C3b-Binding Fusion Proteins

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ST.26 format and is hereby incorporated by reference in its entirety. Said copy, created on Feb. 26, 2024, is named 222237-705301 sequence listing.xml and is 53,248 bytes in size.

TECHNICAL FIELD

The present invention relates to fusion proteins comprising anti C3b antibodies (e.g. single domain antibodies) and recombinant proteins comprising complement control protein (CCP) domains.

BACKGROUND

The complement system constitutes an important arm of the innate immune system. The complement system labels—or opsonizes—intruding pathogens as well as dying host cells for clearance. Excess activation of the complement system may lead to development of a number of diseases. The complement system hence emerges as an attractive target for inhibitors to treat or ameliorate these diseases.

The complement system comprises the three pathways: the classical pathway, the lectin pathway and the alternative pathway (AP). In the initiating steps of the complement system, pattern recognition molecules (PRM) of the classical and lectin pathways binds to the activator. This leads to activation of PRM-associated serine proteases that cleaves complement component C4. One of the cleavage products of C4, C4b covalently attaches to the activator and associates with C2. Upon proteolytic activation of C2, this complex form the C4b2a C3 convertase, that cleaves C3. One of the cleavage products, C3b covalently attaches to the activator and associates with factor B. The C3b-bound factor B may be activated by factor D, which liberates the Ba moiety from the C3b-bound Bb moiety and leads to the formation of the alternative pathway C3 convertase, C3bBb. The AP C3 convertase cleaves C3 and thereby amplifies the outcome of the two other pathways (Merle et al., 2015). In this process, C3 (the substrate) binds C3b in the C3bBb complex (the AP C3 convertase) and the protease (Bb) cleaves C3 into C3b and C3a.

The AP may also initiate independently of the classical and lectin pathways, through spontaneous hydrolysis of an internal thioester of C3. The resulting C3 (H2O) associates with factor B, which is subsequently cleaved by factor D. The resulting complex C3 (H2O) Bb is a fluid phase C3 convertase, that cleaves C3 into C3a and C3b. The nascent C3b may react with nucleophiles on surfaces, whereby the fluid phase C3 convertase, in principle, may lead to opsonization of any surface.

To prevent excess complement activation, the system also comprises a number of regulators. One of these regulators, factor H, is a fluid phase regulator that interacts with host cells and inhibits the complement progression in two ways. Firstly, factor H is a cofactor for the serine protease factor I that degrades and irreversibly inactivates the activation product of the central complement C3b. Secondly, factor H accelerates the decay of the alternative pathway C3 convertase (C3bBb). Factor H comprises 20 CCP domains.

Different minimized versions of factor H have previously been described, including the mini-FH comprising CCP1-4 and CCP19-20 joined by a polypeptide linker (FIG. 1A) (WO 2013/142362 A1). CCP1-3 is believed to be the minimal unit with cofactor activity for C3b proteolysis by factor I (Hocking et al. 2008).

The single domain antibody named hC3Nb1 capable of binding C3 and C3b is disclosed in Jensen et al. 2018. A C3b specific recombinant version of this single domain antibody named EWE-hC3Nb1 is described in WO 2019/238674 A1. This single domain antibody EWE-hC3Nb1 inhibits the alternative pathway (AP) by preventing the binding of factor B (FB) to C3b. hC3Nb1 binds at the interface between MG6 and MG7 domains of C3b and C3 (FIG. 2C). In complex with C3, the N-terminus is in close proximity to the MG6 domain (FIG. 2D). The inventors rationalized that an extension of the N-terminus of hC3Nb1 might render it specific towards C3b. The inventors hence extended the N-terminus of hC3Nb1 by a Glu-Trp-Glu motif, which conferred specificity of the resulting EWE-hC3Nb1 toward C3b (FIGS. 3A-3C). The EWE-hC3Nb1 binds close to, but does not overlap with CCP2 of factor H.

SUMMARY

Regulators of the complement system have long been sought for, since overactivation of the complement system induces tissue damage and sustains chronic inflammation. The inventors of the present disclosure made the surprising discovery that a recombinant linkage of a single domain antibody capable of binding C3b (EWE-hC3Nb1) to a novel minimized version of factor H comprising the CCP2-4 and CCP19-20 domains of factor H, results in an alternative pathway (AP) inhibitor that mediates cleavage of C3b by factor I. As shown in the examples, this novel fusion protein retains both its capabilities from the single domain antibody moiety and surprisingly also the recombinant factor H moiety, thus showing that the inventors have in fact discovered an AP inhibitor that prevents binding of factor B or fragments thereof to C3b and mediates cleavage of C3b by factor I. Having seen the surprising effect of this fusion protein, the inventors of the present disclosure went on to develop an even further minimized version comprising only EWE-hC3Nb1 and CCP2-4. This smaller protein showed an even higher efficiency in promoting C3b cleavage, a slightly lower binding affinity towards C3b and a high capability of being concentrated to a high concentration.

Thus, in a first aspect of the invention, there is provided a fusion protein comprising:

- a) an antibody or antigen-binding fragment thereof that binds to C3b; and
- b) a polypeptide that binds to C3b, wherein the polypeptide comprises: (i) the amino acid sequences according to SEQ ID NO: 11 or a variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 11; and (ii) the amino acid sequences according to SEQ ID NO: 12 or a variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 12; optionally wherein the polypeptide further comprises (iii) the amino acid sequences according to SEQ ID NO: 13 or a variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 13;
  
  wherein the antibody or antigen-binding fragment thereof of the fusion protein does not compete for binding to C3b with the polypeptide of the fusion protein.

In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to C3b and not C3.

In some embodiments, the antibody or antigen-binding fragment thereof does not compete for binding to C3b with factor I.

In some embodiments, the antibody or antigen-binding fragment thereof is capable of inhibiting interaction between C3b and factor B or active Bb and thereby of inhibiting assembly of C3bBb convertase complex and/or the activity of C3bBb convertase.

In some embodiments, the antibody or antigen-binding fragment thereof competes for binding to C3b with factor B.

In some embodiments, the antibody or antigen-binding fragment thereof competes for binding to C3b with factor B, and does not compete for binding to C3b with factor I.

In some embodiments, the antibody or antigen-binding fragment thereof binds an epitope comprised in the MG7 domain of C3b (SEQ ID NO: 20) and the N-terminus region of the C3b alpha chain (e.g., residues 1-30, 1-20 or 4-12 of SEQ ID NO:19).

In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope consisting of the amino acids:

- a) Glu759, Asn760, His918, His919, Phe920 of C3b (corresponding to Glu11, Asn12, His170, His171, Phe172 of the amino acid sequence according to SEQ ID NO: 19);
- b) Glu758, Glu759, Asn760, Arg855, Val916, His918, His919, Phe920 of C3b (corresponding to Glu10, Glu11, Asn12, Arg107, Val168, His170, His171, Phe172 of the amino acid sequence according to SEQ ID NO: 19); or
- c) Asp752, Asp754, Ile755, Ile756, Glu759, Asn760, His918, His919, Phe920 of C3b (corresponding to Asp4, Asp6, Ile7, Ile8, Glu11, Asn12, His170, His171, Phe172 of the amino acid sequence according to SEQ ID NO: 19).

In some embodiments, the antibody or antigen-binding fragment thereof competes for binding to C3b with the CCP1 domain of factor H (SEQ ID NO: 21).

In some embodiments, the antibody or antigen-binding fragment thereof is a single domain antibody.

In some embodiments, the single domain antibody comprises a single variable domain comprising CDR1, CDR2 and CDR3, wherein the CDR1 comprises the amino acid sequence according to SEQ ID NO: 2, the CDR2 comprises the amino acid sequence according to SEQ ID NO: 3 and the CDR3 comprises the amino acid sequence according to SEQ ID NO: 4.

In some embodiments, the single domain antibody specifically binds to C3b and comprises or consists of (a) an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 23 and (b) a N-terminal motif, optionally wherein the single domain antibody comprises or consists of the amino acid sequence of SEQ ID NO: 23 and a N-terminal motif.

In some embodiments, the N-terminal motif creates specificity towards C3b by sterically hindering binding to C3, optionally wherein the N-terminal motif is a 3 amino acid sequence comprising or consisting of the amino acids EWE.

In some embodiments, the single domain antibody comprises or consists of the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the antibody or antigen-binding fragment thereof is connected to the polypeptide by a first linker.

In some embodiments, the first linker is a glycine-serine (GS) linker, such as a GS linker comprising or consisting of an amino acid sequence according to SEQ ID NO: 6.

In some embodiments, the polypeptide is less than 180 amino acids, such as less than 175, 170, 165, 160, 155, 150, 145, 140, 135 or 130 amino acids, optionally wherein the polypeptide is 125 amino acids long.

In some embodiments, the polypeptide comprises or consists of the amino acid sequence according to SEQ ID NO: 10, or a variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 10.

In some embodiments, the polypeptide is less than 240 amino acids, such as less than 235, 230, 225, 220, 215, 210, 205, 200, 195, 190 or 185 amino acids, optionally wherein the polypeptide is 183 amino acids long.

In some embodiments, the polypeptide further comprises: (i) the amino acid sequences according to SEQ ID NO: 14; and/or (ii) the amino acid sequences according to SEQ ID NO: 16.

In some embodiments, the polypeptide comprises: (i) the amino acid sequences according to SEQ ID NO: 11; (ii) the amino acid sequences according to SEQ ID NO: 12; (iii) the amino acid sequences according to SEQ ID NO: 13; (iv) the amino acid sequences according to SEQ ID NO: 14; and (v) the amino acid sequences according to SEQ ID NO: 16.

In some embodiments, the polypeptide comprising or consisting of the amino acid sequence according to SEQ ID NO: 9, or a variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 9.

In some embodiments, the polypeptide is less than 380 amino acids, such as less than 375, 370, 365, 360, 355, 350, 345, 340, 335, 330, 325 or 320 amino acids, optionally wherein the polypeptide is 319 amino acids long.

In some embodiments, the polypeptide does not comprise the CCP1 domain, such as the amino acid sequence according to SEQ ID NO: 21, or a variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 21.

In some embodiments, the polypeptide is capable of recruiting factor I, such as recruiting the amino acid sequence according to SEQ ID NO: 22.

In some embodiments, the fusion protein inhibits the alternative pathway by preventing binding of factor B or fragments thereof to C3b and recruits factor I and/or wherein the fusion protein hinders formation of the alternative pathway C3 convertase, C3bBb and recruits factor I, such as wherein the fusion protein recruits the amino acid sequence according to SEQ ID NO: 22.

In some embodiments, the fusion protein inhibits the alternative pathway by preventing binding of factor B or fragments thereof to C3b and activates degradation of C3b by recruiting factor I.

Having realized that the factor H mediated cofactor activity for C3b proteolysis by factor was retained in the novel minimized version comprising only CCP2-4 and the version comprising CCP2-4 and CCP19-20, the invention in two further aspects relates to: A polypeptide having a length of less than 220 amino acids, wherein the polypeptide comprises the amino acid sequence according to SEQ ID NO: 10 or a variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 10; and

A polypeptide having a length of less than 350 amino acids, wherein the polypeptide comprises the amino acid sequence according to SEQ ID NO: 9 or a variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 9.

In further aspects, the disclosure also relates to a nucleic acid encoding the fusion protein or the polypeptides of the present disclosure.

In a further aspect, the disclosure relates to a vector comprising the nucleic acid of the disclosure.

In a further aspect, the disclosure relates to a method of preparing the vector of the disclosure, the method comprising:

- a) introducing the vector of the disclosure into a cell;
- b) culturing the cell; and
- c) collecting and purifying the vector from the cell;
- d) optionally, lysing the cell to release the vector from the cell.

In a further aspect, the disclosure relates to a host cell comprising the nucleic acid or the vector of the disclosure.

In a further aspect, the disclosure relates to a method of preparing the fusion protein or the polypeptide of the disclosure, the method comprising:

- a) introducing the nucleic acid or the vector of the disclosure into a host cell;
- b) culturing the cell; and
- c) obtaining and optionally purifying the fusion protein or polypeptide from the supernatant;
- d) optionally, lysing the cell prior to purifying the fusion protein or the polypeptide.

In a further aspect, the disclosure relates to a pharmaceutical composition comprising the fusion protein or the polypeptide of the disclosure, optionally comprising a pharmaceutically acceptable carrier.

In a further aspect, the disclosure relates to a method of treating a disorder associated with complement activation, the method comprising administering a therapeutically effective amount of the fusion protein, the polypeptide or the pharmaceutical composition of the disclosure to a subject in need thereof. In some embodiments, the disorder is selected from the group consisting of ocular diseases, neurological diseases, autoimmune and inflammatory disorders, cancers and infectious diseases.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1C Schematic representation of minimized factor H fragments. (FIG. 1A) The previously described mini-FH comprising CCP domains 1˜4 and 19-20 of factor H. (FIG. 1B) The EWEμH fragment (SEQ ID NO: 5) described in the present disclosure, comprising the single domain antibody, EWE-hC3Nb1 (SEQ ID NO: 1), as well as CCP domains 2-4 and 19-20 of factor H (SEQ ID NO: 9). (FIG. 1C) The EWEnH (SEQ ID NO: 7) described in the present disclosure. EWEnH comprises the single domain antibody, EWE-hC3Nb1 (SEQ ID NO: 1) and CCP domains 2-4 of factor H (SEQ ID NO: 10).

FIGS. 2A-2D Structural comparison of hC3Nb1 in complex with C3b and native C3. Structure of the parental hC3Nb1 (black) in complex with (FIG. 2A) C3b (grey) [PDB entry 6EHG] and (FIG. 2B) native C3 (grey) [PDB entry 6RU5]. The C3 molecules of panels A and B are aligned on the macroglobulin (MG)-ring. The binding interface between hC3Nb1 and (FIG. 2C) C3b [PDB entry 6EHG] and (FIG. 2D) native C3 [PDB entry 6RU5]. The structures in panels C and D are aligned on the hC3Nb1 molecule.

When comparing (FIG. 2C) and (FIG. 2D) the figure shows that the MG6 domain is in close proximity to the hC3Nb1 single domain antibody in (FIG. 2D) whereas the MG6 domain is further away in (FIG. 2C). On the basis of this discovery the inventors hypothesized that an additional N-terminal motif might confer specificity towards C3b (FIG. 2C).

FIGS. 3A-3C Validation of EWE-hC3Nb1. (FIG. 3A) SEC-based analysis of the C3b:EWE-hC3Nb1 interaction. C3b in presence or absence of EWE-hC3Nb1 was subjected to SEC. (FIG. 3B) As in panel A, but analyzing the C3:EWE-hC3Nb1 interaction. (FIG. 3C) Non-reducing SDS-PAGE analysis of peak fractions from panels A-B. The data were previously presented in WO 2019/238674 A1.

The figure shows that EWE-hC3Nb1 conferred a clear shift in elution volume, when mixed with C3b (FIG. 3A), but not when mixed with native C3 (FIG. 3B), showing that EWE-hC3Nb1 is specific against C3b. Subsequent SDS PAGE of peak fractions confirmed the formation of the EWE-hC3Nb1:C3b complex (FIG. 3C). On the basis of these data, the inventors concluded that the addition of the N-terminal motif had in fact created specificity against C3b.

FIGS. 4A-4C Design of EWEμH and EWEnH. (FIG. 4A) left: The structure of hC3Nb1 [PDB entry 6EHG] (Jensen et al., 2018) was superimposed onto the structure of C3b:mini-FH [PDB entry 5O35] (Xue et al., 2017) without the CCP1 domain. The dashed line illustrates a flexible linker connecting the C-terminus of EWE-hC3Nb1 and the N-terminus of the CCP2 domain. Right: The structure of hC3Nb1 [PDB entry 6EHG] (Jensen et al., 2018) was superimposed onto the structure of C3b:mini-FH [PDB entry 5O35] (Xue et al., 2017) without the CCP1, and CCP19-20 domains. The dashed line illustrates a flexible linker connecting the C-terminus of EWE-hC3Nb1 and the N-terminus of the CCP2 domain. (FIG. 4B) Interaction site of hC3Nb1 on C3b indicated as footprint [PDB entry 6EHG]. (FIG. 4C) Interaction site of CCP1 of mini-FH on C3b indicated as footprint [PDB entry 5O35]. Amino acid residues within 4.5 Å of hC3Nb1 and CPP1 are indicated in FIGS. 4B and 4C, respectively.

The figure shows how fusion proteins of the disclosure bind to C3b (FIG. 4A) and that EWE-hC3Nb1 and the FH CCP1 contact C3b in the same region and to some extent interact with identical residues on C3b (FIG. 4B) and (FIG. 4C). On the basis of these data, the inventors concluded that EWE-hC3Nb1 and the FH CCP1 domain might compete for binding to C3b.

FIGS. 5A-5C Initial validation of EWEuH. (FIGS. 5A-5B) Factor H-mediated C3b cleavage by factor I. C3b was incubated with 0.2% factor H, 1% factor I and a 1.2-fold molar excess of either (FIG. 5A) EWE-hC3Nb1 or (FIG. 5B) hC3Nb1. The data in panel A were previously presented in WO 2019/238674 A1. The cleavage of C3b was monitored by SDS PAGE. (FIG. 5C) EWEμH cleavage assay. C3b was incubated in presence of factor I and a two-fold molar excess of mini-FH, EWEμH or mini-FH as well as EWE-hC3Nb1. The cleavage of C3b was monitored by SDS-PAGE analysis upon the indicated incubation at 37° C.

The figure shows that EWE-hC3Nb1 (FIG. 5A) and hC3Nb1 (FIG. 5B) delays the cleavage of C3b by factor I. In addition, EWEμH (FIG. 5C) compared to mini-FH (CCP1-4, 19-20) as well as a mix of both EWE-hC3Nb1 and mini-FH (CCP1-4, 19-20) shows that EWEμH sustains the cleavage of C3b, however that the mix of EWE-hC3Nb1 and mini-FH does not. On the basis of these data, the inventors concluded that EWEμH mediates cleavage of C3b by factor I.

FIGS. 6A-6C Comparison of EWEμH and EWEnH. (FIGS. 6A-6B) Comparison of EWEμH and EWEnH. C3b was incubated with 0.5% factor I and a (FIG. 6A) 0.5-fold molar ratio or (FIG. 6B) 2-fold molar excess of EWEμH or EWEnH. The cleavage of C3b by factor I was monitored by SDS-PAGE analysis upon the indicated period of incubation at 37° C. (FIG. 6C) Quantification of cleavage. The intensity of the bands of the α′-chain of C3b in FIGS. 6A-6B were quantified using the ImageJ software. The intensity was normalized to the intensity of the α′-chain of C3b incubated with factor I for 8 h. Average and S.D. (error bars) are shown for n=3 experiments for the 1:2 ratio and n=2 experiments for the 1:0.5 ratio.

The figure shows that EWEnH and EWEμH mediates cleavage of C3b by factor I in molar ratios of C3b: fusion protein of 1:0.5 (FIG. 6A) and 1:2 (FIG. 6B). The quantitive comparison in (FIG. 6C) indicates that EWEnH is more efficient in sustaining C3b cleavage at both tested molar ratios. These data conclude that both fusion proteins are capable of mediating cleavage of C3b, and that EWEnH might be more efficient.

FIGS. 7A-7B Bio-layer interferometry-based analysis of EWE-hC3Nb1. (FIG. 7A) Anti-penta his biosensors were coated with EWE-hC3Nb1 and the sensors were transferred into a dilution series from 1.56-50 nM C3b. (FIG. 7B) Anti-penta his biosensors were coated with EWE-hC3Nb1 and transferred into a dilution series of 3.125-50 nM iC3b.

The figure shows that EWE-hC3Nb1 exhibits similar affinity toward C3b (FIG. 7A) and iC3b (FIG. 7B).

FIGS. 8A-8B Bio-layer interferometry-based analysis of EWEμH. (FIG. 8A) Anti-penta his biosensors were coated with EWEμH. The sensors were transferred into a dilution series from 1.5625-50 nM C3b. (FIG. 8B) Anti-penta his biosensors were coated with EWEμH and transferred into an iC3b dilution series ranging from 3.125-50 nM.

The figure shows that EWEμH exhibits higher affinity towards C3b (FIG. 8A) than towards iC3b (FIG. 8B).

FIGS. 9A-9B Bio-layer interferometry-based analysis of EWEnH. (FIG. 9A) Anti-penta his biosensors were coated with EWEnH. The sensors were transferred into a dilution series from 3.125-100 nM C3b. (FIG. 9B) Anti-penta his biosensors were coated with EWEnH. The iC3b concentration tested ranged from 6.25-100 nM. The figure shows that EWEnH exhibits similar affinity toward C3b (FIG. 9A) and iC3b (FIG. 9B).

FIG. 10 Summary of affinities. The table summarizes the mean of k_a, k_dand K_D±standard deviation from n=3 experiments for C3b kinetics and n=2 experiments for iC3b kinetics.

The figure shows that the fusion EWEμH binds app. 10× stronger to C3b than the single domain antibody EWE-hC3Nb1, whereas it binds with similar affinity towards iC3b. The fusion protein EWEnH shows a slightly lower binding affinity towards C3b and iC3b than the single domain antibody EWE-hC3Nb1. Overall, these data conclude that strong binding affinity towards C3b and iC3b of the single domain antibody EWE-hC3Nb1 is retained when the single domain antibody is comprised in a fusion protein.

FIG. 11 Functional comparison of EWEμH and EWEnH. Inhibition of the AP mediated hemolysis of rabbit erythrocytes. The effects of EWE-hC3Nb1, EWEμH, and EWEnH were assayed in 11% serum. The lysis was normalized to 100% in NHS without nanobodies, and 0% without NHS. The effects were compared to the parental AP inhibitor hC3Nb1 (Jensen et al., 2018), the broad inhibitor hC3Nb2 (Pedersen et al., 2020), and the inactive hC3Nb1 (W102A) mutant (Jensen et al., 2018). The dashed line indicate putative C3 concentrations assuming a C3 concentration of 5.4 UM in undiluted serum (Engstrom et al., 2007). Average and S.D. (error bars) are shown for n=3 experiments.

The figure shows that EWEμH, EWEnH, EWE-hC3Nb1, and hC3Nb1 inhibit the AP mediated C3 fragment deposition. The data confirms that the functional properties of the single domain antibody EWE-hC3Nb1 is retained when the single domain antibody is comprised in a fusion protein.

FIGS. 12A-12B Concentration assay of EWEμH and EWEnH. (FIG. 12A) EWEμH and EWEnH were concentrated and subjected to SEC. (FIG. 12B) SDS-PAGE analysis of peak fractions from FIG. 12A.

The figure shows that the fusion protein EWEnH tolerates ultrafiltration to higher concentrations than EWEμH. On the basis of these data, the inventors conclude that the smaller protein EWEnH tolerates being ultra-filtrated to a surprisingly high concentration and thus might have a higher clinical and commercial value than EWEμH.

FIG. 13 EWEnH and EWEμH mediated degradation of rat C3b. Rat C3b was incubated with 1% w/w factor I and a 2-fold molar excess of EWEμH or EWEnH. The cleavage of rat C3b by factor I was monitored by SDS-PAGE analysis upon the indicated period of incubation at 37° C. The figure shows that both EWEμH and EWEnH are capable of mediating cleavage of rat C3b by factor I.

DETAILED DESCRIPTION

Uncontrolled activation or lack of proper regulation of complement is involved in a range of diseases and the present disclosure therefore provides means for pharmacological regulation of the complement cascade in order to ameliorate disease outcome.

The present invention relates to fusion proteins capable of targeting the complement C3b protein and facilitating its degradation. Generally, the fusion proteins of the disclosure comprise an antibody or antigen-binding fragment thereof (e.g., a single domain antibody), thus when antibodies or antigen-binding fragments thereof (e.g., single domain antibodies) are described further below, they are understood as being comprised in the fusion protein.

In particular, the disclosure relates to a fusion protein comprising (a) an antibody or antigen-binding fragment thereof (e.g., a single domain antibody) that binds to C3b; and (b) a polypeptide comprising complement control protein (CCP) domains, such as μH (micro-H) or nH (nano-H). In some embodiments, the antibody or antigen-binding fragment thereof (e.g., a single domain antibody) is capable of preventing the binding of factor B (FB) (SEQ ID NO:30) to C3b, and the recombinant protein is capable of recruiting factor I (SEQ ID NO: 22). As a result, the fusion protein is capable of inhibiting the AP by preventing binding of factor B or fragments thereof to C3b and by mediating cleavage of C3b by factor I.

The Complement System

The complement system is part of the innate immune system and plays an important role in protection against invading microorganisms and in maintenance of homeostasis. The innate immune system is not adaptable and does not change over the course of an individual's lifetime. More than 50 proteins and protein fragments make up the complement system, including serum proteins, serosal proteins, and cell membrane bound receptors and regulatory proteins. A subset of the complement proteins circulates as inactive precursors (pro-proteins). When stimulated by one of several triggers, proteases in the system cleave specific proteins to initiate an amplifying cascade of further cleavages. The end-result of this activation cascade includes massive amplification of the response, enhanced phagocytosis and pathogen lysis, clearance of immune complexes and apoptotic cells, inflammation, stimulation of adaptive immune responses and assembly of the cell-killing membrane attack complex.

More specifically, the complement system is activated by three different proteolytic pathways: The classical pathway (CP), the lectin pathway (LP) and the alternative pathway (AP). Activation of the complement system results in cleavage of the complement proteins C3 (all pathways) into C3a and C3b. After a certain threshold of C3b density is reached on the complement activating surface, activation of the terminal pathway (TP) results in cleavage of complement C5.

The CP is activated by the C1 complex formed by the pattern recognition molecule C1q and the serine proteases C1r and C1s. C1 recognition leads to activation of the C1 complex. C1 cleaves C4 into C4a and C4b, and C2 can now bind to C4b, and C2 is then cleaved by the C1 complex into C2a and C2b. C4bC2a is the CP C3 convertase that cleaves C3 into C3a and C3b.

The LP is initiated by recognition of carbohydrate or acetylated pathogen-associated molecular patterns (PAMPs) by mannose binding lectin (MBL), the collectin CL-LK and three ficolins, respectively. MBL, CL-LK and the ficolins are associated with MBL-associated serine proteases (MASP)-1 and -2 that are activated upon binding of MBL, CL-LK and ficolins to DAMPs and PAMPs. MASP-1 and MASP-2 activation triggers the same proteolytic cascade as the classical pathway and also leads to assembly of the CP C3 convertase.

The AP may be activated by a spontaneous hydrolysis of an internal thioester in C3, resulting in formation of C3 (H₂O). C3 (H₂O) associates with the protease factor B (FB), which is cleaved by factor D into Bb and Ba. The complex between C3 (H₂O) and Bb is the fluid phase AP C3 convertase that cleaves C3 into C3a and C3b. C3b reacts with nearby nucleophiles on surfaces and become covalently attached to these resulting in activator bound C3b. Such C3b associates with FB that is cleaved and the AP C3 convertase, C3bBb is formed. The AP C3 convertase cleaves C3 into C3b and C3a and more AP convertase is formed in an amplification loop. The activator bound C3b may also originate from C3 cleavage conducted by the CP C3 convertase, and indeed the AP C3 convertase strongly amplifies the initial C3 cleavage taking place in the CP and LP.

C3 and C3b

Complement component 3 (C3) is an immune system protein having a central role in innate immunity and the complement system. Human C3 (SEQ ID NO:33) comprises a 1,663 amino acid sequence (including an N-terminal, 22 amino acid signal peptide). C3 comprises an alpha chain and a beta chain associated through interchain disulfide bonds. C3b is formed from C3 by proteolytic removal of the small anaphylatoxin domain (amino acids 668 to 748) in the alpha chain. Accordingly, amino acids 23 to 667 encode C3b beta chain (SEQ ID NO:24), and amino acids 749 to 1,663 encode C3b alpha chain (SEQ ID NO:19).

C3b is a potent opsonin, targeting pathogens, antibody-antigen immune complexes and apoptotic cells for phagocytosis by phagocytes and NK cells. C3b is also involved in the formation of convertase enzyme complexes for activating and amplifying complement responses. C3b associates with Factor B to form the alternative pathway C3 convertase, C3bBb.

C3b can be processed to an inactive form unable to participate in convertase assembly, designated iC3b. Cleavage is performed by Complement Factor I and is facilitated by co-factors for Complement Factor I. Co-factors for Complement Factor I typically bind to C3b and/or Complement Factor I, and potentiate processing of C3b to iC3b by Complement Factor I. Molecules capable of acting as co-factors for Complement Factor I include Complement Factor H.

As used herein “C3” refers to C3 from any species. In some embodiments, the C3 is mammalian C3 (e.g. cynomolgus, human and/or rodent (e.g. rat and/or murine) C3), preferably human C3. In some embodiments, C3 may be characterized as having the amino acid sequence of SEQ ID NO:33.

As used herein “C3b” refers to C3b from any species. In some embodiments, the C3b is mammalian C3b (e.g. cynomolgus, human and/or rodent (e.g. rat and/or murine) C3b), preferably human C3b. In some embodiments, C3b may be characterized as comprising:

- (a) a C3b alpha chain having the amino acid sequence of SEQ ID NO:19; and
- (b) a C3b beta chain having the amino acid sequence of SEQ ID NO: 24.

In the context of the present disclosure individual complement proteins may be identified as the specific proteins shown in the sequence list further below, however the skilled person will appreciate that the present disclosure is not limited to the specific species or proteins disclosed herein.

Antibodies and Antigen-Binding Fragments Thereof, Including Single-Domain Antibodies

The fusion protein of the present invention comprises an antibody or antigen-binding fragment thereof (e.g., single domain antibody) that binds to C3b. In a preferred embodiment, the antibody or antigen-binding fragment (e.g., single domain antibody) thereof specifically binds to C3b. The term “specifically binds to C3b” refers to an antibody that does not bind to other antigens (such as C3), or does not bind to other antigens with sufficient affinity to produce a physiological effect.

In some embodiments, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) targeting C3b is capable of inhibiting interaction between C3b and the factor B (FB) or the active Bb. This inhibits assembly of the C3bBb convertase complex and/or the activity of the C3bBb convertase. In a preferred embodiment, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) is capable of inhibiting activation of the complement system.

In one embodiment of the present disclosure, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) is capable of inhibiting the alternative pathway by preventing binding of factor B or fragments thereof to C3b. In another embodiment of the present disclosure, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) is capable of hindering formation of the alternative pathway C3 convertase, C3bBb.

In some embodiments, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) is capable of inhibiting interaction between C3b and the factor B (FB) or fragments thereof to C3b by blocking the interaction of the glycan attached to Asn122 of factor B with C3b.

In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) binds to C3b and is capable of preventing binding of the substrate C3 (e.g., amino acid sequence according to SEQ ID NO: 33) to C3b, e.g. C3b alone or C3b of the AP C3 convertase (C3bBb). By blocking binding of C3b to C3, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) is capable of inhibiting the alternative pathway.

The term “antibody” refers to an immunoglobulin (lg) molecule that binds to, or is immunologically reactive with, a particular antigen. Naturally occurring human antibodies are heterotetramers. An antibody generally comprises two “light chains” (LC) and two “heavy chains” (HC). Each heavy chain comprises a heavy chain constant region (CH) and a heavy chain variable region (VH); and each light chain comprises a light chain constant domain (CL) and a light chain variable domain (VL). The heavy chain constant region comprises the heavy chain constant domains CH1, CH2 and CH3 (antibody classes IgA, IgD, and IgG) and optionally the heavy chain constant domain CH4 (antibody classes IgE and IgM).

The variable regions VH and VL further comprises regions of hypervariability, termed complementarity determining regions (CDRs), which are also known as hypervariable regions. Binding between an antibody and its target antigen or epitope is mediated by the CDRs. The CDRs are interspersed with more conserved framework regions (FRs). Generally, each VH and VL of an antibody have three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (from the amino-terminus to the carboxy-terminus).

The constant regions of the heavy chain and the light chain serve as effectors rather than being directly engaged in how an antibody binds to a target. The immunoglobulin can attach to host tissues or factors through the constant regions of antibodies to facilitate immune response. IgA, IgG, IgD, IgE, and IgM are the five main classes of heavy chain constant region, with each having unique effector roles designated by isotype. In certain embodiments, the antibodies or antigen-binding fragments thereof that bind to C3b are IgG isotype. The antibodies or antigen-binding fragments can be any IgG subclass, for example IgG1, IgG2, IgG3, or IgG4 isotype.

In some embodiments, the antibodies of the invention are monoclonal antibodies. A “monoclonal antibody” (mAb) refers to a homogenous antibody population that recognises and binds a single antigenic determinant or epitope. It can be obtained from different ways, including but not limited to hybridoma, phage selection, recombinant expression and transgenic animals.

The antibodies and antigen-binding fragments thereof of the invention may be derived from any species by recombinant means, such as human, rat, mouse, horse, rabbit, goat, swine, bovine, chicken, camelid, donkey, or chimeric versions thereof.

Especially preferred are human or humanized antibodies, especially as recombinant human or humanized antibodies.

The term “antigen-binding fragment” as used herein includes any naturally occurring or artificially constructed configuration of an antigen-binding polypeptide comprising one, two or three heavy chain CDRs, and/or one, two or three light chain CDRs, wherein the polypeptide is capable of binding to the antigen (e.g., C3b).

In some embodiments, the antigen-binding fragment according to the invention can be a single-domain antibody, a minibody, an Fab′, a V NAR domain, an IgNAR, an Fv, an scFv, an Fd, an Fcab, an scFv-Fc, F(ab′)2, a di-scFv, an intrabody, a tetrabody, a triabody, a diabody, an IgG CH2, a bi-specific T-cell engager (BiTE®), a F(ab′)3, a DVD-Ig, an (scFv) 2, a mAb2, a DARPin, or a cyclic peptide.

The strength of binding between receptors and their ligands, for example between an antibody and its antigen can be defined by its affinity towards an antigen. The affinity of an antibody can be defined in terms of the dissociation constant, K_D, which is an equilibrium constant that measures the propensity of a molecular complex to separate (dissociate) reversibly into the molecules forming the complex. The smaller the value of the K_Dof an antibody, the higher affinity that the antibody binds to a target antigen. In one aspect, K_Dis defined as the ratio K_off/K_on, where K_offand K_onare the rate constants for association and dissociation of the molecular complex. Preferably affinity is determined by calculating the dissociation constant K_Dbased on IC₅₀values. Thus, the affinity is measured as an apparent affinity. It is preferred that the antibody or antigen-binding fragment thereof (e.g., single domain antibodies) as described herein bind with an affinity corresponding to a K_Dof about 10⁻⁴M or less, such as about 10⁻⁵M or less, such as about 10⁻⁶M or less, 10⁻⁷M or less, such as about 10⁻⁸M or less, such as about 10⁻⁹M or less, about 10⁻¹⁰M or less, or about 10⁻¹¹M or even less, when measured based on apparent affinities based on EC₅₀values (the effective concentration achieving 50% of maximal binding) in an ELISA assay. In one embodiment, the antibody binds with an affinity corresponding to a K_Dof about 10⁻⁶M or less, such as 10⁻⁶to 10⁻¹⁸M. K_Dvalues for antibodies can be determined using methods in the art in view of the present disclosure. For example, the K_Dof an antibody can be determined by using surface plasmon resonance, such as by using a biosensor system, e.g., a Biacore® system, or by using bio-layer interferometry technology, such as an Octet RED96 system. In one embodiment, the antibody or antigen-binding fragment thereof (e.g., single domain antibodies) binds with an affinity corresponding to a K_Dof 10⁻⁴M to 10⁻¹¹M, 10⁻⁴M to 10⁻¹⁰M, 10⁻⁴M to 10⁻⁹M, 10⁻⁴M to 10⁻⁸M, 10⁻⁴M to 10⁻⁷M, 10⁻⁴M to 10⁻⁶M, 10⁻⁴M to 10⁻⁵M, 10⁻⁵M to 10⁻¹¹M, 10⁻⁵M to 10⁻¹⁰M, 10⁻⁵M to 10⁻⁹M, 10⁻⁵M to 10⁻⁸M, 10⁻⁵M to 10⁻⁷M, 10⁻⁵M to 10⁻⁶M, 10⁻⁶M to 10⁻¹¹M, 10⁻⁶M to 10⁻¹⁰M, 10⁻⁶M to 10⁻⁹M, 10⁻⁶M to 10⁻⁸M, 10⁻⁶M to 10⁻⁷M, 10⁻⁷M to 10⁻¹¹M, 10⁻⁷M to 10⁻¹⁰M, 10⁻⁷M to 10⁻⁹M, 10⁻⁷M to 10⁻⁸M, 10⁻⁸M to 10⁻¹¹M, 10⁻⁸M to 10⁻¹⁰M, 10⁻⁸M to 10⁻⁹M, 10⁻⁹M to 10⁻¹¹M, or 10⁻⁹M to 10⁻¹⁰M as determined by using surface plasmon resonance. In one embodiment, the antibody or antigen-binding fragment thereof (e.g., single domain antibodies) binds with an affinity corresponding to a K_Dof 10⁻⁸M to 10⁻¹⁰M as determined by using surface plasmon resonance.

The antibody may also or alternatively bind their target complement factor with an affinity corresponding to a K_Dthat is at least ten-fold lower, such as at least 100-fold lower, such as at least 1000-fold lower than its affinity for binding to a non-specific antigen (e.g., BSA or casein).

In preferred embodiments, the antibodies or antigen-binding fragments thereof comprised in the fusion proteins provided herein comprise an antigen binding site in a single polypeptide. The antibodies or antigen-binding fragments thereof are therefore herein referred to as “single domain antibodies”. Single domain antibodies are also known as nanobodies.

A single domain antibody is an antibody fragment consisting of a single monomeric variable antibody domain. Like a naturally occurring human antibody, it is able to bind selectively to a specific antigen. Single domain antibodies typically have molecular weights in the range of 12-15 kDa, i.e. much lower than common antibodies, ranging typically from 150 to 160 kDa. Single domain antibodies are also smaller than Fab fragments (˜50 kDa) of heterotetrameric antibodies comprising one light chain and half a heavy chain. The binding site of an immunoglobulin single variable domain is formed by a single VH/VHH or VL domain. Hence, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDRs.

As such, the single variable domain of a single domain antibody may be a light chain variable domain sequence (e.g., a VL-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH-sequence or VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit that essentially consists of the single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit).

The single domain antibody of the present disclosure preferably comprises one or more CDRs. In particular, the CDRs may identify the specificity of the antibody and accordingly it is preferred that the antigen binding site comprises one or more CDRs, preferably at least 1, more preferably at least 2, yet more preferably 3 CDRs. In one specific embodiment, the single domain antibody comprises 3 CDRs.

Given the high importance of the CDR regions in target binding it is generally considered that alterations in the sequence of the antibodies or antigen-binding fragments thereof (e.g., single domain antibodies) are made outside of the CDRs. Thus, in one embodiment of the present disclosure, any sequence variance is outside the complementarity determining regions. The sequence of a CDR region can be annotated by different methods, once the sequence has been obtained. The exact boundaries of CDRs, and framework regions in the present invention have been determined using the Kabat method.

Single domain antibodies can derive from antibodies found in nature, for example in camelids (V_HH) and cartilaginous fishes (V_NAR). New or Nurse Shark Antigen Receptor (NAR) protein exists as a dimer of two heavy chains with no associated light chains. Each chain is composed of one variable (V) and five constant domains. The NAR proteins thus constitute a single immunoglobulin variable-like domain. Single heavy-chain antibodies are also found in camelids, such as such as dromedaries, camels, llamas and alpacas, where the heavy chain has lost one of its constant domains and underwent modifications in the variable domain, both of which are structural elements necessary for the binding of light chains.

However, single domain antibodies can also be engineered by recombinant methods. One approach is to split the dimeric variable domains from common IgG from humans or mice into monomers. Single domains, which are derived from light chains, also bind specifically to target epitopes. Thus, the single domain antibody may be derived from any suitable organism.

Single domain camelid antibodies are equal to regular antibodies in terms of specificity. Single domain antibodies are easily isolated, for example by using phage panning procedures. The smaller size and single domain architecture make these antibodies easier to express as proteins in bacterial cells for large scale production, making them ideal for commercial exploitation. The single domain antibodies of the present disclosure are therefore preferably derived from camelid antibodies, preferably llama antibodies.

In some embodiments, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) is further engineered to obtain specificity. In particular, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) is further engineered to specifically bind C3b and not C3.

In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) comprises a N-terminal motif. In one embodiment of the present disclosure, the N-terminal motif creates specificity towards C3b by sterically hindering binding to C3. In one embodiment of the present disclosure, the N-terminal motif is a protein, such as an IgG. In one embodiment of the present disclosure, the N-terminal motif is the Fc region of a human IgG. In another embodiment, the N-terminal motif may be a bulky N-terminal motif. In one embodiment of the present disclosure, the bulky N-terminal motif is an amino acid sequence consisting of up to 10 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids. In another embodiment of the present disclosure, the bulky N-terminal motif is comprised of bulky amino acids, such as phenylalanine, tyrosine, tryptophan, arginine, histidine, lysine, tyrosine, glutamic acid and glutamine. In a preferred embodiment of the present disclosure, the bulky N-terminal motif is a 3 amino acid sequence consisting of the amino acids EWE.

Single Domain Antibody Species

The single domain antibody of the present invention binds to C3b. In a preferred embodiment, the single domain antibody specifically binds to C3b.

In some embodiments, the single domain antibody targeting C3b is capable of inhibiting interaction between C3b and the factor B (FB) (e.g., amino acid sequence according to SEQ ID NO:30) or the active Bb and thereby of inhibiting assembly of the C3bBb convertase complex and/or the activity of the C3bBb convertase. In a preferred embodiment, the single domain antibody is capable of inhibiting activation of the complement system. In one embodiment of the present disclosure, the single domain antibody is capable of inhibiting the alternative pathway by preventing binding of factor B (e.g., amino acid sequence according to SEQ ID NO:30) or fragments thereof to C3b.

In some embodiments, the single domain antibody is capable of inhibiting interaction between C3b and the factor B (FB) or fragments thereof to C3b by blocking the interaction of the glycan attached to Asn122 of factor B with C3b.

In some embodiments of the present disclosure, the single domain antibody binds to C3b and is capable of preventing binding of the substrate C3 (e.g., amino acid sequence according to SEQ ID NO:33) to C3b, e.g. C3b alone or C3b of the AP C3 convertase (C3bBb).

In some embodiments of the present disclosure, the single domain antibody binds to C3b, wherein the single domain antibody comprises a single variable domain comprising CDR1, CDR2 and CDR3, wherein the CDR1 comprises the amino acid sequence according to SEQ ID NO: 2, the CDR2 comprises the amino acid sequence according to SEQ ID NO: 3 and the CDR3 comprises the amino acid sequence according to SEQ ID NO: 4.

In some embodiments of the present disclosure, the single domain antibody binds to C3b and comprises or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 23. In a preferred embodiment, the single domain antibody comprises or consists of an amino acid sequence having at least 81% sequence identity to the amino acid sequence SEQ ID NO: 23, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity. In a specific embodiment of the present disclosure, the single domain antibody comprises or consists of the amino acid sequence of SEQ ID NO: 23.

In some embodiments of the present disclosure, the single domain antibody comprises a N-terminal motif. In one embodiment of the present disclosure, the N-terminal motif creates specificity towards C3b by sterically hindering binding to C3.

Thus, in some embodiments of the present disclosure, the single domain antibody specifically binds to C3b and comprises or consists of (a) an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 23 and (b) a N-terminal motif. In a preferred embodiment, the single domain antibody comprises or consists of an amino acid sequence having at least 81% sequence identity to the amino acid sequence SEQ ID NO: 23, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity. In a specific embodiment of the present disclosure, the single domain antibody comprises or consists of the amino acid sequence of SEQ ID NO: 23 and a N-terminal motif. In one embodiment of the present disclosure, the N-terminal motif is an amino acid sequence consisting of up to 10 amino acids, such as 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids. In a preferred embodiment of the present disclosure, the N-terminal motif is a 3 amino acid sequence consisting of the amino acids EWE.

In one embodiment of the present disclosure, the single domain antibody specifically binds to C3b and comprises or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 1. In a preferred embodiment, the single domain antibody comprises or consists of an amino acid sequence having at least 81% sequence identity to the amino acid sequence SEQ ID NO: 1, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity. In a specific embodiment of the present disclosure, the single domain antibody comprises or consists of the amino acid sequence of SEQ ID NO: 1.

In one embodiment of the present disclosure, the single domain antibody specifically binds to C3b and comprises or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 25. In a preferred embodiment, the single domain antibody comprises or consists of an amino acid sequence having at least 81% sequence identity to the amino acid sequence SEQ ID NO: 25, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity. In a specific embodiment of the present disclosure, the single domain antibody comprises or consists of the amino acid sequence of SEQ ID NO: 25.

In some embodiments of the present disclosure, the single domain antibody binds to C3b, wherein the single domain antibody comprises a single variable domain comprising CDR1, CDR2 and CDR3, wherein the CDR1 comprises the amino acid sequence according to SEQ ID NO: 27, the CDR2 comprises the amino acid sequence according to SEQ ID NO: 28 and the CDR3 comprises the amino acid sequence according to SEQ ID NO: 29.

In some embodiments of the present disclosure, the single domain antibody binds to C3b and comprises or consists of an amino acid sequence having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 26. In a preferred embodiment, the single domain antibody comprises or consists of an amino acid sequence having at least 81% sequence identity to the amino acid sequence SEQ ID NO: 26, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity. In a specific embodiment of the present disclosure, the single domain antibody comprises or consists of the amino acid sequence of SEQ ID NO: 26.

Given the high importance of the CDR regions in target binding it is generally considered that alterations in the sequence of the single domain antibodies are made outside of the CDRs. Thus, in one embodiment of the present disclosure, any sequence variance is outside the complementarity determining regions. For example, the sequence variation in e.g. SEQ ID NO:1 or SEQ ID NO: 25 is in the framework regions, i.e. outside the amino acid sequences according to SEQ ID NOs: 2, 3 and 4. For example, the sequence variation in e.g. SEQ ID NO:26 is in the framework regions, i.e. outside the amino acid sequences according to SEQ ID NOs: 27, 28 and 29.

Epitope

The antigen binding site of an antibody or antigen-binding fragment thereof (e.g., single domain antibody) is able to bind selectively to a specific antigen. The site on an antigen at which an antibody or antigen-binding fragment thereof (e.g., single domain antibody) binds is referred to as an epitope. The part of an antibody or binding molecule that recognizes the epitope is called a paratope. An epitope can be a linear epitope or a conformational epitope. A linear epitope is an epitope that is recognized by antibodies by its linear sequence of amino acids, or primary structure. A conformational epitope, or a 3-dimensional epitope, has a specific three-dimensional shape and can be comprised of amino acids situated at different sites in a polypeptide chain or even on different polypeptide chains. An epitope can also be located on molecules other than polypeptides, such as saccharides and organic—and inorganic molecules.

One way of determining the epitope of which a protein binds to, is to determine its footprint.

The antibodies or antigen-binding fragments thereof (e.g., single domain antibodies) comprised in the fusion proteins of the disclosure are capable of (e.g. specifically) binding to C3b. An antibody or antigen-binding fragment thereof (e.g., single domain antibodies) that specifically binds to C3b may be cross-reactive with related antigens, in particular C3b of a different species, e.g. cynomolgus, human and/or rodent (e.g. rat and/or murine) C3b. In certain embodiments, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) specifically binds to human C3b and rat C3b.

The epitope on which the antibody binds may be a conformational or a linear epitope. Methods of determining the epitope to which an antibody binds are known in the art. For example, the epitope may be determined by alanine mutagenesis or by X-ray crystallography.

In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) binds an epitope comprised in the amino acid sequence according to SEQ ID NO: 19.

When an antibody is said to bind to an epitope within specified residues, such as SEQ ID NO: 20, for example, what is meant is that the antibody specifically binds to a polypeptide consisting of the specified residues (i.e., SEQ ID NO: 20 in this an example). Such an antibody does not necessarily contact every residue within SEQ ID NO: 20.

In another embodiment, of the present disclosure, the single domain antibody binds an epitope comprised in the MG7 domain of C3b and the N-terminus of the C3b alpha chain, such as an epitope comprised in the amino acid sequence according to SEQ ID NO: 20 and the N-terminus of SEQ ID NO: 19.

In another embodiment, of the present disclosure, the antibody or antigen-binding fragment thereof (e.g., the single domain antibody) binds an epitope comprised in the MG7 domain of C3b (SEQ ID NO: 20) and the N-terminus region of the C3b alpha chain (e.g., residues 1-30, 1-20 or 4-12 of SEQ ID NO:19).

In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) binds to the same binding sites in C3b as that of the CCP1 domain (SEQ ID NO: 21), the CCP4 domain (SEQ ID NO: 16), the CCP19 domain (SEQ ID NO: 14) or the CCP20 domain (SEQ ID NO: 16) of factor H.

In a preferred embodiment, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) binds to the same binding site in C3b as that of the CCP1 domain (SEQ ID NO: 21) of factor H.

In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) binds to the same epitope as that of the single domain antibody as defined in SEQ ID NO: 1 (i.e., EWE-hC3Nb1), SEQ ID NO: 23 (i.e. hC3Nb1), SEQ ID NO: 25 (i.e. IgG-Fc-hC3Nb1) or SEQ ID NO: 26 (i.e. DI62). In a preferred embodiment of the present disclosure, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) binds to the same epitope as that of the single domain antibody as defined in SEQ ID NO: 1 (i.e., EWE-hC3Nb1) or SEQ ID NO: 25 (i.e. IgG-Fc-hC3Nb1). In a more preferred embodiment, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) binds to the same epitope as that of the single domain antibody as defined in SEQ ID NO: 1 (i.e., EWE-hC3Nb1).

In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) binds to an epitope consisting of the amino acids:

- a) Glu759, Asn760, His918, His919, Phe920 of C3b (corresponding to Glu11, Asn12, His170, His171, Phe172 of the amino acid sequence according to SEQ ID NO: 19);
- b) Glu758, Glu759, Asn760, Arg855, Val916, His918, His919, Phe920 of C3b (corresponding to Glu10, Glu11, Asn12, Arg107, Val168, His170, His171, Phe172 of the amino acid sequence according to SEQ ID NO: 19); or
- c) Asp752, Asp754, Ile755, Ile756, Glu759, Asn760, His918, His919, Phe920 of C3b (corresponding to Asp4, Asp6, Ile7, Ile8, Glu11, Asn12, His170, His171, Phe172 of the amino acid sequence according to SEQ ID NO: 19).

In some embodiments, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) binds to the same binding site in C3b as factor B, thereby preventing factor B from binding to C3b. In one embodiment, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) binds to an epitope comprising at least one of the following residues of C3b: Arg94, Gly142 to Arg148, Thr161, Gln177 to Ser181, Pro190 to Glu197, Glu759 to Val762, Glu766, Trp771, Trp773 to Val775, Phe794 to Lys796, Leu867, His868, Arg891, Leu907, Glu909, Glu911, Arg926, Glu977, Asn1293, Ser1302 to Ser1305, Thr1308 to Arg1310, His1312, Glu1322 to Gluy1326, Lys1337, Glu1530, Glu1531, Cys1537, Val1541, Asp1542, Lys1570 to Glu1575 and Val1658 to Asn1663 (corresponding to Arg72, Gly120 to Arg126, Thr139, Gln155 to Ser159 and Pro168 to Glu175 of the amino acid sequence according to SEQ ID NO: 24 and Glu11 to Val14, Glu18, Trp23, Trp25 to Val27, Phe46 to Lys48, Leu119, His120, Arg143, Leu159, Glu161, Glu163, Arg178, Glu229, Asn545, Ser554 to Ser557, Thr560 to Arg562, His564, Glu574 to Glu578, Lys589, Glu782, Glu783, Cys789, Val793, Asp794, Lys822 to Gly827 and Val910 to Asn915 of the amino acid sequence according to SEQ ID NO: 19). The above residues are the putative factor B binding site in C3b (Forneris et al. Science. 2010 Dec. 24; 330 (6012): 1816-20).

In some embodiments, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) binds to the same binding site in C3b as C3, thereby preventing substrate C3 from binding to C3b (e.g. C3b alone or C3b of the AP C3 convertase (C3bBb). In one embodiment, the antibody or antigen-binding fragment thereof (e.g., the single domain antibody) binds an epitope comprised in the MG4 (Thr351 to Ser447 of C3b, corresponding to Thr329 to Ser425 of SEQ ID NO: 24), MG5 (Thr448 to Asp557, corresponding to Thr426 to Asp535 of EQ ID NO: 24) and/or MG7 domain of C3b (SEQ ID NO: 20).

An antigen can comprise many different epitopes. A protein which is not an antibody can also bind to another protein, in some instances it will bind to an epitope with less specificity and less affinity. When two epitopes are situated in close proximity, a situation can occur where only one molecule, is able to bind its epitope, and hence compete for binding with the protein capable of binding said other epitope. In other situations two proteins/antibodies bind to the same epitope, and must compete for binding. In some aspects of the present disclosure, the epitope to which the antibodies or antigen-binding fragments thereof (e.g., single domain antibodies) bind are described by their capability of competing for binding with other proteins.

In certain embodiments, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) does not compete for binding to C3b with the polypeptide of the fusion protein. In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) does not compete for binding to C3b with the amino acid sequence according to SEQ ID NO: 9 or SEQ ID NO: 10.

In one embodiment of the present disclosure, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) does not compete for binding to C3b with factor I, such as the amino acid sequence according to SEQ ID NO: 22.

In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) does not compete for binding to C3b with the CCP2 and/or the CCP3 domain of factor H, such as the amino acid sequence according to SEQ ID NO: 11 and/or SEQ ID NO: 12. In a preferred embodiment of the present disclosure, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) does not compete for binding to C3b with the CCP2 and the CCP3 domain of factor H, such as the amino acid sequence according to SEQ ID NO: 11 and SEQ ID NO: 12.

In a more preferred embodiment of the present disclosure, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) does not compete for binding to C3b with factor I (such as the amino acid sequence according to SEQ ID NO: 22), and the CCP2 and the CCP3 domain of factor H (such as the amino acid sequence according to SEQ ID NO: 11 and SEQ ID NO: 12).

In some embodiments of the present disclosure, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) competes for binding to C3b with factor B (e.g., amino acid sequence according to SEQ ID NO:30).

In a preferred embodiment, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) competes for binding to C3b with the CCP1 domain of factor H, such as the amino acid sequence according to SEQ ID NO: 21.

In another embodiment of the present disclosure, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) competes for binding to C3b with a protein capable of binding to an epitope consisting of the amino acids:

- a) Glu759, Asn760, His918, His919, Phe920 of C3b (corresponding to Glu11, Asn12, His170, His171, Phe172 of the amino acid sequence according to SEQ ID NO: 19);
- b) Glu758, Glu759, Asn760, Arg855, Val916, His918, His919, Phe920 of C3b (corresponding to Glu10, Glu11, Asn12, Arg107, Val168, His170, His171, Phe172 of the amino acid sequence according to SEQ ID NO: 19); or
- c) Asp752, Asp754, Ile755, Ile756, Glu759, Asn760, His918, His919, Phe920 of C3b (corresponding to Asp4, Asp6, Ile7, Ile8, Glu11, Asn12, His170, His171, Phe172 of the amino acid sequence according to SEQ ID NO: 19).

In a preferred embodiment of the present disclosure, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) competes for binding to C3b with the single domain antibody as defined in SEQ ID NO: 1 or SEQ ID NO: 25, i.e., it competes for binding to C3b with EWE-hC3Nb1 or IgG-Fc-hC3Nb1. In a more preferred embodiment of the present disclosure, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) competes for binding to C3b with the single domain antibody as defined in SEQ ID NO: 1, i.e. it competes for binding to C3b with EWE-hC3Nb1.

In one embodiment, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) competes for binding to C3b with factor B (such as the amino acid sequence according to SEQ ID NO: 30), and does not compete for binding to C3b with factor I (such as the amino acid sequence according to SEQ ID NO: 22).

In one embodiment, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) competes for binding to C3b with factor B (such as the amino acid sequence according to SEQ ID NO: 30), and does not compete for binding to C3b with the CCP2 domain (SEQ ID NO: 11) and the CCP3 domain (SEQ ID NO: 12) of factor H.

In a preferred embodiment, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) competes for binding to C3b with factor B (such as the amino acid sequence according to SEQ ID NO: 30), and does not compete for binding to C3b with factor I (such as the amino acid sequence according to SEQ ID NO: 22), and the CCP2 domain (SEQ ID NO: 11) and the CCP3 domain (SEQ ID NO: 12) of factor H.

In a more preferred embodiment, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) competes for binding to C3b with the CCP1 domain of factor H (such as the amino acid sequence according to SEQ ID NO: 21) and factor B (such as the amino acid sequence according to SEQ ID NO: 30), and does not compete for binding to C3b with factor I (such as the amino acid sequence according to SEQ ID NO: 22), and the CCP2 domain (SEQ ID NO: 11) and the CCP3 domain (SEQ ID NO: 12) of factor H.

In one embodiment, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) competes for binding to C3b with C3 (such as the amino acid sequence according to SEQ ID NO: 33), and does not compete for binding to C3b with the CCP2 domain (SEQ ID NO: 11) and the CCP3 domain (SEQ ID NO: 12) of factor H.

In another preferred embodiment, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) competes for binding to C3b with C3 (such as the amino acid sequence according to SEQ ID NO: 33), and does not compete for binding to C3b with factor I (such as the amino acid sequence according to SEQ ID NO: 22), and the CCP2 domain (SEQ ID NO: 11) and the CCP3 domain (SEQ ID NO: 12) of factor H.

Competition between antibodies is determined by an assay in which the antibody under test inhibits specific binding of a reference antibody to a common antigen, i.e. C3b. Numerous types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay; solid phase direct biotin-avidin EIA; solid phase direct labeled assay, solid phase direct labeled sandwich assay; and direct labeled RIA. Typically, such an assay involves the use of purified antigen bound to a solid surface or cells bearing the antigen, an unlabelled test antibody and a labeled reference antibody. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antibody. Usually the test antibody is present in excess. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur. Usually, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 50% or 75%.

Antibody Modifications

The antibodies or antigen-binding fragments thereof (e.g., single domain antibodies) disclosed herein above, may in preferred embodiments comprise modifications, which improve the function and/or usability of the antibody. For example, it is not always desirable to use non-human antibodies for human therapy, and accordingly the single domain antibodies provided herein may be humanized antibodies.

The antibody or antigen-binding fragment thereof (e.g., single domain antibody) according to the disclosure may be a humanized antibody (e.g., humanized single domain antibody). A human antibody as used herein is an antibody, which is obtained from a system using human immunoglobulin sequences. Human antibodies may for example be antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom. Human antibodies may also be isolated from a host cell transformed to express the antibody, e.g., from a transfectoma. Human antibodies may also be isolated from a recombinant, combinatorial human antibody library or directly cloned from human B cells.

Human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis or in vivo somatic mutagenesis and thus the amino acid sequences of the V_Hand V_Lregions of the recombinant antibodies are sequences that, while derived from and related to human germline V_Hand V_Lsequences, may not naturally exist within the human antibody germline repertoire in vivo.

A human antibody is preferably at least 90%, more preferably at least 95%, even more preferably at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by a wild type human immunoglobulin gene.

Said transgenic of transchromosomal animal may contain a human immunoglobulin gene miniloci that encodes unrearranged human heavy (μ and/or γ) and κ light chain immunoglobulin sequences. Furthermore, the animal may contain one or more mutations that inactivate the endogenous heavy and light chain loci.

The antibody or antigen-binding fragment thereof (e.g., single domain antibody) of the disclosure may be a chimeric antibody, i.e. an antibody comprising regions derived from different species. The chimeric antibody may for example comprise variable regions from one species of animal and constant regions from another species of animal. For example, a chimeric antibody can be an antibody having variable regions, which derive from a llama monoclonal antibody and constant regions, which are human. Such antibodies may also be referred to as humanized antibodies. The antibodies or antigen-binding fragments thereof (e.g., single domain antibodies) can advantageously be humanized in order to prevent immunological reactions of the human organism against the antibody.

Thus, the antibodies or antigen-binding fragments thereof (e.g., single domain antibodies) provided herein may be a humanized antibody, which is encoded partly by sequences obtained from human germline immunoglobulin sequences and partly from other sequences. Said other sequences are preferably germline immunoglobulines from other species, which produce antibodies or antigen-binding fragments thereof (e.g., single domain antibodies), most preferably from camelidae species, such as llama. In particular, a humanized antibody may be an antibody in which the antigen binding site is derived from an immunoglobulin from llama, whereas some or all of the remaining immunoglobulin-derived parts of the molecule is derived from a human immunoglobulin. The antigen binding site from said llama may for example consist of a complete V_HH or one or more CDRs grafted onto appropriate human framework regions. Thus, in a humanized antibody, the CDRs can be from camelids or cartilaginous fishes, preferably llama, and the other regions of the antibody are of human origin.

In other embodiments, the antibodies or antigen-binding fragments thereof (e.g., single domain antibodies) are modified by codon optimization or other modifications introduced in order to enhance the function and/or usability of the antibody.

Polypeptide

The present disclosure provides both novel polypeptides and fusion proteins comprising the novel polypeptides. Polypeptides or proteins are complex, three-dimensional structures containing one or more long, folded polypeptide chains. Polypeptide chains are composed of a plurality of small chemical units called amino acids, which are connected in a N-terminal to C-terminal fashion via covalent peptide bonds. Naturally occurring amino acids have an L-configuration. Synthetic peptides can be prepared employing conventional synthetic methods, using L-amino acids, D-amino acids or various combinations of L- and D-amino acids. The term “peptide” describes a combination of two or more amino acids. 20 amino acids exist naturally and are typically designated using the following table:

Letter code

Amino acid
3
1

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
Ile
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

Valine
Val
V

The polypeptides provided in the present disclosure are recombinant proteins derived from natural complement proteins. In one embodiment of the present disclosure, the polypeptide is derived from the complement co-factor protein factor H. In certain embodiments of the present disclosure, the polypeptide comprises specific domains from factor H, such as complement control protein (CCP). Factor H comprises 20 CCP domains (CCP1-CCP20). The CCP domain is an evolutionarily conserved protein domain. It is also known as a sushi domain or short consensus repeats (SCR). The name derives from the visual similarity of the domain to nigiri sushi when the primary structure is drawn showing the loops created by the disulfide bonds. Sushi domains exist in a wide variety of complement and adhesion proteins. The structure is known for this domain; it is based on a beta-sandwich arrangement-one face made up of three β-strands hydrogen-bonded to form a triple-stranded region at its center, and the other face formed from two separate β-strands.

In some aspects of the disclosure, the polypeptides provided are minimized versions of factor H. Such a prior art protein, consisting of CCP domains 1˜4 and 19-20, has previously been referred to as a mini-FH. The minimized versions of the present disclosure may preferably be provided in versions not comprising CCP1 (SEQ ID NO: 21). Thus, in specific embodiments, the present disclosure provides μH (micro-H) and nH (nano-H), wherein μH comprises CCP domains 2-4 and 19-20 and nH comprises CCP domains 2-4.

It is preferred that at least one of the domains CCP2, CCP3 or CCP4 are retained in the polypeptide, such as at least CCP2-3 or CCP3-4. In a preferred embodiment of the disclosure, the domains CCP2-3 are retained in the recombinant variant of the complement protein factor H. In a more preferred embodiment of the disclosure, the domains CCP2-4 are retained in the recombinant variant of the complement protein factor H. When one or more domains are removed, one or more linkers may be added to provide the correct flexibility in the polypeptide chain.

In some aspects of the disclosure, the polypeptide comprises CCP domain 2 (SEQ ID NO: 11) and CCP domain 3 (SEQ ID NO: 12) of Factor H. In some embodiments, the polypeptide comprises (a) an amino acid sequence having at least 80% sequence identity, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity, to the amino acid sequence according to SEQ ID NO: 11 and (b) an amino acid sequence having at least 80% sequence identity, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity, to the amino acid sequence according to SEQ ID NO: 12. In some embodiments, the polypeptide comprises (a) an amino acid sequence having at least 80% sequence identity, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity, to the amino acid sequence according to SEQ ID NO: 11 and (b) an amino acid sequence according to SEQ ID NO: 12. In another embodiment, the polypeptide comprises (a) an amino acid sequence having at least 80% sequence identity, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity, to the amino acid sequence according to SEQ ID NO: 12 and (b) an amino acid sequence according to SEQ ID NO: 11. In some embodiments, the polypeptide is less than 180 amino acids, such as less than 175, 170, 165, 160, 155, 150, 145, 140, 135 or 130 amino acids. In one embodiment, the polypeptide is 125 amino acids long.

In certain embodiments, the polypeptide further comprises CCP domain 4 (SEQ ID NO: 13) of Factor H, or a variant thereof having at least 80%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 13.

In certain embodiments, the polypeptide comprises: (i) the amino acid sequences according to SEQ ID NO: 11; (ii) the amino acid sequences according to SEQ ID NO: 12; and (iii) the amino acid sequences according to SEQ ID NO: 13. In some embodiments, the polypeptide is less than 240 amino acids, such as less than 235, 230, 225, 220, 215, 210, 205, 200, 195, 190 or 185 amino acids. In one embodiment, the polypeptide is 183 amino acids long.

In certain embodiments, the polypeptide further comprises CCP domain 19 (SEQ ID NO: 14) of Factor H, or a variant thereof having at least 80%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 14.

In certain embodiments, the polypeptide further comprises CCP domain 20 (SEQ ID NO: 16) of Factor H, or a variant thereof having at least 80%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity to the amino acid sequence according to SEQ ID NO: 16.

In certain embodiments, the polypeptide further comprises: (i) the amino acid sequences according to SEQ ID NO: 14 or a variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 14; and (ii) the amino acid sequences according to SEQ ID NO: 16 or a variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In certain embodiments, the polypeptide further comprises: (i) the amino acid sequences according to SEQ ID NO: 14; and (ii) the amino acid sequences according to SEQ ID NO: 16.

In certain embodiments, the polypeptide comprises: (i) the amino acid sequences according to SEQ ID NO: 11 or a variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 11; (ii) the amino acid sequences according to SEQ ID NO: 12 or a variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 12; (iii) the amino acid sequences according to SEQ ID NO: 14 or a variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 14; and (iv) the amino acid sequences according to SEQ ID NO: 16 or a variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the polypeptide is less than 380 amino acids, such as less than 375, 370, 365, 360, 355, 350, 345, 340, 335, 330, 325 or 320 amino acids. In one embodiment, the polypeptide is 319 amino acids long.

In certain embodiments, the polypeptide comprises: (i) the amino acid sequences according to SEQ ID NO: 11; (ii) the amino acid sequences according to SEQ ID NO: 12; (iii) the amino acid sequences according to SEQ ID NO: 14; and (iv) the amino acid sequences according to SEQ ID NO: 16. In some embodiments, the polypeptide is less than 380 amino acids, such as less than 375, 370, 365, 360, 355, 350, 345, 340, 335, 330, 325 or 320 amino acids. In one embodiment, the polypeptide is 319 amino acids long.

In certain embodiments, the polypeptide comprises: (i) the amino acid sequences according to SEQ ID NO: 11 or a variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 11; (ii) the amino acid sequences according to SEQ ID NO: 12 or a variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 12; (iii) the amino acid sequences according to SEQ ID NO: 13 or a variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 13; (iv) the amino acid sequences according to SEQ ID NO: 14 or a variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 14; and (v) the amino acid sequences according to SEQ ID NO: 16 or a variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the polypeptide is less than 380 amino acids, such as less than 375, 370, 365, 360, 355, 350, 345, 340, 335, 330, 325 or 320 amino acids. In one embodiment, the polypeptide is 319 amino acids long.

In certain embodiments, the polypeptide comprises: (i) the amino acid sequences according to SEQ ID NO: 11; (ii) the amino acid sequences according to SEQ ID NO: 12; (iii) the amino acid sequences according to SEQ ID NO: 13; (iv) the amino acid sequences according to SEQ ID NO: 14; and (v) the amino acid sequences according to SEQ ID NO: 16. In some embodiments, the polypeptide is less than 380 amino acids, such as less than 375, 370, 365, 360, 355, 350, 345, 340, 335, 330, 325 or 320 amino acids. In one embodiment, the polypeptide is 319 amino acids long.

In certain embodiments, the polypeptide comprises the following combinations of CCP domains or variants thereof:

CCP2
CCP3
CCP4
CCP19
CCP20

(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID
(SEQ ID

Polypeptide
NO: 11)
NO: 12)
NO: 13)
NO: 14)
NO: 14)

P1
X
X

P2
X
X
X

P3
X
X

X

P4
X
X

X

P5
X
X
X

P6
X
X
X
X

P7
X
X
X

X

P8
X
X

X
X

P9
X
X
X
X
X

The said variants in the above embodiments may have at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the corresponding amino acid sequence.

In some embodiments of the present disclosure, the polypeptide is any recombinant variant of the complement protein factor H, wherein the recombinant variant of the complement protein factor H does not comprise CCP1, such as the amino acid sequence according to SEQ ID NO: 21, or a variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 21. In some embodiments, the polypeptide of the present disclosure does not comprise CCP1, such as the amino acid sequence according to SEQ ID NO: 21, or a variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 21.

The full length version of the human orthologue of factor H is a 1231 amino acid polypeptide and the CCP1 domain is a 64 amino acid polypeptide. The CCP2 domain starts at position 83 of the human factor H, thus in one embodiment of the present disclosure, the polypeptide has a maximal length of 1148 amino acids. The average length of the CCP domains of human factor H is 59 amino acids, and thus various recombinant variants of the complement protein factor H can be made where one or more CCP domains are removed.

Since essentially any of the CCP domains can be retained in the polypeptide, the polypeptide can have various lengths of less than 1148 amino acids, such as less than 1140 amino acids, such as less than 1130 amino acids, such as less than 1120 amino acids, such as less than 1110 amino acids, such as less than 1100 amino acids, such as less than 1090 amino acids, such as less than 1080 amino acids, such as less than 1070 amino acids, such as less than 1060 amino acids, such as less than 1050 amino acids, such as less than 1040 amino acids, such as less than 1030 amino acids, such as less than 1020 amino acids, such as less than 1010 amino acids, such as less than 1000 amino acids, such as less than 990 amino acids, such as less than 980 amino acids, such as less than 970 amino acids, such as less than 960 amino acids, such as less than 950 amino acids, such as less than 940 amino acids, such as less than 930 amino acids, such as less than 920 amino acids, such as less than 910 amino acids, such as less than 900 amino acids, such as less than 890 amino acids, such as less than 880 amino acids, such as less than 870 amino acids, such as less than 860 amino acids, such as less than 850 amino acids, such as less than 840 amino acids, such as less than 830 amino acids, such as less than 820 amino acids, such as less than 810 amino acids, such as less than 800 amino acids, such as less than 790 amino acids, such as less than 780 amino acids, such as less than 770 amino acids, such as less than 760 amino acids, such as less than 750 amino acids, such as less than 740 amino acids, such as less than 730 amino acids, such as less than 720 amino acids, such as less than 710 amino acids, such as less than 700 amino acids, such as less than 690 amino acids, such as less than 680 amino acids, such as less than 670 amino acids, such as less than 660 amino acids, such as less than 650 amino acids, such as less than 640 amino acids, such as less than 630 amino acids, such as less than 620 amino acids, such as less than 610 amino acids, such as less than 600 amino acids, such as less than 590 amino acids, such as less than 580 amino acids, such as less than 570 amino acids, such as less than 560 amino acids, such as less than 550 amino acids, such as less than 540 amino acids, such as less than 530 amino acids, such as less than 520 amino acids, such as less than 510 amino acids, such as less than 500 amino acids, such as less than 490 amino acids, such as less than 480 amino acids, such as less than 470 amino acids, such as less than 460 amino acids, such as less than 450 amino acids, such as less than 440 amino acids, such as less than 430 amino acids, such as less than 420 amino acids, such as less than 410 amino acids, such as less than 400 amino acids, such as less than 390 amino acids, such as less than 380 amino acids, such as less than 370 amino acids, such as less than 360 amino acids, such as less than 350 amino acids, such as less than 340 amino acids, such as less than 330 amino acids, such as less than 320 amino acids, such as less than 310 amino acids, such as less than 300 amino acids, such as less than 290 amino acids, such as less than 280 amino acids, such as less than 270 amino acids, such as less than 260 amino acids, such as less than 250 amino acids, such as less than 240 amino acids, such as less than 230 amino acids, such as less than 220 amino acids, such as less than 210 amino acids, such as less than 200 amino acids, such as less than 190 amino acids or such as less than 180 amino acids. In some embodiments, the polypeptide is less than 180 amino acids, such as less than 175, 170, 165, 160, 155, 150, 145, 140, 135 or 130 amino acids. In one embodiment, the polypeptide is 125 amino acids long. In some embodiments, the polypeptide is less than 240 amino acids, such as less than 235, 230, 225, 220, 215, 210, 205, 200, 195, 190 or 185 amino acids. In one embodiment, the polypeptide is 183 amino acids long. In some embodiments, the polypeptide is less than 380 amino acids, such as less than 375, 370, 365, 360, 355, 350, 345, 340, 335, 330, 325 or 320 amino acids. In one embodiment, the polypeptide is 319 amino acids long.

In certain embodiments, the polypeptide comprises at least one linker for linking different CCP domains or variants thereof together. In one embodiment, the linker consists of the amino acid sequence according to SEQ ID NO: 17.

In one embodiment of the present disclosure, the polypeptide comprises or consists of the amino acid sequence according to SEQ ID NO: 10 or a variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 10. In a preferred embodiment, the polypeptide comprises or consists of an amino acid sequence having at least 81% sequence identity to the amino acid sequence SEQ ID NO: 9, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity.

The present disclosure provides in one aspect a polypeptide having a length of less than 220 amino acids, wherein the polypeptide comprises or consists of the amino acid sequence according to SEQ ID NO: 10 or a variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 10. In one embodiment of the present disclosure, the polypeptide is less than 240 amino acids, such as less than 235, 230, 225, 220, 215, 210, 205, 200, 195, 190, 185 amino acids. In one specific embodiment the polypeptide is 183 amino acids long. In a preferred embodiment, the polypeptide comprises or consists of an amino acid sequence having at least 81% sequence identity to the amino acid sequence SEQ ID NO: 10, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity.

In one embodiment of the present disclosure, the polypeptide comprises or consists of the amino acid sequence according to SEQ ID NO: 9 or a variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 9. In a preferred embodiment, the polypeptide comprises or consists of an amino acid sequence having at least 81% sequence identity to the amino acid sequence SEQ ID NO: 9, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity.

In a further aspect of the present disclosure, the disclosure provides a polypeptide having a length of less than 350 amino acids, wherein the polypeptide comprises or consists of the amino acid sequence according to SEQ ID NO: 9 or a variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 9. In one embodiment of the present disclosure, the polypeptide is less than 380 amino acids, such as less than 375, 370, 365, 360, 355, 350, 345, 340, 335, 330, 325, 320 amino acids. In one specific embodiment the polypeptide is 319 amino acids long. In a preferred embodiment, the polypeptide comprises or consists of an amino acid sequence having at least 81% sequence identity to the amino acid sequence SEQ ID NO: 9, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98% or such as at least 99% sequence identity.

In one embodiment of the present disclosure, the polypeptide comprises or consists of:

- a) the amino acid sequence according to SEQ ID NO: 10 or a variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 10;
- b) the amino acid sequence according to SEQ ID NO: 18 or a variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 18; and
- c) a second linker connecting the amino acid sequence according to SEQ ID NO: 10 to the amino acid sequence according to SEQ ID NO: 18, wherein the second linker may consist of the amino acid sequence according to SEQ ID NO: 17.

In some embodiments, the polypeptide of the disclosure is capable of binding to C3b. In some embodiments, the polypeptide that binds to C3b may bind to related antigens, in particular C3b of a different species, e.g. cynomolgus, human and/or rodent (e.g. rat and/or murine) C3b. In certain embodiments, the polypeptide binds to human C3b and rat C3b.

In one embodiment of the present disclosure, the polypeptide is capable of recruiting factor I, such as recruiting the amino acid sequence according to SEQ ID NO: 22. In one embodiment of the present disclosure, the polypeptide is capable of activating degradation of C3b by recruiting factor I, such as recruiting the amino acid sequence according to SEQ ID NO: 22. In one embodiment of the present disclosure, the polypeptide is a co-factor for factor I, such as the co-factor according to the amino acid sequence according to SEQ ID NO: 22. In one embodiment of the present disclosure, the polypeptide does not comprise the amino acid sequence according to SEQ ID NO: 21, or a variant thereof having at least 80% sequence identity to the amino acid sequence according to SEQ ID NO: 21.

In some embodiments of the present disclosure, the polypeptide does not bind to an epitope comprised in the amino acid sequence according to SEQ ID NO: 20.

In certain embodiments, the polypeptide does not compete for binding with the antibody or antigen-binding fragment thereof (e.g., single domain antibody) of the fusion protein.

In some embodiments of the present disclosure, the polypeptide does not compete for binding to C3b with the single domain antibody as defined in SEQ ID NO: 1 (i.e., EWE-hC3Nb1) or SEQ ID NO: 25 (i.e., IgG-Fc-hC3Nb1). In a more preferred embodiment, the polypeptide does not compete for binding to C3b with the single domain antibody as defined in SEQ ID NO: 1 (i.e., EWE-hC3Nb1).

In some embodiments, the polypeptide binds C3b with an affinity corresponding to a K_Dof about 10⁻⁴to about 10⁻⁹M, about 10⁻⁴to about 10⁻⁸M, 10⁻⁴to about 10⁻⁷M, 10⁻⁵to about 10⁻⁹M, 10⁻⁵to about 10⁻⁸M, 10⁻⁵to about 10⁻⁷M, 10⁻⁶to about 10⁻⁹M, 10⁻⁶to about 10⁻⁸M, 10⁻⁶to about 10⁻⁷M, 10⁻⁷to about 10⁻⁹M, 10⁻⁷to about 10⁻⁸M or 10⁻⁸to about 10⁻⁹M as determined by using surface plasmon resonance. In one embodiment, the polypeptide binds C3b with an affinity corresponding to a K_Dof about 10⁻⁸to about 10⁻⁶M as determined by using surface plasmon resonance. In one embodiment, the polypeptide binds C3b with an affinity corresponding to a K_Dof about 10⁻⁷to about 10⁻⁶M as determined by using surface plasmon resonance.

Fusion Proteins

The present disclosure also provides fusion proteins comprising an antibody or antigen-binding fragment thereof (e.g., single domain antibody) and a polypeptide as described above. Such fusion protein can be assembled by methods known to the person skilled in the art. Preferably, the fusion protein is recombinantly designed by fusing gene sequences in vitro. A fusion protein provided herein may consist of an antibody or antigen-binding fragment thereof (e.g., single domain antibody) and a polypeptide. In some embodiments, the fusion protein further comprises a linker connecting the two.

Each of the antibody or antigen-binding fragment thereof (e.g., single domain antibody) and the polypeptide of the fusion protein binds to C3b. Preferably, the polypeptide and the antibody or antigen-binding fragment thereof (e.g., single domain antibody) do not compete with each other for binding to C3b.

In the fusion protein described in the present disclosure, the antibody or antigen-binding fragment thereof (e.g., single domain antibody) can be linked to the polypeptide at any locations. In some embodiments, the C-terminal of the antibody or antigen-binding fragment thereof (e.g., single domain antibody) is linked to the polypeptide. In some embodiments, the C-terminal of the antibody or antigen-binding fragment thereof (e.g., single domain antibody) is linked to the N-terminal of a CCP domain (e.g., CCP2) of the polypeptide. In some embodiments, the N-terminal of the antibody or antigen-binding fragment thereof (e.g., single domain antibody) is linked to the C-terminal of a CCP domain of the polypeptide.