WO1992004053A1 - Immunoglobulines homoconjuguees - Google Patents

Immunoglobulines homoconjuguees Download PDF

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Publication number
WO1992004053A1
WO1992004053A1 PCT/US1991/006195 US9106195W WO9204053A1 WO 1992004053 A1 WO1992004053 A1 WO 1992004053A1 US 9106195 W US9106195 W US 9106195W WO 9204053 A1 WO9204053 A1 WO 9204053A1
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Prior art keywords
homoconjugated
antibodies
antigen
pharmaceutical composition
monoclonal antibodies
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PCT/US1991/006195
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English (en)
Inventor
Edith Ann Wolff
Howard V. Raff
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Bristol-Myers Sqibb Company
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Application filed by Bristol-Myers Sqibb Company filed Critical Bristol-Myers Sqibb Company
Priority to KR1019930700605A priority Critical patent/KR930702029A/ko
Publication of WO1992004053A1 publication Critical patent/WO1992004053A1/fr
Priority to FI930846A priority patent/FI930846A/fi
Priority to NO93930663A priority patent/NO930663L/no

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3015—Breast
    • 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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6881—Cluster-antibody conjugates, i.e. the modifying agent consists of a plurality of antibodies covalently linked to each other or of different antigen-binding fragments covalently linked to each other
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]

Definitions

  • Monoclonal antibodies offer great promise as extremely promise as extremely specific immunotherapeutic agents with potentially minimal side effects.
  • monoclonal antibodies are being developed for a wide variety of applications, such as the treatment of tumors, infectious diseases and autoimmune disorders, regulation of the immune system, and others.
  • few monoclonal antibodies have the qualities that enable.them to successfully make the transition from research and development to clinical regimen.
  • the therapeutic or diagnostic usefulness of a monoclonal antibody results from several factors, in addition to simply binding the desired antigen.
  • the antibody must possess sufficient binding affinity, a measure of the inherent strength of the antibody binding to its corresponding epitope.
  • the monoclonal antibody which is selected must be of an appropriate isotype or subclass thereof to efficiently initiate desired effector functions. These functions include fixation of complement, binding to effector macrophages or polymorphonuclear leukocytes, or other properties that may be required in a particular therapeutic application. Isotype also affects antibody bio-distribution, half-life, transplacental passage, and other characteristics. In general, IgG antibodies would be preferred over IgM antibodies for most therapeutic uses.
  • IgG ⁇ When compared to IgMs, IgG ⁇ typically possess longer in vivo half-lives, are able to cross the placenta to the fetus, and when formulated as a pharmaceutical composition may have a longer shelf life.
  • IgG molecules are monomeric, however, and have only two antigen binding sites so the avidity is much lower than with a comparable IgM antibody, which is pentavalent and has ten antigen binding sites. With conventional technology it is frequently very difficult to identify monoclonal antibodies having the desired antigen binding specificity, -affinity, avidity and effector functions. Recombinant DNA techniques have been developed to avoid the unpredictable and labor intensive method of simply screening large numbers of antibody-producing fused or transformed cells.
  • the avidity of IgG antibodies could be improved by increasing the valency of the molecule to greater than two. More interactions between antibody and antigen would result in tighter binding and would stabilize the antibody-antigen interaction, generally an important attribute for therapeutic use. IgG antibodies of high avidity (via multivalent attachment) and which have the desired effector functions would be greatly preferred over comparable antibodies of low avidity, but to date antibodies having these characteristics have not been described.
  • Ho oconjugated antibodies possess increased therapeutic effectiveness when compared to the corresponding parental antibody monomer. This activity may be due to, inter alia, interactions of higher avidity and increased effector functions. Accordingly, antibodies which bind to the same antigen, and more particularly to the same antigenic determinant, are covalently bonded via cross-linking to one another by synthetic chemical coupling to produce such homoconjugates.
  • the homoconjugates comprise at least two to three antibody molecules, typically of the IgG class.
  • the antibodies are preferably monoclonal antibodies, and may be any of a variety of species. For administration to humans the antibodies will usually be human or murine in origin or have human constant regions.
  • compositions which comprise a pharmaceutically acceptable carrier and at least two IgG antibody molecules, which bind to substantially the same antigenic determinant, chemically bonded to one another by synthetic covalent linkage.
  • the homoconjugated antibodies and pharmaceutical compositions thereof can be used therapeutically in methods of treatment of antigen related diseases to, e.g., protect against infection, such as by ___, coli or group B streptococci, inhibit the growth of tumors, including breast and other tumors, regulate the immune response, and the like.
  • IgG antibodies are able to pass the placenta the preparations can be used to treat a fetus in utero.
  • the invention provides a substantial improvement in methods for therapeutic administration of monoclonal antibodies to a patient for treatment of an antigen related disease.
  • the improvement comprises administering to the patient covalently cross-linked homoconjugated monoclonal antibodies having at least two IgG antibody molecules which bind to the same antigenic determinant of the antigen related to the disease.
  • the antibodies are cross-linked via disulfide bonds.
  • Fig. 1 shows chromatograms of FPLC profiles of the IgG homoconjugate mixtures, with retention time along x-axis and ⁇ 280 along y-axis; Peaks labeled A, B and C, represent trimer, dimer and monomer fractions, respectively;
  • Fig. 2 illustrates the increased binding activity in .EIAs of homoconjugates (dimers or tri ers) compared to initial monomers of monoclonal antibody D3, a human IgG monoclonal antibody which binds to the group carbohydrate of group B streptococci;
  • Fig. 3 illustrates the increased binding activity of homoconjugates (dimers or trimers) compared to initial monomers of monoclonal antibody 5E1-G, a human IgG monoclonal antibody which binds to the capsular carbohydrate of £. coli Kl;
  • Fig. 4 illustrates the increased binding activity in EIAs of homoconjugates (dimers) compared to initial monomers of BR64, a murine IgG monoclonal antibody which binds to a human breast tumor associated antigen;
  • Fig. 5 illustrates the comparative binding activity of homoconjugated chimeric BR96 antibody against tumor cell lines, where Fig. 5A shows binding activity against human breast tumor cell line H3760B, Fig. 5B shows binding activity against human lung tumor cell line H2707, Fig. 5C shows binding activity against human lung tumor cell line H2987, and Fig. 5D is binding activity against human breast tumor cell line H3396;
  • Fig. 6 shows the increased opsonic activity against group B streptococci by dimer and trimer homoconjugates of human monoclonal antibody D3 compared to the initial IgG monomer
  • Fig. 7 shows the enhanced opsonophagocytosis by monoclonal antibody D3 homoconjugates against group B ⁇ treptococcal strains M94 and 1334 compared to the activity by the D3 monomer antibody;
  • Fig. 8 shows the increased opsonic activity against £. coli Kl of dimer and trimer homoconjugates of human monoclonal antibody 5E1-G compared to the initial IgG monomer;
  • Fig. 9 depicts enhanced opsonophagocytosis conferred by homoconjugates of monoclonal antibody 5E1-G against strains H16 and A14 of ___, coli Kl compared to the antibody monomer;
  • Fig. 10 shows increased complement dependent cytotoxicity against breast tumor cell line H3630 by dimer homoconjugates of monoclonal antibody BR64 compared to the initial IgG monomer;
  • Fig. 11 illustrates the cytotoxicity shown by
  • Fig. 12 shows the ___. vivo protection conferred by homoconjugates of monoclonal antibody D3 and control monomer at different concentrations of antibody.
  • the present invention provides homoconjugates of monoclonal antibodies against selected antigens, and methods for preparing such homoconjugates.
  • homoconjugates are prepared which possess increased valency and two or more Fc regions.
  • the invention provides the ability to convert antibodies of perhaps limited in vivo utility to antibodies having characteristics significantly more conducive to a desired therapeutic activity.
  • homoconjugation may serve to convert an IgG monomer of low binding avidity to one of higher avidity and better able to promote effector functions that were perhaps not previously attainable.
  • homoconjugate is meant the covalent association or linking of two, three or more antibody molecules which bind to the same antigenic determinant, thereby forming antibody homodi ers, homotrimers, etc.
  • the homoconjugates may be prepared from two, three or . more different monoclonal antibodies (i.e., those produced by different immortalized cell lines) which bind to the same antigenic determinants (epitopes) on the antigen.
  • the monoclonal antibodies which comprise the ho oconjugate may be different (produced by distinct cell lines) but preferably they are the same, i.e., obtained from the same cell line, and thus constitute a relatively homogeneous preparation of monoclonal antibodies with virtually identical antigen binding specificity.
  • binding to the same or substantially the same epitope is meant to refer to monoclonal antibodies which are capable of reciprocal or non-reciprocal competition with the other for binding to the antigen.
  • competition immunoassays such as by radioimmunoassay or enzyme im unoassay, as generally described in, e.g., U.S. Pat. 3,817,837; Harlow and Lane, Antibodies. A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY (1988); and Day, Advanced Immunochemistry. 2d ed., Wiley-Liss Publications, NY (1990) , each incorporated herein by reference.
  • the Fc regions of the onoclonals used for homoconjugation, or other aspects of the immunoglobulin molecule which do not substantially affect antigen binding specificity, may also be altered to produce desired effector functions. For example, it may be desirable to substitute a Fc domain for protein A binding into a molecule not having that capacity, for ease of purification or the like. Other substitutions may provide for decreased immunogenicity, increased or decreased complement activation, cell receptor binding, control of catabolic rate, placental and gut transfer, ability to participate in antibody-dependent cellular cytotoxicity, and other aspects of immune regulation. A number of antibody functions have been localized to a constant region domain or domains. See. Paul, Fundame ⁇ t?l Ippunolo ⁇ y.
  • the homoconjugated immunoglobulins may be any of the heavy chains and subclasses thereof.
  • the light chains may be either kappa or lambda.
  • homoconjugates of antibodies having gamma heavy chains so as to form homoconjugated multivalent IgG molecules.
  • IgG subclasses of 1, 2, 3 and 4 (human) and 1, 2a, 2b and 3 (murine) human subclasses 1 and 3 and murine subclasses 1, 2a and 2b are generally preferred for applications requiring maximum complement fixation, binding to monocytes, macrophages and polymorphonuclear cells, and the ability to cross the placenta.
  • the effector functions of human IgG, and IgG. antibodies may also be substantially increased by the homoconjugation procedures described herein.
  • antibodies having alpha, mu, epsilon or delta type heavy chains may also be employed for homoconjugation as described herein.
  • the binding affinity of the antibodies for use in homoconjugates will vary, but will generally be at least l ⁇ "4 M, typically at least about 10 ⁇ 6 M to l ⁇ "7 M, and preferably at least about 10 —8 to 10 —9' M or greater.
  • the avidity of the homoconjugates prepared from such antibodies should generally be at least about 10 ⁇ 6 M to 10 ⁇ 7 M, and preferably at least about 10 ⁇ 8 to l ⁇ "*10 M or greater. Means for determining affinity and avidity are known, as described in Day, Advanced Immunochemistrv.
  • homoconjugates may have quantitative increases in avidity, generally the homoconjugates should also have qualitative increases in avidity and effector functions, e.g., those evidenced by antigen binding tests and other functional assays as described herein and as will generally be known to one of ordinary skill in the art.
  • the homoconjugated immunoglobulins may be of any species or combination thereof from which monoclonal antibodies may be prepared. Although it has generally been relatively easy to produce murine monoclonal antibodies of a desired antigen binding specificity, it has been much more difficult to produce human monoclonal antibodies of the desired specificity and having the desired constant region properties. Human monoclonal antibodies are preferable for many applications, especially in vjvp diagnosis and therapy of humans to minimize their recognition as foreign by a patient's immune system.
  • a "human” antibody refers to one that is substantially human in origin but may also contain some non-human and/or non-immunoglobulin sequences.
  • immunoglobulin used synonymously herein with antibody, it will be understood that some non-immunoglobulin sequences may be present in the molecule while retaining the ability to bind antigen.
  • Immunoglobulin refers to both whole immunoglobulins and binding fragments thereof.
  • the antibodies which are used for homoconjugation may be substantially monospecific, i.e., relatively pure preparations of substantially homogeneous antibodies obtained from polyclonal antisera, or may be monoclonal antibodies.
  • Monoclonal antibodies which bind to a desired antigen or epitope thereof are obtained from an established cell line which secretes them.
  • the antibody-producing cell line may be isolated from B cells of several species using conventional fusion, viral transformation or other immortalization techniques well known to those skilled in the art.
  • human monoclonal antibodies may be generated using Epstein- Barr virus (EBV) transformation, hybridoma fusion techniques, or combinations thereof. See, for example, EBV) transformation, hybridoma fusion techniques, or combinations thereof. See, for example,
  • monoclonal antibody an antibody produced by a clonal, immortalized cell line separate from cells producing antibodies with a different antigen binding specificity.
  • monoclonal antibodies are produced and isolated from other monoclonal antibodies and, accordingly, in substantially pure form (relative to other antibodies) and at a concentration generally greater than normally occurring in sera from the animal species which serves as the B cell source.
  • antigen related disease is meant a disease whose manifestation coincide with the clinical presence of a foreign antigen (e.g., bacteria, virus, tumor or tumor associated antigen) or self antigen (as with autoimmune diseases) .
  • a foreign antigen e.g., bacteria, virus, tumor or tumor associated antigen
  • self antigen as with autoimmune diseases
  • monoclonal antibodies have been described in the technical and patent literature, many of which are publicly available from cell depositories, such as the American Type Culture Collection, 12301 Parklawn Dr., Parkville, MD 20852, whose catalogue, ATCC Catalogue of Cell Lines and Hvbridoroas. 6th ed. (1988), is incorporated herein by reference.
  • Representative examples of monoclonal antibodies are described in, e.g., U.S. Pat. Nos. 4,596,769, 4,689,299, 4,753,894, 4,834,975, 4,834,976, 4,925,800, and 4,958,009, each of which is incorporated herein by reference.
  • the methods described herein provide the ability to produce novel cross-linked homoconjugates from immunoglobulins obtained from such cell lines.
  • the chemically linked homoconjugated immunoglobulins will be produced by chemical conjugation of antibodies using well known laboratory procedures, such as by employing cross-linking reagents.
  • chemically linked is meant that the immunoglobulin molecules are synthetically linked, i.e., not produced as such by a cell, to one another by covalent bonds.
  • a preferred method of conjugation is the formation of at least one covalent bond between the immunoglobulin molecules.
  • the immunoglobulin molecules are complexed or chemically bonded together by any of a variety of well known chemical linking procedures.
  • the Fc regions or Fab regions may serve as the site of the linkage.
  • the linkage may be direct, which includes linkages containing a synthetic linking group, or indirect, by which is meant a link having an intervening moiety, such as a protein or peptide, e.g., plasma albumin, or other spacer molecule.
  • the linkage may be by way of heterobifunctional or homobifunctional cross-linkers, e.g., carbodiimide, glutaraldehyde, N-succinimidyl 3-(2- pyridydithio) propionate (SPDP) and derivatives, bis- maleimide, 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) , cross-linking without exogenous cross-linkers by means of groups reactive with the individual molecules, such as carbohydrate, disulfide, carboxyl or amino groups via oxidation or reduction of the native protein, or treatment with an enzyme or the like.
  • heterobifunctional or homobifunctional cross-linkers e.g., carbodiimide, glutaraldehyde, N-succinimidyl 3-(2- pyridydithio) propionate (SPDP) and derivatives, bis- maleimide, 4-(N-maleimidomethyl)cyclo
  • Detection and purification of the homoconjugated immunoglobulins may be accomplished by a variety of techniques, including liquid and affinity chromatography, gradient centrifugation, and gel electrophoresis, among others. Increased activity of the homoconjugates may be measured by quantitative antigen binding assays, antibody competition experiments, opsonophagocytic assays, complement dependent cytotoxicity assays, and the like. These techniques are familiar to those skilled in the art, and are described in, for example, Harlow and Lane, pupra.
  • homoconjugated antibody preparations with increased binding ability will likely be useful in the treatment and diagnosis of a wide variety of conditions referred to herein as antigen related diseases.
  • the homoconjugates will offer significantly improved therapeutic and diagnostic characteristics compared to the unconjugated monomeric antibody. Due to the increased avidity of the homoconjugates, it is now possible in certain instances to convert a previously non-protective or weakly protective IgG antibody to be protective against infection or tumors, for example, or to act as an immunomodulator by potentiating or otherwise regulating a host's immune response to a particular antigen.
  • an IgM antibody to an antigen or particular epitope of the antigen is protective and a monomeric IgG antibody is non-protective or weakly protective
  • a homoconjugate produced using the methods described herein may provide sufficient avidity to confer significant protection against infection, cell killing, etc.
  • an IgG dimer or trimer homoconjugate may possess therapeutic anti-infective qualities that may be found with certain multivalent antibodies such as
  • IgMs but also have qualities inherent to IgG monomers, such as their ability to cross the placenta, to bind to macrophages and PMNs, and the lack of a requirement for complement to mediate opsonization.
  • the IgG homoconjugates may possess other attributes typically associated with IgGs, such as ease of purification, increased stability, increased shelf life, and increased half-life in vivo.
  • the homoconjugate preparations will be useful against a range of targets, such as bacterial and viral antigens, depending of course on the particular specificity of a homoconjugate's antigen binding region, they will be especially useful where the killing of mammalian cells is required.
  • the homoconjugates can be used for the treatment of cancer cells which display particular tumor-associated antigens (e.g., breast or lung tumor associated antigens), the inhibition or killing of mammalian cells infected with viruses or bacteria or cells which express antigens associated with a particular autoimmune disease.
  • the homoconjugates can also be used to eliminate selected cells from bone marrow or in the im unosuppression of graft recipients, etc.
  • the present invention is not limited to antibody homoconjugates which are protective or show other such functional attributes in vivo, as increased avidity also makes feasible an array of diagnostic procedures perhaps not otherwise available to a bivalent monomer of low affinity and/or low avidity.
  • the ability of the resultant antibodies to inhibit a tumor, such as a breast or lung tumor, to act as an immunomodulator, or to protect against challenge by a pathogen, for example, can be measured in a wide variety of in vitro and in vivo systems, as will be known to the skilled artisan.
  • compositions for parenteral administration which comprise a solution of the homoconjugated monoclonal antibody preparations or a cocktail of homoconjugated and monomeric antibodies dissolved in an acceptable carrier, preferably an aqueous carrier.
  • aqueous carriers can be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine and the like.
  • compositions may be sterilized by conventional, well known sterilization techniques.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.
  • the concentration of antibody in these formulations can vary widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected, the condition being treated, e.g., an infectious disease such as a group B ⁇ treptococcal or ___ coli infection, a tumor, such as breast carcinoma, etc., and the subject being treated, i.e., an adult, child or neonate.
  • an infectious disease such as a group B ⁇ treptococcal or ___ coli infection
  • a tumor such as breast carcinoma, etc.
  • the subject i.e., an adult, child or neonate.
  • a typical pharmaceutical composition for intravenous infusion to treat an infection in an adult could be made up to contain 250 ml of sterile Ringer's solution, and about 100 mg to 10 grams of antibody.
  • Actual methods for preparing parenterally or orally administrable compounds will be known or apparent to those skilled in the art and are described in more detail in for example, Remington's Pharmaceutica Science,. 16th ed., Mack Publishing Company, Easton, PA (1982), which is incorporated herein by reference.
  • compositions containing the present homoconjugated antibodies or a cocktail thereof can be administered for prophylactic and/or therapeutic treatments.
  • compositions are administered to a patient already suffering from a disease, in an amount sufficient to cure or at least partially arrest the disease and its complications. An amount adequate to accomplish this is defined as a
  • therapeutically effective dose Amounts effective for this use will depend on the severity of the disease, i.e., infection, tumor, etc., the age of the patient and the general state of the patient's immune system. Generally, the amounts will range from about 0.1 to about 50 mg of antibody per kilogram of body weight per dose, with dosages of from 5 to 25 mg of antibody per kilogram per patient being more commonly used. It must be kept in mind that the materials of the present invention may generally be employed in serious disease states, that is, life-threatening or potentially life threatening situations.
  • compositions containing the present antibodies or cocktails thereof are administered to a patient not already in a disease state to enhance the patient's resistance.
  • Such an amount is defined to be a "prophylactically effective dose.”
  • the precise amounts again depend on the patient's state of health and general level of immunity, but generally range from 0.1 to 25 mg per kilogram, especially 0.5 to 2.5 mg per kilogram.
  • a preferred prophylactic use is for treatment of fetuses and neonates at risk from infection through their mothers. When treatment is dependent on passage through the placenta, the dosage may require adjustment to reflect the percentage of antibody which is able to pass from the blood of the pregnant female to that of the fetus.
  • compositions can be carried out with dose levels and pattern being selected by the treating physician.
  • pharmaceutical formulations should provide a quantity of homoconjugated antibody sufficient to treat the patient.
  • the homoconjugated antibodies of the invention may also find several uses in vitro.
  • the homoconjugated IgG antibodies of Example I below can be used for detecting the presence of group B streptococci or ⁇ __ coli Kl, for vaccine preparation, or the like.
  • the antibodies may be either labeled or unlabeled.
  • Unlabeled homoconjugated antibodies may find particular use in agglutination assays, or they may be used in combination with other labeled antibodies (second antibodies) that are reactive with the homoconjugated antibodies, such as antibodies specific for the Fc regions.
  • the antibody may be directly labeled.
  • labels may be employed, such as radionuclides, particles (e.g. gold, ferritin, magnetic particles, red blood cells), fluors, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (particularly haptens) , etc.
  • immunoassays Numerous types of immunoassays are available and are known to those skilled -in the art, such as competitive and sandwich assays as described in, e.g., U.S. Pat. 4,376,110, incorporated by reference herein, and Harlow and Lane, supra.
  • Kits can also be supplied for use with the subject antibodies in the protection against or detection of the presence of a selected antigen.
  • the subject antibody compositions of the present invention may be provided, usually in lyophilized form in a container, either alone or in conjunction with additional antibodies.
  • the antibodies which may be conjugated to a label or toxin, or unconjugated, are included in the kits with buffers, such as Tris, phosphate, carbonate, etc., stabilizers, biocides, inert proteins, e.g., serum albumin, or the like, and a set of instructions for use. Generally, these materials will be present in less than about 5% wt.
  • a second antibody capable of binding to the homoconjugated antibodies is employed in an assay, this will be present in a separate vial.
  • the second antibody is typically conjugated to a label and formulated in an analogous manner with the antibody formulations described above.
  • This example demonstrates means for preparing homoconjugates of several representative monoclonal antibodies to selected tumor and bacterial antigens. The homoconjugates were then tested in functional assays described in the examples which follow.
  • Monoclonal antibody D3 a human IgG 1 antibody which binds to the group B carbohydrate of group B streptococci.
  • 5E1-G a human IgG, monoclonal antibody which binds to the capsular carbohydrate of ___ coli Kl.
  • BR64 a murine IgG. ⁇ monoclonal antibody which binds to human carcinoma associated antigen, including colon, breast, ovary and lung carcinomas.
  • BR64 is on deposit with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, M.D, as ATCC No. HB 9895.
  • BR96 also on deposit with the American Type Culture Collection as ATCC No. HB 10036, is an IgG human-murine chimeric IgG monoclonal antibody which binds to human lung and breast tumor associated antigens.
  • Homoconjugates of each of the antibodies were prepared using maleimidobutyrloxysuccinimide and i inothiolane according to the following protocol.
  • Antibodies (1 mg/ml) were dialyzed overnight against a coupling buffer (0.1M Na 2 HP0 4 -dibasic, seven-hydrate, 0.1M NaCl, pH 7.5).
  • a coupling buffer 0.1M Na 2 HP0 4 -dibasic, seven-hydrate, 0.1M NaCl, pH 7.5.
  • a second aliquot of the antibody (1 ml) was treated with N-7- maleimidobutyryloxy-succinimide (GMBS) (Calbiochem, La Jolla, CA) , 5 ⁇ l (14 ⁇ g) of GMBS solution (1 mg in 360 ⁇ l dimethylformamide (DMF) .
  • GMBS N-7- maleimidobutyryloxy-succinimide
  • DMF dimethylformamide
  • the ability of the tetravalent and hexavalent monoclonal antibody homoconjugates to bind antigen was compared to the binding activity of the bivalent IgG monomer antibodies.
  • the binding of the anti-GBS homoconjugates was measured against a GBS strain (1334) bound to microtiter wells using poly-L-lysine (PLL) .
  • ___ coli strain HI6 was bound to microtiter wells using poly-L-lysine. Untreated antibody 5E1-G was compared to homoconjugates of IgG dimer and trimer, prepared as described above. Equivalent protein concentrations of antibodies were reacted with the ___ coli. Binding was assayed with biotin labeled anti-human gamma-chain specific antibodies. The results are shown in Fig. 3, where the relative binding activities of the dimer and trimer homoconjugate preparations were significantly greater than the initial IgG monomer.
  • a breast tumor cell line 3396
  • Untreated antibody BR64 was compared to homoconjugates of IgG dimer and trimer.
  • Equivalent protein concentrations of antibodies were reacted with the breast tumor cells. Binding was assayed with biotin labeled anti-murine gamma-chain specific antibodies. The results are shown in Fig. 4, where the relative binding activities of the dimer and trimer homoconjugate preparations were significantly greater than the initial IgG monomer.
  • Carcinoma cell lines (cultured in IMDM with 15% FCS) were trypsinized, washed twice, and resuspended in PBS at 2 x 10 cells/ml.
  • the PLL treated ELISA plates were washed 3 times with saline/Tween (all wash steps done with a gravity flow wash system) .
  • the cell suspension was added at 100 ⁇ l/well (about 20,000 cells/well) and incubated for 1 hr at 37*C. The plates were then washed 3 times with saline/Tween.
  • Antibodies were diluted in specimen diluent (5% nonfat dry milk, 100 ⁇ l/L Foam A, 0.01% w/v thimerosal in PBS) then added to the ELISA plates (100 ⁇ l/well) and incubated for 1 hr at room temp. Following incubation, the plates were washed 3 times with saline/tween, and peroxidase-conjugated goat anti-human or mouse IgG (Tago) diluted in specimen diluent was used as a second step reagent, (100 ⁇ l/well) and incubated for 1 hr at room temp.
  • specimen diluent 5% nonfat dry milk, 100 ⁇ l/L Foam A, 0.01% w/v thimerosal in PBS
  • the monoclonal antibody homoconjugates to GBS were tested in an in vitro opsonophagocytic assay.
  • Homoconjugates to E. coli Kl were tested for functional activity in two types of opsonization assays described below.
  • Homoconjugates of BR64 were tested for in vitro function in a complement dependent cytotoxicity assay, and homoconjugates of BR96 were tested in a complement independent cytotoxicity assay.
  • the opsonophagocytic assays for GBS were performed as follows. Bacteria were prepared by inoculating 10 ml of tryptic soy broth (TSB) with 50 ⁇ l of an overnight broth culture. The tubes were incubated at 37*C on a shaker for 3 hours at which time 1.5 ml of the culture was centrifuged for 1 min. at 10,000 x g, the spent culture media discarded, and the pellet was suspended in 3.5 ml of Hank's balanced salt solution containing 0.1% gelatin and 5 mM HEPES (HBSS/Gel) . The bacterial concentrations were adjusted to about 3 x 10 bacteria/ml by measuring the 0-D.
  • TAB tryptic soy broth
  • the red blood cell (RBC) pellet was washed once with RPMI 1640 medium and resuspended in an equal volume of 37*C PBS. Twenty-five ml of this suspension was added to 25 ml of 2% dextran (in 37*C PBS) and the contents gently but thoroughly mixed end over end. After a 20 in. incubation at 37*C to allow the RBC's to sediment, the supernatant (containing neutrophils) was removed, washed twice in 4*C PBS, once in HBSS/Gel, and suspended in same to 5xl0 7 neutrophils/ml.
  • human serum was thrice adsorbed with live bacteria (Bjornson, A.B. and Michael, J.G., J. Inf. Pis.. 130 Suppl:S119-S126 (1974)) corresponding to the organisms used in the assay.
  • CFU colony forming units
  • homoconjugates prepared with an additional monoclonal antibody to the group B carbohydrate of GBS were tested in in vitro opsonophagocytic assays against two GBS strains, M94 and 1334.
  • the results of the assays are shown in Fig. 7, where it is evident that the anti-GBS D3 homoconjugates resulted in increased opsonization of the GBS human clinical isolates. Again, these results suggest that the homoconjugates will significantly increase the i vivo protective activities of the antibodies when compared to the parental IgG monomeric monoclonal antibodies.
  • heparinized human blood (5 ml) was layered onto 3.0 ml of Mono-Poly Resolving Medium (MPRM, Flow Labs) in polystyrene tubes and centrifuged for 30 minutes at 300 x g at room temp. After centrifugation, three cell layers were evident, with the middle layer containing neutrophils. The serum and top cell layer were removed and discarded, the neutrophils collected and added to a 50 ml tube containing pre-warmed PBS.
  • MPRM Mono-Poly Resolving Medium
  • the neutrophils were centrifuged for 10 minutes at 300 x g at room temp., the supernatant discarded and the cell pellet resuspended with 10 ml tissue culture media (RPMI-1640) containing 0.5% gelatin, and the cell concentration adjusted to 5 x 10 6 cells/ml.
  • 10 ml tissue culture media RPMI-1640
  • target tumor cells H3630
  • 51 Cr 51 Cr
  • tissue culture media 100 ⁇ Ci of 51 Cr for 1 hour at 37°C, 6% C0 2 .
  • 'Amounts are expressed as ⁇ l/well.
  • the percentage kill (% kill) was calculated from the following formula:
  • CPM counts per minute as average of duplicate samples obtained from measurement in gamma counter and He' is Complement Toxicity control.
  • Target tumor cells H3396 at 5 x 10 5 cells/tube were mixed with 100 ⁇ l of test antibody and were incubated at 37*C for 30 minutes. Cells were pelleted and mixed with the appropriate concentration of propidium iodide (Sigma, 10 ⁇ g/tube) . Propidium iodide is a DNA reactive stain that only penetrates the membrane of dead or dying cells. Therefore, by quantitating the number of fluorescent cells within the population, the number of dead cells can be determined (Hellstrom et al.. Cancer Res.. 50:2183-2190 (1990)). After incubation for 10 minutes, the cells were washed in tissue culture media containing 15% fetal calf serum, resuspended in same, and placed on ice.
  • the cells were analyzed for fluorescence on an EPICS Fluorescence Activated Cell Sorter which quantitates live and dead cells on the basis of fluorescence and size (small and large represent dead and live cells, respectively) .
  • the results showed that the BR96 homoconjugate dimers were dramatically more effective in killing the tumor cells than,the initial monomer. These results suggest that the homoconjugates will significantly increase the utility of such antibodies in yivo against tumors.
  • the amount of transplacentally passaged antibody was determined as follows. Anti-human gamma chain antibody was attached to microtiter plates using carbonate buffer. After adding diluted serum samples from the dams or pups, binding was assayed with biotin labeled anti-human gamma chain-specific antibodies. Since one group of dams received only conjugated antibody, any human IgG detected in pup sera should be transplacentally passed homoconjugate.
  • the homoconjugated IgG monoclonal antibody should be useful when administered prophylactically to pregnant females at risk of having a neonate with an increased likelihood of developing a life-threatening infection, such as by ___ coli Kl in the case of the present embodiment.
  • the data also support the use of these homoconjugates in transplacental treatment of a variety of other infections and tumors.
  • the homoconjugated IgG monoclonal antibody is useful administered prophylactically or therapeutically to pregnant females likely to deliver a neonate susceptible to developing or already having an infection, such as by group B streptococci or ___, coli Kl.
  • the present invention also makes possible the use of the homoconjugates in transplacental treatment of a variety of other infections and tumors.
  • This Example describes the use of homoconjugates of the D3 monoclonal antibody to protect against group B streptococcal infection in vivo, consistent with and confirming the results of the in vitro opsonophagocytic assays.
  • Dawley rat pups less than 48 hours old were injected intraperitoneally with approximately 100 GBS organisms two hours after receiving an intraperitoneal injection of either 20, 4, 0.8 or 0.2 ⁇ g of predominantly dimer homoconjugate preparations, 80, 20, or 4 ⁇ g of monomeric D3, or control IgG.
  • rat pups were examined daily for seven days and were scored for symptoms and survival.
  • the results from two experiments demonstrate the increased i vivo protective activity against GBS of the dimer homoconjugates of human monoclonal antibody D3 compared to the initial IgG monomer. As little as 4 ⁇ g of homoconjugated dimer protected animals nearly as well as .that conferred by 80 ⁇ g of monomer.

Abstract

L'invention se rapporte à des anticorps homoconjugués avec forte avidité pour l'antigène, qui possèdent une meilleure activité thérapeutique et qui sont utilisés dans des compositions pharmaceutiques et diagnostiques. Ces homoconjugués, qu'on prépare généralement à partir d'anticoprs monoclonaux de la classe IgG qui se lient au même déterminant antigénique, sont reliés de façon covalente par une réticulation synthétique. Ces homoconjugués se composent d'au moins deux monomères d'immunoglobuline, de façon à produire une molécule de type IgG se caractérisant par sa tétravalence, son hexavalence ou une valence supérieure pour l'antigène sélectionné. Ces homoconjugués ont la capacité de traverser le placenta. Des procédés thérapeutiques utilisant ces homoconjugués sont également décrits.
PCT/US1991/006195 1990-08-31 1991-08-29 Immunoglobulines homoconjuguees WO1992004053A1 (fr)

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KR1019930700605A KR930702029A (ko) 1990-08-31 1991-08-29 동종접합된 면역 글로불린
FI930846A FI930846A (fi) 1990-08-31 1993-02-25 Homokonjugerade immunoglobuliner
NO93930663A NO930663L (no) 1990-08-31 1993-02-25 Homokonjugerte immunglobuliner

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PT98840A (pt) 1992-07-31
NZ239617A (en) 1993-11-25
FI930846A (fi) 1993-04-05
IE913071A1 (en) 1992-03-11
AU8506991A (en) 1992-03-30
CA2090317A1 (fr) 1992-03-01
EP0547137A4 (en) 1993-12-08
IL99363A0 (en) 1992-08-18
JPH06500780A (ja) 1994-01-27
FI930846A0 (fi) 1993-02-25
EP0547137A1 (fr) 1993-06-23
KR930702029A (ko) 1993-09-08

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