WO2007002571A2 - Use of an anti c5 complement antibody to treat patients with sickle cell disease - Google Patents

Use of an anti c5 complement antibody to treat patients with sickle cell disease Download PDF

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Publication number
WO2007002571A2
WO2007002571A2 PCT/US2006/024770 US2006024770W WO2007002571A2 WO 2007002571 A2 WO2007002571 A2 WO 2007002571A2 US 2006024770 W US2006024770 W US 2006024770W WO 2007002571 A2 WO2007002571 A2 WO 2007002571A2
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antibody
sickle cell
complement
antibodies
anemia
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PCT/US2006/024770
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French (fr)
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WO2007002571A3 (en
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Russell Rother
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Alexion Pharmaceuticals, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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

Definitions

  • the present invention relates to methods for the treatment of sickle cell disease.
  • the invention relates to the use of antibodies capable of inhibiting complement as therapeutic agents to treat sickle cell disease.
  • Sickle cell disease also called sickle cell anemia
  • the red blood cells are affected by a mutation in the beta globin gene which affects hemoglobin structure.
  • the mutant hemoglobin causes red blood ceils, which normally are smooth and round, to become hard, sticky, and shaped like sickles or crescents. These red cells tend to get stuck and block the flow of blood in small blood vessels and to lyse which in turn leads to pain, damage and a low blood count or anemia.
  • Sickle cell disease affects about 72,000 Americans, predominantly those of African ancestry.
  • One in every 600 African-American births is a baby with sickle cell disease.
  • One in every 1,000-1,400 Hispanic-American births is a baby with sickle cell disease. Those with the disease usually show some signs and symptoms after four months of age.
  • Blood transfusions do, however, include some risk such as a buildup of excess iron which can damage the heart, liver and other organs.
  • Administration of supplemental oxygen via a breathing mask can be useful during an acute chest syndrome or a sickle cell crisis.
  • Hydroxyurea a prescription drug normally used to treat cancer, is sometimes administered to adults with severe disease. It reduces the frequency of painful crises and may reduce the need for blood transfusions. It appears to work by stimulating production of fetal hemoglobin. Patients who took hydroxyurea over a 9-year period experienced a 40% reduction in deaths. There is concern that long-term use of this drug may cause tumors or leukemia.
  • Drugs being tested include butyric acid (which may increase the amount of fetal hemoglobin in the blood), clotrimazole (normally used to treat fungal infections but which may prevent loss of water from red blood cells and thereby reduce the number of sickle cells that form), and nitric oxide (a gas which helps keep blood vessels open and reduces the stickiness of red blood cells).
  • a search of the ClinicalTrials.gov website using the search term "sickle cell” gave a listing of 55 clinical trials (including completed studies, studies no longer recruiting, studies still recruiting patients, terminated studies, and studies not yet recruiting) related to sickle cell disease or sickle cell trait. This listing is shown as Table 1.
  • Sickle Cell Disease Condition Sickle Cell Anemia
  • Sickle Cell Disease Condition Sickle Cell Disease
  • Sickle Cell Syndromes Condition Sickle Cell Anemia
  • PROPS Completed Penicillin Prophylaxis in Sickle Cell Disease
  • Terminated Bone Marrow transplant from related donor for patients with high risk hemoglobinopathies Conditions Sickle Cell Anemia; Hemoglobinopathy; Thalassemia
  • Kocak et al. (Br. J. Haematol. 92:329-331 (1996)) used the vasodilating Ca +2 channel blocker bencyclane in 18 patients with sickle cell disease. The patients' blood contained fewer irreversibly sickled cells. Inhibition of the sickling-induced fluxes OfNa + , K + and Ca +2 was studied by Joiner et al. using dipyridamole (Blood 97:3976-3983 (2001)). The dipyridamole was found to inhibit sickling by inhibiting the ion flux. Shartava et al. (Am. J. Hematol.
  • complement system acts in conjunction with other immunological systems of the body to defend against intrusion of cellular and viral pathogens.
  • complement proteins There are at least 25 complement proteins, which are found as a complex collection of plasma proteins and membrane cofactors.
  • the plasma proteins make up about 10% of the globulins in vertebrate serum.
  • Complement components achieve their immune defensive functions by interacting in a series of intricate but precise enzymatic cleavage and membrane binding events.
  • the resulting complement cascade leads to the production of products with opsonic, immunoregulatory, and lytic functions.
  • the complement cascade progresses via the classical pathway or the alternative pathway. These pathways share many components and, while they differ in their initial steps, they converge and share the same "terminal complement” components (C5 through C9) responsible for the activation and/or destruction of target cells.
  • the classical complement pathway is typically initiated by antibody recognition of and binding to an antigenic site on a target cell.
  • the alternative pathway is usually antibody independent and can be initiated by certain molecules on pathogen surfaces. Both pathways converge at the point where complement component C3 is cleaved by an active protease (which is different in each pathway) to yield C3a and C3b. Complement activation can also occur via the lectin complement pathway (LCP).
  • Lectins are carbohydrate-binding proteins that recognize oligosaccharide structures present on cell surfaces, the extracellular matrix, and secreted glycoproteins.
  • the LCP also converges at the point of cleavage of complement component C3.
  • C3a is an anaphylatoxin.
  • C3b binds to bacterial and other cells, as well as to certain viruses and immune complexes, and tags them for removal from the circulation.
  • C3b in this role is known as opsonin.
  • the opsonic function of C3b is considered to be the most important anti-infective action of the complement system.
  • C3b also forms a complex with other components unique to each pathway to form classical or alternative C5 convertase, which cleaves C5 into C5a and C5b.
  • C3 is thus regarded as the central protein in the complement reaction sequence since it is essential to both the alternative and classical pathways (Wurzner et al., Complement Inflamm. 8:328-340 (1991)).
  • This property of C3b is regulated by the serum protease Factor I, which acts on C3b to produce iC3b. While still functional as opsonin, iC3b cannot form an active C5 convertase.
  • C5 is a 190 IcDa beta globulin found in normal serum at approximately 75 ⁇ g/mL (0.4 ⁇ M). C5 is glycosylated, with about 1.5-3 percent of its mass attributed to carbohydrate.
  • Mature C5 is a heterodimer of a 999 amino acid 115 kDa alpha chain that is disulfide linked to a 656 amino acid 75 kDa beta chain.
  • C5 is synthesized as a single chain precursor protein product of a single copy gene (Haviland et al., J. Immunol. 146:362-368 (1991)).
  • the cDNA sequence of the transcript of this gene predicts a secreted pro-C5 precursor of 1659 amino acids along with an 18 amino acid leader sequence.
  • the pro-C5 precursor is cleaved after amino acid 655 and 659, to yield the beta chain as an amino terminal fragment (amino acid residues +1 to 655) and the alpha chain as a carboxyl terminal fragment (amino acid residues 660 to 1658), with four amino acids deleted between the two.
  • C5a is cleaved from the alpha chain of C5 by either alternative or classical C5 convertase as an amino terminal fragment comprising the first 74 amino acids of the alpha chain (i.e., amino acid residues 660-733). Approximately 20 percent of the 11 kDa mass of C5a is attributed to carbohydrate. The cleavage site for convertase action is at or immediately adjacent to amino acid residue 733. A compound that would bind at or adjacent to this cleavage site would have the potential to block access of the C5 convertase enzymes to the cleavage site and thereby act as a complement inhibitor. C5 can also be activated by means other than C5 convertase activity.
  • C5a is another anaphylatoxin.
  • C5b combines with C6, C7, and C8 to form the C5b-8 complex at the surface of the target cell.
  • the membrane attack complex (MAC, C5b- 9, terminal complement complex-TCC) is formed.
  • MAC membrane attack complex
  • C5b- 9, terminal complement complex-TCC membrane attack complex
  • C3a and C5a are anaphylatoxins. These activated complement components can trigger mast cell degranulation, which releases histamine and other mediators of inflammation, resulting in smooth muscle contraction, increased vascular permeability, leukocyte activation, and other inflammatory phenomena including cellular proliferation resulting in hypercellularity.
  • C5a also functions as a chemotactic peptide that serves to attract pro-inflammatory granulocytes to the site of complement activation.
  • C3c complement indicative of the alternative pathway
  • C4c indicative of the classical pathway
  • Test and Woolworth (Blood 83:842-852 (1994)) further studied the relationship between sickle cell disease and complement. They first noted that prior work concluded that intravascular hemolysis of sickle cells resulted in complement activation. Test and Woolworth took the opposite view that possibly complement activation was leading to hemolysis. They showed in a series of in vitro assays that regulation of complement in sickle cells was abnormal, especially in that increased binding of C5b-7 and C9 to denser sickle cells was seen and this appeared to increase the susceptibility of sickle cells to lysis. They believe that the increased binding is most likely caused by changes in MIRL (also referred to as CD59) brought about by the sickling process and which is a membrane protein which inhibits lysis by the membrane attack complex.
  • MIRL also referred to as CD59
  • CD59 is a human glycosylphosphatidylinositol-anchored membrane glycoprotein that serves as the principle inhibitor of the cytolytic and pore forming activities of the C5b-9 membrane attack complex (MAC) of human complement.
  • MAC membrane attack complex
  • CD59 protects human blood cells, vascular endothelium and other cells from complement, which is normally present in human plasma and other body fluids.
  • CD59 is known to bind peptide segments of C8 and C9 that become exposed when these complement proteins incorporate into the C5b-9 complex, thereby preventing initiation or propagation of the membrane-inserted C9 polymer that is responsible for membrane damage.
  • Blood cells from patients with paroxysmal nocturnal hemoglobinuria lack the ability to anchor CD59 in their membranes and these cells are very susceptible to lysis via complement. Transducing these cells with a recombinant transmembrane form of CD59 results in the surface expression of CD59 and these transduced cells are protected against classical complement-mediated membrane damage by human serum (Rother et al., Blood 84:2604-2611 (1994)).
  • a method of treating sickle cell disease in a mammal comprising treating said mammal with a therapeutically effective amount of an anti-C5 antibody, wherein said treatment results in decreased lysis of red blood cells in said mammal.
  • said mammal is a human.
  • said antibody is a whole antibody or an antibody fragment.
  • said whole antibody or antibody fragment is selected from the group consisting of: a polyclonal antibody, a monoclonal antibody or antibody fragment, a diabody, a chimerized or chimeric antibody or antibody fragment, a humanized antibody or antibody fragment, a deimmunized human antibody or antibody fragment, a fully human antibody or antibody fragment, a single chain antibody, an Fv, an Fab, an Fab', or an F(ab') 2 .
  • said treatment is chronic. In certain embodiments, said treatment comprises treating an acute episode. In certain embodiments, said mammal is treated by administering said antibody intravenously. [0027] In certain embodiments, said mammal is treated to inhibit or decrease the amount and/or extent of an undesirable physiological condition resulting from excess lysis of red blood cells. In certain embodiments, said undesirable physiological condition is selected from the group consisting of dystonia, a clotting disorder, pulmonary hypertension, systemic hypertension, gastrointestinal contractions, abdominal pain, sternal pain, erectile dysfunction, priapism, inflammation, esophageal spasm, dysphagia, thrombosis, decreased organ perfusion, platelet activation and death.
  • said antibody inhibits activation of the classical pathway, the alternative pathway or the lectin complement pathway.
  • said antibody is an antibody against a member of the complement cascade components such as for example, C5, C5b, C6, C7, C8, and C9.
  • said antibody is an inhibitor of C5 cleavage.
  • said antibody decreases the availability of downstream complement components.
  • said antibody is pexelizumab. In certain embodiments, said antibody is eculizumab.
  • Sickle cell disease is an extremely serious disease affecting approximately 72,000 Americans and many more persons in other countries. At present a cure is almost nonexistent, the only cure being a bone marrow transplant. Gene therapy is a possible cure but is not being performed. In general sickle cell disease patients have their symptoms treated but are not cured. Treatments include treating or preventing infections by administration of antibiotics, blood transfusions to increase the supply of normal red blood cells, drugs to increase the level of fetal hemoglobin which is able to carry oxygen, drugs to prevent loss of water from erythrocytes thereby helping to prevent sideling, and drugs to aid in keeping blood vessels open. Despite these treatments, many patients continue to suffer greatly and many patients die as a result of the disease. More effective treatments are needed.
  • Applicant here proposes that treatment of a sickle cell disease patient with a complement inhibitor will decrease lysis of red blood cells and at least some of the symptoms seen in sickle cell patients. Many of the symptoms are a direct result of the lysis of red blood cells.
  • One aspect of the lysis of red blood cells is the consequent excess free hemoglobin in the bloodstream. This can lead to smooth muscle dystonias involving the gastrointestinal, cardiovascular, pulmonary, and urogenital systems, as well as clotting disorders, these symptoms including gastrointestinal contractions, erectile dysfunction such as priapism, and pulmonary hypertension as well as to clot formation (Rother et al., JAMA 293:1653-1662 (2005), the contents of which are specifically incorporated herein by reference).
  • Eculizumab being a whole antibody, is longer lasting in vivo than is a single-chain antibody and is preferably used although shorter acting inhibitors may be used, especially if used in time of a crisis and is used to relieve the crisis.
  • Eculizumab has been used in clinical studies and found to be well tolerated with minimal side effects.
  • Eculizumab has been administered to patients with paroxysmal nocturnal hemoglobinuria which is a disease in which red blood cells are easily lysed due to a lack of a protein in the red blood cell membrane which protects against lysis by activated complement. Most patients are administered a dose once every two weeks.
  • Suitable anti-C5 antibodies are known to those of skill in the art. Antibodies can be made to individual components of activated complement, e.g., antibodies to C7, C9, etc. (see, e.g., U.S. Patent 6,534,058; published U.S. patent application US 2003/0129187; and U.S. Patent 5,660,825). U.S. Patent 6,355,245 teaches an antibody which binds to C5 and inhibits cleavage into C5a and C5b thereby decreasing the formation not only of C5a but also the down stream complement components.
  • Additional proteins are known which inhibit complement-mediated lysis, including CD59, CD55, CD46 and other inhibitors of C8 and C9 (see, e.g., U.S. Patent 6,100,443). Proteins known as complement receptors and which bind complement are also known (see, Published PCT Patent Application WO 92/10205 and U.S. Patent 6,057,131). Use of soluble forms of complement receptors, e.g., soluble CRl, can inhibit the consequences of complement activation such as neutrophil oxidative burst, complement mediated hemolysis, and C3a and C5a production. Those of skill in the art recognize the above as some, but not all, of the known methods of inhibiting complement and its activation.
  • a preferred method of inhibiting complement activity is to use a monoclonal antibody which binds to complement C5 and inhibits cleavage. This decreases the formation of both C5a and C5b while at the same time allowing the formation of C3a and C3b which are beneficial to the recipient.
  • Such antibodies which are specific to human complement are known (U.S. Patent 6,355,245). These antibodies disclosed in U.S. Patent 6,355,245 include a preferred whole antibody (now named eculizumab). A similar antibody against mouse C5 is called BB5.1 (Frei et al., MoI. Cell. Probes. 1:141-149 (1987)). Antibodies to inhibit complement activity need not be monoclonal antibodies.
  • antibodies can be, e.g., polyclonal antibodies. They may additionally be antibody fragments.
  • An antibody fragment includes, but is not limited to, an Fab, F(ab'), F(ab') 2 , single-chain antibody, and Fv.
  • antibodies can be humanized (Jones et al., Nature 321:522-5 (1986)), chimerized, or deimmunized.
  • the antibodies to be used in the present disclosure may be any of these. It is preferable to use humanized antibodies.
  • a therapeutic agent of the disclosure comprises an antibody or antibody fragment.
  • Antibodies and fragments thereof may be made by any conventional method, such as those methods described herein. Antibodies are found in multiple forms, e.g., IgA, IgG, IgM, etc. Additionally, antibodies can be engineered in numerous ways. They can be made as single-chain antibodies (including small modular immunopharmaceuticals or SMIPsTM), Fab and F(ab') 2 fragments, etc. Antibodies can be humanized, chimerized, deimmunized, or fully human. Numerous publications set forth the many types of antibodies and the methods of engineering such antibodies. For example, see U.S. Patent Nos. 6,355,245; 6,180,370; 5,693,762; 6,407,213; 6,548,640; 5,565,332; 5,225,539; 6,103,889; and 5,260,203.
  • This invention provides fragments of anti-C5 antibodies, which may comprise a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody.
  • antibody fragments include Fab, Fab', F(ab') 2 , and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8:1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual "Fc” fragment, whose name reflects its ability to crystallize readily.
  • Pepsin treatment of an antibody yields an F(ab') 2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
  • Fv usually refers to the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-V L dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although likely at a lower affinity than the entire binding site.
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (CHl) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHl domain including one or more cysteines from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab') 2 antibody fragments originally were produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
  • This disclosure also provides monoclonal anti-C5 antibodies.
  • a monoclonal antibody can be obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts.
  • Monoclonal antibodies are highly specific, being directed against a single antigenic site.
  • each monoclonal antibody is directed against a single determinant on the antigen.
  • the monoclonal antibodies are advantageous in that they are often synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
  • Monoclonal antibodies may also be produced in transfected cells, such as CHO cells and NSO cells.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and does not require production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler et al., Nature 256:495-497 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Patent Nos. 4,816,567 and 6,331,415).
  • the "monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624- 628 (1991) and Marks et al., J. MoI. Biol. 222:581-597 (1991), for example.
  • oligoclonal antibodies refers to a predetermined mixture of distinct monoclonal antibodies. See, e.g., PCT publication WO 95/20401; U.S. Patent Nos. 5,789,208 and 6,335,163.
  • oligoclonal antibodies consisting of a predetermined mixture of antibodies against one or more epitopes are generated in a single cell.
  • oligoclonal antibodies comprise a plurality of heavy chains capable of pairing with a common light chain to generate antibodies with multiple specificities (e.g., PCT publication WO 04/009618).
  • Oligoclonal antibodies are particularly useful when it is desired to target multiple epitopes on a single target molecule (e.g., C5).
  • a single target molecule e.g., C5
  • those skilled in the art can generate or select antibodies or mixture of antibodies that are applicable for an intended purpose and desired need.
  • one or more of the CDRs are derived from an anti-human C5 antibody.
  • all of the CDRs are derived from an anti-human C5 antibody.
  • the CDRs from more than one anti- human C5 antibody are mixed and matched in a chimeric antibody.
  • a chimeric antibody may comprise a CDRl from the light chain of a first anti-human C5 antibody combined with CDR2 and CDR3 from the light chain of a second anti- human C5 antibody, and the CDRs from the heavy chain may be derived from a third anti-human C 5 antibody.
  • the framework regions may be derived from one of the same anti-human C5 antibodies, from one or more different antibodies, such as a human antibody, or from a humanized antibody. Human or humanized antibodies are specific for administration to human patients.
  • Single-chain Fv or “scFv” antibody fragments comprise the V H and V L domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.
  • SMIPs are a class of single-chain peptide engineered to include a target binding region, effector domain (CH2 and CH3 domains). See, e.g., U.S. Patent Application Publication No. 20050238646.
  • the target binding region may be derived from the variable region or CDRs of an antibody, e.g., an anti-C5 antibody of the invention.
  • the target binding region is derived from a protein that binds C5.
  • diabodies refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (V L ) in the same polypeptide chain (V H -VL).
  • VH heavy-chain variable domain
  • V L light-chain variable domain
  • V H -VL polypeptide chain
  • An "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
  • the antibody will be purified to greater than 95% by weight of antibody as determined by the Lowry method, or greater than 99% by weight, to a degree that complies with applicable regulatory requirements for administration to human patients (e.g., substantially pyrogen-free), to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain.
  • Isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step, for example, an affinity chromatography step, an ion (anion or cation) exchange chromatography step, or a hydrophobic interaction chromatography step.
  • the Fc portions of antibodies are recognized by specialized receptors expressed by immune effector cells.
  • the Fc portions of IgGl and IgG3 antibodies are recognized by Fc receptors present on the surface of phagocytic cells such as macrophages and neutrophils, which can thereby bind and engulf the molecules or pathogens coated with antibodies of these isotypes (C. A. Janeway et al., Immunobiology 5th edition, page 147, Garland Publishing (New York, 2001)).
  • single chain antibodies, and chimeric, humanized or primatized (CDR-grafted) antibodies, as well as chimeric or CDR- grafted single chain antibodies, comprising portions derived from different species, are also encompassed by the present disclosure as antigen-binding fragments of an antibody.
  • the various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques.
  • nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein. See, e.g., U.S. Pat. Nos. 4,816,567 and 6,331,415; U.S. Pat. No. 4,816,397; European Patent No.
  • functional fragments of antibodies including fragments of chimeric, humanized, primatized or single chain antibodies, can also be produced.
  • Functional fragments of the subject antibodies retain at least one binding function and/or modulation function of the full-length antibody from which they are derived.
  • Preferred functional fragments retain an antigen-binding function of a corresponding full-length antibody (such as for example, ability of anti-C5 antibody to bind C5).
  • the antibodies can be administered in a variety of unit dosage forms.
  • the dose will vary according to the particular antibody. For example, different antibodies may have different masses and/or affinities, and thus require different dosage levels.
  • Antibodies prepared as Fab fragments will also require differing dosages than the equivalent intact immunoglobulins, as they are of considerably smaller mass than intact immunoglobulins, and thus require lower dosages to reach the same molar levels in the patient's blood.
  • the dose will also vary depending on the manner of administration, the particular symptoms of the patient being treated, the overall health, condition, size, and age of the patient, and the judgment of the prescribing physician.
  • Dosage levels of the antibodies for human subjects are generally between about 1 mg per kg and about 100 mg per kg per patient per treatment, and preferably between about 5 mg per kg and about 50 mg per kg per patient per treatment.
  • the antibody concentrations are preferably in the range from about 25 ⁇ g/mL to about 500 ⁇ g/mL. However, greater amounts may be required for extreme cases and smaller amounts may be sufficient for milder cases.
  • Administration of the antibodies will generally be performed by an intravascular route, e.g., via intravenous infusion by injection. Other routes of administration may be used if desired but an intravenous route will be the most preferable.
  • Formulations suitable for injection are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985). Such formulations must be sterile and non-pyrogenic, and generally will include a pharmaceutically effective carrier, such as saline, buffered (e.g., phosphate buffered) saline, Hank's solution, Ringer's solution, dextrose/saline, glucose solutions, and the like.
  • the formulations may contain pharmaceutically acceptable auxiliary substances as required, such as, tonicity adjusting agents, wetting agents, bactericidal agents, preservatives, stabilizers, and the like.
  • auxiliary substances such as, tonicity adjusting agents, wetting agents, bactericidal agents, preservatives, stabilizers, and the like.
  • antibodies are preferred, especially anti-C5 antibodies which have already been shown to be safe and effective at decreasing the accumulation of downstream complement components in persons, the use of other complement inhibitors is also contemplated by this disclosure.
  • the pharmaceutical formulations and uses of the disclosure may be combined with any known complement inhibitors or sickle cell treatments known in the art (supra).
  • Sickle cell disease patients have episodes of hemolysis in which large amounts of red blood cells are lysed and hemoglobin is released into the plasma. Because of the recurring nature of this disease, such patients will be treated prophylactically by administering anti-C5 antibodies at regular intervals such that active antibody is present in the plasma at any time and can bind to C5 and inhibit its activation, thereby decreasing the amount of lysis of red blood cells. The amount of antibody required will depend upon the severity of the disease. Antibodies are degraded over time and must be replaced.
  • PNH patients are administered an anti-C5 antibody at a dose of 900 mg approximately once every two weeks to maintain an active level of that antibody (Hillmen et al., N. Engl. J. Med. 350:552-559 (2004)) to decrease lysis of red blood cells.
  • the amount delivered depends upon the form of the antibody and its in vivo half-life.

Abstract

A method of treating a sickle cell patient with an anti C5 complement antibody is disclosed. Treatment with said antibody decreases the extent of red blood cell lysis and decreases the consequential clinical sequelae.

Description

USE OF A COMPLEMENT INHIBITOR TO TREAT PATIENTS WITH
SICKLE CELL DISEASE
FIELD OF THE INVENTION
[0001] The present invention relates to methods for the treatment of sickle cell disease. In specific embodiments, the invention relates to the use of antibodies capable of inhibiting complement as therapeutic agents to treat sickle cell disease.
BACKGROUND OF THE INVENTION
[0002] Sickle cell disease (also called sickle cell anemia) is an inherited form of anemia. The red blood cells are affected by a mutation in the beta globin gene which affects hemoglobin structure. The mutant hemoglobin causes red blood ceils, which normally are smooth and round, to become hard, sticky, and shaped like sickles or crescents. These red cells tend to get stuck and block the flow of blood in small blood vessels and to lyse which in turn leads to pain, damage and a low blood count or anemia.
[0003] Sickle cell disease affects about 72,000 Americans, predominantly those of African ancestry. One in every 600 African-American births is a baby with sickle cell disease. One in every 1,000-1,400 Hispanic-American births is a baby with sickle cell disease. Those with the disease usually show some signs and symptoms after four months of age. These include: anemia because the sickle cells are fragile and break apart easily and die leaving a shortage of red blood cells to carry oxygen; periodic episodes of pain referred to as crises which develop when sickle-shaped red blood cells block blood flow through tiny blood vessels to the chest, abdomen and joints, with the pain sometimes occurring in the bones; hand-foot syndrome wherein swollen hands and feet occur when sickle-shaped red blood cells block blood flow out of the hands and feet; jaundice resulting from the liver being overwhelmed by the rapid breakdown of red blood cells; frequent infections because of damage to the spleen caused by the sickle cells; stunted growth because of a shortage of oxygen and nutrients due to a lack of healthy red blood cells; and vision problems which result from damage to blood vessels in the eye caused by sickle cells plugging those blood vessels. Other complications include stroke (caused by sickle cells blocking blood flow to the brain), acute chest syndrome (chest pain, fever and difficulty breathing caused by trapped sickle cells in the lungs); organ damage to not only the spleen and eyes but also the kidneys and liver due to chronic deprivation of oxygen- rich blood; and ulcers on the legs due to a lack of blood vessels to nourish the skin. Gallstones are a possible complication resulting from high levels of bilirubin due to the high level of breakdown of red blood cells. Priapism may also develop if sickle cells prevent blood flow out of an erect penis and this can lead to impotence. Persons with sickle cell disease may also experience fatigue, joint pain, breathlessness, rapid heart rate, abdominal pain, bloody urine (hematuria), excessive urination, and excessive thirst.
[0004] Presently the only cure for sickle cell disease is a bone marrow transplantation. Unfortunately very few sickle cell patients have a suitable donor for transplant. Consequently, treatment of sickle cell patients is usually aimed at avoiding crises, relieving symptoms and preventing complications. Antibiotics such as penicillin are given to children beginning when they are 2-4 months old to help prevent infections such as pneumonia. Vaccines are also given to prevent bacterial infections. Pain relievers, sometimes including narcotics, are commonly administered during a sickle crisis. Red blood cell transfusions are used to increase the number of normal red blood cells in circulation, helping to relieve anemia and its resulting symptoms and helping to prevent stroke. Blood transfusions do, however, include some risk such as a buildup of excess iron which can damage the heart, liver and other organs. Administration of supplemental oxygen via a breathing mask can be useful during an acute chest syndrome or a sickle cell crisis. Hydroxyurea, a prescription drug normally used to treat cancer, is sometimes administered to adults with severe disease. It reduces the frequency of painful crises and may reduce the need for blood transfusions. It appears to work by stimulating production of fetal hemoglobin. Patients who took hydroxyurea over a 9-year period experienced a 40% reduction in deaths. There is concern that long-term use of this drug may cause tumors or leukemia. Other drugs used to activate the production of fetal hemoglobin include 5-azacytidine and 5-aza-2'-deoxycytidine (decitabine) which hypomethylate DNA but, as with hydroxyurea, these can be toxic carcinogens. [0005] Experimental treatments are being performed in an attempt to either cure or better treat sickle cell disease. Because sickle cell disease is caused by a mutation in a single gene, gene therapy is being considered as a treatment to cure the disease. However, gene therapy studies in general for other diseases have not worked well and are fraught with the possibility that the therapy will in turn cause some type of cancer due to indiscriminate insertion of the gene into the genome. Drugs being tested include butyric acid (which may increase the amount of fetal hemoglobin in the blood), clotrimazole (normally used to treat fungal infections but which may prevent loss of water from red blood cells and thereby reduce the number of sickle cells that form), and nitric oxide (a gas which helps keep blood vessels open and reduces the stickiness of red blood cells). A search of the ClinicalTrials.gov website using the search term "sickle cell" gave a listing of 55 clinical trials (including completed studies, studies no longer recruiting, studies still recruiting patients, terminated studies, and studies not yet recruiting) related to sickle cell disease or sickle cell trait. This listing is shown as Table 1.
Table 1
1. Completed A study of the efficacy and safety of ICA- 17043 (with or without hydroxyurea) in patients with sickle cell anemia. Conditions: Sickle Cell Disease; Sickle Cell Anemia
2. Completed Mobilization and Handling of Stem Cells for Transplant from
Healthy Volunteers with Sickle Cell Trait Condition: Sickle Cell Trait
3. Recruiting Nitric Oxide Inhalation to Treat Sickle Cell Pain Crises
Condition: Anemia, Sickle Cell
4. Recruiting Evaluation of Hydroxyurea Plus L-arginine or Sildenafil to
Treat Sickle Cell Anemia Condition: Sickle Cell Anemia
5. Recruiting Natural History of Sickle Cell Disease and Other Hemolytic
Disorders
Conditions: Sickle Cell Anemia; Hemolytic Anemia;
Hemoglobin SC Disease; Hematologic Diseases
6. Recruiting Study of Oral ICL670 (Deferasirox) Relative to Subcutaneous
Deferoxamine in Sickle Cell Disease Patients Conditions: Sickle Cell Disease; Iron Overload; Hemolytic Anemia
7. Recruiting Collection and Storage of Umbilical Cord Stem Cells for
Treatment of Sickle Cell Disease Conditions: Healthy; Sickle Cell Anemia
8. Completed Effects of Nitric Oxide and Nitroglycerin in Patients with
Sickle Cell Anemia
Conditions: Chest Pain; Sickle Cell Anemia
9. Recruiting Secondary Pulmonary Hypertension in Adults with Sickle
Cell Anemia
Conditions: Pulmonary Hypertension; Sickle Cell Anemia
10. Completed Nitric Oxide to Improve Blood Flow in Sickle Cell Disease
Condition: Sickle Cell Anemia
11. Recruiting Bone Marrow Transplantation in Treating Children With
Sickle Cell Disease Condition: Sickle Cell Anemia
12. No longer Hydroxyurea for the Treatment of Patients with Sickle Cell recruiting Anemia
Condition: Sickle Cell Anemia
13. No longer Safety of ICL670 vs. Deferoxamine in Sickle Cell Disease recruiting Patients with Iron Overload due to Blood Transfusions
Condition: Anemia, Sickle Cell
14. No longer Decompression Coring Versus Conservative Therapy in recruiting Patients With Avascular Necrosis of the Hip Related to Sickle
Cell Disease
Conditions: Bone Avascular Necrosis; Sickle Cell Anemia
15. Recruiting Inhaled Nitric Oxide and Transfusion Therapy for Patients with Sickle Cell Anemia and Secondary Pulmonary
Hypertension
Conditions: Sickle Cell Anemia; Pulmonary Hypertension
16. Recruiting Arginine Treatment of Acute Chest Syndrome (Pneumonia) in
Sickle Cell Disease Patients
Conditions: Anemia, Sickle Cell; Pneumonia
17. Recruiting Therapeutic Application of Intravascular Nitrite for Sickle
Cell Disease
Condition: Sickle Cell Anemia
18. Recruiting Atorvastatin Therapy to Improve Endothelial Function in
Sickle Cell Disease Condition: Sickle Cell Disease
19. Completed Cerebrovascular Involvement in Sickle Cell Disease -
Comprehensive Sickle Cell Center
Conditions: Anemia, Sickle Cell; Blood Disease;
Cerebrovascular Accident
20. Completed Prevention of Cerebral Infarction in Sickle Cell Anemia -
Comprehensive Sickle Cell Center
Conditions: Anemia, Sickle Cell; Blood Disease;
Cerebrovascular Disorders; Cerebrovascular Accident
21. Completed A Phase I/II Trial of Recombinant-Methionyl Human Stem
Cell Factor CSCF) in Adult Patients with Sickling Disorders Conditions: hemoglobin SC disease; sickle cell anemia
22. Recruiting Home Based Massage and Relaxation for Sickle Cell Pain
Condition: Sickle Cell Disease
23. Recruiting A Stratified Sickle Event Randomized Trial (ASSERT)
Condition: Sickle Cell Disease
24. Recruiting Study of Clotrimazole and Hydroxyurea in Patients With
Sickle Cell Syndromes Condition: Sickle Cell Anemia
25. No longer Phase III Randomized Study of Poloxamer 188 for Vaso- recruiting Occlusive Crisis of Sickle Cell Disease
Condition: Sickle Cell Anemia
26. Recruiting Phase II Randomized Trial of Arginine Butyrate Plus
Standard Local Therapy in Patients With Refractory Sickle
Cell Ulcers
Conditions: Skin Ulcers; Sickle Cell Anemia
27. Recruiting Phase I/II Randomized Study of Hydroxyurea With or
Without Clotrimazole in Patients With Sickle Cell Anemia Condition: Sickle Cell Anemia
28. Completed Pilot Study of Fructose for Sickle Cell Crisis
Condition: Sickle Cell Anemia
29. Completed Investigation of Selected Patient Groups From The
Cooperative Study of Sickle Cell Disease Conditions: Anemia, Sickle Cell; Blood Disease
30. Completed Neuropsychological Studies of Children with Sickle Cell
Conditions: Blood Disease; Anemia, Sickle Cell; Neurologic Manifestations
31. Recruiiing Stem Cell Transplantation after Reduced-Dose Chemotherapy for Patients with Sickle Cell Disease or Thalassemia Conditions: Hemoglobinopathies; Anemia, Sickle Cell; Hemoglobin SC Disease; Thalassemia; Thalassemia Major
32. Recruiting Induction of Stable Chimerism for Sickle Cell Anemia
Conditions: Blood Disease; Hematopoietic Stem Cell Transplantation; Anemia, Sickle Cell
33. Recruiting L-glutamiiie Therapy for Sickle Cell Anemia
Conditions: Anemia, Sickle Cell; Thalassemia
34. Completed Penicillin Prophylaxis in Sickle Cell Disease (PROPS)
Conditions: Anemia, Sickle Cell; Hematologic Diseases; Hemoglobinopathies; Infection (S. pneumoniae); Pneumonia
35. No longer Multicenter Study of Hydroxyurea in Patients With Sickle recruiting Cell Anemia (MSH)
Conditions: Anemia, Sickle Cell; Hematologic Diseases; Hemoglobinopathies
36. Completed Stroke Prevention in Sickle Cell Anemia (STOP 1)
Conditions: Anemia, Sickle Cell; Cerebral Embolism and Thrombosis; Cerebrovascular Disorders; Hematologic Diseases; Hemoglobinopathies
37. Completed Pediatric Hydroxyurea in Sickle Cell Anemia (PED HLKT)
Conditions: Anemia, Sickle Cell; Hematologic Diseases; Hemoglobinopathies
38. Completed Cooperative Study of The Clinical Course of Sickle Cell
Disease
Conditions: Anemia, Sickle Cell; Blood Disease
39. Completed Indices of Severity and Prognosis for Sickle Cell Disease
Conditions: Anemia, Sickle Cell; Blood Disease
40. Completed Hemostasis in Sickle Cell Disease— Infancy to Adulthood
Conditions: Anemia, Sickle Cell; Blood Disease
41. Recruiting Pediatric Hydroxyurea in Sickle Cell Anemia (BABY HUO
Conditions: Blood Disease; Anemia, Sickle Cell
42. No longer Pain in Sickle Cell Epidemiologic Study recruiting Conditions: Blood Disease; Anemia, Sickle Cell
43. No longer Pediatrics:Chlamydia, Sickle Cell Anemia and Stroke Risk - recruiting Ancillary to STOP II
Conditions: Blood Disease; Anemia, Sickle Cell; Chlamydia Infections; Cerebrovascular Accident
44. Recruiting Silent Cerebral Infarct Multi-Center Clinical Trial
Conditions: Sickle Cell Anemia; Stroke
45. No longer Stroke Prevention in Sickle Cell Anemia (STOP 2) recruiting Conditions: Blood Disease; Cerebrovascular Accident;
Anemia, Sickle Cell
46. Completed Study of Allogeneic Bone Marrow Transplantation Using
Matched, Related Donors in Patients With Nonmalignant Hematologic Disorders
Conditions: Neutropenia; Sickle Cell Anemia; Thalassemia Major; Red-Cell Aplasia, Pure
47. Recruiting Phase I/FI Study of Nonmyeloablative Allogeneic Bone
Marrow Transplantation in Patients With High Risk
Hemoglobinopathy
Conditions: Graft Versus Host Disease; Sickle Cell Anemia;
Thalassemia
48. No longer Transcranial Doppler (TCP) Ultrasound of Subjects Enrolled recruiting jn BABY HUG - Ancillary to BABY HUG
Conditions: Blood Disease; Anemia, Sickle Cell; Cerebrovascular Disorders; Cerebral Embolism and Thrombosis
49. Terminated Bone marrow transplant from donor using less toxic conditioning for patient with high risk hemoglobinopathies Conditions: Sickle Cell Anemia; Hemoglobinopathy; Thalassemia
50. Terminated Bone Marrow transplant from related donor for patients with high risk hemoglobinopathies Conditions: Sickle Cell Anemia; Hemoglobinopathy; Thalassemia
51. Recruiting Improving the Results of Bone Marrow Transplantation for
Patients with Severe Congenital Anemias Conditions: Congenital Hemolytic Anemia; Diamond- Blackfan Anemia
52. Not yet Sibling Donor Cord Blood Banking and Transplantation recruiting Conditions: Blood Disease; Anemia, Sickle Cell; Anemia
(Cooley's); Leukemia; Bone Marrow Transplantation; Hematopoietic Stem Cell Transplantation
53. Recruiting Development of a Hospital-Based Home Program for the Use of Inhaled Nitric Oxide in the Chronic Management of Severe Cardiopulmonary Diseases
Conditions: Pulmonary Hypertension; Lung Disease; Sickle Cell Disease; Cardiac transplant; Lung transplant
54. Recruiting The Effect of Oral Magnesium Pidolate on How Often Painful
Crises Happens in Patients with Hemoglobin SC Disease Condition: Hemoglobin SC Disease
55. No longer Surgery With or Without Chemotherapy in Treating Patients recruiting with Stage 1 Non-small Cell Lung Cancer
Condition: stage I non-small cell lung cancer
[0006] It is seen that numerous of these studies concern the use of hydroxyurea (either alone or in combination with another drug), bone marrow transplantation, nitric oxide, or clotrimazole. A small number of the listed studies are testing other drugs, e.g., hydroxyurea plus L-arginine or sildenafil; nitric oxide plus nitroglycerin; ICA-17043; ICL60 (Deferasirox) as compared to deferoxamine; poloxamer 188; atorvastatin; arginine; L-glutamine; arginine butyrate; fructose; and oral magnesium pidolate.
[0007] In addition to the studies listed above, numerous articles have been published concerning the use of other drugs to treat sickle cell patients. These drugs work in a large variety of ways. Abdulmalik et al. (Br. J. Haematol. 128:552-561 (2005)) used 5-hydroxymethyl-2-furfural which binds to intracellular sickle hemoglobin and inhibits sideling. Vanillin is known to bind to sickle hemoglobin and inhibit sickling, but given orally it is rapidly decomposed. Zhang et al. (Br. J. Haematol. 125:788-795 (2004)) used a vanillin prodrug, MX-1520, which is not rapidly decomposed and found that it reduced the percentage of sickled cells in the blood. DeBellis et al. (Blood Cells MoI. Dis. 31:286-290 (2003)) tested three purine-based antiviral agents (ganciclovir, valacyclovir, and penciclovir) and found that valacyclovir and ganciclovir show anti-sickling activity similar to acyclovir which was known to inhibit aggregation of hemoglobin S and the sickling of erythrocytes. Cordeiro and Oniyangi (Cochrane Database Syst. Rev. 3:CD004448 (2004)) found that a phytomedicine (Niprisan) was effective in reducing episodes of sickle cell disease crisis associated with severe pain. Iyamu et al. (Br. J. Haematol. 122:1001-1008 (2003)) also studied Niprisan (Nix-0699) in mice and found that it inhibited sickling. Manion et al. (Clin. Pharmacol. Ther. 69:346-355 (2001)) studied the use of aspartame for treating sickle cell disease. Aspartame binds to two human Bence Jones proteins (Meg and Sea). In vivo studies in persons showed that aspartame treatment led to decreased sickling. Beddell et al. (Br. J. Pharmacol. 82:397-407 (1984)) designed substituted benzaldehydes to bind preferentially to the oxy conformation of human hemoglobin to stabilize the oxygenated form of hemoglobin and thereby increase its oxygen affinity. At least one such compound was a potent inhibitor at low oxygen pressure of the sickling of erythrocytes. Related work by Arya et al. (Br. J. Haematol. 93:817-821 (1996)) involved the study of Tucaresol (589C80;4[2-formyl-3-hydroxyphenoxymethyl]benzoic acid) which is a substituted benzaldehyde. Administration of tucaresol to sickle cell patients resulted in decreased hemolysis, a decrease in lactate dehydrogenase, and a halving of the irreversibly sickled cell counts. However, 3 of 6 patients developed fever and cervical lymphadenopathy. Riddington and De Franceschi (Cochrane Database Syst. Rev. 4:CD003426 (2002)) reviewed the literature of controlled trials to find trials related to using drugs which aimed to prevent sickle cell related crises by reducing red blood cell dehydration. They found two trials. These tested zinc sulfate and piracetam. The zinc trial showed a significant reduction in the total number of pain, hemolytic, aplastic and sequestration crises over one and a half years. The piracetam trial showed a reduction in pain crises over a one year time span, although blood counts were not significantly changed. Dehydration of red blood cells can occur through the Ca-activated K channel which depends on the parallel movement of Cl ions. Bennekou et al. (Blood 97:1451-1457 (2001)) studied whether Cl-conductance block might prevent dehydration of sickle red blood cells. They used a novel Cl-conductance inhibitor (NS3623). Mice treated with NS3623 showed an increase in hematocrit, a decrease in the mean corpuscular hemoglobin concentration, and a loss of the densest red cell population with a shift from a high proportion of sickled to well-hydrated discoid erythrocytes. Kocak et al. (Br. J. Haematol. 92:329-331 (1996)) used the vasodilating Ca+2 channel blocker bencyclane in 18 patients with sickle cell disease. The patients' blood contained fewer irreversibly sickled cells. Inhibition of the sickling-induced fluxes OfNa+, K+ and Ca+2 was studied by Joiner et al. using dipyridamole (Blood 97:3976-3983 (2001)). The dipyridamole was found to inhibit sickling by inhibiting the ion flux. Shartava et al. (Am. J. Hematol. 62:19-24 (1999)) studied both clotrimazole (which is a specific inhibitor of the Ca+2 activated potassium channel) and the antioxidant N-acetylcysteine. Both were found to inhibit the in vitro formation of high-density sickle cells. The authors concluded that N-acetylcysteine protects the potassium channel from oxidative damage caused by diminished levels of reduced glutathione. Another drug which protects against K+ efflux is bepridil and this was found to significantly inhibit the formation of irreversibly sickled cells in a study by Johnston et al. (Br. J. Haematol. 73:522-526 (1989)). A recent review of established and experimental treatments for sickle cell disease was published by De Franceschi and Corrocher (Haematologica 89:348-356 (2004)), the contents of which are incorporated herein by reference.
[0008] In addition to the numerous studies listed above, basic research has been performed which shows a possible relationship between sickle cell disease and complement. The complement system is described in detail in U.S. Patent 6,355,245. The complement system acts in conjunction with other immunological systems of the body to defend against intrusion of cellular and viral pathogens. There are at least 25 complement proteins, which are found as a complex collection of plasma proteins and membrane cofactors. The plasma proteins make up about 10% of the globulins in vertebrate serum. Complement components achieve their immune defensive functions by interacting in a series of intricate but precise enzymatic cleavage and membrane binding events. The resulting complement cascade leads to the production of products with opsonic, immunoregulatory, and lytic functions. [0009] The complement cascade progresses via the classical pathway or the alternative pathway. These pathways share many components and, while they differ in their initial steps, they converge and share the same "terminal complement" components (C5 through C9) responsible for the activation and/or destruction of target cells.
[0010] The classical complement pathway is typically initiated by antibody recognition of and binding to an antigenic site on a target cell. The alternative pathway is usually antibody independent and can be initiated by certain molecules on pathogen surfaces. Both pathways converge at the point where complement component C3 is cleaved by an active protease (which is different in each pathway) to yield C3a and C3b. Complement activation can also occur via the lectin complement pathway (LCP). Lectins are carbohydrate-binding proteins that recognize oligosaccharide structures present on cell surfaces, the extracellular matrix, and secreted glycoproteins. As with the classical complement pathway and the alternative pathway, the LCP also converges at the point of cleavage of complement component C3. Other pathways activating complement attack can act later in the sequence of events leading to various aspects of complement function. [0011] C3a is an anaphylatoxin. C3b binds to bacterial and other cells, as well as to certain viruses and immune complexes, and tags them for removal from the circulation. C3b in this role is known as opsonin. The opsonic function of C3b is considered to be the most important anti-infective action of the complement system. Patients with genetic lesions that block C3b function are prone to infection by a broad variety of pathogenic organisms, while patients with lesions later in the complement cascade sequence, i.e., patients with lesions that block C5 functions, are found to be more prone only to Neisseria infection, and then only somewhat more prone (Fearon (1983). In Intensive Review of Internal Medicine, 2nd Ed. Fanta and Minaker, eds. Brigham and Women's and Beth Israel Hospitals). [0012] C3b also forms a complex with other components unique to each pathway to form classical or alternative C5 convertase, which cleaves C5 into C5a and C5b. C3 is thus regarded as the central protein in the complement reaction sequence since it is essential to both the alternative and classical pathways (Wurzner et al., Complement Inflamm. 8:328-340 (1991)). This property of C3b is regulated by the serum protease Factor I, which acts on C3b to produce iC3b. While still functional as opsonin, iC3b cannot form an active C5 convertase. [0013] C5 is a 190 IcDa beta globulin found in normal serum at approximately 75 μg/mL (0.4 μM). C5 is glycosylated, with about 1.5-3 percent of its mass attributed to carbohydrate. Mature C5 is a heterodimer of a 999 amino acid 115 kDa alpha chain that is disulfide linked to a 656 amino acid 75 kDa beta chain. C5 is synthesized as a single chain precursor protein product of a single copy gene (Haviland et al., J. Immunol. 146:362-368 (1991)). The cDNA sequence of the transcript of this gene predicts a secreted pro-C5 precursor of 1659 amino acids along with an 18 amino acid leader sequence.
[0014] The pro-C5 precursor is cleaved after amino acid 655 and 659, to yield the beta chain as an amino terminal fragment (amino acid residues +1 to 655) and the alpha chain as a carboxyl terminal fragment (amino acid residues 660 to 1658), with four amino acids deleted between the two.
[0015] C5a is cleaved from the alpha chain of C5 by either alternative or classical C5 convertase as an amino terminal fragment comprising the first 74 amino acids of the alpha chain (i.e., amino acid residues 660-733). Approximately 20 percent of the 11 kDa mass of C5a is attributed to carbohydrate. The cleavage site for convertase action is at or immediately adjacent to amino acid residue 733. A compound that would bind at or adjacent to this cleavage site would have the potential to block access of the C5 convertase enzymes to the cleavage site and thereby act as a complement inhibitor. C5 can also be activated by means other than C5 convertase activity. Limited trypsin digestion (Minta and Man, J. Immunol. 119:1597-1602 (1977); Wetsel and KoIb, J. Immunol. 128:2209-2216 (1982)) and acid treatment (Yamamoto and Gewurz, J. Immunol. 120:2008-2015 (1978); Vogt et al., Molec. Immunol. 26:1133-1142 (1989)) can also cleave C5 and produce active C5b.
[0016] C5a is another anaphylatoxin. C5b combines with C6, C7, and C8 to form the C5b-8 complex at the surface of the target cell. Upon binding and polymerization of several C9 molecules, the membrane attack complex (MAC, C5b- 9, terminal complement complex-TCC) is formed. When sufficient numbers of MACs insert into target cell membranes the openings they create (MAC pores) mediate rapid osmotic lysis of the target cells. Lower, non-lytic concentrations of MACs can produce other effects. In particular, membrane insertion of small numbers of the C5b-9 complexes into endothelial cells and platelets can cause deleterious cell activation. In some cases activation may precede cell lysis. As mentioned above, C3a and C5a are anaphylatoxins. These activated complement components can trigger mast cell degranulation, which releases histamine and other mediators of inflammation, resulting in smooth muscle contraction, increased vascular permeability, leukocyte activation, and other inflammatory phenomena including cellular proliferation resulting in hypercellularity. C5a also functions as a chemotactic peptide that serves to attract pro-inflammatory granulocytes to the site of complement activation.
[0017] In 1994 Chudwin et al. (Clin. Immunol. Immunopathol. 71:199-202 (1994)) reported that red blood cells from sickle cell patients activated complement to a greater extent than did red blood cells from control patients. Additionally they reported that the activation was via the alternative pathway and not the classical pathway. In 1995 Mold et al. (Clin. Immunol. Immunopathol. 76:314-320 (1995)) demonstrated that the alternative pathway activation is initiated by membrane phospholipid changes which occur in sickled erythrocytes. They found that in patients with intermittent pain there was little evidence of complement activation and that there was an increased plasma concentration of C3a during a painful crisis. Also, elevated C3a and C4d levels were observed in patients with continuous pain. Earlier, Tomasko and Chudwin had shown that changes in phospholipid organization in sickle cells may contribute to the chronic alternative complement pathway activation seen in sickle cell disease patients (J. Lab. Clin. Med. 112:248- 253 (1988)). In 1986 De Ceulaer et al. (J. Clin. Lab. Immunol. 21 :37-41 (1986)) published a paper concerning complement levels in sickle cell disease patients and the relation to infection. They reported that complement component C3 levels were consistently lower in sickle cell children and levels of C3d were significantly higher, but they found no difference in the prevalence of infections between the groups. Dieye et al. (Dakar Med. 44:175-179 (1999)) also studied the relationship between complement and infection in sickle cell patients. They found that C3c complement (indicative of the alternative pathway) levels were decreased significantly whereas C4c (indicative of the classical pathway) levels were not. They conclude there is a direct involvement of the complement system in sickle cell disease and the depletion of C3 is a possible cause of increased susceptibility to infections in patients with sickle cell disease.
[0018] Mohamed et al. (Am. J. Trop. Med. Hyg. 49:799-803 (1993)) found that C3d levels are elevated in sickle cell disease patients. They noted that this was not accompanied by altered complement activity in serum in vitro or in C3 reduction. They state that the underlying mechanism of this activation may be the triggering of the complement reaction by the sickled cell, the phospholipid organization of which appears to promote the alternative pathway complement activation. They conclude that the complement activation may contribute to impaired host defense against bacterial infections.
[0019] Test and Woolworth (Blood 83:842-852 (1994)) further studied the relationship between sickle cell disease and complement. They first noted that prior work concluded that intravascular hemolysis of sickle cells resulted in complement activation. Test and Woolworth took the opposite view that possibly complement activation was leading to hemolysis. They showed in a series of in vitro assays that regulation of complement in sickle cells was abnormal, especially in that increased binding of C5b-7 and C9 to denser sickle cells was seen and this appeared to increase the susceptibility of sickle cells to lysis. They believe that the increased binding is most likely caused by changes in MIRL (also referred to as CD59) brought about by the sickling process and which is a membrane protein which inhibits lysis by the membrane attack complex. CD59 is a human glycosylphosphatidylinositol-anchored membrane glycoprotein that serves as the principle inhibitor of the cytolytic and pore forming activities of the C5b-9 membrane attack complex (MAC) of human complement. By inhibiting MAC, CD59 protects human blood cells, vascular endothelium and other cells from complement, which is normally present in human plasma and other body fluids. Although the exact mechanism of its complement inhibitory function remains to be elucidated, CD59 is known to bind peptide segments of C8 and C9 that become exposed when these complement proteins incorporate into the C5b-9 complex, thereby preventing initiation or propagation of the membrane-inserted C9 polymer that is responsible for membrane damage. Blood cells from patients with paroxysmal nocturnal hemoglobinuria (PNH) lack the ability to anchor CD59 in their membranes and these cells are very susceptible to lysis via complement. Transducing these cells with a recombinant transmembrane form of CD59 results in the surface expression of CD59 and these transduced cells are protected against classical complement-mediated membrane damage by human serum (Rother et al., Blood 84:2604-2611 (1994)).
[0020] This work was followed up by Liu et al. (Blood 93:2297-2301 (1999)) who inserted various types of lipids into human erythrocyte membranes. They found that neutral lipids had no effect on C5b-7 uptake or hemolysis by C5b-9, but acidic phospholipids did cause a dose-dependent increase in both lysis and C5b-7 uptake. They concluded that the presence of anionic lipids on the exterior of the membrane increases C5b-7 uptake and subsequent hemolysis. They point out that it is known that sickle cell erythrocytes have increased exposure of phophatidylserine on their external face and are abnormally sensitive to lysis by C5b-9. [0021] Concerning the issue of hemolysis, it is known that in autoimmune hemolytic anemia red blood cell destruction is primarily extravascular. This is largely mediated through either Fc and/or complement receptors expressed by phagocytic effector cells. Experimentally induced autoimmune hemolytic anemia occurs even in the absence of the complement components C3, C4 and C5 but requires the presence of Fc receptors in association with the common FcR γ-chain (Meyer et al., Blood 92:3997-4002 (1998)).
[0022] The prior art as a whole leaves the field quite uncertain as to what role, if any, complement plays in sickle cell disease and more specifically complement's role in lysis of red blood cells in sickle cell disease patients. Mohamed et al. (Am. J. Trop. Med. Hyg. 49:799-803 (1993)) state, "Although some investigators have reported defects in the complement system as the cause for the increased susceptibility to infections, others have not found such defects." They also state that intravascular hemolysis accounts for the destruction of one-third of the siclded erythrocytes, while two-thirds are removed by macrophages in the spleen and other locations and that the hemolytic process in sickle cell anemia may involve several components, including activation of complement, macrophages, and neutrophils. [0023] The publications and other materials used herein to illuminate the background of the disclosure, and in particular, cases to provide additional details respecting the practice, are incorporated herein by reference.
SUMMARY
[0024] Methods for treating patients suffering from sickle cell disease are presented. A method of treating sickle cell disease in a mammal comprising treating said mammal with a therapeutically effective amount of an anti-C5 antibody, wherein said treatment results in decreased lysis of red blood cells in said mammal. In certain embodiments, said mammal is a human.
[0025] In certain embodiments, said antibody is a whole antibody or an antibody fragment. In certain embodiments, said whole antibody or antibody fragment is selected from the group consisting of: a polyclonal antibody, a monoclonal antibody or antibody fragment, a diabody, a chimerized or chimeric antibody or antibody fragment, a humanized antibody or antibody fragment, a deimmunized human antibody or antibody fragment, a fully human antibody or antibody fragment, a single chain antibody, an Fv, an Fab, an Fab', or an F(ab')2.
[0026] In certain embodiments, said treatment is chronic. In certain embodiments, said treatment comprises treating an acute episode. In certain embodiments, said mammal is treated by administering said antibody intravenously. [0027] In certain embodiments, said mammal is treated to inhibit or decrease the amount and/or extent of an undesirable physiological condition resulting from excess lysis of red blood cells. In certain embodiments, said undesirable physiological condition is selected from the group consisting of dystonia, a clotting disorder, pulmonary hypertension, systemic hypertension, gastrointestinal contractions, abdominal pain, sternal pain, erectile dysfunction, priapism, inflammation, esophageal spasm, dysphagia, thrombosis, decreased organ perfusion, platelet activation and death.
[0028] In certain embodiments, said antibody inhibits activation of the classical pathway, the alternative pathway or the lectin complement pathway. In certain embodiments, said antibody is an antibody against a member of the complement cascade components such as for example, C5, C5b, C6, C7, C8, and C9. In certain embodiments, said antibody is an inhibitor of C5 cleavage. In certain embodiments, said antibody decreases the availability of downstream complement components. [0029] In certain embodiments, said antibody is pexelizumab. In certain embodiments, said antibody is eculizumab.
DETAILED DESCRIPTION
[0030] Sickle cell disease is an extremely serious disease affecting approximately 72,000 Americans and many more persons in other countries. At present a cure is almost nonexistent, the only cure being a bone marrow transplant. Gene therapy is a possible cure but is not being performed. In general sickle cell disease patients have their symptoms treated but are not cured. Treatments include treating or preventing infections by administration of antibiotics, blood transfusions to increase the supply of normal red blood cells, drugs to increase the level of fetal hemoglobin which is able to carry oxygen, drugs to prevent loss of water from erythrocytes thereby helping to prevent sideling, and drugs to aid in keeping blood vessels open. Despite these treatments, many patients continue to suffer greatly and many patients die as a result of the disease. More effective treatments are needed. [0031] Several studies dating back several years have shown that complement activation may be affected in sickle cell disease patients. These date back to at least 1967 (Francis and Womack, Am. J. Med. Technol. 33:77-86 (1967)). However the role of complement activation is still unclear. There are conflicting reports as to whether changes in complement result in greater infection. Even if it is proved that infection is greater because of effects to the complement system, such infections are normally treated with inexpensive antibiotics such as penicillin. Other work shows conflicting results as to whether sickle cell disease causes complement activation or whether complement activation causes some of the problems of sickle cell disease. [0032] Applicant here proposes that treatment of a sickle cell disease patient with a complement inhibitor will decrease lysis of red blood cells and at least some of the symptoms seen in sickle cell patients. Many of the symptoms are a direct result of the lysis of red blood cells. One aspect of the lysis of red blood cells is the consequent excess free hemoglobin in the bloodstream. This can lead to smooth muscle dystonias involving the gastrointestinal, cardiovascular, pulmonary, and urogenital systems, as well as clotting disorders, these symptoms including gastrointestinal contractions, erectile dysfunction such as priapism, and pulmonary hypertension as well as to clot formation (Rother et al., JAMA 293:1653-1662 (2005), the contents of which are specifically incorporated herein by reference). Approximately one-third of sickled red blood cells are lysed via intravascular hemolysis (Mohamed et al., Am. J. Trop. Med. Hyg. 49:799-803 (1993)). Applicant here proposes that inhibition of complement activation will decrease the amount of lysis and thereby decrease the amount and/or extent of symptoms related to the lysis of red blood cells in sickle cell disease patients. Inhibitors of members of the complement cascade including for example antibodies to components such as C5, C5b, C6, C7, C8, and C9. In particular embodiments, this application contemplates the use of the anti-C5 antibody eculizumab (a whole antibody) which is known to inhibit cleavage of C5 into C5a and C5b. Treatment of patients with such inhibitors thereby decreases complement mediated lysis of red blood cells. Long lasting inhibitors are preferable. Eculizumab, being a whole antibody, is longer lasting in vivo than is a single-chain antibody and is preferably used although shorter acting inhibitors may be used, especially if used in time of a crisis and is used to relieve the crisis. Eculizumab has been used in clinical studies and found to be well tolerated with minimal side effects. Eculizumab has been administered to patients with paroxysmal nocturnal hemoglobinuria which is a disease in which red blood cells are easily lysed due to a lack of a protein in the red blood cell membrane which protects against lysis by activated complement. Most patients are administered a dose once every two weeks. See Hillmen et al., New Engl. J. Med. 350:552-559 (2004), the contents of which are specifically incorporated herein by reference. [0033] Suitable anti-C5 antibodies are known to those of skill in the art. Antibodies can be made to individual components of activated complement, e.g., antibodies to C7, C9, etc. (see, e.g., U.S. Patent 6,534,058; published U.S. patent application US 2003/0129187; and U.S. Patent 5,660,825). U.S. Patent 6,355,245 teaches an antibody which binds to C5 and inhibits cleavage into C5a and C5b thereby decreasing the formation not only of C5a but also the down stream complement components. [0034] Additional proteins are known which inhibit complement-mediated lysis, including CD59, CD55, CD46 and other inhibitors of C8 and C9 (see, e.g., U.S. Patent 6,100,443). Proteins known as complement receptors and which bind complement are also known (see, Published PCT Patent Application WO 92/10205 and U.S. Patent 6,057,131). Use of soluble forms of complement receptors, e.g., soluble CRl, can inhibit the consequences of complement activation such as neutrophil oxidative burst, complement mediated hemolysis, and C3a and C5a production. Those of skill in the art recognize the above as some, but not all, of the known methods of inhibiting complement and its activation.
[0035] A preferred method of inhibiting complement activity is to use a monoclonal antibody which binds to complement C5 and inhibits cleavage. This decreases the formation of both C5a and C5b while at the same time allowing the formation of C3a and C3b which are beneficial to the recipient. Such antibodies which are specific to human complement are known (U.S. Patent 6,355,245). These antibodies disclosed in U.S. Patent 6,355,245 include a preferred whole antibody (now named eculizumab). A similar antibody against mouse C5 is called BB5.1 (Frei et al., MoI. Cell. Probes. 1:141-149 (1987)). Antibodies to inhibit complement activity need not be monoclonal antibodies. They can be, e.g., polyclonal antibodies. They may additionally be antibody fragments. An antibody fragment includes, but is not limited to, an Fab, F(ab'), F(ab')2, single-chain antibody, and Fv. Furthermore, it is well known by those of skill in the art that antibodies can be humanized (Jones et al., Nature 321:522-5 (1986)), chimerized, or deimmunized. The antibodies to be used in the present disclosure may be any of these. It is preferable to use humanized antibodies.
[0036] In specific embodiments, a therapeutic agent of the disclosure comprises an antibody or antibody fragment. Antibodies and fragments thereof may be made by any conventional method, such as those methods described herein. Antibodies are found in multiple forms, e.g., IgA, IgG, IgM, etc. Additionally, antibodies can be engineered in numerous ways. They can be made as single-chain antibodies (including small modular immunopharmaceuticals or SMIPs™), Fab and F(ab')2 fragments, etc. Antibodies can be humanized, chimerized, deimmunized, or fully human. Numerous publications set forth the many types of antibodies and the methods of engineering such antibodies. For example, see U.S. Patent Nos. 6,355,245; 6,180,370; 5,693,762; 6,407,213; 6,548,640; 5,565,332; 5,225,539; 6,103,889; and 5,260,203.
[0037] This invention provides fragments of anti-C5 antibodies, which may comprise a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8:1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
[0038] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment of an antibody yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
[0039] "Fv" usually refers to the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although likely at a lower affinity than the entire binding site.
[0040] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CHl) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHl domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known. [0041] This disclosure also provides monoclonal anti-C5 antibodies. A monoclonal antibody can be obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are often synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. Monoclonal antibodies may also be produced in transfected cells, such as CHO cells and NSO cells. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and does not require production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler et al., Nature 256:495-497 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Patent Nos. 4,816,567 and 6,331,415). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature 352:624- 628 (1991) and Marks et al., J. MoI. Biol. 222:581-597 (1991), for example. [0042] General methods for the immunization of animals (in this case with C5 and/or C5b, etc.), isolation of antibody producing cells, fusion of such cells with immortal cells (e.g., myeloma cells) to generate hybridomas secreting monoclonal antibodies, screening of hybridoma supernatants for reactivity of secreted monoclonal antibodies with a desired antigen (in this case the immunogen or a molecule containing the immunogen), the preparation of quantities of such antibodies in hybridoma supernatants or ascites fluids, and for the purification and storage of such monoclonal antibodies, can be found in numerous publications. These include: Coligan, et al., eds. Current Protocols In Immunology, John Wiley & Sons, New York, 1992; Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988; Liddell and Cryer, A Practical Guide To Monoclonal Antibodies. John Wiley & Sons, Chichester, West Sussex, England, 1991; Montz et al., Cellular Immunol. 127:337-351 (1990); Wurzner et al., Complement Inflamm. 8:328-340 (1991); and Mollnes et al., Scand. J. Immunol. 28:307-312 (1988).
[0043] A description of the preparation of a mouse anti-human-C5 monoclonal antibody with specific binding characteristics is presented in U.S. Patent Application Publication No. 20050226870. Wurzner et al., Complement Inflamm. 8:328-340 (1991), describe the preparation of other mouse anti-human-C5 monoclonal antibodies referred to as N 19-8 and N20-9.
[0044] Other antibodies specifically contemplated are "oligoclonal" antibodies. As used herein, the term "oligoclonal" antibodies" refers to a predetermined mixture of distinct monoclonal antibodies. See, e.g., PCT publication WO 95/20401; U.S. Patent Nos. 5,789,208 and 6,335,163. In one embodiment, oligoclonal antibodies consisting of a predetermined mixture of antibodies against one or more epitopes are generated in a single cell. In other embodiments, oligoclonal antibodies comprise a plurality of heavy chains capable of pairing with a common light chain to generate antibodies with multiple specificities (e.g., PCT publication WO 04/009618). Oligoclonal antibodies are particularly useful when it is desired to target multiple epitopes on a single target molecule (e.g., C5). In view of the assays and epitopes disclosed herein, those skilled in the art can generate or select antibodies or mixture of antibodies that are applicable for an intended purpose and desired need. [0045] In certain embodiments that include a humanized and/or chimeric antibody, one or more of the CDRs are derived from an anti-human C5 antibody. In a specific embodiment, all of the CDRs are derived from an anti-human C5 antibody. In another specific embodiment, the CDRs from more than one anti- human C5 antibody are mixed and matched in a chimeric antibody. For instance, a chimeric antibody may comprise a CDRl from the light chain of a first anti-human C5 antibody combined with CDR2 and CDR3 from the light chain of a second anti- human C5 antibody, and the CDRs from the heavy chain may be derived from a third anti-human C 5 antibody. Further, the framework regions may be derived from one of the same anti-human C5 antibodies, from one or more different antibodies, such as a human antibody, or from a humanized antibody. Human or humanized antibodies are specific for administration to human patients.
[0046] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore, eds. (Springer-Verlag: New York, 1994), pp. 269-315.
[0047] SMIPs are a class of single-chain peptide engineered to include a target binding region, effector domain (CH2 and CH3 domains). See, e.g., U.S. Patent Application Publication No. 20050238646. The target binding region may be derived from the variable region or CDRs of an antibody, e.g., an anti-C5 antibody of the invention. Alternatively, the target binding region is derived from a protein that binds C5.
[0048] The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).
[0049] An "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In specific embodiments, the antibody will be purified to greater than 95% by weight of antibody as determined by the Lowry method, or greater than 99% by weight, to a degree that complies with applicable regulatory requirements for administration to human patients (e.g., substantially pyrogen-free), to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step, for example, an affinity chromatography step, an ion (anion or cation) exchange chromatography step, or a hydrophobic interaction chromatography step.
[0050] It is well known that the binding to a molecule (or a pathogen) of antibodies with an Fc region assists in the processing and clearance of the molecule (or pathogen). The Fc portions of antibodies are recognized by specialized receptors expressed by immune effector cells. The Fc portions of IgGl and IgG3 antibodies are recognized by Fc receptors present on the surface of phagocytic cells such as macrophages and neutrophils, which can thereby bind and engulf the molecules or pathogens coated with antibodies of these isotypes (C. A. Janeway et al., Immunobiology 5th edition, page 147, Garland Publishing (New York, 2001)). [0051] In certain embodiments, single chain antibodies, and chimeric, humanized or primatized (CDR-grafted) antibodies, as well as chimeric or CDR- grafted single chain antibodies, comprising portions derived from different species, are also encompassed by the present disclosure as antigen-binding fragments of an antibody. The various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein. See, e.g., U.S. Pat. Nos. 4,816,567 and 6,331,415; U.S. Pat. No. 4,816,397; European Patent No. 0,120,694; WO 86/01533; European Patent No. 0,194,276 Bl; U.S. Pat. No. 5,225,539; and European Patent No. 0,239,400 Bl. See also, Newman et al., BioTechnology 10:1455-1460 (1992), regarding primatized antibody. See, erg., Ladner et al., U.S. Pat. No. 4,946,778; and Bird et al., Science 242:423-426 (1988), regarding single chain antibodies.
[0052] In addition, functional fragments of antibodies, including fragments of chimeric, humanized, primatized or single chain antibodies, can also be produced. Functional fragments of the subject antibodies retain at least one binding function and/or modulation function of the full-length antibody from which they are derived. Preferred functional fragments retain an antigen-binding function of a corresponding full-length antibody (such as for example, ability of anti-C5 antibody to bind C5).
Pharmaceutical Formulations and Uses
[0053] Methods of administration of antibodies are well-known to those of skill in the art. To achieve the desired inhibition, the antibodies can be administered in a variety of unit dosage forms. The dose will vary according to the particular antibody. For example, different antibodies may have different masses and/or affinities, and thus require different dosage levels. Antibodies prepared as Fab fragments will also require differing dosages than the equivalent intact immunoglobulins, as they are of considerably smaller mass than intact immunoglobulins, and thus require lower dosages to reach the same molar levels in the patient's blood. The dose will also vary depending on the manner of administration, the particular symptoms of the patient being treated, the overall health, condition, size, and age of the patient, and the judgment of the prescribing physician. Dosage levels of the antibodies for human subjects are generally between about 1 mg per kg and about 100 mg per kg per patient per treatment, and preferably between about 5 mg per kg and about 50 mg per kg per patient per treatment. In terms of plasma concentrations, the antibody concentrations are preferably in the range from about 25 μg/mL to about 500 μg/mL. However, greater amounts may be required for extreme cases and smaller amounts may be sufficient for milder cases.
[0054] Administration of the antibodies will generally be performed by an intravascular route, e.g., via intravenous infusion by injection. Other routes of administration may be used if desired but an intravenous route will be the most preferable. Formulations suitable for injection are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985). Such formulations must be sterile and non-pyrogenic, and generally will include a pharmaceutically effective carrier, such as saline, buffered (e.g., phosphate buffered) saline, Hank's solution, Ringer's solution, dextrose/saline, glucose solutions, and the like. The formulations may contain pharmaceutically acceptable auxiliary substances as required, such as, tonicity adjusting agents, wetting agents, bactericidal agents, preservatives, stabilizers, and the like. Although antibodies are preferred, especially anti-C5 antibodies which have already been shown to be safe and effective at decreasing the accumulation of downstream complement components in persons, the use of other complement inhibitors is also contemplated by this disclosure. The pharmaceutical formulations and uses of the disclosure may be combined with any known complement inhibitors or sickle cell treatments known in the art (supra).
INCORPORATION BY REFERENCE
[0055] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.
[0056] The present methods are described with reference to the following Examples, which are offered by way of illustration and are not intended to limit the disclosure in any manner. Standard techniques well known in the art or the techniques specifically described below are utilized.
EXAMPLE
PROPHYLACTIC TREATMENT OF A SICKLE CELL DISEASE PATIENT WITH ECULIZUMAB [0057] Sickle cell disease patients have episodes of hemolysis in which large amounts of red blood cells are lysed and hemoglobin is released into the plasma. Because of the recurring nature of this disease, such patients will be treated prophylactically by administering anti-C5 antibodies at regular intervals such that active antibody is present in the plasma at any time and can bind to C5 and inhibit its activation, thereby decreasing the amount of lysis of red blood cells. The amount of antibody required will depend upon the severity of the disease. Antibodies are degraded over time and must be replaced. As an example to be used as guidance, PNH patients are administered an anti-C5 antibody at a dose of 900 mg approximately once every two weeks to maintain an active level of that antibody (Hillmen et al., N. Engl. J. Med. 350:552-559 (2004)) to decrease lysis of red blood cells. The amount delivered depends upon the form of the antibody and its in vivo half-life.
[0058] While the invention has been disclosed by reference to the details of preferred embodiments of the invention, it is to be understood that the disclosure is intended in an illustrative rather than in a limiting sense, as it is contemplated that modifications will readily occur to those skilled in the art, within the spirit of the invention and the scope of the appended claims.

Claims

CLAIMS:
1. A method of treating sickle cell disease in a mammal comprising treating said mammal with a therapeutically effective amount of an anti-C5 antibody, wherein said treatment results in decreased lysis of red blood cells in said mammal.
2. The method of claim 1 wherein said mammal is a human.
3. The method of claim 1 wherein said antibody is a whole antibody or an antibody fragment.
4. The method of claim 3 wherein said whole antibody or antibody fragment is selected from the group consisting of: a polyclonal antibody, a monoclonal antibody or antibody fragment, a diabody, a chimerized or chimeric antibody or antibody fragment, a humanized antibody or antibody fragment, a deimmunized human antibody or antibody fragment, a fully human antibody or antibody fragment, a single chain antibody, an Fv, an Fab, an Fab', or an F(ab')2-
5. The method of claim 1 wherein said treatment is chronic.
6. The method of claim 1 wherein said treatment comprises treating an acute episode.
7. The method of claim 1 wherein said mammal is treated by administering said antibody intravenously.
8. The method of claim 1 wherein said mammal is treated to inhibit or decrease the amount and/or extent of an undesirable physiological condition resulting from excess lysis of red blood cells.
9. The method of claim 8 wherein said undesirable physiological condition is selected from the group consisting of dystonia, a clotting disorder, pulmonary hypertension, systemic hypertension, gastrointestinal contractions, abdominal pain, sternal pain, erectile dysfunction, priapism, inflammation, esophageal spasm, dysphagia, thrombosis, decreased organ perfusion, and death.
10. The method of claim 1 wherein said antibody inhibits activation of the classical pathway, the alternative pathway or the lectin complement pathway.
11. The method of claim 1 wherein said antibody is an antibody against any member of the group consisting of the complement components C5, C5b, C6, C7, C8, and C9.
12. The method of claim 1 wherein said antibody is an inhibitor of C5 cleavage.
13. The method of claim 1 wherein said antibody is pexelizumab.
14. The method of claim 1 wherein said antibody is eculizumab.
PCT/US2006/024770 2005-06-23 2006-06-23 Use of an anti c5 complement antibody to treat patients with sickle cell disease WO2007002571A2 (en)

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WO2023023220A1 (en) * 2021-08-20 2023-02-23 Alexion Pharmaceuticals, Inc. Methods for treating sickle cell disease or beta thalassemia using a complement alternative pathway inhibitor

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