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Expression of programmed cell death ligand 1 (PD-L1) within tumors is an important biomarker for guiding immune checkpoint therapies; however, immunohistochemistry-based methods of detection fail to provide a comprehensive picture of PD-L1 levels in an entire patient. To facilitate quantification of PD-L1 in the whole body, we developed a peptide-based, high-affinity PD-L1 imaging agent labeled with [18F]fluoride for positron emission tomography (PET) imaging. The parent peptide, WL12, and the nonradioactive analog of the radiotracer, 19FPy-WL12, inhibit PD-1/PD-L1 interaction at low nanomolar concentrations (half maximal inhibitory concentration [IC50], 26-32 nM). The radiotracer, [18F]FPy-WL12, was prepared by conjugating 2,3,5,6-tetrafluorophenyl 6-[18F]fluoronicotinate ([18F]FPy-TFP) to WL12 and assessed for specificity in vitro in 6 cancer cell lines with varying PD-L1 expression. The uptake of the radiotracer reflected the PD-L1 expression assessed by flow cytometry. Next, we performed the in vivo evaluation of [18F]FPy-WL12 in mice bearing cancer xenografts by PET imaging, ex vivo biodistribution, and blocking studies. In vivo data demonstrated a PD-L1-specific uptake of [18F]FPy-WL12 in tumors that is reduced in mice receiving a blocking dose. The majority of [18F]FPy-WL12 radioactivity was localized in the tumors, liver, and kidneys indicating the need for optimization of the labeling strategy to improve the in vivo pharmacokinetics of the radiotracer.

A majority of cancers fail to respond to immunotherapy with antibodies targeting immune checkpoints, such as cytotoxic T-lymphocyte antigen-4 (CTLA-4) or programmed death-1 (PD-1)/PD-1 ligand (PD-L1). Cancers frequently express transforming growth factor-β (TGFβ), which drives immune dysfunction in the tumor microenvironment by inducing regulatory T cells (Tregs) and inhibiting CD8 + and T H 1 cells. To address this therapeutic challenge, we invent bifunctional antibody–ligand traps (Y-traps) comprising an antibody targeting CTLA-4 or PD-L1 fused to a TGFβ receptor II ectodomain sequence that simultaneously disables autocrine/paracrine TGFβ in the target cell microenvironment ( a -CTLA4-TGFβRII ecd and a -PDL1-TGFβRII ecd ). a -CTLA4-TGFβRII ecd is more effective in reducing tumor-infiltrating Tregs and inhibiting tumor progression compared with CTLA-4 antibody (Ipilimumab). Likewise, a -PDL1-TGFβRII ecd exhibits superior antitumor efficacy compared with PD-L1 antibodies (Atezolizumab or Avelumab). Our data demonstrate that Y-traps counteract TGFβ-mediated differentiation of Tregs and immune tolerance, thereby providing a potentially more effective immunotherapeutic strategy against cancers that are resistant to current immune checkpoint inhibitors. Antitumor T cells can be inhibited by a TGFβ rich tumor microenvironment. The authors develop bifunctional proteins comprising CTLA-4 or PD-L1 immune checkpoint-targeted antibodies fused to a “TGFβ trap” and show that they counteract tumor immune tolerance and enhance the efficacy of these antibodies.

Therapeutics targeting programmed death ligand 1 (PD-L1) protein and its receptor PD-1 are now dominant players in restoring anti-tumor immune responses. PD-L1 detection by immunohistochemistry (IHC) is emerging as a reproducible biomarker for guiding patient stratification for those therapies in some cancers. However, PD-L1 expression in the tumor microenvironment is highly complex. It is upregulated by aberrant genetic alterations, and is highly regulated at the transcriptional, posttranscriptional, and protein levels. Thus, PD-L1 IHC is inadequate to fully understand the relevance of PD-L1 levels in the whole body and their dynamics to improve therapeutic outcomes. Imaging technologies could potentially assist in meeting that need. Early clinical investigations show promising results in quantifying PD-L1 expression in the whole body by positron emission tomography (PET). Within this context, this review summarizes advancements in regulation of PD-L1 expression and imaging agents, and in PD-L1 PET for drug development, and discusses opportunities and challenges presented by these innovations for guiding immune checkpoint therapy (ICT).

Macromolecules such as monoclonal antibodies (mAbs) are likely to experience poor tumor penetration because of their large size, and thus low drug exposure of target cells within a tumor could contribute to suboptimal responses. Given the challenge of inadequate quantitative tools to assess mAb activity within tumors, we hypothesized that measurement of accessible target levels in tumors could elucidate the pharmacologic activity of a mAb and could be used to compare the activity of different mAbs. Using positron emission tomography (PET), we measured the pharmacodynamics of immune checkpoint protein programmed-death ligand 1 (PD-L1) to evaluate pharmacologic effects of mAbs targeting PD-L1 and its receptor programmed cell death protein 1 (PD-1). For PD-L1 quantification, we first developed a small peptide-based fluorine-18–labeled PET imaging agent, [18F]DK222, which provided high-contrast images in preclinical models. We then quantified accessible PD-L1 levels in the tumor bed during treatment with anti–PD-1 and anti–PD-L1 mAbs. Applying mixed-effects models to these data, we found subtle differences in the pharmacodynamic effects of two anti–PD-1 mAbs (nivolumab and pembrolizumab). In contrast, we observed starkly divergent target engagement with anti–PD-L1 mAbs (atezolizumab, avelumab, and durvalumab) that were administered at equivalent doses, correlating with differential effects on tumor growth. Thus, we show that measuring PD-L1 pharmacodynamics informs mechanistic understanding of therapeutic mAbs targeting PD-L1 and PD-1. These findings demonstrate the value of quantifying target pharmacodynamics to elucidate the pharmacologic activity of mAbs, independent of mAb biophysical properties and inclusive of all physiological variables, which are highly heterogeneous within and across tumors and patients.

Identification of highly aggressive tumors from benign phenotypes is an important diagnostic need, with increased expression of hypoxia-inducible factor-1α (HIF-1α) as one factor linked to tumor progression and treatment response. HIF-1α stabilization is closely linked to extracellular pH (pHe) through the regulation of glycolysis, proton transporters and vascular endothelial growth factor (VEGF). Magnetic resonance imaging (MRI)—based pHe measurement has shown promise in differentiating tumor phenotypes. In this study, we evaluated a new protocol designed to enhance tumor acidosis by administering glucose prior to pHe measurements in orthotopic human breast cancer xenografts. To demonstrate the utility of this approach, we used human triple-negative breast cancer (TNBC) MDA-MB-231 cells that were either wild type (231-WT) or genetically engineered to stably express short hairpin RNA (shRNA) against HIF-1α (231-HIF-1α-shRNA) or engineered to overexpress VEGF (231-VEGF). Iopamidol was administered for chemical exchange saturation transfer (CEST) MRI pHe mapping. We observed enhanced differentiation in the pHe maps following glucose administration, with mean pHe of 6.1 ± 0.12 (231-WT), 6.3 ± 0.04 (231-VEGF) and 6.58 ± 0.04 (231-HIF-1α-shRNA). Without glucose stimulation, the corresponding values were 6.31 ± 0.04 (231-WT), 6.30 ± 0.07 (231-VEGF), and 6.55 ± 0.04 (231-HIF-1α-shRNA). These findings were validated by immunoblotting and immunohistochemistry. Collectively our data demonstrate that CEST MRI pHe mapping can effectively differentiates tumors with low HIF-1α expression and that glucose stimulation enhances this differentiation, offering a valuable tool for improved tumor characterization.

Background Chimeric antigen receptor (CAR) T cell therapy is an exciting cell-based cancer immunotherapy. Unfortunately, CAR-T cell therapy is associated with serious toxicities such as cytokine release syndrome (CRS) and neurotoxicity. The mechanism of these serious adverse events (SAEs) and how homing, distribution and retention of CAR-T cells contribute to toxicities is not fully understood. Enabling in vitro methods to allow meaningful, sensitive in vivo biodistribution studies is needed to better understand CAR-T cell disposition and its relationship to both effectiveness and safety of these products. Methods To determine if radiolabelling of CAR-T cells could support positron emission tomography (PET)-based biodistribution studies, we labeled IL-13Rα2 targeting scFv-IL-13Rα2-CAR-T cells (CAR-T cells) with 89 Zirconium-oxine ( 89 Zr-oxine) and characterized and compared their product attributes with non-labeled CAR-T cells. The 89 Zr-oxine labeling conditions were optimized for incubation time, temperature, and use of serum for labeling. In addition, T cell subtype characterization and product attributes of radiolabeled CAR-T cells were studied to assess their overall quality including cell viability, proliferation, phenotype markers of T-cell activation and exhaustion, cytolytic activity and release of interferon-γ upon co-culture with IL-13Rα2 expressing glioma cells. Results We observed that radiolabeling of CAR-T cells with 89 Zr-oxine is quick, efficient, and radioactivity is retained in the cells for at least 8 days with minimal loss. Also, viability of radiolabeled CAR-T cells and subtypes such as CD4 + , CD8 + and scFV-IL-13Rα2 transgene positive T cell population were characterized and found similar to that of unlabeled cells as determined by TUNEL assay, caspase 3/7 enzyme and granzyme B activity assay. Moreover, there were no significant changes in T cell activation (CD24, CD44, CD69 and IFN-γ) or T cell exhaustion (PD-1, LAG-3 and TIM3) markers expression between radiolabeled and unlabeled CAR-T cells. In chemotaxis assays, migratory capability of radiolabeled CAR-T cells to IL-13Rα2Fc was similar to that of non-labeled cells. Conclusions Importantly, radiolabeling has minimal impact on biological product attributes including potency of CAR-T cells towards IL-13Rα2 positive tumor cells but not IL-13Rα2 negative cells as measured by cytolytic activity and release of IFN-γ. Thus, IL-13Rα2 targeting CAR-T cells radiolabeled with 89 Zr-oxine retain critical product attributes and suggest 89 Zr-oxine radiolabeling of CAR-T cells may facilitate biodistribution and tissue trafficking studies in vivo using PET.

Background Molecular simulations are used to provide insight into protein structure and dynamics, and have the potential to provide important context when predicting the impact of sequence variation on protein function. In addition to understanding molecular mechanisms and interactions on the atomic scale, translational applications of those approaches include drug screening, development of novel molecular therapies, and targeted treatment planning. Supporting the continued development of these applications, we have developed the SNP2SIM workflow that generates reproducible molecular dynamics and molecular docking simulations for downstream functional variant analysis. The Python workflow utilizes molecular dynamics software (NAMD (Phillips et al., J Comput Chem 26(16):1781-802, 2005), VMD (Humphrey et al., J Mol Graph 14(1):33-8, 27-8, 1996)) to generate variant specific scaffolds for simulated small molecule docking (AutoDock Vina (Trott and Olson, J Comput Chem 31(2):455-61, 2010)). Results SNP2SIM is composed of three independent modules that can be used sequentially to generate the variant scaffolds of missense protein variants from the wildtype protein structure. The workflow first generates the mutant structure and configuration files required to execute molecular dynamics simulations of solvated protein variant structures. The resulting trajectories are clustered based on the structural diversity of residues involved in ligand binding to produce one or more variant scaffolds of the protein structure. Finally, these unique structural conformations are bound to small molecule ligand libraries to predict variant induced changes to drug binding relative to the wildtype protein structure. Conclusions SNP2SIM provides a platform to apply molecular simulation based functional analysis of sequence variation in the protein targets of small molecule therapies. In addition to simplifying the simulation of variant specific drug interactions, the workflow enables large scale computational mutagenesis by controlling the parameterization of molecular simulations across multiple users or distributed computing infrastructures. This enables the parallelization of the computationally intensive molecular simulations to be aggregated for downstream functional analysis, and facilitates comparing various simulation options, such as the specific residues used to define structural variant clusters. The Python scripts that implement the SNP2SIM workflow are available (SNP2SIM Repository. https://github.com/mccoymd/SNP2SIM , Accessed 2019 February ), and individual SNP2SIM modules are available as apps on the Seven Bridges Cancer Genomics Cloud (Lau et al., Cancer Res 77(21):e3-e6, 2017; Cancer Genomics Cloud [ www.cancergenomicscloud.org ; Accessed 2018 November]).

The CD44 transmembrane glycoproteins play multifaceted roles in tumor progression and metastasis. CD44 expression has also been associated with stem-like breast cancer cells. Hypoxia commonly occurs in tumors and is a major cause of radiation and chemo-resistance. Hypoxia is known to inhibit differentiation and facilitates invasion and metastasis. Here we have investigated the effect of hypoxia on CD44 and two of its isoforms in MDA-MB-231 and SUM-149 triple negative human breast cancer cells and MDA-MB-231 tumors using imaging and molecular characterization. The roles of hypoxia and hypoxia inducible factor (HIF) in regulating the expression of CD44 and its variant isoforms (CD44v6, CD44v7/8) were investigated in human breast cancer cells, by quantitative real-time polymerase chain reaction (qRT-PCR) to determine mRNA levels, and fluorescence associated cell sorting (FACS) to determine cell surface expression of CD44, under normoxic and hypoxic conditions. In vivo imaging studies with tumor xenografts derived from MDA-MD-231 cells engineered to express tdTomato red fluorescence protein under regulation of hypoxia response elements identified co-localization between hypoxic fluorescent regions and increased concentration of (125)I-radiolabeled CD44 antibody. Our data identified HIF-1α as a regulator of CD44 that increased the number of CD44 molecules and the percentage of CD44 positive cells expressing variant exons v6 and v7/8 in breast cancer cells under hypoxic conditions. Data from these cell studies were further supported by in vivo observations that hypoxic tumor regions contained cells with a higher concentration of CD44 expression.

The limited availability of molecularly targeted low‐molecular‐weight imaging agents for monitoring multiple myeloma (MM)‐targeted therapies has been a significant challenge in the field. In response, a first‐in‐class peptide‐based radiotracer, [68Ga]Ga‐AJ206, is developed that can be seamlessly integrated into the standard clinical workflow and is specifically designed to noninvasively quantify CD38 levels and pharmacodynamics by positron emission tomography (PET). A bicyclic peptide, AJ206, is synthesized and exhibits high affinity to CD38 (KD: 19.1 ± 0.99 × 10−9 m) by surface plasmon resonance. Further, [68Ga]Ga‐AJ206‐PET shows high contrast within 60 min and suitable absorbed dose estimates for clinical use. Additionally, [68Ga]Ga‐AJ206 detects CD38 expression in cell line‐derived xenografts, patient‐derived xenografts (PDXs), and disseminated disease models in a manner consistent with flow cytometry and immunohistochemistry findings. Moreover, [68Ga]Ga‐AJ206‐PET successfully quantifies CD38 pharmacodynamics in PDXs, revealing increased CD38 expression in the tumor following all‐trans retinoic acid (ATRA) therapy. In conclusion, [68Ga]Ga‐AJ206 exhibits the salient features required for clinical translation, providing CD38‐specific high‐contrast images in multiple models of MM. [68Ga]Ga‐AJ206‐PET could be useful for quantifying total CD38 levels and pharmacodynamics during therapy to evaluate approved and new therapies in MM and other diseases with CD38 involvement. A novel peptide‐based gallium‐68 labeled radiotracer, [68Ga]Ga‐AJ206, is developed for imaging multiple myeloma using positron emission tomography (PET). [68Ga]Ga‐AJ206 delivers PET images with high contrast within 60 min and detected varying levels of CD38 expression with high sensitivity (left panel). Furthermore, [68Ga]Ga‐AJ206‐PET enables noninvasive quantification of CD38 pharmacodynamics (right panel).