New Paradigm In Cancer Treatment Research Articles

ACTR-47. PHASE-III TRIAL OF STANDARD CHEMOTHERAPY VS. CHEMOTHERAPY GUIDED BY CANCER STEM CELL TEST IN RECURRENT GLIOBLASTOMA (NCT03632135)

Abstract Stupp treatment protocol for patients with glioblastoma (GBM) has improved the median overall survival to 14.6 months. However, no investigations have defined effective strategies against recurrence and the prognosis of recurrent GBM patients remains poor. Personalized medicine with assay-guided treatment targeting chemotherapy resistant cancer stem cells (CSCs) alongside the bulk tumor cells is a new paradigm in cancer treatment that may result in improved patient’s outcome. We are using ChemoID, a CLIA and CAP certified CSC cytotoxicity assay for predicting response to chemotherapeutic agents. Our prospective analysis of 61 GBM patients demonstrated that ChemoID-guided treatment significantly improved tumor response. For every 5% increase in cell kill of CSCs by assay-guided chemotherapy, 12-month patient response (non-recurrence of cancer) increased 2.5-fold, OR=2.3 (p=0.01). We also found that median recurrence time was 20-months versus 3-months for patients with a positive (>40% cell kill) CSC test versus negative, whereas median recurrence time was 13-months versus 4-months for patients with a positive (>55% cell kill) bulk test versus negative. We are conducting a multi-institutional phase-III clinical trial (NCT03632135) to determine the clinical validity of the ChemoID assay as a predictor of clinical response in recurrent GBM. The study has been designed as a parallel group controlled clinical trial and the participants are randomized to either standard of care chemotherapy chosen by the physician or ChemoID-guided therapy. Response to therapy will be measured by MRI imaging using RANO criteria. Primary endpoint of median overall survival (OS) and secondary endpoints of OS at 6, 9, and 12 months, median progression free survival (PFS), PFS at 4, 6, 9, and 12 months, objective tumor response, time to recurrence, and quality of life will be measured. Trial is open and currently 22 subjects have been enrolled. Interim analysis of the trial will be conducted in approximately 12 months.

A systematic analysis of genomics-based modeling approaches for prediction of drug response to cytotoxic chemotherapies

BackgroundThe availability and generation of large amounts of genomic data has led to the development of a new paradigm in cancer treatment emphasizing a precision approach at the molecular and genomic level. Statistical modeling techniques aimed at leveraging broad scale in vitro, in vivo, and clinical data for precision drug treatment has become an active area of research. As a rapidly developing discipline at the crossroads of medicine, computer science, and mathematics, techniques ranging from accepted to those on the cutting edge of artificial intelligence have been utilized. Given the diversity and complexity of these techniques a systematic understanding of fundamental modeling principles is essential to contextualize influential factors to better understand results and develop new approaches.MethodsUsing data available from the Genomics of Drug Sensitivity in Cancer (GDSC) and the NCI60 we explore principle components regression, linear and non-linear support vector regression, and artificial neural networks in combination with different implementations of correlation based feature selection (CBF) on the prediction of drug response for several cytotoxic chemotherapeutic agents.ResultsOur results indicate that the regression method and features used have marginal effects on Spearman correlation between the predicted and measured values as well as prediction error. Detailed analysis of these results reveal that the bulk relationship between tissue of origin and drug response is a major driving factor in model performance.ConclusionThese results display one of the challenges in building predictive models for drug response in pan-cancer models. Mainly, that bulk genotypic traits where the signal to noise ratio is high is the dominant behavior captured in these models. This suggests that improved techniques of feature selection that can discriminate individual cell response from histotype response will yield more successful pan-cancer models.

Nanoscale Bacteria-Enabled Autonomous Drug Delivery System (NanoBEADS) Enhances Intratumoral Transport of Nanomedicine.

Cancer drug delivery remains a formidable challenge due to systemic toxicity and inadequate extravascular transport of nanotherapeutics to cells distal from blood vessels. It is hypothesized that, in absence of an external driving force, the Salmonella enterica serovar Typhimurium could be exploited for autonomous targeted delivery of nanotherapeutics to currently unreachable sites. To test the hypothesis, a nanoscale bacteria‐enabled autonomous drug delivery system (NanoBEADS) is developed in which the functional capabilities of the tumor‐targeting S. Typhimurium VNP20009 are interfaced with poly(lactic‐co‐glycolic acid) nanoparticles. The impact of nanoparticle conjugation is evaluated on NanoBEADS' invasion of cancer cells and intratumoral transport in 3D tumor spheroids in vitro, and biodistribution in a mammary tumor model in vivo. It is found that intercellular (between cells) self‐replication and translocation are the dominant mechanisms of bacteria intratumoral penetration and that nanoparticle conjugation does not impede bacteria's intratumoral transport performance. Through the development of new transport metrics, it is demonstrated that NanoBEADS enhance nanoparticle retention and distribution in solid tumors by up to a remarkable 100‐fold without requiring any externally applied driving force or control input. Such autonomous biohybrid systems could unlock a powerful new paradigm in cancer treatment by improving the therapeutic index of chemotherapeutic drugs and minimizing systemic side effects.

An unsuspected complication with immune checkpoint blockade: a case report

BackgroundImmunotherapy treatment with immune-checkpoint blockade has become a new paradigm in cancer treatment. Despite its efficacy, it has also given rise to a new class of adverse events, immune-related adverse events, which may affect any organ, including the thyroid and the pituitary.Case presentationWe present a case of a 77-year-old Caucasian man with metastatic renal cell carcinoma on immunotherapy treatment who was admitted to our hospital with a severe persistent headache of sudden onset. He had been on corticosteroid therapy for 10 days for suspected immune-related thyroiditis. The patient had tachycardia and mild diarrhea, and his thyroid function tests were compatible with subclinical hyperthyroidism with a suppressed thyroid-stimulating hormone level of 0.01 μIU/ml (0.4–4.5), a raised free T4 level of 2.17 ng/dl (0.7–1.9), and a free T3 level of 4.66 pg/ml (2.27–5). Computed tomography and magnetic resonance imaging revealed an enlargement of the pituitary gland compatible with macroadenoma. In the face of a probable immune-related hypophysitis, high-dose corticosteroid treatment was started. A posterior hormonal evaluation revealed secondary hypothyroidism with a suppressed thyroid-stimulating hormone level of 0.11 μIU/ml (0.4–4.5) and low thyroid hormones, a normal free T4 level of 1.02 ng/dl (0.7–1.9), and a low free T3 level of 1.53 pg/ml (2.27–5). These new findings suggested central hypothyroidism possibly due to pituitary apoplexy as a complication of the macroadenoma. Therefore, levothyroxine substitution was started along with the previously started corticosteroid therapy. The patient’s headache and asthenia gradually resolved, and after a few days, he was released from the hospital with levothyroxine substitution and corticosteroid tapering.ConclusionsThis case emphasizes the importance of the differential diagnosis when dealing with patients on immune checkpoint inhibitors because other non-immune-related events may present. Our patient was finally diagnosed with immune-related hyperthyroidism and a concurrent pituitary macroadenoma. This case also highlights the importance of a prompt start of corticosteroid therapy once immune-related adverse events such as hypophysitis are suspected, because otherwise the outcome would be fatal.

Abstract 5728: DNA demethylating agents and interferons as modulators of Wnt/β-catenin signaling in colorectal cancer

Abstract p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 12.0px 'Times New Roman'} Colorectal cancer (CRC) is the second leading cause of death from cancer in men and the third in women in Canada. There is accumulating evidence suggesting that CRC is organized in a hierarchical manner, at the apex of which are the Cancer-Initiating Cells (CIC). Furthermore, results from xenograft models and human clinical trials indicate a selective enrichment of CICs in tumours that are resistant to therapy, suggesting that targeting CICs may represent a new paradigm in cancer treatment. We have previously reported the treatment with low dose of the DNA demethylating agent, 5-Aza-2'-deoxycytidine (5-AZA-CdR), can target CICs through the activation of the RIG1-MDA5 viral sensing sensing pathway, leading to an anti-viral response in cancer cells. Although it is now known that 5-AZA-CdR induces a state of “viral mimicry” in these cancer cells, the mechanism by which 5-AZA-CdR can specifically target CICs is not well understood. In this study, we propose a novel intersection between RIG1-MDA5-IRF7 and Wnt/β-catenin pathways. We found that treatment of CIC enriched cancer cells with both 5-AZA-CdR and type I and III interferons reduced canonical Wnt/β-catenin signalling. Furthermore, treatment with type I and III interferons showed a reduced CIC frequency in colorectal cancer cells in vitro. Mechanistically, we found that IRF7 and β-catenin interact in the nucleus, giving rise to a novel, non-canonical pathway that can modulate WNT signalling. These findings may explain a novel pathway by which 5-AZA-CdR can specifically target CIC enriched populations in colorectal cancer, and highlights the importance of innate immune pathways in epigenetic therapy. Citation Format: Ilias Ettayebi, Parinaz Mehdipour, Rajat Singhania, Ankur Chakravarthy, Tiago Medina, Shu Yi Shen, Charles Ishak, David Roulois, Daniel De Carvalho. DNA demethylating agents and interferons as modulators of Wnt/β-catenin signaling in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5728.

Abstract 715: Expression of tumor cell-associated immune checkpoint molecules in multiple human solid tumors

Abstract Cancer immunotherapy using monoclonal antibodies targeting inhibitory immune checkpoint molecules is being established as a new paradigm in cancer treatment. This crucial strategy involves inhibition of the interaction between immune checkpoint receptors, expressed on the immune cells, and their respective ligands, expressed on antigen-presenting cells. While this receptor-ligand concept is well accepted in infection immunology, there have been numerous reports on the expression of immune checkpoint molecules in tumor cells. In addition to well-characterized tumor-intrinsic expression of PD11 and PD-L12, many other checkpoint molecules are associated with tumor cells3, conferring important impact on tumor biology and clinical consequences. In this study, we evaluated in situ single-cell expression profiles of immune checkpoint molecules in the tumor microenvironment (TME) of archived FFPE tissues from multiple solid cancers, including breast, kidney, ovarian, stomach, colorectal, and non-small cell lung cancer. Applying the RNAscope® assay, a highly specific and sensitive in situ hybridization (ISH) technology, tissue microarrays consisting of 20-35 FFPE tumors per each cancer type were stained for key therapeutic targets including PD1, PD-L1, TIM3, and LAG3. We observed that, in addition to PD-L1, all evaluated immune checkpoint molecules were expressed in the tumor cells of multiple tumor types though at a lower level than in immune cells. While there was notable heterogeneity of expression in different tumor regions in some samples, the single-cell expression pattern of each checkpoint molecule across all tumor cells in each region suggested a potential tumor-intrinsic mechanism for the expression of these molecules. In selected lung and ovarian cancers, fluorescence multiplex ISH-IHC staining confirmed that multiple different checkpoint molecules were co-expressed together in individual tumor cells. These findings add to the growing evidence that immune checkpoint genes can be expressed in tumor cells in addition to immune cells, which will be missed by gene expression analysis techniques using bulk tissue. The RNAscope® ISH platform is especially well suited for this type of analysis since it allows for assessing cell type-specific expression of any gene within the native tissue architecture even when it is expressed at very low levels. The approach illustrated in this study may help to better understand the anti-tumor activities of immune checkpoint inhibitors and provide new insights into better predictive biomarkers and therapeutic strategies.

Tumor cell-intrinsic expression of immune checkpoint molecules revealed by RNA in situ hybridization

Background: Cancer immunotherapy targeting the PD-1/PD-L1 pathway has been established as a new paradigm in cancer treatment with long lasting clinical benefits in multiple types of cancers. Despite the clinical efficacy observed in some patients, the majority of cancer patients do not respond to PD‐1/PD‐L1 blockades and the development of predictive biomarkers is in critical need to effectively stratify patients for PD-1 blockades. In addition to primary resistance, some responders relapse after a period of response and understanding the mechanism behind both types of resistance are critical to overcome current immunotherapy challenges. Methods: In this study, we evaluated in situ single cell expression profiles of therapeutic checkpoint targets in the tumor microenvironment (TME) of archived FFPE tissues from two cancer types; 30 cases of non-small cell lung cancer (NSCLC) and 30 cases of ovarian cancer. Applying RNAscope® in-situ hybridization (ISH) assays, specific checkpoint target molecules are visualized in highly specific and sensitive manner in individual cells within tissue morphological context. Results: We report that, in addition to PD-L1, multiple other immune checkpoint molecules, including TIM3, LAG3, PD-L2, and GITR (TNFRSF18) are expressed in the tumor cells in subsets of tumors from both NSCLC and ovarian cancer. Furthermore, we observed PD1 is often co-expressed with other therapeutic checkpoint targets, including LAG3, TIM3, and TIGIT, in the same immune cells infiltrated into the microenvironment. Conclusions: These findings demonstrate the utility of the RNAscope® ISH platform in helping to understanding cancer’s mechanism to evade host immune surveillance and ultimately developing resistance against checkpoint blockades. Legal entity responsible for the study: Advanced Cell Diagnostics Inc., 7707 Gateway Blvd., Newark, CA 94560, USA Funding: NA Disclosure: All authors have declared no conflicts of interest.

Abstract 3763: UCART22: allogenic adoptive immunotherapy of leukemia by targeting CD22 with CAR T-cells

Abstract Adoptive immunotherapy using autologous T-cells endowed with chimeric antigen receptors (CARs) has given rise to long-term durable remissions and remarkable objective response rates in patients with refractory leukemia, raising hopes that a wider application of CAR technology may lead to a new paradigm in cancer treatment. However, a limitation of the current autologous approach is that CAR T-cells must be manufactured on a "per patient basis". To overcome this limitation, we have developed a standardized platform for manufacturing T-cells from third-party healthy donors to generate allogeneic "off-the-shelf" engineered CAR+ T-cell-based frozen products. Our allogenic platform utilizes the Transcription Activator-Like Effector Nuclease (TALEN) gene editing technology to inactivate the TCRα constant (TRAC) gene, significantly reducing the potential for T-cells bearing alloreactive TCR’s to mediate Graft-versus-Host Disease (GvHD). We have previously demonstrated the precise and efficient disruption of the the TRAC gene by gene editing, yielding up to 85% of TCRαβ-negative cells. This allows the production of TCRαβ-deficient T-cells that no longer mediate alloreactivity in a xeno-GvHD mouse model. In the clinic, the proof of concept of the applicability of our allogeneic platform was achieved with early compassionate use for patients treated with UCART19, an allogeneic engineered CAR T-cell product directed against CD19. UCART19 clinical trials are currently ongoing. Here, we have developed T-cells targeting CD22 which is expressed on tumor cells from the majority of patients with B-cells leukemia. In a first step, we have screened multiple antigen recognition domains in the context of different CAR architectures to identify effective CAR candidates displaying activity against cells expressing variable levels of the CD22 antigen. As a safety feature, T-cells are engineered to co-express a depletion gene, rendering them sensitive to the monoclonal antibody rituximab. Several constructs of depletion genes have been evaluated in the context of the CD22 CAR. In addition, experiments in an orthotopic ALL mouse model using UCART22 cells demonstrated important anti-tumor activity in vivo. The ability to carry out large scale manufacturing of allogeneic, non-alloreactive CD22 specific T-cells from a single healthy donor can offer the possibility of an off-the-shelf treatment that would be immediately available for administration to a large number of leukemic patients. UCART22 could also offer an alternative to patients who may relapse with CD19-negative tumors after CD19 CAR T-cell treatment. Citation Format: Agnès Gouble, Cécile Schiffer-Mannioui, Severine Thomas, Anne-Sophie Gautron, Laurent Poirot, Julianne Smith. UCART22: allogenic adoptive immunotherapy of leukemia by targeting CD22 with CAR T-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3763. doi:10.1158/1538-7445.AM2017-3763

The Need for Identification Cards in Immuno-Oncology .

Oncology nurses have worked within the three pillars of cancer care-surgery, chemotherapy, and radiation therapy -for decades. Now, immuno-oncologic (I-O) therapy agents are a new paradigm in cancer treatment. On March 28, 2017, an I-O think tank was held at the Oncology Nursing Society (ONS) in Pittsburgh, PA. This think tank was organized in response to the rapid changes in cancer treatment with the advent of I-O therapies. The think tank appraised the needs of patients receiving these agents and the oncology nurses and healthcare providers caring for them. Seventeen nurse experts from around the country joined seven ONS staff members to discuss the challenges faced by patients receiving these therapies and the nurses who administer the treatments and coordinate the care. .

Construction and comparison of different nanocarriers for co-delivery of cisplatin and curcumin: A synergistic combination nanotherapy for cervical cancer.

Co-delivery of two or more drugs into the same cancer cells or tissues in the same nanocarriers provides a new paradigm in cancer treatment. In this study, two kinds of nanocarriers: lipid-polymer hybrid nanoparticles (LPNs) and polymeric nanoparticles (PNPs) were constructed and compared for co-delivery of cisplatin (DDP) and curcumin (CUR). DDP and CUR loaded LPNs (D/C/LPNs) and PNPs (D/C/PNPs) were prepared. Two kinds of nanocarriers were characterized in terms of particle size, zeta potential, drug encapsulation efficiency (EE), and drug release. Their in vitro cytotoxicity and in vivo anti-tumor efficacy was studied on human cervix adenocarcinoma cell line (HeLa cells) and mice bearing cervical cancer model. Compared with D/C/PNPs, D/C/LPNs showed significantly higher cytotoxicity in vitro. D/C/LPNs also displayed the best antitumor activity than other formulations tested in vivo. The results demonstrated that LPNs could improve the anticancer efficacy of drugs to higher levels than PNPs and free drugs, thus could serve as an effective drug system for targeted and synergistic co-delivery nanomedicine for cervical cancer chemotherapy.

Abstract 2748: Targeting DNA repair deficient cancers with the cell-penetrating autoantibody 3E10

Abstract There is growing evidence that many solid tumors have defective DNA repair pathways and cell cycle checkpoints. This has spurred development of therapeutics to specifically target cancer cells with defective DNA repair, but few have been successful outright due to problems such as general toxicity. One novel anti-cancer agent of interest to us is 3E10, a unique cell-penetrating, anti-DNA autoantibody. This molecule is non-toxic to normal cells on its own but has deleterious effects on cells with DNA repair deficiencies. Specifically, 3E10 has been shown to be synthetically lethal in BRCA2-deficient human cancer cells, to be synergistic with radiation and other commonly used DNA damaging therapies, and to reduce the efficiency of Rad51 mediated strand exchange and thus homologous recombination (HR). The goal of this project is to better understand how 3E10 can be used as a new paradigm for cancer treatment. New evidence suggests that 3E10 inhibits Exo1 exonuclease activity. Additionally, preliminary data shows that 3E10 physically interacts with Rad51, ultimately inhibiting Rad51 nuclear localization and foci formation. Additionally, a point mutation in 3E10 that confers increased DNA binding mediates increased cell death in BRCA2-deficient human cancer cells. Furthermore, studies investigating the potential therapeutic effect of 3E10 in patient derived primary melanoma cell lines show that 3E10 is synthetically lethal with PTEN deficiency. This data suggests that 3E10 inhibits HR by binding to DNA at double strand breaks and occupying sites normally processed by core repair machinery, as well as by physically interacting with Rad51. Overall, 3E10 holds great promise as a novel therapeutic agent for various cancers, including PTEN deficient melanomas. Reference: Hansen, J. et al. Targeting cancer with a lupus autoantibody. Science Translational Medicine 4, (2012) Citation Format: Audrey L. Turchick, Peter M. Glazer. Targeting DNA repair deficient cancers with the cell-penetrating autoantibody 3E10. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2748.

Abstract A080: Targeted genome modifications for improved adoptive immunotherapy

Abstract Chimeric antigen receptor (CAR)-redirected T-cells have given rise to long-term durable remissions and remarkable objective response rates in patients with refractory leukemia, raising hopes that a wider application of CAR technology may lead to a new paradigm in cancer treatment. Similarly, transcription activator-like effector nuclease (TALEN™)-mediated gene editing has emerged as powerful strategy to introduce targeted mutations and holds great promise in therapeutics and offers multiple opportunities to improve CAR T-cell therapies. The knockout of the TCR alpha gene eliminates TCR expression and abrogates the donor T-cell's potential for graft-versus-host disease (GvHD) while maintaining a potent anti-tumoral activity. Thus it is possible to manufacture T-cells from third-party healthy donors to generate allogeneic “off-the-shelf” engineered CAR T-cells. Disruption of CD52 or deoxycytidine kinase genes may be a useful approach to makes T-cells compatible with concurrent oncology treatments such as alemtuzumab or Fludarabine while the bypass of key immune checkpoint regulators such PD1/PDL1 by inactivation of T-cells receptors would potentiate the antitumor T-cell response by impairing the interaction of the inhibitory receptor PD-1 on T cells with PD-L1 expressed on tumor cells. Here, we will present in vitro and in vivo proof of concepts demonstrating the potential of TALEN™-mediated gene editing for adoptive T-cell therapy. Citation Format: Julien Valton, Anne-Sophie Gautron, Alexandre Juillerat, Philippe Duchateau, Laurent Poirot. Targeted genome modifications for improved adoptive immunotherapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A080.

Abstract B016: Allogenic CAR T-cells for adoptive immunotherapy

Abstract Chimeric antigen receptor (CAR)-redirected T-cells have given rise to long-term durable remissions and remarkable objective response rates in patients with refractory leukemia, raising hopes that a wider application of CAR technology may lead to a new paradigm in cancer treatment. A limitation of the current autologous approach is that CAR T-cells must be manufactured on a “per patient basis”. We have developed a standardized platform for manufacturing T-cells from third-party healthy donors to generate allogeneic “off-the-shelf” engineered CAR+ T-cell–based frozen products. This platform utilizes Transcription Activator-Like Effector Nuclease (TALEN) gene editing technology to inactivate the TCRα constant (TRAC) gene, eliminating the potential for T-cells bearing alloreactive TCR's to mediate Graft versus Host Disease (GvHD). We have previously demonstrated that editing of the TRAC gene can be achieved at high frequencies, obtaining up to 80% of TCRβα negative cells. This allows us to efficiently produce TCR-deficient T-cells that have been shown to no longer mediate alloreactivity in a xeno-GvHD mouse model. We have adapted this allogeneic platform to the production of T cells targeting CD123, the transmembrane alpha chain of the interleukin-3 receptor, which is expressed in tumor cells of patients with Acute Myeloid Leukemia as well as CS1 a glycoprotein highly expressed on tumor cells of patients with multiple myeloma. We will present both in vitro and in vivo data demonstrating specific anti-tumor activity of engineered CAR T cells against cell lines expressing the targeted antigen. Citation Format: Roman Galetto, Celine Lebuhotel, Isabelle Chion-Sotinel, Patricia Francon, Agnes Gouble, Julianne Smith. Allogenic CAR T-cells for adoptive immunotherapy. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B016.

Abstract IA04: Partnership of radiotherapy and immunotherapy: A new paradigm in cancer treatment

Abstract The success of immune checkpoint inhibitors in inducing tumor regression has demonstrated that specific inhibitory pathways are dominant rate-limiting steps in a significant number of patients with melanoma and other advanced cancers. However, in the majority of patients tumor rejection is hindered by multiple immunosuppressive mechanisms present in the tumor microenvironment. Obstacles to immune-mediated tumor control can be present at both the priming and effector phase of the anti-tumor response, and include defective function and activation of antigen-presenting cells, defective T cell recruitment and infiltration of tumors, and defective recognition and killing of cancer cells by T cells. Ionizing radiation therapy (RT) applied locally to a tumor at therapeutic doses has multiple effects that can potentially overcome each of these obstacles, and we have shown that RT is synergistic with immunotherapy. In pre-clinical models RT converted tumors unresponsive to anti-CTLA-4 mAb into responsive ones, achieving rejection of the irradiated tumor and non-irradiated metastases (abscopal effect) and improved survival [1, 2]. At the effector phase, RT enhanced recruitment of activated T cells to the tumor by induction of chemokines [3], and enhanced immune synapse formation between CD8 T and tumor cells by induction of NKG2D ligands [4]. At the priming phase, we have found that RT has the potential to activate T cell responses to endogenous tumor antigens. However, this potential is not achieved without blocking dominant immunosuppressive pathways. Priming by RT is regulated by positive signals generated by RT and negative signals pre-existing in the tumor microenvironment and/or induced by RT. Positive signals include the induction of an immunogenic tumor cell death and of type I interferon, both of which show some dependency on the dose and/or fractionation of RT. Among negative signals that are exacerbated by RT we have identified TGFβ as a master regulator of the ability of RT to prime CD8 T cell responses to multiple epitopes derived from endogenous tumor antigens, including a transcription factor that regulates epithelial to mesenchymal transition and metastasis. Importantly, priming of CD8 T cells to endogenous tumor antigens is also achieved by blocking immune checkpoint receptors CTLA-4 or PD-1 in combination with RT while each of these treatments used alone is ineffective. Importantly, blocking both TGFβ and PD-1 further improves RT-elicited priming of tumor-specific CD8 T cells. Finally, to obtain a more comprehensive understanding of the ability of RT to cause antigenic spread and elicit T cells to a broad range of tumor antigens possibly including mutated neo-antigens, we are studying the TCR repertoire of intratumoral T cells in mice treated with RT and anti-CTLA-4. Initial results show distinct contributions of RT and anti-CTLA-4 to increasing T cell number and clonality, and changes in clonal representation that are unique to the combination. Overall, data support the concept that RT can be used as a mean to generate an in situ individualized vaccine. We are currently exploring this hypothesis in clinical trials testing the combination of RT and inhibitors of dominant immunosuppressive pathways. Supported by Department of Defense Breast Cancer Research Program, Breast Cancer Research Foundation, Breast Cancer Alliance, The Chemotherapy Foundation.

Molecular mechanisms of Nrf2 regulation and how these influence chemical modulation for disease intervention.

Nrf2 (nuclear factor erytheroid-derived-2-like 2) transcriptional programmes are activated by a variety of cellular stress conditions to maintain cellular homoeostasis. Under non-stress conditions, Nrf2 is under tight regulation by the ubiquitin proteasome system (UPS). Detailed mechanistic investigations have shown the Kelch-like ECH-associated protein 1 (Keap1)–cullin3 (Cul3)–ring-box1 (Rbx1) E3-ligase to be the primary Nrf2 regulatory system. Recently, both beta-transducin repeat-containing E3 ubiquitin protein ligase (β-TrCP) and E3 ubiquitin-protein ligase synoviolin (Hrd1) have been identified as novel E3 ubiquitin ligases that negatively regulate Nrf2 through Keap1-independent mechanisms. In addition to UPS-mediated regulation of Nrf2, investigations have revealed a cross-talk between Nrf2 and the autophagic pathway resulting in activation of Nrf2 in a non-canonical manner. In addition to regulation at the protein level, Nrf2 was recently shown to be regulated at the transcriptional level by oncogenic K-rat sarcoma (Ras). A consequence of these differential regulatory mechanisms is the dual role of Nrf2 in cancer: the canonical, protective role and the non-canonical ‘dark-side’ of Nrf2. Based on the protective role of Nrf2, a vast effort has been dedicated towards identifying novel chemical inducers of Nrf2 for the purpose of chemoprevention. On the other hand, upon malignant transformation, some cancer cells have a constitutively high level of Nrf2 offering a growth advantage, as well as rendering cancer cells resistant to chemotherapeutics. This discovery has led to a new paradigm in cancer treatment; the initially counterintuitive use of Nrf2 inhibitors as adjuvants in chemotherapy. Herein, we will discuss the mechanisms of Nrf2 regulation and how this detailed molecular understanding can be leveraged to develop Nrf2 modulators to prevent diseases, mitigate disease progression or overcome chemoresistance.

Curcumin-guided nanotherapy: a lipid-based nanomedicine for targeted drug delivery in breast cancer therapy

Background: Delivery of anti-cancer drugs into the cancer cells or tissues by multifunctional nanocarriers may provide a new paradigm in cancer treatment. In this study, folate (FA) decorated nanostructured lipid carriers (NLCs) were constructed as nanomedicine for the delivery of curcumin (CUR).Methods: CUR-loaded NLCs (CUR-NLCs) were prepared. FA containing polyethylene glycol (PEG)-distearoylphosphatidylethanolamine (DSPE) (FA-PEG-DSPE) was synthesized and used for the decoration of CUR-NLCs. Their particle size, zeta potential, and drug encapsulation efficiency (EE) were evaluated. In vitro cytotoxicity study FA decorated CUR-NLCs (FA-CUR-NLCs) was tested in MCF-7 human breast cancer cells (MCF-7 cells). In vivo anti-tumor efficacies of the carriers were evaluated on mice bearing breast cancer model.Results: The optimum FA-CUR-NLCs formulations with the particle size of 127 nm and with a +13 mV surface charge. The growth of MCF-7 cells in vitro was obviously inhibited. FA-CUR-NLCs also displayed the best anti-tumor activity than other formulations in vivo.Conclusion: The results demonstrated that FA-CUR-NLCs were efficient in selective delivery to cancer cells over-expressing FA receptors (FRs). Also FA-CUR-NLCs transfer CUR to the breast cancer cells, enhance the anti-tumor capacity. Thus, FA-CUR-NLCs could prove to be a superior nanomedicine to achieve tumor therapeutic efficacy.

Nanostructured lipid carriers based temozolomide and gene co-encapsulated nanomedicine for gliomatosis cerebri combination therapy

Background: Co-delivery of gene and anticancer drug into the same cancer cells or tissues by multifunctional nanocarriers may provide a new paradigm in cancer treatment. In this study, nanostructured lipid carriers (NLCs) were constructed as multifunctional nanomedicine for co-delivery of enhanced green fluorescence protein plasmid (DNA) and temozolomide (TMZ).Methods: TMZ- and DNA-loaded NLCs (TMZ/DNA-NLCs) were prepared. Their particle size, zeta potential, gene-loading capacity (GL) and drug encapsulation efficiency (EE) were evaluated. In vitro cytotoxicity study TMZ/DNA-NLCs was tested in U87 malignant glioma cells (U87 MG cells). In vivo gene transfection and anti-tumor efficacy of the carriers were evaluated on mice bearing malignant glioma model.Results: The optimum TMZ/DNA-NLCs formulations with the particle size of 179 nm and with a +23 mV surface charge; got 91% of GL and 83% of EE. The growth of U87 MG cells in vitro was obviously inhibited. TMZ/DNA-NLCs also displayed the highest gene transfection efficiency and the best antitumor activity than other formulations in vivo.Conclusion: The results demonstrated that TMZ/DNA-NLCs were efficient in selective delivery to malignant glioma cells. Also TMZ/DNA-NLCs transfer both drug and gene to the gliomatosis cerebri, enhance the antitumor capacity and gene transfection efficacy. Thus, TMZ/DNA-NLCs could prove to be a superior co-delivery nanomedicine to achieve therapeutic efficacy and this report could be a new promising strategy for treatment in malignant gliomatosis cerebri.

336. Targeted Genome Modifications for Improved Adoptive Immunotherapy

Chimeric antigen receptor (CAR)-redirected T-cells have given rise to long-term durable remissions and remarkable objective response rates in patients with refractory leukemia, raising hopes that a wider application of CAR technology may lead to a new paradigm in cancer treatment. Similarly, transcription activator-like effector nuclease (TALEN™)-mediated gene editing has emerged as powerful strategy to introduce targeted mutations and holds great promise in therapeutics and offers multiple opportunities to improve CAR T-cell therapies. The knockout of the TCR alpha gene eliminates TCR expression and abrogates the donor T-cell's potential for graft-versus-host disease (GvHD) while maintaining a potent anti-tumoral activity. Thus it is possible to manufacture T-cells from third-party healthy donors to generate allogeneic “off-the-shelf” engineered CAR T-cells. Disruption of CD52 or deoxycytidine kinase genes may be a useful approach to makes T-cells compatible with concurrent oncology treatments such as alemtuzumab or Fludarabine while the bypass of key immune checkpoint regulators such PD1/PDL1 by inactivation of T-cells receptors would potentiate the antitumor T-cell response by impairing the interaction of the inhibitory receptor PD-1 on T cells with PD-L1 expressed on tumor cells. Here, we will present in vitro and in vivo proof of concepts demonstrating the potential of TALEN™-mediated gene editing for adoptive T-cell therapy.

419. Allogenic CAR T-Cells Targeting CD123 for Adoptive Immunotherapy of Acute Myeloid Leukemia (AML)

Chimeric antigen receptor (CAR)-redirected T-cells have given rise to long-term durable remissions and remarkable objective response rates in patients with refractory leukemia, raising hopes that a wider application of CAR technology may lead to a new paradigm in cancer treatment. A limitation of the current autologous approach is that CAR T-cells must be manufactured on a “per patient basis”. We have developed a standardized platform for manufacturing T-cells from third-party healthy donors to generate allogeneic “off-the-shelf” engineered CAR+ T-cell–based frozen products. This platform utilizes Transcription Activator-Like Effector Nuclease (TALEN) gene editing technology to inactivate the TCRα constant (TRAC) gene, eliminating the potential for T-cells bearing alloreactive TCR's to mediate Graft versus Host Disease (GvHD). We have previously demonstrated that editing of the TRAC gene can be achieved at high frequencies, obtaining up to 80% of TCRα negative cells. This allows us to efficiently produce TCR-deficient T-cells that have been shown to no longer mediate alloreactivity in a xeno-GvHD mouse model. In addition to CAR expression, our T-cells are engineered to co-express the RQR8 gene as a safety feature, with the aim of rendering them sensitive to the monoclonal antibody rituximab.In this work we present the adaptation of this allogeneic platform to the production of T cells targeting CD123, the transmembrane alpha chain of the interleukin-3 receptor, which is expressed in tumor cells from the majority of patients with Acute Myeloid Leukemia (AML). To identify an effective CAR targeting CD123, we have screened multiple antigen recognition domains in the context of several different CAR architectures to identify candidates displaying activity against cell lines expressing variable levels of the CD123 antigen. Furthermore, experiments in an AML mouse model using anti-CD123 CAR T cells demonstrate important anti-tumor activity in vivo. The ability to carry out large scale manufacturing of allogeneic, non alloreactive CD123 specific T cells from a single healthy donor will thus offer the possibility of an off-the-shelf treatment that would be immediately available for administration to a large number of AML patients.

MiRNA therapy targeting cancer stem cells: a new paradigm for cancer treatment and prevention of tumor recurrence.

Cancer stem cells (CSCs) are a small subpopulation of cells within tumors that retain the properties of self-renewal and tumorigenicity in vivo. Although CSCs have been reported in multiple cancers, the regulation of CSCs has not been described at the molecular level. miRNAs are endogenous small noncoding RNAs that post-transcriptionally regulate the expression of their target genes via RNA interference and are involved in almost all cellular processes. Since aberrant miRNA expression occurs in CSCs, such dysregulated miRNAs may be promising therapeutic targets. In this review, we summarize the current knowledge regarding miRNAs that regulate CSC properties and discuss an in vivo delivery system for synthetic miRNA mimics and miRNA inhibitors for the development of innovative miRNA therapy against CSCs.