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. Disclosures Locatelli: 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.

Cellular and Molecular Effects of NVP-BKM120 in Lymphoblastic Leukemia Cells

Background: PI3K/AKT/mTOR signaling controls most hallmarks of cancer. Constitutive activation of PIK3 pathway in T cells acute lymphoblastic leukemia (T-ALL) has been reported; in a mouse model, PI3K activation, together with MYC, cooperates in Burkitt lymphoma (BL) pathogenesis. NVP-BKM120 is an orally bioavailable 2,6-dimorpholine pyrimidine derivative, and considered a highly selective pan-class I PI3K inhibitor. In preclinical studies, it has shown efficacy in a variety of malignancies and is currently being investigated in phase I/II/III clinical testing, mainly for advanced solid tumors (clinicaltrials.gov). Aims: Here, we described the effects of the pan-PI3K/AKT/mTOR inhibitor NVP-BKM120 on T-ALL and BL cell lines. Methods: T-ALLcell lines, Jurkat and MOLT-4, and BL cell lines, NAMALWA and Daudi, were obtained from ATCC. NVP-BKM120 was kindly provided by Novartis, and was prepared as a 10mM stock solution in DMSO. Different concentrations of the drug were used as indicated, where cells treated only with DMSO served as control. Cell viability was measured by MTT. Colony formation was carried out in semisolid methyl cellulose medium. The induction of apoptosis was assessed by annexin-V-APC/PI and by caspase cleavage. The cell cycle was analyzed by a PI-staining method. Western blot analysis was performed by standard methods. Vital staining and flow cytometry analysis with acridine orange was performed for the detection and quantification of acidic vesicular organelles (AVOs). Comparisons between the two groups were performed by the t test. Pvalue <0.05 was considered statistically significant. Results: Cell viability decreased in a concentration-dependent manner, with an IC50 range of 7-8 μM and the clonogenic growth was significantly decreased at the concentration of 1μM and at 10µM the colony formation was completely inhibited in all cells tested. After 6 hours of NVP-BKM120 treatment, we observed an increase in apoptotic cells, as well as an increase in the cleavage of procaspase 3, 8 and 9 in Jurkat, MOLT-4 and NAMALWA cells. Compared with DMSO control, NVP-BKM120 does not have any effect during apoptosis induction in the Daudi cell line. NVP-BKM120 treatment also resulted in G2/M arrest, associated with a decrease in the G1population and a decrease in Cyclin B1 protein levels. Immunoblotting analysis of cells treated with the drug revealed decreased phosphorylation, in a dose-dependent manner, of AKT, P70SK6 and 4EBP1, with stable total proteins levels. Additionally, we observed a dose-dependent decrease in BAD phosphorylation, followed by an increase in BAX:BCL2 ratio. Quantification of AVOs showed a dose-dependent increase of AVOs in all cells tested, after NVP-BKM120 treatment. Conclusions: NVP-BKM120 induced apoptosis in a dose-dependent manner in Jurkat, MOLT-4 and NAMALWA cells, while effects of the drug in the Daudi cell line were mainly cytostatic. In those cells, the induction of apoptosis suggested that the death receptor and mitochondrial pathways were activated after drug treatment. In our study, we found that NVP-BKM120 decreased the phosphorylation levels of BAD, which is linked to a pro-apoptotic activity, and up-regulated the BAX:BCL2 ratio. These results are consistent with the activation of caspase-9 and 3, related to the mitochondrial apoptosis. The accumulation of leukemia cells in the G2/M phase of the cell cycle has been associated with enhanced apoptosis. Our results suggest that decreased Cyclin B1 protein expression might be the molecular mechanism through which NVP-BKM120 induces G2/M arrest. The effects of NVP-BKM120 on the PI3K pathway indicate that NVP-BKM120 treatment may overcome rapamycin-induced AKT activation. P70SK6 and 4EBP1 are the two best-characterized substrates of mTOR1. Hence, the decrease in the phosphorylation levels of P70SK6 and 4EBP1 results in impaired oncogenic protein synthesis. Moreover, P70SK6 is one of the kinases whose phosphorylation by mTOR1 results in opposing autophagy. Accordingly, NVP-BKM120 resulted in increased AVOs, which is a characteristic feature of cells engaged in autophagy. In summary, our present study establishes that NVP-BKM120 effectively presents an antitumor activity against T-ALL and BL cell lines. The reduction of proliferation is possibly by down-regulation of Cyclin B1 and the increased BAX:BCL2 ratio is one of the mechanisms involved in the induction of apoptosis. Disclosures Off Label Use: NVP-BKM120 is an orally bioavailable 2,6-dimorpholino pyrimidine derivative, and considered a highly selective pan-class I PI3K inhibitor. .

PARP Inhibitor Selectively Induces Cell Death in E2A-Hlf Positive Leukemia

Background: Defects in homologous recombination repair (HRR) have long been known to contribute to genomic instability leading to tumor development. Poly (ADP-ribose) polymerase (PARP) exerts various cell biological effects, such as maintenance of genomic stability, energy metabolism and cell death. PARP is indispensable in DNA repair machinery, especially in base excision repair (BER). PARP inhibition convert DNA double strand breaks from DNA single strand breaks induced by alkylating agents. These DNA double strand breaks can be repaired by HRR. Therefore, PARP inhibitor induces synthetic lethality in HRR defective cancer cells. Such lethality was successfully shown in BRCA1 or 2 mutated breast cancers. However, only a limited study has been performed other than breast cancers. Some tumors including hematological malignancies are defective in HRR function leading to a possibility to be sensitized to PARP inhibitor. Methods: Sensitivity to PARP inhibitor was screened using 28 leukemia cell lines. HRR activity was measured by DR-GFP HRR assay. Expression of proteins involves HRR was evaluated by cDNA microarray analysis and western blotting. Results: E2A-HLF positive leukemia showed susceptibility to PARP inhibitor. This experiment suggests that expression of E2A-HLF chimeric messenger RNA sensitize the leukemic cell to PARP inhibitor. To elucidate whether E2A-HLF genuinely sensitize the cell for PARP inhibition, E2A-HLF was transduced into PARP inhibitor resistant Burkitt cell line, Daudi, using retrovirus. Compared with mock infected Daudi cell, E2A-HLF expressed Daudi cell showed increased sensitivity to PARP inhibitor. This experiment suggests that E2A-HLF expressed cell is defective HRR pathway. To test this hypothesis, HRR assay using DR-GFP construct was employed. HRR between the two nonfunctional GFP genes to generate a functional GFP gene can be triggered by transient transfection of the I-SceI expression vector, which introduces a DNA double-strand break (DSB) in one of the two GFP genes. HRR proficiency can be determined by the number of cells expressing the GFP protein. DR-GFP HRR assay exhibited defect of HRR function in E2A-HLF expressed cell. Interestingly, expression of BRCA1 was decreased in E2A-HLF transfected cell, which presumably link with decreased HRR activity. Conclusions: Increased sensitivity to PARP inhibitor in E2A-HLF positive leukemia was caused by decreased HRR activity by E2A-HLF expression. PARP inhibitor will be a novel therapeutic approach for refractory leukemia, especially with E2A-HLF translocation. While PARP inhibitor monotherapy is an attractive proposition for treating such as HRR defective E2A-HLF expressed leukemia, combination of HRR inhibitor will be a universal strategy for various types of leukemia. Disclosures No relevant conflicts of interest to declare.

Retargeting of Activated T Cells (ATC) with Gemtuzumab (anti-CD33) Ozogamicin x Anti-CD3 Bispecific Antibody (CD33-OBi) Enhances Cytotoxicity Directed at K562.

Abstract 4094 Poster Board III-1029 Background CD33 is expressed in about 90% of blasts in acute myeloid leukemia but the response rate of gemtuzumab ozogamicin (GO) is about 30%. GO is anti-CD33 hP67.6 linked to calicheamicin. The mechanism of resistance of blasts to GO is not clearly understood. Our previous studies show the anti-CD3 activated T cells (ATC) can be redirected with bispecific antibodies (BiAb) to Her2/neu, CD20, and EGFR. We produced CD33-OBi by chemically heteroconjugating GO with OKT3 (anti-CD3). We determined whether ATC armed with CD33-OBi can enhance the killing of a low expressing GO-resistant cell line K562. Methods ATC were produced by stimulating peripheral blood mononuclear cells (PBMC) with anti-CD3 in the presence of IL-2. K562 cell line, expressing 67% CD33 expression was used as a target given the relative resistance of this cell line to GO. Daudi cell line expressing no CD33 was used as control. Cytotoxicity to K562 and Daudi cell lines was performed using 51Cr labeled cells as targets with different concentrations of GO alone, CD33-OBi, ATC alone and CD33-OBi armed ATC. Armed ATC were washed free of arming BiAb unless otherwise indicated. Various arming doses of the CD33-OBi for ATC were also tested, ADCC with CD33-OBi utilizing fresh PBMC were tested. ADCC with GO was not tested as the mechanism of action for GO is through internalization and DNA damage from the cleaved calicheamicin. Results In antibody doses of 5, 50, 500, and 5000 ng/ml of GO or CD33-OBi alone, specific cytotoxicity was 5,7,14, and 14% for GO and was essentially null at all concentrations for CD33-OBi. At arming doses of 0.5, 5, 50 and 500 ng of CD33-OBi/106 ATC, the plateau of mean specific cytotoxicity directed at K562 cells was 38%, achieved between arming doses of 50 and 500 ng/106ATC at an effector to target ratio (E/T) of 25:1. Daudi CD33-negative control cells were not lysed by either GO or CD33-OBi alone. Subsequently, ATC from 3 normal donors (n=3) armed with CD33-OBi with 50 ng/106 cells at E/T of 25:1, 12.5:1, 6.25:1 and 3.125:1 showed % mean [±SEM] specific cytotoxicity of 34.3[±3.2], 28.5[±3.2], 22[±2.8] and 16.2[±2.1]%, respectively to K562. On the other hand, ATC alone at similar E/T showed % mean specific cytotoxicity of 25[±4.6], 16.3[±3.6], 10.3[±2.4] and 5.3[±1.3].There were significant differences between CD33-OBi-ATC and ATC with p values (Wilcoxan signed rank test) at E/T of 25:1, 12.5:1, 6.25:1 and 3.125:1 that were 0.043, 0.033, 0.014 and 0.012, respectively. There was no specific cytotoxicity directed at Daudi cells under similar conditions. ADCC was performed with CD33-OBi added to co-culture of PBMC and labeled K562 and Daudi cells for 24 hrs which showed background specific cytotoxicity. Conclusions ATC armed with CD33-OBi at 50 ng/106 cells showed enhanced cytotoxicity directed at K562 cells which historically is relatively resistant to GO. The unarmed ATC have been shown to have non MHC restricted cytotoxicity and arming them with the CD33-OBi retargets and enhances the cytotoxicity of ATC to targets that express CD33. Given that patients with CD33 positive blasts may contain a population of CD33 positive leukemia stem cells, this approach may provide a non-toxic strategy to target leukemia stem cells. Testing additional GO susceptible and resistant cell lines under conditions in combination with chemotherapy or hematopoietic stem cell transplant may provide proof of principle for developing this approach. Disclosures: Lum: Transtarget Corporation: Founder.