one that inhibits PCPE-1 activity and is conjugated to a detectable marker, is very suitable for cerasnostic applications.

[0114] Accordingly, the present invention relates to a PCPE-1 specific ligand conjugated to a detectable marker for the cerasnostic use thereof in the treatment and in vivo monitoring of pathological conditions by means of medical images. Preferably, this ligand is a PCPE-1 antagonist.

[0115] Detection kit The present invention also provides a kit for determining the activity and / or amount of PCPE-1 glycoprotein in a biological sample, in vitro or ex vivo, comprising: - a ligand specific for the PCPE-1 glycoprotein according to the present invention, - a detection reagent, - and .

[0116] In particular, this PCPE-1 specific ligand is conjugated to a detectable marker.

[0117] A biological sample refers to any type of sample from a living body, in particular a patient's body, and includes in particular blood, serum, plasma, urine, cerebrospinal fluid and tear fluid.

Examples

[0118] Materials and Methods Protein Production and Purification Proteins PCPE-1 (human native or with an 8his tag at the C-terminus), mini-procollagen I (Mini I, with a twin-strep tag at the N-terminus of the α2 chain), mini-procollagen II (Mini II, with a 6His tag at the N-terminus), mini-procollagen III (Mini III, with a C-myc tag at the N-terminus), and BMP-1 (with a flag tag at the C-terminus) were produced in HEK 293-EBNA or 293-F cells and purified as previously disclosed. The CUB domain of PCPE-1 was prepared by limited proteolysis of CUB1NTR and CUB2NTR as disclosed by Kronenberg et al. CPIII-Long and PCPE-2 (with a 6-his N-terminal tag) were produced by transient transfection of HEK 293T cells and purified as previously disclosed.

[0119] The antigen used for nanobody selection is the CUB1CUB2 region of the PCPE-1 protein (corresponding to amino acids 1 - 279). This was cloned into the pHLsec vector and fused with a 6-His tag and an N-terminal HRV-3C protease cleavage sequence. Then, it was produced by transient transfection of 293-F cells grown at 37 °C, 125 rpm, 8% CO 2 in FreeStyle™ 293 medium (Gibco) according to the procedure described by Pulido et al. CUB1CUB2 was purified on a Ni-excel column (5 mL, Cytiva), and then treated overnight at 4 °C with HRV-3C protease to remove the histidine tag. 20 mM HEPES equilibrated with pH 7.4, 0.3 M NaCl HiLoad Superdex 75 gel filtration was performed on a 16 / 600 SEC (size exclusion chromatography, Cytiva) column, and then the protein was stored at -80 °C in the same buffer. Protein concentration was determined using a Nanodrop 2000 (Thermo Scientific).

[0120] The biotinylated form of CUB1CUB2 was prepared for biopanning and phage ELISA. To achieve this, the sequence encoding the CUB1CUB2 region was cloned into the pHL-Avitag3 vector between the EcoRI and KpnI sites, and then the protein was expressed in 293-T as described above. After purification on cobalt resin, 60 μM of CUB1CUB2avi was biotinylated with 1 μM GST-BirA (biotin-protein ligase) at 30 °C for 5 h in the presence of 10 mM ATP and 50 μM d-biotin in 50 mM bicine buffer, 300 mM potassium glutamate pH 8.3. GST-BirA (cloned into the pGEX vector, provided by Y. Zhao, STRUBI, Oxford, UK) was produced in E. coli BL21(DE3)pLysS bacteria. After purification on cobalt resin, it was gel filtered through a Superdex S75 column (20 mM HEPES buffer (pH 7.4), 0.3 M NaCl) to remove excess BirA and biotin, and a biotinylated protein with a purity of 95% was obtained.

[0121] Nanobody selection in llama Llama nanobodies were generated on the platform of Laboratoire Architecture et Fonction des Macromolecules Biologiques (Marseille, France). Briefly, llamas were immunized by injecting 1 mg of CUB1CUB2 five times consecutively at one-week intervals. After 39 days, blood was collected and a nanobody library was prepared as described above. Two biopanning steps with 50 nM biotinylated CUB1CUB2 were used to enrich phages expressing nanobodies that specifically recognize CUB1CUB2. Forty-eight clones (phagemids) were selected and their affinity for human PCPE-1 was determined by phage ELISA. The positive clones were sequenced, their sequences were aligned, and analyzed using ESPript 3.0. For further characterization, eight nanobodies, I1, I2, I3, I4, I5, I7, I10, and I11, were selected.

[0122] Generation of synthetic nanobodies Synthetic nanobodies were selected by Hybrigenics Services SAS. After three rounds of biopanning against 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 them, H1 - H10, were selected for more detailed characterization.

[0123] Production and purification of nanobodies Synthetic nanobodies were cloned into the pET29b(+) vector and fused with the pelB signal sequence and a C-terminal 6-His tag. The llama nanobodies were cloned into the pHEN6 vector following the pelB sequence and fused with a C-terminal 6-His tag.

[0124] To construct diabodies, a NotI restriction site and a GS 8 sequence (GSGSGSGSGSGSGSGS, SEQ ID NO: 38) were added to the N-terminus, and the VHH-I5 nanobody was amplified by PCR. Then, the GS 8 -VHH-I5 construct was cloned into pET29b(+) by NotI / XhoI digestion to obtain the bivalent nanobody VHH-H4-GS 8 -VHH-I5 (D1).

[0125] Next, the obtained plasmid is transformed into Escherichia coli T7 Express (NEB) bacteria. When the bacterial density reaches OD600 = 0.8, protein expression is induced by treatment with 0.1 mM IPTG (β-D-1-thiogalactopyranoside), and the cells are cultured overnight at 28 °C in Terrific Broth medium containing 0.1% glucose. The cells are then harvested by centrifugation at 4000 rpm for 15 minutes 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 nanobody is 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.

[0126] Also, several nanobodies were cloned into the pHEN vector containing the LPETG sequence (recognition site of sortase, SEQ ID NO: 39) and a 6-His tag at the C-terminal position (provided by Dr. Leo Hanke, Karolinska Institute, Stockholm, Sweden) and produced and purified as disclosed in (Hanke et al., 2020). In parallel, 5M sortase A cloned into the pET30b vector (Addgene #51140) with a C-terminal 6-His tag was produced and purified according to the supplier's instructions. 50 μM of 5M sortase A and 200 μM of GGGK-biotin (Covalab) in 50 mM HEPES pH 7.5, 150 mM NaCl, 10 mM CaCl 2 buffer were used to biotinylate the C-terminus of the nanobody at 25 °C for 2 hours. 5M sortase A and the excess nanobody were removed with Ni-NTA resin (Qiagen, 2 mL), followed by removal of the excess biotin with Zeba spin (7K MWCO, Thermo Fisher Scientific).

[0127] Analysis by SPR Surface plasmon resonance (SPR) experiments were performed on a Biacore T200 (Cytiva). The ligand proteins were dissolved in 10 mM HEPES pH 7.4 buffer for PCPE-1, and in 10 mM sodium acetate pH 4.5 buffer for mini-procollagen III and streptavidin, and immobilized on the CM5 chip by amine coupling. The analyte was diluted in running buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 5 mM CaCl 2 , 0.05% P20) and injected at a flow rate of 30 or 50 μL / min. Competition, epitope binding, and kinetic experiments were all carried out at 25 °C. Regeneration was achieved by continuous injection of 2 M guanidine chloride and 0.5 M EDTA for 30 seconds. Sensorgrams were analyzed using the most appropriate model with Biacore T200 Evaluation v3.2.1 (Cytiva). IC50 was determined by non-linear regression using GraphPad Prism (v8.2.1).

[0128] Activity assay To characterize the effect of the nanobody on PCPE-1 activity, 400 nM CPIII-Long (or Mini I or Mini II) was incubated at 37 °C for 1 hour with 2.5 nM BMP-1 and 75 nM PCPE-1 (if present) in the presence or absence of 2 μM nanobody in the following buffer: 20 mM HEPES pH 7.4, 0.15 M NaCl, 5 mM CaCl2, 0.02% n-octyl-β-D-glucopyranoside. Samples were analyzed by SDS-PAGE on a 4–20% gradient gel (reference, Biorad) and Instant Blue staining (Euromedex). The level of activation by PCPE-1 was evaluated as the ratio between the intensity of the C-propeptide band and the intensity of all long CPIII bands (cleaved and uncleaved) after normalization to the BMP-1 condition only using ImageQuantTL software (Cytiva).

[0129] Effect of the nanobody on C-terminal maturation of fibrillar collagen in vitro Rat cardiac fibroblasts were cultured in DMEM medium, 10% SVF (Eurobio), 1% AAS (antibiotic-antifungal solution, Thermo Fisher) at 37 °C with 5% CO2. At 70% cell confluence, serum was removed and replaced with serum-free medium containing PCPE-1 (5 μg / mL -1 ) or / and 15 μg / mL -1 nanobody. After culturing for 48 hours, the supernatant was collected and procollagen was analyzed by Western blot using an anti-C-propeptide antibody (LF41, provided by Dr. Larry W. Fisher, Bethesda, USA). Bands were quantified using ImageQuantTL software (Cytiva).

[0130] Human skin fibroblasts (provided by the Tissue and Cell Bank of Edouard Herriot Hospital, Lyon) were cultured in the same manner.

[0131] Analysis by ELISA 100 ng of anti-PCPE-1 antibody dissolved in carbonate buffer (pH 9.6) was coated on the bottom of a microplate and left overnight at 4 °C. After blocking with 1% BSA solution, PCPE-1 (purified human protein) was added while increasing the amount, and allowed to contact at room temperature for 1.5 hours. PCPE-1 is detected by adding biotinylated VHH, e.g., VHH-I5 (100 ng) at room temperature for 1 hour, followed by addition of extra avidin-HRP and detection using TMB solution. To detect PCPE-1 in blood, human plasma samples were diluted 50:1 and analyzed as described above.

[0132] Labeling of VHH-H4 and study of its characteristics VHH-H4 is conjugated with NODAGA and then labeled with gallium 68 using the procedure disclosed in (Renard et al., 2020). Its biodistribution is evaluated in rats after an IV injection of 25 μg (12 MBq). For this purpose, nine blood samples (150 - 200 μL) are collected during the 2.5 hours after injection, after which the animals are euthanized. Tissue samples are analyzed by gamma counting.

[0133] Statistical analysis Unless otherwise stated, the data represent the mean ± standard deviation of at least three independent experiments calculated using Graphpad Prism 8.2 software.

[0134] Results Example 1 Selection and characterization of nanobodies against PCPE-1 Figure 1 shows the results of the selection of PCPE-1-binding nanobodies from a llama nanobody library or a synthetic library.

[0135] Since the CUB1 and CUB2 domains of PCPE-1 are necessary and sufficient for its activity, the inventors chose to use as an antigen a protein that contains these two domains but lacks the NTR domain. The labels used for purification were removed to avoid the selection of non-specific ligands. Two strategies were carried out in parallel: in vivo selection of llama nanobodies by animal immunization and in vitro selection from a library of synthetic nanobodies.

[0136] For the immune library, recombinant CUB1CUB2 was injected into llamas 5 times consecutively, followed by 2 rounds of biopanning procedures to generate nanobodies. Forty-eight clones were analyzed by phage-ELISA, and 11 unique clones were obtained after sequencing. These 11 clones can be classified into 6 families according to their CDR3. The first family is the most representative with 5 clones, while the other families have only 1 or 2 variants. Three members of family 1 (VHH-I1, VHH-I5, and VHH-I7) and one member of each of the other families were selected, produced in E. coli, and purified. Their affinity for PCPE-1 was measured by surface plasmon resonance (SPR) (Figure 1A). All except VHH-I10 bind to PCPE-1 (Table 2). Nanobodies belonging to the same family behave very differently. In family 1, the interaction between VHH-I1 and PCPE-1 is less stable than those of VHH-I5 and -I7, resulting in a significantly higher dissociation constant (Table 2). Among other llama nanobodies, only VHH-I3 is a strong ligand for PCPE-1 with a dissociation constant in the nanomolar range.

[0137] Synthetic nanobodies were selected using 3 cycles 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 showed positive signals, corresponding to 24 unique clones after sequencing. When tested against immobilized CUB1NTR, 7 out of 24 clones also showed positive signals. Ten clones (including these 7) were finally produced and purified, and then their affinity for PCPE-1 was measured by SPR (Figure 1B). Of the 10, only half showed a clear interaction with PCPE-1 when injected at 200 nM, and the best results were obtained using VHH-H4 and VHH-H10 (Figure 1B), and its K D is within the nanomolar range (Table 2).

[0138] Measurement of the K D affinity constant of various tested nanobodies.

[0139] Nanobodies from the synthetic nanobody library are designated by names starting with the letter H, and nanobodies from the serum of immunized llamas are designated by names starting with I.

[0140] Figure 1A shows the PCPE-1 binding activities of eight nanobodies derived from llama serum: I1, I2, I3, I4, I5, I7, I10, and I11.

[0141] Figure 1B shows the PCPE-1 binding activities of ten synthetic nanobodies, H1 to H10.

[0142] Table 2 below shows the K D affinity constants of seven llama-derived nanobodies (as described above, the I10 nanobody does not bind at all) and ten synthetic nanobodies.

[0143]

Table 2

[0144] An affinity constant of less than 100 nM represents strong binding, which is true for the nanobodies I3, I5, I7, H4, and H10.

[0145] Example 2 Determination of the region recognized by the nanobody Figure 2 shows the results of the determination of the region of PCPE-1 recognized by the antibody. A nanobody co-injection competition experiment with the CUB1 or CUB2 domain of PCPE-1 was carried out (Figure 2A). When mixed with CUB2, it was found that the nanobodies I3, I5, and I7 could not bind to PCPE-1, but co-injection with CUB1 had substantially no effect, indicating that these nanobodies mainly interact with the CUB2 domain of PCPE-1. In contrast, the interaction between the H4 and H10 synthetic nanobodies and PCPE-1 was prevented by co-injection with CUB1 rather than CUB2, indicating that they interact with the CUB1 domain.

[0146] Next, for the best ligands in each library (I5 and H4), the inventors determined whether other nanobodies have the same epitope or can bind to PCPE-1 simultaneously. To achieve this, the PCPE-1 chip was first saturated with nanobody-I5 or -H4. When VHH-I5 was subsequently co-injected with other llama nanobodies, no change in signal was observed, but co-injection of VHH-I5 and VHH-H4 or -H10 resulted in a sharp increase in signal (Figure 2B), indicating that they can bind simultaneously to distinct PCPE-1 sites. Similarly, when the surface was first saturated with VHH-H4 (Figure 2C), llama nanobodies could still interact with PCPE-1, but VHH-H10 could not bind. These results confirm that the two libraries have different epitopes, but the epitopes are rather conserved within the library. PCPE-2 is a protein of the same family as PCPE-1 and has a 43% identical amino acid sequence. PCPE-2 shares certain activities with PCPE-1 but also has very different specific functions. The specificity of the nanobody interaction for PCPE-1 versus PCPE-2 was analyzed by SPR. To achieve this, VHH-I5 and VHH-H4 were biotinylated at their C-termini using sortase and then immobilized on a streptavidin surface. In both cases, the interaction with PCPE-2 was substantially undetectable (K

[0147] >1 μM), while PCPE-1 bound very strongly (K D = 1.5 nM (VHH-I5) and K D = 3.9 nM (VHH-H4), Figure 2D - E and Table 3 below). Thus, the affinity of VHH-I5 and -H4 for PCPE-1 is at least 250-fold greater than for PCPE-2. D = 3.9 nM (VHH-H4), Figure 2D - E and Table 3 below). Thus, the affinity of VHH-I5 and -H4 for PCPE-1 is at least 250-fold greater than for PCPE-2.

[0148]

Table 3

[0149] Example 3: Antagonistic inhibitory effect of the nanobody according to the present invention against PCPE-1 Figure 3 shows the results of the inhibitory effect of the nanobody according to the present invention on PCPE-1 activity. PCPE-1 is activated by direct interaction with procollagen C-propeptide. Therefore, the inventors used an SPR competition experiment to observe whether the nanobody could prevent the binding of PCPE-1 to mini-procollagen III (Mini III, the C-terminal domain of procollagen III: composed of the end of the triple helix, C-telopeptide and C-propeptide).

[0150] The results show that VHH-I3, -I5, -I7, -H4 and -H10 inhibit the binding of PCPE-1 to Mini III, while VHH-I1, -I2, -I4 and -I11 have no effect (Figure 3A).

[0151] VHH-H4, -H10 and VHH-I5 are the most effective, inhibiting by 50-75% at 250 nM.

[0152] Furthermore, their antagonist effects are additive, and co-injection of a mixture of VHH-I5 and VHH-H4 with PCPE-1 substantially eliminates its interaction with Mini III. This inhibitory effect increases with concentration, and the IC50 is estimated to be approximately 45 nM (VHH-H4), 24 nM (VHH-I5) and 20 nM (mixture of VHH-I5 and VHH-H4) in the presence of 5 nM PCPE-1.

[0153] However, complete inhibition is achieved with a high concentration of VHH-H4, but the addition of VHH-I5 alone does not completely block the PCPE-1 / Mini III interaction, while the combination of VHH-H4 and VHH-I5 results in complete inhibition.

[0154] Since the nanobody prevents the binding of PCPE-1 to procollagen, the next step was to examine whether the nanobody also inhibits PCPE-1 activity in vitro. CPIII-Long (a model substrate of procollagen III) is cleaved by incubation with BMP-1 protease, and this cleavage is increased in the presence of PCPE-1 (Figure 3B, a 2.4-fold increase under these conditions). The addition of VHH-I1, VHH-I2, VHH-I4, or VHH-I11 has little effect on PCPE-1 activity. In contrast, the addition of VHH-I3, VHH-I5, VHH-I7, VHH-H4, or VHH-H10 results in a 25 - 45% decrease in activation, which 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 seem to be changed by the presence of the VHH, despite the fact that BMP-1 also contains three CUB domains.

[0155] Finally, in a cell-based assay, the nanobodies were evaluated for their ability to regulate procollagen I cleavage.

[0156] To mimic the fibrotic state, rat heart fibroblasts were treated with recombinant human PCPE-1 protein (Figure 3C, D). The addition of PCPE-1 to the medium increased the release of procollagen I C-propeptide by approximately 2.5-fold on average. The presence of VHH-I5 and VHH-H4 inhibits the effect of PCPE-1 (by approximately 40%), as shown by the decrease in C-propeptide (Figure 3C). Clearly, the combined treatment with VHH-I5 and VHH-H4 completely blocks the effect of exogenous PCPE-1 and returns the release of C-propeptide to the basal level. Furthermore, treatment of the cells with VHH alone (in the absence of added PCPE-1) has no effect on C-propeptide release.

[0157] All of these results suggest that the selected ligands are potent PCPE-1 antagonists that can block the PCPE-1-mediated stimulation of procollagen C-terminal maturation.

[0158] Example 4: Construction and Characterization of Diabodies (Bivalent Nanobodies) The previous results suggest that combining the I5 nanobody and the H4 nanobody in the same polypeptide chain can further enhance their affinity for PCPE-1 through site cooperativity.

[0159] Figure 4 shows the results obtained for the diab-D1 diabody of SEQ ID NO: 21 produced in bacteria.

[0160] The interaction of diab-D1 with PCPE-1 was evaluated by SPR by injecting increasing concentrations of diab-D1 onto immobilized PCPE-1. The advantage of VHH fusion is clearly manifested in the affinity it provides, as shown by the improvement of the dissociation constant (K D = 0.32 nM) and a substantial delay in dissociation (Figure 4A).

[0161] Furthermore, competition experiments were performed to determine whether diab-D1 has a more potent antagonistic effect than the individually used nanobodies (Figure 4B). The IC 50 calculated for diab-D1 was estimated to be 3.5 nM, which is substantially lower than that of VHH-I5 and VHH-H4 alone or their simultaneous injection (5 - 13-fold) under the same conditions. Similarly, the addition of diab-D1 completely blocks the stimulation of CPIII-Long cleavage by PCPE-1 (Figure 4C).

[0162] Effect of diab-D1 on procollagen I and II cleavage The diab-D1 antibody can also block the PCPE-1 activity against 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 shown in Figure 4D.

[0163] Regulation of collagen maturation by diab-D1 In cell culture (Figures 4E, 4F), diab-D1 can completely block the effect of PCPE-1 addition on the cleavage of endogenous procollagen I by rat heart fibroblasts. Furthermore, in contrast to individual VHHs, the diabody also inhibits C-propeptide cleavage in the absence of added PCPE-1.

[0164] This antibody also inhibits the cleavage of procollagen I produced by human skin fibroblasts, whether in the basal state, under "fibrotic" conditions mimicked by TGF-β stimulation, or in the presence of added exogenous PCPE-1.

[0165] This is shown in Figures 4G and 4H, which show the immunodetection of procollagen I and its C-terminal degradation products in the culture medium of human skin fibroblasts after 72 hours of culture in the absence or presence of PCPE-1, TGF-β, and diab-D1.

[0166] Other measurements were performed by immunofluorescence on cultured human fibroblasts (results not shown). Since the addition of diab-D1 resulted in a decrease in collagen deposition detected by immunofluorescence, it was found that the matrix deposited by the cells was also affected.

[0167] Example 5: Nanobody conjugated to a detectable marker A nanobody containing the LPETG sequence (SEQ ID NO: 39) was conjugated to biotin using sortase as disclosed above. The nanobody conjugated in this way can bind to streptavidin and enables the detection of PCPE1 in complex mixtures by SPR, ELISA, or Western blot.

[0168] Figure 5A shows an SPR study of the interaction between PCPE-1 and biotinylated H4 nanobody captured on a streptavidin-binding chip.

[0169] Figure 5B shows the detection of PCPE-1 using sandwich ELISA. Samples containing PCPE-1 are captured by anti-PCPE-1 antibodies. The addition of biotinylated VHH-I5 enables its detection using a fluorophore such as streptavidin conjugated to HRP or Alexafluor488. This method is highly sensitive and can easily detect 0 - 1 ng of PCPE-1. This makes it possible to determine the amount of PCPE-1 present in human plasma, which is approximately 510 ± 70 ng / mL.

[0170] Alternatively, PCPE-1 can be detected by sandwich ELISA between H4 and I5.

[0171] Also, the nanobody was conjugated to the radioactive marker NODAGA-Ga 68 to.

[0172] To achieve this, VHH-H4 was first conjugated with NODAGA and then labeled with gallium 68 according to the procedure disclosed in (Renard, 2020).

[0173] Then, the biodistribution and pharmacokinetics of the nanobody were evaluated in rats after intravenous injection of 25 μg (12 MBq) of H4-Ga 68 to determine the excretion pathway and accumulation region of the molecule.

[0174] The results are shown in Figure 6A (pharmacokinetics) and 6B (biodistribution in major organs: heart, lung, blood, liver, spleen, kidney, and bladder).

[0175] H4-Ga 68 The nanobody can also be used for the detection of PCPE-1 on tissue sections and can be detected by a phosphor imager. See Figure 6C.

[0176] References U.S. Patent Application Publication No. 20120270246 International Publication No. 2017065206 International Publication No. 2019080284 Henderson NC, Rieder F, Wynn TA. Fibrosis: from mechanisms to medicines. (Fibrosis: from mechanisms to medicines) Nature 2020 November; 587(7835):555 - 566. doi:10.1038 / s41586 - 020 - 2938 - 9. Shi R, Zhang Z, Zhu A, Xiong X, Zhang J, Xu J, Sy MS, Li C. Targeting type I collagen for cancer treatment. (Targeting type I collagen for cancer treatment) Int J Cancer. February 28, 2022. doi:10.1002 / ijc.33985 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. (The basis for procollagen C - proteinase enhancer activity) J Biol Chem. 284, 33437 - 33446 Vadon - Le Goff, S., Kronenberg, D., Bourhis, J.M., Bijakowski, C., Raynal, N., Ruggiero, F., Farndale, R.W., Stocker, W., Hulmes, D.J. & Moali, C. Procollagen C - proteinase enhancer It stimulates procollagen processing by binding to the C-propeptide region only. (The procollagen C-proteinase enhancer stimulates procollagen processing by binding only to the C-propeptide region.) J.Biol.Chem. 286, 38932 - 38938 (2011). Lagoutte P, Bettler E, Vadon-Le Goff S, Moali C. Procollagen C-proteinase enhancer-1 (PCPE-1), a potential biomarker and therapeutic target for fibrosis. (Procollagen C-proteinase enhancer-1 (PCPE-1), a potential biomarker and therapeutic target for fibrosis.) Matrix Biol Plus. April 20, 2021 11:100062. doi:10.1016 / j.mbplus.2021.100062. Lagoutte, P., Oudot, A., Dussoyer, M., Goncalves, V., Bouchot, O., Vandroux, D., Bellaye, P.S., Moali, C. & 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. (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 Desogere P, Montesi SB, Caravan P. Molecular Probes for Imaging Fibrosis and Fibrogenesis. (Molecular probes for imaging fibrosis and fibrogenesis) Chemistry. January 24, 2019 25(5): 1128 - 1141. doi:10.1002 / chem.201801578. Muyldermans S. A guide to: generation and design of nanobodies. (A guide to the generation and design of nanobodies) FEBS J. April 2021 288(7): 2084 - 2102. doi:10.1111 / febs.15515. Gebauer M, Skerra A. Engineering of binding functions into proteins. (Engineering of binding functions into proteins) Curr Opin Biotechnol. December 2019 60: 230 - 241. doi:10.1016 / j.copbio.2019.05.007. Epub June 14, 2019 PMID: 31207556. Needleman SB, Wunsch CD. A general method applicable to the search for similarities in the amino acid sequence of two proteins. (A general method applicable to the search for similarities in the amino acid sequence of two proteins) J Mol Biol. March 1970 48(3): 443 - 53. doi:10.1016 / 0022 - 2836(70)90057 - 4. PMID: 5420325. Kwon NY, Kim Y, Lee JO. Structural diversity and flexibility of diabodies. (Structural diversity and flexibility of diabodies) Methods. February 1, 2019 154: 136 - 142. doi:10.1016 / j.ymeth.2018.09.005. Epub September 25, 2018 PMID: 30261312. Pulido D, Sharma U, Vadon-Le Goff S, Hussain SA, Cordes S, Mariano N, Bettler 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.2018.06.011 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 A universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies. Elife. 2016 Jul 19 5:e16228. doi:10.7554 / eLife.16228. Hanke L, Vidakovics Perez L, Sheward DJ, Das H, Schulte T, Moliner-Morro A, Corcoran M, Achour A, Karlsson Hedestam GB, Hallberg BM, Murrell B, McInerney GM. An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction. (An alpaca nanobody neutralizes SARS-CoV-2 by blocking receptor interaction.) Nat Commun. September 4, 2020 11(1):4420. doi:10.1038 / s41467-020-18174-5. Renard E, Dancer PA, Portal C, Denat F, Prignon A, Goncalves V. Design of Bimodal Ligands of Neurotensin Receptor 1 for Positron Emission Tomography Imaging and Fluorescence-Guided Surgery of Pancreatic Cancer. (Design of Bimodal Ligands of Neurotensin Receptor 1 for Positron Emission Tomography Imaging and Fluorescence-Guided Surgery of Pancreatic Cancer) J Med Chem. March 12, 2020 63(5):2426-2433. doi:10.1021 / acs.jmedchem.9b01407. Epub January 2, 2020 PMID: 31855417.

Claims

**Claim 1** A ligand specific for PCPE-1 glycoprotein, characterized in that it is a nanobody. **Claim 2** A ligand specific for PCPE-1 glycoprotein, characterized by inhibiting the interaction with the C-terminal domain of procollagen. **Claim 3** A ligand specific for the PCPE-1 glycoprotein according to claim 2, characterized in that it is a nanobody. **Claim 4** The nanobody contains three complementarity determining regions (CDRs) with PCPE-1, and among these three CDRs, at least one CD R has at least 80% identity with one of the sequences of SEQ ID NOs: 1 to 15. A ligand specific for the PCPE-1 glycoprotein according to claim 1 or 3. **Claim 5** The three CDRs designated as CDR1, CDR2, and CDR3 are respectively i. CDR1 has at least 80% identity with one of SEQ ID NOs: 1, 4, 7, 10, or 13. ii. CDR2 has at least 80% identity with one of SEQ ID NOs: 2, 5, 8, 11, or 14. iii. CDR3 has at least 80% identity with one of SEQ ID NOs: 3, 6, 9, 12, or 15. A ligand specific for the PCPE-1 glycoprotein according to claim 4, characterized by being defined as such. **Claim 6** The peptide sequence has at least 80% identity with one of the sequences of SEQ ID NOs: 16 to 20. A ligand specific for the PCPE-1 glycoprotein according to any one of claims 1 or 3 to 5. **Claim 7** Specifically, it consists of a combination of two polypeptide sequences each having at least 80% identity with one of the sequences of SEQ ID NOs: 16 to 20, and optionally further contains one or two binders. A ligand specific for 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. **Claim 8** The polypeptide sequence has at least 80% identity with the sequence of SEQ ID NO:

21. A ligand specific for the PCPE-1 glycoprotein according to claim 7. **Claim 9** A nucleic acid encoding the nanobody according to any one of claims 1 or 3 to 6, or encoding a combination of two or three nanobodies according to claim 7 or 8. **Claim 10** A ligand specific for the PCPE-1 glycoprotein according to any one of claims 2 to 8 for use as a medicament.

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

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

13. Use of a ligand specific for the PCPE-1 glycoprotein according to claim 12 for in vivo monitoring of a pathological condition by means of a medical image.

14. A ligand specific for the PCPE-1 glycoprotein according to claim 12 for its theranostic use in the treatment and in vivo monitoring of a pathological condition by means of a medical image.

15. A kit for determining in vitro or ex vivo the activity and / or amount of PCPE-1 glycoprotein in a biological sample, - a ligand specific for the PCPE-1 glycoprotein according to any one of claims 1 to 8 or 12, - a detection reagent, comprising the kit.

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