Gigastasin variants with activity modulating substitutions

Abstract

The present invention relates to polypeptides comprising gigastasin variants in which one or more residues have been substituted compared to the naturally occurring gigastasin of the giant Amazon leech. It further relates to processes for their production, to their lectin pathway inhibition, to their half-life extension, and to their therapeutic use.

Description

[0001] Universitaet Basel P700

[0002] PJG / AK

[0003] 11.12.2025

[0004] Gigastasin Variants with Activity Modulating Substitutions

[0005] FIELD OF THE INVENTION

[0006] The present invention relates to polypeptides comprising gigastasin variants in which one or more residues have been substituted compared to the naturally occurring gigastasin of the giant Amazon leech. It further relates to processes for their production, to their lectin pathway inhibition, to their half-life extension, and to their therapeutic use.

[0007] BACKGROUND

[0008] The complement system provides an innate host defense mechanism by constantly surveying the human body for pathogenic cells. It consists of more than 50 proteins involved in homeostasis, pathogen elimination, and mediation of inflammatory responses.1Initiation of the complement system is achieved through the detection of pathogen- and damage-associated molecular patterns (PAMP and DAMP, respectively) triggering three distinct activation pathways.2In the classical pathway (CP), C1q recognizes and binds to surface-bound IgG and IgM antibodies and activates the associated serine proteases C1r and C1s. Similarly, the lectin pathway (LP) recognizes carbohydrate signatures on cell surfaces via mannose-binding lectin (MBL), ficolins or collectins.3MBL-associated serine proteases ( / .e., MASP-1 and -2) circulate in complex with these pattern recognition molecules and become activated upon target binding. Serine proteases of both pathways are involved in the downstream activation of the cascade by cleaving C4 and C2 to form a C3 convertase complex on target surfaces, which serves to cleave 03 and deposit the opsonin C3b on activating cell surfaces. The alternative pathway (AP) can contribute to complement activation and opsonization via low-level hydrolysis of C3 ( / .e., tick-over), yet also fuels an amplification loop of the complement response. Independent of the initiation route, surface-deposited C3b engages with factor B (FB) and factor D (FD) to form the AP 03 convertase complex and increase the density of C3b on the surface. Opsonization eventually enables downstream effector functions such as opsonophagocytosis and lysis via the membrane attack complex (MAC), whereas the release of anaphylatoxins promotes immuno-inflammatory responses.45 Despite its protective functions, inappropriate complement activation on host cells is frequently described as a contributing factor to various immune and inflammatory diseases.2 6With the growing recognition of complement-mediated disorders, several pathway-directed drugs are in clinical development or have meanwhile reached the market.7These include complement- targeted monoclonal antibodies (mAb) such as ravulizumab (anti-C5; Ultomiris, Alexion) and sutimlimab (anti-C1s; Enjaymo, Sanofi), both of which are approved, and narsoplimab (anti- MASP2; Omeros) that is in late stage clinical development.8These inhibitors are positioned for different, and sometimes multiple, complement-related indications that represent well-defined complementopathies of the rare disease spectrum.9’10However, complement activation is also a driving force in acute thromboinflammatory conditions such as ischemia-reperfusion injury (IRI). IRI occurs after states of occluded blood flow, for example after stroke and myocardial infarction or during organ transplantation. The exposure of DAMPs on hypoxic tissue triggers complement, coagulation, and other defense systems upon restoration of blood circulation, which may affect organ functions and even lead to fatal outcomes.11’12A targeted inhibition of involved activation mechanisms is therefore considered a promising strategy in such conditions, yet difficult to achieve with monospecific inhibitors currently in the clinic. Preparations of human C1 esterase inhibitor (C1-INH), a physiological regulator with broad activities against serine proteases of the CP and LP but also of non-complement pathways {e.g., coagulation proteases), are available but thus far only approved to treat hereditary angioedema.13Moreover, C1-INH is challenging to produce and is either isolated from donor plasma or recombinantly expressed by transgenic rabbits. Therefore, the treatment of IRI and other acute-phase conditions may benefit from serine protease inhibitors with tuned specificity profiles against complement and coagulation pathways and facilitated production.

[0009] Coagulation factors Xia and XI la play crucial roles in the intrinsic pathway of blood coagulation and have been increasingly recognized for their contributions to thromboinflammatory diseases.14On damaged cell surfaces, FXII can be transformed to active FXIIa, which leads to the activation of FXI. Downstream, this induces thrombin generation and enables the formation of fibrin clots. The intrinsic pathway is not only essential for normal hemostasis but also implicated in pathological thrombosis. Elevated levels of FXIa have been associated with increased risks of venous thromboembolism, ischemic stroke, and myocardial infarction.15Similarly, FXIIa, while not essential for hemostasis, contributes to thrombus formation and inflammation. It activates the kallikrein-kinin system, promoting inflammation and vascular permeability. Inhibition of FXIIa has shown potential for reducing thrombosis without significantly affecting hemostasis, making it an attractive target for antithrombotic therapy.16The concept of hemostasis-thrombosis uncoupling has therefore emerged, highlighting the differential roles of the coagulation pathways. The intrinsic pathway is mainly responsible for thrombosis, whereas the extrinsic pathway is primarily involved in hemostasis. This distinction underscores the potential for targeted therapeutic strategies that can mitigate thrombotic events while preserving normal hemostatic functions.17Additional benefit of targeting FXIa in the coagulation cascade is that it can de-risk bleeding events as it already has been shown in FXI deficient populations, that their bleeding events are almost equal to non-deficient subjects.18

[0010] In this regard, many parasites can be considered experts in complement evasion and possess an array of complement- and / or coagulation-targeted inhibitors that may prove valuable as templates or inspiration for therapeutic development.19-22For example, a protease inhibitor from the giant Amazon leech (Haementeria ghillianii), termed gigastasin, has been identified as complement modulator.23-25Initially referred to as BD001 , the protein was shown to potently inhibit the complement protease C1s and, to a lesser degree, FXI la of the coagulation cascade.24 25In the case of C1s, a crystal structure of gigastasin in complex with its target has been solved.23The same study identified MASP-2 as another major target and revealed that gigastasin not only inhibits the CP but even more potently the LP.23Although the functional spectrum of gigastasin renders it an interesting candidate for host defense modulation in biomedical studies, it was not clear whether a protein that features 10 disulfide bridges and several posttranslational modifications could be prepared at sufficient yield, purity and activity to enable experimental and preclinical studies.

[0011] There is an ongoing need for developing improved inhibitors with tuned specificity profiles against complement and coagulation pathways to efficiently prevent or treat IRI and other acute-phase conditions. The inhibitor should have high potency and / or specificity for the lectin pathway and preferably a long plasma half-life.

[0012] SUMMARY OF THE INVENTION

[0013] The inventors of the present invention were able to demonstrate that targeted amino acid substitutions refine binding affinity and / or pathway selectivity of gigastasin variants compared to naturally evolved gigastasin. Although gigastasin of the giant Amazon leech as such is already a very potent serine protease inhibitor downregulating the lectin pathway of complement activation, surprisingly, the introduction of herein described point mutations - alone and in combination - can even further enhance said inhibitory activity. In addition, the resulting modulation of serine protease inhibition can be utilized to tweak selectivity for desired pathways, which may reduce the risk of side effects. Moreover, such new recombinant gigastasin variants (i) can be produced in bacterial expression systems at good yield and purity and can be cleaned off endotoxin and lyophilized for long-term storage; (ii) showed inhibition of other host defense targets, of which coagulation factor Xia seems to be a major intervention point, in addition to their potent activity against C1s and MASP-2; therefore showing a potent inhibition of host defense activation in humans; (iii) can tune the target binding and pathway inhibition selectivity (e.g. target binding in the case of C1s:MASP-2 and pathway inhibition selectivity in CP:LP initiation), potentially decreasing the risk properties for therapeutic treatment in general and / or improving treatment success for certain indications; (iv) can increase the absolute inhibitory activity for one or several of the aforementioned pathways / targets over gigastasin; and / or (v) show improved stability toward thrombin- mediated cleavage / degradation. The successful recombinant production of gigastasin, the impressive stability of the expressed protein, its broad-yet-defined activity against key host defense pathways with clinical relevance, its low predicted immunogenicity potential, and its activity in serum of different species (human, non-human primate, rat, and mouse serum; potentially also in other species) used in preclinical in vitro studies suggest that gigastasin is an interesting therapeutic candidate suitable for preclinical development. Based on this, it is reasonable to expect that enhanced LP selectivity and potency of some gigastasin variants increase the therapeutic potential. In addition, the polypeptides of the present invention may be of use in diagnostics, e.g. as part of a complement activation kit, or in research and process technology, e.g. as detection tools for certain serine proteases, as biosensor or immobilized binding agent in affinity chromatography. Certain mutations may allow improvements in the production process of gigastasin variants by reducing the number of necessary steps or resulting in higher yield.

[0014] The invention provides polypeptides comprising gigastasin variants that adjust activities of gigastasin such as protein stability, target binding, pathway inhibition selectivity and / or potency. The present invention therefore relates to the subject matter defined in the following items [1] to

[0128] ,

[0015]

[0001] A polypeptide comprising a gigastasin variant, wherein the amino acid sequence of said gigastasin variant comprises one or more amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 ; optionally wherein at least one activity of gigastasin is modulated in said polypeptide.

[0016] [2] The polypeptide of item [1], wherein at least some or all of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 are selected from the group consisting of K10D, K10E, W17A, W17F, W17G, W17H, W17L, W17P, W17R, W17Y, D18E, G36A, G36D, G36E, G36R, G36W, Q45K, K53R, L55R, L55E, L55I, L55Q, L55V, T69R, G71 E, G71Q, T84R, Y117D, Y117E, Y117A, Y117R, Y119D, Y119E, Y119A, Y119R, Y121 D, Y121 E, Y121A, and Y121 R.

[0017] [3] The polypeptide of item [1] or [2], wherein at least some or all of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 are selected from the group consisting of D18E, G36A, G36D, G36E, G36R, G36W, Q45K, K53R, L55R, L55E, L55I, L55V, L55Q, T69R, T84R, Y117D, Y117E, Y119D, Y119E, Y121 D, and Y121 E; in particular from the group consisting of G36D, Q45K, L55R, L55E, L55I, L55V, T69R, Y117E, Y119E, and Y121 E.

[0018] [4] The polypeptide of any one of the preceding items, wherein said at least one activity of gigastasin is selected from the group consisting of serine protease inhibitory activity, anticoagulant activity, anti-inflammatory activity, and combinations thereof.

[0019] [5] The polypeptide of item [4], wherein said at least one activity of gigastasin comprises serine protease inhibitory activity, and wherein said serine protease is selected from the group consisting of C1s, FXIa, FXIIa, C1 r, MASP-1 , and MASP-2, in particular is C1s, MASP-2, and / or FXIa.

[0020] [6] The polypeptide of any one of the preceding items, wherein at least some or all of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 are at a position selected from the group consisting of W17, D18, G36, Q45, K53, L55, T69, G71 , F72, T84, Y117, Y119, and Y121 , in particular from the group consisting of D18, G36, Q45, K53, L55, T69, T84, Y117, Y119, and Y121.

[0021] [7] A polypeptide comprising a gigastasin variant, in particular of any one of the preceding items, wherein the amino acid sequence of said gigastasin variant comprises one or more amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 , wherein at least one of said amino acid substitutions is at position G36, Q45, K53, L55, T69 or T84, optionally wherein at least two of said amino acid substitutions are at positions selected from the group consisting of G36, Q45, K53, L55, T69 and T84 and / or wherein at least one amino acid substitution is at position G36.

[0022] [8] The polypeptide of item [7], wherein the at least one amino acid substitution at position G36, Q45, K53, L55, T69 or T84 is selected from the group consisting of G36D, G36E, G36R, Q45K, K53R, L55R, L55I, L55Q, L55V, T69R, and T84R, in particular wherein one substitution is G36D or G36E and at least one further substitutions is selected from the group consisting of Q45K, K53R, L55R, L55I, L55Q, L55V, T69R, and T84R.

[0023] [9] The polypeptide of any one of the preceding items, wherein said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 comprise replacement of one or more of Y117, Y119, and Y121 with glutamic acid or aspartic acid and / or wherein the gigastasin variant has a lesser degree of tyrosine-O-sulfation than naturally occurring gigastasin.

[0024]

[0010] The polypeptide of any one of the preceding items, wherein said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 comprise replacement of Y117 with glutamic acid or aspartic acid, Y119 with glutamic acid or aspartic acid, and Y121 with glutamic acid or aspartic acid.

[0025]

[0011] A polypeptide comprising a gigastasin variant, in particular of any one of the preceding items, wherein the amino acid sequence of said gigastasin variant comprises amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 , wherein three of said amino acid substitutions are at position Y117, Y119, and Y121 , wherein each of these positions is substituted with either glutamic acid or aspartic acid; optionally wherein the gigastasin variant comprises the amino acid sequence as shown in SEQ ID NO: 28 and / or wherein the gigastasin variant has a lesser degree of tyrosine-O-sulfation than naturally occurring gigastasin.

[0026]

[0012] The polypeptide of item

[0011] , wherein the gigastasin variant comprises one to five further amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 selected from the group consisting of D18E, G36A, G36D, G36E, G36R, G36W, Q45K, K53R, L55R, L55E, L55I, L55V, L55Q, T69R, and T84R, in particular from the group consisting of G36D, G36E, G36R, Q45K, K53R, L55R, L55I, L55Q, L55V, T69R, and T84R.

[0027]

[0013] The polypeptide of item

[0011] or

[0012] , wherein the gigastasin variant comprises one to three further amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 selected from the group consisting of D18E, G36A, G36D, G36E, G36R, G36W, Q45K, K53R, L55R, L55E, L55I, L55V, L55Q, T69R, T84R, in particular from the group consisting of G36D, G36E, G36R, Q45K, K53R, L55R, L55I, L55Q, L55V, T69R, and T84R.

[0028]

[0014] The polypeptide of any one of the items

[0011] to

[0013] , wherein any further amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 are selected from the group consisting of W17A, W17F, W17G, W17H, W17L, W17P, W17R, W17Y, D18E, G36A, G36D, G36E, G36R, G36W, Q45K, K53R, L55R, L55E, L55I, L55Q, L55V, T69R, G71 E, G71Q, T84R, in particular selected from the group consisting of D18E, G36A, G36D, G36E, G36R, G36W, Q45K, K53R, L55R, L55E, L55I, L55V, L55Q, T69R, T84R.

[0029]

[0015] A polypeptide comprising a gigastasin variant, in particular of any one of the preceding items, wherein the amino acid sequence of said gigastasin variant comprises at least two amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 ,

[0030] - wherein at least one of said amino acid substitutions is selected from the group consisting of D18E, G36D, G36E, G36R, Q45K, K53R, L55R, L55I, L55Q, L55V, T69R, and T84R, in particular wherein said gigastasin variant comprises a substitution at position G36 and optionally a further substitution selected from the group consisting of D18E, Q45K, K53R, L55R, L55I, L55Q, L55V, T69R, and T84R, and

[0031] - wherein at least one of said amino acid substitutions is a substitution at position Y117, Y119, orY121 with glutamic acid or aspartic acid, in particular wherein each position Y117, Y119, and Y121 is substituted with either glutamic acid or aspartic acid.

[0032]

[0016] The polypeptide of any one of the preceding items, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is at position K10 of SEQ ID NO: 1 , in particular is K10D or K10E.

[0033]

[0017] The polypeptide of any one of the preceding items, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is at position W17 of SEQ ID NO: 1 , in particular is W17A, W17F, W17G, W17H, W17L, W17P, W17R, or W17Y

[0034]

[0018] The polypeptide of any one of the preceding items, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is at position D18 of SEQ ID NO: 1 , in particular is D18W, D18Y, or D18E.

[0035]

[0019] The polypeptide of any one of the preceding items, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is at position G36 of SEQ ID NO: 1 , in particular is G36A, G36D, G36E, or G36R, preferably is G36D or G36E.

[0036]

[0020] The polypeptide of any one of the preceding items, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is at position Q45 of SEQ ID NO: 1 , in particular is Q45K.

[0037]

[0021] The polypeptide of any one of the preceding items, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is at position K53 of SEQ ID NO: 1 , in particular is K53R

[0038]

[0022] The polypeptide of any one of the preceding items, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is at position L55 of SEQ ID NO: 1 , in particular is L55R, L55E, L55I, L55Q, or L55V.

[0039]

[0023] The polypeptide of any one of the preceding items, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is at position T69 of SEQ ID NO: 1 , in particular is T69R.

[0040]

[0024] The polypeptide of any one of the preceding items, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is at position G71 of SEQ I D NO: 1 , in particular is Q71 E or Q71 Q.

[0041]

[0025] The polypeptide of any one of the preceding items, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is at position F72 of SEQ ID NO: 1 , in particular is F72E.

[0026] The polypeptide of any one of the preceding items, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is at position T84 of SEQ ID NO: 1 , in particular is T84R.

[0042]

[0027] The polypeptide of any one of the preceding items, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is at position Y117 of SEQ I D NO: 1 , in particular is Y117D or Y117E.

[0043]

[0028] The polypeptide of any one of the preceding items, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is at position Y119 of SEQ ID NO: 1 , in particular is Y119D or Y119E.

[0044]

[0029] The polypeptide of any one of the preceding items, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is at position Y121 of SEQ ID NO: 1 , in particular is Y121 D or Y121 E.

[0045]

[0030] The polypeptide of any one of the preceding items, wherein three of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 are Y117E, Y119E, and Y121 E or are Y117D, Y119D, and Y121 D, in particular are Y117E, Y119E, and Y121 E.

[0046]

[0031] The polypeptide of any one of the preceding items, having a lectin pathway inhibition potency greater than that of gigastasin.

[0047]

[0032] The polypeptide of any one of the preceding items, having a lower lectin pathway IC50 value as determined via LP ELISA than gigastasin.

[0048]

[0033] The polypeptide of any one of the preceding items, having a classical pathway inhibition potency lower than that of gigastasin.

[0049]

[0034] The polypeptide of any one of the preceding items, having a higher classical pathway IC50 value as determined via CP ELISA than gigastasin.

[0050]

[0035] The polypeptide of any one of the preceding items, wherein at least one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is selected from the group consisting of D18E, G36R, G36W, Q45K, K53R, L55R, T69R, in particular wherein at least two or at least three of said amino acid substitutions are selected therefrom.

[0051]

[0036] The polypeptide of any one of the preceding items, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is D18E.

[0052]

[0037] The polypeptide of any one of the preceding items, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is G36A, G36D, G36W, or G36E, in particular is G36D.

[0053]

[0038] The polypeptide of any one of the preceding items, wherein said polypeptide is less susceptible to thrombin cleavage than gigastasin, in particular wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is G36D or G36E.

[0039] The polypeptide of any one of the preceding items, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is Q45K.

[0054]

[0040] The polypeptide of any one of the preceding items, wherein one of said amino acid substitution relative to the amino acid sequence as shown in SEQ ID NO: 1 is K53R.

[0055]

[0041] The polypeptide of any one of the preceding items, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is L55R, L55I, L55E or L55V, in particular is L55R.

[0056]

[0042] The polypeptide of any one of the preceding items, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is T69R.

[0057]

[0043] The polypeptide of any one of the preceding items, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is T84R.

[0058]

[0044] The polypeptide of any one of the preceding items, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is Y117D or Y117E, in particular is Y117E.

[0059]

[0045] The polypeptide of any one of the preceding items, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is Y119D or Y119E, in particular is Y119E.

[0060]

[0046] The polypeptide of any one of the preceding items, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is Y121 D or Y121E, in particular is Y121E.

[0061]

[0047] The polypeptide of any one of the preceding items, wherein said amino acid substitutions comprise Y117E, Y119E, and Y121E as well as G36D or L55R, in particular Y117E, Y119E, Y121E, G36D and L55R.

[0062]

[0048] The polypeptide of any one of the preceding items, wherein it is a synthetic polypeptide.

[0063]

[0049] The polypeptide of any one of the preceding items, wherein it is a recombinant polypeptide.

[0064]

[0050] The polypeptide of any one of the preceding items, wherein the gigastasin variant is a synthetic and / or recombinant gigastasin variant.

[0065]

[0051] The polypeptide of any one of the preceding items, wherein the gigastasin variant comprises, or consists of, at least 50 amino acids, in particular 70 to 150 amino acids.

[0066]

[0052] The polypeptide of any one of the preceding items, wherein the gigastasin variant comprises, or consists of, 90 to 140 amino acids, in particular 100 to 130 amino acids.

[0067]

[0053] The polypeptide of any one of the preceding items, wherein the gigastasin variant comprises, or consists of, 110 to 125 amino acids, in particular 120 to 124 amino acids.

[0054] The polypeptide of any one of the preceding items, wherein the gigastasin variant comprises a full-length amino acid sequence as shown in SEQ ID NO: 1 with said amino acid substitutions.

[0068]

[0055] The polypeptide of any one of the preceding items, wherein the gigastasin variant comprises less than 20 of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 , in particular less than 15 of said amino acid substitutions.

[0069]

[0056] The polypeptide of any one of the preceding items, wherein the gigastasin variant comprises 3 to 10 of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1.

[0070]

[0057] The polypeptide of any one of the preceding items, wherein the gigastasin variant comprises less than 10 of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 , in particular 8 or less of said amino acid substitutions.

[0071]

[0058] The polypeptide of any one of the preceding items, wherein the gigastasin variant comprises 3 to 8 of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1.

[0072]

[0059] The polypeptide of any one of the preceding items, wherein the gigastasin variant comprises 7 or less of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 , in particular 6 or less of said amino acid substitutions.

[0073]

[0060] The polypeptide of any one of the preceding items, wherein the gigastasin variant comprises 3 to 6 of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1.

[0074]

[0061] The polypeptide of any one of the preceding items, wherein the gigastasin variant comprises 5 or less of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 , in particular 3, 4, or 5 of said amino acid substitutions.

[0075]

[0062] The polypeptide of any one of the preceding items, wherein said amino acid substitutions are the only amino acid substitutions within SEQ ID NO: 1 , in particular wherein the gigastasin variant consists of as many amino acids as SEQ ID NO: 1 .

[0076]

[0063] The polypeptide of any one of the preceding items, having a lectin pathway inhibition potency greater than that of gigastasin; optionally wherein the ratio of the lectin pathway inhibition potency of the polypeptide to the lectin pathway inhibition potency of gigastasin is at least 2, preferably at least 3, more preferably at least 6, most preferably at least 10.

[0077]

[0064] The polypeptide of any one of the preceding items, having a classical pathway inhibition potency lower than that of gigastasin; optionally wherein the ratio of the classical pathway inhibition potency of gigastasin to the classical pathway inhibition potency of polypeptide is at least 2, preferably at least 4, most preferably at least 6.

[0065] The polypeptide of any one of the preceding items, having a classical pathway IC50 value that is higher for the polypeptide than for gigastasin, wherein these IC50 values are determined using CP ELISA.

[0078]

[0066] The polypeptide of any one of the preceding items, having a lectin pathway IC50 value that is lower for the polypeptide than for gigastasin; optionally wherein the ratio of the lectin pathway IC50 value for gigastasin to the lectin pathway IC50 value for the polypeptide is at least 2, preferably at least 3, more preferably at least 6, most preferably at least 10, wherein these IC50 values are determined using CP ELISA.

[0079]

[0067] The polypeptide of any one of the preceding items, having a ratio of classical pathway IC50 value to lectin pathway IC50 value that is higher for the polypeptide than for gigastasin; optionally is at least 2-fold higher, in particular at least 3-fold higher, preferably at least 6-fold higher, wherein the respective IC50 values are determined using CP ELISA and LP ELISA.

[0080]

[0068] The polypeptide of any one of the preceding items, wherein two of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 are selected from the group consisting of D18E / G36R, L55R / G36W, K53R / G36R, and K53R / G36W.

[0081]

[0069] The polypeptide of item

[0068] comprising amino acid substitutions D18E / G36R.

[0082]

[0070] The polypeptide of item

[0068] comprising amino acid substitutions L55R / G36W.

[0083]

[0071] The polypeptide of item

[0068] comprising amino acid substitutions K53R / G36R.

[0084]

[0072] The polypeptide of item

[0068] comprising amino acid substitutions K53R / G36W.

[0085]

[0073] The polypeptide of any one of the preceding items, wherein the binding affinity to C1s of said polypeptide is lower than that of gigastasin; optionally by at least 10 %, or by at least 25 %, or by at least 80 %.

[0086]

[0074] The polypeptide of any one of the items [1] to

[0032] and

[0035] to

[0063] and

[0066] to

[0072] , having a C1s inhibitory activity greater than that of gigastasin.

[0087]

[0075] The polypeptide of item

[0074] , wherein the ratio of the C1s inhibitory activity of said polypeptide to the C1s inhibitory activity of said gigastasin is at least 2.

[0088]

[0076] The polypeptide of item

[0074] or

[0075] , wherein at least one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 comprises a substitution selected from the group consisting of Q45K, T69R, and combinations thereof.

[0089]

[0077] The polypeptide of any one of the preceding items, wherein said at least one amino acid substitution relative to the amino acid sequence as shown in SEQ ID NO: 1 is selected from the group consisting of G36W, G36R, and T84R.

[0090]

[0078] The polypeptide of any one of the preceding items, wherein the amino acids AKKK (SEQ ID NO: 8), in particular AKKKLPKCQK (SEQ ID NO: 9), form or are at the N-terminal end of the polypeptide and / or wherein no molecule is fused to the N-terminal end of the gigastasin variant.

[0079] The polypeptide of any one of items [1] to

[0077] , wherein the amino acids SNAKKK (SEQ ID NO: 19) or MGHHHHHHEN LYFQSNAKKK (SEQ ID NO: 17), in particular SNAKKKLPKCQK (SEQ ID NO: 20) or MGHHHHHHEN LYFQSNAKKK LPKCQK (SEQ ID NO: 18), form, or are at, the N-terminal end of the polypeptide.

[0091]

[0080] The polypeptide of any one of the preceding items, wherein the amino acids YEYDYE (SEQ ID NO: 10), in particular EDPNEEYEYDYE (SEQ ID NO: 11) or CNPESGRCED PNEEEEEDEE (SEQ ID NO.26), form, or are at, the C-terminal end of the polypeptide; and / or wherein no molecule is fused to the C-terminal end of the gigastasin variant.

[0092]

[0081] The polypeptide of any one of the preceding items, comprising a half-life extending moiety (HLEM).

[0093]

[0082] The polypeptide of item

[0081] , wherein the HLEM is a heterologous amino acid sequence fused to the gigastasin variant.

[0094]

[0083] The polypeptide of item

[0082] , wherein said heterologous amino acid sequence comprises or consists of a protein or peptide selected from the group consisting of transferrin and fragments thereof, the C-terminal peptide of human chorionic gonadotropin, an XTEN sequence, homo-amino acid repeats (HAP), proline-alanine-serine repeats (PAS), albumin, afamin, alpha-fetoprotein, Vitamin D binding protein, polypeptides capable of binding under physiological conditions to albumin or immunoglobulin constant regions, polypeptides capable of binding to the neonatal Fc receptor (FcRn), particularly immunoglobulin constant regions and portions thereof, preferably the Fc portion of immunoglobulin, and combinations thereof.

[0095]

[0084] The polypeptide of any one of items

[0081] to

[0083] , wherein the HLEM is human serum albumin (HSA).

[0096]

[0085] The polypeptide of any one of items

[0081] to

[0083] , wherein the HLEM is an Fc portion of an immunoglobulin.

[0097]

[0086] The polypeptide of item

[0081] , wherein the HLEM is conjugated to the polypeptide comprising the gigastasin variant; or wherein the HLEM is conjugated to the gigastasin variant.

[0098]

[0087] The polypeptide of item

[0086] , wherein the HLEM is conjugated to the polypeptide comprising the gigastasin variant via a cleavable linker; or wherein the HLEM is conjugated to the gigastasin variant via a cleavable linker.

[0099]

[0088] The polypeptide of item

[0086] or

[0087] , wherein the HLEM is conjugated to the N- or C- terminus of the polypeptide comprising the gigastasin variant.

[0100]

[0089] The polypeptide of any one of items

[0086] to

[0088] , wherein said HLEM is selected from the group consisting of hydroxyethyl starch (HES), polyethylene glycol (PEG), polysialic acids (PSAs), elastin-like polypeptides, heparosan polymers, hyaluronic acid and non-proteinaceous albumin binding ligands, e.g. fatty acid chains, and combinations thereof.

[0090] The polypeptide of item

[0081] , wherein the HLEM is non-covalently linked to the polypeptide comprising the gigastasin variant.

[0101]

[0091] The polypeptide of item [1] to

[0080] , wherein the polypeptide consists of the gigastasin variant.

[0102]

[0092] A nucleic acid encoding the polypeptide of any one of the preceding items.

[0103]

[0093] A plasmid or vector comprising the nucleic acid of item

[0092] ,

[0104]

[0094] Cells comprising the nucleic acid of item

[0092] or the plasmid or vector of item

[0093] , in particular wherein the cells are E. coli cells.

[0105]

[0095] An isolated gigastasin variant having the same sequence as the gigastasin variant comprised in the polypeptide of any one of the items [1] to

[0091] ,

[0106]

[0096] A method of producing the polypeptide of any one of items [1] to

[0091] , comprising (i) culturing the cells of item

[0094] under conditions that the polypeptide is expressed; and (ii) optionally recovering the polypeptide from the cells or from culture medium.

[0107]

[0097] The method of item

[0096] , wherein the cells are mammalian cells and / or wherein the expressed gigastasin variant comprises a removable N-terminal tag, wherein the N-terminal tag is cleaved after expression.

[0108]

[0098] The method of item

[0096] or

[0097] , wherein the tag is HSA-6xHis-TEV (SEQ ID NO: 12) and removed by TEV cleavage, in particular with subsequent Ni2+-NTA chromatography, after expression.

[0109]

[0099] The method of any one of items

[0097] to

[0098] , wherein the mammalian cells comprise a nucleic acid encoding the polypeptide without amino acid substitutions at positions Y117, Y119, or Y121 ; or the mammalian cells comprise a nucleic acid encoding the polypeptide with amino acid substitutions at positions Y117. Y119, and Y121.

[0110]

[0100] The method of item

[0096] , wherein the cells are non-mammalian cells, in particular wherein the cells are from prokaryotic or lower eukaryotic organisms, such as yeast or bacteria cells.

[0111]

[0101] The method of item

[0096] or

[0100] , wherein the cells comprise a nucleic acid encoding the polypeptide having amino acid substitutions at positions Y117, Y119, and Y121 with Glutamic acid (Glu) and / or Aspartic acid (Asp).

[0112]

[0102] A method of producing a polypeptide, in particular the method according to any of the items

[0096] to

[0101] , comprising

[0113] (i) culturing cells comprising a nucleic acid encoding the polypetide, in particular of any of the items [1] to

[0091] , with amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 with Glutamic acid (Glu) and / or Aspartic acid (Asp) at positions Y117, Y119, and Y121 ; and (ii) recovering the polypeptide from the cells or from culture medium, in particular wherein the cell is a non-mammalian cell, preferably wherein the cell is a bacteria or yeast cell.

[0114]

[0103] The method of any of items

[0096] to

[0102] , wherein the cells are cultured at a temperature of less than 25°C.

[0115]

[0104] The method of any of items

[0096] to

[0103] , wherein the cells are cultured at a temperature of 16 to 20°C, in particularly at about 18°C.

[0116]

[0105] The method of any of items

[0096] to

[0104] , wherein the cells are cultured for at least 30 hours, in particular for at least 48 hours, preferably at least 55 hours.

[0117]

[0106] The method of any of items

[0096] to

[0105] , wherein the cells are cultured for at least 30 hours, in particular for 55 to 80 hours.

[0118]

[0107] The method of any of items

[0096] to

[0106] or the cells of item

[0094] , wherein the cells are E. coli cells that promote an oxidizing environment in the cytoplasm and / or are engineered to allow for enhanced disulfide bond formation.

[0119]

[0108] The method of or the cells of item

[0107] , wherein the cells are E. coli cells that have mutations in the trxB and gor genes promoting an oxidizing environment in the cytoplasm, thus allowing disulfide bonds to form more easily.

[0120]

[0109] The method or the cells of item

[0108] , wherein the cells are E. coli cells selected from the group of SHuffle strains, Origami Strains, and Rosetta-gami 2.

[0121]

[0110] The method of any one of items

[0096] to

[0109] , wherein the method comprises refolding of insoluble inclusion body fractions of the polypeptide to obtain soluble protein fractions of the polypeptide.

[0122]

[0111] The method of any of the items

[0096] to

[0106] or the cells of item

[0094] , wherein the cells are insect cells (e.g. using baculovirus) or yeast cells.

[0123]

[0112] A formulation comprising the polypeptide of any one of items [1] to

[0091] and an excipient.

[0124]

[0113] The polypeptide of any one of items [1] to

[0091] or the formulation of item

[0112] for use as a medicament and / or for use in a method of treating or preventing a disorder associated with complement activation such as a disorder associated with lectin pathway mediated complement activation and / or thrombo-inflammation.

[0125]

[0114] A method of treating or preventing a disorder associated with complement activation, comprising administering to a subject in need thereof an effective amount of the polypeptide of any one of items of items [1] to

[0091] or of the formulation of item

[0112] ,

[0126]

[0115] The polypeptide for use according to item

[0113] , or the method of item

[0114] , wherein the disorder is inflammation or thrombo-inflammation, for example selected from sustained or prolonged inflammation associated with infection [e.g. cerebral malaria], arthritis, autoimmune diseases [such As celiac disease, post-infectious IBS, diabetes mellitus type 1 , Henoch- Schbnlein purpose (HSP) sarcoidosis, systemic lupus erythematosus (SLE), Sjogren syndrome, eosinophilic granulomatosis with polyangiitis, Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenic purpura, Addison's disease, rheumatic arthritis (RA), ankylosing spondylitis, polymyositis (PM), dermatomyositis (DM), Alopecia Areata and ischemia reperfusion injury (IRI) of various organs (liver, colon, etc.), peripheral vascular disease, transfusion-related acute lung injury (TRALI), transplant rejection, pre-eclampsia, severe burns, atherosclerosis, hypertension, antiphospholipid syndrome, sickle cell disease, bacterial and viral infection ischemic restenosis, sepsis, major trauma, and disorders in which platelets modulate cell functions including, without limitation, cancer cells proliferation and / or dissemination.

[0127]

[0116] The polypeptide for use according to item

[0113] , or the method of item

[0114] , wherein said disorder associated with complement activation is selected from the group consisting of ischemia-reperfusion injury, myocardial infarct, stroke, hyperacute rejection, transplant rejection, organ rejection, sepsis, cardiopulmonary bypass, adult respiratory distress syndrome, allergic asthma, restenosis, multiple organ dysfunction syndrome, trauma, hemorrhagic shock, Guillain-Barre syndrome, paroxysmal nocturnal hemoglobinuria, glomerulonephritis, autoimmune disorders, systemic lupus erythematosus, rheumatoid arthritis, age-related and neurodegenerative diseases, Alzheimer's disease, myasthenia gravis, systemic sclerosis, multiple sclerosis, platelet storage, serum sickness, biomaterial- induced thromboinflammatory reactions, atypical hemolytic uremic syndrome (aHUS), hemolytic anemias, and hemodialysis.

[0128]

[0117] The polypeptide for use according to item

[0113] , or the method of item

[0114] , wherein said disorder associated with complement activation is selected from the group consisting of IgA nephropathy, membranous nephropathy, diabetic kidney disease, ischemia reperfusion injury (IRI), renal IRI, myocardial IRI, ischemic stroke, myocardial infarction, stem cell transplantation-associated thrombotic microangiopathy, rheumatoid arthritis, systemic lupus erythematosus, atherosclerosis, COVID-19, acute respiratory distress syndrome, biomaterial- induced inflammatory reactions, adverse effects of hemodialysis treatment, in particular wherein said disorder is associated with lectin pathway activation.

[0129]

[0118] The polypeptide for use according to item

[0113] , or the method of item

[0114] , wherein said disorder associated with complement activation is selected from the group consisting of IgA nephropathy, membranous nephropathy, diabetic kidney disease, ischemia reperfusion injury (IRI), renal IRI, myocardial IRI, ischemic stroke, myocardial infarction, stem cell transplantation-associated thrombotic microangiopathy, rheumatoid arthritis, systemic lupus erythematosus, atherosclerosis, COVID-19, and acute respiratory distress syndrome.

[0130]

[0119] The polypeptide for use according to item

[0113] , or the method of item

[0114] , wherein said disorder associated with complement activation is a disorder resulting from or associated with ischemia, in particular is a disorder selected from the group consisting of ischemia reperfusion injury such as renal IRI or myocardial IRI, ischemic stroke, myocardial infarction, and renal ischemia associated disorders.

[0131]

[0120] The polypeptide for use according to item

[0113] , or the method of item

[0114] , wherein said disorder associated with complement activation is ischemia-reperfusion injury, in particular ischemia-reperfusion injury of the kidney, brain and / or heart, or a disorder causing or that may result from ischemia-reperfusion injury, in particular ischemic stroke, transplant rejection, myocardial infarction, delayed graft function or primary graft dysfunction.

[0132]

[0121] The polypeptide for use according to item

[0113] ,

[0116] to

[0120] , or the method of item

[0114] , wherein said disorder associated with complement activation is stroke, in particular ischemic stroke.

[0133]

[0122] The polypeptide for use according to item

[0113] , or the method of item

[0114] , wherein said disorder associated with complement activation is myocardial infarction.

[0134]

[0123] The polypeptide for use according to item

[0113] , or the method of item

[0114] , wherein said disorder associated with complement activation is stem cell transplantation-associated thrombotic microangiopathy.

[0135]

[0124] The polypeptide for use according to item

[0113] , or the method of item

[0114] , wherein said disorder associated with complement activation is IgA nephropathy.

[0136]

[0125] The polypeptide for use according to item

[0113] , or the method of item

[0114] , wherein said disorder associated with complement activation is Sjogren syndrome, myasthenia gravis, or systemic sclerosis.

[0137]

[0126] The polypeptide for use according to item

[0113] , or the method of item

[0114] , wherein said disorder associated with complement activation is Sjogren syndrome.

[0138]

[0127] The polypeptide for use according to item

[0113] , or the method of item

[0114] , wherein said disorder associated with complement activation is myasthenia gravis.

[0139]

[0128] The polypeptide for use according to item

[0113] , or the method of item

[0114] , wherein said disorder associated with complement activation is systemic sclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0140] Figure 1 : Sequence, structural organization, and C1s interaction of gigastasin. (A) Primary amino acid sequence of gigastasin with reported key residues for C1s interactions marked by boxes. Cysteine residues engaged in disulfide bridges are underlined and residues affected by posttranslational modifications or enzymatic cleavage are indicated. (B) Disulfide bridge pattern and domain organization of gigastasin. (C) Major interaction areas of gigastasin (ribbon) with the C1s protein (surface representation; PDB: 5LIBM).

[0141] Figure 2: Visualization of in s / 7 / co-predicted mutations selected for experimental validation. (A) Amino acid mutations introduced into gigastasin are highlighted and shown in stick representation on the ribbon-structure of the protein. The C1s protein is displayed in surface representation (PDB: 5LIBM). (B) Relevant mutations are depicted in the primary sequence (top) along with the calculated interaction energy (bottom) per residue between gigastasin and C1s. Zero values indicate no binding of this particular gigastasin residue to C1s. Error bars represent standard deviations of n=6 molecular dynamics simulations of 30 ns.

[0142] Figure 3: C1s inhibition of predicted gigastasin mutants as determined by a chromogenic substrate assay. (A) Comparison of C1s inhibition by gigastasin and its mutation derivatives W17G, D18E, K53R, and L55R. (B) Comparison of C1s inhibition by gigastasin and its mutation derivatives G36D, G36E, G36R, and G36W. (C) Comparison of C1s inhibition by gigastasin and its mutation derivatives Q45K, T69R, G71 E, G71Q, and T84R.

[0143] Figure 4: Complement inhibition potency of predicted gigastasin mutants upon classical pathway (CP) or lectin pathway (LP) activation. (A) Comparison of CP inhibition by gigastasin and its mutation derivatives W17G, D18E, K53R, and L55R. (B) Comparison of CP inhibition by gigastasin and its mutation derivatives G36D, G36E, G36R, and G36W. (C) Comparison of CP inhibition by gigastasin and its mutation derivatives Q45K, T69R, G71 E, G71Q, and T84R. (D) Comparison of LP inhibition by gigastasin and its mutation derivatives W17G, D18E, K53R, and L55R. (E) Comparison of LP inhibition by gigastasin and its mutation derivatives G36D, G36E, G36R, and G36W. (F) Comparison of LP inhibition by gigastasin and its mutation derivatives Q45K, T69R, G71 E, G71Q, and T84R. Figure 5: Interaction profiles of gigastasin and selected mutants with its known target C1s and the ortholog C1 r enzyme as measured by surface plasmon resonance. Gigastasin and mutants thereof were immobilized on separate flow cells of SPR sensor chips and a concentration series (4.7-300 nM) of each enzyme was injected into solution.

[0144] Figure 6: C1s inhibition potency of W17 mutants of gigastasin as determined by a chromogenic substrate assay. (A) Amino acids selected as replacement of Trp17 during mutagenesis. (B) 01 s inhibition activity of mutation derivatives W17L, W17P, W17R, W17Y in comparison to parental gigastasin. (C) C1 s inhibition activity of mutation derivatives W17A, W17F, W17G, W17H in comparison to parental gigastasin.

[0145] Figure 7: CP and LP inhibition potency of W17 mutants of gigastasin. (A) CP and LP inhibition potency of gigastasin W17 mutants W17A, W17F, W17G, and W17H. (B) CP and LP inhibition potency of gigastasin W17 mutants W17L, W17P, W17R, and W17Y.

[0146] Figure 8: SPR interaction profiles of gigastasin W17 mutants with C1s and C1 r. Gigastasin mutants were immobilized on separate flow cells of SPR sensor chips and a concentration series (4.7-300 nM) of each enzyme was injected.

[0147] Figure 9: Effect of conservative N-terminal truncation of gigastasin on C1s inhibition. C1 s inhibition activity for a gigastasin mutant missing the first seven amino acids (gigastasin A1-7; expressed in E. coli) as measured by CSA.

[0148] Figure 10: Impact of C-terminal tail modifications on C1s inhibitory activity of gigastasin. (A) Effect of C-terminus truncation (gigastasin A113-122) on 01s inhibition as measured by CSA. (B) Changes in 01 s inhibition as measured by CSA upon mutation of all three C-terminal Tyr residues at position Y117, Y119, and Y121 to Ala (BD3A), Arg (BD3R), or Asp (BD3D).

[0149] Figure 11 : Modulation of LP inhibition activity and / or selectivity by combining individual beneficial mutations. (A-B) Constructs of gigastasin with selected double-mutants were expressed in E. coli. Newly generated inhibitors were tested in CP and LP ELISA and directly compared to parental gigastasin. The selectivity enhancements were visualized by relative inhibition changes for the CP and LP as measured by ELISA. (A) Combinations of individual mutations, which led to weaker potency in CP and LP ELISAs. (B) Combinations of individual mutations, which lead to enhanced LP selectivity with maintained or improved LP inhibition potency.

[0150] Figure 12: Modulation of CP / LP inhibition activity and selectivity by mutagenesis of position Leu55. (A) Comparison of CP inhibition (x-axis) and LP inhibition (y-axis) potency of gigastasin Leu55 mutants as determined by CP and LP ELISA. (B) Table of IC50 values of CP vs. LP inhibition for Leu55 mutations alongside the amino acids of the substituted residues.

[0151] Figure 13: Effect of in vitro sulfation. Evaluation of C1 s-inhibition activity of gigastasin expressed in bacteria before and after in vitro sulfation via recombinant human tyrosylprotein sulfotransferase as measured by CSA.

[0152] Figure 14: Susceptibility of gigastasin to thrombin-mediated cleavage and mitigation of stability risk via mutagenesis of position Gly36. (A) SDS-PAGE analysis of thrombin stability assay samples under non-reducing conditions. (B) SDS-PAGE analysis of thrombin stability assay samples under reducing conditions for gigastasin and its G36D as well as G36E mutants. (C) Comparison of gigastasin and its G36A mutant in the C1s-CSA. (D) Analysis of gigastasin and its G36A and G36D mutants towards the CP as measured by ELISA. (E) Analysis of gigastasin and its G36A and G36D mutants towards the LP as measured by ELISA.

[0153] Figure 15: Complement pathway inhibition of untagged, non-sulfated gigastasin and derivatives thereof containing C-termini with increased negative charges and / or N- terminal HSA fusion tags. (A) Classical pathway inhibition of gigastasin produced in bacteria (E. co / / ; BD001) and its derivative with C-terminal Tyr-to-Glu mutations (BD3E) in comparison with the sulfated HSA-gigastasin fusion protein produced in mammalian cells (HEK) in its intact form (HSA-TEV-Gigastasin) and after removal of the HSA tag by TEV protease (Gigastasin, cleaved tag). (B) Lectin pathway inhibition of gigastasin produced in bacteria (E. co / / ; BD001) and its derivatives with C-terminal Tyr-to-Glu mutations (BD3E) or Tyr-to-Asp mutations (BD3D) in comparison with the sulfated HSA-gigastasin fusion protein produced in mammalian cells (HEK) in its intact form (HSA-TEV-Gigastasin) and after removal of the HSA tag by TEV protease (Gigastasin, cleaved tag).

[0154] Figure 16: Proposed lipidation strategy to improve plasma half-life. (A) Fatty acids or diacids (zigzag line) are coupled to the N- or C-terminus of gigastasin, or to other exposed residues not relevant for target binding, via a suitable linker and spacer. Transient interaction of the lipidated protein with fatty acid binding sites on human serum albumin (HSA) leads to prolonged plasma residence. (B) Example of a lipidation entity comprised of a fatty diacid linked to a glutamoyl linker and PEG spacer.

[0155] Figure 17: Construction of gigastasin-lgG4 Fc fusion proteins. Schematic representation of fusion protein design in (A) traditional gigastasin-Fc and (B) reversed format Fc-gigastasin. (C) SDS-PAGE analysis of gigastasin-Fc fusion constructs after mammalian expression. M) protein marker; 1) sample before FPLC; 2) FPLC flow-through; 3) pierce concentrator flow- through Peak A; 4) reduced sample peak A; 5) non-reduced sample peak A; 6) pierce concentrator flow-through peak B; 7) reduced sample peak B; 8) non-reduced sample peak B.

[0156] Figure 18: Inhibitory activity of gigastasin-Fc fusion expressed in mammalian cells (HEK) in comparison to a gigastasin derivative with C-terminal Tyr-to-Glu mutations (BD3E) expressed in bacteria (E. coli). (A) Chromogenic substrate assay for C1s inhibition. (B) CP activation / inhibition ELISA. (C) LP activation / inhibition ELISA.

[0157] Figure 19: In-silico immunogenicity prediction. (A) Results from Lonza’s tool Epibase predicting MHCII epitopes within the gigastasin sequence with predicted binding affinity (upper section) alongside the allelic coverage within the population (lower section). (B) Visualization of three residues identified as potentially immunogenic within the predicted epitope in ribbon structure of gigastasin.

[0158] Figure 20: Characterization of initial deimmunization mutants of gigastasin using the C1s-CSA. Single mutants Y70F and K77Y were selected based on predicted deimmunization potential and minimal interference with target binding. E106T serves as additional control since this position is not involved in target interaction and does not contribute to any secondary structure elements. Error bars represent the standard deviation of n=3 technical replicates.

[0159] Figure 21 : Summary of mutagenesis strategy performed during SAR studies. Major mutations at identified key positions and their proposed / observed effect as marked in the primary sequence of gigastasin. Mutations Y117E, Y119E, Y121 E, G36D are of particular interest and highlighted in bold font in the boxes. Cysteine residues [CJ are underlined. DETAILED DESCRIPTION

[0160] The present invention relates to a polypeptide comprising a gigastasin variant in which one or more residues have been substituted compared to the naturally occurring gigastasin of the giant amazon leech and in which at least one activity of gigastasin is modulated in said polypeptide.

[0161] Gigastasin and variants thereof

[0162] Gigastasin is a molecule that is derived from the giant Amazonian leech (Haementaria sp.) having the amino acid sequence as shown in SEQ ID NO: 1. Other natural polymorphic variants have been found. Further information is, for example, disclosed in WO2001 / 98365 and WO1999 / 36439 as well as Pang et al. J Immunol, Dec 2017; 199 (11): 3883-3891 , which are incorporated herein by reference. Gigastasin inhibits certain serine proteases involved in coagulation, like factor Xia and factor XI la, and serine proteases of the complement activation pathway, like C1 s, C1 r, MASP-1 , and MASP-2. Complement activation plays a crucial role in the immune defense and is activated by three pathways, namely classical, lectin, and alternative pathway. While the classical pathway is initiated by the C1s and C1r proteases, the lectin pathway is induced by the MASP-1 and MASP-2 proteases.23Gigastasin is known as a potent inhibitor of C1s and MASP-2, to a lesser extent MASP-1 and a weak inhibitor of C1 r.23In its most general form, “potent inhibitor” refers to a substance that is effective at inhibiting a biological or chemical process, usually by binding to its target, such as an enzyme or receptor; in the case of gigastasin it is the inhibition of proteases.

[0163] The term “gigastasin” (also “BD001”) as used herein shall be understood to refer to a molecule that comprises the amino acid sequence as shown in SEQ ID NO: 1. SEQ ID NO: 1 has the amino acid sequence AKKKLPKCQK QEDCGSWDLK CNNVTKKCEC RNQVCGRGCP KERYQRDKYG CRKCLCKGCD GFKCRLGCTY GFKTDKKGCE AFCTCNTKET ACVNIWCTDP YKCNPESGRC EDPNEEYEYD YE. The term “gigastasin” includes naturally occurring gigastasin having the amino acid sequence as shown in SEQ ID NO: 1 and gigastasin produced by recombinant technology having the amino acid sequence as shown in SEQ ID NO: 1. These may differ in posttranslational modifications. For example, naturally occurring gigastasin is sulfated, whereas for recombinant gigastasin this is not necessarily the case (depending on cells used for expression). If herein any activities or properties of gigastasin and gigastasin variants are compared, it is to be understood that the molecules to be compared have different amino acid sequences, but have been produced under substantially the same conditions, i.e. have been recombinantly expressed in the same system using the same cell type for expression (for example using E. coli cells), in particular as described in section 1.3 of this disclosure.

[0164] “Gigastasin variants” as used herein are serine protease inhibitors towards 01 s and / or MASP- 2 with at least 80% sequence identity to SEQ ID NO: 1 , wherein at least one amino acid of gigastasin has been substituted. Gigastasin variants as used herein specifically shall include recombinant gigastasin variants with non-naturally occurring substitutions. In some embodiments, gigastasin variants described herein may have at least 90% sequence identity, in particular at least 95% sequence identity, to SEQ ID NO: 1 of gigastasin. It will be understood and has been demonstrated by extensive experimentation that amino acids of gigastasin can be substituted without elimination of said protease inhibitory activity. However, with some non- naturally occurring substitutions described herein the degree of the protease inhibitory activity as well as complement pathway selectivity can be adjusted. For example, several amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 are described herein that result in enhanced potency and / or selectivity for the lectin pathway of complement activation. Unless otherwise indicated, any amino acid substitutions for gigastasin variants of this disclosure are relative to the amino acid sequence as shown in SEQ ID NO: 1. Any quantitative change of potency, selectivity, stability, or other properties and activities described in this disclosure refer to the comparison of gigastasin variants and gigastasin as shown in SEQ ID NO: 1 under substantially the same production conditions.

[0165] Reference to "about" a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter perse. In certain embodiments, the term "about" includes the indicated amount ± 15%. In other embodiments, the term "about" includes the indicated amount ± 10%, in particular ± 5%. In certain other embodiments, the term "about" includes the indicated amount ± 1 %. Also, the singular forms "a" and "the" include plural references unless the context clearly dictates otherwise. Thus, e.g., reference to "the polypeptide" includes a plurality of such polypeptides and reference to "the assay" includes reference to one or more assays and equivalents thereof known to those skilled in the art.

[0166] An "isolated" polypeptide is one that has been identified, separated, and / or recovered from a component (e.g., natural or recombinant) of its production environment. Preferably, the isolated polypeptide is free of association with all other components from its production environment. Contaminant components of its production environment (such as those resulting from recombinantly transfected cells) are substances that would typically interfere with research, diagnostic, or therapeutic uses for proteins. Typically, isolated polypeptide will be prepared by at least one purification step.

[0167] Activity of Gigastasin

[0168] According to the present invention, at least one activity of gigastasin is modulated in the polypeptide. Activities of gigastasin which may be modulated include serine protease inhibitory activity, anticoagulant activity, anti-inflammatory activity, stability and combinations thereof.

[0169] The most prominent activity of gigastasin is its inhibitory activity on certain serine proteases.

[0170] As mentioned above, when an activity of a polypeptide of the invention is compared to an activity of gigastasin having the amino acid sequence as shown in SEQ ID NO: 1 , it is understood that the molecules to be compared are obtained in substantially the same way, e.g. by substantially the same production process. For example, if the polypeptide of the invention is compared to gigastasin, and the polypeptide of the invention was expressed in E. coli cells, then also the reference molecule gigastasin should be produced and obtained from the same cells.

[0171] The inhibitory activity of an inhibitor can be determined by substrate activity assays and substrate binding assays known in the art. Such methods comprise but are not limited to, biolayer interferometry (BLI), surface plasmon resonance (SPR), ELISA tests, Western blots and immunofluorescence-based assays, protein stability assays, and chromogenic or fluorogenic substrate assays (CSA or FSA, respectively). For example, a standard BLI, SPR, ELISA, or a CSA / FSA can be carried out. A CSA / FSA is commonly used to measure the activity of serine proteases, particularly those involved in coagulation, and makes use of a synthetic peptide comprising a chromogenic group, which gets released after enzymatic cleavage and produces a color. A potent inhibitor binds tightly and effectively to its target, requiring only a small amount to achieve a significant inhibitory effect.

[0172] Unless defined otherwise, the inhibitory activity as used herein is determined as described in the examples. The IC50 value is typically a measure of the inhibition potency (lower IC50 values correspond to increased inhibition potency). The IC50 value for the lectin pathway (LP) is measured using LP ELISA and the IC50 value for the classical pathway (CP) is measured using CP ELISA. Conditions for both assays are described in section 1.7. When IC50 values of gigastasin and gigastasin variants or of different variants are being compared, the same ELISA conditions will be used.

[0173] At least one activity of gigastasin is modulated in the polypeptide. For example, modulation may be an increase or a decrease in activity compared to that of gigastasin. Generally, modulation may be an increase, e.g. of inhibitor activity or stability, by at least 10%, or at least 25%, or at least 50%, or at least 100%, or at least 200%, or at least 500%. Alternatively, modulation may be a decrease by at least 10%, or at least 20%, or at least 30%, or at least 50%, or at least 70%, or at least 90%.

[0174] In one embodiment, the polypeptide of the invention has a lectin pathway inhibition potency greater than that of gigastasin. In such embodiments, the ratio of the lectin pathway inhibition potency of the polypeptide to the lectin pathway inhibition potency of gigastasin is greater than 1 , or at least 2, preferably at least 3, more preferably at least 6, most preferably at least 10. The lectin pathway inhibitory activity can be determined in an ELISA as described in section 1 .7 of the examples. Examples of such embodiments include polypeptides having substitutions at positions D18, G36, Q45K, K53, L55, T69 and T84 relative to gigastasin.

[0175] In other embodiments, the polypeptide of the present invention has a classical pathway inhibition potency lower than that of gigastasin. In such embodiments, the ratio of the classical pathway inhibition potency of gigastasin to the classical pathway inhibition potency of the polypeptide is greater than 1 , or at least 2, preferably at least 4, most preferably at least 6. The classical pathway inhibitory activity can be determined in an ELISA as described in section 1 .7 of the examples.

[0176] In certain embodiments, the polypeptide of the present invention has a C1s inhibitory activity greater than that of gigastasin. Preferably, the polypeptide of the present invention has a C1 s inhibitory activity characterized by an IC50 value of less than 40 nM, determined in a competitive chromogenic substrate assay as described in section 1.6 of the Examples. In some embodiments, the IC50 value of the polypeptide is less than 20 nM, or less than 10 nM, or less than 5 nM. Examples of such embodiments include polypeptides having the substitutions Q45K and T69R relative to gigastasin. Further examples of such embodiments include polypeptides having the substitutions Y117D orY117E, Y119D orY119E, and Y121 D orY121 E relative to gigastasin. In certain embodiments, the polypeptide of the present invention is designed to increase the overall expression and / or production yield when compared to gigastasin. Examples of such embodiments include polypeptides as described herein that have substitutions at positions Y115, Y119 and Y121 relative to gigastasin.

[0177] In certain embodiments, the polypeptide of the present invention is designed to increase the overall potency, stability, and production yield when compared to gigastasin. Examples of such embodiments include polypeptides having the substitutions G36D, Y115E, Y119E and Y121 E relative to gigastasin.

[0178] In certain embodiments, the polypeptide of the present invention is designed to increase the affinity to C1 s and / or MASP-2. Examples of such embodiments include polypeptides having the substitutions K10D or K10E relative to gigastasin.

[0179] Amino Acid Substitutions

[0180] Hereinafter specific amino acid substitutions for gigastasin variants will be described that have been shown to improve and modulate at least one activity of gigastasin and / or may have further beneficial effects.

[0181] In some embodiments, the gigastasin variant comprises the amino acid substitution D18E. This mutation may increase inhibition of the classical and / or lectin pathway in ELISA assay(s), compared to the same gigastasin variant without said substitution.

[0182] In some embodiments, the gigastasin variant comprises the amino acid substitution G36D, G36E, G36R or G36W. Such a mutation may increase inhibition of and / or selectivity for the lectin pathway in ELISA assay(s), compared to the same gigastasin variant without said substitution. G36W may be associated with a stronger increase than G36D, G36E, and G36R. G36D and G36E may be associated with a desirable resistance to thrombin cleavage, in particular G36D.

[0183] In some embodiments, the gigastasin variant comprises the amino acid substitution Q45K. This mutation may increase inhibition of the classical and / or lectin pathway in ELISA assay(s), compared to the same gigastasin variant without said substitution. In some embodiments, the gigastasin variant comprises the amino acid substitution K53R. Such a mutation may increase inhibition of and / or selectivity for the lectin pathway in ELISA assay(s), compared to the same gigastasin variant without said substitution.

[0184] In some embodiments, the gigastasin variant comprises the amino acid substitution L55R, L55E, L55Q or L55V. Such a mutation may increase inhibition of and / or selectivity for the lectin pathway in ELISA assay(s), compared to the same gigastasin variant without said substitution. L55R may have a particularly high increase of selectivity for the lectin pathway with a significant decrease in inhibition of the classical pathway.

[0185] In some embodiments, the gigastasin variant comprises the amino acid substitution T69R. This mutation may increase inhibition of the classical and / or lectin pathway in ELISA assay(s), compared to the same gigastasin variant without said substitution.

[0186] In some embodiments, the gigastasin variant comprises the amino acid substitution T84R. This mutation may increase inhibition of and / or selectivity for the lectin pathway in ELISA assay(s), compared to the same gigastasin variant without said substitution.

[0187] In some embodiments, the gigastasin variant comprises amino acid substitutions at one or more positions selected from Y117, Y119, Y121. It was found that substitutions with Ala and / or Arg are beneficial. Substitutions Y117E, Y119E, Y121 E were found to be particularly desirable, but also Y117D, Y119D and Y121 D show a beneficial effect. Also, any mixtures of these such as Y117D, Y119E, Y121 D or Y117E, Y119D, Y121 D are possible and within the scope of this disclosure. The phrase “wherein each of these positions is substituted with either glutamic acid or aspartic acid’ as used herein shall be understood to also make reference to any such mixtures and is not limited to Y117E, Y119E, Y121 E or Y117D, Y119D, Y121 D alone. Said substitution(s) are of particular relevance, as natural gigastasin contains certain posttranslational modifications that differ in recombinantly generated gigastasin and variants thereof using cells with a low(er) degree of posttranslational modifications, in particular tyrosine-O-sulfation (e.g. many non-mammalian cells, but also certain mammalian cells). Such posttranslational modifications are not only advantageous when producing recombinant gigastasin, but also may enhance complement inhibition. Switching to cells with a lesser degree of posttranslational modifications can represent a challenge. The inventors found that the negative impact from using such cells can be reduced when Ala and / or Arg are substituted for at least some of the positions Y117, Y119, Y121 with a stronger effect when more positions are substituted. This provides access to improved development of numerous gigastasin variants using recombinant expression technology with such cell types, for example with E. coli. Therefore, the proposed substitutions at positions Y117, Y119, Y121 facilitate the general accessibility of respective gigastasin variants. Beyond the herein described therapeutic benefit of these three point mutations, this also may improve the process of generating, exploring and experimentally testing the effect of new point mutations or combinations thereof to identify and confirm technical effects of novel gigastasin variants (many of which are described herein). Therefore, the teaching of substitutions at positions Y117, Y119, Y121 constitutes the backbone of a versatile platform for research, manufacture and development of recombinant gigastasin variants to be used as therapeutic molecules, research tools or other non- therapeutic uses described herein that otherwise may be less accessible.

[0188] It will be understood that the above substitutions may be combined with each other and, in some cases, may result in synergistic effects beyond additive effects. In some embodiments, specific mutations are combined to achieve functional improvements that complement each other, wherein the gigastasin variant comprises three amino acid substitutions at positions Y117, Y119, Y121 as well as a fourth mutation increasing resistance to thrombin-mediated cleavage at position G36 and at least one fifth mutation enhancing LP selectivity and or potency. Suitable mutations at said positions have been described herein. In one embodiment the three amino acid substitutions are Y117E, Y119E, Y121 E, the fourth amino acid substitution is G36D or G36E, in particular G36D, and the fifth amino acid substitution is selected from a group consisting of D18E, Q45K, K53R, L55R, L55I, L55Q, L55V, T69R, and T84R.

[0189] Preferred gigastasin variants of the invention include the following:

[0190] - Gigastasin variants comprising G36E, Y117E, Y119E, and Y121 E; and optionally D18E, Q45K, K53R, L55R, L55I, L55Q, L55V, T69R, or T84R.

[0191] - Gigastasin variants comprising G36D, Y117E, Y119E, and Y121 E; and optionally D18E, Q45K, K53R, L55R, L55I, L55Q, L55V, T69R, or T84R. Examples include variants comprising, or consisting of, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, or SEQ ID NO: 25.

[0192] - Gigastasin variants comprising G36E, Y117D, Y119D, and Y121 D; and optionally D18E, Q45K, K53R, L55R, L55I, L55Q, L55V, T69R, or T84R.

[0193] - Gigastasin variants comprising G36D, Y117D, Y119D, and Y121 D; and optionally D18E, Q45K, K53R, L55R, L55I, L55Q, L55V, T69R, or T84R.

[0194] - Gigastasin variants comprising G36W, Y117E, Y119E, and Y121 E; and optionally D18E, Q45K, K53R, L55R, L55I, L55Q, L55V, T69R, or T84R. Examples include variants comprising, or consisting of, SEQ ID NO: 24. Moreover, it has been shown that many additional substitutions are possible, which may show a smaller degree or no modulation of a gigastasin activity. The inventors contributed a teaching according to which certain substitutions within gigastasin show an enhancement in lectin pathway inhibition, but also gigastasin variants with further improvements in production yields, resistance to thrombin cleavage, classical pathway inhibition, selectivity for certain pathways, or half-life extension. This teaching is not limited to specific gigastasin variants, but instead allows a person skilled in the art to modulate gigastasin activity for the intended purpose, e.g. upregulating lectin pathway inhibition and modulating classical pathway inhibition for various gigastasin variants. Based on this teaching a desired technical effect can be achieved with new variants in a simple and straightforward manner.

[0195] Tag sequence

[0196] The polypeptide of the invention may comprise a tag sequence to facilitate purification and / or detection of the polypeptide of the invention. A suitable tag sequence is the 6xHis-TEV tag which allows purification by Ni2+ columns, and which can be cleaved after affinity purification. Upon cleavage a fragment having the sequence SEQ ID NO: 16 is released. In some embodiments, the tag sequence is removed after the molecule has been produced and / or after a purification step. In some embodiments, two amino acids (SN) of the tag sequence are remaining at the N-terminus of the polypeptide. Other suitable tag sequences are known to the skilled person.

[0197] Binding affinity

[0198] Some polypeptides of the invention exhibit enhanced binding affinity to lectin complement activation pathway enzyme proteins, as determined by surface plasmon resonance (SPR). Gigastasin variants that show enhanced potency in the LP ELISA are also expected to exert improved binding affinity to MASP-2 and / or MASP-1 when compared to gigastasin in SPR assays. The binding affinity may be determined as described in section 1.8 of the examples. After immobilization of gigastasin variants on individual flow cells on an SPR sensor chip, enzymes (e.g. MASP-2) are injected in solution to monitor the interaction profiles. Enhanced binding affinity is determined by a lower dissociation constant (KD), a higher response unit (RU) value and / or a slower dissociation rate. Some polypeptides of the invention exhibit reduced binding affinity to classical complement activation pathway enzyme C1s, as determined by surface plasmon resonance (SPR). The binding affinity is preferably determined as described in section 1.8 of the examples. After immobilization of gigastasin variants on individual flow cells on an SPR sensor chip, enzymes (e.g. C1s) were injected into solution to monitor the interaction profiles. Reduced binding affinity is determined by a higher dissociation constant (KD), a lower response unit (Rll) value and / or a faster dissociation rate.

[0199] In some embodiments, the binding affinity to C1s of the polypeptide is reduced compared to the binding affinity to C1s of gigastasin. For example, the binding affinity may be reduced by at least 10 %, or at least 20 %, or at least 30 %, or at least 40 %, or at least 50 %, or at least 60 %, or at least 70 %, or at least 80 %, or at least 90 %. The polypeptide may comprise the substitutions G36Wor G36R; and / or T84R. The KDvalue of the polypeptide in an affinity assay is preferably less than 144 nm, more preferably less than 125 nm, more preferably less than 100 nM.

[0200] Half-life extending moiety (HLEM)

[0201] In addition to amino acid substitutions in gigastasin, the polypeptide of the invention may in certain embodiments further comprise a half-life extending moiety. The term half-life extending moiety refers to a molecular modification or addition of an agent to the substance that increases its half-life, which refers to the time it takes for half of the substance to be eliminated from the bloodstream of the body. Typically, half-time extending moiety is used to prolong the therapeutic effect of the substance and improve overall drug efficacy together with patient compliance. In the present invention, the half-life-extending moiety may be a heterologous amino acid sequence fused to the gigastasin variant. Alternatively, the half-life-extending moiety may be chemically conjugated to the polypeptide comprising the gigastasin variant by a covalent bond, e.g. by a covalent bond different from a peptide bond.

[0202] In certain embodiments of the invention, the half-life of the polypeptide of the invention is extended by chemical modification, e.g. attachment of a half-life extending moieties such as polyethylene glycol (PEGylation), glycosylated PEG, hydroxyl ethyl starch (HESylation), polysialic acids, elastin-like polypeptides, heparosan polymers or hyaluronic acid. In another embodiment, the polypeptide of the invention is conjugated to an HLEM such as albumin via a chemical linker. The principle of this conjugation technology has been described in an exemplary manner by Conjuchem LLC (see, e.g., US patent No. 7,256,253). In another embodiment, the polypeptide of the invention is conjugated to fatty acids or derived lipidation moieties, or to other albumin-binding moieties to enable transient binding to HSA in blood circulation.

[0203] In other embodiments, the half-life-extending moiety is human serum albumin (HSA) or an immunoglobulin Fc fragment. These may be fused to the N-terminus of the gigastasin variant. The recombinant polypeptide may further comprise a covalent bond positioned between the HSA or the immunoglobulin Fc fragments and the gigastasin variant, or a flexible linker positioned between the HSA or the immunoglobulin Fc fragments and the gigastasin variant, including, but not limited to a 2xGGGGS linker (SEQ ID NO: 4) and a TEV cleavage site (e.g., SEQ ID NO: 15), a 5xGGS linker (SEQ ID NO: 5) or a 2xGGS-6xHis-TEV linker (SEQ ID NO: 6).

[0204] Pharmaceutical compositions

[0205] The polypeptide of the present invention can be formulated into pharmaceutical preparations for therapeutic use. The purified polypeptide may be dissolved in conventional physiologically compatible aqueous buffer solutions to which there may be added, optionally, pharmaceutical excipients to provide pharmaceutical preparations.

[0206] Such pharmaceutical carriers and excipients as well as suitable pharmaceutical formulations, are well known in the art (see for example "Pharmaceutical Formulation Development of Peptides and Proteins", Frokjaer et al., Taylor & Francis (2000) or "Handbook of Pharmaceutical Excipients", 3rd edition, Kibbe et al., Pharmaceutical Press (2000)). Standard pharmaceutical formulation techniques are well known to persons skilled in the art (see, e.g., 2005 Physicians’ Desk Reference®, Thomson Healthcare: Montvale, NJ, 2004; Remington: The Science and Practice of Pharmacy, 20thed., Gennaro et al., Eds. Lippincott Williams & Wilkins: Philadelphia, PA, 2000). In particular, the pharmaceutical composition comprising the polypeptide of the invention may be formulated in lyophilized or stable liquid form. The polypeptide may be lyophilized by a variety of procedures known in the art. Lyophilized formulations are reconstituted prior to use by the addition of one or more pharmaceutically acceptable diluents, such as sterile water for injection or sterile physiological saline solution.

[0207] Formulations of the composition are delivered to the individual by any pharmaceutically suitable means of administration. Various delivery systems are known and can be used to administer the composition by any convenient route. Preferentially, the compositions of the invention are administered systemically. For systemic use, the proteins of the invention are formulated for parenteral (e.g. intravenous, subcutaneous, intramuscular, intraperitoneal, intracerebral, intrapulmonary, intranasal or transdermal) or enteral (e.g., oral, vaginal or rectal) delivery according to conventional methods. The most preferential routes of administration are intravenous and subcutaneous administration. The formulations can be administered continuously by infusion or by bolus injection. Some formulations encompass slow-release systems.

[0208] The polypeptides of the present invention are administered to patients in a therapeutically effective dose, meaning a dose that is sufficient to produce the desired effects, preventing or lessening the severity or spread of the condition or indication being treated without reaching a dose that produces intolerable adverse side effects. The exact dose depends on many factors e.g. the indication, formulation, and mode of administration and has to be determined in preclinical and clinical trials for each respective indication.

[0209] Production of gigastasin variants

[0210] In some embodiments, gigastasin variants are expressed in prokaryotic cells, in particular in E. coli strains with enhanced redox properties to facilitate disulfide formation, e.g., E. coli Rosetta-gami D2, and at low-temperature conditions below 25°C, e.g. at 18 °C, to increase the yield of soluble protein in the cytoplasmic fraction. In some embodiments, mutations of tyrosine with negatively charged residues at the C-terminus, including Y117E, Y117D, Y119E, Y119D, Y121 E or Y121 D, are introduced to increase expression yields.

[0211] In some embodiments, gigastasin variants are expressed in mammalian cells, including but not limited to HEK and CHO cells, as fusion proteins with N-terminal HLEM or other solubilization tags to enable expression. In some embodiments, HLEM or other fusion tags may be linked or conjugated to gigastasin variants via a cleavable polypeptide spacer to allow for post-expression removal of the fusion tag. Such polypeptide spacers are also described herein.

[0212] In some embodiments, a high thermal stability of gigastasin variants is exploited to perform purification via reversed-phase high-performance liquid chromatography (RP-HPLC) using organic solvents, including but not limited to acetonitrile, and ionization reagents, including but not limited to trifluoracetic acid or acetic acid, to remove endotoxin and protein impurities. The production, expression and recovery of gigastasin variants is described in the examples of the present application.

[0213] Diseases to be treated

[0214] The invention further relates to the polypeptide as defined in any one of the items above, for use in a method of treating or preventing complement activation-related conditions in a subject. The term “complement activation- related condition” as used herein relates to disorders, the onset, progression or persistence of one or more symptoms or disease states of which involve the participation of complement activation. Thus, the “complement activation-related condition” as used herein, is any condition / disease of which one or more symptoms or disease states can be treated or prevented by the inhibition of proteases in complement activation. Exemplary complement activation-related conditions include, but are not limited to, ischemia-reperfusion injury, myocardial infarct, stroke, hyperacute rejection, transplant rejection, organ rejection, sepsis, cardiopulmonary bypass, adult respiratory distress syndrome, allergic asthma, restenosis, multiple organ dysfunction syndrome, trauma, hemorrhagic shock, Guillain-Barre syndrome, paroxysmal nocturnal hemoglobinuria, glomerulonephritis, autoimmune disorders, systemic lupus erythematosus, rheumatoid arthritis, age-related and neurodegenerative diseases, Alzheimer's disease, myasthenia gravis, neuromyelitis optica spectrum disorders, antineutrophil cytoplasmic antibody-associated vasculitides, multiple sclerosis, platelet storage, serum sickness, biomaterial-induced thrombo-inflammatory reactions, atypical hemolytic uremic syndrome (aHUS), hemolytic anemias, and hemodialysis-related reactions.

[0215] According to other items above, the invention further relates to the polypeptide as defined in any one of the items above, for use in a method of treating or preventing coagulation-related conditions, such as thrombo-inflammation, including, but not limited to, sustained or prolonged inflammation associated with infection [e.g. cerebral malaria], arthritis, autoimmune diseases [such as celiac disease, post-infectious IBS, diabetes mellitus type 1 , Henoch-Schbnlein purpose (HSP) sarcoidosis, systemic lupus erythematosus (SLE), Sjogren syndrome, eosinophilic granulomatosis with polyangiitis, Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenic purpura, Addison's disease, rheumatoid arthritis (RA), ankylosing spondylitis, polymyositis (PM), dermatomyositis (DM), Alopecia Areata and ischemia reperfusion injury (IRI) of various organs (liver, colon, etc.), peripheral vascular disease, transfusion-related acute lung injury (TRALI), transplant rejection, pre-eclampsia, severe burns, atherosclerosis, hypertension, antiphospholipid syndrome, sickle cell disease, bacterial and viral infection ischemic, restenosis, sepsis, major trauma, and disorders in which platelets modulate cell functions including, without limitation, cancer cells proliferation and / or dissemination.

[0216] In a preferred embodiment, the polypeptide of the invention is used to treat or prevent disorders associated with the lectin pathway. Such disorders include, but are not limited to, IgA nephropathy, stem cell transplantation-associated thrombotic microangiopathy, membranous nephropathy, diabetic kidney disease, ischemia reperfusion injury (IRI), renal IRI, myocardial IRI, ischemic stroke, atherosclerosis, and COVID-19.

[0217] In some embodiments, the polypeptide is used to treat or prevent ischemia-reperfusion injury, in particular ischemia-reperfusion injury of the kidney, brain and / or heart, or a disorder causing or that may result from ischemia-reperfusion injury, in particular ischemic stroke, transplant rejection, myocardial infarction, delayed graft function or primary graft dysfunction. In some embodiments, the polypeptide is used to treat or prevent stroke, in particular ischemic stroke. In some embodiments, the polypeptide is used to treat or prevent myocardial infarction. In some embodiments, the polypeptide is used to treat or prevent stem cell transplantation- associated thrombotic microangiopathy. In some embodiments, the polypeptide is used to treat or prevent IgA nephropathy.

[0218] In some embodiments, the polypeptide is used to treat or prevent Sjogren syndrome, myasthenia gravis, or systemic sclerosis. In some embodiments, the polypeptide is used to treat or prevent Sjogren syndrome. In some embodiments, the polypeptide is used to treat or prevent myasthenia gravis. In some embodiments, the polypeptide is used to treat or prevent systemic sclerosis.

[0219] Non-therapeutic uses

[0220] The polypeptides of the invention can be used for diagnostic purposes and in research. For example, the polypeptides described herein can be comprised in a complement activation kit. They are further useful (also in vitro) as dual pathway inhibitors, or for inhibiting serine proteases. Further applications include the use as detection tool to determine whether serine proteases bind to the polypeptide, and the use as biosensors in affinity chromatography. For affinity chromatography it may be beneficial to have an adjusted (stronger or lesser) retention of molecules that bind the ligand of the stationary phase compared to non-binding molecules, in particular impurities. Such affinity chromatography could for example be used to isolate complement components such as C1s or MASP-2. The present disclosure allows to modulate the affinity as desirable for the intended purpose. Lastly, gigastasin variants as such also can be research tools useful for exploring, refining and confirming the biologic mechanism of action of the natural molecule and derivatives thereof, which may guide development of therapeutic molecules.

[0221] Summary of the sequences in the sequence listing.

[0222] EXAMPLES

[0223] 1. Material and Methods

[0224] 1.1 In-silico mutational scan of gigastasin

[0225] A previously reported co-crystal structure of the active C1s enzyme in complex with gigastasin (PDB: 5LIBM) was used as basis for in silico analyses.23The crystal structure was loaded to Maestro Schrodinger software (Schrodinger, USA) and prepared with the protein preparation wizard. The CCP1 and CCP2 domains of C1s were removed and the missing side chains hydrogen atoms were restored. Lastly, the water molecules were removed and the structure was allowed to minimize, allowing sufficient movement to relax the strained bonds, clashes, and angles. The prepared protein was then loaded into two different software tools, namely BioLuminate (Schrodinger) and FoldX.26Each software was mutating every single residue (except cysteines) of gigastasin to all 20 natural amino acids. The energy change upon mutation was detected and expressed as AAG. The AAG value is generated from a thermodynamic cycle where AG(Wildtype) - AG(Mutant) is calculated, with those values for which AAG < 0 the mutant is more favorable.

[0226] Selected mutants were visually inspected, and solvent-facing residues, as well as residues without direct interaction with C1s, were excluded from this study. In this initial phase, 16 mutants were selected for further expression and testing in biochemical assays. The mutants were named after the residue one-letter code they have in the wildtype, then the residue position in gigastasin, and finally the residue one letter code which they were mutated to. Thereby, the following mutants were selected: W17G, D18E, D18W, D18Y, G36D, G36E, G36R, G36W, Q45K, K53R, L55R, T69R, G71 E, G71Q, F72E, and T84R. After the initial study, the mutants W17A, W17F, W17H, W17P, W17R and W17Y were added to the list and regarded for further testing.

[0227] 1.2 Immunogenicity in-silico analysis

[0228] The in-silico deimmunization of the protein was conducted using the NetMHCHpan 4.127tool to assess immunogenicity, focusing exclusively on MHC class II epitopes due to their relevance in protein immunogenicity. All available human MHC class II alleles were considered in the analysis. For each predicted MHC class II epitope of a length of 15 residues, single point mutations were systematically introduced at each position, substituting all proteinogenic amino acids except cysteine. The immunogenic potential of these mutated peptides was then reevaluated using the same tool. Mutations resulting in reduced predicted immunogenicity were selected for further in vitro validation, with final candidates chosen based on structural considerations and visual inspection. The immunogenic potential of the protein was additionally assessed using the proprietary tool Epibase (Lonza), which identifies MHC class II epitopes. Only the most abundant human MHC class II alleles were included in the analysis. The tool was used to detect potential epitopes without further deimmunization steps.

[0229] 1.3 Cloning, expression, and purification of recombinant gigastasin

[0230] The DNA sequence encoding gigastasin was codon-optimized for E. coli expression and synthesized at ATG:biosynthetics (Merzhausen, Germany).28The construct encoding an N- terminal 6xHis tag followed by a TEV cleavage sequence was generated by PCR and ligated into pET15b vector (Novagene, Lucerne, Switzerland) between Ncol and BamHI restriction sites prior amplification in E. coli DH5a. Derivatives were designed using site-directed mutagenesis by two-step PCR, introducing a point mutation in the wild type gene, and were similarly cloned. After DNA minipreparation, the correctness of the constructs was verified by double-stranded DNA sequencing (Microsynth, Switzerland). For protein expression, plasmids were transformed into a chemo-competent RosettaGami (DE3) strain and grown in LB medium at 37 °C and 180 rpm. At an OD6oo of 0.8-1.0, cells were cooled to 18 °C and supplemented with isopropyl p-D-1 -thiogalactopyranoside (IPTG; Applichem, Darmstadt, Germany; 1 mM final concentration) and further grown for 60 h at 18 °C and 180 rpm. Cells were harvested by centrifugation for 25 min at 5,000 rpm and 4°C, resuspended in binding buffer (150 mM NaH2PO4, 300 mM NaCI, 20 mM imidazole, pH 8.0) and lysed using a cell homogenizer (FPG 12800, Homogenising Systems, Harlow UK) at 110 mbar pressure twice. The lysate was centrifuged at 11 ,000 rpm for 30 min at 4 °C. The supernatant was transferred into ultracentrifugation tubes, centrifuged at 22,000 rpm for 20 min at 4 °C and dialyzed against binding buffer overnight at 4 °C in dialysis tubes with a 7-kDa molecular weight cut-off (Roth, Switzerland). Samples were applied to a 5 mL HisTrap HP column attached to an FPLC system (Akta Pure, Cytiva, Switzerland). After a washing step with binding buffer (150 mM NaH2PO4, 300 mM NaCI, 40 mM imidazole, pH 8.0) the protein was eluted with elution buffer (150 mM NaH2PC>4, 300 mM NaCI, 400 mM imidazole, pH 8.0) and purity was analyzed by SDS-PAGE. Buffer was exchanged to PBS with Pierce Protein Concentrators PES, 10K 5 mL (Thermo Scientific).

[0231] After the initial studies, selected proteins (based on assay performance, expression output, and purity profile) were expressed again and purified via FPLC with a HisTrap HP 5 mL column, with subsequent reverse phase HPLC purification (RP-HPLC) and lyophilization. After lyophilization the products were resuspended in PBS. Presence of the proteins and purity was shown in SDS-PAGE.

[0232] 1.4 In-vitro sulfation of gigastasin

[0233] The in-vitro sulfation protocol has been performed as previously described. The reaction was carried out by incubating 80 pM gigastasin with human tyrosyl protein sulfotransferase 1 (TPST- 1 ; MedChemExpress, HY-P77255-10UG) at a final concentration of 0.05 pg / pL and 800 pM 3'- phosphoadenosine-5'-phosphosulfate (PAPS; 94455, Sigma) in minimal sulfation buffer (40 mM PIPES pH 6.8, 100 mM NaCI, 0.1 % Triton X-100) or manganese sulfation buffer (40 mM Pipes pH 6.8, 300 mM NaCI, 20 mM MnCh, 50 mM NaF, 1 % Triton X-100). The reaction was incubated at 16 °C for 18 h, 26 h and 40 h, followed by SDS-PAGE and western blotting using an anti-sulfotyrosine antibody (Sigma-Aldrich, ZMS1096) to confirm sulfation. Control reactions excluded TPST-1.

[0234] 1.5 Expression of human serum albumin- and lgG4 Fc-fused gigastasin

[0235] 1.5.1 Human albumin fused gigastasin

[0236] The gigastasin complementary DNA sequence was synthesized and extended with a coding sequence for a linker, either 5x[Gly-Gly-Ser] (SEQ ID NO: 5) or 2x[Gly-Gly-Ser]-6xHis-TEV (SEQ ID NO: 6), in its 5 position and inserted into BamH1 and Not sites of plRESpuro3 (BD Biosciences, Heidelberg, Germany). Albumin complementary DNA was amplified by polymerase chain reaction with the forward primer 5’- GCGGCTAGCATGAAGTGGGTAACCTTTATTTCCC-3’ (SEQ ID NO: 13) and the reverse primer 5’-GCGGGATCCTCCTAAGCCTAAGGCAGCTTGACTTG-3’ (SEQ ID NO: 14). The amplicon was digested with Nhe\ and BamH1 and inserted into the Nhel / BamH sites of the gigastasin vector. The resulting vectors, capable of expressing a fusion protein consisting of HSA-gigastasin (SEQ ID NO: 2) or HSA-6xHis-TEV-gigastasin (SEQ ID NO: 3), were grown in E. coli XLIOgold (Agilent, Waldbronn, Germany) and purified using standard protocols (Qiagen, Hilden, Germany). Protein expression was conducted with the Expi293™ Expression System Kit (Thermo Fisher, Waltham MA USA), according to the kit manual. Supernatant was harvested for purification of the produced fusion protein. HSA-gigastasin and HSA-6xHis-TEV- gigastasin were purified by affinity chromatography: cell culture supernatant was applied to an equilibrated CaptureSelect™ Human Albumin (Thermo Fisher, Waltham MA USA) column and the product was eluted with buffer containing 2 M MgCI2.

[0237] 1.5.2 Fc-fused gigastasin

[0238] Two different strategies to link gigastasin to the Fc part of human lgG4 were considered: firstly, gigastasin was coupled to the Fc-region after its C-terminus via a TEV cleavage site followed by a flexible (GGGGS)2 linker (SEQ ID NO: 4). This strategy resembles typical Fc fusion proteins, where a protein is added to the Fc instead of the antigen-binding fragment (Fab) of the antibody. The second strategy was to fuse the Fc region to the N-terminus of gigastasin, as the C-terminus of gigastasin is considered important for complement inhibition, especially via the CP. For this purpose, a flexible linker GGGGG linker (SEQ ID NO: 7) was inserted after the Fc, followed by a TEV cleavage site, so that gigastasin without the Fc could be obtained. Constructs for both variants were successfully produced using the pFUSE-hlgG4-Fc vector (InvivoGen), and then transiently transfected using FugeneHD (Promega) into Freestyle 293- F cells (Gibco). Purification of the expressed Fc-gigastasin was done using a Protein A column (Cytiva) on an FPLC system (Akta Pure, Cytiva, Switzerland). Confirmation of successfully expressed and purified protein was done via SDS-PAGE and western blot (anti-sulfotyrosine and anti-human-Fc antibodies; Sigma-Aldrich).

[0239] 1.6 Chromogenic substrate assays

[0240] The inhibitory activity of gigastasin and derivatives thereof with C1s was evaluated using a competitive chromogenic substrate assay based on previous reports.31First, a serial dilution (0.08 - 10 pM) of gigastasin in PBS-T (0.05 % Tween-20) was mixed in flat-bottom 96-well plates (PolySorp Nunc, Thermo Fischer Scientific) with active C1s enzyme (ComplementTech, Tyler, TX, USA) at a final concentration of 14.5 nM. The reaction mixture was incubated for 10 min at 37 °C prior to the addition of the chromogenic substrate methoxycarbonyl-Lys(Z)-Gly- Arg-pNA (Bachem, 4027448) to a final concentration of 400 pM. C1s plus substrate without inhibitor served as 100 % control, while gigastasin with substrate only and substrate without C1s provided negative controls. The colorimetric reaction was measured at 405 nm on a Synergy HT plate reader (Biotek) at 37 °C for 30 min. Assays were performed in triplicates with two technical replicates each. Results were normalized to the positive and negative controls. Curves were fitted using nonlinear regression to a normalized variable slope model in Prism (v10, GraphPad). Sigmoidal curves representing inhibition of enzymes were fitted to the data using a dose-response model to derive IC50 values.

[0241] 1.7 Complement activation / inhibition assays

[0242] The inhibitory activity of gigastasin and its derivatives to prevent the activation of the classical and lectin pathways of complement was determined by ELISA as described before.32 33ELISA for classical pathway (CP) is herein referred to as CP ELISA and ELISA for lectin pathway (LP) is herein referred to as LP ELISA. Flat-bottom MaxiSorp 96-well plates (Thermo Fischer) were coated overnight at room temperature with 33 pg / mL IgM (Sigma) for classical pathway or 55 pg / mL mannan (Sigma) for lectin pathway activation, and plates were washed with PBS-T. Dilution series of gigastasin and derivatives in 1 % (v / v) pooled normal human serum (NHS) were prepared in HBBT++assay buffer (10 mM HEPES, 150 mM NaCI, pH 7.4 containing 0.5 % BSA, 0.15 mM CaCh, 0.5 mM MgCh, and 0.1 % Tween 20) and samples were diluted 1 :5 in 1 % (v / v) NHS. Negative controls included each inhibitor in assay buffer only, assay buffer only, and 1 % (v / v) serum containing 10 mM EDTA, while HBBT++ containing 1 % (v / v) pooled NHS served as 100 % control. After incubation for 1 h at 37 °C and 700 rpm, plates were washed five times with PBS containing 0.1 % Tween 20. 100 pL of a polyclonal biotinylated mouse anti-human C3b / iC3b antibody (1 :1000; CEDARLANE) were added to each well and incubated at 700 rpm at RT for 1 h. Plates were washed five times with PBST. 100 pL of NeutrAvidin conjugated with HRP (1 :10,000; Invitrogen) were added to each well and incubated for 20 min at RT and 700 rpm. After incubation, plates were washed five times with PBST and 100 pL of 1-StepTM Turbo TMB-ELISA solution (ThermoScientific) was added to each well. The reaction was left to develop until a light blue color was visible and stopped by the addition of 100 pL 2 M sulfuric acid. The colorimetric readout was detected at 450 nm with a Synergy HT plate reader (Biotek). All assays were performed in triplicate and normalized to positive (no inhibitor, 1 % (v / v) serum in buffer) and negative (1 % (v / v) serum in EDTA buffer) controls. Inhibitory curves were fitted using nonlinear regression to a normalized variable slope model in Prism (v9-10, GraphPad).

[0243] 1.8 Binding affinity and kinetic profiles of gigastasin and derivatives

[0244] The kinetic profile of the interaction between gigastasin proteins and proteases of the complement system (C1r, pro-C1s, C1 s) was determined by surface plasmon resonance (SPR).34’35Experiments were performed on a Biacore T200 instrument (Cytiva) at 25 °C using research-grade CM5 sensor chips (Cytiva) and HBS-T (10 mM HEPES pH 7.4, 150 mM NaCI, 0.005 % Tween-20) as running / sample buffer. Gigastasin (1 pM in 10 mM sodium acetate, pH 5.0) was immobilized using standard amine coupling; two flow cells were activated for 7 min with a 1 :1 mixture of 0.1 M sulfo N-hydroxysuccinimide and 0.1 M 3-(N,N- dimethylamino)propyl-N’-ethylcarbodiimide at a flow rate of 10 pL / min. Gigastasin and derivatives were immobilized on single flow cells until a target density of around 1000 Rll was reached, after which flow cells were blocked using 1 M ethanolamine, pH 8.0, for 7 min. To assess the proteases’ binding affinity toward gigastasin and derivatives, concentration series (4.7 - 300 nM) of each protease were injected for 300 s at a flow rate of 10 pL / min, followed by a dissociation phase of 600 s. After each injection cycle, the surface was regenerated using 0.1 M Na2CO3 for 60 s at a flow rate of 10 pL / min. Data was collected at 10 Hz, corrected for the molecular weight of the injected serine protease, evaluated using Biacore T200 Evaluation (v3.2.1 , Cytiva), and plotted using Prism (v10; GraphPad).

[0245] 1.9 Thrombin stability assay

[0246] Thrombin cleavage assays were performed with gigastasin and derivatives that carry singleresidue mutations in the proposed thrombin cleavage site (i.e., p.Gly36Glu, p.Gly36Asp, p.Gly36Ala). Samples were prepared in quadruplicates by mixing substrates on ice as described in Table 1. Table 1 : Reagents used in thrombin cleavage assay

[0247] Thrombin Millipore 1.0 pL

[0248] Thrombin

[0249] Novagen 10x 2.5 plcleavage buffer °

[0250] Protein 3.9 pL

[0251] PBS-T 17.6 pL

[0252] Total 25.0 pL

[0253] Samples were incubated at 37 °C under gentle shaking for 1 , 2, 3, or 4 h, taken out of the incubator, and stored at 4 °C until analysis. 10 pL of each sample were mixed with 10 pL 2x reducing buffer and another 5 pL were mixed with 15 pl 3x non-reducing buffer. The samples were boiled at 99 °C for 5 min and loaded on a Mini-PROTEAN TGX gel (4-20%, Bio-Rad) and the gel was left to run for 35 min at 200 V. The gel was then immersed in QuickBlue Protein Stain (Lubio Science) overnight at RT on the plate shaker. The next day, the gel was de-stained in water before being imaged.

[0254] 2. Results

[0255] 2.1 Example 1 : Recombinant production of high quality functional gigastasin in prokaryotic expression system with target selectivity

[0256] In previous studies, gigastasin (BD001) was either isolated from the salivary glands of the leech or recombinantly expressed in eukaryotic systems.21For the present invention, gigastasin was expressed in E. coli as a soluble and active form. Additionally, the refolding of inclusion bodies was successful, and yield could be increased. The protein was purified to homogeneity and shown to be a potent inhibitor of the complement initiation pathways CP and LP as well as initiation of the intrinsic pathway of the coagulation system. The remarkable stability of recombinant gigastasin enabled stringent purification steps, including RP-HPLC in organic solvents, that not only removed minor protein impurities but also drastically reduced endotoxin contaminants to levels suitable for use in cell-based and in vivo models. This purification step also enabled lyophilization for long-term storage. Importantly, the observation that the functional activity of gigastasin is maintained after heat exposure, HPLC purification, lyophilization and the following activity assays indicate a beneficial stability profile for the production, storage, and distribution of inhibitor preparations. Our characterization of gigastasin’s activity, selectivity and specificity properties confirm and extend on previous reports by showing that the recombinant protein exerts a strong target selectivity for C1s over pro-C1s, a considerable inhibitory preference for the LP over the CP (data not shown).

[0257] 2.2 Example 2: Structure-activity relation study of gigastasin

[0258] In the leech, gigastasin is expressed in the salivary glands as a soluble, single-chain protein of 122 amino acids (Fig. 1).25Previous studies have shown that native gigastasin contains 20 cysteine residues that form 10 disulfide bridges, one glycosylation site, and three tyrosine-O- sulfation sites at the C-terminus of the protein.2425Expression of recombinant gigastasin ( / .e., BD001) in non-mammalian cells indicated that C-terminal sulfation contributes to but is not essential for C1s inhibition activity, leading to an ~10-fold efficacy drop (IC50 of ~3 nM and ~30 nM, respectively).24Glycosylation did not seem to notably contribute to the inhibitory activity. The limited impact of posttranslational modifications, with the exception of disulfide bridges, on gigastasin’s ability to inhibit C1s served as a rationale for producing recombinant gigastasin in bacterial expression systems (E. coir, see above). A published crystal structure of gigastasin in complex with C1s (PDB 5LIBM) revealed a three-domain organization with a central antistasin-like domain (ASD) flanked by a unique N-terminal lobe and a C-terminal tail section.23Gigastasin binds to the catalytic site of C1s primarily via the ASD with contribution of N-lobe residues, whereas the C-terminal tail interacts with an exosite of C1s that mediates binding of natural substrates ( / .e., complement components C4 and C2).23

[0259] While these early functional and structural studies provided important insight into the targetbinding and inhibitory mode of gigastasin, they revealed limited information about the contributions of individual regions or contact residues to C1 s binding and little to no insight into molecular determinants of target selectivity beyond C1 s. A detailed understanding of the target interaction profile is an important part of the potential therapeutic candidate characterization and is expected to open opportunities to use protein engineering to selectively improve PK / PD properties. We therefore employed a panel of in silico and in vitro methods to establish an SAR profile of gigastasin.

[0260] 2.2.1 In-silico affinity prediction using commercial software tools

[0261] Starting with the published gigastasin:C1s crystal structure (PDB 5LIBM),23two different computational suites ( / .e., BioLuminate, FoldX) were utilized with documented features to identify key contact residues in drug:target complexes and perform single-residue mutation scans to predict mutations with enhanced target binding. For the initial SAR study, mutational scans of gigastasin:C1s were performed with both tools to predict single mutants with better C1s affinity. Thereby, the programs mutated each residue in gigastasin to all proteinogenic amino acids (except cysteine) and calculated the change in relative binding energy (AAG). A negative difference in AAG is indicative of an improved target binding affinity of the mutant when compared to the wildtype protein (Table 2).

[0262] Table 2: Selection of mutants based on in-silico predictions and visual inspection.

[0263] 17 TRP GLY 19.7 2.17

[0264] 18 ASP GLU 0.3 0.28

[0265] 18 ASP TRP -0.53 -0.67 Yes

[0266] 18 ASP TYR -11.07 -1.31 Yes

[0267] 36 GLY ARG No

[0268] 36 GLY ASP No

[0269] 36 GLY GLU -0.81 -0.01 Yes

[0270] 36 GLY TRP -4.2 -0.90 No

[0271] 45 GLN LYS -8.29 No

[0272] 53 LYS ARG -0.87 -0.16

[0273] 55 LEU ARG -14.77 0.69 No

[0274] 69 THR ARG -9.63 0.04 No

[0275] 71 GLY GLN -5.66 4.95 No

[0276] 71 GLY GLU 4.56 2.80 Yes

[0277] 72 PHE GLU -2.81 2.71 No

[0278] 84 THR ARG -1.26 -0.05 No

[0279] While each software tool was able to perform an analysis of the gigastasin:C1s complex and predict mutations with proposed enhanced binding, marked differences were observed both concerning the identification of key residues, in the absolute energy differences, and even in the assessment whether a mutation would be beneficial or not (prediction uniformity; Table 2). Structures of mutant complexes for which any of the tools predicted affinity enhancements were therefore inspected visually. Based on computational predictions and visual inspection, 16 mutants were selected for recombinant expression and experimental validation (Fig. 2).

[0280] Only few positions are located in the central ASL domain (residues 59-86) but rather in the N- terminal lobe (residues 1-58). The panel also included mutants, for which both programs predicted impaired C1s binding yet were deemed interesting based on visual inspection. Two amino acids in the N-lobe were previously suggested as key residues for target binding, with Trp-17 supposedly acting as an important anchoring point.23The W17G mutant was therefore included to validate this prediction. In the case of adjacent Asp-18, both tools suggested a beneficial effect for mutations to aromatic residues (Trp, Tyr), yet visual inspection indicated that a side chain extension from Asp to Glu may also improve target contacts. Finally, G36D was included to compare Asp and Glu at this position.

[0281] Although not validated in experimental assays, in silico tools were also employed to predict the effect of two natural polymorphic variants of gigastasin ( / .e., S16T, D60N) that have been reported in the original patent. While Ser-16 is minimally engaged in hydrophobic contacts, the conservative replacement with Thr does not appear to affect target interaction. In the case of Asp-60, a hydrogen bond with a Tyr residue in the C1 s binding site is formed; although the replacement with Asn induces a slight steric effect, this is not expected to have a substantial impact on target binding. As a consequence, both polymorphic versions may be considered functionally intact variants.

[0282] 2.2.2 Experimental validation of affinity-modifying mutants predicted in silico

[0283] 13 mutants were recombinantly expressed and evaluated in a chromogenic substrate assay (OSA) to determine the impact of each mutation on the inhibition of the C1 s catalytic site (Fig.

[0284] 31

[0285] The wildtype form of gigastasin showed an inhibitory activity (IC50 ~ 40 nM) that was in good agreement with previous reports (Fig. 3).24As predicted, the N-lobe mutation W17G led to a complete loss of activity, though the result may have also been influenced by stability issues of the mutant. While the other expressed N-lobe mutant (D18E) still inhibited 01s, its activity was markedly reduced. There was also an affinity drop, yet much less pronounced, for all four mutants at position Gly36; among those, negatively charged residues (Asp, Glu) appear to be better tolerated than positively charged (Arg) or heteroaromatic residues (Trp). Another 5 mutants for which at least one in silico tool predicted an improved target binding (K53R, L55R, G71 E, G71Q, T84R) showed notable activity drops in the experimental assay; similarly to W17G, stability issues may have impacted the result for L55R. Interestingly, there were only two mutants in the panel (Q45K, T69R) that did result in an improved inhibitory activity against 01 s, both in the range of ~2-fold enhancement (Fig. 3). 2.2.3 Functional evaluation of predicted gigastasin mutants in CP and LP ELISA

[0286] While the CSA provides a rapid and reliable assessment of C1s catalytic site inhibition, it does not reflect other changes concerning the mutant’s interaction with C1s or the inhibition of other potential complement protease targets (i.e., C1r, MASP-1 , MASP-2). All mutants were therefore tested in a functional assay under more physiological conditions. In the classical pathway (CP) and lectin pathway (LP) ELISA, they compete with the natural substrates C4 and C2 for binding to the initiating proteases in donor serum. Inhibition of complement- mediated surface opsonization serves as readout (Fig. 4).

[0287] As demonstrated by us and other groups before,23gigastasin showed a much better inhibitory activity towards the LP over the CP (Fig. 4). In the CP ELISA, the activities of the tested mutants generally correlate well with the observed C1s inhibition in the CSA. The exceptions are D18E and K53R, both of which show enhanced inhibition in the ELISA but not in the CSA (Fig. 4). Among the potential explanations is that these mutants may have an effect on C1r in addition to C1s, thereby leading to a stronger CP inhibition overall. In contrast to the complete activity loss observed in the C1s-CSA and the CP ELISA, the W17G mutant did show residual inhibition in the LP ELISA, albeit at ~100-fold reduced activity. The assay-specific difference was even more pronounced in the case of mutant L55R, which profoundly lost activity for C1s and CP inhibition, but maintained a comparable activity to wildtype in the LP ELISA.

[0288] I ntriguingly, these results suggest that mutations in the N-lobe and central lobe may not only affect the inhibitory activity of gigastasin but might also open opportunities to modulate the target selectivity profile of the inhibitor. To corroborate this hypothesis and arrive at an improved understanding of how individual mutations affect the target binding profile, we therefore performed a series of functional and direct binding studies for selected gigastasin mutants that could be expressed well, featured suitable solubility and stability profiles, and demonstrated interesting activity patterns in the initial study. Those mutants were produced at higher quantity and purity to enhance the quantitative accuracy of the evaluation results.

[0289] 2.2.4 Interaction analysis for selected gigastasin mutants binding to CP targets

[0290] Surface plasmon resonance (SPR) studies were performed to determine the binding affinity ( D) and the kinetic association (a) and dissociation rate constants (k ) of selected mutants. In addition to the well-described target C1s, we also included the ortholog enzyme C1 r that is part of the assembled C1 complex (i.e., C1qr2s2) and described to cleave pro-C1s to its active form during CP initiation. All mutants were immobilized on individual flow cells on an SPR sensor chip and enzymes were injected into solution to monitor the interaction profiles (Fig. 5).

[0291] As observed during previous studies, gigastasin showed a strong interaction with C1s whereas the binding to C1r was still notable but with clearly reduced affinity and faster dissociation rate (Fig. 5). When qualitatively assessing the interaction profiles of the mutants with C1s, relatively subtle effects were visible for the G36D, G36E, Q45K, and K53R mutants. Notable reduction in C1s affinity was observed for the G36W, G36R, T84R, and in particular the L55R mutants, latter of which shows a profoundly impaired complex stability. Conversely, T69R was a gigastasin mutant that featured stronger apparent binding to C1s when compared to the parental protein. In general, the mutations appear to have affected C1r binding in similar manners to the interaction with C1s (Fig. 5). Of note, G36W does not strongly improve overall binding to C1r, though it seems to improve complex stability. Most importantly, whereas the L55R mutation showed a more pronounced effect on C1s than C1r binding, the opposite could be observed for T84R that almost completely lost binding to C1r. These three positions may prove particularly accessible for protein engineering efforts to tune the CP activity / selectivity of gigastasin analogues.

[0292] 2.2.5 Impact of gigastasin’s N-lobe domain and its anchoring residue on inhibitory activity

[0293] Even if the central ASL domain of gigastasin is primarily engaged in target contacts and responsible for blocking the catalytic site of C1s, the N-terminal lobe domain (residues 1-58) still contributes to C1s binding.23In fact, in silico predictions identified several residues in this domain that could benefit from improvement, some of which proved valuable in this study. Among N-lobe residues, our own analyses and previous studies identified Trp-17 as particularly important for target interaction.23Indeed, Trp-17 forms extensive van der Waals interactions with the activation loop region at the N-terminus of C1s. This residue may also be responsible for steric clashes when docking gigastasin to C1 r and would thereby define the relative selectivity of the inhibitor for C1 s over C1 r.

[0294] Eight gigastasin derivates with mutations at position 17 were designed, selecting amino acids with distinct physicochemical properties (Fig. 6A) to enable the detection of broad property changes. Derivatives were expressed and purified under same conditions as gigastasin and tested in C1s-CSA, CP and LP ELISA, and with SPR against proCis, C1s, and C1r.

[0295] When exchanging Trp at position 17 with a panel of distinct residues, we generally observe a loss of inhibitory activity towards C1s in the CSA (Fig. 6B). Even changes to other aromatic residues resulted in marked activity losses, from 8-fold for Tyr to 20-fold for Phe and 25-fold for His. The most pronounced drop was observed for W17R, with an inhibitory activity almost 50 times lower than the parental protein. While the current set of mutants does not allow for a clear SAR of the impact of physicochemical properties at position 17, the experiment confirms that Trp-17 is critical for target interaction in the case of C1s.

[0296] To see whether mutations of Trp-17 may have a similar or distinct impact on early complement activation via the CP or LP, we further tested all mutants in CP and LP ELISAs (Fig. 7). In agreement with the CSA, Trp17 mutants also showed a pronounced activity loss in the CP ELISA (up to ~20-fold). Whereas drops in inhibitory activity were also observed in the LP ELISA, the effects were generally less severe when compared to CP inhibition, ranging from 2-fold (W17H) to 10-fold (W17P) - showing enhanced selectivity for the LP compared to CP. Of note, the ranking of activity losses is somewhat different between C1s-CSA and CP ELISA and especially between the CP and LP ELISAs. This confirms that Trp-17 contributes to target interactions, yet with considerably different importance and likely molecular mode for the two complement pathways.

[0297] To further explore whether the observed differences in how Trp-17 mutations affected C1s and CP inhibition (CSA and ELISA, respectively) could be attributed to technical assay properties or distinct target intervention profiles, we employed SPR to monitor their interactions with both CP initiation proteases (C1r, C1s) as well as with the zymogen for of C1s (pro-C1s).

[0298] As expected, based on our previous studies, neither gigastasin nor any of the Trp-17 mutants showed binding to proCis. At the same time, the interaction of gigastasin with Cl rwas notable yet weaker when compared to C1s (Fig. 8). When assessing the C1s interaction of the mutant panel, we observe a good correlation between target affinity and inhibitory activity measured by CSA and ELISA. While W17Y is able to maintain most of its C1s binding capacity, other mutants feature notable affinity drops. Intriguingly, the SPR profile of W17Y for binding to C1s and C1 r looks remarkably similar (Fig. 8). Yet, when considering the strong activity / selectivity difference of parental gigastasin for those two proteases, these results indicate a moderate affinity loss for C1s and a marked affinity gain for C1r in the case of the W17Y mutation. Whether this selectivity shift has an impact on CP modulation profiles in vitro or in vivo remains to be further explored.

[0299] The overall importance of the N-lobe for protein integrity and / or target interaction was further supported by the observation that truncated gigastasin derivatives lacking the entire N-lobe domain or even parts thereof did either not express well in E. coli or were inactive in functional assays (data not shown). In a conservative truncation trial, only the flexible part of the N- terminus up to the first cysteine ( / .e., residues 1-7) was removed. Even though the resulting gigastasin A1-7 could be expressed and showed activity in the C1s-CSA, the inhibitory potency was reduced by a factor 3 (Fig. 9), thereby suggesting that truncation opportunities may be limited.

[0300] Our study has confirmed that the N-lobe with its anchoring Trp-17 residue has a strong impact on C1s binding and CP inhibition, whereas the effect on LP inhibition appears to be less pronounced. While the binding affinity of these derivatives to MASP-1 and -2 remains to be determined, the combination of LP gain-of-function with CP loss-of-function mutants could provide a strategy to enhance selectivity for the LP.

[0301] 2.2.6 Impact on C-terminal tail on inhibitory activities of gigastasin

[0302] Native gigastasin expressed in the salivary glands of the giant Amazon leech contains two major posttranslational modifications, i.e., glycosylation and tyrosine-O-sulfation.2425While advantageous for producing recombinant gigastasin at high yield and for enabling rapid mutagenesis for SAR studies, our prokaryotic expression systems are not capable of performing these modifications. As mentioned above, previous studies suggested that the effect of glycosylation may be negligible, at least for C1s inhibition.24Conversely, missing sulfation of three Tyr at the C-terminal tail of gigastasin was reported to result in a ~10-fold activity drop, which can be explained by the tail’s interaction with a cationic exosite in Cis.2324Although recombinant gigastasin remains a strong complement inhibitor even in the absence of sulfation,24adding or mimicking this modification may influence the target binding and / or selectivity profile of gigastasin. We therefore performed a series of experiments to explore the impact of C-terminal tail more quantitatively as part of our SAR study.

[0303] In view of the substantial contribution of a sulfated C-terminus to C1s inhibition, the question arose whether the impact of a non-sulfated tail would become negligible so that the tail region could be removed. We therefore designed and expressed a truncation analog of gigastasin (A113-122). Although the A113-122 mutant showed residual inhibitory activity in the C1s-CSA, another 10-fold drop was observed when compared to the non-sulfated recombinant gigastasin (Fig. 10A). In relation to the native leech-derived gigastasin, this may theoretically accumulate to a 100-fold loss of inhibitory activity. While these results suggest that the C-terminal tail is not essential for C1s binding, it still appears to contribute even in its non-sulfated form. Whether this results in a similar effect on CP inhibition and / or towards other targets remains to be explored.

[0304] Physicochemically, one difference between a sulfated and non-sulfated tail is a marked decrease in negative charges. We thought that this might potentially provide opportunities to regain some of the lost activity by replacing the C-terminal Tyr with anionic proteinogenic residues ( / .e., Glu, Asp), in a manner compatible with the prokaryotic expression system. We produced a focused derivative library, in which Tyr117, Tyr119, and Tyr121 were all replaced by the same alternative residue, including Glu (BD3E) and Asp (BD3D), to test the hypothesized beneficial charge effect; we also included Ala (BD3A) and Arg (BD3R) mutations to observe the impact of side chain / charge loss and charge reversal as part of our SAR study. Interestingly, the removal of the Tyr side chain in the Ala mutant appeared to slightly improve C1s inhibition, whereas the introduction of a positive charge with Arg resulted in a notable activity loss. A substantial enhancement of inhibitory potency was observed in the mutant carrying three negatively charged Asp or Glu instead of Tyr (Fig. 10B). Of note, the introduction of negative charges at the C-terminus also regularly enhanced the production of soluble recombinant gigastasin derivatives, with up to a notable improvement of expression yields. Together, this part of the SAR study indicates that the positive effect of tyrosine-O-sulfation that gets lost during prokaryotic expression in recombinant gigastasin variants can at least partially be regained by replacing Tyr with Glu or Asp.

[0305] 2.2.7 Extension and improvement of in-silico prediction models

[0306] Our initial in silico screening to predict gigastasin mutants with enhanced C1s binding primarily relied on the published crystal structure of gigastasin:C1s (PDB 5LIBM)23and publicly available software tools (see above). It became evident during analysis and validation that the predictive power of such tools is limited at present, especially if not followed by careful visual inspection of the predicted results. Detailed SAR studies and the successful prediction and / or rational design of engineered gigastasin derivatives critically depend on improved computational models and algorithms. In parallel to the SAR studies described above, we continuously improved the structural model of gigastasin:C1s complex. Building on software tools to quantify per-residue contributions of drug:target interactions previously developed and applied by our groups in other projects,37we extended this functionality to obtain a detailed interaction fingerprint for gigastasin binding to C1s (example in Fig. 2B). While this method could confirm the key residues on gigastasin that largely define C1s interaction as reported in the crystal structure paper (Fig. 1A),23it added a weighing of their relative contributions and identified additional residues with notable target contacts. These fingerprint models prove highly valuable for the rationalization of mutagenesis effects or the identification of areas on gigastasin that may be more accessible for modifications, among other applications.

[0307] Although only a single target complex of gigastasin has so far been published, i.e., C1s,23the structural similarity of serine proteases, or serine protease domains of larger proteins, provides an opportunity to employ the protease interaction fingerprint method to assess gigastasin interactions with other targets as a reasonable estimate. In this approach, crystal structures of selected serine proteases are superimposed with the gigastasin:C1s structure and used as a base to dock the leech inhibitor to the corresponding binding sites. Upon further refinement of the model, computational analysis can be performed. Ideally, targets (or off-targets) for gigastasin of therapeutic interest that were corroborated by such an approach would be experimentally confirmed by dedicated crystal structures, later.

[0308] 2.3 Example 3: SAR-guided engineering of PD properties

[0309] Our initial SAR study based on in silico predictions and experimental validation and exploration using mutagenesis, truncation and other modifications, provided critical insight into the molecular determinants of target binding and selectivity, inhibitory activity, and protein stability, among other properties. They also revealed several structural regions and modification strategies that could be targeted to rationally design gigastasin derivatives with enhanced PD profiles, both for general improvement and for potential activity tuning toward specific indications. In the following sections, we present some of these strategies and provide validation data where already available.

[0310] 2.3.1 Enhancing lectin pathway selectivity through protein engineering

[0311] During the experiments described above, we detected individual mutations that distinctly affected CP and LP inhibition with more or less pronounced activity differences (Table 3). In particular, L55R almost completely lost its activity in the CP ELISA but was still highly active in the LP ELISA. Table 3: Impact of individual gigastasin mutation on LP activity as determined by the SAR study.

[0312] D18E 3-fold better LP inhibition compared to WT

[0313] G36R 2-fold worse in CP and 2-fold better in LP compared to WT selectivity towards LP

[0314] G36W 6-fold worse in CP and 10-fold better in LP compared to WT selectivity towards LP

[0315] Better inhibition in both PW, could lead to stronger inhibition with additional LP Q45K selective mutant

[0316] K53R Equal inhibition in CP, 10-fold better in LP compared to WT

[0317] L55R No inhibition in CP, 6-fold better in LP compared to WT selectivity towards LP

[0318] Better inhibition in both PW, could lead to stronger inhibition with additional LP T69R selective mutant

[0319] Based on these observations, we hypothesized that dual mutation of gigastasin may lead to analogs that feature further enhanced LP selectivity, improved inhibitory potency, or a combination thereof. Constructs were designed for selected double-mutants, which were subsequently expressed in E. coli using the established procedure. Newly generated inhibitors were tested in CP and LP ELISA and directly compared to parental gigastasin. To visualize selectivity enhancements, relative inhibition changes for the CP and LP were plotted on the x- and y-axes, respectively, of a scatter plot (Fig. 11) by using the following equations: x = CP IC50gigastasin LP IC50gigastasin - CP IC50mutant v = - LP IC50mutant ■

[0320] In general, combination of single mutations in one protein did not always lead to an additive positive impact; in fact, some combinations even led to intramolecular changes with opposite effects of what has been observed for the single mutants. For example, while both the Q45K and T69R mutations showed stronger LP inhibition individually, double mutants that included either of those mutations did not show the expected improvements. Some double mutants demonstrated LP pathway selectivity yet at reduced overall potency (Fig. 11 A), which reduces their value for further development. At the same time, four double mutants ( / .e., D18E / G36R, L55R / G36W, K53R / G36R, K53R / G36W) featured a combination of markedly enhanced selectivity for the LP over the CP and up to 10-fold improved inhibitory potency (Fig. 11B).

[0321] As an alternative option to combining single mutations discovered during the initial screening is the focused improvement of individual key residues in gigastasin that appear to define the desired property. In the case of LP selectivity, position 55 appears particularly promising since a single mutation from Leu to Arg almost completely abolished CP inhibition while maintaining LP-directed potency. We therefore evaluated whether a mutation of Leu55 to other amino acid residues with distinct physicochemical properties would result in further modulation of the selectivity and efficacy profile. For an initial screening, we selected lie as the isomeric form of Leu, Vai as another apolar residue with shorter side chain, Gin and Glu as polar and polar / anionic residues with same length, respectively, and Arg as cationic residue that was originally predicted by in silico tools.

[0322] All of the tested derivatives showed improved LP selectivity when compared to parental gigastasin (Fig. 12). However, the substitution of Leu-55 with different amino acids had distinct effects on selectivity and potency. In terms of selectivity, L55R remained the mutant with the most deleterious impact on CP inhibition (>15-fold drop), thereby featuring highly favorable pathway selectivity. At the same time, L55I showed the best potency enhancement with 7-fold better LP inhibition than gigastasin; despite a less pronounced effect on CP inhibition (~2-fold), the LP potency still leads to a marked selectivity shift. The other mutations, i.e., L55V, L55E, and L55Q, demonstrated a similar LP:CP inhibition ration as L55I yet a less pronounced improvement of LP inhibition potencies (3- to 6-fold). These results suggest L55R as most suitable lead analogue for obtaining LP selectivity, whereas L55I emerges as interesting analogue to study options for LP potency enhancement. To further evaluate the promising strategy of optimizing position 55, we suggest testing the designed inhibitors in SPR with C1s (and MASP-1 or -2, if available) to confirm the ELISA data with kinetic data. Combined with structural analyses, this may guide further mutagenesis efforts.

[0323] 2.3.2 Proposed strategies to enhance CP selectivity or modulate other target interactions

[0324] The LP enhancement experiments shown above impressively demonstrated the potential of using SAR-guided protein engineering to tune the target selectivity profile of gigastasin derivatives. While improved LP selectivity may be considered important to address unmet clinical needs, in view of a lack of LP-selective therapeutics in the clinic,38enhanced CP potency and / or selectivity could be of importance in antibody-mediated disorders, including autoimmune diseases and acute rejection of transplants and implants. While not yet experimentally validated, structural analysis employing our protease fingerprint approach suggests several strategies that could be followed to favor CP over LP activity: For example, Glu-80 may act as selectivity filter; in gigastasin, binding to MASP-2 might involve a tight gap that is closed by Met-658, whereas the corresponding pocket in C1 s is larger. Introducing bulkier amino acids at this position could potentially block MASP-2 binding due to steric hindrance, enhancing selectivity for C1 s. Moreover, Leu-66 is buried within a hydrophobic pocket near the catalytic site. Mutating this residue could potentially alter the selectivity profile between 01 s and MASP-2, as the hydrophobic pockets in these enzymes differ, potentially influencing the binding affinity and selectivity. In the case of Lys-10, mutation to Asp or Glu might be possible to form a salt bridge with Arg-437 in 01 s or Arg-444 in MASP- 2; this interaction might further stabilize the binding of the N-terminus. The environment around Arg-31 is potentially different for 01s and MASP-2. While Arg-31 is in close proximity to Glu- 574 and Lys-575 in case of 01 s, for MASP-2 Gln-577 and Arg-578 are the closest residues. This suggests that it might be beneficial to change to a negatively charged amino acid at position 31 for 01 s to form a salt bridge with Lys-575 and a differently sized negative charge in the case of MASP-2. Finally, it is important to see that the 7 N-terminal amino acids of gigastasin are not involved in 01 s binding or form any secondary structure. It might therefore be of interest to extend the N-terminus to reach for example Arg-496 and Lys-499 of 01 s forming a unique contact area, as this additional exosite differs between 01 s and other serine proteases.

[0325] As demonstrated by our SPR studies, some mutations had a notably different impact on 01 r binding when compared to 01s, in particular W17I, G36W, L55R, and T84R. While the benefit of shifting the 01 r:C1 s selectivity for CP inhibition remains to be explored, these residues may provide an important starting point to SAR-guided selectivity tuning.

[0326] Although not yet specifically explored for other targets, it is expected that the same approach with the combination of in silico models, in particular protease fingerprints, and experimental mutagenesis can be extended to other serine proteases, either to enhance the activity of gigastasin derivatives for beneficial targets or abrogate inhibition of off-targets in an indication. Whenever possible, these studies should be supplemented by experimental crystal structures and other biophysical, structural, and functional data.

[0327] 2.3.3 Rescuing activity losses by manipulating the C-terminal tail during or after production

[0328] The expression of gigastasin in lower systems, including bacteria and yeast, restricts the introduction of posttranslational modifications present in the leech-derived protein. This results in a —10-fold loss in C1s inhibition with a change of IC50 from ~3 nM to ~30 nM.24In fact, the impact is similar as in the case of the thrombin inhibitor hirudin, which also contains a sulfated C-terminal tail.39Given that the lack of sulfation still resulted in a potent nanomolar inhibitor, recombinant hirudin derivatives used in clinical preparations {e.g., lepirudin, desirudin) were produced as non-sulfated proteins.40While this approach could be feasible for gigastasin as well, it would still be advantageous to cope for lost sulfation. In principle, expression in baculovirus-insect cells or mammalian cells could naturally introduce sulfation; so far, however, these systems either lacked suitable yields or did not express gigastasin at all. As we showed in our SAR study, the replacement of all three C-terminal Tyr with negatively charged Glu was able to partially regain C1s inhibition activity while preserving the prokaryotic expression option. Currently, this strategy appears to be the one with the best chances for easy implementation as it improves potency and expression yields.

[0329] An alternative option to mutagenesis could be the chemoenzymatic modification of the natural C-terminal tail by human tyrosylprotein sulfotransferases (TPST) in vitro.41-42Initial studies demonstrated that incubation of gigastasin with a TPST reaction solution indeed markedly improved the C1s inhibitory potency in the CSA (Fig. 13).

[0330] 2.3.4 Removal of thrombin cleavage site

[0331] Although therapeutic proteins are intrinsically prone to proteolytic degradation, suitable PK profiles rely on low cleavage susceptibility to common proteases in the therapeutic compartment. While in silico analysis of gigastasin suggest a rather favorable proteolytic stability profile, a natural cleavage site for thrombin at position 37, where the sequence Gly36- Arg37-Gly38may enable thrombin cleavage after arginine. We could demonstrate in vitro that gigastasin is indeed being cleaved by thrombin at 37 °C in a time-dependent manner (Fig. 14A&B); interestingly, the cleavage was only visible on SDS-PAGE under reducing conditions, indicating that the polypeptide chain remains connected via disulfide bridges after thrombin degradation (Fig. 14B). Even though plasma concentrations of active thrombin are typically very low under physiological conditions, they are expected to be elevated in thromboinflammatory disorders. In the worst case, this may lead to partial inactivation of gigastasin at the target site in the body and impact efficacy.

[0332] In addition, mutation experiments had been performed with respect to residue Gly-36. Two of the existing mutants, G36D and G36E, were investigated as to whether the mutations would impact proteolytic stability against thrombin. Indeed, either one of the mutations markedly improved thrombin resistance with little to no degradation being observed after 4 h (Fig. 14A&B). Site-directed mutation of Gly-36 thereby emerges as promising strategy improve the proteolytic stability of gigastasin. We further implore whether a simple methylation of the side chain from Gly to Ala would be sufficient to impair thrombin cleavage, as suggested by in silico analysis using ProtParam thereby avoiding the introduction of additional charges. Using the C1s-CSA, we were also able to demonstrate that G36A fully maintains the inhibitory potency of the parental gigastasin, featuring comparable IC50 values (29.7 and 25.1 nM, respectively; Fig. 14C). However, despite the ProtParam prediction, thrombin cleavage was still observed for the G36A mutant (data not shown); indeed, the sequence corresponds to a thrombin cleavage site in fibrinogen, according to MEROPS.45While the switch from Glu / Arg to Ala was originally intended to better preserve the physicochemical profile of gigastasin, we could meanwhile show that the inhibitory properties of gigastasin are also largely preserved for mutations to anionic residues (Fig. 14D). We are therefore currently recommending G36D or G36E mutations as means to improve thrombin stability, yet option to replace Arg-37 (e.g. with Lys; predicted to abrogate thrombin cleavage by both ProtParam and MEROPS) may provide additional or alternative strategies.

[0333] 2.4 Example 4: Engineering strategies to improve PK properties of gigastasin derivatives

[0334] Even if no in vivo studies have been performed with gigastasin or derivatives thereof, it can be assumed that a small, soluble protein of ~16 kDa would face considerable renal excretion, resulting in a rather short plasma half-life. While this limitation may be less relevant for acutephase clinical settings, in which the compound could be continuously administered by i.v infusion, such profiles could become more restrictive for long-term clinical applications and even for preclinical evaluation in animal models. An improvement of PK parameters, and in particular plasma half-life, is therefore considered advantageous. In early reports of gigastasin, strategies such as PEGylation have been suggested as potential efforts.24However, the expression of gigastasin as fusion protein with PK-modulating domains or its chemical modification with PK-altering entities could also be considered.

[0335] 2.4.1 Gigastasin fusion to N-terminal human serum albumin tags

[0336] Human serum albumin (HSA) is the most abundant plasma protein in the human body and plays a major role in the PK profiles of many low-molecular-weight drugs. Yet, HSA may also be employed to improve the plasma half-life of biopharmaceuticals. Attachment of (small) proteins to HSA increases the overall size and hydrodynamic range, thereby reducing renal filtration and excretion. At the same time, HSA was shown to bind neonatal Fc receptors (FcRn) to engage an endosomal recycling mechanism that may further improve half-life.4647Based on experience with HSA, both as a treatment modality and as a fusion protein in biopharmaceuticals, including insect-derived infestin-4,48it was explored whether mammalian expression of HSA-fusion proteins could be extended to gigastasin. Both native gigastasin and a construct, in which HSA was fused to the N-terminus of the inhibitor via a linker containing 5 Gly-Gly-Ser motif repeats (Table 4), were expressed in HEK cells. As observed in our previous attempts (data not shown), untagged gigastasin could not be successfully expressed in mammalian cells. However, fusion to the HSA tag did result in a stable expression of the inhibitor in HEK cells. In principle, this strategy would enable the production of a larger protein (~80 kDa) with reduced renal clearance, which is sulfated and can be produced under endotoxin-free conditions.

[0337] Table 4: Human serum albumin (HSA)-fusion constructs of gigastasin of this study.

[0338] AKKKLPKCQK QEDCGSWDLK CNNVTKKCEC RNQVCGRGCP KERYQRDKYG

[0339] CRKCLCKGCD GFKCRLGCTY GFKTDKKGCE AFCTCNTKET ACVNIWCTDP

[0340] YKCNPESGRC EDPNEEYEYD YE

[0341] When the inhibitory potencies of the prokaryotical ly expressed gigastasin were compared to the N-terminal HSA-tagged derivative from mammalian production, such HSA-gigastasin indeed showed improved activity in the CP ELISA yet with IC50 values that were 2-3-times better than untagged, non-sulfated gigastasin and in the same range as the 3xYE variant (BD3E; Fig. 15). More surprisingly, the inhibitory capacity of the fusion protein dropped two orders of magnitude below that of untagged gigastasin in the LP ELISA. The fact that the fusion protein did show potent activity for CP inhibition renders the possibility that the effects were based on improper folding or other production issues unlikely. In addition, we could demonstrate that the protein was indeed sulfated (data not shown). A negative impact of the N-terminal HSA-tag regarding LP inhibition therefore emerged as likely hypothesis. The molecular mechanism is not yet known and it remains to be explored whether N-terminal tags diminish LP activity in general or whether the effect depends on tag size and / or spacing. At the same time, such N-terminally HSA-tagged molecules may be beneficial if an enhanced CP selectivity is desired.

[0342] Since the original fusion protein did not allow for the specific removal of the HSA tag and untagged gigastasin could not be expressed under the same conditions, a new construct was designed. This contained a shorter GGS linker but a 6xHis tag followed by a TEV protease cleavage site (Table 4). Thereby, the intact protein maintained the overall size of the original construct but the HSA tag could be removed by TEV cleavage and subsequent Ni2+-NTA chromatography. The prokaryotic gigastasin and its 3xY>E (BD3E) and 3xY>D (BD3D) derivatives, the full-length HSA-6xHis-TEV-gigastasin and its untagged fragment were all subjected to pathway inhibition analysis. In the CP ELISA, it was again observed that the full HSA-fusion protein shows moderately improved potency compared to gigastasin expressed in bacteria in a similar range as its untagged 3xY>E mutant; moreover, the same profound activity drop was detected in the case of LP inhibition (Fig. 15). Intriguingly, removal of the HSA tag from the fusion protein had beneficial consequences for both CP and LP inhibition, yet at distinct rates. In the CP ELISA, untagged mammalian gigastasin demonstrated potent inhibition that was ~10-times stronger than the full-length fusion protein (and the prokaryotic 3xY>E mutant) and ~30-fold stronger than prokaryotic gigastasin. While tag removal largely rescued the profound activity drop of the fusion protein the LP ELISA, the potency of the untagged mammalian gigastasin was still slightly weaker than prokaryotic gigastasin and its C-terminal charge mutants (Fig. 15).

[0343] In addition to the characterization of alternative gigastasin constructs designed to address PK concerns, this study also provides important and somewhat unexpected insight valuable for the SAR assessment of this inhibitor. Based on previous reports regarding C1s inhibition (CSA), the presence of sulfated tyrosines at the C-terminal tail should account for an ~10-fold affinity contribution.24In the CP ELISA, an even stronger improvement of untagged sulfated gigastasin from mammalian production over the comparable non-sulfated counterpart from bacterial expression could indeed be observed. Surprisingly, however, sulfation does not seem to have the same positive impact on LP inhibition potency; in fact, mammalian gigastasin interfered with LP activation even less than bacterial gigastasin. At the same time, the presence of a large N-terminal HSA tag generally had a non-beneficial effect when compared to the untagged fragment, but the impact was far more pronounced for LP than for CP inhibition. It is not yet clear why and how the HSA tag interferes with gigastasin’s LP inhibition capacity. Among potential reasons is that the rather long spacer between HSA and gigastasin ( / .e., 5xGGS or 2xGGS-6xHis-TEV) would allow for unfavorable intramolecular domain-domain interactions in the fusion protein; we therefore currently evaluate the hypothesis that a shorter linker may alleviate some of these effects. Nevertheless, this implies that gigastasin may impair two complement initiation pathways in a similar mode of action but that the target interaction profiles and molecular determinants might be distinct. N-terminal modification may have a stronger impact on LP inhibition whereas C-terminal modifications more directly affect CP inhibition. Latter hypothesis is further supported by the observation that the replacement of C- terminal Tyr residues with anionic amino acids only seem to influence the CP inhibition profile.

[0344] While N-terminal tag interference currently hampers the anticipated PK benefit of HSA-fused gigastasin derivatives, further engineering of the location, spacing, or other aspect of the HSA tag could improve the properties of such constructs. Notwithstanding PK improvement, N- terminal tagging with HSA or other proteins could provide a pathway to enabling gigastasin production in mammalian cells, especially when the tag can be proteolytical ly removed after protein expression (e.g. via TEV protease). Although the impact of C-terminal HSA tags remains to be explored, given the unsuccessful expression in mammalian cells, we hypothesize based on the experience with C-terminal charge effects, that CP activity may be more negatively affected than LP activity. Thereby, C-terminal HSA or other fusion tags could potentially provide avenues to further enhance LP selectivity of the corresponding gigastasin derivatives.

[0345] Alternatively, the PK-modulating effects of HSA could also be harnessed by adding smaller HSA-binding tags to gigastasin.46Among those, lipidation appears to be particularly intriguing as it is increasingly applied as improvement strategy for biopharmaceuticals, including insulin, GLP-1 R agonists, or the complement inhibitor zilucoplan.47In this approach, fatty acids (or diacids) are synthetically attached to the therapeutic protein, which then non-covalently interacts with the HSA pool in circulation to delay renal elimination. Our group has successfully implemented lipidation strategies for in-house peptide drug development projects and considers an extension to gigastasin and other protein therapeutics (Fig. 16).

[0346] 2.4.2 Gigastasin fusion to immunoglobulin Fc fragments

[0347] Alongside HSA tags or HSA-binding moieties, the fusion of therapeutic proteins to the constant fragment of immunoglobulins (Fc-tag) is a frequently applied strategy to improve PK and / or PD properties.49Similar to HSA fusion, the addition of an Fc tag may enhance plasma half-life via both an increase of molecular weight and FcRn binding. In contrast to HSA tags, the active protein is typically expressed as dimer when fused to an Fc tag, which further increases its size but may also (positively or negatively) influence PD properties (through valency, ligation, steric effects).49 50

[0348] To explore whether Fc fusion of gigastasin may enable mammalian expression and investigate the consequences on its PD profile, we designed and produced gigastasin-Fc derivatives. The Fc part of human lgG4 was selected as fusion tag, since lgG4 is known to engage not, or much less, in complement- and Fc receptor-mediated effector functions when compared to IgG 1 or other isoforms.51In the traditional Fc fusion format, the therapeutic protein is placed at the N- terminus of the construct and fused to the N-terminal end of the Fc hinge (Fig. 17A).50While this appears as feasible strategy for gigastasin, there are concerns that applying a tag to the C-terminal tail may negatively affect tyrosine sulfation and / or target interaction, which could in particular hamper CP inhibition efficacy. In an attempt to mitigate this risk, we added a flexible linker (2xGGGGS [SEQ ID NO: 4]) and a TEV cleavage site between gigastasin and the Fc tag. At the same time, we also constructed a reversed format, in which gigastasin is fused to the C-terminus of the lgG4-Fc (Fig. 17B); while less common, such formats have been successfully employed for biopharmaceuticals before.52In principle, this approach should leave the C-terminal tail available for sulfation and target binding but it was unclear whether the N-terminal tag would have similar consequences for LP inhibition as observed for HSA fusion.

[0349] Constructs for both variants were produced using a pFUSE-hlgG4-Fc vector and transiently expressed in suspension HEK cells. SDS-PAGE analysis confirmed that the expression of Fc- gigastasin was successful, whereas the traditional format (gigastasin-Fc) did not express under the same conditions (Fig. 17C). The presence of an Fc tag largely facilitated protein purification via protein A affinity chromatography. Importantly, the presence of sulfotyrosine residues in the fusion protein could be detected via western blot, thereby suggesting that the sulfation sites remain (at least partially) accessible in the reversed format.

[0350] The inhibitory potency of the expressed Fc-gigastasin fusion protein was assessed in the established assays, including the C1s-CSA and the CP and LP activation / inhibition ELISAs. Initial results demonstrate that the Fc-fusion protein retains its ability to inhibit C1s and both complement initiation pathways, yet with distinct outcomes in the different assays. In the C1s- CSA, the IC50 of gigastasin-Fc was ~8-fold weaker than the prokaryotic gigastasin 3xY>E variant (79 vs. 11 nM; Fig. 18A). In the CP ELISA, however, the inhibitory potency was slightly better for the C-terminal tagged construct (ICso of 178 and 297 nM; Fig. 18B). Conversely, N- terminal Fc fusion of gigastasin resulted in a marked loss of potency (~50-fold; 418 nM vs. 9 nM) in the LP ELISA (Fig. 18C), similar to the effect observed to HSA-Fc.

[0351] Our results therefore indicate that N-terminal tagging with large fusion proteins in general rather than the presence of HSA in particular is believed to have a negative impact on LP inhibition of gigastasin. Whether the use of smaller fusion partners and / or shorter spacer units could better conserve LP activity, remains to be investigated. At the same time, N-terminal Fc- fusion may provide an option to enhance CP selectivity of gigastasin derivatives. What may be considered more interesting in this dataset is the observation that Fc fusion appears to interfere less with CP inhibition potency than with the direct inhibition of C1s. Whether this may be caused by the dimeric format that may allow for the simultaneous binding of the inhibitor to two proteases (C1s or C1r) in the C1 complex, remains to be explored. Furthermore, the results for the intact Fc fusion will need to be compared to the untagged fragment after TEV cleavage.

[0352] 2.5 Example 5: Immunogenicity prediction and options to replace / delete high-risk residues

[0353] Immunogenicity, i.e., the potential of a therapeutic reagent to induce an immune response that could result in direct adverse events and / or accelerated elimination due to anti-drug antibodies is a general concern in (bio)pharmaceutical development and should be mitigated whenever possible.53Exogenous proteins in particular, including parasite-derived proteins, bear an intrinsic immunogenicity risk. It should therefore be considered probable that administration of gigastasin could induce some degree of immune response. That said, and reasonable when considering that parasites secrete such proteins in a broad range of hosts, the immunogenicity of at least some parasite-derived inhibitors appears to be low. For example, low-rate antibody formation has been observed in the case of hirudin derivatives but the clinical impact has been reported as being low.54The risk assessment also depends on the anticipated use, with a single short-term application in acute-phase conditions generally being less problematic than a long-term use in chronic diseases. Independently of future applications, however, the immunogenicity potential of gigastasin needs to be investigated to assess the risk during in vivo studies and explore options to mitigate such risks through mutagenesis or other strategies.

[0354] In a first step, general immunogenicity was assessed in silico using state-of-the-art machine learning tools. Both the proprietary tool Epibase (Lonza) and the publicly available tool NetMHCHpan (Technical University of Denmark) were employed to predict potential major histocompatibility complex (MHC) class II epitopes that could lead to an immunogenic response in patients.2755Epibase identified 3 potential epitopes with key residues contributing most to epitope formation located at positions Trp-17, Tyr-44, and Phe-72 (Fig. 19). Notably, not all MHCII alleles are predicted to contribute to these epitopes, with allelic coverage for those sites being less than 25% in the global population. In contrast, panNetMHCll only predicted a single epitope around the residue Phe-72.

[0355] Although both tools identified potential epitopes that could bind to certain MHCII complexes, the number of risk residues was comparatively low. Initial in silico predictions thereby suggest that gigastasin may be considered a low-immunogenic protein. While native gigastasin should therefore be suitable for in vivo studies, during which immunogenicity can be assessed under more relevant conditions, site-directed mutagenesis could be employed to further mitigate such risks if one or more epitopes were to trigger relevant immune responses. Similar to the elimination of thrombin cleavage sites to improve stability (see above), a compromise needs to be found to impair immunogenicity without negatively affecting the PD profile of gigastasin. Owing to its considerable involvement in target binding, replacement of Trp17 may prove more challenging when compared to Tyr44 or Phe72, for which target interaction seems less critical, at least in the case of C1s.

[0356] In silico predictions often have a tendency to overestimate epitope presence, leading to false positive results. To address this, we initially focused on the epitope between residues 67 and 81 , with a particular emphasis on the key amino acid at position 72, which was consistently predicted by both computational tools. panNetMHCll predicted 148 single mutations as potential options to deimmunize the MHCII epitope within this region (Table 5). Given that Phe- 72 is directly involved in target binding, it is unlikely that a different amino acid would be tolerated at this position; we therefore focused our selection strategy to adjacent residues. Structural analysis, including visual inspection and molecular dynamics simulations, identified positions 70 and 77 as the most promising sites for modifying the epitope without altering the overall protein structure.

[0357] Table 5. Potential deimmunization of gigastasin by mutagenesis. panNetMHCll predicted that any of these single point mutations is sufficient to deimmunize the MHCII epitope between residues 67 and 81. Mutations affecting positions 70 and 77 with best-predicted compromise between deimmunization and target interaction interference are marked in bold.

[0358] Gly-67 G67 G67 G67 G67 G67

[0359] M W Y F L

[0360] Thr-69 T69 T69 T69 T69 T69

[0361] W Y F L M

[0362] Tyr-70 Y70 Y70 Y70 Y70 Y70 Y70 Y70 Y70 Y70 Y70 Y70 Y70 Y70 Y70 Y70 Y70 Y70 M F V A I W E Q N G H L P R S T K

[0363] Gly-71 G71 G71 G71 G71 G71 G71 G71

[0364] M l W Y F L P

[0365] Phe-72 F72 F72 F72 F72 F72 F72 F72 F72 F72 F72 F72 F72 F72

[0366] A W Y E Q N G H P R S T K

[0367] Lys-73 K73 K73 K73 K73 K73 K73 K73 K73 K73 K73 K73 K73 K73 K73 M W Y N F G L P S A E Q R T

[0368] Thr-74 T74 T74 T74 T74 T74 T74 T74 T74 T74 T74 T74 T74 T74 W E Q N G H R K M I Y F L

[0369] Asp-75 D75 D75 D75 D75 D75 D75 D75 D75 D75 D75 D75 D75 D75 D75 D75 D75 D75 D7 M V A I W Y E Q N F G H L P R S T 5K Lys-76 K76 K76 K76 K76 K76 K76 K76 K76 K76 K76 K76 K76 M V A I W Y Q F G L R S

[0370] Lys-77 K77 K77 K77 K77 K77 K77 K77 K77 K77 K77 K77 K77 K77 K77 K77 K77 K77 M V A I W Y E Q N F G H L P R S T

[0371] Gly-78 G78 G78 G78 G78 G78 G78 G78 G78 G78 G78 G78 G78 G78 G78 M V I W Y E N F H L P R T K

[0372] Ala-81 A81 A81 A81 A81 A81

[0373] Preliminary de-immunization studies explored the potential of the K77Y and Y70F mutations to serve as candidates in case the wild-type version of gigastasin induces an immunogenic response in in-vitro assays. Initial experiments indicate that these mutations are tolerated by gigastasin, allowing it to retain its ability to inhibit C1 s (Fig.20). 2.6 Example 6: Conclusion and proposed strategy for the development of gigastasin- derived leads

[0374] By performing an extensive set of structural, biophysical, biochemical, and functional assays based on in silico analysis and protein engineering, the characterization of recombinant gigastasin and the expansion of its SAR profile allowed for an ever-growing insight into the mode of action and the PK / PD profile of the leech-derived host defense pathway inhibitor. We could demonstrate that gigastasin can be produced in bacterial expression systems at good yield and purity, and identified strategies to remove endotoxin and lyophilize the protein for long-term storage. Despite a lack of some posttranslational modifications, in particular tyrosine-O-sulfation, recombinant gigastasin showed strong inhibitory activity in target and pathway assays. Our studies indicate that mammalian expression of the native protein is challenging and could not yet be achieved; fusion of N-terminal expression tags {e.g., HSA, lgG4 Fc) appears to profoundly improve mammalian expression, yet such tags so far interfered partially with the functional activity of gigastasin, in particular regarding LP inhibition. Alongside improving PK profiles, such strategically placed fusion tags could also be employed to enhance pathway selectivity, enable mammalian expression, and / or increase the production yield of gigastasin derivatives.

[0375] Comparable to studies with gigastasin from other sources (leech, insect cells, yeast), recombinant gigastasin produced in bacteria showed potent activity against C1s and the classical pathway of complement activation and, in particular, the lectin pathway. In absence of a commercial source of plasma-purified MASP-1 or MASP-2, a direct comparison of CP and LP targets in CSA is not yet possible; we are currently producing recombinant forms of MASP- 1 / 2 to better validate and characterize the effects of LP-relevant gigastasin mutations. Importantly, we were able to demonstrate that gigastasin inhibits other host defense targets, of which coagulation factor Xia seems to be a major intervention point; notable activity could also be shown for C1r and FXIIa, whereas no activity could be detected for FB, FD, and C2 of the complement cascade or FXa, thrombin, and plasma kallikrein of the contact / coagulation system. At least in the case of C1s, gigastasin discriminates effectively between the active enzyme and its zymogen form. While not target-specific, gigastasin and variants thereof thus feature a defined selectivity for certain active serine proteases, notably those involved in the triggering of pathways that contribute to thromboinflammatory conditions. We could further show that gigastasin exerts a species tolerance and potently inhibits complement action in human, non-human primate, rat, and mouse serum (potentially also in other species). While in-vivo immunogenicity remains to be determined, analysis by two in-silico tools indicated a comparatively low immunogenicity risk. The successful recombinant production of gigastasin, the impressive stability of the expressed protein, its broad-yet-defined activity against key host defense pathways with clinical relevance, its low predicted immunogenicity potential, and its activity in animals used in preclinical studies suggest that gigastasin may be an interesting therapeutic candidate suitable for preclinical development.

[0376] Several in-silico prediction and experimental mutagenesis studies have been performed to identify key molecular determinants of gigastasin that shape its PK / PD profile (Fig. 21). We could show that even if a majority of gigastasin’s contact with the active site of C1s is mediated by the central antistasin-like domain, the N-terminal lobe critically contributes to target binding, in particular via the anchoring residue Trp-17. Mutations in the N-lobe did indeed influence the activity of gigastasin, either beneficially or adversely. This explains why truncation derivatives in an attempt to minimize the protein structure have so far been of limited success. Based on our current data sets, it appears that the removal of the flexible regions of the N-terminus (residues 1-7) or the C-terminus (residues 113-122) result in gigastasin derivatives with residual yet profoundly reduced inhibitory activities.

[0377] Intriguingly, several of the hitherto tested mutations not only enhanced or impaired the inhibitory potency of gigastasin in general but resulted in a marked shift in target selectivity, as confirmed by SPR studies. We could show that this insight can be employed to tune the target binding and / or pathway inhibition selectivity, as demonstrated in the case of C1s and CP:LP selectivity, respectively. Some of the current single or double mutants of gigastasin feature a remarkable LP selectivity at high potency. While not yet experimentally validated, the improvement of in-house in silico methods {e.g., protease contact fingerprinting) has provided promising avenues to predict on- / off-target activities of gigastasin and suggest options to tune selectivity profiles. The insight gained in our SAR study about the contributions of the C- terminal tail section may also help in this matter. Also, the lack of tyrosine-O-sulfation leads to a marked activity drop for C1s / CP inhibition but still results in a highly active protein with inhibition in the (low) nanomolar range. Here we could show that some of the lost activity could be regained in a manner compatible with prokaryotic expression by replacing the three C- terminal tyrosine residues with anionic residues (Glu, Asp). In addition to improving CP inhibition, this change also enhanced the expression yield of soluble gigastasin. Most intriguingly and surprisingly, we observed that N- and C-terminal modifications seem to distinctly affect CP and LP activities. The charge status of the C-terminus appears particularly important for CP inhibition (sulfo-Tyr > Glu / Asp > Tyr) but less for LP inhibition. Conversely, the N-terminal addition of large protein fusion tags to enable mammalian expression and potentially improve PK properties, has a more prominent negative impact on LP inhibition of gigastasin derivatives. These observations are expected to guide future experiments to refine the SAR profile and development efforts for the technology.

[0378] Although mammalian expression of gigastasin fusion derivatives containing tags that are expected to prolong plasma half-life remains an important goal, the marked negative impact of such N-terminal tags on complement inhibition, and in particular LP impairment, currently limits this strategy. When considering that many ischemic conditions seem to be at least partially driven by LP activation, and that there is no LP-selective inhibitor in the clinic, LP-directed activity of gigastasin may be regarded an important asset. Among all gigastasin mutants tested to this point, derivatives containing C-terminal Tyr>Glu mutations appear to provide the best compromise between production yields, inhibitory potency, and target selectivity. Although the functional impact of thrombin degradation is not yet clear, the observation that the cleavage site can be mutated without affecting activity, suggests that Gly-36 mutations can be included in future leads and candidates. A gigastasin mutant containing four mutations ( / .e., p.Gly36Asp, p.Tyr117Glu, p.Tyr119Glu, p.Tyr121Glu) emerges as a suitable candidate for experimental, preclinical in vivo studies in rodent PK and disease models.

[0379] The selective modulation of target selectivity profiles may prove highly advantageous fortuning the PD and adverse risk properties of gigastasin derivatives in general or in specific indications. The enhanced LP selectivity and potency of some of our mutants appears particularly intriguing in view of the gap for this activity in the current therapeutic arsenal.38

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Claims

1. Universitaet Basel P700PJG / AK11.12.2025Claims1 . A polypeptide comprising a gigastasin variant, wherein the amino acid sequence of said gigastasin variant comprises amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 , wherein three of said amino acid substitutions are at position Y117, Y119, and Y121 , wherein each of these positions is substituted with either glutamic acid or aspartic acid.

2. The polypeptide of claim 1 , wherein said three amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 are Y117E, Y119E, and Y121 E; or are Y117D, Y119D, and Y121 D; in particular are Y117E, Y119E, and Y121 E.

3. The polypeptide of claim 1 or 2, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is at position G36 of SEQ ID NO: 1 , in particular is G36A, G36D, G36E, or G36R.

4. The polypeptide of any one of the preceding claims, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is G36D or G36E.

5. A polypeptide comprising a gigastasin variant, wherein the gigastasin variant has a lesser degree of tyrosine-O-sulfation than naturally occurring gigastasin.

6. The polypeptide of any one of the preceding claims, having a lectin pathway inhibition potency greater than that of gigastasin.

7. The polypeptide of any one of the preceding claims, wherein at least one of said amino acid substitutions is selected from the group consisting of D18E, Q45K, K53R, L55R, L55I, L55Q, L55V, T69R, and T84R.

8. The polypeptide of any one of the preceding claims, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is Q45K.

9. The polypeptide of any one of the preceding claims, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is selected from the group consisting of K53R, L55R, and L55I.

10. The polypeptide of any one of the preceding claims, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is L55R.

11. The polypeptide of any one of the preceding claims, wherein one of said amino acid substitutions relative to the amino acid sequence as shown in SEQ ID NO: 1 is T69R.

12. The polypeptide of any one of claims 1 to 11 for use in a method of treating or preventing a disorder associated with complement activation, in particular associated with lectin pathway mediated complement activation.

13. A nucleic acid encoding the polypeptide of any one of claims 1 to 11.

14. A plasmid or vector comprising the nucleic acid of claim 13.

15. Cells comprising the nucleic acid of claim 13 or the plasmid or vector of claim 14.

16. A method of producing the polypeptide of any one of claims 1 to 11 , comprising (i) culturing the cells of claim 15 under conditions such that the polypeptide is expressed; and (ii) optionally recovering the polypeptide from the cells or from the culture medium.

17. The method of claim 16, wherein the cells are non-mammalian cell, in particular wherein the cells are from prokaryotic or lower eukaryotic organisms.

18. The method of claim 16 or 17, wherein the cells are cultured at a temperature of less than 25°C.

19. The method of any one of claims 16 to 18 or the cells of claim 15, wherein the cells are non-mammalian cells.

20. The method of claim 19, wherein the cells are E. coli cells, in particular having mutations in the trxB and gor genes promoting an oxidizing environment in the cytoplasm.

21. The method of any one of claims 16 to 20, wherein the method comprises refolding insoluble inclusion body fractions of the polypeptide to obtain soluble protein fractions of the polypeptide.

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