Chimeric antigen receptor-T cells targeting epithelial cell adhesion molecule antigens are effective in the treatment of colorectal cancer.

OBJECTIVE: To construct chimeric antigen receptor (CAR)-T cells targeting epithelial cell adhesion molecule (EpCAM) antigen (anti-EpCAM-CAR-T). METHODS: A third-generation CAR-T cell construct used a single-chain variable fragment derived from monoclonal antibody against human EpCAM. Peripheral blood mononuclear cells were extracted from volunteers. The proportion of cluster of differentiation 8 positive (CD8+) and CD4 + T cells was measured using flow cytometry. Western blot was used to detect the expression of EpCAM-CAR. The killing efficiency was detected using the MTT assay and transwell assay, and the secretion of killer cytokines tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) was detected using the ELISA. The inhibitory effect of EpCAM-CAR-T on colorectal cancer in vivo was detected using xenografts. RESULTS: It was found that T cells expanded greatly, and the proportion of CD3+, CD8 + and CD4 + T cells was more than 60%. Furthermore, EpCAM-CAR-T cells had a higher tumour inhibition rate in the EpCAM expression positive group than in the negative group (P < 0.05). The secretion of killer cytokines TNF-α and IFN-γ in the EpCAM expression positive cell group was higher than that in the negative group (P < 0.05). In the experimental group treated with EpCAM-CAR-T cells, the survival rate of nude mice was higher (P < 0.05), and the tumour was smaller than that in the blank and control groups (P < 0.05). The secretion of serum killer cytokines TNF-α and IFN-γ in tumour-bearing nude mice in the experimental group treated with EpCAM-CAR-T cells was higher than that in the blank and control groups (P < 0.05). CONCLUSION: This study successfully constructed EpCAM-CAR cells and found that they can target and recognise EpCAM-positive tumour cells, secrete killer cytokines TNF-α and IFN-γ and better inhibit the growth and metastasis of colorectal cancer in vitro and in vivo than unmodified T cells.

Fluorescent identification of immunomagnetically captured CTCs using triplex-aptamer-targeted dendritic SiO2@Fe3O4 nanocomposite.

The enumeration of circulating tumor cells (CTCs) in peripheral blood plays a crucial role in the early diagnosis, recurrence monitoring, and prognosis assessment of cancer patients. There is a compelling need to develop an efficient technique for the capture and identification of these rare CTCs. However, the exclusive reliance on a single criterion, such as the epithelial cell adhesion molecule (EpCAM) antibody or aptamer, for the specific recognition of epithelial CTCs is not universally suitable for clinical applications, as it usually falls short in identifying EpCAM-negative CTCs. To address this limitation, we propose a straightforward and cost-effective method involving triplex fluorescently labelled aptamers (FAM-EpCAM, Cy5-PTK7, and Texas Red-CSV) to modify Fe3O4-loaded dendritic SiO2 nanocomposite (dmSiO2@Fe3O4/Apt). This multi-recognition-based strategy not only enhanced the efficiency in capturing heterogeneous CTCs, but also facilitated the rapid and accurate identification of CTCs. The capture efficiency of heterogenous CTCs reached up to 93.33%, with a detection limit as low as 5 cells/mL. Notably, the developed dmSiO2@Fe3O4/Apt nanoprobe enabled the swift identification of captured cells in just 30 min, relying solely on the fluorescently modified aptamers, which reduced the identification time by approximately 90% compared with the conventional immunocytochemistry (ICC) technique. Finally, these nanoprobe characteristics were validated using blood samples from patients with various types of cancers.

Layer-by-layer fabrication of alginate/polyethyleneimine multilayer on magnetic interface with enhanced efficiency in immuno-capturing circulating tumor cells.

BACKGROUND: The technology of capturing circulating tumor cells (CTCs) plays a crucial role in the diagnosis, evaluation of therapeutic efficacy, and prediction of prognosis in lung cancer. However, the presence of complex blood environment often results in severe nonspecific protein adsorption and interferences from blood cells, which negatively impacts the specificity of CTCs capture. There is a great need for development of novel nanomaterials for CTCs capture with prominent anti-nonspecific adsorptions from proteins or blood cells. RESULTS: We present a novel immune magnetic probe Fe3O4@(PEI/AA)4@Apt. The surface of Fe3O4 particles was modified with four layers of PEI/AA composite by layer-by-layer assembly. Furthermore, aptamers targeting epithelial marker EpCAM (SYL3C) and mesenchymal marker CSV (ZY5C) were simultaneously connected on Fe3O4@(PEI/AA)4 to improve the detection of different phenotypic CTCs and reduce false negatives. The results demonstrated that the (PEI/AA)4 coatings not only minimized non-specific protein adsorptions, but also significantly reduced the adsorption rate of red blood cells to a mere 1 %, as a result of which, the Fe3O4@(PEI/AA)4@Apt probe achieved a remarkably high capture efficiency toward CTCs (95.9 %). In the subsequent validation of clinical samples, the probe was also effective in capturing rare CTCs from lung cancer patients. SIGNIFICANCE AND NOVELTY: A (PEI/AA) polymerized composite with controllable layers was fabricated by layer-by-layer self-assembly technique, which displayed remarkable anti-nonspecific adsorption capabilities toward proteins and cells. Importantly, Fe3O4@(PEI/AA)4@Apt probe significantly improved CTCs capture purity in lung cancer patients to 89.36 %. For the first time, this study combined controllable (PEI/AA) layers with magnetic separation to innovatively build a resistant interface that significantly improves the specific capture performances of CTCs, broadening the application of this polymerized composite.

BerEP4-Negative Basal Cell Carcinoma: An Immunophenotype Not to Forget.

ABSTRACT: Basal cell carcinoma (BCC) is the most common cancer worldwide. Although not typically metastatic, BCC can be locally destructive. BerEP4 is an antibody against CD326, an epithelial cell adhesion molecule (EpCAM) that is expressed on epithelial progenitor cells and carcinomas. BerEP4 has been reported to have a 100% positive sensitivity in basal cell carcinomas, but a much lower sensitivity for a variety of other carcinomas, including clear cell renal cell carcinoma and metastatic renal cell carcinoma. A 74-year-old woman presented with a BerEP4-negative, but anti-renal cell antibody-positive BCC, and the stark clinical implications of misdiagnosis. This case stresses the importance of considering BerEP4-negative BCC, even when other abnormal features are present.

Analysis of off-tumour toxicities of T-cell-engaging bispecific antibodies via donor-matched intestinal organoids and tumouroids.

Predicting the toxicity of cancer immunotherapies preclinically is challenging because models of tumours and healthy organs do not typically fully recapitulate the expression of relevant human antigens. Here we show that patient-derived intestinal organoids and tumouroids supplemented with immune cells can be used to study the on-target off-tumour toxicities of T-cell-engaging bispecific antibodies (TCBs), and to capture clinical toxicities not predicted by conventional tissue-based models as well as inter-patient variabilities in TCB responses. We analysed the mechanisms of T-cell-mediated damage of neoplastic and donor-matched healthy epithelia at a single-cell resolution using multiplexed immunofluorescence. We found that TCBs that target the epithelial cell-adhesion molecule led to apoptosis in healthy organoids in accordance with clinical observations, and that apoptosis is associated with T-cell activation, cytokine release and intra-epithelial T-cell infiltration. Conversely, tumour organoids were more resistant to damage, probably owing to a reduced efficiency of T-cell infiltration within the epithelium. Patient-derived intestinal organoids can aid the study of immune-epithelial interactions as well as the preclinical and clinical development of cancer immunotherapies.

The combination therapy with EpCAM/CD3 BsAb and MUC-1/CD3 BsAb elicited antitumor immunity by T-cell adoptive immunotherapy in lung cancer.

Lung cancer remains a global challenge due to high morbidity and mortality rates and poor response to treatment, and there are still no effective strategies to solve it. The bispecific antibody (BsAb) is a novel antibody, which can target two different antigens and mediate specific killing effects by selectively redirecting effector cells to the target cells. In this study, we combined two BsAbs to achieve a dual-target therapy strategy of EpCAM+ and MUC-1+ with high affinity and specificity. The results showed that the combination of two BsAbs against EpCAM and MUC-1 could inhibit the growth of lung cancer more effectively in cell lines and primary tumors. The superior antitumor effect of two BsAbs could be attributable to enhanced CTL and increased production of type I IFNs. At the same time, the combination of EpCAM/CD3 BsAb and MUC-1/CD3 BsAb significantly regulated T population in the TDLNs. Therefore, we have found a potential immunotherapeutic strategy, which was the combination therapy with EpCAM/CD3 BsAb and MUC-1/CD3 BsAb for the treatment of non-small cell lung cancer.

Clinical value of tumor-associated antigens and autoantibody panel combination detection in the early diagnostic of lung cancer.

BACKGROUND: This study aimed to investigate the efficiency of combining tumor-associated antigens (TAAs) and autoantibodies in the diagnosis of lung cancer. METHODS: The serum levels of TAAs and seven autoantibodies (7-AABs) were detected from patients with lung cancer, benign lung disease and healthy controls. The performance of a new panel by combing TAAs and 7-AABs was evaluated for the early diagnosis of lung cancer. RESULTS: The positive rate of 7-AABs was higher than the single detection of antibody. The positive rate of the combined detection of 7-AABs in lung cancer group (30.2%) was significantly higher than that of healthy controls (16.8%), but had no statistical difference compared with that of benign lung disease group (20.8%). The positive rate of 7-AABs showed a tendency to increase in lung cancer patients with higher tumor-node-metastasis (TNM) stages. For the pathological subtype analysis, the positive rate of 7-AABs was higher in patients with squamous cell carcinoma and small cell lung cancer than that of adenocarcinoma. The levels of carcinoembryonic antigen (CEA) and cytokeratin 19 fragment 211 (CYFRA 21-1) were significantly higher than that of benign lung disease and healthy control groups. An optimal model was established (including 7-AABs, CEA and CYFRA21-1) to distinguish lung cancer from control groups. The performance of this model was superior than that of single markers, with a sensitivity of 52.26% and specificity of 77.46% in the training group. Further assessment was studied in another validation group, with a sensitivity of 44.02% and specificity of 83%. CONCLUSIONS: The diagnostic performance was enhanced by combining 7-AABs, CEA and CYFRA21-1, which has critical value for the screening and early detection of lung cancer.

Development of a recombinant anti-VEGFR2-EPCAM bispecific antibody to improve antiangiogenic efficiency.

Tumor progression and metastasis, especially in invasive cancers (such as triple-negative breast cancer [TNBC]), depend on angiogenesis, in which vascular epithelial growth factor (VEGF)/vascular epithelial growth factor receptor [1] has a decisive role, followed by the metastatic spread of cancer cells. Although some studies have shown that anti-VEGFR2/VEGF monoclonal antibodies demonstrated favorable results in the clinic, this approach is not efficient, and further investigations are needed to improve the quality of cancer treatment. Besides, the increased expression of epithelial cell adhesion molecule (EpCAM) in various cancers, for instance, invasive breast cancer, contributes to angiogenesis, facilitating the migration of tumor cells to other parts of the body. Thus, the main goal of our study was to target either VEGFR2 or EpCAM as pivotal players in the progression of angiogenesis in breast cancer. Regarding cancer therapy, the production of bispecific antibodies is easier and more cost-effective compared to monoclonal antibodies, targeting more than one antigen or receptor; for this reason, we produced a recombinant antibody to target cells expressing EpCAM and VEGFR2 via a bispecific antibody to decrease the proliferation and metastasis of tumor cells. Following the cloning and expression of our desired anti-VEGFR2/EPCAM sequence in E. coli, the accuracy of the expression was confirmed by Western blot analysis, and its binding activities to VEGFR2 and EPCAM on MDA-MB-231 and MCF-7 cell lines were respectively indicated by flow cytometry. Then, its anti-proliferative potential was indicated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and apoptosis assay to evaluate inhibitory effects of the antibody on tumor cells. Subsequently, the data indicated that migration, invasion, and angiogenesis were inhibited in breast cancer cell lines via the bispecific antibody. Furthermore, cytokine analysis indicated that the bispecific antibody could moderate interleukin 8 (IL-8) and IL-6 as key mediators in angiogenesis progression in breast cancer. Thus, our bispecific antibody could be considered as a promising candidate tool to decrease angiogenesis in TNBC.

Generating tumor-selective conditionally active biologic anti-CTLA4 antibodies via protein-associated chemical switches.

Anticytotoxic T lymphocyte-associated protein 4 (CTLA4) antibodies have shown potent antitumor activity, but systemic immune activation leads to severe immune-related adverse events, limiting clinical usage. We developed novel, conditionally active biologic (CAB) anti-CTLA4 antibodies that are active only in the acidic tumor microenvironment. In healthy tissue, this binding is reversibly inhibited by a novel mechanism using physiological chemicals as protein-associated chemical switches (PaCS). No enzymes or potentially immunogenic covalent modifications to the antibody are required for activation in the tumor. The novel anti-CTLA4 antibodies show similar efficacy in animal models compared to an analog of a marketed anti-CTLA4 biologic, but have markedly reduced toxicity in nonhuman primates (in combination with an anti-PD1 checkpoint inhibitor), indicating a widened therapeutic index (TI). The PaCS encompass mechanisms that are applicable to a wide array of antibody formats (e.g., ADC, bispecifics) and antigens. Examples shown here include antibodies to EpCAM, Her2, Nectin4, CD73, and CD3. Existing antibodies can be engineered readily to be made sensitive to PaCS, and the inhibitory activity can be optimized for each antigen's varying expression level and tissue distribution. PaCS can modulate diverse physiological molecular interactions and are applicable to various pathologic conditions, enabling differential CAB antibody activities in normal versus disease microenvironments.

Immunotherapy for breast cancer using EpCAM aptamer tumor-targeted gene knockdown.

New strategies for cancer immunotherapy are needed since most solid tumors do not respond to current approaches. Here we used epithelial cell adhesion molecule EpCAM (a tumor-associated antigen highly expressed on common epithelial cancers and their tumor-initiating cells) aptamer-linked small-interfering RNA chimeras (AsiCs) to knock down genes selectively in EpCAM+ tumors with the goal of making cancers more visible to the immune system. Knockdown of genes that function in multiple steps of cancer immunity was evaluated in aggressive triple-negative and HER2+ orthotopic, metastatic, and genetically engineered mouse breast cancer models. Gene targets were chosen whose knockdown was predicted to promote tumor neoantigen expression (Upf2, Parp1, Apex1), phagocytosis, and antigen presentation (Cd47), reduce checkpoint inhibition (Cd274), or cause tumor cell death (Mcl1). Four of the six AsiC (Upf2, Parp1, Cd47, and Mcl1) potently inhibited tumor growth and boosted tumor-infiltrating immune cell functions. AsiC mixtures were more effective than individual AsiC and could synergize with anti-PD-1 checkpoint inhibition.

Highly sensitive electrochemical detection of cancer biomarker based on anti-EpCAM conjugated molybdenum disulfide grafted reduced graphene oxide nanohybrid.

An ultrasensitive, electrochemical biosensor has been fabricated by utilizing molybdenum disulfide (MoS2) grafted reduced graphene oxide (MoS2@rGO) nanohybrid as a sensing platform. Biomolecular-assisted synthetic method was adopted to synthesize MoS2@rGO nanohybrid, where L-cys was used to reduce GO. The MoS2@rGO nanohybrid exhibits improved electrochemical performance when it has been electrophoretically deposited onto the indium tin oxide (ITO) coated glass substrate. Further, epithelialcell adhesion moleculeantibodies (anti-EpCAM) specific to cancer biomarker has been covalently immobilized on the MoS2@rGO/ITO electrodes for label-free detection of EpCAM. Electrochemical results confirm that anti-EpCAM/MoS2@rGO/ITO based biosensor can detect EpCAM in the concentration range of 0.001-20 ng mL-1 with a detection limit of 44.22 fg mL-1 (S/N = 3). The biosensor's excellent analytical performance has been attributed to the efficient immobilization of EpCAM antibodies on the MoS2@rGO surface, which results in high specificity for EpCAM antigen. The fabricated biosensor showed good selectivity, reproducibility, and stability. The successful detection of EpCAM antigen in spiked samples (human saliva, serum and urine) makes this platform an alternative method for early screening of cancer biomarker.

Isolation and characterization of nanobodies against epithelial cell adhesion molecule as novel theranostic agents for cancer therapy.

Epithelial cell adhesion molecule (EpCAM) plays an important role in tumorigenesis. Camelids produce functional antibodies composed of heavy chains only that bind to their antigens via a single domain variable fragment known as nanobody. Nanobodies show multiple advantages over traditional monoclonal antibodies. Isolation of functional anti-EpCAM nanobodies (Nbs) was the main aim of this study. An immune nanobody library containing 108 members was constructed previously. Anti -EpCAM nanobodies were isolated from camel immune library using phage display. Four consecutive rounds of biopanning were performed on immobilized EpCAM. Four nanobodies (Nb4, Nb5, Nb22, and Nb23) with highest signal intensity in monoclonal phage ELISA were selected. Affinity of these selected nanobodies for EpCAM was in the nanomolar range. Selected nanobodies significantly inhibited proliferation of MCF-7 cells. The in vivo study revealed that a significant reduction in tumor size occurred when treated with nanobodies Nb4 and Nb5, after 14 days monitoring. Our data revealed that nanobodies Nb4 and Nb5 could be considered as attractive theranostic agents for EpCAM overexpressing cancers.

N-doped carbon dots modified with the epithelial cell adhesion molecule antibody as an imaging agent for HepG2 cells using their ultra-sensitive response to Al3.

Carbon dots (CDs) are emerging as an ideal multifunctional materials due to their ease of preparation and excellent properties in medical imaging technology, environmental monitoring, chemical analysis and other fields. N-doped CDs modified with the epithelial cell adhesion molecule antibody (anti-EpCAM-NCDs) were synthesized in an ingenious and high-output approach. Due to the fluorescence enhancement effect of the introduced N atoms, the obtained anti-EpCAM-NCDs exhibited a strong green emission with an absolute quantum yield of up to 32.5%. Anti-EpCAM-NCDs have immunofluorescent properties and an active targeting function. The fluorescence effect and fluorescence quenching of anti-EpCAM-NCDs are used to image cells and detect Al3+, respectively. Experimental results show that this probe exhibited a wide linear response to Al3+over a concentration range of 0-100µM with a detection limit and quantification limit of 3 nM and 6 nM, respectively. Significantly, anti-EpCAM-NCDs, which have negligible cytotoxicity, excellent biocompatibility and high photostability, could be used for the intracellular imaging of HepG2 cells and the detection of Al3+in environmental and biological samples. As an efficient multifunctional material, anti-EpCAM-NCDs hold great promise for a number of applications in biological systems.

Development of a novel mammalian display system for selection of antibodies against membrane proteins.

Reliable, specific polyclonal and monoclonal antibodies are important tools in research and medicine. However, the discovery of antibodies against their targets in their native forms is difficult. Here, we present a novel method for discovery of antibodies against membrane proteins in their native configuration in mammalian cells. The method involves the co-expression of an antibody library in a population of mammalian cells that express the target polypeptide within a natural membrane environment on the cell surface. Cells that secrete a single-chain fragment variable (scFv) that binds to the target membrane protein thereby become self-labeled, enabling enrichment and isolation by magnetic sorting and FRET-based flow sorting. Library sizes of up to 109 variants can be screened, thus allowing campaigns of naïve scFv libraries to be selected against membrane protein antigens in a Chinese hamster ovary cell system. We validate this method by screening a synthetic naïve human scFv library against Chinese hamster ovary cells expressing the oncogenic target epithelial cell adhesion molecule and identify a panel of three novel binders to this membrane protein, one with a dissociation constant (KD ) as low as 0.8 nm We further demonstrate that the identified antibodies have utility for killing epithelial cell adhesion molecule-positive cells when used as a targeting domain on chimeric antigen receptor T cells. Thus, we provide a new tool for identifying novel antibodies that act against membrane proteins, which could catalyze the discovery of new candidates for antibody-based therapies.

Efficient Isolation and Phenotypic Profiling of Circulating Hepatocellular Carcinoma Cells via a Combinatorial-Antibody-Functionalized Microfluidic Synergetic-Chip.

As a malignant disease that seriously threatens human health, hepatocellular carcinoma (HCC) lacks effective early screening and prognostic assessment methods. Herein, we developed a method for efficient capture and multiphenotype analysis of circulating tumor cells (CTCs) of hepatocellular carcinoma. The anti-ASGPR antibody and the anti-EpCAM antibody were modified in parallel on a deterministic lateral displacement (DLD)-patterned microfluidic Synergetic-Chip to enhance capture efficiency by a complementary effect. CTCs were detected in 45 out of 45 (100%) HCC patients, with a sensitivity and specificity of 97.8 and 100%, respectively. Patients with more total CTCs and nonepithelial CTCs were in later stages of HCC and had more malignant progression. This strategy proposes a feasible approach for early diagnosis and prognosis of hepatocellular carcinoma.

A Plant-Derived Antigen-Antibody Complex Induces Anti-Cancer Immune Responses by Forming a Large Quaternary Structure.

The antigen-antibody complex (AAC) has novel functions for immunomodulation, encouraging the application of diverse quaternary protein structures for vaccination. In this study, GA733 antigen and anti-GA733 antibody proteins were both co-expressed to obtain the AAC protein structures in a F1 plant obtained by crossing the plants expressing each protein. In F1 plant, the antigen and antibody assembled to form a large quaternary circular ACC structure (~30 nm). The large quaternary protein structures induced immune response to produce anticancer immunoglobulins G (IgGs) that are specific to the corresponding antigens in mouse. The serum containing the anticancer IgGs inhibited the human colorectal cancer cell growth in the xenograft nude mouse. Taken together, antigens and antibodies can be assembled to form AAC protein structures in plants. Plant crossing represents an alternative strategy for the formation of AAC vaccines that efficiently increases anticancer antibody production.

Electrochemical assay for analysis of circulation tumor cells based on isolation of the cell with magnetic nanoparticles and reaction of DNA with molybdate.

A universal strategy was developed for the analysis of circulating tumor cells (CTCs) based on reaction of DNA in the cells with molybdate. Initially, CTCs were enriched and isolated from samples by magnetic nanoparticles. Then, after killing the isolated cells by heat treatment, the cell membrane was raptured, and the DNA molecules contained in the cells were released. The following reaction of the released DNA molecules with molybdate can form redox molybdophosphate, resulting in electrochemical current. This electrochemical assay can be applied to the detection of different CTCs as long as the CTCs can be isolated from the samples, with a universal signal detection method, without additional signal amplification strategies. Breast cancer cell MCF-7 was chosen as a model CTC for this study. At a working potential of 0.2 V vs. Ag/AgCl electrode, the electrochemical current is linearly related to the MCF-7 cell concentration from 5 to 1000 cells mL-1 with a limit of detection of 2 cells mL-1. The assay was successfully applied for detection of MCF-7 in human blood samples. This electrochemical assay can be applied for detection of different CTCs and also for simultaneous detection of CTCs. Graphical abstract A universal strategy was developed for the analysis of circulating tumor cells (CTCs) based on reaction of DNA contained in the cells with molybdate.

Gold nanoparticle-modified black phosphorus nanosheets with improved stability for detection of circulating tumor cells.

Gold nanoparticle (AuNP)-anchored BP nanosheets were synthesized through in situ growth of AuNPs onto BP. Due to the strong chelating ability of P or phosphorus oxides with AuNPs, the stability of BP is improved. As proof-of-concept demonstration of the functionalized BP, electrochemical detection of circulating tumor cells (CTCs) based on BP@AuNPs@aptamer as a probe combined with immunomagnetic separation is reported. The aptamer can specifically bind with CTCs, while the phosphorus oxides including phosphite ion and phosphate ion (PxOy species) on BP and aptamer can react with molybdate to generate an electrochemical current, leading to dual signal amplification. The biosensor is applied to MCF-7 cell detection and displays good analytical performance with a detection limit of 2 cell mL-1. Furthermore, the practicality of this biosensor was validated through sensitive determination of MCF-7 cells in human blood. Therefore, the reported biosensor could be applied to detect other biomarkers, offering an ultrasensitive strategy for clinical diagnostics. Graphical abstract Electrochemical detection of circulating tumor cells based on gold nanoparticle-modified black phosphorus nanosheets is reported.

Influence of antigen density and immunosuppressive factors on tumor-targeted costimulation with antibody-fusion proteins and bispecific antibody-mediated T cell response.

Target expression heterogeneity and the presence of an immunosuppressive microenvironment can hamper severely the efficiency of immunotherapeutic approaches. We have analyzed the potential to encounter and overcome such conditions by a combinatory two-target approach involving a bispecific antibody retargeting T cells to tumor cells and tumor-directed antibody-fusion proteins with costimulatory members of the B7 and TNF superfamily. Targeting the tumor-associated antigens EpCAM and EGFR with the bispecific antibody and costimulatory fusion proteins, respectively, we analyzed the impact of target expression and the influence of the immunosuppressive factors IDO, IL-10, TGF-ß, PD-1 and CTLA-4 on the targeting-mediated stimulation of T cells. Here, suboptimal activity of the bispecific antibody at diverse EpCAM expression levels could be effectively enhanced by targeting-mediated costimulation by B7.1, 4-1BBL and OX40L in a broad range of EGFR expression levels. Furthermore, the benefit of combined costimulation by B7.1/4-1BBL and 4-1BBL/OX40L was demonstrated. In addition, the expression of immunosuppressive factors was shown in all co-culture settings, where blocking of prominent factors led to synergistic effects with combined costimulation. Thus, targeting-mediated costimulation showed general promise for a broad application covering diverse target expression levels, with the option for further selective enhancement by the identification and blockade of main immunosuppressive factors of the particular tumor environment.

Stimuli-Responsive Microfluidic Interface Enables Highly Efficient Capture and Release of Circulating Fetal Cells for Non-Invasive Prenatal Testing.

Circulating fetal nucleated cells (CFCs) carrying whole genomic coding of the fetus in maternal blood have been pursued as ideal biomarkers for noninvasive prenatal testing (NIPT). However, a significant limitation is the need to enrich sufficient cells in quantity and purity for fetal genetic disorder diagnosis. This study for the first time demonstrates a stimuli-responsive ligand enabling interface on array patterned microfluidic chip (NIPT-Chip) for high efficient isolation and release of CFCs in untreated whole blood. Deterministic lateral displacement (DLD)-array was patterned in the chip to increase collision frequency between CFCs and surface-anchored antibody to achieve high efficient cell capture. More importantly, the stimuli-responsive interface enables gentle release of captured CFCs through a thiol exchange reaction for downstream gene analysis of NIPT. With the advantages of simple processing, efficient isolation, and gentle release, NIPT-Chip offers great potential for clinical translation of circulating fetal cell-based NIPT.