Development of a Human Anti-CD27 Antibody with Efficacy in Lymphoma and Leukemia Models by Two Distinct Mechanisms

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2861-2861 ◽  
Author(s):  
Li-Zhen He ◽  
Larry Thomas ◽  
Jeffery Weidlick ◽  
Laura Vitale ◽  
Tom O'Neill ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Cell Lines ◽  
Lymphoid Cell ◽  
Lymphoblastic Leukemia ◽  
Tumor Challenge ◽  
Isotype Control ◽  
Human Lymphoid Cell ◽  
Dose Levels

Abstract Abstract 2861 CD27, a lymphoid cell-specific TNFR superfamily member, is constitutively expressed on the majority of T cells, some NK cells and memory B cells. Through interaction with its ligand CD70, CD27 transduces a co-stimulatory signal promoting T cell and NK cell activation and cytotoxicity. In addition, CD27 is also expressed on many lymphoid-originated hematological neoplastic cells, such as chronic lymphocytic leukemia, mantle cell lymphoma, and Waldenstrom macroglobulinemia, thus being a potential direct target for antibody therapy. To generate potential antibodies for clinical development, we immunized human Ig transgenic mice and developed a panel of CD27 specific human mAbs. Clone 1F5 was identified as a lead based on its high affinity to both human and monkey CD27, enhanced co-stimulation of T cells, and ADCC of CD27-expressing lymphoblastic cell lines. Using SCID mice challenged with CD27-expressing human lymphoid cell lines, we demonstrated that 1F5 mediates conventional antibody effector function. Compared to human IgG1 isotype control (huIgG1), 1F5 at doses ranging 33 μg – 500 μg (x 6) significantly delayed the growth of Burkitt's lymphoma Raji even when administration was initiated 1 week after tumor inoculation. Similar anti-tumor activity was observed against other CD27-expressing tumor lines including, Daudi and T-originated acute lymphoblastic leukemia CCRF-CEM. In order to investigate 1F5 in vivo agonistic activities and T cell-mediated tumor eradication, a human CD27 transgenic mouse model (hCD27-Tg) was generated and backcrossed onto C57BL/6 and BALB/c backgrounds. The expression profile and regulation of the human CD27 transgene driven by its own promoter were similar to that observed with endogenous mouse CD27. In addition to enhancing T cell responses when combined with vaccination, 1F5 treatment was highly effective against syngeneic mouse tumors including lymphoma BCL1 (BALB/c) and thymoma EL4-derived E.G7 (C57BL/6). For the BCL1 model, various dose levels of 1F5 mAb were delivered to mice intraperitoneally on days 3, 5, 7, 9 and 11 after i.v. administration of 107 BCL1 cells to huCD27 Tg and control animals. Controls including hCD27-Tg mice treated with saline or isotype control, or WT mice treated with 1F5 all performed consistently, leading to 50% survival approximately 23 days after tumor challenge. Treatment of mice with mAb, 1F5 substantially improved the 50% survival in a dose dependent fashion to >70 days post tumor challenge at the higher dose levels. Based on the promising efficacy data with anti-CD27 mAb 1F5 in immunocompromised and immunocompetent lymphoma models, a clinical grade product, referred to as CDX-1127 was manufactured and tested for safety. To assess the potential for 1F5 to mediate lymphocyte activation, we investigated its ability to induce proliferation and cytokine release from human PBMC or purified T cell cultures. Consistent with the known biology of CD27 we demonstrated the 1F5 mAb does not lead to direct activation of lymphocytes in the absence of additional signals. However, combining 1F5 with suboptimal levels of T cell receptor stimulation using anti-CD3 mAb (OKT3) was shown to enhance proliferation of human T cells. Two studies were performed using cynomolgus macaques. There were no CDX-1127 related mortalities or changes noted in the clinical condition, food appetence, body weights and body temperature, ophthalmic, electrocardiographic and clinical pathology assessments, organ weights and bone marrow assessments. In addition, there were no major differences in the percentage of lymphocyte populations between control and CDX-1127 treated animals at the end of the study demonstrating that the antibody did not significantly deplete normal CD27-expresssing cells. Based on the pre-clinical studies we are planning a Phase 1 clinical trial of CDX-1127 in patients with hematological malignancies and selected solid tumors. The trial is designed with separate arms to independently assess the safety and activity of CDX-1127 in hematologic malignancies, in which the antibody may act through multiple mechanisms, and in solid tumors where it would be fully dependent on indirect immune mechanisms. Disclosures: He: Celldex Therapeutics, Inc.: Employment. Thomas:Celldex Therapeutics, Inc.: Employment. Weidlick:Celldex Therapeutics, Inc.: Employment. Vitale:Celldex Therapeutics, Inc.: Employment. O'Neill:Celldex Therapeutics, Inc.: Employment. Prostak:Celldex Therapeutics, Inc.: Employment. Sundarapandiyan:Celldex Therapeutics, Inc.: Employment. Marsh:Celldex Therapeutics, Inc.: Employment. Yellin:Celldex Therapeutics, Inc.: Employment. Davis:Celldex Therapeutics, Inc.: Employment. Keler:Celldex Therapeutics, Inc.: Employment.

scholarly journals Dissecting the Immune Cell Progeny of CAR-Expressing Hematopoietic Stem Cells in a Humanized Mouse Model

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4640-4640
Author(s):  
Heng-Yi Liu ◽  
Nezia Rahman ◽  
Tzu-Ting Chiou ◽  
Satiro N. De Oliveira
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
Hematopoietic Stem Cells ◽  
Cell Lines ◽  
Immune Cells ◽  
Research Funding ◽  
Humanized Mice ◽  
Tumor Challenge ◽  
Hematopoietic Stem

Background: Chemotherapy-refractory or recurrent B-lineage leukemias and lymphomas yield less than 50% of chance of cure. Therapy with autologous T-cells expressing chimeric antigen receptors (CAR) have led to complete remissions, but the effector cells may not persist, limiting clinical efficacy. Our hypothesis is the modification of hematopoietic stem cells (HSC) with anti-CD19 CAR will lead to persistent generation of multilineage target-specific immune cells, enhancing graft-versus-cancer activity and leading to development of immunological memory. Design/Methods: We generated second-generation CD28- and 4-1BB-costimulated CD19-specific CAR constructs using third-generation lentiviral vectors for modification of human HSC for assessment in vivo in NSG mice engrafted neonatally with human CD34-positive cells. Cells were harvested from bone marrows, spleens, thymus and peripheral blood at different time points for evaluation by flow cytometry and ddPCR for vector copy numbers. Cohorts of mice received tumor challenge with subcutaneous injection of lymphoma cell lines. Results: Gene modification of HSC with CD19-specific CAR did not impair differentiation or proliferation in humanized mice, leading to CAR-expressing cell progeny in myeloid, NK and T-cells. Humanized NSG engrafted with CAR-modified HSC presented similar humanization rates to non-modified HSC, with multilineage CAR-expressing cells present in all tissues with stable levels up to 44 weeks post-transplant. No animals engrafted with CAR-modified HSC presented autoimmunity or inflammation. T-cell populations were identified at higher rates in humanized mice with CAR-modified HSC in comparison to mice engrafted with non-modified HSC. CAR-modified HSC led to development of T-cell effector memory and T-cell central memory phenotypes, confirming the development of long-lasting phenotypes due to directed antigen specificity. Mice engrafted with CAR-modified HSC successfully presented tumor growth inhibition and survival advantage at tumor challenge with lymphoma cell lines, with no difference between both constructs (62.5% survival for CD28-costimulated CAR and 66.6% for 41BB-costimulated CAR). In mice sacrificed due to tumor development, survival post-tumor injection was directly correlated with tumor infiltration by CAR T-cells. Conclusions: CAR modification of human HSC for cancer immunotherapy is feasible and continuously generates CAR-bearing cells in multiple lineages of immune cells. Targeting of different malignancies can be achieved by adjusting target specificity, and this approach can augment the anti-lymphoma activity in autologous HSC recipients. It bears decreased morbidity and mortality and offers alternative therapeutic approach for patients with no available sources for allogeneic transplantation, benefiting ethnic minorities. Disclosures De Oliveira: National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London: Research Funding; NIAID, NHI: Research Funding; Medical Research Council: Research Funding; CIRM: Research Funding; National Gene Vector Repository: Research Funding.

Adoptive T-Cell Therapy for B-Cell Acute Lymphoblastic Leukemia: Preclinical Studies

Blood ◽  
1999 ◽  
Vol 94 (10) ◽  
pp. 3531-3540 ◽  
Author(s):  
Angelo A. Cardoso ◽  
J. Pedro Veiga ◽  
Paolo Ghia ◽  
Hernani M. Afonso ◽  
W. Nicholas Haining ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Adoptive T Cell Therapy ◽  
Leukemic Cells ◽  
Acute Leukemias ◽  
T Cell Lines

We have previously shown that leukemia-specific cytotoxic T cells (CTL) can be generated from the bone marrow of most patients with B-cell precursor acute leukemias. If these antileukemia CTL are to be used for adoptive immunotherapy, they must have the capability to circulate, migrate through endothelium, home to the bone marrow, and, most importantly, lyse the leukemic cells in a leukemia-permissive bone marrow microenvironment. We demonstrate here that such antileukemia T-cell lines are overwhelmingly CD8+ and exhibit an activated phenotype. Using a transendothelial chemotaxis assay with human endothelial cells, we observed that these T cells can be recruited and transmigrate through vascular and bone marrow endothelium and that these transmigrated cells preserve their capacity to lyse leukemic cells. Additionally, these antileukemia T-cell lines are capable of adhering to autologous stromal cell layers. Finally, autologous antileukemia CTL specifically lyse leukemic cells even in the presence of autologous marrow stroma. Importantly, these antileukemia T-cell lines do not lyse autologous stromal cells. Thus, the capacity to generate anti–leukemia-specific T-cell lines coupled with the present findings that such cells can migrate, adhere, and function in the presence of the marrow microenvironment enable the development of clinical studies of adoptive transfer of antileukemia CTL for the treatment of ALL.

A CXCL10/CXCR3 Driven Thymic Epithelium-Leukemia Cell Crosstalk Augments T Cell Acute Lymphoblastic Leukemia Notch1 Signalling

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2537-2537
Author(s):  
Ashwini M Patil ◽  
Stefanie Kesper ◽  
Vishal Khairnar ◽  
Marco Luciani ◽  
Michael Möllmann ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mouse Model ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Lymphoblastic Leukemia ◽  
Thymic Epithelial Cells ◽  
Hematopoietic Organ ◽  
Notch1 Mutations

Introduction: The thymus is a specialized hematopoietic organ, which is responsible for the generation of T cells. The central thymic cell type controlling T cell development are thymic epithelial cells (TECs). Based on their specific function and anatomic location TECs are separated into cortical and medullary subsets (cTECs and mTECs). cTECs express pivotal NOTCH-ligands such as DLL4 controlling T cell lineage commitment while mTECs play a central role in negative selection of developing T cells. Acquisition of NOTCH1 gain-of-function mutations play a central role in acute T cell lymphoblastic leukemia (T-ALL) development. During T-ALL leukemogenesis aberrant expression of transcription factors such as SCL/TAL1 and LMO1 block T cell differentiation and increase self-renewal while NOTCH1 mutations promote survival and proliferation. Since most acquired NOTCH1 mutations still require ligand binding to exert augmented signaling we propose DLL4-expressing TECs playing a critical role during T-ALL leukemogenesis. Methods: In the present study, we used a Scl/Lmo1 T-ALL transgenic mouse model, murine ANV and TE71 TEC cell lines and human T-ALL cell lines (Jurkat, ALL-SIL, DND-41, and HPB-ALL) to investigate TEC dynamics and function in the T-ALL context. Results: First, we demonstrated T-ALL supporting potential of TEC cell lines in vitro, which was comparable to the mesenchymal cell line OP9. Next, we showed in the Scl/Lmo1 T-ALL mouse model which had a mean survival rate of 90 days that preleukemic thymocytes displayed a striking upregulation of Notch1 target genes. Interestingly, fluorescence microscopy revealed a relative expansion of cortical and a relative reduction of the medullary thymic areas in Scl/Lmo1 thymi (Fig. 1A). Correspondingly, absolute numbers of cTECs expanded while mTEC numbers declined (Fig. 1B). Gene expression profiling of sorted preleukemic Scl/Lmo1 cTECs revealed upregulation of the chemokine CXCL10 (Fig. 1C). Moreover, increased CXCL10 chemokine concentrations were detected in Scl/Lmo1 thymic interstitial fluid (Fig.1D). Strikingly, we demonstrated T-ALL dependence of TEC Cxcl10 upregulation. We showed that Cxcl10 upregulation in TEC cell lines was only induced by direct cellular contact with Scl/Lmo1 thymocytes while wild-type control thymocytes did not alter TEC cell line Cxcl10 expression (Fig. 1E). Next, a high proportion of the CXCL10 receptor CXCR3 expressing cells was revealed in Scl/Lmo1 thymi (Fig. 1F) and by human T-ALL cell lines. Finally, we demonstrated a CXCL10 dependent pro-survival effect within cultured SCL/LMO1 thymocytes (Fig. 1G), which was associated with the activation of NOTCH1 signaling (Fig. 1H). Conclusions: In summary, the data support a novel T-ALL-promoting regulatory circuit in which emerging T-ALL lymphoblasts induce CXCL10 in expanding TECs which positively feeds back to T-ALL cells via the CXCL10 receptor CXCR3. Disclosures Dührsen: Celgene: Research Funding; Takeda: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding; Teva: Honoraria; Novartis: Consultancy, Honoraria; Alexion: Honoraria; Roche: Honoraria, Research Funding; CPT: Consultancy, Honoraria; Janssen: Honoraria. Göthert:Proteros Biostructures: Consultancy; Novartis: Consultancy, Honoraria, Other: Travel support; Pfizer: Consultancy, Honoraria; Incyte: Consultancy, Honoraria, Other: Travel support; Bristol-Myers Squibb: Consultancy, Honoraria, Other: Travel support; AOP Orphan Pharmaceuticals: Honoraria, Other: Travel support.

Expansion and Transformation of Primary Acute Lymphoblastic Leukemia Cells into Antigen-Presenting Cells using a Novel Culturing System Enables the Generation of Leukemia-Reactive T Cell Responses that Are Effective in Vivo.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 303-303
Author(s):  
Bart A. Nijmeijer ◽  
Marianke L.J. Van Schie ◽  
Roel Willemze ◽  
J.H. Frederik Falkenburg
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Primary Cells ◽  
T Cell Anergy ◽  
Cell Anergy ◽  
Antigen Presenting

Abstract Allogeneic cellular immunotherapy is generally ineffective in acute lymphoblastic leukemia (ALL). In vitro studies have suggested that this inefficacy may be the result of a lack of costimulatory molecule expression by ALL cells, resulting in the induction of T cell anergy. Activation of T cells by ALL cells that are transformed into adequate antigen-presenting cells (ALL-APC) may prevent the induction of T cell anergy and result in the generation of competent leukemia-reactive T cell responses for adoptive immunotherapy. However, in vitro modification of ALL cells was hampered by the fact that ALL cells from adult patients could not be cultured in vitro for prolonged periods of time. We have developed a novel serum-free culturing system for B-lineage ALL in which proliferation is initiated and sustained by ALL-cell derived growth factors. Long-term (>2 yrs) proliferation was induced in 12 out of 26 randomly selected primary samples from patients with ALL. The cell cultures ( Leiden cell lines) proliferated with a mean doubling time of 3.0 days (range 2.7–3.6 days). All Leiden cell lines presented the chromosomal abberations observed in the primary cells. The Leiden cell lines displayed an immune phenotype similar to the primary cells, exept for loss of CD34 expression. In vivo characteristics of Leiden cells were evaluated in NOD/scid mice. After intravenous inoculation, Leiden cell lines and primary cells showed identical homing patterns initially involving spleen and bone marrow, followed by the development of overt and progressive leukemia. A comparison of in vivo progression kinetics was performed for one of the Leiden cell lines and the corresponding primary cells. Weekly determination of leukemic cell counts in the blood of engrafted animals revealed that the cell line and the primary cells displayed similar doubling times in vivo of 6.3 and 7.7 days, respectively. To generate cells with improved antigen presentation function, Leiden cell lines were exposed to various activating agents. Stimulation with CpG containing oligonucleotides resulted in induction of CD40 in 9 out of 10 lines. Subsequent ligation of CD40 by culturing CpG-activated Leiden cells on fibroblasts expressing human CD40 ligand resulted in the induction of CD80 or CD86 in 7 of these 10 cell lines. To study the immune stimulatory properties of these Leiden ALL-APC, allogeneic HLA-identical T cells were first activated in vitro by coculturing these cells with either unmodified Leiden cells or with the corresponding Leiden ALL-APC for 3 days, and subsequently infused into groups of 6 leukemic NOD/scid mice. While T cells cocultured with unmodified Leiden cells did not expand in vivo, T cells cocultured with Leiden ALL-APC expanded after infusion in 5 out of 6 animals. This expansion coincided with a 20–75% decrease in leukemic cell numbers in the blood. In conclusion, the novel serum-free culturing system enables long-term culture and manipulation of a significant fraction of primary human ALL. These Leiden cell lines can be modified into ALL-APC that display adequate antigen presenting function, preventing the induction of T cell anergy as demonstrated in vivo in the NOD/scid mouse model.

NK-Like Homeodomain Proteins Regulate NOTCH3-Signaling in T-Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1194-1194
Author(s):  
Stefan Nagel ◽  
Letizia Venturini ◽  
Grzegorz K. Przybylski ◽  
Piotr Grabarczyk ◽  
Corinna Meyer ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Expression Patterns ◽  
Hematopoietic Cells ◽  
Notch Pathway ◽  
T Cell Differentiation ◽  
Homeodomain Proteins

Abstract Three NK-like (NKL) homeobox genes, TLX1/HOX11, TLX3/HOX11L2 and NKX2- 5/CSX, have been implicated in T-cell acute lymphoblastic leukemia (T-ALL). Here we screened further NKL genes in 24 T-ALL cell lines by RT-PCR and identified common expression of MSX2, highlighting this homeobox gene as a potential physiological family member in T-cells. Subsequent quantification of MSX2 confirmed expression in primary hematopoietic cells demonstrating higher levels in CD34+ stem cells when compared to peripheral blood cells or mature CD3+ T-cells. Analysis of core thymic factors in T-ALL cell lines, including IL7, BMP4, TGFbeta, NOTCH and T-cell receptor signaling, suggests their involvement in MSX2 regulation during T-cell differentiation. Chromosomal and genomic analysis of the MSX2 locus (at 5q35) uncovered deletion in t(5;14)(q35;q32) positive T-ALL cell lines associated with low expression levels of MSX2 and ectopic activation of TLX3 or NKX2-5, respectively. For functional analysis we lentivirally transduced T-ALL cells for overexpression of either MSX2 or oncogenic TLX1 and NKX2-5. These cells displayed transcriptional activation of NOTCH3-signaling, as indicated by expression array profiling and real-time PCR analysis of NOTCH3, HES1 and HEY1. The sensitivities to gamma-secretase inhibitor analyzed by MTT-assay of cells overexpressing MSX2, TLX1 or NKX2-5, respectively, were consistently decreased. Furthermore, in addition to MSX2, both TLX1 and NKX2-5 proteins interacted with repressor proteins of the NOTCH-pathway, SPEN/MINT and TLE1/GRG1, as shown by co-immunoprecipitation, probably representing one mechanism of (de)regulation. Elevated expression of NOTCH3 and HEY1 mRNA was detected in TLX1/3 positive T-ALL patients, confirming data obtained from cell lines. In conclusion, we have defined expression patterns, regulation and targets of MSX2 in hematopoietic cells, to reveal a novel modulatory activity in T-cell differentiation operating via NOTCH-signaling, and in leukemogenesis when replaced or supplemented by oncogenic NKL homeodomain proteins.

A Drug Screen in Zebrafish Identifies a New Therapeutic Agent Active in Acute Lymphoblastic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2129-2129
Author(s):  
Nikolaus Trede ◽  
Suzanne Ridges ◽  
Deepa Joshi ◽  
Jon Beck ◽  
Hossein Soffla ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Side Effects ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Apparent Lack ◽  
Zebrafish Larvae ◽  
In Vivo Screen

Abstract Abstract 2129 Objectives: T cell lineage is an independent high risk factor in acute lymphoblastic leukemia (ALL). T-ALL requires high dose multi-agent chemotherapy, conferring many toxic side effects. T-ALL treatment therefore needs new, targeted agents that preserve or improve current treatment efficacy, yet cause fewer side effects than existing chemotherapeutic regimens. To identify such drugs, we pioneered an in vivo screen using transgenic zebrafish with T cell-specific green fluorescent protein (GFP) expression. We reasoned that immature T cells in 5-day-old zebrafish larvae are developmentally analogous to T-ALL lymphoblasts, and likely rely upon similar signaling pathways. Hence, compounds that specifically eliminate T cells in zebrafish larvae might likewise selectively target T-ALL cells. An added benefit of our in vivo screen is that drugs added to the water housing fish larvae must cross an epithelial barrier, can be metabolized by the liver, and can be renally cleared, characteristics not assessed in in vitro-based screening strategies. In addition, by using early larvae as our subjects, drugs lacking T cell specificity will likely impair normal development and/or survival, which we postulate is a predictor of unwanted toxicities. In these ways, our screen mimicked the scenario encountered in patients. Our use of the transgenic p56lck::EGFP zebrafish line facilitated rapid visual assessment of efficacy of a large number of compounds in 96-well format. Materials and Results: In a proof-of-principle experiment, we identified several known anti-T-ALL drugs, most prominently glucocorticoids, from the “Spectrum” library (MicroSource Discovery Systems, Inc., Gaylordsville, CT) of well-characterized compounds. We then screened 39,200 small molecules from the “ChemBridge DIVERSet” combinatorial chemistry library (ChemBridge Corp., San Diego, CA) for those active against zebrafish larval T cells. Of 20 novel “hits” identified, one compound, dubbed Lenaldekar (“LDK”), met additional prioritization criteria. LDK does not impair the cell cycle of developing zebrafish, is well tolerated and orally bioavailable with favorable pharmacokinetic properties in mice. In addition, pilot studies with LDK indicate it is efficacious in treating T-ALL in juvenile and adult fish from an established transgenic rag2::ER-human cMyc T-ALL model. LDK kills all of several murine T-lymphocytic malignancies, induces apoptosis in all human T-ALL lines tested, and shows some activity in human B-ALL lines. However, glioblastoma, colon carcinoma, melanoma, or immortalized human embryonic kidney cell lines are not affected by LDK, even at high concentration (up to 25μM). Using the recently established “phosflow” technique we measured phosphorylation status of key signaling proteins in permeabilized T- and B-ALL lines. Regardless of PTEN status, PDK1, AKT and mTOR downstream targets p4EBP1 and p70S6kinase were dephosphorylated by LDK treatment, as was the p65 subunit of NFκB on serine 529. Results were corroborated by conventional Western blots. However, phosphorylation of STAT3, STAT5, pERK1-2, and p38 were not affected by LDK. LDK's action is distinguished from other AKT/mTOR inhibitors by its lack of activity against AKT-dependent glioblastoma and melanoma cell lines, and its lack of effect on cell size. Finally, LDK decreased tumor burden of human T-ALL in murine xenografts. Conclusions: In view of its apparent lymphocyte selectivity, we posit that LDK modulates a pathway relatively unique to ALL (and immature lymphoblast) cells. This suggests that LDK can serve as a novel molecular tool for studies of normal and malignant lymphocyte biology. Moreover, with its favorable pharmacokinetics, apparent lack of toxicity, and in vivo efficacy in two vertebrate ALL models, LDK is an attractive molecule for development into a targeted treatment for ALL and perhaps other lymphocytic malignancies. Disclosures: No relevant conflicts of interest to declare.

Growth Factor Independent-1 (Gfi1) Is Critically Required for T-Cell Acute Lymphoblastic Leukemia (T-ALL) Tumor Initiation and Maintenance

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3156-3156
Author(s):  
James D. Phelan ◽  
Cyrus Khandanpour ◽  
Shane Horman ◽  
Marie-Claude Gaudreau ◽  
Jinfang Zhu ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Growth Factor ◽  
Cell Lines ◽  
Tumor Regression ◽  
Lymphoblastic Leukemia ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Apoptotic Genes

Abstract Abstract 3156 T cell acute lymphoblastic leukemia (T-ALL) is one of the most common childhood cancers associated with mutations in NOTCH1. The Growth factor independent-1 (Gfi1) transcriptional repressor gene was originally discovered as a common target of Moloney murine leukemia virus (MMLV) proviral insertion in murine T-ALL. In fact, the Gfi1 locus is the most frequently activated gene in MMLV-induced T cell leukemia. Therefore, we investigated whether the most commonly activated gene in MMLV-induced murine T-ALL, Gfi1, could collaborate with the most commonly activated gene in human T-ALL, NOTCH1. Here, we show that GFI1 expression is associated with Notch signaling in human T-ALL (p'0.0003). Functionally, Gfi1 collaborates with Notch-induced murine T-ALL by accelerating an already rapid disease model (p=0.03) without altering the lymphoblastic nature of the disease. Furthermore, inducible deletion of Gfi1 is counter-selected in both Notch-driven retroviral and transgenic mouse models of T-ALL; whereas, constitutive absence of Gfi1 completely prevents transgenic Notch-induced T-ALL (p≤0.04). However, T-ALL tumors can form in Gfi1-/- animals using either ENU-mutagenesis or MMLV-infection, yet tumor formation is delayed (p≤0.02, p≤0.03 respectively). This suggests that Gfi1 deletion does not prevent the formation of the T-ALL initiating cell and that Gfi1 might be absolutely required for Notch-induced T-ALL. Most striking is that Gfi1 is required for T-ALL maintenance in vitro and in vivo. Using three separate Tal1-initiated murine T-ALL cell lines, the overexpression of the Gfi1 dominant-negative, Gfi1N382S, was quickly and completely counter-selected. As Gfi1 has previously been found to regulate pro-apoptotic genes in T cells, we attempted to rescue the above loss of function phenotype by overexpressing the anti-apoptotic factor Bcl2. Notably, counter-selection of Gfi1N382 is not observed or is significantly delayed in all three cell lines. In vivo, inducible deletion of Gfi1 leads to both mutagen- or Notch-induced tumor regression as measured by ultrasound. In fact, levels of Gfi1 expression directly correlate to tumor regression and disease free survival of T-ALL. Finally, targeting Gfi1 enhances the efficacy of radiation therapy and bone marrow transplantation. Deletion of Gfi1 sensitizes T-ALL tumors and T cells to p53-dependent apoptosis after exposure to DNA-damaging agents such as radiation, Etoposide or Daunorubicin by de-repression of the pro-apoptotic Gfi1 target gene Bax. These data extend the role of Gfi1 to human T-ALL and suggest that T-ALL is dependent upon Gfi1 to repress pro-apoptotic genes for tumor survival, ultimately highlighting a new therapeutic target in the fight against lymphoid malignancies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Polycomb Repressor Complex 2 Regulates HOXA9 and HOXA10, Activating ID2 in NK/T-Cell Lines.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1281-1281
Author(s):  
Stefan Nagel ◽  
Letizia Venturini ◽  
Marquez E. Victor ◽  
Corinna Meyer ◽  
Maren Kaufmann ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Nk Cell ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Expression Levels ◽  
Repressor Complex ◽  
T Cell Lines ◽  
T Cell Leukemogenesis

Abstract Abstract 1281 Poster Board I-303 Many oncogenes code for transcription factors involved in regulation of developmental pathways. The activity of these pathways is tissue specific and restricted to certain developmental stages. Here, we searched for T-cell acute lymphoblastic leukemia (T-ALL) oncogenes which physiologically regulate differentiation of natural killer (NK) cells. NK- and T-cells are closely related lymphocytes, sharing the same early progenitors which can differentiate into either lineage. We compared expression profiles of malignant NK- and T-cell lines to identify aberrantly expressed genes in T-ALL. This analysis revealed high expression of HOXA9, HOXA10 and ID2 in NK-cell lines and in one T-ALL line, LOUCY, suggesting leukemic deregulation therein. Subsequently, we analyzed mechanisms underlying their regulation. Overexpression and chromatin immuno-precipitation experiments demonstrated that HOXA9 and HOXA10 directly activate ID2 expression. Analysis of other ALL and acute myeloid leukemia cell lines with and without mixed lineage leukemia (MLL) gene translocations demonstrated a correlated expression of HOXA9/10 and ID2, highlighting ID2 as an indirect target of MLL fusion proteins which deregulate HOXA genes. Furthermore, profiling data of genes coding for chromatin regulators of homeobox genes, including the components EZH2 and HOP of polycomb repressor complex 2 (PRC2), showed downregulation of EZH2 in LOUCY and limited expression of HOP to NK-cell lines. Subsequent treatment of T-ALL cell lines JURKAT and LOUCY with DZNep, an inhibitor of EZH2/PRC2, resulted in elevated and unchanged HOXA9/10 expression levels, respectively, confirming repressive activity of EZH2 in T-cells. Additionally, profiling data and overexpression analysis indicated that reduced expression of E2F cofactor TFDP1 contributed to the lack of EZH2 in LOUCY. Forced expression of HOP in JURKAT cells resulted in reduced HOXA10 and ID2 expression levels, suggesting enhancement of PRC2 repression. Taken together, our results show that major differentiation factors of the NK-cell lineage, including HOXA9, HOXA10 and ID2, were (de)regulated via PRC2 and may contribute to T-cell leukemogenesis. Disclosures No relevant conflicts of interest to declare.

Multi-Omic Based Antigen Discovery for the Immunotherapy of Pediatric Acute T Cell Lymphoblastic Leukemia

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 17-18
Author(s):  
Nikhil Hebbar ◽  
Chunxu Qu ◽  
Hong Wang ◽  
Ying Shao ◽  
Phuong Nguyen ◽  
...  
Keyword(s):  
T Cells ◽  
Plasma Membrane ◽  
T Cell ◽  
Cell Surface ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Differentially Expressed ◽  
Activated T Cells ◽  
Car T Cells ◽  
Car T

Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk disease due to treatment related complications and poor prognosis of patients with relapsed disease. Immunotherapy with monoclonal antibodies (MAbs) and/or chimeric antigen receptor (CAR) T-cells for T-ALL is limited by identification of tumor specific target antigens. Differential expression is necessary to prevent on-target/off-tumor toxicities and fratricide of activated T-cells. Targeting multiple antigens can bypass immune escape and result in improved T-cell effector function, since antigen density correlates with T-cell activation. Here we designed a pipeline (Figure 1) to identify unique surface antigens expressed in T-ALL using proteomic and transcriptomic analyses followed by flow cytometry validation, and functional studies with CAR T cells targeting the identified antigens. We generated an Illumina total stranded RNAseq library from healthy donor myeloid and lymphoid cells of bone marrow, peripheral blood and cord blood (N= 116). We compared data to 265 St. Jude pediatric T-ALL samples and against 53 normal tissue expression data from the GTEx (Genotype-Tissue Expression) project. To analyze the T-cell surface proteome, we isolated plasma membrane fractions from 11 samples including healthy T-cells and T-ALL cell lines using a differential centrifugation-based method. The purity of the plasma membrane fraction was confirmed by western blot. Na+/K+ ATPase and GAPDH were used as controls for the plasma membrane and cytosolic fractions respectively. Following plasma membrane enrichment, the membrane proteins were applied for proteomic analysis using an advanced TMT-L/LC-MS/MS pipeline, and the acquired proteomic data were further processed via the JUMP software suite. 997 unique proteins were quantified from the membrane fractions. Integrated analysis the transcriptomic and proteomic datasets showed significant correlation and yielded a list of candidate genes, which were validated by flow cytometry on a panel of T-ALL cell lines (CCRF, RPMI8402, and MOLT3) and resting and activated T-cells from healthy donors. We identified GRP78 as one of the differentially expressed cell surface antigens and further confirmed its expression on additional T-ALL cell lines (KE37, PF382, PEER, CEMC7) and 3 PDX samples. Finally, we generated GRP78-CAR T cells and demonstrate that GRP78-CAR T cells recognize and kill GRP78+ T-ALL cells and have potent antitumor activity in xenograft and PDX models. We have established an unbiased pipeline to identify differentially expressed antigens on the cell surface of T-ALL blasts and created a healthy tissue RNAseq library. The results from our analyses are encouraging and interrogation of our pipeline has yielded differentially expressed immunotherapy targets for the treatment of relapsed refractory T-ALL. Our results highlight the importance of integrated surface proteomics and transcriptomics analysis. Figure 1: Outline of strategy for target selection: Figure Disclosures Hebbar: St. Jude: Patents & Royalties. Epperly:St. Jude: Patents & Royalties. Gottschalk:Inmatics and Tidal: Membership on an entity's Board of Directors or advisory committees; TESSA Therapeutics: Other: research collaboration; Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; Merck and ViraCyte: Consultancy. Mullighan:AbbVie, Inc.: Research Funding; Illumina: Consultancy, Honoraria, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau. Velasquez:Rally! Foundation: Membership on an entity's Board of Directors or advisory committees; St. Jude: Patents & Royalties.

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