Advances in precision molecular imaging promise to transform our ability to detect, diagnose and treat disease. Here, we describe the engineering and validation of a new cystine knot peptide (knottin) that selectively recognizes human integrin αvβ₆ with single-digit nanomolar affinity. We solve its 3D structure by NMR and x-ray crystallography and validate leads with 3 different radiolabels in pre-clinical models of cancer. We evaluate the lead tracer’s safety, biodistribution and pharmacokinetics in healthy human volunteers, and show its ability to detect multiple cancers (pancreatic, cervical and lung) in patients at two study locations. Additionally, we demonstrate that the knottin PET tracers can also detect fibrotic lung disease in idiopathic pulmonary fibrosis patients. Our results indicate that these cystine knot PET tracers may have potential utility in multiple disease states that are associated with upregulation of integrin α_vβ₆.

The biological impact of exogenous, alternating electric fields (AEFs) and direct-current electric fields has a long history of study, ranging from effects on embryonic development to influences on wound healing. In this article, we focus on the application of electric fields for the treatment of cancers. In particular, we outline the clinical impact of tumor treating fields (TTFields), a form of AEFs, on the treatment of cancers such as glioblastoma and mesothelioma. We provide an overview of the standard mechanism of action of TTFields, namely, the capability for AEFs (e.g., TTFields) to disrupt the formation and segregation of the mitotic spindle in actively dividing cells. Though this standard mechanism explains a large part of TTFields’ action, it is by no means complete. The standard theory does not account for exogenously applied AEFs’ influence directly upon DNA nor upon their capacity to alter the functionality and permeability of cancer cell membranes. This review summarizes the current literature to provide a more comprehensive understanding of AEFs’ actions on cell membranes. It gives an overview of three mechanistic models that may explain the more recent observations into AEFs’ effects: the voltage-gated ion channel, bioelectrorheological, and electroporation models. Inconsistencies were noted in both effective frequency range and field strength between TTFields versus all three proposed models. We addressed these discrepancies through theoretical investigations into the inhomogeneities of electric fields on cellular membranes as a function of disease state, external microenvironment, and tissue or cellular organization. Lastly, future experimental strategies to validate these findings are outlined. Clinical benefits are inevitably forthcoming.

Purpose: Therapeutic checkpoint inhibitors on tumor-infiltrating lymphocytes (TIL) are being increasingly utilized in the clinic. The T-cell immunoreceptor with Ig and ITIM domains (TIGIT) is an inhibitory receptor expressed on T and natural killer cells. The TIGIT signaling pathway is an alternative target for checkpoint blockade to current PD-1/CTLA-4 strategies. Elevated TIGIT expression in the tumor microenvironment correlates with better therapeutic responses to anti-TIGIT therapies in preclinical models. Therefore, quantifying TIGIT expression in tumors is necessary for determining whether a patient may respond to anti-TIGIT therapy. PET imaging of TIGIT expression on TILs can therefore aid diagnosis and in monitoring therapeutic responses. Experimental Design: Antibody-based TIGIT imaging radiotracers were developed with the PET radionuclides copper-64 (64Cu) and zirconium-89 (89Zr). In vitro characterization of the imaging probes was followed by in vivo evaluation in both xenografts and syngeneic tumor models in mouse. Results: Two anti-TIGIT probes were developed and exhibited immunoreactivity of >72%, serum stability of >95%, and specificity for TIGIT with both mouse TIGIT-expressing HeLa cells and ex vivo–activated primary splenocytes. In vivo, the 89Zr-labeled probe demonstrated superior contrast than the 64Cu probe due to 89Zr's longer half-life matching the TIGIT antibody's pharmacokinetics. The 89Zr probe was used to quantify TIGIT expression on TILs in B16 melanoma in immunocompetent mice and confirmed by ex vivo flow cytometry. Conclusions: This study develops and validates novel TIGIT-specific 64Cu and 89Zr PET probes for quantifying TIGIT expression on TILs for diagnosis of patient selection for anti-TIGIT therapies.

Recently, considerable interest has emerged in the development of biosensors to detect biomarkers and immune checkpoints to identify and measure cancer through liquid biopsies. The detection of cancer biomarkers from a small volume of blood is relatively fast compared to the gold standard of tissue biopsies. Traditional immuno-histochemistry (IHC) requires tissue samples obtained using invasive procedures and specific expertise as well as sophisticated instruments. Furthermore, the turnaround for IHC assays is usually several days. To overcome these challenges, on-demand biosensor-based assays were developed to provide more immediate prognostic information for clinicians. Novel rapid, highly precise, and sensitive approaches have been under investigation using physical and biochemical methods to sense biomarkers. Additionally, interest in understanding immune checkpoints has facilitated the rapid detection of cancer prognosis from liquid biopsies. Typically, these devices combine various classes of detectors with digital outputs for the measurement of soluble cancer or immune checkpoint (IC) markers from liquid biopsy samples. These sensor devices have two key advantages: (a) a small volume of blood drawn from the patient is sufficient for analysis, and (b) it could aid physicians in quickly selecting and deciding the appropriate therapy regime for the patients (e.g., immune checkpoint blockade (ICB) therapy). In this review, we will provide updates on potential cancer markers, various biosensors in cancer diagnosis, and the corresponding limits of detection, while focusing on biosensor development for IC marker detection.

Glypican-3 (GPC3) is an attractive diagnostic marker for hepatocellular carcinoma (HCC). We previously reported the potential of an ⁸⁹Zr-labeled murine anti-GPC3 antibody (clone 1G12) for immunoPET imaging of HCC in orthotopic patient-derived xenograft (PDX) mouse models. We now humanized the murine antibody by complementarity determining region (CDR) grafting, to allow its clinical translation for human use. The engineered humanized anti-GPC3 antibody, clone H3K3, retained comparable binding affinity and specificity to human GPC3. H3K3 was conjugated with desferrioxamine (Df) and radiolabeled with ⁸⁹Zr to produce the PET/CT tracer ⁸⁹Zr-Df-H3K3. When injected into GPC3-expressing orthotopic HCC PDX in NOD SCID Gamma (NSG) mice, ⁸⁹Zr-Df-H3K3 showed specific high uptake into the orthotopic PDX and minimal, non-specific uptake into the non-tumor bearing liver. Specificity was demonstrated by significantly higher uptake of ⁸⁹Zr-Df-H3K3 into the non-blocked PDX mice, compared with the blocked PDX mice (which received prior injection of 100 mg of unlabeled H3K3). Region of interest (ROI) analysis showed that the PDX/non-tumor liver ratio was highest (mean ± SD: 3.4 ± 0.31) at 168 h post injection; this ratio was consistent with biodistribution studies at the same time point. Thus, our humanized anti-GPC3 antibody, H3K3, shows encouraging potential for use as an immunoPET tracer for diagnostic imaging of HCC patients.