WO2008140595A2 - Synthetic trivalent haptens, complexes thereof, and uses therefor - Google Patents

Abstract

The invention provides trivalent hapten molecules (trihapten molecules) and complexes thereof. The trivalent hapten molecules are useful for forming relatively stable complexes comprising hapten molecules and divalent ligands (e.g., antibodies) for the hapten molecules. Such trihapten molecules are useful for assays, e.g., of antibodies, or for depleting a ligand from a sample, e.g., for treating a disease by binding an undesirable receptor such as an antibody. Complexes of trivalent hapten molecules are useful for selectively targeting relatively high density, multivalent presentations of haptens, such as occur on cells overexpressing a molecule on their surfaces, such as cancer cells.

Description

SYNTHETIC TRIVALENT HAPTENS, COMPLEXES THEREOF, AND USES THEREFOR

FIELD OF THE INVENTION

This application relates to the field of multivalent binding molecules.

CROSS REFERENCE TO RELATED APPLICATION This application claims priority to U.S. Provisional Application No. 60/872,374, filed December 1, 2006, which is hereby incorporated by reference in its entirety.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT The U.S. Government may have certain rights in this invention pursuant to Grant No. GM30367 awarded by the National Institutes of Health.

BACKGROUND

Multi valency, the simultaneous binding of multiple ligands on one entity to multiple receptors on another, is important throughout biology. As one example, antibodies bind multivalently to their targets. Antibodies are a type of protein that can bind to antigens including antigens on cell surfaces.

SUMMARY

The invention relates to trivalent hapten molecules (trihapten molecules) and complexes thereof. Trivalent hapten molecules can create aggregates when bound to a multivalent ligand, such as a divalent ligand. Antibodies are divalent ligands that specifically bind hapten moieties. It has been found that trihapten molecules as described herein can be used to form stable aggregates of the trihapten molecules and divalent ligands of the molecule. Such molecules are useful, e.g., in laboratory assay applications including diagnosis of disorders, and for treatment of disorders in which it is desirable to bind a ligand (e.g., and effectively inhibit activity of the ligand) such as an antibody that binds the trihapten molecule, in an organism.

Accordingly, the invention relates to the following embodiments. One embodiment of the invention relates to a trihapten molecule of Formula I having flexible anus,

Z Z

Formula I where

X is a trivalent core structure;

Y is a linker moiety that is covalently linked to the core structure; and Z is a hapten moiety that is covalently linked to the linker moiety, where the hapten moieties have the same structure; or a pharmaceutically acceptable salt thereof.

The core structure of the trihapten molecule can have three-fold symmetry. In some cases, the length of the linker (Y) can be from about 1.5 nm to about 9 nm. In one embodiment, X can be:

Formula II; Formula III, Formula IV;

Formula V; Formula VI; Formula VII; Formula VIII; /γ^θχ A

Formula IX Formula X FoiTnula XI; Formula

XII.

where Q is NH, O, or S; or

SiR], wherein Ri is H, -O-Cj-C_ό alkyl, or OH. In a specific case, X can be N.

In another embodiment, Y can include an amino acid or an ethylene glycol. In some cases, Y includes:

where n is an integer from 2 to 20, and m is an integer from 1 to 20. In one embodiment, the trihapten molecule can have an antibody bound to the trihapten molecule, creating a trihapten/antibody complex, in a ratio of trihapten molecule:antibody of 2:3. In a specific embodiment, the /C_/ for the trihapten/antibody complex is smaller than the K_cι for the antibody bound to a monohapten molecule. In one case, the complex includes two trihapten molecules of claim 1 and three antibody molecules bound to the trihapten molecules, where the antibody molecules bind to the haptens of the trihapten molecules.

In some cases, the trivalent molecule includes a hapten or antigen that is a toxin, a peptide, a peptoid, a peptidomimetic, a small non-nucleic acid organic molecule, a small nucleic acid molecule, an aptamer, or a drug. In a specific embodiment, the hapten (Z) can be:

O₉N

a member of the epidermal growth factor receptor family of receptor tyrosine kinases, an anthrax antigen, a Sm antigen, or sialic acid. In some cases (Z) is HER-I, HER-2, HER-3, HER-4, or 2, 4-dinitrophenol.

In a specific embodiment, the invention relates to a trihapten molecule of Formula 1 :

1, or a pharmaceutically acceptable salt thereof.

In another embodiment, the invention relates to a method that includes providing a trihapten molecule of claim 1 having a selected hapten; contacting the trihapten molecule with a sample comprising an antibody that specifically binds the hapten; and determining the amount of antibody bound to the hapten.

Another aspect of the invention relates to a method that includes providing a trihapten molecule of claim 1 , where the trihapten molecule has a selected hapten moiety; contacting the trihapten molecule with an antibody that specifically binds the selected hapten moiety, thereby forming a trihapten/antibody complex; contacting the trihapten-antibody complex with a sample comprising the antibody; and determining the amount of antibody displaced from the trihapten-antibody complex by the sample antibody. The amount of antibody displaced can be determined by assaying a decrease in the trihapten/antibody complex. In one embodiment, the sample antibody, or the trihapten molecule, is labeled. In another embodiment, the invention relates to a method of binding a selected antibody in a stable complex, including providing a trihapten molecule that can bind to the selected antibody; contacting a sample comprising the selected antibody with the trihapten molecule, forming a trihapten/antibody mixture; and incubating the trihapten/antibody mixture under conditions sufficient to permit binding of the trihapten molecule and the antibody and formation of a stable trihapten/antibody complex. In some cases, the selected antibody is from a mammal, such as a human, and the selected antibody can be a disease-associated antibody. In an embodiment, the stable antibody/trihapten complex has a ratio of antibody:trihapten molecule of 3:2.

In yet another embodiment, the invention relates to a method of delivering an antibody to a subject, including providing a complex comprising an antibody and a trihapten molecule; and administering the complex to a subject, where the subject and the trihapten molecule include the same hapten and the antibody is can bind to the hapten.

In some embodiments, the subject is a mammal, such as a rat, a mouse, a dog, a cat, a pig, a goat, a cow, a non-human primate, or a human. In another embodiment, the antibody can bind to an antigen associated with or causing a disease or a symptom of the disease. In some cases, a cell of the subject presents a hapten that can bind to the antibody. The cell can be a disease-associated cell, such as a cancer cell, or more specifically, a tumor cell. In some embodiments, the cell overexpresses the hapten on its surface. And in some cases, the hapten is a disease-associated hapten. The method can employ a complex of antibody and trihapten molecule with a ratio of antibody:trihapten of 3:2, where the antibodies bind to the haptens of the trihapten molecule.

Another embodiment of the invention relates to a method of delivering an agent to a subject, including providing a complex including an antibody, an agent, and a trihapten molecule; and administering the complex to a subject having a disease that the agent can treat where the subject and the trihapten molecule both include a hapten and the antibody is capable of binding to the hapten, he method of claim 36, wherein the subject is a mammal, such as a rat, a mouse, a dog, a cat, a pig, a goat, a cow, a non-human primate, or a human. The agent can be attached to the antibody or can be attached to the trihapten molecule. The agent can also be a label, or a drug, such as a drug for treating cancer or an autoimmune disease. In some aspects of the method, a cell of the subject includes a hapten. The cell can also be a disease-associated cell, such as a cancer cell, such as a tumor cell. The cell can overexpress the hapten on the surface of the cell. The hapten can also be a disease-associated hapten. In some cases, the complex has a ratio of antibodyitrihapten molecule of 3:2, where the antibodies bind to the haptens of the trihapten molecule.

Some other embodiments of the invention relate to a method including providing a trihapten molecule of claim 1 having a selected hapten; contacting the trihapten molecule with a sample comprising an antibody that specifically binds to the hapten; and determining the amount of the antibody bound to the trihapten molecule.

Still other embodiments of the invention relate to a method of determining the relative affinity of a trihapten molecule/antibody complex for a surface, including providing a first surface that has a first plurality of a hapten that can bind to a selected antibody; contacting the first surface with a complex that includes a trihapten molecule/antibody complex, where the antibody of the complex is the selected antibody and the haptens of the complex are the same as the haptens attached to the first surface; determining the amount of the antibody bound to the first surface, providing a first amount; and comparing the first amount with a second amount, such that the second amount is determined by contacting a second surface with a complex that includes the trihapten molecule/antibody complex, where the second surface has a second plurality of haptens, the antibody of the complex is the selected antibody, the haptens of the complex can bind to the selected antibody and are the same as the haptens attached to the second surface, and the density of the second plurality haptens on the second surface is different from the density of the first plurality of haptens son the first surface; and determining the amount of the antibody bound to the second surface, thereby providing a second amount. Yet other embodiments of the invention relate to a method of determining the amount of an antibody in a sample, including providing a sample to be tested for the presence of an antibody against a selected antigen; contacting the sample with a trihapten molecule, such that the hapten of the trihapten molecule can bind an antibody against the selected antigen, providing an assay sample; incubating the assay sample for a time long enough to allow binding of the trihapten molecule and antibody against the selected antigen; and detecting the amount of antibody bound to the trihapten molecule. In some embodiments of the method the sample contains a cell, and/or the hapten is a cell-surface antigen. In some more specific embodiments of the method, the hapten is a Sm antigen, an anthrax antigen, sialic acid,

a member of the epidermal growth factor receptor family of receptor tyrosine kinases, such as HER-I, HER-2, HER-3, or HER-4.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a reproduction of a set of Size Exclusion (SE)-High Performance Liquid Chromatography (HPLC) chromatograms of complexes of IgG^DNP and 1 (at [1] = 0.0-1.2 μM and [IgG^DNP] = 0.6 μM). The top graph calibrates the column against proteins with relevant molecular weights.

Fig. 2 is a graph depicting the mole fraction (lines) produced by fitting the equilibrium model described in the text to the data (markers) from SE-HPLC experiments ([IgG^DNP] was kept constant 0.6 μM). The error bars are from peak integrations of four separate experiments; each datum is the mean of these measurements and the error bars show the maximum deviation.

Fig. 3 A is a bar graph depicting the diameters of the complexes present in solutions (PBS buffer, pH = 7.4, 25 ⁰C) with [l]:[IgG] = 2:3, as measured by DLS, for IgG^DNP alone, for B-E incubation intervals are shown on the plot.

Fig. 3B is a bar graph depicting the diameters of the complexes present in solutions (PBS buffer, pH = 7.4, 25 ⁰C) with [l]:[IgG] = 2:3, as measured by DLS for the incubation interval as shown in the Figure. Fig. 3 C is a bar graph depicting the diameters of the complexes present in solutions (PBS buffer, pH = 7.4, 25 ⁰C) with [l]:[IgG] = 2:3, as measured by DLS for the incubation interval as shown in the Figure.

Fig. 3D is a bar graph depicting the diameters of the complexes present in solutions (PBS buffer, pH = 7.4, 25 ⁰C) with [l]:[IgG] = 2:3, as measured by DLS for the incubation interval as shown in the Figure.

Fig. 3 E is a bar graph depicting the diameters of the complexes present in solutions (PBS buffer, pH = 7.4, 25 ⁰C) with [l]:[IgG] = 2:3, as measured by DLS for the incubation interval as shown in the Figure. Fig. 4 is a pair of graphs depicting the results of an AUC equilibrium experiment of 0.10 μM anti-DNP IgG incubated with 0.067 μM 1 at 6K rpm as observed at 230 nm at 25 ⁰C. The hollow circles are experimental data and the line inside is the fit for a single ideal species. The expected molecular weight is ~ 450 ± 12 kDa and the calculated molecular weight is 464 ± 35 kDa. Fig. 5 is a schematic drawing of the predicted three-dimensional structure of the trivalent molecule and complexes with IgG.

Fig. 6 is a schematic drawing of Scheme 1, the structure proposed for IgG₃Io.

Fig. 7 is a drawing depicting antibodies binding to their antigens (such as a surface receptor) on a normal cell and a target cell. Fig. 8 is a drawing depicting antibodies as bicyclic complexes with a trihapten molecule, binding to a target cell more selectively than to a non-target cell.

Fig. 9 depicts an ELISA assay for selective binding.

Fig. 10 depicts the results of the ELISA assay for selective binding.

DETAILED DESCRIPTION

In one embodiment described herein is a new type of trihapten molecule that can be used to foπii a new type of structured aggregate that is composed of antibodies and trihapten- containing molecules. Antibodies contain paired heavy and light polypeptide chains, and the generic term immunoglobulin is used for all such proteins.

Within this general category, however, five different classes of immunoglobulins IgM, IgD, IgG, IgA, and IgE are known. Antibody molecules are roughly Y-shaped molecules consisting of three equal-sized portions, loosely connected by a flexible tether. Two portions are identical and contain the antigen-binding activity. The Fab fragments ("Fragment antigen binding") correspond to the two identical arms of the antibody molecule. A multivalent ligand can be an antibody. As used herein, "ligand" refers to a molecule that binds to a hapten, e.g. an antigen. In general, such antibodies are described herein with reference to IgG immunoglobulins. However, it is understood that the trihapten-antibody structured aggregate can include any class of immunoglobulin or fragment of an immunoglobulin that can bind two antigenic sites (e.g., haptens). The term antigen and hapten are used interchangeably throughout the specification. The temi antibody/trihapten complex encompasses a bicyclic antibody trihapten aggregate.

Trihapten Molecules

Trivalent hapten molecules are useful for binding multivalent molecules. The hap ten-containing molecule is a molecule having three haptens. The haptens can be equally spaced about the core structure. The haptens can also be attached through flexible arms of the molecule to a trivalent central atom or moiety (a trihapten molecule; THM).

Trihapten molecules of the invention include any molecule of Formula I having flexible amis,

Z

Formula I such that,

X is a trivalent core structure;

Y is a linker moiety that is covalently linked to the core structure; and

Z is a hapten moiety that is covalently linked to the linker moiety, wherein the hapten moieties have the same structure; or a pharmaceutically acceptable salt thereof. In one embodiment, X is any trivalent group or atom to which the linkers can be covalently linked. Examples of such core structures include, without limitation, trivalent cyclic groups such as Formulae II, IV, IX, X, XI, and XII, and trivalent atoms such as Formula III, Formula V, Formula VI, Formula VII and Formula VIII, below. In a more specific embodiment, X is:

Formula II, Formula III, Formula IV,

Formula V, Formula Vl, Formula VII, Formula VIII,

Formula IX, Formula X, Formula XI, or Formula

XII;

where Q is NH, O, or S.

Alternatively, X can be SiRi, wherein R] is H, -O-Cj-C_ό alkyl, or OH. Valency, as it pertains to an atom such as those depicted in the formulae above is a measure of the number of bonds that can be formed by the atom. In the case of trivalent moieties containing more than one atom, valency refers to the number of bonds that can be formed between the moiety and the other chemical groups of the trihapten, such as the Y groups. The core structure may possess 3-fold symmetry, that is, the valency or bonds to three linkers, (Y), are symmetrically distributed about the core structure.

In one embodiment, the trivalent moiety (X) includes a submoiety derived from the covalent linking of (X) with (Y). In an embodiment, the submoiety can be an amide, ether, ester, amine urea, thiourea, or thioamide. In another embodiment, the submoiety is derived from a carboxylic acid including an activated carboxylic acid such as an acid chloride or succinimide; a sulfonyl chloride; an amine; a thiol; an alkylhalide, such as an alkylbromide; an alkylsulfonate, such as tosylate or mesylate; or a cyanate, such as an isocyanate, a thiocyanate, and an isothiocyanate. Each linker (Y) can be attached to (X) through identical or different submoieties.

The linker moiety, (Y), is selected to provide sufficient length and flexibility to the trihapten molecule to permit two haptens of the trihapten molecule to bridge the two binding sites on a single antibody or to bridge binding sites of separate antibodies. The linker also can be selected to have a desired solubility. For example, an oligo ethylene glycol linker may be included to increase the water solubility of the linker and the trihapten molecule. The linker further can include a submoiety derived from the covalent linking of (X) with (Y). In an embodiment, the submoiety can be an amide, ether, ester, amine urea, thiourea, or thioamide. In another embodiment, the submoiety is derived from a carboxylic acid including an activated carboxylic acid such as an acid chloride or succinimide; a sulfonyl chloride; an amine; a thiol; an alkylhalide, such as an alkylbromide; an alkylsulfonate, such as tosylate or mesylate; or a cyanate, such as an isocyanate, a thiocyanate, and an isothiocyanate. Each linker (Y) can be identical or different to the other linkers.

In some embodiments, the linker has a length of about 1.5 nm to about 9 nm, or from about 2 nm to about 5 nm.

In one embodiment, (Y) includes the structure:

Yi-Y₂-Y₃, where

Yi is a submoiety derived from functionality capable of being covalently attached to the core structure (X);

Y₂ is a submoiety linking Yi and Y₃; and

Y₃ is a submoiety derived from functionality capable of being covalently attached to a hapten (Z). In some embodiments, the linker (Y) includes, without limitation, oligo ethylene glycol, oligo amino acids such as oligo sarcosine, oligo glycine, and oligo proline, oligosaccharides, oligo deoxyribonucleic acids (oligo DNA), and oligo ribonucleic acids (oligo RNA). For example, Y₂ can include ethylene glycol; sarcosine; glycine; sugars; amino acids and homologs thereof, such as amino acid chains that include glycine, proline, or lysine; deoxyribonucleic acids (oligo DNA); or ribonucleic acids (oligo RNA) as building blocks. In another embodiment, Y₂ includes chains of such building blocks, such as oligo ethylene glycol, oligo sarcosine, oligo glycine, oligo saccharides, oligo proline, oligo deoxyribonucleic acids (oligo DNA), and oligo ribonucleic acids (oligo RNA).

In another embodiment, the linker (Y) includes a carbon backbone, which may be substituted, such as to aid solubility, add length, or present functionality for attachment to a core structure (X) or hapten (Z). The substituted carbon backbone can include natural or unnatural non-glycine amino acid building blocks such as arginine, lysine, proline, alanine, leucine, isoleucine, valine, serine, aspartate, glutamate, tryptophan, phenylalanine, tyrosine, cysteine, glutamine, or histidine. The carbon backbone can be substituted with such groups as carboxylic acids, sulfonic acids, amines, guanidiniums, or the like.

In specific embodiments, the linker (Y) or YT can include one or more of the following moieties:

wherein n is an integer from 2 to 20, and m is an integer from 1 to 10.

In a specific embodiment, n is 8. In another specific embodiment, m is 1. In another embodiment, at one end the linker (Y) terminates in Yi which derives from functionality that allows the linker to be covalently attached to the core structure (X), and at another end terminates in Y₃ which derives from functionality that allows the linker to be covalently attached to the hapten (Z). In an embodiment, the submoiety can be an amide, ether, ester, amine urea, thiourea, or thioamide. In another embodiment, the submoiety is derived from a carboxylic acid including an activated carboxylic acid such as an acid chloride or succinimide; a sulfonyl chloride; an amine; a thiol; an alkylhalide, such as an alkylbromide; an alkylsulfonate, such as tosylate or mesylate; or a cyanate, such as an isocyanate, a thiocyanate, or an isothiocyanate. Each (Yi) or (Y₃) can be an identical or different submoiety.

In another embodiment, the linker (Y) is about 3.2 nm long. In a specific embodiment, Y₂ is an oligo ethylene glycol linker, such as an ethylene glycol linker having 5 to 10 ethylene glycol units. In another specific embodiment, the linker (Y) is:

In an embodiment, the separation between two hapten molecules energetically favors formation of a bicyclic structure of 2 trihapten molecules and 3 ligands, and a linker is selected accordingly. The linkers (Y), or molecules that can be used to synthesize the linkers (Y), can be purchased commercially such as from Avanti Polar Lipids, made by methods as disclosed in the Examples section below, or made by methods known to a person of skill in the art. Linkers (Y), core structure (X), and haptens (Z) can be assembled into trihapten molecules by methods disclosed herein and by methods known to a person of skill in the art.

The hapten (Z) can be covalently linked to the linker (Y) either directly or through another group. A hapten can be any molecule that can be specifically bound by an antibody or similar molecule (e.g., a Fab fragment or F(ab')2) when the hapten is incorporated into the trihapten molecule. By "specifically binds" is meant that a molecule binds to a particular entity in a sample, but does not significantly bind to other entities in the sample, e.g., a biological sample, that includes the particular entity. In one embodiment, a molecule or complex binds to a particular hapten in a sample, but not significantly to other molecules in the sample. In another embodiment, a molecule or complex binds to a particular presentation of haptens, but not significantly to another different presentation of haptens. One difference in presentation can be the density of haptens on a surface. Useful haptens include haptens that are presented on the surface of a disease-associated cell, such as antigens associated with autoimmune diseases or cancer. Autoimmune antigens include Sm antigen and arthritis antigens. Cancer antigens include members of the epidermal growth factor receptor family of receptor tyrosine kinases, such as human epidermal growth factor receptor 2 (HER-2, also known as Her-2/neu, ErbB2), EGFR (HER-I, ErbBl), HER-2, HER-3 (ErbB3) or HER-4 (ErbB4). Other useful haptens include molecules suitable for assays such as toxins, peptides, peptoids, peptidomimetics, monosaccharides, oligosaccharides, small non-nucleic acid organic molecules such as DNP (2,4-dinitrophenyl), small nucleic acid molecules (natural or synthetic), aptamers, or drugs. Such molecules can be, for example, antigens derived from viruses (e.g., disease viruses), antigens derived from bacteria, or antigens derived from allergens. Other suitable haptens include molecules that bind to antibodies that are to be assayed. Such antibodies include antibodies that are associated with a disease, for example, antibodies that are useful for diagnosis of a disease, for monitoring the treatment of a disease, antibodies whose presence contributes to a disease, or antibodies that are capable of treating a disease. In a specific embodiment, a hapten (Z) includes a submoiety that derives from functionality that allows the hapten to be covalently bonded to a linker (Y). In an embodiment, the submoiety can be an amide, ether, ester, amine urea, thiourea, or thioamide. In another embodiment, the submoiety is derived from a carboxylic acid including an activated carboxylic acid such as an acid chloride or succinimide; a sulfonyl chloride; an amine; a thiol; an alkylhalide, such as an alkylbromide; an alkylsulfonate, such as tosylate or mesylate; or a cyanate, such as an isocyanate, a thiocyanate, or an isothiocyanate. Each hapten (Z) can be bonded to its linker (Y) through a submoiety that is identical or different from the other hapten-linker submoieties.

In another specific embodiment, (Z) is

In yet another specific embodiment, (Z) is human epidermal growth factor receptor 2 (HER-2, also known as Her-2/neu, ErbB2). In some specific embodiments, (Z) is a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, such as EGFR (HER-I , ErbBl), HER-2, HER-3 (ErbB3) or HER-4 (ErbB4).

In still another specific embodiment, (Z) is an anthrax antigen. In another embodiment, (Z) is a Sm antigen, an arthritis antigen, or sialic acid. In general, the separation of the between arms of the trihapten, as measured from hapten to hapten, can be from about 6.0 nm to about 9 nm, e.g., 6.4 nm to 8 nm, although other lengths can be used depending, for example, on the size of the hapten and the ligand. In general, the distance from the core to the hapten is from about 1.5 to 9 nm. Suitable lengths for specific molecules can be determined by identifying lengths that favor formation of bicyclic complexes.

Trihapten molecules of the invention can form stable complexes comprising three hapten-binding molecules, such as antibodies, and two trihapten molecules, i.e., trihapten molecules according to one or more embodiments described herein. Such complexes can be referred to as "antibody/trihapten complexes," and can also be referred to as "bicyclic antibody trihapten aggregates," which term is encompassed by

'"antibody/trihapten complexes." In one instance, when bound to an antibody in a ratio of trihapten molecule:antibody of 2:3 to form a complex, the complex has a Kj that is smaller than the IQ for the ligand bound to a monohapten molecule, i.e. the complex is more stable than the monovalent interaction.

In one instance, as shown in Fig. 7, an antibody binds to its antigen (a surface receptor) whether it is on a normal cell or the target cell. Cells that have the receptor overexpressed on their surfaces (target cells) will accumulate more antibody molecules than normal cells that do not have the receptor overexpressed on their surfaces.

In another embodiment, Fig. 8 depicts antibodies and trihapten molecules as aggregates in the form of complexes, binding to a target cell more selectively than to a non-target cell. The arrows depict the direction of equilibrium favored for each hapten-covered surface. On a surface with lower density of haptens, the equilibrium of the complex favors the receptor being bound to the trihapten molecule instead of the surface. However, on a surface with a higher density of haptens, the antibodies favor binding to the surface instead of to the trihapten molecule. When the antibody is introduced as bicyclic complexes, the delivery of the antibody to the target cell can be more selective, and antibody binding to normal cells and any associated nonspecific toxicity can be reduced. Specific targeting of the target cell over the normal cell can be accomplished if the equilibrium of the targeting agent, such as an antibody/trihapten complex, favors binding to cells presenting a greater density of a hapten.

The ability of trihapten molecules according to one or more embodiments of the invention to form stable complexes with antibodies finds many potential practical applications. Examples of potential practical applications include laboratory assay applications including diagnosis of disorders; treatment of disorders in which it is desirable to remove a ligand, such as an antibody that binds the trimeric molecule, from an organism; increasing the selectivity of binding or delivery of an antibody or an agent that also forms part of stable complex; decreasing non-selective binding of the antibody; selectively targeting cells that express or overexpress a hapten; and improved treatment of diseases using antibodies, including decreasing the nonspecific binding of the antibody and undesirable side effects of antibody treatments.

To test the characteristics of a trihapten molecule, a model system was developed using a rat IgG antibody that binds with high affinity to 2,4-dinitrophenyl (DNP) groups. The IgG^DNP is commercially available, and the synthesis of oligovalent antigens presenting DNP groups can be accomplished using methods known to those in the art, such as are disclosed in the Examples section, below. The IgG^DNP used in the model system has an unusually high affinity for DNP and derivatives. This affinity makes the development of assays for these types of aggregation more straightforward than for more weakly binding systems. Incubation of the trivalent hapten 1 with IgG^DNP yields stable, bicyclic aggregates with stoichiometry IgG₃I₂ (Scheme 1 ; Fig. 6) as one example of an antibody/trihapten complex. These aggregates were characterized using size-exclusion high- performance liquid chromatography (SE-HPLC), analytical ultracentrifugation (AUC) and dynamic light scattering (DLS).

In the model system, the assembly of the stoichiometrically defined supramolecular structure of an antibody/trihapten complex proceeds in competition with polymerization (as established through DLS experiments; Example 2). The structure IgG₃I₂ appears to be the most stable species in the concentration ranges that were examined ([IgG]₀ = 3.3-0.6 μM and [I]₀ = 2.2-0.4 μM).

In the fully extended conformation, the DNP moieties of the trivalent molecule 1 are sufficiently far apart (approximately 6.4 nm) that they can, in principle, bridge the two binding sites on different Fab arms of a single IgG as the average Fab distance upon binding is approximately 8-9 nm, but values as small as approximately 5.5 nm have been observed. Since IgG₃I₂ was the major product of aggregation in this system, the formation of the bicyclic complex was more favorable than the binding of a single molecule of 1 to a single antibody (bridging both Fab arms), at the IgG concentrations used. Furthermore, formation of thermodynamically stable higher aggregates, such as a tricyclic hexameric complex (IgG_f5I₄) was not observed in the model system. The absence of both lower and higher aggregates, and the high yield in conversion to IgG₃I₂, indicates that the trimeric antibody aggregate is the most thermodynamically stable structure.

Some examples provided herein relate to the physical-organic chemistry of such trihapten molecules that can bind to an antibody receptor. The antibody (IgG^DNP) has a high monovalent binding constant (K™"° = 8.0 x 10^'10 M). The increase in the kinetic stability Of IgG₃I₂ relative to IgG(DNP-LyS)₂ was approximated, by estimating the lifetime (τ_orr > 91 min) of the complex from its stability on the SE-HPLC runs (at 0.1 mL/minute flow), to be much greater than a factor of 225. This value is a lower limit. It can be inferred that the kinetic stability of the complex (relative to that of a monomelic aggregate) indicates that multivalency contributes significantly to the stability Of IgG₃Io.

IgG₃I_{2 ^} ^Kd3 - IgG₂I₂ + IgG (1)

IgG₂I₂ ^J_^ 2 IgG l ₍₂₎

Equilibrium 1 , which is the inverse of equilibrium 3 (see Example 2), describes the dissociation of one of the IgGs from the IgG₃I₂ complex. The trivalent molecules are still part of a stable dimeric complex IgG₂I₂- Hence there are two free DNP moieties pre-positioned for a free IgG molecule to bind to. The pre-positioning of the DNP moieties provides a higher avidity of a free IgG molecule for IgG₂Ii than for free DNP. The equilibrium constants obtained from fitting the SE-HPLC data to the equilibrium model (see materials and methods) yield AG₁ = -12.4 kcal/mol, AG₂ = -9.8 kcal/mol and ΔG,^° = -13.6 kcal/mol for the equilibria described by equations 1-3 (see Example 2). The difference in the free energy of binding between equilibrium 1 and equilibrium 3 is ΔΔG,_₃= -1.2 kcal/mol (the free energy of binding for monovalent DNP to IgG^DNP was calculated from fluorescence quenching data, and is equal to - RTInK₁ , where Ki was calculated using our model). This value indicates that binding of an IgG^DNP to the complex IgG₂I₂ is 1.2 kcal/mol more favorable than its binding to DNP. If the free energy of binding of equilibrium 1 is compared to that of equilibrium 2 (dissociation of cyclic dimer to monomer-DNP complexes from equilibrium 2), the ΔΔG,_₃ is calculated to be -3.8 kcal/mol. These interactions are assumed to be directly comparable since two hapten moieties bind to two Fab arms simultaneously in both equilibria, and therefore the difference in the binding strengths can be explained in terms of loss of binding entropy.

In equilibrium 2, the Fab arms and the hapten linkers of the dissociated IgGl complex have more conformational freedom than the free Fab arms and linkers in the IgG₂I₂ form. In the case of equilibrium 1, there is a smaller loss in entropy upon binding than there is for in equilibrium 2, because the DNP moieties for the binding of the third IgG can be pre-positioned by the structure of the complex, and as a result the free energy of binding is more favorable. Taken together, the findings disclosed herein demonstrate that the bivalency of IgGs improves both the thermodynamic and kinetic aspects of their binding, and furthermore, demonstrates that these features of IgGs can be exploited to create aggregates using trihapten molecules that are useful, e.g., for assays, for treatments in which it is desirable to inactivate an antibody, and for selectively targeting a cell or a surface having a relatively high density of a hapten.

The increased stability of the antibody-trihapten aggregates yields an equilibrium of aggregate-bound versus antigen-bound antibodies that favors higher density presentations of the hapten, as shown in Fig. 8. This occurs, for example, on tumor cells, which express higher densities of some antigens on their surface than are expressed on normal cells.

Assays Using Trihapten Molecules

Trihapten molecules can be used to detect a multivalent ligand, such as an antibody, that can bind to the hapten moiety of the trihapten molecule. In such assays, a trihapten molecule is constructed using a hapten that can be bound by the antibody of interest (i.e., the antibody to be detected), the trihapten molecule is incubated with a sample containing the antibody of interest or a sample that may contain an antibody of interest, and the binding of the antibody to the trihapten is evaluated, for example, by detecting trihapten/antibody complexes. In some cases, the binding of the antibody is quantitated, for example, by comparing the amount of antibody bound to the trihapten to a reference assay in which known amounts of antibody are used in a control assay. An advantage of using a trihapten molecule in such an assay is that the stability of the trihapten complex that can form upon binding of antibodies to the trihaptens provides a more sensitive assay than an assay using a hapten alone or the hapten linked to a molecule in a monovalent configuration. The assay can be adapted to detect other multivalent ligands by selecting a hapten that will bind to the multivalent ligand.

Assays using trihapten molecules can be conducted in a liquid phase. In such an assay, the reaction products (i.e., trihapten molecule/antibody complexes) are separated from unreacted components (i.e., unbound trihapten molecules, unbound antibodies, and other components of the tested sample), by any of a number of techniques known in the art, including but not limited to differential centrifugation (see, for example, Rivas et al., 1993, Trends Biochem. Sci. 18:284-7, which is hereby incorporated by reference in its entirety); chromatography (gel filtration chromatography, ion-exchange chromatography); electrophoresis (see, e.g., Ausubel et al., eds. Current Protocols in Molecular Biology, 1999, J. Wiley: New York, which is hereby incorporated by reference in its entirety). Resins and chromatographic techniques used in such methods are known to those skilled in the art. In some embodiments of the invention, trihapten/antibody complexes are assayed without separation from other assay components using detection methods known to those in the art.

Detection of binding can be accomplished using methods known in the art. For example, one component of the assay is labeled with a detectable label. Methods known in the art can be used to generate a detectable label. For example, the trihapten molecule or the antibody can be labeled with 125^ 35g₅ 14Q _or 3j^ either directly or indirectly, trihapten molecule/antibody complexes isolated, and the radioisotope in the complexes detected by direct counting of radioemission or by scintillation counting. In some cases, the detectable label is a fluorescent label. In such cases, the label is incorporated into the molecule being detected or is linked to the molecule, but does not interfere with binding of the molecule as it is used in the assay. Additional examples of methods for detecting binding and complex formation include, without limitation, size exclusion high performance liquid chromatography (SE-HPLC), dynamic light scattering, analytical ultracentrifugation, label-free surface plasmon resonance technology (e.g., BIACore), or fluorescence resonance energy transfer (FRET). Other methods that can be used include assays using microfluidic channels or assays of light scattering, e.g., in a cuvette.

In some cases, an assay is a displacement assay. In one such type of assay, a trivalent molecule/antibody complex is formed in which the antibody contains a detectable label. The complex is incubated with a sample that includes an antibody, hapten, or hapten-containing molecule (e.g., an antigen) corresponding to the hapten moiety or antibody of the complex. The dissociation of antibody and/or ligand from the complex is detected or the decrease in complexes is detected. In some embodiments of the assay, a tetravalent core molecule is used such that three amis are as described herein (i.e., flexible and having three-fold symmetry) and a fourth arm is connected to a fluorescent molecule instead of one more hapten. The complex for a displacement assay can then be incubated in a dialysis membrane. Appearance of the fluorescently labeled trihapten molecule on the outside of the membrane is then assayed and indicates dissociation.

In one embodiment, trihapten molecules or antibody/trihapten complexes can be used to determine the selectivity of binding of an antibody or an antibody/trihapten complex. In another embodiment, trihapten molecules or antibody/trihapten complexes can be used to determine the multivalent binding specificity, or selectivity, of an antibody or an antibody/trihapten complex. "Selectivity" as used herein refers to a relative affinity, such as the ability to preferentially bind one presentation of hapten(s), such as a cell, over another. In an example of a selective binding assay, selective binding experiments can be carried out using monoclonal anti-DNP IgG antibody from rat (IgG ^NP) and trivalent haptens of DNP, such as 1. A trivalent hapten of DNP can be mixed with IgG^DNP at 3 to 2 stoichiometry of IgG^DNP: trivalent hapten to yield bicyclic antibody trimer complexes (IgG₃Ij) as one example of an antibody/trihapten complex. An ELISA assay is used to test the specificity of binding of the bicyclic antibody trimer complexes (IgG₃I₂) (i.e. the IgG^DNP-l antibody/trihapten complex). Plates or slides, such as 96-well plates or glass slides, that present a reactive functionality, such as a polymer containing maleic anhydride or a surface presenting chlorosilane, can be used to covalently attach DNP haptens on the surface of separate loci, such as separate wells, at various densities, as depicted in Fig. 9. Onto a given location, such as a well, the antibody is introduced as either the uncomplexed monomer, or as the antibody/trihapten complex, IgG₃I₂. After washing unbound antibody away from the location, the amount of bound antibody/trihapten complex at each location is determined, such as by ELISA or using other labeled secondary antibodies. A secondary antibody is introduced that binds to the rat IgG^DNP. The secondary antibody can be anti-rat IgG from goat linked to the enzyme horse radish peroxidase (HRP). The amount of IgG^DNP bound to the surface can be quantified by measuring a signal, such as fluorescence, generated by the product of HRP linked to the secondary antibody when HRP is exposed to and reacts with a substrate that generates a signal, such as fluorescence, when reacted with HRP. A HRP kit can be obtained commercially, such as from Molecular Probes. Those in the art will understand how to adapt this assay for use with any antibody/ligand of interest.

Diagnostic Assays The assays described herein can be used to diagnose certain disorders, e.g., autoimmune disorders or other disorders that can be diagnosed by the presence of an antibody against a specific antigen, or disorders that can be diagnosed by the presence of a specific molecule. For example, a hapten associated with a disease (a disease hapten) can be assayed using a trihapten molecule having a hapten moiety corresponding to the disease hapten using a displacement assay. In some cases, the assay is of an antibody that is associated with a disease (a disease antibody). In such assays, a trihapten molecule is used in which the hapten moiety can bind to the disease antibody. In one non-limiting example, lupus is diagnosed by detecting an antibody that binds to the Sm antigen. To conduct the assay, a trihapten molecule is used on which the hapten moiety corresponds to the Sm antigen and the trihapten molecule can specifically bind an antibody directed against the antigen (a Sm trihapten molecule). To conduct an assay, the Sm trihapten molecule is incubated with a sample from a subject suspected of having lupus, and Sm trivalent molecule/antibody complexes are detected. The presence of such complexes indicates that the subject has or is likely to have lupus.

In some cases, a displacement assay is useful as a diagnostic. For example, a trihapten molecule having an anthrax hapten moiety can be used to form a complex with an antibody directed against the anthrax hapten to form an anthrax trihapten molecule/antibody complex. To assay a sample from a subject suspected of having an anthrax infection, the anthrax trihapten molecule/antibody complex is incubated with the sample, and the displacement of the anthrax antibody from the complex is assayed, e.g., by detecting the decrease in trihapten/anthrax antibody complexes. Those in the art will understand how to adapt such assays for use with other selected trihapten molecule/antibody complexes to detect antigens corresponding to the selected hapten of the trihapten molecule. In a non-limiting example, the above assay can be conducted using a sialic acid hapten.

In some embodiments, an antibody/trihapten complex can be used to determine the presence of a disease-associated agent, such as bacteria, viruses, cells, tissues, or antigens. In one non-limiting example, an antibody/trihapten complex can be made where the antibody in the complex preferentially binds to a hapten that is overexpressed on certain cancer cells, such as tumor cells. In one non-limiting example, an antibody-trihapten molecule complex in a ratio of trihapten molecule:antibody of 2:3, with a IQ that is smaller than the IQ for the ligand bound to a monohapten molecule, i.e. the complex is more stable than the monovalent interaction, can preferentially bind to a hapten that is overexpressed on a cell versus a normal cell. This is because the antibody/trihapten complex can preferentially bind to surfaces having a relatively high density of a hapten. Thus, a disease-associated agent, such as a cancer cell, can be labeled preferentially as compared to a normal sample that expresses lower concentrations of an antigen to which an antibody in a trihapten molecule/antibody complex can bind. In such an assay, a sample containing a disease-associated agent is contacted with a trihapten molecule/antibody complex in which the antibody can bind a hapten that is associated with the disease. After incubation, the sample is washed to remove antibody and/or trihapten molecule/antibody complexes that are not bound to a sample component and the amount of antibody binding is determined. Methods of determining the amount of binding are known in the art, including for example, direct labeling of antibody used in the assay or indirect labeling of the antibody after unbound trihapten molecules and complexes are removed. In some cases, the complex can be exposed to a sample containing tumor cells, and the amount of antibody bound to the cells can be determined by the methods described herein and methods known to one in the art. Comparison of the amount of antibody bound to the cells in the sample relative to the amount of antibody bound to a sample of normal (i.e., non-cancerous cells of a corresponding cell type) cells enables the diagnosis of the presence of the disease- associated agent, such as the tumor cells described above. For example, the presence of an abnormal amount of antibody bound to a disease-associated hapten indicates the presence of a disease-associated agent. In another embodiment, antibody/trihapten complexes may be used to reduce the non-specificity of the binding of the antibody in an assay or method of treatment. For example, a complex may be formed of a trihapten molecule and an antibody that binds the haptens. The complex is more stable than a monovalent antibody-hapten interaction, but less stable than multivalent antibody-hapten interactions with a dense presentation of the hapten, as illustrated in Fig. 8. Thus, the antibody in the antibody/trihapten complex can preferentially bind to a dense presentation of the hapten over a sparse presentation of the hapten. This feature of the complex can be used to more specifically target antibodies to their desired, relatively densely multivalent, targets (e.g., cells overexpressing an antigen), while reducing their binding to undesired, less densely multivalent targets (e.g., cells expressing normal amounts of an antigen). Non-limiting examples of these targets include disease- associated cells presenting self-antigens, such as cancer cells and pathogenic cells of the immune system such as in arthritis or lupus. Trihapten molecules and antibody/trihapten complexes can also be used to develop therapeutic agents and screen for toxicity of therapeutic agents. For example, such complexes can be screened for their selectivity for disease-associated cells versus healthy cells. A screen can be accomplished using methods described herein, such as in Diagnostic Assays, Assays Using Trihapten Molecules, and in the Examples section. The generation of many such antibody/trihapten complexes enables high-throughput screening for therapeutic agents.

Pharmaceutical Compositions

Trihapten molecules as described herein can be incorporated into pharmaceutical compositions for use in treating a disease. As used herein, the term

"treatment" is defined as the application or administration of a therapeutic agent to a subject, or application or administration of a therapeutic agent to an isolated tissue or cell line from a subject, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease. In specific embodiments, a disease that can be treated using such pharmaceutical compositions is a disease that can be ameliorated by inactivating a molecule associate with causation or symptoms of a disease (termed herein "a disease-associated" molecule), e.g., an antibody associated with a disease such as an autoimmune disease. Such a pharmaceutical composition includes a trivalent hapten molecule that can bind to the disease-associated molecule, as described herein. In general, the trivalent hapten molecule binds to naturally- occurring molecule that, when bound by the monovalent hapten in a subject, can result in disease or undesirable symptoms. In some cases, the trivalent hapten binds to an undesirable antibody in a subject. In some cases, the disease is caused by undesirable expression of a molecule. In such cases, a trivalent molecule that includes a hapten or a related molecule corresponding to the undesirable molecule is used to treat the disease. Without committing to any specific mode of action, it can be that the trivalent hapten displaces or prevents the undesirable molecule from binding to a receptor and thereby prevents activation of an undesirable physiological process.

In some embodiments, a pharmaceutical composition includes a trihapten molecule that can specifically bind to antibodies that can bind to the hapten of the trihapten molecule, or a pharmaceutically acceptable salt thereof, and also includes a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" includes pharmaceutically acceptable solvents, including such aqueous solvents such as buffers, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, inhalation, transdermal

(topical), transmucosal, and rectal administration; or oral. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or parabens such as methyl, ethyl, or propyl parabens; BHT; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose, additives such as thickeners, like carbomers or celluloses. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the selected particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, such as methyl, ethyl, or propyl parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some cases, one or more isotonic agents are included, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be effected by including in the composition one or more agents that delay absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., the trihapten molecule) in the specified amount in an appropriate solvent with one or a combination of ingredients enumerated above, as needed, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and other ingredients selected from those enumerated above or others known in the art. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are known in the art and include, for example, vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid earner for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, a trihapten molecule is delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the trihapten molecule is formulated into ointments, salves, gels, or creams and may be combined with the above penetrants as generally known in the art. The trihapten molecules can also be prepared in the form of suppositories

(e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the trihapten molecule is prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.

Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811. It is advantageous to formulate oral or parenteral trihapten molecules in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of the trihapten molecule calculated to produce the desired therapeutic effect in association with the selected pharmaceutical carrier.

Toxicity and therapeutic efficacy of a trihapten molecule can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50

(the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Trihapten molecules that exhibit high therapeutic indices are generally used. While trihapten molecules that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such trihapten molecules to the site of affected tissue to minimize potential damage to uninfected cells and, thereby, reduce side effects. While antibody/trihapten complexes that exhibit toxic side effects may also be used, care should be taken to design a delivery system that targets such antibody/trihapten complexes to the site of affected tissue to minimize potential damage to healthy cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such trihapten molecules or such antibody/trihapten complexes generally lies within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any trihapten molecule or antibody/trihapten complex used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test trihapten molecule that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of a trihapten molecule or a trihapten molecule/antibody complex (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, for example, about 0.01 to 25 mg/kg body weight, about 0.1 to 20 mg/kg body weight, or about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The trihapten molecule can be administered one time per week for between about 1 to 10 weeks, for example between 2 to 8 weeks, between about 3 to 7 weeks, or about 4, 5, or 6 weeks. The skilled artisan will appreciate that certain factors may influence the dosage and timing to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a trihapten molecule or a trihapten molecule/antibody complex can include a single treatment or can include a series of treatments.

An antibody that is delivered using a trihapten molecule/antibody complex will generally have a longer half-life than an antibody alone and therefore the dosage may be adjusted accordingly.

A trihapten molecule or an antibody/trihapten complex can be used in the preparation of a medicament for treating a disease associated with the presence of an undesirable antibody.

Methods of Treatment

Trihapten molecules can be used to treat disorders when administered to a subject in need thereof.

In one embodiment, antibody/trihapten complexes enhance the selectivity of tumor cell targeting. In another embodiment, antibody/trihapten complexes reduce the non-specific toxicity of therapeutic antibodies by introducing them as bicyclic complexes.

In another embodiment, antibody/trihapten complexes can be used to selectively target a cell, such as a cell that over expresses a hapten.

In yet another embodiment, trihapten molecules can be useful for, e.g., neutralizing the effects of antibodies that are associated with disease.

In still another embodiment, the introduction of a therapeutic antibody in the form of bicyclic antibody trimer complex can improve the selective targeting for target cells.

In another embodiment, antibody/trihapten complexes may be used to reduce the non-specificity of the binding of the antibody in any assay or method of treatment therewith. For example, a complex may be formed of an antibody and a trihapten molecule. The complex is more stable than a monovalent antibody-hapten interaction, but less stable than multivalent antibody-hapten interactions with a dense presentation of the hapten, as seen in Fig. 8. Thus, the antibody bound in the antibody/trihapten complex can preferentially bind to a dense presentation of the hapten over a sparse presentation of the hapten. This feature of the complex can be used to more specifically target antibodies to their desired, relatively densely multivalent, targets, while reducing their binding to undesired, less densely multivalent targets.

Some embodiments relate to targeting disease-associated cells that express a hapten at a higher density on their surfaces than healthy or non-disease-associated cells. Non-limiting examples include foreign organisms such as bacteria and parasites, and cancer cells. Cancer cells are known to overexpress some molecules on their cell surfaces relative to healthy cells. The likelihood of antibodies in antibody/trivalent hapten complexes for binding to relatively dense presentations of haptens can be exploited as described herein to preferentially target such overexpressing cells and reduce the antibody's binding to healthy cells. Such selective targeting can reduce unwanted side effects, such as toxicity, that may be caused by the binding of therapeutic antibodies to healthy cells.

Therapeutic antibodies used for cancer treatment are typically developed to target cell surface receptors (tumor antigens) that are over expressed on tumor cells. Since healthy cells typically express lower densities of these surface receptors, the degree of antibody targeting of healthy cells is generally lower than targeting of the tumor cells. Nevertheless, therapeutic antibodies yield non-specific toxicity to healthy cells of cancer patients at various levels, depending on the disease and also whether an effector molecule (a cytokine, a toxin or etc.) is attached or not.

A reduction in the non-specific toxicity can be achieved by delivering the therapeutic antibodies as bicyclic complexes which are formed through interaction with trivalent hapten molecules. The trivalent hapten molecule for a particular therapeutic antibody can be synthesized using a mimotope of the antibody's target instead of the actual antigen, such as a tumor antigen. The mimotope can be designed to bind the antibody with an affinity that is weaker than or as tight as the antigen itself. The trivalent molecules that present these mimotopes will be added into a solution of the therapeutic antibody and let to react to form the bicyclic complexes, prior to the delivery of the antibodies to a patient. The antibodies that form these complexes have both binding sites occupied. Hence, when the complex encounters a cell surface that has low density of the receptor — such as when the distances between the surface receptors will only allow monovalent binding; i.e. distances longer than about 9-10 nm — the antibodies will not significantly dissociate from the complex to bind on the cell surface. Meanwhile, when the complex encounters a tumor cell with overexpressed levels of the receptor, since the distance between the surface receptors will now provide antibodies to be able to bind bivalently, an equilibrium between the complex and the cell surface establishes itself, and part of the antibody molecules gets delivered to the tumor cell surface. The magnitude of this delivery can be fine tuned by adjusting the difference between the binding affinity of the surface antigen and the antigen mimic on the trivalent ligand towards the antibody, as well as the kinetics of these interactions. Figures 7 and 8 summarize the described therapeutic antibody delivery technique. Furthermore, the efficiency of delivery can also be adjusted using different length linkers since the stability of the complexes also depend on the length of the linker used.

Treatments Using Trihapten Molecules The present invention provides for therapeutic methods of treating a subject having a disorder associated with aberrant or unwanted expression of an antibody. As used herein, the term "treatment" is defined as the application or administration of a therapeutic agent to a subject, or application or administration of a therapeutic agent to an isolated tissue or cell line from a subject, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease. A therapeutic agent as provided herein includes a trihapten molecule that can bind an antibody associated with a disease. Such trihapten molecules include those that bind to antibodies associated with an autoimmune disorder such as arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, encephalomyelitis, myasthenia gravis, systemic lupus erythematosis; or antibodies associated with other undesirable immune system responses such as allergy.

A trihapten molecule as described herein can be administered to a subject in need thereof at therapeutically effective doses to prevent, treat or ameliorate a disorder associated with undesirable antibody expression. In such methods, the hapten moiety of the trihapten molecule is selected for its ability to bind to the undesirable antibody. A "therapeutically effective" dose refers to that amount of the trihapten molecule sufficient to result in amelioration of symptoms of the disorder. Toxicity and therapeutic efficacy of such molecules can be determined by pharmaceutical procedures known in the art.

In one embodiment, the antibody/trihapten complex further comprises an agent, wherein the agent is capable of treating a disease. In a specific embodiment, the agent is covalently attached or conjugated to the antibody or the trihapten molecule.

Methods of Treating Cancer

In some embodiments, the invention provides methods for treating (including reducing the rate of disease progression) or preventing the progression of cancer, by administering an effective amount of an antibody/trihapten complex to a subject in need thereof. In one embodiment, the methods further include administering an effective amount of another anticancer agent. In another embodiment, an anticancer agent is attached to the antibody/trihapten complex. As an example, in a theory of disease treatment, to which the inventors do not wish to be bound, an antibody/trihapten complex is attached to a drug that is useful in treating the disease. The antibody/trihapten complex targets disease-associated cells as related herein, and delivers the attached drug to the disease-associated cell preferentially to non-disease-associated cells. Examples of cancers include, but are not limited to solid tumors, including but not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic, sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, stomach cancer, oral cancer, nasal cancer, throat cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, small cell lung carcinoma, bladder carcinoma, lung cancer, epithelial carcinoma, skin cancer, melanoma, metastatic melanoma, neuroblastoma, or retinoblastoma; blood-borne cancers, including but not limited to: acute lymphoblastic leukemia ("'ALL"), acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblastic leukemia ("AML"), acute promyelocytic leukemia ('"APL"), acute monoblastic leukemia, acute erythroleukemic leukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acute nonlymphocyctic leukemia, acute undifferentiated leukemia, chronic myelocytic leukemia ("CML"), chronic lymphocytic leukemia ("CLL"), hairy cell leukemia, or multiple myeloma; acute and chronic leukemias including, but not limited to: lymphoblastic, myelogenous, lymphocytic, or myelocytic leukemias; lymphomas, including, but not limited to: Hodgkin's disease, non-Hodgkin's Lymphoma; multiple myeloma,

Waldenstrom's macroglobulinemia, heavy chain disease, polycythemia vera, or central nervous system lymphomas; central nervous system or brain cancers including but not limited to: glioma, pilocytic astrocytoma, astrocytoma, anaplastic astrocytoma, glioblastoma multiforme, niedulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, vestibular schwannoma, adenoma, metastatic brain tumor, meningioma, spinal tumor, or medulloblastoma.

In one embodiment, the cancer is lung cancer, breast cancer, colorectal cancer, prostate cancer, a leukemia, a lymphoma, non-Hodgkin's lymphoma, skin cancer, a brain cancer, a cancer of the central nervous system, ovarian cancer, uterine cancer, stomach cancer, pancreatic cancer, esophageal cancer, kidney cancer, liver cancer, a head and neck cancer, melanoma, or glioma (e.g., pilocytic astrocytoma, astrocytoma, anaplastic astrocytoma or glioblastoma multiforme). In some cases, the cancer a BRCA-I or BRC A-2 deficient cancer, or is deficient in one or more proteins of the Fanconi family.

In still another embodiment, the subject in need of treatment has previously undergone or is presently undergoing treatment for cancer. Such treatments include, but are not limited to, chemotherapy, radiation therapy, surgery or immunotherapy, such as administration of a cancer vaccine. In some embodiments, an antibody/trihapten complex is administered while the subject in need of treatment is undergoing treatment for cancer.

Antibody/trihapten complexes are also useful for treating or preventing a cancer caused by a virus. As an example, in a theory of cancer treatment, to which the inventors do not wish to be bound, an antibody/trihapten complex is attached to a drug that is useful in treating cancer. Alternatively, the antibody in the antibody/trihapten complex can be a therapeutic antibody capable of treating or preventing cancer or a cancer-causing virus. Such viruses include, for example, human papilloma virus, which can lead to cervical cancer (see, e.g., Hernandez- Avila et al, Archives of Medical Research (1997) 28:265-271); Epstein-Barr virus (EBV), which can lead to lymphoma (see, e.g., Herrmann et ah, J Pathol (2003) 199(2): 140- 5); hepatitis B or C virus, which can lead to liver carcinoma (e.g., El-Serag, J Clin Gastroenterol (2002) 35(5 Suppl 2):S72-8); human T cell leukemia virus (HTLV)-I, which can lead to T-cell leukemia (e.g., Mortreux et al., Leukemia (2003) 17:26-38); human herpesvirus-8 infection, which can lead to Kaposi's sarcoma (e.g., Kadow et al., Curr. Opin. Invest. Drugs (2002) 3:1574-9); and Human Immune deficiency Virus (HIV) infection, which can lead to cancer as a consequence of immunodeficiency (e.g., Dal Maso et al., Lancet Oncol. (2003) 4: 1 10-9).

In one embodiment, the antibody/trihapten complex preferentially targets cancer cells that overexpress a hapten over normal cells (cells that do not overexpress the hapten) as related herein, optionally also delivering an attached drug preferentially to the cancer cell.

Antibody/trihapten complexes are also useful for slowing or preventing progression of a cancer, including but not limited to the cancers listed in Table A. Such prophylactic use includes that in which non-neoplastic cell growth such as hyperplasia, metaplasia, or dysplasia has occurred. To slow or prevent progression of a cancer, a trivalent hapten molecule or an antibody/trihapten complex, optionally with an anti-cancer agent attached to the trivalent hapten or antibody/hapten complex, is constructed that can bind to a molecule associated with the onset of the cancer and is administered to the subject in a therapeutically effective amount.

In other embodiments, a subject that has one or more of the following predisposing factors for malignancy can be treated by administration of an effective amount of an antibody/trihapten complex: a chromosomal translocation associated with a malignancy (e.g., the Philadelphia chromosome for chronic myelogenous leukemia; t(14;18) for follicular lymphoma); familial polyposis or Gardner's syndrome; benign monoclonal gammopathy; a first degree kinship with persons having a cancer or precancerous disease showing a Mendelian (genetic) inheritance pattern (e.g., familial polyposis of the colon, Gardner's syndrome, hereditary exostosis, polyendocrine adenomatosis, medullary thyroid carcinoma with amyloid production and pheochromocytoma, Peutz-Jeghers syndrome, neurofibromatosis of Von Recklinghausen, retinoblastoma, carotid body tumor, cutaneous melanocarcinoma, intraocular melano carcinoma, xeroderma pigmentosum, ataxia telangiectasia, Chediak-Higashi syndrome, albinism, Fanconi's aplastic anemia, and Bloom's syndrome); and exposure to carcinogens {e.g., smoking, second-hand smoke exposure, and inhalation of or contacting with certain chemicals).

To treat, prevent, or delay the onset of such disorders, a trivalent hapten molecule or an antibody/trihapten complex is constructed that can bind to a molecule associated with the onset of the disease and is administered to the subject in a therapeutically effective amount.

In one aspect, the present methods for treating or preventing cancer can further include the administration of effective amounts of another anticancer agent and an antibody/trihapten complex to a subject in need thereof. The antibody/trihapten complex and another anticancer agent can be administered concurrently, including within the same composition. In another embodiment, the compositions comprise an effective amount of an anticancer agent, a physiologically acceptable carrier or vehicle, and an effective amount of an antibody/trihapten complex.

Cancers that can be treated or whose progression may be prevented by administering an antibody/trihapten complex and the anticancer agent include, but are not limited to, the list of cancers set forth above in Table A. In one embodiment, the cancer is a tumor.

In an embodiment, the other anticancer agent is interferon-α; interleukin-2; an alkylating agent, such as a nitrogen mustard, a nitrosourea, an alkylsulfonate, a triazene, or a platinum-containing agent.

EXAMPLES

The invention is further illustrated by the following examples. The examples are provided for illustrative purposes only. They are not to be construed as limiting the scope or content of the invention in any way.

Example 1 : Design and Synthesis of a Trihapten Molecule To test the binding and other characteristics of a trihapten molecule, such a molecule was designed and characterized.

Experimental Design Antibody. The rat anti-2,4-DNP IgG binds to the small molecule hapten 2,4- dinitrophenyl (2,4-DNP) with a monovalent dissociation constant of K"l°"° = 8.0 x 10^"

¹ M. This equilibrium value for the monovalent hapten DNP-Lys (K"⁰"⁰ ) was calculated using fluorescence spectrometry. This value of IQ is relatively high for IgGs. The high IQ facilitates analysis performed using SE-HPLC because of the time required for this analysis (about 17 minutes), but is not to be construed as limiting with respect to the IQ of antibodies that are to be bound using trihapten molecules of the invention.

Design of a trivalent hapten. A trihapten molecule was designed to space the haptens (the 2,4-DNP groups) sufficiently far apart so that the assembly of a bicyclic trimer would be sterically feasible, but close enough together to make it energetically unfavorable for a single molecule of 1 to bridge the two Fab arms of a single IgG (Figure 5). Ethylene glycol linkers connect the three DNP molecules to the center of this trivalent system. The depicted linkers are each approximately 3.2 nm long when fully extended; hence the separation between two hapten molecules can be about 6.4 nm. The optimum separation of binding sites in an IgG is approximately 8 nm, although Fab arms can place the binding sites closer.

Materials and Methods

Chemicals. iV-Fmoc-amido-dPEGs -acid was purchased from Quanta BioDesign, Ltd, HBTU from Novabiochem. Tra-succinimidyl amino triacetate was purchased from Molecular Probes, Inc., M;-DNP-Lysine from Sigma- Aldrich Co., yV,jV-diisopropylethylamine (DIEA) was purchased from Sigma, and monoclonal rat anti-2,4-DNP IgG antibodies (IgG^DNP) were purchased from Zymed, Inc (Invitrogen). TV.iV-Dimethylforaiamide (>99.8%) and dimethyl sulfoxide (DMSO) (>99.8%) were purchased from EMD; and acetonitrile (>99.8%) was purchased from Mallinckrodt Chemicals. The IgG^DNP was used without further purification. The purity of the commercial IgG^DNP was estimated to be >94%.

Synthesis. To synthesize the core molecule of 1, 10 mg of 7V-Fmoc-amido- dPEG₈™-acid was dissolved in 2 niL of N,N-dimethylformamide and 1.2 equiv of 2-

( 1 H-benzotriazole- 1 -yl)- 1,1,3 ,3 -tetramethylaminium hexafluorophosphate (HBTU) and 2 equiv of JV,iV-diisopropylethylamine (DIEA) were added (Scheme 2). After 5 minutes, the activated Λ/-Fmoc-amido-dPEGs^T -acid was added to 3 equiv of Nc- DNP-Lysine dissolved in ImL of dimethyl sulfoxide (DMSO). The reaction was ran for 2 hours at room temperature before quenching by the addition of 4 mL of 0.1 % trifluoroacetic acid in water. The product 3 was purified via reversed-phase high pressure liquid chromatography (RP-HPLC) as described in the purification section, below. The Fmoc group on the purified product 3 was removed using 20% piperidine in DMF for 1 hour, and the product 2 was isolated using RP-HPLC followed by lyophilization. The lyophilized product 2 was dissolved in DMF and 2 equiv of

DIEA. The resultant solution was added drop-wise over one hour to 0.3 equivalents of Jrø'-succinimidy] aminotriacetate dissolved in 1 mL of DMF. The reaction was stirred overnight. Analytical HPLC showed a yield of approximately 60% (side products of this reaction are mono- and bi-substituted aminotriacetate). The product 1 was purified via RP-HPLC and characterized using MALDI-TOF mass spectroscopy.

The calculated molecular weight of 1 (C99H _I62Ni₆O₄₈) was 2344 Da; and was found to be 2344 Da (with sodium adduct at 2367 Da, and dehydration products at 2326 Da and 2310 Da).

Scheme 2 Synthesis of 1.

90% purified yield 3

20% Pιperιdιne in DMF

2 80% purified yield

40% purified yield 1

Purification. RP-HPLC purifications were performed on a Vydac Cl 8 column (10 mm x 250 mm, 300 A pore size, 10 μm particle size), using linear solvent gradients of 1% per minute increments in acetonitrile concentration at 2.5 mL/min flow rate on a Dynamax Rainin system. The column eluent was monitored using UV absorbances at 218 nm and 360 ran with a dual wavelength UV detector, Dynamax model UV-D II.

Size Exclusion HPLC (SE-HPLC). SE-HPLC measurements were earned out on a Tosoh TSK-GEL G3000SWXL size-exclusion column using a Varian Pro S tar 400 HPLC system with autosampler. SE-HPLC runs were performed with an isocratic solvent system that was 50 niM phosphate buffer and 370 mM NaCl (to adjust the ionic strength to 0.475 M) at pH 6.8, with a 0.5 mL/min flow rate. The sample peaks were analyzed using a UV/Vis detector, monitored at λ=218 nm. The concentration of antibody was kept constant in all samples (0.2 μM, 0.6 μM, or 1 μM) and incubated the IgGs with different concentrations of 1. The sample concentrations were determined using the reported extinction coefficients for IgGs and DNP. All samples were incubated for 12 hours at 4⁰C prior to injection onto the SE-HPLC column.

Dynamic Light Scattering. DLS experiments were carried out in a 12 μL cuvette on a DynaPro dynamic light scattering instrument at 25 ⁰C. Samples were in 10 mM phosphate (pH 7.40), 137 mM NaCl, 2.7 mM KCl, at 3.33 μM (0.5 mg/mL) anti-DNP IgG and 2.22 μM 1 concentrations in a total volume of 20 μL. To remove dust particles, prior to mixing anti-DNP IgG with synthetic hapten 1, both stock solutions were centrifuged using an Eppendorf Centrifuge™ model 5415 C at 16,000 rcf (relative centrifugal force) for 20 minutes.

Analytical Ultracentrifugation. The molecular weight of the complex in solution was estimated by sedimentation equilibrium in a Beckman XL-I ultracentrifuge. IgG^DNP concentrations in the samples varied from 0.05 μM to 0.5 μM. Samples were dissolved in 10 mM phosphate buffer (pH 7.40), containing 137 mM NaCl and 2.7 mM KCl. The samples were centrifuged at 6,000, 9,000, and

12,000 rpm for 22 hours at 25 ⁰C before absorb ance scans were performed. Data obtained at 25 ⁰C were fit globally to an equation that describes the sedimentation of a homogeneous species using data-analysis programs "Origin" and "Igor" separately. Equation (3) below was used for fits performed using "Igor". Abs = A'exp(H * M[X² - x₀ ² J)+ B (3)

where, Abs = absorbance at radius x, A' = absorb ance at reference radius XQ, H = (I- Vp)ω² 12RT , V = partial specific volume = 0.73 mL/g, p = density of solvent = 1.0017 mL/g, ω~ angular velocity in radians/sec, M = apparent molecular weight, and B = solvent absorbance (blank).

Equilibrium Model Used Fitting the SE-HPLC Data.

Equations 4-8 were developed to account for the mass balance of each species present in solution at each data point. The equations were developed by algebraically rearranging the proposed equations 9-12 (below) for the equilibria in the formation of the complexes that were observed in the SE-HPLC experiments.

K₁ [IgG][I] -[IgGl] = O (4)

[K₂[IgGlY J-[IgG₂I₂] = 0 (5)

(K,[IgG₂ l₂][IgG])-[IgG₃ l₂] = 0 (6)

[IgG ] = [IgG ]_Toml - [I_gG l] - 2[IgG ₂ l₂ ] - 3[IgG , I₂ ] (7)

[I] = [I]₇,,,_/ - [IgG l] - 2[IgG ₂ I₂ ] - 2UgG ₃ I₂ ] (8) The SOLVE function in Mathematica 5.1 was used to solve the equilibrium equations algebraically, and fit the data from the peak integrations of SE-HPLC experiments, yielding polynomial expressions for [IgGl], [IgG₂I₂] and [IgG₃I₂] as functions of [l]_totai_> [IgG] total and the equilibrium constants K₁, K₂ and K₃. Monte Carlo optimization was used to identify optimal K₁, K₂ and K₃ values (Ki = 1.25 x 10⁹

M^"1, K₂ = 2.00 x 10⁷ M^"1, and K₃ - 1.00 x 10¹⁰ M^"1) that fit the data with an R-Square value of 0.988. These equilibrium constants fit the data with R-square values of 0.999929 for the monomer data, 0.999992 for the dimer data and 0.999973 for the trimer data.

Example 2: Results-Structure and Stoichiometry Thermodynamic Analysis of SE-HPLC data.

Compound 1 was prepared as described in Example 1 and mixed with IgG^DNP in phosphate buffered saline (PBS) buffer, the mixture was allowed to equilibrate, and the resulting complexes were analyzed by SE-HPLC. The concentrations of IgG^DNP ranged between 0.5 and 2 μM in different experiments. During the SE-HPLC serial dilution experiments, from 0 to about 2 molar equivalents of 1 per one equivalent of IgG were used, while keeping the antibody concentration constant. Chromatograms of the samples consistently yielded three peaks corresponding to molecular weights of the monomer IgG^DNP, a monocyclic IgG^D dimer, and a bicyclic IgG^DNP trimer (Fig. 1). Integrations of the areas under these peaks established the relative abundances of each one of these species for each serial dilution (Fig. 2). At 0.4 μM 1, conversion of IgG^DNP to the bicyclic IgG^DNP trimer complex occurred in greater than 90% yield at [IgG^DNP] = 0.6 μM. Fig. 5 illustrates the predicted three-dimensional structure of the trivalent molecule and complexes with IgG. The Fab regions (for clarity) (PDB ID# IAOQ) from three IgG molecules are shown on the left portion of Fig. 5 superimposed against a single trihapten molecule 1 to estimate the dimensions potentially available in an IgG₃I₂ aggregate. The Fab region of an IgG antibody at its widest point is approximately 5 nm. Each one of the EGg linkers connecting the antigens to the core of the antigen molecule can extend to approximately 3.2 nm in length. In a fully extended form of the EGs linkers, the Fab regions can adopt the relative distances shown in Fig. 5. This system follows two distinct regimes of behavior determined by the stoichiometrics of 1 to lgG^DNP. At stoichiometrics of l:IgG^DNP <2:3, the major product of aggregation that was observed using SE-HPLC corresponded to a molecular weight of three IgGs; this molecular weight suggests the formation of the complex IgG₃I?. At stoichiometrics of l:IgG^DNP > 2:3, SE-HPLC analysis showed three species with molecular weights corresponding to the bicyclic trimer complex (IgGsIo), monocyclic dimer complex (IgG?!?) and monomer species (IgG, IgGl and/or IgGl₂). The smaller aggregates may emerge as a result of excess 1 competitively dissociating the complex IgG₃I₂ into monocyclic dimer IgG? 1? and monomers IgGl and IgGl₂.

A model was developed to describe this system (Ki, K₂, and K₃ all have units of M^"1; IQ has units of M⁴) and fitted to the data obtained by integrating the peaks of the chromato grams of the SE-HPLC experiments. The following equations are proposed for the formation of the observed complexes in the SE-HPLC experiments: K₁ [igG lj

_{IgG + 1} ,_gG1 *, = __ _{( 1 )}

2 O _T Ig nGtl . ^K2 - _T Ig nG₂ -Ii₂ K is₂ = T L^₂ I T₂₂- J _r (2_nΛ)

[IgG IJ-

IgG₂I? + IgG ÷Jk±± IgG₃I? K, = (3)

The initial reaction combines IgG and 1 , and generates IgGl (equation 1). The next species that forms is IgG₂I₂ (equation 2), which is followed by the addition of the third IgG to this monocyclic antibody dimer, to yield the bicyclic antibody trimer IgG₂I₃ (equation 3). The monocyclic dimer IgG₂I₂, may form by two paths; one is the reaction of two IgGl complexes, and the second is the reaction of a doubly ligand bound IgG, IgGl?, with a free molecule of IgG. We assume that the free energy of these two interactions is indistinguishable since the binding sites are approximately independent.

Equation 4 describes the dissociation of the complex to monomers, and the equilibrium constant relating IgG₃I₂ to IgG and 1 is described by equation 5:

IgG₃I₂ ^- S igG ₊ 2 i ^ = iτfττ W

Peak areas of SE-HPLC chromatograms were integrated using Lorentzian fits. The error bars in Fig. 2 are from peak integrations of four separate experiments, each datum is the mean of these measurements and the error bars show the maximum deviation. Using these values with the SOLVE function in Mathematica 5.1 , we algebraically solved the equilibrium equations (described in Materials and Methods), and generated polynomial expressions for [IgGl], [IgG2l₂] and [IgG₃I?] as functions of [1] total, [IgG] total, ^and for the equilibrium constants Kj, Kj and Ks. Monte Carlo optimization identified optimal values of K;, K₂ and K₃ (Ki = 1.25 x 10⁹ M^"1, /<^"? = 1.50 x 10⁷ M^"1, and K₃ = 1.00 x 10¹⁰ M^"1); these values fit the data with an R-Square value of 0.988. For the initial Ki value we used a value of ^^" J⁰"⁰ that we calculated using fluorescence quenching of Trp residues on the IgG upon ligand binding. The predicted curves (Fig. 2) agree well with the data. When these values are inserted into equation 5, the K_d was calculated to be K₁ = 4.27 x 10^"36 M⁴. When the SE-HPLC experiments were conducted without prior incubation

(injecting the samples within 1 minute of mixing 1 with IgG^DNP), the chromatograms yielded a new peak that eluted in the void volume. A peak at this retention time corresponds to species that are beyond the resolution limits of the column (heavier than 500 IcDa). Injection of the same sample, after five hours of incubation, established that this peak had disappeared and the only major peak remaining was that which belonged to the IgG₃I₂ complex. This observation indicates that the formation Of IgG₃Io proceeds through kinetically formed, thermodynamically less stable, high molecular weight aggregates, which rearrange to the more stable IgG₃I? with time. The rearrangement of these aggregates was examined in greater detail using Dynamic Light Scattering (DLS).

Dynamic Light Scattering (DLS). DLS experiments established the distribution of antibody aggregate sizes during equilibration. DLS was chosen for observing the kinetics of aggregate formation during equilibration since DLS provides real-time information about the aggregate size. Experiments began with 3.33 μM (0.5 mg/mL) anti-DNP IgG (PBS buffer pH 7.4 at 25 ⁰C). We added enough 1 to form IgG₃I₂ (2.22 μM), and measured the scattered light at intervals appropriate for the kinetics of the equilibration of the aggregates. Each datum was an average over 2 minutes. Anti-DNP IgG alone yielded a single species with a radius of 5.4 nm on DLS. It was not expected that the DLS would differentiate between complexes IgG2l2 and IgG₃12 since the hydrodynamic radius of these two species should be very similar. Two minutes after mixing, the solution contained a mixture: a species with an average radius of 11.0 nm (corresponding to the complexes IgG₃^ and IgG2l₂) and larger species with radii ranging from 1 x 10⁴ to 2 x 10⁵ nm corresponding to high molecular weight aggregates (Fig. 3). The conversion of IgG to IgG₃I₂ and IgG₂I₂ at 2 min was -60%. After 15 to 20 minutes of incubation at room temperature, the only observable species (> 99%) was the species having a radius of 11.0 nm. It was concluded that the smaller aggregates are the thermodynamic products, derived by equilibration of unstructured, larger aggregates that are the initial products.

Analytical Ultracentrifugation (A UC) To validate the finding that the thermodynamic product was actually the bicyclic structure IgG₃I₂, sedimentation equilibrium experiments were carried out using a Beckman XL-I ultracentrifuge at rotor speeds of 6,000 rpm, 9,000 rpm, and 12,000 rpm, at 25 ⁰C (Fig. 4). Several samples were observed in PBS buffer at pH 7.4, with a range of antibody concentrations from 0.05 μM to 0.5 μM with 3:2 stoichiometric concentrations of 1.

Sedimentation equilibrium data were collected for 20 hours for each sample. Comparison of the data at 18 hours and 20 hours into the experiment confirmed that the sample had reached equilibrium. The data-analysis software "Origin" was used with a plug-in supplied by Beckman to fit the AUC data. All the AUC data obtained from several equilibrium experiments at various sample concentrations were fit simultaneously. This procedure yielded an estimated molecular weight of 464 ± 35 kDa for the IgG^DNP complex. This result was close to the predicted value of 450 ± 12 kDa for the IgG₃Io species (the molecular weight of an IgG monomer is approximately 150 ± 4 kDa). The AUC experiments carried out with monomelic IgG^DNP yielded a molecular weight of 156 ± 8 kDa. The individual data were fit to the homogenous species model using the data analysis program Igor (as described in Example 1). The molecular weight calculated for the complex using these fits validated the molecular weights obtained from fits performed using "Origin". The results of the ALJC experiments support the conclusions of the SE-HPLC and DLS experiments.

Example 3 : Thermodynamics and Kinetics The DLS experiments established that although monomeric IgG^DNP (0.50 mg/mL, 3.3 μM) immediately forms aggregates on addition of 1, the system reached thermodynamic equilibrium only after 15-20 minutes of incubation at 25 ⁰C. At lower concentrations, the antibody/trivalent hapten mixture may reach thermodynamic equilibrium more rapidly. To measure the rate of ring opening, which was believed to be the slow step of dissociation of the complex, a 1000-fold excess of a competitive monovalent hapten iVε-2,4-DNP-Lysine (DNP-Lys) was added to the preformed bicyclic complex IgG₃Ii. It was predicted that on the opening of one of the rings, the excess DNP-Lys would bind to the free Fab binding site. The resulting aggregate IgG₃I₂ ⁴DNP-LyS would have one of the IgGs bound only monovalently to the rest of the complex. A second dissociation would remove the monovalently bound IgG from the complex, leaving the monocyclic dimer IgG₂I₂, and this dimeric aggregate would in turn dissociate into monomeric IgG units by an analogous mechanism. The counter-intuitive properties of this type of system on dissociation are similar to those identified using a system comprising oligovalent derivatives of vancomycin and D-AIa-D- Ala.

In the present example, a sample was injected onto the SE-HPLC immediately after mixing DNP-Lys with IgG₃I₂. The chromatogram demonstrated that IgG₃I₂ had completely dissociated into monomeric antibody. Assuming that complete dissociation requires five half-lives, and that the sample reaches the column after 60 seconds of mixing, the lower limit for the pseudo first-order rate constant for dissociation is k_off ≤4-2 ^x 10^"2 sec^"1. Although this calculated off-rate for the individual Fab/DNP interaction suggests a lifetime (τ_off) <~0.4 minutes, the IgG₃I₂ complex was kinetically stable over the course of the approximately 17 minute interval required for SE-HPLC. In another experiment, the flow rate of the running buffer was slowed to 0.1 niL/minute (instead of the typical 0.5 mL/minute) in order to increase the length of time the complex spent on the column by about a factor of five. At this flow rate, the retention time for the IgG₃I₂ complex was about 91 minutes. Integrating the peak areas showed that the complex was still completely intact. It is believed that the extra kinetic stability Of IgG₃I₂ (at least a factor of- 225 relative to IgG^βDNP-Lys) reflects the multi valency of the interactions in the aggregate.

Example 4: Selective Binding in an ELISA Assay

The selective binding experiments were earned out using monoclonal anti- DNP IgG antibody from rat (IgG^DNP). The trivalent molecules of DNP (dinitrophenyl) were synthesized as described above. Mixing of IgG^DNP with the trivalent DNP molecule 1 at 3 to 2 stoichiometry yields bicyclic antibody trimer complexes (IgG₃I?), as shown above.

An ELISA assay was used to test the specificity of binding of the bicyclic antibody trimer complexes (IgG₃I₂) (i.e. the IgG^DNP-l antibody/trihapten complex). 96-well plates coated with a polymer terminating in maleic anhydride moieties were used to covalently attach DNP moieties on the surface of separate wells at various densities as described in Fig. 9: Fig. 9. a) average DNP distances calculated, as described below, from the loading of the molecule to the wells are shown for each row on a 96-well plate, b) The DNP molecules are covalently attached to the surface of the 96-well plate through reacting with the maleimide anhydride moieties on the coating polymer. Maleic anhydride functionalized 96-well plates were purchased from Pierce (Product # 15108). Maleic anhydride spontaneously reacts with primary amines to form amide bonds. The beta amine on DNP-Lysine molecules was used to covalently attach these molecules on the functionalized plate surface. The DNP- labeled 96-well plates were then used in an ELISA assay. Specifically, the ELISA assay was conducted as follows: i) anti-DNP was added to the wells either as the uncomplexed monomer, or as the bicyclic complex IgG₃l₂ and incubated for 2 hours; ii) the unbound antibody was washed with 0.05% Tween 20 containing PBS (phosphate buffered saline) buffer; iii) the wells were incubated for 1 hour with HRP (horse radish peroxidase) enzyme linked secondary antibody (anti-Rat IgG from goat); iv) the HRP ligand was added in the wells after the unbound secondary antibody was washed; v) reaction of HRP with its substrate (Amplex Red reagent) in the phosphate buffered reaction buffer containing 0.0035% H₂O₂ produces a fluorescent molecule used to quantify the amount of HRP present on the well by using a plate reader that can measure fluorescence. The HRP kit was obtained from Molecular Probes (product # A22188). The substrate used, Amplex Red reagent, was (10-acetyl-3,7-dihydroxyphenoxazine).

After washing the wells, to remove unbound antibody and trihapten molecule, a secondary antibody was introduced that binds to the rat IgG^DNP. The secondary antibody was anti-rat IgG from goat, and was linked to the horse radish peroxidase enzyme (HRP). The amount of IgG^DNP bound on the surface was quantified by measuring the fluorescence generated by the product of HRP linked to the secondary antibody.

During the ELISA assay, to the wells with different surface densities of DNP, solutions containing a constant concentration of 0.1 11M monomeric antibody or antibody complex were introduced. The ELISA assay established that the monomeric

IgG bound more readily to the DNP moieties on the surface, whereas the complexed IgG required a higher density of DNP on the surface for binding. Furthermore, the amount of binding was always lower for the complexed IgG than for the monomeric IgG. The results of the ELISA assays are given in Fig. 10: The antibody binding is quantified by measuring the increase in fluorescence upon oxidation of using a HRP attached secondary antibody. The dotted lines describe hypothetical cases for different surface density of the target ligands.

The data from ELISA experiments are shown in Figure 10, and are discussed below. In order to interpret the data, the data points were fit to sigmoid curves, where the deflection points show the half point of the maximum antibody binding. According to the averages of seven separate ELISA experiments the mid point for the monomeric antibody occurs when the DNP moieties are separated by 7.1 nm, whereas this separation is as small as 3.4 nm for the antibody/trihapten molecule complexes. Because on average the IgG molecules bind multivalently to their antigens when the antigens are positioned approximately 8 nm apart, this result shows that even the monomeric IgG^DNP prefers to bind bivalently. This is possibly a result of the very fast off-rate for the monovalent DNP-IgG^DNP interaction. The differences seen in this experiments may be more significant if the monovalent off-rate was slower, because this would shift the curve of the monovalent antibody to the right.

To further illustrate the selectivity, consider that normal cells, such as human endothelial cells, can have receptors on their surfaces separated by a distance of 7.1 iim (labeled as Case 1 in Fig. 10). In this case, the monovalent IgG will likely bind to the cell surface at approximately 50% of its maximum capacity, whereas the IgG complex will likely bind only at 20% of its maximum capacity (this number is lower for absolute binding). Thus, non-specific toxicity may be reduced by approximately 30% or more. In the case of an antibody/antigen pair where the monovalent binding has a slower rate, the difference may be more pronounced.

When the DNP molecules have a separation of 11 nm, there may be little benefit in using a complex for the introduction of the antibody (labeled as Case 2 in Fig. 10) to reduce the non-specific toxicity. However, if the antibody/antigen pair had a slower off-rate for monovalent binding, monomelic antibody would have bound to surface at higher quantities and delivery using the complex would have been beneficial.

The average separation between each DNP on the surface of the wells was calculated as follows, and can be readily ascertainable by a person of skill in the art.. Each well of a 96-well plate has a maximum capacity of about 0.360 mL volume of sample, and the total surface area of a single well is 240 mm² (2.4 X 10¹⁴ nm²). If the total area is divided up into equal squares where each DNP molecule sits in the middle of each one of these squares, then the total number of DNP-Lys molecules that are needed to cover the well's surface (through reacting with the maleic anhydride that is coating the surface) at each separation distance, can be calculated by dividing the total area by the square of this distance (which gives the area of the square where

DNP is positioned in the middle). We selected these separation distances to be in a range from 0.8 nm to 100 nm. Using the number of DNP molecules estimated by this method (and assuming only half of the molecules will react and be appropriately positioned for antibody binding) the number of moles of DNP-Lys needed to be added into each row on the 96-well plate to achieve the desired DNP separations was calculated. Volumes of DNP-Lys solution of known concentrations were added to the corresponding wells to achieve the desired number of moles in the wells. The DNP separation distances for each row on the 96-well plate are illustrated in figure 9 a). Example 5: Selective Targeting of Cancer Cells Overexpressing Human Epidermal Growth Factor Receptor 2 (HER-2)

The human epidermal growth factor receptor 2 (HER-2, also known as Her- 2/neu, ErbB2) is a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases, which in humans includes EGFR (HER-I, ErbBl), HER-2, HER-3 (ErbB3) and HER-4 (ErbB4). HER receptors are important in the regulation of cell proliferation and differentiation, therefore, their overexpression and uncontrolled activation is associated with many of the key features of cancer, such as autonomous cell growth, invasion, angiogenic potential and development of distant metastases.

Her-2/neu is overexpressed in -30% of invasive breast cancers and -70% of ductal carcinomata in situ, and in ovarian, renal and colon cancers. As the molecule contains a large extracellular domain and is thus accessible to components of the immune system, a series of monoclonal antibodies targeting Her-2/neu have been generated. The most prominent example is trastuzumab (Herceptin®), a humanized monoclonal antibody.

Multiple antigens have been reported to bind to trastuzumab. One antigenic peptide sequence was selected as a mimotope. This cyclic peptide mimotope will be synthesized on the solid phase and before cleavage, the N-terminus of this cyclic peptide mimotope will be coupled to amido-EGg-carbocylic acid to serve as a linker, yielding NH₂-EG₈-C-QMWAPQWGPD-C) using methods such as in Scheme 2, above, or as are known to a person of skill in the art. After cleavage the amine on the EGs will be used to couple three mimotope-linker molecules to the central molecule (using the method illustrated in Scheme 2, above) to generate a trivalent version of mimotope.

The selective binding of this antibody, either as the uncomplexed monomer or as the bicyclic complex formed upon interaction of the antibody with the trivalent cyclic peptide, will be tested against tumor cells, using non-tumor (normal) cells as controls. We will use several cell lines of epithelial mesenchymal origin that express different levels of HER-2 receptor on their surfaces. These experiments will determine the enhancement in selectivity of targeting for tumor cells as a result of delivering the antibody as the bicyclic complex.

Thus, in one aspect, the bicyclic, trihapten molecule/antibody complexes are useful for the delivery of therapeutic antibodies with enhanced selectivity for tumor cells having surface antigens that are overexpressed. The enhanced selectivity of antibodies delivered in a trihapten molecule/antibody complex leads to reduced nonspecific toxicity and enhanced efficacy.

The following references are hereby incorporated by reference in their entireties:

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It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A trihapten molecule of Formula I having flexible amis,

2

X

Z Z Formula I wherein

X is a trivalent core structure;

Y is a linker moiety that is covalently linked to the core structure; and Z is a hapten moiety that is covalently linked to the linker moiety, wherein the hapten moieties have the same structure; or a pharmaceutically acceptable salt thereof.

2. The trihapten molecule of claim 1, wherein the core structure contains threefold symmetry.

3. The trihapten molecule of claim 1, wherein the length of the linker (Y) is about 1.5 nm to about 9 nm.

4. The trihapten molecule of claim 1 , wherein X is:

Formula II; Formula III, Formula IV; Λ

_/CH o ^{^} >^H.

Formula V; Formula Vl; Formula VII; Formula VIII;

Formula IX; Formula X; Formula XI; Formula XII,

wherein Q is NH, O, or S; or

SiRj, wherein Ri is H, -0-Ci-C₆ alkyl, or OH.

5. The trihapten molecule of claim 4, wherein X is N.

6. The trihapten molecule of claim 1, wherein Y comprises an amino acid or an ethylene glycol.

7. The trihapten molecule of claim 6, wherein Y comprises:

wherein n is an integer from 2 to 20, and m is an integer from 1 to 20.

8. The trihapten molecule of claim 1 , wherein the hapten is a toxin, a peptide, a peptoid, a peptidomimetic, a small non-nucleic acid organic molecule, a small nucleic acid molecule, an aptamer, or a drug.

9. The trihapten molecule of claim 1 , wherein the IQ for an antibody bound to the trihapten molecule in a ratio of trihapten molecule:antibody of 2:3, is smaller than the K_d for the antibody bound to a monohapten molecule.

5 10. A complex comprising two trihapten molecules of claim 1 and three antibody molecules bound to the trihapten molecules, wherein the antibody molecules bind to the haptens of the trihapten molecules.

1 1. The trihapten molecule of claim 1 , wherein (Z) is

a member of the epidermal growth factor receptor family of receptor tyrosine kinases, an anthrax antigen, a Sm antigen, or sialic acid.

12. The trihapten molecule of claim 1, wherein (Z) is HER-I, HER-2, HER-3 or 15 HER-4.

13. The trihapten molecule of claim 1, wherein the hapten is 2, 4-dinitrophenol.

14. A trihapten molecule of Formula 1 :

or a pharmaceutically acceptable salt thereof.

15. A method comprising

(a) providing a trihapten molecule of claim 1 having a selected hapten;

(b) contacting the trihapten molecule with a sample comprising an antibody that specifically binds the hapten; and

(c) determining the amount of antibody bound to the hapten.

16. A method comprising

(a) providing a trihapten molecule of claim 1 , wherein the trihapten molecule has a selected hapten moiety;

(b) contacting the trihapten molecule with an antibody that specifically binds the selected hapten moiety, thereby forming a trihapten/antibody complex;

(c) contacting the trihapten-antibody complex with a sample comprising the antibody; and (d) determining the amount of antibody displaced from the trihapten- antibody complex by the sample antibody.

17. The method of claim 16, wherein the amount of antibody displaced is determined by assaying a decrease in the trihapten/antibody complex.

18. The method of claim 16, wherein the sample antibody is labeled.

19. The method of claim 16, wherein the trihapten molecule or the antibody of the trihapten/antibody complex is labeled.

20. A method of binding a selected antibody in a stable complex, the method comprising

(a) providing a trihapten molecule that can bind to the selected antibody;

((b) contacting a sample comprising the selected antibody with the trihapten molecule, thereby forming a trihapten/antibody mixture; and

(c) incubating the trihapten/antibody mixture under conditions sufficient to permit binding of the trihapten molecule and the antibody and formation of a stable trihapten/antibody complex.

21. The method of claim 20, wherein the selected antibody is from a mammal.

22. The method of claim 21 , wherein the mammal is a human.

23. The method of claim 20, wherein the selected antibody is a disease- associated antibody.

24. The method of claim 20, wherein the stable antibody/trihapten complex has a ratio of antibody:trihapten molecule of 3:2.

25. A method of delivering an antibody to a subject, the method comprising:

(a) providing a complex comprising an antibody and a trihapten molecule of claim 1 ; and

(b) administering the complex to a subject, wherein the subject and the trihapten molecule comprise the same hapten and the antibody is capable of binding to the hapten.

26. The method of claim 25, wherein the subject is a mammal.

27. The method of claim 26, wherein the mammal is a human.

28. The method of claim 25, wherein the antibody can bind to an antigen associated with or causing a disease or a symptom of the disease.

29. The method of claim 25, wherein the subject comprises a cell that comprises a hapten that can bind to the antibody.

30. The method of claim 29, wherein the cell is a disease-associated cell.

31. The method of claim 30, wherein the disease is cancer.

32. The method of claim 31 , wherein the cell is a tumor cell.

33. The method of claim 29, wherein the cell overexpresses the hapten on the cell surface.

34. The method of claim 25, wherein the hapten is a disease-associated hapten.

35. The method of claim 25, wherein the complex has a ratio of antibody:trihapten molecule of 3:2, and wherein the antibodies bind to the haptens of the trihapten molecule.

36. A method of delivering an agent to a subject, the method comprising:

(a) providing a complex comprising an antibody, an agent, and a trihapten molecule of claim 1 ; and (b) administering the complex to a subject having a disease that the agent is capable of treating wherein the subject and the trihapten molecule comprise a hapten and the antibody is capable of binding to the hapten.

37. The method of claim 36, wherein the subject is a mammal.

38. The method of claim 37, wherein the mammal is a human.

39. The method of claim 36, wherein the agent is a label.

40. The method of claim 36, wherein the agent is attached to the antibody.

41. The method of claim 36, wherein the agent is attached to the trihapten molecule.

42. The method of claim 36, wherein the agent is a drug.

43. The method of claim 42, wherein the drug is for treating cancer.

44. The method of claim 36, wherein the subject comprises a cell that comprises the hapten.

45. The method of claim 44, wherein the cell is a disease-associated cell.

46. The method of claim 45, wherein the disease is cancer.

47. The method of claim 46, wherein the cell is a tumor cell.

48. The method of claim 44, wherein the hapten is overexpressed on a surface of the cell.

49. The method of claim 44, wherein the hapten is a disease-associated hapten.

50. The method of claim 36, wherein the complex has a ratio of antibody:trihapten molecule of 3:2, and wherein the antibodies bind to the haptens of the trihapten molecule.

51. A method comprising

(a) providing a trihapten molecule of claim 1 having a selected hapten; (b) contacting the trihapten molecule with a sample comprising an antibody that specifically binds to the hapten; and

(c) determining the amount of the antibody bound to the trihapten molecule.

52. A method of determining the relative affinity of a trihapten molecule/antibody complex for a surface, the method comprising:

(a) providing a first surface comprising a first plurality of a hapten that can bind to a selected antibody; (b) contacting the first surface with a complex comprising a trihapten molecule/antibody complex, wherein the antibody of the complex is the selected antibody and the haptens of the complex are the same as the haptens attached to the first surface;

(c) determining the amount of the antibody bound to the first surface, thereby providing a first amount; and

(d) comparing the first amount with a second amount, wherein the second amount is determined by:

(1) contacting a second surface with a complex comprising the trihapten molecule/antibody complex, wherein the second surface comprises a second plurality of haptens, the antibody of the complex is the selected antibody, the haptens of the complex can bind to the selected antibody and are the same as the haptens attached to the second surface, and the density of the second plurality haptens on the second surface is different from the density of the first plurality of haptens son the first surface; and (2) determining the amount of the antibody bound to the second surface, thereby providing a second amount.

53. A method of determining the amount of an antibody in a sample, the method comprising (a) providing a sample to be tested for the presence of an antibody against a selected antigen;

(b) contacting the sample with a trihapten molecule, wherein the hapten of the trihapten molecule can bind an antibody against the selected antigen, thereby providing an assay sample; (c) incubating the assay sample for a time sufficient to permit binding of the trihapten molecule and antibody against the selected antigen; and

(d) detecting the amount of antibody bound to the trihapten molecule,

54. The method of claim 53, wherein the sample comprises a cell.

55. The method of claim 53, wherein the hapten is a cell-surface antigen.

56. The method of claim 53, wherein the hapten is an Sm antigen, an anthrax antigen, sialic acid,

a member of the epidermal growth factor receptor family of receptor tyrosine kinases.