ALBERT

Description: Introduction: Patients receiving an allogeneic stem cell graft from a cytomegalovirus (CMV) seronegative donor are prone to CMV reactivation with high risk of disease and mortality. Therefore we initiated a clinical phase I trial with a novel vaccine designed by our group: a CMV phosphoprotein 65 (CMVpp65)-derived peptide in water-in-oil emulsion (Montanide™) plus administration of granulocyte-macrophage colony stimulating factor. Material and Methods: 10 patients after allogeneic stem cell transplantation received four vaccines s.c. at a biweekly interval. Patients were monitored for clinical course and CMVpp65 antigenemia. CMV-specific CD8+ and gamma/delta T cells were analyzed by multi-color flow cytometry. Neutralizing anti-CMV antibody assays were established and correlated to clinical parameters. Results: Peptide vaccination was well tolerated, no drug-related serious adverse events were detected. 7 of 9 patients with CMVpp65 antigenemia cleared the CMV with enduring response 〉 1.5 years after 4 vaccinations and are still free from antigenemia until present. 2 patients with CMV reactivation showed persisting CMV antigenemia. One patient received prophylactic vaccination and did not develop antigenemia. An up to 6-fold increase in frequency of both CMV-specific CD8+ T cells or Vdelta2- gamma/delta T cells with a maximum of 1.32% CMV-specific CD8+ T cells ex vivo without priming and 13.7% Vdelta2- gamma/delta T cells of all CD8+ T cells was detected in five patients. Also titers of neutralizing antibodies increased in four patients up to 10-fold over the time of vaccination. Humoral and cellular immune responses correlated with clearance of the CMV load. Conclusion: Administration of CMVpp65 peptide vaccination for patients after allogeneic stem cell transplantation at high risk for CMV reactivation was safe, well tolerated and clinically encouraging. A study in solid-organ transplant patients is ongoing. Disclosures Kuball: Gadeta B.V.: Membership on an entity's Board of Directors or advisory committees. Wuchter:Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Hexal: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Dreger:Gilead: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Gilead: Speakers Bureau; Novartis: Speakers Bureau; Roche: Consultancy. Greiner:BMS: Research Funding.

Description: Background: Chimeric antigen receptor-modified T cells (CARTs) targeting CD19 has illustrated therapeutic efficacy in diffuse large B cell lymphoma (DLBCL). However, severe cytokine release syndrome (sCRS) reaction and CAR-T-cell-related encephalopathy syndrome (CRES) are the main adverse reactions of CART therapy. Because of the concern of severe CRES which is associated with potential mortality, patients with primary central nervous system Lymphoma(PCNSL) are excluded in most of CART clinical trials. CD70 is a promising therapeutic target due to the restricted expression pattern in normal tissues and overexpression in some lymphoma tissues, so CD70 specific CARTs maybe avoid CD19-negative relapse .Here we report a case with refractory and relapsed PCNSL who achieved long-term disease-free survival by combination therapy of CD19 and CD70 specific CARTs without inducing CRS or CRES. Methods: CD3+ cells were selected from the apheresis PBMC and activated before lentiviral 4SCAR infection. The cells were transduced with a caspase 9-inducible, safety-engineered lentivector CAR containing anti-CD19 or -CD70 scFv fused with multiple intracellular signaling domains: CD28/CD27/CD3z-iCasp9 (4SCAR19 or 4SCAR70). The quality of apheresis cells, gene transfer and T cell proliferation efficiencies, and effective CAR T infusion dose were quantitatively scored and documented. Tumor sections were immunohistochemically stained with different antibodies including anti-CD19 and anti-CD70 antibodies to confirm antigen expression. The patient received cyclophosphamide and fludarabine conditioning regimen before the dual CARTs cell infusions. Case presentation: A 67-year-old male who was diagnosed as PCNSL of DLBCL in 2011.The patient received 6 cycles of temozolomide and high does methotrexate, and achieved CR. In December 2016 he relapsed and was treated with right frontal lobe space-occupying resection and multi-course chemotherapy including 2 course of rituximab, high dose methotrexate and temozolomide ,and 6 courses of rituximab, high dose methotrexate and ibrutinib,and he achieved remission again.However, He relapsed again in August 2017, with the clinical symptoms of dizziness , fatigue, left corners of the mouth askew and occlusal difficulties.MRI presented a residual mass on the right side of the postoperative cavity of 26mm*35mm*30mm and stale hemorrhage in left basal ganglia. After confirmation for CD19 and CD70 expression in his tumor specimens, the pt enrolled in the clinicaltrail :"Combination CART cell therapy targeting hematological malignancies"(clinicaltrail.gov registry NCT03125577).He received 4SCAR19 and 4SCAR70 T cells targeting CD19 and CD70 following lymphodepletion chemotherapy with FC regimen conditioning (FLU 30mg/m2, d1-3; CTX 300mg/m2, d1-3) in October 2017. Brain MRI 1 month later showed complete remission and the pt had symptomatic improvement. To date, after more than 8 months of follow-up, the pt remains in CR (figure1) and CART cells still can be detected . There was no CRS or CRES during the treatment and in follow-up period. Conclusions: The study results demonstrate that CARTs are able to pass through the blood-brain barrier without inducing CRS or CRES, suggesting that CNS tumor is not an absolute contraindication to 4sCART therapy. In addition, the combination of CD19 and CD70 specific 4sCARTs could effectively target PCNSL and achieves long-term disease-free survival without sCRS or CRES. Disclosures No relevant conflicts of interest to declare.

Description: Background: Patients (pts) with R/R aggressive large B cell NHL who fail first-line therapy with immunochemotherapy and are ineligible for high-dose chemotherapy and hematopoietic stem cell transplantation (HSCT) have a poor prognosis. Available treatment options include platinum/gemcitabine-based or bendamustine-based regimens in combination with rituximab, with or without radiotherapy, or clinical trials. However, long-term outcomes remain poor due to lack of a curative option. Liso-cel is an investigational, anti-CD19, defined composition, 4-1BB CAR T cell product administered at target doses of CD4+ and CD8+ CAR T cells. In the ongoing TRANSCEND NHL 001 study of liso-cel as third- or later-line treatment for pts with R/R large B cell NHL, preliminary data showed high overall response rates with a low incidence of grade ≥3 cytokine release syndrome (CRS) and neurological events (NEs) (Abramson et al, ASCO 2018). The open-label, phase 2 PILOT study is assessing the safety and efficacy of liso-cel as second-line therapy in TNE pts (NCT03483103). PILOT is the first study evaluating CAR T cell therapy focusing on this pt population. Methods: Eligible pts had R/R large B cell NHL (diffuse large B cell lymphoma [DLBCL], not otherwise specified [NOS], de novo or transformed indolent NHL, high-grade lymphoma with MYC and BCL2 and/or BCL6 [double/triple-hit lymphoma], or follicular lymphoma (FL) grade 3B) and had received only 1 prior line of immunochemotherapy containing an anthracycline and a CD20-targeted agent (eg, R-CHOP). Pts had to be deemed ineligible for high-dose chemotherapy followed by HSCT by meeting at least 1 of the following TNE criteria while still fulfilling the criteria for CAR T cell therapy: age ≥70 years, ECOG PS of 2, and/or impaired pulmonary (DLCO ≤60% but SaO2 ≥92% on room air and CTCAE ≤1 dyspnea), cardiac (LVEF ≥40% and 30 and 2 and ≤5 ×ULN). Liso-cel was administered at a target dose of 100×106 CAR+ T cells after lymphodepletion (LD) with fludarabine/cyclophosphamide for 3 days. Pts could be treated as outpatients at the investigator's discretion. Results: At data cutoff, 10 pts had been leukapheresed, and 9 pts had LD followed by liso-cel infusion; 1 pt is awaiting liso-cel treatment. Liso-cel was manufactured successfully in all pts. Five pts were infused and monitored as outpatients. Median age was 71 (range, 64-79) years; 5 pts were male. Histology included DLBCL NOS (n=7) and transformed FL (n=2); 2 pts had triple-hit, one of whom had transformed from FL. Five pts had relapsed from, and 4 pts had disease refractory to, prior therapy. Median SPD and LDH were 26.6 cm2 and 201 U/L, respectively. Four pts had high tumor burden with SPD ≥50 cm2 (n=4) and/or LDH ≥500 U/L (n=1). The median Hematopoietic Cell Transplantation Comorbidity Index (HCT-CI) score was 3 (range, 0-3). Six pts had 1 or more treatment-emergent adverse events (TEAEs) grade ≥3, which were primarily cytopenias. Three pts had prolonged grade ≥3 cytopenias at Day 29. Two pts had infections of any grade; no pts had grade ≥3 infections. No pts had CRS or NEs, and no pts received tocilizumab, corticosteroids, or vasopressors. There were no cases of macrophage activation syndrome, tumor lysis syndrome, infusion reactions, or grade 5 TEAEs. Among the 5 pts treated and monitored as outpatients, none were admitted to hospital for adverse events within the first 29 days post liso-cel infusion. All 9 pts achieved an objective response. Four pts achieved complete response; all are ongoing. Five pts achieved partial response (PR), with 2 PRs ongoing. Results were similar in inpatient vs outpatient pts. Median follow-up was 3.5 months. Median (range) time to peak CAR T cell expansion was 10 (7-21) days. Conclusions: These preliminary safety and efficacy data from the ongoing phase 2 PILOT study suggest that liso-cel can be successfully administered, including in the outpatient setting, as second-line therapy in pts with R/R aggressive B cell NHL who were ineligible for high-dose chemotherapy and HSCT by prespecified criteria. Updated safety and efficacy data with longer follow-up will be presented. Disclosures Sehgal: Kite/Gilead: Research Funding; Merck: Research Funding; Juno/Celgene: Research Funding. Pribble:Celgene/Juno: Employment. Wang:Celgene Corporation: Employment. Thorpe:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Hildebrandt:Axim Biotechnologies: Equity Ownership; Abbvie: Equity Ownership; GW Pharmaceuticals: Equity Ownership; Endocyte: Equity Ownership; Clovis Oncology: Equity Ownership; Kite Pharma: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other; CVS Health: Equity Ownership; Celgene: Equity Ownership; Axim Biotechnologies: Equity Ownership; Pharmacyclics: Research Funding; Sangamo: Equity Ownership; Cellectis: Equity Ownership; Bluebird Bio: Equity Ownership; Bristol-Myers-Squibb: Equity Ownership; crispr therapeutics: Equity Ownership; IDEXX laboratories: Equity Ownership; Johnson & Johnson: Equity Ownership; Pfizer: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Travel; Procter & Gamble: Equity Ownership; Vertex: Equity Ownership; Scotts-Miracle: Equity Ownership; Takeda: Research Funding; Bayer: Equity Ownership; Astellas: Other: Travel; Kite Pharma: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Travel; Novartis: Equity Ownership; Aetna: Equity Ownership; Juno Therapeutics: Equity Ownership; Cardinal Health: Equity Ownership; Novartis: Equity Ownership; Insys Therapeutics: Equity Ownership; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Travel; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Research Funding; Immunomedics: Equity Ownership.

Description: Background: Relapsed/refractory (R/R) DLBCL is associated with high healthcare resource utilization (HRU) and cost (Danese 2017). Patients with R/R DLBCL have poor outcome with median OS

Description: In mouse models of bone marrow transplantation, irradiation of recipient animals to ablate endogenous hematopoietic tissues is conventionally given by radioisotope-based irradiators. Recently, cabinet-size, X-ray-based irradiators have been advertised as a safe and cost-effective alternative source of radiation. Whereas 137Cesium-generated gamma-rays have an energy of 662 kV, X-rays generated from cabinet irradiators typically have a peak energy of only 130 kV, thus potentially limiting their ability to penetrate tissues. In this study, we performed dosimetry studies of a Faxitron RX650 irradiator operating at peak voltage and tested its effectiveness in ablating murine bone marrow. Thermo-luminescence dosimetry (TLD) chips were placed on and under the skin of freshly sacrificed mice or embedded in the tissue next to the pelvis and long bones. The mice were placed inside a Plexiglas cage at a distance of 16 inches from the X-ray tube and irradiated in a fashion that simulated live mouse irradiation. The TLD chips recorded radiation doses of 150 cGy/min to the skin, 73 cGy/min to the pelvis and 47 cGy/min to the femur, which were dramatically lower than the nominal dose of 527 cGy/min suggested by the manufacturer. This result demonstrated that there was a marked attenuation of radiation by intervening materials and a significant difference in doses delivered to superficial and deep tissues of irradiated mice. Preliminary studies performed on live mice showed that irradiation at a dose high enough to ablate bone marrow cells caused extensive ulceration of back skin, necrosis of ear cartilage and increased mortality rates post-irradiation. To overcome these problems, a 4-point-rotation irradiation procedure was subsequently adopted, whereby the mice to be irradiated were first anesthetized and then placed inside a confinement cage to receive equal fractions of radiation in the supine, prone, left lateral decubitus and right lateral decubitus positions, with four hours between the first and last two fractions. Dosimetry analysis showed that this irradiation protocol gave an effective average dose of 55 cGy/min to hematopoietic tissues of the irradiated mice. To confirm the biological validity of this protocol, four cohorts of mice were given effective doses of either 200, 600, 900 or 1100 cGy and then transplanted with 80,000 bone marrow cells. Twelve days later, prominent spleen colonies (CFU-S) derived from transplanted hematopoietic progenitors were seen in the 900 and 1100 cGy cohorts, whereas no colonies were observed in the 200 and 600 cGy cohorts, indicating successful endogenous bone marrow ablation using the higher radiation doses. Engraftment studies were then performed in which four cohorts of C57BL/6 (B6) mice were given effective doses of either 200, 600, 900 or 1100 cGy, respectively, and transplanted with 5x106 B6.SJL bone marrow cells per mouse. After four weeks, peripheral blood analysis showed donor engraftment rates of 90% in mice irradiated with 900 or 1100 cGy. Our studies showed that X-ray-based irradiators can be used effectively for bone marrow ablation in mice, but careful dosimetry calibration and a 4-point-rotation irradiation procedure is necessary to prevent radiation-associated tissue damage.

Description: Abstract 571 Asymmetric cell division, a proposed mechanism by which hematopoietic progenitor/stem cells (HPSC) maintain a balance between self-renewal and differentiation, has rarely been observed. Here we report the surprising finding that cultured mouse primary HPSC routinely generate pairs of daughter cells with 2 distinct phenotypes after a single round of cell division. Mouse bone marrow cells were cultured on chamber slides in the presence of stem cell factor (SCF). BrdU was added overnight to label dividing cells, and the cells were examined by immunofluorescence microscopy on day 2–4 of culture. In each BrdU+c-Kit+ divided cell doublet, c-Kit was invariably expressed in only 1 of the 2 daughter cells. In contrast, the other daughter cell was negative for c-Kit but positive for the asymmetric cell fate determinant Numb and mature myeloid markers Mac1, Gr1, M-CSFR and F4/80. Similarly, in each BrdU+Sca1+ cell doublet, 1 daughter cell was positive for the stem cell markers Sca1, c-Kit, CD150 and CD201, whereas the other cell was negative for these markers but positive for Numb and the mature myeloid markers. Analysis of 400 such doublets showed that the probability of HPSC undergoing asymmetric division was 99.5% (95% confidence interval 98–100%), indicating that asymmetric division in HPSC is in fact not rare but obligatory. In other model systems, it has been shown that activation of the atypical protein kinase C (aPKC)-Par6-Par3 cell polarity complex and realignment of the microtubule cytoskeleton precede asymmetric cell division. We asked whether similar steps are involved in the asymmetric division of HPSC. We found that c-Kit receptors, upon stimulation by SCF, rapidly capped at an apical pole next to the microtubule-organizing center, followed by redistribution to the same pole of the aPKC-Par6-Par3 complex and microtubule-stabilizing proteins APC, β-catenin, EB1 and IQGAP1. Strikingly, after cell division, the aPKC-Par6-Par3 complex and other polarity markers all partitioned only into the c-Kit+/Sca1+ daughter cell and not the mature daughter cell. The acetylated and detyrosinated forms of stabilized microtubules were also present only in the c-Kit+/Sca1+ cell, as were the Aurora A and Polo-like kinases, 2 mitotic kinases associated with asymmetric cell division. To understand how c-Kit activation triggers downstream polarization events, we studied the role of lipid rafts, cholesterol-enriched microdomains in the cell membrane that serve as organization centers of signaling complexes. These are enriched in phosphatidylinositol 4,5-bisphosphate and annexin 2, putative attachment sites for the aPKC-Par6-Par3 complex. We found that SCF stimulation led to coalescence of lipid raft components at the site of the c-Kit cap, and treatment with a wide range of inhibitors that blocked lipid raft formation abrogated polarization of the aPKC-Par6-Par3 complex and division of the c-Kit+/Sca1+ cells. Because obligatory asymmetric division in cultured HPSC would prevent a net increase in their number, we sought a way to bypass its mechanism. We tested whether inhibition of protein phosphatase 2A (PP2A), a physiological antagonist of aPKC, would enhance aPKC activity and promote self-renewal of HPSC. Treatment of cultured HPSC with okadaic acid or calyculin, 2 well-characterized PP2A inhibitors, increased the percent of c-Kit+/Sca1+ cells undergoing symmetric division from 0% to 23.3% (p30% in mice transplanted with PP2A inhibitor-treated cells. This dramatic increase indicates that PP2A inhibition can effectively perturb the mechanism of asymmetric cell division and promote the self-renewal of HPSC. In summary, our data showed that obligatory asymmetric cell division works to maintain a strict balance between self-renewal and differentiation in HPSC and pharmacological manipulation of the cell polarity machinery could potentially be used to expand HPSC for clinical use. Disclosures: No relevant conflicts of interest to declare.

Description: Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure and predisposition to malignancy. One of the potential therapeutic options for patients with FA is collection of autologous multipotent hematopoietic progenitors prior to the development of severe pancytopenia for autologous transplantation and gene therapy. However, poor engraftment of FA hematopoietic cells represents a major obstacle for effective transplantation. Our current study attempted to investigate the mechanism underlying defective engraftment of FA bone marrow (BM) cells. Using BM cells from patients carrying mutations in the FA complementation group A (FA-A), we demonstrate that SDF (Stromal cell-derived factor)-1alpha – and integrin-mediated migration and adhesion, respectively, is defective in FA primary BM cells compared to those from normal donors (more than 2-fold decrease in both migration and adhesion compared to normal BM cells). Complementation of the FA-A defect by retrovirus gene transfer of FANCA gene almost completely restores the ability of the BM cells to migrate towards the chemokine SDF-1alpha. Similar results are obtained with primary fibroblast cells derived from a FA-A patient, which show 3-fold and 35% decrease in adhesion and migration, respectively, compared to FANCA-corrected cells. Furthermore, when transplanted into immunodeficient Nod/scid recipient mice, the FA-A BM cells exhibited significantly impaired homing function whereas normal cells were efficiently homed in the bone marrow. A significant decrease in the activity of the Rho GTPase Cdc42 in FA-A cells is found associated with the patient cell defective functions. Taken together, these data suggest that the FA proteins play a role in the regulation of cell adhesion and migration in addition to maintaining genomic stability, influencing homing and engraftment, possibly via the small GTPase signaling pathway. These findings may have implications in development of strategies for restoring engraftment function of FA hematopoietic cells.

Description: Introduction: Cutaneous T Cell Lymphoma (CTCL) has an annual incidence of one per 6.4 million persons in the United States and represents 3.9% of all non-Hodgkin lymphomas. Numerous treatment modalities exist, including both oral Bexarotene and topical Bexarotene 1% gel. Bexarotene is a synthetic retinoid that selectively activates retinoid X receptor (RXR) isotypes. Retinoids, natural metabolic derivatives and synthetic analogs of vitamin A, are physiologically important in establishing mucosal immunity. Retinoids induce gut homing in T lymphocytes effectively targeting immune cells away from the skin niche. Although it is appreciated that retinoids activate the retinoic acid receptor (RAR) and retinoid X receptor (RXR) family of nuclear receptors to elicit cellular changes, the molecular details of retinoic acid action remain poorly defined within the context of CTCL therapy. The specific nuclear receptor isotypes involved in CTCL responsiveness and if this responsiveness entails the expression of molecules associated with gut homing is not known. The significance of this knowledge gap is championed by the fact that the full benefit of Bexarotene in CTCL is limited due to adverse metabolic side effects, namely, hypercholesterolemia, hypothyroidism, and hyperlipidemia. The promiscuous pairing nature of RXR with other nuclear receptors such as the liver X receptor and thyroid hormone likely accounts for the detrimental effects. Delineating how retinoids influence CTCL at the molecular level would greatly increase the specificity and efficacy of retinoid-based therapies. Results: Here we employ a battery of agonists and antagonists, including Bexarotene, to delineate the specific nuclear receptors utilized by retinoids to evoke adhesion changes within CTCL cells. When a spectrum of T cell lymphomas/leukemias are cultured with the physiologically relevant all-trans-retinoic acid (atRA) retinoid isomer, only CTCL lineages increase integrin-mediated adhesion to the gut trafficking ligand, Mucosal Adressin Cell Adhesion Molecule (MAdCAM-1). Our data further demonstrate that the individual activation of RAR or RXR nuclear receptors isotypes was sufficient to induce adhesion to MAdCAM-1 within the CTCL cells. Our data also establish that the induced immune cell adhesion was largely attributable to the specific activity of the RAR-alpha receptor isotype. However, longer exposure to other isotype-specific agonists did induce adhesion. Most importantly, we show that the adhesion to MAdCAM-1 was significantly augmented upon activation of both RAR and RXR nuclear receptors in a synergistic manner. In addition, this synergism was only observed with the RAR-alpha isotype. Conclusions: The current study provides molecular resolution as to which nuclear receptors transduce retinoid exposure into biologically relevant CTCL cell adhesion. Our work suggests that Bexarotene therapy can be augmented by combinatorial drug therapy with a RAR-alpha agonist to induce synergistic receptor activation. Decreasing the concentration of Bexarotene required to elicit a desired response and increasing the specificity by targeting distinct receptor isotypes would plausible minimize the adverse side effects associated with retinoids. The significance of this work is underscored by the historical success of retinoid-based therapies such as Bexarotene and the potential undiscovered therapies still embodied within vitamin A. Disclosures No relevant conflicts of interest to declare.

Description: ABSTRACT The prognostic value of IDH1 mutations has been systematically evaluated in acute myeloid leukemia (AML) patients recently. However, the role of IDH1 expression in AML is still under exploration. To investigate the clinical significance, we analyzed the IDH1 expression in 320 patients with cytogenetically normal AML (CN-AML) by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). High expression of IDH1 was predominant in patients with FLT3-ITD and DNMT3A mutations, and less prevalent in cases with CEBPA double allele mutations. Strong association was observed between high IDH1 expression and low expression of micro-RNA 181 family. Prognosis was adversely affected by high IDH1 expression with shorter overall survival (OS) and event free survival (EFS) in the context of clinical characteristics including age, WBC, and gene mutations of NPM1, FLT3-ITD, CEBPA, IDH1, IDH2, and DNMT3A in CN-AML. Moreover, the clinical outcome of IDH1 expression in terms of OS, EFS and complete remission rate still remained in multivariate models in CN-AML. Importantly, the prognostic value was validated using the published microarray data from 79 adult patients treated according to the German AMLCG-1999 protocol. Our results demonstrated that high IDH1expression is associated with a poor prognosis of CN-AML. Disclosures No relevant conflicts of interest to declare.

Description: Background: Chimeric antigen receptor (CAR) engineered T cell therapy is currently revolutionizing the field of cancer immunotherapy. However, further enhancement of the function of CAR T cells either through modification of CAR structure or through combination with other therapies is intensively investigated. Resistance to apoptosis is one of the hallmarks of cancer biology. Overexpression of anti-apoptotic proteins of the Bcl-2 family proteins, such as Bcl-2, Mcl-1 and Bcl-xl is considered primarily responsible for the increased apoptosis resistance and prolonged survival of hematological tumor cells. Apoptosis inhibitor blockade agents (AIBAs) such as Bcl-2, Mcl-1 and COX inhibitors could sensitize malignant cells to apoptosis. Therefore, combining CAR T cell therapy with AIBAs might be a promising approach to increase the therapeutic response. However, the third generation of CAR T cells including a 4-1BB co-stimulatory domain which triggers a signaling cascade with the upregulation of anti-apoptotic molecules might be hampered by AIBAs. Therefore, in this study we analyzed the influence of AIBAs on third generation CD19 CAR T cells. Materials and methods: CD19 CAR T cells were manufactured using a 3rd generation CAR vector containing both CD28 and 4-1BB co-stimulatory domains. The expression of anti-apoptotic Bcl-2 family members in leukemia/lymphoma cell lines and in CD19 CAR T cells have been assessed by quantitative real time polymerase chain reaction (qRT-PCR), western blot (WB) and flow cytometery. The optimal concentrations of inhibitors have been determined by CellTiter Glo™ assay. The effect of inhibitors on CAR T cells and tumor cells has been evaluated by chromium-51 (51Cr) release assay, Calcein AM™ assay and FACS-based apoptosis assay. Intracellular cytokine staining and surface marker staining were performed to determine the function of CAR T cells. Results: qRT-PCR and WB showed that leukemia/lymphoma cell lines 380 and U698M had the highest Bcl-2 (qRT-PCR: 1.73 ± 0.04, p = 0.001; WB: 1.82 ± 0.54, p = 0.016) and Mcl-1 (qRT-PCR: 12.39 ± 1.37, p = 0.000; WB: 1.92 ± 1.08, p = 0.142) expression levels, respectively,. These findings were confirmed by anti-apoptotic BCL-2 family protein intracellular staining. Elevated protein expression of Mcl-1, Bcl-2 and Bcl-xl was observed in CAR T cells upon 380 cell stimulation. The manufactured CD19 CAR T cells exhibited specific killing on CD19+ tumor cells. A slightly enhanced killing efficiency of CAR T cells was observed when 380 cells were co-cultured with CAR T cells in the presence of ABT199 (0 µM vs. 1 µM: 54.21 ± 3.23 vs. 69.06 ± 2.17, p = 0.0041). This result could be explained by a higher sensitivity of 380 cells to ABT199 than CART cells and by an increased activation of CAR T cells. Even though the killing efficiency of CAR T cells was not improved by S63845, the CD107a+TNF-α+IFN-γ+ CAR T cells were increased as well as the proliferation and persistence of CAR T cells were strengthened. Of note, pretreatment of tumor cells with inhibitors could significantly enhance the killing efficiency of CAR T cells via upregulation of CD19 antigen on tumor cells (ABT199-pretreated 380 cells: 0 µM vs.1 µM, 42.59 ± 5.82 vs. 54.58 ± 4.87, p = 0.0493; S63845-pretreated-U698M cells: 0 µM vs. 0.3 µM, 31.88 ± 4.77 vs. 66.35 ± 8.09, p = 0.000). Conclusion: The quantity of CAR T cells can be negatively affected by Apoptosis inhibitor blockade agents. However, the quality of CAR T cells could be improved. The cytotoxic effect of CAR T cells can be further enhanced by pretreatment of tumor cells with inhibitors. Disclosures Müller-Tidow: MSD: Membership on an entity's Board of Directors or advisory committees. Dreger:Neovii, Riemser: Research Funding; MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia; AbbVie, AstraZeneca, Gilead, Janssen, Novartis, Riemser, Roche: Consultancy; AbbVie, Gilead, Novartis, Riemser, Roche: Speakers Bureau. Schmitt:MSD: Membership on an entity's Board of Directors or advisory committees, Other: Sponsoring of Symposia; Therakos Mallinckrodt: Other: Financial Support. Schmitt:Therakos Mallinckrodt: Other: Financial Support.