A matriptase-cleavable substrate

Abstract

The present disclosure generally relates to a matriptase-cleavable substrate comprising the amino acid sequence: X1X2X3X4X5X6 (SEQ ID NO:1), wherein X1 and X2 are independently selected from any amino acid residue; X3 is R or K; X4 is selected from R, S, or G; X5 is R or K; and X6 is selected from S, R, or K.

Description

A MATRIPTASE-CLEAVABLE SUBSTRATETECHNICAL FIELD

[0001] The present disclosure generally relates to a matriptase-cleavable substrate and to a fusion protein comprising the matriptase-cleavable substrate. Furthermore, the present disclosure relates to a conjugate comprising the matriptase- cleavable substrate or the fusion protein for use in diagnosis or in the treatment of disorders connected to an overexpression of matriptase, typically cancer.BACKGROUND

[0002] Proteases are a broad group of enzymes that play a critical role in numerous biological processes by catalyzing the cleavage of proteins into smaller peptides or amino acids. These enzymes are involved in a wide range of cellular activities, including digestion, blood coagulation, immune responses, cell cycle regulation, and apoptosis. Proteases are important targets for therapeutic intervention in various clinical conditions such as cardiovascular diseases, inflammatory disorders, and, particularly, cancer.

[0003] In the context of cancer, proteases contribute to tumor progression by modulating the tumor microenvironment. The expression of proteases is often upregulated in tumor tissues, which has been shown to stimulate tumor cell growth and tumor invasion. Due to their ability to influence cancer cell behavior and tumor microenvironment, proteases are considered promising targets for cancer therapy.

[0004] Matriptase is an epithelial derived transmembrane serine protease of particular interest due to its dysregulated expression in several cancers. Matriptase may promote oncogenic signaling, enhance cell migration, and facilitate extracellular matrix degradation, all of which are important for tumor invasion and metastasis. The expression of matriptase is notably upregulated in many cancer types, including breast, lung, prostate, and colorectal cancers, making it a valuable biomarker and therapeutic target.

[0005] In the last decade, efforts have been made to develop drugs that are selectively active in tumor tissues but remain inert in healthy tissue. Such constructs are named prodrugs and their activation in the tissue of interest can be triggered by proteases, such as matriptase, or other factors from the tumor microenvironment.

[0006] The activation of a prodrug is largely dependent on the ability of the protease to recognize and cleave the prodrug in the tumor microenvironment. A challenge associated with prodrugs is inefficient and slow activation in the tumor microenvironment. Furthermore, the matriptase level may vary among patients and within different tumors in the same patient, which may result in inconsistent drug activation and efficacy.

[0007] Accordingly, there is a need to provide improvements in the field of prodrug activation and to secure that the activation is highly efficient and fast.SUMMARY

[0008] With the aim to improve prodrug activation, the present inventors have identified non-natural matriptase-cleavable substrates that may be added in prodrugs to improve their activation in the tissue of interest.

[0009] The matriptase-cleavable substrates of the present disclosure are specifically designed to be activated by the upregulated matriptase expression characteristic of many aggressive cancers. An improved targeted activation mechanism responding to the tumor-associated protease matriptase may thus be provided. Accordingly, the precision of various cancer therapies may be improved.

[0010] The matriptase-cleavable substrates of the present disclosure are cleaved more effectively compared to previously reported natural and engineered substrates [1,2,3].

[0011] To identify the substrate sequences effectively cleaved by matriptase, the inventors modified a previously described bacterial display method [4]. In this method, protease substrate sequences were expressed on the surface of Escherichia coli (E. coli) in between two different reporter tags. Incubation of the recombinant E. coli with corresponding protease followed by labelling with fluorescent reporter proteins resulted in loss of signal from the outer reporter tag if the sequence was cleaved by the protease.

[0012] A library with more than 4 million variants of the matriptase substrate sequences was designed and displayed on E. coli cells, followed by screening and isolation of the best candidates by flow cytometry sorting (FACS). The most promising substrates were introduced in soluble protein constructs to evaluate their activation upon matriptase treatment in vitro.

[0013] In a first aspect, there is provided a matriptase-cleavable substrate comprising the amino acid sequence:X1X2X3X4X5X6(SEQ ID NO:I), whereinXi and X2are independently selected from any amino acid residue;X3is R or K;X4is selected from R, S, or G;X5is R or K; andX()is selected from S, R, or K.

[0014] The inventors have found that a matriptase-cleavable substrate as defined above is associated with a significantly improved matriptase cleavage efficiency, as demonstrated in the Example section, and as illustrated in figures 5C and 6C.

[0015] The enhanced cleavage efficiency allows for a prodrug incorporating the substrate to be activated faster and at lower concentrations of matriptase. The matriptase-cleavable substrate can thereby improve the potency of a prodrug comprising the substrate. Furthermore, the enhanced activation may make the treatment more effective at lower doses.

[0016] As mentioned hereinbefore, matriptase is often overexpressed in many cancer types. Accordingly, the matriptase-cleavable substrate of the present disclosure may contribute to an effective and safe cancer treatment.

[0017] Besides its impact in cancer therapy, the matriptase-cleavable substrate may be used for other pathological conditions. Matriptase is involved in regulating various signalling pathways by activating or deactivating proteins and peptides.

[0018] The matriptase-cleavable substrate of the present disclosure may also be used for diagnostic purposes, e.g. to study matriptase-related biological processes or to evaluate and / or quantify the level of matriptase expression in a sample.

[0019] For example, the matriptase-cleavable substrates of the present disclosure may be used as biomarkers for diagnosing and monitoring pathological conditions, e.g. cancer. The substrates may thus be useful to identify disease presence or progression based on matriptase activity levels.

[0020] Matriptase belongs to a family of serine proteases known as type II transmembrane serine proteases (TTSPs). Typically, the term “matriptase”, as used herein, means the matriptase encoded by the sti gene, also known as matriptase-i.However, the present disclosure is not limited to cleavage by this specific matriptase, but other matriptases belonging to the TTSP family are also conceivable. For example, the matriptase family includes i.a. matriptase-i (ST14), matriptase-2, matriptase-3, CAP3, and epithin.

[0021] In exemplary embodiments, the matriptase-cleavable substrate comprises no cysteine (C) residue. This may be to prevent the formation of disulfide bridges that could potentially alter the substrate's structure and function.

[0022] In exemplary embodiments, the matriptase-cleavable substrate comprises the amino acid sequence as defined by:X1X2X3RRS (SEQ ID N0:2);XiX2X3SRR (SEQ ID NO:3);X,X2X3GRR (SEQ ID NO:4); orXIX2X >GRK (SEQ ID NO:5), whereinXi and X2are independently selected from any amino acid residue, andX3is R or K.

[0023] The amino acid in position Xi and X2 respectively could be selected from any amino acid. The inventors have found that the amino acid residues in these two positions can vary without affecting the improved cleavage efficiency observed with the substrates.

[0024] In exemplary embodiments, the matriptase-cleavable substrate comprises the amino acid sequence as defined by:XtX2KRRS (SEQ ID NO: 6);XiX2KSRR (SEQ ID NO:y);XiX2RGRR (SEQ ID NO: 8); orXiX2KGRK (SEQ ID NO:9), whereinXi and X2are independently selected from any amino acid residue

[0025] The matriptase-cleavable substrate may comprise the amino acid sequence as defined by:WHKRRS (SEQ ID NO: 10);GAKSRR (SEQ ID NO:ii);VPRGRR (SEQ ID NO: 12);HYKGRK (SEQ ID NO: 13); or a corresponding sequence having at least 83% sequence homology with any one of SEQ ID NO:IO-13.

[0026] The ”% sequence homology” may also be referred to as the “% sequence identity”, and may be calculated as follows. The query sequence is aligned to the target sequence. A comparison is made over the window corresponding to the target sequence. The amino acid residues at each position are compared and the percentage of positions that exhibit identical amino acid residues in the query sequence and the target sequence is reported as % sequence homology.

[0027] The matriptase-cleavable substrate may comprise or consist of the amino acid sequence as defined by any one of SEQ ID NO: 1-13.

[0028] The length of the matriptase-cleavable substrate is typically from 6 to 10 amino acids. Typically, the length of the matriptase-cleavable substrate is 6 amino acids.

[0029] A short substrate is advantageous to secure a more precise and specific cleavage by matriptase. Furthermore, a short substrate is less likely to form secondary structures that could hinder matriptase access or reduce cleavage efficiency. In addition, the use of a short substrate increases the selectivity as it minimizes off-target cleavage by unrelated proteases. Accordingly, the substrate is predominantly recognized and cleaved by matriptase.

[0030] According to another aspect, there is provided a fusion protein comprising the matriptase-cleavable substrate as described hereinbefore and at least one targetbinding domain.

[0031] The target-binding domain may be any peptide or protein having affinity for the target in suit.

[0032] The target-binding domain may e.g. be a tumor-binding domain, a cytokine-binding domain, a receptor-binding domain, a growth-factor binding domain, a pathogen-binding domain, an enzyme-binding domain, or a cell adhesion molecule-binding domain.

[0033] Typically, the target-binding domain is a tumor-binding domain.

[0034] The tumor-binding domain may be a peptide or a protein having high affinity for markers or antigens expressed on the surface of tumor cells.

[0035] The tumor-binding domain may be a HER-binding domain.

[0036] HER-binding domains specifically interact with members of the human epidermal growth factor receptor (HER) family, also known as ErbB receptors. The HER family includes four receptor tyrosine kinases: HER1 (Epidermal Growth Factor Receptor, EGFR), HER2 (Epidermal Growth Factor Receptor 2, ErB2), HER3 (ErB3) and HER4 (ErB4). These receptors play critical roles in cell growth, differentiation, and survival. Overexpression or mutation of HER receptors, particularly HER2 and EGFR, is associated with various cancers, including breast, lung, and gastric cancers.

[0037] The target-binding domain, e.g. tumor-binding domain maybe selected from the group consisting of anticalins, adnectins, knottins, darpins, affitins, affimers, avimers, affilins, antibodies, antibody fragments and / or affibodies.

[0038] Preferably, the target-binding domain is an affibody, an antibody or a fragment of an antibody.

[0039] As used herein, the term “affibody” or “affibody molecule” means a protein based on the three-helix scaffold of the Z domain of Staphylococcal protein A (SpA). The Z domain is the mutated form of the B domain of SpA. The affibody molecule comprises 53-60, typically 58 amino acids and has a molar mass of about 6 kDa. Affibody molecules maybe engineered to bind a large number of target proteins or peptides with high affinity. Specific affibody molecules which bind a desired target can be extracted from pools containing billions of different variants using phage display or E. coli display. The wildtype affibody sequence is defined by SEQ ID N0:i4.

[0040] The affibody sequence defined in SEQ ID N0:i4, may be tailored to enhance the binding to a specific target in suit. An exemplary affibody sequence is defined by SEQ ID NO:15. However, the affibody is by no means limited to this sequence.

[0041] Suitably, the target-binding domain is an antibody, e.g. a monoclonal antibody or a fragment of an antibody, e.g. an antigen-binding fragment.

[0042] Antibody-based prodrugs can be customized to target a wide range of tumor-specific antigens.

[0043] The fusion protein may further comprise a masking domain.

[0044] A “masking domain” is a segment of a protein or a molecule designed to temporarily inactivate or “mask” the functional part of the fusion protein or prodrug until it reaches a specific target site. The masking domain secures that the active component of the fusion protein or prodrug is only activated in specific conditions, e.g. in a tumor-environment where matriptase is overexpressed.

[0045] The masking domain prevents premature activation of the therapeutic agent, e.g. cytotoxic agent, of the prodrug. Hence, undesired side-effects can be avoided.

[0046] The masking domain may be linked to the target-binding domain by a linker and wherein the linker comprises the matriptase-cleavable substrate.

[0047] The linker may be a peptide sequence that covalently links the targetbinding domain to the masking domain. The linker may comprise 6 to 25 amino acid residues, e.g. from 10 to 20 amino acid residues, including the matriptase-cleavable substrate.

[0048] Figure 6A schematically illustrates a target-binding domain (in this case an antibody) linked to a masking domain, wherein the linker comprises the matriptase- cleavable substrate.

[0049] Upon reaching a target site, e.g. a tumor, matriptase triggers the cleavage of the matriptase-cleavable substrate such that the fusion protein or prodrug is “unmasked”. This unmasking event may activate a therapeutic, e.g. cytotoxic agent, and allow it to exert its effect precisely where needed (see figure 6A).

[0050] The invention is by no means limited to a specific masking domain, but any peptide or molecule having the ability to mask the target -binding domain or the therapeutic agent maybe used. For example, masking domains maybe designed using affibodies or antibody fragments that bind to the active site of the therapeutic agent and preventing its action until the masking domain is specifically removed.

[0051] In exemplary embodiments, the masking domain may be an affibody.

[0052] The fusion protein may further comprise a half-life extending domain, e.g. an albumin-binding domain (ABD).

[0053] In exemplary embodiments, the target-binding domain may be an affibody and wherein the fusion protein further comprises a half-life-extending domain.

[0054] The half-life extending domain enhances the half-life of the fusion protein. Accordingly, the stability and circulation time of the fusion protein and the therapeutic agent maybe increased.

[0055] Any half-life extending domain known to the skilled person may be used. Preferably, the half-life-extending domain is an albumin-binding domain (ABD).

[0056] ABD binds to serum albumin, a naturally abundant protein with a long half-life. By binding to albumin, the fusion protein “piggybacks” on the long half-life of albumin, and thereby extends its own circulation time.

[0057] The half-life extending domain may e.g. be an ABD domain defined by the sequence SEQ ID NO: 16. The invention is, however, not limited to this specific sequence.

[0058] As schematically illustrated in figure 5A, the half-life extending domain may be linked to the target-binding domain (in this case an affibody) by a linker, and wherein the linker comprises the matriptase-cleavable substrate.

[0059] The linker may be a peptide sequence that covalently links the targetbinding domain to the half-life extending domain. The linker may comprise 6 to 25 amino acid residues, e.g. from 10 to 20 amino acid residues, including the matriptase-cleavable substrate.

[0060] The fusion protein illustrated in figure 5A may further comprise a masking domain.

[0061] The fusion protein of the present disclosure may comprise a target -binding domain, a masking domain, and / or a half-life-extending domain. Typically, the target-binding domain is linked to the masking domain or the half-life extending domain by a linker, wherein the linker may comprise the matriptase-cleavable substrate. As outlined hereinbefore, the target-binding domain may be an affibody, an antibody, or a fragment of an antibody.

[0062] According to another aspect, there is provided a conjugate comprising the matriptase-cleavable substrate as defined hereinbefore or a fusion protein as defined hereinbefore and a labelling agent.

[0063] The conjugate may be used for diagnostic purposes. For example, the conjugate may e.g. be used in an in vitro assay where a sample (e.g. a tissue biopsy or blood sample) is incubated with the conjugate. Upon cleavage of matriptase, the labelling agent maybe activated or released to produce a measurable signal. The signal may indicate the presence and activity level of matriptase, which can be correlated with certain conditions, e.g. cancer.

[0064] The labelling agent may e.g. be a fluorophore, a chromophore, a radioactive isotope or an enzyme.

[0065] The conjugate may be used to quantify the degree of matriptase expression in vivo or in vitro.

[0066] The quantification may e.g. be based on imaging. In such cases, the labelling agent maybe a radioactive isotope or a radionuclide suitable for imaging. The patient maybe scanned to detect, visualize and / or quantify matriptase expression. The scanning may be a tomography, preferably positron emission tomography (PET) or single-photon emission computed tomography (SPECT). For the latter, a CZT-based camera technology maybe used.

[0067] A radionucleotide suitable for imaging may be selected from the group consisting of18F,124l, 76Br,68Ga, 44Sc,61Cu,64Cu,89Zr, ssCo, 4 Ti,66Ga,86Y,110mIn,123l, 131I, 99mTC)uijn and6?Ga. A preferred group consists of18F,68Ga,<wmTC anq iiqn. Another preferred group consists of18F,68Ga andU1ln.

[0068] According to another aspect, there is provided a therapeutic conjugate comprising the matriptase-cleavable substrate as described hereinbefore or a fusion protein as described hereinbefore and a therapeutic agent.

[0069] The therapeutic conjugate may also be referred to as a prodrug.

[0070] Preferably, the therapeutic agent is a cytotoxic agent.

[0071] The cytotoxic agent is configured to kill and / or inhibit the growth of cancer cells after being released in their vicinity. The cytotoxic agent may be a chemotherapeutic agent.

[0072] The cytotoxic agent may be configured to trigger programmed cell death (apoptosis) in cancer cells, which helps in reducing the tumor size and spread. The cytotoxic agent may also be configured to disrupt the processes required for cell division and thereby prevent cancer cells from proliferating. Alternatively, or in addition, the cytotoxic agent may cause direct damage to the DNA of cancer cells, leading to cell death.

[0073] The present disclosure is not limited to a specific cytotoxic agent, but any cytotoxic agent known to the skilled person maybe used. For example, the cytotoxic agent may be a cytotoxic molecule, peptide, protein or radionuclide.

[0074] The cytotoxic molecule may e.g. be Doxorubicin (DOX), Duocarmycins (DUO), Docetaxel (DTX), Monomethyl auristatin E (MMAE), Monomethyl auristatin F (MMAF), Paclitaxel (PTX), Mertansine (DM1), emtansine (DM1), Ravtansine (DM4), Soravtansine (DM4), Pyrrolobenzodiazepine (PBD), Calicheamicin, DM1, MMAE, MMAF or DM4.

[0075] Examples of cytotoxic proteins are Pseudomonas exotoxin (PE) and engineered variants thereof, including PE38, diphtheria toxin (DT) and deBouganin [8]. Other examples of cytotoxic proteins are targeting domains against immunomodulatory targets such as CD3, CD47, PD-1, PD-L1, CTLA-4, 4-1BB and OX4O

[0015] .

[0076] Examples of cytotoxic radionuclides are ^Lu,9°Y,188Re;186Re;166Ho, ^Sm,6?Cu, ^Cu^Tb,161Tb, 47Sc;22r>Ac;212Pb;218Bi,212Bi,22?Th,223Ra; 58mCo, ^I, 7(>As, 77As and211At.

[0077] According to another aspect, there is provided a therapeutic conjugate as described hereinabove for use in a method of treatment of a disorder.

[0078] The disorder may be any disorder associated with an overexpression of matriptase.

[0079] Typically, the method of treatment may be a method of treatment of a subject suffering from cancer.

[0080] The subject is typically a human.

[0081] The cancer may be selected from the group consisting of lung cancer, preferably non-small cell lung cancer, prostate cancer, breast cancer, colon andrectum cancer, head and neck cancer, esophagogastric cancer, liver cancer, glioblastoma, cervix cancer, ovary cancer, bladder cancer, kidney cancer and pancreatic cancer.

[0082] These cancer types are associated with an overexpression of matriptase.

[0083] In another aspect of the present disclosure, there is provided a pharmaceutical composition comprising the therapeutic conjugate as defined hereinbefore and a pharmaceutically acceptable carrier.

[0084] The composition (or the therapeutic conjugate) may for example be adapted for intravenous or subcutaneous injection.

[0085] According to another aspect, there is provided the use of the matriptase- cleavable substrate as described hereinbefore or the fusion protein as described hereinbefore for the in vitro diagnosis of matriptase activity in a sample.BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 is a schematic representation of the plasmid encoding the matriptase substrate library (A) and the respectively encoded protein displayed in the outer membrane of E. coli cells (B).

[0087] Figure 2 illustrates a table indicating the amino acid frequency in each position of the designed substrate library (A) and the sequenced substrate library (B). The substrate positions are defined as P3, P2, Pi, Pi', P2' and P3'.

[0088] Figure 3 is a schematic representation of the matriptase treatment and library enrichment process by FACS (A) and library sorting outputs analysed by Flow cytometry (B).

[0089] Figure 4 illustrates the analysis of single candidate substrates by cell display using flow cytometry (A) and flow cytometry results plotted indicating % of substrate cleavage after Matriptase treatment (B).

[0090] Figure 5A is a schematic representation of a soluble affibody-ABD fusion protein with a linker comprising the matriptase-cleavable substrate according to an exemplary embodiment of the present disclosure.

[0091] Figure 5B illustrates the analysis of matriptase substrate cleavage in the affibody-ABD fusion proteins by SDS-PAGE.

[0092] Figure 5C illustrates the cleavage efficiency of the inventive substrates compared to two control substrates. The cleavage was performed using three different concentrations (1 nM, 10 nM and 50 nM) and for 10, 30 min or 60 min.

[0093] Figure 6A is a schematic representation of a soluble antibody prodrug comprising the matriptase-cleavable substrate according to an exemplary embodiment of the present disclosure.

[0094] Figure 6B illustrates the analysis of matriptase substrate cleavage in antibody prodrugs by SDS-PAGE.

[0095] Figure 6C illustrates the quantification of the cleavage in the different conditions and the cleavage efficiency of the inventive substrates compared to a control substrate. The cleavage was performed using three different concentrations (1 nM, 10 nM and 50 nM) for 10, 30 min or 60 min.DETAILED DESCRIPTION

[0096] As a first aspect of the present disclosure, there is provided a matriptase- cleavable substrate comprising the amino acid sequence:X1X2X3X4X5X6(SEQ ID NO:I), whereinXi and X2are independently selected from any amino acid residue;

[0097] X3is R or K;

[0098] X4is selected from R, S, or G;

[0099] X5is R or K; and

[0100] X()is selected from S, R, or K.

[0101] As a second aspect of the present disclosure, there is provided a fusion protein comprising the matriptase-cleavable substrate as defined hereinbefore and at least one target-binding domain.

[0102] As a third aspect of the present disclosure, there is provided a conjugate comprising the matriptase-cleavable substrate as defined hereinbefore or a fusion protein as defined hereinbefore and a labelling agent.

[0103] As a fourth aspect of the present disclosure, there is provided a therapeutic conjugate comprising the matriptase-cleavable substrate as defined hereinbefore or a fusion protein as defined hereinbefore and a therapeutic agent.

[0104] As a fifth aspect of the present disclosure, there is provided the therapeutic conjugate as defined hereinbefore for use in a therapeutic method of treatment of a disorder.

[0105] As a sixth aspect of the present disclosure, there is provided a method of treatment of a subject suffering from cancer comprising the step of administration of the therapeutic conjugate as defined hereinbefore.

[0106] As a seventh aspect, there is provided the use of the matriptase-cleavable substrate as described hereinbefore or the fusion protein as described hereinbefore for the in vitro diagnosis of matriptase activity in a sample.EXAMPLESMaterial and methodsCloning and display of matriptase substrate library

[0107] The substrate library was synthetised by Ella Biotech (Ella Biotech GmbH, Germany) and cloned into a previously described pPALU vector [5] by restriction cloning using Ncol and Spel (New England Biolabs, US). The library was transformed into BL21 (DE3) cells by electroporation following a standard protocol [6]. The library size was estimated by spreading dilutions of the transformation sample on carbenicillin agar plates after 1 h incubation. The transformed cells were grown in Luria-Bertani (LB) media supplemented with carbenicillin (100 pg / mL) overnight at 37°C 150 rpm. Next morning, glycerol stocks of the library were made (20% glycerol) and stored at -8o°C. The variability of the library was analysed by deep sequencing using the TrueSeq Miseq Illumina system following the manufacturer recommendations (Illumina, US) (NGI, Sweden).

[0108] For characterisation of the library, fresh LB media was inoculated with a volume of glycerol stock that would cover the library variability. The cells were grown overnight and then diluted in fresh LB media (ODeoo 0.1) grown at 37°C 150 rpm. The cells were induced with arabinose (0.6% final concentration) once they reached anOD6oo of 0.8. The cell cultures were grown overnight at 25°C 150 rpm. The next morning the libraries were ready to be used.Protein labelling

[0109] Recombinant human epidermal growth factor receptor 2 (Her2 / ERBB2 Protein ECD, His Tag, SinoBiologicals, China) was biotinylated using the Biotin-XX Microscale Protein Labeling Kit (Thermo Fisher, US) following the manufacturer recommendations. Human serum albumin (HSA, Albumina Kabi 20%, Kabivitrum, Sweden) was labelled with Alexa-647 fluorophore following the manufacturer recommendations (Alexa Fluor™ 647 NHS Ester (Succinimidyl Ester), Invitrogen, US).Library labelling and sorting by flow cytometry

[0110] The induced cell cultures were taken after an overnight growth and OD()(IOwas measured. A volume of cells corresponding to IOX the library size was washed twice with phosphate-buffered saline ix supplemented with 1% Pluronic acid (PBS-P) and treated with a 100, 50, 25 or 10 nM solution of matriptase protease for 30 min at 37°C. The matriptase concentration was reduced progressively during the selection rounds to increase the sorting stringency. The cells were washed twice with PBS-P and labelled with a 50 nM solution of biotinylated Her2 for ih at RT. Later, the cells were washed once with PBS-P and incubated with 225 nM Alexa Fluor 647-HSA and 33.3 nM Streptavidin, R-Phycoerythrin Conjugate (SAPE, ThermoFisher, US) in PBS- P for 30 min at 4°C.

[0111] Finally, the cells were washed twice with PBS-P and the samples were analysed and sorted using a Cytoflex SRT (Beckman Coulter, USA). Library sizes were estimated by spreading dilutions of the sortings on carbenicillin agar plates after 1 h incubation in media at 37°C 150 rpm. The remaining cells were grown in fresh LB media supplemented with carbenicillin (100 pg / mL) at 37°C 150 rpm. After i6h, the cultures were aliquoted as glycerol stocks (20% glycerol solution) and stored at - 80 °C. Single colony candidates were picked from the agar plates and grown overnight at 37°C 150 rpm. Finally, glycerol stocks of each single candidate were prepared and stored at -8o°C.Deep sequencing analysis

[0112] The overnight culture of the original library was used for plasmid extraction (Qiagen Miniprep Kit, Qiagen, US). The purified plasmids were used as templates for PCR amplification of the substrates with TrueSeq indexes and adapters. The PCR was performed using Phusion polymerase (Thermo Fisher, US) and primers containing binding regions, adapter regions and Trueseq indexes (Illumina, US). The PCR products were purified using a Qiagen gel extraction kit (Qiagen, US) and the concentration was measured by Qubit (Thermo Fisher, US).

[0113] The variability of the unsorted library was analysed by deep sequencing using the TrueSeq Miseq Illumina system following the manufacturer recommendations (Illumina, US) (NGI, Sweden). The resulting sequences were evaluated using PipeBio version 1.0.5 (PipeBio, US) and the data analysis were performed using Microsoft Excel (Microsoft, US).Cell display of individual candidate and analysis by flow cytometry

[0114] Single colony candidates were analysed individually by flow cytometry. Each single clones isolated after FACS was inoculated in 5 mL of media supplemented with Carbenicillin (100 pg / mL) and grown for O / N at 37°C 150 rpm. Afterwards, the cultures were diluted in fresh LB media (ODeoo 0.1) grown at 37°C 150 rpm. The cells were induced with arabinose (0.6% final concentration) once they reached an ODeoo of 0.8. The cell cultures were grown overnight at 25°C 150 rpm

[0115] The next morning, 15 pL of each culture were transferred to individual wells in a conical-shaped 96-well plate. The cells were washed twice with PBS-P and treated with a 10 nM solution of matriptase protease for 10, 30, 60 and 90 min at 37°C. The cells were immediately washed twice with PBS-P and labelled with a 50 nM solution of biotinylated Her2 for ih at RT. Later, the cells were washed once with PBS-P and incubated with 225 nM Alexa Fluor 647-HSA conjugate and 33.3 nM SAPE on PBS-P for 30 min at 4°C. Finally, the cells were washed twice with PBS-P and the samples were analysed using a Cytoflex Flow cytometry instrument (Beckman Coulter, USA).

[0116] The data obtained was used to evaluate the cleavage efficiency of each candidate in comparison to the control substrates and their non-cleaved versions.Soluble substrate production

[0117] The sequences encoding for selected candidates were cloned into plasmids encoding 2 different scaffolds: affibody-ABD dimers and antibody prodrugs. A non- cleavable substrate (defined by the amino acid sequence: ENLYFG (SEQ ID NO: 17) and two previously described matriptase substrates defined by the amino acid sequence: MSGRSANA (control 2, SEQ ID NO: 18) [7] and LSGRSDNH (control 1, SEQ ID NO: 19) [1], respectively, were included for comparison. The cloning was performed using an Infusion kit (Takara, Japan). The plasmids were sequence verified and transformed by heat-shock into E. coli for recombinant protein production following standard protocol [9]. The affibody used in the affibody-ABD dimer was an affibody defined by SEQ ID NO: 15. The ABD domain used in the affibody-ABD dimer was an ABD domain defined by SEQ ID NO: 17.

[0118] The transformed cells were inoculated in 5 mL of Tryptic Soy Broth (TSB) media supplemented with yeast extract (Merck, US) and kanamycin (50 pg / mL). The cells were grown overnight at 37°C 150 rpm.

[0119] After 16 h, the cultures were diluted 1:100 into TSB-Y media supplemented with kanamycin (50 pg / mL). When ODeoo reached 0.7, the cultures were induced with isopropyl P-D-i-thiogalactopyranoside (IPTG) to a final concentration of 1 mM, and incubated O / N at 25°C and 150 rpm. After 16 h, cells were harvested by centrifugation (soooxg, 10 min, 4°C) and resuspended in IMAC equilibration buffer (20 mM Tris-HCl, 300 mM NaCl, pH 7.4). The cells were lysed by sonication for 1.5 minutes (1 sec on:i sec off). Cell debris was removed by centrifugation (25 oooxg, 15 min, 4°C), and the protein-containing supernatant was filtered (0.45 pm), followed by IMAC purification on HisPur Cobalt resin (Thermo Fisher Scientific, US). Briefly, the IMAC resin was washed with 2x10 CV equilibration buffer, followed by sample binding for 30 min, after which the matrix was washed with 3x10 CV wash buffer (20 mM Tris-HCl, 300 mM NaCl, 30 mM imidazole, pH 7.4). Bound protein was eluted by incubation with 2.5 mL elution buffer (20 mM Tris-HCl, 300 mM NaCl, 300 mM imidazole, pH 7.4) for 10 min. Eluted proteins were buffer-exchanged to PBS (pH 7.4) using PD-10 columns (Cytiva, Sweden), according to the manufacturer’s recommendations.

[0120] The proteins were analysed by SDS-PAGE (NuPAGE, Invitrogen, US), and mass spectrometry (MS, 4800 MALDI TOF / TOF, Applied Biosystems / MDS SCIEX).The antibody prodrug encoding plasmids were transfected into ExpiCHO cells using the ExpiFectamine™ CHO Transfection Kit (Thermo Fisher, US) for recombinant antibody expression. After 12 days of production, the cell supernatants were collected (4oooxg, 30 min) and filtered through a 0.2 pm filter. The antibodies were purified using a HiTrap PrismA column in an Akta Start system following the manufacturer recommendations (Cytiva, Sweden). The purified antibodies were buffer-exchanged to PBS ix and kept at 4°C.Soluble substrate cleavage tests

[0121] The affibody-ABD dimers and antibody prodrugs containing the substrates were diluted to a concentration of 50 pg / mL in PBS and treated with Matriptase (1 nM, 10 nM and 50 nM) for 10, 30 and 60 minutes. Immediately after the protease treatment, TCEP was added to the samples, and they were incubated at 9O°C for 10 min to stop the reaction. Finally, the samples were mixed with loading dye (62.5 mM Tris-HCl (pH 6.8), 2% (w / v) SDS, 10% glycerol, 0.01% (w / v) bromophenol blue) and loaded in an SDS-page gel (NuPAGE, Invitrogen). The gel ran for 60 min at 200 V and the staining was performed using GelCode™ Blue Safe Protein Stain (Thermo Fisher, US). The gel images were taken using a GelDoc system (Biorad, US).SDS-page gel quantification

[0122] The 600 dpi SDS-page gel images were analysed by ImageJ using a densitometry tool (ImageJ version 1.54! 03, US). The band quantification reflects the relative amounts of cleaved and intact constructs as a ratio of the protein bands in each lane. The raw data values were analysed using Microsoft Excel (Microsoft, US) and plotted using Prism 8 (GraphPad, US).Results

[0123] As described hereinabove, an in-house developed bacterial display method was used for screening of substrate libraries. The method was based on display of recombinant proteins on E. coli by genetic fusion to an autotransporter protein for anchoring to the outer membrane. The expression cassette was modified to include two reporter tags, binding Her2 (cleavage reporter tag) and albumin (expression reporter tag). The reporter tags were genetically connected via a linker that also contained the matriptase substrate sequence of interest (Figure 1).

[0124] The hypothesis was that, upon protease treatment, the outer reporter tag (anti-Her2) would be cleaved off and no longer be able to bind recombinant Her2. Proteolysis could thus be assessed by flow cytometry after incubation with fluorescently labelled Her2 (see Figure 3 A).

[0125] Since each cell displays around 106copies of the same substrate sequence anchored to its cell membrane, the method is quantitative and fluorescence intensity is inversely correlated with cleavage efficiency. More specifically, the proteolytic cleavage is inversely correlated with the fluorescence intensity from the cleavage reporter (Her 2).Design of matriptase substrate library

[0126] The library was designed based on reported amino acid preferences for matriptase substrates in the literature [5, 10, 11, 12]. In summary, amino acids Arg and Lys were prioritized in position Pi. Non-negatively charged amino acids were prioritized in position Pi’. Small amino acids were prioritized in position P2’, and hydrophobic amino acids were prioritized in position P3’. Cysteines were excluded from all positions to avoid disulphide bond formations. The library design resulted in a theoretical size of 4.7X106variants. The final construct to be displayed in E. coli cells consisted of an Affibody domain with binding capacity to Her2 (ZHer2

[0013] ), a substrate candidate surrounded by 4 GGGS spacers, an albumin binding domain (ABD

[0014] ) and the AIDA-autotransporter [5] (Figure 1).Library display and variability analysis

[0127] The substrate library was subcloned to the surface by the display vector pPALU and transformed to E. coli, yielding 1.26x107 transformants. Deep sequencing was used to analyse the assembled substrate library. The observed frequency of amino acids in each position of the substrate (P3, P2, Pi, Pi’, P2’, P3’) showed a good agreement with the theoretical design (Figures 2A and 2B).Library sorting

[0128] The created library was displayed in E. coli cells and treated with matriptase or PBS before labelling it with Her2-biotin-SAPE and Alexa 647-HSA. The libraries analysed by flow cytometry and the interesting candidates were sorted based on their loss of binding capacity to Her2 caused by the action of matriptase (Figure 3A). The libraries were sorted for a total of 5 cycles to select the population of non-Her2 binding cells, and therefore, enrich the library for variants having a suitable cleavable substrate. The outputs were analysed by flow cytometry indicating the overall loss of binding capacity of the library in subsequent cycles (Figure 3 B).

[0129] The deep sequencing analysis of the sorted libraries revealed the sequences of the enriched candidates.Analysis of single candidates

[0130] A total of 192 single variants were analysed by flow cytometry to study the reduction of Her2 target binding as an indicator of the substrate cleavage efficiency. The loss of target binding is directly proportional to the cleavage efficiency (Figure 4A). The cleavage rate was evaluated using E. coli cells displaying the different variants. A concentration of 10 nM matriptase was used for the assessment at 4 different time points (o, 10, 30 and 60 min) (Figure 4B).

[0131] After individual analysis, four different sequences were selected for their optimal matriptase cleavage. The amino acid sequences of the substrates as well as the control substrates used are shown in Table 1.Table 1: Amino acid sequence of the four substrates selected for optimal cleavage by matriptase and control substratesIn vitro cleavage of soluble proteins

[0132] The in vitro cleavage of soluble proteins was performed in two different soluble protein formats: Affibody-ABD dimer (Figure 5A) and antibody prodrug (Figure 6A). The cleavage was tested using three different concentrations (1 nM, 10 nM and 50 nM) for 10, 30 and 60 min. The results were analysed by SDS-page (Figure 5B, and Figure 6B, respectively). Image J software was used to calculate the relative amounts of cleaved substrate by analysing the band’s density (Figure 5C and Figure 6C, respectively).

[0133] In Figure 5B, the band above 16 kDa corresponds to the full substrate and the bands at 9.9 kDa and 6.0 kDa correspond to the affibody and ABD domain, respectively (Figure 5B). The densitometry analysis of gels was used to quantify the cleavage in different matriptase concentrations and incubation times. The results demonstrate the higher cleavage efficiency of the inventive substrates in comparison to the matriptase substrate controls and a non-cleavable substrate (Figure 5C).

[0134] The inventive substrates (MtpCs, MtpBg, MtpC9, and MtpAi2) could be partially cleaved (>50%) after 10 minutes with 1 nM matriptase. The substrate MtpCs showed the best cleavage efficiency with about 86% cleaved product after 10 minutes with 1 nM matriptase. The Control 2 sample required at least 5 times more matriptase and a significantly longer treatment time (50 nM Matriptase for 30 min). The Control 1 sample could only be cleaved (96.4 %) after 1 hour treatment at the highest matriptase concentration tested (50 nM). The results demonstrate that the inventive substrates (MtpCs, MtpBg, MtpC9, and MtpAi2) were more efficiently cleaved than the control substrates. As expected, the non-cleavable substrate was not cleaved under any condition.

[0135] Next, cleavage was assessed in the antibody prodrug format. The antibody prodrug is schematically illustrated in figure 6A. The antibody used was Cetuximab

[0016] . The masking domain used was an affibody.

[0136] The analysis included the four inventive substrates (MtpCs, MtpBg, MtpC9, and MtpAi2), control 1 substrate for comparison and a non-cleavable linker as negative control. The samples were incubated with different concentrations of matriptase for different times and cleavage was analyzed by SDS-PAGE.

[0137] Different bands could be observed in the SDS-page corresponding to: i) full antibody (150 kDa), ii) antibody heavy chain with substrate and masking affibody domain (58 kDa) (intact), in) antibody heavy chain (50 kDa) (cleaved), iv) antibody light chain (25 kDa), and v) cleaved-off masking affibody domain (7 kDa).

[0138] Again, densitometry analysis of gels was used to quantify the cleavage and the results are shown in Figure 6C, confirming that the inventive substrates were more efficiently cleaved than control substrate 1.

[0139] The results show that 1 nM matriptase was enough to partly cleave the inventive substrates already after 30 min (12.7 - 39.7 %, depending on the candidate). Complete sample cleavage was observed after 30 minutes at 10 nM matriptase for MtpCs, MtpBg, MtpC9, and for 30 minutes and 50 nM matriptase for MtpAi2, compared to no visible cleavage for substrate control (control 1). The substrate control (control 1) required 50 nM of protease and 60 minutes incubation to cleave 19.5% of the sample.

[0140] To summarize, the enhanced cleavage efficiency of the inventive substrates may significantly improve the activation of a prodrug incorporating the substrate(s). Accordingly, the prodrug may be activated more effectively and only in the presence of matriptase.

[0141] Terms, definitions and embodiments of all aspects of the present disclosure apply mutatis mutandis to the other aspects of the present disclosure.

[0142] Even though the present disclosure has been described with reference to specific exemplifying embodiments thereof, many different alterations, modifications and the like will become apparent for those skilled in the art.

[0143] Variations to the disclosed embodiments can be understood and effected by the skilled addressee in practicing the present disclosure, from a study of the drawings, the disclosure, and the appended claims. Furthermore, in the claims, the word "comprising" does not exclude other elements or steps, and the indefinite article "a" or "an" does not exclude a plurality.References[1] Desnoyers, L. R. et al. Tumor-specific activation of an EGFR-targeting probody enhances therapeutic index. Sci. Transl. Med. 5, (2013).[2] Lanchec, E. et al. The type II transmembrane serine protease matriptase cleaves the amyloid precursor protein and reduces its processing to P-amyloid peptide. J. Biol. Chem. 292, (2017).[3] Barre, O. et al. Cleavage specificity analysis of six type II transmembrane serine proteases (TTSPs) using PICS with proteome-derived peptide libraries. PLoS One 9, (2014).[4] Boulware, K. T. & Daugherty, P. S. Protease specificity determination by using cellular libraries of peptide substrates (CLiPS). Proc. Natl. Acad. Sci. U. S. A. 103, (2006).[5] Parks, L., Ek, M., Stahl, S. & Lofblom, J. Investigation of an AIDA-I based expression system for display of various affinity proteins on Escherichia coli. Biochem. Biophys. Res. Commim. 696, (2024).[6] Lessard, J. C. Chapter Twenty Seven - Transformation of E. coli Via Electroporation, in Methods in enzymology vol. Volume 529 (2013).[7] W02022 / 016270A1[8] Antignani et al, Biomolecules; 2020 Sep 17;IO(9):1331.[9] Froger, A. & Hall, J. E. Transformation of Plasmid DNA into E. Coli using the heat shock method. J. Vis. Exp. (2007) doi:io.3791 / 253.

[0010] Igarashi, Y. et al. CutDB: A proteolytic event database. Nucleic Acids Res. 35, (2007).

[0011] Rawlings, N. D. et al. The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database. Nucleic Acids Res. 46, (2018).

[0012] Bateman, A. et al. UniProt: the Universal Protein Knowledgebase in 2023.Nucleic Acids Res. 51, (2023).

[0013] Wllberg, H. et al. Affinity recovery of eight HER2-binding affibody variants using an anti-idiotypic affibody molecule as capture ligand. Protein Expr. Purif. 76, (2011).

[0014] Jonsson, A., Dogan, J., Herne, N., Abrahmsen, L. & Nygren, P. A. Engineering of a femtomolar affinity binding protein to human serum albumin. Protein Eng. Des. Sei. 21, (2008).

[0015] Blanco et al., Clin Cancer Res 2021 Oct 15;27(2O):5457-5464).

[0016] Baselga J. The EGFR as a target for anticancer therapy— focus on cetuximab. Eur J Cancer. 2001 Sep;37 Suppl 4:816-22.

Claims

CLAIMS1. A matriptase-cleavable substrate comprising the amino acid sequence: X1X2X3X4X5X6(SEQ ID NO:1), whereinXi and X2are independently selected from any amino acid residue;X3is R or K;X4is selected from R, S, or G;X5is R or K; andX()is selected from S, R, or K.

2. The matriptase-cleavable substrate according to claim i, comprising the amino acid sequence as defined by:XiX2X3RRS (SEQ ID N0:2);XiX2X3SRR (SEQ ID NO:3);X,X2X3GRR (SEQ ID NO:4); orXIX2X >GRK (SEQ ID NO:5), whereinXi and X2are independently selected from any amino acid residue, and X3is R or K.

3. The matriptase-cleavable substrate according to claim 1 or claim 2, comprising the amino acid sequence as defined by:XtX2KRRS (SEQ ID NO: 6);XiX2KSRR (SEQ ID NO:y);XiX2RGRR (SEQ ID NO: 8); orXiX2KGRK (SEQ ID NO:9), whereinXi and X2are independently selected from any amino acid residue.

4. The matriptase-cleavable substrate according to any one of claims 1-3, comprising the amino acid sequence as defined by:WHKRRS (SEQ ID NO: 10);GAKSRR (SEQ ID NO: 11);VPRGRR (SEQ ID NO: 12);HYKGRK (SEQ ID NO: 13); ora corresponding sequence having at least 83% sequence homology with any one of SEQ ID NO:1O-13.

5. A fusion protein comprising the matriptase-cleavable substrate according to any one of claims 1-4 and at least one target-binding domain.

6. The fusion protein according to claim 5, wherein said target -binding domain is a tumor-binding domain.

7. The fusion protein according to claim 6, wherein said tumor-binding domain is a HER-binding domain.

8. The fusion protein according to any one of claims 5-7, wherein said target-binding domain is an affibody, an antibody or a fragment of an antibody.

9. The fusion protein according to any one of claims 5-8, further comprising a masking domain.

10. The fusion protein according to claim 9, wherein said masking domain is linked to said target-binding domain by a linker and wherein said linker comprises said matriptase-cleavable substrate.

11. The fusion protein according to claim 9 or claim 10, wherein said masking domain is an affibody.

12. The fusion protein according to any one of claims 5-11, wherein said targetbinding domain is an affibody and wherein said fusion protein further comprises a half-life-extending domain, preferably wherein said half-life-extending domain is an albumin-binding domain (ABD).

13. The fusion protein according to claim 12, wherein said half-life extending domain is linked to said tumor-binding domain by a linker, and wherein said linker comprises said matriptase-cleavable substrate.14- A conjugate comprising the matriptase-cleavable substrate according to any one of claims 1-4 or a fusion protein according to any one of claim 5-13 and a labelling agent.

15. The conjugate according to claim 14, wherein said labelling agent is a fluorophore, chromophore, radioactive isotope or an enzyme.

16. A therapeutic conjugate comprising the matriptase-cleavable substrate according to any one of claims 1-4 or a fusion protein according to any one of claim 5-13 and a therapeutic agent.

17. The therapeutic conjugate according to claim 16, wherein said therapeutic agent is a cytotoxic agent.

18. The therapeutic conjugate according to claim 16 or claim 17 for use in a method of treatment of a disorder.

19. The therapeutic conjugate according to claim 18, wherein said disorder is cancer.

20. The therapeutic conjugate according to claim 19, wherein said cancer is selected from the group consisting of lung cancer, preferably non-small cell lung cancer, prostate cancer, breast cancer, colon and rectum cancer, head and neck cancer, esophagogastric cancer, liver cancer, glioblastoma, cervix cancer, ovary cancer, bladder cancer, kidney cancer and pancreatic cancer.

21. Use of the matriptase-cleavable substrate according to any one of claims 1-4, the fusion protein according to any one of claims 5-13 or the conjugate according to any one of claims 14-15 for the in vitro diagnosis of matriptase activity in a sample.

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