EP3737472A1 - Modulators of c1q, in particular of the interaction of apoe with c1q, and uses of the modulators in the therapy of neuronal diseases and inflammation - Google Patents

Abstract

The present invention relates to modulators of complex 1 protein q (C1q) that preferably mimic and/or stabilize the interaction of inhibitor apolipoprotein-E (ApoE) with C1q, and their uses in the prevention or treatment of C1q-related diseases or conditions, such Alzheimer's disease (AD), atherosclerosis, kidney diseases, and ageing.

Description

Modulators of clq, in particular of the interaction of ApoE with clq, and uses of the modulators in the therapy of neuronal diseases and inflammation

The present invention relates to modulators of complex 1 protein q (Clq) that preferably mimic and/or stabilize the interaction of inhibitor apolipoprotein-E (ApoE) with Clq, and their uses in the prevention or treatment of Clq-related diseases or conditions, such Alzheimer's disease (AD), atherosclerosis, and ageing.

Background of the invention

Clq is the first subcomponent of the Cl complex of the classical pathway of complement activation, the complement cascade (CCC). Several functions have been assigned to Clq, which include antibody-dependent and independent immune functions, and are considered to be mediated by Clq receptors present on the effector cell surface. Patients suffering from Lupus erythematosus often have deficient expression of Clq. Clq may also play a central role in the aging of cells, as Clq activates canonical Wnt signaling and promotes aging-related phenotypes.

Human Apolipoprotein-E (ApoE) is a polymorphic multifunctional protein arising from three alleles at a single gene locus. However, a common mode of action of ApoE in physiology and disease has not been identified.

Major clinically significant prototypic human unresolvable diseases, e.g. Alzheimer's disease (AD) and atherosclerosis have been closely linked to ApoE. AD is the most common form of dementia and the ApoE4 isoform predisposes to late onset AD (LOAD), and atherosclerosis is the most common cause of death worldwide.

The human isoforms of ApoE, i.e. ApoE2, ApoE3, and ApoE4 differ by amino acid residues 112 and 158 located outside of the N-terminal receptor- and C-terminal lipid binding domains, respectively, yielding proteins with distinct impacts on tissue homeostasis. The presence of two copies of the E4 allele increases risk by ~l2-fold whereas E2 allele is associated with a ~twofold decreased risk for AD. These data put ApoE central to AD pathophysiology, but it is not yet clear how ApoE alleles modify AD risk.

Roumenina L., et al. (in: Complement Clq-target proteins recognition is inhibited by electric moment effectors. J Mol Recognit. 2007 Sep-0ct;20(5):405-l5) disclose low molecular weight disulphate compounds that bind to the globular (gClq) domain. Betulin disulphate (B2S) and 9,9-bis(4'-hydroxyphenyl)fluorene disulphate (F2S) inhibit the interaction of Clq and its recombinant globular modules with target molecules IgGl, C-reactive protein (CRP) and long pentraxin 3 (PTX3).

Roos, A., et al. (in: Specific Inhibition of the Classical Complement Pathway by Clq- Binding Peptides, J Immunol December 15, 2001, 167 (12) 7052-7059) explored the effects of peptides on complement activation. They selected one peptide that inhibits the classical pathway but not the alternative pathway and present its mechanism of action. Because this peptide inhibits Clq from human, primate, and rodent origin, they propose that this peptide is a promising candidate for further development as a therapeutic Clq inhibitor.

Thielens N.M., et al. (in: Clq: A fresh look upon an old molecule. Mol Immunol. 2017 Sep; 89:73-83) summarize the classical functions of Clq and review novel discoveries within the field.

Hajishengallis G, et al. (in: Novel mechanisms and functions of complement. Nat Immunol. 2017 Nov 16;18(12): 1288-1298) disclose a view of new and previously unanticipated functions of complement and how these affect immunity and disease pathogenesis. They mention neuro inflammation, epilepsy, AD, Ischemia-reperfusion injury, AMD, osteoarthritis, Gaucher‘s disease, and cancer.

Haskard DO, et al. (in: The role of complement in atherosclerosis. Curr Opin Lipidol. 2008 Oct;l9(5):478-82) disclose that Clq has been found to play a protective role in early lesion formation in LDL receptor deficient mice, and Crry-Ig and soluble Cl inhibitor have both been shown to have therapeutic effects in models of vascular injury in ApoE deficient mice.

Liu et al. (in: Targeted mouse complement inhibitor CR2-Crry protects against the development of atherosclerosis in mice. Atherosclerosis. 2014 May; 234(l):237-43) propose a therapeutic potential of targeted complement inhibition.

Yong Shen,et al. (in: What does complement do in Alzheimer’s disease? Old molecules with new insights. Transl Neurodegener. 2013; 2: 21) review the increasing evidence that inflammatory and immune components in brain are important in Alzheimer’s disease (AD) and that anti-inflammatory and immunotherapeutic approaches may be amenable to AD treatment. Complement activation occurs in the brain of patients with AD, and contributes to a local inflammatory state development which is correlated with cognitive impairment.

Speidl WS et al. (in: Complement in atherosclerosis: friend or foe? J Thromb Haemost. 2011 Mar;9(3):428-40) review that atherosclerosis is a chronic inflammatory disease and the complement system plays a central role in innate immunity. Increasing evidence exists that the complement system is activated within atherosclerotic plaques. Whereas complement activation by the classic and lectin pathway may be protective by removing apoptotic cells and cell debris from atherosclerotic plaques, activation of the complement cascade by the alternative pathway and beyond the C3 convertase with formation of anaphylatoxins and the terminal complement complex may be proatherogenic and may play a role in plaque destabilization leading to its rupture and the onset of acute cardiovascular events. They present evidence for complement activation within atherosclerotic plaques and discuss recent data derived from experimental animal models that suggest a dual role of complement in the development of the disease.

US 2015-337030 discloses methods for the treatment and/or prevention of a neurodegenerative disorder, such as non-familial late-onset Alzheimer's disease (LOAD), by using an inhibitor of APOE4, such as an antibody inhibitor, or by using an excess of APOE3 protein. Despite the above approaches, new targets for the therapy of diseases or conditions, such as Alzheimer's disease (AD), atherosclerosis, and ageing are desired, in particular in the context of cellular functions mediated directly or indirectly by the complement cascade. It is therefore an object of the present invention, to provide these new targets and to employ these targets in the development of new and effective therapies. Other objects and aspects of the present invention will become apparent to the person of skill upon reading the following description of the invention.

The above problem is solved in a first aspect by a compound for use in the treatment or prevention of a disease or condition in a mammalian subject, wherein the compound is a modulator of the expression, function, stability of Clq or a variant thereof, and in particular mimics and/or stabilizes the interaction of ApoE with Clq or variants thereof, and wherein said disease or condition is selected from neuronal diseases, cardiovascular diseases, kidney diseases, and ageing. The treatment of the present invention is preferably a method for treating a disease in a subject, comprising a step of administering to the subject a therapeutically effective amount of a modulator of the expression, function, stability of Clq or a variant thereof, and in particular mimics and/or stabilizes the interaction of ApoE with Clq or variants thereof.

In the context of the present invention, the inventors explored the roles of ApoE in the aging choroid plexus (ChP) and the aorta of mammals, in particular mice. The data demonstrate that ApoE is a classical complement cascade (CCC) inhibitor by binding to the Cl complex, and that the CCC-ApoE axis impacts neuronal diseases, cardiovascular diseases and ageing, such as AD and atherosclerosis.

Consequently, Clq provides a new target for the identification of modulators that mimic and/or stabilize the function of Clq in the context of the ApoE-Clq interaction. These modulators should prove beneficial for the treatment or prevention of a disease or condition in a mammalian subject, such as a neuronal disease, cardiovascular disease kidney disease, and ageing, such as AD and atherosclerosis. Other diseases to be treated and/or prevented are IgA nephropathy, vasculitis, SLE nephritis, AMD, trauma, sepsis, ARDS, and SIRS. US 2009-117098 discloses a method of treating glaucoma or elevated intraocular pressure comprising administering a pharmaceutically effective amount of a composition comprising a Complement Clq inhibitor. No specific inhibitor or the function of ApoE binding is mentioned.

Similarly, WO 2005-002513 generally discloses the use of agents that bind and/or inhibit classical complement C 1 subcomponent, Clq, as well as methods of their use. Particularly disclosed is a monoclonal antibody.

The terms“Clq-protein” or“protein of Clq” as used in context of the herein disclosed invention shall preferably pertain to the protein (such as a full-length protein, fusion protein or partial protein) consisting of the subunit chains Clqa, Clqb, and Clqc, comprising the sequence of the Complement Clq subcomponent subunit A (UniProtKB - P02745 for human, SEQ ID NO. 1, UniProtKB - P98086 for mouse, SEQ ID NO. 2); Complement Clq subcomponent subunit B (P02746 for human SEQ ID NO. 3, P14106 for mouse SEQ ID NO. 4); and Complement Clq subcomponent subunit C (P02747 for human SEQ ID NO. 5, Q02105 for mouse SEQ ID NO. 6). The terms shall also refer to the individual or combined one, two or three subunits, as long as they are suitable to maintain Clq function.

The terms shall also refer to a protein comprising the amino acid sequence according to SEQ ID NO: 1 to 6 with any protein modifications. Such protein modifications preferably do not alter the amino acid sequence of the polypeptide chain, but constitute a functional group, which is conjugated to the basic amino acid polymer chain. Protein modifications in context of the invention may be selected from a conjugation of additional amino acid sequences to the amino acid chain, such as ubiquitination, sumolation, neddylation, or similar small protein conjugates. Other protein modifications include, but are not limited to, glycosylation, methylation, lipid- conjugation, or other natural or artificial post-translational modifications known to the skilled person. The terms“protein of a variant of Clq” and the like, shall have the corresponding meaning with respect to a variant of Clq. A variant of Clq is, in some embodiments, a protein comprising an amino acid sequence having at least 60%, 70%, 80%, 90%, preferably at least 80% such as at least 90% sequence identity to SEQ ID NO: 1 to 6, and most preferably at least 95% (such as at least 98%) sequence identity to SEQ ID NO: 1 to 6. In one preferred embodiment of the invention, the variant of Clq comprises an amino acid sequence with at least 80% sequence identity to SEQ ID NO: 1 to 6.

As used herein, the terms“identical” or percent“identity”, when used anywhere herein in the context of two or more nucleic acid or protein/polypeptide sequences, refer to two or more sequences or subsequences that are the same or have (or have at least) a specified percentage of amino acid residues or nucleotides that are the same (i.e., at, or at least, about 60% identity, preferably at, or at least, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% or 94%, identity, and more preferably at, or at least, about 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region - preferably over their full length sequences - , when compared and aligned for maximum correspondence over the comparison window or designated region) as measured using a sequence comparison algorithms, or by manual alignment and visual inspection (see, e.g., NCBI web site). In a particular embodiment, for example when comparing the protein or nucleic acid sequence of Clq to another gene/protein, the percentage identity can be determined by the Blast searches supported at the Human Olfactory Data Explorer (eg, https://genome.weizmann.ac.il/cgi-bin/horde/blastHorde.pl); in particular for amino acid identity, those using BLASTP 2.2.28+ with the following parameters: Matrix: BLOSUM62; Gap Penalties: Existence: 11, Extension: 1; Neighboring words threshold: 11; Window for multiple hits: 40.

“Stabilizing” in the context of the present invention, with respect to binding of ApoE to Clq shall mean an increase, preferably a substantial increase, of the binding affinity and/or the half-life of the complex of the two proteins or fragments thereof. Detecting and measuring of a stabilizing (with or without other molecules involved) are known in the art, and reviewed, for example, in Andrei SA, et al, Stabilization of protein-protein interactions in drug discovery, Expert Opin Drug Discov. 2017 Sep;l2(9):925-940.

A variant of Clq is, in certain embodiments, a functional variant of Clq protein. In other embodiments of the invention, the variant of Clq is selected from the group consisting of an ortho log or paralog of Clq, and a functional fragment of a Clq protein.

Preferred is therefore the compound for use according to the present invention, wherein the variant of Clq is selected from the group consisting of an ortho log or paralog of Clq, and a functional fragment of a Clq protein.

The term“ortholog” refers to homologs in different species that evolved from a common ancestral gene by speciation. Typically, orthologs retain the same, essentially the same or similar function despite differences in their primary structure (mutations). The term“paralog” refers to homologs in the same species that evolved by genetic duplication of a common ancestral gene. In many cases, paralogs exhibit related but not always similar function. The term“splice variant” refers to a related protein expressed from the same genomic locus as a parent protein, but having a different amino acid sequence based on a different exon composition due to differential splicing of the transcribed R A.

In context of the present invention the term“subject” or“patient” preferably refers to a mammal, such as a mouse, rat, guinea pig, rabbit, cat, dog, monkey, or preferably a human, for example a human patient. The subject of the invention may be at danger of suffering or is suffering from neuronal diseases, cardiovascular diseases and ageing, such as AD and atherosclerosis. A more detailed description of medical indications relevant in context of the invention is provided herein below.

Preferred is the compound/modulator for use according to the present invention, wherein said compound is an inhibitor or antagonist of said expression, function, stability of Clq or a variant thereof, and/or mimics and/or stabilizes the interaction of ApoE with Clq or variants thereof. The term“modulator” in context of the present invention shall include inhibitors/antagonists. A preferred embodiment of the invention pertains to inhibitors/antagonists as modulators of the expression, function, stability of Clq or a variant thereof. Preferred modulators are, in context of the invention, inhibitors or antagonists that inhibit (e.g., impair or interfere with) Clq or its variant’s expression, function and/or stability, in particularly specifically and/or selectively, in neuronal cells and tissues showing a pathological aberration, senescence, impaired growth or survival.

In one embodiment, the modulator for use according to the present invention is an antigen binding construct, such as, for example, an antibody, antibody-like molecule or other antigen binding derivative, or an antigen binding fragment thereof), that binds and preferably specifically Clq or a variant thereof, and thereby mimics the preferably specific interaction of Clq with ApoE.

In one embodiment, the modulator for use according to the present invention is a nucleic acid, such as, for example an anti-sense nucleotide molecule such as a siRNA or shRNA molecule that binds to a nucleic acid that encodes or regulates the expression of: (i) Clq or a variant thereof, or (ii) a gene that controls the expression, function and/or stability of Clq or a variant thereof.

Furthermore, particularly preferred modulators (in particular inhibitors/antagonists) of expression, function and/or stability of Clq or a variant thereof, and in particular that mimic and/or stabilizes the preferably specific interaction of Clq with ApoE are in certain embodiments the following specific molecules and/or molecular classes. A compound that is a polypeptide, peptide, glycoprotein, a peptidomimetic, an antigen binding construct (for example, an antibody, antibody-like molecule or other antigen binding derivative, or an or antigen binding fragment thereof), a nucleic acid such as a DNA or RNA, for example an antisense or inhibitory DNA or RNA, a ribozyme, an RNA or DNA aptamer, RNAi, siRNA, shRNA and the like, including variants or derivatives thereof such as a peptide nucleic acid (PNA), a genetic construct for targeted gene editing, such as a CRISPR/Cas9 construct and/or a guide nucleic acid (gRNA or gDNA) and/or tracrRNA. The basic rules for the design of CRISPR/Cas9 mediated gene editing approaches are known to the skilled artisan and for example reviewed in Wiles MV et al.:“CRISPR-Cas9-mediated genome editing and guide RNA design.”, (Mamm Genome. 2015 0ct;26(9-l0):50l-l0) or in Savic N and Schwank G: “Advances in therapeutic CRISPR/Cas9 genome editing.” (Transl Res. 2016 Feb; 168:15-21).

Preferably, said compound is selected from the group consisting of a peptide library, a combinatory library, a cell extract, in particular a plant cell extract, a "small molecular drug", an antisense oligonucleotide, an siRNA, an mRNA, and an antibody or fragment thereof specifically mimicking and/or stabilizing the binding of ApoE to Clq.

An anti-sense nucleotide molecule can, by virtue of it comprising an anti-sense nucleotide sequence, bind to a target nucleic acid molecule (e.g. based on sequence complementarity) within a cell and modulate the level of expression (transcription and/or translation) of Clq (or of a variant of Clq), or it may modulate expression of another gene (e.g. ApoE) that controls the expression, function and/or stability of Clq (or the variant thereof).

As mentioned above, particularly preferred is an isolated antigen binding construct according to the present invention that is capable of specifically binding to Clq or a variant thereof, wherein said antigen binding construct mimics, preferably specifically mimics, and/or stabilizes the interaction of ApoE or variants with Clq or variants thereof.

Preferred antigen binding constructs are antibodies and antibody-like constructs. The term“antibody” includes, but is not limited to, genetically engineered or otherwise modified forms of immunoglobulins, such as intrabodies, chimeric antibodies, fully human antibodies, humanized antibodies, antibody fragments, and heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies, tetrabodies, etc.). The term “antibody” includes cys-diabodies and minibodies. Thus, each and every embodiment provided herein in regard to “antibodies”, or “antibody like constructs” is also envisioned as, bi-specific antibodies, diabodies, scFv fragments, chimeric antibody receptor (CAR) constructs, diabody and/or minibody embodiments, unless explicitly denoted otherwise. A preferred embodiment of the invention pertains to a monoclonal antibody as an (isolated) antigen binding construct. An antibody of the invention may be an IgG type antibody, for example having any of the IgG isotypes.

In particular embodiments of the invention, the antigen binding construct, such as an antibody, is non-natural and/or is not a product of nature. In one of such embodiments, the antigen binding construct may be a non-natural antigen binding construct, such as a synthetic, modified or recombinant antigen binding construct. In another of such embodiments, the antigen binding construct may be first generated following non natural immunization of a (species of) mammal; such as by immunization with an antigen to which such (species of) mammal is not exposed in nature, and hence will not have naturally raised antibodies against.

The term“isolated” as used herein in the context of a polypeptide, such as an antigen binding construct (an example of which could be an antibody), refers to a polypeptide that is purified from proteins or polypeptides or other contaminants that would interfere with its therapeutic, diagnostic, prophylactic, research or other use. An antigen binding construct according to the invention may be a recombinant, synthetic or modified (non natural) antigen binding construct. The term“isolated” as used herein in the context of a nucleic acid or cells refers to a nucleic acid or cells that is/are purified from DNA, R A, proteins or polypeptides or other contaminants (such as other cells) that would interfere with its therapeutic, diagnostic, prophylactic, research or other use, or it refers to a recombinant, synthetic or modified (non-natural) nucleic acid. In this context, a “recombinant” protein/polypeptide or nucleic acid is one made using recombinant techniques. Methods and techniques for the production of recombinant nucleic acids and proteins are well known in the art.

A“functional variant” of ApoE or Clq (such as a functional fragment of an ApoE or Clq protein) is a variant of, such as a fragment of, the protein of ApoE or Clq that provides, possesses and/or maintains one or more of the herein described functions/activities of the non- variant protein of ApoE or Clq, in particular mimics and/or stabilizes the specific binding of the two proteins to each other.

Another aspect of the invention relates to a monoclonal antibody, or a binding fragment thereof, binding to and preferably mimicking and/or stabilizing, ApoE binding to Clq, or vice versa, or the variants of these proteins. Therefore, the present invention relates to the use of Clq as a novel target for the generation of modulating, such as stabilizing, antibodies directed against the respective protein that mimic the binding of ApoE, and thereby modulate the function of these binding partners, specifically Clq. The generation of such antibodies is as such a standard procedure for the skilled artisan. An antibody of the present invention may be a mouse, rat, rabbit, horse, goat, antibody, or a humanized or chimeric antibody.

In another aspect of the invention, a method is provided for identifying a compound suitable for the prevention or treatment of a disease characterized by an undesired function of Clq or a variant thereof, the method comprising the steps of a) contacting at least one of Clq or a variant thereof, a Clq binding fragment of ApoE or a variant thereof, and an ApoE binding fragment of Clq or a variant thereof and/or a cell expressing Clq or a variant thereof with at least one candidate compound that potentially modulates and preferably mimics and/or stabilizes the interaction of ApoE with Clq in a cell, and b) identifying a binding of said candidate compound to Clq.

The terms“ApoE-protein” or“protein of ApoE” as used in context of the herein disclosed invention shall pertain to a protein (such as a full-length protein, fusion protein or partial protein) comprising the sequence of the apo lipoprotein E protein (UniProtKB - E7ERP7 for human, SEQ ID NO. 7, UniProtKB - Q6GTX3-1 for mouse, SEQ ID NO. 8) including the variant amino acids that make up the isoforms, in particular isoform 3 (ApoE3), e.g. amino acid changes at positions 112 and/or 158, respectively (cysl l2, argl58) in the human protein, There are no variants in mouse ApoE.

The terms shall also refer to a protein comprising the amino acid sequence according to SEQ ID NO: 7 or 8 with any protein modifications. Such protein modifications preferably do not alter the amino acid sequence of the polypeptide chain, but constitute a functional group, which is conjugated to the basic amino acid polymer chain. Protein modifications in context of the invention may be selected from a conjugation of additional amino acid sequences to the amino acid chain, such as ubiquitination, sumolation, neddylation, or similar small protein conjugates. Other protein modifications include, but are not limited to, glycosylation, methylation, lipid- conjugation, or other natural or artificial post-translational modifications known to the skilled person. The terms“protein of a variant of ApoE” and the like, shall have the corresponding meaning with respect to a variant of ApoE. A preferred variant of ApoE is a Clq binding fragment, in particular a functional fragment, which also constitutes a compound according to the present invention, i.e. mimics the function of the full-length ApoE regarding the ApoE/Clq interaction in vivo.

Preferred is the method according to the present invention, wherein said modulation is selected from a decrease or an increase of the expression, function, stability and activation of Clq, and in particular mimicking ApoE function on Clq.“Mimicking” ApoE function on Clq shall mean that the candidate compound (or compounds, if several are used together) at least shows a binding to Clq that is substantial the same or the same as the one of ApoE. Ideally, the candidate compound(s) also has/have the same biological function(s) as mediated by ApoE through binding to Clq and as described herein. Thus, a preferred method uses an assay comprising ApoE binding to Clq as a control, in order to identify suitable candidate compounds.

Preferred is the method according to the present invention, wherein said assay is a competitive assay, wherein a modulation is selected from a decrease of the (preferably specific) binding of ApoE or a fragment or variant thereof to Clq or a fragment or variant thereof as measured in the presence of said candidate compound when compared to a control. This will also allow to select compounds that specifically mimic the ApoE binding/function on Clq.

Preferred is the method according to the present invention, wherein said assay is a combined assay, wherein a modulation is selected from an increase of the (preferably specific) binding of ApoE or a fragment or variant thereof to Clq or a fragment or variant thereof as measured in the presence of said candidate compound when compared to a control. This will allow to select compounds that specifically stabilize/promote the ApoE binding/function on Clq.

The reduction (or enhancement) of expression, function and/or stability of Clq or the Clq (or of the variant thereof) and/or the binding of ApoE to Clq, is, preferably, identified by reference to a control method, for example one practiced in the absence of any candidate compound, or with compound having a known effect on such expression, function and/or stability (such as a positive or negative control).

According to the invention, said cell can be selected from the group of neuronal or vascular cells, recombinant host cells suitably expressing ApoE and Clq or binding fragments or variants thereof, yeast cells, and recombinant bacterial cells. Further preferred is a method according to the present invention, wherein said ApoE fragment binds to the Clq stalk, and comprises for example the amino acids 139-152 of the human ApoE polypeptide.

The method according to the present invention as described herein is thus suitable for the identification of compounds that can interact with and/or mimic the binding of ApoE to Clq, and/or activities of Clq, and thus to identify, for example, inhibitors, competitors or modulators of the Clq function and downstream complement amplification loop, in particular, inhibitors, competitors or modulators or mimics and/or stabilizes the binding of ApoE. Preferred are compounds that mimic and/or stabilize the binding of ApoE to Clq. Another aspect is directed at compounds that modulate the expression of Clq in a cell/in cells.

Another aspect is directed at a method according to the present invention, further comprising testing said compound(s) as identified for its activity to specifically bind to the Clq stalk, for example the amino acids 139-152 of the human ApoE polypeptide. Respective assays are known to the person of skill, and can be taken from the respective literature.

Another aspect is directed at a method according to the present invention, further comprising testing said compound(s) as identified for its activity to revert neuronal diseases, inflammation, AD, kidney diseases, atherosclerosis or the effects of ageing. Respective assays are known to the person of skill, and can be taken from the respective literature.

The term "contacting" in the present invention means any interaction between the potentially binding substance(s) (candidate compound) with Clq, whereby any of the two components can be independently of each other in a liquid phase, for example in solution, or in suspension or can be bound to a solid phase, for example, in the form of an essentially planar surface or in the form of particles, pearls or the like. In a preferred embodiment a multitude of different potentially binding substances are immobilized on a solid surface like, for example, on a compound library chip and Clq (or a functional part thereof) is subsequently contacted with such a chip.

Measuring of binding of the compound to Clq (or a functional part thereof) can be carried out either by measuring a marker that can be attached either to the protein or to the potentially interacting compound. Suitable markers are known to someone of skill in the art and comprise, for example, fluorescence markers.

As already described above, the candidate compound is selected from a polypeptide, peptide, glycoprotein, a peptidomimetic, an antigen binding construct (for example, an antibody, antibody-like molecule or other antigen binding derivative, or an or antigen binding fragment thereof), a nucleic acid such as a DNA or RNA, for example an antisense or inhibitory DNA or RNA, a ribozyme, an RNA or DNA aptamer, RNAi, siRNA, shRNA and the like, including variants or derivatives thereof such as a peptide nucleic acid (PNA), a genetic construct for targeted gene editing, such as a CRISPR/Cas9 construct and/or a guide nucleic acid (gRNA or gDNA) and/or tracrRNA.

The thus selected binding compound is then in a preferred embodiment modified in a further step. Modification can be effected by a variety of methods known in the art, which include without limitation the introduction of novel side chains or the exchange of functional groups like, for example, introduction of halogens, in particular F, Cl or Br, the introduction of lower alkyl groups, preferably having one to five carbon atoms like, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n- pentyl or isopentyl groups, lower alkenyl groups, preferably having two to five carbon atoms, lower alkynyl groups, preferably having two to five carbon atoms or through the introduction of, for example, a group selected from the group consisting of NH₂, N0₂, OH, SH, NH, CN, aryl, heteroaryl, COH or COOH group. The thus modified binding substances are then individually tested with a method of the present invention, i.e. they are contacted with Clq (or a functional part thereof) and subsequently binding of the modified compounds to the Clq (or a functional part thereof) polypeptide is measured. In this step, both the binding per se can be measured and/or the effect of the function of the Clq (or a functional part thereof) like, e.g. the binding to ApoE and/or the enzymatic activity of the polypeptide can be measured. If needed the steps of selecting the binding compound, modifying the binding compound, contacting the binding compound with Clq (or a functional part thereof) polypeptide and measuring the binding of the modified compounds to the protein can be repeated a third or any given number of times as required. The above described method is also termed "directed evolution" since it involves a multitude of steps including modification and selection, whereby binding compounds are selected in an "evolutionary" process optimizing its capabilities with respect to a particular property, e.g. its binding activity, its ability to activate or modulate the activity of the Clq polypeptide, and/or its function as an ApoE mimicking and/or stabilizing compound.

Another aspect of the present invention relates to a method for manufacturing a pharmaceutical composition for treating or preventing neuronal diseases, cardiovascular diseases and ageing, comprising the steps of: performing a screening method according to the present invention, and formulating said compound as screened and identified into a pharmaceutical composition.

In a further embodiment of the method of the present invention, the interacting compound identified as outlined above, which may or may not have gone through additional rounds of modification and selection, is admixed with suitable auxiliary substances and/or additives. Such substances comprise pharmacological acceptable substances, which increase the stability, solubility, biocompatibility, or biological half- life of the interacting compound or comprise substances or materials, which have to be included for certain routs of application like, for example, intravenous solution, sprays, band-aids or pills.

Carriers, excipients and strategies to formulate a pharmaceutical composition, for example to be administered systemically or topically, by any conventional route, in particular enterally, e.g. orally, e.g. in the form of tablets or capsules, parenterally, e.g. in the form of injectable solutions or suspensions, topically, e.g. in the form of lotions, gels, ointments or creams, or in nasal or a suppository form are well known to the person of skill and described in the respective literature.

Another aspect of the present invention relates to a pharmaceutical composition comprising the compound for use according to the present invention and a pharmaceutically acceptable carrier, stabilizer and/or excipient.

Another aspect of the present invention pertains to a pharmaceutical composition for use in the prevention or treatment of neuronal diseases, cardiovascular diseases and ageing, in particular AD or atherosclerosis.

The pharmaceutical composition of the invention comprises a compound of the invention (i.e., a modulator of ApoE binding to Clq, or of a variant thereof as described herein), and a pharmaceutical acceptable carrier and/or excipient. As used herein the language“pharmaceutically acceptable” carrier, stabilizer or excipient is intended to include any and all solvents, solubilizers, fillers, stabilizers, binders, absorbents, bases, buffering agents, lubricants, controlled release vehicles, diluents, emulsifying agents, humectants, dispersion media, coatings, antibacterial or antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well-known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary agents can also be incorporated into the compositions.

The pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intrathecal, intra-arterial, intravenous, intradermal, subcutaneous, oral, transdermal (topical) and transmucosal administration.

Solutions or suspensions used for parenteral, intradermal, or subcutaneous application, as well as comprising a compound of the invention as herein can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin; propylene glycol or other synthetic solvents; anti bacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

In certain embodiments, the pharmaceutical composition is formulated for sustained or controlled release of a compound of the invention (e.g., a compound mimicking and/or stabilizing the interaction of ApoE with Clq or a variant thereof). Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polygly colic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art.

It is especially advantageous to formulate oral, rectal or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein includes physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The dosage may vary depending upon the dosage form employed and the route of ad- ministration utilized. For any compound used in the therapeutic approaches of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

An "effective amount" is an amount of the compound(s) or the pharmaceutical composition as described herein that reduces on the expression and/or abundance of Clq, or mimics and/or stabilizes the binding of ApoE to Clq, such as specifically mimics and/or stabilizes the binding of ApoE to Clq.

According to another aspect thereof, the object of the present invention is solved by providing a screening tool for an agent for treating or preventing neuronal diseases, cardiovascular diseases and ageing, in particular a screening tool for screening for a compound that modulates the expression, the biological activity and/or the interaction of Clq, and preferably mimics and/or stabilizes the interaction of ApoE with Clq, comprising an isolated cell expressing Clq and/or expressing an ApoE binding fragment thereof, wherein said cell optionally expresses ApoE and/or an Clq binding fragment thereof. The cell can be a prokaryotic or eukaryotic cell, and the expression construct can be present extrachromosomally or integrated into the chromosome. The polypeptides can be expressed in the form of a fusion protein, for example together with an enzymatically active moiety as reporter-construct, in order to be able to detect the expression product. Preferred host cells are derived from cells selected from the skeletal muscle, liver, adipose tissue, heart, pancreas, kidney, breast tissue, ovarian tissue, and/or hypothalamus. Thus, preferred is a screening tool according to the present invention, wherein said cell is selected from the group of neuronal cells, recombinant host cells expressing Clq and/or an ApoE binding fragment thereof, yeast cells, and recombinant bacterial cells, wherein said recombinant cell optionally expresses ApoE or the Clq binding fragment thereof.

According to yet another aspect thereof, the object of the present invention is solved by providing a screening tool for an agent for treating or preventing neuronal diseases, cardiovascular diseases and ageing, in particular a screening tool for screening for a compound that modulates the expression, the biological activity and/or the interaction of Clq in a cell, and preferably mimics and/or stabilizes the interaction of ApoE with Clq in a cell, wherein said cell as above is part of a non- human transgenic mammal, which preferably overexpresses ApoE and/or Clq optionally as a genetic reporter- construct.. Preferred is a transgenic mouse, rat, pig, goat or sheep, wherein the reporter- construct is preferably expressed in cells selected from the nerves, vasculature, skeletal muscle, liver, adipose tissue, heart, pancreas, kidney, and/or hypothalamus of said animal. Methods to produce these non-human transgenic mammal overexpressing ApoE and/or Clq and/or carrying a ApoE and/or Clq genetic reporter-construct are well known to the person of skill in the art. Preferred is a tool, wherein said Clq binding fragment comprises the amino acids 139-152 of ApoE to bind to the Clq stalk.

Similar to the strategies for identifying compounds that mimic the binding of ApoE to Clq, and/or modulate the biological activity of Clq, compounds can be identified that modulate the expression of Clq in a cell. In preferred strategies, the expression of Clq can be monitored using a genetic reporter-construct for Clq (order to analyze the translation efficiency and/or stability of the Clq polypeptide), for an example a fusion protein comprising a detectable fusion member (such as an enzymatic or fluorophoric group, or GFP as described herein), or the amount of mRNA as present in a cell can be measured, for example, by Northern blot. The expression can also be analyzed and monitored by using chip-analysis or rtPCR. Preferred compounds that modulate the expression of Clq in a cell are selected from specific antisense oligonucleotides, siRNAs, mR As or other preferably mutated nucleic acids encoding Clq. These genetic elements can be used in order to provide/maintain the loss -of- function (e.g. by the truncations as identified) of Clq, or the ApoE binding thereof, in said cell. Another preferred embodiment is the transfer of said genetic elements using gene therapy. Furthermore, encompassed are viral constructs for the introduction of said genetic elements into said cells. Alternatively, also the "naked" nucleic acid can be introduced into the cell(s), e.g. by using particle-mediated technologies. Respective methods are well described in the literature and known to the person of skill.

Further preferred is the screening tool according to the present invention as described herein, wherein said ApoE and/or Clq and/or the variants or fragments thereof are labeled. Fabels and methods for labeling are known to the person of skill, and can be enzymatic labels, dyes, fluorophores, and/or radioactive labels.

According to yet another aspect thereof, the present invention relates to the use of the tools according to the present invention as described herein for screening for a compound that modulates the expression, the biological activity and/or mimics and/or stabilizes the interaction of ApoE with Clq in a cell.

The inventors' data indicate that the interaction of ApoE with Clq can be used as a functional target for a drug (modulator) in order to prevent, treat and/or slow down the course of neuronal diseases, cardiovascular diseases, kidney disease, and ageing, in particular AD in mammals/humans. A construct that mimics and/or stabilizes ApoE binding to Clq can be used as a drug as well.

Thus, another aspect of the present invention then relates to a method for treating or preventing neuronal diseases, cardiovascular diseases and ageing in a patient, comprising administering to said patient an effective amount of a pharmaceutical composition according to the invention as described herein. In general, the attending physician will base a treatment on the compound as identified, and optionally also on other individual patient data (clinical data, family history, DNA, etc.), and a treatment can also be performed based on the combination of these factors. This method of the present invention for example involves integrating individual diagnostic data with patient clinical information and general healthcare statistics to enable, for example, the application of personalized medicine to the patient. Significant information about drug effectiveness, drug interactions, and other patient status conditions can be used, too.

Preferred is a therapeutic method according to the present invention, wherein said mammal to be treated is a mouse, rat or human.

More preferably, the neuronal disease or cardiovascular disease to be treated is AD or atherosclerosis. Preferably, an active agent (modulator) is administered in form of a pharmaceutical composition, such as an antibody, nucleotide or a compound mimicking the ApoE inhibition of Clq.

Treating is meant to include, e.g., preventing, treating, reducing the symptoms of, or curing the disease or condition, i.e. AD, inflammation, atherosclerosis, or ageing.

The terms“prevention” and“preventing” as used herein are used according to their ordinary and plain meaning to mean“acting before”. In the context of a particular disease or health-related condition, those terms refer to administration or application of an agent, drug, or remedy to a subject or performance of a procedure or modality on a subject for the purpose of blocking the onset of a disease or health-related condition. For example, a subject that does not yet meet the clinical criteria or exhibit the symptoms of glaucoma, but does exhibit increases in intraocular pressure may be administered a composition of the present invention to prevent or delay the onset of glaucoma or perhaps reduce the severity of the condition.

The present invention demonstrates in mammals (mice) that ApoE attenuates ChP inflammation and atherosclerosis by inhibiting activation of the CCC through binding to Clq. The inventors present a model in which ApoE binds with high-affinity to Clq which results in a decrease in local accumulation of proinflammatory anaphylatoxin C5a and alleviation from C5b-triggered injury. The data also indicate that Clq- ApoE complexes may act as control nodes in human AD and atherosclerosis (see, e.g., Fig. 3). Each of the currently known vital functions of the ChP requires rigid regulation to avoid brain injury and dysfunction (13-17). Two major interacting molecules of the OL-Clq- ApoE axis identified here, i.e. Clq and ApoE, form a protein-protein complex that acts as a dominant anti-inflammatory module in response to OL-triggered signals. This is in agreement with system analyses which identified prominent complement (including Clqa, Clqb, and Clqc) and innate and adaptive immunity susceptibility genes in LOAD brains (27) and atherosclerosis.

The data are also consistent with the hypothesis that the diseased ChP is a gateway to allow circulating leukocytes to enter the brain parenchyma, and that blood-bom innate immune cells may promote AD plaque formation in the neocortex or atherosclerosis in peripheral arteries.

Thus, therapeutic strategies to target the axis at ApoE including upstream regulators of ApoE (33), the Cl complex, will lead to treatment strategies of diseases that have hitherto termed unresolvable and incurable.

The data indicate that the Clq- ApoE module impacts inflammation in prototypic inflammatory diseases: Clq- ApoE module formation required local activation of na^'ive Clq by any number of inflammatory stimuli including MDA-LDL, oxLDL, Igs, and Ab fibrils; the frequency of the Clq- ApoE module was markedly reduced; the binding affinity of Clq to ApoE was lO-lOOOfold higher than those of other long-established binding partners of Clq or of ApoE; and the three human iso forms of ApoE and its murine counterpart inhibited the CCC activated by MDA-LDL, oxLDL, and Ab fibrils but not by native LDL or soluble Ab. The inventors conclude that the Clq- ApoE module also fine-tunes the activity of the CCC in common inflammatory diseases (Fig. 3).

Inflammatory mediators such as neuritic Ab plaques and hyperlipidemia incite unrelenting activation of the innate immune system of which both Clq and ApoE are ancient and central components. The chronicity of unresolvable inflammation implies ongoing activation of the CCC for extended periods of time and the involvement of adaptive immunity, consistent with a significant lymphocyte component in ChP infiltrates and atherosclerotic plaques in ApoE-/- mice.

This view is consistent with the development of systemic lupus erythematosus in patients afflicted with genetic absence or loss of function mutations in Clq and the presence of autoantibodies against Clq, and of mutations in genes encoding for C2, C4, and other components of the CCC; while previous genetic system analyses identified prominent complement- and immune response-regulating susceptibility genes or gene clusters thereof including Clq and ApoE in LOAD brains (27) and atherosclerosis (28), respectively. The data suggest that the roles of Clq as the initiator of the CCC and the pathobiology of ApoE may be more closely connected than previously anticipated.

The inventors data also extend beyond the identification and role of the Clq- ApoE module in prototypic human inflammatory diseases. All chronic inflammatory diseases are associated with activation of one or more complement pathways and that both Clq and ApoE are induced in response to multiple types of acute and chronic stress-inducing types of tissue injury (1, 3-6, 20, 30, 31, 33). The inventors also consider the body of data in line with recent research in which complement constituents have been demonstrated to function as major regulators of basic cellular processes including inflammasome activation and skewing of T cell immunity (32).

The BBB is a well-recognized critical gateway to allow circulating leukocytes to enter the brain to promote neuritic AD plaque formation; and endothelial cell injury of large arteries may play similar roles to promote atherosclerotic lesion buildup (3-6, 8-11, 13- 17, 29-33).

While the body of the data shows that the Clq- ApoE module plays a role in ChP inflammation, AD, and atherosclerosis, it is prudent to consider the impact of the module in a more comprehensive context: First, each of the two building blocks of the module, Clq and ApoE, may be subject to stress-dependent regulations which may not necessarily occur in parallel but may be dictated by independent events. Although both proteins are ancient molecules having evolved from unicellular organisms they acquired functional domains that may have emerged from independent evolutionary pressures. The ultimate function of the module in AD and atherosclerosis appears to regulate inflammation putting the immune system in a driver seat position. However, the immune system itself is subject to systemic and local controls independent of the module itself including immune senescence, nature of injury, and response of the inflicted tissue parenchyma, and many more. Third, some genetic and environmental risk factors for diseases as varied as AD and atherosclerosis - while overlapping - are also different. For all these reasons, the inventors are led to conclude that the Clq-ApoE module may have two faces in disease acquiring a dichotomic nature. From this reasoning it follows that the mere manifestation of the module in tissues cannot - with certainty - predict the precise role of the module in disease progression: While the present evidence indicates that for ChP inflammation and atherosclerosis, Clq-ApoE module formation seems to be beneficial, the single proteins may be detrimental in AD and other degenerative brain diseases: A large body of data indicates that any ApoE iso form is detrimental as indicated by mouse models in which the absence of ApoE strongly attenuates AD pathology. Clearly, unraveling the precise functioning of the module in each disease requires a better understanding of the tissue environment in which the module acts.

The data as produced for the present invention further indicate that the activity of the classical complement cascade (CCC) initiating molecule, i.e. Clq, exists in three states in vitro and in vivo:

1. In normal human blood Clq is inactive in most instances with no CCC activity.

2. In homeostasis Clq is activated on specific surfaces like modified self surfaces (apoptotic cells) for opsonization of dead cells and enhancing phagocytosis without inducing inflammation. In this case Clq is regulated and the Clq-ApoE complex forms.

3. In the absence of regulator ApoE Clq becomes over-activated- leading to inflammation and infiltration of immune cells.

The Clq molecule as found in human blood or as produced by cells can potentially always activate the classical complement pathway either in response to infections or on specific surfaces of self-cells. Infectious microbes are clear activator surfaces, and no regulation is wanted until the microbe is removed. This is different in homeostasis. On self-surfaces opsonisation (complement activation on level of C3 for opsonization) is desired, but no activation on the C5 level (inflammation). This is the reason why Cl requires regulation. In cases of no clearance of apoptotic cells or accumulation of lipids or plaques, the regulation is overwhelmed, and C5 convertases form, leading to inflammation. The inventors performed co-immunoprecipitation experiments (see below) and the in vitro and data strengthens the previous conclusions regarding the role of ApoE in directly regulating the classical complement pathway by binding to activated Clq.

The exact molecular mechanisms of extracellular lipid accumulation in atherosclerosis or other diseases associated with oxidized lipids have yet to be uncovered. The data show that lipid accumulation in the choroid plexus requires a dual mechanism: Hyperlipidemia and the ApoE4-isoform (or lack of ApoE), whereby the degree of lipid deposition in the choroid plexus correlates with the degree of choroid plexus inflammation and - in human AD patients - with cognitive decline. In the disease situation, much more lipids or aggregates accumulate, and serve as complement activator surfaces. This is the reason why complement becomes over-activated and leads to permanent inflammation, even in the presence of ApoE4. All ApoEs are regulators, but the initial difference is the high presence of oxidized lipids. In consequence, to reduce the plaques and to support ApoE regulatory functions would be a leading strategy for therapy.

The present invention will now be further described in the following examples with reference to the accompanying figures and sequences, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties.

Figure 1: ApoE inhibits CCC initiation by high-affinity binding to Clq.

(a) ApoE inhibits CCC activation but not the alternative pathway. ApoE isoforms ApoE2, ApoE3, or ApoE4 were incubated in normal human serum (NHS), which was activated either via CCC buffer (left) (1% in GVB++) or alternative pathway buffer (right) (20% in MgEGTA); and lysis of sheep or rabbit erythrocytes by TCC was followed by measuring released hemoglobin at 415 nm. (b) ApoE was incubated with NHS in GVB++ buffer or Mg-EGTA buffer or with Clq-deficient serum in GVB++ to activate the alternative and lectin pathways. Survival of E. coli was analyzed counting colony forming units. Survival of E. coli in normal serum was set as 10%. (c) ApoE isoforms inhibit the CCC at the level of TCC and C4b. ApoE isoforms in NHS were added to IgM-coated microtiter plates and TCC or C4b deposition was determined by specific antibodies, respectively (d) Binding of Cl, Clq, Cls, and Clr to ApoE iso forms was determined by bio layer interferometry (e) The binding affinities of ApoE iso forms and Cls to Clq were determined by biolayer interferometry. ApoE proteins and Cls were biotinylated, immobilized on streptavidin-coated sensors, and Clq binding was determined by measuring changes of optical thickness on the sensor (f) The ApoE-Clq interaction is dependent on Ca2+. Data represent means ± SEM of three independent experiments. Two-tailed Student's t-test *P<0.05, ** <0.0l, ***E<0.001. BSA, bovine serum albumin; TCC, terminal complement complex; EfB, microbial inhibitor of the alternative pathway. Vnt: vitronectin. GVB: gelatin veronal buffer.

Figure 2: ApoE139-152 binds to the Clq stalk

(a) Four peptides are depicted in a 3D model of human ApoE3 (PDB ID code: 2L7B) and their corresponding amino acid sequences (b) ApoE4 inhibition was blocked by ApoE peptide P139 - 152 but not by ApoE peptides P30-40, P74-85, P210-232. (c) Binding of ApoE iso forms and corresponding ApoE peptides to Clq were determined by ELISA (d) Binding affinity of P 139- 152 to Clq was determined by MicroScale initial fluorescence analysis (e) Binding of ApoE3 to Clq and LDLR in the presence of SDS or NaCl was determined by ELISA (f) ApoE binds to the stalk of Clq. Clq alone or incubated with biotinylated plasma-purified ApoE3 or biotinylated ApoE peptide P 139- 152 and streptavidin-coated gold particles were visualized by electron microscopy. Bar 20 nm. Data represent means ± SEM of three independent experiments. *P<0.05, ** <0.0l, ***P< 0.001. Two-tailed Student's t-test.

Figure 3: OL-ApoE-Clq complexes are involved in atherosclerosis.

Schematic representation of the Clq- ApoE module. Locally produced and/or serum- recruited Clq is activated in situ by a variety of molecules including oxidized lipid, oxidized LDL, amyloid fibrils, and immunoglobulins. Following activation, Clq acquires the ability to bind ApoE at high affinity and forms the Clq- ApoE module (upper part of the panel) which allows initiation of the classical complement cascade (CCC) with resultant C3 generation and subsequent C5 cleavage to generate C5a and C5b. In the presence of ApoE, however, CCC activity is moderated by the Clq-ApoE module. By contrast, inflammation is amplified in the absence of ApoE (lower panel).

Figure 4: Example of pharmaceutical construct (“Affl”) according to the invention. AFF1 inhibits complement activation

(A) Schematic composition of AFF1. The ApoE binding region that binds to Clq is fused to the regulatory region of factor H (SCR1 to SCR4), the dimerization region of FHR1 (SCR1 to SCR2) and a Histidine tag for purification. (B) Recombinant expression of AFF1 in Pichia pastoris and purification by nickel chelate chromatography revealed a protein with a mobility of 40 kDa (lanes 2,3 and 4 silver staining). Detection of the FHR1 part by monoclonal antibody JHD confirms presence of FHR1 (SCR1 and SCR2)(lanes 5-7, Western blot). (C) Binding of AFF1 to Clq. AFF1 was coated to a microtiter surface and binding of increasing amounts of Clq (10 to 200 nM) were followed by ELISA. A representative assay with three replicates is shown. (D) AFF1 inhibits C3b deposition via the alternative pathway in normal human serum (NHS). AFF1 (100 or 200 nM was added to 20 %NHS EGTA buffer, which inhibits the classical pathway but not eh alternative one. The serum with AFF1 was added to an IgM coated microtiter plate and subsequent deposition of C3b was determined by ELISA. Factor H (FH) shows also inhibition of the alternative pathway. Heat inactivation of serum (iNHS) inhibits complement activation. A representative assay with triplicates is shown.

Figure 5: Co-immunoprecipitation of Clq-ApoE complexes, (a) Anti Clq antisera precipitate Clq-ApoE complexes composed of purified proteins with activated Clq but not with inactivated Clq from normal human serum (NHS) (left and middle panels) (b) Anti- ApoE antisera precipitate Clq-ApoE complexes but not complexes from NHS in which Clq is present in its inactive form. Anti Clq monoclonal antisera (Invitrogen, clone 9A7; in left and middle panels) or anti ApoE monoclonal antisera (Invitrogen, clone 1H4) were immobilized on protein G-coupled Dynabeads (Complement Technology). Clq-deficient serum (dNHS, Complement Technology, 2%), NHS (2%), NHS with added with activated purified Clq (Complement Technology) (NHS+Clq), or NHS with purified ApoE (NHS+ApoE), and activated purified Clq with purified ApoE (Merck) (Clq + ApoE) (lanes 2-5 in a and b) were incubated with antisera-coated Dynabeads, then eluting fractions were examined for Clq and ApoE precipitate; purified Clq was added directly to the first lane of each gel as size marker for WB (Clq in lane 1). NHS or anti-Clq with NHS were incubated with no pre-coated Dynabeads (NHS in lane 6 in a, or anti Clq plus purified ApoE in b, respectively) were used as controls. Proteins were eluted with glycine buffer (pH 2.7), separated by SDS-PAGE and immunoblotted using goat anti-Clq antiserum (Complement Technology) or goat anti ApoE antiserum (Merck) and rabbit anti goat secondary antibody (Dako).

Figure 6: Clq-ApoE complexes are formed on apoptotic cells in vitro. Apoptotic THP-l cells (ATCC® TIB-202™, UV light for 2h) were seeded onto Poly-L-lysine-coated diagnostic slides. Cells were incubated in NHS or Clq-depeleted serum (dNHS) (each 1%) (Complement Technology, Texas, USA) for 30 minutes. Cells were blocked and treated with rabbit anti ApoE antiserum (25 pg/ml; Acris Antibodies, Herford, Germany) and mouse anti-Clq antiserum (25 pg/ml; Thermo Fisher Scientific, Massachusetts, USA). The PLA assay was performed according to the manufacturer’s protocol (Duolink In Situ Red Starter Kit Mouse/Rabbit; Sigma- Aldrich). Images were captured using a Zeiss LSM 710 microscope equipped with ZEN 2011 software. Clq- ApoE complexes (red); nuclei were stained with DAPI (blue). Data represent means ± SEM, Two-tailed Student t-test, *** P <0.001, n= 16 cells for each group. Anti ApoE antisera only was used as negative control. Bar represents 10 pm.

Figure 7: Ca²⁺-dependent binding of ApoE to Clq. (a) Plasma-purified Clq was coated on a sensor chip (CM5) and plasma-derived ApoE (62-1000 nM) was injected into the fluid phase (75 mM NaCl, 5 mM HEPES, 1 mM CaCl₂). Real-time binding of ApoE to Clq was followed using biosensor analyses (b) Binding of ApoE to Clq is reduced in a dose-dependent manner with increasing amounts of EGTA (0.1-3 mM).

Figure 8: Interaction of ApoE with Clq stalks as assayed by EM. ApoE molecules were labeled with 6 nm-sized nanogold particles and incubated with commercial purified activated Clq. Three examples of Clq-ApoE complexes are displayed. Typical Clq molecules with its stalk and 6 globular heads and gold- ApoE binding to the stalk of Clq are shown in enlarged insets. Complexes (white arrows) are schematically encircled in the boxes below to outline the globular heads of Clq surrounding gold-labeled ApoE. Bars represent 40 nm.

Figure 9 shows that Clq-ApoE complexes form in the atherosclerotic plaques.

SEQ ID NO. 1 (Clq human (sub A)):

MEGPRGWLVLCVLAISLASMVTEDLCRAPDGKKGEAGRPGRRGRPGLKGEQG EPGAPGIRTGIQGLKGDQGEPGPSGNPGKVGYPGPSGPLGARGIPGIKGTKGSPG NIKDQPRPAFSAIRRNPPMGGNVVIFDTVITNQEEPYQNHSGRFVCTVPGYYYFT F Q VLSQ WEICLSI V S SSRGQ VRRSLGF CDTTNKGLF Q VVSGGMVLQLQQGDQ V WVEKDPKKGHIYQGSEADSVFSGFFIFPSA

SEQ ID NO. 2 (Clq mouse (sub A)):

METSQGWFVACVFTMTFVWTVAEDVCRAPNGKDGAPGNPGRPGRPGFKGER GEPGAAGIRTGIRGFKGDPGESGPPGKPGNVGFPGPSGPFGDSGPQGFKGVKGN PGNIRDQPRPAFSAIRQNPMTFGNVVIFDKVFTNQESPYQNHTGRFICAVPGFYY FNF Q VISKWDECEFIKS S S GGQPRD SF SF SNTNNKGFF Q VF AGGT VFQFRRGDE V WIEKDP AKGRI Y QGTE AD SIF S GFFIFP S A

SEQ ID NO. 3 (Clq human (sub B)):

MMMKIPWGSIPVLMLLLLLGLIDISQAQLSCTGPPAIPGIPGIPGTPGPDGQPGTP GIKGEKGLPGLAGDHGEFGEKGDPGIPGNPGKVGPKGPMGPKGGPGAPGAPGP KGE S GD YKAT QKI AF S ATRTIN VPLRRD QTIRFDH VITNMNNN YEPRS GKFTCK VPGLYYFTYHASSRGNLCVNLMRGRERAQKVVTFCDYAYNTFQVTTGGMVL KLEQGENVFLQ ATDKNSLLGMEGANSIFSGFLLFPDME A

SEQ ID NO. 4 (Clq mouse (sub B)):

MKTQWGEVWTHLLLLLLGFLHVSWAQSSCTGPPGIPGIPGVPGVPGSDGQPGT PGIKGEKGLPGLAGDLGEF GEKGDPGIPGTPGKV GPKGP V GPKGTPGPSGPRGP KGD S GD Y GAT QKV AFS ALRTINSPLRPN Q VIRFEKVITN ANEN YEPRN GKFT CK VPGLYYFTYHASSRGNLCVNLVRGRDRDSMQKVVTFCDYAQNTFQVTTGGVV LKLEQEE VVHLQ ATDKNSLLGIEG AN SIFTGFLLFPDMD A SEQ ID NO. 5 (Clq human (sub c)):

MD V GPS SLPHLGLKLLLLLLLLPLRGQ ANT GC Y GIPGMPGLPGAPGKDG YDGL PGPKGEPGIPAIPGIRGPKGQKGEPGLPGHPGKNGPMGPPGMPGVPGPMGIPGEP GEEGRYKQKFQSVFTVTRQTHQPPAPNSLIRFNAVLTNPQGDYDTSTGKFTCKV PGLYYFVYH ASHT ANLC VLLYRSGVKVVTFCGHTSKTN Q VNSGGVLLRLQ V G EEVWLAVNDYYDMVGIQGSDSVFSGFLLFPD

SEQ ID NO. 6 (Clq mouse (sub C)):

MVVGPSCQPPCGLCLLLLFLLALPLRSQASAGCYGIPGMPGMPGAPGKDGHDG LQGPKGEPGIPAVPGTRGPKGQKGEPGMPGHRGKNGPRGTSGLPGDPGPRGPP GEPGVEGRYKQKHQSVFTVTRQTTQYPEANALVRFNSVVTNPQGHYNPSTGKF TCEVPGLYYFVYYTSHTANLCVHLNLNLARVASFCDHMFNSKQVSSGGVLLRL QRGDE V WL S VND YN GM V GIEGSNS VF SGFLLFPD

SEQ ID NO. 7 (ApoE human):

MKVLWAALLVTFLAGCQAKVEQAVETEPEPELRQQTEWQSGQRWELALGRF WD YLRWV QTL SEQ V QEELL S S Q VT QELRALMDETMKELKAYKSELEEQLTP V AEETRARL SKELQ AAQ ARLG ADMED V C GRL V Q YRGE V Q AMLGQ STEELRVRL ASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRV RAATVGSLAGQ

SEQ ID NO. 8 (ApoE mouse):

MKALWAVLLVTLLTGCLAEGEPEVTDQLEWQSNQPWEQALNRFWDYLRWVQ TLSDQVQEELQSSQVTQELTALMEDTMTEVKAYKKELEEQLGPVAEETRARLG KEVQAAQARLGADMEDLRNRLGQYRNEVHTMLGQSTEEIRARLSTHLRKMRK RLMRDADDLQKRLAVYKAGAREGAERGVSAIRERLGPLVEQGRQRTANLGAG AAQPLRDRAQ AF GDRIRGRLEE V GN Q ARDRLEE VREHMEE VRSKMEEQTQQIR LQAEIFQARLKGWFEPIVEDMHRQWANLMEKIQASVATNPIITPVAQENQ

EXAMPLES The ChP is the major intracranial neuroimmuno logical interface which produces the cerebrospinal fluid (CSF), forms the blood-CSF barrier, exchanges signals between the brain and the circulation, and is the principal gateway for blood-bom leukocytes to infiltrate the central nervous system in inflammatory and degenerative brain diseases (12-17).

In ApoE-/- ChPs, IgG and IgM colocalized with lipid inside the capillary lumen, the stromal space, and the lipid between the epithelial cells but no Ig binding occurred in lipid-free ChPs. Bell et al. had previously reported that ApoE-deficiency and transgenic expression of ApoE4 in ND ApoE4-KI mice were afflicted with BBB breakdown (1).

Methods:

Proteins and antibodies

Complement components C2, C3, C3b, C4, C4b, Clq, Cls, Factor H, Factor I, and C4BP as well as all primary antibodies were purchased from Complement Technology. Recombinant ApoE iso forms from BioCat; plasma-purified ApoE3 from Biopure. ApoE peptides were generated by Peptide 2.0: ApoE 30-40 LGRFWDYLRWV (SEQ ID No. 9); ApoE 75-85 YKSELEEQLTPV (SEQ ID No. 10); ApoE 139-152 SHLRKLRKRLLRDA (SEQ ID No. 11); ApoE 210-232 WGERLRARMEEMGSRTRDRLDEV (SEQ ID No. 12). LDL and malone dialdehyde- modified LDL (MDA-LDL) were from Cell Biolabs, copper oxidized LDL (oxLDL) from Thermo Fisher (L34357), poA from Athens Research & Technology, vitronectin (Vnt) from Coming. Ab and Ab fibrils from GenSript and HRP-coupled polyclonal antibody against goat and rabbit were from DakoCytomatio. Recombinant EfB was expressed as described (Koch, T. K. et al. Staphylococcus aureus proteins Sbi and Efb recruit human plasmin to degrade complement C3 and C3b. PLoS One 7, e47638 (2012).

Hemolytic assay

Hemolytic assays were performed according to standard complement assays using sheep or rabbit erythrocytes that are sensitive to the complement terminal complex generated in human serum. Lysis of the erythrocytes by this thermal complex indicates complement activation and free hemoglobin is measured by absorbance. Alternative pathway hemolysis assays were performed in a total volume of 100 mΐ containing 20% NHS (pooled from healthy human blood donors), increasing amounts of ApoE, BSA or EfB (each 0.25 - 1.5 mM) and 2x107 rabbit erythrocytes (Fiebig-Nahrstofftechnik) in Mg²⁺-EGTA buffer (20 mM HEPES; 144 mM NaCl; 10 mM EGTA; 7mM MgCl₂; pH 7.4). To analyze inhibition of hemolysis via the classical pathway, 1% NHS, increasing concentrations of ApoE (0.01 - 1.5 mM) or 0.44 mM ApoE4 plus 0.33 mM ApoE peptide together with 2xl0⁷ amboceptor- (Siemens) coated sheep erythrocytes (Fiebig- Nahrstofftechnik) were mixed in gelatin veronal buffer (GVB++; Complement Technology). After pre-incubation of the proteins in NHS for 15' at 37° C the NHS- protein-mix was added to the erythrocytes and incubated for additional 30 min at 37° C. Lysis of erythrocytes was determined by measuring the amount of hemoglobin in the supernatants at 414 nm. The results were calculated as hemolysis rate relative to the level of lysis in absence of ApoE, the latter was set as 100%.

E. coli killing assay

Gram negative bacteria are sensitive for the terminal complement complex and become lysed. Similar to erythrocytes the terminal complement complex forms pores into the bacterial cell wall which kills the microbe. Efficiency of complement activation via the three pathways is tested by different buffers and by survival of the microbes upon plating the treated E. coli on agar plates. Different amounts of ApoE (0.1 - 1 mM) together with 0.2% NHS were pre-incubated for 10 mins at 37° C before adding E. coli pET200/D-TOPO (1000 cells per sample). After an incubation of 30 min at 37° C cells were plated to a LB-agar plate and cultivated o.n. at 37° C before counting the colony forming units. To distinguish which complement pathway was effected the experiment was performed incubating ApoE3 with NHS in GVB++ for all three pathways, with NHS in MgEGTA buffer for the alternative, and with Clq-depleted serum in GVB++ for the lectin and the alternative pathway. Bacterial survival w/o adding ApoE was set as 10%.

Complement activation assay

The effect of ApoE on classical complement pathway C4b and C5b-9 deposition was analyzed by standard ELISA. The classical pathway was activated by coating either IgM (2 pg/ml) or MDA-LDL (1 pg/ml) to a microtiter plate. ApoE (0.25 - 2 mM) or Vnt (0.5 mM) were pre-incubated with 1% NHS in GVB++ buffer for 15 mins at 37° C and added to the coated plates for 1 h at 37° C. Complement activation with NHS alone was set as 100%.

Cleavage and cofactor assay

The influence of ApoE on Cls to cleave C2 and C4 was measured in fluid phase. ApoE3 (5 and 50 pg/ml) was pre-incubated with Cls (20 pg/ml) in GVB++ buffer for 15 mins at 37° C followed with C2 (10 pg/ml) or C4 (10 pg/ml) for 30 mins at 37° C. C4b cleavage activity of ApoE was determined by incubating C4b (20 pg/ml) with ApoE3 (5 and 50 pg/ml) or C4BP (20 pg/ml) for 30 mins at 37°C. C4b cleavage was followed by Western Blotting. Cofactor activity of ApoE was measured by incubating C4b (10 pg/ml) with Factor I (FI) (5 pg/ml), C4BP (20 pg/ml), ApoE3 (10 pg/ml) or combinations thereof (FI together with C4BP and FI together with increasing amounts of ApoE3 (0.1 - 100 pg/ml)) for 30 mins at 37° C. C4b cleavage was analyzed by Western Blotting.

ELISA

Recombinant or plasma-purified ApoE (0.1, 0.4 or 0.5 mM), ApoE peptides (0.1 mM), IgM (1 pg/ml), LDL, oxLDL, and MDA-LDL (each 1 mg/ml) or gelatin (10 pg/ml) were immobilized in carbonate-bicarbonate buffer (Sigma) on microtiter plates (F96 Maxisorb, Nunc-Immuno module) overnight at 4° C. After washing the plate three times with washing buffer (PBS) containing 0.1% Tween 20) wells were blocked (PBS, 1% BSA, 5% milk) for at least 1 h at 37° C. Coated proteins were incubated for 1 h at 37° C with C3, C3b, and Clq (0.1 mM) or Clq (0.02- 5.33 nM) or NHS (0.075 - 10%) in GVB++ buffer. Calcium-dependent binding of Clq to ApoE was determined by diluting Clq in GVB++ buffer or in PBS and adding increasing amounts of EGTA (3 - 12 pM) (Sigma) with a fixed concentration of Clq (2 or 5 nM) to immobilized ApoE proteins. Binding force was analyzed incubating Clq or the LDLR (0.1 mM) with or w/o NaCl (0.5 M) or SDS (1%) on immobilized ApoE3. Clq, C3, and C3b binding was analyzed by specific primary antibodies and followed by HRP-conjugated seconday antibodies. The reaction was developed with TMB (Kementec Diagnostics) or 1,2- phenylenediamine dihydrochloride (OPD tablets, Dako) and the absorbance at 450 nm or 492 nm was measured. Competition assay

To verify binding of ApoE peptide 139-152 to Clq, different amounts of ApoE 139-152 and ApoE 30-40 as a control (6,25 - 100 nM) were incubated with a constant concentration of Clq (0.6 nM) on immobilized ApoE3 (0.1 mM). Binding of Clq to ApoE3 was analyzed using specific antibodies. ELISA was performed to investigate whether ApoE competes with the ClsClr tetramer for binding to Clq. Clq (0.6 nM) together with different amounts of the ClsClr tetramer (1.53-100 nM) in PBS++ were incubated with 0.1 mM immobilized ApoE3 for 1 h at 37°C. After a washing step Clq binding to ApoE was detected. 20 nM Clq were incubated with increasing concentrations of LDLR (0 -20 nM) to immobilized ApoE, the binding of ApoE-Clq and ApoE-LDLR was followed by ELISA. Background binding of anti-Clq and anti- LDLR to immobilized ApoE were set as 0%.

Binding assays - BLItz™

Bio layer interferometry (BLItz™, ForteBio) was used to examine binding of recombinant ApoE iso forms to Cl, C2, C4, Cl complex components Clq, Cls, Clr, and to MBL and the binding of Clq to ApoA. Biotinylated proteins (ligands) are coupled to Streptavidin- coated biosensors and different analytes are added. Streptavidin-coated biosensors (VWR) were hydrated for 10 mins in PBS with Calcium and Magnesium (PBS++, Lonza) before loading 20 pg/ml biotinylated ApoE2, 3 and 4 or ApoA for 120 sec. After a 30 sec baseline step 45 nm of complement protein analyte was associated for 240 sec to the sensor and a 240 sec dissociation step followed. For biotinylation ApoE iso forms as well as ApoA were incubated with biotin (Thermo Scientific) for 30 mins at RT. Samples were separated from unbound biotin using centri pure mini spin columns (Biotech).

Affinity measurement of ApoE binding to Clq

Initial fluorescent analysis (MST) was used to determine the interaction of proteins in fluid phase. This technique measures the movement of proteins over time upon heating a spot of the capillaries with the proteins in solution. Formed complexes have a different pattern of movement than single proteins because of their sizes. The different signals are amplified. Like in biointerometry increasing concentrations of one binding partner is used to determine the on- and off-binding of the proteins. Affinity constant (KD), association (kon), and dissociation (koff) constants of ApoE binding to Clq full length proteins and Cls was determined by BLItz™. Streptavidin-coated biosensors were hydrated for 10 mins in PBS++. Recombinant ApoE2, ApoE3 and ApoE4, plasma- purified ApoE3 or Cls (each 20 pg/ml) were loaded for 120 sec via biotin onto the sensor. After 30 sec baseline, Clq (0.047 nM - 45.65 nM) or MBL (45.45 nM) was associated for 240 sec followed by a 240 sec dissociation step. Affinity values were generated by BLItz™ software analysis as an advanced kinetics experiment using MBL as reference value. Initial fluorescent analysis (NanoTemper) was used to determine the KD of the binding between Clq and ApoE 139-152. Alexa 647-labeled ApoE 136-152 (10 nM) or ApoE 30-40 (30 nM) were incubated with different amounts of Clq (0.073 - 1196.8 nM) in PBS++ for 30' in the dark. After 10 mins centrifugation samples were transferred into standard capillaries and initial fluorescence was measured using a Monolith NT.l l5Pico (LED power 60%, MST power 20%). To test whether the observed fluorescence changes are due to a binding event, an SD-test was performed: The three samples with the highest and the lowest Clq concentration were centrifuged for 10' at l5000xg before removing the supernatant and adding SD-mix (4% SDS, 40 mM DTT). After an incubation step of 5 ' at 95°C, samples were transferred to capillaries and initial fluorescence was measured. The initial fluorescent analysis v2.0.2 was used to determine the KD.

TEM for Clq and ApoE interaction

To visualize single Clq protein particles by TEM Clq (5 pg/ml) was diluted in PBS. In order to gold-label ApoE, biotinylated plasma-purified ApoE (20 pg/ml) was incubated with streptavidin-gold complexes (5 nm gold, British BioCell International Ltd.) diluted 1 :25 in PBS for two hours at RT. Clq (10 pg/ml) was added to the ApoE-streptavidin- gold solution (1 :1 mixture) and incubated under gentle shaking for two hours at RT. Carbon-coated grids were hydrophilized by glow discharge at low pressure in air. Aliquots of Clq alone and Clq-ApoE-streptavidin-gold were adsorbed onto hydrophilic, carbon-coated grids for 1 mins, washed twice with distilled water, and stained on a drop of 2% uranyl acetate in distilled water. Specimens were analyzed with a Zeiss EM902A electron microscope (Carl Zeiss) operated at 80 kV accelerating voltage, and images were recorded with a FastScan-CCD-camera 1,024 x 1,024 (TVIPS).

Statistical Analysis

Significant differences between two groups were analyzed by GraphPad Prism 7 using the two-tailed unpaired Student t-test or one-way ANOVA with multiple testing (Tukey). Values of *p<0.05, **p<0.0l, ***p<0.00l were considered statistically significant.

Results:

ApoE inhibits CCC initiation

The salient absence or low expression of key complement components in the HFD ApoE4-KI ChPs (above) led the inventors to examine a role of ApoE in the classical, the alternative, and lectin pathways (20). ApoE was added to normal healthy serum (NHS), incubated with antibody-coated sheep erythrocytes and pathway-specific activation was followed by lysis of erythrocytes. All three variants, i.e. ApoE2, ApoE3, and ApoE4 reduced CCC activation (Fig. 1 a) but not the alternative pathway (Fig. 1 a/right). Furthermore, in a complement-mediated E. coli killing assay, E. coli remained viable upon ApoE-supplemented NHS challenge, but were killed when complement was activated via the lectin- or alternative pathways (Fig. 1 b), indicates that ApoE inhibits CCC activation, but not the alternative or lectin pathways. All three ApoE iso forms inhibited deposition of C4b and the terminal complement complex (TCC) by ~80% (Fig. 1 c) indicating that ApoEs act at a site of CCC initiation. Malondialdehyde- modified low-density- lipoprotein (MDA-LDL) binds to Clq and activates the CCC. Oxidized-LDL (oxLDL) was reported to activate the CCC21; and the inventors found that purified Clq bound MDA-LDL and oxidized-LDL (oxLDL) but not native LDL. Furthermore, ApoE inhibited the CCC and reduced C4b when the CCC was activated by oxLDL. Moreover amyloid fibrils but not soluble amyloid trigger C3b indicating complement activation.

ApoE inhibits the CCC by high-affinity binding to Clq

During CCC initiation, proteases Cls and Clr bind to Clq, form the Cl complex, then cleave C2 and C4 to form the C3 convertase C4b2a (20) of the CCC. The inventors incubated ApoE3 with C2 or C4 in the presence of the protease Cls. However, ApoE3 failed to inhibit C2 or C4 cleavage by Cls. ApoE3 also lacked co-factor activity for factor I-mediated degradation of C4b. Testing ApoE binding to complement proteins revealed strong binding of ApoE to Cl and Clq, but not to Clr or Cls nor to C2, C3, C3b, or C4, (Fig. 1 d). All recombinant ApoE isoforms and serum-derived ApoE3 bound Clq and Cl. To further delineate ApoE-Clq interactions, the inventors determined the strength of the interaction. All ApoE isoforms bound with similar potencies to Clq and binding constants ranged from to -140-580 pM (Fig. 1 e). The interaction with Clq was specific, as ApoE did not bind to mannan-binding lectin (MBL), a protein initiating the lectin pathway and sharing structural and functional features with Clq; likewise apo lipoprotein A did not interact with Clq. In calcium- free 223 buffer ApoE iso forms bound with low intensity to Clq (Fig. 1 f) indicating 224 that ApoE selectively binds to Clq under CCC-active but not CCC-inactive conditions.

To determine the binding site in ApoE to Clq, four ApoE peptides (Fig. 2 a) were generated and examined for their ability to reduce ApoE4-mediated CCC inhibition. ApoE peptide P139-152 but not P30-40, P74-85, or P210-232, abrogated CCC inhibition by ApoE4 (Fig. 2 b) but P 139- 152 alone did not inhibit CCC activation. Analyzing binding of the four ApoE peptides to Clq showed binding of P 139- 152 to Clq, but not of peptides P30-40, P74-85, or P210-232 (Fig. 2 c). Also, P139-152, but not P30-40 competed with ApoE3 binding to Clq. The binding affinity of P 139- 152 to Clq (by MicroScale thermophoresis) showed a KD of -500 pM (Fig. 2 d).

These data suggested that the Clq binding site in ApoE is located between residues 139-152, which harbors the LDL-receptor (LDLR) binding site (136-150) (22). However, different binding forces for the LDLR and Clq in ApoE were indicated by the observation that ApoE-Clq interaction was strongly reduced by SDS, but not by NaCl, while ApoE-LDLR interaction was affected by NaCl, but not by SDS (Fig. 2 e). Thus, ApoE binding to Clq was mediated by hydrophobic forces. Moreover, LDLR does not compete off ApoE-Clq binding indicating LDLR and Clq do not share the same binding site in ApoE. ApoE interaction with Clq when monitored by electron microscopy showed for gold-labeled ApoE-, as well as gold-labeled ApoE 139- 152 peptide binding to the Clq stalk, but not to the globular heads which mediate target binding (Fig. 2 f). ClsClr tetramers also bind to the Clq stalk (23). However, ApoE and the ClsClr tetramers do not share the same binding site in Clq because the ApoE3- Clq interaction was unaffected by the Cls-Clr tetramer in competition assays. Taken together, these data revealed that ApoE acts as a specific CCC inhibitor by high-affinity binding to Clq.

Example for anti-complement pharmaceutical for the treatment of inflammation in atherosclerosis and Alzheimer’s Disease

The complement system is the central part of the human innate immune system and controls infections and many physiological reactions. The cascade system is spontaneously activated in response to microbial surfaces as well as modified self surfaces and the balanced action of activators and inhibitors directs the newly formed effector components to target surfaces. A number of soluble and membrane bound complement regulators are required to protect intact self-cells and tissue from the damaging effects of complement activation (34-36). Dysregulation of these inhibitors as well as over-activation of the complement system because of mutations/deletion in the corresponding genes have been shown to be associated with a number of different diseases such as atypical Hemolytic Uremic Syndrome, C3-Glomerulopathy or Age related Macular Degeneration (AMD) (34-37). Especially those diseases characterized by cellular deposits containing lipid and proteins but also in trauma and ischemia reperfusion injuries provide so called danger associated molecular patterns (DAMPS) that attract complement proteins as well as circulating natural antibodies (IgM), which in turn also activate complement (38, 39). Natural antibodies that detect epitopes like oxidised phospholipids or annexin IV, have important physiological roles, such as protection from pathogens and the removal of cellular and molecular waste (40, 41). However, subsequent complement activation recruits a number of immune cells that can clear but can also harm the neighbouring tissue and induce sterile inflammation.

In vitro assays identified all ApoE variants to bind with high affinity to activated Clq and to inhibit further activation of exclusively the classical pathway (Fig. 1). These findings indicate that complement is persistently activated in atherosclerosis- and Alzheimer plaques leading to inflammation by recruited immune cells which drive secondary injury. As complement terminal pathway inhibition in ApoE knock out mice with C5-siRNA attenuated complement driven inflammation in choroid plexus and arteries, an ApoE-Clq complex specific recombinant inhibitor was created to inhibit complement amplification induced by Clq similar to ApoE (Fig 4).

The proposed inhibitor construct is composed of an ApoE-derived peptide that recruits the inhibitor to the activated Clq, the site of complement activation. Other regulatory functions may be added to such a construct as schematically indicated.

Additional Examples show that in the interaction of ApoE with Clq, Clq must be activated before the two partner proteins Clq and ApoE can bind, in order to form the high affinity Clq- ApoE complex. The Clq- ApoE complex does not form in the circulation in physiological situations.

In the first example, the inventors immobilized anti-Clq antisera on protein G-coated beads and incubated these beads with: 1. Serum from a normal human Clq-sufficient donor (normal human serum; NHS); or 2. Clq-deficient serum (dNHS); or 3. Purified Clq in its activated form with NHS (Clq+NHS); or 4. Purified Clq and purified ApoE (Clq+ApoE). Beads were washed and eluted proteins were separated by electrophoresis and immunoblots for Clq or ApoE proteins were generated. Both Clq and ApoE can be precipitated by anti-Clq antibody, when beads were incubated with purified activated Clq with NHS (Clq+NHS) or purified activated Clq and purified ApoE (Clq+ApoE) indicating the formation of the Clq- ApoE complex under these experimental conditions (Figure 5). When beads were incubated only with NHS, Clq was identified in the elution fraction. However, no ApoE protein or Clq protein could be observed in the eluting fraction when beads were incubated with dNHS (Figure 5). Similar results were obtained when the inventors immobilized anti ApoE antisera on protein G-coated beads and either incubated with purified activated Clq with ApoE proteins or incubated with NHS (Figure 5). These data show that Clq- ApoE complexes are not present in serum because the Clq is present there in an inactivated/silent form preventing the formation of the complex.

In the second example (data shown in Figure 6), the inventors used cultured human apoptotic cells which provide a binding surface for the inactive Clq in NHS to initiate conversion to its activated form by changing its conformation: The activated form of Clq in turn activates complement by enhancing opsonization with C3b/iC3b and its uptake by macrophages via CR3 (see also Gaboriaud C. et al, The crystal structure of the globular head of complement protein Clq provides a basis for its versatile recognition properties. J Biol Chem. 2003. 2l;278(47):46974-82; Major et al, Calcium- dependent conformational flexibility of a CUB domain controls activation of the complement serine protease Clr. J Biol Chem. 2010. 285(16): 11863-9.; Almitairi JOM et al, Structure of the Clr-Cls interaction of the Cl complex of complement activation. Proc Natl Acad Sci U S A. 2018. H5(4):768-773). The inventors incubated apoptotic cells in complement-sufficient NHS which resulted in binding of Clq to the apoptotic cell surface and its subsequent activation to allow Clq-ApoE complex formation. The formation of the Clq-ApoE complex was demonstrated by the proximity ligation assay (PLA) in situ. This assay provides evidence for a complex formation, as a fluorescent signal is formed exclusively when the proteins are in very close proximity. In contrast, Clq-ApoE complexes were absent in Clq-depleted serum (dNHS) and control groups (only ApoE antiserum; Ctr).

In the third example, purified Clq was immobilized on a sensor chip and real-time binding of ApoE was followed using surface plasmon resonance analysis (Biacore). ApoE bound to Clq in a dose-dependent manner (Figure 7). Complexing of Ca²⁺ by addition of EGTA (to inhibit the CCC) revealed dose-dependent reduction of interaction of the two binding partners (Figure 7) as activated Clq was inactivated in the absence of Ca²⁺.

In order to show that one ApoE molecule binds to one activated Clq molecule, the inventors the performed electron microscopy studies. Commercially purified Clq in its activated form was used to allow complex formation in solution. Clq has a MW ~460 kDa with a tulip-like structure. The MW of 460 kDa allows Clq-ApoE complexes to be visualized by EM (Almitairi JOM et al, Structure of the Clr-Cls interaction of the Cl complex of complement activation. Proc Natl Acad Sci U S A. 2018. H5(4):768-773). In sharp contrast, its binding partner, i.e. ApoE, has a MW ~34 kDa. In conclusion, a 1 : 1 binding was observed. In the example shown in Figure 8, the inventors used recombinant commercially labeled ApoE (40 pg/ml; Aurion). Several dilutions were used to optimize the efficiency of gold-labeled Clq-ApoE complexes and avoid complex aggregates in solution with the purpose to obtain single complexes on the grid surface. Calculations show that the labeling efficiency was ~20% which is in the range of other complexes observed by others. Schematic drawings indicating the ApoE-Clq interactions are shown in rectangle boxes below the EM pictures (Figure 8). Based on these experiments, the inventors conclude that one ApoE molecule bound to one Clq molecule, more than 75% ApoE-gold is bound, there is a 1 :1 interaction of the binding partners, ApoE exclusively binds to the stalk but never to the tulip structures or globular heads of the molecule, gold-labeled peptide ApoEi39_i₅₂ has the same binding characteristics when compared to the full length ApoE protein, and that two different experimental methods show similar results.

Staining the complex by proximity ligation assay in human atherosclerotic plaques clearly restricted the Clq-ApoE complex to the plaques in the human artery, while the intact healthy part of the artery is not stained. This pattern confirms atherosclerotic plaques as complement activator surfaces and that ApoE binds to the activated form of Clq (Figure 9). The inventors conclude that the complex is markedly accumulated within the immediate environment of Ab plaques in APPPS1-21 mouse brains. Altogether, this provides evidence for classical complement over-activation in atherosclerosis and Alzheimer’s disease and that inhibition of complement reduces inflammation in these diseases. The experiments show the important role of ApoE in classical complement inhibition, which suggests inhibition of Clq for treatment of atherosclerosis and Alzheimer’s disease as disclosed herein.

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Claims

1. A compound for use in the treatment or prevention of a disease or condition in a mammalian subject, wherein the compound is a modulator of the expression, function, stability, and activation of Clq or a variant thereof, and in particular mimics and/or stabilizes the interaction of ApoE with Clq or variants thereof, and wherein said disease or condition is selected from neuronal diseases, cardiovascular diseases, kidney diseases, and ageing.

2. The compound for use according to claim 1, wherein the variant of ApoE or Clq is selected from the group consisting of an ortho log or paralog of ApoE or Clq, and a functional fragment of said ApoE or Clq protein, comprising or consisting of for example the amino acids 139-152 of the human ApoE polypeptide.

3. The compound for use according to claim 1 or 2, wherein the compound is an inhibitor or antagonist of said expression, function, stability of Clq or a variant thereof and/or mimics and/or stabilizes the interaction of ApoE with Clq or variants thereof.

4. The compound for use according to any of claims 1 to 3, wherein said compound is an antigen binding construct, such as, for example, an antibody, antibody-like molecule or other antigen binding derivative, or an antigen binding fragment thereof), that binds said Clq or a variant thereof.

5. The compound for use according to any of claims 1 to 3, wherein the compound is a nucleic acid, such as, for example an anti-sense nucleotide molecule such as a siR A or shR A molecule that binds to a nucleic acid that encodes or regulates the expression of: (i) Clq or a variant thereof, or (ii) a gene that controls the expression, function and/or stability of Clq or a variant thereof.

6. A method for identifying a compound suitable for the prevention or treatment of a disease characterized by an undesired function of Clq or a variant thereof, the method comprising the steps of a) contacting at least one of Clq or a variant thereof, a Clq binding fragment of ApoE or a variant thereof, and an ApoE binding fragment of Clq or a variant thereof and/or a cell expressing Clq or a variant thereof, the method comprising the steps of a) contacting at least one of Clq or a variant thereof, a Clq binding fragment of ApoE or a variant thereof, and an ApoE binding fragment of Clq or a variant thereof with at least one candidate compound that potentially modulates and preferably mimics and/or stabilizes the interaction of ApoE with Clq in a cell, and b) identifying a binding of said candidate compound to Clq.

7. The method according to claim 6, wherein said modulation is selected from a decrease or an increase of the expression, function, stability and binding of Clq or a variant thereof, and the stability of binding of ApoE to Clq or a variant thereof.

8. The method according to claim 6 or 7, wherein said compound is selected from the group consisting of a peptide library, a combinatory library, a cell extract, in particular a plant cell extract, a "small molecular drug", an antisense oligonucleotide, an siR A, an mR A and an antibody or fragment thereof specifically mimicking and/or stabilizing the binding of ApoE with Clq.

9. The method according to any of claims 6 to 8, wherein said ApoE binding fragment binds to the Clq stalk, and comprises for example the amino acids 139- 152 of the human ApoE polypeptide.

10. An isolated antigen binding construct, capable of specifically binding to Clq or a variant thereof, wherein said antigen binding construct mimics and/or stabilizes, preferably specifically mimics and/or stabilizes, the interaction of ApoE with Clq or variants thereof.

11. A pharmaceutical composition comprising the compound as used according to any one of claims 1 to 6 or the isolated antigen binding construct according to claim 10; and a pharmaceutically acceptable carrier, stabilizer and/or excipient.

12. A screening tool for screening for an agent for treating or preventing neuronal diseases, cardiovascular diseases and ageing, in particular a screening tool for screening for a compound that modulates the expression, the biological activity and/or the interaction of Cl q in a cell, and preferably mimics and/or stabilizes the interaction of ApoE with Clq in a cell, comprising an isolated cell expressing Clq and/or expressing an ApoE binding fragment thereof, wherein said cell optionally expresses ApoE and/or an Clq binding fragment thereof

13. The screening tool according to claim 12, wherein said ApoE binding fragment binds to the Clq stalk, and comprises for example the amino acids 139-152 of the human ApoE polypeptide.

14. A method for treating or preventing neuronal diseases, cardiovascular diseases, kidney diseases, and ageing in a patient, comprising administering to said patient an effective amount of the pharmaceutical composition according to claim 11.

15. The method according to claim 14, wherein said neuronal disease cardiovascular disease or kidney disease to be treated or prevented is Alzheimer Disease or atherosclerosis or C3 -glomerulopathy.