ALBERT

Description: The cAMP-responsive element binding protein (CREB) is a 43-kDa nuclear transcription factor that regulates cell growth, memory, and glucose homeostasis. We showed previously that CREB is amplified in myeloid leukemia blasts and expressed at higher levels in leukemia stem cells from patients with myeloid leukemia. CREB transgenic mice develop myeloproliferative disease after 1 year, but not leukemia, suggesting that CREB contributes to but is not sufficient for leukemogenesis. Here, we show that CREB is most highly expressed in lineage negative hematopoietic stem cells (HSCs). To understand the role of CREB in hematopoietic progenitors and leukemia cells, we examined the effects of RNA interference (RNAi) to knock down CREB expression in vitro and in vivo. Transduction of primary HSCs or myeloid leukemia cells with lentiviral CREB shRNAs resulted in decreased proliferation of stem cells, cell- cycle abnormalities, and inhibition of CREB transcription. Mice that received transplants of bone marrow transduced with CREB shRNA had decreased committed progenitors compared with control mice. Mice injected with Ba/F3 cells expressing either Bcr-Abl wild-type or T315I mutation with CREB shRNA had delayed leukemic infiltration by bioluminescence imaging and prolonged median survival. Our results suggest that CREB is critical for normal myelopoiesis and leukemia cell proliferation.

Description: Conventional, ex vivo culture of monocytes with recombinant proteins for their differentiation into DCs involves considerable manipulation under “Good Manufacturing Practices” conditions, and is not only more labor intensive but importantly, after ex vivo produced DCs are administered, they lack the stimulatory signals to keep them alive and functional and therefore are short lived. Because of these problems, we have evaluated an one-hit lentiviral transduction approach for genetically modifying monocytes in order to promote autocrine and paracrine production of factors required for their differentiation into immature DCs. High-titer third generation self-inactivating lentiviral vectors expressing granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) efficiently achieved simultaneous and persistent co-delivery of the transgenes into purified human CD14+ monocytes. Co-expression of GM-CSF and IL-4 in monocytes was sufficient to induce their differentiation into lentivirus-modified DCs (“DC/LVs”), as evidenced by their morphology, immunophenotype and immune-function*. Mixed lymphocyte reactions showed that the T-cell stimulating activity of DC/LVs was superior to that of DCs grown by conventional methods. DC/LVs displayed efficient antigen-specific, MHC Class-I restricted stimulation of autologous CD8+ T-cells, as shown by IFN-G production and CTL assays. Importantly, DC/LVs could be maintained metabolically active and viable in culture for 2–3 weeks in the absence of exogenously added growth factors, unlike conventional DCs *. We are now evaluating whether DC/LVs can be re-infused immediately after gene transfer to achieve stable and long-lasting differentiation in vivo. Additionally, the genetic engineering of monocytes is anticipated to generate DCs after one hit of lentiviral transduction, instead of the three consecutive steps for development of DCs (differentiation, maturation, gene delivery of tumor antigens). We have thus established a mouse model for testing DC/LVs in vivo for the treatment of melanoma. Bone marrow cells from C57BL/6 mice transduced with lentiviral vectors expressing GM-CSF and IL-4 recapitulated the same DC/LV morphology and immunophenotype obtained in the human system. Mouse DC/LVs were also more viable in vitro and outperformed conventional mouse DCs in pilot immunization assays as followed by CTL assays and IFN-G ELISPOT. We are currently evaluating the immunotherapeutic efficacy of DC/LVs injected into mice developing B16 melanoma tumors. Co-delivery of a gene for DC maturation (CD40L) and of gene encoding a tumor-associated antigens (MART-1) is being performed. Our goal is to evaluate the implications of simultaneous co-expression of GM-CSF/ IL-4/ CD40L/ MART-1 in DC/LV differentiation and migration to lymph nodes in vivo, immunopotency and safety. Once these pre-clinical considerations are addressed, we foresee a broad clinical application of genetically engineered DCs for vaccination purposes against cancer and chronic infectious diseases.

Description: Introduction: Reactivation of human cytomegalovirus (HCMV) in immune compromised patients after hematopoietic stem cell transplantation (HSCT) is associated with high morbidity and mortality, particularly after cord blood transplantation (CBT). Adoptive transfer of T cells expanded in vitro is currently used as therapy for drug-refractory HCMV disease. A major limitation of this approach is the requirement of HLA-restricted HCMV-specific memory T cells. An alternative approach exploring HLA-independent T cell recognition was sought. Because the HCMV envelope glycoprotein B (gB) is highly expressed during lytic infection and in latently infected cells, we hypothesized that T cells can be redirected to recognize and kill HCMV-specific cells by means of a gB-specific chimeric antigen receptor (CAR). We have synthesized and tested a gB-specific CAR derived from the SM5-1 monoclonal antibody which binds with high affinity (KD 5.7x1011) to a conserved antigenic and non-glycosylated domain of gB. Methods: We generated two codon-optimized SM5-derived scFvs (VH-〉VL and VL-〉VH) and fused with an existing CAR backbone containing an IgG Fc spacer and intracellular signaling domains. CARs containing either CD28.zeta or 4-1BB.zeta were synthesized and expressed in T cells following a standard retroviral transduction protocol yielding 80-90% transduction rate. Expression of the CARs on T cells was confirmed by flow cytometry using goat anti-human immunoglobulin reactive against the IgG Fc region. 293T cells co-expressing gB and dTomato were used for in vitro cytotoxicity assays. Results: T cells expressing gB-CAR/CD28.zeta were cytotoxic against gB+ target cells producing 90% killing of 293T/gB-dTom cells compared with control CD19 CAR/CD28.zeta cells at an effector-to-target ratio 3:1 for 48 h (parental 293T cells were not killed). The cytolytic activity correlated with expansion of CAR T cells and concomitant loss of gB-dTom expression in the remaining viable 293T cells. Sequential co-culture of these gB-CAR T cells with freshly seeded 293T/gB-dTom resulted into further elimination of target cells. We are currently evaluating the effects of different gB-CAR T cell designs in the killing of HCMV-infected cell lines and primary cells using HCMV laboratory strains expressing the GFP and Gaussia Luciferase reporter genes. Pilot experiments indicated that gB-CAR/CD28.zeta cells with the scFv in the VL-〉VH orientation resulted into more clustering and killing of HepG2 cells infected with HCMV-GFP after 24h of co-culture than a control CD19 CAR/CD28.zeta. Humanized mice transplanted with cord blood CD34+ stem cells and challenged with these HCMV laboratory strains will be used to evaluate the in vivo effectivity of cord blood-derived donor-matched gB-CAR-T cells to eliminate acute and latent HCMV infections. Conclusion: These studies explore a novel approach in preventing HCMV reactivation in immunosuppressed patients by redirecting T cells expressing a high-affinity gB-CAR to eliminate HCMV-infected cells in a TCR/MHC-independent manner. Disclosures No relevant conflicts of interest to declare.

Description: In contrast to gene therapy of solid tumors, only a few preclinical studies exist about gene therapy of acute lymphoblastic leukemia. Previously, we showed that vaccination of mice with syngeneic BCR-ABLp185 expressing leukemia cell lines modified to express costimulatory molecules and cytokines induce a systemic immunity against wild type leukemia. However, the difficulties to culture and transfect human leukemia cells limit the clinical application of leukemia cell based vaccines. Thus, we evaluated the pre-immunization of mice with DNA based vaccines subsequently challenged by the cell line BM185. In order to avoid the limitations and serious side effects associated with viral vectors we used nonviral gene delivery methods. Minimalistic immunogenically defined gene expression (MIDGE) vectors encoding a BCR-ABLp185 fusion specific peptide or GM-CSF were used as DNA vaccine and double stem-loop immunomodulators (dSLIM), containing three CpG-motifs were used as immune adjuvant. We provide biometrical and CTL data that shows specific immunization and protection of mice which received the complete vaccine BCR-ABL/GM-CSF/dSLIM by GeneGun delivery of naked DNA. Mean tumor-free survival (p=0.019) and overall survival (p=0.008) were significantly longer compared to non-vaccinated mice. Furthermore we show that BCR-ABL specific sequences are required to prevent Ph+ acute lymphoblastic leukemia by DNA vaccination. However, survival rate was moderate and only 26.7% of vaccinated mice survived and remained tumor-free. While current nonviral genetic tumor vaccine systems are less effective than viral vaccines, in particular when tumor associated antigens are weakly immunogenic, microencapsulation of tumor-specific DNA with poly (β-amino esters) might be a promising, safe and efficient nonviral delivery method for genetic vaccines. These biodegradable cationic polymers can bind and condense DNA into nanoparticles, deliver large DNA payloads and show low toxicity. Therefore, we prepared DNA-polymer complexes of MIDGE-vectors and end-modified poly (butane diol diacrylate co amino pentanol) C32-117 or C32-118 which self-assemble into particles with effective diameters of 200 nm and zeta potentials of 9.8 mV in PBS. We compared the immunization of mice receiving the vaccine BCR-ABL/GM-CSF/dSLIM by intradermal injection as naked DNA or alternatively, complexed with C32-117 or C32-118. We present data about tumor growth, survival rate, tumor free and overall survival of vaccinated and control mice.

Description: Although 70 – 80 % of children with childhood acute lymphoblastic leukemia (ALL) can be cured by poly-chemo-therapy, the prognosis of patients with Philadelphia chromosome positive (Ph+) ALL remains poor. Therefore, new relapse prevention strategies are needed for patients with Ph+ ALL during remission. We have shown previously, that vaccination of mice with leukemia cell lines modified to express costimulatory molecules and cytokines induce a systemic immunity against the syngeneic BCR-ABLp185 expressing cell line BM185. However, the difficulties to culture and transfect human leukemia cells limit the clinical application of leukemia cell based vaccines. Thus, we evaluated the pre-immunization of mice with DNA based vaccines subsequently challenged by the cell line BM185. Ballistic transfer of minimalistic immunogenically defined gene expression (MIDGE) vectors encoding a BCR-ABLp185 fusion specific peptide or GM-CSF were used for in vivo transfection of murine skin. In addition, we used double stem-loop immunomodulators (dSLIM), containing three CpG-motifs as non-specific immune adjuvant. We provide survival data that shows specific immunization and protection of mice which received the complete vaccine BCR-ABL/GM-CSF/dSLIM. Mean tumor-free survival (p=0.019) and overall survival (p=0.008) were significantly longer compared to non-vaccinated mice and 26.7% survived and never developed leukemia. In contrast, tumor-free and overall survival of mice immunized with either dSLIM or GM-CSF alone or both dSLIM and GM-CSF was not significantly longer compared to non-vaccinated mice. Similarly, substitution of BCR-ABL by irrelevant TEL-AML1 sequences abolished the vaccine efficacy of the BCR-ABL/GM-CSF/dSLIM vaccine. The biometrical data were confirmed by CTL assays which showed that specific lysis was significantly higher after vaccination with BCR-ABL/GM-CSF/dSLIM compared to GM-CSF/dSLIM (p

Description: Introduction: Chemotherapy leads to cure of acute myeloid leukemia (AML) in less than half of the patients. Stem cell transplantation can be used as an immunotherapeutic treatment to cure the patient, but carries a high risk of toxicity and mortality. Moreover, not all patients have a suitable donor. We have developed a novel immunotherapeutic treatment, in which we generate in vitro, starting from hematopoietic precursor cells, T-cells that recognize WT1, a tumor antigen that is overexpressed on 70% of the AMLs. Aims: In this study we have evaluated the functionality and specificity of the generated WT1-directed T-cells both in vitro and in vivo. The ultimate goal is to use these cells in patients, as this form of immunotherapy is promising and could be an option for cure in patients who are not eligible for stem cell transplantation. In contrast to the more widely used immunotherapy using TCR-transduced peripheral T-cells, our therapy is expected to be more effective and carry less risk of autoreactivity. Methods: CD34+ cells isolated from cord blood and mobilized peripheral blood mononuclear cells were cultured on OP9-DL1 in the presence of the cytokines IL-7, Flt3-L and SCF, for 2 weeks, until T-cell commitment. Subsequently, they were transduced with a WT1-TCR (H. Stauss) or a CMV-TCR (M. Heemskerk), and again co-cultured until CD4+CD8+ double positive cells were abundantly present (generally after another 2-3 weeks). At that point, the agonist peptide WT1 or CMV resp was added to the culture together with IL-7, and 5 days later cells were harvested and expanded (polyclonally or using agonist peptide), in the presence of IL-2, or IL-7+IL-15. T-cells were evaluated using a 51Chromium release assay, for cytotoxicity against WT1 and HLA-A2 positive and negative targets. Also, upon activation, production of IFN-g was evaluated using ELISA. Immunodeficient 6-8 weeks old NSG mice were irradiated (200 cGy), and 24 hours later injected intravenously with either a luciferase-positive, WT1, HLA-A2 transduced K562 cell line (R. Stripecke), or luciferase-transduced, HLA-A2+, WT1+ primary AML cells (expanded on MS-5 in the presence of cytokines) in the current experiments, and 24 hours later, with 5x106 or 107WT1-TCR T-cells or CMV-TCR T-cells (negative control). Mice were evaluated using the IVIS bioluminescence assay. Results: We observed that a nice mix of WT1-TCR CD8+ and CD4+ T-cells (50%/50%) was generated with cells expanded after harvest from the coculture using the combination of the agonist peptide, IL-7 and IL-15. Using 51Cr release assay and ELISA, we could show that upon activation, the T-cells showed specific cytokine production and efficient killing of tumor cells. We observed that the luciferase+, WT1, HLA-A2 transduced K562 cell line homed to ovaria and brain (female mice) or liver, testes and brain (male mice) when injected intravenously, and these are largely sanctuary sites, not reached by the T-cells, therefore resulting in low efficiency. When this cell line was injected subcutaneously in the hind flank, mice showed significant swelling of the resp limb and needed to be euthanized for ethical reasons before full evaluation was possible. Currently, experiments are ongoing evaluating the efficacy of the WT1 T-cells against luciferase transduced primary AML cells (after long term expansion culture on MS-5), as these cells are expected to home to the bone marrow and blood of the mice, and therefore reflect more the physiological situation, and can be more easily reached by the T-cells. Results of these experiments will be presented at ASH. Summary/Conclusion: We have shown that, using the OP9-DL1 model, we were able to generate large numbers of high-avidity tumor-specific naïve and resting T-cells, after a process similar to thymic positive selection. After expansion (polyclonal or antigen specific -in the presence of the agonist peptide-) and activation, these cells show specificity and functionality in vitro and are currently evaluated in an in vivo immunodeficient mouse model. Disclosures No relevant conflicts of interest to declare.

Description: INTRODUCTION On average 5 recurrent mutations are present in each patient with acute myeloid leukemia (AML). Many mutated genes are implicated as tumor suppressor genes, but their contribution to leukemia stem cell (LSC) survival and chemoresistance is often unknown. We hypothesized that ectopic expression of the wildtype sequence of these genes will restore the function of the tumor suppressor gene and will lead to reduced clonal expansion and increased chemosensitivity. AIM To evaluate the contribution of recurrently mutated genes to leukemia stem cell survival and chemoresistance in human AML cells in vitro and in vivo. METHODS We performed a loss-of-function screening in primary human AML cells and cell lines by lentiviral expression of a pool of 22 wildtype genes associated with AML pathogenesis, which can restore gene function of a repressed pathway or a dysfunctional tumor suppressor gene. The 22 full-length cDNAs were labelled with a genetic barcode, which can be amplified with a common primer for all 22 genes. The readout of the screening was reduced representation of the barcode DNA after in vitro culture or in vivo growth in patient-derived xenograft (PDX) models, which was amplified from DNA and quantified by next-generation sequencing (NGS). Five PDX models with favorable, normal or complex cytogenetics and 3-5 recurrent mutations per model were screened in order to identify cDNAs that could potentially limit the proliferative capacity of LSCs in vivo (5 mice per model). Nine to 16 weeks after transplantation, DNA from blood, bone marrow and spleen were analyzed by NGS. We also screened two CD34+-enriched cord blood samples in vitro. After transduction with the cDNA pool, cells were cultured for 11 days and the barcode representation was analyzed by NGS at days 2, 4, 7, 9 and 11. Finally, we screened the human leukemia cell lines U937and PB14, a newly established cell line from an AML patient with mutations in FLT3, NPM1, RAD21, GSE1, and ROBO2. Cells were treated with cytarabine, doxorubicin or venetoclax for a 3-day period, followed by a 4-day recovery period to allow outgrowth of resistant clones and accumulation of cDNAs that conferred drug resistance. RESULTS Our loss-of-function screening with overexpressed wildtype genes revealed that expression of ETV6 and PTPN11 depleted LSCs in 3 of 5 PDX models and that expression of CEBPA and KDM6A depleted the progeny of normal CD34+ cells in cord blood. ASXL1, EZH2, CUX1, SMC1A and SMC3 had a general negative effect on stem cell self-renewal in both leukemic and normal CD34+ cells. Relative frequencies of the leukemia-specific genes ETV6 and PTPN11 were reduced 3 to 16-fold and 2 to 3-fold, respectively. Both genes were not mutated in the 3 patients' diagnostic samples, but had reduced RNA expression by 10-70% compared to healthy control peripheral blood mononuclear cells. Relative frequencies of cord blood-specific CEBPA was reduced 4-fold and KDM6A 5-fold. We then evaluated whether activation of a repressed pathway can increase sensitivity to cytarabine, doxorubicin or venetoclax in U937 and PB14 cells after 1 or 2 weeks of treatment. All three drugs showed better cytotoxic effects upon p53 expression in both cell lines by a factor of 1.4 to 2.5 fold. Cytarabine and venetoclax improved elimination of leukemic cells that had been transduced with U2AF1 in PB14 cells, which are U2AF1 wildtype. Venetoclax improved elimination of U937 cells that had been transduced with ETV6 or KDM6A, which are wildtype for these genes, while RNA expression was reduced more than 50% in these cells compared to other leukemic cell lines (NB4 and MV4-11). CONCLUSION Functional cDNA screening in PDX models in vivo is feasible and can reveal selective vulnerabilities of leukemic compared to normal stem cells. Our approach was validated by the finding that p53 expression improved chemosensitivity for all drugs tested in two leukemia cell lines, which is expected from the known function of p53 as a critical tumor suppressor gene. Similarly, overexpression of the transcriptional corepressor ETV6 in leukemia cells with low ETV6 expression was found to inhibit leukemia stem cell proliferation in vivo and to sensitize U937 cells to venetoclax. Therefore, activation of ETV6 should be explored as a novel strategy to inhibit LSCs and improve treatment response. Disclosures Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees. Heuser:Janssen: Consultancy; StemLine Therapeutics: Consultancy; Bayer Pharma AG: Consultancy, Research Funding; Tetralogic: Research Funding; Sunesis: Research Funding; Daiichi Sankyo: Research Funding; Karyopharm: Research Funding; BergenBio: Research Funding; Astellas: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding.

Description: Objectives: Epstein-Barr virus (EBV) is associated with lymphoproliferative disease in immunocompromised hosts and with human B-cell lymphomas and carcinomas. Adoptive transfer of virus-specific T cells is not practical when memory T cells from HLA-matched donors are not available. Hence, we designed T cells expressing EBV-specific chimeric antigen receptors (CARs) to bypass the need of matched memory T cells. The surface-bound glycoprotein 350 (gp350) was used as target, because it is abundantly expressed during lytic EBV replication and it can also be detected in EBV-immortalized cells. Methods: gp350-CARs were constructed by fusion of single-chain variable fragments of two high affinity gp530-specific human mABs (7A1 and 6G4) to CAR-backbones containing the CD28/CD3ζ domains. Transduction of human T cells from PBMC and cord blood with γ-retroviral vectors showed higher expression levels of 7A1-gp350-CAR than 6G4-gp350-CAR. Results: We used 293T cells expressing gp350 and B95-8 immortalized cells from a tamarin monkey (6-10% gp350+) and human B cells immortalized with the EBV laboratory strain M81 (30% gp350+) in order to compare the potency of gp350-CAR T cells in vitro. Both 7A1-gp350-CAR and 6G4-gp350-CAR were activated and proliferated in the presence of gp350+ cells, inducing cytotoxicity of the target cells. Pilot experiments in a preclinical humanized mouse model consisting of Nod.Rag mice transplanted with human cord-blood (CB) stem cells and infected with an EBV/fLUC strain, we could confirm persistence of CB-matched 7A1-gp350-CAR T cells in spleen, bone marrow and lung for up to 6 weeks. In some animals, this was correlated with lower EBV dissemination measured by optical imaging and PCR. Conclusions: We showed that EBV-specific CARs can reprogram naïve or memory T cells from PBMC or CB to react against EBV infected cells in an HLA-independent manner. This approach can be translated in the future for the generation of anti-EBV-CAR T cells for patients in Need. Disclosures Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees.