Citations

Abstract Despite its efficacy, chimeric antigen receptor T-cell therapy (CART) is limited by the development of cytokine release syndrome (CRS) and neurotoxicity (NT). While CRS is related to extreme elevation of cytokines and massive T cell expansion, the exact mechanisms for NT have not yet been elucidated. Preliminary studies suggest that NT might be mediated by myeloid cells that cross the blood brain barrier. This is supported by correlative analysis from CART19 pivotal trials where CD14+ cell numbers were increased in the cerebrospinal fluid of patients that developed severe NT (Locke et al, ASH 2017). Therefore, we aimed to investigate the role of GM-CSF neutralization in preventing CRS and NT after CART cell therapy via monocyte control. First, we investigated the effect of GM-CSF blockade on CART cell effector functions. Here, we used the human GM-CSF neutralizing antibody (lenzilumab, Humanigen, Burlingame, California) that has been shown to be safe in phase II clinical trials. Lenzilumab (10 ug/kg) neutralizes GM-CSF when CART19 cells are stimulated with the CD19+ Luciferase+ acute lymphoblastic leukemia (ALL) cell line NALM6, but does not impair CART cell function in vitro. We have found that malignancy associated macrophages reduce CART proliferation. GM-CSF neutralization with lenzilumab results in enhanced CART cell antigen specific proliferation in the presence of monocytes. To confirm this in vivo, NOD-SCID-g-/- mice were engrafted with high disease burdens of NALM6 and treated with low doses of CART19 or control T cells (to induce tumor relapse), in combination with lenzilumab or isotype control antibody. The combination of CART19 and lenzilumab resulted in significant anti-tumor activity and overall survival benefit compared to control T cells (Fig 1A), similar to mice treated with CART19 combined with isotype control antibody, indicating that GM-CSF neutralization does not impair CART cell activity in vivo. This anti-tumor activity was validated in an ALL patient derived xenograft model. Next, we explored the impact of GM-CSF neutralization on CART cell related toxicities in a novel patient derived xenograft model. Here, NOD-SCID-g-/- mice were engrafted with leukemic blasts (1-3x106 cells) derived from patients with high risk ALL. Mice were then treated with high doses of CART19 cells (2-5x106 intravenously). Five days after CART19 treatment, mice began to develop progressive motor weakness, hunched bodies, and weight loss that correlated with massive elevation of circulating human cytokine levels. Magnetic Resonance Imaging (MRI) of the brain during this syndrome showed diffuse enhancement and edema, associated with central nervous system (CNS) infiltration of CART cells and murine activated myeloid cells. This is similar to what has been reported in CART19 clinical trials in patients with severe NT. The combination of CART19, lenzilumab (to neutralize human GM-CSF) and murine GM-CSF blocking antibody (to neutralize mouse GM-CSF) resulted in prevention of weight loss (Fig 1B), decrease in critical myeloid cytokines (Fig 1C-D), reduction of cerebral edema (Fig 1E), enhanced leukemic disease control in the brain (Fig 1F), and reduction in brain macrophages (Fig 1G). Finally, we hypothesized that disrupting GM-CSF through CRISPR/Cas9 gene editing during the process of CART cell manufacturing would result in functional CART cells with reduced secretion of GM-CSF. We designed guide RNA targeting exon 3 of the GM-CSF gene and generated GM-CSFk/o CART19 cells. Our preliminary data suggest that these CARTs produce significantly less GM-CSF upon activation but continue to exhibit similar production of other cytokines and exhibit normal effector functions in vitro (Fig 1H). Using the NALM6 high tumor burden relapse xenograft model as described above, GM-CSFk/o CART19 cells resulted in slightly enhanced disease control compared to CART19 cells (Fig 1I). Thus, modulating myeloid cell behavior through GM-CSF blockade can help control CART mediated toxicities and may reduce their immunosuppressive features to improve leukemic control. These studies illuminate a novel approach to abrogate NT and CRS through GM-CSF neutralization that also potentially enhances CART cell functions. Based on these results, we have designed a phase II clinical trial using lenzilumab as a modality to prevent CART related toxicities in patients with diffuse large B cell lymphoma. Disclosures Ahmed: Humanigen: Employment. Sahmoud:Humanigen: Employment. Durrant:Humanigen: Employment. Russell:Vyriad: Equity Ownership. Kay:Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding. Kenderian:Novartis: Patents & Royalties; Tolero Pharmaceuticals: Research Funding; Humanigen: Research Funding.

Abstract Chimeric antigen receptor T (CAR-T) cell therapy is a new pillar in cancer therapeutics; however, its application is limited by the associated toxicities. These include cytokine release syndrome (CRS) and neurotoxicity. Although the IL-6R antagonist tocilizumab is approved for treatment of CRS, there is no approved treatment of neurotoxicity associated with CD19-targeted CAR-T (CART19) cell therapy. Recent data suggest that monocytes and macrophages contribute to the development of CRS and neurotoxicity after CAR-T cell therapy. Therefore, we investigated neutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) as a potential strategy to manage CART19 cell–associated toxicities. In this study, we show that GM-CSF neutralization with lenzilumab does not inhibit CART19 cell function in vitro or in vivo. Moreover, CART19 cell proliferation was enhanced and durable control of leukemic disease was maintained better in patient-derived xenografts after GM-CSF neutralization with lenzilumab. In a patient acute lymphoblastic leukemia xenograft model of CRS and neuroinflammation (NI), GM-CSF neutralization resulted in a reduction of myeloid and T cell infiltration in the central nervous system and a significant reduction in NI and prevention of CRS. Finally, we generated GM-CSF–deficient CART19 cells through CRISPR/Cas9 disruption of GM-CSF during CAR-T cell manufacturing. These GM-CSFk/o CAR-T cells maintained normal functions and had enhanced antitumor activity in vivo, as well as improved overall survival, compared with CART19 cells. Together, these studies illuminate a novel approach to abrogate NI and CRS through GM-CSF neutralization, which may potentially enhance CAR-T cell function. Phase 2 studies with lenzilumab in combination with CART19 cell therapy are planned.

Abstract Controversy exists surrounding whether heterogeneous disruption of the blood–brain barrier (BBB), as seen in glioblastoma (GBM), leads to adequate drug delivery sufficient for efficacy in GBM. This question is especially important when using potent, targeted agents that have a poor penetration across an intact BBB. Efficacy of the murine double minute-2 (MDM2) inhibitor SAR405838 was tested in patient-derived xenograft (PDX) models of GBM. In vitro efficacy of SAR405838 was evaluated in PDX models with varying MDM2 expression and those with high (GBM108) and low (GBM102) expression were evaluated for flank and orthotopic efficacy. BBB permeability, evaluated using TexasRed-3 kDa dextran, was significantly increased in GBM108 through VEGFA overexpression. Drug delivery, MRI, and orthotopic survival were compared between BBB-intact (GBM108-vector) and BBB-disrupted (GBM108-VEGFA) models. MDM2-amplified PDX lines with high MDM2 expression were sensitive to SAR405838 in comparison with MDM2 control lines in both in vitro and heterotopic models. In contrast with profound efficacy observed in flank xenografts, SAR405838 was ineffective in orthotopic tumors. Although both GBM108-vector and GBM108-VEGFA readily imaged on MRI following gadolinium contrast administration, GBM108-VEGFA tumors had a significantly enhanced drug and gadolinium accumulation, as determined by MALDI-MSI. Enhanced drug delivery in GBM108-VEGFA translated into a marked improvement in orthotopic efficacy. This study clearly shows that limited drug distribution across a partially intact BBB may limit the efficacy of targeted agents in GBM. Brain penetration of targeted agents is a critical consideration in any precision medicine strategy for GBM. Mol Cancer Ther; 17(9); 1893–901. ©2018 AACR.

ABSTRACTHuman cerebral malaria (HCM) is a serious complication ofPlasmodium falciparuminfection. The most severe outcomes for patients include coma, permanent neurological deficits, and death. Recently, a large-scale magnetic resonance imaging (MRI) study in humans identified brain swelling as the most prominent predictor of fatal HCM. Therefore, in this study, we sought to define the mechanism controlling brain edema through the use of the murine experimental cerebral malaria (ECM) model. Specifically, we investigated the ability of CD8 T cells to initiate brain edema during ECM. We determined that areas of blood-brain barrier (BBB) permeability colocalized with a reduction of the cerebral endothelial cell tight-junction proteins claudin-5 and occludin. Furthermore, through small-animal MRI, we analyzed edema and vascular leakage. Using gadolinium-enhanced T1-weighted MRI, we determined that vascular permeability is not homogeneous but rather confined to specific regions of the brain. Our findings show that BBB permeability was localized within the brainstem, olfactory bulb, and lateral ventricle. Concurrently with the initiation of vascular permeability, T2-weighted MRI revealed edema and brain swelling. Importantly, ablation of the cytolytic effector molecule perforin fully protected against vascular permeability and edema. Furthermore, perforin production specifically by CD8 T cells was required to cause fatal edema during ECM. We propose that CD8 T cells initiate BBB breakdown through perforin-mediated disruption of tight junctions. In turn, leakage from the vasculature into the parenchyma causes brain swelling and edema. This results in a breakdown of homeostatic maintenance that likely contributes to ECM pathology.

Background— A portion of sudden cardiac deaths can be attributed to structural heart diseases, such as hypertrophic cardiomyopathy (HCM) or cardiac channelopathies such as long-QT syndrome (LQTS); however, the underlying molecular mechanisms are distinct. Here, we identify a novel CACNA1C missense mutation with mixed loss-of-function/gain-of-function responsible for a complex phenotype of LQTS, HCM, sudden cardiac death, and congenital heart defects. Methods and Results— Whole exome sequencing in combination with Ingenuity variant analysis was completed on 3 affected individuals and 1 unaffected individual from a large pedigree with concomitant LQTS, HCM, and congenital heart defects and identified a novel CACNA1C mutation, p.Arg518Cys, as the most likely candidate mutation. Mutational analysis of exon 12 of CACNA1C was completed on 5 additional patients with a similar phenotype of LQTS plus a personal or family history of HCM-like phenotypes and identified 2 additional pedigrees with mutations at the same position, p.Arg518Cys/His. Whole cell patch clamp technique was used to assess the electrophysiological effects of the identified mutations in Ca V 1.2 and revealed a complex phenotype, including loss of current density and inactivation in combination with increased window and late current. Conclusions— Through whole exome sequencing and expanded cohort screening, we identified a novel genetic substrate p.Arg518Cys/His-CACNA1C, in patients with a complex phenotype including LQTS, HCM, and congenital heart defects annotated as cardiac-only Timothy syndrome. Our electrophysiological studies, identification of mutations at the same amino acid position in multiple pedigrees, and cosegregation with disease in these pedigrees provide evidence that p.Arg518Cys/His is the pathogenic substrate for the observed phenotype.

Abstract Blood–brain barrier (BBB) disruption is a common feature of numerous neurologic disorders. A fundamental question in these diseases is the extent inflammatory immune cells contribute to CNS vascular permeability. We have previously shown that CD8 T cells play a critical role in initiating BBB disruption in the peptide-induced fatal syndrome model developed by our laboratory. However, myelomonocytic cells such as neutrophils have also been implicated in promoting CNS vascular permeability and functional deficit in murine models of neuroinflammatory disease. For this reason, we evaluated neutrophil depletion in a murine model of CD8 T cell-initiated BBB disruption by employing traditionally used anti-granulocyte receptor-1 mAb RB6-8C5 and Ly-6G–specific mAb 1A8. We report that CNS-infiltrating antiviral CD8 T cells express high levels of granulocyte receptor-1 protein and are depleted by treatment with RB6-8C5. Mice treated with RB6-8C5, but not 1A8, display: 1) intact BBB tight junction proteins; 2) reduced CNS vascular permeability visible by gadolinium-enhanced T1-weighted magnetic resonance imaging; and 3) preservation of motor function. These studies demonstrate that traditional methods of neutrophil depletion with RB6-8C5 are broadly immune ablating. Our data also provide evidence that CD8 T cells initiate disruption of BBB tight junction proteins and CNS vascular permeability in the absence of neutrophil support.

Abstract During mitosis, sister chromatids are separated and equally divided over two daughter cells. If missegregation of whole chromosomes occurs, progeny cells with an abnormal number of chromosomes will arise. This is referred to as aneuploidy, a hallmark of most human cancers. We found that inactivation of a single Pten allele in mouse splenocytes and mouse embryonic fibroblasts (MEFs) is sufficient to cause aneuploidy, suggesting a role for Pten in the chromosome segregation process. Upon further study by live cell imaging of haploinsufficient and null Pten MEFs, we indeed found many missegregation errors (59% and 79% respectively, compared to 15% in wildtype MEFs). These abnormalities were independent from the AKT pathway since overexpression of a myristoylated, constitutively active form of AKT in wildtype MEFs resembled a wildtype instead of a Pten heterozygous phenotype. In addition, overexpression of a catalytic mutant Pten (C124S) in Pten+/- MEFs corrected the missegregation errors. We therefore propose a novel tumor-suppressive function of Pten to prevent whole chromosome instability, independent of the increased cell proliferation and survival caused by the activation of AKT. BubR1 is a regulator of kinetochore-microtubule attachment and a potent inhibitor of Cdc20, a key co-activator of the anaphase promoting complex/cyclosome (APC/C) that drives cells into anaphase by targeting the anaphase inhibitors cyclin B1 and securin for degradation by the 26S proteosome. We found that overexpression of BubR1 completely restores accurate chromosome segregation in Pten+/- MEFs, alluding to a specific role for Pten in mitosis. As a first step to test the hypothesis that Pten's mitotic role in preventing aneuploidization represents a key tumor suppressive function of this phosphatase, we bred a Flag-tagged BubR1 transgene into Pten+/- mice. We are currently in the process of studying the impact of the transgene on the development of prostatic intraepithelial neoplasia (PIN). Consistent with our hypothesis, preliminary analysis of two mice shows that BubR1 overexpression considerably decreases PIN lesion multiplicity. Data from the complete analysis of our double transgenic mouse cohort will be presented at the meeting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2992. doi:10.1158/1538-7445.AM2011-2992

Cdc20 is an activator of the anaphase-promoting complex/cyclosome that initiates anaphase onset by ordering the destruction of cyclin B1 and securin in metaphase. To study the physiological significance of Cdc20 in higher eukaryotes, we generated hypomorphic mice that express small amounts of this essential cell cycle regulator. In this study, we show that these mice are healthy and not prone to cancer despite substantial aneuploidy. Cdc20 hypomorphism causes chromatin bridging and chromosome misalignment, revealing a requirement for Cdc20 in efficient sister chromosome separation and chromosome–microtubule attachment. We find that cyclin B1 is newly synthesized during mitosis via cytoplasmic polyadenylation element–binding protein-dependent translation, causing its rapid accumulation between prometaphase and metaphase of Cdc20 hypomorphic cells. Anaphase onset is significantly delayed in Cdc20 hypomorphic cells but not when translation is inhibited during mitosis. These data reveal that Cdc20 is particularly rate limiting for cyclin B1 destruction because of regulated de novo synthesis of this cyclin after prometaphase onset.

Abstract ETS variant 1 (ETV1), also known as ETS-related protein 81, is overexpressed in prostate tumors, but whether and how this transcription factor affects tumorigenesis has remained elusive. Here, we show that ETV1 is primarily overexpressed in the most aggressive human prostate tumors. Transgenic ETV1 mice developed prostatic intraepithelial neoplasia as well as hyperplasia/neoplasia in seminal vesicles. Moreover, ETV1 cooperated with the androgen receptor (AR) to bind to the prostate-specific antigen enhancer and stimulate gene transcription. Consistent with its ability to physically interact with AR, ETV1 rendered an ETV1 binding site–driven reporter androgen inducible, and, on the other hand, ETV1 superinduced transcription from an AR binding site on androgen stimulation. In conclusion, our study substantiates that ETV1 overexpression is an underlying cause in the development of prostate and possibly also seminal vesicle cancer. Its interaction with and activation of AR provides a molecular mechanism on how ETV1 exerts its deleterious function. Thus, inhibiting ETV1 or blocking its interaction with AR may represent novel strategies in prostate cancer therapy. [Cancer Res 2009;69(20):8102–10]