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CSNK2B has recently been implicated as a disease gene for neurodevelopmental disability (NDD) and epilepsy. Information about developmental outcomes has been limited by the young age and short follow-up for many of the previously reported cases, and further delineation of the spectrum of associated phenotypes is needed. We present 25 new patients with variants in CSNK2B and refine the associated NDD and epilepsy phenotypes. CSNK2B variants were identified by research or clinical exome sequencing, and investigators from different centers were connected via GeneMatcher. Most individuals had developmental delay and generalized epilepsy with onset in the first 2 years. However, we found a broad spectrum of phenotypic severity, ranging from early normal development with pharmacoresponsive seizures to profound intellectual disability with intractable epilepsy and recurrent refractory status epilepticus. These findings suggest that CSNK2B should be considered in the diagnostic evaluation of patients with a broad range of NDD with treatable or intractable seizures.

To safely re-open economies and prevent future outbreaks, rapid, frequent, point-of-need, SARS-CoV-2 diagnostic testing is necessary. However, existing field-deployable COVID-19 testing methods require the use of uncomfortable swabs and trained providers in PPE, while saliva-based methods must be transported to high complexity laboratories for testing. Here, we report the development and clinical validation of High-Performance Loop-mediated isothermal Amplification (HP-LAMP), a rapid, saliva-based, SARS-CoV-2 test with a limit of detection of 1.4 copies of virus per µl of saliva and a sensitivity and specificity with clinical samples of > 96%, on par with traditional RT-PCR based methods using swabs, but can deliver results using only a single fluid transfer step and simple heat block. Testing of 120 patient samples in 40 pools comprised of 5 patient samples each with either all negative or a single positive patient sample was 100% accurate. Thus, HP-LAMP may enable rapid and accurate results in the field using saliva, without need of a high-complexity laboratory.

Rapid, scalable, point-of-need, COVID-19 diagnostic testing is necessary to safely re-open economies and prevent future outbreaks. We developed an assay that detects single copies of SARS-CoV-2 virus directly from saliva and swab samples in 30 min using a simple, one-step protocol that utilizes only a heat block and microcentrifuge tube prefilled with a mixture containing the necessary reagents and has a sensitivity and specificity of 97% and 100%, respectively.

Adoptive cell therapy with genetically modified T cells expressing a chimeric antigen receptor (CAR) is a promising therapy for patients with B-cell acute lymphoblastic leukemia. However, CAR-modified T cells (CAR T cells) have mostly failed in patients with solid tumors or low-grade B-cell malignancies including chronic lymphocytic leukemia with bulky lymph node involvement. Herein, we enhance the antitumor efficacy of CAR T cells through the constitutive expression of CD40 ligand (CD40L, CD154). T cells genetically modified to constitutively express CD40L (CD40L-modified T cells) demonstrated increased proliferation and secretion of proinflammatory TH1 cytokines. Further, CD40L-modified T cells augmented the immunogenicity of CD40(+) tumor cells by the upregulated surface expression of costimulatory molecules (CD80 and CD86), adhesion molecules (CD54, CD58, and CD70), human leukocyte antigen (HLA) molecules (Class I and HLA-DR), and the Fas-death receptor (CD95). Additionally, CD40L-modified T cells induced maturation and secretion of the proinflammatory cytokine interleukin-12 by monocyte-derived dendritic cells. Finally, tumor-targeted CD19-specific CAR/CD40L T cells exhibited increased cytotoxicity against CD40(+) tumors and extended the survival of tumor-bearing mice in a xenotransplant model of CD19(+) systemic lymphoma. This preclinical data supports the clinical application of CAR T cells additionally modified to constitutively express CD40L with anticipated enhanced antitumor efficacy.

We developed a new approach to bioluminescent T cell imaging using a membrane-anchored form of the Gaussia luciferase (GLuc) enzyme, termed extGLuc, which we could stably express in both mouse and human primary T cells. In vitro, extGLuc+ cells emitted significantly higher bioluminescent signal when compared to cells expressing GLuc, Renilla luciferase (RLuc) or membrane-anchored RLuc (extRLuc). In vivo, mouse extGLuc+ T cells showed higher bioluminescent signal when compared to GLuc+ and RLuc+ T cells. Application of this imaging approach to human T cells genetically modified to express tumor-specific chimeric antigen receptors (CARs) enabled us to show in vivo CAR-mediated T cell accumulation in tumor, T cell persistence over time and concomitant imaging of T cells and tumor cells modified to express firefly luciferase. This sensitive imaging technology has application to many in vivo cell-based studies in a wide array of mouse models.

On the basis of promising preclinical data demonstrating the eradication of systemic B-cell malignancies by CD19-targeted T lymphocytes in vivo in severe combined immunodeficient-beige mouse models, we are launching phase I clinical trials in patients with chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia. We present here the validation of the bioprocess which we developed for the production and expansion of clinical grade autologous T cells derived from patients with CLL. We demonstrate that T cells genetically modified with a replication-defective gammaretroviral vector derived from the Moloney murine leukemia virus encoding a chimeric antigen receptor (CAR) targeted to CD19 (1928z) can be expanded with Dynabeads CD3/CD28. This bioprocess allows us to generate clinical doses of 1928z+ T cells in approximately 2 to 3 weeks in a large-scale semiclosed culture system using the Wave Bioreactor. These 1928z+ T cells remain biologically functional not only in vitro but also in severe combined immunodeficient-beige mice bearing disseminated tumors. The validation requirements in terms of T-cell expansion, T-cell transduction with the 1928z CAR, biologic activity, quality control testing, and release criteria were met for all 4 validation runs using apheresis products from patients with CLL. Additionally, after expansion of the T cells, the diversity of the skewed Vbeta T-cell receptor repertoire was significantly restored. This validated process will be used in phase I clinical trials in patients with chemorefractory CLL and in patients with relapsed acute lymphoblastic leukemia. It can also be adapted for other clinical trials involving the expansion and transduction of patient or donor T cells using any CAR or T-cell receptor.

The ability to genetically modify human T cells to target tumor antigens through retroviral gene transfer constitutes a potentially powerful approach to cancer immunotherapy. However, low transduction efficiencies may hamper the efficacy of such therapeutic strategies in the clinical setting. Most commonly, gammaretroviral gene transfer into T cells is conducted through spinoculation, that is, centrifugation of retroviral particles and T cells on RetroNectin-coated non-tissue culture vessels. Here we present data investigating the impact of temperature, speed, and frequency of spinoculation on T cell transduction efficiencies. We found that all three variables independently impacted gene transfer, with increasing temperature, speed, and frequency of spinoculation all enhancing the transduction of T cells. These improved conditions were additive, with the greatest proportion of transduced T cells being generated at the highest tested temperature and speed, after daily spinoculation for 2 to 3 days. Under these conditions, enhanced gene transfer was observed in T cells derived from healthy donors, using research-grade vector stocks. Whereas both RetroNectin and spinoculation were critical to optimal gene transduction, preloading of gammaretroviral particles before spinoculation did not enhance gene transfer. Significantly, application of these enhanced transduction conditions to T cells derived from previously treated patients with chronic lymphocytic leukemia allowed for adequate gene transfer under both small-scale and large-scale clinically applicable conditions using either preclinical or current Good Manufacturing Practice-grade gammaretroviral vector stocks.

We report the findings from the first 10 patients with chemotherapy-refractory chronic lymphocytic leukemia (CLL) or relapsed B-cell acute lymphoblastic leukemia (ALL) we have enrolled for treatment with autologous T cells modified to express 19-28z, a second-generation chimeric antigen (Ag) receptor specific to the B-cell lineage Ag CD19. Eight of the 9 treated patients tolerated 19-28z+ T-cell infusions well. Three of 4 evaluable patients with bulky CLL who received prior conditioning with cyclophosphamide exhibited either a significant reduction or a mixed response in lymphadenopathy without concomitant development of B-cell aplasia. In contrast, one patient with relapsed ALL who was treated in remission with a similar T-cell dose developed a predicted B-cell aplasia. The short-term persistence of infused T cells was enhanced by prior cyclophosphamide administration and inversely proportional to the peripheral blood tumor burden. Further analyses showed rapid trafficking of modified T cells to tumor and retained ex vivo cytotoxic potential of CD19-targeted T cells retrieved 8 days after infusion. We conclude that this adoptive T-cell approach is promising and more likely to show clinical benefit in the setting of prior conditioning chemotherapy and low tumor burden or minimal residual disease. These studies are registered at www.clinicaltrials.org as #NCT00466531 (CLL protocol) and #NCT01044069 (B-ALL protocol).

Purpose: Human T cells targeted to the B cell–specific CD19 antigen through retroviral-mediated transfer of a chimeric antigen receptor (CAR), termed 19z1, have shown significant but partial in vivo antitumor efficacy in a severe combined immunodeficient (SCID)-Beige systemic human acute lymphoblastic leukemia (NALM-6) tumor model. Here, we investigate the etiologies of treatment failure in this model and design approaches to enhance the efficacy of this adoptive strategy.Experimental Design: A panel of modified CD19-targeted CARs designed to deliver combined activating and costimulatory signals to the T cell was generated and tested in vitro to identify an optimal second-generation CAR. Antitumor efficacy of T cells expressing this optimal costimulatory CAR, 19-28z, was analyzed in mice bearing systemic costimulatory ligand-deficient NALM-6 tumors.Results: Expression of the 19-28z CAR, containing the signaling domain of the CD28 receptor, enhanced systemic T-cell antitumor activity when compared with 19z1 in treated mice. A treatment schedule of 4 weekly T-cell injections, designed to prolong in vivo T-cell function, further improved long-term survival. Bioluminescent imaging of tumor in treated mice failed to identify a conserved site of tumor relapse, consistent with successful homing by tumor-specific T cells to systemic sites of tumor involvement.Conclusions: Both in vivo costimulation and repeated administration enhance eradication of systemic tumor by genetically targeted T cells. The finding that modifications in CAR design as well as T-cell dosing allowed for the complete eradication of systemic disease affects the design of clinical trials using this treatment strategy.

Abstract 3121 Cellular therapies through allogeneic hematopoietic stem cell transplants (allo-HSCT) and infusion of genetically modified T cells targeted against tumor associated antigens hold great promise for cancer therapy. However, significant risk of graft-versus-host-disease (GvHD) with allo-HSCT and potential toxicities from the transgene products in the case of genetically modified T cells have generated considerable interest in developing safeguards in the form of suicide genes to allow for the efficient in vivo abrogation of infused T cells in case of unanticipated adverse events. We have previously reported the comparable in vitro function of 3 different suicide gene-prodrug combinations (Park et al. ASH 2010 abstract #3771). The three suicide genes examined included herpes simplex virus thymidine kinase (HSV-TK SR39) with the prodrug ganciclovir, inducible caspase 9 (iCasp9) with the chemical inducer of dimerization (CID), and a novel suicide gene nitroreductase (NTR) derived from E.coli combined with the prodrug metronidazole. Here, we report the comparison of in vivo efficacy of these 3 suicide gene-prodrug combinations using our previously published mouse GvHD model (Santos et al. Nat Med 2009). SCID-Beige mice were injected i.v. with 1×106 FACS-sorted major histocompatibility complex mismatched C57/BL6 T cells transduced to express each suicide gene (HSV-TK SR39, iCasp9 or NTR) cloned in frame with external Gaussia Luciferase (extGLuc), separated by the 2A peptide in an SFG-based vector. Seven days after intravenous injection of modified T cells, SCID-Beige mice developed GvHD as evidenced by in vivo expansion of the transferred T cells detected by bioluminescent imaging (BLI) after a bolus injection of coelenterazine. Subsequently, the mice injected with the HSV-TK SR39-transduced T cells were treated intraperitoneally (i.p.) twice daily for 5 days with ganciclovir at 25mg/kg; the mice with the NTR-transduced T cells were treated i.p. three times daily for 5 days with metronidazole at 200μg; the mice with the iCasp9-transduced T cells were treated i.p. daily for 3 days with CID at 50μg; and the mice with the HSV-TK SR39-transduced T cells treated i.p. twice daily for 5 days with normal saline served as a control group. All treated mice were imaged by BLI on days 3, 7, 11, 18, 25, 32, and weekly thereafter for a total of 3 months following the first dose of each prodrug. All of the control mice treated with normal saline developed rapidly progressing GvHD both clinically and by BLI, meeting the criteria for euthanasia by day 18. In contrast, the administration of prodrugs successfully eradicated the modified T cells expressing respective suicide genes by day 3 and no evidence of GvHD was detected. However, a long-term follow-up of these mice revealed the re-expansion of the transferred T cells or relapse of GvHD in all mice in the NTR group (by day 39) and in 2 out of 3 mice in the HSV-TK SR39 group (by day 53), but none in the mice in the iCasp9 group. The relapsed mice in the NTR and HSV-TK SR groups were treated again with the prodrugs, but none responded to the re-treatment. At the end of the 3 month follow-up, all mice in the NTR group and 2 out of 3 mice from the HSV-TK SR39 group died from progressive GvHD, while all of the mice in the iCasp9 group survived with no evidence of GvHD. In conclusion, our results suggest that the long-term safety and efficacy data of iCasp9, together with its lack of immunogenicity and the absence of reported side effects of CID (Di Stasi et al. ASH 2010 abstract #559), might be advantageous as a promising suicide gene-based strategy in ensuring the rapid and sustained eradication of the adoptively transferred T cells in case of unanticipated severe toxicities of cellular therapies. Based on this in vivo data, we are planning to incorporate the iCasp9 suicide gene in our future clinical trials with T cells genetically modified to express a chimeric antigen receptor targeted against a tumor specific antigen. Disclosures: No relevant conflicts of interest to declare.

Abstract 3771 Adoptive infusion of T cells genetically modified to express chimeric antigen receptors (CARs) targeted to tumor associated antigens (TAAs) is a promising approach to cancer therapy. However, since TAAs are often expressed by normal tissues, safeguards are needed in the form of additional transduced suicide genes to allow for the efficient in vivo abrogation of infused T cells in case of unanticipated adverse events which may develop in the clinical setting. To this end, we have investigated the in vitro function of 3 different suicide genes each inserted distal to a CAR gene targeted against CD19 (19-28z) and a 2A linker peptide cloned into the SFG gammaretroviral vector. Specifically, we have tested the herpes simplex virus thymidine kinase (HSV-TK SR39) with the prodrug ganciclovir, inducible caspase 9 (iCasp9) with the chemical inducer of dimerization (CID), and the E.coli derived nitroreductase (NTR) with the prodrug metronidazole. Cell growth of PG13 murine fibroblasts transduced to express 19–28z CAR with NTR, HSV-TK, and iCasp9 was inhibited by 80% at 1mM of metronidazole, 85% at 1μM of ganciclovir, and 90% at 10nM of CID, respectively, when compared to control PG-13 fibroblasts. The drug concentrations tested in these assays were at physiologically achievable concentrations in humans, and did not affect the growth rate of control PG13 fibroblasts. Consistent with these findings in PG13 fibroblasts, we found that human T cells transduced with either 1928z.2A.NTR or 1928z.2A.HSV-TK demonstrated 90% and 88% inhibition, respectively, at similar substrate concentrations. Furthermore, we demonstrate that expression of these suicide genes does not affect the phenotype or function of the 19–28z CAR+ T cells, as assessed in vitro by T cell proliferation and cytotoxicity against CD19-expressing tumor cells. Our studies demonstrate highly effective suicide genes for human T lymphocytes transduced with a tumor targeted CAR, and a novel suicide gene/prodrug (NTR/metronidazole) combination with a comparable efficacy that can potentially serve as a reliable safety mechanism for adoptive T cell immunotherapy. While HSV-TK/ganciclovir has been utilized in various clinical settings, the NTR suicide gene has yet to be used in combination with gene modified tumor-targeted T cells. Furthermore, the NTR suicide gene holds several advantages over the HSV-TK and iCasp9 vectors. First, unlike HSV-TK, the NTR suicide gene is effective in both proliferating and non-proliferating cells. Second, unlike CID that is not commercially available, metronidazole is a widely available antibiotic that is relatively non-toxic. Lastly, metronidazole can be used in patients who may already be taking ganciclovir for cytomegalovirus (CMV) prophylaxis or treatment therefore limiting the application of T cells modified to express the HSV-TK suicide gene. Based on this in vitro data, we are currently testing the function of this suicide gene in vivo in two different animal models. Ultimately we anticipate that further studies with this novel suicide gene/prodrug combination will allow us to enhance safety in future clinical trials utilizing gene modified tumor targeted T cells. Disclosures: No relevant conflicts of interest to declare.

Abstract 3595 Background: Cytokines are posited to play a critical regulatory role on the survival of the B-cell neoplastic clone in Chronic Lymphocytic Leukemia (CLL). AIM: The primary goals of this study were 1) to define additional relevant cytokines, growth factors and chemokines in CLL pathophysiology and 2) to correlate abnormal cytokine levels with disease stage, relevant hematological data and multiple prognostic factors. METHODS: A novel bead-based protein array system was employed to simultaneously measure 38 proinflammatory cytokines in the sera of CLL patients (N=116) and healthy age and sex matched controls (N=30). These results were correlated with Rai stage, β2-microglobulin level, LDH, CD38 expression and cytogenetic abnormalities. RESULTS: We first confirmed previous observations that TNFα, IL-1α, IL-1RA, IL-10, sIL-2Rα, VEGF and sCD40 ligand are significantly elevated in patients with CLL as compared with healthy controls. Expanding on the current literature, we demonstrated perturbations in an additional 15 serum cytokines in affected individuals. Compared to healthy controls, CLL patients had an increase in serum levels of IL-3 (p=0.002), IL-7 (p=0.008), INF-2α (p Disclosures: No relevant conflicts of interest to declare.

Adoptive infusion of T cells genetically modified to express chimeric antigen receptors (CARs) targeted to tumor associated antigens (TAAs) is a promising novel approach to cancer therapy. However, in light of the fact that most TAAs targeted by such modified T cells are expressed, at least to some degree, by normal tissues as well as the fact that clinical experience with this technology is limited, safeguards are needed in the form of additional expression of transduced suicide genes by these T cells to allow for the efficient in vivo abrogation of infused T cells in the setting of unanticipated adverse events which may develop in the context of planned clinical trials. Herein we report the in vitro function of 3 different suicide genes each initially inserted distal to a CAR gene and an IRES (internal ribosome entry site) element cloned into the SFG retroviral vector. Specifically, in this study we evaluated the herpes simplex virus thymidine kinase (HSV-TK SR39), E. coli derived nitroreductase (NTR), and inducible caspase-9 (iCasp-9). We initially found that all 3 suicide genes functioned sub-optimally in the second position of these IRES based bicistronic vectors. In order to enhance suicide gene expression we deleted the IRES elements replacing them with 2A linker peptides, which significantly improved gene expression and the cell growth inhibition upon the addition of respective substrate. In PG13 cells transduced with SFG 1928z. IRES. NTR we found 40% inhibition at 5mM of metronidazole (MTZ), but 90% in PG13 cells transduced with SFG 1928.2A.NTR. Similarly, PG13 (1928z.IRES.HSV-TK) fibroblasts exhibited 60% inhibition at 1µM of ganciclovir compared to 85% inhibition of PG13 (1928z.2A.HSV-TK) fibroblasts. Consistent with these findings, we found that human T cells transduced with either 1928z.2A.NTR or 1928z.2A.HSV-TK demonstrated 90% and 88% inhibition respectively at similar substrate concentrations. In light of this encouraging in vitro data, we are currently in the process of assessing respective suicide gene function in vivo using a syngeneic, immune-competent mouse model recently developed in our laboratory. Ultimately we anticipate that these studies will allow us to rationally select the optimal suicide gene-prodrug combination to be incorporated into future clinical trials utilizing gene modified tumor targeted T cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1944.

Abstract 3436 Poster Board III-324 Patient T cells may be genetically modified to express chimeric antigen receptors (CARs) targeted to antigens expressed on tumor cells. We have initiated a clinical trial treating chemotherapy-refractory chronic lymphocytic leukemia (CLL) patients with autologous T cells modified to express the 19-28z CAR targeted to the CD19 antigen expressed on most B cell malignancies. In the first cohort of this trial, patients were infused with the lowest planned dose of modified T cells alone. All patients treated in this cohort experienced low-grade fevers following modified T cell infusion, and 2 of 3 treated patients exhibited subjective and laboratory evidence of transient reductions in tumor burden. The first patient treated on the second cohort of this study received prior cyclophophamide chemotherapy followed by the same dose of modified T cells administered to the first cohort of patients. This patient experienced persistent fevers, dyspnea, hypotension, renal failure, and died 44 hours following modified T cell infusion, likely secondary to sepsis. Modified T cells were not detectable in the peripheral blood of treated patients at 1 hour following completion of T cell infusion. However, post mortem analyses revealed a rapid infiltration of targeted T cells into anatomical sites of tumor involvement. Serum levels of the inflammatory cytokines IL-5, IL-8, and GM-CSF, but not TNFα, markedly and rapidly increased following infusion of genetically targeted T cells in this patient, mirroring the in vitro cytokine secretion profile of this patient's T cells, and consistent with marked in vivo activation of the modified T cells. Similar cytokine signatures were not found in patients from the first cohort. Significantly, serum cytokine analyses from the second cohort patient revealed a marked increase in the pro-proliferative cytokines IL-2, IL-7, IL-12, and IL-15 following cyclophosphamide therapy, in contrast to the baseline levels found in the first cohort. This report demonstrates the high efficiency trafficking of CD19-targeted T cells and in vivo activation of T cells encoding a second generation CD28/zeta chain-based chimeric antigen receptor. Furthermore, these data highlight mechanisms whereby cyclophosphamide may generate an in vivo milieu that enhances the anti-tumor efficacy of autologous tumor targeted T cells. Disclosures No relevant conflicts of interest to declare.

On 14-16 November 1989, the US Army CECOM Center for Software Engineering hosted the TTCP Workshop on Requirements Engineering and Rapid Prototyping. This event was sponsored by the Technical Cooperation Program (TTCP). The workshop's forty-nine international participants met to share current information on the field, to identify and clarify the most pressing issues, and to provide recommendations to DoD for management, development and research relating to Requirements Engineering. The workshop provided a forum for thirteen technical presentation participants divided into three working groups for small group interaction. These proceedings document the presentations and findings of this workshop and its working groups. Keywords: Software requirements.

On 14-16 November 1989, the US Army CECOM Center for Software Engineering hosted the TTCP Workshop on Requirements Engineering and Rapid Prototyping. This even was sponsored by the Technical Cooperation Program (TTCP) . The workshop's forty-nine international participants met to share current information on the field, to identify and clarify the most pressing issues. and to provide recommendations to DoD for management, development, and research relating to Requirements Engineering. The workshop provided a forum for thirteen technical presentation. Participants divided into three working groups for small-group interaction. These proceedings document the presentations and findings of this workshop and its working groups. Keywords: Software requirements, System requirements.

Current approaches to in vivo bioluminescent imaging (BLI) of T cells utilizing luciferase enzymes may be compromised by poor transduction efficiencies in primary T cells, and low photon emissions. Here we describe a novel and widely applicable approach to in vivo BLI of primary T cells utilizing a membrane-anchored form of the naturally secreted Guassia luciferase (GLuc) enzyme, termed exGLuc. We constructed exGLuc by fusion of the GLuc gene to the coding region of the CD8 transmembrane domain. The resulting exGLuc enzyme is anchored to the cell surface of retrovirally transduced cells. In vitro, cells which expressed the exGLuc enzyme demonstrated a markedly (>9 fold) increased bioluminescent signal when compared to cells which expressed the native GLuc (GLuc), the related Rhenilla luciferase (RLuc), a membrane-anchored form of RLuc (exRLuc), or a green fluorescent protein (GFP)-fire fly luciferase fusion protein. Following injection into SCID-Beige mice, MHC-mismatched C57BL6 T cells transduced to express exGLuc were detected by BLI generating graft versus host disease at an earlier time point and demonstrated a >10 fold increased bioluminescent signal when compared to infused C57BL6 T cells expressing GLuc, RLuc, or exRLuc. We further demonstrate homing of OVA-specific DO11.10 exGLuc+ T cells to A20(OVA) but not A20(GFP) subcutaneous tumors in both SCID-Beige and Balb/c mice. We further successfully applied this strategy to the study of in vivo human T cell homing using a xenogeneic SCID-Beige tumor model. Specifically, we demonstrate that human T cells, retrovirally co-transduced to express both a CD19-specific chimeric antigen receptor (CAR), 19z1, and exGLuc, could be monitored over time, and that these T cells quite rapidly home to subcutaneous CD19+ acute lymphoblastic leukemia (NALM-6) tumors when compared to T cells co-expressing an irrelevant CAR with exGLuc. Furthermore, we were able to demonstrate for the first time specific homing of 19z1/exGLuc+ human T cells to systemic NALM-6 tumor, detecting exGLuc+ T cells in deep tissues including the bone marrow, spleen, and liver. We conclude that the exGLuc enzyme emits a superior bioluminescent signal when compared to other commonly utilized luciferase enzymes; that primary T cells are readily transduced to express the exGLuc enzyme; and that exGLuc+ T cells may be accurately monitored in vivo over time by BLI. Based on these data, we believe that this novel approach to primary T cell in vivo imaging will significantly enhance the study of adoptive T cell immunotherapy in murine models of leukemia and other malignancies.

This panel discussion will focus on software engineering education and training programs: goals, constraints, and methods as seen from different perspectives. Panelists will include both educators and practitioners in the field of software engineering. Following brief statements by each panelist, the discussion will be opened to audience participation in an effort to maximize the interchange of information and ideas. Emphasis will be placed on the interface between formal education and actual practice in software engineering, rather than on the internal details of specific educational programs or software development methodologies. The current status of software engineering curriculum development will be described.