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Purpose: Preclinical studies show that adavosertib, a WEE1 kinase inhibitor, sensitizes TP53-mutant cells to chemotherapy. We hypothesized that adavosertib, plus chemotherapy, would enhance efficacy versus placebo in TP53-mutated ovarian cancer. Patients and Methods: Following safety run-in, this double-blind phase II trial (NCT01357161) randomized women with TP53-mutated, platinum-sensitive ovarian cancer to oral adavosertib (225 mg twice daily for 2.5 days/21-day cycle) or placebo, plus carboplatin (AUC5) and paclitaxel (175 mg/m2), until disease progression or for six cycles. The primary endpoints were progression-free survival (PFS) by enhanced RECIST v1.1 [ePFS (volumetric)] and safety. Secondary/exploratory objectives included PFS by RECIST v1.1 (single dimension), objective response rate, overall survival, and analysis of tumor gene profile versus sensitivity to adavosertib. Results: A total of 121 patients were randomized to adavosertib (A+C; n = 59) and placebo (P+C; n = 62) plus chemotherapy. Adding adavosertib to chemotherapy improved ePFS [median, 7.9 (95% confidence interval (CI), 6.9–9.9) vs. 7.3 months (5.6–8.2); HR 0.63 (95% CI, 0.38–1.06); two-sided P = 0.080], meeting the predefined significance threshold (P < 0.2). Clinical benefit was observed following A+C for patients with different TP53 mutation subtypes, identifying possible response biomarkers. An increase in adverse events was seen with A+C versus P+C: greatest for diarrhea (adavosertib 75%; placebo 37%), vomiting (63%; 27%), anemia (53%; 32%), and all grade ≥3 adverse events (78%; 65%). Conclusions: Establishing an optimal strategy for managing tolerability and identifying specific patient populations most likely to benefit from treatment may increase clinical benefit. Future studies should consider additional adavosertib doses within the chemotherapy treatment cycle and the potential for maintenance therapy.

Background Emerging evidence supports combining immune checkpoint inhibitors (ICIs) with conventional or targeted therapies to enhance ICI antitumour activity and broaden the spectrum of patients who respond to ICIs. Here, we present the safety and preliminary efficacy of ramucirumab, an anti-VEGFR2 IgG1, plus durvalumab, an anti–PD-L1 IgG1, in previously treated patients with advanced non–small-cell lung cancer (NSCLC), gastric/gastro-oesophageal junction adenocarcinoma (gastric/GEJ), or hepatocellular carcinoma (HCC). Patients and methods A 25-centre, phase Ia/b single-arm, non-randomised, multi-cohort study was undertaken in patients with advanced/metastatic disease, Eastern Cooperative Oncology Group performance status, 0–1, progression on prior therapy, no prior ramucirumab or immunotherapy and any PD-L1 status. Patients received ramucirumab (10 mg/kg) plus durvalumab (1125 mg) intravenously Q3W (NSCLC), or ramucirumab (8 mg/kg) plus durvalumab (750 mg) Q2W (gastric/GEJ, HCC). Results Phase Ia treatment was found safe for phase Ib expansion; final enrolment was NSCLC (n = 28), gastric/GEJ (n = 29), HCC (n = 28). Grade ≥3 treatment–related adverse events occurred in 32.1%, 37.9% and 42.9% of patients, respectively. The most common were fatigue (35.7%), hypertension (34.5%) and diarrhoea (28.6%), respectively. Two patients died owing to an adverse event; one was treatment-related (hepatitis acute, HCC cohort). Objective response rate was 11% for NSCLC and HCC and 21% for gastric/GEJ. Median progression-free survival and overall survival were, respectively, 2.7 and 11 months in NSCLC; 2.6 and 12.4 months in gastric/GEJ; 4.4 and 10.7 months in HCC, with more prolonged survival in patients with high PD-L1 expression. Conclusion Ramucirumab/durvalumab exhibited manageable safety. The combination showed antitumour activity in all cohorts, particularly in patients with high PD-L1 expression.

2528 Background: A Phase 1b study (NCT02572687) was conducted to examine the combined effects of Ram (anti VEGFR2) and Durva (anti PD-L1). Methods: Patients (pts) with previously-treated, advanced NSCLC (Cohort [CH] A), G/GEJ adenocarcinoma (CH B), HCC (CH C), ECOG PS 0-1, and no prior Ram or IO therapy, received Ram (10 mg/kg) + Durva (1125 mg) Q3W (CH A) or Ram (8 mg/kg) + Durva (750 mg) Q2W (CH B, C). Primary objective was to assess safety; efficacy was also examined. PD-L1 expression of tumor cells (TC) +/- immune cells (IC) in pretreatment tumor biopsies were assessed using SP263 immunohistochemistry. “High” PD-L1 is ≥25% TC for NSCLC and ≥25% TC or IC for G/GEJ, HCC. Results: CH A, B and C enrolled pts with ECOG PS 1 (%) of 43, 66, 68; and average of 2, 2, 1 prior regimens, respectively. The most common grade 3/4 treatment-emergent adverse events (AE) are hypertension (HTN) (14.3, 17.2, 17.9%), anemia (3.6, 24.1, 21.4%), and fatigue (10.7, 10.3, 10.7%). Grade 3/4 AEs of special interest ( > 5% total pts) for Ram: HTN, bleeding events (3.6, 10.3, 10.7%), Venous thromboembolic events (0, 10.3, 7.1%); for Durva: increase in lipase (10.7, 3.4, 10.6%) and AST (3.6, 3.4, 17.9 %). Data from CH B,C suggest a trend toward increased efficacy in pts with high PD-L1 expressing tumors. Conclusions: Ram + Durva generated no unexpected toxicities and demonstrated antitumor activity. Results in pts with high PD-L1 HCC and G/GEJ cancer warrant further evaluation. Clinical trial information: NCT02572687. [Table: see text]

Purpose: Fulvestrant is an estrogen receptor (ER) antagonist and an approved treatment for metastatic estrogen receptor–positive (ER+) breast cancer. With the exception of ER levels, there are no established predictive biomarkers of response to single-agent fulvestrant. We attempted to identify a gene signature of response to fulvestrant in advanced breast cancer. Experimental Design: Primary tumor samples from 134 patients enrolled in the phase III CONFIRM study of patients with metastatic ER+ breast cancer comparing treatment with either 250 mg or 500 mg fulvestrant were collected for genome-wide transcriptomic analysis. Gene expression profiling was performed using Affymetrix microarrays. An exploratory analysis was performed to identify biologic pathways and new signatures associated with response to fulvestrant. Results: Pathway analysis demonstrated that increased EGF pathway and FOXA1 transcriptional signaling is associated with decreased response to fulvestrant. Using a multivariate Cox model, we identified a novel set of 37 genes with an expression that is independently associated with progression-free survival (PFS). TFAP2C, a known regulator of ER activity, was ranked second in this gene set, and high expression was associated with a decreased response to fulvestrant. The negative predictive value of TFAP2C expression at the protein level was confirmed by IHC. Conclusions: We identified biologic pathways and a novel gene signature in primary ER+ breast cancers that predicts for response to treatment in the CONFIRM study. These results suggest potential new therapeutic targets and warrant further validation as predictive biomarkers of fulvestrant treatment in metastatic breast cancer. Clin Cancer Res; 22(23); 5755–64. ©2016 AACR.

Introduction: Epacadostat, a potent and highly selective oral inhibitor of the indoleamine 2,3- dioxygenase 1 (IDO1) enzyme, plus durvalumab, an anti-PD-L1 antibody, is being studied in patients with advanced solid tumors (NCT02318277). Phase 1 preliminary safety data for the overall population and efficacy data for patients with advanced pancreatic cancer (PC) are reported as of 29 Oct 2017 data cutoff.Methods: Adult patients with PC, melanoma, NSCLC, or SCCHN were enrolled in the 3+3 dose-escalation phase 1. Prior treatment with immune checkpoint inhibitors (in unapproved indications) or IDO inhibitors was not allowed. Patients received epacadostat (25, 50, 75, 100, or 300 mg twice daily [BID]) plus durvalumab (3 or 10 mg/kg every 2 weeks [Q2W]). Safety was assessed in patients receiving ≥1 treatment dose. Response was measured in evaluable patients (≥1 postbaseline scan or discontinued/died before the first scan) per modified RECIST v1.1.Results: Thirty-four patients were enrolled in phase 1. Median age (range) was 68 (46-84) years; most patients were male (62%) and had an ECOG PS of 1 (82%). There was 1 dose-limiting toxicity (grade 3 rash requiring systemic steroids; epacadostat 300 mg BID plus durvalumab 10 mg/kg Q2W) during the 42-day observation period. The most common (≥10%) treatment-related adverse events (TRAEs) were fatigue (32%), pruritus (15%), diarrhea, nausea, and rash (12% each). Grade ≥3 TRAEs occurring in >1 patient included fatigue and rash (n=3 [9%] each). Five patients (15%) discontinued because of TRAEs (grade 1 pneumonitis, grade 2 diarrhea, grade 2 subarachnoid hemorrhage, grade 2 peripheral edema, and grade 3 dyspnea). There were no TRAEs leading to death.Fifteen patients with PC were enrolled across multiple dose levels. Median age (range) was 66 (46-72) years, 67% had liver metastases, and 87% had an ECOG PS of 1. Fourteen patients had received ≥1 prior therapy. PD-L1 expression test results were available in 7/15 patients: 2 had detectable (≥1%) staining of tumor cells; 5 did not. No responses were observed among patients with PC; the disease control rate was 27% (4 SD) per RECIST v1.1 (1 of 4 patients with SD discontinued treatment because of clinical progression). The median duration of disease control was 156 days (95% CI, 91-219).Epacadostat exposure was consistent with previous reports, except in patients with PC where somewhat lower peak exposures (Cmax) were observed.Conclusions: Epacadostat plus durvalumab was generally well tolerated in patients with advanced cancers; the safety profile was consistent with previous reports of durvalumab and epacadostat as monotherapies. In unselected patients with PC, no objective responses were observed; a phase 2 expansion for PC was not conducted. Epacadostat 100 and 300 mg BID are being evaluated in the ongoing phase 2 expansions, including patients with NSCLC, SCCHN, and urothelial carcinoma. Citation Format: Aung Naing, John D. Powderly, Gerald Falchook, Benjamin Creelan, John Nemunaitis, Jose Lutzky, Adi Diab, Judy S. Wang, Naomi Laing, Michelle Niewood, Xiaohua Gong, Gongfu Zhou, Manish Patel. Epacadostat plus durvalumab in patients with advanced solid tumors: preliminary results of the ongoing, open-label, phase I/II ECHO-203 study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT177.

92 Background: Angiogenesis and immunosuppression are hallmarks of tumor growth. This global phase 1 trial evaluates the combination of R (anti-VEGFR2) and D (anti-PD-L1) in pts with G/GEJ by simultaneously targeting these two processes. Methods: This ongoing, multi-cohort, phase 1a/b trial (NCT02572687) enrolled pts with confirmed G/GEJ adenocarcinoma with prior progression on 1 or 2 lines of systemic therapy, measurable disease, ECOG PS 0-1, and baseline tumor tissue. PD-L1 expression was assessed using the SP-263 IHC; MSI status was determined using PCR. Enrolled pts received R (8 mg/kg IV) and D (750 mg IV) every two weeks on a 28-day cycle. Primary objective was to assess safety and tolerability of R+D; preliminary efficacy was also examined. Results: As of 26-May-2017, 29 G/GEJ adenocarcinoma pts were treated. The median age was 55 y; 69% were male; 66% had ECOG PS of 1; 48% had PD-L1 ≥25% expression in tumor or immune cells, 3.5% were MSI-high; and 72% received study treatment as second line for advanced disease. Median duration of treatment was 2.5 mo for R and 3.0 mo for D. Treatment-emergent adverse events (TEAEs) occurred in 29 (100%) pts and 21 (72%) pts experienced grade 3/4 TEAEs, while treatment-related AEs (TRAE) occurred in 24 (83%) pts; none resulted in treatment discontinuation. Ten (35%) pts had grade 3 TRAEs, and no grade 4 or 5. All grade TRAEs occurring in ≥10% of pts were hypertension (34%), fatigue (31%), headache (24%), diarrhea (21%), pyrexia (10%) and decreased appetite (10%). Five pts (17%) reported a serious adverse event related to study treatment. Preliminary efficacy data (RECIST v1.1) showed 5 of 29 pts (17%) achieved a confirmed PR. Only 1 responding pt was MSI high. The overall response rate for pts with PD-L1 ≥25% was 36%. Median PFS was 2.6 mo (95% CI, 1.45 to 6.28). As of data cut-off, 6 pts (21%) remain on treatment. Conclusions: R+D generated no unexpected toxicity, and demonstrated antitumor activity in pts with previously treated advanced G/GEJ adenocarcinoma. Clinical trial information: NCT02572687.

Fulvestrant is a selective estrogen receptor antagonist. Based on the measured growth inhibition of 60 human cancer cell lines (NCI60) in the presence of fulvestrant, as well as the baseline gene expression of the 60 cell lines, a gene expression score that predicts response to fulvestrant was developed. The score is based on 414 genes, 103 of which show increased expression in sensitive cell lines, while 311 show increased expression in the non-responding cell lines. The sensitivity genes primarily sense signaling through estrogen receptor alpha, whereas the resistance genes modulate the PI3K signaling pathway. The latter genes suggest that resistance to fulvestrant can be overcome by drugs targeting the PI3K pathway. The level of this gene expression score and its correlation with fulvestrant response was measured in a panel of 20 breast cancer cell lines. The predicted sensitivity matched the measured sensitivity well (CC = -0.63, P = 0.003). The predictor was applied to tumor biopsies obtained from a Phase II clinical trial. The sensitivity of each patient to treatment with fulvestrant was predicted based on the RNA profile of the biopsy taken before neoadjuvant treatment and without knowledge of the subsequent response. The prediction was then compared to clinical response to show that the responders had a significantly higher sensitivity prediction than the non-responders (P = 0.01). When clinical covariates, tumor grade and estrogen receptor H-score, were included in the prediction, the difference in predicted senstivity between responders and non-responders improved (P = 0.003). Using a pre-defined cutoff to separate patients into predicted sensitive and predicted resistant yielded a positive predictive value of 88% and a negative predictive value of 100% when compared to clinical data. We conclude that pre-screening patients with the new gene expression predictor has the potential to identify those postmenopausal women with locally advanced, estrogen-receptor-positive breast cancer most likely to respond to fulvestrant.

Cisplatin-based chemotherapy is considered the gold standard for patients with advanced bladder cancer. However, despite initial response, many patients will relapse; therefore, novel salvage treatment strategies are desperately needed. Herein, we studied a mechanism based treatment combination using a Smac mimetic with standard chemotherapy. Using a panel of 10 urothelial cancer cell lines, we exposed them to a combination of gemcitabine, cisplatin, and a Smac mimetic. Sensitivity was determined using a DNA fragmentation assay. We determined that three cell lines (UMUC-3, UMUC-13, and RT4v6) were considered sensitive to the combination of gemcitabine and cisplatin and an additional three cell lines were sensitized to gemcitabine and cisplatin with the addition of the Smac mimetic (UMUC-6, UMUC-12, and UMUC-18). We next explored the constitutive expression of selected members of the IAP family (XIAP, cIAP-1, cIAP-2, and Survivin), the BCL family (BCL-2, BCLXL, and BAX) and Smac using gene expression profiling and western blotting. We determined that RNA and protein expression of SMAC, selected members of the IAP family and members of the BCL family did not correlate to drug sensitivity. Lastly, using an in vivo mouse model, we determined that treatment with the Smac mimetic in combination with gemcitabine and cisplatin resulted in increased apoptosis, decreased microvessel density and decreased cellular proliferation. This novel treatment strategy may be effective in patients with advanced urothelial carcinoma and warrants further investigation.

Background: AZD1775 (formerly MK-1775) is a selective inhibitor of WEE1 kinase, which has been shown to sensitize TP53-mutant cancer cells to genotoxic agents such as platinum-based chemotherapies. Mutation of TP53 abrogates the G1/S checkpoint in cells, which may enhance their dependency on the G2/M checkpoint and WEE1 kinase activity for control of the cell cycle and effective repair of DNA damage. The results from a randomized Phase II trial (NCT01357161) evaluating the effects of adding AZD1775 to carboplatin and paclitaxel in patients with TP53-mutant ovarian cancer showed an increase in progression-free survival (PFS) for the AZD1775 arm versus placebo (enhanced RECIST: median PFS 34.14 vs 31.86 weeks; HR = 0.63, 80% CI: 0.45-0.89, P = 0.080; RECIST 1.1: median PFS 42.86 vs 34.86 weeks; HR = 0.55, 80% CI 0.39-0.79, P = 0.030; Oza et al, ASCO 2015). We investigated whether particular genetic factors were associated with an increased response to the combination of AZD1775 and chemotherapy in this trial. Methods: A retrospective analysis was performed to determine whether any specific subtype of TP53 alteration was associated with a greater response to the AZD1775 combination compared with placebo. In addition, next-generation sequencing (NGS) was performed on archival tumors from a subset of patients who provided consent in an effort to identify additional genetic alterations that may predict increased clinical benefit following the addition of AZD1775 to chemotherapy. Results: TP53 data were available for 136 patients (15 patients from an initial open-label safety run-in and 121 patients from the randomized trial) and 133 patients were evaluable for response. Fifty-five patients provided additional consent for NGS of tumor samples. The genetic aberrations observed in the NGS subset of tumors from this trial were heterogeneous, and the total mutational load in cancer-related genes was also variable across tumors. Although the small number of patients with a tumor BRCA mutation limited the statistical power of the comparison between randomized arms, the median PFS was longer in those patients treated with AZD1775 (53.86 weeks, 95% CI 24.43-66.57) versus placebo (45.86 weeks, 95% CI 35.71-55.86) in this BRCA-mutated subgroup. TP53 subgroup analyses showed a similar benefit for patients with missense mutations compared to those with splice site, nonsense and frameshift TP53 mutations. The heterogeneity of the G1/S checkpoint gene aberrations limited the statistical power of the subgroup analysis, but specific genes that warrant further investigation were identified. Conclusions: These results highlight a number of potential candidate genes for increased response to AZD1775 and chemotherapy compared with chemotherapy alone. Citation Format: Naomi Laing, Zhongwu Lai, J. Carl Barrett, Mark J. O’Connor, Amit M. Oza, David Lawrence. Genetic analysis of tumors from a phase II trial evaluating AZD1775, carboplatin and paclitaxel in patients with TP53-mutant ovarian cancer. [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 337.

TPS2608Background: Many cancers are associated with DNA repair and cell cycle mutations that result in G1/S checkpoint deficiencies and high levels of endogenous damage and replication stress. This can lead to dependence on WEE1 kinase, which provides an important G2/M checkpoint, allowing repair of DNA damage prior to mitosis. AZD1775 is a highly selective, small-molecule WEE1 inhibitor being developed for the treatment (tx) of advanced solid tumors. It has previously shown single-agent activity in patients (pts) with BRCA-1/2 mutations (Do, JCO 2015). We described initial safety and efficacy of the dose escalation portion of an open Phase Ib study (NCT02482311) of AZD1775 in pts with refractory solid tumors, and a dose of AZD1775 175 mg PO BID was identified (submitted to AACR 2016). Here we describe the expansion portion of this study in 6 matched cohorts: BRCA-1/2-mutant and wildtype (WT) ovarian cancer (OvC); cyclin-E (CCNE1)-amplified and non-amplified triple-negative breast cancer (TNBC); and any o...

Despite the widespread use of rituximab, a chimeric monoclonal antibody with demonstrated efficacy in the treatment of non-Hodgkin's lymphomas, there is a recognized need to develop new agents with improved efficacy. Towards this end, using XenoMouse technology, a fully human IgG1 anti-CD20 monoclonal antibody was generated. This antibody, denoted mAb 1.5.3, evoked enhanced pro-apoptotic activity in vitro, as compared to rituximab, in the Ramos lymphoma cell line. Also, mAb 1.5.3 mediated both complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) similar to rituximab in human B-lymphoma lines. Interestingly, mAb 1.5.3 demonstrated superior ADCC compared to rituiximab when FcgammaRIIIa F/F allotype donors were profiled and superior cytolytic activity across multiple human B-lymphoma and chronic B-cell leukemia lines in an in vitro whole blood assay. Furthermore, mAb 1.5.3 exhibited enhanced anti-tumor activity in Ramos, Daudi, and Namalwa tumour xenograft models. Lastly, mAb 1.5.3 produced a superior B-cell depletion profile in lymph node organs and bone marrow as compared to rituximab in a primate pharmacodynamic (PD) model. These findings underscore the potential of mAb 1.5.3 to exhibit improved clinical activity in the treatment of B-cell malignancies compared to rituximab.

Introduction: Several multi-gene expression based assays have been developed to assess the prognosis and predict response to endocrine treatments in early stage hormone receptor positive (HR+) breast cancer. Although a significant number of patients with metastatic ER+ disease will not respond to endocrine treatments, molecular assays to predict response in this setting are limited. In addition, tissue specimens of metastatic lesions for molecular studies are not always available. In this study we sought to identify a molecular profile in the primary tumors of patients who developed disease recurrence that could predict response to endocrine treatment in metastatic disease. Methods: We used the primary breast tumor samples from a subgroup of patients participating in the randomized phase III CONFIRM trial, which compared 500mg versus 250mg of fulvestrant in post-menopausal women with HR+ advanced breast cancer. Formalin- fixed paraffin embedded tumors were collected from 130 of the participants and were centrally reviewed for ER, PR, HER2 and Ki67. RNA was sufficient for gene expression profiling in 112 of the cases using the NuGEN Ovation FFPE WTA System and Affymetrix HTA 2.0 GeneChip. The majority of the patients in this analysis developed metastatic disease during adjuvant endocrine treatment (N=55) or had de-novo metastatic disease (N=39) versus relapse after adjuvant treatment (N=18). The association between gene expression and progression free survival (PFS) was investigated using a multivariate Cox proportional hazard model adjusting for statistically significant clinicopatholgical factors. In addition we performed pathway-level analysis and evaluated the PAM50 subtype predictor and Risk of Relapse (ROR) score. Results: The median PFS was 8 months in our cohort. HER2 level by immunohistochemistry above 1+, high PR level, defined as Allred score of above 6, and Ki67 of above 50% were significantly associated with PFS and were included in the multivariate model. Dose of fulvestrant was not associated with PFS in this cohort. We identified a signature of 25 genes that is inversely associated with PFS on fulvestrant treatment (FDR 20%). When compared to other published datasets of breast cancer tumors, these genes are enriched in tumors with poor outcome and triple negative cancers. Pathway analysis revealed an association between activation of the EGFR pathway and decreased PFS (P=0.01). PAM50 subtypes varied with the luminal subtype being the most common (65%) and were generally concordant with the clinical subtype. However, we did not detect a significant trend between PAM50 subtype or ROR score and PFS or overall survival. Conclusions: In this cohort of patients with early and de-novo metastatic disease we identified a gene signature in the primary tumors that is associated with decreased response to fulvestrant treatment in metastatic disease. This signature warrants further validation to determine it’s predictive value and potential to assist in treatment decision making for patients with HR+ metastatic disease. Citation Format: Rinath M Jeselsohn, William T Barry, Jin Zhao, Gilles Buchwalter, Cristina Guarducci, Ilenia Migliaccio, Chiara Biagioni, Martina Bonechi, Naomi Laing, Yuri Rukazenkov, Eric P Winer, Myles Brown, Angelo Di Leo, Luca Malorni. TransCONFIRM: The correlative analysis of breast tumors from patients with advanced hormone receptor positive disease identifies a genetic signature associated with decreased benefit from single agent fulvestrant [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S1-01.

261 Background: Cisplatin-based chemotherapy is considered the gold standard for patients with advanced bladder cancer. Despite initial response, many patients will relapse; therefore, novel salvage treatment strategies are desperately needed. Herein, we studied a mechanism based treatment combination using a Smac mimetic with standard chemotherapy. Methods: A panel of 10 urothelial cancer cell lines was exposed to gemcitabine and cisplatin, a Smac mimetic, and the combination. Sensitivity was determined using a DNA fragmentation assay. Constitutive expression of select members of the IAP family (XIAP, cIAP-1, cIAP-2, Survivin), the BCL family (BCL-2, BCLXL, BAX) and Smac were evaluated by gene expression profiling and Western blotting. Changes in protein expression under treatment conditions were examined using Western blotting. TNF-a blocking antibody was used to explore the contribution of autocrine TNF-a in cellular death. Lastly, a mouse model of bladder cancer was used to determine the effectiveness of the drug combination. Results: UMUC-3, UMUC-13 and RT4v6 were considered sensitive to the combination of gemcitabine and cisplatin based on an apoptosis assay. Three additional cell lines were sensitized to gemcitabine and cisplatin with the addition of the Smac mimetic (UMUC-6, UMUC-12, UMUC-18). The constitutive RNA and protein expression of SMAC, select members of the IAP family, and members of the BCL family did not correlate to drug sensitivity. Autocrine TNF-a demonstrated a partial contribution to cell death in UMUC-12. In an in vivo mouse model, the Smac mimetic alone and in combination with gemcitabine and cisplatin resulted in decreased tumor volume and increased apoptosis compared to the chemotherapy treatment alone. The combination resulted in decreased cellular proliferation and decreased microvessel density. Conclusions: Smac mimetic is able to augment the activity of gemcitabine and cisplatin based chemotherapy in vitro and in vivo. This may be an effective treatment strategy in patients with urothelial carcinoma and warrants further investigation.

In spite of the widespread use of Rituximab, a chimeric monoclonal antibody with demonstrated efficacy in the treatment of non-Hodgkin’s lymphomas, there is a recognized need to develop fully human antibodies with improved efficacy. Towards this end, using XenoMouse™ technology, a fully human IgG1 monoclonal antibody specific to human CD20 was generated. This antibody, denoted mAb 1.5.3, evoked enhanced pro-apoptotic activity in vitro, as compared to Rituximab, in the Ramos human lymphoma cell line. In addition, mAb 1.5.3 was active in mediating complement dependent cytotoxicity (CDC) and elicited improved antibody-dependent cellular cytotoxicity (ADCC) relative to Rituximab in Ramos, Raji, and Daudi human B-lymphoma lines. To recapitulate various aspects of acquired resistance to Rituximab, as observed in a subpopulation of patients, Rituximab-resistant clones were established from lymphoma lines. Interestingly, mAb 1.5.3 demonstrated superior cytolytic activity against engineered Rituximab-refractory lymphoma clones, as well as across multiple human B-lymphoma and chronic B-cell leukemia lines in an in vitro whole blood assay. Furthermore, mAb 1.5.3 exhibited enhanced anti-tumor activity in Rituximab-sensitive cell lines and -refractory engineered lymphoma clones in vivo. Lastly, mAb 1.5.3 produced a superior B-cell depletion profile in lymph node organs and bone marrow as compared to Rituximab in a primate PD model. In contrast to Rituximab, mAb 1.5.3 is a fully human antibody and is thus anticipated to exhibit a longer serum half-life with minimal immunogenicity following repeated administration. In sum, these results demonstrate the superior anti-tumor activity of mAb 1.5.3 relative to Rituximab and its potential for improved clinical activity in the treatment of B-cell malignancies.