Tumor Targeting of 211At-Labeled Antibody under Sodium Ascorbate Protection against Radiolysis.

Astatine-211 (211At) is an alpha emitter applicable to radioimmunotherapy (RIT), a cancer treatment that utilizes radioactive antibodies to target tumors. In the preparation of 211At-labeled monoclonal antibodies (211At-mAbs), the possibility of radionuclide-induced antibody denaturation (radiolysis) is of concern. Our previous study showed that this 211At-induced radiochemical reaction disrupts the cellular binding activity of an astatinated mAb, resulting in attenuation of in vivo antitumor effects, whereas sodium ascorbate (SA), a free radical scavenger, prevents antibody denaturation, contributing to the maintenance of binding and antitumor activity. However, the influence of antibody denaturation on the pharmacokinetics of 211At-mAbs relating to tumor accumulation, blood circulation time, and distribution to normal organs remains unclear. In this study, we use a radioactive anti-human epidermal growth factor receptor 2 (anti-HER2) mAb to demonstrate that an 211At-induced radiochemical reaction disrupts active targeting via an antigen-antibody interaction, whereas SA helps to maintain targeting. In contrast, there was no difference in blood circulation time as well as distribution to normal organs between the stabilized and denatured immunoconjugates, indicating that antibody denaturation may not affect tumor accumulation via passive targeting based on the enhanced permeability and retention effect. In a high-HER2-expressing xenograft model treated with 1 MBq of 211At-anti-HER2 mAbs, SA-dependent maintenance of active targeting contributed to a significantly better response. In treatment with 0.5 or 0.2 MBq, the stabilized radioactive mAb significantly reduced tumor growth compared to the denatured immunoconjugate. Additionally, through a comparison between a stabilized 211At-anti-HER2 mAb and radioactive nontargeted control mAb, we demonstrate that active targeting significantly enhances tumor accumulation of radioactivity and in vivo antitumor effect. In RIT with 211At, active targeting contributes to efficient tumor accumulation of radioactivity, resulting in a potent antitumor effect. SA-dependent protection that successfully maintains tumor targeting will facilitate the clinical application of alpha-RIT.

Radioimmunotherapy with an 211 At-labeled anti-tissue factor antibody protected by sodium ascorbate.

Tissue factor (TF), the trigger protein of the extrinsic blood coagulation cascade, is abundantly expressed in various cancers including gastric cancer. Anti-TF monoclonal antibodies (mAbs) capable of targeting cancers have been successfully applied to armed antibodies such as antibody-drug conjugates (ADCs) and molecular imaging probes. We prepared an anti-TF mAb, clone 1084, labeled with astatine-211 (211 At), as a promising alpha emitter for cancer treatment. Alpha particles are characterized by high linear energy transfer and a range of 50-100 µm in tissue. Therefore, selective and efficient tumor accumulation of alpha emitters results in potent antitumor activities against cancer cells with minor effects on normal cells adjacent to the tumor. Although the 211 At-conjugated clone 1084 (211 At-anti-TF mAb) was disrupted by an 211 At-induced radiochemical reaction, we demonstrated that astatinated anti-TF mAbs eluted in 0.6% or 1.2% sodium ascorbate (SA) solution were protected from antibody denaturation, which contributed to the maintenance of cellular binding activities and cytocidal effects of this immunoconjugate. Although body weight loss was observed in mice administered a 1.2% SA solution, the loss was transient and the radioprotectant seemed to be tolerable in vivo. In a high TF-expressing gastric cancer xenograft model, 211 At-anti-TF mAb in 1.2% SA exerted a significantly greater antitumor effect than nonprotected 211 At-anti-TF mAb. Moreover, the antitumor activities of the protected immunoconjugate in gastric cancer xenograft models were dependent on the level of TF in cancer cells. These findings suggest the clinical availability of the radioprotectant and applicability of clone 1084 to 211 At-radioimmunotherapy.

MR signal changes in superparamagnetic iron oxide nanoparticle-labeled macrophages in response to X irradiation.

To investigate whether MR signals associated with macrophages labeled with superparamagnetic iron oxide nanoparticles (SPIONs) change in response to X irradiation, we performed in vitro MRI of SPION-labeled macrophage-like J774A.1 cells that were subsequently X irradiated. We labeled the cells with ferucarbotran at a concentration of 10 µg iron/mL in culture medium for 16 h and subsequently performed X irradiation at doses of 0, 2, 10, or 20 Gy using a low-energy X-ray unit. On Days 3 and 6, we suspended the cells in agar at a concentration of 2 × 106 cells/mL and acquired multi-gradient echo and multi-spin echo images of the cell samples using a 3 T scanner to estimate R2 * and R2 . In addition, we microscopically investigated the relationship among the MR signal changes, intracellular SPIONs, and acidic organelles. Our data showed that X irradiation of labeled cells caused increased SPION deposition in lysosomes compared with the non-irradiated control. On Day 3, R2 * and R2 values in the 0 to 10 Gy irradiated samples were dose-dependently increased 5.4- and 1.5-fold compared with 17 ± 2 and 13 ± 1/s, respectively, in the non-irradiated control; these values plateaued at more than 10 Gy. Although the increases in R2 *, R2 , and SPION deposition were still observed in the 10 and 20 Gy samples on Days 6 and 7, the 2 Gy samples showed recovery in these parameters as cell growth was restored. Acidic organelles were temporarily increased in the irradiated cells, which suggests that the reduction in lysosomal acidity was not attributable to SPION deposition. In conclusion, X irradiation of macrophages can cause SPION deposition and R2 * and R2 elevation in a specific dose range. MRI of SPION-labeled and subsequently X-irradiated macrophages may be utilized as a novel technique for investigating macrophage responses to X irradiation.

Metallic radionuclide-labeled tetrameric 2,6-diisopropylphenyl azides for cancer treatment.

This study proposes a new method for radionuclide therapy that involves the use of oligomeric 2,6-diisopropylphenyl azides and a chelator to form stable complexes with metallic radionuclides. The technique works by taking advantage of the endogenous acrolein produced by cancer cells. The azides react with the acrolein to give a diazo derivative that immediately attaches to the nearest organelle, effectively anchoring the radionuclide within the tumor. Preliminary in vivo experiments were conducted on a human lung carcinoma xenograft model, demonstrating the feasibility of this approach for cancer treatment.

CdTe XG-Cam: A new high-resolution x-ray and gamma-ray camera for studies of the pharmacokinetics of radiopharmaceuticals in small animals.

BACKGROUND: The recent emergence of targeted radionuclide therapy has increased the demand for imagers capable of visualizing pharmacokinetics in developing radiopharmaceuticals in the preclinical phase. Some radionuclides emit hard x-rays and gamma-rays below 100 keV, in which energy range the performance of conventional NaI scintillators is poor. Multipinhole collimators are also used for small animal imaging with a good spatial resolution but have a limited field of view (FOV). PURPOSE: In this study, a new imager with high sensitivity over a wide FOV in the low-energy band ( < $<$ 100 keV) was developed for the pharmacokinetic study. METHODS: We developed an x-ray and gamma-ray camera for high-resolution spectroscopy, named "CdTe XG-Cam," equipped with a cadmium telluride semiconductor detector and a parallel-hole collimator using a metal 3D printer. To evaluate the camera-system performance, phantom measurements with single and dual nuclides ( 99 m Tc $^{\rm 99m}{\rm Tc}$ , 111 In $^{111}{\rm In}$ , and 125 I ) $^{125}{\rm I)}$ were performed. The performance for in vivo imaging was evaluated using tumor-bearing mice to which a nuclide ( 99 m Tc $^{\rm 99m}{\rm Tc}$ or 125 I ) $^{125}{\rm I)}$  administered. RESULTS: We simultaneously obtained information on 111 In $^{111}{\rm In}$ and 125 I $^{125}{\rm I}$ , which emit emission lines in the low-energy band with peak energies close to each other (23-26 keV for 111 In $^{111}{\rm In}$ and 27-31 keV for 125 I ) $^{125}{\rm I)}$ , and applied an analytical method based on spectral model fitting to determine the individual radioactivities accurately. In the small animal imaging, the distributions of the nuclide in tumors were accurately quantified and time-activity curves in tumors are obtained. CONCLUSIONS: The demonstrated capability of our system to perform in vivo imaging suggests that the camera can be used for applications of pharmacokinetics research.

AP-1 is involved in ICOS gene expression downstream of TCR/CD28 and cytokine receptor signaling.

It has been proposed that sustained ICOS expression in chronic inflammatory immune conditions, such as autoimmunity and allergy, contributes to symptom exacerbation. Therefore modulation of ICOS gene expression could be a potential therapeutic strategy for such immune diseases. However, the precise molecular mechanisms controlling ICOS gene expression remain poorly understood. In this study, we explored transcription factors involving in ICOS gene expression and examined their roles in a physiological situation. Microarray analysis revealed that one AP-1 molecule, Fos-related antigen-2 (Fra2), was highly correlated with ICOS expression. Ectopic expression of Fra2 and other AP-1 molecules upregulated ICOS expression on T cells. We identified an AP-1-responsive site (AP1-RE) within the ICOS promoter region and demonstrated AP-1 actually binds to AP1-RE upon TCR/CD28 stimulation. Meanwhile, we found several cytokines could upregulate ICOS expression on both naïve and effector T cells in a manner independent of TCR/CD28 stimulation. These cytokine stimuli induced AP-1 binding to AP1-RE. Together, our results indicate AP-1 transcription factors are involved in ICOS gene expression downstream of both TCR/CD28 signaling and cytokine receptor signaling, and suggest AP-1 activation via cytokine receptor signaling may be one of the mechanisms maintaining high level ICOS expression in chronic inflammatory immune responses.

Dual-radionuclide in vivo imaging of micro-metastasis and lymph tract with submillimetre resolution.

Multi-radionuclide in vivo imaging with submillimetre resolution can be a potent tool for biomedical research. While high-resolution radionuclide imaging faces challenges in sensitivity, multi-radionuclide imaging encounters difficulty due to radiation contamination, stemming from crosstalk between radionuclides and Compton scattering. Addressing these challenges simultaneously is imperative for multi-radionuclide high-resolution imaging. To tackle this, we developed a high-spatial-resolution and high-energy-resolution small animal single-photon emission computed tomography (SPECT) scanner, named CdTe-DSD SPECT-I. We first assessed the feasibility of multi-tracer SPECT imaging of submillimetre targets. Using the CdTe-DSD SPECT-I, we performed SPECT imaging of submillimetre zeolite spheres absorbed with 125I- and subsequently imaged 125I-accumulated spheroids of 200-400 µm in size within an hour, achieving clear and quantitative images. Furthermore, dual-radionuclide phantom imaging revealed a distinct image of the submillimetre sphere absorbed with 125I- immersed in a 99mTc-pertechnetate solution, and provided a fair quantification of each radionuclide. Lastly, in vivo imaging was conducted on a cancer-bearing mouse with lymph node micro-metastasis using dual-tracers. The results displayed dual-tracer images of lymph tract by 99mTc-phytic acid and the submillimetre metastatic lesion by 125I-, shown to align with the immunofluorescence image.

Influence of Carboxyl Group Ratios on the Design of Breast Cancer Targeting Bimodal MR/NIR-II Imaging Probe from PLGA@Gd-DOTA@PEG Micelles Conjugating Herceptin.

We developed a small MRI/NIR-II probe to target HER2 (tetanucleotide) breast cancer cells. The probe is composed of PLGA-b-PEG micelles encapsulated NIR-II, and Gd-DOTA is conjugated at the border of PLGA/PEG. Herceptin was then conjugated to carboxyl residues of PLGA-b-PEG chains. We examined the influence of carboxyl group ratios on the probe property stability and Herceptin concentration and the binding affinity to HER2(+) cells corresponding to the -COOH ratios. The binding assays demonstrated that the optimal surface ratio of -COOH is 5%, which is less affected by fluorescence reduction and which exhibited the highest antigen-capturing activity.

Protection from contamination by 211At, an enigmatic but promising alpha-particle-emitting radionuclide.

PURPOSE: 211At, a promising alpha-particle-emitting radionuclide, can easily volatilize and contaminate the environment. To safely manage this unique alpha-particle-emitting radionuclide, we investigated the permeability of four types of plastic films and two types of rubber gloves against 211At and identified suitable materials that prevent contamination by 211At. METHODS: Four types of plastic films, polyethylene, polyvinylidene chloride, polyvinyl chloride, and a laminated film, and two types of rubber gloves, latex and nitrile, were examined. Small pieces of filter paper were covered with these materials, and a drop containing 100 kBq of 211At was placed on them. The radioactivity of the pieces of filter paper under the materials was evaluated by measuring counts using a gamma counter and obtaining autoradiograms 3.5 h later. These experiments were also performed using 225Ac, 125I, 111In, 201Tl, and 99mTc. RESULTS: 211At solution easily penetrated polyethylene, polyvinyl chloride, and latex rubber. Similar results were obtained for 125I, while other radionuclides did not penetrate films or gloves. These results suggest that halogenic radionuclides under anionic conditions are likely to penetrate plastic films and rubber gloves. CONCLUSION: Our evaluation revealed that, when 211At solution is used, the protection by polyvinylidene chloride, a laminated film, or nitrile rubber would be more effective than that by polyethylene, polyvinyl chloride, or latex rubber.

The influence of Gd-DOTA conjugating ratios to PLGA-PEG micelles encapsulated IR-1061 on bimodal over-1000 nm near-infrared fluorescence and magnetic resonance imaging.

Multimodal imaging can provide multidimensional information for understanding concealed microstructures or bioprocesses in biological objects. The combination of over-1000 nm near-infrared (OTN-NIR) fluorescence imaging and magnetic resonance (MR) imaging is promising in providing high sensitivity and structural information of lesions. This combination can be facilitated by the development of an imaging probe. The OTN-NIR and MR bimodal fluorescence probes reported to date primarily involve ceramic particles for fluorescence and MRI contrast enhancement effect. In this study, we designed a new bimodal OTN-NIR/MR imaging probe from organic components including an OTN-NIR fluorescent organic dye (IR-1061) encapsulated in the core of a micelle composed of poly(lactic-co-glycolic acid)-block-poly(ethylene glycol) copolymer (PLGA-PEG). For the MRI contrast, gadobutrol (Gd-DOTA) was introduced at the end of the PEG chain at various ratios. Thereafter, the OTN-NIR fluorescence and MR bimodal imaging probes of ca. 20 nm in size were successfully prepared and applied in mouse imaging. The probe exhibited absorption and emission in the OTN-NIR, and T1 contrast enhancement effects on MRI. Moreover, it demonstrated bright OTN-NIR fluorescence and MRI contrast enhancement to depict veins and observe the organs in live mice. The imaging results revealed that the Gd-DOTA introduction ratio is of great importance for controlling the biological response of the probe without reducing the contrast enhancement effect.

Simultaneous visualization of multiple radionuclides in vivo.

The insufficient energy and spatial resolutions of radionuclide imaging with conventional scintillation detectors restrict the visualization of multiple radionuclides and of microstructures in tissue. Here we report the development and performance of an imaging system equipped with a cadmium telluride diode detector that achieves an energy resolution of 1.7% at 140 keV and a spatial resolution of 250 µm. The combination of high-resolution spectra fitted to an X-ray analysis model of the emission lines of the radionuclides in a chosen energy band allowed us to accurately determine individual radiation activities from three radionuclides to simultaneously visualize thyroid tissue (via intravenously administered iodine-125), mandibular lymph nodes (via the intramuscular injection of indium-111) and parotid lymph nodes (via a subcutaneous injection of technetium-99m) in mice. Multi-radionuclide imaging may find advantageous applications in biomedical imaging.

Stabilization of an 211At-Labeled Antibody with Sodium Ascorbate.

211At, an α-particle emitter, has recently attracted attention for radioimmunotherapy of intractable cancers. However, our sodium dodecyl sulfate polyacrylamide gel electrophoresis and flow cytometry analyses revealed that 211At-labeled immunoconjugates are easily disrupted. Luminol assay revealed that reactive oxygen species generated from radiolysis of water caused the disruption of 211At-labeled immunoconjugates. To retain their functions, we explored methods to protect 211At-immunoconjugates from oxidation and enhance their stability. Among several other reducing agents, sodium ascorbate most safely and successfully protected 211At-labeled trastuzumab from oxidative stress and retained the stability of the 211At-labeled antibody and its cytotoxicity against antigen-expressing cells for several days.

Antigen-specific CD4+ effector T cells: analysis of factors regulating clonal expansion and cytokine production: clonal expansion and cytokine production by CD4+ effector T cells.

In order to fully understand T cell-mediated immunity, the mechanisms that regulate clonal expansion and cytokine production by CD4(+) antigen-specific effector T cells in response to a wide range of antigenic stimulation needs clarification. For this purpose, panels of antigen-specific CD4(+) T cell clones with different thresholds for antigen-induced proliferation were generated by repeated stimulation with high- or low-dose antigen. Differences in antigen sensitivities did not correlate with expression of TCR, CD4, adhesion or costimulatory molecules. There was no significant difference in antigen-dependent cytokine production by TG40 cells transfected with TCR obtained from either high- or low-dose-responding T cell clones, suggesting that the affinity of TCRs for their ligands is not primary determinant of T cell antigen reactivity. The proliferative responses of all T cell clones to both peptide stimulation and to TCRbeta crosslinking revealed parallel dose-response curves. These results suggest that the TCR signal strength of effector T cells and threshold of antigen reactivity is determined by an intrinsic property, such as the TCR signalosome and/or intracellular signaling machinery. Finally, the antigen responses of high- and low-peptide-responding T cell clones reveal that clonal expansion and cytokine production of effector T cells occur independently of antigen concentration. Based on these results, the mechanisms underlying selection of high "avidity" effector and memory T cells in response to pathogen are discussed.

Novel Indocyanine Green-Phytate Colloid Technique for Sentinel Node Detection in Head and Neck: Mouse Study.

OBJECTIVE: Sentinel node navigation surgery using real-time, near-infrared imaging with indocyanine green is becoming popular by allowing head and neck surgeons to avoid unnecessary neck dissection. The major drawback of this method is its quick migration through the lymphatics, limiting the diagnostic time window and undesirable detection of downstream nodes. We resolved this problem by mixing indocyanine green (ICG) with phytate colloid to retard its migration and demonstrated its feasibility in a nude mouse study. STUDY DESIGN: Experimental prospective animal study. SETTINGS: Animal laboratory. SUBJECTS AND METHODS: Indocyanine green at 3 concentrations was tested to determine the optimal concentration for sentinel lymph node detection in a mouse model. Effect of indocyanine green with phytate colloid mixture solutions was also analyzed. Indocyanine green or mixture solution at different mixing ratios were injected into the tongue of nude mice and near-infrared fluorescence images were captured sequentially for up to 48 hours. The brightness of fluorescence in the sentinel lymph node and lymph nodes further downstream were assessed. RESULTS: Indocyanine green concentration >50 µg/mL did not improve sentinel lymph node detection. The addition of phytate colloid to indocyanine green extended the period when sentinel lymph node was detectable. Second echelon lymph nodes were not imaged in mice injected with the mixture, while these were visualized in mice injected with indocyanine green alone. CONCLUSION: This novel technique of ICG-phytate colloid mixture allows prolonged diagnostic time window, prevention of downstream subsequent nodes detection, and improved accuracy for the detection of true sentinel lymph nodes.

Suppression of Con A-induced hepatitis induction in ICOS-deficient mice.

Although there is growing evidence that NKT cells play an important role in various immune responses through the invariant T cell receptor, other cell surface molecules responsible for their function are not fully understood. Here we study the role of ICOS, the third member of the CD28 family of costimulatory receptors, in in vivo and in vitro NKT cell responses. To establish its in vivo role in systems dependent on NKT cells, we examined the development of Con A-induced hepatitis in ICOS knockout (ICOS(-/-)) mice. We demonstrated that hepatic injury in ICOS(-/-) mice was greatly suppressed as evidenced by the reduced elevation of serum transaminases, reduced apoptosis of hepatocytes and mild histopathological changes. In investigating the cause of this defect, we first found that the NKT cell population is significantly reduced in the liver and spleen of ICOS(-/-) mice. We made and analyzed mixed bone marrow chimera mice with bone marrow cells from ICOS(+/+) and ICOS(-/-) mice, and demonstrated that the defect in ICOS-mediated costimulation results in a significant defect in the development of NKT cells, especially of Valpha14i NKT cells, in the thymus. When we examined the function of residual NKT cells in ICOS(-/-) mice, we found that their cytokine production following stimulation with alpha-galactosylceramide (alpha-GalCer) was strongly impaired. Based on these findings, we propose that ICOS-mediated costimulation may play a critical role in both the development and the optimal function of NKT cells, and that defective ICOS-mediated costimulation may result in impaired Con A-induced hepatitis in ICOS(-/-) mice.

Potential role of host effector memory CD8+ T cells in marrow rejection after mixed chimerism induction in cynomolgus monkeys.

Mixed hematopoietic chimerism provides a powerful means of achieving transplantation tolerance. We investigated the efficacy of combined blockade of the CD40/CD154 and CD28/B7 costimulation pathways to induce sustained mixed chimerism in cynomolgus monkeys following major histocompatibility complex-mismatched bone marrow (BM) transplants. A nonmyeloablative conditioning regimen of busulfan, intravenous and intraosseous ifosfamide, and anti-thymocyte globulin was used. BM transplantation was followed by a one-week course of CTLA4-Ig/anti-CD154 monoclonal antibodies. Three recipients achieved a wide range of transient chimerism (10.8-79.8%). A rapid proliferation of host effector memory (CD28(low)CD95(high)) CD8(+) T cells was observed in conditioned animals whether or not they received allogeneic BM, and this expansion occurred concurrently with the loss of chimerism in BM recipients. CD8(+) T cells from the recipients had increased reactivity to donor stimulators vs. third-party stimulators. Additional immunosuppression with tacrolimus or deoxyspergualin after transplantation delayed post-transplant proliferation of effector memory CD8(+) T cells but did not promote chimerism. A one-month course of costimulatory blockade also did not prevent marrow rejection. These studies demonstrate that combined CD40/CD154 and CD28/B7 costimulatory blockade supports transient mixed chimerism induction following nonmyeloablative conditioning in primates, but is insufficient to overcome host immune resistance likely mediated by effector memory CD8(+) T cells.

Grb2 and Gads exhibit different interactions with CD28 and play distinct roles in CD28-mediated costimulation.

Although both CD28 and ICOS bind PI3K and provide stimulatory signal for T cell activation, unlike CD28, ICOS does not costimulate IL-2 secretion. CD28 binds both PI3K and Grb2, whereas ICOS binds only PI3K. We have generated an ICOS mutant, which can bind Grb2 by replacement of its PI3K binding motif YMFM with the CD28 YMNM motif, and shown that it induces significant activation of the IL-2 promoter. However, this mutant ICOS was insufficient to activate the NF-kappaB pathway. In this study, we show that Gads, but not Grb2, is essential for CD28-mediated NF-kappaB activation, and its binding to CD28 requires the whole CD28 cytoplasmic domain in addition to the YMNM motif. Mutagenesis experiments have indicated that mutations in the N-terminal and/or C-terminal PXXP motif(s) of CD28 significantly reduce their association with Gads, whereas their associations with Grb2 are maintained. They induced strong activity of the NFAT/AP-1 reporter comparable with the CD28 wild type, but weak activity of the NF-kappaB reporter. Grb2- and Gads-dominant-negative mutants had a strong effect on NFAT/AP-1 reporter, but only Gads-dominant-negative significantly inhibited NF-kappaB reporter. Our data suggest that, in addition to the PI3K binding motif, the PXXP motif in the CD28 cytoplasmic domain may also define a functional difference between the CD28- and ICOS-mediated costimulatory signals by binding to Gads.