Specific Human Antibodies Research Articles

Human cytomegalovirus (HCMV) UL44 and UL57 specific antibody responses in anti-HCMV-positive patients with systemic sclerosis.

The role of human cytomegalovirus (HCMV) has been postulated as a trigger of systemic sclerosis (SSc). The aim of the study was to assess the prevalence of antibodies against HCMV UL44 and UL57 antigens not tested in the past. Sixty SSc patients, 40 multiple sclerosis and 17 normal controls (NCs), all anti-HCMV positive, were tested by immunoblotting. Reactivity to HCMV antigens, expressed as arbitrary units (AUs), was assessed for correlation with clinical and immunological parameters, including types of SSc-related autoantibodies. Anti-UL44 and anti-UL57 HCMV antibodies were present in 3/60 (5%) and 58/60 (96.7%) SSc patients, respectively (p<0.001). Anti-UL57 antibodies were present in 35/40 (87.5%) MS patients and 16/17 (94.1%) NCs (SSc vs MS, MS vs NC, p=ns). Strong (50-75 AU) and very strong (75-100AU) anti-UL57 immunoreactivity was found in 24 (41.4%) and 22 (37.9%) SSc patients, respectively (p=ns). Dilution experiments showed anti-UL57 antibody persistence in up to 1/5000. Overall, there was no difference in the frequency or the magnitude of anti-UL57 immunoreactivity between diffuse cutaneous systemic sclerosis and limited cutaneous systemic sclerosis patients (96.67 vs 96.67%; 65.45±20.19 vs 64.31±21.11AU, p>0.05) but strong anti-UL57 reactivity were more frequent in SSc compared to NCs (p=0.007). Anti-UL57 reactivity was not inhibited by SSc-specific autoantigens. Anti-UL57 seropositivity did not correlate with demographic, clinical or immunological features of SSc. Anti-HCMV UL57 antibodies are universally present in anti-HCMV-positive patients with SSc, while those against UL44 are rarely seen. Because anti-UL57 lack disease specificity and are not involved in cross-reactive responses, their immunopathogenetic potential is to be questioned.

Production of Specific Anti-EGFR Single Chain Antibodies: A Promising Strategy in the Immunotherapy of EGFR Expressing Tumor Tissues

Background: EGFR (Epidermal growth factor receptor) is overexpressed in a number of cancers and plays an important role in several phenomena such as aggressiveness of tumor, decreased survival of the patient, and resistance to treatments such as hormone therapy, chemotherapy, and also radiation. Objectives: The aim of this study is to produce specific human single-chain antibodies against EGFR and evaluate its specificity against the immunodominant epitope in order to offer a new and efficient way in the treatment of EGFR-expressing tumor tissues. Methods: A phage antibody display library of scFv (single chain fragment variable) was panned against an immunodominant epitope of EGFR. In order to select the specific clones, DNA fingerprinting was performed and the common patterns were differentiated. ELISA (Enzyme linked immunosorbent assay) was done to confirm the panning results and show the specificity of the selected clones. Results: Two specific clones with the frequencies of 55% and 30% were differentiated. The clones showed positive ELISA with the corresponding epitope while no positive reaction was observed for negative controls: unrelated peptide, M13KO7 (helper phage), unrelated scFv and no peptide. Conclusions: Immunotherapy against cancer has been a new treatment strategy in the recent years. Small and high affinity scFvs have had a crucial role in this regard. The specific human anti-EGFR scFvs that were selected in this study and reacted with the corresponding epitopehave the potential to be applied as a blocking antibody for interfering with tumor growth in EGFR-expressing tumors. Further studies are needed to evaluate the effects of these antibodies in vitro and in vivo.

Donor specific HLA Antibodies as a Trigger for Ischemic Type Biliary Lesions (ITBL) after Orthotopic Liver Transplantation-A Case Controlled Study

Despite recent surgical developments, biliary complications following orthotopic liver transplantation (OLT) are an important cause of morbidity and mortality. Ischemic Type Biliary Lesions (ITBL) is particularly challenging accounting for a high proportion of bile-duct associated complications following OLT. We aimed to better predict and reduce the incidence of ITBL through detection of donor specific human leucocyte antibodies (DSA). These could be involved in the pathogenesis of this disease. This approach allows to standardized perioperative DSA-screening in OLT-patients.Prospective database of ITBL-patients (n=15) that had undergone OLT from February 2008 to October 2011 was analysed. Clinical parameters, biochemical data including preoperative HLA-status and postoperative DSA-status, complications, morbidity and mortality were studied. The ITBL-patients were matched in a 1:1 ratio accounting for demographic and clinical variables to a control cohort of OLT-patients. Propensity modelling with matched cohort analysis was employed. There was a significance between the detection of de novo DSA and the development of an ITBL (p=0,003) and an asymptomatic elevation of alkaline phosphatase (AP). This suggested a strong relationship between de novo DSA and future development of ITBL. This implies a need for a protocolisation of care with standardized postoperative DSA-status allowing earlier diagnosis and therapeutic intervention.

Disruption of Circulating Extracellular Vesicles as a Novel Therapeutic Strategy against Cancer Metastasis

Metastasis is the main cause of cancer mortality for many types of cancer; however, difficulties remain in effectively preventing metastasis. It has been recently and widely reported that cancer-derived extracellular vesicles (EVs) contribute to cancer metastasis. Thus, therapeutic strategies targeting cancer-derived EVs hold great promise because of the possibility of EVs driving the cancer microenvironment toward metastasis. Here, we provide a novel strategy for therapeutic antibody treatment to target cancer-derived EVs and inhibit the metastasis of breast cancer in a mouse model, establishing a rationale for further clinical investigation. Treatment with human-specific anti-CD9 or anti-CD63 antibodies significantly decreased metastasis to the lungs, lymph nodes, and thoracic cavity, although no obvious effects on primary xenograft tumor growths were observed. In invitro and invivo experiments, the EVs incubated with the targeted antibodies were preferentially internalized by macrophages, suggesting that antibody-tagged cancer-derived EVs would be eliminated by macrophages. Our results suggested that therapeutic antibody administration effectively suppresses EV-triggered metastasis in cancer and that the removal of EVs could be a novel strategy for cancer therapy.

Insulin sensitivity index (ISI 0-120) from oral glucose tolerance test in healthy young adults

Background: Insulin sensitivity (IS) is the body’s systemic responsiveness to glucose and it is the measure of the ability of endogenous insulin to reduce the glucose in extracellular fluids by inhibition of gluconeogenesis and increase peripheral glucose uptake. IS reflects on the efficiency of insulin in response to glucose intake in the body. In diabetics, IS subjects are interpreted to require smaller amounts of insulin to lower blood glucose levels than someone who has low sensitivity. There is a need to detect insulin resistance (IR) in the pre-disease state, i.e., before the onset of impaired glucose tolerance (IGT). Early detection of IR or decreased IS in healthy subjects before the onset of IGT is of importance as it facilitates implementation of preventive measures in subject with such high risk. Aims and Objectives: The objective of the study is detect early occurrence of low Insulin Sensitivity in healthy young adults, using Insulin Sensitivity Index derived from OGTT. Materials and Methods: A total of 80 healthy volunteers in the age group of 18-25 years were recruited for the study, 40 subjects were siblings of diabetics and 40 subjects were siblings of non-diabetics (SND) a standard (75 g) OGTT was performed for the study. Blood samples for determination of plasma glucose and insulin levels drawn at 0 (fasting), 30, 120 min after glucose solution ingestion. Assays of fasting (basal), 30, 120 min venous plasma glucose during OGTT was performed with glucose oxidase method on site using glucose auto analyzer. The serum plasma was stored at −20°C until assayed. Corresponding specific insulin concentration was determined using radioimmunoassay (RIA) with human specific antibody RIA kit IS was calculated using physiological OGTT based mathematical models. QUICKI was derived for fasting insulin and fasting glucose values. ISI 0-120: Uses OGTT values, using only 0 and 120 min post-glucose challenge insulin and glucose concentrations. The reference values for various IR and IS indexes for our urban population with normal OGTT (n = 79) are QUICKI = 0.31 (0.20-0.52) sibling of diabetics (SD) - 0.07, ISI 0-120 = 63.62 (27.37-134.79) SD - 22.71. We observed that SD had significantly lower IS indices ISI 0-120 (56.27, P < 0.002) and a trend toward significance for QUICKI (0.29578, P < 0.056). Results: We observed that the mathematical models ISI 0-120 to be a fairly reliable tool for assessment of IS in normoglycemic young adults, compared to QUICKI. ISI 0-120 take into consideration the all the parameters of the 2 h OGTT glucose and also includes insulin into consideration for evaluation. Simple OGTT based mathematical models can be used as a reasonable alternative to measure IS or IR instead of the cumbersome glucose clamp or other expensive techniques in epidemiological or general clinical settings. Conclusion: Detection of IR in pre-disease condition in healthy individuals, allows the physician to initiate preventive measures, such as lifestyle modification, diet and exercise, thereby preventing the high-risk subjects from progressing to disease state.

Targeting dendritic cells in humanized mice receiving adoptive T cells via monoclonal antibodies fused to Flu epitopes

The targeting of vaccine antigens to antigen presenting cells (APC), such as dendritic cells (DCs), is a promising strategy for boosting vaccine immunogenicity and, in turn, protective and/or therapeutic efficacy. However, in vivo systems are needed to evaluate the potential of this approach for testing human vaccines. To this end, we examined human CD8+ T-cell expansion to novel DC-targeting vaccines in vitro and in vivo in humanized mice. Vaccines incorporating the influenza matrix protein-1 (FluM1) antigen fused to human specific antibodies targeting different DC receptors, including DEC-205, DCIR, Dectin-1, and CD40, elicited human CD8+ T-cell responses, as defined by the magnitude of specific CD8+ T-cells to the targeted antigen. In vitro we observed differences in response to the different vaccines, particularly between the weakly immunogenic DEC-205-targeted and more strongly immunogenic CD40-targeted vaccines, consistent with previous studies. However, in humanized mice adoptively transferred (AT) with mature human T cells (HM-T), vaccines that performed weakly in vitro (i.e., DEC-205, DCIR, and Dectin-1) gave stronger responses in vivo, some resembling those of the strongly immunogenic CD40-targeted vaccine. These results demonstrate the utility of the humanized mouse model as a platform for studies of human vaccines.

Transplantation of Scaffold-Free Cartilage-Like Cell-Sheets Made from Human Bone Marrow Mesenchymal Stem Cells for Cartilage Repair: A Preclinical Study.

The object of this study was to determine culture conditions that create stable scaffold-free cartilage-like cell-sheets from human bone marrow-derived mesenchymal stem cells (hBMSCs) and to assess their effects after transplantation into osteochondral defects in nude rats. (Experiment 1) The hBMSCs were harvested from 3 males, the proliferative and chondrogenic capacities were assessed at passage 1, and the cells were expanded in 3 different culture conditions: (1) 5% fetal bovine serum (FBS), (2) 10% FBS, and (3) 5% FBS with fibroblast growth factor 2 (FGF-2). The cells were harvested and made chondrogenic pellet culture. The cell proliferation rate, glycosaminoglycan/DNA ratio, and safranin-O staining intensity of pellets cultured condition 3 were higher than those of conditions 1 and 2. (Experiment 2) The hBMSCs were expanded and passaged 3 times under culture condition 3, and fabricate the cell-sheets in chondrogenic medium either with or without FBS. The cell-sheets fabricated with FBS maintained their size with flat edges. (Experiment 3) The cell-sheets were transplanted into osteochondral defects in nude rats. Histological analysis was performed at 2, 4, and 12 weeks after surgery. The osteochondral repair was better after sheet transplantation than in the control group and significantly improved Wakitani score. Immunostaining with human-specific vimentin antibody showed that the transplanted cells became fewer and disappeared at 12 weeks. These results indicate that culture with FGF-2 may help to quickly generate sufficient numbers of cells to create stable and reliable scaffold-free cartilage-like cell-sheets, which contribute to the regeneration of osteochondral defects.

Generation of a Bone Organ by Human Adipose-Derived Stromal Cells Through Endochondral Ossification.

: Recapitulation of endochondral ossification (ECO) (i.e., generation of marrow-containing ossicles through a cartilage intermediate) has relevance to develop human organotypic models for bone or hematopoietic cells and to engineer grafts for bone regeneration. Unlike bone marrow-derived stromal cells (also known as bone marrow-derived mesenchymal stromal/stem cells), adipose-derived stromal cells (ASC) have so far failed to form a bone organ by ECO. The goal of the present study was to assess whether priming human ASC to a defined stage of chondrogenesis in vitro allows their autonomous ECO upon ectopic implantation. ASC were cultured either as micromass pellets or into collagen sponges in chondrogenic medium containing transforming growth factor-β3 and bone morphogenetic protein-6 for 4 weeks (early hypertrophic templates) or for two additional weeks in medium supplemented with β-glycerophosphate, l-thyroxin, and interleukin1-β to induce hypertrophic maturation (late hypertrophic templates). Constructs were implanted in vivo and analyzed after 8 weeks. In vitro, ASC deposited cartilaginous matrix positive for glycosaminoglycans, type II collagen, and Indian hedgehog. Hypertrophic maturation induced upregulation of type X collagen, bone sialoprotein, and matrix metalloproteinase13 (MMP13). In vivo, both early and late hypertrophic templates underwent cartilage remodeling, as assessed by MMP13- and tartrate-resistant acid phosphatase-positive staining, and developed bone ossicles, including bone marrow elements, although to variable degrees of efficiency. In situ hybridization for human-specific sequences and staining with a human specific anti-CD146 antibody demonstrated the direct contribution of ASC to bone and stromal tissue formation. In conclusion, despite their debated skeletal progenitor nature, human ASC can generate bone organs through ECO when suitably primed in vitro. Recapitulation of endochondral ossification (ECO) (i.e., generation of marrow-containing ossicles through a cartilage intermediate) has relevance to develop human organotypic models for bone or hematopoietic cells and to engineer grafts for bone regeneration. This study demonstrated that expanded, human adult adipose-derived stromal cells can generate ectopic bone through ECO, as previously reported for bone marrow stromal cells. This system can be used as a model in a variety of settings for mimicking ECO during development, physiology, or pathology (e.g., to investigate the role of BMPs, their receptors, and signaling pathways). The findings have also translational relevance in the field of bone regeneration, which, despite several advances in the domains of materials and surgical techniques, still faces various limitations before being introduced in the routine clinical practice.

Cell-Based Therapy in TBI: Magnetic Retention of Neural Stem Cells In Vivo.

Stem cell therapy is under active investigation for traumatic brain injury (TBI). Noninvasive stem cell delivery is the preferred method, but retention of stem cells at the site of injury in TBI has proven challenging and impacts effectiveness. To investigate the effects of applying a magnetic field on cell homing and retention, we delivered human neuroprogenitor cells (hNPCs) labeled with a superparamagnetic nanoparticle into post-TBI animals in the presence of a static magnetic field. We have previously devised a method of loading hNPCs with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles Molday ION Rhodamine B (MIRB™). Labeling of hNPCs (MIRB-hNPCs) does not affect hNPC viability, proliferation, or differentiation. The 0.6 tesla (T) permanent magnet was placed ∼4 mm above the injured parietal cortex prior to intracarotid injection of 4 × 10(4) MIRB-hNPCs. Fluorescence imaging, Perls' Prussian blue histochemistry, immunocytochemistry with SC121, a human-specific antibody, and T2-weighted magnetic resonance imaging ex vivo revealed there was increased homing and retention of MIRB-hNPCs in the injured cortex as compared to the control group in which MIRB-hNPCs were injected in the absence of a static magnetic field. Fluoro-Jade C staining and immunolabeling with specific markers confirmed the viability status of MIRB-hNPCs posttransplantation. These results show that increased homing and retention of MIRB-hNPCs post-TBI by applying a static magnetic field is a promising technique to deliver cells into the CNS for treatment of neurological injuries and neurodegenerative diseases.

Quantification of MCT1 and CD147 in Red Blood Cells of Arabian and Quarter Horses

During anaerobic glycolysis for energy production, lactate and H+ ions accumulate in muscle fibers and pass into the blood stream. The monocarboxylate transporter isoform 1 (MCT1) and its ancillary protein cluster of differentiation 147 (CD147) transport H+ and lactate ions from the plasma into red blood cells (RBCs), thereby maintaining acid/base homeostasis and retarding systemic acidosis and fatigue. The aim of this study was to compare the levels of MCT1 and CD147 protein in the RBC membranes of Arabian and Quarter Horses with different levels of athletic ability. Blood samples were collected from 40 Arabian and 40 Quarter Horses, both males and females, ranging from 3 to 16 years and 2 to 23 years, respectively. The horses were divided into two groups: 20 animals of low performance and 20 animals of high performance for each breed. The amount of MCT1 and CD147 in the plasma membranes of their RBCs was determined by Western blotting analysis with arbitrary optical density (OD) units, using a human-specific anti-MCT1 and anti-CD147 antibodies that were previously validated for horses. The means ± standard errors were analyzed by repeated measures analysis of variance using the PROC MIXED procedure of SAS software. The effect of age was included as covariate and sex as a class effect in the model. The correlations were analyzed by Pearson correlation test at P < .05. Monocarboxylate transporter isoform 1 with a molecular mass of approximately 52 and 49 kDa was found in the RBC membranes of all the Arabian and Quarter Horses, respectively. Cluster of differentiation 147 also was observed in all Arabian and Quarter Horses at approximately 52 and 48 kDa, respectively. A positive correlation was observed between the total amount of MCT1 and CD147 (r = 0.932, P < .001) in the RBC. The amount of MCT1 was significantly (P < .0001) higher in Quarter Horses (3.03 ± 0.37 OD) than in Arabians (1.02 ± 0.07 OD). Quarter Horses (3.23 ± 0.39 OD) also showed increased contents of CD147 than Arabians (0.89 ± 0.06 OD). However, there was no statistical difference in the amount of the protein between the low- and high-performance groups in either breed. Results indicate that the levels of MCT1 and CD147 are different between Arabian and Quarter Horses and the most probable explanation is that different pathways are used for the production of energy for each breed.

A new data-driven model for post-transplant antibody dynamics in high risk kidney transplantation

The dynamics of donor specific human leukocyte antigen antibodies during early stage after kidney transplantation are of great clinical interest as these antibodies are considered to be associated with short and long term clinical outcomes. The limited number of antibody time series and their diverse patterns have made the task of modelling difficult. Focusing on one typical post-transplant dynamic pattern with rapid falls and stable settling levels, a novel data-driven model has been developed for the first time. A variational Bayesian inference method has been applied to select the best model and learn its parameters for 39 time series from two groups of graft recipients, i.e. patients with and without acute antibody-mediated rejection (AMR) episodes. Linear and nonlinear dynamic models of different order were attempted to fit the time series, and the third order linear model provided the best description of the common features in both groups. Both deterministic and stochastic parameters are found to be significantly different in the AMR and no-AMR groups showing that the time series in the AMR group have significantly higher frequency of oscillations and faster dissipation rates. This research may potentially lead to better understanding of the immunological mechanisms involved in kidney transplantation.

Abstract P2-02-07: Collection, high-resolution imaging, and single cell isolation of circulating tumor cells from patient derived xenograft models using the AccuCyte® – CyteFinder® system

Abstract Background: Patient-derived xenograft (PDX) models of human tumors offer many advantages over traditional cell line xenograft models and other mouse models of cancer. A PDX model may be used to randomize a given patient's tumor to multiple treatment regimens in order to predict treatment responses. When PDX models are grouped, they represent a clinical trial "cohort" for testing new therapies and identifying biomarkers of response. One such biomarker is circulating tumor cells (CTCs), which provide a window of the metastatic process. CTCs have been reported in several PDX models, further supporting their clinical relevance. Thus, PDX models may also be used to study the utility of CTC analysis to inform treatment decisions. However, most current CTC technologies intended for use with human samples cannot be used with the small blood volume from mice. The objective of our study was to adapt the AccuCyte® – CyteFinder® (AC-CF) system to detect CTCs from low volumes of mouse blood, and apply this method for the analysis of CTCs in a PDX model, including individual cell retrieval for molecular analysis. Methods: The AC-CF PDX process was modified to include a red blood cell lysis step instead of the density-based separation for the removal of red blood cells. The isolated cells were spread onto microscope slides using a stabilization solution, stained by multi-color immunofluorescence, and visualized by the CF high-resolution multi-channel fluorescence scanner. Automated image analysis identified CTCs, which was followed by single cell retrieval. For optimization of the assay, BT474 breast cancer cells were spiked into blood from a tumor-free control mouse ( approx. 500 cells in 250 µl). Slides with BT474 cells were used to test sensitivity by using antibodies against human cytokeratins (pan-CK), epithelial cell adhesion molecule (EpCAM), and erbB family growth factor receptors (EGFR and HER2) to detect the spiked-in cells. Assay specificity was tested by using antibodies specific for the mouse isoform of CD45. The antibody panel was tested on blood samples from 6 mice carrying small (300-400 mm3) tumors of the breast cancer PDX model (BCM-4888) previously published to have CTCs. Results: BT474 were identified by their large nuclei, positive staining with human specific antibodies against pan-CK, EpCAM, and EGFR/HER2 markers, and negative staining for mouse CD45. BT474 were detected in approximately the same amount as were spiked in. CTCs were identified in the blood of all 6 PDX mice tested. We found 1-6 CTCs per 330 µl of blood, and clusters of CTCs were also identified in 4 mice. Overall, these findings agree with published data on this PDX model. Single CTCs will be isolated using the CytePicker® retrieval module for single cell sequencing to confirm the human origin of these cells. These results along with ongoing work on additional PDX models will be presented at the meeting. Conclusion: The modified AC-CF process is a simple and sensitive method of analyzing small volumes of blood for CTC detection and isolation, features that are critical for the longitudinal analysis of CTCs in PDX models of cancer. Citation Format: Ramirez AB, Sahay D, Lewis MT, Schiff R, Stilwell JL, Trivedi M, Kaldjian EP. Collection, high-resolution imaging, and single cell isolation of circulating tumor cells from patient derived xenograft models using the AccuCyte® – CyteFinder® system. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-02-07.

Blocking TNF-α inhibits angiogenesis and growth of IFIT2-depleted metastatic oral squamous cell carcinoma cells

Our previous study demonstrated that the depletion of interferon-induced protein with tetratricopeptide repeats 2 (IFIT2) promoted metastasis and was associated with a poor prognosis in patients with oral squamous cell carcinoma (OSCC). Our current study explores the major downstream signaling involved in IFIT2 depletion-induced OSCC metastasis. To this end, we used two cell lines (designated sh-control-xeno and sh-IFIT2-xeno) derived from human OSCC xenografts expressing sh-control and sh-IFIT2, respectively, and one metastatic OSCC subline (sh-IFIT2-meta) from an IFIT2-depleted metastatic tumor. We found that the sh-IFIT2-meta cells proliferated more slowly than the sh-control-xeno cells but exhibited higher migration and chemoresistance. Using microarray technology and Ingenuity Pathway Analysis, we found that TNF-α was one of the major downstream targets in IFIT2-depleted OSCC cells. Quantitative real-time PCR, western blotting, and ELISA results confirmed that TNF-α was upregulated in the sh-IFIT2-meta cells. Blocking TNF-α abolished the angiogenic activity induced by the sh-IFIT2-meta cells. Furthermore, the human-specific TNF-α antibody golimumab significantly inhibited in vivo angiogenesis, tumor growth and metastasis of sh-IFIT2-meta cells. These results demonstrate that IFIT2 depletion results in TNF-α upregulation, leading to angiogenesis and metastasis of OSCC cells.

Optimizing transplantation of sensitized heart candidates using 4 antibody detection assays to prioritize the assignment of unacceptable antigens

The virtual crossmatch relies on the assignment of unacceptable antigens (UAs) to identify compatible donors. The purpose of our study was to identify an algorithm for assignment of UAs such that a negative complement-dependent cytotoxicity (CDC) crossmatch and concomitant negative or weakly positive flow cytometric crossmatch (FXM) are obtained. We used 4 antibody methods: (1) Luminex single antigen (LSA), (2) LSA with a 1:8 serum dilution, (3) C1q LSA, and (4) CDC panel. The UAs were prioritized in the following order: (1) all C1q+/CDC+, (2) LSA 1:8 >7,500 median fluorescence intensity, and (3) LSA >10,000 median fluorescence intensity. Of 295 heart transplants that were performed at our center, 69 (23%) recipients had detectable human leukocyte antigen specific antibody at the time of transplant. All donor specific antibodies (DSAs) were avoided for 44 of 69 (64%) (DSA-). There were 25 recipients who had DSA at the time of transplant: 12 (48%) had negative FXM (DSA+/FXM-), and 13 (52%) had positive T-cell and/or B-cell FXM (DSA+/FXM+). Lower freedom from antibody-mediated rejection was observed for the DSA+/FXM+ group compared with the DSA- group (p < 0.0001). DSA remained detectable after transplant in the sera of 14 recipients, and de novo DSA was detected in 32 recipients. Freedom from antibody-mediated rejection was comparable for both groups (p = 0.53) but was lower than the DSA- group (p < 0.0001). Survival was comparable for all groups at 1,200 days post-transplant. Strategic prioritization of UA assignment has allowed transplantation of highly sensitized patients across the DSA barrier with survival rates comparable to DSA- heart transplant recipients.

Targeting Syndecan-1, a molecule implicated in the process of vasculogenic mimicry, enhances the therapeutic efficacy of the L19-IL2 immunocytokine in human melanoma xenografts.

Anti-angiogenic therapy of solid tumors has until now failed to produce the long lasting clinical benefits desired, possibly due to the complexity of the neoangiogenic process. Indeed, a prominent role is played by "vasculogenic" or "vascular" mimicry (VM), a phenomenon in which aggressive cancer cells form an alternative microvascular circulation, independently of endothelial cell angiogenesis. In this study we observed, in melanoma patient cell lines having vasculogenic/stem-cell like phenotype and in melanoma tumors, the syndecan-1 co-expression with VM markers, such as CD144 and VEGFR-2. We show that melanoma cells lose their ability to form tubule-like structures in vitro after blocking syndecan-1 activity by the specific human recombinant antibody, OC-46F2. Moreover, in a human melanoma xenograft model, the combined therapy using OC-46F2 and L19-IL2, an immunocytokine specific for the tumor angiogenic-associated B-fibronectin isoform(B-FN), led to a complete inhibition of tumor growth until day 90 from tumor implantation in 71% of treated mice, with statistically significant differences compared to groups treated with OC-46F2 or L19-IL2 as monotherapy. Furthermore, in the tumors recovered from mice treated with OC-46F2 either as monotherapy or in combination with L19-IL2, we observed a dramatic decrease of vascular density and loss of VM structures. These findings indicate for the first time a role of syndecan-1 in melanoma VM and that targeting syndecan-1, together with B-FN, could be promising in improving the treatment of metastatic melanoma.

Dll4 Blockade in Stromal Cells Mediates Antitumor Effects in Preclinical Models of Ovarian Cancer

The Notch ligand delta-like 4 (Dll4) has been identified as a promising target in tumor angiogenesis in preclinical studies, and Dll4 inhibitors have recently entered clinical trials for solid tumors, including ovarian cancers. In this study, we report the development of REGN421 (enoticumab), a fully human IgG1 monoclonal antibody that binds human Dll4 with sub-nanomolar affinity and inhibits Notch signaling. Administering REGN421 to immunodeficient mice engineered to express human Dll4 inhibited the growth of several human tumor xenografts in association with the formation of nonfunctional tumor blood vessels. In ovarian tumor xenograft models, Dll4 was expressed specifically by the tumor endothelium, and Dll4 blockade by human-specific or mouse-specific Dll4 antibodies exerted potent antitumor activity, which relied entirely on targeting Dll4 expressed by tumor stromal cells but not by the tumor cells themselves. However, Dll4 blockade reduced Notch signaling in both blood vessels and tumor cells surrounding the blood vessels, suggesting that endothelial-expressed Dll4 might induce Notch signaling in adjacent ovarian tumor cells. The antitumor effects of targeting Dll4 were augmented significantly by simultaneous inhibition of VEGF signaling, whereas this combined blockade reversed normal organ vascular changes induced by Dll4 blockade alone. Overall, our findings deepen the rationale for antibody-based strategies to target Dll4 in ovarian cancers, especially in combination with VEGF blockade.

Application of recombinant severe fever with thrombocytopenia syndrome virus nucleocapsid protein for the detection of SFTSV-specific human IgG and IgM antibodies by indirect ELISA.

BackgroundSevere fever with thrombocytopenia syndrome (SFTS) is an emerging disease that was first reported in China in 2011. It is caused by SFTS virus (SFTSV) which is a member of the Phlebovirus genus in the Bunyaviridae family. SFTSV has been classified as a BSL3 pathogen. There is a need to develop safe and affordable serodiagnostic methods for proper clinical management of infected patients.MethodsThe full length nucleocapsid (N) gene of SFTSV Yamaguchi strain was amplified by RT-PCR and cloned to an expression vector pQE30. The recombinant (r) SFTSV-N protein was expressed by using Escherichia coli (E. coli) expression system and purified under native conditions. rSFTSV-N protein based indirect IgG and IgM enzyme linked immunosorbent assay (ELISA) systems were established to detect specific human IgG and IgM antibodies, respectively. One hundred fifteen serum samples from clinically suspected-SFTS patients were used to evaluate the newly established systems and the results were compared with the total antibody detecting sandwich ELISA system.ResultsThe native form of recombinant (r) SFTSV-N protein was expressed and purified. Application of the rSFTSV-N protein based indirect IgG ELISA to the 115 serum samples showed results that perfectly matched those of the total antibody sandwich ELISA with a sensitivity and specificity of 100 %. The rSFTSV-N protein based indirect IgM ELISA missed 8 positive samples that were detected by the total antibody sandwich ELISA. The sensitivity and specificity of rSFTSV-N-IgM capture ELISA were 90.59 and 100 %, respectively.ConclusionsThe rSFTSV-N protein is highly immunoreactive and a good target for use as an assay antigen in laboratory diagnosis. Its preparation is simpler in comparison with that used for the total antibody sandwich system. Our rSFTSV-N protein-based IgG and IgM ELISA systems have the advantage of distinguishing two types of antibodies and require small volume of serum sample only. They are safe to use for diagnosis of SFTS virus infection and especially fit in large-scale epidemiological investigations.

Abstract 427: Host and tumoral CXCR2 signaling contributes to tumor growth

Abstract Chemokines are essential mediators of leukocyte migration and inflammation. Additionally they play an important role in tumour growth. The G-protein coupled receptor, CXCR2, and its ligands (CXCL 1,2,3,5,6,7 and IL8) have been shown to promote tumour initiation and growth, chemo-resistance, angiogenesis and immune cell infiltration (neutrophil, myeloid derived suppressor cells (MDSCs) and macrophages) into the tumour microenvironment. However the contribution of host and tumour CXCR2 has not been elucidated. We have developed both murine and human specific anti-CXCR2 antibodies alongside a humanised CXCR2 transgenic mouse to address the contribution of host and tumour CXCR2 signalling to tumour growth and maintenance. In pre-clinical tumours, granulocytic cells (neutrophils and MDSCs) within the tumour increase with size, in parallel with alteration in cell numbers observed within the spleen, peripheral blood and bone marrow. Inhibition of host CXCR2 has a dramatic impact on peripheral neutrophil levels, as well as their ability to become activated. In pre-clinical tumour models, inhibition of CXCR2 results in tumour growth inhibition. Contribution of host or tumour cells to the effect of CXCR2 blockade was model dependent. In the EL4 murine lymphoma model, only inhibition of host/peripheral CXCR2 was able to impact tumour growth. Whereas, in other murine syngeneic models (CT26 and B16), both host and tumour CXCR2 play a role in tumour growth. This was further demonstrated in an NSCLC patient derived xenograft, where the contribution of tumour CXCR2 was stronger than the host. Collectively our data show that CXCR2 inhibition (alone or in combination) has potential to influence growth of a number of tumour types. Future work is focused on understanding the mechanisms underlying the effects of CXCR2 inhibition, which remain key to developing it as an effective anti-cancer therapeutic. Citation Format: Danielle Carroll, James Harper, Karen McDaid, Ruth Franks, Catherine Eberline, Jane Kendrew, Richard Sainson, Judith Anderton, Chris Rossant, Karen Coffman, Ching Ching Leow, Ivan Inigo, Mitchell Reville, Jacintha Shenton, Lesley Young, Simon Barry. Host and tumoral CXCR2 signaling contributes to tumor growth. [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 427. doi:10.1158/1538-7445.AM2015-427

Preliminary Tests of Multiplex Immunoassay for Detection of TORCH Infections in Human Blood Serum Using Flow Cytometry

Preliminary tests of multiplex immunoassay for detection of TORCH infections in human blood serum using flow cytometry are presented. The immunoassay was intended to provide qualitative detection of the specific human IgG class antibodies to Toxoplasma gondii, rubella virus, and cytomegalovirus in human blood serum. The multiplex analysis is performed in vitro on polystyrene microbeads labeled with an organic dye. The diagnostic sensitivity of the kit is 97%.

Structural differences of amyloid-β fibrils revealed by antibodies from phage display.

BackgroundBeside neurofibrillary tangles, amyloid plaques are the major histological hallmarks of Alzheimer’s disease (AD) being composed of aggregated fibrils of β-amyloid (Aβ). During the underlying fibrillogenic pathway, starting from a surplus of soluble Aβ and leading to mature fibrils, multiple conformations of this peptide appear, including oligomers of various shapes and sizes. To further investigate the fibrillization of β-amyloid and to have tools at hand to monitor the distribution of aggregates in the brain or even act as disease modulators, it is essential to develop highly sensitive antibodies that can discriminate between diverse aggregates of Aβ.ResultsHere we report the generation and characterization of a variety of amyloid-β specific human and human-like antibodies. Distinct fractions of monomers and oligomers of various sizes were separated by size exclusion chromatography (SEC) from Aβ42 peptides. These antigens were used for the generation of two Aβ42 specific immune scFv phage display libraries from macaque (Macaca fascicularis). Screening of these libraries as well as two naïve human phage display libraries resulted in multiple unique binders specific for amyloid-β. Three of the obtained antibodies target the N-terminal part of Aβ42 although with varying epitopes, while another scFv binds to the α-helical central region of the peptide. The affinities of the antibodies to various Aβ42 aggregates as well as their ability to interfere with fibril formation and disaggregation of preformed fibrils were determined. Most significantly, one of the scFv is fibril-specific and can discriminate between two different fibril forms resulting from variations in the acidity of the milieu during fibrillogenesis.ConclusionWe demonstrated that the approach of animal immunization and subsequent phage display based antibody selection is applicable to generate highly specific anti β-amyloid scFvs that are capable of accurately discriminating between minute conformational differences.