NOD-scid IL2Rgammanull Mice Research Articles

647P - Selective Wnt/β-catenin small-molecule inhibitor CWP232228 impairs tumour growth of colon cancer

BackgroundAberrant activation of Wnt/β-catenin signaling is commonly found in colorectal cancers (CRCs) and is the prime event involved in the rapid proliferation of tumor cells in patients with advanced CRC. However, the limited effects of current therapeutics are unsatisfying, which highlights the importance of novel interventions in CRC chemo-treatment. We designed a highly potent small-molecule inhibitor CWP232228, which antagonizes binding of β-catenin to T cell factor in the nucleus. Thus, the present study aimed to explore the possibility of a selective small molecule β-catenin inhibitor, CWP232228, as a potential therapeutic drug in the treatment of CRCs. MethodsWe observed that CWP232228 at a concentration of as low as 1.0μm induced significant cytotoxic effectsand apoptosis in HCT116. Immunoblot analysis showed that CWP232228 treatment increased the release of cytochrome C, which led to the activation of apical (caspase-9) and effector caspases (caspase-7 and -3) resulting in the increased cleavage of PARP. ResultsIn addition, we observed that CWP232228 caused cell cycle arrest via attenuating the expressions of cyclin D2, D3, and survivin. Thus, we validated decreased expressions of Wnt/β-catenin targeted cell cycle progression regulators, aurora kinase A, c-Myc, cyclin D1, and microphthalmia-associated transcription factor. ConclusionsWe confirmed that CWP232228 targets β-catenin via decreased promoter activities, nuclear expressions of β-catenin. Lastly, we found CWP232228 also inhibited growth of xenografted colon cancer cells in NOD-scid IL2Rgammanull mice. Collectively, results in the current study showed a possibility for CWP232228 as a potential therapeutic drug in CRCs. Legal entity responsible for the studyThe authors. FundingHas not received any funding. DisclosureAll authors have declared no conflicts of interest.

Human cord blood (hCB)-CD34+ humanized mice fail to reject human acute myeloid leukemia cells.

Since their appearance, humanized mice carrying human immune system seemed promising tools to study the crosstalk between cancer and immunity. The NOD-scidIL2Rgammanull (NSG) mice engrafted with human cord blood (hCB)-CD34+ cells have been proposed to be a valuable tool to reproduce human immune system in mouse. However, the lack of solid evidences on the functionality of their human immune components limits their usage in immune-oncology. We report that (hCB)-CD34+ cells lose their ability to propagate and originate bone marrow-derived human immune cells after two serial transplantations in NSG mice. We demonstrate that transplants of bone marrow patient-derived acute myeloid leukemias (hAMLs) grow very similarly in the humanized (hCB)-CD34+ NSG and parental NSG mice. The similar extent of engraftment and development of leukemias in (hCB)-CD34+ NSG and controls suggests a poor human immune response against not compatible hAMLs. Our findings suggest that (hCB)-CD34+ NSG mice are transient and/or incomplete carriers of the human immune system and, therefore, represent a suboptimal tool to study the interaction between tumor and immune cells.

Abstract 2319: Accumulation and anti-tumor effect of chimeric antigen receptor (CAR) NK cells in metastasis uveal melanoma

Abstract Introduction: Metastatic uveal melanoma (MUM) is resistant to currently available treatments and overall prognosis is poor. MUM is an “immune cold” tumor and immune checkpoint blockades have only a marginal effect on this tumor. To bring immune cells to the tumor site and change the tumor microenvironment, we developed Chimeric Antigen Receptor (CAR)-NK cells targeting high molecular weight melanoma associated antigen (HMW-MAA/CPSG4). Methods: NK92 cell line derived from NK cell lymphoma was obtained from the American Type Culture Collection (ATCC). NK92 cells were transfected with retroviral pBMN-I vector co-expressing Green Fluorescent Protein (GFP) and anti-HMW-MAA-CD28-CD3z CAR construct. The efficacy of CAR construct transfection was confirmed by expression of His-Tag and F(ab)2 by using BD FACS Celesta flow cytometry. MUM cell line, TJU-UM001, was established from a hepatic metastasis of a uveal melanoma patient in our institution. TJU-UM001 cells have approximately 37,000 HMW-MAA molecules on their surface and are sensitive to NK92 cell killing. Tdtomato-transfected TJU-UM001 cells (106 cells) were intra-hepatically implanted into NOD-scid IL2Rgammanull (NSG) mice. Once establishment of hepatic tumor was confirmed by IVIS 200 imaging system (PerkinElmer) at Red Fluorescent Protein (RFP) excitation 535nm, 3 x 106 CAR-NK92 cells or NK92 cells without CAR constructs were injected into the spleen. Accumulations of GFP+ NK92 cells in the TJU-UM001 tumors in the liver were analyzed under fluorescence microscopy. In a separate experiment, 107 TJU-UM001 cells were subcutaneously implanted into NSG mice. The size of subcutaneous tumor was measured by Caliper. Once tumor became palpable (7-10 mm), 3 x 106 CAR- or non-CAR NK92 cells were directly injected to the MUM tumors. Forty thousand units of human IL-2 were intra-peritoneally injected into mouse once daily for 4 consecutive days after NK92 cell injections. Results: There were expressions of both His-tag and F(ab)2 on CAR-GFP NK92 cells. GFP and CD54 (ICAM-1) were expressed on 95% and 90% of CAR-GFP NK92 cells, respectively. There was no cross-expression of GFP or human CD54 on mouse cells. Therefore, GFP and CD54 were confirmed to be reliable markers for tracking CAR-GFP NK92 cells in mice. Both CAR- and non-CAR NK92 cells exhibited suppression of TJU-UM001 cell proliferation in in vitro assay. Direct injections of both CAR- and non-CAR NK92 cells showed anti-tumor growth effects on subcutaneously implanted TJU-UM001 tumors. More importantly, via splenic injection, CAR-NK92 cells accumulated selectively in the MUM tumors in the liver, while non-CAR NK92 cells were randomly distributed to normal liver tissue and the MUM tumors. These results support the rationale for hepatic arterial infusion of CAR-NK cells as treatment for uveal melanoma patients with hepatic metastasis. Citation Format: Bao Quoc Lam, Takahito Sugase, Mizue Terai, Meggie Danielson, Nadezhda Anikeyeva, Melissa A. Wilson, Yuri Sykulev, Takami Sato. Accumulation and anti-tumor effect of chimeric antigen receptor (CAR) NK cells in metastasis uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2319.

Abstract 1121: Role of c-Met in colorectal cancer lung metastasis

Abstract Colorectal cancer (CRC) is the second leading cause of cancer deaths in the US; systemic metastasis to the lungs occurs in approximately 10-20% of patients with CRC. Hepatocyte growth factor (HGF) and its tyrosine kinase receptor Met play a pivotal role in the tumor metastatic phenotype and represent attractive therapeutic targets. The present study was designed to determine the role of c-Met in growth and metastasis of CRC cells to the lung. We evaluated patient-derived xenografts (PDX) tumors and CRC cells for c-Met activation (phospho-Met [Tyr1234/1235]), established PDX cell lines with high phospho-Met (Tyr1234/1235) expression from lung or liver metastases, and investigated the role of c-Met expression in CRC lung metastasis. Methods. (1) PDX tumors were implanted into NOD-scid IL2Rgammanull mice; PDX cell lines were established from F3 tumor generation. (2) The protein expression of phospho-Met (Tyr1234/1235) was observed by western blotting and immunohistochemistry. (3). HT29 LungM3 cell line was derived from the human CRC line HT29 following multiple rounds of in vivo selection for lung metastasis in immunodeficient mice. HT29LungM3 cells were transfected with shRNA expression vectors targeting the c-Met gene. HT29LungM3 cells were injected into SCID mice subcutaneously to measure tumor growth or intravenously to examine the role of c-Met in CRC lung metastasis. Results. (1) High phospho-Met (Tyr1234/1235) expression was identified in 6 out of 20 CRC PDX models. Two PDX cell lines with high phospho-Met (Tyr1234/1235) expression, 2377LM (from liver metastasis) and 2387 (from lung metastasis), were established. Metastatic potential of the 2377LM cell line to the liver was confirmed after intrasplenic injection of cancer cells into SCID mice. The metastatic potential of 2387 cells to the lung was confirmed after intravenous injection of cancer cells into SCID mice. (2) Western blot analysis demonstrated high phospho-Met (Tyr1234/1235) expression in HT29LungM3, 2387LM and 2387 cell lines, compared to HCT116 (human CRC), LS174T (human CRC), Caco-2 (human CRC), SK-OV-3 (human ovarian cancer) and JAR (placenta choriocarcinoma) cell lines. (3) HT29LungM3 cells were transfected with shRNA expression vectors targeting the c-Met gene with c-Met knockdown of at least 90%. Surprisingly, c-Met inhibition in the HT29LungM3 cell line had no effect on subcutaneous tumor growth or lung metastasis. Conclusions. Aberrant expression of c-Met is associated with the progression of multiple human malignancies and has been identified as a novel target for the treatment of malignant tumors. Here, we examined expression of phospho-Met (Ty1234/1235) in CRC PDX cell samples and determined that c-Met knockdown does not decrease CRC lung metastasis. These results are important for understanding the mechanism of CRC lung metastasis and proper strategy selection for prevention and treatment of patients with advanced disease. Citation Format: Piotr G. Rychahou, Nick Roller, Eun Y. Lee, Nicole Rychagov, Matthew Melton, Carrigan Wasilchenko, B. Mark Evers. Role of c-Met in colorectal cancer lung metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1121.

Enhanced Bone Marrow Homing of Natural Killer Cells Following mRNA Transfection With Gain-of-Function Variant CXCR4R334X.

Adoptive transfer of natural killer (NK) cells can induce remission in patients with relapsed/refractory leukemia and myeloma. However, to date, clinical efficacy of NK cell immunotherapy has been limited to a sub-fraction of patients. Here we show that steps incorporated in the ex vivo manipulation/production of NK cell products used for adoptive infusion, such as over-night IL-2 activation or cryopreservation followed by ex vivo expansion, drastically decreases NK cell surface expression of the bone marrow (BM) homing chemokine receptor CXCR4. Reduced CXCR4 expression was associated with dampened in vitro NK cell migration toward its cognate ligand stromal-derived factor-1α (SDF-1α). NK cells isolated from patients with WHIM syndrome carry gain-of-function (GOF) mutations in CXCR4 (CXCR4R334X). Compared to healthy donors, we observed that NK cells expanded from WHIM patients have similar surface levels of CXCR4 but have a much stronger propensity to home to BM compartments when adoptively infused into NOD-scid IL2Rgammanull (NSG) mice. Therefore, in order to augment the capacity of adoptively infused NK cells to home to the BM, we genetically engineered ex vivo expanded NK cells to express the naturally occurring GOF CXCR4R334X receptor variant. Transfection of CXCR4R334X-coding mRNA into ex vivo expanded NK cells using a clinically applicable method consistently led to an increase in cell surface CXCR4 without altering NK cell phenotype, cytotoxic function, or compromising NK cell viability. Compared to non-transfected and wild type CXCR4-coding mRNA transfected counterparts, CXCR4R334X-engineered NK cells had significantly greater chemotaxis toward SDF-1α in vitro. Importantly, expression of CXCR4R334X on expanded NK cells resulted in significantly greater BM homing following adoptive transfer into NSG mice compared to non-transfected NK cell controls. Collectively, these data suggest up-regulation of cell surface CXCR4R334X on ex vivo expanded NK cells via mRNA transfection represents a novel approach to improve homing and target NK cell-based immunotherapies to BM where hematological malignancies reside.

Mgta-456 Contains Large Numbers of Expanded Cord Blood (CB) CD34+CD90+ Hematopoietic Stem Cells (HSC) Which Confer All Engraftment Activity and Correlate with Accelerated Neutrophil Recovery after Myeloablative Conditioning in Patients with Hematologic Malignancy

Background: Patient access to well-matched CB containing high doses of stem cells remains a challenge for successful transplants. Low numbers of CD34+ cells in CB has resulted in delayed neutrophil recovery and a risk of graft failure relative to other hematopoietic stem cell (HSC) sources. MGTA-456 is a cell therapy that consists of CD34+ cells expanded in a 15-day culture in the presence of an aryl hydrocarbon receptor antagonist (AHRa) and the CD34 depleted fraction obtained from the same CB unit. Thus far, 40 patients with hematological malignancy (n=36) and non-malignant diseases (n=4) have received MGTA-456 with a median follow-up of 2.5 years (range 0.1 to 5 years) and 75 days (20 to 143 days) respectively. All patients engrafted at a significantly faster rate as compared to similarly treated historical controls (p<0.01). The aim of the current study was to fully characterize the expanded CD34+ cell fraction of MGTA-456 phenotypically and functionally and identify the cell population that correlates with time to neutrophil recovery. We found that the expanded CD34+CD90+ population of MGTA-456 were the cells responsible for engraftment in NOD-scid IL2Rgammanull (NSG) mice. We hypothesized that the dose of CD34+CD90+ cells/kg would have the strongest correlation with time to neutrophil recovery.Methods: The expansion culture consisted of StemSpan SFEM supplemented with SCF, FLT- 3L, TPO and IL-6 (all at 50 ng/mL) and the AHRa without the addition of antibiotics. After a 10 or 15 day culture, the CD34 expanded product was characterized by cell surface markers (CD34, CD90, CD133, CD41, CD71, CD235a, CD3, CD4, CD8, CD14, CD15, CD16, CD11b, CD33, CD19, CD56, and CD10), as well as colony-forming unit (CFU) capacity and engraftment potential in NOD-scid IL2Rgammanull (NSG) mice in a subset of products.Results: The expanded CD34+ cell fraction of MGTA-456 consisted of CD34+CD133-CD90- cells (late progenitors), CD34+CD133+CD90- cells (early progenitors) and CD34+CD133+CD90+ (progenitors and stem cells) as shown in Figure 1. The CD34- cells within the expanded fraction contained erythroid (CD71+) and megakaryocyte progenitors (CD41+), CD33+, CD14+, CD15+, and CD11b+ myeloid cells and CD56+ cells. CD3+, CD8+, CD4+, CD16+, CD19+ as well as CD10+ cells were not present (<1%) in the CD34 expanded fraction of MGTA-456. To identify which of these cell populations contain the NSG engraftment activity, we sorted CD34-, CD34+CD90- and CD34+CD90+ cells from MGTA-456 and transplanted the cell fractions into NSG mice. These studies clearly demonstrated that all NSG engraftment activity resided in the CD34+CD90+ cell subpopulation (Figure 2). Based on this result, we correlated the dose of CD34-, CD34+CD90-, CD34+CD90+ cells, and CFU cells/kg infused with time to neutrophil recovery of patients treated with MGTA-456. The dose of TNC (r2=0.49, P<0.05), CD34+CD90- (r2=0.45, p<0.05), and CD34+CD90+ (r2=0.52, p<0.05) cells/kg all significantly correlated with time to neutrophil recovery. The dose of CD34+CD90+ cells/kg had the strongest correlation with time to neutrophil recovery consistent with the NSG engraftment result.Conclusion: In these studies, we demonstrate that the expanded CD34+ cell fraction of MGTA-456 contains large doses of CD34+CD90+ HSC and progenitors, which are critical for long term engraftment in NSG mice and correlated with rapid neutrophil recovery clinically. [Display omitted] DisclosuresBoitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Goncalves:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Wagner:Magenta Therapeutics: Consultancy, Research Funding; Novartis: Research Funding.

Modeling Sézary Syndrome for Immunophenotyping and Anti-Tumor Effect of Ucart and Long-Acting Interleukin-7 Combination Therapy

Background: Sézary syndrome (SS) is a highly-morbid T cell leukemic lymphoma with no widely-effective treatments and few preclinical models. We demonstrated effective T cell lymphoma therapy with allogeneic gene-edited anti-CD7 CARTs (Cooper et al, Leukemia, 2018). However, SS T cells typically lose CD7 but maintain ubiquitous high CD2 expression. Thus, we generated CD2- and TRAC-deleted anti-CD2 universal CARTs (UCART2) and multiple SS xenograft models (PDXs) as preclinical UCART2 testing platforms. We further tested a stable homodimeric interleukin-7 molecule, the long-acting form of recombinant human interleukin-7 fused with hybrid Fc (rhIL-7-hyFc, NT-I7), to potentiate UCART2 killing of an SS xenograft in vivo.Methods: To generate SS PDX models, we injected NOD scid IL2Rgammanull (NSG) mice expressing SCF, GM-CSF, and IL-3 (NSG-SGM3) with ~2x106 mononuclear cells derived from SS patients. We immunophenotyped SS patient blood and PDX engraftment with two 21-color flow cytometry panels assessing major immune subsets, CTCL, and exhaustion markers (Staser et al, Cytometry A, 2018). To generate UCART2s, we activated human T cells on CD3/CD28 beads, electroporated the T cells with Cas9, a TRAC-targeted gRNA, and a CD2-targeted gRNA followed by viral transduction with an anti-CD2 scFv 3rd generation CAR. For initial UCART2 testing, we injected NSG mice with 5x105 cells from a human Sézary cell line transduced with click beetle red luciferase (HHCBR-GFP) four days prior to UCART2 treatment. Mice were treated with NT-I7 (10mg/kg SC) on days +1, +15 and +29 post UCART2 infusion.Results: SS patient blood showed specific defects in monocyte, monocytic dendritic cell, and natural killer cell differentiation, increased skewing toward granulocytes and non-classical CD16+ monocytes (p<0.01, SS vs. normal PBMCs), and loss of effector memory CD4 cells (8% vs 34%, p<0.001, SS vs. normal PBMCs). SS cells were CD3+CD4+CD2+CD5+CD8- with variable CD7 loss and PD1 gain. Four of six unique human SS samples injected in NSG-SGM3 mice engrafted within ~6 weeks with no signs of xenogeneic GVHD. Following engraftment, SS cells showed near ubiquitous PD1 expression (>90% vs ~20%, p<0.001, SS vs. normal PBMCs), CD7 loss, and increased CD30 and CD26 expression. Immunohistochemistry further revealed atypical CD3+CD4+CD8-CD7- lymphocytes lining the dermo-epidermal junction. Second generation PDXs showed infiltration of the spleens, blood, and bone marrow with CD2+CD7- human cells and developed alopecia, scaling, and subcutaneous and intraperitoneal masses, with immunophenotyping, sequencing, and UCART treatment studies ongoing.To test UCART2's efficacy in killing SS cells in vivo, we injected NSG mice with HHCBR-GFP+ cells. UCART2-treated HHCBR-GFP mice showed dramatically reduced tumor burden as compared to control UCART19-treated HHCBR-GFP mice (BLI; 10^7 vs. 10^11 photon flux/s at 3 weeks, p<0.0001, UCART2 vs. UCART19). Moreover, UCART2-treated HHCBR-GFP mice survived ~40 days as compared to ~21 days in the UCART19 group. Remarkably, UCART2-treated HHCBR-GFP mice receiving NT-I7 showed virtually no tumor burden (maximum 106 photon flux/s vs. 1010 photon flux/s, UCART2+NT-I7 vs. UCART2 only groups) with 100% of UCART2- and NT-I7-treated HHCBR-GFP mice surviving beyond 49 days (Figure 1).Discussion: We describe the generation of physiologically-relevant SS preclinical models, comprehensive immunophenotyping of patient SS samples, clonal SS PDX outgrowth, and the highly effective anti-tumor activity of UCART2- plus NT-I7-mediated killing of SS cells in vivo using an NSG xenograft model. Ongoing studies involve treating primary SS and CTCL PDX models with UCART2 and NT-I7. These preclinical data validate the use of allogeneic “off-the-shelf” adoptive immunotherapy for the treatment of Sézary syndrome, while demonstrating the dramatic enhancement of CART efficacy using a dose-adjustable, clinic-ready long-acting interleukin-7 agonist given in an adjuvant setting. [Display omitted] DisclosuresPark:NeoImmuneTech: Employment. Lee:NeoImmuneTech: Employment. Musiek:Seattle Genetics: Honoraria; Actelion: Other: Scientific Advisory Committee ; Kyowa Kirin: Honoraria.

Abstract 5748: Downregulation of glycine decarboxylase renders ovarian cancer cells less proliferative and more chemoresistant

Abstract Glycine decarboxylase (GLDC) is a very recently identified metabolic oncogene that links glycine metabolism with tumorigenesis. We designed this study to determine the role of GLDC in chemoresistance in ovarian cancer cells. First, we found GLDC is downregulated in paclitaxel-resistant ovarian cancer cells (OVCAR3/PTX). Prompted by decreased glucose transport in OVCAR3/PTX cells, we transplanted OVCAR3 and OVCAR3/PTX onto the back of the NOD-scid IL2Rgammanullmice and found that the OVCAR3/PTX tumor grew less rapidly than paclitaxel-sensitive ovarian cancer cells (OVCAR3). We also found that GLDC is downregulated in OVCAR3/PTX tumor. In vitro study revealed that the inhibitory effect of siGLDC on proliferation was significantly greater in OVCAR3 than in OVCAR3/PTX. We also found that knockdown of GLDC in OVCAR3 cells rendered ovarian cancer cells PTX-resistant. Finally, we determined GLDC expressions in patients with chemosensitive and chemoresistant serous-type ovarian cancer and found that GLDC expression was markedly downregulated in chemoresistant cancer patients. In summary, these results suggest that GLDC is associated with ovarian cancer chemoresistance. Citation Format: So-Jin Shin, Hyewon Chung, Jin Young Kim, Hyera Kim, Chi-Heum Cho, Eunyoung Ha. Downregulation of glycine decarboxylase renders ovarian cancer cells less proliferative and more chemoresistant [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 5748.

Endometrial Mesenchymal Stem/Stromal Cells Modulate the Macrophage Response to Implanted Polyamide/Gelatin Composite Mesh in Immunocompromised and Immunocompetent Mice

The immunomodulatory properties of human endometrial mesenchymal stem cells (eMSC) have not been well characterised. Initial studies showed that eMSC modulated the chronic inflammatory response to a non-degradable polyamide/gelatin mesh in a xenogeneic rat skin wound repair model, but the mechanism remains unclear. In this study, we investigated the immunomodulatory effect of eMSC on the macrophage response to polyamide/gelatin composite mesh in an abdominal subcutaneous wound repair model in C57BL6 immunocompetent and NSG (NOD-Scid-IL2Rgammanull) immunocompromised mice to determine whether responses differed in the absence of an adaptive immune system and NK cells. mCherry lentivirus-labelled eMSC persisted longer in NSG mice, inducing longer term paracrine effects. Inclusion of eMSC in the mesh reduced inflammatory cytokine (Il-1β, Tnfα) secretion, and in C57BL6 mice reduced CCR7+ M1 macrophages surrounding the mesh on day 3 and increased M2 macrophage marker mRNA (Arg1, Mrc1, Il10) expression at days 3 and 7. In NSG mice, these effects were delayed and only observed at days 7 and 30 in comparison with controls implanted with mesh alone. These results show that the differences in the immune status in the two animals directly affect the survival of xenogeneic eMSC which leads to differences in the short-term and long-term macrophage responses to implanted meshes.

Lym-1 Chimeric Antigen Receptor T Cells Exhibit Potent Anti-Tumor Effects against B-Cell Lymphoma.

T cells expressing chimeric antigen receptors (CARs) recognizing CD19 epitopes have produced remarkable anti-tumor effects in patients with B-cell malignancies. However, cancer cells lacking recognized epitopes can emerge, leading to relapse and death. Thus, CAR T cells targeting different epitopes on different antigens could improve immunotherapy. The Lym-1 antibody targets a conformational epitope of Human Leukocyte Antigen-antigen D Related (HLA-DR) on the surface of human B-cell lymphomas. Lym-1 CAR T cells were thus generated for evaluation of cytotoxic activity towards lymphoma cells in vitro and in vivo. Human T cells from healthy donors were transduced to express a Lym-1 CAR, and assessed for epitope-driven function in culture and towards Raji xenografts in NOD-scidIL2Rgammanull (NSG) mice. Lym-1 CAR T cells exhibited epitope-driven activation and lytic function against human B-cell lymphoma cell lines in culture and mediated complete regression of Raji/Luciferase-Green fluorescent protein (Raji/Luc-GFP) in NSG mice with similar or better reactivity than CD19 CAR T cells. Lym-1 CAR transduction of T cells is a promising immunotherapy for patients with Lym-1 epitope positive B-cell malignancies.

Targeting the entry region of Hsp90's ATP binding pocket with a novel 6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl amide

The molecular chaperone Hsp90 plays an important role in cancer cell survival and proliferation by regulating the maturation and stabilization of numerous oncogenic proteins. Due to its potential to simultaneously disable multiple signaling pathways, Hsp90 has emerged as an attractive therapeutic target for cancer treatment. In this study, the design, synthesis, and biological evaluation of a series of Hsp90 inhibitors are described. Among the synthetic compounds, 6,7-dihydrothieno [3,2-c]pyridin-5(4H)-yl amide 19 exhibits a remarkable binding affinity to the N-terminus of Hsp90 in a fluorescence polarization (FP) binding assay (IC50 = 50.3 nM). Furthermore, it effectively inhibits the proliferation of H1975 non-small cell lung cancer (NSCLC) and Skbr3 breast cancer cell lines with GI50 values of 0.31 μM and 0.11 μM, respectively. Compound 19 induces the degradation of the Hsp90 client proteins including EGFR, Her2, Met, c-Raf, and Akt, and consequently promotes apoptotic cancer cell death. Compound 19 also inhibits the growth of H1975 xenografts in NOD-scid IL2R gammanull mice without any apparent body-weight loss. The immunohistologic evaluation indicates that compound 19 decreases the expression of Akt in xenograft tumor tissue via an inhibition of the Hsp90 chaperon function. Additionally, the cytochrome P450 assay indicates that compound 19 has no effect on the activities of five major P450 isoforms (IC50 > 50 μM for 1A2, 2C9, 2C19, 2D6, and 3A), suggesting that clinical interactions between compound 19 and the substrate drugs of the five major P450 isoforms are not expected. Overall, compound 19 represents a new class of Hsp90 inhibitor with its 6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl-amide structure, and it has the therapeutic potential to overcome drug resistance in cancer chemotherapy.

Abstract 1156: Downregulation of glycine decarboxylase contributes to paclitaxel resistance in ovarian cancer cells

Abstract Glycine decarboxylase (GLDC) is a metabolic oncogene that links glycine metabolism with tumorigenesis. In humans, GLDC is part of a multienzyme complex that couples the decarboxylation of glycine to the biosynthesis of serine. The role of GLDC in relation to chemoresistance is unclear. We designed this study to determine the role of GLDC in chemoresistance in ovarian cancer cells. First, we found GLDC is downregulated in paclitaxel resistant ovarian cancer cells (OVCAR3/PTX). Prompted by decreased glucose transport in OVCAR3/PTX cells, we transplanted OVCAR3 and OVCAR3/PTX onto the back of the NOD-scid IL2Rgammanull mice and found that the OVCAR3/PTX tumor grew less rapidly than paclitaxel sensitive ovarian cancer cells (OVCAR3). We also found that GLDC is downregulated in OVCAR3/PTX tumor. In vitro study revealed that the inhibitory effect of siGLDC on proliferation was significantly greater in OVCAR3 than in OVCAR3/PTX. We also found that knockdown of GLDC in OVCAR3 cells rendered ovarian cancer cells PTX-resistant. Finally, we determined GLDC expressions in patients with chemosensitive and chemoresistant serous type ovarian cancer and found that GLDC expression was markedly downregulated in chemoresistant cancer patients. These results suggest GLDC is associated with ovarian cancer chemoresistance. Citation Format: Jin Young Kim, Sojin Shin, Eunyoung HA, Soondo Cha, Chi-Heum Cho. Downregulation of glycine decarboxylase contributes to paclitaxel resistance in ovarian cancer cells. [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 1156.

Anti-hIgE gene therapy of peanut-induced anaphylaxis in a humanized murine model of peanut allergy

Peanuts are the most common food to provoke fatal or near-fatal anaphylactic reactions. Treatment with an anti-hIgE mAb is efficacious but requires frequent parenteral administration. Based on the knowledge that peanut allergy is mediated by peanut-specific IgE, we hypothesized that a single administration of an adeno-associated virus (AAV) gene transfer vector encoding for anti-hIgE would protect against repeated peanut exposure in the host with peanut allergy. We developed a novel humanized murine model of peanut allergy that recapitulates the human anaphylactic response to peanuts in NOD-scid IL2Rgammanull mice transferred with blood mononuclear cells from donors with peanut allergy and then sensitized with peanut extract. As therapy, we constructed an adeno-associated rh.10 serotype vector coding for a full-length, high-affinity, anti-hIgE antibody derived from the Fab fragment of the anti-hIgE mAb omalizumab (AAVrh.10anti-hIgE). In the reconstituted mice peanut-specific IgE was induced by peanut sensitization and hypersensitivity, and reactions were provoked by feeding peanuts to mice with symptoms similar to those of human subjects with peanut allergy. A single administration of AAVrh.10anti-hIgE vector expressed persistent levels of anti-hIgE. The anti-hIgE vector, administered either before sensitization or after peanut sensitization and manifestation of the peanut-induced phenotype, blocked IgE-mediated alterations in peanut-induced histamine release, anaphylaxis scores, locomotor activity, and free IgE levels and protected animals from death caused by anaphylaxis. If this degree of persistent efficacy translates to human subjects, AAVrh.10anti-hIgE could be an effective 1-time preventative therapy for peanut allergy and possibly other severe, IgE-mediated allergies.

734. Pre-Clinical Development and Qualification of ZFN-Mediated Disruption of CCR5 in Human Hematopoietic Stem and Progenitor Cells

INTRODUCTION: Gene therapy for HIV-1 infection is a promising alternative to lifelong combination anti-viral drug treatment. When deleted, chemokine receptor 5 (CCR5), the co-receptor required for R5-tropic HIV-1 infection of human cells, renders cells resistant to HIV-1 infection. This has formed a strategy for the potential cure of HIV-1 infection by disrupting CCR5 genomic sequences in hematopoietic stem/progenitor cells developed using Zinc Finger Nucleases (ZFN). We demonstrate that delivery of CCR5-specific ZFN mRNA (SB-728mR) to HSPC via electroporation results in efficient disruption of CCR5 resulting in a high percentage of cells possessing biallelic modification and yields clinical-scale numbers of CCR5-modified HSPC with minimal cellular cytotoxicity, while maintaining their hematopoietic potential both in vitro and in vivo. METHODS: Pre-clinical experiments were performed at scale using donor HSPC with different concentrations of SB-728mR to optimize the electroporation-mediated CCR5 disruption in CD34+ HSPC for clinical studies in HIV. SB728mR-HSPC were evaluated in vitro for: a) mono- vs bi-allelic disruption and off-target ZFN cutting using MiSeq deep sequencing and the surveyor nuclease assay; b) total yield and viability; and c) hematopoietic function in colony forming unit (CFU) assays. Tumorigenicity studies were performed in NOD-scid-IL2Rgammanull (NSG) mice irradiated with 150 cGy and IV injected with 1e6 SB-728mR-HSPC. equivalent of one clinical dose (150e6 CD34+ cells).Engraftment efficiency was analyzed by %CD45 cells in PBMC at intervals for 5 months. RESULTS: We have demonstrated the specificity and efficacy of ZFN-based CCR5 disruption in HSPC: a) 40-60% CCR5 disruption with 50 to 150 µg/mL mRNA, with off-target modification observed at CCR2 and in 3 additional sites that occur in non-coding (either at intergenic or intronic) sequences; and b) effective disruption of ≤70% CFU from adult mobilized HSPC with maintenance of hematopoietic potential in vitro and in vivo. The tumorigenicity study demonstrated the safety of the gene modified cell product. SB-728mR-HSPC and untransfected control HSPC successfully engrafted as shown by human hematopoietic progeny measured in blood and bone marrow in all animals. Gene modified cells were observed in blood and bone marrow during the course of the study and were generally well tolerated. No ZFN treatment related tumor formation was observed in any animals. CONCLUSIONS: We have developed a method to genetically modify the CCR5 locus in human CD34+ HSPC. This approach is safe, efficient and reproducible. A combination of in vitro and in vivo studies evaluated ZFN specificity and potential tumorigenicity of ZFN modified cells in NSG mice. The established process complies with the regulatory requirements and results of the preclinical studies were used to support the filing of an IND with the FDA. The clinical trial testing SB-728mR-HSPC (NCT02500849@clinicaltirals.gov) is currently enrolling patients with the support of the California Institute for Regenerative Medicine.

Dual Inhibition of PI3K-Delta and Gamma By Duvelisib (IPI-145) Impairs CLL B- and T-Cell Migration, Survival and Proliferation in a Murine Xenograft Model Using Primary Chronic Lymphocytic Leukemia Cells

Duvelisib (IPI-145), a dual inhibitor of phosphoinositide 3-kinase (PI3K)-δ and -γ, has shown clinical activity in treatment-naïve and relapsed/refractory chronic lymphocytic leukemia (CLL) patients. Clinically, duvelisib results in a redistribution of malignant B cells and concomitant reduction in nodal enlargement. These effects are believed to be due to important roles of PI3K- δ and -γ in CXCL12-mediated CLL cell migration (Peluso 2014), cytokine-induced CLL B-cell proliferation, and BCR-stimulated B-cell survival (Balakrishnan 2015). Additional data suggest an effect of duvelisib on the tumor supporting cells of the CLL microenvironment. This includes preclinical studies demonstrating that PI3K-γ inhibition blocks normal T cell migration toward tumor chemokines and prevents murine bone marrow-derived M2 macrophage polarization (Peluso 2014), as well as clinical data in CLL patients receiving duvelisib showing reduced serum levels of myeloid and T cell-secreted cytokines and chemokines (Douglas 2015).To further characterize duvelisib's effect on CLL cells and the tumor microenvironment (TME), a murine xenograft model using primary human CLL cells was employed. We first studied duvelisib's effect on CLL B- and T-cell migration in vivo. CLL PBMCs (n=2; 1 IGHV unmutated (U)-CLL, 1 IGHV mutated (M)-CLL) pre-treated with duvelisib for 48 hours were injected retro-orbitally into NOD-scid IL2Rgammanull (NSG) mice. B- and T-cell localization in tissues and circulation was studied 1 and 24 hours post-injection. Duvelisib treatment (1000 nM) prevented the egress of CLL B and T cells from the circulation into the spleen, indicating impaired homing of CLL B and T cells. To better define the effect of duvelisib on T-cell migration, T cells from CLL patients (n=3; 2 U-CLL, 1 M-CLL) treated ex vivo with duvelisib at 1, 10, 100 and 1000 nM were injected into mice and analyzed for their trafficking 24 hours later. Inhibition of T-cell homing to spleen was dose dependent, with only 100 and 1000 nM having significant effects. Given duvelisib's cellular IC50s for PI3K isoforms, these results suggest that impaired T-cell migration is due to PI3K-γ inhibition, and studies with isoform-selective PI3K-δ and PI3K-γ inhibitors are currently underway to examine this possibility.The effect of duvelisib on CLL T-cell proliferation was evaluated after in vitro activation with anti-CD3/28 Dynabeads plus IL2 (n=6; 3 U-CLL, 3M-CLL). In duvelisib treated cells, CD4+, but not CD8+, T-cell proliferation was inhibited at doses of 100 and 1000 nM, suggesting a role for PI3K-γ. The effects of duvelisib on CLL B- and T-cell growth in vivo (n=4; 2 U-CLL, 2 M-CLL) were then studied. Autologous CLL T cells were stimulated as above and injected with CLL PBMCs into NSG mice. Animals treated orally with duvelisib for 3 weeks at 100 mg/kg/day had preferentially reduced CD4+ T-cell recovery from spleens, thereby decreasing the CD4 to CD8 ratio. In each case, duvelisib treatment reduced the number of splenic CLL B cells. This reduction reflected inhibition of both CLL cell proliferation and survival, since duvelisib treatment decreased the percentage of cycling CLL cells and increased the percentage of apoptotic B cells. Thus, duvelisib may target CLL B-cell growth directly, or indirectly by inhibiting the support of CD4+ T cells in the TME.The potential effect of duvelisib on the tumor-supporting myeloid compartment was also tested. Because of limited human myeloid-cell engraftment in our NSG model, we studied the effect of duvelisib on murine macrophages. Mice receiving duvelisib had reduced numbers of splenic CD11b+ GR-1low LY-6Clow LY-6Gneg macrophages compared to controls, suggesting duvelisib altered macrophage development.Prior in vitro studies demonstrated inhibition of CLL B-cell survival and proliferation by duvelisib, as well as blockade of T-cell migration and M2 macrophage polarization (Balakrishnan 2015; Peluso 2014). Our current in vivo studies further support duvelisib's effect on CLL B-cell growth and survival through inhibition of cellular homing to supportive tissue niches and alterations in the TME. The latter, in part, is through suppression of T-cell support and alterations in the macrophage compartment. Overall, these preclinical results suggest that inhibition of PI3K-δ and PI3K-γ by duvelisib affects CLL cell survival through direct and indirect mechanisms. DisclosuresMcGovern:Infinity Pharmaceuticals, Inc.: Employment. Kutok:Infinity Pharmaceuticals, Inc.: Employment.

MUC1 As a Potential Therapeutic Target in Cutaneous T-Cell Lymphoma

Introduction: MUC1 is an oncoprotein that is aberrantly expressed in many epithelial tumors and hematologic malignancies, conferring cell growth and survival. Expression and functional significance of MUC1 in cutaneous T-cell lymphoma (CTCL) has not been previously characterized. In the present study, we evaluated the potential role that MUC1 mediated signaling plays in the pathogenesis of Mycosis Fungoides (MF) and Sezary Syndrome cells (SS), the 2 common subtypes of CTCL.Methods and results: MUC1 was strongly expressed on the SS cell lines HuT-78 (60%), H9 (77%) and SeAx (59%) and the MF cell lines, Myla (94%) and HuT-102 (87%) when incubated with anti-MUC1 antibody and analyzed by flow cytometry (FCM) . In contrast, B-cell lymphoma (BCL) cell lines (Val, K422, Toledo, SU-DHL-2, OCI-Ly7, OCI-Ly8, SU-DHL-5, SU-DHL-8, OCI-Ly3, RCK8, Ramos, Daudi, JVM-2) expressed minimal levels of MUC1 (0-7%), as did circulating T-cell populations isolated from healthy controls (0-3%). The finding of high MUC1 expression on CTCL and minimal expression on BCL and normal circulating T cells was confirmed byimmunoblotting (IB) with another antibody directed against MUC1-C, the oncogenic subunit. The expression profile of MUC1-C was found to be similar at the mRNA level by RT-PCR analysis. We subsequently examined MUC1 as a therapeutic target in CTCL. We have developed an inhibitor of the MUC1-C receptor subunit, designated GO-203, which blocks MUC1-C dimerization necessary for nuclear translocation and downstream signaling. Growth inhibition was determined using CellTiter-Glo that measures ATP produced by live and proliferating cells and demonstrated that the concentration required to inhibit growth by 50% (IC50) following 72-hour exposure ranged from 3 uM to 3.5 uM in most cell lines (HuT-78, H9 and Myla). Culture of HuT-78, H9 and Myla cells in the presence of 5 uM GO-203 concentration for 72-hours resulted in 70, 80 and 90% cell death respectively as determined by Annexin V and propidium iodide staining by FCM. Cell proliferation, as assessed by incorporation of tritiated thymidine, was inhibited by 70% and 80% in 2 cell lines (HuT-78 and Myla respectively) when treated with GO-203 at concentration of 5 uM for 72-hours. To determine mechanism of action of GO-203 induced apoptosis in vitro, level of hydrogen peroxide (H2O2), a reactive oxygen species (ROS) was measured using FCM based conversion of carboxy-2′7′ dichlorodihydrofluorescein diacetate to 2′7′-dichlorodihydrofluorescein. Cell lysates were analyzed for expression of the protein TIGAR, a fructose-2-6-bisphosphatase p53-inducible regulator of glycolysis and apoptosis by IB. Exposure of Hut-78, Myla and H9 cells to GO-203 for 72-hours was associated with an increase in ROS and down regulation of TIGAR expression. To confirm ROS mediated apoptosis by GO-203, in vitro treatment with the antioxidant N-acetylcysteine (NAC) was performed and levels of H2O2 were measured including IB with anti-TIGAR antibody. Treatment with NAC blocked the decreases in TIGAR levels, supporting the hypothesis that this response was mediated by increases in ROS. The ability of GO-203 to eradicate disease was evaluated in vivo across a spectrum of tumor volumes in 2 xenograft mouse models. Five million HuT-78 and 2.5 million Myla cells were injected subcutaneously (SC) into NOD-SCID IL2Rgammanull (NSG) mice. Treatment with GO-203 was administered daily at the dose of 14mg/kg SC for 3 weeks. Tumor volumetric analysis by three-dimensional ultrasound (3D-US) in HuT-78 and Myla xenograft NSG mouse models demonstrated a statistically significant decrease in tumor volume in mice treated with GO-203 compared to control (In the HuT-78 xenograft, average tumor volume was 707 mm3 in the GO-203 treated arm versus 1171 mm3 in the control arm rendering a p-value of 0.0029; whereas in the Myla xenograft average tumor volume was 184 mm3 in the GO-203 treated arm versus 1080 mm3 in the control arm rendering a p-value of 0.0001).Conclusion: The data demonstratesthat MUC1-C is highly expressed in CTCL cell lines, and that blockade of the oncogenic MUC1-C subunit with the peptide inhibitor GO-203 induces apoptosis in vitro and in vivo. Targeting MUC1-C in in vitro is associated with down-regulation of TIGAR and increases in ROS, indicating that MUC1-C functions in maintaining redox balance. Our results highlight the potential of MUC1-C to be an exciting new therapeutic target in CTCL. DisclosuresKufe:Genus Oncology: Consultancy, Equity Ownership.

Ciglitazone enhances ovarian cancer cell death via inhibition of glucose transporter-1

Ciglitazone is a peroxisome proliferator-activated receptor γ (PPARγ) agonist and improves insulin sensitivity. Apart from antidiabetic activity, ciglitazone elicits inhibitory effects on cancer cell growth. Recent studies indicate that glucose metabolism plays a key role in malignant diseases. Significant increase in glucose consumption is found under malignant conditions. The role of ciglitazone in cancer cell death in relation to glucose metabolism is unclear. Thus we designed this study to determine the effect of ciglitazone on glucose metabolism. First, we found ciglitazone inhibited glucose uptake in ovarian cancer cells but did not affect hexokinase activity. Ciglitazone decreased expression levels of glucose transporter-1 (GLUT-1). We also found that ciglitazone and siGLUT-1 treatments induced cell death in ovarian cancer cells. We identified that ciglitazone decreased expressions of specific protein 1 (Sp-1) and β-catenin while increased phosphorylation levels of AMP-activated protein kinase. In vivo study using NOD-scid IL2Rgammanull mice confirmed that ciglitazone significantly decreased ovarian cancer mass transplanted onto the back of the mice. Finally, we determined GLUT-1 expressions in patients with serous type ovarian cancer and found that GLUT-1 expression was markedly increased in cancer patients and expression level was proportional to the degree of cancer stages. These results suggest that ciglitazone induces apoptosis in ovarian cancer cells by the inhibition of GLUT-1 and provides a possible therapeutic effect of ciglitazone as an adjuvant drug in the treatment of ovarian cancer.

Genetic Profiling By Targeted, Deep Resequencing Confirms That a Murine Xenograft Model Of Acute Myeloid Leukemia (AML) Recapitulates The Mutational Landscape Of The Human Disease and Provides Evidence For Clonal Heterogeneity and Clonal Evolution

BackgroundA growing number of recurrent gene mutations have been identified in AML through novel sequencing techniques. To uncover the functional consequences of these mutations and identify novel therapeutic targets, adequate model systems are needed. Such models should recapitulate the mutational landscape and genetic heterogeneity of AML as closely as possible. In this respect, analyses of primary tumor cells are superior to established permanent cell lines. Xenografts of primary AML blasts in immunodeficient mice thus may represent a valuable platform for functional analyses on a wide spectrum of AML subtypes. However, it has not been determined whether the mutational architecture of AML xenografts faithfully represents the human disease they originate from. MethodsFresh bone marrow or peripheral blood samples were obtained from adult AML patients. One to ten million cells were injected intravenously or intrafemorally into recipient NOD-scid IL2R-gammanull(NSG) mice. Engraftment was monitored by serial immunophenotyping using anti-human CD33 and anti-human CD45 antibodies. Mice showing >35% human cells in the peripheral blood or signs of disease were sacrificed, and xenotransplanted cells re-isolated from femurs and spleens. Using genomic DNA from xenografts and paired primary human specimens, we profiled mutations in 44 genes recurrently mutated in human hematologic malignancies by targeted, deep amplicon resequencing (Agilent Haloplex / Illumina MiSeq). ResultsTo date, 4 pairs of primary AML specimens and matched xenografts have been genetically characterized by targeted resequencing. We obtained between 480k and 1100k paired-end reads (2x250bp) per sample, resulting in >30x coverage for >98.7% of the target sequence. The median coverage for individual target regions ranged from 116-fold (CEBPA) to ∼5200-fold (FLT3).In each patient specimen, 2 to 5 known AML-associated driver mutations were identified in our panel of 44 genes. The Figure shows variant allele frequencies (VAFs) for mutations and known germline polymorphisms (SNPs; dbSNP database v137), in the patient samples and matched xenografts. All mutations that were present in the primary patient samples with a VAF of >10% were also found in the matched xenograft. None of the xenografts acquired new mutations that were undetectable in the original patient specimen. However, each sample pair showed evidence for clonal diversity and clonal evolution. In patient AML361, NPM1, DNMT3A and BCOR mutations were detected at a VAF slightly below .5, consistent with heterozygous mutations present in most cells in the primary specimen. Additionally, a FLT3-internal tandem duplication (ITD) was present at a lower VAF, likely representing a subclonal mutation. In the corresponding xenograft, the FLT3-ITD was observed in a significantly larger fraction of cells, suggesting that the FLT3-mutated clone had a relative growth advantage. Similarly, patient AML393 carried a subclone with mutated BCOR that became the dominant clone in the xenograft. Conversely, in patients AML372 and AML373 we found subclonal NRAS mutations (VAF, 6% and 7%, respectively) that were undetectable in the matched xenografts, indicating that the NRAS-mutated subclones did not engraft. We are currently studying parallel lines of xenografts generated from the same patient, followed by serial re-passaging in NSG mice, to better characterize growth patterns of such patient-derived subclones in our model. Updated results will be presented at the meeting. [Display omitted] Genotypes for known germline SNPs were fully concordant between the human specimens and matched xenografts in 3/4 pairs. The fourth patient (AML393), who had AML relapse after an allogeneic stem cell transplant, showed several SNPs with a low VAF that were not identified in the corresponding xenograft. These SNPs most likely originate from residual donor hematopoiesis in the patient, indicating that non-malignant cells of donor origin did not engraft in the NSG mouse. ConclusionXenografts of primary AML blasts in NSG mice recapitulate the patterns of gene mutations observed in AML patients, and thus provide an opportunity to study the biology of various genetic AML subgroups. Deep, targeted amplicon resequencing can sensitively detect subclonal driver mutations, and in conjunction with the xenograft model can be used to study clonal diversity and clonal evolution in AML. Disclosures:Greif:Illumina:Honoraria.

Abstract 5530: Rejuvenation of dissociated cryopreserved human ovarian papillary serous tumor cells in an in vivo model.

Abstract Banked tumor tissues are a vital source of research material; however, current preservation techniques rely on flash-freezing. While this storage technique maintains the integrity of the nucleic acids and stable proteins, it does not allow for the recovery of live cells from the banked specimen. Serous carcinomas are the most common and deadliest form of gynecologic cancers. These tumors are heterogeneous with a wide array of genetic alterations. Our lab has developed a method which allows banking of live cells derived from primary ovarian serous papillary carcinoma specimens. Here we show that viable cell populations of interest can be isolated from banked cryopreserved tumor samples and grown in vivo as a potential source for analysis of individual tumors. Primary serous tumor samples were subjected to mechanical disruption and enzymatic digestion to produce dissociated cell suspensions. Samples were cryopreserved using controlled temperature reduction and stored in liquid nitrogen vapor. Viability of banked human tumor specimens was evaluated as the ability to establish a xenograft that histologically resembled the primary tumor. Serous tumor epithelia were isolated by flow cytometry using a cell surface marker, epithelial cell adhesion molecule (EpCAM), from this banked cryopreserved cellular population. Xenografts were generated by subcutaneous injection into NOD-scid IL2Rgammanull mice. Growth was monitored by palpation and regenerated tumors were analyzed for histology and marker expression profile. Nine serous tumor samples were banked using this method. To prove the viability of this cryopreservation process, 3 dissociated tumor cell preparations were thawed and injected into NOD-scid mice. Within 6 months, all 3 samples grew as xenografts that closely resembled the primary serous tumor. We then tested if viable cellular fractions of interest could be isolated from these banked serous tumors cells using fluorescent activated cell sorting. A subpopulation of tumors cells expressing EpCAM was collected from the cryopreserved tumor cell population and injected to establish xenografts. In this in vivo assay, xenografts grew as epithelial tumors in 7 of 9 cases. Our findings demonstrate that cryopreservation of dissociated primary tumor cells is a viable option for the generation of live cell tumor banks for papillary serous carcinoma. The advantages of a live cell tumor bank are numerous: 1) characterization of subpopulations of tumor cells in each patient's tumor sample at the RNA, DNA, and protein levels and 2) defining heterogeneity of a serous tumor with a special focus on identifying cancer initiating populations in each patient. We foresee that characterization of each patient's tumor sample using this live tissue bank may help individualize therapeutic strategies in patients with serous cancers. Citation Format: Daniel Y. Paik, Deanna Janzen, Sanaz Memarzadeh. Rejuvenation of dissociated cryopreserved human ovarian papillary serous tumor cells in an in vivo model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5530. doi:10.1158/1538-7445.AM2013-5530

Transplantation of neonatal islets from LEA29Y‐transgenic pigs restores normoglycemia in streptozotocin‐diabetic NSG‐mice

Beta cell replacement therapy represents the only way towards complete restoration of the physiological glucose homeostasis in patients with Type 1 diabetes. Xenotransplantation of transgenic pig islets expressing immunomodulatory molecules might represent a promising approach to overcome the problems of lacking organ donors, adverse effects of the required systemic immunosuppression, and disappointing transplantation outcomes. LEA29Y, a second generation CTLA‐4‐Ig fusion protein exerts potent immunosuppressive effects by selective blockade of the B7 costimulatory pathway. To assess the impact of high local LEA29Y concentrations on xenogeneic islet graft rejection, a transgenic pig model with islet‐specific LEA29Y transgene expression was generated.Islet‐like clusters from neonatal pigs expressing LEA29Y under the control of the porcine insulin promoter and from wild‐type pigs were isolated by collagenase digestion and subsequent in vitro culture. After 6 days 2500 clusters/mouse were transplanted under the kidney capsule of streptozotocin‐diabetic NOD‐scidIL2Rgammanull (NSG) mice. After an initial period of insulin dependency, mice developed stable normoglycemia within 8 weeks after transplantation. Transplanted mice of both groups (Tx, wt; Tx, LEA29Y) exhibited normal and as compared to non‐transplanted normoglycemic NSG mice even improved fasting glucose and glucose tolerance resulting from glucose‐responsive graft‐derived porcine insulin secretion. Beta cell‐specific expression of LEA29Y was detectable in neonatal porcine pancreas, in the grafts of transplanted mice, and was also measurable in the plasma of normoglycemic mice. Furthermore, mice with LEA29Y transgenic grafts exhibited a glucose‐dependent LEA29Y release during glucose tolerance testing.The present study demonstrates that LEA29Y transgenic islet‐like clusters display normal beta cell function and have the same potential as wild‐type islet clusters to restore normoglycemia in streptozotocin‐diabetic NSG mice after an in vivo maturation period towards a functional endocrine tissue. NSG mice with LEA29Y islet grafts may therefore represent a promising model to study the modulation of human‐anti‐pig xenograft rejection in detail. In ongoing experiments xenograft rejection is analyzed in “humanized” mice after the transfer of human PBMCs or isolated CD34+ stem cells.