Targeting CEACAM5-positive solid tumors using NILK-2401, a novel CEACAM5xCD47 κλ bispecific antibody.

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.

CEA-CD3 bispecific antibody cibisatamab with or without atezolizumab in patients with CEA-positive solid tumours: results of two multi-institutional Phase 1 trials.

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.

[Advances of CAR-T cell therapy in treating colorectal cancer].

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.

Ternary electrochemiluminescence quenching effects of CuFe2O4@PDA-MB towards self-enhanced Ru(dcbpy)32+ functionalized 2D metal-organic layer and application in carcinoembryonic antigen immunosensing.

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.

Blockade of interleukin 10 potentiates antitumour immune function in human colorectal cancer liver metastases.

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.

Differential expression of carcinoembryonic antigen-related cell adhesion molecule-5 (CEACAM5) and dipeptidyl peptidase-4 (DPP4) with detection of Middle East respiratory syndrome-coronavirus in peripheral blood.

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.

T Cells Engineered to Express Immunoreceptors Targeting the Frequently Expressed Medullary Thyroid Cancer Antigens Calcitonin, CEA, and RET M918T.

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 ∼40% 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.

Three-dimensional colon cancer organoids model the response to CEA-CD3 T-cell engagers.

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.

An electronic biosensor based on semiconducting tetrazine polymer immobilizing matrix coated on rGO for carcinoembryonic antigen.

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%.

Colorimetric immunosensor constructed using 2D metal-organic framework nanosheets as enzyme mimics for the detection of protein biomarkers.

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.

A highly-specific fully-human antibody and CAR-T cells targeting CD66e/CEACAM5 are cytotoxic for CD66e-expressing cancer cells in vitro and in vivo.

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.

Antibody ligation of CEACAM1, CEACAM3, and CEACAM6, differentially enhance the cytokine release of human neutrophils in responses to Candida albicans.

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.

A pilot trial of vaccination with Carcinoembryonic antigen and Her2/neu peptides in advanced colorectal cancer.

Checkpoint-blockade therapy (CBT) is approved for select colorectal cancer (CRC) patents, but additional immunotherapeutic options are needed. We hypothesized that vaccination with carcinoembryonic antigen (CEA) and Her2/neu (Her2) peptides would be immunogenic and well tolerated by participants with advanced CRC. A pilot clinical trial (NCT00091286) was conducted in HLA-A2+ or -A3+ Stage IIIC-IV CRC patients. Participants were vaccinated weekly with CEA and Her2 peptides plus tetanus peptide and GM-CSF emulsified in Montanide ISA-51 adjuvant for 3 weeks. Adverse events (AEs) were recorded per NIH Common Terminology Criteria for Adverse Events version 3. Immunogenicity was evaluated by interferon-gamma ELISpot assay of in vitro sensitized peripheral blood mononuclear cells and lymphocytes from the sentinel immunized node. Eleven participants were enrolled and treated; one was retrospectively found to be ineligible due to HLA type. All 11 participants were included in AEs and survival analyses, and the 10 eligible participants were evaluated for immunogenicity. All participants reported AEs: 82% were Grade 1-2, most commonly fatigue or injection site reactions. Two participants (18%) experienced treatment-related dose-limiting Grade 3 AEs; both were self-limiting. Immune responses to Her2 or CEA peptides were detected in 70% of participants. Median overall survival (OS) was 16 months; among those enrolled with no evidence of disease (n = 3), median OS was not reached after 10 years of follow-up. These data demonstrate that vaccination with CEA or Her2 peptides is well tolerated and immunogenic. Further study is warranted to assess potential clinical benefits of vaccination in advanced CRC either alone or in combination with CBT.

Covalent Immobilization of Antibodies through Tetrazine-TCO Reaction to Improve Sensitivity of ELISA Technique.

Enzyme-linked immunosorbent assay (ELISA) is routinely used to detect biomolecules related to several diseases facilitating diagnosis and monitoring of these, as well as the possibility of decreasing their mortality rate. Several methods have been carried out to improve the ELISA sensitivity through antibodies immobilization on the microtiter plates. Here, we have developed a strategy of antibodies immobilization to improve the ELISA sensitivity increasing the antibody density surface through the tetrazine (Tz)-trans-cyclooctene (TCO) reaction. For this, we prepared surfaces with tetrazine groups while the captured antibody was conjugated with TCO. The tetrazine surfaces were prepared in two different ways: (1) from aminated plates and (2) from Tz-BSA-coated plates. The surfaces were evaluated using two sandwich ELISA models, one of them using the low-affinity antibody anti-c-myc as a capture antibody to detect the c-myc-GST-IL8h recombinant protein, and the other one to detect the carcinoembryonic human protein (CEA). The sensitivity increased in both surfaces treated with tetrazine in comparison with the standard unmodified surface. The c-myc-GST-IL8h detection was around 10-fold more sensible on both tetrazine surfaces, while CEA ELISA detection increased 12-fold on surfaces coated with Tz-BSA. In conclusion, we show that it is possible to improve the ELISA sensitivity using this immobilization system, where capture antibodies bond covalently to surfaces.

Graphene-PtPd nanocomposite for low-potential-driven electrochemiluminescent determination of carcinoembryonic antigen using Ru(bpy)32.

As well known, the electrochemiluminescence (ECL) of tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)32+) heavily relies on highly positive or negative triggered voltage, prejudicing the detection toward the bio-molecules. In this work, Ru(bpy)32+ could generate enhanced and stable ECL at a low potential of 0.05 V (vs. Ag/AgCl) on graphene-PtPd hybrid, attributing to its excellent electrocatalysis from the synergistic effect between Pt and Pd. The obtained low-potential-driven ECL could be quenched by MoS2 nanoflowers. Based on the quenching effect, a sandwich "signal-off" ECL immunosensor was fabricated to sensitively detect carcinoembryonic antigen (CEA). A linear calibration curve from 1 fg mL-1 to 1 ng mL-1 was obtained along with a low detection limit of 0.54 fg mL-1 (S/N = 3) under optimal conditions. The sensor showed satisfactory specificity, stability, and reproducibility and was successfully applied to determine CEA in actual samples. The recoveries ranged from 98.80 to 100.23%, and the relative standard deviation (RSD) was lower than 5%. Above all, this work explored new materials in low-potential-driven ECL system and provided a reliable sensing strategy for clinical applications.

Pressure-Based Immunoassays with Versatile Electronic Sensors for Carcinoembryonic Antigen Detection.

Pressure-based immunoassays have been studied for point-of-care testing for which increasing the sensitivity is still a challenge. In this study, we described an enhanced pressure-based immunoassay with a versatile electronic sensor for the sensitive biological analysis. The versatile electronic sensor had multifunctional sensing capabilities with temperature and pressure recording. Magnetic bead-modified capture antibody and platinum nanoparticle-labeled detection antibody were used as the biorecognition element of the target carcinoembryonic antigen (CEA) (as a model analyte) and would form a sandwich-type immune complex with CEA. After simple magnetic separation, this complex was transferred into the detection chamber, which contained both hydrogen peroxide (H2O2) and 3,3',5,5'-tetramethylbenzidine (TMB). With the catalytic ability of PtNPs, the "H2O2-TMB-PtNPs" system was catalyzed to generate a large amount of oxygen (O2) and photothermal agent of oxidizer TMB (ox-TMB). Meanwhile, in a sealed chamber, further irradiation with an 808 nm near-infrared laser led to a triple-step signal amplification strategy of pressure increase, temperature increase, and gas thermal expansion to receive a strong electrical signal through the electronic sensor in real time. Thus, the amplified electrical signal from the electronic sensor could reveal the target concentration. In addition, we also verified that the synergistic system with two physical quantities had a lower limit of detection and a wider detection range compared to the detection system with a single physical quantity. In general, this immunoassay not only helped in exploring an effective signal amplification pathway but also offered an opportunity for the development of versatile electronic sensors in point-of-care settings.

Multi-Parameter Quantitative Imaging of Tumor Microenvironments Reveals Perivascular Immune Niches Associated With Anti-Tumor Immunity.

Tumors are populated by a multitude of immune cell types with varied phenotypic and functional properties, which can either promote or inhibit anti-tumor responses. Appropriate localization and function of these cells within tumors is critical for protective immunity, with CD8 T cell infiltration being a biomarker of disease outcome and therapeutic efficacy. Recent multiplexed imaging approaches have revealed highly complex patterns of localization for these immune cell subsets and the generation of distinct tumor microenvironments (TMEs), which can vary among cancer types, individuals, and within individual tumors. While it is recognized that TMEs play a pivotal role in disease progression, a better understanding of their composition, organization, and heterogeneity, as well as how distinct TMEs are reshaped with immunotherapy, is necessary. Here, we performed spatial analysis using multi-parameter confocal imaging, histocytometry, and CytoMAP to study the microanatomical organization of immune cells in two widely used preclinical cancer models, the MC38 colorectal and KPC pancreatic murine tumors engineered to express human carcinoembryonic antigen (CEA). Immune responses were examined in either unperturbed tumors or after immunotherapy with a CEA T cell bispecific (CEA-TCB) surrogate antibody and anti-PD-L1 treatment. CEA-TCB mono and combination immunotherapy markedly enhanced intra-tumoral cellularity of CD8 T cells, dominantly driven by the expansion of TCF1-PD1+ effector T cells and with more minor increases in TCF1+PD1+ resource CD8 T cells. The majority of infiltrating T cells, particularly resource CD8 T cells, were colocalized with dendritic cells (DCs) or activated MHCII+ macrophages, but largely avoided the deeper tumor nest regions composed of cancer cells and non-activated macrophages. These myeloid cell - T cell aggregates were found in close proximity to tumor blood vessels, generating perivascular immune niches. This perivascular TME was present in untreated samples and markedly increased after CEA-TCB therapy, with its relative abundance positively associated with response to therapy. Together, these studies demonstrate the utility of advanced spatial analysis in cancer research by revealing that blood vessels are key organizational hubs of innate and adaptive immune cells within tumors, and suggesting the likely relevance of the perivascular immune TME in disease outcome.

Immobilizing Enzymes on Noble Metal Hydrogel Nanozymes with Synergistically Enhanced Peroxidase Activity for Ultrasensitive Immunoassays by Cascade Signal Amplification.

Enzyme immobilization plays an essential role in solving the problems of the inherently fragile nature of enzymes. Although prominent stability and reuse of enzymes can be achieved by enzyme immobilization, their bioactivity and catalytic efficiency will be adversely affected. Herein, PdCu hydrogel nanozymes with a hierarchically porous structure were used to immobilize horseradish peroxidase (HRP) to obtain PdCu@HRP. In addition to the improvement of stability and reusability, PdCu@HRP displayed synergistically enhanced activities than native HRP and PdCu hydrogels. Not only the specific interactions between PdCu hydrogel nanozymes and enzymes but also the enrichment of substrates around enzymes by electrostatic adsorption of hydrogels was proposed to expound the enhanced catalytic activity. Accordingly, by taking advantage of the excellent catalytic performance of the PdCu@HRP and the glucose oxidase encapsulated in zeolitic imidazolate framework-8, colorimetric biosensing of the carcinoembryonic antigen via catalytic cascade reactions for achieving signal amplification was performed. The obtained biosensor enhanced the detection sensitivity by approximately 6.1-fold as compared to the conventional HRP-based enzyme-linked immunosorbent assay, demonstrating the promising potential in clinical diagnosis.

Rapid tumor-labeling kinetics with a site-specific near-infrared anti-CEA nanobody in a patient-derived orthotopic xenograft mouse model of colon cancer.

BACKGROUND/OBJECTIVES: Nanobodies are the smallest biologic antigen-binding fragments derived from camelid-derived antibodies. Nanobodies effect a peak tumor signal within minutes of injection and present a novel opportunity for fluorescence-guided surgery (FGS). The present study demonstrates the efficacy of an anti-CEA nanobody conjugated to near-infrared fluorophore LICOR-IRDye800CW for rapid intraoperative tumor labeling of colon cancer. METHODS: LS174T human colon cancer cells or fragments of patient-derived colon cancer were implanted subcutaneously or orthotopically in nude mice. Anti-CEA nanobodies were conjugated with IRDye800CW and 1-3 nmol were injected intravenously. Mice were serially imaged over time. Peak fluorescence signal and tumor-to-background ratio (TBR) were recorded. RESULTS: Colon cancer tumors were detectable using fluorescent anti-CEA nanobody within 5 min of injection at all three doses. Maximal fluorescence intensity was observed within 15 min-3 h for all three doses with TBR values ranging from 1.3 to 2.3. In the patient-derived model of colon cancer, fluorescence was detectable with a TBR of 4.6 at 3 h. CONCLUSIONS: Fluorescent anti-CEA nanobodies rapidly and specifically labeled colon cancer in cell-line-based and patient-derived orthotopic xenograft (PDOX) models. The kinetics of nanobodies allow for same day administration and imaging. Anti-CEA-nb-800 is a promising and practical molecule for FGS of colon cancer.

Carcinoembryonic antigen as a specific glycoprotein ligand of rBC2LCN lectin on pancreatic ductal adenocarcinoma cells.

The rBC2LCN lectin, known as a stem cell marker probe that binds to an H type 3 fucosylated trisaccharide motif, was recently revealed to also bind to pancreatic ductal adenocarcinoma (PDAC) cells. A lectin-drug conjugate was generated by fusing rBC2LCN with a cytocidal toxin, and it showed a strong anticancer effect in in vitro and in vivo PDAC models. However, it is unclear which molecules are carrier proteins of rBC2LCN on PDAC cells. In this study, we identified a rBC2LCN-positive glycoprotein expressed in PDAC. Tumor lysates of PDAC patient-derived xenografts (PDXs) were coprecipitated with rBC2LCN lectin and analyzed by liquid chromatography-mass spectrometry. A total of 343 proteins were initially identified. We used a web-based database to select five glycoproteins and independently evaluated their expression in PDAC by immunohistochemistry (IHC). Among them, we focused on carcinoembryonic antigen 5 (CEA) as the most cancer-specific carrier protein in PDAC, as it showed the most prominent difference in expression rate between PDAC cells (74%) and normal pancreatic duct cells (0%, P > .0001). rBC2LCN lectin and CEA colocalization in PDAC samples was confirmed by double-staining analysis. Furthermore, rBC2LCN-precipitated fractions were blotted with an anti-CEA polyclonal antibody (pAb), and CEA pAb-precipitated fractions were blotted with rBC2LCN lectin. The results demonstrate that CEA is in fact a ligand of rBC2LCN lectin.