ALBERT

Description: HGAL is a germinal center (GC)–specific gene that negatively regulates lymphocyte motility and whose expression predicts improved survival of patients with diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin lymphoma (cHL). We demonstrate that HGAL serves as a regulator of the RhoA signaling pathway. HGAL enhances activation of RhoA and its down-stream effectors by a novel mechanism – direct binding to the catalytic DH-domain of the RhoA-specific guanine nucleotide exchange factors (RhoGEFs) PDZ-RhoGEF and LARG that stimulate the GDP-GTP exchange rate of RhoA. We delineate the structural domain of HGAL that mediates its interaction with the PDZ-RhoGEF protein. These observations reveal a novel molecular mechanism underlying the inhibitory effects of GC-specific HGAL protein on the motility of GC-derived lymphoma cells. This mechanism may underlie the limited dissemination and better outcome of patients with HGAL-expressing DLBCL and cHL.

Description: Abstract 316 Identification of genes whose expression correlates with clinical outcome is of paramount importance for designing upfront patient-tailored treatments that will potentially improve the outcome of lymphoma patients. Furthermore, new molecular prognostic biomarkers may lead to the discovery of novel pathophysiological mechanisms underlying lymphoma pathogenesis, potentially highlighting unique biological processes regulated by these proteins in lymphoma cells and in their normal cellular counterparts. HGAL is a novel germinal center (GC)-specific gene whose expression predicts survival of patients with diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin lymphoma (cHL). Recently, we have demonstrated that HGAL is involved in negative regulation of lymphocyte migration, thus potentially constraining lymphocytes to the GC and preventing lymphoma dissemination; however, the molecular mechanism of HGAL effects on cell motility is unknown. Herein, we demonstrated that HGAL serves as a physiological regulator of the RhoA signaling pathway. HGAL over-expression or siRNA-mediated knock-down leads to increased or decreased levels of GTP-bound RhoA, respectively, but does not affect levels of GTP-bound CDC42 and RAC1. HGAL-induced activation of RhoA results in inhibition of lymphocyte and lymphoma cell motility and chemotaxis by activation of its downstream effector ROCK leading to: a) phosphorylation of myosin regulatory light chain (MRLC) that regulates actin-activated Mg-ATPase activity of myosin II; b) phosphorylation of myosin phosphatase (myosin PPTase) subunit MYPT1 inhibiting myosin light chain dephosphorylation; and c) phosphorylation of cofilin leading to cytoskeletal reorganization, as reflected by increased formation of focal adhesions and stress fibers. Opposite effects on RhoA downstream effectors are observed upon knock-down of HGAL expression. Furthermore, HGAL over-expression or knock-down modulate additional functions of RhoA, including transcriptional activation by serum response factor and RhoA transforming potential, as assessed by foci formation in NIH 3T3 cells. Co-immunoprecipitation experiments demonstrated that the effect of HGAL on RhoA is indirect and is mediated by RhoA-specific guanine nucleotide exchange factors (RhoGEFs) PDZ-RhoGEF and LARG that stimulate the GDP-GTP exchange rate. Moreover, we delineate the structural domains of HGAL and PDZ-RhoGEF that mediate the interaction between these proteins. Over-expression of HGAL mutants lacking the PDZ binding domain, mediating the interaction between HGAL and these RhoGEFs, fails to induce activation of RhoA and does not inhibit lymphoma cell motility and chemotaxis. Similarly, knock-downs of PDZ-RhoGEF and/or LARG reverse the inhibitory effects of HGAL on lymphoma cell motility and chemotaxis. These observations reveal a novel molecular mechanism underlying the inhibitory effects of HGAL on the motility of normal GC lymphocyte and GC-derived lymphoma cells. Since HGAL is specifically expressed only during the GC stage of B lymphocyte differentiation, these findings highlight the uniqueness and complexity of cell processes occurring during the GC reaction. They further elucidate the pathophysiologic mechanism underlying the favorable outcome of DLBCL and cHL patients whose tumors express high levels of HGAL protein. Disclosures: No relevant conflicts of interest to declare.

Description: Anti-tumor efficacy of genetically modified T cells depends on in vivo expansion and durable persistence of infused cells. Multiple variables including the structure of the CAR and characteristics of the recipient impact the anti-tumor effect of CAR+ T cells. However, a code for an optimal CAR design that would deliver clinically relevant result is yet to emerge. Here we propose a new measure of "fitness" for CAR+ T cells based on mitochondrial biomass that is quantifiable and could be translated to clinical settings. Spare respiratory capacity (SRC) is defined as the extra mitochondrial capacity available in a cell to produce energy under conditions of increased work or stress. Memory T cells capable of responding to infection has been shown to possess extra SRC (Windt et al., Immunity 2012). We therefore investigated whether subsets of CD19-specific CAR+ T cells after electro-transfer of Sleeping Beauty (SB) plasmids and propagation on activating and propagating cells (AaPC) could be identified based on SRC. Transmission electron microscopy revealed that genetically modified T cells revert to a condensed state of mitochondria after 2 weeks of activation through a second-generation CD19-specific CAR. However, mock-electroporated T cells activated by cross-linking CD3 (using AaPC loaded with OKT3) retain a classic mitochondrial structure. Moreover, antigen-driven numeric expansion in presence of membrane bound IL-15 led to an increase in mitochondrial biomass in CAR+ T cells. We extended these observations to various CAR+ T cells with unique specificity for tumor antigens and found similar changes in mitochondrial structure and distribution. Next, we examined if an increase in mitochondrial biomass influences functionality of genetically modified T cells. By SB mediated transposition CARs were co-expressed along with a fluorescence reporter protein (EYFP-GRX2) constituting yellow fluorescent protein fused to the mitochondrial localization sequence of GRX2 to track mitochondrial distribution in live cells. The genetically modified T cells were selectively propagated by stimulating the CARs using a proprietary monoclonal antibody that binds to a common extracellular stalk motif in CAR construct. CAR+ T cells that signaled through chimeric CD137z exhibited a high mitochondrial mass (EYFPhigh) and had superior rates of expansion ex vivo. In contrast, CAR+ T cells that signaled through chimeric CD28z had a low mitochondrial mass (EYFPdim), elevated levels of apoptosis, and inferior rates of numeric expansion. Confocal microscopy showed EYFP counts were higher for CAR+ T cells that signaled through CD137 signaling domain. We hypothesize that increased survival of CD137z-CAR T cells in a challenging cell culture environment could be due to reserve bio-energetic potential concomitant with the ability to meet metabolic demand of activated T cells. Further, SRC could be quantified using a fluorescent probe for mitochondrial mass pre-infusion which may be a defining criterion attesting to the fitness of CAR+ T cells for human applications. Disclosures Jena: Intrexon: Equity Ownership, Patents & Royalties: Potential royalties (Patent submitted); Ziopharm Oncology: Equity Ownership, Patents & Royalties: Potential roylaties (Patent submitted). Rushworth:Intrexon: Other: Potential Equity ownership; Ziopharam Oncology: Other: Potential Equity Ownership. McNamara:Ziopharm Oncology: Equity Ownership, Patents & Royalties: Potential royalties, Research Funding; Intrexon: Equity Ownership, Patents & Royalties: Potential royalties, Research Funding. Cooper:Ziopharm Oncology: Employment, Equity Ownership, Patents & Royalties, Research Funding; Intrexon: Equity Ownership, Patents & Royalties.

Description: Human epidermal growth factor receptor (EGFR) family consists of four members i.e. EGFR (HER1), HER2 (ErbB2), HER3 (ErbB3,) and HER4 (ErbB4). Overexpression, mutation, or catalytic activation of these proteins can lead to malignancies in breast, ovarian, colorectal, pancreatic and lung. Therapies targeting EGFR-associated proteins to disrupt signaling may fail because of crosstalk within the EGFR family or among downstream pathways. One mechanism of escape is HER3 activation and concomitant heterodimer formation with HER1 causing disease relapse and treatment failure. A bi-specific monoclonal antibody (mAb, MEHD7945A) can specifically bind an epitope shared between HER1-HER3 heterodimer thereby blocking EGFR-HER3 mediated signaling (Schaefer et al., Cancer Cell, 2011). We now report that the specificity of this mAb can be used to redirect the specificity of T cells through enforced expression of a chimeric antigen receptor (CAR) targeting the HER1-HER3 heterodimer, such as expressed on breast cancer cells. A 2nd generation CAR targeting the HER1-HER3 heterodimer was expressed from DNA plasmid constituting scFv (designated DL11f, derived from mAb MEHD7945A) coupled to CD3-zeta fused in frame with chimeric CD28 or CD137 T-cell signaling domains on a clinical-grade Sleeping Beauty (SB) backbone. T cells were electroporated with SB system and numerically expanded on irradiated “universal” activating and propagating cells (uAaPC) (Rushworth et al., J Immunotherapy, 2014). These feeder cells are derived from K-562 cells engineered to co-express a CAR activating ligand (CAR-L, a scFV specific to CAR stalk) to sustain proliferation of genetically modified T cells. We validated CAR expression on genetically modified T cells by flow cytometry and western blot. The specificity of HER1-HER3 specific CAR T cells was confirmed in situ by a proximity ligation-based assay using breast cancer cells. The redirected killing by CAR+ T cells to HER1+HER3+ breast cancer cells was confirmed in vitro and its efficacy evaluated in vivo in NSG mice bearing a breast tumor xenograft. HER1-HER3 specific CAR+ T cells activated via CD137 signaling exhibited superior proliferation compared with T cells expressing CAR with CD28 signaling domain. This is consistent with the ability of CD3-zeta/CD137 endodmain to alter mitochondrial metabolism and to suppress apoptosis leading to proliferation after initial activation. In summary, we report a new CAR design that can interrogate the conformation between two tumor-associated antigens (TAAs). This will likely improve specificity and limit on-target off-tissue side effects compared to CARs targeting only HER-1 or HER-3. Thus, targeting an epitope derived from two TAAs may help distinguish normal cells versus malignant cells and treat HER1+HER3+ malignancies that are resistant to therapies targeting single EGFR family members. These data have immediate translation appeal for targeting solid tumors as we use the SB and AaPC platforms to manufacture CAR+ T cells in our clinical trials. Disclosures Cooper: InCellerate: Equity Ownership; Sangamo: Patents & Royalties; Targazyme: Consultancy; GE Healthcare: Consultancy; Ferring Pharmaceuticals: Consultancy; Fate Therapeutics: Consultancy; Janssen Pharma: Consultancy; BMS: Consultancy; Miltenyi: Honoraria.

Description: Background T-cell receptors (TCRs) can be used to redirect the specificity of T-cells for human application. This has particular appeal for the targeting of neoantigens. However, efficient identification, cloning, and characterization of antigen (Ag)-specific TCRs is needed to enable the timely adoptive transfer of T-cells genetically modified to express therapeutic TCRs. We have harnessed next generation sequencing (NGS) to identify desirable TCRs. This approach enables us to simultaneously identify hundreds of Ag-specific TCRs, along with the expression of genes to characterize functional and phenotypical values of individual Ag-specific T-cells (e.g. related to cytotoxicity, exhaustion, etc...). To take advantage of NGS technology, a sister high-throughput technology was developed to evaluate harvested TCRs. A reporter system was implemented using (immortalized) Jurkat T-ALL cells genetically modified to (i) enforce expression of CD8aβ, (ii) conditionally express GFP under minimal elements of NR4A1 promoter, and (iii) prevent expression of both endogenous TCRα and β chains. Sequenced CDR3 regions were coded within DNA plasmids from Sleeping Beauty (SB) transposons as TCR Vα and Vβ libraries that were expressed on the reporter cell to identify both TCR specificity as well as TCR avidity. Thus, we implemented (i) a TCR cloning system based on CDR3 sequencing of Ag-specific T-cells identified by NGS and (ii) a novel reporter cell based on the TCR-mediated induction of GFP expression. As a proof-of-concept to evaluate the entire platform, we used HLA-A2-restricted CMV peptide (NLVPMVATV: CMV/A2) and NY-ESO-1 peptide (SLLMWITQC: NY-ESO-1/A2) as model Ags. Results The reporter cell was initially genetically modified with either high- or low-avidity TCRs against a NY-ESO-1/A2. Upon stimulation with HLA-A2+ 721.221 immortalized B cells, it was found that the expression of GFP positively correlated with the avidity of TCRs (Figure). Next, we isolated naïve Ag-specific T-cells from umbilical cord blood using CMV/A2 tetramer. Single CMV/A2-specific CD8+ T-cells were sorted and their TCRαβ CDR3 sequences were amplified by reverse transcription and PCR with bar-coded probes. The pooled PCR products were sequenced in MiSeq Sequencer (illumina) to obtain TCRαβ CDR3 regions and analyzed in silico using IMGT (International Immunogenetics Information System). The efficiency of identifying individual TCRαβ pairs was 65% to 76% (using 96-well plate or 384-well plates, respectively). Individual SB-derived DNA transposons expressing TCRαβ constructs were synthesized by Gibson assembly and electroporated, with SB transposase, into the reporter cell. These cells were co-cultured with HLA-A2+ 721.221 stimulator cells loaded with graded doses of cognate peptide. The percentage and intensity of GFP expression was evaluated by high-throughput flow-cytometer (IntelliCyt) which revealed high-avidity Ag-specific TCRαβs. Conclusion Our new high-throughput system can identify and characterize, based on specificity and avidity, Ag-specific TCRαβs within a week. This system will be used to generate neoantigen-specific TCRαβs for human application. Disclosures Zong: ZIOPHARM Oncology, Inc.: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties; Immatics US, Inc: Equity Ownership, Patents & Royalties. Cooper:Ziopharm Oncology: Employment, Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership; City of Hope: Patents & Royalties; Targazyme, Inc.,: Equity Ownership; Immatics: Equity Ownership; Sangamo BioSciences: Patents & Royalties; MD Anderson Cancer Center: Employment; Miltenyi Biotec: Honoraria. McNamara:GeoMcNamara: Consultancy, Other: Consultant in immuno-oncology field; ZIOPHARM Oncology, Inc: Equity Ownership, Patents & Royalties; Intrexon: Equity Ownership, Patents & Royalties. Torikai:intrexon: Equity Ownership, Patents & Royalties; ZIOPHARM Oncology, Inc.: Equity Ownership, Patents & Royalties; Immatics US, Inc: Equity Ownership.

Description: Key Points HGAL protein can be myristoylated and palmitoylated, and these modifications localize HGAL to lipid rafts. Raft localization of HGAL protein facilitates interaction with Syk, and modulation of BCR activation and signaling.

Description: Companion canine cancer provides a clinically-relevant immunotherapy model for human malignancies because of their large size, intra-species genetic diversity, genetic similarity of tumors to human oncology, and spontaneously occurring tumors that develop despite an intact immune system. Canine B-lineage non-Hodgkin lymphoma (NHL) is genetically, molecularly, and physiologically similar to human NHL and the various treatment modalities can be interchanged with exception of antibody-based chemotherapies. Canine NHL represents 25% of all canine cancers diagnosed in the United States among the more than one million pet dogs diagnosed with cancer annually. As in humans, the common presentation is Stage III or Stage IV disease. It is rarely curable in the out-bred dog and even with aggressive chemotherapy the median survival rate is less than a year. Current standard-of-care (SOC) combination chemotherapy, CHOP, induces remission in approximately 85% of dogs. Thus, the companion canine provides a model for human T-cell therapy and addresses an unmet need of dogs diagnosed with NHL. Our initial canine trial (Sci Rep. 2012;2:249) demonstrated a survival advantage upon reconstitution of the immune system with autologous ex vivo-activated CD3+CD8+ T cells after CHOP for pet dogs with NHL. These T cells were propagated on engineered artificial antigen presenting cells (aAPC), which are also used for our human T-cell trials. Our updated survival data shows significant improvement in overall survival (p = 0.01) and tumor-free survival (p = 0.0008) compared with matched historical controls (Figure). We also report initial data on an additional trial infusing donor- or patient-derived canine T cells after allogeneic or autologous hematopoietic stem-cell transplantation (HSCT), respectively. The first allogeneic and autologous recipients with NHL safely received 2 escalating doses of activated T cells and achieved an anti-tumor response. As in our first trial, granzyme B expression in the propagated T cells continues to significantly correlate with prognosis. A third trial tests whether steroid-resistant T cells can persist in pet dogs with NHL receiving systemic dosing of corticosteroids. Fluorescent dyes were used to distinguish infused T cells and demonstrated that steroid-resistant activated T cells survived compared to infused control T cells. In all trials, single-cell studies are underway to determine genetic factors concordant with the survival of infused T cells and treated companion canines. Global peripheral blood T-cell gene signatures, as quantitated by nanostring, were measured at 3 hours, 7 to 14 days, and 35 to 42 days post infusion. The different gene patterns between infused dogs and post infusion time points may reflect changes in en vivo T-cell activation and companion canine prognosis. In conclusion, the add-back of activated CD8+ T cells in dogs with NHL can inform of the immunobiology of adoptive transfer of T cells in humans. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 584 The Human Germinal center Associated Lymphoma (HGAL) gene is exclusively expressed in germinal center (GC) B-lymphocytes and GC-derived lymphomas. In patients with diffuse large B-cell lymphomas (DLBCL), HGAL expression identifies a subgroup of patients with biologically distinct tumors associated with improved survival. Our previous in vitro studies demonstrated that HGAL decreases spontaneous and chemoattractant-induced cell motility by activating the RhoA signaling pathway and by directly interacting and augmenting F-actin and myosin II binding. However, the major function of HGAL in GC lymphocytes remains largely unknown. Based on our previous observation of tyrosine phosphorylation of a modified ITAM motif in the HGAL by Lyn, we hypothesized that HGAL may be involved in B-cell receptor (BCR) signaling. Indeed, following BCR stimulation of two GCB-like lymphoma cell lines (Raji and VAL), we observed marked reduction of Syk, Btk and PLCγ phosphorylation upon knockdown of endogenous HGAL by specific but not control siRNAs. Concordantly, HGAL knockdown in BCR-stimulated Raji cells reduced Ca2+ mobilization and decreased NFAT transcriptional activity as analyzed by a luciferase reporter assay. HGAL expression in the BCR-stimulated HBL1 lymphoma cell line (lacking endogenous HGAL protein) resulted in increased Syk, Btk and PLCγ phosphorylation. Syk plays a major role in coupling BCR activation to downstream effectors. Endogenous HGAL was detected in immunoprecipitates of endogenous Syk and vice versa. Nanoscope microscopy studies confirmed co-localization of HGAL and Syk proteins in cell membranes, which was enhanced following BCR stimulation. In BCR-stimulated cells, Syk kinase activity was markedly increased following addition of HGAL protein as measured by an in vitro Syk kinase activity assay. To comprehensively examine HGAL effects on immune system and BCR signaling, we generated a transgenic mouse model in which HGAL is expressed under the control of the mouse Ly-6E.1 promoter in Sca1+ hematopoietic stem cells and progenitors of C57BL/6 × CBA mice. The Sca1-HGAL transgenic mice showed normal embryonic and post natal development, and at 8 weeks of age demonstrated normal lymphoid development without any significant changes in the major hematopoietic compartments (bone marrow (BM), spleen, thymus and peripheral lymph nodes) and in peripheral blood. They also exhibited normal GC development in response to a T-cell dependent antigen immunization. In contrast, at 12 months of age the Sca1-HGAL mice developed a decrease in BM immature B-cells at the expense of recirculating B-cells (B220+IgDhi) compared to the age-matched normal littermates, suggesting a defect in B-cell lymphopoiesis. All the Sca1-HGAL transgenic mice became ill from approximately 12 months of age and all died between 12 to 22 months of age with statistically shorter survival as compared to the wild type controls. Analysis of these animals showed massive splenomegaly with marked white pulp hyperplasia and presence of multiple, frequently contiguous nodules predominantly composed of polyclonal follicular (B220+CD21intCD23hi) B lymphocytes. Extra-lymphatic infiltration by similar B lymphocytes was observed in the liver, lungs and kidneys of Sca1-HGAL mice with advanced disease. IgG isotype titers in these animals tended to be higher than in the wild-type controls, reaching a statistically significant difference for the IgG1 isotype. Follicular hyperplasia in the Sca1-HGAL transgenic mice is likely attributable to increased RhoA activation and enhanced BCR signalling manifested by increased Syk phosphorylation, Ca2+ mobilization and in vitro B cell proliferation following BCR stimulation, in agreement with similar data observed in human DLBCL cell lines expressing HGAL. Gene expression profiling of lymphoid tissues confirmed significantly enhanced BCR signalling and RhoA pathway activation in Sca1-HGAL transgenic mice, corresponding to similar pathway activation in human lymphoma cell lines over-expressing HGAL. Overall, our findings demonstrate that HGAL, specifically expressed in GC B cells, enhances responsiveness to antigens by stimulating Syk kinase activity that without appropriate regulation may lead to lymphoproliferation. Further studies are needed to examine the role of HGAL in the pathogenesis of GC-derived lymphomas. Disclosures: No relevant conflicts of interest to declare.

Description: A debate has raged in recent years over whether cells within the bone marrow have the capacity to generate non-hematopoietic, specialized tissue in other organs. Previous reports have studied bone marrow transplantation in adult recipients; most models have found that tissue injury was necessary for organ engraftment of bone marrow and at best low levels of engraftment are seen. In the present study, we show complex ductal epithelial structures in the pancreas completely derived from bone marrow transplanted during the neonatal period in the absence of tissue injury. BRDU labeling showed that epithelial cells lining the ducts and islet cells were cycling at significantly higher levels in the neonatal than in the adult pancreas (P

Description: Lineage-specific cell-surface molecules, such as CD19 and CD20 on B-lineage malignancies are the targets for immunotherapies. However, the therapeutic success of CD19-specific T-cell therapy is predicted to be dependent on the continued persistence of adoptively transferred T cells. It is well-recognized that exogenous IL-2 can help sustain the in vivo persistence of ex vivo-propagated CD8+ T cells. Therefore, in order to target biologically active IL-2 to the tumor microenvironment, we have used an immunocytokine (ICK) to deliver this cytokine to binding-sites of a CD20-specific monoclonal antibody (mAb) on malignant B cells. This anti-CD20-IL2 ICK was based on the Leu16 anti-CD20 mAb that was dehumanized to remove T-helper epitopes. Flow cytometry demonstrated that anti-CD20-IL2 ICK specifically bound to CD20+ tumor and IL-2R+ T cells. Thus, we investigated the ability of this ICK to improve the persistence of adoptively transferred B-lineage lymphoma-specific T cells. To obtain CD19-specific T cells that could be non-invasively imaged in vivo, we used non-viral gene transfer to introduce a DNA plasmid to co-express both a CD19-specific immunoreceptor (designated CD19R) and firefly luciferase (ffLuc). The CD19R combines antibody recognition with T-cell effector functions mediated through CD3-ζ . The genetically modified T cells were characterized as differentiated CD8+ effector cells expressing the IL-2 receptor complex, which specifically recognize and lyse CD19+ lymphoma targets. To model the survival of adoptively transferred T cells and treatment of lymphoma in vivo, we generated a CD20+CD19+ ARH-77 tumor line expressing the Renilla luciferase (rLuc) reporter gene. Our data demonstrate that this tumor line is resistant to a CD20-specific mAb, Rituximab, in vivo. Sub-optimal doses of CD19R+ffLuc+ CD8+ T cells, which do not cause complete eradication of tumor by themselves, were infused along with anti-CD20-IL2 ICK and control ICK (with irrelevant specificity) in NOD/scid mice bearing xenografts of the rLuc+ tumor. In vivo non-invasive bioluminescent imaging (BLI) was used to longitudinally measure the persistence of ffluc+ T cells and growth of rluc+ tumor. In our mouse model, the T cells persisted significantly (p