CA2686206A1

CA2686206A1 - Elimination of interference in immunoassays caused by anti-carbohydrate antibodies

Info

Publication number: CA2686206A1
Application number: CA002686206A
Authority: CA
Inventors: David Leroy Wensel; Michel Awwad
Original assignee: Wyeth; David Leroy Wensel; Michel Awwad
Current assignee: Wyeth LLC
Priority date: 2007-05-07
Filing date: 2008-05-06
Publication date: 2008-11-13
Also published as: BRPI0811290A2; EP2142925A1; MX2009012048A; WO2008137885A1; AU2008247352A1; RU2009141159A; MX2009012044A; IL201907A0; KR20100029083A; CN101680880A; JP2010527005A

Abstract

The present invention is directed to assays for the detection of an anti-drug 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.

Description

ELIMINATION OF INTERFERENCE IN IMMUNOASSAYS

CAUSED BY ANTI-CARBOHYDRATE ANTIBODIES
FIELD OF THE INVENTION

[0001] The present invention is directed to assays for the detection of an anti-drug 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.

BACKGROUND OF THE INVENTION

[0002] Glycoproteins are increasingly being used for the treatment and diagnosis of cancer, infections, and immunological diseases. In particular, monoclonal antibodies from various species, as well as chimeric or "humanized" antibodies are the kinds of glycoproteins that are finding increasing clinical use. When administered to a subject 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.

[0003] Regulatory agencies require the monitoring and quantification of human immune response to drugs and or proteins in treated patients. However, pre-dose reactivity of human serum or plasma to monoclonal antibodies, in some cases, is high and may interfere with the monitoring and quantification of the specific immune response to the glycoprotein. Furthermore, it has been noted that subjects that are naive with respect to exposure to certain antibodies used as a therapeutic often demonstrate a pre-treatment serum reactivity with the therapeutic protein.
This reactivity can be attributed to the presence in human serum or plasma of "natural antibodies" reactive with the antibody and in particular with the carbohydrate portion of the administered glycoprotein. (See, e.g., Robert G. Hamilton and N.
Franklin Adkinson. Naturally Occurring Carbohydrate Antibodies: Interference in Solid-Phase Immunoassays. Journal of Immunological Methods 77 (1985) 95-108.);
"Hamilton").
Furthermore, it has been observed that anti-carbohydrate antibodies can act as a source of interference in immunoassays. Justus Adedoyin, S.G.O. Johansson, Hans Gr6nlund, Marianne van Hage. Interference in immunoassays by human IgM with specifi'city for the carbohydrate moiety of animal proteins. Journal of Immunological Methods 310 (2006) 117-125).

[0004] Hamilton discloses that interference caused by these natural antibodies to carbohydrate in immunoassay analysis of human sera can be removed by pre-adsorption of the subject sera with soluble or solid phase carbohydrate reactive with the natural antibodies. Also, U.S. Patent No. 5,856,106 discloses a method for determining the levels of specific immune responsiveness to a glycoprotein in an individual being treated therewith by contacting a body fluid sample obtained from the individual subsequent to glycoprotein treatment with the glycoprotein that has been modified to have an oxidized carbohydrate portion.

[0005] Since the pretreatment serum reactivity with a glycoprotein might either conceal or augment the real immune response to the glycoprotein when administered as a therapeutic, reduction of this reactivity is desired prior to assessing immunoreactivity of a potential therapeutic glycoprotein.

SUMMARY OF THE INVENTION

[0006] 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 pre-treatment or na7ve 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.

[0007] Thus, in one aspect, 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 (iii) 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.

[0008] In another aspect, 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 drug containing a carbohydrate moiety does not comprise a glycoprotein.

[0009] In another aspect, 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.

[0010] In another aspect, 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.

[0011] In one embodiment, 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.

[0012] In another embodiment, 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., naive subjects). In such embodiments, the assay provides information about the presence or absence in a naive subject of endogenous pre-existing antibodies that specifically bind to the drug containing a carbohydrate moiety.

[0013] In some embodiments, information about the presence or absence in a naive 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.

[0014] In some embodiments wherein the assay is performed on a fluid sample from a naive subject, 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.

[0015] In some embodiments wherein the assay is performed on a fluid sample from a naive subject, 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 naive subjecYs sera.

[0016] In another embodiment, 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.

[0017] In another embodiment, 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.

[0018] In another embodiment, 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.

[0019] In another embodiment, 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.

[0020] In another embodiment, the invention provides any one or more of the assays described herein wherein the additional carbohydrate is one or more of bacterial I ipo-polysaacha ride (LPS), a dextran, a levan, pneumococcal polysaccharide, agarose, cellulose, carrageenan, and methylglycoside or a functional fragment of any one of the foregoing carbohydrates.

[0021] In another embodiment, 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/mi and about 0.5 mg/mI. In another embodiment, the additional carbohydrate is present in the combination at a concentration between about ng/ml and about 0.75 mg/mi 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.

[0022] In another embodiment, 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. In some embodiments, 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. In some embodiments, the carrier specifically binds to an antigen expressed on or within cancer cells. In some embodiments 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.

[0023] In another embodiment, the invention provides one or more of the assays described herein wherein the carrier is a monoclonal antibody. In some embodiments, 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.

[0024] In another embodiment, 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.

[0025] In other embodiments, 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, hemiasterlins, and maytansinoids. In some preferred embodiments the cytotoxic agent is a calicheamicin and in some embodiment can be N-acetyl gamma dimethyl hydrazine calicheamicin.

[0026] In another embodiment, 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.

[0027] In some embodiments, the capture agent that specifically binds the anti-drug antibody comprises the drug having a carbohydrate moiety or a portion or functional fragment of the drug containing a carbohydrate moiety.

[0028] In some embodiments, the capture agent that specifically binds the anti-drug 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.

[0029] In some embodiments, the drug containing a carbohydrate moiety and the capture reagent is S-[(2R,4S,6S)-6-({[(2R,4S,5R)-5-({(2S,4S,5S)-5-[acetyl(ethyl)am ino]-4-methoxytetrahydro-2H-pyran-2-yl}oxy)-4-hydroxy-6-methoxy-2-methyltetrahydro-2H-pyran-3-yl]am ino}oxy)-4-hydroxy-2-methyltetrahydro-2H-pyran-3-yl]4-{[(2S, 3R,4R, 5S,6S )-3, 5-d ihyd roxy-4-m ethoxy-6-m ethyltetrahydro-2H-pyran-2-yI]oxy}-3-iodo-5,6-dim ethoxy-2-methylbenzenecarbothioate.

[0030] In some embodiments, the capture agent that specifically binds the drug having a carbohydrate moiety comprises an antibody to the drug containing a carbohydrate moiety. In some embodiments wherein the drug containing a carbohydrate moiety comprises a carrier-drug conjugate, 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.

[0031] In some embodiments wherein the drug containing a carbohydrate moiety comprises a 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.

[0032] In another embodiment, the bound antibody to the drug containing a carbohydrate moiety is detected by binding of a labeled compound to the antibody.
In some preferred embodiments, 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. In some embodiments, the detectable label comprises at least one of an enzyme label, a luminescent label and a radioisotope label.

[0033] In some embodiments, the invention provides an assay as described herein in any one or more of examples one through eight.

[0034] In some embodiments, 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. In some embodiments, 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. In some embodiments, the detectable label comprises at least one label selected from the group consisting of an enzyme label; a luminescent label; and a radioisotope label.

[0035] In some embodiments, 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.

[0036] In some embodiments, 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.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is a graphical representation of the presence of an interfering factor in naive serum having a binding specificity for calicheamicin.

[0038] Figure 2 shows a diagrammatic representation of a calicheamicin as conjugated to an antibody.

[0039] Figure 3 is a graphical representation of the reduction in calicheamicin specific binding in sera titrated with various concentrations of detoxified LPS.

[0040] 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.

[0041] Figure 5 is a graphical representation demonstrating the reduction in calicheamicin specific binding in sera tftrated with various concentrations of detoxified LPS.

[0042] Figure 6 shows a graphical representation of calicheamicin specific IgM
from calicheamicin specific sera binding IgM.

[0043] Figure 7 shows a graphical representation of IgM from Calicheamicin specific sera binding to calicheamicin.

[0044] Figure 8 shows a graphical representation of the presence of calicheamicin specific binding factor in naive human sera.

[0045] Figure 9 shows the chemical structure of S-[(2R,4S,6S)-6-({[(2R,4S,5R)-5-({(2S,4S,5S)-5-[acetyl(ethyl)am ino]-4-methoxytetrahydro-2H-pyran-2-yl}oxy)-hydroxy-6-methoxy-2-methyltetrahydro-2H-pyran-3-yl]am ino}oxy)-4-hydroxy-2-methyltetrahyd ro-2H-pyran-3-yl]4-{[(2S,3R,4R,5S,6S)-3,5-dihydroxy-4-methoxy-6-m ethyltetra hyd ro-2 H-pyra n-2-yl]oxy}-3-iod o-5, 6-d i m ethoxy-2-m ethyl benzeneca rboth ioate).

DETAILED DESCRIPTION OF THE INVENTION

[0046] 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."

[0047] 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. Immunomodulators contain biological response modifiers that are known to have regulatory functions such as lymphokines, growth factors, and complement-activating cobra venom factor (CVF). 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.

[0048] 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 71 (+my1V-region) antibody, which is specific for cell surface antigen CD20, which is expressed on B-cells. These antibody based therapeutics rely either on complement-mediated cytotoxicity (CDCC) or antibody-dependent cellular cytotoxicity (ADCC) against B cells, or on the use of radionuclides, such as 1311 or 90Y, which have associated preparation and use problems for clinicians and patients. Consequently, there is a need for the generation of immunoconjugates that can overcome the shortcomings of current antibody-based therapeutics to treat a variety of malignancies including hematopoietic malignancies like non-Hodgkin's lymphoma (NHL), which can be produced easily and efficiently, and which can be used repeatedly without inducing an immune response.

[0049] 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 y, 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).

[0050] The presence of antibodies in naive sera that specifically bind to the carbohydrate portion of a drug containing a carbohydrate moiety or a carrier drug conjugate wherein the drug portion of the conjugate contains a carbohydrate moiety can interfere with immunoassays to detect (i) antibodies generated in response to the carrier drug conjugate or (ii) the drug or carrier drug conjugate having a carbohydrate. Such interference has now been determined to include endogenous antibodies that specifically bind to carbohydrates that are not associated with a glycoprotein or glycoprotein portion of a carrier drug conjugate.

[0051] As an example, if 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. Also, in an assay where 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.

[0052] It is reported herein that the presence of additional carbohydrate in an assay to detect specific binding between an anti-carrier-drug conjugate antibody and the can-ier-drug conjugate in a fluid sample containing antibody will enable the specific detection of anti-carrier-drug conjugate antibody that is generated in response to the exposure of a subject to the carrier-drug conjugate. The additional carbohydrate does not have to be pre-adsorbed with the fluid sample prior to detecting specific binding between the anti-carrier-drug conjugate antibody and the carrier-drug conjugate. The specific detection is possible because the additional carbohydrate added to the assay will remove any activity that may have been present before exposure to the drug.

[0053] Similarly 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 pre-existing anti-drug antibodies present in the sample.

[0054] 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 naive 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.

[0055] The development of specific binding assay techniques has provided extremely useful analytical methods for determining various substances of diagnostic, medical, environmental and industrial importance which appear in various liquid or solid (e.g., tissue) samples at very low concentrations. Specific binding assays are based on the specific interaction between a bindable analyte under determination and a binding partner therefore, i.e. analyte-specific moiety.
The binding of the analyte-specific moiety and interaction of any additional reagents, if necessary, effect a mechanical separation of bound and unbound labeled analyte or affect the label in such a way as to modulate the detectable signal. The former situation is normally referred to as heterogeneous and the latter as homogeneous, in that the latter does not require a separation step.

[0056] 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. Nos. 5,606,040 and 5,770,710).

[0057] In conventional label conjugate specific binding assay techniques, 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. In the bound-species, 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.

[0058] 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.

[0059] Additional immunoassay formats and protocols are also known in the art and are applicable to use in the present invention. Various binding assays using immobilized or immobilizable materials for the direct immobilization of one of the binding participants in a binding assay reaction, e.g., immobilized antigen or antibody, in order to accomplish the desired separation of the bound and free forms of a labeled reagent, have been proposed. In particular, a number of such binding assays have been described wherein an antibody to an antigen to be detected is bound to an immobilizing material such as the inner wall of a test tube or a plastic or magnetic bead.

[0060] 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.

[0061] Another of such binding assays is described by U.S. Pat. No. 4,230,683 which discloses a method employing a 6 mm polystyrene bead having antigen or antibody bound thereto wherein the antigen or antibody is reacted with a hapten-conjugated antibody to the antigen or antibody. The bound hapten-conjugated antibody is further reacted with labeled anti-hapten antibody in order to determine the amount of antigen or antibody in a test sample.

[0062] Still another of such binding assays is described by U.S. Pat. No.
4,228,237 which discloses a method for the detection and determination of ligands in a liquid medium using enzyme labeled avidin and a biotin labeled reagent in a specific binding process. In this method, the ligand to be detected is contacted with an insoluble phase containing a specific binding substance for the ligand.

[0063] In addition to the direct immobilization techniques heretofore described, indirect immobilization by marking or labeling a binding assay reaction participant to be immobilized with a first binding substance, and then adding an immobilized second binding substance, has been proposed.

[0064] For example, 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. Similarly, 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. Recently, a different approach was disclosed which does not require a separation step and therefore has been referred to as a homogeneous system, in contrast to a heterogeneous system in which separation is essential. 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.

[0065] Still further, the labeled reagent can include other conventional detectable chemical groups. Such 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. (1979)25:353); chromophores; luminescers such as chemiluminescers and bioluminescers (see U.S. Pat. No. 4,380,580);
specifically bindable ligands (e.g., biotin and haptens); electroactive species; and radioisotopes such as 3 H, 35 S, 32 P, 1251, and14 C. Such labels and labeling pairs are detected on the basis of their own physical properties (e.g., fluorescers, chromophores and radioisotopes) or their reactive or binding properties (e.g., enzymes, substrates, coenzymes and inhibitors).

DEFINITIONS

[0066] As used in the specification and the appended claims, the singular forms "a," "an" and "the" include reference to the plural unless the context clearly dictates otherwise. Thus, for example, reference to "an antibody" includes a plurality of such compositions, i.e., "antibodies."

[0067] The term "about" means within 20%, more preferably within 10% and more preferably within 5% of a stated measurement or concentration.

[0068] The term "additional carbohydrate" as used herein, refers to carbohydrates that can compete with carbohydrate that binds to endogenous antibody in serum. The use of bacterial lipopolysaccharide (LPS), methylglycoside, dextran, levan, pneumococcal polysaccharide, agarose, cellulose, or carrageenan in an immunoassay can block the binding of antibodies generally binding to carbohydrates. The addition of 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. In addition, selection of the proper carbohydrate can involve a carbohydrate that specifically binds to the drug-carrier conjugate.

[0069] Unless the context specifically indicates otherwise, the term "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.

[0070] As used herein, the phrase "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. To specifically mimic a carbohydrate portion of a drug containing a carbohydrate moiety the carbohydrate or fragment thereof will specifically bind the same target and receptor compound that binds to the carbohydrate moiety of the drug.

[0071] 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.

[0072] As used herein, the term "capture reagent" is meant to include any composition that specifically binds to a corresponding target. For example, 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. For example, if 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. Furthermore, if 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.

[0073] 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. Thus, in a competitive immunoassay, 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.

[0074] The term "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.
Representative 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, methotrexate, flurouracils, etoposide, taxol, taxol analogs, platins such as cis-platin and carbo-platin, mitomycin, thiotepa, taxanes, vincristine, daunorubicin, epirubicin, actinomycin, authramycin, azaserines, bleomycins, tamoxifen, idarubicin, dolastatins/auristatins, hemiasterlins, esperamicins and maytansinoids.

[0075] As used herein, the term "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 [0076] 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 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.

[0077] The term "drug containing a carbohydrate moiety" as used herein 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. The term 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. Generally, these types of conjugates are referred to as immunoconjugates. In addition, 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. Thus, as used herein, 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-m eth oxytetra hyd ro-2 H-pyra n-2-yl}oxy)-4-hyd roxy-6-m eth oxy-2-m ethyltetra hyd ro-2 H-pyran-3-yl]am ino}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-d im ethoxy-2-methylbenzenecarbothioate).

[0078] 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". Examples of 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. When reference is made to 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.

[0079] Representative non-limiting examples of 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. These drug descriptors are not mutually exclusive, and thus a therapeutic agent may be described using one or more of the above-noted terms.
For example, selected radioisotopes are also cytotoxins. Therapeutic agents may be prepared as pharmaceutically acceptable salts, acids or derivatives of any of the above. Generally, 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. Examples of 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. In addition, conjugates can be made using secondary carriers as the cytotoxic agent, such as liposomes or polymers, for example.

[0080] 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). Representative 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, HALOTESTINO(fluoxymesterone)), progestins (MEGACEO (megestrol acetate), PROVERAO (medroxyprogesterone acetate)), and corticosteroids (prednisone, dexamethasone, hydrocortisone).

[0081] "Luminescent" labels include labels that involve phosphorescence, chemiluminescence andfluorescence. "FluorescenY' 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.

[0082] 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: 3H, 1251 and 35S. The labels, listed above, are detected using a detecting system that is specific to the nature of each of the labels.

[0083] As used herein, the term "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.

[0084] As used herein, the terms "pre-mixing" or "pre-mixed" refer to mixing of two or more components of an assay prior to beginning the assay. For example, "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.

[0085] 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. In a non-competitive sandwich assay, the measurement of the labeled binding event, is directly proportional to the amount of antigen present in the sample.

[0086] The term "specific 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"' M or higher, such as at least about 10$ 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"" 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 10-4 .

[0087] Representative anti-angiogenic agents include inhibitors of blood vessel formation, for example, famesyltransferase 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.

[0088] 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® (1,25-dihydroxycholecalciferol (vitamin D)), aromatase inhibitors (FEMARA® (letrozone)), telomerase inhibitors, iron chelators (e.g., 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (Triapine)), apoptin (viral protein 3--VP3 from chicken aneamia virus), inhibitors of Bcl-2 and Bcl-X(L), TNF-alpha, FAS
ligand, TNF-related apoptosis-inducing ligand (TRAIUApo2L), activators of TNF-alpha/FAS ligand/TNF-related apoptosis-inducing ligand (TRAIUApo2L) signaling, and inhibitors of PI3K-Akt survival pathway signaling (e.g., UCN-01 and geldanamycin).

[0089] In particular embodiments of the invention, the cytotoxic drug having a carbohydrate moiety is an antibiotic such as a calicheamicin, also called the LL-E33288 complex, for example, gamma-calicheamicin,). See U.S. Pat. No.
4,970,198. Early studies with antibody conjugates of gamma calicheamicin hydrazide derivatives showed antigen-based cytotoxicity in vitro and activity in xenograft experiments. 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. Pat. Nos. 4,671,958;
5,053,394;
5,037,651; 5,079,233; and 5,108,912; which are incorporated herein in their entirety.
These compounds contain a methyltrisu[fide that may 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 for conjugating calicheamicin to an antibody. Stabilizing the disulfide bond that is present in all calicheamicin conjugates by adding dimethyl substituents made additional improvements. This led to the choice of 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.

[0090] 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/mI 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° C., 1 hour.
Hydrophobic interaction chromatography (HIC) 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.

[0091] As part of the carrier-drug conjugates, mentioned above, 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. These 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)-({(2S,4S,5S)-5-[acetyl(ethyl)am ino]-4-methoxytetrahydro-2H-pyran-2-yl}oxy)-4-hydroxy-6-methoxy-2-methyltetrahydro-2H-pyran-3-yl]am ino}oxy)-4-hydroxy-2-methyltetrahydro-2H-pyran-3-yl]4-{[(2S,3R,4R,5S,6S )-3,5-dihydroxy-4-methoxy-6-methyltetra hyd ro-2H-pyran-2-yl]oxy}-3-iodo-5,6-d imethoxy-2-methylbenzenecarbothioate) a carbohydrate moiety. This carbohydrate, along with other carbohydrates, can interact with endogenous antibodies in serum that are directed against general carbohydrate moieties.

[0092] All patents, patent applications, and other literature cited herein are hereby incorporated by reference in their entirety.

[0093] The present invention is further illustrated and supported by the following examples. However, these examples should in no way be considered to further limit the scope of the invention. To the contrary, one having ordinary skill in the art would readily understand that there are other embodiments, modifications, and equivalents of the present invention without departing from the spirit of the present invention and/or the scope of the appended claims.

EXAM PLES

INTERFERENCE OF SIGNAL IN NAIVE SERA

[0094] During the development of anti-calicheamicin conjugate antibody assays, initial studies were undertaken on naive human or monkey sera. An ELISA was performed using calicheamicin as a capture reagent immobilized in the wells of a microtiter assay plate and blocked in 4% NFDM/PBST buffer (Non Fat Dry Milk/Phosphate Buffered Saline Tween; 137mM NaCI, 2.7mM KCI, 4.3 mM
Na2HPO4, I mM KH2PO4, pH7.2). The human or monkey sera was diluted in 1%
NFDM/PBST, added to ELISA wells, and then incubated. The wells were washed in THST (50 mM Tris-HCI, 0.5 M NaCI, 1 mM glycine and 0.05% (v/v)Tween 20, pH 8;) and incubated with an anti-species-HRP conjugate. The plates were washed in THST
before developed for a color reaction by the addition of the TMB
(tetramethylbenzidine) reagent.

[0095] The data during these initial assays revealed a high, interfering background signal observed after developing the reaction. In subsequent assays large dilutions, up to 1:160, were made on the sera added to the wells. The background in these assays decreased, as did the sensitivity of the assay. The data suggested that there was an element in the serum contributing to the interference of specific signal in the assay wells. This high background was observed in approximately one-third of the human and 1/3 of the monkey sera that was tested.

[0096] In order to determine what portion of the calicheamicin conjugate was interacting with the interfering serum element, a bridging-type ELISA was perfonned.
Calicheamicin conjugates, CMC-544, CMD-193 and CME-548 and their parental, unconjugated antibodies, anti-CD22 humanized monoclonal antibody G544, anti-Lewis-Y humanized monoclonal G193 and anti-5T4 humanized monoclonal antibody huH8 respectively, were immobilized on plates at a concentration of 100 ng/ml, and blocked in 4% NFDM/PBST.

[0097] 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."

[0098] 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."

[0099] 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."

[0100] Pooled cynomolgus monkey serum was diluted 1:20 in 1% NFDM/PBST
and incubated in wells. Other wells were incubated in buffer without serum as controls. After washing in THST, the assay plate was incubated with calicheamicin-HRP conjugate diluted 1:2000 in PBST. The wells were washed in THST before development with the TMB reagent.

[0101] As seen in Figure 1, there was high background observed only in wells containing the calicheamicin conjugate and not the parental antibody, indicating that the serum element specifically bound only the conjugates, but not their parental antibodies, and "bridged" them to the calicheamicin-HRP. This data suggested that the interfering element in the serum is binding to the calicheamicin moiety and not to the parental unconjugated antibody. Additionally, the ability of the serum element to bridge two different calicheamicin moieties, the calicheamicin conjugated antibodies and the calicheamicin-HRP, suggested a multivalent molecule, namely an immunoglobulin as the interfering serum element.

REDUCTION OF THE INTERFERENCE BY USING ADDITIONAL
CARBOHYDRATES

[0102] In order to determine the nature of the interaction between the interfering element in the sera and the calicheamicin, 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 (na7ve) were diluted in 1 % NFDM/PBST and added to the wells. In an attempt to compete out any interactions with the calicheamicin carbohydrates, detoxified lipopolysaccharide (LPS) was titrated into the sera in concentrations ranging from 1 ng/ml to 100 ng/ml.
Control wells contained no LPS. The assay was then incubated for 1 hour with shaking (standard unless otherwise indicated for all examples herein). The wells were washed in THST and then incubated with calicheamicin-HRP conjugate at a dilution of 1:2000 in PBST. The assay was then developed for a color reaction by the addition of TMB reagent. As shown in Figure 3, increasing amounts of LPS added to the CMC-544 wells caused a decrease in background signal. In the control wells containing CMC-544 without LPS, there was high background observed in all samples.

[0103] Similar tests were done with a secondary ELISA, wherein the CMC-544 monoclonal antibody. Antibody was immobilized in assay wells and then blocked in 4% NFDM/PBST buffer. Sera used in the previously described assays (naive) were diluted in 1% NFDM/PBST and added to the wells. Interfering amounts of either the detoxified portion of LPS or LPS were added to the reaction. CMC-544(biotin) that bound to the CMC-544 on plates was bridged via an antibody in the serum. The CMC-544 biotin was detected with Strepravidin bound to an HRP detector.
Incresaing amounts of LPS and detoxified LPS interfered with this specific binding.
Results are shown in Figure 5.

[0104] These data suggested that the interfering element in the serum interacted with the carbohydrate moiety on calicheamicin and not the antibody portion of the conjugate. The addition of the LPS was able to compete with the carbohydrate on the calicheamicin for the interfering serum element without the need for pre-adsorption of the sera with the LPS.

[0105] A further study was performed to demonstrate the specificity of the serum element for the calicheamicin carbohydrate moiety. Exogenously added methyl glycosideS-[(2R,4S,6S)-6-({[(2R,4S,5R)-5-({(2S,4S,5S)-5-[acetyl(ethyl)amino]-4-m ethoxytetra hyd ro-2 H-pyra n-2-yl}oxy)-4-hyd roxy-6-m ethoxy-2-m ethyltetra hyd ro-2 H-pyran-3-yl]am ino}oxy)-4-hydroxy-2-methyltetrahydro-2H-pyran-3-yl]4-{[( 2 S, 3 R, 4 R, 5S , 6 S)-3 , 5-d i h yd roxy-4-m eth oxy-6-m eth yltetra h yd ro-2 H-py ra n-2-yl]oxy}-3-iodo-5,6-dimethoxy-2-methylbenzenecarbothioate) was tested for its ability to specifically compete out any interactions with the methylglycoside moiety on calicheamicin and therefore to inhibit the interaction with the serum element.
The same assay, as described above for the addition of LPS was performed in separate wells containing CMD-193, CME-548 and CMC-544. Their parental, unconjugated antibodies were used as a control. The above methylglycoside was substituted in this assay for the LPS at various final concentrations ranging from 2 to 2000ng per ml.
As shown in Figure 4, the above indicated methyl glycoside was proven to be a potent inhibitor of the interfering element in the sera tested, with an IC50 in the range of 30-70 nM. These data suggest that the interfering element in sera specifically binds to the carbohydrate methylglycoside S-[(2R,4S,6S)-6-({[(2R,4S,5R)-5-({(2S,4S,5S)-5-[acetyl(ethyl)am ino]-4-methoxytetrahydro-2H-pyran-2-yl}oxy)-4-hydroxy-6-methoxy-2-methyltetrahydro-2H-pyran-3-yl]am ino}oxy)-4-hydroxy-2-methyltetrahyd ro-2H-pyran-3-yl]4-{[(2S, 3R,4R, 5S,6S )-3, 5-dihyd roxy-4-methoxy-6-m ethyltetra hyd ro-2 H-pyra n-2-yl]oxy}-3-iod o-5, 6-d im ethoxy-2-methylbenzenecarbothioate) present on calicheamicin.

SERUM IgG IS NOT THE INTERFERING FACTOR

[0106] As mentioned previously, the bridging capability of the factor indicated an immunoglobulin. Both IgG and IgM were initially examined as possibilities. For IgG, 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.

[0107] The results demonstrated that the purified IgG fractions showed no bridging activity over the buffer-only background, and the IgG-depleted fraction showed the same level of LPS-sensitive bridging activity as untreated serum.
Therefore, IgG does not appear to be responsible for the activity..

IDENTIFYING SERUM FACTOR AS IGM

[0108] To determine the nature of the interfering molecule in the serum binding to the carbohydrate moiety of calicheamicin that could be responsible for the interference observed in the previously described assays, a bridging-type ELISA was performed. Assay wells were coated with the capture reagents: CMC-544, its unconjugated parental antibody G544, or monkey IgM as the positive control.
Milk was used as the negative control. The wells were then blocked in 4% NFDM/PBST
buffer. Serum was diluted in 1% NFDM/PBST and added to the wells. LPS was added to the serum at a concentration of 250 pg/mI and incubated. The wells were washed in THST and incubated with anti-IgM-HRP to detect any IgM bound to the plates. The assay was developed for a color reaction by the addition of the TMB
reagent. As shown in Figure 6, 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.

[0109] In an additional assay, wells were coated with anti-monkey IgM. Anti-mouse IgG was used as a negative control. Dilutions of sera, made in PBST, ranging from .001 to 10% were made and added to the wells. After incubation, calicheamicin-HRP was added. The plates were washed and developed in the TMB reagent. As shown in Figure 7, the serum IgM captured on the plate bound the calicheamicin-HRP in a dose dependent fashion. The wells containing anti-mouse IgG did not.

[0110] These data demonstrate that IgM present in some sera has the ability to specifically bind to the carbohydrate portion of the calicheamicin molecule.
Such binding can give rise to high background signal in assays used to detect antibodies against calicheamicin conjugates or detecting calicheamicin itself. The addition of exogenously added carbohydrates to the assay will aid in the abrogation of the signal generated by the presence of the interfering IgM in the serum.

NAIVE SERUM CONTAINING ANTI-CARBOHYDRATE IGM

[0111] In order to determine the prevalence of anti-carbohydrate antibodies in a random panel of naive human sera, a bridge-type ELISA was performed. CMC-544 was used as a capture reagent and immobilized in the assay wells. The wells were blocked in 4% NFDM/PBST buffer. The naive serum samples were diluted in 1%
NFDM/PBST, added to the wells, and then incubated. The wells were washed in THST and then incubated with calicheamicin-HRP. The assay was then developed for a color reaction by the addition of TMB reagent. No carbohydrate was added to this assay in an effort to determine what percentage of the serum samples examined contained interfering IgM that could bind to the carbohydrate moiety on calicheamicin. As shown in Figure 8, approximately 30% of the serum samples tested showed interference. Similar observations were noted in naive monkey sera, where 2 out of 20 monkeys showed a high level of anti-carbohydrate antibodies and 8 out of 20 showed significant levels of anti-carbohydrate antibodies.

DETECTION OF ANTI CALICHEAMICIN CONJUGATE
ANTIBODIES IN SERUM

[0112] In order to detect the presence of antibodies in serum specifically generated in response to exposure to a calicheamicin-conjugated monoclonal antibody, for example CMC-544, 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)-[acetyl(ethyl)am ino]-4-methoxytetrahydro-2H-pyran-2-yl}oxy)-4-hydroxy-6-methoxy-2-m ethyltetra hyd ro-2 H-pyra n-3-yl]a m i no}oxy)-4-hyd roxy-2-m ethyltetra hyd ro-2 H-pyra n-3-yI]4-{[(2S , 3 R,4 R, 5S , 6S )-3, 5-d i hyd roxy-4-m ethoxy-6-m ethyltetra hyd ro-2 H-pyra n-2-yl]oxy}-3-iodo-5,6-dimethoxy-2-methylbenzenecarbothioate) present on calicheamicin is determined.

[0113] Naive 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 naive sera from a subject being tested for production of anti-drug antibody after exposure to the drug.

[0114] Experimental subjects are injected with or otherwise exposed to CMC-544 and serum is collected. CMC-544, as well as the unconjugated parental antibody G544, are used as capture reagents and immobilized in separate wells at a concentration of 100 ng/ml. The wells are blocked in 4% NFDM/PBST buffer.
Serum is collected from the exposed subjects and mixed with 1% NFDM/PBST, added to the wells, and then incubated. The wells are washed in THST and incubated with calicheamicin-HRP conjugate and the optimal concentration of additional carbohydrate methyl glycoside (S-[(2R,4S,6S)-6-({[(2R,4S,5R)-5-({(2S,4S,5S)-5-[acetyl(ethyl)am ino]-4-methoxytetrahydro-2H-pyran-2-yl}oxy)-4-hydroxy-6-methoxy-2-methyltetrahydro-2H-pyran-3-yl]am ino}oxy)-4-hyd roxy-2-methyltetrahydro-2H-pyran-3-yI]4-{[(2S,3R,4R, 5S,6S)-3,5-d i hydroxy-4-m ethoxy-6-m ethyltetra hyd ro-2 H-pyran-2-yI]oxy}-3-iodo-5,6-dimethoxy-2-methylbenzenecarbothioate) present on calicheamicin). Alternatively, 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.

[0115] To determine if the antibody response is generated against the carbohydrate portion of calicheamicin, 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.

TESTING OF NAIVE SERUM FOR PRESENCE OF ANTIBODY
TO A CARRIER DRUG CONJUGATE WHEREIN THE DRUG HAS A
CARBOHYDRATE MOIETY

[0116] In order to determine the presence of pre-existing interfering IgM in the serum from a candidate subject for treatment with a calicheamicin conjugate, 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 naive 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. The wells are washed in THST
and incubated with calicheamicin-HRP conjugate and the optimal concentration of additional methyl glycoside (S-[(2R,4S,6S)-6-({[(2R,4S,5R)-5-({(2S,4S,5S)-5-[acetyl(ethyl)am ino]-4-methoxytetrahydro-2H-pyran-2-yl}oxy)-4-hydroxy-6-methoxy-2-methyltetrahydro-2H-pyran-3-yl]am ino}oxy)-4-hydroxy-2-methyltetrahydro-2H-pyran-3-yl]4-{[(2S,3R,4R, 5S,6S)-3,5-d ihydroxy-4-methoxy-6-methyltetra hydro-2H-pyra n-2-yl]oxy}-3-iodo-5,6-dimethoxy-2-methylbenzenecarbothioate) present on calicheamicin). Following washing, plates are then developed for a color reaction by the addition of the TMB reagent. A color reaction would indicate Ig bound to the calicheamicin bound in the wells and also bound to the calicheamicin HRP
subsequently added.

[0117] Alternatively, an initial screen of naive sera would have no additional carbohydrate added. Sera would then be subjected to a titration of carbohydrate to verify that carbohydrates are the target of the sera reactivity.

TESTING FOR CARRIER-DRUG CONJUGATE WITH
CALICHEAMICN AS THE DRUG

[0118] In order to detect a calicheamicin conjugate, for example CMC-544, in serum from subjects administered this drug, 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.

Claims

1. An assay to detect the presence of an antibody to a drug containing a carbohydrate moiety in a fluid sample containing an antibody from a subject, 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-mixed 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.

2. An assay to detect the presence of an antibody to a drug containing a carbohydrate moiety in a fluid sample containing antibodies from a subject, 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 drug containing a carbohydrate moiety does not comprise a glycoprotein.

3. The assay of claim 1 or claim 2 wherein the assay is performed after exposure of the subject to the drug containing the carbohydrate moiety.

4. The assay of claim 1 or claim 2 wherein the assay is performed before exposure of the subject to the drug containing the carbohydrate moiety.

5. The assay of any preceding claim wherein the additional carbohydrate is added after the sample and the drug containing a carbohydrate moiety are combined.

6. The assay of any preceding claim wherein the fluid sample from the subject comprises at least one of: whole blood; serum; mucous, saliva, colostrum and plasma.

7. The assay of claim 6 wherein the detecting step comprises at least one of a sandwich assay, a bridging assay and a competitive binding assay.

8. The assay of any preceding claim wherein the detecting step comprises at least one of: detecting a change in refractive index at a solid optical surface in contact with the sample; detecting a change in luminescence; measuring a change in color;
detecting a change in radioactivity; measuring using biolayer interferometry;
measuring using cantilever-detection; measuring using label-free intrinsic Imaging;
and measuring using acoustic-detection.

9. The assay of claim 8 wherein the detecting step comprises an assay selected from the group consisting of: an enzyme-linked immunosorbent assay (ELISA); an electro-chemiluminescent assay (ECL); radioimmunoassay (RIA); solid-phase radioimmunoassay (SPRIA); immunoblotting; immunoprecipitation; Fluorescent Activated Cell Sorting (FACS).

10. The assay of claim 9 wherein the detecting step comprises a bridging assay.

11. The assay of any preceding claim wherein the detecting step comprises an ELISA.

12. The assay of any preceding claim wherein the additional carbohydrate is bacterial lipopolysaccharide (LPS) or a fragment thereof, a dextran or a fragment thereof, a levan or a fragment thereof, pneumococcal polysaccharide or a fragment thereof, agarose or a fragment thereof, cellulose or a fragment thereof, a carrageenan or a fragment thereof, and a methylglycoside or a fragment thereof.

13. The assay of any preceding claim wherein the additional carbohydrate is present in the combination in step a) at a concentration between about 10 ng/ml and about 1 mg/ml

14. The assay of any preceding claim wherein the drug containing a carbohydrate moiety further comprises a carrier conjugated to the drug.

15. The assay of claim 14, wherein 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.

16. The assay of claim 14 or claim 15, wherein the carrier specifically binds to an antigen expressed on the surface of cancer cells.

17. The assay of claim 16, wherein the antigen expressed on 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.

18. The assay of any of claims 15 through 17 wherein the carrier is a monoclonal antibody.

19. The assay of claim 18 wherein the monoclonal antibody is selected from the group consisting of: an anti-CD22 monoclonal antibody; an anti-5T4 monoclonal antibody; an anti-Lewis Y monoclonal antibody: and an anti-CD33 monoclonal antibody.

20. The assay of any preceding claim, wherein the drug containing a carbohydrate moiety is selected from the group consisting of: a cytotoxic agent; a radiotherapeutic;
an immunomodulatory agent; an anti-angiogenic agent; an anti-proliferative agent; a pro-apoptotic agent; a chemotherapeutic agent; and a therapeutic nucleic acid.

21. The assay of claim 20 wherein the drug containing a carbohydrate moiety is a cytotoxic agent.

22. The assay of claim 21 wherein the cytotoxic agent is an inhibitor of tubulin polymerization; an alkylating agent that binds to and disrupts DNA; an inhibitor of protein synthesis; or a tyrosine kinase inhibitor.

23. The assay of claim 21 wherein the cytotoxic agent is selected from calicheamicins, thiotepa, taxanes, vincristine, daunorubicin, doxorubicin, epirubicin, actinomycin, authramycin, azaserines, bleomycins, tamoxifen, idarubicin, dolastatins/auristatins, hemiasterlins, and maytansinoids.

24. The assay of claim 23, wherein the cytotoxic agent is a calicheamicin.

25. The assay of claim 24 wherein the calicheamicin is N-acetyl gamma dimethyl hydrazide calicheamicin.

26. The assay of claim 25 wherein the N-acetyl gamma dimethyl hydrazide calicheamicin is conjugated to a carrier monoclonal antibody and the monoclonal antibody specifically binds to an antigen 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.

27. The assay of any of claims 22 through 24 wherein the additional carbohydrate is 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-methylbenzenecarbothioate).

28. The assay of any preceding claim wherein the detecting step further comprises specific binding of a capture reagent to an antibody in the sample that specifically binds to the drug containing a carbohydrate moiety.

29. The assay of claim 28 wherein the presence of the antibody that specifically binds to the drug containing a carbohydrate moiety is detected by specific binding of a compound to the antibody that specifically binds to the drug containing a carbohydrate moiety.

30. The assay of claim 29 wherein the compound is selected from the group consisting of: the capture reagent further comprising a detectable label; and a second antibody further comprising a detectable label, wherein the second antibody specifically binds to the bound antibody.

31. The assay of claim 30 wherein the detectable label comprises at least one label selected from the group consisting of an enzyme label; a luminescent label; a protein label; a vitamin label; a radioisotope label.

32. The assay of any of claims 28 through 31 wherein the capture reagent comprises a calicheamicin.

33. The assay of claim 32 wherein the capture reagent is the carbohydrate moiety of calicheamicin or a functional fragment thereof.

34. The assay of claim 33 wherein the carbohydrate moiety of calicheamicin is 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-methylbenzenecarbothioate) present on calicheamicin

35. An assay as described in claim 1 and as further described in any of Examples 5, 6 or 7.

36. An assay to detect the presence of 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) a capture reagent that specifically binds to the drug containing a carbohydrate moiety and (iii) additional carbohydrate ; and (b) 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-mixed with the fluid sample prior to detecting whether or not specific binding occurs between the capture reagent and the drug containing a carbohydrate moiety.

37. An assay to detect the presence of 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) a capture reagent that specifically binds to the drug containing a carbohydrate moiety and (iii) additional carbohydrate ; and b) 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.

38. The assay of claim 36 or 37 wherein the presence of the drug bound to the capture reagent is detected with a compound that also specifically binds to the drug.

39. The assay of claim 38 wherein the capture reagent is at least one of an antibody or fragment thereof that specifically binds the drug containing a carbohydrate moiety and a target molecule that specifically binds the drug containing a carbohydrate moiety.

40. The assay of claim 38 or 39 wherein the compound that also specifically binds to the drug is labeled and 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 containing a carbohydrate moiety.

41. The assay of claim 38 or 39 wherein the compound that also specifically binds to the drug is detected by binding of one or more sequential intermediate compounds wherein one or more of the sequential intermediate compounds comprises a detectable label.

42. The assay of claim 40 or 41 wherein the detectable label comprises at least one label selected from the group consisting of: an enzyme label; a luminescent label; a protein label; a vitamin label; and a radioisotope label.

43. The assay of any of the preceding claims 36 through 42 wherein the fluid sample comprises at least one of: whole blood; serum; mucous, saliva; colostrum and plasma.

44. The assay of any of the preceding claims 36 through 43 wherein the detecting step comprises an assay selected from the group consisting of: a sandwich assay; a bridging assay; and a competitive binding assay.

45. The assay of claim 44 wherein the detecting step 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; measuring using biolayer interferometry;
measuring using cantilever-detection; measuring using label-free intrinsic imaging;
and measuring using acoustic detection.

46. The assay of claim 45 wherein the detecting step comprises an assay selected from the group consisting of: an ELISA; an ECL; an RIA; a SPRIA;
immunoblotting;
immunoprecipitation; and immunostaining.

47. The assay of claim 46 wherein the detecting step comprises a bridging assay.

48. The assay of claim 47 wherein the detecting step comprises an ELISA.

49. The assay of any of the preceding claims 36 through 48, wherein the additional carbohydrate is present in the sample at a concentration between about 10 ng/ml and about 1mg/mI.

50. The assay of claim 49, wherein the carbohydrate added to the sample is bacterial lipopolysaccharide (LPS), a dextran, a levan, pneumococcal polysaccharide, agarose, cellulose, carrageenan or methylglycoside.

51. The assay of any of claims 36 through 50, wherein the drug containing a carbohydrate moiety further comprises a carrier conjugated to the drug.

52. The assay of claim 51, wherein 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.

53. The assay of claim 51 or 52, wherein the carrier specifically binds to an antigen expressed on the surface of cancer cells.

54. The assay of claim 53, wherein the antigen expressed on 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.

55. The assay of claim 53 or 54, wherein the carrier is a monoclonal antibody.

56. The assay of claim 55, wherein the monoclonal antibody specifically binds to an antigen 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.

57. The assay of any of claims 36 through 56, wherein the drug containing a carbohydrate moiety is selected from the group consisting of: a cytotoxic agent; a radiotherapeutic; an immunomodulatory agent; an anti-angiogenic agent; an anti-proliferative agent; a pro-apoptotic agent; a chemotherapeutic agent; and a therapeutic nucleic acid.

58. The assay of claim 57 wherein the drug containing a carbohydrate moiety is a cytotoxic agent.

59. The assay of claim 58 wherein the cytotoxic agent is an inhibitor of tubulin polymerization; an alkylating agent that binds to and disrupt DNA; an inhibitor of protein synthesis; or a tyrosine kinase inhibitor.

60. The assay of claim 59 wherein the cytotoxic agent is selected from calicheamicins, thiotepa, taxanes, vincristine, daunorubicin, doxorubicin, epirubicin, actinomycin, authramycin, azaserines, bleomycins, tamoxifen, idarubicin, dolastatins/auristatins, hemiasterlins, and maytansinoids.

61. The assay of claim 57, wherein the cytotoxic agent is a calicheamicin.

62. The assay of claim 61 wherein the calicheamicin is N-acetyl gamma dimethyl hydrazide calicheamicin.

63. The assay of claim 62 wherein the N-acetyl gamma dimethyl hydrazide calicheamicin is conjugated to a carrier monoclonal antibody and the carrier-drug conjugate is selected from the group of compositions consisting of: CMC-544;
CME-548; CMD-193; and gemtuzumab ozogamicin.

64. The assay of any of claims 61 through 63 wherein the additional carbohydrate is a methylglycoside.

65. The assay of any of claims 36 through 64 wherein the capture reagent 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.

66. The assay of any of claims 36 through 64 wherein the capture reagent is an anti-calicheamicin antibody.

67. An assay as in any of claims 36 through 66 and as further described in Example 7.

68. The assay of claim 29 or 30 wherein the compound that binds to the antibody that specifically binds to the drug containing a carbohydrate moiety is detected by binding of one or more sequential intermediate compounds wherein, one or more of the sequential intermediate compounds comprises a detectable label.