Kathleen M. Haines

Control of large, established tumor xenografts with genetically retargeted human T cells containing CD28 and CD137 domains

Mesothelin is a cell-surface molecule over-expressed on a large fraction of carcinomas, and thus is an attractive target of immunotherapy. A molecularly targeted therapy for these cancers was created by engineering T cells to express a chimeric receptor with high affinity for human mesothelin. Lentiviral vectors were used to express a single-chain variable fragment that binds mesothelin and that is fused to signaling domains derived from T-cell receptor zeta, CD28, and CD137 (4–1BB). When stimulated by mesothelin, lentivirally transduced T cells were induced to proliferate, express the antiapoptotic gene Bcl-XL, and secrete multiple cytokines, all features characteristic of central memory T cells. When transferred intratumorally or intravenously into NOD/scid/IL2rγ−/− mice engrafted with large pre-established tumors, the engineered T cells reduced the tumor burden, and in some cases resulted in complete eradication of the tumors at low effector-to-target ratios. Incorporation of the CD137 signaling domain specifically reprogrammed cells for multifunctional cytokine secretion and enhanced persistence of T cells. These findings have important implications for adoptive immunotherapy of cancer, especially in the context of poorly immunogenic tumors. Genetically redirected T cells have promise of targeting T lymphocytes to tumor antigens, confer resistance to the tumor microenvironment, and providing immunosurveillance.

Constitutive non-canonical NFkappaB signaling in pancreatic cancer cells.

Constitutive classical NF-κB activation has been implicated in the development of pancreatic cancer, and inhibition of classical NF-κB signaling sensitizes pancreatic cancer cells to apoptosis. However, the role of the more recently described non-canonical NF-κB pathway has not been specifically addressed in pancreatic cancer. The non-canonical pathway requires stabilization of NIK and IKKα-dependent phosphorylation and processing of NF-κB2/p100 to p52. This leads to the activation of p52-RelB heterodimers that regulate genes encoding lymphoid-specific chemokines and cytokines. We performed qRT-PCR to detect gene expression in a panel of pancreatic ductal adenocarcinoma cell lines (BxPC-3, PCA-2, PANC-1, Capan-1, Hs-766T, AsPC-1, MiaPACA-2) and found only modest elevation of classical NF-κB-dependent genes. In contrast, each of the tumor cell lines displayed dramatically elevated levels of subsets of the non-canonical NF-κB target genes CCL19, CCL21, CXCL12, CXCL13 and BAFF. Consistent with activation of the non-canonical pathway, p52 and RelB co-localized in adenocarcinoma cells in sections of pancreatic tumor tissue, and each of the tumor cell lines displayed elevated p52 levels. Furthermore, p52 and RelB co-immunoprecipitated from pancreatic cancer cells and immunoblotting revealed that NIK was stabilized and p100 was constitutively phosphorylated in a subset of the cell lines. Finally, stable over expression of dominant negative IKKα significantly inhibited non-canonical target gene expression in BxPC-3 cells. These findings therefore demonstrate that the non-canonical NF-κB pathway is constitutively active and functional in pancreatic cancer cells.

A > 200 kb deletion removing the entire β-like globin gene cluster in a family of Irish Descent

We describe a new deletional form of γδβ-thalassemia segregating in two generations of a family of Irish descent. Affected family members present with a β-thalassemia minor phenotype, normal Hb A2 and Hb F levels. Genomic blotting analyses on DNA from affected family members show heterozygosity for a large deletion beginning at least 15 kb upstream of the 5′ endpoint of the γδβ-thalassemia-1 deletion, extending through the entire β-like globin gene cluster, and continuing for at least 10 kb beyond the 3′ endpoint of the deletion associated with the Spanish form of δβd`-thalassemia. This deletion is among the largest described so far, and removes at least 205 kb encompassing the entire β-like globin gene cluster on chromosome 11.

Chinese hamster ovary cell lines selected for resistance to ebolavirus glycoprotein mediated infection are defective for NPC1 expression

Abstract Ebolavirus causes severe hemorrhagic fever in humans and non-human primates. Entry of ebolavirus is mediated by the viral glycoprotein, GP; however, the required host factors have not been fully elucidated. A screen utilizing a recombinant Vesicular Stomatitis Virus (VSV) encoding Zaire ebolavirus GP identified four Chinese Hamster Ovary (CHO) cell lines resistant to GP-mediated viral entry. Susceptibility to vectors carrying SARS coronavirus S or VSV-G glycoproteins suggests that endocytic and processing pathways utilized by other viruses are intact in these cells. A cathepsin-activated form of the ebolaviral glycoprotein did not overcome the entry restriction, nor did expression of several host factors previously described as important for ebolavirus entry. Conversely, expression of the recently described ebolavirus host entry factor Niemann–Pick Type C1 (NPC1) restored infection. Resistant cells encode distinct mutations in the NPC1 gene, resulting in loss of protein expression. These studies reinforce the importance of NPC1 for ebolavirus entry.

Macrophage activation and Fcγ receptor-mediated signaling do not require expression of the SLP-76 and SLP-65 adaptors

The Src‐homology 2 domain‐containing, leukocyte‐specific phosphoprotein of 76 kDa (SLP‐76) is a hematopoietic adaptor that plays a central role during immunoreceptor‐mediated activation of T lymphocytes and mast cells and collagen receptor‐induced activation of platelets. Despite similar levels of expression in macrophages, SLP‐76 is not required for Fc receptor for immunoglobulin G (IgG; FcγR)‐mediated activation. We hypothesized that the related adaptor SLP‐65, which is also expressed in macrophages, may compensate for the loss of SLP‐76 during FcγR‐mediated signaling and functional events. To address this hypothesis, we examined bone marrow‐derived macrophages (BMM) from wild‐type (WT) mice or mice lacking both of these adaptors. Contrary to our expectations, SLP‐76−/− SLP‐65−/− BMM demonstrated normal FcγR‐mediated activation, including internalization of Ig‐coated sheep red blood cells and production of reactive oxygen intermediates. FcγR‐induced biochemical events were normal in SLP‐76−/− SLP‐65−/− BMM, including phosphorylation of phospholipase C and the extracellular signaling‐regulated kinases 1 and 2. To determine whether macrophages functioned normally in vivo, we infected WT and SLP‐76−/− SLP‐65−/− mice with sublethal doses of Listeria monocytogenes (LM), a bacterium against which the initial host defense is provided by activated macrophages. WT and SLP‐76−/− SLP‐65−/− mice survived acute, low‐dose infection and showed no difference in the number of liver or spleen LM colony‐forming units, a measure of the total body burden of this organism. Taken together, these data suggest that neither SLP‐76 nor SLP‐65 is required during FcγR‐dependent signaling and functional events in macrophages.

Ebola virus mediated infectivity is restricted in canine and feline cells.

Ebolaviruses and marburgviruses belong to the Filoviridae family and often cause severe, fatal hemorrhagic fever in humans and non-human primates. The magnitude of the 2014 outbreak in West Africa and the unprecedented emergence of Ebola virus disease (EVD) in the United States underscore the urgency to better understand the dynamics of Ebola virus infection, transmission and spread. To date, the susceptibility and possible role of domestic animals and pets in the transmission cycle and spread of EVD remains unclear. We utilized infectious VSV recombinants and lentivirus pseudotypes expressing the EBOV surface glycoprotein (GP) to assess the permissiveness of canine and feline cells to EBOV GP-mediated entry. We observed a general restriction in EBOV-mediated infection of primary canine and feline cells. To address the entry mechanism, we used cells deficient in NPC1, a host protein implicated in EBOV entry, and a pharmacological blockade of cholesterol transport, to show that an NPC1-dependent mechanism of EBOV entry is conserved in canine and feline cells. These data demonstrate that cells of canine and feline origin are susceptible to EBOV GP mediated infection; however, infectivity of these cells is reduced significantly compared to controls. Moreover, these data provide new insights into the mechanism of EBOV GP mediated entry into cells of canine and feline origin.

Abstract B139: Toxicity testing of EGFRvIII CAR-based immunotherapy of glioblastoma: From bench to bedside

The purpose of this study was to generate a panel of donor-derived primary cells, expand them ex-vivo in to sufficient numbers to utilize as targets for evaluating potential normal tissue toxicity of epidermal growth factor receptor mutation variant three (EGFRvIII)-specific chimeric antigen receptors (CAR) prior to use in clinical trials for patients with glioblastoma (GBM). Nine cell types were obtained, including different epithelial, endothelial, bone, smooth muscle, cardiac, neural, hematopoetic, stem cells, and keratinocytes. Cells were expanded with individual specialized media and protocols for between 8-12 passages. After expansion, primary cell identity was confirmed by morphology, ICC and IHC for characteristic markers. Levels of EGFR and EGFRvIII in each cell type were determined by qRT-PCR. To evaluate potential normal-cell toxicity, CAR T cells were co-cultured with each type of primary cell and function was evaluated in two ways: i) T cell activation was measured by staining and flow cytometry of CD3+ T cells stained intracellularly for CD107a, or GzmB, TNFalpha, IFNgamma, IL-2 cytokines. ii) Target cell lysis was evaluated by labeling primary cells with 51Cr prior to 4 hour co-culture with increasing numbers of CAR T cells, and measuring chromium-release. None of the primary cells showed expression of EGFRvIII, although several, in particular keratinocytes and renal epithelial, had high levels of EGFR. In functional assays, while the EGFR CAR T cells recognized and lysed EGFR expressing cell types, EGFRvIII CARs showed T cell activation and target lysis only of EGFRvIII expressing tumors. EGFRvIII CAR 2173 was selected for use in clinical trials at UPENN and UCSD, treating patients with GBM. To date, 6 patients have been infused with 2173 EGFRvIII CAR T cells, with no observed toxicity. All patients had detectable expansion of CAR T cells in vivo in blood, and one patient with subsequent tumor resection had detectable intra-tumoral CAR T cells. These CARs appear to be safe, persist in vivo and traffic into GBM tumor. An update on the clinical trial will be presented at the conference. Note:This abstract was not presented at the conference. Citation Format: Alexandria P. Cogdill, Alina Boesteanu, Chong Xu, Kathleen Haines, John Scholler, Joseph Fraietta, Yangbing Zhao, Xiaojun Liu, Jennifer Morrissette, Bruce Levine, Simon Lacey, Andreas Loew, Reshma Singh, Jennifer Brogdon, Donald M. O9Rourke, Marcela V. Maus, Carl H. June, Laura A. Johnson. Toxicity testing of EGFRvIII CAR-based immunotherapy of glioblastoma: From bench to bedside. [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 B139.

Abstract B05: A biologic screen to evaluate potential toxicity of chimeric antigen receptor modified T cells against primary normal human tissues

Adoptive cell therapy with chimeric antigen receptor (CAR) engineered T lymphocytes has recently shown great success for the treatment of hematologic malignancies. Similar strategies based on enhancing T cell function and redirecting these lymphocytes against solid tumors are being developed, however, early attempts have resulted in unexpected toxicity of normal cells expressing shared antigen or cross-reactive antigens. A primary challenge in translating new CARs to therapeutic platforms is the lack of robust models to predict potential adverse events pre-clinically. To address this challenge, we developed an in vitro system consisting of thirteen expanded primary human cell types. To detect potential off-tumor on-target effects on normal tissues, we co-cultured CARs directed against human epidermal growth factor receptor (EGFR) in our primary cell panel. Flow cytometric and chromium release assays were used to measure intracellular cytokine production by effectors and specific cell lysis of targets, respectively. T cells expressing a CAR against wild-type EGFR elicited strong effector cytokine production and effectively lysed both tumor and normal cells expressing EGFR. In contrast, T cells engineered to express a humanized CAR against mutant EGFR variant III (EGFRvIII), a tumor-specific antigen, reacted only with tumors and did not react to the panel of primary cell targets. This demonstrated a lack of potential cross-reactivity related to off-tumor on-target effects in the normal cell types represented here, suggesting improved safety for clinical use. The thirteen primary cell types we have generated form a panel of normal cells to test novel CAR T cells for potential toxicity and an adequate therapeutic index prior to first-in-human studies. Citation Format: Alexandria P. Cogdill, Alina Boesteanu, Kathleen Haines, Joseph Fraietta, John Scholler, Andreas Loew, Pramod Thekkat, Jennifer Brogdon, Marcela V. Maus, Carl June, Laura A. Johnson. A biologic screen to evaluate potential toxicity of chimeric antigen receptor modified T cells against primary normal human tissues. [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 B05.