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WO2012042213A1 - Nouvelle interaction entre les protéines sbi et c3d de staphylococcus aureus - Google Patents

Nouvelle interaction entre les protéines sbi et c3d de staphylococcus aureus Download PDF

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Publication number
WO2012042213A1
WO2012042213A1 PCT/GB2011/001414 GB2011001414W WO2012042213A1 WO 2012042213 A1 WO2012042213 A1 WO 2012042213A1 GB 2011001414 W GB2011001414 W GB 2011001414W WO 2012042213 A1 WO2012042213 A1 WO 2012042213A1
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Prior art keywords
sbi
lys
complement inhibitor
ser
ala
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Jean Van Den Elsen
Stefan Bagby
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BATH VENTURES
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BATH VENTURES
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes

Definitions

  • 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.
  • 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).
  • 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
  • SCIN Staphylococcal complement inhibitor
  • the C-terminal fragment of extracellular fibrinogen-binding protein EFb-C and its homologue Ehp bind to the C3d region of C3 , the central
  • 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
  • CHIPS immunoglobulin
  • 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
  • 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.
  • 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.
  • CR2 complement receptor 2
  • 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
  • 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.
  • complex 2 occurs at the convex face of C3d. It is unlike any other known
  • 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 :
  • Sbi-IV component is not more than 45 amino acids in length.
  • Formula I represents the sequence of helix 1 of Sbi-IV
  • 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.
  • substitutions should not disrupt the alpha helical structure of the core sequence or the contacts between the core helix and C3d.
  • any substitutions of other residues of Formula I introduce residues with high helix- forming propensity, e.g. Met, Ala, Leu, Glu or Lys .
  • any such substitutions of other residues of Formula I introduce residues with high helix- forming propensity, e.g. Met, Ala, Leu, Glu or Lys .
  • any such substitutions should not disrupt the alpha helical structure of the core sequence or the contacts between the core helix and C3d.
  • any substitutions of other residues of Formula I introduce residues with high helix- forming propensity, e.g. Met, Ala, Leu, Glu or Lys .
  • any such substitutions of other residues of Formula I introduce residues with high helix- forming propensity, e.g. Met, Ala, Leu, Glu or Lys .
  • 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 .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • 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.
  • 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
  • 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.
  • Residues with hydroxyl groups make excellent transacylation targets for the thioester of C3d.
  • 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.
  • 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:
  • X208 is Asp or an aliphatic side chain with a hydroxyl group (e.g. Ser or Thr) , ⁇
  • 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
  • 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.
  • Glu201, Val205, His207, Val211, Lys212, Asn215 and Ser219 make contacts with the C3d surface. These residues may therefore be referred to as "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.
  • substitutions should not disrupt the alpha helical structure of the core sequence or the contacts between the core helix and C3d.
  • any substitutions of other residues of Formula II introduce residues with high helix- forming propensity, e.g. Met, Ala, Leu, Glu or Lys .
  • any such substitutions of other residues of Formula II introduce residues with high helix- forming propensity, e.g. Met, Ala, Leu, Glu or Lys .
  • any such substitutions should not disrupt the alpha helical structure of the core sequence or the contacts between the core helix and C3d.
  • any substitutions of other residues of Formula II introduce residues with high helix- forming propensity, e.g. Met, Ala, Leu, Glu or Lys .
  • any such substitutions of other residues of Formula II introduce residues with high helix- forming propensity, e.g. Met, Ala, Leu, Glu or Lys .
  • 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.
  • insertions or deletions 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.
  • 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.
  • 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 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) :
  • 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.
  • 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;
  • 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) ;
  • Sbi-IV component is not more than 33 amino acids in length.
  • both X213 has a polar side chain and X214 has a positively charged side chain.
  • 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
  • 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,
  • 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.
  • the core peptide may include one or more of the modifications described above in relation to the second aspect of the
  • 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.
  • Ile204 may be substituted by an aliphatic side chain with a hydroxyl group, e.g. Ser or Thr;
  • Ile204 may be substituted by Tyr,- and/or
  • Asp208 may be substituted by a residue with an aliphatic side chain with a hydroxyl group, e.g. Ser or Thr.
  • 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.
  • substitutions should not disrupt the alpha helical structure of the core sequence or the contacts between the core helix and C3d.
  • any other substitutions of other residues of Formula III introduce residues with high helix- forming propensity, e.g. Met, Ala, Leu, Glu or Lys.
  • any such substitutions are conservative.
  • 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.
  • insertions or deletions 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
  • 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 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.
  • 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.
  • 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 .
  • 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.
  • 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.
  • 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 .
  • 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.
  • 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)
  • the heterologous component may modulate any desired property of the protein, such as stability, activity or
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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) .
  • anti- inflammatory agent may be covalently linked to any part of the complement inhibitor.
  • the antiinflammatory agent may be conjugated to the Sbi-IV component or to another heterologous component (if present) .
  • 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.
  • the invention further provides a nucleic acid encoding a
  • the nucleic acids may be DNA or RNA, in single or double stranded form.
  • 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,
  • 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.
  • the system may be assembled in vitro from
  • isolated proteins e.g. recombinant proteins
  • cells which may be isolated from tissue, or grown in culture
  • the system will contain complement protein C3.
  • Other components of the complement system possibly including
  • 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.
  • 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 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.
  • one or more antibodies may be present.
  • lectin pathway a source of mannose or other carbohydrate bound by MBP or ficolin may be present.
  • LPS lipopolysaccharides
  • 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.
  • 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 play a role in the pathogenesis or symptoms of any inflammatory condition.
  • condition may be considered to be any condition in which
  • activation of the immune system is responsible for or contributes to pathogenesis or symptoms of the condition, either directly or indirectly.
  • 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.
  • septic shock 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)
  • MS multiple sclerosis
  • Alzheimer's disease inflammatory and degenerative diseases of the nervous system
  • ischemia-reperfusion injury e.g. renal ischemic injury, such as acute tubular necrosis
  • trauma such as acute tubular necrosis
  • sepsis the atypical form of haemolytic uretic syndrome (HUS)
  • MGN membrane proliferative glomerulonephritis
  • ARMD age-related macular degeneration
  • RA rheumatoid arthritis
  • SLE systemic lupus erythematosis
  • lupus nephritis antiphospholipid syndrome
  • asthma spontaneous fetal loss.
  • 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.
  • 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) ,
  • IL-1 receptor antagonist e.g. Anakinra
  • 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
  • C3d/C3dg 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) .
  • inhibiting the C3d/C3dg interaction with CR2 may therefore be of use in
  • 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.
  • nucleic acid encoding a complement inhibitor 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.
  • the nucleic acid or expression vector may encode a complement inhibitor having a signal sequence which promotes secretion from mammalian cells.
  • 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
  • ком ⁇ онент administered and have been engineered to be capable of expressing and optionally secreting the complement inhibitor.
  • they may be MHC compatible with the subject.
  • they may be encapsulated in a non- immunogenic material (e.g. alginate) for administration to the subject.
  • complex 2 as a binding mode between Sbi and C3d further leads to new methods of identifying complement inhibitors.
  • compounds which are capable of binding to the convex face of C3d and inhibiting binding of Sbi may find use as complement inhibitors.
  • the invention provides a method of screening for a
  • complement inhibitor comprising contacting a candidate compound with C3d and
  • 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.
  • the reference complement inhibitor may be the Sbi-IV or analogue thereof referred to in (ii) above.
  • 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.
  • 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.
  • the method may comprise providing a parent complement inhibitor which is capable of binding to the convex face of C3d;
  • 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.
  • the performance of the candidate compound and the parent complement inhibitor may both be determined separately and correlated with one another.
  • the performance of the candidate compound may be compared with a previously- determined value for the parent complement inhibitor, e.g.
  • the method may comprise the steps of providing a parent
  • 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 .
  • a parent nucleic acid sequence e.g. a DNA sequence
  • 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.
  • 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.
  • 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.
  • the interacting residues are represented as stick models and are labeled according to full length Sbi and intact pre-pro C3 numbering.
  • FIG. 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.
  • Sbi-IV The interaction surface of Sbi-IV on the convex face of C3d.
  • 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.
  • 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.
  • Anaphylatoxins include the components C3a and C5a.
  • foreign substances such as microorganisms, viruses, etc.
  • opsonins which become covalently bound to hydroxyl and amine groups on the foreign surface.
  • opsonins which include C3b
  • MAC membrane attack complex
  • 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
  • C3 is cleaved at a number of sites during the complement
  • 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.
  • 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..
  • 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..
  • an appropriate target such as a microorganism (e.g. a bacterium), other cell type, virus, etc.
  • 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.
  • 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 ( HCH 3 ) , C3 (H 2 0) , C3b, iC3b, iC3 (NHCH 3 ) , iC3 (H 2 0) and C3dg.
  • the sequence of human C3d is as follows:
  • 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.
  • 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 ⁇ 273, 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.
  • 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.
  • a covalent complex between a candidate molecule or inhibitor and C3d may be
  • the complex may be covalently cross- linked (e.g. by
  • 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.
  • sequence of the Sbi protein including signal sequence, follows :
  • 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.
  • WR putative wall-anchor sequence
  • Sbi-I domain 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.
  • 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
  • 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.
  • 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.
  • 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
  • the complement inhibitors described here may bind C3 ,
  • 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 mammals (e.g. dogs), equines (e.g horses), bovines (e.g. cows), caprines (e.g. goats), ovines (e.g. sheep), other mammals (e.g. dogs), equines (e.g horses), bovines (e.g. cows), caprines (e.g. goats), ovines (e.g. sheep), other mammals.
  • equines e.g horses
  • bovines e.g. cows
  • caprines e.g. goats
  • ovines e.g. sheep
  • 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.
  • 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
  • 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.
  • 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
  • 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.
  • 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 a higher helix forming propensity than others.
  • the amino acids Met, Ala, Leu, Glu and Lys have a higher helix forming propensity than others.
  • the amino acids Met, Ala, Leu, Glu and Lys have a higher helix forming propensity than others.
  • the amino acids Met, Ala, Leu, Glu and Lys have a higher helix forming propensity than others.
  • substitutions of core peptide residues introduce residues with high helix forming propensity.
  • a substitution of a core residue introduces a residue with high helix forming propensity which is also a conservative
  • Gly and Pro have poor helix-forming propensities (although Pro may be found as the first amino acid of an alpha helix) .
  • 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) .
  • 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.
  • 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.
  • 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.
  • 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.
  • the 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.
  • the C-terminal Sbi-IV sequence may have a maximum of 1, 2, 3, 4,5, 6, 7, 8, 9 or 10 substitutions, deletions or insertions relative to the
  • any such mutations are substitutions, e.g.
  • complement inhibitors described in this specification may have additional Sbi sequences upstream (i.e. N-terminal) of the Sbi-IV component.
  • the complement inhibitor may additionally comprise some or all of a Sbi-III domain (i.e.
  • the Sbi-III and Sbi-IV sequences may be separated by linker sequences to introduce conformational flexibility into the molecule.
  • 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.
  • 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
  • the inhibitors described in this specification may further comprise non—Sbi components .
  • 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.
  • 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.
  • aptamers may be RNA or DNA, or synthetic analogs thereof, and are typically oligonucleotides, e.g.
  • 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 .
  • 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.
  • a host cell e.g. within the cytoplasm, within an organelle, or within an inclusion body
  • a signal sequence 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
  • PEG molecules may be conjugated to PEG molecules in order to improve stability in vivo.
  • 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 .
  • 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.
  • 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.
  • 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 .
  • 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.
  • GST glutathione-S-transferase
  • 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.
  • 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.
  • antibody is therefore used herein to encompass any molecule comprising the binding fragment of an antibody.
  • 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.
  • compositions 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
  • 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.
  • compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.
  • 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.
  • 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.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable H, isotonicity and stability.
  • 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.
  • administration is preferably in 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
  • Suitable carriers, adjuvants, excipients, etc. can be found in standard pharmaceutical texts, for example Remington's
  • 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) ,
  • IL-1 receptor antagonist e.g. Anakinra
  • TNF inhibitors including soluble TNF receptor proteins (e.g. Etanercept) .
  • 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
  • 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-
  • 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.
  • 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) .
  • 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.
  • 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 15 N-labelled Sbi-IV for NMR titration experiments was carried out as described before(14). Binding of 15 N- labelled Sbi- IV to unlabelled C3d was followed by recording 1 H- 15 N HSQC spectra as a function of Sbi-IV: C3d ratio. The NMR titration was
  • 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.
  • Synchrotron radiation X- ray scattering data were collected at the X33 beam line of the EMBL, Hamburg Outstation (DORIS III storage ring at DESY) .
  • 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
  • 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.)
  • 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) .
  • 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.
  • 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 are further stabilized by hydrogen bonding interactions involving Sbi-IV helix al residues D208, N215, S219 and D243 and Q251 from helix ⁇ 3 with C3d thioester residues A17/1010, Q20/1013, K78/1071 R79/1072 and Y273/1266 (see Table IV for detailed list of interactions) .
  • C17/1010 thioester-contributing cysteine residue
  • C17/1010 thioester-contributing cysteine residue
  • Sbi-IV D208 is sandwiched between the two thioester- forming residues (Q20/1013 and C17/1010, here mutated to
  • Unit cell dimensions 53.4 A x 81.0 A x 87.7 A, a Space group P2.2.2,
  • 102 123 complex are the radius of gyration, molecular mass (MM), maximum size, Porod volume and excluded volume derived from experimental SAXS data.
  • MM ca i c is the MM calculated from primary sequence.
  • complement modulation protein Sbi reveals two modes of
  • 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.
  • 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
  • complement modulator Sbi also interferes with the link between the innate and adaptive branches of the host immune system.
  • NMR chemical shift analysis has a broad affinity range ( ⁇ - 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 .
  • 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) .
  • VSIEKIV residues which are sometimes considered part of the C-terminal sequence of Sbi-III
  • 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) .

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Abstract

L'invention concerne la protéine Sbi de Staphylococcus aureus et, en particulier, l'interaction entre Sbi et la protéine C3d du complément. La structure cristalline du complexe Sbi-C3d révèle une interaction inattendue entre Sbi et la surface convexe de C3d, ce qui suggère que des fragments et variants particuliers de Sbi peuvent avoir une utilité en tant qu'inhibiteurs de la voie alterne du complément. L'invention concerne de nouveaux peptides et leurs utilisations dans l'inhibition d'états inflammatoires caractérisés par une activation indésirable de la voie du complément.
PCT/GB2011/001414 2010-09-29 2011-09-29 Nouvelle interaction entre les protéines sbi et c3d de staphylococcus aureus Ceased WO2012042213A1 (fr)

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US11771758B2 (en) 2021-03-04 2023-10-03 Helix Nanotechnologies, Inc. Compositions including SBI adjuvants and methods of use thereof

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US12343392B2 (en) 2021-03-04 2025-07-01 Helix Nanotechnologies Inc Compositions including Sbi adjuvants and methods of use thereof

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