ABSTRACT
Carcinoembryonic Antigen (CEA), an acidic glycoprotein with human embryonic antigen properties, is found on the surface of cancer cells that have differentiated from endodermal cells. This paper presents a label-free electrochemical immunoassay for the dual amplification detection of CEA using gold nanoparticles loaded with polypyrrole polydopamine (Au/PPy-PDA) and polymerized polycaprolactone (Ng-PCL) prepared by ring-opening polymerization (ROP). First, the composite Au/PPy-PDA was adhered to the electrode surface. Then, gold nanoparticles form a Au-S bond with the sulfhydryl group in Apt1 to secure it on the electrode surface. Subsequently, the non-specific binding sites on the electrodes surface are closed by bovine serum albumin (BSA). Next, CEA is dropped onto the electrode surface, which is immobilized by antigen-antibody specific recognition, and the carboxyl-functionalized Apt2 forms a "sandwich structure" of antibody-antigen-antibody by specific recognition. Polymeric Ng-PCL is adhered to the electrode surface, leading to an increase in the electrochemical impedance signal, resulting in a complete chain of signal analysis. Finally, the response signal is detected by electrochemical impedance spectroscopy (EIS). Under optimal experimental conditions, the method has the advantages of high sensitivity and wide linear range (1 pg mL-1â¼100 ng mL-1), and the lower limit of detection (LOD) is 0.234 pg mL-1. And it has the same high sensitivity, selectivity and interference resistance for the real samples detection. Thus, it provides a new way of thinking about biomedical and clinical diagnosis.
Subject(s)
Biosensing Techniques , Carcinoembryonic Antigen , Electrochemical Techniques , Gold , Metal Nanoparticles , Polyesters , Polymers , Gold/chemistry , Biosensing Techniques/methods , Metal Nanoparticles/chemistry , Carcinoembryonic Antigen/analysis , Carcinoembryonic Antigen/immunology , Polyesters/chemistry , Electrochemical Techniques/methods , Polymers/chemistry , Humans , Indoles/chemistry , Immunoassay/methods , Limit of Detection , Electrodes , Pyrroles/chemistry , Serum Albumin, Bovine/chemistryABSTRACT
Nanosized sodium bismuth perovskite titanate (NBT) was synthesized and first used as the electrochemical immune sensing platform for the sensitive detection of carcinoembryonic antigen (CEA). Gold nanoparticles (Au NPs) grew on the surface of NBT through forming Au-N bond to obtain Au@NBT, and a label-free electrochemical immunosensor was proposed using Au@NBT as an immunosensing recognizer towards CEA. The well-ordered crystal structure of NBT was not changed at all after the modification of Au NPs outside, but significantly improved the conductivity, catalytic activity, and biocompatibility of the Au@NBT-modified electrode. The unique cubic crystal nanostructure of NBT offered a large active area for both Au NP modification and the subsequent immobilization of biomolecules over the electrode surface, triggering the effective generation of promising properties of the proposed Au@NBT-based electrochemical immunosensor. As expected, favorable detection performances were achieved using this immunosensor towards CEA detection, where a good linear relationship between the current response and CEA concentration was obtained in the concentration range 10 fg mL-1 to 100 ng mL-1 with a low detection limit (LOD) of 13.17 fg mL-1. Also, the significantly enhanced selectivity, and stability guaranteed the promising electrochemical properties of this immunosensor. Furthermore, the analysis of real serum samples verified the high feasibility of this new method in clinical CEA detection. This work opens a new window for the application of nanoperovskite in the early detection of CEA.
Subject(s)
Bismuth , Carcinoembryonic Antigen , Electrochemical Techniques , Gold , Limit of Detection , Metal Nanoparticles , Titanium , Carcinoembryonic Antigen/blood , Carcinoembryonic Antigen/immunology , Titanium/chemistry , Electrochemical Techniques/methods , Humans , Immunoassay/methods , Gold/chemistry , Metal Nanoparticles/chemistry , Bismuth/chemistry , Biosensing Techniques/methods , Oxides/chemistry , Antibodies, Immobilized/immunology , Calcium Compounds/chemistry , ElectrodesABSTRACT
HER2 amplification occurs in approximately 5% of colorectal cancer (CRC) cases and is associated only partially with clinical response to combined human epidermal growth factor receptor 2 (HER2)/epidermal growth factor receptor (EGFR)-targeted treatment. An alternative approach based on adoptive cell therapy using T cells engineered with anti-HER2 chimeric antigen receptor (CAR) proved to be toxic due to on-target/off-tumor activity. Here we describe a combinatorial strategy to safely target HER2 amplification and carcinoembryonic antigen (CEA) expression in CRC using a synNotch-CAR-based artificial regulatory network. The natural killer (NK) cell line NK-92 was engineered with an anti-HER2 synNotch receptor driving the expression of a CAR against CEA only when engaged. After being transduced and sorted for HER2-driven CAR expression, cells were cloned. The clone with optimal performances in terms of specificity and amplitude of CAR induction demonstrated significant activity in vitro and in vivo specifically against HER2-amplified (HER2amp)/CEA+ CRC models, with no effects on cells with physiological HER2 levels. The HER2-synNotch/CEA-CAR-NK system provides an innovative, scalable, and safe off-the-shelf cell therapy approach with potential against HER2amp CRC resistant or partially responsive to HER2/EGFR blockade.
Subject(s)
Colorectal Neoplasms , Receptor, ErbB-2 , Receptors, Chimeric Antigen , Xenograft Model Antitumor Assays , Colorectal Neoplasms/therapy , Colorectal Neoplasms/genetics , Colorectal Neoplasms/immunology , Humans , Receptor, ErbB-2/metabolism , Receptor, ErbB-2/genetics , Animals , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/metabolism , Mice , Cell Line, Tumor , Carcinoembryonic Antigen/immunology , Carcinoembryonic Antigen/genetics , Gene Amplification , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Immunotherapy/methods , Immunotherapy, Adoptive/methods , Disease Models, Animal , FemaleABSTRACT
BACKGROUND: Gastric cancer poses a major diagnostic and therapeutic challenge as surgical resection provides the only opportunity for a cure. Specific labeling of gastric cancer could distinguish resectable and nonresectable disease and facilitate an R0 resection, which could improve survival. METHODS: Two patient-derived gastric cancer lines, KG8 and KG10, were established from surgical specimens of two patients who underwent gastrectomy for gastric adenocarcinoma. Harvested tumor fragments were implanted into the greater curvature of the stomach to establish patient-derived orthotopic xenograft (PDOX) models. M5A (humanized anti-CEA antibody) or IgG control antibodies were conjugated with the near-infrared dye IRDye800CW. Mice received 50 µg of M5A-IR800 or 50 µg of IgG-IR800 intravenously and were imaged after 72 hr. Fluorescence imaging was performed by using the LI-COR Pearl Imaging System. A tumor-to-background ratio (TBR) was calculated by dividing the mean fluorescence intensity of the tumor versus adjacent stomach tissue. RESULTS: M5A-IR800 administration resulted in bright labeling of both KG8 and K10 tumors. In the KG8 PDOX models, the TBR for M5A-IR800 was 5.85 (SE ± 1.64) compared with IgG-IR800 at 0.70 (SE ± 0.17). The K10 PDOX models had a TBR of 3.71 (SE ± 0.73) for M5A-IR800 compared with 0.66 (SE ± 0.12) for IgG-IR800. CONCLUSIONS: Humanized anti-CEA (M5A) antibodies conjugated to fluorescent dyes provide bright and specific labeling of gastric cancer PDOX models. This tumor-specific fluorescent antibody is a promising potential clinical tool to detect the extent of disease for the determination of resectability as well as to visualize tumor margins during gastric cancer resection.
Subject(s)
Adenocarcinoma , Antibodies, Monoclonal, Humanized , Carcinoembryonic Antigen , Fluorescent Dyes , Stomach Neoplasms , Xenograft Model Antitumor Assays , Stomach Neoplasms/surgery , Stomach Neoplasms/pathology , Stomach Neoplasms/immunology , Stomach Neoplasms/diagnostic imaging , Animals , Humans , Mice , Carcinoembryonic Antigen/immunology , Adenocarcinoma/surgery , Adenocarcinoma/pathology , Adenocarcinoma/immunology , Adenocarcinoma/diagnostic imaging , Tumor Cells, Cultured , Female , Indoles , Optical Imaging/methods , Gastrectomy , Mice, Nude , Cell Line, TumorABSTRACT
Background: Blocking the CD47 "don't eat me"-signal on tumor cells with monoclonal antibodies or fusion proteins has shown limited clinical activity in hematologic malignancies and solid tumors thus far. Main side effects are associated with non-tumor targeted binding to CD47 particularly on blood cells. Methods: We present here the generation and preclinical development of NILK-2401, a CEACAM5×CD47 bispecific antibody (BsAb) composed of a common heavy chain and two different light chains, one kappa and one lambda, determining specificity (so-called κλ body format). Results: NILK-2401 is a fully human BsAb binding the CEACAM5 N-terminal domain on tumor cells by its lambda light chain arm with an affinity of ≈4 nM and CD47 with its kappa chain arm with an intendedly low affinity of ≈500 nM to enabling tumor-specific blockade of the CD47-SIRPα interaction. For increased activity, NILK-2401 features a functional IgG1 Fc-part. NILK-2401 eliminates CEACAM5-positive tumor cell lines (3/3 colorectal, 2/2 gastric, 2/2 lung) with EC50 for antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity ranging from 0.38 to 25.84 nM and 0.04 to 0.25 nM, respectively. NILK-2401 binds neither CD47-positive/CEACAM5-negative cell lines nor primary epithelial cells. No erythrophagocytosis or platelet activation is observed. Quantification of the pre-existing NILK-2401-reactive T-cell repertoire in the blood of 14 healthy donors with diverse HLA molecules shows a low immunogenic potential. In vivo, NILK-2401 significantly delayed tumor growth in a NOD-SCID colon cancer model and a syngeneic mouse model using human CD47/human SIRPα transgenic mice and prolonged survival. In cynomolgus monkeys, single doses of 0.5 and 20 mg/kg were well tolerated; PK linked to anti-CD47 and Fc-binding seemed to be more than dose-proportional for Cmax and AUC0-inf. Data were validated in human FcRn TG32 mice. Combination of a CEACAM5-targeting T-cell engager (NILK-2301) with NILK-2401 can either boost NILK-2301 activity (Emax) up to 2.5-fold or allows reaching equal NILK-2301 activity at >600-fold (LS174T) to >3,000-fold (MKN-45) lower doses. Conclusion: NILK-2401 combines promising preclinical activity with limited potential side effects due to the tumor-targeted blockade of CD47 and low immunogenicity and is planned to enter clinical testing.
Subject(s)
Antibodies, Bispecific , CD47 Antigen , Carcinoembryonic Antigen , Animals , Female , Humans , Mice , Antibodies, Bispecific/pharmacology , Antibodies, Bispecific/therapeutic use , Antineoplastic Agents, Immunological/pharmacology , Antineoplastic Agents, Immunological/therapeutic use , Carcinoembryonic Antigen/immunology , CD47 Antigen/immunology , CD47 Antigen/antagonists & inhibitors , Cell Line, Tumor , GPI-Linked Proteins , Macaca fascicularis , Neoplasms/drug therapy , Xenograft Model Antitumor AssaysABSTRACT
Globally, colorectal cancer (CRC) ranks as the third most common cancer and the second leading cause of cancer-related fatalities. According to the World Health Organization, there are over 1.9 million annual cases of CRC diagnosed worldwide, resulting in more than 900 000 deaths. In recent years, chimeric antigen receptor T (CAR-T) cell therapy has shown clinical success in treating certain hematological malignancies and is now being explored for its potential in targeting solid tumors like CRC. Currently, CAR-T cell therapies targeting carcinoembryonic antigen (CEA), natural killer group 2, member D ligand (NKG2DL), and other markers have achieved remarkable results in clinical trials, albeit encountering significant challenges. This review summarizes the promising targets of CAR-T cell therapy for CRC and highlights progress made in clinical trials and preclinical studies. Additionally, the review discusses the challenges faced by CAR-T cell therapy in CRC treatment, including a shortage of tumor-specific antigens, cytokine release syndrome, adverse tumor microenvironment, and limited infiltration of CAR-T cells. In summary, this review provides an overview of the latest research progress and challenges in CAR-T cell therapy for CRC, aiming to contribute fresh insights for the clinical treatment of this disease.
Subject(s)
Colorectal Neoplasms , Immunotherapy, Adoptive , Receptors, Chimeric Antigen , Humans , Colorectal Neoplasms/therapy , Colorectal Neoplasms/immunology , Receptors, Chimeric Antigen/immunology , Carcinoembryonic Antigen/immunology , Tumor Microenvironment , Antigens, Neoplasm/immunology , T-Lymphocytes/immunology , Receptors, Antigen, T-Cell/immunology , AnimalsABSTRACT
Simultaneous sensitive and precise determination of multibiomarkers is of great significance for improving detection efficiency, reducing diagnosis and treatment expenses, and elevating survival rates. However, the development of simple and portable biosensors for simultaneous determination of multiplexed targets in biological fluids still faces challenges. Herein, a unique and versatile immobilization-free dual-target electrochemical biosensing platform, which combines distinguishable magnetic signal reporters with buoyancy-magnetism separation, was designed and constructed for simultaneous detection of carcinoembryonic (CEA) and α-fetoprotein (AFP) in intricate biological fluids. To construct such distinguishable magnetic signal reporters with signal transduction, amplification, and output, secondary antibodies of CEA and AFP were respectively functionalized on methylene blue (MB) and 6-(ferrocenyl)hexanethiol (FeC) modified Fe3O4@Au magnetic nanocomposites. Meanwhile, a multifunctional flotation probe with dual target recognition, capture, and isolation capability was prepared by conjugating primary antibodies (Ab1-CEA, Ab1-AFP) to hollow buoyant microspheres. The target antigens of CEA and AFP can trigger a flotation-mediated sandwich-type immunoreaction and capture a certain amount of the distinguishable magnetic signal reporter, which enables the conversion of the target CEA and AFP quantities to the signal of the potential-resolved MB and FeC. Thus, the MB and FeC currents of magnetically adsorbed distinguishable magnetic reporters can be used to determine the CEA and AFP targets simultaneously and precisely. Accordingly, the proposed strategy exhibited a delightful linear response for CEA and AFP in the range of 100 fg·mL-1-100 ng·mL-1 with detection limits of 33.34 and 17.02 fg·mL-1 (S/N = 3), respectively. Meanwhile, no significant nonspecific adsorption and cross-talk were observed. The biosensing platform has shown satisfactory performance in the determination of real clinical samples. More importantly, the proposed approach can be conveniently extended to universal detection just by simply substituting biorecognition events. Thus, this work opens up a new promising perspective for dual and even multiple targets and offers promising potential applications in clinical diagnosis.
Subject(s)
Biosensing Techniques , Carcinoembryonic Antigen , Electrochemical Techniques , alpha-Fetoproteins , alpha-Fetoproteins/analysis , alpha-Fetoproteins/immunology , Carcinoembryonic Antigen/analysis , Carcinoembryonic Antigen/immunology , Biosensing Techniques/methods , Humans , Immunoassay/methods , Gold/chemistry , Limit of DetectionABSTRACT
Cibisatamab is a bispecific antibody-based construct targeting carcinoembryonic antigen (CEA) on tumour cells and CD3 epsilon chain as a T-cell engager. Here we evaluated cibisatamab for advanced CEA-positive solid tumours in two open-label Phase 1 dose-escalation and -expansion studies: as a single agent with or without obinutuzumab in S1 (NCT02324257) and with atezolizumab in S2 (NCT02650713). Primary endpoints were safety, dose finding, and pharmacokinetics in S1; safety and dose finding in S2. Secondary endpoints were anti-tumour activity (including overall response rate, ORR) and pharmacodynamics in S1; anti-tumour activity, pharmacodynamics and pharmacokinetics in S2. S1 and S2 enrolled a total of 149 and 228 patients, respectively. Grade ≥3 cibisatamab-related adverse events occurred in 36% of S1 and 49% of S2 patients. The ORR was 4% in S1 and 7% in S2. In S2, patients with microsatellite stable colorectal carcinoma (MSS-CRC) given flat doses of cibisatamab and atezolizumab demonstrated an ORR of 14%. In S1 and S2, 40% and 52% of patients, respectively, developed persistent anti-drug antibodies (ADAs). ADA appearance could be mitigated by obinutuzumab-pretreatment, with 8% of patients having persistent ADAs. Overall, cibisatamab warrants further exploration in immunotherapy combination strategies for MSS-CRC.
Subject(s)
Antibodies, Bispecific , Antibodies, Monoclonal, Humanized , CD3 Complex , Carcinoembryonic Antigen , Neoplasms , Humans , Antibodies, Bispecific/therapeutic use , Antibodies, Bispecific/pharmacokinetics , Antibodies, Bispecific/administration & dosage , Antibodies, Bispecific/adverse effects , Antibodies, Bispecific/pharmacology , Antibodies, Monoclonal, Humanized/therapeutic use , Antibodies, Monoclonal, Humanized/pharmacokinetics , Antibodies, Monoclonal, Humanized/administration & dosage , Antibodies, Monoclonal, Humanized/adverse effects , Female , Male , Middle Aged , Aged , CD3 Complex/immunology , Adult , Carcinoembryonic Antigen/immunology , Neoplasms/drug therapy , Neoplasms/immunology , Aged, 80 and over , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/pharmacokineticsABSTRACT
In the last decade, there has been a surge in developing immunotherapies to enhance the immune system's ability to eliminate tumor cells. Bispecific antibodies known as T cell engagers (TCEs) present an attractive strategy in this pursuit. TCEs aim to guide cytotoxic T cells toward tumor cells, thereby inducing a strong activation and subsequent tumor cell lysis. In this study, we investigated the activity of different TCEs on both conventional alpha-beta (αß) T cells and unconventional gamma delta (γδ) T cells. TCEs were built using camelid single-domain antibodies (VHHs) targeting the tumor-associated antigen CEACAM5 (CEA), together with T cell receptor chains or a CD3 domain. We show that Vγ9Vδ2 T cells display stronger in vitro antitumor activity than αß T cells when stimulated with a CD3xCEA TCE. Furthermore, restricting the activation of fresh human peripheral T cells to Vγ9Vδ2 T cells limited the production of protumor factors and proinflammatory cytokines, commonly associated with toxicity in patients. Taken together, our findings provide further insights that γδ T cell-specific TCEs hold promise as specific, effective, and potentially safe molecules to improve antitumor immunotherapies.
Subject(s)
Antibodies, Bispecific , Lymphocyte Activation , Receptors, Antigen, T-Cell, gamma-delta , Humans , Animals , Receptors, Antigen, T-Cell, gamma-delta/immunology , Antibodies, Bispecific/immunology , Lymphocyte Activation/immunology , Immunotherapy/methods , Neoplasms/immunology , Neoplasms/therapy , Single-Domain Antibodies/immunology , Cell Line, Tumor , T-Lymphocytes, Cytotoxic/immunology , Receptors, Antigen, T-Cell, alpha-beta/immunology , Carcinoembryonic Antigen/immunologyABSTRACT
Metastatic colorectal cancer remains a leading cause of cancer-related deaths, with a 5-year survival rate of only 15%. T cell-engaging bispecific antibodies (TCBs) represent a class of biopharmaceuticals that redirect cytotoxic T cells toward tumor cells, thereby turning immunologically "cold" tumors into "hot" ones. The carcinoembryonic antigen (CEA) is an attractive tumor-associated antigen that is overexpressed in more than 98% of patients with colorectal cancer. In this study, we report the comparison of four different TCB formats employing the antibodies F4 (targeting human CEA) and 2C11 (targeting mouse CD3ε). These formats include both antibody fragment-based and IgG-based constructs, with either one or two binding specificities of the respective antibodies. The 2 + 1 arrangement, using an anti-CEA single-chain diabody fused to an anti-CD3 single-chain variable fragment, emerged as the most potent design, showing tumor killing at subnanomolar concentrations across three different CEA+ cell lines. The in vitro activity was three times greater in C57BL/6 mouse colon adenocarcinoma cells (MC38) expressing high levels of CEA compared with those expressing low levels, highlighting the impact of CEA density in this assay. The optimal TCB candidate was tested in two different immunocompetent mouse models of colorectal cancer and showed tumor growth retardation. Ex vivo analysis of tumor infiltrates showed an increase in CD4+ and CD8+ T cells upon TCB treatment. This study suggests that bivalent tumor targeting, monovalent T-cell targeting, and a short spatial separation are promising characteristics for CEA-targeting TCBs.
Subject(s)
Antibodies, Bispecific , Carcinoembryonic Antigen , Colorectal Neoplasms , Antibodies, Bispecific/pharmacology , Antibodies, Bispecific/immunology , Colorectal Neoplasms/immunology , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/pathology , Animals , Carcinoembryonic Antigen/immunology , Humans , Mice , Cell Line, Tumor , T-Lymphocytes/immunology , CD3 Complex/immunology , Xenograft Model Antitumor Assays , Disease Models, AnimalABSTRACT
BACKGROUND: CEACAM5 and CEACAM6 are glycosylphosphatidylinositol (GPI)- linked members of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family, which are frequently upregulated in epithelial cancers where they contribute to invasion, metastasis, immune evasion, and resistance to anoikis. CT109 is a novel antibody with dual specificity to both CEACAM5 and 6. OBJECTIVES: In this study, we aimed to perform the preclinical characterization of CT109 and antibody- drug conjugate (ADCs) derivatives of CT109, focusing on CT109-SN-38. METHODS: CT109's cognate epitope was characterized by scanning mutagenesis. CT109 specificity and internalization kinetics were assessed by immunoblot and flow cytometry, respectively. Cognate antigen expression prevalence in colorectal cancer and normal tissue arrays was determined by immunohistochemistry. CT109 conjugations were generated by the reaction of reduced CT109 cysteines with maleimide-functionalized payload linkers. In vitro cytotoxic activity of CT109 ADCs was characterized on antigen-positive and negative pancreatic ductal adenocarcinoma cell (PDAC) lines using a luminometric viability assay. In vivo efficacy of CT109-SN-38 was assessed on a PDAC tumor xenograft model at 10 and 25 mg/kg concentrations. RESULTS: CT109 was shown to bind a glycoepitope centered on N309. CT109 is internalized in the CEACAM5+/CEACAM6+ double-positive PDAC line, BxPC-3, with a t1/2 of 2.3 hours. CT109 ADCs elicit a dose and antigen-dependent cytotoxic effect, with CT109-SN-38 exhibiting an IC50 value of 21 nM in BxPC-3 cells. In a BxPC-3 tumor xenograft model, CT109-SN-38 reduced tumor growth and induced regression in 3/10 mice at a concentration 25 mg/kg. CONCLUSION: These data suggest that further preclinical and clinical development of CT109-SN-38 is warranted.
Subject(s)
Carcinoembryonic Antigen , Cell Adhesion Molecules , GPI-Linked Proteins , Pancreatic Neoplasms , Animals , Female , Humans , Mice , Antigens, CD/metabolism , Antigens, CD/immunology , Carcinoembryonic Antigen/immunology , Cell Adhesion Molecules/antagonists & inhibitors , Cell Adhesion Molecules/metabolism , Cell Adhesion Molecules/immunology , Cell Line, Tumor , GPI-Linked Proteins/metabolism , GPI-Linked Proteins/antagonists & inhibitors , Immunoconjugates/pharmacology , Irinotecan/pharmacology , Mice, Nude , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/pathology , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Carcinoembryonic antigen (CEA) is a significant glycosylated protein, and the unusual expression of CEA in human serum is used as a tumor marker in the clinical diagnosis of many cancers. Although scientists have reported many ways to detect CEA in recent years, such as electrochemistry, photoelectrochemistry, and fluorescence, their operation is complex and sensitivity is average. Therefore, finding a convenient method to accurately detect CEA is significance for the prevention of malignant tumors. With high sensitivity, quick reaction, and low background, electrochemiluminescence (ECL) has emerged as an essential method for the detection of tumor markers in blood. RESULTS: In this work, a "signal on-off" ECL immunosensor for sensitive analysis of CEA ground on the ternary extinction effects of CuFe2O4@PDA-MB towards a self-enhanced Ru(dcbpy)32+ functionalized metal-organic layer [(Hf)MOL-Ru-PEI-Pd] was prepared. The high ECL efficiency of (Hf)MOL-Ru-PEI-Pd originated from the dual intramolecular self-catalysis, including intramolecular co-reaction between polyethylenimine (PEI) and Ru(dcbpy)32+. At the same time, loading Pd NPs onto (Hf)MOL-Ru-PEI could not only improve the electron transfer ability of (Hf)MOL-Ru-PEI, but also provide more active sites for the reaction of Ru(dcbpy)32+ and PEI. In the presence of CEA, CuFe2O4@PDA-MB-Ab2 efficiently quenches the excited states of (Hf)MOL-Ru-PEI-Pd by PDA, Cu2+, and methylene blue (MB) via energy and electron transfer, leading to an ECL signal decrease. Under optimal conditions, the proposed CEA sensing strategy showed satisfactory properties ranging from 0.1 pg mL-1 to 100 ng mL-1 with a detection limit of 20 fg mL-1. SIGNIFICANCE: The (Hf)MOL-Ru-PEI-Pd and CuFe2O4@PDA-MB were prepared in this work might open up innovative directions to synthesize luminescence-functionalized MOLs and effective quencher. Besides, the ECL quenching mechanism of Ru(dcbpy)32+ by MB was successfully explained by the inner filter effect (ECL-IFE). At last, the proposed immunosensor exhibits excellent repeatability, stability, and selectivity, and may provide an attractive way for CEA and other disease markers determination.
Subject(s)
Biosensing Techniques , Carcinoembryonic Antigen , Humans , Biomarkers, Tumor , Carcinoembryonic Antigen/chemistry , Carcinoembryonic Antigen/immunology , Immunoassay , Metals , Methylene Blue , Ferric Compounds/chemistry , Copper/chemistry , Ruthenium/chemistryABSTRACT
OBJECTIVE: Programmed cell death protein 1 (PD-1) checkpoint inhibition and adoptive cellular therapy have had limited success in patients with microsatellite stable colorectal cancer liver metastases (CRLM). We sought to evaluate the effect of interleukin 10 (IL-10) blockade on endogenous T cell and chimeric antigen receptor T (CAR-T) cell antitumour function in CRLM slice cultures. DESIGN: We created organotypic slice cultures from human CRLM (n=38 patients' tumours) and tested the antitumour effects of a neutralising antibody against IL-10 (αIL-10) both alone as treatment and in combination with exogenously administered carcinoembryonic antigen (CEA)-specific CAR-T cells. We evaluated slice cultures with single and multiplex immunohistochemistry, in situ hybridisation, single-cell RNA sequencing, reverse-phase protein arrays and time-lapse fluorescent microscopy. RESULTS: αIL-10 generated a 1.8-fold increase in T cell-mediated carcinoma cell death in human CRLM slice cultures. αIL-10 significantly increased proportions of CD8+ T cells without exhaustion transcription changes, and increased human leukocyte antigen - DR isotype (HLA-DR) expression of macrophages. The antitumour effects of αIL-10 were reversed by major histocompatibility complex class I or II (MHC-I or MHC-II) blockade, confirming the essential role of antigen presenting cells. Interrupting IL-10 signalling also rescued murine CAR-T cell proliferation and cytotoxicity from myeloid cell-mediated immunosuppression. In human CRLM slices, αIL-10 increased CEA-specific CAR-T cell activation and CAR-T cell-mediated cytotoxicity, with nearly 70% carcinoma cell apoptosis across multiple human tumours. Pretreatment with an IL-10 receptor blocking antibody also potentiated CAR-T function. CONCLUSION: Neutralising the effects of IL-10 in human CRLM has therapeutic potential as a stand-alone treatment and to augment the function of adoptively transferred CAR-T cells.
Subject(s)
Carcinoma , Colorectal Neoplasms , Interleukin-10 , Liver Neoplasms , Receptors, Chimeric Antigen , Receptors, Interleukin-10 , Animals , Humans , Mice , Carcinoembryonic Antigen/immunology , Carcinoma/immunology , Carcinoma/secondary , CD8-Positive T-Lymphocytes/immunology , Colorectal Neoplasms/pathology , Immunotherapy, Adoptive , Interleukin-10/antagonists & inhibitors , Liver Neoplasms/immunology , Liver Neoplasms/secondary , Lymphocyte Activation , Receptors, Antigen, T-Cell/metabolism , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/metabolism , Receptors, Interleukin-10/antagonists & inhibitors , Antibodies, Blocking/immunologyABSTRACT
BACKGROUND: Middle East respiratory syndrome-coronavirus (MERS-CoV) utilizes CD26 (dipeptidyl peptidase-4) and CD66e or CEACAM5 (carcinoembryonic antigen-related cell adhesion molecule 5) receptors for cell infection. Peripheral blood mononuclear cells (PBMCs) play a critical role in mounting adaptive immune response against the virus. This study was performed to assess the expression of CD26 and CD66e on PBMCs and their susceptibility to MERS-CoV infection. METHODS: Surface expression of CD26 and CD66e receptors on PBMCs from MERS-CoV patients (n = 20) and healthy controls (n = 20) was assessed by flow cytometry and the soluble forms were determined by enzyme-linked immunosorbent assay (ELISA). MERS-CoV UpE and Orf1a genes in PBMCs were detected by using Altona diagnostics reverse transcription polymerase chain reaction (RT-PCR) kit. RESULTS: Mean fluorescent intensity (MFI) of CD66e was significantly higher on CD4 + lymphocytes (462.4 ± 64.35 vs 325.1 ± 19.69; p < 0.05) and CD8 + lymphocytes (533.8 ± 55.32 vs 392.4 ± 37.73; p < 0.04) from patients with MERS-CoV infection compared to the normal controls. No difference in MFI for CD66e was observed on monocytes (381.8 ± 40.34 vs 266.8 ± 20.6; p = 0.3) between the patients and controls. Soluble form of CD66e among MERS-CoV patients was also higher than the normal controls (mean= 338.7 ± 58.75 vs 160.7 ± 29.49 ng/mL; p < 0.01). Surface expression of CD26 on PBMCs and its soluble form were no different between the groups. MERS-CoV was detected by RT-PCR in 16/20 (80%) patients from whole blood, among them 8 patients were tested in PBMCs, 4/8 (50%) patients were positive. CONCLUSION: Increased expression levels of CD66e (CEACAM5) may contribute to increased susceptibility of PBMCs to MERS-CoV infection and disease progression.
Subject(s)
Carcinoembryonic Antigen , Dipeptidyl Peptidase 4 , Middle East Respiratory Syndrome Coronavirus , Humans , Carcinoembryonic Antigen/genetics , Carcinoembryonic Antigen/immunology , Coronavirus Infections , Dipeptidyl Peptidase 4/genetics , Dipeptidyl Peptidase 4/immunology , GPI-Linked Proteins/genetics , GPI-Linked Proteins/immunology , Leukocytes, Mononuclear , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/immunologyABSTRACT
Background: Medullary thyroid cancer (MTC) is a rare malignancy originating from the calcitonin-producing C cells of the thyroid. Despite recent therapeutic advances, metastatic MTC remains incurable. Adoptive cell therapy (ACT) using genetically engineered T cells targeting either tissue-restricted tumor-associated antigens or mutated neoantigens has led to durable remissions in other metastatic solid tumors. The majority of MTC express the tumor-associated antigens calcitonin and carcinoembryonic antigen (CEA), and â¼40% of MTC harbor the RET M918T oncogenic driver mutation. Methods: We developed and characterized three immunoreceptors that recognize extracellular CEA, a calcitonin epitope presented by HLA-A*24:02, or an RET M918T neoepitope restricted by HLA-DPB1*04:01/02. The chimeric antigen receptor (CAR) targeting CEA was synthetically designed, while the T cell receptors (TCRs) targeting calcitonin and RET M918T were isolated from a transgenic mouse and patient with MTC, respectively. These immunoreceptors were genetically engineered into peripheral blood T cells and tested for antigen specificity and antitumor activity. Results: T cells expressing the anti-CEA CAR or the calcitonin-reactive TCR produced effector cytokines and displayed cytotoxicity against cell lines expressing their cognate antigen in vitro. In immunodeficient mice harboring a human MTC cell line, the adoptive transfer of T cells engineered to express the anti-CEA CAR or calcitonin-reactive TCR led to complete tumor regression. T cells expressing the HLA-DPB1*04:01/02-restricted TCR targeting RET M918T, which was cloned from peripheral blood CD4+ T cells of a patient with MTC, demonstrated specific reactivity against cells pulsed with the mutated peptide and MTC tumor cells that expressed HLA-DPB1*04:01 and RET M918T. Conclusion: The preclinical data presented herein demonstrate the potential of using genetically engineered T cells targeting CEA, calcitonin, and/or RET M918T to treat metastatic MTC.
Subject(s)
Calcitonin , Carcinoembryonic Antigen , Cell Engineering , Proto-Oncogene Proteins c-ret , Receptors, Antigen, T-Cell , T-Lymphocytes , Animals , Calcitonin/genetics , Calcitonin/immunology , Carcinoembryonic Antigen/genetics , Carcinoembryonic Antigen/immunology , Carcinoma, Neuroendocrine/drug therapy , Carcinoma, Neuroendocrine/therapy , Cell Line, Tumor , Epitopes, T-Lymphocyte/genetics , Epitopes, T-Lymphocyte/immunology , Humans , Mice , Mutation , Proto-Oncogene Proteins c-ret/genetics , Proto-Oncogene Proteins c-ret/immunology , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes/immunology , Thyroid Neoplasms/genetics , Thyroid Neoplasms/metabolism , Thyroid Neoplasms/therapyABSTRACT
Rationale: The CEA-CD3 T cell bispecific antibody cibisatamab (CEA-TCB) is currently undergoing clinical trials. Here we study its performance against three-dimensional tumor organoids in cocultures with T cells as compared to a higher affinity CEACAM5-CD3 (CEACAM5-TCB) bispecific antibody using time-lapse confocal microscopy. Methods: Pre-labelled spheroids derived from colon cancer cell lines and primary organoids derived from four colorectal cancer surgical specimens, which expressed different graded levels of CEA, were exposed in cocultures to T lymphocytes. Cocultures were treated with CEA-CD3 T-cell engagers and were followed by live confocal microscopy. Caspase 3 activation detected in real-time was used as an indicator of tumor cell death. Co-cultures were also set up with autologous tumor-associated fibroblasts to test the co-stimulatory effect of a fibroblast activated protein (FAP)- targeted 4-1BBL bispecific antibody fusion protein currently undergoing clinical trials. Results: Tumor-cell killing of 3D colon carcinoma cultures was dependent on the levels of surface CEA expression, in such a way that the lower affinity agent (CEA-TCB) did not mediate killing by human preactivated T cells below a certain CEA expression threshold, while the high affinity construct (CEACAM5-TCB) remained active on the low CEA expressing organoids. Modelling heterogeneity in the levels of CEA expression by coculturing CEA high and low organoids showed measurable but weak bystander killing. Cocultures of tumor organoids, autologous fibroblasts and T cells allowed to observe a costimulatory effect of anti-FAP-4-1BBL both to release IFNγ and to attain more efficacious tumor cell killing. Conclusion: Three-dimensional tumor cocultures with T cells using live confocal microscopy provide suitable models to test the requirements for colon-cancer redirected killing as elicited by CEA-targeted T-cell engagers undergoing clinical trials and treatment allow combinations to be tested in a relevant preclinical system.
Subject(s)
Antibodies, Bispecific , Carcinoembryonic Antigen , Colonic Neoplasms , T-Lymphocytes , Antibodies, Bispecific/immunology , Antibodies, Bispecific/pharmacology , CD3 Complex/immunology , Carcinoembryonic Antigen/immunology , Colonic Neoplasms/drug therapy , Colonic Neoplasms/immunology , Colonic Neoplasms/metabolism , Humans , Lymphocyte Activation , Organoids/metabolism , T-Lymphocytes/drug effects , T-Lymphocytes/immunologyABSTRACT
Point-of-care devices are expected to play very critical roles in early diagnosis and better treatment of cancer. Here, we report the end-to-end development of novel and portable biosensors for detecting carcinoembryonic antigen (CEA), a cancer biomarker, almost instantly at room temperature. The device uses reduced graphene oxide (rGO) as the base conducting layer and a novel poly[(1,4-phenylene)-alt-(3,6-(1,2,4,5-tetrazine)/3,6-(1,2,4,5-dihydrotetrazine))] (PhPTz) as an immobilizing matrix for the CEA antibodies. Judiciously introduced nitrogen-rich semiconducting PhPTz brings multiple advantages to the device-(1) efficiently immobilizes anti-CEA via synergistic H-bonding with peptide and N-glycal units and (2) transports the charge density variations, originated upon antibody-antigen interactions, to the rGO layer. The CEA was dropped onto the anti-CEA/PhPTz/rGO devices at ambient conditions, to facilitate binding and the change in current flowing through the sensors was measured. A response of 2.75-33.7 µA was observed when the devices were tested for a broad range of concentrations (0.25 pg/mL to 800 ng/mL) of CEA. A portable read-out circuit was assembled using Arduino UNO and a voltage divider circuit, and a simple algorithm was developed for the classification of the CEA concentrations. The prediction accuracy of the interfacing electronics along with the algorithm was found to be 100%.
Subject(s)
Antibodies, Immobilized , Biomarkers, Tumor/analysis , Biosensing Techniques/instrumentation , Carcinoembryonic Antigen/analysis , Graphite , Immunoassay/instrumentation , Algorithms , Biosensing Techniques/methods , Carcinoembryonic Antigen/immunology , Immunoassay/methods , Polymers , Sensitivity and SpecificityABSTRACT
The simple and sensitive detection of protein is of great significance in biological research and medical diagnosis. However, the commonly-used methods, such as enzyme-linked immunosorbent assay (ELISA), usually rely on signal tags labeled on the antibody, which limits the sensitivity and stability. Herein, we have designed and constructed a colorimetric immunosensor in this work for the analysis of protein by taking advantage of 2D metal-organic framework (2D-MOF) nanomaterials as enzyme mimics. The nanomaterial shows a strong peroxidase mimetic activity, and good selectivity after it is modified with a specific aptamer. Therefore, taking carcinoembryonic antigen (CEA) as an example, this immunosensor achieves a good detection performance with a linear range from 1 pg mL-1 to 1000 ng mL-1 and a limit of detection (LOD) of 0.742 pg mL-1. Moreover, the sensor can successfully distinguish the human serum of colorectal cancer patients from healthy people, which suggests that this sensor has great potential in clinical applications. More importantly, the mass production, low cost, stability and ease of transport of the MOFs nanomaterials, as well as the ability for visual detection will make this sensor suitable for point-of-care (POC) testing in remote or resource-poor areas.
Subject(s)
Carcinoembryonic Antigen/blood , Colorimetry/methods , Immunoassay/methods , Metal-Organic Frameworks/chemistry , Nanostructures/chemistry , Antibodies, Immobilized/immunology , Aptamers, Nucleotide/chemistry , Benzidines/chemistry , Biomarkers/blood , Carcinoembryonic Antigen/immunology , Catalysis , Chromogenic Compounds/chemistry , Colorectal Neoplasms/blood , Humans , Immobilized Nucleic Acids/chemistry , Limit of DetectionABSTRACT
Invasive candidiasis is a healthcare-associated fungal infection with a high mortality rate. Neutrophils, the first line of defense during fungal infections, express the immunoregulatory Candida albicans receptors CEACAM1, CEACAM3, and CEACAM6. We analyzed the effects of specific antibodies on C. albicans-induced neutrophil responses. CEACAM6 ligation by 1H7-4B and to some extent CEACAM1 ligation by B3-17, but not CEACAM3 ligation by 308/3-3, resulted in the immediate release of stored CXCL8 and altered transcriptional responses of the C. albicans-stimulated neutrophils. Integrated network analyses and dynamic simulations of signaling cascades predicted alterations in apoptosis and cytokine secretion. We verified that CEACAM6 ligation enhanced Candida-induced neutrophil apoptosis and increased long-term IL-1ß/IL-6 release in responses to C. albicans. CEACAM3 ligation, but not CEACAM1 ligation, increased the long-term release of pro-inflammatory IL-1ß/IL-6. Taken together, we demonstrated for the first time that ligation of CEACAM receptors differentially affects the regulation of C. albicans-induced immune functions in human neutrophils.
Subject(s)
Antigens, CD/immunology , Candida albicans/immunology , Carcinoembryonic Antigen/immunology , Cell Adhesion Molecules/immunology , Neutrophils/immunology , Antibodies, Monoclonal/immunology , Apoptosis/immunology , Candidiasis, Invasive/mortality , Candidiasis, Invasive/pathology , Cytokines/immunology , Female , GPI-Linked Proteins/immunology , Humans , Immunomodulation/immunology , Interleukin-1beta/metabolism , Interleukin-6/metabolism , Interleukin-8/metabolism , MaleABSTRACT
Neuro-endocrine prostate cancer (NEPC) accounts for about 20% of lethal metastatic castration-resistant prostate cancer (CRPC). NEPC has the most aggressive biologic behavior of all prostate cancers and is associated with poor patient outcome. Effective treatment for NEPC is not available because NEPC exhibit distinct cell-surface expression profiles compared to other types of prostate cancer. Recently, the carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) (known as CEA or CD66e) was suggested to be a specific surface protein marker for NEPC. Therefore, we identified a new, fully-human anti-CEACAM5 monoclonal antibody, 1G9, which bound to the most proximal membrane domains, A3 and B3, of CEACAM5 with high affinity and specificity. It shows no off-target binding to other CEACAM family members, membrane distal domains of CEACAM5, or 5800 human membrane proteins. IgG1 1G9 exhibited CEACAM5-specific ADCC activity toward CEACAM5-positive prostate cancer cells in vitro and in vivo. Chimeric antigen receptor T cells (CAR-T) based on scFv 1G9 induced specific and strong antitumor activity in a mouse model of prostate cancer. Our results suggest that IgG1 and CAR-T cells based on 1G9 are promising candidate therapeutics for CEACAM5-positive NEPC and other cancers.