Engineering an antibody with picomolar affinity to DOTA chelates of multiple radionuclides for pretargeted radioimmunotherapy and imaging.

INTRODUCTION: In pretargeted radioimmunotherapy (PRIT), a bifunctional antibody is administered and allowed to pre-localize to tumor cells. Subsequently, a chelated radionuclide is administered and captured by cell-bound antibody while unbound hapten clears rapidly from the body. We aim to engineer high-affinity binders to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelates for use in PRIT applications. METHODS: We mathematically modeled antibody and hapten pharmacokinetics to analyze hapten tumor retention as a function of hapten binding affinity. Motivated by model predictions, we used directed evolution and yeast surface display to affinity mature the 2D12.5 antibody to DOTA, reformatted as a single chain variable fragment (scFv). RESULTS: Modeling predicts that for high antigen density and saturating bsAb dose, a hapten-binding affinity of 100 pM is needed for near-maximal hapten retention. We affinity matured 2D12.5 with an initial binding constant of about 10 nM to DOTA-yttrium chelates. Affinity maturation resulted in a 1000-fold affinity improvement to biotinylated DOTA-yttrium, yielding an 8.2±1.9 picomolar binder. The high-affinity scFv binds DOTA complexes of lutetium and gadolinium with similar picomolar affinity and indium chelates with low nanomolar affinity. When engineered into a bispecific antibody construct targeting carcinoembryonic antigen, pretargeted high-affinity scFv results in significantly higher tumor retention of a (111)In-DOTA hapten compared to pretargeted wild-type scFv in a xenograft mouse model. CONCLUSIONS: We have engineered a versatile, high-affinity, DOTA-chelate-binding scFv. We anticipate it will prove useful in developing pretargeted imaging and therapy protocols to exploit the potential of a variety of radiometals.

Multivalent scaffolds for affinity maturation of small molecule cell surface binders and their application to prostate tumor targeting.

Adamantane scaffolds for affinity maturation of prostate cancer specific ligands of low molecular mass are described. These scaffolds are modular and can be used for conjugation of up to three ligands and an additional effector molecule by standard peptide coupling techniques. The potential of the scaffolds is demonstrated with the multimerization of GPI 1, a prostate cancer specific small molecule. A detailed study of multimerized GPI conjugates with near-infrared fluorophores and their binding properties to different prostate cancer cell lines shows the specific binding of these conjugates to cell types positive for prostate specific membrane antigen (PSMA). We demonstrate that these conjugates allow the sensitive imaging of prostate cancer cells with NIR methodology and suggest that our adamantane scaffolds might be generally useful for affinity maturation of small molecules targeting cell surface epitopes.

Detection of breast cancer microcalcifications using a dual-modality SPECT/NIR fluorescent probe.

Efficient chemical synthesis of a trifunctional, hydroxyapatite-binding molecule, which provides simultaneous in vivo imaging by NIR fluorescence and SPECT/CT has been described. Quantitation by SPECT provides the "gold standard" by which NIR fluorescence tomography of breast cancer microcalcifications can now be compared and optimized.

Microwave-assisted synthesis of near-infrared fluorescent sphingosine derivatives.

Microwave-assisted synthesis of near-infrared fluorescent sphingosine derivatives is described, and the utility of the probes demonstrated by co-localization studies with visible wavelength fluorescent sphingosine derivatives.

Humoral bone morphogenetic protein 2 is sufficient for inducing breast cancer microcalcification.

Microcalcifications are an important diagnostic marker for breast cancer on mammograms, yet the mechanism of their formation is poorly understood. Indeed, there is presently no short-latency, high-yield, syngeneic rodent model of the process. Bone morphogenetic protein 2 (BMP-2) is a key mediator of physiologic bone formation and pathologic vasculature calcification, but its role in breast cancer microcalcification is unknown. In this study, R3230 rat breast tumors were adapted to cell culture, transduced with adenoviral BMP-2, and inoculated into a syngeneic host. Tumor growth and calcium salt deposition were quantified in living animals over time using micro-computed tomography and probed chemically using near-infrared fluorescence. Plasma BMP-2 levels were quantified over time by enzyme-linked immunosorbent assay. Within 3 weeks, 100% of the breast tumors developed microcalcifications, which were absent from all normal tissues. Importantly, when two tumors were initiated in a single host, the ipsilateral tumor expressing BMP-2 was able to induce microcalcification in the contralateral tumor that was not expressing BMP-2, suggesting that BMP-2 can act humorally. Taken together, we describe the first reproducible rodent model of breast cancer microcalcification, prove that BMP-2 expression is sufficient for initiating the process, and lay the foundation for a new generation of targeted diagnostic agents.

Light-directed maskless synthesis of peptide arrays using photolabile amino acid monomers.

Novel photolabile amino acid monomers for photolithographic solid-phase peptide synthesis has been developed and a method for the maskless synthesis of individually addressable peptide microarrays using new building blocks has been described; these peptide microarrays are suitable for repetitive epitope-binding assays.