WO2000033887A2 - Procedes et formulations permettant de reduire des anticorps circulants - Google Patents

Procedes et formulations permettant de reduire des anticorps circulants Download PDF

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WO2000033887A2
WO2000033887A2 PCT/US1999/029336 US9929336W WO0033887A2 WO 2000033887 A2 WO2000033887 A2 WO 2000033887A2 US 9929336 W US9929336 W US 9929336W WO 0033887 A2 WO0033887 A2 WO 0033887A2
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Prior art keywords
αgal
compound
epitope
antibodies
antibody
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PCT/US1999/029336
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WO2000033887A3 (fr
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Richard M. Jack
David S. Jones
Lin Yu
Steven B. Engle
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La Jolla Pharmaceutical Company
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Priority to CA002353620A priority Critical patent/CA2353620A1/fr
Priority to EP99966115A priority patent/EP1135167A2/fr
Priority to AU21735/00A priority patent/AU2173500A/en
Priority to JP2000586377A priority patent/JP2002531531A/ja
Publication of WO2000033887A2 publication Critical patent/WO2000033887A2/fr
Publication of WO2000033887A3 publication Critical patent/WO2000033887A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/595—Polyamides, e.g. nylon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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

Definitions

  • TECHNICAL FIELD This invention relates to reducing of circulating antibodies, particularly disease- associated antibodies, by binding the antibodies to an epitope-presenting carrier.
  • autoimmune disorders such as lupus and idiopathic thrombocytopenia purpura
  • IgE associated disorders There are many conditions and disorders which are associated with circulating antibodies of various classes. Some of these conditions are, for example, autoimmune disorders (such as lupus and idiopathic thrombocytopenia purpura) and IgE associated disorders.
  • One approach to treating, or combating, these disorders is to remove and/or reduce the circulating antibodies, even on a transient basis.
  • apheresis in which blood is removed from an individual and the antibodies are removed extraco ⁇ oreally using an affinity column, such as a protein A column.
  • an affinity column such as a protein A column.
  • the invention provides methods of reducing levels of circulating antibodies, particularly disease or disorder-associated antibodies.
  • the invention provides methods for reducing levels of circulating antibodies in an individual, particularly disease-associated antibodies, comprising administering to the individual an effective amount of an epitope-presenting moiety.
  • the epitope-presenting moiety may be any of a number of embodiments, and is preferably a conjugate comprising a valency platform molecule and an epitope(s).
  • the epitope may be any moiety, as long as it exhibits the requisite binding activity.
  • the invention provides ex vivo methods of reducing circulating antibodies using an eptiope-presenting moiety.
  • the methods provide methods of reducing levels of disease-associated antibodies in an individual, comprising treating the individual's blood (including any component thereof which contains antibody) extracoporeally with an eptiope-presenting carrier under conditions that permit the antibodies to bind the epitope; removing antibody-eptiope-presenting carrier complexes, if any; and returning the blood to the individual.
  • Figure 1 is a reaction scheme illustrating the enzymatic synthesis of the ⁇ Gal epitope, 2-[2-(2-thioethoxy) ethoxyjethyl 3-O-( ⁇ -D-galactopyranosyl)- ⁇ -D- galactopyranoside.
  • Figure 2 is a reaction scheme illustrating the chemical synthesis of the ⁇ Gal epitope, 2-[2-(2-thioethoxy) ethoxy]ethyl 3-O-( ⁇ -D-galactopyranosyl)- ⁇ -D- galactopyranoside.
  • Figure 3 is a reaction scheme illustrating the chemoenzymatic synthesis of the ⁇ Gal epitope, /?-aminophenyl 3-O- ⁇ -D-galactopyranosyl- ⁇ -D-galactopyranoside.
  • Figure 4 illustrates two general synthetic strategies for conjugation chemistry.
  • Figure 5 illustrates a third general synthetic strategy for conjugation chemistry.
  • Figure 6 illustrates two dimeric platforms and four tetrameric platforms.
  • Figure 7 illustrates four octameric platforms.
  • Figure 8 illustrates a monomeric ⁇ Gal conjugate and three dimeric ⁇ Gal conjugates.
  • Figure 9 illustrates two tetrameric ⁇ Gal conjugates as described in Example 3.
  • Figure 10 illustrates two tetrameric ⁇ Gal conjugates as described in Example 3.
  • Figure 11 illustrates two tetrameric conjugates of two ⁇ Gal-isomers as described in Example 3.
  • Figure 12 illustrates an octameric ⁇ Gal conjugate as described in Example 3.
  • Figure 13 illustrates an octameric ⁇ Gal conjugate as described in Example 3.
  • Figure 14 illustrates an octameric ⁇ Gal conjugate as described in Example 3.
  • Figure 15 illustrates an octameric ⁇ Gal conjugate as described in Example 3.
  • Figure 16 is a graph of OD550 versus fraction number and depicts the elution profile of anti- ⁇ Gal from an ⁇ Gal affinity column.
  • FIGS 17A and 17B are graphs depicting affinity purified IgG (17A) and IgM (17B) anti- ⁇ Gal binding to PK15 cells.
  • Flow cytometric analysis results are shown as mean fluorescence intensity (MFI) versus dose, in ⁇ g/ml, of affinity-purified IgG or IgM (solid circles), or column flow-through IgG or IgM (open squares).
  • MFI mean fluorescence intensity
  • Figure 19 is a bar graph of the percent plasma anti- ⁇ Gal IgG following treatment with LJP 712, as described in Example 5.
  • Figure 20 is a graph depicting activation of the classical complement pathway by various substances, as described in Example 5. Symbols are as follows: open circles, LJP 712; open squares, cobra venom factor (CVF); solid triangles, aggregated human gamma globulin (AHG). The dashed line represents results obtained with buffer alone.
  • Figure 21 is a graph depicting activation of the alternative complement pathway by various substances, as described in Example 5. Symbols are as follows: open circles, LJP 712; open squares, CVF; solid triangles, AHG.
  • Figures 22A and 22B are graphs depicting the decrease in plasma anti- ⁇ Gal IgG (22A) and IgM (22B) following treatment with octameric LJP 920 (cpd 46) (circles), compared with PBS (squares), as described in Example 5.
  • Figure 23 is a graph comparing the effect of tetramer LJP 712 (open circles) with octamer LJP 920 (solid circles) on the percent plasma anti- ⁇ Gal IgM, as described in Example 5.
  • PBS solid squares
  • Figure 24 depicts a strategy for the synthesis of compound 29.
  • Figure 25 depicts a strategy for the synthesis of compounds 31 and 32.
  • Figures 26A and 26B depict the synthesis of compound 30.
  • This invention provides effective methods of removing and/or reducing circulating levels of antibodies, particularly disease-associated antibodies. This removal and/or reduction is generally transient, as it is based on binding to circulating antibodies as opposed to causing B cell anergy, although induction of B cell anergy may accompany these methods.
  • reducing and/or "removing" circulating antibodies means that the level of free, or unbound, circulating antibodies has been reduced.
  • antibody by binding of epitope-presenting carrier to antibody, antibody is prevented from being an effector molecule, i.e., binding other targets, and is thus “reduced.”
  • "reducing" circulating antibodies includes clearance of antibody, e.g., physical removal from circulation. One way this way this clearance is effected is clearance of a complex comprising an epitope-presenting carrier and antibody by reticuloendothelial system.
  • An “epitope” is a term well-understood in the art and means any chemical moiety which exhibits specific binding to an antibody.
  • An “epitope” can also comprise an antigen, which is a moiety that contains an epitope, and, as such, also specifically binds to antibody.
  • An epitope or antigen that "specifically binds" to an antibody is a term well understood in the art, and methods to determine such specific binding are also well known in the art.
  • a molecule is said to exhibit “specific binding” if it reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular cell or substance than it does with alternative cells or substances.
  • An antibody "specifically binds" to a target if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances.
  • an “antibody” is an immunoglobulin molecule capable of specific binding to a target, such as a carbohydrate or polypeptide, through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule.
  • a target such as a carbohydrate or polypeptide
  • the term encompasses not only intact antibodies, but also fragments thereof (such as Fab, Fab', F(ab') 2 , Fv), single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion, humanized antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity.
  • a "disease-associated antibody” is an antibody whose production occurs during a disease state and/or whose production is undesireably, such as in autoimmune diseases and transplantation rejection.
  • diseases-associated antibodies include, but are not limited to, anti- double-tranded DNA antibodies (lupus) and anti- ⁇ Gal antibodies (transplantation rejection).
  • Naturally occurring refers to an endogenous chemical moiety, such as a carbohydrate, polynucleotide or polypeptide sequence, i.e., one found in nature. Processing of naturally occurring moieties can occur in one or more steps, and these terms encompass all stages of processing. Conversely, a "non-naturally occurring" moiety refers to all other moieties, i.e., ones which do not occur in nature, such as recombinant polynucleotide sequences and non-naturally occurring carbohydrates.
  • mimetic means a biological or chemical compound which specifically binds to an anti- ⁇ Gal antibody.
  • an " ⁇ Gal epitope” includes mimetics of naturally-occurring ⁇ Gal (such as peptides).
  • a “mimetic” shares an epitope, or binding specificity, with ⁇ Gal.
  • a mimetic may be any chemical substance which exhibits the requisite binding properties, and thus may be, for example, a simple or complex organic or inorganic molecule; a polypeptide; a polynucleotide; a carbohydrate; a lipid; a lipopolysaccharide; a lipoprotein, or any combination of the above, including, but not limited to, a polynucleotide-containing polypeptide; a glycosylated polypeptide; and a glycolipid.
  • the term “mimetic” encompasses the term “mimotope", which is a term well known in the art.
  • mammals include, but are not limited to, farm animals, sport animals, pets, primates, mice and rats.
  • an "effective amount” is an amount sufficient to effect beneficial or desired results, including clinical results.
  • An effective amount can be administered in one or more administrations.
  • an effective amount is an amount sufficient to reduce circulating levels of free antibodies (i.e., unbound to epitope on carrier).
  • a “carrier” is a molecule which contains attachment sites for epitope(s).
  • One preferred example of a carrier is a valency platform molecule.
  • the terms “carrier” and “moiety” are used interchangeably herein.
  • An “epitope presenting carrier” is a carrier which contains attached, or bound, epitopes, at least some of which (at least two of which) are able to bind to an antibody of interest.
  • valency platform molecule means a nonimmunogenic molecule containing sites which allow the attachment of a discrete number of epitopes and/or mimetic(s) of epitopes.
  • a "valency" of a conjugate or valency platform molecule indicates the number of attachment sites per molecule for an epitope(s).
  • the valency of a conjugate is the ratio (whether absolute or average) of epitope to valency platform molecule.
  • Nonimmunogenic when used to describe a carrier (including a valency platform molecule), means that the carrier (such as a valency platform molecule) fails to elicit an immune response (i.e., T cell and/or B cell response), and/or fails to elicit a sufficient immune response, when it is administered by itself to an individual.
  • the degree of acceptable immune response depends on the context in which the valency platform molecule is used, and may be empirically determined.
  • An epitope that is "conjugated" to a carrier is one that is attached to the carrier, either by covalent and/or covalent interactions.
  • a “stable complex” is one that sufficiently persists after its formation to allow subsequent detection and/or removal.
  • a “T cell epitope” means a component or portion thereof for which a T cell has an antigen-specific specific binding site, the result of binding to which activates the T cell. Where an embodiment of the invention is described as "lacking" a T cell epitope, this is taken to mean that a T cell epitope is not detectable using standard assays in the art.
  • an epitope that "lacks" a T cell epitope means that the epitope lacks a T cell epitope which causes T cell activation in the individual(s) to be treated (i.e., who is to receive an epitope-presenting carrier.
  • an epitope may lack a T cell epitope(s) with respect to an individual, or a group of individuals, while possessing a T cell epitope(s) with respect to other individual(s).
  • Methods for detecting the presence of a T cell epitope include assays which detect T cell proliferation (such as thymidine inco ⁇ oration).
  • Polypeptides or other antigens that fail to induce statistically significant inco ⁇ oration of thymidine above background are generally considered to lack T cell epitopes, although it will be appreciated that the quantitative amount of thymidine inco ⁇ oration may vary, depending on the polypeptide (or other antigen) being tested.
  • a stimulation index below about 2-3, more preferably less than about 1 indicates lack of T cell epitopes.
  • the presence of T cell epitopes can also be determined by measuring secretion of T cell-derived lymphokines according to standard methods. Location and content of T cell epitopes, if present, can be determined empirically.
  • blood as used herein is bodily fluid including a cellular component and plasma.
  • Bood means whole blood or a component thereof. Treating an "individual's blood” means that any or all of an individual's blood is treated.
  • a “stable complex” is one that sufficiently persists after its formation to allow subsequent detection and/or removal.
  • the invention provides methods of reducing circulating levels of antibodies, particularly disease-associated antibodies. These methods generally comprise administering an effective amount of an epitope-presenting (which includes an antigen- presenting) carrier (or a composition comprising an epitope-presenting carrier) to an individual. These methods are especially useful for effecting safe (i.e., lack of inflammation and/or other undesirable side effects) and rapid clearance in a more simple and desirable way than using other methods known in the art, such as apheresis.
  • the inventors note that, by binding antibody on multivalent carrier, sufficiently large complexes are likely formed which may effect rapid clearance from circulation without formation of excessively large multi-valent complexes which would cause undesired side effects, such as inflammation, although, as noted below, "reduction" of circulating antibody levels does not require this clearance for pu ⁇ oses of this invention.
  • the initial reduction of circulating antibody is due to binding the epitope- presenting carrier, and further more effective reduction is obtained by clearance.
  • the reduction and/or removal of antibody is preferably selective, i.e., only that antibody for which reduction or removal is desired is affected. However, it may be acceptable that larger classes of antibodies are removed which include the antibody of interest.
  • the extent of cross-reactivity of binding by the epitope will generally govern the types of antibody that are removed from circulation.
  • the reduction is preferably at least about 50%, preferably at least about 60%, preferably at least about 80%>, preferably at least about
  • methods for reducing levels of circulating disease-associated antibodies in an individual, comprising administering to the individual an effective amount of an epitope presenting carrier (or composition comprising an epitope- presenting carrier) comprising a plurality of epitopes conjugated to a carrier which presents the epitopes in a manner effective to adsorb (i.e., bind) the antibodies.
  • an epitope presenting carrier or composition comprising an epitope- presenting carrier
  • a carrier which presents the epitopes in a manner effective to adsorb (i.e., bind) the antibodies.
  • the epitope presenting carrier is multivalent, i.e., is capable of presenting more than one epitope.
  • the valency is at least two.
  • the valency is at least three, at least four, at least six, at least eight, at least 10, at least 12, at least 16, at least 20, at least 24, at least 30, at least 32, at least 36, at least 40, at least 42, at least 46, at least 50.
  • the upper limit of the valency is not necessarily critical, as long as the epitope- presenting carrier effects reduction and/or clearance without undesirable side effects.
  • the valency is two. In other embodiments, the valency is four. In other embodiments, the valency is six. In other embodiments, the valency is any of the following: eight; 10; 12; 16; 20; 22; 24; 26; 28; 30; 32; 34; 36; 38; 40; 42; 44; 46;
  • the carrier may be any chemical moiety, and have any chemical structure, including, but not limited to, organic and inorganic molecules, polypeptides (i.e., polymers of amino acids), nucleic acids, carbohydrates, other polymers, artificial structures, and lipid structures (such as liposomes or micelles) made by standard techniques, or polymerized as described in U.S. Pat. No. 5,512,294.
  • a carrier may be proteinaceous or non-proteinaceous (i.e., organic).
  • proteinaceous platforms include, but are not limited to, albumin, gammaglobulin, immunoglobulin (IgG) and ovalbumin. Borel et al. (1990) Immunol. Methods 126:159-
  • the epitope-presenting carriers are conjugates which comprise a chemically defined valency platform molecule in which a precise valency (as opposed to an average) is provided. See, for example, commonly owned U.S. Pat. Nos. 5,162,515;
  • a defined valency platform is a platform with defined structure, thus a defined number of attachment points and a defined valency.
  • these platforms have the advantage of having a homogeneous (i.e., uniform) molecular weight (as opposed to polydisperse molecular weight), and are thus "chemically defined”.
  • a population of conjugates using these platforms comprise a platform of homogeneous molecular weight or are substantially monodisperse (i.e., have a narrow molecular weight distribution) .
  • polydispersity is used as a measure of the molecular weight homogeneity or nonhomogeneity of a polymer sample. Polydispersity is calculated by dividing the weight average molecular weight (Mw) by the number average molecular weight (Mn). The value of Mw/Mn is unity for a perfectly monodisperse polymer.
  • Polydispersity (Mw/Mn) is measured by methods available in the art, such as gel permeation chromatography.
  • the polydispersity (Mw/Mn) of a sample of platform molecules is preferably less than 2, more preferably, less than 1.5, or less than 1.2, less than 1.07, less than 1.02, or, e.g., about 1.05 to 1.5 or about 1.05 to 1.2.
  • Typical polymers generally have a polydispersity of 2-5, or in some cases, 20 or more.
  • Advantages of the low polydispersity property of the valency platform molecules include improved biocompatibility and bioavailability since the molecules are substantially homogeneous in size, and variations in biological activity due to wide variations in molecular weight are minimized.
  • the low polydispersity molecules thus are pharmaceutically optimally formulated and easy to analyze. Further there is controlled valency of the population of molecules in the sample.
  • Carbamate Linkages An example of a carbamate platform is compound 30, Fig. 7.
  • Preferred valency platform molecules are biologically stabilized, i.e., they exhibit an in vivo excretion half-life often of hours to days to months to confer therapeutic efficacy, and are preferably composed of a synthetic single chain of defined composition. They generally have a molecular weight in the range of about 200 to about 200,000, preferably about 200 to about 50,000 (or less, such as 30,000).
  • Examples of valency platform molecules within the present invention are polymers (or are comprised of polymers) such as polyethylene glycol (PEG), poly-D-lysine, polyvinyl alcohol, polyvinylpyrollidone, D- glutamic acid and D-lysine (in a ratio of 3:2).
  • Preferred polymers are based on polyethylene glycols (PEGs) having a molecular weight of about 200 to about 8,000.
  • Other suitable platform molecules for use in the conjugates of the invention are albumin and IgG.
  • Preferred valency platform molecules suitable for use within the present invention include the chemically-defined, non-polymeric valency platform molecules disclosed in co- owned U.S. Pat. No. 5,552,391. Particularly preferred homogeneous chemically-defined valency platform molecules suitable for use within the present invention are derivatized
  • EDDA 2,2'-ethylenedioxydiethylamine
  • TAG triethylene glycol
  • Suitable valency platform molecules include, but are not limited to, tetraaminobenzene, heptaaminobetacyclodextrin, tetraaminopentaerythritol, 1,4,8,11- tetraazacyclotetradecane (Cyclam) and 1,4,7,10-tetraazacyclododecane (Cyclen).
  • the tetra-bromoacetyl platform PIZ/ID A/TEG platform is used.
  • Derivatives of the PIZ/IDA/TEG (PITG) platform can be prepared as shown below. See PCT/US97/10075 and PCT/US96/09976 for other examples of suitable platforms. Examples of Compatible Cross-linking Groups on PITG Platform
  • an polypeptide epitope is prepared with a thiol linker at the N terminus by chemical or enzymatic synthesis, or by recombinant methods.
  • the linker can be cysteine or an SH containing moiety
  • the modified epitope may then be alkylated by a suitably derivatized platform (such as bromoacetyl or iodoacetyl).
  • PEG must be derivatized and made multivalent, which is accomplished using standard techniques.
  • Some substances suitable for conjugate synthesis, such as PEG, albumin, and IgG are available commercially.
  • valency platforms may be used which, when conjugated, provide an average valency (i.e., these platforms are not chemically defined in terms of their valency).
  • examples of such platforms are polymers such as linear PEG; branched PEG; star PEG; polyamino acids, such as DEK; polylysine; proteins; amino-functionalized soluble polymers.
  • Covalent conjugation of epitope(s) with a carrier such as a valency platform molecule is generally performed using standard chemical techniques. The following are examples of standard chemistry which can be used: 1) thiol substitution; 2) thiol Michael addition; 3) amino alkylation; 4) disulfide bond formation.
  • Figures 4 and 5 provide general, exemplary conjugation strategies.
  • Conjugation of an epitope to a valency platform molecule may be effected in any number of ways, typically involving one or more crosslinking agents and functional groups on the epitope and valency platform molecule.
  • Platforms and epitope(s) must have appropriate linking groups.
  • Linking groups are added to platforms using standard synthetic chemistry techniques. Linking groups may be added to an ⁇ Gal epitope(s) using either standard solid phase synthetic techniques or recombinant techniques (if, for example, the ⁇ Gal epitope is a peptide). Recombinant approaches may require post-translational modification in order to attach a linker, and such methods are known in the art.
  • polypeptides contain amino acid side chain moieties containing functional groups such as amino, carboxyl, or sulfhydryl groups that serve as sites for coupling the polypeptide to the platform. Residues that have such functional groups may be added to the polypeptide if the polypeptide does not already contain these groups. Such residues may be inco ⁇ orated by solid phase synthesis techniques or recombinant techniques, both of which are well known in the peptide synthesis arts. When the polypeptide has a carbohydrate side chain(s), functional amino, sulfhydryl and/or aldehyde groups may be inco ⁇ orated therein by conventional chemistry.
  • functional groups such as amino, carboxyl, or sulfhydryl groups that serve as sites for coupling the polypeptide to the platform. Residues that have such functional groups may be added to the polypeptide if the polypeptide does not already contain these groups. Such residues may be inco ⁇ orated by solid phase synthesis techniques or recomb
  • primary amino groups may be inco ⁇ orated by reaction with ethylenediamine in the presence of sodium cyanoborohydride
  • sulfhydryls may be introduced by reaction of cysteamine dihydrochloride followed by reduction with a standard disulfide reducing agent, while aldehyde groups may be generated following periodate oxidation.
  • the valency platform molecule may also be derivatized to contain functional groups if it does not already possess appropriate functional groups.
  • Hydrophilic linkers of variable lengths are useful for connecting epitopes to valency platform molecules.
  • Suitable linkers include linear oligomers or polymers of ethylene glycol.
  • Such linkers include linkers with the formula
  • These linkers are useful in connecting a molecule containing a thiol reactive group such as haloaceyl, maleiamide, etc., via a thioether to a second molecule which contains an amino group via an amide bond.
  • These linkers are flexible with regard to the order of attachment, i.e., the thioether can be formed first or last. Particular conjugates are described in Example 2 and are depicted in Figures 6-15, which accordingly are provided as embodiments of the invention.
  • liposomes may be used. Liposome technology is known in the art and need not be described in detail herein. As a brief summary, epitopes are appropriately inserted (i.e., inserted so that the binding moiety is available to bind to antibody, which may involve attaching "tails" to the epitope(s) for insertion) into liposomes, which may be of varying size. Mahato et al. (1997) Pharm. Res. 14:853-859. Liposomal preparations include, but are not limited to, cytofectins, multilamellar vesicles and unilamellar vesicles. Polymeric liposomes are described in U.S. Pat. No. 5,512,294.
  • MAPs multiple antigen peptides
  • MAPs have a small immunologically inert core having radially branching lysine dendrites, onto which polypeptide epitopes may be anchored.
  • MAPs may be synthesized using methods known in the art, for example, a solid-phase methods, such as that described in Merrifield et al. (1963) J Am. Chem. Soc. 85:2149.
  • An epitope may be a polypeptide, organic, or inorganic molecule.
  • antibody-binding moieties are known in the art and/or may be developed using standard methods and assays in the art, such as antibody binding assays.
  • Antibodies used for assays to test and/or develop epitopes may be also obtained using standard assays in the art, such as affinity purification (or in some cases may be commercially available).
  • Assays that may be used to determine whether a putative epitope or antigen exhibits requisite binding activity include, but are not limited to, filamentous phage random peptide libraries and screening (by, for example, biopanning, micropanning, phage-capture ELISA, phage-ELISA, colony blot, peptide ELISA, competitive binding peptide ELISA).
  • Suitable epitopes include, but are not limited to, those that bind to: lupus anti-DNA antibodies (see U.S. Pat. Nos. 5,162,515; 5,391,785; 5,276,013; 5,786,512;
  • anti- galactose alpha 1,3 galactosyl ( ⁇ Gal) antibodies anti-cardiolipin antibodies; antiphospholipid antibodies; IgE antibodies; anti-factor VIII antibodies; anti-factor IX antibodies; anti- ⁇ 2 GPI antibodies, particularly to domain 1 ; anti- platelet antibodies; antibodies associated with idiopathic thrombocytopenia pu ⁇ ura (ITP); anti-adenovirus antibodies (which may be problematic when adenovirus is administered as a therapeutic agent); anti-adeno-associated virus (AAV) antibodies (which may be problematic when AAV is administered as a therapeutic agent); anti-alpha chain acetyl choline receptor (myasthenia gravis); anti-RhD antigen antibodies (i.e., Rh disease); anti- thyroid antibodies (for example, in auto-immune thyroiditis).
  • ITP idiopathic thrombocytopenia pu ⁇ ura
  • anti-adenovirus antibodies which may be problematic when adenovirus is administered as a therapeutic agent
  • epitopes that bind any undesired blood group antibody that may interfere with allo- or xenotransplantation.
  • antibodies that bind the structure Gal ⁇ l-3Gal ⁇ l-4GlcNAc ⁇ l-3Gal ⁇ l-4Glc ⁇ l- can be removed using ⁇ Gal epitopes described elsewhere in this application.
  • epitopes can be designed that antigenically resemble the antigenic site on polysaccharides and are suitable for removing antibodies against other carbohydrate-based blood group antigens, including but not limited to those of the ABO system, the MN system, the Lewis system, and the Bombay phenotype.
  • epitopes that bind the anti-polynucleotide (particularly anti- double stranded DNA) antibodies that occur in systemic lupus erythematosis.
  • Preferred platforms for such epitopes are tetrabromoacetyl compounds, and other tetravalent and octavalent valency platform molecules.
  • We have observed that administration of such lupus conjugates in humans have resulted in reduction of circulating anti-ds DNA antibodies. Jones (1995); Weisman et al. (1997) J. Rheum. 24:314-318; Iverson et al. (1998) Lupus 7 (Suppl. 2): S166-S169.
  • a family of carriers can be constructed in which each of the candidates is alternatively displayed on a similar carrier molecule or platform.
  • the composition is then tested for efficacy.
  • an animal model is used in which there are circulating antibodies of the undesired type.
  • the animals can be immunized with an appropriate antigen to initiate the antibody response, if necessary.
  • Test candidates assembled onto a carrier are then used to treat separate animals, either by administration, or by ex vivo use, according to the intended pu ⁇ ose.
  • the animals are bled before and after treatment, and the antibody levels in plasma are determined by standard immunoassay as appropriate for the specific antibody. Efficacy of the candidates is then assessed according to the comparative degree in reduction in the antibody level.
  • the epitope used lacks T cell epitope(s).
  • Methods for detecting T cell epitopes are well known in the art. For example, various assays which detect T cell proliferation (such as thymidine inco ⁇ oration) may be used.
  • the presence of T cell epitopes can also be determined by measuring secretion of T cell-derived lymphokines by methods well known in the art.
  • Antigens that fail to induce statistical significant inco ⁇ oration of thymidine above background i.e., generally p less than 0.05 using standard statistical methods
  • a stimulation index below about 2-3, more preferably less than about 1 indicates lack of T cell epitopes. Location and content of T cell epitopes are determined empirically.
  • This invention further includes methods for reducing levels of disease-associated antibodies in the biological fluid of an individual, comprising contacting the fluid with an epitope-presenting carrier ex vivo under conditions that permit the antibodies to bind epitopes on the carrier.
  • Suitable bodily fluids include those that can be returned to the individual, such as blood, plasma, or lymph.
  • the invention includes methods of reducing levels of disease- associated antibodies in an individual, comprising treating the individual's blood (including any component thereof which contains antibody) extracoporeally (i.e., outside the body or ex vivo) with an eptiope-presenting carrier under conditions that permit the antibodies to bind the epitope; removing antibody-eptiope-presenting carrier complexes, if any; and returning the blood to the individual.
  • the bodily fluid is removed from the individual for extraco ⁇ oreal binding to an epitope-presenting carrier of this invention.
  • an epitope-presenting carrier for example, apparatuses and methods for removing blood and separating it into its constituent components are known in the art (see, e.g., U.S. Patent Nos. 4,086,924; 4,223,672).
  • the blood or portions thereof are then exposed to the carrier.
  • the carrier neutralizes (i.e., binds) the unwanted antibody, and the blood components are then returned to the individual.
  • the antibody-carrier complex is removed before the fluid is returned to the individual. This may be done, for example, by using a carrier attached to a solid phase, or by using a soluble carrier and selectively removing the complex from the treated solution.
  • the carrier is adapted to render it insoluble.
  • the platform can be chemically adapted during synthesis to include an additional reactive group in the core structure.
  • an additional linkage can be added to a triethlyene glycol structure present in the core. The linkage is then used to attach the platform to an insoluble structure, such as a polystyrene or polyethylene bead, a polycellulose membrane, or other desirable structure.
  • matrices include agarose (a neutral linear polysaccharide generally composed of D-galactose and altered 3,6-anhydrogalactose residues, for example SepharoseTM, Pharmacia), activated gels, nitrocellulose, borosilicate, glass fiber filters, silica, polyvinylchloride, polystyrene, and diazotized paper.
  • agarose a neutral linear polysaccharide generally composed of D-galactose and altered 3,6-anhydrogalactose residues, for example SepharoseTM, Pharmacia
  • activated gels nitrocellulose
  • borosilicate borosilicate
  • glass fiber filters silica
  • silica silica
  • polyvinylchloride polystyrene
  • diazotized paper diazotized paper
  • the biological fluid to be treated is contacted with the solid phase, and antibodies in the fluid complex to the solid phase.
  • the supernatant fluid can then be removed from the solid phase for return to the individual.
  • the solid phase can also be cleared of antibody for repeat use by using a suitable wash, providing both the epitope and the carrier is resistant to the washing solution.
  • suitable washing solutions may include 0.1 M glycine buffer, pH 2.4, dilute acetic acid, or 1 M KSCN buffered to ⁇ pH 7.
  • the antibody-carrier complex can be removed from the fluid by any other appropriate method, including but not limited to microfiltration, antibody capture, or precipitation. Solutions suitable to cause precipitation of the complex depend on the solubility of the complex, and may include ammonium sulfate or polyethylene glycol. If the fluid is to be returned to the individual, then the precipitating solution should be chosen so that any that remains in the fluid does not cause an adverse reaction in the individual.
  • the invention also contemplates devices which can be used for reducing the level of antibody in a biological fluid using an epitope-presenting carrier of this invention.
  • the device will be a flow system, comprising the following elements: a) a port that permits biological fluid to flow into the device; b) a chamber in which the fluid is permitted to contact the epitope-bound carrier (optionally in a solid phase); c) a port that permits the treated fluid to flow out of the device.
  • Such devices can be designed as continuous flow systems, and as systems that permit the treatment of a single sample from an individual for pu ⁇ oses of analysis or readministration at a subsequent time.
  • epitope-presenting carrier(s) may be used for administration.
  • the epitope-presenting carrier(s) may be administered neat.
  • the epitope-presenting carrier(s) is in a composition comprising an epitope-presenting carrier(s) and a pharmaceutically acceptable excipient, and may be in various formulations.
  • Pharmaceutically acceptable excipients are known in the art, and are relatively inert substances that facilitate administration of a pharmacologically effective substance. For example, an excipient can give form or consistency, or act as a diluent.
  • Suitable excipients include but are not limited to stabilizing agents, wetting and emulsifying agents, salts for varying osmolarity, encapsulating agents, buffers, and skin penetration enhancers. Excipients as well as formulations for parenteral and nonperenteral drug delivery are set forth in Remington 's Pharmaceutical Sciences 19th Ed. Mack Publishing (1995). Generally, these compositions are formulated for administration by injection (e.g. , intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). Accordingly, these compositions are preferably combined with pharmaceutically acceptable vehicles such as saline, Ringer's solution, dextrose solution, and the like.
  • pharmaceutically acceptable vehicles such as saline, Ringer's solution, dextrose solution, and the like.
  • the epitope-presenting carrier will constitute about 0.01% to 10% by weight of the formulation due to practical, empirical considerations such as solubility and osmolarity.
  • the particular dosage regimen i.e., dose, timing and repetition, will depend on, inter alia, the clinical indication and the particular individual and that individual's medical history.
  • a dose of about 1 ⁇ g to about 100 mg conjugate/kg body weight, preferably about 100 ⁇ g to about 10 mg/kg body weight, preferably about 50 ⁇ g to about 5 mg/kg body weight, preferably about 1 ⁇ g to about 1 g conjugate/kg body weight, preferably about 5 ⁇ g to about 500 mg body weight is administered.
  • Empirical considerations such as the half life, generally will contribute to determination of the dosage.
  • An epitope-presenting carrier may be administered daily, followed by less frequent administrations, such as two times per week, once a week, or even less frequently.
  • an epitope-presenting carrier(s) is administered less frequently, i.e., bi-weekly, weekly, every ten days, or every two weeks.
  • Frequency of administration may be determined and adjusted over the course of therapy, and is based on maintaining the desired level of antibody.
  • Other appropriate dosing schedules may be as frequent as daily or 3 doses per week, or one dose per week, or one dose every two to four weeks, or one dose on a monthly or less frequent schedule depending on the individual or the disease state.
  • Repetitive administrations may be required to achieve and/or maintain the desired level of antibody.
  • sustained continuous release formulations of the compositions may be appropriate.
  • Other formulations include those suitable for oral administration, which may be suitable if the epitope-presenting carrier is able to cross the mucosa.
  • an aerosol formulation may be suitable.
  • more than one epitope-presenting carrier may be present in a composition.
  • Such compositions may contain at least one, at least two, at least three, at least four, at least five different conjugates.
  • Such "cocktails" as they are often denoted in the art, may be particularly useful in treating a broader range of population of individuals. They may also be useful in being more effective than using only one (or fewer than are contained in the cocktail) epitope-presenting carrier(s).
  • the compositions may be administered alone or in conjunction with other forms of agents that serve to enhance and/or complement the effectiveness of an epitope-presenting carrier. Additionally, or alternatively, a dosage regimen may begin with one epitope- presenting carrier, and then switch to another.
  • An individual suitable for administration of an epitope-presenting carrier(s) is one who exhibits undesirable levels of a disease-associated antibody (such as those described above).
  • Levels of disease-associated antibodies may be determined using standard assays in the art such as ELISA.
  • the individual is human. Measurable circulating levels of disease-associated antibody need not be detectable, it may be predictable (due to, for example, risk factors; genetic factors; environmental factors; and other known etiologies) that such antibodies are likely to be produced.
  • the methods of the invention also pertain to those cases in which a prophylactic effect is contemplated.
  • the epitope-presenting carrier (such as a valency platform conjugate) is administered such that the duration of the effect is longer than when compared to other epitope-presenting carrier(s).
  • considerations such as (a) the particular carrier used; (b) valency; (c) type of epitope); (d) dosage regimen; and (e) means of administration may enter into producing this duration.
  • Any one or more of the above factors may provide a basis for the extent of duration of effect (i.e., length of time that the antibodies are reduced). Similarly, any one or more of the above factors may provide a basis for the rapidity of reduction of antibody.
  • Kits for use in conjunction with the methods of the invention also includes kits for use in conjunction with the methods described herein.
  • the kits effect in vivo reduction of circulating antibody levels (i.e., disease-associated antibody).
  • the kits effect extraco ⁇ oreal selective formation and/or removal of immunosorbent-anti -viral antibody complexes (i.e., ex vivo removal).
  • These kits contain components specific for whatever antibody(ies) is targetted for removal.
  • kits and compositions are provided for use in detection of antibody(ies) to be reduced.
  • kits aid in assessing (a) whether an individual is indicated for selective removal of disease-associated antibody (for example, if the titer is considered to be above a requisite threshold); (b) monitoring an individual after treatment (i.e., selective removal) to determine whether further sufficient removal has occurred and/or whether further is indicated (for example, when a period of time has elapsed since removal, and the titer of anti-viral antibody has risen to or past a requisite threshold, and treatment with a viral therapeutic agent is still indicated); (c) which antibody(ies) an individual is producing (this would indicate which antibody or antibodies should be selectively removed).
  • kits contain components specific for antibodies targeted for reduction and/or removal, aiding detection and/or monitoring.
  • kits of the invention comprise an epitope-presenting carrier (preferably, the carrier is a valency platform molecule) that specifically binds the antibody to be removed in suitable packaging.
  • the kit also contains instructions for its use.
  • Appropriate carriers including those conjugated to epitope have been discussed above.
  • kits of the invention may further comprise reagents for testing for the presence (and/or level) of antibody targetted for reduction and/or removal, which would be useful for monitoring pu ⁇ oses.
  • Example 1 Synthesis of ⁇ Gal epitopes
  • the general analytical methods and characterization techniques used in the present disclosure are identified below. NMR spectra wee recorded on a Bruker AC300 spectrometer at 300 MHz for ⁇ and 75 MHz for 13 C.
  • reaction was proceeded at room temperature with gradual addition of donor until 300 mM / nitrophenol had been formed (3 d).
  • the reaction mixture was lyophilized and the residue was suspended in methanol, filtered and concentrated.
  • the unreacted starting material (2.5 g) was recovered by silica gel chromatography (CH 2 Cl 2 /MeOH, 95:5 to 70:30).
  • the products were first purified on P2 Gel filtration column eluted with water and then on reverse phase HPLC column to yield compound 7 (80 mg, 2.0%) and compound 8 (106 mg, 2.6%) as white solids.
  • a solution of compound C in a suitable solvent is treated with thiobenzoic acid and a suitable base.
  • the mixture is extracted with CH 2 C1 2 , and the CH 2 C1 2 layer is dried and concentrated to give crude product which is purified by silica gel chromatography to give compound D.
  • Compound E is treated with trifluoroacetic acid to remove the BOC protecting groups.
  • the mixture is concentrated, and the residue is dissolved in a solution of NaHCO 3 in 1/1 dioxane/water.
  • To the resulting solution is added eight equivalents of bromoacetic anhydride.
  • the mixture is stirred until complete as evidenced by TLC.
  • the mixture is acidified with aqueous H 2 SO 4 and extracted with CH 2 C1 .
  • the CH 2 C1 2 layer is dried and concentrated to give crude product which is purified by silica gel chromatography to give compound 29.
  • FIGS 26A and 26B Compound 30.
  • the bis-hexaethyleneglycolamine (compound 4 ⁇ ) was reacted with di-tert-butyldicarbonate to yield the N-BOC compound (compound JT), which was then reacted with /> ⁇ r ⁇ -nitrophenylchloro formate to yield the para- nitrophenylcarbonate compound (compound 9 ).
  • the jP ⁇ r ⁇ -nitrophenylcarbonate (PNP) group was then converted to a carbamate group by reaction with mono-CBZ-protected piperazine, yielding compound 1CT.
  • the BOC group was removed using trifluoroacetic acid to yield compound I T.
  • Compounds T_ and Y_ were then reacted together to form a
  • Tetraamino platform, compound F was reacted with the N-hydroxysuccinimidyl ester of N ,N ⁇ -bis-CBZ-lysine in a solution of water/acetonitrile which contained Na 2 CO 3 .
  • the acetonitrile was removed under vacuum, and the product precipitated.
  • the precipitate was washed with water and recrystallized from acetonitrile to give G.
  • the CBZ groups were removed from compound G by catalytic hydrogenation using
  • the tert-butylthio protecting group of ⁇ Gal 7 (30 mg, 0.052 mmol) was removed by reducing with tributylphosphine (25 ⁇ L) in 5 mL of water at room temperature overnight. The reaction mixture was concentrated and dried under high vacuum overnight to remove any residual tert-butylthiol. The residue was dissolved in 5 mL of Na 2 CO 3 (10 mg/mL) solution in water/ ACN (1 :1). Tetrameric platform 25 (5.0 mg, 0.0074 mmol) was added and the resulted solution was stirred at room temperature overnight.
  • This conjugate is prepared by following the procedure described above for compound 38 using ⁇ Gal 7 and platform 28.
  • Antibodies Blood was drawn from healthy normal volunteers. Plasma was separated by centrifugation and allowed to clot. Fibrin was removed and plasma was used immediately or stored in aliquots at -70°C. Rhesus monkey serum (California Regional Primate Research Center, Davis, CA) was obtained from blood drawn into vacutainer tubes and allowed to clot. After serum was separated by centrifugation, it was pooled, aliquoted and stored at -20°C or -70°C. In some experiments, sera was heat-inactivated at 56°C for 30 minutes to destroy complement hemolytic activity.
  • Rhesus monkey serum California Regional Primate Research Center, Davis, CA
  • Antibodies to the ⁇ Gal epitope were affinity purified on an ⁇ Gal-Sepharose column, which was prepared by coupling ⁇ Gal-SH to maleimide-Sepharose (Pierce) through Michael addition chemistry at 10 mg/mL resin. Up to 20 mL pooled NHS (or NMS) normal monkey serum was applied to a 2 mL volume of packed ⁇ Gal-Sepharose. After the flow through was collected, the column was washed with 10-20 column volumes or until A 280 reached baseline values and eluted with 0.1 M triethanolamine, pH 11.5 into tubes containing 1M Tris, pH 8.0. The column was immediately washed with 10-20 volumes of phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • ⁇ Gal-SH was coupled to maleimide-BSA (bovine serum albumin) (Pierce) at a 2: 1 ratio (w/w) according to manufacturer's protocol. The ratio of ⁇ Gal molecules coupled per BSA molecule was 10-12:1 or 25:1.
  • ⁇ Gal-BSA or ⁇ Gal-HAS human serum albumin
  • ⁇ Gal-BSA 100 ⁇ l at 5 ⁇ g/mL in PBS
  • Plates were stable for at least 3 months. New lots of plates were compared with binding efficacy of the original lot using pooled standard serum. Pooled standard sera, individual sera or affinity purified anti- ⁇ Gal Ig were titered. Serum (100 ⁇ L neat - 1/256 diluted in HBSA) or affinity-purified anti- ⁇ Gal IgG (100 ⁇ L of serial two-fold dilutions from 2 mg/mL - 1 ⁇ g/mL in Hank's balanced salt solution without Ca +2 or Mg +2 (HBSA)) were incubated in ⁇ Gal-BSA coated wells for 60 minutes at 20°C.
  • Serum 100 ⁇ L neat - 1/256 diluted in HBSA
  • affinity-purified anti- ⁇ Gal IgG 100 ⁇ L of serial two-fold dilutions from 2 mg/mL - 1 ⁇ g/mL in Hank's balanced salt solution without Ca +2 or Mg +2 (HBSA)
  • anti- ⁇ Gal Ig was developed with predetermined saturating concentrations (100 ⁇ L, usually 1 : 1000 dilution) of anti-monkey or anti -human IgG or IgM coupled to alkaline phosphatase for 60 minutes at 20°C. After washing the wells 5 times with wash buffer (1% Tween 20 in PBS), plates were developed with 100 ⁇ L PPMP (phenolphthalein monophosphate) (Sigma) for 5-20 minutes at 20°C. Reactions were stopped by addition of
  • ⁇ Gal (galactose ( ⁇ l, 3, galactose) epitopes were synthesized at a multigram scale as described in Example 1 and were coupled to a well-defined organic platform as described in Example 2.
  • dilution binding concentration was -12.5 ⁇ g/mL.
  • Serum or Ig 50 ⁇ L was incubated with an equal volume of HBSA containing inhibitor which was serially diluted in a two- fold manner from 4 mg/mL to 10 ⁇ g/mL or buffer alone for 60 minutes at 20°C.
  • ELISpot assay Spleens from normal rhesus monkeys were minced and prepared as single cell suspension using deburred frosted glass slides. Contaminating erythrocytes were hypotonically lysed and mononuclear cells (MNC) isolated by Ficoll-hypaque density gradient centrifugation. MNC (100 ⁇ L) were added in serial two-fold dilutions from
  • ExtrAvidin-alkaline phosphatase (Sigma). Alkaline phosphatase substrate B (100 ⁇ L at 1 :100 dilution, Bio-Rad, Hercules, CA) was added and incubation continued overnight at 20°C. Total Ig-producing cells were similarly determined. The footprints were quantified using a Microtek ScanMaker III flat-bed scanner and personal computer utilizing the Image-Pro imaging software (Media Cybernetics, Univ. Rochester Medical School,
  • Cytoxicity assays The porcine kidney epithelial cell line PK-15 and porcine aortic endothelial cells (PAEC) (ATCC, 10801 University Boulevard., Manassas, VA 20110-2209) were cultured as directed. For the assay, cells were removed with trypsin - EDTA and replated subconfluently in 96 well plates or on coverslips in 24-well plates. While still subconfluent (within 2 days of replating), cells were used in cytotoxicity assays. Neat, complement-sufficient serum was incubated with inhibitor as described for 60 minutes at 4°C. Serum was then added to wells containing subconfluent cells from which medium had been aspirated immediately prior to serum addition.
  • PAEC porcine kidney epithelial cell line
  • PAEC porcine aortic endothelial cells
  • Wells were incubated with serum + inhibitor for 60-90 minutes at 37°C. Wells were rinsed and cell death was visualized using a Live/Dead kit (Molecular Probes, Eugene, OR) and quantified microscopically in 10 high powered fields or by counting 250 cells. For some experiments, cells were non- enzymatically removed from flasks with cell dissociation solution (Sigma) and single cell suspensions prepared. Assays were performed as for adherent cells except that cytoxicity was quantified by flow cytometry on a Becton-Dickinson FACScalibur.
  • the synthetically prepared ⁇ Gal epitope (Figure 2) was antigenically active, as demonstrated by its ability (when coupled to Sepharose) to remove >95% of anti- ⁇ Gal Ig from normal rhesus monkey or human serum as measured by FACS.
  • ELISA analysis showed that the ⁇ Gal epitope when presented on a platform as an octamer inhibited both IgG and IgM anti- ⁇ Gal in serum from binding to BSA- ⁇ Gal or to the ⁇ Gal-expressing porcine kidney epithelial cell line PK-15 and was 5-10 fold more efficient at inhibiting the
  • LJP 920 (cpd 46) was well-tolerated in the treated animals with no untoward effects as observed by veterinary staff.
  • IgG anti- ⁇ Gal levels were decreased by 11%, similar to the levels seen with tetramer.
  • Control animals showed little change (Fig. 22A).
  • IgM anti- ⁇ Gal levels in one monkey and 5% in the replicate animal did not change in one animal and increased in the replicate animal, as shown in Figure 22B.
  • That the octamer is more efficient than tetramer at clearing IgM anti- ⁇ Gal is shown in Fig. 23.

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Abstract

L'invention concerne des procédés permettant de réduire la concentration d'anticorps circulant, et notamment des anticorps associés à des maladies. Ces méthodes consistent à administrer à un individu des quantités efficaces de supports présentant des épitopes. Dans d'autres modes de réalisation, l'invention concerne des procédés utilisant des supports présentant des épitopes et permettant de réduire la concentration d'anticorps en circulation.
PCT/US1999/029336 1998-12-08 1999-12-09 Procedes et formulations permettant de reduire des anticorps circulants WO2000033887A2 (fr)

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CA002353620A CA2353620A1 (fr) 1998-12-09 1999-12-09 Procedes et formulations permettant de reduire des anticorps circulants
EP99966115A EP1135167A2 (fr) 1998-12-09 1999-12-09 Procedes et formulations permettant de reduire des anticorps circulants
AU21735/00A AU2173500A (en) 1998-12-09 1999-12-09 Methods and formulations for reducing circulating antibodies
JP2000586377A JP2002531531A (ja) 1998-12-09 1999-12-09 循環している抗体を減少させるための方法および処方物

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US7081242B1 (en) 1999-11-28 2006-07-25 La Jolla Pharmaceutical Company Methods of treating lupus based on antibody affinity and screening methods and compositions for use thereof
EP2583678A2 (fr) 2004-06-24 2013-04-24 Novartis Vaccines and Diagnostics, Inc. Immunopotentiateurs de petites molécules et dosages pour leur détection
US10662212B2 (en) 2014-03-13 2020-05-26 Universitat Basel Carbohydrate ligands that bind to IGM antibodies against myelin-associated glycoprotein
EP3715376A1 (fr) 2019-03-23 2020-09-30 Ablevia biotech GmbH Composé pour la prévention ou le traitement de la myasthénie grave
EP3715374A1 (fr) 2019-03-23 2020-09-30 Ablevia biotech GmbH Composé de séquestration d'anticorps indésirables chez un patient
EP3715375A1 (fr) 2019-03-23 2020-09-30 Ablevia biotech GmbH Composé pour la prévention ou le traitement de la pré-éclampsie
US11091591B2 (en) 2015-09-16 2021-08-17 Universität Basel Carbohydrate ligands that bind to antibodies against glycoepitopes of glycosphingolipids
WO2022063887A1 (fr) 2020-09-23 2022-03-31 Ablevia Biotech Gmbh Composé permettant d'augmenter l'efficacité d'une thérapie de remplacement du facteur viii
WO2022063892A1 (fr) 2020-09-23 2022-03-31 Ablevia Biotech Gmbh Composé pour augmenter l'efficacité de vecteurs viraux
WO2022063880A1 (fr) 2020-09-24 2022-03-31 Ablevia Biotech Gmbh Composé pour prévenir ou traiter la myasthénie grave
WO2022063885A1 (fr) 2020-09-23 2022-03-31 Ablevia Biotech Gmbh Composé pour la séquestration d'anticorps anti-peg indésirables chez un patient
WO2022063882A1 (fr) 2020-09-23 2022-03-31 Ablevia Biotech Gmbh Composé pour la prévention ou le traitement d'affections médiées par des autoanticorps
WO2022063879A1 (fr) 2020-09-23 2022-03-31 Ablevia Biotech Gmbh Composé pour la séquestration d'anticorps indésirables chez un patient
WO2023180502A1 (fr) 2022-03-24 2023-09-28 Ablevia Biotech Gmbh Composé pour augmentation de l'efficacité des virus oncolytiques
US11986536B2 (en) 2019-03-23 2024-05-21 Ablevia Biotech Gmbh Compound for the sequestration of undesirable antibodies in a patient

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WO1993003735A1 (fr) * 1991-08-23 1993-03-04 Alberta Research Council Procedes et compositions d'attenuation de rejet de xenogrephe induit par les anticorps chez les receveurs humains
WO1995007073A1 (fr) * 1993-09-08 1995-03-16 La Jolla Pharmaceutical Company Molecules plates-formes non polymeres a valence, chimiquement definies, et leur conjugues
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Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7081242B1 (en) 1999-11-28 2006-07-25 La Jolla Pharmaceutical Company Methods of treating lupus based on antibody affinity and screening methods and compositions for use thereof
EP2583678A2 (fr) 2004-06-24 2013-04-24 Novartis Vaccines and Diagnostics, Inc. Immunopotentiateurs de petites molécules et dosages pour leur détection
US10662212B2 (en) 2014-03-13 2020-05-26 Universitat Basel Carbohydrate ligands that bind to IGM antibodies against myelin-associated glycoprotein
US11220523B2 (en) 2014-03-13 2022-01-11 Universität Basel Carbohydrate ligands that bind to IgM antibodies against myelin-associated glycoprotein
US11091591B2 (en) 2015-09-16 2021-08-17 Universität Basel Carbohydrate ligands that bind to antibodies against glycoepitopes of glycosphingolipids
EP3960769A1 (fr) 2019-03-23 2022-03-02 Ablevia biotech GmbH Composé de séquestration d'anticorps indésirables chez un patient
US11986536B2 (en) 2019-03-23 2024-05-21 Ablevia Biotech Gmbh Compound for the sequestration of undesirable antibodies in a patient
WO2020193486A1 (fr) 2019-03-23 2020-10-01 Ablevia Biotech Gmbh Composé pour la séquestration d'anticorps indésirables chez un patient
EP3715375A1 (fr) 2019-03-23 2020-09-30 Ablevia biotech GmbH Composé pour la prévention ou le traitement de la pré-éclampsie
EP3715374A1 (fr) 2019-03-23 2020-09-30 Ablevia biotech GmbH Composé de séquestration d'anticorps indésirables chez un patient
EP3715376A1 (fr) 2019-03-23 2020-09-30 Ablevia biotech GmbH Composé pour la prévention ou le traitement de la myasthénie grave
US12011484B2 (en) 2019-03-23 2024-06-18 Ablevia Biotech Gmbh Compound for the sequestration of undesirable antibodies in a patient
WO2020193487A1 (fr) 2019-03-23 2020-10-01 Ablevia Biotech Gmbh Composé destiné à la prévention ou au traitement de myasthénie grave
WO2022063892A1 (fr) 2020-09-23 2022-03-31 Ablevia Biotech Gmbh Composé pour augmenter l'efficacité de vecteurs viraux
WO2022063885A1 (fr) 2020-09-23 2022-03-31 Ablevia Biotech Gmbh Composé pour la séquestration d'anticorps anti-peg indésirables chez un patient
WO2022063882A1 (fr) 2020-09-23 2022-03-31 Ablevia Biotech Gmbh Composé pour la prévention ou le traitement d'affections médiées par des autoanticorps
WO2022063879A1 (fr) 2020-09-23 2022-03-31 Ablevia Biotech Gmbh Composé pour la séquestration d'anticorps indésirables chez un patient
WO2022063887A1 (fr) 2020-09-23 2022-03-31 Ablevia Biotech Gmbh Composé permettant d'augmenter l'efficacité d'une thérapie de remplacement du facteur viii
WO2022063880A1 (fr) 2020-09-24 2022-03-31 Ablevia Biotech Gmbh Composé pour prévenir ou traiter la myasthénie grave
WO2023180502A1 (fr) 2022-03-24 2023-09-28 Ablevia Biotech Gmbh Composé pour augmentation de l'efficacité des virus oncolytiques

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EP1135167A2 (fr) 2001-09-26

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