WO2012088247A2 - Fragments et anticorps anti-c5/c5a/c5adesr - Google Patents

Fragments et anticorps anti-c5/c5a/c5adesr Download PDF

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WO2012088247A2
WO2012088247A2 PCT/US2011/066437 US2011066437W WO2012088247A2 WO 2012088247 A2 WO2012088247 A2 WO 2012088247A2 US 2011066437 W US2011066437 W US 2011066437W WO 2012088247 A2 WO2012088247 A2 WO 2012088247A2
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Prior art keywords
antibody
antigen
amino acid
c5adesr
binding fragment
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PCT/US2011/066437
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English (en)
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WO2012088247A3 (fr
Inventor
Caroline COLLEY
Claire Dobson
Joanne BRENNAN
Sek Fung
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Medimmune, Llc
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Priority to CA2822288A priority Critical patent/CA2822288A1/fr
Publication of WO2012088247A2 publication Critical patent/WO2012088247A2/fr
Publication of WO2012088247A3 publication Critical patent/WO2012088247A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the technology relates in part to antibodies, and in certain embodiments, antibodies that specifically bind to complement system components C5, C5a, and C5adesR (designated for C5adesArg).
  • the complement system is composed of a group of proteins that are normally present in the serum in an inactive state.
  • Complement C5 (190 kD, molecular weight) is present in human serum. It is composed of two polypeptide chains, alpha and beta, with approximate molecular weights of 1 15 kD and 75 kD, respectively. Biosynthesized as a single-chain pro-molecule, C5 is enzymatically cleaved into a two-chain structure during processing and secretion. After cleavage, the two chains are held together by at least one disulphide bond as well as noncovalent interactions.
  • C5 is cleaved into the C5a and C5b fragments during activation of the complement pathways.
  • C5a is the activated form of C5.
  • Complement C5a is a proinflammatory mediator of the complement system, and its actions are mediated via the C5a receptor (C5aR).
  • C5a is also rapidly cleaved to a less active form, C5adesR.
  • C5a augments monocyte production of at least two important proinflammatory cytokines, TNF-alpha and IL-1.
  • COPD chronic obstructive pulmonary disease
  • COPD lungs may also become colonized with Gram negative and Gram positive bacteria which hasten lung function decline and, along with viruses and inhaled pollutants, are major triggers of recurrent debilitating exacerbations.
  • COPD patients suffer recurrent acute exacerbations (AECOPD) caused by both viruses and bacteria.
  • AECOPD recurrent acute exacerbations
  • C5a is closedly linked to the pathogenesis of AECOPD and COPD. Serum and sputum C5a is upregulated in AECOPD patients as compared to severe stable COPD and healthy individuals (Zhang et al. 2008). The increase in C5a levels is positively correlated with increased in systemic inflammatory biomarker C reactive protein (CRP), IL-8, and lung neutrophil and macrophage counts in the lung. The increase in C5a levels correlates with the reduction in lung function as measured by FEVi. These findings are in close agreement with two separate studies by Marc et al. (2010; 2004). Cigarette smoke extract were shown to increase C5aR expression in human bronchial epithelial cells in vitro (Allen-Gipson et al. 2005).
  • Cigarette smoke enhanced C5a- mediated release of IL-8 from human bronchial epithelial cells (Floreani et al. 1998). Together, there is a substantial body evidence to indicate the important roles of C5a in AECOPD and COPD.
  • C5 polypeptide “C5a polypeptide” and “C5adesR polypeptide” are referred to herein as “C5,” “C5a” and “C5adesR,” respectively.
  • the antibody provided herein does not prevent or inhibit the formation of C5b or the resulting membrance attack complex (MAC).
  • the disclosure provides an isolated antibody or antigen-binding fragment thereof which immunospecifically binds to one or more of a C5 polypeptide, a C5a polypeptide and a C5adesR polypeptide, and has one or more of the characteristics selected from the group consisting of:(a) affinity (K D ) for C5a of about 300 pM or less; (b) affinity (K D ) for
  • C5adesR of about 300 pM or less; (c) affinity (K D ) for C5 of about 30 pM or less; (d) binds specifically to one or more of human C5, C5a and C5adesR, but does not bind C5b; (e) binds to cynomolgus C5, C5a and C5adesR, (f) binds to cynomolgus C5a and C5adesR with affinities similar to human C5a and C5adesR; (g) does not block the cleavage of C5 to C5a and C5b; (h) inhibits the binding of the C5a or C5adesR to the C5aR or C5L2 receptor; (i) inhibits C5a and C5adesR mediated signaling in target cells; (j) inhibits C5a and C5adesR mediated calcium mobilization in C5aR and G alpha 16 transfected HEK cells; (k) inhibit
  • the disclosure provides an isolated antibody or antigen-binding fragment thereof which immunospecifically binds to human and cynomolgus C5, C5a and C5adesR with an affinity (K D ) of about 30 pM or less and has one or more of the characteristics selected from the group consisting of: (a) binds specifically to one or more of human C5, C5a and C5adesR, but does not bind C5b; (b) does not block the cleavage of C5 to C5a and C5b; (c) inhibits the binding of the C5a or C5adesR to the C5aR or C5L2 receptor; (d) inhibits C5a and C5adesR mediated signaling in target cells; (e) inhibits C5a and C5adesR mediated calcium mobilization in C5aR and G alpha 16 transfected HEK cells; (f) inhibits C5a mediated chemotaxis of human neutrophils; (g) inhibits chemotaxis of
  • cynomolgus whole blood samples with IC 50 values in the range of about 1 nM to about 50 nM.
  • the disclosure provides an isolated antibody or antigen-binding fragment thereof which immunospecifically binds to human and cynomolgus C5a and C5adesR with an affinity (K D ) of about 300 pM or less and has one or more of the characteristics selected from the group consisting of: (a) binds specifically to one or more of human C5, C5a and C5adesR, but does not bind C5b; (b) does not block the cleavage of C5 to C5a and C5b; (c) inhibits the binding of the C5a or C5adesR to the C5aR or C5L2 receptor; (d) inhibits C5a and C5adesR mediated signaling in target cells; (e) inhibits C5a and C5adesR mediated calcium mobilization in C5aR and G alpha 16 transfected HEK cells; (f) inhibits C5a mediated chemotaxis of human neutrophils. (g) inhibits chemotaxis
  • cynomolgus whole blood samples with IC 50 values in the range of about 1 nM to about 300 nM and about 2 nM to about 200 nM respectively.
  • the isolated antibody or antigen-binding fragment thereof has an affinity (K D ) for human or cynomolgus C5; human or cynomolgus C5a; or human or cynomolgus C5adesR of about 30 pM or less.
  • the isolated antibody or antigen-binding fragment thereof immunospecifically binds to human and cynomolgus C5a and C5adesR with an affinity (K D ) of about 300 pM or less.
  • the isolated antibody or antigen-binding fragment thereof comprises (a) a VH CDR1 having an amino acid sequence identical to, or comprising 1 , 2, or 3 amino acid residue substitutions relative to, any one of SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, or 1 13; (b) a VH CDR2 having an amino acid sequence identical to, or comprising 1 , 2, 3, 4, 5, 6 or 7 amino acid residue substitutions relative to, any one of SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, or 1 14; and (c) a VH CDR3 having an amino acid sequence identical to, or comprising 1 , 2, 3, 4, 5, 6 or 7 amino acid residue substitutions relative to, any one of SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, or 1 15.
  • the isolated antibody or antigen- binding fragment thereof further comprises a VH CDR3 having at least 60.0% identity to the amino acid sequence of SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, or 1 15.
  • the isolated antibody or antigen-binding fragment thereof comprises (a) a VL CDR1 having an amino acid sequence identical to, or comprising 1 , 2, or 3 amino acid residue substitutions relative to, any one of SEQ ID NO: 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, or 1 18; (b) a VL CDR2 having an amino acid sequence identical to, or comprising 1 , 2 or 3 amino acid residue substitutions relative to, any one of SEQ ID NO: 9, 19, 29, 39, 49, 59, 69, 79, 89, 99, 109, or 1 19; (c) a VL CDR3 having an amino acid sequence identical to, or comprising 1 , 2, 3, 4, 5, 6 or 7 amino acid residue substitutions relative to, any one of SEQ ID NO: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10 or 120.
  • the isolated antibody or antigen-binding fragment thereof comprises a VL CDR3 having at least 50.0% identity to the amino acid sequence of SEQ ID NO: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10 or 120.
  • the isolated antibody or antigen-binding fragment thereof comprises (a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, or 1 13; (b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, or 1 14; (c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, or 1 15;(d) a VL CDR1 having the amino acid sequence of SEQ ID NO: 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, or 1 18; (e) a VL CDR2 having the amino acid sequence of SEQ ID NO: 9, 19, 29, 39, 49, 59, 69, 79, 89, 99, 109, or
  • the isolated antibody or antigen-binding fragment further comprises a VH CDR3 having at least 60.0% identity to the amino acid sequence of SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, or 1 15.
  • the isolated antibody or antigen-binding fragment thereof further comprises a VL CDR3 having at least 50.0% identity to the amino acid sequence of SEQ ID NO: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10 or 120.
  • the isolated antibody or antigen-binding fragment thereof comprises a VH chain domain comprising three CDRs and a VL chain domain comprising three CDRs, wherein the three CDRs of the VH chain domain comprise: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, or 1 13; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, or 1 14; and (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, or 1 15.
  • the isolated antibody or antigen-binding fragment further comprises a VH CDR3 having at least 60.0% identity to the amino acid sequence of SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, or 1 15.
  • the isolated antibody or antigen-binding fragment thereof comprises a VH chain domain comprising three CDRs and a VL chain domain comprising three CDRs, wherein the three CDRs of the VL chain domain comprise: (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, or 1 18; (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 9, 19, 29, 39, 49, 59, 69, 79, 89, 99, 109, or 1 19; and
  • VL CDR3 comprising the amino acid sequence of SEQ ID NO: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10 or 120.
  • the isolated antibody or antigen-binding fragment thereof further comprises a VL CDR3 having at least 50.0% identity to the amino acid sequence of SEQ ID NO: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10 or 120.
  • the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable domain having at least about 90% identity to the amino acid sequence of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, or 1 12, wherein said antibody has the activity of inhibiting the binding of one or more of C5a and C5adesR to the C5a receptors.
  • the isolated antibody or antigen-binding fragment thereof comprises a light chain variable domain having at least about 90% identity to the amino acid sequence of SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, or 1 17, wherein said antibody has the activity of inhibiting the binding of one or more of C5a and C5adesR to the C5a receptors.
  • the isolated antibody or antigen-binding fragment thereof further comproses a light chain variable domain having at least about 90% identity to the amino acid sequence of SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, or 1 17.
  • the isolated antibody or antigen- binding fragment thereof further comprises a heavy chain variable domain having at least about 90% identity to the amino acid sequence of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, or 1 12, wherein said antibody has the activity of inhibiting the binding of one or more of C5a and C5adesR to the C5a receptors.
  • an isolated antibody or antigen-binding fragment thereof that has
  • immunospecifically binds to one or more of a human and/or cynomolgus C5 polypeptide, a human and/or cynomolgus C5a polypeptide and a human and/or cynomolgus C5adesR polypeptide and comprises a heavy chain variable domain having at least about 90% identity to the amino acid sequence of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, or 1 12 and a light chain variable domain having at least about 90% identity to the amino acid sequence of SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, or 1 17, wherein said antibody has the activity of inhibiting the binding of one or more of C5a and C5adesR to the C5a receptors.
  • the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable domain of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, or 1 12 and a light chain variable domain of SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, or 1 17.
  • the isolated antibody or antigen-binding fragment thereof comprises a human IgG constant domain having one or more amino acid substitutions relative to a wild-type human IgG constant domain.
  • the human IgG constant domain is lgG1 .
  • the human IgG constant domain is lgG4.
  • the one or more amino acid substitutions is selected from the group consisting of L234F, L235E, P331 S, L235F, L235Y, M252Y, S254T, T256E and S228P.
  • the C5a polypeptide includes an amino acid sequence of SEQ ID NO: 130 or the C5a polypeptide includes cynomolgus C5a having an amino acid sequence of SEQ ID NO: 131.
  • the cyno C5adesR polypeptide includes an amino acid sequence of SEQ ID NO: 132 or SEQ ID NO: 135, or the human C5adesR polypeptide includes an amino acid sequence of SEQ ID NO:134.
  • an isolated antibody or antigen-binding fragment thereof that has
  • an isolated antibody or antigen- binding fragment thereof that immunospecifically binds one or more of a human and/or cynomolgus C5 polypeptide, a human and/or cynomolgus C5a polypeptide and a human and/or cynomolgus C5adesR polypeptide, and comprises (i) a VL CDR3 having an amino acid sequence identical to, (ii) a VL CDR3 having 1 , 2, 3, 4, 5, 6 or 7 amino acid residue substitutions relative to, and/or (iii) a VL CDR3 having at least about 50% identity to, the amino acid sequence of SEQ ID NO: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10 or 120, wherein the antibody or antigen-binding fragment thereof neutralizes the effects of a C5a and/or a C5adesR polypeptide.
  • an isolated antibody or antigen-binding fragment thereof that has
  • an isolated antibody or antigen- binding fragment thereof that immunospecifically binds one or more of a human and/or cynomolgus C5 polypeptide, a human and/or cynomolgus C5a polypeptide and a human and/or cynomolgus C5adesR polypeptide, and comprises (i) a VL CDR3 having an amino acid sequence identical to, (ii) a VL CDR3 having 1 , 2, 3, 4, 5, 6 or 7 amino acid residue substitutions relative to, and/or (iii) a VL CDR3 having at least about 50% identity to, the amino acid sequence of SEQ ID NO: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10 or 120, wherein the antibody or antigen-binding fragment thereof inhibits C5a induced neutrophil activation and/or chemotaxis.
  • an isolated antibody or antigen-binding fragment thereof that has
  • an isolated antibody or antigen-binding fragment thereof that immunospecifically binds one or more of a human and/or cynomolgus C5 polypeptide, a human and/or cynomolgus C5a polypeptide and a human and/or cynomolgus C5adesR polypeptide, and comprises a (i) a VL CDR3 having an amino acid sequence identical to, (ii) a VL CDR3 having 1 , 2, 3, 4, 5, 6 or 7 amino acid residue substitutions relative to, and/or (iii) a VL CDR3 having at least about 50% identity to, the amino acid sequence of SEQ ID NO: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10 or 120, wherein the antibody or antigen-binding fragment thereof inhibits C5a induced calcium mobilization.
  • an isolated antibody or antigen-binding fragment thereof that has
  • an isolated antibody or antigen-binding fragment thereof that immunospecifically binds one or more of a human and/or cynomolgus C5 polypeptide, a human and/or cynomolgus C5a polypeptide and a human and/or cynomolgus C5adesR polypeptide, and comprises a (i) a VL CDR3 having an amino acid sequence identical to, (ii) a VL CDR3 having 1 , 2, 3, 4, 5, 6 or 7 amino acid residue substitutions relative to, and/or (iii) a VL CDR3 having at least about 50% identity to, the amino acid sequence of SEQ ID NO: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10 or 120, wherein the antibody or antigen- binding fragment thereof inhibits C5a- or C5adesR-mediated signaling in target cells.
  • the antibody or antigen-binding fragment thereof is selected from the group consisting of a human antibody, a chimeric antibody, and a humanized antibody.
  • the antibody or antigen-binding fragment thereof of any one of claims 1 -37 which is selected from the group consisting of a naturally-occurring antibody, an scFv fragment, an Fab fragment, an F(ab')2 fragment, a minibody, a diabody, a triabody, a tetrabody, and a single chain antibody.
  • the antibody or antigen-binding fragment thereof is a monoclonal antibody.
  • the disclosure is directed to a composition comprising the isolated antibody or antigen-binding fragment thereof of the disclosure and a carrier.
  • a composition comprising the isolated antibody or antigen-binding fragment thereof of the disclosure and a carrier.
  • the composition further comprises a therapeutic agent.
  • the isolated antibody or antigen-binding fragment thereof is linked to the therapeutic agent via a linker.
  • the composition further comprises a diagnostic agent.
  • the diagnostic agent comprises an imaging agent.
  • the diagnostic agent comprises a detectable label.
  • the isolated antibody or antigen-binding fragment thereof is linked to the diagnostic agent via a linker.
  • the disclosure is directed to a polynucleotide encoding the antibody or antigen-binding fragment thereof of the disclosure. In some embodiments, the disclosure is directed to a vector comprising the polynucleotide of the invention. In another embodiment, the disclosure is directed to a host cell comprising the polynucleotide or a vector of the invention.
  • the invention is directed to a method of producing an antibody or antigen- binding fragment thereof of the disclosure, comprising culturing the host cell of the disclosure, and recovering said antibody or antigen-binding fragment thereof.
  • the invention is directed to an antibody or antigen-binding fragment thereof of the disclosure produced by a method of the invention.
  • the disclosure is directed to a kit comprising (a) the antibody or antigen- binding fragment or the composition of the disclosure; (b) instructions for using the antibody or antigen-binding fragment or composition or directions for obtaining instructions for using the composition.
  • the antibody or antigen-binding fragment or the antibody in the composition is linked to a solid support.
  • the disclosure is directed to a method comprising administering the antibody or antigen-binding fragment or the composition of the disclosure to a subject; and detecting the presence, absence or amount of a biological effect in the subject associated with the administration of the composition.
  • the method comprises administering the antibody or antigen-binding fragment or the composition of the discosure to a subject; and monitoring the condition of the subject.
  • the disclosure is directed to a method for neutralizing a C5 polypeptide, a C5a polypeptide and/or a C5adesR polypeptide by administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof or the composition of the disclosure to neutralize the C5a and/or C5adesR polypeptide without inhibiting the formation of C5b.
  • the disclosure is directed to a method of preventing, treating, or managing a condition mediated by a C5 polypeptide, a C5a polypeptide and/or a C5adesR polypeptide in a subject in need thereof, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof or the composition of the disclosure to prevent, treat or manage the condition.
  • the disclosure is directed to a method for preventing, treating or managing a condition associated with a C5 polypeptide, a C5a polypeptide and/or a C5adesR polypeptide, comprising administering the antibody or antigen-binding fragment thereof or the composition of the disclsoure to a subject in need thereof in an amount effective to inhibit C5a induced neutrophil activation and/or chemotaxis, to inhibit C5a induced calcium mobilization, or to inhibit C5a- or C5adesR-mediated signaling in target cells.
  • the disclosure is directed to a method for treating, preventing or alleviating the symptoms of a disorder mediated by a C5 polypeptide, a C5a polypeptide and/or a C5adesR polypeptide in a subject in need thereof, comprising administering an effective amount of the antibody or antigen-binding fragment thereof or the composition of the disclosure to the subject to treat, prevent or alleviate the symptoms.
  • the disorder is selected from the group consisting of inflammatory disorders, autoimmune disorders, cancer and pain disorders.
  • the disorder is an inflammatory disorder.
  • the inflammatory disorder is COPD.
  • inflammatory disorder is acute exacerbations in chronic obstructive pulmonary disease (AECOPD).
  • the disclosure is directed to a method for diagnosing a disease condition mediated by a C5 polypeptide, a C5a polypeptide and/or a C5adesR polypeptide in a subject, comprising identifying a subject with an increase of C5a polypeptide and/or a C5adesR
  • the subject is a domestic animal. In one embodiment, the subject is a human.
  • an isolated antibody or antigen-binding fragment thereof comprises: (a) a variable heavy (VH) chain CDR1 having an amino acid sequence identical to, or comprising 1 , 2, or 3 amino acid residue substitutions relative to, any one of SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, or 1 13; (b) a VH CDR2 having an amino acid sequence identical to, or comprising
  • an isolated antibody or antigen-binding fragment thereof comprises: (a) a variable light (VL) chain CDR1 having an amino acid sequence identical to, or comprising 1 , 2 or 3 amino acid residue substitutions relative to, any one of SEQ ID NO: 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, or 1 18; (b) a VL CDR2 having an amino acid sequence identical to, or comprising 1 , 2 or 3 amino acid residue substitutions relative to, any one of SEQ ID NO: 9, 19, 29, 39, 49, 59, 69, 79, 89, 99, 109, or 1 19; and (c) a VL CDR3 an amino acid sequence identical to, or comprising 1 ,
  • an isolated antibody or antigen-binding fragment thereof comprises: (a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, or 1 13; (b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, or 1 14; (c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, or 1 15; (d) a variable light (VL) chain CDR1 having the amino acid sequence of SEQ ID NO: 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108
  • an isolated antibody or antigen-binding fragment thereof (i) comprises a VH chain domain comprising three CDRs and a VL chain domain comprising three CDRs and (ii) immunospecifically binds one or more of C5, C5a and C5adesR.
  • the three CDRs of the VH chain domain comprise: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, or 1 13; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, or 1 14; and (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, or 1 15.
  • an isolated antibody or antigen-binding fragment thereof (i) comprises a VH chain domain comprising three CDRs and a VL chain domain comprising three CDRs; and (ii) immunospecifically binds one or more of C5, C5a and C5adesR, where the three CDRs of the VL chain domain comprise: (a) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, or 1 18; (b) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 9, 19, 29, 39, 49, 59, 69, 79, 89, 99, 109, or 1 19; and (c) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10 or 120.
  • an isolated antibody or antigen-binding fragment thereof comprises any combination of SEQ ID NOS presented in Table 20 for each VH CDR1 , VH CDR2, VH CDR3, VL CDR1 , VL CDR2 and VL CDR3. In certain embodiments, an isolated antibody or antigen-binding fragment thereof comprises any combination of SEQ ID NOS presented in Table 21 for each VH and VL chain domain. In some embodiments, an isolated antibody or antigen-binding fragment thereof
  • immunospecifically binds to one or more of a C5 polypeptide, a C5a polypeptide and a C5adesR polypeptide and comprises a heavy chain variable domain having at least about 90% identity to, and/or contains 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12 or 13 amino acid substitutions relative to, the amino acid sequence of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, or 1 12, where the antibody has the activity of inhibiting the binding of one or more of C5a and C5adesR to one or more C5a receptors, C5aR and C5L2.
  • an isolated antibody or antigen- binding fragment thereof immunospecifically binds to one or more of a C5 polypeptide, a C5a polypeptide and a C5adesR polypeptide and comprises a light chain variable domain having at least about 90% identity to, and/or contains 1 , 2, 3, 4, 5, 6, 7 or 8 amino acid substitutions relative to, the amino acid sequence of SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, or 1 17, where the antibody has the activity of inhibiting the binding of one or more of C5a and C5adesR to one or more of the C5a receptors, C5aR and C5L2.
  • an isolated antibody or antigen-binding fragment thereof (i) immunospecifically binds to one or more of a C5 polypeptide, a C5a polypeptide and a C5adesR polypeptide, (ii) comprises a heavy chain variable domain having at least about 90% identity to, and/or contains 1 , 2, 3, 4, 5, 6, 7,8, 9, 10, 1 1 , 12 or 13 amino acid substitutions relative to, the amino acid sequence of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, or 1 12, and (iii) comprises a light chain variable domain having at least about 90% identity to, and/or contains 1 , 2, 3, 4, 5, 6, 7 or 8 amino acid substitutions relative to, the amino acid sequence of SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, or 1 17, where the antibody has the activity of inhibiting the binding of one or more of C5a and C5adesR to one or more of the
  • an isolated antibody or antigen-binding fragment thereof that immunospecifically binds to one or more of C5, C5a and C5adesR and comprises a heavy chain variable domain having about 90% or more identity to, and/or contains 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12 or 13, amino acid substitutions relative to, the amino acid sequence of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, or 1 12.
  • an isolated antibody or antigen-binding fragment thereof that immunospecifically binds to one or more of C5, C5a and C5adesR and comprises a light chain variable domain having 90% or more identity to, and/or contains 1 , 2, 3, 4,5, 6, 7 or 8 amino acid substitutions relative to, the amino acid sequence of SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, or 1 17.
  • an isolated antibody or antigen-binding fragment thereof that immunospecifically binds to one or more of C5, C5a and C5adesR and comprises a heavy chain variable domain having about 90% or more identity to the amino acid sequence of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, or 1 12 and comprises a light chain variable domain having 90% or more identity to the amino acid sequence of SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, or 1 17.
  • an isolated antibody or antigen-binding fragment thereof has the activity of inhibiting the binding of one or more of C5a and C5adesR to one or more of the C5a receptors, C5aR and C5L2.
  • the isolated antibody does not prevent or inhibit the formation of C5b or the resulting MAC complex.
  • an isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable domain of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, or 1 12 and a light chain variable domain of SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, or 1 17.
  • an isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable domain of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, or 1 12 and a light chain variable domain of SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, or 1 17.
  • an isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable domain of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, or 1 12 and a light chain variable domain of SEQ ID NO: 7, 17, 27, 37,
  • the isolated antibody does not prevent or inhibit the formation of C5b or the resulting MAC complex.
  • an isolated antibody or antigen-binding fragment thereof comprising:
  • the isolated antibody does not prevent or inhibit the formation of C5b or the resulting MAC complex.
  • an isolated antibody or antigen-binding fragment thereof further comprises a VH CDR3 having at least 60% identity to the amino acid sequence of SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, or 1 15, and a VL CDR3 having at least 50% identity to the amino acid sequence of SEQ ID NO: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10 or 120, where the antibody or antigen-binding fragment neutralizes C5a and C5adesR.
  • the isolated antibody does not prevent or inhibit the formation of C5b or the resulting MAC complex.
  • an isolated antibody or antigen-binding fragment thereof comprising:
  • an isolated antibody or antigen-binding fragment thereof comprising:
  • an isolated antibody or antigen-binding fragment thereof further comprises a VH CDR3 having at least 60% identity to the amino acid sequence of SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, or 1 15, and a VL CDR3 having at least 50% identity to the amino acid sequence of SEQ ID NO: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10 or 120, where the antibody or antigen-binding fragment inhibits neutophil activation and/or chemotaxis.
  • an isolated antibody or antigen-binding fragment thereof comprising:
  • an isolated antibody or antigen-binding fragment thereof comprising:
  • an isolated antibody or antigen-binding fragment thereof further comprises a VH CDR3 having at least 60% identity to the amino acid sequence of SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, or 1 15, and a VL CDR3 having at least 50% identity to the amino acid sequence of SEQ ID NO: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10 or 120, where the antibody or antigen-binding fragment inhibits C5a- or C5adesR-mediated signalling in target cells.
  • a single chain Fv (scFv) antibody or fragment thereof comprising (i) a heavy chain variable domain amino acid sequence identical to, (ii) a heavy chain variable domain amino acid sequence having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12 or 13 amino acid residue substitutions relative to, and/or (iii) a heavy chain variable domain amino acid sequence having at least about 90% identity to, the amino acid sequence of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, or 1 12.
  • scFv single chain Fv antibody or fragment thereof, comprising (i) a light chain variable domain amino acid sequence identical to, (ii) a light chain variable domain amino acid sequence having 1 , 2, 3, 4, 5, 6, 7 or 8 amino acid residue substitutions relative to, and/or (iii) a light chain variable domain amino acid sequence having 90% or more identity to, the amino acid sequence of SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, or 1 17.
  • a single chain Fv (scFv) antibody or fragment thereof comprising (a)(i) a heavy chain variable domain amino acid sequence identical to, (ii) a heavy chain variable domain amino acid sequence having 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12 or 13 amino acid residue substitutions relative to, and/or (iii) a heavy chain variable domain amino acid sequence having at least about 90% identity to, the amino acid sequence of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, or 1 12, and comprising (b)(i) a light chain variable domain amino acid sequence identical to, (ii) a light chain variable domain amino acid sequence having 1 , 2, 3, 4, 5, 6, 7 or 8 amino acid residue substitutions relative to, and/or (iii) a light chain variable domain amino acid sequence having 90% or more identity to, the amino acid sequence of SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87
  • an isolated antibody or antigen-binding fragment thereof further comprises a human IgG constant domain having one or more amino acid substitutions relative to a wild-type human IgG constant domain.
  • the human IgG constant domain is lgG1.
  • the human IgG constant domain is lgG4.
  • the one or more amino acid substitutions is selected from the group consisting of L234F, L235E, P331 S, L235F, L235Y, and S228P.
  • an isolated antibody or antigen-binding fragment thereof comprising
  • (k) inhibits chemotaxis of human neutrophils at about a molar ratio of 1 :1 between the antibody and human C5a;
  • (j) inhibits C5a mediated neutrophil CD1 1 b expression in human and cynomolgus whole blood samples with IC 50 values in the range of about 1 nM to about 300 nM or about 2 nM to about 200 nM.
  • an isolated antibody or antigen-binding fragment thereof binds an antigen ⁇ e.g., C5, C5a, C5adesR) with an affinity characterized by an equilibrium dissociation constant (K D ) in the range of about 3 pM to about 30 nM.
  • an isolated antibody or antigen-binding fragment thereof binds human C5a with a K D of about 17 nM.
  • an isolated antibody or antigen-binding fragment thereof binds human C5ades R with a K D of about 14 nM.
  • an isolated antibody or antigen-binding fragment thereof binds polyhistindine Flag tagged cynomolgus C5adesR with a K D of about 28 nM. In certain embodiments an isolated antibody or antigen-binding fragment thereof binds human C5a with a K D of about 8 pM or less. In various embodiments an isolated antibody or antigen-binding fragment thereof binds human C5adesR with a K D of about 6 pM. In some embodiments an isolated antibody or antigen-binding fragment thereof binds cynomolgus C5adesR with a K D of about 19 pM.
  • an isolated antibody or antigen-binding fragment thereof binds polyhistindine Flag tagged cynomolgus C5adesR with a K D of about 10 pM. In various embodiments, an isolated antibody or antigen-binding fragment thereof binds human C5a with a K D of about 3pM. In certain embodiments an isolated antibody or antigen-binding fragment thereof binds human C5 with a K D of about 27 pM.
  • the animal is human.
  • C5a or C5adesR activity is selected from one or more of the following events: binding of C5a and C5adesR to C5aR and/or C5L2, activation of C5aR and/or C5L2 expressing effector cells, triggering release of pro-inflammatory cytokines and chemokines from the activated cells and triggering chemotaxis of neutrophils.
  • the C5a activity comprises the activity of a C5a polypeptide.
  • the C5a polypeptide includes an amino acid sequence of SEQ ID NO: 130.
  • the cyno C5a polypeptide includes an amino acid sequne of SEQ ID NO: 131.
  • the C5adesR activity comprises the activity of a C5adesR
  • the C5adesR polypeptide includes an amino acid sequence of SEQ ID NO: 132 or SEQ ID NO: 134, or SEQ ID NO: 135.
  • kits for isolation and purification of C5/C5a/C5adesR comprising an isolated antibody or antigen-binding fragment thereof, and instructions for use.
  • the antibody is coupled to a solid support.
  • the solid support comprises beads, and in certain embodiments, the solid support comprises sepharose beads.
  • the instructions for use include one or more of isolating, purifying, detecting and quantifying a C5 polypeptide, a C5a polypeptide and/or a C5adesR polypeptide.
  • a kit further comprises a buffer, a solid support or a buffer and a solid support.
  • the solid support is one or more of a bead, filter, membrane and multiwell plate.
  • a kit includes instructions for use of an isolated antibody or antigen-binding fragment thereof or directions for obtaining such instructions.
  • a detection kit includes a buffer and membrane suitable for a western blot.
  • a kit includes a loading buffer and an elution buffer.
  • the buffer is suitable for an enzyme-linked
  • an isolated antibody or antigen-binding fragment thereof comprises a therapeutic agent.
  • An antibody described herein sometimes comprises a diagnostic agent.
  • the diagnostic agent may comprise an imaging agent or a detectable label in some embodiments.
  • An isolated antibody or antigen-binding fragment thereof sometimes is linked to a therapeutic agent or diagnostic agent via a linker.
  • a method comprising administering an isolated antibody or antigen-binding fragment thereof and detecting the presence, absence or amount of a biological effect associated with the administration of the antibody.
  • a method that comprises administering an isolated antibody or antigen-binding fragment thereof to a subject; and detecting the presence, absence or amount of a biological effect in the subject associated with the administration of the antibody.
  • a method that comprises administering an isolated antibody or antigen-binding fragment thereof to a subject and monitoring the condition of the subject.
  • a method of preventing, treating, or managing a condition mediated by a C5a polypeptide and/or a C5adesR polypeptide in a subject in need thereof comprising administering to the subject an effective amount an isolated antibody or antigen-binding fragment thereof described herein to prevent, treat or manage the condition.
  • the condition is COPD and/or acute exacerbations of COPD.
  • a method for preventing, treating or managing a condition associated with a C5 polypeptide, a C5a polypeptide and/or a C5adesR polypeptide comprising administering an isolated antibody or antigen-binding fragment thereof described herein to a subject in need thereof in an amount effective to inhibit neutophil activation and/or chemotaxis.
  • a method for preventing, treating or managing a condition associated with a C5 polypeptide, a C5a polypeptide and/or a C5adesR polypeptide comprising administering an isolated antibody or antigen-binding fragment thereof described herein to a subject in need thereof in an amount effective to inhibit calcium mobilisation.
  • the method is a method for preventing the condition.
  • a method for diagnosing a condition mediated by a C5 polypeptide, a C5a polypeptide and/or a C5adesR polypeptide in a subject comprising selecting a subject in need of diagnosis and administering to the subject a diagnostically effective dose of an isolated antibody or antigen-binding fragment thereof described herein.
  • the subject is a domestic animal, and in certain embodiments, the subject is a human. Certain embodiments are described further in the following description, examples, claims and drawings.
  • Figures 1A and 1 B Example of immunoprecipitation results demonstrating that mAb1 lgG2 bound to native, endogenous human C5 and C5a/C5adesR in plasma.
  • Lanes 2 and 8 contain mAb1 lgG2; lanes 3 and 9, isotype matched negative control mAb.
  • For lanes 2-3 normal human plasma was used to determine the binding to plasma C5.
  • Lanes 4 and 10 contained 1 microliter human plasma.
  • Lanes 5 and 1 1 contained 1 microliter zymosan activated human plasma.
  • Lanes 6 and lane 12 contained 100 ng purified human C5 (Complement Technology, A120) and human purified C5a (Biovision, 4995A), respectively.
  • the arrows indicate the bands of native, endogenous C5 and C5a/C5adesR proteins.
  • Figures 2A and 2B Example of immunoprecipitation results demonstrating that mAb1 lgG2 bound to native, endogenous cynomolgus monkey C5 and C5a/C5adesR in cynomolgus monkey plasma.
  • Lanes 2 and 8 contain mAb1 lgG2; lanes 3 and 9, isotype matched negative control mAb.
  • normal cynomolgus plasma was used to determine the binding to plasma C5.
  • lanes 8-9 zymosan-activated cynomolgus plasma was used to determine the binding to plasma
  • Lanes 4 and 10 contained 1 microliter cynomolgus plasma. Lanes 5 and 1 1 contained 1 microliter zymosan activated cynomolgus plasma. Lane 6 and lane 12 contained 100 ng human C5 (Complement Technology, A120) and human C5a (Biovision, 4995A), respectively. The arrows indicate the bands of native, endogenous C5 and C5a/C5adesR proteins. This data is representative of two separate experiments.
  • Figure 3 Example of results showing mAb1 lgG2 had no effect on the activation of the classical complement pathway in hemolytic assays. Eculizumab was used as the positive control.
  • Figure 4 Example of results showing mAb1 lgG2 had no effect on the activation of the alternative complement pathway in hemolytic assays. Eculizumab was used as the positive control.
  • Figure 5 Example of results showing mAb9 lgG2 inhibited neutrophil activation (CD1 1 b upregulation) in the cynomolgus whole blood assay.
  • Figure 6 Example of results showing mAb9 lgG4 S228P inhibited neutrophil activation (CD1 1 b upregulation) in human whole blood assay (pre-incubation condition).
  • Figure 7 Example of results showing mAb9 lgG4 S228P inhibited neutrophil activation in human whole blood assay (no pre-incubation condition).
  • Figures 8A and 8B Example of immunoprecipitation results demonstrating that mAb9 and mAb12 bound to native, endogenous C5 and C5a/C5adesR in human plasma.
  • Lane 1 mAb1 lgG2; lane 2, mAb9 lgG2; lane 3, mAb9 lgG4 S228P; lane 4, mAb12 lgG4 S288P; lane 6, isotype matched lgG4 S288P negative control; lane 9, 100 ng purified human C5 (Complement Technology, A120); lane 9 * , 100 ng purified human C5a (Biovision, 4995A).
  • Figures 9A and 9B Example of immunoprecipitation results demonstrating that mAb9 and mAb12 bound to native, endogenous C5 and C5a/C5adesR in cynomolgus plasma.
  • Lane 1 mAb1 lgG2; lane 2, mAb9 lgG2; lane 3, mAb9 lgG4 S228P; lane 4, mAb12 lgG4 S288P; lane 6, isotype matched lgG4 S288P negative control; lane 9, 100ng purified human C5 (Complement
  • Figure 10 Example of results showing mAb9 lgG4 S228P and mAb12 lgG4 S228P had no effect on the activation of the classical complement pathway.
  • Figure 1 1 Example of results showing mAb9 lgG4 S228P and mAb12 lgG4 S228P had no effect on the activation of the alternative complement pathway.
  • Figures 12A to 12D Heavy chain sequence alignment for mAb1 and derived antibodies showing complimentarity determining region (CDR) 1 -3 and framework region (Fw) 1-4.
  • Figures 13A to 13D Light chain sequence alignment for mAb1 and derived antibodies showing complimentarity determining region (CDR) 1 -3 and framework region (Fw) 1-4.
  • Figure 14 Heavy chain complimentarity determining regions (VH-CDRs) for mAb1 and derived antibodies.
  • Figure 15 Light chain complimentarity determining regions (VL-CDRs) for mAb1 and derived antibodies.
  • antibodies including human, humanized and/or chimeric forms, as well as fragments, derivatives/conjugates and compositions thereof that bind to complement system components C5/C5a/C5adesR.
  • Such antibodies can be useful for detecting and/or visualizing C5, C5a and/or C5adesR, and therefore may be useful in assays and diagnostic methods.
  • Antibodies described herein also interfere with binding between C5a and/or C5adesR and a receptor to which it binds, and therefore can be useful for treating C5a mediated diseases.
  • C5 is cleaved into the C5a and C5b fragments during activation of the complement pathways.
  • Activation of the complement system encompasses mainly three distinct pathways, designated the classical, alternative, and lectin pathways.
  • the classical pathway is a calcium/magnesium- dependent cascade, which is normally activated by the formation of antigen-antibody complexes. It can also be activated in an antibody-independent manner by the binding of C-reactive protein, complexed with ligand, and by many pathogens including gram-negative bacteria.
  • the alternative pathway is a magnesium-dependent cascade which is activated by deposition and activation of C3 on certain susceptible surfaces ⁇ e.g., cell wall polysaccharides of yeast and bacteria, and certain biopolymer materials).
  • the lectin pathway shares overlapping steps with the classical pathway but is triggered through binding of mannan-binding lectin (MBL) to carbohydrate or glycoprotein containing components on microorganisms or damaged tissues. Furthermore, independently from the three complement pathways, activation of C5 to form C5a can be mediated by enzymes, such as thrombin, kallikrein, mast-cell tryptase, and leukocyte derived proteases.
  • enzymes such as thrombin, kallikrein, mast-cell tryptase, and leukocyte derived proteases.
  • complement pathway Activation of the complement pathway generates biologically active fragments of complement proteins, e.g., C3a, C4a and C5a anaphylatoxins and C5b-9 membrane attack complexes (MAC), which mediate inflammatory responses through involvement of leukocyte chemotaxis, activation of macrophages, neutrophils, platelets, mast cells and endothelial cells, increased vascular permeability, cytolysis, and tissue injury.
  • complement proteins e.g., C3a, C4a and C5a anaphylatoxins and C5b-9 membrane attack complexes (MAC), which mediate inflammatory responses through involvement of leukocyte chemotaxis, activation of macrophages, neutrophils, platelets, mast cells and endothelial cells, increased vascular permeability, cytolysis, and tissue injury.
  • MAC membrane attack complexes
  • C5a induces chemotactic migration of neutrophils, eosinophils and monocytes. Both C5a and C5b-9 activate endothelial cells to express adhesion molecules essential for sequestration of activated leukocytes, which mediate tissue inflammation and injury. C5a also mediates inflammatory reactions by causing smooth muscle contraction, increasing vascular permeability, inducing basophil and mast cell degranulation and inducing release of lysosomal proteases and oxidative free radicals. Furthermore, C5a modulates the hepatic acute-phase gene expression and augments the overall immune response by increasing the neutophil activation and/or chemotaxis.
  • C5a modulates the hepatic acute-phase gene expression and augments the overall immune response by increasing the production of TNF alpha, IL-1 beta, IL-6, and IL-8.
  • the human C5a receptor (C5aR) has been cloned. It belongs to a superfamily of seven- transmembrane-domain, G protein-coupled receptors. C5aR is expressed on neutrophils, monocytes, basophils, eosinophils, hepatocytes, lung smooth muscle and endothelial cells, and renal glomerular tissues.
  • the ligand-binding site of C5aR is complex and consists of at least two physically separable binding domains.
  • C5a also binds to an orphan receptor, C5L2.
  • C5L2 is also a seven-transmembrane-domain receptor but it is not coupled to G proteins.
  • C5L2 has been regarded as a non-signalling receptor and that it serves as a decoy, similar to a number of other non-signalling decoy chemoattractant receptors (Lee et al, 2008).
  • C5L2 deficient mice were hypersensitive to LPS-induced septic shock and showed reduced airway hyperresponsiveness and inflammation in an ovalbumin-induced model (Chen et al, 2007). Furthermore, in a mouse model of 'high-grade'sepsis (100% lethality), only blockade of both C5aR and C5L2 provided protection (Rittirsch et al, 2007). Like C5aR, C5L2 is also thought to play a role in the uptake and possibly recycling of C5a by cells.
  • antibody encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies formed from at least two different epitope binding fragments (e.g., bispecific antibodies), human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, single-chain Fvs (scFv), single-chain antibodies, single domain antibodies, domain antibodies, Fab fragments, F(ab')2 fragments, antibody fragments that exhibit the desired biological activity ⁇ e.g., the antigen binding portion), disulfide-linked Fvs (dsFv), and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies herein provided), intrabodies, and epitope-binding fragments of any of the above.
  • monoclonal antibodies including full-length monoclonal antibodies
  • polyclonal antibodies multispecific antibodies formed from at least two different epitope binding fragments (e.g., bispecific antibodies)
  • human antibodies humanized antibodies
  • antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain at least one antigen-binding site.
  • Immunoglobulin molecules can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), subisotype (e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2) or allotype (e.g., Gm, e.g., G1 m(f, z, a or x), G2m(n), G3m(g, b, or c), Am, Em, and Km(1 , 2 or 3)).
  • isotype e.g., IgG, IgE, IgM, IgD, IgA and IgY
  • subisotype e.g., lgG1 , lg
  • Antibodies may be derived from any mammal, including, but not limited to, humans, monkeys, pigs, horses, rabbits, dogs, cats, mice, and the like, or other animals such as birds ⁇ e.g., chickens).
  • Native antibodies generally are heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains (CH).
  • VH variable domain
  • CH constant domains
  • Each light chain has a variable domain at one end (VL) and a constant domain (CL) at its other end.
  • the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
  • Light chains are classified as lambda chains or kappa chains based on the amino acid sequence of the light chain constant region.
  • the variable domain of a kappa light chain may also be denoted herein as VK and the variable domain of a lambda light chain may also be denoted herein as VA.
  • Antibodies provided herein include full length or intact antibody, antibody fragments, native sequence antibody or amino acid variants, human, humanized, post-translationally modified, chimeric or fusion antibodies, immunoconjugates, and functional fragments thereof.
  • the antibodies can be modified in the Fc region, and certain modifications can provide desired effector functions or serum half-life. Where it is desirable to eliminate or reduce effector function, so as to minimize side effects or therapeutic complications, certain other Fc regions may be used.
  • the Fc region of antibodies can be modified to increase the binding affinity for FcRn in endosomes at pH 6.0 and thus increase serum half-life.
  • the Fc region can be conjugated to PEG or albumin to increase the serum half-life, or some other conjugation that results in a desired effect.
  • the C5aR and C5L2 receptors compete for binding or bind substantially to, the same epitope on C5a and C5adesR as antibodies presented herein.
  • Antibodies having one or more biological characteristics e.g., potency, C5/C5a/C5adesR affinity, effector function, ortholog binding affinity, neutralization, and the like
  • Anti-C5/C5a/C5adesR antibodies and fragments can be used for diagnosing and/or treating and/or alleviating one or more symptoms of the C5/C5a/C5adesR associated disease in a mammal. Such diseases include those involving pathologic activation of the complement system.
  • methods provided here are useful for treating a C5a or C5adesR- associated disease/condition and/or preventing and/or alleviating one or more symptoms of the disease or condition in a mammal, comprising administering a therapeutically effective amount of an anti-C5/C5a/C5adesR antibody or fragment to the mammal.
  • the antibody therapeutic compositions can be administered short term (acute) or chronic, or intermittently as directed by a physician.
  • articles of manufacture comprise at least an anti-C5/C5a/C5adesR antibody or antigen binding fragment, such as in sterile dosage form and/or in a kit.
  • a kit containing an anti-C5/C5a/C5adesR antibody or antigen binding fragment can find use, for example, for measuring C5/C5a/C5adesR levels, for purification or immunoprecipitation of C5/C5a/C5adesR from body fluids and tissue or cell samples.
  • a kit can contain an anti-C5/C5a/C5adesR antibody or antigen binding fragment coupled to beads (e.g., sepharose beads).
  • a kit can contain an antibody for detection and quantification of C5/C5a/C5adesR in vitro, e.g., in an ELISA or a western blot.
  • an antibody useful for detection may be provided with a label such as a colorimetric, or luminescent product generating enzyme, a fluorescent molecule or radioisotope.
  • C5adesR polypeptide is referred to herein as an "anti-C5/C5a/C5adesR antibody or fragment” in singular form and as "anti-C5/C5a/C5adesR antibodies and fragments" in plural form.
  • the C5 and C5a and C5adesR polypeptides sometimes are referred to collectively as C5/C5a/C5adesR.
  • Amino acids often are referred to herein by commonly known three letter symbols or by the one- letter symbols recommended by the lUPAC-IUB Biochemical Nomenclature Commission.
  • Nucleotides likewise, often are referred to by commonly accepted single-letter codes.
  • the numbering of amino acids in the variable domain, complementarity determining region (CDRs) and framework regions (FR), of an antibody follow, unless otherwise indicated, the Kabat definition as set forth in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Maryland. (1991 ).
  • the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain.
  • a heavy chain variable domain may include a single amino acid insertion (residue 52a according to Kabat) after residue 52 of H2 and inserted residues ⁇ e.g., residues 82a, 82b, and 82c, etc according to Kabat) after heavy chain FR residue 82.
  • the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard” Kabat numbered sequence. Maximal alignment of framework residues frequently requires the insertion of "spacer" residues in the numbering system, to be used for the Fv region.
  • the identity of certain individual residues at any given Kabat site number may vary from antibody chain to antibody chain due to interspecies or allelic divergence.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment is isolated and/or purified and/or pyrogen-free.
  • purified refers to a molecule of interest, that has been identified and separated and/or recovered from a component of its natural environment.
  • an antibody provided is a purified antibody where it has been separated from one or more components of its natural environment.
  • isolated antibody refers to an antibody which is substantially free of other antibody moleclues having different antigenic specificities (e.g., an isolated antibody that specifically binds to C5, C5a and C5adesR is substantially free of antibodies that specifically bind antigens other than C5, C5a, and C5adesR).
  • a bi- or multi-specific antibody molecule is an isolated antibody when substantially free of other antibody molecules.
  • antibodies provided are isolated antibodies where they have been separated from antibodies with a different specificity.
  • An isolated antibody may be a monoclonal antibody.
  • An isolated antibody that specifically binds to an epitope, isoform or variant of human C5, C5a, and C5adesR may, however, have cross-reactivity to other related antigens, e.g., from other species (e.g., C5, C5a, C5adesR orthologs).
  • An isolated antibody as provided may be substantially free of one or more other cellular materials and/or substantially free of antibodies that specifically bind antigens other than C5, C5a, and C5adesR.
  • a combination of "isolated" monoclonal antibodies is provided, and pertains to antibodies having different specificities and combined in a defined composition. Methods of production and purification/isolation of an anti-C5/C5a/C5adesR antibody or fragment are described herein in more detail.
  • Isolated antibodies presented comprise antibody amino acid sequences disclosed herein, which can be encoded by any suitable polynucleotide. Isolated antibodies sometimes are provided in formulated form.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment binds human C5, C5a and C5adesR and, thereby partially or substantially alters at least one biological activity of C5a and C5adesR, for example, binding of C5a and C5adesR to the receptors C5aR and C5L2.
  • An anti-C5/C5a/C5adesR antibody or fragment often immunospecifically binds to one or more epitopes specific to the C5, C5a and C5adesR proteins, peptide, subunits, fragments, portions or any combination thereof and generally do not specifically bind to other polypeptides.
  • An epitope can comprise at least one antibody binding region that comprises at least one portion of the C5, C5a, and C5adesR proteins.
  • epitope as used herein refers to a protein determinant capable of binding to an antibody.
  • Epitopes generally include chemically active surface groupings of molecules such as amino acids and/or sugar side chains and generally have specific three dimensional structural characteristics, as well as specific chemical characteristics (e.g., charge, polarity, basic, acidic, hydrophobicity and the like). Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents or high temperatures.
  • isolated/purified anti-C5/C5a/C5adesR antibodies and fragments immunospecifically bind to a molecule having the amino acid sequence according to SEQ ID NO: 130 and to a molecule having the amino acid sequence according to SEQ ID NO: 131 , SEQ ID NO:132, SEQ ID NO:134 and SEQ ID NO:135.
  • anti-C5/C5a/C5adesR antibodies and fragments also bind C5, C5a, and C5adesR homologs or orthologs from different species. Species cross-reactive characteristics of anti-C5/C5a/C5adesR antibodies are described herein in more detail.
  • E coli Topi 0 mAb 9 and E coli Top10 mAb 12 were deposited on November 22, 201 1 at the National Collections of Industrial, Marine and Food Bacteria ("NCIMB”; Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, AB21 9YA SCOTLAND) under the Budapest Treaty, and have accession numbers NCIMB 41895 and NCIMB 41896, respectively.
  • NCIMB National Collections of Industrial, Marine and Food Bacteria
  • an anti-C5/C5a/C5adesR antibody or fragment is prepared from the parent antibody mAb1. In some embodiments, the anti-C5/C5a/C5adesR antibody or antigen binding fragment is encompassed within the parent antibody.
  • the term "parent antibody” refers to an antibody that is encoded by an amino acid sequence used for the
  • a parent polypeptide may comprise a native antibody sequence (i.e., a naturally occurring, including a naturally occurring allelic variant) or an antibody sequence with pre-existing amino acid sequence modifications (such as other insertions, deletions and/or substitutions) of a naturally occurring sequence.
  • a parent antibody may be a humanized antibody or a human antibody.
  • anti- C5/C5a/C5adesR antibodies and fragments are variants of the parent antibody.
  • variable refers to an anti-C5/C5a/C5adesR antibody or antigen binding fragment that differs in amino acid sequence from a "parent" anti-C5/C5a/C5adesR antibody or fragment amino acid sequence by virtue of addition, deletion and/or substitution of one or more amino acid residue(s) in the parent antibody sequence.
  • the antigen-binding portion of an antibody comprises one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., C5, C5a, and C5adesR). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full- length antibody.
  • an antigen e.g., C5, C5a, and C5adesR.
  • binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment, which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).
  • a Fab fragment a monovalent fragment consisting of the VL, VH, CL and CH1 domains
  • a F(ab')2 fragment a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region
  • a Fd fragment consisting
  • the two domains of the Fv fragment, VL and VH often are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv)).
  • single chain Fv single chain Fv
  • Such single chain antibodies also are encompassed within the terms "antibody fragment” and "antigen-binding portion” of an antibody.
  • antibody fragments can be obtained using known techniques, and the fragments can be screened for binding activity in the same manner as are intact antibodies.
  • Antigen-binding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment comprises a VH having the amino acid sequence of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, or 1 12, or a sequence having at least about 90%, 90.3%, 95% or 99% identity thereto.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment comprises a VL having the amino acid sequence of SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, or 1 17, or a sequence having at least about 90%, 94.5%, 95% or 99% identity thereto.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment comprises a VH having the amino acid sequence of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, or 1 12, or a sequence having at least about 90%, 90.3%, 95% or 99% identity thereto, and a VL having the amino acid sequence of SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, or 1 17, or a sequence having at least about 90%, 94.5%, 95% or 99% identity thereto.
  • VH is selected from SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, or 1 12.
  • the VL is selected from SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, or 1 17.
  • Tables 20 and 21 provide heavy chain variable regions (VH), light chain variable regions (VL), and complimentarity determining regions (CDRs) for certain embodiments of the antibodies and fragments presented herein.
  • anti-C5/C5a/C5adesR antibodies and fragments comprise a VH and/or VL that has a given percent identify to at least one of the VH and/or VL sequences disclosed in Tables 20 and 21 .
  • percent (%) sequence identity is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical with the amino acid residues or nucleotides in the reference sequences, such as parent antibody 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.
  • Optimal alignment of the sequences for comparison may be produced, besides manually, by means of local homology algorithms known in the art or by means of computer programs which use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin).
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment comprises a VH amino acid sequence having at least about 90% identity, at least about 90.3% identity, at least about 90.5% identity, at least about 91.0% identity, at least about 91.5% identity, at least about 92.0% identity, at least about 92.5% identity, at least about 93.0% identity, at least about 93.5% identity, at least about 94.0% identity, at least about 94.5% identity, at least about 95.0% identity, at least about 95.5% identity, at least about 96.0% identity, at least about 96.5% identity, at least about 97.0% identity, at least about 97.5% identity, at least about 98.0% identity, at least about 98.5% identity, at least about 99.0% identity, at least about 99.5% identity, or about 100% identity to, the amino acid sequence of SEQ ID NOs: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102 and
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment includes a VH amino acid sequence at least about 90%, 90.3%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to, or 100% identical to, the amino acid sequence of SEQ ID NOs: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102 and 1 12.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment comprises 1 -12 amino acid substitutions (e.g., conservative substitutions) in the amino acid sequence of SEQ ID NOs: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102 and 1 12.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment comprising a VH amino acid sequence with a given percent identity to SEQ ID NOs: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102 and 1 12 has one or more characteristics (described in more detail below) selected from the group consisting of:
  • (c) binds specifically to one or more of human C5, C5a and C5adesR, but does not bind
  • (k) inhibits chemotaxis of human neutrophils at about a molar ratio of 1 :1 between the antibody and human C5a;
  • an isolated antibody or antigen-binding fragment thereof binds an antigen ⁇ e.g., C5, C5a, C5adesR) with an affinity characterized by an equilibrium dissociation constant (K D ) in the range of about 3 pM to about 30 nM.
  • an isolate antibody or antigen- binding fragment thereof binds human C5a with a K D of about 17nM.
  • an isolated antibody or antigen binding fragment thereof binds human C5adesR with a K D of about 14 nM. In some embodiments, an isolated antibody or antigen binding fragment thereof binds polyhistidine Flag-tagged cynomolgus (cyno) C5adesR with a K D of about 28 nM. In various embodiments, an isolated antibody or antigen binding fragment thereof binds human C5a with a K D of 8 pM or less. In certain embodiments, an isolated antibody or antigen binding fragment thereof binds human C5adesR with a K D of about 6 pM.
  • an isolated antibody or antigen binding fragment thereof binds cynomolgus C5adesR with a K D of about 19 pM. In other embodiments, an isolated antibody or antigen binding fragment thereof binds polyhistidine Flag- tagged C5adesR with a K D of about 10 pM. In certain embodiments, an isolated antibody or antigen binding fragment thereof binds human C5a with a K D of about 3 pM. In other
  • an isolated antibody or antigen binding fragment thereof binds human C5 with a K D of about 27 pM.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment comprises a VL amino acid sequence having at least about 90% identity, at least about 90.5% identity, at least about 91 .0% identity, at least about 91.5% identity, at least about 92.0% identity, at least about 92.5% identity, at least about 93.0% identity, at least about 93.5% identity, at least about 94.0% identity, at least about 94.5% identity, at least about 95.0% identity, at least about 95.5% identity, at least about 96.0% identity, at least about 96.5% identity, at least about 97.0% identity, at least about 97.5% identity, at least about 98.0% identity, at least about 98.5% identity, at least about 99.0% identity, at least about 99.5% identity, or about 100% identity to, the amino acid sequence of SEQ ID NOs: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107 and 1 17. In some
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment includes a VL amino acid sequence is at least about 90%, 91 %, 92%, 93%, 94%, 94.5%, 95%, 96%, 97%, 98%, 99% identical to, or 100% identical to the amino acid sequence of SEQ ID NOs: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107 and 1 17.
  • the anti-C5/C5a/C5adesR antibody or fragment comprises 1 -10 conservative substitutions in the amino acid sequence of SEQ ID NOs: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107 and 1 17.
  • the anti-C5/C5a/C5adesR antibody or fragment comprising a VL amino acid sequence with a given percent identity to SEQ ID NOs: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107 and 1 17 has one or more characteristics (described in more detail below) selected from the group consisting of:
  • (c) binds specifically to one or more of human C5, C5a and C5adesR, but does not bind
  • (k) inhibits chemotaxis of human neutrophils at about a molar ratio of 1 :1 between the antibody and human C5a;
  • an isolated antibody or antigen-binding fragment thereof as described above binds an antigen ⁇ e.g., C5, C5a, C5adesR) with an affinity characterized by an equilibrium dissociation constant (K D ) in the range of about 3 pM to about 30 nM.
  • an isolate antibody or antigen-binding fragment thereof binds human C5a with a K D of about 17nM.
  • an isolated antibody or antigen binding fragment thereof binds human
  • an isolated antibody or antigen binding fragment thereof binds polyhistidine Flag-tagged cynomolgus (cyno) C5adesR with a K D of about 28 nM. In various embodiments, an isolated antibody or antigen binding fragment thereof binds human C5a with a K D of 8 pM or less. In certain embodiments, an isolated antibody or antigen binding fragment thereof binds human C5adesR with a K D of about 6 pM. In some embodiments, an isolated antibody or antigen binding fragment thereof binds cynomolgus C5adesR with a K D of about 19 pM.
  • an isolated antibody or antigen binding fragment thereof binds polyhistidine Flag-tagged C5adesR with a K D of about 10 pM. In certain embodiments, an isolated antibody or antigen binding fragment thereof binds human C5a with a K D of about 3 pM. In other embodiments, an isolated antibody or antigen binding fragment thereof binds human C5 with a K D of about 27 pM.
  • an antibody or antibody fragment immunospecifically binds to one or more of C5, C5a and C5adesR and comprises a heavy chain variable domain having at least about 90% identity to the amino acid sequence of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, or 1 12 and comprises a light chain variable domain having at least about 90% identity to the amino acid sequence of SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, or 1 17, where the antibody has the activity of inhibiting the binding of one or more of C5a and C5adesR to one of the C5a receptors, C5aR and C5L2.
  • variable domain comprises the antigen-binding region
  • variability is not evenly distributed through the variable domains of antibodies. It is concentrated in segments called Complementarity Determining Regions (CDRs), both in the light chain (VL or VK) and the heavy chain (VH) variable domains.
  • CDRs Complementarity Determining Regions
  • FR framework regions
  • the variable domains of native heavy and light chains each comprise four FR, largely adopting a ⁇ -sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • the CDRs in each chain are held together in close proximity by the FR and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see, Kabat et al., supra).
  • the three CDRs of the heavy chain are designated VH-CDR1 , VH-CDR2, and VH-CDR3, and the three CDRs of the light chain are designated VL-CDR1 , VL-CDR2, and VL-CDR3.
  • This particular region has been described by Kabat et al. (1983) U.S. Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" and by Chothia and Lesk, J. Mol. Biol.
  • Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody.
  • Kabat numbering refers to the numbering system set forth by Kabat et al. (1983) U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest.” Unless otherwise specified, references to the numbering of specific amino acid residue positions in an anti-C5/C5a/C5adesR antibodies and antigen binding fragments thereof of the present invention are according to the Kabat numbering system.
  • the present anti-C5/C5a/C5adesR antibodies and antigen binding fragments comprise at least one antigen binding domain that includes at least one complementarity determining region (CDR1 , CDR2 or CDR3).
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment comprises a VH that includes at least one VH CDR (e.g., VH CDR1 , VHCDR2 or VH CDR3).
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment comprises a VL that includes at least one VL CDR (e.g., VL CDR1 , VL CDR2 or VL CDR3).
  • an isolated antibody or antigen-binding fragment thereof comprises any combination of SEQ ID NOS presented in Table 20 for each VH CDR1 , VH CDR2, VH CDR3, VL CDR1 , VL CDR2 and VL CDR3.
  • an isolated antibody or antigen-binding fragment thereof comprises any combination of SEQ ID NOS presented in Table 21 for each VH and VL chain domain.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment comprises a VH CDR1 having an amino acid sequence identical to, or comprising 1 , 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, or 1 13, a VH CDR2 having an amino acid sequence identical to, or comprising 1 , 2, 3, 4, 5 , 6 or 7 amino acid residue substitutions relative to, SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, or 1 14, and a VH CDR3 having an amino acid sequence identical to, or comprising 1 , 2, 3, 4, 5, 6 or 7 amino acid residue substitutions relative to, SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, or 1 15.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment comprises a VL CDR1 having an amino acid sequence identical to, or comprising 1 , 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, or 1 18, a VL CDR2 having an amino acid sequence identical to, or comprising 1 , 2 or 3 amino acid residue substitutions relative to, SEQ ID NO: 9, 19, 29, 39, 49, 59, 69, 79, 89, 99, 109, or 1 19 and a VL CDR3 having an amino acid sequence identical to, or comprising 1 , 2, 3, 4, 5 , 6 or 7 amino acid residue substitutions relative to, SEQ ID NO: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10 or 120.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment comprises a VH CDR1 having an amino acid sequence identical to, or comprising 1 , 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, or 1 13, a VH CDR2 having an amino acid sequence identical to, or comprising 1 , 2, 3, 4, 5, 6 or 7 amino acid residue substitutions relative to, SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, or 1 14, a VH CDR3 having an amino acid sequence identical to, or comprising 1 , 2, 3, 4 5, 6 or 7 amino acid residue substitutions relative to, SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, or 1 15, a VL CDR1 having an amino acid sequence identical to, or comprising 1 , 2, or 3 amino acid residue substitutions relative to, SEQ ID NO:
  • a VH CDR3 has an amino acid sequence having at least about 60% identity to SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, or 1 15 (e.g., at least about 60% identity, at least about 65% identity, at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 95% identity, at least about 99% identity or at least about 100% identity).
  • a VL CDR3 has an amino acid sequence having at least about 50% identity to SEQ ID NO: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10 or 120 (e.g., at least about 50% identity, at least about 55 identity, at least about 60% identity, at least about 65% identity, at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 95% identity, at least about 99% identity or at least about 100% identity).
  • the anti-C5/C5a/C5adesR antibody or fragment comprises (a) a VH CDR1 having the amino acid sequence of SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, or 1 13; (b) a VH CDR2 having the amino acid sequence of SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, or 1 14 ; (c) a VH CDR3 having the amino acid sequence of SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, or 1 15; (e) a VL CDR1 having the amino acid sequence of SEQ ID NO: 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, or 1 18; (f) a VL CDR2 having the amino acid sequence of SEQ ID NO: 9, 19, 29, 39, 49, 59, 69, 79,
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment immunospecifically binds C5/C5a/C5adesR and comprises (a) a VH CDR1 having an amino acid sequence identical to, or comprising 1 , 2, or 3 amino acid residue substitutions relative to, SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, or 1 13; (b) a VH CDR2 having an amino acid sequence identical to, or comprising 1 , 2, 3, 4, 5, 6 or 7 amino acid residue substitutions relative to, SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, or 1 14; (c) a VH CDR3 having (i) an amino acid sequence identical to, (ii) an amino acid sequence comprising 1 , 2, 3, 4, 5, 6 or 7 amino acid residue substitutions relative to, or (iii) an amino acid sequence having at least about 60% identity to (e.g
  • (c) binds specifically to one or more of human C5, C5a and C5adesR, but does not bind
  • (k) inhibits chemotaxis of human neutrophils at about a molar ratio of 1 :1 between the antibody and human C5a;
  • an isolated antibody or antigen-binding fragment thereof as described above binds an antigen ⁇ e.g., C5, C5a, C5adesR) with an affinity characterized by an equilibrium dissociation constant (K D ) in the range of about 3 pM to about 30 nM.
  • an isolated antibody or antigen-binding fragment thereof binds human C5a with a K D of about 17 nM.
  • an isolated antibody or antigen-binding fragment thereof binds human
  • an isolated antibody or antigen-binding fragment thereof binds polyhistindine Flag tagged cynomolgus C5adesR with a K D of about 28 nM. In certain embodiments an isolated antibody or antigen-binding fragment thereof binds human C5a with a K D of about 8 pM or less. In various embodiments an isolated antibody or antigen-binding fragment thereof binds human C5adesR with a K D of about 6 pM. In some embodiments an isolated antibody or antigen-binding fragment thereof binds cynomolgus C5adesR with a K D of about 19 pM.
  • an isolated antibody or antigen-binding fragment thereof binds polyhistindine Flag tagged cynomolgus C5adesR with a K D of about 10 pM. In certain embodiments an isolated antibody or antigen-binding fragment thereof binds human C5a with a K D of about 3 pM. In certain embodiments an isolated antibody or antigen-binding fragment thereof binds human C5 with a K D of about 27 pM.
  • Table 20 illustrates different sequences that can be selected for each CDR region. These regions can be combined in a variety of combinations as each region can be independently selected for a given antibody.
  • VL CDR3 sequences can be present in any combination with VH and VL CDRs to form a present anti-C5/C5a/C5adesR antibody or fragment.
  • the VH CDR1 is selected from SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, or 1 13
  • the VH CDR2 is selected from SEQ ID NO: 4, 14, 24, 34, 44, 54, 64, 74, 84, 94, 104, or 1 14
  • the VH CDR3 is selected from SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, or 1 15 as depicted in Table 20.
  • VL CDR1 is selected from SEQ ID NO: 8, 18, 28, 38, 48, 58, 68, 78, 88, 98, 108, or 1 18, the VL CDR2 is selected from SEQ ID NO: : 9, 19, 29,
  • VL CDR3 is selected from SEQ ID NO: 10, 20, 30,
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment or antigen-binding fragment thereof comprises a VH CDR3 having (i) an amino acid sequence identical to, (ii) an amino acid sequence comprising 1 , 2, 3, 4, 5, 6 or 7 amino acid residue substitutions relative to, or (iii) an amino acid sequence having at least about 60% identity to (e.g., at least about 60% identity, at least about 65% identity, at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 95% identity), SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, or 1 15.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment comprises a VL CDR3 having (i) an amino acid sequence identical to, (ii) an amino acid sequence comprising 1 , 2, 3, 4, 5, 6 or 7 amino acid residue substitutions relative to, or (iii) an amino acid sequence having at least about at least about 50% identity to (e.g., at least about 50% identity, at least about 55 identity, at least about 60% identity, at least about 65% identity, at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 95% identity), SEQ ID NO: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10 or 120.
  • the remaining portions of the anti-C5/C5a/C5adesR antibodies and fragments may comprise specific sequences disclosed herein or known sequences provided the anti-C5/C5a/C5adesR antibodies and fragments immunospecifically bind to C5/C5a/C5adesR.
  • an isolated antibody or antigen-binding fragment thereof comprising:
  • an isolated antibody or antigen-binding fragment thereof comprising:
  • an isolated antibody or antigen-binding fragment thereof comprising:
  • immunospecifically binds one or more of C5, C5a and C5adesR and comprises a VH CDR3 having (i) an amino acid sequence identical to, (ii) an amino acid sequence comprising 1 , 2, 3, 4, 5, 6 or 7 amino acid residue substitutions relative to, or (iii) an amino acid sequence having at least about 60% identity to (e.g., at least about 60% identity, at least about 65% identity, at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 95% identity), SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, 75, 85, 95, 105, or 1 15, where the antibody or antigen binding fragment inhibits neutophil activation and/or chemotaxis.
  • VH CDR3 having (i) an amino acid sequence identical to, (ii) an amino acid sequence comprising 1 , 2, 3, 4, 5, 6 or 7 amino acid residue substitutions relative to, or (iii) an amino acid sequence having
  • an isolated antibody or antigen-binding fragment thereof comprising:
  • VL CDR3 having (i) an amino acid sequence identical to, (ii) an amino acid sequence comprising 1 , 2, 3, 4, 5, 6 or 7 amino acid residue substitutions relative to
  • an isolated antibody or antigen-binding fragment thereof comprising:
  • an isolated antibody or antigen-binding fragment thereof inhibits C5a- or C5adesR-mediated signalling in target cells.
  • an isolated antibody or antigen-binding fragment thereof inhibits C5a- or C5adesR-mediated signalling in target cells.
  • Anti-C5/C5a/C5adesR antibodies and fragments often comprise one or more amino acid sequences substantially the same as an amino acid sequence described herein.
  • Amino acid sequences that are substantially the same include sequences comprising conservative amino acid substitutions, as well as amino acid deletions and/or insertions.
  • a conservative amino acid substitution refers to the replacement of a first amino acid by a second amino acid that has chemical and/or physical properties (e.g., charge, structure, polarity, hydrophobicity/hydrophilicity) that are similar to those of the first amino acid.
  • Conservative substitutions include replacement of one amino acid by another within the following groups: lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate (E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M), cysteine (C) and glycine (G).
  • Framework regions of the heavy and light chains each comprise four framework regions (in general FR1 , FR2, FR3, FR4 or alternatively FW1 , FW2, FW3, FW4), which are the more highly conserved portions of the variable domains.
  • the four framework regions of the heavy chain are here designated VH-FR1 , VH-FR2, VH-FR3 and VH-FR4, and the four framework regions of the light chain are here designated VL-FR1 , VL-FR2, VL-FR3 and VL-FR4.
  • VH-FR1 begins at position 1 and ends at approximately amino acid 30, VH-FR2 is approximately from amino acid 36 to 49, VH-FR3 is approximately from amino acid 66 to 94 and VH-FR4 is approximately amino acid 103 to 1 13.
  • VL-FR1 begins at amino acid 1 and ends at approximately amino acid 23
  • VL-FR2 is approximately from amino acid 35 to 49
  • VL-FR3 is approximately from amino acid 57 to 88
  • VL-FR4 is approximately from amino acid 98 to 107.
  • the framework regions contain substitutions according to the Kabat numbering system, e.g., insertion at 106A in VL-FR4. In addition to naturally occurring
  • substitutions one or more alterations ⁇ e.g., substitutions) of FR residues also may be introduced in an anti-C5/C5a/C5adesR antibody or antigen binding fragment. In certain embodiments, these alterations result in an improvement or optimization in the binding affinity of the antibody for anti- C5/C5a/C5adesR.
  • Non-limiting examples of framework region residues that can be modified include those that non-covalently bind antigen directly, interact with/effect the conformation of a CDR, and/or participate in the VL-VH interface.
  • a framework region may comprise one or more amino acid changes for the purposes of "germlining".
  • the amino acid sequences of selected antibody heavy and light chains are compared to germline heavy and light chain amino acid sequences and where certain framework residues of the selected VL and/or VH chains differ from the germline configuration (e.g., as a result of somatic mutation of the immunoglobulin genes used to prepare the phage library), it may be desirable to "backmutate” the altered framework residues of the selected antibodies to the germline configuration (i.e., change the framework amino acid sequences of the selected antibodies so that they are the same as the germline framework amino acid sequences).
  • backmutation or "germlining" of framework residues can be
  • variable light and/or heavy chain framework residues are backmutated.
  • a variable heavy chain of an isolated antibody or antigen-binding fragment thereof presented is backmutated.
  • a variable heavy chain of an isolated antibody or antigen-binding fragment thereof comprises at least one, at least two, at least three, at least four, at least five or more backmutations.
  • the VH of an anti-C5/C5a/C5adesR antibody or antigen binding fragment presented herein may comprise FR1 , FR2, FR3 and/or FR4 that has an amino acid sequence identity with the corresponding framework regions (i.e., FR1 of antibody X as compared to FR1 of antibody Y) within SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, or 1 12 that is from about 90% to about 100%.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment comprises a VH FR amino acid sequence (FR1 , FR2, FR3 and/or FR4) at least about 90% identical to, at least about 95% identical to, or 100% identical to, the corresponding FR of VH SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, or 1 12.
  • an anti- C5/C5a/C5adesR antibody or antigen binding fragment comprises a VH FR amino acid sequence (FR1 , FR2, FR3 and/or FR4) at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to, or 100% identical to, the corresponding FR of VH SEQ I D NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, or 1 12.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment may comprise a VH FR (FR1 , FR2, FR3 and/or FR4) having an amino acid sequence identical to, or comprising 1 , 2 or 3 amino acid substitutions relative to, the corresponding FR of VH SEQ I D NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, or 1 12.
  • FR1 , FR2, FR3 or FR4 of the VH may each have an amino acid sequence identical to or comprising 1 , 2 or 3 amino acid
  • the VL of an anti-C5/C5a/C5adesR antibody or antigen binding fragment herein provided may comprise FR1 , FR2, FR3 and/or FR4 that has amino acid sequence identity with the corresponding framework regions (i.e., FR1 of antibody X as compared to FR1 of antibody Y) within the FR of VL SEQ I D NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, or 1 17 (e.g., from about 90% to about 100% sequence identity).
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment comprises a VL FR amino acid sequence (FR1 , FR2, FR3 and/or FR4) at least about 90% identical to, at least about 95% identical to, or 100% identical to, the corresponding FR of VL SEQ I D NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, or 1 17.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment comprises a VL FR amino acid sequence (FR1 , FR2, FR3 and/or FR4) at least about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to, or 100% identical to, the corresponding FR of VL SEQ I D NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, or 1 17.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment comprises a VL FR (FR1 , FR2, FR3 and/or FR4) having an amino acid sequence identical to, or comprising 1 , 2 or 3 amino acid substitutions relative to, the corresponding FR of VL SEQ I D NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, or 1 17.
  • FR1 , FR2, FR3 or FR4 of the VL may each have an amino acid sequence identical to, or comprising 1 , 2 or 3 amino acid substitutions relative to, the corresponding FR1 , FR2, FR3 or FR4 of VH SEQ I D NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, or 1 17.
  • an isolated antibody or antigen-binding fragment thereof thereof
  • VH FR (FR1 , FR2, FR3 and/or FR4) having (i) an amino acid sequence identical to, (ii) an amino acid sequence comprising 1 , 2 or 3 amino acid substitutions relative to, or (iii) an amino acid sequence having at least about 90% identity to (e.g., about 90% 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity to), the corresponding FR of VH SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, or 1 12 and/or VL FR (FR1 , FR2, FR3 and/or FR4) having (i) an amino acid sequence identical to, (ii) an amino acid sequence comprising 1 , 2 or 3 amino acid substitutions relative to, or (iii) an amino acid sequence having about 90% identity to (e.g., about 90%,
  • (c) binds specifically to one or more of human C5, C5a and C5adesR, but does not bind
  • (k) inhibits chemotaxis of human neutrophils at about a molar ratio of 1 :1 between the antibody and human C5a;
  • (j) inhibits C5a mediated neutrophil CD1 1 b expression in human and cynomolgus whole blood samples with IC 50 values in the range of about 1 nM to about 300 nM or about 2 nM to about 200 nM.
  • an isolated antibody or antigen-binding fragment thereof described above binds an antigen ⁇ e.g., C5, C5a, C5adesR) with an affinity characterized by an equilibrium dissociation constant (K D ) in the range of about 3 pM to about 30 nM.
  • an isolated antibody or antigen-binding fragment thereof binds human C5a with a K D of about 17 nM.
  • an isolated antibody or antigen-binding fragment thereof binds human C5ades R with a K D of about 14 nM.
  • an isolated antibody or antigen-binding fragment thereof binds polyhistindine Flag tagged cynomolgus C5adesR with a K D of about 28 nM. In certain embodiments an isolated antibody or antigen-binding fragment thereof binds human C5a with a K D of about 8 pM or less. In various embodiments an isolated antibody or antigen-binding fragment thereof binds human C5adesR with a K D of about 6 pM. In some embodiments an isolated antibody or antigen-binding fragment thereof binds cynomolgus C5adesR with a K D of about 19 pM.
  • an isolated antibody or antigen-binding fragment thereof binds polyhistindine Flag tagged cynomolgus C5adesR with a K D of about 10 pM. In certain embodiments an isolated antibody or antigen-binding fragment thereof binds human C5a with a K D of about 3 pM. In certain embodiments an isolated antibody or antigen-binding fragment thereof binds human C5 with a K D of about 27 pM. Nucleotide sequences encoding anti-C5/C5a/C5adesR antibodies and fragments
  • polynucleotides comprise a nucleotide sequence encoding an anti-C5/C5a/C5adesR antibody or antigen binding fragment described herein or fragments thereof. These include, but are not limited to, nucleotide sequences that code for the above referenced amino acid sequences.
  • polynucleotides that hybridize under stringent or lower stringency hybridization conditions e.g., as defined herein, to polynucleotides that encode an anti-C5/C5a/C5adesR antibody or antigen binding fragment.
  • stringency refers to experimental conditions ⁇ e.g., temperature and salt concentration) of a hybridization experiment to denote the degree of homology between two nucleic acids; the higher the stringency, the higher percent homology between the two nucleic acids.
  • Stringent hybridization conditions include, but are not limited to, hybridization to filter-bound DNA in 6X sodium chloride/sodium citrate (SSC) at about 45 degrees Celsius followed by one or more washes in 0.2X SSC/0.1 % SDS at about 50-65 degrees Celsius, highly stringent conditions such as hybridization to filter-bound DNA in 6X SSC at about 45 degrees Celsius followed by one or more washes in 0.1 X SSC/0.2% SDS at about 65 degrees Celsius, or any other stringent hybridization conditions known.
  • SSC sodium chloride/sodium citrate
  • a nucleic acid or fragment thereof may encode an anti-C5/C5a/C5adesR antibody or antigen binding fragment and hybridize under stringent conditions to a nucleic acid including a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 2, 12, 22, 32, 42, 52, 62, 72, 82, 92, 102, or 1 12.
  • the nucleic acid or fragment thereof may encode an anti-C5/C5a/C5adesR antibody or antigen binding fragment that hybridizes under stringent conditions to a nucleic acid including a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 77, 87, 97, 107, or 1 17.
  • Substantially identical sequences may be polymorphic sequences, i.e., alternative sequences or alleles in a population.
  • An allelic difference may be as small as one base pair.
  • Substantially identical sequences may also comprise mutagenized sequences, including sequences comprising silent mutations.
  • a mutation may comprise one or more residue changes, a deletion of one or more residues, or an insertion of one or more additional residues.
  • the polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides, which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
  • a polynucleotide encoding an antibody may also be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, sometimes polyA+RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody ) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into
  • nucleotide sequence and corresponding amino acid sequence of the antibody may be manipulated using methods known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, and the like, to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.
  • stringent conditions refers to conditions for hybridization and washing. Methods for hybridization reaction temperature condition optimization are known to those of skill in the art. Aqueous and non-aqueous methods are described in that reference and either can be used.
  • Non-limiting examples of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45 degrees Celsius, followed by one or more washes in 0.2X SSC, 0.1 % SDS at 50 degrees Celsius.
  • Another example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45 degrees Celsius, followed by one or more washes in 0.2X SSC, 0.1 % SDS at 55 degrees Celsius.
  • a further example of stringent hybridization conditions is hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45 degrees Celsius, followed by one or more washes in 0.2X SSC, 0.1 % SDS at 60 degrees Celsius.
  • stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45 degrees Celsius, followed by one or more washes in 0.2X SSC, 0.1 % SDS at 65 degrees Celsius. More often, stringency conditions are 0.5M sodium phosphate, 7% SDS at 65 degrees Celsius, followed by one or more washes at 0.2X SSC, 1 % SDS at 65 degrees Celsius.
  • Stringent hybridization temperatures can also be altered (i.e., lowered) with the addition of certain organic solvents, formamide for example.
  • Organic solvents like formamide, reduce the thermal stability of double-stranded polynucleotides, so that hybridization can be performed at lower temperatures, while still maintaining stringent conditions and extending the useful life of nucleic acids that may be heat labile.
  • hybridizing refers to binding of a first nucleic acid molecule to a second nucleic acid molecule under low, medium or high stringency conditions, or under nucleic acid synthesis conditions.
  • Hybridizing can include instances where a first nucleic acid molecule binds to a second nucleic acid molecule, where the first and second nucleic acid molecules are complementary.
  • specifically hybridizes refers to preferential hybridization under nucleic acid synthesis conditions of a primer, to a nucleic acid molecule having a sequence complementary to the primer compared to hybridization to a nucleic acid molecule not having a complementary sequence.
  • specific hybridization includes the hybridization of a primer to a target nucleic acid sequence that is complementary to the primer.
  • primers can include a nucleotide subsequence that may be complementary to a solid phase nucleic acid primer hybridization sequence or substantially complementary to a solid phase nucleic acid primer hybridization sequence (e.g., about 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% identical to the primer hybridization sequence complement when aligned).
  • a nucleotide subsequence that may be complementary to a solid phase nucleic acid primer hybridization sequence or substantially complementary to a solid phase nucleic acid primer hybridization sequence (e.g., about 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
  • a primer may contain a nucleotide subsequence not complementary to or not substantially complementary to a solid phase nucleic acid primer hybridization sequence (e.g., at the 3' or 5' end of the nucleotide subsequence in the primer complementary to or substantially complementary to the solid phase primer hybridization sequence).
  • a primer in certain embodiments, may contain a modification such as inosines, abasic sites, locked nucleic acids, minor groove binders, duplex stabilizers (e.g., acridine, spermidine), Tm modifiers or any modifier that changes the binding properties of the primers or probes.
  • a modification such as inosines, abasic sites, locked nucleic acids, minor groove binders, duplex stabilizers (e.g., acridine, spermidine), Tm modifiers or any modifier that changes the binding properties of the primers or probes.
  • a primer in certain embodiments, may contain a detectable molecule or entity (e.g., a fluorophore, radioisotope, colorimetric agent, particle, enzyme and the like).
  • the nucleic acid can be modified to include a detectable label using any method known to one of skill in the art.
  • the label may be incorporated as part of the synthesis, or added on prior to using the primer in any of the processes described herein. Incorporation of label may be performed either in liquid phase or on solid phase.
  • the detectable label may be useful for detection of targets.
  • the detectable label may be useful for the quantification target nucleic acids (e.g., determining copy number of a particular sequence or species of nucleic acid).
  • detectable label suitable for detection of an interaction or biological activity in a system can be appropriately selected and utilized by the artisan.
  • detectable labels are fluorescent labels such as fluorescein, rhodamine, and others (e.g., Anantha, et al., Biochemistry (1998) 37:2709 2714; and Qu & Chaires, Methods Enzymol.
  • radioactive isotopes e.g., 1251, 1311, 35S, 31 P, 32P, 33P, 14C, 3H, 7Be, 28Mg, 57Co, 65Zn, 67Cu, 68Ge, 82Sr, 83Rb, 95Tc, 96Tc, 103Pd, 109Cd, and 127Xe
  • light scattering labels e.g., U.S. Patent No.
  • chemiluminescent labels and enzyme substrates e.g., dioxetanes and acridinium esters
  • enzymic or protein labels e.g., green fluorescence protein (GFP) or color variant thereof, luciferase, peroxidase
  • other chromogenic labels or dyes e.g., cyanine
  • cofactors or biomolecules such as digoxigenin, strepdavidin, biotin (e.g., members of a binding pair such as biotin and avidin for example
  • a primer may be labeled with an affinity capture moiety.
  • detectable labels are those labels useful for mass modification for detection with mass spectrometry (e.g., matrix-assisted laser desorption ionization (MALDI) mass spectrometry and electrospray (ES) mass spectrometry).
  • MALDI matrix-assisted laser desorption ionization
  • ES electrospray
  • a primer also may refer to a polynucleotide sequence that hybridizes to a subsequence of a target nucleic acid or another primer and facilitates the detection of a primer, a target nucleic acid or both, as with molecular beacons, for example.
  • the term "molecular beacon” as used herein refers to detectable molecule, where the detectable property of the molecule is detectable only under certain specific conditions, thereby enabling it to function as a specific and informative signal.
  • detectable properties are, optical properties, electrical properties, magnetic properties, chemical properties and time or speed through an opening of known size.
  • a molecular beacon can be a single-stranded oligonucleotide capable of forming a stem-loop structure, where the loop sequence may be complementary to a target nucleic acid sequence of interest and is flanked by short complementary arms that can form a stem.
  • the oligonucleotide may be labeled at one end with a fluorophore and at the other end with a quencher molecule.
  • energy from the excited fluorophore is transferred to the quencher, through long-range dipole-dipole coupling similar to that seen in fluorescence resonance energy transfer, or FRET, and released as heat instead of light.
  • molecular beacons offer the added advantage that removal of excess probe is unnecessary due to the self- quenching nature of the unhybridized probe.
  • molecular beacon probes can be designed to either discriminate or tolerate mismatches between the loop and target sequences by modulating the relative strengths of the loop-target hybridization and stem formation.
  • mismatches refers to a nucleotide that is not complementary to the target sequence at that position or positions.
  • a probe may have at least one mismatch, but can also have 2, 3, 4, 5, 6 or 7 or more mismatched nucleotides.
  • An antibody may have one or more characteristics identical to or similar to an antibody described herein, and often possesses one or more of the biological characteristics that distinguishes it from other antibodies that bind to the same antigen, C5/C5a/C5adesR.
  • biological characteristics refers to any one or more of biochemical, binding and functional characteristics, which can be used to select antibodies for therapeutic, research, and diagnostic uses.
  • anti-C5/C5a/C5adesR antibodies and fragments may be the same or different with respect to epitope binding, targeting, affinity, and neutralizing properties of the antibody, for example.
  • Biochemical characteristics of an anti-C5/C5a/C5adesR antibody or fragment include, but are not limited to, isoelectric point (pi) and melting temperature (Tm).
  • the binding characteristics of an anti-C5/C5a/C5adesR antibody or fragment include, but are not limited to, binding specificity; equilibrium dissociation constant (K D ), or its inverse, association constant (K A ), or its component kon (k a ) or koff (k d ) rates; epitope to which it binds; ability to distinguish between various forms and/or preparations of C5/C5a/C5adesR (e.g., recombinant, native, oxidised) and ability to bind soluble and/or immobilized antigen.
  • Functional characteristics of an antibody presented herein include, but are not limited to, inhibition of C5aR and C5L2 receptor binding, inhibition of
  • C5a/C5adesR induced gene expression and target cell responses, and protection and treatment of one or more C5a-related diseases or disorders. Described herein are characteristics of anti- C5/C5a/C5adesR antibodies and fragments and methods for modifying and fine tuning those characteristics. Methods for measuring characteristics of antibodies are known in the art, some of which are detailed hereafter.
  • Antibodies like all polypeptides have an Isoelectric Point (pi), which is generally defined as the pH at which a polypeptide carries no net charge. Protein solubility is typically lowest when the pH of the solution is equal to the isoelectric point (pi) of the protein. As used herein the pi value is defined as the pi of the predominant charge form. The pi of a protein may be determined by a variety of methods including, but not limited to, isoelectric focusing and various computer algorithms. In addition, the thermal melting temperatures (Tm) of the Fab domain of an antibody can be a good indicator of the thermal stability of an antibody and may further provide an indication of the antibody shelf-life.
  • Tm thermal melting temperatures
  • Tm of a protein or protein fragment e.g., a Fab domain
  • Tm of a protein or protein fragment can be measured using any standard method known in the art, for example, by differential scanning calorimetry.
  • anti-C5/C5a/C5adesR antibodies and fragments have certain biochemical characteristics such as a particular isoelectric point (pi) or melting temperature (Tm).
  • pi isoelectric point
  • Tm melting temperature
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment has a pi ranging from 5.5 to 9.5.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment has a pi that ranges from about 5.5 to about 6.0, or about 6.0 to about 6.5, or about 6.5 to about 7.0, or about 7.0 to about 7.5, or about 7.5 to about 8.0, or about 8.0 to about 8.5, or about 8.5 to about 9.0, or about 9.0 to about 9.5.
  • an anti- C5/C5a/C5adesR antibody or antigen binding fragment has a pi that ranges from 5.5-6.0, or 6.0 to 6.5, or 6.5 to 7.0, or 7.0-7.5, or 7.5-8.0, or 8.0-8.5, or 8.5-9.0, or 9.0-9.5.
  • Anti-C5/C5a/C5adesR antibodies and fragments sometimes has a pi of at least 5.5, or at least 6.0, or at least 6.3, or at least 6.5, or at least 6.7, or at least 6.9, or at least 7.1 , or at least 7.3, or at least 7.5, or at least 7.7, or at least 7.9, or at least 8.1 , or at least 8.3, or at least 8.5, or at least 8.7, or at least 8.9, or at least 9.1 , or at least 9.3, or at least 9.5.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment has a pi of at least about 5.5, or at least about 6.0, or at least about 6.3, or at least about 6.5, or at least about 6.7, or at least about 6.9, or at least about 7.1 , or at least about 7.3, or at least about 7.5, or at least about 7.7, or at least about 7.9, or at least about 8.1 , or at least about 8.3, or at least about 8.5, or at least about 8.7, or at least about 8.9, or at least about 9.1 , or at least about 9.3, or at least about 9.5.
  • the antibodies and antigen binding fragments provided herein have a pi of a pi range 7.0-8.0 with a major band at 7.45.
  • solubility by altering the number and location of ionizable residues in the antibody to adjust the pi.
  • the pi of a polypeptide can be manipulated by making appropriate amino acid substitutions (e.g., by substituting a charged amino acid such as a lysine, for an uncharged residue such as alanine).
  • amino acid substitutions of an antibody that result in changes of the pi of the antibody may improve solubility and/or the stability of the antibody.
  • Appropriate amino acid substitutions can be selected for a particular antibody to achieve a desired pi.
  • a substitution is generated in an antibody to alter the pi.
  • substitution(s) of the Fc region that result in altered binding to FcR may also result in a change in the pi.
  • substitution(s) of the Fc region are specifically chosen to effect both the desired alteration in FcR binding and any desired change in pi.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment has a Tm ranging from 64 degrees Celsius to 120 degrees Celsius. In certain embodiments, an anti-
  • C5/C5a/C5adesR antibody or antigen binding fragment has a Tm ranging from about 75 degrees Celsius to about 120 degrees Celsius, or about 75 degrees Celsius to about 85 degrees Celsius, or about 85 degrees Celsius to about 95 degrees Celsius, or about 95 degrees Celsius to about 105 degrees Celsius, or about 105 degrees Celsius to about 1 15 degrees Celsius, or about 1 15 degrees Celsius to about 120 degrees Celsius.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment has a Tm ranging from 75 degrees Celsius to 120 degrees Celsius, or 75 degrees Celsius to 85 degrees Celsius, or 85 degrees Celsius to 95 degrees Celsius, or 95 degrees Celsius to 105 degrees Celsius, or 105 degrees Celsius to 1 15 degrees Celsius, or 1 15 degrees Celsius to 120 degrees Celsius.
  • An anti-C5/C5a/C5adesR antibody or antigen binding fragment sometimes has a Tm of at least about 64 degrees Celsius, or at least about 70 degrees Celsius, or at least about 75 degrees Celsius, or at least about 80 degrees Celsius, or at least about 85 degrees Celsius, or at least about 90 degrees Celsius, or at least about 95 degrees Celsius, or at least about 100 degrees Celsius, or at least about 105 degrees Celsius, or at least about 1 10 degrees Celsius, or at least about 1 15 degrees Celsius, or at least about 120 degrees Celsius.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment has a Tm of at least 64 degrees Celsius, or at least 70 degrees Celsius, or at least 75 degrees Celsius, or at least 80 degrees Celsius, or at least 85 degrees Celsius, or at least 90 degrees Celsius, or at least 95 degrees Celsius, or at least 100 degrees Celsius, or at least 105 degrees Celsius, or at least 1 10 degrees Celsius, or at least 1 15 degrees Celsius, or at least 120 degrees Celsius.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment immunospecifically bind at least one specified epitope or antigenic determinants of the protein, peptide, subunit, fragment, portion or any combination thereof exclusively or preferentially with respect to other polypeptides.
  • epitope or antigenic determinant refers to a protein determinant capable of binding to an antibody, where the term “binding” herein often relates to a specific binding.
  • These protein determinants or epitopes often include chemically active surface groupings of molecules such as amino acids or sugar side chains, often have specific three dimensional structural characteristics, and often have specific charge characteristics.
  • Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
  • discontinuous epitope refers to a conformational epitope on a protein antigen formed from at least two separate regions in the primary sequence of the protein.
  • the epitope is at least 4 amino acid residues, at least 5 amino acid residues, at least 6 amino acid residues, at least 7 amino acid residues, at least 8 amino acid residues or at least 9 amino acid residues to the entire specified portion of contiguous amino acids of the C5/C5a/C5adesR protein.
  • an anti-C5/C5a/C5adesR antibody or fragment immunospecifically binds a C5/C5a/C5adesR polypeptide or antigenic fragments thereof, having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% identity, or 100% identity, to the amino acid sequence of SEQ ID NO: 130.
  • An anti- C5/C5a/C5adesR antibody or fragment may sometimes immunospecifically binds to a
  • C5/C5a/C5adesR polypeptide or antigenic fragments thereof having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, or 100% identity, to the amino acid sequence of SEQ ID NO: 130, 131 , 132, 134 and/or 135.
  • an anti-C5/C5a/C5adesR antibody or fragment may bind an epitope conserved across species. In some embodiments, an anti-C5/C5a/C5adesR antibody or fragment binds murine, non-human primate, rat, bovine, pig or other mammalian C5/C5a and antigenic fragments thereof. In some embodiments an anti-C5/C5a/C5adesR antibody or fragments may bind to one or more C5/C5a/C5adesR orthologs and or isoforms.
  • an anti-C5/C5a/C5adesR antibody or fragment binds to C5/C5a/C5adesR and antigenic fragments thereof from one or more species, including, but not limited to, mouse, rat, monkey, primate, and human.
  • an anti-C5/C5a/C5adesR antibody or fragment may bind an 5 epitope within humans across C5/C5a/C5adesR homologs and/or isoforms and/or conformational variants and/or subtypes. See, e.g., Tables 1 , 2, 5, 6, 7, 8, 9, 1 1 , 12, 13, 14, 16 and 17 for representative IC 50 values of anti-C5/C5a/C5adeR antibodies and fragments described herein.
  • Hydrophobic interactions are a significant driving force for antibody-antigen interactions, and are based on repulsion of water by non-polar groups rather than attraction of molecules. However, certain physical forces also contribute to 15 antigen-antibody binding, for example, the fit or complimentary of epitope shapes with different antibody binding sites. Other materials and antigens may cross-react with an antibody, thereby competing for available free antibody.
  • Binding affinity generally refers to the strength of the sum total of the noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen).
  • binding affinity refers to intrinsic binding affinity that reflects a 1 : 1 interaction between members of 25 a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can
  • K D equilibrium dissociation constant
  • Affinity can be measured by common methods known in the art, including those described and exemplified herein (e.g., BIAcore methods).
  • Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind 30 antigen faster and tend to remain bound longer.
  • a variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present technology.
  • An anti-C5/C5a/C5adesR antibody or antigen binding fragment sometimes has a binding affinity for a C5/C5a/C5adesR epitope characterized by an equilibrium dissociation constant (K D ) of 1 x 10 ⁇ 2 M or less, 1 x 10 ⁇ 3 M or less, 1 x 10 ⁇ 4 M or less, 1 x 10 ⁇ 5 M or less, 1 x 10 ⁇ 6 M or less, 1 x 10 ⁇ 7 M or less, 1 x 10 "8 M or less, 1 x 10 ⁇ 9 M or less, 1 x 10 "10 M or less, 1 x 10 "11 M or less, 1 x 10 "12 M or less, 1 x 10 "13 M or less, 1 x 10 ⁇ 14 M or less or 1 ⁇ 10 ⁇ 15 M or less.
  • K D equilibrium dissociation constant
  • the K D may be from 1 ⁇ 10 ⁇ 15 M to 1 x 10 "2 M, from 1 x 10 "14 M to 1 x 10 "10 M, from 1 x 10 ⁇ 9 M to 1 x 10 "5 M and from 1 x 10 "4 M to 1 x 10 ⁇ 2 M.
  • the K D may be from 3x10 "12 to 30x10 "9 M.
  • an anti-C5/C5a/C5adesR antibodies and fragments is a high-affinity antibody.
  • high-affinity antibody is meant an antibody which binds to a C5/C5a/C5adesR epitope with an affinity less than 10 "9 M (e.g., 10 "10 M, 10 ⁇ 11 M, 10 ⁇ 12 M and the like).
  • an anti-C5/C5a/C5adesR antibodies and fragments is a high-affinity antibody which binds to a C5/C5a/C5adesR epitope with an affinity less than 10 ⁇ 10 M (e.g., 10 ⁇ 11 M, 10 ⁇ 12 M, and the like).
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment is described as having a binding affinity of a specific molarity or better. "Or better” when used herein refers to a stronger binding, represented by a smaller numerical K D value. For example, an antibody which has an affinity for an antigen of "0.6 nM or better", the antibody's affinity for the antigen is ⁇ 0.6 nM, i.e., 0.59 nM, 0.58 nM, 0.57 nM and the like, or any value less than 0.6 nM.
  • an anti-C5/C5a/C5adesR antibody or fragment can bind to a C5/C5a/C5adesR epitope with a koff of less than 10 "3 s "1 , less than 5x10 "3 s “1 , less than 10 "4 s “1 , less than 5x10 "4 s "1 , less than 10 "5 s “1 , less than 5x10 "5 s “1 , less than 10 "6 s "1, less than 5x10 "6 s "1 , less than 10 "7 s “1 , less than 5x10 "7 s “1 , less than 10 " 8 s "1 , less than 5x10 "8 s “1 , less than 10 "9 s "1 ,
  • the present anti-C5/C5a/C5adesR antibodies and fragments bind to a C5/C5a/C5adesR epitope with a kon rate of at least 10 5 M " V 1 , at least 5x10 5 M “ V 1 , at least 10 6 M “ V 1 , at least 5 x 10 6 M “1 s “1 , at least 10 7 M “1 s “1 , at least 5 x 10 7 M “1 s “1 , or at least 10 8 M “ V 1 , or at least 10 9 M “1 s "1 .
  • Determination of binding affinity can be measured using the specific techniques described further in the Example section, see Examples 4 and 20, and methods known in the art.
  • One example of such a method includes measuring the equilibrium dissociation constant "K D " using surface plasmon resonance assays, which can be carried out, for example, using a BIAcoreTM-2000 or a BIAcoreTM T100 instrument (GE Healthcare) essentially as described by Karlsson et al (1991 ).
  • Anti-C5/C5a/C5adesR antibodies or isotype matched negative controls can either be immobilized by protein G' covalently coupled to a CM5 sensor chip via the Fc domain, or covalently coupled by amine linkage to a proprietary CM3 chip surface to a final surface density of approximately 400RU according to manufacturer's instructions.
  • C5a/C5adesR analytes can be prepared at a range of concentrations in HBS-EP buffer (GE Healthcare) and dilutions of each analyte injected sequentially over the antibody surface for 2 minutes, at a constant flow rate (between 30 to 50 microlitres per minute depending on experiment) before surface regeneration with 10 mM glycine, pH 1 .75 - 2, between each injection of antibody.
  • Blank reference flow cell data is subtracted from each IgG dataset and a zero-concentration buffer blank is double-reference subtracted from the main data set to reduce any buffer artefacts.
  • the resulting binding curves for each analyte can be fitted to the 1 :1 Langmuir model (simultaneous ka, kd) using BIAevaluation software.
  • K D the equilibrium dissociation constant
  • K D the equilibrium dissociation constant of anti- C5/C5a/C5adesR antibodies
  • K D the solution phase equilibrium dissociation constant of anti- C5/C5a/C5adesR antibodies
  • K D the equilibrium dissociation constant of anti- C5/C5a/C5adesR antibodies
  • the amount of free antibody is then measured using the KinExa by capturing free antibody using C5a or C5 coated beads, washing away unbound material and detecting bound antibody using a fluorescently labelled species specific antibody.
  • the amount of free antibody detected at each C5a or C5 ligand concentration is plotted against the concentration of C5 or C5a and the KinExa software is used to calculate the equilibrium dissociation constant (K D ).
  • binding assays may be performed as direct binding assays or as competition-binding assays.
  • Binding can be detected for example, by ELISA, western blotting, immunocytochemistry, immunoprecipitation, affinity chromotography, Bio-Layer Interferometry , Octet, ForteBio) and biochemical assays such as Dissociation-Enhanced Lanthanide Fluorescent Immunoassays (DEFLIA ®, Perkin Elmer), Forster resonance energy transfer (FRET) assays (e.g. homogeneous time resolved fluorescence (HTRF®, Cis Biointernational), and radioligand binding assays. Binding can also be detected in cell assays, for example, by flow cytometry and
  • Fluorescent Microvolumetric Assay Technology FMAT®, Applied Biosystems
  • a candidate antibody is tested for binding to C5/C5a/C5adesR antigen.
  • Competition- binding assays assess the ability of a candidate antibody to compete with a known anti-C5/C5a/C5adesR antibody or fragment or other compound, such as the C5a receptors, that binds C5/C5a/C5adesR.
  • any method that permits the binding of an antibody with C5/C5a/C5adesR that can be detected is encompassed with the scope of the present technology for detecting and measuring the binding characteristics of the antibodies. These methods also can be utilized to screen a panel of antibodies for those providing desired characteristics.
  • (c) binds specifically to one or more of human C5, C5a and C5adesR, but does not bind
  • an isolated antibody or antigen-binding fragment thereof binds an antigen ⁇ e.g., C5, C5a, C5adesR) with an affinity characterized by an equilibirium dissociation constant (K D ) in the range of about 3 pM to about 30 nM.
  • an isolated antibody or antigen-binding fragment thereof binds human C5a with a K D of about 17 nM. In various embodiments an isolated antibody or antigen-binding fragment thereof binds human C5ades R with a K D of about 14 nM. In some embodiments an isolated antibody or antigen-binding fragment thereof binds polyhistindine Flag tagged cynomolgus C5adesR with a K D of about 28 nM. In certain embodiments an isolated antibody or antigen-binding fragment thereof binds human C5a with a K D of about 8 pM or less.
  • an isolated antibody or antigen-binding fragment thereof binds human C5adesR with a K D of about 6 pM. In some embodiments an isolated antibody or antigen-binding fragment thereof binds cynomolgus C5adesR with a K D of about 19 pM. In certain embodiments an isolated antibody or antigen-binding fragment thereof binds polyhistindine Flag tagged cynomolgus C5adesR with a K D of about 10 pM. In certain embodiments an isolated antibody or antigen-binding fragment thereof binds human C5a with a K D of about 3 pM. In certain embodiments an isolated antibody or antigen-binding fragment thereof binds human C5 with a K D of about 27 pM.
  • an anti-C5/C5a/C5adesR antibody or fragment alters the biological properties of C5a/C5adesR and/or cells expressing the C5aR and/or C5L2.
  • an anti-C5/C5a/C5adesR antibody or fragment neutralizes the biological activity of C5a/C5adeR by binding to the polypeptide and inhibiting the binding of C5a/C5adesR to its receptor molecule.
  • Neutralization assays can be performed using methods known in the art using, in some circumstances, using commercially available reagents. Neutralization of C5a/C5adesR often is measured with an IC 50 of 1 x 10 "6 M or less, 1 x 10 "7 M or less, 1 x 10 "8 M or less, 1 * 10 "9 M or less, 1 x10 "10 M or less, 1 x10 "11 M or less and 10 "12 M or less.
  • an anti-C5/C5a/C5adesR antibody or fragment neutralizes at least one of chimpanzee C5a/C5adesR, baboon C5a/C5adesR, marmoset C5a/C5adesR, cynomolgus C5a/C5adesR, rhesus C5a/C5adesR, rat C5a/C5adesR, mouse C5a/C5adesR, pig C5a/C5adesR or other mammalian C5a/C5adesR.
  • inhibitor concentration 50% represents the concentration of an inhibitor (e.g., an anti-C5/C5a/C5adesR antibody or fragment provided herein) that is required for 50% inhibition of a given activity of the molecule the inhibitor targets ⁇ e.g., C5a).
  • IC 50 concentration of an inhibitor (e.g., an anti-C5/C5a/C5adesR antibody or fragment provided herein) that is required for 50% inhibition of a given activity of the molecule the inhibitor targets ⁇ e.g., C5a).
  • a lower IC 50 value generally corresponds to a more potent inhibitor.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment inhibits one or more biological activities of C5a/C5adesR.
  • the term “inhibition” as used herein, refers to any statistically significant decrease in biological activity, including full blocking of the activity. For example, “inhibition” can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in biological activity.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment inhibits one or more biological activities of C5a/C5adesR by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment may deplete circulating C5/C5a/C5adesR.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment may achieve at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% depletion of circulating C5/C5a/C5adesR.
  • virtually all detectable C5/C5a/C5adesR is depleted from the circulation.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment may inhibit the expression of one or more C5/C5a/C5adesR inducible genes.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment inhibits the expression of one or more C5/C5a/C5adesR inducible genes by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, by at least 100%, by at least 120%, by at least 140%, by at least 160%, by at least 180%, or by at least 200%.
  • the C5/C5a/C5adesR antigen to be used for production of antibodies may be a C5a protein according to SEQ ID NO: 130 or an antigenic fragment thereof.
  • Examples of nucleotide and amino acid sequences of C5a and C5adesR are available as provided in Table 19.
  • C5, C5a, C5adesR can be produced recombinantly and isolated from, bacterial or eukaryotic cells using standard recombinant DNA methodology.
  • C5a or C5adesR can be expressed as a tagged (e.g., epitope tag) or other fusion protein to facilitate isolation as well as identification in various assays.
  • Antibodies or binding proteins that bind to various tags and fusion sequences are available as elaborated below. Serum purified or chemically synthesised forms of C5a and C5adesR can also be used for generating or characterising antibodies.
  • tag polypeptides and their respective antibodies are known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; flu HA tag polypeptide and its antibody 12CA5; c-myc tag and 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto; and Herpes Simplex virus glycoprotein D (gD) tag and its antibody.
  • the FLAG-peptide is recognized by an anti-FLAG M2 monoclonal antibody. Purification of a protein containing the FLAG peptide can be performed by immunoaffinity chromatography using an affinity matrix comprising the anti-FLAG M2 monoclonal antibody covalently attached to agarose.
  • Other tag polypeptides include the KT3 epitope peptide; an a-tubulin epitope peptide; and the T7 gene 10 protein peptide tag.
  • Polyclonal antibodies to an antigen-of-interest can be produced by various procedures known in the art.
  • a C5, C5a, and/or C5adesR polypeptide or immunogenic fragment thereof can be administered to various host animals including, but not limited to, rabbits, mice, rats, and the like, to induce the production of sera containing polyclonal antibodies specific for the antigen.
  • One or more adjuvants known in the art may be used to increase the immunological response, depending on the host species.
  • Polyclonal antibodies can be raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant.
  • KLH keyhole limpet hemocyanin
  • serum albumin serum albumin
  • bovine thyroglobulin bovine thyroglobulin
  • Conjugates also can be made
  • animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining an appropriate concentration of antigen or conjugate with adjuvant and injecting the solution at multiple sites.
  • the animals are boosted with 1/5 to 1/10 the original amount of antigen or conjugate in adjuvant by subcutaneous injection at multiple sites.
  • the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus.
  • aggregating agents such as alum are suitably used to enhance the immune response.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous or isolated antibodies, e.g., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts.
  • Monoclonal antibodies are highly specific, being directed against a single antigenic site or multiple antigenic sites in the case of multispecific engineered antibodies.
  • polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody is directed against the same determinant on the antigen.
  • the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies.
  • the modifier "monoclonal” is not to be construed as requiring production of the antibody by any particular method. Following is a description of representative methods for producing monoclonal antibodies which is not intended to be limiting and may be used to produce, for example, monoclonal mammalian, chimeric, humanized, human, domain, diabodies, vaccibodies, linear and multispecific antibodies.
  • mice or other appropriate host animals such as hamster
  • lymphocytes may be immunized in vitro.
  • lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent or fusion partner, such as polyethylene glycol, to form a hybridoma cell.
  • the selected myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium that selects against the unfused parental cells.
  • the myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-1 1 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California, USA, and SP-2 and derivatives e.g., X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Maryland, USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies.
  • the clones may be subcloned by limiting dilution procedures and grown by standard methods. Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium.
  • the hybridoma cells may be grown in vivo as ascites tumors in an animal e.g., by i.p. injection of the cells into mice.
  • the monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (e.g., using protein A or protein G-Sepharose) or ion-exchange chromatography, affinity tags, hydroxylapatite chromatography, gel electrophoresis, dialysis, and the like. Exemplary purification methods are described in more detail below. Phage display techniques
  • monoclonal antibodies or antibody fragments can be isolated from antibody phage display libraries expressing CDRs of the desired species as described (McCafferty et al.,
  • the human antibody can be selected from a phage library, where that phage library expresses human antibodies, as descibed in Vaughan et al., 1996, Nat. Biotech., 14:309-314, Sheets et al., 1998, Proc. Nat'l. Acad. Sci., 95:6157-6162, Hoogenboom and Winter,
  • phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
  • phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine).
  • Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
  • Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with scFv, Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein.
  • Affinity maturation strategies and chain shuffling strategies are known in the art and can be employed to generate high affinity human antibodies.
  • Such phage display methods can be used to isolate an anti-C5/C5a/C5adesR antibody or fragment by bringing the phage display library, for example a phage library expressing scFv, into contact with C5a antigen, and selecting one or more antibodies from the library that are able to bind C5a/C5adesR.
  • a phage library expressing scFv for example a phage library expressing scFv
  • nucleic acid encoding antibodies can be isolated and used to generate whole antibodies, including human antibodies, humanized antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below.
  • techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art.
  • Techniques which can be used to produce single-chain Fvs and antibodies are known in the art. Such techniques can be used to generate recombinant antibodies where the binding domain, e.g., ScFv, was isolated from a phage display library.
  • DNA encoding monoclonal antibodies may be readily isolated and/or sequenced using conventional procedures ⁇ e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, Human Embryonic Kidney (HEK) 293 cells or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
  • DNA or genetic material for recombinant antibodies can be obtained from source(s) other than hybridomas to generate an anti-C5/C5a/C5adesR antibody or antigen binding fragment.
  • Recombinant expression of an antibody or variant thereof generally requires construction of an expression vector containing a polynucleotide that encodes the antibody.
  • Vectors for the expression of heavy chains and light chains were originally described in Persic et a ⁇ , 1997.
  • Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy, the entire light chain, or both the entire heavy and light chains.
  • an expression vector is transferred to a host cell by conventional techniques, the transfected cells then are cultured by conventional techniques to produce an antibody.
  • host cells containing a polynucleotide encoding an isolated antibody or antigen-binding fragment thereof presented or fragments thereof, or a heavy or light chain thereof, or portion thereof, or a single-chain antibody herein, operably linked to a heterologous promoter.
  • vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
  • Mammalian cell lines available as hosts for expression of recombinant antibodies are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human embryonic kidney (HEK) 293 cells, and a number of other cell lines.
  • ATCC American Type Culture Collection
  • CHO Chinese hamster ovary
  • HeLa cells HeLa cells
  • BHK baby hamster kidney
  • COS monkey kidney cells
  • human hepatocellular carcinoma cells e.g., Hep G2
  • HEK human embryonic kidney
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the antibody or portion thereof expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NSO (a murine myeloma cell line that does not endogenously produce any functional immunoglobulin chains), SP20, CRL7030 and
  • human cell lines developed by immortalizing human lymphocytes can be used to recombinantly produce monoclonal antibodies.
  • the human cell line PER.C6. (Crucell, Netherlands) can be used to recombinantly produce monoclonal antibodies. Additional cell lines that may be used as hosts for expression of recombinant antibodies include, but are not limited to, insect cells ⁇ e.g., Sf21/Sf9, Trichoplusia ni Bti-Tn5b1 -4) or yeast cells ⁇ e.g., S. cerevisiae, Pichia, US7326681 ; etc), plants cells (US20080066200); and chicken cells.
  • antibodies presented herein are expressed in a cell line with stable expression of the antibody.
  • Stable expression can be used for long-term, high-yield production of recombinant proteins.
  • cell lines which stably express the antibody molecule may be generated.
  • Host cells can be transformed with an appropriately engineered vector comprising expression control elements (e.g., promoter, enhancer, transcription terminators, polyadenylation sites, and the like), and a selectable marker gene. Following the introduction of the foreign DNA, cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • expression control elements e.g., promoter, enhancer, transcription terminators, polyadenylation sites, and the like
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells that stably integrated the plasmid into their chromosomes to grow and form foci which in turn can be cloned and expanded into cell lines.
  • Methods for producing stable cell lines with a high yield are known in the art and reagents are generally available commercially.
  • antibodies presented herein are expressed in a cell line with transient expression of the antibody.
  • Transient transfection is a process in which the nucleic acid introduced into a cell does not integrate into the genome or chromosomal DNA of that cell. A nucleic acid often is maintained as an extrachromosomal element, e.g., as an episome, in the cell.
  • Transcription processes of the nucleic acid of the episome are not affected and a protein encoded by the nucleic acid of the episome is produced.
  • the cell line which can be stable or transiently transfected, is maintained in cell culture medium and conditions known in the art resulting in expression and production of monoclonal antibodies.
  • the mammalian cell culture media is based on commercially available media formulations, including, for example, DMEM or Ham's F12.
  • the cell culture media is modified to support increases in both cell growth and biologic protein expression.
  • the terms "cell culture medium,” “culture medium,” and “medium formulation” refer to a nutritive solution for the maintenance, growth, propagation, or expansion of cells in an artificial in vitro environment outside of a multicellular organism or tissue.
  • Cell culture medium may be optimized for a specific cell culture use, including, for example, cell culture growth medium which is formulated to promote cellular growth, or cell culture production medium which is formulated to promote recombinant protein production.
  • the terms nutrient, ingredient, and component are used interchangeably herein to refer to the constituents that make up a cell culture medium.
  • the cell lines are maintained using a fed batch method.
  • fed batch method refers to a method by which a fed batch cell culture is supplied with additional nutrients after first being incubated with a basal medium.
  • a fed batch method may comprise adding supplemental media according to a determined feeding schedule within a given time period.
  • a “fed batch cell culture” refers to a cell culture where the cells, typically mammalian, and culture medium are supplied to the culturing vessel initially and additional culture nutrients are fed, continuously or in discrete increments, to the culture during culturing, with or without periodic cell and/or product harvest before termination of culture.
  • the cell culture medium used and the nutrients contained therein are known in the art.
  • the cell culture medium comprises a basal medium and at least one hydrolysate, e.g., soy-based, hydrolysate, a yeast-based hydrolysate, or a combination of the two types of hydrolysates resulting in a modified basal medium.
  • the additional nutrients may sometimes include only a basal medium, such as a concentrated basal medium, or may include only hydrolysates, or concentrated hydrolysates.
  • Suitable basal media include, but are not limited to Dulbecco's Modified Eagle's Medium (DMEM), DME/F12, Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, a-Minimal Essential Medium (a-MEM), Glasgow's Minimal Essential Medium (G-MEM), PF CHO (see, e.g., CHO protein free medium (Sigma) or EX- CELLTM 325 PF CHO Serum-Free Medium for CHO Cells Protein-Free (SAFC Bioscience), and Iscove's Modified Dulbecco's Medium.
  • DMEM Dulbecco's Modified Eagle's Medium
  • MEM Minimal Essential Medium
  • BME Basal Medium Eagle
  • RPMI 1640 F-10, F-12
  • a-MEM a-Minimal Essential Medium
  • G-MEM Glasgow's Minimal Essential Medium
  • PF CHO see, e.g., CHO protein free medium
  • basal media which may be used in the technology herein include BME Basal Medium; Dulbecco's Modified Eagle Medium (DMEM, powder) (Gibco-lnvitrogen (# 31600).
  • the basal medium may be is serum-free, meaning that the medium contains no serum (e.g., fetal bovine serum (FBS), horse serum, goat serum, or any other animal-derived serum known in the art) or animal protein free media or chemically defined media.
  • FBS fetal bovine serum
  • horse serum horse serum
  • goat serum or any other animal-derived serum known in the art
  • animal protein free media or chemically defined media.
  • the basal medium may be modified to remove certain non-nutritional components found in standard basal medium, such as various inorganic and organic buffers, surfactant(s), and sodium chloride. Removing such components from basal cell medium allows an increased concentration of the remaining nutritional components, and may improve overall cell growth and protein expression.
  • omitted components may be added back into the cell culture medium containing the modified basal cell medium according to the requirements of the cell culture conditions.
  • the cell culture medium contains a modified basal cell medium, and at least one of the following nutrients, an iron source, a recombinant growth factor; a buffer; a surfactant; an osmolarity regulator; an energy source; and non-animal hydrolysates.
  • modified basal cell medium may optionally contain amino acids, vitamins, or a combination of both amino acids and vitamins.
  • modified basal medium further contains glutamine, e.g., L-glutamine, and/or methotrexate.
  • antibody production is conducted in large quantity by a bioreactor process using fed-batch, batch, perfusion or continuous feed bioreactor methods known in the art.
  • Large- scale bioreactors have at least 1000 liters of capacity, sometimes about 1 ,000 to 100,000 liters of capacity. These bioreactors may use agitator impellers to distribute oxygen and nutrients.
  • Small scale bioreactors refers generally to cell culturing in no more than approximately 100 liters in volumetric capacity, and can range from about 1 liter to about 100 liters.
  • single-use bioreactors may be used for either large-scale or small scale culturing.
  • Temperature, pH, agitation, aeration and inoculum density may vary depending upon the host cells used and the recombinant protein to be expressed.
  • a recombinant protein cell culture may be maintained at a temperature between 30 and 45 degrees Celsius.
  • the pH of the culture medium may be monitored during the culture process such that the pH stays at an optimum level, which may be for certain host cells, within a pH range of 6.0 to 8.0.
  • An impellor driven mixing may be used for such culture methods for agitation.
  • the rotational speed of the impellor may be approximately 50 to 200 cm/sec tip speed, but other airlift or other mixing/aeration systems known in the art may be used, depending on the type of host cell being cultured.
  • aeration is provided to maintain a dissolved oxygen concentration of approximately 20% to 80% air saturation in the culture, again, depending upon the selected host cell being cultured.
  • a bioreactor may sparge air or oxygen directly into the culture medium.
  • Other methods of oxygen supply exist, including bubble-free aeration systems employing hollow fiber membrane aerators.
  • an antibody molecule may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigens Protein A or Protein G, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigens Protein A or Protein G, and sizing column chromatography
  • centrifugation e.g., centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • the antibodies of the present technology or fragments thereof may be fused to heterologous polypeptide sequences (referred to herein as "tags") described above or otherwise known in the art to facilitate purification.
  • the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Procedures for isolating antibodies which are secreted into the periplasmic space of E. coli are known in the art. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
  • the antibody composition prepared from the cells can be purified using, for example,
  • hydroxylapatite chromatography hydrophobic interaction chromatography, ion exchange chromatography, gel electrophoresis, dialysis, and/or affinity chromatography either alone or in combination with other purification steps.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody.
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available.
  • Mechanically stable matrices such as controlled pore glass or
  • poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
  • the Bakerbond ABX resin J.T. Baker, Phillipsburg, New Jersey
  • Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin, SEPHAROSE chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS- PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.
  • the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, and performed at low salt concentrations (e.g., from about 0-0.25 M salt).
  • antibodies that are substantially purified/isolated.
  • these isolated/purified recombinantly expressed antibodies may be administered to a patient to mediate a prophylactic or therapeutic effect.
  • such isolated/purified antibodies may be used to detect a C5/C5a/C5adesR polypeptide and/or diagnose a C5/C5a/C5adesR mediated disease.
  • Humanized antibodies are humanized antibodies, which are generated using methods known in the art.
  • Humanized antibodies are antibody molecules derived from a non-human species antibody (also referred to herein as a donor antibody) that bind the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule (also referred to herein as an acceptor antibody).
  • CDRs complementarity determining regions
  • framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, and sometimes improve, antigen binding and/or reduce immunogenicity.
  • humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • the FR residues are fully human residues.
  • Humanization can be performed by substituting hypervariable region sequences for the corresponding sequences of a human antibody.
  • humanized antibodies may be prepared by methods known in the art, including CDR grafting approaches, veneering or resurfacing, chain shuffling strategies, molecular modeling strategies and the like. These general approaches may be combined with standard mutagenesis and recombinant synthesis techniques to produce anti-C5/C5a/C5adesR antibodies and fragments with desired properties.
  • CDR grafting often is performed by replacing one or more CDRs of an acceptor antibody (e.g., a human antibody) with one or more CDRs of a donor antibody (e.g., a non-human antibody).
  • acceptor antibody e.g., a human antibody
  • donor antibody e.g., a non-human antibody
  • Acceptor antibodies may be selected based on similarity of framework residues between a candidate acceptor antibody and a donor antibody and may be further modified to introduce similar residues. Following CDR grafting, additional changes may be made in the donor and/or acceptor sequences to optimize antibody binding and functionality.
  • CDR regions include the specificity-determining residues and adjacent amino acids, including those at positions 24-34, 50- 55 and 89-97 in the light chain, and at positions 31 -35b, 50-58, and 95-101 in the heavy chain.
  • SDRs specificity-determining residues
  • SDRs specificity-determining residues
  • Minimally immunogenic polypeptide sequences including contact residues which are referred to as SDRs, are identified and grafted onto human framework regions.
  • Veneering or resurfacing is based on the concept of reducing potentially immunogenic amino acid sequences in a rodent or other non-human antibody by resurfacing the solvent accessible exterior of the antibody with human amino acid sequences.
  • veneered antibodies appear less foreign to human cells.
  • a non-human antibody is veneered by (1 ) identifying exposed exterior framework region residues in the non-human antibody, which are different from those at the same positions in framework regions of a human antibody, and (2) replacing the identified residues with amino acids that typically occupy these same positions in human antibodies.
  • humanized antibodies are chimeric antibodies.
  • Chimeric antibodies are antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while another portion of the chain(s) is identical to or homologous to
  • Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen-binding sequences derived from a nonhuman primate (e.g., Old World Monkey, such as baboon, rhesus or cynomolgus monkey) and human constant region sequences.
  • a nonhuman primate e.g., Old World Monkey, such as baboon, rhesus or cynomolgus monkey
  • the antibodies are fully human antibodies.
  • Human antibodies can be derived by in vivo or in vitro methods known in the art.
  • Human antibodies avoid some of the problems associated with antibodies that possess murine or rat variable and/or constant regions.
  • the presence of such murine or rat derived proteins can lead to the rapid clearance of the antibodies or can lead to the generation of an immune response against the antibody by a patient.
  • fully human antibodies can be generated through phage display using human antibody libraries, or by the introduction of functional human antibody loci into a rodent, other mammal or animal so that the rodent, other mammal or animal produces fully human antibodies.
  • transgenic animals e.g., mice
  • transgenic animals e.g., mice
  • the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice can result in complete inhibition of endogenous antibody production.
  • Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice will result in the production of human antibodies upon antigen challenge.
  • XenoMouse® strains of mice that have been engineered to contain up to but less than 1000 kb-sized germline configured fragments of the human heavy chain locus and kappa light chain locus.
  • the XenoMouse® strains are available from Amgen, Inc.
  • XenoMouse® lines of mice are immunized with an antigen of interest, lymphatic cells (such as B-cells) are recovered from the hyper-immunized mice, and the recovered lymphocytes are fused with a myeloid-type cell line to prepare immortal hybridoma cell lines using techniques described above an known in the art. These hybridoma cell lines are screened and selected to identify hybridoma cell lines that produced antibodies specific to the antigen of interest.
  • minilocus an exogenous Ig locus is mimicked through the inclusion of pieces (individual genes) from the Ig locus.
  • one or more VH genes, one or more DH genes, one or more JH genes, a mu constant region, and usually a second constant region (e.g., a gamma constant region) are formed into a construct for insertion into an animal.
  • a second constant region e.g., a gamma constant region
  • KMTM mice which are the result of cross-breeding of Kirin's Tc mice with Medarex's minilocus (Humab) mice have been generated. These mice possess the human IgH transchromosome of the Kirin mice and the kappa chain transgene of the Genpharm mice.
  • Human antibodies can also be derived by in vitro methods. Suitable examples include but are not limited to phage display, ribosome display, yeast display, and the like.
  • the phage display technology can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.
  • V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle.
  • the filamentous particle contains a single- stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties.
  • the phage mimics some of the properties of the B-cell.
  • Phage display can be performed in a variety of formats.
  • Several sources of V-gene segments can be used for phage display.
  • a diverse array of anti-oxazolone antibodies have been isolated from a small random combinatorial library of V genes derived from the spleens of immunized mice.
  • a repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self- antigens) can be isolated essentially following the techniques known in the art.
  • human antibodies may also be generated by in vitro activated B cells.
  • Immunoglobulin genes undergo various modifications during maturation of the immune response, including recombination between V, D and J gene segments, isotype switching, and hypermutation in the variable regions. Recombination and somatic hypermutation are the foundation for generation of antibody diversity and affinity maturation, but they can also generate sequence liabilities that may make commercial production of such immunoglobulins as therapeutic agents difficult or increase the immunogenicity risk of the antibody.
  • mutations in CDR regions are likely to contribute to improved affinity and function, while mutations in framework regions may increase the risk of immunogenicity. This risk can be reduced by reverting framework mutations to germline while ensuring that activity of the antibody is not adversely impacted.
  • the diversification processes may also generate some structural liabilities or these structural liabilities may exist within germline sequences contributing to the heavy and light chain variable domains. Regardless of the source, it may be desirable to remove potential structural liabilities that may result in instability, aggregation, heterogeneity of product, or increased immunogenicity. Examples of undesirable liabilities include unpaired cysteines (which may lead to disulfide bond scrambling, or variable sulfhydryl adduct formation), N-linked glycosylation sites (resulting in heterogeneity of structure and activity), as well as deamidation ⁇ e.g., NG, NS), isomerization (DG), oxidation (exposed methionine), and hydrolysis (DP) sites.
  • unpaired cysteines which may lead to disulfide bond scrambling, or variable sulfhydryl adduct formation
  • N-linked glycosylation sites resulting in heterogeneity of structure and activity
  • deamidation ⁇ e.g., NG, NS isomerization
  • the antibody sequence can be mutated back to the germline sequence.
  • Such corrective mutations can occur at one, two, three, four, five or more positions, or a combination of any of the mutated positions, using standard molecular biological techniques.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment may include an arginine (R) or glutamine (Q) amino acid at position 79 in the VL domain (e.g., Example 8, Table 5).
  • the present antibodies are antibody fragments or antibodies comprising these fragments.
  • the antibody fragment comprises a portion of the full length antibody, which generally is the antigen binding or variable region thereof.
  • Examples of antibody fragments include Fab, Fab', F(ab')2, Fd and Fv fragments.
  • Diabodies; linear antibodies, single-chain antibody molecules; and multispecific antibodies are antibodies formed from these antibody fragments.
  • these fragments were derived via proteolytic digestion of intact antibodies using techniques known in the art. However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and scFv antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of these fragments.
  • the antibody fragments can be isolated from the antibody phage libraries discussed above.
  • Fab'-SH fragments can also be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments.
  • F(ab')2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments are known.
  • the antibody of choice is a single-chain Fv fragment (scFv).
  • the antibody is a Fab fragment.
  • Fv and scFv are the only species with intact combining sites that are devoid of constant regions; thus, they are suitable for reduced nonspecific binding during in vivo use.
  • scFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an scFv.
  • the present antibodies are domain antibodies, e.g., antibodies containing the small functional binding units of antibodies, corresponding to the variable regions of the heavy (VH) or light (VL) chains of human antibodies.
  • domain antibodies include, but are not limited to, those available from Domantis that are specific to therapeutic targets.
  • Commercially available libraries of domain antibodies can be used to identify anti-C5/C5a/C5adesR domain antibodies.
  • anti-C5/C5a/C5adesR antibodies and fragments comprise a C5/C5a/C5adesR functional binding unit and an Fc gamma receptor functional binding unit.
  • the present antibodies are linear antibodies.
  • Linear antibodies comprise a pair of tandem Fd segments (VH-CH 1-VH-CH 1 ) which form a pair of antigen-binding regions.
  • Linear antibodies can be bispecific or monospecific.
  • Bi-specific antibodies are antibodies that have binding specificities for at least two different epitopes. Bispecific antibodies may bind to two different epitopes of either the C5, C5a and/or C5adesR protein, or may bind to each of the C5 and C5a and C5adesR polypeptides. Other such antibodies may combine a C5/C5a/C5adesR binding site with a binding site for another protein. Bispecific antibodies can be prepared as full length antibodies or antibody fragments ⁇ e.g., F(ab')2 bispecific antibodies). Methods for making bispecific antibodies are known in the art.
  • immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host cell.
  • This provides for greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yield of the desired bispecific antibody. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios have no significant affect on the yield of the desired chain combination.
  • bispecific antibodies are composed of a hybrid
  • immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid
  • the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
  • An interface comprises at least a part of the CH3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains ⁇ e.g., tyrosine or tryptophan).
  • Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones ⁇ e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
  • bispecific antibodies can be prepared using chemical linkage using a procedure where intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives.
  • TAB thionitrobenzoate
  • One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody.
  • the bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.
  • bispecific antibodies have been produced using leucine zippers.
  • the leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion.
  • the antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers.
  • This method can also be utilized for the production of antibody homodimers.
  • the "diabody” technology known in the art has provided an alternative mechanism for making bispecific antibody fragments.
  • the fragments comprise a VH connected to a VL by a linker which is too short to allow pairing between the two domains on the same chain.
  • Antibodies with more than two valencies are contemplated.
  • trispecific antibodies can be prepared.
  • an isolated antibody or antigen-binding fragment thereof presented can be a multivalent antibody (other than of the IgM class) with three or more antigen binding sites ⁇ e.g., tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody.
  • a multivalent antibody can comprise a dimerization domain and three or more antigen binding sites.
  • a dimerization domain comprises (or consists of) an Fc region or a hinge region.
  • the antibody comprises an Fc region and three or more antigen binding sites amino-terminal to the Fc region.
  • a multivalent antibody comprises (or consists of) three to about eight antigen binding sites.
  • a multivalent antibody often comprises at least one polypeptide chain where the polypeptide chain(s) comprise two or more variable domains.
  • the polypeptide chain(s) may comprise VD1-(X1 )n-VD2-(X2)n-Fc, where VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n is 0 or 1 .
  • the polypeptide chain(s) may comprise: VH-CH1 -flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fc region chain.
  • a multivalent antibody can further comprise at least two light chain variable domain polypeptides.
  • a multivalent antibody may, for instance, comprise from about two to about eight light chain variable domain polypeptides.
  • the light chain variable domain polypeptides contemplated here often comprise a light chain variable domain and, optionally, further comprise a CL domain.
  • the present technology also encompasses further modifications and, their variants and fragments thereof, of an anti-C5/C5a/C5adesR antibody or antigen binding fragment comprising one or more of the following: amino acid residue and/or polypeptide substitution, addition and/or deletion in the variable light (VL) domain and/or variable heavy (VH) domain and/or Fc region, and a post translational modification. Included in these modifications are antibody conjugates where an antibody has been covalently attached to a moiety. Moieties suitable for attachment to the antibodies include but are not limited to, proteins, peptides, drugs, labels, and cytotoxins.
  • changes to the antibodies may be made to alter or fine tune characteristics (e.g., biochemical, binding and/or functional) of the antibodies as is appropriate for treatment and/or diagnosis of C5/C5a/C5adesR mediated diseases.
  • Methods for forming conjugates, making amino acid and/or polypeptide changes and post-translational modifications are known in the art, some of which are detailed below. Any combination of deletion, insertion, and substitution can be made to arrive at a final construct, provided that the final construct possesses desired characteristics.
  • the antibodies may have about 50% to about 95% sequence identity to the amino acid sequence of the heavy or light chain variable domain of an anti-C5/C5a/C5adesR antibody or antigen binding fragment as described herein.
  • a modified antibody may have an amino acid sequence having at least 50%, 55%, 60% 65%, 70%, 75%, 80%, 85%, 90%, or 95% amino acid sequence identity or similarity with the amino acid sequence of either the heavy or light chain variable domain of an anti-C5/C5a/C5adesR antibody or antigen binding fragment as described herein.
  • an altered antibody has an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% amino acid sequence identity or similarity with the amino acid sequence of the heavy or light chain CDR1 , CDR2, or CDR3 of an anti-C5/C5a/C5adesR antibody or antigen binding fragment as described herein.
  • An altered antibody may sometimes have an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% amino acid sequence identity or similarity with the amino acid sequence of the heavy or light chain FR1 , FR2, FR3 or FR4 of an anti- C5/C5a/C5adesR antibody or antigen binding fragment as described herein.
  • altered antibodies are generated by one or more amino acid alterations (e.g., substitutions, deletion and/or additions) introduced in one or more of the variable regions of the antibody.
  • the amino acid alterations are introduced in the framework regions.
  • One or more alterations of framework region residues may result in an improvement in the binding affinity of the antibody for the antigen. This may be especially true when these changes are made to humanized antibodies where the framework region may be from a different species than the CDR regions.
  • framework region residues to modify include those which non-covalently bind antigen directly, interact with/effect the conformation of a CDR, and/or participate in the VL-VH interface. In some embodiments, from about one to about five framework residues may be altered.
  • hypervariable region residues may be changed randomly, especially where the starting binding affinity of an anti-C5/C5a/C5adesR antibody or antigen binding fragment for the antigen from the second mammalian species is such that such randomly produced antibodies can be readily screened.
  • alanine scanning mutagenesis One useful procedure for generating altered antibodies is called "alanine scanning mutagenesis".
  • one or more of the hypervariable region residue(s) are replaced by alanine or polyalanine residue(s) to alter the interaction of the amino acids with the C5/C5a/C5adesR.
  • Those hypervariable region residue(s) demonstrating functional sensitivity to the substitutions then are refined by introducing additional or other mutations at or for the sites of substitution.
  • the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined.
  • the Ala-mutants produced this way are screened for their biological activity as described herein.
  • the substitutional variant involves substituting one or more hypervariable region residues of a parent antibody ⁇ e.g., a humanized or human antibody).
  • a parent antibody e.g., a humanized or human antibody
  • the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated.
  • a convenient way for generating such substitutional variants involves affinity maturation using phage. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody mutants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene I I I product of M 13 packaged within each particle. The phage-displayed mutants are then screened for their biological activity (e.g., binding affinity) as herein disclosed.
  • Mutations in antibody sequences may include substitutions, deletions, including internal deletions, additions, including additions yielding fusion proteins, or conservative substitutions of amino acid residues within and/or adjacent to the amino acid sequence, but that result in a "silent" change, in that the change produces a functionally equivalent anti-C5/C5a/C5adesR antibody or fragment.
  • Conservative amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.
  • non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • glycine and proline are residues that can influence chain orientation. Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class.
  • non-classical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the antibody sequence.
  • Non-classical amino acids include, but are not limited to, the D-isomers of the common amino acids, alpha -amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, gamma- Abu, epsilon-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t- butylalanine, phenylglycine, cyclohexylalanine, beta-alanine, fluoro-amino acids, designer amino acids such as beta-methyl amino acids, C-alpha-methyl amino acids, N
  • any cysteine residue not involved in maintaining the proper conformation of an anti-C5/C5a/C5adesR antibody or antigen binding fragment also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking.
  • cysteine bond(s) may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).
  • the protein fused to the portion of an antibody is an enzyme component of Antibody-Directed Enzyme Prodrug Therapy (ADEPT).
  • ADPT Antibody-Directed Enzyme Prodrug Therapy
  • DNA shuffling may be employed to alter the characteristics of the antibody or fragments thereof (e.g., an antibody or a fragment thereof with higher affinities and lower dissociation rates).
  • An antibody can further be a binding-domain immunoglobulin fusion protein as known in the art.
  • modified glycosylation in the variable region can alter the affinity of the antibody for a target antigen.
  • the glycosylation pattern in the variable region of the present antibodies is modified.
  • an aglycoslated antibody can be made (i.e., the antibody lacks
  • glycosylation can be altered to, for example, increase the affinity of the antibody for a target antigen.
  • carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence.
  • one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
  • Such aglycosylation may increase the affinity of the antibody for antigen.
  • One or more amino acid substitutions can also be made that result in elimination of a glycosylation site present in the Fc region (e.g., Asparagine 297 of IgG).
  • glycosylated antibodies may be produced in bacterial cells which lack the necessary glycosylation machinery.
  • variants of the Fc region e.g., amino acid substitutions and/or additions and/or deletions
  • variants of the Fc region can enhance or diminish effector function of an antibody and may alter the
  • an anti- C5/C5a/C5adesR antibody or antigen binding fragment comprises an altered Fc region (also referred to herein as "variant Fc region") in which one or more alterations have been made in the Fc region in order to change functional and/or pharmacokinetic properties of the antibodies.
  • the Fc portion may be mutated to decrease effector function using techniques known in the art. For example, alteration of a constant region domain e.g. by point mutations or amino acid substitutions may reduce Fc receptor binding of the circulating modified antibody thereby minimizing effector cell or complement-mediated clearance.
  • the triple mutation L234F/L235E/P331 S ( ⁇ ') causes a profound decrease in the binding activity of human lgG1 molecules to human C1 q, CD64, CD32A and CD16. See, e.g., Oganesyan et al., Acta Crystallogr D Biol Crystallogr. 64:700-704 (2008).
  • antibody half-life means a pharmacokinetic property of an antibody that is a measure of the mean survival time of antibody molecules following their administration.
  • Antibody half-life can be expressed as the time required to eliminate 50 percent of a known quantity of immunoglobulin from the patient's body (or other mammal) or a specific compartment thereof, for example, as measured in serum, i.e., circulating half-life, or in other tissues.
  • Half-life may vary from one immunoglobulin or class of immunoglobulin to another.
  • an increase in antibody half-life results in an increase in mean residence time (MRT) in circulation for the antibody administered.
  • MRT mean residence time
  • An increase in half-life allows for the reduction in amount of drug given to a patient as well as reducing the frequency of administration.
  • a salvage receptor binding epitope into the antibody (especially an antibody fragment) as known in the art.
  • the term "salvage receptor binding epitope” refers to an epitope of the Fc region of an IgG molecule (e.g., lgG1 , lgG2, lgG3, or lgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.
  • Antibodies with increased half-lives may also be generated by modifying amino acid residues identified as involved in the interaction between the Fc and the FcRn receptor.
  • the introduction of the triple mutation M252Y/S254T/T256E ( ⁇ ') into the C(H)2 domain of human immunoglobulin G (IgG) molecules causes an increase in their binding to the human neonatal Fc receptor (FcRn).
  • FcRn human neonatal Fc receptor
  • the half-life of an anti-C5/C5a/C5adesR antibody or fragment may be increased by conjugation to PEG or Albumin by techniques widely utilized in the art.
  • antibodies comprising Fc variant regions of an anti-C5/C5a/C5adesR antibody have an increased half-life of about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 80%, about 85%, about 90%, about 95%, about 100%, about 125%, about 150% or more as compared to an antibody comprising a native Fc region.
  • antibodies comprising Fc variant regions have an increased half-life of about 2 fold, about 3 fold, about 4 fold, about 5 fold, about 10 fold, about 20 fold, about 50 fold or more, or is between 2 fold and 10 fold, or between 5 fold and 25 fold, or between 15 fold and 50 fold, as compared to an antibody comprising a native Fc region.
  • the technology presented herein provides Fc variants, where the Fc region comprises a modification (e.g., amino acid substitutions, amino acid insertions, amino acid deletions) at one or more positions selected from the group consisting of 228, 234, 235, 236, 237, 238, 239, 240, 241 , 243, 244, 245, 247, 251 , 252, 254, 255, 256, 262, 263, 264, 265, 266, 267, 268, 269, 279, 280, 284, 292, 296, 297, 298, 299, 305, 313, 316, 325, 326, 327, 328, 329, 330, 331 , 332, 333, 334, 339, 341 , 343, 370, 373, 378, 392, 416, 419, 421 , 440 and 443 as numbered by the EU index as set forth in Kabat.
  • the Fc region may comprise a non naturally occurring amino acid residue at additional and/or
  • an Fc variant where the Fc region comprises at least one substitution selected from the group consisting of 228P, 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 234I, 234V, 234F, 235A, 235D, 235E, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 235I, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 240I, 240A, 240T, 240M, 241 W, 241 L, 241 Y, 241 E, 241 R.
  • an Fc variant antibody where the Fc region comprises at least one modification (e.g., amino acid substitutions, amino acid insertions, amino acid deletions) at one or more positions selected from the group consisting of 234, 235 and 331 .
  • the non-naturally occurring amino acids are selected from the group consisting of 234F, 235F, 235E, 235Y, and 331 S.
  • an Fc variant antibody where the Fc region comprises at least one modification (e.g., amino acid substitutions, amino acid insertions, amino acid deletions) at one or more positions selected from the group consisting of 228 and 235.
  • the non-naturally occurring amino acids are selected from the group consisting of 228P and 235E.
  • an Fc variant antibody where the Fc region comprises at least one non-naturally occurring amino acid at position 228. In certain embodiments, the non-naturally occurring amino acid is proline.
  • an Fc variant antibody where the Fc region comprises at least one non-naturally occurring amino acid at one or more positions selected from the group consisting of 252, 254, and 256.
  • the non-naturally occurring amino acids are selected from the group consisting of 252Y, 254T and 256E.
  • an Fc variant antibody where the Fc region comprises at least one non-naturally occurring amino acid at one or more positions selected from the group consisting of 252, 254, 256, 234, 235, 331 .
  • the non-naturally occurring amino acids are selected from the group consisting of 252Y, 254T, 256E, 234F, 235E and 331 S.
  • an Fc variant where the Fc region comprises at least one non-naturally occurring amino acid at one or more positions selected from the group consisting of 239, 330 and 332.
  • the non-naturally occurring amino acids are selected from the group consisting of 239D, 330L and 332E.
  • the antibodies presented herein are conjugated or covalently attached to a substance using methods known in the art.
  • the attached substance is a therapeutic agent, a detectable label (also referred to herein as a reporter molecule) or a solid support.
  • Suitable substances for attachment to antibodies include, but are not limited to, an amino acid, a peptide, a protein, a polysaccharide, a nucleoside, a nucleotide, an oligonucleotide, a nucleic acid, a hapten, a drug, a hormone, a lipid, a lipid assembly, a synthetic polymer, a polymeric microparticle, a biological cell, a virus, a fluorophore, a chromophore, a dye, a toxin, a hapten, an enzyme, an antibody, an antibody fragment, a radioisotope, solid matrixes, semi-solid matrixes and combinations thereof.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment is conjugated to a solid support.
  • Antibodies may be conjugated to a solid support as part of the screening and/or purification and/or manufacturing process.
  • Antibodies may be conjugated to a solid support as part of a diagnostic method or composition.
  • a solid support suitable for use in the present technology is typically substantially insoluble in liquid phases. A large number of supports are available and are known in the art.
  • solid supports include solid and semi-solid matrixes, such as aerogels and hydrogels, resins, beads, biochips (including thin film coated biochips), microfluidic chip, a silicon chip, multi-well plates (also referred to as microtitre plates or
  • microplates membranes, conducting and nonconducting metals, glass (including microscope slides) and magnetic supports. More specific examples of solid supports include silica gels, polymeric membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels, polysaccharides such as Sepharose, poly(acrylate), polystyrene, poly(acrylamide), polyol, agarose, agar, cellulose, dextran, starch, FICOLL, heparin, glycogen, amylopectin, mannan, inulin, nitrocellulose, diazocellulose, polyvinylchloride, polypropylene, polyethylene (including poly(ethylene glycol)), nylon, latex bead, magnetic bead, paramagnetic bead, superparamagnetic bead, starch and the like.
  • solid supports include silica gels, polymeric membranes, particles, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels, polysacchari
  • the solid support may include a reactive functional group, including, but not limited to, hydroxyl, carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea, carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide, sulfoxide, and the like, for attaching the antibodies provided herein.
  • a reactive functional group including, but not limited to, hydroxyl, carboxyl, amino, thiol, aldehyde, halogen, nitro, cyano, amido, urea, carbonate, carbamate, isocyanate, sulfone, sulfonate, sulfonamide, sulfoxide, and the like, for attaching the antibodies provided herein.
  • a suitable solid phase support can be selected on the basis of desired end use and suitability for various synthetic protocols.
  • resins generally useful in peptide synthesis may be employed, such as polystyrene (e.g., PAM-resin obtained from Bachem Inc., Peninsula
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment is conjugated to a label for purposes of diagnostics and other assays where the antibody and/or its associated ligand may be detected.
  • a label conjugated to an antibody and used in the present methods and compositions described herein is any chemical moiety, organic or inorganic, that exhibits an absorption maximum at wavelengths greater than 280 nm, and retains its spectral properties when covalently attached to an antibody.
  • Labels include, without limitation, a chromophore, a fluorophore, a fluorescent protein, a phosphorescent dye, a tandem dye, a particle, a hapten, an enzyme and a radioisotope.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment is conjugated to a fluorophore.
  • a fluorophore that can be used to label an anti-C5/C5a/C5adesR antibody or antigen binding fragment includes, without limitation; a pyrene (including any of the corresponding derivative compounds) , an anthracene, a naphthalene, an acridine, a stilbene, an indole or benzindole, an oxazole or benzoxazole, a thiazole or benzothiazole, a 4-amino-7- nitrobenz-2-oxa-1 , 3-diazole (NBD), a cyanine (including any corresponding compounds), a carbocyanine (including any corresponding compounds), a carbostyryl, a porphyrin, a salicylate, an anthranilate, an azulene, a perylene, a
  • oxazines include resorufins (including any corresponding compounds), aminooxazinones, diaminooxazines, and their benzo-substituted analogs.
  • the fluorophores conjugated to the antibodies described herein include xanthene (rhodol, rhodamine, fluorescein and derivatives thereof) coumarin, cyanine, pyrene, oxazine and borapolyazaindacene.
  • fluorophores are sulfonated xanthenes, fluorinated xanthenes, sulfonated coumarins, fluorinated coumarins and sulfonated cyanines.
  • the fluorophores conjugated to the antibodies described herein include lanthanide cryptates and chelates (Perkin Elmer and Cis Biointernational).
  • the choice of the fluorophore attached to the anti-C5/C5a/C5adesR antibody or fragment will determine the absorption and fluorescence emission properties of the conjugated antibody.
  • Physical properties of a fluorophore label that can be used for antibody and antibody bound ligands include, but are not limited to, spectral characteristics (absorption, emission and stokes shift), fluorescence intensity, lifetime, polarization and photo-bleaching rate, or combination thereof. All of these physical properties can be used to distinguish one fluorophore from another, and thereby allow for multiplexed analysis.
  • the fluorophore has an absorption maximum at wavelengths greater than 320 nm.
  • the fluorophore absorbs at or near 488 nm to 514 nm (particularly suitable for excitation by the output of the argon-ion laser excitation source) or near 546 nm (particularly suitable for excitation by a mercury arc lamp).
  • a fluorophore can emit in the NI R (near infra red region) for tissue or whole organism applications.
  • Other desirable properties of the fluorescent label may include cell permeability and low toxicity, for example if labeling of the antibody is to be performed in a cell or an organism (e.g., a living animal).
  • an enzyme is utilized as a label and is conjugated to an anti- C5/C5a/C5adesR antibody or antigen binding fragment.
  • Enzymes are desirable labels because amplification of the detectable signal can be obtained resulting in increased assay sensitivity.
  • the enzyme itself often does not produce a detectable response but functions to break down a substrate when it is contacted by an appropriate substrate such that the converted substrate produces a fluorescent, colorimetric or luminescent signal.
  • Enzymes amplify the detectable signal because one enzyme on a labeling reagent can result in multiple substrates being converted to a detectable signal.
  • the enzyme substrate is selected to yield the measurable product, e.g., colorimetric, fluorescent or chemiluminescence.
  • colorimetric or fluorogenic substrate and enzyme combination uses oxidoreductases such as horseradish peroxidase and a substrate such as 3,3'-diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC), which yield a distinguishing color (brown and red, respectively).
  • DAB 3,3'-diaminobenzidine
  • AEC 3-amino-9-ethylcarbazole
  • colorimetric oxidoreductase substrates that yield detectable products include, but are not limited to: 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), o- phenylenediamine (OPD), 3,3',5,5'-tetramethylbenzidine (TMB), o-dianisidine, 5-aminosalicylic acid, 4-chloro-1 -naphthol.
  • ABTS 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
  • OPD o- phenylenediamine
  • TMB 3,3',5,5'-tetramethylbenzidine
  • o-dianisidine 5-aminosalicylic acid
  • 4-chloro-1 -naphthol 4-chloro-1 -naphthol.
  • Fluorogenic substrates include, but are not limited to, homovanillic acid or 4-hydroxy-3-methoxyphenylacetic acid, reduced phenoxazines and reduced benzothiazines, including Amplex® Red reagent and its variants and reduced dihydroxanthenes, including dihydrofluoresceins and dihydrorhodamines including dihydrorhodamine 123.
  • Peroxidase substrates that are tyramides represent a unique class of peroxidase substrates in that they can be intrinsically detectable before action of the enzyme but are "fixed in place" by the action of a peroxidase in the process described as tyramide signal amplification (TSA). These substrates are extensively utilized to label targets in samples that are cells, tissues or arrays for their subsequent detection by microscopy, flow cytometry, optical scanning and fluorometry.
  • TSA tyramide signal amplification
  • a colorimetric (and in some cases fluorogenic) substrate and enzyme combination sometimes uses a phosphatase enzyme such as an acid phosphatase, an alkaline phosphatase or a recombinant version of such a phosphatase in combination with a colorimetric substrate such as 5-bromo-6- chloro-3-indolyl phosphate (BCIP), 6-chloro-3-indolyl phosphate, 5-bromo-6-chloro-3-indolyl phosphate, p-nitrophenyl phosphate, or o-nitrophenyl phosphate or with a fluorogenic substrate such as 4-methylumbelliferyl phosphate, 6,8-difluoro-7-hydroxy-4-methylcoumarinyl phosphate (DiFMUP, U.S.
  • a phosphatase enzyme such as an acid phosphatase, an alkaline phosphatase or a recombinant version of such a phosphatase in combination
  • Glycosidases in particular beta-galactosidase, beta-glucuronidase and beta-glucosidase, are additional suitable enzymes.
  • Appropriate colorimetric substrates include, but are not limited to, 5- bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) and similar indolyl galactosides, glucosides, and glucuronides, o-nitrophenyl beta-D-galactopyranoside (ONPG) and p-nitrophenyl beta-D-galactopyranoside.
  • fluorogenic substrates include resorufin beta-D- galactopyranoside, fluorescein digalactoside (FDG), fluorescein diglucuronide and their structural variants, 4-methylumbelliferyl beta-D-galactopyranoside, carboxyumbelliferyl beta-D- galactopyranoside and fluorinated coumarin beta-D-galactopyranosides.
  • Additional enzymes include, but are not limited to, hydrolases such as cholinesterases and peptidases, oxidases such as glucose oxidase and cytochrome oxidases, and reductases for which suitable substrates are known. Enzymes and their appropriate substrates that produce chemiluminescence are uesful for some assays. These include, but are not limited to, natural and recombinant forms of luciferases and aequorins. Chemiluminescence-producing substrates for phosphatases, glycosidases and oxidases such as those containing stable dioxetanes, luminol, isoluminol and acridinium esters are additionally productive.
  • haptens such as biotin
  • Biotin is useful because it can function in an enzyme system to further amplify the detectable signal, and it can function as a tag to be used in affinity chromatography for isolation purposes.
  • an enzyme conjugate that has affinity for biotin is used, such as avidin-HRP. Subsequently a peroxidase substrate is added to produce a detectable signal.
  • Haptens utilized as labels can be selected from hormones, naturally occurring and synthetic drugs, pollutants, allergens, affector molecules, growth factors, chemokines, cytokines, lymphokines, amino acids, peptides, chemical intermediates, nucleotides and the like.
  • fluorescent proteins are conjugated to the antibodies as a label.
  • fluorescent proteins examples include green fluorescent protein (GFP) and the phycobiliproteins and the derivatives thereof.
  • the fluorescent proteins, especially phycobiliprotein, are useful for creating tandem dye labeled labeling reagents. These tandem dyes comprise a fluorescent protein and a fluorophore for the purposes of obtaining a larger stokes shift where the emission spectra is farther shifted from the wavelength of the fluorescent protein's absorption spectra. This may be effective for detecting a low quantity of a target in a sample where the emitted fluorescent light is maximally optimized, in other words little to none of the emitted light is reabsorbed by the fluorescent protein.
  • the fluorescent protein and fluorophore function as a Forster resonance energy transfer pair (FRET) where the fluorescent protein emits at the wavelength that the fluorophore absorbs at and the fluorphore then emits at a wavelength farther from the fluorescent proteins than could have been obtained with only the fluorescent protein.
  • FRET Forster resonance energy transfer pair
  • a functional combination may be lanthanide donors (for example europium cryptates) and phycobiliproteins, phycobiliproteins and sulforhodamine fluorophores or sulfonated cyanine fluorophores as known in the art.
  • the fluorophore sometimes functions as the energy donor and the fluorescent protein is the energy acceptor.
  • a label attached to an antibody or fragment thereof is a radioactive isotope. Examples of suitable radioactive materials include, but are not limited to, iodine
  • the complement system plays a central role in the clearance of immune complexes and in immune responses to infectious agents, foreign antigens, virus infected cells and tumor cells. Inappropriate or excessive activation of complement cascade can lead to uncontrolled C5a which then causes severe inflammation and tissue injury.
  • Harmful, and potentially life-threatening, consequences due to severe inflammation and resulting tissue destruction include various disorders such as bacterial sepsis, acute respiratory distress syndrome (ARDS), acute lung injury (ALI), asthma, myocardial and intestinal ischemia/reperfusion injury, graft rejection, organ failure, nephritis, age-related macular degeneration, diabetic retinopathy, pathological inflammation and autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, urticaria, psoriasis etc.
  • Increased in C5a levels in body fluids and tissue samples can be used as a biomarker diagnosis for C5a- mediated diseases and their severity.
  • anti-C5/C5a/C5adesR antibodies and fragments and compositions herein presented may be used in vivo and/or in vitro for diagnosing C5/C5a/C5adesR associated diseases.
  • diagnosis can be achieved, for example, by contacting a sample to be tested, optionally along with a control sample, with the antibody under conditions that allow for formation of a complex between the antibody and C5/C5a/C5adesR. Complex formation is then detected (e.g., using an ELISA).
  • complex is detected in both samples and any statistically significant difference in the formation of complexes between the samples is indicative of the presence of C5/C5a/C5adesR in the test sample.
  • technology provided herein allows for a method of determining the presence of C5/C5a/C5adesR in a sample suspected of containing C5/C5a/C5adesR, the method comprising exposing the sample to an anti-C5/C5a/C5adesR antibody or antigen binding fragment, and determining binding of the antibody to C5/C5a/C5adesR in the sample where binding of the antibody to C5/C5a/C5adesR in the sample is indicative of the presence of the C5/C5a/C5adesR in the sample.
  • the sample is a biological sample.
  • the biological sample is from a mammal experiencing or suspected of experiencing a
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment may be used to detect the overexpression or amplification of C5/C5a/C5adesR using an in vivo diagnostic assay.
  • the anti-C5/C5a/C5adesR antibody or fragment is added to a sample where the antibody binds the C5/C5a/C5adesR to be detected and is tagged with a detectable label ⁇ e.g., a radioactive isotope or a fluorescent label) and externally scanning the patient for localization of the label.
  • FISH assays such as the INFORMTM (sold by Ventana, Arizona) or PATHVISIONTM (Vysis, Illinois) may be carried out on formalin-fixed, paraffin-embedded tissue to determine the extent (if any) of C5/C5a/C5adesRoverexpression in the tumor.
  • the anti-C5/C5a/C5adesR antibodies and fragments may be used in a method of detecting soluble C5/C5a/C5adesR in blood or serum.
  • the method comprises contacting a test sample of blood or serum from a mammal suspected of experiencing a C5/C5a/C5adesR disorder with an anti-C5/C5a/C5adesR antibody or antigen binding fragment presented herein and detecting an increase in soluble C5/C5a/C5adesR in the test sample relative to a control sample of blood or serum from a normal mammal.
  • the method of detecting is useful as a method of diagnosing a C5/C5a/C5adesR disorder associated with an increase in soluble C5/C5a/C5adesR in blood or serum of a mammal.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment may be administered for prevention and/or treatment of a C5/C5a/C5adesR-mediated condition.
  • methods of preventing, treating, maintaining, ameliorating, and/or inhibiting a C5/C5a/C5adesR-mediated disease or disorder where the methods comprise administering an anti-C5/C5a/C5adesR antibody or antigen binding fragment provided herein.
  • compositions and antibodies as presented herein to treat and prevent a wide range of inflammatory conditions including both chronic and acute conditions, such as, but not limited to, cancer, bacterial sepsis, ARDS, ALI, Stargardt's disease, age-related macular degeneration, diabetic retinopathy, appendicitis, peptic, gastric and duodenal ulcers, peritonitis, pancreatitis, ulcerative,
  • the inflammatory disorder is COPD.
  • the inflammatory disorder is acute exacerbation chronic obstructive pulmonary disorder (AECOPD).
  • the technology herein provides methods of administering and using the disclosed compositions and antibodies to treat and prevent a wide range of autoimmune disease.
  • An "autoimmune disease” is a disease or disorder arising from and directed against an individual's own tissues or a co-segregate or manifestation thereof or resulting condition therefrom.
  • Autoimmune disease and related disorders that can be treated, prevented, or managed by methods and compositions herein presented include but are not limited to arthritis (rheumatoid arthritis such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails, atopy including atopic diseases such as
  • hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis,
  • granulomatosis including lymphomatoid granulomatosis, Wegener's granulomatosis,
  • vasculitides including vasculitis, large-vessel vasculitis (including polymyalgia rheumatica and giant-cell (Takayasu's) arteritis), medium-vessel vasculitis (including Kawasaki's disease and polyarteritis nodosa/periarteritis nodosa), microscopic polyarteritis, immunovasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis such as systemic necrotizing vasculitis, and ANCA-associated vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS) and ANCA-associated small-vessel vasculitis, temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA),
  • autoimmune polyendocrinopathies such as pemphigoid bullous and skin pemphigoid, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, and pemphigus erythematosus), autoimmune polyendocrinopathies, Reiter's disease or syndrome, thermal injury, preeclampsia, an immune complex disorder such as immune complex nephritis, antibody-mediated nephritis, polyneuropathies, chronic neuropathy such as IgM polyneuropathies or IgM-mediated neuropathy, thrombocytopenia (as developed by myocardial infarction patients, for example), including thrombotic thrombocytopenic purpura (TTP), post-transfusion purpura (PTP), heparin
  • neurologic paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis such as allergic encephalomyelitis or encephalomyelitis allergica and experimental allergic
  • EAE encephalomyelitis
  • myasthenia gravis such as thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant-cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphoid interstitial pneumonitis (LIP), bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular derma
  • a primary lymphocytosis which includes monoclonal B cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and channelopathies of the CNS, autism, inflammatory myopathy, focal or segmental or focal segmental glomerulosclerosis (FSGS), endocrine opthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome
  • monoclonal B cell lymphocytosis e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS
  • peripheral neuropathy e.g., benign monoclonal gammopathy and monoclonal
  • erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, flariasis, cyclitis such as chronic cyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic), or Fuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, SCID, acquired immune deficiency syndrome (AIDS), echovirus infection, sepsis, endotoxemia, pancreatitis, thyroxicosis, parvovirus infection, rubella virus infection, post-vaccination syndromes, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dor
  • keratoconjunctivitis sicca epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, transplant organ reperfusion, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway/pulmonary disease, silicosis, aphthae, aphthous stomatitis, arteriosclerotic disorders, aspermiogenese, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, endophthalmia
  • phacoanaphylactica enteritis allergica, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearing loss, haemoglobinuria paroxysmatica, hypogonadism, ileitis regionalis, leucopenia, mononucleosis infectiosa, traverse myelitis, primary idiopathic myxedema, nephrosis, ophthalmia symphatica, orchitis granulomatosa, pancreatitis, polyradiculitis acuta, pyoderma gangrenosum, Quervain's thyroiditis, acquired spenic atrophy, non-malignant thymoma, vitiligo, toxic-shock syndrome, food poisoning, conditions involving infiltration of T cells, leukocyte-adhesion deficiency, immune responses
  • cancers and related disorders that can be treated, prevented, or managed by methods and compositions of the present invention include but are not limited the following cancers: leukemias, such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias, such as, myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia leukemias and myelodysplastic syndrome; chronic leukemias, such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia;
  • leukemias such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias, such as, myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia leukemias and myel
  • lymphomas such as but not limited to Hodgkin's disease, non-Hodgkin's disease; multiple myelomas such as but not limited to smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and
  • extramedullary plasmacytoma Waldenstrom's macroglobulinemia; monoclonal gammopathy of undetermined significance; benign monoclonal gammopathy; heavy chain disease; bone and connective tissue sarcomas such as but not limited to bone sarcoma, osteosarcoma,
  • chondrosarcoma Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma; brain tumors such as but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary brain lympho
  • adenocarcinoma lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease, and inflammatory breast cancer
  • adrenal cancer such as but not limited to pheochromocytom and adrenocortical carcinoma
  • thyroid cancer such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer
  • pancreatic cancer such as but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor
  • pituitary cancers such as but limited to Cushing's disease, prolactin- secreting tumor, acromegaly, and diabetes insipius
  • eye cancers such as but not limited to ocular melanoma such as iris melanoma, choroidal melanoma,
  • choriocarcinoma yolk-sac tumor
  • prostate cancers such as but not limited to, prostatic
  • intraepithelial neoplasia adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma
  • penal cancers oral cancers such as but not limited to squamous cell carcinoma; basal cancers; salivary gland cancers such as but not limited to adenocarcinoma, mucoepidermoid carcinoma, and adenoidcystic carcinoma; pharynx cancers such as but not limited to squamous cell cancer, and verrucous; skin cancers such as but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acral lentiginous melanoma; kidney cancers such as but not limited to renal cell carcinoma, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell cancer (renal pelvis and/
  • myxosarcoma osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma and papillary adenocarcinomas (for a review of such disorders, see Fishman et al., 1985, Medicine, 2d Ed., J. B.
  • the methods and compositions of the invention are also useful in the treatment or prevention of a variety of cancers or other abnormal proliferative diseases, including (but not limited to) the following: carcinoma, including that of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, stomach, cervix, thyroid and skin; including squamous cell carcinoma;
  • hematopoietic tumors of lymphoid lineage including leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Burkitt's lymphoma; hematopoietic tumors of myeloid lineage, including acute and chronic myelogenous leukemias and promyclocytic leukemia; tumors of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; other tumors, including melanoma, seminoma, tetratocarcinoma, neuroblastoma and glioma; tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma, and schwannomas; tumors of mesenchymal origin, including fibrosarcoma, rhabdomyoscarama, and osteosarcoma; and other tumors, including melanoma
  • cancers caused by aberrations in apoptosis would also be treated by the methods and compositions of the invention.
  • Such cancers may include but not be limited to follicular lymphomas, carcinomas with p53 mutations, hormone dependent tumors of the breast, prostate and ovary, and precancerous lesions such as familial adenomatous polyposis, and myelodysplastic syndromes.
  • malignancy or dysproliferative changes are treated or prevented in the skin, lung, colon, breast, prostate, bladder, kidney, pancreas, ovary, or uterus.
  • compositions and antibodies of the invention also have analgesic properties.
  • they are appropriate as analgesics for treating and/or preventing pain associated with the diseases listed herein as well as chronic and acute pain resulting from or associated with wounds, medical procedures, surgeries, injury, trauma, etc.
  • the compositions and antibodies may be used as analgics post-surgery analgesics.
  • They may also be used to treat or prevent pain resulting from or associated with ankylosing spondylitis, inflammatory lower back pain, neuropathic pain, painful neuroma, fibromyalgia, headaches, e.g., chronic head aches and migraines, pancreatitis, spinal nerve compression syndromes and non-malignant skeletal pain, inflammatory osteoarthritic pain, rheumatoid arthritic pain, cancer pain, e.g., bone cancer pain.
  • ankylosing spondylitis inflammatory lower back pain
  • neuropathic pain painful neuroma
  • fibromyalgia fibromyalgia
  • headaches e.g., chronic head aches and migraines
  • pancreatitis e.g., spinal nerve compression syndromes and non-malignant skeletal pain
  • osteoarthritic pain e.g., rheumatoid arthritic pain
  • cancer pain e.g., bone cancer pain.
  • the technology herein provides methods of administering and using the disclosed compositions and antibodies to treat and prevent GVHD, humoral rejection, and post- transplant lymphoproliferative disorder in human transplant recipients, including a bone marrow transplant recipient, a liver transplant recipient, in a renal transplant recipient, cardiac transplant recipient, a lung transplant recipient .
  • the compositions and methods of the invention can be used regardless of the particular indications which gave rise to the need for a transplant.
  • the use of the compositions and methods of the invention for the treatment and prevention of GVHD, humoral rejection, and post-transplant lymphoproliferative disorders is not limited by the particular type of tissue which is intended for transplantation or which has been transplanted.
  • the invention provides compositions and methods for the prevention of humoral rejection in a human transplant recipient wherein the transplant recipient is identified as a patient or patient population at increased risk for developing a humoral rejection.
  • Such patients may also be referred to as "sensitized.”
  • the criteria for the identification of sensitized patients is known to the skilled practitioner. Such criteria may include, for example, patients having detectable levels of circulating antibodies against HLA antigens, e.g., anti-HLA alloantibodies. Such criteria may also include patients who have undergone previous transplantations, a pregnancy, or multiple blood transfusions. Patients who are at an increased risk for humoral rejection also include those having imperfect donor-recipient HLA matching, and those transplantations which are ABO- incompatible. Sensitized individuals are preferred candidates for pretreatment or conditioning prior to transplantation. Sensitized individuals are also preferred candidates for post-transplantation maintenance regimens for the prevention of humoral rejection.
  • the antibodies, compositions, and methods of the invention comprise or are used in combination with a therapeutic regimen for the treatment of an acute or chronic rejection.
  • the rejection is characterized as a Stage I, a Stage II, a Stage III, or a Stage IV humoral rejection.
  • the antibodies, compositions, and methods of the invention comprise or are used in combination with a therapeutic regimen for the treatment of an early stage humoral rejection.
  • the early stage humoral rejection is a Stage I, II, or III rejection.
  • Clinical indications of an early stage humoral rejection are determined according to the knowledge and skill in the art and may include, for example, the development in the patient of circulating donor-specific anti-HLA antibodies, the presence of complement markers of antibody activity such as C4d and C3d deposits in graft biopsies, and the presence of anti-HLA antibodies in graft biopsies.
  • MICA MHC class l-related chain A
  • compositions and methods of the invention comprise or are used in combination with a therapeutic regimen for the treatment of humoral rejection characterized in part by graft dysfunction.
  • the patient or patient population in need of treatment for humoral rejection is identified according to criteria known in the art for graft dysfunction.
  • the patient or patient population in need of treatment for humoral rejection is identified according to other criteria that are particular to the type of tissue graft, such as histological criteria.
  • the immunosuppression necessary for successful transplantation can give rise to a post-transplant lymphoproliferative disorder of B cell origin.
  • a post-transplant lymphoproliferative disorder is associated with Epstein-Barr virus infected cells.
  • Post-transplant lymphoproliferative disorder can range in severity from a benign self-limiting mononucleosis-like syndrome to an aggressive non-Hodgkins lymphoma.
  • the compositions and methods of the present invention may be used to treat PTLD arising from any transplant.
  • the transplant is a solid organ transplant, for example, a heart transplant, a liver transplant, a kidney transplant, or a combined kidney-pancreas transplant.
  • the compositions and methods of the invention are used to treat PTLD as part of a therapeutic regimen that includes a temporary cessation or reduction of other immunosuppressive therapy.
  • an anti-C5/C5a/C5adesR antibody or antigen binding fragment provided herein may be formulated with a pharmaceutically acceptable carrier for a pharmaceutical (therapeutic) composition, which may be administered by a variety of methods known in the art.
  • a pharmaceutical (therapeutic) composition which may be administered by a variety of methods known in the art.
  • the route and/or mode of administration may vary depending upon the desired results.
  • pharmaceutically acceptable carrier means one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredients.
  • Such preparations may contain one or more salts, buffering agents, preservatives, compatible carriers, excipients, diluents, stabilizers, lyoprotectants, saccharides, surfactants, solubilizers, polyols, lipophilic solvents, amino acids, chelators, preservatives, adjuvants and/or other therapeutic agents, and the like.
  • Such pharmaceutically acceptable preparations may also contain one or more compatible solid or liquid fillers, diluents, bulking agents and/or encapsulating substances which are suitable for
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the components of the pharmaceutical compositions also are capable of being co-mingled with the antibodies of the present technology, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
  • agents useful for formulating an antibody or fragment thereof e.g., carriers, excipients, adjuvants and other agents are known in the art.
  • a formulation herein is a liquid formulation, a lyophilized formulation, a reconstituted liquid
  • aqueous formulation a stable liquid formulation, an aqueous formulation, a non-aqueous formulation, or an aqueous formulation where the aqueous carrier is distilled water, for example.
  • a formulation herein comprises an anti-C5/C5a/C5adesR antibody or antigen binding fragment in a concentration resulting in a weight per volume (w/v) appropriate for a desired dose.
  • the anti-C5/C5a/C5adesR antibody or fragment is present in the formulation at a concentration of about 1 mg/ml to about 500 mg/ml.
  • the concentration of anti-C5/C5a/C5adesR antibody or fragment, which is included in the formulation is between about 1 mg/ml and about 25 mg/ml, between about 1 mg/ml and about 200 mg/ml, between about 25 mg/ml and about 200 mg/ml, between about 50 mg/ml and about 200 mg/ml, between about 75 mg/ml and about 200 mg/ml, between about 100 mg/ml and about 200 mg/ml, between about 125 mg/ml and about 200 mg/ml, between about 150 mg/ml and about 200 mg/ml, between about 25 mg/ml and about 150 mg/ml, between about 50 mg/ml and about 150 mg/ml, between about 75 mg/ml and about 150 mg/ml, between about 100 mg/ml and about 150 mg/ml, between about 125 mg/ml and about 150 mg/ml, between about 25 mg/ml and about 125 mg/ml, between about 50 mg/ml and about 150 mg/m
  • a formulation comprising an anti-C5/C5a/C5adesR antibody or antigen binding fragment may further comprise one or more active compounds suitable for the particular indication being treated, sometimes those with complementary activities that do not adversely affect each other.
  • a composition may include one or more anti-inflammatory agents and/or one or more agents useful for treating an autoimmune disorder.
  • additional active compound/s is/are suitably present in combination in amounts that are effective for the purpose intended.
  • a formulation may be isotonic with human blood and may have substantially the same osmotic pressure as human blood. Such isotonic formulations generally have an osmotic pressure from about 250 mOSm to about 350 mOSm.
  • Isotonicity can be measured by, for example, using a vapor pressure or ice-freezing type osmometer.
  • Tonicity of a formulation can be adjusted by the use of tonicity modifiers.
  • “Tonicity modifiers” often are pharmaceutically acceptable inert substances that can be added to a formulation to provide an isotonicity of the formulation.
  • Tonicity modifiers include, but are not limited to, saccharides, salts and amino acids.
  • a formulation is substantially free of endotoxins and/or related pyrogenic substances.
  • Endotoxins include toxins that are confined inside a microorganism and are released only when the microorganisms are broken down or die.
  • Pyrogenic substances also include fever- inducing, thermostable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. These substances can cause fever, hypotension and shock if administered to humans. Due to the potential harmful effects, even low amounts of endotoxins often are removed from intravenously administered pharmaceutical drug solutions.
  • the endotoxin and pyrogen levels in the composition are less then 10 EU/mg, or less then 5 EU/mg, or less then 1 EU/mg, or less then 0.1 EU/mg, or less then 0.01 EU/mg, or less then 0.001 EU/mg.
  • a formulation When used for in vivo administration, a formulation often is sterile.
  • a formulation may be sterilized by various sterilization methods, including sterile filtration, radiation, and the like.
  • an antibody formulation is filter-sterilized with a pre-sterilized 0.22-micron filter.
  • Sterile compositions for injection can be formulated according to conventional pharmaceutical practices, such as practices described in "Remington: The Science & Practice of Pharmacy", 21 st ed., Lippincott Williams & Wilkins, (2005), for example.
  • a formulation often is stored in lyophilized form or in solution.
  • a sterile formulation can be placed into a container having a sterile access port, an intravenous solution bag or vial having an adapter that allows retrieval of the formulation, such as a stopper pierceable by a hypodermic injection needle, for example.
  • compositions or formulation is provided as a pre-filled syringe.
  • Therapeutic compositions of the present technology may be formulated for a particular dosage. Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral and other compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • compositions of the present technology can be formulated for particular routes of administration, such as oral, nasal, pulmonary, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration.
  • routes of administration such as oral, nasal, pulmonary, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form often is in an amount of the composition which produces a therapeutic effect.
  • Formulations of the present technology suitable for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present technology may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level often depends upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present technology employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors known in the art.
  • antibodies as presented herein can be formulated to ensure proper distribution in vivo.
  • the blood-brain barrier excludes many highly hydrophilic compounds.
  • One strategy for addressing distribution in vivo is to formulate an antibody or fragment presented herein in a liposome. Methods of manufacturing liposomes are known.
  • Liposomes may comprise one or more moieties that are selectively transported into specific cells or organs, and thereby enhance targeted drug delivery, which sometimes are referred to as targeting molecules.
  • therapeutic molecules in the liposomes are delivered by bolus injection to a site proximal to a desired area (e.g., a site of inflammation).
  • a liposome composition often is in fluid form and can be administered by a convenient delivery technique (e.g., injection with a syringe and needle).
  • a liposome may be stable under conditions of manufacture and storage and may be preserved against contaminating action of microorganisms such as bacteria and fungi.
  • a therapeutic antibody composition may be administered with a suitable medical device.
  • Medical devices such as implants, delivery systems, and modules, are known, for example.
  • a therapeutic composition can be administered with a needleless hypodermic injection device.
  • implants and modules include: implantable micro-infusion pump (e.g., for dispensing medication at a controlled rate); variable flow implantable infusion apparatus ⁇ e.g., for continuous drug delivery); and osmotic drug delivery systems ⁇ e.g., having multi-chamber compartments).
  • a pharmaceutical pack or kit comprising one or more containers filled with a liquid formulation or lyophilized formulation herein.
  • a container filled with a liquid formulation herein is a pre-filled syringe.
  • the formulations herein comprise anti-C5/C5a/C5adesR antibodies and fragments recombinantly fused or chemically conjugated to another moiety, including but not limited to, a heterologous protein, a heterologous polypeptide, a heterologous peptide, a large molecule, a small molecule, a marker sequence, a diagnostic or detectable agent, a therapeutic moiety, a drug moiety, a radioactive metal ion, a second antibody, and a solid support.
  • the formulations herein are formulated in single dose vials as a sterile liquid.
  • the formulations may be supplied in 3 cc USP Type I borosilicate amber vials (West Pharmaceutical Serices - Part No. 6800-0675) with a target volume of 1 .2 ml_.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • a formulation herein is sometimes supplied in a pre-filled syringe.
  • a container filled with a liquid formulation herein is a pre-filled syringe.
  • a pre-filled syringe Any pre-filled syringe known in the art may be used in combination with a liquid formulation herein.
  • Pre-filled syringes that may be used are known in the art.
  • Pre-filled syringes may be made of various materials.
  • a pre-filled syringe is a glass syringe.
  • a pre-filled syringe is a plastic syringe. The nature and/or quality of the materials used for manufacturing the syringe may influence the stability of a protein formulation stored in the syringe.
  • a pre-filled syringe comprises a silicone based lubricant.
  • a pre-filled syringe sometimes comprises baked on silicone.
  • a pre-filled syringe is free from silicone based lubricants. Small amounts of contaminating elements leaching into the formulation from the syringe barrel, syringe tip cap, plunger or stopper may also influence stability of the formulation. For example, it is understood that tungsten introduced during the manufacturing process may adversely affect formulation stability.
  • a pre-filled syringe may comprise tungsten at a level above 500 ppb. In various embodiments, a pre-filled syringe is a low tungsten syringe. In some embodiments, a pre-filled syringe may comprise tungsten at a level between about 500 ppb and about 10 ppb, between about 400 ppb and about 10 ppb, between about 300 ppb and about 10 ppb, between about 200 ppb and about 10 ppb, between about 100 ppb and about 10 ppb, between about 50 ppb and about 10 ppb, between about 25 ppb and about 10 ppb.
  • kits comprising anti-C5/C5a/C5adesR antibodies and fragments are also provided that are useful for various purposes, e.g., research and diagnostic including for purification or immunoprecipitation of C5/C5a/C5adesR from test samples e.g. serum, plasma, bronchiolar lavage fluid (BAL) and sputum, detection of C5/C5a/C5adesR, and the like.
  • test samples e.g. serum, plasma, bronchiolar lavage fluid (BAL) and sputum
  • the kit may contain an anti-C5/C5a/C5adesR antibody or antigen binding fragment coupled to beads ⁇ e.g., sepharose beads).
  • Kits may be provided which contain the antibodies for detection and quantification of C5/C5a/C5adesR in vitro, e.g., in an ELISA or a western blot.
  • the kit comprises a container and a label or package insert on or associated with the container.
  • the container holds a composition comprising at least one anti-C5/C5a/C5adesR antibody or fragment as herein disclosed.
  • Additional containers may be included that contain, e.g., diluents and buffers, control antibodies.
  • the label or package insert may provide a description of the composition as well as instructions for the intended in vitro or diagnostic use.
  • the present technology also encompasses a finished packaged and labeled pharmaceutical product.
  • This article of manufacture includes the appropriate unit dosage form in an appropriate vessel or container such as a glass vial, pre-filled syringe or other container that is hermetically sealed.
  • the unit dosage form is provided as a sterile particulate-free solution comprising an anti-C5/C5a/C5adesR antibody or antigen binding fragment that is suitable for parenteral administration.
  • the unit dosage form is provided as a sterile lyophilized powder comprising an anti-C5/C5a/C5adesR antibody or antigen binding fragment that is suitable for reconstitution.
  • the unit dosage form is suitable for intravenous, intramuscular, intranasal, oral, topical or subcutaneous delivery.
  • the technology encompasses sterile solutions suitable for each delivery route.
  • the technology further encompasses sterile lyophilized powders that are suitable for reconstitution.
  • the packaging material and container are designed to protect the stability of the product during storage and shipment.
  • the products herein include instructions for use or other informational material that advise the physician, technician or patient on how to appropriately prevent or treat the disease or disorder in question.
  • the article of manufacture includes instruction means indicating or suggesting a dosing regimen including, but not limited to, actual doses, monitoring procedures, and other monitoring information.
  • the technology provides an article of manufacture comprising packaging material, such as a box, bottle, tube, vial, container, pre-filled syringe, sprayer, insufflator, intravenous (i.v.) bag, envelope and the like; and at least one unit dosage form of a pharmaceutical agent contained within the packaging material, where the pharmaceutical agent comprises a liquid formulation containing an antibody.
  • packaging material includes instruction means which indicate how that the antibody can be used to prevent, treat and/or manage one or more symptoms associated with a disease or disorder.
  • Example 1 Isolation of anti-C5/C5a/C5adesR neutralizing antibodies
  • scFv single chain Fv
  • Anti-C5/C5a/C5adesR specific scFv antibodies were isolated from the phage display libraries using a series of selection cycles on biotinylated recombinant human C5a (Sigma Aldrich C5788, biotinylated in house) essentially as previously described (Vaughan et al, 1996; Hawkins et al, 1992).
  • phage in selection buffer (30 mM HEPES, 5 mM potassium chloride, 0.1 mM magnesium chloride, 1.5 mM calcium chloride and 500 mM sodium chloride) containing 3% w/v Marvel were incubated with biotinylated human C5a for 1 hour at room temperature with gentle agitation. ScFv-phage bound to antigen were then captured on streptavidin-coated paramagnetic beads (Dynabeads® M-280) following the manufacturer's recommendations. Unbound phage were removed by a series of wash cycles using selection buffer containing Tween (0.1 % v/v). Bound phage particles were eluted, infected into E.
  • IC 50 values were determined by testing a series of dilutions of purified scFv for neutralization of iodinated human C5a (PerkinElmer, NEX250) binding C5aR expressed on HEK cell membranes (Example 26, Materials and Methods).
  • potency and species cross reactivity of purified scFv were determined in functional cell assays measuring calcium mobilization in response to human C5a (purified from serum, AstraZeneca), human C5adesR (Calbiochem 204902), cynomolgus C5a (purified from serum, AstraZeneca) and HIS Flag tagged cynomolgus C5adesR (mammalian expressed, AstraZeneca).
  • IC 50 values were determined by testing a series of dilutions of purified scFv for neutralization of C5a or C5adesR-mediated calcium mobilization from HEK cells co-transfected with the C5aR and Ga16 using a Fluorometric Imaging Plate Reader (FLIPR) as described in Example 26, Materials and Methods.
  • FLIPR Fluorometric Imaging Plate Reader
  • C5a is a potent neutrophil and monocyte/macrophage chemoattractant (Ward et al, 1969; Snyderman et al, 1971 )
  • the neutralization potency of mAb1 was subsequently characterized by chemotaxis assays performed using two cell types; hC5aR-transfected L1.2 cells (Campbell et al, 1996) and freshly isolated human neutrophils.
  • IC 50 values of mAb1 from both L1 .2 and neutrophil chemotaxis assays are shown in Table 1. These results demonstrated that mAb1 neutralizes human C5a induced chemotaxis of L1 .2 cells and neutrophils. No inhibition was observed with the isotype matched negative control antibody.
  • the neutralization potency of mAb1 lgG2 was also evaluated in a human whole blood model of inflammation (Fung et al, 2003).
  • human blood was collected using an anti-coagulant lepirudin that does not interfere with complement pathways.
  • MAb1 or an isotype matched negative control antibody was briefly mixed with whole human blood and the alternative pathway of complement was then activated by E. coli.
  • the C5a-induced activation of neutrophils in the blood was detected by the expression of cell surface CD1 1 b using flow cytometery as described in Example 26, Materials and Methods.
  • Example results shown in Table 1 demonstrate that mAb1 can inhibit native, endogenously produced C5a induced up-regulation of CD1 1 b on neutrophils. No effect was observed with the isotype matched negative control.
  • Table 2 IC 50 values of C5a, C5adesR, and C5 in the mAb1 binding biotinylated human C5a HTRF® competition assay
  • Example 4 Affinity and cross reactivity by BIAcore
  • K D binding affinity of the lead antibody, mAb1 , to human C5a (Sigma C5788), human C5adesR (Calbiochem 204902), and HIS Flag tagged cynomolgus C5adesR (mammalian expressed, AstraZeneca) was determined by surface plasmon resonance using a BIAcore 2000 biosensor (GE Healthcare) essentially as described by Karlsson et al (1991 ).
  • Protein G' Sigma Aldrich, P4689
  • the Protein G' surface was used to capture a constant amount of purified mAb1 lgG2 antibody or isotype matched control via the Fc domain.
  • Human C5a Sigma Aldrich C5788
  • C5adesR Calbiochem 204902
  • HIS FLAG tagged cynomolgus C5a (AstraZeneca) were prepared at a range of concentrations between 250 nM and 15.6 nM, in HBS-EP buffer (GE Healthcare). Dilutions of each analyte were injected over the antibody surface for 2 minutes, at a flow rate of 30 microlitres per minute before surface regeneration with 10 mM glycine, pH 1 .75, between each injection of antibody.
  • mAb1 lgG2 The specificity of mAb1 lgG2 to native, endogenous human and cynomolgus C5 and C5a was determined in an immunoprecipitation assay using freshly prepared human or cynomolgus plasma, as descibed in Example 26, Materials and Methods. Briefly, normal plasma (containing C5) and zymosan-activated plasma (containing C5 and C5a/C5adesR) were incubated with Dynal protein G beads that were pre-coated with mAb1 lgG2 or an isotype matched negative control antibody. After extensive washes, the bound C5 and C5a/C5adesR were eluted, separated by 4-12% PAGE gel, and transferred onto a nitrocellulose membrane.
  • the nitrocellulose membrane was subsequently probed with a biotinylated anti-C5/C5a dual specificity antibody (Quidel), followed by avidin-HRP detection.
  • the bound C5 and C5a/C5adesR proteins were visualized using a chemilluminescent substrate.
  • Figure 1 shows example results demonstrating that mAb1 lgG2 binds to native, endogenous human C5 (lane 2) and C5a/C5adesR (lane 8). This binding is specific as there is greater binding to mAb1 relative to the isotype matched negative control mAb (Lanes 3 and 9).
  • mAb1 lgG2 showed cross reactivity to native, cynomolgus monkey C5 and
  • FIG. 1 shows example results demonstrating that mAb1 lgG2 binds to native, cynomolgus monkey C5 (lane 2) and C5a (lane 8). This binding is specific as there is greater binding to mAb1 relative to the isotype matched negative control mAb (lanes 3 and 9).
  • Example 6 Target specificity Determination Using an HTRF® Competition Assay
  • the target specificity of mAb1 for C5a relative to complement components C3a and C4a was determined using the mAb1 HTRF® epitope competition assay described in Example 26, Materials and Methods, in which the binding of mAb1 lgG2 to biotinylated human C5a was measured.
  • a series of dilutions of human C3a (Calbiochem 204881 ), human C4a (Calbiochem 204887) and human C5a (Sigma C5788) were tested for competitive inhibition of mAb1 binding to biotinylated C5a.
  • Example results are shown in Table 4. No inhibition was observed with C3a or C4a, indicating that mAb1 is specific for C5a.
  • Table 4 IC 50 values of C3a, C4a and C5a in the mAbl binding biotinylated human C5a HTRF® competition assay
  • mAb1 showed no effect on the activation of the classical pathway (CP).
  • a representative result is shown in Figure 3.
  • the positive control an anti-C5 cleavage mAb
  • AP alternative pathway
  • rabbit red blood cells and normal human serum were used in the presence of GVB-Mg ++ -EGTA buffer.
  • Three experiments were performed. As seen with the classical pathway, mAb1 lgG2 and the isotype matched negative control showed no effect on the activation of the alternative complement pathway.
  • a representative result is shown in Figure 4.
  • the positive control an anti-C5 cleavage mAb shows dose-dependent inhibition of complement- mediated haemolysis as expected.
  • variable heavy chain (VH) and variable light chain (VL) domains of mAb1 were aligned to the known human germline sequences in the VBASE database
  • VH domains of mAb1 this was Vh3_DP-47(3-23) and for the VL domains it was V-lambda-2_DPL1 1.
  • Site directed mutagenesis was used to introduce an amino acid change from arginine at Kabat (Kabat et al, 1991 ) position 79 in the VL domain to glutamine. Vernier residues (Foote et al 1992) were left unchanged.
  • the potency of mAb1 and the closest human germline antibody mAb2 was determined by testing a series of dilutions of purified scFv for inhibition in the mAb1 lgG2 binding biotinylated human C5a HTRF® epitope competition assay (described in Example 26, Materials and Methods).
  • Example potencies are provided in Table 5.
  • variable light chain domain of mAb2 which contains a reversion to a glutamine residue at position 79 in the VL domain, was used in the construction of all light chain libraries for Lead Optimization.
  • MAb2 was optimized for improved affinity to human C5a using a targeted mutagenesis approach and affinity-based phage display selections.
  • Large scFv-phage libraries derived from mAb2 were created by oligonucleotide-directed mutagenesis of the variable heavy (VH) and light (VL) chain complementarity determining regions (CDRs) using standard molecular biology techniques as described by Clackson and Lowman, 2004. Individual libraries were built targeting CDR2 and 3 of the variable heavy chain and CDR3 of the variable light chain. The libraries were subjected to affinity-based phage display selections in order to select variants with higher affinity for C5a.
  • VH variable heavy
  • VL light chain complementarity determining regions
  • the selected scFv-phage particles were then rescued as described previously (Osbourn et al, 1996) and the selection process was repeated in the presence of decreasing concentrations of biotinylated human C5a (200 nM to 200 pM over 4 rounds of selections).
  • VH CDR3 randomised libraries were recombined with either VH CDR2 or VL CDR3 libraries to form two individual libraries.
  • the first library contained clones with a random pairing of individually randomised VH CDR2 and VH CDR3 mutations, while the second library consisted of clones with randomly paired VH and VL CDR3 randomised sequences. Selections were then continued with both libraries, in the presence of decreasing concentrations of biotinylated human C5a (from 200 pM to 20 pM over two rounds of selection) as previously described.
  • a representative number of approximately 5000 individual scFv clones from the selection outputs were grown up in 96-well plates and scFv were expressed in the bacterial periplasm. Unpurified periplasmic scFv were then screened either for increased inhibition compared to the parent mAb1 , or an optimized variant of mAb1 ⁇ e.g., mAb5) in HTRF® epitope competition assays or for reduced dissociation rates compared to mAb5 using BIAcore.
  • Such epitope competition assays are based on the principle that a test periplasmic scFv antibody sample, which recognises a similar epitope to the lead C5a neutralising antibody, will compete with the lead antibody for binding to biotinylated C5a resulting in a reduction in assay signal.
  • Antibodies with increased affinity were identified as giving greater inhibition of lead IgG binding to either biotinylated human C5a (Sigma C5788), and/or HIS Flag tagged cynomolgus C5adesR (mammalian expressed, AstraZeneca) relative to mAb1 scFv or an optimized scFv identified from earlier rounds of screening.
  • biotinylated human C5a Sigma C5788
  • HIS Flag tagged cynomolgus C5adesR mimmalian expressed, AstraZeneca
  • Example 11 Identification of Optimized scFv using BIAcore Following recombination of VH and VL CDR3 mutations, further improvements in affinity of optimized antibodies was detected using an 'off-rate' (k d ) screen by BIAcore.
  • biotinylated human C5a Sigma Aldrich C5788, biotinylated in house
  • IC 50 values were determined by testing serial dilutions of purified scFv for competitive inhibition of the binding of biotinylated human C5a (Sigma C5788, modified in house) or His Flag tagged cynomolgus C5adesR (mammalian expressed, AstraZeneca) to optimized lgG2, in HTRF® biochemical epitope competition assays (described in Example 26, Materials and Methods).
  • Optimized lgG2 used in the assays were either mAb5 or mAb9 identified from earlier rounds of selections.
  • Table 6 Examples of optimized scFv IC 50 values in the HTRF® epitope competition assays measuring the inhibition of the binding of biotinylated human C5a or His Flag tagged cynomolgus C5adesR to optimized lead antibody mAb5
  • Table 7 Examples of optimized scFv IC 50 values in the HTRF® epitope competition assays measuring the inhibition of the binding of biotinylated human C5a or HIS Flag tagged cynomolgus C5adesR to optimized lead antibody mAb9
  • Example 13 Potency of Optimized scFv in C5a-mediated Calcium Mobilization Assays The IC 50 values of the most potent/highest affinity optimized scFv were then determined in the in vitro C5a mediated calcium mobilization assays as described in Example 26, Materials and Methods. Purified scFv neutralization of serum purified human C5a (AstraZeneca) and mammalian expressed HIS Flag tagged cynomolgus C5adesR (AstraZeneca) was assessed in the C5a- mediated calcium mobilization assays (described in Example 26, Materials and Methods) using HEK-293 cells co-transfected with the C5aR and Galpha16. Example potency results for the parent anitbody mAb1 and a panel of optimized scFv are summarized in Tables 8 and 9.
  • Table 8 Examples of parent and optimized scFv potencies in the C5a/C5adesR mediated calcium mobilization assays 3
  • Antibodies were reformatted to lgG2 and lgG4 isotypes to minimize the activation and binding of complement C1 q (Burton et al, 1992), as described in Example 26, Materials and Methods.
  • S228P serine #228 to proline mutation
  • Example 15 Effect of optimised antibodies on C5a and C5adesR binding to the C5aR and C5L2 receptors.
  • C5a binds two receptors, C5aR (CD88) and the C5L2 receptor (gpr77).
  • C5L2 receptor binds C5adesR with a 10 fold higher affinity than C5aR (Monk and Cain, 2002).
  • the functional responses elicited by C5a through the C5aR are well characterised, however the C5a and C5adesR mediated signalling properties of the C5L2 receptor are less well defined.
  • Example 16 Potency of Optimized IgG in HTRF® Epitope Competition Assays The potencies of optimized antibodies were determined by testing a series of dilutions of purified IgG (lgG2 and lgG4 S228P formats) in in vitro biochemical assays.
  • Example results are summarized in Table 1 1 and demonstrated that the optimised antibody panel can bind to human C5a with improved potency relative to the parent antibody mAbl and that the antibodies cross react with cynomolgus C5adesR.
  • Table 1 1 Examples of mAb1 and optimized IgG IC 50 values in the HTRF® epitope competition assays for blocking the binding of biotinylated human C5a or HIS Flag tagged cynomolgus C5adesR to optimized lead antibody mAb9 lgG 2 .
  • Geomean IC 50 values are shown where two or more experiments have been performed. Data from three or more separate experiments are shown with 95% confidence intervals in brackets. A range of ligand concentrations was used as described in Example 26 Materials and Methods.
  • IgG derivates of mAbl can neutralize human C5a, human C5adesR, cynomolgus C5a and HIS Flag tagged cynomolgus C5adesR-mediated calcium mobilization in C5aR-transfected HEK cells, with greater potencies than the parent antibody mAbl .
  • Some of the optimized IgG, for example mAb9 and mAbl 2 can completely inhibit calcium mobilisation at a molar ratio of 1 :1 between the antibody and C5a/C5adesR.
  • Example 18 Potency of Disclosed Optimized IgG in C5a-mediated Neutrophil Chemotaxis Assay
  • the functional neutralization potencies of optimized IgG were determined in the neutrophil chemotaxis assay as described in Example 26, Materials and Methods, using freshly isolated human neutrophils. Representative data shown in Table 13 demonstrates that the optimized IgG show increased C5a neutralization potency relative to the parent antibody mAb1.
  • the disclosed optimized IgG antibodies completely inhibit chemotaxis of human neutrophils at a molar ratio of 1 :1 between the antibody and human C5a.
  • Table 13 IC 50 values of mAb1 and optimized IgG antibodies in the neutrophil chemotaxis assay
  • Optimized IgG antibodies were also tested for potency and specificity in both human and cynomolgus monkey whole blood assays measuring C5a induced neutrophil CD1 1 b expression (described in Example 26, Materials and Methods).
  • Optimized antibodies mAb9 lgG2, mAb9 lgG4 S228P and mAb12 lgG4 S228P were briefly pre-incubated with lepirudin anti-coagulated human whole blood prior to activation of the alternative complement pathway using E. coli.
  • C5a-induced activation of neutrophils in the blood was then detected by measuring the expression of cell surface CD1 1 b using flow cytometery.
  • Example results in Table 14 show that mAb9 lgG2, mAb9 lgG4 S228P and mAb12 lgG4 S228P inhibit C5a-induced activation of neutrophils in human whole blood, demonstrating that these mAbs neutralize endogenously produced human C5a in the presence of physiologically relevant concentrations of C5.
  • pre-incubation of mAb9 lgG2 with cynomolgus whole blood also led to inhibition of E. co//-induced activation of neutrophils ( Figure 5), demonstrating that mAb9 lgG2 can neutralize endogenously produced cynomolgus C5a.
  • Table 14 Example IC 50 values of optimized antibodies in human whole blood assays
  • mAb9 lgG4 S228P was also tested for potency in a modified version of this assay in which antibody and E. coli were added to human whole blood simultaneously prior to measuring C5a induced neutrophil CD1 1 b up-regulation (i.e., no pre- incubation of antibody with whole blood).
  • Example results in Figures 6 and 7 demonstrated that mAb9 lgG4 S228P can neutralize C5a-induced neutrophil activation when pre-incubated with whole blood or when added to whole blood simultaneously with E. coli, respectively.
  • Figures 6 and 7 also showed that the positive control antibody (Kaplan, 2002), an anti- C5 mAb (Eculizumab) which blocks cleavage of C5 to C5a and C5b, was less efficient at blocking C5a-mediated neutrophil activation when it is added to whole blood at the same time as E. coli.
  • mAb9 binds to the C5a portion of C5 and thus neutralizes C5a before it is formed upon C5 cleavage when complement cascade is activated.
  • Example 20 Affinity and Cross Reactivity by BIAcore or KinExa
  • the anti-C5/C5a/C5adesR antibody or an isotype matched control antibody was covalently coupled by amine linkage to a proprietary CM3 chip surface to a final surface density of
  • Each analyte to be assessed was prepared at a range of concentrations from 20 nM to 156 pM in HBS-EP+ buffer (GE Healthcare). Analytes were injected sequentially over the chip surface for 2 minutes and the dissociation phase of the interaction was monitored for 1000 seconds with continuous buffer flow at 50 microlitres per minute. The chip surface was regenerated of C5a analytes between cycles, using a 30 second injection of 10 mM glycine pH 2, without significant loss of IgG binding activity. Blank reference flow cell data was subtracted from each IgG dataset and a zero-concentration buffer blank was double-reference subtracted from the main data set to reduce any buffer artefacts.
  • Table 15 Example kinetic data for the optimized C5/C5a/C5adesR antibody mAb9 lgG4 S228P using BIAcore T100 instrument
  • K D solution phase affinity
  • mAb9 lgG4 S228P for bacterially expressed human C5a (Sigma) and serum purified human C5 (Complement Technology) was determined using a KinExa instrument (Sapidyne Instruments).
  • mAb9 lgG4 S228P was pre-mixed with varying concentrations of each ligand until equilibrium was reached.
  • the amount of free antibody was then measured using the KinExa by capturing free mAb9 lgG4 S228P using C5a or C5 coated beads, washing away unbound material and detecting bound antibody using a fluorescently labelled species specific antibody.
  • the cross reactivity of optimized antibodies to human C5a, cynomolgus C5a (including variants), murine C5a, human and cynomolgus C5adesR and human C5 was assessed in HTRF® competition assays.
  • the degree of cross reactivity was determined by testing a series of dilutions of hC5a/C5adesR/C5 samples for competitive inhibition of biotinylated human C5a binding to optimized antibodies mAb9 (lgG4 S228P and lgG2 formats) or mAb12 lgG4 S228P (Example 26, Materials and Methods).
  • Example IC 50 results are summarised in Table 16 and indicate that the optimized antibodies mAb9 lgG4 S228P and mAb12 lgG4 S228P bind human C5a, C5adesR and C5 with similar potencies (within 4 fold).
  • optimized antibodies mAb9 and mAb12 recognise cynomolgus C5a and C5adesR with similar potencies to human C5a (within 4 fold).
  • MAb9 lgG4 S228P or mAb12 lgG4 S228P (GL) did not bind murine C5a.
  • a polymorphism at amino acid residue 61 was identified by amino acid sequencing, comprising of either a leucine or phenylalanine variant.
  • the cross reactivity of optimized antibodies mAb9 lgG4 S228P or mAb12 lgG4 S228P (GL) to the two cynomolgus C5a variants was assessed using biochemical HTRF® competition assays in which the binding of mAb9 lgG4 S228P or mAb12 lgG4 S228P to HIS Flag tagged cynomolgus C5adesR Leu61 (expressed from mammalian cells, AstraZeneca) was measured (see Example 26, Materials and Methods).
  • Table 17 Example IC 50 values of cynomolgus C5a variants in the mAb9 and mAb12 binding Flag tagged cynomolgus C5adesR HTRF® competition assays
  • mAb9 lgG2, mAb9 lgG4 S228P and mAb12 lgG4 S228P bind to native human C5 (lanes 2, 3, and 4, respectively, left panel) and endogenous C5a (lanes 2, 3, and 4, respectively, right panel) generated by activation of human plasma with zymosan.
  • the signal intensities of the optimized antibodies are greater than that obtained with the parental antibody mAb1 (comparing lane 1 against lanes 2, 3 and 4), in accordance with an increase in affinity of the optimized antibodies.
  • mAb9 (lgG2 and lgG4 S228P formats) and mAb12 lgG4 S228P bind to native cynomolgus C5 ( Figures 9A and 9B, lanes 2, 3 and 4 respectively, left panel) in cynomolgus plasma and native cynomolgus C5a ( Figures 9A and 9B, lanes 2, 3 and 4 respectively, right panel) in zymosan activated cynomolgus plasma.
  • the signal intensities of the optimized antibodies are greater than that obtained with the parental antibody mAb1 in accordance with an increase in affinity of the optimized antibodies to cynomolgus C5/C5a.
  • Example 24 Target Binding Specificity of Optimized Antibodies in DELFIA® Assays
  • Example IC 50 results are summarised in Table 18 and demonstrated that the optimized antibodies mAb9 lgG4 S228P and mAb12 lgG4 S228P are selective for C5a, C5adesR and C5 as they do not recognise other homologous complement components.
  • Example 25 Effect of Optimized Antibodies on C5 Cleavage
  • the optimized antibodies, mAb9 lgG2, mAb9 lgG4 S228P, and mAb12 lgG4 S228P were evaluated for their effect on the activation of both classical and alternative complement pathways, using the haemolytic assays described in Example 26, Materials and Methods. Representative data for mAb9 and mAb12 lgG4 S228P are shown in Figures 10 and 1 1. In all experiments performed mAb9 lgG2, mAb9 lgG4 S228P and mAb12 lgG4 S228P showed no effect on the activation of classical or alternative complement pathways. This indicates that these antibodies do not prevent the cleavage of C5 to C5a and C5b upon activation of the complement cascade, thus leaving the pathways for formation of the membrane attack complex intact.
  • Native human C5a and cynomolgus C5a were purified from human and cynomolgus serum from the CPU unit (AstraZeneca R&D Lund, Sweden) and GeneTex Inc. (Irvine, California), respectively.
  • the endogenous C5a proteins were purified at AstraZeneca R&D Lund, Sweden, with minor changes according to Janatova (1988).
  • the clarified solution was dialyzed into 0.05 M NH 4 CHO pH 6.0 and purified by IEX SepharoseTM Fast Flow SP (GE Healthcare) chromatography in a 0.1 -0.8 M NH 4 CHO pH 6.0 gradient over 4 column volumes.
  • the C5a containing fractions were buffer exchanged into 0.1 M NH 4 CHO pH 6.0 on a HiTrap desalting column (GE Healthcare) and applied to Resource S HiTrap column (GE Healthcare) equilibrated in 0.1 M NH 4 CHO, pH 6.0 and then washed with 0.1 M NH 4 CHO pH 7.0 and eluted in a 0.1 -0.4 M NH4CHO, pH 7.0 gradient over 30 column volumes.
  • the C5a containing fractions were
  • This protocol routinely produced >95% pure monomeric glycosylated C5a as determined by SDS-PAGE, mass-spectrometry and analytical gel filtration, with yields ranging from 0.2-1 milligrams pure protein/Litre serum.
  • the cynomolgus serum derived C5a contained two different protein variants with either Leu or Phe at position 61 as determined by mass-spectrometry. Protein sequences for human and cynomolgus C5a are shown herein. N-terminal 10xHis-Flag tagged cynomolgus C5adesR was expressed in HEK293/EBNA cells at AstraZeneca R&D Lund, Sweden.
  • Cynomolgus C5adesR was cloned from cynomolgus liver and thymus cDNA libraries by RT-PCR and the sequences were identical in all analyzed clones.
  • DNA sequence encoding cynomolgus C5adesR was cloned using standard molecular biology techniques into vectors containing the signal peptide of the human growth hormone to facilitate secretion of the tagged C5adesR into the growth media.
  • the growth media were concentrated in a Pellicon XL ultra filtration device with a 5 kDa MW cut-off (Millipore) and clarified by centrifugation at 1000xg for 10 minutes prior adjustment of pH to 8.0.
  • the concentrated media were batch affinity purified with Ni-NTA agarose (Invitrogen) equilibrated in 25 mM HEPES, 500 mM NaCI, 20 mM imidazole, 5 mM DTT, 10% glycerol, pH 8.0 supplemented with Complete EDTA-free protease inhibitor (Roche) and eluted into the same buffer containing 300 mM imidazole.
  • the C5adesR containing fractions were pooled and applied to a Superdex 75 (GE Healthcare) size exclusion column equilibrated in 2x PBS buffer (Medicago, Uppsala, Sweden) supplemented with 10% glycerol.
  • C5adesR The purity and identity of the C5adesR preparation was confirmed by anti-C5a western blots, Edman N-terminal sequencing and mass-spectrometry, respectively.
  • This protocol routinely produced >95% pure monomeric glycosylated C5adesR as determined by SDS-PAGE, mass- spectrometry and analytical gel filtration with yields ranging from 4-10 milligrams pure protein/Litre growth media. Protein and cDNA sequences for 10xHis-Flag tagged cynomolgus C5adesR are shown herein.
  • SPA Scintillation Proximity Assay
  • the emitted radiation (beta-particles for 3 H or Auger electrons for 125 l) will stimulate the scintillant to produce light that can be detected using a scintillation counter. Any unbound radiolabeled ligand will be too distant from the scintillant to allow the energy transfer, resulting in no signal from unbound ligand. This means there is no requirement for separation of bound and unbound radiolabeled ligand, therefore the assay is homogeneous. Inhibitors of the receptor-radiolabelled ligand interaction will prevent the radiolabel coming into close proximity to the scintillant and will be identified by a reduction in the assay signal.
  • the SPA assay described herein measures the binding of iodinated C5a to C5aR expressed on the surface of HEK 293 membrane preparations. The membrane preparations are captured on the surface of wheat germ agglutinin coated SPA beads.
  • Selection outputs were screened in the receptor-ligand competition assay as single point, diluted, unpurified scFv from periplasmic extracts prepared in; 50 mM MOPS (Sigma M9381 ) buffer pH7.4, 0.5 mM EDTA (BDH 100935V) and 0.5 M sucrose (BDH 102745C). Fifty microlitres of scFv sample was added to a 96 well OptiplateTM (PerkinElmer, 6005290).
  • SPA beads wheat germ agglutinin (polyvinyl toluene) SPA beads (GE Healthcare, RPNQ0001 ) at 1 milligram/millilitre coated with HEK 293 membranes expressing the C5aR.
  • SPA beads were incubated with 25 micrograms/ millilitre membrane preparations for 1 hour at 4 degrees Celsius with continuous mixing.
  • SPA beads were centrifuged and resuspended in fresh buffer to remove unbound membrane from the solution.
  • Twenty five microlitres of 400 pM iodinated C5a (PerkinElmer, NEX250) was then added.
  • assay buffer comprised of 25 mM HEPES buffer (Sigma, H4034) containing 1 mM magnesium chloride (BDH, 101494V), 2 mM calcium chloride (BDH, 190464K) and 0.2% bovine serum albumin (BSA, Sigma A9576) adjusted to pH 7.4.
  • Assay plates were incubated for 3 hours at room temperature, in the dark, prior to reading scintillation using a 2 minutes per well count time on a Topcount plate reader (Perkin Elmer). Data was analyzed by calculating % specific binding as described in Equation 1 , where the non-specific binding (NSB) was defined by the addition of 20 nM unlabelled C5a.
  • IC 5 o values were estimated in the assay by adding 50 microlitres of a series of dilutions of test samples to the assay plate and following the method described above. IC 50 values were determined using
  • Equation 2 GraphPad Prism software by curve fitting using a four-parameter logistic equation
  • X is the logarithm of concentration
  • Y starts at Bottom and goes to Top with a sigmoid shape.
  • variable heavy chain VH
  • VL variable light chain
  • the variable heavy chain was cloned into a mammalian expression vector containing the human heavy chain constant domains and regulatory elements to express whole lgG2 heavy chain in mammalian cells.
  • the variable light chain domain was cloned into a mammalian expression vector for the expression of the human lambda light chain constant domains and regulatory elements to express whole IgG light chain in mammalian cells.
  • the heavy and light chain IgG expressing vectors were transiently transfected into HEK293-EBNA mammalian cells where the antibody was expressed and secreted into the medium. Harvested media was clarified by centrifugation prior to purification. The IgG was purified using Protein A (GE Healthcare). Culture supernatants were loaded onto a 1 ml column pre-equilibrated in 50 mM Tris, 0.15M NaCI, pH 8 buffer. The IgG was eluted from the column using 0.1 M Citrate pH 3 directly into 1 M Tris pH 10.
  • the eluates underwent buffer exchange using NAP-10 buffer exchange columns (GE Healthcare) into 1 x Dulbeccos PBS (DPBS).
  • the purified IgG was 0.2 micrometers sterile filtered, analyzed for endotoxin, characterized by SDS PAGE and the concentration determined by absorbance at 280 nm.
  • G alpha 16 is a 'promiscuous' G protein that is capable of linking a number of predominantly Gq-, Gs- and Gi-coupled receptors to the phospholipase C and calcium mobilization pathways. G alpha 16 therefore acts by coupling the C5aR (Gi coupled GPCR) to intracellular calcium pathways (Kostenis et al, 2005).
  • HEK293 transfected cells were maintained in media containing G418 to maintain stable receptor expression.
  • Cells were exposed to human or cynomolgus C5a or C5adesR in the presence or absence of test antibody and calcium mobilization detected using a Fluorometric Imaging Plate Reader (FLIPR®, Molecular Devices).
  • FLIPR® Fluorometric Imaging Plate Reader
  • C5aR and Ga16 co-transfected HEK293 cells were seeded at 5x10 4 cells/well (100 microlitres per well) in DMEM culture medium (Invitrogen 41966) containing 10% v/v FBS (heat inactivated) and 1 milligram/millilitre G418 (Invitrogen 10131 -027) into 96 well black-walled, Poly-D-Lysine coated plates (Beckton Dickinson 9286) using SelecT automation (TAP).
  • DMEM culture medium Invitrogen 41966
  • 10% v/v FBS heat inactivated
  • G418 Invitrogen 10131 -027
  • Fluo-4AM dye loading solution Component C from the Fluo-4 NW Calcium Assay kit, Invitrogen, F36206, prepared according to the manufacturer's instructions. Cells were loaded with dye by incubation at 37 degrees Celsius, 5% C0 2 for 1 -1.5 hours.
  • concentrations and added to the test sample dilutions in a total volume of 60 microlitres were determined in earlier experiments and based on the signal to noise ratio obtained. A range of ligand concentrations were therefore used in the calcium mobilization assays as follows: 70 pM to 1 15 pM human C5a, 80 pM to 200 pM cynomolgus C5a, 300 pM to 400 pM human C5adesR and 3000 pM - 8000 pM cynomolgus C5adesR.
  • Optimized antibodies were characterized using the FLIPRTETRA®.
  • C5aR and G-alpha-16 co- transfected HEK293 cells were seeded at 2.6x10 4 cells/well (fifty microlitres per well) in DMEM culture medium containing 10% v/v FBS (heat inactivated) and 1 milligram/millilitre G418 into 384 well poly-D-lysine black-walled, flat-bottomed plates (Greiner 781946) using SelecT automation (TAP).
  • TEP SelecT automation
  • Fluo-4AM dye loading solution Component C from the Fluo-4 NW Calcium Assay kit, Invitrogen, F36206, prepared according to the manufacturer's instructions. Cells were loaded with dye by incubation at 37 degrees Celsius, 5% C0 2 for 1 -1.5 hours.
  • C5a or C5adesR ligand used in each assay was similar to those described for lead isolation.
  • Ligand and test sample were mixed and incubated for 30 minutes at room temperature and then 15 microlitres of the mix was transferred to the dye-loaded cells using the FLIPRTETRA®. Calcium mobilization and IC 50 determination were carried out as described for lead isolation.
  • L1 .2 cells are mouse pre-B cells that were transfected with human C5aR (Campbell et al, 1996). The cells were maintained in RPMI media (Invitrogen, 030078DJ) with 10% heat-inactivated FBS (Invitrogen, 26140-079) and 400 micrograms/ millilitre Geneticin (Invitrogen, 10131 -035).
  • Human neutrophils were isolated from blood of healthy donors. Human blood was first centrifuged in LSM (MP Biomedicals, 50494X) as described by the manufacturer. The gradient including peripheral blood leukocytes (PBLs) was removed and the red blood cell pellet was saved. The pellet was resuspended in 1x PBS to bring the volume to the original blood volume. Dextran T-500/saline solution was then added to the final concentration of 1 % v/v (Indofine Chemical Company,
  • the cells were incubated at room temperature for approximately 20 minutes or until a clearly defined surface layer appeared. The upper portion was transferred to a 50 ml- polypropylene centrifuge tube and washed once with PBS.
  • the red blood cells were lysed by the addition of 1.8 millilitres of H 2 0 for 20 seconds (using pipette to mix), followed by 0.2 ml 10x PBS to adjust PBS concentration to 1 x PBS, then 40 ml 1 x PBS was added. Cells were washed 3 times and counted. The neutrophils were obtained with purity >97% and viability >95%.
  • the chemotaxis assays were carried out in 96 well plates (NeuroProbe, 106-5). Purified antibodies were 3-fold diluted with migration buffer, 0.5% BSA (Invitrogen, E01-40001 Q), 10 mM HEPES (Invitrogen, 15630-080) in HBSS (Invitrogen, 14175). The recombinant human C5a (Sigma,
  • IC 50 values were determined using GraphPad Prism software by curve fitting using a four- parameter logistic equation as described in Equation 2.
  • bacteria were used to activate the alternative complement pathway.
  • An E .coli (ATCC, LE392) culture was centrifuged and bacteria washed with 1 x PBS. Washed bacteria were then heat-inactivated in a 60 degrees Celsius water bath for 1 hour and stored at -80 degrees Celsius. Before using, the bacteria were washed 9 times to remove debris.
  • Human blood from a healthy donor (10 millilitres) was collected into a red top vacutainer (BD, 366430) containing 10 microlitres anticoagulant, lepirudin (Bayer) at a final concentration of 50 micrograms/millilitre. The blood was then transferred to a polypropylene tube and kept at room temperature.
  • BD red top vacutainer
  • Purified antibodies (lgG2 or lgG4 S228P formats) were 3-fold diluted with 1x PBS and 10 microlitres was transferred to each well of a 96-well polypropylene plate (Costar, CLS3790). Eighty microlitres of blood was then added to each well from a polypropylene reagent reservoir (VWR, 70341 1 ) using a multi-channel pipette. The plate was vortexed briefly on a 96-well plate shaker and incubated in a 37 degrees Celsius water bath. After 4 minutes incubation, 10 microlitres of E. coli (2x107 CFU) were added to each well.
  • the plate was vortexed and incubated in a 37 degrees Celsius water bath for another 10 minutes with mixing every three minutes.
  • Ten microlitres of PE-anti- CD1 1 b/Mac1 antibody (BD, 555388) was added to each well and incubated at room temperature in the dark for 10 minutes.
  • the blood alone, blood plus PE-anti-CD1 1 b/Mac1 antibody, blood plus E. coli and PE-anti-CD1 1 b/Mac1 antibody (P10, maximal activation) and blood plus PE-anti- CD1 1 b/Mac1 antibody (P0, basal state) were included as controls.
  • One hundred and fifty microlitres of FACS lysing buffer (BD, 349202), was added to each well and the plate incubated for 20 minutes at room temperature.
  • Equation 4 The mean fluorescence intensity was determined and used to calculate the percentage of inhibition as described in Equation 4: Equation 4:
  • An HTRF® assay is a homogeneous assay technology that utilizes fluorescence resonance energy transfer between a donor and acceptor fluorophore that are in close proximity (Mathis 1995). Such assays can be used to measure macromolecular interactions by directly or indirectly coupling one of the molecules of interest to a donor fluorophore, europium (Eu3+) cryptate, and coupling the other molecule of interest to an acceptor fluorophore XL665, (a stable cross linked
  • the degree of cross reactivity was determined by titrating C5a/C5adesR/C5 samples into assays in which the binding of biotinylated human C5a (Sigma C5788, modified in-house) to mAb1 lgG2, mAb9 (as lgG4 S228P and lgG2) or mAb12 lgG4 S228P was measured.
  • assay buffer comprised of Dulbeccos PBS (Invitrogen, 14190185) containing 0.8 M potassium fluoride (BDH 103444T) and 0.2% bovine serum albumin (BSA, Sigma A9576). Assay plates were incubated for 4 hours at room temperature before reading time resolved fluorescence at 620 nm and 665 nm emission wavelengths using an EnVision plate reader (Perkin Elmer).
  • Equation 5 Equation 5:
  • the negative control (non-specific binding) is defined by replacing biotinylated C5a with assay buffer.
  • Equation 7 The % Delta F values were subsequently used to calculate % specific binding as described in equation 7: Equation 7:
  • the same assay was also used to assess the selectivity of mAb1 to C5a over complement components C3a and C4a, by titrating purified human C3a and C4a into the assay.
  • test sample dilutions 5 microlitres were added to 384 well low volume assay plates followed by the addition of 5 microlitres of 2 or 1 nM biotinylated human C5a for the mAb9 lgG4 S228P and the mAb12 lgG4 S228P assays respectively.
  • the cross reactivity of mAb9 and mAb12 to variants of cynomolgus C5a was assessed in HTRF® competition assays in which the binding of optimized antibodies mAb9 lgG4 S228P and mAb12 lgG4 S228P to HIS Flag tagged cynomolgus C5a Leu61 (mammalian expressed, AstraZeneca) was measured.
  • Cross reactivity was assessed by titrating untagged cynomolgus C5a Leu61 and Phe61 variants (Almac Sciences) into each assay to determine the IC 50 of each cynomolgus variant.
  • test sample dilutions 5 microlitres were added to 384 well low volume assay plates followed by the addition of 5 microlitres of 2 nM Flag tagged cynomolgus C5a Leu61 .
  • the binding of antibodies to native, endogenous human and cynomolgus C5 and C5a/C5adesR was determined by immunoprecipitation experiments, using human or cynomolgus plasma.
  • Zymosan A (Sigma-Aldrich, Z2450) was washed twice with phosphate buffered saline (PBS), and then centrifuged for 3000rpm for 5 minutes. The pellet was then resuspended at 50-100 milligrams/millilitre in PBS.
  • PBS phosphate buffered saline
  • BD red blood cells
  • PBLs peripheral blood leukocytes
  • BD vacutainer tubes
  • lepirudin final concentration 50 micrograms/ml
  • Plasma was collected, and activated with 5 milligrams/millilitre zymosan A at 37 degrees Celsius for 15 minutes with constantly shaking.
  • Zymosan A was removed by centrifugation at 3000rpm for 5 minutes, and activated plasma was collected and used immediately.
  • Cynomolgus monkey blood was purchased from Bioreclamation (Hicksville, NY). Cynomolgus blood was collected in EDTA tubes for C5 immunoprecipitation, and heparin tubes (final concentration, 15 units/ml) for zymosan A activation and C5a immunoprecipitation.
  • the membrane was then washed in wash buffer (PBS, 0.1 % Tween) and incubated in a 1 :5000 dilution of avidin-HRP detection (Pierce/ Thermo Scientific, Waltham, Massachusetts, final dilution 1 :5000) at room temperature for 1 hour.
  • the membrane was then washed 5 times with wash buffer, and the C5 and C5a/C5adesR bands were then visualized using a chemiluminescent substrate (Pierce/Thermo Scientific, Waltham, Massachusetts).
  • Purified antibodies were serially diluted and 50 microlitres of each dilution was mixed with an equal volume of diluted normal human serum and incubated at room temperature in a U-bottom 96-well plate (BD 3077).
  • the anti-C5/C5a antibody or fragment (Quidel, A217) was used as a positive control. After 7 minutes incubation, 30 microlitres washed CRBCs were added to the mixture and incubated at 37 degrees Celsius for 40 minutes on a shaker. Serum alone (background), CRBCs alone (background), serum plus CRBCs (total lysis) and CRBCs plus H 2 0 were used as controls.
  • RRBCs rabbit red blood cells
  • GVB-Mg ++ -EGTA buffer Boston BioProducts, IBB-320. Cells were counted using a haemocytometer under an inverted microscope. Two hundred million RRBCs were resuspended in 3 ml cold GVB-Mg ++ -EGTA buffer. Normal human serum (Quidel, A1 12) was freshly thawed from -80 degrees Celsius and diluted 5-fold using GVB-Mg ++ -EGTA buffer.
  • Purified antibodies were serially diluted and 50 microlitres of each dilution was mixed with an equal volume of diluted normal human serum and incubated at room temperature in a U-bottom 96-well plate (BD 3077).
  • A217 Antibody (Quidel) was used as a positive control. After 7 minutes incubation, 30 microlitres of washed RRBCs were added to the mixture and incubated at 37 degrees Celsius for 40 minutes on a shaker. Serum alone (background), RRBCs alone (background), serum plus RRBCs (total lysis) and RRBCs plus H 2 0 were used as controls. The plate was then centrifuged at 1200 rpm for 3 minutes at 4 degress Celsius.
  • a panel of optimized antibodies was identified from selections by screening outputs in single point biochemical epitope competition assays using HTRF® technology.
  • the HTRF® epitope competition assays described measure the binding of a lead antibody in IgG format to either biotinylated human C5a (Sigma cat no.C5788, modified in-house) or HIS Flag tagged cynomolgus C5adesR (mammalian expressed, AstraZeneca).
  • Test antibody samples e.g., either scFv or unlabelled IgG
  • scFv or unlabelled IgG which may recognize a similar epitope to the lead antibody, will compete with the lead antibody for binding to C5a, leading to a reduction in assay signal.
  • Lead antibody can be parent mAb1 lgG2 or an optimized derivative of mAb1 , for example mAb5 lgG2.
  • the epitope competition assays established with these lead antibodies were used to screen optimized selection outputs (as unpurified scFv from periplasm) to enable identification of improved affinity antibodies.
  • the mAb1 lgG2 assay measured the binding of cryptate labeled mAb1 lgG2 to biotinylated human C5a and was used to screen for improved antibodies at the outset of lead optimization.
  • the mAb1 biochemical epitope competition assay was no longer suitable for discriminating high affinity C5a binding scFv. Therefore, more sensitive biochemical HTRF® epitope competition assays were adopted in which mAb1 was replaced with a higher affinity optimized antibody mAb5 lgG2 (identified from screening early optimized selection outputs in the mAb1 epitope competition assay). Binding of mAb5 lgG2 to biotinylated human C5a or HIS Flag tagged cynomolgus C5adesR was measured in these assays.
  • selection outputs were screened as diluted, unpurified scFv from periplasmic extracts prepared in 50 mM MOPS buffer pH 7.4, 0.5 mM EDTA and 0.5 M sorbitol.
  • nM mAb5 lgG2 5 microlitres of 6 nM mAb5 lgG2 was then added to the assay plate prior to the addition of a mixed solution of detection reagents containing 40 nM anti-human IgG XL665 conjugate (Cisbio International, 61 HFCXLB) and 0.4 nM cryptate detection ⁇ e.g., streptavidin cryptate (Cisbio International 610SAKLB) for detection of biotinylated human C5a and anti-Flag cryptate (Cisbio International, 61 FG2KLB) for detection of HIS Flag tagged cynomolgus C5adesR).
  • detection reagents containing 40 nM anti-human IgG XL665 conjugate (Cisbio International, 61 HFCXLB) and 0.4 nM cryptate detection ⁇ e.g., streptavidin cryptate (Cisbio International 610SAKLB) for detection of
  • Antibodies were reformatted to lgG2 and lgG4 isotypes to minimize the activation and binding of complement C1 q (Burton et al, 1992). Additionally, in order to stabilize the intrachain disulphide bond between the lgG4 heavy chains, a serine #228 to proline mutation (S228P) was introduced in the hinge region of the lgG4 molecule (Angal et al, 1993; Schuurman et al, 2001 ).
  • variable heavy chain was cloned into a mammalian expression vector containing the human heavy chain constant domains and regulatory elements to express whole lgG2 heavy chain in mammalian cells.
  • variable light chain domain was cloned into a mammalian expression vector for the expression of the human lambda light chain constant domains and regulatory elements to express whole IgG light chain in mammalian cells.
  • the heavy and light chain IgG expressing vectors were transiently transfected into HEK293-EBNA mammalian cells where the lead antibody was expressed and secreted into the medium.
  • the production of mAb9 and mAb12 as the lgG4 S228P isotype was performed by sub-cloning each VH domain into vector expressing whole human antibody lgG4 heavy chain containing a single amino acid substitution at position 228 from serine to proline.
  • the variable heavy chain was cloned into this mammalian expression vector containing the human heavy chain constant domains incorporating the S228P mutation and regulatory elements to express whole lgG4 S228P heavy chain in mammalian cells.
  • variable light chain domain was cloned into a mammalian expression vector for the expression of the human lambda light chain constant domains and regulatory elements to express whole IgG light chain in mammalian cells.
  • the heavy and light chain IgG expressing vectors were transiently transfected into HEK293-EBNA mammalian cells where the lead antibody was expressed and secreted into the medium.
  • the IgG was purified using an endotoxin-low process using MabSelectSuReTM (GE Healthcare). Clarified supernatants were loaded onto a 1 ml column pre-equilibrated in 50 mM Tris, 0.25 M NaCI pH 8 buffer. The IgG was eluted from the column using 0.1 M citrate pH 3 directly into 1 M Tris pH 10. The eluates underwent buffer exchange using NAP-10 buffer exchange columns into 1 x Dulbeccos PBS.
  • the purified IgG were 0.2 micrometer sterile filtered, analysed for endotoxin, characterised by SDS-PAGE and the concentration determined by optical absorbance at 280 nm.
  • HTRF® epitope competition assays were also used to determine IC 50 values of purified optimized scFv and IgG isolated, to enable potency ranking.
  • IC 50 values of competing antibodies were determined in HTRF® epitope competition assays in which the binding of lead optimized antibodies mAb5 or mAb9 to biotinylated human C5a and His Flag tagged cynomolgus C5adesR ligands were measured.
  • IC 50 values were estimated in the mAb5 HTRF® epitope competition assay by adding 5 microlitres of a series of dilutions of test samples to the assay plate.
  • Biotinylated human C5a or HIS Flag tagged cynomolgus C5adesR, mAb5 lgG2 and the detection reagents were then added to the assay as described herein for the mAb5 epitope competition assay herein.
  • IC 50 values were determined as described in Equation 2.
  • IC 50 values of test purified scFv and IgG were estimated in the mAb9 HTRF® epitope competition assays in which the binding of Mab9 lgG2 to biotinylated human C5a or HIS Flag tagged cynomolgus C5adesR was measured.
  • Two assay formats were used for these studies. The first utilised indirect detection of mAb9 lgG2 with anti-human Fc XL665 conjugate and was used to determine IC 50 values of purified scFv, while the second format made use of mAb9 lgG2 directly labelled with cryptate fluorophore enabling unlabelled IgG IC 50 values to be determined.
  • mAb9 lgG2 HTRF® epitope competition assay in which mAb9 lgG2 was directly labeled with cryptate, 5 microlitres of test sample dilutions was added to a 384 well low volume assay plate (Costar 3676) followed by the addition of either 5 microlitres of 2 nM biotinylated human C5a or HIS Flag tagged cynomolgusC5adesR.
  • FMAT assays utilise a fluorescence based platform for the detection of localised fluorescence on beads or cells which have settled to the bottom of microwell plates.
  • the competition FMAT assays described measure the effect of serial dilutions of antibodies on the binding of Alexafluor labelled C5a or C5adesR ligands (Almac Biosciences, custom labelling) to C5aR or gpr77 transfected HEK cells (cell lines supplied by AstraZeneca).
  • the C5aR cell line was routinely cultured in Dulbecco's Modified Eagle's medium 89.3% (v/v) (Invitrogen); foetal bovine serum 8.9% (v/v) (SAFC Bioscences®) and Geneticin 1.8% (v/v), (Invitrogen).
  • the C5aR transfected cells were cultured overnight in medium containing 6.5 mM sodium butyrate (Sigma, B5887).
  • the C5L2 cell line was routinely cultured in Dulbecco's Modified Eagle's medium 88.5% (v/v); foetal bovine serum 8.8% (v/v); Non-essential amino acids 0.9% (v/v), (Invitrogen) and Geneticin 1 .8% (v/v).
  • C5aR and C5L2 transfected cell lines were harvested immediately before use by incubation with accutase (PAA, L1 1-007) for 5 minutes at 37 degrees Celsius.
  • Accutase was neutralised by the addition of an equal volume of medium and the cells were centrifuged at 1200rpm for 5 minutes at room temperature. The cell pellets were then resuspended in FMAT assay buffer (Hank's Balanced Salt Solution (Sigma, H8264) containing 0.01 % sodium azide, (Sigma, S8032) and 0.1 % BSA (Sigma, A9576)) and the required number of cells was transferred to the required volume of FMAT assay buffer to give a concentration of 2 X 10 5 cells/millilitre.
  • FMAT assay buffer Hort's Balanced Salt Solution (Sigma, H8264) containing 0.01 % sodium azide, (Sigma, S8032) and 0.1 % BSA (Sigma, A9576)
  • Antibody samples for testing were diluted to the required concentration in FMAT assay buffer and titrated 3-fold over II points. Twenty microlitres of each antibody dilution was transferred to a 384 well assay plate (Costar, 3655) in duplicate, using MiniTrakTM automation (Perkinelmer.) For measuring the effect of antibodies on the binding of C5a and C5adesR to cell expressed C5L2 receptor, Alexafluor 647 labelled C5a or C5adesR was diluted to 5 nM in FMAT assay buffer and 10 microlitres added to all wells of the assay plate. Non-specific binding was determined using an excess of unlabelled C5a instead of sample.
  • Non-specific binding is defined by replacing antibody sample with an excess of unlabelled C5a and total binding is defined by replacing sample with assay buffer.
  • Alexafluor 647 labelled C5a was diluted to 25 nM in FMAT assay buffer and 10 microlitres added to all wells of the assay plate. Non-specific binding was determined using an excess of unlabelled C5a instead of sample.
  • 20 microlitres of C5aR expressing cell suspension was added to all wells of the assay plate using a Multidrop Combi (Thermo Scientific), to give 4000 cells/well, and the assay plate was sealed and incubated in the dark at room temperature for 4 hours.
  • DELFIA® Target Specificity Assay The target specificity of optimized antibodies for C5a/C5adesR and C5 over complement components C3, C3a, C4 and C4a was determined by assessing whether complement
  • components C3 and C4 and their derivates C3a and C4a could compete with the lead antibodies mAb9 lgG4 S228P or mAb12 lgG4 S228P for binding to biotinylated human C5a in a DELFIA® (Dissociation-Enhanced Lanthanide Fluorescent Immunoassay, Perkin Elmer,) competition assay format.
  • DELFIA® Dissociation-Enhanced Lanthanide Fluorescent Immunoassay, Perkin Elmer,
  • mAb9 lgG4 S228P or mAb12 lgG4 S228P were diluted in Dulbecco's phosphate buffered saline (dPBS, Invitrogen 14190-086) to a concentration of 2.5 nM and 50 microlitres was added to the wells of a 96 well NuncTM Immuno-Maxisorp microtitre assay plate (Thermo Fisher Scientific, 439454).
  • the purified IgG were adsorbed to the surface of the assay plate for 1.5 hours at room temperature and then unbound IgG was washed away with dPBS containing 0.1 % Tween
  • Unbound biotinylated C5a was removed by washing in dPBS-Tween and 50 microlitres of 100 nanograms/millilitre europium labeled streptavidin detection (Perkin Elmer 1244360) diluted in DELFIA® assay buffer (Perkin Elmer 4002-0010) was added to the plate.
  • the assay plate was incubated with detection reagent for 1 hour at room temperature and then washed in DELFIA® wash buffer (Perkin Elmer 4010-0010) prior to the addition of 50 microlitres of DELFIA® enhancer buffer(Perkin Elmer 4001 - 0010) per well.
  • K D The equilibrium dissociation constant for the interaction of mAb9 lgG4 S228P with human C5 protein was measured using a KinExA (kinetic exclusion assay, Sapidyne Instruments Inc) platform. Briefly, human C5 protein was coated onto Azlactone beads (UltraLink Biosupport, Pierce, Rockford. IL) beads at a concentration of 10 micrograms/millilitre and 25 micrograms /milli litre in 50 mM NaC0 3 buffer, pH9.2, using a protocol supplied by the instrument's
  • Protein/Azlactone beads to a capillary flow cell in the instrument, and then to inject individual sample mixtures over the human C5 protein-coated beads. Following this, unbound sample solution was washed from the beads, and then a species-specific, Cy5-labeled antibody detection reagent (prepared at 0.75 micrograms/millilitre or 1 .0 micrograms/millilitre in sample buffer) was passed over the beads. The beads were again washed to remove excess label, then the amount of fluorescence that remained associated with the beads was measured and the signal converted to percent free IgG. Between samples, the bead pack was flushed from the flow cell, and then replenished with more human C5 protein-coated beads in preparation for the next sample.
  • a species-specific, Cy5-labeled antibody detection reagent prepared at 0.75 micrograms/millilitre or 1 .0 micrograms/millilitre in sample buffer
  • K D The equilibrium dissociation constant for the interaction of mAb9 lgG4 S228P with human C5a protein was measured using a KinExA (kinetic exclusion assay) platform. Briefly, human C5a protein was coated onto Azlactone beads (UltraLink Biosupport, Pierce, Rockford. IL) beads at a concentration of 15 micrograms/millilitre and 100 micrograms/millilitre in 50 mM NaC0 3 buffer, pH 9.2, using a protocol supplied by the instrument's manufacturer. Afterwards, the beads were washed and blocked in 1 M Tris buffer, pH 8, containing 10 mg/millilitre bovine serum albumin (BSA) until used.
  • BSA bovine serum albumin
  • batch volumes of mAb9 lgG4 S228P were prepared at 10 pM and 250 pM concentrations in PBS, pH 7.4, buffer containing 0.02% NaN 3 , 0.1 % BSA and 1 mM CaCI 2 (sample buffer). These were then aliquoted into 13 tubes.
  • human C5a protein was added to one tube from each series, and then serially diluted across eleven of the remaining tubes, leaving one IgG sample tube as an antibody-only control.
  • the final human C5a protein concentrations for the 10 pM and 250 pM mAb9 lgG4 S228P experiments ranged from 7.8 fM to 400 pM, and from 49 fM to 2.5 nM, respectively.
  • the human C5a protein -coated Aziactone bead slurries were diluted to approximately 30 millilitres with instrument buffer (PBS, pH7.4+0.02% NaN 3 +1 mM CaCI 2 ) in a bead vial and attached to the KinExA instrument.
  • instrument buffer PBS, pH7.4+0.02% NaN 3 +1 mM CaCI 2
  • a user-defined timing program was then used to sequentially transfer the human C5a protein /Aziactone beads to a capillary flow cell in the instrument, and then to inject individual sample mixtures over the human C5a protein -coated beads.
  • unbound sample solution was washed from the beads, and then a species-specific, Cy5-labeled antibody detection reagent (prepared at 1.0
  • microgram/mL in sample buffer was passed over the beads.
  • the beads were again washed to remove excess label, then the amount of fluorescence that remained associated with the beads was measured and the signal converted to percent free IgG.
  • the bead pack was flushed from the flow cell, and then replenished with more human C5a protein -coated beads in preparation for the next sample.
  • An isotherm was generated which plotted the amount of free mAb9 lgG4 S228P detected at each concentration of human C5a protein.
  • the resulting two binding curves were then evaluated in a dual curve analysis using the instrument's evaluation software, from which the K D was derived.
  • Figures 12A-12D provide an amino acid sequence alignment of the VH regions of mAb1 and derivatives. Framework regions and CDR's are illustrated.
  • Figures 13A-13D provide an amino acid sequence alignment of the VL regions of mAb1 and derivatives. Framework regions and CDR's are illustrated.
  • Figure 14 provides an amino acid sequence alignment of the VH CDR's of mAb1 and derivatives.
  • Figure 15 provides an amino acid sequence alignment of the VL CDR's of mAb1 and derivatives. Positional sequence differences are shown by the presence of a different amino acid with respect to the comparison sequence of mAb1.
  • Example 29 Amino acid sequence identity determination tables.
  • Sequence alignments of the entire single chain Fv region (scFv), VH, VL, individual CDR's, combined CDR regions and other variations were performed comparing mAb1 and its derivatives.
  • the sequence identities are presented in Tables 22-29 below.
  • the sequence identity or similarity is presented on the right side of the diagonal line formed by the blacked out cells of each table.
  • the numbers to the left of the black diagonal line represent the sequence dissimilarity.
  • the alignments were performed using using Clustal W alignment using DNAStar Lasergene 8 software. Table 22. Sequence Identity determination across entire scFv for mAb1 and derivative antibodies.
  • Table 24 Sequence Identity determination across light chain variable regions for mAb1 and derivative antibodies.
  • Table 25 Sequence Identity determination across all 6 CDR's for mAb1 and derivative antibodies.
  • Table 26 Sequence identity determination across VH CDR's for mAb1 and derivative antibodies.
  • Table 27 Sequence identity determination across VL CDR's for mAb1 and derivative antibodies.
  • Table 28 Sequence identity determination across VH CDR3 for mAb1 and derivative antibodies.
  • Table 29 Sequence identity determination across VL CDR3 for mAb1 and derivative antibodies.

Abstract

La présente invention concerne des compositions, des procédés de fabrication et des procédés d'utilisation se rapportant à des anticorps anti-C5/C5a/C5adesR et à des fragments de liaison à l'antigène.
PCT/US2011/066437 2010-12-22 2011-12-21 Fragments et anticorps anti-c5/c5a/c5adesr WO2012088247A2 (fr)

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