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Simple Summary: The specific mechanisms by which tumors acquire resistance to chimeric antigen receptor (CAR) T cell therapy are not completely understood. The aim of this study was to elucidate the complex interactions between tumor cells and CAR T cells targeting epithelial cell adhesion molecule (EpCAM) in a xenograft model of gastric cancer. Using whole-body CAR T cell imaging and single-cell multiomic analyses, we noticed that within resistant tumors, CAR T cells exhibited a tendency to proliferate, but they were largely dysfunctional, losing their ability to fight cancer effectively. Specifically, most CD8 T cells became exhausted within tumors, while CD4 T cells transformed into regulatory T cells that can dampen the immune response. Additionally, the resistant tumor cells had specific gene changes that could promote cancer growth and make the disease more challenging to cure. This research provides valuable information for understanding how tumors resist CAR T cell therapy and may guide future developments in cancer treatment. Epithelial cell adhesion molecule (EpCAM) is a tumor-associated antigen that is frequently overexpressed in various carcinomas. We have developed chimeric antigen receptor (CAR) T cells specifically targeting EpCAM for the treatment of gastric cancer. This study sought to unravel the precise mechanisms by which tumors evade immune surveillance and develop resistance to CAR T cell therapy. Through a combination of whole-body CAR T cell imaging and single-cell multiomic analyses, we uncovered intricate interactions between tumors and tumor-infiltrating lymphocytes (TILs). In a gastric cancer model, tumor-infiltrating CD8 T cells exhibited both cytotoxic and exhausted phenotypes, while CD4 T cells were mainly regulatory T cells. A T cell receptor (TCR) clonal analysis provided evidence of CAR T cell proliferation and clonal expansion within resistant tumors, which was substantiated by whole-body CAR T cell imaging. Furthermore, single-cell transcriptomics showed that tumor cells in mice with refractory or relapsing outcomes were enriched for genes involved in major histocompatibility complex (MHC) and antigen presentation pathways, interferon-γ and interferon-α responses, mitochondrial activities, and a set of genes (e.g., CD74, IDO1, IFI27) linked to tumor progression and unfavorable disease prognoses. This research highlights an approach that combines imaging and multiomic methodologies to concurrently characterize the evolution of tumors and the differentiation of CAR T cells. [ABSTRACT FROM AUTHOR]

The ability to image adoptively transferred T cells in the body and to eliminate them to avoid toxicity will be vital for chimeric antigen receptor (CAR) T cell therapy, particularly against solid tumors with higher risk of off-tumor toxicity. Previously, we have demonstrated the utility of somatostatin receptor 2 (SSTR2) for CAR T cell imaging, illustrating the expansion and contraction of CAR T cells in tumor as well as off-tumor expansion. Using intercellular adhesion molecule 1 (ICAM-1)-specific CAR T cells that secrete interleukin (IL)-12 as a model, herein we examined the potential of SSTR2 as a safety switch when combined with the SSTR2-specific maytansine-octreotate conjugate PEN-221. Constitutive secretion of IL-12 led to continuous expansion of CAR T cells after rapid elimination of tumors, causing systemic toxicity in mice with intact MHC expression. Treatment with PEN-221 rapidly reduced the abundance of CAR T cells, decreasing the severity of xenogeneic graft-versus-host disease (GvHD), and prolonged survival. Our study supports the development of SSTR2 as a single genetic marker for CAR T cells that is readily applicable to humans both for anatomical detection of T cell distribution and an image-guided safety switch for rapid elimination of CAR T cells. [ABSTRACT FROM AUTHOR]

Background: Anaplastic thyroid cancer (ATC) is a rare cancer with poor prognosis and few treatment options. The objective of this study was to investigate new immune-associated therapeutic targets by identifying ATC-derived, human leukocyte antigen (HLA) class II-presenting peptides. One protein that generated multiple peptides in ATC was chondroitin sulfate-proteoglycan-4 (CSPG4), a transmembrane proteoglycan with increased expression in multiple aggressive cancers, but not yet investigated in ATC. Methods: We applied autologous peripheral blood T cells to ATC patient-derived xenografted mice to examine whether ATC induces a tumor-specific T cell response. We then identified peptide antigens eluted from the HLA-DQ complex in ATC patient-derived cells using mass spectrometry, detecting abundant CSPG4-derived peptides specific to the ATC sample. Next, we analyzed the surface expression level of CSPG4 in thyroid cancer cell lines and primary cell culture using flow cytometry. In addition, we used immunohistochemistry to compare the expression level and localization of the CSPG4 protein in ATC, papillary thyroid cancer, and normal thyroid tissue. We then investigated the correlation between CSPG4 expression and clinicopathological features of patients with thyroid cancer. Results: We found that ATC tissue had a high level of HLA-DQ expression and that the patient's CD4+ T cells showed activation when exposed to ATC. By eluting the HLA-DQ complex of ATC tissue, we found that CSPG4 generated one of the most abundant and specific peptides. CSPG4 expression at the cell surface of thyroid cancer was also significantly high when determined by flow cytometry, with the majority of ATC cell lines exhibiting ∼10-fold higher mean fluorescence intensity. Furthermore, most ATC patient cases expressed CSPG4 in the cytoplasm or membrane of the tumor cells. CSPG4 expression was correlated with tumor size, extrathyroidal extension, and intercellular adhesion molecule-1 (ICAM-1) circumferential expression. CSPG4 mRNA overexpression was associated with worse overall survival in patients with ATC and poorly differentiated thyroid cancer. Conclusions: CSPG4 expression is significantly elevated in aggressive thyroid cancers, with a strong correlation with a poor prognosis. The vast number of HLA-DQ eluted CSPG4 peptides was identified in ATC, demonstrating the potential of CSPG4 as a novel immunotherapeutic target for ATC. [ABSTRACT FROM AUTHOR]

The use of magnetic fluid hyperthermia (MFH) for cancer therapy has shown promise but lacks suitable methods for quantifying exogenous irons such as superparamagnetic iron oxide (SPIO) nanoparticles as a source of heat generation under an alternating magnetic field (AMF). Application of quantitative susceptibility mapping (QSM) technique to prediction of SPIO in preclinical models has been challenging due to a large variation of susceptibility values, chemical shift from tissue fat, and noisier data arising from the higher resolution required to visualize the anatomy of small animals. In this study, we developed a robust QSM for the SPIO ferumoxytol in live mice to examine its potential application in MFH for cancer therapy. We demonstrated that QSM was able to simultaneously detect high level ferumoxytol accumulation in the liver and low level localization near the periphery of tumors. Detection of ferumoxytol distribution in the body by QSM, however, required imaging prior to and post ferumoxytol injection to discriminate exogenous iron susceptibility from other endogenous sources. Intratumoral injection of ferumoxytol combined with AMF produced a ferumoxytol-dose dependent tumor killing. Histology of tumor sections corroborated QSM visualization of ferumoxytol distribution near the tumor periphery, and confirmed the spatial correlation of cell death with ferumoxytol distribution. Due to the dissipation of SPIOs from the injection site, quantitative mapping of SPIO distribution will aid in estimating a change in temperature in tissues, thereby maximizing MFH effects on tumors and minimizing side-effects by avoiding unwanted tissue heating. [ABSTRACT FROM AUTHOR]

[18/19F]-4, an anionic GCPII/PSMA inhibitor for image-guided intervention in prostate cancer, is described. [19F]-4 is radiolabeled with a radiochemical yield that is ≥27% and a molar activity of 190 ± 50 mCi/µmol in a <1 h, one-step, aqueous isotopic exchange reaction. [19F]-4 allows PSMA expression to be imaged by fluorescence (FL) and [18F]-PET. PC3-PIP (PSMA-positive, EC50 = 6.74 ± 1.33 nM) cancers are specifically delineated in mice that bear 3 million (18 mg) PC3-PIP and PC3 (control, PSMA-negative) cells. Colocalization of [18/19F]-4 PET, fluorescence, scintillated biodistribution, and PSMA expression are observed. [ABSTRACT FROM AUTHOR]

Introduction CXCR4 specific [ 18 F]-labeled positron emission tomography (PET) imaging agents are needed which would enable general distribution of the radiotracer for clinical investigation. We sought to synthesize, radiolabel and evaluate [ 18 F]RPS-544, a novel non-peptide CXCR4 antagonist as a CXCR4 specific probe. We compared [ 18 F]RPS-544 with the previously published [ 18 F]-3 ([ 18 F]RPS-510 in this paper) in a bi-lateral tumor model of differential CXCR4 expression for its ability to selectively target CXCR4 expression. Methods Radiolabeling of [ 18 F]RPS-544 and [ 18 F]RPS-510 was performed by aromatic substitution on a 6-nitropyridyl group using no-carrier-added [ 18 F]fluoride under basic conditions. 18 F incorporation was determined by radioHPLC. Semi-preparative HPLC was used to purify the final product prior to reformulation. Imaging and biodistribution was performed in nude mice with bilateral PC3 (CXCR4+ and WT) xenograft tumors at 1, 2 and 4 h post injection. Results RPS-544 bound CXCR4 with an IC50 of 4.9 ± 0.3 nM. [ 18 F]RPS-544 showed preferential uptake in CXCR4+ tumors, with a CXCR4/WT ratio of 3.3 ± 1.3 at 1 h p.i. and 2.3 ± 0.5 at 2 h p.i. Maximum uptake in the CXCR4+ tumors was 3.4 ± 1.2%ID/g at 1 h p.i., significantly greater (p = 0.003) than the uptake in the WT tumor. Tumor/blood ratios were 2.5 ± 0.4 and 3.6 ± 0.3 at 1 and 2 h p.i. Tumor/muscle ratios were >4 at all time-points. Tumor/lung ratios were >2 at 1 h and 2 h p.i. Substantial uptake was observed in the liver (15–25%ID/g), kidneys (25–35%ID/g), the small intestine (1–7%ID/g) and the large intestine (1–12%ID/g). Blood concentrations varied over time (0.5–2%ID/g). All other organs showed uptake of <1%ID/g at all time points studied with clearance profiles similar to blood clearance. Conclusions Here we present, to the best of our knowledge, the first high affinity [18F]-labeled tracer, suitable for in vivo PET imaging of CXCR4. [18F]RPS-544 displayed high affinity for CXCR4 and good tumor uptake with a maximum uptake at 1 h p.i.. CXCR4 dependent uptake was demonstrated using bilateral tumors with differential CXCR4 expression as well as pharmacological blockade using the known CXCR4 antagonist, AMD-3100. Tissue contrast as judged by tumor to normal tissue ratios was positive in several key tissues. The structural and pharmacological similarities between [18F]RPS-544 and the approved drug AMD-3465, combined with the ease of synthesis and high molar activity (>185 GBq/μmol) achieved during radiosynthesis could lead to accelerated translation into the clinic. [ABSTRACT FROM AUTHOR]

The ability to use a systemically injected agent to image tumor is influenced by tumor characteristics such as permeability and vascularity, and the size, shape, and affinity of the imaging agent. In this study, six different imaging biomolecules, with or without specificity to tumor, were examined for tumor uptake and internalization at the whole body, ex-vivo tissue, and cellular levels: antibodies, antibody fragments (Fab), serum albumin, and streptavidin. The time of peak tumor uptake was dependent solely on the size of molecules, suggesting that molecular size is the major factor that influences tumor uptake by its effect on systemic clearance and diffusion into tumor. Affinity to tumor antigen failed to augment tumor uptake of Fab above non-specific accumulation, which suggests that Fab fragments of typical monoclonal antibodies may fall below an affinity threshold for use as molecular imaging agents. Despite abundant localization into the tumor, albumin and streptavidin were not found on cell surface or inside cells. By comparing biomolecules differing in size and affinity, our study highlights that while pharmacokinetics are a dominant factor in tumor uptake for biomolecules, affinity to tumor antigen is required for tumor binding and internalization. [ABSTRACT FROM AUTHOR]

The binding of TATA-binding protein (TBP) to the TATA-box containing promoter region is aided by many other transcriptional factors including TFIIA and TFIIB. The mechanistic insight into the assembly of RNA polymerase II preinitation complex (PIC) has been gained by either directly altering a function of target protein or perturbing molecular interactions using drugs, RNAi, or aptamers. Aptamers have been found particularly useful for studying a role of a subset of PIC on transcription for their ability to inhibit specific molecular interactions. One major hurdle to the wide use of aptamers as specific inhibitors arises from the difficulty with traditional assays to validate and determine specificity, affinity, and binding epitopes for aptamers against targets. Here, using a technique called the bio-layer interferometry (BLI) designed for a label-free, real-time, and multiplexed detection of molecular interactions, we studied the assembly of a subset of PIC, TBP binding to TATA DNA, and two distinct classes of aptamers against TPB in regard to their ability to inhibit TBP binding to TFIIA or TATA DNA. Using BLI, we measured not only equilibrium binding constants (KD), which were overall in close agreement with those obtained by electrophoretic mobility shift assay, but also kinetic constants of binding (kon and koff), differentiating aptamers of comparable KDs by their difference in binding kinetics. The assay developed in this study can readily be adopted for high throughput validation of candidate aptamers for specificity, affinity, and epitopes, providing both equilibrium and kinetic information for aptamer interaction with targets. [ABSTRACT FROM AUTHOR]

SELEX, the process of selecting aptamers, is often hampered by the difficulty of preparing target molecules in their native forms and by a lack of a simple yet quantitative assay for monitoring enrichment and affinity of reactive aptamers. In this study, we sought to discover DNA aptamers against human serum markers for potential therapeutic and diagnostic applications. To circumvent soluble expression and immobilization for performing SELEX, we ectopically expressed soluble growth factors on the surface of yeast cells to enable cell-SELEX and devised a flow cytometry-based method to quantitatively monitor progressive enrichment of specific aptamers. High-throughput sequencing of selected pools revealed that the emergence of highly enriched sequences concurred with the increase in the percentage of reactive aptamers shown by flow cytometry. Particularly, selected DNA aptamers against VEGF were specific and of high affinity (KD = ∼ 1 nM) and demonstrated a potent inhibition of capillary tube formation of endothelial cells, comparable to the effect of a clinically approved anti-VEGF antibody drug, bevacizumab. Considering the fact that many mammalian secretory proteins have been functionally expressed in yeast, the strategy of implementing cell-SELEX and quantitative binding assay can be extended to discover aptamers against a broad array of soluble antigens. [ABSTRACT FROM AUTHOR]

Sensitive detection of the metabolites indicative of a particular disease contributes to improved therapy outcomes. Developing binding reagents for detection of low molecular weight metabolites is hampered by the difficulty with immobilization of targets through appropriate covalent chemical linkage while ensuring that selected reagents retain specificity to unmodified metabolites. To circumvent chemical modification of targets, we employed sol-gel droplets deposited onto a porous silicon chip to entrap a purine metabolite, xanthine, which was found at lower levels in urine samples from patients with non-Hodgkin lymphoma. By sol-gel SELEX (systematic evolution of ligands by exponential enrichment) against xanthine, specific aptamers ( KD ∼ 10 μM) with sensitivity of detection at as low as 1 μM were isolated, which bound to other purine metabolites at more than 100-fold lower affinity. In contrast, we failed to isolate xanthine-specific aptamers when SELEX was performed against xanthine covalently linked to polymer resin. This study demonstrates that the sol-gel platform for entrapping low molecular weight metabolites without chemical modifications can be utilized for SELEX to discover aptamers against clinical metabolite markers for diagnosis application. [ABSTRACT FROM AUTHOR]

Highlights: [•] Isolated temperature-sensitive (TS) Drosophila P-TEFb mutants by yeast two-hybrid screen. [•] Developed a streamlined platform for isolating TS mutants of target protein–protein interactions. [•] Developed strategy can be extended to identify conditional and reversible mutant phenotypes. [ABSTRACT FROM AUTHOR]

Abstract: Clinically approved chemotherapeutic nanoparticles may provide advantages over free drugs by achieving slower clearance and preferential accumulation in tumors. However, the lack of leaky vasculatures can create barriers to the permeation of ∼100nm-sized nanoparticles in solid tumors. We hypothesized that nanoparticles designed to target both tumor and tumor stroma would penetrate deeper into the tumors. To construct such comprehensive drug carriers, we utilized cross-linked amphiphilic polymer nanoparticles and functionalized them to target ICAM-1, a biomarker prevalent in various tumors and inflamed tumor stroma. The targeting moiety was derived from the modular domain present in αL integrin, which was engineered for high affinity and cross-reactivity with human and murine ICAM-1. ICAM-1-selective delivery of paclitaxel produced potent tumor suppression of not only ICAM-1-positive cervical cancer cells but also ICAM-1-negative tumors, presumably by causing cytotoxicity in tumor-associated endothelium (CD31+) and macrophages (CD68+) over-expressing ICAM-1. Contrary to the strategies of targeting only the tumor or specific tumor stromal constituents, we present a strategy in delivering therapeutics to the major cellular components of solid tumors. Drug carriers against inflammation-biomarkers may be effective against many different types of tumors, while being less susceptible to the highly mutable nature of tumor markers. [Copyright &y& Elsevier]

Helix-helix interactions are fundamental to many biological signals and systems and are found in homo- or heteromultimerization of signaling molecules as well as in the process of virus entry into the host. In HIV, virus-host membrane fusion during infection is mediated by the formation of six-helix bundles (6HBs) from homotrimers of gp41, from which a number of synthetic peptides have been derived as antagonists of virus entry. Using a yeast surface two-hybrid (YS2H) system, a platform designed to detect protein-protein interactions occurring through a secretory pathway, we reconstituted 6HB complexes on the yeast surface, quantitatively measured the equilibrium and kinetic constants of soluble 6HB, and delineated the residues influencing homo-oligomeric and hetero-oligomeric coiled-coil interactions. Hence, we present YS2H as a platform for the facile characterization and design of antagonistic peptides for inhibition of HIV and many other enveloped viruses relying on membrane fusion for infection, as well as cellular signaling events triggered by hetero-oligomeric coiled coils. [ABSTRACT FROM AUTHOR]

Intermolecular contacts between integrin LFA-1 (αLβ2) and ICAM-1 derive solely from the integrin αL I domain and the first domain (D1) of ICAM-1. This study presents a crystal structure of the engineered complex of the αL I domain and ICAM-1 D1. Previously, we engineered the I domain for high affinity by point mutations that were identified by a directed evolution approach. In order to examine αL I domain allostery between the C-terminal α7-helix (allosteric site) and the metal-ion dependent adhesion site (active site), we have chosen a high affinity variant without mutations directly influencing either the position of the α7-helix or the active sites. In our crystal, the αL I domain was found to have a high affinity conformation to D1 with its α7-helix displaced downward away from the binding interface, recapitulating a current understanding of the allostery in the I domain and its linkage to neighboring domains of integrins in signaling. To enable soluble D1 of ICAM-1 to fold on its own, we also engineered D1 to be functional by mutations, which were found to be those that would convert hydrogen bond networks in the solvent-excluded core into vdW contacts. The backbone structure of the β-sandwich fold and the epitope for I domain binding of the engineered D1 were essentially identical to those of wild-type D1. Most deviations in engineered D1 were found in the loops at the N- terminal region that interacts with human rhinovirus (HRV). Structural deviation found in engineered D1 was overall in agreement with the function of engineered D1 observed previously, i.e., full capacity binding to αL I domain but reduced interaction with HRV. [ABSTRACT FROM AUTHOR]

Ephrin-B2 is predominately expressed in endothelium of arterial origin, involved in developmental angiogenesis and neovasculature formation through its interaction with EphB4. Despite its importance in physiology and pathological conditions, it has been challenging to produce monoclonal antibodies against ephrin-B2 due to its high conservation in sequence throughout human and rodents. Using a novel approach for antibody selection by panning a phage library of human antibody against antigens displayed in yeast, we have isolated high affinity antibodies against ephrin-B2. The function of one high affinity binder (named as 'EC8') was manifested in its ability to inhibit ephrin-B2 interaction with EphB4, to cross-react with murine ephrin-B2, and to induce internalization into ephrin-B2 expressing cells. EC8 was also compatible with immunoprecipitation and detection of ephrin-B2 expression in the tissue after standard chemical fixation procedure. Consistent with previous reports on ephrin-B2 induction in some epithelial tumors and tumor-associated vasculatures, EC8 specifically detected ephrin-B2 in tumors as well as the vasculature within and outside of the tumors. We envision that monoclonal antibody developed in this study may be used as a reagent to probe ephrin-B2 distribution in normal as well as in pathological conditions and to antagonize ephrin-B2 interaction with EphB4 for basic science and therapeutic applications. [ABSTRACT FROM AUTHOR]

Abstract: Dysregulated host inflammatory response causes many diseases, including cardiovascular and neurodegenerative diseases, cancer, and sepsis. Sensitive detection of the site of inflammation will, therefore, produce a wide-ranging impact on disease diagnosis and treatment. We hypothesized that nanoprobes designed to mimic the molecular interactions occurring between inflamed leukocytes and endothelium may possess selectivity toward diverse host inflammatory responses. To incorporate inflammation-sensitive molecular interactions, super paramagnetic iron oxide nanoparticles were conjugated with integrin lymphocyte function-associated antigen (LFA)-1 I domain, engineered to mimic activated leukocytes in physiology. Whole body optical and magnetic resonance imaging in vivo revealed that leukocyte-mimetic nanoparticles localized preferentially to the vasculature within and in the invasive front of the tumor, as well as to the site of acute inflammation. This study explored in vivo detection of tumor-associated vasculature with systemically injected inflammation-specific nanoparticles, presenting a possibility of tumor detection by inflamed tumor microenvironment. [Copyright &y& Elsevier]

Abstract: Dysregulated inflammation contributes to the pathogenesis of various diseases. Therapeutic efficacy of anti-inflammatory agents, however, falls short against resilient inflammatory responses, whereas long-term and high-dose systemic administration can cause adverse side effects. Site-directed drug delivery systems would thus render more effective and safer treatments by increasing local dosage and minimizing toxicity. Nonetheless, achieving clinically effective targeted delivery to inflammatory sites has been difficult due to diverse cellular players involved in immunity and endogenous targets being expressed at basal levels. Here we exploit a physiological molecular interaction between intercellular adhesion molecule (ICAM)-1 and lymphocyte function associated antigen (LFA)-1 to deliver a potent anti-inflammatory drug, celastrol, specifically and comprehensively to inflamed cells. We found that affinity and avidity adjusted inserted (I) domain, the major binding site of LFA-1, on liposome surface enhanced the specificity toward lipopolysaccharides (LPS)-treated or inflamed endothelial cells (HMEC-1) and monocytes (THP-1) via ICAM-1 overexpression, reflecting inherent affinity and avidity modulation of these molecules in physiology. Targeted delivery of celastrol protected cells from recurring LPS challenges, suppressing pro-inflammatory responses and inflammation-induced cell proliferation. Targeted delivery also blocked THP-1 adhesion to inflamed HMEC-1, forming barriers to immune cell accumulation and to aggravating inflammatory signals. Our results demonstrate affinity and avidity of targeting moieties on nanoparticles as important design parameters to ensure specificity and avoid toxicities. We anticipate that such tunable physiologic interactions could be used for designing effective drug carriers for in vivo applications and contribute to treating a range of immune and inflammatory diseases. [Copyright &y& Elsevier]

Abstract: Botulinum neurotoxins (BoNTs) function by delivering a protease to neuronal cells that cleave SNARE proteins and inactivate neurotransmitter exocytosis. Small (14 kDa) binding domains specific for the protease of BoNT serotypes A or B were selected from libraries of heavy chain only antibody domains (VHHs or nanobodies) cloned from immunized alpacas. Several VHHs bind the BoNT proteases with high affinity (K D near 1 nM) and include potent inhibitors of BoNT/A protease activity (K i near 1 nM). The VHHs retain their binding specificity and inhibitory functions when expressed within mammalian neuronal cells as intrabodies. A VHH inhibitor of BoNT/A protease was able to protect neuronal cell SNAP25 protein from cleavage following intoxication with BoNT/A holotoxin. These results demonstrate that VHH domains have potential as components of therapeutic agents for reversal of botulism intoxication. [ABSTRACT FROM AUTHOR]

Abstract: Site-specific delivery of drugs while minimizing unwanted distribution has been one of the pursued goals in cancer therapy. In this endeavor, we have developed targeted polymeric nanoparticles called amphiphilic urethane acrylate nonionomer (UAN) for encapsulation of diverse water-insoluble drugs and diagnostic agents, as well as for simple and reproducible surface conjugation of targeting ligands. Using monoclonal antibodies or lymphocyte function-associated antigen-1 (LFA-1) I domain engineered for varying affinities to intercellular adhesion molecule (ICAM)-1, we were able to deliver UAN nanoparticles to human cancer cells with the efficiency dependent on the strength of the molecular interactions and the degree of ICAM-1 expression on cell surface. Compared to non-specific uptake of free drugs, targeted delivery of UAN nanoparticles carrying equal amount of drugs produced more potent cytotoxicity. Notably, without the targeting ligands attached, UAN nanoparticles were largely precluded from non-specific uptake by the cells, resulting in much lower toxicity. The versatility of our UAN nanoparticles in both payload encapsulation and presentation of targeting ligands may facilitate developing a robust platform for evaluating various combinations of cancer drugs and molecular interactions toward developing effective cancer therapy formulations. [ABSTRACT FROM AUTHOR]

The immunoglobulin (Ig) superfamily is one of the largest families in the vertebrate genome, found most frequently in cell surface molecules. Intercellular adhesion molecule-1 (ICAM-1) contains five extracellular Ig superfamily domains (D1-D5) of which the first domain, D1, is the binding site for the integrin lymphocyte function-associated antigen-1 (LFA-1) and human rhinovirus. Despite the modular nature of many Ig superfamily domains with respect to domain folding and ligand recognition, D1 does not fold on its own due to the loss of its interaction with the second domain. The goal of this study was to engineer ICAM-1 D1 by introducing mutations that would stabilize the Ig superfamily domain fold while retaining its ability to bind to LFA-1 and rhinovirus. First, with a directed evolution approach, we isolated mutations in D1 that showed binding to conformation-specific antibodies and the ligand binding domain of LFA-1 called the inserted, or I, domain. Then, with a rational design approach we introduced mutations that contributed to the stability of ICAM-1 D1 in solution. The mutations that restored native folding of D1 in isolation were those that would convert hydrogen bond networks in buried regions into hydrophobic contacts. Notably, for most mutations, identical or similar types of substitutions were found in ICAM-1 molecules of different species and other ICAM family members. The systematic approach demonstrated in this study to engineer a single Ig superfamily fold in ICAM-1 can be broadly applicable to the engineering of modular Ig superfamily domains in other cell surface molecules. [ABSTRACT FROM AUTHOR]

A systematic approach to the discovery of conformation-specific antibodies or those that recognize activation-induced ñeoepitopes in signaling molecules and enzymes will be a powerful tool in developing antibodies for basic science and therapy. Here, we report the isolation of antibody antagonists that preferentially bind activated integrin Mac-1 (αMβ2) and are potent in blocking neutrophil adhesion and migration. A novel strategy was developed for this task, consisting of yeast surface display of Mac-1 inserted (I) domain library, directed evolution to isolate active mutants of the I domain, and screening of phage display of human antibody library against the active I domain in yeast. Enriched antibody library was then introduced into yeast surface two-hybrid system for final quantitative selection of antibodies from monomeric antigenantibody interaction. This led to highly efficient isolation of intermediate to high affinity antibodies, which preferentially reacted with the active I domain, antagonized the I domain binding to intercellular adhesion molecule (ICAM)-1, complement (3 fragment iC3b, and fibronectin, and potently inhibited neutrophil migration on fibrinogen. The strategy demonstrated herein can be broadly applicable to developing antibodies against modular domains that switch between inactive and active conformations, particularly toward the discovery of antibody antagonists in therapeutic and diagnostic applications. [ABSTRACT FROM AUTHOR]

OCRL, whose mutations are responsible for Lowe syndrome and Dent disease, and INPP5B are two similar proteins comprising a central inositol 5-phosphatase domain followed by an ASH and a RhoGAP-like domain. Their divergent NH2-terminal portions remain uncharacterized. We show that the NH2-terminal region of OCRL, but not of INPP5B, binds clathrin heavy chain. OCRL, which in contrast to INPP5B visits late stage endocytic clathrin-coated pits, was earlier shown to contain another binding site for clathrin in its COOH-terminal region. NMR structure determination further reveals that despite their primary sequence dissimilarity, the NH2-terminal portions of both OCRL and INPP5B contain a PH domain. The novel clathrin-binding site in OCRL maps to an unusual clathrin-box motif located in a loop of the PH domain, whose mutations reduce recruitment efficiency of OCRL to coated pits. These findings suggest an evolutionary pressure for a specialized function of OCRL in bridging phosphoinositide metabolism to clathrin-dependent membrane trafficking. [ABSTRACT FROM AUTHOR]

A quantitative in vivo method for detecting protein-protein interactions will enhance our understanding of protein interaction networks and facilitate affinity maturation as well as designing new interaction pairs. We have developed a novel platform, dubbed "yeast surface two-hybrid (YS2H)," to enable a quantitative measurement of pairwise protein interactions via the secretory pathway by expressing one protein (bait) anchored to the cell wall and the other (prey) in soluble form. In YS2H, the prey is released either outside of the cells or remains on the cell surface by virtue of its binding to the bait. The strength of their interaction is measured by antibody binding to the epitope tag appended to the prey or direct readout of split green fluorescence protein (GFP) complementation. When two a-helices forming coiled coils were expressed as a pair of prey and bait, the amount of the prey in complex with the bait progressively decreased as the affinity changes from 100 μM to 10 μM. With GFP complementation assay, we were able to discriminate a 6-log difference in binding affinities in the range of 100 pM to 100 μM. The affinity estimated from the level of antibody binding to fusion tags was in good agreement with that measured in solution using a surface plasmon resonance technique. In contrast, the level of GFP complementation linearly increased with the on rate of coiled coil interactions, likely because of the irreversible nature of GFP reconstitution. Furthermore, we demonstrate the use of YS2H in exploring the nature of antigen recognition by antibodies and activation allostery in integrins and in isolating heavy chainonly antibodies against botulinum neurotoxin. [ABSTRACT FROM AUTHOR]

An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]

While the majority of thyroid cancer patients are easily treatable, those with anaplastic or poorly differentiated recurrent thyroid carcinomas have a very poor prognosis with a median survival of less than a year. Previously, we have shown a significant correlation between ICAM-1 overexpression and malignancy in thyroid cancer, and have pioneered the use of ICAM-1 targeted CAR T cells as a novel treatment modality. For clinical translation of this novel modality, we designed CAR T cells possessing micromolar rather than nanomolar affinity to ICAM-1 to avoid cytotoxicity in normal cells with basal levels of ICAM-1 expression. Herein, we report the automated process of CAR T cell manufacturing with CliniMACS Prodigy (Miltenyi Biotec) using cryopreserved peripheral blood leukocytes from apheresis collections. Using Prodigy, thawed leukopak cells were enriched for CD4+ and CD8+ T cells, subjected to double transduction using lentiviral vector, and expanded in culture for a total of 10 days with a final yield of 2–4 × 109 cells. The resulting CAR T cells were formulated for cryopreservation to be used directly for infusion into patients after thawing with no further processing. We examined cross-reactivity of CAR T cells toward both human and murine ICAM-1 and ICAM-1 expression in human and mouse tissues to demonstrate that both efficacy and on-target, off-tumor toxicity can be studied in our preclinical model. Selective anti-tumor activity in the absence of toxicity provides proof-of-concept that micromolar affinity tuned CAR T cells can be used to target tumors expressing high levels of antigen while avoiding normal tissues expressing basal levels of the same antigen. These studies support the initiation of a phase I study to evaluate the safety and potential efficacy of micromolar affinity tuned CAR T cells against newly diagnosed anaplastic and refractory or recurrent thyroid cancers. [ABSTRACT FROM AUTHOR]

Determining chemokine receptor CXCR4 expression is significant in multiple diseases due to its role in promoting inflammation, cell migration and tumorigenesis. [68Ga]Pentixafor is a promising ligand for imaging CXCR4 expression in multiple tumor types, but its utility is limited by the physical properties of 68Ga. We screened a library of >200 fluorine-containing structural derivatives of AMD-3465 to identify promising candidates for in vivo imaging of CXCR4 expression by positron emission tomography (PET). Compounds containing fluoroethyltriazoles consistently achieved higher docking scores. Six of these higher scoring compounds were radiolabeled by click chemistry and evaluated in PC3-CXCR4 cells and BALB/c mice bearing bilateral PC3-WT and PC3-CXCR4 xenograft tumors. The apparent CXCR4 affinity of the ligands was relatively low, but tumor uptake was CXCR4-specific. The tumor uptake of [18F]RPS-534 (7.2 ± 0.3 %ID/g) and [18F]RPS-547 (3.1 ± 0.5 %ID/g) at 1 h p.i. was highest, leading to high tumor-to-blood, tumor-to-muscle, and tumor-to-lung ratios. Total cell-associated activity better predicted in vivo tumor uptake than did the docking score or apparent CXCR4 affinity. By this metric, and on the basis of their high yielding radiosynthesis, high tumor uptake, and good contrast to background, [18F]RPS-547, and especially [18F]RPS-534, are promising 18F-labeled candidates for imaging CXCR4 expression. [ABSTRACT FROM AUTHOR]