Mouse Anti-human Antibody Research Articles

E-selectin targeted immunoliposomes for rapamycin delivery to activated endothelial cells

Activated endothelial cells play a pivotal role in the pathology of inflammatory disorders and thus present a target for therapeutic intervention by drugs that intervene in inflammatory signaling cascades, such as rapamycin (mammalian target of rapamycin (mTOR) inhibitor). In this study we developed anti-E-selectin immunoliposomes for targeted delivery to E-selectin over-expressing tumor necrosis factor-α (TNF-α) activated endothelial cells. Liposomes composed of 1,2-dipalmitoyl-sn-glycero-3.;hosphocholine (DPPC), Cholesterol, and 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000]-maleimide (DSPE-PEG-Mal) were loaded with rapamycin via lipid film hydration, after which they were further functionalized by coupling N-succinimidyl-S-acetylthioacetate (SATA)-modified mouse anti human E-selectin antibodies to the distal ends of the maleimidyl (Mal)-PEG groups. In cell binding assays, these immunoliposomes bound specifically to TNF-α activated endothelial cells. Upon internalization, rapamycin loaded immunoliposomes inhibited proliferation and migration of endothelial cells, as well as expression of inflammatory mediators. Our findings demonstrate that rapamycin-loaded immunoliposomes can specifically inhibit inflammatory responses in inflamed endothelial cells.

Abstract 1612: Production of CD3+ cells using ferrofluids for cell isolation, activation, expansion and subsequent transfection for adoptive cell therapy

Abstract The purpose of the study is to demonstrate whether magnetic nanoparticles functionalized with monoclonal antibodies in conjunction with solution-phase monoclonal antibodies can be used to activate T cells for expansion in vitro. The activation, expansion and transfection/transduction of immune cells—particularly T cells—has become an area of significant interest based on encouraging data generated from employing such genetically modified cells for cancer immunotherapies. The promise of soon being able to cure various types of cancers has resulted from years of methodical experimentation in the fields of oncology, immunology, and genetics, as well as the development of a vast array of enabling technological advances. BioMagnetic Solutions has developed colloidal magnetic nanoparticles—referred to as ferrofluids—which can be used to isolate a cell population through an immunomagnetic separation. These ferrofluids comprise a crystalline core of magnetite (ca. 100 nm in diameter) coated with multilayers of clinical-grade human serum albumin and further functionalized with a clinical-grade rat anti-mouse IgG1 monoclonal antibody. These “common-capture ferrofluids” (ca. 130 nm in diameter) are capable of binding cells which have been labeled with a monoclonal mouse IgG1 antibody; for example, CD3+ cells can be readily isolated from a complex cell mixture by labeling with a monoclonal mouse anti-human CD3 antibody, adding common-capture ferrofluid, and subjecting the sample to a magnetic field gradient. This technology can therefore be employed at the clinical scale to isolate T cells from an apheresis product. In this study, we investigated whether T cells isolated using common-capture ferrofluids could subsequently be activated and induced to expand in vitro. Based on preliminary experiments showing that T cell-bound common-capture ferrofluid has excess antibody-binding capacity, we designed experiments to test the hypothesis that solution-phase CD28 could be added to isolated T cells, which would bind to both CD28 determinants on the cell surface as well as to the cell-bound common-capture ferrofluid. Activation was quantified by measuring CD25 expression after four days in culture and expansion was monitored over 15 days. Following an immunomagnetic separation of CD3+ cells, magnetically isolated cells were re-suspended in expansion medium (STEMCELL) supplemented with 100 IU/mL IL-2 (Gibco) to a total concentration of 106 cells/mL and incubated at 37 °C. The following day, 0.5 µg/mL mouse anti-human CD28 monoclonal antibody (Mabtech) was added, and thereafter, cells were periodically agitated and diluted to 106 cells/mL with expansion media. After four days in culture, 88.4% of cells expressed CD25, while a 311-fold expansion was observed after 15 days. In summary, we have demonstrated that common-capture ferrofluids can be used to activate T cells for expansion in vitro. Citation Format: Dustin W. Ritter, Todor R. Khristov, Paul A. Liberti. Production of CD3+ cells using ferrofluids for cell isolation, activation, expansion and subsequent transfection for adoptive cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1612. doi:10.1158/1538-7445.AM2017-1612

Abstract LB-200: A patient derived antibody targeting the tetraspanin CD9 synergistically inhibits tumor growth with an anti PD1 antibody

Abstract Background. It is generally accepted that immune reactions against cancer cells can be induced by immunotherapy. Here, we investigated the possibility that an antibody response has contributed to the success of the immunotherapy of a cancer patient. Methods. A patient with metastatic melanoma was successfully treated by adoptive transfer of ex vivo expanded autologous tumor reactive T cells1. This patient is still tumor free 9 years after treatment. Peripheral blood memory B cells were immortalized by ectopic Bcl-6 and Bcl-xL expression2 and analyzed for the presence of tumor-reactive B cells. Results. We isolated one B cell clone that produced an antibody, named AT1412, recognizing a novel cell surface epitope on the tetraspanin CD9 which is broadly expressed and involved in multiple cellular activities including proliferation and adherence. AT1412 bound more strongly to melanoma cells than to melanocytes and other healthy tissues indicating that the AT1412 epitope is overexpressed on tumor cells. In addition, AT1412 strongly reacted with other tumor types including colon, pancreas and, breast cancer. Further analysis revealed that AT1412 favors binding to a clustered state of CD9. CD9 clusters are dependent on palmitoylation and known to be present on metastatic cells3. Previously published high affinity mouse antibodies targeting human CD9 were found to trigger aggregation of platelets and thus unfit for therapeutic use4. In sharp contrast, AT1412 does not induce platelet aggregation. To address whether AT1412 fails to aggregate platelets because of its low affinity for CD9 we generated high affinity variants of AT1412 using our highly efficient affinity maturation platform. We obtained AT1412 variants with affinities up to 250 fold higher than the wild type AT1412 comparable to that of mouse anti-human CD9 antibodies. These high affinity variants still did not aggregate platelets indicating that the absence of platelet aggregation by AT1412 is due to the spatial arrangement of the AT1412 epitope present on CD9 and not its low affinity. The antibody was able to reduce growth of melanoma tumors and block metastasis in melanoma xenografted mice. A striking synergistic inhibition of tumor growth was observed in mice that were treated with our CD9 antibody in the presence of a clinically successful anti-PD1 checkpoint inhibitor antibody. Discussion. These data suggest that the antibody contributed to the success of the immunotherapy in this patient. This antibody could act together with tumor-reactive T cells in eradicating circulating tumor cells and/or preventing settlement of metastatic tumor cells. Importantly, no antibody-related adverse effects were observed during and after treatment of this patient indicating that the antibody is safe for use in humans. 1. Verdegaal, Cancer Immunol Immunother, 2011 2. Kwakkenbos, Nat Med, 2010 3. Yang, JBC, 2006 4. Boucheix, FEBS Letter, 1983 Citation Format: Remko Schotte, Pauline van Helden, Daniel Go, Christien Fatmawati, Els Verdegaal, Camile Bru, Julien Villaudy, Koen Wagner, Sjoerd van der Burg, Hergen Spits, Wouter Pos. A patient derived antibody targeting the tetraspanin CD9 synergistically inhibits tumor growth with an anti PD1 antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-200. doi:10.1158/1538-7445.AM2017-LB-200

Antibody-dependent Tumor Phagocytosis Enhances PD-L1 and IDOs Expression on Macrophages, Which Mediate Trastuzumab Resistance in Her2+ Breast Cancer

Abstract The immune response has been linked to the therapeutic effect of trastuzumab, the monoclonal antibody against Her2. However, the underlying mechanism remains unclear. Here, we reported that the infiltration of tumor-associated macrophages is associated with the reduction of CD8+ T cells and NK cells in tumors and poor response of trastuzumab-containing neo-adjuvant chemotherapy in Her2+ breast cancer patients. In vitro, the macrophages suppress proliferation and tumoricidal effect of CD8+ T cells and NK cells after phagocytosis of Her2+ tumor cells mediated by trastuzumab in a CD16 and CD32a-dependent manner. By using global expression screening, we identified the immunosuppression effect is due to the upregulation of PD-L1 and IDOs on macrophages after tumor phagocytosis. Accordingly, optimal response of 4D5, the mouse anti-human Her2 antibody as trastuzumab parental drug, require macrophage depletion or anti-PD-L and IDO inhibition in tumor-bearing mice. In consistent with results form mice, the expression of PD-L1 and IDOs on tumor-associated macrophages increase in clinical samples of patients who received trastuzumab-containing neo-adjuvant chemotherapy, but not in those of patients who received neo-adjuvant chemotherapy without trastuzumab. Collectively, our data suggest that trastuzumab and double immune checkpoint blockade exert non-redundant clinical effects on Her2+ breast cancer patients with dense tumor-associated macrophages infiltration.

The expression of podoplanin protein is a diagnostic marker to distinguish the early infiltration of esophageal squamous cell carcinoma

The esophageal squamous cell carcinoma (ESCC) is usually develped from low-grade intraepithelial neoplasia (LGIEN) and high-grade intraepithelial neoplasia (HGIEN) to infiltrative squamous cell carcinoma. Till now, it remains hard to screen for infiltration at earlier stages, especially the differentiation between HGEIN and early infiltrative carcinoma. The purpose of this study is to determine a role of podoplanin in differentiating between HGEIN and early infiltrative squamous cell carcinoma. Totally 133 patients pathologically diagnosed with early ESCC and/or precancerous lesions were enrolled.The EnVision two-step IHC staining technique was applied using the monoclonal mouse anti-human Podoplanin antibody (clone number: D2-40). The expressions of PDPN protein on the basal layer of squamous epithelium lesions could be divided into three different patterns: complete type, incomplete (non-continuous) type, or missing type. A diagnosis of HGEIN can be made if the basal layer showed non-continuous or complete expression of PDPN and a diagnosis of early infiltration can be made if the expression of PDPN is completely missing. Our study confirmed that PDPN was a potential biomarker to identify the presence of early infiltrative squamous cell carcinoma.

Immunohistochemical differentiation between muscularis mucosae and muscularis propria for improving the staging of bladder cancer in patients undergoing transurethral resection of bladder tumours.

Microscopic differentiation between muscularis mucosae (MM) and muscularis propria (MP) of the bladder in the material obtained during transurethral resection (TUR) remains difficult. The study was aimed at determination of the usefulness of immunohistochemical staining in this context. Forty-seven TUR specimens were stained with 5 mouse anti-human antibodies: anti-desmin, anti-filamin, anti-type IV collagen, anti-smoothelin, and anti-vimentin. Slides were assessed under light microscopy and the intensity of the immune reaction within MM and MP was evaluated on a four-level visual scale as follows: negative (0) and weakly (1), moderately (2), or strongly (3) positive. MM was identified in 27 patients (57.4%). The modal values of reaction intensity in MM and MP was 0 and 2 for desmin (p > 0.05), 2 and 2 for filamin (p = 0.01), 2 and 2 for type IV collagen (p > 0.05), 1 and 2 for smoothelin (p = 0.03), and 2 and 0 for vimentin (p = 0.02), respectively. Identical intensity within MM and MP was observed in 7.1%, 28.6%, 20%, 30.1%, 5.6%, respectively. Immunohistochemistry can help differentiate between MM and MP in TUR specimens. As of yet, no single marker can reliably differentiate between MM and MP; however, a combination of anti-filamin, anti-smoothelin, and anti-vimentin antibodies may be reasonable for diagnostic purposes.

Evaluation of androgen receptor and HER2/neu receptor in tumor tissue of transitional cell carcinoma of urinary bladder and normal bladder mucosa

Background: Transitional cell carcinoma (TCC) is the most prevalent, singly accounting for 90% of all bladder cancer cases. Various markers involved in cell cycle but none of them proven its capacity as a prognostic indicator. The discovery of new biological markers may lead to early clinical prediction and may help in individualization of therapeutic approach in management of bladder cancer patients. The human epidermal growth factor receptors (EGFRs) – HER2/neu play a role in neoplastic cell growth and its role proven in breast and ovarian cancer while its role in bladder cancer still not explored its potential. Bladder cancer incidence is higher in male as compared to female. This gender related difference is still not clear in humans while it is proven in animal studies. In this study, we investigated the potential role and association of HER2/neu and androgen receptors (ARs) in etiology and prognosis of TCC. Patients and Methods: The study included 20 histopathologically confirmed cases of TCC of urinary bladder attending the Department of General Surgery, KGMU, Lucknow and 20 healthy individuals were taken as control. Immunohistochemical analysis for AR and HER2/neu receptor was done using paraffin-embedded tissue blocks treated with monoclonal mouse anti-human antibodies against AR and HER2/neu receptor, respectively. Results: HER2/neu over expression was observed in 14 (73%) of which 12 were males and 2 were females. Among these 16.7%, 33.3%, and 37.5% cases were in Grade I, II, and III, respectively. The AR overexpression was observed in 4 (20%) of which all were males. About 16.7% and 27.3% cases were in Grade II and III, respectively. Peak incidence was observed in the age group of 50–70 years (75%). Conclusions: We conclude that HER2/neu and AR can be considered as bad prognostic factor in TCC. These results suggested that targeting AR and HER2/neu may provide novel chemopreventive and therapeutic approaches for the management of bladder cancer.

Bion-1301: A Novel Fully Blocking APRIL Antibody for the Treatment of Multiple Myeloma

A PRoliferation Inducing Ligand (APRIL, TNFSF13), is a ligand for the receptors BCMA and TACI. APRIL serum levels are enhanced in patients diagnosed with Multiple Myeloma (MM), Chronic Lymphocytic Leukemia (CLL), and Colorectal Carcinoma correlated with poor prognosis. Our anti-APRIL antibody blocked CLL survival and inhibited mouse B1 hyperplasia in vivo (Guadagnoli et al., 2011). APRIL is produced by cells in the bone marrow niche, including myeloid-derived cells, osteoclasts and plasmacytoid dendritic cells. APRIL critically triggers BCMA in vitro and in vivoto drive proliferation and survival of human MM cells (Tai et al., 2016). Importantly, APRIL induces resistance to lenalidomide, bortezomib and other standard-of-care drugs. Furthermore, APRIL drives expression of PD-L1, IL-10, VEGF and TGFβ forcing an immunosuppressive phenotype on BCMA+ cells. As MM survival, resistance to treatment and the immunosuppressive phenotype can be blocked by neutralizing APRIL (Tai et al., 2016), development of an antibody blocking APRIL provides a novel avenue for the treatment of MM.A novel mouse anti-human APRIL antibody hAPRIL.01A (Guadagnoli et al., 2011) initially discovered using Aduro’s B-Select platform, was humanized and further engineered enhancing its stability (designated as BION-1301). The antibody binds to recombinant human APRIL with a KDof 0.4 ± 0.15 nM determined by BioLayer Interferometry and an EC50 of 0.29 ± 0.05 nM by ELISA. The epitope of BION-1301 was mapped to the BCMA and TACI binding site explaining its fully blocking capacity. Blocking potency (IC50) was 1.61 ± 0.78 nM (BCMA) and 1.29 ± 0.89 nM (TACI) respectively, corroborated by potent and complete blockade of APRIL-induced cytotoxicity of BCMA-Fas and TACI-Fas Jurkat transfectants. In vitro, BION-1301 suppressed APRIL-induced B-cell IgA and IgG class switching in a dose-dependent fashion. In vivo, BION-1301 was shown to suppress human APRIL induced T cell-independent B cell responses to NP-Ficoll. Biophysical and functional experiments indicated that BION-1301 recapitulated all characteristics of the mouse parental antibody hAPRIL.01A. To support the clinical development of BION-1301, quantitative assays were developed using several mouse-anti-human APRIL antibodies and shown to detect free and complexed APRIL in human blood samples. Results obtained with assays demonstrate that APRIL can be quantified reproducibly in human sera and overcome the drawbacks of previous assays, such as requirement of polyclonal sera, Ig adsorption, interference by human serum and reduced sensitivity.In conclusion, we have generated and functionally characterized a novel humanized APRIL neutralizing antibody, designated BION-1301. The mechanism-of-action and anti-tumor activity described for the parental antibody hAPRIL.01A in vitro and in vivo strongly support the development of BION-1301 as a single agent or in combination with lenalidomide, bortezomib, and suggest a rationale for combination with checkpoint inhibitors. BION-1301 is expected to enter clinical development in 2017.

Abstract B061: CD81: A new target for therapy of B cell lymphoma

Abstract CD81 belongs to an evolutionary conserved family of proteins named tetraspanins and it was originally described as a B cell target. Tetraspanins associate in multi-molecular complexes in tetraspanin-enriched microdomains (TEMS). Indeed, in B cells CD81 associates with CD19 and CD21, and together function as co-receptor to lower the cell activation threshold initiated by the BCR. Recently, we have demonstrated the importance of CD81 in tumor growth and metastasis of solid tumors1. However, the role of CD81 in B cell malignancies has not been explored. Previously, we have shown that anti-CD81 antibodies had anti-proliferative effects on human B cell lymphoma cell lines in vitro2. Here we tested the therapeutic effect of a mouse anti-human CD81 antibody in vivo against Raji and SUP-B8 B cell lymphomas using a xenograft model in SCID mice. Our studies demonstrated that our anti-CD81 antibody had therapeutic effect comparable to rituximab. Moreover, we found ADCC as one of the mechanism of action. Furthermore, to enhance the anti-CD81-mediated ADCC we engineered chimeric antibodies containing human IgG1ADCC-HIGH and human IgG4 ADCC-LOW Fc constant regions. Indeed, the chimeric human IgG1 anti-CD81 mAb showed a remarkable increase in NK cell-mediated ADCC compared to Rituximab in vitro. These results suggest that CD81 can be a potential therapeutic target on B cell lymphomas by virtue of both its direct cytotoxic effect and as a mediator of ADCC. The Human IgG1 version is being developed as a therapeutic candidate. 1. Vences-Catalan, F., et al. Cancer Res 75, 4517-4526 (2015). 2. Oren, R., Takahashi, S., Doss, C., Levy, R. & Levy, S. Mol Cell Biol 10, 4007-4015 (1990). Citation Format: Felipe Vences-Catalán, Chiung-Chi Kuo, Ranjani Rajapaksa, Ronald Levy, Shoshana Levy. CD81: A new target for therapy of B cell lymphoma [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B061.

Preparation and identification of mouse anti-human B7-H6 monoclonal antibodies

Objective To prepare mouse anti-human monoclonal antibodies against B7-H6 and to identify their biological characteristics. Methods The B7-H6 gene was cloned by RT-PCR from a human lung adenocarcinoma cell line (A549) and then subcloned into the eukaryote expression vector pCMV3 to construct the recombinant vector pCMV3-B7-H6. The recombinant vector pCMV3-B7-H6 that was verified with enzyme digestion and gene sequencing was transfected into NIH/3T3 cells by electroporation. BALB/c mice were immunized with the successfully transfected cells named 2H8 through intraperitoneal injection. The monoclonal antibodies against human B7-H6 with the advantages of high affinity and specificity were prepared by using hybridoma technology. Western blot assay and flow cytometry analysis were used to identify the specificity of prepared monoclonal antibodies. Results The recombinant eukaryotic expression vector encoding B7-H6 was successfully constructed. Two hybridoma clones that stably secreted monoclonal antibodies against B7-H6 were screened out by using flow cytometry analysis and the monoclonal antibodies secreted by them were belonged to IgG2a isotype. Specific reactions between B7-H6 and the secreted monoclonal antibodies were confirmed by Western blot assay and flow cytometry analysis. Conclusion The monoclonal antibodies which recognized B7-H6 specifically were prepared successfully. Key words: B7-H6; Eukaryotic expression; Monoclonal antibody; Specificity

CEACAM1 is overexpressed in oral tumors and related to tumorigenesis.

Carcinoembryonic antigen-related adhesion molecule 1 (CEACAM1) is a type 1 transmembrane glycoprotein belonging to the CEA family, which has been known to exist as either soluble forms in body fluids or membrane-bound forms on the cell surface. Aberrant CEACAM1 expression is associated with tumorigenesis and has been reported in a variety of human tumors, especially malignancies. The aim of this study is to determine the expression of CEACAM1 in oral tumors, trying to study CEACAM1 different expressions as a function of histotype. CEACAM1 expression was observed by immunohistochemistry (IHC) with mouse anti-human antibody for CEACAM1. IHC was performed using avidin-biotin-diaminobenzidine staining. The results were expressed as average score±SD (0=negative/8=highest) for each histotype. Oral tumors expressed more CEACAM1 than normal tissues including squamous and salivary epithelia (P<0.05). In malignancies, the squamous cell carcinoma overexpressed CEACAM1, compared to well-differentiated squamous cell with more membranous expression; the intermediately and poorly differentiated squamous cell carcinoma showed more cytoplasmic expression (P<0.05). In addition, the salivary tumors significantly expressed more CEACAM1 than squamous cell carcinoma (P<0.05). So, we thought oral tumors overexpressed CEACAM1 and the cytoplasmic CEACAM1 might be involved in tumorigenesis, and also CEACAM1 might be regarded as a marker of salivary glandular tumors.

Lymphatic vascular endothelial hyaluronan receptor-1 immunoexpression in placenta of HIV infected pre-eclamptic women

IntroductionLymphangiogenesis is the formation of new vessels from pre-existing lymphatic vessels. Data on lymphangiogenesis in the placenta of HIV-infected pre-eclamptics are sparse and the findings are conflicting. The aim of this novel study was to evaluate LYVE-1 immunoexpression in the placenta of HIV infected normotensive versus pre-eclamptic women. MethodsPlacental tissue was obtained from normotensive and pre-eclamptic women stratified according to their HIV status. The pre-eclamptic group was divided into early (<34 weeks) and late (>34 weeks) onset. Immunohistochemistry utilized mouse anti-human LYVE-1 antibody and was morphometrically evaluated. ResultsLYVE-1 immunostaining was localized within endothelium of the arterial supply and venous drainage of both conducting and exchange villi as well as within medial cells of arteries. LYVE-1 immunostained macrophage-like cells were observed within the fetal and maternal circulation. LYVE-1 immunoexpression was higher (p=0.0001) in HIV positive cohort, regardless of pregnancy and villous type. Irrespective of HIV status and pregnancy type, LYVE-1 immunoexpression was significantly elevated in the conducting compared to the exchange villi (p=0.01). LYVE-1 immunoexpression was higher in N and LOPE compared to EOPE groups for both conducting and exchange villi types respectively (p=0.0001 and p=0.006). There is a decrease of LYVE-1 expression in EOPE+ (conducting villi) and EOPE− (exchange villi) compared to N and LOPE subgroups. ConclusionThis study provides a novel insight into an up-regulation of LYVE-1 expression in the fetal circulation of conducting and exchange villi of HIV-infected pre-eclamptics.

Abstract 3963: Capture of EpCAM-negative mesothelioma cells with a “universal CTC-chip”: Sensitivity test and comparison with EpCAM-based standard method

Abstract Backgrounds: Circulating tumor cells (CTCs), as a surrogate of distant metastasis, can be potentially useful in early diagnosis and monitoring therapeutic effects for patients with malignant tumors. Among a variety of systems for detection of CTCs, an epithelial cell adhesion molecule (EpCAM)-based immuno-magnetic separation system “CellSearch” is the only system approved for clinical use. Our previous study showed that CTC detected by CellSearch was useful in the diagnosis or the prognosis of epithelioid-type malignant pleural mesothelioma, however, the sensitivity was insufficient. Because EpCAM-negative tumor cells, such as those originating from non-epithelial cells and those undergoing epithelial-mesenchymal transition, cannot be captured using the “CellSearch”. Therefore, we have developed a novel polymeric microfluidic device system “universal CTC-chip” that can capture a variety of CTCs with or without EpCAM expression (AACR 2015). In the present study, we assessed its sensitivity for capturing mesothelioma cells and compare with CellSearch. Methods: PC-9, human lung cancer cell line and ACC-MESO-4, human mesothelioma cell line were used in this study. Antibodies were coated on the chip in two steps, first with anti-mouse IgG antibody (“base-antibody”) and then as a “capture-antibody”, either mouse anti-human EpCAM antibody to capture EpCAM-positive cells (“EpCAM chip”) or mouse anti-human podoplanin antibody to capture podoplanin-positive mesothelioma cells (“podoplanin chip”). For sensitivity test, each cell line was suspended as concentration 500, 100, 50, 10cells/mL in phosphate buffered saline (PBS) or in blood. After 1ml of sample was flowed, we counted cells and calculated capture efficiency (number of capture cells /flowed cell). For comparison with CellSearch (CS), each cell line was suspended as concentration 50 or 10 cells /mL in blood and calculated detection efficiency (number of detected cells/ suspended cell). Results: In sensitivity test, capture efficiency of PC-9 or ACC-MESO-4 was 101.1%, 90.7% (500 cells/mL), 94.8%, 95.9% (100 cells/mL), 104.7%, 97.9% (50 cells/mL), 114.3%, 113.75% (10 cells/mL), respectively in PBS. In the blood sample, capture efficiency of PC-9 or MESO-4 was 99.2%, 50% (50 cells/mL), 115.5%, 80.55% (10 cells/mL), respectively. On the other hand, detection efficiency of PC-9 (EpCAM chip, CS) was 110%, 121.3% at 50 cells/mL, 195%, 126% at 10 cells/mL in blood. In case of MESO-4, detection efficiency (podoplanin chip, CS) was 55%, 1.1% at 50 cells/mL, 70%, 0% at 10 cells/mL in blood. Conclusion: The novel “universal CTC-chip” can be a promising CTC detection system. Citation Format: Kazue Yoneda, Yasuhiro Chikaishi, Eri Kawashima, Takashi Ohnaga, Fumihiro Tanaka. Capture of EpCAM-negative mesothelioma cells with a “universal CTC-chip”: Sensitivity test and comparison with EpCAM-based standard method. [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 3963.

Abstract 409: Immunofluorescent digital slide technology to evaluate correlation between Glypican-3 expression and response to codrituzumab

Abstract Background: Codrituzumab/GC33/RO5137382 is a recombinant humanized monoclonal antibody against Glypican-3 (GPC3) which is highly expressed in HCC cells. As part of phase I clinical trial, GC-002US study, evaluating escalating doses of codrituzumab plus sorafenib in HCC, immunofluorescent staining of GPC3 were applied in addition to GPC3 immunohistochemistry (IHC) with conventional DAB staining. The objectives of this work were to compare GPC3 expression levels by different staining methods and evaluate the correlation between these GPC3 scores and codrituzumab activities. Methods: Biopsies specimens (n = 34) collected at baseline in the phase I trial were evaluated by GPC3-IHC and immunofluorescent quantification digital slide (IQD) methods. GPC3-IHC were conducted using mouse anti-human GPC3 antibody, mouse GC33 (Ventana Medical Systems) as reported previously (Ikeda M. et al., Cancer Sci. 2014: 105; 455-462) and H scores were derived by two pathologists. For IQD, biopsy specimens were stained with same primary antibody, and were incubated with Qdot 655-conjugated secondary antibody. Fluorescent images of whole slides were obtained with the Nano-Zoomer (Hamamatsu Photonics K. K.). The IQD intensity score (immunofluorescent intensity per pixel) and cell score (immunofluorescent intensity per cell) were calculated respectively using MatLab (MathWorks Inc.). Results: There was a statistically significant correlation between GPC3 H scores and IQD scores. Among GPC3 scores, only the IQD cell score was correlated with serum GPC3 C-terminus increase by treatment (p = 0.032), whereas in the patients treated with higher dose of GC33 (≥ 10 mg/kg/week) the cytoplasmic H and the IQD intensity score were also correlated with serum GPC3 C-terminus increase by treatment. Interestingly, increasing cut-off values of IQD cell score levels led to lower HR and longer PFS. Best HR with lowest p value was observed at a cut-off value of 66,183 (HR = 0.24, p = 0.038). Conclusion: This study suggested that higher GPC3 expression on HCC cells quantified by IQD method was superior to other scores to predict codrituzumab activity, though this analysis was conducted only with limited number of subjects assessed. Further assessment and validation in clinical study of codrituzumab would be necessary to make a final conclusion on the clinical application of GPC3 IQD. Citation Format: Takayoshi Tanaka, Yasuo Sugitani, Tokiya Abe, Miho Kawaida, Ken Yamazaki, Akinori Hashiguchi, Michiie Sakamoto, Toshihiko Ohtomo. Immunofluorescent digital slide technology to evaluate correlation between Glypican-3 expression and response to codrituzumab. [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 409.

Abstract 4222: Usefulness of VE1 immunohistochemical detection of BRAFV600E in aggressive thyroid cancers (PDCs and UCs)

Abstract Activating BRAF mutations are frequent in thyroid follicular cell carcinogenesis. The BRAFV600E point mutation represents the most common oncogenic event in sporadic papillary thyroid cancer (PTC). Although at a much lower frequency the BRAFV600E mutation is also present among less differentiated, more aggressive, I131 resistant forms of thyroid cancer as poorly differentiated carcinomas (PDCs) and undifferentiated carcinomas (UCs). Small molecule inhibitors targeting either the BRAF V600E protein or upstream or downstream kinases involved in MAPK signalling are currently under pre-clinical or clinical investigation in advanced, metastatic, I131 resistant thyroid cancers. Recently, immunohistochemical (IHC) studies on PTCs, using the VE1 mouse anti-human BRAF V600E antibody have shown to be a reliable means for detecting the BRAFV600E mutation in a clinical setting without molecular genotyping units. In this study we sought to determine the usefulness of the VE1 antibody for detecting the BRAF V600E mutant protein in a series of 103 aggressive thyroid cancers (59 PDCs and 44 UCs) previously characterized by PCR-SSCP for the presence of BRAF mutations in exons 11 and 15. The BRAFV600E mutation was present in 10/59 PDCs (17%) and 11/44 UCs (25%). Immunohistochemistry revealed 9 mutated PDCs (sensitivity 90%) and 9 mutated UCs (82% sensitivity). The staining intensity in BRAF V600E mutated samples ranged from weak to strong. Non valuable results were found in 3 PDCs and 4 UCs. Overall the results indicate that immunohistochemistry with VE1 antibody may be an alternative to molecular biology approaches for the routine detection of BRAFV600E point mutations in clinical settings without molecular genotyping facilities. It may help clinicians in targeted therapy decision making. Citation Format: Noa Feás-Rodríguez, Angela M. Costa, Tomás Alvarez Gago, Juan José Mateos Otero, Jose Manuel Cameselle Teijeiro, Raquel Muñoz, Ginesa Maria Garcia-Rostan. Usefulness of VE1 immunohistochemical detection of BRAFV600E in aggressive thyroid cancers (PDCs and UCs). [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 4222.

B Cell Development in the Bone Marrow Is Regulated by Homeostatic Feedback Exerted by Mature B Cells.

Cellular homeostasis in the B cell compartment is strictly imposed to balance cell production and cell loss. However, it is not clear whether B cell development in the bone marrow is an autonomous process or subjected to regulation by the peripheral B cell compartment. To specifically address this question, we used mice transgenic for human CD20, where effective depletion of B lineage cells is obtained upon administration of mouse anti-human CD20 antibodies, in the absence of any effect on other cell lineages and/or tissues. We followed the kinetics of B cell return to equilibrium by BrdU labeling and flow cytometry and analyzed the resulting data by mathematical modeling. Labeling was much faster in depleted mice. Compared to control mice, B cell-depleted mice exhibited a higher proliferation rate in the pro-/pre-B compartment, and higher cell death and lower differentiation in the immature B cell compartment. We validated the first result by analysis of the expression of Ki67, the nuclear protein expressed in proliferating cells, and the second using Annexin V staining. Collectively, our results suggest that B lymphopoiesis is subjected to homeostatic feedback mechanisms imposed by mature B cells in the peripheral compartment.

The feasibility of adenoviral co-transduction of BMP2 and BMP7 for the expression of recombinant human BMP2/7 heterodimer in rat bone marrow mesenchymal stem cells

To investigate the feasibility of rat bone marrow mesenchymal stem cells (BMSCs) as the target cell of adenovirus-mediated co-transduction of BMP2 and BMP7 genes and then facilitate the expression of recombinant BMP2/7 heterodimer protein. 3 adult male Fischer 344 rats of about 10 weeks of age were used for harvest and in vitro culture of rat BMSCs. Recombinant adenovirus vector carrying BMP2 or BMP7 target genes were constructed with AdMax vector system, and production of high-titer adenoviruses were packaged with HEK293T cells and then concentrated with CSCl2 density-gradient ultra-centrifugation. Rat BMSCs from passage 3 were seeded in 6-well plates at the concentration of 10 000 cells/cm2.After overnight pre-culture, BMSCs were allowed to culture in 200 μl serum-free alpha MEM containing both Ad-BMP2 and Ad-BMP7 adenovirus (100 MOI of each virus). After 7 days in vitro culture, conditioned cell culture supernatants were collected and followed by immunoprecipitation through immune protein G columns pre-loaded with mouse anti-human BMP7 antibody. The resulted protein immune-precipitates were used to assay the expression of BMP2/7 heterodimers via Western Blot and ELISA assay. As a negative control, Rat BMSCs were also genetically transduced with Ad-GFP virus at a concentration of 200 MOI. Our data demonstrated that recombinant adenoviruses carrying BMP2 or BMP7 target gene was successfully reconstructed, packaged, and confirmed via Western Blot assay, which as respected, presented as an unique band at 55 000 size for BMP2 or 49 000 size for BMP7.Adenovirus Ad-GFP was used to verify the integrity of recombinant virus and its transfection efficiency in rat BMSCs, which showed well cell attachment to culture plate and had no cytotoxicity. Green fluorescent protein in BMSCs was also noted eminently under fluorescent microscope. Combined transduction with AdBMP2 plus Ad-BMP7 resulted in the formation of BMP2/7 heterodimers from rat BMSCs. Analysis of conditioned medium via Western Blot assay showed a single protein band of about 47 000 size, just as expected. Quantitative ELISA analysis presented a prominent expression of about (4.33 ± 0.42) ng/ml for recombinant BMP2/7 heterodimers. A paired t test showed significant difference (P ＜ 0.05),when compare to control groups of (0.08 ± 0.02) ng/ml. As an ideal cell source for tissue engineering, rat BMSCs can be genetically modified with Ad-BMP2 plus Ad-BMP7 mediated co-transduction strategy, and efficiently produce recombinant human BMP2/7 heterodimers in vitro, which should facilitate further studies on the beneficial effect of BMP2/7 heterodimers to ex vivo osteogenesis of BMSCs

Human nasal epithelial cells derived from multiple subjects exhibit differential responses to H3N2 influenza virus infection in vitro

Human nasal epithelial cells derived from multiple subjects exhibit differential responses to H3N2 influenza virus infection in vitro

Fourier plane colorimetric sensing using broadband imaging of surface plasmons and application to biosensing

We demonstrate an optical technique for refractive index and thickness sensing of sub-wavelength-thick dielectric analytes. The technique utilizes the broadband, multimode, directional leakage radiation arising from the excitation of hybrid mode surface plasmons (SP) on low aspect ratio periodic plasmonic substrates with period ≈λ. The approach requires relaxed fabrication tolerances compared to extra ordinary transmission-based sensing techniques, wherein minor shifts in the fabricated dimensions result in a very large change from the designed resonant wavelength. We show that refractive index perturbations due to about 10-nm-thick dielectric can be captured optically by the usage of carefully designed plasmonic substrates, a halogen lamp source, free-space optical components, polarizers, and a low-end, consumer-grade charge coupled device camera. The plasmonic substrates were designed for converting the signature of hybrid mode SP excitation into a transmission peak by utilizing a thin homogeneous metal layer sandwiched between the periodic plasmonic structures and the substrate. The resonance is highly sensitive to the refractive index and thickness of the analyte superstrate. The excitation of hybrid mode SP results in a polarization rotation of 90° of the leaked radiation at resonant wavelength. In order to eliminate the problem of image registration (i.e., placing the same feature in the same pixel of the image, for comparison before and after a change in refractive index) for sensing, we perform the color analysis in the Fourier plane. The change in color of the bright emitted spot with highest momentum, corresponding to the leakage of fundamental SP mode, was used to measure the changes in refractive index, whereas the number and color of spots of lower momenta, corresponding to higher-order Fabry Perot modes, was used to measure the variation in thickness. We further show that the Fourier plane analysis can also be used to sense the index of thicker dielectrics, where real plane image analysis may fail to sense index perturbations, simply due to superposition of different modes in the real plane images of such substrates. Control experiments and analysis revealed a refractive index resolution of 10–5 RIU. The results were correlated with simulations to establish the physical origin of the change in the fundamental mode and higher-order modes due to the refractive index and thickness of analyte. As a demonstration of an application and to test the limits of sensing, the substrates were used to image the surface functionalization using 2-nm-thick 11-mercaptoundecanoic acid and immobilization of 7-nm-thick mouse anti-human IgG antibody. In biological systems, where a priori knowledge about a process step is available, where accurate chemical composition testing is not necessary or possible, the presented method could be used to study the surface changes using a label-free sensing mechanism.

Low Expression of p53 Protein in Bone Marrow after Hematopoietic Stem Cell Transplantation Indicates Risk of Relapse in Pediatric Patients with Acute Lymphoblastic Leukemia

Advances in treatment over the last decades have resulted in almost 90% 5-year survival of children with acute lymphoblastic leukemia (ALL). Nonetheless, a minor group with unfavorable prognosis becomes candidates for hematopoietic stem cell transplantation (HSCT). After HSCT, blast count, minimal residual disease (MRD) and chimerism analysis are used as markers for successful engraftment and risk of relapse. However, additional markers are needed to complement the existing ones, as 30 % of ALL-patients still experience relapse after HSCT. Mutations in the tumor suppressor gene TP53 are detected in 2% at diagnosis of pediatric ALLs, but in 11-28 % after relapse. The aim of this study was to evaluate if alterations in the expressions of the TP53 encoded protein p53, analyzed by immunohistochemistry, could act as a prognostic marker in pediatric ALL patients post HSCT.Paraffin-embedded bone marrow samples were collected retrospectively from 49 children diagnosed with pre-B ALL and, treated with HSCT at Karolinska University Hospital between 1997-2010. Patients samples were requested from six different times during the course of the disease; time of diagnosis, before HSCT and at routine controls after HSCT at 0-3, >3-6, >6-12 and >12-24 months. For no patient data was available from all time periods. In total 176 samples were collected. A control group consisted of fifty-five children, where a bone marrow biopsy had been performed due to suspected bone marrow disorder, but a malignant blood disease had been excluded.Tissue micro array (TMA) prepared microscope slides were stained for the p53 protein using a monoclonal mouse anti-human antibody (clone DO-7, Leica), which detects both the wild-type and mutant forms of p53. Protein expression was examined by two independent examiners in a light microscope at high magnification (40X). Cells positive for the p53-protein identified by brown nuclear staining, were counted and calculated as a percentage of total cells per sample for each antibody respectively. Mean number of cells counted per sample was 695. Some samples were lost prior to final analysis due to either; sample core lost during TMA (n=12) or, sample had <100 cells which was minimum number of cells for inclusion in analysis (n=10). The final analysis included 154 bone marrow samples from 49 patients and 55 control samples.Data was analyzed in Statsoft Statistica 12.7. Nonlinear logistic regression was used to evaluate predictive value of the protein. Data was analyzed independently at each of the following times; diagnosis, time of HSCT and at 0-3, >3-6, >6-12 and >12-24 months post HSCT. A p value < 0.05 was considered significant.Interestingly, we found that a low percentage of cells positive for p53 protein expression in the bone marrow at >3-6 months after HSCT was associated with increased risk of relapse in pediatric ALL patients, odds ratio 0.82 (confidence interval 0.68-0.99) p= 0.041. Figure I shows the range of p53 expression in the two groups individually at each time.At >3-6 months after HSCT, the cut off value was calculated to 3.3% p53 positive cells, and was correct for 16 of 20 non-relapse cases and for 9 of 14 relapse cases (overall 73.5% correct). All relapses occurred between 6-50 months after HSCT (mean=20, median=14).The lower amount of p53 positive cells in the relapsed patients compared to the non-relapsed patients could possibly be due to an underlying mutation in TP53 that has altered the ability of producing a protein. This would be in similarity with the literature where it is suggested that TP53 mutations are more common at relapse in pediatric ALL patients.In summary, a lower expression of p53 protein in the bone marrow at >3-6 months after HSCT was significantly associated with increased risk of relapse. Evaluation of p53 protein expression by immunohistochemistry, in bone marrow from pediatric ALL-patients that undergo HSCT may be a potential additional marker for predicting relapse. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.