WO2016118007A1 - Means and methods for counteracting complement-mediated hemolytic anemia - Google Patents

Means and methods for counteracting complement-mediated hemolytic anemia Download PDF

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WO2016118007A1
WO2016118007A1 PCT/NL2016/050047 NL2016050047W WO2016118007A1 WO 2016118007 A1 WO2016118007 A1 WO 2016118007A1 NL 2016050047 W NL2016050047 W NL 2016050047W WO 2016118007 A1 WO2016118007 A1 WO 2016118007A1
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sequence
antibody
sequence identity
light chain
functional equivalent
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Frank Baas
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Regenesance B.V.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/472—Complement proteins, e.g. anaphylatoxin, C3a, C5a
    • 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
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the invention relates to the fields of biology, immunology and medicine.
  • Hemolytic anemia involves the lysis of erythrocytes. This can occur within blood vessels (intravascular) or at other sites within the body (extr avascular). It has many possible causes, which are either inherited or acquired.
  • One disorder involving hemolytic anemia is haemolytic uremic syndrome (HUS).
  • HUS is typically defined by the simultaneous occurrence of microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury.
  • the most common cause of HUS is the Shiga toxin-producing Escherichia coli (STEC).
  • SEEC Shiga toxin-producing Escherichia coli
  • aHUS atypical HUS
  • Primary causes for aHUS are dysregulation of complement by either mutations or antibodies. Secundary causes of aHUS are infections.
  • PNH paroxysmal nocturnal hemoglobinuria
  • PNH Marchiafava-Micheli syndrome.
  • PNH is a genetically acquired life -threatening disorder involving lysis of erythrocytes by the complement system.
  • Complement components assemble at the surface of erythrocytes and form the Membrane Attack Complex (MAC), resulting in lysis.
  • MAC Membrane Attack Complex
  • This effect is due to an impaired formation of functional surface proteins CD55 and/or CD59 on the surface of erythrocytes, which normally protect the erythrocytes from complement activity.
  • CD55 which is also known as complement decay accelerating factor
  • CD55 and CD59 are linked to erythrocytes via a glycosylphosphatidylinositol (GPI) anchor.
  • GPI glycosylphosphatidylinositol
  • PNH patients exhibit a defect in the assembling of these glycolipid-protein structures, so that CD55 and/or CD59 are not, or to a lesser extent, coupled to erythrocytes.
  • the most common defective enzyme in PNH is phosphatidylinositol glycan A (PIGA), one of several enzymes needed to make GPL PNH is considered an intravascular hemolytic anemia, since the complement cascade attacks the erythrocytes within the circulatory system. Symptoms of PNH are, amongst other things, fatigue, shortness of breath and palpitations.
  • the most characteristic sign of PNH is a red discoloration of the urine, which is a result of free hemoglobulin and hemosiderin, released from lysed erythrocytes.
  • Eculizumab (trade name Soliris), which is a humanized monoclonal antibody against
  • complement component C5 is used in order to decrease PNH symptoms and the need for blood transfusions.
  • Drawbacks of eculizumab are its high costs and the occurrence of various side effects such as an increased sensitivity for infections.
  • C5a is essential for regeneration of liver cells. Therefore, if an individual has liver damage, eculizumab will inhibit liver repair.
  • the present invention provides such alternative means and methods for counteracting complement-mediated hemolytic anemia.
  • the invention provides a method for at least in part preventing or treating complement- mediated hemolytic anemia, the method comprising administering an inhibitor of human complement component C6 to a human individual in need thereof.
  • Some embodiments provide an inhibitor of human complement component C6 for use in a method for at least in part treating or preventing complement-mediated hemolytic anemia.
  • Some embodiments provide a use of an inhibitor of human complement component C6 for the preparation of a medicament for at least in part treating or preventing complement-mediated hemolytic anemia.
  • complement-mediated hemolytic anemia embraces disorders involving abnormal lysis of erythrocytes, either intravascular or extravascular, wherein complement activation plays a role. This means that lysis of erythrocytes is, either directly or indirectly, a result of complement activity.
  • disorders involving complement-mediated hemolytic anemia are PNH, HUS and aHUS.
  • said hemolytic disorder is PNH.
  • said hemolytic disorder is aHUS.
  • preventing complement-mediated hemolytic anemia for instance embraces delaying the onset of disease, or the course of the disease, or reducing the severity of at least one aspect of the disease, such as for instance the degree of erythrocyte lysis, or alleviating at least one symptom of the disease, such as for instance fatigue, wherein the C6 inhibitor is administered before onset of the disease or before worsening of the symptoms of the disease, so that the onset of disease will be delayed, and/or an individual's course of disease will be milder, as compared to a situation without at least partial prevention according to the invention.
  • the term "at least in part treating complement- mediated hemolytic anemia” for instance embraces reducing at least one aspect of the disease, such as for instance the degree of erythrocyte lysis, and/or delaying the course of the disease, and/or alleviating at least one symptom of the disease, so that a patient's condition will improve as compared to the situation before treatment.
  • the C6 inhibitor may be administered at any stage of the disease. For the patient's well-being, administration at an early stage is preferred.
  • counteracting complement-mediated hemolytic anemia encompasses reducing at least one aspect of the disease, such as for instance the degree of erythrocyte lysis, and/or alleviating at least one symptom of the disease, and/or reducing the severity of at least one aspect of the disease, and/or delaying the onset or course of the disease.
  • counteracting complement- mediated hemolytic anemia embraces at least in part treating or preventing complement-mediated hemolytic anemia.
  • the complement system a highly conserved part of the innate immune system, consists of a cascade of self-cleaving proteases that act as a first line of defence against pathogens.
  • the complement system a key component of innate immunity, defends against infections and disposes of dead or dying cells. Because complement can harm self-tissue, activation is tightly controlled by regulators to eliminate pathogens or damaged cells without injuring the host. In addition, complement activation may link innate to adaptive immune responses.
  • the complement system can be activated via three pathways, the classical, lectin and alternative pathway.
  • the classical and lectin pathways are initiated by pattern recognition proteins (PRPs). PRPs recognize repeating patterns on membranes, so called pathogen associated membrane patterns (PAMP). In case of the classical activation pathway this is done by Clq which recognize
  • antigen/antibody complexes Upon recognition the proteins become active and associate with proteolytic components, Clr and Cls. The lectin pathway is activated by the binding of mannose binding lectin, which recognizes mannose (sugar) patterns. Upon binding the membrane associated serine protease 2 (MASP2), a proteolytic component, becomes active. The proteases, Clq-r-s and MBL-MASP2 complexes, cleave C2 and C4 protein that can in turn associate and form the C3 convertase (C4bC2b complex).
  • MASP2 membrane associated serine protease 2
  • C3 is the main effector molecule of the complement cascade. All pathways converge at the level of C3 cleavage.
  • C3 convertase cleaves C3 in C3a and C3b
  • C3b associates with the C4bC2b complex forming
  • C5 convertase (C4bC2bC3b) which cleaves C5 into C5a and C5b.
  • C5b acts as a anchor molecule for the membrane attack complex (MAC or C5b-9), a pore forming structure consisting of C5b-C6-C7- C8 and C9.
  • MAC or C5b-9 membrane attack complex
  • C5b-9 membrane attack complex
  • MAC or C5b-9 membrane attack complex
  • a pore forming structure consisting of C5b-C6-C7- C8 and C9.
  • anaphylatoxins C3a and C5a are powerful inducers of proinflammatory cytokines and induce attraction of phagocytes.
  • the activators of both the classical and the lectin pathway are membrane bound.
  • the alternative pathway is activated in fluid phase by spontaneous conversion of the C3 molecule into its active component.
  • the C3b alone is highly unstable, but can associate with factor B, which is cleaved by factor D, to form the active C3bBb convertase.
  • This C3bBb is a C3 convertase, which binds to membranes; this binding is stabilized by Properdin.
  • the amount of C3bBb rapidly increases, but is strictly regulated on self-targets.
  • Regulatory complement components either induce an accelerated decay of the convertase or act as cofactor for Factor I to degrade activated complement fragments.
  • CD59 prevents formation of the MAC by inserting between the C8 and C9 subunits of the C5b-9 polymer.
  • a potent regulator of the alternative pathway is factor H, which induces a fast turn-over of the C3bBb complex, by breaking the link between C3b and the Bb proteins.
  • factor H also works on membrane-bound C3bBb by allowing binding of yet another regulator of the complement system factor I, which degrades C3b (for a detailed review on the complement system see Ricklin et al., 2010).
  • the complement system is a highly conserved part of the innate immune system that acts as a first line of defence against pathogens, involving a complex mechanism of activating and deactivating complement factors.
  • complement activation causes injury and contributes to pathology in various diseases.
  • activation of the alternative pathway accounts for approximately 80% of the complement activity, even if the initial activation occurred via either the classical or the lectin pathway.
  • the inventor has for the first time provided proof of principle that human patients suffering from complement-mediated hemolytic anemia, such as PNH or HUS patients, can be treated with a C6 inhibitor.
  • the inventor has shown that inhibition of human C6 in an environment that is indicative for PNH results in diminished erythrocyte lysis.
  • erythrocytes containing diminished levels of functional CD55 and/or CD59 at their surface are attacked and lysed by complement via formation of the membrane attack complex (MAC).
  • MAC membrane attack complex
  • PNH patients contain erythrocytes that are vulnerable to complement-mediated lysis.
  • the current inventor has demonstrated that in vivo inhibition of human C6 in rats results in diminished lysis of erythrocytes that are vulnerable to complement-mediated lysis.
  • the current inventor has also demonstrated that inhibition of C6 in normal human blood results in diminished MAC formation and lysis of erythrocytes that are vulnerable to complement- mediated lysis. According to the present invention, this is indicative for treatment of complement-mediated hemolytic anemia, such as PNH or HUS, by counteracting human C6 activity.
  • complement-mediated hemolytic anemia such as PNH or HUS
  • the current inventor has taken a different approach. Instead of C6-deficient animals, the inventor has used C6 knock-out rats that were supplemented with human C6.
  • This human C6 is capable of complexing with rat C5, C7, C8 and C9 in order to form a functional membrane attack complex (MAC).
  • MAC membrane attack complex
  • the rats had a functional complement system, in contrast to the C6- knockout animals that have been used before the present invention. Subsequently, these rats were provided with antibodies that are specific for human C6 (and which are unable to block rat complement factors such as rat C5 or rat C6).
  • Blood from these rats was subsequently used in order to test lysis of erythrocytes that were rendered vulnerable to complement lysis (like the CD55- and CD59-deprived erythrocytes of a PNH patient).
  • As a positive control blood was used from the above-mentioned human C6-containing rats, that had not received an anti-C6 antibody.
  • the hemolytic activity of a normal functioning complement system against vulnerable, PNH-like, erythrocytes was tested, and it was established that administration of a C6 inhibitor could effectively counteract this hemolytic activity.
  • C6 inhibitors in a method according to the present invention is preferred over the use of the currently used anti-C5 antibody eculizumab.
  • C6 is barely produced locally in inflamed nervous tissue, whereas C5 gives a very strong signal in case of neuroinflammation.
  • C6 has only one function, i.e. formation of the membrane attack complex (MAC).
  • MAC membrane attack complex
  • C5 on the other hand, has additional roles.
  • the cleaved product C5a plays an important role in immunity.
  • the C5 antibody eculizumab blocks cleavage of C5, thereby blocking the production of both the anaphylatoxin C5a and the anchor for MAC formation, C5b. Blocking C5b production counteracts the formation of MAC, which provides the desired anti- lysis effect.
  • the simultaneous reduction of C5a results in many side effects such as reduced chemotaxis after an infection has occurred.
  • maintaining C5a production is also important because signaling of C5a through the C5aReceptor plays a role in regeneration and liver repair. With the use of C6 inhibitors, these beneficial roles of C5a are maintained, thereby reducing side effects.
  • advantages of the methods according to the present invention are, amongst other things, that C5a production is maintained, resulting in less side effects.
  • inhibitor of human complement component C6 also referred to as “inhibitor of C6” or “C6 inhibitor” means a compound that is able to at least partly counteract C6 activity.
  • said C6 inhibitor is able to at least partly counteract the role of C6 in the formation of the MAC.
  • Such C6 inhibitor is, therefore, particularly suitable for use in directly or indirectly diminishing lysis of erythrocytes in PNH or HUS patients.
  • Various inhibitors of human complement component C6 are suitable for performing the methods according to the present invention.
  • said inhibitor is selected from the group consisting of a C6 antagonist, a peptide, a polypeptide, an antisense nucleic acid molecule, a small molecule, or a C6 receptor.
  • a C6 antagonist binds C6 so that C6 can no longer bind to complement component C5b, C7, C8 and/or C9, which at least in part inhibits MAC formation.
  • a C6 antagonist binds a C6 receptor, for instance present on C5b, which also counteracts MAC formation.
  • a peptide or polypeptide comprises a plurality of amino acids, whereby a given amino acid residue is typically bound to an adjacent amino acid residue via a peptide bond.
  • non-natural bonds and/or non-natural amino acid residues may also be present in a C6 inhibiting peptide or polypeptide as referred to herein.
  • a C6 inhibiting (poly)peptide is for instance capable of binding C6, or binding a C6 receptor that is for instance present on C5b, which binding counteracts MAC formation.
  • a small molecule is defined herein as a chemical compound, typically having a size of at most 900 daltons.
  • a C6 inhibiting small molecule as referred to herein is capable of at least in part inhibiting the assembly of the MAC.
  • a C6 inhibiting C6 receptor as referred to herein may be present in isolated or recombinant form, or as part of a larger complex such as, for instance, on a truncated form of C5b. Binding of such C6 receptor to C6 counteracts the role of C6 in MAC formation.
  • the use of a C6 receptor on natural C5b is not encompassed by the present invention, since natural C5b does not inhibit MAC formation.
  • a binding compound and “a nucleic acid sequence” encompass one or more binding compounds, and one or more nucleic acid sequences, respectively.
  • a method according to the invention wherein said inhibitor of human complement component C6 is an antibody, or a functional part or a functional equivalent thereof, or a nucleic acid sequence encoding therefore.
  • antibody refers to an immunoglobulin protein comprising at least a heavy chain variable region (VH), paired with a light chain variable region (VL), that is specific for a target epitope.
  • VH heavy chain variable region
  • VL light chain variable region
  • a “functional part of an antibody” is defined herein as a part that has at least one shared property as said antibody in kind, not necessarily in amount. Said functional part is capable of binding the same antigen as said antibody, albeit not necessarily to the same extent.
  • a functional part of an antibody comprises at least a heavy chain variable domain (VH).
  • VH heavy chain variable domain
  • Non-limiting examples of a functional part of an antibody are a single domain antibody, a single chain antibody, a nanobody, a unibody, a single chain variable fragment (scFv), a Fab fragment and a F(ab')2 fragment.
  • a “functional equivalent of an antibody” is defined herein as an artificial binding compound, comprising at least one CDR sequence of an antibody, preferably a heavy chain CDR3 sequence.
  • Said functional equivalent preferably comprises the heavy chain CDR3 sequence of an antibody, as well as the light chain CDR3 sequence of said antibody. More preferably, said functional equivalent comprises the heavy chain CDR1, CDR2 and CDR3 sequences of an antibody, as well as the light chain CDR1, CDR2 and CDR3 sequences of said antibody.
  • a functional equivalent of an antibody is for instance produced by altering an antibody such that at least an antigen-binding property of the resulting compound is essentially the same in kind, not necessarily in amount. This is done in many ways, for instance through conservative amino acid substitution, whereby an amino acid residue is substituted by another residue with generally similar properties (size, hydrophobicity, etc), such that the overall functioning of the antibody is essentially not affected.
  • a heavy chain of an antibody is the larger of the two types of chains making up an immunoglobulin molecule.
  • a heavy chain comprises a constant domain and a variable domain, which variable domain is involved in antigen binding.
  • a light chain of an antibody is the smaller of the two types of chains making up an immunoglobulin molecule.
  • a light chain comprises a constant domain and a variable domain. The variable domain is often, but not always, together with the variable domain of the heavy chain involved in antigen binding.
  • Complementary- determining regions are the hypervariable regions present in heavy chain variable domains and light chain variable domains. In case of whole antibodies, the CDRs 1-3 of a heavy chain and the CDRs 1-3 of the connected light chain together form the antigen-binding site.
  • an antibody or functional part or functional equivalent is also referred to as "a binding compound”.
  • the terms “specific for” and “capable of specifically binding” are used herein interchangeably and refer to the interaction between an antibody, or functional part or functional equivalent thereof, and its epitope. This means that said antibody, or functional part or functional equivalent thereof, preferentially binds to said epitope over other antigens or amino acid sequences.
  • the antibody, functional part or equivalent may non-specifically bind to other antigens or amino acid sequences, the binding affinity of said antibody or functional part or functional equivalent for its epitope is significantly higher than the non-specific binding affinity of said antibody or functional part or functional equivalent for other antigens or amino acid sequences.
  • An antibody or functional part or functional equivalent according to the invention that is able to bind a particular epitope of C6 can also be specific for another protein or (poly)peptide, if said C6 epitope happens to be also present on such other protein or (polypeptide.
  • an antibody referred to herein as being specific for C6 is also specific for such other protein or (poly)peptide comprising the same epitope.
  • a nucleic acid molecule encoding a C6-inhibiting antibody or functional part or functional equivalent as referred to herein preferably comprises a chain of nucleotides, more preferably DNA, cDNA or RNA.
  • such nucleic acid molecule comprises other kinds of nucleic acid structures such as for instance a DNA/RNA helix, peptide nucleic acid (PNA), locked nucleic acid (LNA) and/or a ribozyme.
  • PNA peptide nucleic acid
  • LNA locked nucleic acid
  • nucleic acid molecule thus encompasses a chain comprising non-natural nucleotides, modified nucleotides and/or non-nucleotide building blocks which exhibit the same function as natural nucleotides.
  • the percentage of identity of an amino acid or nucleic acid sequence, or the term “% sequence identity” is defined herein as the percentage of residues in a candidate amino acid or nucleic acid sequence that is identical with the residues in a reference sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity.
  • Methods and computer programs for the alignment are well known in the art, for example Align 2, Align Plus, Clustal in the PC/Gene program (which is available from Intelligenetics, Mountain View, California), and GAP, BESTFIT, BLAST, FASTA and TFASTA in the Wisconsin Genetics Software Package of Genetics Computer Group, version 10 (available from Accelrys, San Diego, California), as explained in more detail on page 12 of WO 2010/005310.
  • Non-limiting examples of mathematical algorithms for determining the percentage sequence identity between two nucleic acid sequences or amino acid sequences are the algorithm of Myers and Miller (1988) CABIOS 4: 11- 17, the local homology algorithm of Smith et al. (1981) Adv.Appl.Math.
  • a human patient suffering from, or at risk of suffering from, complement-mediated hemolytic anemia such as a PNH or HUS or aHUS patient
  • This is a novel antibody, provided by the present inventor, that is selected for its excellent capacity of counteracting MAC formation and lysis of red blood cells, as shown in the Examples.
  • Antibody 7E5 comprises heavy chain CDR3 sequence PSTEALFAY and light chain CDR3 sequence MQASHAPYT.
  • antibody 7E5 comprises heavy chain CDR1 sequence DYYMA and heavy chain CDR2 sequence TINYDGSSTYYRESVKG, as well as light chain CDR1 sequence
  • the sequences of antibody 7E5 are depicted in Figure 10. Based on the CDR sequences of antibody 7E5, it is possible to produce an antibody or functional part or functional equivalent thereof comprising at least one CDR sequence of 7E5, which is specific for C6.
  • a method for treating complement-mediated hemolytic anemia preferably PNH, or HUS or aHUS, the method comprising administering to an individual in need thereof an isolated, recombinant and/or synthetic antibody, or a functional part or functional equivalent thereof, or a nucleic acid molecule encoding therefore, comprising at least one CDR sequence of antibody 7E5.
  • Said antibody or functional part or functional equivalent preferably comprises the heavy chain CDR3 sequence PSTEALFAY and the light chain CDR3 sequence MQASHAPYT of antibody 7E5.
  • said individual is provided with a nucleic acid molecule or functional equivalent thereof, or a vector, which encodes any of the antibodies or functional parts or functional equivalents recited herein.
  • a vector comprising) one or more nucleic acid molecule(s) or functional equivalent(s) encoding such antibody or functional part or functional equivalent is/are administered to an individual suffering from, or at risk of suffering from, complement-mediated hemolytic anemia such as PNH, or HUS or aHUS
  • the nucleic acid molecule(s) or functional equivalent(s) will be translated in vivo into a C6-inhibiting binding compound.
  • Such produced binding compounds are then capable of preventing and/or counteracting erythrocyte lysis.
  • an antibody or functional part or functional equivalent comprising the heavy chain CDR3 sequence PSTEALFAY and the light chain CDR3 sequence MQASHAPYT, or at least one nucleic acid molecule or functional equivalent encoding therefore, for use in a method for at least in part treating or preventing complement-mediated hemolytic anemia such as PNH, or HUS or aHUS.
  • an antibody or functional part or functional equivalent comprising the heavy chain CDR3 sequence PSTEALFAY and the light chain CDR3 sequence MQASHAPYT, or at least one nucleic acid molecule or functional equivalent encoding therefore, for the preparation of a medicament for at least in part treating or preventing complement-mediated hemolytic anemia such as PNH, or HUS or aHUS.
  • binding compounds are used that comprise at least two CDRs, more preferably at least three CDRs, of the heavy and light chains of antibody 7E5.
  • at least two or three CDRs of the heavy and light chains of antibody 7E5 are jointly present in one binding compound in a method according to the invention.
  • a binding compound is used that comprises all three heavy chain CDRs and all three light chain CDRs of antibody 7E5.
  • the invention therefore further provides a method for at least in part preventing or treating complement-mediated hemolytic anemia, preferably PNH, or HUS or aHUS, the method comprising administering to an individual in need thereof an isolated, synthetic or recombinant antibody or a functional part or a functional equivalent thereof, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises:
  • complement-mediated hemolytic anemia preferably PNH, or HUS or aHUS
  • said antibody or functional part or functional equivalent comprises a heavy chain CDR1 sequence comprising the sequence DYYMA and a heavy chain CDR2 sequence comprising the sequence TINYDGSSTYYRESVKG and a heavy chain CDR3 sequence comprising the sequence PSTEALFAY and a light chain CDR1 sequence comprising the sequence RSSQSLLNDVGNTYLY and a light chain CDR2 sequence comprising the sequence LVSDLGS and a light chain CDR3 sequence comprising the sequence
  • the above-mentioned at least one nucleic acid molecule or functional equivalent comprise(s) the 7E5 VH and VL nucleic acid sequences as depicted in Figure 10.
  • At least one of said CDR sequences is optimized, thereby generating a variant binding compound, preferably in order to improve the C6 binding efficacy, the selectivity, or the stability of the resulting binding compound.
  • This is for instance done by mutagenesis procedures where after the stability and/or binding efficacy of the resulting compounds are preferably tested and an improved C6-specific binding compound is selected.
  • a skilled person is well capable of generating variants comprising at least one altered 7E5 CDR sequence. For instance, conservative amino acid substitution is applied. Examples of a
  • conservative amino acid substitution include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another hydrophobic residue, and the substitution of one polar residue for another polar residue, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine.
  • an antibody or functional part or functional equivalent is used in a method according to the invention that comprises a CDR sequence which is at least 90% identical to a 7E5 CDR sequence, so that the favorable C6- binding and MAC formation-inhibiting characteristic of 7E5 is maintained or even improved.
  • variant binding compounds comprising an amino acid sequence which is at least 90% identical to a 7E5 CDR sequence is therefore also within the scope of the present invention.
  • said variant binding compounds comprise heavy chain CDRl-3 and light chain CDR 1-3 sequences which are at least 90% identical to the heavy and light chain CDR 1-3 sequences of 7E5.
  • the CDR sequences of the variant binding compounds differ in no more than three, preferably in no more than two, preferably in no more than one amino acid from the original CDR sequences of antibody 7E5.
  • At least one sequence in at least one of the framework regions of an immunoglobulin variable region can also be optimized. This is preferably done in order to improve stability and/or to reduce immunogenicity.
  • the rat framework regions of antibody 7E5 are preferably humanized in order to reduce the chance of inducing an immune response in a human patient and/or in order to improve the half life of the resulting binding compound.
  • Framework sequences are for instance optimized by mutating a nucleic acid molecule encoding such framework sequence where after the characteristics of the resulting antibody - or functional part or functional equivalent - are preferably tested. This way, it is possible to obtain improved binding compounds.
  • human germline sequences are used for framework regions in antibodies that are based on 7E5.
  • the use of human germline sequences minimizes the risk of immunogenicity of said antibodies in humans.
  • a method according to the invention wherein a synthetic or recombinant antibody is used that comprises at least one non-natural mutation in a framework region.
  • a synthetic or recombinant antibody or functional part or functional equivalent is used in a method according to the invention that comprises at least one non- natural mutation in a constant region.
  • a non-natural mutation is meant that the resulting amino acid sequence does not occur in nature. Instead, it has been artificially produced.
  • a C6-inhibiting binding compound is used for counteracting complement-mediated hemolytic anemia, preferably PNH, or HUS or aHUS, wherein said binding compound is a chimeric, humanized or human antibody.
  • a chimeric or humanized antibody is used that comprises the CDR regions of antibody 7E5 and that further comprises human framework and/or constant region sequences.
  • a binding compound for use according to the invention preferably comprises at least a heavy chain CDR3 sequence having at least 90% sequence identity with the heavy chain CDR3 sequence of antibody 7E5 and a light chain CDR3 sequence having at least 90% sequence identity with the light chain CDR3 sequence of antibody 7E5.
  • said antibody or functional part or functional equivalent comprises a heavy chain CDR3 sequence having at least 90% sequence identity with the sequence PSTEALFAY and a light chain CDR3 sequence having at least 90% sequence identity with the sequence MQASHAPYT.
  • said sequence identity is at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%.
  • at least 1, 2 or 3 amino acid residues of a given CDR sequence may vary while retaining the same binding specificity (in kind, not necessarily in amount).
  • a C6-specific binding compound is preferably used for treatment of complement- mediated hemolytic anemia, preferably PNH, or HUS or aHUS, wherein the binding compound contains a heavy chain CDR3 sequence wherein at most 3, preferably at most 2, more preferably at most 1 amino acid deviates from the sequence PSTEALFAY (the heavy chain CDR 3 sequence of antibody 7E5), and wherein the binding compound contains a light chain CDR3 sequence wherein at most 3, preferably at most 2, more preferably at most 1 amino acid deviates from the sequence MQASHAPYT (the light chain CDR 3 sequence of antibody 7E5).
  • the binding compound contains a heavy chain CDR3 sequence wherein at most 3, preferably at most 2, more preferably at most 1 amino acid deviates from the sequence PSTEALFAY (the heavy chain CDR 3 sequence of antibody 7E5), and wherein the binding compound contains a light chain CDR3 sequence wherein at most 3, preferably at most 2, more preferably at most 1 amino acid deviates from the sequence MQASHAP
  • a binding compound or at least one nucleic acid molecule or functional equivalent encoding therefore, is used in a method according to the invention, wherein said binding compound comprises heavy chain CDRl-3 sequences and light chain CDRl-3 sequences that have at least 90% sequence identity with the heavy and light chain CDRl-3 sequences of antibody 7E5 as depicted in Figure 10.
  • the invention therefore further provides a method according to the invention for treating complement-mediated hemolytic anemia, preferably PNH, or HUS or aHUS, wherein said antibody or functional part or functional equivalent comprises:
  • antibody or functional part or functional equivalent comprises:
  • a light chain CDR3 sequence having a sequence which has at least 90% sequence identity with the sequence MQASHAPYT, for use in a method for at least in part preventing or treating complement-mediated hemolytic anemia, preferably PNH, HUS or aHUS.
  • an isolated, synthetic or recombinant antibody or functional part or functional equivalent comprising a heavy chain CDRl sequence having a sequence which has at least 90% sequence identity with the sequence DYYMA and a heavy chain CDR2 sequence having a sequence which has at least 90% sequence identity with the sequence TINYDGSSTYYRESVKG and a heavy chain CDR3 sequence having a sequence which has at least 90% sequence identity with the sequence PSTEALFAY and a light chain CDRl sequence having a sequence which has at least 90% sequence identity with the sequence RSS Q S LLND VGNTYLY and a light chain CDR2 sequence having a sequence which has at least 90% sequence identity with the sequence LVSDLGS and a light chain CDR3 sequence having a sequence which has at least 90% sequence identity with the sequence MQASHAPYT for the preparation of a medicament for at least in part preventing or treating complement-mediated hemolytic anemia, preferably PNH, HUS or aHUS.
  • the above mentioned antibody or functional part or equivalent comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences that are at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical to the above recited CDR sequences.
  • at least 1, 2 or 3 amino acid residues in the recited CDR sequences may vary while retaining the same binding activity (in kind, not necessarily in amount).
  • said heavy and light chain CDR 1, 2 and 3 sequences preferably deviate in no more than three, preferably no more than two, more preferably no more than one amino acid from the recited 7E5 CDR sequences.
  • an antibody or functional part or functional equivalent that comprises a variable heavy chain sequence and/or a variable light chain sequence of antibody 7E5, or a sequence which has at least 80% sequence identity thereto.
  • a modified variable heavy chain and/or variable light chain sequence contains humanized framework regions, which is preferred for medical use in humans, as explained before.
  • the variable heavy and light chain sequences of antibody 7E5 are
  • variable light chain sequence having at least 80% sequence identity with the sequence
  • DWLTQTPSTLSATIGQSVSISCRSSQSLLNDVGNTYLYWYLQRPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISGVEAEDLGIYYCMQASHAPYTFGAGTNL ELK or sequences that are at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100% identical to said heavy chain or light chain sequences.
  • variable light chain sequence having at least 80% sequence identity with the sequence
  • heavy and light chain CDRl-3 sequences of said binding compound differ in no more than 3, preferably in no more than 2, more preferably in no more than 1 amino acid residues from the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10.
  • said heavy and light chain CDRl-3 sequences are identical to the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10.
  • At least one nucleic acid molecule or functional equivalent encoding for any of the above-recited antibodies or functional parts or functional equivalents is used in a method or use according to the invention.
  • Said at least one nucleic acid molecule or functional equivalent preferably comprise a nucleic acid sequence that has at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100% sequence identity with the 7E5 VH and/or 7E5 VL nucleic acid sequences as depicted in Figure 10.
  • Some embodiments provide an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or
  • variable light chain sequence having at least 80% sequence identity with the sequence
  • complement-mediated hemolytic anemia preferably comprises PNH, HUS or aHUS.
  • an antibody or functional part or functional equivalent or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
  • variable light chain sequence having at least 80% sequence identity with the sequence
  • complement-mediated hemolytic anemia preferably comprises PNH, HUS or aHUS.
  • VHs preferred heavy chain variable regions
  • VLs light chain variable regions
  • VHs and VLs for treatment of human patients suffering from or at risk of suffering from complement-mediated hemolytic anemia, such as PNH, HUS or aHUS, is preferred because the humanized framework regions will reduce the immunogenicity and increase the half life of the antibodies or antibody variants within the human body. As shown in Example 7 and Figure 7A, any combination of these VHs and VLs is able to reduce
  • complement-mediated hemolytic anemia preferably PNH, HUS or aHUS
  • the method comprising administering an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, to a human individual in need thereof, wherein said antibody or functional part or functional equivalent comprises: - a variable heavy chain sequence having at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100%, sequence identity with a VH sequence selected from the group consisting of the VH sequences
  • variable light chain sequence having at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100%, sequence identity with a VL sequence selected from the group consisting of the VL sequences 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, 7F02 and 7G08 as depicted in Figure 10.
  • an antibody or functional part or functional equivalent or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with a VH sequence selected from the group consisting of the VH sequences 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, 7F02 and 7C02 as depicted in Figure 10, and wherein said antibody or functional part or functional equivalent further comprises a variable light chain sequence having at least 80% sequence identity with a VL sequence selected from the group consisting of the VL sequences 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, 7F02 and 7G08 as depicted in Figure 10, for the preparation of a medicament for at least in part preventing or treating
  • Some embodiments provide an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with a VH sequence selected from the group consisting of the VH sequences 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, 7F02 and 7C02 as depicted in Figure 10, and wherein said antibody or functional part or functional equivalent further comprises a variable light chain sequence having at least 80% sequence identity with a VL sequence selected from the group consisting of the VL sequences 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, 7F02 and 7G08 as depicted in Figure 10, for use in a method for at least in part preventing or treating complement-mediated hemolytic anemia.
  • Said % sequence identity is preferably at least 85%, more preferably at least 86%, more
  • the heavy and light chain CDRl-3 sequences of said binding compound preferably differ in no more than 3, preferably in no more than 2, more preferably in no more than 1 amino acid residues from the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10.
  • said heavy and light chain CDRl-3 sequences are identical to the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10.
  • VL 8G09 EVQLVESDGGLVQPGGSLRLSCVASGFTFSDYYMAWVRQAPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKRTLYLQMDSLRAEDTAVYYCARPSTEALFAY WGQGTLVTVSS and the amino acid sequence of VL 8G09 is
  • DIVLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTRL EIK Further provided is therefore a method for at least in part preventing or treating complement-mediated hemolytic anemia, the method comprising administering an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, to a human individual in need thereof, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
  • variable light chain sequence having at least 80% sequence identity with the sequence
  • an antibody or functional part or functional equivalent or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
  • variable light chain sequence having at least 80% sequence identity with the sequence
  • Some embodiments provide an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
  • variable light chain sequence having at least 80% sequence identity with the sequence
  • DIVLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTRL EIK for use in a method for at least in part preventing or treating complement- mediated hemolytic anemia.
  • Said % sequence identity of the above-mentioned VH and VL sequences is preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100%.
  • the heavy and light chain CDRl-3 sequences of said binding compound preferably differ in no more than 3, preferably in no more than 2, more preferably in no more than 1 amino acid residues from the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10.
  • said heavy and light chain CDRl-3 sequences are identical to the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10.
  • Said complement-mediated hemolytic anemia preferably comprises PNH, HUS or aHUS.
  • VQ LVE S D GGLVQ PGGS LKLS C AAS GFTFS D YYMAWVRQ GPGKGLE WVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRAEDTATYYCARPSTEALFAY WGHGTLVTVSS and the amino acid sequence of VL 7E 12 is
  • DWLTQTPSTLSVTPGQPASISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGIYYCMQASHAPYTFGQGTNL EIK Further provided is therefore a method for at least in part preventing or treating complement-mediated hemolytic anemia, the method comprising administering an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, to a human individual in need thereof, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
  • variable light chain sequence having at least 80% sequence identity with the sequence DWLTQTPSTLSVTPGQPASISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGIYYCMQASHAPYTFGQGTNL EIK.
  • VQ LVE S D GGLVQ PGGS LKLS C AAS GFTFS D YYMAWVRQ GPGKGLE WVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRAEDTATYYCARPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
  • Some embodiments provide an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
  • VQ LVE S D GGLVQ PGGS LKLS C AAS GFTFS D YYMAWVRQ GPGKGLE WVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRAEDTATYYCARPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
  • DWLTQTPSTLSVTPGQPASISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGIYYCMQASHAPYTFGQGTNL EIK for use in a method for at least in part preventing or treating complement- mediated hemolytic anemia.
  • Said % sequence identity of the above-mentioned VH and VL sequences is preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100%.
  • the heavy and light chain CDRl-3 sequences of said binding compound preferably differ in no more than 3, preferably in no more than 2, more preferably in no more than 1 amino acid residues from the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10.
  • said heavy and light chain CDRl-3 sequences are identical to the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10.
  • Said complement-mediated hemolytic anemia preferably comprises PNH, or HUS or aHUS.
  • VQ LVE S D GGLVQ PGGS LRLS C AAS GFTFSD YYMAWVRQ GPTKGLE WVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMDSLRAEDTAVYYCARPSTEALFAY WGHGTLVTVSS and the amino acid sequence of VL 7G09 is
  • DIVLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTRL EIK Further provided is therefore a method for at least in part preventing or treating complement-mediated hemolytic anemia, the method comprising administering an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, to a human individual in need thereof, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
  • VQ LVE S D GGLVQ PGGS LRLS C AAS GFTFSD YYMAWVRQ GPTKGLE WVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMDSLRAEDTAVYYCARPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
  • an antibody or functional part or functional equivalent or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
  • variable light chain sequence having at least 80% sequence identity with the sequence
  • Some embodiments provide an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
  • VQ LVE S D GGLVQ PGGS LRLS C AAS GFTFSD YYMAWVRQ GPTKGLE WVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMDSLRAEDTAVYYCARPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
  • DIVLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTRL EIK for use in a method for at least in part preventing or treating complement- mediated hemolytic anemia.
  • Said % sequence identity of the above-mentioned VH and VL sequences is preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100%.
  • the heavy and light chain CDRl-3 sequences preferably differ in no more than 3, preferably in no more than 2, more preferably in no more than 1 amino acid residues from the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10.
  • said heavy and light chain CDRl-3 sequences are identical to the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10.
  • Said complement-mediated hemolytic anemia preferably comprises PNH, or HUS or aHUS.
  • VL 8F07 EVQLVESGGGLVQPGGSLRLSCAASGFSFSDYYMAWVRQGPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRSEDTATYYCARPSTEALFAY WGHGTLVTVSS and the amino acid sequence of VL 8F07 is
  • DWLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPDRFSGSGSGTDFTLKISGVEAEDVGVYYCMQASHAPYTFGAGTKL EIK Further provided is therefore a method for at least in part preventing or treating complement-mediated hemolytic anemia, the method comprising administering an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, to a human individual in need thereof, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
  • variable light chain sequence having at least 80% sequence identity with the sequence
  • an antibody or functional part or functional equivalent or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
  • variable light chain sequence having at least 80% sequence identity with the sequence
  • Some embodiments provide an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
  • variable light chain sequence having at least 80% sequence identity with the sequence
  • DWLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPDRFSGSGSGTDFTLKISGVEAEDVGVYYCMQASHAPYTFGAGTKL EIK for use in a method for at least in part preventing or treating complement- mediated hemolytic anemia.
  • Said % sequence identity of the above-mentioned VH and VL sequences is preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100%.
  • the heavy and light chain CDRl-3 sequences preferably differ in no more than 3, preferably in no more than 2, more preferably in no more than 1 amino acid residues from the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10.
  • said heavy and light chain CDRl-3 sequences are identical to the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10.
  • Said complement-mediated hemolytic anemia preferably comprises PNH, or HUS or aHUS.
  • the amino acid sequence of VH 7F06 is
  • VL 7F06 EVQLVESGGGLVQPGGSLKLSCAASGFTFRDYYMAWVRQGPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNSLYLQMDSLRAEDTAVYYCARPSTEALFAY WGHGTLVTVSS and the amino acid sequence of VL 7F06 is DWLTQTPLTLSVTPGQPVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTRL ELK.
  • a method for at least in part preventing or treating complement-mediated hemolytic anemia comprising administering an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, to a human individual in need thereof, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
  • variable light chain sequence having at least 80% sequence identity with the sequence
  • an antibody or functional part or functional equivalent or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
  • variable light chain sequence having at least 80% sequence identity with the sequence
  • Some embodiments provide an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
  • variable light chain sequence having at least 80% sequence identity with the sequence
  • Said % sequence identity of the above-mentioned VH and VL sequences is preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100%.
  • the heavy and light chain CDRl-3 sequences preferably differ in no more than 3, preferably in no more than 2, more preferably in no more than 1 amino acid residues from the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10.
  • said heavy and light chain CDRl-3 sequences are identical to the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10.
  • Said complement-mediated hemolytic anemia preferably comprises PNH, or HUS or aHUS.
  • VQ LVE S D GGLVQ PGGS LKLS C AAS GFTFS D YYMAWVRQ GPTKGLE WVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCSRPSTEALFAY WGHGTLVTVSS and the amino acid sequence of VL 7F11 is
  • DWLTQTPSTLSVTPGQPVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISGVEAEDVGVYYCMQASHAPYTFGAGTRL EIK Further provided is therefore a method for at least in part preventing or treating complement-mediated hemolytic anemia, the method comprising administering an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, to a human individual in need thereof, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
  • VQ LVE S D GGLVQ PGGS LKLS C AAS GFTFS D YYMAWVRQ GPTKGLE WVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCSRPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
  • an antibody or functional part or functional equivalent or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
  • VQ LVE S D GGLVQ PGGS LKLS C AAS GFTFS D YYMAWVRQ GPTKGLE WVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCSRPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
  • Some embodiments provide an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
  • VQ LVE S D GGLVQ PGGS LKLS C AAS GFTFS D YYMAWVRQ GPTKGLE WVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCSRPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
  • DWLTQTPSTLSVTPGQPVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISGVEAEDVGVYYCMQASHAPYTFGAGTRL EIK for use in a method for at least in part preventing or treating complement- mediated hemolytic anemia.
  • Said % sequence identity of the above-mentioned VH and VL sequences is preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100%.
  • the heavy and light chain CDRl-3 sequences preferably differ in no more than 3, preferably in no more than 2, more preferably in no more than 1 amino acid residues from the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10.
  • said heavy and light chain CDRl-3 sequences are identical to the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10.
  • Said complement-mediated hemolytic anemia preferably comprises PNH, or HUS or aHUS.
  • VL 7E11 EVQLVESGGGLVQPGGSLRLSCVASGFTFSDYYMAWVRQAPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMDSLRAEDTAVYYCARPSTEALFAY WGQGTLVTVSS and the amino acid sequence of VL 7E11 is
  • DIVLTQTPLSLSATPGQSVSISCRSSQSLLNDVGNTYLYWYLQRPGQSPQLLIYL VSDLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTNL EIK Further provided is therefore a method for at least in part preventing or treating complement-mediated hemolytic anemia, the method comprising administering an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, to a human individual in need thereof, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
  • variable light chain sequence having at least 80% sequence identity with the sequence
  • an antibody or functional part or functional equivalent or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
  • variable light chain sequence having at least 80% sequence identity with the sequence
  • Some embodiments provide an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
  • variable light chain sequence having at least 80% sequence identity with the sequence
  • DIVLTQTPLSLSATPGQSVSISCRSSQSLLNDVGNTYLYWYLQRPGQSPQLLIYL VSDLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTNL EIK for use in a method for at least in part preventing or treating complement- mediated hemolytic anemia.
  • Said % sequence identity of the above-mentioned VH and VL sequences is preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100%.
  • the heavy and light chain CDRl-3 sequences preferably differ in no more than 3, preferably in no more than 2, more preferably in no more than 1 amino acid residues from the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10.
  • said heavy and light chain CDRl-3 sequences are identical to the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10.
  • Said complement-mediated hemolytic anemia preferably comprises PNH, or HUS or aHUS.
  • VL 7F02 EVQLVESGGGLVQPGGSLKLSCAASGFTFSDYYMAWVRQGPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNSLYLQMNSLRSEDTAVYYCARPSTEALFAY WGHGTLVTVSS and the amino acid sequence of VL 7F02 is
  • DWMTQTPSTLSATPGQSASISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGIYYCMQASHAPYTFGAGTRL ELK Further provided is therefore a method for at least in part preventing or treating complement-mediated hemolytic anemia, the method comprising administering an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, to a human individual in need thereof, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
  • variable light chain sequence having at least 80% sequence identity with the sequence
  • an antibody or functional part or functional equivalent or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
  • variable light chain sequence having at least 80% sequence identity with the sequence
  • Some embodiments provide an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
  • variable light chain sequence having at least 80% sequence identity with the sequence
  • Said % sequence identity of the above-mentioned VH and VL sequences is preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100%.
  • the heavy and light chain CDRl-3 sequences preferably differ in no more than 3, preferably in no more than 2, more preferably in no more than 1 amino acid residues from the heavy and light chain CDRl-3 regions of antibody 7E5, as depicted in Figure 10.
  • said heavy and light chain CDRl-3 sequences are identical to the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10.
  • Said complement-mediated hemolytic anemia preferably comprises PNH, or HUS or aHUS.
  • the amino acid sequence of VH 7C02 is
  • VL 7G08 EVQLVESGGGLVQPGGSLKLSCAASGFTFSDYYMAWVRQAPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPSTEALFAY WGHGTLVTVSS and the amino acid sequence of VL 7G08 is
  • DIVMTQTPLSLSATPGQPASISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQASHAPYTFGQGTKL EIK Further provided is therefore a method for at least in part preventing or treating complement-mediated hemolytic anemia, the method comprising administering an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, to a human individual in need thereof, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with
  • variable light chain sequence having at least 80% sequence identity with
  • an antibody or functional part or functional equivalent or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with
  • variable light chain sequence having at least 80% sequence identity with
  • Some embodiments provide an antibody or functional art or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with
  • variable light chain sequence having at least 80% sequence identity with
  • DIVMTQTPLSLSATPGQPASISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQASHAPYTFGQGTKL EIK for use in a method for at least in part preventing or treating complement- mediated hemolytic anemia.
  • Said % sequence identity of the above-mentioned VH and VL sequences is preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100%.
  • the heavy and light chain CDRl-3 sequences preferably differ in no more than 3, preferably in no more than 2, more preferably in no more than 1 amino acid residues from the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10.
  • said heavy and light chain CDRl-3 regions are identical to the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10.
  • Said complement-mediated hemolytic anemia preferably comprises PNH, or HUS or aHUS.
  • nucleic acid molecule(s) or functional equivalent(s) thereof encoding for any of the above-recited VHs and/or VLs is/are used in a method or use according to the invention.
  • Said nucleic acid molecule(s) or functional equivalent(s) preferably comprise:
  • nucleic acid sequence that has at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100% sequence identity with a VH nucleic acid sequence selected from the group consisting of the VH nucleic acid sequences of 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, 7F02 and 7C02 as depicted in Figure 10, and - a nucleic acid sequence that has at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%
  • antibody 7E5 is able to bind amino acid residues 835-854 of the human C6 sequence as depicted in Figure 11.
  • C6-specific binding compounds that are able to bind these amino acid residues are therefore preferred binding compounds for the methods and uses according to the present invention, because these binding compounds bind and inhibit human C6 so that erythrocyte lysis is at least in part diminished.
  • a method for at least in part preventing or treating complement-mediated hemolytic anemia comprising administering an antibody or functional part or functional equivalent, or a nucleic acid molecule encoding therefore, to a human individual in need thereof, wherein said antibody or functional part or functional equivalent is able to bind amino acid residues 835-854 of the human C6 sequence as depicted in Figure 11.
  • an antibody or functional part or functional equivalent or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent is able to bind amino acid residues 835-854 of the human C6 sequence as depicted in Figure 11, for the preparation of a medicament for at least in part preventing or treating complement-mediated hemolytic anemia.
  • embodiments provide an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent is able to bind amino acid residues 835-854 of the human C6 sequence as depicted in Figure 11, for use in a method for at least in part preventing or treating complement- mediated hemolytic anemia.
  • said complement-mediated hemolytic anemia preferably comprises aHUS or PNH.
  • a use of a human or humanized antibody is preferred for therapeutic applications in humans, since such human or humanized antibodies typically elicit a diminished immune response, and have a longer half life, as compared to antibodies from non-human animals.
  • complement-mediated hemolytic anemia such as PNH, HUS or aHUS
  • antibodies or functional parts or functional equivalents thereof that do not, or to a little extent, induce complement- dependent cytotoxicity (CDC), since this activates the complement system.
  • CDC is most strongly mediated by the Fc regions of IgG3 and IgGl antibodies.
  • IgG4 antibodies do not or barely induce CDC. Therefore, in a preferred embodiment, a method or use according to the invention is provided wherein an antibody is used that is of the IgG4 isotype.
  • binding compounds are used in a method or use according to the invention wherein the binding compounds are monoclonal antibodies.
  • a monoclonal antibody is an antibody consisting of a single molecular species.
  • An advantage of monoclonal antibodies is the fact that they can be produced in large quantities by monoclonal antibody-producing cells or
  • a C6-specific antisense nucleic acid molecule is used against complement-mediated hemolytic anemia.
  • Non-limiting examples include oligomers, aptamers, short interfering RNA (siRNA), microRNA (miRNA) and ribozymes.
  • oligomer means a single stranded or double stranded nucleic acid molecule that contains natural and/or non-natural nucleotides which are bound to each other via backbone linkages, thereby forming an oligonucleotide.
  • Non-limiting examples of oligomers are DNA, RNA, DNA/RNA helix, peptide nucleic acid (PNA) and locked nucleic acid (LNA).
  • WO 2010/005310 which is incorporated herein by reference. This patent application discloses preferred C6-specific oligomers and is from the same (co)-inventor as the current application.
  • WO 2010/005310 concerns neurological applications for the C6-specific oligomers and does not concern treatment of complement-mediated hemolytic anemia.
  • C6-specific oligomers as described in WO 2010/005310 are also suitable for counteracting complement-mediated hemolytic anemia such as PNH, HUS or aHUS.
  • oligonucleotide refers to a polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or analogues thereof.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally- occurring portions.
  • modified or substituted oligonucleotides are used in view of their preferred properties such as, for instance, increased stability and enhanced cellular uptake.
  • an oligomer is used in a method or use according to the present invention, wherein the oligomer has a length of between 10 to 50 nucleotides and wherein the oligomer has a contiguous nucleic acid sequence with at least 80% sequence identity to a complementary region of the human C6 sequence as depicted in Figure 11.
  • such oligomers are particularly suitable for counteracting C6 activity.
  • a method for at least in part preventing or treating complement-mediated hemolytic anemia comprising administering to a human individual in need thereof an oligomer of between 10 to 50 nucleotides in length having a contiguous nucleic acid sequence with at least 80% sequence identity to a complementary region of the human C6 sequence as depicted in Figure 11. Also provided is a use of an oligomer of between 10 to 50 nucleotides in length having a contiguous nucleic acid sequence with at least 80% sequence identity to a complementary region of the human C6 sequence as depicted in Figure 11, for the preparation of a medicament for at least in part preventing or treating complement-mediated hemolytic anemia.
  • Some embodiments provide an oligomer of between 10 to 50 nucleotides in length having a contiguous nucleic acid sequence with at least 80% sequence identity to a complementary region of the human C6 sequence as depicted in Figure 11, for use in a method for at least in part preventing or treating complement-mediated hemolytic anemia.
  • said sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least
  • Said complement-mediated hemolytic anemia preferably comprises PNH, or HUS or aHUS.
  • a method or use according to the invention wherein the oligomer comprises a moiety that facilitates liver uptake.
  • This will increase the in vivo accumulation of the oligomer in the liver, thereby enhancing C6 inhibition since human C6 is predominantly produced in the liver.
  • a non-limiting example of a moiety that facilitates liver uptake is triantennary N-acetyl galactosamine (GalNac or GN3), as described in Prakash et al.,
  • said inhibitor against human complement component C6 is an oligomer of between 10 to 50 nucleotides in length having a contiguous nucleic acid sequence with at least 80% sequence identity to a complementary region of the human C6 sequence as depicted in Figure 11, which oligomer is coupled to a triantennary N-acetyl galactosamine moiety.
  • Preferred oligomers include at least one nucleotide analogue.
  • a method or use according to the invention is provided wherein the oligomer comprises a modified internucleoside linkage.
  • the oligomer comprises a modified nucleobase.
  • said nucleotide analogue is a modified sugar moiety selected from the group consisting of: 2'-0-methoxyethyl modified sugar moiety, a 2'-methoxy modified sugar moiety, a 2'-0-alkyl modified sugar moiety, and a bicyclic sugar moiety.
  • the bicyclic sugar moiety is a locked nucleic acid (LNA) monomer.
  • LNA is a modified RNA nucleotide wherein the ribose moiety contains an extra bridge connecting the 2' oxygen and 4' carbon. This additional bridge "locks" the ribose in the 3'-endo (North) conformation.
  • the use of LNA typically increases the hybridization sensitivity and specificity of the oligomer.
  • the modified internucleoside linkage is a phosphorothioate internucleoside linkage.
  • the modified nucleobase is 5-methylcytosine.
  • C6-specific oligomers that target nucleotides 112-152, 433-473, 546-586, 706-746, or 1015- 1055 from the ATG start site of the human C6 sequence as depicted in Figure 11 (which is referred to in
  • WO 2010/005310 as SEQ ID NO: l) are preferred since it has been demonstrated that oligomers that target these specified C6 regions have particular good in vivo C6 inhibiting activity. This is exemplified in WO 2010/005310 by five
  • oligomers 1008, 1009, 1010, 1011 and 1012, shown in Table 1 on page 64 of WO 2010/005310) are all very efficient in reducing C6 mRNA levels in vivo, thereby reducing C6 activity.
  • targeting the particular C6 regions as recited above is particularly preferred for reducing C6 activity. This enables treatment of disorders associated with the formation of the membrane attack complex (MAC), such as PNH, or HUS or aHUS.
  • MAC membrane attack complex
  • a method for at least in part preventing or treating complement-mediated hemolytic anemia comprising administering to a human individual in need thereof an oligomer of between 10 to 50 nucleotides in length having a contiguous nucleic acid sequence with at least 80% sequence identity to a complementary region of the human C6 sequence as depicted in Figure 11, wherein the oligomer is targeted to about nucleotides 112- 152, 433-473, 546-586, 706-746, or 1015-1055 from the ATG start site of the human C6 sequence as depicted in Figure 11.
  • Some embodiments provide an oligomer of between 10 to 50 nucleotides in length having a contiguous nucleic acid sequence with at least 80% sequence identity to a complementary region of the human C6 sequence as depicted in Figure 11, wherein the oligomer is targeted to about nucleotides 112-152, 433- 473, 546-586, 706-746, or 1015-1055 from the ATG start site of the human C6 sequence as depicted in Figure 11, for use in a method for at least in part preventing or treating complement-mediated hemolytic anemia.
  • the oligomer comprises at least one nucleotide analogue.
  • the oligomer is capable of reducing the level of C6 mRNA expression in a mammal by at least 20% as determined by a qPCR assay.
  • said sequence identity is preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% or even 100%.
  • Said complement-mediated hemolytic anemia preferably comprises PNH, or HUS or aHUS.
  • Said oligomers preferably comprise a sequence selected from the group consisting of TTGTCTCTGTCTGCTC (oligo 1008 of WO 2010/005310),
  • TAACTTGCTGGGAATA (ohgo 1009 of WO 2010/005310), CCCATCAGCTGCACAC (oligo 1010 of WO 2010/005310), TTCT ATAGTTT GTAC C (oligo 1011 of
  • WO 2010/005310 and GTTGTATTCTAAAGGC (oligo 1012 of WO 2010/005310).
  • at least one of the nucleotides of these oligomers are LNA.
  • the first three nucleotides and the last three nucleotides of these oligomers are LNA.
  • an "individual” or “subject” is preferably a human individual. Said individual preferably suffers from, or is at risk of suffering from, a disorder involving complement-mediated hemolytic anemia, such as PNH, or HUS or aHUS.
  • a disorder involving complement-mediated hemolytic anemia such as PNH, or HUS or aHUS.
  • a C6 inhibitor can be administered to a human individual in a method according to the invention using any suitable route for administration.
  • a C6 inhibitor is administered to an individual orally, by aerosol or as a suppository.
  • liposomes are also practically useful.
  • a C6 inhibitor is administered via one or more injections, such as intraperitoneal, intravascular or intramuscular injections.
  • Dose ranges of C6 inhibitors to be used in the therapeutic applications as described herein are typically designed on the basis of rising dose studies in clinical trials for which rigorous protocol requirements exist.
  • Typical doses of administration of a C6 inhibitor in a method according to the invention are between 0.1 and 50 mg per kg body weight, preferably between 0.1 and 35 mg per kg body weight.
  • treatment is started with between one and ten high dose
  • the C6 inhibitor is administered daily, weekly, two-weekly or 4-6 weekly.
  • treatment with C6- inhibiting oligomers is preferably started with daily dosages of between 1- 10 mg per kg body weight, followed by weekly, two-weekly or 4-6 weekly dosages of between 0.1 and 1 mg per kg body weight.
  • a C6 inhibitor is combined with a pharmaceutically acceptable carrier, adjuvant, diluent and/or excipient.
  • suitable carriers for instance comprise keyhole limpet haemocyanin (KLH), serum albumin (e.g. BSA or RSA) and ovalbumin.
  • KLH keyhole limpet haemocyanin
  • BSA serum albumin
  • ovalbumin ovalbumin.
  • suitable adjuvants oil -based and water-based, are known to a person skilled in the art.
  • said adjuvant comprises Specol.
  • said suitable carrier comprises a solution like for example saline.
  • Some embodiments provide a use of an inhibitor of human complement component C6 for counteracting hemolysis. This encompasses in vivo uses of such C6 inhibiting compound, for instance for PNH, HUS or aHUS treatment.
  • hemolysis is counteracted ex vivo, for instance in a hemolytic assay in order to determine complement activity against erythrocytes.
  • ex vivo procedure is for instance useful for determining whether erythrocytes are prone to lysis by complement.
  • erythrocytes from a human individual who is suspected of PNH are subjected to complement, either in the presence or absence of a C6-inhibiting compound. If these erythrocytes appear to be lysed by complement, and if a C6 inhibiting compound appears to be able to reduce such lysis, it is concluded that the erythrocytes are prone to complement lysis.
  • a blood sample from which the erythrocytes are derived is preferably typed as containing erythrocytes that are prone to complement lysis.
  • complement mediated hemolytic anemia for instance PNH, HUS or aHUS
  • the current application may describe features as parts of the same embodiment or as parts of separate embodiments, the scope of the present invention also includes embodiments comprising any combination of all or some of the features described herein.
  • Figure 1A is a bar graph showing the results of a haemolytic assay using supernatants from 38 hybridoma from two different rats immunized with human C6.
  • Figure IB is a bar graph showing the results of a mannan-activated
  • Figure 2 is a graph showing the kinetics for recombinant rat 7E5 binding to human C6 as measured with Label-free surface plasmon resonance (Biacore).
  • Figure 3A-D are figures showing data of the epitope cross-blocking experiment between 27B1 mAb and 7E5 mAb competing for binding for C6 as measured with Label-free surface plasmon resonance (Biacore). The results show that 7E5 occupies a different epitope on C6 than 27B1.
  • Figure 4A is an alignment of the human C6 and rat C6 partial amino acid sequences showing the location of peptide 418.
  • Figure 4B is a diagram of the human C6 protein with the location of peptide 418 indicated by an arrow.
  • Figure 5 shows the results of an in vivo experiment in rats proving that 7E5 blocks C6 in vivo in C6 deficient rats supplemented with human C6, as measured by haemolytic assay.
  • Rats received 2mg of human C6 suppletion (intravenously) and subsequent 12 mg of antibody 7E5 (intravenously).
  • Complement activity is plotted on the Y-axis, whereby O.D. 1.0 indicates maximum lysis of sheep erythrocytes (as measured in the supernatant at 405nm using a spectrophotometer), and an O.D. 0 indicates absence of lysis.
  • Figure 6A is the alignment of the amino acid sequences of the heavy chain variable regions of rat anti-C6 7E5 mAb and human VH3_1 germline, with differences indicated. Indicated below the alignment are the amino acid exchanges targeted for humanization.
  • Figure 6B is the alignment of the amino acid sequences of the light chain variable regions of rat anti-C6 7E5 mAb and human Vk2_5 germline, with differences indicated. Indicated below the alignment are the amino acid exchanges targeted for humanization.
  • Figure 7 shows the results of haemolytic assays demonstrating the inhibitory activity on erythrocyte lysis of all 81 possible combinations of the 9 humanized 7E5 variant VH chains (shown in Figure 7A) and 9 humanized 7E5 variant VL chains (shown in figure 7B) .
  • Figure 8A depicts an alignment of the amino acid sequence of the 7E5 heavy chain variable region with the heavy chain amino acid sequences of the humanized 7E5 variants 7C02, 7E11, 7E12, 7F02, 7F06, 7F11, 7G09, 8F07 and 8G09. The conserved CDR1, 2 and 3 regions are indicated.
  • Figure 8B depicts the amino acid sequence of the 7E5 light chain variable region aligned with the light chain amino acid sequences of the humanized 7E5 variants 7E11, 7E12, 7F02, 7F06, 7F11, 7G08, 7G09, 8F07 and 8G09. The conserved CDR1, 2 and 3 regions indicated.
  • Figure 9 depicts the affinity as measured with Label-free surface plasmon resonance (Biacore) of 8 humanized F'Abs for human C6 (coated on the biacore chip) in comparison to the affinity of the wild type 7E5 rat F'Ab.
  • Biacore Label-free surface plasmon resonance
  • Figure 10 depicts the amino acid and nucleic acid sequences of the heavy and light chain CDR 1-3 regions and the VH and VL regions of antibody 7E5. Also depicted are the sequences of several humanized VHs and VLs.
  • Figure 11 depicts a nucleic acid sequence encoding human C6 mRNA (Genbank Ref. NM_000065.2). The ATG start site is indicated.
  • Figure 12 shows that antibody 7E5 counteracts anti CD59 induced lysis. The experiment was performed in 1.5% human serum. Examples
  • Example 1 Generation of Rat Anti-Human C6 Monoclonal Antibodies Five rats (PVG C6 -/-) were immunized against human C6 protein. C6 deficient rats were chosen because according to the current understanding in the field it is extremely difficult to generate functional C6 antibodies in normal rodents. For unknown reasons immunization against C6 is not efficient in wild type animals. The idea is that in C6 deficient animals the antibody response is more robust because these animals have no functional C6 protein in circulation and are likely to consider C6 as completely "foreign.” Human C6 was purified from whole human serum by means of affinity chromatography using 23D1 mouse monoclonal antibody 23D1 (described in detail in L. Clayton (2005) Ph.D. Thesis, Cambridge University) coupled to Sepharose (GE Healthcare Cat No. 17-0717-01).
  • a pre- immunization bleed was performed on the rats by collecting 100 ⁇ of blood from the tail vein.
  • rats were injected at four locations subcutaneously (s.c.) with 100 ⁇ g C6 antigen in Complete Freund's Adjuvant (CFA), in a volume of 250 ⁇ per injection.
  • CFA Complete Freund's Adjuvant
  • Booster injections were performed on Days 14 and 21, again at four s.c. locations with 50 ⁇ g C6 antigen in Incomplete Freund's Adjuvant (IFA) in a volume of 250 ⁇ per injection.
  • Test bleeds were performed on Day 36 by collecting 100 ⁇ of blood from the tail vein for in vitro tests.
  • test bleeds were analyzed in a C6 ELISA, a C6 Western blot and in a haemolytic assay (described further below), which showed that all five rats had a positive immune response against human C6: all five rats had antibodies that blocked hemolysis in the haemolytic assay and all five rats had antibodies that recognized purified C6 on Western blot (denaturing conditions).
  • a pre-fusion booster was performed on Day 62 by injection of 100 ⁇ g antigen in 250 ⁇ PBS intraperi tone ally. Finally, a pre-fusion booster was performed on Day 64 by injection of 100 ⁇ g antigen in 250 ⁇ PBS intravenously (tail vein).
  • Hybridoma Preparation Hybridomas were prepared by fusion of the splenocytes from the human C6-immunized rats with Y3-Agl.2.3 fusion partner cells using standard polyethylene glycol (PEG)-mediated fusion essentially as described in Luk, J. M. et al. (1990) J. Immunol. Methods 129:243-250.
  • PEG polyethylene glycol
  • erythrocyte lysis can be quantified by measuring the OD at 405 or 415 nm in the supernatant and is a direct
  • Complement inhibitors can be tested in this system because if they are effective they will prevent erythrocyte lysis in a quantitative fashion.
  • a haemolytic system ready to use was obtained commercially (Virion/Serion GmbH, Wurzburg, Gemany) along with CFT buffer (Virion/Serion GmbH, Wurzburg, Gemany).
  • the CFT buffer was prepared according to the manufacturer's instructions.
  • the haemolytic system was placed on a rollerbank in a coldroom to thoroughly mix the erythrocytes.
  • To prepare a CFT serum cocktail 100 ⁇ of human serum was added to 5 ml of CFT buffer.
  • test inhibitors Dilutions of test inhibitors, in a volume of 50 ⁇ , were added to round bottom 96- well plates, 50 ⁇ of CFT serum cocktail was added to each well and mixed carefully while pipetting and the plates were incubated at 37° C for 30 minutes. Positive controls was EDTA. Negative control was serum free or C6 deficient serum. After incubation, plates were spun down at 2000 rpm for 5 minutes (Hettich table top centrifuge) and 80 ⁇ of supernatant was transferred to flat bottom plate for measurement at 405 or 415 nm. The OD was measured within 10 minutes of transfer.
  • Test supernatants were added in dilution in the haemolytic assay to determine whether they prevent erythrocyte lysis. Exemplary results are shown in Figure 1A, which demonstrates that certain of the supernatants exhibited stronger inhibitory activity than others.
  • supernatants #6- 12 exhibited stronger inhibition than the other supernatants, with supernatants #11 and #12 showing the strongest inhibition.
  • this assay shows that under conditions that are indicative for PNH, supernatants #6-12 are well capable of inhibiting erythrocyte lysis.
  • the supernatants (1:50 dilution) were also tested in the haemolytic assay using rat serum as complement source and no inhibitory effect was observed, demonstrating that the inhibitory activity of the antibodies was specific for human C6.
  • MAC ELISA Assay We used a second assay to determine whether the supernatants are able to block MAC activity.
  • the ELISA wells in the plate are coated with either Mannan or IgG as trigger for either the Lectin or the Classical pathway of complement, respectively, in the presence of serum.
  • the serum contains the components of the complement system which are activated through either pathway when they are exposed to the coated plate.
  • the MAC is formed as part of the terminal complement system and MAC will be deposited on the ELISA plate. MAC deposition on the plate can be detected by HRP-conjugated antibodies and visualized by enzymatic reaction in the presence of a chromogen and substrate.
  • This reaction produces a color that can be quantified by measuring the OD at 450 or 655 nm.
  • the OD is a direct measurement of the amount of MAC formation.
  • Complement inhibitors can be tested in this system because if they are effective they will prevent or inhibit deposition of MAC on the plate.
  • a mannan activated complement ELISA assay ELISA plates were coated with mannan and diluted hybridoma supernatant and human serum was added. Complement components that form a complex on the mannan coated plate can be detected using antibodies. In this particular assay we looked for detection of C9 which indicates formation of MAC. If less C9 is detected this indicates MAC inhibition.
  • the positive controls in this experiment are EDTA, because the reaction is calcium dependent.
  • coating buffer (15 mM Na 2 C0 3 , 35 mM NaHCOs, 15 mM NaN 3 , pH 9.6), blocking buffer (1 mg/ml BSA/HAS, 10 mM Tris/HCl, pH 7.4, 145 mM NaCl, 15 nM NaN 3 , pH 7.4) wash buffer (1 x TBS, 0.05% Tween 20, 5 mM CaC ) and dilution buffer (4 mM barbital, 145 mM NaCl, 2 mM CaC , 1 mM MgC , 0.3% BSA, 0.02% Tween 20) were used.
  • TMB Sigma T2885; stock solution prepared of 10 mg/ml TMB in DMSO
  • 10 ⁇ 3% H202 were added to 5 ml NaAc buffer (8.2 gm Natrium Acetate, 21 gm Citric Acid Monohydrate in 1 liter H2O) and distributed to the 96-well plates. The reaction was stopped with 25 ⁇ 1 M H2SO4 and the OD was measured with a
  • the surface was blocked with a 7 minute injection of 1 M ethanolamine, pH 8.0.
  • Flow cell 1 was immobilized with an antibody from an earlier experiment (avWWF; 987 RU) and served as a reference surface.
  • the analytes (anti C6- antibodies, 150 kDa) in 10 mM HEPES, 150 mM NaCl, 0.005% P20, pH 7.4, were injected over the two flow cells at a flow rate of 30 ⁇ /min and at a temperature of 25°C. The injected concentrations differ per antibody. Data were collected at a rate of 1 Hz. The complex was allowed to associate and dissociate for 90 and 300 seconds, respectively. The surfaces were regenerated with a 10 second injection of 0.1 M HC1. Duplicate injections (in random order) of each sample and a buffer blank were flowed over the two surfaces.
  • Table 3 Time to 5% Dissociation for 7E5 Binding kd (s- 1) Time (min) Time (hour) R (RU)
  • Table 4 Time to 95% Dissociation for 7E5 Binding kd (s-1) Time (min) Time (hour) R (RU)
  • the 7E5-C6 complex on the chip was washed with buffers with decreasing pH. Hepes buffered saline ((HBS) was used for pH 7.4, 7.0 and 6.5. lOmM Sodium Acetate was used for pH 6.0, 5.5, 5.0, 4.5 and 4.0. It was observed that the stability of the complex is not sensitive for low pH.
  • HBS Hepes buffered saline
  • lOmM Sodium Acetate was used for pH 6.0, 5.5, 5.0, 4.5 and 4.0. It was observed that the stability of the complex is not sensitive for low pH.
  • Example 3 Epitope Mapping of 7E5 Monoclonal Antibody
  • peptide arrays Consecutive overlapping 16mer peptides (peptides 16 amino acids long, overlapping 14 amino acids) from the C6 protein sequence were synthesized and spotted in a grid pattern on a membrane. The membrane was then incubated with 7E5 antibody to detect which peptide was recognized by the antibody.
  • the primary peptide sequence recognized by 7E5 was GS CQD GRQLE WGLERT (peptide 418).
  • Figure 4A shows the sequence of peptide 418 and surrounding area in human and rat C6. As illustrated schematically in Figure 4B, peptide 418 is partially located at the end of the first FIM domain of C6.
  • a Biacore cross blocking experiment was conducted in which the C6 antigen was coupled to the chip, followed by flow of the analyte(s), which was either a single anti-C6 antibody alone (as a control) or a first anti-C6 antibody (Antibody 1) followed by a second anti-C6 antibody (Antibody 2) to determine cross- blocking.
  • the results for the cross -blocking experiment to determine whether the mouse mAb 27B1 binds the same epitope as the rat mAb 7E5 are shown in Figure 3A-D, wherein Fig. 3A shows the results with 27B1 as Antibody 1 and 7E5 as Antibody 2, Fig. 3B shows the results with 7E5 as Antibody 1 and 27B1 as
  • Serum from a wild type PVG rat and a non-treated C6 deficient rat were used as references for maximal and minimal haemolytic activity.
  • the half-life of human C6 in the rats was estimated to be about 48 hrs.
  • VH and VL sequences of the 7E5 mAb were determined using standard technology known in the art.
  • the nucleotide sequence of the VH region is as follows: gaggtgcagctggtggagtctgatggaggcttagtgcagcctggagggtccctgaaactctcctgtgtagcctcaggattctctttt cagtgactattacatggcctgggtccgccagggtccaacgaaggggctggagtgggtcgcaaccattaattatgatggtagtag tacttactatcgagagtccgtgaagggccgattcactatctccagagataatgcgaaacgcaccctatacctgcaaatggacag tctgaggtctgaggacacggccacttattactgttcaagaccttctacggaggcctgtttgtttgtttgctttactggggccacgg
  • amino acid sequence of the VH region is as follows:
  • amino acid sequences of the VH CDR1, CDR and CDR3 are as follows:
  • the nucleotide sequence of the VL region is as follows:
  • amino acid sequences of the VL CDR1, CDR and CDR3 are as follows:
  • the closest human germline is human VK2 family member 5.
  • the closest human JH germline appeared to be IGKJ2 and IGKJ5.
  • the alignment against these germline segments is shown in Figure 6B.
  • the FRs and CDRs are indicated, which enables the identification of FR residues deviating from the human germlines.
  • the 7E5 light chain amino acid sequence is also shown in Figure 10.
  • Humanized 7E5 Fab Library Construction For the construction of the final humanized 7E5 Fab phage display library, we initially constructed two different sub-libraries:
  • Human C6 was biotinylated and QCed by SDS-PAGE, Western Blot and ELISA using the anti-human C6 antibody 7E5 to detect the biotinylated C6 captured on neutravidin coated plates.
  • phages incubated with neutravidin- captured human C6 were also incubated in the presence of an excess of non- biotinylated C6 for 2 hours or overnight (off-rate selections) in an attempt to, after several washings, get rid of high off-rate binding clones.
  • coli TGI infected with the eluted phage pools obtained after the second and third round of off-rate selections were grown at 37 °C for 8 hours in two 96 well plates (Master plates) containing 100 ⁇ of 2TYGlucose2% Carbenicillin 100 pg/ml, stored in 20% glycerol at -80 °C and used for later sequencing, and periplasmic extract production.
  • Master plates 100 ⁇ of 2TYGlucose2% Carbenicillin 100 pg/ml, stored in 20% glycerol at -80 °C and used for later sequencing, and periplasmic extract production.
  • a total of two master plates (MPs) were generated with clones from the second round selections and from the third round selections. From these MPs, bacterial extracts containing soluble
  • monoclonal Fabs (periplasmic extracts) were produced. Monoclonal bacterial small-scale cultures were induced at ⁇ of 0.8 by adding isopropyl-b-D- thiogalactopyranoside (IPTG) to a final concentration of 1 mM. The periplasmic extracts (P.E.s) containing Fabs were then prepared by freezing- thawing of the bacterial pellet in PBS and subsequent centrifugation to remove cell debris.
  • IPTG isopropyl-b-D- thiogalactopyranoside
  • P.E.s at 1:5 dilution were tested for binding to 10 nM of biotinylated hC6 captured on neutravidin-coated Maxisorp plate.
  • P.E. prepared from rat 7E5 WT Fab was used as positive control.
  • Blank P.E. (prepared from non-inoculated well in the MP) was used as negative controls.
  • Binding of P.E.s to the target was detected with an anti-c-myc mouse antibody conjugated to Horseradish peroxidase (HRP). A binding hit rate of 40% was obtained for both MPs and binding signals (O.D. 450nm values) of the positive clones were comparable to the signal obtained with the parental rat 7E5 Fab.
  • a Biacore 3000 (GE Healthcare) was used. For that purpose, 50 pg/ml of hC6 in acetate buffer pH 4.5 was immobilized on a CM5 sensor chip (GE Healthcare BR- 1000- 12) to approximately 2000 RU. Regeneration conditions were tested and 2 x 10 ⁇ of 10 mM NaOH and 1 M NaCl were used for the regeneration between sample injections. 30 ⁇ of P.E.s, prepared as described above, were diluted in 120 ⁇ of HBS-EP buffer and from this 60 ⁇ were injected with a flow of 30 ⁇ /min. Dissociation was measured during 400 seconds and the off-rate was determined by applying the 1: 1 Langmuir dissociation fitting model.
  • a lead panel of eight clones that had good binding, off-rate and human identity and homology data were selected, referred to as 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11 and 7F02.
  • the complete nucleotide and amino acid sequences of the variable domains of the heavy and light chains of the lead panel of eight humanized clones are shown below:
  • FIG 8B An alignment of the amino acid sequence of the rat 7E5 light chain variable region to the amino acid sequences of the light chain variable regions of the humanized 7E5 variants 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11 and 7F02 is shown in Figure 8B, with CDR1, 2 and 3 indicated.
  • the heavy chain CDR1, 2 and 3 sequences for the eight humanized variants of the rat 7E5 antibody are the same as those in the rat 7E5 mAb (the amino acid sequences of which are shown in Figure 10).
  • the light chain CDR1, 2 and 3 sequences for the eight humanized variants of the rat 7E5 antibody are the same as those in the rat 7E5 mAb (the amino acid sequences of which are also shown in Figure 10).
  • Fab Expression, Purification and QC In order to characterize some of the 7E5 humanized variants in further assays (i.e., complement mediated lysis of pre- sensitized erythrocytes assay, affinity determination, melting temperatures and aggregation behavior assays), soluble Fabs were produced and purified from the lead panel of eight clones described above.
  • the Fab genes of all 8 humanized clones plus the 7E5 WT control were cloned into pCB4 expression vector (very similar to pCB13 but without the gene 3 codifying sequence) via Sfil/Notl digestion and transformed into TGI E.coli strain via heat shock. The sequences were confirmed using the CLC Main Workbench Software.
  • Production of P.E.s containing soluble Fabs from the pCB4-cloned 7E5 humanized variants as well as from 7E5 WT was performed in 800 ml of 2xYT supplemented with 0.1% of glucose and Carbenicillin at 100 pg/ml. After induction at OD600 of 0.5-0.8 with IPTG to a final concentration of 1 mM, the culture was incubated at 24 °C for at least 20 hours. The soluble Fabs were purified with TALON metal affinity resin.
  • Biacore affinity analysis was performed on the eight selected humanized Fabs (7E12, 7E11, 7F2, 7F6, 7F11, 7G9, 8F7 and 8F9) as compared to the (parental) wild-type rat 7E5 mab and to the mouse 27B1 mAb. The results are shown in Figure 9. The results indicate that humanization of 7E5 did not alter the specificity or activity of the antibody.
  • Example 7 "Mix & Match" Characterization of Humanized anti-C6
  • the humanized VH chains used were the eight VH chains described in Example 6 (8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11 and 7F02), as well as a ninth chain, 7C02, the amino acid sequence of which is shown in Figure 8A. An alignment of these nine chains is shown in Figure 8A.
  • the humanized VL chains used were the eight VH chains described in Example 6 (8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11 and 7F02), as well as a ninth chain, 7G08, the amino acid sequence of which is shown in Figure 8B. An alignment of these nine chains is shown in Figure 8B.
  • the heavy and light chain nucleotide sequences were cloned into expression vectors to create coding sequences for full-length chains having a stabilized IgG4 (S228P) constant region.
  • the 9 heavy chains and 9 light chains were co-expressed as pairs in every possible combination in CHO host cells. Thus, all 81 possible "mix and match" combinations of the 9 heavy chains and 9 light chains were evaluated. The 81 pairs were each tested in the hemolytic assay and in the MAC ELISA. For each assay, 4 ⁇ g of humanized 7E5 mAb from CHO
  • human erythrocytes were incubated with a blocking monoclonal antibody (Mab) against CD59. This rendered the erythrocytes susceptible to hemolysis by complement, like the CD55- and/or CD59-deprived erythrocytes of a PNH patient.
  • the Mab that was used is a IgG2A which activates complement.
  • 1.5% human serum was added to the erythrocytes. This resulted in complement-mediated lysis of the vulnerable erythrocytes.
  • a C6 inhibitor like 7E5 is able to counteract complement-mediated lysis of vulnerable erythrocytes like the CD55- and/or CD59- deprived erythrocytes of a PNH patient.

Abstract

The invention provides means and methods for counteracting complement-mediated hemolytic anemia, which involves the use of an inhibitor of human complement component C6.

Description

Title: Means and methods for counteracting complement-mediated hemolytic anemia
The invention relates to the fields of biology, immunology and medicine.
Hemolytic anemia involves the lysis of erythrocytes. This can occur within blood vessels (intravascular) or at other sites within the body (extr avascular). It has many possible causes, which are either inherited or acquired. One disorder involving hemolytic anemia is haemolytic uremic syndrome (HUS). HUS is typically defined by the simultaneous occurrence of microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. The most common cause of HUS is the Shiga toxin-producing Escherichia coli (STEC). Research over the last 20 years has shown that complement dysregulation accounts for most of the non- STEC cases of HUS such as atypical HUS (aHUS). Primary causes for aHUS are dysregulation of complement by either mutations or antibodies. Secundary causes of aHUS are infections.
Another severe disorder involving hemolytic anemia is paroxysmal nocturnal hemoglobinuria (PNH), which was previously referred to as
Marchiafava-Micheli syndrome. PNH is a genetically acquired life -threatening disorder involving lysis of erythrocytes by the complement system. Complement components assemble at the surface of erythrocytes and form the Membrane Attack Complex (MAC), resulting in lysis. This effect is due to an impaired formation of functional surface proteins CD55 and/or CD59 on the surface of erythrocytes, which normally protect the erythrocytes from complement activity. Normally, CD55, which is also known as complement decay accelerating factor
(DAF), protects erythrocytes by degrading C3, thereby counteracting the formation of MAC at the surface of the erythrocyte. CD59, which is also known as MAC- inhibitory protein (MAC-IP), is able to bind complement factors C8 and C9 and to inhibit C9 polymerization. As a result, the MAC cannot insert into the cell membrane, so that osmolytic pore formation is prevented. In healthy individuals, CD55 and CD59 are linked to erythrocytes via a glycosylphosphatidylinositol (GPI) anchor. PNH patients exhibit a defect in the assembling of these glycolipid-protein structures, so that CD55 and/or CD59 are not, or to a lesser extent, coupled to erythrocytes. The most common defective enzyme in PNH is phosphatidylinositol glycan A (PIGA), one of several enzymes needed to make GPL PNH is considered an intravascular hemolytic anemia, since the complement cascade attacks the erythrocytes within the circulatory system. Symptoms of PNH are, amongst other things, fatigue, shortness of breath and palpitations. The most characteristic sign of PNH is a red discoloration of the urine, which is a result of free hemoglobulin and hemosiderin, released from lysed erythrocytes.
Currently, PNH patients are treated with blood transfusions. Eculizumab (trade name Soliris), which is a humanized monoclonal antibody against
complement component C5, is used in order to decrease PNH symptoms and the need for blood transfusions. Drawbacks of eculizumab, however, are its high costs and the occurrence of various side effects such as an increased sensitivity for infections. Moreover, C5a is essential for regeneration of liver cells. Therefore, if an individual has liver damage, eculizumab will inhibit liver repair.
There is, therefore, a need for alternative treatment options for complement- mediated hemolytic anemia disorders, such as HUS and PNH.
The present invention provides such alternative means and methods for counteracting complement-mediated hemolytic anemia. In one aspect, the invention provides a method for at least in part preventing or treating complement- mediated hemolytic anemia, the method comprising administering an inhibitor of human complement component C6 to a human individual in need thereof. Some embodiments provide an inhibitor of human complement component C6 for use in a method for at least in part treating or preventing complement-mediated hemolytic anemia. Some embodiments provide a use of an inhibitor of human complement component C6 for the preparation of a medicament for at least in part treating or preventing complement-mediated hemolytic anemia. As used herein, the term "complement-mediated hemolytic anemia" embraces disorders involving abnormal lysis of erythrocytes, either intravascular or extravascular, wherein complement activation plays a role. This means that lysis of erythrocytes is, either directly or indirectly, a result of complement activity. Non-limiting examples of disorders involving complement-mediated hemolytic anemia are PNH, HUS and aHUS. In some preferred embodiments, said hemolytic disorder is PNH. In some preferred embodiments, said hemolytic disorder is aHUS.
The term "at least in part preventing complement-mediated hemolytic anemia" for instance embraces delaying the onset of disease, or the course of the disease, or reducing the severity of at least one aspect of the disease, such as for instance the degree of erythrocyte lysis, or alleviating at least one symptom of the disease, such as for instance fatigue, wherein the C6 inhibitor is administered before onset of the disease or before worsening of the symptoms of the disease, so that the onset of disease will be delayed, and/or an individual's course of disease will be milder, as compared to a situation without at least partial prevention according to the invention. The term "at least in part treating complement- mediated hemolytic anemia" for instance embraces reducing at least one aspect of the disease, such as for instance the degree of erythrocyte lysis, and/or delaying the course of the disease, and/or alleviating at least one symptom of the disease, so that a patient's condition will improve as compared to the situation before treatment. The C6 inhibitor may be administered at any stage of the disease. For the patient's well-being, administration at an early stage is preferred.
The term "counteracting complement-mediated hemolytic anemia" encompasses reducing at least one aspect of the disease, such as for instance the degree of erythrocyte lysis, and/or alleviating at least one symptom of the disease, and/or reducing the severity of at least one aspect of the disease, and/or delaying the onset or course of the disease. Hence, the term "counteracting complement- mediated hemolytic anemia" embraces at least in part treating or preventing complement-mediated hemolytic anemia.
The complement system, a highly conserved part of the innate immune system, consists of a cascade of self-cleaving proteases that act as a first line of defence against pathogens. The complement system, a key component of innate immunity, defends against infections and disposes of dead or dying cells. Because complement can harm self-tissue, activation is tightly controlled by regulators to eliminate pathogens or damaged cells without injuring the host. In addition, complement activation may link innate to adaptive immune responses. The complement system can be activated via three pathways, the classical, lectin and alternative pathway. The classical and lectin pathways are initiated by pattern recognition proteins (PRPs). PRPs recognize repeating patterns on membranes, so called pathogen associated membrane patterns (PAMP). In case of the classical activation pathway this is done by Clq which recognize
antigen/antibody complexes. Upon recognition the proteins become active and associate with proteolytic components, Clr and Cls. The lectin pathway is activated by the binding of mannose binding lectin, which recognizes mannose (sugar) patterns. Upon binding the membrane associated serine protease 2 (MASP2), a proteolytic component, becomes active. The proteases, Clq-r-s and MBL-MASP2 complexes, cleave C2 and C4 protein that can in turn associate and form the C3 convertase (C4bC2b complex).
C3 is the main effector molecule of the complement cascade. All pathways converge at the level of C3 cleavage. C3 convertase cleaves C3 in C3a and C3b, C3b associates with the C4bC2b complex forming C5 convertase (C4bC2bC3b), which cleaves C5 into C5a and C5b. C5b acts as a anchor molecule for the membrane attack complex (MAC or C5b-9), a pore forming structure consisting of C5b-C6-C7- C8 and C9. Additionally, anaphylatoxins C3a and C5a are powerful inducers of proinflammatory cytokines and induce attraction of phagocytes. The activators of both the classical and the lectin pathway are membrane bound. In contrast, the alternative pathway is activated in fluid phase by spontaneous conversion of the C3 molecule into its active component. The C3b alone is highly unstable, but can associate with factor B, which is cleaved by factor D, to form the active C3bBb convertase. This C3bBb is a C3 convertase, which binds to membranes; this binding is stabilized by Properdin. On foreign cells the amount of C3bBb rapidly increases, but is strictly regulated on self-targets. Regulatory complement components either induce an accelerated decay of the convertase or act as cofactor for Factor I to degrade activated complement fragments. Decay accelerating factor (DAF/CD55) and C4-binding protein (C4BP) accelerate decay of the convertase; membrane cofactor protein (MCP/CD46) acts together with Factor I to degrade C3b to its inactive form iC3b; complement receptor 1 (CR1/CD35) and Factor H can do both. In addition, CD59 prevents formation of the MAC by inserting between the C8 and C9 subunits of the C5b-9 polymer. A potent regulator of the alternative pathway is factor H, which induces a fast turn-over of the C3bBb complex, by breaking the link between C3b and the Bb proteins. Additionally, factor H also works on membrane-bound C3bBb by allowing binding of yet another regulator of the complement system factor I, which degrades C3b (for a detailed review on the complement system see Ricklin et al., 2010).
In conclusion, the complement system is a highly conserved part of the innate immune system that acts as a first line of defence against pathogens, involving a complex mechanism of activating and deactivating complement factors. When this balance between activation and inhibition is disrupted, complement activation causes injury and contributes to pathology in various diseases. Of note, activation of the alternative pathway accounts for approximately 80% of the complement activity, even if the initial activation occurred via either the classical or the lectin pathway. The inventor has for the first time provided proof of principle that human patients suffering from complement-mediated hemolytic anemia, such as PNH or HUS patients, can be treated with a C6 inhibitor. The inventor has shown that inhibition of human C6 in an environment that is indicative for PNH results in diminished erythrocyte lysis. In PNH patients, erythrocytes containing diminished levels of functional CD55 and/or CD59 at their surface are attacked and lysed by complement via formation of the membrane attack complex (MAC). Hence, PNH patients contain erythrocytes that are vulnerable to complement-mediated lysis. As shown in the Examples, the current inventor has demonstrated that in vivo inhibition of human C6 in rats results in diminished lysis of erythrocytes that are vulnerable to complement-mediated lysis. Moreover, the current inventor has also demonstrated that inhibition of C6 in normal human blood results in diminished MAC formation and lysis of erythrocytes that are vulnerable to complement- mediated lysis. According to the present invention, this is indicative for treatment of complement-mediated hemolytic anemia, such as PNH or HUS, by counteracting human C6 activity.
Prior art experiments have been described wherein C6-knockout non-human animals are tested for the lack of hemolytic activity of their serum. In such cases, animals with an impaired complement system are artificially produced, for instance via knockout of their C6 genes, and it is subsequently established that their complement system is, indeed, impaired. This is clearly not indicative for a human PNH or HUS patient, which contains a normal, well functioning
complement system. Therefore, the current inventor has taken a different approach. Instead of C6-deficient animals, the inventor has used C6 knock-out rats that were supplemented with human C6. This human C6 is capable of complexing with rat C5, C7, C8 and C9 in order to form a functional membrane attack complex (MAC). Hence, the rats had a functional complement system, in contrast to the C6- knockout animals that have been used before the present invention. Subsequently, these rats were provided with antibodies that are specific for human C6 (and which are unable to block rat complement factors such as rat C5 or rat C6). Blood from these rats was subsequently used in order to test lysis of erythrocytes that were rendered vulnerable to complement lysis (like the CD55- and CD59-deprived erythrocytes of a PNH patient). As a positive control, blood was used from the above-mentioned human C6-containing rats, that had not received an anti-C6 antibody. Hence, the hemolytic activity of a normal functioning complement system against vulnerable, PNH-like, erythrocytes was tested, and it was established that administration of a C6 inhibitor could effectively counteract this hemolytic activity. It is clear that this hemolytic assay reflects the situation in human PNH and HUS patients, whereas the prior art use of C6-knockout animals does not, since in PNH and HUS patients vulnerable erythrocytes are attacked by a normal functioning complement system. The inventor therefore for the first time provides proof of principle for the use of a C6 inhibitor in a patient suffering from complement- mediated hemolytic anemia, such as a PNH or HUS patient.
The use of C6 inhibitors in a method according to the present invention is preferred over the use of the currently used anti-C5 antibody eculizumab. C6 is barely produced locally in inflamed nervous tissue, whereas C5 gives a very strong signal in case of neuroinflammation. C6 has only one function, i.e. formation of the membrane attack complex (MAC). For treatment of complement-mediated hemolytic anemia, such as PNH or HUS, blocking of this MAC formation is contemplated, so that lysis of erythrocytes is diminished. C5, on the other hand, has additional roles. The cleaved product C5a plays an important role in immunity. The C5 antibody eculizumab blocks cleavage of C5, thereby blocking the production of both the anaphylatoxin C5a and the anchor for MAC formation, C5b. Blocking C5b production counteracts the formation of MAC, which provides the desired anti- lysis effect. However, the simultaneous reduction of C5a results in many side effects such as reduced chemotaxis after an infection has occurred. Furthermore, maintaining C5a production is also important because signaling of C5a through the C5aReceptor plays a role in regeneration and liver repair. With the use of C6 inhibitors, these beneficial roles of C5a are maintained, thereby reducing side effects.
Hence, advantages of the methods according to the present invention are, amongst other things, that C5a production is maintained, resulting in less side effects.
As used herein, the term "inhibitor of human complement component C6", also referred to as "inhibitor of C6" or "C6 inhibitor", means a compound that is able to at least partly counteract C6 activity. Preferably, said C6 inhibitor is able to at least partly counteract the role of C6 in the formation of the MAC. Such C6 inhibitor is, therefore, particularly suitable for use in directly or indirectly diminishing lysis of erythrocytes in PNH or HUS patients. Various inhibitors of human complement component C6 are suitable for performing the methods according to the present invention. In some embodiments, said inhibitor is selected from the group consisting of a C6 antagonist, a peptide, a polypeptide, an antisense nucleic acid molecule, a small molecule, or a C6 receptor. In some embodiments, a C6 antagonist binds C6 so that C6 can no longer bind to complement component C5b, C7, C8 and/or C9, which at least in part inhibits MAC formation. In other embodiments, a C6 antagonist binds a C6 receptor, for instance present on C5b, which also counteracts MAC formation.
A peptide or polypeptide comprises a plurality of amino acids, whereby a given amino acid residue is typically bound to an adjacent amino acid residue via a peptide bond. However, non-natural bonds and/or non-natural amino acid residues may also be present in a C6 inhibiting peptide or polypeptide as referred to herein. A C6 inhibiting (poly)peptide is for instance capable of binding C6, or binding a C6 receptor that is for instance present on C5b, which binding counteracts MAC formation.
A small molecule is defined herein as a chemical compound, typically having a size of at most 900 daltons. A C6 inhibiting small molecule as referred to herein is capable of at least in part inhibiting the assembly of the MAC.
A C6 inhibiting C6 receptor as referred to herein may be present in isolated or recombinant form, or as part of a larger complex such as, for instance, on a truncated form of C5b. Binding of such C6 receptor to C6 counteracts the role of C6 in MAC formation. The use of a C6 receptor on natural C5b is not encompassed by the present invention, since natural C5b does not inhibit MAC formation.
As used herein, the term "a" means "at least one", so that both singular and plural forms are embraced. For instance, the terms "a binding compound" and "a nucleic acid sequence" encompass one or more binding compounds, and one or more nucleic acid sequences, respectively.
In a preferred embodiment a method according to the invention is provided, wherein said inhibitor of human complement component C6 is an antibody, or a functional part or a functional equivalent thereof, or a nucleic acid sequence encoding therefore.
The term "antibody" as used herein, refers to an immunoglobulin protein comprising at least a heavy chain variable region (VH), paired with a light chain variable region (VL), that is specific for a target epitope.
A "functional part of an antibody" is defined herein as a part that has at least one shared property as said antibody in kind, not necessarily in amount. Said functional part is capable of binding the same antigen as said antibody, albeit not necessarily to the same extent. In one embodiment a functional part of an antibody comprises at least a heavy chain variable domain (VH). Non-limiting examples of a functional part of an antibody are a single domain antibody, a single chain antibody, a nanobody, a unibody, a single chain variable fragment (scFv), a Fab fragment and a F(ab')2 fragment.
A "functional equivalent of an antibody" is defined herein as an artificial binding compound, comprising at least one CDR sequence of an antibody, preferably a heavy chain CDR3 sequence. Said functional equivalent preferably comprises the heavy chain CDR3 sequence of an antibody, as well as the light chain CDR3 sequence of said antibody. More preferably, said functional equivalent comprises the heavy chain CDR1, CDR2 and CDR3 sequences of an antibody, as well as the light chain CDR1, CDR2 and CDR3 sequences of said antibody. A functional equivalent of an antibody is for instance produced by altering an antibody such that at least an antigen-binding property of the resulting compound is essentially the same in kind, not necessarily in amount. This is done in many ways, for instance through conservative amino acid substitution, whereby an amino acid residue is substituted by another residue with generally similar properties (size, hydrophobicity, etc), such that the overall functioning of the antibody is essentially not affected.
As is well known by the skilled person, a heavy chain of an antibody is the larger of the two types of chains making up an immunoglobulin molecule. A heavy chain comprises a constant domain and a variable domain, which variable domain is involved in antigen binding. A light chain of an antibody is the smaller of the two types of chains making up an immunoglobulin molecule. A light chain comprises a constant domain and a variable domain. The variable domain is often, but not always, together with the variable domain of the heavy chain involved in antigen binding.
Complementary- determining regions (CDRs) are the hypervariable regions present in heavy chain variable domains and light chain variable domains. In case of whole antibodies, the CDRs 1-3 of a heavy chain and the CDRs 1-3 of the connected light chain together form the antigen-binding site.
As used herein, the term "an antibody or functional part or functional equivalent" is also referred to as "a binding compound". The terms "specific for" and "capable of specifically binding" are used herein interchangeably and refer to the interaction between an antibody, or functional part or functional equivalent thereof, and its epitope. This means that said antibody, or functional part or functional equivalent thereof, preferentially binds to said epitope over other antigens or amino acid sequences. Thus, although the antibody, functional part or equivalent may non-specifically bind to other antigens or amino acid sequences, the binding affinity of said antibody or functional part or functional equivalent for its epitope is significantly higher than the non-specific binding affinity of said antibody or functional part or functional equivalent for other antigens or amino acid sequences.
An antibody or functional part or functional equivalent according to the invention that is able to bind a particular epitope of C6 can also be specific for another protein or (poly)peptide, if said C6 epitope happens to be also present on such other protein or (polypeptide. In that case an antibody referred to herein as being specific for C6 is also specific for such other protein or (poly)peptide comprising the same epitope.
As used herein, a nucleic acid molecule encoding a C6-inhibiting antibody or functional part or functional equivalent as referred to herein preferably comprises a chain of nucleotides, more preferably DNA, cDNA or RNA. In other embodiments such nucleic acid molecule comprises other kinds of nucleic acid structures such as for instance a DNA/RNA helix, peptide nucleic acid (PNA), locked nucleic acid (LNA) and/or a ribozyme. Such other nucleic acid structures are referred to as functional equivalents of a nucleic acid molecule. The term "functional equivalent of a nucleic acid molecule" thus encompasses a chain comprising non-natural nucleotides, modified nucleotides and/or non-nucleotide building blocks which exhibit the same function as natural nucleotides. The percentage of identity of an amino acid or nucleic acid sequence, or the term "% sequence identity", is defined herein as the percentage of residues in a candidate amino acid or nucleic acid sequence that is identical with the residues in a reference sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for the alignment are well known in the art, for example Align 2, Align Plus, Clustal in the PC/Gene program (which is available from Intelligenetics, Mountain View, California), and GAP, BESTFIT, BLAST, FASTA and TFASTA in the Wisconsin Genetics Software Package of Genetics Computer Group, version 10 (available from Accelrys, San Diego, California), as explained in more detail on page 12 of WO 2010/005310. Non-limiting examples of mathematical algorithms for determining the percentage sequence identity between two nucleic acid sequences or amino acid sequences are the algorithm of Myers and Miller (1988) CABIOS 4: 11- 17, the local homology algorithm of Smith et al. (1981) Adv.Appl.Math. 2:482, the homology alignment algorithm of Needleman and Wunsch (1970) J..Mol..Biol. 48: 443-453, the search-for-similarity-method of Pearson and Lipman (1988) Proc.Natl.Acad.Sci. 85:2444-2448, and the algorithm of Karlin and Altschul (1993) Proc.Natl.Acad.Sci. USA 90: 5873-5877, as discussed on page 12 of
WO 2010/005310.
In preferred embodiments a human patient suffering from, or at risk of suffering from, complement-mediated hemolytic anemia, such as a PNH or HUS or aHUS patient, is treated with an antibody that at least comprises the heavy chain CDR3 region and the light chain CDR3 region of antibody 7E5. This is a novel antibody, provided by the present inventor, that is selected for its excellent capacity of counteracting MAC formation and lysis of red blood cells, as shown in the Examples. Antibody 7E5 comprises heavy chain CDR3 sequence PSTEALFAY and light chain CDR3 sequence MQASHAPYT. Furthermore, antibody 7E5 comprises heavy chain CDR1 sequence DYYMA and heavy chain CDR2 sequence TINYDGSSTYYRESVKG, as well as light chain CDR1 sequence
RSSQSLLNDVGNTYLY and light chain CDR2 sequence LVSDLGS. The sequences of antibody 7E5 are depicted in Figure 10. Based on the CDR sequences of antibody 7E5, it is possible to produce an antibody or functional part or functional equivalent thereof comprising at least one CDR sequence of 7E5, which is specific for C6. Provided is therefore a method for treating complement-mediated hemolytic anemia, preferably PNH, or HUS or aHUS, the method comprising administering to an individual in need thereof an isolated, recombinant and/or synthetic antibody, or a functional part or functional equivalent thereof, or a nucleic acid molecule encoding therefore, comprising at least one CDR sequence of antibody 7E5. Said antibody or functional part or functional equivalent preferably comprises the heavy chain CDR3 sequence PSTEALFAY and the light chain CDR3 sequence MQASHAPYT of antibody 7E5.
In some embodiments, said individual is provided with a nucleic acid molecule or functional equivalent thereof, or a vector, which encodes any of the antibodies or functional parts or functional equivalents recited herein. When (a vector comprising) one or more nucleic acid molecule(s) or functional equivalent(s) encoding such antibody or functional part or functional equivalent is/are administered to an individual suffering from, or at risk of suffering from, complement-mediated hemolytic anemia such as PNH, or HUS or aHUS, the nucleic acid molecule(s) or functional equivalent(s) will be translated in vivo into a C6-inhibiting binding compound. Such produced binding compounds are then capable of preventing and/or counteracting erythrocyte lysis. Further provided is therefore an antibody or functional part or functional equivalent comprising the heavy chain CDR3 sequence PSTEALFAY and the light chain CDR3 sequence MQASHAPYT, or at least one nucleic acid molecule or functional equivalent encoding therefore, for use in a method for at least in part treating or preventing complement-mediated hemolytic anemia such as PNH, or HUS or aHUS. Also provided is a use of an antibody or functional part or functional equivalent comprising the heavy chain CDR3 sequence PSTEALFAY and the light chain CDR3 sequence MQASHAPYT, or at least one nucleic acid molecule or functional equivalent encoding therefore, for the preparation of a medicament for at least in part treating or preventing complement-mediated hemolytic anemia such as PNH, or HUS or aHUS.
In some embodiments, binding compounds are used that comprise at least two CDRs, more preferably at least three CDRs, of the heavy and light chains of antibody 7E5. Hence, preferably at least two or three CDRs of the heavy and light chains of antibody 7E5 are jointly present in one binding compound in a method according to the invention.
In a particularly preferred embodiment a binding compound is used that comprises all three heavy chain CDRs and all three light chain CDRs of antibody 7E5. The invention therefore further provides a method for at least in part preventing or treating complement-mediated hemolytic anemia, preferably PNH, or HUS or aHUS, the method comprising administering to an individual in need thereof an isolated, synthetic or recombinant antibody or a functional part or a functional equivalent thereof, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises:
- a heavy chain CDR1 sequence comprising the sequence DYYMA; and
- a heavy chain CDR2 sequence comprising the sequence TINYDGSSTYYRESVKG; and
- a heavy chain CDR3 sequence comprising the sequence PSTEALFAY; and
- a light chain CDR1 sequence comprising the sequence RSSQSLLNDVGNTYLY; and
- a light chain CDR2 sequence comprising the sequence LVSDLGS; and
- a light chain CDR3 sequence comprising the sequence MQASHAPYT.
Also provided is the recited antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, for use in a method for at least in part preventing or treating
complement-mediated hemolytic anemia, preferably PNH, or HUS or aHUS, as well as a use of an isolated, synthetic or recombinant antibody, or a functional part or a functional equivalent thereof, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a heavy chain CDR1 sequence comprising the sequence DYYMA and a heavy chain CDR2 sequence comprising the sequence TINYDGSSTYYRESVKG and a heavy chain CDR3 sequence comprising the sequence PSTEALFAY and a light chain CDR1 sequence comprising the sequence RSSQSLLNDVGNTYLY and a light chain CDR2 sequence comprising the sequence LVSDLGS and a light chain CDR3 sequence comprising the sequence
MQASHAPYT, for the preparation of a medicament for at least in part preventing or treating complement-mediated hemolytic anemia, preferably PNH, or HUS or aHUS. In some embodiments, the above-mentioned at least one nucleic acid molecule or functional equivalent comprise(s) the 7E5 VH and VL nucleic acid sequences as depicted in Figure 10.
Optionally, at least one of said CDR sequences is optimized, thereby generating a variant binding compound, preferably in order to improve the C6 binding efficacy, the selectivity, or the stability of the resulting binding compound. This is for instance done by mutagenesis procedures where after the stability and/or binding efficacy of the resulting compounds are preferably tested and an improved C6-specific binding compound is selected. A skilled person is well capable of generating variants comprising at least one altered 7E5 CDR sequence. For instance, conservative amino acid substitution is applied. Examples of a
conservative amino acid substitution include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another hydrophobic residue, and the substitution of one polar residue for another polar residue, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine. Preferably, an antibody or functional part or functional equivalent is used in a method according to the invention that comprises a CDR sequence which is at least 90% identical to a 7E5 CDR sequence, so that the favorable C6- binding and MAC formation-inhibiting characteristic of 7E5 is maintained or even improved. The use of variant binding compounds comprising an amino acid sequence which is at least 90% identical to a 7E5 CDR sequence is therefore also within the scope of the present invention. Preferably, said variant binding compounds comprise heavy chain CDRl-3 and light chain CDR 1-3 sequences which are at least 90% identical to the heavy and light chain CDR 1-3 sequences of 7E5. Preferably, the CDR sequences of the variant binding compounds differ in no more than three, preferably in no more than two, preferably in no more than one amino acid from the original CDR sequences of antibody 7E5.
Besides optimizing CDR sequences in order to improve C6 binding efficacy or specificity, at least one sequence in at least one of the framework regions of an immunoglobulin variable region can also be optimized. This is preferably done in order to improve stability and/or to reduce immunogenicity. For instance, for human medical applications, the rat framework regions of antibody 7E5 are preferably humanized in order to reduce the chance of inducing an immune response in a human patient and/or in order to improve the half life of the resulting binding compound. Framework sequences are for instance optimized by mutating a nucleic acid molecule encoding such framework sequence where after the characteristics of the resulting antibody - or functional part or functional equivalent - are preferably tested. This way, it is possible to obtain improved binding compounds. In a preferred embodiment, human germline sequences are used for framework regions in antibodies that are based on 7E5. The use of human germline sequences minimizes the risk of immunogenicity of said antibodies in humans. Further provided is therefore a method according to the invention, wherein a synthetic or recombinant antibody is used that comprises at least one non-natural mutation in a framework region. Additionally, or alternatively, a synthetic or recombinant antibody or functional part or functional equivalent is used in a method according to the invention that comprises at least one non- natural mutation in a constant region. By a "non-natural mutation" is meant that the resulting amino acid sequence does not occur in nature. Instead, it has been artificially produced.
In some embodiments, a C6-inhibiting binding compound is used for counteracting complement-mediated hemolytic anemia, preferably PNH, or HUS or aHUS, wherein said binding compound is a chimeric, humanized or human antibody. For instance, a chimeric or humanized antibody is used that comprises the CDR regions of antibody 7E5 and that further comprises human framework and/or constant region sequences. From the above it is clear that, besides antibody 7E5, variant binding compounds based on the CDR regions of 7E5 can also be generated, using techniques known in the art such as for instance mutagenesis. This is for instance shown in the Examples. Typically, CDR sequence variations between 90 and 99% and VH and VL sequence variations between 80 and 99% are tolerated while maintaining a certain antigen specificity. A use of a binding compound in a method or use according to the invention, wherein the binding compound comprises a sequence that has at least 90% sequence identity to at least one CDR sequence of antibody 7E5, is therefore also provided herein. Since the antigen specificity of an antibody is typically dominated by the CDR3 sequences, a binding compound for use according to the invention preferably comprises at least a heavy chain CDR3 sequence having at least 90% sequence identity with the heavy chain CDR3 sequence of antibody 7E5 and a light chain CDR3 sequence having at least 90% sequence identity with the light chain CDR3 sequence of antibody 7E5. Further provided is, therefore, a method or use according to the invention, wherein said antibody or functional part or functional equivalent comprises a heavy chain CDR3 sequence having at least 90% sequence identity with the sequence PSTEALFAY and a light chain CDR3 sequence having at least 90% sequence identity with the sequence MQASHAPYT. Preferably, said sequence identity is at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%. Typically, at least 1, 2 or 3 amino acid residues of a given CDR sequence may vary while retaining the same binding specificity (in kind, not necessarily in amount). Hence, a C6-specific binding compound is preferably used for treatment of complement- mediated hemolytic anemia, preferably PNH, or HUS or aHUS, wherein the binding compound contains a heavy chain CDR3 sequence wherein at most 3, preferably at most 2, more preferably at most 1 amino acid deviates from the sequence PSTEALFAY (the heavy chain CDR 3 sequence of antibody 7E5), and wherein the binding compound contains a light chain CDR3 sequence wherein at most 3, preferably at most 2, more preferably at most 1 amino acid deviates from the sequence MQASHAPYT (the light chain CDR 3 sequence of antibody 7E5). Preferably, a binding compound, or at least one nucleic acid molecule or functional equivalent encoding therefore, is used in a method according to the invention, wherein said binding compound comprises heavy chain CDRl-3 sequences and light chain CDRl-3 sequences that have at least 90% sequence identity with the heavy and light chain CDRl-3 sequences of antibody 7E5 as depicted in Figure 10. The invention therefore further provides a method according to the invention for treating complement-mediated hemolytic anemia, preferably PNH, or HUS or aHUS, wherein said antibody or functional part or functional equivalent comprises:
- a heavy chain CDR1 sequence having a sequence which has at least 90% sequence identity with the sequence DYYMA; and
- a heavy chain CDR2 sequence having a sequence which has at least 90% sequence identity with the sequence TINYDGSSTYYRESVKG; and
- a heavy chain CDR3 sequence having a sequence which has at least 90% sequence identity with the sequence PSTEALFAY; and
- a light chain CDRl sequence having a sequence which has at least 90% sequence identity with the sequence RSSQSLLNDVGNTYLY; and
- a light chain CDR2 sequence having a sequence which has at least 90% sequence identity with the sequence LVSDLGS; and
- a light chain CDR3 sequence having a sequence which has at least 90% sequence identity with the sequence MQASHAPYT.
Also provided is an isolated, synthetic or recombinant antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises:
- a heavy chain CDRl sequence having a sequence which has at least 90% sequence identity with the sequence DYYMA; and
- a heavy chain CDR2 sequence having a sequence which has at least 90% sequence identity with the sequence TINYDGSSTYYRESVKG; and
- a heavy chain CDR3 sequence having a sequence which has at least 90% sequence identity with the sequence PSTEALFAY; and
- a light chain CDRl sequence having a sequence which has at least 90% sequence identity with the sequence RSSQSLLNDVGNTYLY; and
- a light chain CDR2 sequence having a sequence which has at least 90% sequence identity with the sequence LVSDLGS; and
- a light chain CDR3 sequence having a sequence which has at least 90% sequence identity with the sequence MQASHAPYT, for use in a method for at least in part preventing or treating complement-mediated hemolytic anemia, preferably PNH, HUS or aHUS.
Further provided is a use of an isolated, synthetic or recombinant antibody or functional part or functional equivalent comprising a heavy chain CDRl sequence having a sequence which has at least 90% sequence identity with the sequence DYYMA and a heavy chain CDR2 sequence having a sequence which has at least 90% sequence identity with the sequence TINYDGSSTYYRESVKG and a heavy chain CDR3 sequence having a sequence which has at least 90% sequence identity with the sequence PSTEALFAY and a light chain CDRl sequence having a sequence which has at least 90% sequence identity with the sequence RSS Q S LLND VGNTYLY and a light chain CDR2 sequence having a sequence which has at least 90% sequence identity with the sequence LVSDLGS and a light chain CDR3 sequence having a sequence which has at least 90% sequence identity with the sequence MQASHAPYT for the preparation of a medicament for at least in part preventing or treating complement-mediated hemolytic anemia, preferably PNH, HUS or aHUS. Alternatively, or additionally, one or more nucleic acid molecule(s) encoding the recited antibody or functional part or functional equivalent is/are used.
Preferably, the above mentioned antibody or functional part or equivalent comprises heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences that are at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% identical to the above recited CDR sequences. As described herein before, at least 1, 2 or 3 amino acid residues in the recited CDR sequences may vary while retaining the same binding activity (in kind, not necessarily in amount). Hence, said heavy and light chain CDR 1, 2 and 3 sequences preferably deviate in no more than three, preferably no more than two, more preferably no more than one amino acid from the recited 7E5 CDR sequences.
In some embodiments, an antibody or functional part or functional equivalent is used that comprises a variable heavy chain sequence and/or a variable light chain sequence of antibody 7E5, or a sequence which has at least 80% sequence identity thereto. Preferably, such modified variable heavy chain and/or variable light chain sequence contains humanized framework regions, which is preferred for medical use in humans, as explained before. The variable heavy and light chain sequences of antibody 7E5 are
E VQ LVE S D GGLVQ PGGS LKLS C VAS GFS FSD YYMAWVRQ GPTKGLE WVATINY DGSSTYYRESVKGRFTISRDNAKRTLYLQMDSLRSEDTATYYCSRPSTEALFAY WGHGTLVTVSS and
DWLTQTPSTLSATIGQSVSISCRSSQSLLNDVGNTYLYWYLQRPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISGVEAEDLGIYYCMQASHAPYTFGAGTNL ELK, respectively (shown in Figure 10). Also provided is, therefore, a method or use according to the invention, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
EVQLVESDGGLVQPGGSLKLSCVASGFSFSDYYMAWVRQGPTKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKRTLYLQMDSLRSEDTATYYCSRPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DWLTQTPSTLSATIGQSVSISCRSSQSLLNDVGNTYLYWYLQRPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISGVEAEDLGIYYCMQASHAPYTFGAGTNL ELK, or sequences that are at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100% identical to said heavy chain or light chain sequences. The higher the sequence identity, the more closely an antibody resembles antibody 7E5. Sequence variability is typically allowed, and even preferred, in the framework regions, for instance for humanization purposes. Also provided is therefore a method or use according to the invention, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
EVQLVESDGGLVQPGGSLKLSCVASGFSFSDYYMAWVRQGPTKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKRTLYLQMDSLRSEDTATYYCSRPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DWLTQTPSTLSATIGQSVSISCRSSQSLLNDVGNTYLYWYLQRPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISGVEAEDLGIYYCMQASHAPYTFGAGTNL ELK, and wherein the heavy and light chain CDRl-3 sequences of said binding compound differ in no more than 3, preferably in no more than 2, more preferably in no more than 1 amino acid residues from the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10. In one preferred embodiment, said heavy and light chain CDRl-3 sequences are identical to the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10. In some embodiments, at least one nucleic acid molecule or functional equivalent encoding for any of the above-recited antibodies or functional parts or functional equivalents is used in a method or use according to the invention. Said at least one nucleic acid molecule or functional equivalent preferably comprise a nucleic acid sequence that has at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100% sequence identity with the 7E5 VH and/or 7E5 VL nucleic acid sequences as depicted in Figure 10. Some embodiments provide an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
EVQLVESDGGLVQPGGSLKLSCVASGFSFSDYYMAWVRQGPTKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKRTLYLQMDSLRSEDTATYYCSRPSTEALFAY WGHGTLVTVSS and a variable light chain sequence having at least 80% sequence identity with the sequence
DWLTQTPSTLSATIGQSVSISCRSSQSLLNDVGNTYLYWYLQRPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISGVEAEDLGIYYCMQASHAPYTFGAGTNL ELK,and wherein the heavy and light chain CDRl-3 sequences of said binding compound are identical to the heavy and light chain CDRl-3 sequences of antibody 7E5 as depicted in Figure 10, for use in a method for at least in part preventing or treating complement-mediated hemolytic anemia. Said complement-mediated hemolytic anemia preferably comprises PNH, HUS or aHUS.
Also provided is a use of an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
EVQLVESDGGLVQPGGSLKLSCVASGFSFSDYYMAWVRQGPTKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKRTLYLQMDSLRSEDTATYYCSRPSTEALFAY WGHGTLVTVSS and a variable light chain sequence having at least 80% sequence identity with the sequence
DWLTQTPSTLSATIGQSVSISCRSSQSLLNDVGNTYLYWYLQRPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISGVEAEDLGIYYCMQASHAPYTFGAGTNL ELK and wherein the heavy and light chain CDRl-3 sequences of said binding compound are identical to the heavy and light chain CDRl-3 sequences of antibody 7E5 as depicted in Figure 10, for the preparation of a medicament for at least in part treating or preventing complement-mediated hemolytic anemia. Said complement-mediated hemolytic anemia preferably comprises PNH, HUS or aHUS.
In the Examples non-limiting examples are provided of preferred heavy chain variable regions (VHs) and light chain variable regions (VLs) that comprise the heavy and light chain CDRl-3 regions of antibody 7E5 and humanized framework regions. These are the VHs named 8G09, 7E 12, 7G09, 8F07, 7F06, 7F11, 7E11, 7F02 and 7C02 and the VLs named 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, 7F02 and 7G08. The use of these VHs and VLs for treatment of human patients suffering from or at risk of suffering from complement-mediated hemolytic anemia, such as PNH, HUS or aHUS, is preferred because the humanized framework regions will reduce the immunogenicity and increase the half life of the antibodies or antibody variants within the human body. As shown in Example 7 and Figure 7A, any combination of these VHs and VLs is able to reduce
complement-mediated hemolytic anemia. Further provided is therefore a method for at least in part preventing or treating complement mediated hemolytic anemia, preferably PNH, HUS or aHUS, the method comprising administering an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, to a human individual in need thereof, wherein said antibody or functional part or functional equivalent comprises: - a variable heavy chain sequence having at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100%, sequence identity with a VH sequence selected from the group consisting of the VH sequences 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, 7F02 and 7C02 as depicted in Figure 10, and
- a variable light chain sequence having at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100%, sequence identity with a VL sequence selected from the group consisting of the VL sequences 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, 7F02 and 7G08 as depicted in Figure 10.
Also provided is a use of an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with a VH sequence selected from the group consisting of the VH sequences 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, 7F02 and 7C02 as depicted in Figure 10, and wherein said antibody or functional part or functional equivalent further comprises a variable light chain sequence having at least 80% sequence identity with a VL sequence selected from the group consisting of the VL sequences 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, 7F02 and 7G08 as depicted in Figure 10, for the preparation of a medicament for at least in part preventing or treating
complement-mediated hemolytic anemia. Some embodiments provide an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with a VH sequence selected from the group consisting of the VH sequences 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, 7F02 and 7C02 as depicted in Figure 10, and wherein said antibody or functional part or functional equivalent further comprises a variable light chain sequence having at least 80% sequence identity with a VL sequence selected from the group consisting of the VL sequences 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, 7F02 and 7G08 as depicted in Figure 10, for use in a method for at least in part preventing or treating complement-mediated hemolytic anemia. Said % sequence identity is preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least
90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100%. As explained before, in order to maintain the C6-binding capacity of the binding compounds, the heavy and light chain CDRl-3 sequences of said binding compound preferably differ in no more than 3, preferably in no more than 2, more preferably in no more than 1 amino acid residues from the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10. In one preferred embodiment, said heavy and light chain CDRl-3 sequences are identical to the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10.
The amino acid sequence of VH 8G09 is
EVQLVESDGGLVQPGGSLRLSCVASGFTFSDYYMAWVRQAPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKRTLYLQMDSLRAEDTAVYYCARPSTEALFAY WGQGTLVTVSS and the amino acid sequence of VL 8G09 is
DIVLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTRL EIK. Further provided is therefore a method for at least in part preventing or treating complement-mediated hemolytic anemia, the method comprising administering an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, to a human individual in need thereof, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
EVQLVESDGGLVQPGGSLRLSCVASGFTFSDYYMAWVRQAPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKRTLYLQMDSLRAEDTAVYYCARPSTEALFAY WGQGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DIVLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTRL EIK. Also provided is a use of an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
EVQLVESDGGLVQPGGSLRLSCVASGFTFSDYYMAWVRQAPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKRTLYLQMDSLRAEDTAVYYCARPSTEALFAY WGQGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DIVLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTRL EIK, for the preparation of a medicament for at least in part preventing or treating complement-mediated hemolytic anemia. Some embodiments provide an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
EVQLVESDGGLVQPGGSLRLSCVASGFTFSDYYMAWVRQAPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKRTLYLQMDSLRAEDTAVYYCARPSTEALFAY WGQGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DIVLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTRL EIK, for use in a method for at least in part preventing or treating complement- mediated hemolytic anemia. Said % sequence identity of the above-mentioned VH and VL sequences is preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100%. Again, as explained before, in order to maintain the C6-binding capacity of the binding compounds, the heavy and light chain CDRl-3 sequences of said binding compound preferably differ in no more than 3, preferably in no more than 2, more preferably in no more than 1 amino acid residues from the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10. In one preferred embodiment, said heavy and light chain CDRl-3 sequences are identical to the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10. Said complement-mediated hemolytic anemia preferably comprises PNH, HUS or aHUS.
The amino acid sequence of VH 7E12 is
E VQ LVE S D GGLVQ PGGS LKLS C AAS GFTFS D YYMAWVRQ GPGKGLE WVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRAEDTATYYCARPSTEALFAY WGHGTLVTVSS and the amino acid sequence of VL 7E 12 is
DWLTQTPSTLSVTPGQPASISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGIYYCMQASHAPYTFGQGTNL EIK. Further provided is therefore a method for at least in part preventing or treating complement-mediated hemolytic anemia, the method comprising administering an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, to a human individual in need thereof, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
E VQ LVE S D GGLVQ PGGS LKLS C AAS GFTFS D YYMAWVRQ GPGKGLE WVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRAEDTATYYCARPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence DWLTQTPSTLSVTPGQPASISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGIYYCMQASHAPYTFGQGTNL EIK. Also provided is a use of an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
E VQ LVE S D GGLVQ PGGS LKLS C AAS GFTFS D YYMAWVRQ GPGKGLE WVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRAEDTATYYCARPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DWLTQTPSTLSVTPGQPASISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGIYYCMQASHAPYTFGQGTNL EIK, for the preparation of a medicament for at least in part preventing or treating complement-mediated hemolytic anemia. Some embodiments provide an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
E VQ LVE S D GGLVQ PGGS LKLS C AAS GFTFS D YYMAWVRQ GPGKGLE WVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRAEDTATYYCARPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DWLTQTPSTLSVTPGQPASISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGIYYCMQASHAPYTFGQGTNL EIK, for use in a method for at least in part preventing or treating complement- mediated hemolytic anemia. Said % sequence identity of the above-mentioned VH and VL sequences is preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100%. Again, as explained before, in order to maintain the C6-binding capacity of the binding compounds, the heavy and light chain CDRl-3 sequences of said binding compound preferably differ in no more than 3, preferably in no more than 2, more preferably in no more than 1 amino acid residues from the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10. In one preferred embodiment, said heavy and light chain CDRl-3 sequences are identical to the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10. Said complement-mediated hemolytic anemia preferably comprises PNH, or HUS or aHUS.
The amino acid sequence of VH 7G09 is
E VQ LVE S D GGLVQ PGGS LRLS C AAS GFTFSD YYMAWVRQ GPTKGLE WVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMDSLRAEDTAVYYCARPSTEALFAY WGHGTLVTVSS and the amino acid sequence of VL 7G09 is
DIVLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTRL EIK. Further provided is therefore a method for at least in part preventing or treating complement-mediated hemolytic anemia, the method comprising administering an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, to a human individual in need thereof, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
E VQ LVE S D GGLVQ PGGS LRLS C AAS GFTFSD YYMAWVRQ GPTKGLE WVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMDSLRAEDTAVYYCARPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DIVLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTRL EIK. Also provided is a use of an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
EVQLVESDGGLVQPGGSLRLSCAASGFTFSDYYMAWVRQGPTKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMDSLRAEDTAVYYCARPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DIVLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTRL EIK, for the preparation of a medicament for at least in part preventing or treating complement-mediated hemolytic anemia. Some embodiments provide an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
E VQ LVE S D GGLVQ PGGS LRLS C AAS GFTFSD YYMAWVRQ GPTKGLE WVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMDSLRAEDTAVYYCARPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DIVLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTRL EIK, for use in a method for at least in part preventing or treating complement- mediated hemolytic anemia. Said % sequence identity of the above-mentioned VH and VL sequences is preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100%. Again, as explained before, in order to maintain the C6-binding capacity of the binding compounds, the heavy and light chain CDRl-3 sequences preferably differ in no more than 3, preferably in no more than 2, more preferably in no more than 1 amino acid residues from the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10. In one preferred embodiment, said heavy and light chain CDRl-3 sequences are identical to the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10. Said complement-mediated hemolytic anemia preferably comprises PNH, or HUS or aHUS.
The amino acid sequence of VH 8F07 is
EVQLVESGGGLVQPGGSLRLSCAASGFSFSDYYMAWVRQGPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRSEDTATYYCARPSTEALFAY WGHGTLVTVSS and the amino acid sequence of VL 8F07 is
DWLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPDRFSGSGSGTDFTLKISGVEAEDVGVYYCMQASHAPYTFGAGTKL EIK. Further provided is therefore a method for at least in part preventing or treating complement-mediated hemolytic anemia, the method comprising administering an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, to a human individual in need thereof, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
EVQLVESGGGLVQPGGSLRLSCAASGFSFSDYYMAWVRQGPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRSEDTATYYCARPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DWLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPDRFSGSGSGTDFTLKISGVEAEDVGVYYCMQASHAPYTFGAGTKL EIK. Also provided is a use of an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
EVQLVESGGGLVQPGGSLRLSCAASGFSFSDYYMAWVRQGPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRSEDTATYYCARPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DWLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPDRFSGSGSGTDFTLKISGVEAEDVGVYYCMQASHAPYTFGAGTKL EIK, for the preparation of a medicament for at least in part preventing or treating complement-mediated hemolytic anemia. Some embodiments provide an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
EVQLVESGGGLVQPGGSLRLSCAASGFSFSDYYMAWVRQGPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRSEDTATYYCARPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DWLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPDRFSGSGSGTDFTLKISGVEAEDVGVYYCMQASHAPYTFGAGTKL EIK, for use in a method for at least in part preventing or treating complement- mediated hemolytic anemia. Said % sequence identity of the above-mentioned VH and VL sequences is preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100%. Again, as explained before, in order to maintain the C6-binding capacity of the binding compounds, the heavy and light chain CDRl-3 sequences preferably differ in no more than 3, preferably in no more than 2, more preferably in no more than 1 amino acid residues from the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10. In one preferred embodiment, said heavy and light chain CDRl-3 sequences are identical to the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10. Said complement-mediated hemolytic anemia preferably comprises PNH, or HUS or aHUS. The amino acid sequence of VH 7F06 is
EVQLVESGGGLVQPGGSLKLSCAASGFTFRDYYMAWVRQGPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNSLYLQMDSLRAEDTAVYYCARPSTEALFAY WGHGTLVTVSS and the amino acid sequence of VL 7F06 is DWLTQTPLTLSVTPGQPVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTRL ELK. Further provided is therefore a method for at least in part preventing or treating complement-mediated hemolytic anemia, the method comprising administering an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, to a human individual in need thereof, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
EVQLVESGGGLVQPGGSLKLSCAASGFTFRDYYMAWVRQGPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNSLYLQMDSLRAEDTAVYYCARPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DWLTQTPLTLSVTPGQPVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTRL ELK. Also provided is a use of an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
EVQLVESGGGLVQPGGSLKLSCAASGFTFRDYYMAWVRQGPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNSLYLQMDSLRAEDTAVYYCARPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DWLTQTPLTLSVTPGQPVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTRL ELK, for the preparation of a medicament for at least in part preventing or treating complement-mediated hemolytic anemia. Some embodiments provide an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
EVQLVESGGGLVQPGGSLKLSCAASGFTFRDYYMAWVRQGPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNSLYLQMDSLRAEDTAVYYCARPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DWLTQTPLTLSVTPGQPVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTRL ELK, for use in a method for at least in part preventing or treating complement- mediated hemolytic anemia. Said % sequence identity of the above-mentioned VH and VL sequences is preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100%. Again, as explained before, in order to maintain the C6-binding capacity of the binding compounds, the heavy and light chain CDRl-3 sequences preferably differ in no more than 3, preferably in no more than 2, more preferably in no more than 1 amino acid residues from the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10. In one preferred embodiment, said heavy and light chain CDRl-3 sequences are identical to the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10. Said complement-mediated hemolytic anemia preferably comprises PNH, or HUS or aHUS.
The amino acid sequence of VH 7F11 is
E VQ LVE S D GGLVQ PGGS LKLS C AAS GFTFS D YYMAWVRQ GPTKGLE WVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCSRPSTEALFAY WGHGTLVTVSS and the amino acid sequence of VL 7F11 is
DWLTQTPSTLSVTPGQPVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISGVEAEDVGVYYCMQASHAPYTFGAGTRL EIK. Further provided is therefore a method for at least in part preventing or treating complement-mediated hemolytic anemia, the method comprising administering an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, to a human individual in need thereof, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
E VQ LVE S D GGLVQ PGGS LKLS C AAS GFTFS D YYMAWVRQ GPTKGLE WVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCSRPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DWLTQTPSTLSVTPGQPVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISGVEAEDVGVYYCMQASHAPYTFGAGTRL EIK. Also provided is a use of an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
E VQ LVE S D GGLVQ PGGS LKLS C AAS GFTFS D YYMAWVRQ GPTKGLE WVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCSRPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DWLTQTPSTLSVTPGQPVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISGVEAEDVGVYYCMQASHAPYTFGAGTRL EIK, for the preparation of a medicament for at least in part preventing or treating complement-mediated hemolytic anemia. Some embodiments provide an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
E VQ LVE S D GGLVQ PGGS LKLS C AAS GFTFS D YYMAWVRQ GPTKGLE WVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCSRPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DWLTQTPSTLSVTPGQPVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISGVEAEDVGVYYCMQASHAPYTFGAGTRL EIK, for use in a method for at least in part preventing or treating complement- mediated hemolytic anemia. Said % sequence identity of the above-mentioned VH and VL sequences is preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100%. Again, as explained before, in order to maintain the C6-binding capacity of the binding compounds, the heavy and light chain CDRl-3 sequences preferably differ in no more than 3, preferably in no more than 2, more preferably in no more than 1 amino acid residues from the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10. In one preferred embodiment, said heavy and light chain CDRl-3 sequences are identical to the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10. Said complement-mediated hemolytic anemia preferably comprises PNH, or HUS or aHUS.
The amino acid sequence of VH 7E11 is
EVQLVESGGGLVQPGGSLRLSCVASGFTFSDYYMAWVRQAPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMDSLRAEDTAVYYCARPSTEALFAY WGQGTLVTVSS and the amino acid sequence of VL 7E11 is
DIVLTQTPLSLSATPGQSVSISCRSSQSLLNDVGNTYLYWYLQRPGQSPQLLIYL VSDLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTNL EIK. Further provided is therefore a method for at least in part preventing or treating complement-mediated hemolytic anemia, the method comprising administering an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, to a human individual in need thereof, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
EVQLVESGGGLVQPGGSLRLSCVASGFTFSDYYMAWVRQAPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMDSLRAEDTAVYYCARPSTEALFAY WGQGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DIVLTQTPLSLSATPGQSVSISCRSSQSLLNDVGNTYLYWYLQRPGQSPQLLIYL VSDLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTNL EIK. Also provided is a use of an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
EVQLVESGGGLVQPGGSLRLSCVASGFTFSDYYMAWVRQAPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMDSLRAEDTAVYYCARPSTEALFAY WGQGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DIVLTQTPLSLSATPGQSVSISCRSSQSLLNDVGNTYLYWYLQRPGQSPQLLIYL VSDLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTNL EIK, for the preparation of a medicament for at least in part preventing or treating complement-mediated hemolytic anemia. Some embodiments provide an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
EVQLVESGGGLVQPGGSLRLSCVASGFTFSDYYMAWVRQAPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMDSLRAEDTAVYYCARPSTEALFAY WGQGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DIVLTQTPLSLSATPGQSVSISCRSSQSLLNDVGNTYLYWYLQRPGQSPQLLIYL VSDLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTNL EIK, for use in a method for at least in part preventing or treating complement- mediated hemolytic anemia. Said % sequence identity of the above-mentioned VH and VL sequences is preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100%. Again, as explained before, in order to maintain the C6-binding capacity of the binding compounds, the heavy and light chain CDRl-3 sequences preferably differ in no more than 3, preferably in no more than 2, more preferably in no more than 1 amino acid residues from the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10. In one preferred embodiment, said heavy and light chain CDRl-3 sequences are identical to the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10. Said complement-mediated hemolytic anemia preferably comprises PNH, or HUS or aHUS.
The amino acid sequence of VH 7F02 is
EVQLVESGGGLVQPGGSLKLSCAASGFTFSDYYMAWVRQGPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNSLYLQMNSLRSEDTAVYYCARPSTEALFAY WGHGTLVTVSS and the amino acid sequence of VL 7F02 is
DWMTQTPSTLSATPGQSASISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGIYYCMQASHAPYTFGAGTRL ELK. Further provided is therefore a method for at least in part preventing or treating complement-mediated hemolytic anemia, the method comprising administering an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, to a human individual in need thereof, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
EVQLVESGGGLVQPGGSLKLSCAASGFTFSDYYMAWVRQGPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNSLYLQMNSLRSEDTAVYYCARPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DWMTQTPSTLSATPGQSASISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGIYYCMQASHAPYTFGAGTRL ELK. Also provided is a use of an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
EVQLVESGGGLVQPGGSLKLSCAASGFTFSDYYMAWVRQGPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNSLYLQMNSLRSEDTAVYYCARPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DWMTQTPSTLSATPGQSASISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGIYYCMQASHAPYTFGAGTRL ELK, for the preparation of a medicament for at least in part preventing or treating complement-mediated hemolytic anemia. Some embodiments provide an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
EVQLVESGGGLVQPGGSLKLSCAASGFTFSDYYMAWVRQGPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNSLYLQMNSLRSEDTAVYYCARPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DWMTQTPSTLSATPGQSASISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGIYYCMQASHAPYTFGAGTRL ELK, for use in a method for at least in part preventing or treating complement- mediated hemolytic anemia. Said % sequence identity of the above-mentioned VH and VL sequences is preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100%. Again, as explained before, in order to maintain the C6-binding capacity of the binding compounds, the heavy and light chain CDRl-3 sequences preferably differ in no more than 3, preferably in no more than 2, more preferably in no more than 1 amino acid residues from the heavy and light chain CDRl-3 regions of antibody 7E5, as depicted in Figure 10. In one preferred embodiment, said heavy and light chain CDRl-3 sequences are identical to the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10. Said complement-mediated hemolytic anemia preferably comprises PNH, or HUS or aHUS. The amino acid sequence of VH 7C02 is
EVQLVESGGGLVQPGGSLKLSCAASGFTFSDYYMAWVRQAPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPSTEALFAY WGHGTLVTVSS and the amino acid sequence of VL 7G08 is
DIVMTQTPLSLSATPGQPASISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQASHAPYTFGQGTKL EIK. Further provided is therefore a method for at least in part preventing or treating complement-mediated hemolytic anemia, the method comprising administering an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, to a human individual in need thereof, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with
EVQLVESGGGLVQPGGSLKLSCAASGFTFSDYYMAWVRQAPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with
DIVMTQTPLSLSATPGQPASISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQASHAPYTFGQGTKL EIK. Also provided is a use of an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with
EVQLVESGGGLVQPGGSLKLSCAASGFTFSDYYMAWVRQAPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with
DIVMTQTPLSLSATPGQPASISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQASHAPYTFGQGTKL EIK, for the preparation of a medicament for at least in part preventing or treating complement-mediated hemolytic anemia. Some embodiments provide an antibody or functional art or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with
EVQLVESGGGLVQPGGSLKLSCAASGFTFSDYYMAWVRQAPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with
DIVMTQTPLSLSATPGQPASISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQASHAPYTFGQGTKL EIK, for use in a method for at least in part preventing or treating complement- mediated hemolytic anemia. Said % sequence identity of the above-mentioned VH and VL sequences is preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100%. Again, as explained before, in order to maintain the C6-binding capacity of the binding compounds, the heavy and light chain CDRl-3 sequences preferably differ in no more than 3, preferably in no more than 2, more preferably in no more than 1 amino acid residues from the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10. In one preferred embodiment, said heavy and light chain CDRl-3 regions are identical to the heavy and light chain CDRl-3 sequences of antibody 7E5, as depicted in Figure 10. Said complement-mediated hemolytic anemia preferably comprises PNH, or HUS or aHUS.
In some embodiments, one or more nucleic acid molecule(s) or functional equivalent(s) thereof encoding for any of the above-recited VHs and/or VLs is/are used in a method or use according to the invention. Said nucleic acid molecule(s) or functional equivalent(s) preferably comprise:
- a nucleic acid sequence that has at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100% sequence identity with a VH nucleic acid sequence selected from the group consisting of the VH nucleic acid sequences of 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, 7F02 and 7C02 as depicted in Figure 10, and - a nucleic acid sequence that has at least 80%, preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, or even 100% sequence identity with a VL nucleic acid sequence selected from the group consisting of the VL nucleic acid sequences of 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11, 7F02 and 7G08 as depicted in Figure 10.
As further shown in the Examples, antibody 7E5 is able to bind amino acid residues 835-854 of the human C6 sequence as depicted in Figure 11. C6-specific binding compounds that are able to bind these amino acid residues are therefore preferred binding compounds for the methods and uses according to the present invention, because these binding compounds bind and inhibit human C6 so that erythrocyte lysis is at least in part diminished. Further provided is therefore a method for at least in part preventing or treating complement-mediated hemolytic anemia, the method comprising administering an antibody or functional part or functional equivalent, or a nucleic acid molecule encoding therefore, to a human individual in need thereof, wherein said antibody or functional part or functional equivalent is able to bind amino acid residues 835-854 of the human C6 sequence as depicted in Figure 11. Also provided is a use of an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent is able to bind amino acid residues 835-854 of the human C6 sequence as depicted in Figure 11, for the preparation of a medicament for at least in part preventing or treating complement-mediated hemolytic anemia. Some
embodiments provide an antibody or functional part or functional equivalent, or at least one nucleic acid molecule or functional equivalent encoding therefore, wherein said antibody or functional part or functional equivalent is able to bind amino acid residues 835-854 of the human C6 sequence as depicted in Figure 11, for use in a method for at least in part preventing or treating complement- mediated hemolytic anemia. Again, said complement-mediated hemolytic anemia preferably comprises aHUS or PNH.
As already explained herein before, a use of a human or humanized antibody is preferred for therapeutic applications in humans, since such human or humanized antibodies typically elicit a diminished immune response, and have a longer half life, as compared to antibodies from non-human animals.
When counteracting complement-mediated hemolytic anemia such as PNH, HUS or aHUS, it is the intention to reduce complement activity and MAC formation. It is, therefore, preferred to use antibodies or functional parts or functional equivalents thereof that do not, or to a little extent, induce complement- dependent cytotoxicity (CDC), since this activates the complement system. CDC is most strongly mediated by the Fc regions of IgG3 and IgGl antibodies. IgG4 antibodies do not or barely induce CDC. Therefore, in a preferred embodiment, a method or use according to the invention is provided wherein an antibody is used that is of the IgG4 isotype.
In some embodiments, binding compounds are used in a method or use according to the invention wherein the binding compounds are monoclonal antibodies. A monoclonal antibody is an antibody consisting of a single molecular species. An advantage of monoclonal antibodies is the fact that they can be produced in large quantities by monoclonal antibody-producing cells or
recombinant DNA technology. Furthermore, monoclonal antibodies are preferred for medical regulation procedures.
In some embodiments, a C6-specific antisense nucleic acid molecule is used against complement-mediated hemolytic anemia. Non-limiting examples include oligomers, aptamers, short interfering RNA (siRNA), microRNA (miRNA) and ribozymes. As used herein, the term "oligomer" means a single stranded or double stranded nucleic acid molecule that contains natural and/or non-natural nucleotides which are bound to each other via backbone linkages, thereby forming an oligonucleotide. Non-limiting examples of oligomers are DNA, RNA, DNA/RNA helix, peptide nucleic acid (PNA) and locked nucleic acid (LNA).
Reference is made to WO 2010/005310, which is incorporated herein by reference. This patent application discloses preferred C6-specific oligomers and is from the same (co)-inventor as the current application. WO 2010/005310 concerns neurological applications for the C6-specific oligomers and does not concern treatment of complement-mediated hemolytic anemia. However, according to the present invention, C6-specific oligomers as described in WO 2010/005310 are also suitable for counteracting complement-mediated hemolytic anemia such as PNH, HUS or aHUS.
As stated in WO 2010/005310, the term "oligonucleotide" refers to a polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or analogues thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally- occurring portions. In some embodiments, modified or substituted oligonucleotides are used in view of their preferred properties such as, for instance, increased stability and enhanced cellular uptake.
Preferably an oligomer is used in a method or use according to the present invention, wherein the oligomer has a length of between 10 to 50 nucleotides and wherein the oligomer has a contiguous nucleic acid sequence with at least 80% sequence identity to a complementary region of the human C6 sequence as depicted in Figure 11. As described in WO 2010/005310, such oligomers are particularly suitable for counteracting C6 activity. Further provided is therefore a method for at least in part preventing or treating complement-mediated hemolytic anemia, the method comprising administering to a human individual in need thereof an oligomer of between 10 to 50 nucleotides in length having a contiguous nucleic acid sequence with at least 80% sequence identity to a complementary region of the human C6 sequence as depicted in Figure 11. Also provided is a use of an oligomer of between 10 to 50 nucleotides in length having a contiguous nucleic acid sequence with at least 80% sequence identity to a complementary region of the human C6 sequence as depicted in Figure 11, for the preparation of a medicament for at least in part preventing or treating complement-mediated hemolytic anemia. Some embodiments provide an oligomer of between 10 to 50 nucleotides in length having a contiguous nucleic acid sequence with at least 80% sequence identity to a complementary region of the human C6 sequence as depicted in Figure 11, for use in a method for at least in part preventing or treating complement-mediated hemolytic anemia. Preferably, said sequence identity is at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least
88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% or even 100%. Said complement-mediated hemolytic anemia preferably comprises PNH, or HUS or aHUS.
In some embodiments, a method or use according to the invention is provided wherein the oligomer comprises a moiety that facilitates liver uptake. This will increase the in vivo accumulation of the oligomer in the liver, thereby enhancing C6 inhibition since human C6 is predominantly produced in the liver. A non-limiting example of a moiety that facilitates liver uptake is triantennary N-acetyl galactosamine (GalNac or GN3), as described in Prakash et al.,
Nuc.Ac.Res. (2014): 1- 12. Further provided is therefore a method or use according to the invention, wherein said inhibitor against human complement component C6 is an oligomer of between 10 to 50 nucleotides in length having a contiguous nucleic acid sequence with at least 80% sequence identity to a complementary region of the human C6 sequence as depicted in Figure 11, which oligomer is coupled to a triantennary N-acetyl galactosamine moiety.
Preferred oligomers include at least one nucleotide analogue. For instance, in some embodiments a method or use according to the invention is provided wherein the oligomer comprises a modified internucleoside linkage. In some embodiments, the oligomer comprises a modified nucleobase. Reference is again made to WO 2010/005310 which describes preferred nucleotide analogues. In some embodiments, said nucleotide analogue is a modified sugar moiety selected from the group consisting of: 2'-0-methoxyethyl modified sugar moiety, a 2'-methoxy modified sugar moiety, a 2'-0-alkyl modified sugar moiety, and a bicyclic sugar moiety.
In a particularly preferred method or use according to the invention, the bicyclic sugar moiety is a locked nucleic acid (LNA) monomer. LNA is a modified RNA nucleotide wherein the ribose moiety contains an extra bridge connecting the 2' oxygen and 4' carbon. This additional bridge "locks" the ribose in the 3'-endo (North) conformation. The use of LNA typically increases the hybridization sensitivity and specificity of the oligomer. In other preferred embodiments, the modified internucleoside linkage is a phosphorothioate internucleoside linkage. In some embodiments, the modified nucleobase is 5-methylcytosine.
As shown in WO 2010/005310, C6-specific oligomers that target nucleotides 112-152, 433-473, 546-586, 706-746, or 1015- 1055 from the ATG start site of the human C6 sequence as depicted in Figure 11 (which is referred to in
WO 2010/005310 as SEQ ID NO: l) are preferred since it has been demonstrated that oligomers that target these specified C6 regions have particular good in vivo C6 inhibiting activity. This is exemplified in WO 2010/005310 by five
representative oligomers (i.e. oligomers 1008, 1009, 1010, 1011 and 1012, shown in Table 1 on page 64 of WO 2010/005310). These representative oligomers are all very efficient in reducing C6 mRNA levels in vivo, thereby reducing C6 activity. Hence, it has been demonstrated that targeting the particular C6 regions as recited above is particularly preferred for reducing C6 activity. This enables treatment of disorders associated with the formation of the membrane attack complex (MAC), such as PNH, or HUS or aHUS.
Further provided is therefore a method for at least in part preventing or treating complement-mediated hemolytic anemia, the method comprising administering to a human individual in need thereof an oligomer of between 10 to 50 nucleotides in length having a contiguous nucleic acid sequence with at least 80% sequence identity to a complementary region of the human C6 sequence as depicted in Figure 11, wherein the oligomer is targeted to about nucleotides 112- 152, 433-473, 546-586, 706-746, or 1015-1055 from the ATG start site of the human C6 sequence as depicted in Figure 11. Also provided is a use of an oligomer of between 10 to 50 nucleotides in length having a contiguous nucleic acid sequence with at least 80% sequence identity to a complementary region of the human C6 sequence as depicted in Figure 11, wherein the oligomer is targeted to about nucleotides 112-152, 433-473, 546-586, 706-746, or 1015-1055 from the ATG start site of the human C6 sequence as depicted in Figure 11, for the preparation of a medicament for at least in part preventing or treating complement-mediated hemolytic anemia. Some embodiments provide an oligomer of between 10 to 50 nucleotides in length having a contiguous nucleic acid sequence with at least 80% sequence identity to a complementary region of the human C6 sequence as depicted in Figure 11, wherein the oligomer is targeted to about nucleotides 112-152, 433- 473, 546-586, 706-746, or 1015-1055 from the ATG start site of the human C6 sequence as depicted in Figure 11, for use in a method for at least in part preventing or treating complement-mediated hemolytic anemia. In some embodiments, the oligomer comprises at least one nucleotide analogue. In some embodiments, the oligomer is capable of reducing the level of C6 mRNA expression in a mammal by at least 20% as determined by a qPCR assay. Again, said sequence identity is preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99% or even 100%. Said complement-mediated hemolytic anemia preferably comprises PNH, or HUS or aHUS. Said oligomers preferably comprise a sequence selected from the group consisting of TTGTCTCTGTCTGCTC (oligo 1008 of WO 2010/005310),
TAACTTGCTGGGAATA (ohgo 1009 of WO 2010/005310), CCCATCAGCTGCACAC (oligo 1010 of WO 2010/005310), TTCT ATAGTTT GTAC C (oligo 1011 of
WO 2010/005310) and GTTGTATTCTAAAGGC (oligo 1012 of WO 2010/005310). Preferably, at least one of the nucleotides of these oligomers are LNA. Preferably, the first three nucleotides and the last three nucleotides of these oligomers are LNA.
As used herein, an "individual" or "subject" is preferably a human individual. Said individual preferably suffers from, or is at risk of suffering from, a disorder involving complement-mediated hemolytic anemia, such as PNH, or HUS or aHUS.
A C6 inhibitor can be administered to a human individual in a method according to the invention using any suitable route for administration. For instance, a C6 inhibitor is administered to an individual orally, by aerosol or as a suppository. For mucosal administration, liposomes are also practically useful. In some embodiments, a C6 inhibitor is administered via one or more injections, such as intraperitoneal, intravascular or intramuscular injections. Dose ranges of C6 inhibitors to be used in the therapeutic applications as described herein are typically designed on the basis of rising dose studies in clinical trials for which rigorous protocol requirements exist. Typical doses of administration of a C6 inhibitor in a method according to the invention are between 0.1 and 50 mg per kg body weight, preferably between 0.1 and 35 mg per kg body weight. In some embodiments, treatment is started with between one and ten high dose
administrations (such as for instance between 15 and 50 mg per kg body weight), followed by lower dose administrations (such as for instance between 0.1 and 10 mg per kg body weight. In some embodiments, the C6 inhibitor is administered daily, weekly, two-weekly or 4-6 weekly. In some embodiments, treatment with C6- inhibiting oligomers is preferably started with daily dosages of between 1- 10 mg per kg body weight, followed by weekly, two-weekly or 4-6 weekly dosages of between 0.1 and 1 mg per kg body weight.
In some embodiments a C6 inhibitor is combined with a pharmaceutically acceptable carrier, adjuvant, diluent and/or excipient. Non-limiting examples of suitable carriers for instance comprise keyhole limpet haemocyanin (KLH), serum albumin (e.g. BSA or RSA) and ovalbumin. Many suitable adjuvants, oil -based and water-based, are known to a person skilled in the art. In one embodiment said adjuvant comprises Specol. In another embodiment, said suitable carrier comprises a solution like for example saline.
Some embodiments provide a use of an inhibitor of human complement component C6 for counteracting hemolysis. This encompasses in vivo uses of such C6 inhibiting compound, for instance for PNH, HUS or aHUS treatment.
Alternatively, or additionally, hemolysis is counteracted ex vivo, for instance in a hemolytic assay in order to determine complement activity against erythrocytes. Such ex vivo procedure is for instance useful for determining whether erythrocytes are prone to lysis by complement. For instance, erythrocytes from a human individual who is suspected of PNH are subjected to complement, either in the presence or absence of a C6-inhibiting compound. If these erythrocytes appear to be lysed by complement, and if a C6 inhibiting compound appears to be able to reduce such lysis, it is concluded that the erythrocytes are prone to complement lysis. In such case, a blood sample from which the erythrocytes are derived is preferably typed as containing erythrocytes that are prone to complement lysis. This way, complement mediated hemolytic anemia (for instance PNH, HUS or aHUS) can be diagnosed. While the current application may describe features as parts of the same embodiment or as parts of separate embodiments, the scope of the present invention also includes embodiments comprising any combination of all or some of the features described herein.
The invention is further explained in the following examples. These examples do not limit the scope of the invention, but merely serve to clarify the invention. Brief description of the drawings
Figure 1A is a bar graph showing the results of a haemolytic assay using supernatants from 38 hybridoma from two different rats immunized with human C6. Figure IB is a bar graph showing the results of a mannan-activated
complement ELISA using supernatants from the same 38 hybridomas depicted in figure la. Analysis of both figures led us to conclude that supernatant 11
(producing the 7E5 mAb) has the strongest inhibitory effect.
Figure 2 is a graph showing the kinetics for recombinant rat 7E5 binding to human C6 as measured with Label-free surface plasmon resonance (Biacore).
Figure 3A-D are figures showing data of the epitope cross-blocking experiment between 27B1 mAb and 7E5 mAb competing for binding for C6 as measured with Label-free surface plasmon resonance (Biacore). The results show that 7E5 occupies a different epitope on C6 than 27B1.
Figure 4A is an alignment of the human C6 and rat C6 partial amino acid sequences showing the location of peptide 418. Figure 4B is a diagram of the human C6 protein with the location of peptide 418 indicated by an arrow.
Figure 5 shows the results of an in vivo experiment in rats proving that 7E5 blocks C6 in vivo in C6 deficient rats supplemented with human C6, as measured by haemolytic assay. Rats received 2mg of human C6 suppletion (intravenously) and subsequent 12 mg of antibody 7E5 (intravenously). Complement activity is plotted on the Y-axis, whereby O.D. 1.0 indicates maximum lysis of sheep erythrocytes (as measured in the supernatant at 405nm using a spectrophotometer), and an O.D. 0 indicates absence of lysis.
Figure 6A is the alignment of the amino acid sequences of the heavy chain variable regions of rat anti-C6 7E5 mAb and human VH3_1 germline, with differences indicated. Indicated below the alignment are the amino acid exchanges targeted for humanization. Figure 6B is the alignment of the amino acid sequences of the light chain variable regions of rat anti-C6 7E5 mAb and human Vk2_5 germline, with differences indicated. Indicated below the alignment are the amino acid exchanges targeted for humanization.
Figure 7 shows the results of haemolytic assays demonstrating the inhibitory activity on erythrocyte lysis of all 81 possible combinations of the 9 humanized 7E5 variant VH chains (shown in Figure 7A) and 9 humanized 7E5 variant VL chains (shown in figure 7B) .
Figure 8A depicts an alignment of the amino acid sequence of the 7E5 heavy chain variable region with the heavy chain amino acid sequences of the humanized 7E5 variants 7C02, 7E11, 7E12, 7F02, 7F06, 7F11, 7G09, 8F07 and 8G09. The conserved CDR1, 2 and 3 regions are indicated. Figure 8B depicts the amino acid sequence of the 7E5 light chain variable region aligned with the light chain amino acid sequences of the humanized 7E5 variants 7E11, 7E12, 7F02, 7F06, 7F11, 7G08, 7G09, 8F07 and 8G09. The conserved CDR1, 2 and 3 regions indicated.
Figure 9 depicts the affinity as measured with Label-free surface plasmon resonance (Biacore) of 8 humanized F'Abs for human C6 (coated on the biacore chip) in comparison to the affinity of the wild type 7E5 rat F'Ab.
Figure 10 depicts the amino acid and nucleic acid sequences of the heavy and light chain CDR 1-3 regions and the VH and VL regions of antibody 7E5. Also depicted are the sequences of several humanized VHs and VLs.
Figure 11 depicts a nucleic acid sequence encoding human C6 mRNA (Genbank Ref. NM_000065.2). The ATG start site is indicated.
Figure 12 shows that antibody 7E5 counteracts anti CD59 induced lysis. The experiment was performed in 1.5% human serum. Examples
Example 1: Generation of Rat Anti-Human C6 Monoclonal Antibodies Five rats (PVG C6 -/-) were immunized against human C6 protein. C6 deficient rats were chosen because according to the current understanding in the field it is extremely difficult to generate functional C6 antibodies in normal rodents. For unknown reasons immunization against C6 is not efficient in wild type animals. The idea is that in C6 deficient animals the antibody response is more robust because these animals have no functional C6 protein in circulation and are likely to consider C6 as completely "foreign." Human C6 was purified from whole human serum by means of affinity chromatography using 23D1 mouse monoclonal antibody 23D1 (described in detail in L. Clayton (2005) Ph.D. Thesis, Cardiff University) coupled to Sepharose (GE Healthcare Cat No. 17-0717-01).
Antigen and Immunization: One week before immunization, a pre- immunization bleed was performed on the rats by collecting 100 μΐ of blood from the tail vein. On Day 1 of the immunization, rats were injected at four locations subcutaneously (s.c.) with 100 μg C6 antigen in Complete Freund's Adjuvant (CFA), in a volume of 250 μΐ per injection. Booster injections were performed on Days 14 and 21, again at four s.c. locations with 50 μg C6 antigen in Incomplete Freund's Adjuvant (IFA) in a volume of 250 μΐ per injection. Test bleeds were performed on Day 36 by collecting 100 μΐ of blood from the tail vein for in vitro tests. These test bleeds were analyzed in a C6 ELISA, a C6 Western blot and in a haemolytic assay (described further below), which showed that all five rats had a positive immune response against human C6: all five rats had antibodies that blocked hemolysis in the haemolytic assay and all five rats had antibodies that recognized purified C6 on Western blot (denaturing conditions). A pre-fusion booster was performed on Day 62 by injection of 100 μg antigen in 250 μΐ PBS intraperi tone ally. Finally, a pre-fusion booster was performed on Day 64 by injection of 100 μg antigen in 250 μΐ PBS intravenously (tail vein). Spleens from two rats were harvested on Day 66 (with the other three rats left as backup) and the isolated splenocytes used for hybridoma preparation. Hybridoma Preparation: Hybridomas were prepared by fusion of the splenocytes from the human C6-immunized rats with Y3-Agl.2.3 fusion partner cells using standard polyethylene glycol (PEG)-mediated fusion essentially as described in Luk, J. M. et al. (1990) J. Immunol. Methods 129:243-250.
Supernatants were harvested and used for initial screening for anti-human C6 antibodies via ELISA using 96-well plates coated with human C6 antigen. Positive clones were selected and subcloned. Thirty-eight positive clones were selected for further analysis. Haemolytic Assay: These 38 supernatants and control supernatants were further tested in a haemolytic assay at a 1:50 dilution using human serum as complement source. In this assay, sheep erythrocytes were coated with a polyclonal rabbit serum directed against sheep erythrocytes (sometimes called an
amboceptor). This resulted in antibody-bound erythrocytes that are vulnerable to lysis by complement, like the CD55- and/or CD59-deficient erythrocytes of human PNH patients. These vulnerable erythrocytes were incubated in the presence of normal human serum. The serum contains the components of the complement system, which are activated through the classical pathway when the vulnerable erythrocytes are encountered. Hence, this hemolytic assay is indicative for PNH patients, since in both cases erythrocytes that are vulnerable to complement- mediated lysis are exposed to a functional complement system. The Membrane Attack Complex (MAC) is formed as part of the terminal complement system and MAC initiates lysis of the erythrocytes. Erythrocyte lysis can be quantified by measuring the OD at 405 or 415 nm in the supernatant and is a direct
measurement of the activity of the MAC. Complement inhibitors can be tested in this system because if they are effective they will prevent erythrocyte lysis in a quantitative fashion.
To perform the assay, a haemolytic system ready to use was obtained commercially (Virion/Serion GmbH, Wurzburg, Gemany) along with CFT buffer (Virion/Serion GmbH, Wurzburg, Gemany). The CFT buffer was prepared according to the manufacturer's instructions. The haemolytic system was placed on a rollerbank in a coldroom to thoroughly mix the erythrocytes. To prepare a CFT serum cocktail, 100 μΐ of human serum was added to 5 ml of CFT buffer. Dilutions of test inhibitors, in a volume of 50 μΐ, were added to round bottom 96- well plates, 50 μΐ of CFT serum cocktail was added to each well and mixed carefully while pipetting and the plates were incubated at 37° C for 30 minutes. Positive controls was EDTA. Negative control was serum free or C6 deficient serum. After incubation, plates were spun down at 2000 rpm for 5 minutes (Hettich table top centrifuge) and 80 μΐ of supernatant was transferred to flat bottom plate for measurement at 405 or 415 nm. The OD was measured within 10 minutes of transfer.
Test supernatants were added in dilution in the haemolytic assay to determine whether they prevent erythrocyte lysis. Exemplary results are shown in Figure 1A, which demonstrates that certain of the supernatants exhibited stronger inhibitory activity than others. In particular, supernatants #6- 12 exhibited stronger inhibition than the other supernatants, with supernatants #11 and #12 showing the strongest inhibition. Hence, this assay shows that under conditions that are indicative for PNH, supernatants #6-12 are well capable of inhibiting erythrocyte lysis. The supernatants (1:50 dilution) were also tested in the haemolytic assay using rat serum as complement source and no inhibitory effect was observed, demonstrating that the inhibitory activity of the antibodies was specific for human C6.
MAC ELISA Assay: We used a second assay to determine whether the supernatants are able to block MAC activity. In this assay, the ELISA wells in the plate are coated with either Mannan or IgG as trigger for either the Lectin or the Classical pathway of complement, respectively, in the presence of serum. The serum contains the components of the complement system which are activated through either pathway when they are exposed to the coated plate. The MAC is formed as part of the terminal complement system and MAC will be deposited on the ELISA plate. MAC deposition on the plate can be detected by HRP-conjugated antibodies and visualized by enzymatic reaction in the presence of a chromogen and substrate. This reaction produces a color that can be quantified by measuring the OD at 450 or 655 nm. The OD is a direct measurement of the amount of MAC formation. Complement inhibitors can be tested in this system because if they are effective they will prevent or inhibit deposition of MAC on the plate. In a mannan activated complement ELISA assay, ELISA plates were coated with mannan and diluted hybridoma supernatant and human serum was added. Complement components that form a complex on the mannan coated plate can be detected using antibodies. In this particular assay we looked for detection of C9 which indicates formation of MAC. If less C9 is detected this indicates MAC inhibition. The positive controls in this experiment are EDTA, because the reaction is calcium dependent.
For the assays, coating buffer (15 mM Na2C03, 35 mM NaHCOs, 15 mM NaN3, pH 9.6), blocking buffer (1 mg/ml BSA/HAS, 10 mM Tris/HCl, pH 7.4, 145 mM NaCl, 15 nM NaN3, pH 7.4) wash buffer (1 x TBS, 0.05% Tween 20, 5 mM CaC ) and dilution buffer (4 mM barbital, 145 mM NaCl, 2 mM CaC , 1 mM MgC , 0.3% BSA, 0.02% Tween 20) were used. The wells of a flat -bottomed high binding 96-well plate were coated with 100 μΐ coating buffer containing 10 μg/ml Mannan (Sigma, Cat. No. M7504) or IgG and incubated overnight at 4° C. Plates were blocked with 200 μΐ blocking buffer for 1 hour at room temperature. Human serum in dilution buffer (1: 100) was diluted with supernantant (1:50) in round bottom plates and 50 μΐ per well was added to the flat bottom high binding plates. The plates were incubated for 1 hour at 37° C, followed by washing 3 times with wash buffer. Anti- C5b-9neo (clone aEl l; DAKO, Cat. No. M0777) was diluted 1: 100 in dilution buffer and 50 μΐ was added per well. The plates were incubated for 1 h at room
temperature, followed by washing 3 times with wash buffer. Anti-mouse HRP (DAKO, Cat. No. P0447) was diluted 1:2000 in dilution buffer, 50 μΐ was added per well and the plates were incubated for 30 minutes at room temperature, followed by washing three times with wash buffer. To develop, 50 μΐ TMB chromogen
(TMB: Sigma T2885; stock solution prepared of 10 mg/ml TMB in DMSO) and 10 μΐ 3% H202 were added to 5 ml NaAc buffer (8.2 gm Natrium Acetate, 21 gm Citric Acid Monohydrate in 1 liter H2O) and distributed to the 96-well plates. The reaction was stopped with 25 μΐ 1 M H2SO4 and the OD was measured with a
spectrophotometer at 450 nm/655 nm.
Exemplary results of this assay are shown in Figure IB, which
demonstrates that two supernatants, #11 and #17 had significantly better inhibitory ability than the other 36 supernatants, with supernatant #11 having by far the most superior inhibitory ability of all of the clones analyzed.
Since supernatant #11 exhibited the strongest inhibition in both the haemolytic assay and in the MAC ELISA assay, this hybridoma was selected for further characterization. The monoclonal antibody produced by this hybridoma is referred to herein as 7E5.
Example 2: Characterization of 7E5 Monoclonal Antibody
We wanted to know whether C6 was bound by 7E5 in serum of human and cynomolgus monkeys (M.fascicularis). We tested the ability of 7E5 to bind C6 in serum on Western blot under denaturing conditions. Human serum and serum from cynomolgus monkeys were used for PAGE (10% gel) and standard Western blotting. 7E5 was incubated on the blots for 1 hour in a 1:500 dilution. Detection was done using anti-rat horse radish peroxidase (HRP) (DAKO, 1: 1000) and Lumilight (Roche) in a LAS3000 (Fuji) darkbox imaging system. The results show that 7E5 recognizes C6 in human serum and in serum from cynomolgus monkeys. To investigate the kinetics of 7E5 binding to C6 we used surface plasmon resonance measurement in a BIACORE 2000 (GE Healthcare) apparatus equipped with a research-grade CM5 sensor chip. The ligand (C6, 113 kDa) was immobilized using amine -coupling chemistry. The surface of flow cell two was activated for 7 minutes with a 1: 1 mixture of 0.1 M NHS (N-hydroxysuccinimide) and 0.4 M EDC (3-(N,N-dimethylamino) propyl-N-ethylcarbodiimide) at a flow rate of 5 μΐ/min. The ligand at a concentration of 10 pg/ml in 10 mM sodium acetate, pH 5.0, was immobilized at a density of 955 RU. The surface was blocked with a 7 minute injection of 1 M ethanolamine, pH 8.0.
Flow cell 1 was immobilized with an antibody from an earlier experiment (avWWF; 987 RU) and served as a reference surface.
To collect kinetic binding data, the analytes (anti C6- antibodies, 150 kDa) in 10 mM HEPES, 150 mM NaCl, 0.005% P20, pH 7.4, were injected over the two flow cells at a flow rate of 30 μΐ/min and at a temperature of 25°C. The injected concentrations differ per antibody. Data were collected at a rate of 1 Hz. The complex was allowed to associate and dissociate for 90 and 300 seconds, respectively. The surfaces were regenerated with a 10 second injection of 0.1 M HC1. Duplicate injections (in random order) of each sample and a buffer blank were flowed over the two surfaces.
The data were fit to a simple 1: 1 interaction model using the global data analysis option available within BiaEvaluation 4.1 software. The Biacore kinetic results are shown in Figure 2. Results from a representative experiment also are summarized in Tables 1-4 below.
Table 1: Kinetics of 7E5 Binding Determined by Surface Plasmon Resonance
Figure imgf000056_0001
Table 2: Complex Half Life for 7E5 Binding kd (s- 1) ti/2 (sec) ti/2 (min) ti/2 (hour)
4.27 x lO 6 s-1 162330 2705.5 45.1
Table 3: Time to 5% Dissociation for 7E5 Binding kd (s- 1) Time (min) Time (hour) R (RU)
4.27 x lO 6 s- 1 200.2 3.3 34
Table 4: Time to 95% Dissociation for 7E5 Binding kd (s-1) Time (min) Time (hour) R (RU)
4.27 x lO 6 s-1 11692.9 194.9 2 The KD of 7E5 is calculated as 2.5 x 10 10 M. This high affinity is primarily caused by the high antibody-antigen complex half-life (45 hrs). Thus, 7E5 binding is very stable, with the half- life of the 7E5-C6 complex estimated at over 40 hrs.
To determine whether the antigen-antibody complex could be released in the endosomes and lysosomes after binding and cellular uptake by the Fc gamma receptor, the sensitivity of the 7E5 -C6 complex was tested in low pH. Since in lysomes the pH is about 4.8, complex stability was tested up to pH=4. In this BIACORE experiment the 7E5-C6 complex on the chip was washed with buffers with decreasing pH. Hepes buffered saline ((HBS) was used for pH 7.4, 7.0 and 6.5. lOmM Sodium Acetate was used for pH 6.0, 5.5, 5.0, 4.5 and 4.0. It was observed that the stability of the complex is not sensitive for low pH.
Effect of Pre-Incubation on Haemolytic Assay: Since BIACORE
experiments revealed that the slow release of 7E5 from C6 is a primary
determinant of the KD of 7E5 we investigated whether pre-incubation of 7E5 with the complement source (human serum) prior to adding the erythrocytes can increase inhibitory efficacy in the haemolytic assay. 7E5 was pre-incubated with human serum for 30, 90 or 180 minutes at room temperature (20°C) before the erythrocytes were added and the reaction was started at 37°C. The results showed that increasing the pre-incubation time up to 3 hours did not result in
improvement of inhibition of haemolysis.
Example 3: Epitope Mapping of 7E5 Monoclonal Antibody To discover the epitope of 7E5 in C6 we used peptide arrays. Consecutive overlapping 16mer peptides (peptides 16 amino acids long, overlapping 14 amino acids) from the C6 protein sequence were synthesized and spotted in a grid pattern on a membrane. The membrane was then incubated with 7E5 antibody to detect which peptide was recognized by the antibody. The primary peptide sequence recognized by 7E5 was GS CQD GRQLE WGLERT (peptide 418).
Subsequently, an alanine scan in which alanine is used to replace amino acids one by one on selected peptides was performed to help pinpoint the epitope. In in addition to modifications of peptide 418, also the peptide with 4 amino acids shifted relative to 418, peptide 420 (DGRQLEWGLERTRLSS) and some of its alanine modifications showed binding of 7E5. We conclude that the main epitope of 7E5 is expected to be within this peptide sequence: 418-420
GSCQDGRQLEWGLERTRLSS.
Figure 4A shows the sequence of peptide 418 and surrounding area in human and rat C6. As illustrated schematically in Figure 4B, peptide 418 is partially located at the end of the first FIM domain of C6.
To determine whether other antibodies bind to the same epitope as the 7E5 antibody, a Biacore cross blocking experiment was conducted in which the C6 antigen was coupled to the chip, followed by flow of the analyte(s), which was either a single anti-C6 antibody alone (as a control) or a first anti-C6 antibody (Antibody 1) followed by a second anti-C6 antibody (Antibody 2) to determine cross- blocking. The results for the cross -blocking experiment to determine whether the mouse mAb 27B1 binds the same epitope as the rat mAb 7E5 are shown in Figure 3A-D, wherein Fig. 3A shows the results with 27B1 as Antibody 1 and 7E5 as Antibody 2, Fig. 3B shows the results with 7E5 as Antibody 1 and 27B1 as
Antibody 2, Fig. 3C shows the results for 27B1 alone and Figure 3D shows the results for 7E5 alone. Example 4: In Vivo Efficacy of rat 7E5 Monoclonal Antibody
To test whether 7E5 is able to block C6 in a living animal we used C6 deficient PVG rats supplemented with human C6. We used this approach because 7E5 is specific for human C6 and is unable to block rat C6. In the C6 deficient rats we can inject human C6 to restore full complement system functionality and MAC activity, and measure the effect of 7E5 without confounding effects caused by rat C6.
First, we tested this approach by determining the haemolytic activity in two rats injected with human C6. By taking several blood samples in time after injection of C6 we could estimate the half- life of human C6 in the rats to be about 48 hours. Two C6 deficient rats were injected IV with 4 mg/kg of human C6. Blood samples were taken 10 minutes, 24 hours and 48 hours post injection of C6. After coagulation of all blood samples serum was isolated by spinning down the coagulate (13,000 rpm in an Eppendorf table top centrifuge for 10 minutes at room temperature). The serum was used in the haemolytic assay described in Example 1 to determine MAC activity. Serum from a wild type PVG rat and a non-treated C6 deficient rat were used as references for maximal and minimal haemolytic activity. Using the haemolytic assay, the half-life of human C6 in the rats was estimated to be about 48 hrs.
In a pilot experiment, one female C6 deficient PVG rat (220 grams body weight) was injected with a high 12 mg dose of 7E5 intraperi tone ally (IP) and supplemented with 2 mg of human C6 (intravenous injection). Human C6 was isolated from human serum using affinity purification with C6 antibody coated columns. C6 was dosed 1 mg 24 hours before and 1 mg 5 minutes after 7E5 bolus injection. The control rat (same weight as the 7E5 treated rat) received only the C6 injection. Blood for the haemolytic assay was drawn 60 minutes after injection of 7E5. The results are shown in Figure 5. The results show that haemolytic activity was blocked by 7E5 60 minutes after 7E5 dosing thus proving that 7E5 can block MAC formation in vivo.
In a subsequent experiment we injected 1 mg C6 in two C6 deficient female rats (PVG strain). Blood samples were taken before C6 injection and after C6 injection (IV 1 mg) to establish normal and supplemented haemolytic activity. 10 minutes after C6 injection, 7E5 was dosed at either 8 mg IP or 2 mg IV. 60 minutes after 7E5 dosing, blood samples were taken to assess the effect of 7E5 on haemolytic activity of the same kind of sheep erythrocytes that had been rendered vulnerable to complement-mediated lysis by a rabbit polyclonal anti-erythrocyte serum as described in Example 1 (Hemolytic System; order number HS-050 (Labor Dr. Merk & Kollegen GmbH); obtained from Serion GmbH). Both dosing strategies blocked MAC activity in the blood. Then another 1 mg of C6 was injected IV in the same rats. Blood sampling in the 15 minutes after the new C6 supplementation showed only modest increase in haemolytic activity inferring that haemolytic activity remained inhibited in both rats by free circulating 7E5.
These experiments show that C6-mediated erythrocyte lysis is counteracted after in vivo administration of 7E5. Therefore, these experiments provide proof of principle that complement-mediated haemolytic anemia such as PNH, HUS and aHUS can be counteracted by administering a C6-inhibitor to individuals in need thereof.
Example 5: Sequencing and Recombinant Expression of 7E5 Monoclonal Antibody
The VH and VL sequences of the 7E5 mAb were determined using standard technology known in the art.
The nucleotide sequence of the VH region is as follows: gaggtgcagctggtggagtctgatggaggcttagtgcagcctggagggtccctgaaactctcctgtgtagcctcaggattctcttt cagtgactattacatggcctgggtccgccagggtccaacgaaggggctggagtgggtcgcaaccattaattatgatggtagtag tacttactatcgagagtccgtgaagggccgattcactatctccagagataatgcgaaacgcaccctatacctgcaaatggacag tctgaggtctgaggacacggccacttattactgttcaagaccttctacggaggccctgtttgcttactggggccacggcactctggt cactgtctcctca
The amino acid sequence of the VH region is as follows:
EVQLVESDGGLVQPGGSLKLSCVASGFSFSDYYMAWVRQGPTKGLEWVATINYDGSSTY YRESVKGRFTISRDNAKRTLYLQMDSLRSEDTATYYCSRPSTEALFAYWGHGTLVTVSS
The amino acid sequences of the VH CDR1, CDR and CDR3 are as follows:
CDR1: DYYMA
CDR2: TINYDGSSTYYRESVKG
CDR3: PSTEALFAY
The nucleotide sequence of the VL region is as follows:
Gatgttgtgctgacccagactccatccacattatcggctaccattggacaatcggtctccatctcttgcaggtcaagtcagagtctc ttaaatgatgttggaaacacctatttatattggtatctacagaggcctggccaatctccacagcttctaatttatttggtctccgacctg ggatctggggtccccaacaggttcagtggcagtgggtcaggaacagatttcacactcaaaatcagtggagtggaggctgagg atttgggaatttattactgcatgcaagctagtcatgctccgtacacgtttggagctgggaccaacctggaactgaaa The amino acid sequence of the VL region is as follows:
DVVLTQTPSTLSATIGQSVSISCRSSQSLLNDVGNTYLYWYLQRPGQSPQLLIYLVSDLGS GVPNRFSGSGSGTDFTLKISGVEAEDLGIYYCMQASHAPYTFGAGTNLELK
The amino acid sequences of the VL CDR1, CDR and CDR3 are as follows:
CDR1: RS SQ S LLND VGNTYLY
CDR2: LVSDLGS
CDR3: MQASHAPYT
Following introduction of appropriate restriction sites for cloning and optimization of the coding sequence for expression in the production cell line (Hek- 293 cells), expression cassettes were prepared. The synthesized heavy and light chain variable domains of 7E5 were cloned into the pMQR eukaryotic expression vector set (pMQR-hlgGl and pMQR-hlgK), thus generating a human-rat chimeric recombinant antibody. Sequence analysis of the resulting clones indicated that both sequences were cloned correctly. The pMQR eukaryotic expression vectors harboring both 7E5 variable domains were transfected into Hek-293 cells and these cells were allowed to produce the recombinant antibody. Following production, hlgGl/hlgK antibodies were detected in the spent supernatant by means of capture ELISA. The transfection supernatant was shown to contain recombinant 7E5 at 0.019 mg/ml.
Example 6: Humanization of 7E5 Monoclonal Antibody
As an alternative to antibody humanization methods based on cycles of site- directed mutagenesis, we humanized the rat 7E5 mAb using a humanization approach based on CDR-homology between human and murine antibodies as described by Hwang and colleagues (Methods. 2005. 36:35-42). This method is based on the principle that if a non-human and a human antibody have similarly structured CDRs, the human frameworks will also support the non-human CDRs, with good retention of affinity. In this method, the human framework sequences are chosen from the set of human germline genes based on the structural similarity of the human CDRs to those of the antibody to be humanized (same Chothia canonical structures). A phage display library of Fab variant sequences, containing deviating FR residues, is generated. After affinity- driven selections, individual clones are screened for binding and off-rate and the sequence human identity and homology is determined.
The process to humanize 7E5 rat antibody applied in this work consisted of the following steps:
1- Design of humanization library: Identification of the closest human germlines and identification of the rat VH and VK FR residues deviating from these human germlines.
2- Assembly of the 7E5 gene libraries (using overlapping oligonucleotides to synthetically generate the variable heavy (VH) and light (VL) chain encoding genes via PCR).
3- Cloning of these gene libraries into a phagemid (pCB13-CKl/3) containing the human constant heavy (CHI) and light (CK) chain (library construction).
4- Selection of the functional Fabs using phage display and affinity selection.
5- Screening for off-rate (Biacore) and sequencing.
6- Selection of the Fabs with the highest human identity and homology without loss of binding to hC6.
7- Production and purification of eight humanized leads to be used in further affinity measurements and in functional assays. Design of Humanization Library: Using the nucleotide and amino acid sequences of the variable domains of the rat 7E5 antibody and public databases and tools, we confirmed that 7E5 uses IGHV5S45*01, IGHD 1-6*01, IGHJ3*01 and IGKV2S27*01, IGKJ2-3*01 as germline segments. We also concluded that the canonical fold combinations for CDR HI and CDR H2 of 7E5 is 1-3 and for CDR LI and CDR L2 of 7E5 is 4-1.
Comparison of the 7E5 VH sequence with human germlines with the identical canonical fold combination 1-3 for CDR1 and CDR2 revealed human germline VH3 family member 1 as the closest match. The closest human JH germline appeared to be IGHJ4. The alignment against these germline segments is shown in Figure 6A. The FRs and CDRs are indicated, which enables the identification of FR residues deviating from the human germlines. The 7E5 heavy chain amino acid sequence is also shown in Figure 10.
Using a similar analysis we determined that for the 7E5 VK sequence the closest human germline is human VK2 family member 5. The closest human JH germline appeared to be IGKJ2 and IGKJ5. The alignment against these germline segments is shown in Figure 6B. The FRs and CDRs are indicated, which enables the identification of FR residues deviating from the human germlines. The 7E5 light chain amino acid sequence is also shown in Figure 10.
As shown in Figures 6A and 6B there were 13 positions for 7E5 VH sequences and 16 for 7E5 VK, respectively, for which the human residues were incorporated for the humanization libraries but also the rat residue in case the change would be detrimental for antigen binding. Taking into account the number of positions to mutate and the number of variants per position, the library size to cover the introduced diversity would be 8.2 x 103 and 9.8 x 104 for humanized VH and VK libraries respectively.
Humanized 7E5 Fab Library Construction: For the construction of the final humanized 7E5 Fab phage display library, we initially constructed two different sub-libraries:
1- VH Humanized Fab sub-library, in which the humanized 7E5 VH gene was cloned together with the WT 7E5VK into pCB13-CK3 phagemid, containing the genes coding for the human constant domains CHI and CK.
2- VL Humanized Fab sub-library, in which the humanized 7E5 VK gene was cloned together with the WT 7E5VH into phagemid vectorspCB13-CKl and pCB13-CK3, containing the genes coding for the human constant domains CHI and CK.
Due to the cloning strategy and sequences of the two different phagemids used, the residues in positions 104 to 107 of the Light chain V domain of clones produced from pCB13-CKl would correspond to LEIK (Humanized 7E5 sequence), while V domain light chains of clones produced in pCB13-CK3 would show in the same positions amino acids LELK (7E5 WT VK sequence).
The two resulting sub-libraries were panned against human C6 and binding clones were recovered to proceed to final Fab library construction in which both heavy and light chain were humanized.
Synthetic Gene Assembly: To construct the different humanized heavy and light chain sub-libraries, the humanized 7E5 VH and VK genes were generated by gene assembly (Cherry, J. et al. (2008) J Biochem Biophys Methods, 70:820-2; Stemmer, W.P. et al. (1995) Gene, 164:49-53)..
Humanized 7E5 VH and VK Sub-libraries Construction: For the
construction of the 7E5 VH Fab sub-library, we cloned the synthetic VH genes of approximately 400 bp generated by gene assembly and a DNA fragment codifying for 7E5 VK WT into the phagemid pCB13-CK3 (containing the human constant heavy and kappa light chain encoding genes).
For the construction of the 7E5 VK Fab sub-library, we cloned the synthetic VK genes of approximately 400 bp generated by gene assembly and a DNA fragment codifying for 7E5 VH WT via ApaLI/ Xhol sites and Ncol/Nhel respectively into an equimolar mixture of phagemids pCB13-CKl and pCB13-CK3 (containing the human constant heavy and kappa light chain encoding genes).
We transformed the new vectors resulting from the cloning process by electroporation into E. coli TGI cells. The size of the libraries was calculated from 5 μΐ spots of TGI transformed cells on LBA Carbenicillin (100pg/ml), Glucose2% and the percentage of Fab inserts was determined by colony PCR. The size and insert percentage of the sub-libraries is summarized below in Table 5. Table 5: Size and Insert Percentage Obtained for Humanized 7E5 Sub-Libraries
Figure imgf000065_0001
Sub-libraries were also QCed by DNA sequence analysis of 48 clones per library. Amino acid sequences were extracted using CLC Main Workbench
Software. Analysis of valid VH and VK sequences and of the frequency of WT or mutated residue per position revealed that the sub-libraries were successfully designed and constructed with the ratio of WT/mutation of approximately 50/50 (33/33/33 for position 103 in the VK gene) and the average number of FR mutations was obtained as designed.
Panning Selections of Humanized 7E5 VH and VK Sub-Libraries: We prepared phages from the two sub-libraries and used them for a first round selection on coated human C6. The aim of this round of selection was to clean up the sub-libraries from non-binding Fabs and therefore no stringent conditions were applied.
For the panning selections 5 and 0 pg/ml of human C6 were coated in 96- well Maxisorp plate (Nunc) and blocked with low-fat milk powder (Marvell 4% in PBS). After 2 hours of incubation with sub-library phage and subsequent washes, trypsin elution (10 mg/ml) was performed at room temperature. Protease activity was immediately neutralized by applying 16 mM protease inhibitor ABSF.
All phage outputs were infected into logarithmically grown E .coli TGI cells and 5 μΐ of the infected bacteria were plated on agar plates
(LBAGluc2%Carbl00pg/ml) for analysis of outputs and for enrichment
determination. We calculated enrichment as the ratio between the number of phage eluted from human C6 versus those eluted from the no protein conditions.
Very good enrichments compared to background (PBS) for both Humanized 7E5 VK and VH Fab sub-libraries were observed. Construction of Final Humanized 7E5 Fab Phage Display Library: The final humanized 7E5 Fab library was constructed by combining the recovered humanized heavy chains (VHCH) from clones selected from the 7E5 VH Fab sub- library with the recovered humanized light chains (VKCK) selected from the 7E5 VK Fab sub-library. We calculated the size of the resulting library from 5 μΐ spots of TGI transformed cells on LBA Carbenicillin (lOOpg/ml), Glucose 2% and we determined the percentage of Fab inserts by colony PCR. Selections of Humanized 7E5 Fab Library: In order to select humanized variants with no loss of affinity, or even with improved affinities, when compared to the rat WT 7E5 antibody, in-solution phage display selections with the humanized 7E5 Fab library were performed using biotinylated hC6 antigen.
Human C6 was biotinylated and QCed by SDS-PAGE, Western Blot and ELISA using the anti-human C6 antibody 7E5 to detect the biotinylated C6 captured on neutravidin coated plates. We performed three consecutive rounds of affinity driven selections in which the antigen concentration was decreased from round to round, as well as the phage input was also decreased from round 1 to round 2. In the second and third round of selections, phages incubated with neutravidin- captured human C6 were also incubated in the presence of an excess of non- biotinylated C6 for 2 hours or overnight (off-rate selections) in an attempt to, after several washings, get rid of high off-rate binding clones. As control, in parallel, similar selections were performed where the phage were incubated with neutravidin-captured human C6 and PBS instead of non-biotinylated hC6 (no off- rate selections).
All phage selection outputs were infected into logarithmically grown E .coli TGI cells and 5 μΐ of the infected bacteria were plated on agar plates
(LBAGluc2%Carbl00 pg/ml) for analysis of outputs and for enrichment determination. Enrichment was calculated as the ratio between the number of phage eluted from human C6 versus those eluted from the no protein conditions. Very good enrichments compared to background (PBS) were obtained. Binding Screening of Clones Selected from Humanized 7E5 Fab Library: Individual colonies of E. coli TGI infected with the eluted phage pools obtained after the second and third round of off-rate selections were grown at 37 °C for 8 hours in two 96 well plates (Master plates) containing 100 μΐ of 2TYGlucose2% Carbenicillin 100 pg/ml, stored in 20% glycerol at -80 °C and used for later sequencing, and periplasmic extract production. A total of two master plates (MPs) were generated with clones from the second round selections and from the third round selections. From these MPs, bacterial extracts containing soluble
monoclonal Fabs (periplasmic extracts) were produced. Monoclonal bacterial small-scale cultures were induced at ΟΌβοο of 0.8 by adding isopropyl-b-D- thiogalactopyranoside (IPTG) to a final concentration of 1 mM. The periplasmic extracts (P.E.s) containing Fabs were then prepared by freezing- thawing of the bacterial pellet in PBS and subsequent centrifugation to remove cell debris.
In order to determine the target binding capacity of the selected clones, P.E.s at 1:5 dilution were tested for binding to 10 nM of biotinylated hC6 captured on neutravidin-coated Maxisorp plate. P.E. prepared from rat 7E5 WT Fab was used as positive control. Blank P.E. (prepared from non-inoculated well in the MP) was used as negative controls. Binding of P.E.s to the target was detected with an anti-c-myc mouse antibody conjugated to Horseradish peroxidase (HRP). A binding hit rate of 40% was obtained for both MPs and binding signals (O.D. 450nm values) of the positive clones were comparable to the signal obtained with the parental rat 7E5 Fab.
Off-Rate Screening and Analysis of Human Identity and Homology of Selected Clones Binding Human C6: For the positive binding clones, the off-rate for hC6 was determined using the SPR method and in parallel, the DNA coding for the variable domain of the heavy and of the light chains was sequenced.
To determine the off-rate a Biacore 3000 (GE Healthcare) was used. For that purpose, 50 pg/ml of hC6 in acetate buffer pH 4.5 was immobilized on a CM5 sensor chip (GE Healthcare BR- 1000- 12) to approximately 2000 RU. Regeneration conditions were tested and 2 x 10 μΐ of 10 mM NaOH and 1 M NaCl were used for the regeneration between sample injections. 30 μΐ of P.E.s, prepared as described above, were diluted in 120 μΐ of HBS-EP buffer and from this 60 μΐ were injected with a flow of 30 μΐ/min. Dissociation was measured during 400 seconds and the off-rate was determined by applying the 1: 1 Langmuir dissociation fitting model.
In parallel, to analyze human identity and homology the DNA coding for the variable heavy chain and light chain of clones that showed specific binding to hC6 was sequenced. Amino acid sequences were extracted using CLC Main Workbench Software. The VK and the VH sequences were aligned separately against the reference sequence (7E5 WT). All sequences were analyzed to determine the percentage of human identity (fraction of framework residues which is found in the closest matching germline) and human homology (fraction of framework residues which is found in the closest matching germline or other germlines of the same subclass) using the Abligner software.
Overall a good correlation was observed between the ELISA and the Biacore data, and also good human identity and homology percentage values varying from 88-99%.
A lead panel of eight clones that had good binding, off-rate and human identity and homology data were selected, referred to as 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11 and 7F02. The complete nucleotide and amino acid sequences of the variable domains of the heavy and light chains of the lead panel of eight humanized clones are shown below:
8G09 VH and VL Nucleotide Sequences:
8G09 VH
GAGGTGTAGCTGGTGGAGTCTGATGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAGACTCTCCTGTGT AGCCTCAGGATTCACTTTCAGTGACTATTACATGGCCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGG AGTGGGTCGCAACCATTAATTATGATGGTAGTAGTACTTACTATCGAGAGTCCGTGAAGGGCCGATTCA CTATCTCCAGAGATAATGCGAAACGCACCCTATACCTGCAAATGGACAGTCTGAGGGCTGAGGACACG G CCGTTTATT ACTGTG C AAG ACCTTCT ACG G AG G CCCTGTTTG CTTACTG G G G CCA AG G C ACTCTG GTC ACTGTCTCCTCA 8G09 VK
GATATTGTGCTGACCCAGACTCCATTGACATTATCGGTTACCCCTGGACAATCGGTCTCCATCTCTTGCA GGTCAAGTCAGAGTCTCTTAAATGATGTTGGAAACACCTATTTATATTGGTATCTACAGAAGCCTGGCC AATCTCCACAGCTTCTAATTTATTTGGTCTCCGACCTGGGATCTGGGGTCCCCAACAGGTTCAGTGGCA GTGGGTCAGGAACAGATTTCACACTCAAAATCAGTAGAGTGGAGGCTGAGGATGTGGGAGTTTATTAC TGCATGCAAGCTAGTCATGCTCCGTACACGTTTGGAGCGGGGACCAGACTCGAGATCAAA 7E12 VH and VL Nucleotide Sequences:
7E12 VH
GAGGTGTAGCTGGTGGAGTCTGATGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGC AGCCTCAGGATTCACTTTCAGTGACTATTACATGGCCTGGGTCCGCCAGGGTCCAGGGAAGGGGCTGG AGTGGGTCGCAACCATTAATTATGATGGTAGTAGTACTTACTATCGAGAGTCCGTGAAGGGCCGATTCA CTATCTCCAGAGATAATGCGAAAAACACCCTATACCTGCAAATGAACAGTCTGAGGGCTGAGGACACG GCCACTTATTACTGTGCAAGACCTTCTACGGAGGCCCTGTTTGCTTACTGGGGCCACGGCACTCTGGTC ACTGTCTCCTCA 7E12 VK
GATGTTGTGCTGACCCAGACTCCATCGACATTATCGGTTACCCCTGGACAACCGGCCTCCATCTCTTGCA GGTCAAGTCAGAGTCTCTTAAATGATGTTGGAAACACCTATTTATATTGGTATCTACAGAAGCCTGGCC AATCTCCACAGCTTCTAATTTATTTGGTCTCCGACCTGGGATCTGGGGTCCCCAACAGGTTCAGTGGCA GTGGGTCAGGAACAGATTTCACACTCAAAATCAGTAGAGTGGAGGCTGAGGATGTGGGAATTTATTAC TGCATGCAAGCTAGTCATGCTCCGTACACGTTTGGACAGGGGACCAACCTCGAGATCAAA
7G09 VH and VL Nucleotide Sequences:
7G09 VH
GAGGTGTAGCTGGTGGAGTCTGATGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAGACTCTCCTGTGC AGCCTCAGGATTCACTTTCAGTGACTATTACATGGCCTGGGTCCGCCAGGGTCCAACGAAGGGGCTGG AGTGGGTCGCAACCATTAATTATGATGGTAGTAGTACTTACTATCGAGAGTCCGTGAAGGGCCGATTCA CTATCTCCAGAGATAATGCGAAAAACACCCTATACCTGCAAATGGACAGTCTGAGGGCTGAGGACACG G CCGTTTATT ACTGTG C AAG ACCTTCT ACG G AG G CCCTGTTTG CTTACTG G G G CC ACG G C ACTCTG GTC ACTGTCTCCTCA
7G09 VK
GATGTTGTGCTGACCCAGACTCCATCGTCATTATCGGTTACCCCTGGACAATCGGCCTCCATCTCTTGCA GGTCAAGTCAGAGTCTCTTAAATGATGTTGGAAACACCTATTTATATTGGTATCTACAGAAGCCTGGCC AATCTCCACAGCTTCTAATTTATTTGGTCTCCGACCTGGGATCTGGGGTCCCCGACAGGTTCAGTGGCA GTGGGTCAGGAACAGATTTCACACTCAAAATCAGTAGAGTGGAGGCTGAGGATTTGGGAATTTATTAC TGCATGCAAGCTAGTCATGCTCCGTACACGTTTGGACAGGGGACCAAACTCGAGCTGAAA
8F07 VH and VL Nucleotide Sequences:
8F07 VH
GAGGTGTAGCTGGTGGAGTCTGGTGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAGACTCTCCTGTGC AGCCTCAGGATTCTCTTTCAGTGACTATTACATGGCCTGGGTCCGCCAGGGTCCAGGGAAGGGGCTGG AGTGGGTCGCAACCATTAATTATGATGGTAGTAGTACTTACTATCGAGAGTCCGTGAAGGGCCGATTCA CTATCTCCAGAGATAATGCGAAAAACACCCTATACCTGCAAATGAACAGTCTGAGGTCTGAGGACACG GCCACTTATTACTGTGCAAGACCTTCTACGGAGGCCCTGTTTGCTTACTGGGGCCACGGCACTCTGGTC ACTGTCTCCTCA
8F07 VK
GATGTTGTGCTGACCCAGACTCCATTGACATTATCGGTTACCCCTGGACAATCGGTCTCCATCTCTTGCA GGTCAAGTCAGAGTCTCTTAAATGATGTTGGAAACACCTATTTATATTGGTATCTACAGAAGCCTGGCC AATCTCCACAGCTTCTAATTTATTTGGTCTCCGACCTGGGATCTGGGGTCCCCGACAGGTTCAGTGGCA
GTGGGTCAGGAACAGATTTCACACTCAAAATCAGTGGAGTGGAGGCTGAGGATGTGGGAGTTTATTAC
TGCATGCAAGCTAGTCATGCTCCGTACACGTTTGGAGCGGGGACCAAACTCGAGATCAAA
7F06 VH and VL Nucleotide Sequences:
7F06 VH
GAGGTGTAGCTGGTGGAGTCTGGTGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGC AGCCTCAGGATTCACTTTCAGGGACTATTACATGGCCTGGGTCCGCCAGGGTCCAGGGAAGGGGCTGG AGTGGGTCGCAACCATTAATTATGATGGTAGTAGTACTTACTATCGAGAGTCCGTGAAGGGCCGATTCA CTATCTCCAGAGATAATGCGAAAAACAGCCTATACCTGCAAATGGACAGTCTGAGGGCTGAGGACACG G CCGTTTATT ACTGTG C AAG ACCTTCT ACG G AG G CCCTGTTTG CTTACTG G G G CC ACG G C ACTCTG GTC ACTGTCTCCTCA 7F06 VK
GATGTTGTGCTGACCCAGACTCCATTGACATTATCGGTTACCCCTGGACAACCGGTCTCCATCTCTTGCA GGTCAAGTCAGAGTCTCTTAAATGATGTTGGAAACACCTATTTATATTGGTATCTACAGAAGCCTGGCC AATCTCCACAGCTTCTAATTTATTTGGTCTCCGACCTGGGATCTGGGGTCCCCAACAGGTTCAGTGGCA GTGGGTCAGGAACAGATTTCACACTCAAAATCAGTAGAGTGGAGGCTGAGGATGTGGGAGTTTATTAC TGCATGCAAGCTAGTCATGCTCCGTACACGTTTGGAGCGGGGACCAGACTCGAGCTGAAA
7F11 VH and VL Nucleotide Sequences:
7F11 VH
GAGGTGTAGCTGGTGGAGTCTGATGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGC AGCCTCAGGATTCACTTTCAGTGACTATTACATGGCCTGGGTCCGCCAGGGTCCAACGAAGGGGCTGG AGTGGGTCGCAACCATTAATTATGATGGTAGTAGTACTTACTATCGAGAGTCCGTGAAGGGCCGATTCA CTATCTCCAGAGATAATGCGAAAAACACCCTATACCTGCAAATGAACAGTCTGAGGGCTGAGGACACG G CCGTTTATT ACTGTTC AAG ACCTTCT ACG GAG G CCCTGTTTG CTTACTG G G G CC ACG G C ACTCTG GTC A CTGTCTCCTCA
7F11 VK
GATGTTGTGCTGACCCAGACTCCATCGACATTATCGGTTACCCCTGGACAACCGGTCTCCATCTCTTGCA GGTCAAGTCAGAGTCTCTTAAATGATGTTGGAAACACCTATTTATATTGGTATCTACAGAAGCCTGGCC AATCTCCACAGCTTCTAATTTATTTGGTCTCCGACCTGGGATCTGGGGTCCCCAACAGGTTCAGTGGCA GTGGGTCAGGAACAGATTTCACACTCAAAATCAGTGGAGTGGAGGCTGAGGATGTGGGAGTTTATTAC TGCATGCAAGCTAGTCATGCTCCGTACACGTTTGGAGCGGGGACCAGACTCGAGATCAAA
7E11 VH and VL Nucleotide Sequences:
7E11 VH
GAGGTGCAGCTGGTGGAGTCTGGTGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAGACTCTCCTGTGT AGCCTCAGGATTCACTTTCAGTGACTATTACATGGCCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGG AGTGGGTCGCAACCATTAATTATGATGGTAGTAGTACTTACTATCGAGAGTCCGTGAAGGGCCGATTCA CTATCTCCAGAGATAATGCGAAAAACACCCTATACCTGCAAATGGACAGTCTGAGGGCTGAGGACACG G CCGTTTATT ACTGTG C AAG ACCTTCT ACG G AG G CCCTGTTTG CTTACTG G G G CCA AG G C ACTCTG GTC ACTGTCTCCTCA
7E11 VK
GATATTGTGCTGACCCAGACTCCATTGTCATTATCGGCTACCCCTGGACAATCGGTCTCCATCTCTTGCA GGTCAAGTCAGAGTCTCTTAAATGATGTTGGAAACACCTATTTATATTGGTATCTACAGAGGCCTGGCC AATCTCCACAGCTTCTAATTTATTTGGTCTCCGACCTGGGATCTGGGGTCCCCGACAGGTTCAGTGGCA GTGGGTCAGGAACAGATTTCACACTCAAAATCAGTAGAGTGGAGGCTGAGGATGTGGGAGTTTATTAC TGCATGCAAGCTAGTCATGCTCCGTACACGTTTGGAGCGGGGACCAACCTCGAGATCAAA
7F02 VH and VL Nucleotide Sequences:
7F02 VH
GAGGTGCAGCTGGTGGAGTCTGGTGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGC AGCCTCAGGATTCACTTTCAGTGACTATTACATGGCCTGGGTCCGCCAGGGTCCAGGGAAGGGGCTGG AGTGGGTCGCAACCATTAATTATGATGGTAGTAGTACTTACTATCGAGAGTCCGTGAAGGGCCGATTCA CTATCTCCAGAGATAATGCGAAAAACAGCCTATACCTGCAAATGAACAGTCTGAGGTCTGAGGACACG G CCGTTTATT ACTGTG C AAG ACCTTCT ACG GAG G CCCTGTTTG CTTACTG G G G CC ACG G C ACTCTG GTC ACTGTCTCCTCA 7F02 VK
GATGTTGTGATGACCCAGACTCCATCGACATTATCGGCTACCCCTGGACAATCGGCCTCCATCTCTTGCA GGTCAAGTCAGAGTCTCTTAAATGATGTTGGAAACACCTATTTATATTGGTATCTACAGAAGCCTGGCC AATCTCCACAGCTTCTAATTTATTTGGTCTCCGACCTGGGATCTGGGGTCCCCAACAGGTTCAGTGGCA GTGGGTCAGGAACAGATTTCACACTCAAAATCAGTAGAGTGGAGGCTGAGGATGTGGGAATTTATTAC TGCATGCAAGCTAGTCATGCTCCGTACACGTTTGGAGCGGGGACCAGACTCGAGCTGAAA
8G09 VH and VL Amino Acid Sequences:
8G09 VH
EVQLVESDGGLVQPGGSLRLSCVASGFTFSDYYMAWVRQAPGKGLEWVATI NYDGSSTYYRESVKGRFTIS RDNAKRTLYLQM DSLRAEDTAVYYCARPSTEALFAYWGQGTLVTVSS
8G09 VK
DIVLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYLVSDLGSGVPNRFSGSGSGT DFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTRLEIK
7E12 VH and VL Amino Acid Sequences:
7E12 VH
EVQLVESDGGLVQPGGSLKLSCAASGFTFSDYYMAWVRQGPGKGLEWVATI NYDGSSTYYRESVKGRFTIS RDNAKNTLYLQM NSLRAEDTATYYCARPSTEALFAYWGHGTLVTVSS
7E12 VK
DVVLTQTPSTLSVTPGQPASISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYLVSDLGSGVPNRFSGSGSG TDFTLKISRVEAEDVGIYYCMQASHAPYTFGQGTNLEIK 7G09 VH and VL Amino Acid Sequences:
7G09 VH
EVQLVESDGGLVQPGGSLRLSCAASGFTFSDYYMAWVRQGPTKGLEWVATI NYDGSSTYYRESVKGRFTIS RDNAKNTLYLQM DSLRAEDTAVYYCARPSTEALFAYWGHGTLVTVSS
7G09 VK
DIVLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYLVSDLGSGVPNRFSGSGSGT DFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTRLEIK 8F07 VH and VL Amino Acid Sequences:
8F07 VH
EVQLVESGGGLVQPGGSLRLSCAASGFSFSDYYMAWVRQGPGKGLEWVATI NYDGSSTYYRESVKGRFTIS RDNAKNTLYLQM NSLRSEDTATYYCARPSTEALFAYWGHGTLVTVSS 8F07 VK
DVVLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYLVSDLGSGVPDRFSGSGSG TDFTLKISGVEAEDVGVYYCMQASHAPYTFGAGTKLEIK
7F06 VH and VL Amino Acid Sequences:
7F06 VH
EVQLVESGGGLVQPGGSLKLSCAASGFTFRDYYMAWVRQGPGKGLEWVATINYDGSSTYYRESVKGRFTIS RDNAKNSLYLQM DSLRAEDTAVYYCARPSTEALFAYWGHGTLVTVSS
7F06 VK
DVVLTQTPLTLSVTPGQPVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYLVSDLGSGVPN RFSGSGSG TDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTRLELK
7F11 VH and VL Amino Acid Sequences:
7F11 VH
EVQLVESDGGLVQPGGSLKLSCAASGFTFSDYYMAWVRQGPTKGLEWVATI NYDGSSTYYRESVKGRFTIS RDNAKNTLYLQM NSLRAEDTAVYYCSRPSTEALFAYWGHGTLVTVSS
7F11 VK
DVVLTQTPSTLSVTPGQPVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYLVSDLGSGVPN RFSGSGSG TDFTLKISGVEAEDVGVYYCMQASHAPYTFGAGTRLEIK
7E11 VH and VL Amino Acid Sequences:
7E11 VH
EVQLVESGGGLVQPGGSLRLSCVASGFTFSDYYMAWVRQAPGKGLEWVATI NYDGSSTYYRESVKGRFTIS RDNAKNTLYLQM DSLRAEDTAVYYCARPSTEALFAYWGQGTLVTVSS 7E11 VK
DIVLTQTPLSLSATPGQSVSISCRSSQSLLNDVGNTYLYWYLQRPGQSPQLLIYLVSDLGSGVPDRFSGSGSGT DFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTN LEIK
7F02 VH and VL Amino Acid Sequences:
7F02 VH
EVQLVESGGGLVQPGGSLKLSCAASGFTFSDYYMAWVRQGPGKGLEWVATI NYDGSSTYYRESVKGRFTIS RDNAKNSLYLQM NSLRSEDTAVYYCARPSTEALFAYWGHGTLVTVSS
7F02 VK
DVVMTQTPSTLSATPGQSASISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYLVSDLGSGVPNRFSGSGS GTDFTLKISRVEAEDVGIYYCMQASHAPYTFGAGTRLELK An alignment of the amino acid sequence of the rat 7E5 heavy chain variable region to the amino acid sequences of the heavy chain variable regions of the humanized 7E5 variants 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11 and 7F02 is shown in Figure 8A, with CDR1, 2 and 3 indicated. An alignment of the amino acid sequence of the rat 7E5 light chain variable region to the amino acid sequences of the light chain variable regions of the humanized 7E5 variants 8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11 and 7F02 is shown in Figure 8B, with CDR1, 2 and 3 indicated. The heavy chain CDR1, 2 and 3 sequences for the eight humanized variants of the rat 7E5 antibody are the same as those in the rat 7E5 mAb (the amino acid sequences of which are shown in Figure 10). Likewise, the light chain CDR1, 2 and 3 sequences for the eight humanized variants of the rat 7E5 antibody are the same as those in the rat 7E5 mAb (the amino acid sequences of which are also shown in Figure 10).
Fab Expression, Purification and QC: In order to characterize some of the 7E5 humanized variants in further assays (i.e., complement mediated lysis of pre- sensitized erythrocytes assay, affinity determination, melting temperatures and aggregation behavior assays), soluble Fabs were produced and purified from the lead panel of eight clones described above. The Fab genes of all 8 humanized clones plus the 7E5 WT control were cloned into pCB4 expression vector (very similar to pCB13 but without the gene 3 codifying sequence) via Sfil/Notl digestion and transformed into TGI E.coli strain via heat shock. The sequences were confirmed using the CLC Main Workbench Software.
Production of P.E.s containing soluble Fabs from the pCB4-cloned 7E5 humanized variants as well as from 7E5 WT was performed in 800 ml of 2xYT supplemented with 0.1% of glucose and Carbenicillin at 100 pg/ml. After induction at OD600 of 0.5-0.8 with IPTG to a final concentration of 1 mM, the culture was incubated at 24 °C for at least 20 hours. The soluble Fabs were purified with TALON metal affinity resin.
When 500 ng of the resulting purification products were run on a SDS- PAGE several extra bands apart from the Fab specific bands (50 KDa and aproximately 25 KDa under non-reduced and under reduced conditions
respectively) were observed. To further purify these samples, a resin from Life Technologies that contains a VHH that specifically binds to human CHI domain (CaptureSelectTM Affinity resin IgG-CHl, cat# 194320005) was used according to manufacturer instructions. The concentration of the resulting purified protein was estimated by measuring the OD280nm using a micro-volume spectrophotometer and assuming a molar extinction coefficient on £=1.53. SDS-PAGE analysis of the purified samples showed a high level of purity. The functionality of the purified Fab was confirmed in ELISA where binding of serial dilutions of these Fabs to 10 nM of biotinylated hC6 captured on neutravi din-coated Maxisorp plate was examined. All eight purified Fabs exhibited effective binding to hC6.
Biacore Analysis: In order to determine whether humanization of 7E5 altered the binding specificity or activity of the resultant humanized antibodies, Biacore affinity analysis was performed on the eight selected humanized Fabs (7E12, 7E11, 7F2, 7F6, 7F11, 7G9, 8F7 and 8F9) as compared to the (parental) wild-type rat 7E5 mab and to the mouse 27B1 mAb. The results are shown in Figure 9. The results indicate that humanization of 7E5 did not alter the specificity or activity of the antibody. Example 7: "Mix & Match" Characterization of Humanized anti-C6
Antibodies
In this experiment, a panel of humanized VH chains and humanized VL chains from the selected humanized anti-C6 antibodies were expressed as full- length antibodies in mammalian cells in various combinations and were evaluated for their functional activity.
The humanized VH chains used were the eight VH chains described in Example 6 (8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11 and 7F02), as well as a ninth chain, 7C02, the amino acid sequence of which is shown in Figure 8A. An alignment of these nine chains is shown in Figure 8A.
The humanized VL chains used were the eight VH chains described in Example 6 (8G09, 7E12, 7G09, 8F07, 7F06, 7F11, 7E11 and 7F02), as well as a ninth chain, 7G08, the amino acid sequence of which is shown in Figure 8B. An alignment of these nine chains is shown in Figure 8B.
The heavy and light chain nucleotide sequences were cloned into expression vectors to create coding sequences for full-length chains having a stabilized IgG4 (S228P) constant region. The 9 heavy chains and 9 light chains were co-expressed as pairs in every possible combination in CHO host cells. Thus, all 81 possible "mix and match" combinations of the 9 heavy chains and 9 light chains were evaluated. The 81 pairs were each tested in the hemolytic assay and in the MAC ELISA. For each assay, 4 μg of humanized 7E5 mAb from CHO
supernatant was used. The results for the hemolytic assay are shown in Figure 7A. The results for the MAC ELISA are shown in Figure 7B. The results demonstrate that all 81 possible "mix and match" combinations of the 9 VH and 9VL chains exhibited strong inhibitory activity in both assays.
Example 8
In order to mimic a PNH situation, human erythrocytes were incubated with a blocking monoclonal antibody (Mab) against CD59. This rendered the erythrocytes susceptible to hemolysis by complement, like the CD55- and/or CD59-deprived erythrocytes of a PNH patient. The Mab that was used is a IgG2A which activates complement. Subsequently, 1.5% human serum was added to the erythrocytes. This resulted in complement-mediated lysis of the vulnerable erythrocytes.
Importantly, addition of 7E5 (original rat anti human C6 on a human IgG4 tail) appeared to block this complement-mediated lysis in a dose dependent way. This was assayed in a standard haemolytic titration assay (see Figure 12).
From this result it is concluded that a C6 inhibitor like 7E5 is able to counteract complement-mediated lysis of vulnerable erythrocytes like the CD55- and/or CD59- deprived erythrocytes of a PNH patient.

Claims

Claims
1. A method for counteracting complement-mediated hemolytic anemia, the method comprising administering an inhibitor of human complement component C6 to a human individual in need thereof.
2. A method according to claim 1, wherein said hemolytic anemia is paroxysmal nocturnal hemoglobinuria (PNH).
3. A method according to claim 1, wherein said hemolytic anemia is haemolytic uremic syndrome (HUS) or atypical haemolytic uremic syndrome (aHUS).
4. A method according to any one of claims 1-3, wherein said inhibitor of human complement component C6 is selected from the group consisting of a C6 antagonist, a peptide, a polypeptide, an antisense nucleic acid molecule, a small molecule, or a C6 receptor.
5. A method according to any one of claims 1-3, wherein said inhibitor of human complement component C6 is an antibody, or a functional part or a functional equivalent thereof, or a nucleic acid molecule encoding therefore.
6. A method according to claim 5, wherein said antibody or functional part or functional equivalent comprises a heavy chain CDR3 sequence having at least 90% sequence identity with the sequence PSTEALFAY, and a light chain CDR3 sequence having at least 90% sequence identity with the sequence MQASHAPYT.
7. A method according to claim 5 or 6, wherein said antibody or functional part or functional equivalent comprises:
- a heavy chain CDR1 sequence having a sequence which has at least 90% sequence identity with the sequence DYYMA; and
- a heavy chain CDR2 sequence having a sequence which has at least 90% sequence identity with the sequence TINYDGSSTYYRESVKG; and
- a heavy chain CDR3 sequence having a sequence which has at least 90% sequence identity with the sequence PSTEALFAY; and
- a light chain CDR1 sequence having a sequence which has at least 90% sequence identity with the sequence RSSQSLLNDVGNTYLY; and
- a light chain CDR2 sequence having a sequence which has at least 90% sequence identity with the sequence LVSDLGS; and - a light chain CDR3 sequence having a sequence which has at least 90% sequence identity with the sequence MQASHAPYT.
8. A method according to any one of claims 5-7, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
E VQ LVE S D GGLVQ PGGS LKLS C VAS GFS FSD YYMAWVRQ GPTKGLE WVATINY DGSSTYYRESVKGRFTISRDNAKRTLYLQMDSLRSEDTATYYCSRPSTEALFAY WGHGTLVTVSS and a variable light chain sequence having at least 80% sequence identity with the sequence
DWLTQTPSTLSATIGQSVSISCRSSQSLLNDVGNTYLYWYLQRPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISGVEAEDLGIYYCMQASHAPYTFGAGTNL ELK.
9. A method according to any one of claims 5-7, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
EVQLVESDGGLVQPGGSLRLSCVASGFTFSDYYMAWVRQAPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKRTLYLQMDSLRAEDTAVYYCARPSTEALFAY WGQGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DIVLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTRL EIK.
10. A method according to any one of claims 5-7, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
E VQ LVE S D GGLVQ PGGS LKLS C AAS GFTFS D YYMAWVRQ GPGKGLE WVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRAEDTATYYCARPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DWLTQTPSTLSVTPGQPASISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGIYYCMQASHAPYTFGQGTNL EIK.
11. A method according to any one of claims 5-7, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
E VQ LVE S D GGLVQ PGGS LRLS C AAS GFTFSD YYMAWVRQ GPTKGLE WVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMDSLRAEDTAVYYCARPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DIVLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTRL EIK.
12. A method according to any one of claims 5-7, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
EVQLVESGGGLVQPGGSLRLSCAASGFSFSDYYMAWVRQGPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRSEDTATYYCARPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DWLTQTPLTLSVTPGQSVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPDRFSGSGSGTDFTLKISGVEAEDVGVYYCMQASHAPYTFGAGTKL EIK.
13. A method according to any one of claims 5-7, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
EVQLVESGGGLVQPGGSLKLSCAASGFTFRDYYMAWVRQGPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNSLYLQMDSLRAEDTAVYYCARPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DWLTQTPLTLSVTPGQPVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTRL ELK.
14. A method according to any one of claims 5-7, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence E VQ LVE S D GGLVQ PGGS LKLS C AAS GFTFS D YYMAWVRQ GPTKGLE WVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCSRPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DWLTQTPSTLSVTPGQPVSISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISGVEAEDVGVYYCMQASHAPYTFGAGTRL EIK.
15. A method according to any one of claims 5-7, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
EVQLVESGGGLVQPGGSLRLSCVASGFTFSDYYMAWVRQAPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNTLYLQMDSLRAEDTAVYYCARPSTEALFAY WGQGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DIVLTQTPLSLSATPGQSVSISCRSSQSLLNDVGNTYLYWYLQRPGQSPQLLIYL VSDLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQASHAPYTFGAGTNL EIK .
16. A method according to any one of claims 5-7, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with the sequence
EVQLVESGGGLVQPGGSLKLSCAASGFTFSDYYMAWVRQGPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNSLYLQMNSLRSEDTAVYYCARPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with the sequence
DWMTQTPSTLSATPGQSASISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPNRFSGSGSGTDFTLKISRVEAEDVGIYYCMQASHAPYTFGAGTRL ELK .
17. A method according to any one of claims 5-7, wherein said antibody or functional part or functional equivalent comprises a variable heavy chain sequence having at least 80% sequence identity with
EVQLVESGGGLVQPGGSLKLSCAASGFTFSDYYMAWVRQAPGKGLEWVATINY DGSSTYYRESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARPSTEALFAY WGHGTLVTVSS and/or a variable light chain sequence having at least 80% sequence identity with
DIVMTQTPLSLSATPGQPASISCRSSQSLLNDVGNTYLYWYLQKPGQSPQLLIYL VSDLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQASHAPYTFGQGTKL EIK.
18. A method according to any one of claims 5- 17, wherein said antibody or functional part or functional equivalent is able to bind amino acid residues 835-854 of the human C6 sequence as depicted in Figure 11.
19. A method according to any one of claims 5- 18, wherein said antibody is human or humanized.
20. A method according to any one of claims 5- 19, wherein said antibody is of the IgG4 isotype.
21. A method according to any one of claims 1-3, wherein said inhibitor of human complement component C6 is an oligomer of between 10 to 50 nucleotides in length having a contiguous nucleic acid sequence with at least 80% sequence identity to a complementary region of the human C6 sequence as depicted in Figure 11.
22. A method according to claim 21, wherein the oligomer comprises a modification that facilitates liver uptake.
23. A method according to claim 21 or 22, wherein the oligomer comprises a modified internucleoside linkage.
24. A method according to any one of claims 21-23, wherein the oligomer further comprises a modified nucleobase.
25. A method according to any one of claims 21-24, wherein the nucleotide analogue is a modified sugar moiety selected from the group consisting of: 2'-0- methoxyethyl modified sugar moiety, a 2'-methoxy modified sugar moiety, a 2'-0- alkyl modified sugar moiety, and a bicyclic sugar moiety.
26. A method according to claim 25, wherein the bicyclic sugar moiety is a locked nucleic acid (LNA) monomer.
27. A method according to claim 23, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.
28. A method according to claim 24, wherein the modified nucleobase is
5 -methylcytosine .
29. Use of an inhibitor of human complement component C6 for counteracting complement-mediated hemolysis.
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