Integrin-associated protein (CD47) is a multiply membrane spanning member of the immunoglobulin superfamily that regulates some adhesion-dependent cell functions through formation of a complex with αvβ3 integrin and trimeric G proteins. Cholesterol is critical for the association of the three protein components of the supramolecular complex and for its signaling. The multiply membrane spanning domain of IAP is required for complex formation because it binds cholesterol. The supramolecular complex forms preferentially in glycosphingolipid-enriched membrane domains. Binding of mAb 10G2 to the IAP Ig domain, previously shown to be required for association with αvβ3, is affected by both the multiply membrane spanning domain and cholesterol. These data demonstrate that cholesterol is an essential component of the αvβ3/IAP/G protein signaling complex, presumably acting through an effect on IAP conformation.

Integrin-associated protein (IAP, CD47) is a plasma membrane receptor for thrombospondins and signal regulatory proteins (SIRPs) that has an essential role in host defense through its association with integrins. The IAP gene encodes alternatively spliced carboxyterminal cytoplasmic tails that have no previously described function. IAP cytoplasmic tails can bind two related proteins that mediate interaction between IAP and vimentin-containing intermediate filaments, named proteins linking IAP with cytoskeleton (PLICs). Integrins interact with PLICs indirectly, through IAP. Transfection of PLICs induces redistribution of vimentin and cell spreading in IAP-expressing cells. This novel connection between plasma membrane and cytoskeleton is likely to be significant in many adhesion-dependent cell functions.

A series of multivalent, functional polymer nanoparticles with diagnostic/imaging units and targeting ligands for molecular targeting were synthesized with the loading of the chain end functionalized, GRGDS peptide targeting sequence (model system based on integrin αvβ3) ranging from 0 to 50%. Accurate structural and functional group control in these systems was achieved through a modular approach involving the use of multiple functionalized macromonomer/monomer units combined with living free radical polymerization. In cellulo results show an increase in uptake in αvβ3 integrin-positive U87MG glioblastoma cells with increasing RGD loading and a possible upper limit on the effectiveness of the number of RGD peptides for targeting αvβ3 integrin. Significantly, this increased targeting efficiency is coupled with in vivo biodistribution results which show decreased blood circulation and increased liver uptake with increasing RGD loading. The results demonstrate the importance of controlling ligand loading in order to achieve optimal performance for therapeutic and imaging applications for multivalent nanoparticle based systems.

Recently, the somatostatin receptor subtype 2 (SSTR2) selective antagonist sst 2 -ANT was determined to have a high affinity for SSTR2. Additionally, 111 In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-sst 2 -ANT showed high uptake in an SSTR2-transfected, tumor-bearing mouse model and suggested that radiolabeled SSTR2 antagonists may be superior to agonists for imaging SSTR2-positive tumors. This report describes the synthesis and evaluation of 64 Cu-CB-4,11-bis(carboxymethyl)-1, 4,8,11-tetraazabicyclo[6.6.2]hexadecane-sst 2 -ANT ( 64 Cu-CB-TE2A-sst 2 -ANT) as a PET radiopharmaceutical for the in vivo imaging of SSTR2-positive tumors. Methods: Receptor-binding studies were performed to determine the dissociation constant of the radiopharmaceutical 64 Cu-CB-TE2A-sst 2 -ANT using AR42J rat pancreatic tumor cell membranes. The internalization of 64 Cu-CB-TE2A-sst 2 -ANT was compared with that of the 64 Cu-labeled agonist 64 Cu-CB-TE2A-tyrosine 3 -octreotate ( 64 Cu-CB-TE2A-Y3-TATE) in AR42J cells. Both radiopharmaceuticals were also compared in vivo through biodistribution studies using healthy rats bearing AR42J tumors, and smallanimal PET/CT of 64 Cu-CB-TE2A-sst 2 -ANT was performed. Results: The dissociation constant value for the radiopharmaceutical was determined to be 26 6 2.4 nM, and the maximum number of binding sites was 23,000 fmol/mg. 64 Cu-CB-TE2A-sst 2 -ANT showed significantly less internalization than did 64 Cu-CB-TE2A-Y3-TATE at time points from 15 min to 4 h. Biodistribution studies revealed that the clearance of 64 Cu-CB-TE2A-sst 2 -ANT from the blood was rapid, whereas the clearance of 64 Cu-CB-TE2A-sst 2 -ANT from the liver and kidneys was more modest at all time points. Tumor-to-blood and tumor-to-muscle ratios were determined to be better for 64 Cu-CB-TE2A-sst 2 -ANT than those for 64 Cu-CB-TE2A-Y3-TATE at the later time points, although liver and kidney uptake was significantly higher. Small-animal imaging using 64 Cu-CB-TE2A-sst 2 -ANT revealed excellent tumor-to-background contrast at 4 h after injection, and standardized uptake values remained high even after 24 h. Conclusion: The PET radiopharmaceutical 64 Cu-CB-TE2A-sst 2 -ANT is an attractive agent, worthy of future study as a PET radiopharmaceutical for the imaging of somatostatin receptorpositive tumors.

Pertuzumab is a monoclonal antibody that binds to HER2 and is used in combination with another HER2–specific monoclonal antibody, trastuzumab, for the treatment of HER2+ metastatic breast cancer. Pertuzumab binds to an HER2 binding site distinct from that of trastuzumab, and its affinity is enhanced when trastuzumab is present. We aim to exploit this enhanced affinity of pertuzumab for its HER2 binding epitope and adapt this antibody as a PET imaging agent by radiolabeling with 89Zr to increase the sensitivity of HER2 detection in vivo. Here, we investigate the biodistribution of 89Zr-pertuzumab in HER2–expressing BT-474 and HER2–nonexpressing MDA-MB-231 xenografts to quantitatively assess HER2 expression in vivo. In vitro cell binding studies were performed resulting in retained immunoreactivity and specificity for HER2–expressing cells. In vivo evaluation of 89Zr-pertuzumab was conducted in severely combined immunodeficient mice, subcutaneously inoculated with BT-474 and MDA-MB-231 cells. 89Zr-pertuzumab was systemically administered and imaged at 7 days postinjection (p.i.) followed by terminal biodistribution studies. Higher tumor uptake was observed in BT-474 compared to MDA-MB-231 xenografts with 47.5 ± 32.9 and 9.5 ± 1.7% ID/g, respectively at 7 days p.i (P = 0.0009) and blocking studies with excess unlabeled pertuzumab showed a 5-fold decrease in BT-474 tumor uptake (P = 0.0006), confirming the in vivo specificity of this radiotracer. Importantly, we observed that the tumor accumulation of 89Zr-pertuzumab was increased in the presence of unlabeled trastuzumab, at 173 ± 74.5% ID/g (P = 0.01). Biodistribution studies correlate with PET imaging quantification using max SUV (r = 0.98, P = 0.01). Collectively, these results illustrate that 89Zr-pertuzumab as a PET imaging agent may be beneficial for the quantitative and noninvasive assessment of HER2 expression in vivo especially for patients undergoing trastuzumab therapy.