The developmental processes of lymphopoiesis generate mature B lymphocytes from hematopoietic stem cells through increasingly restricted intermediates. Networks of transcription factors regulate these cell fate choices and are composed of both ubiquitously expressed and B lineage-specific factors. E-protein transcription factors are encoded by the three genes E2A, E2-2 (SEF2-1), and HEB. The E2A gene is required for B cell development and encodes the alternatively spliced proteins E12 and E47. During B lymphocyte development, the cells have to pass several checkpoints verifying the functionality of their antigen receptors. Early in the development, the expression of a pre-B cell receptor (pre-BCR) with membrane-bound immunoglobulin (Ig) heavy chain protein associated with surrogate light chain (SLC) proteins is a critical checkpoint that monitors for functional Ig heavy chain rearrangement. Signaling from the pre-BCR induces survival and a limited clonal expansion. Here it is shown that pre-BCR signaling rapidly down-regulates the SLCs l5 and VpreB and also the co-receptor CD19. Ca2+ signaling and E2A were shown to be essential for this regulation. E2A mutated in its binding site for the Ca2+ sensor protein calmodulin (CaM), and thus with CaM-resistant DNA binding, makes l5, VpreB and CD19 expression resistant to the inhibition following pre-BCR stimulation. Thus, Ca2+ down-regulates SLC and CD19 gene expression upon pre-BCR stimulation through inhibition of E2A by Ca2+/CaM. A general negative feedback regulation of the pre-BCR proteins as well as many co-receptors and proteins in signal pathways from the receptor was also shown. After the ordered recombination of Ig heavy chain gene segments, also Ig light chain gene segments are recombined together to create antibody diversity. The recombinations are orchestrated by the recombination activating gene (RAG) enzymes, other enzymes that cleave/mutate/assemble DNA of the Ig loci, and the transcription factor Pax5. A key feature of the immune system is the concept that one lymphocyte has only one antigen specificity that can be selected for or against. This requires that only one of the alleles of genes for Ig chains is made functional. The mechanism of this allelic exclusion has however been an enigma. Here pre-BCR signaling was shown to down-regulate several components of the recombination machinery including RAG1 and RAG2 through CaM inhibition of E2A. Furthermore, E2A, Pax5 and the RAGs were shown to be in a complex bound to key sequences on the IgH gene before pre-BCR stimulation and instead bound to CaM after this stimulation. Thus, the recombination complex is directly released through CaM inhibition of E2A. Upon encountering antigens, B cells must adapt to produce a highly specific and potent antibody response. Somatic hypermutation (SH), which introduces point mutations in the variable regions of Ig genes, can increase the affinity for antigen, and antibody effector functions can be altered by class switch recombination (CSR), which changes the expressed constant region exons. Activation-induced cytidine deaminase (AID) is the mutagenic antibody diversification enzyme that is essential for both SH and CSR. The AID enzyme has to be tightly controlled as it is a powerful mutagen. BCR signaling, which signals that good antibody affinity has been reached, was shown to inhibit AID gene expression through CaM inhibition of E2A.  SH increases the antigen binding strength by many orders of magnitude. Each round of SH leads to one or a few mutations, followed by selection for increased affinity. Thus, BCR signaling has to enable selection for successive improvements in antibodies (Ab) over an extremely broad range of affinities. Here the BCR is shown to be subject to general negative feedback regulation of the receptor proteins as well as many co-receptors and proteins in signal pathways from the receptor. Thus, the BCR can down-regulate itself to enable sensitive detection of successive improvements in antigen affinity. Furthermore, the feedback inhibition of the BCR signalosome and most of its protein, and most other gene regulations by BCR stimulation, is through inhibition of E2A by Ca2+/CaM. Differentiation to Ab-secreting plasmablasts and plasma cells is antigen-driven. The interaction of antigen with the membrane-bound Ab of the BCR is critical in determining which clones enter the plasma cell response. Genome-wide analysis showed that differentiation of B cells to Ab-secreting cell is induced by BCR stimulation through very fast regulatory events, and induction of IRF-4 and down-regulation of Pax5, Bcl-6, MITF, Ets-1, Fli-1 and Spi-B gene expressions were identified as immediate early events. Ca2+ signaling through CaM inhibition of E2A was essential for these rapid down-regulations of immediate early genes after BCR stimulation in initiation of plasma cell differentiation.

No description available.

No description available.

A imunoterapia utilizando linfócitos T modificados com receptor quimérico de antígenos (CAR) tem se mostrado eficaz no tratamento de leucemia e linfomas resistentes à quimioterapia. A proteína CD19 é considerada um alvo ideal porque é expressa na maioria dos tumores de linfócitos B e linfócitos B normais, mas não em outras células. Estudos clínicos recentes mostraram excelentes respostas de linfócitos T-CAR em uma variedade de tumores de células B. Os vetores lentivirais são o método mais comumente utilizado para modificação genética em ensaios clínicos. Este estudo teve como objetivo desenvolver uma plataforma eficiente para a produção de lentivírus e testar a funcionalidade desses vetores para que possam ser usados para modificar geneticamente linfócitos T. A transfecção transiente de céulas HEK293T com plasmídeos na proporção de 3:1:1:1 (transgene:gag-pol:VSV-G:rev) utilizando lipossomos catiônicos e 5 mM de butirato de sódio resultou nos títulos virais mais elevados. Isso representa um aumento de 17 vezes no título viral da transfecção com polietilenoimina (PEI). Três métodos para concentracao lentiviral foram utilzados nesse trabalho, ultracentrifugação, filtração tangencial e ultrafiltração. A ultrafiltração sobre membrana com corte de peso molecular (MWCO) de 100 kDa resultou na maior taxa de recuperação de partículas virais viáveis, aproximadamente 82%. As partículas virais produzidas por este processo demonstraram ser funcionais para a transdução de linfócitos T. Além disso, o receptor quimérico (CAR) se mostrou específico contra o antígeno CD19 de células B, resultando na ativação dos linfócitos T-CAR e gerando citotoxicidade contra células CD19+ in vitro. Houve uma redução de aproximadamente 87% das células alvo, quando analisado por citometria de fluxo e uma citotoxicidade média de 50% foi observada por ensaios colorimétricos. / Immunotherapy using T cells modified with chimeric antigen receptor (CAR) has been proven effective in the treatment of leukemia and lymphomas resistant to chemotherapy. CD19 protein has been shown to be an ideal target because it is expressed on most B-cell tumors and normal B cells, but not in other cells. Recent clinical studies have shown excellent responses of CAR T-cells in a variety of B-cell tumors. Lentiviral vectors are the most commonly used method for genetic modification in clinical trials. This study aimed to develop an efficient platform for lentiviral production and to test the functionality of those vectors so that they can be used in to genetically modify T cells. Transient transfection of HEK293T cells with plasmids in a 3:1:1:1 ratio (transgene:gag-pol:VSV-G:rev) using cationic liposomes and 5 mM sodium butyrate resulted in the highest viral titers. That represents a 17-fold increase in viral titer from polyethylenimine (PEI) transfection. Three methods for lentiviral concentration were used in this work, ultracentrifugation, tangential filtration and ultrafiltration. Membrane ultrafiltration with 100 kDa molecular weight cutoff (MWCO) resulted in the highest recovery rate of viable viral particles, approximately 82%. The viral particles produced by this process have been shown to be functional for the transduction of T cells. In addition, the chimeric receptor (CAR) was shown to be specific against the B cell antigen CD19, resulting in the activation of CAR-T cells and generating cytotoxicity against CD19+ cells in vitro. There was a reduction of approximately 87% of the target cells when analyzed by flow cytometry and an average cytotoxicity of 50% was observed by colorimetric assays.

Ciltacabtagene autoleucel (cilta-cel) is a Chimeric antigen receptor T-cell therapy with the potential for long-term disease control in heavily pre-treated patients with relapsed/refractory multiple myeloma (RRMM). As cilta-cel was assessed in the single-arm CARTITUDE-1 clinical trial, we used an external cohort of patients from the Therapie Monitor registry fulfilling the CARTITUDE-1 inclusion criteria to evaluate the effectiveness of cilta-cel for overall survival (OS) and time to next treatment (TTNT) vs. real-world clinical practice. Individual patient data allowed us to adjust the comparisons between both cohorts, using the inverse probability of treatment weighting (IPW; average treatment effect in the treated population (ATT) and overlap population (ATO) weights) and multivariable Cox proportional hazards regression. Outcomes were compared in intention-to-treat (HR, IPW-ATT: TTNT: 0.13 (95% CI: 0.07, 0.24); OS: 0.14 (95% CI: 0.07, 0.25); IPW-ATO: TTNT: 0.24 (95% CI: 0.12, 0.49); OS: 0.26 (95% CI: 0.13, 0.54)) and modified intention-to-treat (HR, IPW-ATT: TTNT: 0.24 (95% CI: 0.09, 0.67); OS: 0.26 (95% CI: 0.08, 0.84); IPW-ATO: TTNT: 0.26 (95% CI: 0.11, 0.59); OS: 0.31 (95% CI: 0.12, 0.79)) populations. All the comparisons were statistically significant in favor of cilta-cel. These results highlight cilta-cel’s potential as a novel, effective treatment to address unmet needs in patients with RRMM.

Glioblastoma is the most commonly diagnosed central nervous system primary malignancy and it is considered a terminal diagnosis with few treatment options available. Glioblastoma tumors frequently develop treatment resistance due in part to their highly heterogenic nature. The heterogeneity of glioblastoma is partially attributed to the presence of glioma stem-like cells (GSC), which are highly invasive and resistant to chemotherapy and irradiation treatments. Signaling of the receptor tyrosine kinase MET is a known regulator of GSC. Glioblastoma patients have an increasingly poor prognosis that corresponds with increasing MET expression. Both GSC and MET are known to contribute to treatment resistance in glioblastoma and several MET alterations have been observed in glioblastoma. In these studies, we investigated MET alterations that are commonly found in glioblastoma. Using patient-derived xenograft (PDX) lines, the MET alterations were characterized and confirmed to be MET positive, MET amplified, or harbor a PTPRZ1-MET fusion. We also included a MET null glioblastoma PDX line. The PDX lines demonstrated markers for GSC potential with all showing neurosphere formation, the ability to initiate tumor growth in immune-compromised mice, and expression of GSC markers GFAP, Sox2, and nestin. The MET alterations were further examined by examining tyrosine kinase inhibitors' effect on viability and MET signaling. Oncogene addiction through MET amplification was found to have the best response to inhibition. The MET fusion bearing line demonstrated less sensitivity to inhibition than has been shown in other studies, indicating a need for further research into co-mutations that increase sensitivity to MET inhibition. We also investigated the efficacy of novel MET-targeting chimeric antigen receptor T cells (MET.CART cells). The MET.CART cells were able to specifically target and successfully kill MET-expressing glioblastoma cells. Together these results imply the need for more personalized treatment of glioblastoma based on the molecular biology of the tumor.

Up to 60% of patients with aggressive B-cell lymphoma who receive chimeric antigen receptor (CAR) T-cell therapy experience treatment failure and subsequently have a poor prognosis. Allogeneic hematopoietic stem cell transplantation (alloHSCT) remains a potentially curative approach for patients in this situation. Induction of a deep response prior to alloHSCT is crucial for long-term outcomes, but the optimal bridging strategy following relapse after CAR T-cell therapy has not yet been established. Polatuzumab vedotin, an antibody drug conjugate targeting CD79b, is a novel treatment option for use in combination with rituximab and bendamustine (Pola-BR) in relapsed or refractory disease. Patients: We report two heavily pretreated patients with primary refractory diffuse large Bcell lymphoma (DLBCL) and primary mediastinal B-cell lymphoma (PMBCL) respectively who relapsed after therapy with CAR T-cells with both nodal and extranodal manifestations of the disease. After application of three courses of Pola-BR both patients achieved a complete metabolic remission. Both patients underwent alloHSCT from a human leukocyte antigen (HLA)-mismatched donor following conditioning with busulfan and fludarabine and are disease free 362 days and 195 days after alloHSCT respectively. We conclude that Pola-BR can be an effective bridging therapy before alloHSCT of patients relapsing after CAR T-cell therapy. Further studies will be necessary to define the depth and durability of remission of this salvage regimen before alloHSCT.

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Roman&quot;,&quot;serif&quot;;mso-fareast-font-family:Calibri;mso-fareast-theme-font:minor-latin;color:black;mso-ansi-language:EN-US;mso-fareast-language:EN-US;mso-bidi-language:AR-SA">Karcinom dojke je heterogena bolest koju karakteri&scaron;u različita morfologija, imunohisto-hemijski profil, klinički tok i terapijski odgovor. Ki-67 proliferativni indeks je jedan od markera sa prognostičkim i prediktivnim značajem, čije metodolo&scaron;ko određivanje i analiza jo&scaron; uvek nisu standardizovani. <b>Cilj: </b>Utvrditi graničnu (&ldquo;cut-off&rdquo;) prognostičku vrednost Ki-67 indeksa, kao i povezanost vrednosti Ki-67 u ranom luminalnom karcinomu dojke sa prognostičkim i prediktivnim parametrima karcinoma dojke, kao &scaron;to su životna dob bolesnica, veličina tumora, histolo&scaron;ki gradus (HG) i nivo tumorske ekspresije receptora estrogena (ER) i progesterona (PR). Takođe, cilj istraživanja je i utvrđivanje značajnosti razlike u vrednosti Ki-67 proliferativnog indeksa u odnosu na pojavu lokalnog recidiva, udaljenih metastaza i dužinu preživljavanja u toku petogodi&scaron;njeg perioda praćenja pacijentkinja. <b>Metode: </b>Retrospektivno je analizirano 120 patohistolo&scaron;kih izve&scaron;taja bolesnica kojima je u periodu od 01.01.2009. godine do 31.12.2011. godine na Institutu za onkologiju Vojvodine imunohistohemijskom analizom dokazan luminalni karcinom dojke (pozitivan ER i PR, negativan HER2), bez metastaza u aksilarnim limfnim čvorovima. <b>Rezultati: </b>Metodama deskriptivne statistike prosečna starost pacijentkinja je iznosila 57,42&plusmn;10,17 godina; prosečna veličina tumora 17,98&plusmn;6,97mm; recidiv je registrovan kod 8 (6,7%) pacijentkinja uz prosečan vremenski period do pojave recidiva od 49&plusmn;20,23 meseci. Vrednost &ldquo;cut off&rdquo; indeksa Ki-67 od prognostičkog značaja za vremenski period bez recidiva je iznosio 20,75%. Nije dokazana signifikantna veza između vrednosti Ki-67 i godina starosti pacijentkinja (p=0,401, odnosno p=0,293), kao i jačine ekspresije ER (p=1,00, p=0,957) i PR (p=0,273, p=0,189). Ustanovljena je signifikantna povezanost Ki-67 postoji sa veličinom (p=0,035, p=0,20) i HG tumora (p=0,041, p=0,20). Prosečan period praćenja bolesnica iznosio je 72,92&plusmn;8,38 meseci; nije registrovana pojava udaljenih metastaza, kao ni smrtni ishod. U odnosu na pojavu lokalnog recidiva, Kaplan-Majerovom analizom i Koksovom regresionom analizom proliferativni indeks Ki-67 se pokazao kao signifikantan prediktor za procenu ponovnog javljanja bolesti, lokalnog recidiva (Log rank (df = 1) = 2,73; p=0,045). Takođe je ustanovljeno da je statistički značajan prediktor za procenu recidiva bolesti i starosna dob bolesnica (Log rank (df = 1) = 6,885; p=0,009). Intenzitet pozitivnosti ER i PR, veličina tumora i histolo&scaron;ki gradus se nisu pokazali kao prediktori za pojavu recidiva luminalnih karcinoma dojke (p &gt; 0,05). <b>Zaključak: </b>Zbog heterogene prirode oboljenja, kori&scaron;ćenjem standardnih histopatolo&scaron;kih faktora i biomarkera te&scaron;ko je predvideti tok i ishod karcinoma dojke. Ki-67 je proliferativni marker, čija visoka vrednost korelira sa faktorima lo&scaron;e prognoze.</span></p> / <p><!--[if gte mso 9]><xml> <o:DocumentProperties> <o:Author>Tanja Lakic</o:Author> <o:Version>12.00</o:Version> </o:DocumentProperties></xml><![endif]--><!--[if gte mso 9]><xml> <w:WordDocument> <w:View>Normal</w:View> <w:Zoom>0</w:Zoom> <w:TrackMoves/> <w:TrackFormatting/> <w:PunctuationKerning/> <w:ValidateAgainstSchemas/> <w:SaveIfXMLInvalid>false</w:SaveIfXMLInvalid> <w:IgnoreMixedContent>false</w:IgnoreMixedContent> <w:AlwaysShowPlaceholderText>false</w:AlwaysShowPlaceholderText> <w:DoNotPromoteQF/> <w:LidThemeOther>EN-US</w:LidThemeOther> <w:LidThemeAsian>X-NONE</w:LidThemeAsian> <w:LidThemeComplexScript>X-NONE</w:LidThemeComplexScript> <w:Compatibility> <w:BreakWrappedTables/> <w:SnapToGridInCell/> <w:WrapTextWithPunct/> <w:UseAsianBreakRules/> <w:DontGrowAutofit/> <w:SplitPgBreakAndParaMark/> <w:DontVertAlignCellWithSp/> <w:DontBreakConstrainedForcedTables/> 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Normal";mso-tstyle-rowband-size:0;mso-tstyle-colband-size:0;mso-style-noshow:yes;mso-style-priority:99;mso-style-qformat:yes;mso-style-parent:"";mso-padding-alt:0cm 5.4pt 0cm 5.4pt;mso-para-margin-top:0cm;mso-para-margin-right:0cm;mso-para-margin-bottom:10.0pt;mso-para-margin-left:0cm;line-height:115%;mso-pagination:widow-orphan;font-size:11.0pt;font-family:"Calibri","sans-serif";mso-ascii-font-family:Calibri;mso-ascii-theme-font:minor-latin;mso-hansi-font-family:Calibri;mso-hansi-theme-font:minor-latin;mso-bidi-font-family:"Times New Roman";mso-bidi-theme-font:minor-bidi;}</style><![endif]--></p><p class="Default"><b><span style="font-size:11.0pt">Introduction: </span></b><span style="font-size:11.0pt">Breast cancer is a heterogeneous disease characterized by different morphology, immunohistochemical profile, clinical course and response to applied therapy. Ki-67 proliferative index is one of the prognostic and predictive factors, whose methodological determination and analysis are still unstandardized. <b>Objective: </b>Determination of cut-off value for Ki-67 index, its corelation in luminal breast carcinoma with patient&#39;s age, tumor size, histological grade (HG) and expression of estrogen (ER) and progesterone (PR). Also, the aim of the study was to determine the significance of the difference in the value of the Ki-67 proliferative index in relation to the occurrence of local relapse, distant metastases and survival rates during the five-year follow-up period of the patient. <b>Methods: </b>Retrospectively, we analysed 120 pathohistological reports of patients who were treated in the period from 01.01.2009 until 31.12.2011 at the Oncology Institute of Vojvodina, and to whom immunohistochemically was proven luminal breast cancer (positive ER and PR, negative HER2), without axillary lymph node metastases. </span><b><span style="font-size:11.0pt">Results: </span></b><span style="font-size:11.0pt">The average patient&rsquo;s age was 57.42&plusmn;10.17 years; average tumor size 17.98&plusmn;6.97mm; recurrence was registered in 8 (6.7%) patients with average recurrence time of 49&plusmn;20.23 months. &quot;Cut off&quot; Ki-67 value of prognostic significance for period without recurrence was 20.75%. Test didn&rsquo;t show significant relationship between Ki-67 and patient&rsquo;s age (p=0.401 and p=0.293), as well as the strength of expression ER (p=1.00, p=0.957) and PR (p=0.273, p=0.189). Significant correlation was present for Ki-67 with size (p=0.035, p=0.20) and tumor&rsquo;s HG (p=0.041, p=0.20). The average follow-up period for patients was 72.92&plusmn;8.38 months; there was no registered occurrence of distant metastases or fatal outcome. In relation to the occurrence of local relapse, Kaplan-Meier analysis and Cox regression analysis, the proliferative index Ki-67 proved to be a significant predictor for the assessment of recurrence of the disease, local relapse (Log rank (df = 1) = 2.73; p = 0.045). Also, it was founded that a statistically significant predictor for assessing the recurrence of the disease is the age of the patients (Log rank (df = 1) = 6.885; p = 0.009). The intensity of ER and PR expression, tumor size and histological grade have not been shown to be predictors of the recurrence of luminal breast carcinoma (p&gt; 0.05). </span><b><span style="font-size:11.0pt">Conclusion: </span></b><span style="font-size:11.0pt">Breast carcinoma is heterogeneous disease, so it is difficult to predict its course and outcome using standard histopathological factors and biomarkers. Ki-67 is proliferative marker whose high value correlates with factors of bad prognosis. </span></p>

Nous avons produit des CART-cells et des LT-mock ont été produits ex-vivo. L'efficacité de transduction est mesuré par l'expression CD3+/CD19+ (82% en moyenne, n=S), en cytométrie en flux. L'expression protéique de CAR a été vérifiée par western blot et en cytométrie en flux attestant de son expression membranaire. Nous avons testé la fonctionnalité de la cassette suicide iCaspase9/ AP1903 sur CART-cells après 48h de traitement à I' AP1903. Nous avons montré que plus de 90% des CART-cells entrent en apoptose (Marquage 7-AAD en cytométrie en flux). Nous avons ensuite réalisé des tests fonctionnels in-vitro des CART-cells contre des lignées LMC exprimant naturellement IL-lRAP ou contre des lignées transfectées pour exprimer la forme membranaire d'IL-lRAP. Après co­culture des CART-cells en présence des cellules cibles IL-lRAP+ nous avons montré qu'ils prolifèrent (test CFSE), qu'ils sécrètent de l'interféron y et des cytokines inflammatoires. Enfin, par marquage 7-AAD, nous avons démontré la cytotoxicité des CART-cells contre les cellules ILl-RAP+. Finalement, nous avons montré l'efficacité des CART-cells in vivo, dans un modèle de xénogreffe tumorales dans des souris immunodéficiences NSG greffées par la lignée K562-IL1RAP+ / Luciférase +. On constate une diminution de la masse tumorale 4 jours après injection des CART-cells, jusqu'à disparition totale. Cette preuve de concept laisse entrevoir des perspectives thérapeutiques alternatives dans le traitement de la LMC ou LAM, qui devront être optimiser dans des modèles murins (séquence et nombres d'injection, nombres de cellules, associations avec ITKS ou autres chimiothérapies) afin de conduire un essai clinique de phase I, pour démontrer la faisabilité et la sécurité de l'approche pour envisager une démonstration d'efficacité chez l'homme. La sécurisation par la cassette suicide permet d'envisager l'utilisation des CART-cells en situation autologue ou allogénique (Donor Lymphocytes infusion, DU) / Chronic myeloid leukemia (CML) is a myeloproliferative disorder and is characterized by the presence of a Philadelphia chromosome (Phl) encoding the BCR-ABL fusion protein with constitutive tyrosine kinase activity. The treatment by Tyrosine Kinase lnhibitors (ITK) is not curative. The problem today is ta target leukemic stem cells (HSCs) to prevent relapse of patients after stopping treatment with TKI and permanently eradicate the disease. Studies have identified the interleukin-1 accessory protein receptor (IL-lRAP) as a potential target for killing CSL (BCR-ABL1 positive) in CML or in acute myeloid leukemia (AML). We therefore propose to develop a cell therapy approach targeting IL-lRAP, using T cells (LT) redirected by a CAR (Chimeric Antigen Receptor) in the treatment of CML and AML. We produced a specific anti-lL-1RAP murine monoclonal antibody (mAb) called # A3C3. We tested the specificity of the antibody # A3C3 in flow cytometry, in Western blot, by immunohistochemistry or confocal microcopy, on positive human cell lines IL-1RAP (KU812) or IL-1RAP negative (Raji). Moreover, by using this antibody, we have demonstrated by immunohistochemistry, on 20 different normal tissues that our antibody does not recognize or very few non-specific (off-target) targets. Finally, we demonstrated that this antibody is able to detect positive IL-1RAP CSLs in primary bone marrow or peripheral blood samples of LMC patients at diagnosis or after ITK treatment. By molecular biology, we have sequenced and identified rearrangements of the heavy (lgH) and light {lgL) chains coding region for hypervariable regions of# A3C3. The coding sequence for the "single chain variable fragment" (scFv), lin king the 2 lgH and lgl chains, was cloned into a 3rd generation lentiviral skeleton securised by a suicide gene, inducible caspase 9 (iCASP9). We produced CART-cells and LT-mock ex-vivo. The transduction efficiency is measured by CD3 + / CD19 + expression (82% on average, n = 5), in flow cytometry. The functionality of the suicide cassette iCaspase9/AP1903 on CART-cells after 48h treatment with AP1903 was tested. We have shown the death of more than 90% of CART-cells (7-AAD labeling in flow cytometry). We then performed in-vitro functional tests of CART-cells against LMC lines naturally expressing IL-1RAP or against transfected lines to express the membrane form of IL-1RAP. After co-culture of CART-cells in the presence of IL-1RAP + target cells, we have shown that they proliferate (CFSE test), that they secrete interferon y and inflammatory cytokines (intracellular labeling and Cytokines Bead Array assay). They are able to degranulate (CD107a & b labeling). Finally, by 7 AAD labeling, we demonstrated the cytotoxicity of CART-cells against IL1-RAP + cells. Finally, we showed the effectiveness of CART-cells in vivo, in a tumor xenograft model in NSG immunodeficiency mice engrafted by the K562-IL1RAP+/Luciferase+ line. There is a decrease in the tumor load 4 days after injection of CART-cells, until complete disappearance.This proof of concept suggests alternative therapeutic perspectives in the treatment of CML or AML, which should be optimized in murine models (sequence and injection numbers, cell numbers, associations with ITKS or other chemotherapies) in order to Phase I clinical trial, to demonstrate the feasibility and safety of the approach to consider a demonstration of efficacy in human. Securing by the suicide cassette makes it possible to consider the use of CART-cells in an autologous or allogeneic situation (Donor Lymphocytes infusion, DU)

Die vorliegende Arbeit beinhaltet die Generierung und Evaluation von Natürlichen Killerzellen, die EGFRvIII-positive und SDF-1alpha sekretierende primäre Glioblastomzellen aufspüren, erkennen und effizient abtöten können. Die Kombination der gelenkten Zytotoxizität mit einer optimierten Migration von Effektorzellen des Immunsystems wird auf Grundlage der in dieser Arbeit gewonnenen Daten als ein vielversprechender Ansatz für eine zukünftige Therapie des primären Glioblastoms vorgeschlagen.