scholarly journals Leukemia Stem Cells Are Characterized By CLEC12A Expression and Chemotherapy Refractoriness That Can be Overcome By Targeting with Chimeric Antigen Receptor T Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 766-766 ◽  
Author(s):  
Saad S Kenderian ◽  
Marco Ruella ◽  
Olga Shestova ◽  
Michael Klichinsky ◽  
Miriam Y Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Stem Cell ◽  
Single Dose ◽  
Cell Line ◽  
Induction Chemotherapy ◽  
Chimeric Antigen Receptor ◽  
Research Funding ◽  
Tail Vein Injection ◽  
Equity Ownership

Abstract Chemo-refractory acute myeloid leukemia (AML) is associated with poor prognosis and treatment options are extremely limited. Most of these patients are ineligible for allogeneic stem cell transplantation. Chemo-refractory AML is thought to arise due to selection pressure of resistant clones from prior use of chemotherapy or in some cases pre-exist due to properties of the leukemic stem cells (LSC). CLEC12A (also known as CLL1) has previously been described as being selectively over expressed in LSCs. Successful modalities to target CLEC12A and eradicate the LSC would overcome chemo-refractoriness in AML and would represent a vertical advance in the field. In this study, we confirm that CLEC12A is heterogenously expressed on AML blasts and over-expressed on AML LSC. We also show that CLEC12A is overexpressed on bone marrows from patients with AML that fail to achieve a complete remission after induction chemotherapy, suggesting that it could be a marker for residual disease that is refractory to chemotherapy. We then separated AML blasts into CLEC12A positive or negative cells by magnetic sorting. CLEC12A positive blasts selected from AML patients were more resistant to chemotherapy compared to CLEC12A negative blasts (20% killing of CLEC12A positive AML cells versus 43% of CLEC12A negative AML cells when cultured with cytarabine 10 µg/ml, P=0.01). This finding was confirmed by using the AML MOLM14 cell line engineered to overexpress CLEC12A. CLEC12Ahigh MOLM14 cells were more resistant to chemotherapy compared to wild type MOLM14 cells (P=0.003). We then evaluated CLEC12A resistance to chemotherapy in a patient derived AML xenograft model. We found a relative increase in CLEC12A positive cells post Ara-C induction chemotherapy in AML xenograft models (Figure 1). The observation that CLEC12A positive cells are more resistant to chemotherapy provided a solid rationale to target CLEC12A with chimeric antigen receptor T (CART) cells. We therefore developed a second generation CLEC12A directed CAR construct using CD3z and 41BB costimulatory domains and generated CLEC12A CART cells by lentiviral transduction with this construct. Upon incubation with primary AML samples or AML cell lines, CLEC12A CART cells resulted in modest effector functions, due to the heterogeneity of CLEC12A expression on AML blasts. However when CLEC12A overexpressed MOLM14 cell line or CLEC12Apos selected leukemic cells were used as targets, CLEC12A-CART cells resulted in potent cytotoxicity, proliferation and cytokine production, indicating that CLEC12A-CART cells are more specific for LSC. To test the in vivo anti-leukemic activity of CLEC12A CARTs, we used primary human AML blasts xenografted into NSG-S mice (NOD-SCID-γc-/-, additionally transgenic for human stem cell factor, IL3 and GM-CSF). Treatment with CLEC12A CART (single dose, 1x105 total T cells via tail vein injection) resulted in modest activity against AML when employed as monotherapy. To investigate the potential role of CLEC12A CART cells in eradication of MRD and LSC, mice were treated first with chemotherapy (cytarabine 60 mg/kg intraperitoneal injection daily for 5 days) followed by a single dose (1x105 total T cells via tail vein injection) of either CLEC12A CARTs or control untransduced T cells (UTD). Treatment with CLEC12A CART cells resulted in eradication of leukemia and prolonged survival in these mice (overall survival at 200 days of 100% after CLEC12A CARTs compared to 20% after UTD, p=0.01, Figure 2). In conclusion, our preclinical studies reveal that CLEC12A positive cells in leukemia are resistant to chemotherapy and can be successfully targeted with CART cells. CLEC12A CART cells can potentially be employed as a consolidation regimen after induction chemotherapy to eradicate LSC and MRD in AML. Disclosures Kenderian: Novartis: Patents & Royalties, Research Funding. Ruella:novartis: Patents & Royalties: Novartis, Research Funding. Singh:Novartis: Employment. Richardson:Novartis: Employment, Patents & Royalties, Research Funding. June:Tmunity: Equity Ownership, Other: Founder, stockholder ; Immune Design: Consultancy, Equity Ownership; Novartis: Honoraria, Patents & Royalties: Immunology, Research Funding; University of Pennsylvania: Patents & Royalties; Celldex: Consultancy, Equity Ownership; Johnson & Johnson: Research Funding; Pfizer: Honoraria. Gill:Novartis: Patents & Royalties, Research Funding.

scholarly journals CD200 Is a Stem Cell-Specific Immunosuppressive Target in AML

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2768-2768
Author(s):  
Shelley Herbrich ◽  
Keith Baggerly ◽  
Gheath Alatrash ◽  
R. Eric Davis ◽  
Michael Andreeff ◽  
...  
Keyword(s):  
Gene Expression ◽  
Flow Cytometry ◽  
T Cells ◽  
Stem Cell ◽  
Board Of Directors ◽  
Research Funding ◽  
Advisory Committees ◽  
Cd200 Receptor ◽  
Blast Cells ◽  
Equity Ownership

Abstract Acute myeloid leukemia (AML) stem cells (LSC) are an extremely rare fraction of the overall disease (likely <0.3%), largely quiescent, and capable of both long-term self-renewal and production of more differentiated leukemic blasts. Besides their role in disease initiation, they are also hypothesized as the likely source of deadly, relapsed leukemia. Due to the quiescent nature of the LSCs, they are capable of evading the majority of chemotherapeutic agents that rely on active cell-cycling for cytotoxicity. Therefore, novel therapeutic approaches specifically engineered to eradicate LSCs are critical for curing AML. We previously introduced a novel bioinformatics approach that harnessed publically available AML gene expression data to identify genes significantly over-expressed in LSCs when compared to their normal hematopoietic stem cell (HSC) counterparts (Herbrich et al Blood 2017 130:3962). These datasets contain gene expression arrays on human AML patient samples sorted by leukemia stem, progenitor, and blast cells (with normal hematopoietic cell subsets for comparison). We have since expanded our statistical model to identify targets that are both significantly overexpressed in AML LSCs when compared to HSC as well as LSCs compared to their corresponding, more differentiated blast cells. Instead of traditional methods for multiple testing corrections, we looked at the intersection of genes that met the above criteria in 3 independently generated datasets. This resulted in a list of 30 genes, 28 of which appear to be novel markers of AML LSCs. From this list, we first chose to focus on CD200, a type-1 transmembrane glycoprotein. CD200 is broadly expressed on myeloid, lymphoid, and epithelial cells, while the CD200 receptor (CD200R) expression is strictly confined to myeloid and a subset of T cells. CD200 has been shown to have an immunosuppressive effect on macrophages and NK cells and correlates with a high prevalence FOXP3+ regulatory T cells (Coles et al Leukemia 2012; 26:2146-2148). Additionally, CD200 has been implicated as a poor prognostic marker in AML (Damiani et al Oncotarget 2015; 6:30212-30221). To date, we have screened 20 primary AML patient samples by flow cytometry, 90% of which are positive for CD200. Expression is significantly enriched in the CD34+/CD123+ stem cell compartment. To examine the role of CD200 in AML, we established two in vitro model systems. First, we used CRISPR/Cas9 to knockout the endogenous CD200 protein in Kasumi-1. Further, we induced CD200 in the OCI-AML3 cell line that had no expression at baseline. Both cell lines did not express the CD200 receptor before or after manipulation, negating any autocrine signaling. In both systems, CD200 manipulation did not affect the proliferation rate or viability of the cells. To examine the immune function of CD200 in AML, we performed a series of mixed lymphocyte reactions. We cultured normal human peripheral blood mononuclear cells (PBMCs) with the CD200+ or CD200- cells from each line both. Cells were incubated in the culture media for 4-48 hours before being harvested and measured by flow cytometry for apoptosis or intracellular cytokine production. The presence of CD200 on the cell surface reduced the rate of immune-specific apoptosis among these leukemia cells. The difference in cell killing was most likely attributable to a CD200-specific suppression of CD107a, a surrogate marker or cytotoxic activity. In the OCI-AML3 model, PBMCs co-cultured with CD200+ cells produced approximately 40% less CD107a when compared to the CD200- co-culture. Additionally, we characterized our new cell lines using RNA sequencing. By comparing the CD200+ to the CD200- cells within each line, we observed that CD200+ cells significantly downregulate genes involved in defining an inflammatory response as well as genes regulated by NF-κB in response to TNFα. This indicates that CD200 may have an undiscovered intrinsic role in suppressing the immune microenvironment of AML LSCs. In conclusion, we have expanded our novel bioinformatics approach for robustly identifying AML LSC-specific targets. Additionally, we have shown that one of these markers, CD200, has a potential role as a stem cell-specific immunosuppressive target by reducing immune-mediated apoptosis and transcriptionally suppressing inflammatory cell processes. We are extending our study to explore CD200 in primary patient samples using a CD200-blocking antibody. Disclosures Andreeff: SentiBio: Equity Ownership; Amgen: Consultancy, Research Funding; Oncolyze: Equity Ownership; Reata: Equity Ownership; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Jazz Pharma: Consultancy; Astra Zeneca: Research Funding; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy. Konopleva:Stemline Therapeutics: Research Funding.

scholarly journals A Phase I Study Evaluating the Safety and Persistence of Allorestricted WT1-TCR Gene Modified Autologous T Cells in Patients with High-Risk Myeloid Malignancies Unsuitable for Allogeneic Stem Cell Transplantation

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1367-1367
Author(s):  
Emma C Morris ◽  
Rita Tendeiro-Rego ◽  
Rachel Richardson ◽  
Thomas A Fox ◽  
Francesca Sillito ◽  
...  
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
High Risk ◽  
Phase I ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Post Infusion ◽  
Equity Ownership ◽  
Acute Myeloid

Background:Patients with acute myeloid leukemia (AML), myelodysplasia (MDS) or tyrosine kinase inhibitor resistant chronic myeloid leukemia (CML) who are unsuitable for consolidative allogeneic stem cell transplantation (alloSCT) have high relapse rates following chemotherapy. Wilms' tumor 1 (WT1) is highly expressed in the majority of acute myeloid leukemias (AML) and in many subtypes of myelodysplasia (MDS) as well as other hematological and solid tumors. WT1 is an intracellular antigen, which makes it difficult to target using current Chimeric Antigen Receptor (CAR)-T cell technologies. The use of genetically modified T cells expressing WT1-specific α/β T cell receptors can re-direct T cell specificity via the recognition of intracellular peptides presented by MHC molecules on the malignant cell surface. Phase I clinical trials of WT1-TCR gene-modified T cells have been conducted in the settings of relapsed disease and post-alloSCT and preliminary data suggests this treatment approach is safe and potentially clinically effective in these cohorts (Tawara et al. Blood. 2017;130(18):1985-94; Chapuis et al, Nat Med. 2019;25(7):1064-72). Methods:We report a phase I/II safety and dose escalation study evaluating WT1-TCR gene-modified autologous T cells in HLA-A*0201 positive patients with AML, MDS and CML, unsuitable for alloSCT (NCT02550535) (Fig 1A). Patient T cells were harvested by leucapheresis and transduced with a retroviral vector construct encoding the codon optimised variable and constant a and bchains of the human pWT126-specific TCR separated by a self-cleaving 2A sequence (Fig 1B). Bulk transduced T cells were analysed by flow cytometry (CD3, CD8 and Vb2.1) prior to infusion and at regular intervals post-infusion. A quantitative PCR assay was developed to identify WT1-TCR expressing T cells in the peripheral blood post infusion. Patients received minimal conditioning with fludarabine and methylprednisolone prior to transfer of transduced T cells. All subjects were followed for a minimum of 12 months or until death. Results:A total of 10 patients (6 AML, 3 MDS and 1 TKI- resistant CML) were recruited. The mean age was 71.3 years (range 64-75) and all had high risk disease (by cytogenetic or clinical criteria). All AML patients were in complete morphological remission at the time of trial entry, whilst MDS patients had ≤ 15% blasts on bone marrow examination. All 10 patients received the gene-modified T cells in dose escalation cohorts (seven patients received £2x107/kg and three patients received £1x108/kg bulk WT1 TCR transduced cells). No adverse events directly attributable to the investigational product were recorded apart from one possible cytokine release syndrome, which was managed without tociluzimab. Transferred T cells demonstrated in vivoproliferation commensurate with maintenance of functional capacity despite ex vivo manipulation (Fig 1C and 1D). The TCR-transduced T cells were detectable in all patients at 28 days and in 7 patients persisted throughout the study period (Fig 1E). All 6 AML patients were alive at last follow up (median 12 months; range 7-12.8 months). The 3 patients with MDS had a median survival of 3 months (range 2.1-3.96 months) post T cell infusion. 2 died from progressive disease and one from other causes. 2 patients discontinued the study early due to disease progression. Conclusions: This is the second reported phase I/II clinical trial of autologous WT1-TCR gene-modified T cells for treatment of AML and MDS in a high-risk cohort of patients not suitable for alloSCT. We have shown that the WT1-TCR T cells demonstrated a strong safety profile without detectable on-target, off-tumour toxicity and no severe adverse events in the ten patients treated. An important cause of treatment failure for adoptive cellular therapies is the lack of persistence of transferred T cells leading to loss of disease specific effects. We demonstrated that autologous WT1-TCR T cells proliferated in vivoand persisted for many months. Recent work within our group (in press) has shown that TCRs modified to include key framework residues, show increased TCR expression and functional improvement. These modifications could be incorporated into future studies to improve efficacy. This data supports the rationale for a larger, phase II trial of WT1-TCR T cells in myeloid malignancies in patients for whom alloSCT is not appropriate, in order to assess clinical efficacy. Figure 1 Disclosures Morris: Quell Therapeutics: Consultancy, Other: Scientific Founder,stock; Orchard Therapeutics: Consultancy. Qasim:CellMedica: Research Funding; Bellicum: Research Funding; UCLB: Other: revenue share eligibility; Autolus: Equity Ownership; Orchard Therapeutics: Equity Ownership; Servier: Research Funding. Mount:Gamma Delta Therapeutics: Employment. Inman:Cellmedica: Employment. Gunter:Cellmedica: Employment. Stauss:Cell Medica: Other: I have stock; Quell Therapeutics: Consultancy, Other: I have stock.

scholarly journals Chimeric Antigen Receptor-Modified T Cells for the Treatment of Acute Myeloid Leukemia Expressing CD33

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4058-4058 ◽  
Author(s):  
Degang Song ◽  
Michael H. Swartz ◽  
Steve G. Biesecker ◽  
Fernando Borda ◽  
Rutul R. Shah ◽  
...  
Keyword(s):  
T Cells ◽  
Acute Myeloid Leukemia ◽  
Chimeric Antigen Receptor ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Employment Equity ◽  
Car T Cells ◽  
Car T ◽  
Equity Ownership ◽  
Acute Myeloid

Abstract Relapsed acute myeloid leukemia (AML) is an aggressive disease with very poor outcomes. Redirection of T-cell specificity via chimeric antigen receptor (CAR) has shown promising anti-tumor activity in clinical trials, particularly for B cell linage malignancies. CD33 is a transmembrane protein expressed on normal and malignant myeloid-derived cells as well (as on subsets of activated T cells and NK cells). Since this protein is commonly expressed on AML cells, we sought to evaluate the efficacy of targeting AML with CD33-specific CAR-T cells. We generated a lentiviral construct to co-express CD33-specific CAR and a kill switch based on a tag derived from the epidermal growth factor receptor. The latter allows for the conditional elimination of CAR-T cells in vivo. Following transduction of primary T cells, we confirmed CAR and kill switch co-expression by flow cytometry and western blot analyses. Elimination of genetically modified T cells was demonstrated using the clinically-available antibody, cetuximab. CD33 CAR-T cells demonstrated specific cytotoxicity to CD33+ target cell lines. CD33 CAR-T cells were also activated to produce IFNg, TNF, and IL-2 cytokines in response to CD33+ target cells. Furthermore, adoptive transfer of CD33 CAR-T in immunocompromised (NSG) mice bearing established CD33+(CD19neg) AML (MOLM-13) tumor resulted in reduction of tumor burden and improvement of overall survival, compared to control mice receiving CD19 CAR-T cells or no immunotherapy (Figure). Sampling of blood demonstrated the persistence of the CD33 CAR-T cells with no detection of AML (MOLM-13) tumor cells. These pre-clinical data demonstrate the effectiveness of CD33 CAR-T cells in targeting CD33+ AML tumor cells and provide a rationale for future clinical evaluation in AML patients with unmet medical need. Disclosures Song: Intrexon Corporation: Employment, Equity Ownership. Swartz:Intrexon Corporation: Employment, Equity Ownership. Biesecker:Intrexon Corporation: Employment, Equity Ownership. Borda:Intrexon Corporation: Employment. Shah:Intrexon Corporation: Employment, Equity Ownership. Wierda:Genentech: Research Funding; Gilead: Research Funding; Abbvie: Research Funding; Novartis: Research Funding; Acerta: Research Funding. Cooper:MD Anderson Cancer Center: Employment; Intrexon: Equity Ownership; Sangamo BioSciences: Patents & Royalties; Targazyme,Inc.,: Equity Ownership; City of Hope: Patents & Royalties; ZIOPHARM Oncology: Employment, Equity Ownership, Patents & Royalties; Miltenyi Biotec: Honoraria; Immatics: Equity Ownership. Chan:Intrexon Corporation: Employment, Equity Ownership.

Alloreactive CD8+ Effector T Cells Proliferate and Persist Upon Chronic Exposure to Alloantigens through Reactivation of Stem Cell Transcriptional Programs.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2444-2444
Author(s):  
Koji Kato ◽  
Shuaiying Cui ◽  
Rork Kuick ◽  
Shin Mineishi ◽  
Elizabeth Hexner ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Proliferation ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Chronic Exposure ◽  
Research Funding ◽  
Effector T Cells ◽  
T Cell Proliferation ◽  
Effector T Cell

Abstract Abstract 2444 Poster Board II-421 Alloreactive effector T cells are the central to graft-versus-host disease (GVHD), a life-threatening complication after allogeneic hematopoietic stem cell transplantation (HSCT). In GVHD host antigens are never cleared and alloreactive effector T cells are continuously generated over a period of several months or longer, but their suppression and control have proven to be difficult in practice. Using mouse models of GVHD directed against minor histocompatibility antigens (miHAs), we demonstrate that alloreactive effector T cells proliferate and persist upon chronic exposure to alloantigens via reactivation of stem cell transcriptional programs normally expressed in embryonic stem cells and neural stem cells. Many activated stem cell genes in effector T cells were distinct from those in memory T cells and were maintained at high levels upon T cell receptor activation, suggesting a specific role in chronically activated effector T cells. One of these genes, Ezh2, encodes a chromatin modifying enzyme essential to the proliferation, survival and differentiation of stem cells, was upregulated in CD8+ effector T cells upon antigenic stimulation and downregulated when the antigen was withdrawn. Pharmacologically inactivation of EZH2 with 3-Deazaneplanocin A inhibited effector T cell proliferation and survival. Silencing Ezh2 independently validated that Ezh2 was important for regulating effector T cell proliferation and expression of many stem cell genes. To further evaluate whether alloreactive CD8+ effector T cells obtained stem cell-like properties, e.g. the ability to self-renew to continually generate effector cells, we adoptively transferred highly purified miHA H60-specific (H60+) CD8+ effector T cells into secondary allogeneic and congenic recipients, respectively. As compared to congenic recipients, allogeneic recipients had 80-fold more proliferating H60+CD8+ effector T cells. These donor H60+CD8+ effector T cells expressed high levels of CD122, CD69, CXCR3, PD1, IFNγ and Granzyme B, required miHA H60 stimulation to sustain their replication with effector function and expression of stem cell genes, and caused severe GVHD in secondary allogeneic recipients. These results indicate that stem cell transcriptional programs expressed in embryonic and neural stem cells may play important roles in effector T cells. Among these stem cell genes, Ezh2 emerges as an important therapeutic target in modulating alloreactive T cell-mediated GVHD. Disclosures: Zhang: University of Michigan Comprehensive Cancer Center: Research Funding; Damon Runyon Cancer Research Foundation: Research Funding.

scholarly journals Efficient Gene Transduction and Reprogramming of Hematopoietic Cells Including T-Cells By Using a Non-Integrating Measles Virus Vector

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3494-3494
Author(s):  
Jiyuan Liao ◽  
Yasushi Soda ◽  
Ai Sugawara ◽  
Yoshie Miura ◽  
Takafumi Hiramoto ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Gene Transfer ◽  
Research Funding ◽  
Measles Virus ◽  
Gene Transduction ◽  
Efficient Gene ◽  
Car T ◽  
Ipsc Generation ◽  
Equity Ownership

Abstract By the ectopic expression of reprogramming genes OCT, KLF4, SOX2 and MYC (OKSM), somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs). Human iPSCs are considered a promising cell source to provide an import tool for the basic investigation and the advanced medicine including gene therapy and regenerative medicine. To establish iPSCs, integration-free Sendai virus (SV) vectors have been most widely used do far, but transduction and reprogramming of T cells without stimulation is still very challenging. On the other hand, a great success of chimeric antigen receptor T cell (CAR-T) therapies highlighted the importance of anti-cancer immunity for the cancer treatment. Particularly, many refractory patients with acute lymphoblastic leukemia and B-cell lymphoma were successfully treated with CD19-CAR-T therapies, however, some patients died before receiving the treatment due to long preparation time of CAR-Ts. Therefore, rapid production systems of CAR-Ts are desired, and for this purpose, efficient and safe gene transduction systems to T cells should be developed. In this study, we developed a new non-integrating measles virus (MV) vector-based delivery system with F deletion to eliminate cell membrane fusion-associated cytotoxicity. MV vectors transduced genes through MV receptors including CD46 and signaling lymphocyte activation molecule (CD150/SLAM). First, we examined transduction efficiencies of MV vectors and SV vectors in hematopoietic cells by using GFP expression vectors (MV-Gs and SV-Gs). Compared to SV-Gs, our MV-Gs allowed more efficient gene transfer into most hematopoietic cell type including T (3-fold) and B cells (7-fold) (Fig. 1). Furthermore, at the same multiplicity of infection (MOI) of viral transduction, MV-Gs induced less apoptosis in T cell subset compared to SV-Gs (Fig. 2) due to the slower kinetics of viral RNA amplification in the transduced cells 24 h ,48 h and 72 h post transduction. Those results encouraged us to examined if MV vectors are more potent than SV vectors in iPSC generation from unstimulated T cells. To address this question, we developed MV vectors harboring four reprogramming genes (MV-OKSMGs) and compared with SV vectors harboring these genes (SV-OKSMGs). As expected, with the MV-OKSMGs, we could generate high-quality iPSCs with the similar morphology, pluripotency markers, karyotype and differentiation capacity as human embryonic stem cells. Upon the less cytotoxicity, iPSC generation efficiency of MV-OKSMGs was much higher than that of SV-OKSMGs for unstimulated T cells (0.47 ± 0.25% vs 0.008 ± 0.009%). Considering the safe history of MV vaccine, carrying capabilities of multiple genes, more flexible receptors and higher transduction efficiency for resting T cells, our exclusive MV vector would be a potential gene transfer system for iPSC generation and lymphocyte-based-immunotherapies such as CAR-T therapies. Disclosures Liao: neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding. Soda:Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Sugawara:neopharma Japan Co. Ltd: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Miura:neopharma Japan Co. Ltd: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Tahara:TAKARA BIO, INC.: Research Funding. Takishima:neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding. Hirose:TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding. Hijikata:Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Miyamoto:Shinnihonseiyaku Co., Ltd: Research Funding; TAKARA BIO, INC.: Research Funding; neopharma Japan Co. Ltd: Research Funding. Takeda:TAKARA BIO, INC.: Research Funding. Tani:neopharma Japan Co. Ltd: Research Funding; Oncolys BioPharma Inc.: Equity Ownership; SymBio Pharmaceuticals Limited: Equity Ownership; TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding.

CXCR4 Pepducins in Stem Cell Mobilization.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2440-2440 ◽  
Author(s):  
Katie M O'Callaghan ◽  
Mo-Ying Hsieh ◽  
Richard A VanEtten ◽  
Lidija Covic ◽  
Athan Kuliopulos
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Peripheral Blood ◽  
Research Funding ◽  
Calcium Flux ◽  
Intracellular Loop ◽  
Cell Penetrating ◽  
Equity Ownership ◽  
Cell Mobilization

Abstract Abstract 2440 Poster Board II-417 Hematopoietic stem cells (HSCs) and progenitor cells (HPCs) reside in the bone marrow and give rise to blood-forming cells. Mobilization of these cells from the bone marrow is clinically important as these cells can be harvested from the peripheral blood, and used in transplantation. G-CSF (granulocyte colony stimulating factor) is used clinically and is considered the ‘gold standard' for human progenitor cell mobilization. However, there is broad variability in mobilization between G-CSF-treated patients, with some heavily-treated patients exhibiting very poor mobilization. Therefore, novel agents to mobilize HSCs and HPCs are urgently required. The G protein-coupled chemokine receptor, CXCR4, and its ligand, stromal derived factor-1a (SDF-1a), regulate a diverse array of cellular processes, including hematopoiesis, stem cell homing to the bone marrow, and survival of HSCs and HPCs. SDF-1a secreted from stromal cells in the bone marrow acts on CXCR4-expressing HSCs and HPCs to maintain these cells within the bone marrow microenviroment. Therefore, disruption of the interaction between SDF-1a and CXCR4 is a potential strategy for mobilization of these cells to the peripheral blood. In this study, we employed cell-penetrating lipopeptide ‘pepducins' which interact with the intracellular loops of CXCR4 to specifically antagonize SDF-1a-mediated CXCR4 activity. We characterized a number of pepducins that are targeted to different intracellular loop regions of CXCR4. We demonstrated that two pepducins based on the first intracellular (i1) loop, PZ-218 and PZ-305, significantly inhibited SDF-1a-mediated calcium flux and chemotaxis of human neutrophils. A pepducin targeted to the third intracellular loop (i3), PZ-210, also inhibited calcium flux and migration of neutrophils, and caused a conformational change in CXCR4 that inhibited binding of the 12G5 CXCR4 antibody. Conversely, no such conformational change was elicited by the i1 pepducins PZ-218 and PZ-305, suggesting that different pepducins have distinct modes of action on CXCR4 signaling. To examine the efficacy of these pepducins in mobilization of stem cells, colony forming unit (CFU) assays and transplantation studies were performed. We determined that, when used in combination with G-CSF, the i1 and i3-loop pepducins mobilized up to three-fold more progenitor cells from the bone marrow than G-CSF treatment alone. Furthermore, lethally irradiated mice were rescued with peripheral blood from pepducin-treated donor mice, and cells mobilized by pepducin treatment had long-term repopulating ability in donor mice. These data demonstrate the potential therapeutic application of cell-penetrating pepducins in CXCR4 antagonism for the purpose of stem cell mobilization and modulation of the effects of SDF-1a on hematopoietic cells. Disclosures: O'Callaghan: Ascent Therapeutics Inc: Research Funding. Covic:Ascent Therapeutics Inc: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Kuliopulos:Ascent Therapeutics Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Inflammatory Cell Tissue Extravasation Pathways Can Be Regulated by Adherent Stem Cells by Inhibiting Selectin Ligand and Adhesion Receptor Expression On T Cells and Activated Endothelium.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3631-3631
Author(s):  
Wouter Van't Hof ◽  
Amy Raber ◽  
Juliana Woda ◽  
Nicholas Lehman ◽  
Rochelle Cutrone ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Endothelial Cells ◽  
Stem Cell ◽  
Cell Surface ◽  
Inflammatory Cells ◽  
Employment Equity ◽  
Activated T Cells ◽  
Tnf Α ◽  
Equity Ownership

Abstract Abstract 3631 Poster Board III-567 MultiStem is an adult bone marrow derived stem cell product with immune-modulatory activity that falls within a class of adherent stem cell platforms emerging as modalities for allogeneic therapy for modulation of GVHD. Despite initial preclinical and clinical success there remains significant need for mechanistic understanding of stromal cell interaction with inflammatory cells, particularly relating to biodistribution and the microenvironments encountered after intravenous administration. Successful GVHD treatment or prophylaxis regimens will likely by optimized by a better appreciation of adherent stem cell interactions with GVHD initiating T cells in the tissue microenvironment or in secondary lymphoid tissues. In this study we evaluated interactions of MultiStem with activated peripheral blood cells as well as activated endothelial cells using in vitro co-culture systems that permit cross-talk via soluble factors. In co-culture experiments with activated T cells we observed that MultiStem strongly inhibits gene expression of Fut7, the enzyme responsible for expression of Lewis antigen (CD15s). The resultant lack of CD15s expression on the cell surface of activated T cells was shown to result in their reduced ability to bind to endothelium activated with TNFa, relative to T cells activated in the absence of MultiStem. Furthermore, we found that co-culture of MultiStem with endothelial cells prevents upregulation of E-selectin, V-CAM, and to a lesser degree, I-CAM, to the cell surface of endothelial cells upon activation with TNF-α or interleukin-1α. Inhibition of E-selectin expression by co-culture with MultiStem was not caused by increased cleavage of E-selectin from the cell surface. Instead, MultiStem modulates cell surface induction through decreasing transcription of V-CAM, E-selectin and ICAM. This reduction in cell surface adhesion molecule expression results in decreased neutrophil binding to endothelial cells compared with untreated controls. This activity does not appear to be common to all bone marrow derived adherent stem cells as mesenchymal stem cells (MSC) are unable to modulate V-CAM, E-selectin or I-CAM cell surface upregulation upon activation with TNF-α. These results suggest MSC and MultiStem have distinct secretion profiles. These novel observations indicate that, in addition to immunomodulation activity, MultiStem has unique potential to deter GVHD by impacting various arms of the extravasation process of GVHD pathology causing inflammatory cells. Disclosures: Van't Hof: Athersys: Employment, Equity Ownership. Raber:Athersys: Employment, Equity Ownership. Woda:Athersys: Employment, Equity Ownership. Lehman:Athersysa: Employment, Equity Ownership. Cutrone:Athersys: Employment. Ting:Athersys: Employment, Equity Ownership. Deans:Athersys: Employment, Equity Ownership.

scholarly journals Deciphering the Mechanisms of Osteoblast-Induced Resistance of Leukemic Stem Cell (LSC) in Ph+ CML: Role of PI3-Kinase, BRD4 and MYC and Development of Strategies to Overcome Osteoblast-Induced Resistance

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1481-1481
Author(s):  
Yueksel Filik ◽  
Karin Bauer ◽  
Barbara Peter ◽  
Emir Hadzijusufovic ◽  
Georg Greiner ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Drug Resistance ◽  
Endothelial Cells ◽  
Stem Cell ◽  
Cell Line ◽  
Induced Resistance ◽  
Research Funding ◽  
Mtor Inhibitor ◽  
Pi3 Kinase

Abstract Chronic myeloid leukemia (CML) is a stem cell neoplasms characterized by the chromosome translocation t(9;22) and the related BCR-ABL1 fusion gene. Therapy with BCR-ABL1 kinase inhibitors is highly effective in the treatment of CML and deep molecular responses are achieved in most patients. However, not all patients respond to these drugs due to resistance of leukemic stem cells (LSC). Recent data suggest that the disease-related microenvironment, known as the stem cell niche contributes to drug resistance and relapse in CML. So far, little is known about the resistance mechanisms protecting niche cells in the bone marrow of patients with CML. We have recently shown that osteoblasts are a major site of LSC-mediated resistance against BCR-ABL1-targeting drugs in CML. In the current study, we screened for drugs that are able to suppress growth and viability of osteoblasts and/or other niche-related cells and can thereby overcome drug resistance of CML LSC. Proliferation was analyzed by determining 3H-thymidine uptake in niche-related cells and apoptosis was measured by Annexin-V/DAPI-staining and flow cytometry. We found that the dual PI3 kinase (PI3K) and mTOR inhibitor BEZ235 and the selective pan-PI3K inhibitor copanlisib suppress proliferation of primary osteoblasts (BEZ235 IC 50: 0.05 µM; copanlisib IC 50: 0.05 µM), the osteoblastic cell line CAL-72 (BEZ235 IC 50: 0.5 µM; copanlisib IC 50: 1 µM), primary human umbilical vein endothelial cells (BEZ235 IC 50: 0.5 µM; copanlisib IC 50: 0.5 µM) and the endothelial cell line HMEC-1 (BEZ235 IC 50: 1 µM; copanlisib IC 50: 1 µM), whereas no comparable effects were seen with the mTOR inhibitor rapamycin. As determined by flow cytometry, BEZ235 and copanlisib suppressed the expression of phosphorylated (p) pAKT and pS6 in endothelial cells and osteoblasts whereas rapamycin downregulated pS6 expression but did not decrease expression of pAKT. Moreover, we found that BEZ235 and copanlisib cooperate with nilotinib and ponatinib in suppressing growth and viability of osteoblasts and endothelial cells. Furthermore, BEZ235 and copanlisib were found to overcome osteoblast-induced resistance of K562, KU812 cells, and primary CD34 +/CD38 − CML LSC against nilotinib and ponatinib. This effect was also seen when CAL-72 cells were first exposed to BEZ235 or copanlisib and washed before co-cultures with CML cells and BCR-ABL1 inhibitors were prepared, suggesting that osteoblast inhibition was a crucial event capable of disrupting LSC resistance in these co-cultures. Of all other drugs tested, only the BRD4-targeting drug JQ1 was found to suppress CAL72-induced resistance in the CML cell lines KU812 and K562, suggesting that osteoblast-induced resistance of CML cells is also mediated by a BRD4-MYC pathway. In a next step, we examined the expression of resistance-mediating immune checkpoint molecules on CML cells (KU812, K562, LSC) and on osteoblasts by flow cytometry. We found that CML cells and CAL72 cells constitutively express PD-L1 and that interferon-gamma (IFN-G) promotes the expression of PD-L1 in all cell types tested. Moreover, we found that the BRD4 blocker JQ1 and the BRD4-degrader dBET6 suppress the IFN-G-induced upregulation of PD-L1 in CML LSC and osteoblasts. In conclusion, our data show that osteoblast-induced resistance of CML stem cells is mediated by a PI3K-dependent pathway and BRD4/MYC, and that BRD4-inhibition or BRD4-degradation counteracts osteoblast-induced resistance of CML (stem) cells against BCR-ABL1 inhibitors and PD-L1 expression on CML LSC and osteoblasts. We hypothesize that checkpoint inhibition may assist in drug-induced eradication of CML LSC and thus in the development of curative drug therapies in Ph + CML. Disclosures Hoermann: Novartis: Honoraria. Gleixner: Pfizer: Honoraria; Abbvie: Honoraria; BMS: Honoraria; Incyte: Honoraria; Novartis: Honoraria. Sperr: AbbVie, BMS-Celgene, Daiichi Sankyo, Deciphera, Incyte, Jazz, Novartis, Pfizer, StemLine, Thermo Fisher: Honoraria, Research Funding. Valent: Novartis: Honoraria; Pfizer: Honoraria, Research Funding; Celgene/BMS: Honoraria, Research Funding; Incyte: Honoraria, Research Funding; OAP Orphan Pharmaceuticals: Honoraria.

scholarly journals F-Deletion Non-Integrating Measles Virus Vector: A Promising Tool for T-Cell Engineering and Naive iPSCs Generation

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5750-5750
Author(s):  
Jiyuan Liao ◽  
Yasushi Soda ◽  
Ai Sugawara ◽  
Yoshie Miura ◽  
Takafumi Hiramoto ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Measles Virus ◽  
Transduction Efficiency ◽  
Promising Tool ◽  
Primed State ◽  
Equity Ownership ◽  
T Cell Engineering

Although great successes of chimeric antigen receptor T-cell (CAR-T) therapy highlighted the importance of anti-cancer immunity for cancer treatment, there are still some problems remained, i.e., long preparation time, extremely high cost and potential risk by insertional mutagenesis. To developmore rapid and safer T-cell engineering systems than current procedures using retrovirus or lentiviral vectors, we have long been focusing on measles virus as a new vector because of its high infectivity to T cells including resting state and rapid gene expression without chromosome integration. Presently, Sendai virus vectors (SVs), which is also a Paramyxoviridaevirus-based vector, are widely used for gene delivery and induced pluripotent stem cells (iPSCs), but the transduction to undifferentiated T cells (UTs) is a big challenge for SVs. We, therefore, compared the gene transduction efficiency between our measles vector (MVs) and SVs. We also compared iPSCs generation efficiencies between MVs and SVs. We engineered our non-replicating and non-integrating measles virus vectors (MV)with F deletion to eliminate cell membrane fusion-associated cytotoxicity.Based on the original property of measles virus,our recombinant MVsallowed more efficient gene transduction to various hematopoietic cells including UTs and B cells than SVs. Importantly, MVs induced less apoptosis compared withSVs due to their slower amplification of viral RNA in transduced cells. Moreover, we could establish iPSCs from UTs with MVs harboring reprogramming genes 50 times more efficiently than SVs harboring the same reprogramming genes. MV-induced iPSCs derived from CD3+T cells (MV-TiPSCs) were similar to regular human pluripotent stem cells (hPSCs: embryonic stem cells and iPSCs), which are in primed state, in morphology, the expressions of pluripotent markers and the ability to differentiate into three germ layers. On the other hand, without using naive induction culture condition, MV-induced iPSCs derived from CD34+hematopoietic progenitor cells (MV-HPC-iPSCs) presented a dome shape and showed a transcriptome profile close to naive iPSCs. To further confirm naive-like properties of MV-HPC-iPSCs, we evaluated gene expression patterns of these cells for 22 common genes most differently expressed in naive and primed hPSCs reported in previous reports (Fig.1).As expected, MV-HPC-iPSCs were clustered in naive hPSCs group while MV-HPC-iPSCs after culturing in primed induction condition showed primed-like features (Fig.2). Moreover, whole genome bisulfite sequencing analysis showed that MV-HPC-iPSCs had lower methylation than primed MV-HPC-iPSCs. These results strongly suggested that MV could induce naive-like iPSCs directly, and primed induction culture changed the cells to primed state with increasing genomic methylation. Considering the very safe history of MV vaccine, the capabilities of simultaneous expressions of multiple genes and the high transduction efficiency for hematopoietic cells including UTs, our MVs will be useful to directly induce naive state iPSCs, and be a promising tool for developing new T-cell immunotherapies. Disclosures Liao: TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding. Soda:TAKARA BIO, INC.: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; neopharma Japan Co. Ltd: Research Funding. Miura:Neoprecision therapeutics: Research Funding. Tahara:TAKARA BIO, INC.: Research Funding. Miyamoto:neopharma Japan Co. Ltd: Research Funding; Shinnihonseiyaku Co., Ltd: Research Funding; TAKARA BIO, INC.: Research Funding. Takeda:TAKARA BIO, INC.: Research Funding. Tani:Oncolys BioPharma Inc.: Equity Ownership; SymBio Pharmaceuticals Limited: Equity Ownership; TAKARA BIO, INC.: Research Funding; Neoprecision therapeutics: Equity Ownership, Research Funding; neopharma Japan Co. Ltd: Research Funding; Shinnihonseiyaku Co., Ltd: Equity Ownership.

Construction and Pre-Clinical Evaluation of a New Anti-CD19 Chimeric Antigen Receptor

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1627-1627
Author(s):  
Anne Marijn Kramer ◽  
Sara Ghorashian ◽  
Gordon Weng-Kit Cheung ◽  
Winston Vetharoy ◽  
Dale Moulding ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Chimeric Antigen Receptor ◽  
Research Funding ◽  
Antigen Receptor ◽  
Target Cells ◽  
Functional Avidity ◽  
Car T ◽  
Low Levels

Abstract Relapsed and refractory B-lineage acute lymphoblastic leukemia remain the leading cause of cancer related death in children and young adults. Clinical studies of adoptive cell immunotherapy, re-directing T cells against CD19 by endowing them with a chimeric antigen receptor (CAR), have shown considerable clinical responses. To date, 3 different binding domains (scFv) targeting CD19 have been used in CARs taken forward in clinical trials and we have constructed a new CD19-CAR, derived from a different anti-human CD19 antibody, clone CAT. Whether different binding affinities of the CD19 targeting domain, when significantly different, could affect CAR-mediated T cell functionality has not been evaluated in depth. We therefore investigated the impact of scFv affinity on CAR-mediated T cell function in vitro, as well as on anti-tumour efficacy in vivo. We have generated 3 CD19-CARs only differing in their scFv, which were derived from 3 anti-human CD19 antibodies (Clones FMC63, 4G7 & CAT) respectively. All other structural variables of the CAR and the use of the 4-1BB endodomain were identical. The Kd values obtained by Biacore Surface Plasmon resonance (SPR) analysis ranged from 8.8 x 10-10 to 1.1 x 10-7. Differences in affinity were predominantly determined by the off-rates, leading to significantly quicker dissociation from its target in CAT scFv compared to FMC63 and 4G7. CAT-CAR transduced T-cells showed enhanced cytotoxic responses to the CD19+ cell line SUPT1-CD19 in 51Cr release assays (p<0.001) compared to 4G7 and FMC63. Moreover, CAT+ T-cells demonstrate an increased proliferative capacity following antigen specific stimulation and an increased capacity to produce IL-2 and TNFα (p<0.001). A quick dissociation rate has been described to be of particular importance when targeting cells with low levels of antigen expression, as T cell functional avidity can be detrimentally affected when dissociation is prolonged (Thomas et al, Blood 2011). We therefore investigated cytotoxicity of CAR transduced T cells against a cell line engineered to express CD19 at very low levels. This demonstrated increased cytotoxicity by CAT+ T-cells as well as greater CD107a degranulation in response to low CD19 expressing targets compared to FMC63 or 4G7-transduced T cells. Similarly, CAT+ T-cells showed greater killing of NALM 6 cells at very low effector:target ratios, reflecting the ability of serial killing by CAT+ T-cells by virtue of their rapid dissociation from target cells. Live cell imaging studies by confocal microscopy analysis confirmed a higher number of serial engagements by CAT+ T-cells (p<0.001), as well as greater motility (p<0.001). We are now studying the relative potency in a xenogeneic model of ALL, using a CAR-T cell dose that is purposefully lowered to a suboptimal range to study kinetic differences and tumor clearance. Preliminary data suggests that, transferred after exposure to leukemia, CAT+ T cells have a less exhausted phenotype and higher effector:target ratios 2 weeks after infusion. Further experiments, in which recipient mice are re-challenged with the same tumor, will assess differences in the ability of adoptively-transferred CAR T cells to form memory. In conclusion, we have developed a novel CD19-CAR which confers enhanced cytotoxicity and proliferative responses compared to existing CD19-CARs. Our work indicates that the scFv binding kinetics impacts the functional avidity of CAR-transduced T cells, providing important implications for the design of future CARs, especially when tumour cells expressing low levels of antigen are targeted. Disclosures Onuoha: Autolus Ltd: Employment, Research Funding. Pule:Autolus Ltd: Employment, Equity Ownership, Research Funding; UCL Business: Patents & Royalties; Amgen: Honoraria; Roche: Honoraria.

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