WO2019186084A1

WO2019186084A1 - Anti-complement histones

Info

Publication number: WO2019186084A1
Application number: PCT/GB2018/050789
Authority: WO
Inventors: Simon Timothy ABRAMS; Cheng Hock Toh; Guozheng Wang
Original assignee: The University Of Liverpool
Priority date: 2018-03-26
Filing date: 2018-03-26
Publication date: 2019-10-03

Abstract

The invention relates to histone proteins and modified histone proteins for use in inhibiting the activity of complement, which also includes the medical use of modified histones in the treatment or prevention of diseases associated with enhanced complement activity. The invention further relates to modified histone proteins themselves, and to non-medical uses of histones for inhibiting complement activity in components of cell culture media.

Description

ANTI-COMPLEMENT HISTONES

FIELD OF THE INVENTION

The present invention relates to histone proteins and modified histone proteins for use in inhibiting the activity of complement proteins. The present invention further relates to medical uses of such histones in the treatment or prevention of diseases associated with enhanced complement activity. The present invention further relates to non-medical uses of such histones for inhibiting complement activity in components of cell culture media and to the modified histones themselves.

INTRODUCTION

The complement system is a key innate immune defence against infection and an important driver of inflammation. Complement enhances the ability of antibodies and phagocytic cells to clear microbes and damaged cells from an organism. The complement system consists of three cascading pathways - the classical pathway, the MBL or lectin pathway, and the alternative pathway. Each pathway is made up of a number of different complement proteins and fragments. Most of these complement proteins exist in blood plasma as inactive pre proteins that must be cleaved by proteases to become active. Once activates by various factors such as antibody-antigen complexes, an amplifying complement cascade is begun which results in complement activation/fixation. The pathways eventually converge to produce the Membrane Attack Complex (MAC) which can pierce the membranes of pathogens to cause lysis.

Although essential for immune response, the complement system has the potential to be very damaging to the tissues of an organism. Inappropriate or uncontrolled activation of complement can cause local or systemic inflammation, tissue damage, and disease. Therefore the pathways are normally tightly regulated. However, there are certain disorders in which deregulation or enhanced expression of the components of the complement pathway occurs, such that activation of the complement pathway is enhanced beyond normal levels. In addition, many common diseases have been found to be linked to complement. These disorders and diseases can be difficult to manage because the complement pathway relies on cascades in which one event will trigger another and in which the effects are amplified at each step. Furthermore, complement is essential to human immunity, therefore attempting to suppress the activation of complement poses a risk of infection. Anti-complement therapies are an emerging area. The recent interest in anti-complement therapies has been instigated by the demonstration of genetic associations that firmly link complement to common diseases. However, there is only one commercially available drug which targets complement. Eculizumab is a C5-specific monoclonal antibody therapy which is marketed for the treatment of specific complement disorders; paroxysmal nocturnal haemoglobinuria and atypical uraemic syndrome. Other drugs for targeting different specific components of complement are in development but have not yet reached market. For example, the C3-specific blocker compstatin and an antibody binding fragment specific for Factor D are undergoing clinical trials for the treatment of age-related macular degeneration.

Accordingly, there is a need for further anti-complement treatments directed towards other complement disorders and directed towards other components of the complement system. These is especially a need for anti-complement treatments which can selectively target each of the three complement pathways individually. At present, there is no anti-complement therapeutic that can target a specific complement pathway or part of a pathway, rather than all three at the same time. The currently available Eculizumab affects all three complement pathways because it targets the common component C5, leaving patients dangerously exposed to an increased risk of infections. Therefore, there is a lack of flexibility and a lack of tailored therapies when treating people with increased complement activity or at risk of developing increased complement activity.

Histones are highly alkaline proteins found in eukaryotic cell nuclei that package and order DNA into structural units called nucleosomes. Beside their usual functional within cells, upon trauma or damage to cells, histones are released and can act extracellularly as damage- associated molecular pattern molecules triggering inflammation and toxic responses in cells. Mimicking this activity, when histones are administered intravenously to mice they cause widespread damage by tissue damage, cell lysis, inflammatory cytokine release and coagulation activation. Histones are therefore considered to be cytotoxic to organisms. For this reason, histones are not considered to be therapeutic, and thus far have not been considered for medical usage.

It is an object of one or more of the aspects of the present invention to address the above- mentioned problems in the art.

SUMMARY OF THE INVENTION According to a first aspect of the present invention there is provided a histone protein or modified histone protein, for use in inhibiting complement activity.

According to a second aspect of the present invention there is provided a modified histone protein for use in inhibiting complement activity.

In one embodiment, there is provided a use of a histone protein or modified histone protein in the inhibition of complement activity.

According to a third aspect of the present invention there is provided a modified histone protein for use in the inhibition of complement activity in the treatment or prevention of a disease associated with enhanced complement activity, wherein the modified histone protein has reduced cytotoxicity as compared to a corresponding wild-type histone protein.

According to a fourth aspect of the present invention there is provided a method of inhibiting complement activity in a subject in need thereof, comprising the following steps:

(a) Providing the subject with a therapeutically effective amount of a modified histone protein;

wherein the modified histone protein has reduced cytotoxicity as compared to a corresponding wild-type histone protein.

According to a fifth aspect of the present invention there is provided a method of treatment or prevention of a disease associated with enhanced complement activity in a subject in need thereof, comprising the following steps:

(b) Providing the subject with a therapeutically effective amount of a modified histone protein;

In one embodiment, the method is for inhibiting complement activity in the treatment or prevention of a disease associated with enhanced complement activity.

In one embodiment, the subject in need thereof is a subject having, or at risk of developing, a disease associated with enhanced complement activity. According to a sixth aspect of the present invention there is provided a histone protein or a modified histone protein, for use in inhibiting complement activity in a cell culture media component.

According to a seventh aspect of the present invention there is provided a modified histone protein for use in inhibiting complement activity in a cell culture media component.

According to an eighth aspect of the present invention, there is provided a method of inhibiting complement activity in a cell culture media component comprising the following steps:

(a) Contacting a cell culture media component with a histone protein or a modified histone protein

According to a ninth aspect of the present invention, there is provided a method of inhibiting complement activity in a cell culture media component comprising the following steps:

(a) Contacting a cell culture media component with a modified histone protein

According to a tenth aspect of the present invention there is provided a modified histone protein wherein the modified histone protein is at least 70% identical to one of the following sequences: SEQ ID NO.1 , SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5.

According to an eleventh aspect of the present invention there is provided a modified histone protein wherein the modified histone protein is at least 70% identical to one of the following sequences: SEQ ID NO. 6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 , SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID No.15, SEQ ID NO.16 and SEQ ID NO.17.

In a twelfth aspect, the present invention relates to an expression vector comprising a nucleic acid encoding a modified histone protein according to the tenth or eleventh aspects.

BRIEF DESCRIPTION OF THE DRAWINGS

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

Figure 1 Shows the Identification of Complement component 4 (C4) as a histone binding protein. (A) Using histone-conjugated Sepharose, a number of proteins were pulled down. Among them, were 2 major bands detected by Coomassie blue stained gel (B) and (C) Typical spectra of the two major proteins are presented. (D) complement proteins were subjected to SDS-PAGE and stained with coomassie blue, (E) Western blotting with HRP- histones, shows that a mixture of wild type Histones also bind to C1q, C3, C4, C5 and C8, (F) Western blotting with H2BN modified histone protein showing binding to C3, C4, C5 and C8, (G) Western blotting with H3C modified histone protein showing binding to C3, C4, C5 and C8.

Figure 2 shows that C4 binds to individual histones. (A) 3.5 mM individual histones and 7mM S100P as a control were subjected to SDS-PAGE. One gel was transferred onto PVDF membrane and probed with HRP-conjugated C4 protein (upper panel). The other gel was stained with Coomassie brilliant blue (lower panel). (B-F) SPR analysis: Individual histones were first immobilized on streptavidin surfaces. Different concentrations of C4 were then applied onto each surface and typical binding curves are presented. No binding of histones to S100P was observed.

Figure 3 shows that Histones inhibit complement activation. (A) Classical, lectin and alternative complement pathways were activated by IgM, mannan and LPS, respectively, in the absence or presence of different concentrations of a mixture of calf thymus histones (0- 50 pg/ml). MAC formation was detected by anti-human C5b-9 antibodies. Complement activity was expressed as a percentage of control (without histones) which was set to 100%. The MeansiSD of relative activities are presented. (B) MeansiSD of relative total complement activity activated by zymosan, in the presence of different concentration of calf thymus histones (0-50 pg/ml). (C) Means ± SD of relative Classical pathway activation in the presence of different concentrations of individual histones (0-50 pg/ml). (D) Means ± SD of relative effect of 20pg/ml individual histones on the Classical pathway activation. (E) Means ± SD of relative activities of the lectin pathway in the presence of different concentration of individual histones (0-50 pg/ml). (F) Means ± SD of relative effect of 20pg/ml individual histones on lectin pathway. Means ± SD were calculated from at least 3 independent experiments. (G) (H) and (I) show that histone-derived modified peptides also inhibit complement activation in vitro. The effect of full length and truncated histones is shown on complement activation. Classical, lectin and alternative complement pathways were activated by IgM, mannan and LPS, respectively, in the absence or presence of 20 pg/ml full length histones, Nterminal [Start of histone protein, containing tail region and positive charge] and C-terminal [End of histone protein, containing protein core] truncated histone proteins or histone-derived H1 M and H3M modified peptides (Mut). ELISA was used to measure C5b-9 (MAC) complex formation (complement activity) in each of the individual pathways. All peptide treatments were compared to normal serum samples without treatment (UT). Effect of histone-derived peptides on classical, lectin and alternative pathway activation are represented as a percentage compared to UT serum (set to 100%).

Figure 4 shows anti-histone treatment rescues complement activity. (A) and (B) nonanticoagulant heparin (20 pg/ml) was used to rescue complement activities of classical (A) and lectin (B) pathways, which have been inhibited by individual histones (20 pg/ml). (C) and (D): Antihistone H4 antibody (12 pg/ml) were used to rescue complement activities of Classical (C) and lectin (D) inhibited by H4 (20 pg/ml). Means ± SD of relative activities from at least 3 independent experiments are presented.

Figure 5 shows histones have no effect on C4 cleavage but significantly reduce C3 and C5 convertase activities. (A) In vitro cleavage of C4 by C1s in the presence or absence of histones. C4 (250 pg/ml) was incubated with C1s (50 pg/ml) ± calf thymus histones (100 pg/ml) at 37°C for 30 mins. Protein bands were visualized under Coomassie brilliant blue staining following separation by gradient (8-18%) SDS-PAGE. (B) Western blot with antiC4a antibody (Upper panel). Densitometry from 3 independent experiments demonstrates fold changes compared sample without (-Hist) to sample with histones (+Hist) (Lower panel). (C) 0.25 pM C4, C4b, C4a and S100P (as a control) were subjected to blotting with HRP conjugated calf thymus histones. A typical blot is presented from 3 independent experiments. (D-F) Complement in serum was activated by IgM (Classical pathway, CP), mannan (Lectin), LPS (Alternative pathway, AP) or zymosan in the absence or presence of a mixture of calf thymus histones (50 pg/ml), treated for 1 hr at 37_° C. Levels of C3a (D), C5a (E) or MAC (F) were detected by ELISA. MeansiSD from at least 3 independent experiments are presented.

Figure 6 shows the effect of the addition of complement C4 on histone activities. (A) Classical and (B) lectin pathways of complement activation in the presence of increasing C4 (0-300 pg/ml) and H4 (20 pg/ml). (C) Zymosan-activated complement activity in the absence or presence of H4 (20 pg/ml) and C4 (300 pg/ml) (1 :1 molar ratio).

Figure 7 shows the cytotoxicity of wild type and modified histones after culturing the histones with cells and determining the cell viability as a percentage of untreated (UT) cells, which was set to 100%. Students T-test shows no significant difference between UT cells and those treated with H1M and H3M modified peptides (n=3). Figure 8 shows the dose response of mixed wild-type histone protein toxicity in vivo. (A) Intravenous histones at doses of up to 50 g/kg had no effects on mortality, whereas doses of 75 mg/kg led to death of all mice within 24 hours, with LD50 of around 60 mg/kg. (B) Circulating histone levels achieved in mice injected with 20, 30, 50, 60, and 75 mg/kg of histones were 40.2 ± 7.6, 65.5 ± 13.4, 87.3 ± 22.2, 107.3 ± 38.9, and 236.8 ± 21.7 pg/mL, respectively.

Figure 9 shows the kinetics of modified histone peptides H1 M and H3M after administration to mice. The maximal circulating levels of H1M (A) and H3M (B) either subcutaneously (s.c) or intravenously (i.v). Absolute circulating levels of each protein were calculated over time.

Figure 10 shows that modified histones inhibit complement activity in vivo. C57/BL mice were injected with zymosan (150ul/100grams) without (to activate complement) or with subcutaneous administration of histones (ctHist) (positive control for complement inhibition). Mice were also treated with Zymosan without or with either subcutaneous (s.c) or intravenous (i.v) H1 M or H3M infusion. Global complement activation (C5b-9 complexes) was measured at 4 hrs by ELISA. The levels of C5-9 complexes (MAC) enhanced by zymosan were set as 100%. (5 mice per group)*P<0.05.

Figure 11 shows the urine albumin of rats injected with anti-GBM ± modified histone proteins H1 M or H3M. 24h urines were collected and albumin was measured by ELISA. Total urine albumin per 24 hours is presented in the nephritis model without and with either H1 M or H3M.

Figure 12 shows blood urea nitrogen (BUN) at 7 days after anti-GBM injection. *P<0.05 when compared with nephritis model (anti-GBM) alone.

Figure 13 shows anti-complement C3 and anti-lgG staining of kidney sections. A and B are sections from the model rat kidney and stained with anti-complement C3 antibody. Arrows indicate positive staining. C: model+H1 M treatment. D: Model+H3M treatment. E: Model + calf thymus histone treatment. C-E were all stained with anti-complement C3. F: Model + H1 M treatment and stained with anti-lgG-HRP. Bar=20pm.

DETAILED DESCRIPTION The present invention is based upon the inventor’s finding that histones are able to bind components of the complement system and inhibit complement activity.

Histones have not been associated with complement or indeed any immune functions in organisms. The typical function of histones is in the storage of genomic DNA within nucleosomes inside the nucleus of cells. It is known that in this format histones are stable, but outside of this context, in an extracellular environment histones are known to cause widespread damage to cells and are considered to be cytotoxic. Accordingly, until now, histones were believed to have the normal function of DNA storage, and a secondary abnormal function of alerting the organism by acting as damage-associated molecular pattern molecules upon their release from damaged cells.

Surprisingly, the inventors have discovered that histones have activity on the complement system. The inventors have shown that in vitro, histones can bind to various complement proteins. In doing so, histone proteins have the ability to inhibit complement activity by inhibiting one or more of the complement pathways. Furthermore, the inventors have found that certain histones only inhibit one complement pathway, others inhibit two of the complement pathways, and some can inhibit all three, giving rise to various non-medical and medical uses of histones that have not previously been envisaged.

Advantageously, the inventors have found that histone proteins (non-modified and modified) have non-medical applications in the laboratory. As described further herein, the inventors have found that histones can be used to prepare cell culture media in which complement is typically undesirable. Currently, time consuming preparation must be undertaken in which such media is heat treated to denature complement proteins. In doing so, many other desirable proteins are also affected and denatured. The present invention provides a way to prepare cell culture media components using histones without the need for heat treatment in a targeted manner which only reduces the complement activity without affecting other desirable proteins.

Furthermore, histones have not previously been considered for use as therapeutic molecules due to their demonstrated cytotoxicity in vivo. However, the inventors envisaged that their inhibitory activity on complement could have medical applications for treating those with disorders in complement regulation. Surprisingly, the inventors have further discovered how to modify histone proteins to reduce their toxicity, and have produced modified histone proteins which are completely non-toxic to cells. The inventors discovered that the histone proteins have certain parts that give rise to the cytotoxic effect by binding to phospholipids, which form a large proportion of many cell membranes, and disrupting these membranes to cause cell lysis. Accordingly, the inventors have produced modified histone proteins in which said cytotoxic portions have been removed or varied and tested them in vitro and in vivo cellular models. The inventors have demonstrated that these modified histone proteins have reduced cytotoxicity compared to non-modified histones, and in many cases 100% cell survival in their presence. They have further demonstrated that such modified histone proteins are effective in vivo to treat diseases in which complement activity is enhanced. Accordingly, the inventors have developed modified histones proteins for medical applications which can be safely administered to an organism, and which are effective in the treatment of enhanced complement related disorders.

Furthermore, the inventors have found that each of the modified histones have varied activity on the three complement pathways. The inventors have discovered that different modified histones act on different complement pathways and proteins meaning that each modified histone is effective for the treatment of a selective set of diseases resulting from activity of specific complement proteins or pathways. Therefore, the modified histones described herein provide a collection of more tailored therapies for the treatment of enhanced complement disorders wherein a medical professional can select the most appropriate modified histone for the relevant complement pathway which is overactive. This is a great improvement over the single therapy for enhanced complement activity on the market, Eculizumab, which targets all three complement pathways at once leaving medical professionals with no flexibility in treatment and leaving the patient open to serious infections. The present invention allows for treatment of enhanced complement activity without the risk of infections by targeting only one or two complement pathways and leaving the other/s active. Additionally, the present invention provides viable alternative treatments to Eculizumab which do target all three complement pathways and which could be used when patients are non-responsive to Eculizumab or who have adverse reactions to this treatment. In addition, histones are small molecules, typically much smaller than antibodies. Therefore, they are easier to produce and deliver into the organism than other therapies.

Furthermore, the inventors believe that the modified histone proteins of the invention, due to their small size, are much less likely to be immunogenic. As such, the inventors believe that the modified histone proteins of the invention are a promising alternative treatment for patients who have developed immunogenic responses and/or antibodies to other anti complement treatments. Further beneficial uses of the modified histone proteins are seen in vitro in laboratory settings. As noted above, the inventors have discovered that non-modified or wild type histones can be used to prepare cell cultures in which complement is typically undesirable. Modified histones can also be used in a similar manner to prepare cell culture components. However, due to their reduced cytotoxicity, the modified histones have the further advantage that they can be added directly to cell cultures in which cells are present to inhibit complement activity. Thereby entirely removing the need for a step of preparation of the cell culture media to inhibit complement prior to plating cells.

The invention, and certain terms used in the disclosure of the present invention, will now be defined and described further.

Histone Proteins

The present invention is based upon the finding that histone proteins and modified histone proteins are able to bind components of the complement system and inhibit complement activity.

The term‘histone’ or‘histone protein’ as referred to herein indicates a protein that is typically found in eukaryotic cell nuclei that is capable of packaging and ordering DNA into structural units called nucleosomes.

The histones described herein may be wild type histone proteins, or may be modified histone proteins.

‘Wild-type’ as used herein refers to a naturally occurring form of a histone protein (i.e. a histone protein which has not been modified), and may also include naturally occurring variant forms of histone proteins. References to a‘corresponding wild-type histone protein’ as used herein indicate a comparison of a modified histone protein with the same histone protein in its naturally occurring form which has not been modified. For example, when considering a modified H3 histone protein, the relevant corresponding wild-type histone protein is histone H3 which may be as defined in SEQ ID NO.4.

Known naturally occurring histone proteins are histone H1 also known as histone H5 in some species (SEQ ID NO.1), histone H2A (SEQ ID NO.2), histone H2B (SEQ ID NO.3), histone H3 (SEQ ID NO.4), histone H4 (SEQ ID NO.5). Naturally occurring variants of said histone proteins are also known. Examples of naturally occurring variant histones are follows: H3.1 , H3.2, TS H3.4 , H3.3, centromeric H3, H2AZ, H2AB, H2AW, H2AL, H2AP, H2A1 , H2AX, H2B1 , H2BW, and H2BE. Further naturally occurring histone proteins and variant histone proteins may be found on the database "HistoneDB 2.0”.

Suitably, a naturally occurring variant of a histone protein may be at least 70%, at least 75%, at least 80%, be at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 97%, at least 98%, at least 99%, or more, identical to a sequence of naturally occurring histone proteins, for example histone H3 (SEQ ID NO: 4) or histone H4 (SEQ ID NO: 5).

Suitably, the histone protein may be any wild type histone protein or variant thereof.

Suitably, the histone protein may be selected from any of the following: histone H1 , histone H2A, histone H2B, histone H3, and histone H4, or variants thereof.

Suitably, the histone protein may be at least 80% identical to one of the following sequences: SEQ ID NO.1 , SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5.

Suitably, the histone protein may be at least 85%, at least 90%, at least 95%, at least 99% identical to one of the following sequences: SEQ ID NO.1 , SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5.

In one embodiment, the histone protein consists of one of the following sequences: SEQ ID NO.1 , SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5.

‘Modified’ as used herein refers to a histone protein that has been changed such that its amino acid sequence is not the same as the amino acid sequence of the corresponding wild- type histone protein. It will be appreciated that a modified histone protein is, by definition, not a wild-type histone protein.

The sequence of the modified histone protein may be changed by man-made modifications or otherwise. Such modifications may include truncations or deletions (for example single amino acid deletions, deletions of 2 or more contiguous amino acids, and/or truncations); sequence mutations including inversions, substitutions, repeats, reversals; amino acid modifications including tagging, phosphorylation, methylation and biotinylation; and the like. One or more modifications may be present in a modified histone protein sequence. One or more different types of modification may be present in a modified histone protein sequence. Suitably, the modified histone proteins described herein have reduced cytotoxicity in comparison with the corresponding wild-type histone protein. ‘Reduced cytotoxicity’ is defined hereinbelow.

Suitably, therefore, the modified histone protein has been modified to reduce its cytotoxicity. Suitably, the modified histone protein has been modified to reduce its cytotoxicity yet retain its ability to inhibit complement activity. Suitably, the modified histone protein has been modified to reduce its cytotoxicity yet retains at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 99% at least 100% of its ability to inhibit complement activity in comparison with a corresponding wild-type histone protein.

In one embodiment, there is provided a modified histone protein wherein the modified histone protein has reduced cytotoxicity as compared to a corresponding wild-type histone protein.

Suitably, the modified histone protein has been modified to remove one or more cytotoxic portions of its amino acid sequence.

Suitably, the modified histone protein may be a fragment and/or may comprise one or more amino acid sequence mutations.

‘Fragment’ as used herein refers to an incomplete histone protein as compared to the corresponding wild-type histone protein. Such fragments may include a truncated form of a wild type histone protein, or a wild type histone protein that has one or more domains, sections, or parts of its amino acid sequence missing, or that have been removed.

Suitably, the modified histone protein is a fragment of the corresponding wild-type histone protein. Suitably the modified histone protein is a truncated histone protein. Suitably, the modified histone protein consists of a truncation of the corresponding wild-type histone protein amino acid sequence.

It will be appreciated that fragments of this sort may share at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with the corresponding portion of the wild-type histone amino acid sequence. Suitably such a fragment may, consist of approximately 20%-95%, or 30%-90%, or 40% to 60%, or 45% to 55% of the total length of the corresponding wild-type histone amino acid sequence. Suitably such a fragment may consist of approximately 30%-50% of the total length of the corresponding wild-type histone amino acid sequence.

Suitably, the modified histone protein has been modified to remove at least a part of the N- terminal and/or C-terminal region.

In one embodiment, the modified histone protein is a truncated histone protein.

Suitably, the truncated histone proteins lacks all or part of the N-terminal tail region.

Suitably, the truncated histone proteins lacks all or a part of the C-terminal region.

In an embodiment where the modified histone protein is a truncated histone protein lacking a part of the C-terminal region, suitably the modified histone protein may consist of amino acids 1-120, 1-110, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, or 1-40 of the corresponding wild- type histone amino acid sequence, and any integer value therebetween.

In an embodiment where the modified histone protein is a truncated histone protein lacking a part of the N-terminal tail region, suitably the modified histone protein may consist of amino acids 40 onwards, 50 onwards, 60 onwards, 70 onwards, 80 onwards, 90 onwards, 100 onwards, 110 onwards, or 120 onwards of the corresponding wild-type histone amino acid sequence, and any integer value therebetween.

Suitably, the modified histone protein may be a truncated histone protein lacking a part of the C-terminal region of SEQ ID NO.1 , SEQ ID NO.4 SEQ ID NO.3, SEQ ID NO.4, or SEQ ID NO.5.

Suitably, the modified histone protein may be a truncated histone protein lacking a part of the N-terminal tail region of SEQ ID NO.1 , SEQ ID NO.4 SEQ ID NO.3, SEQ ID NO.4, or SEQ ID NO.5.

Suitably, the modified histone protein may be a truncated histone protein lacking amino acids 40 onwards, 50 onwards, 60 onwards, 70 onwards, 80 onwards, 90 onwards, 100 onwards, 110 onwards, 120 onwards or any integer value therebetween of SEQ ID NO.1 , SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, or SEQ ID NO.5. Suitably, the modified histone protein may be a truncated histone protein lacking amino acids 1-120, 1-110, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40 or any integer value therebetween of SEQ ID NO.1 , SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, or SEQ ID NO.5.

Suitably, the modified histone protein may comprise one or more amino acid sequence mutations (such mutations being assessed with reference to the corresponding wild type histone protein amino acid sequence).

Suitably the mutations are selected from one of: deletions, inversions, substitutions, repeats, or reversals of the histone protein amino acid sequence. In one embodiment, the modified histone protein comprises one or more point mutations. In one embodiment, the modified histone protein comprises one or more substitution mutations.

Suitably, the modified histone protein comprises one or more amino acid mutations to remove positively charged amino acid residues. Suitably, the positively charged amino acid residues are removed by substitution mutations. Suitably, the positively charged amino acid residues are removed by substitution with a neutral or negatively charged amino acid residue. Positively charged amino acid residues which may be substituted include: lysine, arginine, and/or histidine.

Suitably the modified histone protein comprises one or more lysine substitution mutations. Suitably, the modified histone protein comprises one or more lysine to alanine substitution mutations.

Merely by way of example, in the context of histone H4 (as set out in SEQ ID NO: 5), the modification may comprise a substitution of one or more amino acids selected from the group consisting of Lys31 , Arg35, Arg39 and Arg45.

Suitably, the modified histone protein may comprise at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, or more amino acid mutations, which may suitably be substitutions, when compared to the corresponding wild-type histone protein amino acid sequence.

Suitably, the modified histone protein may comprise up to 50, up to 40, up to 30, up to 20, up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or 1 amino acid mutations, which may suitably be substitutions, when compared to the corresponding wild type histone protein amino acid sequence.

Suitably the modified histone protein comprises up to 30 amino acid substitutions when compared to the corresponding wild type histone protein amino acid sequence.

In one embodiment, the modified histone protein comprises between 5 to 20 amino acid substitutions when compared to the corresponding wild type histone protein amino acid sequence.

In one embodiment, the modified histone protein comprises 6 lysine to alanine substitution mutations when compared to the corresponding wild type histone protein amino acid sequence.

Suitably, the positively charged amino acid residues which are substituted are located in an exposed part of the histone protein structure. Suitably, the positively charged amino acid residues which are substituted are outside of any tertiary structure in the histone protein such as alpha helices or beta sheets. Suitably, the positively charged amino acid residues which are substituted are outside of the histone protein core domain. Suitably, the positively charged amino acid residues which are substituted are not located in the alpha helices of the histone protein structure. Suitably, the positively charged amino acid residues which are substituted are located in the N-terminal tail region of the histone protein.

Suitably, the modified histone protein may be a fragment and further comprise one or more amino acid sequence mutations.

Suitably, the modified histone protein may be a truncated histone protein lacking a part of the N-terminal tail region and a part of the C-terminal region and further comprising multiple amino acid sequence mutations.

Suitably, fragments of histone proteins and histone proteins comprising amino acid sequence mutations share the biological activity of the corresponding wild-type histone proteins. In particular, the fragmented or mutated versions should share at least the ability to bind complement proteins and to inhibit at least one or a part of a complement pathway. Suitably these fragments or mutated histone proteins may also share the reduced cytotoxicity characteristic that makes the modified histone proteins suitable for use in therapeutic applications. Suitably, the modified histone protein retains the ability to inhibit complement activity. Suitably, the modified histone protein retains the ability to bind at least one complement protein.

Suitably, the modified histone protein retains at least two alpha helices, suitably three alpha helices. Suitably, the modified histone protein retains a globular/core domain.

In accordance with the invention, the modified histone protein is at least 70% identical to one of the following sequences: SEQ ID NO.1 , SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5.

Suitably, the modified histone protein may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to one of the following sequences: SEQ ID NO.1 , SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5.

Suitably, the modified histone protein is not a wild-type histone protein.

Furthermore, in accordance with the invention, the modified histone protein is at least 70% identical to one of the following sequences: SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO. 6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 , SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, and SEQ ID No.15.

Suitably, the modified histone protein may be at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to one of the following sequences: SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO. 6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 , SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, and SEQ ID No.15.

Suitably, wherein the modified histone protein still retains the ability to inhibit complement activity.

Suitably, wherein the modified histone protein still comprises reduced toxicity as compared to a corresponding wild-type histone protein.

In one embodiment, the modified histone protein consists of an amino acid sequence which is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% to one of the following sequences: SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO. 6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 , SEQ ID N0.12, SEQ ID NO.13, SEQ ID NO.14, and SEQ ID No.15.

In one embodiment, the modified histone protein consists of one of the following sequences: SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO. 6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 , SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, and SEQ ID No.15.

Reduced Cytotoxicity

The present invention relates to modified histone proteins having reduced cytotoxicity when compared with a corresponding wild-type histone protein.

The term‘cytotoxicity’ refers to the toxicity of the relevant histone protein, modified or not, to living cells, either in vivo or in vitro. Toxicity to living cells may be measured by contacting living cells with the relevant agent, for a period of time and determining the number of living cells before and after the contact has been made, and calculating the difference in this number. If the number of cells that are alive has fallen in this time by a statistically significant amount, then the agent is generally regarded as cytotoxic. If the number of cells that are alive does not fall by a statistically significant amount, then the agent is generally regarded as non-cytotoxic.

Therefore‘reduced cytotoxicity’ as used herein means that the modified histone protein is less cytotoxic to living cells than a corresponding wild-type histone protein. Suitably, this is determined by directly comparing the cytotoxicity of the relevant modified histone protein to a corresponding wild-type histone protein in a toxicity assay.

For example, a typical toxicity assay may be conducted as demonstrated in the examples herein by culturing endothelial cells with 20pg/ml of the relevant histone protein, modified or not, for 1 hour at 37°C and under 5% CO2. Then determining cell viability by staining the cells with propidium iodide and quantifying the number of alive cells using FACS. Cell viability is then expressed as a percentage of the untreated cells which is set to 100%.

Suitably the modified histone proteins have a cytotoxicity of less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, suitably less than 15%, suitably less than 10%, suitably less than 8%, suitably less than 5%, suitably less than 3%, suitably less than 1 %, suitably 0%. In one embodiment, the modified histone proteins have a cytotoxicity of between 0% and 1 %.

Suitably the modified histone proteins retain the cell viability of over 80%, suitably over 85%, suitably over 90%, suitably over 92%, suitably over 95%, suitably over 97%, suitably over 99%, suitably 100% of cells brought into contact with the modified histone protein.

In one embodiment, the modified histone proteins retain the cell viability of between 99% and 100% of cells brought into contact with the modified histone protein.

Suitably the modified histone proteins are non-cytotoxic.

Suitably, cytotoxicity and/or cell viability are calculated after culturing the modified histone protein for a period of time with live cells.

Suitably, cytotoxicity and/or cell viability may be calculated after culturing 20pg/ml of the modified histone protein with live cells for 1 hour at 37°C with less than 5% CO2.

Complement Activity

The present invention refers to the use of histone proteins and modified histone proteins to inhibit complement activity.

The inhibition of complement activity may comprise the inhibition, inactivation or downregulation of one or more complement proteins. The inhibition of complement activity may comprise the inhibition, inactivation or downregulation of the whole or part of a complement pathway. The inhibition, inactivation or deactivation of the whole or part of a complement pathway may result from the inhibition, inactivation or deactivation of more than one complement protein, or indeed from a single complement protein.

Suitably, the inhibition of complement activity may be partial or full inhibition of complement activity, therefore inhibition of complement activity may be described or present as suppression of complement activity.

Suitably, the inhibition of complement activity may be measured by the number of Membrane Attack Complexes that are formed (MAC) in a sample. Suitably, histone proteins or modified histone proteins inhibit MAC formation. Suitably, histone proteins or modified histone proteins inhibit MAC formation by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. In one embodiment, histone proteins or modified histone proteins abolish MAC formation.

Suitably, complement activity can be measured by known methods such as those described in the examples herein. For example, complement activity may be measured by a complement functional screen which determines MAC production. One example is the COMPL300 Total Complement Functional Screen kit (Wielisab, Sweden). References to ‘complement activity’ may be used interchangeably herein with ‘MAC formation’. Complement activity and MAC formation may be expressed as a percentage of normal complement activity/MAC formation as measured in a suitable reference sample.

Suitably, therefore, the histone proteins or modified histone proteins inhibit complement activity by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. In one embodiment, the histone proteins or modified histone proteins abolish complement activity.

Suitably the complement pathway is selected from any known complement pathway such as: the classical pathway, the lectin pathway, and the alternative pathway. Suitably, the histone proteins or modified histone proteins may inhibit the whole or a part of any of the classical pathway, the lectin pathway, and the alternative pathway, or any combination thereof.

Suitably, the histone proteins or modified histone proteins are monospecific, bispecific or trispecific for the complement pathways. In other words, the histone protein or modified histone protein may only inhibit the whole or a part of one complement pathway, the whole or a part of two complement pathways, or the whole or a part of all three complement pathways.

Suitably, the histone proteins or modified histone proteins may inhibit the classical pathway monospecifically and are selected from SEQ ID NO. 6 (H1 N).

Suitably, the histone proteins or modified histone proteins may inhibit the lectin pathway monospecifically and are selected from SEQ ID NO. 8 (H2AN).

Suitably, the histone proteins or modified histone proteins inhibit the classical and lectin pathway bispecifically and are selected from SEQ ID NO. 1 (H1), SEQ ID NO.7 (H1C), SEQ ID NO.2 (H2A), SEQ ID NO.3 (H2B), SEQ ID NO.10 (H2BN), SEQ ID NO.4 (H3), SEQ ID NO.12 (H3N), SEQ ID NO.5 (H4).

Suitably, the histone proteins or modified histone proteins inhibit the classical, lectin and alternative pathway trispecifically and are selected from SEQ ID NO. 13 (H3C), SEQ ID NO.16 (H1 M), SEQ ID NO.17 (H3M).

Suitably, the histone proteins or modified histone proteins may inhibit complement activity by binding to one or more complement proteins. Suitably the histone proteins or modified histone proteins may bind to any complement proteins selected from any of the known proteins of the complement system such as: C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, MBL, ficolin, MASP, Factor B, Factor H, Factor I, CFH-related protein 1 , C4 binding protein, protein S, and Factor D.

Suitably, the histone proteins or modified histone proteins inhibit complement activity by binding to one or more of the complement proteins selected from: C1q, C3, C4, C5 and C8.

Suitably, the histone protein or modified histone protein binds to C3 and is selected from: SEQ ID NO.10 (H2BN) and SEQ ID NO.13 (H3C).

Suitably, the histone protein or modified histone protein binds to C4 and is selected from: SEQ ID NO. 4 (H3), SEQ ID NO.5 (H4), SEQ ID NO.1 (H1), SEQ ID NO.3 (H2B), SEQ ID NO.10 (H2BN) and SEQ ID NO.13 (H3C).

Suitably, the histone protein or modified histone protein binds to C5 and is selected from: SEQ ID NO.10 (H2BN) and SEQ ID NO.13 (H3C).

Suitably, the histone protein or modified histone protein binds to C8 and is selected from: SEQ ID NO. SEQ ID NO.10 (H2BN) and SEQ ID NO.13 (H3C).

Suitably, the histone proteins or modified histone proteins inhibit complement activity by reducing the enzymatic activity of C3 convertase and/or C5 convertase. Suitably, the reduction in enzymatic activity of C3 convertase and/or C5 convertase may be caused by histone proteins or modified histone proteins binding to complement components of the convertase enzymes such as C3b and C4b. Reduction in the enzymatic activity of convertases may be measured by measuring the rate of conversion of complement proteins. Suitably, measuring the enzymatic activity of C3 convertase may be achieved by measuring the rate of conversion of C3 into C3a and C3b. Suitably, measuring the enzymatic activity of C5 convertase may be achieved by measuring the rate of conversion of C5 to C5a and C5b.

Diseases associated with enhanced complement activity

The present invention relates to the use of modified histone proteins having reduced cytotoxicity for the treatment or prevention of diseases associated with enhanced complement activity.

It will be appreciated that the modified histones have been modified to reduce their cytotoxicity, thereby making the modified histones suitable for therapeutic use.

As used herein the terms“treat”,“treating” or“treatment” refer to a clinical improvement of enhanced complement activity. Such a clinical improvement may be demonstrated by an improvement of the pathology and/or the symptoms associated with the disease. In a suitable embodiment an improvement of the symptoms may be demonstrated by reducing the frequency at which the symptoms occur and/or the severity of the symptoms.

The term“prevention” as used herein, refers to prophylactic use of modified histone proteins of the invention.

The treatment of diseases associated with enhanced complement activity may include the treatment of the physiological symptom of enhanced complement activity in itself without the presence of a clinical disease diagnosis.

Prevention of diseases associated with enhanced complement activity may include the provision of a modified histone protein to a subject who is at risk of, or who is suspected of, developing enhanced complement activity, suitably before the subject develops enhanced complement activity. Prophylactic use of modified histone proteins may be of particular relevance to an asymptomatic subject. For example a subject with a primary disorder which may lead to secondary enhanced complement activity, a subject exposed to environmental factors which lead to enhanced complement activity, and/or a subject known to carry a mutation which increases the subject’s likelihood of developing a disease associated with enhanced complement activity.

In one embodiment, the subject may be provided with a modified histone protein as a first line treatment for a disease associated with enhanced complement activity. In such an embodiment, the subject would have not been provided with any other treatment prior to treatment in accordance of the present invention.

However, a modified histone protein may also be used to treat a disease associated with enhanced complement activity in which other treatments were found ineffective. In such embodiments, the subject may have received another treatment prior to treatment in accordance of the present invention. Merely by way of example, the subject may have already received another treatment such as Eculizumab. Thus, suitably a modified histone protein may be employed in the use or method of the invention as a second line treatment for a disease associated with enhanced complement activity.

Diseases associated with enhanced complement activity may be regarded as pathological conditions presenting with an increase in activity of at least a part of at least one complement pathway above the normal level of complement activity. This may include the over-activation or de-regulation of one or more individual complement proteins. In this case, the‘normal level’ of complement activity is defined as the level of complement activity in a suitable reference sample.

A reference sample in this case may be a sample from a comparable healthy subject. A sample in this case may refer to any suitable biological material in which complement proteins will be present and in which their activity can be measured, such as blood or serum for example.

In one embodiment, enhanced complement activity may be regarded as an increase in the formation of MAC above the normal level. Suitably, complement activity may be measured by the number of Membrane Attack Complexes (MAC) that are formed, for example in a complement functional screen as described herein.

Suitably, enhanced complement activity may be acquired or may be genetic. Suitably, acquired enhanced complement activity is caused by disease. Suitably, genetic enhanced complement activity is caused by a genetic predisposition, which may then lead to disease. Suitably, genetic enhanced complement activity may be inherited. Acquired enhanced complement activity may be as a result of a different (primary) disorder, or may be induced. Acquired enhanced complement activity caused by a different (primary) disorder, may be referred to as secondary enhanced complement activity.

Suitably, the modified histone is for use in the treatment or prevention of diseases associated with enhanced complement activity selected from any of the following classes: kidney diseases; autoimmune diseases; degenerative diseases; transplant induced diseases; transfusion induced diseases; dialysis induced diseases; systemic inflammatory diseases.

Suitably, kidney diseases associated with enhanced complement activity may be selected from: glomerulopathy or Atypical haemolytic uremic syndrome, for example. Suitably degenerative diseases associated with enhanced complement activity may be selected from: Alzheimer’s disease, age-related macular degeneration, for example. Suitably, autoimmune diseases associated with enhanced complement activity may be selected from: Paroxysmal nocturnal haemoglobinuria, Systemic lupus erythematosus, Antiphospholipid antibody syndrome, Myasthenia gravis, ANCA-associated vasculitis, auto-immune haemolytic anaemia, primary cold agglutinin disease, secondary cold agglutinin syndrome (which may be associated with malignant and acute infections), paroxysmal cold haemoglobinuria, for example. Suitably, transplant induced diseases associated with enhanced complement activity may be selected from: Antibody-induced rejection after transplantation, graft vs. host disease, Ischaemia-reperfusion injury (IRI), for example. Suitably, transfusion or dialysis induced diseases associated with enhanced complement activity may be selected from: Thrombotic microangiopathies, Haemodialysis-related complications, for example.

Merely by way of example, the subject may have a genetic predisposition to enhanced complement activity or diseases associated therewith if the subject has mutations in genes encoding proteins involved with one or more of the complement pathways.

Suitably, the modified histone is for use in the treatment or prevention of diseases associated with enhanced complement activity which are caused by a genetic predisposition.

Suitably, the modified histone is for use in the treatment or prevention of diseases associated with enhanced complement activity caused by a genetic mutation.

Suitably, the modified histone is for use in the treatment or prevention of diseases associated with enhanced complement activity caused by a mutation in the genes encoding any of the following complement proteins: Factor H, Factor B, C3, Factor I, C1 , thrombomodulin.

Suitably, diseases associated with enhanced complement activity may also be caused by a mutation in the genes encoding other non-complement proteins that are related to a complement pathway.

Suitably, the modified histone is for use in the treatment or prevention of diseases associated with enhanced complement activity caused by a mutation in the genes encoding any of the following non-complement proteins: membrane cofactor proteins; complement inhibitor proteins; PIGA; and anti-complement antibodies.

Suitably, the modified histone is for use in the treatment or prevention of diseases associated with enhanced complement activity caused by a mutation in any of the following proteins: Factor H, Factor B, C3, Factor I, C1 , C1 -inhibitor, thrombomodulin, membrane cofactor proteins; complement inhibitor proteins, PIGA, and anti-complement antibodies

Suitably, the modified histone is for use in the treatment or prevention of diseases associated with enhanced complement activity caused by a mutation in any of the following proteins: Factor H, Factor B, C3, Factor I, C1 , thrombomodulin, membrane cofactor proteins.

In one embodiment, the modified histone is for use in the treatment or prevention of atypical haemolytic uremic syndrome.

Suitably, the modified histone is for use in the treatment or prevention of diseases associated with enhanced complement activity caused by a mutation in any of the following proteins: Factor H, Factor B, C3.

In one embodiment, the modified histone is for the use in the treatment or prevention of age- related macular degeneration.

Suitably, the modified histone is for use in the treatment or prevention of diseases associated with enhanced complement activity caused by a mutation in the protein PIGA.

In one embodiment, the modified histone is for use in the treatment or prevention of Paroxysmal nocturnal haemoglobinuria. Suitably, the medical use or treatment in accordance with the present invention may make use of the modified histone protein in conjunction with a second treatment. Merely by way of example, a suitable second treatment may be selected from a group consisting of: a chemical; or biopharmaceutical, such as for example, a protein/peptide, a vaccine, an antibody, a nucleic acid etc.

Suitably, the treatment in accordance with the present invention may make use of the modified histone protein in conjunction with a treatment for preventing coagulation.

By‘preventing coagulation’ we mean decreasing coagulation as compared to a suitable control.

Suitably, coagulation may be decreased by downregulating the clotting cascade. Suitably, the clotting cascade may be downregulated by decreasing the amount or activity of one or more components of the clotting cascade. It will be appreciated that decreasing the activity of one or more components of the clotting cascade may result in an increase in the reaction time of one or more reactions of the clotting cascade. Suitably, the component and/or the reaction of the clotting cascade may be one of the intrinsic and/or extrinsic pathway.

Suitably, coagulation may be prevented ex vivo (for example in vitro) or in vivo.

In a suitable embodiment, coagulation may be considered to be prevented if viscosity of blood and/or plasma is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or more as compared to a reference value.

In a suitable embodiment, coagulation may be considered to be prevented if the rate of active thrombin generation is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or more as compared to a reference value.

In a suitable embodiment, coagulation may be considered to be prevented if the amount of thrombin is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or more as compared to a reference value. In a suitable embodiment, coagulation may be considered to be prevented if prothrombin cleavage is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or more as compared to a reference value.

In a suitable embodiment, coagulation may be considered to be prevented if the rate of fibrin generation and/or the amount of fibrin is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or more as compared to a reference value.

Suitably, coagulation may be considered to be prevented if the amount and/or activity of a component of the intrinsic and/or extrinsic coagulation pathways selected from the group consisting of thrombin (activated tissue factor II), prothrombin (tissue factor II), fibrinogen (tissue factor I), fibrin (activated tissue factor I), tissue factor III, tissue factor V, activated tissue factor V, tissue factor VII, activated tissue factor VII, tissue factor VIII, activated tissue factor VIII, tissue factor IX, activated tissue factor IX, tissue factor X, activated tissue factor X, tissue factor XI, activated tissue factor XI, tissue factor XII, activated tissue factor XII, tissue factor XIII, and activated tissue factor XIII is decreased. An amount and/or activity is considered to be decreased if it is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% less than compared to a reference value.

A suitable reference value may be obtained from a sample or subject in the absence of a histone modified protein. The sample may be a blood and/or plasma sample. A skilled person will be able to determine a suitable reference value without difficulty.

Suitable treatments for preventing coagulation may include any anti-coagulant therapy available, such as: warfarin, heparin, vitamin K antagonists and direct inhibitors to thrombin or factor Xa.

Surprisingly the inventors have further discovered that some of the modified histone proteins described herein have anti-coagulant properties.

Accordingly, suitable treatments for preventing coagulation may include a further modified histone protein. Suitably the further modified histone protein is an anti-coagulant. Suitably the further modified histone protein has reduced cytotoxicity compared to a corresponding wild-type histone proteins as described elsewhere herein. In one embodiment, there is provided a modified histone protein for use in preventing coagulation, wherein the modified histone protein has reduced cytotoxicity compared to a corresponding wild-type histone proteins as described elsewhere herein.

Suitably, the modified histone protein is for use in preventing coagulation in the treatment or prevention of a coagulation disorder. Suitably the coagulation disorder results in thickening of the blood, and/or excessive clotting and/or increased predisposition to thickening and/or clotting of the blood. In a suitable embodiment, a coagulation disorder may be genetic or acquired.

Suitably the modified histone protein is for use in preventing coagulation in the treatment or prevention of a coagulation disorder selected from, for example: Antiphospholipid syndrome, Arteriosclerosis, Atherosclerosis, Antithrombin deficiency, Deep vein thrombosis (DVT), Protein C deficiency, Protein S deficiency, Elevated homocysteine, Elevated Factor VIII, Elevated lipoprotein, or Factor V Leiden.

In one embodiment, the modified histone protein is at least 70% identical to one of the following sequences: SEQ ID NO.5, SEQ ID NO.4, SEQ ID NO.3, SEQ ID NO.2, and SEQ ID NO.1. Suitably, wherein the further modified histone protein is not a wild-type histone protein.

In one embodiment, the further modified histone protein is at least 70% identical to one of the following sequences: SEQ ID NO.6 (H1 N), SEQ ID NO.7 (H1C), SEQ ID NO.8 (H2AN), SEQ ID NO.10 (H2BN), SEQ ID NO.12 (H3N), SEQ ID NO.14 (H4N), SEQ ID NO.16 (H1M).

Suitably, the further modified histone protein is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to one of the following sequences: SEQ ID NO.1 , SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5.

Suitably, the further modified histone protein is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identical to one of the following sequences: SEQ ID NO.6 (H1 N), SEQ ID NO.7 (H1C), SEQ ID NO.8 (H2AN), SEQ ID NO.10 (H2BN), SEQ ID NO.12 (H3N), SEQ ID NO.14 (H4N), SEQ ID NO.16 (H1M).

Suitably, wherein the further modified histone protein still retains the ability to prevent coagulation. Suitably, wherein the further modified histone protein still comprises reduced toxicity as compared to a corresponding wild-type histone protein.

In one embodiment, the further modified histone protein consists of one of the following sequences: SEQ ID NO.6 (H1 N), SEQ ID NO.7 (H1C), SEQ ID NO.8 (H2AN), SEQ ID NO.10 (H2BN), SEQ ID NO.12 (H3N), SEQ ID NO.14 (H4N), SEQ ID NO.16 (H1 M).

In one embodiment, there is provided a modified histone protein for use in inhibiting complement activity in combination with a further modified histone protein for use in preventing coagulation.

Suitably the modified histone protein and the further modified histone protein are different proteins. However, it will be appreciated that some modified histone proteins have both anti complement and anti-coagulant properties.

Providing

The term“providing” as used herein encompasses any techniques by which the subject receives a therapeutically effective amount of the modified histone protein.

In a suitable embodiment, the modified histone protein may be provided to the subject either directly or indirectly.

In one embodiment, the subject may be provided directly with the modified histone protein. For example the subject may be provided with a pharmaceutical composition comprising the modified histone protein itself.

In another embodiment the subject may be provided indirectly with the modified histone protein. In such an embodiment, the subject may, for example, be provided with a nucleic acid encoding such a protein. Methods by which the modified histone protein may be indirectly provided to the subject will be known to those skilled in the art. Merely by way of example, a modified histone protein may be provided to the subject with the use of an expression vector comprising a nucleic acid sequence encoding such a protein.

It will be appreciated that there are various routes in which the subject may be provided with a therapeutically effective amount of the modified histone protein. Such suitable routes may be selected from the group consisting of: oral, parenteral, intravenous, intraperitoneal, intramuscular, intravascular, intranasal, intraperitoneal, rectal, subcutaneous, transdermal and percutaneous.

In one embodiment, the subject is provided a therapeutically effective amount of the modified histone protein intravenously or subcutaneously.

In such an embodiment, the subject may be provided with a pharmaceutical composition comprising the modified histone protein.

It will be appreciated that the most appropriate route of providing the modified histone protein may be determined with reference to the type of disease to be treated.

Therapeutically Effective Amount

The term“therapeutically effective amount” as used herein, refers to the amount of modified histone protein, that when provided to the subject, is sufficient to treat or prevent a disease associated with enhanced complement activity.

Therefore, a therapeutically effective amount is an amount of a modified histone protein which will result in inhibition of complement activity in a subject.

A therapeutically effective amount is also an amount of a modified histone protein which will result in a clinical improvement in a subject having a disease associated with enhanced complement activity, or which will result in a clinically stable state in a subject at risk of developing enhanced complement activity.

Clinical improvement may be demonstrated by an improvement of the pathology and/or symptoms associated with the disease. A clinically stable state may be demonstrated by the absence of the pathology and/or symptoms associated with the disease. Suitably, clinical improvement may be demonstrated by a decrease in complement activity. Suitably, a clinically stable state maybe demonstrated by restoration of complement activity to a normal level. Complement activity may be measured or determined as described elsewhere herein.

It will be appreciated that the therapeutically effective amount will vary depending on various factors, such as the subject’s body weight, sex, diet and route by which the modified histone protein is provided. Such a therapeutically effective amount may be provided to the subject in a single or multiple doses or by continuous infusion. Suitably, the therapeutically effective amount of the modified histone protein is provided to the subject in multiple doses. Suitably, the therapeutically effective amount of the modified histone protein is provided to the subject once every month, twice every month, once every two weeks, once every week, once every few days, once every two days, once per day, more than once per day, twice per day, three times per day, four times per day, once every few hours, once every 6 hours, once every 5 hours, once every 4 hours, once every 3 hours, once every 2 hours, or once every hour.

In one embodiment, the therapeutically effective amount of the modified histone protein is provided to a subject once every four hours.

It will also be appreciated that the therapeutically effective amount will vary depending on the type of disease associated with enhanced complement activity. The skilled person will also recognise that the therapeutically effective amount will vary depending on the severity, or stage of said disease.

Suitably, the therapeutically effective amount of modified histone protein may be between 0.1 and 100 mg/kg of body weight. Suitably, the therapeutically effective amount of a modified histone protein may be approximately 0.1 , 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100mg/kg. Suitably, the therapeutically effective amount may be between 1 and 90 mg/kg, between 5 and 80 mg/kg, between 7.5 and 70 mg/kg, between 10 and 60 mg/kg, or between 12 and 58 mg/kg, or any value therebetween. More suitably, the therapeutically effective amount may be between 15 and 55 mg/kg, or between 20 and 50 mg/kg.

Suitably, the therapeutically effective amount may be around 20 mg/kg, 25mg/kg, 30mg/kg, 35mg/kg, 40mg/kg, 45mg/kg, or 50 mg/kg.

In one embodiment, the therapeutically effective amount is around 20 mg/kg.

It will be appreciated that a therapeutically effective amount of modified histone protein may be determined in vitro or in vivo, using techniques known to the skilled person.

Subject As used herein the term“subject” refers to an individual. Suitably, the subject may be a mammal. Merely by way of example, the subject may be a selected from a group consisting of: a human, a primate, a dog, a cat, a rat and a mouse.

In one embodiment, the subject is human. Suitably, the subject may be male or female. Suitably, the subject may be an adult or a child.

Suitably, the subject has enhanced complement activity or is at risk of developing enhanced complement activity. Suitably, the subject has a disease associated with enhanced complement activity or is identified to be at risk of developing a disease associated with enhanced complement activity.

A subject may be, for example, identified to be at risk of developing enhanced complement activity or a disease associated therewith if the subject has a familial history of such diseases, or if the subject has a genetic predisposition to enhanced complement activity as defined above. By way of example, a subject at risk of developing enhanced complement activity may have a primary disorder which may lead to a secondary complement disorder/disease, be exposed to environmental factors which may lead to an acquired complement disorder/disease, and/or have a genetic mutation associated with a coagulation disorder/disease. Suitably, a subject at risk of developing enhanced complement activity or a disease associated therewith may be asymptomatic.

An expression vector

As touched upon elsewhere in this specification, the modified histone protein may be provided to the subject with the use of an expression vector comprising a nucleic acid sequence encoding such a protein.

Accordingly, the twelfth aspect of the invention relates to an expression vector comprising a nucleic acid sequence encoding a modified histone protein.

Suitably, the expression vector may comprise a nucleic acid encoding one or more amino acid sequences selected from: SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. Suitably, the expression vector may comprise a nucleic acid encoding an amino acid sequence sharing at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 91% identity, at least 92% identity, at least 93% identity, at least 94% identity, or at least 95% identity with one or more of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15. Suitably, the expression vector may comprise a nucleic acid encoding an amino acid sequence sharing at least 96% identity, at least 97% identity, at least 98% identity with, or at least 99% identity with one or more of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15.

Suitably, the expression vector may be viral or non-viral. By way of example, a suitable viral expression vector may be derived from a virus selected from the group consisting of paramyxovirus, retrovirus, adenovirus, lentivirus, pox virus, alphavirus, and herpes virus. Suitably, the virus is paramyxovirus. An example of a particularly suitable paramyxovirus is Sendai virus. Other viral expression vectors suitable for providing the modified histone protein to the subject are known in the art.

Suitable non-viral expression vectors may be selected from the group consisting of inorganic particle expression vectors (such as calcium phosphate, silica, and gold), lipid based particle expression vectors (for example cationic lipids, lipid nano emulsions, and solid lipid nanoparticles) and polymer based particle expression vectors (for example peptides, polyethylenimine, chitosan, and dendimers). Other suitable non-viral expression vectors will be known to those skilled in the art.

Methods of delivering expression vectors to a cell are also well known in the art. Merely by way of example, such methods include viral transfection, electroporation and sonoporation.

Cell Culture Media Component

The present invention further relates to non-medical uses of histone proteins, or modified forms thereof, in the preparation of media for cell cultures.

In accordance with the invention, there is provided a method of inhibiting complement activity in a cell culture media component comprising the following steps: (a) Contacting a cell culture media component with a histone protein or a modified histone protein

Suitably, the cell culture media component may be contacted with a histone protein or modified histone protein in the presence of cells or in the absence of cells. Suitably contacting in the presence of cells may be during cell culture. Suitably contacting in the absence of cells may be before cell culture.

Suitably, when the cell culture media component is contacted with a histone protein in the absence of cells, the histone protein may be wild-type or modified.

Suitably, when the cell culture media component is contacted with a histone protein in the presence of cells, the histone protein is modified. Suitably, the modified histone has been modified to reduce its cytotoxicity as compared to a corresponding wild-type histone protein. Suitably, therefore, the modified histone protein is then suitable for use in the presence of cells. Suitable modifications of histone proteins are discussed hereinabove.

Suitably, contacting the cell culture media component with a modified histone protein in the presence of cells may comprise the addition of an effective amount of the modified histone protein to a cell culture. Suitably, the effective amount of the modified histone may be regarded as the amount which is effective to inhibit complement activity in the cell culture by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100%. Suitably, the effective amount of the modified histone may be regarded as the amount which is effective to inactivate complement in the cell culture. Suitably the effective amount of the modified histone may be between 0.1 and 100 ug/ml. Suitably, the effective amount may be between 1 and 90ug/ml, between 5 and 80 ug/ml, between 7.5 and 70 ug/ml, between 10 and 60 ug/ml, or between 12 and 58 ug/ml. More suitably, the effective amount may be between 15 and 55 ug/ml, or between 20 and 50 ug/ml.

In one embodiment, contacting the cell culture media component with a modified histone protein in the presence of cells may comprise the addition of 20ug/ml of the modified histone protein to a cell culture.

Suitably, contacting the cell culture media component with a modified histone protein in the absence of cells may comprise passing the cell culture media component over the modified histone protein. Suitably, the modified histone protein may be located upon a support. Suitably, the modified histone may be immobilized on or in a support. Non-limiting examples of a support include microcarriers such as polymer spheres, beads, and disks that can be porous or non-porous, cross-linked beads (e.g., dextran) which may be charged with specific chemical groups (e.g., tertiary amine groups), 2D supports including nonporous polymer fibers, plates, or sheets, 3D supports including hollow fibers, tubes, multicartridge reactors, semi-permeable membranes that can comprise porous fibers, or columns. Supports can be fabricated from materials such as dextran, gelatin, glass, sepharose and cellulose.

Suitably the support may comprise a column or plate or the like upon which the modified histone protein may be immobilised. Suitably, therefore, contacting the cell culture media component with a modified histone protein in the absence of cells may comprise passing the cell culture media component through a column upon which is immobilised the modified histone protein. Alternatively, or additionally, the modified histones may be immobolized within particles such as beads. Suitably, therefore, contacting the cell culture media component with a modified histone protein in the absence of cells may comprise contacting particles comprising modified histones with a cell culture media component.

Suitably, the contact between the cell culture media component and the histone protein or modified histone protein is for a sufficient time to allow the histone protein to inhibit complement activity.

Suitably, a sufficient amount of time is the amount of time required to inhibit complement activity in the cell culture media component by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100%. Suitably, a sufficient amount of time may be regarded as the amount time required to inactivate complement activity in the cell culture media component. Suitably, the cell culture media component is contacted with a histone protein or a modified histone protein for at least at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes.

Suitably, the cell culture media component is contacted with a histone protein or a modified histone protein for less than 1 hour.

In one embodiment, contacting a cell culture media component with a histone protein or a modified histone protein is for a least 1 hour to allow the histone protein to inhibit complement activity.

Suitably these steps of the method may equally be applied to the use defined in the sixth aspect. The invention will now be described with reference to the following non-limiting examples.

EXAMPLES

1. Materials and Methods

Human samples

Plasma was isolated from blood drawn from patients admitted to the Intensive Care Unit at the Royal Liverpool University Hospital (RLUH) by centrifugation at 2600 g for 20 min. Ethical approval was granted by the Northwest and RLUH Research Ethics Committees (Ref: 13/NW/0089).

Methods of generation modified histone proteins

Histone peptides were expressed in C41 (DE3) Escherichia coli, using pET-16b expression vector, purified using Histag resin (Qiagen) and assessed by SDS-PAGE.

Isolation of histone binding proteins from human plasma

Citrated plasma was diluted with 2 ^c phosphate buffered saline (PBS) (v/v) and centrifuged to eliminate insoluble contents. Harvested supernatant was then pre-cleared using blank Sepharose resin and then loaded on CNBr-activated Sepharose 4B (GE Healthcare, Little Chalfont, UK) column conjugated with calf thymus histones (Roche, West Sussex, UK). After very stringent washing with PBS+0.5% (v/v) Tween-20 (Sigma-Aldrich, Dorset, UK) followed by PBS, histone-binding proteins were eluted and separated by gel electrophoresis. Gel slices from SDS-PAGE were washed (2 x 30 min) with 50% acetonitrile, 0.2M ammonium bicarbonate (pH 8.9) and then dried in a rotary evaporator. The slices were rehydrated in rehydrate buffer [2 M urea, 0.2 M ammonium bicarbonate pH 7.8] containing 0.2 pg trypsin and incubated at 37°C overnight. Excess buffer was then removed and peptides were extracted from the gel slices with 60% acetonitrile, 0.1 % TFA. The total peptide extract was concentrated in a rotary evaporator and then desalted using C18 ZipTips, according to the manufacturer’s instructions.

Identification of histone binding protein

Liquid chromatography-mass spectrometry (LC-MS/MS) analysis was performed using a MALDI-Tof instrument (Waters-Micromass) using a saturated solution of alpha-cyano-4 hydroxy-cinnaminic acid in 50% acetonitrile/0.1% trifluoroacetic acid. Further to identification of C4 as the putative histone-binding protein, confirmatory experiments were performed. C4 (Fitzgerald Industries International, USA) or calf thymus histones were conjugated using Lighting-Link HRP conjugation kit (Innova bioscience, Babraham, Cambridge, UK), according to manufacturer’s instructions. In further experiments, 3.5 mM of H1 , H2A, H2B, H3 and H4, or 0.25 mM of C4, C4b, and C4a were subjected to SDS-PAGE and probed with HRP-conjugated C4 or histones, respectively. Bands were visualized with the Pierce ECL (Thermo Scientific).

Surface Plasmon Resonance (SPR) measurements

The binding parameters of C4 to individual histones, including the equilibrium dissociation constant (KD), on-rates (kon) and off-rates (koff) were measured by SPR analysis using a Proteon XPR36 system (Bio-Rad, UK) as described previously (Mould AP, Askari JA, Byron A, Takada Y, Jowitt TA, Humphries MJ. Ligand-induced Epitope Masking: DISSOCIATION OF INTEGRIN adbI-FIBRONECTIN COMPLEXES ONLY BY MONOCLONAL ANTIBODIES WITH AN ALLOSTERIC MODE OF ACTION. Journal of Biological Chemistry. 2016;291 (40):20993-21007. Briefly, chips coated with 20 pg/ml streptavidin (GLH, Bio-Rad, UK), which could directly interact with histones, (Bailey LM, Ivanov RA, Wallace JC, Polyak SW. Artifactual detection of biotin on histones by streptavidin. Anal Biochem. 2008;373(1):71-77 were used for immobilizing individual histones and measuring binding affinities to C4. Running buffer (10 mM HEPES pH 7.4, 150 mM NaCI, 0.05% Tween 20 and regeneration buffer (0.1 M glycine pH 2.2) were used throughout the assays. Five pg/mL of each recombinant histone (H1 , H2A, H2B, H3 or H4) in running buffer was captured only on the surface of flow cells L2-L6 with L1 (streptavidin only) used as a reference. For kinetic measurements, a concentration series of C4 was injected at a flow rate of 100 pl/min (60s association time, 300s dissociation time) over both captured histone surface and reference surface at 25°C. All binding sensorgrams were collected, processed, and analysed using the integrated ProteOn Manager software (Bio-Rad Laboratories) assuming 1 :1 interaction kinetics. All results shown are representative of at least three separate experiments.

Complement activity assay

The effect of histones on complement activity in the three pathways was measured using COMPL300 Total Complement Functional Screen kit (Wielisab, Sweden). Briefly, mixtures of the reaction were added to strips of wells for the classical pathway (coated with IgM), alternative pathway (coated with LPS) and lectin pathway (coated with mannan). Normal human sera were diluted with assay specific kit buffers to ensure that the specific pathway to be investigated was activated (Seelen MA, Roos A, Wieslander J, et al. Functional analysis of the classical, alternative, and MBL pathways of the complement system: standardization and validation of a simple ELISA. J Immunol Methods. 2005;296(1-2):187-198. After one hour incubation at 37°C and washing of the strips, alkaline phosphatase-conjugated anti human C5b-9 was added before incubation at room temperature for 30 mins. Additional washing was performed and substrate added for 30 min. Absorbance values were then determined at 405 nm. In each assay, standard positive and negative control sera, provided in the kit were used. The complement activity for each pathway was expressed as a percentage of the activity of the calibrating serum. Measurements of C3a and C5a were performed using C3a and C5a ELISA kits (e-Bioscience UK). C5b-9 induced by zymosan (Comp Tech, USA) was measured by ELISA (Quidel Corporation, USA).

Antibody and heparin blocking assay

To inhibit histone activity, 20 pg/ml non-anticoagulant heparin (Sigma, UK) with 20 pg/ml H1 , H2A, H2B, H3 or H4 proteins, or 12 pg/ml anti-histone H4 antibody (Abrams ST, Zhang N, Manson J, et al. Circulating Histones Are Mediators of Trauma-associated Lung Injury. Am J Respir Crit Care Med. 2013; 187(2): 160-169. with 20 pg/ml H4 was incubated prior to complement activation using Wieslab COM PL CP310 kit. Percentage changes were compared to untreated samples (100%).

C4 cleavage assay

C1s (50 pg/ml, Comp Tech, USA) was incubated with C4 (250 pg/ml) in the presence or absence of histones (100 pg/ml) at 37°C for 30 mins. SDS loading buffer was then added and boiled for 10 mins prior to SDS-PAGE. The gel was stained with Coomassie brilliant blue or subjected to Western blotting with anti-C4a antibody (Comp Tech). C4a band intensities were measured using GeneSnap software (version 7.05) (Syngene) and fold changes were calculated.

Cell viability assay

Cell viability was assessed using EAhy926 (endothelial cells) following 1 hour incubation with or without histones (20 pg/ml). Flow cytometric analysis of propidium iodide-stained cells clearly separated damage cells compared to untreated controls. Viability of untreated cells was set as 100% for comparison.

2. Results

Identification of C4 as a histone-binding protein

Histone-conjugated Sepharose beads were used to pull down human plasma proteins. Following extensive washing, proteins bound to histone-beads were eluted. Multiple proteins were visualised on Coomassie blue stained gels with two major protein bands at approximately 70 kDa and 25 kDa (Figure 1A). Following LC-MS/MS analysis, complement C4 and CRP were identified (Figure 1 B, C). CRP was previously reported to be a major histone-binding protein, which is able to neutralise histone toxicities (Abrams ST, Zhang N, Dart C, et al. Human CRP defends against the toxicity of circulating histones. J Immunol. 2013; 191 (5):2495-2502. The same experiment was repeated with equal molar of complement proteins and S100P (2x molar ratio as negative control) were subjected to SDS-PAGE and stained with Coomassie blue (Figure 1 D). Furthermore, Western blotting was conducted with the same complement proteins and HRP-histones, showing that a mixture of histones could bind to C1q, C3, C4, C5 and C8 (Figure 1 E), and that modified histones can still bind complement proteins, specifically C3, C4, C5 and C6 (Figures 1 F and 1G).

Individual histones bind to complement C4 with different affinities

To determine the relative binding of individual histones to C4, equal molar concentrations of individual histones were subjected to gel overlay assay (Figure 2A upper), along with Coomassie blue staining to demonstrate equal loading (Figure 2A lower). Figure 2A shows that H3 and H4 predominantly bound to C4, with H1 and H2B binding to a lesser extent. To determine the comparative binding strengths to C4 under physiological conditions, surface plasmon resonance (SPR) (ProteOn XPR-36) (Figure 2B-F) was used. The results (Table 1) show that H3 and H4 (had much higher binding affinity than H1 and H2B with weakest binding to H2A.

Tabfe 1 ; Klae ics C4 Mii iisg te IIKIIV &I fifelO S

aAssoci8liom rate; ^Dissociation rale: ^s Equilibrium issociation eo isnt.

Some histones significantly inhibit classical and lectin but not alternative pathways

To investigate the functional consequences of histone-C4 binding on the activation of classical, lectin and alternative pathways, the Complement functional screen kit was used. Pre-incubation of different concentrations of calf thymus histones with human serum significantly reduced the production of MAC by activated classical and lectin pathways. Significant reduction occurred at 10 pg/ml histones with 50 pg/ml histones virtually abolishing MAC formation (Figure 3A). In contrast, histones showed much less effect on the alternative pathway and 50 pg/ml histones reduced MAC formation by 20%. To evaluate the overall effect of different histones on complement activation in human serum, zymosan was used to activate complement in the presence or absence of histones. Figure 3B shows that 50 pg/ml of a mixture of histones could significant inhibit MAC production induced by zymosan. As to the role of individual histones in the activation of the classical (Figure 3C, D) and lectin (Figure 3E, F) pathways, both were significantly inhibited by 20 pg/ml individual histones.

The effect of N-terminal and C-terminal modified truncated histone proteins on the complement pathways was further tested. Either N-terminal [Start of histone protein, containing tail region and positive charge] or C-terminal [End of histone protein, containing protein core] truncated modified histones, along with the further modified H1 M and H3M histone proteins, were tested for activity on the three complement activation pathways (classical, lectin and alternative). The activity of complement in each pathway was measured using the same method described above and screened using COMPL300 Total Complement Functional Screen kit (Wielisab, Sweden). The results are shown in figures 3G, 3H and 3I.

These data demonstrate that all full length wild-type histones can significantly inhibit both the classical and lectin binding pathways, but have much less effect on the alternative pathway (Figures 3A-F). Furthermore, the modified histone proteins H1 N, H1C, H2BN, H3N and H3C significantly inhibit classical pathway activation. The modified histone proteins H1C, H2AN, H2BN, H2N and H3C significantly inhibit the lectin-binding pathway. Of these modified truncated peptides, H3C significantly inhibited all complement activation pathways, including the alternative pathway. Similarly, both H1 M and H3M modified histone peptides could significantly inhibit complement activation by all 3 pathways.

Anti-histone reagents can rescue complement activity

To demonstrate the specificity of histones on complement activation, anti-histone H4 and non-anticoagulant heparin that have been shown to specifically inhibit histone toxicity both in vitro and in vivo (Abrams ST, Zhang N, Manson J, et al. Circulating Histones Are Mediators of Trauma-associated Lung Injury. Am J Respir Crit Care Med. 2013;187(2):160-169 Wildhagen KC, Garcia de Frutos P, Reutelingsperger CP, et al. Non-anticoagulant heparin prevents histone-mediated cytotoxicity in vitro and improves survival in sepsis. Blood. 2013), were used. Both could reverse the inhibition of classical and lectin pathways by individual histones (Figure 4A-D). These data demonstrate that the effect of histones on complement activation is specific.

Histones do not affect C4 cleavage but significantly reduce C3 and C5 convertase activity To attempt to clarify the molecular mechanisms of histone-inhibited complement activation through interactions with C4, the cleavage of C4 to C4a and C4b by C1s was investigated as this is a key process of C4 activation. Histones showed no effect on the production of C4a (Figure 5 A, B) to indicate that histone binding does not affect C1s cleavage of C4. Figure 5C shows that histones bind to C4b but not C4a. The presence of histones significantly reduced the production of C3a and C5a in classical and lectin pathways but not the alternative pathway (Figure 5D-F). This suggests that histone-bound C4b are not as efficient as C4b alone in forming active C3 and C5 convertases. The overall C3a, C5a and C5b-9 (MAC) production induced by zymosan (Figure 5D-F) was significantly reduced by histones, mainly due to the suppression of both classical and lectin pathways by histone inhibition of the C3 and C5 convertases.

Excess C4 only partially rescues histone-inhibited complement activation

Using C4 up to 300 pg/ml, only one third of the maximal complement activity of classical and lectin pathways could be recovered in the presence of up to 20 pg/ml mixed histones (Figure 6A, B). However, zymosan-induced complement activation could be recovered by 300 pg/ml C4 [from 25% to 70% of total activity] in the presence of 20 pg/ml H4 (Figure 6C). This suggests that histones may target other components of the complement system in addition to C4.

Histones can be modified to significantly reduce cell toxicity

Figure 7 shows that all full-length wild-type histone proteins are cytotoxic to cells. In contrast, modified N and C-terminal truncated peptides produced herein i.e. , H1 N, H2AC, H2BN and C, H3C and H4N and H4C, are no longer cytotoxic in vitro. The further modified histone- derived peptides that have cell membrane-binding properties removed (H1 M and H3M) are also non-toxic.

Cytotoxicity was measured by culturing EAhy926 cells were cultured without or with full length wild-type histones (NewEngland Biolabs), N-terminal [Start of histone protein, containing tail region and positive charge] and C-terminal [End of histone protein, containing protein core] truncated histone peptides as produced herein, or H1 M and H3M peptides with further point mutations as produced herein at a concentration of 20pg/ml for 1 hour at 37°C under 5% C02. Cell viability was determined by propidium iodide (PI) staining and quantified using Fluorescence-activated cell sorting (FACS). Cell viability is expressed as a percentage of untreated (UT) cells, which was set to 100%. Students T-test shows no significant difference between UT cells and those treated with H1 M and H3M peptides (n=3).

In vivo Suitability of Modified Histone Proteins

For kinetic analysis, we based our dosing on previous data generated using mixed histones. 20mg/kg of H1M or H3M modified histone protein were injected into C57/BL male 12 weeks old either subcutaneously or intravenously and blood was collected at 0, 1 , 2, 4, 6 hour after injection.

Peptide concentrations were detected by ELISA (testing histags). We found that both peptides were cleared efficiently in around 2 hours of intravenous injection (Figure 9). If injected subcutaneously, maximal concentration was achieved around 2 hours and cleared around 6h after injection. This indicates suitability of the modified histones for use as a therapy.

For toxicity, the dose response of wild-type mixed histone toxicity in vivo was measured. As shown in Figure 8A Intravenous histones at doses of up to 50 mg/kg had no effects on mortality, whereas doses of 75 mg/kg led to death of all mice within 24 hours, with LD50 of around 60 mg/kg. In Figure 8B it is shown that circulating histone levels achieved in mice injected with 20, 30, 50, 60, and 75 mg/kg of histones were 40.2 ± 7.6, 65.5 ± 13.4, 87.3 ± 22.2, 107.3 ± 38.9, and 236.8 ± 21.7 pg/mL, respectively. Mice were further injected with H1 M and H3M modified histones up to 50mg/kg and blood was collected at 2 hours and 6 hours. WBC and platelet counts, thrombin-antithrombin (TAT) were determined. Mice were euthanized at 6h after injection and organs were fixed. Pathological changes were examined by H&E staining. In line with in vitro cytotoxicity tests described above, there were no obvious toxic effects found phenotypically, physiologically and microscopically (see figure 13). This indicates that doses of 50mg/kg or under are appropriate for use in therapy.

In vivo testing of modified histones in a Mouse Zymosan-infusion Model

Zymosan is a glucan with repeating glucose units connected by B-1 ,3-glycosidic linkages and has been demonstrated to activate all 3 complement pathways both in vitro and in vivo. Since we have shown that histone-derived peptides are able to interact with mouse complement component 4 (C4), a zymosan-infusion mouse model was chosen. Five mice per group were injected with zymosan alone, or zymosan + H1 M or H3M. C57/BL mice were injected with zymosan (150ul/100grams) without (to activate complement) or with subcutaneous administration of histones (ctHist) (positive control for complement inhibition). Mice were also treated with Zymosan without or with either subcutaneous (s.c) or intravenous (i.v) H1 M or H3M infusion. Global complement activation % (indicated by C5b-9 complexes) was measured at 4 hrs by ELISA to evaluate complement activity in vivo. The levels of C5-9 complexes enhanced by zymosan were set as 100%. (5 mice per group)*P<0.05. These data demonstrate that H1M and H3M are very effective at abrogating complement activation in vivo by different routes of administration (Figure 10). In vivo testing of modified histones in a Rat anti-GBM induced nephritis model Anti-glomerular basement membrane (GBM) antibody-mediated glomerular injury rat model has been extensively used as a model because complement activation has been well demonstrated to play important roles in kidney injury caused by autoantibodies against GBM. Since no commercial anti-mouse GBM antiserum is available and histone-derived peptides also interact with rat C4 and inhibit complement activation in vitro, we used a rat model with sheep nephrotoxic antiserum against rat GBM from Probetex. Before the injection of anti-GBM antiserum, rats were monitored for baseline albuminuria, serum albumin and urea concentrations. Rats received an injection of 500mI of sheep anti-GBM antiserum intravenously on 3 consecutive days. Treatment groups were injected intravenously with histone derived modified peptides H1M and H3M at 20mg/kg every 4 hours for 7 days, following the first infusion of anti-GBM antibodies. Urine and blood was collected at days 3,7and 14 after the first injection of anti-GBM antiserum. Rats were euthanized at day 17 and their kidneys and other organs were harvested for histological analysis. Urine albumin concentrations were measured by ELISA and the 24-h urinary albumin excretion was calculated. All the rats injected with anti-GBM became very ill after 2 days but none of them died.

Figure 11 shows the changes in urine albumin levels measured by ELISA. All rats injected with anti-GBM showed significant increase in urinary protein excretion indicating the induced nephritis. At day 3, H1M and H3M treated rats had lower levels of urine albumin than the model alone showing that the modified histones were able to improve the nephritis. At day 7, all rats treated with H1M or H3M showed lower levels of albuminuria than the nephritis model alone (Anti-GMB) with the difference remaining stable at day 14 despite stopping H1 M or H3M treatments on day 7. This shows that treatment with modified histones can reduce the effects of nephritis and that the treatment can remain effective for a long period of time.

Blood urea nitrogen (BUN) was measured at day 7 to evaluate renal function. Anti-GBM elevated BUN levels whilst modified histone peptide H1 M and H3M treatments reduced them to indicate a protective effect of modified histone peptides against renal injury (Figure 12). *P<0.05 when compared with nephritis model (anti-GBM) alone.

Kidneys were collected and sections stained using anti-complement C3 antibody and anti- IgG-HRP (Figure 13). More obvious C3 precipitation was found in kidneys of mice from the nephritis model alone (Figure 13A and B) whilst much less C3 precipitation were found in kidneys of mice that were also treated with H1 M (Figure 13C), H3M (Figure 13D) or calf thymus histones (Figure 13E) indicating the modified histones act to reduce complement activation in vivo and provide therapeutic effects therefrom.

The in vivo data demonstrate that various modified histone peptides are able to inhibit activation of all 3 complement pathways without inducing toxic effects. No off-target procoagulant effects were found. In vivo experimentation in mice as well as rats using two different models of physio/pathological complement activation consolidate the in vitro findings presented hereinabove. These results extend the rationale of such modified histone peptides as therapeutic candidates for the effective treatment of diseases associated with enhanced complement activity. The data herein also points to the viability of using these peptides by subcutaneous injection in addition to the intravenous route.

While specific embodiments of the invention have been described herein for the purpose of reference and illustration, various modifications will be apparent to a person skilled in the art without departing from the scope of the invention as defined by the appended claims. Preferred, suitable, and optional features of any one particular aspect or embodiment of the present invention are also preferred, suitable, and optional features of any other aspect or embodiment described herein.

All documents filed herewith are hereby incorporated by reference into the present specification.

SEQUENCE INFORMATION

SEQ ID NO.1 - Histone H1

MSETAPAETA TPAPVEKSPA KKKATKKAAG AGAAKRKATG PPVSELITKA VAASKERNGL SLAALKKALA AGGYDVEKNN SRKLGLKSL VSKGTLVQTK GTGASGSFKL NKKAASGEAK PKAKKAGAAK AKKPAGATPK KAKKAAGAKK AVKKTPKKAK KPAAAGVKKV AKSPKKAKAA AKPKKATKSP AKPKAVKPKA AKPKAAKPKA AKPKAAKAKK AAAKKK

SEQ ID N0.2 - Histone H2A

MSGRGKQGGK ARAKAKTRS S RAGLQFPVGR VHRLLRKGNY AERVGAGAPV YLAAVLEYLT AEILELAGNA ARDNKKTRII PRHLQLAIRN DEELNKLLGK VTIAQGGVLP NIQAVLLPKK TESHHKAKGK

SEQ ID N0.3 - Histone H2B

MPEPAKSAPA PKKGSKKAVT KAQKKDGKKR KRSRKESYSI YVYKVLKQVH PDTGISSKAM GIMNSFVNDI FERIAGEASR LAHYNKRSTI TSREIQTAVR LLLPGELAKH AVSEGTKAVT KYTSSK

SEQ ID N0.4 - Histone H3

M ARTKQT ARK STGGKAPRKQ LATKAARKSA PATGGVKKPH RYRPGTVALR EIRRYQKSTE LLIRKLPFQR LVREIAQDFK TDLRFQSSAV MALQEACEAY LVGLFEDTNL CAIHAKRVTI MPKDIQLARR IRGERA SEQ ID N0.5 - Histone H4

MSGRGKGGKG LGKGGAKRHR KVLRDNIQGI TKPAIRRLAR RGGVKRISGL IYEETRGVLK WLENVIRDA VTYTEHAKRK TVTAMDVVYA LKRQGRTLYG FGG

SEQ ID NO.6 - Modified Histone H1 N

MSETAPAETA TPAPVEKSPA KKKATKKAAG AGAAKRKATG PPVSELITKA VAASKERNGL SLAALKKALA AGGYDVEKNN SRKLGLKSL VSKGTLVQTK GTGASGSFKL

SEQ ID NO.7 - Modified Histone H1C

NKKAASGEAK PKAKKAGAAK AKKPAGATPK KAKKAAGAKK AVKKTPKKAK KPAAAGVKKV AKSPKKAKAA AKPKKATKSP AKPKAVKPKA AKPKAAKPKA AKPKAAKAKK AAAKKK

SEQ ID NO.8 - Modified Histone H2AN

MSGRGKQGGK ARAKAKTRS S RAGLQFPVGR VHRLLRKGNY AERVGAGAPV YLAAVLEYLT AE

SEQ ID NO.9 - Modified Histone H2AC

EILELAGNA ARDNKKTRII PRHLQLAIRN DEELNKLL GK VTIAQGGVLP NIQAVLLPKK TESHHKAKGK

SEQ ID NO.10 - Modified Histone H2BN

MPEPAKSAPA PKKGSKKAVT KAQKKDGKKR KRSRKESYSI YVYKVLKQVH PDTGISSKAM GIM

SEQ ID NO.11 - Modified Histone H2BC

YNKRSTI TSREIQTAVR LLLPGELAKH AVSEGTKAVT KYTSSK

SEQ ID NO.12 - Modified Histone H3N

M ARTKQT ARK STGGKAPRKQ LATKAARKSA PATGGVKKPH RYRPGTVALR EIRRYQKSTE LLIRKLPFQR LVRE

SEQ ID NO.13 - Modified Histone H3C

IAQDFK TDLRFQSSAV MALQEACEAY LVGLFEDTNL CAIHAKRVTI MPKDIQLARR IRGERA

SEQ ID NO.14 - Modified Histone H4N

SGRGKGGKG LGKGGAKRHR KVLRDNIQGI TKPAIRRLAR R

SEQ ID NO.15 - Modified Histone H4C

IYEETRGVLK VFLENVIRDA VTYTEHAKRK TVTAMDVVYA LKRQGRTLYG FGG

SEQ ID NO.16 - Modified Histone H1M

GLSLAALKAALAAGGYDVEKNNSRIKLGLKSLVSKGTLVQTKGTGASGSFKLNKAAASGEAKPKAKAAGAAKAA

KPAGATPKAAAKAAGA

SEQ ID NO.17 - Modified Histone H3M

GLPFQRLVREIAQDFKTDLRFQSSAVMALQEACEAYLVGLFEDTNLCAIHAKRVTIMPKDIQLARRIRGERA

Claims

1. A histone protein or modified histone protein, for use in inhibiting complement activity.

2. The histone protein or modified histone protein according to claim 1 , wherein the histone protein or modified histone protein is a wildtype histone protein or variant thereof.

3. The histone protein or modified histone protein according to claims 1 or 2 wherein the histone protein is selected from histone H1 , histone H2A, histone H2B, histone H3, and histone H4, or variants thereof.

4. The histone protein or modified histone protein according to any of claims 1-3 wherein the histone protein is at least 70% identical to one of the following sequences: SEQ ID NO.1 , SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5.

5. The histone protein or modified histone protein according to claim 1 , wherein the histone protein is modified.

6. The modified histone protein according to claim 5, wherein the modified histone protein has reduced cytotoxicity in comparison with a corresponding wild-type histone protein.

7. The modified histone protein according to claim 5 or 6, wherein the modified histone protein has been modified to remove one or more cytotoxic portions of its amino acid sequence.

8. The modified histone protein according to any of claims 5-7 wherein the modified histone protein has been modified to remove at least a part of the N-terminal and/or C-terminal region.

9. The modified histone protein according to any of claims 5-8 wherein the modified histone protein is a fragment of a wild-type histone protein.

10. The modified histone protein according to claim 9, wherein the fragment consists of approximately 20%-95%, or 30%-90%, or 40% to 60%, or 45% to 55% of the total length of the wild-type histone amino acid sequence.

11. The modified histone protein according to any of claims 5-9, wherein the modified histone protein is a truncated histone protein lacking a part of the C-terminal region.

12. The modified histone protein according to claim 11 , wherein the modified histone protein consists of amino acids 1-120, 1-110, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, or 1-40 of a wild-type histone amino acid sequence, and any integer value therebetween.

13. The modified histone protein according to claims 11 or 12, wherein the modified histone protein is a truncated histone protein lacking a part of the C-terminal region of SEQ ID NO.1 , SEQ ID NO.4 SEQ ID NO.3, SEQ ID NO.4, or SEQ ID NO.5.

14. The modified histone protein according to claims 11-13, wherein the modified histone protein is a truncated histone protein lacking amino acids 1-120, 1-110, 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40 or any integer value therebetween of SEQ ID NO.1 , SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, or SEQ ID NO.5.

15. The modified histone protein according to any of claims 5-10, wherein the modified histone protein is a truncated histone protein lacking a part of the N-terminal region.

16. The modified histone protein according to claim 15, wherein the modified histone protein consists of amino acids 40 onwards, 50 onwards, 60 onwards, 70 onwards, 80 onwards, 90 onwards, 100 onwards, 110 onwards, or 120 onwards of a wild-type histone amino acid sequence, and any integer value therebetween.

17. The modified histone protein according to claims 15 or 16, wherein the modified histone protein is a truncated histone protein lacking a part of the N-terminal tail region of SEQ ID NO.1 , SEQ ID NO.4 SEQ ID NO.3, SEQ ID NO.4, or SEQ ID NO.5.

18. The modified histone protein according to claims 15-17, wherein the modified histone protein is a truncated histone protein lacking amino acids 40 onwards, 50 onwards, 60 onwards, 70 onwards, 80 onwards, 90 onwards, 100 onwards, 110 onwards, 120 onwards or any integer value therebetween of SEQ ID NO.1 , SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, or SEQ ID NO.5.

19. The modified histone protein according to any of claims 5-18, wherein the modified histone protein comprises an amino acid mutation to remove a positively charged amino acid residue.

20. The modified histone protein according to claim 19, wherein the positively charged amino acid residue is removed by substitution.

21. The modified histone protein according to claim 20, wherein the positively charged amino acid residue is substituted with a neutral or negatively charged amino acid residue.

22. The modified histone protein according to claims 19-21 , wherein the modified histone protein comprises up to 50, up to 40, up to 30, up to 20, up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or 1 amino acid substitutions when compared to a corresponding wild type histone protein amino acid sequence.

23. The modified histone protein according to claims 19-22, wherein the positively charged amino acid residue is located in an exposed part of the histone protein structure.

24. The modified histone protein according to claims 19-23, wherein the positively charged amino acid residue is located in the N-terminal tail region of the histone protein.

25. The modified histone protein according to any of claims 5-24 wherein the modified histone proteins have a cytotoxicity of less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, less than 8%, less than 5%, less than 3%, suitably less than 1%, as compared to a corresponding wild-type histone.

26. The modified histone protein according to any of claims 5-25 wherein the modified histone protein is non-cytotoxic.

27. The histone protein or modified histone protein according to any preceding claim, wherein the histone protein or modified histone protein inhibits complement activity by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%.

28. The histone protein or modified histone protein according to any preceding claim, wherein the histone protein or modified histone protein inhibits complement activity by inhibiting the whole or a part of any of the complement pathways selected from the classical pathway, the lectin pathway, and the alternative pathway, or any combination thereof.

29. The histone protein or modified histone protein according to any of claims 1-28, wherein the histone protein or modified histone protein inhibits the classical pathway monospecifically and is preferably selected from SEQ ID NO. 6.

30. The histone protein or modified histone protein according to any of claims 1-28, wherein the histone protein or modified histone protein inhibits the lectin pathway monospecifically and is preferably selected from SEQ ID NO. 8.

31. The histone protein or modified histone protein according to any of claims 1-28, wherein the histone protein or modified histone protein inhibits the classical and lectin pathway bispecifically and is preferably selected from SEQ ID NO.7, SEQ ID NO.10, SEQ ID NO.12, SEQ ID NO. 1 , SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5.

32. The histone protein or modified histone protein according to any of claims 1-28, wherein the histone proteins or modified histone protein inhibits the classical, lectin and alternative pathway trispecifically and is preferably selected from SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO. 13.

33. The histone protein or modified histone protein according to any of claims 1-32, wherein the histone protein or a modified histone protein is for use in inhibiting complement activity in a cell culture media component.

34. A modified histone protein for use in the inhibition of complement activity in the treatment or prevention of a disease associated with enhanced complement activity, wherein the modified histone protein has reduced cytotoxicity as compared to a corresponding wild-type histone protein.

35. A modified histone protein for the use according to claim 34, wherein the modified histone protein is as defined in any of claims 6-33.

36. A modified histone protein for the use according to claim 35, wherein the disease associated with enhanced complement activity is acquired or genetic.

37. A modified histone protein for the use according to claims 35 or 36 wherein the disease associated with enhanced complement activity is selected from any of the following classes: kidney diseases; autoimmune diseases; degenerative diseases; transplant induced diseases; transfusion induced diseases; dialysis induced diseases; systemic inflammatory diseases.

38. A modified histone protein for the use according to any of claims 35-37, wherein the disease associated with enhanced complement activity is selected from: glomerulopathy, atypical haemolytic uremic syndrome, alzheimer’s disease, age- related macular degeneration, paroxysmal nocturnal haemoglobinuria, systemic lupus erythematosus, antiphospholipid antibody syndrome, myasthenia gravis, ANCA-associated vasculitis, auto-immune haemolytic anaemia, primary cold agglutinin disease, secondary cold agglutinin syndrome (which may be associated with malignant and acute infections), paroxysmal cold haemoglobinuria, antibody- induced rejection after transplantation, graft vs. host disease, ischaemia-reperfusion injury (IRI), thrombotic microangiopathies, and haemodialysis-related complications.

39. A method of inhibiting complement activity in a cell culture media component comprising the following steps:

(a) Contacting a cell culture media component with a histone protein or a modified histone protein.

40. The method according to claim 39, wherein the histone protein or modified histone protein is as defined in any of claims 2-33.

41. The method according to claims 39 or 40, wherein the cell culture media component is contacted with a histone protein or modified histone protein in the presence of cells or in the absence of cells.

42. The method according to claim 41 , wherein the cell culture media component is contacted with a histone protein in the presence of cells and the histone protein is modified.

43. The method according to clam 42, wherein the method comprises the addition of an effective amount of the modified histone protein to a cell culture.

44. The method according to claim 43, wherein the cell culture media component is contacted with a histone protein in the absence of cells and the histone protein is wild-type or modified.

45. The method according to claim 44, wherein the method comprises passing cell culture media component over the histone protein.

46. The method according to any of claims 39-45, wherein contacting the cell culture media component and the histone protein or modified histone protein is for a sufficient time to allow the histone protein to inhibit complement activity.

47. The method according to clam 46, wherein the cell culture media component is contacted with a histone protein or a modified histone protein for at least 5 minutes, at least 10 minutes, at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes.

48. The method according to claims 46 or 47 wherein the cell culture media component is contacted with a histone protein or a modified histone protein for less than 1 hour.

49. A modified histone protein wherein the modified histone protein is at least 70% identical to one of the following sequences: SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO. 6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 , SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, and SEQ ID NO.15.

50. A modified histone protein according to claim 49, wherein the modified histone protein consists of one of the following sequences: SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO. 6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 , SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, and SEQ ID No.15.

51. A modified histone protein wherein the modified histone protein is at least 70% identical to one of the following sequences: SEQ ID NO.1 , SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, and SEQ ID NO.5.

52. An expression vector comprising a nucleic acid encoding a modified histone protein according to any of claims 49-51.