Intercellular Adhesion Molecule-1 Antibody Research Articles

ICAM1 antibody drug conjugates exert potent antitumor activity in papillary and anaplastic thyroid carcinoma

Treatment options for anaplastic thyroid cancer (ATC) and refractory papillary thyroid carcinoma (PTC) are limited and outcomes remain poor. In this study, we determined via bioinformatic expression analyses and immunohistochemistry staining that intercellular adhesion molecule-1(ICAM1) is an attractive target for ATC and PTC. We designed and engineered two ICAM1-directed antibody-drug conjugate (I1-MMAE and I1-DXd), both of which potently and selectively ablate multiple human ATC and PTC cell lines without affecting non-plastic cells invitro. Furthermore, I1-MMAE and I1-DXd mediated a potent tumor regression in ATC and PTC xenograft models. To develop a precision medicine, we also explored magnetic resonance imaging (MRI) as a non-invasive biomarker detection method to quantitatively map ICAM1 antigen expression in heterogeneous thyroid tumors. Taken together, this study provides a strong rationale for the further development of I1-MMAE and I1-DXd as promising therapeutic candidates to treat advanced PTC and ATC.

CD38 x ICAM1 Bispecific Antibody Is a Novel Approach for Treating Multiple Myeloma and Lymphoma

Targeting cluster of differentiation 38 (CD38) with monoclonal antibodies has resulted in outstanding responses in patients with multiple myeloma (MM). However, a significant portion of patients failed to respond and nearly all patients eventually relapsed. Furthermore, daratumumab, an anti-CD38 antibody, only showed limited monotherapy activity in relapsed/refractory Non-Hodgkin lymphoma (NHL) patients in a phase 2 study. One of the mechanisms of resistance has been partially attributed to lower CD38 expression. Intercellular adhesion molecule 1 (ICAM1), an immunoglobulin (Ig)-like cell adhesion molecule, is highly expressed in multiple myeloma and lymphoma. Antibody against ICAM1, bersanlimab (BI505, BioInvent), was well-tolerated, but only showed limited clinical efficacy in MM patients. Here, we generated bispecific CD38 x ICAM1 antibody to target ICAM1 + tumor types with low to medium CD38 expression. RNA sequencing (RNAseq) results from the Cancer Cell Line Encyclopedia (CCLE) database showed that ICAM1 is highly expressed on myeloma and lymphoma cell lines. ICAM1 expression levels for selected myeloma and lymphoma cell lines were then validated using flow cytometry. The CD38 x ICAM1 bispecific antibody was constructed by paring a novel CD38 antibody and a novel ICAM1 antibody through an asymmetric three chain knob-into-hole format. The bispecific antibody showed potent in vitro antibody-dependent cellular cytotoxicity (ADCC) activities on ICAM1 + tumor cells with medium to low CD38 levels, where daratumumab has low or minimal effect. The bispecific antibody also showed potent in vitro antibody-dependent cellular phagocytosis (ADCP) activities on cell lines with a range of CD38 expression. The CD38 x ICAM1 bispecific antibody further demonstrated potent tumor inhibition activities in in vivo myeloma and lymphoma cell line-derived xenograft (CDX) models, including cell lines with low to medium CD38 expression. We then evaluated CD38 and ICAM1 expressions in lymphoma patient-derived xenograft (PDX) samples by RNAseq. Among the 37 PDX samples, 27 of them showed ICAM1 expression above 2 5 fragments per kilobase of transcript per million map reads (FPKM). On the contrary, there is a wide range of CD38 expression levels with only 6 samples having CD38 expression above 2 5 FPKM. The ICAM1 and CD38 expressions in the selected PDX samples were further validated with IHC staining. Most importantly, the CD38 x ICAM1 bispecific antibody showed complete tumor inhibition in a rituximab-resistant lymphoma PDX model, whereas daratumumab only showed minimal efficacy. In conclusion, the CD38 x ICAM1 bispecific antibody demonstrated improved efficacy and specificity toward CD38 + and ICAM1 + tumor cells and represents a novel approach for treating multiple myeloma and lymphoma. DisclosuresNo relevant conflicts of interest to declare.

Abstract 1918: Coxsackievirus A11 elicits a potent oncolytic activity in human malignant pleural mesothelioma via ICAM-1 receptor

Abstract Malignant pleural mesothelioma (MPM) is an aggressive solid cancer with dismal prognosis. Oncolytic virotherapy is a promising anticancer treatment that directly lyses tumor cells. Coxsackievirus A11 (CVA11) is an emerging oncolytic virus against solid cancers. Herein we tested whether CVA11 infection has an oncolytic activity in human MPM cell lines and identified its cognate receptor. We found that CVA11 infection shows a remarkable oncolytic activity in six of six human MPM cell lines. MPM cells having higher intercellular adhesion molecule-1 (ICAM-1) expression showed higher CVA11-induced cytotoxicity, and that identified ICAM-1 as its surface receptor because coinfection with neutralizing ICAM-1 antibody substantially abrogated CVA11-triggered cytotoxicity in MPM cells. In addition, we unraveled that the activations of both phosphoinositide 3-kinase/Akt and mitogen-activated protein (MAP)/extracellular signal-regulated (ERK) kinase (MEK) signaling pathways partially but significantly contributed to the CVA11-triggered cytotoxicity of CVA11 as evidenced by treating with respective inhibitors. Taken together, our findings show a novel oncolytic virotherapy using CVA11 could be an alternative therapeutic modality for human MPM. Citation Format: Koji Okamura, Hiroyuki Inoue, Yuki Ikematsu, Rie Furukawa, Keiichi Ota, Yasuto Yoneshima, Eiji Iwama, Kentaro Tanaka, Isamu Okamoto. Coxsackievirus A11 elicits a potent oncolytic activity in human malignant pleural mesothelioma via ICAM-1 receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1918.

Tissue Targeting and Ultrasound-Targeted Microbubble Destruction Delivery of Plasmid DNA and Transfection In Vitro.

Ultrasound-targeted microbubble destruction (UTMD) has been shown a promising approach for target-specific gene delivery and treatment of many diseases in the past decade. To improve the therapeutic potential of UTMD, the gene carrier of microbubbles should possess adequate DNA condensation capability and (or) specific cell or tissue selectivity. The tissue-targeted and ultrasound-targeted cationic microbubbles were developed to meet gene therapy. A tissue-targeted stearic acid-inserted cationic microbubbles (SCMBs) were prepared for ultrasound-targeted gene delivery. Branched PEI was modified with stearic acid and further mixed with 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and biot-1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (ammonium salt) (Biot-DSPE-PEG2000), intercellular adhesion molecule-1 (ICAM-1) antibody and plasmid DNA to prepare cationic microbubbles through ultrasonic hydration. The ICAM-1 antibody and plasmid DNA were expected to assemble to the surface of SCMBs via biotin-avidin interaction and electrostatic interaction, respectively. It was found that the SCMBs had higher zeta potential compared with neutral microbubbles (NMBs) and cationic microbubbles (CMBs). In contrast, DNA incorporated SCMBs4 showed negative potential, exhibiting good DNA-binding capacity. Confocal images showed that the HeLa cells were attached around by the SCMBs4 from the view of green fluorescence of fluorescein isothiocyanate-loaded IgG which conjugated to ICAM-1 antibody on their surface. After ultrasound treatment, HeLa cells treated with SCMBs exhibited slightly stronger red fluorescence under confocal laser scanning microscope, indicating a synergistic promotion for transfection efficiency. This tissue- and ultrasound-targeted cationic microbubble demonstrated here showed a promising strategy for improving gene therapy in the future.

The Association of ICAM-1 Detected by Immunohistochemical Staining with Triple Negative and Non-Triple Negative Breast Carcinoma

Metastasis of tumour cell greatly contributes to the cause of mortality. Tumour-associated macrophage (TAM) and the intercellular adhesion molecule 1 (ICAM-1) were associated with metastases of breast carcinoma. However, the relationship between lymphatic vessel densities and invasion with TAM and ICAM-1 remained unclear. The aim of this study was to investigate the relationship of lymphovascular densities and invasions with patient’s clinicopathological data. The roles of TAM and ICAM-1 influencing lymphovascular invasions were also investigated. Haematoxylin and eosin (H&E) and immunohistochemical (IHC) staining on a consecutive section of 37 formalin fixed-paraffin embedded (FFPE) breast invasive carcinoma samples were carried out. The D2-40, CD34, CD163, and ICAM-1 antibodies were used to stain lymphatic vessel, blood vessel, TAM, and ICAM-1 receptor, respectively. The total lymphatic vessel density (LVD) was significantly reduced on increased tumor size (p=0.045). The increase of intra-tumoral LVD and lymphatic vessel invasion (LVI) was significantly associated with human epidermal growth factor receptor 2 (HER2) negative status (p=0.022 and p=0.05, respectively). The percentage of LVI was higher than blood vessel invasion (BVI) in 18.5%. Lymphovascular invasions detected in H&E were missed in 49.76% compared with those detected in IHC-stained tissues (206/410). ICAM-1 scores were significantly associated with non-triple negative breast cancer (non-TNBC) (p=0.008). ICAM-1 is significantly overexpressed on non-TNBC sample. Therefore, ICAM-1 might be clinically useful as a targeted molecule for non-TNBC patients. In histological reporting, in addition to H&E staining, IHC staining using D2-40 and CD34 should be considered to increase the accuracy of diagnosis.

Combining vascular targeting and the local first pass provides 100-fold higher uptake of ICAM-1-targeted vs untargeted nanocarriers in the inflamed brain

New advances in intra-arterial (IA) catheters offer clinically proven local interventions in the brain. Here we tested the effect of combining local IA delivery and vascular immunotargeting. Microinjection of tumor necrosis factor alpha (TNFα) in the brain parenchyma causes cerebral overexpression of Inter-Cellular Adhesion Molecule-1 (ICAM-1) in mice. Systemic intravenous injection of ICAM-1 antibody (anti-ICAM-1) and anti-ICAM-1/liposomes provided nearly an order of magnitude higher uptake in the inflamed vs normal brain (from ~0.1 to 0.8%ID/g for liposomes). Local injection of anti-ICAM-1 and anti-ICAM-1/liposomes via carotid artery catheter provided an additional respective 2-fold and 5-fold elevation of uptake in the inflamed brain vs levels attained by IV injection. The uptake in the inflamed brain of respective untargeted IgG counterparts was markedly lower (e.g., uptake of anti-ICAM-1/liposomes was 100-fold higher vs IgG/liposomes). These data affirm the specificity of the combined effect of the first pass and immunotargeting. Intravital real-time microscopy via cranial window revealed that anti-ICAM-1/liposomes, but not IgG/liposomes bind to the lumen of blood vessels in the inflamed brain within minutes after injection. This straightforward framework provides the basis for translational efforts towards local vascular drug targeting to the brain.

Bioresponsive Nanoparticles Targeted to Infectious Microenvironments for Sepsis Management.

Sepsis is a life-threatening disease resulted from a dysregulated host immune response to bacterial infections, continuing to cause high morbidity and mortality worldwide. Despite discoveries of many potential therapeutic targets, effective treatments of sepsis are lacking. Here, a strategy is reported to target infectious microenvironments (IMEs) via bioresponsive nanoparticles that simultaneously eliminate bacteria and alleviate the host inflammation response, thus managing sepsis in mice. The nanoparticle is made of copolymers sensitive to pH and bacterial enzymes to self-assemble into a micelle loaded with both an antibiotic (ciprofloxacin) and an anti-inflammatory agent ((2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide). In addition, the nanoparticle is conjugated with intercellular adhesion molecule-1 antibodies to target IMEs. Nanoparticle targeting to IMEs and local cues as triggers to deliver therapeutics in on-demand manners is demonstrated using an acute lung bacterial infection mouse model. In the sepsis mouse model induced by peritonitis at a lethal dose of bacterial invasion, it is shown that concurrently targeting pathogens and excessive inflammation pathways is valuable to manage the sepsis. The study illustrates not only the development of a new delivery system but also the mechanism-based therapy of nanomedicine for infectious diseases.

In vivo imaging of endothelial cell adhesion molecule expression after radiosurgery in an animal model of arteriovenous malformation.

Focussed radiosurgery may provide a means of inducing molecular changes on the luminal surface of diseased endothelium to allow targeted delivery of novel therapeutic compounds. We investigated the potential of ionizing radiation to induce surface expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) on endothelial cells (EC) in vitro and in vivo, to assess their suitability as vascular targets in irradiated arteriovenous malformations (AVMs). Cultured brain microvascular EC were irradiated by linear accelerator at single doses of 0, 5, 15 or 25 Gy and expression of ICAM-1 and VCAM-1 measured by qRT-PCR, Western, ELISA and immunocytochemistry. In vivo, near-infrared (NIR) fluorescence optical imaging using Xenolight 750-conjugated ICAM-1 or VCAM-1 antibodies examined luminal biodistribution over 84 days in a rat AVM model after Gamma Knife surgery at a single 15 Gy dose. ICAM-1 and VCAM-1 were minimally expressed on untreated EC in vitro. Doses of 15 and 25 Gy stimulated expression equally; 5 Gy was not different from the unirradiated. In vivo, normal vessels did not bind or retain the fluorescent probes, however binding was significant in AVM vessels. No additive increases in probe binding were found in response to radiosurgery at a dose of 15 Gy. In summary, radiation induces adhesion molecule expression in vitro but elevated baseline levels in AVM vessels precludes further induction in vivo. These molecules may be suitable targets in irradiated vessels without hemodynamic derangement, but not AVMs. These findings demonstrate the importance of using flow-modulated, pre-clinical animal models for validating candidate proteins for vascular targeting in irradiated AVMs.

Abstract 2207: Using AFM to predict the specificity of tumor targeting nanomedicine in vivo

Abstract Most cancer target and biomarker studies focus on the identification of overexpressed genes or proteins of cancer cells. However, their potential feasibility as tumor-specific antigens targeted by antibody-guided nanomedicine has not consistently been based on quantitative assessments of tumor specificity and binding strength. Cancer cell membrane surfaces possess hundreds of different functionally versatile proteins (receptors and ligands) which are heterogeneously upregulated or downregulated to promote cancer development and progression. Selection of appropriate drug targets is pivotal to the successful development of tumor-targeted therapeutics because tumor-specific affinity of potential cancer targets is a key factor modulating cellular binding and uptake, and has the potential to translate to therapeutic bioactivity. To date, the quantitative measurements of cancer target antibody-antigen interactions are often limited by dynamic biophysical and biomechanical methods for probing such interactions. To address this challenge, we used flow cytometric analyses to compare 40 potential cancer cell surface target candidates for triple negative breast cancer (TNBC), an aggressive malignancy with limited treatment options. We selected intercellular adhesion molecule-1 (ICAM-1) as the “optimized” TNBC target for nanomedicine. Reasoning that adhesion forces between the ICAM-1 antibody and live human breast cancer cells could be used to guide nanomedicine delivery to TNBC tumors, we used atomic force microscopy (AFM) to evaluate the binding strength between the ICAM-1 antibody and antigen on live human TNBC cells and non-neoplastic cells. Adhesion forces between the ICAM-1 antibody and live human TNBC MDA-MB-231 cells (523 ± 113 pN) were significantly higher than those of non-neoplastic MCF10A cells (336 ± 33 pN). We next validated these findings by engineering ICAM-1-targeted immunoliposomes and then tested their TNBC-specific binding affinity and cytotoxicity on three TNBC cell lines. In vivo imaging experiments further confirmed that the ICAM-1-targeted immunoliposomes preferentially accumulated in orthotopic TNBC tumors, in comparison with nonspecific, IgG-conjugated liposomes. These data support the conclusion that ICAM-1 antibody-conjugated immunoliposomes containing chemotherapeutic drugs have the potential to preferentially target and destroy TNBC cells, and may also be applicable to a range of metastatic cancers that demonstrate an overexpression of ICAM-1. These findings also support the use of AFM as a valuable tool in the characterization of surface antigen-antibody interactions that predict in vitro and in vivo binding affinity. Acknowledgements: D. Auguste acknowledges the support of NIH 1DP2CA174495. M. Moses acknowledges the support of NIH R01CA185530 and the Breast Cancer Research Foundation. Citation Format: Peng Guo, Biran Wang, Daxing Liu, Jiang Yang, Marsha Moses, Debra Auguste. Using AFM to predict the specificity of tumor targeting nanomedicine in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2207.

Celecoxib increases lung cancer cell lysis by lymphokine-activated killer cells via upregulation of ICAM-1.

The antitumorigenic mechanism of the selective cyclooxygenase-2 (COX-2) inhibitor celecoxib is still a matter of debate. Using lung cancer cell lines (A549, H460) and metastatic cells derived from a lung cancer patient, the present study investigates the impact of celecoxib on the expression of intercellular adhesion molecule 1 (ICAM-1) and cancer cell lysis by lymphokine-activated killer (LAK) cells. Celecoxib, but not other structurally related selective COX-2 inhibitors (i.e., etoricoxib, rofecoxib, valdecoxib), was found to cause a substantial upregulation of ICAM-1 protein levels. Likewise, ICAM-1 mRNA expression was increased by celecoxib. Celecoxib enhanced the susceptibility of cancer cells to be lysed by LAK cells with the respective effect being reversed by a neutralizing ICAM-1 antibody. In addition, enhanced killing of celecoxib-treated cancer cells was reversed by preincubation of LAK cells with an antibody to lymphocyte function associated antigen 1 (LFA-1), suggesting intercellular ICAM-1/LFA-1 crosslink as crucial event within this process. Finally, celecoxib elicited no significant increase of LAK cell-mediated lysis of non-tumor bronchial epithelial cells, BEAS-2B, associated with a far less ICAM-1 induction as compared to cancer cells. Altogether, our data demonstrate celecoxib-induced upregulation of ICAM-1 on lung cancer cells to be responsible for intercellular ICAM-1/LFA-1 crosslink that confers increased cancer cell lysis by LAK cells. These findings provide proof for a novel antitumorigenic mechanism of celecoxib.

Abstract 4410: An ICAM-1-targeted, Lcn2 siRNA-encapsulating liposome as a potent anti-angiogenic agent for triple-negative breast cancer

Abstract Introduction: The significant growth and metastasis of triple negative breast cancer (TNBC) tumors, coupled with limited treatment options, have made TNBC a clinically challenging disease. We have previously shown that the intercellular adhesion molecule-1 (ICAM-1) is highly overexpressed in human TNBC tissues and cell lines, suggesting that ICAM-1 could be a potential TNBC molecular target. Lipocalin 2 (Lcn2), a 25-kDa protein belonging to the lipocalin protein superfamily. We have recently reported that Lcn2 is a regulator of angiogenesis. We have also found that down-regulation of TNBC-secreted Lcn2 is associated with reduced tumor angiogenesis in preclinical studies of breast cancer, making it a promising TNBC anti-angiogenic target. Here, we describe a novel liposomal drug delivery system that integrates TNBC targeting via ICAM-1, with Lcn2 siRNA silencing, to synergistically inhibit TNBC angiogenesis in vitro and in vivo, representing a novel anti-angiogenic therapeutic approach for the treatment of TNBC. Materials and Methods: ICAM-1 antibody, or non-targeted IgG conjugated, Lcn2 siRNA encapsulating liposomes (ICAM-Lcn2-LPs or IgG-Lcn2-LPs (non-targeting control)) were prepared by the extrusion method. Lcn2 siRNA silencing in TNBC cells was determined by qRT-PCR, and the anti-angiogenic functions of ICAM-Lcn2-LP were evaluated via a series of in vitro angiogenic assays including: endothelial cell proliferation, endothelial cell migration, and endothelial cell tube formation. These in vitro anti-angiogenic results were further confirmed in vivo using the chick chorioallantoic membrane (CAM) assay. Results: We have engineered a liposomal siRNA delivery system to target triple negative breast cancer (TNBC) via a novel molecular target, ICAM-1, and selectively silenced a TNBC-overexpressing angiogenic mediator, Lcn2 using siRNA. This ICAM-1 targeted, Lcn2 siRNA delivery liposome binds human TNBC MDA-MB-231 cells 3.1-fold greater than non-neoplastic MCF-10A cells. Potent Lcn2 knockdown by ICAM-Lcn2-LP led to a significant reduction in VEGF secretion by MDA-MB-231 cells, which led to reduced TNBC-induced angiogenesis both in vitro and in vivo. Conclusions: We have successfully engineered an immunoliposome that synergistically couples TNBC-targeting with Lcn2 siRNA silencing. Synthesized ICAM-Lcn2-LPs can significantly suppress in vitro and in vivo angiogenic activities of TNBC cells by regulating VEGF secretion. The modular design introduced here provides a basis for future TNBC-targeted therapeutics. Acknowledgements: This research is supported by NIH(NCI 1DP2CA174495) and the Breast Cancer Research Foundation. Citation Format: Peng Guo, Jiang Yang, Marsha Moses, Debra Auguste. An ICAM-1-targeted, Lcn2 siRNA-encapsulating liposome as a potent anti-angiogenic agent for triple-negative breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4410. doi:10.1158/1538-7445.AM2015-4410

Antibody conjugates bispecific for intercellular adhesion molecule 1 and allergen prevent migration of allergens through respiratory epithelial cell layers

Antibody conjugates bispecific for intercellular adhesion molecule 1 and allergen prevent migration of allergens through respiratory epithelial cell layers

Clinicopathological features of sarcoidosis manifesting as generalized chronic myopathy.

Although chronic myopathy has been reported to affect skeletal muscle in sarcoidosis, its clinicopathological features have not been fully elucidated. We characterized the clinical, histopathological, and prognostic features of eleven patients with sarcoidosis manifesting with chronically progressive, generalized myopathy that was confirmed with muscle biopsy. Generalized muscle weakness extending to the four extremities and trunk was the cardinal feature of these cases. Muscle atrophy was evident in nine patients, particularly in the lower limbs, whereas myalgia was reported in only two patients. Myopathy was the first manifestation of sarcoidosis in five patients. Only six patients showed elevated plasma creatine kinase levels. Using skeletal muscle computed tomography, the distribution of muscle atrophy was predominant in the hip adductors, knee flexors and ankle plantarflexors. Radiological assessments, including magnetic resonance imaging, gallium scintigraphy, and fluorodeoxyglucose positron emission tomography-computed tomography imaging, revealed findings suggestive of skeletal muscle inflammation in only half of the patients examined. However, in all patients, muscle biopsy specimens revealed an active inflammatory process, as observed by focal non-caseating epithelioid granuloma with predominant CD4-positive lymphocytic infiltration. Sarcolemmas were diffusely stained with HLA-ABC, HLA-DR and intercellular adhesion molecule-1 antibodies, suggesting diffuse and active antigen presentation. Functional improvement after immunomodulatory treatment was significantly better in patients with short disease durations (p < 0.05). The therapeutic response was poor in patients with long disease durations; thus, early diagnosis and early initiation of treatment are important.

Cannabinoids increase lung cancer cell lysis by lymphokine-activated killer cells via upregulation of ICAM-1

Cannabinoids have been shown to promote the expression of the intercellular adhesion molecule 1 (ICAM-1) on lung cancer cells as part of their anti-invasive and antimetastatic action. Using lung cancer cell lines (A549, H460) and metastatic cells derived from a lung cancer patient, the present study addressed the impact of cannabinoid-induced ICAM-1 on cancer cell adhesion to lymphokine-activated killer (LAK) cells and LAK cell-mediated cytotoxicity. Cannabidiol (CBD), a non-psychoactive cannabinoid, enhanced the susceptibility of cancer cells to adhere to and subsequently be lysed by LAK cells, with both effects being reversed by a neutralizing ICAM-1 antibody. Increased cancer cell lysis by CBD was likewise abrogated when CBD-induced ICAM-1 expression was blocked by specific siRNA or by antagonists to cannabinoid receptors (CB1, CB2) and to transient receptor potential vanilloid 1. In addition, enhanced killing of CBD-treated cancer cells was reversed by preincubation of LAK cells with an antibody to lymphocyte function associated antigen-1 (LFA-1) suggesting intercellular ICAM-1/LFA-1 crosslink as crucial event within this process. ICAM-1-dependent pro-killing effects were further confirmed for the phytocannabinoid Δ9-tetrahydrocannabinol (THC) and R(+)-methanandamide (MA), a hydrolysis-stable endocannabinoid analogue. Finally, each cannabinoid elicited no significant increase of LAK cell-mediated lysis of non-tumor bronchial epithelial cells, BEAS-2B, associated with a far less pronounced (CBD, THC) or absent (MA) ICAM-1 induction as compared to cancer cells. Altogether, our data demonstrate cannabinoid-induced upregulation of ICAM-1 on lung cancer cells to be responsible for increased cancer cell lysis by LAK cells. These findings provide proof for a novel antitumorigenic mechanism of cannabinoids.

Attenuation of Inflammatory Response and Reduction in Infarct Size by Postconditioning Are Associated With Downregulation of Early Growth Response 1 During Reperfusion in Rat Heart

Early growth response 1 (EGR-1) works as a master regulator that plays a key role in triggering inflammation-induced tissue injury after ischemia and reperfusion. This study tested the hypothesis that postconditioning (Postcon) or anti-inflammatory compound, curcumin, ameliorates inflammatory responses and further reduces infarct size by normalizing EGR-1 expression during reperfusion. In the control group, male Sprague-Dawley rats were subjected to 30-min ischemia and 180-min reperfusion. Postcon with four cycles of 10-s/10-s reperfusion/ischemia was applied at the onset of reperfusion. Curcumin (150 mg/kg per day) was fed 5 days before ischemia. Relative to the control, Postcon reduced expression of EGR-1 mRNA and protein, as further identified by less EGR-1 immunoreactivity in myocardial nuclei and microvessels during reperfusion. Along with EGR-1 downregulation, levels of plasma and myocardial tumor necrosis factor α and interleukin 6 (IL-6) were significantly decreased. Upregulated P-selectin and intercellular adhesion molecule 1 mRNA and protein as well as their immunoreactivity at area at risk myocardium were significantly attenuated. Neutrophil extravasation identified by myeloperoxidase immunohistochemical staining was inhibited. Infarct size, determined with triphenyltetrazolium chloride staining, was smaller in the Postcon group than that in the control. The protection achieved with pretreatment with curcumin was comparable to the benefits gained by Postcon in all end points measured. In H9C2 rat cardiomyoblast cell line, EGR-1 siRNA downregulated hydrogen peroxide-induced EGR-1 mRNA expression and subsequently reduced tumor necrosis factor α mRNA level. These results suggest that EGR-1 seems to play a critical role in myocardial reperfusion injury because downregulation of EGR-1 either by Postcon or the use of pharmacological intervention reduces infarct size, most likely through an inhibition of inflammation-mediated processes.

Progressive Changes in Inflammatory and Matrix Adherence of Bronchial Epithelial Cells with Persistent Respiratory Syncytial Virus (RSV) Infection (Progressive Changes in RSV Infection)

In addition to the acute manifestations of respiratory syncytial virus (RSV), persistent infection may be associated with long-term complications in the development of chronic respiratory diseases. To understand the mechanisms underlying RSV-induced long-term consequences, we established an in vitro RSV (strain A2) infection model using human bronchial epithelial (16HBE) cells that persists over four generations and analyzed cell inflammation and matrix adherence. Cells infected with RSV at multiplicity of infection (MOI) 0.0067 experienced cytolytic or abortive infections in the second generation (G2) or G3 but mostly survived up to G4. Cell morphology, leukocyte and matrix adherence of the cells did not change in G1 or G2, but subsequently, leukocyte adherence and cytokine/chemokine secretion, partially mediated by intercellular adhesion molecule-1 (ICAM-1), increased drastically, and matrix adherence, partially mediated by E-cadherin, decreased until the cells died. Tumor necrosis factor-α (TNF-α) secretion was inhibited by ICAM-1 antibody in infected-16HBE cells, suggesting that positive feedback between TNF-α secretion and ICAM-1 expression may be significant in exacerbated inflammation. These data demonstrate the susceptibility of 16HBE cells to RSV and their capacity to produce long-term progressive RSV infection, which may contribute to inflammation mobilization and epithelial shedding.

An Anti-Human ICAM-1 Antibody Inhibits Rhinovirus-Induced Exacerbations of Lung Inflammation

Human rhinoviruses (HRV) cause the majority of common colds and acute exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Effective therapies are urgently needed, but no licensed treatments or vaccines currently exist. Of the 100 identified serotypes, ∼90% bind domain 1 of human intercellular adhesion molecule-1 (ICAM-1) as their cellular receptor, making this an attractive target for development of therapies; however, ICAM-1 domain 1 is also required for host defence and regulation of cell trafficking, principally via its major ligand LFA-1. Using a mouse anti-human ICAM-1 antibody (14C11) that specifically binds domain 1 of human ICAM-1, we show that 14C11 administered topically or systemically prevented entry of two major groups of rhinoviruses, HRV16 and HRV14, and reduced cellular inflammation, pro-inflammatory cytokine induction and virus load in vivo. 14C11 also reduced cellular inflammation and Th2 cytokine/chemokine production in a model of major group HRV-induced asthma exacerbation. Interestingly, 14C11 did not prevent cell adhesion via human ICAM-1/LFA-1 interactions in vitro, suggesting the epitope targeted by 14C11 was specific for viral entry. Thus a human ICAM-1 domain-1-specific antibody can prevent major group HRV entry and induction of airway inflammation in vivo.

Targeted Nanoparticles that Mimic Immune Cells in Pain Control Inducing Analgesic and Anti-inflammatory Actions: A Potential Novel Treatment of Acute and Chronic Pain Conditions

Background: The peripheral immune-derived opioid analgesic pathway has been well established as a novel target in the clinical pain management of a number of painful pathologies, including acute inflammatory pain, neuropathic pain, and rheumatoid arthritis. Objective: Our objective was to engineer targeted nanoparticles that mimic immune cells in peripheral pain control to deliver opioids, in particular loperamide HCl, specifically to peripheral opioid receptors to induce analgesic and anti-inflammatory actions for use in painful inflammatory conditions. This peripheral analgesic system is devoid of central opioid mediated side effects (e.g., respiratory depression, sedation, dependence, tolerance). Study Design: A randomized, double blind, controlled animal trial. Methods: Thirty-six adult male Wistar rats (200 - 250 g) were randomly divided into 6 groups: loperamide HCl-encapsulated anti-ICAM-1 immunoliposomes, naloxone methiodide + loperamide HCl-encapsulated antiICAM-1 immunoliposomes, loperamide HCl-encapsulated liposomes, empty anti-ICAM-1 immunoliposomes, empty liposomes, and loperamide solution. Animals received an intraplantar injection of 150 µL Complete Freund’s Adjuvant (CFA) into the right hindpaw and experiments were performed 5 days post-CFA injection, which corresponded to the peak inflammatory response. All formulations were administered intravenously via tail vein injection. The dose administered was 200 µL, which equated to 0.8 mg of loperamide HCl for the loperamide HCl treatment groups (sub-therapeutic dose). Naloxone methiodide (1 mg/kg) was administered via intraplantar injection, 15 minutes prior to loperamide-encapsulated anti-ICAM-1 immunoliposomes. An investigator blinded to the treatment administered assessed the time course of the antinociceptive and antiinflammatory effects using a paw pressure analgesiometer and plethysmometer, respectively. Biodistribution studies were performed 5 days post-CFA injection with anti-ICAM-1 immunoliposomes and control liposomes via tail vein injection using liquid scintillation counting (LSC). Results: Administration of liposomes loaded with loperamide HCl, and conjugated with antibody to intercellular adhesion molecule-1 (anti-ICAM-1), exerted analgesic and anti-inflammatory effects exclusively in peripheral painful inflamed tissue. These targeted nanoparticles produced highly significant analgesic and anti-inflammatory effects over the 48 hour time course studied following intravenous administration in rats with Complete Freund’s Adjuvant-induced inflammation of the paw. All control groups showed no significant antinociceptive or anti-inflammatory effects. Our biodistribution study demonstrated specific localization of the targeted nanoparticles to peripheral inflammatory tissue and no significant uptake into the brain. Limitations: In vivo studies were performed in the well-established rodent model of acute inflammatory pain. We are currently studying this approach in chronic pain models known to have clinical activation of the peripheral immune-derived opioid response. Conclusions: The study presents a novel approach of opioid delivery specifically to injured tissues for pain control. The study also highlights a novel anti-inflammatory role for peripheral opioid targeting, which is of clinical relevance. The potential also exists for the modification of these targeted nanoparticles with other therapeutic compounds for use in other painful conditions. Key words: Pain, inflammation; immune cells, opioids, ICAM-1, loperamide, targeted drug delivery, immunoliposomes

Development of a Homogeneous High-Throughput Screening Assay for Biological Inhibitors of Human Rhinovirus Infection

Infection with human rhinovirus (HRV) is thought to result in acute respiratory exacerbations of chronic obstructive pulmonary disorder (COPD). Consequently, prevention of HRV infection may provide therapeutic benefit to these patients. As all major group HRV serotypes infect cells via an interaction between viral coat proteins and intercellular adhesion molecule–1 (ICAM-1), it is likely that inhibitors of this interaction would prevent or reduce infections. Our objective was to use phage display technology in conjunction with naive human antibody libraries to identify anti–ICAM-1 antibodies capable of functional blockade of HRV infection. Key to success was the development of a robust, functionally relevant high-throughput screen (HTS) compatible with the specific challenges of antibody screening. In this article, we describe the development of a novel homogeneous time-resolved fluorescence (HTRF) assay based on the inhibition of soluble ICAM-1 binding to live HRV16. We describe the implementation of the method in an antibody screening campaign and demonstrate the biological relevance of the assay by confirming the activity of resultant antibodies in a cell-based in vitro HRV infection assay.

Cannabidiol inhibits lung cancer cell invasion and metastasisviaintercellular adhesion molecule‐1

Cannabinoids inhibit cancer cell invasion via increasing tissue inhibitor of matrix metalloproteinases-1 (TIMP-1). This study investigates the role of intercellular adhesion molecule-1 (ICAM-1) within this action. In the lung cancer cell lines A549, H358, and H460, cannabidiol (CBD; 0.001-3 μM) elicited concentration-dependent ICAM-1 up-regulation compared to vehicle via cannabinoid receptors, transient receptor potential vanilloid 1, and p42/44 mitogen-activated protein kinase. Up-regulation of ICAM-1 mRNA by CBD in A549 was 4-fold at 3 μM, with significant effects already evident at 0.01 μM. ICAM-1 induction became significant after 2 h, whereas significant TIMP-1 mRNA increases were observed only after 48 h. Inhibition of ICAM-1 by antibody or siRNA approaches reversed the anti-invasive and TIMP-1-upregulating action of CBD and the likewise ICAM-1-inducing cannabinoids Δ(9)-tetrahydrocannabinol and R(+)-methanandamide when compared to isotype or nonsilencing siRNA controls. ICAM-1-dependent anti-invasive cannabinoid effects were confirmed in primary tumor cells from a lung cancer patient. In athymic nude mice, CBD elicited a 2.6- and 3.0-fold increase of ICAM-1 and TIMP-1 protein in A549 xenografts, as compared to vehicle-treated animals, and an antimetastatic effect that was fully reversed by a neutralizing antibody against ICAM-1 [% metastatic lung nodules vs. isotype control (100%): 47.7% for CBD + isotype antibody and 106.6% for CBD + ICAM-1 antibody]. Overall, our data indicate that cannabinoids induce ICAM-1, thereby conferring TIMP-1 induction and subsequent decreased cancer cell invasiveness.