Co-Stimulatory Receptor Signaling in CAR-T Cells.

T cell engineering strategies have emerged as successful immunotherapeutic approaches for the treatment of human cancer. Chimeric Antigen Receptor T (CAR-T) cell therapy represents a prominent synthetic biology approach to re-direct the specificity of a patient's autologous T cells toward a desired tumor antigen. CAR-T therapy is currently FDA approved for the treatment of hematological malignancies, including subsets of B cell lymphoma, acute lymphoblastic leukemia (ALL) and multiple myeloma. Mechanistically, CAR-mediated recognition of a tumor antigen results in propagation of T cell activation signals, including a co-stimulatory signal, resulting in CAR-T cell activation, proliferation, evasion of apoptosis, and acquisition of effector functions. The importance of including a co-stimulatory domain in CARs was recognized following limited success of early iteration CAR-T cell designs lacking co-stimulation. Today, all CAR-T cells in clinical use contain either a CD28 or 4-1BB co-stimulatory domain. Preclinical investigations are exploring utility of including additional co-stimulatory molecules such as ICOS, OX40 and CD27 or various combinations of multiple co-stimulatory domains. Clinical and preclinical evidence implicates the co-stimulatory signal in several aspects of CAR-T cell therapy including response kinetics, persistence and durability, and toxicity profiles each of which impact the safety and anti-tumor efficacy of this immunotherapy. Herein we provide an overview of CAR-T cell co-stimulation by the prototypical receptors and discuss current and emerging strategies to modulate co-stimulatory signals to enhance CAR-T cell function.

Indoleamine 2,3-dioxygenase 1 is essential for sustaining durable antibody responses.

Humoral immunity is essential for protection against pathogens, emphasized by the prevention of 2-3 million deaths worldwide annually by childhood immunizations. Long-term protective immunity is dependent on the continual production of neutralizing antibodies by the subset of long-lived plasma cells (LLPCs). LLPCs are not intrinsically long-lived, but require interaction with LLPC niche stromal cells for survival. However, it remains unclear which and how these interactions sustain LLPC survival and long-term humoral immunity. We now have found that the immunosuppressive enzyme indoleamine 2,3- dioxygenase 1 (IDO1) is required to sustain antibody responses and LLPC survival. Activation of IDO1 occurs upon the engagement of CD80/CD86 on the niche dendritic cells by CD28 on LLPC. Kynurenine, the product of IDO1 catabolism, activates the aryl hydrocarbon receptor in LLPC, reinforcing CD28 expression and survival signaling. These findings expand the immune function of IDO1 and uncover a novel pathway for sustaining LLPC survival and humoral immunity.

Electrochemical Detection of Fertility Hormones.

Fertility hormone levels are constantly changing, but it is crucial for a woman to be able to monitor her fertility levels if she is interested in conceiving. Women and physicians often have a difficult time determining ovulation windows due to fluctuating menstrual cycles and inaccurate interpretations of hormone levels. Current methods of fertility monitoring include physical or vaginal exams, laparoscopy, ultrasound scans, as well as evaluation of hormone levels. A rapid, at-home fertility monitoring tool can help alleviate the apprehensiveness associated with routine screenings and give women the privacy desired when trying to conceive. Herein, we discuss the development of an electrochemical biosensor for quantification of three fertility hormones: beta-estradiol, progesterone, and FSH. Each biomarker's MRE was immobilized onto a gold disk electrode through the use of self-assembled monolayer linking chemistry. Using electrochemical impedance spectroscopy (EIS), the biomarker concentration was correlated to impedance magnitude. An optimal binding frequency was identified for each biomarker, permitting simplistic hardware requirements and investigation into multimarker detection. Analytes were tested in both purified solutions and 1%-90% whole blood. Each biomarker exhibited a unique imaginary impedance peak and optimal binding frequency. The determination was made by assessing the response parameters including the linear fit correlation across the physiological hormone ranges. The existence of unique optimal frequencies permits for simultaneous detection of multiple hormones in a single test. Additionally, the identified frequency was robust across purified and complex solutions. Response characteristics were negatively impacted by the introduction of blood-based contaminants. However, the introduction of Nafion membranes, similar to ones used in commercial glucose sensors, is both feasible and beneficial.

An Experimental Platform for Characterizing Cancer Biomarkers with Capabilities in Noninvasive and Continuous Screening.

Early detection is crucial to the proper and effective treatment of two metastatic cancers, prostate cancer and small cell lung cancer. Currently, preventative screenings for these conditions are restricted to high-risk populations and extremely expensive. The discovery of clinically indicative biomarkers has been revolutionary in advancing screening and diagnostic capabilities. Prostate-specific antigen (PSA), an extracellular secreted protein of the prostate gland, and neuron-specific enolase (NSE), an enzyme of neuronal origin, have reported reputable specificity for prostate cancer and small cell lung cancer (SCLC). Current efforts are underway to develop a rapid, label-free means of measuring both PSA and NSE levels in a clinical environment for early screening applications of highly metastatic cancers. Electrochemical impedance spectroscopy (EIS) and impedance time (Z-t) are rapid, sensitive electrochemical techniques previously validated in the detection of several clinically relevant biomarkers, including cardiovascular disease and diabetes mellitus. Herein, we determine the optimal frequencies of PSA (81.38 Hz) and NSE (14.36 Hz) using EIS that are robust across analytical platforms and in the presence of potentially interfering species. The reported empirical evidence supports the prevalence of electrostatic interactions in electrochemical systems and provides alternative theoretical support of previous findings. Finally, Z-t was implemented for its utility in continuous monitoring applications and to lay the foundation for future improvements to continuous sensor platforms.

Facilitating Earlier Diagnosis of Cardiovascular Disease through Point-of-Care Biosensors: A Review.

Cardiovascular disease (CVD) accounts for 30% of all global deaths and is predicted to dominate in the coming years, despite vast improvements in medical technology. Current clinical methods of assessing an individual's cardiovascular health include blood tests to monitor relevant biomarker levels as well as varying imaging modalities such as electrocardiograms, computed tomography, and angiograms to assess vasculature. As informative as these tools are, they each require lengthy scheduling, preparation, and highly trained personnel to interpret the results before any information is accessible to patients, often leading to delayed treatment, which can be fatal. A point-of-care (POC) sensor platform is thus paramount in rapid and early diagnosis of CVD. Among the many POC detection platforms, including established optical and mechanical methods, electrochemical-based detection mechanisms have become increasingly desirable because of their superior sensitivity, low cost, and label-free detection. Specifically, electrochemical impedance spectroscopy (EIS) has demonstrated remarkable abilities in low-level (femtomolar) detection of several clinically useful biomarkers and has been reported in CVD diagnostic applications. In this review, we provide an in-depth overview of prevalent CVD diseases and clinically relevant proteomic biomarkers for assessing them. Subsequently, we discuss the ongoing development of POC sensors for CVD, highlighting the current clinical gold standard, potential alternative modalities, and electrochemical methodologies previously successful in quantifying specific biomarkers approved by the Food and Drug Administration (FDA). A discussion of EIS highlighting the attributes and capabilities of novel analysis algorithms is included to showcase the possibility of simultaneous dual-marker detection.

Direct Measurement of a Biomarker's Native Optimal Frequency with Physical Adsorption Based Immobilization.

The optimal frequency (OF) of a biomarker in electrochemical impedance spectroscopy (EIS) is the frequency at which the EIS response best reflects the binding of the biomarker to its molecular recognition element. Commonly, biosensors rely on complicated immobilization chemistry to attach biological molecules to the sensor surface, making the direct study of a biomarker's native OF a challenge. Physical adsorption presents a simple immobilization strategy to study the native biomarker's OF, but its utility is often discouraged due to a loss in biological activity. To directly study a biomarker's native OF and investigate the potential of OF to overcome the limitations of physical adsorption, a combination of EIS and glutaraldehyde-mediated physical adsorption was explored. The experimental sensing platform was prepared by immobilizing either anti-lactoferrin (Lfn) IgG or anti-immunoglobulin E (IgE) onto screen printed carbon electrodes. After characterizing the native OFs of both biomarkers, investigation of the platform's specificity, stability, and performance in complex medium was found to be sufficient. Finally, a paper-based tear sampling component was integrated to transform the testing platform into a prototypical point-of-care dry eye diagnostic. The investigation of native OFs revealed a correlation between the native OFs (57.44 and 371.1 Hz for Lfn and IgE, respectively) and the molecular weight of the antibody-antigen complex. Impedance responses at the native OFs have enabled detection limits of 0.05 mg/mL and 40 ng/mL for Lfn and IgE, respectively, covering the clinically relevant ranges. The native OFs were found to be robust across various testing mediums and conditions.