N-terminal peptide fragments for complement regulation

Abstract

The present invention provides for N-terminal peptide fragments for complement regulation, their use for the treatment of complement-mediated diseases, and their use in methods of modulation of the complement system.

Description

[0001] P7187PC00

[0002] N-terminal peptide fragments for complement regulation

[0003] Background

[0004] The complement system is a complex proteolytic cascade which is a critical component of the innate immune response, playing a pivotal role in host defence and the maintenance of homeostasis. It consists of a complex network of proteins that, upon activation, lead to the opsonization of pathogens, recruitment of inflammatory cells, and direct lysis of target cells. The complement cascade can be activated through three major pathways: the classical, alternative, and / or lectin (MBL) pathways. While this system is essential for combating infections, dysregulation of complement activity can cause local and / or systemic inflammation, tissue damage and disease and is implicated in a variety of diseases, particularly autoimmune disorders such as systemic lupus erythematosus (SLE) and rheumatoid arthritis, as well as kidney diseases including IgA nephropathy and focal segmental glomerulosclerosis (FSGS) (Ricklin et al., 2010).

[0005] In recent years, the therapeutic regulation of complement activity has emerged as a major target for treating these diseases. Numerous complement modulators are currently being investigated in clinical trials, aiming to modulate different components of the complement cascade to prevent tissue damage and inflammation. Most of these inhibitors are monoclonal antibodies or synthetic compounds designed to specifically target complement proteins or their activation fragments. For example, Eculizumab (Soliris) is a monoclonal antibody that targets the complement protein C5, preventing its cleavage and thereby inhibiting the formation of the membrane attack complex (MAC). Eculizumab has been approved for the treatment of several rare and severe conditions, including paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), and generalized myasthenia gravis. Ravulizumab (Ultomiris) is a longer-acting C5 inhibitor that offers similar therapeutic benefits with less frequent dosing. Further, Avacopan inhibits the complement component C5a receptor and has shown promise in treating ANCA-associated vasculitis. Additionally, small molecules like Danicopan, which inhibits factor D and thereby the alternative pathway, are also advancing through clinical trials, offering new options for targeting specific pathways within the complement system. However, while these approaches show promise, they often come with limitations such as high production costs, potential immunogenicity, and the need for repeated administration (Morgan et Harris., 2015). P7187PC00

[0006] One of the major challenges in developing effective complement modulators lies in their specificity and ability to efficaciously modulate the complement system without causing widespread immune suppression. The currently available inhibitors primarily focus on targeting complement components that are common to multiple pathways, which can lead to broad immunosuppressive effects. Moreover, none of the inhibitors in clinical development are derived from naturally occurring human proteins, which could offer a more physiological and potentially safer approach to modulating complement activity.

[0007] Summary

[0008] It is therefore an objective of the present disclosure to provide protein fragments that modulate the complement system, specifically targeting the pathways involved in complement-mediated diseases. These novel fragments offer a more specific and potent approach to complement modulation with the potential for dual functional activity combining complement pathway modulation with targeted modulation of cytokine receptor signalling, and in turn enhanced therapeutic outcomes in the treatment of complement-mediated diseases, such as SLE and related disorders.

[0009] The present disclosure relates to peptide fragments comprising at least 10 amino acids and at the most 200 amino acids, wherein the peptide fragment comprises a sequence comprising at least one of the following sequences; SEQ ID NO: 1 to 58 or functional homologues of any of the aforementioned sharing at least 90% sequence identity therewith, for use in a method of treatment of complement-mediated disorders.

[0010] In another aspect, the present disclosure provides for peptide fragments comprising at least 10 amino acids and at the most 40 amino acids, wherein the peptide fragment comprises a sequence comprising at least one of the following sequences; SEQ ID NO: 1 or SEQ ID NO 3-56 or functional homologues thereof sharing at least 90% sequence identity therewith.

[0011] The disclosed peptide fragments represent a novel approach to modulating the complement system, which is implicated in various autoimmune and kidney diseases. One advantage of the peptides of the present invention is that by utilizing sequences P7187PC00 derived from human complement components, these peptides offer the potential for highly specific and potent modulation of complement activity.

[0012] The invention provides for peptides that can target specific pathways within the complement system, such as the classical, alternative, and / or lectin pathways. The ability to modulate these pathways selectively allows for a more refined therapeutic approach, potentially reducing unwanted side effects associated with broad complement inhibition. In particular, the invention includes methods of treating complement-mediated disorders by using these peptides, which may modulate specific components of the complement cascade depending on the concentration and context in which they are used.

[0013] A further advantage of the peptides of the present invention is that they achieve exerting robust inhibitory effects at low, physiologically relevant concentrations, as illustrated in the Examples herein.

[0014] Another advantage of the peptides of the present invention is that they achieve specificity in modulation of specific components of pathways within the complement system, such as the classical, alternative, and / or lectin pathways whilst concomitantly addressing other inflammatory mechanisms. This is demonstrated in the Examples herein, wherein the C3 fragment C3-LHF1 (SEQ ID NO:2) fragment is shown to selectively inhibit the classical and / or lectin complement pathways in the C9 deposition assays of Example 6, and exhibits targeted modulation of cytokine receptor signaling, such as IL6ST-mediated pathways, as demonstrated in Examples 7 and 8.

[0015] In some embodiments, the peptide fragments of the invention also exhibit a dual mechanism of action, for example combining selective inhibition of the complement pathways, such as the classical and / or lectin complement pathways, with targeted modulation of cytokine receptor signalling, such as IL6ST-mediated pathways, as demonstrated in the Examples herein.

[0016] The disclosed methods of modulating the complement system involve providing compositions that include human complement factors, regulators, or receptors, which are then contacted with the disclosed N-terminal peptide fragments. This interaction between the peptide fragments and complement components can result in the desired P7187PC00 modulation of complement activity, offering therapeutic benefits in complement- mediated diseases such as systemic lupus erythematosus, rheumatoid arthritis, and various forms of nephritis.

[0017] Overall, the disclosed peptide fragments and methods offer a promising new strategy for managing complement-mediated disorders by leveraging the body's own complement system, with potential advantages in specificity, safety, and efficacy over existing therapeutic approaches.

[0018] Description of Figures

[0019] Figure 1. Workflow to access proteolytic events in human plasma. (A) Workflow of the N-terminome enrichment protocol. A new, shorter High-efficiency Undecanal-based N Termini EnRichment (SHUNTER) workflow was established. (B) Dimethylation efficiency, N-termini purity (pullout efficiency), and quantified N-termini (n=3 technical replicates of human plasma) are compared using the old HUNTER and the new SHUNTER workflow for N-termini enrichment.

[0020] Figure 2. (A): Application of SHUNTER workflow to patient plasma samples with Shiga Toxin producing E. coli-induced hemolytic uremic syndrome (STEC-HUS), treated with anti-C5 antibody (Eculizumab) and quantification of N-termini after day 8s and 30 of treatment, compared to baseline (day 1 of Eculizumab treatment). (B & C): Profiling the proteolytic regulation during Eculizumab treatment in STEC-HUS. Comparison of N-terminome after day 8 (B) or day 30 (C) of Eculizumab (anti-C5) treatment vs baseline (day 1). Significantly decreased and increased (|log2FC| > 1 & p- value 0.05) N-termini are depicted by white circles. Alterations in the N-terminome are more diverse and pronounced on day 8, whereas on day 30, hemopexin cleavages are increasingly present. The C5 N-terminus C5_1381 is significantly up-regulated at day 8 and substantially up-regulated at day 30 of Eculizumab treatment (marked in by black star). (D): Significantly regulated complement N-termini (|log2FC| > 1 & p-value < 0.05) during Eculizumab treatment feature a strongly regulated N-terminus for C5 (C5_1381) after day 8 of Eculizumab treatment .The X-axis represents the Iog2 fold change (log2FC) in N-termini expression levels, indicating the magnitude of regulation, while the Y-axis represents the -Iog10 moderated p-value, indicating the statistical significance of the changes observed. (E & F) Scatterplot of the log2FC N-terminome P7187PC00 vs. the log2FC of total proteome after 8 days (E) or 30 days of treatment (F) of treatment. Black datapoints correspond to complement proteins and show a lower correlation between N-termini and proteome abundance. The corresponding correlation is given as Pearson’s correlation coefficient (R). (G-l): In vitro profiling of major human complement initiating proteases, processed with GluC. Volcano plots and motifs of protease-induced proteolytic patterns for the alternative (MASP-3, G), lectin (MASP-1 , H), and classical pathway (C1r / C1s, I). After recombinant protease digestion (2h, 37 °C) and dimethyl labeling, proteins were enzymatically digested using GluC as an alternative protease for complementary analysis of proteolytic pattern. iceLogos generated from significantly cleaved (| log2FC| > 1 & p-value < 0.05) N-terminal cleavage windows of the protein N-termini display a different cleavage preference for the tested protease. The X-axis represents the Iog2 fold change (log2FC) in N-termini expression levels, indicating the magnitude of regulation, while the Y-axis represents the -Iog10 moderated p-value, indicating the statistical significance of the changes observed. The top cleavages in the complement system are presented in the tables and displayed as black stars on the volcano plots.

[0021] Figure 3. In vitro profiling of major human complement initiating proteases. (A), Workflow for the in vitro assays: incubation of recombinant proteases with heat- inactivated human plasma to determine specific substrates for key proteases of the complement system by subsequent SHUNTER N-termini enrichment. (B) Overview of the identified substrates for the individually evaluated proteases. (C) For all four proteases, cleavages take mainly place in complement component C3. (D) In vitro substrate specificity for key complement proteases; iceLogos generated from significantly cleaved (|log2FC| > 1 & p-value < 0.05) N-terminal cleavage windows of the protein N-termini display a different cleavage preference for the tested four proteases. The top cleavages in the complement system are given. (E) Substrate network of significantly cleaved complement proteins by the investigated proteases.

[0022] Figure 4. Identification of human proteolytic patterns in systemic lupus erythematosus (SLE). (A), Workflow for identification of SLE-specific inflammatory proteolysis. (B) Volcano plot and corresponding tables demonstrating significant alteration (|log2FC|> 0.35 & p-value < 0.05) of N-termini in SLE (increased / decreased cleavages in the complement system are displayed as black dots). The bubble size codes the number of identifications in different samples. Tables display the top P7187PC00 significantly altered N-termini in SLE. (C) Multi-omics factor analysis (MOFA) of SLE cohort demonstrates variance can be mainly explained by N-terminome. Certain factors show a significant correlation to clinical parameters relevant to SLE (* = p-value < 0.05, * = p-value < 0.01). (D) Proteolytic cleavages driving N-terminome factors in SLE with a focus on anticoagulant treatment (factor 5) and eGFR (factor 7), sorted by the alteration in SLE / CTRL. Grouped into increased vs decreased proteolytic in SLE as denoted by the labels on the Y axis.

[0023] Figure 5. Integration of multi-layered proteolytic processing landscape in SLE.

[0024] (A) Circle plot demonstrating known protein fragments in grey (layer one, outer layer), presence and regulation in cross-sectional SLE cohort (layer 2, black / white = increased / decreased cleavage in SLE), regulation in lupus nephritis patients (layer 3, black / white = increased / decreased cleavage in active vs. remission), and in vitro substrates (layer 4, 5, 6, 7), as well as total frequency of the N-termini identification in the center. Two MOFA-prioritized candidate N-termini are highlighted with dotted lines.

[0025] (B), Lupus human fragments (LHF), comprising the C-terminal part of C3, including the C345C domain (C3-LHF1), and the distal 26 amino acids of C8b (C8b-LHF2). (C) Alignment of the C-terminal region of human and mouse C3 depicting the identified / regulated cleavages in the cross-sectional SLE cohort (red / blue arrows) and the persistent cleavages after 3 weeks of isotope labeling in mouse serum (maroon arrows). The identified C3-LHF1 is highlighted in grey and corresponds to a region of stable C3 N-termini in the isotope labeled mouse serum.

[0026] Figure 6. Top N-terminome & proteome weights for all MOFA factors. (A), The top positive weights with strongest influence on each of the ten MOFA factor were assessed for the N-terminome of the cross-sectional SLE cohort, filtered for a minimal log2FC of 0.35, and sorted by increased or decreased abundance in SLE, as denoted on the Y-axis labels. The candidate N-termini C3-LHF1 (C3_1534) and C8b-LHF2 (C8B_565) are prominently present in factors 3, 5 and 7 (bold). (B), Correspondingly, the top positive weights for the proteome of the cross-sectional SLE cohort are given.

[0027] Figure 7. Sequence validation of C3-LHF1 & C3-LHF1 pull-down information. (A), Recombinant C3-LHF1 was processed with the two different digestion enzymes trypsin and GluC, and subsequently analyzed by nano-LC-MS / MS. Almost full sequence coverage (bold) was achieved in database searches against the host CHO proteome, P7187PC00 which was used for protein production, as a background. (B), Cell painting profiles for C3-LHF1 displays similarities to some compounds which display DNA-binding and / or cell growth / cytoskeleton regulatory function.

[0028] Figure 8. Characterization of the bioactivity for the C3 fragment C3-LHF1 (1514- 1663). (A) C3-LHF1 inhibits complement activity as measured by C9 deposition in reference to untreated vehicle control of the same patient (n=6 for SLE / MBL, otherwise n=7 ± SE, significance determined by paired t-test: * = p < 0.05, ** = p < 0.01 , *** = p < 0.001), particularly the mannose-binding lectin (MBL) and classical pathway. (B), Activation of TLR signaling by C3-LHF1, particularly on TLR7 / 5 chimera, TLR1 / 2 and TLR10 as determined by luciferase-reporter HEK293T cell lines. (C) Significant targets (log2FC > 1 , p-value < 0.05, and quantification in 3 out of 4 replicates, no imputation) for hC3Nb2 and C3-LHF1 pull-downs are given. The C3 nanobody has a very narrow purification range, while C3-LHF1 purifies a greater number of proteins. The majority of the identified putative interaction partners are specific for CTRL or SLE plasma and can be categorized as cytoskeletal organization, signaling proteins and members of the translation apparatus. (D), C3-LHF1 induces CD62L-shedding in granulocytes; the median fluorescence intensity (MFI) of detected CD62L on granulocytes from five donors is illustrated for treatment with PBS, LPS at 0.19 mg / mL (pos. control), C3- LHF1 at 1.2 mg / mL, or POD2 control peptide (same manufacturer, same synthesis strategy) at 1.7 mg / mL. CD62L expression was detected using a fluorescent-labeled monoclonal antibody.

[0029] Figure 9. Characterization of the bioactivity of the C-terminal C8b fragment C8b- LHF2. (A) C8b-LHF2 inhibits complement activity (n=6 for SLE / MBL assay, otherwise n=7 ± SE, significance determined by paired t-test: p < 0.05: *, p < 0.01 : **, p < 0.001: ***) of all three pathways at higher concentrations. (B) TLR signaling was moderately activated by C8b-LHF2 in rapidly and in a concentration-dependent manner. (C)C8b- LHF2 induces CD62L-shedding in granulocytes in healthy blood donors; the MFI of detected CD62L from n=5 donors are illustrated for treatment with PBS, LPS at 0.19 mg / mL, C8b-LHF2 at 2.7 mg / mL, or POD2 at 1.7 mg / mL. The CD62L expression was detected using a fluorescent-labeled monoclonal antibody.

[0030] Figure 10: Characterization of the bioactivity of the C3 fragment C3-LHF1. a, Pulldowns with native and denatured (heat-inactivated and carbamidomethylated) C- P7187PC00 terminally His-tagged C3-LHF1 were performed on human kidney homogenate, b, Significantly enriched (adj. p-value < 0.05, grey data points with black borders) proteins in the kidney pull-downs included the IL6 signaling component IL6ST (gp130). c, Thermal proteome profiling by proteome integral solubility alteration assay (TPP-PISA) in human kidney lysates to identify putative C3-LHF1 interacting proteins (n=3). Only sign, altered (adj. p-value < 0.05) and plasma membrane-resident proteins are displayed after addition of 1 or 10 pM C3-LHF1 (1 h, 37 °C). Proteins stabilized by interactions with C3-LHF1 display a higher abundance in the treated samples (grey data points with black border, IL6ST depicted as filled black circle), d, C3-LHF1 effect on IL6R / IL6ST signaling was tested in a HEK-Blue IL6Ra / IL6ST reporter line (incubation o / N at 37 °C). C3-LHF1 effect was concentration dependent, and activating (black), unlike bulk C3 (light grey, n=4, ± SE - IL6 used at 3x10-6pg / mL, dark grey), e, In HEK-Blue IL6Ra / IL6ST cell line with both IL6 and C3-LHF1 (o / N at 37 °C, dashed line with triangles), a partial competition can be observed at IL6 concentrations of >1x 10'4pg / mL (n=4, ±SE). f, Tocilizumab (TOC, 2 pg / mL for 3h at 37 °C, black striped bars) did not inhibit the C3-LHF1 response in the IL6Ra / IL6ST cell line (black bars), unlike the IL6 response at 1x10-4pg / mL (grey bars, grey striped bars are with tocilizumab inhibition; n=4, ±SE).

[0031] Figure 11 : Innate immune signaling activity of C3-LHF1 on human kidney organoid and cell systems, a, Rationale for assessing the activity of C3-LHF1 on kidney organoids, b, Baseline alterations in the kidney organoid proteome upon application of 18 pM C3-LHF1 for 48h (significance threshold: adj. p-value < 0.05, | log2FC| > 0.58), corresponding significant entries are displayed as grey or black data points with a black border, (n=4): a reduction (grey data points with black border) of CMTM4 and an increase (black data points) of JAK3 / STAT3 can be observed, c, Proteome alterations upon TNFa (25 ng / mL) treatment and combined TNFa (25 ng / mL) and C3-LHF1 (18 pM) treatment for 48h (sign, threshold: adj. p-value < 0.05, |log2FC| > 0.58, n=4). d, Combined analysis of C3-LHF1 / PBS and C3-LHF1+TNFa / TNFa reveals significantly C3-LHF1 affected proteins (adj. p-value < 0.05): podocyte markers are down-regulated by C3-LHF1 application (KIRREL1 , NPHS1, NPHS2, PTPRO, PODXL - grey data points with black borders), while the inflammatory response by the IL6ST-JAK3-STAT3 axis is up-regulated (STI NG 1, IL6ST, JAK3, STAT3 - black filled data points), e, CXCL10 secretion as a measure of inflammatory response was assayed in human kidney organoid supernatants and revealed an increase in CXCL10 P7187PC00 secretion after 48h of C3-LHF1 incubation (n=4 ± SE, * = p < 0.05, *** = p < 0.001). f, Combined proteomic and phosphoproteomic analysis (n=4) reveals significant changes in the IL6ST-JAK3-STAT3 signaling axis upon C3-LHF1 application in the absence and presence of TNFa (up-regulation indicated by upward triangles, down-regulation by downward triangles), g, Regulatory tyrosine (Y) phosphosites in the human kidney organoid phosphoproteome upon combined C3-LHF1+TNFa treatment for 48h. STAT3 phosphorylation at Y705 is the main induced phosphosite by C3-LHF1 in the presence of TNFa.

[0032] Figure 12: Mechanistic analysis of C3-LHF1 -induced IL6 signaling in HEK-Blue IL6 reporter cells demonstrates IL6ST dependency and IL6Ra independence.

[0033] HEK-Blue IL6 reporter cells were pre-treated with the corresponding inhibitors for 2h at 37 °C. Applied inhibitors were sarilumab (SARI, 1 pg / mL) and tocilizumab (TOC, 1 pg / mL) for IL6Ra or SC-144 (20 pM) and anti-IL6ST (1 pg / mL) for IL6ST (gp130). Subsequently, cells were treated o / N at 37 °C with 10'4pg / mL IL6 agonist (a, black bars) or 20 pM C3-LHF1 (SEQ ID NO:2) (b, white bars) and the secretion of the reporter SEAP quantified at 620nm (n=4 ± SD). Significant inhibition in comparison to the pure IL6 or C3-LHF1 (SEQ ID NO:2) treatment is indicated (p < 0.01 : *, p < 0.01: **, p < 0.001 : ***). IL6Ra inhibition does not block C3-LHF1 activation of IL6 signaling (a, SARI / TOC), whereas IL6ST inhibition reduces the activation significantly (b, SC-144, a- IL6ST).

[0034] Detailed description

[0035] Definitions

[0036] As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly states otherwise. Thus, for example, reference to “an antibody” includes a plurality of such constructs.

[0037] As used herein, the term “about” refers to a value or range that is approximate and allows for inherent variations in measurement, experimental error, and rounding. When applied to a numerical value, such as “about 90% sequence identity,” it encompasses values that are within the range that a person skilled in the art would consider equivalent in the given technical context, for example within ±0.5%, ±1%, ±2%, ±3%, ±4%, or ±5% of the stated value, unless otherwise specified. P7187PC00

[0038] The term “some embodiments” can include one, or more than one embodiment.

[0039] The terms “protein” or “polypeptide” as defined herein are used interchangeably and refer to molecules mainly consisting of a chain of amino acids linked by peptide bonds, without reference to a specific mode of action, size, 3-dimensional structure or origin. A fragment or portion of a protein may thus still be referred to as a "protein". An "isolated protein" is used to refer to a protein which is no longer in its natural environment, for example in vitro. A “heterologous protein” refers to a protein which is not naturally present in the type of cell in which it is expressed, for example a heterologous protein may be expressed in a recombinant bacterial or plant host cell, whereas it is not expressed in the corresponding wild type bacteria or plant cell. An enzyme is a protein having enzymatic activity.

[0040] The term “sequence identity”, with respect to a polynucleotide or polypeptide, refers to the percentage of nucleic acids or amino acids in the candidate sequence that are identical to the residues of a corresponding native polynucleotide or polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity, and considering any conservative substitutions according to the NCIIIB rules (http: / / www.chem. qmul.ac.uk / iubmb / misc / naseq.html; NC-llIB, Eur J Biochem (1985)) as part of the sequence identity. In particular, the percentage of similarity refers to the percentage of residues conserved with similar physiochemical properties. Neither 5' or 3' extensions nor insertions (for nucleic acids) or N’ or C’ extensions nor insertions (for polypeptides) result in a reduction of identity. Methods and computer programs for the alignments are well known in the art. Generally, a given identity between two sequences implies that the identity between these sequences is at least equal to the similarity; for example, if two sequences are 70% identical to one another, they cannot be less than 70% similar to one another - but could be sharing 80% similarity or more. As used herein, the expression “at least X% sequence identity” refers to when a candidate amino acid or nucleic acid sequence is aligned with a reference sequence, the candidate sequence shares at least the recited percentage of identical residues with the reference sequence. Thus, by way of example, the term “at least 90% sequence identity” is to be understood as encompassing sequences having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the reference sequence. More generally, the expression “at least X% sequence identity” is P7187PC00 to be interpreted as including X% identity and all greater percentages of identity up to 100%. Conversely, the expression “at the most X% sequence identity” is intended to mean that the candidate sequence shares no greater than the recited percentage of identical residues with the reference sequence. Thus, by way of example, the term “at the most 90% sequence identity” is to be understood as encompassing sequences having 90%, 89%, 88%, 87%, 86%, 85% and so on down to 0%. More generally, the expression “at the most X% sequence identity” is to be interpreted as including X% identity and all lower percentages of identity down to 0%.

[0041] The term “functional homologue” or “functional variant” refers herein to functional variants of a parent polypeptide, such as an enzyme, which retain at least some of the activity of the parent polypeptide, such as the parent enzyme; the term does not necessarily imply that the functional variant shares significant homology with the parent polypeptide. Thus, a functional variant of a given polypeptide can catalyse the same conversion as the enzyme from which it is derived, although the efficiency of reaction may be different, e.g. the efficiency is decreased or increased compared to the parent enzyme, the substrate specificity is modified, the longevity or turnover of the enzyme is modified, the cellular localisation of the enzyme is modified. How to test whether the functionality of the functional variant is modified compared to the original polypeptide is well within reach of the skilled person. It can for example be assessed by purifying the functional variant, e.g. using an affinity tag, and comparing its activity on a given substrate in vitro to the activity of the parent polypeptide.

[0042] The term "complement-mediated disorder" refers to any pathological condition or disease in which the dysregulation or aberrant activation of the complement system plays a central role in the disease's onset, progression, or severity. These disorders arise when the complement system, which is typically involved in immune defence and inflammation, becomes overactive, insufficiently controlled, or misdirected, leading to tissue damage, inflammation, and / or immune-mediated injury. Examples of complement-mediated disorders include, but are not limited to, autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis, and various forms of nephritis, including IgA nephropathy. The present invention targets these conditions by modulating complement activity through the use of specific peptide fragments, thereby offering a novel therapeutic approach to managing such disorders. P7187PC00

[0043] Peptide fragments of the present disclosure

[0044] The term “peptide fragment” refers to refers to a short chain of amino acids, typically ranging from a few to several dozen residues preferably 10 to 40 residues, that is derived from a larger protein or polypeptide. A peptide fragment may be modified, wherein the peptide fragment comprises any of the modifications described in the below section entitled ‘peptide fragment modifications’. The peptide fragment retains a portion of the primary structure of the original or primary protein and may exhibit specific and / or distinct biological or functional properties. Peptide fragments can be generated naturally through enzymatic cleavage, synthesized chemically, or produced recombinantly. The peptide fragment may encompass not only the exact sequences derived from the parent protein but also variants and analogues with similar functional characteristics, provided they maintain a significant degree of sequence identity or exhibit comparable biological activity.

[0045] In one aspect, the present invention provides for a peptide fragment comprising at least 10 amino acids and the most 200 amino acids, wherein the peptide fragment comprises a sequence comprising at least one of the following sequences; SEQ ID NO: 1-58 or functional homologues thereof sharing at least 90% sequence identity therewith, for use in a method of treatment of complement-mediated disorders.

[0046] It may be preferred that the peptide fragment of the present invention comprises at least 10 amino acids, such as at least 20 amino acids, such as at least 30 amino acids, such as at least 40 amino acids, such as at least 50 amino acids, such as at least 60 amino acids, such as at least 70 amino acids, such as at least 80 amino acids, such as at least 90 amino acids, such as at least 100 amino acids, such as at least 110 amino acids, such as at least 120 amino acids, such as at least 130 amino acids, such as at least 140 amino acids, such as at least 150 amino acids, such as at least 160 amino acids, such as at least 170 amino acids, such as at least 180 amino acids, such as at least 190 amino acids, such as at least 200 amino acids.

[0047] It may be preferred that that peptide fragment of the present invention comprises at the most 200 amino acid, such as at the most 190 amino acids, such as at the most 180 amino acids, such as at the most 170 amino acids, such as at the most 160 amino acids, such as at the most 150 amino acids, such as at the most 140 amino acids, such as at the most 130 amino acids, such as at the most 120 amino acids, such as at the most 110 amino acids, such as at the most 100 amino acids, such as at the most 90 P7187PC00 amino acids, such as at the most 80 amino acids, such as at the most 70 amino acids, such as at the most 60 amino acids, such as at the most 50 amino acids, such as at the most 40 amino acids, such as at the most 30 amino acids, such as at the most 20 amino acids, such as at the most 10 amino acids.

[0048] It may be preferred that the peptide fragment comprises at least 10 amino acids and at the most 200 amino acids, such as at least 10 amino acids and at the most 180 amino acids, such as at least 10 amino acids and at the most 160 amino acids, such as at least 10 amino acids and at the most 140 amino acids, such as at least 10 amino acids and at the most 120 amino acids, such as at least 10 amino acids and at the most 100 amino acids, such as at least 10 amino acids and at the most 80 amino acids, such as at least 10 amino acids and at the most 60 amino acids, such as at least 10 amino acids and at the most 40 amino acids, such as at least 10 amino acids and at the most 20 amino acids. It may be preferred that the peptide fragment comprises at least 20 amino acids and at the most 200 amino acids, such as at least 20 amino acids and at the most 180 amino acids, such as at least 20 amino acids and at the most 160 amino acids, such as at least 20 amino acids and at the most 140 amino acids, such as at least 20 amino acids and at the most 120 amino acids, such as at least 20 amino acids and at the most 100 amino acids, such as at least 20 amino acids and at the most 80 amino acids, such as at least 20 amino acids and at the most 60 amino acids, such as at least 20 amino acids and at the most 40 amino acids. It may be preferred that the peptide fragment comprises at least 30 amino acids and at the most 200 amino acids, such as at least 30 amino acids and at the most 180 amino acids, such as at least 30 amino acids and at the most 160 amino acids, such as at least 30 amino acids and at the most 140 amino acids, such as at least 30 amino acids and at the most 120 amino acids, such as at least 30 amino acids and at the most 100 amino acids, such as at least 30 amino acids and at the most 80 amino acids, such as at least 30 amino acids and at the most 60 amino acids. It may be preferred that the peptide fragment comprises at least 40 amino acids and at the most 200 amino acids, such as at least 40 amino acids and at the most 180 amino acids, such as at least 40 amino acids and at the most 160 amino acids, such as at least 40 amino acids and at the most 140 amino acids, such as at least 40 amino acids and at the most 120 amino acids, such as at least 40 amino acids and at the most 100 amino acids, such as at least 40 amino acids and at the most 80 amino acids. It may be preferred that the peptide fragment comprises at least 50 amino acids and at the most 200 amino acids, such as at least P7187PC00

[0049] 50 amino acids and at the most 180 amino acids, such as at least 50 amino acids and at the most 160 amino acids, such as at least 50 amino acids and at the most 140 amino acids, such as at least 50 amino acids and at the most 120 amino acids, such as at least 50 amino acids and at the most 100 amino acids. It may be preferred that the peptide fragment comprises at least 60 amino acids and at the most 200 amino acids, such as at least 60 amino acids and at the most 180 amino acids, such as at least 60 amino acids and at the most 160 amino acids, such as at least 60 amino acids and at the most 140 amino acids, such as at least 60 amino acids and at the most 120 amino acids. It may be preferred that the peptide fragment comprises at least 70 amino acids and at the most 200 amino acids, such as at least 70 amino acids and at the most 180 amino acids, such as at least 70 amino acids and at the most 160 amino acids, such as at least 70 amino acids and at the most 140 amino acids. It may be preferred that the peptide fragment comprises at least 80 amino acids and at the most 200 amino acids, such as at least 80 amino acids and at the most 180 amino acids, such as at least 80 amino acids and at the most 160 amino acids. It may be preferred that the peptide fragment comprises at least 90 amino acids and at the most 200 amino acids, such as at least 90 amino acids and at the most 180 amino acids. It may be preferred that the peptide fragment comprises at least 100 amino acids and at the most 200 amino acids.

[0050] The term “comprises at the most n amino acids” as used herein should be understood as that said peptide fragment contains no more than n amino acids, however the peptide fragment may comprise other moieties, which are not amino acids, e.g. any of the modifications described herein below. A peptide fragment comprising at the most n amino acids may consist of n amino acids, or fewer.

[0051] The term “comprises at least one of the following sequences; SEQ ID NO: 1-58” as used herein should be understood as that said peptide fragment comprises or consists of SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 and so forth. The same understanding should be applied to similar sentences referring to different SEQ ID numbers.

[0052] In another aspect, the present invention provides for a peptide fragment comprising at least 10 amino acids and at the most 200 amino acids, wherein the peptide fragment comprises a sequence comprised within at the most 30 amino acids of the C-terminus of a primary protein comprising any one of SEQ ID NO: 60-99. P7187PC00

[0053] In another aspect, the present invention provides for a peptide fragment comprising at least 10 amino acids and at the most 200 amino acids, wherein the peptide fragment comprises a sequence comprising at least one of the following sequences; SEQ ID NO: 1-58 or functional homologues thereof sharing at least 90% sequence identity therewith, wherein the peptide fragment comprises a sequence comprised within at the most 30 amino acids of the C-terminus of a primary protein comprising any one of SEQ ID NO: 60-99, for use in a method of treatment of complement-mediated disorders.

[0054] In another aspect, the present invention provides for a peptide fragment comprising at least 10 amino acids and at the most 40 amino acids, wherein the peptide fragment comprises a sequence comprising at least one of the following sequences; SEQ ID NO: 1 or SEQ ID NO 3-56 or functional homologues thereof sharing at least 90% sequence identity therewith.

[0055] In another aspect, the present invention provides for a peptide fragment comprising at least 10 amino acids and at the most 40 amino acids, wherein the peptide fragment comprises a sequence comprised within at the most 30 amino acids of the C-terminus of a primary protein comprising any one of SEQ ID NO: 60-99.

[0056] In another aspect, the present invention provides for a peptide fragment comprising at least 10 amino acids and at the most 40 amino acids, wherein the peptide fragment comprises a sequence comprising at least one of the following sequences; SEQ ID NO:

[0057] 1 or SEQ ID NO 3-56 or functional homologues thereof sharing at least 90% sequence identity therewith, wherein the peptide fragment comprises a sequence comprised within at the most 30 amino acids of the C-terminus of a primary protein comprising any one of SEQ ID NO: 60-99.

[0058] In another aspect, the present invention provides for a peptide fragment which comprises a sequence comprising at least one of the following sequences; SEQ ID NO:

[0059] 2 (LHF1) or any one of SEQ ID NO: 1 or 3-58or functional homologues thereof sharing at least 95% sequence identity therewith, such as sharing 100% sequence identity therewith. P7187PC00

[0060] In a further aspect, the peptide fragment comprises at least one of the following sequences; SEQ ID NO: 1 and / or SEQ ID NO: 3-35, or functional homologues thereof preferably sharing at least 90% sequence identity therewith, more preferably sharing at least 95% sequence identity therewith, even more preferably sharing at least 100% sequence identity therewith.

[0061] In another aspect, the present invention provides for a peptide fragment comprising at least 120 amino acids and at the most 200 amino acids, wherein the peptide fragment comprises a sequence comprising at least one of the following sequences; SEQ ID NO: 2 (LHF1) or SEQ ID NO: 58, or functional homologues thereof sharing at least 90% sequence identity therewith, such as sharing 100% sequence identity therewith. It may be preferred that the peptide fragment of the present invention comprises at least 130 amino acids. It may be preferred that the peptide fragment of the present invention comprises at the most 190 amino acids, such as at the most 180 amino acids, such as at the most 175 amino acids, such as 170 amino acids, such as 160 amino acids, such as 150 amino acids, such as 149 amino acids. It may be preferred that the peptide fragment of the present invention comprises at least 130 amino acids and at the most 180 amino acids, such as at least 130 amino acids and at the most 175 amino acids. It may be preferred that the peptide fragment comprises or consists of 149 amino acids. It may be preferred that the peptide fragment comprises or consists of 169 amino acids.

[0062] In another aspect, the present invention provides for a peptide fragment comprising at least 10 amino acids and at the most 40 amino acids, wherein the peptide fragment comprises a sequence comprising at least one of the following sequences; SEQ ID NO: 1 (LHF2), or functional homologues thereof sharing at least 90% sequence identity therewith, such as sharing 100% sequence identity therewith.

[0063] It may be preferred that the peptide fragment of the present invention comprises at least 20 amino acids, such as at least 25 amino acids, such as at least 26 amino acids. It may be preferred that the peptide fragment of the present invention comprises at the most 35 amino acids, such as at the most 30 amino acids, such as at the most 28 amino acids, such as at the most 27 amino acids, such as at the most 26 amino acids. It may be preferred that the peptide fragment comprises at least 20 amino acids and at the most 35 amino acids, such as at least 20 amino acids and at the most 30 amino P7187PC00 acids, such as at least 20 amino acids and at the most 28 amino acids, such as at least 20 amino acids and at the most 27 amino acids, such as at least 20 amino acids and at the most 26 amino acids. It may be preferred that the peptide fragment comprises at least 25 amino acids and at the most 35 amino acids, such as at least 25 amino acids and at the most 30 amino acids, such as at least 25 amino acids and at the most 28 amino acids, such as at least 25 amino acids and at the most 27 amino acids, such as at least 25 amino acids and at the most 26 amino acids. It may be preferred that the peptide fragment comprises at least 26 amino acids and at the most 35 amino acids, such as at least 26 amino acids and at the most 30 amino acids, such as at least 26 amino acids and at the most 28 amino acids, such as at least 26 amino acids and at the most 27 amino acids. It may be preferred that the peptide fragment comprises or consists of 26 amino acids.

[0064] TM-scores

[0065] The term “TM-score” or “template modelling score” refers to a recognized metric used in the field of bioinformatics to assess the similarity between two protein structures. The TM-score quantitatively measures this similarity on a scale from 0 to 1. Typically, scores below 0.20 indicate randomly chosen, unrelated proteins, whereas scores above 0.5 suggest that the structures share roughly the same fold. A detailed methodology for calculating the TM-score is provided in the publication "Zhang Y and Skolnick J (2004). Scoring function for automated assessment of protein structure template quality. Proteins. 57 (4): 702-710." The TM-score can be calculated, for example, by uploading two three-dimensional structures in PDB format to the online resource available at https: / / zhanggroup.org / TM-score / . In situations where a pair of three-dimensional structures for comparison is not available, established methods for predicting the three-dimensional structure of a polypeptide are well known to those skilled in the art. For instance, a neural network trained for this specific task, such as AlphaFold, can be employed.

[0066] 3D structures of proteins and / or domains thereof are available through different sources. Thus, 3D structures to be used with the present invention may be available through various databases or can be predicted structures. AlphaFold3, referred to herein as AlphaFold, represents a state-of-the-art artificial intelligence (Al) system developed by DeepMind for predicting the three-dimensional (3D) structures of proteins from their amino acid sequences. This system is detailed in the publication: Jumper, J., P7187PC00

[0067] Evans, R., Pritzel, A. et al. "Highly accurate protein structure prediction with AlphaFold." Nature 596, 583-589 (2021). https: / / doi.org / 10.1038 / s41586-021-03819-2. AlphaFold DB is an online database which hosts over 200 million entries, encompassing the human proteome as well as the proteomes of 47 other key organisms relevant to research and global health. These entries are freely accessible at https: / / alphafold.ebi.ac.uk / .

[0068] The (predicted) structure of the peptide fragments of the invention can be retrieved by inputting identifiers such as the protein name, gene name or UniProt accession number as set forth in the below ‘sequence overview’ section. Structures of polypeptides not included in the AlphaFold DB may for example be predicted using the source code available at https: / / github.com / google-deepmind / alphafold, and / or a Colab notebook accessible at https: / / colab.research.google.com / github / deepmind / alphafold / blob / main / notebooks / Alp haFold.ipynb. To generate a 3D structure using the Colab notebook, the amino acid sequences of said peptide fragments, as set forth in the section ‘sequence overview’ can be inserted.

[0069] In one aspect, the three dimensional structure of the peptide fragment or its functional variant has a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of a peptide as set forth in any one of SEQ ID NOs: 1-58.

[0070] Groups of peptides

[0071] The charge of peptide fragments plays a crucial role in determining their functional properties, particularly in their interactions with components of the complement system. The net charge of a peptide fragment, which is influenced by the distribution of acidic and basic amino acids within its sequence, can affect its binding affinity to complement proteins, regulatory factors, or receptors. For instance, positively charged fragments may have a higher affinity for negatively charged surfaces or protein domains, facilitating specific interactions that can either enhance or inhibit complement activation. Conversely, negatively charged or neutral fragments may preferentially bind to different molecular targets or exhibit distinct functional outcomes. The charge of P7187PC00 these peptide fragments not only influences their binding interactions but also their solubility, stability, and overall bioactivity within the physiological environment. By carefully modulating the charge of these fragments, it is possible to optimize their efficacy as modulators of the complement system, enabling precise therapeutic intervention in complement-mediated disorders.

[0072] In one aspect, the peptide fragment is negatively charged. In particular, said negatively charges peptide fragment may comprise at least one of the following sequences; SEQ ID NO: 1 and / or SEQ ID NO: 3-17, or functional homologues thereof preferably sharing at least 90% sequence identity therewith, more preferably sharing at least 95% sequence identity therewith, even more preferably sharing 100% sequence identity therewith.

[0073] In one aspect, the peptide fragment is non-charged. In particular, said non-charged peptide fragment may comprise at least one of the following sequences; SEQ ID NO: 18-24 or functional homologues thereof preferably sharing at least 90% sequence identity therewith, more preferably sharing at least 95% sequence identity therewith, even more preferably sharing at least 100% sequence identity therewith.

[0074] In one aspect, the peptide fragment is positively charged. In particular, said positively charge peptide fragment may comprise at least one of the following sequences; SEQ ID NO: 25-35 or functional homologues thereof preferably sharing at least 90% sequence identity therewith, more preferably sharing at least 95% sequence identity therewith, even more preferably sharing 100% sequence identity therewith. It may be preferred the peptide fragment is positively charged.

[0075] In another aspect, the peptide fragment comprises at least one of the following sequences; SEQ ID NO: 2 or SEQ ID NO: 36-56 or SEQ ID NO: 58, or functional homologues thereof preferably sharing at least 90% sequence identity therewith, more preferably sharing at least 95% sequence identity therewith, even more preferably sharing 100% sequence identity therewith. It may be preferred that the peptide fragment is detected in the plasma of at least 20% of patients suffering from a complement-associated disease, such as SLE. It may also be preferred that the peptide fragment is increased by at least 50% in the plasma of patients suffering from a P7187PC00 complement-associated disease compared to the plasma of healthy patients, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%.

[0076] In some aspects, the peptide fragment comprises at least one of the following sequences; SEQ ID NO: 2 or SEQ ID NO: 36-42 or SEQ ID NO: 58, preferably sharing at least 90% sequence identity therewith, more preferably sharing at least 95% sequence identity therewith, even more preferably sharing 100% sequence identity therewith. It may be preferred that the peptide fragment is negatively charged.

[0077] The C345C domain (also referred to as the NTR domain) is a conserved structural module found at the C-terminus of several complement proteins, including C3, 04, 05, and factor B, as characterised in Thai & Ogata (2003). In complement component 03, the C345C domain forms part of the p-chain and contributes to the binding interface for other complement proteins during convertase assembly. Structural and functional studies have shown that this domain participates in interactions with components of the classical, lectin, and alternative pathway 03 convertases, including C4b, C2a, Bb, and properdin, and can influence the stability and activity of these enzyme complexes. The C345C fold is typically cysteine-rich and contains multiple disulfide bonds that stabilise its tertiary structure, making it relatively resistant to proteolytic degradation.

[0078] In some embodiments, the peptide fragment comprises or consists of at least one of the following sequences; SEQ ID NO: 2 or any one of SEQ ID NO: 36-42 or SEQ ID NO: 58, or functional homologues thereof preferably sharing at least 90% sequence identity therewith, more preferably sharing at least 95% sequence identity therewith, even more preferably sharing 100% sequence identity therewith, and the peptide fragment further comprises the C345C domain of 03. In some embodiments, the C345C domain of 03 comprises a sequence comprising or consisting of amino acid positions 1518-1663 of 03 (SEQ ID NO: 126). In some embodiments, the C345C domain of 03 is as set forth in SEQ ID NO: 127. Accordingly, this facilitates the peptide fragments to interact selectively with complement pathway proteins, particularly inhibiting complement activation at an early stage.

[0079] In another aspect, the peptide fragment comprises at least one of the following sequences; SEQ ID NO: 43-45 or functional homologues thereof preferably sharing at least 90% sequence identity therewith, more preferably sharing at least 95% sequence P7187PC00 identity therewith, even more preferably sharing 100% sequence identity therewith. It may be preferred that the peptide fragment is non-charged.

[0080] In another aspect, the peptide fragment comprises at least one of the following sequences; SEQ ID NO: 46-56 or functional homologues thereof preferably sharing at least 90% sequence identity therewith, more preferably sharing at least 95% sequence identity therewith, even more preferably sharing 100% sequence identity therewith. It may be preferred that the peptide fragment is positively charged.

[0081] It may be preferred that the peptide fragment comprises SEQ ID NO: 1 and / or SEQ ID NO: 2 or functional homologues thereof sharing at least 90% sequence identity therewith. It may also be preferred that the peptide fragment comprises SEQ ID NO: 1 and / or SEQ ID NO: 2 or functional homologues thereof sharing at least 95% sequence identity therewith, such as sharing 100% sequence identity therewith.

[0082] It may be preferred that the peptide fragment comprises SEQ ID NO: 1 or functional homologues thereof sharing at least 90% sequence identity therewith. It may also be preferred that the peptide fragment comprises SEQ ID NO: 1 or functional homologues thereof sharing at least 95% sequence identity therewith, such as sharing 100% sequence identity therewith. It may be preferred that the three dimensional structure of the peptide fragment, for example of the functional homologue or functional variant of SEQ ID NO:1 has a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of a peptide as set forth in SEQ ID NO: 1. In particular, it may be preferred that the three structure of the peptide fragment has a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure defined by the structural coordinates as set forth in Table 1.

[0083] Table 1. The structural coordinates of the three dimensional structure of a peptide as set forth in SEQ ID NO: 1. P7187PC00 P7187PC00

[0084] It may be preferred that the peptide fragment comprises or consists of SEQ ID NO: 2 or functional homologues thereof sharing at least 90% sequence identity therewith. It may also be preferred that the peptide fragment comprises or consists of SEQ ID NO: 2 or functional homologues thereof sharing at least 95% sequence identity therewith, such as sharing 100% sequence identity therewith. It may be preferred that the three dimensional structure of the peptide fragment, for example of the functional homologue or functional variant of SEQ ID NO:2 has a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of a peptide as set forth in SEQ ID NO: 2. In particular, it may be preferred that the three dimensional structure of the peptide fragment has a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure defined by the structural coordinates as set forth in Table 2.

[0085] It may be preferred that the peptide fragment comprises or consists of SEQ ID NO: 58 or functional homologues thereof sharing at least 90% sequence identity therewith. It may also be preferred that the peptide fragment comprises or consists of SEQ ID NO: 58 or functional homologues thereof sharing at least 95% sequence identity therewith, such as sharing 100% sequence identity therewith. P7187PC00

[0086] Table 2. The structural coordinates of the three dimensional structure of a peptide as set forth in SEQ ID NO: 2. P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00

[0087] Peptide fragment modifications

[0088] The presently disclosed peptide fragments may optionally comprise one or more modifications. The term “modification” as used herein includes any functionalisation of the polypeptide, e.g. via formation of a covalent bond between any atom in the polypeptide and an atom in another compound, or a covalent bond between two atoms in the polypeptide itself, or to another similar to different polypeptide.

[0089] In one aspect, the peptide fragment disclosed herein comprises incorporation of non- natural amino acids or D-amino acids, cyclization, stapling, conjugation to a biomolecule, chelation, lipidation, N-terminal acetylation, C-terminal amidation, PEGylation and / or glycosylation. It may be preferred that the peptide fragment comprises a modification of conjugation to a biomolecule, such as biotin, such as an amino acid, such as an un-natural amino acid, such as a peptide, such as a protein, such as a carbohydrate, such as a mono saccharide, such as a disaccharide, such as P7187PC00 a disaccharide, such as a polysaccharide, such as a lipid, such as a fatty acid, such as a saturated fatty acid, such as a unsaturated fatty acid, such as a triglyceride, such as a steroid, such as a PEG, such as a fluorophore. It may be preferred that the modification comprises conjugation to a tag, such as a chemical tag, such as a protein tag, such as fluorescent tag, such as an isotopic tag. It may be preferred that the peptide fragment is conjugated to a carrier molecule that enhances cellular uptake. In particular, it may be preferred that the carrier molecule is selected from the group consisting of liposomes, nanoparticles, and polymer conjugates.

[0090] Modulation of complement activity

[0091] The presently disclosed peptide fragments are capable of modulating complement activity. In some embodiments, the peptide fragments modulate the activity of the complement system. The term "modulation of the complement system" refers to the alteration or regulation of the activity of the complement cascade, which is a key component of the immune system involved in pathogen defence, inflammation, and immune complex clearance. Modulation can include either the inhibition or activation of specific pathways within the complement system, such as the classical, alternative, and / or lectin (MBL) pathways. This modulation can affect various components of the complement system, including complement proteins, receptors, and regulatory factors, ultimately influencing the downstream effects such as inflammation, cell lysis, or opsonization. How to measure complement system modulation is well within the reach of the skilled person, and may include methods such as Enzyme-Linked Immunosorbent Assays (ELISA), Time-Resolved Immunofluorometric Assays (TRIFMA-Based Assays and / or DELFIA), Hemolytic Assays (e.g., CH50 and / or AH50), Nephelometry, Turbidimetry, Flow Cytometry, Mass Spectrometry (e.g., LC / MS), Western Blotting, Surface Plasmon Resonance (SPR), Cytokine Profiling, Reverse transcription polymerase chain reaction (RT-PCR), Immunohistochemistry and / or Immunofluorescence Microscopy.

[0092] Accordingly, it may be preferred that modulating complement activity is measured using one or more of the following techniques: enzyme-linked immunosorbent assays (ELISA), time-resolved immunofluorometric assays (TRIFMA and / or DELFIA), Mass Spectrometry, liquid chromatography-mass spectrometry (LC / MS), haemolytic assays, Nephelometry, Turbidimetry, flow cytometry, surface plasmon resonance, P7187PC00 immunoassay, immunofluorescence microscopy, Cytokine Profiling, western blot, Immunohistochemistry and / or RT-PCR.

[0093] In one aspect, modulation of complement activity by the peptide fragments of the invention comprises modulation of the expression and / or activity and / or function of the classical complement pathway, the alternative complement pathway and / or the mannose-binding lectin (MBL) pathway or components thereof. It may be preferred that the modulation of complement activity comprises modulation of the expression and / or activity of any one or more of the following: the C1 complex, C1q, C1r, C1s, C2, C2a, C2b, C4, C4a, C4b, C3, C3a, C3b, C3c, C3d, C3 convertase, C5, C5a, C5b, C5 convertase, C6, C7, C8, C9, the membrane attack complex (MAC), factor B, factor D, factor H, factor I, factor P (properdin), MASP-1 , MASP-2, MASP-3, C4b-binding protein (C4BP), complement receptor 1 (CR1), complement receptor 2 (CR2), complement receptor 3 (CR3), complement receptor 4 (CR4), decay-accelerating factor (DAF), CD59, and / or any fragments or subunits thereof. In some embodiments modulation of complement activity comprises modulation of the expression and / or activity of any one or more of the following: the C1 complex, C1q, C1r, C1s, C2, C2a, C2b, C4, C4a, C4b, C3, C3a, C3b, C3c, C3d, C3 convertase, C5, C5a, C5b, C5 convertase, C6, C7, C8, C9, the membrane attack complex (MAC), factor B, factor D, factor H, factor I, factor P (properdin), MASP-1 , MASP-2, MASP-3, C4b-binding protein (C4BP), complement receptor 1 (CR1), complement receptor 2 (CR2), complement receptor 3 (CR3), complement receptor 4 (CR4), decay-accelerating factor (DAF), CD59, and / or any fragments or subunits thereof, wherein said modulation essentially is determined as described herein below in Examples 5 and 6 disclosed herein. In particular, it may be preferred that modulation of complement activity comprises modulation of the expression and / or activity of complement factor C9, wherein said modulation essentially is determined as described herein below in Examples 5 and 6 disclosed herein.

[0094] In one aspect, modulation of complement activity by the peptide fragments of the invention comprises inhibition of the expression and / or activity and / or function of; the classical complement pathway, the alternative complement pathway and / or the mannose-binding lectin (MBL) pathway, or components thereof. In some embodiments, modulation of complement activity by the peptide fragments of the invention comprise inhibition of the expression and / or activity and / or function of; the classical complement P7187PC00 pathway and / or the mannose-binding lectin (MBL) pathway, or components thereof. How to test whether the peptide fragments of the invention inhibit the expression and / or activity and / or function of; the classical complement pathway, the alternative complement pathway and / or the mannose-binding lectin (MBL) pathway, or components thereof compared with control or vehicle is well within the reach of the skilled person. It can for example be assessed as described herein this section and / or Examples 5 and 6 disclosed herein.

[0095] It may be preferred that modulating complement activity by the peptide fragments of the invention comprises inhibition of the classical complement pathway of the components thereof and mannose-binding lectin (MBL) pathway or components thereof. It may also be preferred that the peptide fragment the classical complement pathway of the components thereof and mannose-binding lectin (MBL) pathway or components thereof by at least 5% compared with vehicle, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99%. How to test whether the peptide fragments of the invention inhibit the expression and / or activity and / or function of the classical complement pathway of the components thereof and mannose-binding lectin (MBL) pathway or components thereof compared with control or vehicle is well within the reach of the skilled person. It can for example be assessed as described herein this section and / or Example 5 disclosed herein.

[0096] It may be preferred that modulating complement activity by the peptide fragments of the invention comprises inhibition of the mannose-binding lectin (MBL) pathway or components thereof. It may also be preferred that the peptide fragment inhibits the mannose-binding lectin (MBL) pathway or components thereof by at least 5% compared with vehicle, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99%. How to test whether the peptide fragments of the invention inhibit P7187PC00 the expression and / or activity and / or function of; the classical complement pathway, the alternative complement pathway and / or the mannose-binding lectin (MBL) pathway, or components thereof compared with control or vehicle is well within the reach of the skilled person. It can for example be assessed as described herein this section and / or Examples 5 and 6 disclosed herein.

[0097] It may be preferred that modulating complement activity by the peptide fragments of the invention comprises inhibition of the classical complement pathway or components thereof. It may also be preferred that peptide fragment inhibits the classical complement pathway or components thereof by at least 5% compared with vehicle, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99%. How to test whether the peptide fragments of the invention inhibit the expression and / or activity and / or function of the classical complement pathway or components thereof compared with control or vehicle is well within the reach of the skilled person. It can for example be assessed as described herein this section and / or Examples 5 and 6 disclosed herein.

[0098] It may be preferred that modulating complement activity by the peptide fragments of the invention comprises inhibition of the alternative pathway or components thereof. It may also be preferred that the peptide fragment inhibits the alternative pathway or components thereof by at least 5% compared with vehicle, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99%. How to test whether the peptide fragments of the invention inhibit the expression and / or activity and / or function of the alternative pathway or components thereof compared with control or vehicle is well within the reach of the skilled person. It can for example be assessed as described herein this section and / or Examples 5 and 6 disclosed herein. P7187PC00

[0099] It is preferable that the peptide fragments comprises a consecutive sequence comprised within at the most 30 amino acids of the C-terminus of a primary protein comprising any one of SEQ ID NO: 60-99. The term “primary peptide” or “primary protein” or “primary plasma peptide” or “primary plasma protein” refers to the full length protein encoded by a given gene. This peptide serves as the substrate from which smaller, functional peptide fragments are generated through enzymatic cleavage. These fragments, often referred to as N-terminal peptides or complement N-termini, may retain specific bioactivity and may play roles in modulating the complement system. The "primary peptide" thus represents the original, unprocessed form from which bioactive fragments are derived.

[0100] It also preferable that the presently disclosed peptide fragments comprises complement N-termini, which has been generated by proteolytic processing of a primary plasma peptide. The term “complement N-termini” or “N-terminal peptides” refer to the exposed N-terminal region of a complement protein after it has been processed or activated, such as via proteolytic cleavage during the complement cascade. For example, when complement proteins such as C3 or C5 are cleaved to produce C3a and C3b or C5a and C5b, new N-terminal residues are exposed in the cleavage products, which are crucial for their respective functions in the immune response. These N-terminal regions may have specific biological activities, such as binding to receptors or other components of the immune system, mediating inflammation, or promoting phagocytosis. In particular, it may be preferable that the primary plasma peptide is selected from SEQ ID NO: 60-99.

[0101] It may be preferred that the peptide fragment is naturally found in the plasma of a patient suffering from a complement-mediated disorder. It may also be preferred that the peptide fragment is part of the complement system.

[0102] It is preferable that the peptide fragments are part of the N-terminome of a subject. The term “N-terminome" refers to the complete set of N-terminal peptide sequences present within a biological sample, such as plasma, in a subject. These N-terminal peptides are usually generated through post-translational proteolytic processing of proteins, and their study, known as N-terminomics, provides insights into the specific proteolytic events occurring within the body. The N-terminome reflects the dynamic state of proteolysis in the sample and includes peptides that may play significant roles in P7187PC00 biological processes, including modulation of the complement system. It may also be preferable that the peptide fragment is part of the N-terminome of a patient suffering from a complement-mediated disease. In particular, it may be preferable that the peptide fragment is part of the N-terminome of a patient suffering from a complement- mediated disease, for example Systemic Lupus Erythematosus (SLE), lupus nephritis, Hemolytic Uremic Syndrome (HUS), Atypical Hemolytic Uremic Syndrome (aHUS), Paroxysmal Nocturnal Hemoglobinuria (PNH), Age-Related Macular Degeneration (AMD), C3 Glomerulopathy, Dense Deposit Disease (DDD), Rheumatoid Arthritis (RA), Membranoproliferative Glomerulonephritis (MPGN), Neuromyelitis Optica (NMO), Myasthenia Gravis (MG), Cryoglobulinemia, Autoimmune Hemolytic Anemia (AIHA), Vasculitis, Ankylosing Spondylitis, Cold Agglutinin Disease, Immune Complex Glomerulonephritis, Glomerulonephritis, IgA nephropathy (IgAN), Sepsis, Meningococcal Disease, Recurrent Bacterial Infections, Alzheimer’s Disease, Multiple Sclerosis, Uveitis, Complement Component Deficiencies such as C2, C3, and / or C4 deficiencies, Hereditary Angioedema (HAE), Atherosclerosis, Myocardial Infarction (Ml), Dermatitis Herpetiformis, Bullous Pemphigoid, Ischemia-Reperfusion Injury, Transplant Rejection, or Idiopathic Thrombocytopenic Purpura (ITP).

[0103] In particular, it may be preferable that the peptide fragment is part of the N-terminome of a patient suffering from systemic Lupus Erythematosus (SLE). In particular, it may be preferable that the peptide fragment is part of the N-terminome of a patient suffering from Hemolytic Uremic Syndrome (HUS) or Atypical Hemolytic Uremic Syndrome (aHUS). In particular, it may be preferable that the peptide fragment is part of the N- terminome of a patient suffering from lupus nephritis. In particular, it may be preferable that the peptide fragment is part of the N-terminome of a patient suffering from Glomerulonephritis.

[0104] Indications

[0105] In one aspect, the peptide fragments of the invention, as disclosed in any of the preceding sections of the detailed description, are provided for use in a method of treatment of complement-mediated disorders. It may be preferable that the complement-mediated disorder is a complement-mediated disorder of the kidneys. It may be preferable that the complement-mediated disorder is Systemic Lupus Erythematosus (SLE), lupus nephritis, inflammatory kidney disease, cytokine-driven nephropathy, acute kidney disease (CKD), chronic kidney disease (CKD), Hemolytic P7187PC00

[0106] Uremic Syndrome (HUS), Atypical Hemolytic Uremic Syndrome (aHUS), Paroxysmal Nocturnal Hemoglobinuria (PNH), Age-Related Macular Degeneration (AMD), C3 Glomerulopathy, Dense Deposit Disease (DDD), Rheumatoid Arthritis (RA), Glomerulonephritis, Membranoproliferative Glomerulonephritis (MPGN), IgA nephropathy (IgAN), Neuromyelitis Optica (NMO), Myasthenia Gravis (MG), Cryoglobulinemia, Autoimmune Hemolytic Anemia (AIHA), Vasculitis, Ankylosing Spondylitis, Cold Agglutinin Disease, Immune Complex Glomerulonephritis, Sepsis, Meningococcal Disease, Recurrent Bacterial Infections, Alzheimer’s Disease, Multiple Sclerosis, Uveitis, Complement Component Deficiencies such as C2, C3, and / or C4 deficiencies, Hereditary Angioedema (HAE), Atherosclerosis, Myocardial Infarction (Ml), Dermatitis Herpetiformis, Bullous Pemphigoid, Ischemia-Reperfusion Injury, Transplant Rejection, or Idiopathic Thrombocytopenic Purpura (ITP), juvenile idiopathic arthritis, giant cell arteritis, adult-onset Still’s disease, Sjogren’s syndrome, systemic sclerosis, psoriatic arthritis, Castleman’s disease, multiple myeloma, cytokine release syndrome, haemophagocytic lymphohistiocytosis, complement-mediated glomerulopathy, acute kidney injury associated with systemic inflammation, sepsis- associated cytokine storm, and COVID-19-associated hyperinflammation.

[0107] In particular, it may be preferable that the complement-mediated disorders is systemic Lupus Erythematosus (SLE). In particular, it may be preferable that the complement- mediated disorders is Hemolytic Uremic Syndrome (HUS) or Atypical Hemolytic Uremic Syndrome (aHUS). In particular, it may be preferable that the complement-mediated disorder is lupus nephritis. In particular, it may be preferable that the complement- mediated disorder is Glomerulonephritis.

[0108] It may be preferred that the peptide fragment is formulated for administration by oral administration, injection, inhalation, local administration, rectal administration, nasal administration, buccal administration, vaginal administration, or through an implanted drug reservoir.

[0109] It may also be preferred that the method comprises administration of at least one additional therapeutic agent effective in treating complement-mediated disorders. In particular, it may also be preferred that the other therapeutic agent is an inhibitor of a complement pathway component. P7187PC00

[0110] It may be preferred that the method of treatment of a complement-mediated disease comprises administration of a therapeutically effective amount of the peptide fragment as described in the section ‘peptide fragments of the present disclosure’.

[0111] The subject to be treated may be any warm-blooded animal but is, in particular, a mammal, and more in particular a human being. As will be clear to the skilled person, the subject to be treated will, in particular, be a person suffering from, or at risk of, the diseases and disorders mentioned herein.

[0112] Compositions, formulations

[0113] As used herein, the term "pharmaceutical composition" refers to the combination of an active agent (e.g., a composition of the present invention) with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo. The term "pharmaceutical composition" can be a formulation containing a composition of the present invention and / or an agent for use in a method of treatment of complement-mediated disorders. It may be preferable that the pharmaceutical composition of the present invention further comprises one or more pharmaceutically acceptable excipients.

[0114] The present invention includes peptide fragments as described in the ‘peptide fragments of the invention’ section, compositions and methods for use in treatment or a complement-mediated disorders, as described in the ‘indications’ section. Administration of the peptide fragments and / or compositions according to the present invention will typically be via any common route. This includes, but is not limited to parenteral, orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal, or intravenous injection. Accordingly, the pharmaceutical composition of the peptides fragments of the present invention is formulated for delivery via oral, intravenous, subcutaneous, or intramuscular administration. Additional formulations which are suitable for other modes of administration include oral formulations. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, or powders and contain about 10% to about 95% of active ingredient, preferably about 25% to about 70%. P7187PC00

[0115] Typically, compositions of the invention are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective. The quantity to be administered depends on the subject to be treated. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner.

[0116] Modulation of IL6ST-Mediated Signalling

[0117] In one embodiment, the peptide fragment or pharmaceutical composition according to any of the preceding sections in the detailed description, are provided for use in a method of treating or preventing a renal disorder associated with IL6ST (gp130) signalling, wherein said renal disorder is selected from the group comprising: inflammatory kidney disease, lupus nephritis, complement-mediated glomerulopathy, proteinuric kidney disease, acute kidney disease and chronic kidney disease.

[0118] As used herein, the term “IL6ST mediated signalling” refers to cellular signal transduction pathways initiated through activation of the interleukin-6 signal transducer (IL6ST, also known as gp130), whether directly or indirectly, leading to downstream intracellular responses. Such signalling may occur via interaction of IL6ST with its cognate cytokine receptors, co-receptors, or ligands, or through direct engagement of IL6ST in the absence of IL6R. IL6ST mediated signalling encompasses activation or modulation of downstream effectors, including but not limited to STAT3 phosphorylation (for example at Y705), IL6ST phosphorylation (for example at Y814), nephrin phosphorylation (for example at Y1176), modulation of CXCL10 secretion, and / or upregulation of STI NG 1. The term also includes both agonistic and antagonistic modulation of IL6ST activity, whether partial or complete, and is not limited to naturally occurring ligand-receptor interactions.

[0119] As used herein, the terms “abnormal signalling” and dysregulated signalling” refer to any deviation from the normal or homeostatic pattern, magnitude, duration, or context of a cellular signalling pathway, resulting in an altered biological response. Such deviation may include excessive (hyperactive) signalling, insufficient (hypoactive) signalling, temporally inappropriate signalling, signalling in an inappropriate cell type or tissue, or activation of non-canonical pathways. The terms further encompass signalling in which the normal regulatory mechanisms controlling activation, amplitude, P7187PC00 duration, or termination are impaired or overridden, including sustained activation, delayed deactivation, loss of feedback control, or inappropriate receptor-ligand engagement. In the context of the present disclosure, abnormal or dysregulated signalling may include inappropriate activation or inhibition of IL6ST (gp130) mediated signalling or related cytokine pathways, and may be associated with inflammatory, autoimmune, complement-mediated, or other pathological conditions.

[0120] One advantage of the peptide fragments of the invention, such as C3-derived LHF1 fragments, is that they provide a dual functional role wherein not only to potently inhibit activation of the classical and mannose-binding lectin pathways of the complement system at low micromolar concentrations (see examples 5 and 6), but also to engage IL6ST (gp130) directly to modulate downstream cytokine signalling. Indeed, as shown in the present application, experimental data (see examples 7 and 8) in HEK-Blue IL6 reporter cells demonstrate that this IL6ST-mediated activation occurs independently of I L6R, as inhibition with IL6Ra-targeted antibodies did not reduce the LHF1 response, whereas IL6ST-specific inhibitors abolished it. Accordingly, in some embodiments, the peptide fragments of the invention modulate the activity of the classical complement pathway, the alternative complement pathway and / or the mannose-binding lectin (MBL) pathway or components thereof, and modulates IL6ST mediated signalling. In particular, the C3-derived fragment LHF1 comprises or consists of an amino acid sequence selected from SEQ ID NO: 2, or functional homologues thereof.

[0121] As used herein, the term “partial agonist” refers to a molecule that binds to and activates a receptor but produces a sub-maximal biological response compared to a full agonist, and may occur even when occupying all available receptor sites. Partial agonists may modulate signalling output by activating certain downstream pathways selectively, or by eliciting weaker activation than the natural ligand, thereby competitively reducing overactivation by full agonists. In the present disclosure, a partial agonist of IL6ST may induce STAT3 activation to a limited degree compared to IL-6 or other full agonists, potentially conferring therapeutic benefit by fine-tuning cytokine responses.

[0122] In some embodiments, the peptide fragment acts as a partial agonist of IL6ST and induces STAT3 activation. In some embodiments, the peptide fragment induces STAT3 activation. In some embodiments, the peptide fragment modulates CXCL10 secretion, P7187PC00

[0123] STING1 upregulation, and / or phosphorylation of STAT3 at Y705, IL6ST at Y814, or nephrin at Y1176. In some embodiments, the peptide fragment causes a reduction in the expression of podocyte markers, including NPHS1 (nephrin), NPHS2 (podocin), PODXL (podocalyxin), and PTPRO.

[0124] In some embodiments, modulation of IL6ST mediated signalling by the peptide fragments occurs independently of IL6Ra engagement. In some embodiments, modulation of IL6ST mediated signalling by the peptide fragment occurs via one or more alternative co-receptors other than IL6Ra.

[0125] In some embodiments, the peptide fragments or pharmaceutical compositions as disclosed herein, are provided for use in the treatment or prevention of a complement- mediated disease associated with abnormal or dysregulated IL-6 signalling.

[0126] In some embodiments, the complement-mediated disease or disorder associated with abnormal IL-6 signalling, is selected from the group comprising rheumatoid arthritis, juvenile idiopathic arthritis, giant cell arteritis, adult-onset Still’s disease, systemic lupus erythematosus, lupus nephritis, Sjogren’s syndrome, systemic sclerosis, vasculitis, psoriatic arthritis, ankylosing spondylitis, Castleman’s disease, multiple myeloma, cytokine release syndrome, haemophagocytic lymphohistiocytosis, complement- mediated glomerulopathy, membranoproliferative glomerulonephritis, IgA nephropathy, acute kidney injury associated with systemic inflammation, sepsis-associated cytokine storm, or COVID-19-associated hyperinflammation. In some embodiments, the a complement-mediated disease associated with abnormal IL-6 signalling is characterised by dysregulation of IL6ST (gp130) mediated signalling.

[0127] In some embodiments, the peptide fragments or pharmaceutical composition as disclosed herein, are provided for use in a method of treating or preventing a renal disorder associated with IL6 signalling, wherein said renal disorder is selected from the group comprising: inflammatory kidney disease, lupus nephritis, complement-mediated glomerulopathy, proteinuric kidney disease, acute kidney disease and chronic kidney disease. P7187PC00

[0128] Methods of modulating the complement system

[0129] In one aspect, the present invention provides for methods of modulating the complement system, said method comprising: a. Providing a composition comprising a human complement factor, a regulator of the complement system, or a receptor involved in complement activity selected from the group comprising the C1 complex, C1q, C1r, C1s, C2, C2a, C2b, C4, C4a, C4b, C3, C3a, C3b, C3c, C3d, C3 convertase, C5, C5a, C5b, C5 convertase, C6, C7, C8, C9, the membrane attack complex (MAC), factor B, factor D, factor H, factor I, factor P (properdin), MASP-1 , MASP-2, MASP-3, C4b-binding protein (C4BP), complement receptor 1 (CR1), complement receptor 2 (CR2), complement receptor 3 (CR3), complement receptor 4 (CR4), decay-accelerating factor (DAF), CD59, and / or any fragments, proteolytic derivatives or subunits thereof, b. Contacting said composition with the peptide fragment as defined in the section ‘peptide fragments of the present disclosure’.

[0130] It may be preferred that the composition comprising a human complement factor is serum, plasma, blood or cerebrospinal fluid. It may also be preferred that the method is an in vitro method. It may also be preferred that the method is an in vivo method.

[0131] Items

[0132] 1. A peptide fragment comprising at least 10 amino acids and at the most 200 amino acids, wherein the peptide fragment comprises a sequence comprising at least one of the following sequences; SEQ ID NO: 1-58 or functional homologues thereof sharing at least 90% sequence identity therewith, for use in a method of treatment of complement-mediated disorders.

[0133] 2. A peptide fragment comprising at least 10 amino acids and at the most 200 amino acids, wherein the peptide fragment comprises a sequence comprising at least one of the following sequences; SEQ ID NO: 1-58 or functional homologues thereof sharing at least 90% sequence identity therewith, wherein the peptide fragment modulates the activity of the complement system, for use in a method of treatment of complement-mediated disorders. P7187PC00

[0134] 3. A peptide fragment comprising at least 10 amino acids and at the most 200 amino acids, wherein the peptide fragment comprises a sequence comprised within at the most 30 amino acids of the C-terminus of a primary protein comprising any one of SEQ ID NO: 60-99.

[0135] 4. A peptide fragment comprising at least 10 amino acids and at the most 200 amino acids, wherein the peptide fragment comprises a sequence comprising at least one of the following sequences; SEQ ID NO: 1-58 or functional homologues thereof sharing at least 90% sequence identity therewith, wherein the peptide fragment comprises a sequence comprised within at the most 30 amino acids of the C-terminus of a primary protein comprising any one of SEQ ID NO: 60-99, for use in a method of treatment of complement-mediated disorders.

[0136] 5. The peptide fragment according to item 2 or the peptide fragment for the use according to any of items 1 , 2 or 4, wherein the peptide fragment comprises at least 10 amino acids, such as at least 20 amino acids, such as at least 30 amino acids, such as at least 40 amino acids, such as at least 50 amino acids, such as at least 60 amino acids, such as at least 70 amino acids, such as at least 80 amino acids, such as at least 90 amino acids, such as at least 100 amino acids, such as at least 110 amino acids, such as at least 120 amino acids, such as at least 130 amino acids, such as at least 140 amino acids, such as at least 150 amino acids, such as at least 160 amino acids, such as at least 170 amino acids, such as at least 180 amino acids, such as at least 190 amino acids, such as at least 200 amino acids.

[0137] 6. The peptide fragment according to any of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment comprises at the most 200 amino acid, such as at the most 190 amino acids, such as at the most 180 amino acids, such as at the most 170 amino acids, such as at the most 160 amino acids, such as at the most 150 amino acids, such as at the most 140 amino acids, such as at the most 130 amino acids, such as at the most 120 amino acids, such as at the most 110 amino acids, such as at the most 100 amino acids, such as at the most 90 P7187PC00 amino acids, such as at the most 80 amino acids, such as at the most 70 amino acids, such as at the most 60 amino acids, such as at the most 50 amino acids, such as at the most 40 amino acids, such as at the most 30 amino acids, such as at the most 20 amino acids, such as at the most 10 amino acids.

[0138] 7. A peptide fragment comprising at least 10 amino acids and at the most 40 amino acids, wherein the peptide fragment comprises a sequence comprising at least one of the following sequences; SEQ ID NO: 1 or SEQ ID NO 3-56 or functional homologues thereof sharing at least 90% sequence identity therewith.

[0139] 8. A peptide fragment comprising at least 10 amino acids and at the most 40 amino acids, wherein the peptide fragment comprises a sequence comprised within at the most 30 amino acids of the C-terminus of a primary protein comprising any one of SEQ ID NO: 60-99.

[0140] 9. A peptide fragment comprising at least 10 amino acids and at the most 40 amino acids, wherein the peptide fragment comprises a sequence comprising at least one of the following sequences; SEQ ID NO: 1 or SEQ ID NO 3-56 or functional homologues thereof sharing at least 90% sequence identity therewith, wherein the peptide fragment comprises a sequence comprised within at the most 30 amino acids of the C-terminus of a primary protein comprising any one of SEQ ID NO: 60-99.

[0141] 10. The peptide fragment according to any of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment comprises a sequence comprising at least one of the following sequences; SEQ ID NO: 1-58 or functional homologues thereof sharing at least 95% sequence identity therewith.

[0142] 11. The peptide fragment according to any of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment comprises a sequence comprising at least one of the following sequences; SEQ ID NO: 1-58 or functional homologues thereof sharing about 100% sequence identity therewith. P7187PC00

[0143] 12. The peptide fragment according to any of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the three dimensional structure of the peptide fragment has a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of a peptide as set forth in any one of SEQ ID NOs: 1-58.

[0144] 13. The peptide fragment according to any of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment comprises at least one of the following sequences; SEQ ID NO: 1 and / or SEQ ID NO: 3-35, or functional homologues thereof preferably sharing at least 90% sequence identity therewith, more preferably sharing at least 95% sequence identity therewith, even more preferably sharing at least 100% sequence identity therewith.

[0145] 14. The peptide fragment according to item 13, wherein the peptide fragment comprises a sequence comprised within at the most 30 amino acids of the C- terminus of a primary protein comprising any one of SEQ ID NO: 60-99.

[0146] 15. The peptide fragment according to any of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment comprises at least one of the following sequences; SEQ ID NO: 1 and / or SEQ ID NO: 3-17, or functional homologues thereof preferably sharing at least 90% sequence identity therewith, more preferably sharing at least 95% sequence identity therewith, even more preferably sharing at least 100% sequence identity therewith.

[0147] 16. The peptide fragment according to item 15, wherein the peptide fragment is negatively charged.

[0148] 17. The peptide fragment according to any of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the P7187PC00 peptide fragment comprises at least one of the following sequences; SEQ ID NO: 18-24 or functional homologues thereof preferably sharing at least 90% sequence identity therewith, more preferably sharing at least 95% sequence identity therewith, even more preferably sharing at least 100% sequence identity therewith.

[0149] 18. The peptide fragment according to item 17, wherein the peptide fragment is non-charged.

[0150] 19. The peptide fragment according to any of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment comprises at least one of the following sequences; SEQ ID NO: 25-35 or functional homologues thereof preferably sharing at least 90% sequence identity therewith, more preferably sharing at least 95% sequence identity therewith, even more preferably sharing at least 100% sequence identity therewith.

[0151] 20. The peptide fragment according to item 19, wherein the peptide fragment is positively charged.

[0152] 21. The peptide fragment according to any of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment comprises at least one of the following sequences; SEQ ID NO: 2 or SEQ ID NO: 36-56 or SEQ ID NO: 58, or functional homologues thereof preferably sharing at least 90% sequence identity therewith, more preferably sharing at least 95% sequence identity therewith, even more preferably sharing at least 100% sequence identity therewith.

[0153] 22. The peptide fragment according to item 21 , wherein the peptide fragment is detected in the plasma of at least 20% of patients suffering from a complement- associated disease, such as SLE.

[0154] 23. The peptide fragment according to any one of items 21-22, wherein the peptide fragment is increased by at least 50% in the plasma of patients suffering from a complement-associated disease compared to the plasma of healthy patients, P7187PC00 such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%.

[0155] 24. The peptide fragment according to any of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment comprises at least one of the following sequences; SEQ ID NO: 2 or SEQ ID NO: 36-42 or SEQ ID NO: 58, preferably sharing at least 90% sequence identity therewith, more preferably sharing at least 95% sequence identity therewith, even more preferably sharing at least 100% sequence identity therewith.

[0156] 25. The peptide fragment according to item 24, wherein the peptide fragment is negatively charged.

[0157] 26. The peptide fragment according to any of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment comprises at least one of the following sequences; SEQ ID NO: 43-45 or functional homologues thereof preferably sharing at least 90% sequence identity therewith, more preferably sharing at least 95% sequence identity therewith, even more preferably sharing at least 100% sequence identity therewith.

[0158] 27. The peptide fragment according to item 26, wherein the peptide fragment is non-charged.

[0159] 28. The peptide fragment according to any of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment comprises at least one of the following sequences; SEQ ID NO: 46-56 or functional homologues thereof preferably sharing at least 90% sequence identity therewith, more preferably sharing at least 95% sequence identity therewith, even more preferably sharing at least 100% sequence identity therewith.

[0160] 29. The peptide fragment according to item 27, wherein the peptide fragment is positively charged. P7187PC00

[0161] 30. The peptide fragment according to any of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment comprises SEQ ID NO: 1 (LHF2) and / or SEQ ID NO: 2 (LHF1) or functional homologues thereof sharing at least 90% sequence identity therewith.

[0162] 31 . The peptide fragment according to any of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment comprises SEQ ID NO: 1 (LHF2) and / or SEQ ID NO: 2 (LHF1) or functional homologues thereof sharing at least 95% sequence identity therewith, such as sharing 100% sequence identity therewith.

[0163] 32. The peptide fragment according to any of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment comprises SEQ ID NO: 1 (LHF2) or functional homologues thereof sharing at least 90% sequence identity therewith.

[0164] 33. The peptide fragment according to any of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment comprises SEQ ID NO: 1 (LHF2) or functional homologues thereof sharing at least 95% sequence identity therewith, such as sharing 100% sequence identity therewith.

[0165] 34. The peptide fragment according to any of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the three dimensional structure of the peptide fragment has a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1 .0, compared to the three dimensional structure of a peptide as set forth in SEQ ID NO: 1.

[0166] 35. The peptide fragment according to any one of items or the peptide fragment for the use according to any of the preceding items, wherein the three structure of P7187PC00 the peptide fragment has a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure defined by the structural coordinates as set forth in Table 1.

[0167] 36. The peptide fragment according to any of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment comprises SEQ ID NO: 2 (LHF1) or functional homologues thereof sharing at least 90% sequence identity therewith.

[0168] 37. The peptide fragment according to any of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment comprises SEQ ID NO: 2 (LHF1) or functional homologues thereof sharing at least 95% sequence identity therewith, such as sharing 100% sequence identity therewith.

[0169] 38. The peptide fragment according to any of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the three dimensional structure of the peptide fragment has a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1 .0, compared to the three dimensional structure of a peptide as set forth in SEQ ID NO: 2. .

[0170] 39. The peptide fragment according to any of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the three dimensional structure of the peptide fragment has a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure defined by the structural coordinates as set forth in Table 2. P7187PC00

[0171] 40. The peptide fragment according to any of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment comprises SEQ ID NO: 58 or functional homologues thereof sharing at least 90% sequence identity therewith.

[0172] 41 . The peptide fragment according to any of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment comprises SEQ ID NO: 58 or functional homologues thereof sharing at least 95% sequence identity therewith, such as sharing 100% sequence identity therewith.

[0173] 42. The peptide fragment according to any of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment comprises at least one of the following sequences; SEQ ID NO: 2 or any one of SEQ ID NO: 36-42 or SEQ ID NO: 58 and comprises a C345C domain of C3, wherein the C345C domain of C3 is as set forth in SEQ ID NO: SEQ ID NO:127.

[0174] 43. The peptide fragment according to any of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment comprises at least one of the following sequences; SEQ ID NO: 2 or any one of SEQ ID NO: 36-42 or SEQ ID NO: 58 and comprises a C345C domain of C3, wherein the C345C domain of C3 comprises a sequence comprising or consisting of amino acid positions 1518-1663 of C3 (SEQ ID NO: 126).

[0175] 44. The peptide fragment according to any one of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment further comprises a modification.

[0176] 45. The peptide fragment according to item 44, wherein the modification comprises incorporation of non-natural amino acids or D-amino acids, cyclization, stapling, conjugation to a biomolecule, chelation, lipidation, N-terminal acetylation, C- terminal amidation, PEGylation and / or glycosylation. P7187PC00

[0177] 46. The peptide fragment according to any one of items 44-45, wherein the modification comprises conjugation to a biomolecule, such as biotin, such as an amino acid, such as an un-natural amino acid, such as a peptide, such as a protein, such as a carbohydrate, such as a mono saccharide, such as a disaccharide, such as a disaccharide, such as a polysaccharide, such as a lipid, such as a fatty acid, such as a saturated fatty acid, such as a unsaturated fatty acid, such as a triglyceride, such as a steroid, such as a PEG, such as a fluorophore.

[0178] 47. The peptide fragment according to any one of items 44-46, wherein the modification comprises conjugation to a chemical tag, such as a chemical tag, such as a protein tag, such as fluorescent tag, such as an isotopic tag.

[0179] 48. The peptide fragment according to any one of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment is conjugated to a carrier molecule that enhances cellular uptake.

[0180] 49. The peptide fragment according to item 48, wherein the carrier molecule is selected from the group consisting of liposomes, nanoparticles, and polymer conjugates.

[0181] 50. The peptide fragment according to any of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment is capable of modulating complement activity.

[0182] 51 . The peptide fragment according to any one of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment modulates the activity of the complement system.

[0183] 52. The peptide fragment according to item 50, wherein modulating complement activity is measured using one or more of the following techniques: enzyme- linked immunosorbent assays (ELISA), time-resolved immunofluorometric assays (TRIFMA and / or DELFI A), liquid chromatography-mass spectrometry P7187PC00

[0184] (LC / MS), haemolytic assays, flow cytometry, surface plasmon resonance, immunoassay, immunofluorescence microscopy, western blot, and / or RT-PCR.

[0185] 53. The peptide fragment according to any one of items 50-52, wherein modulating complement activity comprises modulation of the expression and / or activity and / or function of the classical complement pathway, the alternative complement pathway and / or the mannose-binding lectin (MBL) pathway or components thereof.

[0186] 54. The peptide fragment according to any of items 50-53, wherein modulating complement activity comprises modulation of the expression and / or activity of any one or more of the following: the C1 complex, C1 q, C1 r, C1 s, C2, C2a, C2b, C4, C4a, C4b, C3, C3a, C3b, C3c, C3d, C3 convertase, C5, C5a, C5b, C5 convertase, C6, C7, C8, C9, the membrane attack complex (MAC), factor B, factor D, factor H, factor I, factor P (properdin), MASP-1 , MASP-2, MASP-3, C4b-binding protein (C4BP), complement receptor 1 (CR1), complement receptor 2 (CR2), complement receptor 3 (CR3), complement receptor 4 (CR4), decay-accelerating factor (DAF), CD59, IL6ST (gp130), JAK3, STAT3 and / or any fragments or subunits thereof.

[0187] 55. The peptide fragment according to any one of items 50-54, wherein modulating complement activity comprises inhibition of the expression and / or activity and / or function of; the classical complement pathway, the alternative complement pathway and / or the mannose-binding lectin (MBL) pathway, or components thereof.

[0188] 56. The peptide fragment according to any one of items 50-54, wherein modulating complement activity comprises inhibition of the expression and / or activity and / or function of; the classical complement pathway, and / or the mannose-binding lectin (MBL) pathway, or components thereof.

[0189] 57. The peptide fragment according to any one of items 50-55, wherein the peptide fragment is capable of modulating complement activity, wherein modulating complement activity comprises inhibition of the mannose-binding lectin (MBL) pathway or components thereof. P7187PC00

[0190] 58. The peptide fragment according to any one of items 50-57, wherein the peptide fragment inhibits the mannose-binding lectin (MBL) pathway or components thereof by at least 5% compared with vehicle, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99%.

[0191] 59. The peptide fragment according to any one of items 32-35 and 50-58, wherein the peptide fragment inhibits the mannose-binding lectin (MBL) pathway or components thereof by at least 5% compared with vehicle, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99%.

[0192] 60. The peptide fragment according to any one of items 50-55, wherein the peptide fragment is capable of modulating complement activity, wherein modulating complement activity comprises inhibition of the classical complement pathway or components thereof.

[0193] 61 . The peptide fragment according to any one of items 50-55 and 60, wherein the peptide fragment inhibits the classical complement pathway or components thereof by at least 5% compared with vehicle, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99%. P7187PC00

[0194] 62. The peptide fragment according to any one of items 32-35, 50-55 and 60, wherein the peptide fragment inhibits the classical complement pathway or components thereof by at least 5% compared with vehicle, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99%.

[0195] 63. The peptide fragment according to any one of items 36-39, 50-55 and 60, wherein the peptide fragment inhibits the classical complement pathway or components thereof by at least 5% compared with vehicle, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99%.

[0196] 64. The peptide fragment according to item 55, wherein the peptide fragment is capable of modulating complement activity, wherein modulating complement activity comprises inhibition of the alternative pathway or components thereof.

[0197] 65. The peptide fragment according to any one of items 50-55 or 64, wherein the peptide fragment inhibits the alternative pathway or components thereof by at least 5% compared with vehicle, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99%.

[0198] 66. The peptide fragment according to any one of items 32-35, 50-55 and 60, wherein the peptide fragment inhibits the classical complement pathway or P7187PC00 components thereof by at least 5% compared with vehicle, such as at least 10%, such as at least 15%, such as at least 20%, such as at least 25%, such as at least 30%, such as at least 35%, such as at least 40%, such as at least 45%, such as at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 99%.

[0199] 67. The peptide fragment according to any one of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment comprises complement N-termini, which has been generated by proteolytic processing of a primary plasma peptide.

[0200] 68. The peptide fragment according to item 67, wherein the primary plasma peptide is selected from the group consisting of SEQ ID NO: 60-99.

[0201] 69. The peptide fragment according to any one of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment is naturally found in the plasma of a patient suffering from a complement-mediated disorder.

[0202] 70. The peptide fragment according to any one of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment is part of the complement system.

[0203] 71 . The peptide fragment according to any one of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment is part of the N-terminome of a subject.

[0204] 72. The peptide fragment according to any one of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment is part of the N-terminome of a patient suffering from a complement-mediated disease. P7187PC00

[0205] 73. The peptide fragment according to any one of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment is part of the N-terminome of a patient suffering from a complement-mediated disease selected from the group comprising Systemic Lupus Erythematosus (SLE), lupus nephritis, inflammatory kidney disease, cytokine-driven nephropathy, acute kidney disease (CKD), chronic kidney disease (CKD), and kidney diseases associated with dysregulated IL6, IL6ST or JAK-STAT signalling, Hemolytic Uremic Syndrome (HUS), Atypical Hemolytic Uremic Syndrome (aHUS), Paroxysmal Nocturnal Hemoglobinuria (PNH), Age- Related Macular Degeneration (AMD), C3 Glomerulopathy, Dense Deposit Disease (DDD), Rheumatoid Arthritis (RA), Membranoproliferative Glomerulonephritis (MPGN), Neuromyelitis Optica (NMO), Myasthenia Gravis (MG), Cryoglobulinemia, Autoimmune Hemolytic Anemia (AIHA), Vasculitis, Ankylosing Spondylitis, Cold Agglutinin Disease, Immune Complex Glomerulonephritis, Glomerulonephritis, IgA nephropathy (IgAN), Sepsis, Meningococcal Disease, Recurrent Bacterial Infections, Alzheimer’s Disease, Multiple Sclerosis, Uveitis, Complement Component Deficiencies such as C2, C3, and / or C4 deficiencies, Hereditary Angioedema (HAE), Atherosclerosis, Myocardial Infarction (Ml), Dermatitis Herpetiformis, Bullous Pemphigoid, Ischemia-Reperfusion Injury, Transplant Rejection, and Idiopathic Thrombocytopenic Purpura (ITP).

[0206] 74. The peptide fragment according to any one of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment is part of the N-terminome of a patient suffering from a complement-mediated disease selected from the group comprising Systemic Lupus Erythematosus (SLE), lupus nephritis, inflammatory kidney disease, cytokine-driven nephropathy, acute kidney disease (CKD), chronic kidney disease (CKD), Atypical Hemolytic Uremic Syndrome (aHUS), Paroxysmal Nocturnal Hemoglobinuria (PNH), Age-Related Macular Degeneration (AMD), C3 Glomerulopathy, Dense Deposit Disease (DDD), Rheumatoid Arthritis (RA), Glomerulonephritis, Membranoproliferative Glomerulonephritis (MPGN), IgA nephropathy (IgAN), Neuromyelitis Optica (NMO), Myasthenia Gravis (MG), Cryoglobulinemia and / or Autoimmune Hemolytic Anemia (AIHA). P7187PC00

[0207] 75. The peptide fragment according to any one of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment is part of the N-terminome of a patient suffering from systemic Lupus Erythematosus (SLE).

[0208] 76. The peptide fragment according to any one of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment is part of the N-terminome of a patient suffering from Hemolytic Uremic Syndrome (HUS) or Atypical Hemolytic Uremic Syndrome (aHUS).

[0209] 77. The peptide fragment according to any one of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment is part of the N-terminome of a patient suffering from lupus nephritis.

[0210] 78. The peptide fragment according to any one of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment is part of the N-terminome of a patient suffering from Glomerulonephritis.

[0211] 79. A pharmaceutical composition comprising the peptide fragment according to any of the preceding items or a pharmaceutical composition comprising the peptide fragment according to any of the preceding items for use according to any one of the preceding items, wherein the composition further comprises one or more pharmaceutically acceptable excipients.

[0212] 80. The pharmaceutical composition according to item 79, wherein the pharmaceutical composition is formulated for delivery via oral, intravenous, subcutaneous, or intramuscular administration.

[0213] 81. The pharmaceutical composition according to item 79, wherein the pharmaceutical composition is formulated for oral administration, injection, inhalation, local administration, rectal administration, nasal administration, P7187PC00 buccal administration, vaginal administration, or through an implanted drug reservoir.

[0214] 82. The peptide fragment or pharmaceutical composition for use according to any of the preceding items, wherein the complement-mediated disorders is selected from the group comprising Systemic Lupus Erythematosus (SLE), lupus nephritis, inflammatory kidney disease, cytokine-driven nephropathy, acute kidney disease (CKD), chronic kidney disease (CKD), Hemolytic Uremic Syndrome (HUS), Atypical Hemolytic Uremic Syndrome (aHUS), Paroxysmal Nocturnal Hemoglobinuria (PNH), Age-Related Macular Degeneration (AMD), C3 Glomerulopathy, Dense Deposit Disease (DDD), Rheumatoid Arthritis (RA), Glomerulonephritis, Membranoproliferative Glomerulonephritis (MPGN), IgA nephropathy (IgAN), Neuromyelitis Optica (NMO), Myasthenia Gravis (MG), Cryoglobulinemia, Autoimmune Hemolytic Anemia (AIHA), Vasculitis, Ankylosing Spondylitis, Cold Agglutinin Disease, Immune Complex Glomerulonephritis, Sepsis, Meningococcal Disease, Recurrent Bacterial Infections, Alzheimer’s Disease, Multiple Sclerosis, Uveitis, Complement Component Deficiencies such as C2, C3, and / or C4 deficiencies, Hereditary Angioedema (HAE), Atherosclerosis, Myocardial Infarction (Ml), Dermatitis Herpetiformis, Bullous Pemphigoid, Ischemia-Reperfusion Injury, Transplant Rejection, Idiopathic Thrombocytopenic Purpura (ITP), juvenile idiopathic arthritis, giant cell arteritis, adult-onset Still’s disease, Sjogren’s syndrome, systemic sclerosis, psoriatic arthritis, Castleman’s disease, multiple myeloma, cytokine release syndrome, haemophagocytic lymphohistiocytosis, complement-mediated glomerulopathy, acute kidney injury associated with systemic inflammation, sepsis-associated cytokine storm, and COVID-19- associated hyperinflammation.

[0215] 83. The peptide fragment or pharmaceutical composition for use according to any of the preceding items, wherein the complement-mediated disorders is selected from the group comprising Systemic Lupus Erythematosus (SLE), lupus nephritis, inflammatory kidney disease, cytokine-driven nephropathy, acute kidney disease (CKD), chronic kidney disease (CKD), Hemolytic Uremic Syndrome (HUS), Atypical Hemolytic Uremic Syndrome (aHUS), Paroxysmal Nocturnal Hemoglobinuria (PNH), Age-Related Macular Degeneration (AMD), P7187PC00

[0216] C3 Glomerulopathy, Dense Deposit Disease (DDD), Rheumatoid Arthritis (RA), Glomerulonephritis, IgA nephropathy (IgAN), Membranoproliferative Glomerulonephritis (MPGN), Neuromyelitis Optica (NMO), Myasthenia Gravis (MG), Cryoglobulinemia and / or Autoimmune Hemolytic Anemia (AIHA).

[0217] 84. The peptide fragment or pharmaceutical composition for use according to any of the preceding items in a method of treatment of complement-mediated disorders, wherein the complement-mediated disorders is systemic Lupus Erythematosus (SLE).

[0218] 85. The peptide fragment or pharmaceutical composition for use according to any of the preceding items in a method of treatment of complement-mediated disorders, wherein the complement-mediated disorders is Hemolytic Uremic Syndrome (HUS) or Atypical Hemolytic Uremic Syndrome (aHUS).

[0219] 86. The peptide fragment or pharmaceutical composition for use according to any of the preceding items in a method of treatment of complement-mediated disorders, wherein the complement-mediated disorders is lupus nephritis.

[0220] 87. The peptide fragment or pharmaceutical composition for use according to any of the preceding items in a method of treatment of complement-mediated disorders, wherein the complement-mediated disorders is Glomerulonephritis.

[0221] 88. The peptide fragment or pharmaceutical composition for use according to any of the preceding items, wherein the peptide fragment is formulated for administration by oral administration, injection, inhalation, local administration, rectal administration, nasal administration, buccal administration, vaginal administration, or through an implanted drug reservoir.

[0222] 89. The peptide fragment or pharmaceutical composition for use according to any one of the preceding items, wherein the method comprises administration of at least one additional therapeutic agent effective in treating complement- mediated disorders. P7187PC00

[0223] 90. The peptide fragment of item 89, wherein the other therapeutic agent is an inhibitor of a complement pathway component.

[0224] 91. A method of treatment of a complement-mediated disease comprising administration of a therapeutically effective amount of the peptide fragment of any of the preceding items to an subject in need thereof.

[0225] 92. An in vitro method of treatment of a complement-mediated disease comprising administration of a therapeutically effective amount of the peptide fragment of any of the preceding items to an subject in need thereof.

[0226] 93. A method of modulating the complement system, said method comprising: a. Providing a composition comprising a human complement factor, a regulator of the complement system, or a receptor involved in complement activity selected from the group comprising the C1 complex, C1q, C1r, C1s, C2, C2a, C2b, C4, C4a, C4b, C3, C3a, C3b, C3c, C3d, C3 convertase, C5, C5a, C5b, C5 convertase, C6, C7, C8, C9, the membrane attack complex (MAC), factor B, factor D, factor H, factor I, factor P (properdin), MASP-1 , MASP-2, MASP-3, C4b-binding protein (C4BP), complement receptor 1 (CR1), complement receptor 2 (CR2), complement receptor 3 (CR3), complement receptor 4 (CR4), decay-accelerating factor (DAF), CD59, and / or any fragments, proteolytic derivatives or subunits thereof, b. Contacting said composition with the peptide fragment of any one of the preceding claims.

[0227] 94. An in vitro method of modulating the complement system, said method comprising: a. Providing a composition comprising a human complement factor, a regulator of the complement system, or a receptor involved in complement activity selected from the group comprising the C1 complex, C1q, C1r, C1s, C2, C2a, C2b, C4, C4a, C4b, C3, C3a, C3b, C3c, C3d, C3 convertase, C5, C5a, C5b, C5 convertase, C6, C7, C8, C9, the membrane attack complex (MAC), factor B, factor D, factor H, factor I, factor P (properdin), MASP-1 , MASP-2, MASP-3, C4b-binding P7187PC00 protein (C4BP), complement receptor 1 (CR1), complement receptor 2 (CR2), complement receptor 3 (CR3), complement receptor 4 (CR4), decay-accelerating factor (DAF), CD59, and / or any fragments, proteolytic derivatives or subunits thereof, b. Contacting said composition with the peptide fragment of any one of the preceding claims.

[0228] 95. The method according to item 93, wherein said composition is serum, plasma, blood or cerebrospinal fluid.

[0229] 96. The method according to items 93-95, wherein the method is an in vitro method.

[0230] 97. The method according to items 93-95, wherein the method is an in vivo method.

[0231] 98. The peptide fragment according to any one of the preceding items or the peptide fragment for the use according to any of the preceding items wherein the peptide fragment modulates the activity of the classical complement pathway, the alternative complement pathway and / or the mannose-binding lectin (MBL) pathway or components thereof, and modulates IL6ST mediated signalling.

[0232] 99. The peptide fragment according to any one of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment acts as a partial agonist of IL6ST and induces STAT3 activation.

[0233] 100. The peptide fragment according to any one of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment induces STAT3 activation.

[0234] 101. The peptide fragment according to any one of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment modulates CXCL10 secretion, STI NG 1 upregulation, and / or phosphorylation of STAT3 at Y705, IL6ST at Y814, or nephrin at Y1176. P7187PC00

[0235] 102. The peptide fragment according to any one of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein the peptide fragment causes a reduction in the expression of podocyte markers, including NPHS1 (nephrin), NPHS2 (podocin), PODXL (podocalyxin), and PTPRO.

[0236] 103. The peptide fragment according to any one of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein modulation of IL6ST mediated signalling by the peptide fragment occurs independently of IL6Ra engagement.

[0237] 104. The peptide fragment according to any one of the preceding items or the peptide fragment for the use according to any of the preceding items, wherein modulation of IL6ST mediated signalling by the peptide fragment occurs via one or more alternative co-receptors other than IL6Ra.

[0238] 105. The peptide fragment or pharmaceutical composition according to any one of the preceding items, for use in the treatment or prevention of a complement-mediated disease associated with abnormal IL-6 signalling.

[0239] 106. The peptide fragment or pharmaceutical composition according to any one of the preceding items, for use in the treatment or prevention of a complement-mediated disease or disorder associated with abnormal IL-6 signalling, wherein the disease or disorder is selected from the group comprising rheumatoid arthritis, juvenile idiopathic arthritis, giant cell arteritis, adult-onset Still’s disease, systemic lupus erythematosus, lupus nephritis, Sjogren’s syndrome, systemic sclerosis, vasculitis, psoriatic arthritis, ankylosing spondylitis, Castleman’s disease, multiple myeloma, cytokine release syndrome, haemophagocytic lymphohistiocytosis, complement-mediated glomerulopathy, membranoproliferative glomerulonephritis, IgA nephropathy, acute kidney injury associated with systemic inflammation, sepsis-associated cytokine storm, or COVID-19-associated hyperinflammation. P7187PC00

[0240] 107. The peptide fragment or pharmaceutical composition according to any one of the preceding items, for use in the treatment or prevention of a complement-mediated disease associated with abnormal IL-6 signalling, wherein said disorder is characterised by dysregulation of IL6ST (gp130) mediated signalling.

[0241] 108. The peptide fragment or pharmaceutical composition according to any one of the preceding items, for use in a method of treating or preventing a renal disorder associated with IL6ST (gp130) signalling, wherein said renal disorder is selected from the group comprising: inflammatory kidney disease, lupus nephritis, complement-mediated glomerulopathy, proteinuric kidney disease, acute kidney disease and chronic kidney disease.

[0242] 109. A method of treating a complement-mediated disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptide fragment according to any one of items 1-81.

[0243] 110. The method of item 109, wherein the complement-mediated disorder is selected from the group comprising Systemic Lupus Erythematosus (SLE), lupus nephritis, inflammatory kidney disease, cytokine-driven nephropathy, acute kidney disease (CKD), chronic kidney disease (CKD), Hemolytic Uremic Syndrome (HUS), Atypical Hemolytic Uremic Syndrome (aHUS), Paroxysmal Nocturnal Hemoglobinuria (PNH), Age-Related Macular Degeneration (AMD), C3 Glomerulopathy, Dense Deposit Disease (DDD), Rheumatoid Arthritis (RA), Glomerulonephritis, Membranoproliferative Glomerulonephritis (MPGN), IgA nephropathy (IgAN), Neuromyelitis Optica (NMO), Myasthenia Gravis (MG), Cryoglobulinemia, Autoimmune Hemolytic Anemia (AIHA), Vasculitis, Ankylosing Spondylitis, Cold Agglutinin Disease, Immune Complex Glomerulonephritis, Sepsis, Meningococcal Disease, Recurrent Bacterial Infections, Alzheimer’s Disease, Multiple Sclerosis, Uveitis, Complement Component Deficiencies such as C2, C3, and / or C4 deficiencies, Hereditary Angioedema (HAE), Atherosclerosis, Myocardial Infarction (Ml), Dermatitis Herpetiformis, Bullous Pemphigoid, Ischemia-Reperfusion Injury, Transplant Rejection, Idiopathic Thrombocytopenic Purpura (ITP), juvenile idiopathic P7187PC00 arthritis, giant cell arteritis, adult-onset Still’s disease, Sjogren’s syndrome, systemic sclerosis, psoriatic arthritis, Castleman’s disease, multiple myeloma, cytokine release syndrome, haemophagocytic lymphohistiocytosis, complement-mediated glomerulopathy, acute kidney injury associated with systemic inflammation, sepsis-associated cytokine storm, and COVID-19- associated hyperinflammation.

[0244] 111. The method of any one of items 108 and 110, wherein the complement- mediated disorder is selected from the group comprising SLE, lupus nephritis, inflammatory kidney disease, cytokine-driven nephropathy, acute kidney disease (CKD), chronic kidney disease (CKD), HUS, aHUS, PNH, AMD, C3 Glomerulopathy, DDD, RA, Glomerulonephritis, MPGN, IgAN, NMO, MG, Cryoglobulinemia, and AIHA.

[0245] 112. The method of any one of items 109-111 , wherein the complement- mediated disorder is systemic Lupus Erythematosus (SLE).

[0246] 113. The method of any one of items 109-111 , wherein the complement- mediated disorder is Hemolytic Uremic Syndrome (HUS) or Atypical Hemolytic Uremic Syndrome (aHUS).

[0247] 114. The method of any one of items 109-111 , wherein the complement- mediated disorder is lupus nephritis.

[0248] 115. The method of any one of items 109-111 , wherein the complement- mediated disorder is Glomerulonephritis.

[0249] 116. The method of any one of items 109-115, wherein the peptide fragment is formulated for administration by oral administration, injection, inhalation, local administration, rectal administration, nasal administration, buccal administration, vaginal administration, or through an implanted drug reservoir.

[0250] 117. The method of any one of items 109-116, wherein the method further comprises administration of at least one additional therapeutic agent effective in treating complement-mediated disorders. P7187PC00

[0251] 118. The method of item 117, wherein the other therapeutic agent is an inhibitor of a complement pathway component.

[0252] 119. A method of treating or preventing a complement-mediated disease associated with abnormal IL-6 signalling in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptide fragment or pharmaceutical composition according to any one of items 1-81.

[0253] 120. The method of item 119, wherein the disease or disorder is selected from the group comprising rheumatoid arthritis, juvenile idiopathic arthritis, giant cell arteritis, adult-onset Still’s disease, systemic lupus erythematosus, lupus nephritis, Sjogren’s syndrome, systemic sclerosis, vasculitis, psoriatic arthritis, ankylosing spondylitis, Castleman’s disease, multiple myeloma, cytokine release syndrome, haemophagocytic lymphohistiocytosis, complement- mediated glomerulopathy, membranoproliferative glomerulonephritis, IgA nephropathy, acute kidney injury associated with systemic inflammation, sepsis- associated cytokine storm, or COVID-19-associated hyperinflammation.

[0254] 121. The method of any one of items 119-120, wherein said disorder is characterised by dysregulation of IL6ST (gp130) mediated signalling.

[0255] 122. A method of treating or preventing a renal disorder associated with IL6ST (gp130) signalling in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptide fragment or pharmaceutical composition according to any one of items 1-80, wherein said renal disorder is selected from the group comprising: inflammatory kidney disease, lupus nephritis, complement-mediated glomerulopathy, proteinuric kidney disease, acute kidney disease and chronic kidney disease.

[0256] Examples P7187PC00

[0257] Example 1 - Development of an optimised workflow (SHUNTER) to assess proteolytic events in human plasma

[0258] Aim

[0259] To improve existing methods of enriching protein cleavages and peptides with protease-induced N-termini, to facilitate investigation of the complex proteolytic modification of proteins in human plasma.

[0260] Methods

[0261] The High-efficiency Undecanal-based N Termini EnRichment (HUNTER) protocol (Weng et al., 2019) was optimised through a systematic iteration of incubation and labelling conditions, to develop the Short High efficiency Undecanal-based N-termini EnRichment (SHUNTER) protocol.

[0262] SHUNTER method development: The established High-efficiency Undecanal-based N-Termini EnRichment (HUNTER) method for N-termini enrichment was optimised to be faster and more efficient. Starting from the HUNTER protocol (Weng et al., 2019), a label-free “simplex” approach was established to expand the N-termini methodology to larger, cohort-sized scales.

[0263] Previously, differential treatments or groups were isotopically labelled with distinct formaldehyde variants on protein-level (Fig. 1a). Labelling reactions were performed at 37 °C during this workflow for long timespans (1h for each labelling round). At first, a single formaldehyde labelling strategy was applied, where in vivo protein N-termini and lysines were labelled with13CD2O to discriminate them from tryptically-generated peptide N-termini later during the trypsin digestion step. Applying only one formaldehyde variant enables “label-free-like” unpaired N-terminomics.

[0264] Within the improved, faster SHUNTER workflow (Fig. 1a), the labelling reactions were reduced to 5 minutes at 95 °C without compromising labelling efficiency (Fig. 1b). Additionally, the switch to SP3 bead-based peptide cleanup improved yields and led to faster turn-around times, avoiding potential losses due to the previous second vacuum concentration step after C18 StageTip purification (Fig. 1a, last step). P7187PC00

[0265] Liquid chromatography-mass spectrometry (LC-MS): Samples were measured on a Thermo Ultimate 3000 nanoHPLC system coupled online to an Exploris480 mass spectrometer. A total of 1 g of purified N-terminal peptides were separated in a 60- minute total runtime binary LC gradient on a two-column setup with a Thermo PepMap 100 Cis (#164535) pre-column and an Aurora Ultimate 25 cm column (#AUR3- 25075C18). For most SHUNTER samples, data-independent acquisition (DIA) was used. The DIA gradient ranged from 5-25% B (A: H2O + 0.1% FA, B: ACN + 0.1 %FA) for 35 minutes at 280 nL / min, heated at 50 °C. DIA MS acquisition was performed with the FAIMS Pro device (Standard Resolution mode with 3.8 L / minute gas flow) at two alternating compensation voltages of -40 and -61V throughout the total runtime of 60 minutes with MS1 acquisition being operated in positive profile mode at a resolution of 120 000 in the scan range of 350-1350 m / z and a normalized AGC target of 300% (RF Lens 40%). DIA MS2 data was recorded in positive profile mode at a resolution of 30 000 with 40 windows from 350-1350 m / z, 25 m / z each and a normalized AGC target of 300% (RF Lens 50%). Preferred charge states were set to 2-6. Parts of the runs, especially from the method development period, were performed in data-dependent mode (DDA) on the same setup.

[0266] The LC / MS methodology was optimised to improve the depth of the N-terminome and enable high-throughput analysis of our cohorts. Beginning with long-gradient separations of 150 minutes for data-dependent acquisition (DDA; datasets l-lll), the LC gradient was optimised to only 60 minutes with comparable depth as for the 150- minute gradient in combination with data-independent acquisition (DIA; dataset IV). Significant improvements in identification rates, even with reduced starting proteome amounts, were observed.

[0267] Data analysis: DDA and DIA runs were recorded and analyzed by FragPipe v20.0 and the built-in DiaNN v1.8.1beta258 to validate the improved SHUNTER workflow (Fig. 1a). To determine the labelling efficiency for the dimethyl labelling and the pullout efficiency for the undecanal tagging of the tryptic peptides, searches were performed a) with trypsin specificity and the variable dimethylation on lysines (+34.0631 Da for 13CD2O / NaBH3CN) for the preSHUNTER samples or b) with ArgC specificity and variable acetylation (+42.0210), dimethylation (+34.0631) and pyro-Glu formation (- 17.0265 on Q / C or -18.0106 on E) on N-termini, fixed dimethylation on lysines P7187PC00

[0268] (+34.0631 Da), carbamidomethylation on cysteines (+57.02146 Da) and variable oxidations on methionine (+15.9949 Da).

[0269] The efficiency of dimethylation on lysines was determined by comparing the percentage of labelled lysines to all lysines in the corresponding peptides containing lysine in each replicate. The pullout efficiency was defined as the percentage of labelled / protected protein N-termini compared to all identified N-termini, which do not feature any N-terminal modification. To assess the degree of protein modifications generated by the old HUNTER or the new SHUNTER protocol, the open search strategy was applied in FragPipe59,60. In brief, due to the large search windows of - 150 to 500 Da, any potential modification could be detected in these searches, and identified putative modifications are analyzed with PTMShepherd61 to determine the most abundant modifications and their putative localization to distinct amino acids. The information herein can be used to monitor the degree of undesired side reactions or biases in the methods HUNTER vs. SHUNTER.

[0270] Results

[0271] The High-efficiency Undecanal-based N Termini EnRichment (HUNTER) protocol (Weng et al., 2019) was optimised through a systematic iteration of incubation and labelling conditions (Fig. 1a). Protease-generated protein N-termini were retrieved as dimethylated lysine residues after depletion of all free amine groups. The dimethylation efficiency on lysines, purity of N-termini in the sample, and the number of totally identified N-termini were markedly increased by the SHUNTER protocol (Fig. 1b). The SHUNTER protocol demonstrated overlap of N-termini () with less acetylated protein N- termini ().

[0272] Both data-independent (DIA) and particularly data-dependent acquisition (DDA) of N- termini showed markedly increased numbers of identifications (1.28-fold for DIA and > 3-fold for DDA) compared to previously published HUNTER protocols (Weng et al., 2019,). An open search of mass spectra data for modifications revealed a reduction of losses with the mass of -57.021 Da, most likely corresponding to an improved carbamidoylation in the new protocol (). Furthermore, unwanted side-reactions (lysine acetylation, guanidination), as well as a side-chain addition of 7.96 Da were reduced with the SHUNTER protocol. P7187PC00

[0273] Conclusion

[0274] The optimised SHUNTER increased the depth and purity of N-termini preparation for maximizing proteolysis detection in human plasma, compared with the HUNTER protocol (Weng et al., 2019).

[0275] Example 2 - Identification of human proteolytic patterns in haemolytic uremic syndrome patients

[0276] Aim

[0277] To apply the optimised SHUNTER workflow to a complement system-associated clinically relevant setting, for the investigation of the complex proteolytic modification of proteins in human plasma.

[0278] Methods

[0279] The SHUNTER workflow and LC-MS methods as described in Example 1 were utilised. Plasma samples from patients, which experienced an episode of hemolytic uremic syndrome, induced by Shiga toxin producing Escherichia coli (STEC-HUS) (Obrig et al., 2012) were utilised. Plasma was obtained at baseline (day 1, defined as active disease) before, as well as day 8 and 30 after Eculizumab treatment (300 mg on day one and day eight), an inhibitor of complement protease C5 (Fig. 2a).

[0280] Eculizumab treatment of patients and plasma collection: As part of the ECUSTEC trial (EudraCT Number: 2016-000997-39), UK pediatric patients presenting with Shiga Toxin producing Escherichia Coli (STEC) Hemolytic Uremic Syndrome (HUS) (STEC HUS) were given Eculizumab at Day 1 and Day 8 (or a placebo) following hospital admission. Blood and urine samples were collected up to 1 month post admission, alongside clinical data. Prior to inclusion, patients provided written informed consent. The trial was conducted in accordance with the principles of the Helsinki Declaration and was approved by North East - Newcastle and North Tyneside 1 Research Ethics Committee. The EDTA-plasma samples had never been thawed before.

[0281] Structural mapping: Mutations were mapped onto the structures of native C3 from the Protein Data Bank (PDB) entry 6ru5, C3b (entry 6ehg), C4 (entry 4jpm), C4b (6ysg) and C8a,b,c (entry 3ojy) using pymol. P7187PC00

[0282] Results

[0283] In total, 4789 protein N-termini were identified, with 476 N-termini stemming from 130 gene products significantly regulated (|log2FC| > 1 & p-value < 0.05) by C5 inhibition (Fig. 2B & 2C).

[0284] In all three patients, Eculizumab caused an early increase in the N-terminus of C5 at 11381 , followed by a reduction of C3, C9, and C4 termini (Fig. 2D), suggesting that there was a reduction and feedback loop of C5 inhibition (Fig. 2B-2D).

[0285] Structural mapping of the 11381 cleavage site on C5 allocated it proximal to flexible loop in the MG8 domain and the ANA domain released by C5 convertase, suggesting that this proteolysis may lead to partial unfolding of the MG8 domain.

[0286] Total proteins assessed as #+the input to the SHUNTER workflow showed differential regulation compared to the N-termini quantified by MS / MS (Fig. 2E & 2F). Only 20 out of the total 314 identified N-termini from the complement system corresponded to previously described fragments. Thus, the workflow enabled the discovery of undescribed proteolysis products and signals involving complement proteins.

[0287] Data annotation - Proteolytic targets of major complement proteases

[0288] To annotate this novel type of data, a library of in vitro targets of the four major regulators and initiating proteases of the human complement system was generated: MASP-1 for the lectin, MASP-3 for the alternative, and C1 r and C1 s for the classical pathway.

[0289] Heat-treated plasma from four healthy donors was sampled and subsequently incubated with purified active versions of the recombinant proteases (Fig. 3A). Statistical criteria for significance were used (Iog2 fold change >1, moderated p-value < 0.05) to define protease-generated Neo-N-termini. Following this process, 7.5% to 10.4% of all protease-generated serum protein N-terminal fragments were from complement proteins (Fig. 3B).

[0290] All four complement proteases resulted in new fragments of C3, with MASP-3 being the most active towards C3 (Fig. 3C). 01 s and 01 r both resulted in several auto-digestion cleavages. Complement Factor H (CFH) was split following incubation with 01 s and MASP-3, whereas 05 was cut following incubation with active MASP-1. Position- weighted matrices demonstrated the motif of the N-termini of the increased stable P7187PC00 cleavage sites, indicating protease preference (Fig. 3D). MASP-1 cleaved after residues K / R, supporting its known basic preference. C1r had a less clear spectrum. MASP-3 was also directed towards basic residues; however, it was in conjunction with acidic residues in proximity to the cleavage site. The E-directed cleavage site of MASP- 3, and the motifs of the other proteases were confirmed using a second protein-N- termini recovery strategy with GluC as a sample preparation enzyme (Fig. 2G-2I). The generated proteolytic fragments in a network with the four active proteases were visualised.

[0291] Conclusion

[0292] The SHUNTER workflow enabled the discovery of undescribed proteolysis products and signals involving complement proteins. The analysis revealed known but also novel potential protease-substrate relationships on human complement proteins (Fig. 3E). Proteolytic targets of major complement proteases were annotated.

[0293] Example 3 - Identification of human proteolytic patterns in systemic lupus erythematosus (SLE)

[0294] Aim

[0295] To investigate the complex proteolytic modification of proteins in the plasma of patients with systemic lupus erythematosus.

[0296] Methods

[0297] The SHUNTER workflow and LC-MS methods as described in Example 1 were utilised. Sample preparation methods were utilised as described in Example 2.

[0298] Human SLE cohort: Patients diagnosed with SLE and under the care of the outpatient clinic at the Department of Rheumatology, Aarhus University Hospital (AUH), were consecutively enrolled between 2015 and 2017. Detailed inclusion and exclusion criteria and comprehensive clinical data have been previously outlined (Troldborg et al., 2018). Prior to inclusion, patients provided written informed consent. The study was conducted in accordance with the principles of the Helsinki Declaration. It was approved by both the Danish Data Protection Agency and the Central Denmark Region Committees on Health Research Ethics (approval number: #1-10-72-214-13). The P7187PC00 biobanked EDTA-plasma samples had been stored at -80 °C and had never been thawed.

[0299] N-termini sample preparation: Human EDTA-plasma samples were processed in a semi-automated manner on an Opentrons OT-2 pipetting robot (Opentrons, NYC, USA) from a SLE cohort consisting of 143 SLE patients from the AUH SLE cohort and age- matched healthy control subjects (n=23). In brief, previously unthawed EDTA plasma was diluted 1 :8 with 4% SDS / 0.1 M HEPES pH 7.4 / 5 mM EDTA, supplemented with Roche complete protease inhibitor cocktail (1x), and was denatured for 5 minutes at 95 °C. Subsequently, the protein concentration was determined by the Pierce bicinchoninic acid (BCA) protein assay (Thermo Fisher) in a 1 :10 dilution and 300 pg of 1 :10 diluted human EDTA-plasma was processed as follows: cysteines were carbamidomethylated with 10 mM tris(2-carboxyethyl)phosphine) (TCEP) and 50 mM chloroacetamide (CAA) for 5 minutes at 95°C. Excess CAA was quenched with a second round of 10 mM TCEP for another 5 minutes at 95 °C. Proteins were purified using SP3 paramagnetic beads (hydrophobic) in the final 80% ethanol (EtOH) for 10 minutes and washed two times with 200 pL acetonitrile (ACN) 90 %. After resuspension in 100 pL 4%SDS / 0.1 M HEPES pH 7.4 / 5 mM EDTA, the proteins were dimethyl-labeled at lysine residues, and the protein N-termini in two rounds with 30 mM13CD2O and 15 mM NaBHsCN for 5 minutes at 95 °C each. Consequently, the13CD2O artificial dimethyl label labels all in vivo proteolytical ly processed protein N-termini. Dimethyl-labeling was stopped by addition of final 100 mM Tris-HCI pH 7.4 for 5 minutes at 95 °C and a second round of SP3 cleanup was performed with two washes of 200 pL ACN 90% and an additional, third round of washing with 100 pL ACN 90%. After air-drying the beads for a brief amount of time (1 minute), the beads were resuspended in 60 pL 50 mM HEPES pH 7.4 / 2.5 mM CaCh, supplemented with 1 :100 trypsin (Serva #37286.03, Heidelberg, Germany) and digested o / N at 37 °C at 1000 rpm. Following digestion o / N, a bulk proteome sample was obtained by taking a 10% preSHUNTER aliquot, purified by Cis Stage-Tip cleanup. The remaining 90% was used for hydrophobic tagging of the newly generated tryptic peptide N-termini with the aldehyde undecanal (20:1 undecanal-to-protein ratio) in final 40% EtOH with 30 mM NaBHsCN for 5 minutes at 95 °C. Afterwards, a second 30 mM NaBHsCN was added for 5 minutes at 95 °C, followed by quenching of the aldehyde reaction with final 100 mM Tris-HCI pH 7.4 for 5 minutes at 95 °C. To purify the original dimethyl-labeling blocked protein-N-termini from the aldehyde-labeled tryptic peptide N-termini, a C18 P7187PC00 depletion was performed using HR-X spin columns (20 mg, Macherey-Nagel) in 40% EtOH. The combined flow-through is evaporated in a vacuum concentrator and purified using SP3 beads. The final N-terminal peptides were re-suspended in 20 pL H2O + 0.1% foric acid (FA), and peptide concentration was determined spectroscopically using NanoDrop One (Thermo Fisher) based measurement at A280.

[0300] Data validation with MOFA: The quantified data was validated with the MOFA2 (Argelaguet et al., 2020) package v1.10.0 in R. Log10-transformed intensities for N- termini, and proteins were used as quantitative traits and the corresponding gene ID as a feature for proteins. On the other hand, N-termini were identified by a feature-tag consisting of a “gene:start_position-end_position” (e.g., 03:755-780) identifier. MOFA2 model generation was restricted to ten factors, and the convergence level for model generation was “slow” to yield the highest quality for the MOFA2 model. Putative candidate cleavages were selected by a substantial regulation in the SLE cohort versus the controls of at least 27% (| log2FC| > 0.35, cf.) and a substantial contribution to individual MOFA2 model factors (within the top 25%).

[0301] Data Analysis: Acquired RAW files for SHUNTER and preSHUNTER runs of the SLE cohorts were analyzed with FragPipe with a fixed N-termini strategy, where only dimethylated lysine and peptide N-termini (+34.0631 Da for13CD2O / NaBH3CN) in addition to carbamidomethylation on cysteines (+57.02146 Da) were required as fixed modifications. Variable modifications were limited to oxidations on methionine (+15.9949 Da).

[0302] Results

[0303] The number of proteolytic events per protein demonstrates that many cleavages occur in fibrinogen, the most abundant serum protein albumin, but also particularly in the C3 protein. C3 was by far the most abundantly cleaved protein of the complement system . The quantification of N-termini in lupus patients compared to age-matched healthy controls (n=23) revealed a substantial alteration of N-termini in SLE (Fig. 4B), with many complement N-termini found in most of the patients (bigger circle size in Fig. 4B). Beyond that, reduced proteolytic processing of inter-alpha-trypsin inhibitor heavy chain H1 (ITIH1) at A126 (log2FC SLE / CTRL -1.19) was observed, a protease inhibitor or of PROS1 at F92 (log2FC SLE / CTRL -1.66), an anticoagulant protein with immunosuppressive abilities (Ubil et al., 2018), suggesting direct interplay between P7187PC00 coagulation and immunity. Analysis of the bulk proteome was performed in parallel , and the N-termini abundance was independent of regulation on the proteome level .

[0304] Using Multi-omics-factor (MOFAplus; Argelaguet et al., 2020) analyses, the N- terminomic and proteomics dimensions of patient heterogeneity on a clinical level were investigated. N-terminomic data could explain most of the variance in the dataset, contributing almost exclusively to factors 1 , 3, 5, 7, and 10 (Fig. 4C). Complete clinical data covering disease severity score, renal function, and biomarker profiles were linked to these patients. N-terminome-driven factors were strongly associated with patient factors, such as CXCL10 cytokine (factor 1), chloroquine treatment (factor 3), anticoagulant treatment (Factor 5), and estimated glomerular filtration rate (eGFR, factor 7), all with significant interactions (adj. p-value < 0.05, Fig. 4C). An overview of the two individual factors 5 and 7 illustrate the strongest contributors to each MOFA factor, filtered for a substantial alteration in N-termini abundance (> 27%,), presence at a relevant scale (> 20% of all SLE cohort patients) and sorted by log2FC SLE / CTRL (Fig. 4D). Closer examination of the factors revealed that termini of complement proteins, particularly in the C-terminal part of C3 (amino acid 1514, 1534) and C8b (565) contributed to the variance explained by Factor 3, 5 and 7.

[0305] Conclusion

[0306] Using patients with deep clinical phenotyping (n=143), several regulated protein N- termini, which can be coupled to the disease state in SLE, were identified. On a global scale, strong dysregulation of plasma proteolysis in lupus patients was observed, with significant regulation in several known fragments of the human complement system. Several relevant cleavage products were observed, such as an increase of the C3g and C3dg fragments starting at E955 (C3_955, C3_1481, C3_960), a novel and superior marker of complement activation in SLE patients (Fig. 4B; Troldborg et al., 2018). Most other products were decreased including several cuts in the C-terminal region of C3 (D1525, D1534, E1538 and Y1561, Fig. 4B).

[0307] Further integration with MOFA factors revealed the N-terminome to be a bigger driving force than the bulk proteome (Fig. 4C, top) for the clinical and molecular variance presents in the SLE cohort (Fig. 4C, center). It allowed for further breakdown into factors of global (Table 3) or individual factor variance. Several undescribed P7187PC00 complement N-termini emerged from this additional factorization into ten MOFA factors correlating with patient information (Fig. 4C, bottom).

[0308] Example 4 - Integration of multi-layered proteolytic processing landscape in SLE

[0309] Aim

[0310] To further investigate the complex proteolytic modifications in the plasma of patients with biopsy proven lupus nephritis, the most important major organ manifestation of SLE. Additionally, to integrate the multi-layered proteolytic processing landscape of the cross-sectional SLE cohort and active vs. remission lupus nephritis.

[0311] Methods

[0312] The SHUNTER workflow, LC-MS, the Human SLE cohort and Data validation with MOFA methods as described in Example 1 were utilised. Sample preparation methods were utilised as described in Example 2.

[0313] Longitudinal analysis of lupus nephritis patient plasma active disease vs. remission: Patients with lupus nephritis from the Hamburg Glomerulonephritis registry were used for monitoring the lupus nephritis active vs. remission states. Prior to inclusion, patients provided written informed consent. The study was conducted in accordance with the principles of the Helsinki Declaration and was approved by both the Hamburg Committee on Health Research Ethics (approval number: PV4806). Only patients with biopsy proven class IV OR lll / IV OR lll / IV with or without class V were chosen. Initial serum samples were taken at a time when patients had active disease. Subsequent samples were taken when patients had reached complete renal response (CRR) according to KDIGO (reduction in proteinuria <0.5 g / g measured by urinary Protein Creatinine Ratio (uPCR) and stabilization or improvement in kidney function (±10%-15% of baseline). Clinical data was gathered at the same time as samples for analysis of proteolysis.

[0314] Lupus nephritis sample preparation: To monitor the modulation of proteolytic cleavages in active vs. remission lupus nephritis, n=6 SLE patients from a second site (University Hospital Hamburg-Eppendorf, Hamburg, Germany). EDTA-plasma samples were available for active and remission states of the same patient and were subsequently processed as the cross-sectional SLE cohort from AUH. The regulation P7187PC00 of proteolytic processing was quantified as the alteration in the N-termini abundance between active / remission states.

[0315] Shiny application: The Shiny app was developed using R version 4.3.2 and Shiny version 1.8.0 and deployed on shinyapps. io. The heatmap visualization was generated with ComplexHeatmap version 2.18.0. In-vitro values were filtered based on log2FC > 1 and p-value < 0.05, with non-qualifying values replaced by NA. The app's code is available on GitHub at adhutz / fatihs-app. The website can be accessed at: https: / / ahutz.shinyapps.io / proteolysee /

[0316] Results

[0317] Plasma from six patients with biopsy proven lupus nephritis, the most important major organ manifestation of SLE, was analysed. Initial samples were obtained at the time of active disease. Later samples were obtained when patients, after induction therapy, had complete renal remission (CRR as per KDIGO; Rovin et al., 2024). Consistent differences in the active versus remission stages were observed, particularly in the complement system .

[0318] All data (Table 3) from both in vitro and patient profiling samples were integrated as a circle plot, highlighting several relevant proteolytic fragments (Fig. 5A). These data also demonstrated that the analyses covered virtually all described fragments that clustered in hotspots.

[0319] Table 3. Identified N-Termini observed in the SLE cohort. P7187PC00 P7187PC00

[0320] Furthermore, several novel cleavage sites were observed consistently across different human datasets and attributed as in vitro substrates of one of the four studied complement-initiating proteases (Fig. 5A). For full data accessibility, aggregated patient information data in a shiny application is presented, providing a resource for circulating plasma protein proteolytic processing. The proteolySee app is to be found at https: / / ahutz.shinyapps.io / proteolySee /

[0321] Two proteolytic cleavages in C3 and C8b (Fig. 5B) were associated with clinical parameters in the cross-sectional SLE cohort for MOFA factors 3 (chloroquine treatment), 5 (anticoagulant treatment), and 7 (eGFR, Fig. 4C).

[0322] The functions of said proteolytic cleavages in C3 and C8b, were further investigated. New N-termini starting in the C-terminal part of the protein (at position 1514 and 1534) of C3 were strongly associated with several individual factors (factor 3 / 5 in MOFA analysis, Fig. 6A), but not associated on a global proteome scale with those factors (Fig. 6B). These 1514 and 1534 C3 cleavage sites were also correspondingly differentially regulated in active versus remission among the human lupus nephritis patients . In vitro, these N-termini were generated by recombinant MASP-3, and, to a lesser degree, MASP-1 (Fig. 3D). Analysis of the domain structure of C3 (Fig. 5B) revealed that the fragment comprises the C345C domain. Further visualization of the C3 structure revealed that all selected C3 sites with weight in the MOFA analysis were accessible to proteolytic cleavage and not buried in the molecule. A second fragment was observed in the C-terminal part of complement C8b. This fragment was also accessible. P7187PC00

[0323] Conclusion

[0324] Consistent differences in the complex proteolytic modifications in the plasma of patients with biopsy proven lupus nephritis in the active versus remission stages were observed, particularly in the complement system.

[0325] Several novel cleavage sites were observed consistently across different human datasets and attributed as in vitro substrates of one of the four studied complementinitiating proteases.

[0326] Two proteolytic cleavages in C3 and C8b were associated with clinical parameters in the cross-sectional SLE cohort, the functions of which were investigated.

[0327] Example 5 - Characterization of the bioactivity for the C3 fragment C3-LHF1 (1514- 1663).

[0328] Aim

[0329] To analyse the potential relevance of the exact identified N-terminus C3_1514 from the SLE cohort, and characterize the bioactivity of the expressed recombinant C3 fragment (termed C3-LHF1 ; SEQ ID NO:2) , corresponding to residues 1514-1663 of the C-terminal region of human C3.

[0330] Methods

[0331] C3-LHF1 fragment was produced in CHO cells with additional stringent endotoxin removal and was sequence validated by MS (Fig. 7A).

[0332] Synthesis of recombinant fragments: Selected protein N-termini identified in the SLE cohort were synthesized as recombinant LHF (Lupus Human Fragment) protein fragments or peptides.

[0333] C3-LHF1 is a 175 aa-long C3 fragment comprising SEQ ID NO: 2, and further comprising a C-terminal His-tag and an N-terminal tag for secretion, expressed in a mammalian CHO protein expression system (pos. 1514: MGWSCIILFLVATATGVHS AEENCFIQKSDDKVTLEERLDKACEPGVDYVYKTRLVKVQLSNDFDEYIMAIEQTIKSG SDEVQVGQQRTFISPIKCREALKLEEKKHYLMWGLSSDFWGEKPNLSYIIGKDTWVEH WPEEDECQDEENQKQCQDLGAFTESMWFGCPNHHHHHH (SEQ ID NO: 59) - P7187PC00 additional tags for secretion and purification underlined) and was correspondingly purified. Endotoxins were removed and validated to be <0.1 Ell / mg by the manufacturer. Production of the exact C3 C-terminal N-terminus at position 1534 (SEQ ID NO: 57) in CHO cells yielded low purity & yield, but more successful production of the recombinant protein could be obtained by extending the fragment N-terminally to position 1514, so the final C3-LHF1 protein fragment was from 1514-1663 (SEQ ID NO: 58). The LHF2 peptide represents a C-terminal fragment of complement C8b (pos. 565: QRQCNNPPPQNGGSPCSGPASETLDCS; SEQ ID NO:1). Both fragments were synthesized by Genscript (Rijswijk, Netherlands) and had a purity > 97% (C8b-LHF2) or > 99% (C3-LHF1) as validated by SEC & MS. The LHF2 peptide exhibited solubility in PBS (with moderate heating) or DMSO.

[0334] The SHUNTER workflow and LC-MS methods as described in Example 1 were utilised.

[0335] Cell painting assay: Since the human complement system executes several physiological functions, the fragments displayed toxicity or bioactivity in human cancer cells via the cell painting assay, a five-plex staining followed by an automated validation that can determine the bioactivities of molecules.

[0336] Human bone osteosarcoma cells, U-2OS (ATCC HTB-96) were cultured in McCoy’s 5A (Sigma cat. no. M9309) supplemented with 10% fetal bovine serum (FBS) (Gibco cat. no. A3160802) and 1% penicillin / streptomycin. Cells were cultured at 37 °C in a humidified atmosphere (5% CO2) and passaged when 70-90% confluence was reached. To passage cells, they were washed in Dulbecco’s Phosphate Buffered Saline (PBS) (2x5 mL, Sigma, cat. no. D8537) and detached from the culture flask by trypsin-EDTA (Sigma, cat. no. T4049) and one portion of cells are reseeded in fresh full growth medium in a T75 flask (Thermo Scientific cat. no. 130190). The cell painting protocol is adapted to a 96-well plate format.

[0337] Cells (4000 cells / well) were seeded into the inner 60 wells of a 96-well plate with optical bottom (Corning Cat# 3603) in complete medium (75 pL) and incubated (37 °C, 5% CO2, humid) for 24 h. C3-LHF1 is dissolved in water. To ensure comparability, stocks of C3-LHF1 were first diluted into media containing DMSO (50 pL C3-LHF1 stock + 75 pL 3.33% DMSO in media) to make 4X solutions with 2% DMSO (final DMSO = 0.5%). All other compounds were dissolved in DMSO and diluted 50 times in media to make 4X solutions with 2% DMSO. Compounds or DMSO (for negative P7187PC00 control) were dosed in the designated culture plates in quadruplicates, distributed over

[0338] 4 plates, in 25 pL medium with a normalized DMSO concentration (0.5%). Twelve DMSO control wells were included on each plate for normalization. After 24 h, 75 pL medium was removed and replaced with 75 pL complete medium containing 500 nM MitoTracker Deep Red (final C = 325 nM), and plates were incubated in the dark for 30 min. Wells were then aspirated, 75 pL medium was added, before adding 25 pL 16% paraformaldehyde (Electron Microscopy Sciences 15710-S) (final PFA = 4%), and plates were incubated in the dark for 20 min. Plates were washed once with 1X HBSS (Invitrogen Cat#: 14065-056) and 75 pL 0.1% (vol / vol) Triton X-100 (BDH Cat#: 306324N) in 1X HBSS was added, and incubated for 15 min in the dark. Plates were washed twice with 1X HBSS before addition of 75 pL multiplex staining solution (Hoechst 33342: 5 pg / mL; Concanavalin-Alexa Fluor 488 conjugate: 35 pg / mL; SYTO 14 Green Fluorescence Nuclei Acid Stain: 3 pM; Phalloidin-Alexa Fluor 568 conjugate:

[0339] 5 pL / mL; Wheat-Germ agglutinin-Alexa Fluor 555 conjugate: 1.5 pg / mL) in HBSS containing 1% BSA (Sigma-Aldrich Cat# A9647) and incubation for 30 min in the dark. Plates were washed three times with 1X HBSS with no final aspiration and imaged immediately in a Zeiss Celldiscoverer 7 automated microscope.

[0340] Nine images are acquired in each well with 2x2 binning using the AxioCam 702 CMOS 12-bit camera with 4x analog gain in Zen 3.0 software for Celldiscoverer 7 using the following imaging settings: P7187PC00

[0341] In short, CellProfiler 2.1.1 was used to correct images for uneven illumination, followed by image segmentation and extraction of 1476 features across nuclei, cytoplasm, and the whole cell on a per-cell basis. Features were then averaged to per-well profiles, after which the data was normalized on a per-plate basis followed by per-treatment aggregation, which affords the final profiles using the cytominer 0.1.0 package67in R 3.6.068.

[0342] The heatmap of morphological profiles is visualized with heatmap.2 in the gplots 3.0.3 package. The Pearson correlation matrix was calculated using the stats package in R 3.6.0 and visualized using the corrplot 0.84 package.

[0343] Hierarchical clustering of the correlation matrix uses the stats package and Pearson correlation coefficients as distance metric and average linkage method.

[0344] The activity score was calculated as the intra-replicate correlation, which is the Pearson correlation between technical replicates. Significant activity is determined as intra-replicate correlation > 0.6, as determined by evaluation of in-house generated profiles in consolidation with the mp-value and Mahalanobis distance.

[0345] Complement ELISA assays: To assess any modulation exhibited by the LHF1 / 2 fragments synthesized, we used the SVAR WIESLAB® Complement System Screens (SVAR Life Science, Malmo, Sweden) for the Alternative, Classical, and MBL pathways. The assay detects the deposition of complement factor C9 on the three different surfaces used in the kit. In brief, freshly thawed human serum was preincubated with increasing concentrations of C3-LHF1 (1 , 2, 5, 10, and 20 pM), C8b- LHF2 (0.02, 0.2, 0.5, 1 , and 2 mM) or 1x PBS as vehicle control, all diluted to the same volume with the corresponding diluent buffers from each kit for 30 min. at RT. Subsequently, the assay was performed as instructed by the manufacturer, and the ELISA plate read at 405nm. Corresponding samples with no detectable activity in each pathway were eliminated for further data analysis. The percent modulation of complement activity was calculated in relation to the vehicle PBS control treatment for each corresponding plasma sample separately and means were calculated.

[0346] Toll-like receptor (TLR) activation in Flp-ln™-293 NF-KB cells: Human embryonic kidney (HEK) cells stably expressed an NF-KB responsive response element linked to a Firefly luciferase gene. Additionally, plasmids coding for Toll-like receptors (TLR) and essential cofactors were stably or transiently transfected into the cells (stable: TLR1 / 2, TLR6 / 2, TLR5, TLR4, TLR7; transient: TLR10). TLR1 and TLR6 were co-transfected P7187PC00 with TLR2. The intracellular TLR7 includes intracellular and transmembrane domains of TLR5 to bring the receptor to the cell’s outer membranes. Details of cloning and transfecting procedures have been described elsewhere (Hassan et al, 2021).

[0347] 5 x 104TLR NF-KB cells were seeded per well of a 96-well plate (Greiner Bio-one, Frickenhausen, Germany) in 145 pl DMEM containing 4.5 g / l glucose, 10% (v / v) FCS, 1% (v / v) Pen / Strep, 1 mM L-Glutamine, and 250 pM Luciferin D (Promega, Madison, USA). Cells were incubated 12-16 h in a humidified incubator at 37 °C under 5% (v / v) CO2 to adhere to the bottom of the plate. The plate was sealed prior to the measurement in a TopCountONXT (PerkinElmer, Waltham, USA) microplate luminescence counter at 37 °C. The measurement occurred without stimulation for 2 h (pre-run) before adding C3-LHF1 protein / C8b-LHF2 peptide / PBS vehicle control. Luminescence measurement continued for an additional 20 to 28 h at 37 °C. Luciferase values were given in “counts per second” (cps). Cps were normalized by the PBS vehicle control at each time-point and the data was presented as the mean of n=3 individually measured wells.

[0348] Granulocyte CD62L shedding assay: Activation of granulocytes (CD62L shedding assay) was conducted using samples of four milliliters of sodium heparin-stabilized whole blood obtained from healthy blood donors at the blood bank of Aarhus University Hospital, Denmark. Experiments commenced within one hour of blood collection. The samples were gently mixed, and fifty microliters were transferred into five-milliliter polystyrene tubes. Subsequently, each tube was treated with C3-LFH1, C8b-LFH2, POD2 (irrelevant control peptide), lipopolysaccharide (LPS, sourced from Escherichia coli 0111 :B4, 'tlrl-3pelps', Invivogen, San Diego, CA, USA), or sterile phosphate- buffered saline (PBS, pH 7.4). All compounds were dissolved in sterile PBS, with final concentrations as specified in the text. After gentle mixing, the tubes were incubated in a water bath at 37°C for one hour.

[0349] For each donor, all tubes, except one treated with PBS, received 5 pL of allophycocyanin-labeled mouse monoclonal IgG anti-human CD62L (Clone: DREG-56, Catalog No: 559772, BD Pharmingen, Franklin Lakes, NJ, USA). After gentle mixing, the tubes were incubated in the dark for 15 minutes at ambient temperature. Subsequently, each tube was treated with 1 mL of erythrocyte lysis buffer (composed of 155 mM ammonium chloride, 10 mM potassium hydrogen carbonate, and 0.1 mM EDTA, pH 7.3), gently mixed, and then incubated in the dark for an additional 15 minutes at ambient temperature. P7187PC00

[0350] Whole blood samples from healthy blood donors (n = 5) were treated with PBS (unlabeled control and vehicle control), LPS (positive control) at 0.19 mg / mL, C3-LHF1 at 1.2 mg / mL (= 66 pM), C8b-LHF2 at 2.7 mg / mL (= 1 mM), or POD2 (negative control) at 1.7 mg / mL ( = 1 mM). The presence of surface CD62L on granulocytes was then assessed using flow cytometry. Cell suspensions were analyzed following incubation using a NovoCyte 3000 Flow Cytometer (ACEA Biosciences, CA, USA).

[0351] Allophycocyanin was excited at 640 nm, and emissions were detected using a 675 / 30 nm bandpass filter. Analysis was conducted on 100 microliters from each tube. Granulocytes were gated based on forward- and side-scatter signals. Allophycocyanin fluorescence was analyzed as height signals.

[0352] Pull-down assays from human plasma with C3-LHF1. Human EDTA-plasma (n=4 healthy controls + n=4 SLE patients, each 2x male and 2x female) was diluted with 1x PBS, 0.02% Tween-20, 1x complete protease inhibitors without EDTA (Roche) to the same protein concentrations and pre-cleared on NTA-Co2+-based magnetic beads (Invitrogen) for 30 min with gentle rotation (15 rpm) at RT. Meanwhile, the beads for the pull-down were prepared by immobilizing i) recombinant His-tagged C3-LHF1, ii) His- tagged nanobody against C3 (hC3Nb2), and iii) a blank negative control (NC) on NTA- Co2+-based magnetic beads for 10 min. at RT. After the pre-clearing, each unbound human plasma was equally split into three assays for C3-LHF1, hC3Nb2 and NC and a small aliquot was kept for proteomic analysis of each pre-cleared lysate. The pull-down was performed for 1h with gentle rotation (15 rpm) and beads subsequently washed 4x with 1x PBS, 0.02% Tween-20, 600 mM NaCI, 1x complete protease inhibitors without EDTA. Elution was performed with 1x Lammli buffer + 0.5M imidazole for 30 min. at 37 °C and the eluate was purified with SP3 beads for proteomic sample processing with an overnight digestion with trypsin at 37 °C.

[0353] Results

[0354] The analysis revealed that the LHF1 (SEQ ID NO:2) fragment had no effect on viability but demonstrated significant (mp-value < 0.001) bioactivity with concentrations above 10 pM. Further analysis of the profile revealed correlations to known compounds that are DNA binders (Fig. 7B). P7187PC00

[0355] Subsequently, the impact of C3-LHF1 (SEQ ID NO:2) on the regulation of the human complement system in vitro was analysed. After activating the alternative, classical, or MBL pathways and relevant surfaces, the MAC deposition on the surfaces was probed. C3-LHF1 (SEQ ID NO:2) was highly active in suppressing the classical and MBL pathway, already at low micromolar concentrations (Fig. 8A).

[0356] The capacity of the LHF1 (SEQ ID NO:2) fragment to activate toll-like receptors (TLRs) and NFKB signaling was investigated. C3-LHF1 (SEQ ID NO:2) activated NFKB signaling via TLR1 / 2, TLR7 / 5 chimeras, and to a lesser degree TLR10 signaling with the application of 10 / 20 pM of C3-LHF1 (Fig. 8B).

[0357] C3-LHF1 (SEQ ID NO:2) binding partners by pull-down from diluted human CTRL and SLE plasma (n=4 each) were analysed. C3-LHF1 with a nanobody purifying the entire C3 (hC3Nb2) (Kishimoto et al., 1989) and characterized both pull-downs by mass spectrometry in comparison to negative control (NC) pull-downs (Fig. 8C). Baseline differences in SLE vs. CTRL bulk proteome did not drive the corresponding pull-downs. Both hC3Nb2 and C3-LHF1 pulled out complement C4 from CTRL plasma but not from SLE plasma (Fig. 8C, Common).

[0358] In contrast, C3-LHF1 (SEQ ID NO:2) and hC3Nb2 pulled down Nidogenl only from the SLE patients, but not CTRL plasma. In addition, C3-LHF1 pull-downs displayed a distinct pattern from the corresponding nanobody pulldown for C3: C3-LHF1 specifically binds DNA-binding proteins (Fig. 8C, C3-LHF1-specific), several of which are potentially linked to lupus (CSRP1 , RPL22, WDR1). To proof bioactivity in native human granulocytes, the effect of C3-LHF1 on surface CD62L shedding in granulocytes was investigated (Fig. 8D), indicating an activating effect on these cells.

[0359] Conclusion

[0360] The complement ELISA assay (Fig. 8A) demonstrated that C3-LHF1 (SEQ ID NO:2) is highly active in suppressing the classical and MBL pathway, already at low micromolar concentrations.

[0361] The functional effects of a recombinant C3-LHF1 (SEQ ID NO:2) fragment (residues 1514-1663) was studied. Cleavage at residue A1514 is associated with the top N- termini driving the MOFA analysis factor 7, which correlates significantly with eGFR in P7187PC00 the clinical cohort (Fig. 4C & 4D). The fragment has an ability to suppress the C9 deposition from the MBL and classical pathway but not the alternative pathway (Fig. 8A). Searching for interacting proteins for the fragment, we could identify cytoskeletal organization proteins associated with C3-LHF1, but not to the C3 binding nanobody hC3Nb2 - whereas both C3 and C3-LHF1 were associated with C4 (Fig. 8C).

[0362] The C3-LHF1 -specific interactors in the pull-down (Fig. 8C) were several autoimmunity-linked proteins: CSRP1 was identified in a genetic screen for IFNa regulated genes in different lupus mouse models, WDR1 was investigated as a cause for spontaneous autoinflammatory disease in mice and RPL22 was identified as one of the few consistent differentially expressed genes in a bioinformatic approach to predict lupus disease activity by gene expression data.

[0363] Example 6 - Characterization of the bioactivity for the C8b fragment C8b-LHF2, (565- 589).

[0364] Aim

[0365] To characterize the bioactivity of the expressed recombinant C8b fragment (termed C8b-LHF2; SEQ ID NO:1), corresponding to residues 565-589 of the C-terminal region of human C8b.

[0366] Methods

[0367] C3-LHF2 fragment (QRQCNNPPPQNGGSPCSGPASETLDCS; SEQ ID NO: 1) was produced in CHO cells with additional stringent endotoxin removal and was sequence validated by MS, as described in Example 5. The SHUNTER workflow and LC-MS methods as described in Example 1 were utilised. The cell painting assay, complement ELISA assays, Toll-like receptor (TLR) activation in Flp-ln™-293 NF-KB cells, and Granulocyte CD62L shedding assay, as described in Example 6 were utilised.

[0368] Results

[0369] The fragment of C8b was chemically synthesized, comprising the last 26 C-terminal amino acids of C8b (C8b-LHF2, Residues 565-589). The C8-C-terminus is responsible for anchoring the MAC complex to the membrane. This fragment will still likely be linked to the rest of C8b due to the disulfide bridge C557-C590 but the structure of C8b implicates a C568-C580 disulfide bond, which could yield a cyclic peptide. P7187PC00

[0370] The complement ELISA assay (Fig 9A) demonstrated that C8b-LHF2 has the capacity to inhibit all three pathways of the complement system. C8b-LHF2 was able to suppress the deposition of C9 via the AP pathway, as well as the MBL pathway and interestingly increased C9 deposition from the MBL pathway at lower concentration only in SLE patients (Fig. 9A).

[0371] The cell painting assay utilising the C8b-LHF2 peptide demonstrated a reduced activity and a slight viability reduction, though only with very high concentrations.

[0372] TLR activation by the C8b-LHF2 peptide was observed (Fig. 9B). Granulocyte activation indicated by CD62L shedding was achieved with high concentrations of C8b- LHF2 (Fig. 9C).

[0373] Conclusion

[0374] C8b-LHF2 has the capacity to inhibit all three pathways of the complement system.

[0375] The C-terminal peptide containing residues 565-589 from C8b, forming complement C8 together with C8a and C8g was a strong driver of renal-related clinical parameters. The fragment induced upon cleavage of the peptide bond 563-564 in C8b will probably remain together with the rest of C8b due to the disulfide bridge 557-590. Proteolytic cleavage of the 563-564 peptide bond is anticipated to disrupt the secondary structure of the C-terminal thrombospondin repeat in C8b. If such a cleavage occurs within the C5b-8 complex, it will likely interfere with the successful incorporation of C9 into the MAC.

[0376] Example 7 - C3-LHF1 interacts and activates human gp130 (IL6ST).

[0377] Aim

[0378] To elucidate the molecular targets of C3-LHF1 (SEQ ID NO: 2) in human renal tissue and determine whether C3-LHF1 interacts with the interleukin-6 signal transducer gp130 (IL6ST), thereby influencing IL6 signalling pathways.

[0379] Methods

[0380] Pull-down assays from human kidney with C3-LHF1. Healthy human kidney tissue was homogenized in 1x RIPA buffer supplemented with Roche complete protease P7187PC00 inhibitors and lysed for 30 min. on ice before centrifugation at 15000g at 4 °C for 15min. The lysate was split into n=3 technical replicates and pre-cleared on NTA-Co2+- based magnetic beads (Invitrogen) for 30 min. with gentle rotation (15 rpm) at RT as for the human plasma pull-down assays. The pre-cleared, non-bound lysates were equally split among bead control, C3-LHF1 and C3-LHF1DNpull-downs and incubated for 30 min. with gentle rotation (15 rpm) at RT. After excessive washing with 50 mM KPj pH 8.0 / 300 mM NaCI / 0.01% Tween-20, beads were resuspended in 50 pL 50 mM HEPES pH 7.4 / 2.5 mM CaCh and pull-downs digested o / N on bead with Serva trypsin at 37 °C. Digested peptides were cleaned up for MS analysis with SP3 beads.

[0381] Thermal proteome profiling - proteome integral solubility alteration assay for C3- LHF1: Thermal proteome profiling - proteome integral solubility assays (TPP-PISA) were performed on human kidney tissue (n=3) lysed in 50 mM HEPES pH 7.4 / 100 mM NaCI / 2.5 mM EDTA pH 8.0 / Roche complete protease inhibitors / 20 pg / mL DNAse I. Treatments with 1 or 10 pM C3-LHF1 or 1xPBS control were carried out at 37 °C for 1h and subsequent thermal denaturation was performed at three different temperatures (53 / 56 / 59 °C) for 5 min. before the samples were combined. Combined proteins were solubilized with 0.8% NP-40 / 50 mM HEPES pH 7.4 / 100 mM NaCI / 2.5 mM EDTA pH 8.0 / Roche complete protease inhibitors (30 min. at 4 °C) and the soluble proteins were separated by centrifugation at 18 000 g for 30 min. (4 °C). An aliquot of 50 pg from the soluble proteome supernatant was lysed in final 2% SDS / 50 mM HEPES pH 7.4 / 2.5 mM EDTA for 5 min. at 95 °C, carbamidomethylated with 5 mM TCEP / 20 mM CAA for 5 min. at 95 °C, purified with paramagnetic SP3 beads and digested o / N at 37 °C with trypsin (protease: proteome ratio 1 :50). Subsequently, the peptides were purified using Cis StageTips before being analyzed via HPLC / MS.

[0382] LC-MS methods as described in Example 1 were utilised.

[0383] IL6 reporter assays: HEK-Blue IL-6 reporter line cells (Invivogen #hkb-hil6) reconstituting the IL6Ra / IL6ST / STAT3 axis and exhibiting a Secreted embryonic alkaline phosphatase (SEAP) response to activation were cultivated according to the manufacturer’s instructions. In brief, cells were seeded in a volume of 100 pL DMEM media supplemented with glutamine and high glucose onto a 96 well plate. Incubations were performed with 100 pL of fresh media, supplemented with recombinant human IL6 (R&D systems #7270-1 L / CF) as a positive control, reconstituted in 1x PBS, or C3- P7187PC00

[0384] LHF1 o / N at 37° C, 5% CO2. Subsequently, 180 pL of QUANTI-Blue solution were added to 20 pL of the cells, the reaction incubated for 1h at 37 °C and the colorimetric SEAP product measured at 639nm using a spectrophotometer. Inhibition of the IL6Ra response was performed by pre-incubating some wells with Tocilizumab at 2pg / mL or vehicle for 3h at 37 °C before performing the subsequent treatment. Inhibiting the C3- LHF1 activity was achieved by pre-incubation of C3-LHF1, hC3Nb3 or a combination of C3-LHF1 with the hC3Nb3 nanobody in a 1 :1 w / w ratio at 37 °C for 1h before treatments were started on the cells.

[0385] To identify interaction partners of C3-LHF1 in human kidney tissue, pull-down experiments were conducted using bead-bound C3-LHF1 as bait. Human kidney lysates were incubated with either native C3-LHF1, vehicle control, or denatured C3- LHF1 (C3-LHF1DN; carbamidomethylated and heat-denatured). Proteins captured by the beads were analysed by unbiased mass spectrometry (Fig. 10a).

[0386] To confirm and expand on these findings, thermal proteome profiling (TPP-PISA) was performed using 1 pM or 10 pM C3-LHF1 with human kidney lysates, evaluating protein stabilisation across temperature gradients (53 °C, 56 °C, 59 °C) (Fig. 10c).

[0387] For functional analysis, IL6Ra / IL6ST cell-based reporter assays were employed. Cells were stimulated with varying concentrations of C3-LHF1 and IL6, in the presence or absence of the IL6Ra inhibitor Tocilizumab or the C3-specific nanobody hC3Nb3. Reporter activity dependent on STAT3 signalling was recorded (Fig. 10d-f).

[0388] Results

[0389] Mass spectrometric analysis revealed that C3-LHF1 specifically bound to IL6ST (gp130), a key signal transducer in IL6-mediated signalling (Fig. 10b). In the TPP-PISA assay, IL6ST emerged as the most thermally stabilised protein upon C3-LHF1 treatment, with a log2fold change (log2FC) of +1.32 at 1 pM C3-LHF1 (Fig. 10c). IL6Ra / IL6ST reporter assays demonstrated that C3-LHF1 dose-dependently engaged the IL6 receptor complex, consisting of IL6ST and IL6Ra - this leads to reporter activation, which depends on engagement of STAT3. Thus, C3-LHF1 dose- dependently activated STAT3 signalling, indicating receptor engagement. With descending concentrations of IL6, partial inhibition of IL6 signaling was observed. Activation was observed in the presence of low IL6 concentrations (Fig. 10e), suggesting that C3-LHF1 acts as a partial agonist on IL6ST (gp130). Tocilizumab, an P7187PC00 antibody and IL6Ra inhibitor, did not abrogate the C3-LHF1-induced reporter response, indicating that C3-LHF1 may signal independently of IL6Ra (Fig. 10f), and directly activate IL6ST.

[0390] Conclusion

[0391] C3-LHF1 directly interacts with IL6ST (gp130) in human kidney tissue and functions as a partial agonist of the IL6 receptor complex. This activation occurs independently of IL6Ra, revealing a novel mode of cytokine receptor engagement by a complement- derived peptide fragment.

[0392] Example 8 - C3-LHF1 activates IL6ST and JAK-STAT signalling in kidney organoids. Aim

[0393] To investigate the effects of the C-terminal complement fragment C3-LHF1 on IL6ST (gp130)-mediated signalling and downstream responses in human kidney organoids, including the JAK-STAT signalling axis and related proinflammatory mediators.

[0394] Methods

[0395] Human kidney organoids: Kidney organoids were derived from the human female induced pluripotent stem cell (iPSC) line UKEi001-A (cellosaurus ID number: CVCL_A8PR) using a modified Takasato protocol. Human dermal fibroblasts were obtained via skin biopsy from control patients. To generate organoids, hiPSCs were dissociated into single cells using Accutase (Gibco), seeded onto Matrigel-coated (Corning) 6-well plates (Nunc) at a density of 12,000 cells / cm2in E8 media (Thermo Fisher Scientific) with Y-27632 (10 pM, Biorbyt), and incubated overnight at 37 °C and 5% CO2. This hiPSC monolayer was cultured in E6 media (Thermo Fisher Scientific) supplemented with 7 pM CHIR99021 (Sigma) from day 1 to day 4, followed by 200 ng / ml FGF9 (Peprotech), 1 pg / ml heparin (Stemcell Technologies), and 1 pM CHIR99021 from days 5 to 7. To form organoids, cells were then dissociated using Trypsin (Gibco), washed with E6 media, and centrifuged at 200 g. The cell pellet was resuspended in Stage 1 media [E6 media containing 200 ng / mL FGF9, 1 pg / mL heparin, 1 pM CHIR99021 , 0.1% PVA (Sigma), 0.1 % MC (Sigma), 10 pM Y-27632] and transferred to 6-well plates pre-treated with Pluronic-F12 (Sigma) for low adhesion conditions (day 7 + 0). Cell aggregates spontaneously formed after rotating the culture dishes on an orbital shaker (Thermo Fisher Scientific) at 70 rpm, incubated at 37 °C and 5% CO2 for 24 h. The medium was switched to Stage 2 [E6 media containing P7187PC00

[0396] 200 ng / ml FGF9, 1 pg / ml heparin, 1 pM CHIR99021, 0.1% PVA, 0.1% MC] for another 4 days (7 + 1 to 7 + 4). From day 7 + 5 onwards, organoids were cultured in Stage 3 media [E6 media containing 0.1% PVA, 0.1% MC] until the end of the experiment. Organoids were stimulated with TN Fa, and cell pellets and media were collected and analyzed as detailed above. Experiments were done in three independent differentiations, and at the organoid age of day 23-25.

[0397] Phosphoproteomics and proteomics analysis of C3-LHF1 signaling in organoids: Human kidney organoids were generated as described above and treated on D23 with TNFa (50ng / mL) or C3-LHF1 (18 pM) or combinations of both for a total duration of 48h. Supernatants were taken after 24h and 48h and the final organoid cell pellets (n=4 replicates) were processed for proteomics and phosphoproteomics. Lysis was performed with 300 pL 4% SDS / 0.1M HEPES pH 7.4 / 5 mM EDTA (supplemented with Roche complete protease inhibitors) for 5 min. at 95 °C and the protein concentration determined with the BCA assay (Thermo Scientific). Nucleic acids in the samples were broken down with 50U benzonase / sample for 30 min. at 37 °C and cysteines carbamidomethylated with 5 mM TCEP and 20 mM CAA for 5 min at 95 °C. Subsequently, proteins were purified with SP3 beads for o / N digestion with trypsin (Serva) at 37 °C in a 1:50 protease: proteome ratio. For phosphoproteomics, > 1 mg of digested peptides were purified by Oasis HLB Cis cartridges (Waters), dried down and re-suspended in 200 pL of loading buffer (80% AON, 1M glycolic acid, 5% TFA). Phosphopeptides were bound to MagReSyn Ti-IMAC beads (ReSyn Bio) for 20 min. at RT and washed with two subsequent washes of loading buffer, 80% AON + 1% TFA, and 10% ACN+0.2% TFA before being eluted with 1% ammonia. Enriched phosphopeptides and proteome samples were purified with Cis StageTips and measured as described in Lasse et al., 2023.

[0398] Human kidney organoids derived from induced pluripotent stem cells (iPSCs) were employed, which have previously been shown to exhibit transcriptional and proteomic features analogous to those observed in inflammatory renal disease (Lasse et al., 2023). Organoids were treated with C3-LHF1 (18 pM for 48 h), in both the presence and absence of tumour necrosis factor-alpha (TNFa). Untreated and TNFa-only treated organoids served as controls. P7187PC00

[0399] Whole-proteome profiling was performed, enabling quantification of 10,582 proteins. To assess the functional consequences of protein-level changes, secretion of CXCL10 (a STING1 target) was measured in organoid supernatants. Additionally, phosphoproteomic analysis was conducted with a total depth of 72,874 phosphosites, and statistical significance was applied at | log2FC| > 0.58 and adjusted p < 0.05. Specific attention was given to changes in phosphorylation of key signalling proteins, including IL6ST, STAT3, and nephrin.

[0400] Results

[0401] Proteomic profiling revealed that C3-LHF1 treatment upregulated JAK3 and STAT3, key components of the IL6ST downstream signalling cascade (Fig. 11 b). This upregulation was observed both in the presence and absence of TNFa (Fig. 11c).

[0402] The perturbation caused by C3-LHF1 also led to increased levels of STI NG 1 , an innate immune sensor known to orchestrate inflammatory responses (Fig. 11 d).

[0403] Correspondingly, increased secretion of CXCL10, a known target gene of STING1 , was detected in organoid culture supernatants following C3-LHF1 treatment (Fig. 11e). Phosphoproteomic analysis identified IL6ST as the most differentially phosphorylated receptor. Enhanced phosphorylation was observed at TBK-sensitive serine / threonine sites and at the autophosphorylation site Y814, which is linked to impaired tissue regeneration (Fig. 11f) (Shkhyan et al., 2023).

[0404] Among tyrosine residues, STAT3 phosphorylation at Y705 was the most significantly upregulated phosphosite in organoids treated with C3-LHF1 in the presence of TNFa (log2FC +2.84; Fig. 11g). Moreover, nephrin phosphorylation was increased at a known activation, previously implicated in glomerular damage in proteinuric disease site (Verma et al., 2006).

[0405] Further supporting a detrimental effect of C3-LHF1 , markers of podocyte identity — including NPHS1 (nephrin), NPHS2 (podocin), PODXL (podocalyxin), and PTPRO — were among the most downregulated proteins in response to C3-LHF1 (Fig. 11 d).

[0406] To investigate whether the IL6ST pathway activation observed in organoids was a direct effect of C3-LHF1 , mechanistic studies were performed in HEK-Blue IL6 reporter cells. Pre-treatment with IL6Ra-targeting antibodies (sarilumab, tocilizumab) blocked IL6-induced signalling but had no effect on C3-LHF1-induced signalling, whereas IL6ST-specific inhibitors (SC-144, anti-IL6ST antibody) significantly reduced the C3- LHF1 response. These findings are consistent with a mechanism in which C3-LHF1 P7187PC00 may engage IL6ST in a manner that does not require IL6Ra, suggesting signalling predominantly through IL6ST.

[0407] Conclusion C3-LHF1 robustly activates IL6ST and downstream JAK-STAT signalling in human kidney organoids, driving phosphorylation changes associated with inflammatory responses and renal damage. This activation occurs via IL6ST independently of IL6Ra and may involve alternative or partial receptor complexes. Such a mechanism supports a distinct mode of cytokine receptor modulation in addition to complement pathway inhibition. The findings suggest that C3-LHF1 exacerbates inflammatory signalling and negatively affects podocyte marker expression, consistent with a pathogenic role in kidney disease, while also opening consideration for indications linked to IL-6 pathway dysregulation. Sequence overview P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00 P7187PC00

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Claims

P7187PC00Claims1. A peptide fragment comprising at least 10 amino acids and at the most 200 amino acids, wherein the peptide fragment comprises a sequence comprising at least one of the following sequences; SEQ ID NO: 2 (LHF1) or any one of SEQ ID NO: 1 or 3-58 or functional homologues thereof sharing at least 90% sequence identity therewith, for use in a method of treatment of complement- mediated disorders.

2. A peptide fragment comprising at least 10 amino acids and at the most 200 amino acids, wherein the peptide fragment comprises a sequence comprising at least one of the following sequences; SEQ ID NO: 2 (LHF1) or any one of SEQ ID NO: 1 or 3-58 or functional homologues thereof sharing at least 90% sequence identity therewith, wherein the peptide fragment modulates the activity of the complement system, for use in a method of treatment of complement-mediated disorders.

3. A peptide fragment comprising at least 10 amino acids and at the most 200 amino acids, wherein the peptide fragment comprises a sequence comprised within at the most 30 amino acids of the C-terminus of a primary protein comprising any one of SEQ ID NO: 60-99.

4. The peptide fragment according to any of the preceding claims or the peptide fragment for the use according to any of claims 1 to 2, wherein the peptide fragment comprises at least 10 amino acids, such as at least 20 amino acids, such as at least 30 amino acids, such as at least 40 amino acids, such as at least 50 amino acids, such as at least 60 amino acids, such as at least 70 amino acids, such as at least 80 amino acids, such as at least 90 amino acids, such as at least 100 amino acids, such as at least 110 amino acids, such as at least 120 amino acids, such as at least 130 amino acids, such as at least 140 amino acids, such as at least 150 amino acids, such as at least 160 amino acids, such as at least 170 amino acids, such as at least 180 amino acids, such as at least 190 amino acids, such as at least 200 amino acids.P7187PC005. The peptide fragment according to any of the preceding claims or the peptide fragment for the use according to any of the preceding claims, wherein the peptide fragment comprises at the most 200 amino acid, such as at the most 190 amino acids, such as at the most 180 amino acids, such as at the most 170 amino acids, such as at the most 160 amino acids, such as at the most 150 amino acids, such as at the most 140 amino acids, such as at the most 130 amino acids, such as at the most 120 amino acids, such as at the most 110 amino acids, such as at the most 100 amino acids, such as at the most 90 amino acids, such as at the most 80 amino acids, such as at the most 70 amino acids, such as at the most 60 amino acids, such as at the most 50 amino acids, such as at the most 40 amino acids, such as at the most 30 amino acids, such as at the most 20 amino acids, such as at the most 10 amino acids.

6. The peptide fragment according to any of the preceding claims or the peptide fragment for the use according to any of the preceding claims, wherein the three dimensional structure of the peptide fragment has a TM-score of at least 0.50, such as at least 0.55, at least 0.60, at least 0.65, at least 0.70, at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.91 , at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or even 1.0, compared to the three dimensional structure of a peptide as set forth in any one of SEQ ID NOs: 1-58.

7. The peptide fragment according to any of the preceding claims or the peptide fragment for the use according to any of the preceding claims, wherein the peptide fragment comprises at least one of the following sequences; SEQ ID NO: 2 (LHF1) or SEQ ID NO: 36-56 or SEQ ID NO: 58, or functional homologues thereof sharing at least 90% sequence identity therewith.

8. The peptide fragment according to any of the preceding claims or the peptide fragment for the use according to any of the preceding claims, wherein the peptide fragment comprises at least one of the following sequences; SEQ ID NO: 2 (LHF1) or any one of SEQ ID NO: 36-56 or SEQ ID NO: 58, or functional homologues thereof sharing at least 95% sequence identity therewith.P7187PC009. The peptide fragment according to any of the preceding claims or the peptide fragment for the use according to any of the preceding claims, wherein the peptide fragment comprises at least one of the following sequences; SEQ ID NO: 2 (LHF1) or any one of SEQ ID NO: 36-56 or SEQ ID NO: 58, or functional homologues thereof sharing at least about 100% sequence identity therewith.

10. The peptide fragment according to any of the preceding claims or the peptide fragment for the use according to any of the preceding claims, wherein the peptide fragment comprises SEQ ID NO: 2 (LHF1) or functional homologues thereof sharing at least 90% sequence identity therewith.

11. A peptide fragment comprising at least 10 amino acids and at the most 40 amino acids, wherein the peptide fragment comprises a sequence comprising at least one of the following sequences; SEQ ID NO: 1 (LHF2) or any one of SEQ ID NO: 3-56 or functional homologues thereof sharing at least 90% sequence identity therewith.

12. A peptide fragment comprising at least 10 amino acids and at the most 40 amino acids, wherein the peptide fragment comprises a sequence comprising at least one of the following sequences; SEQ ID NO: 1 (LHF2) or any one of SEQ ID NO: 3-56 or functional homologues thereof sharing at least 90% sequence identity therewith, wherein the peptide fragment modulates the activity of the complement system.

13. The peptide fragment according to any of the preceding claims, wherein the peptide fragment comprises at least one of the following sequences; SEQ ID NO: 1 (LHF2) and / or any one of SEQ ID NO: 3-35, or functional homologues thereof sharing at least 90% sequence identity therewith.

14. The peptide fragment according to any of the preceding claims, wherein the peptide fragment comprises at least one of the following sequences; SEQ ID NO: 1 (LHF2) and / or any one of SEQ ID NO: 3-35, or functional homologues thereof sharing at least 95% sequence identity therewith.P7187PC0015. The peptide fragment according to any of the preceding claims, wherein the peptide fragment comprises at least one of the following sequences; SEQ ID NO: 1 (LHF2) and / or any one of SEQ ID NO: 3-35, or functional homologues thereof sharing at least about 100% sequence identity therewith.

16. The peptide fragment according to any of the preceding claims, wherein the peptide fragment comprises SEQ ID NO: 1 (LHF2) or functional homologues thereof sharing at least 90% sequence identity therewith.

17. The peptide fragment according to any one of the preceding claims or the peptide fragment for the use according to any of the preceding claims, wherein the peptide fragment further comprises a modification.

18. The peptide fragment according to any one of the preceding claims or the peptide fragment for the use according to any of the preceding claims, wherein the peptide fragment modulates the activity of the complement system.

19. The peptide fragment according to any one of the preceding claims or the peptide fragment for the use according to any of the preceding claims, wherein modulating complement activity comprises modulation of the expression and / or activity and / or function of the classical complement pathway, the alternative complement pathway and / or the mannose-binding lectin (MBL) pathway or components thereof.

20. The peptide fragment according to any one of the preceding claims or the peptide fragment for the use according to any of the preceding claims, wherein modulating complement activity comprises inhibition of the expression and / or activity and / or function of; the classical complement pathway, and / or the mannose-binding lectin (MBL) pathway, or components thereof.

21. The peptide fragment according to any one of the preceding claims or the peptide fragment for the use according to any of the preceding claims, wherein the peptide fragment comprises complement N-termini, which has been generated by proteolytic processing of a primary plasma peptide.P7187PC0022. The peptide fragment according to any one of the preceding claims or the peptide fragment for the use according to any of the preceding claims, wherein the peptide fragment modulates the activity of the classical complement pathway, the alternative complement pathway and / or the mannose-binding lectin (MBL) pathway or components thereof, and modulates IL6ST mediated signalling.

23. A pharmaceutical composition comprising the peptide fragment according to any of the preceding claims, wherein the composition further comprises one or more pharmaceutically acceptable excipients.

24. A pharmaceutical composition comprising the peptide fragment according to any of the preceding claims, for use in a method of treatment of complement- mediated disorders, wherein the composition further comprises one or more pharmaceutically acceptable excipients.

25. The peptide fragment or pharmaceutical compositions for the use according to any of the preceding claims, wherein the complement-mediated disorders is selected from the group comprising Systemic Lupus Erythematosus (SLE), lupus nephritis, Hemolytic Uremic Syndrome (HUS), Atypical Hemolytic Uremic Syndrome (aHUS), Paroxysmal Nocturnal Hemoglobinuria (PNH), Age-Related Macular Degeneration (AMD), C3 Glomerulopathy, Dense Deposit Disease (DDD), Rheumatoid Arthritis (RA), Glomerulonephritis, Membranoproliferative Glomerulonephritis (MPGN), IgA nephropathy (IgAN), Neuromyelitis Optica (NMO), Myasthenia Gravis (MG), Cryoglobulinemia, Autoimmune Hemolytic Anemia (AIHA), Vasculitis, Ankylosing Spondylitis, Cold Agglutinin Disease, Immune Complex Glomerulonephritis, Sepsis, Meningococcal Disease, Recurrent Bacterial Infections, Alzheimer’s Disease, Multiple Sclerosis, Uveitis, Complement Component Deficiencies such as C2, C3, and / or C4 deficiencies, Hereditary Angioedema (HAE), Atherosclerosis, Myocardial Infarction (Ml), Dermatitis Herpetiformis, Bullous Pemphigoid, Ischemia-Reperfusion Injury, Transplant Rejection, and Idiopathic Thrombocytopenic Purpura (ITP).142P7187PC0026. The peptide fragment or pharmaceutical composition according to any one of the preceding claims, for use in the treatment or prevention of a complement- mediated disease associated with abnormal IL-6 signalling.

27. An in vitro method of modulating the complement system, said method comprising: a. Providing a composition comprising a human complement factor, a regulator of the complement system, or a receptor involved in complement activity selected from the group comprising the C1 complex, C1q, C1r, C1s, C2, C2a, C2b, C4, C4a, C4b, C3, C3a, C3b, C3c, C3d, C3 convertase, C5, C5a, C5b, C5 convertase, C6, C7, C8, C9, the membrane attack complex (MAC), factor B, factor D, factor H, factor I, factor P (properdin), MASP-1 , MASP-2, MASP-3, C4b-binding protein (C4BP), complement receptor 1 (CR1), complement receptor 2 (CR2), complement receptor 3 (CR3), complement receptor 4 (CR4), decay-accelerating factor (DAF), CD59, and / or any fragments, proteolytic derivatives or subunits thereof, b. Contacting said composition with the peptide fragment of any one of the preceding claims.

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