Synthesis and Evaluation of a Cathepsin B-Recognizing Trifunctional Chelating Agent to Improve Tumor Retention of Radioimmunoconjugates.

Cathepsin B (CTSB) is a lysosomal protease that is overexpressed in tumor cells. Radioimmunoconjugates (RICs) composed of CTSB-recognizing chelating agents are expected to increase the molecular weights of their radiometabolites by forming conjugates with CTSB in cells, resulting in their improved retention in tumor cells. We designed a novel CTSB-recognizing trifunctional chelating agent, azide-[111In]In-DOTA-CTSB-substrate ([111In]In-ADCS), to synthesize a RIC, trastuzumab-[111In]In-ADCS ([111In]In-TADCS), and evaluated its utility to improve tumor retention of the RIC. [111In]In-ADCS and [111In]In-TADCS were synthesized with satisfactory yield and purity. [111In]In-ADCS was markedly stable in murine plasma until 96 h postincubation. [111In]In-ADCS showed binding to CTSB in vitro, and the conjugation was blocked by the addition of CTSB inhibitor. In the internalization assay, [111In]In-TADCS exhibited high-level retention in SK-OV-3 cells, indicating the in vitro utility of the CTSB-recognizing unit. In the biodistribution assay, [111In]In-TADCS showed high-level tumor accumulation, but the retention was hardly improved. In the first attempt to combine a CTSB-recognizing unit and RIC, these findings show the fundamental properties of the CTSB-recognizing trifunctional chelating agent to improve tumor retention of RICs.

SPECT/CT imaging of inflammation and calcification in human carotid atherosclerosis to identify the plaque at risk of rupture.

BACKGROUND: Calcification and inflammation are atherosclerotic plaque compositional biomarkers that have both been linked to stroke risk. The aim of this study was to evaluate their co-existing prevalence in human carotid plaques with respect to plaque phenotype to determine the value of hybrid imaging for the detection of these biomarkers. METHODS: Human carotid plaque segments, obtained from endarterectomy, were incubated in [111In]In-DOTA-butylamino-NorBIRT ([111In]In-Danbirt), targeting Leukocyte Function-associated Antigen-1 (LFA-1) on leukocytes. By performing SPECT/CT, both inflammation from DANBIRT uptake and calcification from CT imaging were assessed. Plaque phenotype was classified using histology. RESULTS: On a total plaque level, comparable levels of calcification volume existed with different degrees of inflammation and vice versa. On a segment level, an inverse relationship between calcification volume and inflammation was evident in highly calcified segments, which classify as fibrocalcific, stable plaque segments. In contrast, segments with little or no calcification presented with a moderate to high degree of inflammation, often coinciding with the more dangerous fibrous cap atheroma phenotype. CONCLUSION: Calcification imaging alone can only accurately identify highly calcified, stable, fibrocalcific plaques. To identify high-risk plaques, with little or no calcification, hybrid imaging of calcification and inflammation could provide diagnostic benefit.

Imaging inflammation in atherosclerotic plaques, targeting SST2 with [111In]In-DOTA-JR11.

BACKGROUND: Imaging Somatostatin Subtype Receptor 2 (SST2) expressing macrophages by [DOTA,Tyr3]-octreotate (DOTATATE) has proven successful for plaque detection. DOTA-JR11 is a SST2 targeting ligand with a five times higher tumor uptake than DOTATATE, and holds promise to improve plaque imaging. The aim of this study was to evaluate the potential of DOTA-JR11 for plaque detection. METHODS AND RESULTS: Atherosclerotic ApoE-/- mice (n = 22) fed an atherogenic diet were imaged by SPECT/CT two hours post injection of [111In]In-DOTA-JR11 (~ 200 pmol, ~ 50 MBq). In vivo plaque uptake of [111In]In-DOTA-JR11 was visible in all mice, with a target-to-background-ratio (TBR) of 2.23 ± 0.35. Post-mortem scans after thymectomy and ex vivo scans of the arteries after excision of the arteries confirmed plaque uptake of the radioligand with TBRs of 2.46 ± 0.52 and 3.43 ± 1.45 respectively. Oil red O lipid-staining and ex vivo autoradiography of excised arteries showed [111In]In-DOTA-JR11 uptake at plaque locations. Histological processing showed CD68 (macrophages) and SST2 expressing cells in plaques. SPECT/CT, in vitro autoradiography and immunohistochemistry performed on slices of a human carotid endarterectomy sample showed [111In]In-DOTA-JR11 uptake at plaque locations containing CD68 and SST2 expressing cells. CONCLUSIONS: The results of this study indicate DOTA-JR11 as a promising ligand for visualization of atherosclerotic plaque inflammation.

A Radiolabeled Self-assembled Nanoparticle Probe for Diagnosis of Lung-Metastatic Melanoma.

Melanoma is a highly malignant skin cancer that frequently metastasizes to the lung, bone, and brain at an early phase. Therefore, noninvasive detection of metastasized melanoma could be beneficial to determine suitable therapeutic strategies. We previously reported a biocompatible ternary anionic complex composed of plasmid DNA (pDNA), polyethyleneimine (PEI), and γ-polyglutamic acid (γ-PGA) based on an electrostatic interaction, which was highly taken up by melanoma cells (B16-F10), even if it was negatively charged. Here, we developed a radiolabeled γ-PGA complex by using indium-111 (111In)-labeled polyamidoamine dendrimer (4th generation; G4) instead of pDNA and iodine-125 (125I)-labeled PEI instead of native PEI, and evaluated its effectiveness as a melanoma-targeted imaging probe. This ternary complex was synthesized at a theoretical charge ratio; carboxyl groups of 111In-diethylenetriaminepentaacetic acid (DTPA)-G4 : amino groups of 125I-PEI : carboxyl groups of γ-PGA was 1 : 8 : 16, and the size and zeta potential were approximately 29 nm and -33 mV, respectively. This complex was taken up by B16-F10 cells with time. Furthermore, a biodistribution study, using normal mice, demonstrated its accumulation in the liver, spleen, and lung, where macrophage cells are abundant. Almost the same level of radioactivity derived from both 111In and 125I was observed in these organs at an early phase after probe injection. Compared with the normal mice, significantly higher lung-to-blood ratios of radioactivity were observed in the B16-F10-lung metastatic cancer model. In conclusion, the radiolabeled γ-PGA complex would hold potentialities for nuclear medical imaging of lung metastatic melanoma.

Preparation, quality control, and biodistribution assessment of [111 In]In-DOTA-PR81 in BALB/c mice bearing breast tumors.

In this study, [111 In]In-DOTA-PR81 was developed, and its preliminary preclinical qualifications were assessed for single photon emission computed tomography (SPECT) imaging of breast cancer. DOTA-NHS-ester was practiced and successively purified by molecular filtration. The chelate:mAb ratio was determined by spectrophotometry. DOTA-PR81 was radiolabeled with In-111 and its radiochemical yield, in vitro stability, in vitro internalization, and immunoreactivity tests were performed. SPECT imaging and tissue counting were applied to evaluate the tissue distribution of [111 In]In-DOTA-hIgG and [111 In]In-DOTA-PR81 in BALB/c mice bearing breast tumors. The radiochemical yield of [111 In]In-DOTA-PR81 complex was >95.0 ± 0.5% (ITLC), and the specific activity was 170 ± 44 MBq/mg. Conjugation reaction resulted in the average number of chelators attached to a mAb (c/a) of 3.4 ± 0.3:1. The radioimmunoconjugate showed immunoreactivity towards MCF7 cell line and MUC1 antigen while its significant in vitro and in vivo stability were investigated over 48 h, respectively (93.0 ± 1.2% in phosphate-buffered saline (PBS) and 84.0 ± 1.3% in human serum). The peak concentration of internalized activity of [111 In]In-DOTA-PR81 was between 4 to 6 h. In comparison with control probes, the complex was accumulated with high specificity and sensitivity at the tumor site. Achieved results indicated that [111 In]In-DOTA-PR81 could be contemplated as an appropriate radiotracer for prognostic imaging of antigens in oncology.

Evaluation of Nanotargeted 111In-Cyclic RGDfK-Liposome in a Human Melanoma Xenotransplantation Model.

Nanotargeted liposomes may be modified with targeting peptide on the surface of a prepared liposome to endow specificity and elevate targeting efficiency. The aim of this study was to develop a radioactive targeted nanoparticle, the 111In-cyclic RGDfK-liposome, and its advantage of recognizing the αVß3 integrin was examined. The cyclic RGDfK modified liposomes were demonstrated the ability to bind the αVß3 integrin expressed on the surface of human melanoma cell in vitro and in vivo. The effects of the cyclic RGDfK-liposome on the functioning of phagocytes was also examined, showing no considerable negative effects on the engulfment of bacteria and the generation of reactive oxygen species. Based upon these findings, the cyclic RGDfK- liposome is said to be a promising agent for tumor imaging.

Molecular Imaging and Preclinical Studies of Radiolabeled Long-Term RGD Peptides in U-87 MG Tumor-Bearing Mice.

The Arg-Gly-Asp (RGD) peptide shows a high affinity for αvß3 integrin, which is overexpressed in new tumor blood vessels and many types of tumor cells. The radiolabeled RGD peptide has been studied for cancer imaging and radionuclide therapy. We have developed a long-term tumor-targeting peptide DOTA-EB-cRGDfK, which combines a DOTA chelator, a truncated Evans blue dye (EB), a modified linker, and cRGDfK peptide. The aim of this study was to evaluate the potential of indium-111(111In) radiolabeled DOTA-EB-cRGDfK in αvß3 integrin-expressing tumors. The human glioblastoma cell line U-87 MG was used to determine the in vitro binding affinity of the radiolabeled peptide. The in vivo distribution of radiolabeled peptides in U-87 MG xenografts was investigated by biodistribution, nanoSPECT/CT, pharmacokinetic and excretion studies. The in vitro competition assay showed that 111In-DOTA-EB-cRGDfK had a significant binding affinity to U-87 MG cancer cells (IC50 = 71.7 nM). NanoSPECT/CT imaging showed 111In-DOTA-EB-cRGDfK has higher tumor uptake than control peptides (111In-DOTA-cRGDfK and 111In-DOTA-EB), and there is still a clear signal until 72 h after injection. The biodistribution results showed significant tumor accumulation (27.1 ± 2.7% ID/g) and the tumor to non-tumor ratio was 22.85 at 24 h after injection. In addition, the pharmacokinetics results indicated that the 111In-DOTA-EB-cRGDfK peptide has a long-term half-life (T1/2λz = 77.3 h) and that the calculated absorbed dose was safe for humans. We demonstrated that radiolabeled DOTA-EB-cRGDfK may be a promising agent for glioblastoma tumor imaging and has the potential as a theranostic radiopharmaceutical.

Preliminary Study of a 1,5-Benzodiazepine-Derivative Labelled with Indium-111 for CCK-2 Receptor Targeting.

The cholecystokinin-2 receptor (CCK-2R) is overexpressed in several human cancers but displays limited expression in normal tissues. For this reason, it is a suitable target for developing specific radiotracers. In this study, a nastorazepide-based ligand functionalized with a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator (IP-001) was synthesized and labelled with indium-111. The radiolabeling process yielded >95% with a molar activity of 10 MBq/nmol and a radiochemical purity of >98%. Stability studies have shown a remarkable resistance to degradation (>93%) within 120 h of incubation in human blood. The in vitro uptake of [111In]In-IP-001 was assessed for up to 24 h on a high CCK-2R-expressing tumor cell line (A549) showing maximal accumulation after 4 h of incubation. Biodistribution and single photon emission tomography (SPECT)/CT imaging were evaluated on BALB/c nude mice bearing A549 xenograft tumors. Implanted tumors could be clearly visualized after only 4 h post injection (2.36 ± 0.26% ID/cc), although a high amount of radiotracer was also found in the liver, kidneys, and spleen (8.25 ± 2.21%, 6.99 ± 0.97%, and 3.88 ± 0.36% ID/cc, respectively). Clearance was slow by both hepatobiliary and renal excretion. Tumor retention persisted for up to 24 h, with the tumor to organs ratio increasing over-time and ending with a tumor uptake (1.52 ± 0.71% ID/cc) comparable to liver and kidneys.

Lesson of the month: novel method to quantify neutrophil uptake in early lung cancer using SPECT-CT.

Neutrophils play an important role in the lung tumour microenvironment. We hypothesised that radiolabelled neutrophils coupled to single-photon emission CT (SPECT) may non-invasively quantify neutrophil uptake in tumours from patients with non-small cell lung cancer. We demonstrated increased uptake of radiolabelled neutrophils from the blood into tumours compared with non-specific uptake using radiolabelled transferrin. Moreover, indium-111-neutrophil activity in the tumour biopsies also correlated with myeloperoxidase (MPO)-positive neutrophils. Our data support the utility of imaging with In-111-labelled neutrophils and SPECT-CT to quantify neutrophil uptake in lung cancer.

Site-Specific 89Zr- and 111In-Radiolabeling and In Vivo Evaluation of Glycan-free Antibodies by Azide-Alkyne Cycloaddition with a Non-natural Amino Acid.

Antibody-drug conjugates (ADCs) are a class of targeted therapeutics consisting of a monoclonal antibody coupled to a cytotoxic payload. Various bioconjugation methods for producing site-specific ADCs have been reported recently, in efforts to improve immunoreactivity and pharmacokinetics and minimize batch variance-potential issues associated with first-generation ADCs prepared via stochastic peptide coupling of lysines or reduced cysteines. Recently, cell-free protein synthesis of antibodies incorporating para-azidomethyl phenylalanine (pAMF) at specific locations within the protein sequence has emerged as a means to generate antibody-drug conjugates with strictly defined drug-antibody-ratio, leading to ADCs with markedly improved stability, activity, and specificity. The incorporation of pAMF enables the conjugation of payloads functionalized for strain-promoted azide-alkyne cycloaddition. Here, we introduce two dibenzylcyclooctyne-functionalized bifunctional chelators that enable the incorporation of radioisotopes for positron emission tomography with 89Zr (t1/2 = 78.4 h, ß+ = 395 keV (22%), γ = 897 keV) or single photon emission computed tomography with 111In (t1/2 = 67.3 h, γ = 171 keV (91%), 245 keV (94%)) under physiologically compatible conditions. We show that the corresponding radiolabeled conjugates with site-specifically functionalized antibodies targeting HER2 are amenable to targeted molecular imaging of HER2+ expressing tumor xenografts in mice and exhibit a favorable biodistribution profile in comparison with conventional, glycosylated antibody conjugates generated by stochastic bioconjugation.

Copper-64 labeled nanoparticles for positron emission tomography imaging: a review of the recent literature.

INTRODUCTION: Nuclear medicine plays a crucial role for personalized therapy, mainly in oncology. Chemotherapy and radiotherapy present some disadvantages and research is shifting toward nanotechnology with significant improvements in therapy and diagnosis of several cancers. Indeed, nanoparticles can be tagged with different radioisotopes for single photon emission computed tomography (SPECT) and positron emission tomography (PET) imaging and for therapy. This review describes the current state of the art of 64Copper-labeled nanoparticles for PET imaging of cancer. EVIDENCE ACQUISITION: We performed a systematic analysis of literature using the terms "64CuCl2," "64Cu," "Copper" AND "nanoparticle" AND "PET" in online databases: i.e. PubMed/MEDLINE and Scopus. The search was limited to English papers and original articles. We excluded articles not in English language, abstracts, case reports, review articles and meeting presentations. EVIDENCE SYNTHESIS: Amongst the 116 articles retrieved, 88 were excluded because reviews, or not in English, or only in-vitro studies or meeting presentations. We considered only 28 original papers. The most used nanoparticles are liposomes and they are mainly used in breast cancer although other animal models of cancer have been also investigated. CONCLUSIONS: The results showed that nanoparticles can be considered a promising radiopharmaceutical for PET imaging of different type of cancer.

Correlation of 18F-FDG uptake and thyroid cancer stem cells.

BACKGROUND: 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography (18F-FDG PET) has the potential to detect various types of cancers, including thyroid cancer (TC), at a potentially curable stage. Increased uptake of 18F-FDG was observed in anaplastic and poorly differentiated thyroid cancer cells, and PET-positive tumors are more likely to be resistant to 131I treatment. As cancer stem cells (CSCs) possess a dedifferentiated phenotype and are resistant to many anticancer therapies, we hypothesized that the expression of CSC-related markers is correlated with the ability of tumor cells in TC to uptake FDG. METHODS: The present study cohort included 12 patients with TC, who underwent 18F-FDG PET/CT imaging before surgery. Quantitative polymerase chain reaction (QPCR) and immunohistochemical (IHC) staining were performed to analyze the expression patterns of gene markers related to embryonic stem (ES) cells and CSCs in TC. RESULTS: The mRNA expression levels of CSC- (CD133 and CD44) and ES-related genes (Oct4 and Nanog) were higher in TC tissue than in normal thyroid tissue, whereas the mRNA expression levels of thyroid-specific genes (Tg, TSHR, and TTF1) were higher in normal thyroid tissue than in TC tissue. There was a positive and statistically significant correlation between FDG uptake (SUVmax) of tumor and relative mRNA levels of CD133, CD44, Oct4, and Nanog. The IHC results demonstrated that CD133 and Nanog were expressed in TC tissue but not in normal thyroid tissue, however, CD44 expression was observed in both TC and normal thyroid tissue. Comparisons of the clinicopathological parameters between TC tissues with low and high SUVmax demonstrated significant differences in protein level of CD133 but not in that of Nanog. CONCLUSIONS: The pre-therapeutic tumor SUVmax obtained from 18F-FDG PET/CT may be a potential predictor for evaluating the proportion of CSC population in individual patients with TC.

Evaluation of 111In-DOTA-F56 peptide targeting VEGFR1 for potential non-invasive gastric cancer xenografted tumor mice Micro-SPECT imaging.

Non-invasive imaging of vascular endothelial growth factor receptor 1 (VEGFR1) remains a great challenge in the early diagnosis of tumors, especially in gastric cancer. Here, we designed and evaluated a novel 111In-DOTA-F56 peptide as a radioactive analogue of F56 (peptide WHSDMEWWYLLG) to bind VEGFR1. It was obtained by radiolabeling DOTA-F56 with 111InCl3 with 98% radiochemical purity and 1.4 ± 0.4 GBq/µmol specific activity. 111In-DOTA-F56 was obtained by the reaction of DOTA-F56 (10 µg) with 111InCl3 in pH 4.0 sodium acetate buffer at 85 °C for 20 min. 111In-DOTA-F56 shows good stability in 0.01 M Phosphate Buffered Saline (PBS) and 5% Human Serum Albumin (HSA). 111In-DOTA-F56 has a high binding affinity for human gastric cancer BGC-823 cells. Bio-distribution studies of 111In-DOTA-F56 were performed in nude mice xenografted with human gastric cancer BGC-823 cells and the results revealed tumor uptake accumulation. A blocking dose of DOTA-F56 significantly reduced the tumor uptake of 111In-DOTA-F56. Tumors were observed with Micro-SPECT images, and the uptake in the tumor increased with time from 4 h to 24 h. The MIP of the Micro-SPECT also showed that the excess DOTA-F56 can specifically block 111In-DOTA-F56 in a mouse tumor model. We successfully synthesized the 111In-DOTA-F56 VEGFR1-targeted peptide as a non-invasive molecule with fine radiochemical properties. Micro-SPECT indicates tumor uptake, which can be further blocked by excess of the F56 peptide, indicating that 111In-DOTA-F56 peptide has potential for early detection of VEGFR1 positive gastric cancer and is worthy of further clinical investigations.

Synthesis and evaluation of indium-111-labeled imidazothiadiazole sulfonamide derivative for single photon emission computed tomography imaging targeting carbonic anhydrase-IX.

Carbonic anhydrase-IX (CA-IX) is a zinc enzyme overexpressed in the hypoxic regions of many types of solid tumors; therefore, in vivo imaging of CA-IX may contribute to cancer diagnosis. In this study, we newly designed and synthesized an 111In-labeled CA-IX imaging agent based on an imidazothiadiazole sulfonamide (IS) scaffold conjugated with a chelating moiety, DO3A ([111In]DO3A-IS1), and evaluated its utility for imaging of CA-IX high-expressing tumors. [111In]DO3A-IS1 was successfully synthesized at a 76% radiochemical yield by reacting its precursor with 111InCl3 in acetate buffer. In in vitro assays, [111In]DO3A-IS1 showed marked stability in murine plasma and greater binding to CA-IX high-expressing (HT-29) cells (118 ± 21% initial dose/mg protein) than CA-IX low-expressing (MDA-MB-231) cells (1.4 ± 0.3% initial dose/mg protein). Moreover, in an in vivo biodistribution assay, [111In]DO3A-IS1 showed marked accumulation in the HT-29 tumor (8.71 ± 1.41% injected dose/g at 24 h postinjection). In addition, in a single photon emission computed tomography (SPECT) study, [111In]DO3A-IS1 clearly and selectively visualized the HT-29 tumor as compared with the MDA-MB-231 tumor. These results indicate that [111In]DO3A-IS1 may serve as a useful SPECT imaging agent with the novel scaffold targeting CA-IX.

Chelation in One Fell Swoop: Optimizing Ligands for Smaller Radiometal Ions.

[44/47Sc]Sc3+, [68Ga]Ga3+, and [111In]In3+ are the three most attractive trivalent smaller radiometalnuclides, offering a wide range of distinct properties (emission energies and types) in the toolbox of nuclear medicine. In this study, all three of the metal ions are successfully chelated using a new oxine-based hexadentate ligand, H3glyox, which forms thermodynamically stable neutral complexes with exceptionally high pM values [pIn (34) > pSc (26) > pGa (24.9)]. X-ray diffraction single crystal structures with stable isotopes revealed that the ligand is highly preorganized and has a perfect fit to size cavity to form [Sc(glyox)(H2O)] and [In(glyox)(H2O)] complexes. Quantitative radiolabeling with gallium-68 (RCY > 95%, [L] = 10-5 M) and indium-111 (RCY > 99%, [L] = 10-8 M) was achieved under ambient conditions (RT, pH 7, and 15 min) with very high apparent molar activities of 750 MBq/µmol and 650 MBq/nmol, respectively. Preliminary quantitative radiolabeling of [44Sc]ScCl3 (RCY > 99%, [L] = 10-6 M) was fast at room temperature (pH 7 and 10 min). In vitro experiments revealed exceptional stability of both [68Ga]Ga(glyox) and [111In]In(glyox) complexes against human serum (transchelation <2%) and its suitability for biological applications. Additionally, on chelation with metal ions, H3glyox exhibits enhanced fluorescence, which was employed to determine the stability constants for Sc(glyox) in addition to the in-batch UV-vis spectrophotometric titrations; as a proof-of-concept these complexes were used to obtain fluorescence images of live HeLa cells using Sc(glyox) and Ga(glyox), confirming the viability of the cells. These initial investigations suggest H3glyox to be a valuable chelator for radiometal-based diagnosis (nuclear and optical imaging) and therapy.

Thermal combination therapies for local drug delivery by magnetic resonance-guided high-intensity focused ultrasound.

Several thermal-therapy strategies such as thermal ablation, hyperthermia-triggered drug delivery from temperature-sensitive liposomes (TSLs), and combinations of the above were investigated in a rhabdomyosarcoma rat tumor model (n = 113). Magnetic resonance-guided high-intensity focused ultrasound (MR-HIFU) was used as a noninvasive heating device with precise temperature control for image-guided drug delivery. For the latter, TSLs were prepared, coencapsulating doxorubicin (dox) and [Gd(HPDO3A)(H2O)], and injected in tumor-bearing rats before MR-HIFU treatment. Four treatment groups were defined: hyperthermia, ablation, hyperthermia followed by ablation, or no HIFU. The intratumoral TSL and dox distribution were analyzed by single-photon emission computed tomography (SPECT)/computed tomography (CT), autoradiography, and fluorescence microscopy. Dox biodistribution was quantified and compared with that of nonliposomal dox. Finally, the treatment efficacy of all heating strategies plus additional control groups (saline, free dox, and Caelyx) was assessed by tumor growth measurements. All HIFU heating strategies combined with TSLs resulted in cellular uptake of dox deep into the interstitial space and a significant increase of tumor drug concentrations compared with a treatment with free dox. Ablation after TSL injection showed [Gd(HPDO3A)(H2O)] and dox release along the tumor rim, mirroring the TSL distribution pattern. Hyperthermia either as standalone treatment or before ablation ensured homogeneous TSL, [Gd(HPDO3A)(H2O)], and dox delivery across the tumor. The combination of hyperthermia-triggered drug delivery followed by ablation showed the best therapeutic outcome compared with all other treatment groups due to direct induction of thermal necrosis in the tumor core and efficient drug delivery to the tumor rim.

Radiolabeled Human Monoclonal Antibody 067-213 has the Potential for Noninvasive Quantification of CD73 Expression.

BACKGROUND: CD73 is an ectonucleotidase regulating extracellular adenosine concentration and plays an important role in adenosine-mediated immunosuppressive pathways. The efficacy of CD73-targeted therapy depends on the expression levels of CD73; therefore, monitoring CD73 status in cancer patients would provide helpful information for selection of patients who would benefit from CD73-targeted therapy. Here, we evaluated the ability of 111In-labeled antibody 067-213, which has high affinity for human CD73, to act as a noninvasive imaging probe. METHODS: Cell binding and competitive inhibition assays for 111In-labeled 067-213 were conducted using MIAPaCa-2 (high CD73 expression) and A431 (low CD73 expression) cells. For in vivo assessments, biodistribution and SPECT/CT studies were conducted in MIAPaCa-2 and A431 tumor-bearing mice. To estimate the absorbed dose in humans, biodistribution and SPECT/CT studies were conducted in healthy rats. RESULTS: 111In-labeled 067-213 bound to MIAPaCa-2 and A431 cells in a CD73-dependent manner and the affinity loss after 111In-labeling was limited. Biodistribution and SPECT/CT studies with 111In-labeled 067-213 in mice showed high uptake in MIAPaCa-2 tumors and lower uptake in A431 tumors. In rats, the probe did not show high uptake in normal organs, including endogenously CD73-expressing organs. The estimated absorbed doses in humans were reasonably low. CONCLUSIONS: 111In-labeled 067-213 showed CD73-expression-dependent tumor uptake and low uptake in normal organs and tissues. Radiolabeled 067-213 holds promise as an imaging probe for noninvasive evaluation of CD73 expression levels in patients. Our data encourage further clinical studies to clarify a role for CD73 monitoring in patients receiving CD73-targeted immune therapy.

Influence of Residualizing Properties of the Radiolabel on Radionuclide Molecular Imaging of HER3 Using Affibody Molecules.

Human epidermal growth factor receptor type 3 (HER3) is an emerging therapeutic target in several malignancies. To select potential responders to HER3-targeted therapy, radionuclide molecular imaging of HER3 expression using affibody molecules could be performed. Due to physiological expression of HER3 in normal organs, high imaging contrast remains challenging. Due to slow internalization of affibody molecules by cancer cells, we hypothesized that labeling (HE)3-ZHER3:08698-DOTAGA affibody molecule with non-residualizing [125I]-N-succinimidyl-4-iodobenzoate (PIB) label would improve the tumor-to-normal organs ratios compared to previously reported residualizing radiometal labels. The [125I]I-PIB-(HE)3-ZHER3:08698-DOTAGA was compared side-by-side with [111In]In-(HE)3-ZHER3:08698-DOTAGA. Both conjugates demonstrated specific high-affinity binding to HER3-expressing BxPC-3 and DU145 cancer cells. Biodistribution in mice bearing BxPC-3 xenografts at 4 and 24 h pi showed faster clearance of the [125I]I-PIB label compared to the indium-111 label from most tissues, except blood. This resulted in higher tumor-to-organ ratios in HER3-expressing organs for [125I]I-PIB-(HE)3-ZHER3:08698-DOTAGA at 4 h, providing the tumor-to-liver ratio of 2.4 ± 0.3. The tumor uptake of both conjugates was specific, however, it was lower for the [125I]I-PIB label. In conclusion, the use of non-residualizing [125I]I-PIB label for HER3-targeting affibody molecule provided higher tumor-to-liver ratio than the indium-111 label, however, further improvement in tumor uptake and retention is needed.

New Recombinant Carriers Binding Specifically to the Epidermal Growth Factor Receptor.

New recombinant carriers-modular nanotransporters (MNTs)-with N-terminal ligand module to the epidermal growth factor receptor (EGFR) were developed and characterized. Human epidermal growth factor (hEGF) and antibody-like protein Z1907 were used as a ligand module. We demonstrated that MNTs are able to internalize in a receptor-specific manner into the target cancer cells and to accumulate in the target cell nuclei. Conjugation of MNTs with the Auger electron emitter 111In significantly enhanced the cytotoxic effect of 111In on the target cells. It was found that the transfer of EGF from the C-terminus to the N-terminus of the MNT enhanced the proliferation of target cells, whereas the use of Z1907 did not have a similar effect.

Therapeutic efficacy evaluation of radioimmunotherapy with 90 Y-labeled anti-podoplanin antibody NZ-12 for mesothelioma.

Podoplanin is a type I transmembrane sialomucin-like glycoprotein that is highly expressed in malignant mesothelioma. The rat-human chimeric antibody NZ-12 has high affinity for human podoplanin and antibody-dependent cellular cytotoxicity and is applicable for radioimmunotherapy (RIT) to enhance the antitumor effect. In the present study, we evaluated the in vivo and in vitro properties of radiolabeled NZ-12 and the antitumor effect of RIT with 90 Y-labeled NZ-12 in an NCI-H226 (H226) malignant mesothelioma xenograft mouse model. 111 In-labeled NZ-12 bound specifically to H226 cells with high affinity, and accumulation was high in H226 tumors but low in major organs. RIT with 90 Y-labeled NZ-12 significantly suppressed tumor growth and prolonged survival without body weight loss and obvious adverse effects. Higher podoplanin expression levels were observed in human mesothelioma specimens, suggesting higher tumor accumulation of 90 Y-labeled NZ-12 in patients compared with the H226 tumor xenografts. Our findings suggest that 90 Y-labeled NZ-12 is a promising RIT agent as a new therapeutic option for malignant mesothelioma that warrants further clinical studies to evaluate the dosimetry and efficacy in patients.