WO2004031240A1 - Anticorps monoclonal anti-c3-2 dirige contre le troisieme composant du complement c3 et son utilisation dans des procedes d'inhibition de l'activation du complement - Google Patents

Anticorps monoclonal anti-c3-2 dirige contre le troisieme composant du complement c3 et son utilisation dans des procedes d'inhibition de l'activation du complement Download PDF

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WO2004031240A1
WO2004031240A1 PCT/EP2003/010989 EP0310989W WO2004031240A1 WO 2004031240 A1 WO2004031240 A1 WO 2004031240A1 EP 0310989 W EP0310989 W EP 0310989W WO 2004031240 A1 WO2004031240 A1 WO 2004031240A1
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activation
antibody
complement
human
mab
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PCT/EP2003/010989
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Hilde De Winter
Marie-Ange Buysse
Erik Hack
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Stichting Sanquin Bloedvoorziening
Sanquin Blood Supply Foundation Amsterdam
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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
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

  • This invention is in the field of immunology/biochemistry, and describes a method to inhibit inflammatory reactions in vivo, more specifically the activation of the complement system.
  • the invention consists of the identification and inhibition of a novel functional domain on the third component of complement, C3, which domain is essential for the activation of C3. Inhibition of conformational changes of the identified domain prevents the activation of C3, and hence the generation of biologically active peptides such as C3a and C5a, and the formation of membrane attack complexes.
  • the preferred inhibitor is a monoclonal antibody (mAb), a humanised monoclonal antibody or a human monoclonal antibody against the identified domain, or functional fragments derived theref om, or peptides complementary to the identified domain.
  • Activation of the complement system plays a key role in the normal mflammatory response to injury.
  • This system consists of a set of proteins, which circulate in blood as inactive precursor proteins, also known as factors. During activation of the system one factor activates the subsequent one by limited proteolysis and so on. This activation process resembles a cascade system, and, therefore, the complement system is also considered as one of the major plasma cascade systems, the other being the coagulation, the fibrinolytic and the contact systems.
  • the physiological role of the complement system is to defend the body against invading micro-organisms.
  • the complement system can be activated via three pathways, the classical, the mannose- binding lectin (MBL) and the alternative pathway, all activating a common terminal pathway leading to the formation of the membrane-attack complex (Walport MJ, 2001; Fujita T., 2002; Turner, 1996; Cooper N.R., 1985; Muller-Eberhard H.J. et al, 1980; Muller-Eberhard H.J., 1992, In: Gallin JI, Goldstein IM, Snyderman R (eds): Inflammation: Basic Principles and Clinical Correlates, New York, Raven Press Ltd, p.33).
  • MBL mannose- binding lectin
  • proinflainmatory peptides like anaphylatoxins C3a and C5a are generated and the membrane attack complex, C5b-9, is formed.
  • Complement activation products especially the anaphylatoxines, elicit a number of biological effects such as chemotaxis of leukocytes, degranulation of phagocytic cells, mast cells and basophils, smooth muscle contraction and the increase of vascular pe ⁇ neability (Hugh, 1986).
  • generation of toxic oxygen radicals and the induction of synthesis and release of arachidonic acid metabolites and cytokines lead to the amplification of the inflammatory response.
  • complement is an important line of defence against pathogenic organisms, its activation can also lead to host cell damage.
  • Complement-mediated tissue injury has been reported in a wide variety of inflammatory diseases, including sepsis and septic shock, toxicity induced by the in vivo administration of cytokines or rnAbs, ⁇ nmune complex diseases as rheumatoid arthritis, systemic lupus erythematosus and vasculitis, multiple trauma, ischaemia- reperfusion injuries, myocardial infarction, and so on.
  • the pathogenic role of complement activation in these conditions is likely related, in some way or another, to the aforementioned biological effects of its activation products. Inhibition of complement activation may therefore be beneficial in these conditions.
  • Human C3 is a 190-kD glycoprotein consisting of an enchain (110 kD) and a ⁇ -chain (77 kD), held together by disulpbide and non-covalent bonds (Lambris JD, 1988; Janotova J, 1986; De Brain MHL, et al. 1985). Its normal plasma concentration is 1.0-1.6 g/L (Muller-Eberhard H.J., 1992, In: Gallin JI, Goldstein IM, Snyderman R (eds): Inflammation: Basic Principles and Clinical Correlates, New York, Raven Press, p.33).
  • C3 can be synthesised by many cells, of which hepatocytes are the main producers of plasma C3 (Alper CA, et al. 1969). C3 is synthesised as a single chain precursor, pro-C3, which proteolytically is processed into its two peptide-chains (Morris KM, et al. 1982). The complete amino acid sequence of C3 has been derived from the complete cDNA coding sequence (De Bruin MHL, et al. 1985).
  • Each of the three complement pathways generates a C3 convertase by a different route, nl. classical pathway C3 convertase (C4b2a), alternative pathway C3 convertase (C3b,Bb) and MBL pathway C3 convertase (C4b2a).
  • C4b2a classical pathway C3 convertase
  • C3b,Bb alternative pathway C3 convertase
  • MBL pathway C3 convertase C4b2a
  • Two of the three convertases are identical and all are homologous and have the same activity, nl. proteolytic activation of C3, thereby generating C3a and C3b.
  • the principal effector molecules of complement activation and the late events are the same for all three pathways.
  • Proteolytic activation of C3 leads to cleavage of C3 onto an anaphylatoxic peptide C3a and an opsonic f agment C3b.
  • Covalent attachment of metastable C3b to target cells undergoing complement attack results in generation of C5a and formation of C5b-9 membrane attack complex.
  • the tissue injury that results from complement activation is directly mediated by the membrane attack complex, C5b-9, and indirectly by the generation of anaphylatoxic peptides C3a and C5a. These peptides induce damage through their effect on neutrophils and mast cells.
  • neutrophils Upon stimulation with C5a, neutrophils produce a serine elastase that causes tissue injury.
  • C5a also triggers the generation of toxic oxygen-derived free radicals from neutrophils, and both C3a and C5a stimulate rapid and enhanced production of leukotrienes from IL-13-primed basophils.
  • the activation of complement in vivo is tightly regulated at several levels. Both plasma and membrane proteins provide regulation at the levels of C3 and C4 involvement.
  • the plasma protein inhibitors are factor H and C4 binding protein, and the regulatory membrane-bound proteins located on cell surfaces are CR1, DAF and MCP.
  • the proteins with inhibitory activity prevent the release of the anaphylatoxic peptides C3a and C5a by inhibiting the C3 and C5 convertases, by promoting dissociation of the multisubunit complexes, and/or by inactivation of the complexes by proteolysis (Sahu et al., 1998; Campbell et al., 1988).
  • complement-mediated pathology has been reported in several diseases. It is well known that activation of one pathway (classical, alternative or lectin binding pathway) leads to recruitment of the others. For example, activation of the classical pathway results in activation of the alternative pathway. Similarly, activation of the lectin pathway supports the activation of the alternative pathway. Thus, in most clinical conditions multiple pathways are activated. These results suggest the usefulness of a complement inhibitor that blocks all three pathways. The three pathways converge at the C3 activation step. Blocking this step would result in total shutoff of the complement cascade including the generation of C3a, C5a and MAC formation.
  • Other methods for inhibiting complement can be achieved by neutralising the action of complement derived anaphylatoxin C5a, by interfering with complement receptor 3 (CR3, CD18/l lb)-mediated adhesion of inflammatory cells to the vascular endothelium or by incorporation of membrane-bound complement regulators (DAF-CD55, MCP-CD46, CD59) (Kirschfink, 1997).
  • compstatin small molecule inhibitor of C3, called " compstatin” , which was isolated from a phage-display peptide library by screening for binding to C3b.
  • Compstatin also binds to native C3, thereby preventing activation of C3.
  • the present invention however clearly identifies the presence of a new functional domain exposed on native C3, which is essential for the activation and proteolytic cleavage of C3 and is different from the binding site of compstatin.
  • native C3 the third component of complement, contains a novel functional domain, which is essential for the activation and proteolytic cleavage of C3 into C3a and C3b.
  • This domain in part located on the 23kD-o ( -chain-fragment of C3c, is expressed preferentially on native C3. Blocking this domain of C3 prevents the generation of
  • the present invention anus at providing a molecule binding a functional domain expressed on native C3, which is capable of inhibiting the generation of biologically active peptides such as C3a and C3b, thereby inhibiting complement activation.
  • exemplary molecules may be a mAb against the identified functional domain of C3, or a humanised or human mAb against the identified functional domain of C3, or peptides complementary to the identified functional domain of C3.
  • the present invention also contemplates the use of said molecule for inhibiting complement activation.
  • the present invention further aims at providing molecules capable of inhibiting the activation of native C3 by binding to the identified new functional domain for use in the treatment of complement-mediated diseases. Furthermore, the present invention aims at providing a prophylactic or therapeutic method to inhibit activation of complement in vivo, which method comprises administering an inhibitor of the novel functional domain of C3.
  • Another aim of the present invention is to provide for a prophylactic or therapeutic method to treat complement-mediated diseases, which method comprises administering a molecule as described above capable of neutralising the novel functional domain of C3, thereby inhibiting complement activation.
  • the present invention aims at providing pharmaceutical compositions comprising a molecule capable of inhibiting the generation of biologically active peptides such as C3a and C3b, thereby inhibiting complement activation.
  • the present invention also aims at providing methods for the preparation of said molecules capable of inhibiting C3 activation.
  • FIG. 1 Inhibition of the hemolytic activity of complement via the classical [A] or alternative pathway [B] by mAb anti-C3-2 or its F(ab) 2 fragments in human serum.
  • Mab anti-C3-l l specifically reacting with C3d, is used as control mAb.
  • Cells used in [A] are antibody-sensitized sheep red blood cells and in [B] rabbit erythrocytes. Results are expressed as % lysis of the cells.
  • the indicated mAb anti-C3-2 concentration represents that in the sample added to the sample of fresh serum. Insets show the dose response-curves of serum in the respective assay.
  • the volume of serum in [A] was 1.7 ⁇ l and in [B] 23 ⁇ l.
  • FIG. 4 Influence of mAb anti-C3-2 of the generation of C3b/bi/c (A), C4b/bi/c (B) and soluble C5b-C9 (C) complexes in human serum by zyrhosan. Results are expressed as nmol/L (A and B) or ⁇ g/ml (C). The experiments were performed as described in the legend to figure 3, except that zymosan instead of AHG was used.
  • Figure 5. Influence of mAb anti-C3-2 of the generation of C3b/bi/c (A), C4b/bi/c (B) and soluble C5b-C9 (C) complexes in human seram by E.coli bacteria in human seram. Results are expressed as nmol/L (A and B) or ⁇ g/ml (C). Experiments are similar as those described in Figure 3 except that E.Coli, instead of AHG, were tested.
  • FIG. 6 Inhibition by mAb anti-C3-2 of the generation of C3a by E.coli in human serum. Results are expressed as nmol/L. Fresh serum samples incubated with either one vol of mAb anti-C3-2 and one vol of E.coli, or one vol of PBS and one vol of E.coli, or with 2 vol of PBS, were tested for the presence of C3 a by radioimmunoassay after the reaction had been stopped by the addition of one vol of EDTA. A serum sample, which was first incubated with one vol of E.coli for 30 min at 37°C, after which one vol of mAb anti-C3-2 and one vol of EDTA were added, served as control.
  • FIG. 8 Cleavage of the ⁇ -chain of human C3 by trypsin is not prevented by mAb anti-C3-2.
  • C3 was digested by limited amounts of trypsin in the presence or absence of mAb anti-C3-2.
  • Lanes 1 and 2 high and low molecular weight markers, respectively;
  • lane 3 C3 incubated with trypsin only (SBTI added after incubation);
  • lanes 4 and 5 C3 incubated with trypsin in the presence of 25 or 250 ⁇ g mAb anti-C3-2, respectively;
  • lane 6 C3 incubated with trypsin in the presence of SBTI;
  • lane 7 C3 alone.
  • Figure 9 Cleavage of the cx-chain of human C3 by the purified C3 -convertase, C3(H 2 O)Bb, is prevented by mAb anti-C3-2.
  • Figure 10 Cleavage of the cx-chain of human C3 by the purified C3 -convertase, C3(H 2 O)Bb, was prevented by mAb anti-C3-2.
  • the epitope for mAb anti-C3-2 is 10-fold better expressed on native C3 than on C3 with a disrupted thioester. Binding of 125 I-anti-C3-2 to C3-Sepharose was inhibited by fluid phase C3 species. Results are given as % of the added 125 I-labeled anti-C3c antibodies that were bound.
  • MAb anti-C3-2 does not prevent the conformational changes of C3 following disruption of the thioester.
  • Plasma C3 incubated with methylamine in the presence of mAb anti- C3-2 was tested in the radioimmunoassay for iC3. Results are expressed as % of the input of labeled antibodies bound to the Sepharose in the assay.
  • FIG. 12 The epitope for mAb anti-C3-2 is in part located on the 23kD- ⁇ -chain fragment of C3c.
  • [A] Shows an immunoblot of C3 species (and of C4 as a control) incubated with mAb anti-C3-2. Electrophoresis was done under reducing conditions.
  • [B] Peptide-chain specific ELISA. Chains of C3c were purified by preparative SDS-PAGE, fixed onto ELISA plates and incubated with mAb anti-C3-2.
  • FIG. 13 Inhibition of activation of C3 by pre-treatment with mAb anti-C3-2 in 2 baboons challenged with a lethal dose of E.coli.
  • Levels of C3b/bi/c were assessed in blood samples collected at T+0 (i.e. the start of the E.coli infusion), +30, +60, +120, +180, + 240, + 300, + 360 and + 1440 minutes.
  • Open symbols represent levels in the anti-C3-2 treated baboons, filled symbols are mean and SD of 6 baboons that did not receive mAb anti-C3-2.
  • Figure 14 Nucleic acid and amino acid sequence of the variable kappa light region of the anti-C3-2 mAb (A) and the variable heavy region (B) of the anti-C3-2 mAb.
  • Figure 16 Interaction of C3-2 with methylamine-treated C3 (in crude plasma).: A: interaction of C3-2 with 0.4M methylamine-treated plasma; B. interaction of C3-2 with 1.2M methylamine-treated plasma; C. interaction of C3-2 with active C3 in plasma; D. control sample: running buffer (hepes buffered saline) containing 0.4M methylamine.
  • Figure 17 Amino acid sequence of the mouse anti-C3 heavy and light chain variable domains: Italic: primer-induced sequence; : Sequence confirmed by N-terminal protein sequence analysis; : Sequence confirmed by 'nested' -PCR; VL/VH: Consensus sequence; CDR prediction.
  • Figure 18 Augments of mouse and humanised anti-C3 heavy and light chain variable domains: Mouse: CDR prediction; Humanised: Humanised resiudes.
  • Figure 19 Interaction of humanised anti-C3-2 mAb with native C3.
  • the figure shows the binding of different concentrations of C3 (present in recalcified plasma) to the humanized C3-2 antibody immobilized onto the sensorchip.
  • FIG. 20 Anti-C3-2 antibody prevents human complement-mediated damage of the rabbit isolated heart.
  • Basic for the present invention is the realisation that the third component of complement, C3, contains a novel functional domain which is essential for the activation of C3 and that of the subsequent complement cascade, and that activation of C3 can be inhibited by binding of a mAb to this novel functional domain.
  • this invention be constructed so narrowly, virtually every method to inhibit the function of the identified novel domain on C3 is intended to come into the scope of this invention.
  • the present invention relates to an inhibitor of complement activation characterised in that said inhibitor specifically binds on a functional domain expressed on native human C3, thereby inhibiting the generation of biologically active peptides such as C3a and C3b, inhibiting the activation of C5 and subsequent factors.
  • the new functional domain is located on the 23kD- ⁇ -chain fragment of C3c, as indicated in the examples, and is clearly different from the binding site of compstatin (data not shown).
  • inhibitor refers to "a molecule capable of inhibiting” . Both terms are used interchangeably.
  • molecule encompasses, but is not limited to, an antibody and fragments thereof, a diabody, a triabody, a tetravalent or other multivalent antibody, a peptide, a low molecular weight non-peptide molecule (also referred to as "small molecules”) specifically binding said functional domain on native C3.
  • a molecule specifically binding on the functional domain refers to a molecule, which is capable of forming a complex with the functional domain in an environment where other substances in the same environment are not complexed with the same functional domain.
  • the inhibitor is an antibody binding the functional domain expressed on native C3, which is essential for the activation of C3.
  • antibody refers to polyclonal antibodies, monoclonal antibodies, antibodies
  • monoclonal antibody refers to an antibody composition having a homogeneous antibody population. The term is not intended to be limited by the manner in which it is made.
  • a monoclonal antibody typically displays a single binding affinity for a particular polypeptide with which it immunoreacts.
  • the monoclonal antibody used is further characterised as immunoreacting with a specific polypeptide.
  • said antibody is the murine monoclonal antibody anti-C3-2 produced by a hybridoma that is deposited according to the Budapest Treaty at the collection of DSMZ under the accession number DSM ACC2562.
  • the antibodies of the present invention may be altered as described further, without affecting their functional activity, i.e. neutralising the functional domain of native C3, and thereby inhibiting C3 activation.
  • the antibodies of the present invention can be prepared by using a technique which provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Kohler and Milstein (Kohler and Milstein, 1975).
  • the preparation of high titer neutralising polyclonal antibody can be achieved by immunising rabbits, sheeps, goats, horses or other species, and employing one of several ii munization schemes including those using peptides or peptide conjugates.
  • rabbits can be immunised with 25 ⁇ g of purified human C3 in complete Freund's adjuvant by subcutaneous injection.
  • the animals are then repeatedly boosted at 3 week-intervals, each booster consisting of a subcutaneous injection of 25 ⁇ g of human C3 together with incomplete Freund's adjuvant.
  • plasma can be obtained from the animals by plasmaphoresis to yield about 100 ml of plasma each week.
  • Polyclonal anti-C3 antibodies of the present invention can also be generated in vivo by administering C3 protein or fragments thereof in combination with at least one foreign T- cell epitope, as an immunogen.
  • C3 protein or fragments thereof in combination with at least one foreign T- cell epitope, as an immunogen.
  • Several methods for inducing an antibody response in vivo against self-proteins have been described in the art.
  • One example is the method described in the international patent application WO 9505849.
  • Monoclonal antibodies of the present invention can be obtained by isolating immune cells from an animal immunised with human C3, and immortalisation of these cells to yield antibody secreting cell lines such as hybridomas.
  • Cell lines that produce the desired antibodies can be identified by screening culture supernatants for the presence of antibody activity, and by establishment of the effect of the selected antibody on the functional activity of the complement system, and in particular that of C3.
  • Human C3 isolated according to a variety of purification methods may be used to immunise an appropriate host animal.
  • the preferred purification scheme is that described by
  • a variety of immunization protocols may be employed, and may consist of intravenous, subcutaneous, or intraperitoneal immunization, followed by one or more boosts.
  • a suitable adjuvant is Freund's adjuvant.
  • the precise schedule of administration of the human C3 to the host animal in general is not well defined.
  • the choice of the immunization procedure is more dependent on host animal antibody responses to the administered C3, as measured by a suitable assay (vide infra).
  • a preferred immunization procedure is hyperimmunization with human C3 as described (Hack CE, et al. 1988).
  • lymphocytes human or murine or other
  • An alternative approach for immunization comprises the use of synthetic peptides that mimic the domain of C3 essential for activation as recognised by mAb anti-C3-2 (vide infra).
  • the methods of making antibodies against peptides are well-known in the art and generally require coupling of the peptides to a suitable carrier molecule, for example bovine serum albumin or keyhole limpet hemocyanin.
  • a suitable carrier molecule for example bovine serum albumin or keyhole limpet hemocyanin.
  • the peptides can be made according to procedures well known in the art. The procedure also may use commercially available peptide synthesiser machines.
  • the hybrid cell line that produces the antibody may be used as a source of DNA or mRNA encoding the desired antibody, which may be isolated and transferred to cells by known genetic techniques to produce genetically engineered antibody.
  • Monoclonal antibodies can also be produced in various other ways with techniques well understood by those having ordinary skill in the art. Details of these techniques are described in Antibodies: A Laboratory Manual, Harlow et al. Cold Spring Harbor Publications, p. 726 (1988), or are described by Campbell, A.M. ("Monoclonal Antibody Technology Techniques in Biochemistry and Molecular Biology," Elsevier Science Publishers, Amsterdam, The Netherlands (1984)) or by St. Groth et al. (1980)). These other techniques include, but are not limited to techniques for recombinant production of monoclonal antibodies. Monoclonal antibodies of any mammalian species, including humans, can be used in this invention. Accordingly, the antibodies according to this embodiment may be human monoclonal antibodies.
  • Such human monoclonal antibodies may be prepared, for instance, by the generation of hybridomas, derived from immunised transgenic animals, containing large sections of the human immunoglobulin (Ig) gene loci in the germline, integrated by the yeast artificial chromosomal (YAC) technology (Mendez et al., 1997).
  • hybridomas derived from immunised transgenic animals, containing large sections of the human immunoglobulin (Ig) gene loci in the germline, integrated by the yeast artificial chromosomal (YAC) technology (Mendez et al., 1997).
  • the te ⁇ n "humanised antibody” means that at least a portion of the framework regions of an immunoglobulin or engineered antibody construct is derived from human immunoglobulin sequences. It should be clear that any method to humanise antibodies or antibody constructs, as for example by variable domain resurfacing (Roguska et al., 1994) or CDR grafting or reshaping (Hurle and Gross, 1994), can be used.
  • chimeric antibody refers to an engineered antibody construct comprising of variable domains of one species (such as mouse, rat, goat, sheep, cow, lama or camel variable domains), which may be humanised or not, and constant domains of another species (such as non-human primate or human constant domains) (for review see Hurle and Gross (1994)). It should be clear that any method known in the art to develop chimeric antibodies or antibody constructs can be used.
  • variable domains of one species such as mouse, rat, goat, sheep, cow, lama or camel variable domains
  • constant domains of another species such as non-human primate or human constant domains
  • fragment refers to F(ab), F(ab)'2, Fv, scFv and other fragments which retain the antigen binding function and specificity of the parent antibody.
  • the methods for producing said fragments are well known to a person skilled in the art and can be found, for example, in Antibody Engineering, Oxford University Press, Oxford (1995) (1996) and Methods in Molecular Biology, Humana Press, New Jersey (1995).
  • single chain Fv also termed scFv, refers to engineered antibodies prepared by isolating the binding domains (both heavy and light chains) of a binding antibody, and supplying a linking moiety which permits preservation of the binding function.
  • Cell lines that secrete antibody against human C3 can be identified by assaying culture supernatants, ascitic fluid, etc., for the presence of antibody.
  • the preferred screening procedure comprises two sequential steps, the first being identification of hybridomas that secrete mAb against human C3, the second being determination of the ability of the mAb to inhibit activation of C3.
  • the initial screening step of culture supernatants of hybridomas obtained by fusion of lymphocytes of mice immunised with C3, parts thereof, or with C3 peptides, with an appropriate fusion partner is preferably done by an ELISA or a RIA.
  • Both assays are known to those skilled in the art, and consist of coupling of human C3 to a solid-phase matrix, and assaying for antibody binding to C3 by a second, labelled antibody.
  • peptides are used for immunization, peptides coupled to a solid-phase matrix, also can be used in these assays.
  • the preferred assay is an ELISA in which purified human C3 (Tack BF, et al. 1976) is used for coating, and which is further carried out according to the procedure described by Smeenk RTJ, et al. (1987).
  • Alternative assays may be those described by Langone J, et al. (1983).
  • an alternative screening procedure may be used to assess whether the selected antibody may bind C3 in solution. This is achieved by a method in which an anti- immunoglobulin agent is coupled to a solid-phase matrix, and bound antibodies against C3 are specifically detected using labelled purified C3.
  • the preferred RIA procedure for screening of C3 antibodies may be that described byhack CE, et al. (1988).
  • solutions containing C3 may be incubated with the antibody coupled to a solid-phase matrix via an anti-im unoglobulin reagent. The matrix is then washed, and bound C3 is dissociated from it. The eluted C3 may then be measured by SDS-PAGE followed by Western blotting.
  • any construct of an antibody or a fragment is also a subject of current invention.
  • the term "construct” relates to diabodies, triabodies, tetravalent antibodies, pepta- or hexabodies, and the like, that are derived from an anti-human C3 antibody according to the present invention.
  • Said multivalent antibodies, comprising at least one hypervariable domain from an anti-C3 antibody according to the present invention can be mono-, bi- or multispecific.
  • the present invention further also relates to C3 -binding peptides and low molecular weight nonpeptides capable of binding the functional domain on C3 homologous to the epitope of the anti-C3-2 mAb.
  • Said diabodies, triabodies, tetravalent antibodies, C3-binding peptides and low molecular weight nonpeptide molecules can be produced by the following methods:
  • hybridomas derived from immunised transgenic mice, containing large sections of the human immunoglobulin (Ig) gene loci in the germ line, integrated by the yeast artificial chromosomal (YAC) technology, resulting in effective blocking antibodies as described by Mendez et al (1997).
  • C3-binding peptides or fragments refers to any peptide (i.e. a polymer composed of at least two amino acids) which cross-links, or reacts with the functional domain on native human C3 homologous to the epitope of the mAb anti-C3-2.
  • low-molecular weight nonpeptide molecules refers to any molecule which is not a peptide and which cross-links, or reacts with the functional domain on native human C3 homologous to the epitope of the mAb anti-C3-2.
  • the inhibitor of the functional domain on native human C3 is any molecule capable of binding a functional domain on native human C3 essential in the activation of C3 which comprises an epitope for the mAb anti-C3-2 or said inhibitor can be any molecule competing with the mAb anti-C3-2 for the binding on said functional domain expressed on native human C3.
  • the small molecule inhibitor of C3, compstatin is not competing with the mAb anti-C3-2 for binding on native C3, indicating that both compstatin and anti-C3-2 mAb have different binding sites on native C3 (data not shown).
  • molecule competing with the mAb anti-C3-2 means that said molecule has the same or comparable specificity for the functional domain exposed on native
  • a further embodiment of the present invention relates to an inliibitor comprising the variable region or the humanised variable region of the mAb anti-C3-2 or fragments thereof, capable of binding the functional domain exposed on native C3 thereby inhibiting the activation of C3.
  • the inhibitors described in the present invention are characterised by their ability to inhibit activation of C3. Said inhibitors can be selected by the assessment of their effect on the hemolytic activity of the complement system in human serum, as well as on the generation of complement activation products by complement activators in serum.
  • C3 inhibitors more particular C3 antibodies may be tested by adding these to fresh human serum, followed by measurement of the hemolytic activity of the mixture in hemolytic assays.
  • hemolytic assays are well known in the art.
  • serial dilutions of fresh human seram are added to a constant number of erythrocytes optimally sensitised with IgG/M antibodies, in the presence of veronal buffered saline (VBS) containing CaCl 2 and Mg Cl 2 , and incubated under shaking at 37°C.
  • VBS veronal buffered saline
  • the intact erythrocytes are pelleted by centrifugation, and hemolysis is assessed by measuring hemoglobin content of the supernatant spectrophotometrically.
  • a similar type of assay may be used except that non-sensitised rabbit erythrocytes instead of antibody- sensitised red blood cells, and VBS containing MgEGTA rather than CaCl 2 and MgCl 2 , are used.
  • the effect of the selected C3 inhibitor, more particular C3 antibody, on the generation of complement activation products in human serum can be analysed by adding purified inhibitor, more particular antibody, to serum, followed by incubation at 37°C of the mixture with complement activators such as aggregated IgG, cobra venom factor, E.coli bacteria or zymosan. After this incubation EDTA is added to prevent further activation and the mixture is tested for the presence of complement activation products such as C3a, C4a, C5a, C3b/bi/c, C4b/bi/c or C5b-C9. Assays for these complement activation products are well known in the art and can be obtained commercially. The preferred assays are those described by hack CE, et al. (1988), Ralph CE, et al. (1990) and Wolbink GJ, et al. (1993).
  • the inhibitors described in the present invention are according to another embodiment further characterised by specifically binding on a new functional domain expressed on native human C3.
  • the new functional domain present on native human C3 is characterised by comprising the epitope of the mAb anti-C3-2, being clearly different from the C3 -convertase binding site and is in part located on the 23 kD- ⁇ -chain fragment of C3c.
  • Neutralising said functional domain results in the inactivation of C3, thereby inhibiting the generation of biologically active peptides such as C3a and C3b, inhibiting the activation of C5 and subsequent factors.
  • the present invention also relates to a functional domain exposed on native human C3 characterised in that said functional domain is neutralised by binding an inhibitor of the present invention, thereby preventing the generation of the biologically active peptides C3a and C3b.
  • the inhibitors of the present invention can be used for the preparation of a medicament for inhibiting C3 activation, thereby inhibiting the generation of the biologically active peptides C3a and C3b, inhibiting the activation of C5 and subsequent factors.
  • the inhibitors of the present invention can be used for the preparation of a medicament for inhibiting complement activation in vivo.
  • the inhibitors can be used alone or in combination with other drags.
  • inhibitors of the present invention can be used alone or in combination with other drags, for the preparation of a medicament to treat a host organism suffering of a complement mediated disease, or at risk with respect to such a disease.
  • autoimmune diseases such as experimental allergic neuritis, type II collagen-induced arthritis, myasthenia gravis, hemolytic anemia, glomeralonephritis, rheumatoid arthritis, systemic lupus erythematosus and immune complex-induced vasculitis, adult respiratory distress syndrome, stroke, xenotransplantation, multiple sclerosis, burn injuries, extracorporeal dialysis and blood oxygenation, inflammatory disorders, including sepsis and septic shock, toxicity induced by the in vivo administration of cytokines or mAbs, multiple trauma, ischaemia-reperfusion injuries, myocardial infarction.
  • autoimmune diseases such as experimental allergic neuritis, type II collagen-induced arthritis, myasthenia gravis, hemolytic anemia, glomeralonephritis, rheumatoid arthritis, systemic lupus erythematosus and immune complex-induced vasculitis, adult respiratory distress syndrome, stroke, xenotransplantation
  • patients suffering from a disease involving complement- mediated damage can be administered an effective amount of an inhibitor as described so that complement activation is inhibited.
  • effective amount it is meant a concentration of inhibitor, which is capable of inhibiting complement activation.
  • the present invention relates to a method for inhibiting complement activation comprising the step of administering an inhibitor as described by the present invention.
  • the present invention also relates to a method for preventing or treating diseases mediated by activation of complement comprising the step of administering an inhibitor as described by the present invention.
  • Treatment will generally consist of administering the inhibitors parenterally, preferably intravenously.
  • the dose and administration regimen will depend on the extent of inhibition of complement activation aimed at.
  • the amount of antibody given will be in the range of 5 to 20 mg per kg of body weight.
  • the inhibitor will be formulated in an injectable form combined with a pharmaceutically acceptable parenteral vehicle.
  • Such vehicles are well-known in the art and examples include saline, dextrose solution, Ringer's solution and solutions containing small amounts of human serum albumin.
  • the inhibitor will be formulated in such vehicles at a concentration of about 100 mg per ml.
  • the inhibitor is given by intravenous injection. It will, of course, be understood that intended to come within the scope of this invention is virtually every method of administering C3 inhibitors as described by the present invention, to yield sufficiently high levels either in the circulation or locally.
  • compositions comprising an inhibitor as described above in a pharmaceutical acceptable carrier.
  • the pharmaceutical compositions according to the invention may be fo ⁇ nulated in accordance with routine procedures for administration by any route, such as oral, topical, parenteral, sublingual, transdermal or by inhalation.
  • the compositions may be in the form of tablets, capsules, powders, granules, lozenges, creams or liquid preparations, such as oral or sterile parenteral solutions or suspensions or in the form of a spray, aerosol or other conventional method for inhalation.
  • the term 'pharmaceutical acceptable carrier' relates to carriers or excipients, which are inherently nontoxic and nontherapeutic.
  • excipients examples include, but are not limited to, saline, Ringer's solution, dextrose solution and Hank's solution.
  • Nonaqueous excipients such as fixed oils and ethyl oleate may also be used.
  • a preferred excipient is 5% dextrose in saline.
  • the excipient may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, including buffers and preservatives.
  • the present invention also relates to the use of the mAb anti-C3-2 for identifying molecules capable of binding the new functional domain on native C3. Said molecules can be identified by their capability of competing with anti-C3-2 mAb for binding on the new functional domain on native C3, in a competition assay.
  • Example I hmnunization with human C3 or peptide immunogens and the production of hybridomas.
  • mice with purified human C3 with the aim to isolate lymphocytes from the immunised mice and to produce murine hybridomas. It will be further appreciated that the procedure can be employed to produce antibodies against C3 fragments or peptides.
  • mice were immunised by repeated intraperitoneal injections of 25 ⁇ g of purified human C3 given at three- week intervals. The first C3 gift was mixed with complete Freund's adjuvant, the subsequent with incomplete Freund's adjuvant.
  • spleens were removed from the immunised mice and the splenocytes were fused with the murine myeloma cell-line SP2/0-Agl4, according to the procedure first described by Kohler G, et al. (1975), except that feeder cells were replaced by IL-6, formerly called hybridoma growth factor (Aarden La, et al. 1985).
  • mice were sacrificed and splenocytes teased from the spleens, and washed in serum free Dulbecco's Modified Eagles medium. Similarly, SP2/0-Agl4 myeloma cells were washed, and added to the splenocytes yielding a 5:1 ratio of splenocytes to myeloma cells. The cells were then pelleted, and the supernatant was removed. One ml of a 40 % (v/v) solution of polyethylene glycol 1500 was then added dropwise over a 60 sec period, after wliich the cells were incubated for another 60 sec at 37°C.
  • Dulbecco's Modified Eagles medium Nine ml of Dulbecco's Modified Eagles medium was then added with gentle agitation. The cells were pelleted, washed to remove residual polyethylene glycol, and finally plated at a concentration of 10 5 cells per well in Dulbecco's Modified Eagles medium containing 10% (v/v) fetal calf serum ( lOO ⁇ l per well). After 24 hours, 100 ⁇ l of a 2x solution of hypoxanthine/azaserine selection medium was added to each well. At day 4 hypoxanthine/azaserine selection medium was replenished, at day 7 it was replaced by Dulbecco's Modified Eagles medium containing 10% (v/v) fetal calf seram.
  • monocyte derived or recombinant human IL-6 was present in the culture at concentrations of approximately 10 pg/ml. About 80% of the wells exhibited cell growth at day 10.
  • Example 2 ELISA for the detection of C3 antibodies.
  • the wells were screened for the presence of antibody to C3 using an enzyme-linked immuno sorbent assay, in which purified human native C3 was used for coating (2 ⁇ g/ml in PBS, pH 7.4 (PBS); 100 ⁇ l/well). Residual non-specific binding sites were then blocked by a 30 minutes incubation at room temperature with PBS/0.1% (w/v) Tween 20 (PBS-T) containing 0.2 % (w/v) gelatin (PBS-TG). Then, after a wash procedure (5 times with PBS- T), the plates were incubated for 120 min at 37°C with 20 ⁇ l of hybridoma supernatant together with 80 ⁇ l of PBS-TG.
  • PBS-T PBS/0.1% (w/v) Tween 20
  • PBS-TG 0.2 % gelatin
  • Example 3 Preparation of purified mAb anti-C3-2 and its F(ab)' 2 -fragments.
  • Antibody may be produced in vitro from the hybridoma anti-C3-2 by culturing the cells in 1 litre roller-bottles in Iscove's Modified Dulbecco medium supplemented with 2% (v/v) fetal calf serum, 10 pg/ml IL-6, 50 ⁇ M 2-mercaptoethanol, and penicillin and streptomycin. The cells were grown to a density of > 10 6 cells per ml, and one to two weeks later the supernatants were collected. Solid ammonium sulphate was added to yield 50% saturation (i.e., approximately 2M), and an antibody-enriched fraction was obtained by centrifugation for 30 min at 1,300 g.
  • the precipitate was dissolved in 1.5 M NaCl/0.75 M glycine, pH 8.9, and put onto a protein A-Sepharose column (Pharmacia). The column was washed with PBS, and then mAb anti-C3-2 was eluted off with glycine-HCl, pH 2.5. Fractions were neutralised instantaneously with 2M TRIS, pH 8.0, and those containing protein were pooled and dialysed against PBS.
  • F(ab)' 2 -fragments of mAb anti-C3-2 were prepared by incubating overnight at 37°C 2 vol of anti-C3-2 in PBS with one vol of 0.1 M sodium acetate containing pepsin (Cooper Biomedical; 2500 U/mg) to yield a final ratio of pepsin to anti-C3-2 of 1:50, and a pH 4.1. Thereafter, one vol of 1 M TRIS, pH 8.1, was added, and the preparation was dialysed against PBS. On SDS-PAGE the preparation appeared to contain approximately 10-20% uncleaved anti-C3-2, which was removed by passing the preparation over a protein A-column (Pharmacia).
  • Pepsin-digestion did not affect the binding of anti-C3-2 to purified C3 coated onto plastic plates as was assessed with an enzyme-linked sorbent assay, in wliich peroxidase- conjugated rat mAb against mouse kappa-light chain was used to detect bound anti-C3-2 mAb or fragments thereof. Using a similar assay with rat mAb against the heavy chain of mouse IgGl, it was demonstrated that the digested preparation contained less then 1% of uncleaved anti-C3-2 mAb.
  • Example 4 Inhibition of the hemolytic activity of seram and of purified C3 by mAb anti-C3-
  • mAb anti-C3-l l tested as a control, was not able to inhibit lysis of the antibody-sensitised red blood cells by fresh serum. Complete inhibition of hemolytic activity by mAb anti-C3-2 was not observed at any of the concentrations tested. Most likely, this residual hemolytic activity represented the C3-bypass by C4 (Masaki T, et al. 1991).
  • mAb anti-C3-2 inhibited alternative pathway mediated lysis in a dose dependent fashion yielding 90% inhibition of lysis at a concentration of 3.8 ⁇ M.
  • intact mAb anti-C3-l l also inhibited this alternative pathway mediated lysis, its F(ab)' 2 -fragments did not (Fig IB).
  • Table 1 monoclonal antibody anti-C3-2 inhibits the hemolytic activity of human C3.
  • Results were corrected for background lysis (15%) and are given as % lysis. (Note that the concentrations ofC3 used in this experiment are sufficient to yield complete lysis of the added sensitised cells.)
  • Example 5 Inhibition of fixation of C3 to aggregated IgG by mAb anti-C3-2.
  • Aggregated human IgG (AHG; Ralph CE, et al. (1981)) was incubated overnight in 2 ml-polystyrene tubes at a concentration of 10 ⁇ g/ml in PBS (final volume 0.5 ml). Then, the tubes were washed twice with PBS-T. Thereafter, the tubes were incubated with 500 ⁇ l of VBS/0.1% (w/v) Tween 20 (VBS-T) containing 2 ⁇ l of fresh normal human serum as well as varying amounts mAb anti-C3-2 or control antibodies, for 2 hours at 37°C.
  • VBS-T VBS/0.1% (w/v) Tween 20
  • Example 6 Inhibition by mAb anti-C3-2 of the generation of complement activation products in serum by several complement activators.
  • Fig 3 shows the inhibition by mAb anti- C3-2 of the generation of C3b/bi/c (nM) and soluble C5b-C9 complexes (mg/L) in human serum by aggregated IgG (AHG; at concentrations of 1 and 0.2 mg/ml).
  • Fig 4 shows a similar type of experiment, using zymosan (Sigma Chem Co., St. Louis, MO) washed with PBS as an activator.
  • zymosan Sigma Chem Co., St. Louis, MO
  • PBS PBS
  • Fig 5 shows the inhibition by mAb anti-C3-2 of the generation of C3b/bi/c and soluble C5b-C9 complexes in human serum by E.coli bacteria.
  • the experiment was performed in a similar way as those described above, except that E.coli organisms (Hinshaw LB, et al. (1983)) at concentrations of 10 10 or 10 9 organisms per ml were used to activate the complement system in serum. After the incubation at 37°C and the addition of EDTA, the mixtures were centrifuged for 10 min at 1,300 g to remove the E.coli organisms. Again, mAb anti-C3-2 significantly inhibited the generation of C3b/bi/c and C5b-C9 complexes in seram.
  • Mab anti-C3-2 not only inhibited the generation of C3b/bi/c, but also that of C3a as is shown in Fig 6: one vol of mAb anti-C3-2 (15 ⁇ M in PBS) was added to one vol of fresh human serum. Then, one vol of E.coli (10 10 organisms per ml in PBS) was added and the mixture was incubated for 30 min at 37°C. Finally, one vol of 0.2 M EDTA, pH 7.5, was added and serial dilutions of the mixtures were tested for the presence of C3a(desarg) by a specific radioimmunoassay (Hack CE, et al. 1988). Results are expressed in nM.
  • mAb anti-C3-2 dose-response effect of mAb anti-C3-2 on the generation of C3b/bi/c in human seram by aggregated IgG, zymosan, or E.coli was investigated.
  • One vol of varying concentrations of mAb anti-C3-2 (1.9-15 ⁇ M in PBS) or one vol of PBS, was added to one vol of fresh human serum.
  • one vol of AHG (0.2 mg/ml), zymosan (1 mg/ml) or E.coli (10 9 organisms/ml) was added and the mixture was incubated for 20 min at 37°C.
  • Example 7 mAb anti-C3-2 prevents cleavage of the ⁇ -chain of C3 by a C3 -convertase, but not that by trypsin.
  • anti-C3-2 its epitope (partially) overlapped the cleavage site on the ⁇ -chain for trypsin, which under limiting conditions also cleaves the bond between arg-748 and ser-749. Therefore, purified human C3 (25 ⁇ g; Tack BF, et al. 1976), mAb anti-C3-2 (25 or 250 ⁇ g), trypsin (0.25 ⁇ g; Sigma Chem Co, St Louis, MO) in PBS (final volume 116 ⁇ l) were incubated for 3 min at 37°C. Then, soybean trypsin inhibitor (SBTI; 25 ⁇ l containing 25 ⁇ g; Sigma), were added.
  • SBTI soybean trypsin inhibitor
  • Fig 8 Lanes 1 and 2: high and low molecular weight markers, respectively; lane 3: C3 incubated with trypsin only (SBTI added after incubation); lanes 4 and 5: C3 incubated with trypsin in the presence of 25 or 250 ⁇ g mAb anti-c3-2, respectively; lane 6: C3 incubated with trypsin in the presence of SBTI; lane 7: C3 alone.
  • mAb anti-C3-2 could not prevent cleavage of the ⁇ -chain by trypsin (Fig.8; the upper arrow on the left indicates the position of the uncleaved ⁇ -chain, the lower that of the (cleaved) ⁇ '-chain).
  • mAb anti-C3-2 could not prevent cleavage of the ⁇ -chain by trypsin, it did prevent cleavage by a C3 -convertase. Therefore, mAb anti-C3-2 does not bind to the C3 -convertase cleavage site, but rather recognises a hitherto unknown domain on C3, which is essential for cleavage by a C3-convertase.
  • Example 8 Mab anti-C3-2 binds to native C3 and is not able to prevent the conformational changes induced by disruption of the thioester.
  • Mab anti-C3-2 (and as a control mAb anti-C3-l, which is directed against an epitope on the ⁇ -chain expressed equally well on activated and non-activated C3 species) was purified from hybridoma supernatant using protein G affinity chromatography following the instructions of manufacturer (Pharmacia), and labelled with I25 I with the Chloramin T method to yield a specific activity of 5-7 ⁇ Ci/ ⁇ g protein.
  • I25 I-mAb was then incubated with 0.5 ml of a 1 mg/ml suspension of Sepharose 4B to which native C3 was coupled, in the presence of varying concentrations of native C3, C3b (prepared by limited trypsin digestion of native C3), C3c (purified from aged serum as described by hack CE, et al. 1988, and C3 in which the thioester was disrupted by incubation (60 min at 37°C) with 0.4 M methylamine, pH 7.5 (iC3). After an overnight incubation at room temperature, the beads were washed and bound 125 I-mAb was assessed by counting the radioactivity bound to the beads using a multichannel gammacounter.
  • mAb anti-C3-2 The influence of mAb anti-C3-2 on the conformational changes of C3 following disraption of the thioester, was studied by adding 20 ⁇ g of mAb anti-C3-2 in 10 ⁇ l PBS (or 10 ⁇ l of PBS only, as a control) to 100 ⁇ l of fresh human plasma (1 to 50 diluted in PBS or PBS only) and incubating the mixture for 60 min at room temperature. Dilutions of the mixtures were then tested in the radioimmunoassay for iC3 (uncleaved C3 with a disrupted thioester) as described (Hack CE, et al. 1990).
  • Example 9 The epitope for mAb anti-C3-2 is in part located on the 23kD- ⁇ chain fragment ofC3c. Initially we attempted to map the epitope for mAb anti-C3-2 using immunoblotting of
  • C3 species Samples of C3 species (5 or 1 ⁇ g) purified as described (Hack CE, et al. 1988), were incubated for 5 min at 100°C in reducing SDS sample buffer and thereafter electrophoresed on SDS-polyacrylamide gels (5-20 %, w/v). Purified C4 (Hessing M, et al. 1993) was included as a control. Proteins were then transferred to nitrocellulose sheets. The sheets were incubated in PBS-T containing 5% (w/v) de-lipidated protein powder (Protifar) for 30 min at 37°C, followed by an incubation of biotinylated mAb anti-C3-2 (approximately 2 ⁇ g/ml) in the same buffer.
  • Example 10 Effect of mAb anti-C3-2 on complement activation in vivo.
  • Haematologic parameters were assessed in blood samples collected at T+0 (i.e. the start of the E.coli infusion), +30, +60, +120, +180, +240, +360 and +1440 minutes. In addition, at each of these time points also 5 ml blood samples were collected in 10 mM EDTA/100 ⁇ g/ml SBTI/10 mM benzamidine (final concentrations). Levels of C3b/bi/c in these samples were determined as described (De Boer JP, et al. 1993). The results, shown in Fig 13, were expressed as % of C3b/bi/c of the standard which consisted of normal baboon serum aged, i.e.
  • the C3-2 antibody was shown to interact specifically with native C3, containing an intact thioester bond and not with an inactive form, containing a hydrolysed ester bond. This was shown using Surface Plasmon Resonance-analysis with the BIACORE 3000 ® (Biacore AB,
  • SPR surface plasmon resonance
  • a protein e.g. antibody
  • a binding protein e.g. antigen
  • Binding association and dissociation is monitored with mass sensitive detection.
  • BIACORE ® experiments were performed in which the C3-2 antibody was immobilised onto a CM5 sensorchip (Biacore AB).
  • the C3-2 antibody was immobilised using amine coupling according to the manufacturer's procedure. Briefly, the antibodies were diluted to 5 ⁇ g/ml in 10 mM acetatebuffer pH4.8 and injected at 5 ⁇ l/min until an immobilisationlevel of +/- 500 RU was reached. Injecting 0.1M ethanolamine pH 8.5 blocked residual unreacted ester groups. An irrelevant antibody of the same subclass was immobilised to the same level and was used for subtraction of non-specific binding of C3 to the mlgGl surface.
  • Native C3 (present in human plasma) was injected at 10-300 ⁇ g/ml (concentration active C3 in plasma: 1.2 mg/ml). 150 ⁇ l C3 solution was injected at 50 ⁇ l/min. The surface was regenerated with 2 pulses of 60 ⁇ l 0.2M Na2CO3 pHl l (50 ⁇ l/min). Binding to the irrelevant IgGl surface was subtracted as a blank. Kinetic constants were calculated using the BIAevaluation software 3.1. Results of a typical experiment are shown in figure 15. These data show that the C3-2 antibody interacts with high affinity with active C3 present in plasma.
  • Inactive C3 (iC3) was prepared by treating human plasma with the nucleophilic reagent methylamine. This results in the cleavage of an intramolecular thioester bond and induces C3b-like properties (Pangburn et al., 1981). Treatment was done by incubating 1 volume of plasma with 1 volume of methylamine. HC1 (either 0.4 or 1.2M) during 1 hour at 37°C. The treated plasma was injected over the C3-2 coated sensorchip and binding to the immobilized C3-2 was monitored. Results are shown in figure 16. These data show that treatment with 0.4M methylamine resulted in a decreased association rate combined with no change in dissociation rate, indicating a reduction in concentration of active C3.
  • a pellet of approx. 10 ⁇ subcultured hybridoma cells was prepared and total RNA was prepared (QIAGEN Rneasy procedure) for subsequent cDNA synthesis (QIAGEN OneStep RT-PCR) of both variable regions (heavy and light) with 'gene-specific' oligonucleotides.
  • the mouse VH genes were amplified using the primers VH1BACK and VH1-FOR2. These are consensus primers that cover the majority of mouse heavy chain gene families.
  • the primer VK2BACK is used in combination with a mix of four J region primers (MJK1FONX, MJK2FONX, MJK4FONX and MJK5FONX) to amplify the light chain kappa families.
  • the VK2B ACK is a consensus primer that covers most of the mouse kappa families.
  • PCR fragments of approx. 350 bp were obtained (with VH slightly larger than VL).
  • the specific PCR fragments of the expected size were excised from gel, purified, cloned into pGEM-T vector and sequenced.
  • VH in total 14 clones were sequenced, of which 10 were completely identical.
  • 3 clones differed from the previous 10 at one position within framework regions and 1 clone had an undetermined nucleotide in CDR3.
  • VL in total 16 clones were sequenced, of which 14 were completely identical. 2 clones differed from the previous 14 at several positions or had some undetermined nucleotides.
  • the first 8 amino acids of the light and heavy chain variable regions were uncertain since the nucleotide sequence at these positions is primer-induced.
  • N-tenr ⁇ nal protein sequencing of both antibody chains demonstrated that a correction was required for residues 1 (D -> E) and 3 (E->V) of the light chain and for residues 1 (* -> D) and 3 (K - > Q) of the heavy chain variable region.
  • the N-terminal protein sequencing confirmed the correct sequence of the residues of the first framework region and CDR1 of both variable regions.
  • both heavy and light chains need to be preceded by suitable signal peptides for correct processing and transport. Therefore, both domains were fused to naturally occuring signal peptide sequences, as obtained from characterisation of human antibodies. The created cleavage site was analysed and predicted to have a high probability for correct cleavage.
  • correponding nucleotide sequences for both heavy and light chain humanised variable regions including their signal peptide, consensus Kozak sequences and endonuclease restriction sites for cloning purposes, were made by PCR-based gene assembly and synthesis.
  • the coding sequence for the humanised VL domain was cloned to a human Ig Kappa light chain constant region resulting in the coding sequence for the complete humanised anti-C3 light chain.
  • the coding sequence for the humanised VH domain was cloned to a human IgGl heavy chain constant region resulting in the coding sequence for the complete humanised anti-C3 heavy chain.
  • Humanised antibody was produced by a transient transfection of COS-7 cells. Serum-free conditioned medium was harvested at 48 hr and purified using standard chromatographic procedures (protein A affinity chromatography). The humanized C3-2 interacted with native C3 with comparable kinetics as the murine antibody as was shown using BIACORE-analysis (figure 19).
  • Example 13 Anti-C3-2 antibody prevents human complement-mediated damage of. the rabbit isolated heart.
  • Langendorff preparation Male NZW rabbits (1.8-2.4 kg) were anaesthetized; after administration of heparin, hearts were excised and flushed with NaCl 0.9% through a catheter in the aorta. Hearts were attached to a modified Langendorff perfusion apparatus, and perfused in a retrograde manner.
  • the filtered solution was gassed continuously with a mixture of 95% 0 2 /5% CO 2 to achieve the desired oxygen partial pressure of 225 to 300 mm Hg (Micro 13 pH/blood gas analyzer; Instrumentation Laboratory, Lexington, MA).
  • Recalcified human plasma as a source of the complement components was added to the perfusate as described below. Retrograde perfusion was performed at a constant pressure and a recirculating perfusate volume of 500 ml.
  • the hearts were paced via electrodes attached to the right atrium with square wave stimuli from a laboratory stimulator (165 impulses/min, 5 ms duration, 4 V; Grass SD-5, Quincy, MA).
  • the physiologic parameteres monitored included the aortic flow, the isovolumic left ventricular pressure and its first derivative, dP/dt.
  • the intraventricular fluid-filled latex balloon used for the ventricular pressure measurements was filled to achieve an end-diastolic pressure of 12 to 15 mm Hg. Measurements were recorded continuously (Grass polygraph model 79D), and stored on a digital archive and analysis system (Po-Ne-Mah HD-4, Storrs, CT).
  • Instrumentation of the heart included cannulation of the pulmonary artery, pulmonary vein ligation, and closure of the left atrial appendage incision around the shunt, thermistor, and ballon tubing to prevent fluid leakage.
  • Treatment protocol Six treatment groups were used to determine the ability of anti-C3-2 antibody to inhibit the consequences of complement activation on the measured functional parameters in the rabbit isolated perfused heart: 1) control; perfusion with Krebs-Henseleit buffer; 2) control; perfusion with 6% heat-inactivated human plasma; 3) perfusion with 6% normal human plasma; 4) perfusion with 6% normal human plasma in the presence of 3.5 mg anti-C3-2 antibody; 5) perfusion with 6% normal human plasma in the presence of 7.5 mg anti-C3-2 antibody; 6) perfusion with 6% normal human plasma in the presence of 15 mg anti-C3-2 antibody.
  • Hearts were excised, instrumented and their function was allowed to stabilize for 20 minutes with plasma-free buffer perfusion medium. Base-line functional parameters, as well as the coronary flow were recorded. In treatment groups 4 to 6, anti-C3-2 antibody was added to the reservoir containing the perfusion medium.10 minutes later, 2% (v/v) human plasma was added to the the perfusion medium. Additional human plasma (2% per time point) was added at 15 and 20 minutes after administration of the anti-C3-2 antibody to achieve a final concentration of 6% human plasma in the perfusion medium. The monitored parameters were recorded at 5 minutes intervals through 90 minutes after addition of the anti-C3 -antibody, the end of the protocol.
  • Human plasma was obtained by venapuncture from fasted donors and frozen. After thawing overnight at 4°C, the plasma was reconstituted with CaCl 2 to a final concentration of 10 mM. Afterwards, the plasma was incubated for 30 minutes at 37° C, to allow a fibrinogen clot to form, which was removed. The plasma was centrifuged (3000 rpm, 15 min) at 4° C; after which it was collected andd stored at -80° C, until shipment on dry ice to the center where the Langendorff experiments were conducted. Heat-inactivated human plasma was prepared by heating (56° C, 1 hour).

Abstract

L'invention concerne un procédé d'inhibition de réactions inflammatoires in vivo, plus particulièrement de l'activation d'un système complément. L'invention vise à identifier et inhiber un nouveau domaine fonctionnel sur le troisième composant indigène du complément C3 dont le domaine est essentiel pour l'activation de C3. L'inhibition de modifications conformationnelles du domaine identifié évite l'activation de C3 et, de ce fait, la génération de peptides biologiquement actifs, notamment C3a et C5a, ainsi que la formation de complexes d'attaque membranaire. L'inhibiteur préféré est un anticorps monoclonal (mAb), un anticorps monoclonal humanisé ou un anticorps monoclonal humain dirigé contre le domaine identifié ou des fragments fonctionnels dérivés de celui-ci, ou encore des peptides complémentaires au domaine identifié.
PCT/EP2003/010989 2002-10-04 2003-10-03 Anticorps monoclonal anti-c3-2 dirige contre le troisieme composant du complement c3 et son utilisation dans des procedes d'inhibition de l'activation du complement WO2004031240A1 (fr)

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US8652475B2 (en) 2004-02-10 2014-02-18 Musc Foundation For Research Development Inhibition of factor B, the alternative complement pathway and methods related thereto
US9066925B2 (en) 2009-07-02 2015-06-30 Musc Foundation For Research Development Methods of stimulating liver regeneration
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US8652475B2 (en) 2004-02-10 2014-02-18 Musc Foundation For Research Development Inhibition of factor B, the alternative complement pathway and methods related thereto
US8703140B2 (en) 2004-02-10 2014-04-22 Musc Foundation For Research Development Inhibition of factor B, the alternative complement pathway and methods related thereto
EP1885398A1 (fr) * 2005-05-26 2008-02-13 The Regents of the University of Colorado Inhibition de la voie de complément alternative pour le traitement de lésions traumatiques du cerveau, de lésions de la moelle épinière et de conditions apparentées
EP1885398A4 (fr) * 2005-05-26 2011-12-14 Univ Colorado Inhibition de la voie de complément alternative pour le traitement de lésions traumatiques du cerveau, de lésions de la moelle épinière et de conditions apparentées
US8911733B2 (en) 2005-05-26 2014-12-16 Musc Foundation For Research Development Inhibition of the alternative complement pathway for treatment of traumatic brain injury, spinal cord injury and related conditions
US9096677B2 (en) 2007-03-14 2015-08-04 Alexion Pharmaceuticals, Inc. Humaneered anti-factor B antibody
US9066925B2 (en) 2009-07-02 2015-06-30 Musc Foundation For Research Development Methods of stimulating liver regeneration
US9803005B2 (en) 2012-05-24 2017-10-31 Alexion Pharmaceuticals, Inc. Humaneered anti-factor B antibody
WO2019195136A1 (fr) * 2018-04-03 2019-10-10 Ngm Biopharmaceuticals, Inc. Agents de liaison à c3 et procédés d'utilisation associés
JP2021520195A (ja) * 2018-04-03 2021-08-19 エヌジーエム バイオファーマシューティカルス，インコーポレーテッド Ｃ３結合薬及びその使用方法
US11136381B2 (en) 2018-04-03 2021-10-05 Ngm Biopharmaceuticals, Inc. Anti-C3 antibodies
US20220119508A1 (en) * 2018-04-03 2022-04-21 Ngm Biopharmaceuticals, Inc. C3-binding agents and methods of use thereof
RU2802307C2 (ru) * 2018-04-03 2023-08-24 ЭнДжиЭм БАЙОФАРМАСЬЮТИКАЛЗ, ИНК. C3-связывающие агенты и способы их применения
US11767359B2 (en) 2018-04-03 2023-09-26 Ngm Biopharmaceuticals, Inc. C3-binding agents and methods of use thereof
JP7477457B2 (ja) 2018-04-03 2024-05-01 エヌジーエム バイオファーマシューティカルス，インコーポレーテッド Ｃ３結合薬及びその使用方法
WO2022171771A1 (fr) * 2021-02-12 2022-08-18 Boehringer Ingelheim International Gmbh Protéines de liaison à l'antigène c3 du complément
WO2024086555A1 (fr) 2022-10-17 2024-04-25 Ngm Biopharmaceuticals, Inc. Utilisations d'anticorps anti-c3

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