Anti-carcinoembryonic Antigen Monoclonal Antibody Research Articles

Large-area grown ultrathin molybdenum oxides for label-free sensitive biomarker detection.

The rise of two-dimensional (2D) materials has provided a confined geometry and yielded methods for guiding electrons at the nanoscale level. 2D material-enabled electronic devices can interact and transduce the subtle charge perturbation and permit significant advancement in molecule discrimination technology with high accuracy, sensitivity, and specificity, leaving a significant impact on disease diagnosis and health monitoring. However, high-performance biosensors with scalable fabrication ability and simple protocols have yet to be fully realized due to the challenges in wafer-scale 2D film synthesis and integration with electronics. Here, we propose a molybdenum oxide (MoOx)-interdigitated electrode (IDE)-based label-free biosensing chip, which stands out for its wafer-scale dimension, tunability, ease of integration and compatibility with the complementary metal-oxide-semiconductor (CMOS) fabrication. The device surface is biofunctionalized with monoclonal anti-carcinoembryonic antigen antibodies (anti-CEA) via the linkage agent (3-aminopropyl)triethoxysilane (APTES) for carcinoembryonic antigen (CEA) detection and is characterized step-by-step to reveal the working mechanism. A wide range and real-time response of the CEA concentration from 0.1 to 100 ng mL-1 and a low limit of detection (LOD) of 0.015 ng mL-1 were achieved, meeting the clinical requirements for cancer diagnosis and prognosis in serum. The MoOx-IDE biosensor also demonstrates strong surface affinity towards molecules and high selectivity using L-cysteine (L-Cys), glycine (Gly), glucose (Glu), bovine serum albumin (BSA), and immunoglobulin G (IgG). This study showcases a simple, scalable, and low-cost strategy to create a nanoelectronic biosensing platform to achieve high-performance cancer biomarker discrimination capabilities.

PEGylated Fluorescent Anti-carcinoembryonic Antigen Antibody Labels Colorectal Cancer Tumors in Orthotopic Mouse Models

Colorectal cancer (CRC) patients often develop liver metastasis. However, curative resection of liver metastasis is not always possible due to poor visualization of tumor margins. The present study reports the characterization of a humanized anti-carcinoembryonic antigen monoclonal antibody conjugated to a PEGylated near-infrared dye, that targets and brightly labels human CRC tumors in metastatic orthotopic mouse models. The hT84.66-M5A (M5A) monoclonal antibody was conjugated with a polyethylene glycol (PEG) chain that incorporated a near infrared (NIR) IR800 dye to establish M5A-IR800 Sidewinder (M5A-IR800-SW). Nude mice with CRC orthotopic primary tumors and liver metastasis both developed from a human CRC cell line, were injected with M5A-IR800-SW and imaged with the Pearl Trilogy Imaging System. M5A-IR800-SW targeted and brightly labeled CRC tumors, both in primary-tumor and liver-metastasis models. M5A-IR800-SW at 75μg exhibited highly-specific tumor labeling in a primary-tumor orthotopic model with a median tumor-to-background ratio of 9.77 and in a liver-metastasis orthotopic model with a median tumor-to-background ratio of 7.23 at 96h. The precise labeling of the liver metastasis was due to lack of hepatic accumulation of M5A-IR800-SW in the liver. M5A-IR800-SW provided bright and targeted NIR images of human CRC in orthotopic primary-tumor and liver-metastasis mouse models. The results of the present study suggest the clinical potential of M5A-IR800-SW for fluorescence-guided surgery including metastasectomies for CRC. The lack of hepatic NIR signal is of critical importance to allow for precise labeling of liver tumors.

First-In-Human Pilot PET Immunoimaging Study of 64Cu-Anti-Carcinoembryonic Antigen Monoclonal Antibody (hT84.66-M5A) in Patients with Carcinoembryonic Antigen-Producing Cancers.

Background: PET imaging using radiolabeled immunoconstructs shows promise in cancer detection and in assessing tumor response to therapies. The authors report the first-in-human pilot study evaluating M5A, a humanized anti-carcinoembryonic antigen (CEA) monoclonal antibody (mAb), radiolabeled with 64Cu in patients with CEA-expressing malignancies. The purpose of this pilot study was to identify the preferred patient population for further evaluation of this agent in an expanded trial. Methods: Patients with CEA-expressing primary or metastatic cancer received 64Cu-DOTA-hT84.66-M5A with imaging performed at 1 and 2 days postinfusion. 64Cu-DOTA-hT84.66-M5A PET scan findings were correlated with CT, MRI, and/or FDG PET scans and with histopathologic findings from planned surgery or biopsy performed postscan. Results: Twenty patients received 64Cu-DOTA-hT84.66-M5A. Twelve patients demonstrated positive images, which were confirmed in 10 patients as tumor by standard-of-care (SOC) imaging, biopsy, or surgical findings. Four of the 8 patients with negative imaging were confirmed as true negative, with the remaining 4 patients having disease demonstrated by SOC imaging or surgery. All 5 patients with locally advanced rectal cancer underwent planned biopsy or surgery after 64Cu-DOTA-hT84.66-M5A imaging (4 patients imaged 6-8 weeks after completing neoadjuvant chemotherapy and radiation therapy) and demonstrated a high concordance between biopsy findings and 64Cu-DOTA-hT84.66-M5A PET scan results. Three patients demonstrated positive uptake at the primary site later confirmed by biopsy and at surgery as residual disease. Two patients with negative scans each demonstrated complete pathologic response. In 5 patients with medullary thyroid cancer, 64Cu-DOTA-hT84.66-M5A identified disease not seen on initial CT scans in 3 patients, later confirmed to be disease by subsequent surgery or MRI. Conclusions: 64Cu-DOTA-hT84.66-M5A demonstrates promise in tumor detection, particularly in patients with locally advanced rectal cancer and medullary thyroid cancer. A successor trial in locally advanced rectal cancer has been initiated to further evaluate this agent's ability to define tumor extent before and assess disease response after neoadjuvant chemotherapy and radiotherapy. clinical trial.gov (NCT02293954).

NIR fluorochrome labeled anti-carcinoembryonic antigen monoclonal antibody for gastric cancer-specific image guided surgery.

340 Background: Carcinoembryonic antigen (CEA) is a widely known tumor marker that is clearly expressed in gastrointestinal tract cancer. We utilized a CEA-specific chimeric antibody conjugated to a near infrared (NIR) fluorophore to facilitate CEA-targeted fluorescence image–guided surgery (FGS) of gastric cancer. The anti-CEA antibody, SGM-101 is conjugated with NIR dye BM-105, which has an absorbance band centered at 705 nm. Methods: RNA sequencing data of 34 gastric cancer cell lines from Cancer Cell Line Encyclopedia were screened and validated by qPCR and western blotting. Flow cytometry and confocal microscopy were performed by SGM-101, Alexa Fluor-680, Isotype-101 and Isotype-680 to quantify fluorescence intensity. SGM-101(n = 5) or Isotype-101(n = 2) was injected to mouse xenografts through a tail vein which had been subcutaneously implanted with MKN-45, SNU-16, and SNU-668. IVIS Spectrum quantified radiant efficiency of fluorescence in the region of interest at serial time points. The extracted tumor in peak time was analyzed by confocal imaging for microdistribution. In addition, 85As2mLuc were injected intraperitoneally in 6-week-old female BALB/c-nu mice for peritoneal carcinomatosis. Bioluminescence/fluorescence imaging was performed with IVIS Spectrum at peak time and analyzed via Living Image. Histologic evaluations were processed with H&E and Immunohistochemistry (IHC) data by a pathologist. Results: RNA expression of ceacam5 and protein expression of CEA in gastric cell lines was measured by RNA sequencing, qPCR, and western blotting. CEA expression patterns displays similar with fluorescence intensity patterns which were quantified through flow cytometry and immunocytochemistry show that CEA localized in membranes. In subcutaneously implanted model, the radiant efficiency of each group shows that the accumulation of SGM-101 has significantly higher fluorescence signal in the high CEA expressing group (MKN-45) and medium expressing group (SNU-16) while no fluorescence signal was observed in the CEA negative group (SNU-668) via IVIS Spectrum. Biodistribution of SGM-101 indicates that the maximum peak accumulation point was 48 hours after tail vein injection. Frozen tissue which was extracted at peak detection time shows micro-distribution of SGM-101 and expression of extracted tissue CEA expression was validated with IHC by pathological analysis. In the peritoneal carcinomatosis model, the imaging of fluorescence detection patterns corresponds with bioluminescence imaging and histological evaluation. Conclusions: CEA expression corresponded with intensity of in vitro fluorescence immunodetection and a tumor area accumulation in gastric cancer xenografts by SGM-101. This study indicates that NIR tumor specific imaging can be a feasible tool for image-guided surgery.

Physiologically Based Modeling of the Pharmacokinetics of “Catch-and-Release” Anti-Carcinoembryonic Antigen Monoclonal Antibodies in Colorectal Cancer Xenograft Mouse Models

Engineered monoclonal antibodies (mAbs) with pH-sensitive target release, or "catch-and-release" (CAR) binding, have shown promise in decreasing the extent of target-mediated mAb elimination, increasing mAb exposure, and increasing dose potency. This study developed a mechanistic physiologically based pharmacokinetic (PBPK) model to evaluate the effects of pH-sensitive CAR target binding on the disposition of anti-carcinoembryonic antigen (CEA) mAbs in mouse models of colorectal cancer. The PBPK model was qualified by comparing model-predicted plasma concentration-time data with data observed in tumor-bearing mice following the administration of T84.66, a "standard" anti-CEA mAb that demonstrates strong binding at pH 7.4 and 5.5. Further simulations evaluated the effects CAR pH-dependent binding, with decreasing CEA affinity with decreasing pH, on anti-CEA mAb plasma pharmacokinetics. Simulated data were compared with data observed for a novel CAR mAb, 10H6. The PBPK model provided precise parameter estimates, and excellent data characterization (median prediction error 18.4%) following fitting to T84.66 data. Simulations well predicted 10H6 data (median prediction error 21.4%). Sensitivity analyses demonstrated that key determinants of the disposition of CAR mAbs include the following: antigen binding affinity, the rate constant of mAb-CEA dissociation in acidified endosomes, antigen concentration, and the tumor vasculature reflection coefficient.

"Catch-and-Release" Anti-Carcinoembryonic Antigen Monoclonal Antibody Leads to Greater Plasma and Tumor Exposure in a Mouse Model of Colorectal Cancer.

In this study, we examined the effects of target expression, neonatal Fc receptor (FcRn) expression in tumors, and pH-dependent target binding on the disposition of monoclonal antibodies (mAbs) in murine models of colorectal cancer. A panel of anti-carcinoembryonic antigen (CEA) mAbs was developed via standard hybridoma technology and then evaluated for pH-dependent CEA binding. Binding was assessed via immunoassay and radioligand binding assays. 10H6, a murine IgG1 mAb with high affinity for CEA at pH = 7.4 (KD = 12.6 ± 1.7 nM) and reduced affinity at pH = 6.0 (KD = 144.6 ± 21.8 nM), and T84.66, which exhibits pH-independent CEA binding (KD = 1.1 ± 0.11 and 1.4 ± 0.16 nM at pH 7.4 and 6.0), were selected for pharmacokinetic investigations. We evaluated pharmacokinetics after intravenous administration to control mice and to mice bearing tumors with (MC38CEA+, LS174T) and without (MC38CEA-) CEA expression and with or without expression of murine FcRn, at doses of 0.1, 1, and 10 mg/kg. 10H6 displayed linear pharmacokinetics in mice bearing MC38CEA+ or MC38CEA- tumors. T84.66 displayed linear pharmacokinetics in mice with MC38CEA- tumors but dose-dependent nonlinear pharmacokinetics in mice bearing MC38CEA+ In addition to the improved plasma pharmacokinetic profile (i.e., linear pharmacokinetics, longer terminal half-life), 10H6 exhibited improved exposure in MC38CEA+ tumors relative to T84.66. In mice bearing tumors with CEA expression, but lacking expression of murine FcRn (LS174T), 10H6 demonstrated nonlinear pharmacokinetics, with rapid clearance at low dose. These data are consistent with the hypothesis that pH-dependent CEA binding allows mAb dissociation from target in acidified endosomes, enabling FcRn-mediated protection from target-mediated elimination in mice bearing MC38CEA+ tumors.

Detection of Micrometastases Using SPECT/Fluorescence Dual-Modality Imaging in a CEA-Expressing Tumor Model.

Intraoperative dual-modality imaging can help the surgeon distinguish tumor from normal tissue. This technique may prove particularly valuable if small tumors need to be removed that are difficult to detect with the naked eye. The humanized anticarcinoembryonic antigen (anti-CEA) monoclonal antibody, labetuzumab, can be used as a tumor-targeting agent in colorectal cancer, since CEA is overexpressed in approximately 95% of colorectal cancer. Dual-labeled labetuzumab, labeled with both a near-infrared fluorescent dye (IRDye800CW) and a radioactive label (111In), can be used as a tracer for dual-modality imaging. This study aimed to assess whether dual-modality imaging using 111In-diethylenetriaminepentaacetic acid (DTPA)-labetuzumab-IRDye800CW can detect pulmonary micrometastases in a mouse model. Methods: Pulmonary GW-39 human colonic carcinoma microcolonies were induced in athymic BALB/c mice by intravenous injection of 100 μL of a GW-39 cell suspension. After 1, 2, 3, and 4 wk of tumor growth, dual-modality imaging was performed 3 d after intravenous injection of 111In-DTPA-labetuzumab-IRDye800CW (10 μg, 25 MBq). Small-animal SPECT images and optical images were acquired, and image-guided surgery was performed. Finally, the biodistribution of the dual-labeled tracer was determined. Formalin-fixed sections of the lungs were analyzed using fluorescence imaging, autoradiography, and immunohistochemistry. Results: Submillimeter pulmonary tumor colonies could be visualized with both small-animal SPECT and fluorescence imaging from the first week of tumor growth, before they became visible to the naked eye. Furthermore, dual-modality imaging could be used to guide resection of tumors. Mean uptake (percentage injected dose per gram) of the dual-labeled tracer in tumors was 17.2 ± 5.4 and 16.5 ± 4.4 at weeks 3 and 4, respectively. Immunohistochemical analysis of the tumorous lungs showed that the distribution of the radioactive and fluorescent signal colocalized with CEA-expressing tumors. Conclusion: Dual-modality imaging after injection of 111In-labetuzumab-IRDye800CW can be used to detect submillimeter CEA-expressing pulmonary tumors before they become visible to the naked eye, supporting the added value of this technique in the resection of small tumors.

Sorafenib Decreases Tumor Exposure to an Anti-carcinoembryonic Antigen Monoclonal Antibody in a Mouse Model of Colorectal Cancer.

In this investigation, we test the hypothesis that treatment with sorafenib, an anti-angiogenic agent, decreases tumor vascularization and, consequently, hinders the delivery of monoclonal antibodies (mAb) to xenograft tumors. Severe combined immunodeficiency mice bearing carcinoembryonic antigen (CEA) expressing tumor xenografts were divided into control and sorafenib-treated groups. Sorafenib was administered to the latter group at 50mg/kg IP every 48h, starting 4days post-tumor implantation. When tumors attained a size of 200-300mm(3), mice were evaluated for (a) tumor microvessel density (using immunohistochemical analysis), (b) tumor macromolecular extravasation (using Evans Blue Dye (EBD)), (c) pharmacokinetics of an anti-CEA mAb, T84.66, following an intravenous dose of 10mg/kg, and (d) intra-tumoral spatial distribution of T84.66 (using autoradiography). Sorafenib treatment resulted in a substantial reduction in tumor growth rate, a visible reduction in tumor microvessel density, and in a 46.4% decrease in EBD extravasation in tumor tissue (p < 0.0455). For control and treated mice, no significant difference was found for the area under the mAb plasma concentration-time curve (AUC(0-7d): 1.67 × 10(3) ± 1.28 × 10(2) vs. 1.76 × 10(3) ± 1.75 × 10(2) nM × day, p = 0.51). However, tumor AUC(0-7d) was reduced by 40.8% in sorafenib-treated mice relative to that observed in control mice (5.61 × 10(2) ± 4.27 × 10(1) vs. 9.48 × 10(2) ± 5.61 × 10(1) nM × day, p < 0.001). Sorafenib therapy was also found to markedly alter mAb tumor spatial distribution. The results collectively suggest that sorafenib treatment causes a significant reduction in mAb delivery to, and distribution within, solid tumors.

Combination of antibody targeting and PTD-mediated intracellular toxin delivery for colorectal cancer therapy.

The bottlenecks of current chemotherapy in the treatment of colorectal cancer lie in the ineffectiveness of the existing anti-cancer small molecule drugs as well as the dose-limiting toxicity caused by the nonselective action on normal tissues by such drugs. To address these problems, we introduce a novel therapeutic strategy based on tumor targeting using a non-internalizing anti-carcinoembryonic antigen (CEA) monoclonal antibody (mAb) and intracellular delivery of the extremely potent yet cell-impermeable protein toxin gelonin via the aid of a cell-penetrating peptide (also termed as protein transduction domain; PTD). A chimeric TAT-gelonin fusion protein was genetically engineered, and it displayed remarkably enhanced anti-cancer activity against human colorectal cancer cells, with IC50 values being several orders of magnitude lower than the unmodified gelonin. On the other hand, a chemically synthesized conjugate of heparin and a murine anti-CEA mAb, T84.66 (termed T84.66-Hep) was found able to bind highly specifically to CEA over-expressing LS174T colorectal cancer cells. When mixing together, TAT-gelonin and T84.66-Hep could associate tightly and automatically through an electrostatic interaction between the cationic TAT and anionic heparin. In preliminary in vivo studies using LS174T s.c. xenograft tumor bearing mouse, selective and significantly augmented (58-fold) delivery of TAT-gelonin to the tumor target was observed, when compared with administration of TAT-gelonin alone. More importantly, efficacy studies also revealed that only the TAT-gelonin/T84.66-Hep complex yielded a significant inhibition of tumor growth (46%) without causing gelonin-induced systemic toxicity. Overall, this study suggested a generic strategy to effectively yet safely deliver potent PTD-modified protein toxins to the tumor.

Successful Fluorescence-Guided Surgery on Human Colon Cancer Patient-Derived Orthotopic Xenograft Mouse Models Using a Fluorophore-Conjugated Anti-CEA Antibody and a Portable Imaging System

Fluorescence-guided surgery (FGS) can enable successful cancer surgery where bright-light surgery often cannot. There are three important issues for FGS going forward toward the clinic: (a) proper tumor labeling, (b) a simple portable imaging system for the operating room, and (c) patient-like mouse models in which to develop the technology. The present report addresses all three. Patient colon tumors were initially established subcutaneously in nonobese diabetic (NOD)/severe combined immune deficiency (SCID) mice immediately after surgery. The tumors were then harvested from NOD/SCID mice and passed orthotopically in nude mice to make patient-derived orthotopic xenograft (PDOX) models. Eight weeks after orthotopic implantation, a monoclonal anti-carcinoembryonic antigen (CEA) antibody conjugated with AlexaFluor 488 (Molecular Probes Inc., Eugene, OR) was delivered to the PDOX models as a single intravenous dose 24 hours before laparotomy. A hand-held portable fluorescence imaging device was used. The primary tumor was clearly visible at laparotomy with the 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. The tumor was completely resected under fluorescence navigation. Histologic evaluation of the resected specimen demonstrated that cancer cells were not present in the margins, indicating successful tumor resection. The FGS animals remained tumor free for over 6 months. The results of the present report indicate that FGS using a fluorophore-conjugated anti-CEA antibody and portable imaging system improves efficacy of resection for CEA-positive colorectal cancer. These data provide the basis for clinical trials.

Cytotoxicity of recombinant immunotoxin containing lectin A chain from Korean mistletoe

Toxin moiety in immunotoxin (IT) plays an important role in cytotoxicity of target cells. We constructed a recombinant immunotoxin (RIT), which is composed of a recombinant lectin A chain from Korean mistletoe (AKM) and a single-chain Fv (scFv) of an anti-carcinoembryonic antigen (CEA) monoclonal antibody. This single-chain IT was expressed in E. coli, purified, and refolded as a soluble protein. The resulting recombinant protein displayed strong cytotoxicity against CEA-expressing cancer cells but only minimal cytotoxicity against non CEA-expressing cells. The present study, we suggest that AKM may be a potential toxin candidate for development of an IT.

Flt3 ligand enhances anti-tumor effects of antibody therapeutics

Fms-like tyrosine kinase 3 ligand ([Flt3 ligand], FL) stimulates proliferation and development of a wide range of hematopoietic cells including hematopoietic stem cells and myeloid and lymphoid progenitor cells. FL also has been shown to have anti-tumor effects in a variety of in vivo tumor models. In this study, the effect of FL against tumor growth was investigated in the COLO-205 human colon tumor xenograft model. FL was delivered in vivo by the "hydrodynamics-based gene delivery of naked DNA" method. In this experimental setting, FL and/or the therapeutic antibody anti-carcinoembryonic antigen (CEA) monoclonal antibody was administered. FL alone or anti-CEA antibody alone induced significant growth inhibition; furthermore, FL plus antibody treatment produced synergistic anti-tumor effects. This study is the first demonstration of a synergistic anti-tumor effect between FL and antibody therapeutics.

Pretargeted immuno-PET of CEA-expressing intraperitoneal human colonic tumor xenografts: a new sensitive detection method

BackgroundIn this study, pretargeted immuno-positron-emission tomography [PET] with a bispecific monoclonal anti-carcinoembryonic antigen [CEA] (CEACAM5) × anti-hapten antibody (bispecific monoclonal antibody [bsmAb]) and a small (1.5 kD) peptide labeled with 68Ga was compared to fludeoxyglucose [18F-FDG]-PET for detecting intraperitoneal [i.p.] CEA-expressing human colonic tumor xenografts in nude mice.MethodsTwo groups of female BALB/c nude mice were inoculated with LS174T human colonic tumor cells i.p. One group received 5 MBq 18F-FDG, and the other received intravenous injections of the bsmAb, followed 16 h later with 5 MBq of 68Ga-labeled peptide. One hour after the radiolabeled peptide or FDG was given, micro-PET/computed tomography images were acquired. Thereafter, the uptake of the 68Ga or 18F in dissected tissue was determined.ResultsWithin 1 h, high uptake of the 68Ga-labeled peptide in the tumor lesions (23.4 ± 7.2% ID/g) and low background activity levels were observed (e.g., tumor-to-intestine ratio, 58 ± 22). This resulted in a clear visualization of all intra-abdominal tumor lesions ≥ 10 μL and even some tumors as small as 5 μL (2 mm diameter). 18F-FDG efficiently localized in the tumors (8.7 ± 3.1% ID/g) but also showed physiological uptake in various normal tissues (e.g., tumor-to-intestine ratio, 3.9 ± 1.1).ConclusionsPretargeted immuno-PET with bsmAb and a 68Ga-labeled peptide could be a very sensitive imaging method for imaging colonic cancer, disclosing occult lesions.

Abstract 5282: Pretargeted immunoPET for imaging human colonic cancer in a mouse model

Abstract Objective: Molecular imaging with pretargeting bispecific antibodies allows rapid and specific targeting of tumors for PET imaging. In this study, pretargeted immunoPET with a bispecific monoclonal anti-carcinoembryonic antigen (CEA; CEACAM5) × anti-hapten antibody (bsMAb) and a small (1.5 kD) peptide labeled with a short-lived positron-emitter 68Ga was compared to FDG for detection of intraperitoneal CEA-expressing human colonic tumor xenografts in nude mice. Methods: In BALB/c nude mice LS174T human colonic tumor cells were injected intraperitoneally. One group (n=5) received 5 MBq FDG i.v., and another group (n=5) received the bsMAb i.v. and 5 MBq of 68Ga-labeled peptide i.v. 16 h later. One hour after the injection of the peptide or FDG, PET/CT images were acquired. One day later the same procedure was repeated vice versa, and after microPET imaging the uptake of the 68Ga or 18F in dissected tissue was determined. Results: Within one hour, specific tumor targeting of 68Ga-labeled peptide (23.4 ± 7.2% ID/g) combined with the low background activity levels resulted in the clear visualization of the small intra-abdominal tumor lesions (1.5-5 mm). High tumor/background radioactivity concentration ratios were obtained (e.g. tumor/intestines 57.5 ± 22.4). FDG localized efficiently in the tumors (8.7 ± 3.1 % ID/g), but with physiological uptake in various normal tissues (ratio tumor/intestines 4.0 ± 0.9). Conclusion: Pretargeted immunoPET performs exceptionally well with short-lived radionuclides, and is a highly sensitive procedure for dectection of small tumor deposits that is also more specific than FDG-PET. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5282. doi:10.1158/1538-7445.AM2011-5282

Synthesis of Fluorescent Silica Nanoparticles and Their Applications as Fluorescence Probes

This paper presents the synthesis of organic dye molecules embedded silica nanoparticles by Stöber method and their applications as fluorescence probes in cell imaging. By modifying the surface of fluorescent silica nanoparticles (FSNs) with amino, biologically functionalized and monodisperse FSNs can be obtained. In this work, FSNs were conjugated with monoclonal anti-Carcinoembryonic Antigen (anti-CEA) antibody via covalent binding. The antibody-conjugated FSNs can be used to label the SPCA-1 cells successfully, demonstrating that the application of FSNs as fluorescence probes in fluorescence imaging and bioassay would be feasible.

Abstract A32: Pretargeted immunoPET of CEA-expressing in intraperitoneal human colonic tumor xenografts in nude mice

Abstract Objective: Radiolabeled antibodies were being developed commercially for the detection of several cancer types. However, imaging with radiolabeled antibodies require a relatively long interval between injection and imaging acquisition for adequate contrast to develop. The pretargeting method is a 2-step method: a unlabeled bsMAb with affinity for the tumor and a small radiolabeled molecule is injected, followed by the a radiolabeled compound. This aims to optimize the imaging system by specific tumor accumulation and fast blood clearance of the radiopharmacon, which allows imaging within one hour. Short-lived positron emitting radionuclides, e.g. 68Ga (half life: 68 min) match the pharmacokinetics of the small molecule. In this study, pretargeted immunoPET with a bispecific monoclonal anti-carcinoembryonic antigen (CEA) x anti-histamine-succinyl-glycine (HSG) antibody, TF2, and a radiolabeled peptide, IMP288, was compared to 18F-FDG in detecting CEA-expressing tumors in nude mice. Methods: Groups of five BALB/c nude mice were inoculated with CEA-expressing human colonic tumor cells intraperitoneally. One group received 5 MBq 18F-FDG intravenously (i.v.). Another group received TF2 and 5 MBq 68Ga-IMP288 i.v. 16 h later. After one hour PET/CT images were acquired. One day later this was repeated vice versa. Uptake of 68Ga-IMP288 or 18F-FDG in the tumors and other dissected tissues was determined. Results: High, specific tumor uptake of 68Ga-IMP288 (23.4 ± 7.2% ID/g) was observed, with very low accretion in the normal tissues (intestines: 0.50 ± 0.19 % ID/g; liver: 1.83 ± 0.45 % ID/g). This resulted in high tumor-to-background ratios (e.g. tumor-to-intestines 57.5 ± 22.4) and in the clear visualization of the small intra-abdominal tumor lesions (1.5–5 mm), as soon as one hour after the injection of 68Ga-IMP288. 18F-FDG localized efficiently in the tumors (8.7 ± 3.1 % ID/g), but with physiological uptake in various normal tissues (intestines: 2.15 ± 0.61 % ID/g; liver: 2.78 ± 0.52 % ID/g). The low tumor-to-background ratio (e.g. tumor-to-intestines 4.0 ± 0.9) complicated the detection of the tumors. Conclusions: This study indicated that pretargeted immunoPET with TF2 and 68Ga-IMP288 is a specific and sensitive method for detecting colon cancer. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A32

Synthesis of magnetic and fluorescent bifunctional nanocomposites and their applications in detection of lung cancer cells in humans

We developed a novel strategy to detect lung cancer cells by utilizing magnetic and fluorescent bifunctional nanocomposites (BNPs) in combination with monoclonal anti-carcinoembryonic antigen (CEA) antibodies. The BNPs, consisting of silica-coated superparamagnetic nanoparticles and quantum dots (QDs), exhibited high luminescence and were easily separated in an external magnetic field. The binding specificity of the antibody-conjugated BNPs (immunonanoparticles) were confirmed via incubating with human lung adenocarcinoma SPCA-1 cells, human leukemic K562 cells and human embryonic lung fibroblasts MRC-5 cells. Further experiments demonstrated that the as-prepared immunonanoparticles can efficiently capture and detect cancer cells in pleural effusion from lung cancer patients. These results suggest that this method, of which the detection procedures are completed within 1h, could be applied to the rapid and cost-effective monitoring of cancer cells in clinical samples.

Toxicity and efficacy of combined radioimmunotherapy and bevacizumab in a mouse model of medullary thyroid carcinoma

Significant antitumor effects were previously observed with radioimmunotherapy (RIT) using an anti-carcinoembryonic antigen (CEA) monoclonal antibody (F6) labeled with iodine-131 in medullary thyroid cancer (MTC)-bearing nude mice. Nevertheless, no complete response was achieved. Because angiogenesis is critical for tumor growth, bevacizumab is used to treat solid tumor in clinical practice. The present pilot study evaluated toxicity and efficacy of RIT combined with bevacizumab in mice subcutaneously grafted with TT MTC cells. Groups of 4-6 nude mice were treated with 5 microg/g bevacizumab twice weekly during 4 weeks and/or 100 MBq of (131)I-F6. For combined therapy, bevacizumab was given at Day 0 followed by (131)I-F6 at Day 30. The control group received no treatment. Animal weight, hematological toxicity, tumor volume, and serum calcitonin were monitored for 2 or 4 months. Bevacizumab alone induced no cytopenia and no significant weight loss. A weight loss of 12 +/- 1% and 15 +/- 2% was observed in mice treated by RIT alone or bevacizumab + RIT, respectively. RIT alone and combined treatment induced leukopenia and anemia. RIT alone and RIT plus bevacizumab induced tumor responses with minimum relative tumor volume of 0.38 +/- 0.24 and 0.15 +/- 0.07%, respectively, and time to progression of 35 +/- 5 and 56 +/- 11 days, respectively. Pretreatment with bevacizumab improved RIT efficacy, with similar toxicity as compared as RIT alone.

Radioimmunotherapy of Human Colon Cancer Xenografts with 131I-Labeled Anti-CEA Monoclonal Antibody

Radioimmunotherapy (RIT) is a promising approach for the treatment of a wide variety of malignancies. The aim of this study was to investigate the therapeutic efficacy of (131)I-labeled anticarcinoembryonic antigen (CEA) monoclonal antibody CL58 in a human colon cancer mouse model. In vitro and in vivo characteristics of (125)I-CL58 were evaluated in LS180 human colon cancer cells and the nude mouse model. (131)I-CL58 was prepared and its in vivo therapeutic efficacy was tested. (125)I-CL58 showed high affinity to LS180 cells, as well as high tumor uptake and long tumor retention in LS180 tumor xenografts. (131)I-CL58 exhibited dose-dependent inhibition of LS180 tumor growth. With the excellent in vitro and in vivo characteristics, and the effective therapy for colon cancer in animal model, (131)I-CL58 is a promising agent for RIT of CEA-positive tumors including colon cancer.

Animal biodistribution and pharmacokinetics study of ~(131)I-labelled rch24

Objective To evaluate biodistribution and pharmacokinetics pattern of ~(131)I-labeled rch24which is the region-grafted (humanized) anti-carcinoembryonic antigen (CEA) monoclonal antibody in nude mice. Methods Nude mice bearing cancer xenografts received intravenous injections of ~(131)I- rch24, then blood, plasma, heart, liver, spleen, lung, kidney, tumor and other tissues were taken at different time point for determination the concentration of radioactivity and calculate the T/NT value. Nude mice were packeted randomly to four group of high, medium, low dose and continuous administration, blood drug concentration was detected by ELISA method at the different intervals. Then, draw the concentration-time curve and calculate the pharmacokinetics paramete. Results After administration, radioactivity of the tumour was significantly enhanced whereas radioactivity of normal tissues decreased gradually. For single administration, at the dose of low to medium, pharmacokinetics pattern was linearity -kinetics whereas for high dose group,pharmacokinetics paramete shown some behavior of non-linearity-kinetics. Conclusion Our results suggest that the ~(131)I-labeled region-grafted (humanized) anti-CEA monoclonal antibody rch24 exhibit a considerable targeting activity so as to ~(131)I radioisotopes can be concentrated specifically in tumor. The pharmacokinetics pattern of this medicine was different at different dose. Key words: Carcinoembryonic antigen; Antibodies; monoclonal; ~(131)I; Tissue distribution; Pharmacokinetics