PSMA-reactive NB7 single domain antibody fragment: A potential scaffold for developing prostate cancer theranostics.

INTRODUCTION: Single domain antibody fragments (sdAbs) are an appealing scaffold for radiopharmaceutical development due to their small size (~15 kDa), high solubility, high stability, and excellent tumor penetration. Previously, we developed NB7 sdAb, which has very high affinity for an epitope on PSMA that is different from those targeted by small molecule PSMA inhibitors. Herein, we evaluated NB7 after radioiodination using [*I]SGMIB (1,3,4-isomer) and iso-[*I]SGMIB (1,3,5-isomer), as well as their 211At-labeled analogues. METHODS: [*I]SGMIB, iso-[*I]SGMIB, [211At]SAGMB, and iso-[211At]SAGMB conjugates of NB7 sdAb were synthesized and their binding affinity, cell uptake and internalization were assessed in PSMA+ PC3 PIP and PSMA- PC3 flu cells. Biodistribution studies were performed in mice bearing PSMA+ PC3 PIP xenografts. First, a single-label experiment evaluated the tissue distribution of a NB7 bearing a His6-tag (NB7H6) and labeled with iso-[125I]SGMIB. Three paired-label experiments then were performed to compare: a) NB7 labeled using [*I]SGMIB and iso-[*I]SGMIB, b) 131I- vs 211At-labeled NB7 conjugates and c) [125I]SGMIB-NB7H6 to the small molecule PSMA inhibitor [131I]YF2. RESULTS: All NB7 radioconjugates bound specifically to PSMA with dissociation constants, Kd, in the low nM range (1.4-6.4 nM). An initial biodistribution study demonstrated good tumor uptake for iso-[125I]SGMIB-NB7H6 (7.2 ± 1.5 % ID/g at 1 h) and no deleterious effect of the His6-tag on renal activity levels, which declined to 3.1 ± 1.1 % ID/g by 4 h. Paired-label biodistribution found no distinction between the two SGMIB isomer NB7 conjugates with the [131I]SGMIB-NB7-to-iso-[125I]SGMIB-NB7 tumor uptake ratios not significantly different from unity: 1.06 ± 0.08 at 1 h, 1.04 ± 0.12 at 4 h, and 1.07 ± 0.09 at 24 h. Both isomer conjugates cleared rapidly from normal tissues and exhibited very low uptake in thyroid, lacrimal and salivary glands. Paired-label biodistribution of [131I]SGMIB-NB7H6 and [211At]SAGMB-NB7H6 demonstrated similar tumor uptake and kidney clearance for the two radioconjugates. However, levels of 211At in thyroid, stomach, salivary and lacrimal glands were significantly higher (P < 0.05) that those for 131I suggesting greater dehalogenation for [211At]SAGMB-NB7H6. Finally, co-administration of [125I]SGMIB-NB7H6 and [131I]YF2 demonstrated good tumor uptake for both with considerably more rapid renal clearance for the NB7 radioconjugate. CONCLUSION: NB7 radioconjugates exhibited good accumulation in PSMA-positive xenografts with rapid clearance from kidney and other normal tissues. We conclude that NB7 is a potentially useful scaffold for developing PSMA-targeted theranostics with different characteristics than current small molecule and antibody-based approaches.

Nanobodies Targeting Prostate-Specific Membrane Antigen for the Imaging and Therapy of Prostate Cancer.

The repertoire of methods for the detection and chemotherapeutic treatment of prostate cancer (PCa) is currently limited. Prostate-specific membrane antigen (PSMA) is overexpressed in PCa tumors and can be exploited for both imaging and drug delivery. We developed and characterized four nanobodies that present tight and specific binding and internalization into PSMA+ cells and that accumulate specifically in PSMA+ tumors. We then conjugated one of these nanobodies to the cytotoxic drug doxorubicin, and we show that the conjugate internalizes specifically into PSMA+ cells, where the drug is released and induces cytotoxic activity. In vivo studies show that the extent of tumor growth inhibition is similar when mice are treated with commercial doxorubicin and with a 42-fold lower amount of the nanobody-conjugated doxorubicin, attesting to the efficacy of the conjugated drug. These data highlight nanobodies as promising agents for the imaging of PCa tumors and for the targeted delivery of chemotherapeutic drugs.

A dual-specific macrophage colony-stimulating factor antagonist of c-FMS and αvß3 integrin for osteoporosis therapy.

There is currently a demand for new highly efficient and specific drugs to treat osteoporosis, a chronic bone disease affecting millions of people worldwide. We have developed a combinatorial strategy for engineering bispecific inhibitors that simultaneously target the unique combination of c-FMS and αvß3 integrin, which act in concert to facilitate bone resorption by osteoclasts. Using functional fluorescence-activated cell sorting (FACS)-based screening assays of random mutagenesis macrophage colony-stimulating factor (M-CSF) libraries against c-FMS and αvß3 integrin, we engineered dual-specific M-CSF mutants with high affinity to both receptors. These bispecific mutants act as functional antagonists of c-FMS and αvß3 integrin activation and hence of osteoclast differentiation in vitro and osteoclast activity in vivo. This study thus introduces a versatile platform for the creation of new-generation therapeutics with high efficacy and specificity for osteoporosis and other bone diseases. It also provides new tools for studying molecular mechanisms and the cell signaling pathways that mediate osteoclast differentiation and function.

Engineering a monomeric variant of macrophage colony-stimulating factor (M-CSF) that antagonizes the c-FMS receptor.

Enhanced activation of the signaling pathways that mediate the differentiation of mononuclear monocytes into osteoclasts is an underlying cause of several bone diseases and bone metastasis. In particular, dysregulation and overexpression of macrophage colony-stimulating factor (M-CSF) and its c-FMS tyrosine kinase receptor, proteins that are essential for osteoclast differentiation, are known to promote bone metastasis and osteoporosis, making both the ligand and its receptor attractive targets for therapeutic intervention. With this aim in mind, our starting point was the previously held concept that the potential of the M-CSFC31S mutant as a therapeutic is derived from its inability to dimerize and hence to act as an agonist. The current study showed, however, that dimerization is not abolished in M-CSFC31S and that the protein retains agonistic activity toward osteoclasts. To design an M-CSF mutant with diminished dimerization capabilities, we solved the crystal structure of the M-CSFC31S dimer complex and used structure-based energy calculations to identify the residues responsible for its dimeric form. We then used that analysis to develop M-CSFC31S,M27R, a ligand-based, high-affinity antagonist for c-FMS that retained its binding ability but prevented the ligand dimerization that leads to receptor dimerization and activation. The monomeric properties of M-CSFC31S,M27R were validated using dynamic light scattering and small-angle X-ray scattering analyses. It was shown that this mutant is a functional inhibitor of M-CSF-dependent c-FMS activation and osteoclast differentiation in vitro Our study, therefore, provided insights into the sequence-structure-function relationships of the M-CSF/c-FMS interaction and of ligand/receptor tyrosine kinase interactions in general.

Protein Engineering by Combined Computational and In Vitro Evolution Approaches.

Two alternative strategies are commonly used to study protein-protein interactions (PPIs) and to engineer protein-based inhibitors. In one approach, binders are selected experimentally from combinatorial libraries of protein mutants that are displayed on a cell surface. In the other approach, computational modeling is used to explore an astronomically large number of protein sequences to select a small number of sequences for experimental testing. While both approaches have some limitations, their combination produces superior results in various protein engineering applications. Such applications include the design of novel binders and inhibitors, the enhancement of affinity and specificity, and the mapping of binding epitopes. The combination of these approaches also aids in the understanding of the specificity profiles of various PPIs.

Combinatorial and Computational Approaches to Identify Interactions of Macrophage Colony-stimulating Factor (M-CSF) and Its Receptor c-FMS.

The molecular interactions between macrophage colony-stimulating factor (M-CSF) and the tyrosine kinase receptor c-FMS play a key role in the immune response, bone metabolism, and the development of some cancers. Because no x-ray structure is available for the human M-CSF · c-FMS complex, the binding epitope for this complex is largely unknown. Our goal was to identify the residues that are essential for binding of the human M-CSF to c-FMS. For this purpose, we used a yeast surface display (YSD) approach. We expressed a combinatorial library of monomeric M-CSF (M-CSFM) single mutants and screened this library to isolate variants with reduced affinity for c-FMS using FACS. Sequencing yielded a number of single M-CSFM variants with mutations both in the direct binding interface and distant from the binding site. In addition, we used computational modeling to map the identified mutations onto the M-CSFM structure and to classify the mutations into three groups as follows: those that significantly decrease protein stability; those that destroy favorable intermolecular interactions; and those that decrease affinity through allosteric effects. To validate the YSD and computational data, M-CSFM and three variants were produced as soluble proteins; their affinity and structure were analyzed; and very good correlations with both YSD data and computational predictions were obtained. By identifying the M-CSFM residues critical for M-CSF · c-FMS interactions, we have laid down the basis for a deeper understanding of the M-CSF · c-FMS signaling mechanism and for the development of target-specific therapeutic agents with the ability to sterically occlude the M-CSF·c-FMS binding interface.