Scaffold proteins and uses thereof

Abstract

The present invention relates to a protein comprising the defined amino acid sequence of SEQ ID NO: 115, wherein within said amino acid sequence there is present a sequence of the general formula Ih-mod:GX1CX 1V X2X3X4X5 (wherein X1 is any of F, Y, L, P, Q, M, V, W, A, or T; X 1V is R or K; X2 is A, G, S, or T; X3 is a 17-set of amino acids (wherein the 17-set comprises A, I, L, F, and Y); X4 is any of K, I, Q, R, H, S, F, M, N, L, or V; and X5 is any of R, V, I, K, M, Q, E, F, L, N, Y, D, S, and H; and b) at position 34 of the amino acid sequence of SEQ ID NO: 115, it contains an amino acid selected from the 34-set (wherein the 34-set comprises Y, I, F, G, V, and S). The present invention further relates to the use of said protein in pharmaceuticals, kits, and screening methods. The protein is suitable for inhibiting the MASP-2 enzyme.

Description

[Technical Field]

[0001] The present invention relates to a class of novel scaffold proteins, methods for making proteins with such scaffolds, and the use of such proteins in the manufacture of pharmaceuticals. [Background technology]

[0002] The complement system is a highly conserved part of the innate immune system of vertebrates, including humans. It is a network of distinct proteins, including serine proteases, soluble and membrane-bound receptors, and regulatory factors. In the blood, it is evolutionarily and functionally closely related to other protein networks, such as those involved in blood coagulation and fibrinolysis. As part of the immune system, the complement system can recognize labels and eliminate invading pathogenic microorganisms (viruses, bacteria, and fungi) and dangerous altered self-structures, such as virus-infected cells, apoptotic / necrotic cells, and cancer cells. The complement system forms one of the first lines of defense against pathogenic microorganisms. In addition to being a key effector arm of the innate immune system, the complement system also connects the innate and adaptive immune responses in numerous ways. Although it is a network of protein molecules, it can stimulate and regulate various cellular processes (Merle 2015a; Merle 2015b; Hajishengallis 2017).

[0003] The complement system consists of more than 30 protein components. Major components are serine proteases that activate each other in a cascade-like manner (Sim 2004). Activation involves limited proteolysis of proenzyme proteases by activated subcomponents. Other components include pattern recognition molecules, inhibitors, regulators, and cell surface receptors. A key event during complement activation is the cleavage of the C3 component. C3 is cleaved into two fragments, C3a and C3b, by the C3 convertase enzyme complex. As a result of a significant conformational change, a thioester bond is exposed on the surface of the C3b molecule, through which it can be deposited on activated surfaces (e.g., bacterial cells, immune complexes) (Geisbrecht 2022). C3b not only acts as an opsonin, promoting the clearance of dangerous structures by phagocytes, but also serves as a platform for further complement activation and the initiation of the final pathway leading to the destruction of invading cells. The C3a component is an anaphylatoxin that induces inflammation through the activation of immune cells (Ricklin 2016).

[0004] There are three routes by which the C3 convertase complex forms: the classical pathway, the lectin pathway, and the alternative pathway. In the classical and lectin pathways, a pattern recognition molecule binds to an activating surface, and an associated serine protease initiates a proteolytic cascade. The pattern recognition molecule in the classical pathway is C1q (Thielen 2017). C1q consists of three types of polypeptide chains (A, B, and C chains) and has a structure resembling a bouquet of six tulips. The A, B, and C chains form trimers with collagen-like arms in the N-terminal half and a globular head in the C-terminal half of the molecule. Six such trimers combine to form the complete C1q structure. In the circulation, C1q associates with two types of serine proteases: C1r and C1s. Two C1r molecules and two C1s molecules form a tetramer (C1s-C1r-C1r-C1s) that binds to the collagen arms of C1q (Zwarthoff 2021). The resulting C1 complex is the initiation complex of the classical pathway. Upon activation, the globular head of C1q binds to an activator structure. Typical activators of the classical pathway are immune complexes, C-reactive protein, and apoptotic cells (Diebolder 2014, Sharp 2019). In the C1 complex, C1r and C1s serine proteases exist in zymogen form and become activated after C1q binds to the activator surface. During activation, the Arg-Ile bond is cleaved at the activation peptide of the serine protease domain. The resulting two polypeptide chains are held together by a disulfide bond. The first enzymatic event in classical pathway activation is autoactivation of C1r. In the next step, active C1r cleaves and activates the zymogen C1s, the executive protease of the C1 complex. It cleaves the C4 and C2 components to generate the classical pathway C3 convertase C4b2a (Gal 2009).

[0005] The same C3 convertase complex is generated via the lectin pathway. Five distinct pattern recognition molecules exist in the lectin pathway: MBL (mannose-binding lectin), ficolin-1 / -2 / -3, and CL-LK (Holmskov 2003). The global structure of MBL and ficolins is similar to that of C1q, but the trimeric subunits consist of a single polypeptide chain. While C1q has a well-defined hexameric structure, the polymerization states of MBL and ficolins vary between dimers and pentamers. The most compact polymeric form is a tetramer. Two collectins, collectin kidney 1 (CL-K1) and collectin liver 1 (CL-L1), form heterotrimeric subunits containing one CL-L1 polypeptide chain and two CL-K1 polypeptide chains. There are three MBL-related serine proteases (hereafter referred to as MASPs): MASP-1, MASP-2, and MASP-3 (Dobo 2016a). MASP-1 and MASP-2 exist in proenzyme forms and are associated with pattern recognition molecules. MASP-1 and MASP-2 are the initiator proteases of the lectin pathway. MASP-1 and MASP-2 form homodimers via their N-terminal non-catalytic regions. These homodimers bind to pattern recognition molecules. Tetrameric pattern recognition molecules typically bind to a single MASP dimer. Most activation complexes of the lectin pathway contain one type of MASP. During lectin pathway activation, pattern recognition molecules bind to activator surfaces (e.g., bacterial surfaces). MBL binds to Ca 2+Ficolins bind to carbohydrate arrays on bacterial surfaces in a phospho-dependent manner. Ficolins bind to acetylated compounds, typically acetylated sugars (e.g., N-acetyl-glucosamine). On the surface, MBL-MASP-1 and MBL-MASP-2 complexes are deposited side-by-side (Degn 2014). The first enzymatic event during activation of the lectin pathway is autoactivation of MASP-1. Activated MASP-1 is the exclusive activator of MASP-2 (Heja 2012a). Only activated MASP-2 can cleave C4, while C2 is cleaved by both MASP-1 and MASP-2. The resulting C3 convertase complex (C4b2a) is identical to that in the canonical pathway. A third complement pathway, the alternative pathway, is closely related to the lectin pathway. MASP-3, an alternative splice product of the MASP1 gene, is responsible for activating pro-factor D (pro-FD) (Dobo 2016b). MASP-3 constitutively activates pro-FD, even in the absence of any danger signals (PAMPs or DAMPs). Due to the constant proteolytic activity of MASP-3, FD exists in an activated form in plasma. When C3b is deposited on activated surfaces, it binds factor B (FB), a serine protease component of the alternative pathway C3 convertase. FD has extremely narrow substrate specificity; C3-bound FB is the sole substrate for FD. After FD mediates cleavage of FB, the larger fragment Bb remains bound to C3b, while the smaller fragment (Ba) dissociates. The resulting alternative pathway convertase (C3bBb) cleaves other C3 molecules, which can serve as platforms for more C3 convertase complexes. As a result, the alternative pathway forms a positive feedback mechanism to permit complement activation independent of the initiation pathway (Harboe 2004).

[0006] Just as the classical or lectin pathway generates the first C3b molecule, the alternative pathway initiates and provides an amplification loop. The alternative pathway can also initiate itself due to a so-called "tick-over" mechanism (Pangburn 1981). C3 is slowly hydrolyzed in the fluid phase. The resulting C3(H2O) has a C3b-like conformation and binds FB. After FD-mediated cleavage, the resulting C3(H2O)Bb is a fluid-phase C3 convertase. This fluid-phase convertase subsequently deposits C3b on nearby surfaces. In the case of self-tissues, membrane-bound complement inhibitors prevent complement activation. However, on pathogenic surfaces, a positive amplification loop for the alternative pathway is established. In this way, the "tick-over" mechanism of the alternative pathway can distinguish between self and non-self structures without the use of pattern recognition molecules.

[0007] C3 cleavage is a turning point in the complement cascade. This is the point where the three activation routes (classical, lectin, and alternative) converge and initiate a common terminal pathway. As the density of deposited C3b increases on the surface, the substrate specificity of C3 convertase switches from C3 to C5 (Mannes 2021). C5 convertases (C4b2a (C3b)n and C3bBb (C3b)n) cleave C5 into C5b and C5a. The smaller fragment (C5a) is a highly potent anaphylatoxin that stimulates various cells (endothelial cells, lymphocytes, monocytes, etc.) via G protein-coupled receptors and triggers inflammatory responses. The larger fragment (C5b) binds C6 and C7. The C5b-7 complex binds to the cell membrane and binds C8. After a conformational change, the C5b-8 complex penetrates itself through the pathogen's membrane (e.g., a bacterial membrane) and recruits a large number (approximately 20) of C9 molecules. The C5b-9n complex (commonly used in the scientific literature: TCC = terminal complement complex (also known as MAC = membrane attack complex)) forms a pore (approximately 240 Å in diameter) in the cell membrane, resulting in cell destruction via osmotic shock and lysis (Tegla 2011). This defense mechanism is crucial for Gram-negative bacteria, particularly Neisseria species (Petersen 1979; Lewis 2014).

[0008] The classical pathway C1r and C1s and the lectin pathway MASP-1 / -2 / -3 form a group of proteases with identical domain organization and related functions (Gal 2009). Members of the C1r / C1s / MASPs family consist of six domains: five non-catalytic domains in the N-terminal half of the molecule and one serine protease domain at the C-terminus. The co-catalytic domain is responsible for protein-protein interactions (e.g., dimerization, tetramer formation, and binding to pattern recognition molecules), while the trypsin-like serine protease domain possesses enzymatic (proteolytic) activity. In the N-terminal region, there is a CUB domain (CUB = C1r / C1s, sea urchin Uegf, and bone morphogenetic protein-1), an EGF (EGF = epidermal growth factor) domain, another CUB domain, and two CCP (CCP = complement control protein) domains. The CUB1-EGF-CUB2 fragment mediates the dimerization of MASPs and the binding of pattern recognition molecules to the collagen arms. The serine protease domain binds and cleaves substrates, while the CCP domain contributes to substrate specificity by providing an additional binding site (exosite) for the substrate (Kidmose 2012). The CCP1-CCP2-SP fragment is enzymatically identical to the full-length molecule. These proteases (except MASP-3) exist in the circulatory system as proenzymes and are activated only after the pattern recognition portion of the complex binds to an activating surface. In vitro, MASP-1 and MASP-2 can autoactivate, but under physiological conditions, MASP-2 does not exhibit this ability. In blood, MASP-1 is the exclusive activator of MASP-2 (Heja 2012a). The serum concentration of MASP-1 is 143 nM (11 μg / ml), while that of MASP-2 is 6 nM (0.4 μg / ml). The low concentration and low autoactivation potential of MASP-2 preclude autoactivation in blood. At the activation surface, MBL-MASP-1 and MBL-MASP-2 complexes are juxtaposed, and due to the high concentration of MASP-1, each MASP-2 molecule is surrounded by numerous MASP-1 molecules (Degn 2014). This arrangement and the enzymatic properties of MASP-1 ensure that MASP-1 acts as the exclusive activator of MASP-2.MASP-1 has a highly potent autoactivation capacity and cleaves the proenzyme MASP-2 very efficiently (Megyeri 2013). On the other hand, MASP-2 is highly competent in cleaving C4 (more competent than C1s). MASP-1 cannot cleave C4 and therefore cannot initiate the lectin pathway by itself.

[0009] Both MASP-1 and MASP-2 are essential for lectin pathway activation; therefore, inhibition of either MASP-1 or MASP-2 inhibits lectin pathway activation. Low concentrations of MASP-2 make them a more attractive drug target than MASP-1 in situations where lectin pathway inhibition is beneficial. MASP-2 has very narrow substrate specificity (cleaving C4 and C2), whereas MASP-1 has a large number of substrates, all of which are linked to innate immune responses (Dobo 2016a). MASP-1 has thrombin-like activity (cleaving fibrinogen, prothrombin, factor XIII, and protease-activated receptors). Thus, MASP-1 may contribute to thrombus formation and directly activate endothelial cells and leukocytes. Furthermore, MASP-1 can release bradykinin from high-molecular-weight kininogen, affecting vascular permeability (Dobo 2011; Debreczeni 2019). Recently, it has been shown that MASP-2 also promotes thrombus formation, and high plasma concentrations of MASP-2 increase the risk of future venous thromboembolism (Damoah 2022). Both the MASP1 and MASP2 genes have alternative splicing products. The MASP1 gene encodes three distinct proteins: MASP-1, MASP-3, and MAp44. The non-catalytic regions (first five domains) of MASP-1 and MASP-3 are identical, but their serine protease domains differ. Although these proteases bind to the same pattern recognition molecules (MBL, ficolins), the different SP domains result in different enzymatic properties and biological roles for MASP-1 and MASP-3. MASP-3 cannot autoactivate, and its only known physiological substrate is pro-FD. In blood, most MASP-3 (approximately 80%) exists in an activated form, so other proteases must be responsible for its activation (Oroszlan 2017). Recently, it has been discovered that human proprotein convertases (PCSK6, PC5A, and furin) can activate the proenzyme MASP-3 (Oroszlan 2021). Because secreted PCSK6 (also known as PACE4) is present in the blood, it is likely to be the major or exclusive activator of MASP-3.MASP-3 has no role in lectin pathway activation. Permanent inhibition of MASP-3 results in inhibition of the alternative pathway (Cummings 2017). MASP-3 may exert some inhibitory effect on the lectin pathway by competing with MASP-1 and MASP-2 for recognition molecules. A similar inhibitory function has been attributed to another alternative splice product of the MASP1 gene: MAp44 (also known as MAP-1) (Pavlov 2012). MAp44 consists of the first four non-catalytic domains of MASP-1 (CUB1-EGF-CUB2-CCP1). The MASP2 gene also has an alternative splice product: MAp19 (MAP-2, also known as sMAP). MAp19 contains the first two N-terminal domains of MASP-2 (CUB1-EGF), and its biological function is unknown (Stover 1999). Theoretically, it would function as an inhibitor of the lectin pathway (similar to MAp44), but its low serum concentrations and its weak binding to pattern recognition molecules make this speculation questionable.

[0010] An intact complement system is essential for maintaining the body's immune homeostasis. It prevents inflammation and autoimmune disease through the removal of immune complexes and cellular debris. It is a potent killing mechanism that efficiently targets invading microorganisms while sparing healthy human cells. Numerous fluid-phase and cell-surface inhibitors ensure the safe operation of the complement cascade in human blood. However, if the strict regulatory control of complement activation is impaired for any reason, inappropriate or uncontrolled activation of the complement system can result in local and / or systemic inflammation, autologous tissue damage, and the development of serious pathologies. The complement system is now recognized as an attractive therapeutic target for treating various diseases (Dobo 2018, Mastellos 2019, Ricklin 2019).

[0011] Ischemia-reperfusion injury (IRI) is a severe autoimmune reaction in which complement activation plays a key role. When blood flow in an organ is temporarily restricted or interrupted for some reason (e.g., vascular occlusion), the loss of oxygen (hypoxia) renders the tissue susceptible to complement-mediated attack after blood return (reperfusion). During reperfusion, the immune system recognizes ischemic cells as damaged self cells (DAMPs, or damage-associated molecular patterns), initiating a complex inflammatory response in which the complement system plays a pivotal role. IRI significantly contributes to tissue damage in cases of myocardial infarction and stroke and can also be a cause of complications during coronary artery bypass surgery and organ transplantation (Markiewsky 2007). The lectin pathway is predominantly involved in this process, as pattern recognition molecules (MBL, collectin 11) can recognize certain carbohydrate traces on ischemic cells (Collard 2000; Nauser 2018). Natural IgM antibodies also bind to certain neoantigens exposed on ischemic tissue, and these IgMs trigger the lectin pathway (Chan 2004, Zhang 2006, McMullen 2006). The alternative pathway amplifies complement deposition on infected tissue. Several animal model experiments have demonstrated that abolishing lectin pathway activation reduces the severity and consequences of IRI (Jordan 2001, Hart 2005, La Bonte 2009). Inhibition of MASP-2 is a promising approach for treating or preventing IRI. In mouse models, targeting (eliminating or blocking) MASP-2 has been shown to protect against myocardial and gastrointestinal IRI (Schwaeble 2011, Clark 2018). While this protection is MASP-2 dependent, C4 has not been shown to play a role in MASP-2-mediated IRI in this mouse model. Similarly, in a mouse model, tissue damage in renal IRI was MASP-2 dependent but C4 independent (Asgari 2014). A natural endogenous lectin pathway inhibitor (MAp44) was also effective in attenuating myocardial IRI (Pavlov 2012). MAp44 (as a non-catalytic fragment of MASP-1 / 3) can displace MASP-1 and MASP-2 from pattern recognition molecules.In a mouse model (animals expressing human MBL), anti-MBL antibodies reduced the size of myocardial IRI (Pavlov 2015). The protective effect of lectin pathway inhibition was also demonstrated in a renal IRI model in pigs (Castellano 2010). Furthermore, targeting MASP-2 in mice similarly mediated protection against postischemic brain injury (Orsini 2016). In line with this preclinical finding, MBL-deficient patients after ischemic stroke also showed smaller infarct size and better functional outcomes (Osthoff 2011). In summary, the above results strongly suggest that inhibition of the lectin pathway, and in particular inhibition of the MASP-2 enzyme, can prevent or alleviate IRI in different organ systems in various animal models as well as in human patients.

[0012] Traumatic and septic tissue injury also lead to strong complement activation. Complement overactivation and the "protease storm" during sepsis fuel a vicious hyperinflammatory cycle leading to multiple organ failure. Complement inhibition may be beneficial (Liu 2007), and C1-inhibitor is being tested in trauma (Igonin 2012, van Erp 2021). C1-inhibitor is a major inhibitor of the classical and lectin pathways and also inhibits plasma kallikrein. Plasma kallikrein and MASP-1 are responsible for the release of the vasoactive peptide bradykinin from high molecular weight kininogen (Dobo 2011).

[0013] Artificial materials used in modern medicine, such as polymer plastics, metal alloys, and nanoparticles (contrast agents and drug carriers, especially liposomes and lipid nanoparticle vaccines), can activate the complement system, resulting in an allergic-like reaction called CARPA (Complement Activation-Associated Pseudoallergy) (Szebeni 2005, Dezsi 2022). CARPA is independent of IgE, but its mechanism is not yet fully understood. As pattern recognition molecules may recognize artificial surfaces, inhibition of the lectin pathway should be a therapeutic option.

[0014] Hemodialysis is an essential treatment for patients with various kidney diseases or end-stage renal disease (ESRD). During hemodialysis, the patient's blood comes into intensive contact with the artificial surfaces of the hemodialysis filter unit. This contact can cause complement activation, exacerbate inflammation, and contribute to cardiovascular disease (Ekdahl 2017). The introduction of synthetic polymers in hemodialysis has reduced the level of inflammation compared to cellulose-based filters, but unwanted complement activation remains a problem (Ekdahl 2011). The application of complement inhibitors during hemodialysis can alleviate inflammatory symptoms and improve patients' quality of life.

[0015] Pathological complement activation is a critical factor in many renal diseases. The kidneys are particularly vulnerable to complement-mediated attack due to their unique anatomical and functional characteristics. In cases of C3 glomerulopathy (C3G), uncontrolled complement activation leads to C3 deposition in the glomeruli without immunoglobulin deposition (Fakhouri 2010, Pickering 2013). C3G has two major subgroups: dense deposit disease (DDD) and C3 glomerulonephritis (C3GN). In cases of membranoproliferative glomerulonephritis, classical pathway activation contributes to C3 deposition, as immunoglobulins and C1q are also deposited in the kidney. In cases of IgA nephropathy, polymeric IgA1 triggers activation of the lectin and alternative pathways.

[0016] Primary membranous nephropathy (pMN) is the most common cause of nephrotic syndrome in nondiabetic Caucasian adults over the age of 40. It has an estimated incidence of 8–9 cases per million. Recently, activation of the lectin pathway has been shown to play a key role in the pathogenesis of pMN. In pMN, anti-PLA2R1 antibodies induce proteolysis of two essential podocyte proteins, synaptopodin and NEPH1, resulting in perturbation of the podocyte cytoskeleton. Anti-PLA2R1 IgG4 directly binds to MBL in a glycosylation-dependent manner. Increased levels of galactose-deficient IgG4 were identified in a cohort of pMN patients, which correlated with anti-PLA2R1 titers and podocyte damage induced by patient sera. Abnormally glycosylated IgG4-induced lectin pathway activation is abolished by MASP-1- and MASP-2-specific inhibitors. This suggested that blocking the lectin pathway is a novel and effective therapeutic option for the treatment of pMN (Haddad 2021).

[0017] Atypical hemolytic uremic syndrome (aHUS) is a complement-mediated disorder manifesting as microangiopathic hemolytic anemia, thrombocytopenia, vascular damage with thrombosis, and organ damage, typically in the kidney (Noris 2009). The complement system attacks the renal endothelium, promoting the formation of microthrombi in the renal microvasculature. The development of aHUS is associated with uncontrolled complement activation due to mutations in factor H (the master regulator of the alternative pathway) (Neumann 2003) or autoantibodies against factor H (Hofer 2014).

[0018] Hemolytic uremic syndrome (HUS) is also caused by bacterial infection. Bacterial toxins (e.g., Shiga toxin) impair the regulation of the complement cascade, resulting in uncontrolled activation of the complement system (Conway 2015). Inhibition of the lectin pathway of complement activation provided protection against HUS in a mouse model of HUS (Ozaki 2016).

[0019] Besides the kidney, another organ highly vulnerable to pathological complement activation is the eye. Age-related macular degeneration (AMD) is a chronic inflammatory disease of the retina and the leading cause of irreversible visual field loss in developed countries (Geerlings 2017). It is estimated to affect a large number of people worldwide (approximately 100 million cases of AMD). In the central retina of AMD patients, immune deposits called drusen accumulate beneath the retinal pigment epithelium. Drusen (containing activated complement components) disrupt the transport of oxygen and nutrients to photoreceptors, facilitating their degradation. The application of complement inhibitors in the treatment of AMD is currently undergoing clinical trials (Fritsche 2016).

[0020] Improvement of lectin pathway activation is also involved in the pathogenesis of rheumatoid arthritis (Ammiztboll 2012) and juvenile idiopathic arthritis (Petri 2015). Rheumatoid arthritis is a highly complex disorder, the pathological mechanisms of which remain unclear. The proteolytic activity of MASP-1 and MASP-2 contributes to the progression of this disease (Holers 2018).

[0021] Excessive activity of the complement system also plays a role in the development and maintenance of various neurodegenerative diseases (e.g., Alzheimer's disease, Huntington's disease, Parkinson's disease, and multiple sclerosis) (Tichaczek-Goska 2012; Ingram 2009). The complement system is responsible for synapse elimination during normal postnatal brain development. If this process is pathologically regulated during adulthood, it can lead to the development of neurodegenerative diseases (Presumey 2017). Pathological activation of the lectin pathway in the central nervous system similarly contributes to the development of schizophrenia (Mayilyan 2006).

[0022] Paroxysmal nocturnal hemoglobinuria (PNH) is a rare complement-related disorder (Hill 2017). Acquired somatic mutations in genes responsible for membrane anchor synthesis result in deficiency of two membrane-associated regulator proteins: DAF (CD55) and CD59. In PNH patients, red blood cells become more susceptible to (bystander) complement attack, leading to their lysis (intravascular hemolysis) and contributing to thrombotic complications. Complement inhibition is an effective strategy for treating PNH (Gavriilaki 2022).

[0023] Inhibition of the lectin pathway of complement activation also mediates protection against graft rejection after organ transplantation (Fildes 2008; Ibernon 2014).

[0024] Uncontrolled and excessive complement activation can lead to severe complications during viral infections. While the complement system contributes to the elimination of viruses and virus-carrying cells, overactivation of the complement system can trigger harmful inflammatory responses. One example is COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The primary cause of death in COVID-19 is severe respiratory failure. Pathological activation of the lectin pathway leads to arterial thrombosis and severe endothelial damage in lung tissue (Magro 2020). The lectin pathway plays a key role in this process (Goetz 2022). The coronavirus spike (S) protein and nucleocapsid (N) protein have been shown to activate the lectin pathway. The N protein has been reported to directly activate MASP-2 (Ali 2021). Heavy MASP-2 deposits have been detected in the lung and kidney tissues of affected patients (Niederreiter 2022). Inhibition of MASP-2 may be beneficial in coronavirus infections through the reduction of inflammation, epithelial damage, and thrombosis (Rambaldi 2020, Flude 2021).

[0025] Because the complement system is potentially harmful, it is tightly regulated in the blood. Both fluid-phase and membrane-anchored inhibitors ensure that the complement system does not harm self-cells. The primary inhibitor of the early proteases of the classical and lectin pathways is C1-inhibitor (Davis 2010). C1-inhibitor is a serpin (serine protease inhibitor) that acts as a pseudosubstrate and forms an irreversible covalent complex with the protease. C1-inhibitor inhibits C1r, C1s, MASP-1, and MASP-2. MASP-3 is not inhibited by C1-inhibitor, and there are no known physiological inhibitors of this protease. C1-inhibitor has anti-inflammatory properties but is not specific; it inhibits several pathways simultaneously. In addition to the complement system, C1-inhibitor inhibits the coagulation, contact, and fibrinolytic systems. While beneficial in some cases, it can cause complete immunosuppression. In many cases, pathway-specific inhibition is preferable because, while the disease-causing pathway is blocked, other pathways can maintain their protective function against infection. Preventing lectin pathway activation can prevent the development of certain diseases without interfering with the protective functions of the classical and alternative pathways. Other serpins (antithrombin) are also effective inhibitors of the lectin pathway in the presence of heparin (Parej 2013). Heparin itself can attenuate lectin pathway activation to some extent. α2-macroglobulin has been suggested to inhibit the lectin pathway, but this topic remains controversial. The standard blood-derived inhibitor TFPI (tissue factor pathway inhibitor) is a very weak inhibitor of MASP-2 (Keizer 2015). Given its low serum concentration (2.5 nM), it has negligible effect on lectin pathway activation in physiological settings.

[0026] As mentioned above, inhibition of unwanted and inappropriate complement activation would have therapeutic benefit in many clinical situations. Trypsin-like complement proteases are effectively inhibited by small organic molecules (e.g., benzamidine, NPGB, FUT-175). However, these compounds are not specific enough; they inhibit all trypsin-like proteases (thrombin, plasmin, and kallikrein) in other cascade systems in the blood. In many cases, pathway-specific inhibition is required to treat certain diseases without serious side effects. Peptide and protein inhibitors create multiple contacts with the protease, ensuring specific and effective inhibition. Inhibition of the lectin pathway occurs via inhibition of either MASP-1 or MASP-2. Due to its low serum concentration, MASP-2 is an ideal target for lectin pathway inhibition.

[0027] International Patent Application WO2010 / 136831 discloses oligopeptides that are inhibitors of MASP enzymes (selectively inhibiting the lectin pathway). Some of them were selective inhibitors of MASP-2 over MASP-1 enzymes, while some of them were not selective between MASP-1 and MASP-2. The oligopeptides described in the prior art were of plant origin; i.e., they were designed by phage display from the 14-amino acid long sunflower trypsin inhibitor (SFTI) and named SFTI-based MASP inhibitors (SFMI) (Kocsis 2010).

[0028] International Patent Application WO2012 / 007777 discloses proteins with certain MASP inhibitory sequences. These sequences were designed by phage display using the inhibitory loop of the desert locust (S. gregaria) chymotrypsin inhibitor (SGCI). The MASP inhibitors described in the prior art and named SGCI-based MASP inhibitors (SGMIs) are insect-derived and selective for MASP-1 or MASP-2 (Heja 2012b).

[0029] International patent application WO2011 / 047346 discloses MASP-2 inhibitors for the treatment of complement-mediated blood coagulation disorders.

[0030] Inhibitory oligopeptides are often inserted into proteins, i.e., host proteins, to maintain the functional structure of the peptide and prevent degradation by proteases or other factors. A commonly used choice of host protein of this type is a protease inhibitor called a Kunitz domain type protein or short Kunitz domain. Kunitz domain type proteins are widely used for this purpose because they are stable and easy to produce. Such modified Kunitz domains are useful biopharmaceuticals that act as specific protease inhibitors. US 5,994,125 A discloses Kunitz domain type proteins that inhibit the serine protease human plasma kallikrein. US 5,994,125 A describes the definition, characteristics, and specifications of Kunitz domains and is therefore incorporated herein by reference in its entirety.

[0031] International Patent Application WO2018 / 127719 discloses human protein-based compounds that are effective and selective inhibitors of the human MASP-2 enzyme. Unlike International Patent Applications WO2010 / 136831 and WO2012 / 007777 (both of which disclose non-human peptide or protein-based MASP-2 inhibitor compounds), the human protein-based compounds of WO2018 / 127719 should be expected to pose a significantly lower risk of immunogenicity in humans.

[0032] The protein according to the invention disclosed in WO2018 / 127719 is based on a Kunitz-type scaffold of the second domain (residues 121-178) of the human tissue factor pathway inhibitor-1 protein (TFPI-1; UniProt ID P10646), and is hereinafter referred to as TFPI-D2 (SEQ ID NO: 116).

[0033] The general Kunitz domain sequence (SEQ ID NO: 1) defined in patent application US5994125A is as follows: [ka]

[0034] As disclosed in WO2018 / 127719, the bolded segments (xCxxxxx) within the generic Kunitz domain sequence indicate positions that are replaced by a specific amino acid sequence, resulting in a protein that inhibits the human MASP-2 enzyme. The one-residue-long Kunitz domain segment GxCxxxxx in the Kunitz domain forms a disulfide-stabilized surface loop. This (the so-called canonical inhibitory loop) occupies the substrate-binding groove of MASP-2, as shown below.

[0035] The Kunitz domain family belongs to a large group of substrate-like reversible protease inhibitors (with at least 18 independently evolved families). Each family has a distinct scaffold, but they all share essentially the same surface inhibitory loop in their canonical conformation that blocks the substrate-binding groove of the enzyme.

[0036] The residue positions of substrate-like canonical inhibitory loops are designated by the nomenclature of Schechter & Berger 1967 (originally introducing peptide substrates). A fugitive peptide bond is formed between the carbonyl group of the P1 residue and the amino group of the P1' residue. The canonical inhibitory loop is generally located within a P4-P4' segment, which is eight residues long. In the case of the general Kunitz domain sequence (SEQ ID NO: 1), the P4-P4' segment corresponds to GxCxxxxx (see Table 100), where the highly conserved x12 glycine (Kunitz numbering in SEQ ID NO: 1) occupies the P4 position and the highly conserved x14 cysteine ​​(Kunitz numbering in SEQ ID NO: 1) occupies the P2 position. Both of these conserved Kunitz domain residues are important for forming and stabilizing the canonical conformation of the inhibitory loop.

[0037] [Table 100] Numbering concept of P4-P4' segments in Kunitz domains TIFF2025534392000003.tif39153 *:x denotes a variable position.

[0038] Among the inhibitors disclosed in WO2018 / 127719, the most potent human MASP-2 inhibitory compound (SEQ ID NO: 11 in WO2018 / 127719) binds human MASP-2 with an inhibitory binding constant (K I ) value of 2 nM, but hardly inhibited rat MASP-2 (K I The inhibitor has the canonical inhibitory loop sequence of GPCRAVKR (K value 640 nM; making it unusable in rat animal models). The other inhibitor with the inhibitory loop sequence of GPCRAVKR of SEQ ID NO: 14 disclosed in WO2018 / 127719 is only an approximately 4-fold weaker inhibitor of human MASP-2, with a K I It has a K value of 7.9 nM, but is an equally good inhibitor of rat MASP-2 (K Ivalue 7.2nM) Summary of the Invention [Problem to be solved by the invention]

[0039] Although the nanomolar inhibitory binding constants of the two inhibitors mentioned above are generally considered to represent high affinity, they are still not high enough for all types of drug development purposes.

[0040] Therefore, the goal of the present invention is to develop compounds with significantly higher binding affinities and, therefore, lead molecules with significantly higher potency. To achieve this goal, a structure-based directed evolution campaign was carried out starting from the above inhibitor (shown as SEQ ID NO: 14 in WO2018 / 127719; herein referred to as EVO2 (SEQ ID NO: 2)). These efforts indeed led to the development of a set of amino acid residues at specific positions outside the canonical inhibitory loop, which dramatically increased the affinity and inhibitory potency of the previously developed MASP-2 inhibitors. That is, the essence of the present invention lies not in the canonical inhibitory loop, but in the host protein sequence outside the canonical inhibitory loop. [Means for solving the problem]

[0041] The present invention relates, in certain respects, to modified Kunitz domain proteins having a general amino acid sequence defined within the general sequence of SEQ ID NO::115.

[0042] The present invention is based on the surprising finding that two Kunitz domain residues at precisely defined Kunitz domain positions can synergistically enhance MASP-2 inhibitory potency compared to compounds disclosed in WO 2018 / 127719. Furthermore, positions in the Kunitz domain have been identified where certain amino acids further enhance potency.

[0043] One of the two positions mentioned above is non-conserved position 17 according to the numbering of SEQ ID NO::1 (i.e., general Kunitz sequence numbering), which occupies the P2' position of the P4-P4' segment (see Table 100 above). This position corresponds to the general formula Ih: GX1CRX2X3X4X5 In this X3 position, the general formula Ih takes into account the amino acid residues V, A, I, L, M, D, H and S.

[0044] In another aspect of the present invention, different but overlapping sets of amino acid residues are found at position 17 to be favorable for human MASP-2 binding. Based on higher than expected average 5% codon-normalized frequencies in 290 human MASP-2-binding clones, the predominant amino acid types were found to be L (14%), I (14%), F (13%), Y (11%), A (10%), M (7%), V (6%), and H (6%), as shown in Table 9. Of these eight amino acid types, L, I, F, Y, and A are the most favorable.

[0045] On the one hand, the present invention restricts the original set described in WO2018 / 127719 to A, I, and L (i.e., excluding V, M, D, H, and S). On the other hand, the present invention expands this set by two amino acids, namely F and Y. As a result, the set with five amino acids is clearly hydrophobic. For the purposes of the present invention, this modified amino acid set is defined as the "17-set" (meaning that at position 17 according to the numbering of SEQ ID NO: 1, the following amino acids can be present: A, I, L, F, or Y).

[0046] Therefore, due to the modified X3 amino acid set (compared to WO2018 / 127719), i.e. the new (17-set), for the purposes of the present invention, the present invention defines the modified general formula Ih-mod as follows: GX1CX 1V X2X3X4X5(Ih-mod) (where, X1 is F, Y, L, P, Q, M, V, W, A, or T; X 1V is R or K; X2 is A, G, S, or T; X3 is A, I, L, F, or Y; X4 is K, I, Q, R, H, S, F, M, N, L, or V; and X5 is R, V, I, K, M, Q, E, F, L, N, Y, D, S, H; (Here, the possible amino acids at the X3 position form a 17-set.) As detailed below, the amino acid sequence of the general formula Ih-mod is found within the general Kunitz domain sequence (starting at position 12 and ending at position 19 of SEQ ID NO: 1).

[0047] The other position is non-conserved position 34 according to the numbering of SEQ ID NO: 1 (i.e., general Kunitz sequence numbering). This position is not contiguous with the P4-P4' segment and is therefore referred to as a non-active position.

[0048] As shown in Table 3, when position 17 contains only L or V, human MASP-2 prefers the following six amino acid types at position 34: Y (17%), I (14%), S (11%), F (10%), L (8%), and H (7%); whereas, as shown in Table 9, when position 17 can contain any of 20 amino acids, human MASP-2 prefers the following five amino acid types at position 34: Y (18%), I (17%), F (14%), G (6%), and V (6%). The combined set contains the following eight preferred amino acid types at position 34: Y, I, F, G, V, S, L, and H. Six residues from this set, namely, Y, I, F, G, V, and S, are most preferred at position 34. This set of amino acids is significantly hydrophobic, similar to the 17-set amino acids described above, except for S34 (which, just like Y34, has a hydroxyl group, suggesting a complex-stabilizing ability of the hydroxyl group). For the purposes of the present invention, this set of amino acids is defined as the "34-set" (meaning that the following amino acids can be present at position 34 according to the numbering of SEQ ID NO: 1: Y, I, F, G, V, and S).

[0049] Surprisingly, the inventors found that when any of the tested 17-set and 34-set combinations from the 30 pairs were introduced into the Kunitz domain-based proteins disclosed in WO2018 / 127719, they increased the MASP-2 binding affinity and lectin pathway inhibitory ability of these compounds by 40-fold or more.

[0050] Therefore, the present invention provides a method for the production of a medicament comprising the amino acid sequence of SEQ ID NO: 115, wherein the variable positions in the amino acid sequence of SEQ ID NO: 115 are restricted as follows: x1 to x4, x58, x57, and x56 are variable or absent; x6~x11, x13, x15~x20, x24~x29, x31~x32, x34, x39, x41~x42, x44, x46~x50, x52~x54 are variable; x21 is F, Y, or W; x22 is Y or F; x23 is Y or F; x35 is Y or W; x36 is G or S; x40 is G or A; x43 is N or G; and x45 is F or Y), said protein comprising: i) General formula Ih-mod starting at position 12 and ending at position 19 of SEQ ID NO: 115: GX1CX 1V X2X3X4X5(Ih-mod) (where, X1 is any of F, Y, L, P, Q, M, V, W, A, or T; X 1V is R or K; X2 is either A, G, S, or T; X3 is any amino acid of the 17-set (wherein the 17-set comprises A, I, L, F, and Y); X4 is either K, I, Q, R, H, S, F, M, N, L, or V; and X5 is R, V, I, K, M, Q, E, F, L, N, Y, D, S, or H) having an amino acid sequence segment of ii) at position 34 of the amino acid sequence of SEQ ID NO: 115, containing a 34-set of amino acids (wherein the 34-set comprises Y, I, F, G, V, and S); and salts, esters, and pharmaceutically acceptable prodrugs of said protein.

[0051] According to a preferred embodiment, the protein, its salts, esters, and pharmaceutically acceptable prodrugs are I It is a human MASP-2 inhibitor with a potency of less than 100 nM.

[0052] According to a preferred embodiment, the protein comprises an amino acid sequence having at least 70%, or at least 80%, or at least 90%, or at least 95% similarity, more preferably at least 98% similarity, even more preferably at least 70%, or at least 80%, or at least 90%, or at least 95% identity, and most preferably 98% identity to the amino acid sequence of SEQ ID NO: 116, with the proviso that i) the amino acid segment starting at position 12 and ending at position 19 has a sequence defined by the general formula Ih-mod, and ii) at position 34 of the amino acid sequence of SEQ ID NO: 115, an amino acid selected from the 34-set is contained.

[0053] According to a preferred embodiment, the protein comprises an amino acid sequence, wherein the amino acid pairs from the 17-set and the 34-set are selected from the group consisting of A / Y, A / I, A / F, A / G, A / V, A / S, I / Y, I / I, I / F, I / G, I / V, I / S, L / Y, L / I, L / F, L / G, L / V, L / S, F / Y, F / I, F / F, F / G, F / V, F / S, Y / Y, Y / I, Y / F, Y / G, Y / V, Y / S (in x17 / x34 format).

[0054] According to a more preferred embodiment, the amino acid pairs from the 17-set and 34-set are selected from the group consisting of A / Y, A / I, A / F, A / V, I / Y, I / I, I / F, I / G, I / V, I / S, L / Y, L / I, L / F, L / G, L / V, L / S, F / I, F / G, F / V, F / S, Y / Y, Y / I, Y / G, Y / V, Y / S (in x17 / x34 format).

[0055] According to a further preferred embodiment, the protein comprises an amino acid sequence, wherein at position 9 of the amino acid sequence of SEQ ID NO: 115, the protein is any of a 9-set of amino acids, wherein the 9-set consists of N and E.

[0056] According to a further preferred embodiment, the protein comprises an amino acid sequence, wherein at position 39 of the amino acid sequence of SEQ ID NO: 115, the protein is any of the 39-set of amino acids, wherein the 39-set consists of F and L.

[0057] According to a further preferred embodiment, the protein comprises an amino acid sequence, wherein at position 46 of the amino acid sequence of SEQ ID NO: 115, the protein is any of a 46-set of amino acids, wherein the 46-set consists of V and E.

[0058] According to another preferred embodiment, the protein is selected from proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:55, and SEQ ID NO:56.

[0059] According to a further preferred embodiment, the protein comprises an amino acid sequence having at least 70%, or at least 80%, or at least 90%, or at least 95% similarity, more preferably at least 98% similarity, even more preferably at least 70%, or at least 80%, or at least 90%, or at least 95% identity, and most preferably 98% identity, or is completely identical to any of the amino acid sequences set out in SEQ ID NO: 3 to SEQ ID NO: 22 and SEQ ID NO: 24 to SEQ ID NO: 32, with the proviso that the amino acid segment starting at position 12 and ending at position 19 has a sequence defined by the general formula Ih-mod and contains an amino acid selected from the 34-set at position 34 of the amino acid sequence of SEQ ID NO: 115.

[0060] According to another preferred embodiment, the protein is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32. According to an even more preferred embodiment, the protein is selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, with the proviso that the amino acid segment beginning at position 12 and ending at position 19 has a sequence defined by the general formula Ih-mod and comprises an amino acid sequence containing an amino acid selected from the 34-set at position 34 of the amino acid sequence of SEQ ID NO: 115.

[0061] According to another embodiment of the present invention, the protein is in the form of a fusion protein, the fusion protein comprising: i) SEQ ID NO: 115, wherein the variable positions in the amino acid sequence of SEQ ID NO: 115 are restricted as follows: x1 to x4, x58, x57, and x56 are variable or absent; x6 to x11, x13, x15 to x20, x24 to x29, x31 to x32, x34, x39, x41 to x42, x44, x46 to x50, and x52 to x54 are variable; x21 is F, Y, or W; x22 is Y or F; x23 is Y or F; x35 is Y or W; x36 is G or S; x40 is G or A; x43 is N or G; and x45 is F or Y; GX1CX 1V X2X3X4X5(Ih-mod) (wherein X1 is F, Y, L, P, Q, M, V, W, A, or T; and X 1V is R or K, X2 is any of A, G, S, or T, X3 is any amino acid from a 17-set (wherein the 17-set comprises A, I, L, F, and Y), X4 is any of K, I, Q, R, H, S, F, M, N, L, or V, and X5 is any of R, V, I, K, M, Q, E, F, L, N, Y, D, S, or H); and b) an amino acid sequence of SEQ ID NO: 115, at position 34 of the amino acid sequence containing an amino acid selected from a 34-set (wherein the 34-set comprises Y, I, F, G, V, and S); and ii) an antibody Fc-domain, preferably a human antibody Fc-domain The compound comprises:

[0062] According to a preferred embodiment, the fusion protein comprises an amino acid sequence that has at least 70%, or at least 80%, or at least 90%, or at least 95% similarity, more preferably at least 98% similarity, even more preferably at least 70%, or at least 80%, or at least 90%, or at least 95% identity, and most preferably 98% identity or is completely identical to SEQ ID NO: 114.

[0063] The present invention also relates to a pharmaceutical product containing at least one protein of the present invention, a pharmaceutically acceptable salt, a pharmaceutically acceptable ester, or a pharmaceutically acceptable prodrug thereof, and at least one excipient. The at least one protein is preferably selected from proteins defined by any of the amino acid sequences of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56; more preferably, the at least one protein is selected from proteins defined by any of the amino acid sequences of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56; NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: selected from proteins comprising any of 32 amino acid sequences;Most preferably, the at least one protein is selected from proteins comprising any of the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26;

[0064] The additive is preferably a matrix that ensures a controlled release of the active ingredient.

[0065] The pharmaceutical preparation according to the present invention is preferably in the form of drops, tablets, powders, granules, suppositories, injections, syrups, inhalants, or nasal delivery agents.

[0066] A nucleic acid encoding any of the proteins of the present invention as defined above.

[0067] A vector comprising the nucleic acid.

[0068] A kit comprising at least one protein of the invention, a salt or ester thereof, and instructions for use or a reference to such instructions.

[0069] A method for screening compounds that potentially inhibit MASP-2 enzyme, preferably human MASP-2 enzyme: in the process, i) a labeled form of the protein according to the present invention, its salt or ester is added to a solution containing the MASP-2 enzyme, preferably human MASP-2 enzyme, then ii) a solution containing one or more test compounds is added to the solution, and iii) the amount of released labeled protein is measured.

[0070] Use of the protein according to the present invention, its salt, ester or prodrug for inhibiting the MASP-2 enzyme, preferably the human MASP-2 enzyme.

[0071] Use of the protein according to the present invention, a pharmaceutically acceptable salt, a pharmaceutically acceptable ester, or a pharmaceutically acceptable prodrug thereof in the manufacture of a medicament suitable for the treatment or prevention of a disease that is treated by inhibiting the complement system.

[0072] The disease is preferably selected from the following list: (1) ischemia-reperfusion (IR) injuries, including myocardial infarction (e.g., treated by percutaneous coronary intervention or thrombolysis), coronary artery bypass surgery, graft IR injury in organ transplants, gastrointestinal IR injury, renal IR injury, post-ischemic encephalopathy, stroke, and disorders occurring after thrombosis affecting any area of ​​the body (especially those associated with recanalization after arterial occlusion due to thrombosis or other occlusive disease); (2) autoimmune nephritis (including dense deposit disease and C3 glomerulonephritis), IgA nephropathy, membranous nephropathy, rheumatoid arthritis (RA), juvenile idiopathic arthritis, age-related macular degeneration, systemic lupus erythematosus (SLE), and atypical hemolytic uremic syndrome (aHUS). Inflammatory and autoimmune conditions involving excessive activation of the complement system, including thrombotic microangiopathy (TMA), post-infectious hemolytic uremic syndrome (HUS), pseudoallergy resulting from complement activation (CARPA), paroxysmal nocturnal hemoglobinuria (PNH), multiple trauma, and graft rejection after organ transplantation; (3) neurodegenerative diseases, preferably Alzheimer's disease, Huntington's disease, Parkinson's disease, multiple sclerosis, and age-related macular degeneration; (4) complement hyperactivation due to infection with viruses, such as COVID-19 (SARS-CoV-2), acute respiratory distress syndrome (ARDS), and complement-related microangiopathy and thrombosis due to severe COVID-19 infection.

[0073] A method for isolating human MASP-2 enzyme: during the process, i) a carrier containing one or more immobilized proteins of the present invention, or their pharmaceutically acceptable salts or esters, is contacted with a solution containing the human MASP-2 enzyme, and ii) the preparation is washed. [Brief explanation of the drawings]

[0074] [Figure 1] 1 is a schematic representation of the phage display method used to evolve inhibitors of the present invention. [Figure 2] 1 shows the DNA and amino acid sequences of the fusion gene created for display of TFPI-D2 on the surface of M13 bacteriophage. [Figure 3] A sequence logo diagram is shown representing the set of unique sequences obtained in the first stage of evolution by selecting the first library for human or rat MASP-2 enzyme. [Figure 4] A sequence logo diagram showing the set of unique sequences obtained in the second stage of evolution by selecting a second library for the human or rat MASP-2 enzyme is shown. [Figure 5] A sequence logo diagram representing the set of unique sequences obtained in the third stage of evolution by selecting the third library for human or rat MASP-2 enzyme is shown, with additional sequence logos corresponding to all human MASP-2 binding clones, regardless of whether human or rat MASP-2 was selected. [Figure 6] The cumulative side chain volume distribution of the x17 / x34 residue pair is shown. [Figure 7] 1 shows the vector map of the pS100A4-EVO24 bacterial expression plasmid. [Figure 8] 1 shows the vector map of the pEW-EVO24L bacterial expression plasmid. [Figure 9] The DNA and protein sequences of the EVO24L chimeric protein are shown. [Figure 10] Figure 1 shows the pharmacodynamic effects of selected compounds in rats. DETAILED DESCRIPTION OF THE INVENTION

[0075] Inhibition of the complement system (including the lectin system) would be an efficient tool in the fight against human diseases that arise as a result of abnormal activity of the complement system.

[0076] Currently known selective lectin pathway blocker standard inhibitors have plant-derived SFTI peptide structures (see WO2010 / 136831, in which SFTI-based SFMIs and inhibitors are described), or insect-derived pacifastin protein structures (see WO2012 / 007777, in which pacifastin-based SGMI inhibitors are described), or human Kunitz domain scaffolds (see WO2018 / 127719). Although the most recently mentioned TFMI inhibitors clearly pose a lower risk of immunogenicity in human hosts than previous inhibitors with non-human scaffolds, it is highly likely that their efficacy can be significantly improved by a limited number of amino acid substitutions, including scaffold positions different from those occupied by residues of the sequence of general formula Ih described in WO2018 / 127719. The inventors pursued and achieved this goal by identifying amino acid substitutions that increase the MASP-2 binding strength (decreasing the KD) of the corresponding compounds and enhance their lectin pathway blocking potency.

[0077] The inventors have surprisingly found that Kunitz domain protein compounds having a sequence of the modified general formula Ih-mod, optionally in combination with the 34-set, further optionally with the 9-set, 39-set and / or 46-set, are suitable for the purposes of the present invention, i.e. they are more efficient inhibitors of the human MASP-2 enzyme than those described in WO2018 / 127719. As used herein, a Kunitz domain protein has the general Kunitz domain sequence (SEQ ID NO: 1), as defined in US5994125A: xxxxCxxxxxxGxCxxxxxxXXXxxxxxxCxxFxXXGCxXxxXxXxxxxxCxxxCxxx (where, x1 to x4, x58, x57, and x56 are variable or absent; x6~x11, x13, x15~x20, x24~x29, x31~x32, x34, x39, x41~x42, x44, x46~x50, x52~x54 are variable; X21 = Phe, Tyr, Trp; X22 = Tyr or Phe; X23 = Tyr or Phe; X35 = Tyr or Trp; X36 = Gly or Ser; X40 = Gly or Ala; X43 = Asn or Gly; and X45 = Phe or Tyr) It has.

[0078] The protein of the present invention is defined via the amino acid segment of SEQ ID NO: 115 with certain restrictions.

[0079] The present invention relates to human MASP-2 inhibitors as well as to research into rat MASP-2 inhibitors. The latter research aims to determine whether any human MASP-2 inhibitors can also inhibit rat MASP-2. These bispecific inhibitors are useful for in vivo studies conducted in rats as an animal model. The research activities cannot be divided into a "human" part and a "rat" part. Nevertheless, in addition to describing the development of human MASP-2 inhibitors, the following description also includes reference to the development of rat MASP-2 inhibitors. The primary purpose of the information related to rat MASP-2 is to provide sufficient support for the present invention.

[0080] In this supplemental specification, even if a "sequence" is stated without the prefix "amino acid" or "nucleic acid," it must be understood as an amino acid sequence.

[0081] As used herein, the general formula Ih-mod, along with the 9-set, 34-set, 39-set, and 46-set, describes an amino acid sequence or set of amino acids using one-letter abbreviations for amino acid residues known to those skilled in the art. The positions of the 8-unit long P4-P4' segment of the general formula Ih-mod (see Table 100) are represented by X1 through X5 (X), in the case where the amino acid at that position is variable. 1V In the case of invariant positions, X1 to X5 are designated by a single letter abbreviation (including ). In the case of invariant positions, X2 is designated by a single letter abbreviation (e.g., G, C, or R). The possibilities at positions X1 to X5 are designated by a single letter abbreviation. For example, in the case of the general formula Ih-mod, X2 is designated as A, G, S, or T, meaning that alanine, glycine, serine, and threonine are options at position X2. The IUPAC recommendations (Nomenclature of α-amino acids, Recommendations, 1974 - Biochemistry, 14(2), 1975) were used to label the amino acid side chains in a given sequence.

[0082] The present invention relates to Kunitz domain proteins. The Kunitz family or Kunitz domain should be understood within the scope of the present invention as follows: The previously referenced US Pat. No. 5,994,125A provides a detailed description of Kunitz domains. Briefly, Kunitz domains refer to homologs of bovine pancreatic trypsin inhibitor (hereinafter, BPTI (not Kunitz soybean trypsin inhibitor)). A Kunitz domain is a protein domain having at least 51 amino acids (up to about 61 amino acids) containing at least two, preferably three disulfides. Here, all Kunitz domain residues are numbered from 1 to 58 with reference to the 58-amino acid residue mature form of BPTI (the amino acid sequence of which is disclosed as SEQ ID NO: 21 in US Pat. No. 5,994,125A). It should be noted that the full-length prepro-form of BPTI contains 100 amino acid residues, and the 58-residue mature segment corresponds to the segment 39 to 93 according to the full-length protein numbering system. It is noted here that the sequence of mature BPTI disclosed as SEQ ID NO: 2 in Table 2 of US 5,994,125A contains a Met at (mature) position 44, whereas in some published BPTI sequences an Asn is present at this position (e.g., Uniprot P00974 (see residue 79 according to full length numbering)). However, this difference does not affect the definition of a Kunitz domain from the perspective of the present invention. Thus, the first cysteine ​​residue is residue 5 and the last cysteine ​​is residue 55. For the purposes of the present invention, an amino acid sequence is considered a Kunitz domain even if it aligns to the sequence of SEQ ID NO: 1 with three or fewer mismatches. SEQ ID NO: In 1, "x" matches any amino acid, and "X" matches the type listed for position. Disulfide bonds link at least two of 5 to 55, 14 to 38, and 30 to 51. The number of disulfides can be reduced by one, but no canonical cysteines are left unpaired.When a cysteine ​​is changed in this way, a compensatory cysteine ​​is added at the appropriate position, or the matching cysteine ​​is also replaced with a non-cysteine ​​(the latter is generally preferred). For example, the Drosophila funebris male accessory gland protease inhibitor does not have a cysteine ​​at position 5, but does have a cysteine ​​at position -1 (just before position 1); presumably, this forms a disulfide with Cys55. If Cys14 and Cys18 are replaced, the requirements for Gly12, (Gly or Ser)37, and Gly36 are waived. Zero to many residues (including additional domains, including other Kunitz domains) can be attached to either end of the Kunitz domain.

[0083] The general sequence of a Kunitz domain is as follows (SEQ ID NO: 1): xxxxCxxxxxxGxCxxxxxxXXXxxxxxxCxxFxXXGCxXxxXxXxxxxxCxxxCxxx (where, x1 to x4, x58, x57, and x56 are variable or absent; x6~x11, x13, x15~x20, x24~x29, x31~x32, x34, x39, x41~x42, x44, x46~x50, x52~x54 are variable; X21 = Phe, Tyr, Trp; X22 = Ty or Phe; X23 = Tyr or Phe; X35 = Tyr or Trp; X36 = Gly or Ser; X40 = Gly or Ala; X43 = Asn or Gly; and X45 = Phe or Tyr) where "x" matches any amino acid and "X" matches the type listed for that position.

[0084] As explained, the present invention provides a method for the production of a medicament comprising the amino acid sequence of SEQ ID NO: 115, wherein the variable positions in the amino acid sequence of SEQ ID NO: 115 are restricted as follows: x1 to x4, x58, x57, and x56 are variable or absent; x6~x11, x13, x15~x20, x24~x29, x31~x32, x34, x39, x41~x42, x44, x46~x50, x52~x54 are variable; x 21 is F, Y, or W; x22 is Y or F; x23 is Y or F; x35 is Y or W; x36 is G or S; x40 is G or A; x43 is N or G; and x45 is F or Y), said protein comprising: i) The general formula starting at position 12 and ending at position 19 of SEQ ID NO: 115: GX1CX 1V X2X3X4X5(Ih-mod) (where, X1 is any of F, Y, L, P, Q, M, V, W, A, or T; X 1V is R or K; X2 is either A, G, S, or T; X3 is any amino acid of the 17-set (wherein the 17-set comprises A, I, L, F, and Y); X4 is either K, I, Q, R, H, S, F, M, N, L, or V; and X5 is R, V, I, K, M, Q, E, F, L, N, Y, D, S, or H) and ii) at position 34, containing any amino acid of the 34-set (wherein the 34-set comprises Y, I, F, G, V, and S); The present invention also relates to a salt, ester, or pharmaceutically acceptable prodrug of the protein.

[0085] The amino acid sequence of SEQ ID NO: 115 is the framework of the general Kunitz sequence (SEQ ID NO: 1), with the variable amino acids defined as follows, as originally disclosed for the Kunitz domain in US5994125: x1 to x4, x58, x57, and x56 are variable or absent; x6~x11, x13, x15~x20, x24~x29, x31~x32, x34, x39, x41~x42, x44, x46~x50, x52~x54 are variable; x21 is F, Y, or W; x22 is Y or F; x23 is Y or F; x35 is Y or W; x36 is G or S; x40 is G or A; x43 is N or G; and x45 is F or Y. When reference is made herein to a protein having the amino acid sequence of SEQ ID NO: 115, the limitations defined in this paragraph must be understood to be valid for said protein, unless expressly disclosed otherwise.

[0086] As a result, the protein of SEQ ID NO: 115 exhibits all the characteristics of a Kunitz domain, i.e., it is a Kunitz domain protein. The proteins of the present invention are defined through SEQ ID NO: 115 (wherein some variable portions of SEQ ID NO: 115 are restricted to achieve a set of proteins of the present invention). One of these constraints concerns the 8-unit-long P4-P4' segment occupying positions 12 to 19 within the protein of SEQ ID NO: 115 (see Table 100). This P4-P4' segment can be any of the amino acid sequences previously defined under the general formula Ih-mod. Position X3 in Ih-mod is position P2' of the P4-P4' segment, which is position 17 of the amino acid sequence SEQ ID NO: 115. Because this position X3 has exceptional importance in terms of the present invention compared to the other positions in the P4-P4' segment, the possible amino acids at this position (i.e., A, I, L, F, or Y) are, for practical reasons, shown throughout this specification as a 17-set.

[0087] Another restriction in SEQ ID NO: 115 relates to position 34, which is outside the P4-P4' segment towards the C-terminus. According to the present invention, the possible amino acids at this position are Y, I, F, G, V, and S. Again, for practical reasons, these amino acids are collectively designated as the 34-set throughout this specification.

[0088] The basic concept of the present invention is that when both of the above-mentioned constraints are applied to a protein having the amino acid sequence scaffold of SEQ ID NO: 115, it leads to a set of proteins that effectively inhibit human MASP-2 protein.

[0089] A protein sequence defined in this way (i.e., SEQ ID NO: 115 with the two aforementioned essential restrictions) can be a portion of a larger protein. Proteins with two or more Kunitz domains also fall within the scope of the present invention, provided that at least one of the Kunitz domains meets the above amino acid sequence criteria.

[0090] As will be apparent to those skilled in the art, the proteins of the present invention may be in the form of salts, esters, or pharmaceutically acceptable prodrugs, and these variants of the proteins also fall within the scope of the present invention.

[0091] Those skilled in the art will understand that the sequence portions corresponding to the P4-P4' segment defined by the general formula Ih-mod and the defined sets (i.e., the required 17-set and 34-set, and the optional 9-set, 39-set, and 46-set) are essential to the present invention. However, those skilled in the art will also understand that other portions of the Kunitz domain proteins of the present invention are also important for providing the molecular environment necessary for the effective spatial arrangement of the atoms of these elements. Additional functions of protein portions beyond these elements include, for example, providing the necessary solubility of the protein in formulations; carrying other molecular objects, such as labels, anchoring elements, and providing beneficial pharmacokinetic and pharmacological properties to formulations.

[0092] Those skilled in the art will understand that to ensure these functions of a protein, additional molecular elements may be required, such as further amino acid sequence development at either of the terminal portions, modified amino acids, carbohydrate moieties, special small molecules or biomolecular compounds, etc. Such types of modified proteins are also within the scope of the present invention, keeping in mind the above-mentioned essence of the present invention.

[0093] The present invention relates to Kunitz domain proteins and protein derivatives that selectively inhibit the human MASP-2 enzyme. By selective inhibition, we primarily understand selectivity with respect to MASP-1 and other proteolytic cascades in the blood, such as enzymes of the classical and alternative complement pathways and the extrinsic and intrinsic coagulation pathways. It will be clear to those skilled in the art that the protein environment of the amino acid sequence defined by SEQ ID NO: 115 will influence effective inhibition.

[0094] Furthermore, those skilled in the art will understand that the modified domain proteins of the present invention can be incorporated into other proteins, such that the Kunitz domain protein maintains its MASP-2 inhibitory and lectin pathway blocking properties in the resulting chimeric protein. With the above-described nature of the invention in mind, such chimeric proteins are also within the scope of the present invention. One such chimeric protein is EVO24L (SEQ ID NO: 114), which is provided as a non-limiting example (see E.3 in Example E below for details).

[0095] The scope of the present invention also includes proteins into which elements that ensure detectability (e.g., fluorescent groups, radioactive atoms, etc.) have been incorporated. This type of labeling is advantageous according to current state of the art in diagnostics, research practice, etc.

[0096] Furthermore, the scope of protection of the present invention includes proteins that, in addition to those defined by the amino acid sequence scaffold of SEQ ID NO: 115, contain additional amino acids, amino acid sequences, or protein domains at their N-terminus, C-terminus, or both, provided that these additional elements do not have a significant negative effect on the MASP-2 inhibitory activity of the original sequence. The purpose of such additional elements located at the termini is to facilitate immobilization, ensure the possibility of linking with other reagents, influence solubility, absorption, in vivo stability, pharmacokinetic and pharmacodynamic properties, and other properties.

[0097] The present invention also relates to salts of the protein of the present invention. For example, when the protein is administered to the human body in the context of pharmaceutical applications, pharmaceutically acceptable salts are preferred. Pharmaceutically acceptable salts are salts that do not cause undue toxicity, irritation, allergic reactions, or similar phenomena upon contact with corresponding human tissues. Non-limiting examples of acid addition salts include acetate, citrate, aspartate, benzoate, benzenesulfonate, butyrate, digluconate, hemisulfate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, methanesulfonate, oxalate, propionate, succinate, tartrate, phosphate, and glutamate. Non-limiting examples of base addition salts include salts based on alkali metals or alkaline earth metals (lithium, potassium, sodium, calcium, magnesium, aluminum), quaternary ammonium salts, and amine cations (methylamine, ethylamine, diethylamine, etc.).

[0098] The ester of the protein according to the present invention includes all esters known to those skilled in the art. In cases where the protein is applied to the human body, for example, within the framework of pharmaceutical applications, pharmaceutically acceptable esters are preferred. A pharmaceutically acceptable ester means one that does not cause unnecessary toxicity, irritation, allergic symptoms, or similar phenomena when in contact with corresponding human tissues. Methods for forming esters using surface functional groups of proteins are within the general knowledge of those skilled in the art.

[0099] In the context of the present invention, a prodrug is a compound that converts in vivo into a protein according to the present invention. The conversion occurs during enzymatic hydrolysis, for example, in blood. In the prodrug form, the compound is inactive: it cannot perform its function. For example, if any of the amino acid residues in the inhibitory loop are covalently modified with a bulky compound, the loop cannot efficiently interact with any protease, including MASP-2. If the chemical modification can be removed by a chemical reaction, for example, hydrolysis catalyzed by a host enzyme, the prodrug is converted into an active drug. Protein modifications that produce prodrugs are known to those skilled in the art (Tobin 2014, Gou 2016).

[0100] According to a preferred embodiment, the protein has a K of 100 nM or less. I The compound is a human MASP-2 inhibitor with a K value for the interaction. I If a value of 100 nM or less is measured, it is considered a human MASP-2 inhibitor within the meaning of the present invention. This corresponds to an inhibitory capacity that can provide a biologically relevant degree of MASP-2 inhibition. A person skilled in the art will recognize a protein as a human MASP-2 inhibitor only if it physically interacts with human MASP-2, thereby interfering with the binding of substrates to human MASP-2 and / or interfering with the function of the catalytic center of human MASP-2. The inventors have determined the equilibrium inhibition constant, i.e., K I The inhibitory potency is strictly defined through measurements of the K, which corresponds to the human MASP-2-inhibitor interaction. IWithin the context of the present invention, the values ​​should be understood as having been determined using a suitable enzyme inhibition kinetic evaluation method that measures the concentration of uninhibited active human MASP-2 as a function of the concentration of the applied inhibitor with at least as much reliability as that achieved by the modified version of the method of Empie and Laskowski (Empie, 1982) detailed in Szakacs 2019. By active human MASP-2 is meant the protein with UniProt ID O00187 (which has undergone proteolytic cleavage of the peptide bond after Arg-444), or a shorter but equally enzymatically active fragment of said protein. I For details of the measurement method, see Example F.2 below.

[0101] The present invention also relates to domain proteins and protein derivatives similar to the disclosed sequences of the present invention, i.e., as defined by the amino acid sequence scaffold of SEQ ID NO: 115 and similar biological activity when compared to the proteins of the present invention. Those skilled in the art will recognize that certain side chain modifications or amino acid substitutions can be made without altering the biological function of the protein in question. Such modifications are based on similarities in amino acid side chains, e.g., similarities in size, charge, hydrophobicity, hydrophilicity, etc. The aim of such changes may be to increase the stability of the protein against enzymatic degradation or to improve certain pharmacodynamic or other parameters.

[0102] The similarity between two proteins is defined as the percentage of similar or identical amino acids. To determine these percentage values, the two amino acid sequences are first aligned for optimal comparison. For example, for optimal alignment, gaps are introduced into one or both of the first and second amino acid sequences, and non-similar sequences, such as the Fc sequence portion of a fusion protein according to the present invention, are ignored for comparison purposes. Optimal alignment is measured as the best score using the GAP program in the GCG software package with a Blosum 62 scoring matrix, a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. In the present invention, the generic Kunitz domain sequence (SEQ ID NO: 1) is a good starting point for alignment. Once the amino acid sequences are aligned, the amino acid residues at corresponding amino acid positions are compared. If a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, the amino acids at that position are identical. The percentage identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap (which need to be introduced for optimal alignment of the two sequences). Importantly, in the present invention, the sequence segment of the Ih-mod segment, and optionally positions 9, 34, 39, and 46 according to the Kunitz numbering in SEQ ID NO: 1, are not counted when determining the percentage of similarity and identity. That is, only aligned amino acid pairs of the two sequences (outside the Ih-mod segment and not present at positions 9, 34, 39, and 46) are counted. For example, taking the proteins EVO215 (SEQ ID NO: 26) and TFPI-D2 (SEQ ID NO: 116) as examples, the percentage of identity is determined in the present invention as follows: In this calculation example, only positions 34 and 9 of the P4-P4' segment (i.e., Ih-mod) are defined for the sake of specificity. EVO215 (SEQ ID NO: 26): [ka] TFPI-D2 (SEQ ID NO: 116): [ka]

[0103] Although the P4-P4' segment (i.e., Ih-mod), positions 34, and 9 are defined in this example, they are not used in the identity calculation and are underlined. Thus, amino acids 58-10 of the 58 are used to select 48 positions (i.e., positions not underlined). There is one amino acid position, i.e., position 46, which has a difference (see bold text). As a result, there are identical amino acids at 47 positions out of 48 positions (100%), resulting in 97.9% amino acid sequence identity. In another preferred embodiment (where positions 9 and 46 are also defined in addition to the essential positions defined by Ih-mod and position 34), rather, position 46 is not counted, and 47 positions out of a total of 47 positions are identical, resulting in 100% sequence identity in the present invention.

[0104] One preferred Kunitz domain that has proven useful in the context of the present invention is the TFPI-D2 protein, i.e., the second domain (SEQ ID NO: 116) of human tissue factor pathway inhibitor-1 (TFPI-1; UniProt ID P10646). TFPI-D2 has proven useful when modifications corresponding to the two aforementioned constraints are applied to its amino acid sequence (i.e., the P4-P4' segment is defined as Ih-mod and the amino acid at position 34 must be one of the 34-set amino acids). Those skilled in the art will understand that, in addition to these constraints (which are essential for the purposes of the present invention), a certain degree of flexibility is allowed for the amino acid composition of the Kunitz protein, excluding the positions of these constraints, in order to preserve the functionality of the MASP-2 inhibitor. That is, some amino acids that are not part of the P4-P4' segment and are not at position 34 can be substituted by other amino acids without risking significant changes in function, stability, etc. According to a preferred embodiment of the present invention, positions 9, 39, and 46 are also defined by the present invention, without leaving room for substitution with similar amino acids. Consequently, proteins that exhibit a certain level of similarity to said portion of the TFPI-D2 protein (i.e., the P4-P4' segment and the portion falling outside position 34, and according to a preferred embodiment, the portion falling outside positions 9, 39, and 46) also fall within the scope of the present invention. Similarity in this context allows for conservative substitution of amino acid residues that have similar physicochemical properties when the protein indicated to exhibit similarity is aligned with the TFPI-D2 protein (SEQ ID NO: 116). In the present invention, this similarity is at least 70%, or at least 80%, or at least 90%, or at least 95%, and preferably at least 98%.That is, those proteins that exhibit at least 70%, or at least 80%, or at least 90%, or at least 95%, preferably at least 98%, amino acid sequence similarity with proteins comprising the two claimed limitations (i.e., the definition according to general formula Ih-mod and the 34-set definition) and optionally limitations at positions 9, 39, and 46 fall within the scope of the present invention, and said level of similarity is measured over the portion of the sequence outside the portion affected by said limitations.

[0105] A subset of similar proteins is determined by identity. In this sense, those proteins fall within the scope of the present invention and exhibit at least 70%, at least 80%, or at least 90%, or at least 95%, preferably at least 98% identity with the TFPI-D2-derived proteins of the present invention, i.e., with the TFPI-D2-derived proteins having the two restrictions at the P4-P4' segment and position 34 (optionally, also the restrictions at positions 9, 39, and 46), and beyond these positions, i.e., outside the P4-P4' segment and position 34 (optionally, the restrictions at positions 9, 39, and 46), the remaining sequence exhibits at least 70%, at least 80%, or at least 90%, or at least 95%, preferably at least 98% identity.

[0106] Proteins defined by percentage similarity or percentage identity in this way are within the scope of the present invention, even if they are part of a larger protein. Proteins having two or more Kunitz domains are also within the scope of the present invention, provided that at least one of the Kunitz domains meets the above-mentioned criteria for degree of similarity and percentage identity. It is obvious to those skilled in the art to use protein alignment algorithms to align larger proteins with the TFPI-D2-derived proteins of the present invention, taking into account the general Kunitz domain sequence (SEQ ID NO: 1).

[0107] As defined above, the 17-set and 34-set contain five and six possible amino acids, respectively. Consequently, there are 5 x 6 total, or 30, possible combinations, not counting other variable positions in the protein. These 30 combinations are as follows (in x17 / x34 format): A / Y, A / I, A / F, A / G, A / V, A / S, I / Y, I / I, I / F, I / G, I / V, I / S, L / Y, L / I, L / F, L / G, L / V, L / S, F / Y, F / I, F / F, F / G, F / V, F / S, Y / Y, Y / I, Y / F, Y / G, Y / V, Y / S. For example, the combination A / Y means that there is an A (i.e., alanine) in each protein at position 17 (i.e., X3 in the generic sequence Ih-mod) and a Y (i.e., tyrosine) at inactive position 34. The inventors found that all of the 30 pairwise combinations of the 17-set and 34-set are positively selected for binding to MASP-2 via their characteristic combined hydrophobicity and cumulative side chain size ranges favorable to MASP-2, which is why they used the 17-set and 34-set segments in proteins that proved essential.

[0108] Of the 30 possible x17 / x34 combinations as working pairs, the following 25 combinations are particularly preferred: A / Y, A / I, A / F, A / V, I / Y, I / I, I / F, I / G, I / V, I / S, L / Y, L / I, L / F, L / G, L / V, L / S, F / I, F / G, F / V, F / S, Y / Y, Y / I, Y / G, Y / V, and Y / S. These combinations are particularly preferred because their positive selection is more reliable, as inferred from their observed frequencies (listed in Table 11 and plotted in Figure 6, which clearly exceed the frequency values ​​of the corresponding cumulative size ranges in the starting library before selection). Five of the other 30 x17 / x34 combinations exhibit either very small or very large cumulative side chain sizes. For purely combinatorial reasons, the starting frequencies of these sets are inherently low in the starting library, and their active selection usually requires a higher number of selection cycles.

[0109] As mentioned above, selecting certain amino acids from the 17-set and one from the 34-set is essential to obtain a protein according to the present invention. However, during the investigation, other sites with the potential for certain amino acids were identified, the use of which can enhance the efficacy of the MASP-2 inhibitors of the present invention. For these sites, arbitrary sets were defined during the investigation. Some amino acids in these arbitrary sets are found in natural Kunitz domain proteins (e.g., as in SEQ ID NO: 116), or such amino acids are not present at these positions in natural Kunitz domain proteins.

[0110] The 9-set defines any amino acid for position 9, and this 9-set comprises N and E. This means that in a preferred embodiment of the invention, N or E is at position 9 of SEQ ID NO 200.

[0111] The 39-set defines any amino acid for position 39, and this 39-set comprises F and L. This means that in a preferred embodiment of the invention, F or L is at position 39 of SEQ ID NO 200.

[0112] The 46-set defines any amino acid for position 46, and this 46-set comprises V and E. This means that in a preferred embodiment of the invention, V or E is at position 46 of SEQ ID NO 200.

[0113] According to a more preferred embodiment, the present invention relates to proteins selected from the list below. These sequences, i.e., SEQ ID NO: 33 to SEQ ID NO: 52 and SEQ ID NO: 54 to SEQ ID NO: 56 (23 sequences), are general amino acid sequences (where x and X are defined as above in relation to the general Kunitz domain sequence of SEQ ID NO: 1). However, certain positions in these sequences have strict amino acids. These precisely defined positions are either conserved residues or residues that have been obtained as a result of research activities. Bold and underlined positions indicate positions that are essential or optional, but are defined within the meaning of the present invention. For example, in SEQ ID NO: 33, at position 9, N is shown in bold and underlined, and any 9-set is N or E, as defined above; at position 34, Y is shown in bold and underlined, and any 34-set is Y, I, F, G, or V, as defined above; at position 39, F is shown in bold and underlined, and any 39-set is F or L, as defined above; and at position 46, V is shown in bold and underlined, and any 46-set is V or E, as defined above. The generic sequence Ih-mod is GPCRALKR in SEQ ID NO: 33, and it is seen from SEQ ID NO: 33 that there is an L at position 17, and the required 17-set is A, I, L, F, or Y. Each of these sequences, SEQ ID NO: 33 to SEQ ID NO: 52 and SEQ ID NO: 54 to SEQ ID NO: 56, is a generalized version of a given protein that was developed during research activities and proved effective (see below). For each amino acid sequence, the original protein is shown in parentheses. SEQ ID NO: 33 (generalized from VO23): [ka] SEQ ID NO: 34 (generalized from EVO211: [ka] SEQ ID NO: 35 (generated from EVO23a): [ka] SEQ ID NO: 36 (generalized from EVO22a): [ka] SEQ ID NO: 37 (generalized from EVO22): [ka] SEQ ID NO: 38 (generalized from EVO214): [ka] SEQ ID NO: 39 (generalized from EVO21b): [ka] SEQ ID NO: 40 (generalized from EVO22d): [ka] SEQ ID NO: 41 (generalized from EVO25): [ka] SEQ ID NO: 42 (generalized from EVO21): [ka] SEQ ID NO: 43 (generalized from EVO21c): [ka] SEQ ID NO: 44 (generalized from EVO212): [ka] SEQ ID NO: 45 (generalized from EVO24): [ka] SEQ ID NO: 46 (generalized from EVO21d): [ka] SEQ ID NO: 47 (generalized from EVO214a): [ka] SEQ ID NO: 48 (generalized from EVO222): [ka] SEQ ID NO: 49 (generalized from EVO223): [ka] SEQ ID NO: 50 (generalized from EVO211a): [ka] SEQ ID NO: 51 (generalized from EVO221): [ka] SEQ ID NO: 52 (generalized from EVO22b): [ka] SEQ ID NO: 54 (generalized from EVO21a): [ka] SEQ ID NO: 55 (generalized from EVO2c): [ka] SEQ ID NO: 56 (generalized from EVO215): [ka]

[0114] According to a further preferred embodiment, the amino acid sequence of the protein has at least 70%, or at least 80%, or at least 90%, or at least 95% similarity, more preferably 98% similarity, even more preferably at least 70%, or at least 80%, or at least 90%, or at least 95% identity, and most preferably 98% identity, or is completely identical to any of the amino acid sequences set out in SEQ ID NO:3 to SEQ ID NO:22 and SEQ ID NO:24 to SEQ ID NO:32, with the proviso that the amino acid sequence starting at position 12 and ending at position 19 has a sequence defined by the general formula Ih-mod. The percentage of similarity and identity will be calculated within the framework of the present invention as described above.

[0115] During the course of research leading to the present invention, 29 new MASP-2 inhibitors were developed, synthesized, and tested, which are related to the TFMI-2b of the invention disclosed in WO2018 / 127719 and are hereinafter referred to as EVO2 (SEQ ID NO: 2). These proteins of the invention were partially synthesized, for example, to create highly improved MASP-2 inhibitors and lectin pathway inhibitors, and to predict sequences for activity algorithm associations with the improved MASP-2 inhibitors. The names, SEQ ID NOs (SEQ ID NOs: 3 to 22 and SEQ ID NOs: 24 to 32, in descending order), sequences, and their human lectin pathway inhibitory abilities of these 29 proteins of the invention are listed in Table 1, with the original EVO2 protein shown in the first row for reference. [Table 1] [Table 1] TIFF2025534392000030.tif21673

[0116] Of these 29 proteins of the present invention, 23 are human lectin pathway inhibitors with 47-fold to 6-fold greater potency than EVO2. The 23 inhibitors, SEQ ID NO: 3 to SEQ ID NO: 22 and SEQ ID NO: 24 to SEQ ID NO: 26, which are modified TFPI-D2 proteins, are particularly preferred embodiments of the present invention, since their increased inhibitory potency reciprocally allows for reduced applied doses, thereby reducing the burden of off-target binding-related side effects.

[0117] The present invention also relates to a protein in which the sequence determined by the scaffold of SEQ ID NO: 115 forms a fusion protein together with an antibody Fc domain.

[0118] Using genetic engineering techniques well known to those skilled in the art, two pairs of proteins or protein domains can be fused together to produce a contiguous polypeptide (a fusion protein). In such fusion proteins, the fusion proteins or protein domains can retain their original structural and functional properties, including the ability to interact with their original binding partners (be they macromolecules, small molecules, ions, or atoms). Thus, structurally, the fusion protein construct retains the properties and functions useful for peptide- or protein-based inventions and combines them with other useful functions provided by the fusion partner. For example, a fusion partner that binds to a specific cell surface molecule of a tissue can transport a peptide- or protein-based drug to that tissue during compounding, providing increased specificity and efficacy. The Fc domain of an antibody is often used as a fusion partner for protein- or peptide-based drugs for several different purposes. Depending on their type and glycosylation status, the Fc domain can bind to a variety of soluble and cell surface proteins, thereby providing specific biological functionality. For example, one such binding partner is the plasma protein C1q, which is a pattern recognition molecule of the classical complement pathway (see the "Background" section for details). Binding of C1q to surface-located Fc triggers complement activation.

[0119] Other binding partners are cell surface Fc receptor proteins that capture Fc-fusion proteins and trigger specific cellular responses. These interactions usually require an appropriately glycosylated Fc domain.

[0120] In the present invention, an IgG Fc fusion partner was used to produce a recombinant fusion protein in E. coli. As a result, the Fc domain was aglycosylated, reducing its binding ability to most Fc-binding proteins, with the exception of the neonatal Fc receptor (FcRn).

[0121] The main function of FcRn is to dramatically extend the plasma half-life of immunoglobulins and albumin. All plasma proteins, immunoglobulins and albumin, are constantly taken up by endothelial cells and various leukocytes via pinocytosis. While most proteins are rapidly degraded via the lysosomal pathway, immunoglobulins and albumin are captured by FcRn in early endosomes and targeted to the cell surface, where they are released into the plasma.

[0122] According to a preferred embodiment, the fusion protein of the present invention is a protein exhibiting at least 70%, or at least 80%, or at least 90%, or at least 95% similarity, more preferably at least 98%, and even more preferably at least 70%, or at least 80%, or at least 90%, or at least 95% identity, and most preferably 98% identity, or completely identical to SEQ ID NO: 114, i.e., EVO24L. Percentages of similarity and identity will be calculated within the framework of the present invention as described above. The protein EVO24L of the present invention has proven to be very useful, as detailed in Example F.9.2 below. Briefly, the Fc fusion partner of EVO24L dramatically prolonged the presence of EVO24L in the rat blood circulation compared to EVO24 lacking the Fc fusion segment.

[0123] The present invention also relates to a pharmaceutical product containing at least one protein of the present invention, a pharmaceutically acceptable salt, a pharmaceutically acceptable ester, or a pharmaceutically acceptable prodrug thereof, and at least one excipient. The at least one protein is preferably selected from proteins comprising any of the amino acid sequences of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56; more preferably, the protein is selected from proteins comprising any of the amino acid sequences of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56; 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: selected from proteins comprising any of 32 amino acid sequences;Most preferably, the protein is selected from proteins comprising any of the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26;

[0124] The proteins of the present invention are used in pharmaceuticals suitable for treating organisms, such as mammals, including humans, that possess MASP-2 proteins. Pharmaceuticals for humans are particularly preferred. Such pharmaceuticals contain at least one additive in addition to the protein.

[0125] The additive is necessary to achieve the appropriate biological effect. Such a formulation is, for example, a pharmaceutical product combined with a matrix that ensures the controlled release of an active agent, which is well known to those skilled in the art. Generally, the matrix that ensures the controlled release of an active agent is a polymer (e.g., polylactic acid, polyglycolide) that degrades upon entering the appropriate tissue (e.g., plasma), for example, by enzymatic or acid hydrolysis. The additive is preferably a matrix that ensures the controlled release of an active agent.

[0126] Other additives known in the art may also be used in the human pharmaceutical preparations according to the invention, such as diluents, fillers, pH adjusters, disintegration promoters, color additives, antioxidants, preservatives, isotonicity agents, etc. These additives are known in the art.

[0127] The pharmaceutical preparation according to the present invention is preferably in the form of drops, tablets, powders, granules, suppositories, injections, syrups, inhalants, or nasal delivery agents.

[0128] Preferably, the human pharmaceuticals according to the invention can enter tissues via parenteral administration (intravenous, intramuscular, subcutaneous, nasal, inhalation, etc.). In view of this, preferred pharmaceuticals are aqueous or non-aqueous solutions, dispersions, suspensions, emulsions, or solid (e.g., powdered) preparations, which are converted into one of the above-mentioned fluids immediately before use. In such fluids, suitable vehicles, carriers, diluents, or solvents are, for example, water, ethanol, different polyols (e.g., glycerol, propylene glycol, polyethylene glycol, and similar substances), carboxymethylcellulose, different (vegetable) oils, organic esters, and mixtures of all these substances.

[0129] Preferred dosage forms of the pharmaceuticals according to the present invention include, inter alia, drops, tablets, powders, suppositories, injections, syrups, inhalants, and nasal delivery. One of the preferred administration routes for proteins and peptides is nasal delivery, which bypasses the blood-brain barrier (Meredith 2015). Therefore, preferred formulations include nasal delivery systems such as cyclodextrins, inhalable solutions, and the like.

[0130] The dose to be administered depends on the disease, the patient's sex, age, and weight, and on the severity of the disease. In the case of oral administration, the preferred daily dose varies, for example, between 0.01 and 1 g of active agent, and in the case of parenteral administration (e.g., intravenously administered formulations), the preferred daily dose varies between 0.001 and 1 g. Those skilled in the art will recognize that the dose to be selected will depend greatly on the molecular weight of the given protein used.

[0131] Additionally, the pharmaceuticals may also be formulated in liposomes or microcapsules as known in the art.

[0132] Nucleic acids encoding any of the proteins of the present invention are also within the scope of the present invention. The proteins of the present invention are introduced into target tissues by state-of-the-art means using natural or modified RNA or DNA molecules encoding the proteins of the present invention. The proteins are produced through the action of a suitable transcription and translation system in the target organism. Such nucleic acids (e.g., in the form of mRNA) are delivered to cells or cell lines for the purpose of producing the proteins of the present invention. Alternatively, delivery to the mammalian, preferably human, body is carried out, for example, in the form of an mRNA vaccine. Once the amino acid sequence of the protein in question is known, one skilled in the art can determine the corresponding DNA or RNA nucleic acid sequence based on the well-known genetic code. The degeneracy of the genetic code makes it possible to adapt the DNA or RNA sequence to specific needs.

[0133] Such nucleic acids are incorporated into vectors for gene transfer. Based on the sequence information of the nucleic acids, those skilled in the art will recognize how to design, synthesize, and use such transfection vectors.

[0134] The present invention also relates to kits comprising at least one protein of the present invention, a salt or ester thereof, and instructions for use or reference to such instructions. These kits are used to measure and / or localize the MASP-2 enzyme. Such uses extend to competitive and non-competitive tests, radioimmunoassays, bioluminescent and chemiluminescent tests, fluorescent tests, immune-linked assays (e.g., ELISA), immunocytochemical assays, etc.

[0135] According to the present invention, these kits are particularly preferred and are suitable, for example, for testing potential inhibitors of the human MASP-2 enzyme in competitive binding assays. Such kits can be used to measure the ability of potential inhibitors to displace the proteins of the present invention from the MASP-2 enzyme. To detect them, the proteins of the present invention must be labeled in some way (e.g., by incorporating fluorescent groups or radioactive atoms, or other labeling means known in the art).

[0136] Kits according to the present invention may include other solutions, means, and raw materials needed to prepare the solutions and reagents, and instructions, which are understood to include simple reference to an online manual.

[0137] The present invention further relates to a method for screening compounds that potentially inhibit MASP-2 enzyme, preferably human MASP-2 enzyme, in which the method comprises the steps of: i) adding a protein according to the present invention, its salt or ester (labeled form) to a solution containing the MASP-2 enzyme, preferably human MASP-2 enzyme; then ii) adding a solution containing one or more test compounds to the solution; and iii) measuring the amount of released labeled protein. In such a screening method, the protein of the present invention is used in labeled (fluorescent, radioactive, etc.) form to ensure detectability at a later time point. A preparation containing such a protein is added to a solution containing MASP-2 enzyme or a sample containing surface-immobilized MASP-2 enzyme, during which the labeled protein binds to the MASP-2 enzyme. Following a suitable incubation period, a solution containing the test compound is added to the preparation, followed, generally, by further incubation. Binding of a compound to the MASP-2 enzyme (the test compound binds partially or completely to the same site where the sequence according to the present invention is located, i.e., in a competitive manner, or somewhere else (but where its binding alters the conformation of the MASP-2 enzyme so that it loses its ability to bind to the protein), i.e., in a non-competitive manner) displaces the labeled protein from the MASP-2 enzyme to the extent of their inhibitory ability. The concentration of displaced protein is measured by using any method suitable for detecting the label (e.g., fluorescent or radioactive) used for the protein molecule of the present invention. Incubation periods, washing conditions, detection methods and other parameters are optimized in a manner known to those skilled in the art. The screening method according to the present invention can also be used in high-throughput screening (HTS) methods, as will be obvious to those skilled in the art. The MASP-2 enzyme used in the screening method is preferably human MASP-2 enzyme.

[0138] The present invention further relates to the use of the proteins, their pharmaceutically acceptable salts, esters or prodrugs in inhibiting MASP-2 protein, preferably human MASP-2 protein. It will be apparent to those skilled in the art that inhibitors (i.e., proteins of the present invention) can be used in a number of applications in which inhibition of a target protein (i.e., MASP-2) is useful. These applications include, for example, screening methods, drug development processes (e.g., lead optimization, use as reference compounds), potential health conditions and diseases, the relevance of mutations in MASP-2 protein, etc.

[0139] The present invention also relates to the use of proteins according to the invention, their pharmaceutically acceptable salts, esters or prodrugs in the manufacture of medicaments suitable for the treatment or prevention of diseases in which inhibition of the functioning of the complement system has a desirable effect. Such medicaments are used in organisms that possess the MASP-2 enzyme, generally mammals. However, the most preferred medicaments of the present invention are human medicaments. The use of proteins in the manufacture of medicaments is well known to those skilled in the art.

[0140] The disease is preferably selected from the following non-limiting group: (1) ischemia-reperfusion (IR) injury (particularly associated with recanalization after arterial occlusion due to thrombosis or other occlusive disease), including myocardial infarction (e.g., treated by percutaneous coronary intervention or thrombolysis), coronary artery bypass surgery, graft IR injury in organ transplants, gastrointestinal IR injury, renal IR injury, post-ischemic encephalopathy, stroke, and diseases occurring after thrombosis affecting any region of the body; (2) autoimmune nephritis (including dense deposit disease and C3 glomerulonephritis), IgA nephropathy, membranous nephropathy, rheumatoid arthritis (RA), juvenile nephropathy, and the like. Inflammatory and autoimmune conditions involving excessive activation of the complement system, including idiopathic arthritis, age-related macular degeneration, systemic lupus erythematosus (SLE), atypical hemolytic uremic syndrome (aHUS), thrombotic microangiopathy (TMA), post-infectious hemolytic uremic syndrome (HUS), pseudoallergy resulting from complement activation (CARPA), paroxysmal nocturnal hemoglobinuria (PNH), multiple trauma, and graft rejection after organ transplantation; (3) neurodegenerative diseases, preferably Alzheimer's disease, Huntington's disease, Parkinson's disease, multiple sclerosis, and age-related macular degeneration; (4) complement hyperactivation due to viruses, such as COVID-19 (SARS-CoV-2) infection, acute respiratory distress syndrome (ARDS), and complement-related microangiopathy and thrombosis due to severe COVID-19 infection.

[0141] The protein according to the present invention is useful in the treatment of the above diseases. The present invention also relates to a method for isolating human MASP-2 enzyme, in which i) a carrier having one or more immobilized proteins, their pharmaceutically acceptable salts, or esters is contacted with a solution containing the human MASP-2 enzyme, and ii) the preparation is washed. During the method, a protein according to the present invention is immobilized and the immobilized protein is contacted with a solution that presumably contains human MASP-2 enzyme. If this solution actually contains human MASP-2 enzyme, it will be firmly immobilized via the immobilized protein. This method is suitable for both analytical and preparative purposes. The solution containing human MASP-2 enzyme can be a pure protein solution, an extract of varying degrees of purification, a tissue specimen, etc.

[0142] [Example] The present invention will be described in detail below based on examples, but these examples are not intended to limit the present invention.

[0143] [Example A] Concepts about amino acid sequences The conservation laws observed within the Kunitz family are listed in Table 14 of US5994125A. These conservation laws were also taken into consideration in the research activities of the present invention. For illustration, the most important features are shown in the following sequence of EVO2 (SEQ ID NO: 2) (the amino acid sequence of which is described in WO2018 / 127719 as SEQ ID NO: 14, and the protein is named TFMI-2b): [ka] Shown in. In SEQ ID NO: 2 above, bold highlights completely conserved residues, and italics indicate positions represented by only 2-3 amino acid types within the Kunitz family. Positions randomized with respect to the invention described in WO2018 / 127719 are double underlined. Positions randomized with respect to the present invention are single or double underlined. Only non-conserved residues were randomized.

[0144] To better understand the above concept of the present invention, the sequence of the present invention is taken as an example, GPCRALKR as one of the preferred sequences of the general formula Ih-mod, N as one of the 9-set of preferred amino acids, G as one of the 34-set of preferred amino acids, L as one of the 39-set of preferred amino acids, and V as one of the 46-set of preferred amino acids, and SEQ ID NO: 42: [ka] As a host protein for obtaining the protein, the general Kunitz domain scaffold defined by SEQ ID NO: 115 is used.

[0145] If the same amino acid combination is introduced into the corresponding residue positions in EVO2 (SEQ ID NO: 2), SEQ ID NO: 12 (the sequence of EVO21, a potent MASP-2 inhibitor of the present invention) is obtained: [ka] (Here, the double underlined portion indicates the sequence portion of the general formula Ih-mod.) That is, SEQ ID NO: 42 is a general amino acid sequence generalized from the exact sequence of EVO21.

[0146] That is, the preferred general amino acid sequences of the present invention, i.e., SEQ ID NO: 33 to SEQ ID NO: 52 and SEQ ID NO: 54 to SEQ ID NO: 56, can be derived in a similar manner from the exact amino acid sequences of SEQ ID NO: 3 to SEQ ID NO: 22 and SEQ ID NO: 24 to SEQ ID NO: 26, as will be apparent to those skilled in the art.

[0147] [Example B] Structural identification of EVO2 residues evolved to improve MASP-2 inhibitory potency The protein EVO2 (described as TFMI-2b in WO2018 / 127719) is a ~7 nM inhibitor of both human and rat MASP-2. We solved the crystal structure of EVO2 in complex with rat MASP-2 (unpublished) and analyzed which positions in EVO2 outside of already evolved regions either harbor residues that contact MASP-2 or have evolved to become novel contact positions.

[0148] The five positions in the EVO2 protein were identified as x9 (E), x10 (D), x34 (K), x39 (L), and x46 (E) according to the Kunitz domain numbering system. The nomenclature of MASP-2 surface loops follows the nomenclature of serine protease loops introduced by Perona and Craik (1997). Analysis led to the following important results: positions x9 and x10 can contact MASP-2 loop 3 from one side, while x39 can contact loop 3 from the other side; position x34 has the potential to contact MASP-2 loop D; position x46 is in contact with MASP-2 loop A; and positions x34 and x39 are located on the same loop, which structurally supports the functional structure of the canonical binding loop, in part, via the C14-C38 disulfide that connects these loops.

[0149] This suggests that at least some positions in the canonical inhibitory and supporting loops influence each other's function, allowing pairs of residues to act synergistically.

[0150] [Example C] A three-stage directed evolution campaign using phage display This potential source of affinity improvement was successfully explored and exploited in a three-stage directed evolution campaign using phage display. The logic of the strategy is outlined below, and the main conclusions are presented here. To better understand how phage display works, the third of the three stages of the campaign is detailed in Example D.

[0151] Three-stage directed evolution led to new insights and significantly improved MASP-2 inhibitors.

[0152] [Example C.1] Stage 1 In the first stage, we started from the EVO2 gene and simultaneously randomized the Kunitz positions x9, x10, x34, x39, and x46, as well as x13 (corresponding to the P3 position of the P4-P4' segment). In addition, we applied the two-way randomization allowances L and V at the x17 position (corresponding to the P2' position of the P4-P4' segment).

[0153] Parallel selections were performed on human and rat MASP-2 as detailed in Example D, "Phage Display," sections D.1 and D.2. Statistical analysis of selected clones suggested that at position x9, N was slightly favored in place of the original amino acid E, x10 degenerated to wild-type D, L was preferred over V in both human and rat MASP-2 at the X13 P2' position, and at position x34, both human and rat MASP-2 selected over wild-type K, with the human enzyme preferring Y, whereas the rat enzyme preferred G / N / Y in that order. At position x39, the human enzyme slightly preferred F over wild-type L, whereas the rat enzyme strongly preferred L over F. At position x46, there was no strong preference for either amino acid type, but V was the amino acid slightly favored by both enzymes.

[0154] At position x13, i.e., the P3 position in the P4-P4' segment, the original campaign described in WO2018 / 127719 resulted in a preference for F / Y / L for human MASP-2 and a preference for P / V / I for rat MASP-2. In the evolutionary campaign of the present invention, the original preferences of the rat enzyme were maintained, while the human enzyme tolerated many amino acid residues (with a slight preference for P / Y / A), indicating that the newly evolved positions act synergistically with the residue at position P3 in inhibiting human MASP-2.

[0155] Based on these findings, we designed and generated the following five new EVO2-based mutants as recombinant proteins: EVO21 (SEQ ID NO: 12): [ka] EVO22 (SEQ ID NO: 7): [ka] EVO23 (SEQ ID NO: 3): [ka] EVO24 (SEQ ID NO: 15): [ka] EVO25 (SEQ ID NO: 11): [ka]

[0156] [Example C.2] Stage 2 For the second stage, we started with mutant EVO22 (SEQ ID NO: 7) (based on findings in the first stage, x10 was kept as wild-type D and x39 was kept as wild-type L, while x9 was N, x34 was Y, and x46 was V).

[0157] For this new amino acid sequence, essentially the same type of evolution described in WO2018127719 was repeated, fully randomizing the x13 (P3), x15 (P1), x16 (P1'), x17 (P2'), x18 (P3'), and x19 (P4') positions. Libraries were again independently selected for both human and rat MASP-2.

[0158] The findings show that at the P3 site, human MASP-2 prefers W / L / M / F / Y, which is more similar to the results of the first evolution campaign described in WO2018 / 127719 than the pattern selected in the first stage of this campaign (further supporting that some of the newly evolved positions act synergistically with residues at the P3 position in terms of inhibiting human MASP-2). In contrast, the P3 preference of the rat enzyme remained essentially the same (V / P / I) as in the first stage of the campaign.

[0159] At the P1' position, the consensus preference for A for both enzymes remained the same as described in WO2018 / 127719.

[0160] At the P3' and P4' positions, both enzymes appear to be quite promiscuous, with no clear preference pattern.

[0161] In contrast, a clear degree of dependence was observed at P2'. In the original evolutionary campaign for K34 described in WO2018 / 127719, human MASP-2 preferred V / A / I / L at the P2'(x17) position, whereas the rat enzyme preferred L / Y / I / M. In the first stage of the campaign (position x34 was fully randomized), binary L / V randomization at P2'(x17) resulted in an L preference. In the second stage, fixing Y34 again changed the pattern at x17. At the same time, both enzymes mostly preferred P2'(x17)A, and the preference for L remained unchanged, while the preference for V disappeared. This suggests that the A17 / Y34 and L17 / Y34 combinations increase MASP-2 binding affinity, while the V17 / Y34 combination decreases it.

[0162] Based on these findings, four additional mutants were generated as recombinant proteins to test the role of the P3 position and the functional coupling of the P2' (x17) and x34 positions. EVO221 (SEQ ID NO: 21): [ka] EVO222(SEQ ID NO: 18): [ka] EVO223 (SEQ ID NO: 19): [ka] EVO224 (SEQ ID NO: 28): [ka]

[0163] [Example C.3.] Testing of variants from the first and second stages Five new variants from the first stage and four new variants from the second stage were tested for binding affinity to human and rat MASP-2 in surface plasmon resonance assays, and their human and rat lectin pathway inhibitory potencies were measured in serum assays. Based on the obtained results summarized in Table 15 (for SPR data) and Table 16 (for lectin pathway inhibition data), 13 additional proteins of the invention were designed, produced as recombinant proteins, and tested in immune functional assays. These data are summarized in Tables 15 and 16 and described in Examples F.3, F.4.1, and F.5.1. The 13 new variants are as follows: EVO214 (SEQ ID NO: 8): [ka] EVO211 (SEQ ID NO: 4): [ka] EVO22a (SEQ ID NO: 6): [ka] EVO212 (SEQ ID NO: 14): [ka] EVO22b (SEQ ID NO: 22): [ka] EVO215 (SEQ ID NO: 26): [ka] EVO2a (SEQ ID NO: 32): [ka] EVO2b (SEQ ID NO: 31): [ka] EVO2c (SEQ ID NO: 25): [ka] EVO2d (SEQ ID NO: 30): [ka] EVO21a (SEQ ID NO: 24): [ka] EVO213 (SEQ ID NO: 27): [ka] EVO216 (SEQ ID NO: 29): [ka]

[0164] [Example C.4.] Stage 3 In the third directed evolution stage using phage display, we considered the possible combinations of 20 amino acids at positions P3 (x13), P2' (x17), and x34, and considered the occurrence of R / K / T at the P1 position in the initial library, allowing us to test whether K is a better P1 residue than R for several sequences.

[0165] These randomizations were performed using the modified EVO214 sequence (containing E at x9, F at x39, and V at x46, the latter two providing slightly higher affinity for human MASP-2): EVO214a (SEQ ID NO: 17) [ka] The survey was conducted on the following points.

[0166] Libraries were generated and independently selected for binding to human and rat MASP-2, as detailed below. Selected clones were tested for binding to both rat and human MASP-2. Clones that were able to bind to the human MASP-2 enzyme (selected regardless of which enzyme) were analyzed to determine the optimal x17 / x34 amino acid combination.

[0167] Based on this new information, seven additional new variants were designed and combinations were generated and tested for human and rat lectin pathway inhibitory potency in serum tests. The results are summarized in Table 16, Example F, Section F.4.1.

[0168] These variants are: EVO21b (SEQ ID NO: 9): [ka] EVO21c (SEQ ID NO: 13): [ka] EVO21d (SEQ ID NO: 16): [ka] EVO214a (SEQ ID NO: 17): [ka] EVO211a (SEQ ID NO: 20): [ka] EVO22d (SEQ ID NO: 10): [ka] EVO23a (SEQ ID NO: 5): [ka]

[0169] Overall, based on the sequence characteristics of the clones selected in the three-stage directed evolution campaign, together with the functional characteristics of all 29 new proteins of the present invention, it was surprisingly found that Kunitz domain protein-based compounds having a sequence of the modified general formula Ih-mod combined with the 34-set, and optionally further combined with the 9-set, 39-set, and / or 46-set, are suitable for the purposes of the present invention, i.e., they are significantly more effective inhibitors of the human MASP-2 enzyme than those described in WO2018 / 127719.

[0170] [Example D] phage display The phage display method described below was used to develop proteins according to the present invention.

[0171] Phage display is suitable for achieving directed in vitro evolution of proteins and peptides. The main steps of the state-of-the-art method (Smith 1985) are shown in Figure 1. In this process, the gene for the protein of interest is linked to a bacteriophage envelope protein gene. Throughout this specification, the term "phage" or "bacteriophage" refers to a class I filamentous phage, such as M13 bacteriophage. When a bacteriophage is created in this way, a fusion protein is generated that is integrated into the surface of the phage. The phage particle carries the heterologous protein inside and displays it on its surface. The protein and its gene are physically linked via the phage. For directed protein evolution, carefully determined codons are altered in the gene encoding it. Using combinatorial mutagenesis based on a mixture of synthetic oligonucleotides, multiple codons are mutated simultaneously. The positions of mutations and the variability per position are simultaneously measured.

[0172] Phage protein libraries are typically created by creating a DNA library containing billions of variants and then injecting it into bacteria. Each phage displays only one type of protein variant and carries only the gene for this variant. Individual variants are separated from each other using methods similar to affinity chromatography based on their ability to bind to a predetermined target molecule chosen by the researcher. Typically, the target molecule is linked or attached to a surface, which acts as the stationary phase in affinity chromatography. At the same time, in contrast to simple protein affinity chromatography, the so-called protein-phages selected in this way and carrying the target-binding variants of the displayed protein have two important distinctive properties: on the one hand, they can grow in E. coli cells, and on the other hand, these particles also display the selected variants of the displayed protein and carry the coat gene packaged within the phage particle.

[0173] During evolution, instead of testing individual mutants, billions are actually tested simultaneously. The binding mutants are cultured, and after several cycles of selection and propagation, a population enriched in functional mutants is obtained. From this population, individual phage clones displaying a selected variant of the evolved protein are tested in functional tests. Phage protein variants that prove suitable during testing are identified by sequencing of physically linked genes. In addition to individual measurements, sequence analysis of a large number of appropriately functionally selected clones also reveals which amino acid sequences are capable of performing the function. In this way, a database based on actual tests is created, which allows the refinement of sequence-function algorithms. The variants that prove best by this criterion are generated as independent proteins, and these are then tested in further, more accurate tests.

[0174] Vectors suitable for phage display were developed from commercially available vectors, which are described below.

[0175] [Example D.1.] Phage-displayed protein evolution As described above, the present invention was developed in three successive directed evolution stages. The first stage was a modified TFPI-D2 protein (herein designated TFPI-D2 as the second domain (SEQ ID NO: 116, residues 121-178) of the human tissue factor pathway inhibitor-1 protein (TFPI-1; UniProt ID P10646)). This modified TFPI-D2 is the TFMI-2b protein of the invention described in WO2018 / 127719, designated EVO2 (SEQ ID NO: 2). In the first stage, five previously untested and potential human MASP-2 contact positions based on the EVO2:rat MASP-2 structure, x9, x10, x34, x39, and x46 (according to the general Kunitz domain sequence (SEQ ID NO: 1)), were fully randomized, and x13 (the P3 position of the canonical inhibitory loop) was also fully randomized. Additionally, binary randomization was also performed at x17 (the P2' position of the canonical inhibitory loop (i.e., the P4-P4' segment)). The library was selected independently from both human and rat MASP-2. Based on this selection, five proteins of the present invention were designed and produced as recombinant proteins, and among these, EVO22 (SEQ ID NO: 7) was used as the starting mutant for the second stage of directed evolution. On the external surface of the sequence, the canonical inhibitory loop EVO22 differs from EVO2 in that it contains residues N9 / L39 / V46.

[0176] For the second stage of evolution, we used this new amino acid sequence context to essentially repeat the same type of canonical inhibitory loop described in WO2018 / 127719, i.e., fully randomized positions x13 (P3), x15 (P1), x16 (P1'), x17 (P2'), x18 (P3'), and x19 (P4'). Again, we independently selected libraries for both human and rat MASP-2.

[0177] Based on the obtained sequence patterns of the selected clones, a new protein, EVO214a (SEQ ID NO: 17), was designed and used as the starting sequence for the third stage of evolution (particularly focusing on the synergistic interaction at positions x17 and x34).

[0178] [Example D.2.] Creating a Library The phagemid vector construct applied in the invention described in WO2018127719 was used in all three stages of directed evolution leading to the present invention. The vector displays Kunitz domain inhibitors fused to M13 phage p8 protein in a monovalent manner, i.e., only a single copy of the inhibitor is displayed on the phage particle. This is essential for the selection of high-affinity binding molecules, as higher copy numbers can lead to binding activity, i.e., the phage particle simultaneously binds to the surface via multiple individual inhibitor / target molecule pairs.

[0179] In the system used, a phage-TFPI-D2 mutant library was created via a glycine-serine linker as an N-terminal fusion to the p8 main envelope protein.

[0180] A linear epitope tag recognized by a monoclonal antibody, the so-called "Flag-tag," was inserted at the N-terminus of each TFPI-D2 library member using an appropriate spacing peptide linker, with the goal of ensuring good display of each library member, even if it does not bind to the target protease, MASP-2 (or other proteases of interest).

[0181] The following example shows how three phage libraries were created for the three-stage evolution (Example D.2.1). To avoid unnecessary repetition of the methodology, phage selection is described only for the third stage of evolution (Example D.2.2), but results are presented for all three evolutionary stages (Example D.2.3). Example D.2.4 describes a method for heterologous expression of inhibitors, and Example D.2.5 describes how to test the inhibitors of the present invention for quality and efficacy.

[0182] The oligonucleotides required for phage display-based protein evolution are summarized in Table 2. [Table 2] [Table 2]

[0183] [Example D.2.1.] Creation of phage library 1 D.2.1.1. Construction of phage display vectors This construct was based on the vector originally introduced and described in detail in WO 2018 / 127719. The vector contains a codon-optimized version of the DNA encoding TFPI-D2 (the protein is flanked at both ends by Ser / Gly linkers and displayed as a p8 coat protein fusion on the surface of bacteriophage M13). Furthermore, the construct provides the displayed protein with an N-terminal FLAG tag for easy evaluation of display efficiency. The coding DNA is located between the Kpn2I (BspEI) and SacI sites, as shown in Figure 2 (which illustrates the DNA sequence (SEQ ID NO: 117) and amino acid sequence (SEQ ID NO: 118) of the fusion gene engineered to display TFPI-D2 on the surface of M13 bacteriophage).

[0184] The different functional parts of the fusion protein are as follows (numbering corresponds to the nucleotides in Figure 2): positions 4-81 are the malE periplasmic signal sequence, positions 85-108 are the FLAG-tag, positions 111-135 are the first Ser-Gly linker, positions 136-309 are the TFPI-D2 domain, positions 310-342 are the second Ser-Gly linker, and positions 343-492 are the M13 phage major coat protein (p8). The restriction enzyme cleavage sites used during the construction of the fusion gene are shown above the sequences of those cleavage sites in Figure 2. The residues targeted by directed evolution according to the prior invention are shown at nucleotide positions 172-174 and 178-192.

[0185] For the first stage of evolution, the following synthetic DNA (designated EVO2-stage-1-stop (SEQ ID NO: 57)) was designed: [ka]

[0186] SEQ ID NO: 57 contains a codon-optimized and stop codon-containing coding DNA encoding EVO2 modified by replacing the TFPI-D2 gene between the BspEI (TCCGGA (italics)) and SacI (GAGCTC (italics)) sites. The EVO2-encoding DNA is modified to carry a TAA stop codon at codon positions corresponding to the x9, x10, x13, x17, x34, x39, and x46 amino acid positions. Continuous dashed lines indicate segments that serve in reverse complement form as template regions for library mutagenesis oligonucleotides, as described below.

[0187] The following library mutagenesis oligonucleotides were used: EVO2-stage-1-lib-1(SEQ ID NO: 58)(85-mer): [ka] and EVO2-stage-1-lib-2(SEQ ID NO: 59)(86-mer): [ka] (Here, the degenerate codon replacing the stop codon is shown in bold.) For the second stage of evolution, the following synthetic DNA was designed: EVO2-stage-2-stop (SEQ ID NO: 60). [ka] (Here, bold indicates the stop codons replacing the amino acid codons at positions x13, x15, x16, x17, x18, and x19; wavy lines indicate the exosite positions with newly introduced mutations at x9 (N9), x34 (Y34), and x46 (V46); and dotted lines indicate the segments that act as template regions for the library mutagenesis oligonucleotides described below.)

[0188] The following library mutagenesis oligonucleotides were used: EVO2-stage-2-lib(SEQ ID NO: 61) [ka] (Here, the degenerate codon replacing the stop codon is shown in bold.)

[0189] For the third stage of evolution, the same synthetic DNA was used as the template used for the first stage of evolution. EVO2-stage-1-stop(SEQ ID NO: 57): [ka] The continuous dashed lines indicate segments that acted in reverse complement form as template regions for mutagenic oligonucleotides (SEQ ID NO: 62 and SEQ ID NO: 63), which were used as suitable stop templates for subsequent sequence library mutagenesis.

[0190] Stop template generating oligonucleotide EVO2-stage-3-stop-1 (SEQ ID NO: 62): [ka] Stop template generating oligonucleotide EVO2-stage-3-stop-2 (SEQ ID NO: 63): [ka]

[0191] When these oligonucleotides are used together as mutagenesis primers on a template containing only the reverse complement of SEQ ID NO: 57, they create a modified version of the coding DNA of the original EVO214a (SEQ ID NO: 17) that encodes an E at x9, an F at x39, and a V at x46 (the latter two providing slightly higher affinity for human MASP-2). However, the resulting gene will have stop codons at the P3 (x13), P1 (x15), P2' (x17), and x34 coding codon positions.

[0192] The resulting gene sequence shown below is EVO214a-STOP (SEQ ID NO: 64): [ka] The continuous dashed lines indicate the segments that act in reverse complement form as template regions for the library mutagenesis oligonucleotides described below.

[0193] Library mutagenesis oligonucleotide EVO2-stage-3-lib-1 (SEQ ID NO: 65): [ka] Library mutagenesis oligonucleotide EVO2-stage-3-lib-2 (SEQ ID NO: 66): [ka] In the library mutagenesis oligonucleotides, bold indicates the degenerate codons NNK, which encode the complete set of 20 amino acids at positions x13 (P3), x17 (P2'), and x34, and AVA, which represents the codon sets AAA, AGA, and ACA, thereby encoding the amino acids K, R, and T at x15, which is the P1 position.

[0194] These oligonucleotides, taken together, create the coding DNA for a randomized version of EVO214a (SEQ ID NO: 17) that allows all possible combinations of the 20 natural amino acids at the P3 (x13), P2' (x17), and x34 positions, and that has been modified to allow for the occurrence of R / K / T amino acids at the P1 position of the initial library. This allows testing whether K is a more preferred P1 residue than R at some sequence point. Threonine is tolerated to see if it can be completely removed during binding selection, i.e., selection is performed.

[0195] Now that we have described the essential DNA constructs and mutagenic oligonucleotides, we provide examples of how these tools can be used for phage display-based directed evolution. While the present invention is based on three stages of directed evolution, in terms of technical realization, the same methodology is applied to all three stages in the same order. To avoid unnecessary repetition, these methods and steps are always detailed for one of the three evolutionary stages, avoiding unnecessary repetition of the same technical description. In the following sections, we present the directed evolution stages of a three-stage evolution campaign. (Example D.2.1.2. was intentionally omitted.)

[0196] D.2.1.3. DNA library creation D.2.1.3.1. Creation of stop mutant phagemids by Kunkel mutagenesis. D.2.1.3.1.1. Transformation of E. coli strain CJ 236 Raw DNA solution: 1 μl (~100 ng) pTFPI-D2-pro-lib phagemid 4μl 5xKCM (0.5M KCl, 0.15M CaCl2, 0.25M MgCl2) 15 μl distilled water The DNA solution was cooled on ice. 20 μl of CJ236 cells (NEB) was added to the DNA solution, and the sample was incubated on ice for 20 minutes. The cells were then left at room temperature for 10 minutes, and 200 μl of LB medium was added, followed by shaking at 37°C for 30 minutes. The cells were spread onto LB-agar + ampicillin (100 μg / ml) plates and grown overnight at 37°C.

[0197] D.2.1.3.1.2. Generation and isolation of uracil-containing phages From another colony, cells were inoculated into 2 ml of 2YT / ampicillin (100 μg / ml), chloramphenicol (5 μg / ml). The next day, 30 μl of the culture was inoculated into 3 ml of medium of the same composition. 600 nm As soon as the optical dispersion of the cell suspension reached 0.4 (measured by HPLC), the cells were infected with M13-KO7 fel per phage (NEB) to an average of at least 10 phages per E. coli cell. After shaking at 37°C for 30 minutes, the cells were added to 30 ml of 2YT / ampicillin (100 μg / ml), kanamycin (25 μg / ml) medium. The cells were shaken at 37°C for 16 hours or more. The cells were then isolated from the culture by centrifugation (10,000 rpm, 10 minutes, 4°C) to form a phage-containing supernatant. The phage was precipitated in a clean centrifuge tube by adding 1 / 5 (by volume) of a PEG / NaCl solution (20% PEG8000, 2.5 M NaCl). After thorough mixing in the precipitant, the sample was left at room temperature for 20 minutes. The phage particles were then sedimented by centrifugation (12,000 rpm, 10 min, 4°C). The supernatant was carefully poured off and returned to the tube in the same position. The liquid adhering to the tube wall was then collected by brief centrifugation (1,000 rpm, 1 min, 4°C) and then removed with a pipette. The phage were suspended in 800 μl PBS, and remaining cell fragments were removed from the sample by centrifugation in a microcentrifuge (12,000 rpm, 10 min), and the supernatant was transferred to a clean microcentrifuge tube. The supernatant thus obtained contained pure phage.

[0198] D.2.1.3.1.3. Isolation of single-stranded DNA from phages Single-stranded DNA (ssDNA) was isolated from approximately 800 μl of phage using a QIAprep® Spin Miniprep Kit (#27106) supplemented with an in-house prepared solution of 2.8 M citric acid, 1 M sodium perchlorate, and 30% (v / v) isopropanol, according to the manufacturer's instructions. This supplemented kit replaces the original QIAgen Spin M13 kit (dedicated for M13 DNA isolation but discontinued by the manufacturer). The amount of pure ssDNA was determined based on ultraviolet light absorbance at 260 nm.

[0199] D.2.1.3.1.4. Kunkel mutagenesis Oligonucleotides for stop mutations: EVO2-stage-3-stop-1(SEQ ID NO: 62): [ka] and EVO2-stage-2-stop-2(SEQ ID NO: 63): [ka] It was introduced in.

[0200] D.2.1.3.1.4.1. Oligophosphorylation Both oligonucleotides were phosphorylated in separate reactions as follows: 2 μl mutagenesis oligo (330 ng / μl) 2μl 10X TM buffer (0.5M Tris-HCl, pH7.5, 0.1M MgCl2) 2 μl 10 mM ATP 1 μl 100 mM DTT 12 μl distilled water 1 μl Polynucleotide kinase (NEB, 10 U / μl) The reaction mixture was incubated at 37°C for 30 minutes.

[0201] D.2.1.3.1.4.2. Oligo-template annealing 1 μg ssDNA 2 μl of product from kinase oligo reaction of both mixtures 2.5μl 10X TM buffer Distilled water (to make the final volume 25 μl) Incubation: 90°C for 1 minute, 50°C for 3 minutes, then briefly centrifuged and placed on ice.

[0202] D.2.1.3.1.4.3. Polymerization and Ligation To the DNA solution from D.2.1.3.1.4.2., the following reagents were added: 1 μl 10 mM ATP 1.5 μl 100 mM DTT 0.6μl T4 ligase (NEB, 400U / μl) The reaction mixture was incubated at room temperature for 30 minutes to facilitate ligation of the two oligonucleotides. The following reagents were added to achieve second strand synthesis: 1 μl 25 mM dNTPs, 0.6 μl T7 polymerase (NEB, 10 U / μl).

[0203] The reaction mixture was incubated at 37°C for 2 hours. The entire mixture was run on a 1% agarose gel, and products of the desired size were excised from the gel. The Kunkel product was isolated from this gel slice in 30 μl of elution buffer (EB) using a QIAgen Gel Extraction kit (cat. no. 28706). As described in D.2.1.3.1.1, the Kunkel product was transformed into XL1 Blue cells using 1 μl of DNA. From individual colonies, cell cultures were grown in LB / ampicillin (100 μg / ml) medium. Phagemids were isolated from the cells using a QIAprep® Spin Miniprep Kit (#27106) according to the manufacturer's instructions. Identification and quantification of the DNA constructs were tested using the Big Dye Terminator v3.1 cycle Sequencing Kit (Applied Biosystems; cat. no. 4336917) system, which was used for sequencing PCR reactions. The products of the sequencing reaction were run by BIOMI Kft. (Goedöllöe, Hungary). The vector thus created was named pEVO214a-STOP phagemid, and the functionally relevant part of its sequence corresponds to SEQ ID NO: 64.

[0204] D.2.1.3.2. Library Kunkel mutagenesis Library mutagenesis was performed as described above in Section D.2.1.3.1.4., but using 10-fold the amount of reagents described therein. The library oligos were similar to the stop mutagenesis oligos, except that in the latter case, degenerate NNK and / or AVA triplets (using IUPAC coding relative to degenerate oligonucleotides) were present in place of the TAA stop codon. The sequences of the library mutagenesis oligonucleotides were as follows: Library mutagenesis oligonucleotide EVO2-stage-3-lib-1 (SEQ ID NO: 65) [ka] and Library mutagenesis oligonucleotide EVO2-stage-3-lib-2 (SEQ ID NO: 66) [ka] Oligophosphorylation was performed as described above in D.2.1.3.1.4.1. To create the library, a 10-fold increase in the amount of oligos was used in the oligo-template annealing, thus using all of the oligos created during the kinase reaction. The template for mutagenesis was uracil-containing ssDNA with a stop codon, which was created from the pEVO214a-STOP phagemid obtained as a result of the method detailed above by infection of CJ236 cells with M13K07 helper phage. To create the 10-fold library, 20 μg of template was used, and the volume of the annealing reaction was increased 10-fold to 250 μl, while the final concentrations of the components were maintained the same. The incubation period was extended to 90°C for 2 minutes and 50°C for 5 minutes. As described above, separate ligation reactions were applied to ligate the two mutagenic oligonucleotides at room temperature for 30 minutes, and then after adding dNTPs and T7 polymerase, the mixture was incubated at 37°C for 3 hours.

[0205] The product was purified using a Qiagen Gel Extraction kit. It was not isolated from the gel but was purified using two columns. Elution was performed in 2 x 30 μl USP distilled water.

[0206] D.2.1.4. Electroporation and propagation of phage libraries The DNA library was introduced into supercompetent cells via electroporation, with the goal of introducing the phagemid into as many cells as possible, thereby allowing the library to grow to 10 8 ~10 9 The aim is to contain each transformed cell.

[0207] The DNA library dissolved in USP distilled water (salt-free) was added to 2 x 350 μl of supercompetent cells. 30 μl of the DNA library was electroporated into 350 μl of supercompetent cells, and the process was repeated with the other half of the DNA library. The procedure was performed in an electroporation cuvette with a gap size of 0.2 cm, according to the following protocol: 2.5 kV, 200 ohms, 25 μF.

[0208] After electroporation, the cells were carefully transferred to 2 x 25 ml SOC medium and incubated at 37 °C with shaking at 200 rpm for 30 minutes. A 10 μl sample was then taken and serially diluted 10-fold, 8-member dilutions were performed. 10 μl from each dilution was spotted onto LB plates, LB plates with 100 μg / ml ampicillin, and LB plates with 10 μg / ml tetracycline, and grown overnight at 37 °C. After the samples were taken, the remaining 2 x 25 ml of medium was infected with 2 x 250 μl of M13KO7 helper phage (1 x 10 PFU / ml) and shaken at 37 °C with shaking at 220 rpm for 30 minutes. The entire product was then inoculated into 2 x 500 ml of 2YT plates with 100 μg / ml ampicillin and 25 μg / ml kanamycin. The cultures were grown in two 2 L baffled Erlenmeyer flasks at 37°C and 220 rpm for 18 hours.

[0209] Based on titration, the library was 2.5 x 10 9 It contained the mutations.

[0210] [Example D.2.2.] Library selection for human MASP-2 enzyme and, independently, in parallel, for rat MASP-2

[0211] D.2.2.1. Target enzyme The MASP-2 target enzyme consists of one serine protease (SP) domain and two complement control protein domains (CCP1 and CCP2) (Gal 2007). These are recombinant fragment products that retain the catalytic activity of the intact molecule ("catalytic fragments"). The protein was produced in inclusion bodies, from which the biologically active conformation was obtained by renaturation. Purification was performed by anion and cation exchange separation. The activity of the protein was also tested in solution and in ELISA plate-bound form. (For details on the amino acid sequence and preparation of the human MASP-2 target, see Ambrus 2003.) The catalytic fragment of rat MASP-2 begins at Gln298 and ends at Phe685 according to UniProt numbering (entry Q9JJS8). Cloning was performed as in the case of human MASP-2 described in Ambrus 2003. As a result of cloning, a recombinant protein with an extra Met-Thr dipeptide segment at the N-terminus was generated. Rat recombinant MASP-2 protein was expressed, refolded, and purified according to methods previously used for the human protein fragment.

[0212] The target data used during selection are: Human MASP-2cf CCP1-CCP2-SP: Mw = 44,017 Da, C stock =3.0g / l Rat MASP-2cf CCP1-CCP2-SP: Mw=42309 Da, C stock =3.6g / l

[0213] D.2.2.2. Selection process

[0214] D.2.2.2.1. Isolating phages At the end of the procedure described in chapter D.2.1.3., phages were produced in 18 hours in 2 x 500 ml of culture medium and isolated in a first step of selection to immediately use the library for selection.

[0215] The cell culture was centrifuged at 8,000 rpm for 10 minutes at 4°C. The supernatant (containing the bacteriophage) was poured into a clean centrifuge tube, and 1 / 5 of its volume of precipitant (2.5 M NaCl; 20% PEG-8000) was added. Precipitation was allowed to proceed at room temperature for 20 minutes. The tube was then centrifuged again at 10,000 rpm for 10 minutes at 4°C. The supernatant was discarded, and the tube was again briefly centrifuged, and the remaining liquid was removed with a pipette. The white precipitate was dissolved in 25 ml of [PBS; 5 mg / ml BSA; 0.05% Tween-20] buffer. To remove residual cell debris, the tube was again centrifuged at 12,000 rpm for 10 minutes, and the supernatant was transferred to a clean tube.

[0216] D.2.2.2.2. First Selection Cycle a) Immobilization: Target molecules were immobilized in separate wells on a 96-well Nunc Maxisorp ELISA plate (cat#442404). During immobilization, the concentrations of human MASP-2cf and rat MASP-2cf were 20 μg / ml. Both proteins were diluted in immobilization buffer [200 mM Na2CO3; pH 9.4], and 100 μl was placed in each well. Both human and rat MASP-2cf were incubated overnight at 4°C. In the first selection cycle, 12 wells were used per target protein. Each second column was left empty. As a negative control, immobilization buffer alone was placed in one column. This column was then treated in the same way as the one covered with the target protein. b) Blocking: The fixation solution was removed and 200 μl / well of blocking buffer [PBS; 5 mg / ml BSA] was placed on the plate and incubated at room temperature with mixing at 150 rev / min for at least 1 hour. c) Washing: ELISA plates were washed four times with 1 L of wash buffer [PBS; 0.05% Tween-20]. d)Select: The phages of the library isolated as described above were pipetted onto the plate at 100 μl per well, and the plate was incubated at room temperature with mixing at 110 rev / min for 3 hours. e) Cultivation of E. coli XL1 Blue: During the selection period, XLI Blue cells (freshly harvested previously using an inoculation loop) were inoculated from the plate into 2 x 30 ml of [2YT; 10 μg / ml tetracycline] medium. These cells were to be infected with phage eluted from the target protein. At the time of infection, the cells should be in exponential growth phase. OD 600 nm A medium with an NA of ~0.3-0.5 (obtained by growing E. coli at 37°C and 220 rpm for 2-3 hours) was required. f) Washing: The ELISA plate was washed 12 times using 3 L of wash buffer. g) Elution: Elution was performed using 100 μl / well of 100 mM HCl solution. The acid solution was applied and shaken for 5 minutes, after which the acid solution was removed from each well one by one. Phages eluted from individual target proteins were collected in tubes to which 12×15 μl of 1 M Tris-base buffer had been previously introduced to rapidly neutralize the acid solution containing the phages. The tubes were immediately mixed and placed on ice. h) Infection: 4.5 ml of exponentially growing XL1 Blue culture was placed in a test tube and infected with 500 μl of phage solution eluted from the target protein. A total of three infections were performed with phage eluted from human MASP-2, rat MASP-2, and a negative control material. The culture was incubated at 37°C and 220 rpm for 30 minutes. i) Titration: A 20 μl sample was taken from each infected culture and diluted 10-fold in volume with 2YT medium, and sequences were prepared by further diluting 10 times. Each point was spotted onto an LB agar plate (100 μg / ml ampicillin) and grown overnight at 37°C. j) Infection with helper phage: Immediately after sampling, add 50 μl of M13KO7 (1x10 13 PFU / ml) helper phage was added and the medium was incubated for an additional 30 minutes. k) All infected cultures were transferred to 200 ml of [2YT; 100 μg / ml ampicillin; 30 μg / ml kanamycin] medium and incubated at 37° C. with mixing at 220 rpm for 18 hours. l) Concentration: The next morning, the titration results were evaluated. The number of phage eluted from human MASP-2, i.e., the number of phage-infected and thus ampicillin-resistant colonies, was approximately 100-fold higher than the number of phage eluted from the BSA background, whereas in the case of rat MASP-2, this ratio was approximately 10. This ratio is referred to as the enrichment value.

[0217] D.2.2.2.3. Second Selection Cycle In this cycle, the same steps as in the first selection cycle were repeated, except that eight wells were used per target, where each target protein had its own control substance (eight wells), and phage eluted and propagated in the previous cycle were placed over both the target and control proteins.

[0218] Phage produced at 18 hours were isolated as described above, except that at the end they were dissolved in 10 ml of sterile PBT buffer. After the second selection cycle, fresh exponentially growing XL1 Blue cells were infected with 300 μl of eluted phage. In all four cases (two target proteins + two control substances), titration was performed, and the cultures, also infected with helper phage, were then transferred to 30 ml of [2YT; 100 μg / ml ampicillin; 30 μg / ml kanamycin] medium.

[0219] After the second selection cycle, the number of clones eluted from specific target-coated, BSA-blocked wells was determined and divided by the number of clones eluted from target-free, BSA-blocked wells. Enrichment values ​​of 310-470 for human MASP-2 and 120-130 for rat MASP-2 were obtained.

[0220] D.2.2.3. Testing individual clones using phage ELISA assay In the ELISA test, phage clones were selected that could bind strongly to their respective target proteins while displaying significantly lower signals on the BSA-coated control material.

[0221] a) Infection: In both cases of human MASP-2 and rat MASP-2, phage eluted from selection cycle 1 and selection cycle 2 were used to generate colony-similar phage solutions. To this end, 10 μl of eluted phage from selection cycle 1 and 10 μl of eluted phage from selection cycle 2 were added separately to 250 μl of log-phase XL1 Blue culture. To ensure a large excess of cells during infection, the eluted phage was prediluted to contain 200–400 phage per 10 ml. The infected cells were incubated at 37°C for 30 min while the suspension was mixed at 220 rpm. The cells were then plated onto [LB; 100 μg / ml ampicillin] plates and grown overnight at 37°C. b) Inoculation: [2YT; 100 μg / ml ampicillin; 10 10 Individual colonies were inoculated into so-called "single loose" tubes (National Scientific Supply-TN0933-C01) containing 500 μl of medium [phage / ml M13KO7 helper phage]. These tubes were arranged similarly to a 96-well ELISA plate configuration, and were rotated (with mixing at 300 rpm) in a plate incubator at 37°C, allowing samples within the tubes to be vigorously aerated and suitable for generating small-volume cultures. c) Immobilization: As described in the relevant sections above, both human and rat MASP-2 proteins were immobilized at a concentration of 10 μg / ml on Nunc ELISA Maxisorp plates in a volume of 50 μl / well. Each clone was tested against its own target protein, against other target proteins, and against a BSA-coated control surface. d) After 18 hours, the "single loose" tubes were centrifuged at 2,500 rpm for 10 minutes at 4°C in a rotor suitable for accommodating ELISA plates, and the supernatants were pipetted into clean tubes. An aliquot from each tube was tested in the phage-ELISA, after which the remaining supernatant was heated at 65°C for 2 hours to kill any contaminating E. coli. Samples were then stored at -20°C until used for sequencing. e) Blocking: The liquid was removed from the fixed samples, and 200 μl / well of [PBS; 5 mg / ml BSA] blocking buffer was added to each well. Incubation was carried out for at least 1 hour at room temperature with mixing at 150 rev / min. f) Washing: The plate was washed four times with 1 L of wash buffer. g) Application of phage: 50-50 μl of phage, generated and isolated as described above, was placed in each well. From the same clone, samples were pipetted into three wells: one containing the target, one containing the other target, and one containing the BSA control. The incubation was carried out for 1 hour at room temperature with mixing at 110 rev / min. h) Washing: The plate was washed six times with 1.5 L of wash buffer. i) Anti-M13 antibody: 50 μl of monoclonal anti-M13 HRP-conjugated antibody (Amersham, cat#27-9421-01) diluted 5,000-fold in buffer [PBS; 5 mg / ml BSA; 0.05% Tween-20] was added to the wells and incubated for 30 minutes at room temperature with mixing at 110 rev / min. j) Washing: The plate was washed six times with 1.5 L of wash buffer, then twice with PBS. k) Signal generation: 50 μl of 1-Step Ultra TMB-ELISA material (Pierce, cat#34028) was added to each well and shaken briefly, after which the reaction was stopped by adding 50 μl of 1 M HCl to each well. l) Reading: Absorbance was measured at 450 nm using a BioTek Epoch (Agilent) plate-reading photometer. Phage supernatant samples that displayed low-intensity signals in the BSA background but at least twice as strong signals for their target proteins were collected and used for sequencing. Two microliters of supernatant was used for sequencing PCR reactions using the Big Dye Terminator v3.1 cycle Sequencing Kit (Applied Biosystems; cat#4336917). Samples were analyzed by BIOMI Kft (Goedöllöe, Hungary).

[0222] [Example D.2.3.] result This example describes the results of the tests described in Examples D.2.1. and D.2.2., i.e., the sequences obtained.

[0223] From the phage eluted from human MASP-2, 224 clones (112 from section cycle 1 and 112 from section cycle 2) were tested using ELISA, ultimately yielding 192 individual sequences. In the case of rat MASP-2, 128 clones were tested, of which 108 were ELISA-positive, corresponding to 98 individual sequences. When interpreting the results, it had to be taken into account that the NNK codon pattern used in constructing the DNA library did not necessarily guarantee the same initial frequency for each of the 20 individual amino acids. In the NNK codon pattern, an amino acid can have one, two, or three codons. Therefore, codon normalization was performed by dividing all amino acid frequencies by the number of codons. A given amino acid is represented in the NNK set.

[0224] After data normalization, sequence logo diagrams were generated for the sequences with the aid of WebLogo (Crooks 2004), which is accessible on the internet (http: / / weblogo.berkeley.edu / logo.cgi).

[0225] A sequence logo is a graphic display of the information content and distribution of amino acids per position in a set of aligned sequences, using single-letter amino acid abbreviations. At each position, the height of the logo's columns indicates the occurrence of the element (in the present invention, 20 different types of amino acids for x13, x17, and x34, and 3 different types for x15). The lower the occurrence, the higher the column. In the case of perfect distribution (all allowed amino acids occur in equal proportions), the height is zero. The maximum value belongs to the case in which only one type of element (amino acid) occurs. Within a column, individual amino acids are arranged based on their frequency of occurrence, with the most frequent one at the top. The height of the letter representing an amino acid is proportional to its relative frequency of occurrence at a given position (e.g., a 50% occurrence is half the height of the column). In the case of color diagrams, amino acids with similar chemical properties are generally shown in the same or similar colors (different shades of gray are used in the figures accompanying this specification).

[0226] On the horizontal axis of a typical sequence logo diagram, the number of individual positions in the randomized region is found, with site P1 corresponding to position x15, and on the vertical axis the information content of the position is measured in bits.

[0227] In the modified version of the sequence logo diagram, all column heights are set equal to allow better visualization of rarely selected amino acids, especially at positions with low conversion levels, so that the column heights and the letters stacked therein do not make the letters less difficult to read.

[0228] Logos for all three phage display evolution stages are shown in Figures 3, 4 and 5.

[0229] The codon-normalized frequencies of each amino acid at each randomized position are displayed in Tables 3-9 for all three stages.

[0230] [Table 3] Stage 1 phage display evolution: Codon-normalized amino acid frequencies representing a set of 64 human MASP-2-binding clones selected for the human MASP-2 enzyme TIFF2025534392000080.tif223154

[0231] [Table 4] Stage 1 phage display evolution: Codon-normalized amino acid frequencies representing a set of 55 rat MASP-2-binding clones selected for rat MASP-2 enzyme TIFF2025534392000081.tif197155

[0232] [Table 5] Stage 2 phage display evolution: Codon-normalized amino acid frequencies representing a set of 54 human MASP-2-binding clones selected for the human MASP-2 enzyme TIFF2025534392000082.tif177153

[0233] [Table 6] Stage 2 phage display evolution: Codon-normalized amino acid frequencies representing a set of 68 rat MASP-2-binding clones selected for rat MASP-2 enzyme TIFF2025534392000083.tif175153

[0234] [Table 7] Stage 3 phage display evolution: Codon-normalized amino acid frequencies representing a set of 192 human MASP-2-binding clones selected for the human MASP-2 enzyme TIFF2025534392000084.tif229153

[0235] [Table 8] Stage 3 phage display evolution: Codon-normalized amino acid frequencies representing a set of 98 rat MASP-2-binding clones selected for rat MASP-2 enzymes TIFF2025534392000085.tif201153

[0236] [Table 9] Stage 3 phage display evolution: Codon-normalized amino acid frequencies representing a set of 290 human MASP-2-binding clones selected for either rat or human MASP-2 TIFF2025534392000086.tif222153

[0237] Using the logo and table, we examined which amino acids are preferred at each position and how they differ depending on whether they are derived from human MASP-2 or rat MASP-2.

[0238] For each phage display evolution stage, the results are shown in the normal and modified sequence logo diagram versions and tables described above. For each phage display evolution stage, one normal logo and one modified logo pair are shown for both human MASP-2 and rat MASP-2 selection. Tables 3 and 4 and corresponding Figure 3 show the results of the first evolution stage, while Tables 5 and 6 and corresponding Figure 4 show the results of the second evolution stage. For the third evolution stage, in addition to separate analyses of human MASP-2 and rat MASP-2 selected clones, a third set corresponding to all human MASP-2 binding clones, regardless of whether they were selected for human MASP-2 or rat MASP-2, was also analyzed. Therefore, for this final evolution stage, three tables, namely Tables 7, 8, and 9, are disclosed herein, with one additional logo pair shown in Figure 5.

[0239] Attention is now drawn to the results shown in Table 9 and the corresponding Figure 5, which are most relevant for the present invention, i.e., as they relate to the complete set of human MASP-2 binding clones, whether originally selected for the human or rat MASP-2 enzyme.

[0240] The logo diagram shows the choices that can be made at each location.

[0241] At x15 (P1 position), other than the allowed R / K / T, T (threonine) was not selected and R (arginine) was selected in 79%, while K (lysine) in 21% of clones. When K is part of the domain, R at the P1 position is the most favored amino acid for the S1 pocket of human MASP-2.

[0242] At x13 (position P3), the human MASP-2 binder closely reproduces the findings of the original invention provided in WO2018 / 127719, i.e., the entire set of general (Ih) sequences of WO2018 / 127719 at position x13 (F, Y, L, P, Q, M, V, Q, A, T) is represented in the set of amino acids occurring at x13 of the human MASP-2 binder, and the three most selected amino acids, F / Y / P, constitute a subset of the four most selected amino acids, F / Y / L / P, of the original invention shown in WO2018 / 127719.

[0243] This suggests that simultaneous randomization of the x13 / x15 / x17 / x34 positions did not change the optimal x13 (P3) amino acid set of Kunitz domain proteins in terms of binding to human MASP-2, compared to the case in WO2018127719 in which x34 was fixed as K.

[0244] In contrast, at x34, when the x13 / x15 / x17 / x34 positions were simultaneously randomized and evolved according to the present invention, the human MASP-2-binding clone set was enriched in variants with a significantly hydrophobic set of Y / I / F / G / V amino acids, while the original hydrophobic and charged K was unfavorable. This suggests that the physicochemical properties of the original K34 were suboptimal for human MASP-2 binding. Furthermore, simultaneous evolution of the x13 / x15 / x17 / x34 positions also characteristically alters the set of amino acids at x17 that is optimal for human MASP-2 binding. In the invention described in WO20181 / 27719, this set was V, A, I, L, M, D, H, S, which, when co-evolved with x34, became I, L, F, Y, A.

[0245] Compared to their original amino acid preferences revealed in the invention described in WO2018 / 127719, positions x13 and x15 do not show significantly altered amino acid preferences in the present invention, and from these four positions, we conclude that only x17 and x34 are functionally strongly linked, i.e., the specific x17 / x34 amino acid residue pair synergistically boosts MASP-2 binding affinity.

[0246] The inventors analyzed what the common physicochemical properties of the most favorable x17 / x34 pairs were and identified a combination of two properties: combined hydrophobicity and cumulative side chain volume.

[0247] To calculate the cumulative side chain volume of the x17 / x34 side chain pair, the side chain volumes from the Harpaz 1994 study (volume excess compared to glycine is considered, which is zero for Gly as shown in Table 10) were used.

[0248] [Table 10] Amino acid side chain volume data from the Harpaz 1994 study TIFF2025534392000087.tif128153*:The volume of C corresponds to the reduced form.

[0249] The cumulative side-chain volumes were measured for all 290 clones capable of binding to human MASP-2, regardless of whether they were selected for human or rat MASP-2. Then, rounding to two decimal places, this set was distributed into groups according to cumulative side-chain volume. Prior to selection, the baseline cumulative side-chain volume distribution in the starting library was estimated as follows: The initial library had a x17 / x34 amino acid pair, corresponding to 32x32 codon pairs derived from the NNK codon set. (The translation of the TAG codon is considered Q as a result of the supE44 mutation in the XL-1 Bluekink strain.) Equivalent cumulative volumes were assigned and classified into the sets, and the 960 data were normalized to 290 clones to harmonize the number of human MASP-2-binding clones. The observed number of clones for each cumulative size range approximates the density distribution of the selected population and is compared to the density distribution calculated for the initial library (see Table 11 and Figure 6).

[0250] [Table 11] Number of x17 / x34 amino acid pairs in each cumulative side chain volume group (bold indicates cumulative size preferred by human MASP-2) [Table 3]

[0251] We found eight cumulative side chain size groups favorable for human MASP-2 binding: 100, 130, 160, 180, 200, 210, 230, and 270 (numbers are in Å). 3 corresponds to).

[0252] The five most selected amino acid types at x17 and x34 yielded 25 (x17 / x34) amino acid pairs, a 1 / 16 subset of the 400 possible amino acid pairs. Of these 25 pairs, 21 have cumulative side chain sizes that account for a large proportion of human MASP-2-binding clones, as shown in Figure 6. The remaining four pairs essentially represent low abundance sets in the initial library, with A17 / G34 representing a very small size category and F17 / F34, F17 / Y34, and Y17 / F34 representing very large size categories. Because the number of predicted amino acid pairs in the initial library was essentially small, only heterogeneously strong positive selection could produce a statistically significant large proportion of these amino acid combinations.

[0253] For production, an expression system created by the inventors was used, for details of which see Example E.

[0254] [Example E] Heterologous expression of inhibitors All enzymes and reagents were obtained from Fermentas / Thermo Scientific. Reactions were performed according to the manufacturer's instructions. Between PCR reactions, annealing was performed at 50°C for 30 minutes, followed by 30 cycles. The PCR was performed using an Esco Swift Mini instrument. For DNA isolation, the Fermentas / Thermo Scientific GeneJet PCR purification kit (#K0701), Gel extraction kit (#K0691), and Plasmid miniprep kit (#K0502) were used according to the manufacturer's instructions. All DNA constructs were verified by Sanger sequencing using the ABI PRISM BigDye Terminator v3.1 Ready Reaction Cycle Sequencing Kit according to the manufacturer's instructions. Sequencing reaction products were analyzed by BIOMI Kft. (Goedöllöe, Hungary). The sequences of the oligonucleotides used in Section D.2.4.1 are listed in Table 12.

[0255] [Table 12] [Table 4] TIFF2025534392000090.tif242104

[0256] E.1. Creation of expression vectors for the production of proteins of the invention The genes for all precise proteins of the invention were expressed in the same bacterial expression vector construct applied in the invention described in WO2018 / 127719. The original vector is called pS100A4. Depending on the number and location of mutations, genes for many different proteins of the invention were constructed by four different methods as described in sections E.1.1. to E.1.4., in each case placing the gene between the unique BamHI and XhoI sites of the vector. All approaches used the following configuration: His6-tag-S100A4 protein-linker peptide-TEV cleavage site-protein of the present invention The same type of fusion gene construct was generated encoding a fusion protein having the following structure:

[0257] The construct allows high-level expression of the fusion protein in E. coli, purification by immobilized metal ion affinity chromatography via the His6-tag, and release of the protein of the invention by TEV protease (Tobacco Etch Virus protease).

[0258] E.1.1. To create expression vectors for the production of the following variants: EVO21 (SEQ ID NO: 12), EVO22 (SEQ ID NO: 7), EVO23 (SEQ ID NO: 3), EVO24 (SEQ ID NO: 15), and EVO25 (SEQ ID NO: 11). The five proteins of the present invention, described below under heading E.1.2, were generated by a three-step megaprimer mutagenesis method using the pS100A4-EVO2 expression vector (acting as a template) containing the gene for the EVO2 (SEQ ID NO: 2) mutant.

[0259] In the first polymerase chain reaction (PCR) step, the NL_3' mutagenesis primer (SEQ ID NO: 67) paired with the S100A4_seq primer (SEQ ID NO: 68) was used to create the modified EVO2 gene segment (to be shared by these proteins of the present invention). The product was purified using the GeneJet PCR Purification Kit. This purified product was used as a forward megaprimer with the T7rev primer (SEQ ID NO: 69) in a reaction using pS100A4-EVO2 as a template. The reaction generated a modified EVO2 gene with a mutation in its first half. The PCR was purified using the GeneJet PCR Purification Kit.

[0260] In the second PCR step, five separate PCRs were performed. Each reaction contained one mutant-specific mutagenesis primer (GLV for EVO21) paired with a common T7rev primer (SEQ ID NO: 69). 5' (SEQ ID NO: 70); YLV for EVO22 5' (SEQ ID NO: 71); YFV for EVO23 5' (SEQ ID NO: 72); YLE for EVO24 5' (SEQ ID NO: 73); and YFE for EVO25 5' (SEQ ID NO: 74)). The sequences of the primers are listed in Table 12. The products from five separate PCRs were individually purified using the GeneJet PCR Purification Kit and used as megaprimers in the third PCR step.

[0261] In the third PCR step, the purified product from the first PCR step was used as a template, and the S100A4_seq primer (SEQ ID NO: 67) was used as the forward primer in five separate reactions, paired with one of the five purified megaprimers from the second step to generate the final mutant genes. These mutant genes were cloned into the pS100A4 fusion protein expression vector using BamHI and XhoI enzymes as follows: The mutant PCR product and pS100A4 vector were digested with BamHI (10 U) and XhoI (20 U) in 1X BamHI buffer at 37°C. The digested DNA was run on an agarose gel, and the appropriate size fragment was excised and isolated. The DNA was eluted from the column in 30 μl of 0.1xEB. The concentration of the isolated DNA was measured using a BioTek Epoch reader, a Take3 Trio microvolume plate, and Gene5 software. Genes with amino acid sequences according to EVO21 (SEQ ID NO: 12), EVO22 (SEQ ID NO: 7), EVO23 (SEQ ID NO: 3), EVO24 (SEQ ID NO: 15), and EVO25 (SEQ ID NO: 11) were ligated into the vector using T4 DNA ligase. A 5-fold molar excess of PCR product was present in the ligase reaction.

[0262] XL1 Blue cells were transformed with the product of the ligase reaction as described in Section D.2.1.3.1.1. and plated on LB / agar + ampicillin (100 μg / ml). Plates were incubated at 37° C. for 16 hours.

[0263] Individual colonies of transformed cells were picked into LB + ampicillin (100 μg / ml) and incubated at 37°C with shaking at 220 rpm for 16 hours. Plasmid DNA was isolated from the culture. DNA was eluted from the column with 50 μl of 0.1xEB.

[0264] E.1.2. To create expression vectors for the production of proteins according to EVO221 (SEQ ID NO: 21), EVO222 (SEQ ID NO: 18), EVO223 (SEQ ID NO: 19), and EVO224 (SEQ ID NO: 28). The protein of the present invention described under the heading E.1.2. was generated by a two-step megaprimer mutagenesis method using the pS100A4-EVO22 expression vector containing the gene with the amino acid sequence according to EVO22 (SEQ ID NO: 7) as a template.

[0265] In the first PCR step, the S100A4_seq primer (SEQ ID NO: 68) was used in pairs with one of four mutant-specific mutagenesis primers: VRAAAV_3' (SEQ ID NO: 75), LRAAAV_3' (SEQ ID NO: 76), PRAAAV_3' (SEQ ID NO: 77), and VRALAV_3' (SEQ ID NO: 78) to generate megaprimers for the next step. The primer sequences are shown in Table 12. Products from four separate PCRs were purified using a GeneJet PCR Purification Kit.

[0266] In the second PCR step, pS100A4-EVO22 was used as a template, and the purified product of the first PCR step was used as a forward megaprimer paired with the T7rev primer (SEQ ID NO: 69). The products of these four separate PCRs were purified using the GeneJet PCR Purification Kit.

[0267] The mutant genes were cloned into the pS100A4 fusion expression vector using BamHI and XhoI enzymes. The mutated PCR products and the pS100A4 vector were digested with BamHI (10 U) and XhoI (20 U) in 1X BamHI buffer at 37°C for 3 hours. The digested DNA was run on an agarose gel, and fragments of the appropriate size were excised and isolated. The DNA was eluted from the column with 30 μl of 0.1xEB. The concentration of the isolated DNA was determined using a BioTek Epoch reader, a Take3 Trio microvolume plate, and Gene5 software. Genes with amino acid sequences according to EVO221 (SEQ ID NO: 21), EVO222 (SEQ ID NO: 18), EVO223 (SEQ ID NO: 19), and EVO224 (SEQ ID NO: 28) were ligated into the vector using T4 DNA ligase. A 5-fold molar excess of PCR product was present in the ligase reaction. XL1 Blue cells were transformed with the products of the ligase reaction as described in Section D.2.1.3.1.1. and plated onto LB / agar + ampicillin (100 μg / ml) plates. Plates were incubated at 37°C for 16 hours.

[0268] Individual colonies of transformed cells were picked into LB + ampicillin (100 μg / ml) and incubated at 37°C with shaking at 220 rpm for 16 hours. Plasmid DNA was isolated from the culture. DNA was eluted from the column with 50 μl of 0.1xEB.

[0269] E.1.3. To create expression vectors for the production of proteins according to EVO2a (SEQ ID NO: 32), EVO2b (SEQ ID NO: 31), EVO2c (SEQ ID NO: 25), EVO2d (SEQ ID NO: 30), EVO21a (SEQ ID NO: 24), EVO211 (SEQ ID NO: 4), EVO212 (SEQ ID NO: 14), EVO213 (SEQ ID NO: 27), EVO214 (SEQ ID NO: 8), EVO215 (SEQ ID NO: 26), EVO216 (SEQ ID NO: 29), EVO22a (SEQ ID NO: 6), and EVO22b (SEQ ID NO: 22) The proteins of the invention described under the heading E.1.3. were generated by the QuikChange mutagenesis method using the pS100A4-EVO2 expression vector (40 ng) containing the gene with the amino acid sequence according to EVO2 (SEQ ID NO: 2) as a template. The names of the corresponding mutagenesis primers include the name of the inhibitor mutant. For example, the forward and reverse primers for generating EVO2a are named EVO2a_f (SEQ ID NO: 79) and EVO2a_r (SEQ ID NO: 80), respectively. All QuikChange primers are listed in Table 12. Mutants EVO21a (SEQ ID NO: 24), EVO211 (SEQ ID NO: 4), EVO212 (SEQ ID NO: 14), EVO213 (SEQ ID NO: 27), EVO214 (SEQ ID NO: 8), EVO215 (SEQ ID NO: 26), and EVO216 (SEQ ID NO: 29) were generated by QuikChange mutagenesis using the pS100A4-EVO2 expression vector containing the gene with the amino acid sequence according to EVO21 (SEQ ID NO: 12) as a template (40 ng). The corresponding QuikChange primers are listed in Table 12.

[0270] Mutants EVO22a (SEQ ID NO: 6) and EVO22b (SEQ ID NO: 22) were generated by QuikChange mutagenesis using the pS100A4-EVO2 expression vector containing the gene with the amino acid sequence according to EVO22 (SEQ ID NO: 7) as a template (40 ng). The corresponding QuikChange primers are listed in Table 12.

[0271] PCR was performed using 0.5 μM forward and reverse mutagenic primers (primer sequences are shown in Table 12), 1.25 U KOD polymerase (Sigma-Aldrich), with an annealing temperature of 70°C and an extension time of 6 minutes (20 cycles).

[0272] The reaction product was treated with 0.5 U DpnI at 37°C for 1 hour to digest the methylated template DNA.

[0273] XL1 Blue cells were transformed with the products of the DpnI reaction as described in section 1.3.1.1 and plated on LB / agar + ampicillin (100 μg / ml). Plates were incubated at 37° C. for 16 hours.

[0274] Individual colonies of transformed cells were picked into LB + ampicillin (100 μg / ml) and incubated at 37°C with shaking at 220 rpm for 16 hours. Plasmid DNA was isolated from the culture. DNA was eluted from the column with 50 μl of 0.1xEB.

[0275] E.1.4. E To create expression vectors for the production of proteins according to EVO21b (SEQ ID NO: 9), EVO21c (SEQ ID NO: 13), EVO21d (SEQ ID NO: 16), EVO211a (SEQ ID NO: 20), EVO214a (SEQ ID NO: 17), EVO22d (SEQ ID NO: 10), and EVO23a (SEQ ID NO: 5). The coding genes for mutants EVO21b (SEQ ID NO: 9), EVO21c (SEQ ID NO: 13), EVO21d (SEQ ID NO: 16), EVO211a (SEQ ID NO: 20), EVO214a (SEQ ID NO: 17), EVO22d (SEQ ID NO: 10), and EVO23a (SEQ ID NO: 5) were purchased as synthetic genes and introduced into the pS100A4-EVO2 expression vector by cassette exchange. The sequences of the sense strands of the synthetic genes were designated EVO21b_DNA (SEQ ID NO: 107), EVO21c_DNA (SEQ ID NO: 108), EVO21d_DNA (SEQ ID NO: 109), EVO211a_DNA (SEQ ID NO: 110), EVO214a_DNA (SEQ ID NO: 111), EVO22d_DNA (SEQ ID NO: 112), and EVO23a_DNA (SEQ ID NO: 113). The sequences of the sense strands of the synthetic genes are shown in Table 13.

[0276] [Table 13] Synthetic genes of EVO21b_DNA (SEQ ID NO: 107), EVO21c_DNA (SEQ ID NO: 108), EVO21d_DNA (SEQ ID NO: 109), EVO211a_DNA (SEQ ID NO: 110), EVO214a_DNA (SEQ ID NO: 111), EVO22d_DNA (SEQ ID NO: 112), and EVO23a_DNA (SEQ ID NO: 113) TIFF2025534392000091.tif230153

[0277] The mutated gene was cloned into the pS100A4 fusion expression vector using BamHI and XhoI enzymes. The mutated PCR product and the pS100A4-EVO2 vector were digested with BamHI (10 U) and XhoI (20 U) in 1X BamHI buffer at 37°C for 3 hours. The digested DNA product was run on an agarose gel, and fragments of the appropriate size were excised and isolated. The DNA was eluted from the column in 30 μl of 0.1xEB. The concentration of the isolated DNA molecules was determined using a BioTek Epoch reader, a Take3 Trio microvolume plate, and Gene5 software. DNAs designated VO221_DNA, EVO222_DNA, EVO223_DNA, and EVO224_DNA were ligated to the vector using T4 DNA ligase. A 5-fold molar excess of PCR product was present in the ligase reaction.

[0278] XL1 Blue cells were transformed with the product of the ligase reaction as described in Section D.2.1.3.1.1. and plated on LB / agar + ampicillin (100 μg / ml). Plates were incubated at 37°C for 16 hours.

[0279] Individual colonies of transformed cells were picked into LB + ampicillin (100 μg / ml) and incubated at 37°C with shaking at 220 rpm for 16 hours. Plasmid DNA was isolated from the culture. DNA was eluted from the column with 50 μl of 0.1xEB. Individual colonies of transformed cells were picked into LB + ampicillin (100 μg / ml) and incubated at 37°C with shaking at 220 rpm for 16 hours. Plasmid DNA was isolated from the culture. DNA was eluted from the column with 50 μl of 0.1xEB.

[0280] The expression vector constructs used for refolding all 29 single domain proteins of the present invention are shown in Figure 7, using EVO24 as an example.

[0281] E.2. Bacterial production of recombinant proteins For protein expression, we used Escherichia coli Shuffle B (NEB, C3028J), a strain genetically engineered to allow disulfide bond formation in the cytoplasm. This strain also expresses the disulfide bond isomerase and chaperone protein DsbC in the cytoplasm to aid protein folding by supporting the formation of the most stable native disulfide bond pattern (Lobstein 2012).

[0282] E.2.1. transformation One μl of expression vector and 100 μl of Shuffle B competent cells were used. The cells were incubated on ice for 30 minutes and then heat-shocked at 42°C for 1 minute. 200 μl of LB medium was added to the cells, shaken at 37°C for 30 minutes, and then plated onto an LB / agar + ampicillin (100 μg / ml) plate. The plate was incubated at 30°C overnight.

[0283] E.2.2. Biomass Production To serve as inoculum, the cells on the plate were washed into 30 ml of LB + ampicillin (75 μg / ml) and shaken overnight at 30°C. One liter of Terrific Broth (TB) medium (12 g trypsin, 24 g yeast extract, 4 ml glycerol in 900 ml water, to which 100 ml of a buffer containing 0.72 M KHPO and 0.17 M KHPO) was added into a 2.8 L Fernbach flask and supplemented with ampicillin to a final concentration of 75 μg / ml. The flask was preincubated at 30°C with shaking at 180 rpm, after which the inoculum was added and the culture was allowed to stand for 1 hour. 600 nm The flasks were shaken until a pH value of 0.8 was reached. At this point, expression of the recombinant gene was induced by adding IPTG solution to a final concentration of 0.1 mM, and the cultures were shaken at 18°C ​​for an additional 16–20 hours. The cells were then pelleted by centrifugation (5 minutes, 7,500 x g, 4°C), the supernatant was discarded, the wet mass of the cell pellet was measured, and the cell pellet was suspended in an appropriate volume of 50 mM Tris-HCl, 500 mM NaCl buffer at a cell pellet wet mass / cell suspension volume ratio of 1 g / 5 ml.

[0284] E.2.3. Protein purification Cells were disrupted by sonication, and the sample was centrifuged (20 min, 48,000 x g) to remove cell debris. The supernatant, containing the fusion protein and other soluble cytoplasmic components, was loaded onto an IMAC column (10 ml BioRad Profinity IMAC resin) containing immobilized nickel ions. The column was equilibrated with 50 mM Tris-HCl, 500 mM NaCl, pH 8.0 buffer (IMAC buffer). After loading the sample onto the column, the column was washed with 10 column volumes of IMAC buffer. The His-tagged S100A4-fusion inhibitor was eluted with 50 mM Tris-HCl, 250 mM imidazole, 300 mM NaCl, pH 8.0 buffer (IMAC elution buffer).

[0285] To reduce the concentration of imidazole in the sample, the eluted fusion protein was dialyzed against 20 mM Tris-HCl pH 8.0, 150 mM NaCl (dialysis buffer) for 3 h at room temperature using a dialysis tubing cellulose membrane with a cutoff of 12–14 kDa (Sigma D9527).

[0286] E.2.4. Protein Processing Protease cleavage was carried out in a buffer containing 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM reduced glutathione, and 0.1 mM oxidized glutathione (proteases were added at molar ratios of 1:50 to 1:100).

[0287] The reaction was incubated for 16 hours at room temperature. A His-tagged version of TEV protease was produced in-house as described by van den Berg (2006) with modifications, and the purified enzyme was stored at -80°C in the presence of 1 mM TCEP. The extent of degradation was tested by SDS-PAGE on a 15% Tris-Tricine gel.

[0288] E.2.5. Isolation of the Protein of the Invention At this stage of the procedure, the major components of the sample are the processed protein (without the His tag), His6-tagged S100A4, His6-tagged TEV protease, and possible minor amounts of unprocessed fusion protein. The sample was centrifuged to remove any precipitate and reloaded onto an IMAC column equilibrated with dialysis buffer. The His6-tagged protein component of the sample was captured by the immobilized nickel ions of the resin, while the processed protein was present in the flow-through fraction.

[0289] The flow-through fraction was collected and dialyzed against 150 mM acetate / NH4 (gel filtration buffer) at room temperature for 3 hours using a dialysis tubing cellulose membrane (Thermo 68035) with a 3.5 kDa cutoff. After lyophilization, the protein of the present invention was resuspended in gel filtration buffer and reloaded onto a Superdex 30 HiLoad 16 / 60 column equilibrated with gel filtration buffer. The major peak was collected. The molar concentration of the protein of the present invention sample was determined based on UV absorption at 280 nm, and the sample was divided into aliquots, lyophilized, and stored at 4°C.

[0290] E.2.6. Isolation of the Proteins of the Invention for In Vivo Studies For samples prepared for in vivo studies, a cation exchange column chromatography step was introduced prior to the gel filtration step to further minimize nucleic acid and endotoxin contamination. The flow-through fraction from the second IMAC step containing the protein of the invention was dialyzed against 8 mM acetic acid / NH₄ / 42 mM acetic acid, pH 4.0 buffer, and the sample was loaded onto a 5 ml HiTrap SP HP cation exchange chromatography column (15 ml / min) (Cytiva 17115201) equilibrated with the same buffer. After extensive washing with dialysis buffer (15 ml / min), the protein of the invention was eluted with a gradient generated by switching to 150 mM acetic acid / NH₄, the gel filtration buffer (8 CV, 2 ml / min).

[0291] After lyophilization, the protein of the present invention was resuspended in gel filtration buffer and loaded onto a Superdex 30 HiLoad 16 / 60 column equilibrated with gel filtration buffer. The major peak was collected. The molar concentration of the protein of the present invention sample was determined based on UV absorption at 280 nm, and the sample was divided into aliquots, lyophilized, and stored at 4°C.

[0292] E.3. To generate an Fc-fused version of EVO24 (SEQ ID NO: 15), designated EVO24L (SEQ ID NO: 114). It will be apparent to those skilled in the art that the Kunitz domain proteins of the present invention can be combined with other proteins or portions of other proteins, so that the Kunitz domain protein maintains its function according to the present invention, but, as expected, in the context of a chimeric protein, acquires new or enhanced beneficial properties provided by the other protein portions.

[0293] One of the most widely known examples of this approach is to equip a peptide or protein of the invention with an antibody Fc domain. This Fc domain can be intact or modified and can confer a variety of predictable properties through protein-protein interactions. These predictable properties are strictly controlled by the amino acid sequence and the presence or absence of various post-translational modifications of a particular Fc domain. For example, some Fc domains can form stable monomers, homodimers, heterodimers, or larger multimers; some can bind to the nascent Fc-receptor protein (FcRn), resulting in extended in vivo half-life of the chimeric protein; and some can bind to other host proteins and mediate various functional implications, such as classical complement pathway activation or immune cell activation. Those skilled in the art are aware that bacterial expression produces non-glycosylated proteins, and that loss of Fc-glycosylation does not impair the half-life-extending properties of such Fc domains but eliminates many of their immunostimulatory effector functions.

[0294] In this example, the above-described principle was applied by expressing the EVO24 (SEQ ID NO: 15) of the present invention fused to an IgG1-type Fc domain that forms a stable homodimer, the protein being designated EVO24L (SEQ ID NO: 114).

[0295] E.3.1. DNA construct enabling expression of EVO24L (SEQ ID NO: 114) The starting version of the DNA encoding the EVO24-Fc domain fusion was obtained as a synthetic gene and introduced into a bacterial expression vector that provides an N-terminal His-tag coding sequence that is cut off by the WELQut (also known as SplB) protease. In this DNA construct, designed restriction enzyme sites allow for easy substitution of a linker between the N-terminal EVO24 Kunitz segment and the C-terminal Fc domain, along with segments within the Fc domain that affect the monomeric / dimeric nature of the domain.

[0296] Starting from this synthetic DNA construct, simple recombinant DNA methods were used to express the optimized construct presented in this example. The relevant portions of the final DNA construct and the encoded protein are shown in Figures 8 and 9, respectively.

[0297] The amino acid sequence of the WELQut-processed form of EVO24L corresponds to SEQ ID NO:114.

[0298] E.3.2. Bacterial production and isolation of EVO24L The protocol for the production and isolation of EVO24L (SEQ ID NO: 114) was very similar to that detailed in the corresponding section in E.2. Therefore, only the differences are described. Proteolytic removal of the His-tag was performed using His-tagged WELQut protease, which we produced as a recombinant protein. Unlike TEV protease, the enzyme does not require reducing conditions for its activity; therefore, proteolytic processing can be carried out in a standard oxidizing environment, making it ideal for processing disulfide-containing proteins. After the second IMAC step, the pH of the flow-through fraction containing processed EVO24L (SEQ ID NO: 114) was adjusted to 7.0 with 1 M NaH2PO4 buffer, and the sample was loaded onto a 50 ml XK 26 / 20 column containing Cytiva HiTrap Q HP anion exchange resin (#17101401) equilibrated with a buffer (AEX buffer A) of the same composition as the pH-adjusted dialysis buffer. The column was washed with AEX buffer. At this pH, EVO24L flowed through, while the majority of contaminants, including nucleic acids and other endotoxins, were captured by the column. The AEX flow-through was concentrated using Pierce™ Protein Concentrator PES, 10K MWCO, 5-20 ml (#88527), and the concentrated sample was loaded onto a Cytivia HiLoad 26 / 600 Superdex 200 size exclusion column equilibrated with vehicle buffer containing 25 mM Na phosphate pH 6.3, 100 mM NaCl, and 2.5% (wt / vol) sucrose. The EVO24L-containing peak was collected, and the sample was reconcentrated into a new Pierce™ Protein Concentrator PES, 10K MWCO, 5-20 ml (#88527) tube. The sample concentration (ε 280 =41.995 M -1 cm -1 ) and adjusted to 1 mM. Samples were tested by analytical gel filtration and C4 deposition serology assay for lectin pathway inhibitory potency.

[0299] E.3.3. Lectin pathway inhibitory potency of EVO24L (SEQ ID NO: 114) As described in 5.4.1, the IC of the EVO24L sample 50 The IC value of EVO24L was measured. The internal standard was EVO24 (SEQ ID NO: 15). The molar concentration of the Kunitz domain portion of dimeric EVO24L was examined. 50 This value is 2.5 times higher than that of EVO24, suggesting that although the domain portion of Kunitz is fully functional, in a given test only one Kunitz domain can bind to the immobilized MASP-2 target at a time.

[0300] [Example F] Functional characteristics of inhibitors F.1. LC-MS analysis of the protein of the present invention The accurate molecular weight of the protein of the present invention was verified by mass spectrometry experiments on a high-resolution hybrid quadrupole time-of-flight mass spectrometer (Waters Select Series Cyclic IMS, Waters Corp., Wilmslow, UK). The mass spectrometer was operated in positive W mode. Leucine enkephalin was used as the lock mass standard. Ionization was performed using a ZSpray ion source operated under the following parameters: capillary voltage: 2 kV, cone gas flow rate: 20 L / h, desolvation gas flow rate: 800 L / h, desolvation temperature: 400°C, nebulizer gas: 6 bar, source temperature: 120°C. Chromatographic separation was performed on a Waters Acquity I-Class UPLC system directly coupled to the mass spectrometer. RPLC-MS analysis was performed on a Waters Acquity BEH300 C4 UPLC column (2.1 x 150 mm, 1.7 μm) under the following parameters: mobile phase A: 0.1% trifluoroacetic acid in water; mobile phase B: 0.1% trifluoroacetic acid in acetonitrile; flow rate: 400 μl / min; column temperature: 80 °C; gradient: 2 min: 5% B, 8 min: 45% B, 8.5 min: 90% B, 9 min: 90% B, 9.1 min: 5% B, 12 min: 5% B. UV detection was performed at 220 and 280 nm. The m / z range was 300–2000. Data collection and analysis were performed using MassLynx 4.2 software. Mass analysis accuracy was better than 5 ppm.

[0301] F.2. K for human and rat MASP-2 I Measuring Constants The equilibrium inhibition constants (K) of the nine proteins of the present invention designed and generated based on the first and second stages of the directed evolution campaign IThe activity of human MASP-2 (5 nM) and rat MASP-2 (2 nM) was measured for the following variants: EVO21 (SEQ ID NO: 12), EVO22 (SEQ ID NO: 7), EVO23 (SEQ ID NO: 3), EVO24 (SEQ ID NO: 15), EVO25 (SEQ ID NO: 11), EVO221 (SEQ ID NO: 21), EVO222 (SEQ ID NO: 18), EVO223 (SEQ ID NO: 19), and EVO224 (SEQ ID NO: 28).

[0302] K of the protein of the invention for MASP enzymes I For the measurement of ZL-Lys-SBzl, a catalytic enzyme fragment containing three C-terminal domains: CCP1, CCP2, and SP was used. The synthetic substrate used in the device was ZL-Lys-SBzl hydrochloride (Sigma, C3647), from which a 10 mM stock solution was prepared. The reaction was carried out in a volume of 0.2 ml at room temperature in a buffer solution containing 20 mM HEPES, 145 mM NaCl, 5 mM CaCl2, and 0.05% Triton-X100, pH 7.4. Free thiols were generated upon enzyme-catalyzed hydrolysis of the thioester bond in the substrate, which reacted with the co-substrate 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent, Sigma, D8130), present in solution in a twofold excess relative to ZL-Lys-SBzl. This resulted in the release of a chromogenic group, which was monitored via the increase in absorbance at 410 nm using a BioTek Synergy H4 multimode microplate reader.

[0303] Serial dilutions were prepared from individual inhibitors, and after addition of the enzyme, complex formation was continued for 2 hours at room temperature. Samples were transferred onto a 96-well microplate (Nunc 269620). The reaction was initiated by adding a mixture of substrate and co-substrate to the sample at final concentrations of 250 μM and 500 μM, respectively. The substrate concentration and data collection time were optimized to achieve less than 10% substrate consumption within the measurement time frame, i.e., to maintain a substantially constant rate of product formation.I To measure the δ values, we used a method developed for the characterization of tight-binding (slow-dissociating) inhibitors (Empie 1982), later modified (Szakacs 2019). The rate of product formation was measured by measuring the change in absorbance, which is a linear function of product concentration as a function of reaction time.

[0304] The reaction rate measured for the inhibitor-containing samples was divided by the reaction rate corresponding to the inhibitor-free samples. This ratio was multiplied by the total enzyme concentration to obtain the free enzyme concentration in the inhibitor-containing samples. These free enzyme concentration data were then plotted as a function of total inhibitor concentration and nonlinearly fitted to the equation: I The values ​​were calculated:

number

[0305] [Table 14] Equilibrium inhibition constant values ​​of the protein of the present invention for human and rat MASP-2 TIFF2025534392000093.tif120153

[0306] The results demonstrated that eight of the nine proteins of the present invention were more potent inhibitors of human MASP-2 than EVO2 (SEQ ID NO: 2). In fact, for these eight proteins, binding was too strong to be accurately measured. Therefore, an independent method, surface plasmon resonance (SPR), was used.

[0307] F.3. Surface plasmon resonance-based kinetic parameters and affinity of the proteins of the present invention for human and rat MASP-2 To accurately measure the affinity of the strongest binding inhibitors according to the present invention for human and rat MASP-2 catalytic fragments, surface plasmon resonance spectroscopy was applied using a Bio-Rad ProteOn™ XPR36 Protein Interaction Array System.

[0308] The K of the nine proteins of the present invention described above I Based on these values, 13 additional proteins were designed, generated, and isolated, some of which were designed to better understand protein sequence-activity relationships, and others to identify additional ultra-potent MASP-2 inhibitors.

[0309] Therefore, a total of 22 proteins of the present invention, including the nine previous proteins and 13 novel proteins, were tested together with EVO2 by the SPR method. The list of 22 proteins is as follows: EVO21 (SEQ ID NO: 12), EVO22 (SEQ ID NO: 7), EVO23 (SEQ ID NO: 3), EVO24 (SEQ ID NO: 15), EVO25 (SEQ ID NO: 11), EVO221 (SEQ ID NO: 21), EVO222 (SEQ ID NO: 18), EVO223 (SEQ ID NO: 19), and EVO224 (SEQ ID NO: 28), EVO2a (SEQ ID NO: 32), EVO2b (SEQ ID NO: 31), EVO2c (SEQ ID NO: 25), EVO2d (SEQ ID NO: 30), EVO21a (SEQ ID NO: 24), EVO211 (SEQ ID NO: 4), EVO212 (SEQ ID NO: 5). ID NO: 14), EVO213 (SEQ ID NO: 27), EVO214 (SEQ ID NO: 8), EVO215 (SEQ ID NO: 26), EVO216 (SEQ ID NO: 29), EVO22a (SEQ ID NO: 6), and EVO22b (SEQ ID NO: 22).

[0310] In addition to the binding affinity, the rate of complex formation and dissociation, the SPR method also measures the delivery association rate coefficient (k on ) and dissociation rate coefficient (k off ) values ​​were also quantitatively evaluated.

[0311] Human or rat MASP-2 catalytic fragments were covalently immobilized to a Bio-Rad ProteOn™ GLC Sensor Chip (15 μg / ml in 10 mM Na acetate pH 4.5) to a ligand density of 2500 RU. Inhibitors were injected onto the chip in two-fold serial dilutions over five points, supplemented with a buffer control, using a running buffer containing 20 mM HEPES pH 7.4, 150 mM NaCl, 2 mM CaCl2, 0.5 mM MgCl2, 0.005% Tween-20, and 6 mM NaN3. Rate coefficients were obtained by global fitting of the double-reference association and dissociation phases using a 1:1 Langmuir model. To control for measurement reproducibility, EVO2 (SEQ ID NO: 2) was injected periodically throughout the course of the experiment as an internal standard. K for repeated measurements of EVO2 was calculated. d and R max The relative standard deviations for both were acceptable (<30%). The results are shown in Table 15.

[0312] [Table 15] SPR-based rate and affinity values ​​for proteins of the invention [Table 5]

[0313] F.4. Effects of the protein of the present invention on three complement activation pathways in human serum

[0314] F.4.1. Inhibitory potency of the proteins of the present invention on the human lectin pathway As outlined above, the complement system is activated through three pathways (which converge at the level of the C3 convertase). The three activation pathways are the classical pathway, the lectin pathway, and the alternative pathway. MASP-1 and MASP-2 are lectin pathway-specific proteases, and both are key enzymes in lectin pathway activation. Complete inhibition of either of these proteases completely blocks lectin pathway activation. The protein inhibitors of the present invention are therefore expected to block lectin pathway activation but not affect the convertase enzymes of the other two pathways or the common complement route.

[0315] The so-called WIELISA kit (Euro-Diagnostica AB, COMPL300) was developed to selectively measure activation of the three complement pathways. The kit is compatible with three different conditions, each in which only one of the three pathways is activated, while the other two remain inactive. The kit detects the C9 neoepitope in the C5-9 complex, the final component of complement activation in the merged route of the three pathways. However, Kocsis et al. (2010) developed another assay for the same purpose. This assay follows the principle of the WIELISA kit. Pathway activation is measured by detecting activated C3 or C4 fragments or the C5-9 neoepitope via antibodies specific for the aforementioned complement components. This method was used to evaluate the inhibitory potency of the protein of the present invention as a MASP-2 inhibitor.

[0316] The assay was performed using normal human serum (Quidel Corporation, A113). A 5 ml aliquot was thawed on ice, divided into aliquots, and stored at -80°C until use. The assay was performed as described by Kocsis et al. (Kocsis 2010) with modifications. A 96-well Greiner high-binding ELISA plate (cat. no. 655061) was coated overnight at 4°C with 100 μl / well of 10 μg / ml mannan dissolved in coating buffer (50 mM sodium carbonate, pH 9.6). Control wells contained coating buffer only. The walls were blocked for at least 1 hour at 37°C with 200 μl / well of 10 mg / ml bovine serum albumin (BSA) dissolved in 50 mM Tris pH 7.4, 150 mM NaCl buffer.

[0317] Normal human serum was thawed on ice and diluted in 20 mM HEPES pH 7.4, 5 mM CaCl2, 5 mM MgCl2, 150 mM NaCl, 0.1% Tween-20 (serum dilution buffer). Serial dilutions of inhibitors were made in serum dilution buffer and added to the diluted serum samples to reach a final serum dilution of 50. Samples were incubated at room temperature for 30 minutes.

[0318] The ELISA was thoroughly washed with 50 mM Tris pH 7.4, 5 mM CaCl2, 150 mM NaCl, 0.1% Tween-20 (wash buffer), and then the pre-incubated serum samples were transferred to the plates. Two negative controls were performed. In one, diluted serum samples without inhibitor were transferred to a surface treated with BSA only. In the other negative control, diluted serum was transferred to a mannan-coated surface supplemented with EDTA (ethylenediaminetetraacetic acid) to a final concentration of 20 mM. EDTA is a Ca 2+ and Mg 2+Ion-dependent "downstream" complement activation is prevented through chelating these ions. To assess maximal complement activity, 50-fold diluted serum without inhibitors was also transferred to the mannan-coated plate. The plate was incubated for 30 minutes at 37°C, washed with wash buffer, and 100-100 μl of α-human C4c antibody (rabbit) (DakoCytomation-Q0369) diluted 3000-fold in wash buffer containing 10 mg / ml BSA (antibody buffer) was pipetted into the wells. The plate was incubated for 60 minutes at 37°C. The plate was washed again, and 100 μl / well of peroxyconjugated α-rabbit IgG monoclonal antibody (mouse) (Sigma-A1949) diluted 40,000-fold in antibody buffer was transferred to the plate. The plate was then incubated for 30 minutes at 37°C. The plate was then rinsed with wash buffer. Next, 100 μl / well of 1 mg / ml o-phenylenediamine dihydrochloride (OPD, Sigma-P9029) peroxidase substrate diluted in 50 mM citrate pH 5.0, 0.1% H2O2 buffer was transferred to the plate to generate a photometric signal proportional to the amount of C4 deposited on the surface. After signal development, the reaction was stopped by adding 50 μl / well of 1 M sulfuric acid. Absorbance at 490 nm was recorded using a BioTek Synergy H4 Hybrid reader. Three parallel measurements were performed for each data point. 0% serum activity was represented by a serum sample containing 20 mM EDTA, and 100% activity was represented by a serum sample without added inhibitor.

[0319] Data were analyzed using Origin Pro 8 software, and the inhibitor concentration providing 50% inhibition of C4 deposition (IC 50 ) was determined by fitting the data with the DoseReso function (a pharmacology built-in equation set). In this experiment, the complement inhibitory potency of the proteins of the invention was compared to that of EVO2. All measurements were performed from a single thawed serum sample at the same time and on the same plate, and the IC of EVO2 measured on the same plate. 50 In comparison with the IC value, the IC50 was evaluated.

[0320] I C 50 Since values ​​may depend on the actual serum sample used, the data were normalized via the following steps: i) IC of the mutants used in the experiment 50 The value is the IC of EVO2 in a given experiment. 50 expressed as a fraction of the value; ii) EVO2 IC from measurements 50 Calculate the average of the values ​​and use the "Average IC" 50 value"; iii) IC of the protein of the present invention used in the experiment 50 Average IC values 50 Recalculate the "normalized IC" as a fraction of (step i) 50 "

[0321] Through this normalization process, the inhibitory potency of each protein of the present invention could be directly compared to the others to determine the most potent lectin pathway inhibitors. The results are shown in Table 16.

[0322] The data demonstrated that the further evolved proteins of the invention are up to 48-fold more efficient inhibitors of the human lectin pathway than EVO2. The IC of the most effective variant 50 The values ​​range from 2 to 10 nM.

[0323] [Table 16] Ability of the protein of the present invention to inhibit the lectin pathway [Table 6]

[0324] F.4.2. Evaluating the effects of the proteins of the present invention on human classical and alternative pathway activation Based on their high inhibitory potency against both human and rat lectin pathway activation, four proteins of the invention were selected to test their pathway specificity: EVO21 (SEQ ID NO: 12), EVO214 (SEQ ID NO: 8), EVO23 (SEQ ID NO: 3), and EVO24 (SEQ ID NO: 15). Evaluation was performed similarly to that described in F.4.1., with the following modifications: i) for selective classical pathway activation, 10 μg / ml aggregated human IgG was immobilized on ELISA plates at 100 μl / well; ii) the final dilution of serum was 60-fold for classical pathway measurements and 6-fold for alternative pathway measurements.

[0325] For alternative pathway measurements, 20 mM HEPES pH 7.4, 5 mM MgCl, 20 mM EGTA, 150 mM NaCl, and 0.1% Tween-20 were used instead of serum dilution buffer. The EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid) component chelates calcium ions, which specifically blocks classical and lectin pathway activation (which, unlike the alternative pathway, are calcium-dependent). iii) Pathway activity was assessed using α-human anti-C3c antibody (rabbit) (DakoCytomation-A0062) at a 1:2000 dilution for the classical pathway and a 1:5000 dilution for the alternative pathway.

[0326] In the first experiment, the proteins of the invention were tested at a fixed concentration of 10 μM. Negative and positive controls were prepared as described in 5.4.1. The four proteins of the invention were tested for their lectin-based inhibitory IC 50 At concentrations three orders of magnitude higher than the RI values, the proteins exerted low to moderate inhibition of the classical pathway and negligible inhibition of the alternative pathway, demonstrating that these proteins of the present invention are lectin pathway specific. The results are shown in Table 17.

[0327] [Table 17] Effects of four selected proteins of the present invention at a concentration of 10 μM on human classical and alternative complement pathway activity TIFF2025534392000096.tif61153

[0328] The lack of classical and alternative pathway inhibition demonstrates that these proteins of the invention do not inhibit the following serine proteases: C1r, C1s, factor D, factor B (i.e., C3bBb-type C3-convertase), and C2 (i.e., C4b2a-type C3-convertase), demonstrating the high target specificity of EVO21 (SEQ ID NO: 12), EVO214 (SEQ ID NO: 8), EVO23 (SEQ ID NO: 3), and EVO24 (SEQ ID NO: 15).

[0329] F.5. The effects of these proteins of the present invention on the three complement activation pathways in rat serum

[0330] F.5.1. Inhibitory ability of the protein of the present invention on the rat lectin pathway The efficacy of the proteins of the invention to inhibit the lectin system in rat serum was tested essentially as described for the human lectin pathway in F.4.1., with two modifications: rat serum was used at a 60-fold dilution and a 2000-fold dilution for the detection of deposited C4 fragments. α-Human C4c antibody (DakoCytomation-Q0369) recognizes rat C4 fragments. Pooled rat serum was used. Data evaluation was as described in F.4.1.

[0331] All but two proteins of the invention, EVO215 (SEQ ID NO: 26) and EVO216 (SEQ ID NO: 29), are potent inhibitors of the lectin pathway in rat serum. The results are shown in Table 16 in Example F, section F.4.1.

[0332] F.5.2. Evaluating the effects of the proteins of the present invention on classical and alternative pathway activation in rats The specificity of the four selected proteins of the present invention was evaluated in human blood as described in F.4.2. The results are shown in Table 18.

[0333] [Table 18] Effects of four selected proteins of the present invention at a concentration of 10 μM on rat classical and alternative complement pathway activity TIFF2025534392000097.tif66153

[0334] Proteins EVO214 (SEQ ID NO: 8) and EVO23 (SEQ ID NO: 3) showed significant inhibitory activity against the alternative pathway in rat serum, so the two inhibitors were evaluated by serial dilution and the IC values ​​of these inhibitors were calculated. 50 The values ​​were determined using the method described in F.4.2. The results are shown in Table 19 and discussed at the end of this section.

[0335] [Table 19] EVO214 (SEQ ID NO: 8) and EVO23 (SEQ ID NO: 3) inhibit the rat alternative pathway only at high concentrations TIFF2025534392000098.tif49156

[0336] EVO23 (SEQ ID NO: 3) provides greater than 80% inhibition of the alternative pathway in rat serum, but only at high (10 μM) concentrations, while EVO214 (SEQ ID NO: 8) provides only 39% inhibition, neither of which exerts significant inhibition of the alternative pathway at concentrations below 5 μM.

[0337] Overall, the proteins of the present invention are potent inhibitors of the lectin pathway in rat serum, -7 ~10 -9 IC in the range of M 50The four selected proteins, EVO21 (SEQ ID NO: 12), EVO214 (SEQ ID NO: 8), EVO23 (SEQ ID NO: 3), and EVO24 (SEQ ID NO: 15), did not inhibit the alternative pathway in rat serum and exerted inhibitory effects on the alternative pathway only at high (10 μM) concentrations. Because this inhibitory effect was observed at a 6-fold dilution of serum, it is assumed that this effect would be even less pronounced in vivo in whole blood. Considering these factors, the four proteins are considered to be specific lectin pathway inhibitors in rat serum.

[0338] The lack of significant classical and alternative pathway inhibition demonstrates that these four selected proteins of the present invention do not inhibit the following serine proteases: C1r, C1s, factor D, factor B (i.e., C3bBb-type C3-convertase), and C2 (i.e., C4b2a-type C3-convertase), demonstrating the high target specificity of EVO21 (SEQ ID NO: 12), EVO214 (SEQ ID NO: 8), EVO23 (SEQ ID NO: 3), and EVO24 (SEQ ID NO: 15).

[0339] F.6. Effect of the protein of the present invention on human and rat blood coagulation The effect of the proteins of the invention on the blood clotting process was tested in three standard tests: thrombin time (testing any direct effect on thrombin), prothrombin time (testing any effect on the extrinsic pathway), and activated partial thromboplastin time (testing any effect on the intrinsic pathway).

[0340] F.6.1. Measurement of human blood coagulation After informed consent, blood was collected from healthy individuals by venipuncture. Blood was treated with sodium citrate (3.8% (wt / vol)) and centrifuged. All three assays were performed on an automated Sysmex CA-1500 (Sysmex) using Innovin reagents (Dale Behring, Marburg, Germany).

[0341] The proteins of the invention were applied in two-fold serial dilutions in 1.4% (wt / vol) sodium bicarbonate (vehicle) to a final highest concentration of 10 μM (3.4 μM for EVO24 (SEQ ID NO: 15)) and a final lowest concentration of 156 nM (53 nM for EVO24 (SEQ ID NO: 15)).

[0342] All measurements were performed in duplicate, and a vehicle control was also tested. The highest concentration values ​​were the K values ​​of the four proteins of the present invention for human MASP-2. D is about five orders of magnitude larger than the value The results are shown in Tables 20a to 20d.

[0343] [Table 20] Effects of EVO21 (SEQ ID NO: 12), EVO24 (SEQ ID NO: 15), EVO23 (SEQ ID NO: 3), and EVO214 (SEQ ID NO: 8) on human blood coagulation Table 20a TIFF2025534392000099.tif74153 Table 20b TIFF2025534392000100.tif75153 Table 20c TIFF2025534392000101.tif74153 Table 20d TIFF2025534392000102.tif64153

[0344] F.6.2. Measurement of blood coagulation in rats Rat coagulation assessment was performed using Wistar rat plasma on a Sysmex CA-660 coagulation analyzer. All proteins of the present invention were tested on two plasma aliquots from three rats. Proteins of the present invention were dissolved in a 1.4% (wt / vol) sodium bicarbonate vehicle. EVO24 (SEQ ID NO: 15) was tested at a plasma concentration of 3.4 μM, and EVO21 (SEQ ID NO: 12), EVO214 (SEQ ID NO: 8), and EVO23 (SEQ ID NO: 3) were tested at a plasma concentration of 10 μM. The protein of the present invention was dissolved in a sodium bicarbonate vehicle. The results are shown in Table 21.

[0345] [Table 21] Effects of EVO214 (SEQ ID NO: 8), EVO21 (SEQ ID NO: 12), EVO23 (SEQ ID NO: 3), and EVO24 (SEQ ID NO: 15) on rat blood coagulation (in three standard blood coagulation tests: APTT: activated partial thromboplastin time, PT: prothrombin time, TT: thrombin time). TIFF2025534392000103.tif203153

[0346] F.6.3. Blood clotting test conclusions Even at the highest concentrations, the protein of the present invention had no or negligible effect in the PT and TT tests in both human and rat evaluations. Based on the results, it can be clearly stated that the protein of the present invention does not inhibit with any appreciable affinity the following blood coagulation proteases: thrombin, fVIIa, and fXa.

[0347] On the other hand, all four of the proteins of the present invention prolong the APTT time at a concentration of 1 μM or higher (K ​​of the four proteins of the present invention for human and rat MASP-2). D (approximately 3-4 orders of magnitude longer than the value).

[0348] Nevertheless, the effect on APTT indicates that the protein of the present invention is able to inhibit, at least weakly, at least one of the following blood coagulation enzymes: fIXa, fXIa, and fXIIa.

[0349] Therefore, these proteins of the present invention were also tested in vitro on these enzymes.

[0350] F.7. Testing the efficacy of EVO21 (SEQ ID NO: 12), EVO214 (SEQ ID NO: 8), EVO23 (SEQ ID NO: 3), and EVO24 (SEQ ID NO: 15) of the present invention against MASP-1 and against human blood coagulation factors fIXa, fXIa, and fXIIa As recent research has shown that MASP-1 also contributes to the physiological clotting of human blood (Golomingi 2022), the inventors also tested four compounds for their MASP-1 inhibitory ability.

[0351] Measurements were performed using non-binding microtiter plates (Greiner; #655901) with a final volume of 100 μl. After several pilot experiments to find the appropriate inhibitor concentration range, the maximum inhibitor concentrations were set at 20 μM for MASP-1 inhibition, 10 μM for fXIa inhibition, and 40 μM for fIXa and fXIIa inhibition. Proteins of the present invention were added to reach the previously optimized final concentrations of 10 μM for MASP-1, 50 nM for fIXa, 3.3 nM for fXIa, and 27.5 nM for fXIIa.

[0352] After adding substrates (150 μM Z-Lys-S-benzyl and 300 μM 4,4′-dithiodipyridine (DTDP) (cosubstrates) for MASP-1; 150 μM Z-Gly-Arg-S-benzyl and 300 μM DTDP (cosubstrates) for fIXa; 300 μM Cbz-GPR-pNA for fXIa; and 100 μM HD-PFR-pNA for fXIIa), the samples were incubated at room temperature for 10 min. The reaction buffer was 50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl, 0.1% PEG-8000 pH 7.4 for MASP-1; 20 mM HEPES, 145 mM NaCl, 0.05% Triton-X100 pH 7.4 for fIXa and fXIa; and this was supplemented with 5 mM CaCl for fXIIa.

[0353] The potency of the proteins of the invention was determined as IC , which corresponds to the inhibitor concentration that provides 50% inhibition. 50 The results are shown in Table 22.

[0354] [Table 22] IC of EVO21, EVO214, EVO23, and EVO24 against the indicated proteases 50 value TIFF2025534392000104.tif78153

[0355] Overall, the four selected proteins of the present invention inhibit MASP-1 with an IC of approximately 10 μM. 50 It inhibits fIXa and fXIIa at IC values ​​between 3 and 100 μM, with negligible inhibition of both. Only fXIa is inhibited at IC values ​​below micromolar. 50 Although the K values ​​of the four proteins of the present invention for human and rat were inhibited by D is about 3 to 4 orders of magnitude higher than the value.

[0356] Nevertheless, potential off-target effects for fXIa are expected. It is important to note that complete deficiency of fXI is associated with reduced bleeding during trauma. Furthermore, besides fXIIa, fXIa is considered an optimal target for the development of novel antithrombotic drugs that are safer than currently available compounds (Mohammed 2018), (Al-Horani 2016).

[0357] F.8. Measurement of the stability of the protein of the present invention To evaluate the in vivo lectin pathway inhibitory potency of four selected proteins: EVO21 (SEQ ID NO: 12), EVO23 (SEQ ID NO: 3), EVO24 (SEQ ID NO: 15), and EVO214 (SEQ ID NO: 8), we aimed to store and use them at a high (1.5 mM) concentration in 1.4% (wt / vol) sodium bicarbonate buffer, a suitable vehicle for animal studies. To test the stability of these four proteins, lyophilized samples from a sample batch were compared. One sample was dissolved in 1.4% sodium bicarbonate buffer and stored at 4°C for 5 weeks. Another aliquot of the sample was freshly dissolved in vehicle buffer. The lectin pathway inhibitory potency of these samples was assessed simultaneously in the same plate as described in F.5.1.

[0358] IC between freshly thawed samples and samples stored for 5 weeks 50 No significant difference in values ​​was observed, demonstrating that the proteins of the present invention are stable in the vehicle buffer for at least 5 weeks.

[0359] The stability of EVO24L (SEQ ID NO: 114) was evaluated by IC of freshly prepared EVO24L samples and EVO24L samples stored at 4°C for 5 weeks. 50 The IC values ​​were measured between freshly prepared samples and samples stored for 5 weeks. 50No significant differences were observed in the values, demonstrating that EVO24L is stable in vehicle buffer for at least 5 weeks.

[0360] F.9. The extent and time course of the in vivo complement lectin pathway inhibitory efficacy of the proteins of the present invention administered to rats is monitored by measuring residual lectin pathway activity in serum samples ex vivo. Two such campaigns were conducted. In the first campaign, EVO21 (SEQ ID NO: 12), EVO24 (SEQ ID NO: 15), EVO23 (SEQ ID NO: 3), and EVO214 (SEQ ID NO: 8) of the present invention were tested against EVO2 (SEQ ID NO: 2). In the second campaign, an Fc-fused form of EVO24, designated EVO24L (SEQ ID NO: 114), was tested. Section F.9.1 onward details the general procedure, with specific data corresponding to the first campaign. Significant differences in the second campaign are explained in section F.9.2.

[0361] F.9.1. Pharmacodynamic testing of EVO21 (SEQ ID NO: 12), EVO23 (SEQ ID NO: 3), EVO24 (SEQ ID NO: 15), and EVO214 (SEQ ID NO: 8) Healthy male Wistar-Hanover rats (4 rats / test protein) were anesthetized by intraperitoneal (ip) injection of sodium pentobarbital, with repeated administration to maintain anesthesia. Body temperature was maintained reliably with a heating pad. The animals' ECGs (leads I-II-III) were monitored throughout the experiment. In the event of cessation of spontaneous breathing or a significant decrease or arrhythmia in heart rate, the animals were orally intubated and connected to a Rodent Ventilator (Ugo Basile, Model 7025, Varese, Italy) for artificial ventilation at a rate and stroke volume according to the manufacturer's recommendations. After the onset of anesthesia, blood samples were collected by cannulating the right carotid artery. Arterial blood was collected via a polyethylene cannula and allowed to drip directly into a tube containing a clot activator (VACUETTE® TUBE 2 ml Z Serum Clot Activator).

[0362] The test substance (1.5 mM) or vehicle was administered as a slow intravenous (iv) bolus injection lasting 1 minute, followed immediately by an intravenous bolus injection of the two components together at a volume of 1.5 ml / kg (corresponding to a dose of 4.5 μmol / kg and approximately 32 mg / kg). For serum sample preparation, blood samples were collected 20 and 5 minutes before administration of the test substance or vehicle, and 5, 30, 60, 120, and 240 minutes after administration of the test substance or vehicle. The blood samples were incubated at room temperature for 30±5 minutes to allow blood to clot. The clotted blood samples were centrifuged at 4000 rpm at 20°C for 15 minutes. Two 100 μl serum samples were collected from each blood sample and pipetted accurately into 0.5 ml Eppendorf tubes. The serum samples were frozen in liquid nitrogen and stored at -80°C until evaluation.

[0363] The activity of the complement lectin pathway was assessed by activation of serum samples on mannan-coated surfaces and measurement of C4b deposition by ELISA. Briefly, the surface of a microtiter plate was coated with mannan (10 μg / ml) overnight at 4°C. After washing, the walls were blocked with a 1% BSA solution to prevent nonspecific protein binding. Rat serum samples were applied to the mannan-coated surface at a 32-fold dilution and incubated at 37°C for 30 minutes. During this time, MASP-2 was activated, cleaving the C4 component and covalently depositing the C4b fragment on the surface of the microtiter plate.

[0364] In the case of a 32-fold serum dilution, activation of the lectin pathway occurs efficiently in rat serum, but the alternative pathway is not initiated. Activation of the alternative pathway would not generate cleaved C4 and therefore would not contribute to the measurement signal. However, if the alternative pathway were activated, the densely deposited excess C3b component would compete with C4b for free surfaces.

[0365] After washing, the primary antibody (anti-human C4c polyclonal rabbit antibody (DakoCytomation-Q0369)) was first applied to the plate surface at a 1:2000 dilution. After another wash, an anti-rabbit IgG horseradish peroxidase (HRP)-conjugated monoclonal mouse antibody (Sigma-A1949) was used at a 1:40,000 dilution. The HRP component of the conjugated antibody catalyzes the chemical reaction between OPD (ortho-phenylenediamine) peroxidase and the enzyme's hydrogen peroxide substrate at a rate proportional to the amount of immobilized HRP. The enzymatic reaction was allowed to continue for 8 minutes at room temperature, and then stopped by adding 1 M sulfuric acid solution. The absorbance at 490 nm was measured using a spectrophotometer.

[0366] Because absorbance is proportional to the amount of HRP (which is itself proportional to the deposited C4b), the level of lectin pathway activity could be estimated. Serum samples obtained from each animal were evaluated in triplicate on the same plate. For pharmacodynamic (PD) endpoint readings, 100% (control serum) activity was given by the lectin pathway activity of serum containing no protein of the present invention (pre-dose sample collected at -5 min), and 0% (minus background absorbance at the time of complete inhibition) was provided by EDTA-treated control serum. To measure the inhibitory effect of the administered test substance (i.e., protein of the present invention) or vehicle, C4b deposition values ​​measured in serum samples collected at different time points were expressed as a percentage of the absorbance value, subtracted from the background of the -5 min pre-dose sample from the same rat. These percentage lectin pathway activity values ​​were subjected to descriptive statistical evaluation and plotting.

[0367] As Figure 10 shows, EVO21, EVO23, EVO24, and EVO214 are all significantly more efficient in vivo lectin pathway inhibitors than EVO2, based on the magnitude of maximum lectin pathway inhibitory potency at 5 minutes after administration. When administered at 32 mg / kg, EVO2 provides 56% pathway inhibition, while the four test proteins of the present invention provide 86-91% inhibition. The most efficient protein is EVO24, providing 80% inhibition after 1 hour and approximately 50% inhibition after 4 hours. The corresponding data for EVO2 are 40% and 20% inhibition, respectively.

[0368] Based on these data, an Fc-fused version of EVO24 was generated to reduce its clearance rate from the blood and thereby improve its pharmacodynamic properties.

[0369] F.9.2. Pharmacodynamic testing of EVO24L (SEQ ID NO: 114) For EVO24L, the method was the same as that described in F.9.1., except that a total dose of 4.5 μmol / kg was administered to rats as a slow intravenous (iv) bolus infusion over 2 minutes, and no intraperitoneal administration was applied. In this case, 5 animals / protein (vehicle or EVO24L) were used.

[0370] As Figure 10 shows, the Fc fusion of EVO24L dramatically enhanced the potency of the compound. At the first sampling time point (5 min), EVO24L provided over 98% lectin pathway inhibition, and even at the last time point (4 h), lectin pathway inhibition was approximately 98%. It should be noted that the fact that C4 deposition was completely inhibited clearly demonstrates that the non-glycosylated nature of the IgG1 fusion tag blocks IgG-dependent activation of the classical complement pathway.

[0371] Overall, due to the Fc fusion tag, a single administration of EVO24L can provide near-complete pathway inhibition for at least several hours. With repeated administration and the use of a slow-release device, it has been clearly demonstrated that complete and long-lasting lectin pathway blockade can be achieved in this type of embodiment of the present invention.

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Claims

1. the amino acid sequence of SEQ ID NO: 115, wherein the variable positions in the amino acid sequence of SEQ ID NO: 115 are x1 to x4, x58, x57, and x56 are variable or absent; x6 to x11, x13, x15 to x20, x24 to x29, x31 to x32, x34, x39, x41 to x42, x44, x46 to x50, x52 to x54 are variable; x21 is F, Y, or W; x22 is Y or F; x23 is Y or F; x35 is Y or W; x36 is G or S; x40 is G or A; x43 is N or G; and x45 is F or Y and i) a compound of the general formula Ih-mod starting at position 12 and ending at position 19 of SEQ ID NO: 115: GX 1 CX 1V X 2 X 3 X 4 X 5 (where, X 1 is either F, Y, L, P, Q, M, V, W, A, or T; X 1V is R or K; X 2 is either A, G, S, or T; X 3 is any amino acid from the 17-set (wherein the 17-set comprises A, I, L, F, and Y); X 4 is either K, I, Q, R, H, S, F, M, N, L, or V; and X 5 is R, V, I, K, M, Q, E, F, L, N, Y, D, S, or H) and ii) containing any of the amino acids of the 34-set (wherein the 34-set comprises Y, I, F, G, V, and S) at position 34 of the amino acid sequence of SEQ ID NO: 115; A protein, or a salt, ester, or pharmaceutically acceptable prodrug thereof, characterized in that:

2. K below 100 nM I The protein described in claim 1, or a salt, ester, or pharmaceutically acceptable prodrug thereof, characterized in that it is a human MASP-2 inhibitor having therapeutic value.

3. 3. The protein according to claim 1 or 2, or a salt, ester, or pharmaceutically acceptable prodrug thereof, characterized in that the protein comprises an amino acid sequence having at least 70%, or at least 80%, or at least 90%, or at least 95% similarity, more preferably at least 98% similarity, even more preferably at least 70%, or at least 80%, or at least 90%, or at least 95% identity, and most preferably 98% identity, with the amino acid sequence of SEQ ID NO: 116, with the proviso that i) the amino acid segment starting at position 12 and ending at position 19 has a sequence defined by the general formula Ih-mod, and ii) at position 34 of the amino acid sequence of SEQ ID NO: 115, contains an amino acid selected from the 34-set.

4. 2. The protein according to claim 1, or a salt, ester, or pharmaceutically acceptable prodrug thereof, characterized in that the protein comprises an amino acid sequence (wherein the amino acid pairs from the 17-set and 34-set are selected from the group consisting of A / Y, A / I, A / F, A / G, A / V, A / S, I / Y, I / I, I / F, I / G, I / V, I / S, L / Y, L / I, L / F, L / G, L / V, L / S, F / Y, F / I, F / F, F / G, F / V, F / S, Y / Y, Y / I, Y / F, Y / G, Y / V, Y / S (in x17 / x34 format)).

5. 5. The protein of claim 4, or a salt, ester, or pharmaceutically acceptable prodrug thereof, characterized in that the amino acid pairs from the 17-set and 34-set are selected from the group consisting of A / Y, A / I, A / F, A / V, I / Y, I / I, I / F, I / G, I / V, I / S, L / Y, L / I, L / F, L / G, L / V, L / S, F / I, F / G, F / V, F / S, Y / Y, Y / I, Y / G, Y / V, and Y / S (in x17 / x34 format).

6. 2. The protein of claim 1, or a salt, ester, or pharmaceutically acceptable prodrug thereof, characterized in that the protein comprises an amino acid sequence (wherein at position 9 of the amino acid sequence of SEQ ID NO: 115, an amino acid of any of the 9-set (the 9-set consists of N and E) is contained).

7. The protein according to claim 1 or 6, or a salt, ester, or pharmaceutically acceptable prodrug thereof, characterized in that the protein comprises an amino acid sequence (wherein the amino acid sequence of SEQ ID NO: 115 contains any amino acid of the 39-set (the 39-set consists of F and L) at position 39).

8. The protein according to claim 1 or 6, or a salt, ester, or pharmaceutically acceptable prodrug thereof, characterized in that the protein comprises an amino acid sequence (wherein the amino acid sequence of SEQ ID NO: 115 contains any amino acid of the 46-set (the 46-set consists of V and E) at position 46).

9. The protein of claim 7, or a salt, ester, or pharmaceutically acceptable prodrug thereof, characterized in that the protein comprises an amino acid sequence (wherein the amino acid sequence of SEQ ID NO: 115 contains any of the 46-set amino acids (the 46-set consists of V and E) at position 46).

10. 2. The protein of claim 1, or a salt, ester, or pharmaceutically acceptable prodrug thereof, characterized in that the protein is selected from proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO:

56.

11. 2. The protein of claim 1, or a salt, ester, or pharmaceutically acceptable prodrug thereof, characterized in that the protein has at least 70%, or at least 80%, or at least 90%, or at least 95% similarity, more preferably at least 98% similarity, even more preferably at least 70%, or at least 80%, or at least 90%, or at least 95% identity, and most preferably 98% identity, or is completely identical to any of the amino acid sequences set forth in SEQ ID NO: 3 to SEQ ID NO: 22 and SEQ ID NO: 24 to SEQ ID NO: 32, with the proviso that the amino acid segment starting at position 12 and ending at position 19 has a sequence defined by the general formula Ih-mod, and that at position 34 of the amino acid sequence of SEQ ID NO: 115, an amino acid sequence containing an amino acid selected from the 34-set is used.

12. The proteins are represented by SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO:

12. The protein of claim 11, or a salt, ester, or pharmaceutically acceptable prodrug thereof, characterized in that it comprises an amino acid sequence having at least 95% similarity, more preferably at least 98% similarity, even more preferably at least 95% identity, and most preferably 98% identity, or complete identity, with any of the amino acid sequences set forth in SEQ ID NO: 26, with the proviso that the amino acid segment starting at position 12 and ending at position 19 has a sequence defined by the general formula Ih-mod, and at position 34 of the amino acid sequence of SEQ ID NO: 115, an amino acid sequence containing an amino acid selected from the 34-set.

13. The protein is in the form of a fusion protein, said fusion protein comprising: i) SEQ ID NO: 115, wherein the variable positions in the amino acid sequence of SEQ ID NO: 115 are restricted as follows: x1 to x4, x58, x57, and x56 are variable or absent; x6 to x11, x13, x15 to x20, x24 to x29, x31 to x32, x34, x39, x41 to x42, x44, x46 to x50, x52 to x54 are variable; x21 is F, Y, or W; x22 is Y or F; x23 is Y or F; x35 is Y or W; x36 is G or S; x40 is G or A; x43 is N or G; and x45 is F or Y; GX 1 CX 1V X 2 X 3 X 4 X 5 (where X 1 is one of F, Y, L, P, Q, M, V, W, A, or T, and X 1V is R or K, and X 2 is either A, G, S, or T, and X 3 is any amino acid of the 17-set (wherein the 17-set comprises A, I, L, F, and Y), and X 4 is either K, I, Q, R, H, S, F, M, N, L, or V, and X 5 is any of R, V, I, K, M, Q, E, F, L, N, Y, D, S, and H; and b) an amino acid sequence of SEQ ID NO: 115, comprising at position 34 an amino acid selected from a 34-set, wherein the 34-set comprises Y, I, F, G, V, and S; and ii) an antibody Fc-domain, preferably a human antibody Fc-domain 2. The protein of claim 1, or a salt, ester, or pharmaceutically acceptable prodrug thereof, comprising:

14. 14. The protein of claim 13, or a salt, ester, or pharmaceutically acceptable prodrug thereof, characterized in that the fusion protein comprises an amino acid sequence having at least 70%, or at least 80%, or at least 90%, or at least 95% similarity, more preferably at least 98% similarity, even more preferably at least 70%, or at least 80%, or at least 90%, or at least 95% identity, and most preferably 98% identity, or completely identical to SEQ ID NO:

114.

15. 2. A method for producing a pharmaceutical composition comprising at least one protein according to claim 1, a pharmaceutically acceptable salt, a pharmaceutically acceptable ester or a pharmaceutically acceptable prodrug thereof, and at least one additive, wherein the at least one protein is preferably selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: and more preferably, the at least one protein is selected from the group consisting of proteins defined by any of the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32;Most preferably, the at least one protein is selected from the group consisting of proteins comprising any of the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO:

26.

16. 16. A pharmaceutical product according to claim 15, characterized in that the additive is preferably a matrix which ensures a controlled release of the active agent.

17. 17. The pharmaceutical product according to claim 15 or 16, characterized in that it is preferably in the form of drops, tablets, powders, granules, suppositories, injections, syrups, inhalants or nasal delivery agents.

18. A nucleic acid encoding the protein of claim 1.

19. A vector comprising the nucleic acid of claim 18.

20. 10. A kit comprising at least one protein, salt or ester thereof according to claim 1 and a manual for use or a reference to such a manual.

21. A method for screening compounds that potentially inhibit MASP-2 enzyme, preferably human MASP-2 enzyme, in the process of which i) a labeled form of the protein, its salt or ester described in claim 1 is added to a solution containing the MASP-2 enzyme, preferably the human MASP-2 enzyme, then ii) a solution containing one or more test compounds is added to the solution, and iii) the amount of labeled protein released is measured.

22. Use of the protein, its salt, ester or prodrug described in claim 1 for inhibiting the MASP-2 enzyme, preferably human MASP-2 enzyme.

23. Use of the protein described in claim 1, its pharmaceutically acceptable salt, pharmaceutically acceptable ester, or pharmaceutically acceptable prodrug in the manufacture of a pharmaceutical suitable for the treatment or prevention of a disease that is treated by inhibiting the complement system.

24. The disease is preferably selected from the following list: (1) ischemia-reperfusion (IR) injury (particularly associated with recanalization after arterial occlusion due to thrombosis or other occlusive disease), including myocardial infarction, coronary artery bypass surgery, graft IR injury in organ transplantation, gastrointestinal IR injury, renal IR injury, post-ischemic brain injury, stroke, and diseases occurring after thrombosis affecting any region of the body; (2) autoimmune nephritis (including dense deposit disease and C3 glomerulonephritis), IgA nephropathy, membranous nephropathy, rheumatoid arthritis (RA), juvenile idiopathic arthritis, age-related macular degeneration, systemic erythematosus, and the like. (3) inflammatory and autoimmune conditions accompanied by excessive activation of the complement system, including systemic lupus erythematosus (SLE), atypical hemolytic uremic syndrome (aHUS), thrombotic microangiopathy (TMA), post-infectious hemolytic uremic syndrome (HUS), pseudoallergy resulting from complement activation (CARPA), paroxysmal nocturnal hemoglobinuria (PNH), multiple trauma, and graft rejection after organ transplantation; (3) neurodegenerative diseases, preferably Alzheimer's disease, Huntington's disease, Parkinson's disease, multiple sclerosis, and age-related macular degeneration; (4) complement hyperactivation due to infection with a virus, such as COVID-19 (SARS-CoV-2), acute respiratory distress syndrome (ARDS), and complement-related microangiopathy and thrombosis due to severe COVID-19 infection.

25. A method for isolating human MASP-2 enzyme, the process comprising: i) contacting a support having immobilized thereon one or more proteins described in claim 1 or 13, or their pharmaceutically acceptable salts or esters, with a solution containing the human MASP-2 enzyme; and ii) washing the preparation.

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