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Supplemental Figure 1: Activation of iCO molecules in T cells enhances NF-κB signaling;Supplemental Figure 2: Activation of iCO molecules enhances cytokine production in T cells;Supplemental Figure 3: CID dose-dependent IL2 production of HER2ζ.iCO T cells can be controlled by CID;Supplemental Figure 4: iCO activation enhances pro-inflammatory cytokine secretion of HER2ζ.iCO T cells;Supplemental Figure 5: Signal 1 is critical for HER2ζ.iCO T-cell effector function;Supplemental Figure 6: HER2ζ.iCO and HER2.CD28ζ T-cell lines have similar T-cell subset composition;Supplemental Figure 7: HER2ζ.iCO T cells have reduced PD-1 surface expression;Supplemental Figure 8: Recurrent tumors from HER2ζ.iCO T-cell + CID and HER2.CD28 ζ T-cell treated mice express HER2;Supplemental Figure 9: HER2ζ.iCO and HER2.CD28ζ T-cell lines have similar T-cell subset composition in vivo;Supplemental Figure 10: Anatomic location of tumors in treated mice;Supplemental Figure 11: Chemokine secretion of A549 cells, and chemokine receptor expression of T cells;Supplemental Figure 12: PD-L1 expression in tumor cell lines;Supplemental Figure 13: Generation of T-cells expressing HER2.CD28ζ.iCO;Supplemental Figure 14: iCO activation enhances expansion and pro-inflammatory cytokine secretion of HER2ζ.iCO and HER2.CD28ζ.iCO T-cells;Supplemental Figure 15: iCO activation enhances cytolytic activity of HER2ζ.iCO and HER2.CD28ζ.iCO T-cells; Supplemental Figure 16: Second CID injection eliminates recurrent tumors in 2/3 mice

Supplementary Figure 1 Legend from Suppression of Melanotroph Carcinogenesis Leads to Accelerated Progression of Pituitary Anterior Lobe Tumors and Medullary Thyroid Carcinomas in Rb+/− Mice

Supplementary Figure 1 from Pleiotropic Biological Activities of Alternatively Spliced TMPRSS2/ERG Fusion Gene Transcripts

In vivo imaging advances have greatly expanded the use of animal cancer models. Herein, we describe two new models that permit prostate imaging ex vivo, in vivo, and in utero. Further, we show the use of these models for detecting small metastasis and testing reagents that modulate the androgen receptor (AR) axis. A luciferase reporter gene was directed to the prostate epithelium using three composite promoters called human kallikrein 2 (hK2)-E3/P, PSA-E2/P, and ARR2PB, derived from hK2, PSA, and rat probasin regulatory elements, to generate the EZC1, EZC2, and EZC3-prostate mice, respectively. EZC2 and EZC3-prostate display robust expression in the prostate with only minimal detectable expression in other organs, including testes and epididymis. Luciferase expression was detected as early as embryonic day 13 (E13) in the urogenital track. To image prostate cancer progression, lines of EZC mice were bred with prostate cancer models TRAMP and JOCK1, and imaged longitudinally. When crossed with prostate cancer models, EZC3 facilitated detection of metastatic lesions although total prostate luciferase expression was static or reduced due to weakening of AR-regulated promoters. Castration reduced luciferase expression by 90% and 97% in EZC2 and EZC3 mice, respectively, and use of GnRH antagonist also led to extensive inhibition of reporter activity. The EZC-prostate model permits prostate imaging in vivo and should be useful for imaging prostate development, growth, metastasis, and response to treatment noninvasively and longitudinally. These models also provide powerful new reagents for developing improved drugs that inhibit the AR axis. (Cancer Res 2006; 66(12): 6199-209)

PDF file - 1959K, Figure S1. Schematic diagram representing the function of the transgenic systems. Figure S2. Analysis of inducible LRP5 transgene expression. Figure S3. Pathological Matrix. Figure S4. Murine Reactive Stroma Hallmarks. Figure S5. CID-Dependent Seminal Vesicle Occlusion. Figure S6. Immunohistochemical Characterization of FGFR1 Epithelia - WNT Stroma Lesions. Figure S7. LCM qPCR Validation. Figure S8. Distinctive Wnt signature in human reactive stroma grade 3.

Supplementary Methods from Enhanced Activation of Human Dendritic Cells by Inducible CD40 and Toll-like Receptor-4 Ligation

Supplementary Figures 1-8 from Enhanced Activation of Human Dendritic Cells by Inducible CD40 and Toll-like Receptor-4 Ligation

The reactive stroma surrounding tumor lesions performs critical roles ranging from supporting tumor cell proliferation to inducing tumorigenesis and metastasis. Therefore, it is critical to understand the cellular components and signaling control mechanisms that underlie the etiology of reactive stroma. Previous studies have individually implicated fibroblast growth factor receptor 1 (FGFR1) and canonical WNT/β-catenin signaling in prostate cancer progression and the initiation and maintenance of a reactive stroma; however, both pathways are frequently found to be coactivated in cancer tissue. Using autochthonous transgenic mouse models for inducible FGFR1 (JOCK1) and prostate-specific and ubiquitously expressed inducible β-catenin (Pro-Cat and Ubi-Cat, respectively) and bigenic crosses between these lines (Pro-Cat × JOCK1 and Ubi-Cat × JOCK1), we describe WNT-induced synergistic acceleration of FGFR1-driven adenocarcinoma, associated with a pronounced fibroblastic reactive stroma activation surrounding prostatic intraepithelial neoplasia (mPIN) lesions found both in in situ and reconstitution assays. Both mouse and human reactive stroma exhibited increased transforming growth factor-β (TGF-β) signaling adjacent to pathologic lesions likely contributing to invasion. Furthermore, elevated stromal TGF-β signaling was associated with higher Gleason scores in archived human biopsies, mirroring murine patterns. Our findings establish the importance of the FGFR1–WNT–TGF-β signaling axes as driving forces behind reactive stroma in aggressive prostate adenocarcinomas, deepening their relevance as therapeutic targets. Cancer Res; 74(2); 609–20. ©2013 AACR.

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Supplementary Figures 1-4 from Versatile Prostate Cancer Treatment with Inducible Caspase and Interleukin-12

To establish optimized conditions for immunity against prostate cancer, we compared the efficacy of multiple approaches in autochthonous and s.c. transgenic adenocarcinoma of the mouse prostate (TRAMP)-based models. Mice immunized with interleukin (IL)-12–containing apoptotic, but not necrotic TRAMP-C2 cell–based, vaccines were resistant to TRAMP-C2 tumor challenge and re-challenge, independently of the route of vaccination (s.c. or i.p.). Administration of γ-irradiated TRAMP-C2 cells preinfected with adenovirus containing both B7-1 and IL-12 genes, unlike adenovirus containing B7-1 alone, considerably protected C57BL/6 mice from TRAMP-C2 tumor growth and extended the life span of TRAMP mice. Vaccines that included dendritic cells, instead of IL-12, were equally efficient. Whereas injections of ligand-inducible caspase-1– and IL-12–containing adenoviruses cured small s.c. TRAMP-C2 tumors, nanopump-regulated delivery of viruses led to elimination of much larger tumors. The antitumor immune responses involved CD4+-, CD8+-, and natural killer cells and were strengthened by increasing the number of vaccinations. Intraprostatic administration of inducible caspase-1– and IL-12–containing adenoviruses resulted in local cell death and improved survival of adenocarcinoma-bearing TRAMP mice. Thus, tumor cell apoptosis induced by caspase in situ and accompanied by IL-12 is efficient against prostate cancer in a preclinical model.

Supplementary Figures 1-3 from EZC-Prostate Models Offer High Sensitivity and Specificity for Noninvasive Imaging of Prostate Cancer Progression and Androgen Receptor Action

Mice with a single copy of the retinoblastoma gene (Rb+/−) develop a syndrome of multiple neuroendocrine neoplasia. They usually succumb to fast-growing, Rb-deficient melanotroph tumors of the pituitary intermediate lobe, which are extremely rare in humans. Thus, full assessment of Rb role in other, more relevant to human pathology, neoplasms is complicated. To prevent melanotroph neoplasia while preserving spontaneous carcinogenesis in other types of cells, we have prepared transgenic mice in which 770-bp fragment of pro-opiomelanocortin promoter directs expression of the human RB gene to melanotrophs (TgPOMC-RB). In three independent lines, transgenic mice crossed to Rb+/− background are devoid of melanotroph tumors but develop the usual spectrum of other neoplasms. Interestingly, abrogation of melanotroph carcinogenesis results in accelerated progression of pituitary anterior lobe tumors and medullary thyroid carcinomas. A combination of immunologic tests, cell culture studies, and tumorigenicity assays indicates that α-melanocyte–stimulating hormone, which is overproduced by melanotroph tumors, attenuates neoplastic progression by decreasing cell proliferation and inducing apoptosis. Taken together, we show that cell lineage–specific complementation of Rb function can be successfully used for refining available models of stochastic carcinogenesis and identify α-melanocyte–stimulating hormone as a potential attenuating factor during progression of neuroendocrine neoplasms.

Supplementary Table 1 from Pleiotropic Biological Activities of Alternatively Spliced TMPRSS2/ERG Fusion Gene Transcripts

Supplementary Figure 1 from Suppression of Melanotroph Carcinogenesis Leads to Accelerated Progression of Pituitary Anterior Lobe Tumors and Medullary Thyroid Carcinomas in Rb+/− Mice

Despite the potency of dendritic cells (DC) as antigen-presenting cells for priming adaptive immunity, DC-based cancer vaccines have been largely insufficient to effectively reduce tumor burden or prevent tumor progression in most patients. To enhance DC-based vaccines, we used the combination of a synthetic ligand-inducible CD40 receptor (iCD40) along with Toll-like receptor-4 (TLR-4) ligation in human monocyte-derived DCs. The iCD40 receptor permits targeted, reversible activation of CD40 in vivo, potentially bypassing the essential role of CD4+ T cells for activation of DCs. As a rigorous preclinical study of this approach, we evaluated key parameters of DC activation and function. Whereas neither iCD40 nor TLR-4 signaling alone led to high levels of interleukin (IL)-12p70 and IL-6, using iCD40 in combination with lipopolysaccharide (LPS) or monophosphoryl lipid A led to strongly synergistic production of both. Furthermore, this approach led to high expression of DC maturation markers, epitope-specific CTL and T helper 1 responses, as well as DC migration in vitro and in vivo. Moreover, use of iCD40-modified and LPS-stimulated DCs led to targeted expansion of autologous T cells against tumor-associated antigens, including prostate-specific membrane antigen, and elimination of preestablished tumors, supporting this technology as a potent strategy for DC-based cancer immunotherapy. [Cancer Res 2007;67(21):10528–10]

Allogeneic, off-the-shelf (OTS) chimeric antigen receptor (CAR) cell therapies have the potential to reduce manufacturing costs and variability while providing broader accessibility to cancer patients and those with other diseases. However, host-versus-graft reactivity can limit the durability and efficacy of OTS cell therapies requiring new strategies to evade adaptive and innate-immune responses. Human herpes virus-8 (HHV8) maintains infection, in part, by evading host T and natural killer (NK) cell attack. The viral K3 gene encodes a membrane-tethered E3 ubiquitin ligase that discretely targets major histocompatibility complex (MHC) class I components, whereas K5 encodes a similar E3 ligase with broader specificity, including MHC-II and the MHC-like MHC class I polypeptide-related sequence A (MIC-A)- and sequence B (MIC-B)-activating ligands of NK cells. We created γ-retroviruses encoding K3 and/or K5 transgenes that efficiently transduce primary human T cells. Expression of K3 or K5 resulted in dramatic downregulation of MHC-IA (human leukocyte antigen [HLA]-A, -B, and -C) and MHC class II (HLA-DR) cell-surface expression. K3 expression was sufficient for T cells to resist exogenously loaded peptide-MHC-specific cytotoxicity, as well as recognition in one-way allogeneic mixed lymphocyte reactions. Further, in immunodeficient mice engrafted with allogeneic T cells, K3-transduced T cells selectively expanded in vivo. Ectopic K5 expression in MHC class I(−), MIC-A(+)/B(+) K562 cells also reduced targeting by primary NK cells. Coexpression of K3 in prostate stem cell antigen (PSCA)-directed, inducible MyD88/CD40 (iMC)-enhanced CAR-T cells did not impact cytotoxicity, T cell growth, or cytokine production against HPAC pancreatic tumor target cells, whereas K5-expressing cells showed a modest reduction in interleukin (IL)-2 production without effect on cytotoxicity. Together, these results support application of these E3 ligases to advance development of OTS CAR-T cell products.

Natural killer (NK) cells expressing chimeric antigen receptors (CARs) are a promising anticancer immunotherapy, leveraging both innate NK cell antitumor activity and target-specific cytotoxicity. Inducible MyD88/CD40 (iMC) is a potent, rimiducid-regulated protein switch that has been deployed previously as a T-cell activator to enhance proliferation and persistence of CAR-modified T cells. In this study, iMC was extended to CAR-NK cells to enhance their growth and augment cytotoxicity against tumor cells. iMC-activated NK cells substantially increased cytokine and chemokine secretion and displayed higher levels of perforin and granzyme B degranulation. In addition, iMC activation could be coupled with ectopic interleukin-15 (IL-15) to further enhance NK cell proliferation. When coexpressed with a target-specific CAR (CD123 or BCMA), this IL-15/iMC system showed further augmented antitumor activity through enhanced CAR-NK cell expansion and cytolytic activity. To protect against potential toxicity from engineered NK cells, an orthogonal rapamycin-regulated Caspase-9 (iRC9) was included in a 4-gene, dual-switch platform. After infusion of dual-switch NK cells, pharmacologic iRC9 dimerization led to rapid elimination of a majority of expanded transduced NK cells. Thus, CAR-NK cells utilizing dual molecular switches provide an innovative and effective approach to cancer immunotherapy with controlled specificity, efficacy, and safety.

During daily fishing operations, spanner crab Ranina ranina catch rates can fluctuate substantially, but the environmental drivers responsible for these fluctuations largely remain unresolved. Earlier research suggests that spanner crab catchability increases with strengthening currents, but uncertainties surround the magnitude of the measured current speeds and, consequently, their relationship with catch rates. Here, we explore the effects of bottom currents on spanner crab catch rates in South East Queensland, Australia. Using generalized additive mixed modeling, our results indicated that strengthening current speeds increased catch rates until reaching approximately 0.15 m/s, at which point the catch rates began to gradually decline. Results from a general linear regression model also showed that between fishing periods carried out on the same day, catch rates increased or decreased concurrently with current speeds. We conclude that bottom current speed should be considered in future stock assessment models. Better understanding the processes responsible for changes in bottom current speed will enable more accurate estimates of spanner crab population densities in the Australian fishery and will benefit the economic efficiency of commercial crabbing operations. Furthermore, future studies that investigate the effects of current speed on catch rates for other crab species should consider differences in locomotory characteristics and how they may impact the foraging efficiency of crabs under different flow conditions.

Use of chimeric antigen receptors (CARs) as the basis of targeted adoptive T cell therapies has enabled dramatic efficacy against multiple hematopoietic malignancies, but potency against bulky and solid tumors has lagged, potentially due to insufficient CAR-T cell expansion and persistence. To improve CAR-T cell efficacy, we utilized a potent activation switch based on rimiducid-inducible MyD88 and CD40 (iMC)-signaling elements. To offset potential toxicity risks by this enhanced CAR, an orthogonally regulated, rapamycin-induced, caspase-9-based safety switch (iRC9) was developed to allow in vivo elimination of CAR-T cells. iMC costimulation induced by systemic rimiducid administration enhanced CAR-T cell proliferation, cytokine secretion, and antitumor efficacy in both in vitro assays and xenograft tumor models. Conversely, rapamycin-mediated iRC9 dimerization rapidly induced apoptosis in a dose-dependent fashion as an approach to mitigate therapy-related toxicity. This novel, regulatable dual-switch system may promote greater CAR-T cell expansion and prolonged persistence in a drug-dependent manner while providing a safety switch to mitigate toxicity concerns. Keywords: CAR-T, dimerizer, cell therapy, costimulation switch, apoptosis, rapamycin, rimiducid, iMC, iRC9, safety switch

Successful adoptive chimeric antigen receptor (CAR) T-cell therapies against hematological malignancies require CAR-T expansion and durable persistence following infusion. Balancing increased CAR-T potency with safety, including severe cytokine-release syndrome (sCRS) and neurotoxicity, warrants inclusion of safety mechanisms to control in vivo CAR-T activity. Here, we describe a novel CAR-T cell platform that utilizes expression of the toll-like receptor (TLR) adaptor molecule, MyD88, and tumor-necrosis factor family member, CD40 (MC), tethered to the CAR molecule through an intentionally inefficient 2A linker system, providing a constitutive signal that drives CAR-T survival, proliferation, and antitumor activity against CD19+ and CD123+ hematological cancers. Robust activity of MC-enhanced CAR-T cells was associated with cachexia in animal models that corresponded with high levels of human cytokine production. However, toxicity could be successfully resolved by using the inducible caspase-9 (iC9) safety switch to reduce serum cytokines, by administration of a neutralizing antibody against TNF-α, or by selecting “low” cytokine-producing CD8+ T cells, without loss of antitumor activity. Interestingly, high basal activity was essential for in vivo CAR-T expansion. This study shows that co-opting novel signaling elements (i.e., MyD88 and CD40) and development of a unique CAR-T architecture can drive T-cell proliferation in vivo to enhance CAR-T therapies.

Species catchability is an important parameter used to help optimise stock assessment modelling and the economic efficiency of commercial fishing operations. Previous studies have shown several physical oceanographic parameters, including ambient temperature, waves and currents, affect the catchability of spanner crabs (Ranina ranina) throughout the Indo-Pacific. Most notably in the Australian fishery, where oceanographic processes vary over space and time, a positive relationship between bottom boundary layer temperature (BBLT) and catch rates was observed. Here, we aimed to better understand how localised oceanographic processes affected this relationship in the southernmost South-East Queensland (SEQ) sector of the Australian fishery at seasonal and short temporal scales. Our results show cooler BBLT, upwelling-favourable alongshore wind stress and increased catch rates occurred during mating season in austral spring. At the end of austral summer, BBLT began warming, downwelling-favourable winds were dominant, and catch rates declined around the post-moult period. Outputs from the generalised linear models (GLMs) that separated these effects in each season show that, at shorter temporal scales, daily catch rates also increased with episodic BBLT cooling and upwelling-favourable alongshore wind stress, but only during austral autumn and winter. These new findings suggest that region-specific, short-term and seasonal variability of oceanographic processes responsible for changes in BBLT play an important role in influencing the catchability of spanner crabs. We suggest that the effects of region-specific physical oceanographic processes must be considered in future work when investigating the catchability of commercially important fisheries species fished over large spatial domains.

To assist with developing an understanding of the nutrient supply dynamics in the Great Australian Bight (GAB) the vertical turbulent nitrate flux was calculated at stations along two cross-shelf transects using direct measurements of turbulence and nitrate concentrations. Coincident hydrographic profiling conducted at the onset of the summertime upwelling season revealed upwelling onto the shelf in the eastern GAB (eGAB) and downwelling at the shelf slope in the central GAB (cGAB). Under these conditions, examination of the strength of competing vertical temperature and salinity gradients using the Turner angle suggested a high potential for double diffusive convective mixing. However, corresponding estimates of the mixing efficiency derived from microstructure profiling indicated turbulence was the main mixing process driving vertical fluxes across the region. The average upward nitrate flux between the surface mixed layer depth(MLD) and the base of the euphotic layer (92 m depth) was O(10−6−10−5) mmol N m−2 s−1 in the cGAB, with the peak flux observed at the upper-slope station (~ 400 m total water depth). In the eGAB, the magnitude of flux estimates at the shelf-slope and offshore stations were similar to those measured in the cGAB. An enhanced nitrate flux O(10−4) mmol N m−2 s−1 was observed on the shelf in the eGAB (~ 100 m total water depth) as the result of enhanced turbulence and an increased vertical nitrate gradient. Application of Redfield stoichiometry suggests vertical turbulent nitrate fluxes play a significant role in supporting up to 50% of the primary production in the GAB. We hypothesize that mixing processes resulting from interactions between upwelled and downwelled water masses and new water masses formed on the shelf during summer play an important role in maintaining the bands of subsurface chlorophyll a maxima observed below the MLD at the nitricline during periods of strong stratification. The snapshot provided by this study, emphasizes the need to better understand the variability and influence of mixing processes on nutrient supply and productivity across the GAB marine ecosystem.

Adoptive immunotherapy with T cells expressing chimeric antigen receptors (CAR) has had limited success for solid tumors in early-phase clinical studies. We reasoned that introducing into CAR T cells an inducible costimulatory (iCO) molecule consisting of a chemical inducer of dimerization (CID)–binding domain and the MyD88 and CD40 signaling domains would improve and control CAR T-cell activation. In the presence of CID, T cells expressing HER2–CARζ and a MyD88/CD40–based iCO molecule (HER2ζ.iCO T cells) had superior T-cell proliferation, cytokine production, and ability to sequentially kill targets in vitro relative to HER2ζ.iCO T cells without CID and T cells expressing HER2–CAR.CD28ζ. HER2ζ.iCO T cells with CID also significantly improved survival in vivo in two xenograft models. Repeat injections of CID were able to further increase the antitumor activity of HER2ζ.iCO T cells in vivo. Thus, expressing MyD88/CD40–based iCO molecules in CAR T cells has the potential to improve the efficacy of CAR T-cell therapy approaches for solid tumors. Significance: Inducible activation of MyD88 and CD40 in CAR T cells with a small-molecule drug not only enhances their effector function, resulting in potent antitumor activity in preclinical solid tumors, but also enables their remote control post infusion. Cancer Discov; 7(11); 1306–19. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 1201

This phase I trial reports the safety and activity of BPX101, a second-generation antigen-targeted autologous antigen presenting cell (APC) vaccine in men with metastatic castration-resistant prostate cancer (mCRPC). To manufacture BPX101, APCs collected in a single leukapheresis were transduced with adenoviral vector Ad5f35 encoding inducible human (ih)-CD40, followed by incubation with protein PA001, which contains the extracellular domain of human prostate-specific membrane antigen. The ih-CD40 represents a modified chimeric version of the dendritic cell (DC) co-stimulatory molecule, CD40, which responds to a bioinert membrane-permeable activating dimerizer drug, rimiducid (AP1903), permitting temporally controlled, lymphoid-localized, DC-specific activation. Eighteen men with progressive mCRPC following ≤1 prior chemotherapy regimen were enrolled to evaluate three doses of BPX101 (4 × 106, 12.5 × 106 and 25 × 106 cells) administered intradermally every 2–4 weeks followed by rimiducid (0.4 mg/kg) intravenous (IV) infusion 24 h after each BPX101 dose. There were no dose-limiting toxicities. Immune upregulation as well as anti-tumor activity was observed with PSA declines, objective tumor regressions and robust efficacy of post-trial therapy. This novel antigen-targeted and in vivo activated immunotherapy platform may warrant further development as monotherapy and as a component of rational combinations.

This review examines the influence of physical oceanographic processes on catchability of spanner crab (Ranina ranina) in northeast Australia. Physical oceanographic processes may affect crab catchability by influencing their activity levels and ability to detect bait. Bottom temperature, current velocity, and swell intensity appear to influence catches of spanner crab. At this stage, it appears warmer temperatures enhance catchability of spanner crab. Spanner crabs were more catchable in stronger currents, and crabs were observed to arrive from down-current of baited traps. However, a decline in catch was observed following periods of intense swell. Data derived from Waverider buoys suggest that occasionally these periods create strong wave-induced seabed current velocities, lead to at depths of 70 m. The oscillatory motion of wave-induced seabed velocities may cause higher suspended sediment concentrations. These observations corroborate the views of local fishermen that spanner crabs avoid ‘murky’ water. The effect of turbidity on catchability requires further research. Overall, we advocate that studies employ robust methodologies to measure physical oceanographic processes to accurately predict catchability. Moreover, large-scale physical oceanographic processes may also play an important role in catchability of spanner crab; including upwelling, eddies, and the East Australian Current. Integrating physical oceanography and fisheries interactions will considerably benefit commercial fishermen as well as provide valuable information for evidence-based management of these valuable resources.

Hydrodynamic dispersion has a significant impact on the mass transport of sediments and contaminants within coastal waters. In this study apparent horizontal dispersion in a tidally-dominated shallow estuary was investigated using field observations and a numerical model. A cluster of four Lagrangian drifters was released in two shallow regions inside Moreton Bay, Australia: between two small islands and in an open water area. During a 16-h tracking period, the drifters generally showed similar behaviour, initially moving with the dominant current and remaining together before spreading apart at the change of tide. Two dispersion regimes were identified, a slow dispersion during the earlier stage and a rapid dispersion during the latter stage of deployment. Such change in regime typically occurred during the succeeding ebb or flow tides, which may be attributable to residual eddies breaking down during reversal of tidal direction. In addition, a power function of the squared separation distance over the apparent dispersion coefficient produced an R2 exceeding 0.7, indicating a significant relationship between them. By applying a three-dimensional hydrodynamic model, the trajectories of artificial particles spreading in the bay were simulated, which allowed the calculation of dispersion coefficients throughout the entire bay. The study results demonstrate that the tidal effects on dispersion were dependent on the effect of tidal excursion and residual current. The tide was found to be the most dominant driver of dispersion in the bay when unobstructed by land; however, bathymetric and shoreline characteristics were also significant localised drivers of dispersion between the two islands as a result of island wake.

Background: Effective chimeric antigen receptor (CAR) T cell therapy against solid tumors must overcome a hostile, tumor microenvironment that includes tumor-associated macrophages (TAM). Pancreatic adenocarcinomas (PDAC) are commonly infiltrated with TAMs polarized to a tumor-promoting M2 phenotype rather than a T cell-stimulatory and tumor-inhibitory M1 phenotype. The GoCAR platform combines an inducible MyD88/CD40 (iMC) costimulation protein with a 1st generation CAR. Our previously published results demonstrated that iMC costimulation, activated by the small molecule dimerizer, rimiducid (Rim), enhanced CAR-T proliferation and anti-tumor efficacy. Here, we examined the extrinsic effects of iMC signaling on CAR-T cell immune-activating ligands, cytokine production and their ability to polarize M2 macrophage to an anti-tumor phenotype. Methods: Macrophages were prepared from peripheral blood monocytes of four or more random blood donors from the Gulf Coast Regional Blood Center (Houston, TX) and differentiated in vitro to an M2 phenotype with TGF-β and IL-10, or an M1 phenotype (as a positive control). GoCAR-T cells targeting prostate stem cell antigen (PSCA) were prepared by retroviral transduction from the autologous donors. To test the effects of iMC activation on macrophage polarization, contact-dependent or independent (i.e., separation of cell populations with transwell inserts) coculture assays were performed with and without activation of iMC with 1 nM Rim and/or surface-bound PSCA. Anti-tumor cytotoxicity was measured by coculture with PSCA+ Panc1-GFP cells. Results: CAR activation by PSCA antigen recognition or iMC activation with Rim decreased CD163, an M2 macrophage marker, and increased CD80, expressed on M1 macrophage. Full activation of the GoCAR-T cells with both Rim and the target antigen fully repolarized M2 macrophage to an M1 marked phenotype (CD163lowCD80high). This repolarization could be directed partially in the absence of cell-cell contact by diffusion of soluble factors through Transwell membranes. M2 macrophages repolarized by conditioning media from activated GoCAR-T cells also exhibited the functionality of M1 macrophages and acquired cytotoxicity against tumor cells. Furthermore, cultures of conditioned macrophages and limiting dilutions of GoCAR-T cells demonstrated a cooperative enhancement of the cytotoxicity of PSCA GoCAR-T cells toward Panc-1 targets. This cooperation was more effective for GoCAR-T cells than CD28- or 4-1BB-enhanced 2nd generation PSCA CAR-T cells. Conclusions: These results predict that GoCAR-T activation with Rim will convert TAMs within a solid tumor microenvironment from T cell inhibitors to tumor-caustic agents. Citation Format: Xiaohong Wang, Konrad Gabrusiewicz, David M. Spencer, Aaron E. Foster, J. Henri Bayle. Small molecule inducible MyD88/CD40 (iMC) in CAR-T cells can repolarize M2 macrophage to an anti-tumor M1 phenotype [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2193.

BACKGROUND: Accurate pathologic nodal staging improves early-stage non-small-cell lung cancer survival. In an ongoing implementation study, we measured the impact of a surgical lymph node specimen collection kit and a more thorough pathologic gross dissection method, on attainment of guideline-recommended pathologic nodal staging quality. METHODS: We prospectively collected data on curative-intent non-small cell lung cancer resections from 2009–2016 from 11 hospitals in 4 contiguous Dartmouth Hospital Referral Regions. We categorized patients into 4 groups based on exposure to the two interventions in our staggered implementation study design. We used Chi-squared tests to examine the differences in demographic and disease characteristics and surgical quality criteria across implementation groups. RESULTS: Of 2,469 patients, 1,615 (65%) received neither intervention; 167 (7%) received only the pathology intervention; 264 (11%) received only the surgery intervention; 423 (17%) had both. Rates of non-examination of lymph nodes reduced sequentially in the order of no intervention, novel dissection, kit, and combined interventions, including non-examination of: any lymph nodes, hilar/intrapulmonary and mediastinal nodes (p

To test the feasibility of a single T-cell manipulation to eliminate alloreactivity while sparing antiviral and antitumor T cells, we infused 12 haploidentical hematopoietic stem cell transplant patients with increasing numbers of alloreplete haploidentical T cells expressing the inducible caspase 9 suicide gene (iC9-T cells). We determined whether the iC9-T cells produced immune reconstitution and if any resultant graft-versus-host disease (GVHD) could be controlled by administration of a chemical inducer of dimerization (CID; AP1903/Rimiducid). All patients receiving >10(4) alloreplete iC9-T lymphocytes per kilogram achieved rapid reconstitution of immune responses toward 5 major pathogenic viruses and concomitant control of active infections. Four patients received a single AP1903 dose. CID infusion eliminated 85% to 95% of circulating CD3(+)CD19(+) T cells within 30 minutes, with no recurrence of GVHD within 90 days. In one patient, symptoms and signs of GVHD-associated cytokine release syndrome (CRS-hyperpyrexia, high levels of proinflammatory cytokines, and rash) resolved within 2 hours of AP1903 infusion. One patient with varicella zoster virus meningitis and acute GVHD had iC9-T cells present in the cerebrospinal fluid, which were reduced by >90% after CID. Notably, virus-specific T cells recovered even after AP1903 administration and continued to protect against infection. Hence, alloreplete iC9-T cells can reconstitute immunity posttransplant and administration of CID can eliminate them from both peripheral blood and the central nervous system (CNS), leading to rapid resolution of GVHD and CRS. The approach may therefore be useful for the rapid and effective treatment of toxicities associated with infusion of engineered T lymphocytes. This trial was registered at www.clinicaltrials.gov as #NCT01494103.

Anti-tumor efficacy of T cells engineered to express chimeric antigen receptors (CARs) is dependent on their specificity, survival, and in vivo expansion following adoptive transfer. Toll-like receptor (TLR) and CD40 signaling in T cells can improve persistence and drive proliferation of antigen-specific CD4+ and CD8+ T cells following pathogen challenge or in graft-versus-host disease (GvHD) settings, suggesting that these costimulatory pathways may be co-opted to improve CAR-T cell persistence and function. Here, we present a novel strategy to activate TLR and CD40 signaling in human T cells using inducible MyD88/CD40 (iMC), which can be triggered in vivo via the synthetic dimerizing ligand, rimiducid, to provide potent costimulation to CAR-modified T cells. Importantly, the concurrent activation of iMC (with rimiducid) and CAR (by antigen recognition) is required for interleukin (IL)-2 production and robust CAR-T cell expansion and may provide a user-controlled mechanism to amplify CAR-T cell levels in vivo and augment anti-tumor efficacy.

Adoptive transfer of donor-derived T lymphocytes expressing a safety switch may promote immune reconstitution in patients undergoing haploidentical hematopoietic stem cell transplant (haplo-HSCT) without the risk for uncontrolled graft versus host disease (GvHD). Thus, patients who develop GvHD after infusion of allodepleted donor-derived T cells expressing an inducible human caspase 9 (iC9) had their disease effectively controlled by a single administration of a small-molecule drug (AP1903) that dimerizes and activates the iC9 transgene. We now report the long-term follow-up of 10 patients infused with such safety switch-modified T cells. We find long-term persistence of iC9-modified (iC9-T) T cells in vivo in the absence of emerging oligoclonality and a robust immunologic benefit, mediated initially by the infused cells themselves and subsequently by an apparently accelerated reconstitution of endogenous naive T lymphocytes. As a consequence, these patients have immediate and sustained protection from major pathogens, including cytomegalovirus, adenovirus, BK virus, and Epstein-Barr virus in the absence of acute or chronic GvHD, supporting the beneficial effects of this approach to immune reconstitution after haplo-HSCT. This study was registered at www.clinicaltrials.gov as #NCT00710892.

Spencer, D., Lemckert, C.J., Yu, Y., Gustafson, J., Lee, S.Y., Zhang, H., 2014. Quantifying Dispersion in an Estuary: A Lagrangian Drifter Approach. In: Green, A.N. and Cooper, J.A.G. (eds.), Proceedings 13th International Coastal Symposium (Durban, South Africa), Journal of Coastal Research, Special Issue No. 70, pp. 029–034, ISSN 0749-0208. Describing the physical drivers of water bodies plays an integral role in the marine environment. Lagrangian drifters are used to describe hydrodynamics, where tracking groups (clusters) of drifting floats is performed to determine the dispersion behaviour in the water. Using two types of Lagrangian drifters released in Moreton Bay, Australia, the dispersion behaviour is determined over the duration of a semi-diurnal spring tide cycle. A Self-Locating Datum Marker Buoy (SLDMB) was designed as the first drifter type. The second drifter (Small Drifter) was designed to be approximately half the size of the SLDMB. SLDMB and Small Drifter clusters were deployed f...

In the Australian spanner crab (Ranina ranina) fishery, management and industry are looking for improvements to the existing indicators of stock abundance. Prior research linked several oceanographic indices to the catchability of spanner crabs; however, it was unclear whether nearshore (e.g. river-runoff) or region-specific oceanographic features (e.g. eddies and the East Australian Current) are responsible for these effects on catch rates. Using satellite remote sensing and fishery-independent survey data, we analysed the influence of oceanographic and environmental indices on spanner crab catch rates in southern Queensland. Outputs from Generalised Additive Models (GAM) show that catch rates exhibit a large amount of variability between different regions of the fishery, with highest catch rates at fishing grounds within 40 km from the shelf break. Offshore oceanic waters, transported into various regions by different oceanographic processes, were linked to an increase in catch rates. Lower concentrations of surface chlorophyll a were also correlated with higher catch rates, but only in survey regions exposed to the effects of the Fraser Gyre and at the mouth of bays. Overall, results highlighted that the effects of environmental indices on catch rates were not homogeneous across the fishery. Rather, relationships were linked to region-specific (

The reactive stroma surrounding tumor lesions performs critical roles ranging from supporting tumor cell proliferation to inducing tumorigenesis and metastasis. Therefore, it is critical to understand the cellular components and signaling control mechanisms that underlie the etiology of reactive stroma. Previous studies have individually implicated fibroblast growth factor receptor 1 (FGFR1) and canonical WNT/β-catenin signaling in prostate cancer progression and the initiation and maintenance of a reactive stroma; however, both pathways are frequently found to be coactivated in cancer tissue. Using autochthonous transgenic mouse models for inducible FGFR1 (JOCK1) and prostate-specific and ubiquitously expressed inducible β-catenin (Pro-Cat and Ubi-Cat, respectively) and bigenic crosses between these lines (Pro-Cat × JOCK1 and Ubi-Cat × JOCK1), we describe WNT-induced synergistic acceleration of FGFR1-driven adenocarcinoma, associated with a pronounced fibroblastic reactive stroma activation surrounding prostatic intraepithelial neoplasia (mPIN) lesions found both in in situ and reconstitution assays. Both mouse and human reactive stroma exhibited increased transforming growth factor-β (TGF-β) signaling adjacent to pathologic lesions likely contributing to invasion. Furthermore, elevated stromal TGF-β signaling was associated with higher Gleason scores in archived human biopsies, mirroring murine patterns. Our findings establish the importance of the FGFR1–WNT–TGF-β signaling axes as driving forces behind reactive stroma in aggressive prostate adenocarcinomas, deepening their relevance as therapeutic targets. Cancer Res; 74(2); 609–20. ©2013 AACR.

OBJECTIVES: Flow cytometry (FC) aids in characterization of cellular and molecular factors involved in pathologic immune responses. Although FC has potential to facilitate early drug development in inflammatory bowel disease, interlaboratory variability limits its use in multicenter trials. Standardization of methods may address this limitation. We compared variability in FC-aided quantitation of T-cell responses across international laboratories using three analytical strategies. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated from three healthy donors, stimulated with phorbol 12-myristate 13-acetate and ionomycin at a central laboratory, fixed, frozen, and shipped to seven international laboratories. Permeabilization and staining was performed in triplicate at each laboratory using a common protocol and centrally provided reagents. Gating was performed using local gating with a local strategy (LGLS), local gating with a central strategy (LGCS), and central gating (CG). Median cell percentages were calculated across triplicates and donors, and reported for each condition and strategy. The coefficient of variation (CV) was calculated across laboratories. Between-strategy comparisons were made using a two-way analysis of variance adjusting for donor. RESULTS: Mean interlaboratory CV ranged from 1.8 to 102.1% depending on cell population and gating strategy (LGLS, 4.4-102.1%; LGCS, 10.9-65.6%; CG, 1.8-20.9%). Mean interlaboratory CV differed significantly across strategies and was consistently lower with CG. CONCLUSIONS: Central gating was the only strategy with mean CVs consistently lower than 25%, which is a proposed standard for pharmacodynamic and exploratory biomarker assays

SummaryThe role of Notch signaling in the maintenance of adult murine prostate epithelial homeostasis remains unclear. We found that Notch ligands are mainly expressed within the basal cell lineage, while active Notch signaling is detected in both the prostate basal and luminal cell lineages. Disrupting the canonical Notch effector Rbp-j impairs the differentiation of prostate basal stem cells and increases their proliferation in vitro and in vivo, but does not affect luminal cell biology. Conversely, ectopic Notch activation in adult prostates results in a decrease in basal cell number and luminal cell hyperproliferation. TGFβ dominates over Notch signaling and overrides Notch ablation-induced proliferation of prostate basal cells. However, Notch confers sensitivity and positive feedback by upregulating a plethora of TGFβ signaling components including TgfβR1. These findings reveal crucial roles of the self-enforced positive reciprocal regulatory loop between TGFβ and Notch in maintaining prostate basal stem cell dormancy.

Recent modest successes in ex vivo dendritic cell (DC) immunotherapy have motivated continued innovation in the area of DC manipulation and activation. Although ex vivo vaccine approaches continue to be proving grounds for new DC manipulation techniques, the intrinsic limits of ex vivo therapy, including high cost, minimal standardization, cumbersome delivery, and poor accessibility, incentivizes the development of vaccines compatible with in vivo DC targeting. We describe here a method to co-deliver both tumor-specific antigen (TSA) and an iMyD88/CD40 adjuvant (iMC), to DCs that combines toll-like receptor (TLR) and CD40 signaling. In this study, we demonstrate that simple TSA delivery via adenoviral vectors results in strong antitumor immunity. Addition of iMC delivered in a separate vector is insufficient to enhance this effect. However, when delivered simultaneously with TSA in a single bicistronic vector (BV), iMC is able to significantly enhance antigen-specific cytotoxic T-cell (CTL) responses and inhibit established tumor growth. This study demonstrates the spatial-temporal importance of concurrent DC activation and TSA presentation. Further, it demonstrates the feasibility of in vivo molecular enhancement of DCs necessary for effective antitumor immune responses.

Cellular therapies could play a role in cancer treatment and regenerative medicine if it were possible to quickly eliminate the infused cells in case of adverse events. We devised an inducible T-cell safety switch that is based on the fusion of human caspase 9 to a modified human FK-binding protein, allowing conditional dimerization. When exposed to a synthetic dimerizing drug, the inducible caspase 9 (iCasp9) becomes activated and leads to the rapid death of cells expressing this construct.We tested the activity of our safety switch by introducing the gene into donor T cells given to enhance immune reconstitution in recipients of haploidentical stem-cell transplants. Patients received AP1903, an otherwise bioinert small-molecule dimerizing drug, if graft-versus-host disease (GVHD) developed. We measured the effects of AP1903 on GVHD and on the function and persistence of the cells containing the iCasp9 safety switch.Five patients between the ages of 3 and 17 years who had undergone stem-cell transplantation for relapsed acute leukemia were treated with the genetically modified T cells. The cells were detected in peripheral blood from all five patients and increased in number over time, despite their constitutive transgene expression. A single dose of dimerizing drug, given to four patients in whom GVHD developed, eliminated more than 90% of the modified T cells within 30 minutes after administration and ended the GVHD without recurrence.The iCasp9 cell-suicide system may increase the safety of cellular therapies and expand their clinical applications. (Funded by the National Heart, Lung, and Blood Institute and the National Cancer Institute; ClinicalTrials.gov number, NCT00710892.).

The activation of innate and adaptive immunity is always balanced by inhibitory signalling mechanisms to maintain tissue integrity. We have identified the E3 ligase c-Cbl--known for its roles in regulating lymphocyte signalling--as a modulator of dendritic cell activation. In c-Cbl-deficient dendritic cells, Toll-like receptor-induced expression of proinflammatory factors, such as interleukin-12, is increased, correlating with a greater potency of dendritic-cell-based vaccines against established tumours. This proinflammatory phenotype is accompanied by an increase in nuclear factor (NF)-κB activity. In addition, c-Cbl deficiency reduces both p50 and p105 levels, which have been shown to modulate the stimulatory function of NF-κB. Our data indicate that c-Cbl has a crucial, RING-domain-dependent role in regulating dendritic cell maturation, probably by facilitating the regulatory function of p105 and/or p50.

Background Natural Killer (NK) lymphocytes possess innate anti-tumor activity that has the potential to be used as an allogeneic cell therapy due to reduced GvHD risk relative to αβ T cells. Despite their potential, adoptive NK cell immunotherapies have been limited by poor expansion in vivo. Using our previously developed Chimeric Antigen Receptor-T cell (CAR-T) strategy that relies on rimiducid-based dimerization of inducible MyD88/CD40 (iMC) to regulate T cell expansion and survival, we demonstrate that iMC can also be applied to NK cell growth and anti-tumor efficacy in vitro and in vivo. Moreover, a rapamycin-inducible Caspase-9 (iRC9) was used to provide an orthogonally regulated safety switch. Methods and Results CD56+ NK cells were isolated from peripheral blood of human donors, stimulated overnight with IL-15 then activated by seeding with K562 erythroleukemia target cells. NK cells were then transduced with γ-retrovirus encoding control iRC9-2A-ΔCD19, iRC9-2A-ΔCD19-2A-iMC (dual-switch NK) or iRC9-2A-IL-15-2A-ΔCD19-2A-iMC (dual-switch/IL-15 NK). ΔCD19 marked transduced cells in 50:50 cocultures with untransduced NK cells. NK cells containing only iRC9 grew at the same rate as untransduced cells, but iMC-expressing NK cells displayed enhanced growth that was further augmented by 1 nM rimiducid treatment. In cocultures with THP1 acute myeloid leukemia cells at increasing Target:Effector (T:E) ratios, presence (P < 0.001, two way ANOVA) and activation (P To study in vivo anti-tumor activity, immunodeficient NSG mice were engrafted with dual-switch NK cells with or without autocrine IL-15 expression in the presence or absence of THP-1 tumor targets. When tumor was present, unstimulated iMC with IL-15 or activation of iMC without IL-15 expression supported modest NK cell expansion, but rimiducid stimulation of iMC plus autocrine IL-15 showed enhanced NK expansion in vivo. Furthermore, in tumor-free animals only dual-switch/IL-15 NK cells with weekly rimiducid stimulation expanded and persisted in vivo (up to 7 weeks). Cotransduction of a first generation CD123-targeted CAR to produce dual-switch/IL-15 CD123CAR-NK cells led to rimiducid-dependent control of THP1 tumor outgrowth in vivo beyond 40 days. Conversely, temsirolimus-mediated activation of the iRC9 safety switch rapidly (< 24 hours) ablated dual-switch NK cells in vivo. Conclusions Inducible MyD88/CD40 is an activation switch that supports NK cell expansion, persistence and anti-tumor activity. When paired with autocrine IL-15 expression, this platform supports NK expansion and persistence in vivo, and AML tumoricidal activity that can be further activated by target-specific CAR expression. Moreover, the fast-acting, orthogonally regulated proapoptotic switch, iRC9, mitigates the risk of off-tumor targeting. Therefore, we describe a novel, regulated NK cell platform that solves many of the challenges of NK cell-based therapy and should be amenable to a readily translatable off-the-shelf cellular therapy for malignancies. Disclosures Wang: Bellicum Pharmaceuticals: Employment, Equity Ownership. Chang:Bellicum Pharmaceuticals: Employment, Equity Ownership. Jasinski:Bellicum Pharmaceuticals: Employment, Equity Ownership. Medina:Bellicum Pharmaceuticals: Employment, Equity Ownership. Zhang:Bellicum Pharmaceuticals: Employment, Equity Ownership. Foster:Bellicum: Employment, Equity Ownership. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership. Bayle:Bellicum Pharmaceuticals: Employment, Equity Ownership.

Background: Adoptive transfer of allogeneic donor T cells can be an effective treatment for hematological malignancies through recognition of leukemia-associated antigens (LAAs) on tumor cells or through alloreactivity. However, alloreactive T cells can also cause graft-versus-host disease (GvHD) limiting their use as an immunotherapy. To leverage the anti-tumor effects of allogeneic polyclonal T cells while minimizing GvHD, we have genetically modified donor T cells with the inducible caspase-9 (iC9) safety switch, which induces apoptosis following exposure to the small molecule ligand rimiducid. Here we show that iC9-modified allogeneic T cells (BPX-501) persist, expand and contain functional LAA-specific T cells in children receiving an alpha/beta TCR and CD19-depleted HLA-haploidentical hematopoietic stem cell transplant (haplo-HSCT) for the treatment of myeloid malignancies. Methods: Pre-infusion products (BPX-501: donor T cells modified with the bicistronic retroviral vector encoding iC9 and truncated CD19 (ΔCD19)) and patient peripheral blood mononuclear cells (PBMCs) were analyzed from twelve patients (AML (10), MDS (1), JMML (1)) receiving BPX-501 (1x106 cells/kg) following an alpha/beta T cell and CD19 B cell-depleted haplo-HSCT (BP-004U: NCT03301168). Engraftment and persistence were measured by coexpression of CD3 and CD19 by flow-cytometry. Endogenous and gene-modified T cells were also phenotyped for CD4:CD8 ratios, memory cell composition (TN, TCM, TEM, TEMRA; CD45RA and CD62L) and T cell receptor Vβ diversity. BPX-501 products and post-treatment samples were characterized for LAA-specific T cells using IFN-γ ELISpot against peptide pools (15 aa overlapping by 5 aa) derived from WT1, PRAME, MAGE (A1, C1, C3), NE and PR3, with and without exposure to 10 nM rimiducid to determine the anti-leukemic contribution of BPX-501. Results: BPX-501 was infused at a median time of 22.5 days after HSCT (range 12-34, one patient was infused at day 89 and one patient was infused at day 147). BPX-501 cells (CD3+CD19+) were detectable in the peripheral blood at 1-2 weeks after infusion in all 12 patients, reaching a peak expansion frequency of a median of 24% ± 17% of total CD3+ T cells, and an absolute cell number of 66.9 ± 112 cells/µl at 2 months post-infusion and could be detected for up to 24 months. BPX-501 T cells showed a CD8-skewed phenotype whereas endogenous T cells exhibited a more balanced CD4:CD8 ratio. BPX-501 were predominantly CD45RA-CD62L+ and CD45RA-CD62L- central and effector memory T cells, respectively. In BPX-501 products, we detected LAA-specific T cells by ELISpot using overlapping peptide pools to WT1, PRAME, MAGE, NE and PR3, and in peripheral blood samples obtained 2 to 5 months post-T cell infusion. Importantly, LAA-reactivity was greatly diminished with exposure to iC9-activating rimiducid. Further, we measured the TCR Vβ usage and observed highly-skewed TCR repertoire in BPX-501 T cells compared to endogenous T cells in 6 months after HSCT indicating selection and expansion of TCR clones. Three patients engrafted BPX-501 were treated with rimiducid to control GvHD resulting in a rapid decrease (62% ± 12%) of CD3+CD19+ T cells in the peripheral blood. In patients treated with rimiducid, CD3+CD19+ T cells recover without further instances of GvHD suggestive of in vivo depletion of alloreactive T cell clones using iC9. Summary: Allogeneic T cells engineered with the iC9 safety switch engraft, expand and demonstrate long-term persistence following adoptive transfer into patients receiving a haplo-HSCT. LAA-specific T cells and alloreactive T cells within the BPX-501 product are detectable in the peripheral blood following infusion and likely contribute to elimination of myeloid malignancies. Disclosures Shaw: Bellicum Pharmaceuticals: Employment, Equity Ownership. Zhou:Bellicum Pharmaceuticals: Employment, Equity Ownership. Lu:Bellicum Pharmaceuticals: Employment, Equity Ownership. Aldinger:Bellicum Pharmaceuticals, Inc.: Employment. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership. Locatelli:Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Foster:Bellicum: Employment, Equity Ownership.

Adoptive transfer of antigen-specific cytotoxic T lymphocytes has shown promise for the therapy of cancer. However, tumor-specific T cells are susceptible to diverse inhibitory signals from the tumor microenvironment. The Akt/protein kinase B plays a central role in T-cell proliferation, function, and survival and we hypothesized that expression of constitutively active Akt (caAkt) in T cells could provide resistance to many of these tumor-associated inhibitory mechanisms. caAkt expression in activated human T cells increased proliferation and cytokine production, a likely result of their sustained expression of nuclear factor-κB (NF-κB) and provided resistance to apoptosis by upregulating antiapoptotic molecules. caAkt expressing T cells (caAkt-T-cells) were also relatively resistant to suppression by and conversion into regulatory T cells (Tregs). These characteristics provided a survival advantage to T cells cocultured with tumor cells in vitro; CD3/28-stimulated T cells expressing a chimeric antigen receptor (CAR) specific for disialoganglioside (GD2) that redirected their activity to the immunosuppressive, GD2-expressing neuroblastoma cell line, LAN-1, resisted tumor-induced apoptosis when co-expressing transgenic caAkt. In conclusion, caAkt-transduced T cells showed resistance to several evasion strategies employed by tumors and may therefore enhance the antitumor activity of adoptively transferred T lymphocytes.

SummaryFibroblast Growth Factor Receptor-1 (FGFR1) is commonly overexpressed in advanced prostate cancer (PCa). To investigate causality, we utilized an inducible FGFR1 (iFGFR1) prostate mouse model. Activation of iFGFR1 with chemical inducers of dimerization (CID) led to highly synchronous, step-wise progression to adenocarcinoma that is linked to an epithelial-to-mesenchymal transition (EMT). iFGFR1 inactivation by CID withdrawal led to full reversion of prostatic intraepithelial neoplasia, whereas PCa lesions became iFGFR1-independent. Gene expression profiling at distinct stages of tumor progression revealed an increase in EMT-associated Sox9 and changes in the Wnt signaling pathway, including Fzd4, which was validated in human PCa. The iFGFR1 model clearly implicates FGFR1 in PCa progression and demonstrates how CID-inducible models can help evaluate candidate molecules in tumor progression and maintenance.