WO2012042213A1

WO2012042213A1 - Novel interaction between staphylococcus aureus sbi and c3d proteins

Info

Publication number: WO2012042213A1
Application number: PCT/GB2011/001414
Authority: WO
Inventors: Jean Van Den Elsen; Stefan Bagby
Original assignee: BATH VENTURES
Current assignee: BATH VENTURES
Priority date: 2010-09-29
Filing date: 2011-09-29
Publication date: 2012-04-05
Anticipated expiration: 2013-03-29
Also published as: GB201016403D0

Abstract

The invention relates to the Sbi protein of Staphylococcus aureus, and in particular to the interaction between Sbi and the complement protein C3d. The crystal structure of the Sbi-C3d complex reveals an unexpected interaction between Sbi and the convex surface of C3d, which suggests that particular fragments and variants of Sbi may have utility as inhibitors of the alternative complement pathway. The invention provides novel peptides and their use in the inhibition of inflammatory conditions characterised by undesirable activation of the complement pathway.

Description

Novel interaction between Staphylococcus aureus Sbi and C3d proteins

Field of the Invention

The invention relates to the Sbi protein of Staphylococcus aureus, and in particular to the interaction between Sbi and the complement protein C3d. The crystal structure of the Sbi-C3d complex reveals an unexpected interaction between Sbi and the convex surface of C3d, which suggests that particular fragments and variants of Sbi may have utility as inhibitors of the alternative complement pathway.

Background to the Invention

Many bacterial pathogens have evolved ways to adapt to their host environment and to survive host immune system attack by producing a variety of immuno-modulating factors. Gram-positive human pathogen Staphylococcus aureus, for example, is a leading cause of hospital and community acquired infections (1) and is a master of immune evasion. S. aureus has a vast arsenal of intrinsic factors that can regulate both adaptive and innate immune systems in a variety of hosts and in addition has evolved elements that enable the bacterium to hijack host immuno- regulators enabling it to persist in the host environment. Sbi, for instance binds to factor H in a tri -partite complex (2) and ClfA binds to and activates the regulatory protease factor I (3).

Currently six intrinsic complement modulators secreted by

Staphylococcus aureus have been identified and characterized. They include Staphylococcal complement inhibitor (SCIN) (4) , which binds to the classical (C4b2a) and alternative (C3bBb) pathway C3 convertases at a bacterial surface, stabilizing them and

inhibiting their enzymatic activity (5) . The C-terminal fragment of extracellular fibrinogen-binding protein EFb-C and its homologue Ehp bind to the C3d region of C3 , the central

complement component . Efb and Ehp interact with the C3d fragment in native C3 as well as within activated C3b, thereby inhibiting C3b deposition on target surfaces (6-8) . Efb and Ehp binding to C3 has been proposed to induce a conformational change in native C3 so that it can no longer participate in the propagation of downstream activation processes in the cascade amplification pathway (7 , 8 ) . Staphylococcal superantigen-like protein 7 (SSL7) affects the terminal pathway by binding to C5 and in so doing inhibits the complement-mediated bactericidal activity of human serum(9), most likely by preventing C5 cleavage by C5

convertases. Chemotaxis inhibitory protein of S. aureus

(CHIPS) (10) binds to the C5a receptor presented on phagocytes in a way that prevents signaling via the inflammatory anaphylatoxin C5a. Finally, S. aureus binder of immunoglobulin (Sbi) , the most recently characterized member of immuno modulators, affects the adaptive immune system by sequestering host IgG through the formation of insoluble complexes (11) . In addition to

immunoglobulin binding domains I and II, Sbi contains two further domains (Sbi-III and IV) that can bind C3d (in native C3 , iC3b and C3dg) and in concert cause futile fluid phase consumption of C3 , the most abundant complement component, through activation of the alternative pathway (12) . The four N-terminal Sbi domains (I- IV; also referred to as Sbi-E) form a very elongated molecule with a diameter of ~155A(12) , followed by a proline repeat- containing linker and a tyrosine-rich region of unknown

architecture. Although the C-terminal region of Sbi lacks an LPXTG cell -wall anchoring sequence it was previously thought to be attached to the staphylococcal cell wall (13). More recently it has been demonstrated that Sbi is secreted in the surrounding medium (12) . Complement subversion by S. aureus displays a high level of redundancy, involving complement inhibitors with very similar binding modes. For example, Sbi domain IV possesses significant structural and functional similarities with Efb-C and Ehp. The three molecules display a common three-helix bundle fold (7, 8, 14) and share the two most prominent C3d anchoring residues (R131 and N138, in Efb-C; R75 and N82, in Ehp, R231 and N238 in Sbi) despite minimal overall sequence identity. In addition they inhibit the alternative complement pathway and through their interaction with the same residues on the acidic concave surface on C3d they block the binding of C3d to complement receptor 2 (CR2) (15,16), thereby interfering with the vital link between the adaptive and innate branches of the human immune system. On the other hand, the mechanism through which Sbi (Sbi-E and Sbi-III- IV) interferes with alternative pathway is very different from that of Efb-C or Ehp. In the presence of domain III, Sbi-IV induces futile fluid phase consumption of complement component C3 , involving the formation of a covalent adduct with activated C3b(12) . More recently, Sbi has been implied in the hijacking of host complement regulator protein factor H. Alternative pathway regulators factor H (fH) and factor H-like-1 (FHL-1) were shown to bind to Sbi in complex with C3d, forming a tripartite

comple (2) .

Summary of the Invention

The inventors have determined the crystal structure of Sbi-IV bound to C3d. Surprisingly the structure shows two molecules of Sbi-IV interacting with each molecule of C3d. The first

interaction (referred to herein as complex 1) occurs at the concave surface of C3d and resembles the interaction of C3d with other Staphylococcal complement inhibitors such as Efb-C and Ehp. The second interaction (referred to herein as complex 2) occurs at the convex face of C3d. It is unlike any other known

interaction with C3d and may represent the covalent adduct formed between Sbi and C3 in serum. In complex 2, helix 1 of Sbi-IV makes substantial contacts with C3d, suggesting that helix 1 may itself be capable of binding to C3d and acting as an inhibitor of the alternative complement pathway . Thus, in a first aspect the present invention provides a

complement inhibitor comprising an Sbi-IV component capable of binding to C3d, wherein the Sbi-IV component comprises a core peptide of Formula I :

Val Ser lie Glu Lys Ala lie Val Arg His Asp Glu Arg Val Lys Ser Ala Asn Asp Ala lie Ser Lys Leu Asn or having

(i) up to 6 deletions relative to Formula I, which may be independently at the N or C terminal ends of Formula I; and/or (ii) up to 5 substitutions relative to Formula I;

and wherein the Sbi-IV component is not more than 45 amino acids in length.

Formula I represents the sequence of helix 1 of Sbi-IV,

encompassing residues Vall98-Asn222 of the full length Sbi protein sequence shown below. For consistency throughout this specification, residues of Sbi and substituents thereof will be referred to by their position in that full-length sequence. The core sequence preferably adopts an alpha-helical conformation in solution along all or substantially all of its length, e.g. over at least 15, 16, 17, 18, 19 or 20 contiguous amino acids, preferably over at least 20 contiguous amino acids. It is believed that Glu201, Ile204, Val205, His207, Asp208, Val211, Lys212, Asn215 and Ser219 make contacts with the C3d surface. These residues may therefore be referred to as "contact residues" . Preferably not more than five of the contact residues Glu201,

Ile204, Val205, His207, Asp208, Val211, Lys212, Asn215 and Ser219 are substituted or deleted, e.g. not more than four, not more than three, not more than two, or not more than one of the contact residues are substituted or deleted. Any substitutions of these residues are preferably conservative substitutions. In some embodiments all of these residues are conserved, i.e. are not substituted or deleted.

Desirably, substitutions should not disrupt the alpha helical structure of the core sequence or the contacts between the core helix and C3d. Preferably any substitutions of other residues of Formula I introduce residues with high helix- forming propensity, e.g. Met, Ala, Leu, Glu or Lys . Preferably any such

substitutions are conservative. Preferably the core peptide does not contain Pro or Gly, since those residues have particularly poor helix- forming propensity. If Pro or Gly are present, then they desirably occur within the N- or C-terminal 3 amino acids of the core sequence, i.e. N-terminal of Glu201 or C-terminal of Ser21 .

It is also desirable that the relative spacing of these residues and hence the relative orientation of their side chains around the alpha-helix is maintained. Thus, the core peptide sequence preferably has no internal insertions or deletions relative to the sequence of Formula I. If insertions or deletions are present, then they are preferably not within the region defined by the contact residues. Thus they desirably occur within the N- or C-terminal 3 amino acids of Formula I, i.e. N-terminal of Glu201 or C-terminal of Ser219. Within the region defined by the contact residues, i.e. between Glu201 and Ser219, an internal deletion or substitution may be compensated by an internal substitution or deletion respectively in the same region of the core peptide. The substitution and deletion should preferably be as close together as possible in order to minimise disruption to the helical structure of the core peptide and maintain relative positioning of the contact residues. For example, they may be adjacent to one another, or within one, two, three or four residues of one another. The Sbi-IV component may comprise additional Sbi-IV sequence C- terminal of the core peptide sequence. The additional Sbi-IV sequence may be wild type Sbi-IV sequence or may differ from the wild type sequence at one or more positions. The additional sequence typically has at least 80% sequence identity to the corresponding portion of the wild type Glu223-Ala266 sequence.

In some embodiments, the Sbi-IV component comprises not more than 40, not more than 35, or not more than 30 contiguous amino acids of Sbi-IV, e.g. not more than 29, 28, 27, 26 or 25 contiguous amino acids of wild type Sbi-IV sequence.

In total, the Sbi-IV component is not more than 45 amino acids in length, e.g. not more than 40, not more than 35, or not more than 30 amino acids in length, e.g. not more than 29, 28, 27, 26, 25, 24, 23, 22, 21, 20 or 19 amino acids in length.

As described in more detail below, the complement inhibitor may additionally comprise further Sbi sequence in addition to the Sbi-IV component. Such further Sbi sequence will typically be located N-terminal of the core peptide sequence, e.g. it may comprise a Sbi-III domain or a fragment thereof.

In certain embodiments, it may be desirable that the complement inhibitor comprise not more than 100 contiguous amino acids of Sbi, e.g. not more than 90, 80, 70, 60, 50, 40 or 30 amino acids of Sbi, e.g. not more than 29, 28, 27, 26 or 25 contiguous amino acids of Sbi .

In some embodiments, the complement inhibitor itself is not more than 100 contiguous amino acids in length, e.g. not more than 90, 80, 70, 60, 50, 40 or 30 amino acids in length, e.g. not more than 29, 28, 27, 26, 25, 24, 23, 22, 21, 20 or 19 amino acids in length.

Several features of complex 2 suggest ways in which the

interaction between C3d and helix 1 of Sbi-IV can be enhanced. Firstly, in complex 2, Sbi-IV is very close to the location of the thioester bond between residues CyslOlO and Glnl013 of C3d. (The complex whose structure is described herein contains a C3d molecule in which CyslOlO has been replaced by Ala.) It is believed that a transacylation reaction between Sbi-IV and C3d in the fluid phase leads to formation of a covalent adduct between the two molecules and futile consumption of C3 in serum. This transacylation reaction could be enhanced by certain

modifications at helix 1 of Sbi-IV.

Secondly, the structure of complex 2 shows a glycerol molecule bound between the C3d and Sbi molecules. Modifications to helix 1 of Sbi-IV which mimic the interaction between the glycerol molecule and C3d can therefore be used to enhance the binding between Sbi-IV and C3d.

Replacement of the residue at position 204 with Tyr provides an inhibitor which is capable of mimicking the additional

interaction with C3d provided by the bound glycerol molecule in complex 2, thus strengthening the interaction with C3d.

Residues with hydroxyl groups make excellent transacylation targets for the thioester of C3d. Thus replacement of the residue at position 204 or 208 with such a residue provides an inhibitor having increased reactivity with the C3d thioester and consequently an improved ability to inhibit or inactivate C3d.

Thus, in a second aspect, the invention provides a complement inhibitor comprising an Sbi-IV component capable of binding to C3d, wherein the Sbi-IV component comprises a core peptide of Formula II:

Val Ser He Glu Lys Ala X204 Val Arg His X208 Glu Arg Val Lys Ser Ala Asn Asp Ala He Ser Lys Leu Asn; wherein X204 is lie or an aliphatic side chain with a hydroxyl group (e.g. Ser or Thr) or Tyr; and

X208 is Asp or an aliphatic side chain with a hydroxyl group (e.g. Ser or Thr) ,·

or having

(i) up to 6 deletions relative to Formula II, which may be independently at the N or C terminal ends of Formula II; and/or

(ii) up to 5 substitutions relative to Formula II;

with the proviso that X204 and X208 are as defined and are not simultaneously lie and Asp respectively.

It may be desirable to create combinations of these different modifications in the same inhibitor. For example, it may be desirable that the core peptide has both a residue with an aliphatic side chain with a hydroxyl group at position 208 and a Tyr residue at position 204.

Formula II represents the sequence of helix 1 of Sbi-IV

(encompassing residues Vall98-Asn222 of the full length Sbi protein sequence) with appropriate modifications.

As described above in relation to the first aspect of the invention, the core sequence preferably adopts an alpha-helical conformation in solution along all or substantially all of its length, e.g. over at least 15, 16, 17, 18, 19 or 20 contiguous amino acids, preferably at least 20 contiguous amino acids.

In addition to residues X204 and X208, it is believed that

Glu201, Val205, His207, Val211, Lys212, Asn215 and Ser219 make contacts with the C3d surface. These residues may therefore be referred to as "contact residues" .

Preferably not more than five of the contact residues Glu201, Val205, His207, Val211, Lys212, Asn215 and Ser219 are substituted or deleted, e.g. not more than four, not more than three, not more than two, or not more than one of the contact residues are substituted or deleted. Any substitutions of these residues are preferably conservative substitutions. In some embodiments all of these residues are conserved, i.e. are not substituted or deleted.

Desirably, substitutions should not disrupt the alpha helical structure of the core sequence or the contacts between the core helix and C3d. Preferably any substitutions of other residues of Formula II introduce residues with high helix- forming propensity, e.g. Met, Ala, Leu, Glu or Lys . Preferably any such

substitutions are conservative. Preferably the core peptide does not contain Pro or Gly, since those residues have particularly poor helix- forming propensity. If Pro or Gly are present, then they desirably occur within the N- or C-terminal 3 amino acids of the core sequence, i.e. N-terminal of Glu201 or C-terminal of Ser219.

It is also desirable that the relative spacing of these residues and hence the relative orientation of their side chains around the alpha-helix is maintained. Thus, the core sequence

preferably has no internal insertions or deletions relative to the sequence of Formula II. If insertions or deletions are present, then they are preferably not within the region defined by the contact residues. Thus they desirably occur within the N- or C-terminal 3 amino acids of Formula II, i.e. N-terminal of Glu201 or C-terminal of Ser219. Within the region defined by the contact residues, i.e. between Glu201 and Ser219, an internal deletion or substitution may be compensated by an internal substitution or deletion respectively in the same region of the core peptide. The substitution and deletion should preferably be as close together as possible in order to minimise disruption to the helical structure of the core peptide and maintain relative positioning of the contact residues. For example, they may be adjacent to one another, or within one, two, three or four residues of one another. The Sbi-IV component of the complement inhibitor comprises at least the core peptide, which corresponds to a modified form of helix 1 of Sbi-IV. However it may further comprise additional Sbi-IV sequence C- terminal of the core peptide sequence, up to an entire Sbi-IV domain. The additional Sbi-IV sequence may be wild type Sbi-IV sequence or may differ from the wild type sequence at one or more positions. The additional sequence typically has at least 80% sequence identity to the corresponding portion of the wild type Glu223-Ala266 sequence.

Thus the Sbi-IV component may comprise an additional 5, 10, 15, 20, 25 or more contiguous amino acids located C-terminal of the core peptide. It may be up to 65 amino acids in length, e.g. up to 60, up to 50, up to 45, up to 40, up to 35, up to 30, or up to 25 amino acids in length.

It may comprise a full Sbi-IV domain (residues Vall98-Ala266 , with appropriate modif cations) , or a sequence having at least 80% identity to Sbi-IV over the entire length of the Sbi-IV domain .

The complement inhibitor may comprise further Sbi sequence, such as an Sbi-III domain (residues 150-197 of the full length Sbi sequence shown herein) or a sequence having at least 80% identity to Sbi-III over the entire length of the Sbi-III domain, or a fragment of either.

The Sbi-IV component of the complement inhibitor may comprise or consist of one of the following core peptide sequences:

Val Ser lie Glu Lys Ala Tyr Val Arg His Asp Glu Arg Val Lys Ser Ala Asn Asp Ala lie Ser Lys Leu Asn; Val Ser lie Glu Lys Ala Ser Val Arg His Asp Glu Arg Val Lys Ser Ala Asn Asp Ala lie Ser Lys Leu Asn; Val Ser lie Glu Lys Ala Thr Val Arg His Asp Glu Arg Val Lys Ser Ala Asn Asp Ala lie Ser Lys Leu Asn; Val Ser lie Glu Lys Ala lie Val Arg His Ser Glu Arg Val Lys Ser Ala Asn Asp Ala lie Ser Lys Leu Asn;

Val Ser lie Glu Lys Ala lie Val Arg His Thr Glu Arg Val Lys Ser Ala Asn Asp Ala lie Ser Lys Leu Asn;

Val Ser lie Glu Lys Ala Tyr Val Arg His Ser Glu Arg Val Lys Ser Ala Asn Asp Ala lie Ser Lys Leu Asn; or

Val Ser lie Glu Lys Ala Tyr Val Arg His Thr Glu Arg Val Lys Ser Ala Asn Asp Ala lie Ser Lys Leu Asn.

The Sbi-IV component of the complement inhibitor may comprise or consist of one of the following full-length Sbi-IV sequences, or a sequence having at least 80% identity to any one of these sequences (while still satisfying the requirements for the definition of the core peptide, above) :

Val Ser He Glu Lys Ala Tyr Val Arg His Asp Glu Arg Val Lys Ser

Ala Asn Asp Ala He Ser Lys Leu Asn Glu Lys Asp Ser He Glu Asn

Arg Arg Leu Ala Gin Arg Glu Val Asn Lys Ala Pro Met Asp Val Lys

Glu His Leu Gin Lys Gin Leu Asp Ala Leu Val Ala Gin Lys Asp Ala

Glu Lys;

Val Ser He Glu Lys Ala Ser Val Arg His Asp Glu Arg Val Lys Ser

Ala Asn Asp Ala He Ser Lys Leu Asn Glu Lys Asp Ser He Glu Asn

Arg Arg Leu Ala Gin Arg Glu Val Asn Lys Ala Pro Met Asp Val Lys

Glu His Leu Gin Lys Gin Leu Asp Ala Leu Val Ala Gin Lys Asp Ala

Glu Lys;

Val Ser He Glu Lys Ala Thr Val Arg His Asp Glu Arg Val Lys Ser

Ala Asn Asp Ala He Ser Lys Leu Asn Glu Lys Asp Ser He Glu Asn Arg Arg Leu Ala Gin Arg Glu Val Asn Lys Ala Pro Met Asp Val Lys

Glu His Leu Gin Lys Gin Leu Asp Ala Leu Val Ala Gin Lys Asp Ala

Glu Lys;

Val Ser He Glu Lys Ala He Val Arg His Ser Glu Arg Val Lys Ser

Ala Asn Asp Ala He Ser Lys Leu Asn Glu Lys Asp Ser He Glu Asn

Arg Arg Leu Ala Gin Arg Glu Val Asn Lys Ala Pro Met Asp Val Lys

Glu His Leu Gin Lys Gin Leu Asp Ala Leu Val Ala Gin Lys Asp Ala

Glu Lys;

Val Ser He Glu Lys Ala He Val Arg His Thr Glu Arg Val Lys Ser

Ala Asn Asp Ala He Ser Lys Leu Asn Glu Lys Asp Ser He Glu Asn

Arg Arg Leu Ala Gin Arg Glu Val Asn Lys Ala Pro Met Asp Val Lys

Glu His Leu Gin Lys Gin Leu Asp Ala Leu Val Ala Gin Lys Asp Ala Glu Lys;

Val Ser He Glu Lys Ala Val Arg His Ser Glu Arg Val Lys Ser

Ala Asn Asp Ala He Ser Lys Leu Asn Glu Lys Asp Ser He Glu Asn

Arg Arg Leu Ala Gin Arg Glu Val Asn Lys Ala Pro Met Asp Val Lys

Glu His Leu Gin Lys Gin Leu Asp Ala Leu Val Ala Gin Lys Asp Ala

Glu Lys; or

Val Ser He Glu Lys Ala ΊΣΣ Val Arg His Thr Glu Arg Val Lys Ser

Ala Asn Asp Ala He Ser Lys Leu Asn Glu Lys Asp Ser He Glu Asn

Arg Arg Leu Ala Gin Arg Glu Val Asn Lys Ala Pro Met Asp Val Lys

Glu His Leu Gin Lys Gin Leu Asp Ala Leu Val Ala Gin Lys Asp Ala

Glu Lys.

The structure of complex 1 shows contacts between helix 2 of Sbi- IV and the concave face of C3d, primarily via residues Arg231, Arg235, Asn238 and Lys239. The proximity of these residues to helix 1 suggests that helix 1 could be modified to make similar contacts with the concave face of C3d while also maintaining its capacity to interact with the convex surface of C3d. This enables a single modified helix to be designed which is capable of mimicking both binding modes between Sbi-IV and C3d. Binding to the concave surface of C3d may enable inhibition of further aspects of C3d function, such as its binding to CR2.

Thus, in a third aspect, the invention provides a complement inhibitor comprising an Sbi-IV component capable of binding to C3d, wherein the Sbi-IV component comprises a core peptide of Formula III:

Val Ser lie Glu Lys Ala X204 Val Arg His X208 Glu Arg Val Lys X213 X214 Asn Asp Ala lie Ser Lys Leu Asn;

wherein

X213 is Ser or a residue with a polar side chain (e.g. Asn); and X214 is Ala or a residue with a positively charged side chain (e.g. Lys or Arg) ;

or having

(i) up to 6 deletions relative to Formula III, which may be independently at the N or C terminal ends of Formula III; and/or

(ii) up to 5 substitutions relative to Formula III;

with the proviso that X213 and X214 are as defined and are not simultaneously Ser and Ala respectively;

and wherein the Sbi-IV component is not more than 33 amino acids in length.

In certain embodiments, it may be desirable that both X213 has a polar side chain and X214 has a positively charged side chain.

Thus, for example, the inhibitor may have Asn at position 213 and Lys at position 214, or may have Asn at position 213 and Arg at position 214. Formula III represents the sequence of helix 1 of Sbi-IV

As described above in relation to the first and second aspects of the invention, the core sequence preferably adopts an alpha- helical conformation in solution along all or substantially all of its length, e.g. over at least 15, 16, 17, 18, 19 or 20 contiguous amino acids, preferably at least 20 contiguous amino acids . It is believed that Glu201, Ile204, Val205, His207, Asp208,

Val211, Lys212, Asn215 and Ser219 also make contacts with the C3d surface. These residues may therefore be referred to as "contact residues . Preferably not more than five of the contact residues Glu201,

The core peptide may include one or more of the modifications described above in relation to the second aspect of the

invention. This may allow further control over the interaction with C3d, particularly with the convex face of C3d, e.g. via transacylation and/or interactions mimicking the glycerol molecule of complex 2.

Thus, for example, amongst the contact residues:

(i) Ile204 may be substituted by an aliphatic side chain with a hydroxyl group, e.g. Ser or Thr;

(ii) Ile204 may be substituted by Tyr,- and/or

(iii) Asp208 may be substituted by a residue with an aliphatic side chain with a hydroxyl group, e.g. Ser or Thr.

In certain embodiments the core peptide may have both a residue having an aliphatic side with a hydroxyl group at position 208 and a Thr residue at position 204. Desirably, substitutions should not disrupt the alpha helical structure of the core sequence or the contacts between the core helix and C3d. Preferably any other substitutions of other residues of Formula III introduce residues with high helix- forming propensity, e.g. Met, Ala, Leu, Glu or Lys. Preferably any such substitutions are conservative. Preferably the core peptide does not contain Pro or Gly, since those residues have particularly poor helix- forming propensity. If Pro or Gly are present, then they desirably occur within the N- or C-terminal 3 amino acids of the core sequence, i.e. N-terminal of Glu201 or C- terminal of Ser219.

preferably has no internal insertions or deletions relative to the sequence of Formula III. If insertions or deletions are present, then they are preferably not within the region defined by the contact residues. Thus they desirably occur within the N- or C-terminal 3 amino acids of Formula III, i.e. N-terminal of

Glu201 or C-terminal of Ser219. Within the region defined by the contact residues, i.e. between Glu201 and Ser219, an internal deletion or substitution may be compensated by an internal substitution or deletion respectively in the same region of the core peptide. The substitution and deletion should preferably be as close together as possible in order to minimise disruption to the helical structure of the core peptide and maintain relative positioning of the contact residues. For example, they may be adjacent to one another, or within one, two, three or four residues of one another.

Thus the Sbi-IV component may comprise or consist of the sequence : Val Ser He Glu Lys Ala He Val Arg His Asp Glu Arg Val Lys Asn Lys Asn Asp Ala He Ser Lys Leu Asn The Sbi-IV component may comprise additional Sbi-IV sequence C- terminal of the core peptide sequence. The additional Sbi-IV sequence may be wild type Sbi-IV sequence or may differ from the wild type sequence at one or more positions. The additional sequence typically has at least 80% sequence identity to the corresponding portion of the wild type Glu223 -Ala266 sequence.

In total, the Sbi-IV component is not more than 33 amino acids in length, e.g. not more than 30 amino acids in length, e.g. not more than 29, 28, 27, 26, 25, 24, 23, 22, 21, 20 or 19 amino acids in length.

The complement inhibitor may further Sbi sequence in addition to the Sbi-IV component. Such further Sbi sequence will typically be located N-terminal of the Sbi-IV component, e.g. it may comprise an Sbi-III domain or a fragment thereof.

In all aspects of the invention, the complement inhibitor is typically 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.

The identification of complex 2 as a binding mode between C3d and Sbi suggests that other molecules which are capable of binding to the convex face of C3d may be used as complement inhibitors.

Typically such molecules will bind to the convex face of C3d and thus inhibit binding of Sbi to the same C3d molecule, e.g. by steric interference .

Thus a further example of a complement inhibitor according to the invention is an antibody specific for the convex face of C3d, or a fragment thereof comprising a functional antigen binding site. A further example is a nucleic acid molecule (also known as an aptamer) capable of binding specifically to the convex face of C3d. Such aptamers may be composed of R A or DNA, or synthetic analogs thereof, and are typically oligonucleotides, e.g. of 20 nucleotides or fewer.

A complement inhibitor 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. The heterologous component may comprise a non-proteinaceaous molecule chemically linked to the complement-binding protein. Examples include polyethylene glycol (PEG) , and poly-sialic acid.

Alternatively (where appropriate) the heterologous component may comprise a proteinaceous moiety, e.g. an antibody Fc region. A proteinaceous heterologous component may be expressed as a fusion protein with the complement inhibitor.

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.

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 .

Additionally or alternatively, the heterologous component may be, or may comprise, a further anti-inflammatory agent covalently coupled to the complement inhibitor. The anti-inflammatory agent may be a drug suitable for treating the inflammatory condition from which an intended recipient is suffering. Suitable agents are well known to the skilled person and include steroidal and non-steroidal anti- inflammatory agents, and anti-inflammatory proteins. Specific examples include, but are not limited to, Erythropoietin (EPO) , recombinant human IL-1 receptor antagonist (e.g. Anakinra) and TNF inhibitors, including soluble TNF receptor proteins (e.g. Etanercept) .

Regardless of the nature of the anti- inflammatory agent, it may be covalently linked to any part of the complement inhibitor.

For example, it may be conjugated to the Sbi-IV component or to another heterologous component (if present) . When the antiinflammatory agent is a protein, it may be provided as a fusion protein with the complement inhibitor, optionally via a peptide linker as described above.

Fusion (or chemical conjugation) to other proteins such as albumin may also be useful to extend half-life in vivo. The invention further provides a nucleic acid encoding a

complement inhibitor as described above . The nucleic acids may be DNA or RNA, in single or double stranded form. Also provided is an expression vector comprising such a nucleic acid. The expression vector will typically comprise a nucleic acid of the invention in which the open reading frame encoding the complement inhibitor is operably linked to regulatory sequences (e.g. promoter, enhancer and transcriptional terminator sequences, as well as translational control sequences) to allow transcription and translation of the peptide in a desired cell type. The open reading frame may further encode a signal sequence so that the complement inhibitor can be secreted from the cell in which it is expressed. The signal sequence may be cleaved as part of the secretion process. The skilled person will be able to design suitable vectors depending on the desired cell type, which may include bacterial, yeast, insect or

mammalian cells. Also provided is a host cell comprising a nucleic acid or expression vector as described above. Suitable host cells include bacterial, yeast, insect and mammalian host cells. In certain embodiments the host cell is capable of expressing and optionally secreting the complement inhibitor.

The invention further provides a method for inhibiting complement activation in a biological system, comprising contacting the system with a complement inhibitor as described in relation to any of the above aspects of the invention.

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. 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 inhibitor may comprise a Sbi-III domain capable of binding to C3 protein. If it is desirable specifically to inhibit the alternative pathway, the complement inhibitor typically will not comprise a functional Sbi-III domain, i.e. one capable of binding to C3 protein.

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 ..

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 Clq, Clr, Cls, C2 , C4 and C3. The lectin pathway will typically include mannose-binding protein (MBP) or ficolin, and the proteases MASP-1 and MASP-2.

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. 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.

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 .

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.

Thus the invention provides a method of treating an inflammatory condition in an individual, comprising administering a complement inhibitor as described above to said individual .

The invention also provides use of a complement inhibitor as described above in the preparation of a medicament for the treatment of an inflammatory condition. The invention also provides a complement inhibitor as described above for use in the treatment of an inflammatory condition. 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) .

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.

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.

It may be desirable to treat these conditions using a complement inhibitor which lacks a functional Sbi-III domain. This strategy could avoid unnecessary inhibition of the other complement pathways, so maintaining as much of the patient's normal immune function as possible. The complement inhibitor may be administered in conjunction with a further anti-inflammatory agent, which may be a drug suitable for treating the inflammatory condition from which an intended recipient is suffering. The complement inhibitor and the further anti-inflammatory agent may be provided in the same composition or in separate

compositions and may be formulated for administration together or separately. In some embodiments, the anti-inflammatory agent and the complement inhibitor may be covalently linked, e.g. the anti- inflammatory agent may be conjugated to the complement inhibitor or provided as fusion protein with it .

Suitable anti-inflammatory agent are well known to the skilled person and include steroidal and non-steroidal anti-inflammatory agents, and anti-inflammatory proteins. Specific examples include, but are not limited to, Erythropoietin (EPO) ,

recombinant human IL-1 receptor antagonist (e.g. Anakinra) and TNF inhibitors, including soluble TNF receptor proteins (e.g. Etanercept) .

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. Certain of the inhibitors described herein, especially those capable of binding to the concave face of C3d (i.e. those of the third aspect of the invention) may be capable of inhibiting 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 such a complement inhibitor to said individual. The invention further provides the use of such a complement inhibitor in the preparation of a medicament for inhibiting B cell proliferation and/or antibody production.

The invention further provides such a complement inhibitor fir use in a method of inhibiting B cell proliferation and/or antibody production.

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 .

The interaction between C3d/C3dg and CR2 has also been implicated in HIV infection of 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 (Dopper 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.

Thus the invention provides a method of treatment or prophylaxis of HIV infection in an individual comprising administering a complement inhibitor as described above to said individual . The invention further provides the use of such a complement inhibitor in the preparation of a medicament for treatment or prophylaxis of HIV infection. The invention further provides such a complement inhibitor for use in treatment or prophylaxis of HIV infection.

It will be appreciated that all therapeutic aspects of the invention may equally make use of a nucleic acid encoding a complement inhibitor of the invention, or an expression vector comprising such a nucleic acid, as described above. Nucleic acids and expression vectors may be used in so-called DNA vaccination techniques, in which the nucleic acid is administered directly to a subject, and is taken up by cells or tissues of that subject and the encoded protein expressed therein. It may be desirable that the protein is secreted from the cell. Thus the nucleic acid or expression vector may encode a complement inhibitor having a signal sequence which promotes secretion from mammalian cells.

Alternatively a cell comprising such a nucleic acid or expression vector, which is capable of expressing and optionally secreting the complement inhibitor, may be used therapeutically. The cells may be derived from the subject to whom they are to be

administered and have been engineered to be capable of expressing and optionally secreting the complement inhibitor. Alternatively they may be MHC compatible with the subject. Alternatively, they may be encapsulated in a non- immunogenic material (e.g. alginate) for administration to the subject.

The identification of complex 2 as a binding mode between Sbi and C3d further leads to new methods of identifying complement inhibitors. As explained above, compounds which are capable of binding to the convex face of C3d and inhibiting binding of Sbi may find use as complement inhibitors. Thus the invention provides a method of screening for a

complement inhibitor comprising contacting a candidate compound with C3d and

(i) determining the ability of the candidate compound to bind to the convex face of C3d; and/or

(ii) determining the ability of the candidate compound to inhibit binding of Sbi-IV or an analogue thereof to C3d, e.g. to the convex face of C3d. The performance of the candidate compound may be compared to a reference complement inhibitor known to bind to the convex face of Sbi-IV. The reference complement inhibitor may comprise or consist of Sbi-IV, or a complement inhibitor as described elsewhere in this specification.

The method may be performed as a competitive assay, in which C3d is contacted with both the candidate compound and the reference complement inhibitor. In such embodiments, the reference complement inhibitor may be the Sbi-IV or analogue thereof referred to in (ii) above.

Particularly for non-competitive assay formats, the performance of the candidate compound and the reference complement inhibitor may both be determined separately and correlated with one another. Determination may be performed at substantially the same time . Alternatively the performance of the candidate compound may be compared with a previously-determined value for the reference complement inhibitor, e.g. determined under similar or substantially identical assay conditions.

The candidate compound may also be a complement inhibitor as described elsewhere in this specification. However it may be any other molecular entity such as a small molecule (e.g. less than 500 Da), a protein (e.g. composed of greater than 50 amino acids) such as an antibody or a fragment thereof comprising a functional antigen binding domain, a peptide (e.g. composed of 50 amino acids or less), a nucleic acid (which may be DNA or RNA, e.g. an aptamer) , a sugar, oligosaccharide or polysaccharide, etc..

The ability of the various compounds to bind the convex face of C3d may be determined directly or indirectly, and may include determining non-covalent binding to C3d, formation of a covalent adduct with C3d (e.g. by transacylation) , and/or the ability to inhibit complement activation. Suitable methods may include crystallisation of the candidate molecule with C3d followed by determination of the structure of the complex (e.g. by X-ray crystallography) . MR methods can be used to analyse binding in solution, as can methods such as surface plasmon resonance. Immunological methods such as ELISA may also be used. The skilled person will be capable of designing a suitable assay format to suit their needs.

The method may be applied to optimise a known complement inhibitor. Thus the method may comprise providing a parent complement inhibitor which is capable of binding to the convex face of C3d;

providing a candidate compound which is a modified form of said parent complement inhibitor;

contacting said candidate compound with C3d; and

(i) comparing the ability of the candidate compound to bind to the convex face of C3d with that of the parent complement inhibitor; and/or

(ii) comparing the ability of the candidate compound to inhibit binding of Sbi-IV, or an analogue thereof to the convex face of C3d with that of the parent complement inhibitor.

The parent complement inhibitor may be a complement inhibitor as described elsewhere in this specification. The method may be performed as a competitive assay, in which C3d is contacted with both the candidate compound and the parent complement inhibitor. Alternatively the performance of the candidate compound and the parent complement inhibitor may both be determined separately and correlated with one another. Alternatively the performance of the candidate compound may be compared with a previously- determined value for the parent complement inhibitor, e.g.

determined under similar or substantially identical assay conditions .

The method may comprise the steps of providing a parent

complement inhibitor, and modifying the parent complement inhibitor to generate the candidate compound.

The modification may involve the steps of providing a parent nucleic acid sequence (e.g. a DNA sequence) encoding the parent complement inhibitor and modifying the nucleic acid sequence to generate a variant nucleic acid sequence encoding the candidate molecule .

Since helix 1 of Sbi-IV is implicated in the interaction between Sbi and C3d, the parent complement inhibitor may have an Sbi component comprising helix 1 of Sbi-IV or a variant thereof, and the candidate compound may have an Sbi component having a modified form of helix 1 compared with the parent complement inhibitor . The parent complement inhibitor may comprise an Sbi-IV domain, a variant thereof having at least 80% sequence identity therewith, or a fragment of either capable of binding to C3d, and should include a sequence corresponding to helix 1 of Sbi-IV, i.e.

residues Vall98 -Asn222 in the sequence provided herein, or an analogue thereof . In some embodiments the candidate molecule may differ from the parent complement inhibitor at just one amino acid, or at 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. In some embodiments it may differ from the parent complement inhibitor only in the region corresponding to helix 1 of Sbi-IV.

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

Figure 1. Crystal structures of the two Sbi-IV-C3d complexes. Ribbon representations of the two Sbi-IV-C3d complexes in different C3d side view orientations (A and B) . The positions of the concave and convex faces of C3d are indicated. The N- and C- termini of each molecule are indicated and the interacting a helices of C3d and both Sbi-IV molecules are specified. All molecular figures were prepared using MacPyMOL (www.pymol.org) . Figure 2. Close-up view of the interacting C3d and Sbi-IV residues in complex 1. A. Ribbon diagrams of C3d and Sbi-IV highlighting the main amino acids involved in the interactions between the two molecules at the concave surface of C3d. The interacting residues are represented as stick models and are labeled according to full length Sbi and intact pre-pro C3 numbering. B. Detailed view of the interactions involving the two ^■C3d anchoring' Sbi-IV residues, R231 and N238.

Figure 3. Close-up view of the interactions between Sbi-IV and the C3d thioester region observed in complex 2. A. Ribbon representation of the newly discovered Sbi-IV contact interface at the convex surface of C3d. In this orientation, amino acids at the N- terminus of helix al in Sbi-IV are seen interacting with the C3d thioester region residues. B. Detailed view of the interactions made by the C-terminal Sbi-IV residues, including ones from helices al and 3. Two glycerol molecules were also found to bind to the thioester region of C3d (glycerol was used as a cryo-protectant during data collection) .

Figure 4. Diagrammatic representation of various forms of C3 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. (See O2007/138238 for more details.) Figure 5. The interaction surface of Sbi-IV on the convex face of C3d.

Detailed Description of the Invention

Complement

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. 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 between CyslOlO and Glnl013 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.

C3 is cleaved at a number of sites during the complement

activation process, giving rise to various proteolytic fragments including C3a, C3b, iC3b, C3c and C3dg. These are illustrated in Figure 4. 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.

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..

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 inhibitors 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..

Thus the complement inhibitors 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 . [Seelen, M. A. 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 296, 187-98 (2005).]

The inhibitors described in this specification are said to be capable of binding to C3d. They may therefore be capable of binding to isolated C3d, or to any C3 molecule containing C3d, including native C3 , C3 ( HCH₃) , C3 (H₂0) , C3b, iC3b, iC3 (NHCH₃) , iC3 (H₂0) and C3dg. The sequence of human C3d is as follows:

1 MLDAERLKHLIVTPSGCGEQNMIG TPTVIAVHYLDETEQWEKFGLEKRQGALELIKKGY

61 TQQLAFRQPSSAFAAFV RAPST LTAYWKVFSLAV LIAIDSQVLCGAV WLILEKQK

121 PDGVFQEDAPVIHQE IGGLRNNNE DMALTAFVLISLQEAKDICEEQV SLPGSITKAG

181 DFLEANYMNLQRSYTVAIAGYALAQMGRLKGPLLNKFLTTA D NRWEDPG QLYNVEAT

241 SYALLIALLQLKDFDFVPPVVRWLNEQRYYGGGYGSTQATFMVFQAIJAQYQKDAP

Its structure is described by Nagar et al . (1998) (Ref. 17) . The term C3d in this specification can be construed to refer to a molecule having this sequence, or a variant thereof having at least 80% sequence identity to this sequence, e.g. at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to this sequence across its full length, or a fragment of either capable of binding to Sbi-IV. C3d molecules from other species may be employed.

In the C3d variant used for crystallisation in the examples, the Cys at position 17 (position 1010 of full-length C3) was replaced by Ala, but the sequence was otherwise identical to that shown above .

The convex face of C3d with which Sbi-IV interacts involves residues from C3d loop regions connecting helices alphal and alpha2, alpha3 and alpha4, and alphas and alpha6 (Nagar et al . (1998)) . It can be broadly described as the area bounded by residues S15, K78, P130, R141 and Y273 (see Fig. 5) . It includes residues Glu 19, Q20, F76, R79, 1132 and H133. C17 can also be considered part of the convex face of the molecule although it is generally not accessible to water molecules at the surface of the molecule.

A molecule which binds the convex face of C3d will therefore make contacts with one or more of residues S15, C17, Q20, F76, K78, R79, P130, 1132, H133 R141 and Υ273, or residues at structurally equivalent positions in C3d variants (e.g. A17 in the molecule used in the examples) or in C3d from other species. Equivalence may be determined by sequence alignment and/or comparison with the structure described by Nagar et al . (2008) . Contacts may be via non-covalent (e.g. via polar, ionic or van der Waals interactions) or covalent interactions. For example, a molecule may bind covalently to C17 or Q20 as part of a

transesterification reaction with the thiolactone ring of C3d. Inhibitors which do not transesterify with the C17 or Q20 residues may nevertheless sterically inhibit reaction of the C3d thiolactone (transesterification) with other molecules, e.g. on pathogen surfaces, and so inhibit the function of C3d.

Interaction of a candidate molecule or inhibitor with the convex face of C3d may be assessed, for example, by crystallisation of a complex between the molecules followed by X-ray crystallography, or by N R, e.g. as described herein. Mass spectroscopic

techniques may also be useful. For example, a covalent complex between a candidate molecule or inhibitor and C3d may be

subjected to proteolysis and the resulting fragments analysed by mass spectroscopy to determine which fragments are associated with one another. If the molecules do not interact covalently, then the complex may be covalently cross- linked (e.g. by

irradiation, or by use of a suitable chemical cross- linking reagent) before proteolysis and mass spectroscopy. Functional assays may also be employed, e.g. by assessing the ability of a candidate molecule or inhibitor to inhibit covalent binding of another molecule to C3d, such as a molecule on the surface of a pathogen, by transesterification with the C3d thiolactone.

The convex surface of C3d with which Sbi-IV interacts comprises the residues Q105, E167, Q168, D103, S104, A101, 1164, 1102, D1S3, V97, 1100, Q50, N98, L99, R49, H33, D36, L46, K291 and E37. A molecule which binds the concave face of C3d will therefore make contacts with one or more of those residues or residues at structurally equivalent positions in C3d variants or in C3d from other species. Equivalence may be determined by sequence alignment and/or comparison with the structure described by Nagar et al .. Interaction with C3d may be determined as described above for the convex face of C3d, or by functional assays such as inhibition of binding between C3d and CR2.

Sbi protein

The sequence of the Sbi protein, including signal sequence, follows :

Met Lys Asn Lys Tyr He Ser Lys Leu Leu Val Gly

1 5 10

Ala Ala Thr He Thr Leu Ala Thr Met He Ser Asn

15 20

Gly Glu Ala Lys Ala Ser Glu Asn Thr Gin Gin Thr

25 30 35

Ser Thr Lys His Gin Thr Thr Gin Asn Asn Tyr Val

40 45

Thr Asp Gin Gin Lys Ala Phe Tyr Gin Val Leu His

50 55 60

Leu Lys Gly He Thr Glu Glu Gin Arg Asn Gin Tyr

65 70

He Lys Thr Leu Arg Glu His Pro Glu Arg Ala Gin

75 80

Glu Val Phe Ser Glu Ser Leu Lys Asp Ser Lys Asn

85 90 95

Pro Asp Arg Arg Val Ala Gin Gin Asn Ala Phe Tyr

100 105

Asn Val Leu Lys Asn Asp Asn Leu Thr Glu Gin Glu

110 115 120

Lys Asn Asn Tyr He Ala Gin He Lys Glu Asn Pro

125 130

Asp Arg Ser Gin Gin Val Trp Val Glu Ser Val Gin

135 140

Ser Ser Lys Ala Lys Glu Arg Gin Asn He Glu Asn

145 150 155

Ala Asp Lys Ala He Lys Asp Phe Gin Asp Asn Lys

160 165

Ala Pro His Asp Lys Ser Ala Ala Tyr Glu Ala Asn

170 175 180

Ser Lys Leu Pro Lys Asp Leu Arg Asp Lys Asn Asn

185 190

Arg Phe Val Glu Lys Val Ser He Glu Lys Ala He

195 200

Val Arg His Asp Glu Arg Val Lys Ser Ala Asn Asp

205 210 215

Ala He Ser Lys Leu Asn Glu Lys Asp Ser He Glu

220 225

Asn Arg Arg Leu Ala Gin Arg Glu Val Asn Lys Ala

230 235 240

Pro Met Asp Val Lys Glu His Leu Gin Lys Gin Leu

245 250

Asp Ala Leu Val Ala Gin Lys Asp Ala Glu Lys Lys Val Ala Pro Lys Val Glu Ala Pro Gin He Gin Ser

265 270 275

Pro Gin lie Glu Lys Pro Lys Val Glu Ser Pro Lys

280 285

Val Glu Val Pro Gin He Gin Ser Pro Lys Val Glu

290 295 300

Val Pro Gin Ser Lys Leu Leu Gly Tyr Tyr Gin Ser

305 310

Leu Lys Asp Ser Phe Asn Tyr Gly Tyr Lys Tyr Leu

315 320

Thr Asp Thr Tyr Lys Ser Tyr Lys Glu Lys Tyr Asp

325 330 335

Thr Ala Lys Tyr Tyr Tyr Asn Thr Tyr Tyr Lys Tyr

340 345

Lys Gly Ala He Asp Gin Thr Val Leu Thr Val Leu

350 355 360

Gly Ser Gly Ser Lys Ser Tyr He Gin Pro Leu Lys

365 370

Val Asp Asp Lys Asn Gly Tyr Leu Ala Lys Ser Tyr

375 380

Ala Gin Val Arg Asn Tyr Val Thr Glu Ser He Asn

385 390 395

Thr Gly Lys Val Leu Tyr Thr Phe Tyr Gin Asn Pro

400 405

Thr Leu Val Lys Thr Ala He Lys Ala Gin Glu Thr

410 415 420

Ala Ser Ser He Lys Asn Thr Leu Ser Asn Leu Leu

425 430

Ser Phe Trp Lys .

435

From N- to C-terminus, the protein is composed of a leader peptide, domains Sbi-I, II, III and IV, a putative wall-anchor sequence (WR) and a so-called Y region.

In this specification, by "Sbi-I domain" is meant a polypeptide sequence comprising at least amino acids 42 to 90 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.

By "Sbi-II domain" is meant a polypeptide sequence comprising at least amino acids 92 to 149 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 .

By "Sbi -III domain" is meant a polypeptide sequence comprising at least amino acids 150 to 197 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.

By "Sbi-IV domain" is meant a polypeptide sequence comprising at least amino acids 198 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.

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.

The complement inhibitors described here may bind C3 ,

particularly C3d, 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 bind human C3 protein.

Core peptide sequence and Sbi-IV component

The complement inhibitors described herein contain an "Sbi-IV component" which comprises or consists of a "core peptide" having the sequence of helix 1 of Sbi-IV, or a sequence corresponding to helix 1 of Sbi-IV. Such core peptide sequences include, for example, those of Formulae I, II and III. Native or wild type human Sbi-IV helix 1 consists of residues Vall98-Asn222 of Sbi, having the sequence

Val Ser lie Gin Lys Ala lie Val Arg His Asp Glu Arg Val Lys Ser Ala Asn Asp Ala lie Ser Lys Leu Asn. Residues "corresponding to" helix 1 are those residues of the Sbi component of the inhibitor which align with the residues of helix 1 in native Sbi-IV when the two sequences are optimally aligned.

The core sequence is at least 19 amino acids in length, and preferably 20, 21, 22, 23, 24 or 25 amino acids in length.

Typically it has at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or at least 20 amino acids which are identical to the

corresponding residues of native helix 1 when the sequences are optimally aligned, although this may be higher (e.g. 21, 22, 23, 24 or 25 identical amino acids) . A number of residues of helix 1 are believed to form contacts with the surface of C3d and are thus designated "contact

residues". These include Glu201, Ile204, Val205, His207, Asp208, Val211, Lys212, Asn215 and Ser219, numbered according to the full-length Sbi sequence.

If substitutions are made at these residues (apart from those designated in Formulae I, II and III) , then it may be preferable that these are conservative substitutions.

Conservative substitutions may be defined as substitutions within the following groups of amino acids:

I. Asp and Glu (acidic amino acids);

II. Arg, Lys and His (basic amino acids);

III. Asn, Gin, Ser, Thr and Tyr (uncharged polar amino acids);

IV. Ala, Gly, Val, Leu, lie, Pro, Phe, Met, Trp and Cys (non- polar amino acids) . Preferably the sequence corresponding to helix 1 itself adopts an alpha helical conformation. An alpha helix is a well known secondary structural motif in which every backbone carbonyl oxygen forms a hydrogen bond with the backbone NH group of the amino acid four residues later (i.e. in the C-terminal direction) along the peptide chain, to form a helix having 3.6 amino acid residues per turn. Preferably the core sequence forms at least 5 turns of helix, more preferably at least 6 turns of helix. For example, it may adopt an alpha helical conformation over at least 15, 16, 17, 18, 19 or 20 contiguous amino acids, preferably at least 20 contiguous amino acids.

Certain amino acids have a higher helix forming propensity than others. The amino acids Met, Ala, Leu, Glu and Lys have

particularly high helix- forming propensity. Thus it may be desirable that substitutions of core peptide residues (other than the contact residues) introduce residues with high helix forming propensity. In some embodiments a substitution of a core residue (other than a contact residue) introduces a residue with high helix forming propensity which is also a conservative

substitution for the corresponding residue in helix 1.

Gly and Pro have poor helix-forming propensities (although Pro may be found as the first amino acid of an alpha helix) . Thus the inhibitors of the invention preferably do not contain Gly or Pro within the core peptide sequence.

Certain pairs of residues are capable of forming intramolecular bonds between their side chains. These may be capable of stabilising an alpha helical structure. Such interactions normally take place between residues separated by three amino acids in the linear peptide chain (i.e. between residues X and X+4) . For example a pair of residues comprising an acidic residue (Glu or Asp) and a basic residue (Lys or Arg) may form a salt bridge . Lys is also capable of forming a lactam ring with either Glu or Asp. Tyr is capable of forming a lactone ring with Glu or Asp.

Thus the core peptide sequence may comprise a pair of residues capable of forming an intramolecular bond between their side chains. Typically, neither of these residues is a contact residue.

The Sbi-IV component of the inhibitor is capable of binding to the convex face of C3d. The Sbi-IV component may also be capable of binding to the concave face of C3d.

It consists of the core peptide sequence (as the most N-terminal part of the Sbi-IV component) with additional Sbi-IV sequence optionally present C-terminal of the core peptide sequence. Thus the additional Sbi-IV sequence typically corresponds to residues 223 onwards of Sbi-IV; that is to say, it aligns with residues 223 onwards of wild type Sbi-IV when the sequences of he Sbi-IV component and the wild type Sbi-IV sequence are optimally aligned.

The Sbi-IV component has a maximum length of 65 amino acids, i.e. the same length as the wild type Sbi-IV sequence. However, in some aspects of the invention, there are further restrictions on the length of the Sbi-IV component. For example, in the first aspect of the invention the Sbi-IV component may only be up to 45 amino acids in length. In the third aspect of the invention, the Sbi-IV component may only be up to 33 amino acids in length.

The C-terminal sequence of the Sbi-IV component can be wild type Sbi-IV sequence or may be a variant thereof, e.g. it may contain one or more mutations (substitutions, deletions or insertions) relative to the corresponding wild type Sbi-IV sequence.

Typically it has at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity to the corresponding portion of the wild type Sbi-IV sequence, e.g. 95%, 96%, 97%, 98% or 99% identity to the corresponding portion of the wild type Sbi-IV sequence.

For example (depending on the length of the C-terminal Sbi-IV sequence) it may have a maximum of 1, 2, 3, 4,5, 6, 7, 8, 9 or 10 substitutions, deletions or insertions relative to the

corresponding wild type sequence. In certain embodiments it may be desirable that any such mutations are substitutions, e.g.

conservative substitutions.

Further components of the inhibitor Certain complement inhibitors described in this specification may have additional Sbi sequences upstream (i.e. N-terminal) of the Sbi-IV component. For example, the complement inhibitor may additionally comprise some or all of a Sbi-III domain (i.e.

residues 150-197 of the full length Sbi sequence shown herein) or a sequence having at least 80% identity to Sbi-III over the entire length of the Sbi-III domain, or a fragment of either.

The Sbi-III and Sbi-IV sequences 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.

Without wishing to be bound by any particular theory, it is believed that inhibitors containing an Sbi-III domain 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. The inhibitors described in this specification may further comprise non—Sbi components . Thus the Sbi component may be expressed as a fusion protein with one or more heterologous components such as antibody Fc regions, or any other desired fusion partner. A "heterologous" component is not derived from Sbi, e.g. it does not have more than 40% identity with any contiguous sequence of comparable length of the full length Sbi protein .

Heterologous components may be located N-terminal or C-terminal of the Sbi-IV component (or other Sbi sequence present in the molecule) . The invention also extends to complement inhibitors which do not contain Sbi sequence but nevertheless are capable of binding the convex face of C3d and inhibiting binding of Sbi-IV thereto.

Uses of such inhibitors are also envisaged. Such non-Sbi inhibitors may include antibodies specific for the convex face of C3d, or fragments thereof comprising functional antigen binding sites. Further examples include nucleic acid molecules (also known as aptamers) capable of binding specifically to the convex face of C3d. Such aptamers may be RNA or DNA, or synthetic analogs thereof, and are typically oligonucleotides, e.g.

composed of 20 nucleotides or fewer.

Whatever the nature of the inhibitor, it is preferred that the inhibitors 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. It may be that the inhibitors do not include Sbi sequence downstream (C-terminal) of residue 266 of the Sbi sequence .

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.

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. 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.

Screening and other assay methods

The invention provides methods of screening for compounds capable of inhibiting the interaction between Sbi and C3.

Interactions between a complement inhibitor or candidate molecule and C3d may be studied in vitro by immobilising one member of the pair on a solid support and bringing the other member of the pair into contact with it .

The immobilised member is generally contacted with a sample containing the other member under appropriate conditions which allow the two 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 .

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.

Preferred assay formats include immunological techniques such as ELISA assays .

Alternatively, techniques such as surface plasmon resonance may be used to monitor binding between the two members of the binding pair directly, without the need for either component to be labelled.

The member 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.

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, W093/8472, W095/18376/

W095/18377, W095/24649 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.

As already described, such assay methods may be used to screen for compounds capable of inhibiting binding between C3d 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.

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 CHI domains; (ii) the Fd fragment consisting of the VH and CHI 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) .

Pharmaceutical compositions

The complement inhibitors 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.

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.

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 H, 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.

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

Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.

The complement inhibitor may be administered in conjunction with a further anti- inflammatory agent, which may be a drug suitable for treating the inflammatory condition from which an intended recipient is suffering.

The complement inhibitor and the further anti-inflammatory agent may be provided in the same composition or in separate

compositions and may be formulated for administration together or separately. Suitable anti -inflammatory agent are well known to the skilled person and include steroidal and non-steroidal anti -inflammatory agents, and ant -inflammatory proteins. Specific examples include, but are not limited to, Erythropoietin (EPO) ,

Experimental procedures

Cloning, expression and purification of Sbi-IV and C3d.

DNA coding for Sbi-IV was amplified using as template the previously described Sbi III-IV pHIS-Parallel plasmid (12) . The following oligonucleotide primers were used: Sbi-IV forward primer (BamHI) CGG GAT CC GTT TCA ATT GAA AAA GCA ATC; Sbi-IV reverse primer (Hindlll) CCC AAG CTT TCA TTA CGC CAC TTT CTT TTC AGC. Following sequential restriction digestions with BamHI and Hindlll, the Sbi-IV fragment was ligated into a pQE30 vector. Sbi-IV expression in E. coli BL21(DE3) (Stratagene) was induced with 1 mM IPTG for 3 hours at 37 'C. The cells were harvested by centrifugation (6000 g for 10 minutes at 4 °C) and resuspended in His buffer A (50 mM Tris, 300 mM sodium chloride, 20 mM

imidazole, pH 8.0) to give a total volume of 10 ml. The cells were then kept on ice and sonicated (80% amplitude in 0.5 second pulses with 3 seconds between pulses for a total sonication time of 1 minute) using a Branson 450 Digital Sonifier. A further -20 ml His buffer A was added and cleared supernatant containing soluble protein was produced by centrifuging at 60,000 g for 30 minutes at 4 °C. Cleared lysate was loaded into a 50 ml superloop and connected to an AktaPurifier 10 chromatography unit (GE Healthcare) . The 6 -His tagged protein was purified on a 5 ml His Trap FF column (GE Healthcare) using an imidazole gradient (0-500 mM) with His buffer B (50 mM Tris, 300 mM sodium chloride, 500 mM imidazole, pH 8.0) . C3d, comprised of amino acids 996-1303 of C3 , was previously cloned into the pET15b vector to enable purification by ion- exchange chromatography (17) . The N-terminal His tag coding sequence was deleted from this vector such that C3d was expressed without the His tag. The plasmid was transformed into E. coli BL21(DE3) cells (Stratagene) . 1L secondary cultures were induced with 1 mM IPTG overnight at 18 °C. Cleared lysate was subjected to ion-exchange chromatography on a 5 ml Hi-

Trap (SP FF) column (G.E. Healthcare) using HEPES buffer A (50 mM HEPES pH 5.5) as the loading buffer and eluting with a linear NaCl gradient to 500 mM in the same buffer.

The Sbi IV-C3d complex was prepared on a 1 ml His trap HP column (GE Healthcare) using the AktaPurifier . Purified Sbi IV was applied to a 1 ml His Trap HP column (GE Healthcare) equilibrated with His buffer A. The column was then washed with His buffer A and then purified C3d was applied to the column. After a further wash the complex was eluted using an imidazole gradient of 0 - 500 mM using His buffer B (50 mM Tris, 300 mM sodium chloride, 500 mM imidazole, pH 8.0). Purified protein complex was then buffer exchanged into 50 mM Tris pH 7.0 prior to crystal trials.

Crystallisation, data collection and structure analysis.

Sbi-IV-C3d complex (in 50 mM Tris pH 7.0 at -15 mg/ml) was subjected to a ProPlex screen and a tacsimate screen, using the 'sitting drop' vapour diffusion method. The Sbi-IV-C3d complex produced small needle- like crystals in various conditions of the ProPlex screen within 7 days. Crystals grew in the following ProPlex-96 conditions: 100 mM Tris pH 8.0, 20% w/v PEG 4000; 200 mM sodium chloride, 100 mM Tris pH 8.0, 20% w/v PEG 4000 and in 100 mM sodium HEPES pH 7.0 , 1 M sodium citrate. Large crystals suitable for X-ray diffraction analysis were obtained in 100 mM Tris pH 8.0, 200 mM NaCl, 20 % PEG 4000, using micro seading (Sead Bead, Hampton Research) .

X-ray diffraction data were collected at the Diamond Light Source (Oxfordshire, UK) on an ADSC Q315 3x3 CCD detector on station 104 (at wavelength λ=0.9702 A) . A crystal of the Sbi-IV-C3d complex was removed from the crystallisation drop using a cryoloop and was placed into cryoprotectant (20% (v/v) glycerol) containing reservoir solution for 1 minute. The crystal was then removed from the drop using a micromount and held in a stream of gaseous nitrogen to facilitate freezing of the crystal. 360 images were collected at an oscillation angle of 1°. Data were processed using the HKL2000 package (18) . Results obtained with this crystal are described in more detail below. Molecular replacement was carried out with Balbes(19), using the structure of C3d (Protein Data Bank accession code C3D1) as a search model. Automated rebuilding and refinement was carried out by Arp/wArp(20) . Model building was done with Coot (21) followed by rounds of refinement using Refmac5, part of the ccp4i

software (22 ) .

The final coordinates of the Sbi-IV-C3d complex have been deposited to the Protein Data Bank (PDB accession codes: 2wy7 and 2wy8) .

A further crystal was obtained by crystallisation of the same complex in 0.1 M HEPES buffer pH 7.5, 20% polyethylene glycol (PEG) 2000 and 0.4 mM methyl-acrylamide phenylboronic acid (MPBA) at 18 °C. It was analysed as described above.

NMR sample preparations and titration experiments. Preparation of uniformly ¹⁵N-labelled Sbi-IV for NMR titration experiments was carried out as described before(14). Binding of ¹⁵N- labelled Sbi- IV to unlabelled C3d was followed by recording ¹H-¹⁵N HSQC spectra as a function of Sbi-IV: C3d ratio. The NMR titration was

performed as previously described (23,24). Briefly, two initial NMR samples were prepared in 0.5 ml NMR buffer (5 mM MES, 100 mM sodium chloride, 1 mM EDTA, 1 mM benzamidine and 10% D₂0 pH 5.5) . Sample 1 contained 0.6 mM Sbi IV (1:0 molar ratio of Sbi IV:C3d) . Sample 2 contained 0.6 mM Sbi IV, 1.2 mM C3d (1:2 molar ratio of Sbi IV: C3d) . The buffer composition of both samples was identical as both samples were extensively exchanged into the same batch of sample buffer. Throughout the titration the concentration of Sbi-IVwas maintained at a constant 0.6 mM and the C3d concentration was varied to give a series of Sbi-IV:C3d molar ratios from 1.0:0.0 to 1:2. A ¹H-¹⁵N HSQC spectrum was acquired at each titration point with 512 complex ¹H points and 192 complex ¹⁵N points with 16 scans per increment and spectral widths of 8000 Hz in 4i and 1219 Hz in ¹⁵N. The initial NMR samples represented the end points of the titration.

Intermediate values of Sbi-IV:C3d were obtained by simultaneously taking equal aliquots from both sample 1 and sample 2 and then transferring the aliquots to the other NMR tube (i.e., from tube 1 to tube 2 and vice versa) . This procedure was repeated until a series of twelve ¹H-¹⁵N HSQC experiments at Sbi-IV:C3d molar ratios between 1:0 and 1:2 was completed.

NMR experiments were performed on a Varian Unity INOVA

spectrometer operating at a nominal proton frequency of 600 MHz, using a room temperature triple resonance 5 mm probe equipped with pulse field gradients (PFG) along the z axis. All NMR data processing was performed using NMRPipe/NMRDraw(25) . NMR data were analysed with Analysis (26) .

Small-angle X-ray scattering analysis. Synchrotron radiation X- ray scattering data were collected at the X33 beam line of the EMBL, Hamburg Outstation (DORIS III storage ring at DESY) .

Solutions of Sbi-E, Sbi-III-IV, Sbi-IV, C3d and complexes of the Sbi protein constructs with C3d were measured at protein

concentrations of ~2, ~5, and -10 mg/ml (sample temperature 10 °C) , using a MAR345 image plate detector and sample detector distance of 2.7 m and wavelength λ = 1.56 A, covering the momentum transfer range 0.08 < s < 0.45 nm^"1 (s = 4n sin(0)/X where 20 is the scattering angle) . Complexes of Sbi constructs were prepared in a 1:1 ratio at concentrations mentioned above. Prior to data collection, dynamic light scattering analysis

(Nano-S Zetasizer, Malvern) was used to ensure the monodispersity of the protein samples. To check for radiation damage, two successive 2 minute exposures taken on the same sample were compared; no radiation effects were observed. The data were processed using standard procedures and extrapolated to zero solute concentration using the program package PRIMUS (27) .

The forward scattering 1(0) and the radius of gyration R_g were computed from the entire scattering patterns using the indirect transform package GNOM(28), which also provided the intraparticle distance distribution function p(r) and the maximum dimension

Ana_X. The molecular mass of the solute was evaluated by comparison of the forward scattering with that from a reference solution of bovine serum albumin (molecular mass 66 kDa) . The estimation of excluded volume V_ex and low resolution ab initio models of Sbi-E, Sbi-III-IV, Sbi-IV, C3d and complexes thereof were obtained using the program DAMMIF (29 , 30 ) . The program employs simulated

annealing to build a compact interconnected configuration of beads inside a sphere with the diameter D_max that fits

experimental data minimizing the discrepancy:

0{S_j) where N is the number of experimental points, c is a scaling factor, Jexp(Sj) and I_Caic(Sj) are experimental and calculated intensities respectively, and a{Sj) is the experimental error at the momentum transfer Sj . Ten DAMMIF runs were performed to check the stability of the solution, and the results were averaged using the program DAMAVER (31) to yield the most probable models. Rigid body modeling was performed using the program SAXREF(32) .

RESULTS

Structures of the Sb±-IV~C3d complexes.

To gain understanding of the molecular details of the recognition and inhibition of human complement protein C3 by staphylococcal complement modulator Sbi we determined the three-dimensional structure of the complex between C3 binding domain Sbi-IV and the thioester containing fragment C3d of complement component C3. The crystal structure of the Sbi-IV-C3d complex was determined at a resolution of 1.7A with a single molecule of Sbi-IV bound to one molecule of C3d in the asymmetric unit (for data collection statistics see Table I) . This complex was refined to 1.7A resolution with R and Rtree values of 16.9% and 20.5%,

respectively (see Table II) . During initial analysis of the structure it became evident that the interaction between C3d and Sbi-IV involved two separate binding modes including a second symmetry- related Sbi-IV molecule. This second binding mode was treated as a separate complex (complex 2) and refined, with respective R values of 17.2% (R_cryst ) and 20.5% (R_free ) ·

While the structure of C3d in both complexes, when compared with a previous uncomplexed structure (PDB accession code 1C3D(17)), revealed no significant structural changes in its classical α-α6 barrel fold (alignment within 3.5 A, r.m.s.d. 0.668 A), notable structural changes were observed between the previously described solution structure of Sbi-IV (PDB accession code 1JVH (14)) and C3d-bound Sbi-IV. This is reflected in the structural alignment of 54 residues in the free and bound state of Sbi-IV revealing a 14-residue gap at the C-terminal end of the molecule (alignment of residues within 3.5 A, r.m.s.d. 1.838 A) . In the crystal structure, helix 3 is positioned significantly closer to the al and a.2 helices, resulting in a more compact three-helix bundle fold. The structural alignment further reveals several smaller structural differences between the solution and X-ray structure, including residues R210 and V211 located within the al helix, N222 and E223 (a2 - a3 loop) , and residues E246 and H247 of the N-terminal part of the a3 helix. (Not shown.)

Sbl-IV - C3d interactions.

Similar to complexes of staphylococcal complement inhibitors Efb- C and Ehp with C3d(7,8) , in complex 1 Sbi-IV binds to the edge of the acidic residue- lined concave surface of dome- shaped C3d

(Figure 1A and IB) . In complex 2 Sbi-IV contacts the convex face of C3d containing the thioester, an interaction that has not been observed before with any staphylococcal or other complement inhibitors. Both interfaces show high shape complementarity with comparable buried surface areas (b.s.a.) of 1,535A² (complex 1) and 1,267A² (complex 2), accounting for 31% and 26% of the Sbi-IV surface, respectively. These b.s.a. values are comparable to that of the Efb-C-C3d complex (1,658 A²; calculated using PISA(33)) . Below we describe the interactions observed in both complexes in more detail .

Complex 1: Sbi-IV interactions with the concave surface of C3d.

Sbi-IV interacts with the concave surface of C3d mainly through its helix a2 residues (1228, E229, R230, R231, Q234, R235, N238 intact Sbi numbering) with additional contributions from amino acids in helices l (R209 and R213) and 3 (K245) (detailed in Figure 2A) . C3d contributions to the interface involve residues from the acidic 30's-40's cluster, connecting helices <x2 and a3 (including D36/1029, E37/1030 and R49/1042; C3d numbering/intact pre-pro C3 numbering) , loop residues connecting helices a and a5 (N98/1091, L99/1092, 1100/1093, 1102/1095 and S104/1097) and helices a6 and a7 (D163/1156 and E167/1160, the acidic 160's cluster) . In the structure of complex 1, Sbi-IV helix a2 residue R231 anchors deeply into a pocket in the acidic 30's-40's cluster in C3d, stabilized by a structural water molecule. On the C- terminal side of helix 2, N238 is involved in an elaborate hydrogen bond network with main-chain atoms of residues in the loop connecting a4 and a5 of C3d, including V97/1090, 1100/1093, 1102/1095 and S104/1097. All complex 1 interactions are listed in Table III .

Complex 2: Sbi-IV interactions with the convex surface of C3d.

The crystal structure of Sbi-IV in complex with C3d reveals another binding mode that is not observed in the structures of C3d complexed with Efb-C and Ehp. In the Efb-C-C3d and Ehp-C3d complexes, the highly conserved thioester region is obscured by the formation of a dimer of two C3d molecules in the crystal, whereas in complex 2 Sbi-IV helices al and 3 form a highly complementary interface with this hydrophobic region. The thioester region interface seen in complex 2 includes residues from loop regions connecting C3d helices al and a2, 3 and a4, and oc5 and a6. At the core of the interface lie intimate

hydrophobic contacts between residues V211, V244 and L248 in Sbi- IV and 11125 of C3d. These interactions are strengthened by van der aals stacking interactions between K212 (Sbi-IV) and

F76/1069 (C3d) , as is shown in Figure 3A and 3B. These

hydrophobic contacts are further stabilized by hydrogen bonding interactions involving Sbi-IV helix al residues D208, N215, S219 and D243 and Q251 from helix α3 with C3d thioester residues A17/1010, Q20/1013, K78/1071 R79/1072 and Y273/1266 (see Table IV for detailed list of interactions) . In the C3d construct used in our experiments the thioester-contributing cysteine residue (C17/1010) is substituted by an alanine. In the complex 2 structure Sbi-IV D208 is sandwiched between the two thioester- forming residues (Q20/1013 and C17/1010, here mutated to

A17/1010) that are modified in native C3 forming the thiolactone ring that mediates covalent attachment (Figure 3A) .

Results from site-directed mutagenesis in C3d(16) and in Sbi- IV (12, 14) together with surface plasmon resonance (SPR) and isothermal calorimetry titration (ITC) validate the formation of complex 1. More so, the results from these techniques indicate that complex 1 may be the sole species formed under these experimental conditions. To expand on these observations, we analyzed the complexes of Sbi-E, Sbi-III-IV and Sbi-IV with C3d using a two additional structural techniques, small-angle X-ray scattering (SAXS) and MR chemical shift analysis. The latter technique was chosen also because of its broad affinity range.

SAXS analysis of the complexes of C3d with Sbi-E, Sbi-III-IV and Sbi-IV.

For further examination of the two C3d binding modes of Sbi-IV observed in the crystal structure, we used small angle X-ray scattering analysis to study complexes of C3d with Sbi constructs Sbi-E, Sbi-III-IV and Sbi-IV in solution. The results from these analyses are listed in Table V. In contrast with the dual C3d binding mode seen in the crystal structure, Sbi-IV and all other Sbi constructs form complexes with C3d in a 1:1 stoichiometry.

When the atomic coordinates of both complexes 1 and 2 were fitted with the SAXS data, using the program CRYSOL, the resulting chi values (complex 1, χ=2.7; complex 2, χ=2.5; Table VI) are inconclusive as to which complex is formed, although there is a slight preference for the model based on complex 2. In

concordance with these results, the ab initio models and the atomic coordinates of the Sbi-IV C3d complexes can be

superimposed with similarities of 1.5 (complex 1) and 1.4

(complex 2) . (Not shown.)

NMR titration analysis of the Sbi-IV residues involved in the interaction with C3d.

We used NMR titration to gain further understanding of the two interaction modes between Sbi-IV and C3d by examining the complex from the Sbi-IV perspective. ^-" HSQC spectra of ¹⁵N- labelled Sbi-IV were recorded in the presence of differing concentrations of C3d. As expected from the interactions observed in the complex 1 structure, the most prominent chemical shift changes involve Sbi-IV anchoring residues R231 and N238. Helix l residue R210, an additional contact residue in the complex 1 structure also showed a considerable chemical shift perturbation. Notably, peaks arising from other residues in Sbi-IV helices oil and a3 were also perturbed in the Sbi-IV-C3d titration, albeit to a lesser extent than R231, N238 and R210. Chemical shift perturbations were observed for V244 (located in 2- 3 loop), L248 (a3) and al residues E201, V205 and E209. Although these residues can be seen interacting in Sbi-IV-C3d complex 2, they are not detected by either ITC or SPR. (Data not shown.) Table I. Data collection statistics

Unit cell dimensions 53.4 A x 81.0 A x 87.7 A, a Space group P2.2.2,

Molecules/ Asymmetric Unit 2

Wavelength 0.9702 A

Resolution limits 1.7 A

Temperature 100

Total reflections 1369401

Unique reflections (1.70 - 1.76A) 41 186 (3710)

Ι/σΙ (1.70 - 1.76A) 30.4 (3.3)

Completeness (1.70 - 1.76A) 96.0% (87.6%)

Table II. Re nement statistics

Table III. Sbi-IV interactions with the concave surface of C3d (complex 1)

10

15

20

25

MTable V. Overall arameters evaluated rom SAXS data

Sbi-IV 1.65 1 1.2 10.9 7.5 23 25

Sbi-HI-IV 3.15 17.2 16.5 1 1.5 32 38

Sbi-E 3.87 30.6 30.7 14.2 67 78

Sbi-IV-C3d

2.72 44.0 45.6 8.0 71 70 complex

Sbi-III-IV-

3.10 52.6 51.2 12.0 77 75 C3d complex

Sbi-E-C3d

3.67 67.0 65.4 13.5 102 123 complex

are the radius of gyration, molecular mass (MM), maximum size, Porod volume and excluded volume derived from experimental SAXS data. MM_cai_c is the MM calculated from primary sequence.

Table VI. Summar o SAXS data modelin ts

Table VII. Com arison o C3d bindin data rom Sbi-IV Ε -C and Eh

Identical interactions between C3d and SbilV were subsequently observed in another crystal (space group P21; dimensions (A) a=48.8; b=86.1; c=49.2; alpha=90°; beta=117°; gamma=90°;

diffraction to 1.85A resolution) providing further confirmation that complex 2 is not a crystallisation artefact but represents a real physiological interaction between Sbi and C3d. (Data not shown . )

0

DISCUSSION

The crystal structure presented here of the complexes between complement fragment C3d and domain IV of staphylococcal

complement modulation protein Sbi reveals two modes of

5 interaction: 1) charge-driven interactions with the acidic

concave surface of C3d, mainly through helix a2 of Sbi-IV

(complex 1) ; 2) hydrophobic interactions with the C3d thioester region, via Sbi-IV helices ocl and 3 (complex 2) .

Complex 1.

The interactions observed in this complex resemble the binding between C3d and staphylococcal complement inhibitors Efb-C(7) and Ehp(8). Although there is only 19% sequence identity between Sbi- IV and Efb-C and Ehp, 5 of the 8 identical residues are in the interacting α2 helix of Sbi-IV. These residues include R231 and N238 that have been shown to be essential for the interaction with C3d in all three inhibitors (7,8,14) . In Efb-C and Ehp, these residues (R131/R75 and N138/N82 supported by H130/85) are involved in an intricate network of 7 hydrogen bonds with C3d (Table VII) . Sbi-IV-C3d complex 1 is stabilized by 9 hydrogen bonds involving R231 and N238, assisted by R20S, R235 and K245. While in Efb-C and Ehp this hydrogen bond network is supported with a single salt-bridge (involving R131/75) , in Sbi-IV there are 9 ionic bonds with C3d involving the above mentioned hydrogen bond network residues.

Although an intricate interaction network consisting of 9 hydrogen bonds and 9 salt-bridges would suggest that Sbi-IV-C3d binding should be higher affinity than C3d with inhibitors Efb-C and Ehp, ITC results prove otherwise. As is shown in Table VII, Efb-C and Ehp bind C3d with a 2 and 3 orders of magnitude higher affinity than Sbi-IV, respectively. This is also reflected by their ability to inhibit the alternative complement pathway by two orders of magnitude. Even though the enthalphy changes seen in the binding of C3d by Efb-C and Ehp point to a more optimal ionic interaction and network of hydrogen bonds, the larger number of Sbi-IV residues contributing to hydrogen and ionic bonds includes charged residues (R231, R206, R235 and K245) , containing large aliphatic moieties that may add significantly to the entropically favorable hydrophobic interactions. Also, not all H-bonds and salt bridges will contribute equally to the complex (34) and based on the individual b.s.a. contributions of Sbi-IV residues to the complex we predict that R231 (144 A²) , R235 (100 A²) and N238 (77 A²) are the largest contributors, followed by K245 (39 A²) and R206 (33 A²) . While in Efb-C the simultaneous loss of the anchoring residues R131 and N138 results in a non- functional Efb-C protein, the individual mutants still form stable 1:1 complexes with no significant structural effects in their complexes with C3d(35) . The equivalent residues R231 and N238 in Sbi-IV appear to play a more prominent role in their interactions with C3d as the R231A/N238A double mutant, as well as the individual mutants, completely abolish its C3d binding capacit (12, 14) .

In complex 1, N238 forms intricate hydrogen bond interactions with the C3d main chain, while R231 makes ionic interactions with the side chain of D36/1029. The latter observation is in

excellent agreement with recent results from mutational mapping analyses which revealed that the loss of charge mutations in the acidic 30's-40's cluster (D36/1029 and R49/1042) lead to a complete loss of Sbi-IV binding activity(16) , while alanine mutations in the other acidic cluster (D163/1156 and 1164/1157) caused moderate defects in binding of Sbi-IV. The C3d amino acids that were tested in the mutational analysis show a substantial overlap with the interaction boundaries observed in complex 1. The importance of C3d residues D36/1029 and R49/1042 determined by the mutational analysis are reflected in the complex structure by their individual b.s.a. contributions in the complex (37 A² and 53 A², respectively) . Amino acids D163/1156 and 1164/1157, causing moderate binding defects in the mutational analysis, also show to be large surface contributors in complex 1 (58 A² and 38 A², respectively) . The complex 1 structure identifies an

additional 5 residues, omitted in the mutational analyses, with considerable b.s.a. contributions. These include 40s cluster residue L46/1039 (48 A²) and 100s cluster residues V97/1090 (54 A²) N98/1091 (103 A²); A101/1094 (A²) and 1167/1160 (70 A²).

The above-mentioned mutagenesis studies also revealed the C3d binding sites Sbi-IV overlaps with that of CR2. These data were confirmed in the earlier studies in which was shown that Sbi-III- IV (12) and also Sbi-IV (16) have the capability to competitively inhibit the binding of C3dg to CR2(12), indicating that

complement modulator Sbi also interferes with the link between the innate and adaptive branches of the host immune system.

Similar to Efb-C (12 , 15) , the interaction of Sbi-IV with the acidic concave surface of C3d observed in Sbi-IV-C3d complex 1 therefore explains the observed inhibition of the crucial C3d-CR2 interaction by Efb-C and Sbi-IV. However, the discovery of overlapping binding sites for Sbi-IV and CR2 does not fit with the CR2 binding site on C3d observed in a crystal structure of this complex (36) . The Sbi-IV molecule in complex 1 (or in complex 2) does not interfere with the interactions between CR2 and C3d in structure described by Szakonyi and co-workers. This

controversy has recently been resolved with the finding that zinc ions, at concentrations used in the crystallization conditions, obstruct the binding between C3d and CR2, casting doubt on the physiological relevance of the C3d-CR2 interface observed in the crystal structure of this complex (16) . It is more likely that the observed interactions are crystallization artifacts, stabilized by the presence of the divalent cations . The crystal structures of native C3(37) and C3b(38,39) have been very helpful in gaining understanding in the observed Sbi-IV binding modes. Alignment of Sbi-IV-C3d complex 1 with the structures of native C3(37) and C3b(38,39) was used to provide the structural basis for previously observed binding preferences for C3 and its proteolytic cleavage products. While good binding has been reported between Sbi-IV and intact C3 , the strongest interactions were observed with iC3b and C3dg and the weakest binding to C3c and C3b(12) . As was shown for Efb(7) , the superpositioning of the C3d structure in complex 1 with that of the thioester domain (TED) in C3 caused no structural

interference between Sbi-IV and C3 , with opportunities for additional contacts between Sbi-IV helix a3 and C3 a₂- macroglobulin domain 2 (MG2) . (Not shown.) The previously observed low affinity of Sbi-IV for C3b(12) can equally be elucidated when complex 1 is superposed onto the structure of C3b. Sbi-IV helix a3 now appears to cause a significant steric clash with C3b a₂-macroglobulin domain I (MG1) that would result in disruption of the structure of active C3b. (Not shown.) The high affinity of Sbi for C3 cleavage product iC3b on the other hand indicates that in this species the complex 1 Sbi-IV binding site on C3d becomes more accessible. Covaplex 2. The presented results from SAXS analysis of the interaction between Sbi and C3d point to the formation of a 1:1 complex between the two proteins, confirming previous SPR and ITC binding experiments with Sbi-IV and C3d (12,14) . Although it is not possible to distinguish between complex 1 and complex 2 using SAXS, perhaps because of the spherical nature of C3d, results from binding studies with Sbi- IV (14) in combination with

mutational mapping studies in C3d(16) identify the interactions observed in complex 1 as the sole functionally relevant species.

NMR chemical shift analysis has a broad affinity range (ΙΟΟηΜ - lOmM) because it can reliably detect even a small percentage of bound ligand (40) . Even low binding affinities in the high millimolar range can be detected, which are beyond the detection limits of ITC or SPR. Our chemical shift analyses of the Sbi- IV: C3d complex clearly show interactions of C3d with both faces of the Sbi-IV molecule that are in concordance with both of the binding modes observed in the structure . These results indicate that in addition to the high affinity interaction between C3d and Sbi-IV seen in complex 1, another C3d binding site is present on Sbi-IV, represented by complex 2, which is of such low affinity that it can only be detected by NMR and X-ray crystallography. Another feature of the complex 2 binding mode that points to a specific interaction is the significant shape and charge

complementarity between the C3d and Sbi-IV molecules, displaying a buried surface area that is on a par with that observed in complex 1, involving 16 residues interacting via 7 hydrogen bonds and covering over a quarter of the available Sbi-IV surface area. For comparison, the b.s.a. observed in complex 2 (1,267 A²) is significantly larger than the crystal packing surface between CR2 and C3d in the structure of the latter complex (797 A²) , covering only 8% of the CR2 molecule (16,36). The crystal structure presented here in combination with the chemical shift NMR analyses indicates that a low affinity short- distance attraction exists between Sbi-IV and C3d, resulting in the interaction seen in complex 2. Perhaps this complex reflects an interaction that is required for the formation of the covalent adduct that is formed between Sbi and C3 when incubated human serum. Even though Sbi- III is needed for activation of C3 it is possible that Sbi-IV forms a significant part of the C3b

transacylation target. Interestingly, the N-terminus of the Sbi- IV construct that was used in these studies partly includes seven residues which are sometimes considered part of the C-terminal sequence of Sbi-III (VSIEKIV, residues 199-205) . Although previous NMR solution analysis of Sbi-III and Sbi-IV (14) show that this region is disordered in both molecules, in the Sbi- IV:C3d structure it is fully folded and a-helical, with three residues {E201, 1204 and V205) contributing to interactions with the thioester region of C3d (Table IV) . These specific

interactions are also confirmed in the NMR chemical shift analysis .

Any suspicion that complex 2 represents a crystallisation artefact is further reduced by the observation of identical interactions between C3d and Sbi-IV in an independently-generated crystal having different unit cell characteristics.

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. Lowy, F. D. (1998) N Engl J Med 339, 520-532

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Claims

1. A complement inhibitor comprising an Sbi-IV component capable of binding to C3d, wherein the Sbi-IV component comprises a core peptide of Formula I:

and wherein the Sbi-IV component is not more than 45 amino acids in length.

2. A complement inhibitor according to claim 1 wherein said core peptide adopts an alpha-helical conformation in solution.

3. A complement inhibitor according to claim 1 or claim 2 wherein not more than five of contact residues Glu201, Ile204, Val205, His207, Asp208, Val211, Lys212, Asn215 and Ser219 are substituted or deleted.

4. A complement inhibitor according to claim 3 wherein any substitutions of the contact residues are conservative

substitutions .

5. A complement inhibitor according to claim 3 wherein all contact residues are conserved.

6. A complement inhibitor according to claim 1 or claim 2 wherein the core peptide has the sequence of Formula I.

7 .A complement inhibitor according to any one of claims 1 to 6 wherein the Sbi-IV component comprises additional Sbi-IV sequence C-terminal of the core peptide sequence.

8. A complement inhibitor according to claim 7 wherein the additional Sbi-IV sequence is wild type Sbi-IV sequence.

9. A complement inhibitor according to any one of the

preceding claims which comprises further Sbi sequence N-terminal of the core peptide.

10. A complement inhibitor according to claim 9 wherein said further Sbi sequence comprises a Sbi- III domain or a fragment thereof .

11. A complement inhibitor according to any one of the

preceding claims which comprises not more than 100 contiguous amino acids of Sbi, e.g. not more than 90, 80, 70, 60, 50, 40 or 30 amino acids of Sbi, e.g. not more than 29, 28, 27, 26 or 25 contiguous amino acids of Sbi.

12. A complement inhibitor according to any one of the

preceding claims wherein the complement inhibitor is not more than 100 contiguous amino acids in length, e.g. not more than 90, 80, 70, 60, 50, 40 or 30 amino acids in length, e.g. not more than 29, 28, 27, 26, 25, 24, 23, 22, 21, 20 or 19 amino acids in length.

13. A complement inhibitor comprising an Sbi-IV component capable of binding to C3d, wherein the Sbi-IV component comprises a core peptide of Formula II: Val Ser He Glu Lys Ala X204 Val Arg His X208 Glu Arg Val Lys Ser Ala Asn Asp Ala He Ser Lys Leu Asn; wherein X204 is He or an aliphatic side chain with a hydroxyl group (e.g. Ser or Thr) or Tyr; and X208 is Asp or an aliphatic side chain with a hydroxyl group (e.g. Ser or Thr) ;

or having

(ii) up to 5 substitutions relative to Formula II;

14. A complement inhibitor according to claim 13 wherein the core peptide has a residue with an aliphatic side chain with a hydroxyl group at position 208 and a Tyr residue at position 204.

15. A complement inhibitor according to claim 13 or claim 14 wherein said core peptide adopts an alpha-helical conformation in solution.

16. A complement inhibitor according to any one of claims 13 to 15 wherein not more than five of contact residues Glu201, Val205, His207, Val211, Lys212, Asn215 and Ser219 are substituted or deleted.

17. A complement inhibitor according to claim 16 wherein any substitutions of the contact residues are conservative

substitutions.

18. A complement inhibitor according to any one of claims 13 to 15 wherein all contact residues are conserved.

19. A complement inhibitor according to any one of claims 13 to 15 wherein the core peptide has the sequence of Formula I.

20. A complement inhibitor according to any one of claims 13 to 19 wherein the Sbi-IV component comprises additional Sbi-IV sequence C-terminal of the core peptide sequence.

21. A complement inhibitor according to claim 20 wherein the additional Sbi-IV sequence is wild type Sbi-IV sequence.

22. A complement inhibitor according to claim 20 or claim 21 wherein the Sbi-IV component is up to 65 amino acids in length, e.g. up to 60, up to 50, up to 45, up to 40, up to 35, up to 30, or up to 25 amino acids in length.

23. A complement inhibitor according to any one of claims 13 to 22 which comprises further Sbi sequence N-terminal of the core peptide .

24. A complement inhibitor according to claim 23 wherein said further Sbi sequence is a Sbi-III domain or a fragment thereof.

25. A complement inhibitor according to any one of claims 13 to

24 wherein the core peptide sequence is :

Val Ser lie Glu Lys Ala Tyr Val Arg His Asp Glu Arg Val Lys Ser Ala Asn Asp Ala lie Ser Lys Leu Asn;

Val Ser lie Glu Lys Ala Ser Val Arg His Asp Glu Arg Val Lys Ser Ala Asn Asp Ala lie Ser Lys Leu Asn; Val Ser lie Glu Lys Ala Thr Val Arg His Asp Glu Arg Val Lys Ser Ala Asn Asp Ala lie Ser Lys Leu Asn;

Val Ser lie Glu Lys Ala lie Val Arg His Ser Glu Arg Val Lys Ser Ala Asn Asp Ala lie Ser Lys Leu Asn;

Val Ser lie Glu Lys Ala Tyr Val Arg His Ser Glu Arg Val Lys Ser Ala Asn Asp Ala lie Ser Lys Leu Asn; or Val Ser lie Glu Lys Ala Tyr Val Arg His Thr Glu Arg Val Lys Ser Ala Asn Asp Ala lie Ser Lys Leu Asn.

26. A complement inhibitor according to any one of claims 13 to 25 wherein the Sbi-IV component has the sequence:

Val Ser lie Glu Lys Ala Tyr Val Arg His Asp Glu Arg Val Lys Ser

Ala Asn Asp Ala He Ser Lys Leu Asn Glu Lys Asp Ser He Glu Asn

Arg Arg Leu Ala Gin Arg Glu Val Asn Lys Ala Pro Met Asp Val Lys Glu His Leu Gin Lys Gin Leu Asp Ala Leu Val Ala Gin Lys Asp Ala

Glu Lys ;

Val Ser He Glu Lys Ala Ser Val Arg His Asp Glu Arg Val Lys Ser

Glu His Leu Gin Lys Gin Leu Asp Ala Leu Val Ala Gin Lys Asp Ala

Glu Lys ;

Val Ser He Glu Lys Ala Thr Val Arg His Asp Glu Arg Val Lys Ser Ala Asn Asp Ala He Ser Lys Leu Asn Glu Lys Asp Ser He Glu Asn

Arg Arg Leu Ala Gin Arg Glu Val Asn Lys Ala Pro Met Asp Val Lys

Glu His Leu Gin Lys Gin Leu Asp Ala Leu Val Ala Gin Lys Asp Ala

Glu Lys;

Val Ser He Glu Lys Ala He Val Arg His Ser Glu Arg Val Lys Ser

Ala Asn Asp Ala He Ser Lys Leu Asn Glu Lys Asp Ser He Glu Asn

Arg Arg Leu Ala Gin Arg Glu Val Asn Lys Ala Pro Met Asp Val Lys

Glu His Leu Gin Lys Gin Leu Asp Ala Leu Val Ala Gin Lys Asp Ala

Glu Lys;

Val Ser He Glu Lys Ala He Val Arg His Thr Glu Arg Val Lys Ser

Ala Asn Asp Ala He Ser Lys Leu Asn Glu Lys Asp Ser He Glu Asn

Arg Arg Leu Ala Gin Arg Glu Val Asn Lys Ala Pro Met Asp Val Lys

Glu His Leu Gin Lys Gin Leu Asp Ala Leu Val Ala Gin Lys Asp Ala Glu Lys; Val Ser lie Glu Lys Ala Tyr Val Arg His Ser Glu Arg Val Lys Ser

Ala Asn Asp Ala He Ser Lys Leu Asn Glu Lys Asp Ser He Glu Asn

Arg Arg Leu Ala Gin Arg Glu Val Asn Lys Ala Pro Met Asp Val Lys

Glu His Leu Gin Lys Gin Leu Asp Ala Leu Val Ala Gin Lys Asp Ala

Glu Lys; or

Val Ser He Glu Lys Ala Tyr Val Arg His Thr Glu Arg Val Lys Ser

Ala Asn Asp Ala He Ser Lys Leu Asn Glu Lys Asp Ser He Glu Asn

Arg Arg Leu Ala Gin Arg Glu Val Asn Lys Ala Pro Met Asp Val Lys

Glu His Leu Gin Lys Gin Leu Asp Ala Leu Val Ala Gin Lys Asp Ala

Glu Lys .

27. A complement inhibitor comprising an Sbi-IV component capable of binding to C3d, wherein the Sbi-IV component comprises a core peptide of Formula III:

Val Ser He Glu Lys Ala X204 Val Arg His X208 Glu Arg Val Lys

X213 X214 Asn Asp Ala He Ser Lys Leu Asn;

wherein X213 is Ser or a residue with a polar side chain (e.g. Asn); and X214 is Ala or a residue with a positively charged side chain (e.g. Lys or Arg) ;

or having

(ii) up to 5 substitutions relative to Formula III;

and wherein the Sbi-IV component is not more than 33 amino acids in length.

28. A complement inhibitor according to claim 27 wherein X213 has a polar side chain and X214 has a positively charged side chain .

29. A complement inhibitor according to claim 28 wherein X213 is Asn and X214 is Lys, or X213 is Asn and X214 is Arg.

30. A complement inhibitor according to any one of claims 27 to 29 wherein said core peptide adopts an alpha-helical conformation in solution.

31. A complement inhibitor according to any one of claims 27 to

30 wherein not more than five of contact residues Glu201, Ile204, Val205, His207, Asp208, Val211, Lys212, Asn215 and Ser219 are substituted or deleted.

32. A complement inhibitor according to any one of claims 27 to

31 wherein:

(i) Ile204 is substituted by an aliphatic side chain with a hydroxyl group, e.g. Ser or Thr; or

(ii) Ile204 is substituted by Tyr; and/or

(iii) Asp208 is substituted by a residue with an aliphatic side chain with a hydroxyl group, e.g. Ser or Thr.

33. A complement inhibitor according to claim 29 which

comprises or consists of the sequence :

Val Ser lie Glu Lys Ala lie Val Arg His Asp Glu Arg Val Lys Asn Lys Asn Asp Ala lie Ser Lys Leu Asn.

34. A complement inhibitor according to any one of claims 27 to 33 wherein the Sbi-IV component comprises additional Sbi-IV sequence C-terminal of the core peptide sequence.

35. A complement inhibitor according to claim 34 wherein the additional Sbi-IV sequence is wild type Sbi-IV sequence.

36. A complement inhibitor according to any one of claims 27 to 35 which comprises further Sbi sequence N-terminal of the core peptide .

37. A complement inhibitor according to claim 36 wherein said further Sbi sequence is a Sbi-III domain or a fragment thereof.

38. A complement inhibitor according to any one of the

preceding claims further comprising a heterologous component.

39. A complement inhibitor according to claim 38 wherein the heterologous component is a polymeric component such as

polyethylene glycol (PEG) or poly-sialic acid.

40. A complement inhibitor according to claim 38 wherein the heterologous component is a protein, e.g. an antibody Fc region.

41. A nucleic acid encoding a complement inhibitor according to any one of the preceding claims.

42. An expression vector comprising a nucleic acid according to claim 41.

43. A host cell comprising a nucleic acid according to claim 41 or expression vector according to 42, which is capable of expressing and optionally secreting the complement inhibitor.

44. A method for inhibiting complement activation in a

biological system, comprising contacting the system with a complement inhibitor according to any one of claims 1 to 40.

45. A method of treating an inflammatory condition in an individual, comprising administering a complement inhibitor according to any one of claims 1 to 40, or a nucleic acid, expression vector or host cell according to any one of claims 41 to 43 , to said individual .

46. Use of a complement inhibitor according to any one of claims 1 to 40, or a nucleic acid, expression vector or host cell according to any one of claims 41 to 43, in the preparation of a medicament for the treatment of an inflammatory condition.

47. A complement inhibitor according to any one of claims 1 to 40, or a nucleic acid, expression vector or host cell according to any one of claims 41 to 43, for use in the treatment of an inflammatory condition.

48. A method according to claim 45, use according to claim 46, or complement inhibitor, nucleic acid, expression vector or host cell according to claim 47, wherein said condition is selected from 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.

49. A method of inhibiting B cell proliferation and/or antibody production in an individual, comprising administering a

complement inhibitor according to any one of claims 27 to 37 to said individual .

50. Use of a complement inhibitor according to any one of claims 27 to 37, a nucleic acid encoding a complement inhibitor according to any one of claims 27 to 37, an expression vector comprising such a nucleic acid, or a host cell comprising such a nucleic acid or expression vector, in the preparation of a medicament for inhibiting B cell proliferation and/or antibody production .

51. A complement inhibitor according to any one of claims 27 to 37, a nucleic acid encoding a complement inhibitor according to any one of claims 27 to 37, an expression vector comprising such a nucleic acid, or a host cell comprising such a nucleic acid or expression vector, for use in a method of inhibiting B cell proliferation and/or antibody production.

52. A method of treatment or prophylaxis of HIV infection in an individual comprising administering a complement inhibitor according to any one of claims 27 to 37, a nucleic acid encoding a complement inhibitor according to any one of claims 27 to 37, an expression vector comprising such a nucleic acid, or a host cell comprising such a nucleic acid or expression vector to said individual .

53. Use of a complement inhibitor according to any one of claims 27 to 37, a nucleic acid encoding a complement inhibitor according to any one of claims 27 to 37, an expression vector comprising such a nucleic acid, or a host cell comprising such a nucleic acid or expression vector, in the preparation of a medicament for treatment or prophylaxis of HIV infection.

54. A complement inhibitor according to any one of claims 27 to 37, a nucleic acid encoding a complement inhibitor according to any one of claims 27 to 37, an expression vector comprising such a nucleic acid, or a host cell comprising such a nucleic acid or expression vector, for use in treatment or prophylaxis of HIV infection.

55. A method of screening for a complement inhibitor comprising contacting a candidate compound with C3d and (i) determining the ability of the candidate compound to bind to the convex face of C3d; and/or

(ii) determining the ability of the candidate compound to inhibit binding of Sbi-IV or an analogue thereof to C3d, e.g. to the convex face of C3d.

56. A method according to claim 55 comprising comparing the performance of the candidate compound to a reference complement inhibitor known to bind to the convex face of Sbi-IV.

57. A method according to claim 56 wherein the reference complement inhibitor comprises or consists of Sbi-IV, or a complement inhibitor according to any one of claims 1 to 40.

58. A method according to claim 56 or claim 57 comprising contacting C3d with both the candidate compound and the reference complement inhibitor .

59. A method according to any one of claims 55 to 58 wherein the candidate compound is a complement inhibitor according to any one of claims 1 to 40.

60. A method according to any one of claims 55 to 58 wherein the candidate compound is a small molecule, a protein (e.g. an antibody or a fragment thereof comprising a functional antigen binding domain) , a peptide, a nucleic acid, a sugar,

oligosaccharide or polysaccharide.

61. A method according to claim 55 comprising providing a parent complement inhibitor which is capable of binding to the convex face of C3d;

contacting said candidate compound with C3d; and (i) comparing the ability of the candidate compound to bind to the convex face of C3d with that of the parent complement inhibitor; and/or

(ii) comparing the ability of the candidate compound to inhibit binding of Sbi-IV, or an analogue thereof to the convex face of

C3d with that of the parent complement inhibitor.

62. A method according to claim 61 wherein the parent

complement inhibitor is a complement inhibitor according to any one of claims 1 to 40.

63. A method according to claim 61 or claim 62 wherein which C3d is contacted with both the candidate compound and the parent complement inhibitor.

64. A method according to any one of claims 61 to 63 comprising the steps of providing a parent complement inhibitor, and modifying the parent complement inhibitor to generate the candidate compound.

65. A method according to claim 64 comprising the steps of providing a parent nucleic acid sequence (e.g. a DNA sequence) encoding the parent complement inhibitor and modifying the nucleic acid sequence to generate a variant nucleic acid sequence encoding the candidate molecule .

66. A method according to claim 64 or claim 65 wherein the parent complement inhibitor comprises an Sbi-IV component comprising helix 1 of Sbi-IV or a variant thereof, and the candidate compound has an Sbi-IV component having a modified form of helix 1 compared with the parent complement inhibitor.

67. A method according to any one of claims 64 to 66 wherein the candidate compound differs from the parent complement inhibitor only in the region corresponding to helix 1 of Sbi-IV.