NL2037390B1 - Selection and characterization of a peptide-based complement activator - Google Patents

Abstract

The present invention relates to complement component C1q binders (“C1q binders”) comprising a peptide that binds to a globular head domain of complement component C1q and by this can either activate or inhibit complement. Further disclosed are pharmaceutical compositions and kits comprising the C1q binders according to the invention. The invention a|so relates to the medical uses of such C1q binders and compositions. In particular, the C1q binders or compositions of the invention may be used in the treatment, prevention and diagnosis of diseases or disorders. The invention a|so relates to nuc|eic acids encoding the peptides.

Description

SELECTION AND CHARACTERIZATION OF A PEPTIDE-BASED COMPLEMENT

ACTIVATOR

FIELD OF THE INVENTION

The present invention relates to complement component C1q binders (“C1q binders”) comprising a peptide that binds to a globular head domain of complement component C1q and by this can either activate or inhibit complement. Further disclosed are pharmaceutical compositions and kits comprising the C1q binders according to the invention. The invention also relates to the medical uses of such C1q binders and compositions. In particular, the C1q binders or compositions of the invention may be used in the treatment, prevention and diagnosis of diseases or disorders. The invention also relates to nucleic acids encoding the peptides.

BACKGROUND

The human complement pathway is part of our innate immune system and protects us against infections, but also mediates clearance of cellular debris and helps to regulate autoimmunity.

The classical complement pathway can be initiated when the first component of complement, the C1 complex (Fig. 1a), binds to antibodies, specifically Immunoglobulin (Ig) M or IgG subclass 1, 2 or 3 (1). IgM circulates as an obligate pentamer or hexamer (2, 3), and IgG1 and

IgG3, although monomers in solution, both form hexameric platforms upon antigen binding (Fig. 1b) (4, 5). The hexameric platforms comprise the antibody fragment crystallizable (Fc) regions, to which the C1 complex can bind (6). The C1 complex comprises the C1q ligand binding and scaffolding complex (composed of a hexamer of heterotrimeric proteins), which encapsulates the serine proteases C1r and C1s, that form a heterotetrameric C1r252 platform.

Binding of the six globular head domains of C1q (gC1q) to the Fc platforms induces activation of

Clr, which then cleaves C1s to form the active serine protease. Active C1 initiates a cascade of proteolytic events (Fig. 1a), as C1s proceeds to cleave the soluble proteins C4 and C2 to progress the complement pathway. The pathway deposits the opsonins C4b, C3b and C5b onto the surrounding surfaces, which bind to receptors on phagocytic cells to enhance phagocytosis (7, 8). The pathway terminates with the formation of the membrane attack complex (MAC), which forms a pore that lyses the targeted membrane. Anaphylatoxins are also released upon cleavage of C4, C3 and C5, known as C4a, C3a and C5a, which attract phagocytic cells.

Complement is emerging as a focus for therapeutic intervention (7, 9). In particular, complement-targeting macrocyclic peptides are being developed to block C1 activation (10, 11), complement-mediated hemolysis (12) and inflammatory signalling (13), which are being developed to treat hypoxic ischemic encephalopathy (14), paroxysmal nocturnal hemoglobinuria, and myasthenia gravis (15), respectively. These treatments are all antagonistic; they block complement activation or progression. In contrast, a new generation of therapeutics are being developed to activate complement as a route to achieve targeted cell killing via complement-dependent cytotoxicity (CDC).

CDC has been exemplified by antibody-based immunotherapeutics, such as Alemtuzumab,

Daratumumab and Rituximab (16-19). This was seen as an adjunct of monoclonal antibody therapy. Recently, however, CDC has been actively pursued as a desired therapeutic mechanism on its own. Hexamerizing mutants of IgG comprise a platform of CDC-inducing immunotherapeutics (20, 21), which are now being directed towards CD20 and CD52 for depletion of hematological cells. More recently, nanobodies that recruit C1 to specific cell types are being developed to target epidermal growth factor receptor (EGFR) and the HIV-1 envelope protein (22, 23).

Knowledge of the structural details and constraints of C1 binding and activation have been instrumental in these developments (6, 24). The cryo-electron microscopy (cryoEM)-derived structures of antigen-bound to IgM, IgG1 and IgG3 all showed a common structural motif present during C1 binding (Fig. 1b) (2, 4, 5). These data revealed that interaction between the 766 kDa C1 complex and the hexameric Fc platforms (900-1000 kDa) is mediated by small motifs at the periphery of the Fc domains (Fig. 1c) (2, 4). These structural motifs derive from homologous sequences in IgG and IgM (Fig. 1d), and contain a conserved LPxP (leucine, proline, glycine/serine, proline) sequence that adopts a B-turn-B hairpin at the periphery of the

Fc platform. This motif is present in all human antibody (sub)classes that are able to activate complement (Fig. 1d) (1).

The six gC1q domains mediate binding of the C1 complex to antibodies and other DAMPs.

However, antibodies are big molecules coming along with difficulties in their handling.

Therefore, the present invention refers to the selection and characterization of small cyclic peptides that bind to the gC1q domains. These peptides are capable of recruiting C1 from human serum. In solution, these peptides can inhibit complement activation. Furthermore, when immobilised on surfaces, these peptides can activate the C1s proteases and cause MAC pore formation on cell-mimetic lipid membranes.

These peptides have a similar activating potential as IgG1 antibodies. Whereas antibodies are ~150 kDa in mass, and form hexameric platforms >900 kDa in mass when activating complement, the peptides according to the invention are less than 2 kDa in mass. The present invention provides small, drug-like molecules with ~0.2% of the mass of an antibody complex yet remains able to activate complement.

Peptides are beneficial over whole antibodies due to their ease of manufacture and the ability to tag other proteins or molecules. The peptides according to the invention are able to imbue other molecules, peptides and proteins with complement-activating abilities.

SUMMARY OF THE INVENTION

In a first embodiment, the invention provides a complement component C1q binder comprising a peptide comprising the amino acid sequence YXiXoTFYXaXaXsFTLQFIXsX7 (SEQ ID NO. 1), wherein

Xs is absent, T, or K,

Xz is absent or V,

X3 is any amino acid,

Xa is Dor N,

Xs is any amino acid,

Xs is absent, A,E or T, and

Xz is C, S or a stop codon, on the proviso that a maximum of two of X+, Xz, and Xs are absent, wherein the peptide binds to a globular head domain of mammalian complement component C1q.

Suitably, Xs and/or Xs may be P.

Suitably, the peptide may comprise the sequence YTVTFYPDPFTLQFIAX7, (SEQ ID NO: 2) wherein X7 is C or S.

Suitably, the peptide may be surface-bound and is capable of activating complement.

Suitably, the peptide may be in soluble form and is capable of inhibiting complement activation.

Suitably, the peptide may be a cyclic peptide, wherein the N-terminal end of the peptide binds to the C-terminal end of Xs.

Suitably, the N-terminal tyrosine may be chloroacetylated (CI-Ac).

Suitably, the C-terminal and/or N-terminal end may be configured to conjugate to one or more distinct moieties.

Suitably, the one or more distinct moiety may be a GS-linker selected from the group consisting of GS, GSGS (SEQ ID NO. 43), GGGS (SEQ ID NO. 44), and GSGGSG (SEQ ID NO. 45).

Suitably, the peptide may comprise one of the sequences selected from the group consisting of

YTVTFYXaDXsFTLQFIACGSK (SEQ ID NO. 86), wherein X3 and/or Xs is/are any amino acid,

YTVTFYPDXsFTLQFIACGSK (SEQ ID NO. 87), wherein Xs is any amino acid,

YTVTFYX:DPFTLQFIACGSK (SEQ ID NO. 88), wherein Xs is any amino acid.

Suitably, the peptide may consist of one of the sequences selected from the group consisting of

YTVTFYXsDXsFTLQFIACGSK (SEQ ID NO. 86), wherein Xs and/or Xs is/are any amino acid,

YTVTFYPDXsFTLQFIACGSK (SEQ ID NO. 87), wherein Xs is any amino acid,

YTVTFYXasDPFTLQFIACGSK (SEQ ID NO. 88), wherein Xs is any amino acid.

Suitably, the N-terminal tyrosine may be chloroacetylated (CI-Ac).

Suitably, the peptide may be a linear peptide, wherein X; is any amino acid other than C and/or wherein the N-terminal tyrosine is not chloroacetylated (CI-Ac).

Suitably, the peptide may comprise the sequence YTVTFYPDPFTLQFIAX; (SEQ ID NO. 105), wherein X7 is any amino acid other than C and/or wherein the N-terminal tyrosine is chloroacetylated (CI-Ac).

Suitably, the peptide may consist of the sequence YTVTFYPDPFTLQFIAX; (SEQ ID NO. 105), wherein X7 is any amino acid other than C and/or wherein the N-terminal tyrosine is chloroacetylated (CI-Ac).

Suitably, the N-terminal tyrosine may be L-tyrosine.

In a second embodiment, the invention provides an isolated nucleic acid comprising a sequence encoding a peptide comprised by a complement component C1q binder according to the invention.

In a third embodiment, the invention provides an isolated cell comprising a nucleic acid according to the invention.

In a fourth embodiment, the invention provides a pharmaceutical composition comprising a complement component C1q binder according to the invention, a nucleic acid according to the invention or a cell according to the invention and a pharmaceutically acceptable excipient, adjuvant, diluent, or carrier.

In a fifth embodiment, the invention provides a kit comprising a complement component C1q 5 binder according to the invention, a nucleic acid according to the invention, a cell according to the invention or a pharmaceutical composition according to the invention and instructions for using the peptide to treat, prevent or diagnose a disease or disorder in a subject.

In a sixth embodiment, the invention provides a complement component C1q binder according to the invention, a nucleic acid according to the invention, a cell according to the invention or a pharmaceutical composition according to the invention, for use as a medicament or a diagnostic.

In a seventh embodiment, the invention provides a complement component C1q binder according to the invention, a nucleic acid according to the invention, a cell according to the invention or a pharmaceutical composition according to the invention, for use in the treatment, diagnosis, or prevention of a disease or disorder associated with complement activation or inhibition involving C1q.

In an eighth embodiment, the invention provides a complement component C1q binder according to the invention, a nucleic acid according to the invention, a cell according to the invention or a pharmaceutical composition according to the invention, for use in the treatment, diagnosis, or prevention of infectious disease, neurological disease, neurodegenerative disease, graft versus host disease, auto-immune disease or cancer.

Suitably, wherein the peptide or pharmaceutical composition may be formulated for use with one or more other pharmaceutical compositions, detectable agents or therapeutic agents, preferably wherein the one or more other pharmaceutical compositions, detectable agents or therapeutic agents is selected from the group consisting of a: complement activating compound, complement inhibiting compound, tracer, label, radioactive compound, toxin, CRISPR- associated protein (Cas), preferably wherein the Cas is Cas9, and nucleic acid, preferably wherein the nucleic acid is a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA), more preferably wherein the RNA is selected from the group consisting of a: small interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA (asRNA), aptamer, circular RNA (circRNA), self-amplifying RNA (saRNA), catalytic RNA, anti-miRNA, long non-coding RNA (IncRNA), single-guide RNA (sgRNA) and mRNA encoding a Cas, or wherein the DNA is selected from the group consisting of a: antisense oligonucleotide (ASO), aptamer, episome and catalytic DNA.

In a ninth embodiment, the invention provides a method of treating a subject, comprising administering a therapeutically effective amount of a complement component C1q binder according to the invention, a nucleic acid according to the invention, a cell according to the invention or a pharmaceutical composition according to the invention to a subject in need thereof.

In a tenth embodiment, the invention provides a method of producing a peptide comprised by a complement component C1q binder according to the invention, the method comprising expressing a nucleic acid in accordance to the invention in a host cell.

It will be appreciated that, except for where the context requires otherwise, the considerations set out in this disclosure should be considered to be applicable to all aspects of the invention.

Various aspects of the invention are described in further detail below.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

Figure 1 describes the classical complement pathway, which is initiated by defined structural motifs. a) Overview of the classical complement pathway. b) CryoEM-derived structures of antibody-C1 complexes. The interfaces between the gC1q domains and antibodies are highlighted as black spheres. Structures were built into maps with accession codes EMD-4232,

EMD-16241, and EMD-4878 (lgG1, IgG3 and IgM, respectively). ¢) Structures of the interfaces (dashed boxes in b) between gC1q and ALPAP (lgG1 and IgG3; top) and DLPSP (IgM; bottom). d) Sequence comparison of the B-turn-B motif of human antibody (sub)classes, and their ability to activate complement.

Figure 2 depicts RaPID selection, sequence analysis and peptide screening. a) gPCR quantifying the DNA recovery after a selection round. cDNA conjugated peptides were either incubated with SCgC1g-presenting beads (positive) or beads without protein (negative). b)

Phylogenetic tree representation of the top 100 sequences. c} Representative sequences obtained from each cluster in b. Lines connecting N-terminal tyrosine and cysteine denote the position of cyclization, and (bio) denotes the C-terminal lysine analogue that was modified with biotin. d} ELISA detecting C1q binding to immobilised peptides after incubation with purified

C1q. e) ELISA detecting C1q binding to immobilised peptides after incubation with human serum. f) Structure of cL3, which was modified with either a biotin or azide moiety.

Figure 3 shows activation of complement via C1q binding to cL3. a) ELISA detecting C1q was performed to evaluate the ability of cL3 to bind C1 from 1% human serum. b) ELISA detecting

C5b-9 deposition to evaluate complement activation by cL3. In both a and b, streptavidin-coated plates are used and cL3 is bound directly via a biotin linker. €) As in a, but this time the azide- cL3 variant was used to immobilise cL3, before detection of C1q. d) As in b, but this time the azide-cL3 variant was used to immobilise cL3, before detection of C5b-9. e) Deletion mutants of cL3 binding were assessed to determine recruitment of C1q from 1% human serum. Lys(N3) denotes the lysine residue with the sidechain amine replaced by an azide, and * denotes that the cysteine has been cyclized via the N-terminal chloroacetyl modification. All error bars display the standard deviation.

Figure 4 shows activation of complement on lipid bilayer surfaces. a) Liposomes displaying cL3 can activate the C1s proteases. Raw fluorescence data is shown in the left. On the right, the fluorescence data with baseline activity of C1s in the presence of unmodified liposomes is set to 1, and displays a significant difference to the upregulated activity by cL3 modified liposomes (****, p < 0.001). b) Liposomes displaying cL3 can be lysed via complement activation, resulting in an increase in fluorescence. Blanks are liposomes that do not display cL3.

Figure 5 shows Inhibition of antibody-dependent complement activation by cL3. a) The ability of cL3 to inhibit complement activation by IgG antibody platforms was assessed. Human serum, 10%, was incubated with 100 uM of cL3 10 min, and fluorescence increase caused by liposome lysis was monitored. b) A titration of cL3 was performed under the same conditions as a (n=4 technical replicates). A one-way ANOVA, followed by a Tuckey test was performed comparing the fluorescent data after 20 minutes to the IgG condition {p < 0.05 *, p < 0.01 **, p < 0.001 ***). c) Competition ELISAs, performed in 1% human serum, determining cL3-mediated inhibition of

C1q and C5b-9 deposition by pooled IgG. Samples are normalized to the DMSO control (maximal signal, 100%) and no serum samples (minimal signal, 0%). d) Competition ELISAs as in ¢ determining inhibition of C5b-9 deposition by cL3-deletion mutants.

Figure 6 shows cL3 variants effect on complement modulation. a) ELISA determining the activation of complement in 1% human serum by azide modified inv-cL3 detecting C1q and

C5b-9. b) Competition ELISA showing inv-cL3 inhibiting C1q and C5b-9 deposition by blocking binding to pooled IgG. €} Competition ELISA showing lin-cL3 inhibiting C1q and C5b-9 deposition by blocking binding to pooled IgG. d) The ability of lin-L3 to inhibit complement activation by IgG was assessed in liposome lysis assays.

Figure 7 shows competition assays between cL3 and C1q binders. a) Competition ELISAs where either cL3 (left) or C1qNB75 (right) are immobilised and competed against solution phase

C1gNB75 (100 nM) or cL3 (20 pM), respectively, in the presence of 1% human serum. Binding of complement component C1q and deposition of C5b-9 was measured. b) ELISAs determining cL3-mediated inhibition of C1q binding and C5b-9 deposition by CRP. 1% human serum was incubated with cL3 before adding to the wells containing CRP. Samples are normalized to the

DMSO control (maximal signal, 100%) and no serum samples (minimal signal, 0%). ¢) Binding locations of CRP (grey) and C1gNB75 (black) on gC1q (white). d) C1 complex showing potential cL3 binding locations (black).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides new cyclic and linear peptides that bind to a globular head domain of mammalian complement component C1q and are able to either activate or inhibit complement.

The invention is based upon the inventors’ surprising finding that the short peptides according to the invention are able to bind to a globular head domain of mammalian complement component

C1q and that depending on whether they are surface-bound or in soluble form, can activate or inhibit complement, respectively.

General definitions

Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular

Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins

Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionary of many of the terms used in the invention.

Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein.

Accordingly, the terms defined immediately below are more fully described by reference to the

Specification as a whole. Also, as used herein, the singular terms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise. It is to be understood that this invention is not limited to the particular methodology, protocals, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

The term “complement component C1q binder” or “C1q binder” are used interchangeably herein and refer to any compound or construct that comprises the peptides disclosed herein. The construct can besides the peptide, for example, comprise a protein, oligo-/polypeptides, linker, drugs, peptides, conjugates, chemical modifications, combinations thereof, etc. In other words the peptides disclosed herein can form a part of a C1q binder together with other components or the peptide itself is the C1q binder in cases when no other components are comprised. In cases where the peptides disclosed herein form the C1q binder the terms “C1q binder according to the invention” and “peptide according to the invention” are used interchangeably.

The term “peptide” as used herein refers to a chain of amino acids linked by peptide bonds with a length of up to 50 amino acids, up to 40 amino acids, up to 30 amino acids, or up to 20 amino acids, and includes oligopeptides, dipeptides, tripeptides, and tetrapeptides. In cases of more than 20 amino acids the peptide may also be termed “polypeptide”. Preferably the peptide has a length of up to 20 amino acids. In one example the peptide is no more than 19 amino acids long, no more than 18 amino acids long, no more than 17 amino acids long, no more than 16 amino acids long, no more than 15 amino acids long, or no more than 14 amino acids long. The term “peptide” as used herein refers to the chain of amino acids without covering by its meaning any linkers, conjugates, or labels that can be attached to the peptide. In case of a cyclic peptide the term “peptide” refers to the part that forms the ring. Any further amino acids would not be considered to fall under the definition peptide but may be a linker. The peptide according to the invention can also be part of a protein and can be introduced into the protein at the N-terminus, at the C-terminus, or between the N- and the C-terminus in a way that it imbues the protein with

C1q binding properties.

The N-terminus of a peptide (also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) is the start of a peptide terminated by an amino acid with a free amine group (-NH2). By convention, peptide sequences are written N-terminus to C-terminus (from left to right). The C-terminus (also known as the carboxyl-terminus, carboxy-terminus, C-terminal tail,

C-terminal end, or COOH-terminus) is the end of an amino acid chain (protein or peptide), terminated by a free carboxyl group (-COOH) for linear peptides. In (macro)cyclic peptides the

C-terminus is may, for example, be amidated without having a free carboxyl group, and the N- terminus may be part of the cyclic ring.

The term “amino acid” as used herein refers to organic compounds that contain both amino and carboxylic acid functional groups, including proteinogenic (protein-building, natural), non- proteinogenic (non-natural), and modified amino acids. The term “amino acid” as used herein includes alpha, non-alpha, L, D, essential and non-essential amino acids. A skilled person will appreciate that when two or more amino acids combine to form a peptide, the elements of water are removed, and what remains of each amino acid is called an amino-acid residue. The amino acid residue is the part of an amino acid that makes it unique from all the others. As such, reference herein to an 'amino acid’ in the context of an amino acid sequence contained within a peptide will be understood to refer to the respective amino acid residue as appropriate. The residues may be a "naturally occurring amino acid residue" (i.e. encoded by the genetic code) and selected from the group consisting of: alanine (Ala, A); arginine (Arg, R}); asparagine (Asn,

N); aspartic acid (Asp, D); cysteine (Cys, C); glutamine (Gln, Q); glutamic acid (Glu, E); glycine (Gly, G); histidine (His, H); isoleucine (lle, I): leucine (Leu; L); lysine (Lys, K); methionine (Met,

M); phenylalanine (Phe, F); proline (Pro, P); serine (Ser, S); threonine (Thr, T); tryptophan (Trp,

W); tyrosine (Tyr, Y); and valine (Val, V). “Non-proteinogenic amino acids” as used herein refers to D-amino acids, homo amino acids, beta-homo amino acids, N-methyl amino acids, alpha-methyl amino acids, non-natural side chain variant amino acids and other unusual amino acids. Further examples for “non- proteinogenic amino acids” are 6-azido-L-lysine, biotinylated lysine, or alkyne-derivatised amino acids.

The “(mammalian) complement component C1q (C1q) ” as used herein refers to the ligand- binding unit of the C1 complex of complement and is a pattern recognition molecule. C1q is a 460-kDa hexameric protein assembled from six heterotrimeric collagen-like fibers, each being prolonged by a C-terminal globular domain which mediates the recognition function of C1. The terms “globular head domain of C1q”, “globular domain of C19”, “globular head of C19", “gC1q”, or “C1q globular domain” are used interchangeably herein and refer to a heterotrimeric association of protein modules known as gC1q domains, which comprise the non-collagenous

C-terminal region of C1q.

The term “complement activation” as used herein means any way of triggering or inducing the cascade of an immune response associated with complement at any stage of the cascade (see

Fig. 1a). In particular, the activation of C1 is considered to correspond to the complement activation as used herein. Several methods for determining if complement has been activated are known in the art. For example, activation of the C1s protease was assayed using Boc-Leu-

Gly-Arg-Amino Methyl Cumarin (LGR-AMC), which becomes fluorescent upon cleavage by C1s (Fig. 4a). Further examples are described in the Example section. For example, C1 activation leads of deposition of complement components C5b-9, which can be detected using ELISA (e.g., Fig. 3b). Similarly, complement activation can lead to the formation of the MAC pore, which can be detected in liposome lysis assays (e.g., Fig. 4b).

The term “complement inhibition” as used herein means any way of stopping or blocking the cascade of an immune response associated with complement at any stage of the cascade (see

Fig. 1a). The inhibition of the complement can for example result in a reduced activity of the C1 complex or lead to no or reduced formation of the membrane attack complex (MAC).

The term "nucleic acid" as used herein typically refers to an oligomer or polymer (preferably a linear polymer) of any length composed of nucleotides. A nucleotide unit commonly includes a heterocyclic base, a sugar group, and at least one, e.g. one, two, or three, phosphate groups, including modified or substituted phosphate groups. Heterocyclic bases may include inter alia purine and pyrimidine bases such as adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U) which are widespread in naturally-occurring nucleic acids, other naturally-occurring bases (e.g., xanthine, inosine, hypoxanthine) as well as chemically or biochemically modified (e.g., methylated), non-natural or derivatised bases. Sugar groups may include inter alia pentose (pentofuranose) groups such as preferably ribose and/or 2-deoxyribose common in naturally-occurring nucleic acids, or arabinose, 2-deoxyarabinose, threose or hexose sugar groups, as well as modified or substituted sugar groups. Nucleic acids as intended herein may include naturally occurring nucleotides, modified nucleotides or mixtures thereof. A modified nucleotide may include a modified heterocyclic base, a modified sugar moiety, a modified phosphate group or a combination thereof. Modifications of phosphate groups or sugars may be introduced to improve stability, resistance to enzymatic degradation, or some other useful property. The term nucleic acid further preferably encompasses DNA, RNA and DNA RNA hybrid molecules, specifically including hnRNA, pre-mRNA, mRNA, cDNA, genomic DNA, amplification products, oligonucleotides, and synthetic {e.g., chemically synthesised) DNA, RNA or DNA RNA hybrids. A nucleic acid can be naturally occurring, e.g., present in or isolated from nature; or can be non-naturally occurring, e.g., recombinant, i.e., produced by recombinant DNA technology, and/or partly or entirely, chemically or biochemically synthesised. A nucleic acid can be double-stranded, partly double stranded, or single-stranded. Where single-stranded, the nucleic acid can be the sense strand or the antisense strand. In addition, nucleic acid can be circular or linear. The terms “polynucleotide” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof.

Certain sequences provided herein are described using percent identity to a sequence with a defined amino acid or nucleic acid sequence. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes may be at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. Preferably, the percent identity between two amino acid sequences is determined using the Needleman et al. (1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a BLOSUM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

Preferably, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a

NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is within a sequence identity or homology limitation of the invention) are a BLOSUM 82 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

Alternatively, the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers et al. (1989) CABIOS 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-410). BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Altschul et al. (1997, Nucl. Acids Res. 25:3389-3402). When using BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

See <http://www.ncbi.nlm.nih. gov>.

A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) of “sequence identity” to another sequence. This means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Ausubel et al. eds. (2007) Current Protocols in Molecular

Biology. Preferably, default parameters are used for alignment. One alignment program is

BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE;

Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations +

SwissProtein + SPupdate + PIR. Details of these programs can be found at the following

Internet address: ncbi.nlm.nih.gov/cgi-bin/BLAST. The terms “xx % sequence identity” and “xx % identity” are used interchangeably herein. A nucleic acid of the invention may be a vector.

The term “vector” is well known in the art, and as used herein refers to a nucleic acid molecule, e.g. double-stranded DNA. In one example, the vector has an exogenous nucleic acid sequence inserted into it. A vector can suitably be used to transport an inserted nucleic acid molecule into a suitable host cell. A vector typically contains all of the necessary elements that permit transcribing the insert nucleic acid molecule, and, preferably, translating the transcript into a polypeptide. A vector typically contains all of the necessary elements such that, once the vector is in a suitable host cell, the vector can replicate independently of, or coincidental with, the host chromosomal DNA; several copies of the vector and its inserted nucleic acid molecule may be generated. Vectors of the present invention can be episomal vectors (i.e., that do not integrate into the genome of a host cell} or can be vectors that integrate into the host cell genome. This definition includes both non-viral and viral vectors. Non-viral vectors include but are not limited to plasmid vectors (e.g. pMA-RQ, pUC vectors, bluescript vectors (pBS) and pBR322 or derivatives thereof that are devoid of bacterial sequences (minicircles)) transposons-based vectors (e.g. PiggyBac (PB) vectors or Sleeping Beauty (SB) vectors), etc. Larger vectors such as artificial chromosomes (bacteria (BAC), yeast (YAC), or human (HAC)) may be used to accommodate larger inserts. In one particular example, a vector described herein may therefore be a plasmid vector. Such plasmid vectors may be present within a cell. In one example, therefore a cell may be provided which comprises a vector (e.g. a plasmid as described herein) comprising a nucleic acid sequence described herein. A cell may therefore be provided comprising a nucleic acid sequence of the invention.

A vector as defined herein may also be a viral vector. A “viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors (AAV), alphavirus vectors and the like. Typically, but not necessarily, viral vectors are replication-deficient as they have lost the ability to propagate in a given cell since viral genes essential for replication have been eliminated from the viral vector. However, some viral vectors can also be adapted to replicate specifically or preferentially in a given cell, such as e.g. a cancer cell, and are typically used to trigger the (cancer) cell-specific (onco)lysis. These viral vectors are referred to herein as “oncolytic viruses”. Virosomes are a non-limiting example of a vector that comprises both viral and non-viral elements, in particular they combine liposomes with an inactivated HIV or influenza virus (Yamada et al., 2003). Another example encompasses viral vectors mixed with cationic lipids.

While it is possible to use in vitro translation or chemical (solid-phase) peptide synthesisers to produce a peptide as disclosed herein, cellular expression systems may be used in the routine.

Suitable host cells for cloning or expression/secretion of peptide-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, peptides may be produced in bacteria, in particular when glycosylation is not needed (see, e.g., US 5,648,237, US 5,789,199 and US 5,840,523, Charlton, Methods in Molecular Biology 248 (2003) 245-254 (B.K.C. Lo, (ed.), Humana Press, Totowa, NJ), describing expression of antibody fragments in E. coll.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeasts are suitable cloning or expression hosts for polypeptide-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of a polypeptide with a partially or fully human glycosylation pattern (see e.g. Gerngross, Nat.

Biotech. 22 (2004) 1409- 1414, and Li, et al, Nat. Biotech. 24 (2006) 210-215).

Suitable host cells for the expression of peptides are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells.

Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts (see, e.g., US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978 and US 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants)).

Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are the COS-7 cell line (monkey kidney CVI cell transformed by SV40); the HEK293 cell line (human embryonic kidney); the BHK cell line (baby hamster kidney); the TM4 mouse Sertoli cell line (TM4 cells as described, e.g., in Mather, Biol. Reprod. 23 (1980) 243-251); the CVI cell line (monkey kidney cell); the VERO-76 cell line (African green monkey kidney cell); the HELA cell line (human cervical carcinoma cell); the MDCK cell line (canine kidney cell); the BRL-3A cell line (buffalo rat liver cell); the W138 cell line (human lung cell); the HepG2 cell line (human liver cell); the MMT 060562 cell line (mouse mammary tumor cell); the TRI cell line (e.g. described in Mather, et al, Anal. N.Y. Acad. Sci. 383 (1982) 44-68); the MRC5 cell line; and the

FS4 cells-line. Other useful mammalian host cell lines include the CHO cell line (Chinese hamster ovary cell), including DHFR negative CHO cell lines (see e.g. Urlaub, et al, Proc. Natl.

Acad. Sci. USA 77 (1980) 4216), and myeloma cell lines such as YO, NSO and Sp2/0 cell line.

For a review of certain mammalian host cell lines suitable for peptide production, see, e.g.,

Yazaki, and Wu, Methods in Molecular Biology, Antibody Engineering 248 (2004) 255-268 (B.K.C. Lo, (ed.), Humana Press, Totowa, NJ).

As understood by a person skilled in the medical art, the terms, "treat" and "treatment," refer to medical management of a disease, disorder, or condition of a subject (i.e., patient) (see, e.g.,

Stedman's Medical Dictionary). In general, an appropriate dose and treatment regimen provide the inhibitor in an amount sufficient to provide therapeutic and/or prophylactic benefit.

Therapeutic benefit for subjects to whom the inhibitor described herein is administered, includes, for example, an improved clinical outcome, wherein the object is to prevent or slow or retard (lessen) an undesired physiological change associated with the disease, or to prevent or slow or retard (lessen) the expansion or severity of such disease. As discussed herein, effectiveness of the inhibitor may include beneficial or desired clinical results that comprise, but are not limited to, abatement, lessening, or alleviation of symptoms that result from or are associated with the disease to be treated; decreased occurrence of symptoms; improved quality of life; longer disease-free status (i.e., decreasing the likelihood or the propensity that a subject will present symptoms on the basis of which a diagnosis of a disease is made); diminishment of extent of disease; stabilized (i.e., not worsening) state of disease; delay or slowing of disease progression; amelioration or palliation of the disease state; and remission {whether partial or total), whether detectable or undetectable; and/or overall survival.

As used herein, the phrase “a disorder associated with complement activation or inhibition involving C1q” refers to but is not limited to any disease or disorder resulting directly or indirectly from and/or completely or partially from complement activation or inhibition involving C1q.

Herein the terms “diseases”, “disorders”, and “conditions” are used interchangeably and refer to a disorder of structure or function in a human or animal, especially one that produces specific symptoms or that affects a specific location and is not simply a direct result of physical injury.

Diseases that can be treated and/or prevented with the peptide described herein are described elsewhere herein in detail.

As used herein the term “subject” refers to a mammalian individual, e.g., a human, dog, cat, pig, horse, mouse, cow, rat etc having or at risk of having a specified condition, disorder or symptom. The subject may be a patient i.e., a subject in need of treatment in accordance with the invention. The subject may have received treatment for the condition, disorder or symptom.

Alternatively, the subject has not been treated prior to treatment in accordance with the present invention. The subject is preferably in need of administration of a peptide of the invention.

As used herein, the “administration” or “administering” of a (pharmaceutical) composition described herein to a subject includes any route of introducing or delivering to a subject which allows for the composition to perform its intended function. Administration can be carried out by any suitable route, including orally, intranasally, intraocularly, ophthalmically, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), or topically. Administration includes self-administration and the administration by another. The composition can be administered as a therapeutically effective amount. As used herein, the phrase “therapeutically effective amount” means a dose or plasma concentration in a subject that provides the specific pharmacological effect for which the described compositions are administered, e.g., to treat a disease of interest in a target subject. The therapeutically effective amount may vary based on the route of administration and dosage form, the age and weight of the subject, and/or the disease or condition being treated.

A composition may be a pharmaceutical composition or formulation that comprises the peptide, a sequence encoding the peptide, or cell comprising the nucleic acid encoding the peptide and a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier. Pharmaceutical compositions or formulations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents or compounds.

As used herein, "pharmaceutically acceptable” refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected peptide without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

Excipients are natural or synthetic substances formulated alongside an active ingredient (e.g. a neurotoxin as provided herein), included for the purpose of bulking-up the formulation or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption or solubility. Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life. Pharmaceutically acceptable excipients are well known in the art. A suitable excipient is therefore easily identifiable by one of ordinary skill in the art. By way of example, suitable pharmaceutically acceptable excipients include water, saline, aqueous dextrose, glycerol, ethanol, and the like.

Adjuvants are pharmacological and/or immunological agents that modify the effect of other agents in a formulation. Pharmaceutically acceptable adjuvants are well known in the art and include cell-penetrating peptides. A suitable adjuvant is therefore easily identifiable by one of ordinary skill in the art.

Diluents are diluting agents. Pharmaceutically acceptable diluents are well known in the art and include water or saline. A suitable diluent is therefore easily identifiable by one of ordinary skill in the art.

Carriers are non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients of the formulation. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. Pharmaceutically acceptable carriers are well known in the art and include serum albumin. A suitable carrier is therefore easily identifiable by one of ordinary skill in the art.

Peptides

According to a first aspect of the invention a complement component C1q binder is provided comprising a peptide comprising the amino acid sequence YX Xo TFYXaX4XsFTLQFIXsX7 (SEQ

ID NO. 1), wherein

Xs is absent, T, or K,

Xz is absent or V,

X3 is any amino acid,

Xa is Dor N,

Xs is any amino acid,

Xs is absent, A,E or T, and

Xz is C, S or a stop codon, on the proviso that a maximum of two of X4, Xz, and Xs are absent, wherein the peptide binds to a globular head domain of mammalian complement component C1q.

In one example X3 or Xs is P. In one example X3 and Xs are P.

In one example X; is T.

In one example Xz is V.

In one example X4 is D.

In one example Xs is A.

In one example Xz is C.

In one example X; is T, and Xz is V.

In one example X; is T, Xo is V, and Xa is P.

In one example X; is T, Xz is V, Xs is P, and Xs is D.

In one example X; is T, X2 is V, X3 is P, Xs is D, and Xs is P.

In one example X; is T, X2 is V, A3 is P, Xs is D, Xs is P, and Xs is A.

In one example the peptide comprises the sequence YTVTFYPDPFTLQFIAX; (SEQ ID NO. 2), wherein X7 is C or S.

In one example the peptide is YTVTFYPDPFTLQFIAC (SEQ ID NO. 3).

In one example the peptide is YVTFYPDPFTLQFIAC (SEQ ID NO. 4).

In one example the peptide is YTFYPDPFTLQFIAC (SEQ ID NO. 5).

In one example the peptide is YPDPFTLQFIAC (SEQ ID NO. 6).

In one example the peptide is YDPFTLQFIAC (SEQ ID NO. 7).

In one example the peptide is YFTLQFIAC (SEQ ID NO. 8).

In one example the peptide is YTVTFYPDPFTLQFIC (SEQ ID NO. 9).

In one example the peptide is YTVTFYPDPFTLQFC (SEQ ID NO. 10).

In one example the peptide is YTVTFYPDPFTLC (SEQ ID NO. 11).

In one example the peptide is YTVTFYPDPFC (SEQ ID NO. 12).

In one example the peptide is YTVTFYPDPFTLQFIAS (SEQ ID NO. 13).

In one example the peptide is YVTFYPDPFTLQFIAS (SEQ ID NO. 14).

In one example the peptide is YTFYPDPFTLQFIAS (SEQ ID NO. 15).

In one example the peptide is YPDPFTLQFIAS (SEQ ID NO. 18).

In one example the peptide is YDPFTLQFIAS (SEQ ID NO. 17).

In one example the peptide is YFTLQFIAS (SEQ ID NO. 18).

In one example the peptide is YTVTFYPDPFTLQFIS (SEQ ID NO. 19).

In one example the peptide is YTVTFYPDPFTLQFS (SEQ ID NO. 20).

In one example the peptide is YTVTFYPDPFTLS (SEQ ID NO. 21).

In one example the peptide is YTVTFYPDPFS (SEQ ID NO. 22).

A peptide of the invention may carry a detectable label. Suitable labels include radioisotopes, fluorescent labels, enzyme labels, or other protein labels such as biotin.

In one example the peptide is surface-bound and is capable of activating complement.

The term “surface-bound” as used herein refers to any form of immobilization of the peptide on any surface of any molecule, device, compound etc. As soon as the peptide is bound to something else and is no longer just the peptide itself, it is considered as surface-bound according to the present invention. Appropriate “surfaces” to which the peptide may be bound may be cells, cell blebs or fragments, lipid membranes, hydrogels, or solid surfaces such as beads or plastic adsorbent surfaces or ELISA/detection plates.

By “activating complement” a cascade of proteolytic and binding events is initiated which pathway can terminate with the formation of the membrane attack complex (MAC). “Complement activation” by the C1 complex involves activation of the C1s protease, cleavage of

C4 by C1s, deposition of opsonins, formation of C3 and C5 convertases, and MAC pore formation, and can stop at any of these points.

In one example the peptide is in soluble form and is capable of inhibiting complement activation.

The term “inhibiting complement activation” as used herein refers to any reduction, abolishment, prevention, blocking, suppression, slowing, or interference of the classical complement pathway as shown in Fig. 1a. Inhibition may be reversible or irreversible.

In a particular example, the peptide inhibits complement activation by inhibiting the binding of

C1q to its ligand (as it is known to a person skilled in the art, C1q as the ligand-binding unit of the C1 complex of complement, is a pattern recognition molecule with the unique ability to sense a huge variety of targets, e.g., IgG or C-reactive protein (CRP)).

In another example, the peptide may allow binding of a C1q binding ligand to C1q but still inhibits complement activation.

In some examples, the inhibition may reduce complement activation by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% as compared to the activity of a control (e.g., activity in the absence of the inhibitor).

Functional tests to determine inhibition of complement activation are known to a person skilled in the art. Examples for such tests are provided in the Examples section herein.

In some examples, the peptide inhibits, reduces, slows, halts, blocks, supresses, abolishes and/or prevents complement activation.

In one example the peptide is a cyclic peptide, wherein the N-terminal end of the peptide binds to the C-terminal end of Xs.

The cyclic form provides the peptides with a higher resistance to proteases than linear peptides leading to an increased stability of cyclic peptides.

The term “cyclic peptide” as used herein refers to a ring structure formed by the peptide obtained through binding between Y as the first amino acid forming the N-terminal end of the peptide with X7 as the last amino acid forming the C-terminal end of the peptide. Typically an amino terminus and a carboxy terminus (so called head-to-tail cyclisation), an amino terminus and a sidechain (so called head-to-sidechain cyclisation), carboxy terminus and a sidechain (so called sidechain-to-tail cyclisation), or a side chain and a side chain (so called sidechain-to- sidechain cyclisation) may be linked with a covalent bond to form a cyclic peptide. Head-to-tail cyclic peptides may typically be formed by amide bond formation. Sidechain-to-sidechain cycles may typically be formed via Cys-Cys disulfide bridge formation or amide bond formation within a cyclic peptide. Alternatively, an amino terminus, a carboxy terminus or a side chain may be linked with a covalent bond to the peptide backbone to form a cyclic peptide. A person skilled in the art would be well aware that there are various ways of how the N-terminal and/or C-terminal end of the peptide could be designed in order to obtain a cyclisation or the peptide. A possible way could be that an N-terminal lysine at position 1 reacts with a glutamic acid at position 7 to form a side-chain to side-chain amide bond (also known as a lactam).

In one example the N-terminal tyrosine is chloroacetylated (CI-Ac).

In one example the peptide is (CI-Ac)YTVTFYPDPFTLQFIAC (SEQ ID NO. 23).

In one example the peptide is (CI-Ac)YVTFYPDPFTLQFIAC (SEQ ID NO. 24).

In one example the peptide is (CI-Ac)YTFYPDPFTLQFIAC (SEQ ID NO. 25).

In one example the peptide is (CI-Ac)YPDPFTLQFIAC (SEQ ID NO. 26).

In one example the peptide is (CI-Ac)YDPFTLQFIAC (SEQ ID NO. 27).

In one example the peptide is (CI-Ac)YFTLQFIAC (SEQ ID NO. 28).

In one example the peptide is (CI-Ac)YTVTFYPDPFTLQFIC (SEQ ID NO. 29).

In one example the peptide is (CI-Ac)YTVTFYPDPFTLQFC (SEQ ID NO. 30).

In one example the peptide is (CI-Ac)YTVTFYPDPFTLC (SEQ ID NO. 31).

In one example the peptide is (CI-Ac)YTVTFYPDPFC (SEQ ID NO. 32).

In one example the peptide is (CI-Ac)YTVTFYPDPFTLQFIAS (SEQ ID NO. 33).

In one example the peptide is (CI-Ac)YVTFYPDPFTLQFIAS (SEQ ID NO. 34).

In one example the peptide is (CI-AC)YTFYPDPFTLQFIAS (SEQ ID NO. 35).

In one example the peptide is (CI-AC)YPDPFTLQFIAS (SEQ ID NO. 36).

In one example the peptide is (CI-AC)YDPFTLGFIAS (SEQ ID NO. 37).

In one example the peptide is (CI-Ac)YFTLQFIAS (SEQ ID NO. 38).

In one example the peptide is (CI-Ac)YTVTFYPDPFTLQFIS (SEQ ID NO. 39).

In one example the peptide is (CI-Ac)YTVTFYPDPFTLQFS (SEQ ID NO. 40).

In one example the peptide is (CI-AC)YTVTFYPDPFTLS (SEQ ID NO. 41).

In one example the peptide is (CI-Ac)YTVTFYPDPFS (SEQ ID NO. 42).

The chloroacetylation of the N-terminal end can lead to a cyclisation of the peptide by a reaction between the chloroacetyl group and a thiol moiety, such as found on a cysteine residue.

In one example Xz, i.e. the C-terminal end, is C.

In one example Xs, i.e. the C-terminal end, is C and the chloroacetylated N-terminal Y reacts with and binds to the cysteine residue by covalent bond formation, resulting in a cyclisation of the peptide.

In one example the C-terminal and/or N-terminal end is configured to conjugate to one or more distinct moieties. In another example other amino acids of the peptide are configured to conjugate to one or more distinct moieties.

The term “distinct” as used herein refers to any moiety that is not part of the described peptide, i.e. any moiety that is added to the peptide YX X2TFYXsXeXsFTLQFIXeX7 (SEQ ID NO. 1).

The term “moiety” as used herein refers to any possible binding partner. The following is a non- exhaustive list of examples for possible moieties: amino acids, linkers, polymers, chemicals,

DNA-sequences, RNA-sequences, nucleotide sequences, peptides, compounds, drugs. For example, targeting molecules such as nanobodies, antibody fragments such as Fab domains or single-chain Fv fragments, whole antibodies, DNA nanostructure platforms, or protein scaffolds, or combinations thereof. Further examples are described in “DNA nanostructure-templated antibody complexes provide insights into the geometric requirements of human complement cascade activation”, Abendstein et al., bioRxiv,Oct. 2023.

In one example a combination of distinct moieties binds to the C-terminal end. The combination can be in sequential order, like a linker followed by one or more amino acids.

In one example the one or more distinct moiety is a linker.

The term “linker” as used herein can refer to any other linker known to a person skilled in the art that connects the peptide with the distinct moiety to form a conjugate. It can be a cleavable or uncleavable linker. These include, but are not limited to, natural or non-natural amino acids, polyethylene glycol linkers of various length, mono- or bi-functional linkers.

The term “linker” as used herein may refer to any amino acid sequence able to link the peptide to another further distinct moiety. Thereby the linker is chosen in a way to support the desired purpose, for example by exposing the peptide or the moiety further distinct moiety. A person skilled in the art would be well aware of how to choose an appropriate linker. Suitable linker sequences could be identified by literature search.

In one embodiment, the C-terminal linker sequence has a length of at least 2 amino acids, preferably at least 3 amino acids, more preferably of at least 4 amino acids.

In one example the one or more distinct moiety is a GS-linker selected from the group consisting of GS, GSGS (SEQ ID NO. 43), GGGS (SEQ ID NO. 44), and GSGGSG (SEQ ID

NO. 45).

In one example the peptide is YTVTFYPDPFTLQFIACGSK (SEQ ID NO. 48).

In one example the peptide is YVTFYPDPFTLQFIACGSK (SEQ ID NO. 47).

In one example the peptide is YTFYPDPFTLQFIACGSK (SEQ ID NO. 48).

In one example the peptide is YPDPFTLQFIACGSK (SEQ ID NO. 49).

In one example the peptide is YDPFTLQFIACGSK (SEQ ID NO. 50).

In one example the peptide is YFTLQFIACGSK (SEQ ID NO. 51).

In one example the peptide is YTVTFYPDPFTLQFICGSK (SEQ ID NO. 52).

In one example the peptide is YTVTFYPDPFTLQFCGSK (SEQ ID NO. 53).

In one example the peptide is YTVTFYPDPFTLCGSK (SEQ ID NO. 54).

In one example the peptide is YTVTFYPDPFCGSK (SEQ ID NO. 55).

In one example the peptide is YTVTFYPDPFTLQFIASGSK (SEQ ID NO. 56).

In one example the peptide is YVTFYPDPFTLQFIASGSK (SEQ ID NO. 57).

In one example the peptide is YTFYPDPFTLQFIASGSK (SEQ ID NO. 58).

In one example the peptide is YPDPFTLQFIASGSK (SEQ ID NO. 59).

In one example the peptide is YDPFTLQFIASGSK (SEQ ID NO. 60).

In one example the peptide is YFTLQFIASGSK (SEQ ID NO. 61).

In one example the peptide is YTVTFYPDPFTLQFISGSK (SEQ ID NO. 62).

In one example the peptide is YTVTFYPDPFTLQFSGSK (SEQ ID NO. 63).

In one example the peptide is YTVTFYPDPFTLSGSK (SEQ ID NO. 64).

In one example the peptide is YTVTFYPDPFSGSK (SEQ ID NO. 65).

In one example the peptide is (CI-Ac)YTVTFYPDPFTLQFIACGSK (SEQ ID NO. 66).

In one example the peptide is (CI-Ac)YVTFYPDPFTLQFIACGSK (SEQ ID NO. 67).

In one example the peptide is (CI-Ac)YTFYPDPFTLQFIACGSK (SEQ ID NO. 68).

In one example the peptide is (CI-Ac)YPDPFTLQFIACGSK (SEQ ID NO. 69).

In one example the peptide is (CI-Ac)YDPFTLQFIACGSK (SEQ ID NO. 70).

In one example the peptide is (CI-Ac)YFTLQFIACGSK (SEQ ID NO. 71).

In one example the peptide is (CI-AC)YTVTFYPDPFTLQFICGSK (SEQ ID NO. 72).

In one example the peptide is (CI-AC)YTVTFYPDPFTLQFCGSK (SEQ ID NO. 73).

In one example the peptide is (CI-AC)YTVTFYPDPFTLCGSK (SEQ ID NO. 74).

In one example the peptide is (CI-Ac)YTVTFYPDPFCGSK (SEQ ID NO. 75).

In one example the peptide is (CI-Ac)YTVTFYPDPFTLQFIASGSK (SEQ ID NO. 78).

In one example the peptide is (CI-Ac)YVTFYPDPFTLQFIASGSK (SEQ ID NO. 77).

In one example the peptide is (CI-Ac)YTFYPDPFTLQFIASGSK (SEQ ID NO. 78).

In one example the peptide is (CI-Ac)YPDPFTLQFIASGSK (SEQ ID NO. 79).

In one example the peptide is (CI-Ac)YDPFTLQFIASGSK (SEQ ID NO. 80).

In one example the peptide is (CI-Ac)YFTLQFIASGSK (SEQ ID NO. 81).

In one example the peptide is (CI-Ac)YTVTFYPDPFTLQFISGSK (SEQ ID NO. 82).

In one example the peptide is (CI-AcC)YTVTFYPDPFTLQFSGSK (SEQ ID NO. 83).

In one example the peptide is (CI-AC)YTVTFYPDPFTLSGSK (SEQ ID NO. 84).

In one example the peptide is (CI-Ac)YTVTFYPDPFSGSK (SEQ ID NO. 85).

In one example the peptide comprises one of the sequences selected from the group consisting of YTVTFYX3DXsFTLQFIACGSK (SEQ ID NO. 86), wherein Xz and/or Xs is/are any amino acid,

YTVTFYPDXsFTLQFIACGSK (SEQ ID NO. 87), wherein Xs is any amino acid,

YTVTFEYX3DPFTLQFIACGSK (SEQ ID NO. 88), wherein Xs is any amino acid.

In one example the peptide comprises one of the sequences selected from the group consisting of YTVTFYXsDXsFTLQFIACGSK (SEQ ID NO. 89), wherein X3 and/or Xs is/are any amino acid and wherein the N-terminal Y is chloroacetylated, YTVTFYPDXsFTLQFIACGSK (SEQ ID NO. 90), wherein Xs is any amino acid and wherein the N-terminal Y is chloroacetylated,

YTVTFYX3DPFTLQFIACGSK (SEQ ID NO. 91), wherein Xs is any amino acid and wherein the

N-terminal Y is chloroacetylated.

In one example the peptide comprises one of the sequences selected from the group consisting of YTVTFYX:DXsFTLQFIACGSK (SEQ ID NO. 92), wherein X3 and/or Xs is/are any amino acid and wherein the N-terminal Y is chloroacetylated and wherein the peptide is cyclic, YTVTFYPDXsFTLQFIACGSK (SEQ ID NO. 93), wherein Xs is any amino acid wherein the

N-terminal Y is chloroacetylated and wherein the peptide is cyclic,

YTVTFYX:DPFTLQFIACGSK (SEQ ID NO. 94), wherein Xs is any amino acid, wherein the N- terminal Y is chloroacetylated and wherein the peptide is cyclic.

In one example the peptide comprises the sequence YTVTFYPDPFTLQFIACGSK (SEQ ID NO. 95), wherein the N-terminal Y is chloroacetylated and wherein the peptide is cyclic.

In one example the peptide consists of one of the sequences selected from the group consisting of YTVTFYXsDXsFTLQFIACGSK (SEQ ID NO. 86), wherein Xs and/or Xs is/are any amino acid,

YTVTFYPDXsFTLQFIACGSK (SEQ ID NO. 87), wherein Xs is any amino acid,

YTVTFYX:DPFTLQFIACGSK (SEQ ID NO. 88), wherein Xs is any amino acid.

In one example the peptide consists of one of the sequences selected from the group consisting of YTVTFYX:DXsFTLQFIACGSK (SEQ ID NO. 89), wherein X3 and/or Xs is/are any amino acid and wherein the N-terminal Y is chloroacetylated, YTVTFYPDXsFTLQFIACGSK (SEQ ID NO. 90), wherein Xs is any amino acid wherein the N-terminal Y is chloroacetylated,

YTVTFYXasDPFTLQFIACGSK (SEQ ID NO. 91), wherein Xs is any amino acid and wherein the

N-terminal Y is chloroacetylated.

In one example the peptide consists of one of the sequences selected from the group consisting of YTVTFYXsDXsFTLQFIACGSK (SEQ ID NO. 92), wherein X3 and/or Xs is/are any amino acid and wherein the N-terminal Y is chloroacetylated and wherein the peptide is cyclic, YTVTFYPDXsFTLQFIACGSK (SEQ ID NO. 93), wherein Xs is any amino acid wherein the

N-terminal Y is chloroacetylated and wherein the peptide is cyclic,

YTVTFYXasDPFTLQFIACGSK (SEQ ID NO. 94), wherein Xs is any amino acid and wherein the

N-terminal Y is chloroacetylated and wherein the peptide is cyclic.

In one example the cyclic peptide consists of the sequence YTVTFYPDPFTLQFIACGSK (SEQ

ID NO. 95), wherein the N-terminal Y is chloroacetylated, and wherein the peptide is cyclic.

For the avoidance of doubt, when the peptide is described herein as consisting of a specific sequence, this still encompasses the peptide being bound by additional (distinct) moieties, such as an additional linker, detection agent etc.

In one example the peptide is a linear peptide.

In one example the C1q binder comprises a protein and the linear peptide.

The term “linear” as used herein refers to a peptide wherein the comprised amino acid only bind to the adjacent N-terminal and C-terminal amino acid, and the two amino acids at the N- and C- terminal end do not bind to each other. For example, the amino acid at position 3 of the peptide disclosed herein only binds to the amino acids at positions 2 and 4 in order to form a peptide but does not bind to the amino acids at positions 1, and 5 to 17. By this any cyclic structures within the peptide itself are excluded and avoided. A person skilled in the art would be well aware how to avoid cyclisation of the peptide and how to obtain a linear peptide.

In one example the peptide is a linear peptide, wherein X7 is any amino acid other than C and/or wherein the N-terminal tyrosine is not chloroacetylated (CI-Ac). This means that by X7 not being

C or by not chloroacetylating the N-Terminal end a linear peptide can be obtained since a cyclisation is not possible if one or both of the alternatives is/are fulfilled.

In one example the linear peptide comprises one of the sequences selected from the group consisting of YTVTFYX3DXsFTLQFIAX;7 (SEQ ID NO. 98), wherein X3 and/or Xs is/are any amino acid and wherein X7 is any amino acid other than C and/or wherein the N-terminal tyrosine is not chloroacetylated (CI-Ac), YTVTFYPDXsFTLQFIAX; (SEQ ID NO. 97), wherein Xs is any amino acid and wherein X7 is any amino acid other than C and/or wherein the N-terminal tyrosine is not chloroacetylated (CI-Ac), YTVTFYX3DPFTLQFIAX; (SEQ ID NO. 98), wherein X3 is any amino acid and wherein X7 is any amino acid other than C and/or wherein the N-terminal tyrosine is not chloroacetylated (CI-Ac).

In one example the linear peptide comprises one of the sequences selected from the group consisting of YTVTFYX3DXsFTLQFIAX;7 (SEQ ID NO. 99), wherein X3 and/or Xs is/are any amino acid, wherein Xz is any amino acid other than C and wherein the N-terminal Y is chloroacetylated, YTVTFYPDXsFTLQFIAX; (SEQ ID NO. 100), wherein Xs is any amino acid, wherein X7 is any amino acid other than C and wherein the N-terminal Y is chloroacetylated,

YTVTFEYX3DPFTLQFIAX;7 (SEQ ID NO. 101), wherein Xs is any amino acid, wherein X7is any amino acid other than C, and wherein the N-terminal Y is chloroacetylated.

In one example the linear peptide comprises one of the sequences selected from the group consisting of YTVTFYX3DXsFTLQFIAC (SEQ ID NO. 102), wherein Xs and/or Xs is/are any amino acid and wherein the N-terminal Y is not chloroacetylated, YTVTFYPDXsFTLQFIAC

(SEQ ID NO. 103), wherein Xs is any amino acid and wherein the N-terminal Y is not chloroacetylated, YTVTFYX3DPFTLQFIAC (SEQ ID NO. 104), wherein Xs is any amino acid and wherein the N-terminal Y is not chloroacetylated.

In one example the linear peptide comprises one of the sequences selected from the group consisting of YTVTFYX3DX5FTLQFIAC (SEQ ID NO. 107), wherein Xs and/or Xs is/are any amino acid and wherein the N-terminal Y is chloroacetylated, YTVTFYPDXsFTLQFIAC (SEQ ID

NO. 108), wherein Xs is any amino acid and wherein the N-terminal Y is chloroacetylated,

YTVTFYX3DPFTLQFIAC (SEQ ID NO. 109), wherein Xs is any amino acid and wherein the N- terminal Y is chloroacetylated.

In one example the peptide comprises the sequence YTVTFYPDPFTLQFIAX; (SEQ ID NO. 105), wherein X7is any amino acid other than C and/or wherein the N-terminal tyrosine is chloroacetylated (CI-Ac).

In one example the linear peptide consists of the sequences selected from the group consisting of YTVTFYX:DXsFTLQFIAX; (SEQ ID NO. 96), wherein Xs and/or Xs is/are any amino acid and wherein X7 is any amino acid other than C and/or wherein the N-terminal tyrosine is not chloroacetylated (CI-Ac), YTVTFYPDXsFTLQFIAX7 (SEQ ID NO. 97), wherein Xs is any amino acid and wherein X7 is any amino acid other than C and/or wherein the N-terminal tyrosine is not chloroacetylated (CI-Ac), YTVTFYX3DPFTLQFIAX7 (SEQ ID NO. 98), wherein Xa is any amino acid and wherein X7 is any amino acid other than C and/or wherein the N-terminal tyrosine is chloroacetylated (CI-Ac).

In one example the linear peptide consists of the sequences selected from the group consisting of YTVTFYX:DXsFTLQFIAX; (SEQ ID NO. 99), wherein Xs and/or Xs is/are any amino acid, wherein X7 is any amino acid other than C and wherein the N-terminal Y is chloroacetylated,

YTVTFYPDXsFTLQFIAX; (SEQ ID NO. 100), wherein Xs is any amino acid, wherein Xz is any amino acid other than C and wherein the N-terminal Y is chloroacetylated,

YTVTFEYX3DPFTLQFIAX7 (SEQ ID NO.101), wherein X3 is any amino acid, wherein X7 is any amino acid other than C, and wherein the N-terminal Y is chloroacetylated.

In one example the linear peptide consists of the sequences selected from the group consisting of YTVTFYX:DXsFTLQFIAC (SEQ ID NO. 102), wherein X3 and/or Xs is/are any amino acid and wherein the N-terminal Y is not chloroacetylated, YTVTFYPDXsFTLQFIAC (SEQ ID NO. 103), wherein Xs is any amino acid and wherein the N-terminal Y is not chloroacetylated,

YTVTFYX3DPFTLQFIAC (SEQ ID NO. 104), wherein Xs is any amino acid and wherein the N- terminal Y is not chloroacetylated.

In one example the peptide consists the sequence YTVTFYPDPFTLQFIAX; (SEQ ID NO. 105), wherein X7 is any amino acid other than C and/or wherein the N-terminal tyrosine is chloroacetylated (CI-Ac).

For the avoidance of doubt, when the peptide is described herein as consisting of a specific sequence, this still encompasses the peptide being bound by additional (distinct) moieties, such as an additional linker, detection agent etc.

In case of the peptide being linear any linker, distinct moieties, etc can be located at the N- and/or C-terminus. Alternatively, a linear peptide according to the invention can be a part (at the

N- or C-terminus or in between) of a protein sequence.

In one example the linear peptide comprises one of the sequences selected from the group consisting of YTVTFYX3DXsFTLQFIAX7GSK (SEQ ID NO. 99), wherein Xs and/or Xs is/are any amino acid, wherein X7 is any amino acid other than C and/or wherein the N-terminal tyrosine is chloroacetylated (CI-Ac), YTVTFYPDX:FTLQFIAX7GSK (SEQ ID NO. 100), wherein Xs is any amino acid, wherein X7 is any amino acid other than C and/or wherein the N-terminal tyrosine is chloroacetylated (CI-Ac), YTVTFYX3DPFTLQFIAX7GSK (SEQ ID NO. 101), wherein Xs is any amino acid, wherein Xz is any amino acid other than C and/or wherein the N-terminal tyrosine is chloroacetylated (CI-Ac).

In one example the N-terminal tyrosine is D-tyrosine.

In one example all the amino acids of the peptide are present in the D-conformation.

In one example all the amino acids are present in the L-conformation.

In one example some amino acids are present in the L-conformation and some are present in the D-conformation.

Definitions for the C1q binder, peptide, modifications of the peptide, amino acids, nucleic acids, cells etc. might be provided elsewhere herein and equally apply here. Definitions, embodiments, examples etc. herein for one aspect of the invention equally apply for all the other aspects of the invention. Unless it is apparent from the context, each of the embodiments and examples listed herein can be applied for use in any of the aspects of the invention.

Nucleic acids, vectors, and cells

According to another aspect of the invention a nucleic acid is provided comprising a sequence encoding a peptide according to the invention.

The nucleic acid of the invention may be a DNA molecule encoding a peptide of the invention.

Alternatively, the nucleic acid of the invention may be an RNA molecule, encoding a peptide of the invention. A person skilled in the art would be well aware of methods to obtain a nucleic acid sequence encoding the disclosed peptides.

It will be appreciated the nucleic acids of the invention may be incorporated in larger nucleic acid sequences, which will comprise regions that do not encode the peptides of the invention.

Merely by way of example, a nucleic acid of the invention may be incorporated in an expression plasmid, such as pFUSE-hlgG1-Fc-TP-LH309/310CL or pFUSE-hIgG1-Fe-TP-L310H.

According to another aspect of the invention a vector is provided comprising the nucleic acids described herein.

According to another aspect of the invention an isolated cell is provided comprising a nucleic acid according to the invention.

Suitable vectors and cells are described elsewhere herein.

Definitions for the C1q binder, peptide, modifications of the peptide, amino acids, nucleic acids, cells etc. might be provided elsewhere herein and equally apply here. Definitions, embodiments, examples etc. herein for one aspect of the invention equally apply for all the other aspects of the invention. Unless it is apparent from the context, each of the embodiments and examples listed herein can be applied for use in any of the aspects of the invention.

Pharmaceutical compositions, uses, treatments, and methods for production

According to another aspect of the invention a pharmaceutical composition is provided comprising a C1q binder according to the invention, a nucleic acid according to the invention or a cell according to the invention and a pharmaceutically acceptable excipient, adjuvant, diluent, or carrier.

In one embodiment the pharmaceutical composition comprises two or more peptides according to the invention.

Possible combinations of peptides may be combinations of the peptides disclosed in the

Example section. In particular, cL3 with one or more of cL3-delN1, cL3-delN2, cL3-delC1, and cL3-delC2.

In one embodiment the pharmaceutical composition comprises two or more cyclic peptides according to the invention.

In one embodiment the pharmaceutical composition comprises two or more linear peptides according to the invention.

In one embodiment the pharmaceutical composition comprises a linear and a cyclic peptide according to the invention.

According to another aspect of the invention the C1q binder/peptide according to the invention, a nucleic acid according to the invention, a cell according to the invention or a pharmaceutical composition according to the invention is provided for treating, preventing or diagnosing a disease or disorder in a subject.

In one example the C1q binder/peptide according to the invention, the nucleic acids according to the invention, the cells according to the invention, or the pharmaceutical compositions according to the invention are used as a medicament or a diagnostic tool.

By using the C1q binder/peptide according to the invention, the presence of auto-antibodies to

C1q can be detected. These autoantibodies are present in patients suffering from autoimmune disease, like Systemic lupus erythematosus, Hypocomplementemic Urticarial Vasculitis

Syndrome and Rheumatoid Vasculitis,. By this it is possible to contribute to the diagnosis of such diseases or disorders in a subject.

In one example the C1q binder/peptide according to the invention, the nucleic acids according to the invention, the cells according to the invention, or the pharmaceutical compositions according to the invention are used for the treatment, diagnosis, or prevention of a disease or disorder associated with complement activation or inhibition involving C1q.

In one example the C1q binder/peptide according to the invention, the nucleic acids according to the invention, the cells according to the invention, or the pharmaceutical compositions according to the invention are used for the treatment, diagnosis, or prevention of infectious disease, neurological disease, neurodegenerative disease, graft versus host disease, auto- immune disease or cancer. Specific examples include rheumatoid arthritis, B and T cell lymphomas and leukemias, HER2-positive cancers, or bacterial, fungal or viral infections.

In one example the C1q binder/peptide, nucleic acid, cell, or pharmaceutical composition as disclosed herein, may be formulated for use with one or more other pharmaceutical compositions, detectable agents or therapeutic agents, preferably wherein the one or more other pharmaceutical compositions, detectable agents or therapeutic agents is selected from the group consisting of a: complement activating compound, complement inhibiting compound, tracer, label, radioactive compound, toxin, CRISPR-associated protein (Cas), preferably wherein the Cas is Cas9, and nucleic acid, preferably wherein the nucleic acid is a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA), more preferably wherein the RNA is selected from the group consisting of a: small interfering RNA (siRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger

RNA (mRNA), antisense RNA (asRNA), aptamer, circular RNA {(circRNA), self-amplifying RNA (saRNA), catalytic RNA, anti-miRNA, long non-coding RNA (IncRNA), single-guide RNA (sgRNA) and mRNA encoding a Cas, or wherein the DNA is selected from the group consisting of a: antisense oligonucleotide (ASO), aptamer, episome and catalytic DNA.

According to another aspect of the invention a method for treating a subject is provided comprising administering a therapeutically effective amount of a C1q binder/peptide according to the invention, a nucleic acid according to the invention, a cell according to the invention or a pharmaceutical composition according to the invention to a subject in need thereof.

According to another aspect of the invention a method of producing a C1q binder/peptide according to the invention is provided, the method comprising expressing a nucleic acid in a host cell.

In one embodiment a C1q binder/peptide of the invention may be produced from or delivered in the form of a polynucleotide which encodes, and is capable of expressing, it. Such polynucleotides can be synthesized according to methods well known in the art, as described by way of example in Sambrook et al (1989, Molecular Cloning - a laboratory manual; Cold Spring

Harbor Press). Such polynucleotides may be used in vitro or in vivo in the production of a peptide of the invention. Such polynucleotides may therefore be administered or used in the treatment of a disease or disorder as described herein.

Since the peptides of the invention are intended for use in pharmaceutical compositions it will readily be understood that they are each preferably provided in substantially pure form, for example at least 60% pure, more suitably at least 75% pure and preferably at least 85%, especially at least 98% pure (% are on a weight for weight basis). Impure preparations of the compounds may be used for preparing the more pure forms used in the pharmaceutical compositions; these less pure preparations of the compounds should contain at least 1 %, more suitably at least 5% and preferably from 10 to 59% of a compound of the invention.

Definitions for the C1q binder, peptide, modifications of the peptide, amino acids, nucleic acids, cells etc. might be provided elsewhere herein and equally apply here. Definitions, embodiments, examples etc. herein for one aspect of the invention equally apply for all the other aspects of the invention. Unless it is apparent from the context, each of the embodiments and examples listed herein can be applied for use in any of the aspects of the invention.

Kits

According to another aspect of the invention a kit is provided comprising a C1q binder/peptide according to the invention, a nucleic acid according to the invention, a cell according to the invention or a pharmaceutical composition according to the invention and instructions for using the peptide to treat, prevent or diagnose a disease or disorder in a subject.

In one example the disease or disorder is an infectious disease, neurological disease, neurodegenerative disease, graft versus host disease, tissue or organ rejection, auto-immune disease, cancer, or a disease or disorder associated with complement activation or inhibition involving C1q.

In one example to kit is used to treat or prevent an infectious disease, neurological disease, neurodegenerative disease, graft versus host disease, tissue or organ rejection, auto-immune disease, cancer, or a disease or disorder associated with complement activation or inhibition involving C1q, in a subject in need of such treatment.

Optionally, the kits may contain one or more control samples or references.

However, the kits are not so limited and variations with will apparent to one of ordinary skill in the art.

The kits may comprise buffers. The individual components may be housed in separate containers, e.g. tubes.

Said kit comprises instructions regarding the use of the contained components.

The components of the kit may be housed in a container that is suitable for transportation.

Definitions for the C1q binder, peptide, modifications of the peptide, amino acids, nucleic acids, cells etc. might be provided elsewhere herein and equally apply here. Definitions, embodiments, examples etc. herein for one aspect of the invention equally apply for all the other aspects of the invention. Unless it is apparent from the context, each of the embodiments and examples listed herein can be applied for use in any of the aspects of the invention.

Aspects of the invention are demonstrated by the following non-limiting examples.

Examples

Abstract

The human complement pathway plays a pivotal role in immune defence, homeostasis, and autoimmunity regulation, and complement-based therapeutics have emerged as promising interventions, with both antagonistic and agonistic approaches being explored. The classical pathway of complement is initiated when the C1 complex binds to hexameric antibody platforms. Recent structural data revealed that C1 binds to small, homogeneous interfaces at the periphery of the antibody platforms. Here, we have developed a novel strategy for complement activation using macrocyclic peptides designed to mimic the interface between antibodies and the C1 complex. In vitro selection utilizing the RaPID system identified a cyclic peptide (cL3) that binds to the C1 complex via the globular head domains of C1q. Notably, when immobilized on surfaces, cL3 effectively recruits C1 from human serum, activates C1s proteases, and induces lysis of cell-mimetic lipid membranes. This represents the first instance of a peptide capable of activating complement by binding C1 when immobilized. Further characterization revealed critical residues within cL3 essential for C1 binding and activation.

Deletion mutants elucidated the importance of macrocycle size and specific amino acids for efficient complement activation. Importantly, cL3 also demonstrated the ability to inhibit complement-mediated lysis without affecting C1 binding, highlighting its potential as a therapeutic modality to prevent complement-dependent cytotoxicity whilst promoting cellular phagocytosis and cell clearance. In summary, this study introduces the concept of "Peptactins” - peptide-based activators of complement - and underscores the potential of macrocyclic peptides for complement modulation, offering potential advantages over traditional biologicals in terms of size, production, and administration.

Introduction

Knowledge of the structural details and constraints of C1 binding and activation have been instrumental in these developments (6, 24). The cryo-electron microscopy (cryoEM)-derived structures of antigen-bound to IgM, IgG1 and IgG3 all showed a common structural motif present during C1 binding (Fig. 1b) (2, 4, 5). These data revealed that interaction between the 766 kDa C1 complex and the hexameric Fc platforms (800-1000 kDa) is mediated by small motifs at the periphery of the Fc domains (Fig. 1¢) (2, 4). These structural motifs derive from homologous sequences in IgG and IgM (Fig. 1d), and contain a conserved LPxP (leucine, proline, glycine/serine, proline) sequence that adopts a B-turn-B hairpin at the periphery of the

Fc platform. This motif is present in all human antibody (sub)classes that are able to activate complement (Fig. 1d) (1).

Using the random nonstandard peptides integrated discovery (RaPID) system (25-27), a cyclic peptide that binds to the C1 complex via the gC1q domains has been identified. In solution, this peptide can inhibit complement activation. Furthermore, when immobilised on surfaces, this peptide is capable of recruiting C1 from human serum, activating the C1s proteases, and inducing MAC pore formation on cell-mimetic lipid membranes. Competition assays have revealed a novel mechanism for complement inhibition that does not affect C1 binding.

Results

Selection of a macrocyclic peptide able to recruit C1q from human serum

A single-chain mutant of gC1q (SCgC1q) was immobilized fused to a triple-step tag on streptactin-coated beads and these were used to perform RaPID selection (25-27). In an attempt to mimic the B-turn-B structure of the native antibody motifs, obligate PxP codons were introduced into the mRNA library used for selection, resulting in 17-amino acid peptides with the sequence Y'x5PxPx7C, where x corresponds to any amino acid except methionine. Here, the

N-terminal methionine has been recoded to an N-chloroacetylated-L-tyrosine residue (Y’), which cyclises with either the C-terminal cysteine residue, or a cysteine encoded within the random stretch of residues. The mRNA library therefore corresponds to ~1012 potential different peptide sequences (27, 28), which are tagged during in vitro translation with their encoding mRNA.

These were panned against the immobilised SCgC1q with 7 rounds of positive selection, and 5 rounds of negative selection against beads without SCgC1q (Fig. 2a). The resulting library was subjected to next-generation sequencing and cluster analysis to identify peptides that bind with measurable affinity to gC1q. Analysis of the top 100 most abundant unique sequences identified

7 sequences or families that were selected by comparing relative abundance and intersequence differences (Fig. 2b).

For each family the most abundant sequence was chemically synthesized (Fig. 2c). For any sequences with two cysteines, the most C-terminal residue was mutated to an alanine, resulting in smaller macrocycles. These sequences were synthesised with a biotin on a C-terminal lysine sidechain after a GS linker, and the crude peptides were cyclised by reacting the N-terminal chloroacetylated tyrosine with the single cysteine residue. Biotinylated peptides were immobilised on a streptavidin-coated ELISA plate and recruitment of C1q analysed (Fig. 2d).

Successful binding of full-length purified C1q was measured for all peptides, albeit with different efficiencies. However, analysis of C1q recruitment from human serum indicated that only cyclic-

L3 (cL3) was able to efficiently bind (Fig. 2e,f).

Surface-bound cyclic-L3 activates complement

Peptactin cL3 was resynthesized at a larger scale and purified. Biotinylated cL3 was again bound to a streptavidin-coated plate, but this time the ability of cL3 to both recruit C1q from human serum and activate complement was assessed. Activation was measured by detecting

C5b-8, which are the components of the terminal complement pathway that comprise the MAC (Fig. 1a), a pore that lyses lipid membranes. Again, cL3 displayed efficient C1q recruitment, but this time also showed C5b-9 deposition (Fig. 3a,b), indicating successful C1 activation.

Peptactin cL3 was also synthesised with an azide moiety in place of the biotin molecule on the lysine sidechain (Fig. 2f). A biotin-dibenzocyclooctyne (DBCO) molecule was reacted with the azide-functionalised cL3 as an alternative mechanism for biotinylation of the peptide. This also showed cL3 was able to bind and activate C1q (Fig. 3c,d). Both of these biotinylated cL3 molecules gave similar EC50 values of ~3-6 uM.

This data demonstrates the ability of macrocyclic peptides to activate the human complement system, and so this class of molecule was named “Peptactin”, for peptide-based activator of the complement system.

Deletion mutants of peptactin L3 determine the structural constraints of the macrocycle

The macrocycle of cL3 is relatively large as compared to other macrocyclic drugs (29).

Therefore, systematic deletions from cL3 were implemented to alter the ring size, and determine which residues were important for gC1q binding and complement activation. Seven deletion mutants were synthesised by removing up to 8 residues from the N- and C-termini (not including the N-terminal tyrosine or C-terminal cysteine; Fig. 3e), and their ability to bind to C1q in human serum was determined. Removal of one amino acid from the N-terminus (cL3-delN1), which corresponds to threonine2, did not appear to affect C1q binding at low concentration, but somehow caused reduced binding at higher concentrations, indicating a slight disruption compared to cL3. However, removal of two residues (cL3-delN2) caused much weaker binding, requiring ~15x more peptactin to recruit C1q. Removal of one residue from the C terminus (cL3- delC1) also caused much weaker binding, whilst any other deletion seemingly abolished C1q binding.

Peptactin cL3 can activate complement on lipid membranes

Complement activation occurs on cell membranes so, to move towards more native systems, liposomes were produced to act as cell mimetics. Liposomes were synthesised with a DBCO functional group displayed at 1 mol % on the surface of the membrane. Azide-functionalised cL3 was then added; DBCO reacts specifically and rapidly with azide moieties, forming a covalent bond between the lipid bilayer and the azide-cL3. Liposomes now displaying cL3 were purified before addition of pure C1 complex (composed of C1q, C1r and C1s). Binding of C1q leads to activation of C1r, which in turn activates C1s. Activated C1s is a serine protease that is responsible for propagating the complement pathway. To assess C1s activation, a non- fluorescent substrate was added, Boc-Leu-Gly-Arg-Amino Methyl Cumarin (LGR-AMC), which becomes fluorescent upon cleavage by C1s (30). Membrane-bound cL3 was able to significantly activate C1s when compared to liposomes without cL3 (Fig. 4a).

Next, the ability of cL3 to activate complement and cause membrane lysis via formation of the

MAC pore was assessed. Liposomes were synthesised displaying DBCO as above, but this time were formed encapsulating a high concentration of sulforhodamine B, which is self- quenched and non-fluorescent. Upon complement activation and MAC pore formation, the sulforhodamine B becomes diluted and fluoresces. Purified liposomes containing sulforhodamine B and displaying cL3 were mixed with human serum and the fluorescence monitored. A fluorescence increase was seen only on liposomes displaying cL3 and in the presence of human serum (Fig. 4b), indicating that peptactin cL3 is able to activate complement and cause MAC pore formation on lipid bilayers.

Solution-phase cL3 can inhibit complement activation

The classical complement pathway is initiated upon C1 binding to ligands, which include IgG antibodies, and it is known that C1 modulators can display both inhibitory and agonistic properties depending on how these molecules act in solution (22, 24). The ability of peptactin cL3 to inhibit complement activation was investigated further using antigenic liposomes.

Liposomes were prepared to displaying antigens based on a CD52 mimotope (2). CD52- presenting liposomes were synthesized containing self-quenching sulforhodamine B. Upon addition of anti-CD52 1gG1 and human serum to these liposomes, complement activation occurs, as measured by an increase in fluorescence upon MAC pore formation (Fig. 5a).

However, preincubating the serum with 100 uM of cL3 for 10 minutes results in statistically- relevant inhibition of complement-mediated liposome lysis (Fig. 5a). Subsequently, a titration of cL3 was performed, giving an approximate IC50 of ~60 uM (Fig. 5b).

To determine how peptactin cL3 was inhibiting complement activation, pooled human IgG was coated on ELISA plates and the ability of cL3 to inhibit either C1q binding or C5b-9 deposition was assessed (Fig. 5c). Although cL3 was not able to inhibit C1q binding to pooled IgG, it did inhibit complement activation, as measured by reduced C5b-9 deposition, with an IC50 of ~0.3

MM in this system. The cL3 deletion mutants were also evaluated for their capacity to inhibit 5b- 9 deposition (Fig. 5d). As before for the C1q recruitment data (Fig. 3e), any deletion was detrimental for inhibition, but here maximal inhibition with full-length cL3 was observed, followed by cL3-delN1, cL3-delC1, and cL3-delC2, respectively.

This competition assay was utilised to determine the importance of other structural features of cL3 by synthesising analogues of cL3. Peptactin cL3 is cyclised between the N-terminal chloroacetyl group and the cysteine residue at position 17 (Fig. 2f). To determine the importance of the conformational state of L3 on C1q binding and complement activation, peptactin L3 was synthesised with the N-terminal L-form tyrosine residue exchanged for a D- isomer, henceforth named inv-cL3. Changing this stereochemistry has been previously shown to impact the efficacy of macrocyclic peptides (31, 32). Inv-cL3 was assessed for its agonistic properties, revealing an approximate 10-fold decrease in EC50 compared to cL3 (Fig. 6a).

Peptactin L3 is therefore more potent when composed entirely of L-form amino acids. Next, the ability of inv-cL3 to inhibit C1q binding to pooled IgG was determined. Here, inv-cL3 demonstrated potential inhibition of C1q binding to pooled IgG at high concentrations (Fig. 6b), but more apparent was the ability of inv-cL3 to inhibit C5b-9 deposition, which was inhibited with an IC50 of ~2 uM (Fig. 6b). This was significantly worse than full-length cL3, and in line with activation experiments. Finally, a linear variant of L3 (lin-L3) was synthesised by replacing cysteine17 with a serine, and omitting the chloroacetyl group during synthesis. Lin-L3 proved able to inhibit C1 binding to pooled IgG, and C5b-9 deposition (Fig. 6c), but was much less effective than cL3 or inv-cL3. Furthermore, lin-L3 was not able to inhibit MAC pore formation, even after preincubating the serum with 100 uM of lin-L3 for 1 hour (Fig. 6d).

Mapping the binding site of cL3 onto gC1q

To gain insights into the binding location on gC1q, competition assays were performed with C1 ligands and binders. The nanobody C1gNB75 binds gC1q at a known position (24), and can therefore be used to help map the binding site of cL3 on C1q. A competition ELISA, using immobilised cL3 and solution-phase C1gNB75, showed that cL3 does not compete with

C1gNB75 (Fig. 7a). However, when C1gNB75 was immobilised instead of cL3, solution-phase cL3 showed inhibition of only C5b-9 deposition but not C1 binding (Fig. 7a), similarly to IgG (Fig. 5c).

As well as being initiated by antibodies, the classical complement pathway can also be activated upon C1 binding to the pentraxin C-reactive protein (CRP) (33, 34). The binding site of gC1q to CRP has been determined using a combination of mutagenesis studies and cryo- electron tomography (35, 36), and so this can be used to determine possible binding locations of cL3 to gC1q. CRP was coated on ELISA plates and the ability of cL3 to inhibit either C1q binding or C5b-9 deposition was assessed. This revealed that cL3 was able to inhibit CRP- mediated complement activation via both reduced C1q binding and C5b-9 deposition, with IC50 values of ~5 uM and 0.3 uM, respectively (Fig. 7b).

Together, these data show that cL3 binds in the region proximal to CRP, at the bottom of the gC1q domain (36), and distal to the region where both IgG-Fc domains and the C19qNB75 bind (Fig. 7c), resulting in a map of potential cL3 binding sites on the complete C1 complex (Fig. 7d).

Discussion

Therapeutic development of C1 agonists has mainly focussed on biologicals. Engineered antibodies and nanobodies have shown promise for several clinical targets and some of these protein engineering techniques, such as the development of the Hexabody platform, are now under clinical evaluation (20, 22). However, biologicals have inherent downsides, mainly due to their size, which pose challenges in administering to patients and costly production. Synthetic drugs such as peptides and small molecules offer solutions to these disadvantages. CryoEM structures of various antibodies in complex with C1 revealed that, while antibodies assemble into large ~900-1000 kDa platforms, the actual interface of the platforms with gC1q comprised small homologous peptide sequences, of ~5 amino acids (Fig. 1) (2, 4, 5). Therefore a strategy was pursued to mimic these interfaces by utilising peptide design and selection. The term “Peptactin”, or peptide activator of the immune system, is introduced for peptides that fall into this category.

RaPID selection was used to obtain peptides that enable complement activation. For this reason, two conserved prolines found in the native C1-antibody interface were introduced, within a PxP motif, into the peptide library (Fig. 1d). The resulting peptides all displayed the ability to bind pure C1q (Fig. 2d), but only one, cL3, was capable of doing so in human serum (Fig. 2e). Presumably, the other peptides bind non-specifically to other proteins or components found in human serum, which then blocks recruitment of C1q. Synthesis of cL3 modified with either a biotin or an azide at the C-terminus indicated that the peptide was amenable for modification and targeting (Fig. 3). In particular, the azide moiety can be used for conjugation of diverse molecules and proteins via click-chemistry, such as potential drug delivery strategies for administering the peptactin (37). Displaying cL3 on cell membrane mimetics led to efficient complement activation in the presence of human serum (Figs. 3 and 4), resulting in the first small-molecule activator of complement.

Deletion mutants were synthesised to explore the impact of the composition and size of the macrocycle itself. Deletion of one amino acid from either end of the peptide led to reduced binding and activation (Figs. 3e and 5d), indicating that these residues are important for binding, either via direct interaction with gC1q, or due to a necessity for the macrocycle to be 17 residues in length. Deletion of more than two residues abolished binding, but single deletions, from either the N- or the C-termini, were still able to activate complement, albeit with much reduced activity compared to the full-length cL3 (Fig. 5d). To determine whether tyrosine or cysteine17, which are both crucial for cyclisation of the peptide, are also involved with binding, variants without cysteine were synthesised (which then formed a linear peptide; lin-L3), or with

L-tyrosine1 exchanged for D-tyrosine1 (inv-cL3). Both of these were worse than cL3 (Fig. 5), indicating that the peptactin is more effective when cyclised, but also that the tyrosine may be important for binding. However, changing amino acid stereochemistry has been shown to impact peptide structure (38), which may account for the worse binding observed here.

Next the ability of cL3 to inhibit complement activation was evaluated by performing competition assays against C1q binders. By incubating human serum with cL3, complement-mediated liposome lysis by inhibition of C5b-9 deposition was successfully reduced (Fig. 5a,b).

Surprisingly, however, C1q binding was not inhibited (Fig. 5c). These data indicate that C1q binding to IgG is not affected, but complement activation is inhibited. IgG is the native ligand for

C1, and activates complement on cells by forming hexameric Fc platforms (6). A possible explanation for this is that cL3 is unable to inhibit all 6 headgroups of hexameric C1q from binding to IgG, therefore no inhibition of C1 binding was seen as some of the gC1q domains are still available to bind. However, if cL3 does bind to some of the 6 gC1q headgroups, it may inhibit complement activation without inhibiting C1q binding, as observed here. Alternatively, it has been posited that a structural rearrangement may occur after C1q binding to IgG, but before

C1r and C1s activation (2, 4, 39). It is conceivable that cL3 binds to a region on gC1q and impedes rotation of gC1q relative to the C1q collagen arms, which has been implied from antibody binding studies that identified a cryptic epitope accessible only on antibody-bound C1q (39). Interestingly, inhibition of C5b-9, but not C1 binding, is also observed for the nanobody

C1gNB75 (Fig. 7b,c). C1gNB75 is known to bind at the same location as antibodies (24), and so the ability of cL3 to inhibit only complement activation, and not C1 binding, is conserved for other C1q binders. In contrast, cL3 inhibited C1q binding to CRP as well as complement activation (Fig. 7a). This is likely due to the binding site(s) for C1q to CRP being different to that of IgG (Fig. 7d) (36).

Complement inhibition has been extensively explored as a therapeutic route over the past decade; indeed, six complement inhibitors are now approved for clinical use (12, 40-44). Two of these inhibitors target the C1r and C1s proteases (40, 41), including administration of native human C1-inhibitor (C1-INH) to treat hereditary angioedema (40), or the humanized monoclonal antibody Sutimlimab to treat cold agglutinin disease (41). Similarly, peptide inhibitor of complement C1 (PIC1) also inhibits the binding of the C1 proteases to C1q (11), and this family of inhibitors are currently being investigated in animal models and clinical trials as a treatment for acute and delayed hemolytic transfusion reactions, cystic fibrosis and diabetic wound healing (45, 46). To the best of our knowledge, peptide 2J is the only peptide inhibitor of complement that has been developed to bind to the gC1q domains (47). In contrast to peptactin cL3, peptide 2J inhibited binding of C1q to IgG, leading to reduced complement activation. From the available data, peptactin cL3 compares favourably to these other peptide-based complement inhibitors in terms of IC50. Peptactin cL3 could therefore be used to expand the complement therapy toolbox, and initial tests performed here showcase potential to prevent

CDC via a novel route of complement inhibition without affecting C1 binding (Fig. 5a,c). C1is a ligand for complement receptor 1, which mediates opsonophagocytosis; clearance of tagged material by professional phagocytic cells (48). The ability of cL3 to allow binding of C1 to IgG- coated cells without activating complement may also therefore be a method to promote clearance of material via phagocytosis without inducing the inflammatory complement cascade.

Improving complement activation has been achieved by glycoengineering (49, 50), antibody isotype engineering (51, 52), Fc engineering (53, 54), and enhancing IgG hexamerization (6, 20, 21). Indeed, it is likely that all of these approaches affect antibody oligomerization and the formation of an Fc platform to which C1 can bind. Cause-and-effect relationships of antibody platform to C1 binding can be hard to isolate, since both antibody assembly and C1 binding must occur prior to activation. This adds a level of abstraction, which makes data interpretation complex. Furthermore, protein engineering can be labour intensive and costly; peptide synthesis overcomes those disadvantages. A monomeric binder such as cL3 could yield additional insights into the additive effect of ligand affinity and avidity, providing valuable data into the intricacies of C1 activation, thereby augmenting the design and development of complement-modulating therapies.

Materials and Methods

RaPID selection on gC1q

Selections were performed on streptactin XT magnetic beads (Iba Lifesciences), which were used to immobilize purified single chain C1q (SCgC1q) modified with a triple Strep-tag, ordered from uProtein Express BV (Netherlands) (25, 55). SCgC1q was produced in HEK293E-253 cells as previously described (55). Procedures of the selection are described in detail in

Thijssen et al. (27). Briefly, a DNA library was ordered from IDT (USA), which encoded a semi- random library (X2. XSP8X8P7X... X15) of NNK codons. L-tyrosine cyanomethyl esters were charged with tRNAcau such as described in Goto et al. (56). The peptide sequence contains the modified tyrosine at position 1, and a cysteine residue at position 17, which is followed by a

GSGSGS (SEQ ID NO. 108) linker. RaPID selection comprises a series of sequential rounds of i} RNA generation, ii) peptide synthesis, in which the peptides are covalently linked to the RNA encoding them via a puromycin linker, iii) generation of cDNA to the RNA tag on the peptide to stabilize the complex iv) exposing library to gC1q in in phosphate buffered saline (PBS) plus 0.1% Tween-20, v) washing and vi) QC of the selection via qPCR. The pool cDNA that remained after binding to gC1q was amplified and subjected to another round of selection. After two rounds with only positive selection, a negative control selection was performed where peptides were exposed to beads without bound SCgC1q. Recovery percentages, as shown in the results section, are the percentage of output sample compared to sample input, as determined by qPCR. The process was repeated until the positive selection displayed superior enrichment compared to the negative selection. DNA output was then sequenced using an lumina ISeq platform using a 2*150 bp V2 reagent kit at the VUMC Medical Genomics sequencing facility (Amsterdam, NL).

Peptide synthesis

Peptide synthesis for the initial selection of the various RaPID family sequences was performed at 25 uM scale on a Syro Multisyntech Automated Peptide synthesizer (SYRO robot; Part Nr.

Syro II; Serial: 2015-05-01 Syro II; Multisyntech GmbH, Germany, under inert gas (N2) application). The machine was washed with NMP and the resin was swelled with NMP for 5 minutes. Fmoc deprotections were performed using 20% piperidine in NMP. Coupling reactions were performed using PyBOP and DIPEA. After the last coupling steps the resin was washed manually using DCM and diethyl ether. The resin was left to dry. Coupling of chloroacetic acid and subsequent cyclization was performed as described in general procedure B, C and D in the work of van Haren et al. (26). For the initial selection crude peptides were used. For all subsequent syntheses, procedures were identical and peptides were purified via reverse phase

HPLC and freeze-dried. The deletion scan products were ordered from GenScript (United

Kingdom) at a purity of at least 75% after freeze-drying to a white powdered form.

Protein expression

The C1gNB75 plasmid was a gift from Nick Laursen (24). C1gNB75 was produced using BL21

DE3, which were grown to an OD600 of 0.6 in lysogeny broth. Samples were oxygenated by shaking at 200 rpm and kept at 37°C. After the appropriate OD was reached, 0.5 mM of

Isopropyl B-D-1-thiogalactopyranoside (IPTG) (VWR chemicals, NL) was added to induce protein synthesis and bacteria were incubated at 20°C with shaking for approximately 16 hours.

Bacteria were collected in a pellet by centrifugation. Cells were lysed in cold wash buffer (300 mM NaCl, 20 mM Tris-HCI, and 20 mM imidazole, pH 8) using probe sonication and debris removed at 24000g for 40 minutes at 4°C. HisPurTM NiNTA (Thermofisher Scientific) beads were used to purify the protein. Columns were equilibrated with 10 column volumes of wash buffer after which the supernatant was added to the column. Wash buffer with increasing concentrations of imidazole was added to the column until a concentration of 250 mM imidazole was reached. The eluted protein fractions were collected and purified further using size exclusion chromatography (S200 Superdex® prep grade), which had been equilibrated with

PBS. Samples were concentrated using Amicon® spin filters and stored at -80°C. Alemtuzumab (antiCD52 1gG1) was a gift from the Trouw lab (LUMC, NL).

Enzyme-linked immunosorbent assay (ELISA) for complement activation

Maxisorb Nunc Immunoplates (Thermofisher Scientific, Massachusetts, USA) were coated with streptavidin (Thermofisher Scientific) at 10 pg/ml in 0.1 M Na2CO3, 0.1M NaHCO3 (coating buffer), pH 9.6 at 50 pl per well and allowed to incubate either at room temperature overnight, or 37°C for 1 hour. Plates were washed with PBS with 0.05% tween-20 three times. For each following step, samples were incubated at 37°C for 1 hour and subsequently washed with the

PBS-T solution. After streptavidin binding, wells were blocked with 100 pl of 0.1 M spermidine (Thermofisher Scientific) in distilled water. Peptides were incubated in 50 ul PBS with 0.05%

Tween-20 (PBS-T) to create a peptide presenting surface. In all the following steps, 50 pl was added to the plates. In all activation assays, peptides are modified with a biotin to achieve binding to streptavidin. Note that in certain instances the peptides are initially modified with an azide. In such conditions a biotin-PEG4-DBCO (Jena Bioscience GmbH, Germany) was co- incubated with the peptides at 25 mol% excess of biotin linker to create a biotin modified peptide in situ. Hereafter the wells were incubated with 1% normal human serum (NHS) (Complement Technology, Inc., Texas, USA) in RPMI medium (Roswell Park Memorial Institute 1640 medium). Following these steps, complement components C1q or C5b-9 were detected.

Primary Rabbit anti-C1q and Mouse anti C5b-9 (Dako, Denmark) was added to PBS, 0.05%

Tween-20, 0.1% BSA (PBS-BT) in 1:2000 and 1:333 dilution, respectively. Next, goat anti-rabbit and anti-mouse antibodies (Dako, Denmark} modified with horseradish peroxidase (HRP) were diluted 1:5000 in PBS-T and added to the plates. To detect the HRP on the secondary antibody 2.5 mg/ml ABTS (2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) was added in citric acid buffer (0.15 M, pH 4.2) (ABTS buffer) containing 0.15 (v/v %) H202 was used and samples were incubated at room temperature for 30 minutes. Absorption measurements at 415 nm were performed on a CLARIOstar microplate reader (BMG Labtech, Offenburg, Germany).

Competition ELISAs

Materials, including buffers and blocking agent, were the same as for the activation assay, unless stated otherwise. To determine the competition with pooled human IgG (Sigma-Aldrich,

USA), Maxisorb plates were coated with 10 pg/ml of IgG pooled from human serum, incubated,

washed and blocked. Peptides were incubated with 1% normal human serum (pooled,

Complement Technology) for at least 30 minutes before adding to the IgG coated plates. DMSO controls and no serum controls were added. Human serum with and without peptides was added to the plates and subsequent complement inhibition was determined by detecting the presence of C1q and C5b-9. Data shown in the graphs is normalized to the maximum signal of the DMSO control and the minimal signal of the no serum control. Absorption measurements at 415 were performed on a CLARIOstar microplate reader (BMG Labtech, Offenburg, Germany).

To perform the competition assays to assess overlap in the binding region of gC1q to cL3 and either C1gNB75 or CRP, buffers, materials and sample volumes were as described above.

C1gNB75 or CRP were added to the Maxisorb plates at 10 pg/ml in binding buffer and incubated at 37°C for one hour, then washed three times with PBS-T. In all further steps plates were incubated at 37°C and then washed each time. After coating the plates with the proteins, wells were blocked with spermidine. Then human serum, 1% in RPMI was added to the plates in presence or absence of 20 uM of cL3 for the antibodies. For CRP a titration of cL3 was performed. Subsequently complement components C1q and C5b-9 were detected and absorbance as a consequence of ABTS conversion was measured.

To immobilise cL3, wells were coated with streptavidin 10 ug/ml in coating buffer. Then wells were blocked with spermidine and subsequently 20 uM of cL3 was added to the plates, incubated and washed. Then human serum (1% in RPMI) was added to the wells in the presence or absence of 100 nM of C1gNB75. Complement components C1q of C5b-9 were detected and ABTS conversion measured.

Liposome preparation

Liposomes were prepared using DMPC:DMPC:CHOL:DBCO (1,2-dimyristoyl-sn-glycero-3- phosphocholine: 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt}:Cholesterol (ovine): 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-dibenzocyclooctyl) 44:5:50:1 molar %. Liposomes were ordered from Avanti Polar lipids (Pelham, USA). The lipids were dissolved in glass vials in a chloroform/methanol mixture 9:1 v/v mix. Lipids were then added to 1 mg of total lipid mass and the organic solvent mix was evaporated using N2 for at least 2 hours. Aqueous buffer was used to rehydrate the lipids, as specified in the relevant sections below. The vials were incubated in a water bath at 50-60°C for one hour and agitated by pipetting to resuspend. Liposomes were extruded through with a 400 nm polycarbonate membrane using a mini-extruder (Avanti Polar Lipids). These liposomes were stored at 4°C until further use for up to two weeks.

Conjugation of the peptides onto the liposomes surfaces was achieved using copper-free click chemistry between the azide handle on the peptides 100 pM with the DBCO-coated liposomes by incubating overnight. Unreacted peptide was removed from the sample by centrifugation at

20000 g for 15 minutes at 4°C, supernatant was removed and liposomes resuspended in the relevant assay buffer.

C1s activity assay

Using either peptide modified liposomes or blank liposomes, C1s enzymatic activity of was monitored using a substrate conversion assay. C1s substrate Boc-Leu-Gly-Arg-AMC (Amino

Methyl Coumarin) — PeptaNova GmbH (Sandhausen, Germany) (LGR-AMC) was incubated with purified C1 protein (Complement Technology, Texas, USA). Pure C1 was buffer exchanged using 100 kDa spin filters (Amicon) into 150 mM NaCl 50 mM Tris-HCI pH 7.5, 5 mM CaCl2 (assay buffer). After all components were mixed, the substrate was added at 500 uM, diluted from a 10 mM stock (5% DMSO final concentration). C1 protein concentration was kept at 40 nM for all these experiments. It is important to note that when using purified C1 protein (from

Complement Technology), the sample has to be buffer exchanged from the manufacturer storage solution. Liposomes were formed in PBS and, after conjugation of the peptide using copper-free click chemistry, buffer exchanged to assay buffer via centrifugation as described above. Fluorescent signal over time from the AMC over was monitored on a CLARIOstar microplate reader (BMG Labtech, Offenburg, Germany). Measurements were taken every minute for a period of 5 hours with excitation and emission set at 360 and 460 nm, respectively.

Liposome lysis assay

The liposome lysis assay monitors the formation of the membrane attack complex upon complement activation. Peptide-modified liposomes were exposed to 10% human serum (Complement Technology) in PBS and then peptides recruit C1 to the membrane leading to activation of C1 and the downstream complement pathway. Liposomes were prepared to contain 20 mM Sulforhodamine B (SRB) (Sigma Aldrich, St louis, MO, USA), in PBS. The content release of SRB is monitored over time (every 15 seconds) on a CLARIOstar microplate reader (BMG Labtech, Offenburg, Germany).

Complement inhibition assay

A cell mimetic system was used as in the liposome lysis assay. Liposomes encapsulating SRB (20 mM in PBS) were synthesised displaying 1 mol% cholesterol-modified CD52 mimotope (2).

Liposomes were pre-incubated with 500 nM of anti-CD52 IgG1 (alemtuzumab). Before adding 10% human serum to the liposomes, various concentrations of peptide were added to the serum and incubated for 10 minutes to 1 hour. For the titrations samples were incubated for 10 minutes. Buffer and DMSO controls were added and control sample with no antibody was added to act as baseline signal. Data was collected as in the liposome lysis assay. Statistical analysis for the complement inhibition assay was performed using GraphPad Prism (version 9).

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NL 2037390 2024-04-03 P352986NL Academisch Ziekenhuis Leiden (h.o.d.n.

LUMC) SELECTION AND CHARACTERIZATION OF A PEPTIDE-BASED COMPLEMENT

ACTIVATOR 136 17 AA PAT source 1..17 mol_type protein organism synthetic construct VARIANT 2 note absent, T, or K VARIANT 3 note absent or V VARIANT 7 note any amino acid VARIANT 9 note any amino acid VARIANT 16 note absent, A, E or T VARIANT 17 note C, S or a stop codon SITE 2..16 note a maximum of two of residues 2, 3 and 16 are absent YXXTFYXBXFTLQFIXX 17 AA PAT source 1..17 mol_type protein organism synthetic construct VARIANT 17 note Cor S

YTVTFYPDPFTLQFIAX 17 AA PAT source 1..17 mol_type protein organism synthetic construct YTVTFYPDPFTLQFIAC 16 AA PAT source 1..16 mol_type protein organism synthetic construct YVTFYPDPFTLQFIAC 15 AA PAT source 1..15 mol_type protein organism synthetic construct YTFYPDPFTLQFIAC 12 AA PAT source 1..12 mol_type protein organism synthetic construct YPDPFTLQFIAC 11 AA PAT source 1..11 mol_type protein organism synthetic construct YDPFTLQFIAC 9 AA PAT source 1..9 mol_type protein organism synthetic construct YFTLQFIAC 16 AA PAT source 1..16 mol_type protein organism synthetic construct YTVTFYPDPFTLQFIC 15 AA PAT source 1..15 mol_type protein organism synthetic construct YTVTFYPDPFTLQFC 13

AA PAT source 1..13 mol_type protein organism synthetic construct

YTVTFYPDPFTLC 11 AA PAT source 1..11 mol_type protein organism synthetic construct YTVTFYPDPFC 17 AA PAT source 1..17 mol_type protein organism synthetic construct YTVTFYPDPFTLQFIAS 16 AA PAT source 1..16 mol_type protein organism synthetic construct YVTFYPDPFTLQFIAS 15 AA PAT source 1..15 mol_type protein organism synthetic construct YTFYPDPFTLQFIAS 12 AA PAT source 1..12 mol_type protein organism synthetic construct YPDPFTLQFIAS 11 AA PAT source 1..11 mol_type protein organism synthetic construct YDPFTLQFIAS 9 AA PAT source 1..9 mol_type protein organism synthetic construct YFTLQFIAS 16 AA PAT source 1..16 mol_type protein organism synthetic construct YTVTFYPDPFTLQFIS 15 AA PAT source 1..15 mol_type protein organism synthetic construct YTVTFYPDPFTLQFS 13

AA PAT source 1..13 mol_type protein organism synthetic construct

YTVTFYPDPFTLS 11 AA PAT source 1..11 mol_type protein organism synthetic construct YTVTFYPDPFS 17 AA PAT source 1..17 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YTVTFYPDPFTLQFIAC 16

AA PAT source 1..16 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YVTFYPDPFTLQFIAC 15 AA PAT source 1..15 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine

YTFYPDPFTLQFIAC 12 AA PAT source 1..12 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YPDPFTLQFIAC 11 AA PAT source 1..11 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YDPFTLQFIAC 9 AA PAT source 1..9 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YFTLQFIAC 16 AA PAT source 1..16 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YTVTFYPDPFTLQFIC 15 AA PAT source 1..15 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YTVTFYPDPFTLQFC 13 AA

PAT source 1..13 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YTVTFYPDPFTLC 11 AA PAT source 1..11 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YTVTFYPDPFC 17 AA PAT source 1..17 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YTVTFYPDPFTLQFIAS 16 AA PAT source 1..16 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine

YVTFYPDPFTLQFIAS 15 AA PAT source 1..15 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YTFYPDPFTLQFIAS 12 AA PAT source 1..12 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YPDPFTLQFIAS 11 AA PAT source 1..11 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YDPFTLQFIAS 9

AA PAT source 1..9 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YFTLQFIAS 16 AA PAT source 1..16 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine

YTVTFYPDPFTLQFIS 15 AA PAT source 1..15 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YTVTFYPDPFTLQFS 13 AA PAT source 1..13 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YTVTFYPDPFTLS 11 AA PAT source 1..11 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YTVTFYPDPFS 4

AA PAT source 1..4 mol_type protein organism synthetic construct GSGS 4 AA PAT source 1..4 mol_type protein organism synthetic construct GGGS 6 AA PAT source 1..6 mol_type protein organism synthetic construct GSGGSG 20 AA PAT source 1..20 mol_type protein organism synthetic construct YTVTFYPDPFTLQFIACGSK 19

AA PAT source 1..19 mol_type protein organism synthetic construct

YVTFYPDPFTLQFIACGSK 18 AA PAT source 1..18 mol_type protein organism synthetic construct YTFYPDPFTLQFIACGSK 15 AA PAT source 1..15 mol_type protein organism synthetic construct YPDPFTLQFIACGSK 14 AA PAT source 1..14 mol_type protein organism synthetic construct YDPFTLQFIACGSK 12 AA PAT source 1..12 mol_type protein organism synthetic construct YFTLQFIACGSK 19 AA PAT source 1..19 mol_type protein organism synthetic construct

YTVTFYPDPFTLQFICGSK 18 AA PAT source 1..18 mol_type protein organism synthetic construct YTVTFYPDPFTLQFCGSK 16 AA PAT source 1..16 mol_type protein organism synthetic construct YTVTFYPDPFTLCGSK 14 AA PAT source 1..14 mol_type protein organism synthetic construct YTVTFYPDPFCGSK 20 AA PAT source 1..20 mol_type protein organism synthetic construct YTVTFYPDPFTLQFIASGSK 19

AA PAT source 1..19 mol_type protein organism synthetic construct

YVTFYPDPFTLQFIASGSK 18 AA PAT source 1..18 mol_type protein organism synthetic construct YTFYPDPFTLQFIASGSK 15 AA PAT source 1..15 mol_type protein organism synthetic construct YPDPFTLQFIASGSK 14 AA PAT source 1..14 mol_type protein organism synthetic construct YDPFTLQFIASGSK 12 AA PAT source 1..12 mol_type protein organism synthetic construct YFTLQFIASGSK 19 AA PAT source 1..19 mol_type protein organism synthetic construct

YTVTFYPDPFTLQFISGSK 18 AA PAT source 1..18 mol_type protein organism synthetic construct YTVTFYPDPFTLQFSGSK 16 AA PAT source 1..16 mol_type protein organism synthetic construct YTVTFYPDPFTLSGSK 14 AA PAT source 1..14 mol_type protein organism synthetic construct YTVTFYPDPFSGSK 20 AA PAT source 1..20 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YTVTFYPDPFTLQFIACGSK 19 AA PAT source 1..19 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine

YVTFYPDPFTLQFIACGSK 18 AA PAT source 1..18 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YTFYPDPFTLQFIACGSK 15

AA PAT source 1..15 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YPDPFTLQFIACGSK 14 AA PAT source 1..14 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine

YDPFTLQFIACGSK 12 AA PAT source 1..12 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YFTLQFIACGSK 19 AA PAT source 1..19 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YTVTFYPDPFTLQFICGSK 18 AA PAT source 1..18 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine

YTVTFYPDPFTLQFCGSK 16 AA PAT source 1..16 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YTVTFYPDPFTLCGSK 14

AA PAT source 1..14 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YTVTFYPDPFCGSK 20 AA PAT source 1..20 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine

YTVTFYPDPFTLQFIASGSK 19 AA PAT source 1..19 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YVTFYPDPFTLQFIASGSK 18 AA PAT source 1..18 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YTFYPDPFTLQFIASGSK 15 AA PAT source 1..15 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine

YPDPFTLQFIASGSK 14 AA PAT source 1..14 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YDPFTLQFIASGSK 12 AA PAT source 1..12 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YFTLQFIASGSK 19 AA PAT source 1..19 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine

YTVTFYPDPFTLQFISGSK 18 AA PAT source 1..18 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YTVTFYPDPFTLQFSGSK 16

AA PAT source 1..16 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine YTVTFYPDPFTLSGSK 14 AA PAT source 1..14 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine

YTVTFYPDPFSGSK 20 AA PAT source 1..20 mol_type protein organism synthetic construct VARIANT 7 note any amino acid VARIANT 9 note any amino acid

YTVTFYXDXFTLQFIACGSK 20 AA PAT source 1..20 mol_type protein organism synthetic construct VARIANT 9 note any amino acid YTVTFYPDXFTLQFIACGSK 20

AA PAT source 1..20 mol_type protein organism synthetic construct VARIANT 7 note any amino acid YTVTFYXDPFTLQFIACGSK 20 AA PAT source 1..20 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine

VARIANT 7 note any amino acid VARIANT 9 note any amino acid

YTVTFYXDXFTLQFIACGSK 20 AA PAT source 1..20 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine VARIANT 9 note any amino acid YTVTFYPDXFTLQFIACGSK 20 AA PAT source 1..20 mol_type protein organism synthetic construct VARIANT 7 note any amino acid SITE 1 note chloroacetylated tyrosine YTVTFYXDPFTLQFIACGSK 20 AA PAT source 1..20 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine VARIANT 7 note any amino acid VARIANT 9 note any amino acid REGION 1..20 note the peptide is cyclic YTVTFYXDXFTLQFIACGSK 20 AA PAT source 1..20 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine VARIANT 9 note any amino acid REGION 1..20 note the peptide is cyclic

YTVTFYPDXFTLQFIACGSK 20 AA PAT source 1..20 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine VARIANT 7 note any amino acid REGION 1..20 note the peptide is cyclic YTVTFYXDPFTLQFIACGSK 20 AA

PAT source 1..20 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine REGION 1..20 note the peptide is cyclic

YTVTFYPDPFTLQFIACGSK 17 AA PAT source 1..17 mol_type protein organism synthetic construct VARIANT 7 note any amino acid VARIANT 9 note any amino acid

VARIANT 17 note any amino acid other than C YTVTFYXDXFTLQFIAX 17 AA PAT source 1..17 mol_type protein organism synthetic construct VARIANT 9 note any amino acid VARIANT 17 note any amino acid other than C YTVTFYPDXFTLQFIAX 17

AA PAT source 1..17 mol_type protein organism synthetic construct VARIANT 7 note any amino acid VARIANT 17 note any amino acid other than C

YTVTFYXDPFTLQFIAX 17 AA PAT source 1..17 mol_type protein organism synthetic construct VARIANT 7 note any amino acid VARIANT 9 note any amino acid VARIANT 17 note any amino acid other than C SITE 1 note chloroacetylated tyrosine

YTVTFYXDXFTLQFIAX 17 AA PAT source 1..17 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine VARIANT 9 note any amino acid

VARIANT 17 note any amino acid other than C YTVTFYPDXFTLQFIAX 17 AA PAT source 1..17 mol_type protein organism synthetic construct VARIANT 7 note any amino acid VARIANT 17 note any amino acid other than C SITE 1 note chloroacetylated tyrosine YTVTFYXDPFTLQFIAX 17 AA PAT source 1..17 mol_type protein organism synthetic construct VARIANT 7 note any amino acid VARIANT 9 note any amino acid YTVTFYXDXFTLQFIAC 17 AA PAT source 1..17 mol_type protein organism synthetic construct VARIANT 9 note any amino acid

YTVTFYPDXFTLQFIAC 17 AA PAT source 1..17 mol_type protein organism synthetic construct VARIANT 7 note any amino acid YTVTFYXDPFTLQFIAC 17 AA PAT source 1..17 mol_type protein organism synthetic construct VARIANT 17 note any amino acid other than C SITE 1 note chloroacetylated tyrosine YTVTFYPDPFTLQFIAX 6 AA

PAT source 1..6 mol_type protein organism synthetic construct GSGSGS 17 AA PAT source 1..17 mol_type protein organism synthetic construct SITE 1 note chloroacetylated tyrosine VARIANT 7 note any amino acid VARIANT 9 note any amino acid YTVTFYXDXFTLQFIAC 17 AA PAT source 1..17 mol_type protein organism synthetic construct VARIANT 9 note any amino acid SITE 1 note chloroacetylated tyrosine YTVTFYPDXFTLQFIAC 17 AA PAT source 1..17 mol_type protein organism synthetic construct VARIANT 7 note any amino acid SITE 1 note chloroacetylated tyrosine YTVTFYXDPFTLQFIAC 5 AA PAT source 1..5 mol_type protein note IgM (Fig 1d) organism synthetic construct DLPSP 5 AA PAT source 1..5 mol_type protein note IgG1 (Fig 1d) organism synthetic construct ALPAP 5 AA PAT source 1..5 mol_type protein note IgG2 (Fig 1d) organism synthetic construct

GLPAP 5 AA PAT source 1..5 mol_type protein note IgG3 (Fig 1d) organism synthetic construct ALPAP 5 AA PAT source 1..5 mol_type protein note IgG4 (Fig 1d) organism synthetic construct GLPSS 5 AA PAT source 1..5 mol_type protein note IgA1 (Fig 1d) organism synthetic construct ESKTP 5 AA PAT source 1..5 mol_type protein note IgA2 (Fig 1d) organism synthetic construct ELKTP 5 AA PAT source 1..5 mol_type protein note IgD (Fig 1d) organism synthetic construct SLPPQ

AA PAT source 1..5 mol_type protein note IgE (Fig 1d) organism synthetic construct HLPRA 21 AA PAT source 1..21 mol_type protein note cL1 (Fig 2c) organism synthetic construct SITE 1 note N-chloroacetylated-L-tyrosine, cyclised to cysteine residue 17 SITE 20 note biotin-modified lysine SITE 17 note cyclised to N- chloroacetylated-L-tyrosine residue 1 YYFKYVPYPTGLYNVYCGSKG 21 AA PAT source 1..21 mol_type protein note cL2 (Fig 2c) organism synthetic construct SITE 1 note

N-chloroacetylated-L-tyrosine, cyclised to cysteine residue 6 SITE 20 note biotin- modified lysine SITE 6 note cyclised to N-chloroacetylated-L-tyrosine residue 1

YYWAGCPSPLSDSGYRAGSKG 21 AA PAT source 1..21 mol_type protein note cL3 (Fig 2c) organism synthetic construct SITE 1 note N-chloroacetylated-L-tyrosine, cyclised to cysteine residue 17 SITE 20 note biotin-modified lysine SITE 17 note cyclised to N-chloroacetylated-L-tyrosine residue 1 YTVTFYPDPFTLQFIACGSKG 21

AA PAT source 1..21 mol_type protein note cL4 (Fig 2c) organism synthetic construct SITE 1 note N-chloroacetylated-L-tyrosine, cyclised to cysteine residue 6

SITE 20 note biotin-modified lysine SITE 6 note cyclised to N-chloroacetylated-L- tyrosine residue 1 YYWAGCSNLYINAGSRAGSKG 21 AA PAT source 1..21 mol_type protein note cL8 (Fig 2¢) organism synthetic construct SITE 1 note N- chloroacetylated-L-tyrosine, cyclised to cysteine residue 11 SITE 20 note biotin- modified lysine SITE 11 note cyclised to N-chloroacetylated-L-tyrosine residue 1

YYTSKGPNPFCLLWRDAGSKG 21 AA PAT source 1..21 mol_type protein note cL11 (Fig 2c) organism synthetic construct SITE 1 note N-chloroacetylated-L-tyrosine, cyclised to cysteine residue 17 SITE 20 note biotin-modified lysine SITE 17 note cyclised to N-chloroacetylated-L-tyrosine residue 1 YFLNLKPSPFNWWNNYCGSKG 21

AA PAT source 1..21 mol_type protein note cL13 (Fig 2c) organism synthetic construct SITE 1 note N-chloroacetylated-L-tyrosine, cyclised to cysteine residue 17

SITE 20 note biotin-modified lysine SITE 17 note cyclised to N-chloroacetylated-L- tyrosine residue 1 YWLQSRPNPFQIEELWCGSKG 21 AA PAT source 1..21 mol_type protein note cL3 (Fig. 3e) organism synthetic construct SITE 1 note N- chloroacetylated-L-tyrosine, cyclised to cysteine residue 17 SITE 17 note cyclised to

N-chloroacetylated-L-tyrosine residue 1 SITE 20 note lysine residue with the sidechain amine replaced by an azide YTVTFYPDPFTLQFIACGSKG 19 AA PAT source 1..19 mol_type protein note cL3-delN1 (Fig. 3e) organism synthetic construct SITE 1 note N-chloroacetylated-L-tyrosine, cyclised to cysteine residue 16 SITE 16 note cyclised to N-chloroacetylated-L-tyrosine residue 1 SITE 19 note lysine residue with the sidechain amine replaced by an azide YVTFYPDPFTLQFIACGSK 18 AA PAT source 1..18 mol_type protein note cL3-delN2 (Fig. 3e) organism synthetic construct SITE 1 note N-chloroacetylated-L-tyrosine, cyclised to cysteine residue 15

SITE 15 note cyclised to N-chloroacetylated-L-tyrosine residue 1 SITE 18 note lysine residue with the sidechain amine replaced by an azide YTFYPDPFTLQFIACGSK

AA PAT source 1..15 mol_type protein note cL3-delN5 (Fig. 3e) organism synthetic construct SITE 1 note N-chloroacetylated-L-tyrosine, cyclised to cysteine residue 12 SITE 12 note cyclised to N-chloroacetylated-L-tyrosine residue 1 SITE 15 note lysine residue with the sidechain amine replaced by an azide

YPDPFTLQFIACGSK 14 AA PAT source 1..14 mol_type protein note cL3-delN6 (Fig. 3e) organism synthetic construct SITE 1 note N-chloroacetylated-L-tyrosine, cyclised to cysteine residue 11 SITE 11 note cyclised to N-chloroacetylated-L- tyrosine residue 1 SITE 14 note lysine residue with the sidechain amine replaced by an azide YDPFTLQFIACGSK 12 AA PAT source 1..12 mol_type protein note cL3- delN8 (Fig. 3e) organism synthetic construct SITE 1 note N-chloroacetylated-L- tyrosine, cyclised to cysteine residue 9 SITE 9 note cyclised to N-chloroacetylated-

L-tyrosine residue 1 SITE 12 note lysine residue with the sidechain amine replaced by an azide YFTLQFIACGSK 19 AA PAT source 1..19 mol_type protein note cL3- delC1 (Fig. 3e) organism synthetic construct SITE 1 note N-chloroacetylated-L- tyrosine, cyclised to cysteine residue 16 SITE 16 note cyclised to N- chloroacetylated-L-tyrosine residue 1 SITE 19 note lysine residue with the sidechain amine replaced by an azide YTVTFYPDPFTLQFICGSK 18 AA PAT source 1..18 mol_type protein note cL3-delC2 (Fig. 3e) organism synthetic construct SITE 1 note

N-chloroacetylated-L-tyrosine, cyclised to cysteine residue 15 SITE 15 note cyclised to N-chloroacetylated-L-tyrosine residue 1 SITE 18 note lysine residue with the sidechain amine replaced by an azide YTVTFYPDPFTLQFCGSK 16 AA PAT source 1..16 mol_type protein note cL3-delC4 (Fig. 3e) organism synthetic construct SITE 1 note N-chloroacetylated-L-tyrosine, cyclised to cysteine residue 13 SITE 13 note cyclised to N-chloroacetylated-L-tyrosine residue 1 SITE 16 note lysine residue with the sidechain amine replaced by an azide YTVTFYPDPFTLCGSK 14 AA PAT source 1..14 mol_type protein note cL3-delC6 (Fig. 3e) organism synthetic construct SITE 1 note N-chloroacetylated-L-tyrosine, cyclised to cysteine residue 11 SITE 11 note cyclised to N-chloroacetylated-L-tyrosine residue 1 SITE 14 note lysine residue with the sidechain amine replaced by an azide YTVTFYPDPFCGSK 17 AA PAT source 1..17 mol_type protein note page 34 organism synthetic construct SITE 1 note N- chloroacetylated-L-tyrosine VARIANT 2..6 note any amino acid except methionine

VARIANT 8 note any amino acid except methionine VARIANT 10..16 note any amino acid except methionine YXXXXXPXPXXXXXXXC

Claims (26)

Conclusions A complement component C1q binder comprising a peptide comprising the amino acid sequence YX1X2TFYX3X4X5FTLQFIX6X7 (SEQ ID NO. 1), wherein X; is absent, or is T or K, X; is absent, or is V, Xs is any amino acid 1s, X4 is D or N, Xs is any amino acid 1s, Xs is absent, or is A, E, or T, and X; is C, S, or a stop codon, provided that a maximum of two of X;, Xs, and Xe are absent, wherein the peptide binds to a globular head domain of mammalian complement component C1q. Complement component Clq binder according to claim 1, wherein X3 and/or X5 is/are P. The complement component C1q binder of claim 1 or 2, wherein the peptide comprises the sequence YTVTFYPDPFTLQFIAX7, (SEQ ID NO: 2), wherein X7 is C or Sis. Complement component Clq binder according to any one of the preceding claims, wherein the peptide is surface-bound and capable of activating complement. 5. Complement component Clq binder according to any one of the preceding claims, wherein the peptide is present in soluble form and is capable of inhibiting complement activation. 6. The complement component Clq binder of any preceding claim, wherein the peptide is a cyclic peptide Xs, wherein the N-terminus of the peptide binds to the C-terminus of Xs. 7. Complement component Clq binder according to claim 6, wherein the N-terminus tyrosine is chloroacetylated (Cl-Ac) IS. 8. The complement component Clq binder of claim 6 or 7, wherein the C-terminus and/or the N-terminus is/are configured to conjugate with one or more different groups. The complement component C1q binder of claim 8, wherein the one or more different groups is or are a GS linker selected from the group consisting of GS, GSGS (SEQ ID NO. 43), GGGS (SEQ ID NO. 44), and GSGGSG (SEQ ID NO. 45). The complement component C1q binder of any preceding claim, wherein the peptide comprises one of the sequences selected from the group consisting of YTVTFYX3DX5FTLQFIACGSK (SEQ ID NO. 86), wherein X; and/or X; is any amino acid, YTVTFYPDX5FTLQFIACGSK (SEQ ID NO. 87), wherein X; is any amino acid, YTVTFYX3DPFTLQFIACGSK (SEQ ID NO. 88), wherein Xs any amino acid 1s. Complement component C1q according to any one of the preceding claims, wherein the peptide consists of one of the sequences selected from the group consisting of YTVTFYX3DX5FTLQFIACGSK (SEQ ID NO. 86), wherein Xs and/or X; is/are any amino acid, YTVTFYPDX5FTLQFIACGSK (SEQ ID NO. 87), wherein Xs is any amino acid, YTVTFYX3DPFTLQFIACGSK (SEQ ID NO. 88), wherein Xa is any amino acid. Complement component Clq binder according to claim 10 or 11, wherein the N-terminus tyrosine is chloroacetylated (Cl-Ac) 1s. The complement component C1q binder of any one of claims 1 to 5, and 8 to 11, wherein the peptide is a linear peptide, wherein X; is any amino acid other than C and/or wherein the N-terminus tyrosine is not chloroacetylated (Cl-Ac) 1s. The complement component C1q binder of claim 13, wherein the peptide comprises the sequence YTVTFYPDPFTLQFIAX7 (SEQ ID NO. 105), wherein X7 is any amino acid other than C and/or wherein the N-terminus tyrosine is chloroacetylated (Cl-Ac). The complement component C1q binder of claim 13 or 14, wherein the peptide consists of the sequence YTVTFYPDPFTLQFIAX7 (SEQ ID NO. 105), wherein X is any amino acid other than C and/or wherein the N-terminus tyrosine is chloroacetylated (Cl-Ac) IS. The complement component Clq binder according to any preceding claim, wherein the N-terminus tyrosine is L-tyrosine. 17. Isolated nucleic acid comprising a sequence encoding a peptide that is part of a complement component Clq binder according to any one of the preceding claims. An isolated cell comprising a nucleic acid according to claim 17. A pharmaceutical composition comprising a complement component Clq binder according to any one of claims 1 to 16, a nucleic acid according to claim 17, or a cell according to claim 18, as well as a pharmaceutically acceptable excipient, a pharmaceutically acceptable adjuvant, a pharmaceutically acceptable diluent, or a pharmaceutically acceptable carrier. A kit comprising a complement component C1q binder according to any one of claims 1 to 16, a nucleic acid according to claim 17, a cell according to claim 18, or a pharmaceutical composition according to claim 19, as well as instructions for using the peptide to treat, prevent, or diagnose a disease or condition in a subject. 21. A complement component Clq binder according to any one of claims 1 to 16, a nucleic acid according to claim 17, a cell according to claim 18, or a pharmaceutical composition according to claim 19, for use as a medicament or as a diagnostic agent. 22. The complement component Clq binder of any one of claims 1 to 16, a nucleic acid of claim 17, a cell of claim 18, or a pharmaceutical composition of claim 19, for use in the treatment, diagnosis or prevention of a disease or condition associated with complement activation or inhibition in which Clq plays a role. A complement component Clq binder according to any one of claims 1 to 16, a nucleic acid according to claim 17, a cell according to claim 18, or a pharmaceutical composition according to claim 19, for use in the treatment of an infectious disease, a neurological disease, a neurodegenerative disease, a transplant-versus-host disease, an autoimmune disease, or cancer. 24. The complement component C1q binder or pharmaceutical composition for use according to any one of claims 21 to 23, wherein the peptide or pharmaceutical composition is formulated for use with one or more other pharmaceutical compositions, detectable agents, or therapeutic agents, preferably wherein the one or more other pharmaceutical compositions, detectable agents, or therapeutic agents is/are selected from the group consisting of: complement activating compound, complement inhibiting compound, tracer, label, radioactive compound, toxin, CRISPR-associated protein (Cas), preferably wherein the Cas is CAS9, and a nucleic acid, preferably wherein the nucleic acid is a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA), more preferably wherein the RNA is selected from the group consisting of: small interfering RNA (siRNA), asymmetric interfering RNA (a1RNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), antisense RNA (asRNA), aptamer, circular RNA (circRNA), self-amplifying RNA (saRNA), catalytic RNA, anti-miRNA, long non-coding RNA (IncRNA), single-conducting RNA (sgRNA), and mRNA encoding a Cas, or where the DNA is selected from the group consisting of: an antisense oligonucleotide (ASO), aptamer, episome, and catalytic DNA. A method of treating a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a complement component Clq binder according to any one of claims 1 to 16, a nucleic acid according to claim 17, a cell according to claim 18, or a pharmaceutical composition according to claim 19. A method for producing a peptide that is part of a complement component Clq binder according to any one of claims 1 to 16, the method comprising expressing in a host cell a nucleic acid according to claim 17.