anti-CEA Monoclonal Antibody Research Articles

Biliary Carcinoembryonic Antigen (CEA) Levels: The Role in Detection of Occult Hepatic Metastases in Colorectal Carcinoma

Objective: Colorectal carcinoma (CRC) is a major public health concern, often complicated by hepatic metastasis. Despite the widespread use of serum Carcinoembryonic Antigen (CEA) for postoperative monitoring, early detection of hepatic metastasis remains elusive. This study aims to evaluate the prognostic significance of CEA levels in both peripheral blood and gallbladder bile for predicting hepatic metastasis in CRC patients, and to explore its potential utility for personalized treatment regimens. Materials and Methods: A prospective randomized study was conducted over three years, enrolling 31 participants, 21 of whom underwent curative operations for CRC. Preoperative and postoperative CEA levels were assessed with rigorous diagnostic imaging and histological examinations. Patients were stratified into groups based on liver metastasis, postoperative chemotherapy, and CEA levels in both blood and bile. Results: A statistically significant difference was found in biliary CEA levels between patients with (mean = 129.22 ng/ml) and without intraoperative liver metastasis (mean = 33.16 ng/ml), p-value = 0.01. Serum and biliary CEA levels were robustly correlated, p > 0.05. Biliary CEA values differed significantly across Duke's stages, with particular significance between the control group and Duke's stage D (p < 0.001). Among patients without intraoperative liver metastasis but with biliary CEA >10 ng/ml, 80% developed liver metastasis postoperatively despite undergoing chemotherapy. Conclusion: This research highlights the pivotal role of biliary CEA levels in diagnosing latent hepatic metastases in colorectal cancer. Demonstrating superior sensitivity compared to traditional serum tests, biliary CEA presents a potential shift in early detection. Elevated levels suggest expanded therapeutic options, from timely hepatectomies to adopting radiofrequency ablation and anti-CEA monoclonal antibodies, enhancing the likelihood of better patient outcomes. Essentially, these findings enhance our understanding of colorectal cancer, emphasizing the importance of personalized care, early diagnosis, and customized treatments, thereby directing us toward better patient outcomes.

Air-brush spray coated Ti3C2-MXene-graphene nanohybrid thin film based electrochemical biosensor for cancer biomarker detection

Cancer is a significant health hazard worldwide and poses a greater threat to the quality of human life. Quantifying cancer biomarkers with high sensitivity has demonstrated considerable potential for compelling, quick, cost-effective, and minimally invasive early-stage cancer detection. In line with this, efforts have been made towards developing an f-graphene@Ti3C2-MXene nanohybrid thin-film-based electrochemical biosensing platform for efficient carcinoembryonic antigen (CEA) detection. The air-brush spray coating technique has been utilized for depositing the uniform thin films of amine functionalized graphene (f-graphene) and Ti3C2-MXene nanohybrid on ITO-coated glass substrate. The chemical bonding and morphological studies of the deposited nanohybrid thin films are characterized by advanced analytical tools, including XRD, XPS, and FESEM. The EDC-NHS chemistry is employed to immobilize the deposited thin films with monoclonal anti-CEA antibodies, followed by blocking the non-specific binding sites with BSA. The electrochemical response and optimization of biosensing parameters have been conducted using CV and DPV techniques. The optimized BSA/anti-CEA/f-graphene@Ti3C2-MXene immunoelectrode showed the ability to detect CEA biomarker from 0.01 pg mL−1 to 2000 ng mL−1 having a considerably lower detection limit of 0.30 pg mL−1.

Construction of Magnetic Core-Large Mesoporous Satellite Immunosensor for Long-Lasting Chemiluminescence and Highly Sensitive Tumor Marker Determination.

Chemiluminescence immunoassay exhibits high sensitivity and signal-to-noise ratio, thus attracting great attention in the early diagnosis and dynamic monitoring of diseases. However, the collection of conventional flash-type chemiluminescence signal (<5s) relies heavily on automatic sampling and reading instrument. Herein, a novel core-satellite multifunctional chemiluminescence immunosensor is designed for the efficient enrichment and highly sensitive determination of cancer biomarker carcinoembryonic antigen (CEA) with enhanced and long-lasting output signal that can be conveniently recorded by a simple microplate plate reading instrument. Anti-CEA monoclonal antibody 2 (Ab2) modified Fe3 O4 @SiO2 microspheres (Fe3 O4 @SiO2 -Ab2, 370nm in diameter) are synthesized as the core for selectively capturing and enriching target CEA in solution, and anti-human CEA monoclonal antibody 1 (Ab1) and horseradish peroxidase (HRP) co-immobilized dendritic large-mesoporous silica nanospheres (MSNs-HRP/Ab1, 80nm in diameter, pore size: 17nm) are synthesized as the satellite for efficient immunological recognition and signal amplification. The as-designed core-satellite magnetic chemiluminescence immunosensors exhibit a broad linear range of 0.01-20ngmL-1 and a low detection limit of 3.0pgmL-1 for the convenient, highly specific, and sensitive determination of CEA in human serum. Such core-satellite chemiluminescence immunosensors are expected to act as a powerful tool for in vitro detection of various biomarkers, overcome the defect of conventional chemiluminescence relying heavily on expensive and bulky automatic instruments and popularize chemiluminescence analysis to primary medical institutions and remote areas.

In Vitro Characterization of 177Lu-DOTA-M5A Anti-Carcinoembryonic Antigen Humanized Antibody and HSP90 Inhibition for Potentiated Radioimmunotherapy of Colorectal Cancer.

Carcinoembryonic antigen (CEA) is an antigen that is highly expressed in colorectal cancers and widely used as a tumor marker. 131I and 90Y-radiolabeled anti-CEA monoclonal antibodies (mAbs) have previously been assessed for radioimmunotherapy in early clinical trials with promising results. Moreover, the heat shock protein 90 inhibitor onalespib has previously demonstrated radiotherapy potentiation effects in vivo. In the present study, a 177Lu-radiolabeled anti-CEA hT84.66-M5A mAb (M5A) conjugate was developed and the potential therapeutic effects of 177Lu-DOTA-M5A and/or onalespib were investigated. The 177Lu radiolabeling of M5A was first optimized and characterized. Binding specificity and affinity of the conjugate were then evaluated in a panel of gastrointestinal cancer cell lines. The effects on spheroid growth and cell viability, as well as molecular effects from treatments, were then assessed in several three-dimensional (3D) multicellular colorectal cancer spheroid models. Stable and reproducible radiolabeling was obtained, with labeling yields above 92%, and stability was retained at least 48 h post-radiolabeling. Antigen-specific binding of the radiolabeled conjugate was demonstrated on all CEA-positive cell lines. Dose-dependent therapeutic effects of both 177Lu-DOTA-M5A and onalespib were demonstrated in the spheroid models. Moreover, effects were potentiated in several dose combinations, where spheroid sizes and viabilities were significantly decreased compared to the corresponding monotherapies. For example, the combination treatment with 350 nM onalespib and 20 kBq 177Lu-DOTA-M5A resulted in 2.5 and 2.3 times smaller spheroids at the experimental endpoint than the corresponding monotreatments in the SNU1544 spheroid model. Synergistic effects were demonstrated in several of the more effective combinations. Molecular assessments validated the therapy results and displayed increased apoptosis in several combination treatments. In conclusion, the combination therapy of anti-CEA 177Lu-DOTA-M5A and onalespib showed enhanced therapeutic effects over the individual monotherapies for the potential treatment of colorectal cancer. Further in vitro and in vivo studies are warranted to confirm the current study findings.

Ti3C2-MXene-assisted signal amplification for sensitive and selective surface plasmon resonance biosensing of biomarker

Surface plasmon resonance (SPR) technology has become one of the powerful tools for real-time bioassay of various targets without needing labels. However, SPR biosensing faces great challenge with the extension of application fields due to the limited sensitivity. Herein, we report an ultrasensitive SPR biosensing strategy for detecting carcinoembryonic antigen (CEA) with the introduction of amino-functionalized Ti3C2-MXene (N-Ti3C2-MXene) nanosheets. The ultrathin Ti3C2-MXene nanosheets were prepared by hydrochloric acid (HF) etching and further modified by (3-aminopropyl) triethoxysilane (APTES) to obtain amino terminals for conjugating polyclonal anti-CEA antibody (Ab2) via covalent bonding. Staphylococcal protein A (SPA) decorated Au film was utilized as sensing platform to orientedly immobilize monoclonal anti-CEA antibody (Ab1) via its Fc region. The N-Ti3C2-MXene-Ab2 nanoconjugates were introduced to the sensing system after capture of CEA, forming sandwiched immunocomplexes on the SPR chip. A signal amplification strategy of Ti3C2-MXene-induced spontaneous gold reductive deposition on Ti3C2-MXene nanosheets was employed to further enhance sensitivity. The proposed biosensor exhibits a wide linear range for CEA determination of 10−11 to 10−6 g mL−1 with a detection limit of 1.7 pg mL−1 (S/N=3). Moreover, good reproducibility and high specificity were demonstrated by serum samples analysis, which indicates the present method holds potential in evaluating CEA in human serum for early diagnosis and monitoring of cancer.

An immunosensor for the determination of carcinoembryonic antigen by Surface Plasmon Resonance imaging

Carcinoembryonic antigen (CEA) is one of the biomarkers most commonly used to determine tumor activity. In this work, a Surface Plasmon Resonance imaging (SPRi) immunosensor was developed. The immunosensor consists of a cysteamine linker attached to a gold chip and mouse monoclonal anti-CEA antibody bonded by the “EDC/NHS protocol”. The formation of successive immunosensor layers was confirmed by AFM measurements. The concentration of the antibody was optimized. The linear response range of the developed immunosensor is between 0.40 and 20 ng mL−1, and it is suitable for CEA measurement in both blood cancer patients and healthy individuals. Only 3 μL of serum or plasma sample is required, and no preconcentration is used. The method has a precision of 2–16%, a recovery of 101–104% depending on CEA concentration, a detection limit of 0.12 ng mL−1 and a quantification limit of 0.40 ng mL−1. The method is selective (with respect to albumin, leptin, interleukin 6, metalloproteinase-1, metallopeptidase inhibitor 1 and CA 125/MUC16) and it was validated by comparison with the standard electrochemiluminescence method on a series of colorectal cancer blood samples.

Ultrasensitive and Selective Determination of Carcinoembryonic Antigen Using Multifunctional Ultrathin Amino-Functionalized Ti3C2-MXene Nanosheets

Herein, we report on a two-dimensional amino-functionalized Ti3C2-MXene (N-Ti3C2-MXene)-based surface plasmon resonance (SPR) biosensor for detecting carcinoembryonic antigen (CEA) utilizing a sandwich format signal amplification strategy. Our biosensor employs an N-Ti3C2-MXene nanosheet-modified sensing platform and a signal enhancer comprising N-Ti3C2-MXene-hollow gold nanoparticles (HGNPs)-staphylococcal protein A (SPA) complexes. Ultrathin Ti3C2-MXene nanosheets were synthesized and functionalized with aminosilane to provide a hydrophilic-biocompatible nanoplatform for covalent immobilization of the monoclonal anti-CEA capture antibody (Ab1). The N-Ti3C2-MXene/HGNPs nanohybrids were synthesized and further decorated with SPA to immobilize the polyclonal anti-CEA detection antibody (Ab2) and serve as signal enhancers. The capture of CEA followed by the formation of the Ab2-conjugated SPA/HGNPs/N-Ti3C2-MXene sandwiched nanocomplex on the SPR chip results in the generation of a response signal. The fabricated N-Ti3C2-MXene-based SPR biosensor exhibited a linear detection range of 0.001-1000 PM with a detection limit of 0.15 fM. The proposed biosensor showed high sensitivity and specificity for CEA in serum samples, which gives it application potential in the early diagnosis and monitoring of cancer. We believe that this work also opens new avenues for development of MXene-based highly sensitive biosensors for determining various biomolecules.

Impact of Different Selectivity between Soluble and Membrane-bound Forms of Carcinoembryonic Antigen (CEA) on the Target-mediated Disposition of Anti-CEA Monoclonal Antibodies.

Carcinoembryonic antigen (CEA) is a tumor-specific antigen overexpressed in multiple cancers. CEA is expressed as a membrane protein, a part of which is cleaved from the cell membrane and secreted into blood. The soluble form of CEA (sCEA) has been shown to accelerate the clearance of anti-CEA antibody, which limits the antibody distribution in the tumor. To overcome this issue, we developed an anti-CEA monoclonal antibody, 15-1-32, which shows a strong affinity for membrane-bound CEA (mCEA) and relatively weak affinity for sCEA. In this study, we compared the effect of sCEA on the pharmacokinetics of 15-1-32 in mice with that of another anti-CEA monoclonal antibody, labetuzumab, showing less selectivity to mCEA than 15-1-32. As expected, the effect of sCEA on the serum concentration of 15-1-32 was much smaller than that of labetuzumab. The decrease in the area under the curve (AUC) of serum concentration was 22.5% for 15-1-32 when it was coadministered with sCEA, while that of labetuzumab was 79.9%. We also compared the pharmacokinetics of these two antibodies in CEA-positive tumor-bearing mice. The AUCs of 15-1-32 and labetuzumab were decreased in tumor-bearing mice compared with non-tumor-bearing mice to a similar extent (approximately 40% decrease). These results suggested that mCEA also contributes to the clearance of anti-CEA antibodies in CEA-positive tumor-bearing mice. Although the increased selectivity to mCEA minimized the effect of sCEA on the pharmacokinetics of 15-1-32, it may be insufficient to improve the pharmacokinetics in CEA-positive cancer patients. SIGNIFICANCE STATEMENT: Because previous studies reported the rapid clearance of anti-CEA antibodies mediated by soluble CEA, we obtained a monoclonal antibody, 15-1-32, selective to membrane-bound CEA and evaluated the effects of CEA on its pharmacokinetics. Although the effect of soluble CEA on the serum concentration of 15-1-32 was very small, the clearance of 15-1-32 in CEA-positive tumor-bearing mice was still rapid, suggesting membrane-bound CEA also contributes to the clearance of anti-CEA antibodies. These results indicated that increasing selectivity to membrane-bound CEA is not enough to improve the pharmacokinetics of anti-CEA antibody.

A 2D transition metal carbide MXene-based SPR biosensor for ultrasensitive carcinoembryonic antigen detection

Surface plasmon resonance (SPR) has become a leading technique for in situ bioaffinity assay of diverse targets without need of fluorescent or enzymatic labeling. Nanomaterials-enhanced SPR sensors have developed rapidly and widened the application scope of SPR sensing technology. In this report we describe an ultrasensitive SPR biosensor for detecting carcinoembryonic antigen (CEA). Our SPR biosensor utilizes a Ti3C2-MXene-based sensing platform and multi-walled carbon nanotube (MWCNTs)-polydopamine (PDA)-Ag nanoparticle (AgNPs) signal enhancer. Ti3C2-MXene, a new class of two-dimensional (2D) transition metal carbides, offers a large hydrophilic-biocompatible surface ideal for SPR biosensing. Ti3C2-MXene/AuNPs composites after synthesis are then decorated with staphylococcal protein A (SPA) to orient and immobilize monoclonal anti-CEA antibody (Ab1) through its Fc region. By introducing MWCNTs-PDA-AgNPs-polyclonal anti-CEA antibody (MWPAg-Ab2) conjugate combined with a sandwich format, the present method provides a dynamic range for CEA determination of 2×10-16 to 2×10-8 M and a detection limit of 0.07 fM. This biosensing approach demonstrates good reproducibility and high specificity for CEA in real serum samples providing a promising method to evaluate CEA in human serum for early diagnosis and monitoring of cancer.

Safety and effectiveness of SGM-101, a fluorescent antibody targeting carcinoembryonic antigen, for intraoperative detection of colorectal cancer: a dose-escalation pilot study

Tumour-targeted fluorescence imaging has the potential to advance current practice of oncological surgery by selectively highlighting malignant tissue during surgery. Carcinoembryonic antigen (CEA) is overexpressed in 90% of colorectal cancers and is a promising target for colorectal cancer imaging. We aimed to assess the tolerability of SGM-101, a fluorescent anti-CEA monoclonal antibody, and to investigate the feasibility to detect colorectal cancer with intraoperative fluorescence imaging. We did an open-label, pilot study in two medical centres in the Netherlands. In the dose-escalation cohort, we included patients (aged ≥18 years) with primary colorectal cancer with increased serum CEA concentrations (upper limit of normal of ≥3 ng/mL) since diagnosis, who were scheduled for open or laparoscopic tumour resection. In the expansion cohort, we included patients (aged ≥18 years) with recurrent or peritoneal metastases of colorectal cancer, with increasing serum concentrations of CEA since diagnosis, who were scheduled for open surgical resection. We did not mask patients, investigators, or anyone from the health-care team. We assigned patients using a 3 + 3 dose design to 5 mg, 7·5 mg, or 10 mg of SGM-101 in the dose-escalation cohort. In the expansion cohort, patients received a dose that was considered optimal at that moment of the study but not higher than the dose used in the dose-escalation cohort. SGM-101 was administered intravenously for 30 min to patients 2 or 4 days before surgery. Intraoperative imaging was done to identify near-infrared fluorescent lesions, which were resected and assessed for fluorescence. The primary outcome was tolerability and safety of SGM-101, assessed before administration and continued up to 12 h after dosing, on the day of surgery, the first postoperative day, and follow-up visits at the day of discharge and the first outpatient clinic visit. Secondary outcomes were effectiveness of SGM-101 for detection of colorectal cancer, assessed by tumour-to-background ratios (TBR); concordance between fluorescent signal and tumour status of resected tissue; and diagnostic accuracy in both cohorts. This trial is registered with the Nederlands Trial Register, number NTR5673, and ClinicalTrials.gov, number NCT02973672. Between January, 2016, and February, 2017, 26 patients (nine in the dose-escalation cohort and 17 in the expansion cohort) were included in this study. SGM-101 did not cause any treatment-related adverse events, although three possibly related mild adverse events were reported in three (33%) of nine patients in the dose-escalation cohort and five were reported in three (18%) of 17 patients in the expansion cohort. Five moderate adverse events were reported in three (18%) patients in the expansion cohort, but they were deemed unrelated to SGM-101. No changes in vital signs, electrocardiogram, or laboratory results were found after administration of the maximum dose of 10 mg of SGM-101 in both cohorts. A dose of 10 mg, administered 4 days before surgery, showed the highest TBR (mean TBR 6·10 [SD 0·42] in the dose-escalation cohort). In the expansion cohort, 19 (43%) of 43 lesions were detected using fluorescence imaging and were not clinically suspected before fluorescent detection, which changed the treatment strategy in six (35%) of 17 patients. Sensitivity was 98%, specificity was 62%, and accuracy of fluorescence intensity was 84% in the expansion cohort. This study presents the first clinical use of CEA-targeted detection of colorectal cancer and shows that SGM-101 is safe and can influence clinical decision making during the surgical procedure for patients with colorectal cancer. Surgimab.

FRET on lateral flow test strip to enhance sensitivity for detecting cancer biomarker

Fluorescence resonance energy transfer (FRET) between fluorescein isothiocyanate (FITC) and gold nanoparticles (Au NPs) is introduced in the lateral flow strip to detect cancer biomarker CEA with the color and fluorescence dual-readout. Anti-CEA monoclonal antibody coated Au NPs were on the conjugate pad and FITC labelled antibody (FITC-Ab) for CEA was coated on the test line. All the reagents were general in the lateral flow strip or commercially available and no new materials or technique were involved, which make our proposal a more universal method and easier to operate. With the addition of CEA on the sample pad, anti-CEA monoclonal antibody coated Au NPs-CEA-FITC-Ab complex formed on the test line, leading to a megascopic red line and simultaneous quenched fluorescence of FITC via FRET. The visual limit of detection (LOD) through distinguishing red color change was 10ng/mL and the LOD by differentiating fluorescence intensity was 0.1ng/mL, which was two orders of magnitude lower than that without considering fluorescence in the strip. And the linear range changed from 10–80ng/mL to 5–80ng/mL with the analysis of fluorescence change. Meanwhile, the feasibility of our method applied in real clinical samples was also confirmed.

Optical transformation of a CdTe quantum dot-based paper sensor for a visual fluorescence immunoassay induced by dissolved silver ions.

This work designs a simple low-cost visual fluorescence immunoassay for disease-related biomarkers (carcinoembryonic antigen, CEA, used as a model protein) in biological fluids, based on the structural and optical transformation of CdTe quantum dots (QDs) immobilized on paper induced by dissolved silver ions (Ag+) from silver nanoparticles (AgNPs) via a cation-exchange reaction. A sandwich-type immunoreaction was initially carried out in a removable polystyrene high-binding microplate coated with monoclonal anti-CEA capture antibody by using AgNP-labeled polyclonal anti-CEA antibody as the detection antibody. Thereafter, the carried AgNPs accompanying the sandwiched immunocomplexes were dissolved by acid to release numerous silver ions, which induced the ion-exchange reaction with the immobilized CdTe QDs on the paper (attached onto the microplate lid) for the Cd-to-Ag transformation, thus resulting in the quenching of the visual fluorescence from the CdTe QDs owing to Ag2Te formation. Under optimal conditions, the fluorescence intensity decreased with the increasing CEA concentration from 0.02 to 50 ng mL-1 with a detection limit of 5.6 pg mL-1. Further, a visual assay based on a CdTe QD-based paper sensor was developed for CEA detection, and 5.0 pg mL-1 CEA could be discriminated with the naked eye. In addition, our strategy displayed high specificity, good reproducibility and acceptable accuracy for analyzing human serum specimens with consistent results obtained using the commercialized enzyme-linked immunosorbent assay (ELISA) method.

PTD-Modified ATTEMPTS for Enhanced Toxin-based Cancer Therapy: An In Vivo Proof-of-Concept Study.

To investigate the feasibility of applying PTD-modified ATTEMPTS (Antibody Targeted Triggered Electrically Modified Prodrug-Type Strategy) for enhanced toxin therapy for the treatment of cancer. A heparin-functionalized murine anti-CEA monoclonal antibody (mAb), T84.66-heparin (T84.66-Hep), was chemically synthesized and characterized for specific binding to CEA overexpressed cells. The T84.66-Hep was then applied to the PTD-modified ATTEMPTS approach and the crucial features of the drug delivery system (DDS), 'antibody targeting' and 'heparin/protamine-based prodrug', were evaluated in vitro to examine whether it could selective delivery a PTD-modified toxin, recombinant TAT-gelonin chimera (TAT-Gel), to CEA high expression cancer cells (LS174T). Furthermore, the feasibility of the drug delivery system (DDS) was assessed in vivo by biodistribution and efficacy studies using LS174T s.c. xenograft tumor bearing mice. T84.66-Hep displayed specific binding, but limited internalization (35% after 48h incubation) to CEA high expression LS174T cells over low expression HCT116 cells. When mixed together with TAT-Gel, the T84.66-Hep formed a strong yet reversible complex. This complex formation provided an effective means of active tumor targeting of TAT-Gel, by 1) directing the TAT-Gel to CEA overexpressed tumor cells and 2) preventing nonspecific cell transduction to non-targeted normal cells. The cell transduction of TAT-Gel could, however, be efficiently reversed by addition of protamine. Feasibility of in vivo tumor targeting and "protamine-induced release" of TAT-Gel from the T84.66-Hep counterpart was confirmed by biodistribution and preliminary efficacy studies. This study successfully demonstrated in vitro and in vivo the applicability of PTD-modified ATTEMPTS for toxin-based cancer therapy.

Abstract B01: A portable imaging system for fluorescence-guided surgery on a human colon cancer patient-derived orthotopic xenograft (PDOX™) nude mouse model

Abstract In this study, we determined the effectiveness of fluorescence-guided surgery (FGS) using a fluorophore-conjugated anti-carcinoembryonic antigen (CEA) antibody on a colon-cancer patient-derived orthotopic xenografts (PDOX™) nude mouse model. The patient tumor was established in NOD/SCID mice and passed orthotopically in nude mice to make PDOX™ models. Eight weeks after orthotopic implantation in nude mice, a monoclonal anti-CEA antibody conjugated with Alexa 488 was delivered to the PDOX™ models as a single intravenous dose 24 hours before laparotomy. The tumor was completely resected under fluorescence guidance using a hand-held portable fluorescence imaging system. Frozen section microscopy of the resected specimen demonstrated that the anti-CEA antibody selectively labeled cancer cells in the colon cancer PDOX™. Histologic evaluation of the resected specimen demonstrated that cancer cells were not present in the margins. The results of the present report predict that FGS using a fluorophore-conjugated anti-CEA antibody and hand-held portable imaging system should improve intraoperative staging and efficacy of resection for colorectal cancer in the clinic. Citation Format: Yukihiko Hiroshima, Ali Maawy, Sharmeela Kaushal, Yong Zhang, Fuminari Uehara, Shinji Miwa, Shuya Yano, Sho Sato, Takashi Murakami, Masashi Momiyama, Takashi Chishima, Kuniya Tanaka, Michael Bouvet, Itaru Endo, Robert M. Hoffman. A portable imaging system for fluorescence-guided surgery on a human colon cancer patient-derived orthotopic xenograft (PDOX™) nude mouse model. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B01.

Fractionated Radioimmunotherapy with 90Y-Labeled Fully Human Anti-CEA Antibody

Carcinoembryonic antigen (CEA) is an attractive target molecule of radioimmunotherapy (RIT). To enhance RIT's therapeutic efficacy, the fractionation of radiolabeled antibody doses is an attractive strategy. In this study, a fully human anti-CEA monoclonal antibody (mAb) C2-45 was selected by virtue of its lack of immunogenicity, and the effectiveness of fractionated RIT with yttrium-90 (⁹⁰Y)-labeled mAb C2-45 was evaluated. In LS180 tumor-bearing mice, indium-111 (¹¹¹In)-labeled mAb C2-45 showed high and persistent tumor accumulation. Therapeutic studies were performed with single doses of ⁹⁰Y-mAb C2-45 (100 or 200 μCi) or double doses of 100 μCi ⁹⁰Y-mAb C2-45 at different intervals (5, 10, and 15 days). All ⁹⁰Y-mAb C2-45-treated mice showed inhibition of tumor progression, while the time to tumor progression was much longer in both the 200-μCi-treated group and the double 100-μCi-treated group than in the single 100-μCi-treated group. The therapeutic effect of the double 100 μCi administration at days 0 and 15 lasted significantly longer than that in the other treatment groups. These findings indicate that ⁹⁰Y-mAb C2-45 may be a promising agent for the treatment of CEA-positive cancer and that the fractionation of ⁹⁰Y-labeled antibody doses could enhance the therapeutic effect if performed according to an appropriate protocol.

Improved EIS Performance of an Electrochemical Cytosensor Using Three-Dimensional Architecture Au@BSA as Sensing Layer

An ultrasensitive electrochemical cytosensor for quantitative determination of carcinoembryonic antigen (CEA)-positive tumor cells was developed using three-dimensional (3D) architecture Au@BSA microspheres as sensing layer with the conjugation of targeting molecule monoclonal anti-CEA antibody (anti-CEA). The prepared Au@BSA microspheres exhibited satisfactory biocompatibility for cell proliferation via evaluation from thiazolyl blue tetrazolium bromide (MTT) assay, providing a suitable platform for cell adhesion study. Attributed to the excellent electroconductivity of Au@BSA, amplified electrochemical signals could be obtained and resulted in the greatly enhanced detection sensitivity. Electrochemical testing techniques including electrochemical impedance spectroscopy (EIS), differential pulse voltammetry (DPV), and cyclic voltammetry (CV) were applied to assess the optimal conditions, specificity, and detection performance of as-fabricated cytosensor. The attachment of CEA-positive BXPC-3 cells onto the anti-CEA immobilized sensing layer led to the increased EIS responses, which changed linearly in the cell concentration range from 5.2 × 10(1) to 5.2 × 10(7) cells mL(-1) with a detection limit of 18 cells mL(-1). This proposed cytosensing strategy revealed high specificity to CEA-positive cells, acceptable intra-assay precision, excellent fabrication reproducibility with the RSD of 3.5%, and good stability owing to the outside BSA biocompatible layer, developing a promising technique for early monitoring of tumor cells at a lower level.

Abstract 728: Fluorescence-guided surgery for human-patient colon cancer tumorgrafts using a fluorophore-conjugated anti-CEA antibody.

Abstract One of the most important prognostic factors for overall survival in colorectal cancer patients is the complete resection of tumor. We have previously demonstrated the effective fluorescence-guided surgery of primary and metastatic cancer with the use of fluorescent probes in mouse models. In this study, we determined the effectiveness of fluorescence-guided surgery using a fluorophore-conjugated anti-CEA antibody for human-patient colon cancer tumorgrafts in nude mouse models. Colon-cancer-patient tumor specimens were initially established subcutaneously in NOD/SCID mice immediately after surgery. The patient tumors were then harvested from NOD/SCID mice and passed orthotopically in nude mice. Eight weeks after orthotopic implantation of human colon cancer tumorgraft, a monoclonal anti-CEA antibody conjugated with Alexa 488 was delivered to tumor-bearing mice as a single intravenous dose 24 hours before laparotomy. Using fluorescence imaging, the primary tumor was clearly visible at laparotomy. The tumor was completely resected under fluorescence navigation. Histologic evaluation of the resected specimen revealed there were no cancer cells in the margin. The cancer cells were strongly stained by anti-CEA antibody conjugated with Alexa488. Fluorescence-guided surgery using a fluorophore-conjugated anti-CEA antibody should improve intraoperative staging and efficacy of resection for CEA-positive colorectal cancer. Citation Format: Yukihiko Hiroshima, Ali Maawy, Yong Zhang, Takashi Chishima, Kuniya Tanaka, Michael Bouvet, Itaru Endo, Robert M. Hoffman. Fluorescence-guided surgery for human-patient colon cancer tumorgrafts using a fluorophore-conjugated anti-CEA antibody. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 728. doi:10.1158/1538-7445.AM2013-728

A Novel Homogeneous Time-Resolved Fluoroimmunoassay for Carcinoembryonic Antigen Based on Water-Soluble Quantum Dots

Quantum dots are not widely used in clinical diagnosis. However, the homogeneous time-resolved fluorescence assay possesses many advantages over current methods for the detection of carcinoembryonic antigen (CEA), a primary marker for many cancers and diseases. Therefore, a novel luminescent terbium chelates- (LTCs) and quantum dots-based homogeneous time-resolved fluorescence assay was developed to detect CEA. Glutathione-capped quantum dots (QDs) were prepared from oil-soluble QDs with a 565nm emission peak. Conjugates (QDs-6F11) were prepared with QDs and anti-CEA monoclonal antibody. LTCs were prepared and conjugates (LTCs-S001) were prepared with another anti-CEA monoclonal antibody. The fluorescence lifetime of QDs was optimized for sequential analysis. The Förster distance (R0) was calculated as 61.9Å based on the overlap of the spectra of QDs-6F11 and LTCs-S001. Using a double-antibody sandwich approach, the above antibody conjugates were used as energy acceptor and donor, respectively. The signals from QDs were collected in time-resolved mode and analyzed for the detection of CEA. The results show that the QDs were suitable for time-resolved fluoroassays. The spatial distance of the donor-acceptor pair was calculated to be 61.9Å. The signals from QDs were proportional to CEA concentration. The standard curve was LogY = 2.75566 + 0.94457 LogX (R = 0.998) using the fluorescence counts (Y) of QDs and the concentrations of CEA (X). The calculated sensitivity was 0.4ng/mL. The results indicate that water-soluble QDs are suitable for the homogenous immunoassay. This work has expanded future applications of QDs in homogeneous clinical bioassays. Furthermore, a QDs-based homogeneous multiplex immunoassay will be investigated as a biomarker for infectious diseases in future research.

Tumor-selective near-infrared-photoimmunotherapy using a human anti-CEA monoclonal antibody

Tumor-selective near-infrared-photoimmunotherapy using a human anti-CEA monoclonal antibody

Nanoplatinum-enclosed gold nanocores as catalytically promoted nanolabels for sensitive electrochemical immunoassay

Here we designed a new electrochemical immunoassay protocol for determination of carcinoembryonic antigen (CEA) using nanoplatinum-enclosed gold nanocores (Pt@Au) as catalytically promoted nanolabels on the carbon nanospheres and graphene-modified immunosensor. The Pt@Au nanolabels were synthesized and functionalized with monoclonal anti-CEA antibodies and glucose oxidase (GOx). Using the functional Pt@Au nanolabels as molecular tags, the assay was implemented relative to glucose–hydroquinone system with a sandwich-type immunoassay. Initially, the added glucose was oxidized to gluconolactone and H2O2 by the labeled GOx, and then the generated H2O2 was reduced with the help of platinum nanoparticles, leading to the production of oxygen. The self-produced oxygen could promote the re-oxidation of the glucose, thus resulting in the dual amplification of the electrochemical signal. Several nanolabels, such as multiarmed star-like platinum nanowires, hollow platinum nanospheres and Pt@Au nanostructures, were investigated for CEA detection and improved analytical features were obtained with the Pt@Au nanostructures. Under optimal conditions, the Pt@Au-based immunoassay displayed a wide working range from 0.001 to 120ngmL−1 with a low detection limit of 0.5pgmL−1 CEA at 3sB. Intra- and inter-assay coefficients of variation were <10.9%. The system was evaluated with 10 clinical serum samples, receiving good accordance with results from enzyme-linked immunosorbent assay method.