Bicistronic Retroviral Vector Research Articles

CD52/FLAG and CD52/HA Fusion Proteins as Novel Magnetic Cell Selection Markers.

At present, the magnetic selection of genetically modified cells is mainly performed with surface markers naturally expressed by cells such as CD4, LNGFR (low affinity nerve growth factor receptor), and MHC class I molecule H-2Kk. The disadvantage of such markers is the possibility of their undesired and poorly predictable expression by unmodified cells before or after cell manipulation, which makes it essential to develop new surface markers that would not have such a drawback. Earlier, modified CD52 surface protein variants with embedded HA and FLAG epitope tags (CD52/FLAG and CD52/HA) were developed by the group of Dr. Mazurov for the fluorescent cell sorting of CRISPR-modified cells. In the current study, we tested whether these markers can be used for the magnetic selection of transduced cells. For this purpose, appropriate constructs were created in MigR1-based bicistronic retroviral vectors containing EGFP and DsRedExpress2 as fluorescent reporters. Cytometric analysis of the transduced NIH 3T3 cell populations after magnetic selection evaluated the efficiency of isolation and purity of the obtained populations, as well as the change in the median fluorescence intensity (MFI). The results of this study demonstrate that the surface markers CD52/FLAG and CD52/HA can be effectively used for magnetic cell selection, and their efficiencies are comparable to that of the commonly used LNGFR marker. At the same time, the significant advantage of these markers is the absence of HA and FLAG epitope sequences in cellular proteins, which rules out the spurious co-isolation of negative cells.

Selection of Cell Populations with High or Low Surface Marker Expression Using Magnetic Sorting.

Magnetic cell sorting technology stands out because of its speed, simplicity, and ability to process large cell numbers. However, it also suffers from a number of drawbacks, in particular low discrimination power, which results in all-or-none selection outcomes limited to a bulk separation of cell populations into positive and negative fractions, as well as the modest purity of the selected cells and the inability to select subpopulations of cells with high expression of a surface marker. In the present study, we developed a simple solution to this problem and confirmed the effectiveness of this approach by multiple experiments with the magnetic selection of transduced cell populations. Murine NIH 3T3 cells were transduced with the bicistronic retroviral vector constructs co-expressing fluorescent reporter proteins EGFP (enhanced green fluorescent protein) or DsRed-Express 2 and LNGFR (low-affinity nerve growth factor receptor) as surface selection markers. The effects of the magnetic selection of transduced cells with anti-LNGFR Micro Bead (MB) doses ranging from 0.5 to 80 µL have been assessed. Low doses of MBs favored the depletion of weakly positive cells from the population, resulting in the higher expression levels of EGFP or DsRed-Express2 reporters in the selected cell fractions. Low MB doses also contributed to the increased purity of the selected population, even for samples with a low initial percentage of positive cells. At the same time, high MB doses resulted in the increased yield and a more faithful representation of the original expression profiles following selection. We further demonstrate that for populations with fairly narrow distribution of expression levels, it is possible to achieve separation into high- and low-expressing subsets using the two-stage selection scheme based on the sequential use of low and high doses of MBs. For populations with broad expression distribution, a one-stage selection with low or high doses of MBs is sufficient for a clear separation of low- and high-expressing subsets in the column-retained and flow-through fractions, respectively. This study substantially extends the potential of magnetic cell sorting, and may open new possibilities in a number of biomedical applications.

Abstract 887: Killer cell immunoglobulin-like receptor 2DL2 (KIR2DL2) immune checkpoint modulates CAR-T cell effector function

Abstract Significance: A modulatory role of killer immunoglobulin-like receptor 2DL2 (KIR2DL2) during T cell receptor (TCR) late signaling events has been proposed. However, its role in CAR-T cell effector function has not yet been studied. Our work aims to understand the differential modulatory effect of KIR2DL2 immune checkpoint on the effector function of therapeutic T cells used for the treatment of liquid and solid tumors. Methods: We generated CAR-T cells, with or without KIR2DL2 overexpression, using a bicistronic retroviral vector based on the MSGV1 backbone. HLA-C1-deficient tumor cells (HLA-C1−/−) were generated by CRISPR/Cas-based knockout of the B2M gene. We designed gRNAs targeting KIR2DL2, validated their cleavage efficiency and proved the feasibility to abrogate its expression in primary T cells. We tested the inhibitory role of KIR2DL2 in vitro in HLA-C1+ or HLA-C1−/− tumor cells using impedance- and bioluminescence-based cytotoxic assays at different effector:target ratios. We evaluated cytokine production after coculture of CAR-T and target cells using the ELLA system. To assess the modulatory role of KIR2DL2 in vivo, we generated HLA-C1+ or HLA-C1− subcutaneous xenografts of human pancreatic adenocarcinoma (HPAC) cells, then treated the mice with KIR2DL2+ or KIR2DL2− PSCA-CAR-T cells. We assessed the effect of KIR2DL2 in a lymphoblastic leukemia (ALL) xenograft model (NALM6 cells), treated with either KIR2DL2+ or KIR2DL2-deficient CD19-CAR-T cells (i.v.). Results: In vitro, KIR2DL2+ CAR-T cells were significantly less cytotoxic in an HLA-C1-dependent manner, regardless of their target antigen. Cytokine analysis after coculture showed that overexpression of KIR2DL2 in CAR-T cells impaired their ability to produce IFN-γ, IL-2 and TNF-α in presence of HLA-C1. In vivo, KIR2DL2-overexpressing PSCA-CAR-T cells injected in NSG mice harboring PSCA+/HLA-C1+ pancreatic tumors were not able to eliminate tumors. In contrast, KIR2DL2-overexpressing CAR-T cells transferred to mice harboring PSCA+/HLA-C1−/− tumor cells performed as well as their KIR2DL2− counterparts. Two gRNAs targeting KIR2DL2 were validated in primary T cells by means of an enzymatic cleavage assay and flow cytometry. Current efforts are focused on characterizing the impact of KIR2DL2 ablation on the anti-leukemia activity of CAR-T cells in vivo. Conclusions: KIR2DL2 impairs CAR-T cell effector function and cytokine secretion in vitro in both pancreatic and ALL tumor models, and in vivo in the pancreatic model. We implemented abrogation of KIR2DL2 by CRISPR/Cas9 during CD19-CAR-T cell manufacturing. Ongoing efforts focus on testing the impact of KIR2DL2 ablation on the in vivo performance of CAR-T cells. These results strengthen the notion of KIR2DL2 as an immune checkpoint with a relevant role in T cell immunosurveillance and may constitute an actionable target to enhance adoptive cell therapies. Citation Format: Miguel Gomez Fontela, Sebastian Snedal, Elena Martinez Planes, Daniel Abate-Daga. Killer cell immunoglobulin-like receptor 2DL2 (KIR2DL2) immune checkpoint modulates CAR-T cell effector function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 887.

Inducible MyD88/CD40 (iMC) Costimulation Enhances Polyclonal Epstein-Barr Virus-Specific Cytotoxic T Lymphocyte (EBV-CTL) Proliferation and Anti-Tumor Activity

Introduction: Adoptive T cell transfer with antigen-specific cytotoxic T lymphocytes (CTLs) have shown promise for the treatment of cancer. Antitumor efficacy correlates with T cell expansion and persistence following infusion, suggesting that enhancing T cell proliferation and survival may increase the potency of T cell therapies. Here, we demonstrate that genetic modification of EBV-specific T cells with inducible MyD88/CD40 (iMC) provides user-controlled costimulation that amplifies IL-2 production, CTL proliferation and cytotoxicity against EBV-transformed B cells, suggesting that iMC may be used to augment EBV-CTL against EBV-associated malignancies.Methods: Peripheral blood mononuclear cells (PBMCs) from healthy donors were used to generate EBV lymphoblastoid cells (LCLs) by transforming B cells with B95-8 virus. EBV-CTL were subsequently generated by stimulating PBMCs with irradiated (40 Gy) autologous LCLs at a T cell to LCL ratio of 40:1 (day 0) and then subsequently expanded for an additional 2 weeks by restimulation (4:1 CTL:LCL ratio) in the presence of 50 U/ml IL-2. EBV-CTL were then transduced with bicistronic retroviral vectors encoding iMC and truncated CD19 (ΔCD19) (SFG-iMC-ΔCD19) or a control vector lacking the MC costimulatory domains (SFG-FKBP-ΔCD19). EBV specificity and transduction efficiency of EBV-CTL was assessed by EBV-specific dextramers and CD3+CD19+ expression by flow cytometry, respectively. In vitro antitumor activity and T cell proliferation was measured using a 7 day live cell imaging (IncuCyte) coculture assays with transduced EBV-CTL and autologous or HLA-disparate LCL with or without 1 nM rimiducid to activate the iMC costimulatory molecule at effector to target (E:T) ratios of 40:1, 20:1, and 10:1. Cytokine production was measured by ELISA.Results: EBV-CTL were successfully generated from EBV-seropositive donors and demonstrated specificity against EBV lytic antigens (EBNA3A; BZLF1) and showed specific recognition of autologous EBV-LCL, but not HLA-mismatched LCL. EBV-CTL were also efficiently transduced with iMC and control (FKBP) retroviral vectors (93% and 82%, respectively). Cytokine production (IL-2 and IL-6) by gene-modified EBV-CTL targeting autologous LCL was significantly enhanced by iMC activation with rimiducid (48-fold increase compared to FKBP-ΔCD19-transduced EBV-CTL and iMC-ΔCD19-transduced EBV-CTLs without rimiducid stimulation). In vitro live cell imaging demonstrated at high E:T ratios (40:1) that EBV-CTLs efficiently killed matched LCLs. However, at lower E:T ratios (20:1 and 10:1), only EBV-CTLs transduced with iMC-ΔCD19 and stimulated with rimiducid were able to control LCL outgrowth over 7 days, showing a 10-fold increase inhibition in tumor growth.Conclusion: iMC provides a flexible, costimulatory module that can be used to enhance EBV-specific CTL to increase anti-tumor activity. Activation of iMC with rimiducid, in combination with recognition of cognate EBV antigens, induces IL-2 production and promotes CTL proliferation and survival. These data suggest that genetic modification of EBV-CTL, or other virus or tumor-associate antigen-specific T cells, including tumor infiltrating lymphocytes (TILs), with iMC may provide necessary costimulation on demand to increase T cell proliferation and persistence leading to increased anti-tumor efficacy. DisclosuresKhalil:Bellicum Pharmaceuticals: Employment. Yvon:Bellicum Pharmaceuticals: Equity Ownership. Mahendravada:Bellicum Pharmaceuticals: Employment. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership, Other: stockholders . Foster:Bellicum Pharmaceuticals: Employment, Other: stockholders .

Optimized gene engineering of murine CAR-T cells reveals the beneficial effects of IL-15 coexpression.

Limited clinical benefit has been demonstrated for chimeric antigen receptor (CAR) therapy of solid tumors, but coengineering strategies to generate so-called fourth-generation (4G) CAR-T cells are advancing toward overcoming barriers in the tumor microenvironment (TME) for improved responses. In large part due to technical challenges, there are relatively few preclinical CAR therapy studies in immunocompetent, syngeneic tumor-bearing mice. Here, we describe optimized methods for the efficient retroviral transduction and expansion of murine T lymphocytes of a predominantly central memory T cell (TCM cell) phenotype. We present a bicistronic retroviral vector encoding both a tumor vasculature-targeted CAR and murine interleukin-15 (mIL-15), conferring enhanced effector functions, engraftment, tumor control, and TME reprogramming, including NK cell activation and reduced presence of M2 macrophages. The 4G-CAR-T cells coexpressing mIL-15 were further characterized by up-regulation of the antiapoptotic marker Bcl-2 and lower cell-surface expression of the inhibitory receptor PD-1. Overall, this work introduces robust tools for the development and evaluation of 4G-CAR-T cells in immunocompetent mice, an important step toward the acceleration of effective therapies reaching the clinic.

Phase I Alexander study of AUTO3, the first CD19/22 dual targeting CAR T cell therapy, with pembrolizumab in patients with relapsed/refractory (r/r) DLBCL.

8001 Background: CD19 directed CAR T cells are effective in patients with r/r DLBCL, however relapses due to CD19 loss or PDL1 upregulation are common. In this study, we evaluate the safety and efficacy of AUTO3, a CAR T targeting CD19/22 with limited duration of PD-1 blockade. Methods: We constructed a bicistronic retroviral vector encoding both an anti-CD19 (OX40 co-stim) and an anti-CD22 (41BB co-stim) CAR with humanized binders. The cell product was manufactured in a semi-automated and closed process using CliniMACS Prodigy. Patients (≥ 18 years) with r/r DLBCL (NOS) or transformed (tDLBCL); ECOG <2, adequate organ function are eligible. Lymphodepletion was Flu/Cy prior to AUTO3. Bridging therapy was allowed. The three dose levels explored are 50, 150, and 450 x 10^6 CAR T cells. Patients received AUTO3 alone, or with 3 doses of pembrolizumab (pem) 200 mg q 3 wks starting on D14 (regimen A), or with a single dose of pem 200 mg on D-1 (regimen B). The primary endpoint is frequency of DLTs and grade (G) 3-5 adverse events (AE) and secondary endpoints included ORR, CRR, and biomarkers. Results: As of Jan 21, 2020, 28 patients underwent leukapheresis, 27 successfully manufactured, 1 being manufactured, and 19 patients treated with AUTO3. The median age was 57 (28 - 71) and median number of prior therapies was 3 (2 - 10). 89% had refractory disease, 74% were DLBCL NOS, and 26% were tDLBCL. Dose escalation from 50 to 450 x 106 cells with pem regimen A and B have been completed without DLTs. G > 3 treatment emergent AEs that occurred > 15% were neutropenia (89%), thrombocytopenia (58%), anemia (47%), febrile neutropenia (16%), and hypophosphataemia (16%). Across all dose levels, there were 0% sCRS with primary infusion and 5% severe neurotoxicity (sNT) (1/19), which resolved. There were no cases of sCRS and no neurotoxicity of any grade at > 50 x 106 cells. Eighteen patients were evaluable for efficacy. Among the 11 treated at dose > 50 x 106, the ORR and CRR were 64% and 55%, and all CRs are ongoing (1-12 mth). Two out of 3 patients achieved CR at 450 x 106 cells on pem regimen B. Additional patients and longer follow up, as well as biomarkers, will be presented. Conclusions: AUTO3 at > 50 x 106 CAR T cells with pembrolizumab induces CRs without severe CRS or neurotoxicities of any grade. Clinical trial information: NCT03287817 .

CAR T Cells Redirected to CD44v6 Control Tumor Growth in Lung and Ovary Adenocarcinoma Bearing Mice.

The main challenge of adoptive therapy with Chimeric Antigen Receptor modified T cells (CAR T) is the application to the field of solid tumors, where the identification of a proper antigen has emerged as one of the major drawbacks to CAR T cell treatment success. CD44 is a glycoprotein involved in cell-cell and cell-matrix interactions. The isoform containing the variant domain 6 of CD44 gene (CD44v6) has been implicated in tumorigenesis, tumor cell invasion and metastasis and represents an attractive target for CAR T cell therapies. Targeting CD44v6 antigen has been shown to control tumor growth in acute myeloid leukemia and multiple myeloma mouse models. While CAR T approach for the treatment of B cell malignancies has shown great success, response rates among patients with solid cancer are less favorable. The purpose of our study was to test the efficacy of CD44v6.CAR T cells, produced in compliance with Good Manufacturing Practice (GMP), in adenocarcinoma tumor models. We generated a bicistronic retroviral vector containing the CD44v6 CAR and the HSV-TK Mut2 suicide gene to enhance the safety of the proposed CAR T cell therapy. CD44v6 transduced CAR T cells were homogeneously positive for ΔLNGFR selection marker, were enriched in T central memory (TCM) and T memory stem cells (TSCM) and displayed a highly activated phenotype. In vitro assays revealed antigen-specific activation and cytotoxicity of human CD44v6.CAR T cells against CD44v6 expressing tumor cell lines. When infused in immunodeficient tumor bearing mice, human CD44v6.CAR T cells were able to reach, infiltrate and proliferate at tumor sites, finally resulting in tumor growth control. Next, we checked if cells produced in compliance with GMP grade standards retained the same antitumor activity of those produced with research grade materials and protocols. Noteworthy, no differences in the potency of the CAR T obtained with the two manufacturing processes were observed. In conclusion, our preclinical results suggest that CD44v6.CAR T based adoptive therapy could be a promising strategy in solid cancer treatment.

Phase 1/2 Study of AUTO3 the First Bicistronic Chimeric Antigen Receptor (CAR) Targeting CD19 and CD22 Followed By an Anti-PD1 in Patients with Relapsed/Refractory (r/r) Diffuse Large B Cell Lymphoma (DLBCL): Results of Cohort 1 and 2 of the Alexander Study

Introduction: CD 19 CAR T cell therapies have shown significant activity in patients with r/r DLBCL, however relapses due to CD19 loss or PDL1 upregulation are common. In this study, we are evaluating the safety and efficacy of AUTO3, a CAR T cell therapy designed to target CD19 and CD22 simultaneously followed by 3 doses of anti-PD1 monoclonal antibody pembrolizumab (Pem) treatment. The incorporation of dual targeting and anti-PD1 to target relapses due to antigen loss and PDL1 upregulation is novel and first in its kind. Methods & Patients: We constructed a novel bicistronic retroviral vector encoding both an anti-CD19 CAR and an anti-CD22 CAR. Antigen binding domains were humanized incorporating an OX40 co-stimulatory domain for the CD19 CAR and a 41BB co-stimulatory domain for the CD22 CAR. The CD22 CAR was enhanced by incorporating a novel pentameric spacer. The cell product was manufactured in a semi-automated and closed process. Patients (≥ 18 years) with r/r DLBCL not otherwise specified (NOS), high grade, or transformed from indolent histology; Eastern Cooperative Oncology Group Performance Status <2, adequate renal, hepatic, cardiac function and an absolute lymphocyte count ≥0.5 x 10e9/L are eligible. Patients with CNS disease, prior allogeneic stem cell transplant, prior CD19 or CD22 directed therapy are excluded, as well as patients with any contraindication to receiving Pem. All patients received lymphodepletion with 30 mg/m2/day fludarabine and 300 mg/m2/day cyclophosphamide for 3 days prior to AUTO3 infusion. Patients in cohort 1 received 50 x 10e6 and patients in cohort 2 received 150 x 10e6 transduced CAR T-cells. The first 3 patients in lowest dose cohort received AUTO3 alone, subsequently all patients should receive AUTO3 followed by 3 doses of Pem 200 mg given every 3 weeks starting day 14. The primary endpoints of phase 1 are frequency of dose-limiting toxicities (DLTs) and grade (G) 3-5 toxicity. Key secondary endpoints include overall response rate, complete response rate, duration of response, disease free survival, overall survival, and biomarker endpoints such as the level of AUTO3 cells in blood and duration of B cell aplasia. Results: As of the data cut-off date (05-July, 2019), 24 patients have been enrolled, 4 patients were screen failures, 1 patient is in screening, and 19 patients underwent leukapheresis. 16/16 patients had product successfully manufactured and 3 patient products in the manufacturing process. 5/16 patients discontinued before dosing and 11 patients were dosed with AUTO3 in cohort 1 and 2. Four with AUTO3 alone, and 7 with AUTO3 followed by Pem. Seven patients received 50x10e6 AUTO3, and 4 patients received 150 x 10e6 AUTO3. Median age was 49, median 3 prior lines of treatment, and 27% had prior autologous transplant. Seven patients had DLBCL NOS and 4 patients had transformed DLBCL from marginal zone or follicular lymphoma. Eleven patients in cohort 1 and 2 had a minimum of 4 week follow up and were evaluable for safety and efficacy analysis. No AUTO3 related deaths and no DLTs were observed. G > 3 treatment emergent adverse events > 10 % were neutropenia (73%), thrombocytopenia (64%), anemia (64%), and infection (18%). Treatment emergent adverse events > 25% regardless of grades were pyrexia (82%), thrombocytopenia (82%), neutropenia (73%), anemia (64%), nausea (36%), vomiting (36%), fatigue (36%), headache (36%), constipation (36%), dyspnea (27%), and cough (27%). 27% experienced cytokine release syndrome (CRS), all were grade 1, no ≥ G2 CRS were noted. Only one case (9%) of neurotoxicity was noted which was grade 3 and treated with steroids and tocilizumab. Dose levels 50 and 150 x 10e6 have been cleared without DLTs. At the lowest dose level of 50 x 10e6 cells, the objective response rate (ORR) was 57% and complete remission rate (CRR) was 29%. The ORR and CRR at dose 150 x 10e6 cells was 50%. Updated follow up and additional patient data at higher dose levels as well as cellular kinetics, product characteristics and relevant biomarkers will be presented. Conclusions: AUTO3, a novel bicistronic anti CD19/CD22 CAR T therapy, has a manageable safety profile in combination with Pem. Notable is the lack of severe CRS (0%). The efficacy data with 2/4 patients achieving CR at dose 150 x 10e6 cells is promising. The study continues to enroll patients at higher dose levels of AUTO3 followed by Pem. Disclosures Al-Hajj: Autolus Therapeutics: Employment, Equity Ownership. Thomas:Autolus: Employment, Equity Ownership. Faulkner:Autolus Therapeutics: Employment, Equity Ownership. Kotsopoulou:Autolus Therapeutics: Employment, Equity Ownership. Pule:Autolus: Employment, Equity Ownership, Patents & Royalties. Peddareddigari:Autolus Therapeutics: Employment, Equity Ownership. Khokhar:Autolus Therapeutics: Employment, Equity Ownership. Jonnaert:Autolus Therapeutics: Employment. Chen:Autolus Therapeutics: Employment. Osborne:Novartis: Other: Travel; Roche: Consultancy, Honoraria, Other: Travel, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; MSD: Consultancy; Servier: Consultancy; Gilead: Consultancy; Takeda: Consultancy, Honoraria, Other: Travel, Speakers Bureau.

Phase I Study of AUTO3, a Bicistronic Chimeric Antigen Receptor (CAR) T-Cell Therapy Targeting CD19 and CD22, in Pediatric Patients with Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia (r/r B-ALL): Amelia Study

Introduction CAR T-cell therapies directed against CD19 or CD22 have shown remarkable activity in r/r B-ALL but relapse due to target antigen down-regulation/loss has been the major cause of treatment failure. To address this, we developed AUTO3, a CAR T-cell therapy designed to target CD19 and CD22 simultaneously. Preliminary results of this study showed an acceptable safety profile and encouraging efficacy in pediatric r/r B-ALL (all 6 patients treated in active doses ≥3 x 106 CAR T-cells/ Kg achieved complete remission (CR) with negative minimal residual disease (MRD) (Amrolia et al, Blood 2018 132:279). Here we present the updated results of CAR naïve patients treated at the active doses. Methods & Patients We constructed a bicistronic retroviral vector encoding both an anti-CD19 CAR and an anti-CD22 CAR. This second-generation CAR incorporated an OX40 co-stimulatory domain for the CD19 CAR and a 41BB for the CD22 CAR. The cell product was manufactured on a semi-automated/closed process. Patients (aged 1‒24 years) with high risk relapsed (IBFM criteria) or refractory B-ALL, adequate performance score/organ function, an absolute lymphocyte count ≥0.5 x 109/L are eligible. Patients with CNS Grade 3 disease, active graft versus host disease are excluded. Patients receive lymphodepletion with 30 mg/m2/day fludarabine x 4 days and 500 mg/m2/day cyclophosphamide x 2 days prior to AUTO3 infusion. Three dose levels were explored (1 x 106, 3 x 106, and 5 x 106 cells/kg), CAR T cells are infused as a single (for <25% blasts) or split (for ≥25% blasts) dose based on leukemia burden. Bridging therapy is allowed during the manufacturing period. The primary endpoint is the frequency of dose-limiting toxicities (DLTs) and key secondary endpoints include morphological/MRD negative CR rate, disease-free survival, overall survival, as well as AUTO3 levels and persistence in blood and bone marrow. Results As of the data cut-off date (June 17, 2019), 10 patients received AUTO3 at 3 x 106 cells/Kg (n= 5, of whom 1 received split dose) or 5 x 106 CAR T-cells/Kg (n= 5, all of them received single infusion). The median transduction efficiency was 15.5% (range 8.6‒39.3%). Median age was 8.5 years (range 5‒16 years) and 5 (50%) patients had prior haemopoietic stem cell transplant (HSCT). One patient (10%) had prior anti-CD19 CAR-T cells. The disease burden at Day ‒7 ranged from 0 to 38% (median 7.5%) blasts. Among the 10 treated patients, 2 have not completed the 30 days post-infusion DLT observation period as of the cut-off date. No deaths or DLTs were observed. MTD has not yet been reached. The most common grade (Gr) ≥3 adverse events were neutropenia (60%), anaemia (50%), pyrexia (40%), febrile neutropenia (40%) and thrombocytopenia (30%). Eight patients (80%) had Gr 1 cytokine release syndrome (CRS), one (10%) had Gr 2 CRS; no ≥Gr 3 CRS was observed. Only one patient was treated with tocilizumab and none required admission to ICU due to CRS. One patient (10%) experienced Gr 1 neurotoxicity; no ≥ Gr 2 neurotoxicity was reported. Among the 9 CAR naïve patients, 7 (4 in the 3 x 106 cells/Kg dose cohort, 3 in the 5 x 106 cells/Kg dose cohort) had a minimum of 8 weeks' follow up and were evaluable for efficacy analysis. All 7 patients achieved CR/CRi (100%) following AUTO3 infusion as well as molecular negative remission (100%). After a median follow-up of 8 months (range 2-12), emergence of MRD by PCR occurred in four patients, lack of persistence of circulating CAR T-cells was observed in 3 of the 4 patients. Three relapses were reported including one with CD19 negative/CD22 low expression at 1 year after treatment. One patient in ongoing molecular remission proceeded to HSCT. All the remaining 4 patients in ongoing CR/CRi maintain B-cell aplasia. The median CAR T-cell expansion (expressed as vector copy number per microgram of DNA) at peak was 102K (range 56-128). The median persistence of CAR-T cells in blood was 180 days (range 21-330). Updated data with longer follow up and additional patient data will be presented. Conclusion This interim data analysis demonstrates that AUTO3 at ≥3 x 106 cells dose achieved 100% molecular remission rate with a favourable safety profile, no ≥ Gr 3 CRS or ≥ Gr 2 neurotoxicity was reported. The most common cause of relapse was antigen positive relapse due to lack of CAR T cell persistence. Evaluation of patients with a modified manufacturing process is planned. Disclosures Amrolia: UCLB: Patents & Royalties. Clark:Autolus Ltd: Employment, Equity Ownership. Al-Hajj:Autolus Therapeutics: Employment, Equity Ownership. Cordoba:Autolus: Employment, Equity Ownership. Kotsopoulou:Autolus Therapeutics: Employment, Equity Ownership. Khokhar:Autolus Therapeutics: Employment, Equity Ownership. Pule:Autolus: Employment, Equity Ownership, Patents & Royalties. Peddareddigari:Autolus Therapeutics: Employment, Equity Ownership.

Study of AUTO3, the First Bicistronic Chimeric Antigen Receptor (CAR) Targeting CD19 and CD22, Followed By Anti-PD1 Consolidation in Patients with Relapsed/Refractory (r/r) Diffuse Large B Cell Lymphoma (DLBCL): Alexander Study

Introduction CAR T-cell therapies directed against CD19 have shown significant activity in patients with r/r DLBCL; however, a significant number of patients relapse due to target antigen loss or upregulation of PDL1. In this study, we evaluate the safety and efficacy of AUTO3, a CAR T-cell therapy designed to target CD19 and CD22 simultaneously in order to reduce the likelihood of relapse due to antigen loss, followed by 3 doses of anti-PD1 monoclonal antibody pembrolizumab (pembro) consolidation treatment. Methods & Patients We constructed a novel bicistronic retroviral vector encoding both an anti-CD19 CAR and an anti-CD22 CAR. Antigen binding domains were humanized and both CARs are in "2nd generation" format (incorporating an OX40 co-stimulatory domain for the CD19 CAR and a 41BB for the CD22 CAR). The performance of the CD22 CAR was optimized by incorporating a novel pentameric spacer. The cell product was manufactured on a semi-automated and closed process using CliniMACS® Prodigy (Miltenyi Biotec). Patients (≥18 years) with r/r DLBCL not otherwise specified (NOS) or transformed from indolent histology; have chemorefractory disease or relapse after at least 2 prior therapies or autologous stem cell transplantation (ASCT); Eastern Cooperative Oncology Group performance status All patients receive lymphodepletion with 30 mg/m 2 /day fludarabine and 300 mg/m 2 /day cyclophosphamide for 3 days prior to AUTO3 infusion. Three dose levels are being explored (50 x 10e6; 150 x 10e6, and 300 x 10e6 transduced CAR T cells). The first 3 patients in lowest dose cohort receive AUTO3 alone; subsequently, all patients receive AUTO3 followed by 3 doses of pembro 200 mg given every 3 weeks. The primary endpoint of Phase I is frequency of dose-limiting toxicities (DLTs) and grade (Gr) 3‒5 toxicity; key efficacy endpoints include overall response rate (ORR) based on Lugano classification, duration of response, and overall survival, as well as biomarker endpoints such as the level of AUTO3 cells in blood and duration of B-cell aplasia. Results As of the data cut-off date (July 20, 2018), 6 patients have been enrolled and dosed on study: 3 with AUTO3 alone and 2 with AUTO3 followed by pembro (1 patient is awaiting pembro dosing). All patients had product successfully manufactured and received 50 x 10e6 cells. The transduction efficiency was 17% (range 16‒29%). Median age was 35 years (range 28‒60), median prior lines of treatment was 3 (range 2‒4), 1 patient (17%) had prior ASCT, 4 patients (67%) had chemorefractory disease. Four patients had DLBCL NOS at initial diagnosis; 2 patients had transformed DLBCL from marginal zone and follicular lymphoma, respectively. Five patients had a minimum of 4 weeks9 follow up and were evaluable for safety and efficacy analysis. No AUTO3-related deaths and no DLTs were observed. The most common Gr ≥3 adverse events were neutropenia in 5 patients (100%), platelet count decreased in 2 patients (40%), and hypophosphatemia in 2 patients (40%). One patient (20%) experienced Gr 1 cytokine release syndrome (CRS); no Gr 2 or higher CRS was observed. One patient (20%) who received AUTO3 alone had Gr 3 neurotoxicity that fully recovered. No immune adverse events related to pembro were observed and no patients required ICU admission. Four of the 5 patients had a response with ORR of 80% (95% CI 28.4‒99.5%). Two patients had a CR (40%; 95% CI 5.3‒85.3%) and continue to be in CR at the time of data cut off, with longest follow up of 3 months. CAR T-cell expansion was seen consistently in all patients. Conclusions This first study of simultaneously targeting CD19 and CD22 with a novel bicistronic CAR, AUTO3, demonstrates a manageable safety profile in combination with pembro. Early efficacy, even at the lowest dose of 50 x 10e6 cells, is promising with an ORR of 80% and 2 patients attaining a CR. The ALEXANDER study continues to enroll patients with AUTO3 followed by pembro. Updated follow up and additional patient data at higher dose levels, as well as cellular kinetics, relevant product characteristics, and biomarkers, will be presented. Disclosures Ardeshna: Celgene: Consultancy, Honoraria; ADC Therapeutics: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Gilead: Consultancy, Honoraria. Osborne: Novartis: Other: Travel to conference; Pfizer: Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; MSD: Consultancy; Servier: Consultancy; Celgene: Consultancy. Al-Hajj: Autolus Ltd: Equity Ownership; Autolus Ltd: Employment. Thomas: Autolus Ltd: Employment, Equity Ownership, Patents & Royalties. Faulkner: Autolus Ltd: Employment, Equity Ownership; GlaxoSmithKline: Equity Ownership. Pule: Autolus Ltd: Employment, Equity Ownership, Other: Salary contribution paid for by Autolus, Research Funding; University College London: Patents & Royalties: Patent with rights to Royalty share through UCL. Peddareddigari: Autolus Therapeutics plc: Employment; Autolus Therapeutics plc: Patents & Royalties; Autolus Therapeutics plc: Equity Ownership. Khokhar: Autolus Ltd: Employment; Autolus Ltd: Equity Ownership.

Patient-Derived Chimeric Antigen Receptor T-Cell Production Based on a Gammaretroviral Vector Platform Is Not Associated with Generation of CAR+ Leukemia Blasts

In view of the exciting results reported in patients with CD19+ malignancies given CAR T cells, it is expected that a continuously growing number of patients will be offered this treatment and, thus, will be exposed to gene-modified products. Since the techniques of gene manipulation are relatively new, some of the risks associated to CAR T therapy may be unpredictable.Recently, two patients who relapsed with CD19-, CAR-expressing leukemia were reported, this observation being interpretable in light of an inadvertent leukemic cell transduction with the second generation CAR.CD19 lentivirus during CAR T cell manufacturing (Lacey, ASH, 2016 128:281). Immunoglobulin heavy chain sequencing analysis of 17 additional infusion products also identified the leukemic clonotypes in six additional products (86%). In vitro and in vivo experiments proved that these CAR+ leukemic clones were not killed by CAR.CD19 T cells (Ruella, ASH, 2017 130:4463).Since lentiviruses proved to be superior for transduction of quiescent hematopietic stem cells due to their ability to infect non-dividing cells, we hypothesized that CAR-T cell manufacturing based on the genetic modification of T cells by gammaretroviral vector could theoretically represent a safe approach.Peripheral blood or bone marrow (BM)-derived mononuclear cells of patients with >40% of blasts at diagnosis (CD45dim+/CD34+/CD19+/CD22+/CD10+), were transduced with a retrovirus encoding for a second generation CAR.CD19.41bb.z in frame with a suicide gene (i.e., inducible caspase 9, iC9) employed in the academic Clinical Trial (NCT03373071) run at the Bambino Gesù Children's Hospital, Rome, Italy. Patient-derived CAR-T cells showed a phenotype not significantly different from that found on CAR-T cells generated by healthy-donors (data not shown). In particular, we demonstrated that both flow-cytofluorimetry and RealTime-quantitative PCR (with a sensitivity up to 10-5) failed to identify leukemic cells in the final CAR-T cell products generated from Bcp-ALL patients.To generate an in vitro model of CAR+ leukemic cells, we genetically modified CD19+ RAJI and DAUDI cell lines with the bicistronic retroviral vector carrying both second generation CAR.CD19 and the suicide gene iC9 (iC9.CAR-RAJI and iC9.CAR-DAUDI). We demonstrated the possibility of promptlyeliminating CAR+ leukemic cells, through exposure to 20nM of AP1903 of iC9.CAR-DAUDI and iC9.CAR-RAJI cells. Indeed, very early activation (6 hours) of the suicide gene iC9 resulted into a significant reduction in the percentage of CAR+ RAJI leukemic cells (Fig.A).The presence of iC9.CAR.CD19 molecule on leukemic cells precluded the detection of the CD19 antigen, whereas cells retain the expression of all other specific B-lineage markers. CD19 antigen started to be detectable 72 hours after AP1903 exposurewhen CAR negative leukemic cells become preponderant. To demonstrate that CD19 antigen was not down-regulated, but only masked by CAR molecule in iC9.CAR-RAJI and iC9.CAR-DAUDI cell lines, we measured CD19 mRNA, showing no significant modification with respect to wild-type (WT) RAJI and DAUDI cell lines. Moreover, iC9.CAR-RAJI and iC9.CAR-DAUDI cell lines were effectively eliminated by CAR.CD19 T cells (12.5±13.7% and 3.4±4.3% residualleukaemia, respectively) at the same extent of WT cell line (0% and 0.08±0.1%, residual leukaemia, respectively; p>0.05 Fig.B).To assess if patient-derived iC9.CAR.CD19 T cells were able to generate leukemia in vivo mouse model, NSG female mice were infused i.v. with 10x106 CAR-T cells and control NT-T cells. Mice were monitored for a total period of 250 days, by recurrent bleed. Simultaneously, another cohort of mice was infused with patient-derived BM cells (5x106) and monitored for the same time. Mice infused with Bcp-ALL BM cells developed leukemia-phenotype,with 82% of cells expressing hCD45dim and hCD19. By contrast, mice receiving patient-derived CAR-T cells showed a lowpercentage of CD45+ cells (0.1±0.01%), all CD3+. Despite the long period of observation, we did not detect any expansion of hCD19+ cells in this animal cohort.Taken together these data suggest that the use of a retroviral platform, associated with the presence of iC9 suicide gene, contributes to the genesis of a highly functional and safe CAR-T product, even when the production starts from a biological material characterized by high contamination of leukemic blasts. [Display omitted] DisclosuresLocatelli:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; bluebird bio: Consultancy; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Simultaneous Targeting of CD19 and CD22: Phase I Study of AUTO3, a Bicistronic Chimeric Antigen Receptor (CAR) T-Cell Therapy, in Pediatric Patients with Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia (r/r B-ALL): Amelia Study

Simultaneous Targeting of CD19 and CD22: Phase I Study of AUTO3, a Bicistronic Chimeric Antigen Receptor (CAR) T-Cell Therapy, in Pediatric Patients with Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia (r/r B-ALL): Amelia Study

Characterization of Allogeneic T Cells Expressing Inducible Caspase-9 Following Adoptive Transfer in Children Receiving an HLA-Haploidentical Hematopoietic Stem Cell Transplant for the Treatment of Myeloid Malignancies

▪Background: Adoptive transfer of allogeneic donor T cells can be an effective treatment for hematological malignancies through recognition of leukemia-associated antigens (LAAs) on tumor cells or through alloreactivity. However, alloreactive T cells can also cause graft-versus-host disease (GvHD) limiting their use as an immunotherapy. To leverage the anti-tumor effects of allogeneic polyclonal T cells while minimizing GvHD, we have genetically modified donor T cells with the inducible caspase-9 (iC9) safety switch, which induces apoptosis following exposure to the small molecule ligand rimiducid. Here we show that iC9-modified allogeneic T cells (BPX-501) persist, expand and contain functional LAA-specific T cells in children receiving an alpha/beta TCR and CD19-depleted HLA-haploidentical hematopoietic stem cell transplant (haplo-HSCT) for the treatment of myeloid malignancies.Methods: Pre-infusion products (BPX-501: donor T cells modified with the bicistronic retroviral vector encoding iC9 and truncated CD19 (ΔCD19)) and patient peripheral blood mononuclear cells (PBMCs) were analyzed from twelve patients (AML (10), MDS (1), JMML (1)) receiving BPX-501 (1x106 cells/kg) following an alpha/beta T cell and CD19 B cell-depleted haplo-HSCT (BP-004U: NCT03301168). Engraftment and persistence were measured by coexpression of CD3 and CD19 by flow-cytometry. Endogenous and gene-modified T cells were also phenotyped for CD4:CD8 ratios, memory cell composition (TN, TCM, TEM, TEMRA; CD45RA and CD62L) and T cell receptor Vβ diversity. BPX-501 products and post-treatment samples were characterized for LAA-specific T cells using IFN-γ ELISpot against peptide pools (15 aa overlapping by 5 aa) derived from WT1, PRAME, MAGE (A1, C1, C3), NE and PR3, with and without exposure to 10 nM rimiducid to determine the anti-leukemic contribution of BPX-501.Results: BPX-501 was infused at a median time of 22.5 days after HSCT (range 12-34, one patient was infused at day 89 and one patient was infused at day 147). BPX-501 cells (CD3+CD19+) were detectable in the peripheral blood at 1-2 weeks after infusion in all 12 patients, reaching a peak expansion frequency of a median of 24% ± 17% of total CD3+ T cells, and an absolute cell number of 66.9 ± 112 cells/µl at 2 months post-infusion and could be detected for up to 24 months. BPX-501 T cells showed a CD8-skewed phenotype whereas endogenous T cells exhibited a more balanced CD4:CD8 ratio. BPX-501 were predominantly CD45RA-CD62L+ and CD45RA-CD62L- central and effector memory T cells, respectively. In BPX-501 products, we detected LAA-specific T cells by ELISpot using overlapping peptide pools to WT1, PRAME, MAGE, NE and PR3, and in peripheral blood samples obtained 2 to 5 months post-T cell infusion. Importantly, LAA-reactivity was greatly diminished with exposure to iC9-activating rimiducid. Further, we measured the TCR Vβ usage and observed highly-skewed TCR repertoire in BPX-501 T cells compared to endogenous T cells in 6 months after HSCT indicating selection and expansion of TCR clones. Three patients engrafted BPX-501 were treated with rimiducid to control GvHD resulting in a rapid decrease (62% ± 12%) of CD3+CD19+ T cells in the peripheral blood. In patients treated with rimiducid, CD3+CD19+ T cells recover without further instances of GvHD suggestive of in vivo depletion of alloreactive T cell clones using iC9.Summary: Allogeneic T cells engineered with the iC9 safety switch engraft, expand and demonstrate long-term persistence following adoptive transfer into patients receiving a haplo-HSCT. LAA-specific T cells and alloreactive T cells within the BPX-501 product are detectable in the peripheral blood following infusion and likely contribute to elimination of myeloid malignancies. DisclosuresShaw:Bellicum Pharmaceuticals: Employment, Equity Ownership. Zhou:Bellicum Pharmaceuticals: Employment, Equity Ownership. Lu:Bellicum Pharmaceuticals: Employment, Equity Ownership. Aldinger:Bellicum Pharmaceuticals, Inc.: Employment. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership. Locatelli:Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Foster:Bellicum: Employment, Equity Ownership.

Development and Evaluation of an Optimal Human Single-Chain Variable Fragment-Derived BCMA-Targeted CAR T Cell Vector.

B cell maturation antigen (BCMA) has recently been identified as an important multiple myeloma (MM)-specific target for chimeric antigen receptor (CAR) Tcell therapy. In CAR Tcell therapy targeting CD19 for lymphoma, host immune anti-murine CAR responses limited the efficacy of repeat dosing and possibly long-term persistence. This clinically relevant concern can be addressed by generating a CAR incorporating a human single-chain variable fragment (scFv). We screened a human B cell-derived scFv phage display library and identified a panel of BCMA-specific clones from which human CARs were engineered. Despite a narrow range of affinity for BCMA, dramatic differences in CAR Tcell expansion were observed between unique scFvs in a repeat antigen stimulation assay. These results were confirmed by screening in a MM xenograft model, where only the top preforming CARs from the repeat antigen stimulation assay eradicated disease and prolonged survival. The results of this screening identified a highly effective CAR Tcell therapy with properties, including rapid invivo expansion (>10,000-fold, day 6), eradication of large tumor burden, and durable protection to tumor re-challenge. We generated a bicistronic construct including a second-generation CAR and a truncated-epithelial growth factor receptor marker. CAR Tcell vectors stemming from this work are under clinical investigation.

PIL6-TRAIL-engineered umbilical cord mesenchymal/stromal stem cells are highly cytotoxic for myeloma cells both in vitro and in vivo

BackgroundMesenchymal/stromal stem cells (MSCs) are favorably regarded in anti-cancer cytotherapies for their spontaneous chemotaxis toward inflammatory and tumor environments associated with an intrinsic cytotoxicity against tumor cells. Placenta-derived or TRAIL-engineered adipose MSCs have been shown to exert anti-tumor activity in both in-vitro and in-vivo models of multiple myeloma (MM) while TRAIL-transduced umbilical cord (UC)-MSCs appear efficient inducers of apoptosis in a few solid tumors. However, apoptosis is not selective for cancer cells since specific TRAIL receptors are also expressed by a number of normal cells. To overcome this drawback, we propose to transduce UC-MSCs with a bicistronic vector including the TRAIL sequence under the control of IL-6 promoter (pIL6) whose transcriptional activation is promoted by the MM milieu.MethodsUC-MSCs were transduced with a bicistronic retroviral vector (pMIGR1) encoding for green fluorescent protein (GFP) and modified to include the pIL6 sequence upstream of the full-length human TRAIL cDNA. TRAIL expression after stimulation with U-266 cell conditioned medium, or IL-1α/IL-1β, was evaluated by flow cytometry, confocal microscopy, real-time PCR, western blot analysis, and ELISA. Apoptosis in MM cells was assayed by Annexin V staining and by caspase-8 activation. The cytotoxic effect of pIL6-TRAIL+-GFP+-UC-MSCs on MM growth was evaluated in SCID mice by bioluminescence and ex vivo by caspase-3 activation and X-ray imaging. Statistical analyses were performed by Student’s t test, ANOVA, and logrank test for survival curves.ResultspIL6-TRAIL+-GFP+-UC-MSCs significantly expressed TRAIL after stimulation by either conditioned medium or by IL-1α/IL-1β, and induced apoptosis in U-266 cells. Moreover, when systemically injected in SCID mice intratibially xenografted with U-266, those cells underwent within MM tibia lesions and significantly reduced the tumor burden by specific induction of apoptosis in MM cells as revealed by caspase-3 activation.ConclusionsOur tumor microenvironment-sensitive model of anti-MM cytotherapy is regulated by the axis pIL6/IL-1α/IL-1β and appears suitable for further preclinical investigation not only in myeloma bone disease in which UC-MSCs would even participate to bone healing as described, but also in other osteotropic tumors whose milieu is enriched of cytokines triggering the pIL6.

Interleukin-7 Mediates Selective Expansion of Tumor-redirected Cytotoxic T Lymphocytes (CTLs) without Enhancement of Regulatory T-cell Inhibition

The antitumor activity of chimeric antigen receptor (CAR)-redirected CTLs should be enhanced if it were possible to increase their proliferation and function after adoptive transfer without concomitantly increasing the proliferation and function of regulatory T cells (Treg). Here, we explored whether the lack of IL-7Rα in Treg can be exploited by the targeted manipulation of the interleukin-7 (IL-7) cytokine-cytokine receptor axis in CAR-engrafted Epstein-Barr Virus-specific CTLs (EBV-CTLs) to selectively augment their growth and antitumor activity even in the presence of Treg. We generated a bicistronic retroviral vector encoding a GD2-specific CAR and the IL-7Rα subunit, expressed the genes in EBV-CTLs, and assessed their capacity to control tumor growth in the presence of Treg in vitro and in vivo when exposed to either interleukin-2 (IL-2) or IL-7 in a neuroblastoma xenograft. We found that IL-7, in sharp contrast with IL-2, supports the proliferation and antitumor activity of IL-7Rα.CAR-GD2(+) EBV-CTLs both in vitro and in vivo even in the presence of fully functional Treg. IL-7 selectively favors the survival, proliferation, and effector function of IL-7Rα-transgenic/CAR-redirected EBV-CTLs in the presence of Treg both in vitro and in vivo. Thus, IL-7 can have a significant impact in sustaining expansion and persistence of adoptively CAR-redirected CTLs.

Isolation of Tumor Antigen-Specific Single-Chain Variable Fragments Using a Chimeric Antigen Receptor Bicistronic Retroviral Vector in a Mammalian Screening Protocol

The clinical potential of chimeric antigen receptors in adoptive cellular therapy is beginning to be realized with several recent clinical trials targeting CD19 showing promising results in advanced B cell malignancies. This increased efficacy corresponds with improved engineering of the chimeric receptors with the latest-generation receptors eliciting greater signaling and proliferation potential. However, the antigen-binding single-chain variable fragment (scFv) domain of the receptors is critical in determining the activity of the chimeric receptor-expressing T cells, as this determines specificity and affinity to the tumor antigen. In this study, we describe a mammalian T cell line screening protocol employing a 2A-based bicistronic retroviral vector to isolate functional scFvs. This approach involves expression of the scFv library in a chimeric antigen receptor, and is based on selection of clones capable of stimulating CD69 upregulation in a T cell line and has a number of advantages over previously described methods in that the use of a 2A cassette ensures the exclusion of nonexpressing scFvs and the screening using a chimeric receptor in a mammalian T cell line ensures selection in the optimum context for therapeutic use. Proof-of-principle experiments show that the protocol was capable of a 10(5)-fold enrichment of positive clones after three rounds of selection. Furthermore, an antigen-specific clone was successfully isolated from a partially enriched scFv library, confirming the strength of the protocol. This approach has the potential to identify novel scFvs of use in adoptive T cell therapy and, potentially, wider antibody-based applications.

A Bicistronic Retroviral Vector Expressing IL-15 and Herpes Simplex Virus Thymidine Kinase for the Transduction of Human T Lymphocytes for Adoptive Cell Transfer Therapies

A Bicistronic Retroviral Vector Expressing IL-15 and Herpes Simplex Virus Thymidine Kinase for the Transduction of Human T Lymphocytes for Adoptive Cell Transfer Therapies

Loss of Hypermethylated in Cancer 1 (HIC1) in Breast Cancer Cells Contributes to Stress-induced Migration and Invasion through β-2 Adrenergic Receptor (ADRB2) Misregulation

The transcriptional repressor HIC1 (Hypermethylated in Cancer 1) is a tumor suppressor gene inactivated in many human cancers including breast carcinomas. In this study, we show that HIC1 is a direct transcriptional repressor of β-2 adrenergic receptor (ADRB2). Through promoter luciferase activity, chromatin immunoprecipitation (ChIP) and sequential ChIP experiments, we demonstrate that ADRB2 is a direct target gene of HIC1, endogenously in WI-38 cells and following HIC1 re-expression in breast cancer cells. Agonist-mediated stimulation of ADRB2 increases the migration and invasion of highly malignant MDA-MB-231 breast cancer cells but these effects are abolished following HIC1 re-expression or specific down-regulation of ADRB2 by siRNA treatment. Our results suggest that early inactivation of HIC1 in breast carcinomas could predispose to stress-induced metastasis through up-regulation of the β-2 adrenergic receptor.

Evaluation of Residual Promoter Activity in γ-Retroviral Self-inactivating (SIN) Vectors

Therapeutic gene delivery mediated by retroviral vectors has the advantage of stable integration into the host genome. A major safety concern for gene delivery achieved by murine leukemia virus (MLV)-based retroviral vectors is the activation of adjacent cellular genes including oncogenes following integration into the host genome. Self-inactivating (SIN) vectors lacking viral enhancers/promoters in their 3' long terminal repeat (LTR) have been proposed as a means of overcoming this safety concern. However the MLV-based SIN vectors currently used by laboratories to assess insertional mutagenesis, integration site selection, and the potency of transgene expression are not uniform in the composition of their 3' LTRs. We constructed a series of SIN vectors representative of the currently employed vectors, but lacking an internal promoter. Green fluorescent protein (GFP) was used as a reporter gene. Target cells exposed to these vectors were evaluated for number of integrants and GFP expression at the messenger RNA (mRNA) level and protein level. We found that viral promoter activity in the 3' LTR is not attenuated in many currently employed SIN vectors. These results suggest that the influence of strong residual promoter activity should be taken into consideration when interpreting experimental results obtained using SIN vectors in gene therapy research.