[0001] Novel applications for Staphylococcus aureus Sbi protein
[0002]Field of tlie Invention
[0003]The invention relates to the Sbi protein of Staphylococcus aureus, and in particular to newly-identified functions of the Sbi protein which open up new methods for regulating the immune system, treatment of S. aureus infection, and identifying new compounds for treatment of S. aureus infection.
[0004]Background to the Invention
[0005]Staphylococcus aureus is a member of the human commensal flora that, when host mucosal and/or immune defences are weakened, causes numerous diseases associated with pneumonia and sepsis. The ability of S. aureus to evade the host's adaptive immune response has long been recognised1. Cell wall-associated Protein A (SpA) , for instance, binds immunoglobulin G Fc fragment, thereby blocking phagocytosis, and it interacts with certain Fab fragments, thus characterising SpA as a B-cell superantigen2'3.
[0006]The importance of the immunoglobulin binding activity mediated by SpA to S. aureus virulence is unclear. In S. aureus mutants with an inactivated SpA gene, virulence is only slightly impaired compared to wild-type strains10'n.
[0007]Recently, a second staphylococcal immunoglobulin-binding protein has been identified, designated Sbi8. It is present in many S. aureus strains (including MSSA and MRSA strains), and may therefore help to clarify the role of immunoglobulin binding in staphylococcal virulence. Sbi is a 436 amino acid cell surface protein that contains one functional immunoglobulin-binding domain and a second predicted immunoglobulin-binding motif, both with sequence similarity to the five immunoglobulin-binding repeats (E, A, B, C and D) of SpA (see Figure Ia and Ib) , but no other significant sequence similarity to known proteins. In addition, Sbi has been shown to bind another plasma component,
adhesion protein β2-glycoprotein I (β2-GPI)8'9, a membrane protein which has been implicated in blood coagulation9'12.
[0008]Summary of the Invention The inventors have found that the S. aureus Sbi protein is able to bind the C3 complement protein and various proteolytic fragments thereof. It is also able to inhibit the three pathways of the complement system, namely the classical pathway, alternative pathway, and the lectin pathway (also known as the mannose-binding lectin pathway, or MBLP) . Without wishing to be bound by any particular theory, it is believed that the ability to inhibit the complement system is a result of its ability to bind C3 protein.
[0009]This finding has clear implications for the pathogenesis and virulence of S. aureus. It also suggests new methods and compositions for regulating the immune system.
[0010]In a first aspect, the present invention provides a method for inhibiting complement activation in a biological system, comprising contacting the system with a complement-binding protein comprising a Sbi-III domain capable of binding to C3 protein and/or a Sbi-IV domain capable of binding to C3 protein.
[0011]The method may be applied in any appropriate biological system containing components of one or more of the classical, alternative or lectin complement pathways, whether in vivo, ex vivo or in vitro.
[0012]If it is desirable to inhibit activation of all three complement pathways, or there is no need to inhibit one pathway in preference, to the others, the complement-binding protein may comprise a Sbi-III domain capable of binding to C3 protein.
The complement-binding protein may additionally comprise a Sbi-IV domain capable of binding to C3 protein. For example, it may comprise a- Sbi-III-IV polypeptide.
[0013]If it is desirable specifically to inhibit the alternative pathway, the complement-binding protein may comprise a Sbi-IV domain capable of binding to C3 protein, in the absence of a functional Sbi-III domain.
[0014]An in vitro system may comprise an isolated biological sample, such as a blood, plasma or serum sample, or a fraction thereof. Alternatively, the system may be assembled in vitro from individual components such as isolated proteins (e.g. recombinant proteins), cells (which may be isolated from tissue, or grown in culture), etc..
[0015]In general, the system will contain complement protein C3. Other components of the complement system (possibly including inhibitors) will also be present, but (at least in vitro) the precise components present may depend on the particular complement pathway under examination and the assay being performed. Components of the alternative pathway include C3, properdin, Factor B, Factor D, Factor H and Factor I. Components of the classical pathway include CIq, CIr, CIs, C2, C4 and C3. The lectin pathway will typically include mannose-binding protein (MBP) or ficolin, and the proteases MASP-I and MASP-2.
[0016]The system may also comprise a stimulus to initiate the complement cascade and also a target for lysis, opsonisation and/or phagocytosis, which may be a cell, liposome, virus, protein, or other appropriate component. The effects exerted on the target may provide a suitable read-out in an assay for complement activation. Suitable "target" cells may be prokaryotic or eukaryotic and include microorganisms such as bacteria, or erythrocytes, which are commonly used in assays for
complement function and complement activators. The stimulus and the target may be the same or different.
[0017]Where the system comprises components of the classical pathway, one or more antibodies may be present. For study of the lectin pathway, a source of mannose or other carbohydrate bound by MBP or ficolin may be present. For study of the alternative pathway, lipopolysaccharides (LPS) may be present.
[0018]Additionally or alternatively the system may contain "responder" cells capable of responding to one or more products of complement activation, such as anaphylatoxins (C3a and C5a) or opsonised targets (which may or may not be cells) carrying opsonins such as C3b. Such "responder" cells are typically cells of the immune system and include basophils, neutrophils, mast cells, and macrophages .
[0019]The invention also provides a method of inhibiting C3a anaphylatoxin activity in a biological system, comprising contacting the system with a complement-binding protein comprising a Sbi-IV domain capable of binding to C3 protein.
[0020]If desired, the complement-binding protein may further comprise a Sbi-III domain capable of binding to C3 protein. For example, it may comprise a Sbi-III-IV polypeptide. However, use of the Sbi- IV domain alone may be preferred.
[0021]This method may be applied in isolation, or in conjunction with a method of inhibiting complement activation. In such cases, complement activation may be inhibited using a complement binding protein as described herein comprising a Sbi-III domain or a fragment or derivative thereof capable of binding to C3 protein.
[0022]The methods of the invention may also be applied in vivo in situations where complement is activated inappropriately, to an
excessive degree, or in an otherwise undesirable manner. Such complement activation may play a- role in the pathogenesis or symptoms of any inflammatory condition. An inflammatory condition may be considered to be any condition in which activation of the immune system (whether the innate immune system, acquired immune system, or both) is responsible for or contributes to pathogenesis or symptoms of the condition, either directly or indirectly.
[0023]Thus the invention provides a method of treating an inflammatory condition in an individual, comprising administering a complement-binding protein comprising a Sbi-III domain capable of binding to C3 protein and/or a Sbi-IV domain capable of binding to C3 protein to said individual. The complement-binding protein may comprise a Sbi-III-IV polypeptide.
[0024]The invention also provides use of a complement-binding protein comprising a Sbi-III domain capable of binding to C3 protein and/or a Sbi-IV domain capable of binding to C3 protein in the preparation of a medicament for the treatment of an inflammatory condition. The complement-binding protein may comprise a Sbi- III-IV polypeptide.
[0025]Conditions in which complement has been specifically identified as contributing to pathogenesis or symptoms include conditions characterised by circulating immune complexes or deposition of immune complexes in tissues such as rheumatoid arthritis (RA) and systemic lupus erythematosis (SLE), lupus nephritis, ischemia- reperfusion injury and post-ischemic inflammatory syndrome (e.g. renal, intestinal and myocardial reperfusion injury) , systemic inflammatory response syndrome (SIRS) and acute respiratory distress syndrome (ARDS) , septic shock, trauma, burns, acid aspiration to the lungs, immune-mediated diseases of the kidney and the eye (including the atypical form of haemolytic uretic syndrome (HUS), membrane proliferative glomerulonephritis (MPGN),
IgA nephropathy and age-related macular degeneration (ARMD) ) , inflammatory and degenerative diseases of the nervous system such as multiple sclerosis (MS) and Alzheimer's disease, arteriosclerosis, transplant rejection, inflammatory complications following cardiopulmonary bypass and haemodialysis, antiphospholipid syndrome, asthma, and spontaneous fetal loss. For reviews see Thurman and Holers, J. Immunol. 176: 1305-1310 (2006); Seelen et al., Journal of Nephropathy 18(6): 642-653 (2005) .
[0026]In certain conditions, it may be possible to achieve significant therapeutic benefit by specifically inhibiting the alternative pathway in preference to the classical pathway and lectin pathway.
[0027]For example, in some of the above conditions, alternative pathway activation is believed to contribute much more significantly to pathogenesis or symptoms than activation of the other pathways. Such conditions include ischemia-reperfusion injury (e.g. renal ischemic injury, such as acute tubular necrosis) , trauma, sepsis, the atypical form of haemolytic uretic syndrome (HUS) , membrane proliferative glomerulonephritis (MPGN) , age-related macular degeneration (ARMD) , rheumatoid arthritis (RA) , systemic lupus erythematosis (SLE) , lupus nephritis, antiphospholipid syndrome, asthma, and spontaneous fetal loss.
[0028]It may be desirable to treat these conditions using a complement- binding protein comprising an Sbi-IV domain capable of binding to C3 protein, in the absence of a functional Sbi-III domain. This strategy could avoid unnecessary inhibition of the other
[0029]' complement pathways, so maintaining as much of the patient's normal immune function as possible.
[0030]The anaphylatoxin C3a exerts powerful pro-inflammatory activity. Thus in many inflammatory disorders, it may be desirable to
inhibit C3a anaphylatoxin activity. This may be achieved using a complement-binding protein Comprising an Sbi-IV domain capable of binding to C3 protein. Again, it may be desirable that' the complement-binding protein lacks a functional Sbi-III domain, so as to maintain as much of the patient' s normal immune function as possible. Inflammatory conditions treatable in this manner include airway hyper-responsiveness.
[0031]The complement receptor 2 (CR2, also designated CD21) is present on the surface of B cells and follicular dendritic cells, and binds to complexes of antigen with C3d or C3dg. This interaction stimulates B cell proliferation and antibody production and may enhance B cell responses to low levels of antigen which might not otherwise stimulate B cell responses.
[0032]Thus the complement binding proteins described herein may be used to inhibit this interaction between CR2 and C3d/C3dg. The invention therefore provides a method of inhibiting B cell proliferation and/or antibody production in an individual, comprising administering a complement-binding protein comprising a Sbi-III domain capable of binding to C3 protein to said individual.
[0033]The invention further provides the use of a complement-binding protein comprising a Sbi-III domain capable of binding to C3 protein in the preparation of a medicament for inhibiting B cell proliferation and/or antibody production.
[0034]The complement-binding protein may additionally contain a Sbi-IV domain capable of binding to C3 (for example, the protein may comprise a Sbi-III-IV polypeptide) , or it may lack a functional Sbi-IV domain.
[0035]Inhibition of B cell proliferation and/or antibody production may be useful in any inflammatory condition in which antibody
production contributes to the pathogenesis or symptoms experienced. These include the inflammatory conditions set out above .
[0036]The interaction between C3d/C3dg and CR2 has also been implicated in HIV infection of CD4+ T cells. C3d/C3dg bound to HIV virions is thought to interact with CR2 on B cells and follicular dendritic cells and mediate transfer of the virion to CD4 T cells (Dδpper et al., Eur. J. Immunol. 2003, 33:2098-2107). Without wishing to be bound by any particular theory, inhibiting the C3d/C3dg interaction with CR2 may therefore be of use in treatment of HIV by limiting infection of T cells.
[0037]Thus the invention provides a method of treatment or prophylaxis of HIV infection in an individual comprising administering a complement-binding protein comprising a Sbi-III domain capable of binding to C3 protein to said individual.
[0038]The invention further provides the use of a complement-binding protein comprising a Sbi-III domain capable of binding to C3 protein in the preparation of a medicament for treatment or prophylaxis of HIV infection.
[0039]The complement-binding protein may additionally contain a Sbi-IV domain capable of binding to C3 (for example, it may comprise a Sbi-III-IV polypeptide) , or it may lack a functional Sbi-IV domain .
[0040]The finding that Sbi interacts with C3 and inhibits complement activation suggests that this may be a mechanism by which S. aureus evades the innate immune system. Inhibiting this interaction may therefore provide a means to treat S. aureus infection. It may also be possible to use inhibitors of the interaction prophylactically, to reduce the ability of S. aureus to establish an infection.
Thus in a further aspect the invention provides a method of prophylaxis or treatment of S. aureus infection, comprising administering an inhibitor of the interaction between C3 and Sbi.
[0041]The invention further provides the use of an inhibitor of the interaction between C3 and Sbi in the preparation of a medicament for prophylaxis or treatment of S. aureus infection.
[0042]Typically, the inhibitor will be a substance capable of binding to Sbi and inhibiting binding between Sbi and C3. For example, the inhibitor may be an antibody specific for Sbi protein and capable of inhibiting its interaction with C3. The antibody may bind to any epitope on Sbi as long as it possesses the required inhibitory activity, but will typically bind to an epitope on Sbi-III, an epitope on Sbi-IV, or an epitope on Sbi-III-IV.
[0043]It may be desirable to use combinations of antibodies. For example, in certain embodiments at least a first antibody specific for Sbi-III which is capable of preventing Sbi-III binding to C3 may be administered in conjunction with a second antibody specific for Sbi-IV which is capable of preventing Sbi- IV binding to C3. The two antibodies may be administered together or separately, in the same or different compositions.
[0044]The invention also provides methods by which compounds may be screened for an ability to inhibit the interaction between Sbi and C3. As described above, such compounds may have therapeutic utility in the treatment or prophylaxis of S. aureus infection.
[0045]Thus in a further aspect, there is provided a method of testing a candidate compound for an ability to inhibit the interaction between Sbi and C3, comprising contacting the candidate compound with (i) C3 protein or a fragment thereof; and
(ii) a complement-binding protein comprising a Sbi-III domain capable of binding to said C3 protein or fragment thereof and/or a Sbi-IV domain capable of binding to said C3 or fragment thereof; and determining binding between (i) and (ii) .
[0046]When the complement-binding protein comprises Sbi-III, the C3 protein or fragment preferably comprises the C3d or C3dg fragment. Examples include C3, C3(NHCH3), C3 (H20) , C3b, iC3b, iC3(NHCH3), iC3 (H2O) , C3d and C3dg.
[0047]When the complement-binding protein comprises Sbi-IV, the C3 protein or fragment preferably comprises the C3a fragment. Examples include C3, C3(NHCH3), C3 (H20) , iC3(NHCH3), iC3 (H2O) and C3a. See Figure 8 and the Examples for further illustration.
[0048]However it will be understood that if the complement-binding protein comprises both Sbi-III and Sbi-IV (e.g. if it comprises a Sbi-III-IV polypeptide) , the C3 protein or fragment may preferably comprise either the C3d/C3dg fragment and/or the C3a fragment .
[0049]For the purposes of the screening method, either the C3 protein or fragment or the complement-binding protein is preferably immobilised on a solid phase, and contacted with a sample containing the other protein. The candidate compound under test may be introduced before, concurrently with, or after the sample containing the other protein component.
[0050]Any suitable format may be used for the assay, including high- throughput formats .
[0051]The invention further provides methods for detection or isolation of C3 and fragments thereof, e.g. in or from biological samples.
Thus in a further aspect there is provided a method of detecting C3 or a fragment thereof in a sample, or isolating C3 or a fragment thereof from a sample, comprising contacting said sample with a complement-binding protein comprising a Sbi-III domain capable of binding to said C3 or fragment thereof and/or a Sbi-IV domain capable of binding to said C3 or fragment thereof.
[0052]The complement-binding protein is preferably immobilised on a suitable solid phase.
[0053]Typically the sample is a biological sample comprising blood, plasma or serum.
[0054]Whether a method of detecting or isolating C3 (or fragment thereof) , the method will typically comprise a step of removing any bound impurities or contaminants, e.g. by suitable washing.
[0055]A method of detecting C3 (or fragment thereof) will typically comprise the step of determining the amount of C3 bound to the complement binding protein.
[0056]A method of isolating C3 (or fragment thereof) will typically comprise a step of eluting the bound C3 or fragment from the complement-binding protein.
[0057]In a further aspect, the invention provides a complement-binding protein comprising a Sbi-III domain capable of binding C3 protein, and/or a Sbi-IV domain capable of binding C3 protein, in the absence of a functional Sbi-I domain and/or a functional Sbi- II domain.
[0058]Thus the complement-binding protein may comprise a Sbi-III domain in the absence of Sbi-IV, or may comprise a Sbi-IV domain in the absence of Sbi-III, or may comprise a Sbi-III domain and a Sbi-IV domain; for example, it may comprise a Sbi-III-IV polypeptide.
Preferably the complement-binding protein lacks both a Sbi-I domain and a Sbi-II domain. It may contain fragments of either or both of' these domains, but any sequences derived from Sbi-I or Sbi-II are preferably non-functional in that they lack the ability to bind immunoglobulin.
[0059]In preferred embodiments, the complement-binding protein is soluble, i.e. it is not anchored in a cell wall or cell membrane, and thus preferably does not contain a cell wall-anchoring sequence or cell membrane-anchoring sequence. This may facilitate recombinant expression and purification as well as use as a therapeutic agent. In preferred embodiments the protein is expressed in a suitable host cell and secreted from that cell into the culture medium from where it can be purified. Any signal sequence required for secretion may or may not be cleaved during the secretion process. However in some circumstances it may be desirable to express the protein within a host cell (e.g. within the cytoplasm, within an organelle, or within an inclusion body) and isolate it from the host cell.
[0060]A complement-binding protein as described in this specification may be associated with one or more heterologous (i.e. non-Sbi) components. The heterologous component may modulate any desired property of the protein, such as stability, activity or immunogenicity . For example, the heterologous component may be used to increase or reduce half-life in vitro or in vivo.
[0061]The heterologous component may be a non-proteinaceaous molecule chemically linked to the complement-binding protein. Examples include polyethylene glycol (PEG) , and poly-sialic acid.
[0062]Alternatively (where appropriate) the heterologous component may be expressed as a fusion protein with the complement binding protein.
In the case of fusion proteins, a flexible peptide linker is typically included between the two components to allow the two components to interact freely with one another without steric hindrance.' The skilled person is perfectly capable of designing a suitable linker. Conventionally, such linkers are between 12 and 20 amino acids in length, and have a high proportion of small and hydrophilic amino acid residues (e.g. glycine and serine) to provide the required flexibility without compromising aqueous solubility of the molecule.
[0063]Antibody hinge regions may serve as peptide linkers, and also contain cysteine residues which mediate disulphide bridge formation between between pairs of heavy chains in the intact native antibody. Thus if the hinge regions are present in the fusion proteins described herein, disulphide bonds will typically be formed between pairs of fusion proteins (under oxidising conditions), leading to covalently linked dimers .
[0064]Fusion (or chemical conjugation) to other proteins such as albumin may also be useful to extend half-life in vivo.
[0065]The invention further provides a nucleic acid encoding a complement-binding protein comprising a Sbi-III domain capable of binding C3 protein, and/or a Sbi-IV domain capable of binding C3 protein, in the absence of a functional Sbi-I domain and/or a functional Sbi-II domain.
[0066]The encoded protein may comprise a Sbi-III domain in the absence of Sbi-IV, or may comprise a Sbi-IV domain in the absence of Sbi- III, or may comprise a Sbi-III domain and a Sbi-IV domain. For example, it may comprise a Sbi-III-IV polypeptide. Preferably the encoded protein lacks both a Sbi-I domain and a Sbi-II domain.
The nucleic acid may encode a fusion protein comprising the complement-binding protein and a heterologous component (and any intervening linker sequence), as described above.
[0067]The invention further provides a vector (e.g. an expression vector) comprising a nucleic acid of the invention.
[0068]The invention also provides a host cell comprising a nucleic acid or a vector of the invention. The host cell may be prokaryotic or eukaryotic as desired.
[0069]The invention also provides a complement-binding protein, nucleic acid, vector or host cell as described above for use in a method of medical treatment.
[0070]The invention further provides a pharmaceutical composition comprising a complement-binding protein, nucleic acid, vector or host cell as described above and a pharmaceutically acceptable carrier.
[0071]The invention further provides" a complement-binding protein comprising a Sbi-III domain capable of binding to C3 protein and/or a Sbi-IV domain capable of binding to C3 protein, or a nucleic acid encoding the same, or a vector or host cell containing such a nucleic acid, for use in a method of medical treatment. The complement-binding protein may comprise a Sbi-III domain in the absence of Sbi-IV, or may comprise a Sbi-IV domain in the absence of Sbi-III, or may comprise a Sbi-III domain and a Sbi-IV domain. For example, it may comprise a Sbi-III-IV polypeptide.
[0072]The invention will now be described in more detail, by way of example and not limitation, by reference to the accompanying drawings .
Description of the Figures
[0073]Figure Ia. Schematic drawing of the domain structure of Sbi compared with the structure of SpA. Indicated are the positions of the signal peptide sequence (S) , ligand binding domains (SpA: E, D, A, B and C; Sbi: predicted domains I, II, III and IV), cell wall-spanning regions (Wr and Wc) and membrane spanning region M. The position of the cell wall-anchoring LPXTG motif in SpA is indicated. The predicted cell wall-spannning proline-repeat region (Wr) in Sbi8 is also shown, as is the C-terminal tyrosine- rich region (Y) , which has been implicated in IgG-mediated signal transduction30. Ib Amino acid sequence alignment of SpA' s five immunoglobulin-binding domains E, D, A, B, C and Sbi-I and Sbi- II. The entire sequence of SpA-D is given and identical amino acids in o.ther domains are depicted with a dot (.) . Positions of the three helices in the domain structure of SpA-D are overlaid. Fc-interacting residues are indicated with dashed hatching and Fab-interacting SpA-D residues are indicated with cross-hatching. Amino acid numbering follows the Sbi-I sequence.
[0074]Figure 2a. Experimental scattering curve of Sbi-E with error bars (1) and the scattering from the ab in±tio model (2) . The plot displays the logarithm of the scattering intensity as a function of momentum transfer s. The distance distribution function of Sbi-E calculated from the experimental data by the program GNOM is presented in the inset figure. 2b. A typical ab initio bead model of Sbi-E determined by DAMMIN14. The 4 major globular domains are depicted with Roman numerals. A homology model of Sbi domains I and II (shown in surface and ribbon representations) is superimposed onto the ab initio SAXS structure.
[0075]Figure 3a. Double immunodiffusion assay with recombinant SpA and Sbi against human serum IgG and IgG Fc fragment. The spur of precipitation, indicating partial identity between the IgG binding epitopes of SpA and Sbi, is indicated by an arrow. 3b.
Surface plasmon resonance analysis of the binding of human IgG subclasses IgGl, IgG2, IgG3 and IgG4 by monovalent Sbi~I and Sbi- II domains .
[0076]Figure 4a. Serum affinity pull-down assay with Sbi-III-IV. Shown in lane 1 is the SDS-PAGE analysis of the human serum protein component polypeptide chains (prey proteins) eluted from an NHS-sepharose column with immobilised Sbi-III-IV as bait protein. Lane M shows a protein ladder of molecular weight markers. Gel bands yielding peptide fragments of complement component C3, identified by MALDI-TOF analysis, are indicated with asterisks. Also indicated is the predominant chain-origin of the C3 peptide fragments. 4Jb. SDS-PAGE analysis of an affinity pull-down assay with human serum comparing Sbi-III-IV (lane 3) with NHS-sepharose immobilised Sbi-I (lane 1) , Sbi-III (Iane2) and Sbi-IV (lane 4) . 4c. SDS-PAGE analysis comparing affinity pull-down assays with human serum (lane 1) and sera from cow (lane 2), calf (lane 3), chicken (lane 4), goat (lane 5), horse (lane 6) , mouse (lane 7) and rabbit (lane 8) , using NHS- immobilised Sbi-III-IV in all cases.
[0077]Figure 5a. Sensorgrams showing the relative binding between sensorchip imobilised Sbi-III-IV (-1200 RU) and complement C3 derivatives: native 03, C3b, iC3b, C3c, C3dg, methylamine-reacted C3 (03(NHCH3)), iC3 (NHCH3) and trypsinised 03 (NHCH3) (denoted
[0078]"C3b") (see Figure 8 for a diagrammatic representation of each C3 derivative) . The experiment was done at physiologic ionic strength. To the right of the graph, the complement fragments are listed in decreasing order corresponding to the order of peak heights in the graph. 5Jb. Sensorgrams showing the relative binding between sensor chip-immobilised Sbi-III-IV (-1800 RU) , Sbi-III (-800 RU) and Sbi-IV (-800 RU) with the complement C3 derivatives: native C3, C3b, iC3b, C3c, C3dg, methylamine-reacted 03 (03(NHCH3)), iC3 (NHCH3) and trypsinized C3(NHCH3) (denoted as "C3b") under physiologic NaCl concentration. 5c. Steady state
analysis of the interaction of C3dg with sensor chip-bound Sbi- III-IV (-1200 RU) . RU values determined from the steady state plateau region were plotted as a function of analyte (C3dg) concentration, and the data were fit by nonlinear regression according to the single site Langmuir binding model. 5d Results from complement inhibition assays for the classical (CP) , mannose-binding lectin (MBLP) and alternative (AP) complement pathways with constructs Sbi-E, Sbi-I, Sbi-III-IV and Sbi-IV. Complement activity of human serum pre-incubated with the Sbi fragment for 30 min at 37 °C. The activity detected for the positive control (human serum) in all the three pathways was set at 100%.
[0079]Figure 6. Schematic representation of the Sbi constructs used in the experiments described in this paper. The engineering of the Sbi-E, Sbi-I and Sbi-II constructs is based on sequence homology with SpA. The boundaries of Sbi-IV are based on the minimal β2- GPI domain identified by Zhang et al.9
[0080]Figure 7. Experimental scattering curve of Sbi-E (open circles) and the scattering from the model obtained with DAMMIN (solid line) . The plot displays the logarithm of the scattering intensity as a function of momentum transfer s = 4π sin(θ)/λ where 2Θ is the scattering angle and λ = 1.5 A is the X-ray wavelength.
[0081]Figure 8. Diagrammatic representation of C3 derivatives used in this study showing both their polypeptide chain composition and the state of the side chains involved in the C988-Q991 thioester bond. Dark grey shading denotes the C3a domain, light grey shading the C3d domain and hatched box the "g" segment of the C3dg fragment. The molecular masses of the constituent chains are indicated. Methylamine treatment of native C3 results in a g-glutamylmethyamide adduct of the Q991 side chain carbonyl
originally part of the intramolecular thioester bond with the sulphydryl of C988.
[0082]Figure 9. Inhibition of the binding of C3dg to CR2 by Sbi-III-IV. Shown is the displacement of a standard C3dg saturation curve (closed circles) binding to a sensordisc with immobilised CR2 (~1500RU) in the presence of a constant concentration (5 μM) of Sbi-III-IV as co-analyte (open circles) . Aλlag phase' in the saturation curve is observed when the molar ratio of C3dg to Sbi- III-IV is less than 1:1. At C3dg concentrations with a molar excess over Sbi-III-IV, the maximal saturation level overlaps with the standard C3dg binding curve, indicating that the inhibitory effect of Sbi-III-IV is due to direct binding to C3dg as opposed to an interaction between Sbi-III-IV and CR2.
[0083]Figure 10. Sensorgrams of (a) Sbi-III-IV binding to C3a- immobilised sensordiscs, compared to (b) a sham-activated and deactivated sensordisc. The net response, after subtracting the a-specific binding from the specific binding is shown in (c) . 1Od shows a plot of the RU values at the end of the injection phase versus the concentration of the analyte (Sbi-III-IV) . The data were fit by nonlinear regression according to a single site Langmuir binding model, yielding a KΛ value of 5χlO4 M"1.
[0084]Figure 11. Phosphoimage of SDS-PAGE of12SI-C3 spiked serum samples incubated with various Sbi constructs (Sbi-E, Sbi-III-IV, Sbi-III and Sbi-IV) . Also shown are sham-activated serum samples (no Sbi fragment added) , heat-aggregated IgG positive control, and standards for C3 and its break-down products C3b, iC3b and C3 after incubation with factor H and I. The positions of the various C3 α and β chain break-down products are indicated
Figure 12 Phosphoimage of SDS-PAGE of12SI-C3 spiked serum samples incubated with Sbi . constructs Sbi-E and Sbi-III-IV showing a complement activation time course.
[0085]Detailed Description of the Invention Complement
[0086]The complement system is a crucial part of the innate immune system and consists of a group of approximately 20 proteins, mostly found in the serum. When the system is activated, a cascade of sequential enzyme activation takes place, in which the product of one reaction is itself an enzyme which catalyses the next stage of activation. The cascade thus contains a number of points at which exponential signal amplification occurs, potentially resulting in a massive response from a very small initial stimulus .
[0087]The system has three known activation mechanisms, referred to as the classical pathway, the alternative pathway, and the lectin pathway. In simple terms, these three pathways converge into a common downstream effector pathway. Activation by any one of the three mechanisms has three main effects. Firstly, it results in generation of small protein fragments called anaphylatoxins, which serve as chemoattractants to recruit immune cells to the site of activation. Anaphylatoxins include the components C3a and C5a. Secondly, foreign substances (such as microorganisms, viruses, etc.) which trigger the complement cascade are marked for destruction by coating with so-called opsonins, which become covalently bound to hydroxyl and amine groups on the foreign surface. These opsonins (which include C3b) are recognised by phagocytic cells such as neutrophils. Thirdly, a complex called the membrane attack complex (MAC) , comprising the complement components C5b-C9, may be formed in the membrane of foreign cells leading to membrane lysis. This involves cleavage of C5 (into C5a and C5b) and recruitment of C6, C7, C8 and C9 to form the complex itself.
The protein C3 is a crucial component of all three complement activation pathways. In its intact form it consists of an alpha chain and a beta chain linked by a disulphide bridge. The alpha chain contains an unusual thioester bond which can be cleaved by hydrolysis,, or by nucleophilic attack from a suitable group on the surface of a foreign substance (or "target") such as an invading microorganism. This cleavage event is an important part of the activation process.
[0088]C3 is cleaved at a number of sites during the complement activation process, giving rise to various proteolytic fragments including G3a, C3b, iC3b, C3c and C3dg. These are illustrated in Figure 7. C3a is an anaphylatoxin. C3b is an opsonin and also participates in the formation of an enzyme capable of further C3 cleavage (a "C3 convertase") . iC3b is an inactivated form of C3b formed when C3b is cleaved by a control protein which prevents excessive activation of the complement cascade. C3c and C3dg are further downstream cleavage products of iC3b.
[0089]Thus, activation of complement at a given site typically results in the generation of complement activation products such as anaphylatoxins and opsonins, which provide signals to various immune cell types here termed "responder" cells. Responder cells are primarily cells of the immune system such as basophils, neutrophils, mast cells and macrophages. Anaphylatoxins and opsonins trigger various functions in these cell types such as chemotaxis (towards the site of complement activation) , mast cell degranulation, activation of respiratory burst, phagocytosis of opsonised targets, etc..
[0090]Any system comprising C3 always shows a low level of complement activation via spontaneous hydrolysis of C3 (referred to as "tick-over" C3 activation) . However the cascade is normally kept in check by powerful regulatory mechanisms.
The complement-binding proteins described in this specification are capable of inhibiting the increased level of activation of the full enzyme cascade which may triggered by an appropriate stimulus or presence of an appropriate target, such as a microorganism (e.g. a bacterium), other cell type, virus, etc..
[0091]Thus the complement-binding proteins described herein may be used to inhibit any one or more of the effects of complement which occur as a result of C3 activation or downstream of C3 activation. These include C3 cleavage to C3a and C3b, C5 cleavage to C5a and C5b, opsonisation of targets by C3b, phagocytosis of opsonised targets, assembly of MAC, and/or lysis of target cells. Assays for determination of complement activity in vitro are described by Seelen et al.29.
[0093]The sequence of the Sbi protein, including signal seguence, is as follows :
[0094]Met Lys Asn Lys Tyr lie Ser Lys Leu Leu VaI GIy
[0096]Ala Ala Thr lie Thr Leu Ala Thr Met lie Ser Asn
[0097]15 20 GIy GIu Ala Lys Ala Ser GIu Asn Thr Gin GIn Thr 25 30 35
[0098]Ser Thr Lys His GIn Thr Thr Gin Asn Asn Tyr VaI
[0100]Thr Asp GIn GIn Lys Ala Phe Tyr Gin VaI Leu His 50 55 60
[0101]Leu Lys GIy He Thr GIu GIu Gin Arg Asn GIn Tyr
[0103]He Lys Thr Leu Arg GIu His Pro GIu Arg Ala GIn
[0104]75 80 GIu VaI Phe Ser GIu Ser Leu Lys Asp Ser Lys Asn 85 90 95
[0105]Pro Asp Arg Arg VaI Ala GIn GIn Asn Ala Phe Tyr
[0107]Asn VaI Leu Lys Asn Asp Asn Leu Thr GIu Gin GIu HO 115 120
[0108]Lys Asn Asn Tyr He Ala GIn He Lys GIu Asn Pro 125 130
Asp Arg Ser GIn GIn VaI Trp VaI GIu Ser VaI GIn
[0110]Ser Ser Lys Ala Lys GIu Arg GIn Asn lie GIu Asn 145 150 155 Ala Asp Lys Ala lie Lys Asp Phe Gin Asp Asn Lys
[0112]Ala Pro His Asp Lys Ser Ala Ala Tyr GIu Ala Asn 170 175 180
[0113]Ser Lys Leu Pro Lys Asp Leu Arg Asp Lys Asn Asn' 185 190
[0114]Arg Phe VaI GIu Lys VaI Ser lie GIu Lys Ala lie
[0116]VaI Arg His Asp GIu Arg VaI Lys Ser Ala Asn Asp 205 210 215 Ala lie Ser Lys Leu Asn GIu Lys Asp Ser lie GIu
[0118]Asn Arg Arg Leu Ala GIn Arg GIu VaI Asn Lys Ala 230 235 240
[0119]Pro Met Asp VaI Lys GIu His Leu Gin Lys GIn Leu 245 250
[0120]Asp Ala Leu VaI Ala GIn Lys Asp Ala GIu Lys Lys
[0122]VaI Ala Pro Lys VaI GIu Ala Pro GIn He GIn Ser 265 270 275 Pro GIn He GIu Lys Pro Lys VaI GIu Ser Pro Lys
[0124]VaI GIu VaI Pro GIn He GIn Ser Pro Lys VaI GIu 290 295 300
[0125]VaI Pro GIn Ser Lys Leu Leu GIy Tyr Tyr GIn Ser 305 310
[0126]Leu Lys Asp Ser Phe Asn Tyr GIy Tyr Lys Tyr Leu
[0128]Thr Asp Thr Tyr Lys Ser Tyr Lys GIu Lys Tyr Asp 325 330 335 Thr Ala Lys Tyr Tyr Tyr Asn Thr Tyr Tyr Lys Tyr
[0130]Lys GIy Ala He Asp GIn Thr VaI Leu Thr VaI Leu 350 355 360
[0131]GIy Ser GIy Ser Lys Ser Tyr He Gin Pro Leu Lys 365 370
[0132]VaI Asp Asp Lys Asn GIy Tyr Leu Ala Lys Ser Tyr
[0134]Ala Gin VaI Arg Asn Tyr VaI Thr GIu Ser He Asn 385 390 395 Thr GIy Lys VaI Leu Tyr Thr Phe Tyr Gin Asn Pro
[0136]Thr Leu VaI Lys Thr Ala He Lys Ala GIn GIu Thr 410 415 420
[0137]Ala Ser Ser He Lys Asn Thr Leu Ser Asn Leu Leu 425 430
[0138]Ser Phe Trp Lys. 435
The domain structure of this protein, as elucidated by the present inventors, is illustrated in Figure Ia.
[0139]In this specification, by "Sbi-I domain" is meant a polypeptide sequence comprising at least amino acids 42 to 94 of the Sbi sequence shown above, or a variant or fragment thereof having at least 80% sequence identity therewith. The domain may have the ability to bind immunoglobulin.
[0140]By "Sbi-II domain" is meant a polypeptide sequence comprising at least amino acids 92 to 156 of the Sbi sequence shown above, or a variant or fragment thereof having at least 80% sequence identity therewith. The domain may have the ability to bind immunoglobulin.
[0141]By "Sbi-III domain" is meant a polypeptide sequence comprising at least amino acids 150 to 196 of the Sbi sequence, a variant thereof having at least 80% sequence identity therewith which retains the ability to bind to C3 protein, or a fragment of either which retains the ability to bind C3 protein. The fragment may be at least 30, at least 35, at least 40, or at least 45 amino acids in length.
[0142]By "Sbi-IV domain" is meant a polypeptide sequence comprising at least amino acids 197 to 266 of the Sbi sequence, or a variant thereof having at least 80% sequence identity therewith which retains the ability to bind to C3 protein, or a fragment of either which retains the ability to bind C3 protein. The fragment may be at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, or at least 65 amino acids in length.
An "Sbi-III-IV" polypeptide is a polypeptide which comprises at least amino acids 150 to 266 of the Sbi protein, or a variant thereof having at least 80% sequence identity therewith.
[0143]The Sbi-III and Sbi-IV domains may bind C3 from any mammalian species, including rodents (e.g. mice, rats), lagomorphs (e.g. rabbits), felines (e.g. cats), canines (e.g. dogs), equines (e.g horses), bovines (e.g. cows), caprines (e.g. goats), ovines (e.g. sheep) , other domestic, livestock or laboratory animals, or primates (monkeys, apes or humans), but preferably binds human C3 protein.
[0144]Percent (%) amino acid sequence identity with respect to a reference sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. % identity values may be determined by WU-BLAST-2 (Altschul et al . , Methods in Enzymology, 266:460-480 (1996)). WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11. A % amino acid sequence identity value is determined by the number of matching identical residues as determined by WU-BLAST-2, divided by the total number of residues of the reference sequence (gaps introduced by WU-BLAST-2 into the reference sequence to maximize the alignment score being ignored), multiplied by 100.
[0145]Certain complement-binding proteins described in this specification possess a Sbi-III domain as defined above capable of binding C3 protein. They may additionally possess a Sbi-IV domain as defined above capable of binding C3 protein. The Sbi-
III and Sbi-IV domains may be separated by linker sequences to introduce conformational flexibility into the molecule. Alternatively the proteins may comprise a Sbi-III-IV polypeptide sequence as defined above. These complement-binding proteins may be used to inhibit complement activation via any of the three pathways. They may also be used to inhibit the interaction between C3d/C3dg and CR2 and may also inhibit the anaphyϊatoxin activity of C3a especially if a Sbi-IV domain is present.
[0146]Without wishing to be bound by any particular theory, it is believed that complement-binding proteins containing an Sbi-III domain, more specifically those containing an Sbi-III domain and an Sbi-IV domain, such as an Sbi-III-IV protein, may act to cause breakdown of C3. It is believed that this leads to depletion or consumption of C3 without the normal activation of those components downstream of C3 in the complement cascade, references to inhibition of complement activation may be construed accordingly.
[0147]Other complement binding proteins described in this specification possess a Sbi-IV domain as defined above, in the absence of a functional Sbi-III domain. That is to say, they do not possess a Sbi-III domain or derivative or fragment thereof which is capable of binding C3 protein. They may comprise sequences from the Sbi- III domain as described above, as long as those Sbi-III sequences do not retain a substantial ability to bind C3. Such complement- binding proteins are capable of specifically inhibiting the alternative complement activation pathway. They may also inhibit the anaphylatoxin activity of C3a.
[0148]Either type of complement-binding protein may possess further Sbi sequences derived from upstream (N-terminal) of the Sbi-III domain, including a Sbi-II domain, and/or a Sbi-I domain. Additionally or alternatively, they may possess further Sbi sequence derived from downstream (C-terminal) of the Sbi-IV
domain, such as the cell wall-spanning sequence and/or Y region. However it is generally preferred that they do not possess Sbi-I or Sbi-II domains, or the cell wall-spanning or Y domains.
[0149]Preferably the complement-binding proteins are soluble; i.e. they are not anchored in a cell wall or cell membrane, and thus preferably do not contain a cell wall-anchoring sequence or cell membrane-anchoring sequence. This may facilitate recombinant expression and purification as well as use as a therapeutic agent. Soluble complement-binding proteins preferably do not include Sbi sequence downstream (C-terminal) of residue 266 of the Sbi sequence.
[0150]In preferred embodiments the protein is expressed in a 'suitable host cell and secreted from that cell into the culture medium from where it can be purified. Any signal sequence required for secretion may or may not be cleaved during the secretion process. However in some circumstances it may be desirable to express the protein within a host cell (e.g. within the cytoplasm, within an organelle, or within an inclusion body) and isolate it from the host cell at a later stage.
[0151]If a signal sequence is present, it may be the Sbi signal sequence illustrated above or may be a heterologous signal sequence. The skilled person will be able to select a suitable signal sequence in order to achieve satisfactory secretion from any chosen host cell.
[0152]The complement-binding proteins described in this specification may be expressed as fusion protein with heterologous components such as antibody Fc regions, or any other desired fusion partner.
[0153]Additionally or alternatively, they may be chemically derivatised in order to modify their pharmacokinetic and/or activity
properties. For example, they may be conjugated to PEG molecules in order to improve stability in vivo.
[0154]Screening and other assay methods The invention provides methods of screening for compounds capable of inhibiting the interaction between Sbi and C3. Also provided are assay methods for determining the presence of C3 or a fragment thereof in a sample, such as a biological sample comprising blood, serum or plasma.
[0155]Interactions between the complement-binding protein and C3 protein (or fragment) may be studied in vitro by immobilising one on a solid support and bringing the other into contact with it.
[0156]The immobilised protein is generally contacted with a sample containing the other protein under appropriate conditions which allow the two proteins to bind to one another (or would allow such binding in the absence of any inhibitor or candidate inhibitor) . The fractional occupancy of the binding sites on the immobilised component can then be determined either directly or indirectly, e.g. by labelling the component in the sample or by using a developing agent or agents to arrive at an indication of the presence or amount of the component in the sample.
[0157]Typically, the developing agents are directly or indirectly labelled (e.g. with radioactive, fluorescent or enzyme labels, such as horseradish peroxidase) so that they can be detected using techniques well known in the art. Directly labelled developing agents have a label associated with or coupled to the agent. Indirectly labelled developing agents may be capable of binding to a labelled species (e.g. a labelled antibody capable of binding to the developing agent) or may act on a further species to produce a detectable result. Thus, radioactive labels can be detected using a scintillation counter or other radiation counting device, fluorescent labels using a laser and confocal
microscope, and enzyme labels by the action of an enzyme label on a substrate, typically to produce a colour change. In further embodiments, the developing agent or analyte may be tagged to allow its detection, e.g. linked to a nucleotide sequence which can be amplified in a PCR reaction. The developing agent (s) can be used in a competitive method in which the developing agent . competes with the analyte for occupied binding sites of the binding agent, or non-competitive method, in which the labelled developing agent binds analyte bound by the binding agent or to occupied binding sites. Both methods provide an indication of the number of the binding sites occupied by the analyte, and hence the concentration of the analyte in the sample, e.g. by comparison with standards obtained using samples containing known concentrations of the analyte.
[0158]Preferred assay formats include immunological techniques such as ELISA assays.
[0159]Alternatively, techniques such as surface plasmon resonance may be used to monitor binding between the two proteins directly, without the need for either component to be labelled.
[0160]The protein which is immobilized may be immobilized using an antibody against that protein bound to a solid support or via other technologies which are known per se, including simply coating the protein on a suitable surface, such as a well of a microtiter plate. A preferred in vitro interaction may utilise a fusion protein including glutathione-S-transferase (GST) , which may be immobilized on glutathione agarose beads.
[0161]There is also an increasing tendency in the diagnostic field towards miniaturisation of such assays, e.g. making use of binding agents (such as antibodies or nucleic acid sequences) immobilised in small, discrete locations (microspots) and/or as arrays on solid supports or on diagnostic chips. These
approaches can be particularly valuable as they can provide great sensitivity (particularly through the use of fluorescent labelled reagents), require only very small amounts of biological sample from individuals being tested and allow a variety of separate assays can be carried out simultaneously. This latter advantage can be useful as it provides an assay employing a plurality of analytes to be carried out using a single sample. Examples of techniques enabling this miniaturised technology are provided in WO84/01031, WO88/1058, WO89/01157, WO93/8472, WO95/18376/ WO95/18377, WO95/24649 and EP 0 373 203 A. Thus, in a further aspect, the present invention provides a kit comprising a support or diagnostic chip having immobilised thereon a plurality of binding agents capable of specifically binding different protein markers or antibodies, optionally in combination with other reagents (such as labelled developing reagents) needed to carrying out an assay. In this connection, the support may include binding agents specific for analytes such as vimentin, e.g. as disclosed in US Patent No: 5,716,787.
[0162]As already described, such assay methods may be used to screen for compounds capable of inhibiting binding between C3 and Sbi proteins. Candidate agents identified by such screens may be subjected to one or more rounds of modification and re-testing in order to identify further agents having improved properties. The skilled person will be aware of numerous suitable screening methods and will be able to design appropriate protocols for identification of candidate binding agents.
[0163]Antibodies It has been shown that fragments of a whole antibody can perform the function of binding antigens. The term "antibody" is therefore used herein to encompass any molecule comprising the binding fragment of an antibody. Examples of binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CHl domains; (ii) the Fd fragment consisting of the VH and CHl
domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E. S. et al., Nature 341, 544-546 (1989)) which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv) , wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding member (Bird et al, Science, 242, 423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883, 1988) .
[0164]Pharmaceutical compositions
[0165]The complement-binding proteins and other therapeutic agents described in this specification can be formulated in pharmaceutical compositions. These compositions may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes or topical application.
[0166]Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
[0167]For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will
be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
[0168]Whether it is a polypeptide, antibody, peptide, nucleic acid molecule, small molecule or other pharmaceutically useful compound according to the present invention that is to be given to an individual, administration is preferably in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical . doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Suitable carriers, adjuvants, excipients, etc. can be found in standard pharmaceutical texts, for example Remington's
[0169]Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.
[0171]Here we reveal Sbi's extracellular domain organisation, determine the specific function of the individual domains and describe their unique role in 5. aureus' evasion of both adaptive and innate immune systems in humans. To investigate the arrangement of the domains in solution, we cloned, expressed and purified the
extracellular part of Sbi, adjacent to the predicted cell wall- spanning proline-rich repeat region8 (Sbi-E, residues 28-266, Fig. Ia and Figure 6) and subjected the fragment to small-angle X-ray scattering (SAXS) , a technique well suited to study flexible macromolecules in solution13. Based on the SAXS structure we then engineered four recombinant Sbi fragments, spanning the N-terminal region of the protein (Sbi-I, Sbi-II, Sbi-III-IV' and Sbi-IV, as shown in Figure 6) , and examined them in various assays for immunoglobulin binding. Other human serum proteins that interact with Sbi were identified by affinity pulldown and MALDI-TOF mass spectrometry, and candidates were further investigated in direct binding assays.
[0172]Results and Discussion Domain structure of Sbi's extracellular region
[0173]We investigated the domain organisation of Sbi's extracellular ligand-binding region by SAXS using a 31 kDa construct of Sbi (Sbi-E), containing the N-terminal segment adjacent to the proline-rich region. The X-ray scattering curve for Sbi-E (Figure 7) yields a molecular mass estimate of 33 + 3 kDa, indicating that the protein is monomeric in solution. The experimental radius of gyration R9 and the maximum size, Draax, were 46 ± 1 A and 160 ± 10 A, respectively, exceeding the values expected for a compact globular protein of such molecular mass by nearly a factor of two. The distance distribution function of Sbi-E (Figure 2a, inset) has a skewed profile characteristic for elongated particles (Svergun and Koch, 2003) . These results indicate that Sbi-E is a highly elongated molecule in solution. The low resolution shape of Sbi, reconstructed ah initio using the bead modelling program DAMMIN14, neatly fits the experimental data with discrepancy χ=l .2 (Figure 7). A typical reconstructed model, displayed in Figure 2b, is indeed very elongated (155 A in diameter) and depicts four bead domains connected by thinner loops. Given the low resolution of SAXS, this model should not be considered as the unique model of Sbi-E, but the elongated
multi-domain appearance (also observed in other independent ab initio reconstructions) clearly points to the existence of four structured domains, joined by (flexible) linkers. A homology model of Sbi's immunoglobulin binding domains I and II, based on SpA domain D, or on domains B and E, superimposes well with the presented ab initio domain organization (Figure 2b) . This four- domain structure of the extracellular region of Sbi has also been incorporated in Figure Ia, with the two novel domains labelled as Sbi-III and Sbi-IV.
[0174]Sbi-E binds Fc fragment of human IgG, not Fab As well as Fc binding, all individual SpA domains display an additional interaction with certain (VH3+) Fab fragments, via their heavy chain, that does not interfere with the antibody's antigen-binding site15. Sequence alignment (Figure Ib) reveals that almost all amino acids involved in Fc-binding in SpA (involving helices 1 and 2)16 are identical in Sbi domains Sbi-I and Sbi-II. The lack of sequence identity between Sbi and SpA' s Fab-binding region (involving helices 2 and 3)17, highlighted in Figure Ib, suggests that either Sbi has a different VH family heavy chain Fab-specificity or that it does not bind Fab. To test this we performed a double immunodiffusion assay with recombinant SpA and Sbi-E against human serum IgG. An arched line of precipitate in the diffusion equivalence zone indicates that SpA and Sbi share some identical binding epitopes on human IgG (Figure 3a) . However, the spur formation of precipitate towards the Sbi well indicates that SpA possesses binding specificity for a human IgG epitope that is not shared by Sbi. In the absence of Fab, SpA' s capacity to form insoluble complexes with human IgG Fc is completely eliminated, as has been reported previously18, while precipitation with Sbi remains unchanged (Figure 3a) . It is therefore unlikely that Sbi has a VH family Fab specificity different from SpA, ruling out its possible role as a B cell super antigen.
Domains Sbi~I and Sbi-II both bind immunoglobulins Constructs of functional immunoglobulin-binding domain Sbi-I (residues 42-94) and predicted immunoglobulin-binding domain Sbi- II (residues 92-156) were tested individually in SPR experiments with human IgG subclasses 1, 2, 3 and 4. In Figure 3b we show that both domains bind human IgG with a high specificity for subclass IgGl, displaying affinity constants (Ka= 3.5XlO7M"1 for Sbi-I, 4.3xlO7 M"1 for Sbi-II) that are an order of magnitude higher than those found for the binding of human FcI fragments by mono-valent SpA domains (reported Ka values in the range of 105- 106 M"1 19"21) . We found that both domains bind IgG4 with similar affinity (7.6xlO6 M"1 for Sbi-I, 6.3XlO6M"1, for Sbi-II). For IgG2, Sbi-I has a significantly higher affinity than Sbi-II (4xlO7 M"1 versus 3xlO6 M"1) . As with SpA, neither Sbi-E nor the individual- immunoglobulin binding domains show any measurable affinity for IgG3.
[0175]SJbi fragments III-IV and IV bind complement component C3
[0176]In search of additional serum components that interact with Sbi, an affinity pull-down assay was performed with human serum, using recombinant constructs Sbi-III-IV (residues 150-266), Sbi-III and Sbi-IV as bait proteins. SDS-PAGE analysis of the serum proteins contained in the samples eluted from the Sepharose-immobilised Sbi-III-IV column is shown in Figure 4a. An identical but fainter SDS-PAGE profile was found using a Sepharose-immobilised Sbi-IV column (Figure 4b, lane 4) . The polypeptides labelled with asterisks were identified as fragments of complement component C3 by MALDI-TOF mass spectrometry. From the SDS-PAGE profile and the origin of the peptides in the gel fractions, as identified by mass spectrometry, it appears that Sbi-III-IV binds native C3 (consisting of a 119 kDa α-chain and a 75 kDa β-chain) and some C3 cleavage products (see Figure 8 for a diagrammatic representation of the various C3 degradation products referred to below) . Intriguingly, no significant protein band can be
identified with a molecular weight similar to β2-GPI, the previously identified ligand for Sbi (Zhang et al., 1999). The peptides originating from the C3 α-chain present in the gel fractions running at approximately 65 kDa, accompanied by the C- terminal α-chain fragments running at 43 and 40 kDa, suggest that Sbi binds the C3 cleavage product iC3b. The hallmarks of fragments C3b (a 110 kDa α-chain fragment) and C3c (a 29 kDa α- chain) are absent from the SDS-PAGE profile. Finally, one can infer from the non-stoichiometric staining intensities of the C3- derived bands of the pull-down experiment that in addition to intact C3 and iC3b, an iC3b-like species, iC3 (H2O) , which originates from thioester-hydrolysed, but peptide chain-intact C3, also binds to Sbi. The presence in iC3 (H2O) of an -74 kDa chain consisting of C3a joined to α-65 would contribute to the higher than expected β-chain intensity relative to α-chain, as would its α-40 chain contribute to the higher than expected intensity of this band relative to the α-65 band from the iC3b that is pulled down.
[0177]Because of the high relative amounts of factor I-cleaved C3 products pulled down from human serum by insolubilized Sbi-III- IV, the possibility existed that Sbi-III-IV possessed the factor I cofactor activity. However, when this was assessed using thioester-cleaved 03(NHCH3) as the cleavage target for factor I, even in the presence of an equimolar concentration of Sbi-III-IV to 03(NHCH3), no factor I-mediated cleavages took place, whereas complete cleavage to iC3 (NHCH3) was observed using a catalytic amount of factor H (data not shown) . Thus Sbi-III-IV does not possess- factor I cofactor activity.
[0178]SDS-PAGE and MALDI-TOF mass spectrometry analysis of the serum proteins contained in the samples eluted from Sepharose- immobilised Sbi-I (see Figure 4b, lane 1) and Sbi-II columns were identified as immunoglobulins. Affinity pull-down experiments
using a Sepharose-immobilised Sbi-III column yielded no protein bands that were identifiable with MALDI-TOF (see Figure 4b, lane 2), localizing Sbi's C3-binding properties to the C-terminal Sbi- IV domain. We also found that Sbi's complement C3 binding characteristics are not unique to human C3.' C3 and its degradations products were identified by MALDI-TOF analysis in affinity pull down experiments with sera from cow, calf, goat, horse, mouse and rabbit (see Figure 4c) . Intriguingly, in these experiments a weak band with a molecular weight similar to β2-GPI (~45 kDa) can be seen in some lanes on the gel shown in Figure
[0179]4c. Although it is specifically prominent in cow serum (lane 2), it could not be identified as β2-GPI by MALDI-TOF. Interestingly, Zhang and co-workers first discovered the interaction between Sbi and β2-GPI using bovine serum (Zhang et al., 1999). In their paper an SDS-PAGE analysis of proteins from human serum, bound by an Sbi fragment lacking the IgG binding regions, shows a band with a high staining intensity at a molecular weight similar to C3 and several faint bands that could be C3 degradation products .
[0180]To verify the binding of Sbi to C3 and its degradation products, and to identify its site of interaction, constructs Sbi-E, Sbi- III, Sbi-IV and Sbi-III-IV were immobilised on biosensordiscs and subjected to SPR binding experiments with native C3 and sequential proteolytic cleavage products C3b, iC3b, C3c and C3dg. Sbi-E and Sbi-III-IV binding was weakest to C3c and C3b, and strongest to iC3b and C3dg, suggesting that the main binding site for Sbi in intact C3 may be located within C3dg and becomes more accessible in the smaller C3dg fragment.
[0181]Sensorgrams of the binding experiments with Sbi-III-IV is shown in Figure 5a and in Figure 5b, left panel. When approximately the same number of molecules of the constructs Sbi-III-IV, Sbi- III and Sbi-IV were immobilized in each flow cell very little
binding of C3 and its degradation products was observed with the Sbi-III construct (Figure 5b, middle panel) , confirming the results from the affinity pull-down experiments. Sbi-III-IV (Figure 5b, left) and Sbi-IV (Figure 5b, right) essentially display the same C3 interaction characteristics with the weakest binding to C3c and C3b, and strongest to iC3b and C3dg, suggesting that the main binding site for Sbi in intact C3 may be located within C3dg and becomes more accessible in the smaller C3dg fragment. Similar binding data were obtained when the C3 fragment scan was done on a chip bearing Sbi-E (data not shown) . The C3 fragment specificity was further confirmed by the lack of binding of bovine serum albumin, and the human complement C4- derived fragments C4b and C4dg, the latter being a structural homologue of the binding-competent C3dg fragment (data not shown) .
[0182]This binding behaviour shows similarities with complement receptor CR2, except that CR2 does not bind native C322.
[0183]Interestingly, the interaction between C3dg and chip-immobilized CR2 is hindered when Sbi-III-IV is present as a co-analyte (Figure 9) . Specifically, axlag phase' in the saturation curve is observed when the molar ratio of C3dg to Sbi-III-IV is less than 1:1. At C3dg concentrations with a molar excess over Sbi- III-IV, the maximal saturation level overlaps that of the standard C3dg binding curve, indicating that the inhibitory effect of Sbi-III-IV is due to direct binding to C3dg, as opposed to an interaction between Sbi-III-IV and CR2.
[0184]Sbi association with most C3 fragments seemed to follow two phases; an initial fast binding followed by a slow binding phase. Only the binding of C3dg could be fit well to a single-site binding model. These latter interactions were of moderately-high affinity with KD values for Sbi-E of 0.9 μM and 1.6 μM at half-
and full-physiological NaCl concentrations, respectively, and for Sbi-III-IV of 0.7 μM and 1.4 μM under the same two ionic strength conditions. Only the binding of C3dg reached a clear steady- state plateau during the injection phase. This equilibrium state binding data could be well fit to a single-class Langmuir binding site binding model, an example of which for Sbi-III-IV is shown in Figure 5c. The interactions with C3dg were of moderately-high affinity with KA values for Sbi-E of 1.IxIO6M"1 and 6.3XlO5M"1 at half- and full-physiological NaCl concentrations, respectively, and for Sbi-III-IV of 1.4XlO6M"1 and 7.IxIO5M"1 under the same two ionic strength conditions.
[0185]When injected at the same concentrations over sensordisc surfaces with immobilised Sbi-E, Sbi-III-IV and Sbi-IV, all three Sbi constructs bound C3 better than C3b (data for Sbi-III-IV shown in Figure 5a) . Particularly noteworthy was the very slow dissociation rate displayed by native C3, suggesting that quite a stable complex had formed. To explore whether this difference in binding behaviour is dependent on conformation or because of the presence of the C3a anaphylatoxin fragment, purified native C3 was treated with methylamine to- produce a thioester-cleaved form (03(NHCH3)) that takes on a C3b-like conformation23 but with the C3a fragment still attached. C3 (NHCH3) showed considerably higher binding than did C3b (Figure 5a) , indicative of a role for C3a in the binding. When C3 (NHCH3) is trypsinised to release C3a, this "C3b" showed poor binding analogous to C3b generated from C3. Similarly, binding of iC3b by Sbi was significantly enhanced by the presence of C3a in the methylamine-treated C3 conversion product iC3 (NHCH3) .
[0186]The interaction between Sbi-III-IV and C3a was probed in saturation experiments with Sbi-III-IV binding to sensordiscs bearing a recombinant C3a. Results are shown in Figure 10.
Attempts to analyse the kinetics in terms of a simple 1:1 Langmuir binding model did not yield acceptable fits, especially for the dissociation phase where some rebinding of Sbi-III-IV to the C3a surface may have been occurring. Although a steady-state plateau has not been fully reached, a plot of RU at the end of the injection phase versus concentration of Sbi-III-IV, shown in Figure 1Od, indicates that the binding is saturable. Analysis of these data in terms of a single class of binding site suggests an affinity of Sbi-III-IV for C3a of at least 5 x 104 M"1, at physiologic ionic strength, although this is likely an underestimate given that equilibrium had not been fully reached.
[0187]Sbi domain Ill-TV inhibits activation of the three pathways of the complement system To determine whether Sbi's C3 binding properties interfere with complement activation we examined the effect of the constructs Sbi-E, Sbi-I, Sbi-III-IV and Sbi-IV on activation of the■classical pathway (CP), the mannose-binding lectin (MBL) pathway and the alternative pathway (AP) . Preincubation of human serum with two of the three recombinant constructs that bind C3 (Sbi-E and Sbi-III-IV) dramatically reduced the activity of all three pathways (CP: 4% and 3% activity; MBLP: 2% and 1% activity; AP: 0% activity with both Sbi-E and Sbi-III-IV, respectively) , as is shown in Figure 5d. Remarkably, the Sbi-IV fragment, containing Sbi' s minimal β2-GPI-binding region, lacks the C3-mediated inhibition of the three complement pathways but specifically blocks the alternative pathway. These results indicate that Sbi also interferes with the interaction between C3 and other factors involved in the alternative pathway, such as Factors B and D, or directly binds to these factors. Although we have not been able to show a direct interaction between Sbi domain III and complement component C3, our results indicate that the union of both domains III and IV is essential for Sbi's interference with all three complement pathways. Sbi domain IV, containing Sbi's
minimal C3-binding region, perhaps hinders the interaction between C3 and propagating components of the alternative pathway.
[0188]The complement system is one of the key components of the innate immune defence against microbial pathogens by promoting phagocytosis and local inflammatory responses. Activation of complement component C3 is central to all three complement pathways of the innate immune system. Several small excreted factors preventing complement mediated defense mechanisms, expressed by human pathogen S. aureus, have been identified in recent studies24. They include: extracellular fibrinogen binding protein Efb4'5, chemotaxis inhibitory protein CHIPS6, and staphylococcal complement inhibitor SCIN7. Our data establish that S. aureus also expresses a cell wall-associated protein, Sbi, that in addition to interfering directly with the adaptive immune system via two immunoglobulin-binding domains, also utilizes two novel domains in blocking the action of complement component C3 by interacting with two of its functionally crucial subdomains . Not only would Sbi be able to inhibit complement activation, but it may also bind to, and thereby functionally inhibit C3 split products resulting from complement activation that does occur. C3a is a powerful mediator of inflammation25 and the C3d portion of C3dg is not only involved in mediating covalent attachment of nascently-activated C3b to the target pathogen26, but also provides a crucial link between the innate and adaptive immune systems through its interaction with CR2 on B cells and follicular dendritic cells27. By inhibiting the interaction between C3dg and CR2 (see Figure 9) we have found that Sbi interferes with this crucial immunological link.
[0189]Sbi causes breakdown of C3
[0190]Monitoring125I-C3 in serum incubated with Sbi fragments reveals that Sbi-E and Sbi-III-IV both cause breakdown of C3, leading to a consumption of C3 (Figure 11) after 5 minutes of incubation
(Figure 12) . By contrast, neither Sbi-III nor Sbi-IV on their own have this effect.
[0191]Insights gained here will impact our understanding of S. aureus infections and immune evasion in humans and in animals of economic importance as well as advancing the design of S. aureus vaccines. .Inhibition of complement activation by Sbi constructs may offer a promising target for development of therapies for complement-associated inflammatory diseases.
[0192]Materials and Methods
[0193]Cloning and expression of recombinant Sbi constructs
[0194]Five recombinant fragments of the N-terminal region of Sbi
[0195](adjacent to the poly-proline region) were engineered: Sbi-E (amino acids 28-266) , Sbi-I (amino acids 42-94) , Sbi-II (amino acids 92-156), Sbi-III-IV (amino acids 150-266) , Sbi-III and Sbi-IV (amino acids 197-266). The Sbi gene constructs were amplified by PCR using 5. aureus strain Mu50 genomic DNA as a template. The following oligonucleotide primers were used for Sbi-E, Sbi-I, Sbi-II, Sbi-III-IV, Sbi-III and Sbi-IV, respectively: 5'-CAT GCC ATG GCG AGT GAA AAC ACG CAA CAA-3' (forward primer) and 5'- CCG CTC GAG TCA TTA CGC CAC TTT CTT TTC AGC-3' (reverse primer); 5'-CAT GCC ATG GGA ACT CAA AAC AAC TAC GTA ACA-3' (forward primer) and 5'-CCG CTC GAG TCA CTA GCT GTC TTT AAG TGA TTC AGA-3' (reverse primer); 5'-CAT GCC ATG GAC AGC AAG ACC CCA GAC CGA-3' (forward primer) and 5'-CCG CTC GAG GAG TCA TTA ATT TTC AAT ATT TTG ACG-3' (reverse primer); 5'-CAT GCC ATG GAA CGT CAA AAT ATT GAA AAT GCG-3' (forward primer) 5'-CCG CTC GAG TdA TTA CGC CAC TTT CTT TTC AGC-3' (reverse primer); 5'- CAT GCC ATG GAA CGT CAA AAT ATT GAA AAT GCG-3' (forward primer); 5'-CCG CTC GAG TCA TTA AAC GAT'TGC TTT TTC AAT TGA-3' (reverse primer); 5'-CAT GCC ATG GTT TCA ATT GAA AAA GCA ATC GTT-3' (forward primer) 5'-CCG CTC GAG TCA TTA CGC CAC TTT CTT TTC Aces' .(reverse primer) . All primers were obtained from MWG biotech AG. The resulting amplified fragments were subsequently cloned
into the pET-based "parallel" vector28 (using the incorporated Ncol (forward primer) and Xhol (reverse primer) restriction sites) producing the constructs psbi-e, psbi-i, psbi-ii, psbi- iii-iv and psbi-iv, respectively.
[0196]Sbi constructs were expressed in E. coli strains BL21(DE3), BL21 (DE3) -Star and Rosetta. Freshly transformed E. coli cells were grown in a shaker at 37 °C in Luria Burtani broth (LB), containing ampicillin, until they reached an A600 of 0.6. Isopropyl-β-D-thiogalactopyranoside (Melford) was added to a final concentration of 0.2 itiM, and the cells were incubated at 28°C for an additional 4 hours. Cells from a 11 culture were harvested by centrifugation, resuspended in 10 ml binding buffer (20 mM Tris HCl, 0.5 M NaCl, 20 itiM imidazole, pH 8.0), and lysed by sonication. The lysate was centrifuged at 40,000 g for 15 minutes and the supernatant filtered through a 0.45-μm filter. The proteins were purified using nickel-ion chelating chromatography by either applying the filtered supernatant to a Sartobind membrane (Sartorius) or a ImI HiTrap column attached to an AKTA purifier (Amersham Biosciences) . Next, the column was washed with binding buffer and the bound proteins were eluted with a buffer containing 1 M Imidazole, for the Sartobind purification, or a 0.05-1 M imidazole gradient for the HiTrap purification. Purified protein was dialysed into a buffer solution, typically 2OmM Tris pH 8.0, 10OmM NaCl and stored at - 800C until use. Protein concentration was determined using a Bradford protein assay (Bio-Rad Laboratories) and absorbance at 280 nm.
[0197]Double immunodiffusion assay
[0198]Double immunodiffusion experiments were performed on Petri dishes containing a 1% agarose gel. Wells were punched in the agar and individual wells filled with 100 μl of Sbi-E at lmg/ml in PBS; human serum IgG (Sigma) or IgG Fc (Bethyl Laboratories) at lmg/ml
in PBS; or recombinant SpA (Pierce) at lmg/ml in PBS and left to incubate for 48 hours at room temperature. Insolubleλimmune complexes' formed at the zone of equivalence were visualised by Coomassie staining.
[0199]Serum affinity pull-down assay
[0200]Purified Sbi-I, Sbi-II, Sbi-III, Sbi-IV and Sbi-III-IV were each covalently coupled to a ImI NHS-activated sepharose High Performance column (Amersham Biosciences) according to the manufacturer's instructions. After equilibration with PBS, 5ml human serum (Carnbrex) was applied to the column. Columns were washed with PBS and bound proteins were eluted with a 0-1 M NaCl gradient over 10 ml. The binding of serum proteins by bait proteins Sbi-III-IV, Sbi-III and Sbi-IV was analysed using SDS- PAGE, using immobilised Sbi-I as a negative control. Size exclusion chromatography, with a Suρerdex-200 gel filtration column (Amersham Biosciences) , was used for further purification of the bound fragments. Trypsinised protein fragments from excised gel slices were analysed by MALDI-TOF. ProteinLynx software was used for protein identification.
[0202]SpA-D structures were used as a template for the generation of a homology model of Sbi domains I and II, using the program MOE (Chemical Computing Group, Inc) .
[0203]Small angle X-ray scattering (SAXS) experiments and data analysis Synchrotron radiation X-ray scattering data were collected on the X33 beam line at the EMBL, Hamburg Outstation (on the DORIS III storage ring, at DESY) . Solutions of Sbi-E were measured on a
[0204]MAR345 image plate detector at protein concentrations of 2.8, 8.4 and 16.7 mg/ml and sample-detector distance of 2.4 m and wavelength λ = 1.5 A, covering the momentum transfer range 0.013 < s < 0.45 A"1. To check for radiation damage, two 2-minute
exposures were compared; no radiation effects were observed. The data were processed using standard procedures using the program package PRIMUS31.
[0205]The forward scattering 1(0) and the radius of gyration R9 were evaluated using the Guinier approximation32 assuming that at very small angles (s < 1.3/Rg) the intensity is represented as I(s) = 1(0) exp (- (sRg)2/3) . These parameters were also computed from the entire scattering patterns using the indirect transform package GNOM33, which also provided the maximum particle dimension Dmaκ.
[0206]The molecular mass (MM) of the solute was evaluated by comparison of the forward scattering with that from reference solutions of bovine serum albumin (MM = 66 kDa) . The low resolution ab initio model of Sbi was constructed using the program DAMMIN14, which represents the protein by an assembly of densely packed beads. Simulated annealing was employed to build a compact interconnected configuration of beads inside a sphere with the diameter Dmax that fits the experimental data Iexp(s) minimizing the discrepancy:
[0207]
where N is the number of experimental points, c is a scaling factor and Icaχc[Sj) and σfs-,-) are the calculated intensity and the experimental error at the momentum transfer Sj, respectively.
[0208]Surface plasmon resonance (SPR)
[0209]Immunoglobulin binding SPR measurements were recorded using an Autolab ESPRIT instrument (EcoChemie) , with two flow cells. Dextran-NTA derivatised SPR sensordiscs (Xan Tec Bioanalytics) were coated with nickel by dispensing 80ul of a NiCl containing buffer (20 mM Tris pH 8.0, 100 mM NaCl, 300 μM NiCl, 0.005% surfactant Tween 20) into each channel and mixing for approximately 5 min. Excess NiCl was expelled by washing the
cuvette with wash buffer (20 mM Tris pH 8.0, 100 mM NaCl 0.005% Tween 20) . Constructs Sbi-E, Sbi-I or Sbi-II were bound to the chip via their N-terminal 6xHis tag. A baseline was established for a minimum 60 s before binding experiments with the four human IgG subclasses were performed. IgG solutions were applied to channel 1, with an association time of 400 s and dissociation time of 20 s. BSA was applied to channel 2 as a negative control. Data was extracted using the differential data (channel 1 - channel 2) . Following each measurement, the sensordisc was regenerated with 0.35 M EDTA. All data analysis was performed using kinetic evaluation software (EcoChemie, version 4.1.0 2004) . The difference in binding between the two channels was fitted to a monophasic association curve in order to determine Jc0n, which is the sum of the kinetic association constant kass and the disassociation constant, kdiss: kon = kass[lqG] + kdiss.
[0210]Measurements of Jf0n were made with a minimum of five and a maximum of 10 different IgG concentrations. A linear plot of Jc0n vs. IgG concentration thus yielded both kass and kdiss.
[0211]Complement binding experiments were performed on a BIACore 3000 Instrument (BIACORE, Life Sciences), which has four flow cells. Each experiment was run in 10 mM HEPES, pH 7.2, 3mM EDTA, 0.02% surfactant P20 with either physiological salt (150 mM NaCl), or half physiological salt (75 mM NaCl) at a flow rate of 20 μl/ml. Ligands molecules were covalently coupled to a CM5 sensordisc via amino groups, with flow cell 1 being the sham-activated and deactivated reference channel. Each experiment consisted of a 60 second analyte injection, followed by a 60 second buffer flow over the surface before the injection loop and flow cell were washed. The sensordisc surface was regenerated between experiments with a 60s pulse of 2M NaCl, which brought the sensorgram signal back to baseline. Each injection flowed over all four flowcell surfaces in series and gave three sets of data (the signal of flowcell 1 being subtracted from the other
sensorgrams) . Control proteins C4Bdg, C4b and BSA were used to validate the specificity of C3 binding to the sensordiscs bearing Sbi-E and Sbi-III-IV. Affinity constants for C3dg binding to Sbi- E and Sbi-III-IV were determined by fitting the steady-state plateau RU values of a series of C3dg analyte injections to a single site Langmuir binding isotherm model using MacCurvefit vl .5.5 non-linear regression software. Inhibition of the interaction between C3dg and CR2 by Sbi-III-IV was probed in saturation experiments with C3dg binding to sensordiscs bearing a recombinant two domain CR2 construct (CCP 1-2), in the presence of a constant concentration (5 μM) of Sbi-III-IV.
[0212]Complement components used as analytes were prepared as follow: C3, purified from pooled human plasma34 was subjected to a final chromatographic step on Mono S FPLC to separate thioester-intact native C3 from thioester-hydrolysed confomational isoforms35. C3b was generated by limited trypsinizaton of native C323 and conversion of C3b to iC3b employed fl (l%,w/w), with fH (2% w/w) as the I-cofactor36. To generate C3c, C3b was incubated overnight at 22°C with fl (1%, w/w) and soluble CRl (2% w/w, a gift from Avant Immunotherapeutics, Needham, MA) . Treatment of native C3 with 100 mM CH3NH2 at pH 8 for 6 h at 370C yields a thioester carbonyl-derivatised form of C3 denoted C3 (NHCH3) , which adopts a C3b-like conformation23. Treatment with fl and fH yields an iC3b- like species, iC3 (NHCH3) in which C3a is still present. All of the aforementioned digestion products were purified via chromatography on Mono Q FPLC using previously described elution condition37. C3dg and C4dg were recombinantly produced in E. coli as described previously38'39. All complement proteins used as analytes in SPR experiments were exchanged into the SPR running buffer, and separated from any minor oligomeric species formed during storage, by FPLC gel filtration on Superose 6 (Superdex 200 for C3dg and C4dg) within 24 h of use.
Complement activity assays
[0213]The Wielisa Total Complement System Screen (Wieslab) , described by Seelen and co-workers29, was used to detect inhibition, by recombinant fragments Sbi-E, Sbi-I, Sbi-III-IV and Sbi-IV, of the classical (CP), rαannose-binding lectin (MBLP) and alternative
[0214](AP) complement pathways. lμg of each protein fragment was added per 1 μl of human serum (positive control serum, supplied with the kit) , and assayed for complement activity either directly or after 30 min incubation at 370C. The assay was completed in duplicate, according to the manufacturers instructions, and included a blank, a positive control (human serum from healthy individuals) and a negative control (heat inactivated serum). Activity inhibition was quantified from the absorbance at 405 nm using the equation: (sample-negative control) / (positive control/negative control) x 100%.
[0215]Factor I cofactor activity assay
[0216]Twenty micrograms of C3 (NHCH3) were incubated for 18 hours at
[0217]370C with 0.2 μg of factor I and 2 μg of Sbi-III-IV in a volume of 40 μl of phosphate-buffered saline, pH 7.2. This quantity of Sbi-III-IV was in slight stoichiometric excess (1.15:1) relative to the target C3 derivative. Cleavage to iC3 (NHCH3) was assessed on reduced SDS-PAGE via the disappearance of intact a-chain, the appearance of a-40/a-43 fragments and an increase in the intensity of the b-chain region due to the co-migration of a-75 fragment.' Substitution of 0.2 μg of factor H for Sbi-III-IV served as a positive control as quantitative conversion to iC3 (NHCH3) occurred using this substoichiometric amount (0.013:1) of cofactor. See Figure 4 for a diagrammatic representation of the various C3 degradation products.
[0218]Radiochemical assay
[0219]Human serum was spiked with125I-C3, so as to give ~5000 cpm/μl.
[0220]2.5 μl of SM fragment (2 mg/ml Sbi-E, Sbi-III-IV, Sbi-III or
Sbi-IV) was added to 5 μl of 80% serum (dilution due to spiking) ± 10 mM EDTA or EGTA and incubated at 37 "C for 30 minutes. Minus EDTA samples had EDTA added at the end of the incubation. Subsequently SDS sample buffer (with DDT) was added to the samples followed by incubation at 100 °C for 10 minutes. The positive control contained 5 μl of125I-C3-spiked serum with 2.5 μl heat-aggregated IgG (4.5 mg/ml) . Spontaneous activation controls consisted of125I-C3-spiked serum ± EDTA or EGTA with 2.5 μl of buffer, incubated at either 37 °C or 4 °C. Samples were analysed via SDS-PAGE followed by overnight development using a phosphoimager .
[0221]While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention. All documents cited herein are expressly incorporated by reference.
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