Search

Movie 2. CAR.OT-I cells co-cultured with MC57-OVA257 cells.

Supplementary Figure 2. OVA257 peptide dose does not affect effector cell maturation status.

Movie 4. Serial killing of tumor targets by CAR.OT-I effector cells following engagement through the TCR

Movie 3. CAR.OT-I cells co-cultured with MC57-HER2 cells.

Supplementary Figure 1. TCR activated OT-I and CAR. OT-I cells display similar expression levels of cytotoxic granules.

Adoptive therapy with chimeric antigen receptor (CAR) T cells shows great promise clinically. However, there are important aspects of CAR-T-cell biology that have not been explored, particularly with respect to the kinetics of activation, immune synapse formation, and tumor cell killing. Moreover, the effects of signaling via the endogenous T-cell receptor (TCR) or CAR on killing kinetics are unclear. To address these issues, we developed a novel transgenic mouse (designated CAR.OT-I), in which CD8+ T cells coexpressed the clonogenic OT-I TCR, recognizing the H-2Kb–presented ovalbumin peptide SIINFEKL, and an scFv specific for human HER2. Primed CAR.OT-I T cells were mixed with SIINFEKL-pulsed or HER2-expressing tumor cells and visualized in real-time using time-lapse microscopy. We found that engagement via CAR or TCR did not affect cell death kinetics, except that the time from degranulation to CAR-T-cell detachment was faster when CAR was engaged. We showed, for the first time, that individual CAR.OT-I cells can kill multiple tumor cells (“serial killing”), irrespective of the mode of recognition. At low effector:target ratios, the tumor cell killing rate was similar via TCR or CAR ligation over the first 20 hours of coincubation. However, from 20 to 50 hours, tumor cell death mediated through CAR became attenuated due to CAR downregulation throughout the time course. Our study provides important insights into CAR-T–tumor cell interactions, with implications for single- or dual receptor–focused T-cell therapy. Cancer Immunol Res; 3(5); 483–94. ©2015 AACR.See related commentary by June, p. 470

Movie 5. Serial killing of tumor targets by CAR.OT-I effector cells following engagement through the CAR

Supplementary Methods. Time lapse live video (TLLV) microscopy

Background Diffuse intrinsic pontine glioma (DIPG) and other diffuse midline gliomas (DMG) of the thalamus and spinal cord are rare but devastating high-grade glial tumors of childhood with no curative treatment. Despite aggressive treatment attempts the prognosis has remained poor. Chimeric antigen receptor (CAR) T cell therapy has been identified as a promising new approach in the treatment of DMG tumors; however, additional targets are urgently required given known tumor heterogeneity and the prospect of antigen escape of this cancer. Methods Using cell surface mass spectrometry, we detected high HER2 cell surface protein across a panel of patient-derived DIPG cells, thereby identifying an existing CAR T cell therapy for use in DIPG. Primary human T cells were transduced to express a second-generation HER2 CAR and interrogated for efficacy against patient-derived DIPG cells. Results HER2 CAR T cells demonstrated potent and antigen-specific cytotoxicity and cytokine secretion when co-cultured with patient-derived DIPG cells. Furthermore, HER2 CAR T cells provided a significant regression in intracranial DIPG xenograft tumors. Conclusions HER2 CAR T cells are already in clinic development and are well tolerated in pediatric patients. Here we provide strong preclinical evidence for the inclusion of DIPG patients in future pediatric CNS tumor HER2 CAR T cell clinical trials.

Glioblastoma multiforme (GBM) is the most common and aggressive form of brain cancer, with treatment options often constrained due to inherent resistance of malignant cells to conventional therapy. We investigated the impact of triggering programmed cell death (PCD) by using BH3 mimetic drugs in human GBM cell lines. We demonstrate that co-targeting the pro-survival proteins BCL-XL and MCL-1 was more potent at killing six GBM cell lines compared to conventional therapy with Temozolomide or the bromodomain inhibitor JQ1 in vitro. Enhanced cell killing was observed in U251 and SNB-19 cells in response to dual treatment with TMZ or JQ1 combined with a BCL-XL inhibitor, compared to single agent treatment. This was reflected in abundant cleavage/activation of caspase-3 and cleavage of PARP1, markers of apoptosis. U251 and SNB-19 cells were more readily killed by a combination of BH3 mimetics targeting BCL-XL and MCL-1 as opposed to dual treatment with the BCL-2 inhibitor Venetoclax and a BCL-XL inhibitor. The combined loss of BAX and BAK, the essential executioners of intrinsic apoptosis, rendered U251 and SNB-19 cells refractory to any of the drug combinations tested, demonstrating that apoptosis is responsible for their killing. In an orthotopic mouse model of GBM, we demonstrate that the BCL-XL inhibitor A1331852 can penetrate the brain, with A1331852 detected in both tumour and healthy brain regions. We also investigated the impact of combining small molecule inducers of ferroptosis, erastin and RSL3, with BH3 mimetic drugs. We found that a BCL-XL or an MCL-1 inhibitor potently cooperates with inducers of ferroptosis in killing U251 cells. Overall, these findings demonstrate the potential of dual targeting of distinct PCD signalling pathways in GBM and may guide the utility of BCL-XL inhibitors and inducers of ferroptosis with standard of care treatment for improved therapies for GBM.

De novo-designed receptor transmembrane domains (TMDs) present opportunities for precise control of cellular receptor functions. We developed a de novo design strategy for generating programmed membrane proteins (proMPs): single-pass α-helical TMDs that self-assemble through computationally defined and crystallographically validated interfaces. We used these proMPs to program specific oligomeric interactions into a chimeric antigen receptor (CAR) that we expressed in mouse primary T cells and found that both in vitro CAR T cell cytokine release and in vivo antitumor activity scaled linearly with the oligomeric state encoded by the receptor TMD, from monomers up to tetramers. All programmed CARs stimulated substantially lower T cell cytokine release relative to the commonly used CD28 TMD, which we show elevated cytokine release through lateral recruitment of the endogenous T cell costimulatory receptor CD28. Precise design using orthogonal and modular TMDs thus provides a new way to program receptor structure and predictably tune activity for basic or applied synthetic biology.

Objectives The increasing success of Chimeric Antigen Receptor (CAR) T cell therapy in haematological malignancies is reinvigorating its application in many other cancer types and with renewed focus on its application to solid tumors. We present a novel CAR against glioblastoma, an aggressive, malignant glioma, with a dismal survival rate for which treatment options have remained unchanged for over a decade. Methods We use the human Retained Display (ReD) antibody platform (Myrio Therapeutics) to identify a novel single‐chain variable fragment (scFv) that recognises epidermal growth factor receptor mutant variant III (EGFRvIII), a common and tumor‐specific mutation found in glioblastoma. We use both in vitro functional assays and an in vivo orthotopic xenograft model of glioblastoma to examine the function of our novel CAR, called GCT02, targeted using murine CAR T cells. Results Our EGFRvIII‐specific scFv was found to be of much higher affinity than reported comparators reverse‐engineered from monoclonal antibodies. Despite the higher affinity, GCT02 CAR T cells kill equivalently but secrete lower amounts of cytokine. In addition, GCT02‐CAR T cells also mediate rapid and complete tumor elimination in vivo. Conclusion We present a novel EGFRvIII‐specific CAR, with effective antitumor functions both in in vitro and in a xenograft model of human glioblastoma., We have made novel chimeric antigen receptor T (CART) cells specific for glioblastoma, a devastating brain tumor with poor survival. This receptor is of extremely high affinity, and the CAR T cells can completely clear brain tumors in mice.

De novo designed receptor transmembrane domains (TMDs) present opportunities for precise control of cellular receptor functions. We developed a de novo design strategy for generating programmed membrane proteins (proMPs): single-pass α-helical TMDs that self-assemble through computationally defined and crystallographically validated interfaces. We used these proMPs to program specific oligomeric interactions into a chimeric antigen receptor (CAR) and found that both in vitro CAR T cell cytokine release and in vivo antitumor activity scaled linearly with the oligomeric state encoded by the receptor TMD, from monomers up to tetramers. All programmed CARs (proCARs) stimulated substantially lower T cell cytokine release relative to the commonly used CD28 TMD, which we show elevated cytokine release through lateral recruitment of the endogenous T cell costimulatory receptor CD28. Precise design using orthogonal and modular TMDs thus provides a new way to program receptor structure and predictably tune activity for basic or applied synthetic biology.

Oncology immunotherapy has been a significant advancement in cancer treatment and involves harnessing and redirecting a patient’s immune response towards their own tumour. Specific recognition and elimination of tumour cells was first proposed over a century ago with Paul Erlich’s ‘magic bullet’ theory of therapy. In the past decades, targeting cancer antigens by redirecting T cells with antibodies using either bispecific T cell engagers (BiTEs) or chimeric antigen receptor (CAR) T cell therapy has achieved impressive clinical responses. Despite recent successes in haematological cancers, linked to a high and uniformly expressed CD19 antigen, the efficacy of T cell therapies in solid cancers has been disappointing, in part due to antigen escape. Targeting heterogeneous solid tumours with T cell therapies will require the identification of novel tumour specific targets. These targets can be found among a range of cell-surface expressed antigens, including proteins, glycolipids or carbohydrates. In this review, we will introduce the current tumour target antigen classification, outline existing approaches to discover novel tumour target antigens and discuss considerations for future design of antibodies with a focus on their use in CAR T cells.

Significance Davenport et al. discovered that the chimeric antigen receptor (CAR) immune synapse structure is different from the T cell receptor (TCR) synapse. The CAR immune synapse formed a disorganized pattern of Lck and more rapidly recruited lytic granules compared with the TCR. The differing immune synapse correlated with faster killing of tumor target cells and detachment from dying tumor cells by CAR-T cells. These findings provide a mechanism whereby CAR-T cells can effectively reduce large tumor burden in patients. This study will form a basis upon which to compare future receptor design to modulate signaling and programming of cytotoxic CAR-T cells to improve treatment of solid cancers., Chimeric antigen receptor T (CAR-T) cells are effective serial killers with a faster off-rate from dying tumor cells than CAR-T cells binding target cells through their T cell receptor (TCR). Here we explored the functional consequences of CAR-mediated signaling using a dual-specific CAR-T cell, where the same cell was triggered via TCR (tcrCTL) or CAR (carCTL). The carCTL immune synapse lacked distinct LFA-1 adhesion rings and was less reliant on LFA to form stable conjugates with target cells. carCTL receptors associated with the synapse were found to be disrupted and formed a convoluted multifocal pattern of Lck microclusters. Both proximal and distal receptor signaling pathways were induced more rapidly and subsequently decreased more rapidly in carCTL than in tcrCTL. The functional consequence of this rapid signaling in carCTL cells included faster lytic granule recruitment to the immune synapse, correlating with faster detachment of the CTL from the target cell. This study provides a mechanism for how CAR-T cells can debulk large tumor burden quickly and may contribute to further refinement of CAR design for enhancing the quality of signaling and programming of the T cell.

The clinical success of chimeric antigen receptor (CAR) T cell immunotherapy in the treatment of haematological cancers has encouraged the extensive development of CAR design to improve their function and increase their applicability. Advancements in protein engineering have seen modifications to both the ecto- and endo-domains of the CAR, with recent designs targeting multiple antigens and including inducible elements. These developments are likely to play an important role in inducing effective CAR T cell responses in a solid tumour context, where clinical responses have not been effective to date. This review highlights the spectrum of novel strategies being employed in CAR design, including for example variations in targeting tumour antigens by utilising different ectodomain designs such as dual chain CARs, natural receptor or ligand-based CARs, and T cell receptor fusion constructs, and also reviews some of the innovative approaches to a “universal” CAR and various multi-antigen targeting CAR strategies. We also explore how choices in the endodomain impact CAR function and how these need to be considered in the overall CAR design.

Skin integrity requires an ongoing replacement and repair orchestrated by several cell types. We previously investigated the architecture of the skin of avian myeloblastosis viral oncogene homolog (Myb) knock-out (KO) embryos and wound repair in Myb(+/)(-) mice revealing a need for Myb in the skin, attributed to fibroblast-dependent production of collagen type 1. Here, using targeted Myb deletion in keratin-14 (K14) positive cells we reveal further Myb-specific defects in epidermal cell proliferation, thickness and ultrastructural morphology. This was associated with a severe deficit in collagen type 1 production, reminiscent of that observed in patients with ichthyosis vulgaris and Ehlers-Danlos syndrome. Since collagen type 1 is a product of fibroblasts, the collagen defect observed was unexpected and appears to be directed by the loss of Myb with significantly reduced tumor growth factor beta 1 (Tgfβ-1) expression by primary keratinocytes. Our findings support a specific role for Myb in K14+ epithelial cells in the preservation of adult skin integrity and function.

Chimeric antigen receptor (CAR) T cells have been highly successful in treating hematological malignancies, including acute and chronic lymphoblastic leukemia. However, treatment of solid tumors using CAR T cells has been largely unsuccessful to date, partly because of tumor-induced immunosuppressive mechanisms, including adenosine production. Previous studies have shown that adenosine generated by tumor cells potently inhibits endogenous antitumor T cell responses through activation of adenosine 2A receptors (A2ARs). Herein, we have observed that CAR activation resulted in increased A2AR expression and suppression of both murine and human CAR T cells. This was reversible using either A2AR antagonists or genetic targeting of A2AR using shRNA. In 2 syngeneic HER2+ self-antigen tumor models, we found that either genetic or pharmacological targeting of the A2AR profoundly increased CAR T cell efficacy, particularly when combined with PD-1 blockade. Mechanistically, this was associated with increased cytokine production of CD8+ CAR T cells and increased activation of both CD8+ and CD4+ CAR T cells. Given the known clinical relevance of the CD73/adenosine pathway in several solid tumor types, and the initiation of phase I trials for A2AR antagonists in oncology, this approach has high translational potential to enhance CAR T cell efficacy in several cancer types.

The translation of basic research into improved therapies for breast cancer patients requires relevant preclinical models that incorporate spontaneous metastasis. We have completed a functional and molecular characterisation of a new isogenic C57BL/6 mouse model of breast cancer metastasis, comparing and contrasting it with the established BALB/c 4T1 model. Metastatic EO771.LMB tumours were derived from poorly metastatic parental EO771 mammary tumours. Functional differences were evaluated using both in vitro assays and spontaneous metastasis assays in mice. Results were compared to non-metastatic 67NR and metastatic 4T1.2 tumours of the 4T1 model. Protein and transcript levels of markers of human breast cancer molecular subtypes were measured in the four tumour lines, as well as p53 (Tp53) tumour-suppressor gene status and responses to tamoxifen in vivo and in vitro. Array-based expression profiling of whole tumours identified genes and pathways that were deregulated in metastatic tumours. EO771.LMB cells metastasised spontaneously to lung in C57BL/6 mice and displayed increased invasive capacity compared with parental EO771. By immunohistochemical assessment, EO771 and EO771.LMB were basal-like, as was the 4T1.2 tumour, whereas 67NR had a luminal phenotype. Primary tumours from all lines were negative for progesterone receptor, Erb-b2/Neu and cytokeratin 5/6, but positive for epidermal growth factor receptor (EGFR). Only 67NR displayed nuclear estrogen receptor alpha (ERα) positivity. EO771 and EO771.LMB expressed mutant p53, whereas 67NR and 4T1.2 were p53-null. Integrated molecular analysis of both the EO771/EO771.LMB and 67NR/4T1.2 pairs indicated that upregulation of matrix metalloproteinase-3 (MMP-3), parathyroid hormone-like hormone (Pthlh) and S100 calcium binding protein A8 (S100a8) and downregulation of the thrombospondin receptor (Cd36) might be causally involved in metastatic dissemination of breast cancer.

Adoptive therapy with chimeric antigen receptor (CAR) T cells shows great promise clinically. However, there are important aspects of CAR-T-cell biology that have not been explored, particularly with respect to the kinetics of activation, immune synapse formation, and tumor cell killing. Moreover, the effects of signaling via the endogenous T-cell receptor (TCR) or CAR on killing kinetics are unclear. To address these issues, we developed a novel transgenic mouse (designated CAR.OT-I), in which CD8+ T cells coexpressed the clonogenic OT-I TCR, recognizing the H-2Kb–presented ovalbumin peptide SIINFEKL, and an scFv specific for human HER2. Primed CAR.OT-I T cells were mixed with SIINFEKL-pulsed or HER2-expressing tumor cells and visualized in real-time using time-lapse microscopy. We found that engagement via CAR or TCR did not affect cell death kinetics, except that the time from degranulation to CAR-T-cell detachment was faster when CAR was engaged. We showed, for the first time, that individual CAR.OT-I cells can kill multiple tumor cells (“serial killing”), irrespective of the mode of recognition. At low effector:target ratios, the tumor cell killing rate was similar via TCR or CAR ligation over the first 20 hours of coincubation. However, from 20 to 50 hours, tumor cell death mediated through CAR became attenuated due to CAR downregulation throughout the time course. Our study provides important insights into CAR-T–tumor cell interactions, with implications for single- or dual receptor–focused T-cell therapy. Cancer Immunol Res; 3(5); 483–94. ©2015 AACR. See related commentary by June, p. 470

Cancers can be addicted to continued and relatively high expression of nuclear oncoproteins. This is evident in colorectal cancer (CRC) where the oncoprotein and transcription factor MYB is over expressed and essential to continued proliferation and tumour cell survival. Historically, targeting transcription factors in the context of cancer has been very challenging. Nevertheless, we formulated a DNA vaccine to generate a MYB-specific immune response in the belief MYB peptides might be aberrantly presented on the cell surface of CRC cells. MYB, like many tumour antigens, is weakly immunogenic as it is a 'self' antigen and is subject to tolerance. To break tolerance, a fusion vaccine was generated comprising a full-length MYB complementary DNA (cDNA) flanked by two potent CD4-epitopes derived from tetanus toxoid. Vaccination was achieved against tumours initiated by two distinct highly aggressive, syngeneic cancer cell lines (CT26 and MC38) that express MYB. This was done in BALB/c and C57BL/6 mouse strains respectively. We introduced multiple inactivating mutations into the oncogene sequence for safety and sub-cloned the cDNA into a Food and Drug Administration (FDA)-compliant vector. We used low dose cyclophosphamide (CY) to overcome T-regulatory cell immune suppression, and anti-program cell death receptor 1 (anti-PD-1) antibodies to block T-cell exhaustion. Anti-PD-1 administered alone slightly delayed tumour growth in MC38 and more effectively in CT26 bearing mice, while CY treatment alone did not. We found that therapeutic vaccination elicits protection when MC38 tumour burden is low, mounts tumour-specific cell killing and affords enhanced protection when MC38 and CT26 tumour burden is higher but only in combination with anti-PD-1 antibody or low dose CY, respectively.

Chimeric antigen receptor T cells (CAR T) re-directed to CD19, have induced remarkable responses in clinical trials for patients with B-cell malignancies. Patients have responded to therapy with a CAR T dose which is a fraction of the pre-existing tumor burden. Explanations for this observation include studies which show the proliferative potential of the CAR T cells (Kalos M et al Sci Transl Med 2011), as well as our recent study which showed that individual CAR T cells can serial kill tumor cells (Davenport AJ et al CIR 2015, Figure 6). Using our dual antigen receptor model (OT-I T cell receptor and 2nd generation HER-2 CAR in the same T cell, termed CAR.OTI cells), we also observed a reproducible and significantly shorter time from CAR- vs TCR-mediated activation to detachment from dying tumor cells (Davenport AJ et al CIR 2015, Figure 4D)suggesting that CAR-mediated individual killing events are actually faster. To explore how this may occur, we examined the immune synapse structure at 20 minutes of CAR.OTI CTL co-culture with tumor cells expressing the cognate antigen for either the CAR or TCR. At this timepoint, CAR.OTI CTL, activated via the TCR, formed a conventional bull's eye immune synapse with accumulation of LCK and actin clearance (Figure 1). Surprisingly, CAR.OTI CTL activated via their CAR had an immune synapse with no or diffuse LCK and small actin rings (Figure 1). At the same timepoint, CAR-activated CAR.OTI CTL conjugates with tumor cells were characterized by a microtubule organizing center (MTOC) distant from the immune synapse LCK accumulation. In contrast TCR activated CAR.OTI CTL conjugates consistently had the MTOC proximal to the LCK accumulation (Figure 2A). Despite this, CAR-mediated CAR.OTI CTL killing of tumor targets was inhibited by a protein kinase C zeta inhibitor and is, therefore, MTOC dependent (Figure 2B). The MTOC circumnavigates the activated CTL nucleus and moves to the immune synapse, bringing cytotoxic granules with it. Using time lapse live video (TLLV) microscopy, we compared CAR.OTI CTL cytotoxic granule movement when the CAR.OTI were activated via the TCR vs CAR. We showed that following CAR vs TCR activation, CAR.OTI cytotoxic granules moved with a significantly higher velocity, and had a shorter time lapse to reach the immune synapse following activation (Ca2+ flux), and a significantly shorter time to detachment from the dying tumor cell (Figure 2C). We then re-examined immune synapse formation at an earlier timepoint, to explore whether the data from Figures 1-2 could be explained by a more rapid CTL response following CAR-mediated signaling. In contrast to our observations at 20 minutes, at five minutes we showed CAR-activated CAR.OTI CTL formed conjugates with tumor targets and the immune synapse showed distinct LCK accumulation and actin clearance (Figure 3A). Finally, we explored CAR.OTI CTL signaling and showed that CAR-mediated activation induced a significantly lower number of Ca2+ fluxes, however each Ca2+ flux amplitude was not different (Figure 3B). We also examined changes in proximal (phospho-LCK, pLCK) and distal (phospho-ERK, pERK) signals in CAR- versus TCR- activated CAR.OTI cells, and showed that CAR-mediated activation induced more rapid proximal and distal activation signals (Figure 3C). In conclusion, this study showed that compared to activation by TCR ligation, T cells respond to CAR ligation with faster phospho-protein signaling, Ca2+ flux, formation of an immune synapse and a more rapid movement of the MTOC and delivery of the cytotoxic granules to kill the tumor cells. Furthermore, LFA-1 did not accumulate at the immune synapse following CAR activation, therefore, reduced adhesion may facilitate the observed rapid detachment from the dying tumor cell, and enable the CAR T to rapidly move onto the next tumor target for 'bigger, stronger, faster' killing. Download : Download high-res image (152KB) Download : Download full-size image Download : Download high-res image (156KB) Download : Download full-size image Download : Download high-res image (175KB) Download : Download full-size image Disclosures No relevant conflicts of interest to declare.

We present a comprehensive average velocity field for Alaska, based on repeated GPS surveys covering the period 1992―2007, and review the major results of previously published papers that used subsets of this data. The spatially and temporally complex pattern of crustal deformation in Alaska results from the superposition of several processes, including postseismic deformation after the 1964 earthquake, spatial variations in plate coupling/slip deficit, translation and rotation of large crustal blocks or plates, and a large slow-slip event in Cook Inlet. Postseismic deformation from the 1964 earthquake continues today, mainly caused by viscoelastic relaxation, and causes trenchward motion. The behavior of the shallow seismogenic zone along the Alaska-Aleutian megathrust is characterized by dramatic along-strike variability. The width of the inferred seismogenic zone varies over along-strike distances that are short compared to the width. The along-strike distribution of locked and creeping regions along the megathrust is consistent with the persistent asperity hypothesis. A large slow-slip event occurred in upper Cook Inlet in 1998―2001, and a smaller event in the same area in 2005―2006. No sign of slow-slip events has been found in segments that are dominated by creep, which suggests that creep there occurs quasi-statically. The overriding plate in Alaska is subject to considerable internal deformation, and can be described in terms of the independent motions of at least four blocks: the Bering plate, the Southern Alaska block, the Yakutat block, and the Fairweather block.

Interior Alaska is an actively deforming landscape that is part of a broad zone of deformation extending hundreds of kilometers inboard of the convergent plate boundary. This chapter provides an overview of recent studies on the seismicity, tectonic geomorphology, and crustal motions from GPS measurements in interior Alaska and the surrounding regions. Although the combined data sets do not point to one single mechanism of deformation for interior Alaska, we suggest that the interactions between North America and two crustal blocks, the Bering and Wrangell, are responsible for the broad diffuse belt of deformation that characterizes this region. We propose that the deformation zone extending along the NNE-striking, left-lateral seismic zones north of the Denali fault and along the Susitna seismic zone south of the fault represents the eastern extent of the distributed boundary between the Bering block and the North American plate. Double-difference earthquake relocations document the fine details of seismic structures and help define the seismogenic part of the crust in interior Alaska. Frequency―magnitude and stress analysis provide insights into faulting parameters and earthquake behavior in the region. Stream channel morphologies and stream profile perturbations help define active surface deformation in interior Alaska. GPS measurements show that the relative motions across the region are different than what would be expected for a stable plate interior, and include the effects of multiple deformation sources. Results from the study show that three dominant types of structures characterize interior Alaska: right-lateral strike-slip faults, left-lateral strike-slip seismic zones, and thrust faults.

Heat induces Hsp70.1 (HSPA1) and Hsc70 (HSPA8) to form complex detergent insoluble cytoplasmic and nuclear structures that are distinct from the cytoskeleton and internal cell membranes. These novel structures have not been observed by earlier immunofluorescence studies as they are obscured by the abundance of soluble Hsp70.1/Hsc70 present in cells. While resistant to detergents, these Hsp70 structures display complex intracellular dynamics and are efficiently disaggregated by ATP, indicating that this pool of Hsp70.1/Hsc70 retains native function and regulation. Hsp70.1 promotes the repair of proteotoxic damage and cell survival after stress. In heated fibroblasts expressing Hsp70.1, Hsp70.1 and Hsc70 complexes are efficiently disaggregated before the cells undergo-heat induced apoptosis. In the absence of Hsp70.1, fibroblasts have increased rates of heat-induced apoptosis and maintain stable insoluble Hsc70 structures. The differences in the intracellular distribution of Hsp70.1 and Hsc70, combined with the ability of Hsp70.1, but not Hsc70, to promote the disaggregation of insoluble Hsp70.1/Hsc70 complexes, indicate that these two closely related proteins perform distinctly different cellular functions in heated cells.

The presence of tumor immune infiltrating cells (TILs), particularly CD8(+) T-cells, is a robust predictor of outcome in patients with colorectal cancer (CRC). We revisited TIL abundance specifically in patients with microsatellite stable (MSS) CRC without evidence of lymph node or metastatic spread. Examination of the density of CD8(+) T-cells in primary tumors in the context of other pro-oncogenic markers was performed to investigate potential regulators of TILs. Two independent cohorts of patients with MSS T2-4N0M0 CRC, enriched for cases with atypical relapse, were investigated. We quantified CD8(+) and CD45RO(+) -TILs, inflammatory markers, NFkBp65, pStat3, Cyclo-oxygenase-2 (COX2) and GRP78 as well as transcription factors (TF), β-catenin and MYB. High CD8(+) TILs correlated with a better relapse-free survival in both cohorts (p = 0.002) with MYB and its target gene, GRP78 being higher in the relapse group (p = 0.001); no difference in pSTAT3 and p65 was observed. A mouse CRC (CT26) model was employed to evaluate the effect of MYB on GRP78 expression as well as T-cell infiltration. MYB over-expressing in CT26 cells increased GRP78 expression and the analysis of tumor-draining lymph nodes adjacent to tumors showed reduced T-cell activation. Furthermore, MYB over-expression reduced the efficacy of anti-PD-1 to modulate CT26 tumor growth. This high MYB and GRP78 show a reciprocal relationship with CD8(+) TILs which may be useful refining the prediction of patient outcome. These data reveal a new immunomodulatory function for MYB suggesting a basis for further development of anti-GRP78 and/or anti-MYB therapies.

Chimeric antigen receptor (CAR) T cells have shown clinical efficacy in refractory B cell malignancies. Despite these exciting clinical results, our fundamental understanding of CAR-T cell biology is limited, possibly limiting the broader application of CAR-T cells to additional haemopoetic and solid cancers. To date, the mechanism of CAR-T immunological synapse formation with tumor cells and kinetics of subsequent serial killing by CAR-T cells has not been explored. Here, we investigated the kinetics of CAR-T cell activation and cytotoxicity, including immune synapse formation, and kinetics of tumor cell killing, closely comparing to activation and cytotoxicity via the T cell receptor (TCR). To address this, we developed a mouse model in which the CD8+ T cells (termed CAR.OT-I cells) co-expressed two antigen receptors, the clonogenic OT-I TCR, and a second generation CAR comprising a scFV to human HER2, CD28 and CD3ζ signaling domains. Effector CAR.OT-I cells were activated via their antigen receptors using either SIINFEKL-pulsed or HER-2 expressing tumor cells, the interactions between the effector CAR.OT-I cells and tumor cells were then assessed by time lapse live microscopy. CAR.OT-I cell activation via the endogenous TCR or the CAR did not affect tumor killing kinetics, except the time taken from CAR.OT-I activation to detachment (from the dying tumor cell) was significantly slower when the endogenous TCR was engaged. Subsequently, we showed for the first time, that CAR.OT-I cells have serial killing capacity, which is important to consider when therapeutic numbers of CAR-T cells are likely to be outnumbered by tumor targets. Individual CAR.OT-I cells killed multiple tumor cells, whether activated via the endogenous TCR or the CAR. We further explored whether these findings have implications for killing of tumor cells using low effector:target cell ratio in short versus long-term killing assays, chromium release and xCELLigence killing assays respectively. We observed, no matter which antigen receptor was activated, the effector CAR.OT-I cells were equivalent killers of tumor cells in short term assays (4-8 hours). However, over a period of 50 hours, CAR.OT-I cells activated via the CAR killed tumor cells at a lower rate than when activated via the TCR. This was due to CAR.OT-I CAR expression down-regulation from 20-50 hours. This study highlights that fundamental differences occur in the way CAR-T cells kill tumor cells, depending on how the effector CAR-T cell is activated. Furthermore, the study provides important insights for CAR-T cell activation in vivo with implications for single- or dual-receptor-focused CAR-T cell therapy and improved clinical benefit. Disclosures No relevant conflicts of interest to declare.

Introduction: Chimeric antigen receptor (CAR) T cells have had spectacular success in the clinic, particularly in B cell malignancies. This clinical success was built on years of pre-clinical development; despite this there remains aspects of CAR-T cell responses to antigen activation, which have not been fully explored. In this study we compared the kinetics of CAR- versus TCR-mediated activation, immune synapse formation and tumor cell killing. This study has particular clinical relevance to CAR-T cell therapy, including conventional CAR-T cells and the more recently described dual antigen receptor T cells. Experimental procedures/strategy: To address these questions, we developed an in vitro model system where T cells could be activated via a CAR or TCR in response to cognate antigen on tumor cells. We used CD8+ T cells from a novel transgenic mouse model where both the CAR and OT-I TCR were constitutively expressed (CAR.OT-I cells), the CAR comprised an scFv directed to human HER-2 (hHER-2) and intracellular signaling domains (CD28 and CD3ζ). Effector CAR.OT-I cells were activated via their TCR using MC57 cells pulsed with SIINFEKL (MC57 OVA257), or via their CAR using MC57 cells, which expressed hHER-2. Interactions between individual effector CAR.OT-I cells and the tumor cells were monitored via time lapse live video (TLLM) microscopy and the kinetics of effector cell activation, delivery of lethal hit, tumor cell killing and effector cell detachment recorded. In addition, short term (four and eight hour Cr-release assay) and long term (50 hour xCELLigence assay) killing assays were performed to compare killing capacity and kinetics. Key data: When effector CAR.OTI cells recognized targets via their TCR or CAR, they displayed the same kinetics for activation, delivery of lethal hit and tumor cell killing to OTI CTL. In contrast, time for CAR.OT-I cell detachment from tumor cells was significantly shorter when activated via the CAR rather than the TCR. Short term killing assays (4-8 hours) showed no difference in bulk CAR.OT-I cell killing of tumor cells. In contrast, longer term killing assays (50 hours) showed there was a significant difference in tumor cell killing rate between CAR- versus TCR-activated. The difference in killing rate was only apparent from 20-50 hours, when CAR-activated CAR.OT-I cells had a significantly reduced tumor cell killing rate compared to TCR-activated CAR.OT-I cells. Finally, we showed for the first time, that individual CAR. OT-I cells were capable of killing multiple tumor target cells (serial killing). This occurred whether the CAR.OT-I cells were activated via the CAR or TCR. Conclusion: In conclusion, this study provides novel information regarding CAR-T cell killing of tumor cells and has practical implications for the use of CAR-T cells in the clinic, whether the therapy is using single or dual antigen receptor T cells. Citation Format: Alex J. Davenport, Misty R. Jenkins, Ryan S. Cross, S Yong Carmen, H Miles Prince, David S. Ritchie, Joseph A. Trapani, Michael H. Kershaw, Phillip K. Darcy, Paul J. Neeson. Individual CAR-T cells, when activated via their CAR or TCR, kill tumor cells with similar kinetics and can kill multiple sequential tumor targets. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B10.

MYB oncogene upregulation is associated with estrogen receptor (ER)-positive breast cancer, but disease requirements for MYB function in vivo have not been explored. In this study, we provide evidence of a critical requirement for MYB functions in models of human and murine breast cancer. In human breast cancer, we found that MYB expression was critical for tumor cell growth both in vitro and in vivo in xenograft settings. In transgenic knockout mice, tissue-specific deletion of the murine MYB gene caused a transient defect in mammary gland development that was reflected in delayed ductal branching and defective apical bud formation. In mouse mammary tumor virus (MMTV)-NEU mice where tumors are initiated by activation of HER2, MYB deletion was sufficient to abolish tumor formation. In the more aggressive MMTV-PyMT model system, MYB deletion delayed tumorigenesis significantly. Together, the findings in these transgenic knockout models implied that MYB was critical during an early window in mammary development when it was essential for tumor initiation, even though MYB loss did not exert a lasting impact upon normal mammary function. Two important MYB-target genes that promote cell survival, BCL2 and GRP78/BIP, were each elevated compared with nontransformed mammary epithelial cells, thereby promoting survival as confirmed in colony formation assays in vitro. Taken together, our findings establish a role for MYB at the hub of ER- and HER2-dependent pathways in mammary carcinogenesis. Cancer Res; 71(22); 7029–37. ©2011 AACR.

[1] For the past 300 years, southeastern Alaska has undergone rapid ice-melting and land uplift attributable to global warming. Corresponding crustal deformation (3 cm/yr) caused by the Little Ice Age retreat is detectable with modern geodetic techniques such as GPS and tidal gauge measurements. Geodetic deformation provides useful information for assessing ice-melting rates, global warming effects, and subcrustal viscosity. Nevertheless, integrated geodetic observations, including gravity measurements, are important. To detect crustal deformation caused by glacial isostatic adjustment and to elucidate the viscosity structure in southeastern Alaska, Japanese and U.S. researchers began a joint 3-year project in 2006 using GPS, Earth tide, and absolute gravity measurements. A new absolute gravity network was established, comprising five sites around Glacier Bay, near Juneau, Alaska. This paper reports the network's gravity measurements during 2006–2008. The bad ocean model in this area hindered ocean loading correction: Large tidal residuals remain in the observations. Accurate tidal correction necessitated on-site tidal observation. Results show high observation precision for all five stations

[1] In the paper ‘‘Evidence for and implications of a Bering Plate based on geodetic measurements from the Aleutians and western Alaska’’ by Ryan S. Cross and Jeffrey T. Freymueller (Journal of Geophysical Research, 113, B07405, doi:10.1029/2007JB005136, 2008), the magnitudes of the estimated angular velocities for the Bering plate were off by two orders of magnitude (or by a factor of 100) because of a unit conversion error. The same error affected all forward and inverse calculations, so only the Bering plate rotation rate was incorrect; the pole location and all other calculations in the paper are correct. The correct angular speed for the best model is 0.064 ± 0.033 degrees/ Ma. An updated Table 3 is given below, with the correct rates for all models tested.

[1] Global Positioning System (GPS) measurements are used to examine the hypothesis of a clockwise rotating Bering plate. Originally proposed on the basis of seismicity, the Bering plate encompasses the Bering Sea, western Alaska, and the Aleutian Islands. GPS measurements from the Bering plate's interior show south to southwest motions of 3–5 mm/a (where a is years). We construct elastic dislocation models to determine the spatial distribution and intensity of locked patches on the Aleutian subduction interface. We use these to remove interseismic strain from the GPS observation and determine an arc translation velocity for each region of the Aleutians, revealing south and southwest motions of 4–8 mm/a. We combine the arc translation rates with measurements from the Bering plate's interior sites and estimate the Euler pole for the Bering plate relative to North America to be located at 42.5°N, 121.3°E with an angular speed of 6.0°/Ma. The clockwise rotation of the Bering plate may cause left lateral faulting in interior Alaska. The Bering plate's interaction with south-central Alaska may be responsible for the decreased slip rate on the western Denali fault and for contraction across the central Alaska Range. We analyze slip partitioning along the Aleutian arc on the basis of both GPS measurements and slip azimuths of thrust earthquakes and find a systematic discrepancy between plate convergence direction and slip azimuths. We conclude that slip partitioning in the back arc only develops west of Amchitka Pass, whereas slip partitioning in the fore arc is present throughout the arc.

[1] We use GPS measurements in the first geodetic study of plate coupling on the Andreanof segment, Aleutian subduction zone. Convergence of 7.3 cm/yr at the subduction zone results in northwestward displacements greater than 1 cm/yr at stations located on the west end of the Andreanof region, but velocities at the east end are smaller in magnitude and oriented west-southwest. These velocity variations are caused by differences in plate coupling along the subduction zone, which correlate with the rupture zones of the 1986 and 1996 Mw8 earthquakes. We construct a dislocation model to estimate the velocity of the arc in the Andreanof region relative to North America, and fault plane coupling coefficients. Our best model shows a southwestward velocity of 7.8 mm/yr, a high degree of coupling in the main thrust zone at the west end of the subduction zone, and little to no coupling at the east end.