Abstract Background Heterogeneity in the treatment of a disease is a marker of suboptimal quality of care. Recent studies suggest that there is a marked heterogeneity in the management of inflammatory bowel disease. The aim of this study is to evaluate the heterogeneity in the treatment used and the outcomes (rate of hospitalization and surgery) for Crohn’s disease (CD) in the different health areas of Catalonia. Methods All patients with CD included in the Catalan Health Surveillance System (CHSS) including data on more than 7 million individuals from 2011 to 2017 were identified using with the ICD-9-CM codes. The different Catalonian health areas were grouped into 19 groups according to the reference hospital (figure 1). Exposures to different treatments were retrieved from the electronic dispensation records. Data on hospitalizations and surgeries were also extracted from the CHSS according to ICD-9-CM codes. Treatment used rates (systemic corticosteroids, non-biological and biological immunosuppressant) and outcome rates (hospitalization and surgery) were calculated. Results The use of systemic corticosteroids presented a decreasing trend over the study period, with an average rate of use of the total number of patients in the different territories between 10% and 16% (figure 2). The use of non-biological immunosuppressant treatment has remained stable, with an average rate of use ranging from 20% to 40% (figure 3). On the other hand, the use of biological immunosuppressant treatment increased, with an average rate of use in the different territories ranging from 10 to 22% (figure 4). Hospitalizations for any reason showed an increasing trend between 2011–2017 with an average rate between 19% and 30% per year depending on the area. Contrarily, hospitalizations for CD presented a decreasing trend, with an average rate between 5% and 10% per year. Surgical treatment (both resections and ostomies) remained stable over time. Rates per year were between 1% and 2%. Conclusion In this population study we appreciated an important heterogeneity in the use of non-biological and biological immunosuppressant treatments, identifying use rates of almost the double in some of the areas. There is also a remarkable variability in the rate of hospitalizations for CD between certain areas of Catalan territory.

Apoptosis through the TRAIL receptor pathway can be induced via agonistic. IgG to either TRAIL-R1 or TRAIL-R2. Here we describe the use of phage display to isolate a substantive panel of fully human anti-TRAIL receptor single chain Fv fragments (scFvs); 234 and 269 different scFvs specific for TRAIL-R1 and TRAIL-R2 respectively. In addition, 134 different scFvs that were cross-reactive for both receptors were isolated. To facilitate screening of all 637 scFvs for potential agonistic activity in vitro, a novel high-throughput surrogate apoptosis assay was developed. Ten TRAIL-R1 specific scFv and 6 TRAIL-R2 specific scFv were shown to inhibit growth of tumor cells in vitro in the absence of any cross-linking agents. These scFv were all highly specific for either TRAIL-R1 or TRAIL-R2, potently inhibited tumor cell proliferation, and were antagonists of TRAIL binding. Moreover, further characterization of TRAIL-R1 agonistic scFv demonstrated significant anti-tumor activity when expressed and purified as a monomeric Fab fragment. Thus, scFv and Fab fragments, in addition to whole IgG, can be agonistic and induce tumor cell death through specific binding to either TRAIL-R1 or TRAIL-R2. These potent agonistic scFv were all isolated directly from the starting phage antibody library and demonstrated significant tumor cell killing properties without any requirement for affinity maturation. Some of these selected scFv have been converted to IgG format and are being studied extensively in clinical trials to investigate their potential utility as human monoclonal antibody therapeutics for the treatment of human cancer.

OBJECTIVES Tumor necrosis factor-related apoptosis-inducing tigand (TRAIL) induces apoptosis in a variety of tumor cells through two of its receptors: TRAIL-RI and TRAIL-R2. In this study, we investigated the susceptibility of human prostate cancer and bladder cancer cells to HGS-ETR2, a human monoclonal agonistic antibody specific for TRAIL-R2.METHODS The cell surface expression of TRAIL-RI and TRAIL-R2 on prostate cancer and bladder cancer cells was determined using flow cytometry. Cytotoxicity was assessed by 3-(4,5-dimethylthiazot-2-yl)-2,5-diphenyltetrazolium bromide assay, and caspase activities were measured by a quantitative colorimetric assay.RESULTS HGS-ETR2 effectively induced apoptotic cell death in DU145, PC3, and LNCaP human prostate cancer cells and J82 and T24 human bladder cancer cells. The increased effectiveness of HGS-ETR2 for inducing cell death might have been affected by differences in the cell surface expression of the two TRAIL receptors, in that TRAIL-R2, but not TRAIL-RI, was frequently expressed in the prostate cancer and bladder cancer cells. HGS-ETR2 significantly activated the caspase cascade, including caspase-3, -6, -8, and -9, which were the downstream molecules of the death receptors in prostate cancer cells. Caspase-3, -6, and -9 were also significantly activated with HGS-ETR2-induced apoptosis in the bladder cancer cells.CONCLUSIONS These findings suggest the potential utility of TRAIL-R2 antibody as a novel therapeutic agent against prostate cancer and bladder cancer.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in a variety of tumor cells through two of its receptors: TRAIL-R1 and TRAIL-R2. We investigate the susceptibility of human renal cell carcinoma (RCC) cells to TRM-1 and HGS-ETR2, 2 human monoclonal agonistic antibodies specific for TRAIL-R1 and TRAIL-R2, respectively. HGS-ETR2 effectively induced apoptotic cell death in 10 of 11 cell cultures, including 2 human RCC cell lines and 9 human primary RCC cell cultures, with a more pronounced effect after preincubation with anti-human IgG Fc. In contrast, TRM-1 was effective in only 1 primary RCC cell culture. The increased effectiveness of HGS-ETR2 for inducing cell death might have been affected by differences in the cell-surface expression of the 2 TRAIL receptors, namely that TRAIL-R2 but not TRAIL-R1 was frequently expressed in most of the RCC cells tested. The activities of caspase-9, -8, -6, and -3 were increased with HGS-ETR2-induced apoptosis, and cell death could be blocked by specific caspase inhibitors for caspase-9, -8, and -3, and the general caspase inhibitor. In vivo administration of HGS-ETR2 with or without cross-linker significantly suppressed tumor growth of subcutaneously inoculated human RCC xenografts in immunodeficient mice. These results suggest the potential utility of TRAIL-R2 antibody as a novel therapeutic agent in RCC.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a death protein that preferentially kills tumour cells while sparing normal cells. TRAIL has four exclusive receptors, two of which (TRAIL-R1, TRAIL-R2) are death receptors. Both TRAIL/Apo2L and agonistic antibodies to the TRAIL death receptors are currently being explored for cancer therapy. Although the activity of TRAIL/Apo2L in a variety of haematological malignancies has been examined, the activity of anti-TRAIL receptor agonistic antibodies in primary and cultured lymphoma cells has not. Using two fully human selective agonistic monoclonal antibodies to the TRAIL death receptors TRAIL-R1 (HGS-ETR1) and TRAIL-R2 (HGS-ETR2) this study demonstrated that both monoclonal antibodies activated caspase-8 and induced cell death in five of nine human lymphoma cell lines, and induced > 10% cell death in 67% and 70%, respectively, of 27 primary lymphoma cells, and > 20% cell death in at least one-thirds of the samples. HGS-ETR1 and HGS-ETR2 demonstrated comparable activity in the fresh tumour samples, which was independent of TRAIL receptor surface expression, Bax, cFLIP, or procaspase-8 expression, or exposure to prior therapy. Furthermore, both antibodies enhanced the killing effect of doxorubicin and bortezomib. Our data demonstrate that HGS-ETR1 and HGS-ETR2 monoclonal antibodies can induce cell death in a variety of cultured and primary lymphoma cells, and may have therapeutic value in lymphoma.

Purpose: Substantial evidence indicates that supraoligomerization of the death receptors for Fas ligand and tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is necessary for efficient activation of the apoptotic pathway. Bivalent IgG antibodies can induce the efficient apoptosis by mimicking the natural ligands but only after these antibodies are further oligomerized by cross-linking. In this study, we generated a novel agonist antibody to TRAIL receptor 2 (TRAIL-R2) capable of inducing apoptosis without cross-linking and elucidated its mode of action and efficacy. Experimental Design: A fully human antibody to TRAIL-R2, KMTR2, was generated from KM Mouse immunized with TRAIL-R2 ectodomain. Apoptosis-inducing activities of unfractionated or purified monomeric IgG of KMTR2 was evaluated in the presence or absence of cross-linkers, secondary antibodies or Fc receptor–expressing effector cells, against human colorectal adenocarcinoma Colo205. Oligomerization of TRAIL-R2 was analyzed by size exclusion chromatography and confocal microscopy, and in vivo efficacy was examined in Colo205 xenograft model. Results: KMTR2 specifically recognized TRAIL-R2 and induced apoptosis with or without cross-linking. Size exclusion chromatography showed that the apoptosis activity coeluted with monomeric IgG and was effective independent of secondary antibody or Fc receptor–expressing effector cells. The antibody formed supracomplexes with soluble recombinant and membrane-anchored TRAIL-R2 and enhanced clustering of TRAIL-R2 on cell surface without cross-linking. KMTR2 was dramatically efficacious in reducing established human tumor. Conclusion: Our findings indicate that novel agonist antibody KMTR2 can direct antibody-dependent oligomerization of TRAIL-R2 and initiates efficient apoptotic signaling and tumor regression independent of host effector function. Thus, the direct agonist would be a lead candidate for cancer therapeutics.

Background: Tumor necrosis factor (TNF) related apoptosis inducing ligand (TRAIL/Apo2L) is a member of TNF superfamily that preferentially induces apoptosis in cancer cells, while sparing normal cells.Because of the potential therapeutic application of this pathway.Furthermore, although several mechanisms of resistance have been proposed for TRAIL, similar mechanisms have not been identified for these selective agonistic antibodies.Methods: we evaluated the in vitro activity of two selective fully human agonistic monoclonal antibodies to the TRAIL death receptors TRAIL-R1 (HGS-ETR1) and TRAIL-R2 (HGS-ETR2) in 9 lymphoma cell lines and 27 primary lymphoma tumor samples.Cell proliferation and apoptosis were determined by the MTS and Annexin-V binding assays.Cell surface receptor expression was determined by Western blot, flow cytometry, and immunohistochemistry. Results: HGS-ETR1 and HGS-ETR2 antibodies demonstrated antiproliferative activity in 5 of 9 cell lines which was associated with caspase 8 activation and induction of apoptosis.Both antibodies induced cell death in two-thirds of the primary lymphoma samples, irrespective of prior exposure to chemotherapy.Sensitivity to these antibodies could not be predicted by the level of surface TRAIL-R1 or TRAIL-R2 receptor expression, or by the intracellular levels of cFLIP, caspase-8, or Bax.However, the absence of Bid expression in the lymphoma cell lines correlated with resistance to both antibodies (P = 0.0159).Conclusions: Our data demonstrate that HGS-ETR1 and HGS-ETR2 monoclonal antibodies to the TRAIL death receptors TRAIL-R1 and TRAIL-R2 can induce cell death in a variety of cultured and primary lymphoma cells, and therefore may have therapeutic value in lymphoma.Phase II study of HGS-ETR1(TRM-1) antibody is currently enrolling patient to evaluate the activity of this antibody in vivo.

4388 Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) death receptors are abundantly expressed on the cell surface of a majority of human tumor cells. Activation of this pathway rapidly leads to programmed cell death. These characteristics make the TRAIL receptor (TRAIL-R) pathway an attractive target for cancer therapeutic agents. Binding of agonist monoclonal antibodies (mAbs) or the ligand TRAIL, to either functional death receptor (TRAIL-R1, TRAIL-R2) induces activation of a protease-mediated signaling cascade that culminates in programmed cell death. A fully human, agonist, TRAIL-R2 monoclonal antibody (mAb), HGS-TR2J (also known as KMTR-2), is being developed for use as a cancer therapeutic agent. This mAb is currently in Phase I clinical safety trials. Here we have evaluated the ability of this mAb to activate the TRAIL-R2 signaling cascade and assessed the in vitro and in vivo efficacy of HGS-TR2J in human tumor cell lines alone or in combination with various chemotherapeutic agents. Tumor cell lines were evaluated for TRAIL-R expression by flow cytometry and sensitivity to HGS-TR2J and chemotherapeutic agents in vitro. Human tumor cell lines from different tumor types expressed TRAIL-R2 on the cell surface and displayed moderate to significant sensitivity (50-90% cell death) to HGS-TR2J in vitro. In several different human tumor cell lines, HGS-TR2J enhanced the cytotoxic activity of chemotherapeutic agents. Importantly, some combinations elicited a synergistic effect on cell viability. Pre-established NSCLC and colon adenocarcinoma xenografts rapidly regressed after treatment with HGS-TR2J. Tumor volume was significantly reduced (90-75%) after the first dose of HGS-TR2J. The inhibition of tumor growth persisted throughout the duration of treatment and generated complete elimination of tumor in several animals. The rapid decrease in tumor volume was associated with a concomitant increase in the number of apoptotic cells in the tumor and an increase in levels of active caspases, an indicator of TRAIL-R pathway activity. In addition, there appeared to be fluctuations in TRAIL-R2 expression in xenografts as determined by immunohistochemistry. In summary, these data reveal HGS-TR2J has significant anti-tumor activity, alone and in combination with various chemotherapeutic agents, in an array of human tumor cell lines. This anti-tumor activity induces a concomitant increase in active caspases, an indicator of TRAIL-R pathway activity and apoptosis. Sensitivity to this TRAIL-R specific mAb extends beyond the requirement for cell surface receptor expression. These studies illuminate the potential of HGS-TR2J as a therapeutic for the treatment of human cancer.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a variety of tumour cells through activation of TRAIL-R1 and TRAIL-R2 death signalling receptors. Here, we describe the characterisation and activity of HGS-ETR1, the first fully human, agonistic TRAIL-R1 mAb that is being developed as an antitumour therapeutic agent. HGS-ETR1 showed specific binding to TRAIL-R1 receptor. HGS-ETR1 reduced the viability of multiple types of tumour cells in vitro, and induced activation of caspase 8, Bid, caspase 9, caspase 3, and cleavage of PARP, indicating activation of TRAIL-R1 alone was sufficient to induce both extrinsic and intrinsic apoptotic pathways. Treatment of cell lines in vitro with HGS-ETR1 enhanced the cytotoxicity of chemotherapeutic agents (camptothecin, cisplatin, carboplatin, or 5-fluorouracil) even in tumour cell lines that were not sensitive to HGS-ETR1 alone. In vivo administration of HGS-ETR1 resulted in rapid tumour regression or repression of tumour growth in pre-established colon, non-small-cell lung, and renal tumours in xenograft models. Combination of HGS-ETR1 with chemotherapeutic agents (topotecan, 5-fluorouracil, and irinotecan) in three independent colon cancer xenograft models resulted in an enhanced antitumour efficacy compared to either agent alone. Pharmacokinetic studies in the mouse following intravenous injection showed that HGS-ETR1 serum concentrations were biphasic with a terminal half-life of 6.9-8.7 days and a steady-state volume of distribution of approximately 60 ml kg(-1). Clearance was 3.6-5.7 ml(-1) day(-1) kg(-1). These data suggest that HGS-ETR1 is a specific and potent antitumour agent with favourable pharmacokinetic characteristics and the potential to provide therapeutic benefit for a broad range of human malignancies.

6595 Bacground: TRAIL is a death protein that has a promising anti-cancer activity. Recent studies demonstrated that agonisitic antibodies to the TRAIL death receptors R1 and R2 can mimick the activity of TRAIL protein, and therefore may be of therapeutic value. The activity of these novel antibodies have been previously examined in primary lymphoma specimens. Methods: We evaluated the in vitro activity of two fully human agonistic monoclonal antibodies to the TRAIL death receptors R1 (HGS-ETR1) and R2 (HGS-ETR2) in 5 lymphoma cell lines and 27 primary lymphoid malignancy samples. Results: Although R1 and R2 receptors proteins were expressed in the majority of the samples as determined by Western blot, the surface receptor expression in the same sample frequently did not correlate with receptor protein expression suggesting that not all the receptors are present on the surface. Both ETR1 and ETR2 inhibited cell prolifeation and induced apoptosis in a dose and time dependent manner. However, ETR1 and ETR2 were not equally effective in different samples, and their activity did not correlate with the level of surface receptor expression. When the analysis is restricted to the 13 cases of primary non-Hodgkin's lymphoma samples, ETR1 induced at least 10% cell death in 62% of the samples, while ETR2 was effective in 77% of the samples. In the 14 primary chronic lymphocytic leukemia samples, ETR1 was effective in 71% of the samples, while ETR2 was effective in 64% of the samples. In some cases, ETR1 and ETR2 synergized with bortezomib (Velcade) and doxorubicin chemotherapy. However, this synergistic activity was not observed in all samples. Conclusions: We conclude that ETR1 and ETR2 monoclonal antibodies to the TRAIL death receptors R1 and R2 can induce cell death in a wide variety of cultured and primary tumor cells of lymphoid orgin origin. This preclinical data provide the basis for evaluating these novel antibodies for the treatment of patients with lymphoma and chronic lymphocytic leukemia. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Human Genome Sciences

4957 Targeted induction of apoptosis is an attractive approach for development of novel oncology therapeutics. One method of inducing apoptosis is through the TRAIL-Receptor family, which has five related members only two of which are pro-apoptotic, TRAIL-R1 (DR4) and TRAIL-R2 (DR5). These receptors have a cytoplasmic death-domain and signal to induce the extrinsic cell death pathway; they are also broadly expressed on many human tumors. Agonist antibodies specific for TRAIL-R1 and TRAIL-R2 are in development as potential anti-cancer therapeutics; however, the relationship of receptor expression to response to these agonist antibodies has not been established. To allow for evaluation of this relationship, immunohistochemistry (IHC) assays that detect expression of TRAIL-R1 and TRAIL-R2 in formalin-fixed, paraffin-embedded (FFPE) human tumor tissues were developed. It was critical that these assays detect TRAIL-R1 and TRAIL-R2 with great specificity and a high level of sensitivity, as tumors with low expression may be very significant as an agonist antibody target. Affinity purified rabbit polyclonal antibodies, specific for TRAIL-R1 and TRAIL-R2, were optimized using a two step assay utilizing the EnVision+® visualization system. The optimal antigen retrieval was determined to be heat induced epitope retrieval (HIER). The optimal antibody concentrations were established while employing an incubation time of 30 minutes for the TRAIL-R1 assay and 60 minutes for the TRAIL-R2 assay. The reactions were visualized with 3’3-diaminobenzidine (DAB+). Sections from FFPE pellets from cell lines previously characterized by flow cytometry for surface expression of TRAIL-R1 and TRAIL-R2 demonstrated optimal IHC staining using both assays. Similarly, tumor-enriched xenograft models showed IHC staining with minimal background staining of the surrounding non-reactive mouse tissue. Optimal staining for both TRAIL-R1 and TRAIL-R2 was observed on a wide range of human tumor specimens, including colon, lung and breast carcinoma. Staining of the tumor cells was favorable with minimal background staining in the stroma. In conclusion, sensitive and specific IHC assays have been developed for demonstration of TRAIL-R1 and TRAIL-R2 expression for use on FFPE human tumor samples. These assays allow for distinguishing between expression of TRAIL-R1 and TRAIL-R2, and are suitable for the further exploration of the relationship between TRAIL-R1 and TRAIL-R2 expression and therapeutic response to agonist antibodies.