Haptens, hapten conjugates, compositions thereof and method for their preparation and use

BACKGROUND

Generally, only large molecules, infectious agents, and insoluble foreign ma tier can elicit an immune response in an animal. However, haptens, which are small molecules, can in certain instances be induced to elicit an immune response if they are first coupled to a large carrier (such as a protein) to form an immunogen. Haptens in combination with anti-hapten antibodies that are raised against the immunogens and isolated are usefiil for detecting particular molecular targets. For example, specific binding moieties such as primary antibodies and nucleic acid probes can be labeled with one or more hapten molecules, and once these specific binding moieties are bound to their molecular targets they can be detected using an anti-hapten antibody conjugate that includes a detectable label such as an enzyme or a fluorescent label. Binding of the detectable anti-hapten antibody conjugate to a sample indicates the presence of the target in a sample.

Digoxigenin, present exclusively in Digitalis plants as a secondary metabolite, is an example of a hapten that has been utilized in a variety of molecular assays. U.S. Patent No. 4,469,797, entitled Digoxigenin Immunogens, Antibodies,

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Label Conjugates and Related Derivatives, discloses using immunoassays to determine digoxin concentrations in blood samples based upon the specific binding of antidigoxin antibodies to the drug in the test sample. U.S. Patent No. 5,198,537, entitled Digoxigenin Derivatives and Use Thereof, describes a number of additional digoxigenin derivatives that have been used in immunological tests, such as immunoassays.

Other haptens have been developed for analytical procedures including biotin and fluorescein. However, each of these haptens has specific drawbacks that have made dioxigenin the hapten of choice for sensitive immunoassays. In the case of biotin, certain biological samples include endogenous biotin that can lead to background interference. Similarly, fluorescein, a fluorescent molecule, can lead to background fluorescence in a fluorescent immunoassay. For in situ assays such as immunohistochemical (IHC) assays and in situ hydribization (ISH) assays of tissue and cytological samples, especially multiplexed assays of such samples, it is highly desirable to identify and develop new haptens and anti-hapten antibodies (and conjugates thereof) to provide additional assay flexibility, especially since it is becoming clear that samples can best be characterized through simultaneous detection of multiple targets.

A primary goal of cancer therapy is to selectively kill, or inhibit uncontrolled growth of] malignant cells while not adversely affecting normal cells. Traditional chemotherapeutic drugs are highly cytotoxic, and while preferably having greater affinity for malignant cells than for normal cells, nevertheless typically adversely affect normal cells. New therapeutics are now being developed that target the growth factor and nutrient pathways that regulate cell growth and metabolism in response to intracellular and environmental cues. These signaling pathways often are altered or dysregulated in cancer. For example, certain growth factors (such as EGF, a growth factor that activates protein-receptor tyrosine kinase (“RTK”) activity to initiate a signal transduction cascade resulting in changes in cell growth, proliferation and differentiation) are involved in the pathogenesis and progression of different cancers. Such pathways and associated signaling molecules provide attractive targets for therapeutic intervention, but it is becoming increasingly evident that different populations of patients have tumors that appear to be dysregulated in 2

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PCT/US2007/023140 different manners. For example, a particular therapeutic target (or combination of therapeutic targets) may only be present in tumors from certain populations of patients, and thus identifying such certain populations having the target (or combination of targets) can be used to stratify patients into potential non-responders and potential non-responders to a therapeutic (or combination of therapeutics) directed toward the target (or targets). The use of companion diagnostics to stratify patients in this manner is a first step toward personalizing the treatment of cancer in individual patients. Increased individualization of treatments will certainly involve multiplexed assays for multiple therapeutic targets.

Unfortunately, in recent years there has been little research directed to developing additional classes of haptens against which sensitive and specific antibodies can be raised in order to enable highly multiplexed assays. Such highly multiplexed assays would be useful for monitoring the response of individuals to a given therapeutic regimen and for companion diagnostic applications. Identifying additional classes of haptens and methods for their use in analytical and therapeutic applications would substantially advance the state of the art in this field.

SUMMARY

Thus, based on the above, a need exists in the art for additional haptens, and hapten conjugates, that are useful for diagnostic and/or therapeutic applications. Accordingly, certain disclosed embodiments of the present invention concern new classes of haptens, hapten conjugates and compositions thereof.

Embodiments of a method for performing a multiplexed diagnostic assay, such as for two or more different targets in a sample, are described. One embodiment comprised contacting the sample with two or more specific binding moieties that bind specifically to two or more different targets. The two or more specific binding moieties are conjugated to different haptens, and at least one of the haptens is an oxazole, a pyrazole, a thiazole, a nitroaryl compound, a benzofurazan, a triterpene, a urea, a thiourea, a rotenoid, a coumarin or a cyclolignan. The sample is contacted with two or more different anti-hapten antibodies that can be detected separately. The two or more different anti-hapten antibodies may be conjugated to different detectable labels. In some embodiments, the two or more different anti

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PCT/US2007/023140 hapten antibodies are from different mammalian species. The method may further comprise contacting the two or more different anti-hapten antibodies with two or more anti-antibodies that specifically bind the two or more different anti-hapten antibodies. For such embodiments, the two or more anti-antibodies may be conjugated to different detectable labels.

Certain embodiments of the hapten are azoles having the following general chemical formula r₂

<img file="CA2995467C_D0001.tif" />

where R1-R4 independently are selected from hydrogen, acyl, aldehydes, alkoxy, aliphatic, substituted aliphatic, heteroaliphatic, oxime, oxime ether, alcohols, amido, amino, amino add, aryl, alkyl aryl, carbohydrate, monosaccharides, disaccharides, oligosaccharides, polysaccharides, caibonyl, carboxyl, carboxylate, cyclic, cyano, ester, ether, exomethylene, halogen, heteroaryl, heterocyclic, hydroxyl, hydroxylamine, aliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, and combinations thereof at least one of the R1-R4 substituents being bonded to a linker or is a reactive group suitable for coupling to a linker or a carrier molecule, X independently is nitrogen or carbon, Y is oxygen, sulfur or nitrogen, and if Y is oxygen or sulfur, then there is no Ri group, and if Y is nitrogen, then there is at least one Ri group. One specific example of such a hapten has the following structure.

<img file="CA2995467C_D0002.tif" />

Another class of haptens is the nitroaiyl compounds having the following general chemical formula

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r₆

R₅‘

<img file="CA2995467C_D0003.tif" />

r₂

R3

R4 where at least one of Ri-R« is nitro, and the remaining Ri-Rs ring substituents independently are selected from hydrogen, acyl, aldehydes, alkoxy, aliphatic heteroaliphatic, oxime, oxime ether, alcohols, amido, amino, amino acid, aryl, alkyl aryl, carbohydrate, monosaccharides, disaccharides, oigosaccharides, polysaccharides, carbonyl, carboxyl, carboxylate, clic, heterocyclic, cyano, ester, ether, halogen, heteroaryl, hydroxyl, hydroxlyamine, keto, sulfhydryl, sulfonyl, sulfoxide, exomethylene, or two or more of the Ri-Rc substituents are atoms in a ring system, and at least one of the Ri-Rt substituents is bonded to a linker or is a reactive group suitable for coupling to the linker.

Another class of haptens is the benzofurazans or derivatives thereof, such as compounds having a formula

R4 where the RpRj substituents independently are selected from hydrogen, acyl, aldehydes, alkoxy, aliphatic, substituted aliphatic, heteroaliphatic, oxime, oxime ether, alcohols, amido, amino, amino acid, aryl, alkyl aryl, carbohydrate, monosaccharides, disaccharides, oligosaccharides, polysaccharides, carbonyl, carboxyl, carboxylate, cyclic, cyano, ester, ether, exomethylene, halogen, heteroaryl, heterocyclic, hydroxyl, hydroxytamine, aliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, and combinations thereof, or two or more of the R)-R&lt; substituents are atoms in a ring system bonded or fused to the compounds having the illustrated general formula, at least one of the Ri-R* substituents being bonded to the linker or is the reactive group, and Y is oxygen, sulfur or a carbon atom having Rj and Rs

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PCT/US2007/023I40 substituents, where Rj and Rs are as stated for R1-R4. One specific example of such a hapten has the following structure.

<img file="CA2995467C_D0004.tif" />

Another class of haptens is cyclic terpenes having a formula

<img file="CA2995467C_D0005.tif" />

where Ri-Rji independently are selected from hydrogen, acyl, aldehydes, alkoxy, aliphatic, substituted aliphatic, heteroaliphatic, oxime, oxime ether, alcohols, amido, amino, amino acid, aryl, alkyl aryl, carbohydrate, monosaccharides, disaccharides, oligosaccharides, polysaccharides, carbonyl, carboxyl, carboxylate, cyclic, cyano, ester, ether, exomethylene, halogen, heteroaryl, heterocyclic, hydroxyl, hydroxylamine, aliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, and combinations thereof, at least one of the R1-R21 substituents being bonded to a linker or is a reactive group suitable for coupling to the linker or a carrier molecule, and where two or more of R1-R21 substituents may be atoms in a ring system bonded or fused to the compounds having the illustrated general fonnula, Y is a bond, thereby defining a 5-membered ring, or is a carbon atom bearing R22 and R» substituents, where R22 and R23 are as stated for R1-R21· One specific example of such a hapten has the following structure.

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<img file="CA2995467C_D0006.tif" />

Another class of haptens is ureas and thioureas having a formula

Y

<img file="CA2995467C_D0007.tif" />

R2 R3 where R1-R3 independently are hydrogen, aliphatic, substituted aliphatic, cyclic, heterocyclic, aryl and heteroaryl, and Y is oxygen or sulfur. Particular examples of ureas or thioureas are aryl ureas or thioureas having a formula

<img file="CA2995467C_D0008.tif" />

where R1-R7 independently are independently are selected from hydrogen, acyl, aldehydes, alkoxy, aliphatic, substituted aliphatic, heteroaliphatic, oxime, oxime ether, alcohols, amido, amino, amino acid, aryl, alkyl aryl, carbohydrate, monosaccharides, disaccharides, oligosaccharides, polysaccharides, carbonyl, carboxyl, carboxylate, cyclic, cyano, ester, ether, exomethylene, halogen, heteroaryl, heterocyclic, hydroxyl, hydroxylamine, aliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, and combinations thereof, at least one of the R1-R7 substituents is bonded to a linker or is a reactive group, and where two or more of R1-R7 substituents may be atoms in a ring system bonded or fused to the compounds having the illustrated general formula, and Y is oxygen or sulfur. One specific example of such a hapten has a formula

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<img file="CA2995467C_D0009.tif" />

I I

H H

Another class of haptens is the rotenoids having a formula

<img file="CA2995467C_D0010.tif" />

where Ri-Ri&lt; independently arc hydrogen, acyl, aldehydes, alkoxy, aliphatic, substituted aliphatic, heteroaliphatic, oxime, oxime ether, alcohols, amido, amino, amino acid, aryl, alkyl aryl, carbohydrate, monosaccharides, disaccharides, oligosaccharides, polysaccharides, carbonyl, carboxyl, carboxylate, cyclic, cyano, ester, ether, exomethylene, halogen, heteroaryl, heterocyclic, hydroxyl, hydroxylamine, aliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, and combinations thereof, at least one of the R|-R|« substituents is coupled to a linker or is a reactive group, and where two or more of R|-Ri4 substituents may be atoms in a ring system bonded or fused to the compounds having the illustrated general formula. One specific example of such a hapten has a formula

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<img file="CA2995467C_D0011.tif" />

The rotenone haptens also include rotenone isoxazolines, which typically have a formula

<img file="CA2995467C_D0012.tif" />

With reference to the rotenone isoxazolines, R-R$ independently are hydrogen, aldehyde, alkoxy, aliphatic, particulary lower aliphatic, including all branched chain isomers, such as isoprene, and all stereoisomers, substituted aliphatic, heteroaliphatic, e.g., organic chains having heteroatoms, such as oxygen, nitrogen, sulfur, alkyl, particularly alkyl having 20 or fewer carbon atoms, and even more typically lower alkyl having 10 or fewer atoms, such as methyl, ethyl, propyl, isopropyl, and butyl, substituted alkyl, such as alkyl halide (e.g. -CXj where X is a halide, and combinations thereof, either in the chain or bonded thereto) amino, amino acid, amido, cyano (-CN), halogen, hydroxyl, hydroxylamine, oxime (HON=), oxime ether (e.g., methoxyimine, CHj-O-N=) alkyl hydroxyl, particularly lower alkyl hydroxyl, carbonyl, keto, such as aliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, carboxyl, carboxylate (and salts thereof, such as Group 1 metal or ammonium ion carboxylates) ester, alkyl ester, acyl, exomethylene, ether, cyclic, heterocyclic, aryl, alkyl aryl, such as benzyl, heteroaryl, polysaccharides, carbohydrate, monosaccharides, such as glucose and fructose, disaccharides, such as

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PCT/US2007/023140 sucrose and lactose, oligosaccharides and polysaccharides, and combinations thereof. At least one of the R-Rs substituents also is bonded to a linker or to a carrier molecule. Y is oxygen, nitrogen, or sulfur.

Another class of haptens is oxazoles or thiazoles having a formula

R₂

R_t r₃ where Ri-Rj independently are selected from hydrogen, acyl, aldehydes, alkoxy, aliphatic, substituted aliphatic, heteroaliphatic, oxime, oxime ether, alcohols, amido, amino, amino acid, aryl, alkyl aryl, carbohydrate, monosaccharides, disaccharides, oligosaccharides, polysaccharides, carbonyl, carboxyl, carboxylate, cyclic, cyano, ester, ether, exomethylene, halogen, heteroaryl, heterocyclic, hydroxyl, hydroxylamine, aliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, and combinations thereof, at least one of the R1-R3 substituents is coupled to a linker or is a reactive group, and where Rj-Rj substituents may be atoms in a ring system bonded or fused to the compounds having the illustrated general formula, and Y is oxygen or sulfur. A specific example of such a hapten has the following chemical structure.

<img file="CA2995467C_D0013.tif" />

Another class of haptens is the coumarins or coumarin derivatives having a formula

<img file="CA2995467C_D0014.tif" />

where Ri-R« independently are selected from hydrogen, acyl, aldehydes, alkoxy, aliphatic, substituted aliphatic, heteroaliphatic, oxime, oxime ether, alcohols, amido, amino, amino acid, aryl, alkyl aryl, carbohydrate, monosaccharides, disaccharides,

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PCT/US2007/023140 oligosaccharides, polysaccharides, carbonyl, carboxyl, carboxylate, cyclic cyano ester, ether, exomethylene, halogen, heteroaryl, heterocyclic, hydroxyl, hydroxylamine, aliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide’ and combinations thereof, or two or more of the R.-Re substituents available for fonning 5 such compounds also may be atoms in a ring system bonded or fused to the compounds having the illustrated general fonnula, « least one of the R,-R₆substituents is coupled to a linker or is a reactive group, and y _{js oxygen}. _{njtrogen or}sulfur.

Another class of haptens is the cyclolignans having a fonnula

Ra Rc OR

<img file="CA2995467C_D0015.tif" />

where R,-_R|I independently are selected from hydrogen, acyl, aldehydes alkoxy aliphatic, substituted aliphatic, heteroaliphatic, oxime, oxime ether, alcohols, amido ammo, amino acid, aryl, alkyl ary1, carbohydrate, monosaccharides, disaccharides, ’ oligosaccharides, polysaccharides, carbonyl, carboxyl, carboxylate, cyclic, cyano, «ter, ether, exomethylene, halogen, heteroaryl, heterocyclic, hydroxyl, hydroxylamine, aliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide’ and combinations thereof, or two or more of the R,-R_i2 substituents available for forming such compounds also may be atoms in a ring system bonded or fused to the compounds having the illustrated general formula, at least one of the R_rR_I220 ^su*^>si^^uents is coupled to a linker or is a reactive group. Specific exampies of cyclolignan haptens include

CA 299R4«1 ·>ηιο_Λζ.

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<img file="CA2995467C_D0016.tif" />

<img file="CA2995467C_D0017.tif" />

Another general class of haptens of the present invention is heterobicyclic/biaryl compounds, typically phenyl quinolines and quinoxalines. The heterobicyclic/biaryl compounds typically have a first general chemical formula as below.

<img file="CA2995467C_D0018.tif" />

R i -R.2 substituents independently are selected from: hydrogen, acyl, aldehydes, alkoxy, aliphatic, particulary lower aliphatic, substituted aliphatic, heteroaliphatic, e.g., organic chains having heteroatoms, such as oxygen, nitrogen, sulfur, alkyl,

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PCT/US2007/023140 particularly alkyl having 20 or fewer carbon atoms, and even more typically lower alkyl having 10 or fewer atoms, such as methyl, ethyl, propyl, isopropyl, and butyl, substituted alkyl, such as alkyl halide (e.g. -CX3 where X is a halide, and combinations thereof, cither in the chain or bonded thereto,), oxime, oxime ether (e.g., methoxyimine, CHj-O-N=) alcohols (i.e. aliphatic or alkyl hydroxyl, particularly lower alkyl hydroxyl) amido, amino, amino acid, aryl, alkyl aryl, such as benzyl, alkoxy aryl, such as methoxy and ethoxy, carbohydrate, monosaccharides, such as glucose and fructose, disaccharides, such as sucrose and lactose, oligosaccharides and polysaccharides, carbonyl, carboxyl, carboxylate (including salts thereof, such as Group I metal or ammonium ion carboxylates), cyclic, heterocyclic, cyano (-CN), ester, alkyl ester, ether, halogen, heteroaryl, hydroxyl, hydroxylamine, oxime (HO-N=), keto, such as aliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, exomethylene, and combinations thereof. Two or more of the R1-R2 substituents, most typically plural R| substituents, also may be atoms, typically carbon atoms, in a ring system bonded or fused to the compounds having the illustrated general formula. At least one of the R1-R2 substituents is bonded to a linker or directly to a carrier. Y is oxygen, nitrogen or sulfur, typically nitrogen. If Y is nitrogen, then the formula also can include double bonds to the one or more nitrogen atoms.

Compounds having a single heteroatom are exemplified by phenylquinolines, as follows.

<img file="CA2995467C_D0019.tif" />

Compounds having two heteroatoms are represented by quinoxalines, as indicated by the general formula below.

<img file="CA2995467C_D0020.tif" />

Particular examples include 2-(3,4-dimethoxyphenyl)quinoline-4-carboxylic acid)

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CO₂H

<img file="CA2995467C_D0021.tif" />

OCH₃ och₃ and 3-hydroxy-2-quinoxalinecarbamide.

<img file="CA2995467C_D0022.tif" />

OH

NH₂

Another general class of haptens is azoaryi compounds, such as azobenzenes, having a first general chemical formula as below.

N=N z^R1

R1-R2 substituents independently are selected from: hydrogen, acyl, aldehydes, alkoxy, aliphatic, particular/ lower aliphatic, substituted aliphatic, heteroaliphatic, e.g., organic chains having heteroatoms, such as oxygen, nitrogen, sulfur, alkyl, particularly alkyl having 20 or fewer carbon atoms, and even more typically lower alkyl having 10 or fewer atoms, such as methyl, ethyl, propyl, isopropyl, and butyl, substituted alkyl, such as alkyl halide (e.g. -CX3 where X is a halide, and combinations thereof, either in the chain or bonded thereto,), oxime, oxime ether (e.g., methoxyimine, CHj-O-N=) alcohols (i.e. aliphatic or alkyl hydroxyl, particularly lower alkyl hydroxyl) amido, amino, amino acid, aryl, alkyl aryl, such as benzyl, alkoxy aryl, such as methoxy and ethoxy, carbohydrate, monosaccharides, such as glucose and fructose, disaccharides, such as sucrose and lactose, oligosaccharides and polysaccharides, carbonyl, carboxyl, carboxylate (including salts thereof, such as Group I metal or ammonium ion carboxylates), cyclic,

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PCT7US2007/02314Û heterocyclic, cyano (-CN), ester, alkyl ester, ether, halogen, heteroaryl, hydroxyl, hydroxylamine, oxime (HO-N=), keto, such as aliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, sulfonyl, exomethylene, and combinations thereof. Two ore more R₂ substituents also may be atoms, typically carbon atoms, in a ring system bonded or fused to the compounds having the illustrated general formula. For example, 2 R₂ substituents may form a fused phenyl ring, or a fused heterocyclic or heteroaryl structure. A particular azoaryl haptens, 4-(dimethylamino)azobenzene*4'sulfonyl chloride, has the formula provided below.

<img file="CA2995467C_D0023.tif" />

Another class of haptens is benzodiazepine having a first general formula as indicated below.

<img file="CA2995467C_D0024.tif" />

R1-R5 independently are selected from: acyl, aldehydes, alkoxy, aliphatic, particulary lower aliphatic, substituted aliphatic, heteroaliphatic, e.g., organic chains having heteroatoms, such as oxygen, nitrogen, sulfur, alkyl, particularly alkyl having 20 or fewer carbon atoms, and even more typically lower alkyl having 10 or fewer atoms, such as methyl, ethyl, propyl, isopropyl, and butyl, substituted alkyl, such as alkyl halide (e.g. -CXj where X is a halide, and combinations thereof, either in the chain or bonded thereto,), oxime, oxime ether (e.g., methoxyimine, CHj-O-N=) alcohols (i.e. aliphatic or alkyl hydroxyl, particularly lower alkyl hydroxyl) amido, amino, amino acid, aiyl, alkyl aryl, such as benzyl, carbohydrate, monosaccharides,

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PCT/ÜS2007/023140 such as glucose and fructose, disaccharides, such as sucrose and lactose, oligosaccharides and polysaccharides, carbonyl, carboxyl, carboxylate (including salts thereof, such as Group I metal or ammonium ion carboxylates), cyclic, cyano (CN), ester, ether, exomethylene, halogen, heteroaryl, heterocyclic, hydrogen, hydroxyl, hydroxylamine, oxime (HO-N=), keto, such as aliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, sulfonyl, and combinations thereof. Two or more of the Rj substituents also maybe atoms, typically carbon atoms, in a ring system bonded or fused to the compounds having the illustrated general formula. At least one of the R1-R5 positions is bonded to a linker or is occupied by a functional group suitable for coupling to a linker or a carrier molecule. R1-R5 most typically are aliphatic, aryl, hydrogen, or hydroxyl, even more typically alkyl, hydrogen or phenyl. Y is oxygen or sulfur, most typically oxygen. A particular example of a benzodiazepine hapten, -(2-hydroxyphenyl)-lH-benzo[bJ[l,4]diazepine-2(3H)-one, is provided below.

<img file="CA2995467C_D0025.tif" />

The present disclosure also provides embodiments of a compound having a formula (hapten)_re-(linker)n-(reactive groupé where the hapten is an oxazole, pyrazole, thiazole, nitroaryl, benzofurazan, triterpene, urea, thiourea, rotenoid, coumarin, cycloiignan, or combinations thereof, m is from 1 to about 200, n is 0 to about 200, and 0 is from 1 to 200. In certain embodiments m is 1 to about 100, n is from 0 to about 5, and o is from about 1 to about 5, and in other embodiments m, n and 0 are 1.

The present disclosure also describes hapten-carrier conjugates comprising a hapten coupled to a carrier where the hapten is an oxazole, pyrazole, thiazole, nitroaryl, benzofurazan, triterpene, urea, thiourea, rotenoid, coumarin, cycloiignan, or combinations thereof. Certain embodiments of such conjugates have a formula

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PCT7US2007/023140 (hapten)_m-(linker)_n-(carrier)p where m is from 1 to about 200, n is 0 to about 200 and p is from 1 to about 10. For other embodiments m is from 1 to about 100, n is 1 to 100, and p is from 1 to about 5, and yet other embodiments m, η, o and p are 1. For certain embodiments, the linker is heteroaliphatic, such as alkyl or alkylene oxides, 5 with one particular embodiment comprising an ethylene glycol linker having from 1 to about 15 ethylene glycol units. The carrier may be a specific binding carrier, such as a protein, a nucleic acid, or an antibody. The carrier also may be an immunogenic carrier.

The present disclosure also concerns antibodies that specifically bind to a 10 hapten selected from an oxazole, pyrazole, thiazole, nitroaryl, benzofiirazan, triterpene, urea, thiourea, rotenoid, coumarin, or cyclolignan.

Pharmaceutical compositions also are described. One embodiment of a pharmaceutical composition comprised diagnostically or therapeutically effective amounts of a hapten-carrier conjugate comprising a hapten coupled to a carrier 15 where the hapten is an oxazole, pyrazole, thiazole, nifroaryl, benzofurazan, triterpene, urea, thiourea, rotenoid, coumarin, cyclolignan, or combinations thereof. For such compositions, the hapten-carrier conjugate may have a formula (hapten)_m(linker)_n-(reactive group)₀-(carrier)p where the hapten is an oxazole, pyrazole, thiazole, nitroaryl, benzofurazan, triterpene, urea, thiourea, rotenoid, coumarin, 20 cyclolignan, or combinations thereof.

Multiplexed arrays also are described. For example, disclosed embodiments of a multiplexed assay comprised a hapten selected from oxazoles, pyrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarins, cyclolignans, and combinations thereof.

Kits for use in an enzyme immunoassay are disclosed. Certain kit embodiments comprise a hapten-conjugated antibody or hapten conjugated to a nucleic acid probe, the hapten being selected from oxazoles, pyrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarins, cyclolignans, and combinations thereof. Such kits also typically include an anti30 hapten antibody conjugated to a detectable label.

Embodiments of an immunoassay process are described. For example, the immunoassay process may comprise providing a redive hapten conjugate suitable 17

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PCT/US2007/023140 for performing the immunoassay, the hapten being selected from oxazoles, pyrazoles, thiazoles, nilroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarins, cyclolignans, and combinations thereof. The hapten conjugate is then used in at least one step of the immunoassay. The hapten conjugate may be a hapten-linker conjugate. Alternatively, the hapten conjugate may be a hapten-carrier conjugate, where the carrier might be an immunogenic carrier or a specific binding carrier.

A method for identifying a mammalian tumor is disclosed. One embodiment comprises assaying a sample obtained from the mammalian tumor to detect a pattern 10 of expression, phosphorylation or both expression and phosphorylation, using a hapten conjugate where the hapten is selected from oxazoles, pyrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarins, cyclolignans, and combinations thereof.

A method for assessing a response to drug therapy in an individual also is disclosed. One embodiment of the method comprised obtaining a first tissue or cell sample from the individual before exposing the individual to a drug therapy. A second tissue or cell sample is obtained from the individual after exposing the individual to the drag therapy. A biochemical product and/or process affected by the therapy is detected from the first sample and the second sample, where detecting comprises using a hapten conjugate having a hapten selected from oxazoles, pyrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarins, cyclolignans, and combinations thereof. The results for the first sample are compared to the second sample to determine whether the drug therapy had a positive, negative or null effect.

A method for making a conjugate comprising a hapten also is disclosed. One embodiment of the method comprised providing a hapten selected from oxazoles, pyrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarins, cyclolignans, and combinations thereof The hapten is then coupled to a linker or a carrier.

A method for detecting a molecule of interest in a biological sample also is disclosed. One embodiment of the method comprised contacting the biological sample with a hapten-antibody conjugate comprising an antibody linked to the

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PCT/US2007/023140 hapten using a heterobi functional PEG linker, the hapten being selected from oxazoles, pyrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarins, podophyllotoxin-based compounds, and combinations thereof. A signal generated by the hapten-antibody conjugate is detected after treatment with an anti-hapten antibody having at least one detectable label.

The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing illustrating one embodiment of a multiplexed in situ hybridization process.

FIG. 2 is a schematic drawing illustrating one embodiment of a method for using enzymes as signal generating moieties.

FIG. 3 is a schematic drawing illustrating direct detection of a target using a primary antibody comprising a detectable signal generating label.

FIG. 4 is a schematic diagram illustrating one embodiment of a method for amplifying detection signals.

FIG. 5 is a schematic drawing illustrating one embodiment of haptenquantum dot immunohistochemistry detection according to the present invention.

FIG. 6 is a photomicrograph depicting IHC positive staining of anti-hapten antibody detection using a primary antibody conjugated to a disclosed embodiment of a class of haptens according to the present invention.

FIG. 7 is a photomicrograph depicting IHC negative staining using an antihapten antibody using a primary antibody conjugated to a disclosed embodiment of a class of haptens according to the present invention.

FIG. 8 is a schematic diagram illustrating one embodiment of a disclosed method for multiplexed detection of multiple targets in a sample using plural different haptens and plural different signal generating moieties to generate plural different detectable signals.

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FIG. 9 is a photomicrograph depicting using multiple haptens and antibodies thereto, such as antibiotin and antidinitrophenyl, for detection of sample, such as a protein, in tissue.

FIG. 10 schematically illustrates one embodiment of multiplexed detection of two different classes of targets, namely gene and protein targets.

FIG. 11 is a photomicrograph depicting detection of protein and 2 genes, such as by using an antidinitrophenyl antibody.

FIG. 12 illustrates hybridoma fusion product ELISA results for mouse IgG monoclonal antibodies against one embodiment of a disclosed benzofurazan hapten.

FIG. 13 illustrates hybridoma fusion product ELISA results for mouse IgG monoclonal antibodies against one embodiment of a disclosed benzoforazan hapten.

FIG. 14 illustrates hybridoma fusion product ELISA results for mouse IgG monoclonal antibodies against one embodiment of a disclosed benzoforazan hapten.

FIG. 15 illustrates hybridoma fusion product ELISA results for mouse IgG monoclonal antibodies against one embodiment of a disclosed benzofurazan hapten.

FIG. 16 illustrates hybridoma fusion product ELISA results for mouse IgG monoclonal antibodies against one embodiment of a disclosed benzoforazan hapten.

FIG. 17 is a graph of percent nucleotide versus DNP concentration.

FIG. 18 is a graph of salmon DNA concentration on percent nucleotide labeled.

FIG. 19 is a chromogenic IHC staining of CD20-biotin-labeled primary antibodies with anti-biotin HRP conjugates on normal tonsil tissue.

FIG. 20 is a chromogenic IHC staining of a CD45 thiazole sulfonamidebased hapten-labeled primary antibodies with anti-thiazole sulfonamide HRP conjugates on normal tonsil tissue.

FIG. 21 is a chromogenic IHC staining of a Ki-67 benzoforazan-based hapten-labeled primary antibodies with anti- benzofurazan HRP conjugates on normal tonsil tissue.

FIG. 22 is a chromogenic IHC staining of a CD34 nitropyrazole-based hapten-labeled primary antibodies with anti- nitropyrazole HRP conjugates on normal tonsil tissue.

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FIG. 23 is a chromogenic IHC staining of a Kappa dinitrophenyl-based hapten-labeled primary antibodies with anti- dinitrophenyl HRP conjugates on normal tonsil tissue.

FIG. 24 is a chromogenic IHC staining of Lambda rotenone-based haptenS labeled primary antibodies with anti- rotenone HRP conjugates on normal tonsil tissue.

FIG. 25 is a fluorescent IHC staining in normal tonsil using anti-lambda attached to biotin and detected with anti-biotin antibody QDot conjugate.

FIG. 26 is a fluorescent IHC staining in normal tonsil using antil-lambda attached to thiazole sulfonamide-based hapten and detected with anti- thiazole sulfonamide antibody QDot conjugate.

FIG. 27 is a fluorescent IHC staining in normal tonsil using antil-lambda attached to benzofurazan-based hapten and detected with anti- benzofurazan antibody QDot conjugate.

FIG. 28 is a fluorescent IHC staining in normal tonsil using antil-lambda attached to dinitrophenyl-based hapten and detected with anti- dinitrophenyl antiaabody QDot conjugate.

FIG. 29 is a fluorescent IHC staining in normal tonsil using antil-lambda attached to nitropyrazole-based hapten and detected with anti- nitropyrazole 20 antibody QDot conjugate.

FIG. 30 is a fluorescent IHC staining in normal tonsil using antil-lambda attached to rotenone-based hapten and detected with anti- rotenone antibody QDot conjugate.

FIG. 31 is a fluorescent IHC staining of biotin labeled CD20 primary antibody and anti-biotin QDot 525 conjugate on normal tonsil tissue.

FIG. 32 is a fluorescent IHC staining of thiazole sulfonamide-based hapten labeled CD45 primary antibody and anti-thiazole sulfonamide antibody QDot 565 conjugate on normal tonsil tissue.

FIG. 33 is a fluorescent IHC staining of benzofurazan-based hapten labeled 30 K.i67 primary antibody and anti-benzofiirazan antibody QDot 585 conjugate on normal tonsil tissue.

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FIG. 34 is a fluorescent IHC staining of dinitrophenyl-based hapten labeled Kappa primary antibody and anti-dinitrophenyl antibody QDot 605 conjugate on normal tonsil tissue.

FIG. 35 is a fluorescent IHC staining of nitropyrazole-based hapten labeled CD34 primary antibodies anti-nitropyrazole antibody QDot 655 conjugate on normal tonsil tissue.

FIG. 36 is a fluorescent IHC staining of rotenone-based hapten labeled Lambda primary antibodies and anti-rotenone antibody QDot 705 conjugate on normal tonsil tissue.

FIG. 37 is multiplexed staining composite using a mixture of the primary antibody-hapten conjugates and sequentially visualized with a mixture of anti-hapten antibody QDot conjugates on normal tonsil tissue as stated in FIGS. 31-36 and in Example 34.

FIG. 38 is an image extracted from a multiplexed staining composite that represents anti CD45-thiazole sulfonamide-conjugate and hapten primary antibody and anti-thiazole sulfonamide antibody QDot 565 conjugate on normal tonsil tissue.

FIG. 39 is an image extracted from a multiplexed staining composite from FIG. 37 that represents the staining from the Ki67 benzofurazan-conjugate and hapten primary antibody and anti-benzofitrazan-based antibody QDot 585 conjugate on normal tonsil tissue.

FIG. 40 is an image extracted from a multiplexed staining composite from FIG. 37 that represents anti Kappa-dinitrophenyl-conjugate and hapten primary antibody and anti-dinitrophenyl-based hapten anatibody QDot 605 conjugates on normal tonsil tissue.

FIG. 41 is an image extracted from a multiplexed staining composite from FIG. 37 with anti CD34 nitropyrazole-conjugate and hapten primary antibody and anti-nitropyrazole-bascd hapten antibody QDot 655 conjugate on normal tonsil tissue.

FIG. 42 is an image extracted from a multiplexed staining composite from FIG. 37 with anti CD20 biotin primary antibody QDot 525 conjugate on normal tonsil tissue.

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FIG. 43 is an image extracted from a multiplexed staining composite from FIG. 37 with Lambda-rotenone-conjugate and hapten primary antibody and antirotenone-based hapten antibody QDot 705 conjugate on normal tonsil tissue.

FIG. 44 is a graph of wavelength versus relative fluorescence that represents the wavelengths used to extract individual QDot signals from the multiplexed staining composite of FIG. 37 and shows the signal is above the nominal autofluorescence of the tonsil tissue.

DETAILED DESCRIPTION

I. Terms and Introduction

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genas Vll, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, 15 published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John &amp; Sons, Inc., 1995 (ISBN 0471186341); and other similar references.

As used herein, the singular terms “a,” “an,” and “the” indude plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Also, as used herein, the term “comprises means “includes.” Hence “comprising A or B” means induding A, B, or A and B. It is further to be understood that all nucleotide sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic adds or polypeptides or other compounds are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disdosurc, suitable methods and materials are described below.

In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

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In order to facilitate review of the various examples of this disclosure, the following explanations of specific terms are provided:

Adjuvant: A substance that non-specifically enhances the immune response to an antigen. Development of vaccine adjuvants for use in humans is reviewed in

Singh et al. (Nat. Biotechnol. )7:1075-1081, 1999), which discloses that, at the time of its publication, aluminum salts, such as aluminum hydroxide (Amphogel, Wyeth Laboratories, Madison, NJ), and the MF59 microemulsion are the only vaccine adjuvants approved for human use. An aluminum hydrogel (available from Brentg Biosector, Copenhagen, Denmark is another common adjuvant).

In one embodiment, an adjuvant includes a DNA motif that stimulates immune activation, for example the innate immune response or the adaptive immune response by T-cells, B-cells, monocytes, dendritic cells, and natural killer cells. Specific, non-limiting examples of a DNA motif that stimulates immune activation include CpG oligodeoxynucleotides, as described in U.S. Patent Nos. 6,194,388;

6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199. Amplification: Certain embodiments of the present invention allow a single target to be detected using plural visualization complexes, where the complexes can be the same or different, to facilitate identification and/or quantification of a particular target.

Analog, Derivative or Mimetic: An analog is a molecule that differs in chemical structure from a parent compound, for example a homolog (differing by an increment in the chemical structure, such as a difference in the length of an alkyl chain), a molecular fragment, a structure that differs by one or more functional groups, a change in ionization. Structural analogs are often found using quantitative structure activity relationships (QSAR), with techniques such as those disclosed in Remington (The Science and Practice of Pharmacology, 19th Edition (1995), chapter 28). A derivative is a biologically active molecule derived from the base structure. A mimetic is a molecule that mimics the activity of another molecule, such as a biologically active molecule. Biologically active molecules can include chemical structures that mimic the biological activities of a compound.

Animal: Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human 24

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PCT/US2007/023M0 and non-human mammals. Similarly, the term “subject” includes both human and veterinary subjects, for example, humans, non-human primates, dogs, cats, horses, and cows.

Antibody: “Antibody” collectively refers to immunoglobulins or immunoglobulin-like molecules (including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, for example, in mammals such as humans, goats, rabbits and mice) and antibody fragments that specifically bind to a molecule of interest (or a group of highly similar molecules of interest) to the substantial exclusion of binding to other molecules (for example, antibodies and antibody fragments that have a binding constant for the molecule of interest that is at least 10⁵ M'* greater, at least 10⁴ M * greater or at least 10⁵ M’¹ greater than a binding constant for other molecules in a biological sample.

More particularly, “antibody” référés to a polypeptide ligand comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen. Antibodies are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (V_H) region and die variable light (VJ region. Together, the Vh region and the Vl region are responsible for binding the antigen recognized by the antibody.

This includes intact immunoglobulins and the variants and portions of them well known in the art. Antibody fragments include proteolytic antibody fragments [such as F(ab’)2 fragments, Fab’ fragments, Fab’-SH fragments and Fab fragments as are known in the art}, recombinant antibody fragments (such as sFv fragments, dsFv fragments, bispecific sFv fragments, bispecific dsFv fragments, F(ab)'₂fragments, single chain Fv proteins (“scFv”), disulfide stabilized Fv proteins (dsFv”), diabodies, and triabodies (as are known in the art), and camelid antibodies (see, for example, U.S. Patent Nos. 6,015,695; 6,005,079- 5,874,541; 5,840,526; 5,800,988; and 5,759,808). A scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains. The term also includes genetically engineered forms such as chimeric antibodies (for

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PCTAJS2007/023140 example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3^,li Ed., W.H. Freeman &amp; Co., New York, 1997.

Typically, a naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. There arc two types of light chain, lambda (λ) and kappa (k). There are five main heavy drain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region and a variable region, (the regions are also known as ‘'domains”). In combination, the heavy and the light chain variable regions specifically bind the antigen. Light and heavy drain variable regions contain a framework“ region interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs”. The extent of the framework region and CDRs have been defined (see, Kabat et al., Sequences of

Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991 ). The Kabat database is now maintained online. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of 20 an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.

The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified 25 by the chain in which the particular CDR is located. Thus, a Vh CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, whereas a Vl CDRI is the CDR1 from the variable domain of the light chain of the antibody in which it is found. An antibody that binds RET will have a specific V_H region and the Vl region sequence, and thus specific CDR sequences. Antibodies with different 30 specificities (i.e. different combining sites for different antigens) have different CDRs. Although it is the CDRs that vary from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen

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PCT/US2007/023140 binding. These positions within the CDRs are called specificity determining residues (SDRs).

Antigen: A compound, composition, or substance that may be specifically bound by the products of specific humoral or cellular immunity, such as an antibody molecule or T-cell receptor. Antigens can be any type of molecule including, for example, haptens, simple intermediary metabolites, sugars (e.g., oligosaccharides), lipids, and hormones as well as macromolecules such as complex carbohydrates (e.g., polysaccharides), phospholipids, nucleic acids and proteins. Common categories of antigens include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoa and other parasitic antigens, tumor antigens, antigens involved in autoimmune disease, allergy and graft rejection, toxins, and other miscellaneous antigens. In one example, an antigen is a Bacillus antigen, such as yPGA.

Avidin: Any type of protein that specifically binds biotin to the substantial exclusion of other small molecules that might be present in a biological sample. Examples of avidin include avidins that are naturally present in egg white, oilseed protein (e.g., soybean meal), and grain (e.g., com/maize) and streptavidin, which is a protein of bacterial origin.

Binding affinity: The tendency of one molecule to bind (typically noncovalently) with another molecule, such as the tendency of a member of a specific binding pair for another member of a specific binding pair. A binding affinity can be measured as a binding constant, which binding affinity for a specific binding pair (such as an antibody/antigen pair or nucleic acid probe/nuclcic acid sequence pair) can be at least 1 x 10^s M’¹, such as at least 1 x 10⁶ M'¹, at least 1 x 10⁷ M’’ or at least 1 x 108 M*¹. In one embodiment, binding affinity is calculated by a modification of the Scatchard method described by Frankel et al., Mol. Immunol., 16:101-106,1979. In another embodiment, binding affinity is measured by an antigen/antibody dissociation rate. In yet another embodiment, a high binding affinity is measured by a competition radioimmunoassay. In several examples, a high binding affinity for an antibody/antigen pair is at least about 1x10* M'¹. In other embodiments, a high binding affinity is at least about 1.5 x 10⁸ M’¹, at least about 2.0 x 10⁸ M'¹, at least

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PCT/ÜS2007/023140 about 2.S x 10* M¹, at least about 3.0 x 10⁸ M’¹, at least about 3.5 x 10⁸ M'¹, at least about 4.0 x IO⁸ M¹, at least about 4.5 x 10⁸ M¹, or at least about 5.0 x 10⁸ M’¹.

Carrier: A molecule to which a hapten or an antigen can be bound. Carrier molecules include immunogenic carriers and specific-binding carriers. When bound 5 to an immunogenic carrier, the bound molecule may become immunogenic. Immunogenic carriers may be chosen to increase the immunogenicity of the bound molecule and/or to elicit antibodies against the carrier, which are diagnostically, analytically, and/or therapeutically beneficial. Covalent linking of a molecule to a carrier can confer enhanced immunogenicity and T-cell dependence (Pozsgay et al., 10 PNAS 96:5194-97, 1999; Lee el al., J. Immunol. 116:1711-18,1976; Dintziset al., PM4S 73:3671-75,1976). Usefill carriers include polymeric carriers, which can be natural (for example, proteins from bacteria or viruses), semi-synthetic or synthetic materials containing one or more functional groups to which 8 reactant moiety can be attached. Specific binding carriers can by any type of specific binding moiety, 15 including an antibody, a nucleic acid, an avidin, a protein-nucleic add.

Examples of suitable immunogenic carriers are those that can increase the immunogenicity of a hapten and/or help elicit antibodies against the hapten which are diagnostically, analytically, and/or therapeutically beneficial. Useful carriers include polymeric carriers, which can be natural (such as proteins like ovalbumin or 20 keyhole limpet hemocyanin) or derived from a natural polymer isolated from any organism (including viruses), semi-synthetic or synthetic materials containing one or more functional groups, for example primary and/or secondary amino groups, azido groups, hydroxyl groups, or carboxyl groups, to which a reactant moiety can be attached. The carrier can be water soluble or insoluble, and in some embodiments is 25 a protein or polypeptide. Carriers that fulfill these criteria are generally known in the art (see, for example, Fattom et al., Infect. Immun. 58:2309-12,1990; Devi et al., PNAS 88:7175-79,1991; Szu et al., Infect. Immun. 59:4555-61,1991; SzaetaI.,J. Exp. Med. 166:1510-24,1987; and Pavltakova et al., Infect. Immun. 68:2161-66, 2000).

'ihe immunogenic carrier can be a polypeptide, such as a polypeptide of a rotavirus, or of a virus other than a rotavirus. A non limiting, and far from exhaustive list of such other viruses includes Adeno-associated virus, Adenovirus,

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Avian infectious bronchitis virus, Baculovirus, Chicken pox, Corona virus, Cytomegalovirus, Distemper, Enterovirus, Epstein Barr virus, Feline leukemia virus, Flavivirus, Foot and mouth disease virus, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis E, Herpes species, Herpes simplex, Influenza virus, HIV-1, HIV-2, HTLV I, Influenza A and B, Kunjin virus, Lassa fever virus, LCMV (lymphocytic choriomeningitis virus), lentivirus, Measles, Mengo virus, Morbillivirus, Myxovirus, Papilloma virus, Parovirus, Parainfluenza virus, Paramyxovirus, Parvovirus, Poko virus, Polio virus, Polyoma tumour virus, pseudorabics, Rabies virus, Reovirus, Respiratory syncytial virus, retrovirus, rhinovirus, Rinderpest, Rotavirus, Semliki forest virus, Sendai virus, Simian Virus 40, Sindbis virus, SV5, Tick bome encephalitis virus, Togavirus (rubella, yellow fever, dengue fever), Vaccinia virus, Venezuelan equine encephalomyelitis, Vesicular stomatis virus, metapneumovirus, norovirus, SARS virus, smallpox virus, picomaviruses, varicella zoster, and West Nile virus.

Alternatively, tire immunogenic carrier polypeptide can be that of a bacteria or other pathogenic organism. Exemplary bacterial polypeptides include those of Achromobacter xylosoxidans, Acinetobacter calcoaceticus, preferably A. anitratus, A. haemolyticus, A. alcaligenes, and A. Iwoffii, Actinomyces israelii. Aeromonas hydrophilia. Alcaligenes species, preferably A.faecalis, A. odorans and A. deniirificans. Arizona hinshawii. Bacillus anthracis, Bacillus cereus, Bacteroides fragilis. Bacteroides melaninogenicus, Bordetella pertussis, Borrelia burgdorferi, Borrelia recurrentis. Brucella species, preferably B. abortus. B, suis, B. melitensis and B. cants, Calymmatobacterium granulomatis, Campylobacter coli (e.g., the CjaA polypeptide), Campylobacter fetus ssp. intestinalis, Campylobacter fetus ssp. jejuni. Chlamydia species, preferably C. psittaci and C. trachomatis. Chromobacterium violaceum, Citrobacter species, preferably C. freundii and C. di versus. Clostridium botulinum, Clostridium perfringens, Clostridium difficile, Clostridium tetani, Corynebacterium diphtherias. Corynebacterium. preferably C. ulcerans, C. haemolyticum and C. pseudotuberculosis. Coxiella burnetii, Edwardsiella tarda, Eikenella corrodens, Enterobacter. preferably E. cloacae. E. aerogenes. E. hafniae (also named Hajhia alvei) and E. agglomérons, Erysipelothrix rhusiopathiae, Escherichia coli. Flavobacterium meningosepticum, Francisella

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PCT/US2007/023140 tularensis, Fusobacterium nucleatum. Gardnerella vaginalis, Haemophilus ducreyi. Haemophilus influenzae, Helicobacter species (e.g., the UreB polypeptide of H. pylori), Klebsiella species, preferably K_ pneumoniae, K. ozaenae og K. rhinoscleromatis, Legionella species, Leptospira interrogans. Listeria monocytogenes, Moraxella species, preferably M. lacunata and M osloensis, Mycobacterium bovis. Mycobacterium leprae, Mycobacterium tuberculosis (e.g., the CFP10 polypeptide), Mycoplasma species, preferably M. pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia species, preferably N. asteroides and N. brasiliensis. Pasteurella haemolytica, Pasteurella muhocida, Peptococcus magnus, Plesiomonas shigelloides. Pneumococci, Proteus species, preferably P. mirabilis, P. vulgaris, P. rettgeri and P. morganii (also named Providencia rettgeri and Morganella morganii respectively), Providencia species, preferably P. alcalifaciens, P. stuartii and P. rettgeri (also named Proteus rettgeri), Pseudomonas aeruginosa, Pseudomonas mallei. Pseudomonas pseudomallei, Rickettsia,

Rochalimaia henselae. Salmonella species, preferably S. entendis. S. typhi and S. derby, and most preferably Salmonella species of the type Salmonella DTI 04, Serratia species, preferably S. marcescens, Shigella dysenteriae, S. flexneri, S. boydii and S. sonnei. Spirillum minor. Staphylococcus aureus, Staphylococcus epidermidis. Staphylococcus saprophyticus, Streptobacillus moniliformis,

Streptococcus, preferably S. faecalis, S. faecium and S. durons, Streptococcus agalactiae. Streptococcus pneumoniae. Streptococcus pyogenes (e.g., the Sfbl polypeptide), Treponema carateum. Treponema pallidum, Treponema pertenue, preferably T. pallidum. Ureaplasma urealylicum, Vibrio cholerae. Vibrio parahaemolyticus. Yersinia enterocolitica, and Yersinia pestis.

Parasitic immunogenic carriers may for example be isolated and/or derived from Malaria (Plasmodium falciparum. P. vivax, P. malariae), Schistosomes, Trypanosomes. Leishmania. Filarial nematodes, Trichomoniasis. Sarcosporidiosis, Taenia (T. saginata, T. solium), Leishmania, Toxoplasma gondii, Trichinelosis (Trichinella spiralis) or Coccidiosis (Eimeria species).

Illustrative fungal immunogenic carriers can be isolated and/or derived from a fungus selected from Cryptococcus neoformans, Candida albicans, Aspergillus fumigatus and Coccidioidomycosis.

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Specific, non-limiting examples of water soluble polypeptide immunogenic carriers include, but are not limited to, natural, semi-synthetic or synthetic polypeptides or proteins from bacteria or viruses. In one embodiment, bacterial products for use as carriers include bacterial wall proteins and other products (for example, streptococcal or staphylococcal cell walls), and soluble antigens of bacteria. In another embodiment, bacterial products for use as carriers include bacterial toxins. Bacterial toxins include bacterial products that mediate toxic effects, inflammatory responses, stress, shock, chronic sequelae, or mortality in a susceptible host.

Specific, non-limiting examples of water insoluble polymericcarriers include, but are not limited to, aminoalkyi agarose (for example, aminopropyi or aminohexyl SEPHAROSE; Pharmacia Inc., Piscataway, N.J.), aminopropyl glass, cross-linked dextran, and the like, to which a reactive moiety can be attached. Other carriers can be used, provided that a functional group is available for covalently attaching a reactive group.

Chimeric antibody: An antibody that has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species, such as a murine antibody that specifically binds RET.

Conjugating, joining, bonding or linking; Covalently linking one molecule to another molecule to make a larger molecule. For example, making two polypeptides into one contiguous polypeptide molecule, or to covalently attaching a hapten or other molecule to a polypeptide, such as an scFv antibody. In the specific context, the terms include reference to joining a ligand, such as an antibody moiety, to an effector molecule (“EM”). The linkage can be either by chemical or recombinant means. “Chemical means” refers to a reaction between the antibody moiety and the effector molecule such that there is a covalent bond formed between the two molecules to form one molecule.

Coupled: When applied to a first atom or molecule being coupled to a second atom or molecule can be both directly coupled and indirectly coupled. A secondary antibody provides an example of indirect coupling. One specific example of indirect coupling is a rabbit anti-hapten primary antibody that is bound by a

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PCT7US2007/023140 mouse anti-rabbit IgG antibody, that is in turn bound by a goat anti-mouse IgG antibody that is covalently linked to a detectable label.

Epitope: An antigenic determinant. These are particular chemical groups or contiguous or non-contiguous peptide sequences on a molecule that are antigenic, that is, that elicit a specific immune response. An antibody binds a particular antigenic epitope.

Hapten: A molecule, typically a small molecule that can combine specifically with an antibody, but typically is substantially incapable of being immunogenic except in combination with a carrier molecule.

Homopoiymer: This term refers to a polymer formed by the bonding together of multiple units of a single type of molecular species, such as a single monomer (for example, an amino acid).

Humanized antibody: An antibody comprising a humanized light chain and a humanized heavy chain immunoglobulin. A humanized antibody binds to the same antigen as the donor antibody that provides the CDRs. The acceptor framework of a humanized immunoglobulin or antibody may have a limited number of substitutions by amino acids taken from the donor framework. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. Humanized immunoglobulins can be constructed by means of genetic engineering (see for example, U.S. Patent No. 5,585,089).

Humanized immunoglobulin: an immunoglobulin including a human framework region and one or more CDRs from a non-human (for example a mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is termed a “donor,” and the human immunoglobulin providing the framework is termed an “acceptor.” In one embodiment, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they must be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to ««responding parts of natural human immunoglobulin sequences.

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Immune Response: A response of a cell of the immune system, such as a B-cell, T-cell, macrophage or polymorphonucleocyte, to a stimulus. An immune response can include any cell of the body involved in a host defense response for example, an epithelial cell that secretes interferon or a cytokine. An immune response includes, but is not limited to, an innate immune response or inflammation.

Immunogenic Conjugate or Composition: A term used herein to mean a composition useful for stimulating or eliciting a specific immune response (or immunogenic response) in a vertebrate. In some embodiments, the immunogenic response is protective or provides protective immunity, in that it enables the 10 vertebrate animal to better resist infection or disease progression from the organism against which the immunogenic composition is directed. One specific example of a type of immunogenic composition is a vaccine.

Immunogen: A compound, composition, or substance which is capable, under appropriate conditions, of stimulating the production of antibodies or a T-cell 15 response in an animal, including compositions that are injected or absorbed into an animal.

Immunologically Effective Dose: An immunologically effective dose of the disclosed conjugates of the disclosure is therapeutically effective and will prevent, treat, lessen, or attenuate the severity, extent or duration of a disease or 20 condition.

Immunologically reactive conditions: Includes reference to conditions which allow an antibody raised against a particular epitope to bind to that epitope to a detectably greater degree than, and/or to the substantial exclusion of, binding to substantially all other epitopes. Immunologically reactive conditions are dependent 25 upon the format of the antibody binding reaction and typically are those utilized in immunoassay protocols or those conditions encountered in vivo. See Harlow &amp; Lane, supra, for a description of immunoassay formats and conditions. The immunologically reactive conditions employed in the methods are physiological conditions which include reference to conditions (such as temperature, osmolarity, 30 pH) that are typical inside a living mammal or a mammalian cell. While it is recognized that some organs are subject to extreme conditions, the intra-organismal and intracellular environment normally lies around pH 7 (i.e., from pH 6.0 to pH

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8.0, more typically pH 6.5 to 7.5), contains water as the predominant solvent, and exists at a temperature above 0°C and below 50°C. Osmolarity is within the range that is supportive of cell viability and proliferation.

Inhibiting or Treating a Disease: Inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease such as anthrax. “Treatment” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. As used herein, the term “ameliorating,” with reference to a disease, pathological condition or symptom, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the number of relapses of the disease, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular disease.

Isolated: An “isolated” microorganism (such as a virus, bacterium, fungus, or protozoan) has been substantially separated or purified away from microorganisms of different types, strains, or species. Microorganisms can be isolated by a variety of techniques, including serial dilution and culturing.

An “isolated” biological component (such as a nucleic acid molecule, protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism in which the component naturally occurs, such as other chromosomal and extra-chromosomal DNA and RNA, proteins, and organelles. Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids or proteins, or fragments thereof.

Detectable Label: A detectable compound or composition that is attached directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule. Specific, non-limiting examples of labels include fluorescent tags, enzymes, and radioactive isotopes.

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Linker peptide: A peptide within an antibody binding fragment (such as an Fv fragment) which serves to indirectly bond the variable heavy chain to the variable light chain. “Linker” can also refer to a peptide serving to link a targeting moiety, such as a scFv, to an effector molecule, such as a cytotoxin or a detectable label.

Mammal: This term includes both human and non-human mammals.

Similarly, the term “subject” includes both human and veterinary subjects.

Molecule of interest or Target: A molecule for which the presence, location and/or concentration is to be determined. Examples of molecules of interest include proteins and nucleic acid sequences tagged with haptens.

Monoclonal antibody: An antibody produced by a single clone of

B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

Multiplex, -ed, -ing: Embodiments of the present invention allow multiple targets in a sample to be detected substantially simultaneously, or sequentially, as desired, using plural different conjugates. Multiplexing can include identifying and/or quantifying nucleic acids generally, DNA, RNA, peptides, proteins, both 20 individually and in any and all combinations. Multiplexing also can include detecting two or more of a gene, a messenger and a protein in a cell in its anatomic context.

Nanoparticle: A nanoscale particle with a size that i$ measured in nanometers, for example, a nanoscopic particle that has at least one dimension of 25 less than about 100 nm. Examples of nanoparticles include paramagnetic nanoparticles, superparamagnetic nanoparticles, metal nanoparticles, fullerene-like materials, inorganic nanotubes, dendrimers (such as with covalently attached metal chelates), nanofibers, nanohoms, nano-onions, nanorods, nanoropes and quantum dots. A nanoparticle can produce a detectable signal, for example, through 30 absorption and/or emission of photons (including radio frequency and visible photons) and plasmon resonance.

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Neoplasia and Tumor: The process of abnormal and uncontrolled growth of cells. Neoplasia is one example of a proliferative disorder.

The product of neoplasia is a neoplasm (a tumor), which is an abnormal growth of tissue that results from excessive cell division. A tumor that does not 5 metastasize is referred to as “benign.” A tumor that invades the surrounding tissue and/or can metastasize is referred to as “malignant.” Examples of hematological tumors include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), 10 chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous ceil carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cardnoma, medullary thyroid cardnoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary cardnoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical 25 cancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma).

Pharmaceutically Acceptable Carriers: The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical 36

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PCT/US2007/023I40 delivery of one or more therapeutic compounds or molecules, such as one or more SARS-CoV nucleic acid molecules, proteins or antibodies that bind these proteins, and additional pharmaceutical agents. The term “pharmaceutically acceptable carrier” should be distinguished from “carrier” as described above in connection with a hapten/carrier conjugate or an antigen/carrier conjugate.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pili, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

Polypeptide: A polymer in which the monomers are amino acid residues which are joined together through amide bonds. When the amino acids are alphaamino acids, either the L-optical isomer or the D-optical isomer can be used. The terms “polypeptide” or “protein” as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. The term “polypeptide” is specifically intended to cover naturally occurring proteins, as well as those which are recombinantiy or synthetically produced.

The term “residue” or “amino acid residue” includes reference to an amino acid that is incorporated into a protein, polypeptide, or peptide.

Protein: A molecule, particularly a polypeptide, comprised of amino acids.

Purified: The tom “purified” does not require absolute purity, rather, it is intended as a relative term. Thus, for example, a purified peptide, protein, conjugate, or other active compound is one that is isolated in whole or in part from proteins or other contaminants. Generally, substantially purified peptides, proteins, conjugates, or other active compounds for use within the disclosure comprise more than 80% of all macromolecular species present in a preparation prior to admixture 37

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PCT/IIS2007/023I40 or formulation of the peptide, protein, conjugate or other active compound with a pharmaceutical carrier, excipient, buffer, absorption enhancing agent, stabilizer, preservative, adjuvant or other co-ingredient in a complete pharmaceutical Formulation for therapeutic administration. More typically, the peptide, protein, conjugate or other active compound is purified to represent greater than 90%, often greater than 95% of all macromolecular species present in a purified preparation prior to admixture with other formulation ingredients. In other cases, the purified preparation may be essentially homogeneous, wherein other macromolecular species are not detectable by conventional techniques.

Quantum dot: A nanoscale particle that exhibits size-dependent electronic and optical properties due to quantum confinement. Quantum dots have, for example, been constructed of semiconductor materials (e.g., cadmium selenide and lead sulfide) and from crystallites (grown via molecular beam epitaxy), etc. A variety of quantum dots having various surface chemistries and fluorescence characteristics are commercially available from Invitrogen Corporation, Eugene, OR (see, for example, U.S. Patent Nos. 6,815,064,6,682596 and 6,649,138.

Quantum dots are also commercially available from Evident Technologies fTroy, NY). Other quantum dots include alloy quantum dots such as ZnSSe, ZnSeTe, ZnSTe, CdSSe, CdSeTe, 20 ScSTe, HgSSe, HgSeTe, HgSTe, ZnCdS, ZnCdSe, ZnCdTe, ZnHgS, ZnHgSe, ZnHgTe, CdHgS, CdHgSe, CdHgTe, ZnCdSSe, ZnHgSSe, ZnCdSeTe, ZnHgSeTe, CdHgSSe, CdHgSeTe, InGaAs, GaAlAs, and InGaN quantum dots (Alloy quantum dots and methods for making the same are disclosed, for example, in US Application Publication No. 2005/0012182 and PCT Publication WO 2005/001889).

. 25 Reactive Groups: Formulas throughout this application refer to reactive groups,” whcich can be any of a variety of groups suitable for coupling a first unit to a second unit as described herein. For example, the reactive group might be an amine-reactive group, such as an isothiocyanate, an isocyanate, an acyl azide, an NHS ester, an acid chloride, such as sulfonyl chloride, aldehydes and glyoxals, epoxides and oxiranes, carbonates, arylating agents, imidoesters, carbodiimides, anhydrides, and combinations thereof. Suitable thiol-reactive functional groups include haloacetyl and alkyl halides, maleimides, aziridines, acryloyl derivatives, 38

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PCT/US2007/023140 arylating agents, thiol-disulfide exchange reagents, such as pyridyl disulfides, TNBthiol, and disulfide reductants, and combinations thereof. Suitable carboxylatereactive functional groups include diazoalkanes, diazoacetyl compounds, carbonyldiimidazole compounds, and carbodiimides. Suitable hydroxyl-reactive functional groups include epoxides and oxiranes, carbonyldiimidazole, N.N'disuccinimidyl carbonates or N-hydroxysuccinimidyl chloroformâtes, periodate oxidizing compounds, enzymatic oxidation, alkyl halogens, and isocyanates. Aldehyde and ketone-reactive functional groups include hydrazines, Schiff bases, reductive amination products, Mannich condensation products, and combinations thereof. Active hydrogen-reactive compounds include diazonium derivatives, mannich condensation products, iodination reaction products, and combinations thereof. Photoreactive chemical functional groups include aryl azides, halogenated aryl azides, benzophonones, diazo compounds, diazirine derivatives, and combinations thereof.

] 5 Sample: A biological specimen containing genomic DNA, RNA (including mRNA), protein, or combinations thereof, obtained from a subject. Examples include, but are not limited to, peripheral blood, urine, saliva, tissue biopsy, surgical specimen, amniocentesis samples and autopsy material. In one example, a sample includes a biopsy of an adenocarcinoma, a sample of noncancerous tissue, a sample 20 of normal tissue (from a subject not afflicted with a known disease of disorder).

Specific binding moiety: A member of a specific-binding pair. Specific binding pairs are pairs of molecules that are characterized in that they bind each other to the substantial exclusion of binding to other molecules (for example, specific binding pairs can have a binding constant that is at least 10³ M’¹ greater, 10⁴25 M‘¹ greater or 10^s M'¹ greater than a binding constant for either of the two members of the binding pair with other molecules in a biological sample). Particular examples of specific binding moieties include specific binding proteins (for example, antibodies, lectins, avidins such as streptavidins, and protein A), nucleic acids sequences, and protein-nucleic acids. Specific binding moieties can also 30 include the molecules (or portions thereof) that are specifically bound by such specific binding proteins.

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Therapeutically Effective Amount: A quantity of a specified agent sufficient to achieve a desired effect in a subject being treated with that agent. For example, this may be the amount of a conjugate useful in increasing resistance to, preventing, ameliorating, and/or treating infection and disease. Ideally, a therapeutically effective amount of an agent is an amount sufficient to increase resistance to, prevent, ameliorate, and/or treat infection and without causing a substantial cytotoxic effect in the subject The effective amount of an agent useful for increasing resistance to, preventing, ameliorating, and/or treating infection and disease in a subject will be dependent on the subject being treated, the severity of the 10 affliction, and the manner of administration of the therapeutic composition.

Vaccine: A vaccine is a pharmaceutical composition that elicits a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective response. Typically, a vaccine elicits an antigenspecific immune response to an antigen of a pathogen, for example, a bacterial or 15 viral pathogen, or to a cellular constituent correlated with a pathological condition.

A vaccine may include a polynucleotide, a peptide or polypeptide, a polysaccharide, a virus, a bacteria, a cell or one or more cellular constituents. In some cases, the virus, bacteria or cell may be inactivated or attenuated to prevent or reduce the likelihood of infection, while maintaining the immunogenicity of the vaccine constituent.

The antigenic polypeptide can be that of a rotavirus, or of a virus other than a rotavirus. A non limiting, and far from exhaustive list of such other viruses includes Adeno-associated virus, Adenovirus, Avian infectious bronchitis virus, Baculovirus, Chicken pox, Corona virus, Cytomegalovirus, Distemper, Enterovirus, Epstein Barr 25 virus, Feline leukemia virus, Fiavivirus, Foot and mouth disease virus, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis E, Herpes species, Hetpes simplex, Influenza virus, HIV-1, HIV-2, HTLV 1, Influenza A and B, Kunjin virus, Lassa fever virus, LCMV (lymphocytic choriomeningitis virus), lentivirus, Measles, Mengo virus, Morbillivirus, Myxovirus, Papilloma virus, Parovirus, Parainfluenza virus, 30 Paramyxovirus, Parvovirus, Poko virus, Polio virus, Polyoma tumour virus, pseudorabies, Rabies virus, Reovirus, Respiratory syncytial virus, retrovirus, rhinovinis, Rinderpest, Rotavirus, Semliki forest virus, Sendai virus, Simian Virus

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40, Sindbis virus, SV5, Tick bome encephalitis virus, Togavirus (rubella, yellow fever, dengue fever), Vaccinia virus, Venezuelan equine encephalomyelitis, Vesicular stomatis virus, metapncumovirus, norovirus, SARS virus, smallpox virus, picomaviruses, varicella zoster, and West Nile virus.

Alternatively, the antigenic polypeptide can be that of a bacteria or other pathogenic organism. Exemplary bacterial polypeptides include those of Achromobacter xylosoxidans. Acinetobacter calcoaceticus, preferably A. anitratus, A. baemoiyticus, A. alcaligenes, and A. Iwoffii, Actinomyces israelii, Aeromonas hydrophilia, Alcaligenes species, preferably A.faecalis. A. odorans and A.

denitrificans, Arizona hinshawii, Bacillus anthracis. Bacillus cereus, Bacteroides fragilis, Bacteroides melanihogenicus, Bordetella pertussis, Borrelia burgdorferi, Borrelia recurrentis. Brucella species, preferably B. abortus, B. suis, B. melitensis and B. canis, Calymmatobacterium granulomatis, Campylobacter coli (e.g, the CjaA polypeptide). Campylobacter fetus ssp. intestinalis, Campylobacter fetus ssp.

jejuni. Chlamydia species, preferably C. psittaci and C. trachomatis. Chromobacterium violaceum, Citrobacter species, preferably C. freundii and C. diversus, Clostridium botulinum, Clostridium perfringens, Clostridium difficile, Clostridium tetani, Corynebacterium diphtheriae, Corynebacterium, preferably C.

' ulcerans, C. haemolyticum and C. pseudotuberculosis, Coxiella burnetii,

Edwardsiella tarda, Eikenella corrodens, Enterobacter. preferably E. cloacae, E. aerogenes, E. hafniae (also named Hafnia alvei) and E. agglomérons, Erysipelothrix rhusiopathiae. Escherichia coli, Flavobacterium meningosepticum. Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis. Haemophilus ducreyi, Haemophilus influenzae. Helicobacter species (e.g., the UreB polypeptide of H.

pylori), Klebsiella species, preferably K. pneumoniae, K. ozaenae og K rhinoscleromatis, Legionella species, Leptospira interrogans. Listeria monocytogenes, Moraxella species, preferably M. lacunata and M. osloensis, Mycobacterium bovis, Mycobacterium leprae. Mycobacterium tuberculosis (e.g., the CFP10 polypeptide), Mycoplasma species, preferably M. pneumoniae, Neisseria gonorrhoeae. Neisseria meningitidis, Nocardia species, preferably N. asteroides and N. brasiliensis, Pasleurella haemolytica, Pasleurella multocida, Peptococcus magnus, Plesiomonas shigelloides. Pneumococci, Proteus species, preferably P.

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PCT/ÜS2007/023140 mirabilis, P. vulgaris. P. rettgeri and P. morganii (also named Providencia rettgen and Morganella morganii respectively), Providencia species, preferably P. alcalifaciens, P. stuartii and P. rettgeri (also named Proteus rettgeri), Pseudomonas aeruginosa, Pseudomonas mallei, Pseudomonas pseudomallei, Rickettsia, Rochalimaia henselae. Salmonella species, preferably S. entendis, S. typhi and 5. derby, and most preferably Salmonella species of the type Salmonella DTI 04, Serratia species, preferably S. marcescens, Shigella dysenteriae, S. flexneri, S. boydii and S. sonnei, Spirillum minor. Staphylococcus aureus, Staphylococcus epidermidis. Staphylococcus saprophyticus. Streptobacillus moniliformis. Streptococcus, preferably S. faecalis, S. faecium and S. durons, Streptococcus agalactiae. Streptococcus pneumoniae. Streptococcus pyogenes (e.g., the Sfbl polypeptide), Treponema carateum, Treponema pallidum, Treponema perlenue, preferably T. pallidum. Ureaplasma urealyticum. Vibrio cholerae, Vibrio parahaemolyticus. Yersinia enterocolitica, and Yersinia pestis.

Parasitic haptens or antigens may for example be selected from Malaria (Plasmodium falciparum, P. vivax, P. malariae), Schistosomes, Trypanosomes, Leishmania, Filarial nematodes, Trichomoniasis. Sarcosporidiosis, Taenia (T. saginata, T. solium), Leishmania. Toxoplasma gondii, Trichinelosis (Trichinella spiralis) or Coccidiosis (Eimeria species).

Illustrative fungal haptens or antigens could be derived a fungus selected from Cryptococcus neoformans, Candida albicans. Aspergillus jumigatus and Coccidioidomycosis.

The hapten or antigen may also be derived from any animal, including for example vertebrates. For example the hapten or antigen may comprise components derived from ovalbumin, keyhole limpet hemocyanin and sperm-whale myoglobulin.

Examples of suitable carriers are those that can increase the immunogenicity of the conjugate and/or elicit antibodies against the carrier which are diagnostically, analytically, and/or therapeutically beneficial. Useful carriers include polymeric carriers, which can be natural, semi-synthetic or synthetic materials containing one or more functional groups, for example primary and/or secondary amino groups, azido groups, hydroxyl groups, or carboxyl groups, to which a reactant moiety can 42

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PCT/US2007/023I40 be attached. The carrier can be water soluble or insoluble, and in some embodiments is a protein or polypeptide. Carriers that fulfill these criteria are generally known tn the art (see, for example, Fattom et al., Infect. Immun 58:230912,1990; Devi et aL, PNAS 88:7175-79, 1991; Szu et al., Infect. Immun. 59:45555 61,1991; Szu et al., J. Exp. Med. 166:1510-24, 1987; and Pavliakova et al., Infect.

Immun. 68:2161-66,2000). A carrier can be useful even if the antibody that it induces is not of benefit by itself.

Specific, non-limiting examples of water soluble polypeptide carriers include, but are not limited to, natural, semi-synthetic or synthetic polypeptides or 10 proteins from bacteria or viruses. In one embodiment, bacteria! products for use as carriers include bacterial wall proteins and other products (for example, streptococcal or staphylococcal cell walls), and soluble antigens of bacteria. In another embodiment, bacterial products for use as carriers include bacterial toxins. Bacterial toxins include bacterial products that mediate toxic effects, inflammatory 15 responses, stress, shock, chronic sequelae, or mortality in a susceptible host.

Specific, non-limiting examples of water insoluble carriers include, but are not limited to, aminoalkyl agarose (for example, aminopropyi or aminohexyl SEPHAROSE; Pharmacia Inc., Piscataway, NJ.), aminopropyl glass, cross-linked dextran, and the like, to which a reactive moiety can be attached. Other carriers can 20 be used, provided that a functional group is available for covalently attaching a reactive group.

II. Haptens

Disclosed embodiments of such haptens include pyrazoies, particularly nitropyrazoles; nitrophenyl compounds; benzofurazans; triterpenes; ureas and thioureas, particularly phenyl ureas, and even more particularly phenyl thioureas; rotenone and rotenone derivatives, also referred to herein as rotenoids; oxazole and thiazoles, particularly oxazole and thiazole sulfonamides; coumarin and coumarin derivatives; cyclolignans, exemplified by Podophyliotoxin and Podophyllotoxin derivatives; and combinations thereof.

For the general formulas provided below, if no substituent is indicated, a person of ordinary skill in the art will appreciate that the substituent is hydrogen. A 43

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PCT7US2007/023140 bond that is not connected to an atom, but is shown, for example, extending to the interior of a ring system, indicates that the position of such substituent is variable. A curved line drawn through a bond indicates that some additional structure is bonded to that position, typically a linker or the functional group or moiety used to couple 5 the hapten to a carrier. Moreover, if no stereochemistry is indicated for compounds having one or more chiral centers, all enantiomers and diasteromers are included. Similarly, for a recitation of aliphatic or alkyl groups, all structural isomers thereof also are included.

1. Azoles

A first general class of haptens of the present invention is azoles, typically oxazoles and pyrazoles, more typically nitro oxazoles and nitro pyrazoles, having the following general chemical formula.

r₂ r/ *

With reference to this general formula, n is 0-2, most typically 0 or 1. R1-R4 can be any organic group that does not interfere with, and potentially facilitates, the function as a hapten. More specifically, R1-R4 independently are selected from: hydrogen, acyl, aldehydes, alkoxy, aliphatic, particulary lower aliphatic, substituted aliphatic, heteroaliphatic, e.g., organic chains having heteroatoms, such as oxygen, nitrogen, sulfur, alkyl, particularly alkyl having 20 or fewer carbon atoms, and even more typically lower alkyl having 10 or fewer atoms, such as methyl, ethyl, propyl, isopropyl, and butyl, substituted alkyl, such as alkyl halide (e.g. -CXj where X is a halide, and combinations thereof, either in the chain or bonded thereto,), oxime, oxime ether (e.g., methoxyimine, CHj-O-N=) alcohols (i.e. aliphatic or alkyl hydroxyl, particularly lower alkyl hydroxyl) amido, amino, amino acid, aryl, alkyl aryl, such as benzyl, carbohydrate, monosaccharides, such as glucose and fructose, disaccharides, such as sucrose and lactose, oligosaccharides and polysaccharides, carbonyl, carboxyl, carboxylate (including salts thereof, such as Group 1 metal or

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PCT/US2007/023140 ammonium ion carboxylates), cyclic, cyano (-CN), esta·, ether, exomethylene, halogen, heteroaryl, heterocyclic, hydroxyl, hydroxylamine, oxime (HO-N=), keto, such as aliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, and combinations thereof. Two or more of these Rplti substituents also may be atoms, typically carbon atoms, in a ring system bonded or fused to the compounds having the illustrated general formula. At least one of the R1-R4 substituents is bonded to a linker or is a functional group suitable for coupling to a linker or a carrier molecule. R1-R4 most typically are aliphatic, hydrogen or nitro groups, even more typically alkyl, hydrogen or nitro, and still even more typically lower (10 or fewer carbon atoms) alkyl, hydrogen, nitro, or combinations thereof. The number of nitro groups can vary, but most typically there are 1 or 2 nitro groups. X independently is nitrogen or carbon. Y is oxygen, sulfur or nitrogen. If Y is oxygen or sulfur, then there is no Ri group, and n=0. If Y is nitrogen, then there is at least one Ri group.

A person of ordinary skill in the art will appreciate that, for compounds having 2 or more W groups, the relative positions thereof is variable. For example, a diazole could have nitrogen atoms at the 1 and 2 positions, or the 1 and 3 positions. Moreover, more than two heteroatoms also are possible, such as with triazines.

At least one of R1-R4 for these azole compounds is bonded to some other group or is a variable functional group. For example, the illustrated compounds can be coupled either directly to a carrier or to a linker at any of the suitable positions about the azole ring.

Working embodiments typically were mono- or dinitro pyrazole derivatives, such that at least one of R1-R4 is a nitro group, and perhaps two of R1-R4 are nitro groups, with the remaining R1-R4 being used to couple the hapten to a linker or a carrier.

r₂

One particular compound had the following structure.

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<img file="CA2995467C_D0026.tif" />

2. Nitroaryl

A second general class of haptens of the present invention are nitroaryl compounds. Exemplary nitroaryl compounds include, without limitation, nitrophenyl, nitrobiphenyl, nitrotriphenyl, etc., and any and all heteroaryl counterparts, having the following general chemical formula.

<img file="CA2995467C_D0027.tif" />

With reference to this general formula, such compounds have at least one, and optionally plural, nitro groups. Thus, at least one of Ri-R^ is nitro. If more than one of R)-R$ is nitro, all combinations of relative ring positions of plural nitro substituents, or nitro substituents relative to other ring substituents, are included within this class of disclosed haptens. Dinitroaryl compounds are most typical. A person of ordinary skill in the art will appreciate that as the number of nitro groups increases, the number of remaining ring substituents in the general formula decreases. These substituents independently are selected from: hydrogen, acyl, aldehydes, alkoxy, aliphatic, particulary lower aliphatic, substituted aliphatic, heteroaliphatic, e.g., organic chains having heteroatoms, such as oxygen, nitrogen, sulfur, alkyl, particularly alkyl having 20 or fewer carbon atoms, and even more typically lower alkyl having 10 or fewer carbon atoms, such as methyl, ethyl, propyl, isopropyl, and butyl, substituted alky), such as alkyl halide (e.g. -CXj where X is a halide, and combinations thereof, either in the chain or bonded thereto), oxime, oxime ether (e.g., methoxyimine, CHj-O-N=) alcohols (i.e. aliphatic or alkyl hydroxyl, particularly lower alkyl hydroxyl) amido, amino, amino acid, aryl, alkyl

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PCT/US2007/023140 aryl, such as benzyl, carbohydrate, monosaccharides, such as glucose and fructose, disaccharides, such as sucrose and lactose, oligosaccharides and polysaccharides, carbonyl, carboxyl, carboxylate (including salts thereof, such as Group I metal or ammonium ion carboxylates), cyclic, heterocyclic, cyano (-CN), ester, ether, halogen, heteroaryl, hydroxyl, hydroxlyamine, oxime (HO-N=), keto, such as aliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, exomethylene, and combinations thereof. At least one of the RrR« substituents is bonded to a linker or is a functional group suitable for coupling to a linker or a carrier molecule.

Two or more of the Ri-R« substituents also may be atoms, typically carbon atoms, in a ring system, such as napthalene (shown below) or anthracene type derivatives. Ring systems other than 6-membered ring systems can be formed, such as fused 6-5 ring systems.

Re Ri

Rel^l^³Rs R4

Again, at least on of the ring positions occupied by R|-Rj is bonded to a linker or is a variable functional group suitable for coupling, such as by covalent bonding, to a carrier molecule. For example, nitroaryl compounds of the present invention can include a functional group for coupling to a carrier, or to a linker, at various optional ring locations.

Working embodiments are exemplified by nitrophenyl compounds. Solely by way of example, mononitroaryl compounds are exemplified by nitrocinnamide compounds. One embodiment of a nitrocinnamide-based compound is exemplified by 4,5-dimethoxy-2-nitrocinnamide, shown below.

H₃CO

NO?

H₃CO

<img file="CA2995467C_D0028.tif" />

nh₂

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The nitrophenyl class of compounds also is represented by dinitrophenyl compounds. At least one of the remaining carbon atoms of the ring positions not having a nitro group is bonded to a functional group, to a linker, or directly to a carrier. Any and all combinations of relative positions of these groups are included within the class of disclosed haptens.

O_: o₂n

Working embodiments are more particularly exemplified by 2,4-dinitrophenyl compounds coupled to a linker, as illustrated below.

NO₂ r₂

IO

Ri-Rj are as stated above.

3. Benzofurazans

Benzofurazans and derivatives thereof are another class of haptens within the scope of the present invention. A general formula for the benzofurazan-type compounds is provided below.

Ri

<img file="CA2995467C_D0029.tif" />

R₂ r₃

R4

R1-R4 substituents independently are selected from: hydrogen, acyl, aldehydes, alkoxy, aliphatic, particulary lower aliphatic, such as isoprene, substituted aliphatic, heteroaliphatic, e.g., organic chains having heteroatoms, such as oxygen, nitrogen, sulfur, alkyl, particularly alkyl having 20 or fewer carbon atoms, and even more typically lower alkyl having IO or fewer atoms, such as methyl, ethyl, propyl,

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PCT/US2007/023140 isopropyl, and butyl, substituted alkyl, such as alkyl halide (e.g. -CXj where X is a halide, and combinations thereof, either in the chain or bonded thereto,), oxime, oxime ether (e.g., methoxyimine, CHj-O-N=) alcohols (i.e. aliphatic or alkyl hydroxyl, particularly lower alkyl hydroxy!) amido, amino, amino acid, aryl, alkyl aryl, such as benzyl, carbohydrate, monosaccharides, such as glucose and fructose, disaccharides, such as sucrose and lactose, oligosaccharides and polysaccharides, carbonyl, carboxyl, carboxylate (including salts thereof, such as Group I metal or ammonium ion carboxylates), cyclic, heterocyclic, cyano (-CN), ester, alkyl ester, ether, halogen, heteroaryl, hydroxyl, hydroxylamine, oxime (HO-N=), keto, such as aliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, exomethylene, and combinations thereof. Two or more of these R1-R4 substituents also maybe atoms, typically carbon atoms, in a ring system bonded or fused to the compounds having the illustrated general formula. At least one of the R1-R4 substituents is bonded to a linker or directlyt to a carrier. Y is a carbon atom having R$ and R^ substituents, where R$ and R« are as stated for R1-R4, oxygen or sulfur, typically oxygen. Compounds where Y is oxygen are more particularly exemplified by compounds having the following structure, where R1-R4 are as stated above, and most typically are independently hydrogen and lower alkyl.

<img file="CA2995467C_D0030.tif" />

One working embodiment of a compound according to this class of haptens had the following chemical structure.

<img file="CA2995467C_D0031.tif" />

4. Triter penes

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Triterpenes arc another class of haptens within the scope of the present invention. The basic ring structure common to the cyclic triterpenes has four sixmembered fused rings, A-D, as indicated below.

<img file="CA2995467C_D0032.tif" />

A number of publications discuss naturally occurring, semi-synthetic and synthetic triterpene species within the genus of triterpenes useful for practicing the present invention, including: J.C. Connolly and R. A. Hill, Triterpenoids, Nat. Prod. Rep., 19,494-513 (2002); Baglîn et al., A Review of Natural and Modified Beculinic, Ursolic and Echinocystic Acid Derivatives as Potential Antitumor and Anti-HIV Agents, Mini Reviews in Medicinal Chemistry, 3, 525-539; W.N. and M.C. Setzer, Plant-Derived Triterpenoids as Potential Antineoplastic Agents, Mini Reviews in Medicinal Chemistry, 3,540-556 (2003); and Baltina, Chemical Modification of Glycyrrhizic Acid as a Route to New Bioactive Compounds for Medicine, Current Medicinal Chemistry, 10, 155-171 92003),

Based on the present disclosure and working embodiments thereof, as well as disclosures provided by these prior publications, and with reference to this first general formula, Ri-Rji independently are selected from: hydrogen, acyl, aldehydes, alkoxy, aliphatic, particular/ lower aliphatic, such as isoprene, substituted aliphatic, heteroaliphatic, e.g., organic chains having heteroatoms, such as oxygen, nitrogen, sulfur, alkyl, particularly alkyl having 20 or fewer carbon atoms, and even more typically lower alkyl having 10 or fewer atoms, such as methyl, ethyl, propyl, isopropyl, and butyl, substituted alkyl, such as alkyl halide (e.g. -CX3 where X is a halide, and combinations thereof, either in the chain or bonded thereto,), oxime, oxime ether (e.g., methoxyimine, CHj-O-N^) alcohols (i.e.

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PCT/US2007/023140 aliphatic or alkyl hydroxyl, particularly lower alkyl hydroxyl) amido, amino, amino acid, aryl, alkyl aryl, such as benzyl, carbohydrate, monosaccharides, such as glucose and fructose, disaccharides, such as sucrose and lactose, oligosaccharides and polysaccharides, carbonyl, carboxyl, carboxylate (including salts thereof, such as Group I metal or ammonium ion carboxylates), cyclic, heterocyclic, cyano (-CN), ester, alkyl ester, ether, halogen, heteroaryi, hydroxyl, hydroxylamine, oxime (HON=), keto, such as aliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, exomethylene, and combinations thereof. Two or more of these R1-R21 substituents also may be atoms, typically carbon atoms, in a ring system bonded or fused to the 10 compounds having the illustrated general formula. At least one of the R|-R₂i substituents is bonded to a linker or is a functional group suitable for coupling to a linker or a carrier molecule. Y is a bond, thereby defining a 5-membered ring, or is a carbon atom bearing R22 and R23 substituents, where these R groups are as stated above.

Disclosed embodiments of triterpenes exemplifying this class of haptens also may include an E ring, and this E ring can be of various ring sizes, particulary rings having 5-7 atoms, typically carbon atoms, in the ring. For example, the E ring might be a 6-membered ring, as indicated by the following general formula, where R1-R31 are as stated above for R|-R₂1.

R26

<img file="CA2995467C_D0033.tif" />

H3C CH₃ r₂₁ R₂o

The following general formual indicates that the Rd substituent may be an acyl group bearing an R33 substituent selected from hydrogen, hydroxyl, ester, i.e. 51

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OR34 where R34 is aliphatic, typically alkyl or substituted alkyl, and even more typically lower alkyl, amido, including primary amide (-NH2), secondary amide (NHR35) and tertiary amide (-NR35R36), where R35 and R36 arc aliphatic, typically lower aliphatic, more typically alkyl, substituted alkyl, and even more typically 5 lower alkyl or substituted lower alkyl. This general formula also indicates that the

Ri substituent often is an OR32 substituent, where R32 is hydrogen or aliphatic, more typically alkyl or substituted alkyl, ane even more typically lower alkyl. The remaining R groups are as stated above with reference to the first general formula.

<img file="CA2995467C_D0034.tif" />

The E ring also may be a 5 membered ring, as indicated by the formula below where the R|-R₂₉ groups are as stated above for Ri-R?t-

<img file="CA2995467C_D0035.tif" />

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<img file="CA2995467C_D0036.tif" />

With reference to these general formulae, the R.1-R29 groups are as stated above for R1-R21.

As with exemplary compounds where the E ring is a 6-membered ring, compounds where the E ring is a 5-membered ring also can include substituents at Ri and Rn as discussed above. Specifically, this general fonnual indicates that the Rtj substituent may be an acyl group bearing an Rjj substituent selected from hydrogen, hydroxyl, ester, i.e. -OR34 where R34 is aliphatic, typically alkyl or substituted alkyl, and even more typically lower alkyl, amido, including primary amide (-NH2), secondary amide (-NHRj$) and tertiary amide (-NRjjR^), where R35 and R36 are aliphatic, typically lower aliphatic, more typically alkyl, substituted alkyl, and even more typically lower alkyl or substituted lower alkyl. This general formula also indicates that the Ri substituent often is an OR32 substituent, where R32 is hydrogen or aliphatic, more typically alkyl or substituted alkyl, ane even more typically lower alkyl.

Exemplary compounds also include 5-membered rings as both the A aid the E ring. General formulae for such exemplary compounds are provided below, where the R1-R29 substituents are as stated above.

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<img file="CA2995467C_D0037.tif" />

Again, the Rt and R13 substituents can be oxygen-based functional groups. The Ru substituent may be an acyl group bearing an R33 substituent selected from hydrogen, hydroxyl, ester, i.e. -OR34 where R_M is aliphatic, typically alkyl or substituted alkyl, and even more typically lower alkyl, amido, including primary amide (-NH2), secondary amide (-NHR35) and tertiary amide (-NRjjRjé), where Rj$ and Rjs are aliphatic, typically lower aliphatic, more typically alkyl, substituted alkyl, and even more typically lower alkyl or substituted lower alkyl. This general formula also indicates that the R_t substituent often is an OR32 substituent, where R32 is hydrogen or aliphatic, more typically alkyl or substituted alkyl, and even more typically lower alkyl.

<img file="CA2995467C_D0038.tif" />

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Exemplary triterpenes of the present invention also may include one or more sites of unsaturation in one or more of the A-E rings. Exemplary compounds often have at least one site of unsaturation in the C ring, such as the double bond in the C ring as indicated below.

<img file="CA2995467C_D0039.tif" />

The site of unsaturation may be an alpha, beta unsaturated ketone, such as illustrated below for the C ring.

<img file="CA2995467C_D0040.tif" />

The triterpenes also have a number of stereogenic carbon atoms. A person of 10 ordinary skill in the art will appreciate that particular enantiomers are most likely to occur naturally. While the naturally occurring enantiomer may be most available, and/or effective, for practicing disclosed embodiments, all other possible

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PCT/US2007/023140 stereoisomers are within the scope of the present invention. Moreover, other naturally occuring triterpenes, or synthetic derivatives thereof, or fully synthetic compounds, may have (1) different stereochemistry, (2) different substituents, and further may be substituted at positions that are not substituted in the naturally 5 occurring compounds. The general formulae provided above do not indicate stereochemistry at the chiral centers. This is to signify that both enantiomers at each chiral center, and all diastereomeric isomer combinations thereof, are within the scope of the present invention.

Particular working embodiments of the present invention are exemplified by 10 the following general formula, in which the substituents are as stated above.

<img file="CA2995467C_D0041.tif" />

The stereochemistry and substituents for a naturally occuring triterpene useful as a hapten for practicing the present invention are shown below.

<img file="CA2995467C_D0042.tif" />

The hydroxyl group in the A ring typically is oxidized to a carbonyl functional group in working embodiments. As a result, the carbon atom bearing the carbonyl group is no longer a chiral center.

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5. Ureas and Thioureas

Ureas and thioureas, particularly aryl and heteroaryl ureas and thioureas, are another class of haptens within the scope of the present invention. A general formula for urea-based haptens of the present invention is provided below.

<img file="CA2995467C_D0043.tif" />

R2 R3

With reference to this general formula, R1-R3 are independently hydrogen, aliphatic, substituted aliphatic, typically alkyl, substituted alkyl, and even more typically lower alkyl and substituted lower alkyl, cyclic, heterocyclic, aryl and heteroaryl. More specifically, R| typically is aryl or aliphatic, often having at least one site of unsaturation to facilitate chromophoric activity. R2 and Rj most typically are independently hydrogen and lower alkyl. Y is oxygen (urea derivatives) or sulfur (thioureas).

Aryl derivatives typically have the following formula.

<img file="CA2995467C_D0044.tif" />

R.1-R7 independently are selected from: hydrogen, acyl, aldehydes, alkoxy, aliphatic, particulary lower aliphatic, such as isoprene, substituted aliphatic, heteroaliphatic, e.g., organic chains having heteroatoms, such as oxygen, nitrogen, sulfur, alkyl, particularly alkyl having 20 or fewer carbon atoms, and even more typically lower alkyl having 10 or fewer atoms, such as methyl, ethyl, propyl, isopropyl, and butyl, substituted alkyl, such as alkyl halide (e.g. -CXj where X is a halide, and combinations thereof, either in the chain or bonded thereto,), oxime, oxime ether (e.g., methoxyimine, CHj-O-N=) alcohols (i.e. aliphatic or alkyl hydroxyl, particularly lower alkyl hydroxyl) amido, amino, amino acid, aryl, alkyl aryl, such as benzyl, carbohydrate, monosaccharides, such as glucose and fructose, disaccharides, such as sucrose and lactose, oligosaccharides and polysaccharides, carbonyl,

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PCT/US2007/023140 carboxyl, carboxylate (including salts thereof, such as Group I metal or ammonium ion carboxylates), cyclic, heterocyclic, cyano (-CN), ester, alkyl ester, ether, halogen, heteroaryl, hydroxyl, hydroxylamine, oxime (HO-N=), keto, such as aliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, exomethylene, and combinations thereof. At least one of the R3-R7 substituents also is bonded to a linker or to a carrier molecule. Two or more of these R3-R7 substituents available for such bonding also may be atoms, typically carbon atoms, in a ring system bonded or fused to the compounds having the illustrated general formula.

Additional rings also can be present, as indicated by the exemplary structures 10 provided below. The R groups are as stated above for R1-R7 and Y is oxygen or sulfur.

<img file="CA2995467C_D0045.tif" />

A particular subclass of thioureas is represented below.

<img file="CA2995467C_D0046.tif" />

^R1 ^r2

With reference to this general formula, n is I to 5, typically 1-2, R| and R2 are independently hydrogen or lower alkyl, and X independently is a halide or combinations of different halides.

One example of a working embodiment of a phenyl thiourea is provided 20 below.

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<img file="CA2995467C_D0047.tif" />

The trifluoromethyl groups arc shown in the 2 and 4 positions relative to the thiourea moiety. A person of ordinary skill in the art will appreciate that compounds having all relative positions for disubstituted compounds, such as 2,3, and compounds having more than two trihaloalkyl substituents, at all possible relative positions of such plural trihaloalkyl substituents, also are within the scope of the present invention. A particular example of a rhodamine thiourea hapten has the following formula.

<img file="CA2995467C_D0048.tif" />

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6. Rotenones

Rotenone and rotenone-based haptens, collectively referred to as rotenoids, provide another class of haptens within the scope of the present invention. A first general formula for rotenone, and rotenone-based haptens, is provided below.

<img file="CA2995467C_D0049.tif" />

A number of publications discuss naturally occurring, semi-synthetic and synthetic rotenoids that are useful for describing the genus of rotenoids useful for practicing the present invention, including: Leslie Crombie and Donald Whiting, Biosynthesis in the Rotenoids Group of Natural Products: Application of Isotope Methodology, 10 Phytochemistry, 49,1479-1507 (1998); and Nianbai Fang, and John Casida, Cube

Resin Insecticide: Identification and Biolgoical Activity of 29 Rotenoid Constituents. Based on the present disclosure and working embodiments, as well as disclosures provided by these prior publications, and with reference to this first general formula, R|.R|&lt; 15 independently are hydrogen, aldehyde, alkoxy, aliphatic, particulary lower aliphatic, such as isoprene, substituted aliphatic, heteroaliphatic, e.g., organic chains having heteroatoms, such as oxygen, nitrogen, sulfiir, alkyl, particularly alkyl having 20 or fewer carbon atoms, and even more typically lower alkyl having 10 or fewer atoms, such as methyl, ethyl, propyl, isopropyl, and butyl, substituted alkyl, such as alkyl 20 halide (e.g. -CX3 where X is a halide, and combinations thereof, either in the chain or bonded thereto) amino, amino acid, amido, cyano (-CN), halogen, hydroxyl, hydroxylamine, oxime (HO-N=), oxime ether (e.g., methoxyimine, CH3-O-N=) alkyl hydroxyl, particularly lower alkyl hydroxyl, carbonyl, keto, such as aliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, carboxyl, carboxylate (and salts

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PCT/ÜS2007/023140 thereof, such as Group I metal or ammonium ion carboxylates) ester, alkyl ester, acyl, exomethylene, ether, cyclic, heterocyclic, aryl, alkyl aryl, such as benzyl, heteroaryl, polysaccharides, carbohydrate, monosaccharides, such as glucose and fructose, disaccharides, such as sucrose and lactose, oligosaccharides and polysaccharides, and combinations thereof. Two or more of these R₍.R m substituents also may be atoms, typically carbon atoms, in a ring system bonded or fused to the compounds having the illustrated general formula. At least one of the RiJRi4 substituents also is bonded to a linker or to a carrier molecule.

While Ri and R7 can be as stated above, such substituents more typically independently are hydrogen, OR $, where R] 5 is hydrogen, aliphatic, substituted aliphatic, typically alkyl, substituted alkyl, and even more typically lower alkyl and substituted lower alkyl, such as lower alkyl halides, cyclic, heterocyclic, aryl and heteroaryl, -NR21, where R21 is hydrogen, aliphatic, substituted aliphatic, typically alkyl, substituted alkyl, and even more typically lower alkyl and substituted lower alkyl, such as lower alkyl halides, cyclic, heterocyclic, aryl and heteroaryl, or N-LRG, where L is a linker or a reactive group, such as an amine, as discussed in more detail herein.

R« and R7 also can form a double bond, such as a double bond to an oxygen to form a carbonyl. If Rg and/or R? are not -L-RG, then at least one of the R substituents is bonded to a linker or to a carrier molecule.

The B ring also can include at least one additional site of unsaturation. For example, Rs and Ru can form a double bond.

Rio and Rh can be joined in a 5· or 6-membered ring. For example, Rio and Ri 1 may define a pyran or furan ring, and more particularly is a substituted and/or 25 unsaturated pyran or furan ring.

Certain exemplary rotenone-based haptens of the present invention also typically satisfy the following second general fonnula.

«1

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<img file="CA2995467C_D0050.tif" />

With reference to this second general formula, the R substituents are as stated above. If Re or R? is not -L-RG, then at least one of the remaining R groups is bonded to a linker or to a carrier.

Rio and R₍ i can be joinied in a 5- or 6-membered ring, such as a pyran or furan, and more particularly a substituted and/or unsaturated pyran or furan ring. Thus, a third general formula useful for describing certain rotenone-based haptens of the present invention is provided below, where the R substituents are as stated above.

<img file="CA2995467C_D0051.tif" />

Y is a bond, thereby defining a 5-membered ring, or is a carbon atom in a 6membered ring bearing R₎₉ and R» substituents, as shown below, where the R substituents arc as stated above.

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<img file="CA2995467C_D0052.tif" />

<img file="CA2995467C_D0053.tif" />

Rs and R_t2 at the ring juncture are shown without indicating particular stereochemistry. The naturally occurring compound has a c/s-ring juncture, but racemic mixtures also are useful for practicing the present invention. Also, the trans stereoisomer likely quickly equilibrates to form the racemic mixture.

Working embodiments of compounds within this class more typically satisfy the following third general formula.

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<img file="CA2995467C_D0054.tif" />

With referent» to this general formula, R,^ hydrogen, _a|_{kyI or}define a double bond, such as to oxygen to form a carbonyl. R„ and R„ independently are hydrogen and aliphatic, typteaHy |₀„„ aliphatic one example of which is isoprene, as shown below.

<img file="CA2995467C_D0055.tif" />

Again, a particular enantiomer is shown in the above formula, but a person of ordinary skill iu the an will appreciate that the scope of the present invemion is tatted to the particular enantiomer shown. inet»), ,|| stereo,'somm that ad as 10 i^,aP^tensalsoarewlthinthescopeofthedtsclosure. All substitutions discussed above for this class of compounds applies to this particular compound. Other substitutions also are readily , _{of 0Klirao},_skil| „ example, the methoxy groups ontheAriug can be any alkoxy compound, particular ower alkoxy groups. The isoprene unit also provides an olefin that can be synthetically modified, perhaps to provide an alternative position, or at least a second position, for coupling the hapten to a linker or a carrier molecule. For example, the olefin could be converted to an alcohol by hydroboration, ft _alSo could

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WO 2008/063378 be convened &lt;0 . halide or an epoxide either for use as a hapten or as intennedia.es useful for further transformation.

A fourth general formula for describing rotenone-based haptens of the present invention is particularly directed to rotenone isoxaxolines, as provtded 5 below.

<img file="CA2995467C_D0056.tif" />

R.R, independently are hydregen. aldehyde. atoxy. *4 '<™“ aliphatic, including all branched chain isomère, such as tsoprene, and all stereoisomers, subs.tn.ted aliphatic. haeroaliphado. e.g. orgmic dretm> hnvmg heteroatoms, such as oxygen, nitrogen, sulfur, alkyl.

fewer carbon atoms, and even more typically lower alkyl having 10 or fewer atoms, halide (e g -CX, where X is . halide, and combinations thereof, etto m the ehtun „ bonded thereto) amino. ™ hydros* hydroxylamine, oxime (HO-N-), oxime ether (eg. methoxyimme CH,-O-N=) X hydroxyl, particdariy low« « bydrox* enrimn,!. keto, such as ahphahe ketones, mho. sulfhydryl. suilbnyl, sulfoxide carttoxyl. earboxyiate (and sate thereof, such as Group I metal or ammonium ion caiboxylates) ester, al y erter, acyl exomelhylene, «her, cydie heterooyelic, myl. alkyl «*.·»* as benzyl heteroaryl, polysacchandes, «rbohydrate. monosaeehmides. such » glucose and fructose, disaccharides, such as sucrose and l.«ose, oligosacdtandes mi polysaccharides, aM combinations thereof. A. leas, one of the R-R, substitue^ .to is bonded to a tinker or to a eartier moioouie Y is oxygen, nitrogen, or «fc.

A particular working embodiment of a rotenone-based hapten sauslyrngthts fourth general formula is provided below.

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<img file="CA2995467C_D0057.tif" />

7. Oxazoles and Thiazoles

Oxazole and thiazole sulfonamides provide another class of haptens within the scope of the present invention. A general formula for oxazole and thiazole sulfonamides is provided below.

<img file="CA2995467C_D0058.tif" />

With reference to this first general formula Ri-Rj independently are selected from: hydrogen, acyl, aldehydes, alkoxy, aliphatic, particulary lower aliphatic, such as isoprene, substituted aliphatic, heteroaliphatic, e.g., organic chains having heteroatoms, such as oxygen, nitrogen, sulfiur, alkyl, particularly alkyl having 20 or fewer carbon atoms, and even more typically lower alkyl having 10 or fewer atoms, such as methyl, ethyl, propyl, isopropyl, and butyl, substituted alkyl, such as alkyl halide (e.g. -CXj where X is a halide, and combinations thereof, either in the chain or bonded thereto,), oxime, oxime ether (e.g., methoxyimine, CHj-O-N=) alcohols (i.e. aliphatic or alkyl hydroxyl, particularly lower alkyl hydroxyl) amido, amino, amino acid, aryl, alkyl aryl, such as benzyl, carbohydrate, monosaccharides, such as glucose and fructose, disaccharides, such as sucrose and lactose, oligosaccharides and polysaccharides, carbonyl, carboxyl, carboxylate (including salts thereof, such as Group I metal or ammonium ion carboxylates), cyclic, heterocyclic, cyano (-CN), ester, alkyl ester, ether, halogen, heteroaryl, hydroxyl, hydroxylamine, oxime (HO N=), keto, such as aliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide,

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For certain exemplary working embodiments, Ri has been amido, such as the amide derivatives shown below. R_z provides a position for coupling to a linker or to a carrier molecule, although the positions indicated by Ri and Rz also provide alternative or additional positions for coupling to a linker and/or carrier molecule. R₂, for certain working embodiments, has been -SO₂, and has been used to couple linkers by forming a sulfonamide. Thus, a second general fonnula for working embodiments of haptens exemplifying this class of haptens is indicated below, where the Rj-Re substituents and Y are as stated above.

For certain working embodiments R« has been alkyl, particularly lower alkyl, such as methyl, and Y has been sulfur.

One working embodiment of a compound according to this class of haptens had the following chemical structure.

The thiazole or oxazole might also be part of a larger ring system. For example, the 5-membered oxazole or thiazole might be coupled to at least one additional ring, such as a phenyl ring, as indicated below.

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<img file="CA2995467C_D0059.tif" />

While the Ri-Rs groups generally can be as stated above, such compounds also provide a position for coupling to a linker and/or to a carrier molecule, such as a R$. One possible sulfonamide derivative is provided below.

<img file="CA2995467C_D0060.tif" />

8. Coumarins

Coumarin and coumarin derivatives provide another class of haptens within the scope of the present invention. A general formula for coumarin and coumarin derivatives is provided below.

<img file="CA2995467C_D0061.tif" />

With reference to this general formula, Ri-Re independently are selected from: hydrogen, acyl, aldehydes, alkoxy, aliphatic, particulary lower aliphatic, such as isoprene, substituted aliphatic, heteroaliphatic, e.g., organic chains having heteroatoms, such as oxygen, nitrogen, sulfur, alkyl, particularly alkyl having 20 or fewer carbon atoms, and even more typically lower alkyl having 10 or fewer atoms, such as methyl, ethyl, propyl, isopropyl, and butyl, substituted alkyl, such as alkyl halide (e.g. -CX3 where X is a halide, and combinations thereof, either in the chain or bonded thereto,), oxime, oxime ether (e.g., methoxyimine, CH₃-O-N=) alcohols (i.e. aliphatic or alkyl hydroxyl, particularly lower alkyl hydroxyl) amido, amino,

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PCT/US2007/023140 amino acid, aryl, alkyl aryl, such as benzyl, carbohydrate, monosaccharides, such as glucose and fructose, disaccharides, such as sucrose and lactose, oligosaccharides and polysaccharides, carbonyl, carboxyl, carboxylate (including salts thereof, such as Group I metal or ammonium ion carboxylates), cyclic, heterocyclic, cyano (-CN), 5 ester, alkyl ester, ether, halogen, heteroaryl, hydroxyl, hydroxylamine, oxime (HOhl=), keto, such as aliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, exomethylene, and combinations thereof. At least one of the Ri-R« substituents also typically is bonded to a linker or a carrier molecule. Certain working embodiments have used the position indicated as having an Rj substituent for coupling toa linker or carrier molecule. The 4 position can be important if fluorescence is used to detect these compounds. Substituents other than hydrogen at (he 4 position are believed to quench fluorescence, although such derivatives still may be chromophores. Y is oxygen, nitrogen or sulfur. Two or more of the R|-R« substituents available for forming such compounds also may be atoms, typically carbon atoms, in a ring $ system bonded or fused to the compounds having the illustrated genera! formula. Exemplary embodiments of these types of compounds are provided below.

<img file="CA2995467C_D0062.tif" />

<img file="CA2995467C_D0063.tif" />

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<img file="CA2995467C_D0064.tif" />

A person of ordinary skill in the art will appreciate that the rings also could be heterocyclic and/or heteroaryl.

Working embodiments typically were fused A-D ring systems having at least 5 one carrier molecule coupling position, with one possible coupling position being indicated below.

<img file="CA2995467C_D0065.tif" />

With reference to this general formula, the R and Y variable groups are as stated above. Most typically, R|-Rm independently are hydrogen or lower alkyl. Particular 10 embodiments of coumarin-based haptens include 2,3,6,7-tetrahydro-l 1-oxo1 H,5H,11 H-[ 1 ]benzopyrano[6,7,8-ij]&lt;|uinoli2ine-10-carboxylic acid

<img file="CA2995467C_D0066.tif" />

and diethyl coumarin

<img file="CA2995467C_D0067.tif" />

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9. Cyclolignans r₅

<img file="CA2995467C_D0068.tif" />

Rl2 r₇-Re ~R₉

R10

R11

Lignin-based compounds, particulary cyclolignans, such as Podophyllotoxin and derivatives (hereof, provide another class of haptens within the scope of the present invention. A first general formula for these cyclolignin-based derivatives is provided below.

R&lt; R₃\JTa

Ri

A number of publications discuss naturally occuring, semi-synthetic and synthetic cyclolignans that are useful for describing the genus of cyclolignans useful for practicing the present invention, including: Stephanie Desbene and Sylviane GiorgiRenault, Drugs that Inhibit Tubulin Polymerization: The Particuar Case of Podophyllotoxin and Analogues, Curr. Med. Chem. - Anti-Cancer Agents, 2,71-90 (2002); M. Gordaliza el al., Podophyllotoxin: Distribution, Sources, Applications and New Cytotoxic Derivatives, Toxicon, 44,441-459 (2004); Phillipe Meresse et ai., Etoposide: Discovery and Medicinal Chemistry, Current Medicinal Chemistry,

11,2443-2466 (2004); M. Pujol el ah. Synthesis and Biological Activity of New Class of Dioxygenated Anticancer Agents, Curr. Med. Chem. - Anti-Cancer Agents, 5,215-237 (2005); and Youngjae You, Podophyllotoxin. Derivatives: Current Synthetic Approaches for New Anticancer Agents, Current Pharmaceutical Design, 20 11,1695-1717(2005). Based on the present disclosure and working embodiments, as well as disclosures provided by these prior publications, and with reference to this first general formula, R|-Ru typically are selected from hydrogen, aldehyde, alkoxy, aliphatic, particulary lower aliphatic, such as isoprene, substituted aliphatic, heteroaliphatic, e.g., organic chains 25 having heteroatoms, such as oxygen, nitrogen, sulfur, alkyl, particularly alkyl having or fewer carbon atoms, and even more typically lower alkyl having 10 or fewer atoms, such as methyl, ethyl, propyl, isopropyl, and butyl, substituted alkyl, such as alkyl halide (e.g. -CX3 where X is a halide, and combinations thereof, either in the 71

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PCT/US2007/023140 chain or bonded thereto,) amino, amino acid, amido, cyano (-CN), halogen, hydroxyl, hydroxylamine, oxime, oxime ether (e.g., methoxyimine, CHj-O-N=) alkyl hydroxyl, particularly lower alkyl hydroxyl, carbonyl, keto, such as aliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, carboxyl, carboxylate (and salts thereof, such as Group I metal or ammonium ion carboxylates) ester, alkyl ester, acyl, exomethylene, ether, cyclic, heterocyclic, aryl, alkyl aryl, such as benzyl, heteroaryl, polysaccharides, carbohydrate, monosaccharides, such as glucose and fructose, disaccharides, such as sucrose and lactose, oligosaccharides and polysaccharides, and combinations thereof. At least one of R1-R12 provides a position for coupling the compound to a linker or to a carrier molecule. Furthermore, certain of the R groups may be atoms in a ring system. For example, R₂ and R₃, as well as two of R7-R10, can be joined together in a ring system. At least one of R|2 and Rn also often is an aryl group, such as a benzene ring or a substituted benzene ring.

Certain working embodiments also satisfied the following second general formula, where the R substituents are as stated above.

<img file="CA2995467C_D0069.tif" />

Exemplary compounds where at least one of R| 1 and R12 is an aryl group have the following general formula, where the R substituents are as stated above.

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<img file="CA2995467C_D0070.tif" />

R16-R20 are generally as stated above, but more typically independently are hydrogen or alkoxy, typically lower alkoxy, such as methoxy, as sfiown below.

<img file="CA2995467C_D0071.tif" />

At least one of the R substituents typically is bonded to a linker, is a reactive functional group capable of reacting with a linker, or is -L-RG. For example, R$ often is -L-RG.

Rj and R$ also may form a double bond, such as a double bond to oxygen to form a carbonyl functional group or a double bond to a nitrogen atom to form an imine. Certain exemplary compounds where Rs and R« form a double bond had the following general formula, where the remaining R substituents are as stated above. Y is selected from nitrogen, oxygen or sulfur. If Y is nitrogen, then the nitrogen 73

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PCT/US2Ô07/023140 atom may further have bonded thereto hydrogen, or some atom, functional group or chemical moiety other than hydrogen. For example, the nitrogen may have an aliphatic substituent, such an alkyl group, an aryl or heteroaryl substituent, or a substituted aryl or heteroaryl substituent, such as alkyl and/or alkoxy substituted aryl 5 or heteroaryl substituent.

<img file="CA2995467C_D0072.tif" />

R16-R20 independently are selected from hydrogen and alkoxy, more typically lower alkoxy, such as methoxy, as indicated below.

<img file="CA2995467C_D0073.tif" />

As with all hapten conjugates of the present invention, at least one of the R substituents typically is bonded to a linker, is a reactive functional group capable of

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PCT7US2007/023140 reacting with a linker, is -L-RG, or is directly bonded to a carrier. For example, R« often is -L-RG.

The chemical structure for Podophyllotoxin, a compound exemplifying this cyclolignan class of haptens, is provided below.

<img file="CA2995467C_D0074.tif" />

_s OCH₃

Podophyllotoxin, also referred to as podofilox, is a non-alkaloid toxin having a molecular weight of 414.40 and a compositional fonnula of C22H22OS. Podophyllotoxin is present at concentrations of 0.3 to 1.0% by mass in the rhizome of American Mayapple Podophyllum peltatum. The melting point of

Podophyllotoxin is 183.3 - 184,0 °C.

Accordingly, cyclolignans according to the present invention based substantially on the Podophyllotoxin structure have the following general formula, where Y is selected from nitrogen, oxygen or sulfur.

<img file="CA2995467C_D0075.tif" />

A specific example of a cyclolignan hapten according to the present invention is shown below.

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<img file="CA2995467C_D0076.tif" />

This compound was made starting with Podophyllotoxin. The hydroxyl group of Podophyllotoxin was oxidized to a ketone. The ketone was then reacted with a substituted hydrazine to produce the compound indicated above. The hydrazine 5 reagent can be substituted as desired, including aliphatic and aryl substituents.

10. Heterobiaryl

Another general class of haptens of the present invention is heterobiaryl compounds, typically phenyl quinolines and quinoxalines. Disclosed heterobiaryl 10 compounds have a first general chemical formula as below.

<img file="CA2995467C_D0077.tif" />

With reference to this general fonnual, A-D are selected from carbon, nitrogen, oxygen, and sulfur, and any and all combinations thereof. Most typically A-D arc carbon or nitrogen. Ri-Rz substituents independently are selected from: hydrogen, 15 acyl, aldehydes, alkoxy, aliphatic, particulary lower aliphatic, substituted aliphatic, heteroaliphatic, e.g., organic chains having heteroatoms, such as oxygen, nitrogen, sulfur, alkyl, particularly alkyl having 20 or fewer carbon atoms, and even more typically lower alkyl having 10 or fewer atoms, such as methyl, ethyl, propyl, isopropyl, and butyl, substituted alkyl, such as alkyl halide (e.g. -CXj where X is a

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PCT/US2007/023140 halide, and combinations thereof, either in the chain or bonded thereto,), oxime, oxime ether (e.g., methoxyimine, CH₃-O-N=) alcohols (i.e. aliphatic or alkyl hydroxyl, particularly lower alkyl hydroxyl) amido, amino, amino acid, aryl, alkyl aryl, such as benzyl, alkoxy aryl, such as methoxy and ethoxy, carbohydrate, monosaccharides, such as glucose and fructose, disaccharides, such as sucrose and lactose, oligosaccharides and polysaccharides, carbonyl, carboxyl, carboxylate (including salts thereof, such as Group I metal or ammonium ion carboxylates), cyclic, heterocyclic, cyano (-CN), ester, alkyl ester, ether, halogen, heteroaryl, hydroxyl, hydroxylamine, oxime (HO-N=), keto, such as aliphatic ketones, nitro, 10 sulfhydryl, sulfonyl, sulfoxide, exomethylene, and combinations thereof. Two or more of the Ri-R₂ substituents, most typically plural Rt substituents, also may be atoms, typically carbon atoms, in a ring system bonded or fused to the compounds having the illustrated general formula. At least one of the Ri-R₂ substituents typically is bonded to a linker or directly to a carrier.

Particular embodiments of the heterobiaryl compounds have the following formula.

<img file="CA2995467C_D0078.tif" />

R1 and R2 are as stated above for the first general formula. Y is oxygen, nitrogen or sulfur, typically nitrogen. If Y is nitrogen, then the formula also can include double 20 bonds to the one or more nitrogen atoms.

Compounds having a single heteroatom are exemplified by phenylquinolines, such as follows.

<img file="CA2995467C_D0079.tif" />

More particular embodiments include aryl substituted haptens, exemplified by the following general formula.

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Ri

<img file="CA2995467C_D0080.tif" />

H-r₃

With reference to this general formula, R1-R3 are as indicated above. More typically, Ri is hydrogen, R₂ is acyl, and Rj is alkoxy. A particular example, 2-(3,4dimethoxyphenyl)quinoline-4-carboxylic acid, is provided below.

<img file="CA2995467C_D0081.tif" />

Compounds having two heteroatoms are represented by quinoxalines, as indicated by the general formula below.

Ri

<img file="CA2995467C_D0082.tif" />

Rj

A particular example of biaryl-diheteroatom hapten of the present invention is exemplified by 3-hydroxy-2-quinoxalinecarbamide, below. Again, the Ri and R₂substituents are as stated above with respect to this class of haptens.

<img file="CA2995467C_D0083.tif" />

OH

<img file="CA2995467C_D0084.tif" />

OH

11. Azoaryl

Another general class of haptens of the present invention is azoaryl compounds, such as azobenzenes, having a first general chemical formula as below.

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N=N z^R1 r₂—J

RpRj substituents independently are selected from: hydrogen, acyl, aldehydes, alkoxy, aliphatic, particulary lower aliphatic, substituted aliphatic, heteroaliphatic, e.g., organic chains having heteroatoms, such as oxygen, nitrogen, sulfur, alkyl, particularly alkyl having 20 or fewer carbon atoms, and even more typically lower alkyl having 10 or fewer atoms, such as methyl, ethyl, propyl, isopropyl, and butyl, substituted alkyl, such as alkyl halide (e.g. -CXj where X is a halide, and combinations thereof, either in the chain or bonded thereto,), oxime, oxime ether (e.g., methoxyimine, CHj-O-N=) alcohols (i.e. aliphatic or alkyl hydroxyl, particularly lower alkyl hydroxyl) amido, amino, amino acid, aryl, alkyl aryl, such as benzyl, alkoxy aryl, such as methoxy and ethoxy, carbohydrate, monosaccharides, such as glucose and fructose, disaccharides, such as sucrose and lactose, oligosaccharides and polysaccharides, carbonyl, carboxyl, caiboxylate (including salts thereof, such as Group I metal or ammonium ion carboxylates), cyclic, heterocyclic, cyano (-CN), ester, alkyl ester, ether, halogen, heteroaryl, hydroxyl, hydroxylamine, oxime (HO-N=), keto, such as aliphatic ketones, nitro, sulfhydiyl, sulfonyl, sulfoxide, sulfonyl, exomethylene, and combinations thereof. Two ore more Rz substituents also may be atoms, typically carbon atoms, in a ring system bonded or fused to the compounds having the illustrated general formula. For example, 2 Rj substituents may form a fused phenyl ring, or a fused heterocyclic or heteroaryl structure.

Certain disclosed azoaryl compounds have a first amine substituent and a second aryl substituent. These compounds typically have the following fbimula.

<img file="CA2995467C_D0085.tif" />

Aryl

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With refererence to this general formula, R2-R4 are as stated above with respect to this class of haptens, with particular embodiments having R2-R3 aliphatic, particularly alkyl, more particularly lower alkyl, and R4 hydrogen.

A third general formula for describing azoaryl compounds is provided below.

<img file="CA2995467C_D0086.tif" />

R2-R5 are as stated above for this particular class of haptens. At least one of R2-R5 defines a position for coupling a linker or carrier to the azoaryi hapten to form a conjugate. For example, Rj may be a sulfonyl halide functional group. Sulfonyl 10 halides, such as that shown below, are usefol functional groups for coupling linkers to the azoaryl haptens.

<img file="CA2995467C_D0087.tif" />

With reference to this formula, R2-R5 are as stated above. X is a halide. A particular embodiment of these azoaryl haptens, 4-(dimethylamino)azobenzene-4*sulfonyl chloride, has the formula provided below.

<img file="CA2995467C_D0088.tif" />

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12. Benzodiazepines

Another class of haptens according to the present invention is the benzodiazepine haptens, having a first general formula as indicated below.

Rs

<img file="CA2995467C_D0089.tif" />

Ri-Rs independently are selected from: acyl, aldehydes, alkoxy, aliphatic, particulary lower aliphatic, substituted aliphatic, heteroaliphatic, e.g., organic chains having heteroatoms, such as oxygen, nitrogen, sulfur, alkyl, particularly alkyl having 20 or fewer carbon atoms, and even more typically lower alkyl having 10 or fewer atoms, such as methyl, ethyl, propyl, isopropyl, and butyl, substituted alkyl, such as alkyl halide (e.g. -CX₃ where X is a halide, and combinations thereof, either in the chain or bonded thereto,), oxime, oxime ether (e.g., methoxyimine, CH₃-O-N=) alcohols (i.e. aliphatic or alkyl hydroxyl, particularly lower alkyl hydroxyl) amido, amino, amino acid, aryl, alkyl aryl, such as benzyl, carbohydrate, monosaccharides, such as glucose and fructose, disaccharides, such as sucrose and lactose, oligosaccharides and polysaccharides, carbonyl, carboxyl, caiboxylate (including salts thereof, such as Group I metal or ammonium ion carboxylates), cyclic, cyano (CN), ester, ether, exomethylene, halogen, heteroaryl, heterocyclic, hydrogen, hydroxyl, hydroxylamine, oxime (HO-N=), keto, such as aliphatic ketones, nitro, sulfhydryl, sulfonyl, sulfoxide, and combinations thereof. Two or more of the R$ substituents also may be atoms, typically carbon atoms, in a ring system bonded or fused to the compounds having the illustrated general formula. At least one of the Ri-R₃ positions is bonded to a linker or is occupied by a functional group suitable for coupling to a linker or a carrier molecule. Ri-Rs most typically are aliphatic, aryl, hydrogen, or hydroxyl, even more typically alkyl, hydrogen or phenyl. Y is oxygen or sulfur, most typically oxygen.

Particular embodiments of the benzodiazepine haptens have Ri aryl, as indicated below.

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PCT/US2007/023140 r₅

<img file="CA2995467C_D0090.tif" />

For these embodiments, R_z-Rs are as stated above for this class of haptens, more typically such substituents are independently selected from aliphatic, particular 5 alkyl, hydrogen and hydroxyl. Certain disclosed embodiments are phenyl compounds, as illustrated below.

<img file="CA2995467C_D0091.tif" />

Again, R2-R4 are as stated above, but more typically such substituents are independently selected from aliphatic, particularly alkyl, hydrogen and hydroxyl. Certain disclosed embodiments are phenyl compounds, as illustrated below. A particular embodiment, 4-(2-hydroxyphenyi)-lH-benzo{b](l,4]dia2epine-2(3H)-one, is provided below.

<img file="CA2995467C_D0092.tif" />

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III. Linkers 1. General

As indicated by the general formula hapten-optional linker-earner conjugates of the present application may include linkers. Any linker currently known for this purpose, or developed in the future, can be used to form conjugates of the present invention by coupling to the haptens disclosed herein. Useful linkers can either be homo- or heterobifunclional, but more typically are heterobifunctional.

2. Aliphatic

Solely by way of example, and without limitation, a first class of linkers suitable for forming disclosed hapten conjugates are aliphatic compounds, such as aliphatic hydrocarbon chains having one or more sites of unsaturation, or alkyl chains. The aliphatic chain also typically includes terminal functional groups, including by way of example and without limitation, a carbonyl-reactive group, an amine-reactive group, a thiol-reactive group or a photo-reactive group, that facilitate coupling to haptens and other desired compounds, such as specific binding moieties. The length of the chain can vary, but typically has an upper practical limit of about 30 atoms. Chain links greater than about 30 carbon atoms have proved to be less effective than compounds having smaller chain links. Thus, aliphatic chain linkers 20 typically have a chain length of from about 1 carbon atom to about 30 carbon atoms.

However, a person of ordinary skill in the art will appreciate that, if a particular linker has greater than 30 atoms, and still operates efficiently for linking the hapten to a carrier molecule coupling unit, and the conjugate still functions as desired, then such chain links are still within the scope of the present invention.

3. Alkylene Oxides

A second class of linkers useful for practicing the present invention are the alkylene oxides. The alkylene oxides are represented herein by reference to glycols, such as ethylene glycols. Hapten conjugates of the present invention have proved 30 particularly useful if the hydrophilicity of the linker is increased relative to their hydrocarbon chains. As a result, the alkylene oxides, such as the glycols, have proved useful for practicing this invention. A person of ordinary skill in the art will

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PCT/ÜS2007/023140 appreciate that, as the number of oxygen atoms increases, the hydrophilicity of the compound also may increase. Thus, linkers of the present invention typically have a formula of (-OCHjCH^O-),, where n is from about 2 to about 15, but more particularly is from about 2 to about 8.

Heterobifunctiona! polyalkyleneglycol linkers useful for practicing certain disclosed embodiments of the present invention are described in assignee’s copending applications, including Nanoparticle Conjugates, U.S. Patent Application No.l 1/413,778, filed April 28,2006; Antibody Conjugates, U.S. Application No. 11/413,415, filed April 27,2006; and “Molecular Conjugate,” U.S. Provisional Patent Application No. 60/739,794, filed November 23,2005.

A person of ordinary skill in the art will appreciate that the linkers disclosed in these applications can be used to link specific binding moieties, signal generating moieties and haptens in any and all desired combinations. Heterobifimctional polyalkyleneglycol linkers are disclosed below, and their use exemplified by reference to coupling specific binding moieties, sudt as antibodies and nucleic acids, to haptens and detectable labels. In particular, conjugates of anti-hapten antibodies and detectable labels and conjugates of primary antibodies or nucleic acids with haptens are exemplified below.

One particular embodiment of a linker for use with disclosed conjugates is a heterobifimctional polyalkyleneglycol linker having the general structure shown below:

<img file="CA2995467C_D0093.tif" />

wherein A and B include different reactive groups, x is an integer from 2 to 10 (such as 2,3 or 4), and y is an integer from 1 to 50, for example, from 2 to 30 such as from 3 to 20 or from 4 to 12. One or more hydrogen atoms can be substituted for additional functional groups such as hydroxyl groups, alkoxy groups (such as methoxy and ethoxy), halogen atoms (F, Cl, Br, I), sulfate groups and amino groups (including mono- and di-substituted amino groups such as dialkyi amino groups.

A and B of the linker can independently include a carbonyl-reactive group, an amine-reactive group, a thiol-reactive group or a photo-reactive grouHeerop, but are not the same. Examples of carbonyl-reactive groups include aldehyde- and

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PCT/US2007/023I40 ketone-reactive groups like hydrazine derivatives and amines. Examples of aminereactive groups include active esters such as NHS or sulfo-NHS, isothiocyanates, isocyanates, acyl azides, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, anhydrides and the like. Examples of thiol5 reactive groups include non-polymerizable Michael acceptors, haloacetyl groups (such as iodoacetyl), alkyl halides, maleimides, aziridines, acryloyl groups, vinyl sulfones, benzoquinones, aromatic groups that can undergo nucleophilic substitution such as fluorobenzene groups (such as tetra and pentafluorobenzene groups), and disulfide groups such as pyridyl disulfide groups and thiols activated with Ellman’s 10 reagent. Examples of photo-reactive groups include aryl azide and halogenated aiyl azides. Alternatively, A and/or B can be a functional group that reacts with a specific type of reactive group. For example, A and/or B can be an amine group, a thiol group, or a carbonyl-containing group that will react with a corresponding reactive group (such as an amine-reactive group, thiol-reactive group or carbonyl15 reactive group, respectively) that has been introduced or is otherwise present on a hapten and/or a carrier. Additional examples of each of these types of groups will be apparent to those skilled in the art. Further examples and information regarding reaction conditions and methods for exchanging one type of reactive group for another are provided in Hermanson, “Bioconjugate Techniques,” Academic Press, 20 San Diego, 1996. In a particular embodiment, a thiol-reactive group is other than vinyl sulfone.

In some embodiments, a thiol-reactive group of the heterobifunctional linker is covalently attached to a specific-binding moiety and an amine-reactive group of the heterobifunctional linker is covalently attached to an amine-reactive group of a 25 hapten derivative (such as an activated ester formed by reacting a carboxylic acid group with SMCC), the nanoparticle, or vice versa. For example, a thiol-reactive group of the heterobifunctional linker can be covalently attached to a cysteine residue (such as following reduction of cystine bridges) of the specific-binding moiety or a thiol-reactive group of the heterobifiinctional linker can be covalently 30 attached to a thiol group that is introduced to the specific-binding moiety, and the amine-reactive group is attached to an activated hapten derivative having an amine reactive group such as an activated ester. Where the conjugate includes an anti

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PCT/ÜS2007/023140 hapten antibody conjugated to a detectable label, a thiol-reactive group of the heterobifiinctional linker can be covalently attached to the antibody and an amine reactivde group of the heterobifunctional linker can be covalently attached to the antibody and an amine reactive group of the heterobifunctional linker can be covalently attached to the detectable label or vice versa.

Alternatively, an aldehyde-reactive group of the heterobifiinctional linker can be covalently attached to a specific binding moiety and a either a functional group or a different reactive group of the linker is attached to a hapten. Where the specific binding moiety is an anti-hapten antibody and the antibody is conjugated to a 10 detectable label, an aldehyde-reactive group of the heterobifunctional linker can be covalently attached to the antibody and an amine-reactive group of the heterobifiinctional linker can be covalently attached to the detectable label, or vice versa. In a particular embodiment, an aldehyde-reactive group of the heterobifiinctional linker can be covalently attached to an aldehyde formed on a 15 glycosylated portion of an anti-hapten antibody, and an amine-reactive group of the linker is attached to the detectable label. In yet other embodiments, an aldehydereactive group of the heterobifiinctional linker is covalently attached to the antihapten antibody and a thiol-reactive group of the heterobifiinctional linker is attached to the detectable label, or vice versa.In yet other embodiments, an aldehyde-reactive group of the heterobifiinctional linker is covalently attached to the specific-binding moiety and a thiol-reactive group of the heterobifiinctional linker is attached to the nanoparticle, or vice versa.

In some embodiments the heterobifiinctional linker has the formula:

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A---X---[(CH₂)_x-o]--Y-----B wherein A and B are different reactive groups and are as stated above; x and y are as stated above, and X and Y are additional spacer groups, for example, spacer groups having between 1 and 10 carbons such as between 1 and 6 carbons or between 1 and

4 carbons, and optionally containing one or more amide linkages, ether linkages, ester linkages and the like. Spacers X and Y can be the same or different, and can be straight-chained, branched or cyclic (for example, aliphatic or aromatic cyclic structures), and can be unsubstituted or substituted. Functional groups that can be substituents on a spacer include carbonyl groups, hydroxyl groups, halogen (F, Ci, 10 Br and I) atoms, alkoxy groups (such as methoxy and ethoxy), nitro groups, and sulfate groups.

In particular embodiments, the heterobifunctional linker comprises a heterobifunctional polyethylene glycol linker having the formula:

<img file="CA2995467C_D0094.tif" />

whereinn = l to50,forexample,n=2to30suchasn = 3to20orn = 4to 12. In more particular embodiments, a carbonyl of a succinimide group of this linker is covalently attached to an amine group on a detectable label and a maleitnide group of the linker is covalently attached to a thiol group of an anti-hapten antibody, or vice versa. In other more particular embodiments, an average of between about 1 and about 10 specific-binding moieties are covalently attached to a nanoparticle, such as semiconductor nanocrystals (such as quantum dots, obtained for example, from Invitrogen Corp., Eugene, OR; see, for example, U.S. Patent Nos. 6,815,064, 6,682,596 and 6,649,138, paramagnetic nanoparticles, metal nanoparticles, and superparamagnetic nanoparticles.

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In other particular embodiments, the heterobifunctional linker comprises a heterobifunctional polyethylene glycol linker having the formula:

<img file="CA2995467C_D0095.tif" />

wherein m - 1 to 50, for example, m= 2 to 30 such as m = 3 to 20 or m = 4 to 12. In more particular embodiments, a hydrazide group of the linker is covalently linked with an aldehyde group of an anithapten antibody and a maleimide group of the linker is covalently linked wtih a thiol group of a detectable label, or vice versa. In even more particular embodiments, the aldehyde group of the specific-binding moiety is an aldehyde group formed in an Fc portion of an anti-hapten antibody by 10 oxidation of a glycosylated region of the Fc portion of the antibody. In other even more particular embodiments, an average of between about 1 and about 10 antihapten antibodies are covalently attached to a nanoparticle. Briefly, maleimide/hydrazide PEG-linkcrs of the formula above can be synthesized from corresponding maleimide/active ester PEG linkers (which are commercially available, for example, from Quanta Biodesign, Powell, OH) by treatment with a protected hydrazine derivative (such as a Boc-protected hydrazine) followed by treatment with acid.

A conjugate of a specific binding moiety (SBM) and one or more of the disclosed haptens is provided. The SBM in these conjugates can indude, for 20 example, an antibody, a nucleic acid, a lectin or an avidin such as streptavidin. If the SBM includes an antibody, the antibody can specifically bind any particular molecule or particular group of highly similar molecules, for example, an antibody that specifically binds a particular protein that may be present in a sample. Alternatively, the antibody can be an anti-antibody antibody that can be used as a 25 secondary antibody in an immunoassay. For example, the antibody can comprise an anti-IgG antibody such as an anti-mouse IgG antibody, an anti-rabbit IgG antibody or an anti-goat IgG antibody.

In particular embodiments, a disclosed antibody conjugate has the formula:

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<img file="CA2995467C_D0096.tif" />

wherein Ab is an antibody, n = 1 to 50 (such as n = 2 to 30, n = 3 to 20 or n = 4 to 12) and j = 1 to 10 (such as j = 2 to 6 or j = 3 to 4). X is a spacer group suitable for spacing the hapten from the remainder of the conjugate and allowing the hapten to be coupled to the remainder of the conjugate. For example, a spacer group may be an aliphatic or aromatic group, typically an aliphatic group, and even more typically an alkyl or substituted alkyl group having from about 1 to about 10 carbon atoms, such as between 1 and 6 carbons or between 1 and 4 carbons. The spacer also may include atoms other than carbon, such as heteroatoms, including but not limited to, halides, nitrogen, oxygen, sulfur, and combinations thereof. Such additional atoms can define functional groups. For example, the spacer group optionally may include one or more amide linkages, ether linkages, ester linkages, amine linkages and the like. The structure of the spacer will depend on the chemistry used to couple the hapten to the linker, and specific examples of such linkages are later dicussed with regard to specifically disclosed haptens, but in general the group X can, for example, be formed by reacting an amine on the linker with an amine reactive group added to the hapten (or vice versa) or a carbonyl on the linker with a carbonyl reactive group added to the hapten (or vice versa.

Alternatively a conjugate of an antibody with one or more of die disclosed haptens can have the following formula:

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<img file="CA2995467C_D0097.tif" />

wherein Ab is an antibody, m= 1 to 50 (such as m = 2 to 30, m = 3 to 20 or n = 4 to 12) and k = 1 to 10 (such as k = 2 to 6 or kj = 3 to 4) and X is again a spacer group, for example, a spacer group having between 1 and 10 carbon atoms, such as between 1 and 6 carbons or between I and 4 carbons, and optionally containing one or more amide linkages, ether linkages, ester linkages, amine linkages and the like.

In other embodiments, the specific binding moiety linked to one or more haptens is a nucleic acid. In a particular embodiment, such a conjugate can have the fonnula:

<img file="CA2995467C_D0098.tif" />

wherein Nuc is any nucleic acid base containing compound, including a nucleoside, nucleotide, nucleotide phosphate (such as a nucleotide triphosphate), an oligonucleotide, or a polynucleotide, and m can be, for example, from about I to 500 such as m = 1 to 100 or m -1 to 50) and X is yet again a spacer group, for example,. a spacer group having between 1 and 10 carbon atoms, such as between 1 and 6 carbons or between 1 and 4 carbons, and optionally containing one or more amide linkages, ether linkages, ester linkages, amine linkages and the like.

Also provided is a conjugate of an antibody that specifically binds a disclosed hapten. In particular embodiments, such a conjugate can have the following fonnula:

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<img file="CA2995467C_D0099.tif" />

wherein AHAb is an anti-hapten antibody, DL is a detectable label such as an enzyme„n= 1 to50(suchasn~2to30,n = 3 to20orn = 4to 12)ando= I to 10 (such as o = 2 to 6 or o = 3 to 4); or

<img file="CA2995467C_D0100.tif" />

wherein AHAb is an anti-hapten antibody, DL is a detectable label such as a nanoparticle,n = 1 to 50(such as n = 2 to30, n == 3 to20orn = 4to 12) and p= 1 to 10 (such asp= 2 to 6 or p = 3 to 4).

In yet other particular embodiments, a disclosed conjugate comprises a conjugate having the formula:

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<img file="CA2995467C_D0101.tif" />

wherein AHAb is an anti-hapten antibody, DL is a detectable label such as a nanoparticle, n = 1 to50(such asn-2 to 30,n = 3 to 20 or n« 4 to 12) and q = 1 to 10 (such as q - 2 to 6 or q = 3 to 4); or

<img file="CA2995467C_D0102.tif" />

wherein AHAb is an anti-hapten antibody, DL is a detectable label such as an enzyme and n = 1 to 50 (such as n = 2 to 30, n = 2 to20orn = 4 to 12) and r= 1 to 10 (such as r = 2 to 6 or r = 3 to 4).

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In still other particular embodiments, a heterobitunctional PEG-linked specific-binding moiety-nanoparticle conjugate comprises a conjugate having the formula:

<img file="CA2995467C_D0103.tif" />

wherein AHAb is an anti-hapten antibody, DL is a detectable label such as an enzyme, m = 1 to 50 (such as m = 2 to 30, m = 3 to 20 or m = 4 to 12) and s = 1 to 10 (such as s = 2 to 6 or s = 3 to 4); or

<img file="CA2995467C_D0104.tif" />

wherein AHAb is an anti-hapten antibody, DL is a detectable label such as a nanoparticle, m = 1 to 50 (such as m = 2 to 30,2 to 20 or 4 to 12) and t = 1 to 10 (such as t = 2 to 6 or t = 3 to 4).

In still further particular embodiments, a heterobifunctional PEG-linked specific-binding moiety-nanoparticle conjugate comprises a conjugate having the formula:

<img file="CA2995467C_D0105.tif" />

wherein AHAb is an antihapten antibody, DL is a detectable label, m = 1 to 50 (such asm = 2 to30, m = 3 to 20or m = 4 to 12)and u = 1 to 10 (suchasu = 2 to 6oru = 3 to 4); or

<img file="CA2995467C_D0106.tif" />

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PCT/US2007/023140 wherein SBM is a specific-binding moiety, NP is a nanoparticle, m = 1 to 50 (such asm = 2 to 30,m = 2 to 20orm = 4 to 12) and v = 1 to 10 (such as v = 2 to 6 or v = 3 to 4).

Disclosed conjugates can be utilized for detecting one or more molecules of interest in a biological sample in any type of assay, including immunohistochemical assays and in situ hybridization assays.. In one embodiment, the disclosed conjugates are used as a hapten-labeled antibody in an immunoassay, for example, a hapten-labeled primary antibody directed to a particular molecule that is then contacted with an anti-hapten antibody conjugate including a detectable label. Alternatively, a hapten-labeled nucleic acid probe bound to a target nucleic acid is then contacted with an anti-hapten antibody conjugate including a detectable label. The biological sample can be any sample containing biomolecules (such as proteins, nucleic acids, lipids, hormones etc.), but in particular embodiments, the biological sample includes a tissue section (such as obtained by biopsy) or a cytology sample (such as a Pap smear or blood smear). Other types of assays in which the disclosed conjugates can be used are readily apparent to those skilled in the art, and particular examples are discussed below.

In another aspect, a method is disclosed for preparing a specific-binding moiety-hapten conjugate, the method including forming a thiolated specific-binding moiety from a specific-binding moiety; reacting a hapten having an amine group with a malcimide/active ester bifunctional linker to form an activated hapten; and reacting the thiolated specific-binding moiety with the activated hapten to foim the specific-binding moiety-hapten conjugate.

A thiolated specific-binding moiety can be formed by reacting the specificbinding moiety with a reducing agent to form the thiolated specific-binding moiety, for example, by reacting the specific-binding moiety with a reducing agent to form a thiolated specific-binding moiety having an average number of thiols per specificbinding moiety of between about 1 and about 10. The average number of thiols per specific-binding moiety can be determined by titration. Examples of reducing agents include reducing agents selected from the group consisting of 2mercaptoethanol, 2-mercaptoethylamine, DTT, DTE and TCEP, and combinations thereof. In a particular embodiment the reducing agent is selected from the group

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PCT/ÜS2007/023140 consisting of DTT and DTE, and combinations thereof, and used at a concentration of between about 1 mM and about 40 mM.

Alternatively, forming the thiolated specific-binding moiety includes introducing a thiol group to the specific-binding moiety. For example, the thiol 5 group can be introduced to the specific-binding moiety by reaction with a reagent selected from the group consisting of 2-lminothiolane, SATA, SATP, SPDP, N~ Acetylhomocysteinethiolactone, SAMSA, and cystamine, and combinations thereof (see, for example, Hermanson, “Bioconjugate Techniques,” Academic Press, San Diego, 1996. In a more particular embodiment, introducing the thiol group to the specific-binding moiety includes reacting the specific-binding moiety with an oxidant (such as periodate) to convert a sugar moiety (such as in a glycosylated portion of an antibody) of the specificbinding moiety into an aldehyde group and then reacting the aldehyde group with cystamine. In another more particular embodiment, the specific binding moiety includes streptavidin and introducing the thio! group comprises reacting the streptavidin with 2-iminothiolane (Traut reagent).

In other particular embodiments, reacting the hapten with a maleimide/active ester bifiuictional linker to form an activated nanoparticle includes reacting the hapten with a PEG maleimide/active ester having the formula:

<img file="CA2995467C_D0107.tif" />

wherein n = 1 to 50, for example, n = 2 to 30 such as n=3 to 20 or n=4 to 12.

In a further aspect, a method is disclosed for preparing a specific-binding moiety-hapten conjugate composition that includes reacting a specific-binding moiety with an oxidant to form an aldehyde-bearing specific-binding moiety;

reacting the aldehyde-bearing specific-binding moiety with a malcimide/hydrazide bifunctional linker to form a thiol-reactive specific-binding moiety; and reacting the thiol-reactive specific-binding moiety with a thiolated hapten to form the specificbinding moiety-nanoparticle conjugate. In à particular embodiment, the specificbinding moiety is an antibody and reading the specific-binding moiety with an

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PCT/US2007/023140 oxidant to form the aldehyde-bearing specific-binding moiety includes oxidizing (such as with periodate, h, Br₂, or a combination thereof, or neuramidase/ galactose oxidase) a glycosylated region of the antibody to form the aldehyde-bearing antibody. In a more particular embodiment, reacting an antibody with an oxidant to 5 form an aldehyde-bearing antibody includes introducing an average of bet ween about 1 and about 10 aldehyde groups per antibody, in a more particular embodiment, the maleimide/hydrazide bi functional linker has the formula:

<img file="CA2995467C_D0108.tif" />

wherein m = 1 to 50, for example, m = 2 to 30 such as m=3 to 20 or m = 4 to 12. A 10 thiolated hapten can be formed from a hapten by introducing a thiol group to the hapten (for example, by reacting a hapten with a reagent selected from the group consisting of 2-lminothiolane, SATA, SATP, SPDP, NAcetylhomocysteinethiolactone, SAMSA, and cystamine, and combinations thereof).

In other embodiments, a hapten-linker conjugate having a hydrazide reactive group is reacted with a carbonyl group of an aldehyde formed on an antibody to form a hapten-linker-antibody conjugate. Hapten-linker conjugates having hydrazide reactive group are discussed further below.

4. Commercially Available Linkers

Additional linkers also are commercially available. Pierce Biotechnology Inc., of Rockford, Illinois, provides certain linkers that are useful for practicing the present invention. For example, Pierce provides sulfosuccinimidyl-4-(Nmaleimidomethyl) cyclohexane-l-carboxylate(suIfo-SMCC). Pierce also sells a 25 sutfonamidyl compound without the sulfo group, referred to as SMCC, which also is a useful linker. Sulfo-SMCC is a water soluble, non-cleavable membrane, impermeable cross-linker. NH esters of this compound can react readily with primary amines at pH 7-9 to form stable amide bonds. Malemides react with sulfydryl groups at pH between about 6 to 8, more typically from about 6.5 to about

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7.5, to form stable thioether bonds. For use in coupling the haptens to a carrier comprising a free amine, the sulfo-SMCC can react with the free amine of the carrier to provide a malemide-activated carrier. The carrier type compound then can be reacted with a hapten, such as a hapten having a free sulfidryl group or hydroxyl 5 group, to form a conjugate according to the present invention.

Pierce also provides additional exemplary linkers, as well as additional information concerning the length for each potentially suitable for practical use.. For example, for functional groups reactive with amines, Pierce provides the following compounds: EGS (ethylene glycol bisfsuccinimidylsuccinate]); Sulfo10 EGS (ethylene glycol Zrô[sulfosuccinimidylsuccinate]); DTSSP (3,3 dithiobûfsulfosuccinimidylpropionate]); DSS (disuccinimidyl suberate); BS (bzsfsulfosuccinimidyl] suberate); DSG (disuccinimidyl glutarate); and MSA (methyl N-succinimidyl adipate. Examples of sulfhydryl reactive linkers include DPDPB (l,4-di-[3’-(2'-pyridyldithio)-propionamido]butane); BM[PEO)j(l,l 1-bis15 maleimidotriethyleneglycol); BMH (bis-maleimidohexane); BM[PEOj₂(l,8-0tsmaleimîdodiethyleaeglycol); HBVS (1,6-hexane-bîs-vinylsulfone); DTME (dithiobzs-maleimidoethane); BMDB (l,4-b/s-maleimidyl-2,3-dihydroxybutane); BMB (1,4-bis-maleimidiobutane; and BMOE (bis-maleimidoethane). Photoreactive compounds also are available from Pierce, including BASED (b£s-(b-(420 azido$alicylamido)ethyl]disulfide) and APG (p-azidophenyl glyoxal monohydrate).

5. Carbodiimide Coupling

Carbodiimides [R-N=C=N-Ril can be used to couple haptens directly to target molecules, including amino acids, proteins, nucleotides, and oligonucleotides. 25 See, for exampl e, New Biotinylating Reagent Utilizing Carbodiimide Function, Nucleic Acid Symposiums Series, No. 34,69-70 (1995).

Alternatively, carbodiimide functionalities can be incorporated into or coupled to a linker as discussed above for forming hapten-linker conjugates. A general synthetic scheme for making hapten-dPEGx-carbodiimides is provided 30 below in the working examples. Using this general synthetic scheme, several working embodiments of hapten-carbodiimides have been made, including nitropyrazole-dPEGx-carbodiimide, benzofurazan-dPEGx-carbodiimide,

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PCT/US2007/023140 dinitrophenyl-dPEGx-carbodiimidc, thiazolesulfonamide-dPEGx-carbodiimideand rotenoid-dPEGx-carbodiimide.

IV. Hapten-Linker Conjugates

Compounds of the present invention, also referred to as conjugates, typically comprise a hapten typically coupled to a linker. The hapten conjugate also may indude a carrier, such as a polypeptide, protein, mononucleotide, dinucleotide, trinucleotide, oligonucleotide, or nucleic acid(s), either coupled directly to the hapten, or coupled to linker. Particular examples of carriers indude immunogenic carriers, antibodies and nucleic acid probes. The hapten, carrier and/or linker may include one or more functional groups or moieties, typically electrophiles and electrophile/nucleophile pairs that are useful for coupling a hapten to a carrier, either directly or indirectly through a linker. Thus, a first general formula describing certain embodiments of the present disclosure is hapten-carrier. Such compounds also optionally, and most typically, include a linker. Embodiments having a linker satisfy the formula hapten-linker-carrier. A combined formula therefore is (haptenjk-flinkerjm-carriera where k is 1, m and n are 0 or l,and at least one of m or n is 1. A person of ordinary skill in the art will understand that, for the general formula, k=l, m=l or n=l implies no limitation on the number or structure of the hapten, the linker or the carrier. For example, a carrier can have multiple linkers attached and the multiple linkers can be attached to multiple haptens to provide a conjugate of the combined formula. Furthermore, a linker can include plural subunits or be formed from various subcomponents. For example, both a carrier and a hapten can include attached linkers, wherein the linkers can then be reacted to couple the hapten and the carrier together.

In a particular embodiment, a conjugate according to the disclosure has the general structure (specific-binding moiety)-linker-hapten, and more particularly (specific-binding moiety)-(iinker-hapten)p where p=l-200, for example p= 1-50 such as 1 -10. In one example, the linker comprises a PEG linker. In more particular embodiments, the specific binding moiety is an antibody or a nucleic acid. In another example, the specific-binding moiety is an antibody and the linker includes a carbonyl reactive group covalently linked to an aldehyde group of an oxidized sugar

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PCT/US2007/023140 moiety of an Fc region of the antibody. In yet another example, the specific-binding moiety is an antibody and the linker includes a sulfhdryl reactive group covalently linked to a thiol group of the antibody, wherein the thiol group is generated by reducing a disulfide bond in the antibody. In still another example, the specific binding moiety is a nucleic acid and the linker includes a carbonyl reactive group covalently linked to a cytosine residue of the nucleic add.

In another particular embodiment, a conjugate according to the disclosure has the general structure hapten-linker-RG, wherein RG refers to a reactive group, such as a carbonyl reactive group, a thiol reactive group or an amine reactive group.

Although typically one hapten will be linked to one linker bearing a reactive group, it is possible to have multiple haptens attached to one linker haring a reactive group, or to have multiple linkers having reactive groups attached to one hapten. Hapten linker conjugates such as these are particularly useful for attaching a hapten to an antibody (such as discussed in the previous paragraph) and also for attaching a hapten to an immunogenic carrier such as KLH to provide an immunogen that can be used to stimulate an animal to produce an antibody that specifically binds to the hapten. Thus, an antibody that specifically binds to a hapten is an aspect of the disclosure.

Iri yet another aspect, a conjugate is disclosed that includes an anti-hapten antibody (such as can be produced using a disclosed immunogen) and a detectable label (such as a quantum dot or enzyme). Thus, a general fonnula for this type of conjugate is (anti-hapten antibody^ - (detectable label), where t and s can each independently be 1-100, but more typically, t = 1 ands= 1-10. Conjugates of antihapten antibodies with detectable labels can be used in conjunction with other hapten-carrier conjugates (such as hapten labeled nucleic acid probes for target genomic sequences and hapten-labeled primary antibodies that specifically bind to target proteins) to allow multiplexed assays of multiple targets in a single sample.

Conjugates of the present invention may be formed by coupling a disclosed exemplary linker or linkers to a disclosed hapten or haptens. Many of the haptens 30 have plural locations to which a linker may be coupled. Suitable linker positions with respect to the general formulae provided for disclosed haptens are indicated below, as are general formulae for hapten-linker conjugates. Particular hapten99

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PCT/US2007/023140 linker conjugates are provided by reference to PEG linkers, as are protocols for synthesizing these compounds.

1. Oxazoles and Pyrazoles

A first general class of haptens of the present invention are oxazoles and pyrazoles, most typically nitro oxazoles and nitro pyrazoles, having the following general chemical formula, as discussed in more detail herein.

^r2 _xXx^xR3^x, ».

Ri

R&lt;

Any one or more of the R1-R4 positions can be coupled to a linker. The position occupied by the R? substituent is quite suitable for coupling linkers to this class of haptens, as indicated below, where L is a linker and RG is a reactive functional group.

<img file="CA2995467C_D0109.tif" />

Hapten-linker conjugates have been formed using PEG-based linkers. One example of such a compound, 5-nitro-3-pyrazole carbamide, is shown below. For this and subsequent embodiments, X is from about 2 to about 24.

<img file="CA2995467C_D0110.tif" />

A particular embodiment where X is 4 is provided below.

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Ο ο

<img file="CA2995467C_D0111.tif" />

νο₂

This example satisfies the formula hapten-L-RG where L is a PEG 4 (4 ethylene oxy units) and the reactive group is a carboxylic acid functional group. The carboxylic acid functional group has been converted to other reactive functional groups in 5 working embodiments. For example, the carboxylic acid functional group can be converted to an activated ester, such as an NHS ester, as shown below.

<img file="CA2995467C_D0112.tif" />

And, the activated ester can be converted to other useful reactive functional group, such as a hydrazide, as illustrated below.

<img file="CA2995467C_D0113.tif" />

/

2. Nitroaryl

A second general class of haptens of the present invention are nitroaryl compounds having the following general chemical formula.

<img file="CA2995467C_D0114.tif" />

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Such compounds have at least one, and optionally plural, nitro groups so that at least one of R|-Rj is nitro. Any of the Rj-R^ positions not coupled to a nitro group is a potential position for coupling linkers to the aryl ring. Mononitrophenyl compounds are represented by nitrocinnamide hapten conjugates as illustrated below.

H₃CO

NO, _H

I

Yr

H3CO

<img file="CA2995467C_D0115.tif" />

O

Working embodiments also are exemplified by 2,4-dinitrophenyl compounds. Exemplary hapten conjugates of this class are illustrated below, where R1-R3 are as stated above.

<img file="CA2995467C_D0116.tif" />

Hapten-linker conjugates have been formed using PEG-based linkers, of such a compound is shown below.

One example

<img file="CA2995467C_D0117.tif" />

This example therefore satisfies the formula hapten-L-RG where L is a PEG 4 (4 ethylene oxy units) and the reactive group is a carboxylic acid functional group. The carboxylic acid functional group has been converted to other reactive functional

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PCT/ÜS2007/023140 groups in working embodiments. For example, the carboxylic acid functional group can be converted to an activated ester, such as an NHS ester, as shown below.

<img file="CA2995467C_D0118.tif" />

The activated ester can be converted to other useful reactive functional group, such as a hydrazide, as illustrated below.

<img file="CA2995467C_D0119.tif" />

3. Benzofurazan and Related Compounds

Benzofurazans and derivatives thereof are in another class of haptens of the present invention. A general formula for the benzofurazan-type compounds is provided below.

<img file="CA2995467C_D0120.tif" />

The R1-R4 and Y substituents are as stated above. At least one of the R1-R4 substituents is bonded to a linker, to a carrier, or is a functional group suitable for coupling to a linker or a carrier. The R₂ and Rj positions are most likely used to couple the linker to this class of haptens (R₂ and R₃ may be substantially identical in terms of reactivity, particularly if R| and R« are the same). Such hapten conjugates are exemplified by the general formula provided below.

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<img file="CA2995467C_D0121.tif" />

Hapten-linker conjugates have been formed using PEG-based linkers. One example of such a compound, 2,1,3-benzoxadiazole-5-carbamide, is shown below. O

<img file="CA2995467C_D0122.tif" />

A particular embodiment had the following formula.

O

<img file="CA2995467C_D0123.tif" />

Ulis example satisfies the formula hapten-L-RG where L is a PEG 4 (4 ethylene oxy units) and the reactive group is a carboxylic acid functional group. The carboxylic acidfpnctional group has been converted to other reactive functional groups in working embodiments. For example, the carboxylic acid functional group can be converted to an activated ester, such as an NHS ester, as shown below.

<img file="CA2995467C_D0124.tif" />

The activated ester can be converted to other useful reactive functional group, such as a hydrazide, as illustrated below.

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Ο Ο

<img file="CA2995467C_D0125.tif" />

4. Triterpenes

Tri terpenes are another class of haptens within the scope of the present invention. The basic ring structure common to the triterpenes has four sixmembered fused rings, A-D, as indicated below, where theRj-Rji and Y substituents are as stated above.

<img file="CA2995467C_D0126.tif" />

Disclosed embodiments of triterpenes exemplifying this class of haptens also may include an E ring, that can be of various ring sizes. For example, the E ring might be a 5- or 6-membered ring. Quite often, these compounds include an alpha-beta unsaturated ketone, such as illustrated below for the C ring.

105

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<img file="CA2995467C_D0127.tif" />

A person of ordinary skill in the art will appreciate that many of the positions occupied by the R groups in these general formulae may be useful for coupling the haptens to a linker to form a reactive conjugate. With reference to the alpha-beta 5 unsaturated compounds, particular linker positions ate indicated below using arrows.

<img file="CA2995467C_D0128.tif" />

For example, hapten conjuagates of the present inveiotn A particular reactive conjugate according to this dass has the following formula.

106

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Ο

<img file="CA2995467C_D0129.tif" />

Other exemplary triterpene structures and potential linker coupling positions are provided below.

<img file="CA2995467C_D0130.tif" />

Betulioic, Ursolic and Echinocystic Acid Core Structures

<img file="CA2995467C_D0131.tif" />

Alkyl chain (prenyl derivative), gammalactone ring, or furan

Cucurbitacin Core

Hapten-linker conjugates have been formed using PEG-based linkers. One example of such a compound is shown below.

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<img file="CA2995467C_D0132.tif" />

This example therefore satisfies the formula hapten-L-RG where L is a PEG 4 (4 ethylene oxy units) and the reactive group is a carboxylic acid functional group. The 5 carboxylic acid functional group can be converted to an activated ester, such as an NHS ester, as shown below.

<img file="CA2995467C_D0133.tif" />

The illustrated activated ester has been coupled directly to a protein carrier.

Alternatively, the activated ester could be converted into a different reactive functional group, such as a hydrazide by treatement with a protected, e.g. a BOCprotected hydrazine reagent, if desired.

S. Ureas and Thioureas

Ureas/thioureas, particularly aryl and heteroaryl ureas and thioureas, is another class of haptens within the scope of the present invention. Aryl derivatives typically have the following formula.

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<img file="CA2995467C_D0134.tif" />

At least one of the R3-R7 substituents also may be bonded to a linker, to a carrier, or is a functional group suitable for coupling to a linker and/or to a carrier molecule. Alternatively, the urea/thiourea functional group can be used to couple a linker to this class of disclosed haptens. An exemplary hapten conjugate, with particular reference to thioureas, is provided below.

<img file="CA2995467C_D0135.tif" />

RG

Hapten-linker conjugates have been formed using PEG-based linkers. One example of such a compound is shown below.

OFj

<img file="CA2995467C_D0136.tif" />

CH

This example therefore satisfies the formula hapten-L-RG where L is a PEG 8 (8 ethylene oxy units) and the reactive group is a carboxylic acid functional group. The carboxylic acid functional group can be converted to an activated ester, such as an NHS ester, as shown below.

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<img file="CA2995467C_D0137.tif" />

The activated ester can be converted to other useful reactive functional group, such as a hydrazide, as illustrated below.

CFj

<img file="CA2995467C_D0138.tif" />

Rhodamine thiourea hapten conjugates according to the present invention typically have the following formula.

<img file="CA2995467C_D0139.tif" />

With reference to this formula, R typically is independently selected from hydrogen, 10 aliphatic, particulary alkyl, heteroaliphatic, substituted aliphatic, such as alkyl halide, aryl, heteroaryl, and combinations thereof. R₍ typically is independently selected from hydrogen, aliphatic, particulary alkyl, heteroaliphatic, substituted aliphatic, such as alkyl halide, alcohol, amine, substituted amine, such as lower alkyl amine, one example being diethyl amine, aryl, heteroaryl, halogen, hydroxyl, and no

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PCT/US2007/023140 combinations thereof. Rj typically is independently selected from hydrogen, aliphatic, particulary alkyl, heteroaliphatic, substituted aliphatic, such as alkyl halide, alcohol, amine, substituted amine, aryl, heteroaryl, halogen, hydroxyl, and combinations thereof. Y is oxygen, nitrogen or sulfur. A particular embodiment of 5 a rhodamine B thiourea had the following fonnula.

<img file="CA2995467C_D0140.tif" />

6. Rotenone and Rotenone-based Haptens

Rotenone, and rotenone-based haptens, define another class of haptens within the scope of the present invention. General formulas for rotenone, and rotenone-based haptens, are provided below.

<img file="CA2995467C_D0141.tif" />

Formula 1

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<img file="CA2995467C_D0142.tif" />

Formula 2

Any of the Ri-Rm positions can be used to couple linkers to this class of haptens. Certain particular compounds of Formula 1 have Re and R? form a double bond, such as a double bond to oxyten to form a carbonyl or a double bond to an nitrogen ro form an imine. Specific exemplary linker coupling positions with reference to these hapten conjugates is provided below.

<img file="CA2995467C_D0143.tif" />

A person of ordinary skill in the art will appreciate that the carbonyl compound can be used to bond to the linker. This class of exemplary hapten conjugates is exemplified by the general formulas provided below.

<img file="CA2995467C_D0144.tif" />

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PCT7US2007/023140 the general formulas provided below.

<img file="CA2995467C_D0145.tif" />

One example of a hapten-linker conjugate having a PEG-based linker is shown below.

<img file="CA2995467C_D0146.tif" />

A particular embodiment had the following formula.

<img file="CA2995467C_D0147.tif" />

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This example satisfies the fonnula hapten-L-RG where Lisa PEG 8 (8 ethylene oxy units) and the reactive group is a carboxylic acid functional group. The carboxylic acid functional group can be converted into a different reactive functional group, as desired, such as an activated ester, including the NHS ester shown below.

<img file="CA2995467C_D0148.tif" />

The activated ester can be converted to other useful reactive functional group, such

<img file="CA2995467C_D0149.tif" />

For rotenone isoxazolines, exemplary hapten-linker conjugates had the following fonnula.

<img file="CA2995467C_D0150.tif" />

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7. Oxazole and Thiazole Sulfonamides

Oxazole and thiazole sulfonamides provide another class of haptens within the scope of the present invention. A general formula for oxazole and thiazole sulfonamides is provided below.

<img file="CA2995467C_D0151.tif" />

Any one or more of the R1-R3 positions can be used to couple a linker or carrier to this class of haptens to form hapten conjugates. For certain exemplary working embodiments, Ri has been amido, such as the amide derivatives shown below. For these compounds, the R₂ and Rj positions are suitable for coupling to a linker. R₂, 10 for certain working embodiments, has been -SO₂, and has been used to couple linkers by forming a sulfonamide. Thus, a second general formula for working embodiments of haptens exemplifying this class of haptens is indicated below.

<img file="CA2995467C_D0152.tif" />

Exemplary hapten conjugates based on this general formula include those having the following formula.

<img file="CA2995467C_D0153.tif" />

Hapten-linker conjugates have been formed using PEG-based linkers. One example of such a compound, 2-acetamido-4-methyl-5-thiazolesulfonamide, is shown below.

<img file="CA2995467C_D0154.tif" />

A particular embodiment had the following structure.

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<img file="CA2995467C_D0155.tif" />

This example satisfies the formula hapten-L-RG where L is a PEG 8 (8 ethylene oxy 5 units) and the reactive group is a carboxylic acid functional group. The carboxylic acid functional group can be converted into a different reactive functional group, as desired, such as an activated ester, including the NHS ester shown below.

<img file="CA2995467C_D0156.tif" />

The activated ester can be converted to other useful reactive functional group, such as a hydrazide, as illustrated below.

<img file="CA2995467C_D0157.tif" />

8. Coumarins

Coumarin and coumarin derivatives provide another class of haptens within the scope of the present invention. A general formula for coumarin and coumarin derivatives is provided below.

<img file="CA2995467C_D0158.tif" />

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Any of the Ri-Rê positions also typically is bonded to a linker, to a carrier or is a functional group suitable for coupling to a linker or a carrier molecule. Certain working embodiments have used the position indicated as having an R$ substituent for coupling to linkers. The position occupied by the R« substituent can be important if fluorescence is used to detect these compounds. Substituents other than hydrogen at the position occupied by R$ in the general formula are believed to quench fluorescence, although such derivatives still may be chromophores. Exemplary hapten conjugates made using this formula have the following general formula.

<img file="CA2995467C_D0159.tif" />

RG

R4

Working embodiments typically wen fused A-D ring systems, as indicated below.

R₂ Rl Rl4 Rt3

<img file="CA2995467C_D0160.tif" />

Rg Rio

Hapten conjugates exemplifying this class are provided below.

O

<img file="CA2995467C_D0161.tif" />

O

OH

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<img file="CA2995467C_D0162.tif" />

<img file="CA2995467C_D0163.tif" />

These examples satisfy the formula hapten-L-RG where L is a PEG linker and the reactive group is a carboxylic acid functional group. The carboxylic acid functional group can be converted into a different reactive functional group, as desired, such as 5 an activated ester, including the NHS esters shown below.

<img file="CA2995467C_D0164.tif" />

The NHS esters can be converted to other usefill reactive functional group, such as a 10 hydrazide, as illustrated below.

<img file="CA2995467C_D0165.tif" />

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<img file="CA2995467C_D0166.tif" />

9. Cyclolignans

Cyclolignans provide another class of haptens within the scope of the present 5 invention. A first general formula, discussed herein in detail, is provided below.

Rjt Rs Re ^R3\ -JC Z^R? rirr-Re

JL· JÇ^r9 ^R2 |^ Rio Ri R12

At least one of the R)-R_I2 substituents typically is bonded to a linker or to carrier, or is a reactive functional group capable of reacting with a linker or carrier. At least one of Ru and R| > also often is an aryl group, such as a benzene ring or a substituted IO benzene ring. Exemplary compounds where at least one of Rn and R₎₂ is an aryl group typically have the following general formula, where the R substituents are as stated above.

<img file="CA2995467C_D0167.tif" />

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R9 often is -L-RG, and R16-R20 independently are hydrogen and alkoxy, typically lower alkoxy, such as methoxy, as shown below. The following general molecular formula indicates likely positions for coupling linkers to this class of haptens.

<img file="CA2995467C_D0168.tif" />

Another general formula useful for describing species of compounds within this class is as follows.

<img file="CA2995467C_D0169.tif" />

As with all hapten conjugates of the present invention, at least one of the R substituents typically is bonded to a linker, is a reactive functional group capable of 10 reacting with a linker, or is -L-RG. For example, R? often is -L-RG, as indicted below.

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<img file="CA2995467C_D0170.tif" />

Hapten-L-RG conjugates exemplifying this class are provided below.

<img file="CA2995467C_D0171.tif" />

<img file="CA2995467C_D0172.tif" />

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<img file="CA2995467C_D0173.tif" />

OCH₃

<img file="CA2995467C_D0174.tif" />

OCH3

These examples satisfy the fonnula hapten-L-RG where L is a PEG linker and the reactive group is a carboxylic acid functional group. .The carboxylic acid functional group can be converted into a different reactive functional group, as desired, such as 5 an activated ester, including the NHS esters shown below.

<img file="CA2995467C_D0175.tif" />

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<img file="CA2995467C_D0176.tif" />

och₃

<img file="CA2995467C_D0177.tif" />

_meNHS~>e —

<img file="CA2995467C_D0178.tif" />

nh₂

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<img file="CA2995467C_D0179.tif" />

<img file="CA2995467C_D0180.tif" />

10. Heterobiaryi Hapten Conjugates

Heterobiaryl hapten conjugates provide another class of haptens within the scope of the present invention. This general class of haptens has a first general chemical formula as below.

<img file="CA2995467C_D0181.tif" />

With reference to this general formual, A-D are selected from carbon, nitrogen, 10 oxygen, and sulfur, and most typically are carbon or nitrogen.

<img file="CA2995467C_D0182.tif" />

At least one of the Rt-Rj substituents typically is bonded to a linker or to carrier, or is a reactive functional group capable of reacting with a linker or carrier.

A particular example of a monoheteroatombiaryl hapten conjugate is illustrated below. R typically is hydroxyl or carboxyl. For hydroxyl conjugates, the hydroxyl group can be converted to a halide, and subsequently displaced using an aminocarbodiimide to produce a carbodiimide compound suitable for directly

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PCT/US2007/023140 labeling biomolecules. The carboxyl group can be activated, such as by formation of the acid halide or NHS esta^- for further reaction, such as with a protected hydrazide. The synthesis of these compounds is decribed in further detail below.

<img file="CA2995467C_D0183.tif" />

Another general class of haptens of the present invention is heterobicyclic/biaryl compounds, typically phenyl quinolines and quinoxalines, having a first general chemical formula as below.

<img file="CA2995467C_D0184.tif" />

Ri'Rj substituents are as stated above for this class of haptens. A particular example of a diheteroatombiaryl hapten conjugate is illustrated below.

<img file="CA2995467C_D0185.tif" />

As with the monoheteroatom derivatives, R typically is hydroxyl or carboxyl. These 15 functional groups can be used to form additional conjugates as disclosed herein. R typically is hydroxyl or carboxyl. For hydroxyl conjugates, the hydroxyl group can be converted to a halide, and subsequently displaced using an aminocatbodiimide to produce a carbodiimide compound suitable for directly labeling biomolecules. The carboxy) group can be activated, such as by formation of the acid halide or NHS 20 ester for further reaction, such as with a protected hydrazide. The synthesis of these compounds is decribed in further detail below.

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11. Azoaryl Conjugates

Certain disclosed embodiments of azoaryl hapten conjugates had a fonnula as provided below.

<img file="CA2995467C_D0186.tif" />

Linker or Carrier

R2-R4 are as stated above. The linker or carrier may be, for example, coupled to the azoaryl hapten by reaction with a sulfonyl halide. One embodiment of such a conjugate, 4&lt;dimethylamino)azobenzene-4'-sulfonamide, has the fonnula provided below.

<img file="CA2995467C_D0187.tif" />

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12. Benzodiazepine Conjugates

Particular embodiments of the benzodiazepine haptens have R| aryl, as indicated below, where a linker or carrier is coupled to the aryl group. For example, benzodiazepine hapten linker conjugates have a formula as indicated below.

<img file="CA2995467C_D0188.tif" />

Linker

R2-R5 are as stated above. More typically such substituents are independently selected from aliphatic, particular alkyl, hydrogen and hydroxyl. Certain disclosed embodiments are phenyl compounds, as illustrated below.

<img file="CA2995467C_D0189.tif" />

Again, R2-R4 are as stated above. A particular embodiment, (E>2-(2(2-oxo-23dihydror-lH-benxo{b][l,4]diazepin-4yl)phenoxy)acetamidc, is provided below.

<img file="CA2995467C_D0190.tif" />

R typically is hydroxyl or carboxyl. These functional groups can be used to form additional conjugates as disclosed herein. R typically is hydroxyl or carboxyl. For hydroxyl conjugates, the hydroxyl group can be converted to a halide, and subsequently displaced using an aminocarbodiimide to produce a carbodiimide

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V. Carriers

A person of ordinary skill in the art will recognize that the hapten linker conjugates of the present application also may include a carrier that is coupled to the hapten, or hapten-linker, by a suitable functional group. Carriers can be, by way of 10 example, and without limitation, amino acids, polypeptides, proteins, and portions thereof; nucleosides, nucleotides, nucleotide drains, nucleic acids, DNA, RNA, mRNA, etc.; and combinations thereof. Typically earner molecules are proteins, DNA, RNA, or combinations thereof.

Suitable carriers also are disclosed in published patent documents. For 15 example, Waggoner et al., U.S. patent application number 2004/0057958, discloses additional suitable carriers. Carriers may be used to enhance the immunogenicity of a hapten, or any other antigenic compound that is immunogenic, non-immunogcnic, or weakly immunogenic when not associated with the carrier. Certain properties of potential carriers also can be 20 considered when selecting a particular earner, such as physiochemical qualities including bang non-immunogenic, non-allergenic, non-antigenic, being metabolizable, molecular weight, solubility, particularly in aqueous physiological solutions, such as phosphate buffered saline, for example, and capable of being conjugated (e.g., covalently bound) or associated (e.g., admixed with or associated 25 through charge-charge interactions) with the antigenic compound.

A single carrier can be used, as well as mixtures of different carriers. Different carriers include, for example, polymers of different lengths, such, as, for example, two or more different length homopolymers, as well as mixtures of two or more different carriers or polymers of the invention. Using a single carrier requires 30 produding only one carrier-hapten complex, whereas using multiple carriers obviously is more difficult. Using more than one carrier may be advantageous if the immune response generated against a particular hapten or epitope varies, such as in

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PCT7US2007/023140 magnitude or specificity, for example, depending upon the particular carrier used, and the most optimal carrier is not known or has not yet been experimentally determined.

The carrier may be a synthetic or natural polymer, substantially antigenic, substantially non-antigenic or biodegradable, br both. Examples of suitable polymers useful as carriers include, but are not limited to, poly(diene), a poly(alkene), a poly(acrylic), a poly(methacryiic), a poly(vinyl ether), a polyfvinyl alcohol), a polyfvinyl ketone), a polyfvinyl halide), a polyfvinyl nitrile), a polyfvinyl ester), a polyfstyrene), a polycarbonate), a polyfester), a polyforthoester), a polyfesteramide), a polyfanhydride), a polyfurethane), a polyfamide), a cellulose ether, a cellulose ester, a polyfsaccharide), polyflactide-co-glycolide), a polyflactide), a polyfglycolide), a copolyoxalate, a polycaprolactone, a polyflactideco-caprolactone), a polyfesteramide), a polyorthoester, a polyfa-hydroxybutyric acid), a polyanhydride or a mixture thereof. In another embodiment, the polymers may be a polymer or oligomer derived from the polymerization or oligomerization of at least one monomer selected from an alpha hydroxycarboxylic acid or acids (such as alpha hydroxycarboxylic acid comprises glycolic acid, lactic acid, ahydroxy butyric acid, a-hydroxyisobutyricacid, a-hydroxyvaleric acid, ahydroxyisovaleric acid, a-hydroxy caproic acid, a-hydroxy-a-ethylbutyric add, a20 hydroxyisocaproic acid, a-hydroxy-3-methylvaleric acid, a-hydroxyheptanoic acid, a-hydroxyoctanoic add, a-hydroxydecanoic acid, a-hydroxymysristic add, ahydroxystearic acid, a-hydroxyligoceric add), a lactone (3-propiolactone, tetramethyleneglycolide, b-butyrolactone, 4-butyrolactone, pivalactone), a diene, an alkene, an acrylate, a methacrylate, a vinyl ether, a vinyl alcohol, a vinyl ketone, a vinyl halide, a vinyl nitrile, a vinyl ester, styrene, a carbonate, an ester, an orthoester, an esteramide, an anhydride, a urethane, an amide, a cellulose ether, a cellulose ester, a saccharide, an alpha hydroxycarboxylic add, a lactone, an esteramide, or a mixture thereof.

The polymer may be derived from one or more amino acids, including both homopolymers and heteropolymers thereof. For example, polyglutamate derived from L-glumatic acid, D-glumatic acid or mixtures, e.g., racemates, of these L and D isomers are used. L and/or D glutanyl, aspartly, glycyl, seryl, threonyl, and cysteinyl 129

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PCT/US2007/023140 are all examples of amino acids that may be used. The polymer also may be a block, graft or random copolymer. These include, for example, copolymers containing at least one other amino acid, such as aspartic acid, serine, tyrosine, glycine, ethylene glycol, ethylene oxide, (or an oligomer or polymer of any of these) or polyvinyl alcohol. Glutamic acid may, of course, cany one or more substituents and the polymers include those in which a proportion or all of the glutamic acid monomers are substituted. Particular polymer examples include, but are not limited to, polyflglutamic acid), poly(d-giutamic acid), poly(di-glutamic acid), poly(i-aspartic acid), poly(d-aspartic acid), poly(dl-aspartic acid), poly(l-serine), poly(d-serine), polyolserine), poly(l-tyrosine), poly(d-tyrosine), poly(dl-tyrosine), poly(l-glysine), poly(dglysine), poly(dl-glysine), poly(l-lhreonine), poly(d-threonine), poly(dl-threonine), poly(d-cysteine), poly(l-cysteine), and poly(dl-cysteine). In further embodiments, the polymers are copolymers, such as block, graft or random copolymers, of the above listed poly(amino acids) with polyethylene glycol, polycaprolactone, polyglycolic acid and polylactic acid, as well as poly(2-hydroxyethyl 1-glutamine), chitosan, carboxymethyl dextran, hyaluronic acid, human serum albumin and alginic acid, with poly-glutamic acids being particularly preferred.

The molecular weight of suitable polymers may vary. Typically, however, the molecular weight is from about 1,000 kilodaltons molecular weight to less than 10,000,000 kilodaltons.

Working embodiments exemplifying protein carriers included bovine thyroglobulin, keyhole limpet hemocyanin, or bovine serum albumin.

Hapten conjugates of the present application include a reactive functional groups for coupling the hapten to a carrier, or the hapten to a linker to form a hapten-linker compound. For protein coupling, proteins include various functional groups, typically nucleophiles, that can be coupled to suitable electrophilic functional groups to form hapten conjugates. For example, a free amine (-NH₂) or secondary amine can be used to couple the protein to the hapten or hapten-linker compound to form amides by reaction with carboxylic acids or carboxylic acid derivatives, such as acid halides (-CO), succinimide ester, etc.; alkyl halides can be used to foim amines; carbonyl compounds, such as ketones and aldehydes, can be used to form imines; and combinations thereof. Certain amino acids include other 130

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PCT/US2007/023J40 reactive functional groups suitable for coupling carriers of haptens, including reactive hydroxyl and/or sulfhydryl groups. Exemplary couplings include ester and lactone formation by reaction with a carboxylic acid or carboxylic acid derivative; ether formation; and combinations thereof.

VI. Signal Generating Moieties

Conjugates comprising signal generating moieties, such as conjugates of specific-binding moieties and signal-generating moieties, can be used in assays for detecting specific target molecules in biological samples. The signal-generating 10 portion is utilized to provide a detectable signal that indicates the presence/and or location of the target. Examples of signal-generating moieties include, by way of example and without limitation: enzymes, such as horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, β-galactosidase, β-glucuronidase or β-lactamase. Horseradish peroxidase is widely used as a label for immunoglobulins 15 in many different immunochemistry applications including ELISA, inununobiotting and immunohistochemistry. In addition to other possible disclosed embodiments, HRP can be conjugated to antibodies by several different methods including glutaraldehyde, periodate oxidation, through disulfide bonds, and also via amino and thiol directed cross—linkers. HRP is the smallest and most stable of the three most 20 popular enzyme labels (HRP, alkaline phosphatase, and B-galactosidase) and its glycosylation leads to lower non-specific binding; fluorescent molecules.(such as fluoresceins, coumarins, BODIPY dyes, resorufins, rhodamines; additional examples can be found in The Handbook—A Guide to Fluorescent Probes and Labeling Technologies, Invitrogen Corporation, Eugene, OR), detectable constructs (such as 25 fluorescent constructs like quantum dots, which can be obtained, for example, from Invitrogen Corporation, Eugene, OR; see, for example, U.S. Patent Nos. 6,815,064, 6,682596 and 6,649,138, metal chelates (such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd³⁺) and liposomes (such as liposomes sequestering fluorescent 30 molecules.

When the signal-generating moiety includes an enzyme, a chromagenic compound, fluorogénic compound, or luminogenic compound can be used to

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PCT/US2007/023140 generate a detectable signal (a wide variety of such compounds are available, for example, from Invitrogen, Eugene OR). Particular examples of chromogenic compounds include di-aminobenzidine (DAB), 4-nitrophenylphospate (pNPP), fast red, bromochloroindolyl phosphate (BCIP), nitro blue tétrazolium (NBT),

BCIP/NBT, fast red, AP Orange, AP blue, tetramethylbenzidine (TMB), 2,2*-azinodi-(3-ethylbcnzothiazolinesulphonate] (ABTS), o -dianisidine, 4-chloronaphthol (4CN), nitrophenyl-P-D-galactopyranoside (ONPG), o-phenylenediamine (OPD), 5bromo-4-chloro-3-indolyl-p-galactopyranoside (X-Gal), methylumbelliferyl-p-Dgalactopyranoside (MU-Gal), p-nitorphenyl-a-D-galactopyranoside (PNP), 510 bromo-4-chloro-3-indolyl- β -D-glucuronide (X-Gluc), 3-amino-9-ethyl cafbazol (AEC), fuchsin, iodonitrotetrazolium (INT), tétrazolium blue and tétrazolium violet.

Labeled secondary antibodies can be purchased from a number of sources, such as, but not limited to, Pierce Co. Amersham and Evident Technologies provide a broad range of conjugated antibody possibilities. CyDye, EviTag Quantum Dot, 15 fluorescein (FITC), and rhodamine can be utilized. These conjugates span a variety of applications, colors, and emission ranges. The EviTag Quantum Dots from Evident Technologies offer photo-stability and multicolor fluorescence in a variety of wavelengths, with the advantage over organic fluorophores of improved photostability, color multiplexing, and single source excitation. Each Evitag generates a sharp emission wavelength making them ideal for multiplexing in intact cell environments.

The Amersham CyDyes offer superior photostability over a broad range of pH values.

One type of detectable conjugate is a covalent conjugate of an antibody and a fluorophore. Directing photons toward the conjugate that are of a wavelength absorbed by the fluorophore stimulates fluorescence that can be detected and used to qualitate, quantitate and/or locate the antibody. A majority of the fluorescent moieties used as fluorophores are organic molecules having conjugated pi-electron systems. While such organic fluorophores can provide intense fluorescence signals,

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PCT/US2007/023140 they exhibit a number of properties that limit their effectiveness, especially in multiplex assays and when archival test results are needed.

Organic fluorophores can be photo-bleached by prolonged illumination with an excitation source, which limits the time period during which maximal and/or detectable signals can be retrieved from a sample. Prolonged illumination and/or prolonged exposure to oxygen can permanently convert organic fluorophores into non-fluorescent molecules. Thus, fluorescence detection has not been routinely used when an archival sample is needed.

Chromophoric and/or fluorescent semiconductor nanocrystals, also often referred to as quantum dots, can be used for identifying complexes. Nanocrystalline quantum dots are semiconductor nanocrystalline particles, and without limiting the present invention to use with particle light emitters of a particular size, typically measure 2-10 nm in size (roughly the size of typical proteins). Quantum dots typically are stable fluorophores, often are resistant to photo bleaching, and have a wide range of excitation, wave-length and narrow emission spectrum. Quantum dots having particular emission characteristics, such as emissions at particular wavelengths, can be selected such that plural different quantum dots having plural different emission characteristics can be used to identify plural different targets. Quantum dot bioconjugates are characterized by quantum yields comparable to the brightest traditional dyes available. Additionally, these quantum dot-based fluorophores absorb 10-1000 times more light than traditional dyes. Emission from the quantum dots is narrow and symmetric, which means overlap with other colors is minimized, resulting in minimal bleed through into adjacent detection channels and attenuated crosstalk, in spite of the fact that many more colors can be used simultaneously. Symmetrical and tuneable emission spectra can be varied according to the size and material composition of the particles, which allows flexible and close spacing of different quantum dots without substantial spectral overlap. In addition, their absorption spectra are broad, which makes it possible to excite all quantum dot color variants simultaneously using a single excitation wavelength, thereby minimizing sample autofluorescence.

Furthermore, it has been found that pegylation, the introduction of polyethylene glycol groups onto the quantum dot conduits, can substantially 133

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PC17US2007/023140 decrease non-specific protein.quantum dot interaction. Certain quantum dots are commercially available, such as from Quantum Dot Corp., of Hayward, California, and Invitrogen.

Standard fluorescence microscopes are an inexpensive tool for the detection of quantum dot bioconjugates. Since quantum dot conjugates are virtually photostable, time can be taken with the microscope to find regions of interest and adequately focus on the samples. Quantum dot conjugates are useful any time bright photo-stable emission is required and are particularly usefill in multicolor applications where only one excitation source/filter is available and minimal crosstalk among the colors is required. For example, quantum dots have been used to form conjugates of Streptavidin and IgG to label cell surface markers and nuclear antigens and to stain microtubules and actin (Wu, X. et al. (2003). Nature Biotech. 21,41-46).

As an example, fluorescence can be measured with the multispectral imaging system Nuance™ (Cambridge Research &amp; Instrumentation, Woburn, MA). As another example, fluorescence can be measured with the spectral imaging system SpectrView™ (Applied Spectral Imaging, Vista, CA). Multispectral imaging is a technique in which spectroscopic information at each pixel of an image is gathered and the resulting data analyzed with spectral image-processing software. For example, the Nuance system can take a series of images at different wavelengths that are electronically and continuously selectable and then utilized with an analysis program designed for handling such data. The Nuance system is able to obtain quantitative information from multiple dyes simultaneously, even when the spectra of the dyes are highly overlapping or when they are co-localized, or occurring at the same point in the sample, provided that the spectral curves are different. Many biological materials autofluoresce, or emit lower-energy light when excited by higher-energy light. This signal can result in lower contrast images and data. Highsensitivity cameras without multispectral imaging capability only increase the autofluorescence signal along with the fluorescence signal. Multispectral imaging can unmix, or separate out, autofluorescence from tissue and, thereby, increase the achievable signal-to-noise ratio.

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Haptens can be conjugated to quantum dots, and quantum dot fluorescence can be stimulated, such as by using fluorescence resonance energy transfer (FRET) whereby low-wavelength light stimulates quantum dot fluorescence. Invitrogen has determined that biotin-conjugated quantum dots had a 100-fold lower limit of detection for the biotin derivative biocytin than anti-biotin Alexa Fluor. Fully biotinylated quantum dots were 10-fold less sensitive than quantum dots with 25 percent biotin coverage.

Quantum dot use has so far been limited by their lack of biocompatibility. New advances in surface coating chemistry, however, have helped to overcome these problems. See, for example, Wu, X. et al. Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots, Nature Biolechnol. 21,41-46 (2003); Jaiswal, J. K., Mattoussi, H., Mauro, J. M. &amp; Simon, S. M. Long-term multiple color imaging of live cells using quantum dot bioconjugates, Nature Biotechnol. 21,47—51 (2003); and Dubertret, B. et al. In vivo imaging of quantum dots encapsulated in phospholipid micelles. Science 298,17591762 (2002).

Quantum dots also have been conjugated to biorecognition molecules, Id., such as streptavidin. These conjugates have been used on both fixed cells and tissue sections. In addition, cell-surface proteins and the endocytic compartments of live cells have been labelled with quantum dot bioconjugates.

Fluorescent proteins also can be used as a carrier, or can be coupled to a carrier, to facilitate visualization. For example, green fluorescent protein (GFP) was originally isolated from the light-emitting organ of the jellyfish Aequorea victoria. Chimeric GFP fusions can be expressed in situ by gene transfer into cells, and can be localized to particular sites within the cell by appropriate targeting signals. Spectral variants with blue, cyan and yellowish-green emissions have been. successfully generated from the Aequorea GFP, but none exhibit emission maxima longer than 529 nm. GFP-like proteins have been isolated from Anthozoa (coral animals) that significantly expanded the range of colors available for biological applications. The family of'GFP-like proteins' deposited in sequence databases now includes approximately 30 significantly different members. Fluorescent proteins

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PCT/US2007/023140 refers to proteins that can become spontaneously fluorescent through the autocatalytic synthesis of a chromophore.

Proteins that fluoresce at red or far-red wavelengths (red fluorescent proteins or RFPs) are known. RFPs can be used in combination with other fluorescent proteins that fluoresce at shorter wavelengths for both multicolour labelling and fluorescence resonance energy transfer (FRET) experiments. Commercially available RFPs are derived from two wild-type GFP-like proteins. DsRed (drFPS83) has excitation and emission maxima at 558 nm and 583 nm, respectively. A far-red fluorescent protein was generated by mutagenesis of a chromoprotein that absorbs at

571 nm. HcRedl (Clontech) has excitation and emission maxima at 588 nm and

618 nm, respectively. The fluorescent protein that emits fluorescence at the longest wavelength (without any mutations being introduced) is eqFP611, cloned from the sea anemone Entacmaea quadricolor. This protein absorbs at 559 nm and emits at 611 nm. As many spectral variants have emerged, more investigators are becoming interested in the simultaneous imaging of multiple fluorophores and/or FRET signals.

Fusion proteins also can be used to form hapten conjugates of the present invention. There are at least three points to consider when creating a functional fluorescent protein: the fluorescent protein must fold correctly to fluoresce; the host 20 protein must fold correctly to be functional; and the integrity of the chimeric protein must be maintained.

The length and sequence of any linker between the fluorescent protein and host protein should be optimized for each specific application. The most widely used linker designs have sequences that primarily consist of glycine (Gly) and serine 25 (Ser) stretches, Ser residues being interspersed to improve the solubility of a polyGly stretch.

The decision of whether to fuse a fluorescent protein to the amino or carboxy) terminus of a protein depends on the properties of the protein. For example, a particular terminus might need to be preserved to retain proper protein 30 function or to ensure correct localization. This decision might also be made on the basis of structural aspects of the particular fluorescent protein. For example, Aequorea GFP has a floppy carboxyl terminal tail of approximately ten amino acids, 136

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PCT/US2007/023140 which makes its fusion to the amino terminus of other proteins possible without the addition of a linker. By contrast, DsRed is more successfully fused to the carboxyl terminus of proteins of interest, because the amino termini project fully from a tetrameric complex of DsRed. If neither end of a host protein can be modified, it is 5 possible to insert the fluorescent protein into the middle of the protein.

Citrine and Venus, two bright versions of a yellow-emitting mutant of GFP ( YFP) that mature efficiently, have recently been developed.

Two recently developed varieties of DsRed, known as Tland E57, display improved maturation, making them preferable for use in dual-color experiments.

Fluorescence of some GFP variants can be *photoactivated‘ by specific illumination, which provides the advantage that fluorescence can be turned on at a chosen time point. Three fluorescent proteins that undergo photochemical modification in or near the chromophore have been developed, PA-GFP, Kaede ami KJFP1, that enable selective activation of fluorescence signals after specific

1S illumination, and can be used to fluorescently mark individual cells, organelles or proteins.

Table 1 provides additional examples of signal generating moieties and conjugates comprising such moieties.

Table 1

Exemplary Antibody-Detectable Label Conjugates

V ·±· L.rbeP Label

.....i . iitiboil) onjugati ,.. Recommended lor.. ·· . _^ηΐ.^Π€ Excitation Emission

Γ Λ V^bt·! ·..···. ··, · 'Ϊ/ ’e ··' V \ . S^01ur (nrn) &lt; (nm)

<td> Lake Placid Blue (EviTag™ Quantum Dot)</td><td> Flow cytometry, immunoblots, and fluorescent microscopy</td><td></td><td> &lt;450</td><td> 490</td>

<td> Fluorescein (Le. FITC)</td><td> Flow cytometry, incL BD FACS systems and Guava System, and fluorescent microscopy</td><td></td><td> 494</td><td> $18</td>

<td> Adirondack Green (EviTag™ Quantum Dot)</td><td> Flow cytometry, immunoblots, and fluorescent microscopy </td><td></td><td> &lt;450</td><td> 520</td>

<td> Rhodamine Green</td><td> Fluorescent microscopy</td><td></td><td> 502</td><td> 527</td>

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<td> Catskill Green (EviTag™ Quantum Dot)</td><td> Fluorescent microscopy</td><td></td><td> &lt;450</td><td> 540</td>

<td> Rhodamine 6G</td><td> Flow cytometry, immunoblots, and fluorescent microscopy</td><td></td><td> 525</td><td> 555</td>

<td> Hops Yellow (EviTag™ Quantum Dot)</td><td> Flow cytometry, immunoblots, and fluorescent microscopy</td><td> LI</td><td> &lt;450</td><td> 560</td>

<td> Amersham Cy3</td><td> Fluorescent microscopy</td><td></td><td> 550</td><td> 565</td>

<td> R-Phycoerythrin (PE)</td><td> Flow cytometry, Euminex® and Guava systems, FRET assays, and capillary electrophoresis; use with F1TC for double labeling</td><td></td><td> (495)565</td><td> 575</td>

<td> Rhodamine Red</td><td> Flow cytometry, fluorescent microscopy</td><td> LI</td><td> 560</td><td> 580</td>

<td> Birch Yellow (EviTag™ Quantum Dot)</td><td> Fluorescent microscopy</td><td></td><td> &lt;450</td><td> 580</td>

<td> Amersham Cy3.S</td><td> Fluorescent microscopy</td><td></td><td> 581</td><td> 596</td>

<td> Fort Orange (EviTag™ Quantum Dot)</td><td> Flow cytometry, immunoblots, and fluorescent microscopy</td><td></td><td> &lt;450</td><td> 600</td>

<td> SulfoRhodamine (Alias Texas Red®)</td><td> Flow cytometry and fluorescent microscopy</td><td></td><td> 596</td><td> 61S</td>

<td> Amereham Cy5</td><td> Immunoblot, incl.. Amersham ' Typhoon System, and immunofluorescent applications</td><td></td><td> 650</td><td> 670</td>

<td> Allophycocyanin (APC)</td><td> FRET assays and HTRF assays</td><td></td><td> 652</td><td> 670</td>

<td> Amersham Cy5-5</td><td colspan="2"> Immunoblot, especially LI-COR Odyssey systems |PWBS!</td><td> 675</td><td> 694</td>

<td> Biotin</td><td colspan="2"> Flow cytometry and other Β|μΚ·Ι fluorescent applications</td><td> -</td><td></td>

Many of these labels can be used with multiple antibodies that do not cross-react to create custom multiplexed assays.

VII. Processes for Forming Hapten Conjugates - Reaction Schemes

The following schemes provide exemplary embodiments of a method useful for making conjugates of the present invention. Other synthetic methodologies also 5 are useful for making such conjugates, and the following schemes should not be construed to limit the method to the particular synthetic methodologies depicted.

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1. Nitropyrazole Conjugates

Scheme 1 illustrates one meihod suitable for coupling exemplary nitropyrazole haptens to an alkylene oxide linker, and subsequently to a protein carrier.

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<img file="CA2995467C_D0191.tif" />

MAL-—dPEGg-NHS

<img file="CA2995467C_D0192.tif" />

Ο

1) BOC-EDA

2) TFA/DCM

<img file="CA2995467C_D0193.tif" />

<img file="CA2995467C_D0194.tif" />

O

<img file="CA2995467C_D0195.tif" />

Scheme 1

Scheme 1 illustrates coupling the exemplary nitropyrazole hapten is to an exemplary ethylene glycol linker via the pendent carboxylic acid functional group. The first step is forming an N-hydroxysuccinimide (NHS) ester from nitropyrazole. This activates

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PCT/ÜS2007/023140 the ester for subsequent reaction with a nucleophile. Formation of the NHS ester was achieved in working embodiments using dicyclohexylcarbodiimide (DCC) as a coupling reagent. Dichloromethane was used as the solvent, and triethylamine was added as a base. The NHS ester is then ready for coupling to a nucleophile.

A first possible synthetic path is reacting the activated ester with a diamine to produce an amide having a terminal amine. The diamine can be a protected diamine, as illustrated in Scheme 1 where the BOC-ethylene diamine compound is used. The BOC-protected amide is then deprotected using trifluoroacetic acid (TFA) in dichloromethane. The deptrotected compound can then be reacted with a maleimide10 PEG-NHS ester to couple a linker to the hapten. The linker also includes a reactive functional group at the terminal end.

As another alternative, the hapten having an activated ester can be coupled to a linker to form an amide having either a terminal carboxylic acid functional group or a terminal hydroxyl group. A second DCC coupling reaction can be performed to again 15 activate the carboxylic acid functional group, which is reacted with the illustrated protected hydrazine reagent. Deprotection in hydrochloric acid produced the illustrated hydrazide. Alternatively, nitropyrazole hapten-PEG linker having an activated ester pendent functional group is suitable for reacting with a carrier protein to form an immunogen.

Another alternative synthetic path is illustrated using the hapten-PEG linker having a terminal hydroxyl group. The hydroxyl terminated compound can be reacted with mesyl chloride, followed by reaction with iodide to provide the iodo-substituted derivative. This compound can be reacted with the illustrated dimethyl amine carbodiimide to produce a compound useful for direct labeling of a biomolecule.

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2. Nitrophenyl Conjugates

Scheme 2 illustrates exemplary dinitrophenyl haptens coupled to an alkylene oxide (PEG) linker. These hapten-linker conjugates subsequently can be derivatized as desired or directly coupled to a carrier.

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<img file="CA2995467C_D0196.tif" />

^h?n^nh₂

<img file="CA2995467C_D0197.tif" />

<img file="CA2995467C_D0198.tif" />

<img file="CA2995467C_D0199.tif" />

<img file="CA2995467C_D0200.tif" />

Scheme 2

The exemplary dinitrophenyl hapten was coupled to an alkylene oxide linker, namely a bifimctional polyethylene glycol having both a pendent free acid and amine.

In a first approach, the dinitrophenyl hapten was coupled to ethylene diamine via

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PCT/US2007/023140 substitution, with the ring position occupied by fluorine being activated for nucleophilic substitution by the presence of the ortho and para nitro groups. The resulting compound includes a terminal nucleophilic amine for coupling to a maleimide-PEG-NHS ester.

Alternatively, the dinitrophenyl hapten can be reacted with an amino-PEG compound having either a terminal carboxlic acid or hydroxyl group. With reference to the carboxylic acid derivative, this compound was reacted with the dinitrophenyl hapten to substitute for fluorine. An NHS ester was then formed using DCC in dichloromethane. The activated ester is suitable for derivatizing as desired, such as by reaction with the illustrated protected hydrazine reagent, followed by deprotection using an acid, such as hydrochloric or trifluoracetic acid. Alternatively, the activated ester is suitable for coupling to a carrier protein to form an immunogen.

As yet another alternative, the dinitrophenyl hapten can be reacted with an amino-PEG linker to produce a compound having a terminal hydroxyl group. The 15 hydroxyl terminated compound can be reacted with mesyl chloride, followed by reaction with iodide to provide an iodo-substituted derivative. This compound can be reacted with the illustrated dimethyl amine carbodiimide to produce a compound useful for direct labeling of a biomolecule.

A second example of a synthetic pathway for making cinnamide-based conjugates is provided below in Scheme 3. The exemplary nitrophenyl hapten was converted to the corresponding NHS ester using DCC. The NHS ester was reacted with ethylene diamine. The resulting compound includes a terminal nucleophilic amine for coupling to a maleimide-PEG-NHS ester.

Alternatively, the nitrophenyl hapten can be reacted with an amino-PEG compound having either a terminal carboxlic acid or hydroxyl group. With reference to the carboxylic acid derivative, this compound was reacted with the nitrophenyl. An NHS ester was then formed using DCC in dichloromethane. The activated ester is suitable for derivatizing as desired, such as by reaction with the illustrated protected hydrazine reagent, followed by deprotection in hydrochloric acid. Alternatively, the activated ester is suitable for coupling to a carrier protein to form an immunogen.

As yet another alternative, the nitrophenyl hapten can be reacted with a aminoPEG linker to produce a compound having a terminal hydroxyl group. The hydroxyl 144

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PC77US2007/023140 terminated compound can be reacted with mesyl chloride, followed by reaction with iodide to provide an iodo-substituted derivative. This compound can be reacted with the illustrated dimethyl amine carbodiimide to produce a compound useful for direct labeling of a biomoleculc.

<img file="CA2995467C_D0201.tif" />

<img file="CA2995467C_D0202.tif" />

<img file="CA2995467C_D0203.tif" />

<img file="CA2995467C_D0204.tif" />

. 3M HO in dioxane

<img file="CA2995467C_D0205.tif" />

<img file="CA2995467C_D0206.tif" />

<img file="CA2995467C_D0207.tif" />

Scheme 3

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3. Benzofurazan Conjugates

Scheme 4 illustrates synthetic methodologies suitable for coupling exemplary benzofurazan haptens to an alkylene oxide linker.

<img file="CA2995467C_D0208.tif" />

<img file="CA2995467C_D0209.tif" />

MAL— dPEG_e—NHS

<img file="CA2995467C_D0210.tif" />

<img file="CA2995467C_D0211.tif" />

<img file="CA2995467C_D0212.tif" />

Scheme 4

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With reference to Scheme 4, the exemplary benzofurazan hapten includes a carboxylic acid functional group. The first step is activation of the carboxylic acid functional group by reaction with NHS using DCC as a coupling agent to form an activated ester, or by formation of an acid chloride. As a first option, the activated ester can be reacted with a diamine to produce a terminal amine. In certain embodiments, the diamine is a protected diamine, such as a BOC-protected diamine, as illustrated below in Scheme 5. Following the coupling reaction, the BOC protecting group can be removed in acid, such as trifluoroacetic acid. The deprotected compound is then reacted with a maleimide-PEG-NHS ester to couple a linker to the hapten.

As a second alternative illustrated by Scheme 4, the activated ester is now ready for coupling with a linker, if desired, such as the exemplary bifunctional alkylene oxide linkers, i.e. PEG linkers. Exemplary PEG linkers may have both an amine and a carboxylic acid group or an amine and a hydroxyl group. Reaction of activated ester compound with linker provides either a carboxlic acid terminated compound or a hydroxyl group terminated compound. The carboxylic acid can be converted to an activated ester by reaction with NHS using DCC as a coupling agent This activated ester can be reacted with the illustrated protected hydrazine reagent, followed by deprotection in hydrochloric acid.

Alternatively, the hydroxyl terminated compound can be reacted with mesyl chloride, followed by reaction with iodide to provide the iodo-substituted compound. This compound can be reacted with the illustrated dimethyl amine carbodiimide to produce a compound useful for direct labeling of a biomolecule.

Another alternative synthesis path for making a maleimide-dPEGg conjugate is provided below in Scheme 5. The acid chloride is then mated with a BOC-protected hydrazide, followed by deprotection using trfilfooroacetic acid. The hydrazide is then reacted with maleimide-dPEG8-NHS to produce the illustrated conjugate.

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<img file="CA2995467C_D0214.tif" />

Scheme 5

4. Triterpene-Linker Conjugates and Triterpene Immunogens

Scheme 6 illustrates one method suitable for coupling exemplary triterpene haptens to an alkylene oxide linker to form hapten-linker conjugates. The haptenlinker conjugates can be further derivatized as desired, or can be directly coupled to a protein carrier molecule.

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<img file="CA2995467C_D0215.tif" />

<img file="CA2995467C_D0216.tif" />

itnmunoQenc protein carte* (Sovin· thynapiebutni, Keytiei»

Limpci Hemocyanin or Bovine Sena* Afeumin)

MIUNOGEN

Scheme 6

With reference to Scheme 6, starting compound 40 was oxidized to ketone 41 using pyridinium dichromate (PDC). The NHS activated ester 42 was then formed using DCC coupling in dichloromethane. Activated ester 42 was then reacted with a bifunctional PEG-4 linker 43 comprising both an amine and carboxylic acid functional group to form amide 44. The carboxylic acid functional group of compound 44 was converted to the activated ester 45 again using NHS and DCC. Activated ester 45 was 10 then coupled to an immunogenic protein carrier to fonn immunogen 46.

S. Urea- and Thio urca-Based Hapten-Linker Conjugates and Immunogens

Scheme 7 illustrates one method suitable for coupling exemplary urea and thioureas-based haptens to an alkylene oxide linker, and subsequently to a protein carrier molecule.

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<img file="CA2995467C_D0217.tif" />

<img file="CA2995467C_D0218.tif" />

<img file="CA2995467C_D0219.tif" />

cf₃ o

OCC. NHS

DCM

<img file="CA2995467C_D0220.tif" />

<img file="CA2995467C_D0221.tif" />

Scheme 7

With reference to Scheme 7, starting isothiocyanate compound 51 was reacted with a PEG-4 linker 52 comprising both an amine and a carboxylic acid functional group to form thiourea 53. The carboxylic acid functional group of compound 53 was converted to the activated ester 54 using NHS and DCC. Activated ester 54 was then coupled to protected hydrazine reagent 55, followed by deprotection in 3M hydrochloric acid, to form compound 56. Alternatively, activated ester 54 can be coupled to a carrier to form immunogen 58.

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PCT/US2M7«2314Û

6. Rotenone-Based Hapten-Linker Conjugates and Immunogens

Scheme 8 illustrates one method suitable for coupling exemplary rotenonebased haptens to an alkylene oxide linker, and subsequently to a protein carrier molecule

<img file="CA2995467C_D0222.tif" />

<img file="CA2995467C_D0223.tif" />

<img file="CA2995467C_D0224.tif" />

<img file="CA2995467C_D0225.tif" />

Schemes

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Starting compound 60 was treated with NHjOH-HCI to form an intermediate oxime, which was then reacted with alpha bromoacetic acid, compound 61, to form oxime 62. The carboxylic acid functional group of compound 62 was converted into 5 an NHS ester using DCC to form compound 63. Compound 63 was then coupled to an exemplary PEG-4 linker 64, having both an amine and a carboxylic acid functional group, to produce compound 65. The carboxylic acid functional group of compound 65 was converted to the NHS ester 66 by reaction with NHS using DCC as a coupling agent. Compound 66 was then treated with BOC-protected hydrazine compound 67, and then deprotected using 3M hydrochloric acid in dioxane, to produce compound 68. A person of ordinary skill in the art will appreciate that compound 66 also could be coupled to a carrier, such as a protein carrier, as disclosed herein to form an immunogen.

Scheme 9 illustrates synthetic paths used with rotenone isoxazolines.

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<img file="CA2995467C_D0226.tif" />

<img file="CA2995467C_D0227.tif" />

H 3M HCl in dioxane

<img file="CA2995467C_D0228.tif" />

H

Scheme 9

Scheme 9 illustrates making rotenone isoxazoline conjugates by sequentially treating the starting compound with ammonium hydroxide, followed by bromoacetic acid. This results in ring rearrangement to produce rotenone isoxazolines having a

1S3

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PCT/US2007/023140 terminal carboxylic acid functional group. The NHS ester was produced using N-3dimethylaminopropyl-N’-ethylcarbodiimide (EDAC). As a first option, the NHS ester can be reacted with a diamine to produce an amide having a terminal amine. This compound can then be reacted with a maleimide-PEG-NHS ester to couple to the hapten a PEG linker having a reactive terminus.

As a second alternative illustrated by Scheme 9, the the NHS ester is ready for coupling with a linker, if desired, such as the exemplary bifunctional alkylene oxide linkers, i.e. PEG linkers. These exemplary PEG linkers have plural reactive functional groups, such as an amine and a carboxylic acid group or an amine and a hydroxyl group. Reacting the NHS ester with a linker provides either a carboxlic acidterminated compound or a hydroxyl group-terminated compound. The carboxylic acid can be converted to an NHS ester using DCC as a coupling agent. The NHS ester can be reacted with the illustrated protected hydrazine reagent, followed by deprotection in hydrochloric acid, to produce the amino-tenninated amide.

Alternatively, the hydroxyl terminated compound can be reacted with mesyl chloride, followed by reaction with a halide, such as iodide, to provide the halidesubstituted compound. This compound can be reacted with the illustrated dimethyl amine carbodiimide to produce a compound useful for direct labeling of a biomolecule.

7. Oxazole- and Thiazole-Based Conjugates

Scheme 10 illustrates one method suitable for coupling exemplary oxazole- and thiazole-based haptens to an exemplary alkylene oxide linker. The hapten-linker conjugate then can be derivatized as desired, or can be directly coupled to a protein carrier molecule to form an immunogen.

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<img file="CA2995467C_D0229.tif" />

1) 8OC-5DA

2) TFA/DCM

<img file="CA2995467C_D0230.tif" />

H₂N—dPEGe-COjH

NHrdPEGj-OH

<img file="CA2995467C_D0231.tif" />

DCC. NHS

<img file="CA2995467C_D0232.tif" />

<img file="CA2995467C_D0233.tif" />

H

ί. 3M HCI in dioxane

<img file="CA2995467C_D0234.tif" />

<img file="CA2995467C_D0235.tif" />

Scheme 10

With reference to Scheme 10, the exemplary thiazole hapten, having a reactive sulfonyl chloride functional group, was reacted with either ethylene diamine or an exemplary bi functional PEGs linker. As a first option, the thiazole can be reacted with 155

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PCT/US2007/023140 a BOC-protected ethylene diamine, followed by deprotection using TFA, to produce an amide having a terminal amine. This compound can then be reacted with a maleimide-PEG-NHS ester to couple a linker to the hapten. Alternatively, the compound can be coupled to directly tocarrier protein to form an immunogen.

Alternatively, the thiazole hapten can be reacted with a amino-dPEG linker having either a terminal hydroxyl group or a terminal carboxylic acid group. The carboxylic acid-terminated linker can be converted to the NHS ester using DCC, followed by reaction with a BOC protected hydrazide. The BOC group can be removed using an acid, such as 3M HC1, to form the hydrazide terminated conjugate.

The hydroxyl-terminated thiazole sulfonamide PEG conjugate can be reacted with mesyl chloride, followed by reaction with a halide, such as iodide, to provide the bal ide-substituted compound. This compound can be reacted with the illustrated dimethyl amine carbodiimide to produce a compound useful for direct labeling of a biomolecule.

8. Coumarin-Based Hapten-Linker Conjugates and Immunogens Scheme 11 illustrates one method suitable for coupling exemplary coumarinbased haptens to an exemplary alkylene oxide linker. The resulting hapten-linker conjugate can be derivatized further as desired, or can be coupled to a carrier, such as 20 a protein carrier, to form an immunogen.

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<img file="CA2995467C_D0236.tif" />

<img file="CA2995467C_D0237.tif" />

<img file="CA2995467C_D0238.tif" />

<img file="CA2995467C_D0239.tif" />

With reference to Scheme J1, the starting compound includes a carboxylic acid functional group that was converted to an NHS using DCC as a coupling agent. As a 5 first option, the NHS ester can be reacted with ethylene diamine to produce an amide

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PCT7US2007/023140 having a terminal amine. This compound can then be reacted with a maleimide-PEGNHS ester to couple to the hapten a linker having a reactive terminal functional group.

Alternatively, the NHS ester can be coupled with the exemplary bifiinctional PEGg to produce amides having either a terminal carboxylic acid or hydroxyl functional group. The carboxylic acid functional group can be converted to an NHS ester using DCC as a coupling agent. The NHS ester was then reacted with the protected hydrazine compound, followed by deprotection in 3M hydrochloric acid in dioxane, to produce the hydrazide. Alternatively, the NHS ester can be coupled to an immunogenic protein to produce an immunogen.

The hydroxyl-terminated coumarin PEG conjugate can be reacted with mesyl chloride, followed by reaction with a halide, such as iodide, to provide the halidesubstituted compound. This compound can be reacted with the illustrated dimethyl amine carbodiimide to produce a compound useful for direct labeling of a biomolecule.

9. Cydolignan-Linker Conjugates and Immunogens

Scheme 12 illustrates one method suitable for coupling exemplary Podophyllotoxin-based haptens to an exemplary alkylene oxide linker. The haptenlinker conjugate then can be further derivatized as desired, or directly coupled to a carrier molecule, such as a protein carrier molecule.

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PCT/US2OO7AI23M(I

<img file="CA2995467C_D0240.tif" />

With reference to Scheme 12, the starting alcohol was oxidized to the corresponding ketone using manganese dioxide oxidation in dichloroethane. The 5 ketone was then converted to an intermediate oxime (not shown), followed by ring

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PCT/US2007/023140 rearrangement to the 5-membered heterocycle, thereby producing a compound having a carboxylic acid functional group by ring opening of the lactone. This compound was converted to the NHS ester, using either DCC or EDAC. As a first option, the NHS ester can be reacted with ethylene diamine to produce an amide having a terminal 5 amine. This compound can then be reacted with a maleimide-PEG-NHS ester to couple a linker to the hapten.

Alternatively, the NHS ester was coupled with a PEGg linker to produce an amide having either a terminal carboxylic acid or hydroxyl functional group. The carboxylic functional group of the amide was converted to the NHS ester. This 10 compound was then coupled to a protein carrier to produce an immunogen.

Alternatively, the NHS ester was reacted with a BOC-protected hydrazine reagent, followed by deprotection in 3M hydrochloric acid in dioxane, to produce the hydrazide.

The hydroxyl-teiminated PEG conjugate can be reacted with mesyl chloride, followed by reaction with a halide, such as iodide, to provide the halide-substituted compound. This compound can be reacted with the illustrated dimethyl amine carbodiimide to produce a compound useful for direct labeling of a biomolecule.

10. Heteroaryl Conjugates

Schemes 13 and 14 illustrate one method suitable for coupling exemplary heteroaryl-based haptens to an exemplary alkylene oxide linker. The hapten-linker conjugate then can be further derivatized as desired, or directly coupled to a carrier molecule, such as a protein carrier molecule.

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<img file="CA2995467C_D0241.tif" />

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<img file="CA2995467C_D0242.tif" />

<img file="CA2995467C_D0243.tif" />

MAL—dPEGj—NHS

<img file="CA2995467C_D0244.tif" />

<img file="CA2995467C_D0245.tif" />

Scheme 14

<img file="CA2995467C_D0246.tif" />

With reference to Schemes 13 and 14, the starting compounds each include a carboxyhc acid functional group. In a first approach, the carboxylic acid can be 5 convened to an ethyleneamino amide by reaction with BOC-protected ethylene

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PCT/US2007/023140 diamine using HOBT/EDAC. The BOC protecting group is removed using an acid, such as TFA in dichloromethane. This compound can then be reacted with a maleimide-PEG-NHS ester to couple a linker to the hapten.

Alternatively, the NHS ester can be coupled to a PEGt linker to produce an amide having either a terminal carboxylic acid or hydroxyl functional group. The carboxylic functional group of the amide can be converted to an NHS ester using DCC as coupling agent. The NHS ester was reacted with a BOC-protected hydrazine reagent, followed by deprotection in 3M hydrochloric acid in dioxane, to produce the hydrazide. Alternatively, the NHS ester can be coupled to a protein carrier to produce 10 an immunogen.

The hydroxyl-terminated PEG conjugate can be reacted with mesyl chloride, followed by reaction with a halide, such as iodide, to provide the halide-substituted compound. This compound can be reacted with the illustrated dimethyl amine carbodiimide to produce a compound useful for direct labeling of a biomolccule.

11. Azoaryl Conjugates

Scheme 15 illustrates one method suitable for coupling exemplary azoarylbased haptens to an exemplary alkylene oxide linker. The hapten-linker conjugate then can be further derivatized as desired, or directly coupled to a carrier molecule, 20 such as a protein carrier molecule.

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<img file="CA2995467C_D0247.tif" />

MAL— dPEG_#-NHS

Λ

<img file="CA2995467C_D0248.tif" />

<img file="CA2995467C_D0249.tif" />

i) CI-S-CH3

<img file="CA2995467C_D0250.tif" />

Scheme IS

With reference to Scheme 15, the exemplary azoaryl hapten, having a reactive sulfonyl chloride functional group, was reacted with either ethylene diamine or an an 5 exemplary bifunctional PEGg linker. As a first option, the azoaryl compound can be reacted with ethylene diamine to produce a sulfamide having a terminal amine. This

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PCT/US2007/023140 compound can then be reacted with a maleimide-PEG-NHS ester to couple to the hapten a linker having a reactive terminal functional group.

Alternatively, lhereative sulfonyl chloride can be reacted with a PEGg linker to produce a sulfamide having either a terminal carboxylic acid or hydroxyl functional 5 group. The carboxylic functional group of the amide can be converted to an NHS ester using DCC as coupling agent. The NHS ester was reacted with a BOC-protected hydrazine reagent, followed by deprotection in 3M hydrochloric acid in dioxane, to produce the hydrazide. Alternatively, the NHS ester can be coupled to a protein carrier to produce an immunogen.

The hydroxyl-terminated PEG conjugate can be reacted with mesyl chloride, followed by reaction with a halide, such as iodide, to provide the halide-substituted compound. This compound can be reacted with the illustrated dimethyl amine carbodiimide to produce a compound useful for direct labeling of a biomolecule.

12. Benzodiazepine Conjugates

Scheme 16 illustrates one method suitable for coupling exemplary benzodiazepine-based haptens to an exemplary alkylene oxide linker. The haptenlinker conjugate then can be further derivatized as desired, or directly coupled to a carrier molecule, such as a protein carrier molecule.

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<img file="CA2995467C_D0251.tif" />

<img file="CA2995467C_D0252.tif" />

<img file="CA2995467C_D0253.tif" />

Scheme 16

With reference to Scheme 16, the exemplary benzodiazepine hapten includes a hydroxyl group. This group was reacted with ethyliodoacetate, followed by treatment 5 with sodium hydroxide to produce a compound having a terminal carboxylic acid functional group. The carboxylic acid was converted to an NHS ester using EDAC as a coupling agent. The NHS ester wa« reacted with either ethvlene diamine or an

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PCT/IÎS2007/023140 exemplary bifiinctional PEG» linker. As a first option, the azoaryl compound can be reacted with ethylene diamine to produce an amide having a terminal amine. This compound can then be reacted with a maleimide-PEG-NHS ester to couple a linker to the hapten.

Alternatively, the reative NHS ester can be reacted with a PEGg linker to produce an amide having either a terminal carboxylic acid or hydroxyl functional group. The carboxylic functional group of the amide can be converted to an NHS ester using DCC as coupling agent. The NHS ester was reacted with a BOC-protected hydrazine reagent, followed by deprotection in 3M hydrochloric acid in dioxane, to produce the hydrazide. Alternatively, the NHS ester can be coupled to a protein carrier to produce an immunogen.

The hydroxyl-terminated PEG conjugate can be reacted with mesyl chloride, followed by reaction with a halide, such as iodide, to provide the halide-substituted compound. This compound can be reacted with the illustrated dimethyl amine carbodiimide to produce a compound usefol for direct labeling of a biomolecule.

13. Maleimide/Hydrazide PEG-linker Synthesis

Scheme 17 shows a general method for preparing maleimide/hydrazide heterobifunctional PEG linkers. Briefly, a maleimide/active ester PEG linker 102 (such as obtained from Quanta Biodesign) is reacted with a protected hydrazine derivative 104 to produce compound 106. Compound 106 was then deprotected with acid to yield the maleimide/hydrazide PEG linker 108.

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<img file="CA2995467C_D0254.tif" />

Scheme 17

A specific synthesis of a maleitnide/hydrazide PEG&lt; linker is outlined tn

Scheme 16 below. To the active ester 110 (116mg, 1.0 eq.) in 5 ml dry dioxane was added 30 mg (1.0 eq.) of the Boc protected hydrazine 112 in 5 ml of dry dioxane over I hour. The reaction was then stirred at ambient temperature under dry nitrogen for 16 hours The reaction mixture was fractionated by HPLC utilizing a Waters Delta 600 HPLC fitted with a 2996 photo-diode array detector and a

Phenomenex luna 10 μ, Cl 8(2), 100A, 250 x 30 mm column. The column was eluted with 30-60% ACN / water over 30 min at a flow rate of 12 mL / min. The desired Boc protected-PEGi-maleimide 114 eluted at 38 minutes giving 50 mg of a thick yellow oil after drying under high vaccum. The final deprotected hydrazide 116 was then obtained by stirring the residue with 6 ml of anhydrous 2 N HCL !

dioxane under dry nitrogen for 45 minutes. Concentration via rotory evaporation then gave 55 mg of the hydrazide-PEG^maleimide HCL salt.

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<img file="CA2995467C_D0255.tif" />

<img file="CA2995467C_D0256.tif" />

14. Linker-Detectable Label Conjugates

Certain embodiments of the present invention concern forming conjugates using linkers. The following non-limiting examples are provided to illustrate embodiments of the method by reference to embodiments for forming detectable label conjugates using maleimide PEG active esters to exemplify the process. A person of 10 ordinary skill in the art will appreciate that the illustrated embodiments can be used to form other types of conjugates as disclosed hererin.

In one embodiment, a disclosed specific-binding moiety nanoparticle conjugate is prepared according to the processes described in Schemes 19 to 22 below, wherein the heterobifimctional poiyalkyiene glycol linker is a polyethylene 15 glycol linker having an amine-reactive group (active ester) and a thiol-reactive group (maleimide). As shown in Scheme 19, a nanoparticle (such as a quantum dot) that has one or more available amine groups is reacted with an excess of the linker to form an activated nanoparttcle.

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<img file="CA2995467C_D0257.tif" />

<img file="CA2995467C_D0258.tif" />

<img file="CA2995467C_D0259.tif" />

Scheme 19

Thiol groups may be introduced to the antibody by treating the antibody with a reducing agent such as DTT as shown in Scheme 20. For a mild reducing agent such as DTE or DTT, a concentration of between about 1 mM and about 40 mM, for example, a concentration of between about 5 mM and about 30 mM and more typically between about 15 mM and about 25 mM, is utilized to introduce a limited number of thiols (such as between about 2 and about 6) to the antibody while keeping the antibody intact (which can be determined by size-exclusion chromatography). A suitable amount of time for the reaction with a solution of a particular concentration can be readily determined by titrating the number of thiols produced in a given amount of time, but the reaction is typically allowed to proceed from 10 minutes to about one day, for example, for between about 15 minutes and about 2 hours, for example between about 20 minutes and about 60 minutes.

w AVReduction * SH

-- SH

Scheme 20

The components produced according to Schemes 19 and 20 are then combined to give a conjugate as shown in Scheme 21.

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..Μ.

--SH

<img file="CA2995467C_D0260.tif" />

<img file="CA2995467C_D0261.tif" />

Scheme 21

Although Schemes 19-21 illustrate an optimal process for maleimide PEG active esters, wherein the nanoparticle is first activated by reacting an amine group(s) with the active ester of the linker to form an activated nanoparticle, it also is possible to first activate the antibody by reacting either an amine(s) or a thiol(s) on the antibody with the linker and then react the activated antibody with the nanoparticle [having either a thiol(s) or an amine(s) to react with the remaining reactive group on the linker as appropriate).

Thus, in an alternative embodiment, an antibody is activated for conjugation and then conjugated to a nanoparticle as shown in Schemes 22 and 23 below. In Scheme 23, the antibody is activated instead of the nanoparticle as was shown in Scheme 19. In the particular embodiment of Scheme 22, a sugar moiety (such as

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PCT/US2007/023140 located in a glycosylated region of the Fc portion of the antibody) is first oxidized to provide an aldehyde group, which is then reacted with an aldehyde-reactive group of the linker (such as a hydrazide group of the illustrated maleimide/hydrazide PEG linker).

<img file="CA2995467C_D0262.tif" />

Oxidation

- CHO

<img file="CA2995467C_D0263.tif" />

<img file="CA2995467C_D0264.tif" />

Then, as shown in Scheme 23, a thiol-reactive group of the linker portion of the activated antibody (such as a maleimide group as illustrated) is then reacted with a thiol group on the nanoparticle. Again, the process can be reversed, wherein the 10 linker is first reacted with an aldehyde group on the nanoparticle (formed, for example, by oxidation of a sugar moiety) to form an activated nanoparticle, and then the activated nanoparticle can be reacted with a thiol group on the antibody.

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<img file="CA2995467C_D0265.tif" />

<img file="CA2995467C_D0266.tif" />

<img file="CA2995467C_D0267.tif" />

Scheme 23

Although schemes 17-2 above and 22 that follows show particular examples of conjugates for illustrative purposes, it is to be understood that the ratio of specificbinding moiety (in this case, antibody) to nanoparticle in the disclosed conjugates can vary from multiple (such as 5,10,20 or more) specific binding moieties per nanoparticle to multiple nanoparticles per specific-binding moiety (such as 5,10,20 or more).

IS. Introduction of Thiols to Antibodies

To activate an antibody for conjugation, for example, an anti-mouse IgG or anti-rabbit IgG antibody, the antibody can be incubated with 25 mmol DTT at ambient temperature (23 - 25 °C) for about 25 minutes. After purification across a 15 PD-10 SE column, DTT-free antibody, typically with two to six free thiols, is obtained (Scheme2). The exemplary procedure outlined for preparing goat antimouse IgG thiol is generally applicable to other antibodies. The number of thiols per antibody can be determined by titration, for example, by using the thiol assay

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PCT7IJS2007/023140 described in U.S. Provisional F’atent Application No. 60/675759, filed April 28, 2005.

16. Conjugates of Immunoglobulins and Streptavidin with CdSe/ZnS Quantum Dots for Ultrasensitive (and Multiplexed) Immunohistochemical and In Situ HybridizationDetection in Tissue Samples.

One embodiment of a method for incorporating an immunoglobulin into a quantum dot shell is described in this example. This embodiment involves: 1) functionalization of amine-terminated quantum dot capping groups with a suitable heterobifimctional NHS ester-(PEG)x- maleimide; (x=4,8,12) 2) reduction of native disulfides by treatment with dithiothreitol (DTT); 3) derivatizing maleimideterminated quantum dots with these thiolated immunoglobulins; and 4) purifying the conjugates using suitable techniques, such as size-exclusion chromatography. The process is depicted in Scheme 24.

<img file="CA2995467C_D0268.tif" />

Scheme 24

A streptavidin conjugate can be made by substituting a thiolated streptavidin for the thiolated immunoglobulin in the process. For example, a streptavidin molecule treated with 2-iminothiolane.

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The quantum dots used in this example were protected by an electrostatically bound shell of trioctyl phosphine oxide (TOPO) and an intercalating amphiphilic polymer to induce water solubility. This polymer has approximately 30 terminal amine groups for further functionalization. See E.W. Williams, et. al. “Surface5 Modified Semiconductive and Metallic Nanoparticles Having Enhanced Dispersibility in Aqueous Media”, U.S. Patent No. 6,649,138.

In order to form highly sensitive quantum dot conjugates, antibodies were attached to the quantum dots with varying ratios. The chemistry is similar to that described in U.S. Provisional Patent Application No. 60/675759, filed 10 April 28,2005.

This methodology is advantageous due to the need for few reagents because native disulfides are used. Additionally, the antibody remains discrete and does not form fragments. This allows for two binding sites from each tethered antibody. Furthermore, highly stable and bright conjugates are produced. The brightness surpasses commercially available streptavidin-QD conjugates (Invitrogen Corporation, Eugene, OR) on the same tissue. Goat anti-biotin and rabbit anti-DNP antibodies conjugated to quantum dots of differing wavelengths of emission were produced, thereby permitting multiplex assays. HPV detection through FISH was demonstrated with the disclosed quantum dot conjugates.

VHI. Embodiments of a Method for Using Disclosed Haptens, Hapten Conjugates, and Compositions Thereof

A. In Situ Hybridization

Certain exemplary embodiments of the present invention are disclosed herein with reference to the attached drawings. FIG. I illustrates one embodiment of an in situ hydridization scheme 10 that can be implemented with various embodiments of disclosed haptens. A sample having a target 12, such as a protein, is selected. A probe 14 useful for detecting the target 12, such as an antibody, also is selected. At least one hapten 16 of the classes of haptens disclosed herein is conjugated to the probe 14. Target 12 is treated with the probe 14 conjugated to the hapten 16 in a manner effective to form a complex that can be visualized using any suitable means.

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FIG. 1 depicts treating the target 12 complexed with the probe-hapten conjugate with an anti-hapten antibody 18 having a detectable label 20. A person of ordinary skill in the art wilt appreciate that the detectable label 20 can be any of the variety of signal generating moieties disclosed herein or that would be known to a person of ordinary skill in the art, or combinations thereof, such as an enzyme, an organic chromophore, such as a flourphore, chromophoric nanoparticles, such as fluorescent quantum dots, etc. The detectable label 20 is used to visualize the complex. For example, if the detectable label 20 is an enzyme, a substrate for the enzyme is provided, thereby producing a uniquely identifiable precipitate, such as a colored 10 precipitate.

FIG. 1 also illustrates using at least one, and typically plural probes, where the probe or probes is conjugated to at least one hapten, and potentially plural different haptens, to simultaneously visualize multiple targets in a sample. FIG. 1 illustrates a sample having a particular target 22 that is recognized by a probe 24.

Hapten 26 is conjugated to the probe 24. Hapten 26 may be the same or different from hapten 16. The sample is then treated with an anti-hapten antibody 28 conjugated to a detectable label 30. This process can continue, as illustrated for targets 32 and 42.

Signal generating moieties 20,30,40 and 50 as illustrated in FIG. 1 can be the same label, such as an enzyme. In this situation, the process may comprise adding anti-hapten antibodies 18,28,38 and 48 sequentially. After each application a different precipitate is formed by adding a different substrate. Substrates used for previous visualization reactions are washed from the samples before second and subsequent substrates are added.

FIG. 2 illustrates antibody 60 coupled to detectable label, such as an enzyme

62. An enzyme substrate 64 is added to produce a detectable enzymatic product 66. One specific embodiment of this process is Silver In Situ Hydridization (S1SH). One suitable enzyme 62 for SISH is horseradish peroxidase, using silver ions and hydrogen peroxide as a substrate. The detectable product 66 is elemental silver particles.

As another example, enzyme 62 might be alkaline phosphatase. Substrate 64 is a source of silver ions and a phosphate-protected reductant. Again, the visually 176

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PCT/US2007/023H0 detectable product 66 is elemental silver. Silver can be detected by any suitable means, including bright field microscopy.

The embodiment illustrated in FIG. 2 also can be used to implement Chromogenic In Situ Hydridization. In this process, an enzyme 62 is again selected, 5 with suitable examples including those disclosed herein or are otherwise known to those of ordinary skill in the art, with horseradish peroxidase and alkaline phosphatase being used to exemplify particular embodiments. A substrate is then . selected suitable for producing a colored precipitate product 66 that can be detected using techniques known in the art, including bright field microscopy. The chromogenic compound can be fluorogenic. Suitable fluorogenic compounds are commercially available from various sources. For example, beta-lactamase and fluorogenic beta-lactamase substrates are available from Invitrogen Detection. The substrate can be made fluorogenic by the enzymatic action, or a fluorogenic substrate can be rendered non-fluorescent. Quantum dots also can be used to visualize immunohistochemical interactions too. FFourescent probes and quantum dots typically are monitored using a fluorescence microscope.

FIG. 3 illustrates one embodiment of a direct detection process. For this process, a primary antibody 70 is selected for a particular target. For example, the primary antibody 70 might be a monoclonal antibody, such as mouse monoclonal 20 IgG antibody. Primary antibody 70 also typically includes a detectable label 72, as discussed above.

Alternatively, an amplification process can be used, as is illustrated schematically in FIG. 4. This embodiment also can be used for diagnostic tests. A target is selected. A primary antibody 80 is added to the sample in a manner to 25 allow complexation of the target and primary antibody. A secondary antibody 82 against the primary antibody 80 is added to the sample. Antibody 82 includes a detectable label that can be used to identify, particulary visually or by visual means, such as microscopy, the complexed target using a substrate, as discussed herein. Antibody 82 can be any suitable antibody, including by way of example and without 30 limitation, a labeled rabbit anti-mouse IgG antibody. Moreover, a secondary antibody 86 to the primary antibody 80 also can be added to the sample. Antibody 86 can be any suitable antibody against the primary antibody, such as an antibody 177

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PCT/US2007/023140 from a different species. For example, antibody 86 might be, by way of example and without limitation, a goat antibody raised against the primary antibody, such as a mouse IgG antibodies. FIG. 4 illustrates adding! at least one additional antiantibody 88 having a detectable label 90 to the sample to amplify the signa) produced by the detected target. In this exemplary process, the antibody 88 might be a labeled rabbit anti-goat IgG antibody. Antibody 88 can be added simultaneously with, or subsequent to, as the labeled antibody 84.

Certain embodiments of the present invention are facilitated by using antihapten monoclonal antibodies. FIG. 5 schematically represents one embodiment of 10 the present invention useful for hybridoma screening. As with preceding examples, a particular target is selected. For example, a target situated in a tissue 100, such as the illustrated lambda epitope 102, is identified. A primary antibody 104 directed to the target 102 is administered in a manner effective for the antibody to recognize the target. One example of a suitable primary antibody 104 for the illustrated sysem is 15 anti-Lambda rabbit antibody. As indicted in FIG. 5, antibody 104 has at least one, and potentially plural, haptens 106 conjugated thereto, as with the illustrated embodiment. A person of ordinary skill in die art will recognize first that the number of haptens conjugated to the antibody can vary, but this number typically is from 1 to about 5 haptens, but more typically is 2 to 3. Furthermore, a person of 20 ordinary skill in the art will appreciate that the haptens conjugated to the primary antibody can be the same or different.

Tissue sample 100 is treated with anti-hapten antibodies 108. For example, in the embodiment illustrated in FIG. 5, haptens 106, coupled to the primary antibody 104, then effectively become coupled to an anti -haptenantibody 108, such 25 as may be provided from a hybridoma mouse monoclonal antibody. Thus, for each hapten 106 coupled to the primary antibody 104, there will be a secondary antibody 108. The complex formed by the anti-hapten antibody 108, such as a mouse monoclonal antibody, then must be identified. One method is to now treat the composition with an antibody that recognizes the mouse antibody, such as a goat 30 antibody. In the illustrated embodiment of FIG. 5, goat antibodies 110 are conjugated to a detectable label, such as an enzyme, including the illustrated horseradish peroxidase (HRP) enzymes 112. This complex is then incubated with an 178

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HRP substrate, as is known to persons of ordinary skill in the art, to form detectable, e.g. colored, precipitates. This process can be used for screening, such as hybridoma screening.

To screen for antihapten monoclonal antibodies, a tissue sample, such as normal human tonsil tissue is obtained. The sample may be embedded in paraffin, and if so, the tissue sample is deparaffinized, such as by using VMSI EZPrep solution. Cell conditioning and antigen retrieval is then performed using VMSI CCI. A primary polyclonal antibody, such as human anti-lamba (available from Dako), was conjugated to embodiments of haptens disclosed in the present application. Conjugation typically occurred at the Fc region of the antibody. Conjugating to the Fc region reduces the likelihood that the binding will affect the antibody specificity. A solution comprising an effective amount of the primaty antibody is applied to the tissue for an effective period of time. For working embodiments the effective concentration has been about 10 gg/ml of the primary antibody, and the effective time period has been about 60 minutes. The tissue sample is then washed. Thereafter, a potential anti-hapten antibody (e.g. KLHCGTI-1.1+5-27F09-02E01) is applied to the tissue sample for an effective period of time, such as about 60 minutes. The antibody is then detected using any suitable means, such as VMSI Omni Map DAB stain.

Automated immunohistochemistry (1HC) screening of potential anti-hapten antibodies was performed using a VMSI Discovery XT and formalin-fixed, paraffinembedded human tonsil tissue on glass slides. Tissue samples first undergo depariffination, antigen retrieval, followed by the addition of a primary antibody linked to the hapten of interest, the potential anti-hapten antibody and a detection antibody. The detection antibody is visualized using a chromogen detection reagent from VMSI. Stained slides were manually screened under a microscope. Samples having a correct primary antibody staining pattern were selected as potential anti-hapten candidates. To test for selectivity and specificity, candidate anti-hapten cell fusion products are further screened using primary antibodies conjugated to a hapten of a different chemical class under the same staining method detailed above.

FIG. 6 is a photomicrograph depicting IHC positive staining of anti-hapten antibody detection using a primary antibody conjugated to a disclosed embodiment 179

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PCT71JS2007/023140 of a nitropyrazole hapten according to the present invention. FIG. 6 clearly demonstrates visualization of a target in a sample using haptens according to the present invention coupled to a detectable label.

FIG. 7 is a photomicrograph depicting IHC negative staining using an antihapten antibody, such as an anti-nitropyrazole antibody, and a primary antibody conjugated to a disclosed embodiment of a class of haptens according to the present invention, such as a phhenylthioureà. FIG. 7 clearly demonstrates visualization of a target in a sample using haptens according to the present invention coupled to a detectable label.

Embodiments of the present invention are useful for multiplexing, i.e. simultaneous detection of multiple targets in a sample. One embodiment of this approach is illustrated schematically in FIG. 8. FIG. 8 illustrates that a sample, such as tissue sample 120, may have multiple targets, including: Ki-67 (122) (a protein antigen that accumulates from G1 -phase to mitosis, where it is found at its highest content. During interphase the Ki-67 protein is predominantly associated with the nucleoli. During mitosis it shows a close association with the chromosomes. Ki-67 is present in nuclei of proliferating (G1 -, S-, G2-phase and mitosis) cells, but not in nuclei of quiescent or resting cells (G0-phase). Recently it was demonstrated that the Ki-67 protein belongs to the family of MPM-2 antigens and that phosphorylation of the Ki-67 protein during mitosis is associated with the condensation of the chromosomes and the separation of sister chromatids. A C-terminal domain of Ki67 protein can bind to all three members of the mammalian heterochromatin protein ! (HP1) family in vitro and in vivo suggesting a role for Kt-67 protein in the control of higher order chromatin structure; CD3 antigen (124) [CD3 is a protein complex composed of three distinct chains (CD3y, CD38 and CD3s) in mammals, that associate with T-cell receptors (TCR) to generate an activation signal in Tlymphocytes. The TCR, ζ-chain and CD3 molecules together comprise the TCR complex. The CD3y, CD35 and CD3e chains are highly related cell surface proteins]; Kappa protein (126); CD20 (128) [an antigen expressed on normal and malignant human B cells that is thought to function as a receptor during B cell activation]; CD-68 antigen (130) [a 110 kDa highly glycosylated transmembrane protein which is mainly located in lysosomes]; and lambda protein (132). An

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2018-02-23 antibody for each of the targets 122, 124, 126, 128, 130 and 132 is then selected, and added to (he tissue sample 120 in a manner effective to allow antibody recognition of the target. For example, Ki-67 (122) may be recognized by a primary antibody 134 conjugated to BF hapten 136. An anti-BF monoclonal antibody 138 is then added to the sample in a manner effective to allow recognition of the BF hapten by the anti-BF antibody 138. Anti-BF monoclonal antibody 138 includes a detectable label 140, such as Qdot 585.

Qdot 585 produces a yellow-orange light. A person of ordinary skill in the art will appreciate that this process can be varied from that described. For example, hapten 136 need not be BF, nor does the detectable label 140 need to be Qdot 585, nor even a Qdot. Rather, all various combinations of haptens and signal generating moieties as described herein and as would be known to a person of ordinary skill in the art can be used to practice the invention.

With continued reference to FIG. 7, CD3 ( 124) may be recognized by a primary antibody 142 conjugated to biotin 144. This feature illustrates another embodiment of the present invention where known agents, such as biotin, can be used in combination with disclosed embodiments of haptens, hapten conjugates, and compositions thereof. An anti-biotin monoclonal antibody 146 is then added to the 20 sample in a manner effective to allow recognition of biotin by the anti-biotin antibody 146. Anti-biotin monoclonal antibody 146 includes a detectable label 148, such as Qdot 525, which produces a bluish green color.

Kappa (126) may be recognized by a primary antibody 150 conjugated to dinitrophenyl hapten 152. An anti-DNP monoclonal antibody 154 is then added to 25 the sample in a manner effective to allow recognition of the DNP hapten by the antiDNP antibody 154. Anti-biotin monoclonal antibody 154 includes a detectable label 156, such as Qdot 605, which produces an orange color.

CD20 (128) may be recognized by a primary antibody 158 conjugated to nitrophenyl hapten 160. An anti-NP monoclonal secondary antibody 162 is then 30 added to the sample in a manner effective to allow recognition of NP 160 by the anti-NP antibody 162. Anti-NP monoclonal antibody 162 includes a detectable label 167, such as Qdot 655, which produces a light red color.

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CD-68 (130) may be recognized by a primary antibody 166 conjugated to TS hapten 168. An anti-TS monoclonal secondary antibody 170 is then added to the sample in a manner effective to allow recognition of TS 168 by the anti-TS antibody 170. Anti-TS monoclonal antibody 170 includes a detectable label 172, such as

Qdot 565, which produces a light green color.

Lambda (132) may be recognized by a primary antibody 174 conjugated to rotenone hapten 176. An anti-Rot monoclonal secondary antibody 178 is then added to the sample in a manner effective to allow recognition of Rot 176 by the anti-Rot antibody 178. Anti-Rot monoclonal antibody 178 includes a detectable label 180, 10 such as Qdot 705, which produces a dark red color. Thus, by using different signal generating moieties, several different sample targets can be visualized susbstantiaily simultaneously, or sequentially, as may be desired.

Working embodiments have used multiple different haptens, and antibodies thereto, to visualize a detectable target. FIG. 9 illustrates the results of such an 15 approach. FIG. 9 is a staining image produced using multiple haptens and antibodies thereto. FIG. 9 clearly shows visualization of the protein.

Embodiments of the present invention also are useful for implementing a different type of multiplexing, i.e. simultaneous detection of multiple different types of targets, such as protein and nucleic acid targets, in a sample. This is illustrated 20 schematically in FIG. 10 with reference to Her2 (human epidermal growth factor receptor 2). Her2 is a gene that helps control how cells grow, divide, and repair themselves. The Her2 proto-oncogene encodes a transmembrane glycoprotein of 185 kDa with intrinsic tyrosine kinase activity. Amplification of the Her2 gene and overexpression of its product induce cell transformation. Numerous studies have 25 demonstrated the prognostic relevance of pl 85(Her2), which is overexpressed in

10% to 40% of human breast tumors.

FIG. 10 illustrates fluorescent imaging. As illustrated tn FIG. 10, a hapten labeled Her2 probe 200 is added to a sample in a manner effective to allow the probe to complex with the Her2 gene. Probe 200 includes a hapten 202 that can be any 30 known hapten, including embodiments of haptens disclosed herein. FIG. 10 illustrates using dinitrophenyl hapten 202. The complexed gene is then treated with

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An anti-Her2 protein antibody 208, such as Anti-her2 4B5 rabbit antibody, is added to the sample in a manner effective to allow recognition of the Her2 protein. The anti-Her2 antibody 208 includes at least one hapten 210, and potentially plural haptens 210, which may be the same or different. The embodiment illustrated in FIG. 10 illustrates the process using biotin. An anti-hapten secondary antibody 212 is then added to the sample in a manner effective to allow complexation of the secondary antibody2!2 and hapten(s) 210. Anti-haptcn secondary antibody 212 includes a detectable label 214, such as a Qdot 655. Thus, the embodiment illustrated in FIG. 10 allows multiplexed detection of gene and gene product.

FIG. 11 illustrates the results of such a multiplexed chromogenic detection. FIG. 11 is a staining image depicting detection of protein and 2 genes, such as by using anti-biotin and anti-dinitrophenyl antibodies.

IX. Test Kits

Disclosed embodiments of the present invention provide, in part, kits for carrying out various embodiments of the method of tire invention. Examples of such kits include those useful for cholesterol analyses, pregnancy kits, cancer diagnostic kits, etc. Test kits of the present invention typically have a hapten conjugate according to the present invention, such as at least one hapten-specific binding molecule conjugate, including hapten-antibody conjugates and/or hapten-nucleic acid probe conjugates, and an anti-hapten antibody, particularly an anti-hapten antibody conjugated to a detectable label

As a specific example, kits are provided for characterizing a mammalian tumor’s responsiveness to drug therapies, such as inhibitors. Particular examples include, without limitation, an inhibitor of the mTOR pathway or a dual mTOR pathway inhibitor and an EGR pathway inhibitor comprising at least two reagents, preferably antibodies, that can detect the expression, phosphorylation, or both of polypeptides in the EGF pathway, the mTOR pathway, or both. For example, the kit can contain at least two, three, or four reagents that bind to a phosphorylated form of ERK, that bind to the phosphorylated form of MEK, that bind to HIF-Ια, or that

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PCT/US200 7/023140 bind to mTOR. Further, the kit can include additional components other then the above-identified reagents, including but not limited to additional antibodies. Such kits may be used, for example, by a clinician or physician as an aid to selecting an appropriate therapy for a particular patient.

X. Automated Embodiments

A person of ordinary skill in the art will appreciate that embodiments of the method disclosed herein for using hapten conjugates can be automated. Ventana Medical Systems, Inc. is the assignee of a number of United States patents disclosing systems and methods for performing automated analyses, including U.S. Patent Nos. 5,650,327,5,654,200,6,296,809,6,352,861,6,827,901 and 6,943,029, and U.S. published application Nos. 20030211630 and 20040052685.

Particular embodiments of hapten staining procedures were conducted using various automated processes. Additional detail concerning exemplary working embodiments are provided in the working examples.

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XI. Working Examples

The following examples are provided to illustrate certain specific features of working embodiments. The scope of the present invention is not limited to those features exemplified by the following examples.

Materials

DTT was purchased from Aldrich and quantum dots were purchased from Quantum Dot, Co. and used as received. NHj-dPEGg-COjH, NHj-dPEGg-OH, NHS-dPEG|2-MAL and NHS-dPEGi-MAL were purchased from Quanta BioDesign. Goat anti*biotin was received lyophilized from Sigma. Antibody concentrations were calculated using ε^ο = 1.4 ml mg 'em¹. Quantum dot concentrations were determined using ε«οΐ(*3) = 650 000 M 'cm'¹ for 605 nm emitting quantum dots (QDgos) and e«s(±3) = 700 000 W'cm’¹ for QD 655. Deionized water was passed through a Milli-Q Biocel System to reach a resistance of 18.2 ΜΏ. Buffer exchange was performed on PD-10 columns (GE Biosciences). Size-exclusion chromatography (SEC) was performed on Akta purifiers (GE Biosciences) which was calibrated with protein standards of known molecular weight. The flow rate was 0.9 ml/nain on a Superdex 200 GL 10/300 (GE Biosciences) running PBS, pH 7.5.

Example 1

This example concerns the synthesis of Rotenone isoxazoline.

<img file="CA2995467C_D0269.tif" />

Rotenone (20.00 g, 50.7 mmol, 1.0 eq.) and hydroxylamine hydrochloride (35.20 g, 507 mmol, 10.0 eq.) were suspended/dissolved in absolute ethanol (600 mL). A solution of sodium hydroxide (24.30 g, 608 mmol, 12 eq.) in water (120

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PCT7US2007/023140 mL) was added to the stirred suspension and refluxed for three hours. After the reaction was cooled to room temperature, the solution was filtered and the filtrate reduced in vacuo to approximately 150 mL volume. The reduced filtrate was diluted with water (200 mL) and extracted three times with methylene chloride (200 mL each). The methylene chloride washes were combined, dried over anhydrous magnesium sulfate, filtered and the solvent removed under vacuum. The resulting material (20 g) was taken up in methylene chloride (10 mL) and purified by flash chromatography (Isco Combi fl ash) using a 330 gram column and eluting using a methylene chloride to 20% methanol/methylene chloride gradient. The desired compound (6.17 g, 30%) was isolated as the earlier eluting fraction. Purity was determined by HPLC and structure by ’H/^I3C-NMR and MS.

Example 2

This example concerns the synthesis of Rotenone isoxazoline acetic acid.

<img file="CA2995467C_D0270.tif" />

Rotenone isoxazoline (3.53 g, 8.62 mmol, 1.0 eq.) was stirred/suspended in anhydrous dimethylformamide (80 mL). 'Bromoacetic acid (28.10 g, 86.2 mmol, 10.0 eq.) was added under nitrogen. Cesium carbonate (28.1 g, 86.2 mmol, 10.0 eq.) and silver oxide (2.99 g, 12.9 mmol, 1.5 eq.) were added and the reaction stirred under nitrogen at ambient temperature for 2] hours. The reaction mixture was diluted with methylene chloride (120 mL), filtered and the solvent removed in vacuo. The residue was taken up in methylene chloride (15 mL) and chromatographed (Isco CombiFlash) using a 120 gram Redisep column with a 0 to 10% methanol gradient in methylene chloride. The fractions containing the desired compound were combined and concentrated under vacuum to give 2.98 g (74%). Purity was determined by HPLC and structure by *H/^,3C-NMR and MS.

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Examplc3

This example concerns the synthesis of diazapinone ester.

<img file="CA2995467C_D0271.tif" />

Ethyl iodoacetate KOtBu

DMF/THF

<img file="CA2995467C_D0272.tif" />

l,3-Dihydro-4-(2-hydroxyphenyl)-2H-l,5'benzodiazapin*2-one (2.837 g, 11.2 mmol, 1.0 eq) was stirred-suspended in 40 mL of DMF, 34 mL (34 mmol, 3.0 eq) of a 1.0 M (in THF) solution of potassium tert-butoxide added, 1.6 mL (13.5 mmol, 1.2 eq) of ethyl iodoacetate added, and reaction stirred under N2 for 3 hours. Then an additional 1.6 mL (13.5 mmol, 1.2 eq) of ethyl iodoacetate was added, and stirring continued for 2 hours. The reaction was then poured into 100 mL of water and extracted with EtOAc (3 x 100 mL). The EtOAc extracts were combined, dried over MgSO4, solvent removed in vacuo, and resulting oil purified by flash chromatography, eluting with ElOAc/Hexane (20/80). Obtained 806 mg (21% yield). Purity was determined by HPLC and structure by NMR and MS.

Example 4

This example concerns the synthesis of diazapinone acid.

<img file="CA2995467C_D0273.tif" />

The diazapine ester (999 mg, 2.95 mmol, 1.0 eq) was dissolved in 30 mL of MeOH, 944 mg (23.6 mmol, 8.0 eq) of sodium hydroxide dissolved in 15 mL of water added, and reaction stirred for 40 minutes. The reaction was then diluted with 75 mL of water, pH adjusted to less than 4 with 6 M HC1, and extracted with EtOAc (3 x 75 mL). The EtOAc extracts were combined, dried over MgSO4, and solvent removed in vacuo. Obtained 865 mg (94% yield). Purity was determined by HPLC and structure by NMR and MS.

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Example 5

This example concerns the synthesis of oxopodophyllotoxin from podophyllotoxin.

<img file="CA2995467C_D0274.tif" />

Podophyllotoxin (3.15 g, 7.61 mmol, 1.0 eq) was dissolved in 50mLof DCE, manganese(I V) oxide (6.6 g, 76 mmol, 10 eq.) added, and the reaction mixture was refluxed for 1 hour. Additional manganese(lV) oxide (6.6 g, 76 mmol, 10 eq.) was added, and refluxing continued for 5 more hours, then reaction stirred at room temperature for 60 hours. The reaction was then filtered through celite to give a redbrown solution, filtrate solvent removed in vacuo, and resulting residue recrystallized from EtOH. Obtained 1.968 g (63% yield). Purity was determined by HPLC and structure by NMR and MS.

Example 6

This example concerns the synthesis of pyrazopodophyllic acid.

<img file="CA2995467C_D0275.tif" />

Oxopodophyllotoxin (200 mg, 0.485 mmol, 1.0 eq) was stirred-suspended in mL of EtOH. Methoxyphenyl hydrazine hydrochloride (110 mg, 0.631 mmol,

1.3 eq) was added, pyridine (0.300 mL, 3.71 mmol, 7.6 eq) was added, and the reaction mixture stirred under N_2> at 95 °C, for 18 hours. The reaction was then

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PCT/US2007/023140 allowed to cool, poured into 20 mL of saturated sodium bicarbonate, and extracted with EtOAc (3 x 20 mL). The EtOAc extracts were combined, dried over MgSO₄, solvent removed in vacuo, and resulting residue purified by flash chromatography, eluting with DCM/MeOH (98/2). Obtained 121 mg (47% yield). Purity was determined by HPLC and structure by NMR and MS.

Example 7

This example concerns an exemplary synthesis of hapten carboxylic acid Nhydroxysuccinimidyl esters.

Hapten OH

DCC or EDAC. NHS

Hapten

The hapten carboxylic acid (5.0 mmol, 1.0 eq.) was taken up in 10 ml of dry DCM in a 50 ml round bottom flask. The solution was blanketed with dry nitrogen and NHS (5.5 mmol, 1.1 eq.) was added followed by 1.0 M DCC in DCM (6.0 mmol, 1.2 eq.), and triethylamine (6.0 mmol, 1.2 eq.). The reaction was allowed to stir at room temperature under dry nitrogen for 16 hours at which point the solvent was removed under vacuum. The residue was taken up in 2 ml of dry DCM and filtered to remove the urea byproduct. The filter cake was then washed 2 times with 0.5 ml of dry DCM. The combined DCM portions were then dried under vacuum to give the hapten NHS ester which was used without fiirther purification.

Example 8

This example concerns an exemplary synthesis of hapten-dPEGg-carboxylic acids.

Hapten Cl

OR

O

<img file="CA2995467C_D0276.tif" />

OH

Hapten'

O

The hapten NHS ester or hapten acyl chloride (5.0 mmol, 1.0 eq.) was taken up in 10 ml of dry DCM in a 50 ml round bottom flask. The solution was blanketed

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PCT/US2007/023140 with dry nitrogen and amino-dPEGg-carboxylic acid (5.5 mmol, 1.1 eq.) was added followed by tricthylamine (6.0 mmol, 1.2 eq.). The reaction was allowed to stir at room temperature under dry nitrogen for 16 hours at which point the solvent was removed under vacuum. The residue was taken up in minimal methanol and purified by preparative HPLC. The appropriate fractions were then pooled and dried under high vacuum to give the pure hapten-dPEGg-acid.

<img file="CA2995467C_D0277.tif" />

OH

Alternatively, haptens that have a sulfonyl chloride moiety, such as the thiazolebased haptens in Scheme 10 and the azoaryl-based haptens in Scheme 15, could be 10 directly coupled to amino-dPEGg-carboxylic acid under the same stoichiometry and reaction conditions to produce hapten sulfamide-dPEGg-carboxylic acid.

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Example 9

This example concerns an exemplary synthesis of hapten-dPEGg-carboxylic acid /V-hydroxysuccinimidyl esters.

<img file="CA2995467C_D0278.tif" />

The hapten-dPEGg-carboxylic acid (5.0 mmol, 1.0 eq.) was taken up in 10 ml of dry DCM in a 50 ml round bottom flask. The solution was blanketed with dry nitrogen and NHS (5.5 mmol, 1.1 eq.) was added, followed by 1.0 M DCC in DCM (6.0 mmol, 1.2 eq.), and triethylamine (6.0 mmol, 1.2 eq.). The reaction was allowed to stir at room temperature under dry nitrogen for 16 hours at which point the solvent was removed under vacuum. The residue was taken up in 2 ml of dry DCM and filtered to remove the urea byproduct The filter cake was then washed 2 times with 0.5 ml of dry DCM. The combined DCM portions were then dried under vacuum to give the hapten-dPEGg-NHS ester which was used without further purification.

Example 10

This example concerns an exemplary synthesis of hapten-dPEGg-hydrazides.

⁰ I

Ο O 0

------— Hapten^x^'N(^^'°)^x>x~Y^N'NH₂

H ⁸ O ) ' «. 3M HCl h dioxane h ₈ J

The hapten-dPEGg-NHS ester (1.0 mmol, 1.0 eq.) was taken up in 5 ml of dry DCM in a 25 ml round bottom flask. The solution was blanketed with dry nitrogen and BOC hydrazide (12 mmol, 1.2 eq.) was added. The reaction was allowed to stir at room temperature under dry nitrogen for 16 hours at which point 25 the solvent was removed under vacuum. The residue was then taken up in 10 ml of 4N HC1 in dioxane and stirred at room temperature for three hours. The solvent was then removed under vacuum and the residue purified by preparative HPLC to give the pure hapten-dPEGg-hydrazide.

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Example Π

The following examples concern an exemplary synthesis of haptenethylamines by reacting ethylene diamine with a hapten-NHS, -sulfonyl chloride, 5 acid chloride or l-fluoro-2,4-dinitrobenzene.

0^,0

V> r'^s'n^^NHz rx-S--- U

<img file="CA2995467C_D0279.tif" />

<img file="CA2995467C_D0280.tif" />

<img file="CA2995467C_D0281.tif" />

<img file="CA2995467C_D0282.tif" />

<img file="CA2995467C_D0283.tif" />

<img file="CA2995467C_D0284.tif" />

The hapten-NHS ester, hapten-sulfonyl chloride, hapten-acid chloride, or 1f)uoro-2,4-dinitrobcnzcnc (1 mmol, 1.0 cq.) was dissolved in anhydrous methylene chloride (10 mL) and added dropwise to a solution of ethylene diamine (20 mmol, 10 20 eq.) in anhydrous methylene chloride ( 10 mL) under nitrogen and ambient conditions. The reaction mixture was stirred for one hour and the solvent removed in vacuo. The residue was taken up in an appropriate solvent and chromatographed on flash silica gel or by preparative HPLC. Typical yields were 30-60%. Purity was determined by HPLC and structure by 'H/^,3C-NMR and MS.

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Example 12

This examples concerns reacting N-butoxycarbonyl ethylene diamine with a hapten-NHS ester, -sulfonyl chloride, -acid chloride or l-fluoro-2,4-dinitrobenzene.

O,, zP h r^xS'n^^x^ⁿ'boc

R^-CI «

<img file="CA2995467C_D0285.tif" />

H₂N BOC »

<img file="CA2995467C_D0286.tif" />

<img file="CA2995467C_D0287.tif" />

<img file="CA2995467C_D0288.tif" />

<img file="CA2995467C_D0289.tif" />

The hapten-NHS ester, hapten-sulfonyl chloride, hapten-acid chloride, or 1fluoro-2,4-dinitrobenzene (1.0 mmol, 1.0 eq.) was dissolved in anhydrous methylene chloride (10 mL) and added dropwise to a solution of N-butoxycarbonyl ethylene diamine (1.0 mmol, 1.0 eq.) in anhydrous methylene chloride (10 mL) under nitrogen and ambient conditions. The reaction mixture was stirred for two hours and the solvent removed in vacuo. The residue was taken up in an appropriate solvent and chromatographed on flash silica gel or by preparative HPLC. Typical yields were 20-40%. Purity was determined by HPLC and structure by ’H/^C-NhlR and MS.

Example 13

This example concerns deprotecting hapten-BOC-ethylene diamine compounds.

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PCT/US2007/023140 θ' &lt;θ Η

R'%—%OC

H

<img file="CA2995467C_D0290.tif" />

NH₂

H

<img file="CA2995467C_D0291.tif" />

H ⁿ'boc

TFA

<img file="CA2995467C_D0292.tif" />

<img file="CA2995467C_D0293.tif" />

<img file="CA2995467C_D0294.tif" />

The hapten-BOC-ethylene diamine (1.0 mmol, 1.0 eq.) was dissolved in anhydrous methylene chloride (2.0 mL). Trifluoroacetic acid (2.0 mL) was added under ambient conditions and stirred for 30 minutes. The solvent was removed 5 under vacuum to constant weight and the material used without purification.

Typical yields were 90-95%. Purity was determined by HPLC and structure by 'H/^,3C-NMRandMS.

Example 14

This example concerns an exemplary synthesis of hapten-dPEG»maleimides.

R'%—^NH2 H

<img file="CA2995467C_D0295.tif" />

<img file="CA2995467C_D0296.tif" />

MAL—dPEG_e—NHS

--------------------->.

<img file="CA2995467C_D0297.tif" />

<img file="CA2995467C_D0298.tif" />

<img file="CA2995467C_D0299.tif" />

The hapten-ethylene diamine derivative ( 1.0 mmol, ! .0 eq.) was disso! ved in 15 anhydrous dimethyl formamide (5.0 mL) and triethylamine (4.0 mmol, 4.0 eq.) was added and stirred at ambient conditions under nitrogen. M AL-dPEGj-NHS (1.0 mmol, 1.0 eq., Quanta BioDesign) was dissolved in anhydrous dimethyl formamide

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PCT/ÜS2007/023140 (5.0 mL) and added to the hapten-ethylene diamine solution. The reaction was stin-ed at ambient conditions under nitrogen overnight. The solvent was removed under vacuum and purified by preparative HPLC. Typical yields were 70-90%. Purity was determined by HPLC and structure by 'H/ⁿC-NMR and MS.

Example 15

This example concerns an exemplary synthesis of hapten dPEGj-alcohois.

CL zP 'si Hapten Cl

O_NZO 'si Hapten N

H

<img file="CA2995467C_D0300.tif" />

<img file="CA2995467C_D0301.tif" />

<img file="CA2995467C_D0302.tif" />

NHa-dPEGeOH

O

Hapten'^'CI

<img file="CA2995467C_D0303.tif" />

OjiN

<img file="CA2995467C_D0304.tif" />

<img file="CA2995467C_D0305.tif" />

In a 25 ml RB flask, the hapten-NHS ester, hapten-sulfonyl chloride, hapten10 acid chloride, or l-fluoro-2,4-dinitrobenzene (1.0 eq., 2.7 mmol) is reacted with amino-dPEGr-alcohol (1.0 eq., 2.7 mmol, Quanta BioDesign) in 5 ml of dry DMF. The reaction is then blanketed with dry nitrogen and stirred at room for 16 hours. The solvent is removed under vacuum and the target alcohol purified by either silica gel chromatography or preparative HPLC. Product purity and identity was determined by HPLC, MS, and 'H/^,:5C-NMR.

Example 16

This example concerns an exemplary procedure of hapten-dPEGî-mesylates.

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<img file="CA2995467C_D0306.tif" />

OH

<img file="CA2995467C_D0307.tif" />

OMs

Οχ 'sC Hapten N

<img file="CA2995467C_D0308.tif" />

<img file="CA2995467C_D0309.tif" />

Οχ ,0 'S' Hapten N

<img file="CA2995467C_D0310.tif" />

The hapten-dPEGî-alcohol was taken up in anhydrous DMF (7mL) in a 25 ml RB flask. The flask was purged with dry nitrogen and mesyl chloride (1.1 eq.) was added via syringe. The solution was stirred at room temperature for two minutes before adding anhydrous triethylamine (2.2 eq.) over approximately 20 minutes. The reaction was stirred for 16 hours at room temperature before removing the solvent under vaccum. The residue was taken up in dry DCM and purified via silica gel chromatoghraphy to afford the mesylate after removing the solvent under vacuum. Product purity and identity was determined by HPLC, MS, and *H/^,3C·

NMR.

Example 17

This example concerns an exemplary procedure of hapten-dPEGriodides.

<img file="CA2995467C_D0311.tif" />

<img file="CA2995467C_D0312.tif" />

<img file="CA2995467C_D0313.tif" />

<img file="CA2995467C_D0314.tif" />

<img file="CA2995467C_D0315.tif" />

<img file="CA2995467C_D0316.tif" />

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The haptcn-dPEGi-mesylate was dissolved in dry acetone (10 mL) and converted to the iodide by refluxing in the presence of sodium iodide (10 eq.) for three hours. The pure iodide was obtained after silica gel chromatography. Product purity and identity was determined by HPLC, MS, and 'H/'³C-NMR.

Example 18

This example concerns an exemplary procedure of hapten-dPEGicarbodiimides.

<img file="CA2995467C_D0317.tif" />

<img file="CA2995467C_D0318.tif" />

<img file="CA2995467C_D0319.tif" />

<img file="CA2995467C_D0320.tif" />

<img file="CA2995467C_D0321.tif" />

The hapten-dPEG7-iodide was dissolved in dry DMF (10 mL), and treated with ethyl-N.N-dimethyl propyl carbodiimide (10 eq.) under nitrogen. After stirring at room temperature under dry nitrogen for 16 hours, the solvent was removed under vacuum to give a biphasic system composed of the desired carbodiimide and the excess ethyl-A/,A/-dimethylpropyl carbodiimide. The later was decanted off and the desired product dried under high vacuum. Product purity and identity was determined by HPLC, MS, and 'H/¹³C-NMR.

Example 19

This example concerns an exemplary procedure of generating a 4-aminodeoxycytidine triphosphate-dPEGg-hapten.

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<img file="CA2995467C_D0322.tif" />

4-Amino-deoxycytidine triphosphate (1.0 eq. as the triethylammonium salt) was dissolved in anhydrous DMSO to produce a 0.01 M solution. A solution of the hapten-dPEGg-NHS (l.l eq) in anhydrous DMSO was added to the 4-aminodeoxycytidine triphosphate and stirred for 16-24 hrs. under nitrogen. The 4-aminodeoxycytidine triphosphate-dPEGg-hapten was purified by preparative HPLC using a.Waters Sunfire OBD Preparative column (10 pm, C18,50 x 250mm) and eluting with a gradient of acetonitrile:water:0.8M triethylammonium carbonate (1:83:16'to 25:59:16 over 30min). The pure fractions were combined, lyophilized, and redissolved in a minimal amount of DI water. The water solution was passed through a sodium ion-exchange column (SP Sephadex C-25, GE Lifesciences). The sodium salt of 4-amino-deoxycytidine triphosphate-dPEGg-hapten was lyophilized to constant weight, and characterized by HPLC, ‘H/ⁱ⁵C-NMR and MS.

Example 20

This example concerns an exemplary procedure of generating an immunogen with an immunogenic carrier protein and hapten-dPEGg-NHS. A lyophilized immunogenic carrier protein, such as keyhole limpet hemocyanin (KLH), bovine thyroglobulin (BtG), or bovine serum albumin (BSA), was reconstituted in 1.0 mL PBS, pH 7.2 to give approximately a 10 mg/mL protein solution. The haptendPEGg-NHS (300 eq. for KLH, 150 eo. for BtG, or 60 cq. for BSA) was dissolved in 198

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100 μΕ DMF, added to the protein solution and rotated at room temperature overnight. The reaction was passed through a 0.2 pm GHP syringe filter and purified by SEC chromatography on an AKTA Purifier running at 0.9 mL/min. over a GE Lifesciences Superdex 200 10/300 GL column with PBS, pH 7.2. Fractions were pooled and collected corresponding to the monomeric immunogenic protein. The hapten-labeled protein was characterized by BCA protein assay (Pierce) for protein concentration and fiuorescamine lysine assay (Bio-Tek) for hapten loading.

Example 21

This example concerns an exemplary procedure of conjugating a primary antibody with a hapten-dPEGg-NHS. A polyclonal or monoclonal antibody in PBS, pH 7.0-7.5, was treated with a' solution of hapten-dPEGj-NHS (20 eq.) in anhydrous DMSO to give a final DMSÔ concentration not to exceed 10% v/v. The reaction was rotated 18 hours in an amber vial at room temperature and filtered (0.2jrm GHP syringe filter) prior to purification by SEC chromatography on an AKTA Purifier running at 0.9 mL/min. over a GE Lifesciences Superdex 200 10/300 GL column with PBS, pH 7.2. Typical yields were 70-80% with hapten coverage of 4-6 haptens per antibody.

Example 22 .

This example concerns an exemplary procedure of conjugating to the Fcregion on a primary antibody with a hapten-dPEGg-NHS. A polyclonal or monoclonal antibody in PBS, pH 7.0-7.5, was treated with an unbuffered, aqueous solution of lOOmM sodium periodate to give a final concentration of20mM periodate. The solution was rotated at room temperature in an amber rial for two hours. Tire antibody was desalted and buffer exchanged by passing through a Sephadex G-25 column (PD-10, GE Lifcscienccs) with ABS (0.1M acetate, 0.15M NaCl, pH 5.5). The oxidized antibody solution was reacted with an unbuffered, aqueous solution of polyacrylamide hydrazide (50 eq) (farther detail concerning using polyacrylamide hydrazide is provided by assignee's copending application No. 60/931,546, which was filed on May 23,2007) and incubated for one hour at ambient temperature. Sodium

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PCI7ÜS2007/023140 cyanoborohydride (100 eq.) was added and the reaction was rotated overnight The PAH-Ab conjugate was purified by SEC chromatography on an AKTA Purifier running at 0.9 mL/min. over a GE Lifesciences Superdex 200 10/300 GL column with ABS, pH 5.5. The hapten-dPEGg-NHS (20 eq.) in anhydrous DMSO was added to give a final DMSO concentration not to exceed 10% v/v. The hapten* dPEGg-PAH-Ab conjugate was purified by SEC chromatography on an AKTA Purifier running at 0.9 mL/min. over a GE Lifesciences Superdex 200 10/300 GL column with PBS, pH 7.2.

Example 23

This example concerns an exemplary procedure of conjugating to the Fcregion on a primary antibody with a hapten-dPEGg-hydrazide. A polyclonal or monoclonal antibody in PBS, pH 7.0-7.5, was treated with an unbuffered, aqueous solution of lOOmM sodium periodate to give a final concentration of 20mM periodate. The solution was rotated at room temperature in an amber vial for two hours. The antibody was desalted and buffer exchanged by passing through a Sephadex G-25 column (PD-10, GE Lifesciences) with ABS (0.1 M acetate, 0.15 M NaCl, pH 5.5). The oxidized antibody solution was reacted for one hour at room temperature with a solution of the hapten-dPEG-hydrazide (20 eq.) in DMSO, such that the final concentration of DMSO did not exceed 10% v/v. Sodium cyanoborohydride (100 eq.) was added and the reaction was rotated overnight. The hapten-dPEGg-Ab conjugate was purified by SEC chromatography on an AKTA Purifier running at 0.9 mL/min. over a GE Lifesciences Superdex 200 10/300 GL column with PBS, pH 7.2.

Example 24

This example concerns an exemplary procedure of conjugating a primary antibody with a hapten-dPEGg-maleimide. To a solution of polyclonal or monoclonal antibody in 100 mM phosphate, ImM EDTA, pH 6.5 buffer was added 30 DTT at a final concentration of 25 mM. This mixture was.rotated for precisely 25 minutes before desalting on a Sephadex G25 (PD-10, GE Lifesciences) in lOOmM phosphate, ImM EDTA, pH 6.5 buffer. Hapten-dPEGg-maleimide(50eq.) was

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PCT/US2007/023140 added as a DMF solution, such that the final concentration of DMF did not exceed

10% v/v. The reaction mixture was rotated overnight in an amber vial under ambient conditions. The hapten-dPEGg-Ab conjugate was purified by SEC chromatography on an AK.TA Purifier running at 0.9 mL/min. over a GE

Lifcscicnces Superdex 200 10/300 GL column with PBS, pH 7.5.

Example 25

This example concerns an exemplary procedure of conjugating singlestranded DNA with a hapten-dPEGj-carbodiimide. DNA (100 pg) was taken up in 10 TE buffer at 1 mg/ml in a tube and heated to 98 °C for one minute. The reaction mixture was frozen in dry ice-ethanol and 10Q pL 0.5 M borate buffer, pH 9.5 was added. The reaction mixture was warmed to room temp and the hapten-dPEGg-EDC ( 100 pL of 1.0 mM in DMSO) was added. The mixture was incubated at 60 °C for one hour, then added salt and precipitated with isopropanol. The precipitate was washed three times with 80% EtOH.

The results of the ssDNA labeling with DNP-dPEGg-EDC are provided in FIGS. 17 and 18. FIG. 17 indicates that the percent of nucleotide labeling increases, substantially linearly, with increased hapten conjugate. FIG. 17 illustrates that the percent nucleotide labeled decreased with increasing DNA stock concentration.

Example 26

This example concerns an exemplary procedure of labeling DNA with a hapten-dPEGg-amino-dCTP. The incorporation of the hapten label onto DNA was accomplished by the nick translation procedure described in Rigby, PW;

Dieckmann, M; Rhodes, C. and Berg, P., “Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I”, J, Mol. Biol.. V113:237-251, 1977. Labeling efficiency was 2-6% based on comparison of the 260nm absorbance of DNA and k_m0]I and e (extinction coefficient) of the specific hapten.

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Example 27

This example concerns an exemplary procedure of screening anti-hapten hybridomas. Benzofurazan-dPEGg-BSA (VMS1-1357-98) was coated onto microplatcs. The dilution buffer used was 0.15 molar phosphate buffered saline (PBS). The concentration of the Benzofurazan-dPEGg-BSA was 2 pg/ml, and the well concentration was 50 pL. These samples were incubated al 4 *C overnight. 1% Nonfat dry milk (NFDM) (10 mg/mL, 300 pL/well) was used as a blocking reagent, followed by incubation at 37 *C for 120 minutes. Plates were washed, as deemed necessary, using 0.15M PBS comprising 0.05% Tween 20. Each tested hapten then was used to produce mouse antisera. A total concentration of 80 pL per well mouse antisera diluted with 1 % NFDM in 0.15 M PBS was provided using the dilutions and plate design protocol indicated below in Table 6. The plates were then incubated at 37 *C for 150 minutes. A goat antimouse-horseradish peroxidase conjugate (Gl-a-Mu-HRP, Pierce) was used as a secondary antibody at a concentration of 1:10,000 in 0.15 M PBS with 0.05% Tween 20 to provide a total well volume of 50 pL/well. The plates were then incubated for 60 minutes at 37 *C. The ELISA set up and results are summarized below in Table 2.

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Table 2

<td colspan="14"> ELISA Results</td>

<td> Accession:</td><td></td><td colspan="2"> ί Customer</td><td></td><td> I</td><td colspan="2"></td><td colspan="3"> Sample tested:</td><td colspan="3"> mouse antisera</td>

<td> 500421</td><td> {</td><td colspan="2"> .‘Ventana</td><td></td><td> } 1</td><td></td><td> • ·</td><td colspan="2"> 1</td><td colspan="4"> I ! *</td>

<td> Assay parameters:</td><td></td><td colspan="4"> 1 i I</td><td colspan="2"> 5 !</td><td colspan="2"> |</td><td colspan="4"> i ‘ ’ ; i · i</td>

<td colspan="2"> Step</td><td colspan="2"> Reagent</td><td colspan="2"> Serial dilution</td><td colspan="2"> Dilution buffer</td><td colspan="2"> Concentration</td><td colspan="2"> VolumelWeM</td><td colspan="2"> Incubation</td>

<td colspan="2"> Ao coating</td><td colspan="2"> VMSt-1357-98</td><td colspan="2"></td><td colspan="2"> 0.15M PBS</td><td colspan="2"> 2ng(mL</td><td colspan="2"> 50 uL</td><td colspan="2"> 0N@4C</td>

<td colspan="2"> Blocking</td><td colspan="2"> 1%NFOM</td><td colspan="2"></td><td colspan="2"> 0.15M PBS</td><td colspan="2"> 10 mgfmL</td><td colspan="2"> 300 pL</td><td colspan="2"> 2hr.©37C</td>

<td colspan="2"> sample Dilution</td><td colspan="2"> see below</td><td colspan="2"> 5X</td><td colspan="2"> txNFDMtaaSMPeS</td><td colspan="2"> starting @ 1:50</td><td colspan="2"> 80pL</td><td colspan="2"> 2.5to.$37C</td>

<td colspan="2"> Secondary Ab</td><td colspan="2"> Gt-nUAtHRP</td><td colspan="2"></td><td colspan="2"> .8M PBS wr · «X</td><td colspan="2"> 1:10000</td><td colspan="2"> 50 pL</td><td colspan="2"> 1tr.@37C</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td> I</td>

<td></td><td></td><td colspan="3"> Plate design:</td><td></td><td colspan="2"> L</td><td></td><td colspan="2"> 1</td><td></td><td></td><td> i</td>

<td> Sample dilution</td><td> -&gt;</td><td colspan="2"> 1:50</td><td colspan="2"> 1:250</td><td colspan="3"> 1:1250 · 1:6250</td><td colspan="2"> 1:31250</td><td colspan="2"> 1:156250</td><td></td>

<td></td><td></td><td colspan="2"> 1~I 2</td><td> 3</td><td> 4</td><td> 5</td><td> LLL? .</td><td> 8</td><td> 9,</td><td> 10</td><td> 11</td><td> 12</td><td></td>

<td> Mui1</td><td> A</td><td> 1.89</td><td> 0.01</td><td> 1.81</td><td colspan="3"> 0.001 1.82i 0.00Î 1.60</td><td colspan="2"> 0.00! 1.06</td><td> 0.00</td><td> 0.56</td><td colspan="2"> o.ooi</td>

<td> Mu #2</td><td> 8</td><td colspan="2"> 1.76! 0.01</td><td> 1.67</td><td> 0.00</td><td colspan="2"> 1.73! 0.00} 1.25</td><td colspan="2"> Ô.00Î Ô.60</td><td> 0.00</td><td> Ô.25</td><td> 000</td><td> ....</td>

<td> Mu *3</td><td> C</td><td colspan="2"> 1.70! 0 01</td><td> 1.83</td><td> 0.00</td><td colspan="2"> 1.87! O.Ooj 1.50</td><td colspan="2"> O.OOl 0.95</td><td> 0.01</td><td colspan="2"> 0.37 j 0.00</td><td></td>

<td> Mu »4</td><td> 0</td><td> 1.75</td><td> 0.01</td><td colspan="2"> 1.73' 0.02</td><td colspan="2"> 1.83Î 0.001 1.49</td><td> 0.01</td><td colspan="2"> 0.98' 0.00</td><td> 0.42</td><td> 0.00</td><td></td>

<td> Mu #5</td><td> E</td><td> 1.62</td><td> 0.01</td><td> 1.58</td><td> 0.00</td><td colspan="2"> 1.721 0001 1.35</td><td> 0.00</td><td> 0.74</td><td> 0.00</td><td> 0.33</td><td> 0.00</td><td></td>

<td> Prebieed pool</td><td> F</td><td> 0.06</td><td> 0.01</td><td> 0.01</td><td> 0.00</td><td colspan="2"> 0.00* 0.011 0.00</td><td> 0.00</td><td colspan="2"> 0.00! ₀₀₀</td><td> 0.00</td><td> 0.00</td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td colspan="2"> !</td><td></td><td></td><td></td>

<td colspan="2"> I 1</td><td></td><td></td><td></td><td></td><td></td><td> 1</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td> Xj</td><td></td><td> X</td><td></td><td> X !</td><td> X</td><td></td><td> X</td><td></td><td> X</td><td></td>

<td> Antigen:</td><td colspan="7"> VMSH 357-98 Lot #C0508l610 j</td><td colspan="6"> x= blank (no antigen)</td>

<td> Wash Buffer</td><td colspan="6"> 0.15M PBS with 0.05% Tween 20</td><td> J</td><td> NFÛM</td><td colspan="3"> £ non-fat dried milk</td><td></td><td></td>

<td> Secondary Ab:</td><td colspan="7"> KRP-goat-a-mu IgO Fc specific min. x-react#60312</td><td colspan="2"> Bleed date: !</td><td></td><td></td><td colspan="2"> 0/12/2005</td>

<td> Substrate:</td><td colspan="4"> TMB lot #P502807</td><td> ;</td><td></td><td></td><td colspan="2"> Assay dale: i</td><td></td><td></td><td colspan="2"> 9/13/2005</td>

The results shown in Table 2 indicate that each of the mice tested is suitable for raising an antibody response, and further that such haptens can be visualized to confirm a response. With respect to the particular hapten tested, mouse number 1 appears to provide the best response over all dilutions tested.

Example 28

This example concerns an exemplary procedure of conjugation of anti-hapten antibodies to horseradish peroxidase (HRP). Images produced using such conjugates are provided by FIGS. 19-24.

Activation of HRP

To a 4 mL amber vial was added 78.8 mg (100 eq.) of MAL-dPEG₄™ NHS ester (Quanta Biodesign, Powell, OH, F. W. = 513.50), followed by 2.46 mL (61.5 mg, 1.53 μΜ) of HRP (Horseradish Peroxidase, Pierce, Rockford, IL Lot FJ925901) as a 25 mg / mL solution in 0.1 M sodium phosphate, pH 7.5. The vial was then placed on an autorotator in the dark at ambient temperature (23 - 25 °C), and the

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PCT/US2007/023140 amide bond forming reaction was allowed to proceed for one hour. A 400 μΙ aliquot was then removed for purification, and the remainder of the solution was temporarily stored at 4 °C. Pure HRP-PEG₄-ma!eimide was then obtained by fractionating the sample on an AK.TA Purifier fitted with a Superdex 10/300 column (GB Lifesciences) eluted with 0.1 M sodium phosphate, pH 7.5 at 1.0 mL / min. The HRP containing fractions were pooled to give 2.0 ml of a 4.52 mg / mL solution of HRP-PEGi-maleimide (90 % recovery) as measured by UV/VÏS spectrophotometry using an extinction coefficient at 280 nm of a 1% solution (pH 6.5) of 6.52.

Introduction of Thiols to Antibodies

To a 8 mL amber vial was added 3.0 mL of a mouse anti-hapten monoclonal antibody as a 2.1 mg/mL solution in 0.1 M sodium phosphate, 1.0 mM EDTA, pH 6.5. To this solution was then added 216 pL of a freshly prepared 500 mM solution of the reducing agent DTT (1,4-Dithiothreitol, Sigma-Aldrich, St. Louis, MO). The vial was placed in the dark on an autorotator and the disulfide reduction was allowed to proceed for 25 minutes. The reaction solution was split into four equal volumes (due to the limited capacity of a desalting column used), and excess DTT was removed by passing each of the fractions across a PD-10 desalting column (GE Lifesciences) eluted with 0.1 M sodium phosphate, 1.0 mM EDTA, pH 6.5. The antibody containing fractions were combined to give 8.0 mL of a 0.8 mg/mL solution of reduced mouse anti-hapten antibody (71 % recovery) as measured by UV/VIS spectrophotometry using an extinction coefficient at 280 nm of a 1% solution at pH 6.5 of 14.

HRP-Antibody Conjugation

To the reduced antibody (such as mouse anti-nitropyrazole monoclonal antibody), is added a three fold molar excess of HRP-PEG-rmaleimide. The reaction is then incubated at ambient temperature (23 - 25 °C) for 16 hours. After purification across a Superdex 200 10/300 GL SE column a conjugate, typically with an average of 2 or 3 HRPs per antibody, is obtained. The number of HRPs per

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PCT7US2007/023140 antibody was determined by measuring the ratio of absorbances at 280 ran / 403 ran of the conjugate. The conjugate was then stored in a cold room at 4°C until use.

Example 29

This example concerns an exemplary procedure of conjugation of anti-hapten antibodies to quantum dots (QD).

Reduction of Inter-Chain Disulfides on Antibodies

To a solution of polyclonal or monoclonal antibody in 100 mM phosphate, ImM EDTA, pH 6.5 buffer was added DTT at a final concentration of 25 mM. This mixture was rotated for precisely 25 minutes before desalting on a Sephadex G25 (PD-10, GE Lifesciences) in lOOmM phosphate, ImM EDTA, pH 6.5 buffer.

Synthesis of QD-dPEGn-MAL

To a solution of quantum dots (8-9 μΜ in 50 mM borate buffer, pH 8.0) was added NHS-dPEGn-MAL (50 eq.) and rotated for two hours. The maleimidefunctionalized quantum dots (QD-dPEGn-MAL) were purified by desalting on a Sephadex G25 column (PD-10, GE Lifesciences) in 0.1 M phosphate, 0.1 M NaCl, pH 7.0 buffer.

Synthesis of QD-MAL-Antibody Conjugate

The purified QD-maleimide was combined with the purified thiolated antibody in molar ratios of 4:1 antibodies to QD and rotated for a 16 hour period. The QD-Ab conjugate was purified by SEC chromatography on an AKTA Purifier running at 0.9 mL/min. over a GE Lifesciences Superdex 200 10/300 GL column with 50 mM borate buffer, pH 8.0.

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Example 30

This example demonstrates the ability of primary antibody-hapten conjugates to be visualized by chromogenic immunohistochemistry (IHC). Stainings representative of this approach are provided by FIGS. 19-24. FIG. 6 also is a staining representative of this approach, where methods for conjugating a primary antibody with a hapten are described in Examples 21-24. FIG. 7 is a control, whereby tissue treated with an antibody other than the appropriate anti-hapten antibody. FIG. 7 establishes the specificy of the method as no visualization occurs unless the appropriate anti-hapten antibody is used.

Tonsil tissue sections were treated with an anti-lambda polyclonal antibody (Dako) conjugated with haptens by the method in Examples 21,22,23 or 24. The slides were developed using standard protocols for HRP signal generation (by addition of DAB) on an automated stainer (BenchMark® XT, Ventana Medical Systems, Inc, Tucson, AZ). A typical automated protocol includes deparaffinization, several rinse steps, addition of a reaction buffer, addition of the primary antibody (anti-lambda:hapten conjugate), addition of the secondary antibody (anti-hapten:HRP conjugate), addition of DAB and hydrogen peroxide, and addition of a counterstain.

Manual scoring was conducted by Board-certified pathologists. Staining intensities, percentage of reactive cells, and cellular localization were recorded. For qualitative stain intensity, 0 is the most negative and 3+ is the most positive. Slides were reviewed and scored by the pathologist prior to quantitation by optical imaging.

For optical imaging, a digital application (VMS1) with image quantification 25 based on the intensity (expressed as average optical density, or avg. OD) of the stain converted to a numerical score was utilized. A high-resolution image was captured for each sample and the OD value was determined based on specific classifiers for the shape and color range for positively stained cells. At least three different areas per specimen were captured using either a 20x or 40x objective lens. In some cases, 30 a “combined score” or multiplicative index was derived that incorporates both the percentage of positive cells and the staining intensity according to the following formula: Combined score = (% positive) X (optical density score).

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Example 31

This example demonstrates the ability of primary antibody-hapten conjugates to be visualized by fluorescent (Quantum Dot) immunohistochemistry (IHC).

Tonsil tissue sections were treated with an anti-Kappa polyclonal antibody (Dako) conjugated with haptens by the method in Examples 21,22,23 or 24. The slides were developed using a standard protocol for an automated Stainer (BenchMark* XT, Ventana Medical Systems, Inc, Tucson, AZ). A typical automated protocol is as follows: the paraffin coated tissue on the slide was heated 10 to 75°C for 8 minutes and treated twice with EZPrep (VMS!), volume adjusted at

75°C before application of the Liquid Cover Slip or LCS (VMS1). After two 8 minute incubation times at 75°C, the slide was rinsed and EZPrep volume was adjusted, followed with LCS to deparaffinize the tissue. The slide was cooled to 37°C, incubated for 2 minutes and rinsed once with Reaction Buffer (VMS1). The slide was treated with Cell Conditioner (VMS1) twice, followed by LCS. The slide was heated to 95°C for 8 minutes, followed by LCS, and was heated to 100°C for 4 minutes, followed by LCS. Cell Conditioner, incubate for 4 minutes, apply LCS, this incubation process with Cell Conditioner was repeated 9 times at 100°C. The slide was cooled down for 8 minutes, rinsed with Reaction Buffer, volume adjusted, 20 and followed by another dispense of LCS. The slide was heated to 37°C for 2 minutes and rinsed two times before the addition of anti-Kappa:hapten conjugate (LOO gL at 1.0 mg/mL) followed by LCS and incubation at 37°C for 32 minutes. The slide was rinsed twice with Reaction Buffer followed by the application of liquid cover slip and the addition of QDotranti-hapten conjugate (100 pL, 20-50 25 nmol) and incubated at 37°C for 32 minutes. The slide was rinsed two times with buffer followed by LCS. The slide was removed from the instrument and treated to a detergent wash before manual application of a cover slip. Results were interpreted using a light microscope and aid in the differential diagnosis of pathophysiological processes, which mayor may not be associated with a particular antigen.

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Example 32

This example demonstrates the ability of hapten-labeled DNA to be visualized by chromogenic in situ hybridization (ISH). Automated silver or diaminobenzidine (DAB) in-situ hybridization protocols for detection ofHER2 gene 5 copy number were developed on the Ventana Medical Systems Benchmark XT instrument. Staining is completed on formalin fixed paraffin embedded tissue on glass slides within nine hours. The steps of the procedure are as follows: deparaffination, cell conditioning using VMS1 protease 3, addition of the haptenlabeled HER2 DNA probe (from Example 26), tissue and probe denaturation, hybridization of four hours, and detection with chromogenic silver catalyzed by HRP. Specifically, a Nitropyrazole labeled HER2 probe was hybridized in an automated fashion on formalin-fixed breast tissue, followed by detection with antiNitropyrazole Ab-HRP conjugate. Detection can be accomplished by use of the UltraView detection kit (Ventana Medical Systems) or through silver deposition using the HER2 SISH (Ventana Medical Systems) automated protocol. Results were interpreted using a light microscope and aid in the differential diagnosis of pathophysiological processes, which may or may not be associated with a particular antigen.

Example 33

This example demonstrates the ability of hapten-labeled DNA to be visualized by fluorescent (Quantum Dot) in situ hybridization (ISH). Automated fluorescent (via Quantum Dots) in-situ hybridization protocols for detection of HER2 gene copy number were developed on the Ventana Medical Systems . Benchmark XT instrument Staining is completed on formalin fixed paraffin embedded tissue on glass slides within nine hours. The steps of the procedure are as follows: deparaffination, cell conditioning using VMSI protease 3, addition of the hapten-labeled HER2 DNA probe (from Example 26), tissue and probe denaturation, hybridization of four hours, and detection with anti-hapten Ab:Quantum Dot conjugates (from Example 29). Specifically, a Benzofurazan labeled HER2 probe was hybridized in an automated fashion, followed by detection with anti208

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Benzoforazan Ab-Quantum Dot 655 conjugate. Imaging was performed on a Nikon fluorescence scope.

Example 34

This example demonstrates the ability to multiplex primary antibody-hapten conjugates and detect by multiplex fluorescent (Quantum Dot) immunohistochemistry (1HC). This approach is schematically illustrated in FIG. 8. Tonsil tissue sections were treated with a mixture of primary antibody-hapten conjugates: anti-CD20 Ab-biotin, anti-CD34 Ab-nitropyrazole, anti-CD45 Abthiazoiesulfonamide, anti-Kappa Ab-dinitrophenyl, anti-Lambda Ab-rotenone, and anti-Ki67 Ab-benzoforazan. The anti-CD20-biotin conjugate was made by coupling antibody thiol functional groups to maleimide-dPEGl 1-biotin used in a 20-fold excess (described in Example 24). The anti-CD34-nitropyrazole conjugate was formed by coupling to the Fc portion of the antibody using polyacrylamide hydrazide and a 20-fold excess of NHS-dPEG8-NP (described in Example 22). This resulted in 12 nitropyrazole haptens per antibody. The anti-CD45thiazolesulfonamide conjugate was made by reacting antibody lysines with a 20-fold excess of NHS-dPEG8-TS (described in Example 21). This resulted in 10 thiazolesulfonamide haptens per antibody. The Kappa-dinitrophenyl conjugate was formed by coupling to the Fc portion of the antibody using polyacrylamide hydrazide and a 100-fold excess of NHS-dPEG8-DNP (described in Example 22). This resulted in 62 dinitrophenyl haptens per antibody. Both the Lambda rotenone conjugate and the Ki67 benzoforazan conjugate were made by reacting anibody lysines to NHS-dPEG8-ROT and NHS-dPEG8-BF respectively (described in Example 21). This resulted in 0.3 rotenone haptens per antibody, and 2 benzoforazan haptens per antibody. The tonsil sections were then treated with a cocktail of secondary antibodies that were synthesized using the procedure in Example 29: Gt α-biotin polyAb:QD 525 (300 nM); Ms α -NP mAb:QD 655 (50 nM); Ms a -TS mAb:QD 565 (300 nM); Rb a-DNP polyAB:QD 605 (100 nM); Ms a-ROT mAb:QD 705 (200 nM); and Ms aBF mAb:QD 585 (300 nM).

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Fluorescence Microscopy

Imaging was performed on a Nikon fluorescence scope. Unmixing of fluorescence spectra was achieved utilizing a CRi camera. DAPI was used for counterstaining for the multiplexed tonsil sections.

FIG. 37 is multiplexed staining composite that was produced using a mixture of primary antibody-hapten conjugates and sequentially visualized with a mixture of anti-hapten antibody QDot conjugates as stated in FIGS. 31-36 and in this Example 34.

FIGS. 38-43 are images extracted from the multiplexed staining of FIG. 37.

FIG. 44 is a graph of wavelength versus relative fluorescence that represents the wavelengths used to extract individual QDot signals from the multiplexed staining composite of FIG. 37. FIG. 44 also establishes that the the fluorescent signal is above the nominal autofluorescence of the tonsil tissue.

VII. Compositions Comprising One or More

Haptens and/or Hapten Conjugates

The conjugates disclosed herein may be included in diagnostic and/or pharmaceutical compositions (including therapeutic and prophylactic formulations), typically combined together with one or more pharmaceutically acceptable vehicles and, optionally, other therapeutic ingredients (for example, antibiotics, or antiinflammatories).

Such pharmaceutical compositions can be administered to subjects by a variety of methods, such as mucosal administration modes, including by oral, rectal, intranasal, intrapulmonary, or transderma! delivery, or by topical delivery to other surfaces. Optionally, the conjugate can be administered by non-mucosal routes, including by intramuscular, subcutaneous, intravenous, intra-atrial, intra-articular, intraperitoneal, or parenteral routes. In other alternative embodiments, the conjugate can be administered ex vivo by direct exposure to cells, tissues or organs originating from a subject.

To formulate the pharmaceutical compositions, the conjugate can be combined with various pharmaceutically acceptable additives, as well as a base or vehicle for dispersion of the conjugate. Desired additives include, but are not limited to, pH control agents, such as arginine, sodi··”' hydroxide, glycine, hydrochloric acid, citric

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PCT/US2007/023140 acid, and (he like. In addition, local anesthetics (for example, benzyl alcohol), isotonizing agents (for example, sodium chloride, mannitol, sorbitol), adsorption inhibitors (for example, Tween 80), solubility enhancing agents (for example, cyclodextrins and derivatives thereof), stabilizers (for example, serum albumin), and reducing agents (for example, glutathione) can be included. Adjuvants, such as aluminum hydroxide (for example, Amphogel, Wyeth Laboratories, Madison, NJ), Freund’s adjuvant, MPL™ (3-O-deacylated monophosphoryl lipid A; Corixa, Hamilton ΓΝ) and IL-12 (Genetics Institute, Cambridge MA), among many other suitable adjuvants well known in the art, can be included in the compositions.

The conjugate can be dispersed in a base or vehicle, which can include a hydrophilic compound having a capacity to disperse the conjugate, and any desired additives. The base can be selected from a wide range of suitable compounds, including but not limited to, copolymers of polycarboxylic acids or salts thereof, carboxylic anhydrides (for example, maleic anhydride) with other monomers (for example, methyl (meth)acrylate, acrylic acid and the like), hydrophilic vinyl polymers, such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives, such as hydroxymethylcellulose, hydroxypropylcellulose and the like, and natural polymers, such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid, and nontoxic metal salts thereof. Often, a biodegradable polymer is selected as a base or vehicle, for example, polylactic acid, poly(lactic acid-giycolic acid) copolymer, polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid) copolymer and mixtures thereof. Alternatively or additionally, synthetic fatty acid esters such as polyglycerin fatty acid esters, sucrose fatty acid esters and the like can be employed as vehicles. Hydrophilic polymers and other vehicles can be used alone or in combination, and enhanced structural integrity can be imparted to the vehicle by partial crystallization, ionic bonding, cross-linking and the like. The vehicle can be provided in a variety of forms, including, fluid or viscous solutions, gels, pastes, powders, microspheres and films for direct application to a mucosal surface.

The conjugate can be combined with the base or vehicle according to a variety of methods, and release of the conjugate can be by diffusion, disintegration of the vehicle, or associated formation of water channels. In some circumstances, the conjugate is dispersed in microcapsules (microspheres) or nanocapsules (nanospheres) 211

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PCT/US2007/023140 prepared from a suitable polymer, for example, isobutyl 2-cyanoacrylate (see, for example, Michael el al., J. Pharmacy Pharmacol. 43:1-5, 1991), and dispersed in a biocompatible dispersing medium, which yields sustained delivery and biological activity over a protracted time.

The compositions of the disclosure can alternatively contain as pharmaceutically acceptable vehicles substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. For solid compositions, conventional nontoxic pharmaceutically acceptable vehicles can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.

Pharmaceutical compositions for administering the conjugate also can be formulated as a solution, microemulsion, or other ordered structure suitable for high concentration of active ingredients. The vehicle can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity for solutions can be maintained, for example, by the use of a 20 coating such as lecithin, by the maintenance of a desired particle size in the case of dispersible formulations, and by the use of surfactants. In many cases, it will be desirable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol and sorbitol, or sodium chloride in the composition. Prolonged absorption of the conjugate can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin.

In certain embodiments, the conjugate can be administered in a time release formulation, for example in a composition which includes a slow release polymer. These compositions can be prepared with vehicles that will protect against rapid release, for example a controlled release vehicle such as a polymer, microencapsulated delivery system or bioadhesive gel. Prolonged delivery in various compositions of the disclosure can be brought about by including in the composition agents that delay absorption, for example, aluminum monostearate hydrogels and gelatin. When

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PCT/US2007/023140 controlled release formulations are desired, controlled release binders suitable for use in accordance with the disclosure include any biocompatible controlled release material which is inert to the active agent and which is capable of incorporating the conjugate and/or other biologically active agent. Numerous such materials are known 5 in the art. Useful control led-release binders are materials that are metabolized slowly under physiological conditions following their delivery (for example, at a mucosal surface, or in the presence of bodily fluids). Appropriate binders include, but are not limited to, biocompatible polymers and copolymers well known in the art for use in sustained release formulations. Such biocompatible compounds are non-toxic and inert to surrounding tissues, and do not trigger significant adverse side effects, such as nasal irritation, immune response, inflammation, or the like. They are metabolized into metabolic products that are also biocompatible and easily eliminated from the body.

Exemplary polymeric materials for use in the present disclosure include, but are not limited to, polymeric matrices derived from copolymeric and homopolymeric polyesters having hydrolyzable ester linkages. A number of these are known in the art to be biodegradable and to lead to degradation products having no or low toxicity. Exemplary polymers include polyglycolic acids and polylactic acids, poly(DL-Iactic acid-co-glycolic acid), poly(D-lactic acid-co-glycolic acid), and poly(L-lactic acid-co20 glycolic acid). Other useful biodegradable or bioerodable polymers include, but are not limited to, such polymers as poly(epsilon-caprolactone), poly(epsilon-apro!actoneCO-lactic acid), poly(epsilon.-aprolactone-CO-glycolic acid), poly(beta-hydroxy butyric acid), poly(aIkyl-2-cyanoacrilatek hydrogels, such as polyfliydroxyethyl methacrylate), polyamides, poly(amino acids) (for example, L-leucine, glutamic acid, 25 L-aspartic acid and the like), poly(cstcr urea), poly(2-hydroxyethyl DL-aspartamide), polyacetal polymers, polyorthoesters, polycarbonate, polymaleamides, polysaccharides, and copolymers thereof. Many methods for preparing such formulations are well known to those skilled in the art (see, for example, Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, 30 Inc., New York, 1978). Other useful formulations include controiled-release microcapsules (U.S. Patent Nos. 4,652,441 and 4,917,893), lactic acid-glycolic acid copolymers useful in making microcapsules and other formulations (U.S. Patent Nos.

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4,677,191 and 4,728,721) and sustained-release compositions for water-soluble peptides (U.S. Patent No. 4,675,189).

The pharmaceutical compositions of the disclosure typically are sterile and stable under conditions of manufacture, storage and use. Sterile solutions can be prepared by incorporating the conjugate in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the conjugate and/or other biologically active agent into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders, methods of preparation include vacuum drying and freeze-drying which yields a powder of the conjugate plus any additional desired ingredient from a previously sterile-filtered solution thereof. The prevention of the action of microorganisms can be accomplished by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

In accordance with the various treatment methods of the disclosure, the conjugate can be delivered to a subject in a manner consistent with conventional methodologies associated with management of the disorder for which treatment or prevention is sought. In accordance with the disclosure herein, a prophylactically or therapeutically effective amount of the conjugate and/or other biologically active agent is administered to a subject in need of such treatment for a time and under conditions sufficient to prevent, inhibit, and/or ameliorate a selected disease (for example, anthrax) or condition or one or more symptom(s) thereof.

VIII. Drug Development and Drug Therapy Embodiments

Disclosed embodiments of the haptens, hapten conjugates, and compositions thereof, can be used as reagents useful for identifying and detecting expression or activation of biological markers associated with diseases, therapeutic efficacy, tumorigenesis in cells and tissue samples from cancer patients, etc. The methods provided herein are useful for predicting or assessing a response of an individual patient to a particular treatment regimen. These embodiments shall be particularly exemplified herein by reference to cancer and cancer treatment, but a person of

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PCT/ÜS2007/023140 ordinary skill in the art will appreciate that the scope of the invention is not limited to cancer diagnosis and treatment, but instead can be applied to other diseases, drug development and drug therapies too.

One embodiment of the method comprises first selecting a therapeutic. At least one hapten is conjugated to the drug, which is then administered to a subject.

Drug distribution, metabolite production, etc., then can be monitored by following the hapten-therapeutic conjugate, such as by using an anti-hapten antibody having a detectable label.

One embodiment of the method involves identifying a mammalian tumor by assaying a sample obtained from a mammalian tumor. For example, the sample may be assayed for inhibitors of a pathway, such as an mTOR pathway inhibitor, an EGF pathway inhibitor, or a dual inhibitor. The method may comprise assaying a sample obtained from the mammal to detect genes being expressed or over expressed, proteins produced from such genes, pattern of expressions, cellular processes, such as phosphorylation, or combinations thereof. “Cell or tissue sample” as used in the present context means biological samples comprising cells, oten tumor cells, that are isolated from body samples, such as, but not limited to, smears, sputum, biopsies, secretions, cerebrospinal fluid, bile, blood, lymph fluid, urine and faeces, or tissue which has been removed from organs, such as breast, lung, intestine, skin, cervix, prostate, and stomach. For example, a tissue samples can comprise a region of functionally related cells or adjacent cells.

For the exemplary embodiment concerning mammalian cancer, patterns of expression, phosphorylation or both expression and phosphorylation can be used. For example, disclosed embodiments of the present invention can be used to analyze 25 a panel of two or more polypeptides consisting of:

(a) at least one polypeptide of the EGF pathway, and (b) at least one polypeptide of the mTOR pathway

A detected pattern of expression, phosphorylation or both expression and phosphorylation identifies mammalian tumors in need of dual mTOR pathway 30 inhibitor and EGF pathway inhibitor therapy. In certain embodiments, the at least one polypeptide of the EGF pathway includes the phosphorylated ERK polypeptide; the phosphorylated MEK polypeptide, or both the phosphorylated ERK polypeptide 215

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PCT/ÜS2007/023140 and the phosphorylated MEK polypeptide. In other embodiments, the at least one polypeptide of the mTOR pathway comprises HIF-Ια polypeptide, mTOR pol ypeptide, or both HIF-1 a and mTOR polypeptide.

In certain embodiments, patterns of expression, proteins expressed, cellular processes, such as phosphorylation, and combinations thereof can be determined in a control. Detected patterns and/or products can be compared to those in a patient. For example, a detected pattern of expression, phosphorylation or both expression and phosphorylation of the panel of polypeptides is compared to the pattern of expression, phosphorylation or both expression and phosphorylation of the panel of polypeptides in a control. Increased levels of tbe pane! of polypeptides in the sample as compared to the levels of the panel of polypeptides in the control indicates that the mammalian tumor is in need of dual mTOR pathway inhibitor and EOF pathway inhibitor therapy.

Certain embodiments of the present invention provide methods for predicting 15 response in patients to a treatment regimen, such as cancer subjects to cancer therapy. Predictive biomarkers can be identified in patients for whom administering a particular therapeutic agent will be most effective, including a dual inhibitor therapy. For example, predictive biomarkèrs can be identified for assessing or monitoring the efficacy of dual therapeutic agents targeted to members of the EGF 20 pathway or the mTOR pathway.

Timely identification of non-responsive patients to a treatment regimen allows clinicians to limit a cancer patient’s exposure to unnecessary side-effects of treatment and to institute alternative treatments. Unfortunately, methods present in the art, including histological examination, are insufficient for such a timely and 25 accurate identification. The present invention provides embodiments of a method for developing more informed and effective regimes of therapy that can be administered to patients with an increased likelihood of an effective outcome (i.e., reduction or elimination of the tumor).

A diagnosis, both an initial diagnosis of disease and subsequent monitoring 30 of the disease course (before, during, or after treatment), often is confirmed using histological examination of cell or tissue samples removed from a patient. For tumors, clinical pathologists need to be able to accurately determine whether such

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PCT/US2007/023140 samples are benign or malignant and to classify the aggressiveness of tumor samples deemed to be malignant. These determinations often form the basis for selecting a suitable course of patient treatment. Similarly, the pathologist needs to be able to detect the extent to which a cancer has grown or gone into remission, particularly as a result of or consequent to treatment, most particularly treatment with chemotherapeutic or biological agents.

Histological examination involves tissue-staining procedures as disclosed herein alone or in combination with other known technologies that permit morphological features of a sample to be readily observed, such as under a light microscope. A pathologist, after examining the stained sample, typically makes a qualitative determination of whether the tumor sample is malignant. Ascertaining a tumor’s aggressiveness merely by histological examination is difficult. A tumor’s aggressiveness often is a result of the biochemistry of the cells within the tumor, such as protein expression, suppression and protein/or phosphorylation, which may or may not be reflected by sample morphology. Assessing the biochemistry of the cel Is within a tumor sample using disclosed embodiments of the present invention alone, and optionally in combination with other known techniques is desirable, as is observing, and potentially quantitating, both gene expression and protein phosphorylation of tumor-related genes or proteins, or more specifically cellular components of tumor-related signaling pathways.

Cancer therapy can be based on molecular profiling of tumors rather than simply their histology or site of the disease. Elucidating the biological effects of targeted therapies in tumor tissue and correlating these effects with clinical response helps identify the predominant growth and survival pathways operative in tumors, thereby establishing a pattern of likely responders and conversely providing a rational for designing strategies to overcome resistance. Successful diagnostic targeting of a growth factor receptor determinea if tumor growth or survival is being driven by the targeted receptor or receptor family, by other receptors not targeted by the therapy, and whether downstream signaling suggests that another oncogenic pathway is involved. Furthermore, where more than one signaling pathway is implicated, members of those signaling pathways can be used as diagnostic targets to determine if a dual inhibitor therapy will be or is effective.

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Effective chemotherapeutic medications destroy tumor cells and not adjacent normal cells. This is accomplished using medications that affect cell activities predominantly occurring in cancer cells but not in normal cells. One difference between normal and tumorous cells is the amount of oxygen in the cells. Many tumorous cells are “hypoxic,” i.e. oxygen deficient.

Mammalian cells have an array of responses to balance the requirement for oxygen as an energy substrate and the inherent risk of oxidative damage to cellular macromolecules. Molecular bases for a variety of cellular and systemic mechanisms of oxygen homeostasis have been idenfied, and the mechanisms have been found to occur at every regulatory level, including gene transcription, protein translation, posttranslational modification, and cellular localization (Harris, 2002, Nat Rev. 2:38-47).

Embodiments of the present invention can be used to analyze these products and/or processes. One disclosed embodiment first comprises identifying tumors. A panel of diagnostics of each tumor is used to find suitable, and preferably the best, candidate for each therapy. For example, treatment by an mTOR pathway-targeted therapy, such as rapamycin or PX-478, may not be effective unless an EGF pathway inhibitor is used in combination. Where there are high expression levels of EGF pathway components, such as pERK. and pMEK, an mTOR pathway-targeted therapy is not effective. Disclosed embodiments of the present a clinician to identify a more effective combination of targeted therapies.

Automated (computer-aided) image analysis systems known in the art can augment visual examination of tumor samples. In a representative embodiment, a cell or tissue sample is exposed to at least one disclosed hapten, hapten conjugate, such as a hapten-antibody conjugate, or composition thereof, having a detectable label, or that is recognized by an anti-hapten antibody having a detectable label. These hapten-based reagents and processes can be specific for a particular biological marker, such as those disclosed herein. An image, typically a magnified image, of the sample is then processed by a computer that receives the image, typically from a charge-coupled device (CCD) or camera such as a television camera. Such a system can be used, for example, to detect and measure expression and activation levels of desired targets, such as HIF-Ια, pMEK, pERK, mTOR, pmTOR, pAKT, pTSC2,

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PCT/US2007/023140 pS6, and p4EBPl in a sample, or any additional diagnostic biomarkers. Thus, disclosed embodiments of the present invention provide a more accurate cancer diagnosis and better characterization of gene expression in histologically identified cancer cells, most particularly with regard to expression of tumor marker genes or 5 genes known to be expressed in particular cancer types and subtypes (e.g., having different degrees of malignancy). This information allows a clinician to determine a more effective therapy regimen, and to monitor the results of an implemented therapy regimen. For example, drugs with clinical efficacy for certain tumor types or subtypes can be administered to patients whose cells are so identified.

Another drawback of conventional therapies is that the efficacy of specific therapeutic agents in treating a particular disease in an individual human patient is unpredictable. This unpredictability has to date substantially precluded determining, prior to starting therapy, whether one or more selected agents would be active or to render an accurate prognosis or course of treatment in an individual patient. This is ] 5 especially important because a particular disease presents the clinician with a choice of treatment regimens, without any current way of assessing which regimen will be most efficacious for a particular individual. Disclosed embodiments of the present invention are able to better assess the expected efficacy of a proposed therapeutic agent (or combination of agents) in an individual patient. Disclosed embodiments 20 are advantageous for the additional reasons that they are both time- and costeffective in assessing the efficacy of chemotherapeutic regimens and are minimally traumatic to cancer patients.

As a result, disclosed embodiments of the present method can be used to identify a disease that will respond to the proposed treatment, such as a mammalian 25 tumor that responds to particular inhibitor, such as an mTOR pathway inhibitor, or a dual mTOR pathway inhibitor and EGF pathway inhibitor therapy. Further, disclosed embodiments of the invention can be used to select a patient for a particular treatment, or can be used to identify a disease that does not respond to directed therapies. Further, methods of this invention can be used to select a subject 30 that will not likely be responsive to a proposed treatment.

Patterns of expression, or other cellular processes, such as phosphorylation, cellular products, etc., arc detected and optionally quantified using disclosed

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PCT/US2007/023140 embodiments of the present invention. For example, expression and/or phosphorylation patterns of polypeptides can be detected using biodetection reagents specific for the polypeptides. Exemplary biodetection reagents include antibodies and nucleic acid probes, typically a collection of one or more nucleic acid fragments whose hybridization to a sample can be detected. The antibody and probe may be unlabeled or labeled so that its binding to the target or sample can be detected. For example, the antibody or probe might be conjugated to at least one disclosed hapten, alone or in combination with other disclosed or known haptens. An anti-hapten antibody having a detectable label can be administered to a sample in a manner effective to allow the anti-hapten antibody to complex with the hapten. The complex is then visualized.

Nucleic acid probes mahy be from a source of nucleic acids from one or more particular (preselected) portions of the genome, e.g., one or more clones, an isolated whole chromosome or chromosome fragment, or a collection of polymerase 15 chain reaction (PCR) amplification products. The nucleic acid probe also may be isolated nucleic acids immobilized on a solid surface (e.g., nitrocellulose, glass, quartz, fused silica slides), as in an array. The probe may be a member of an array of nucleic acids as described, for instance, in WO 96/17958. Techniques capable of producing high density arrays can also be used for this purpose (see, e.g., Fodor 20 (1991) Science 767-773; Johnston (1998) Curr. Biol. 8: R171-R174; Schummer (1997) Biotechniques 23: 1087-1092; Kern (1997) Biotechniques 23:120-124; U.S. Pat. No. 5,143,854).

One of ordinary skill in the art will recognize that the precise sequence of the particular probes can be modified to a certain degree to produce probes that are substantially identical, but retain the ability to specifically bind to (i.e., hybridize specifically to) the same targets or samples as the probe from which they were derived. The term nucleic acid refers to a deoxyribonucleotide or ribonucleotide in either single- or double-stranded form. The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogues of natural nucleotides that have similar or improved binding properties, for the purposes desired, as the reference nucleic acid. The term also includes nucleic acids which are metabolized in a manner similar to naturally occurring nucleotides or at rates that are improved for 220

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PCT/US2007/023140 the purposes desired. The term also encompasses nucleic-acid-like structures with synthetic backbones. One of skill in the art would recognize how to use a nucleic acid probes for screening of cancer cells in a sample by reference, for example, to U.S. Patent 6,326,148, directed to screening of colon carcinoma cells.

Polypeptides associated with cancer can be quantified by image analysis using a suitable primary antibody against biomarkers, including but not limited HIFlo, pMEK, pERK, mTOR, pmTOR, pAKT, pTSC2, pS6, and p4EBPl, detected directly or using an appropriate secondary antibody (such as rabbit anti-mouse IgG when using mouse primary antibodies) and/or a tertiary avidin (or Strepavidin) 10 biotin complex (“ABC”).

Examples of reagents useful in the practice of the methods of the invention as exemplified herein include antibodies specific for HIF-la, including but not limited to the mouse monoclonal antibody VMSI 760-4285, obtained from Ventana Medical Systems, Inc. (Tucson, AZ). Other reagents useful in the practice of the 15 methods of this invention indude, but are not limited to, rabbit polyclonal antibody

Abeam 2732 specific to mTOR, rabbit polyclonal antibody CST 2971 specific to pmTOR, rabbit polyclonal antibody CST 3614 specific to mTSC2, rabbit polyclonal antibody CST 2211 specific to pS6, rabbit monoclonal antibody CST 3787 specific to pAKT, rabbit polyclonal antibody CST 9121 specific to pMEK, rabbit polyclonal 20 antibody VMSI 760-4228 specific to mERK (p44/p42), and rabbit polyclonal antibody CST 9455 specific to m4EBPl.

Further, predictive patterns or products, such as peptides or phosphorylation thereof can be compared to a sample that has not received treatment, such as a nontumor tissue or cell sample. The non-tumor tissue or cell sample can be obtained 25 from a non-tumor tissue or cell sample from the same individual, or alternatively, a non-tumor tissue or cell sample from a different individual. A detected pattern for a polypeptide is referred to as decreased in the mammalian tumor, tissue, or cell sample, if there is less polypeptide detected as compared to the a non-tumor tissue or cell sample. A detected pattern for a polypeptide is referred to as increased in the 30 mammalian tumor, tissue, or cell sample, if there is more polypeptide detected as compared to the a non-tumor tissue or cell sample. A detected pattern for a polypeptide is referred to as normal in the mammalian tumor, tissue, or cell sample, 221

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PCT/US2007/023140 if there is the same, or approximately the same, polypeptide detected as compared to a non-tumor tissue or cell sample.

Target protein amounts may be quantified by measuring the average optical density of the stained antigens. Concomitantly, the proportion or percentage of total 5 tissue area stained can be readily calculated, for example as the area stained above a control level (such as an antibody threshold level) in the second image. Following visualization of nuclei containing biomarkers, the percentage or amount of such cells in tissue derived from patients after treatment are compared to the percentage or amount of such cells in untreated tissue. For purposes of the invention, “determining” a pattern of expression, phosphorylation, or both expression and phosphorylation of polypeptides is understood broadly to mean merely obtaining the expression level information on such polypeptide(s), either through direct examination or indirectly from, for example, a contract diagnostic service.

IX. Miscellaneous Utilities

Although the examples presented herein to exemplify the invention primarily pertain to immunohistochemical assays, the disclosed haptens, the disclosed haptenlabeled probes and the disclosed detection methods can be applied to any type of immunoassay, nucleic acid-based assay or peptide nucleic acid (PNA)-based assay.

For example, the disclosed haptens can form a component of a detection scheme for enzyme-linked immunosorbent assays (ELISA); protein, nucleic acid and PNA microarray assays; and, for flow cytometric assays. Furthermore, immunohistochemical assays such as those specifically detailed herein also can be applied for detection of target molecules in tissue arrays.

The disclosed haptens, antibodies conjugated to haptens and detection methods can be utilized for detection of target molecules in standard (indirect), sandwich and competitive format ELISA assays. In a standard format ELISA, target molecules arenon-specifically adhered to a substrate (such as a nitrocellulose substrate) and are subsequently detected by one or more primary antibodies that specifically bind to the desired target or targets. In one embodiment, the primary antibodies are labeled with different haptens as disclosed herein, and disclosed antihapten antibodies conjugated to detectable labels (more specifically, enzymes in an 222

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ELISA, but other detectable labels such as quantum dots could be substituted) are subsequently added for visualization of the presence of target molecules adhered to the substrate. Alternatively, in the sandwich format, capture antibodies specific to one or more target molecule are adhered (covalently or non-covalently) to a substrate and a sample is added, allowing any target molecules present to also become adhered to the substrate through the interaction with the capture antibodies. After washing to remove non-target molecules that are non-specificaily bound to the substrate, one or more detection antibodies that bind to the desired targets at a different site from the capture antibodies are added. In one embodiment, the detection antibody or antibodies are labeled with one or more disclosed haptens and then one or more disclosed anti-hapten antibodies conjugated to the same or different detectable label are added for subsequent visualization of the targets adhered to the.substrate. In any format, amplification of the visualization signal is possible utilizing additional intermediate antibodies such as species-specific anti15 antibodies.

In other embodiments, disclosed haptens, disclosed hapten-labeled antibodies and hapten-labeled nucleic acids, and disclosed detection methods can be employed for target detection in blot assays, wherein proteins or nucleic adds that are separated by electrophoresis are blotted non-spedfically to a substrate, which 20 substrate is then queried for the presence of particular nucleic acid sequences or proteins. For example, in a Southern Blot assay, nucleic acids are separated by agarose gel electrophoresis and blotted from the gel in relative position to one another onto a nitrocellulose filter, which filter can then be probed with hapten. labeled nucleic acid probes that can then be detected by utilizing disclosed anti25 hapten antibodies conjugated to detectable labels. Detection of targets in Northern (RNA) and Western (Protein) blots also are possible utilizing the disclosed haptens.

The disclosed haptens, hapten-labeled probes and detection methods also can be utilized in target detection schemes using microatrays, such as for normal and reverse phase protein microarrays and for nucleic acid microarrays (including cDNA 30 and oligonucleotide microarrays). For example, in a reverse phase protein microarray, samples are spotted individually onto a substrate where proteins in the samples become non-specifically bound to the substrate. Subsequently, multiple

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PCT/US2007/023140 different antibodies (such as disclosed antibody-hapten conjugates) are used to probe the spots for the presence of particular proteins. Each spot can be probed for a plurality of different proteins simultaneously, or alternatively each spot can be probed for a different protein, or each spot can be probed for the same protein (such as where each spot is from a different sample taken from a subject at different times, for example, following administration of a drug to the subject). In the case of nucleic acid microarrays, disclosed hapten labeled probes can be utilized in a detection scheme.

Tissue microarrays are advantageously used to implement disclosed embodiments of the invention, to rapidly screen multiple tissue samples under uniform staining and scoring conditions. (Hoos et al., 2001, Am J Pathol. 158: 1245-51). Scoring of the stained arrays can be accomplished manually using the standard 0 to 3+ scale, or by an automated system that accurately quantifies (he staining observed. For example, with disclosed drug therapy embodiments, this analysis can be used to identify biomarkers that best predict patient outcome following treatment. Patient “probability of response” ranging from 0 to 100 percent can be predicted based upon the expression, phosphorylation or both of a small set of ligands, receptors, signaling proteins or predictive combinations thereof. Additional samples from cancer patients can be analyzed, either as an alternative to or in addition to tissue microarray results. For example, analysis of samples from breast cancer patients can confirm the conclusions from the tissue arrays, if the patient’s responses correlate with a specific pattern of receptor expression and/or downstream signaling.

The disclosed haptens, hapten-labeled probes and detection methods also find utility in flow cytometry, where cells are probed for (he presence of one or more target molecules (e.g. particular proteins or nucleic acid sequences) and possibly sorted according to the presence or absence of one or more target molecules (such as in fluorescence assisted cell sorting, FACS). In one embodiment, one or more disclosed hapten-labeled antibodies or hapten-labeled nucleic acid probes are contacted to a plurality of cells, and then the cells are contacted with one or more anti-hapten antibodies that are conjugated to one or more differentially detectable labels.

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X. Antigen/Antibody Recognition and Target Generally

Any antibody that specifically binds the hapten of interest, or an epitope from the antigen of interest, can be used in the methods disclosed herein. In one example the sequence of the specificity determining regions of each complementarity determining region (CDR) of an antibody that specifically binds the antigen of interest is determined. Residues that are outside the specificity determining region (SDR, non-ligand contacting sites) may be substituted. For example, at most one, two or three amino acids can be substituted. The production of chimeric antibodies, which include a framework region from one antibody and the CDRs from a different antibody, is well known in the art. For example, humanized antibodies can be routinely produced. The antibody or antibody fragment can be a humanized immunoglobulin having complementarity determining regions (CDRs) from a donor monoclonal antibody that specifically binds the antigen of interest and immunoglobulin and heavy and light, chain variable region frameworks from human acceptor immunoglobulin heavy and light chain frameworks. Generally, the humanized immunoglobulin specifically binds to RET with an affinity constant of at least 10’ M¹, such as at least 10^s M'¹ at least 5 X 10⁸ M'¹ or at least IO⁹ NT¹.

Humanized monoclonal antibodies can be produced by transferring donor complementarity determining regions (CDRs) from heavy and light variable chains of the donor mouse immunoglobulin into a human variable domain, and then substituting human residues in the framework regions when required to retain affinity. The use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of the constant regions of the donor antibody. Techniques for producing humanized monoclonal antibodies are described, for example, by Jones el al., Nature 321:522, 1986; Riechmann el al., Nature 332:323, 1988; Verhoeyen et al., Science 239:1534, 1988; Carteret al., Proc. Natl. Acad. Sci. U.S.A. 89:4285, 1992; Sandhu, Crit. Rev. Biotech. 12:437, 1992; and Singerez al., J. Immunol. 150:2844, 1993. The antibody may be of any isotype, but in several embodiments the antibody is an IgG, including but not limited to, IgGi, lgG₂, JgGj and IgG«.

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In one embodiment, the sequence of the humanized immunoglobulin heavy chain variable region framework can be at least about 65% identical to the sequence of the donor immunoglobulin heavy chain variable region framework. Thus, the sequence of the humanized immunoglobulin heavy chain variable region framework 5 can be at least about 75%, at least about 85%, at least about 99% or at least about 95%, identical to the sequence of the donor immunoglobulin heavy chain variable region framework. Human framework regions, and mutations that can be made in a humanized antibody framework regions, are known in the art (see, for example, in U.S. Patent No. 5,585,089.

Exemplary human antibodies are LEN and 21/28 CL. These are framework regions that are used in a variety of antibodies that bind tumor markers. A person of ordinary skill in the art will appreciate that others could be used, and that these regions are exemplary only. The sequences of the heavy and light chain frameworks are known in the art.

Antibodies, such as murine monoclonal antibodies, chimeric antibodies, and humanized antibodies, include full length molecules as well as fragments thereof, such as Fab, F(ab*)2, and Fv which include a heavy chain and light chain variable region and are capable of binding the epi topic determinant. These antibody fragments retain some ability to selectively bind with their antigen or receptor.

These fragments include:

(1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact Iigjhit chain and a portion of one heavy chain;

(2) Fab‘, the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;

(3) (Fab'Jj, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab*)2 is a dimer of two Fab' fragments held together by two disulfide bonds;

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PCT/US2007/023140 (4) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody (such as scFv), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.

Methods of making these fragments are known in the art (see for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). To produce these antibodies, the V« and the Vl can be expressed from two individual nucleic acid constructs in a host cell. If the V» and the V_L are expressed non-contiguously, the chains of the Fv antibody are typically held together by noncovalent interactions. However, these chains tend to dissociate upon dilution, so methods have been developed to crosslink the chains through glutaraldehyde, intermolecular disulfides, or a peptide linker. Thus, in one example, the Fv can be a disulfide stabilized Fv (dsFv), wherein the heavy chain variable region and the light chain variable region are chemically linked by disulfide bonds.

In an additional example, the Fv fragments comprise Vh and Vl chains connected by a peptide linker. These single-chain antigen binding proteins (scFv) are prepared by constructing a structural gene comprising DNA sequences encoding the Vh and Vl domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as £. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing scFvs are known in the art (see Whitlow et al., Methods: a Companion to Methods in Enzymology, Vol. 2, page 97,1991 ; Bird et al., Science 242:423,1988; U.S. Patent No. 4,946,778; Pack et al., Bio/Technology 11:1271,1993; and Sandhu, supra).

Antibody fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by 227

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PCT/US2067/023140 enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted Ffab'h- This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab’ monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab* fragments and an Fc fragment directly (see U.S. Patent No. 4,036,945 and U.S. Patent No. 4,331,647, and references contained therein; Nisonhoff et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., Methods in Enzymology, Vol. 1, page 422, Academic Press, 1967; and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

One of ordinary skill in the art will realize that conservative variants of the antibodies can be produced. Such conservative variants employed in antibody fragments, such as dsFv fragments or in scFv fragments, will retain critical amino acid residues necessary for correct folding and stabilizing between the Vh and the Vl regions, and will retain the charge characteristics of the residues in order to preserve the low pl and low toxicity of the molecules. Amino acid substitutions (such as at most one, at most two, at most three, at most four, or at most five amino acid substitutions) can be made in the Vh and the Vl regions to increase yield. Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (1), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

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Thus, one of skill in the art can readily review SEQ ID NOs: 1-2 and 5-10, locate one or more of the amino acids in the brief table above, identify a conservative substitution, and produce the conservative variant using well-known molecular techniques.

Effector molecules, such as therapeutic, diagnostic, or detection moieties can be linked to an antibody that specifically binds an antigen of interest, using any number of means known to those of skill in the art. Both covalent and noncovalent attachment means may be used. The procedure for attaching an effector molecule to an antibody varies according to the chemical structure of the effector. Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (-ΝΗ_Ζ) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of the effector molecule. Alternatively, the antibody is derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of any of a number of linker molecules such as those available from Pierce Chemical Company, Rockford, IL. The linker can be any molecule used to join the antibody to the effector molecule. The linker is capable of forming covalent bonds to both the antibody and to the effector molecule. Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where the antibody and the effector molecule are polypeptides, the linkers may be joined to the constituent amino adds through their side groups (such as through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.

In view of the large number of methods that have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, label (such as enzymes or fluorescent molecules) drugs, toxins, and other agents to antibodies one skilled in the art will be able to determine a suitable method for attaching a given agent to an antibody or other polypeptide.

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XI. Methods of Producing Antibodies using DNA Generally

Exemplary nucleic acids encoding sequences encoding an antibody that specifically binds an antigen of interest can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques, and instructions sufficient to direct persons of skill through many cloning exercises are found in Sambrook et al., supra, Berger and Kimmel (eds.), supra, and Ausubel, supra. Product information from manufacturers of biological reagents and experimental equipment also provide useful information. Such manufacturers include the SIGMA Chemical Company (Saint Louis, MO), R&amp;D Systems (Minneapolis, MN), Pharmacia Amersham (Piscataway, NJ), CLONTECH Laboratories, Inc. (Palo Alto, CA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, WI), Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg, MD), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland), Invitrogen (San Diego, CA), and Applied Biosystems (Foster City, CA), as well as many other commercial sources known to one of skill.

Nucleic acids can also be prepared by amplification methods. Amplification methods include polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (3SR). A wide variety of cloning methods, host cells, and in vitro amplification methodologies are well known to persons of skill.

In one example, an antibody of use is prepared by inserting the cDNA which encodes a variable region from an antibody that specifically binds an antigen of interest into a vector.

Once the nucleic acids encoding the antibody or fragment thereof are isolated and cloned, the protein can be expressed in a recombinantly engineered cell such as bacteria, plant, yeast, insect and mammalian cells. One or more DNA sequences encoding the antibody or fragment thereof can be expressed in vitro by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that ail progeny may not be identical to the parental cell since there may be mutations that occur during replication. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.

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Polynucleotide sequences encoding the antibody or fragment thereof can be operatively linked to expression control sequences. An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences. The expression control sequences include, but are not limited to appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.

The polynucleotide sequences encoding the antibody or fragment thereof can be inserted into an expression vector including, but not limited to a plasmid, virus or other vehicle that can be manipulated to allow insertion or incorporation of sequences and can be expressed in either prokaryotes or eukaryotes. Hosts can include microbial, yeast, insect and mammalian organisms. Methods of expressing

DNA sequences having eukaryotic or viral sequences in prokaryotes are well known in the art. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art.

Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art. Where the 20 host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl₂ method using procedures well known in the art. Alternatively, MgCh or RbCI can be used. Transformation can also be performed after forming a protoplast of the host cell if desired, or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate coprecipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used. Eukaryotic cells can also be cotransformed with polynucleotide sequences encoding the antibody or fragment thereof, and a second 30 foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform 231

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PCT/US2007/023140 eukaryotic cells and express the protein (see for example, Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). One of skill in the art can readily use an expression systems such as plasmids and vectors of use in producing proteins in cells including higher eukaryotic cells such as the COS, CHO, HeLa and 5 myeloma cell lines.

Isolation and purification of recombinanlly expressed polypeptide can be carried out by conventional means including preparative chromatography and immunological separations. Once expressed, the recombinant antibodies can be purified according to standard procedures of the art, including ammonium sulfate 10 precipitation, affinity columns, column chromatography, and the like (see, generally, R. Scopes, Protein Purification, Springer-Verlag, N.Y., 1982). Substantially pure compositions of at least about 90 to 95% homogeneity are disclosed herein, and 98 to 99% or more homogeneity can be used for pharmaceutical purposes. Once purified, partially or to homogeneity as desired, if to be used therapeutically, the polypeptides should be substantially free of endotoxin.

Methods for expression of single chain antibodies and/or refolding to an appropriate active form, including single chain antibodies, from bacteria such as E. coli have been described and arc well-known and are applicable to the antibodies disclosed herein. See, Buchner et al., Anal. Biochem. 205:263-270,1992;

Pluckthun, Biotechnology 9:545, 1991; Huse et al., Science 246:1275,1989 and Ward et al., Nature 341:544,1989.

Often, functional heterologous proteins from E. coli or other bacteria are isolated from inclusion bodies and require solubilization using strong denaturants, and subsequent refolding. During the solubilization step, as is well known in the art, 25 a reducing agent must be present to separate disulfide bonds. An exemplary buffer with a reducing agent is: 0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (di thioerythritol). Rcoxidation of the disulfide bonds can occur in the presence of low molecular weight thiol reagents in reduced and oxidized form, as described in Saxena et al., Biochemistry 9: 5015-5021,1970, and especially as described by Buclmer et al., supra.

Renaturation is typically accomplished by dilution (for example, 100-fold) of the denatured and reduced protein into refolding buffer. An exemplary buffer is 0.1 232

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M Tris, pH 8.0,0.5 M L-arginine, 8 mM oxidized glutathione (GSSG), and 2 mM EDTA.

As a modification to the two chain antibody purification protocol, the heavy and light chain regions are separately solubilized and reduced and then combined in the refolding solution. An exemplary yield is obtained when these two proteins are mixed in a molar ratio such that a 5 fold molar excess of one protein over the other is not exceeded. It is desirable to add excess oxidized glutathione or other oxidizing low molecular weight compounds to the refolding solution after the redox-shuffling is completed.

In addition to recombinant methods, the antibodies disclosed herein can also be constructed in whole or in part using standard peptide synthesis. Solid phase synthesis of the polypeptides of less than about 50 amino acids in length can be accomplished by attaching the C-terminal amino acid of the sequence to an insoluble support followed by sequential addition of the remaining amino acids in the sequence. Techniques for solid phase synthesis are described by Barany &amp; Merrifield, The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A. pp. 3-284; Merrifield et al., J. Am. Chem. Soc. 85:21492156, 1963, and Stewart et al., Solid Phase Peptide Synthesis, 2nded., Pierce Chem. Co., Rockford, Ill., 1984. Proteins of greater length maybe synthesized by condensation of the amino and carboxyl termini of shorter fragments. Methods of forming peptide bonds by activation of a carboxyl terminal end (such as by the use of the coupling reagent N, N'-dicycylohexylcarbodiimide) arc well known in the art.

XII. Antigens

Exemplary antigens of interest include those listed below:

Table 1

Exemplary antigens of interest (target antigens)

<td> Viral Target Antigens</td><td> Exemplary Target Antigen Sequences from the Target Antigens</td><td> SEQ1D NO:</td>

<td> BK</td><td> TLYKKMEQDVKVAHQ GNLPLMRKAYLRKCK TFSRMKYNICMGKCI</td><td> 1 22 23</td>

<td> JC</td><td> SITEVECFL</td><td> 2</td>

<td> Epstein-Barr (EBV)</td><td> QPRAPIRPI</td><td> 3</td>

<td> cytomegalovirus (CM V)</td><td> NLVPMVATV</td><td> 4</td>

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<td> HPV</td><td> YMLDLQPET(T)</td><td> 5</td>

<td> Influenza A</td><td> G1LGFVFTL</td><td> 6</td>

<td></td><td></td><td></td>

<td> Tumor Target Antigens and their derivative peptides</td><td></td><td></td>

<td> PRAME</td><td> LYVDSLFFL</td><td> 7</td>

<td> WTl</td><td> RMFPNAPYL</td><td> 8</td>

<td> Survivin</td><td> ELTLGEFLKL</td><td> 9</td>

<td> AFP</td><td> GVALQTMKQ</td><td> 10</td>

<td> ELF2M</td><td> ETVSEQSNV</td><td> 11</td>

<td> proteinase 3 and its peptide PR1</td><td> VLQELNVTV</td><td> 12</td>

<td> neutrophil elastase</td><td> VLQELNVTV</td><td> 13</td>

<td> MAGE</td><td> EADPTGHSY</td><td> 14</td>

<td> MART</td><td> AAGIGILTV</td><td> 15</td>

<td> tyrosinase</td><td> rhrplqevypeanapighnre</td><td> 16</td>

<td> GP100</td><td> WNRQLYPEWTEAQRLD</td><td> 17</td>

<td> NY-Eso-1</td><td> VLLKEFTVSG</td><td> 18</td>

<td> Herceptin</td><td> KIFGSLAFL</td><td> 19</td>

<td> carcino-embryonic antigen (CEA)</td><td> HLFGYSWYK</td><td> 20</td>

<td> PSA</td><td> FLTPKKLQCV</td><td> 21</td>

<td></td><td></td><td></td>

<td> Fungal Target Antigen</td><td></td><td></td>

<td> Blastomyces dermatitidis</td><td> CELDNSHEDYNWNLWFKWCSGHGR TGHGKHFYDCDWDPSHGDYSWYLW DPSHGDYSWYLWDYLCGNGHHPYD DYLCGNGHHPYDCELDNSHEDYSW dpyncdwdpyhekydwdlwnkwcn KYDWDLWNKWCNKDPYNCDWDPYH</td><td> 24 25 26 27 28 29</td>

Table 2 Exemplary tumors and their tumor antigens

<td> Tumor</td><td> Tumor Associated Target Antigens</td>

<td> Acute myelogenous leukemia</td><td> Wilms tumor 1 (WT1 ), preferentially expressed antigen of melanoma (PRAME), PR1, proteinase 3, elastase, cathepsin G</td>

<td> Chronic myelogenous leukemia</td><td> WTl, PRAME» PR 1» proteinase 3» elastase, cathepsin G</td>

<td> Myelodysplastic syndrome</td><td> WTl, PRAME, PR1, proteinase 3, elastase, cathepsin G</td>

<td> Acute lymphoblastic leukemia</td><td> PRAME</td>

<td> Chronic lymphocytic leukemia</td><td> Surviving</td>

<td> Non-Hodgkin’s lymphoma</td><td> Surviving</td>

<td> Multiple myeloma</td><td> New Yorkesophageous 1 (NY-Esol)</td>

<td> Malignant melanoma</td><td> MAGE, MART, Tyrosinase, PRAME GP100</td>

<td> Breast cancer</td><td> WTl, herccptin</td>

<td> Lung cancer</td><td> WTl</td>

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<td> Prostate cancer</td><td> Prostate-specific antigen (PSA)</td>

<td> Colon cancer</td><td> Carcinoembryonic antigen (CEA)</td>

<td> Renal cell carcinoma (RCC)</td><td> Fibroblast growth factor 5 (FGF-5)</td>

Any antigenic peptide (such as an immunogenic fragment) from an antigen of interest can be used to generate a population of T cells specific for that antigen of interest. Numerous such antigenic peptides are known in the art, such as viral and tumor antigens. This disclosure is not limited to using specific antigen peptides. Particular examples of antigenic peptides from antigens of interest, include, but are not limited to, those antigens that are viral, fungal, and tumor associated, such as those shown in Table 1. Additional antigenic peptides are known in the art (for example see Novellino et al., Cancer Immunol. Immunother. 54(3):187-207,2005, and Chen et al., Cytotherapy, 4:41-8,2002.

Although Table 1 discloses particular fragments of full-length antigens of interest, one skilled in the art will recognize that other fragments or the full-length protein can also be used in the methods disclosed herein. In one example, an antigen of interest is an “immunogenic fragment of a full-length antigen sequence. An 15 “immunogenic fragment” refers to a portion of a protein which can be used to induce an immune response, such as a B cell response, such as the production of antibodies. Typically, such fragments'are 8 to 12 contiguous amino acids of a full length antigen, although longer fragments may of course also be used. In particular examples, the immunogenic fragment is 8-100 contiguous amino acids from a fiill20 length target antigen sequence, such as 8-50 amino acids, 8-20 amino acids, or 10, 20, 30,40, 50, 100 or 200 contiguous amino acids from a full-length target antigen sequence.

Through the study of single amino acid substituted antigen analogs and the sequencing of endogenously bound, naturally processed peptides, critical residues 25 that correspond to motifs required to produce antigenic molecules have been identified (see, for example, Southwood et al., J. Immunol. 160:3363,1998; Rammensee et al., Immunogenetics 41:178, 1995; Ratnmensee et al., J. Curr. Opin. Immunol. 10:478, 1998; Engelhard, Curr. Opin. Immunol. 6:13,1994; Setteand . Grey, Curr. Opin. Immunol. 4:79, 1992).

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In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention.