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T cells engineered to express chimeric antigen receptors (CARs) targeting CD19 have demonstrated impressive activity against relapsed or refractory B cell cancers yet fail to induce durable remissions for nearly half of patients treated. Enhancing the efficacy of this therapy requires detailed understanding of the molecular circuitry that restrains CAR-driven anti-tumor T cell function. We developed and validated an in vitro model that drives T cell dysfunction through chronic CAR activation and interrogated how CAR costimulatory domains, central components of CAR structure and function, contribute to T cell failure. We found that chronic activation of CD28-based CARs results in activation of classical T cell exhaustion programs and development of dysfunctional cells that bear the hallmarks of exhaustion. In contrast, 41BB-based CARs activate a divergent molecular program and direct differentiation of T cells into a novel cell state. Interrogation of CAR T cells from a patient with progressive lymphoma confirmed activation of this novel program in a failing clinical product. Further, we demonstrate that 41BB-dependent activation of the transcription factor FOXO3 is directly responsible for impairing CAR T cell function. These findings identify that costimulatory domains are critical regulators of CAR-driven T cell failure and that targeted interventions are required to overcome costimulation-dependent dysfunctional programs.

While chimeric antigen receptor (CAR) T cells targeting CD19 can cure a subset of patients with B cell malignancies, most patients treated will not achieve durable remission. Identification of the mechanisms leading to failure is essential to broadening the efficacy of this promising platform. Several studies have demonstrated that disruption of CD19 genes and transcripts can lead to disease relapse after initial response; however, few other tumor-intrinsic drivers of CAR T cell failure have been reported. Here we identify expression of the Golgi-resident intramembrane protease Signal peptide peptidase-like 3 (SPPL3) in malignant B cells as a potent regulator of resistance to CAR therapy. Loss of SPPL3 results in hyperglycosylation of CD19, an alteration that directly inhibits CAR T cell effector function and suppresses anti-tumor cytotoxicity. Alternatively, over-expression of SPPL3 drives loss of CD19 protein, also enabling resistance. In this pre-clinical model these findings identify post-translational modification of CD19 as a mechanism of antigen escape from CAR T cell therapy.

Chimeric antigen receptor-engineered T cells targeting CD19 (CART19) have revolutionized the management of relapsed and refractory B cell malignancies. Despite high initial response rates, many patients with acute lymphoblastic leukemia (ALL) ultimately relapse after CART19. In contrast, most patients with non-Hodgkin lymphoma experience only partial or no responses. Collectively, To identify pathways responsible for enabling tumor-intrinsic resistance to CART19 we performed a genome-wide loss-of-function screen in the Nalm6 ALL cell line. The second-most enriched gene in this screen was SPPL3 (Figure 1a), encoding a Golgi-resident aspartyl protease. Previous studies have determined that SPPL3 functions to broadly limit protein glycosylation by cleaving glycosyltransferases from the Golgi membrane, impairing their ability to add complex glycans to proteins as they pass through the Golgi (Voss M. et al. EMBO, 2014). Using targeted genomic disruption, we confirmed that loss of SPPL3 results in resistance to CART19 in human ALL and non-Hodgkin lymphoma models (Figures 1b-c). CART19 cells exposed to SPPL3KO ALL demonstrated significantly lower expression of CD69, PD1, Tim3 and CD107a, as well as less activation of the central T cell transcription factors NFAT and NFκB, indicating a global suppression of T cell stimulation. Consistent with its known function, loss of SPPL3 resulted in increased addition of complex glycans to CD19. Surface staining of SPPL3KO cells revealed that CD19 antibodies were less capable of binding this hyperglycosylated CD19. This included decreased binding of the antibody used to construct the anti-CD19 CAR (clone FMC63). Protein modeling revealed that an asparagine residue known to be normally glycosylated on CD19 (N125) is in close physical proximity to the FMC63 binding site (Figure 1d), suggesting that the addition of complex glycans at this site may be responsible for disruption of CAR binding that led to impaired T cell activation. We next turned our attention to CD22, another B cell antigen that is normally glycosylated and the target of CAR therapy. In contrast to CD19, loss of SPPL3 had no impact on CD22 glycosylation or antibody binding. Similarly, loss of SPPL3 did not enable resistance to CD22-targeted CAR T cells. These findings substantiated our hypothesis loss of SPPL3 lead to CART19 failure directly via modifying CD19 glycosylation, and not through another CD19-independent mechanism. To further validate the impact of CD19 glycosylation in regulating CART19 efficacy, we over-expressed SPPL3 in ALL cells, previously shown to promote global hypoglycosylation. We confirmed decreased glycosylation of CD19 (Figure 1e), and found that this resulted in loss of FMC63 binding to CD19 and complete resistance to CART19 activity (Figure 1f). In summary, our findings identify that changes to CD19 glycosylation, either enhanced or decreased, impair the ability of CARs to bind and initiate T cell effector function against malignant B cells. Further, these data identify post-translational protein modification as a novel mechanism of antigen escape from CAR-based T cell immunotherapy. Figure 1 Figure 1. Disclosures Ruella: AbClon: Consultancy, Research Funding; viTToria biotherapeutics: Research Funding; BMS, BAYER, GSK: Consultancy; Novartis: Patents & Royalties; Tmunity: Patents & Royalties. Gill: Interius Biotherapeutics: Current holder of stock options in a privately-held company, Research Funding; Novartis: Other: licensed intellectual property, Research Funding; Carisma Therapeutics: Current holder of stock options in a privately-held company, Research Funding.

The chemokine receptors CXCR1/2 and their ligand CXCL8 are essential for the activation and trafficking of inflammatory mediators as well as tumor progression and metastasis. The CXCL8-CXCR1/2 signaling axis is involved in the pathogenesis of several diseases including chronic obstructive pulmonary diseases (COPD), asthma, cystic fibrosis and cancer. Interaction between CXCL8 secreted by select cancer cells and CXCR1/2 in the tumor microenvironment is critical for cancer progression and metastasis. The CXCL8-CXCR1/2 axis may play an important role in tumor progression and metastasis by regulating cancer stem cell (CSC) proliferation and self-renewal. During the past two decades, several small-molecule CXCR1/2 inhibitors, CXCL8 releasing inhibitors, and neutralizing antibodies against CXCL8 and CXCR1/2 have been reported. As single agents, such inhibitors are expected to be efficacious in various inflammatory diseases. Several preclinical studies suggest that combination of CXCR1/2 inhibitors along with other targeted therapies, chemotherapies, and immunotherapy may be effective in treating select cancers. Currently, several of these inhibitors are in advanced clinical trials for COPD, asthma, and metastatic breast cancer. In this review, we provide a comprehensive analysis of the role of the CXCL8-CXCR1/2 axis and select genes co-expressed in this pathway in disease progression. We also discuss the latest progress in developing small-molecule drugs targeting this pathway.

The chemokine receptor CXCR2 is expressed on various immune cells and is essential for neutrophil recruitment and angiogenesis at sites of acute and chronic inflammation caused by tissue injury or infection. CXCR2 and its ligand, CXCL8, are implicated in a number of inflammation-mediated diseases such as chronic obstructive pulmonary disease, cystic fibrosis, and cancer. Though the development of CXCR2-specific small-molecule inhibitors as anti-inflammatory agents has been pursued by pharmaceutical companies within the past decade, there are currently no clinically approved CXCR2 inhibitors. A pharmacophore model based on previously reported CXCR2 antagonists was developed to screen a database of commercially available compounds. Small-molecule compounds identified from the pharmacophore screening were selected for in vitro screening in a cell-based CXCR2-mediated β-arrestin-2 recruitment assay and further characterized in several cell-based assays and lipopolysaccharide (LPS)-induced lung inflammation studies in mice. CX compounds identified from pharmacophore modeling inhibited cell migration, lung and colon cancer cell proliferation, and colony formation. Mechanistic studies of CX4152 showed that this compound inhibits CXCR2 signaling through downregulation of surface CXCR2. Additionally, CX4152 significantly inhibits CXCL8-mediated neutrophil migration and LPS-induced lung inflammation in mice. Using a CXCR2-inhibitor-based pharmacophore model, we identified a novel set of sulfonamides from a diverse library of small molecules. These compounds inhibit CXCR2/β-arrestin-2 association, cell migration and proliferation, and acute inflammation in mouse models. CX compounds identified from our pharmacophore models are potential leads for further optimization and development as anti-inflammatory and anticancer agents.

Background and Purpose Since the CXC chemokine receptor CXCR2 and its cognate ligand CXCL8 (IL-8) critically regulate neutrophil trafficking during inflammation, they have been implicated in a number of inflammatory lung diseases. Several CXCR2 antagonists have been described and the blockade of CXCR2 has shown promise in pre-clinical disease models and early clinical trials. However, given its potential, there are fewer distinct classes of antagonists of CXCR2 than of other clinically relevant molecular targets. Thus, we sought to identify additional classes of compounds that alter CXCR2 function. Experimental Approach We used the CXCR2 TangoTM assay to screen an in-house library of highly diverse chemical compounds. CX4338 [2-(benzylamino)-4,4-dimethyl-6-oxo-N-phenylcyclohex-1-enecarbothioamide] was identified from our screen and additional studies to characterize the compound were performed. Receptor internalization and second-messenger assays were used to assess the effects of CX4338 on CXCR2-mediated signalling. Wound healing, transwell cell migration and LPS-induced lung inflammation in mice were used to determine the in vitro and in vivo effects of CX4338. Key Results CX4338 selectively inhibited CXCR2-mediated recruitment of β-arrestin-2 and receptor internalization, while enhancing CXCR2-mediated MAPK activation. Additionally, CX4338 inhibited CXCL8-induced chemotaxis in CXCR2-overexpressing cells and human neutrophils. In vivo, CX4338 significantly reduced neutrophils in bronchoalveolar lavage induced by LPS in mice. Conclusions and Implications A novel compound CX4338 inhibited CXCR2-mediated cell migration with a mechanism of action not previously reported. Also, selective inhibition of CXCR2-mediated β-arrestin-2 activation is sufficient to inhibit CXCL8-mediated chemotaxis.

Despite the recent focus on unconventional resources such as shale gas and tight oil in North America, large unrecovered volumes of oil remain in conventional reservoirs making them viable candidates for chemical enhanced oil recovery processes. Replacing or following traditional water flooding with aqueous chemicals that use both surfactant & alkali to reduce interfacial tension and polymer to improve sweep efficiency has been successful in recovering incremental oil from these reservoirs. However, designing the chemical injection scheme is complex, and must be tailored to specific reservoir rock and fluid properties. A strategic design methodology can help provide an optimal, well-performing chemical formulation, even for challenging reservoirs. Currently, there are around ten Canadian chemical injection projects in operation with the latest being the Little Bow Upper Mannville "I" Pool. An ASP EOR project was initiated in this reservoir in March 2014 in a multiphase development plan. When the project was initiated, Little Bow oil (Phase 1&2) production was around 350 bbl/day. Successful implementation of this project is expected to result in incremental recovery of 5.2 million barrels of oil (12% of the OOIP) over the base waterflood. As of January 2016, 6.648 million barrels of ASP solution has been injected in the reservoir and the field is now showing first signs of incremental production. This paper presents a workflow that integrates laboratory results, geological and geophysical data and production history into an effective forecasting model. Complex geology and a long production history of the partly depleted Little Bow reservoir have presented a challenge for history matching the primary and water-flood production. Through an iterative process, a full field model was built, history matched, then used as a base case to determine an optimal operational design for the full field. A multidisciplinary team including geologists, exploitation and reservoir engineers collaborated to develop a 3D geological model and achieve the history match using an iterative approach; resulting in an idealized workflow and a superior history matched model. Using this model and an advanced optimization algorithm, a full field ASP operation design was optimized (based on NPV) for slug sizes, chemical concentrations, pattern design for injection/production wells locations, and drilling & workover locations. The optimized ASP injection scheme is implemented and some field results from January till June 2016 are presented in the paper.

Overcoming drug resistance with remarkable cytotoxic activity by anthracene-9,10-dione derivatives would offer a potential therapeutic strategy. In this study, we report the synthesis and the cytotoxicity of a novel set of anthraquninones. (4-(4-Aminobenzylamino)-9,10-dioxo-9,10-dihydroanthracen-1-yl-4-methylbenzenesulfonate) (3) has excellent in vitro cytotoxicity against doxorubicin-resistant cancer cell line (IC50=0.8 μM), 20-fold higher than doxorubicin. The cytotoxic effect via G2/M arrest does not appear to be ROS.

Chemokine receptor CXCR2 is expressed on various immune cells and is essential for neutrophil recruitment and angiogenesis at sites of acute and chronic inflammation caused by tissue injury or infection. Because of its role in inflammation, it has been implicated in a number of immune-mediated inflammatory diseases such as psoriasis, arthritis, COPD, cystic fibrosis, asthma, and various types of cancer. CXCR2 and its ligands are up-regulated in cancer cells as well as the tumor microenvironment, promoting tumor growth, angiogenesis, and invasiveness. Although pharmaceutical companies have pursued the development of CXCR2-specific small-molecule inhibitors as anti-inflammatory agents within the last decades, there are currently no clinically approved CXCR2 inhibitors. Using a high-throughput, cell-based assay specific for CXCR2, we screened an in-house library of structurally diverse compounds and identified a class of pyrimidine-based compounds that alter CXCR2-mediated second messenger signaling. Our lead compound, CX797, inhibited IL8-mediated cAMP signaling and receptor degradation while specifically up-regulating IL8-mediated β-arrestin-2 recruitment. CX797 also inhibited IL8-mediated cell migration. Mechanistic comparison of CX797 and a previously reported CXCR2 inhibitor, SB265610, show these two classes of compounds have a distinct mechanism of action on CXCR2.

Modern assisted history matching tools allow an engineer to specify the uncertain parameters in a simulation dataset then perform an optimization to minimize the difference between observed field data and the corresponding simulation outputs. During this optimization, multiple simulations are run, with the uncertain parameters varying within the ranges specified by the engineer. The difference between the observed field data and the simulation output is measured by an objective function. Standard objective functions have been reported in the literature for the difference between observed and simulated production and injection rates, and for measurements in time and space done at observation wells. In this work we incorporate an additional objective function term that measures the difference between the observed and simulated steam chamber location and shape. In addition, the differences in 3D volumes were visualized, which lead to a better physical understanding of what parameters should be adjusted during a history match.For a steam injection process like Steam Assisted Gravity Drainage (SAGD), 4D seismic may be used to determine where a steam chamber is located in a reservoir at a point in time. The objective function developed in this work measures the difference between an observed chamber's shape and location in the reservoir and the corresponding shape and location for the chamber indicated in the simulation output. The objective function is a binary mismatch function, checking each simulation grid block to see if the seismic chamber and the simulation chamber agree or disagree, and calculating the ratio of the total volume of disagreements over the total volume of agreements.This steam chamber mismatch function was included in an assisted history match performed on a well pair from Suncor Energy's SAGD project at Firebag. The inclusion of this additional information added additional constraints to the simulation model, leading to a conceptually more dependable history match and a better geological and dynamic characterization of the reservoir.

Since the CXC chemokine receptor CXCR2 and its cognate ligand CXCL8 (IL-8) critically regulate neutrophil trafficking during inflammation, they have been implicated in a number of inflammatory lung diseases. Several CXCR2 antagonists have been described and the blockade of CXCR2 has shown promise in pre-clinical disease models and early clinical trials. However, given its potential, there are fewer distinct classes of antagonists of CXCR2 than of other clinically relevant molecular targets. Thus, we sought to identify additional classes of compounds that alter CXCR2 function.We used the CXCR2 Tango(TM) assay to screen an in-house library of highly diverse chemical compounds. CX4338 [2-(benzylamino)-4,4-dimethyl-6-oxo-N-phenylcyclohex-1-enecarbothioamide] was identified from our screen and additional studies to characterize the compound were performed. Receptor internalization and second-messenger assays were used to assess the effects of CX4338 on CXCR2-mediated signalling. Wound healing, transwell cell migration and LPS-induced lung inflammation in mice were used to determine the in vitro and in vivo effects of CX4338.CX4338 selectively inhibited CXCR2-mediated recruitment of β-arrestin-2 and receptor internalization, while enhancing CXCR2-mediated MAPK activation. Additionally, CX4338 inhibited CXCL8-induced chemotaxis in CXCR2-overexpressing cells and human neutrophils. In vivo, CX4338 significantly reduced neutrophils in bronchoalveolar lavage induced by LPS in mice.A novel compound CX4338 inhibited CXCR2-mediated cell migration with a mechanism of action not previously reported. Also, selective inhibition of CXCR2-mediated β-arrestin-2 activation is sufficient to inhibit CXCL8-mediated chemotaxis.

Herein, we report for the first time the design and synthesis of a novel cyclotide able to efficiently inhibit HIV-1 viral replication by selectively targeting cytokine receptor CXCR4. This was accomplished by grafting a series of topologically modified CVX15 based peptides onto the loop 6 of cyclotide MCoTI-I. The most active compound produced in this study was a potent CXCR4 antagonist (EC50 ≈ 20 nM) and an efficient HIV-1 cell-entry blocker (EC50 ≈ 2 nM). This cyclotide also showed high stability in human serum thereby providing a promising lead compound for the design of a novel type of peptide-based anti-cancer and anti-HIV-1 therapeutics.

Background: Chemokine receptor, CXCR2 is expressed on various immune cells and is essential for neutrophil recruitment and angiogenesis at sites of acute and chronic inflammation caused by tissue injury or infection. Due to its role in inflammation, it has been implicated in a number of inflammation-mediated diseases such as psoriasis, arthritis, COPD, cystic fibrosis, and asthma. More recently, it is also implicated in various types of cancer. CXCR2 and its ligands are up-regulated in cancer cells as well as the tumor microenvironment and have been shown to promote tumor growth, angiogenesis and invasiveness. Though pharmaceutical companies have pursued the development of CXCR2-specific small-molecule inhibitors as anti-inflammatory agents within the two past decades, there are currently no clinically approved CXCR2 inhibitors. Methodology/Principal Findings: Using a high-throughput, cell-based, CXCR2 assay, we screened an in-house library of structurally diverse compounds and identified a novel class of CXCR2 biased ligand. Our lead compound, CX797, inhibited IL8-mediated cyclic AMP signaling and receptor degradation, while specifically up-regulating IL8-mediated β-arrestin recruitment and MAP kinase signaling. CX797 also inhibited Jurkat cell proliferation via G0/G1 cell cycle arrest. Conclusions/Significance: In our studies we have identified a novel class of CXCR2 ligand, CX797, and the first to report biased CXCR2 ligand signaling. Mechanistic studies of CX797 and previously reported CXCR2 inhibitor, SB265610, show these two classes have distinct mechanism of action on CXCR2. CX797 is a potential candidate for further optimization and development for various inflammatory diseases and CXCR2-expressing cancer. Citation Format: Helen Ha, Nouri Neamati. Discovery and mechanistic studies of novel class of CXCR2 inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4558. doi:10.1158/1538-7445.AM2013-4558

Background Only a limited number of studies have performed comprehensive investigations of coding variation in relation to breast cancer risk. Given the established role of estrogens in breast cancer, we hypothesized that coding variation in steroid receptor coactivator and corepressor genes may alter inter-individual response to estrogen and serve as markers of breast cancer risk. Methods We sequenced the coding exons of 17 genes (EP300, CCND1, NME1, NCOA1, NCOA2, NCOA3, SMARCA4, SMARCA2, CARM1, FOXA1, MPG, NCOR1, NCOR2, CALCOCO1, PRMT1, PPARBP and CREBBP) suggested to influence transcriptional activation by steroid hormone receptors in a multiethnic panel of women with advanced breast cancer (n = 95): African Americans, Latinos, Japanese, Native Hawaiians and European Americans. Association testing of validated coding variants was conducted in a breast cancer case-control study (1,612 invasive cases and 1,961 controls) nested in the Multiethnic Cohort. We used logistic regression to estimate odds ratios for allelic effects in ethnic-pooled analyses as well as in subgroups defined by disease stage and steroid hormone receptor status. We also investigated effect modification by established breast cancer risk factors that are associated with steroid hormone exposure. Results We identified 45 coding variants with frequencies ≥ 1% in any one ethnic group (43 non-synonymous variants). We observed nominally significant positive associations with two coding variants in ethnic-pooled analyses (NCOR2: His52Arg, OR = 1.79; 95% CI, 1.05–3.05; CALCOCO1: Arg12His, OR = 2.29; 95% CI, 1.00–5.26). A small number of variants were associated with risk in disease subgroup analyses and we observed no strong evidence of effect modification by breast cancer risk factors. Based on the large number of statistical tests conducted in this study, the nominally significant associations that we observed may be due to chance, and will need to be confirmed in other studies. Conclusion Our findings suggest that common coding variation in these candidate genes do not make a substantial contribution to breast cancer risk in the general population. Cataloging and testing of coding variants in coactivator and corepressor genes should continue and may serve as a valuable resource for investigations of other hormone-related phenotypes, such as inter-individual response to hormonal therapies used for cancer treatment and prevention.