Search Results (60)

We have previously reported on a novel monoclonal antibody (mAb) we designated F5, which was raised against a glycopeptide derived from the tandem repeat (TR) region of Mucin-4 (MUC4), a heavily O-glycosylated protein that is overexpressed in many pancreatic cancer cells. This mAb was highly specific for the MUC4 glycopeptide antigen in glycan microarrays, ELISA and SPR assays, selectively stained tissue derived from advanced-stage tumors, and bound MUC4⁺ tumor cells in flow cytometry assays. The mAb was also unique in that it did not cross-react with other commercial anti-MUC4 mAbs that were raised in a similar but non-glycosylated TR sequence. Here we describe the selective conjugation of a novel near-infrared dye to this mAb and in vivo biodistribution of this labeled mAb to various MUC4-expressing tumors in mice. The labeled mAb were selectively distributed to both cell-derived xenograft (CDX) flank tumors and patient-derived xenograft (PDX) tumors that expressed MUC4 compared to those that were MUC4-negative. Organ distribution analysis showed high uptake in MUC4⁺ relative to MUC4⁻ tumors. These results suggest that mAb F5 may be used to develop MUC4-targeted, passive antibody-based immunotherapies against Pancreatic Ductal Adenocarcinomas (PDACs) which are notorious for being refractory to many chemo- and radiotherapies Full article

Near-infrared photoimmunotherapy (NIR-PIT) is a promising cancer treatment that uses near-infrared light to activate a conjugate of a monoclonal antibody (mAb) and a photoactivatable silica phthalocyanine dye (IRDye700DX: IR700). Unlike conventional photodynamic therapy (PDT), NIR-PIT selectively destroys targeted tumor cells while preserving the surrounding normal tissue and providing superior tissue penetration. Recently, NIR-PIT has been approved for the treatment of unresectable recurrent head and neck cancers in Japan. It induces highly selective cancer cell death; therefore, it is expected to be a new curative treatment option for various cancers, including brain tumors. In this review, we compare the principles of NIR-PIT and PDT and discuss the potential applications of NIR-PIT for brain tumors. We selected targetable proteins across various types of brain tumors and devised a strategy to effectively pass the mAb–IR700 conjugate through the blood–brain barrier (BBB), which is a significant challenge for NIR-PIT in treating brain tumors. Innovative approaches for delivering the mAb–IR700 conjugate across the BBB include exosomes, nanoparticle-based systems, and cell-penetrating peptides. Small-molecule compounds, such as affibodies, are anticipated to rapidly accumulate in tumors within intracranial models, and our preliminary experiments demonstrated rapid uptake. NIR-PIT also induces immunogenic cell death and activates the anti-tumor immune response. Overall, NIR-PIT is a promising approach for treating brain tumors. It has the potential to overcome the limitations of conventional therapies and offers new hope to patients with brain tumors. Full article

Background/Objectives: The Ca²⁺-activated K⁺ channel K_Ca3.1 is not only involved in physiological processes such as immune reactions and control of vascular tone, but is highly expressed in various tumor entities. Thus, imaging of K_Ca3.1 channels comes into focus for the localization of high channel density, i.e., for tumor diagnosis. In particular, the physicochemical properties of the fluorescent probes should be improved compared to existing probes. Methods: The small molecule inhibitor of the K_Ca3.1 channel, senicapoc, was used as a warhead and was coupled with different fluorescent dyes. After synthesis of the novel probes, their physicochemical properties (lipophilicity, photophysical properties) and their ability to image K_Ca3.1 channels in A549-3R lung tumor cells were determined. Results: In order to increase the polarity and quantum yield of reported fluorescent probes, three strategies were followed: (1) An F-atom at the B-atom of bodipy-labeled senicapoc derivatives 9a, 9b, and 15a was replaced by a OCH₃ moiety, which decreased the logP value by one log-unit. (2) The p-phenylene moiety of the linker was replaced by an aliphatic tetramethylene linker decreasing the lipophilicity by 0.3–0.5 log-units. (3) Instead of bodipy dyes, fluorescein was coupled with the senicapoc warhead resulting in very polar probes 21a and 21b with low logP values of 1.5 and 1.3, respectively. Introduction of an ethyl moiety at the bodipy core increased the quantum yield, which resulted in the best punctate staining pattern of fixed and living A549-3R lung tumor cells with the ethylbodipy-labeled senicapoc derivative 10b. The specificity was shown by various control experiments. Co-staining with 10b and an antibody did not result in overlapping signals. Conclusions: The well-balanced lipophilicity and fluorescent quantum yield render the ethylbodipy-labeled senicapoc derivative 10b a very good probe to image selectively K_Ca3.1 ion channels in fixed and living tumor cells. It was hypothesized that the antibody binds selectively at the closed channel (58.5%), whereas the senicapoc–bodipy conjugate 10b binds selectively at the open channel (41.5%). The ratio 58.5:41.5 reflects the ratio of the ion channel in closed and open conformations. Full article

The development of accurate and high-throughput tools for cancer biomarker detection is crucial for the diagnosis, monitoring, and treatment of diseases. In this study, we developed a simple and rapid fluorescence-linked immunosorbent assay (FLISA) using fluorescent dye-conjugated antibody fragments against programmed cell death ligand 1 (PDL1) and human epithelial growth factor receptor 2 (HER2). We optimized key steps in the FLISA process, including antigen immobilization, blocking, and antibody reaction, reading the assay time to 3 h—significantly faster compared to the 23 h duration of usual FLISA. The limit of detection for the rapid FLISA in detecting PDL1 was lower than that of FLISA, and the detection of HER2 was similar between the two methods, indicating that the rapid FLISA provides a fast and accurate approach for detecting PDL1 and HER2. This robust platform can be readily adapted for various fluoroimmunoassays targeting other antigens of interest. Full article

Background/Objectives: Photoimmunotherapy (PIT) is an innovative approach for the targeted therapy of cancer. In PIT, photosensitizer dyes are conjugated to tumor-specific antibodies for targeted delivery into cancer cells. Upon irradiation with visible light, the photosensitizer dye is activated and induces cancer-specific cell death. In the present article, we describe the PIT of prostate cancer (PC) as a therapeutic option for the targeted treatment of localized prostate cancer. Methods: We conjugated the silicon phthalocyanine dye WB692-CB2 to recombinant cysteine-modified anti-CD44 and anti-EpCAM antibodies via a maleimide linker and tested the antibody dye conjugates for PIT on PC cells and prostate cancer stem cell (PCSC)-like cells. Results: The anti-CD44 and anti-EpCAM antibody dye conjugates showed specific binding and high cytotoxicity against PC and PCSC-like cells following irradiation with red light. Combined treatment with both conjugates led to enhanced cytotoxic effects. Conclusions: PIT with our dye WB692-CB2 can serve as an effective focal therapy against prostate cancer, preserving the prostate gland and minimizing side effects. It can be employed during radical prostatectomy (RP) to treat residual tumor cells or lymph node metastases in areas where further surgical intervention is not feasible. Utilizing multiple conjugates against antigens expressed on differentiated PC and PCSC-like cells, such as CD44 and EpCAM, could be an effective method to eradicate residual cancer cells in heterogeneous tumors. This approach could reduce the risk of local recurrence after RP and thus increase the therapeutic outcome of PC patients. Full article

Background/Objectives: Pancreatic cancer is the third leading cause of death related to cancer. The only possible cure presently is complete surgical resection; however, this is limited by difficulty in clearly defining tumor margins. Enhancement of the visualization of pancreatic ductal adenocarcinoma (PDAC) tumor margins using near-infrared dye-conjugated tumor-specific antibodies was pioneered by using anti-CEA, anti-CA19.9, and anti-MUC5AC in orthotopic mouse models of pancreatic cancer. Recently, an antibody to Mucin 4 (MUC4) conjugated to a fluorescent probe has shown promise in targeting colon tumors in orthotopic mouse models. Methods: In the present study, we targeted pancreatic cancer using an anti-MUC4 antibody conjugated to IRDye800 (anti-MUC4-IR800) in orthotopic mouse models. Two pancreatic cancer human cell lines were used, SW1990 and CD18/HPAF. Results: Anti-MUC4-IR800 targeted the two pancreatic cancer cell line tumors in orthotopic mouse models with high tumor-to-pancreas ratios and high tumor-to-liver ratios, with greater targeting seen in SW1990. Conclusions: The present results suggest anti-MUC4-IR800’s potential to be used in fluorescence-guided surgical resection of pancreatic cancer. Full article

Background: The present study aimed to validate the accuracy of a tumor-specific antibody to target liver metastases of colorectal cancer. Methods: A humanized anti-CEA antibody conjugated to a fluorescent dye (M5A-IR800) was tested for targeting human colorectal cancer liver metastases (CRLMs) expressing luciferase in an orthotopic mouse model. Orthotopic mouse models of CRLMs were established by implanting fragments of a luciferase-expressing human colorectal cancer cell line, LS174T, in the liver of nude mice. Mice received 50 µg M5A-IR800 72 h prior to imaging. To test co-localization, bioluminescence imaging was performed using D-luciferin, which was given via intraperitoneal injection just prior to imaging. Results: Tumors were able to be visualized non-invasively through the skin with the luciferase–luciferin signal. Intra-abdominal imaging showed accurate labeling of CRLMs with M5A-IR800, which co-localized with the luciferase–luciferin signal. Conclusions: The present results validate the accuracy of a tumor-specific anti-CEA antibody in targeting liver metastases of colorectal cancer. Full article

The first example of sonodynamic therapy (SDT) with a cyanine dye–antibody conjugate is reported. The aim of this study was to evaluate the sonodynamic efficacy of a trastuzumab-guided diiodinated heptamethine cyanine-based sensitizer, 2ICy7–Ab, versus its non-iodinated counterpart, Cy7–Ab, in a human epidermal growth factor receptor 2-positive (HER2+) xenograft model. In addition, the combined sonodynamic and photodynamic (PDT) effects were investigated. A single intravenous injection of 2ICy7–Ab followed by sonication or combined sonication and photoirradiation in mice resulted in complete tumor growth suppression compared with the nontreated control and showed no detectable toxicity to off-target tissues. In contrast, Cy7–Ab provided only a moderate therapeutic effect (~1.4–1.6-fold suppression). SDT with 2ICy7–Ab resulted in a 3.5-fold reduction in tumor volume within 45 days and exhibited 13-fold greater tumor suppression than PDT alone. In addition, 2ICy7–Ab showed more durable sonostability than photostability. The sonotoxicity of the iodinated versus noniodinated counterparts is attributed to the increased generation of hydroxyl radicals, superoxide, and singlet oxygen. We observed no significant contribution of PDT to the efficacy of the combined SDT and PDT, indicating that SDT with 2ICy7–Ab is superior to PDT alone. These new findings set the stage for the application of cyanine–antibody conjugates for fluorescently monitored targeted sonodynamic treatment of cancer. Full article

Brain metastasis is the most common intracranial malignancy in adults. The prognosis is extremely poor, partly because most patients have more than one brain lesion, and the currently available therapies are nonspecific or inaccessible to those occult metastases due to an impermeable blood–tumor barrier (BTB). Phosphatidylserine (PS) is externalized on the surface of viable endothelial cells (ECs) in tumor blood vessels. In this study, we have applied a PS-targeting antibody to assess brain metastases in mouse models. Fluorescence microscopic imaging revealed that extensive PS exposure was found exclusively on vascular ECs of brain metastases. The highly sensitive and specific binding of the PS antibody enables individual metastases, even micrometastases containing an intact BTB, to be clearly delineated. Furthermore, the conjugation of the PS antibody with a fluorescence dye, IRDye 800CW, or a radioisotope, ¹²⁵I, allowed the clear visualization of individual brain metastases by optical imaging and autoradiography, respectively. In conclusion, we demonstrated a novel strategy for targeting brain metastases based on our finding that abundant PS exposure occurs on blood vessels of brain metastases but not on normal brain, which may be useful for the development of imaging and targeted therapeutics for brain metastases. Full article

Near-infrared photoimmunotherapy (NIR-PIT) is a new cancer therapy that uses NIR light and conjugates of a tumor-targeting monoclonal antibody and phthalocyanine dye. In clinical practice, frontal and cylindrical diffusers are the only options for NIR illumination. However, illumination in a narrow space is technically difficult with such diffusers. Therefore, we evaluated a lateral illumination system using a lateral emitting laser (LEL) fiber. The LEL fiber illuminated a certain area in a lateral direction. NIR-PIT with an LEL fiber reduced luciferase activity in a light-dose-dependent manner in A431-GFP-luc cells in vitro and significantly suppressed tumor proliferation in a xenograft mouse model. To evaluate the usefulness of the LEL fiber in the illumination of a narrow space, a tumor was illuminated from the inside of a cylinder, mimicking a narrow space, and the fluorescence intensity in the tumor was monitored. In the frontal diffuser, NIR light was unevenly delivered and little light reached a distal tumor area from the illuminated side. By contrast, the LEL fiber allowed a uniform illumination of the entire tumor, and a loss of fluorescence was observed even in distal areas. These findings suggested that the LEL fiber can be used for NIR-PIT and is suitable for NIR light illumination in a narrow space. Full article

Methylene blue (MB) is a chemical dye which is used as an alternative drug for malachite green. In this study, based on computational chemistry, a novel hapten (TM) of MB was designed by comparing the conformational and electronic properties of MB. TM was synthesized and further conjugated with bovine serum albumin (BSA) and Ovalbumin (OVA). Then, the polyclonal antibodies were obtained by immunization with the immunogen of TM-BSA. Under optimal conditions, a sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed for the determination of MB, with an IC₅₀ value of 41.5 μg L⁻¹ and displaying a cross reaction of 78.2~88.9% with three different MB metabolins. The limit of detection (LOD) and the limit of quantitation (LOQ) for MB were validated to be 4.8 µg/kg and 6.0 µg/kg, respectively. Spiking experiments showed recoveries between 82.3% and 84.3%, with a relative standard deviation (RSD) of ≤6%. The results showed the generated polyclonal antibodies from the new hapten TM were reliable and could be used for detecting MB in fishery productions. Full article

Near-infrared photoimmunotherapy (NIR-PIT) is a novel cancer therapy based on a monoclonal antibody (mAb) conjugated to a photosensitizer (IR700Dye). The conjugate can be activated by near-infrared light irradiation, causing necrotic cell death with high selectivity. In this study, we investigated NIR-PIT using a small protein mimetic (6–7 kDa, Affibody) which has more rapid clearance and better tissue penetration than mAbs for epidermal growth factor receptor (EGFR)-positive salivary gland cancer (SGC). The level of EGFR expression was examined in vitro using immunocytochemistry and Western blotting. Cell viability was analyzed using the alamarBlue assay. In vivo, the volume of EGFR-positive tumors treated with NIR-PIT using the EGFR Affibody–IR700Dye conjugate was followed for 43 days. It was found that NIR-PIT using the EGFR Affibody–IR700Dye conjugate induced the selective destruction of EGFR-positive SGC cells and restricted the progression of EGFR-positive tumors. We expect that NIR-PIT using the EGFR Affibody–IR700Dye conjugate can efficiently treat EGFR-positive SGC and preserve normal salivary function. Full article

Immunosorbent turnip vein clearing virus (TVCV) particles displaying the IgG-binding domains D and E of Staphylococcus aureus protein A (PA) on every coat protein (CP) subunit (TVCV_PA) were purified from plants via optimized and new protocols. The latter used polyethylene glycol (PEG) raw precipitates, from which virions were selectively re-solubilized in reverse PEG concentration gradients. This procedure improved the integrity of both TVCV_PA and the wild-type subgroup 3 tobamovirus. TVCV_PA could be loaded with more than 500 IgGs per virion, which mediated the immunocapture of fluorescent dyes, GFP, and active enzymes. Bi-enzyme ensembles of cooperating glucose oxidase and horseradish peroxidase were tethered together on the TVCV_PA carriers via a single antibody type, with one enzyme conjugated chemically to its Fc region, and the other one bound as a target, yielding synthetic multi-enzyme complexes. In microtiter plates, the TVCV_PA-displayed sugar-sensing system possessed a considerably increased reusability upon repeated testing, compared to the IgG-bound enzyme pair in the absence of the virus. A high coverage of the viral adapters was also achieved on Ta₂O₅ sensor chip surfaces coated with a polyelectrolyte interlayer, as a prerequisite for durable TVCV_PA-assisted electrochemical biosensing via modularly IgG-assembled sensor enzymes. Full article

The development of new neurotherapeutics depends on appropriate animal models being chosen in preclinical studies. The cuprizone model is an effective tool for studying demyelination and remyelination processes in the brain, but blood–brain barrier (BBB) integrity in the cuprizone model is still a topic for debate. Several publications claim that the BBB remains intact during cuprizone-induced demyelination; others demonstrate results that could explain the increased BBB permeability. In this study, we aim to analyze the permeability of the BBB for different macromolecules, particularly antibody conjugates, in a cuprizone-induced model of demyelination. We compared the traditional approach using Evans blue injection with subsequent dye extraction and detection of antibody conjugates using magnetic resonance imaging (MRI) and confocal microscopy to analyze BBB permeability in the cuprizone model. First, we validated our model of demyelination by performing T2-weighted MRI, diffusion tensor imaging, quantitative rt-PCR to detect changes in mRNA expression of myelin basic protein and proteolipid protein, and Luxol fast blue histological staining of myelin. Intraperitoneal injection of Evans blue did not result in any differences between the fluorescent signal in the brain of healthy and cuprizone-treated mice (IVIS analysis with subsequent dye extraction). In contrast, intravenous injection of antibody conjugates (anti-GFAP or non-specific IgG) after 4 weeks of a cuprizone diet demonstrated accumulation in the corpus callosum of cuprizone-treated mice both by contrast-enhanced MRI (for gadolinium-labeled antibodies) and by fluorescence microscopy (for Alexa488-labeled antibodies). Our results suggest that the methods with better sensitivity could detect the accumulation of macromolecules (such as fluorescent-labeled or gadolinium-labeled antibody conjugates) in the brain, suggesting a local BBB disruption in the demyelinating area. These findings support previous investigations that questioned BBB integrity in the cuprizone model and demonstrate the possibility of delivering antibody conjugates to the corpus callosum of cuprizone-treated mice. Full article

Near-infrared photoimmunotherapy (NIR-PIT) is a new phototherapy that utilizes a monoclonal antibody (mAb) against cancer antigens and a phthalocyanine dye, IRDye700DX (IR700) conjugate (mAb-IR700). Photodynamic therapy (PDT) is a combination therapy that utilizes photoreactive agents and light irradiation as well as NIR-PIT. In the present study, we compared these therapies in vitro. The characterization of cellular binding/uptake specificity and cytotoxicity were examined using two mAb-IR700 forms and a conventional PDT agent, talaporfin sodium, in three cell lines. As designed, mAb-IR700 had high molecular selectivity and visualized target molecule-positive cells at the lowest concentration examined. NIR-PIT induced necrosis and damage-associated molecular patterns (DAMPs), a surrogate maker of immunogenic cell death. In contrast, talaporfin sodium was taken up by cells regardless of cell type, and its uptake was enhanced in a concentration-dependent manner. PDT induced cell death, with the pattern of cell death shifting from apoptosis to necrosis depending on the concentration of the photosensitizer. Induction of DAMPs was observed at the highest concentration, but their sensitivity differed among cell lines. Overall, our data suggest that molecule-specific NIR-PIT may have potential advantages compared with PDT in terms of the efficiency of tumor visualization and induction of DAMPs. Full article