WO2000035483A1 - Techniques et produits permettant de reguler l'activation du complement associee a la voie du complement a lectine - Google Patents

Techniques et produits permettant de reguler l'activation du complement associee a la voie du complement a lectine Download PDF

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Publication number
WO2000035483A1
WO2000035483A1 PCT/US1999/029919 US9929919W WO0035483A1 WO 2000035483 A1 WO2000035483 A1 WO 2000035483A1 US 9929919 W US9929919 W US 9929919W WO 0035483 A1 WO0035483 A1 WO 0035483A1
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Prior art keywords
mbl
isolated
peptide
monoclonal antibody
antibody
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PCT/US1999/029919
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English (en)
Inventor
Gregory L. Stahl
Charles D. Collard
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The Brigham And Women's Hospital, Inc.
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Priority to JP2000587802A priority Critical patent/JP2002532079A/ja
Priority to CA002347734A priority patent/CA2347734A1/fr
Priority to EP99967362A priority patent/EP1140171A4/fr
Publication of WO2000035483A1 publication Critical patent/WO2000035483A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A61P7/08—Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock
    • 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
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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 and products for regulating lectin 15 complement pathway (LCP) associated complement activation.
  • the invention relates to methods for inhibiting LCP associated complement activation by contacting a mammalian cell having a mannose binding lectin (MBL) ligand with an MBL inhibitor.
  • MBL mannose binding lectin
  • the invention also relates to products which are MBL inhibitors, such as an MBL binding peptide.
  • the immune system functions to defend the body against pathogenic bacteria, viruses and parasites. Immunity against foreign pathogens usually involves the complement system.
  • the complement system is a cascade of 18 sequentially activated serum proteins
  • Complement which functions to recruit and activate other cells of the immune system, effect cytolysis of target cells and induce opsonization of foreign pathogens.
  • Complement can be activated by the presence of either antibody/antigen complexes, as in the classical complement pathway, or microbial surfaces, as in the alternative complement pathway. Complement activation can also occur via the lectin complement pathway (LCP).
  • Lectins are carbohydrate-binding
  • the LCP is an antibody-independent cascade that is initiated by binding of mannan-
  • MBL mannose binding lectin
  • MBL Mannose binding lectin associated serine protease
  • MASP-1 and MASP-2 which show striking homology to the two Clq-associated serine proteases of the classical complement pathway, Clr and Cls (Thiel S, et al., "A second serine protease associated with mannan- binding lectin that activates complement", Nature 1997;386:506-510).
  • N-acetyl-D-glucosamine GluNAc
  • mannose N- acetylmannosamine
  • fucose maltose > glucose » galactose and N- acetylgalactosamine
  • Thiel S, et al. "A second serine protease associated with mannan- binding lectin that activates complement", Nature 1997;386:506-510; Turner MW, "Mannose-binding lectin: The pluripotent molecule of the innate immune system", Immunol.Today, 1996;17:532-540).
  • Binding of the MBL/MASP complex to cell surface carbohydrates activates the LCP, which in turn activates the classical complement pathway independently of Clq, Clr, Cls or antibodies (Fig. 1). Most if not all the carbohydrate moieties to which MBL binds are not normally expressed by unperturbed human tissue.
  • the present invention relates to methods and products for regulating lectin complement pathway (LCP) associated complement activation.
  • LCP lectin complement pathway
  • MBL mannan-binding lectin
  • MASP-1 and MASP-2 two associated serine proteases
  • MBL deficiencies can result in medical disorders.
  • a disease known as MBL deficiency in which children are deficient in MBL, renders the children prone to the development of infectious diseases.
  • the present invention is based upon the surprising discovery that MBL recognizes specific carbohydrates or peptides on the surface of mammalian endothelial cells, causing complement deposition through activation of the LCP.
  • MBL does not recognize the cell wall of human and animal cells.
  • MBL does recognize specific sequences on the surface of mammalian cells.
  • MBL deposition on the surface of mammalian cells results in activation of LCP, contributing to the development of diseased or damaged tissue.
  • the invention is a method for inhibiting LCP-associated complement activation.
  • the method includes the step of contacting a mammalian cell having a surface exposed MBL ligand with an effective amount of an MBL inhibitor to inhibit cellular MBL deposition and LCP-associated complement activation.
  • the method is an in vitro screening assay.
  • the invention is a method for inhibiting a cellular injury mediated by LCP-associated complement activation.
  • the method includes the step of administering to a subject in need thereof an effective amount of an MBL inhibitor to inhibit LCP- associated complement activation.
  • the MBL inhibitor is an isolated MBL binding peptide.
  • the isolated MBL binding peptide has an MBL binding CDR3 region or functional variant thereof.
  • the isolated MBL binding peptide is an antibody fragment. In other embodiments, the isolated MBL binding peptide is an antibody.
  • the MBL inhibitor is an isolated MASP binding peptide.
  • the isolated MASP binding peptide may bind to either MASP-1 or MASP-2 or both, preventing MASP from participating in the LCP.
  • the cellular injury mediated by LCP-associated complement activation may contribute to the development of injured tissue associated with a variety of disorders.
  • the cellular injury is associated with atherosclerosis.
  • the cellular injury is associated with arthritis, myocardial infarction, ischemia and reperfusion, transplantation, CPB, stroke, ARDS, SLE, Lupus, or dialysis.
  • the MBL inhibitor may be administered to the subject by any route known in the art.
  • the MBL inhibitor may be administered to the subject by an aerosol route of delivery.
  • an MBL inhibitor is provided.
  • the MBL inhibitor is an isolated peptide that selectively binds to a human MBL epitope and inhibits LCP-associated complement activation.
  • the invention is a hybridoma cell line.
  • the hybridoma cell line is the cell line deposited under ATCC accession number HB- 12621.
  • the hybridoma cell line is the cell line deposited under ATCC accession number HB- 12620.
  • the hybridoma cell line is the cell line deposited under ATCC accession number HB-12619.
  • the invention is a composition of an MBL inhibitor, wherein the MBL inhibitor is an isolated binding peptide that selectively binds to a human
  • the composition is a pharmaceutical composition including an effective amount for treating an MBL mediated disorder of the isolated MBL binding peptide and a pharmaceutically acceptable carrier.
  • the composition also includes a drug for the treatment of an MBL mediated disorder.
  • the isolated MBL binding peptide has an MBL binding CDR3 ⁇ region or a functional variant thereof of a monoclonal antibody produced by hybridoma cell line 3F8 deposited under ATCC accession number HB- 12621. In another embodiment the isolated MBL binding peptide has an MBL binding CDR3 2 region or a functional variant thereof of a monoclonal antibody produced by hybridoma cell line 2A deposited under
  • the isolated MBL binding peptide has an MBL binding CDR3 2 region or a functional variant thereof of a monoclonal antibody produced by hybridoma cell line hMBL i. 2 deposited under ATCC accession number
  • the isolated peptide may be an intact soluble monoclonal antibody.
  • the isolated peptide is monoclonal antibody (3 F 8 ) produced by the hybridoma cell line deposited under ATCC Accession No. HB- 12621.
  • the isolated peptide is monoclonal antibody ( A9 ) produced by the hybridoma cell line deposited under ATCC Accession No. HB- 12620.
  • the isolated peptide is monoclonal antibody hMBL i. 2 produced by the hybridoma cell line deposited under ATCC Accession No. HB-12619.
  • the isolated peptide is a humanized monoclonal antibody.
  • the isolated peptide is an antibody fragment.
  • the isolated peptide may be a monoclonal antibody fragment selected from the group consisting of an F(ab') 2 fragment, Fd fragment, and an Fab fragment.
  • the isolated peptide may also be a peptide having a light chain CDR2 region selected from the group consisting of a CDR2 (3 F 8) of a monoclonal antibody produced by hybridoma( 3F8) deposited under ATCC Accession No. HB- 12621, a CDR2 (2 A ) of a monoclonal antibody produced by hybridoma 2A deposited under ATCC Accession No. HB-12620, and a CDR2(h MBL i.
  • the isolated peptide has a light chain CDR1 region selected from the group consisting of a CDR1 (3 F 8) of a monoclonal antibody produced by hybridoma ⁇ F 8) deposited under ATCC Accession No. HB-12621 , a CDR1 ( A 9) of a monoclonal antibody produced by hybridoma ( A9) deposited under ATCC Accession No. HB-12620, and a CDRl (h BL i.
  • the invention is a composition, wherein the MBL inhibitor is an anti-MBL antibody that: (i) selectively binds to a human MBL epitope, and (ii) prevents LCP activation.
  • the invention is a method for screening a subject for susceptibility to treatment with an MBL inhibitor.
  • the method includes the steps of isolating a mammalian cell from a subject, and detecting the presence of an MBL on a surface of the mammalian cell, wherein the presence of the MBL indicates that the cell is susceptible to LCP-associated complement activation and that the subject is susceptible to treatment with an MBL inhibitor.
  • the method includes the step of contacting the MBL with a detection reagent that selectively binds to the MBL to detect the presence of the MBL.
  • the detection reagent in one embodiment is an isolated MBL binding peptide.
  • a method for screening a subject for susceptibility to treatment with MBL inhibitor includes the steps of contacting a mammalian cell from a subject with a labeled isolated MBL binding peptide, and detecting the presence of an MBL on the surface of the mammalian cell, wherein the presence of the MBL indicates that the cell is susceptible to LCP-associated complement activation and that the subject is susceptible to treatment with an MBL inhibitor.
  • the mammalian cell is an endothelial cell.
  • Figure 1 is a schematic depicting the antigen/antibody-dependent classical complement pathway and the antibody-independent alternative and lectin complement pathways. All three pathways merge at C3 and lead to the formation of the terminal complement complex (C5b-9).
  • Figure 2 depicts a flow cytometry printout to demonstrate MBL deposition on
  • MBL deposition on HUVECs subjected to zero (normoxia) or 24 hours of hypoxia was studied by flow cytometry using a monoclonal anti-human MBL antibody.
  • Figure 3 is a graph depicting MBL deposition on HUVECs (ELISA). MBL deposition on HUVECs subjected to zero (normoxia) or 24 hours of hypoxia followed by 3 hours of reoxygenation was examined by ELISA using a monoclonal anti-human MBL antibody. MBL deposition on hypoxic HUVECs reoxygenated in the presence of 30% human serum (vehicle) was significantly greater than normoxic HUVECs or hypoxic
  • HUVECs reoxygenated in 30% human serum treated with 30 mmol/L GluNac.
  • Figure 4a is a graph depicting iC3b deposition following competitive inhibition of
  • iC3b deposition was studied by ELISA on HUVECs reoxygenated in the presence of 30%) human serum treated with 30 mmol/L GluNAc, D-mannose, or L-mannose. Deposition of iC3b on hypoxic HUVECs reoxygenated in 30%> human serum (vehicle) or 30%) human serum treated with L-mannose was significantly greater than normoxic HUVECs. iC3b deposition, however, on HUVECs reoxygenated in 30%> human serum treated with GluNAc or D-mannose did not significantly differ from normoxic controls.
  • Figure 4b is a graph depicting iC3b deposition following depletion of MBL from human serum.
  • HUVECs were reoxygenated in the presence of MBL-depleted human serum to inhibit the lectin complement pathway.
  • Deposition of iC3b (ELISA) on hypoxic HUVECs reoxygenated in 30%> human serum was significantly greater (p ⁇ 0.05) than normoxic HUVECs.
  • iC3b deposition, however, on hypoxic HUVECs reoxygenated in 30%> MBL-depleted human cell was significantly less (p ⁇ 0.05) than hypoxic HUVECs reoxygenated in 30% human serum.
  • MBL was added back to the MBL-depleted human serum
  • iC3b deposition on the hypoxic/reoxygenated HUVECs was significantly greater than normoxic HUVECs.
  • Figure 5 is a graph depicting percent hemolysis as an indicator of classical complement pathwa ⁇ ' activity. No significant differences in the serum complement hemolytic assay (CH 50 ) were observed between human serum or MBL-depleted human serum, indicating that depletion of MBL did not inhibit or deplete classical complement pathway activity;
  • Figure 6 depicts a Western blot analysis of C3 activation following hypoxia reoxygenation using purified C2, C3, C4, and MBL.
  • Western blot analysis of the C3 and C3b ⁇ ' -chain was performed under reduced conditions with a polyclonal anti- human C3 antibody on the supernatants of normoxic and hypoxic (12 hours) HUVECs reoxygenated for 3 hours in the presence of purified C2, C3, C4, and MBL.
  • Lanes 1 and 2 represent normoxic HUVECs supernatant, lanes 3 and 4, hypoxic HUVECs supernatant, lane 5, purified C3 standard, and lane 6, purified C3b standard.
  • the results demonstrate an increased band density of C3b ⁇ ' -chain in the hypoxic/reoxygenated supernatants compared to the normoxic supernatants.
  • the Figure is representative of five experiments.
  • Figure 7 is a scan of a Western blot analysis of human MBL.
  • Monoclonal antibodies 3F8, hMBL1.2, 2A9 or 1C10 were used for western blot analysis of reduced MBL.
  • Lanes 1, 2. 3 and 4 represent staining of reduced human MBL with 10 ⁇ g/ml of mAb
  • Figure 8 is a graph depicting C3 deposition with inhibitors.
  • Figure 9 is a graph depicting inhibition of VCAM-1 expression. Reoxygenation of hypoxic HUVECs in 30% HS treated with PBS (Vehicle) induced a significant increase in
  • VCAM-1 expression compared to normoxic cells incubated with 30% HS.
  • the invention relates to methods and products for regulating and manipulating lectin complement pathway (LCP)-associated complement activation.
  • LCP lectin complement pathway
  • the invention is based on the finding that LCP-associated complement activation plays a role in complement induced cellular injury of mammalian cells.
  • MBL interacts with carbohydrates or peptides on the surface of mammalian cells in vitro and in vivo.
  • the surface associated MBL leads to the accumulation of complement on the surface of the cell, ultimately leading to cell injury or death.
  • LCP-associated complement activation was predominantly associated with infectious microorganisms, suggesting that MBL deposition should be promoted in order to enhance the killing of infectious microorganisms.
  • LCP LCP-associated complement activation
  • the LCP is not necessary for eradication of infectious microorganisms in adult mammals, and in fact, it contributes to cellular injury associated with several types of disorders, such as atherosclerosis, arthritis, myocardial infarction, ischemia and reperfusion, transplantation, CPB, stroke, ARDS, SLE, Lupus, or dialysis.
  • the invention is a method for inhibiting LCP-associated complement activation.
  • the method includes the steps of contacting a mammalian cell having surface exposed MBL ligand with an effective amount of an MBL inhibitor to inhibit LCP- associated complement activation.
  • the methods of the invention are useful for inhibiting LCP-associated complement activation on the surface of a mammalian cell having surface exposed MBL ligand (carbohydrate or peptide groups) recognized by MBL.
  • the mammalian cell may be any cell in which the cell surface carbohydrates or peptides interact with MBL.
  • the mammalian cell is an endothelial cell having a surface exposed MBL ligand.
  • vascular endothelial cells have been shown in subjects that have sustained ischemic/reperfusion injury to express an MBL ligand.
  • Mammalian cells having MBL ligands can easily be identified.
  • an MBL binding assay e.g., such as those described below can be used to identify MBL ligands.
  • the method for inhibiting LCP-associated complement activation may be used for a variety of in vitro and in vivo purposes.
  • the method may be used, for instance, as an in vitro screening assay.
  • the in vitro screening assay may be used to identify compounds which function as an MBL inhibitor, such as the assay described above, to identify mammalian cells having surface exposed MBL ligands, or to detect susceptibility of a subject to treatment with MBL inhibitor.
  • a cell is isolated from the subject and the presence of MBL or the ability of MBL to bind to the surface is detected.
  • MBL is present on the surface of a cell or is able to bind to the surface of a cell
  • the cell is susceptible to LCP- associated complement activation. If this is the case, then the subject is susceptible to treatment with an MBL inhibitor.
  • the methods of the invention are also useful in vivo when it is desirable to inhibit
  • MBL deposition on a mammalian cell surface For instance, the methods of the invention are useful for treating an MBL mediated disorder.
  • the MBL inhibitors can be used alone as a primary therapy or in combination with other therapeutics as an adjuvant therapy to enhance the therapeutic benefits of other medical treatments.
  • the mammalian cell is contacted with an MBL inhibitor.
  • the step of "contacting” as used herein refers to the addition of the MBL inhibitor to a medium containing a mammalian cell.
  • the medium may be an in vitro tissue culture or a biological specimen, an ex vivo sample, or in vivo.
  • the step of contacting refers to the addition of the MBL inhibitor in such a manner that it will prevent LCP-associated complement activation associated with the mammalian cell.
  • An "MBL mediated disorder” as used herein is a disorder which involves cellular injury caused by LCP-associated complement activation.
  • MBL disorders include, for instance, atherosclerosis, arthritis, myocardial infarction, ischemia and reperfusion, transplantation, CPB, stroke, ARDS, SLE, Lupus, or dialysis. Each of these disorders is well-known in the art and is described, for instance, in Harrison's Principles of Internal Medicine (McGraw Hill, Inc., New York).
  • I/R injury can lead to ischemia-reperfusion (I/R) injury.
  • I/R-induced injury is the hypoxic and reoxygenated environments created in affected tissues. Fluctuations in oxygen content as observed in these instances can create oxygen free radicals which have been reported to, among other things, modulate endothelial cell surface profile.
  • the invention also is useful for treating cellular injury arising from ischemia/reperfusion associated with atherosclerosis and/or cardio-vascular remodeling.
  • Injury to the vascular system can lead to a number of undesirable health conditions, including, for example, forms of atherosclerosis and arteriosclerosis that are associated with unwanted vascular smooth muscle cell proliferation.
  • a common injury to the vascular system occurs as a side effect of a medical procedure for treating ischemic heart disease.
  • Ischemia refers to a lack of oxygen due to inadequate perfusion of blood.
  • Ischemic heart disease is characterized by a disturbance in cardiac function due to an inadequate supply of oxygen to the heart. The most common form of this disease involves a reduction in the lumen of coronary arteries, which limits coronary blood-flow. Under these conditions the carbohydrate or peptide residues of the cell surface become exposed or an MBL ligand is synthesized, allowing MBL to associate with the cell surface and initiate the LCP associated complement activation.
  • ischemic heart disease When ischemic heart disease becomes very serious, then management must be invasive. Until recently, ischemic heart disease was treated by coronary-artery, bypass surgery. Less invasive procedures, however, now have been developed. These procedures involve the use of catheters introduced into the narrowed region of the blood vessel ("the stenosis”) for mechanically disrupting, laser ablating or dilating the stenosis.
  • the stenosis the narrowed region of the blood vessel
  • compositions may be administered in combination with other therapeutic treatments.
  • the most widely used method to achieve revascularization of a coronary artery is percutaneous transluminal coronary angioplasty.
  • a flexible guide wire is advanced into a coronary artery and positioned across the stenosis.
  • a balloon catheter then is advanced over the guide wire until the balloon is positioned across the stenosis.
  • the balloon then is repeatedly inflated until the stenosis is substantially eliminated.
  • This procedure as compared to heart surgery, is relatively noninvasive and can result in hospital stays of only three days. The procedure is an important tool in the management of serious heart conditions.
  • MBL inhibitor is a compound that prevents LCP-associated complement activation.
  • the MBL inhibitor may function by blocking MBL deposition on the surface of a mammalian cell or by blocking the association of MASP-1 or MASP-2 or C3b associated with MBL deposition.
  • the ability of an MBL inhibitor to block MBL deposition or prevent association of MASP-1, MASP-2, or C3b with MBL can be detected using routine in vitro binding assays, such as the following assay (also described in the Examples).
  • MBL deposition (or association with MASP-1, MASP-2, or C3b) can be measured by ELISA on normoxic HUVECs and HUVECs subjected to 24 hr of hypoxia followed by 3 hr of reoxygenation in the presence of 30%> human serum (HS) or 30% HS treated with 3, 30, or 300 mmol/L of N-acetyl-D-glucosamine (GluNAc) or with the putative binding peptide to inhibit competitively MBL deposition.
  • HS human serum
  • GluNAc N-acetyl-D-glucosamine
  • C3 and MBL specific cell surface ELISAs can be performed using peroxidase- conjugated polyclonal goat anti-human C3 antibody (Cappel, West Chester, PA) and monoclonal anti-human MBL antibody (Biodesign, Kennebunk, ME, clone #131-1), respectively.
  • HUVECs are grown to confluence on 0.1 %> gelatinized 96-well plastic plates
  • the cell media are aspirated and 100 ⁇ l of one the following is added to each well: 1) 30% HS, 2) Hank's balanced salt solution, 3) 30%) HS + 3, 30, or 300 mmol/L GluNAc, 4) 30% HS + 3, 30, or 300 mmol/L D-mannose, 5) 30% HS + 3, 30, 300 mmol/L L-mannose, 6) 30% MBL-depleted HS 7) 30% MBL-depleted HS + 0.6 ⁇ g/ml MBL or 8) 30% HS + 3, 30, or 300 mmol/L putative MBL binding peptide.
  • the MBL ELISA plates are then washed and incubated for 1 hr at 4 °C with 50 ⁇ l of peroxidase-conjugated polyclonal goat anti-mouse IgG secondary antibody (1 :1000 dilution). After washing the cells, the plates are developed with 50 ⁇ l of ABTS (2,2 " -azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)), and read (Molecular Devices, Sunnyvale, CA) at 405 nm. Background controls for the C3 ELISA consist of cells to which only the anti-human C3 antibody is added (i.e., no HS) or cells incubated with 30% heat-inactivated HS.
  • the assay may be performed, however, using MBL containing HS and adding an MBL binding peptide and/or a control peptide.
  • the MBL inhibitor is an isolated MBL binding peptide.
  • An "isolated MBL binding peptide" as used herein is a peptide which binds to MBL and inhibits LCP associated complement activation.
  • One method by which MBL binding peptides inhibit LCP associated complement activation is by binding to MBL and inhibiting MBL association with surface exposed MBL ligands.
  • the MBL binding peptide may bind to MBL and inhibit the association between MBL and MASP-1 or -2 and/or C3b.
  • MBL Human serum mannose- binding lectin-associated serine protease- 1
  • compositions of the invention include an MBL inhibitor which is an isolated binding peptide that selectively binds to a human MBL epitope and that inhibits
  • a "human MBL epitope” as used herein is a portion of MBL which when contacted with an MBL-binding peptide inhibits LCP- associated complement activation by preventing the association between MBL and the MBL ligand or MASP-1 or - 2 and/or C3b.
  • the MBL epitope is a region of the MBL which interacts with any of the three deposited monoclonal antibodies.
  • the MBL inhibitor is an isolated MASP binding peptide.
  • isolated MASP binding peptide refers to a peptide which binds to MASP-1 or MASP-2 and prevents LCP-associated complement activation by preventing MASP-1 or MASP-2 from forming a complex with MBL on the surface of a cell thereby preventing the resulting C3b deposition associated with the MBL-MASP complex.
  • the MBL inhibitor is a mannan binding peptide.
  • a "mannan binding peptide” as used herein is a peptide which binds to the MBL ligand on the surface of a mammalian cell, preventing its interaction with the MBL-MASP complex.
  • the MBL inhibitors may easily be prepared or identified by those of ordinary skill in the art using routine experiments since MBL, MASP, mannan and C3b are all well known compounds which have been characterized and described extensively in the prior art.
  • the MBL, MASP, and mannan binding peptides of the invention can be identified using routine assays, such as the binding and LCP complement activation assays described above and elsewhere throughout this patent application.
  • the peptides of the invention are isolated peptides.
  • isolated peptides means that the peptides are substantially pure and are essentially free of other substances with which they may be found in nature or in vivo systems to an extent practical and appropriate for their intended use.
  • the peptides are sufficiently pure and are sufficiently free from other biological constituents of their hosts cells so as to be useful in, for example, producing pharmaceutical preparations or sequencing.
  • an isolated peptide of the invention may be admixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the peptide may comprise only a small percentage by weight of the preparation.
  • the peptide is nonetheless substantially pure in that it has been substantially separated from the substances with which it may be associated in living systems.
  • MBL binding peptides also may easily be synthesized or produced by recombinant means by those of skill in the art. Methods for preparing or identifying peptides which bind to a particular target are well known in the art. Molecular imprinting, for instance, may be used for the de novo construction of macromolecular structures such as peptides which bind to a particular molecule. See for example Kenneth J. Shea, Molecular Imprinting of Synthetic Network Polymers: The De Novo synthesis of Macromolecular Binding and Catalytic Sites, TRIP Vol. 2, No. 5, May 1994; Klaus Mosbach, Molecular Imprinting, Trends in Biochem.
  • One method for preparing mimics of MBL binding peptides involves the steps of: (i) polymerization of functional monomers around a known MBL binding peptide or the binding region of an anti-MBL antibody (such as the deposited antibodies) (the template) that exhibits a desired activity; (ii) removal of the template molecule; and then (iii) polymerization of a second class of monomers in the void left by the template, to provide a new molecule which exhibits one or more desired properties which are similar to that of the template.
  • an anti-MBL antibody such as the deposited antibodies
  • MBL binding molecules which are MBL inhibitors such as polysaccharides, nucleosides, drugs, nucleoproteins, lipoproteins, carbohydrates, glycoproteins, steroids, lipids, and other biologically active materials can also be prepared.
  • MBL inhibitors such as polysaccharides, nucleosides, drugs, nucleoproteins, lipoproteins, carbohydrates, glycoproteins, steroids, lipids, and other biologically active materials.
  • This method is useful for designing a wide variety of biological mimics that are more stable than their natural counterparts, because they are typically prepared by the free radical polymerization of functional monomers, resulting in a compound with a nonbiodegradable backbone.
  • Other methods for designing such molecules include for example drug design based on structure activity relationships which require the synthesis and evaluation of a number of compounds and molecular modeling.
  • Peptides which bind to the MBL may also be identified by conventional screening methods such as phage display procedures (e.g., methods described in Hart, et al., J. Biol. Chem. 269:12468 (1994)).
  • Hart et al. report a filamentous phage display library for identifying novel peptide ligands for mammalian cell receptors.
  • phage display libraries using, e.g., Ml 3 or fd phage are prepared using conventional procedures such as those described in the foregoing reference.
  • the libraries display inserts containing from 4 to 80 amino acid residues.
  • the inserts optionally represent a completely degenerate or a biased array of peptides.
  • Ligands that bind selectively to MBL are obtained by selecting those phages which express on their surface a ligand that binds to the MBL. These phages then are subjected to several cycles of reselection to identify the peptide ligand-expressing phages that have the most useful binding characteristics. Typically, phages that exhibit the best binding characteristics (e.g., highest affinity) are further characterized by nucleic acid analysis to identify the particular amino acid sequences of the peptides expressed on the phage surface and the optimum length of the expressed peptide to achieve optimum binding to the MBL. Alternatively, such peptide ligands can be selected from combinatorial libraries of peptides containing one or more amino acids.
  • Such libraries can further be synthesized which contain non-peptide synthetic moieties which are less subject to enzymatic degradation compared to their naturally-occurring counterparts.
  • any known binding assay may be employed.
  • the peptide may be immobilized on a surface and then contacted with a labeled MBL.
  • the amount of MBL which interacts with the peptide or the amount which does not bind to the peptide may then be quantitated to determine whether the peptide binds to MBL.
  • a surface having the deposited monoclonal antibody immobilized thereto may serve as a positive control.
  • Screening of peptides of the invention also can be carried out utilizing a competition assay. If the peptide being tested competes with the deposited monoclonal antibody, as shown by a decrease in binding of the deposited monoclonal antibody, then it is likely that the peptide and the deposited monoclonal antibody bind to the same, or a closely related, epitope. Still another way to determine whether a peptide has the specificity of the deposited monoclonal antibody of the invention is to pre-incubate the deposited monoclonal antibody with MBL with which it is normally reactive, and then add the peptide being tested to determine if the peptide being tested is inhibited in its ability to bind MBL.
  • the peptide being tested is inhibited then, in all likelihood, it has the same, or a functionally equivalent, epitope and specificity as the deposited monoclonal antibody.
  • anti-idiotypic antibodies which can be used to screen other antibodies to identify whether the antibody has the same binding specificity as the deposited monoclonal antibodies of the invention.
  • anti-idiotypic antibodies can be used for active immunization (Herlyn, et al., Science, 232:100, 1986).
  • anti-idiotypic antibodies can be produced using well-known hybridoma techniques (Kohler and Milstein, Nature, 256:495, 1975).
  • An anti-idiotypic antibody is an antibody which recognizes unique determinants present on the deposited monoclonal antibodies. These determinants are located in the hypervariable region of the antibody.
  • An anti-idiotypic antibody can be prepared by immunizing an animal with the deposited monoclonal antibodies. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing deposited monoclonal antibodies and produce an antibody to these idiotypic determinants. By using the anti-idiotypic antibodies of the immunized animal, which are specific for the deposited monoclonal antibodies of the invention, it is possible to identify other clones with the same idiotype as the deposited monoclonal antibody used for immunization.
  • Idiotypic identity between monoclonal antibodies of two cell lines demonstrates that the two monoclonal antibodies are the same with respect to their recognition of the same epitopic determinant.
  • anti-idiotypic antibodies it is possible to identify other hybridomas expressing monoclonal antibodies having the same epitopic specificity.
  • an anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region which is the image of the epitope bound by the first monoclonal antibody.
  • the anti- idiotypic monoclonal antibody can be used for immunization, since the anti-idiotype monoclonal antibody binding domain effectively acts as an antigen.
  • Activation assays also can be used to assess the relative inhibitory concentrations of a peptide in an activation assay and to identify those peptides which inhibit activation by at least, e.g., 75%).
  • the peptide that inhibits the activation of MBL is an antibody or a functionally active antibody fragment.
  • Antibodies are well known to those of ordinary skill in the science of immunology. As used herein, the term "antibody” means not only intact antibody molecules but also fragments of antibody molecules retaining MBL binding ability. Such fragments are also well known in the art and are regularly employed both in vitro and in vivo.
  • the term "antibody” means not only intact immunoglobulin molecules but also the well-known active fragments F(ab') 2 , and Fab. F(ab') 2 , and Fab fragments which lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al., J. Nwc/. Med. 24:316-325 (1983)).
  • the complementarity determining regions (CDRs) of an antibody are the portions of the antibody which are largely responsible for antibody specificity. The CDR's directly interact with the epitope of the antigen (see, in general, Clark, 1986; Roitt, 1991).
  • FR1 through FR4 framework regions
  • CDRl through CDR3 complementarity determining regions
  • the framework regions (FRs) maintain the tertiary structure of the paratope, which is the portion of the antibody which is involved in the interaction with the antigen.
  • the CDRs, and in particular the CDR3 regions, and more particularly the heavy chain CDR3 contribute to antibody specificity. Because these CDR regions and in particular the CDR3 region confer antigen specificity on the antibody these regions may be incorporated into other antibodies or peptides to confer the identical antigen specificity onto that antibody or peptide.
  • MBL inhibitors of the present invention encompass in some embodiments of the invention MBL binding peptides which include a MBL binding region which specifically binds to human MBL and inhibits LCP associated complement activation, e.g., by preventing MBL from interacting with MBL ligands.
  • MBL ligands as used herein are carbohydrates or peptides with which MBL can interact.
  • the MBL binding region is a MBL binding CDR3 region.
  • a "MBL binding CDR3 region” as used herein is a CDR3 peptide sequence derived from the monoclonal antibodies produced by the hybridomas deposited with the ATCC under ATCC Accession No. (HB- 12621), ATCC Accession No.
  • Hybridoma 3F8 produces monoclonal (3F8 ) having binding specificity for MBL.
  • Monoclonal antibody 3F8 includes the CDR3 3FS region within its sequence.
  • CDR3 (3F8 ) includes the CDR3 region of monoclonal antibody( 3 F 8 ).
  • Hybridoma( A 9 ) produces monoclonal antibody ( A 9) having binding specificity for MBL.
  • Monoclonal antibody ( A 9) includes the CDR3( 2A9) region within its sequence.
  • CDR3 ( A9 ) includes the CDR3 region of monoclonal antibody 2 A 9 .
  • Hybridoma(hMBLi 2) produces monoclonal antibody ( MBLi i) having binding specificity for MBL.
  • Monoclonal antibody ( MBLi 2) includes the CDR3 (n MBLi 2) region within its sequence.
  • CDR3(hMBLi 2) includes the CDR3 region of monoclonal antibody ( MBLi 2)- Each of monoclonal antibody F 8 , monoclonal antibody 2 A9, and monoclonal antibody (hMBLi 2) specifically bind to MBL and prevent MBL from interacting with an MBL ligand.
  • the "MBL binding CDR3 region” refers to the CDR3( 3F8 ), CDR3( 2A9 ) and CDR3( hMB Li 2) peptide sequences.
  • the peptides of the invention include functional variants of the MBL binding CDR3 region.
  • a "functional variant” as used herein is a peptide having the sequence of the CDR3( 3 p 8 ), CDR3( 2A9 ), or CDR3(hMBLi 2) regions with conservative substitutions therein.
  • conservative substitution refers to an amino acid substitution which does not alter the relative charge or size characteristics of the peptide in which the amino acid substitution is made.
  • amino acids include substitutions made amongst amino acids with the following groups: (1) M,I,L,V; (2) F.Y.W: (3) K,R,H; (4) A,G; (5) S,T; (6) Q,N; and, (7) E,D.
  • substitutions can be made by a variety of methods known to one of ordinary skill in the art.
  • amino-acid substitutions may be made by PCR-directed mutation, site-directed mutagenesis according to the method of Kunkel (Kunkel, Proc. Nat. Acad. Sci. U.S.A. 82: 488-492,
  • deposited monoclonal antibody is used to refer to each of the monoclonal antibodies (monoclonal antibody ( 3 F 8 ), monoclonal antibody ( A ), or monoclonal antibody(hMBLi.2) produced by the ATCC deposited hybridomas.
  • monoclonal antibody 3 F 8
  • monoclonal antibody A
  • monoclonal antibody(hMBLi.2) produced by the ATCC deposited hybridomas.
  • the peptide of the invention is an intact soluble anti- MBL monoclonal antibody in an isolated form or in a pharmaceutical preparation.
  • An intact soluble monoclonal antibody as is well known in the art, is an assembly of polypeptide chains linked by disulfide bridges. Two principle polypeptide chains, referred to as the light chain and heavy chain, make up all major structural classes (isotypes) of antibody. Both heavy chains and light chains are further divided into subregions referred to as variable regions and constant regions.
  • the term "monoclonal antibody” refers to a homogenous population of immunoglobulins which specifically bind to an epitope (i.e. antigenic determinant) of human MBL.
  • the peptide of the invention in one embodiment is, for example, the deposited monoclonal antibody.
  • the preparation and use of the deposited monoclonal antibody is described more fully in the attached Examples.
  • the peptide of the invention is an intact antibody having the binding characteristics of the deposited monoclonal antibody.
  • An antibody having the binding characteristics of the deposited monoclonal antibody is one which binds to MBL and inhibits MBL from interacting with MBL ligands.
  • One of ordinary skill in the art can easily identify antibodies having the binding characteristics of the deposited monoclonal antibody using the screening and binding assays set forth in detail below.
  • the peptide useful according to the methods of the present invention is an intact humanized anti-MBL monoclonal antibody in an isolated form or in a pharmaceutical preparation.
  • the following examples of methods for preparing humanized monoclonal antibodies that interact with MBL and inhibit LCP associated complement activation are exemplary and are provided for illustrative purposes only.
  • a "humanized monoclonal antibody” as used herein is a human monoclonal antibody or functionally active fragment thereof having human constant regions and a MBL binding CDR3 region from a mammal of a species other than a human.
  • Humanized monoclonal antibodies may be made by any method known in the art. Humanized monoclonal antibodies, for example, may be constructed by replacing the non-CDR regions of a non-human mammalian antibody with similar regions of human antibodies while retaining the epitopic specificity of the original antibody. For example, non-human CDRs and optionally some of the framework regions may be covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody.
  • a first replicable expression vector including a suitable promoter operably linked to a DNA sequence encoding at least a variable domain of an Ig heavy or light chain and the variable domain comprising framework regions from a human antibody and a CDR region of a murine antibody is prepared.
  • a second replicable expression vector is prepared which includes a suitable promoter operably linked to a DNA sequence encoding at least the variable domain of a complementary human Ig light or heavy chain respectively.
  • a cell line is then transformed with the vectors.
  • the cell line is an immortalized mammalian cell line of lymphoid origin, such as a myeloma, hybridoma. trioma, or quadroma cell line, or is a normal lymphoid cell which has been immortalized by transformation with a virus.
  • the transformed cell line is then cultured under conditions known to those of skill in the art to produce the humanized antibody.
  • the DNA sequence encoding the domain may be prepared by oligonucleotide synthesis.
  • Another method involves the preparation of the DNA sequence encoding the variable CDR containing domain by oligonucleotide site-directed mutagenesis. Each of these methods is well known in the art. Therefore, those skilled in the art may construct humanized antibodies containing a murine CDR region without destroying the specificity of the antibody for its epitope.
  • the humanized antibodies of the invention are human monoclonal antibodies including at least the MBL binding CDR3 region of the deposited monoclonal antibody.
  • humanized antibodies may be produced in which some or all of the FR regions of deposited monoclonal antibodies have been replaced by homologous human FR regions.
  • the Fc portions may be replaced so as to produce IgA or IgM as well as human IgG antibodies bearing some or all of the CDRs of the deposited monoclonal antibody.
  • Such humanized antibodies will have particular clinical utility in that they will specifically recognize human MBL but will not evoke an immune response in humans against the antibody itself.
  • a murine CDR is grafted into the framework region of a human antibody to prepare the " humanized antibody.” See, e.g., L. Riechmann et al.. Nature 332, 323 (1988); M. S. Neuberger et al., Nature 314, 268 (1985) and EPA 0 239 400 (published Sep. 30, 1987).
  • antibodies e.g., anti-MBL, anti-MASP, anti-mannan-like antibodies
  • anti-MBL anti-MBL
  • anti-MASP anti-mannan-like antibodies
  • MBL monoclonal antibody specific for MBL
  • Murine monoclonal antibodies may be made by any of the methods known in the art utilizing MBL as an immunogen.
  • An example of a method for producing murine monoclonals useful according to the invention is the following: Female Balb/C mice were
  • mice 10 initially inoculated (i.p.) with 250 ul of the following mixture: 250 ⁇ l Titermax mixed with 100 ⁇ g human MBL in 250 ⁇ l PBS. The following week and for three consecutive weeks the mice were injected with 50 ⁇ g hMBL in 250 ⁇ lPBS. On the 4th week the mice were injected with 25 ⁇ g MBL in 250 ⁇ l PBS and the mice were fused 4 days later.
  • the fusion protocol is adapted from Current Protocols in Immunology.
  • splenocytes were fused 1 :1 with myelinoma fusion partner P301 from ATCC using PEG 150 at 50%) w/v.
  • the fused cells were plated at a density of 1.25xl0 6 / m. with 100 ⁇ l/well of a 96 well microtiter plate.
  • the fusion media consisted of Deficient DME high glucose, Pen/Strep (50,000 U pen, 50,000 ⁇ g strep per liter), 4 mM L-glutamine, 20% fetal bovine serum, 10% thyroid enriched media, 1% OPI, 1% NEAA, 1% HAT, and 50 ⁇ M 2-
  • Human monoclonal antibodies may be made by any of the methods known in the art, such as those disclosed in US Patent No. 5,567,610, issued to Borrebaeck et al., US Patent No. 565,354, issued to Ostberg, US Patent No. 5,571,893, issued to Baker et al, Kozber, J. Immunol. 133: 3001 (1984), Brodeur, et al., Monoclonal Antibody Production Techniques and Applications, p. 51-63 (Marcel Dekker, Inc, new York, 1987), and Boerner el al., J Immunol., 147: 86-95 (1991).
  • such antibodies may also be prepared by immunizing transgenic animals that are capable of producing human antibodies (e.g., Jakobovits et al., PNAS USA, 90: 2551 (1993), Jakobovits et al., Nature, 362: 255-258 (1993), Bruggermann et al., Year in Immuno., 7:33 (1993) and US Patent No. 5,569,825 issued to Lonberg).
  • transgenic animals e.g., Jakobovits et al., PNAS USA, 90: 2551 (1993), Jakobovits et al., Nature, 362: 255-258 (1993), Bruggermann et al., Year in Immuno., 7:33 (1993) and US Patent No. 5,569,825 issued to Lonberg).
  • PBL Peripheral Blood Lymphocytes
  • B, T and accessory (A) cells described methods such as (Danielsson, L., Moller, S. A. & Borrebaeck, C.A.K. Immunology 61, 51-55 (1987)).
  • PBL are fractionated into T and non-T cells by rosetting with 2-amino ethyl (isothiouronium bromide) - treated sheep red corpuscles, and the latter cell population is incubated on Petri dishes coated with fibronectin or autologous plasma.
  • Non-adherent cells are decanted, and adherent cells (accessory cells) are removed by lOmM EDTA.
  • the B cells are stimulated with 50 ⁇ g Staphylococcus aureus Cowan I/ml and irradiated (2000R) T cells with 10 ⁇ g PWM/ml overnight.
  • the accessory cells are stimulated with 5 IU gamma interferon/ml and 10 ⁇ m indomethacin.
  • the cell populations are cultured in supplemented RPMI 1640 which contains 10% human AB serum at a cell ratio of 2: 1 :0.4 (Ti:B:A) for a total of 6 days.
  • the antigenic dose of MBL is 1 ⁇ g/ml.
  • the culture is supplemented with recombinant IL-2 (5 U/ml) and sPWM-T (25 %> by vol.), produced by described methods such as (Danielsson, L., Moller, S.A. & Borrebaeck C.A.K. Immunology 61, 51-55 (1987)).
  • T cells (10 cells/ml) suspended in serum-free RPMI 1640 are incubated with 2.5 mM freshly prepared Leu-OMe for 40 min at room temperature. The cells are then washed 3 times in RPMI 1640 containing 2%> human antibody serum.
  • the peptide containing a MBL binding region is a functionally active antibody fragment.
  • the paratope is involved in the binding of the antibody to its epitope (see, in general, Clark, W.R. (1986) 77ze Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential
  • the pFc' and Fc regions of the antibody are effectors of the complement cascade but are not involved in antigen binding.
  • An isolated F(ab') fragment is referred to as a bivalent monoclonal fragment because of its two antigen binding sites.
  • an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region designated an Fab fragment
  • Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd (heavy chain variable region).
  • the Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.
  • the term "functionally active antibody fragment” means a fragment of an antibody molecule including a MBL binding peptide of the invention which retains the LCP associated complement inhibitory activity of an intact antibody having the same specificity such as the deposited monoclonal antibodies. Such fragments are also well known in the art and are regularly employed both in vitro and in vivo. In particular, well- known functionally active antibody fragments include but are not limited to F(ab')2, Fab, Fv and Fd fragments of antibodies. These fragments which lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)).
  • single-chain antibodies can be constructed in accordance with the methods described in U.S. Patent No. 4,946,778 to Ladner et al. Such single-chain antibodies include the variable regions of the light and heavy chains joined by a flexible linker moiety.
  • Methods for obtaining a single domain antibody (“Fd") which comprises an isolated variable heavy chain single domain also have been reported (see, for example, Ward et al., Nature 341 :644-646 (1989), disclosing a method of screening to identify an antibody heavy chain variable region (V H single domain antibody) with sufficient affinity for its target epitope to bind thereto in isolated form).
  • Functionally active antibody fragments also encompass "humanized antibody fragments.” As one skilled in the art will recognize, such fragments can be prepared by traditional enzymatic cleavage of intact humanized antibodies. If, however, intact antibodies are not susceptible to such cleavage, because of the nature of the construction involved, the noted constructions can be prepared with immunoglobulin fragments used as the starting materials: or, if recombinant techniques are used, the DNA sequences, themselves, can be tailored to encode the desired "fragment” which, when expressed, can be combined in vivo or in vitro, by chemical or biological means, to prepare the final desired intact immunoglobulin fragment. In addition to the identification of peptides from libraries etc.
  • the peptides of the invention including those containing the MBL binding CDR3 region may easily be synthesized or produced by recombinant means. Such methods are well known to those of ordinary skill in the art. Peptides can be synthesized for example, using automated peptide synthesizers which are commercially available. The peptides can be produced by recombinant techniques by incorporating the DNA expressing the peptide into an expression vector and transforming cells with the expression vector to produce the peptide.
  • the sequence of the CDR regions may be determined by methods known in the art.
  • the heavy chain variable region is a peptide which generally ranges from 100 to 150 amino acids in length.
  • the light chain variable region is a peptide which generally ranges from 80 to 130 amino acids in length.
  • the CDR sequences within the heavy and light chain variable regions which include only approximately 3-25 amino acid sequences may easily be sequenced by one of ordinary skill in the art.
  • the peptides may even be synthesized by commercial sources such as by the Scripps Protein and Nucleic Acids Core Sequencing Facility (La Jolla California).
  • variable region cDNA can be prepared by polymerase chain reaction from the deposited hybridoma RNA using degenerate or non-degenerate primers (derived from the amino acid sequence). The cDNA can be subcloned to produce sufficient quantities of double stranded DNA for sequencing by conventional sequencing reactions or equipment.
  • nucleic acid sequences of the heavy chain Fd and light chain variable domains of the deposited MBL monoclonal antibody are determined, one of ordinary skill in the art is now enabled to produce nucleic acids which encode this antibody or which encode the various antibody fragments, humanized antibodies, or peptides described above. It is contemplated that such nucleic acids will be operably joined to other nucleic acids forming a recombinant vector for cloning or for expression of the peptides of the invention.
  • the present invention includes any recombinant vector containing the coding sequences, or part thereof, whether for prokaryotic or eukaryotic transformation, transfection or gene therapy. Such vectors may be prepared using conventional molecular biology techniques, known to those with skill in the art, and would comprise DNN coding sequences for the
  • CDR3 region and additional variable sequences contributing to the specificity of the antibodies or parts thereof, as well as other non-specific peptide sequences and a suitable promoter either with (Whittle et al., Protein Eng. 1 :499, 1987 and Burton et al., Science 266:1024-1027, 1994) or without (Marasco et al., Proc. Natl. Acad. Sci. (USA) 90:7889, 1993 and Duan et al., Proc. Natl. Acad. Sci. (USA) 91 :5075-5079,1994) a signal sequence for export or secretion.
  • Such vectors may be transformed or transfected into prokaryotic (Huse et al., Science 246:1275, 1989, Ward et al., Nature 341: 644-646, 1989; Marks et al., J. Mol. Biol. 222:581, 1991 and Barbas et al., Proc. Natl. Acad. Sci. (USA) 88:7978, 991) or eukaryotic (Whittle et al., 1987 and Burton et al., 1994) cells or used for gene therapy (Marasco et al., 1993 and Duan et al., 1994) by conventional techniques, known to those with skill in the art.
  • prokaryotic Hos et al., Science 246:1275, 1989, Ward et al., Nature 341: 644-646, 1989; Marks et al., J. Mol. Biol. 222:581, 1991 and Barbas et al., Proc. Natl. Acad.
  • a "vector" may be any of a number of nucleic acids into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell.
  • Vectors are typically composed of D ⁇ A although R ⁇ A vectors are also available.
  • Vectors include, but are not limited to, plasmids and phagemids.
  • a cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired D ⁇ A sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell.
  • replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase.
  • An expression vector is one into which a desired D ⁇ A sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an R ⁇ A transcript.
  • Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector.
  • Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., ⁇ -galactosidase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques.
  • Preferred vectors are those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined.
  • the expression vectors of the present invention include regulatory sequences operably joined to a nucleotide sequence encoding one of the peptides of the invention.
  • regulatory sequences means nucleotide sequences which are necessary for or conducive to the transcription of a nucleotide sequence which encodes a desired peptide and/or which are necessary for or conducive to the translation of the resulting transcript into the desired peptide. Regulatory sequences include, but are not limited to, 5' sequences such as operators, promoters and ribosome binding sequences, and 3' sequences such as polyadenylation signals.
  • the vectors of the invention may optionally include 5' leader or signal sequences, 5' or 3' sequences encoding fusion products to aid in protein purification, and various markers which aid in the identification or selection of transformants. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art. The subsequent purification of the peptides may be accomplished by any of a variety of standard means known in the art.
  • a preferred vector for screening peptides is a recombinant DNA molecule containing a nucleotide sequence that codes for and is capable of expressing a fusion peptide containing, in the direction of amino- to carboxy-terminus, (1) a prokaryotic secretion signal domain, (2) a peptide of the invention, and, optionally, (3) a fusion protein domain.
  • the vector includes DNA regulatory sequences for expressing the fusion peptide, preferably prokaryotic regulatory sequences.
  • Such vectors can be constructed by those with skill in the art and have been described by Smith et al. (Science 228:1315-1317, 1985), Clackson et al.
  • a fusion peptide may be useful for purification of the peptides of the invention.
  • the fusion domain may. for example, include a poly-His tail which allows for purification on
  • the 5 fusion domain is a filamentous phage membrane anchor. This domain is particularly useful for screening phage display libraries of monoclonal antibodies but may be of less utility for the mass production of antibodies.
  • the filamentous phage membrane anchor is preferably a domain of the cpIII or cpVIII coat protein capable of associating with the matrix of a filamentous phage particle, thereby incorporating the fusion peptide onto the phage surface,
  • the secretion signal is a leader peptide domain of a protein that targets the protein membrane of the host cell, such as the periplasmic membrane of gram negative bacteria.
  • a preferred secretion signal for E. coli is a pelB secretion signal.
  • the ribosome binding site includes an initiation codon (AUG) and a sequence 3-9 nucleotides long located 3-1 1 nucleotides upstream from the initiation codon (Shine, et al., Nature 254:34, 1975).
  • Binding of the ribosome to mRNA and the sequence at the 3' end of the mRNA can be affected by several factors: (I) The degree of complementarity between the SD sequence and 3' end of the
  • the nucleotide sequence following the AUG, which affects ribosome binding (Taniguchi, et al., J. Mol. Biol., 118:533, 1978).
  • the 3' regulatory sequences define at least one termination (stop) codon in frame with and operably joined to the heterologous fusion peptide.
  • the vector utilized includes a prokaryotic origin of replication or replicon, i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extra-chromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
  • a prokaryotic origin of replication or replicon i.e., a DNA sequence having the ability to direct autonomous replication and maintenance of the recombinant DNA molecule extra-chromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith.
  • a prokaryotic host cell such as a bacterial host cell, transformed therewith.
  • origins of replication are well known in the art.
  • Preferred origins of replication are those that are efficient in the host organism.
  • a preferred host cell is E. coli.
  • a preferred origin of replication is Col ⁇ l found in pBR322 and a variety of other common plasmids. Also preferred is the pl5A origin of replication found on pACYC and its derivatives.
  • Col ⁇ l and pl5A replicons have been extensively utilized in molecular biology, are available on a variety of plasmids and are described by
  • those embodiments that include a prokaryotic replicon preferably also include a gene whose expression confers a selective advantage, such as drug resistance, to a bacterial host transformed therewith.
  • Typical bacterial drug resistance genes are those that confer resistance to ampicillin, tetracycline, neomycin/kanamycin or chloramphenicol.
  • Vectors typically also contain convenient restriction sites for insertion of translatable DNA sequences.
  • Exemplary vectors are the plasmids pUC18 and pUC19 and derived vectors such as pcDNAII available from Invitrogen, (San Diego, CA).
  • pcDNAII available from Invitrogen, (San Diego, CA).
  • the peptide of the invention is an antibody including both heavy chain and light chain sequences
  • these sequences may be encoded on separate vectors or, more conveniently, may be expressed by a single vector.
  • the heavy and light chain may, after translation or after secretion, form the heterodimeric structure of natural antibody molecules.
  • Such a heterodimeric antibody may or may not be stabilized by disulfide bonds between the heavy and light chains.
  • a vector for expression of heterodimeric antibodies is a recombinant DNA molecule adapted for receiving and expressing translatable first and second DNA sequences. That is, a DNA expression vector for expressing a heterodimeric antibody provides a system for independently cloning (inserting) the two translatable DNA sequences into two separate cassettes present in the vector, to form two separate cistrons for expressing the first and second peptides of a heterodimeric antibody.
  • the DNA expression vector for expressing two cistrons is referred to as a dicistronic expression vector.
  • the vector comprises a first cassette that includes upstream and downstream DNA regulatory sequences operably joined via a sequence of nucleotides adapted for directional ligation to an insert DNA.
  • the upstream translatable sequence preferably encodes the secretion signal as described above.
  • the cassette includes DNA regulatory sequences for expressing the first antibody peptide that is produced when an insert translatable DNA sequence (insert DNA) is directionally inserted into the cassette via the sequence of nucleotides adapted for directional ligation.
  • the dicistronic expression vector also contains a second cassette for expressing the second antibody peptide.
  • the second cassette includes a second translatable DNA sequence that preferably encodes a secretion signal, as described above, operably joined at its 3' terminus via a sequence of nucleotides adapted for directional ligation to a downstream DNA sequence of the vector that typically defines at least one stop codon in the reading frame of the cassette.
  • the second translatable DNA sequence is operably joined at its 5' terminus to DNA regulatory sequences forming the 5' elements.
  • the second cassette is capable, upon insertion of a translatable DNA sequence (insert DNA), of expressing the second fusion peptide comprising a secretion signal with a peptide coded by the insert
  • the peptides of the present invention may also, of course, be produced by eukaryotic cells such as CHO cells, human hybridomas, immortalized B-lymphoblastoid cells, and the like.
  • eukaryotic cells such as CHO cells, human hybridomas, immortalized B-lymphoblastoid cells, and the like.
  • a vector is constructed in which eukaryotic regulatory sequences are operably joined to the nucleotide sequences encoding the peptide.
  • the design and selection of an appropriate eukaryotic vector is within the ability and discretion of one of ordinary skill in the art.
  • the subsequent purification of the peptides may be accomplished by any of a variety of standard means known in the art.
  • the present invention provides host cells, both prokaryotic and eukaryotic, transformed or transfected with, and therefore including, the vectors of the present invention.
  • a coding sequence and regulatory sequences are said to be "operably joined" when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences.
  • two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein.
  • a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired peptide.
  • regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribing and 5' non-translating sequences involved with initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like.
  • 5' non-transcribing regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene.
  • compositions may be administered in a pharmaceutically acceptable composition.
  • pharmaceutically-acceptable carriers for monoclonal antibodies, antibody fragments, and peptides are well-known to those of ordinary skill in the art.
  • a pharmaceutically-acceptable carrier means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients, i.e., the ability of the MBL inhibitor to inhibit LCP associated complement activation.
  • Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers and other materials which are well-known in the art.
  • peptides of the invention may be formulated into preparations in solid, semi-solid, liquid or gaseous forms such as tablets, capsules, powders, granules, ointments, solutions, depositories, inhalants (e.g., aerosols) and injections, and usual ways for oral, parenteral or surgical administration.
  • the invention also embraces locally administering the compositions of the invention, including as implants.
  • compositions can be administered by injection by gradual infusion over time or by any other medically acceptable mode.
  • the administration may. for example, be intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous or transdermal.
  • Preparations for parenteral administration includes sterile aqueous or nonaqueous solutions, suspensions and emulsions.
  • nonaqueous solvents are propylene glycol, polyethylene glycol, vegetable oil such as olive oil, an injectable organic esters such as ethyloliate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers. electrolyte replenishers, (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. Those of skill in the art can readily determine the various parameters for preparing these alternative pharmaceutical compositions without resort to undue experimentation.
  • the compositions of the invention When the compositions of the invention are administered for the treatment of pulmonary disorders the compositions may be delivered for example by aerosol.
  • the compositions of the invention are administered in therapeutically effective amounts.
  • an "effective amount" of the inhibitor of the invention is a dosage which is sufficient to inhibit the increase in, maintain or even reduce the amount of undesireable LCP associated complement activation.
  • the effective amount is sufficient to produce the desired effect of inhibiting associated cellular injury until the symptoms associated with the MBL mediated disorder are ameliorated or decreased.
  • an effective amount of the peptide is an effective amount for preventing cellular injury.
  • a therapeutically effective amount may vary with the subject's age, condition, and sex, as well as the extent of the disease in the subject and can be determined by one of skill in the art. The dosage may be adjusted by the individual physician or veterinarian in the event of any complication.
  • a therapeutically effective amount typically will vary from about 0.01 mg/kg to about 500 mg/kg, were typically from about 0.1 mg/kg to about 200 mg/kg, and often from about 0.2 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or several days (depending of course of the mode of administration and the factors discussed above).
  • a preferred concentration of the inhibitor is a concentration which is equimolar to the concentration of MBL in the plasma of a subject.
  • the normal plasma concentration of MBL can be assessed clinically.
  • a normal range of MBL is l-2 ⁇ g/ml MBL/plasma.
  • an effective amount of an inhibitor is by screening the ability of the inhibitor to inhibit the LCP associated complement activation in an in vitro assay.
  • the activity of the inhibitor can be defined in terms of the ability of the inhibitor to inhibit LCP associated complement activation.
  • An exemplary assay for measuring the ability of a putative inhibitor of the invention to inhibit LCP associated complement activation is provided in the Examples and has been discussed above.
  • the exemplary assay is predictive of the ability of an inhibitor to inhibit LCP associated complement activation in vivo and, hence, can be used to select inhibitors for therapeutic applications.
  • the MBL inhibitors may be administered in a physiologically acceptable carrier.
  • physiologically-acceptable refers to a non-toxic material that is compatible with the biological systems such of a tissue or organism.
  • the physiologically acceptable carrier must be sterile for in vivo administration. The characteristics of the carrier will depend on the route of administration. The characteristics of the carrier will depend on the route of administration.
  • the invention further provides detectably labeled, immobilized and toxin conjugated forms of the peptides, antibodies and fragments thereof.
  • the antibodies may be labeled using radiolabels, fluorescent labels, enzyme labels, free radical labels, avidin-biotin labels, or bacteriophage labels, using techniques known to the art (Chard, Laboratory Techniques in Biology, "An Introduction to Radioimmunoassay and Related Techniques," North Holland Publishing Company (1978).
  • Typical fluorescent labels include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, and fluorescamine.
  • Typical chemiluminescent compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, and the oxalate esters.
  • Typical bioluminescent compounds include luciferin, and luciferase.
  • Typical enzymes include alkaline phosphatase, ⁇ -galactosidase, glucose-6-phosphate dehydrogenase, maleate dehydrogenase, glucose oxidase, and peroxidase.
  • the invention also includes methods for screening a subject for susceptibility to treatment with an MBL inhibitor. In one aspect, the method is accomplished by isolating a mammalian cell from a subject and detecting the presence of an MBL or an MBL ligand on a surface of the mammalian cell. The presence of the MBL indicates that the cell is susceptible to LCP-associated complement activiation, and that the subject is susceptible to treatment with an MBL inhibitor.
  • the mammalian cell may be isolated by any method known in the art, for instance by a biopsy.
  • Another method for accomplishing the screening assay involves the steps of contacting a mammalian cell from the subject with a labeled isolated MBL binding peptide and detecting the presence of an MBL on the surface of the mammalian cell. This assay may be performed in vitro, ex vivo, or in vivo. Many labels which can be used to observe the MBL binding peptide interacting with the mammalian cell are known in the art under each of these conditions. For instance, radioactive compounds can be used in vitro, and other biocompatible labels can be used ex vivo or in vivo. Once the subjects are identified which are susceptible to treatment with an MBL inhibitor, the subjects can then be treated according to the methods of the invention.
  • Example 1 MBL and Complement Deposition on Human Coronary Arteries.
  • MBL and associated MASPs were purified from human plasma.
  • MBL was isolated from human plasma as previously described ⁇ Tan, Chung, et al. 1996 Biochem. J. 319, 329-332 ⁇ . Briefly, human plasma was mixed with 7%> PEG3500 (w:v). The pellet was collected by centrifugation and resuspended in TBS-Ca 2+ [50 mM Tris, 150 mM NaCl, 0.05% Tween 20 and 20 mM CaCl 2 at pH 7.8]. The supernatant was applied to a mannan-Sepharose column (25 ml, Sigma).
  • the column was washed with TBS-Ca 2+ with 109mM EDTA].
  • the protein containing supernatant was calcified to 40 mM calcium and then applied to a maltose-Sepharose column (5 ml).
  • the column was washed with TBS-Ca 2+ and then eluted with TBS-Ca 2+ containing 100 mM N- acetylglucosamine.
  • Western analysis and SDS-PAGE established purity for MBL, and the absence of IgG and IgM. Purified MBL and associated MASPs were analyzed by SDS/PAGE. Western blotting was performed to rule out IgG and/or IgM contamination.
  • Anti-Human MBL Antibodies Purified human MBL was used to immunize rabbits to produce polyclonal anti-human MBL antibodies (Harlow E, et al., Antibodies: A laboratory manual. Cold Spring Harbor, NY, Cold Spring Harbor Laboratory, 1988). Adult rabbits were injected with 100 ⁇ g of MBL emulsified in complete Freund's adjuvant. Booster immunizations (100 ⁇ g of MBL in incomplete Freund's adjuvant) were started 4 wk after the priming immunization and continued at 4 wk intervals. Polyclonal IgG anti-human MBL antibody (R2.2) was purified from sera by protein G affinity chromatography. Human Coronary Artery Immunohistochemistry. Immunohistochemical analysis of
  • MI myocardial infarction
  • Infarcted myocardium was identified macroscopically at autopsy by discolor, pallor, and hyperemia. To improve macroscopic diagnosis, a slice of non-fixed myocardium was incubated in nitroblue tetrazolium solution that leaves the damaged myocardium unstained. Histopathological first signs of infarction were wavy myocardial fibers and myocytolysis followed by signs of coagulation necrosis (i.e., edema, hemorrhage,
  • tissue samples were then incubated for 30 min at 22 °C with either polyclonal rabbit anti-human C3d (Dakopatts, Glostrup, Denmark), MBL (polyclonal R2.2), IgG, IgM, transferrin, or haptoglobin antibody (all from Behringwerke AG, Germany). After washing with PBS, the specimens were then stained with an appropriate fluorescein isothiocyanate (FITC)-conjugated secondary antibody. Controls consisted of specimens incubated with either polyclonal rabbit anti-human C3d (Dakopatts, Glostrup, Denmark), MBL (polyclonal R2.2), IgG, IgM, transferrin, or haptoglobin antibody (all from Behringwerke AG, Germany). After washing with PBS, the specimens were then stained with an appropriate fluorescein isothiocyanate (FITC)-conjugated secondary antibody. Controls consisted of specimens incubated with
  • Atherosclerotic coronary arteries obtained from patients suffering from acute MI demonstrated specific MBL and C3d deposits on the endothelium, intima, and
  • MBL and C3d deposition appeared to be greatest in the lipid core and surrounding areas of this core in atherosclerotic lesions.
  • No MBL deposits were seen on normal coronary arteries, although the basement membrane sometimes appeared to stain lightly for MBL.
  • antisera against human transferrin, haptoglobin, IgG, and IgM did not stain normal human coronary arteries or atherosclerotic lesions in vessels obtained from acute MI patients.
  • no staining was observed in control experiments in which human coronaries were stained with non- immune rabbit serum or with the secondary antibody only.
  • the cells were initially seeded in either 75 cm 2 flasks or 100 mm
  • Endothelial cell purity was assessed by phase microscopic "cobblestone appearance", uptake of fluorescent acetylated low-density lipoprotein and the presence of von Willebrand factor. All experiments were conducted on HUVECs during passages 1-3.
  • HS MBL-depleted Human Serum
  • HS was treated with 2 mmol/L ethylenediamine tetraacetate (EDTA) and phenylmethanesulfonyl fluoride (PMSF) to inhibit complement activation and was applied to a mannan column equilibrated with loading buffer (1.25 mmol/L NaCl, 10 mmol/L imidazole, 20 mmol/L CaCl 2, pH 7.8).
  • loading buffer (1.25 mmol/L NaCl, 10 mmol/L imidazole, 20 mmol/L CaCl 2, pH 7.8.
  • the resultant eluent was dialyzed overnight in Hank's buffered salt solution containing Mg 2+ and Ca 2+ .
  • HUVECs were grown to confluence in 100-mm Petri dishes coated with gelatin. MBL deposition was measured by flow cytometry in normoxic HUVECs and HUVECs subjected to 24 hr of hypoxia followed by 3 hr of reoxygenation in the presence of 30% HS. After washing the cells in Ca free or sufficient buffer, the cells were fixed, scraped, and then incubated with 20 ⁇ g/ml of monoclonal anti-human MBL antibody (Biodesign, Kennebunk, ME, clone #131-1) or isotype control monoclonal antibody to porcine C5a for 1.5 hr at 4 °C.
  • monoclonal anti-human MBL antibody Biodesign, Kennebunk, ME, clone #131-1
  • isotype control monoclonal antibody to porcine C5a for 1.5 hr at 4 °C.
  • C3 and MBL specific cell surface ELISAs were developed using peroxidase-conjugated polyclonal goat anti-human C3 antibody (Cappel, West Chester, PA) and monoclonal anti-human MBL antibody (Biodesign, Kennebunk, ME, clone #131-1), respectively.
  • HUVECs were grown to confluence on 0.1% gelatinized 96-well plastic plates (Corning Costar, Cambridge, MA). The plates were then subjected to 0 (normoxia) or 24 hr of hypoxia.
  • hypoxic stress was maintained using a humidified sealed chamber (Coy Laboratory Products, Inc., Grass Lake, MI) at 37 °C gassed with 1% O 2, 5% CO 2 , balance N 2 (Collard CD, et al., "Reoxygenation of hypoxic human umbilical vein endothelial cells activates the classical complement pathway", Circulation 1997;96:326-333).
  • the cell media were aspirated and 100 ⁇ l of one of the following was added to each well: 1) 30% HS, 2) Hank's balanced salt solution, 3) 30% HS + 3, 30, or 300 mmol/L GluNAc, 4) 30% HS + 3, 30, or 300 mmol/L D-mannose, 5) 30% HS + 3, 30, 300 mmol/L L-mannose, 6) 30% MBL-depleted HS + 3F8 (0, 20, 50 ⁇ g/ml)or 7) 30% MBL-depleted HS + 0.6 ⁇ g/ml MBL.
  • the cells were then washed and incubated at 4 °C for 1.5 hr with 50 ⁇ l of peroxidase- conjugated polyclonal goat anti-human C3 antibody (1 :1000 dilution) or monoclonal anti- human MBL antibody (1 :1000 dilution).
  • the MBL ELISA plates were then washed and incubated for 1 hr at 4 °C with 50 ⁇ l of peroxidase-conjugated polyclonal goat anti-mouse IgG secondary antibody (1 :1000 dilution).
  • MBL deposition was measured by ELISA on normoxic HUVECs and HUVECs subjected to 24 hr of hypoxia followed by 3 hr of reoxygenation in the presence of 30%) HS or 30%> HS treated with 3, 30, or 300 mmol/L of N-acetyl-D-glucosamine (GluNAc) to competitively inhibit MBL deposition.
  • MBL deposition on hypoxic HUVECs reoxygenated in 30%> HS was significantly greater (approximately 3-fold increase; p ⁇ 0.05) than on normoxic HUVECs or HUVECs reoxygenated in HS treated with GluNAc ( Figure 3).
  • hypoxic/reoxygenated HUVECs assuming 2xl0 5
  • hypoxia/reoxygenation increased endothelial MBL deposition.
  • Example 3 Deposition of iC3b Following Competitive Inhibition of MBL.
  • HUVEC cell culture and quantitation of iC3b deposition by ELISA were performed as outlined in Example 2. Results. HUVECs were subjected to 0 or 24 hr of hypoxia followed by 3 hr of reoxygenation in the presence of 30% HS or 30%> HS treated with 3, 30, or 300 mmol/L GluNAc, D-mannose or L-mannose in order to inhibit MBL deposition, LCP activation and iC3b deposition.
  • Example 4 Deposition of iC3b Following MBL Depletion and Reconstitution.
  • HUVEC cell culture and quantitation of iC3b deposition by ELISA were carried out as in Example 2.
  • HUVECs were subjected to 0 or 24 hr of hypoxia followed by 3 hr of reoxygenation in the presence of 30%> HS, 30% MBL-depleted HS or 30% MBL-depleted
  • iC3b deposition on hypoxic HUVECs reoxygenated in MBL-depleted HS was significantly less (p ⁇ 0.05) than on hypoxic HUVECs reoxygenated in HS.
  • MBL was added back to the MBL-depleted HS
  • hypoxia and 3 hr of reoxygenation was restored.
  • Example 5 Complement hemolytic assay (CH 50 ) of MBL-depleted HS.
  • Example 6 Western blot analysis of C3 activation following hypoxia/reoxygenation using purified C2, C3, C4, and MBL.
  • HUVECs were grown to confluence in 96 well plates and then subjected to normoxia or hypoxia (24 hr). The cells were then washed with GVB+ and reoxygenated for 3 hr in the presence of 50 ⁇ l of the following complement cocktail: MBL (1.2 ⁇ g/ml), C2 (8 ⁇ g/ml), C3 (400 ⁇ g/ml), and C4 (200 ⁇ g/ml) (C2, C3, and C4 were purchased from Advanced Research Technologies; San Diego, CA). These complement concentrations were representative of the concentrations normally present in 30% HS. Following reoxygenation, the supernatants were collected and the protein concentration determined (BioRad, Hercules, CA).
  • Example 7 Microphysiometer evaluation of HUVEC receptor-ligand activation.
  • HUVEC extracellular acidification rate were evaluated by use of a Cytosensor microphysiometer (Molecular Devices, Sunnyvale, CA). HUVECs were grown to 75% confluence on gelatin-coated (1%>) transwell capsules and subjected to 24 hr of hypoxia followed by 3 hr of reoxygenation. Following 30 min of equilibration in modified RPMI containing 1 mmol/L phosphate buffer (Molecular Devices,
  • HUVECs were perfused with 300-1500 ng/ml of purified MBL (dialyzed in the modified RPMI) for 30 sec before the first rate measurement and perfusion was maintained for 40 min.
  • MBL purified MBL
  • histamine histamine
  • Example 8 Preparation and Characterization of Monoclonal Antibodies to Human MBL.
  • mice Female Balb/C mice were initially inoculated (i.p.) with 250 ul of the following mixture: 250 ⁇ l Titermax mixed with 100 ⁇ g human MBL in 250 ⁇ l PBS. The following week and for three consecutive weeks the mice were injected with 50 ⁇ g hMBL in 250 ⁇ lPBS. On the 4th week the mice were injected with 25 ⁇ g MBL in 250 ⁇ l PBS and the mice were fused 4 days later. The fusion protocol was adapted from Current Protocols in Immunology. The splenocytes were fused 1 :1 with myelinoma fusion partner P301 from ATCC using PEG 150 at 50% w/v. The fused cells were plated at a density of 1.25x10 6 / m.
  • the fusion media consisted of Deficient DME high glucose, Pen Strep (50,000 U pen, 50,000 ⁇ g strep per liter), 4 mM L-glutamine, 20% fetal bovine serum, 10% thyroid enriched media, 1% OPI, 1% NEAA, 1% HAT, and
  • the functional screen for inhibition of MBL function by anti-human MBL antibodies was adapted from the literature ⁇ Super, Levinsky, et al., The level of mannan- binding protein regulates the binding of complement-derived opsonins to mannanand zymosan at low serum concentrations, Clin. Exp. Immunol. 1990; 79:144-150 ⁇ . Briefly, 100 ⁇ l of meannan (0.5 mg/ml in sodium carbonate/bicarbonate buffer, pH 9.6) was added to RIA/EIA plates at 4C overnight. The plates were then washed 3 times in PBS/0.5%> Tween pH 7.3, once in PBS and finally in veronal-buffered saline.
  • meannan 0.5 mg/ml in sodium carbonate/bicarbonate buffer, pH 9.6
  • Human serum is diluted to 4%> in VBS containing 5 mM Ca 2+ and Mg 2+ . Diluted sera and tissue culture supernatant or purified antibody (various concentrations) are then 1 :1 to a mannan-coated well to yield a final volume or 100 ⁇ l at a concentration of 2%o human sera. The plate is then incubated at 37C for 30 min. Positive and negative controls consist of human sera without and with 100 mM N-acetlglucosamine (GluNac). The plates are then washed four times in PBS/Tween. The plates are then incubated with an anti-human C3 polyclonal antibody coupled with HRP
  • GluNAc N- acetylglucosamine
  • 2A9 and hMBL 1.2 inhibited C3 deposition with and EC50 of approximately 30 and 50 nM, respectively.
  • An isotype control antibody that recognizes MBL by solid phase ELISA did not inhibit MBL dependent C3 deposition.
  • these antibodies are approximately 10 5 - 10 6 times more potent than GluNAc.
  • the data represent 3 separate experiments with at least 4 observations per experiment. HUBECs were hypoxic for 24 hours and then reoxygenated in 30%) human sera.
  • iC3b deposition was then normalized to normoxic cells. An approximate 190%) increase in iC3b deposition on hypoxic cells was observed following reoxygenation (Figure 8). 3F8 attenuated iC3b deposition on hypoxic/reoxygenated HUVECs in a dose- dependent manner.
  • Example 9 Complement activation and deposition following HUVEC oxidative stress. To characterize further the functional properties of these novel mAbs and to demonstrate specifically the role of MBL in complement activation following oxidative stress of human endothelial cells, we assessed MBL and C3 deposition on hypoxic human endothelial cells following reoxygenation in human sera.
  • hypoxic HUVECs were reoxygenated in human sera treated with PBS (vehicle), 3F8, hMBLi.2, 2A9, or 1C10 (50 ⁇ g/ml final concentration).
  • Cell membrane bound proteins were resolved by SDS-PAGE under reduced conditions, transferred to membranes, and analyzed for human C3dg (i.e., part of the ⁇ -chain of iC3b).
  • the ⁇ - and ⁇ -chain of iC3b were the only C3 stainable bands present on the cellular membranes.
  • C3 and MBL staining were observed under normoxic conditions, confirming our finding of low level C3 deposition under normoxic conditions, confirming our finding of low level C3 deposition under normoxic conditions.
  • C3 and MBL staining on hypoxic/reoxygenated HUVECs was significantly greater than normoxic HUVECs.
  • Clone 1C10 failed to inhibit C3 or MBL deposition following oxidative stress.
  • C3 and MBL staining was significantly decreased on hypoxic/reoxygenated HUVECs treated with mAb 3F8 (5 ⁇ g/ml) to levels below those observed under normoxic conditions (similar results were observed with mAbs hMBL1.2 or 2A9).
  • VCAM-1 ELISA VCAM-1 ELISA. Briefly, HUVECs were grown to confluence on 0.1% gelatinized 96-well plastic plates and then subjected to 0 or 12 hr of hypoxia. The cell media was then aspirated and HBSS, 30% HS or 30% HS treated with 3F8 (5 ⁇ g/ml) was added to each well. The cells were then reoxygenated for 3 hr at 37°C in 95% air and 5% C02. The cells were washed, fixed, washed again, and incubated at 4°C for 1.5 hr with the anti-human VCAM-1 mAb (clone 6G10 obtained from the Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, I A).

Abstract

L'invention concerne des techniques et des produits permettant de réguler l'activation du complément associée à la voie du complément à lectine (LCP). Ces techniques comprennent des techniques in vitro and in vivo qui permettent d'inhiber l'activation du complément associée à la LCP. Ces techniques consistent à mettre en contact une cellule de mammifère possédant un ligand de la lectine mannose-spécifique (MBL) exposé en surface avec une quantité efficace d'un inhibiteur de la lectine mannose-spécifique, afin d'inhiber l'activation du complément associée à la LCP. L'inhibiteur de la lectine mannose-spécifique peut être administré à un sujet, afin d'empêcher une lésion cellulaire induite par l'activation du complément associée à la LCP. Les produits de l'invention sont des compositions d'un inhibiteur de la lectine mannose-spécifique. L'inhibiteur de la lectine mannose-spécifique est un peptide isolé se fixant à ladite lectine mannose spécifique, qui se lie sélectivement à un épitope de la lectine mannose-spécifique humaine, et qui inhibe l'activation du complément associée à la LCP. Les produits comprennent également des lignées cellulaires hybridomes et des compositions pharmaceutiques.
PCT/US1999/029919 1998-12-15 1999-12-15 Techniques et produits permettant de reguler l'activation du complement associee a la voie du complement a lectine WO2000035483A1 (fr)

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CA002347734A CA2347734A1 (fr) 1998-12-15 1999-12-15 Techniques et produits permettant de reguler l'activation du complement associee a la voie du complement a lectine
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