Enhancement of Antibody Selectivity via Bicyclic Complex FormationJared F. Stefanick,†,∥ Tanyel Kiziltepe,†,§,∥ Michael W. Handlogten,† Nathan J. Alves,†

and Basar Bilgicer*,†,‡,§

†Department of Chemical and Biomolecular Engineering, ‡Department of Chemistry and Biochemistry, and §Advanced Diagnosticsand Therapeutics, University of Notre Dame, Notre Dame, Indiana 46556, United States

*S Supporting Information

ABSTRACT: This study describes a strategy where antibodyselectivity for high antigen-density surfaces is enhanced byforming a thermodynamically stable bicyclic complex. Thebicyclic complex was formed via multivalent interactions of theantibody with a synthetic trivalent mimotope at a 3:2 molarratio. Complex formation was analyzed using dynamic lightscattering and analytical ultracentrifugation, showing a hydro-dynamic radius of ∼22 nm and a calculated molecular weightof 397 kDa, depicting a trimeric complex formation. Thecomplex has high thermodynamic stability and results in a 10-fold higher binding affinity for the trivalent mimotope (Kd =0.14 μM) compared to the monovalent mimotope (Kd = 1.4μM). As bicyclic complexes, the antibodies showed ∼18% binding of the monomeric form to low antigen-density surfaces. Athigh antigen-density, antibody binding was equal whether delivered as a complex or a monomer. These results establish bicycliccomplex selectivity for high antigen-density surfaces and suggest a potential method to enhance therapeutic antibody selectivityfor diseased cells.

SECTION: Biophysical Chemistry

In this study, we describe a strategy where the selectivity of amonoclonal antibody for its target surface is enhanced by

delivering it as a thermodynamically stable bicyclic complex. Intherapeutic applications, monoclonal antibodies target a cellsurface receptor that is expressed at several folds (up to 100)higher density on target cells than healthy cells to provideselectivity.1−7 Nevertheless, nonselective binding and theassociated nonspecific toxicity still remains a problem depend-ing on the breadth of occurrence of the receptor on healthycells.8−11 This is well-exemplified in the case of trastuzumab,which is a therapeutic antibody that targets the human HER2receptor. Although trastuzumab has significantly improved theprognosis of breast cancer patients overexpressing HER2,12,13 ithas been associated with significant cardiac toxicity attributedto its nonselective binding to HER2 receptors on adult hearttissue.13−17 This underlines the need for a more selectivetargeting approach.In previous studies, we showed that bivalent binding of an

IgG antibody (anti-DNP IgG; IgGDNP) to the correspondingsynthetic trivalent ligand (tris-DNP) generates a bicyclicantibody complex with a high degree of thermodynamic andkinetic stability: IgGDNP showed ∼104-fold higher affinity forcomplex over its monovalent affinity for DNP.18,19 In thisstudy, we extend and apply these findings to trastuzumab andestablish that its selectivity for high antigen-density surfaces canbe enhanced by delivering it as part of such a bicyclic complex(Figure 1). Antibodies bind to low antigen-density surfacesmonovalently, whereas they can bind to high antigen-density

surfaces bivalently, which in turn amplifies their affinity. Theselectivity arises from the differences in free energy ofassociation of the antibody with the bicylic complex (ΔG°comp

tri ),low antigen-density surface (monovalent binding, ΔG°surfmono),and the high antigen-density surface (bivalent binding,ΔG°surfbi ). For the bicyclic complex to show selectivity for ahigh density surface, the formation of a bicyclic complex shouldbe more favorable than monovalent binding to a low antigen-density surface and less favorable than bivalent binding to ahigh antigen-density surface; in other words: ΔG°surfmono >ΔG°comptri > ΔG°surfbi . Therefore, when the complex is designed tomeet these criteria, antibody dissociates and binds to a surfaceonly when the surface has high antigen-density to enablebivalent binding (as in target diseased cells) (Figure 2).To demonstrate the selectivity achieved by a bicyclic complex

in an in vitro model system, trastuzumab and a peptide mimicof HER2 antigen (mimotope) were used as the antibody/hapten pair. A trivalent version of the mimotope (T) wassynthesized and allowed to react with trastuzumab to facilitatethe formation of bicyclic complexes (Figure 1).20 The bicycliccomplex stability primarily depends on the mimotope’s affinityfor trastuzumab. The peptide mimotope used in this study wasselected from the most potent peptide mimotopesdiscovered

Received: December 22, 2011Accepted: February 9, 2012Published: February 9, 2012

Letter

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through different phage display librariesreported to bindtrastuzumab.21−23 In our hands, the sequence that was reportedby Jiang et al.21 (LLGPYELWELSH) had the highest affinityfor trastuzumab (Kd

mono = 1.4 μM, see the SupportingInformation for methods); therefore, this mimotope sequenceis used in the reported study. Another factor that determinesthe thermodynamic stability of the bicyclic complex is theflexibility, length, and chemistry of the linker used in trivalentmimotope synthesis. In previous studies, we established thatethylene glycol exhibited the most favorable linker properties,including water solubility, flexibility, and minimal nonspecificinteractions with antibodies.18,24 Accordingly, in the synthesisof trivalent mimotope (T), three ethylene glycol linkers of sixrepeating units were used to conjugate each mimotopesequence to a tertiary amine core (Supporting InformationFigures 1−5). This design yields a maximum separationdistance of ∼5 nm between the mimotopes when fullyextended, providing enough space for binding of threeantibodies to one trivalent molecule simultaneously withoutsteric concerns. In our previous report, we established that theoptimal bicyclic complex formation was achieved using 3:2stoichiometry of antibody to trivalent hapten.18 In line withthese previous findings, we determined by dynamic lightscattering (DLS) that the 3:2 stoichiometric ratio was alsooptimal for bicyclic complex formation with trastuzumab andwas therefore used in the rest of the study.To characterize the biophysical properties of the bicyclic

complex, we first measured the apparent binding constant(Kd

tri) of the interaction between the trivalent mimotope andtrastuzumab using a fluorescence quenching technique (seeSupporting Information for methods). For trivalent mimotope(T) binding to trastuzumab, we measured an apparent bindingconstant of Kd

tri = 0.14 μM, which is a ∼10-fold enhancement

over the monovalent mimotope affinity (Kdmono = 1.4 μM;

Figure 3A). Binding interactions of monovalent and trivalentmimotopes with trastuzumab were also measured usingisothermal titration calorimetry (ITC), which delivered similarvalues and also determined a ∼10-fold enhancement (2.1 and0.24 μM respectively, Supporting Information Figure 6).Furthermore, ITC data for trastuzumab titration with a trivalentmimotope established a molar ratio of 0.63:1 of trivalentmimotopes to antibodies, consistent with an expected ratio of0.67 (two trivalent mimotopes/three antibodies), validating thestoichiometry of the interaction. Next, we verified theformation of the bicyclic complex (IgG3T2) by analyzing theaggregate formation with DLS measurements. According toDLS, monomeric trastuzumab, in the absence of trivalentmimotope, has a hydrodynamic radius of ∼11 nm. Upon theaddition of a stoichiometric amount of trivalent mimotope totrastuzumab, we observed that its hydrodynamic radiusincreased to ∼22 nm (Figure 3B). Because we do not expecta difference in the hydrodynamic radii between a monocyclicand a bicyclic complex, DLS results verified the formation ofcyclic complexes, however, not necessarily the formation ofbicyclic ones. Therefore, we further analyzed the system usinganalytical ultracentrifugation (AUC) equilibrium sedimenta-tion. These experiments helped determine the apparentmolecular weight (MW) for the monomeric antibody (expected145 kDa, measured 132 kDa [confidence interval: 130, 134kDa]). The equilibrium analysis of the antibody as a bicycliccomplex upon reaction with the trivalent mimotope revealedthat the bicyclic complex was the dominant species (expected435 kDa, measured 397 kDa [confidence interval: 386, 408kDa]; Figure 3C). AUC results together with DLS confirmedformation of the bicyclic complex (IgG3T2) upon reaction oftrastuzumab (IgG) with the trivalent peptide mimotope (T).

Figure 1. Equilibria of bicyclic complex formation. Bicyclic trastuzumab complex was formed by mixing stoichiometric ratios of antibody with thesynthetic trivalent mimotope (T). At 3:2 stoichiometry of antibody to trivalent mimotope, mostly bicyclic complex was observed.

Figure 2. Cartoon schematics describing enhanced selectivity of bicyclic complex. (A) When delivered as a monomer, antibody binds to the targetreceptor with high specificity. High antigen-density surfaces (i.e., diseased cells) accumulate more antibody molecules; nevertheless, significantbinding to low antigen-density surfaces (i.e., healthy cells) still takes place, resulting in nonspecific toxicity. (B) When the antibody is delivered aspart of a bicyclic complex, binding to low antigen-density surfaces is reduced as a result of the thermodynamic equilibrium established between thecomplex and surface antigen. A high antigen-density surface is required to shift the equilibrium toward binding to the target surface.

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Next, we evaluated the selectivity of the bicyclic trastuzumabcomplex for surfaces with high antigen density by synthesizingsurfaces with various HER2 mimotope densities to mimichealthy and tumor cell surfaces. In healthy cells, HER2receptors are expressed at (2 to 5) × 104 copies per cell,whereas HER2 overexpressing breast carcinomas can bear asmany as (2 to 5) × 106 copies per cell (100-fold increase).13 Onthe basis of calculations performed by assuming a ∼10 μmdiameter for a mammalian cell and a homogeneous receptordistribution over the cell surface, these values suggest anaverage HER2−HER2 distance of ∼80−125 nm for healthycells and ∼8−13 nm for cancer cells. It is noteworthy thatHER2 receptors are usually found as clusters in cancer cells;therefore, these distances are most likely underestimates.Hence, we generated surfaces with various mimotope densities,ranging from 1 to 100 nm separations, by conjugating the

peptide to maleic-anhydride-coated 96-well plate surfaces usingamide coupling. The yield of mimotope conjugation to eachsurface was calculated by measuring the amount of unreactedmimotope remaining in each solution using a fluorescentlylabeled peptide mimotope, which was consistently >95%. Theaverage distance between each mimotope on this syntheticsurface was calculated from the total number of mimotopemolecules attached to the surface divided by the total surfacearea of the well. It is noteworthy that indeed we expect somefraction of the surface-conjugated mimotopes to be unavailablefor antibody binding due to the inhomogeneous topography ofthe polymer-coated surface, yielding larger than calculatedseparation distances. Therefore, the distances reported in thisstudy are approximate values, and the results of our analysisestablish trends for trastuzumab binding to surfaces. Con-sequently, it is important to confirm that the synthesized highantigen-density surfaces can accommodate bivalent interactionsbetween the antibody and the antigen. For this purpose, weperformed a competitive inhibition experiment where trastu-zumab binding to high antigen-density surfaces (2 nmseparation) was competitively inhibited using mono- andtrivalent mimotopes. We measured the IC50 for the monovalentmimotope to be 180 μM (Supporting Information Figure 7).This value is equal to the effective molarity of the bivalentinteraction. Using this IC50, we calculated the dissociationconstant of the antibody for the high antigen-density surface(Kd

bi) to be equal to 11 nM, which is an enhancement of morethan 100-fold over the antibody binding to monovalentmimotope (we measured a similar enhancement using SPR,Supporting Information Figure 8). The enhancement in theaffinity strongly suggests that antibody molecules bivalentlyinteract with the high antigen-density surface.Finally, the bicyclic complex selectivity was compared against

the monomeric antibody using various antigen-density-functionalized surfaces. To 96-well plate surfaces, wheremimotope separation distances ranged from 1 to 100 nm,trastuzumab was delivered in the form of either monomer orbicyclic complex. After 90 min of incubation, unboundtrastuzumab was washed from wells, a secondary antibodytargeting trastuzumab was introduced, and surface-boundtrastuzumab was quantified via enzyme-linked immunosorbantassay (ELISA). On surfaces where antigen-density was very low,we did not observe any trastuzumab binding from either thecomplex or the monomer due to the low affinity of thisinteraction. As antigen-density on the surface increased, theaverage separation distance between mimotopes decreased, andsurface-bound trastuzumab increased for both forms. However,there is a significant shift in the observed binding curve for thebicyclic complex compared with the monomeric trastuzumab(Figure 4A). The shift of the binding curve demonstrates theshift in binding behavior of the antibody when delivered in theform of a bicyclic complex. It is important to point out that thisparticular binding curve is not a traditional binding curve, and itcannot be used to calculate Kd for two reasons. First, instead ofchanging the concentration of the antibody in solution, like in atraditional titration, the ligand density on the surface is altered.Second, as the ligand density changes, the antibody’s affinity forthe surface changes, hence the value of Kd for collected data isdifferent for each data point. The shift between the two bindingcurves demonstrates one important point: the bicyclictrastuzumab complex requires higher mimotope density onthe surface for binding than its monomeric form.

Figure 3. (A) Binding constants for the interaction of the antibodywith the mono- and trivalent peptide mimotopes were determined by afluorescence quenching assay. Red solid line with squares is trivalent(Kd = 0.14 ± 0.01 μM); blue dashed line with diamonds is monovalent(Kd = 1.4 ± 0.1 μM). Experiments were performed in triplicate. Datarepresent means ± SD. Bicyclic complex formation was observed using(B) size determination by DLS (blue bar monomer antibody, red barsbicyclic complex) and (C) sedimentation equilibrium analysis ofbicyclic complex by AUC.

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The selectivity introduced by bicyclic complexes is bestdemonstrated by a bar graph (Figure 4B). For high antigen-density surfaces (representative of tumor cells), efficientbinding was observed whether trastuzumab was delivered as amonomer or as a bicylic complex. In other words, there is noapparent difference in surface binding between delivering theantibody as a monomer or bicyclic complex for high antigen-density surfaces. When the antigen density was reduced(representative of healthy cells), however, antibody bindingfrom the bicyclic complex decreased to ∼53 and ∼18% of thatof monomeric trastuzumab for a separation distance of 5 and 10nm, respectively.25 Taken together, these results establish thatthe bicyclic complex has enhanced selectivity for surfaces withhigh antigen-density.

From a thermodynamic perspective, these results all fallwithin our expectations. The antibody forms bivalentinteractions under two circumstances: (i) as part of a bicycliccomplex and (ii) when binding to a high antigen-densitysurface. When part of a bicyclic complex, bivalent bindingenhances the avidity by 10-fold, and when binding to a highantigen-density surface, the enhancement is more than 100-fold(Table 1). A 10-fold difference in binding affinity reflects as a1.36 kcal/mol difference in free energy. The binding enthalpy isadditive in these noncooperative binding events.26,27 Forbicyclic complex formation, the enthalpy of antibody bindingto trivalent-mimotope (ΔH° tri) is equal to three times that ofthe monovalent mimotope (ΔH° mono), which we verified byITC measurements (ΔH° mono = −12.7 kcal/mol, ΔH° tri =−36.7 kcal/mol). Assuming that antibody does not make anyfavorable or unfavorable interactions with the surface, theenthalpy of antibody binding to high antigen-density surfaces(ΔH° bi) is equal to twice ΔH°mono (ΔH° bi = −25.4 kcal/mol),which is less favorable than binding to the trivalent mimotope(ΔH° tri). Despite being enthalpically not as favorable, the freeenergy of antibody binding to high antigen-density surfaces(ΔG°surfbi = −10.9 kcal/mol, calculated from Kd

bi) is still morefavorable than that of binding to a trivalent mimotope(ΔG°comptri = −9.3 kcal/mol). Hence, the difference in bindingenhancement is a direct result of the differences in bindingentropies (ΔS° bi = −48.8 cal/mol·K vs ΔS° tri = −91.6 cal/mol·K).28 The higher entropic cost observed for bicycliccomplex formation is presumably due to the translational(ΔS° trans) as well as the conformational (ΔS° conf) componentsof entropy. The difference in enhancement of binding affinity,ΔG°, is a combination of different enthalpic and entropiccomponents for the relevant interaction. Taken together, theseresults indicate that for the bicyclic complex of trastuzumabantibody binding to a high antigen-density surface will alwaysbe more favorable than complex formation, and, therefore, thecomplex will always show more selectivity for surfaces wherethe antibody can bind bivalently.These results suggest a potential application for bicyclic

complex strategy in the delivery of therapeutic antibodies withenhanced selectivity for tumor cells with overexpressed surfacereceptors. For a successful implementation of this approach in abiological system, bicyclic complexes should be synthesizedusing a high affinity mimotope (one possessing a Kd

complementary to that of HER2 for trastuzumab). Deliveryof antibodies as bicyclic complexes would potentially amplifytheir selectivity for the target cells, which in turn may reducenonselective targeting of healthy tissue and the associatednonspecific toxicity. Currently, this work is in progress in ourlaboratory.

Figure 4. (A) Surfaces with various mimotope densities weresynthesized, and antibody binding was measured using an ELISAassay. Blue dashed line is trastuzumab as monomer, and red solid lineis the bicyclic complex. The shift in the binding curve establishes theenhanced selectivity of bicyclic complexes for high antigen-densitysurfaces. (B) Bar plot representations of relative surface-boundtrastuzumab normalized to the monomeric form clearly display theselectivity introduced by the bicylic complex. No difference intrastuzumab binding was observed between delivering the antibodyas a monomer (blue bars) or bicyclic complex (red bars) for highantigen-density surfaces, whereas complex showed selectivity forsurfaces with high mimotope density. All experiments were performedin triplicate. Data represent means ± SD. *P < 0.001 based onStudent’s t test.

Table 1. Summary of Thermodynamic Properties of Antibody Binding to Monovalent Ligand, Trivalent Ligand, and Surface-Immobilized Ligand

ΔH° (kcal·mol−1) ΔS° (cal·mol−1·K−1) ΔG° (kcal·mol−1) Kd (M)

mimotope −12.7 −15.8 (−16.6)a −8.0 (−7.8)a 1.4 × 10−6 (2.1 × 10−6)a

trivalent mimotope (T) −36.7 −91.6 (−92.7)a −9.3 (−9.0)a 1.4 × 10−7 (2.4 × 10−7)a

high antigen-density surface −25.4b −48.8b −10.9b 1.1 × 10−8

aΔH°mono, ΔH°tri, and parenthetical values are obtained from ITC experiments. bΔG°bi is calculated using ΔG°bi = −RT ln KA, where KA = 1/Kdbi.

ΔH°bi is calculated from ΔH°bi = 2ΔH°mono. ΔS°bi is calculated using ΔG°bi = ΔH°bi − TΔS°bi.

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■ ASSOCIATED CONTENT

*S Supporting InformationExperimental protocols and supplementary data. This materialis available free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATION

Corresponding Author*Address: Department of Chemical and Biomolecular Engi-neering, Department of Chemistry and Biochemistry, Uni-versity of Notre Dame, 165 Fitzpatrick Hall, Notre Dame, IN46556-5637. Telephone: 574-631-1429. E-mail: bbilgicer@nd.edu.

Author Contributions∥These authors contributed equally.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSGenentech has provided us with trastuzumab as a gift tosupport the investigation of our hypothesis. We would like tothank the Center for Environmental Science and Technologyfor usage of the DLS, Dr. Frank Castellino for usage of theanalytical ultracentrifuge, and Dr. Brian Baker for usage ofBiacore at the University of Notre Dame.

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