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WO1992007003A1 - MACROGLOBULINES-α2 MODIFIEES ET LEUR UTILISATION COMME INHIBITEURS POUR DES CYTOKINES ET DES FACTEURS DE CROISSANCE - Google Patents

MACROGLOBULINES-α2 MODIFIEES ET LEUR UTILISATION COMME INHIBITEURS POUR DES CYTOKINES ET DES FACTEURS DE CROISSANCE Download PDF

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Publication number
WO1992007003A1
WO1992007003A1 PCT/CA1990/000356 CA9000356W WO9207003A1 WO 1992007003 A1 WO1992007003 A1 WO 1992007003A1 CA 9000356 W CA9000356 W CA 9000356W WO 9207003 A1 WO9207003 A1 WO 9207003A1
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WIPO (PCT)
Prior art keywords
tgf
methylamine
tnf
cytokine
plasmin
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PCT/CA1990/000356
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English (en)
Inventor
Anthony M. Hayes
Steven L. Gonias
Jonathan Lamarre
Gordon K. Wollenberg
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University Of Guelph
University Of Virginia Alumni Patent Foundation
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Application filed by University Of Guelph, University Of Virginia Alumni Patent Foundation filed Critical University Of Guelph
Priority to PCT/CA1990/000356 priority Critical patent/WO1992007003A1/fr
Priority to CA 2070682 priority patent/CA2070682A1/fr
Publication of WO1992007003A1 publication Critical patent/WO1992007003A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4717—Plasma globulins, lactoglobulin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to the production and use of chemically modified ⁇ 2 -macroglobulins in the treatment of diseases caused by either an excess or an inappropriate growth factor or cytokine activity.
  • cytokines are naturally occurring polypeptides secreted in very small amounts by living organisms. A lack or an excess of those polypeptides may considerably affect, either positively or negatively, the given organisms. Cytokines are small polypeptides that initiate cellular responses by interacting with specific cell-surface receptors. In vi vo, important cellular activities such as proliferation and differentiated function require an intricate cytokine "language" in which an appropriate balance of many different cytokines and other influences function simultaneously. Therefore, mechanisms which influence the availability and activity of cytokines to specific cells must play a pivotal role in determining cellular physiology.
  • cytokine activity cannot be regulated or localized solely by differences in receptor expression. Instead, the regulation of cytokine activity is apparently accomplished in part by complex collections of binding molecules and inhibitors analogous to the well-defined systems of coagulation and complement.
  • physiologic anti- inflammatory mechanisms which reduce or eliminate cytokine signals once the homeostatic "objective" has been achieved.
  • cytokines including TGF- ⁇ 1, Il- ⁇ 1, EGF, FGFs and CSF-1 are transferred to the cellular membrane or secreted from the cell as inactive precursor molecules. Activation of precursor cytokines is thenaccomplished through proteolysis, pH extremes and possibly by enzymes which remove carbohydrates (glycosidases). Little is known about these enzymatic reactions, however, enzyme activators and inhibitors that limit the activity of proteinases and glycosidases in solution or at the cell surface are almost certainly involved.
  • the serpin family of proteinase inhibitors includes molecules such as protease nexins and plasminogen activator inhibitor- (PAI-1); these inhibitors have frequently been implicated in cell growth. Whether these modifiers of enzyme activity regulate cytokine activation remains to be determined. However, the activity of plasminogen activators and plasminogen activator inhibitors is itself regulated by TGF- ⁇ and FGF suggesting a complex feedback process.
  • cytokine-receptor interactions clearly represents an important level at which cytokine activity is regulated.
  • receptor affinity (K D ) and receptor binding capacity (B max ) for specific cytokines can be modified by the same cytokine (homologous modulation), by different cytokines (heterologous modulation) or by other signalling molecules.
  • these types of regulatory mechanisms include the IL-1-induced increase in IL-2 receptor expression on T lymphocytes, and the modulation of EGF receptors on hepatocytes by glucocorticoids, insulin and norepinephrine.
  • IL-1RA naturally-occurring receptor antagonist for IL-1
  • IL-1RA a naturally-occurring receptor antagonist for IL-1
  • the protein has. recently been cloned from human monocytes and expressed in E. coli .
  • the roles of this molecule in vivo have yet to be determined, however, IL-1RA or other cytokine receptor antagonists should provide a novel approach to the study of cytokine activity in complicated systems involving multiple cytokines.
  • Examples include: betaglycan, decorin and high molecular weight proteins from rat glomeruli, all of which bind TG1 ⁇ s , heparin and glycosamine glycans which bind FGFs. Mechanisms for the direct or indirect removal or catabolism of most active cytokines have yet to be identified. Recently, the high-affinity hepatocyte receptor for EGF has been implicated in the direct clearance of EGF from plasma by EGF-receptor-mediated endocytosis (RME).
  • RME EGF-receptor-mediated endocytosis
  • TGF- ⁇ s The transforming growth factors- ⁇ (TGF- ⁇ s) are a family of potent, polypeptide growth factors secreted as inactive precursors by many cells, particularly stromal cells and platelets. Several forms of TGF- ⁇ have been characterized. TGF- ⁇ 1 and TGF- ⁇ 2 are 70% homologous by amino acid sequence and have similar biological effects on most cells, including normal and neoplastic hepatocytes. The precursor forms are activated by pH extremes and some proteinases , conditions likely to occur in the extracellular milieu of tissue necrosis.
  • TGF- ⁇ 1 Transforming growth factor- ⁇ 1
  • TGF- ⁇ 1 is a 25- kDa disulfide linked homodimer. It is secreted as an inactive high-molecular weight precursor with an N- terminal pro-region.
  • the activity of TGF- ⁇ 1 may be regulated in part by molecules that influence the rate of precursor activation. Plasmin and cathepsin-D both react with the TGF- ⁇ 1 precursor yielding the active 25- kDa dimer. The dimers then bind to cell surface receptors or to certain soluble proteins including betaglycan, fibronectin and ⁇ 2 -macroglobulin ( ⁇ 2 M) .
  • These TGF- ⁇ 1 carrier molecules may transfer TGF- ⁇ 1 from one tissue to another or direct the activity to a specific cell type.
  • TGF- ⁇ binds to cell surface receptors and elicit a range of responses depending on the nature and differentiated state of the target cell and its environmental influences. Constitutive receptors with different affinities for TGF- ⁇ 1 and TGF- ⁇ 2 have been recognized, implying that separate biological functions may exist. Recent evidence that TGF- ⁇ 1 but not TGF- ⁇ 2 inhibits the proliferation of hematopoietic progenitor cells and bovine aortic endothelial cells appears to support this hypothesis.
  • TGF- ⁇ 1 The active form of TGF- ⁇ 1 is a multifunctional regulator of cell growth demonstrating both proliferative and antiproliferative activities. TGF- ⁇ 1 modulates the immune response, regulates cellular secretion of extracellular matrix proteins and affects cellular production of proteinases and proteina ⁇ e inhibitors. All TGF- ⁇ 1 activities result from interaction with specific cell surface receptors, the most important of which is probably a 53-kDa glycoprotein. Since numerous normal and neoplastic cells secrete TGF- ⁇ 1 and express the TGF- ⁇ 1 receptor, it is not clear whether physiologic mechanisms exist to direct TGF- ⁇ 1 to specific target cells.
  • cytokines may mediate harmful tissue responses.
  • experimental glomerulonephritis is associated with excess TGF- ⁇ activity; TNF- ⁇ has been implicated in the development of experimental pulmonary fibrosis and endotoxic shock is associated with elevated activity of TNF- ⁇ and IL-1.
  • TNF- ⁇ has been implicated in the development of experimental pulmonary fibrosis
  • endotoxic shock is associated with elevated activity of TNF- ⁇ and IL-1.
  • These diseases may be controlled by strategies that counteract the production of the damaging cytokines, or accelerate cytokine inactivation and clearance from the affected sites.
  • disorders or deficiencies in any of the normal containment mechanisms might be partly responsible for inappropriate cytokine effects in some disorders. Therefore, molecules that bind cytokines including natural carriers and neutralizing anti-cytokine antibodies are potentially useful in the therapy of cytokine-dependent inflammatory and proliferative disorders.
  • TGF- ⁇ 1 and - ⁇ 2 transforming growth factor- ⁇ 1 and - ⁇ 2
  • bFGF basic fibroblast growth factor
  • PDGF platelet-derived growth factor
  • NGF nerve growth factor
  • IL-1 ⁇ interleukin- 1 ⁇
  • IL-6 interleukin-6
  • cytokines When bound to ⁇ 2 M, different cytokines behave non-uniformly with respect to specific cellular receptor-interactions and biological activity.
  • PDGF, NGF and IL-6 remain biologically active at least partially when complexed with ⁇ 2 M in various cell culture assays.
  • the TG ⁇ - ⁇ 1 / ⁇ 2 M complex from human serum is a biologically latent form of the growth factor while IL-1 ⁇ and bFGF demonstrate markedly decreased activity in the presence of excess ⁇ 2 M.
  • O'Connor-McCourt and Wakefield in (1987), J. Biol. Chem.
  • TGF- ⁇ 2 transforming growth factor- ⁇ 2
  • ⁇ 2 M might modify the biological activity of the TGF- ⁇ s differently under specific circumstances. Furthermore, the inhibition of the biological activity of some cytokines after complex formation with ⁇ 2 M strongly suggests that ⁇ 2 M might be an important modulator of cytokine activity in vi vo.
  • Native ⁇ 2 M undergoes a specific conformational change following reaction with proteinases or primary amines. This conformational change increases its mobility from a "slow” to a “fast” migrating form during non-denaturing polyacrylamide gel electrophoresis.
  • primary amines such as methylamine react with ⁇ 2 M thiol ester bonds causing a conformational change similar to that induced by proteinase.
  • the proteinase is trapped and a nondissociable complex is formed.
  • the change in ⁇ 2 M structure generates a site that binds to specific cell surface receptors on hepatocytes, macrophages and fibroblasts.
  • the growth factor or cytokine binding activity of ⁇ 2 M may be increased or decreased coincident with this conformational change, depending on the growth fa ⁇ tor/cytokine involved and on the type of conformational change induced.
  • the binding of 125 I-TGF- ⁇ 1 and 125 I-bFGF to ⁇ 2 M is increased relative to native ⁇ 2 M by reaction with methylamine but is either reduced (TGF- ⁇ 1) or unchanged (bFGF) by reaction with trypsin.
  • reaction of ⁇ 2 M with methylamine reduces its tendency to form a complex with PDGF.
  • IL-l ⁇ apparently does not bind to the native form of ⁇ 2 M but does bind to ⁇ 2 M-trypsin or ⁇ 2 M-methylamine.
  • ⁇ 2 M Relative to native ⁇ 2 M, some forms of "fast" ⁇ 2 M bind greater amounts of TGF- ⁇ 1, TGF- ⁇ 2, bFGF and IL-1 ⁇ suggesting that proteinase-modified forms of ⁇ 2 M may scavenge some cytokines more efficiently.
  • "fast" forms of ⁇ 2 M bind to specific cell surface receptors on hepatocytes, macrophages and fibroblasts and are rapidly endocytosed and degraded. Conformationally transformed ⁇ 2 M is rapidly cleared from the systemic circulation primarily by the liver, whereas native ⁇ 2 M is cleared at a much slower rate.
  • TGF- ⁇ 1 In the plasma, essentially all TGF- ⁇ 1 is associated with ⁇ 2 M. However, TGF- ⁇ 1 bound to ⁇ 2 M- plasmin or ⁇ 2 M-methylamine is removed from the systemic circulation by the same receptors which clear conformationally transformed ⁇ 2 M.
  • ⁇ ji receptors have been studied extensively using in vi vo model systems.
  • receptor binding and endocytosis causes the rapid plasma clearance of ⁇ 2 M-proteinase complexes and ⁇ 2 M-methylamine (t 1 ⁇ 2 3-5 min).
  • the major organ responsible for ⁇ 2 M-proteinase clearance is the liver; the primary cell type is the hepatocyte.
  • Native ⁇ 2 M has a relatively long circulating half-life due to the inability to interact with ⁇ 2 M receptors.
  • the clearance of ⁇ 2 M in mice is species independent; ⁇ -macroglobulins from multiple species compete for ⁇ 2 M receptor sites in vi vo.
  • ⁇ 2 M may represent an effective mechanism for removing both proteinase-reacted forms of ⁇ 2 M and growth factor- cytokine/ ⁇ 2 M complexes from the systemic circulation.
  • ⁇ 2 M does not, at present, have the ability to regulate the activity of all growth factors and cytokines.
  • Mechanisms for the regulation of cytokine activity in vi vo are obviously very complex, involving the simultaneous modulation of cytokine mRNA expression, secretion, activation, and receptor expression in addition to the cytokine scavenging systems discussed.
  • the complexity of these control mechansism suggests that modification of any single mechanism to prevent or treat cytokine-mediated disease may meet with limited success.
  • the recent use of specific anti-cytokine antibodies to identify the causal associations between cytokines and certain disease states suggests that, under some circumstances, the use of antagonists to active cytokines may have therapeutic benefits.
  • ⁇ 2 M The binding and clearance of multiple cytokines in association with ⁇ 2 M supports a potential therapeutic role for ⁇ 2 M in the treatment of some of cytokine- mediated disease states.
  • ⁇ 2 Ms rather than neutralizing anti-cytokine antibodies to ameliorate the adverse effects of excess TGF- ⁇ ,. TNF and IL-1.
  • Anti- cytokine antibodies are monospecific but pathologic states in which they have been experimentally evaluated involve many potentially injurious cytokines interacting in a "cascade" fashion. Although combinations of multiple neutralizing antibodies specific for IL-1 ⁇ , IL- 1 ⁇ , TNF- ⁇ , TNF- ⁇ and other mediators might be used, the efficacy of such polyvalent antibody therapy has not yet been examined.
  • the multi-specific cytokine-binding properties of exogenously administered activated ⁇ 2 Ms might be more effective in counteracting adverse processes in some inflammatory diseased, providing there is no underlying problem with the ⁇ 2 M- RME pathway for their clearance.
  • RME of activated ⁇ 2 Ms likely represents one of the "normal" physiologic containment systems for cytokines, administration of activated ⁇ 2 Ms should allow liminted dissociation of cytokines by heparin and similar molecules where specifically necessary for tissue repair and host defence mechanisms.
  • ⁇ ji is present in high concentrations as the native molecule in all individuals, and can be removed, appropriately activated, and reinfused as an autologous cytokine scavenger, thereby reducing the risks inherent in the use of heterologous proteins and blood products.
  • cytokine binding proteins such as ⁇ 2 M have not been evaluated. Because of the complexity of multiple, overlapping cytokine activities in normal physiology and disease it is difficult to speculate on specific disorders which may arise secondary to the removal of important cytokines und «r some circumstances. Clearly, the high concentration of native ⁇ 2 M in normal plasma suggests that toxic effects should be limited to activated forms of ⁇ 2 M with high cytokine-binding activity. The rapid receptor-mediated clearance of cytokine-binding forms of ⁇ 2 M in normal individuals should serve to limit the detrimental effects of such treatments.
  • ⁇ 2 M is a potent extracellular binder of various cytokines and growth factors, sometimes inhibiting the biological activity of some important regulatory factors in vi tro. This extracellular function of ⁇ 2 M is thought to be an important physiological mechanism restricting the activity of various regulatory factors in vi vo.
  • a method for preventing the interaction of growth factors or cytokines with their specific cell receptors in mammals comprises administering to the mammal a therapeutic amount of a chemically modified a 2 -macroglobulin having the ability to form a ⁇ 2 -macroglobulin-growth factor or-cytokine complex with a given growth factor or cytokine.
  • the complex preferentially undergoes receptor-mediated modification, preferably endocytosis by ⁇ 2 -macroglobulin receptors, thereby reducing the availability of the active growth factor or cytokine to its specific cell surface receptors.
  • the present invention also relates to a method for the treatment of diseases caused by excess or inappropriate growth factor or cytokine activity.
  • the method comprises administering to a patient a therapeutic amount of the above-noted modified ⁇ 2 - macroglobulin.
  • the chemically modified ⁇ 2 - macroglobulin is selected from the group consisting of ⁇ 2 M-thrombin, ⁇ 2 M-plasmin, ⁇ 2 M-methylamine, ⁇ 2 M- methylamine cis -DDP and ⁇ 2 M-methylamine papain .
  • the method of the present invention is particularly useful for reducing the activity of TGF- ⁇ 1, TGF- ⁇ 2, TNF- ⁇ , IL-1 ⁇ and b-FGF in vi vo. It has been demonstrated that some forms of modified ⁇ 2 Ms may play an important role in growth regulation by triggering the formation of "scavenger" molecules that bind growth factors or cytokines with high affinity. As a result of this high affinity binding, the biological activity of these growth factors or cytokines are considerably reduced by preventing their association with cell surface receptors on target cells.
  • TGF- ⁇ 1 and TGF- ⁇ 2 these growth factors are supplied at the site of injury following tissue necrosis through plasma leakage and platelet activation.
  • Free, active TGF- ⁇ is a potent chemoattractant for monocytes and a mitogen for stromal cells.
  • Native ⁇ 2 M and ⁇ 2 M-thrombin may act as carriers of TGF- ⁇ , available locally by heparin dissociation but not systemically because of hepatic endocytosis of ⁇ 2 M- proteinase complexes.
  • ⁇ 2 M-plasmin generated during fibrinolysis, counteracts the activity of TGF- ⁇ by acting as a more efficient "scavenger" for the TGF- ⁇ , increasing the overall transport of TGF- ⁇ (complexed with ⁇ 2 M) to the liver for clearance.
  • Variations in the relative concentrations of ⁇ 2 M with different binding capacities for the TGF- ⁇ may be particularly important in the environment of post-necrotic liver repair where the factors involved in these studies are produced and where TGF- ⁇ has been implicated in normal regenerative processes and hepatic fibrosis.
  • TNF- ⁇ the binding of TNF- ⁇ by specific proteinase-reacted forms of ⁇ 2 M, especially ⁇ 2 M- plasmin, may be used to modify the biological action of TNF- ⁇ .
  • ⁇ 2 M/plasmin complexes are formed in plasma in vi vo under some conditions in which plasminogen is converted to plasmin by urokinase.
  • the rapid receptor- mediated removal and metabolism of reacted forms of ⁇ 2 M by hepatocytes, macrophages and fibroblasts suggest that removal of ⁇ 2 M/TNF- ⁇ complexes might occur by a similar mechanism. These clearance mechanisms would facilitate the removal of ⁇ 2 M complexes locally at the site of inflammation by macrophages and fibroblasts.
  • TNF- ⁇ resembles other cytokines in its multipotent effects in different cell types, these local containment mechanisms are important in preventing the dissemination of TNF- ⁇ and other factors away from their intended site of paracrine action.
  • TNF- ⁇ complexed to some reacted forms of ⁇ 2 M are possible mechanisms to inhibit the development of endotoxic shock or disease-induced cachexia resulting from elevated levels of circulating TNF- ⁇ .
  • Figure 1 represents the non-denaturing gel electrophoresis and autoradiography of native ⁇ 2 M, ⁇ 2 M- trypsin and ⁇ 2 M-methylamine/ 125 I-TGF- ⁇ 2 complexes.
  • Figure 2a represents the cross competition assays between 125 I-TGF- ⁇ 1 and unlabeled TGF- ⁇ 2 or 125 l-
  • TGF- ⁇ 2 and unlabeled TGF- ⁇ 1 for binding to ⁇ 2 M- methylamine TGF- ⁇ 2 and unlabeled TGF- ⁇ 1 for binding to ⁇ 2 M- methylamine.
  • Figure 2b represents the results of the direct competition assay between 125 I-TGF- ⁇ 2 and unlabeled TGF- ⁇ 2 for binding to ⁇ 2 M-methylamine.
  • I-TGF- ⁇ 1 to native ⁇ 2 M, ⁇ 2 M-plasrain, ⁇ 2 M-thrombin and ⁇ 2 M-trypsin.
  • Figure 4 represents the competition between native ⁇ 2 M and ⁇ 2 M-trypsin for 125 I-TGF- ⁇ binding.
  • Figure 5 represents the competition between ⁇ 2 M- plasmin for 125 I-TGF- ⁇ s in native ⁇ 2 M/ 125 I-TGF- ⁇ complexes.
  • Figure 6 represents the dissociating effect of heparin on native ⁇ 2 M and ⁇ 2 M-proteinase/TGF- ⁇ complexes.
  • Figure 7 represents the plasma clearance of native ⁇ 2 M as well as various types of modified ⁇ 2 Ms-
  • Figure 8 represents the plasma clearance of free 1 25 I-TGF- ⁇ 1 and 125 I-TGF- ⁇ 1 bound to various forms of ⁇ 2 M.
  • Figure 9 represents clearance competition studies between ⁇ 2 M-methylamine- 125 I-TGF- ⁇ 1 and ⁇ 2 M- methylaraine or ⁇ 2 M-trypsin.
  • Figure 10 represents delayed clearance competition studies between ⁇ 2 M-methylamine- 125 I-TGF- ⁇ 1 and ⁇ 2 M-methylamine.
  • Figure 11 represents 125 I-TNF- ⁇ complex formation with ⁇ 2 M-methylamine , ⁇ 2 M-plasmin ( human ) and ⁇ 2 M-plasmin ( bovine ) following 5% native polyacrylamide gel electrophoresis .
  • Figure 12 represents the binding of 125 I-TNF- ⁇ to ⁇ 2 M-plasmin (human) and ⁇ 2 M-methylamine after gel filtration FPLC.
  • Figure 13 represents the preferential complex formation of 125 I-TNF- ⁇ with ⁇ 2 M-human plasmin and ⁇ 2 M- methylamine in human serum and plasma.
  • Figure 14 represents the clearance time of TNF- ⁇ and TNF- ⁇ / ⁇ 2 M-plasmin in CD-1 mice.
  • Figure 15 represents the inhibition of clearance of TNF- ⁇ / ⁇ 2 M-plasmin by ⁇ 2 M-trypsin injected at 10 minutes after TNF- ⁇ / ⁇ 2 M-plasmin and TNF- ⁇ / ⁇ 2 M-plasmin in
  • Figure 16 represents the effect of co-injection of ⁇ 2 M-plasmin on TNF- ⁇ clearance.
  • Figure 17 represents the inhibition of clearance of TNF- ⁇ / ⁇ 2 M-plasmin after reaction with cisplat and during ⁇ 2 M-trypsin competition in CD-1 mice.
  • the present invention relates to the use of chemically modified ⁇ 2 Ms to reduce the availability of active growth factors or cytokines to their specific cell surface receptors.
  • Various chemically modified forms of ⁇ 2 M have been found to play an important role in the regulation of various cytokines and growth factors in vi vo. This regulation appears to involve the conversion of native ⁇ 2 M to "scavenger" forms with higher affinity that bind the targeted cytokine or growth factor.
  • cytokines and growth factors bind preferentially to some forms of ⁇ 2 M that have previously undergone some type of conformational change.
  • ⁇ 2 M may be viewed as an "intercellular shuttle", mediating the clearance of TGF- ⁇ from the circulation or directing the cytokine to specific cell types that express ⁇ 2 M receptors (macrophages and hepatocytes).
  • the present invention provides for the first time a process through which certain forms of modified ⁇ 2 M may act as "scavenger” molecules for limiting the in vi vo activity of various cytokines or growth factors.
  • TGF- ⁇ 1, TGF- ⁇ 2 and TNF- ⁇ will hereafter be illustrated, although it is to be understood that the present invention is not limited to those cytokines or growth factors.
  • the present invention deals with the use of modified ⁇ 2 - macroglobulins as "scavenger" molecules that will play an important role in a major extracellular, physiologic binding and clearance mechanism for various growth factors and cytokines. This is accomplished through known ⁇ 2 -macroglobulins but more importantly, through novel specific modified forms of ⁇ 2 M which retain high capacity binding properties for growth factors and cytokines while being cleared by ⁇ 2 M scavenging receptors.
  • therapeutic administration of high capacity scavenging forms of ⁇ 2 M may be used to facilitate rapid clearance of ⁇ 2 M/growth factor or ⁇ 2 M/cytokine complexes, thus preventing interaction with cellular growth factor/cytokine receptors and subsequent undesirable biological effects.
  • examples include: systemic administration to counteract TNF- ⁇ activity in endotoxic shock or TGF- ⁇ activity in hepatic fibrosis intrauterine administration in cases of infertility due to chronic endometrial inflammation and intra-articular administration in many joint diseases.
  • TNF- ⁇ and TGF- ⁇ are related to the binding of TNF- ⁇ and TGF- ⁇ to various forms of ⁇ M.
  • other cytokines and growth factors such as 11-1 ⁇ , 11-6, bFGF, PDGF and NGF have also been shown to bind to ⁇ M and may therefore be considered as similar candidates for therapeutic use.
  • Papain, methylamine HCl, and p-nitrophenyl-p'- guanadinobenzoate-HCL (NPGB) and trypsin were purchased from Sigma Chemical Co., St-Louis, MO. Coomassie blue stain was purchased from Bio-Rad Laboratories, Richmond, CA. Cis-dichlorodiamineplatinum (II) (Cis-DDP) was purchased from Aldrich Chemical Co. (Milwaukee, WI.). Proteins
  • Native ⁇ 2 M was purified from fresh human plasma as described by Imber et al. in (1981), J. Biol. Chem. 256, 8134-8139. Trypsin (from bovine pancreas) and heparin (from porcine intestine) were purchased from Sigma. Purified human or bovine plasmin (2U/rog) and thrombin (30U/mg) were purchased from Boehringer- Manheim, Dorval, Quebec. In some experiments, highly purified human plasminogen was prepared as described by Deutsch and Mertz in (1970) Science (Washington D.C.)
  • TGF- ⁇ 1, TGF- ⁇ 2, 125 I-TGF- ⁇ 1, and 125 I-TGF- ⁇ 2 were purchased from R&D Systems, Minneapolis, MN. The specific activity was between 30 and 100 ⁇ Ci/ ⁇ g. The concentration of active enzyme in trypsin preparations was determined by the method of Chase and Shaw (supra).
  • ⁇ 2 M-methylamine was reacted with 1.67 mM cis-DDP in PBS for 6 hours at 37°C and then dialyzed against PBS at 4°C. Under these conditions, the cis-DDP reacts with a critical methionine residue in the ⁇ 2 M receptor recognition site, forming a stable complex ( ⁇ 2 M-M Pt ) which does not bind to ⁇ 2 M receptors.
  • ⁇ 2 M-M Pt Each ⁇ 2 M-M Pt preparation was screened using an in vi tro rat hepatocyte binding assay as described elsewhere. ⁇ 2 M- M Pt did not displace 125 I- ⁇ 2 M-methylamine from hepatocyte receptors at 4°C.
  • ⁇ 2 M-methylamine was treated with papain at pH 5.0 as described by Sottrup-Jensen et al. in (1986) FEBS Lett. 205: 20-24.
  • Papain digests ⁇ 2 M-methylamine into one major fragment (600-kDa) and four equivalent 18-kDa peptides representing the C-termini of the four ⁇ 2 M subunits.
  • the 600-kDa fragment retains the basic structural features of ⁇ 2 M-methylamine as shown by electron microscopy but lacks the receptor binding domains.
  • the 600-kDa fragment was purified by chromatography on Ultrogel AcA-22. In hepatocyte binding assays, 100 nM 600-kDa fragment did not displace
  • Native, proteinase-reacted or methylaminereacted forms of ⁇ M (0.15 ⁇ M) or 5 ⁇ l of normal human serum were incubated for 1 hour at 37°C with 0.8 nM 125 I-TGF- ⁇ 1 or 125 I-TGF- ⁇ 2 in 100 ⁇ l 50 mM Tris Hcl, pH 7.6.
  • heparin 100 ⁇ g/ml was added to the solutions.
  • Radioiodinated TGF- ⁇ 1 was incubated with native ⁇ 2 M, ⁇ 2 M-methylamine, ⁇ 2 M-M Pt or the 600-kDa fragment.
  • the ⁇ 2 M and TGF- ⁇ 1 concentrations were 1.4 ⁇ M and 1.2 nM, respectively.
  • TGF- ⁇ 1- ⁇ 2 M-methylamine complex was separated from free TGF- ⁇ 1 by FPLC on Superose-6, stored at 4°C for 48 hours, and then subjected to FPLC again to probe for complex dissociation. Greater than 98% of the radioactivity remained associated with the high- molecular weight ⁇ 2 M peak.
  • 0.8 nM 125 I- TGF- ⁇ 1 or 125 I-TGF- ⁇ 2 was incubated with a reaction mixture containing 0.15 ⁇ M native ⁇ 2 M and the following concentrations of ⁇ 2 M-plasmin: 0, 0.0015 ⁇ M, 0.003 ⁇ M,
  • Figure 1 shows the denaturing gel electrophoresis and autoradiography of native ⁇ 2 M, ⁇ 2 M- trypsin and ⁇ 2 M-methylamine/ 125 I-TGF- ⁇ 2 complexes.
  • Reaction mixtures containing 125 I-TGF- ⁇ 2 only (lane 1), or 125 I-TGF- ⁇ 2 with native ⁇ 2 M (lane 2), ⁇ 2 M-trypsin (lane 3) or ⁇ 2 M-methylamine (lane 4) were subjected to non-denaturing gel electrophoresis, Coomassie blue staining (panel A) and autoradiography (panel B) as described previously.
  • 125 I-TGF- ⁇ 2 formed complexes with purified native ⁇ 2 M (Fig.
  • Figure 2a shows the cross competition between
  • ⁇ 2 M- methylamine was incubated with 125 I-TGF- ⁇ 1 and a 0-
  • Figure 2b shows the direct competition between 125 I-TGF- ⁇ 2 and unlabeled TGF- ⁇ 2 for binding to ⁇ 2 M- methylamine.
  • ⁇ 2 M-methylamine was incubated with 125 I-
  • Figure 3 shows the relative binding of 125 I-
  • TGF- ⁇ 1 to native ⁇ 2 M, ⁇ 2 M-plasmin, ⁇ 2 M -thrombin and ⁇ 2 M- trypsin.
  • 125 I-TGF- ⁇ 1 was incubated for 2 h. with identical concentrations of native ⁇ 2 M (lane 1), ⁇ 2 M- methylamine (lane 2), ⁇ 2 M-plasmin (lane 3), ⁇ 2 M-thrombin (lane 4) or ⁇ 2 M-trypsin.
  • Figure 4 shows the competition between native ⁇ 2 M and ⁇ 2 M-plasmin for 125 I-TGF- ⁇ binding.
  • 125 I-TGF- ⁇ 1 (A) or 125 I-TGF- ⁇ 2 (B) was incubated with a fixed concentration of native ⁇ 2 M (0.15 ⁇ M) and increasing concentrations of ⁇ 2 M-plasmin in the following ratios ( ⁇ 2 M-plasmin:native) : 0 (lanes 1), 1:100 (lanes 2), 1:50 (lanes 3), 1:25 (lanes 4), 1:10 (lanes 5), 1:5 (lanes 6), 1:2 (lanes 7) and 1:1 (lanes 8).
  • Figure 5 shows the competition by ⁇ 2 M-plasmin for 125 I-TGF- ⁇ s in native ⁇ 2 M/ 125 I-TGF- ⁇ complexes.
  • 125 I-TGF- ⁇ 1 was incubated with native ⁇ 2 M (0.15 ⁇ M) for 1 hour. After the incubation period, an identical quantity of ⁇ 2 M-plasmin was added and incubated for an additional 1 hour. Aliquots of the mixtures or controls containing native ⁇ 2 M or ⁇ 2 M-plasmin and 125 I-labeled
  • TGF- ⁇ 1 were subjected to electrophoresis, Coomassie blue staining (A) and autoradiography (B) as described earlier.
  • Lane 1 125 I-TGF- ⁇ 1+native ⁇ 2 M (1 h. inbucation).
  • Lane 2 [ 125 I-TGF- ⁇ 1+native ⁇ 2 M (1 h. incubation)]+ ⁇ 2 M-plasmin (additional 1 hour).
  • Lane 3 125 I-TGF- ⁇ 1+native ⁇ 2 M (1 h. inbucation).
  • TGF- ⁇ 2 which was allowed to form a complex with native ⁇ 2 M (1 hour incubation) subsequently became bound to ⁇ 2 M- plasmin when ⁇ 2 M-plasrain was added to the reaction mixture prior to electrophoresis (Fig. 5).
  • ⁇ 2 M-plasmin also incorporated additional radiolabelled growth factor from the incubation mixture as indicated by the greater density of the ⁇ 2 M-plasmin band on autoradiography (Fig. 5).
  • Figure 6 shows the dissociating effect of heparin on native ⁇ 2 M and ⁇ 2 M-proteinase/TGF- ⁇ complexes.
  • Aliquots from incubation mixtures containing 125 I-TGF- ⁇ 1 (A) or 125 I-TGF- ⁇ 2 (B) and native ⁇ 2 M (lanes 1,2), ⁇ 2 M-throrabin (lanes 3,4), ⁇ 2 M-plasmin (lanes 5,6) or a 2 M- methylamine (lanes 7,8) were subjected to non-denaturing gel electrophoresis, Coomassie blue staining (A) and autoradiography (B) as described earlier.
  • 6 and 8 also contain heparin (100 ⁇ g/ral).
  • 125 I-TGF- ⁇ 2 and native, methylamine- or proteinase- reacted forms of ⁇ 2 M were less susceptible to dissociation by heparin (Fig. 6b).
  • TGF- ⁇ 2 complexes with ⁇ 2 M-plasmin were only weakly dissociated by heparin concentrations as high as 100 ⁇ g/ml (Figs. 6a and b).
  • TGF- ⁇ 1 and TGF- ⁇ 2 interacted with ⁇ 2 M complexes that were stable during non-denaturing gel electrophoresis.
  • the reaction of ⁇ 2 M with different proteinases and methylamine markedly altered the binding properties of ⁇ 2 M for 125 I-TGF- ⁇ 1 and 125 I-TGF- ⁇ 2.
  • ⁇ 2 M-plasmin and ⁇ 2 M-methylamine demonstrated greater apparent binding capacities for TGF- ⁇ 1 and TGF- ⁇ 2 than native ⁇ 2 M, ⁇ 2 M-thrombin or ⁇ 2 M- trypsin.
  • the binding of 125 I-TGF- ⁇ 2 to purified human ⁇ 2 M and ⁇ 2 M in plasma or serum was similar to that previously demonstrated for 125 I-TGF- ⁇ 1.
  • Huang et al. in (1988), J. Biol. Chem. 263, 1535-1541 first demonstrated that the electrophoretic conformation affects the TGF- ⁇ -binding activity of ⁇ 2 M. This hypothesis was based on studies with ⁇ 2 M-methylamine which does not occur naturally. The present studies confirm those of Huang et al. supra and extend the findings by identifying ⁇ 2 M-plasmin as a naturally occurring, high affinity binding protein for TGF- ⁇ that may be responsible for a significant part of the TGF- ⁇ - carrier function of ⁇ 2 M in vi vo.
  • TGF- ⁇ 2 from ⁇ 2 M-plasmin. Furthermore, the complexes between 125 I-TGF- ⁇ 2 and all forms of a 2 M examined are less susceptible to heparin-induced dissociation than the equivalent complexes with 125 I-TGF- ⁇ 1.
  • TGF- ⁇ 1 and TGF- ⁇ 2 The binding of TGF- ⁇ 1 and TGF- ⁇ 2 by ⁇ 2 M-plasmin is particularly interesting in light of evidence from others that plasmin activates the latent precursor form of TGF- ⁇ 1 secreted by fibroblasts in culture.
  • the proteolytic activity of plasmin itself is modified by ⁇ 2 M, therefore, plasmin may act at two or more levels to control TGF- ⁇ activity.
  • TGF- ⁇ has recently been demonstrated to increase the expression and secretion of plasminogen activator inhibitors and increase or decrease the expression and secretion of plasminogen activators depending on cell type. These factors in turn control the amount of active plasmin present locally and, indirectly, the local concentration of ⁇ 2 M- plasmin when plasma a 2 M is available.
  • Plasmin has been shown to activate collagenases and other metalloproteinases involved in cell growth. The experiments described above would appear to suggest for the first time an additional role for plasmin in growth regulation by triggering the formation of a "scavenger" molecule that binds TGF- ⁇ s with high affinity.
  • Plasma clearance studies were performed in 20 week old CD-1 mice (Charles River) as previously described by Gorias et al. in 1984, Ann. N.Y. Acad. Sci. 421, 457-471. Briefly, 125 I-TGF- ⁇ 1, 125 I- ⁇ 2 M, or various FPLC- purified ⁇ 2 M- 125 I-TGF- ⁇ 1 complexes were injected into the lateral tail veins of anesthetized mice. The total injection volume was 400 ⁇ L. Blood samples (25 ⁇ L) were taken, beginning at 10 s, from the retroorbital venous plexus using heparinized hematocrit tubes. Radioactivity in each blood sample was determined in a gamma counter and plotted as a percentage of the radioactivity present in the 10 s sample. Each experiment was performed at least in triplicate.
  • both 125 I- ⁇ 2 M-tryps ⁇ n and 125 I- ⁇ 2 M-methylamine cleared rapidly after intravenous injection.
  • elimination from the vascular compartment was described by a first order process with a half-life of 3-5 minutes.
  • the saturability of the ⁇ 2 M-proteinase clearance pathway can be easily demonstrated in vi vo.
  • a large molar excess of ⁇ 2 M-proteinase or ⁇ 2 M-methylamine causes a dose-dependent decrease in the clearance rate of 125 I- ⁇ 2 M-trypsin or 125 I- ⁇ 2 M-methylamine.
  • Figure 8 shows the plasma clearance of free
  • 125 I-TGF- ⁇ 1 ( ) and 125 I-TGF- ⁇ 1 bound to various forms of ⁇ 2 M including ⁇ 2 M-methylamine ( ), ⁇ 2 M-M pt ( ), and the 600-kDa fragment ( ).
  • TGF- ⁇ 1- ⁇ 2 M complex was resolved from free TGF- ⁇ 1 by chromatography on Superose-6. The error bars represent one standard deviation.
  • TGF- ⁇ 1 The plasma clearance of TGF- ⁇ 1 in mice, reported here, is comparable to that determined in rats by Coffey et al in (1987), J. Clin. Invest. 89:750-757.
  • TGF- ⁇ 1 complex differed significantly from the clearance of the free growth factor.
  • the clearance curve was monophasic and adequately described by a first order process with a half-life of 4 min. By 30 min., residual radioligand in the blood samples reached background levels (less than 2%).
  • mice 1 25 I-TGF- ⁇ 1 or FPLC-purified ⁇ 2 M-methylamine- 1 25 I-TGF- ⁇ 1 complex were injected intravenously in mice. After 45 minutes, the animals were sacrificed. The major body organs were recovered intact, rinsed in normal saline, blotted to remove surface moisture and weighed. The radioactivity was then determined in each organ and expressed as a percentage of the total recovered radioactivity. The results were normalized for organ size by dividing the percent of recovered radioactivity in each organ by the organ mass.
  • organ mass was standardized for organ mass , the highest density of
  • TGF- ⁇ 1 complex was significantly different. Over 90% of the radioactivity was recovered in the liver. The nearly exclusive role of the liver was evident even after correcting the data for organ mass. The percentage of recovered radioactivity in each organ was within 2% of that reported for 125 I- ⁇ 2 M-methylamine elsewhere.
  • the TGF- ⁇ 1 binding activity of ⁇ 2 M-trypsin is not increased compared with native ⁇ 2 M.
  • Figure 9 summarizes the clearance competition studies. ⁇ 2 M-methylamine- 125 I-TGF- ⁇ 1 complex was purified by chromatography and injected simultaneously with a large dose of ⁇ 2 M-methylamine ( ) or ⁇ 2 M-trypsin
  • TGF- ⁇ 1 clearance requires available ⁇ 2 M receptors in vi vo.
  • the TGF- ⁇ 1 binding activity of ⁇ 2 M-trypsin is not greater than that of native ⁇ 2 M. Therefore, the competing ligands did not affect the clearance of ⁇ 2 M- methylamine- 125 I-TGF- ⁇ 1 complex by interacting with the radioligand in vi vo. Since ⁇ 2 M-methylaraine and ⁇ 2 M- trypsin almost completely arrested ⁇ 2 M-methylamine- 125 I- TGF- ⁇ 1 clearance, pathways for the plasma elimination of the radiolabelled complex other than the ⁇ 2 M receptor probably do not exist in mice. Delaved competition experiments
  • Plasma clearance of a radioligand results from reversible association with cell-surface receptors or irreversible processes such as endocytosis.
  • delayed competition experiments were performed. ⁇ 2 M- methylamine- 125 I-TGF- ⁇ 1 complex was injected intravenously in mice. After 15 min, 1.0 mg of ⁇ 2 M- methylamine was administered in a second intravenous injection. Blood sampling was conducted throughout. If a radio-ligand is reversibly bound to cellular receptors, the second injection causes redistribution of radioactivity back into the plasma. This result has been observed with asialotransferin.
  • TGF- ⁇ 1 can be displaced back into the vascular compartment after clearance occurs.
  • Figure 10 summarizes these experiments. ⁇ 2 M- methylamine- 125 I-TGF- ⁇ 1 complex was injected alone and allowed to clear for 15 min. A large molar excess of ⁇ 2 M-methylamine was then administered intravenously in a second injection. The time of the second injection is shown by the arrow.
  • TGF- ⁇ 1 since active forms of TGF- ⁇ 1 must be able to bind to specific TGF- ⁇ receptors, ⁇ 2 M-TGF- ⁇ 1 complexes are latent or inactive because they are not TGF- ⁇ -receptor recognized. Free TGF- ⁇ 1 was rapidly eliminated from the vascular compartment, most likely due to interaction with TGF- ⁇ receptors located diffusely in multiple organs. By contrast, clearance of ⁇ 2 M-methylamine-TGF- ⁇ 1 complex was dependent on the ⁇ 2 M receptor exclusively.
  • ⁇ JM-methylamine provides a useful model to study the structure and function of ⁇ 2 M after conformational change. Based on numerous physico- chemical studies, differences in the structoure of ⁇ 2 M- methylaraine and ⁇ 2 M-proteinase are minimal, ptinaps no greater than the variability reported for different ⁇ 2 M- proteinase complexes. From the standpoint of the current investigation, ⁇ 2 M-methylamine provides an excellent model to study ⁇ 2 M receptor interactions; however, this particular form of ⁇ 2 M is not physiologically significant.
  • ⁇ 2 M in the plasma originates primarily from the liver; however, macrophages can also secrete ⁇ 2 M at sites of inflammation or an immune response. Therefore, complexes of ⁇ 2 M with TGF- ⁇ 1 may form both inside and outside the vascular compartment. Since the hepatocyte is the primary cell responsible for clearing ⁇ 2 M-methylamine and ⁇ 2 M-proteinase complexes from the circulation, it is likely that hepatocytes bind ⁇ 2 M- methylamine-TGF- ⁇ 1 complex as well. At this time, it is not clear whether TGF- ⁇ 1 retains any activity when presented to hepatocytes as an ⁇ 2 M-methylamine-TGF- ⁇ 1 complex by the ⁇ 2 M receptor. Huang et al.
  • ⁇ 2 M-TGF- ⁇ 1 complexes isolated from plasma are latent (inactive) and suggested that these complexes may include only receptor-recognized forms of ⁇ 2 M (proteinase complexes). If this hypothesis is correct, the data presented here suggests that the plasma half-life of the la Itent ⁇ 2 M-TGF- ⁇ 1 complex is very short. Alternatively, a long circulating half-life is predicted for TGF- ⁇ 1 that binds to native ⁇ 2 M. Binding of modified ⁇ 2 Ms to TNF- ⁇ .
  • Methylamine HCl, trypsin (from bovine pancreas) and heparin (from porcine intestine) and p- nitrophenyl-p'-guanadino benzoate-HCL (NPGB) were purchased from Sigma Chemical Co., St. Louis, MO.; purified bovine plasmin (2U/rag), human plasmin (8U/mg) and bovine thrombin (30U/mg) from Boehringer-Mannheim, Dorval, Quebec, Canada; Coomassie brilliant blue stain and sodium dodecyl sulfate (SDS) from Bio-Rad Laboratories, Richmond, CA.; and recombinant human 125 I-
  • TNF- ⁇ (15-30 TBq/mmol) from Amersham, Oakville, Canada.
  • Rat plasma was collected from healthy adult volunteers. Rat plasma was collected terminally from the aorta of normal Fisher 344 rats (275-300 g) purchased from Charles River Laboratories, St. Constant, Quebec and maintained under routine housing conditions.
  • Native ⁇ 2 M was purified from fresh human plasma by zinc-Sepharose-4B affinity chromatography using the method of Imber and Pizzo supra. Purified (1 ⁇ M) ⁇ 2 M was incubated with 4 ⁇ M of human plasmin, bovine plasmin, thrombin or trypsin in 50 mM Tris HCl, pH 7.6 at 37oC. Residual proteinase activity was inhibited after two hours by the addition of NPGB (100 ⁇ M). ⁇ 2 M, human plasma and serum were reacted with methylamine using the method of Gonias et al set forth in (1982) Biochem. Biophys. Acta 705:306-314.
  • Reaction products were subjected to PAGE under nondenaturing conditions as previously described.
  • Kodak X-Omat AR-5 film was exposed to dried gels at - 70°C using Lanex regular intensifying screens.
  • Figure 11 represents 125 I-TNF- ⁇ complex formation with ⁇ 2 M-methylamine, ⁇ 2 M-plasmin (human) and ⁇ 2 M-plasmin (bovine) following 5% native polyacrylamide gel electrophoresis. Reaction mixtures of 125 I-TNF- ⁇
  • TNF- ⁇ to ⁇ 2 M was demonstrated with ⁇ 2 M-plasmin (human), ⁇ 2 M-plasmin (bovine) or ⁇ 2 M-methylamine as shown in Figures 1 and 2.
  • binding of 125 I-TNF- ⁇ to the native a 2 M was minimal.
  • the extent of 125 I-TNF- ⁇ binding to ⁇ 2 M-methylamine, ⁇ 2 M-plasmin (human) and a 2 M- plasmin (bovine) was comparable by autoradiography of native gels.
  • the more sensitive size exclusion FPLC method demonstrated that more 125 I-TNF- ⁇ associated with the ⁇ 2 M-plasmin (human) than with the ⁇ 2 M- methylamine.
  • Figure 13 represents the preferential complex formation of 125 I-TNF- ⁇ with ⁇ 2 M-human plasmin and ⁇ 2 M- methylamine in human serum and plasma.
  • 125 I-TNF- ⁇ was incubated for 2 hours at 37°C with 2 ⁇ l of rat plasma
  • 125 I-TNF- ⁇ binds preferentially to specific reacted forms of ⁇ .M rather than other serum or plasma proteins .
  • 125 I-TNF- ⁇ was incubated with 2 ⁇ l of serum or plasma containing either native or reacted forms of ⁇ 2 M.
  • 125 I-TNF- ⁇ bound preferentially to the constitutive a 2 M which had been converted to the "fast" form by reaction of human plasma or serum with methylamine ( Figure 13, lanes 2 and 3 respectively).
  • 125 I-TNF- ⁇ bound preferentially to methylamine-converted "fast" ⁇ - macroglobulins in rat, mouse, swine and bovine serum or plasma; in all of these species, only minimal binding of 1 25 I-TNF- ⁇ was associated with "slow" migrating forms.
  • 125 I-TNF- ⁇ also bound preferentially to ⁇ 2 M-plasmin ( Figure 3, lanes 6 and 7) or ⁇ 2 M-methylamine which had been added to human serum and plasma ( Figure 13, lanes
  • TNF- ⁇ resembles TGF- ⁇ 1 and TGF- ⁇ 2 in that these cytokines bind preferentially to ⁇ 2 M-plasmin as compared to their binding to native ⁇ 2 M. Similarly, ⁇ 2 M- methylamine complexes with greater amounts of TNF- ⁇ ,
  • TGF- ⁇ 1, TGF- ⁇ 2, bFGF and IL- ⁇ 1 than does native a 2 M .
  • cytokines bind preferentially to reacted forms of ⁇ 2 M.
  • native ⁇ 2 M demonstrates the greatest affinity for PDGF.
  • TNF- ⁇ is not bound covalently to ⁇ 2 M-plasmin, since greater than 95% of the TNF- ⁇ / ⁇ 2 M-plasmin complexes dissociate in the presence of SDS. This is in contrast to PDGF which binds covalently to ⁇ 2 M. TNF- ⁇ / ⁇ 2 M-plasmin complexes are also insensitive to dissociation by heparin, in contrast to certain TGF- ⁇ / ⁇ 2 M complexes.
  • DMEM fetal bovine serum
  • FBS fetal bovine serum
  • actinomycin D Gibco
  • mice 10 6 cpm in 400 ⁇ l) with or without a 20 times molar excess of ⁇ 2 M-trypsin were injected into the tail vein of mice anesthetized with Isoflurane (Anaquest, Pointe Claire, Canada). Blood samples were collected from the retro-orbital venous plexus using heparinized hematocrit tubes at 10 s, 5, 10 and 20 min after injection.
  • Radioactivity in each 25 ⁇ l sample was determined in a gamma counter and values are expressed as a percentage of the radioactivity in the 10 s sample.
  • TNF- ⁇ / ⁇ 2 M complexes (Table IV), indicating that ⁇ 2 M receptors are involved in plasma clearance of 125 I-TNF- a/ ⁇ 2 H complexes.
  • FPLC purified 125 I-TNF- ⁇ / ⁇ 2 M-plasmin complexes (approximately 10 6 cpm in 400 ⁇ l) with or without a 20 times molar excess of ⁇ 2 M-trypsin were injected into the tail of anesthetized 18 wk CD1 female mice.
  • Blood samples (25 ⁇ l) were collected from the retro-orbital venous plexus using heparinlzed hematocrit tubes at 10 s and the indicated times after injection and counted in a gamma counter.
  • * values are the mean radioactivity ( ⁇ SD) in the blood samples at various times determined in three independent clearance experiments and are expressed percent of the radioactivity in the initial sample, 10 s after injection.
  • 125 I-cytokine complexes with ⁇ 2 M-MA or ⁇ 2 M-proteinase are injected in 400 ⁇ l PBs into the tail vein of mice under light isoflurance anesthesia. Blood samples (25 ⁇ l) are taken with mi ⁇ rohematocrit tubes from the retro-orbital venous plexus at 10 seconds and various times after injection, as previously described for TGF- ⁇ 1.
  • Radioactivity present in circulation is determined by gamma counter and expressed as a percentage of the sample drawn at 10 seconds.
  • the major body organs are collected intact, rinsed .with normal saline, blotted, weighed, and counted. The radioactivity in each organ is expressed as a percentage of the total recovered radioactivity.
  • TNF- ⁇ injected in the uncomplexed form rapidly disappears from circulation, but not by the ⁇ 2 M-receptor clearance pathway as shown in Figure 14. This likely indicates distribution of active TNF- ⁇ to functional receptors on various target cells, some of which can react adversely to excess TNF- ⁇ in endotoxic or septic shock. TNF- ⁇ bound to ⁇ 2 Ms distributes predominantly to the liver (see Table VI similar to the distribution of TGF- ⁇ s).
  • Competition studies for binding to ⁇ 2 M receptors in vivo are performed by injecting 1.0 mg of unlabelled ⁇ 2 M-MA or ⁇ 2 M-trypsin (20-fold excess) in 250 ⁇ l PBS simultaneously with or at 10 minutes after injecting the 125 I-cytokine/ ⁇ 2 M complex.
  • ⁇ 2 M-receptor-dependent clearance of label is verified by demonstrating persistence of label in plasma when animals are given a large pulse of a 2 M- trypsin, which saturates the ⁇ 2 M-RME pathway but does not itself bind TNF- ⁇ . Int'ernalization of ⁇ 2 M in vi vo is verified by a minimal displacement (or so called "bump") of recently cleared label into plasma by a pulse of unlabelled ⁇ 2 M-trypsin administered 10-15 minutes after the ⁇ 2 M-cytokine complex. Results are shown in Figures 15 to 17.
  • 125 I-TNF- ⁇ alone is not cleared for circulation by the ⁇ 2 M receptor pathway, because clearance is not blocked by saturation of ⁇ 2 M-receptors by ⁇ 2 M-trypsin.
  • Radioactivity recovered from each organ is expressed as a percent of the total radioactivity in the organ listed. Values are the mean ⁇ SD for 3 experiments.

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Abstract

Cette invention concerne un procédé permettant d'empêcher l'interaction des facteurs de croissance ou des cytokines avec leurs récepteurs cellulaires spécifiques chez les mammifères. Ledit procédé comprend l'administration au mammifère d'une quantité thérapeutique d'une macroglobuline-α2 modifiée chimiquement ayant la capacité de former un facteur de croissance de macroglobuline-α2 ou un complexe de cytokine avec un facteur de croissance donné ou une cytokine donnée. Le complexe subit de préférence une modification du récepteur par administration de médicament, de préférence l'endocytose par des récepteurs de macroglobuline-α2, ce qui réduit ainsi la disponibilité du facteur de croissance actif ou de la cytokine active pour son récepteur de surface cellulaire spécifique. Les complexes sont tout particulièrement utiles dans le traitement du choc endotoxique ou du choc septique grâce à l'inhibition du TNF-α.
PCT/CA1990/000356 1990-10-18 1990-10-18 MACROGLOBULINES-α2 MODIFIEES ET LEUR UTILISATION COMME INHIBITEURS POUR DES CYTOKINES ET DES FACTEURS DE CROISSANCE WO1992007003A1 (fr)

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CA 2070682 CA2070682A1 (fr) 1990-10-18 1990-10-18 .alpha.2-macroglobulines modifiees et leur utilisation pour l'elimination des cytokines et des facteurs de croissance

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994014976A1 (fr) * 1992-12-18 1994-07-07 Duke University Complexe modulateur de la reponse immunitaire, et ses utilisations
WO1999000409A1 (fr) * 1997-06-30 1999-01-07 The University Of Virginia Patent Foundation Macroglobulines a chimiquement modifiees, leurs procedes de fabrication et leurs procedes d'utilisation dans une therapie anti-cytokine

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Biological Abstracts, volume 87, no. 12, Schlesinger et al.: "Covalent binding to x-macroglobulins of a protein with free sulhydryl groups produced by activated B cells: blocking by D-penicillamine and gold compounds" *
Science, volume 218, 5 November 1982, AAAS, W.J. Johnson et al.: "Receptors for maleylated proteins regulate secretion of neutral proteases by murine macro-phages", pages 574-576 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994014976A1 (fr) * 1992-12-18 1994-07-07 Duke University Complexe modulateur de la reponse immunitaire, et ses utilisations
WO1999000409A1 (fr) * 1997-06-30 1999-01-07 The University Of Virginia Patent Foundation Macroglobulines a chimiquement modifiees, leurs procedes de fabrication et leurs procedes d'utilisation dans une therapie anti-cytokine

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