[go: up one dir, main page]

WO2008137885A1 - Elimination d'interférences provoquées par des anticorps antiglucides dans le cadre de dosages immunologiques - Google Patents

Elimination d'interférences provoquées par des anticorps antiglucides dans le cadre de dosages immunologiques Download PDF

Info

Publication number
WO2008137885A1
WO2008137885A1 PCT/US2008/062739 US2008062739W WO2008137885A1 WO 2008137885 A1 WO2008137885 A1 WO 2008137885A1 US 2008062739 W US2008062739 W US 2008062739W WO 2008137885 A1 WO2008137885 A1 WO 2008137885A1
Authority
WO
WIPO (PCT)
Prior art keywords
assay
antibody
carbohydrate moiety
drug
carbohydrate
Prior art date
Application number
PCT/US2008/062739
Other languages
English (en)
Inventor
David Leroy Wensel
Michel Awwad
Original Assignee
Wyeth
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wyeth filed Critical Wyeth
Priority to BRPI0811290-8A2A priority Critical patent/BRPI0811290A2/pt
Priority to CN200880015303A priority patent/CN101680880A/zh
Priority to JP2010507595A priority patent/JP2010527005A/ja
Priority to CA002686206A priority patent/CA2686206A1/fr
Priority to MX2009012048A priority patent/MX2009012048A/es
Priority to AU2008247352A priority patent/AU2008247352A1/en
Priority to MX2009012044A priority patent/MX2009012044A/es
Priority to EP08755078A priority patent/EP2142925A1/fr
Publication of WO2008137885A1 publication Critical patent/WO2008137885A1/fr
Priority to IL201907A priority patent/IL201907A0/en

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements

Definitions

  • the present invention is directed to assays for the detection of an antidrug antibody in general and in particular to the detection of an anti-drug antibody wherein the drug has a carbohydrate moiety.
  • the invention is also directed to assays for the detection of a drug in general and in particular to the detection of a drug wherein the drug has a carbohydrate moiety.
  • the invention is further directed to methods for identifying appropriate subjects for treatment with a drug containing a carbohydrate moiety.
  • Glycoproteins are increasingly being used for the treatment and diagnosis of cancer, infections, and immunological diseases.
  • monoclonal antibodies from various species, as well as chimeric or "humanized” antibodies are the kinds of glycoproteins that are finding increasing clinical use.
  • an immune response is sometimes generated by patients against these antibodies. The response is defined by the difference between the reactivity of the pre-treatment and that of the post-treatment serum or plasma with the infused antibody.
  • the present disclosure reports the existence of pretreatment serum reactivity with a drug having a carbohydrate moiety, wherein the carbohydrate moiety is not directly associated with a glycoprotein.
  • Drug-carrier conjugates comprising the drug calicheamicin conjugated to a monoclonal antibody which targets the calicheamicin to a particular cell type or types have been found to react with pretreatment or na ⁇ ve human and monkey serum.
  • the pre-treatment reactivity is specific for the calicheamicn portion of the drug-carrier conjugate and in particular is directed to a carbohydrate portion of the calicheamicin. It has further been found that providing additional carbohydrate to pre-treatment sera removes background reactivity of the sera with calicheamicin.
  • the invention provides an assay to detect the presence of an antibody to a drug containing a carbohydrate moiety in a fluid sample containing an antibody, the assay comprising the following steps: combining (i) the sample with (ii) the drug containing a carbohydrate moiety and (Ni) additional carbohydrate; and detecting whether or not specific binding occurs between an antibody in the sample and the drug containing a carbohydrate moiety; wherein the drug containing a carbohydrate moiety does not comprise a glycoprotein.
  • the invention provides an assay to detect the presence of a drug containing a carbohydrate moiety in a fluid sample containing an antibody, the assay comprising: combining (i) the sample with (ii) a capture reagent that specifically binds to the drug containing a carbohydrate moiety and (Hi) additional carbohydrate; and detecting whether or not specific binding occurs between the drug containing a carbohydrate moiety in the sample and the capture reagent; wherein the drug containing a carbohydrate moiety does not comprise a glycoprotein.
  • the invention provides an assay to detect the presence of an antibody to a drug containing a carbohydrate moiety in a fluid sample containing an antibody, the assay comprising the following steps: a) combining (i) the fluid sample with (ii) the drug containing a carbohydrate moiety and (iii) additional carbohydrate; and b) detecting whether or not specific binding occurs between an antibody in the sample and the drug containing a carbohydrate moiety; wherein the additional carbohydrate is not pre-adsorbed with the fluid sample prior to detecting whether or not specific binding occurs between an antibody in the sample and the drug containing a carbohydrate moiety.
  • the invention provides an assay to detect the presence of a drug containing a carbohydrate moiety in a fluid sample containing an antibody, the assay comprising: combining (i) the sample with (ii) a capture reagent that specifically binds to the drug containing a carbohydrate moiety and (iii) additional carbohydrate; and detecting whether or not specific binding occurs between the drug containing a carbohydrate moiety in the sample and the capture reagent; wherein the additional carbohydrate is not pre-adsorbed with the fluid sample prior to detecting whether or not specific binding occurs between the capture reagent and the drug containing a carbohydrate moiety.
  • the invention provides any one or more of the assays described herein wherein the assay is performed on a fluid sample from a subject after exposure of the subject to the drug containing the carbohydrate moiety.
  • the invention provides any one or more of the assays described herein wherein the assay is performed on a fluid sample prior to exposure of the subject to the drug containing the carbohydrate moiety (i.e., na ⁇ ve subjects).
  • the assay provides information about the presence or absence in a na ⁇ ve subject of endogenous pre-existing antibodies that specifically bind to the drug containing a carbohydrate moiety.
  • information about the presence or absence in a na ⁇ ve subject of endogenous pre-existing antibodies that specifically bind to the drug containing a carbohydrate moiety can be used to identify subjects that are more likely to respond to treatment with the drug containing a carbohydrate moiety.
  • the assay can further comprise a comparative analysis between the sample from a subject before the subject has been exposed to a drug and a sample from the same subject after the subject has been exposed to the drug, wherein the amount of antibody to a drug containing a carbohydrate moiety in a sample following exposure is indicative of an immune response to the drug.
  • the additional carbohydrate is added to a final concentration that removes any reactivity with the drug containing a carbohydrate moiety that is a result of endogenous pre-treatment anti-carbohydrate antibody present in the na ⁇ ve subjects sera.
  • the invention provides any one or more of the assays described herein wherein the fluid sample from the subject comprises at least one of: whole blood; serum; mucous, saliva, colostrum and plasma.
  • the invention provides any one or more of the assays described herein wherein the detecting step comprises one or more of a sandwich assay, a bridging assay and a competitive binding assay.
  • the invention provides any one or more of the assays described herein wherein the detecting step comprises at least one of: detecting or measuring a change in refractive index at a solid optical surface in contact with the sample; detecting or measuring a change in luminescence; detecting or measuring a change in color; detecting or measuring a change in radioactivity; detecting or measuring using biolayer interferometry; detecting or measuring using cantilever-detection; detecting or measuring using label-free intrinsic imaging; and detecting or measuring using acoustic detection.
  • the invention provides any one or more of the assays described herein wherein the detecting step comprises an assay selected from the group consisting of: an enzyme-linked immunosorbent assay (ELISA); an electro-chemiluminescent assay (ECL); a radioimmunoassay (RIA); solid-phase radioimmunoassay (SPRIA); immunoblotting; immunoprecipitation; Fluorescent Activated Cell Sorting (FACS): and immunostaining.
  • an enzyme-linked immunosorbent assay ECL
  • ECL electro-chemiluminescent assay
  • RIA radioimmunoassay
  • SPRIA solid-phase radioimmunoassay
  • FACS Fluorescent Activated Cell Sorting
  • the invention provides any one or more of the assays described herein wherein the additional carbohydrate is one or more of bacterial lipo-polysaacharide (LPS), a dextran, a levan, pneumococcal polysaccharide, agarose, cellulose, carrageenan, and methylglycoside or a functional fragment of any one of the foregoing carbohydrates.
  • LPS bacterial lipo-polysaacharide
  • a dextran a dextran
  • a levan pneumococcal polysaccharide
  • agarose agarose
  • cellulose cellulose
  • carrageenan methylglycoside or a functional fragment of any one of the foregoing carbohydrates.
  • the invention provides any one or more of the assays described herein wherein the additional carbohydrate is present in the combination at a concentration between about 10 ng/ml and about 1 mg/ml. In another embodiment, the additional carbohydrate is present in the combination at a concentration between about 10 ng/ml and about 0.5 mg/ml. In another embodiment, the additional carbohydrate is present in the combination at a concentration between about 100 ng/ml and about 0.5 mg/ml.
  • the additional carbohydrate is present in the combination at a concentration between about 100 ng/ml and about 0.75 mg/ml In another embodiment, the additional carbohydrate is present in the combination at a concentration between about 100 ng/ml and about 0.25 mg/ml and any ranges in between, including fractions thereof.
  • the invention provides any one or more of the assays described herein wherein the drug containing a carbohydrate moiety further comprises a carrier conjugated to the drug.
  • the carrier is selected from the group consisting of: mono- and polyclonal antibodies and their chemically or genetically manipulated counterparts; their antigen-recognizing fragments and their chemically or genetically manipulated counterparts; small modular immunopharmaceuticals (SMIPs) and their chemically or genetically manipulated counterparts; nanobodies and their chemically or genetically manipulated counterparts; soluble receptors and their chemically or genetically manipulated counterparts; growth factors and their chemically or genetically manipulated counterparts; aptamers; liposomes; non-glycosylated proteins; and nanoparticles.
  • SMIPs small modular immunopharmaceuticals
  • the carrier specifically binds to an antigen expressed on or within cancer cells.
  • the antigen expressed on or within the cancer cells is selected from the group consisting of: 5T4; CD19; CD20; CD22; CD33; Lewis Y; HER-2; type I Fc receptor for immunoglobulin G (Fc gamma R1); CD52; epidermal growth factor receptor (EGFR); vascular endothelial growth factor (VEGF); DNA/histone complex; carcinoembryonic antigen (CEA); CD47; VEGFR2 (vascular endothelial growth factor receptor 2 or kinase insert domain- containing receptor, KDR); epithelial cell adhesion molecule (Ep-CAM); fibroblast activation protein (FAP); Trail receptor-1 (DR4); progesterone receptor; oncofetal antigen CA 19.9; and fibrin.
  • 5T4 CD19; CD20; CD22; CD33; Lewis Y; HER-2; type I Fc receptor for immunoglobulin G (Fc
  • the invention provides one or more of the assays described herein wherein the carrier is a monoclonal antibody.
  • the monoclonal antibody is selected from the group consisting of: an anti-CD22 monoclonal antibody; an anti-5T4 monoclonal antibody; anti-CD33 antibody; and an anti-Lewis Y monoclonal antibody.
  • the invention provides one or more of the assays described herein wherein the drug containing a carbohydrate moiety is selected from the group consisting of: a cytotoxic agent; a radiotherapeutic agent; an immunomodulatory agent; an anti-angiogenic agent; an anti-proliferative agent; a pro-apoptotic agent; a chemotherapeutic agent; a therapeutic nucleic acid; an inhibitor of tubulin polymerization; an alkylating agent that binds to and disrupt DNA; an inhibitor of protein synthesis; and a tyrosine kinase inhibitor.
  • the assays described herein can be performed on fluid samples containing or exposed to more than one drug containing a carbohydrate moiety.
  • the assays described herein can include portions or functional fragments of the drug containing a carbohydrate moiety.
  • the invention provides any one or more of the assays described herein wherein the drug containing a carbohydrate moiety is a cytotoxic agent is selected from calicheamicins, thiotepa, taxanes, vincristine, daunorubicin, doxorubicin, epirubicin, actinomycin, authramycin, azaserines, bleomycins, tamoxifen, idarubicin, dolastatins/auristatins, hemiasteriins, and maytansinoids.
  • cytotoxic agent is selected from calicheamicins, thiotepa, taxanes, vincristine, daunorubicin, doxorubicin, epirubicin, actinomycin, authramycin, azaserines, bleomycins, tamoxifen, idarubicin, dolastatins/auristatins, hemiasteriins,
  • the cytotoxic agent is a calicheamicin and in some embodiment can be N-acetyl gamma dimethyl hydrazine calicheamicin.
  • the invention provides an assay as described herein wherein the antibodies to a drug having a carbohydrate moiety or the drug containing a carbohydrate moiety is detected by binding to a capture agent.
  • the capture agent that specifically binds the antidrug antibody comprises the drug having a carbohydrate moiety or a portion or functional fragment of the drug containing a carbohydrate moiety.
  • the capture agent that specifically binds the antidrug antibody is the carbohydrate portion of the drug containing a carbohydrate moiety or a functional fragment or portion of the drug containing a carbohydrate moiety.
  • the drug containing a carbohydrate moiety and the capture reagent is S-[(2R,4S,6S)-6-( ⁇ [(2R,4S,5R)-5-( ⁇ (2S,4S,5S)-5-
  • the capture agent that specifically binds the drug having a carbohydrate moiety comprises an antibody to the drug containing a carbohydrate moiety.
  • the capture agent that specifically binds the drug having a carbohydrate moiety comprises a target that specifically binds the carrier portion of the carrier-drug conjugate.
  • the capture agent that specifically binds the drug having a carbohydrate moiety comprises at least a portion of 5T4; CD19; CD20; CD22; CD33; Lewis Y; HER-2; type I Fc receptor for immunoglobulin G (Fc gamma R1 ); CD52; epidermal growth factor receptor (EGFR); vascular endothelial growth factor (VEGF); DNA/histone complex; carcinoembryonic antigen (CEA); CD47; VEGFR2 (vascular endothelial growth factor receptor 2 or kinase insert domain- containing receptor, KDR); epithelial cell adhesion molecule (Ep-CAM); fibroblast activation protein (FAP); Trail receptor-1 (DR4); progesterone receptor; oncofetal antigen CA 19.9; and fibrin, wherein the portion specifically binds to the carrier portion of the carrier-drug conjugate.
  • EGFR epidermal growth factor receptor
  • VEGF vascular endothelial growth factor
  • CEA carcino
  • the bound antibody to the drug containing a carbohydrate moiety is detected by binding of a labeled compound to the antibody.
  • the labeled compound comprises the capture reagent further comprising a detectable label; or a second antibody or antibody fragment further comprising a detectable label, wherein the second antibody specifically binds to the bound antibody.
  • the detectable label comprises at least one of an enzyme label, a luminescent label and a radioisotope label.
  • the invention provides an assay as described herein in any one or more of examples one through eight.
  • the presence of drug containing a carbohydrate moiety bound to the capture reagent is detected with a labeled compound that also specifically binds to the drug.
  • the labeled compound is selected from the group consisting of: the capture reagent further comprising a detectable label; and an antibody further comprising a detectable label, wherein the antibody specifically binds to the drug.
  • the detectable label comprises at least one label selected from the group consisting of an enzyme label; a luminescent label; and a radioisotope label.
  • the assay to detect the presence of an antibody to a drug containing a carbohydrate moiety or the presence of a drug containing a carbohydrate moiety in a fluid sample containing antibodies comprises at least one of: measuring a change in refractive index at a solid optical surface in contact with the sample; measuring a change in luminescence; measuring a change in color; and measuring a change in radioactivity.
  • the assay to detect the presence of an antibody to a drug containing a carbohydrate moiety or the presence of a drug containing a carbohydrate moiety in a fluid sample containing antibodies comprises an assay selected from the group consisting of: an ELISA; an ECL; an RIA; a SPRIA; immunoblotting; immunoprecipitation; and immunostaining.
  • Figure 1 is a graphical representation of the presence of an interfering factor in na ⁇ ve serum having a binding specificity for calicheamicin.
  • Figure 2 shows a diagrammatic representation of a calicheamicin as conjugated to an antibody.
  • Figure 3 is a graphical representation of the reduction in calicheamicin specific binding in sera titrated with various concentrations of detoxified LPS.
  • Figure 4 is a graphical representation of the reduction in calicheamicin specific binding in sera titrated with various concentrations of the carbohydrate moiety of calicheamicin, methyl glycoside.
  • Figure 5 is a graphical representation demonstrating the reduction in calicheamicin specific binding in sera titrated with various concentrations of detoxified LPS.
  • Figure 6 shows a graphical representation of calicheamicin specific IgM from calicheamicin specific sera binding IgM.
  • Figure 7 shows a graphical representation of IgM from Calicheamicin specific sera binding to calicheamicin.
  • Figure 8 shows a graphical representation of the presence of calicheamicin specific binding factor in na ⁇ ve human sera.
  • Figure 9 shows the chemical structure of S-[(2R,4S,6S)-6-( ⁇ [(2R,4S,5R)-
  • Drug conjugates developed for systemic pharmacotherapy are target- specific cytotoxic agents. The concept involves coupling a therapeutic agent to a carrier molecule with specificity for a defined target cell population. As used herein, such drug conjugates are also referred to as “carrier-drug conjugates.”
  • Antibodies with high affinity for antigens are a natural choice as targeting moieties or as the "carrier" portion of a carrier-drug conjugate. With the availability of high affinity monoclonal antibodies, the prospects of antibody-targeting therapeutics have become promising. Toxic substances that have been conjugated to monoclonal antibodies include toxins, low-molecular-weight cytotoxic drugs, biological response modifiers, and radionuclides. Antibody-toxin conjugates are frequently termed immunotoxins, whereas immunoconjugates consisting of antibodies and low- molecular-weight drugs such as methothrexate and Adriamycin are called chemoimmunoconjugates.
  • Radioimmunoconjugates consist of radioactive isotopes, which may be used as therapeutics to kill cells by their radiation or used for imaging.
  • Antibody-mediated specific delivery of cytotoxic drugs to tumor cells is expected to not only augment their anti-tumor efficacy, but also prevent nontargeted uptake by normal tissues, thus increasing their therapeutic indices.
  • a number of antibody-based therapeutics for treating a variety of diseases including cancer and rheumatoid arthritis have been approved for clinical use or are in clinical trials for a variety of malignancies including B-cell malignancies such as Non-Hodgkin's lymphoma.
  • One such antibody-based therapeutic is rituximab (Rituxan.TM.), an unlabelled chimeric human ⁇ 1 (+m ⁇ 1V-region) antibody, which is specific for cell surface antigen CD20, which is expressed on B-cells.
  • Immunoconjugates comprising a member of the potent family of antibacterial and antitumor agents, known collectively as the calicheamicins or the LL-E33288 complex, (see U.S. Pat. No. 4,970,198 (1990)), were developed for use in the treatment of myelomas.
  • One of the most potent of the calicheamicins is designated ⁇ i which is herein referenced simply as gamma.
  • These compounds contain a methyltrisulfide that can be reacted with appropriate thiols to form disulfides, at the same time introducing a functional group such as a hydrazide or other functional group that is useful in attaching a calicheamicin derivative to a carrier. (See U.S. Pat. No. 5,053,394).
  • a drug is used as a target to capture antibodies specific for the drug, there may be a false positive reaction (or increased quantity) if an endogenous anti-carbohydrate moiety antibody binds to the capture agent.
  • a capture reagent for the drug is used in conjunction with a labeled compound to detect the drug in sera, there may be false negatives (or a decrease in quantity) due to anti-carbohydrate antibody binding to the drug, which prevents or reduces specific binding of a labeled compound to the drug if the bound antibody interferes with binding of the labeled compound.
  • the presence of additional carbohydrate in an assay to detect specific binding between an capture reagent and a carrier-drug conjugate in a fluid sample containing antibody will enable the specific detection of the carrier-drug conjugate antibody.
  • the additional carbohydrate does not have to be pre-adsorbed with the fluid sample prior to detecting specific binding between the capture reagent and the carrier-drug conjugate.
  • the specific detection is possible because the additional carbohydrate added to the assay will remove any masking activity of preexisting anti-drug antibodies present in the sample.
  • the additional carbohydrate is added to the serum in order to negate the effect of binding of endogenous antibodies in the serum to the drug containing a carbohydrate moiety.
  • the amount of additional carbohydrate can be titrated with na ⁇ ve serum in order to determine the optimal amount to add when seeking to reduce background. If analysis and detection of the drug containing carbohydrate moiety will rely on a capture reagent that does not bind to the carbohydrate moiety, then additional carbohydrate may be added to excess.
  • MYLOTARG ® (Sievers, E. L. et al (1999) Blood: 93, 3678-3684), also referred to as CMA-676 or CMA, is a commercially available drug that comprises calicheamicin bound to a monoclonal antibody as the carrier.
  • MYLOTARG ® (gemtuzumab ozogamicin) is currently approved for the treatment of acute myeloid leukemia in elderly patients.
  • the drug consists of an antibody against CD33 that is bound to calicheamicin by means of an acid-hydrolyzable linker.
  • the disulfide analog of the semi-synthetic N-acetyl gamma calicheamicin was used for conjugation (U.S. Pat.
  • a sample of the liquid medium to be assayed is combined with various reagent compositions.
  • Such compositions include a label conjugate comprising a binding component incorporated with a label.
  • the binding component in the conjugate participates with other constituents, if any, of the reagent composition and the ligand in the medium under assay to form a binding reaction system producing two species or forms of the conjugate, e.g., a bound-species (conjugate complex) and a free- species.
  • a bound-species conjugate complex
  • the binding component of the conjugate is bound by a corresponding binding partner whereas in the free species, the binding component is not so bound.
  • the amount or proportion of the conjugate that results in the bound species compared to the free species is a function of the presence (or amount) of the analyte to be detected in the test sample.
  • An alternative format for specific binding assays is the "sandwich" assay protocol in which the target analyte is bound between a first specific binding partner, which is fixed directly or through a linkage group to a solid matrix, and a second specific binding partner, which is associated with a signal generating or labeling system.
  • U.S. Pat. No. 4,243,749 discloses a competitive binding assay wherein a reaction is carried out in a test tube having a specific antibody to a hapten under determination insolubilized or immobilized on the inner wall of the test tube.
  • the reaction includes a labeled hapten conjugate wherein the quantity of the labeled hapten conjugate which becomes bound to the test tube wall is inversely proportional to the amount of the hapten under determination.
  • U.S. Pat. No. 4,298,685 discloses an enzyme immunoassay wherein a sample containing a biological substance under determination is mixed with antibodies to the biological substance tagged with biotin and with an enzyme- labeled form of the substance under assay. An immobilized form of avidin is then added wherein the avidin binds to the biotin-tagged antibody to immobilize the antibody-bound fraction of the enzyme-labeled reagent.
  • United Kingdom Patent Application No. GB 2,084,317A discloses an antigen-linked competitive enzyme immunoassay using avidin bound to a solid material and a biotin-labeled antigen. Radiolabeled and enzyme-tagged immunoassays require some type of separation step.
  • U.S. Pat. No. 3,817,837 discloses a competitive binding assay method involving the steps of combining the liquid to be assayed with a soluble complex consisting of an enzyme as a labeling substance covalently bound to the ligand to be detected and with a soluble receptor, usually an antibody, for the ligand; and analyzing for the effect of the liquid to be assayed on the enzymatic activity of the enzyme in the complex.
  • the labeled reagent can include other conventional detectable chemical groups.
  • detectable chemical groups can be any material having a detectable physical or chemical property. Such materials have been well developed in the field of immunoassays and in general any label useful in such methods can be applied to the present invention.
  • Particularly useful are enzymatically active groups, such as enzymes (see Clin. Chem. (1976)22:1243), enzyme substrates (see U.S. Pat. No. 4,492,751 ), prosthetic groups or coenzymes (see U.S. Pat. Nos. 4,230,797 and 4,238,565), and enzyme inhibitors (see U.S. Pat. No. 4,134,792); spin labels; fluorescers (see Clin. Chem.
  • additional carbohydrate refers to carbohydrates that can compete with carbohydrate that binds to endogenous antibody in serum.
  • LPS bacterial lipopolysaccharide
  • methylglycoside methylglycoside
  • dextran methylglycoside
  • levan pneumococcal polysaccharide
  • agarose agarose
  • cellulose cellulose
  • carrageenan in an immunoassay can block the binding of antibodies generally binding to carbohydrates.
  • any of these carbohydrates will allow specific binding of other reagents in the assay to bind to non-carbohydrate regions of N-acetyl gamma dimethyl hydrazide alone or as part of a carrier-drug conjugate, thus reducing non-specific background signal generated from anti-carbohydrate antibodies in serum.
  • selection of the proper carbohydrate can involve a carbohydrate that specifically binds to the drug-carrier conjugate.
  • antibody as used herein is meant to include one or more of mono- and polyclonal antibodies and their chemically or genetically manipulated counterparts; their antigen-recognizing fragments and their chemically or genetically manipulated counterparts; and synthetic molecules comprising their antigen-recognizing fragments.
  • additional carbohydrate is also meant to encompass the same carbohydrate that is attached to the drug containing a carbohydrate moiety or a fragment thereof as well as other carbohydrates or fragments thereof that specifically mimic the carbohydrate portion of the drug containing a carbohydrate moiety.
  • carbohydrate or fragment thereof will specifically bind the same target and receptor compound that binds to the carbohydrate moiety of the drug.
  • a “bridging assay” refers to an immunoassay wherein the capturing system is an immunocomplex containing the sample analyte formed in solution and then captured by a bridging element which links the immunocomplex to a solid phase. A detectable label covalently coupled to an element binding to the bridging element is used for the detection of a binding event.
  • capture reagent is meant to include any composition that specifically binds to a corresponding target.
  • a capture reagent for a corresponding target that is a drug containing a carbohydrate moiety includes but is not limited to an antibody that specifically binds the drug containing a carbohydrate moiety and any specific binding partner for the drug containing a carbohydrate moiety.
  • a drug containing a carbohydrate moiety further comprises an antibody conjugated to the drug
  • the target of the antibody's antigen-binding domain is also a capture reagent for the drug containing a carbohydrate moiety.
  • the drug containing a carbohydrate moiety has a corresponding receptor binding partner, or comprises a soluble form of a receptor that has a corresponding binding partner, the receptor and receptor binding partner are capture reagents for each other.
  • the term "competition assay” refers to an assay where an unlabeled analyte in a heterogeneous sample is measured by its ability to compete with an element, primarily an antibody or antigen, covalently attached to a detectable label.
  • the unlabeled analyte and the labeled molecule will compete with each other for the binding to a capture reagent immobilized on a solid phase.
  • the unlabeled analyte blocks the ability of the labeled molecule to bind because the binding site on the capture reagent is already occupied.
  • less label measured in the assay means more of the unlabeled analyte is present.
  • the amount of antigen in the heterogenous sample is inversely related to the amount of label measured.
  • cytotoxin or "cytotoxic agent” generally refers to an agent that inhibits or prevents the function of cells and/or results in destruction of cells.
  • Representative cytotoxins include antibiotics, inhibitors of tubulin polymerization, tyrosine kinase inhibitors, alkylating agents that bind to and disrupt DNA, and agents that disrupt protein synthesis or the function of essential cellular proteins such as protein kinases, phosphatases, topoisomerases, enzymes, and cyclins.
  • cytotoxins include, but are not limited to, doxorubicin, daunorubicin, idarubicin, aclarubicin, zorubicin, mitoxantrone, epirubicin, carubicin, nogalamycin, menogaril, pitarubicin, valrubicin, cytarabine, gemcitabine, trifluridine, ancitabine, enocitabine, azacitidine, doxifluridine, pentostatin, broxuridine, capecitabine, cladribine, decitabine, floxuridine, fludarabine, gougerotin, puromycin, tegafur, tiazofurin, adriamycin, cisplatin, carboplatin, cyclophosphamide, dacarbazine, vinblastine, vincristine, mitoxantrone, bleomycin, mechlorethamine, prednisone, procarbazine
  • carrier or carrier molecule includes, but is not limited to: mono- and polyclonal antibodies and their chemically or genetically manipulated counterparts; their antigen-recognizing fragments and their chemically or genetically manipulated counterparts; small modular immunopharmaceuticals (SMIPs) and their chemically or genetically manipulated counterparts; nanobodies and their chemically or genetically manipulated counterparts; soluble receptors and their chemically or genetically manipulated counterparts; growth factors and their chemically or genetically manipulated counterparts; aptamers; liposomes; non- glycosylated proteins; and nanoparticles; aptamers; liposomes; non-glycosylated proteins; and nanoparticles
  • a "detectable label” refers to any substance or group of substances which is either directly or indirectly involved in the production of a detectable signal and may be covalently attached to any antibody or antigen used in any of the immunoassays of this invention.
  • Detectable labels conventionally used are, for example, enzymatic, radioactive, fluorescent, chemiluminescent or phosphorescent labels.
  • a preferred label is an enzyme label, and still more preferred is horseradish peroxidase (HRP) which results in a colorimetric assay upon addition of the enzyme substrate which, in the case of HRP, is a solution of hydrogen peroxide and 3, 3', 5.5'- tetramehylbinzidine-2 HCI (TMB).
  • HRP horseradish peroxidase
  • HRP can be used in a direct manner when covalently attached to an antibody or antigen that will specifically bind to an already bound captured analyte. HRP can also be added to an immunoassay in an indirect manner when added as a secondary reagent, for example, if an antibody or antigen is labeled with biotin and avidin-HRP is subsequently added.
  • drug containing a carbohydrate moiety refers to any substance having biological or detectable activity, for example therapeutic agents, detectable labels, binding agents, etc., and prodrugs, which are metabolized to an active agent in vivo.
  • drug containing a carbohydrate moiety also includes drug derivates, wherein a drug containing a carbohydrate moiety has been functionalized to enable conjugation with an antibody or another carrier molecule.
  • these types of conjugates are referred to as immunoconjugates.
  • the term is meant to refer to a portion or functional fragment of a drug containing a carbohydrate moiety that is able to bind to antibodies that specifically bind to the carbohydrate moiety when it is part of the drug or carrier-drug conjugate.
  • the term drug containing a carbohydrate moiety can be applied to the drug portion of a carrier-drug conjugate (e.g., a monoclonal carrier bound to calicheamicin) as well as the calicheamicin portion of a drug or the methylglycoside portion of the calicheamicin (i.e., S- [(2R,4S,6S)-6-( ⁇ [(2R,4S,5R)-5-( ⁇ (2S,4S,5S)-5-[acetyl(ethyl)amino]-4- methoxytetrahydro-2H-pyran-2-yl ⁇ oxy)-4-hydroxy-6-methoxy-2-methyltetrahydro-2H- pyran-3-yl]amino ⁇ oxy)-4-hydroxy-2-methyltetrahydro-2H-pyran-3-yl]4- ⁇ [(2S,3R,4R,5S,6S)-3,5-dihydroxy
  • a drug containing a carbohydrate moiety functionalized to enable conjugation with an antibody as used herein is also meant to include the "drug" portion of a "carrier-drug conjugate".
  • the antibodies, as part of the carrier-drug conjugate are specific for CD22, 5T4, CD33 and Lewis-Y antigens. These antibodies are specific for receptors that specifically binds to an antigen expressed on cancer cells.
  • a drug containing a carbohydrate moiety that "does not comprise a glycoprotein” such reference is meant to include the situation wherein the carrier portion of the carrier-drug conjugate can include a glycoprotein.
  • therapeutic drugs useful as part of a carrier-drug conjugate include cytotoxins, radioisotopes, chemotherapeutic agents, immunomodulatory agents, anti-angiogenic agents, anti-proliferative agents, pro-apoptotic agents, and cytostatic and cytolytic enzymes (e.g., RNAses).
  • a drug may also include a therapeutic nucleic acid, such as a gene encoding an immunomodulatory agent, an anti-angiogenic agent, an anti-proliferative agent, or a pro-apoptotic agent.
  • radioisotopes are also cytotoxins.
  • Therapeutic agents may be prepared as pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • conjugates having a radioisotope as the drug are referred to as radioimmunoconjugates and those having a chemotherapeutic agent as the drug are referred to as chemoimmunoconjugates.
  • suitable drugs for use in immunoconjugates include the taxanes, maytansines, CC-1065 and the duocarmycins, the calicheamicins and other enediynes, and the auristatins.
  • Other examples include the anti-folates, vinca alkaloids, and the anthracyclines.
  • Plant toxins other bioactive proteins, enzymes (i.e., ADEPT), radioisotopes, photosensitizers (i.e., for photodynamic therapy) can also be used in immunoconjugates.
  • conjugates can be made using secondary carriers as the cytotoxic agent, such as liposomes or polymers, for example.
  • an "immune response” is meant to refer to any response to an antigen or antigenic determinant by the immune system of a vertebrate subject, including humoral immune responses (e.g. production of antigen-specific antibodies) and cell- mediated immune responses (e.g. lymphocyte proliferation).
  • humoral immune responses e.g. production of antigen-specific antibodies
  • cell- mediated immune responses e.g. lymphocyte proliferation
  • immunomodulatory agents include cytokines, xanthines, interleukins, interferons, and growth factors (e.g., TNF, CSF, GM-CSF and G-CSF), and hormones such as estrogens (diethylstilbestrol, estradiol), androgens (testosterone, HALOTESTIN®(fluoxymesterone)), progestins (MEGACE® (megestrol acetate), PROVERA® (medroxyprogesterone acetate)), and corticosteroids (prednisone, dexamethasone, hydrocortisone).
  • TNF TNF
  • CSF CSF
  • GM-CSF GM-CSF
  • G-CSF growth factors
  • hormones such as estrogens (diethylstilbestrol, estradiol), androgens (testosterone, HALOTESTIN®(fluoxymesterone)), progestins (MEGACE® (megestrol a
  • Luminescent labels include labels that involve phosphorescence, chemiluminescence andfluorescence.
  • Fluorescent label refers to a fluorophore that when covalently attached to a protein (or other compound) will, when excited with short wavelengths, show optimal energy transfer between fluorophores and can be read at specific wavelengths, primarily ultraviolet.
  • the most common fluorescent labels are derivatives of fluorescein and rohodamine, FITC and TRITC respectively. These fluorophores are covalently attached to antibodies or antigens and are used for detection of a specific binding event.
  • a chemiluminescent label is one in which it is induced to emit light by a developing reagent causing the formation of an excited state molecule that decays, thereby emitting detectable light.
  • the most commonly used chemiluminescent labels are: acrodinium, luminol and dioxetane. Acrodinium and luminol are excited by peroxidase enzyme reactions and can be used in immunoassays that employ an HRP label.
  • a phosphorescent label is one in which there is an emission of light from a substance exposed to radiation and persisting as a glow after the excitatory radiation is removed.
  • An example of a phosphorescent detectable label are derivatives of p-isothiocyanatophenyl Pt(ll)-and Pd(II)- coproporphyrin I.
  • a radioactive label refers to a radionuclide that when covalently attached to a protein will decay, the presence of a binding event then determined by the detection of the radiation that is emitted.
  • Common radionuclides used as detectable labels are: 3 H, 125 I and 35 S.
  • the labels, listed above, are detected using a detecting system that is specific to the nature of each of the labels.
  • portion or functional fragment thereof when used to identify a capture reagent, antibody, or drug containing a carbohydrate moiety is meant to include any portion of the compound in question that is able to undergo specific binding with a target partner in a manner the same or similar to the parent molecule.
  • pre-mixing refers to mixing of two or more components of an assay prior to beginning the assay.
  • pre-adsorption of sera and additional carbohydrate or “preadsorption of the sera and additional carbohydrate” as used in the assays described herein would means pre-mixing or pre-incubation of the sera and additional carbohydrate prior to mixing either the sera or additional carbohydrate with the drug containing a carbohydrate moiety.
  • the term "sandwich assay” refers to a non-competitive assay format that provides a high level of sensitivity and specificity.
  • the format is referred to as a sandwich due to the fact that the analyte, comprising a heterogeneous sample of different antibodies and antigens, is bound (sandwiched) between two highly specific reagents.
  • the first specific reagent is a capture reagent that is a purified antibody or antigen that is bound to a solid phase typically attached to the bottom of a plate well.
  • the heterogeneous analyte is exposed to the capture reagent and any constituent of the analyte having affinity for the capture reagent will specifically bind to it.
  • a second, highly specific binding element is added.
  • the binding element has a covalently attached detectable label.
  • the complex usually antibody-antigen, can then be detected.
  • the measurement of the labeled binding event is directly proportional to the amount of antigen present in the sample.
  • binding refers to an affinity between two molecules, for example specific binding between amolecule and its binding partner results in preferential binding in a heterogeneous sample comprising the molecule and its binding partner and one or more different molecules. Binding in IgG antibodies, e.g., is generally characterized by an affinity of at least about 10 "7 M or higher, such as at least about 10 "8 M or higher, or at least about 10 "9 M or higher, or at least about 10 '10 or higher, or at least about 10 11 M or higher, or at least about 10 "12 M or higher. IgM molecules are known in some instances to have affinities as low as 10"*.
  • Representative anti-angiogenic agents include inhibitors of blood vessel formation, for example, farnesyltransferase inhibitors, COX-2 inhibitors, VEGF inhibitors, bFGF inhibitors, steroid sulphatase inhibitors (e.g., 2-methoxyoestradiol bis-sulphamate (2-MeOE2bisMATE)), interleukin-24, thrombospondin, metallospondin proteins, class I interferons, interleukin 12, protamine, angiostatin, laminin, endostatin, and prolactin fragments.
  • farnesyltransferase inhibitors e.g., COX-2 inhibitors
  • VEGF inhibitors e.g., VEGF inhibitors
  • bFGF inhibitors e.g., VEGF inhibitors
  • bFGF inhibitors e.g., VEGF inhibitors
  • bFGF inhibitors e.g., VEGF inhibitors
  • Anti-proliferative agents and pro-apoptotic agents include activators of PPAR-gamma (e.g., cyclopentenone prostaglandins (cyPGs)), retinoids, triterpinoids (e.g., cycloartane, lupane, ursane, oleanane, friedelane, dammarane, cucurbitacin, and limonoid triterpenoids), inhibitors of EGF receptor (e.g., HER4), rampamycin, CALCITRIOL.RTM. (1 ⁇ -dihydroxycholecalciferol (vitamin D)), aromatase inhibitors (FEMARA.RTM.
  • PPAR-gamma e.g., cyclopentenone prostaglandins (cyPGs)
  • retinoids e.g., cycloartane, lupane, ursane, oleanane, friedelane, dammar
  • telomerase inhibitors include iron chelators (e.g., 3- aminopyridine-2-carboxaldehyde thiosemicarbazone (Triapine)), apoptin (viral protein 3--VP3 from chicken aneamia virus), inhibitors of Bcl-2 and BcI-X(L), TNF-alpha, FAS ligand, TNF-related apoptosis-inducing ligand (TRAI L/Apo2L), activators of TNF- alpha/FAS ligand/TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) signaling, and inhibitors of PI3K-Akt survival pathway signaling (e.g., UCN-01 and geldanamycin).
  • iron chelators e.g., 3- aminopyridine-2-carboxaldehyde thiosemicarbazone (Triapine)
  • apoptin viral protein 3--VP3 from chicken aneami
  • the cytotoxic drug having a carbohydrate moiety is an antibiotic such as a calicheamicin, also called the LL- E33288 complex, for example, gamma-calicheamicin ⁇ 1 ).
  • a calicheamicin also called the LL- E33288 complex
  • gamma-calicheamicin ⁇ 1 gamma-calicheamicin ⁇ 1
  • Stabilizing the disulfide bond that is present in all calicheamicin conjugates by adding dimethyl substituents made additional improvements. Additional examples of calicheamicins suitable for use in preparing antibody/drug conjugates of the invention are disclosed in U.S.
  • N-acetyl gamma calicheamicin dimethyl hydrazide or NAc-gamma DMH, as one of the optimized derivatives for conjugation.
  • Disulfide analogs of calicheamicin can also be used, for example, analogs described in U.S. Pat. Nos. 5,606,040 and 5,770,710, which are incorporated herein in their entirety.
  • Representative methods for preparing antibody/drug conjugates include those described for preparation of CMC-544 in U.S. patent application Publication No. 2004-0082764A1 and U.S. patent application Publication No. 2004-0192900, which are incorporated herein in their entirety.
  • Conjugation may be performed using the following conditions: 10 mg/ml antibody, 8.5% (w/w) calicheamicin derivative, 37.5 mM sodium decanoate, 9% (v/v) ethanol, 50 mM HEPBS (N-(2- Hydroxyethyl)piperazine-N'-(4-butanesulfonic acid)), pH 8.5, 32.degree. C, 1 hour.
  • Hydrophobic interaction chromatography may be performed using a butyl sepharose FF resin, 0.65 M potassium phosphate loading buffer, 0.49 M potassium phosphate wash buffer, and 4 mM potassium phosphate elution buffer. Buffer exchange may be accomplished by size exclusion chromatography, ultrafiltration/diafiltration, or other suitable means.
  • N-acetyl gamma dimethyl hydrazide was conjugated to monoclonal antibodies that specifically bind to the CD22 receptor, the 5T4 receptor and the Lewis-Y antigen, all expressed on cancer cells.
  • carrier-drug conjugates as part of the N-acetyl gamma dimethyl hydrazide molecule, have methylglycoside (i.e., S-[(2R,4S,6S)-6-( ⁇ [(2R,4S,5R)-5- ( ⁇ (2S,4S,5S)-5-[acetyl(ethyl)amino]-4-methoxytetrahydro-2H-pyran-2-yl ⁇ oxy)-4- hydroxy-6-methoxy-2-methyltetrahydro-2H-pyran-3-yl]amino ⁇ oxy)-4-hydroxy-2- methyltetrahydro-2H-pyran-3-yl]4- ⁇ [(2S,3R,4R,5S,6S)-3,5-dihydroxy-4-methoxy-6- methyltetrahydro-2H-pyran-2-yl]oxy ⁇ -3-iodo-5,6-dimethoxy-2- methylbenzene
  • THST 50 mM Tris-HCI, 0.5 M NaCI, 1 mM glycine and 0.05% (v/v)Tween 20, pH 8;
  • TMB tetramethylbenzidine
  • CMD-544 is a calicheamicin-monoclonal antibody drug conjugate described in U.S. patent application US2004082764.
  • the monoclonal antibody portion of this carrier drug conjugate has a target specificity for human CD22 antigen, with the parental antibody designated herein as "G-544.”
  • CMD-193 is a calicheamicin-monoclonal antibody drug conjugate described in U.S. patent application Publication No. US20060002942.
  • the monoclonal antibody portion of this carrier drug conjugate has a target specificity for Lewis Y antigen, with the parental antibody designated herein as "G-193.”
  • CME-548 is a calicheamicin-monoclonal antibody drug conjugate described in U.S. patent application Publication No. US2006008522.
  • the monoclonal antibody portion of this carrier drug conjugate has a target specificity for human 5T4 receptor antigen, with the parental antibody designated herein as "huH8.”
  • a bridging-type ELISA was performed.
  • Calicheamicin has, as part of the molecule, a carbohydrate moiety that could be bound by pre-existing anti-carbohydrate antibodies in some serum samples.
  • CMC- 544 was used as a capture reagent, immobilized in assay wells and then blocked in 4% NFDM/PBST buffer.
  • Sera used in the previously described assays (na ⁇ ve) were diluted in 1% NFDM/PBST and added to the wells.
  • LPS lipopolysaccharide
  • the bridging capability of the factor indicated an immunoglobulin.
  • IgG and IgM were initially examined as possibilities.
  • protein A resin was used to purify the IgG fraction from monkey serum known to contain the interfering factor.
  • SDS-PAGE indicated successful isolation of IgG (not shown; L38054-17).
  • Both the IgG-enriched and IgG-depleted fractions of the serum were tested for bridging capability after adjusting concentrations to account for inevitable dilution of sample during the batch-binding chromatography process.
  • the wells were washed in THST and incubated with anti-lgM-HRP to detect any IgM bound to the plates.
  • the assay was developed for a color reaction by the addition of the TMB reagent.
  • CMC-544 captured large amounts of IgM from serum, with significantly less signal obtained in the presence of lipopolysaccharide. Very little IgM bound to the parental unconjugated antibody G544.
  • a bridging-type ELISA is performed after the optimal concentration of methyl glycoside S-[(2R,4S,6S)-6-( ⁇ [(2R,4S,5R)-5-( ⁇ (2S,4S,5S)-5- [acetyl(ethyl)amino]-4-methoxytetrahydro-2H-pyran-2-yl ⁇ oxy)-4-hydroxy-6-methoxy-2- methyltetrahydro-2H-pyran-3-yl]amino ⁇ oxy)-4-hydroxy-2-methyltetrahydro-2H-pyran- 3-yl]4- ⁇ [(2S,3R,4R,5S,6S)-3,5-dihydroxy-4-methoxy-6-methyltetrahydro-2H-pyran-2- yl]oxy ⁇
  • Na ⁇ ve sera is titrated as above to determine the minimum (and thus optimum) concentration of carbohydrate that will remove endogenous anti- carbohydrate background noise from the na ⁇ ve sera from a subject being tested for production of anti-drug antibody after exposure to the drug.
  • the additional carbohydrate can be added directly to the wells with the serum in the initial incubation with the capture reagent and control wells. Following washing, plates are then developed for a color reaction by the addition of the TMB reagent. The presence of a color reaction after development will indicate the presence of antibodies against a calicheamicin conjugate in the serum that are not a result of endogenous anti-carbohydrate antibody but are instead a result of the production of an immune reaction in response to the carrier-drug conjugate.
  • the sera from an exposed subject is then titrated with the calicheamicin-specific methyl glycoside to determine if the antibody reactivity returns to background levels. If the antibody response is titrated back to background levels, then the antibody response generated in response to the carrier- drug conjugate is due to the carbohydrate moiety of the calicheamicin.
  • a bridging- type ELISA is performed.
  • Calicheamicin is used as a capture reagent and immobilized in the assay wells. The wells are blocked in 4% NFDM/PBST buffer. Serum, collected from the drug na ⁇ ve subject is diluted in 1% NFDM/PBST, added to the assay and incubated. The optimum concentration of methyl glycoside is determined as described above.
  • a titration of carbohydrates can also include a no carbohydrate control test for each serum sample.
  • an ELISA is performed. Subjects are injected with or otherwise exposed to CMC-544 and serum is collected. CD22, the antigen that is specifically bound by parental antibody G544, is immobilized in assay wells and used as a capture reagent. The wells are then blocked in 4% NFDM/PBST buffer. Serum is diluted in 1% NFDM/PBST and added to the wells, and then incubated. Since the additional carbohydrate should not interfere with capture of CMC-544 by CD22-specific binding, a vast excess of carbohydrate can be added here to block antibody present in the sera that might bind to the CMC-544.
  • the plate is washed in THST and incubated with anti-calicheamicin-HRP conjugate and the plates are then developed for a color reaction by the addition of the TMB reagent. A color reaction would indicate the presence of the calicheamicin conjugate in the sera.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biophysics (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention concerne des dosages permettant la détection d'un anticorps anti-médicament en général et, plus particulièrement, la détection d'un anticorps anti-médicament, ledit médicament comportant un groupe glucidique. L'invention concerne également des dosages permettant la détection d'un médicament en général et, plus particulièrement, la détection d'un médicament comportant un groupe glucidique. L'invention concerne encore des procédés d'identification de sujets pouvant bénéficier d'un traitement par un médicament contenant un groupe glucidique.
PCT/US2008/062739 2007-05-07 2008-05-06 Elimination d'interférences provoquées par des anticorps antiglucides dans le cadre de dosages immunologiques WO2008137885A1 (fr)

Priority Applications (9)

Application Number Priority Date Filing Date Title
BRPI0811290-8A2A BRPI0811290A2 (pt) 2007-05-07 2008-05-06 Eliminação de interferência em imunoensaios causada por anticorpos anticarboidrato
CN200880015303A CN101680880A (zh) 2007-05-07 2008-05-06 免疫检定中由抗碳水化合物抗体引起的干扰的消除
JP2010507595A JP2010527005A (ja) 2007-05-07 2008-05-06 抗炭水化物抗体によって生じるイムノアッセイにおける妨害の除去
CA002686206A CA2686206A1 (fr) 2007-05-07 2008-05-06 Elimination d'interferences provoquees par des anticorps antiglucides dans le cadre de dosages immunologiques
MX2009012048A MX2009012048A (es) 2007-05-07 2008-05-06 Eliminacion de interferencia en inmunoensayos ocasionada por anticuerpos de anti-carbohidrato.
AU2008247352A AU2008247352A1 (en) 2007-05-07 2008-05-06 Elimination of interference in immunoassays caused by anti-carbohydrate antibodies
MX2009012044A MX2009012044A (es) 2007-05-07 2008-05-06 Eliminacion de interferencia en inmunoensayos causada por anticuerpos anti-carbohidrato.
EP08755078A EP2142925A1 (fr) 2007-05-07 2008-05-06 Elimination d'interférences provoquées par des anticorps antiglucides dans le cadre de dosages immunologiques
IL201907A IL201907A0 (en) 2007-05-07 2009-11-03 Elimination of interference in immunoassays caused by anti-carbohydrate antibodies

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US92814407P 2007-05-07 2007-05-07
US60/928,144 2007-05-07
US92843807P 2007-05-08 2007-05-08
US60/928,438 2007-05-08

Publications (1)

Publication Number Publication Date
WO2008137885A1 true WO2008137885A1 (fr) 2008-11-13

Family

ID=39666131

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2008/062739 WO2008137885A1 (fr) 2007-05-07 2008-05-06 Elimination d'interférences provoquées par des anticorps antiglucides dans le cadre de dosages immunologiques

Country Status (11)

Country Link
EP (1) EP2142925A1 (fr)
JP (1) JP2010527005A (fr)
KR (1) KR20100029083A (fr)
CN (1) CN101680880A (fr)
AU (1) AU2008247352A1 (fr)
BR (1) BRPI0811290A2 (fr)
CA (1) CA2686206A1 (fr)
IL (1) IL201907A0 (fr)
MX (2) MX2009012044A (fr)
RU (1) RU2009141159A (fr)
WO (1) WO2008137885A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011135024A1 (fr) 2010-04-29 2011-11-03 Biomedical Diagnostics Sa Procédés de détection d'anticorps
CN106164670A (zh) * 2014-02-11 2016-11-23 建新公司 用于检测抗药物抗体的存在或抗药物抗体的量的测定
CN108535490A (zh) * 2017-03-06 2018-09-14 苏州和锐生物科技有限公司 生物制剂血药浓度检测方法及试剂
CN113514632A (zh) * 2021-04-20 2021-10-19 中国科学技术大学 基于纳米抗体的微悬臂梁免疫传感方法
US11320423B2 (en) 2015-12-01 2022-05-03 Roche Diagnostics Operations, Inc. Method for reduction of interferences in immunoassays

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB201522839D0 (en) * 2015-12-23 2016-02-03 Randox Lab Ltd And Randox Teoranta Method
EP4119944A1 (fr) * 2016-06-23 2023-01-18 Ablynx N.V. Analyse amélioré pharmacocinétique pour des domaines variables simples de l'immunoglobuline
CA3098206A1 (fr) * 2018-07-10 2020-01-16 Regeneron Pharmaceuticals, Inc. Procedes d'attenuation d'interference de cible medicamenteuse dans un dosage immunologique d'anticorps anti-medicament (ada)
JP2025514738A (ja) * 2022-04-19 2025-05-09 ソングァン メディカル ファンデーション 血液中の抗薬物抗体濃度測定による生物学的薬物に対する治療反応性の予測方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997016727A1 (fr) * 1995-11-01 1997-05-09 Biotransplant, Inc. Reduction de l'activite de fond pour un dosage, a l'aide de glycoproteines comportant des glucides oxydes
US5705353A (en) * 1995-06-07 1998-01-06 Beckman Instruments, Inc. Method of reducing interferences in assays

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5705353A (en) * 1995-06-07 1998-01-06 Beckman Instruments, Inc. Method of reducing interferences in assays
WO1997016727A1 (fr) * 1995-11-01 1997-05-09 Biotransplant, Inc. Reduction de l'activite de fond pour un dosage, a l'aide de glycoproteines comportant des glucides oxydes

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HAMILTON R G AND ADKINSON N F: "Naturally ocurring carbohydrate antibodies: Interference in solid-phase immunoassays", JOURNAL OF IMMUNOLOGICAL METHODS, vol. 77, 1985, pages 95 - 108, XP002491456 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011135024A1 (fr) 2010-04-29 2011-11-03 Biomedical Diagnostics Sa Procédés de détection d'anticorps
CN106164670A (zh) * 2014-02-11 2016-11-23 建新公司 用于检测抗药物抗体的存在或抗药物抗体的量的测定
CN106164670B (zh) * 2014-02-11 2019-06-14 建新公司 用于检测抗药物抗体的存在或抗药物抗体的量的测定
US11215623B2 (en) 2014-02-11 2022-01-04 Genzyme Corporation Assays for detecting the presence or amount of an anti-drug antibody
US11927596B2 (en) 2014-02-11 2024-03-12 Genzyme Corporation Assays for detecting the presence or amount of an anti-drug antibody
US11320423B2 (en) 2015-12-01 2022-05-03 Roche Diagnostics Operations, Inc. Method for reduction of interferences in immunoassays
CN108535490A (zh) * 2017-03-06 2018-09-14 苏州和锐生物科技有限公司 生物制剂血药浓度检测方法及试剂
CN113514632A (zh) * 2021-04-20 2021-10-19 中国科学技术大学 基于纳米抗体的微悬臂梁免疫传感方法

Also Published As

Publication number Publication date
BRPI0811290A2 (pt) 2014-10-21
IL201907A0 (en) 2010-06-16
JP2010527005A (ja) 2010-08-05
CA2686206A1 (fr) 2008-11-13
CN101680880A (zh) 2010-03-24
MX2009012048A (es) 2010-02-24
EP2142925A1 (fr) 2010-01-13
AU2008247352A1 (en) 2008-11-13
KR20100029083A (ko) 2010-03-15
MX2009012044A (es) 2009-11-18
RU2009141159A (ru) 2011-06-20

Similar Documents

Publication Publication Date Title
WO2008137885A1 (fr) Elimination d'interférences provoquées par des anticorps antiglucides dans le cadre de dosages immunologiques
CN101371140B (zh) 抗药物抗体的测定
JP6678792B2 (ja) 多重特異性結合物の結合パートナーを検出するための方法
CN105378480B (zh) 检测血清样品中抗药物抗体的干扰抑制性免疫测定
JPH0814579B2 (ja) 特異的結合性物質の測定法及び試薬
CN101896820A (zh) 区分测定
US12099055B2 (en) Methods and compositions for removing biotin interference from assays using conjugated molecular traps
EP3857224B1 (fr) Piége moléculaire pour éliminer l'interférence de la biotine dans des dosages
US20070184501A1 (en) Blocker reagent for reduction of heterophile interferences
CN112703400B (zh) 用于从使用环糊精阱的测定除去生物素干扰的方法和组合物
JP7273195B2 (ja) 干渉抑制薬物動態イムノアッセイ
JP2014221745A (ja) タンパク質標識抗体およびそれを含む組成物、ならびにそれらの製造方法
EP4317172A1 (fr) Procédé d'analyse immunologique pour un télopeptide c-terminal de collagène de type i
JP3749636B2 (ja) メダラシンの免疫学的測定方法
HK1217759B (en) Interference-suppressed immunoassay to detect anti-drug antibodies in serum samples
HK1208731B (en) Method for the detection of a multispecific binder

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 200880015303.3

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08755078

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2686206

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 2008755078

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2010507595

Country of ref document: JP

Ref document number: 7178/DELNP/2009

Country of ref document: IN

Ref document number: MX/A/2009/012044

Country of ref document: MX

Ref document number: MX/A/2009/012048

Country of ref document: MX

WWE Wipo information: entry into national phase

Ref document number: 2008247352

Country of ref document: AU

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2008247352

Country of ref document: AU

Date of ref document: 20080506

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 20097025396

Country of ref document: KR

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2009141159

Country of ref document: RU

ENP Entry into the national phase

Ref document number: PI0811290

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20091106