Leukemic Stem Cells of Monocytic AMLs Are Not-Resistant to BCL-2 Inhibition

Abstract Treatment with Hypomethylating agents (HMA) such as 5-Azazytidine (AZA) in combination with the BCL-2 inhibitor Venetoclax (VEN) has recently become the standard of care for AML patients unsuitable for intensive induction chemotherapy and shows results superior to treatment with AZA alone (DiNardo et al., 2020, NEJM). However upfront resistance and relapse following initial response remain major obstacles. It has recently been proposed that monocytic differentiation predicts resistance to AZA/VEN treatment in AML (Pei et al., 2020 Cancer Discovery). This appears to be due to increased expression of other anti-apoptotic proteins such as MCL-1 in monocytic AMLs, which conveys resistance to AZA/VEN therapy, as survival of leukemic cells in these patients is no longer dependent on BCL-2. However, an independent study found no impaired outcome in patients with monocytic AMLs treated with HMA/VEN (Maiti et al., 2020, Blood, ASH abstract). Here, we show that monocytic AML cell lines and bulk cells of monocytic primary AML cells are indeed intrinsically resistant to AZA/VEN treatment. However, in a collective of 30 patients treated with HMA/VEN at Heidelberg University Medical Center between 2018 and 2020, monocytic differentiation assessed by flow cytometry was not an independent risk factor for refractory disease. We hypothesized that the conflicting data may be caused by intra-patient heterogeneity of AZA/VEN sensivitity and assessed killing efficiency in various immunophenotypic subpopulations of 12 primary AML patient samples in vitro. The CD64 +CD11b +, differentiated blast population made up >50% of leukemic cells in monocytic and <20% in primitive samples and showed high levels of resistance to AZA/VEN therapy in both primitive and monocytic leukemias but did not engraft when transplanted into NSG mice, arguing they do not contain leukemic stem cells (LSC). In contrast, we found immature CD64 -CD11b - GPR56 + LSC to be sensitive to AZA/VEN treatment irrespective whether they were derived from monocytic or primitive types of primary AMLs. As expected, LSCs from either monocytic or primitive AMLs initiated disease in NSG mice, highlighting that targeting LSCs is essential for the effect of AML therapy. Next, we investigated expression of BCL-2, MCL-1 and BCL-xL in the same primary patient samples and observed high MCL-1 expression in monocytic AML samples. However, MCL-1 expression was restricted to the CD64 +CD11b + population whereas in the LSC sub-populations robust expression of BCL-2 but low levels of MCL-1 and BCL-xL were detected, independent of whether monocytic or primitive AMLs were analyzed. To further validate the sensitivity of LSCs of monocytic AML to BCL-2-I, we established a platform combining BH-3 profiling with multi-color flow cytometry, allowing for single cell assessment of cellular dependencies on independent apoptotic pathways. We found that LSCs of both AML types show high VEN/BAD but low MS-1 induced apoptosis, functionally confirming the expression patterns of BCL-2 and MCL-1. As LSCs are rare in monocytic samples, investigation of samples in bulk are dominated by MCL-1 expressing and resistant non-LSCs, explaining the overall higher MCL-1 expression/survival of monocytic compared to immature AML cells. However, our data uncovers sensitivity of LSCs to AZA/VEN independent of overall monocytic or primitive sample classification and provide a mechanistic explanation for the clinical data of Maiti et al. and our Heidelberg AML collective, which found no increased resistance of monocytic AMLs to AZA/VEN treatment. Disclosures Unglaub: JazzPharma: Consultancy, Other: travel costs/ conference fee; Novartis: Consultancy, Other: travel costs/ conference fee. Schlenk: Abbvie: Honoraria; Agios: Honoraria; Astellas: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria; Daiichi Sankyo: Honoraria, Research Funding; Hexal: Honoraria; Neovio Biotech: Honoraria; Novartis: Honoraria; Pfizer: Honoraria, Research Funding, Speakers Bureau; Roche: Honoraria, Research Funding; AstraZeneca: Research Funding; Boehringer Ingelheim: Research Funding. Müller-Tidow: Janssen: Consultancy, Research Funding; Bioline: Research Funding; Pfizer: Research Funding.

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Identification of Small Molecules Selectively Targeting Leukemic Stem Cells within Their Stromal Niche

Blood ◽

10.1182/blood.v120.21.413.413 ◽

2012 ◽

Vol 120 (21) ◽

pp. 413-413

Author(s):

Alissa R. Kahn ◽

Kimberly A. Hartwell ◽

Peter G. Miller ◽

Benjamin L. Ebert ◽

Todd R. Golub ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Cord Blood ◽

Leukemic Cells ◽

Leukemic Stem Cells ◽

Minimal Toxicity ◽

Nsg Mice ◽

Cd34 Cells

Abstract Abstract 413 Current therapies for acute myeloid leukemia (AML) are highly toxic, yet the relapse rate remains high. New therapies are needed to improve cure rates while decreasing toxicity. Because therapies may be affected by the tumor niche, we aimed to test new compounds on leukemic stem cells (LSCs) within their stromal microenvironment. A niche-based high throughput screen identified candidate small molecules potentially toxic to MLL-AF9 murine leukemic stem cells (LSCs) while sparing normal hematopoietic stem cells (HSCs) and bone marrow stroma (Hartwell et al, Blood 118, Abs 760, 2011.) Three such compounds, including a selective serotonin receptor antagonist highly specific for the 5-HT1B receptor, SB-216641, and two antihelminthics, parbendazole and methiazole, were found to be effective and selected for studies on human leukemias. We first examined SB-216641, studying the effects of this compound on 7 human primary AML samples. We began by assessing the compound's effect on LSCs using the week 5 cobblestone area forming cell (CAFC) assay, a standard in vitro stem cell assay. CD34+ cells were isolated with immunomagnetic beads. The leukemic cells were pulse treated for 18 hours and washed prior to placement on MS-5 murine stroma. We performed serial drug dilutions using the CAFC assay with the human primary samples as well as with HSCs derived from cord blood. All human leukemic samples formed cobblestone areas in the control setting (46-200 CAFCs/106 cells plated). IC50 for the human primary leukemia CAFCs was 630 nm, and at 10 μM all LSCs were killed while normal human HSCs had 100% survival. A combination of the AML cell line HL60 transduced with GFP-luciferase and normal cord blood CD34+ cells (1:200) were then pre-incubated overnight with SB-216641 at 5 and 10 μM and injected into Nod Scid IL2R-gamma null (NSG) mice. The control mice had leukemic engraftment by luciferase imaging and flow cytometry and the mice that received treated cells had no leukemic engraftment but normal multilineage engraftment of cord blood. Primary patient AML samples were also pre-incubated overnight with SB-216641 at 10 μM and injected into NSG mice. As shown by flow cytometry, control mice engrafted with leukemia and mice that received pre-treated cells had no engraftment following exposure to SB-216641. Finally, an in vivo study was completed on NSG mice injected intraperitoneally with 20 mg/kg/day beginning on day 1 or day 8 after inoculation with HL60 (500 cells). The mice were imaged at 2 and 3 week time points and both treatment groups had significantly less leukemia on imaging than the control group with minimal toxicity noted. Another specific 5-HT1B receptor antagonist, SB-224289, was found to have similar activity to SB-216641 against leukemic cells and to spare HSCs in preliminary studies. Similar CAFC studies with serial dilutions on primary AML samples were performed on the two anti-helminthic agents. IC50 for parbendazole was 1.25 μM and for methiazole 5 μM. As shown by luciferase imaging and flow cytometry, when injected with combined HL60 and cord blood pre-incubated overnight at 5 and 10 μM with each compound as described above, the control mice engrafted with leukemia and the mice that received treated cells had no leukemic engraftment but normal multilineage engraftment of cord blood. NSG mice were then injected with primary AML pretreated overnight with parbendazole at 10 μM. As shown by flow cytometry, control mice engrafted with leukemia and mice that received pre-treated cells had significantly lower engraftment following exposure to parbendazole (p = 0.01). Two new avenues of leukemia therapy were discovered warranting further investigation. SB-216641, an agent with a completely novel receptor target in leukemia therapy, has shown both in vitro success in human leukemia as well as preliminary success in vivo with minimal toxicity. We aim to move forward with this agent while also testing parbendazole in vivo, as this compound is already known to have good pharmacokinetics and minimal toxicity in animals. The high toxicity to LSCs and sparing of normal HSCs give both these agents an attractive profile for future clinical trials. Disclosures: Ebert: Genoptix: Consultancy; Celgene: Consultancy.

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FLT3 Splice Variant (FLT3Va) As a Potential Immunotherapeutic Target in Patients with Acute Myeloid Leukemia (AML)

Blood ◽

10.1182/blood.v128.22.1681.1681 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1681-1681

Author(s):

Sophia Adamia ◽

Jeffrey Nemeth ◽

Shruti Bhatt ◽

Sarah R Walker ◽

Natalie I Voeks ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Surface ◽

Board Of Directors ◽

Research Funding ◽

Leukemic Cells ◽

Advisory Committees ◽

Gm Csf ◽

Nsg Mice ◽

Blocking Antibodies ◽

Pdx Models

Abstract Alternative pre-mRNA splicing (AS) is a normal epigenetic phenomenon, a key regulator of gene expression, yields multiple transcripts and thus a variety of proteins from a single gene. Mutations in the spliceosome components resulting in aberrant splicing isoforms are common in AML, and other myeloid neoplasms, and may generate leukemia-specific neoantigens targetable with an antibody-drug conjugates (ADCs) or blocking antibodies. Our previous studies revealed that the FLT3 cell surface receptor is one of the most commonly misspliced genes in AML (54-63% of ~400 AML patients). We conducted cloning and sequencing analyses in AML cells and identified multiple aberrant splice-variants of FLT3 that resulted from either skipping of one or more exons or activation of cryptic splicing sites. Transfection of cDNA with three of these variants in TF-1 (AML cell line) cells resulted in expression of Flt3 variant proteins on the cell surface. We successfully generated rabbit polyclonal antiserum against a unique peptide sequence present in the most commonly expressed abnormal splice variant, which we termed Flt3Va. Immunoblots performed with the polyclonal antibody identified a ~160 kDa protein expressed by TF-1 cells transfected with FLT3Va, and the antibody did not react with untransfected TF-1 cell lysate. Using standard techniques, we generated rabbit hybridomas and evaluated the clones by flow cytometry and western blotting experiments. Based on these data, we selected one antibody clone (15-7) for further experiments. The 15-7 anti-Flt3Va rabbit monoclonal antibody identified Flt3Va protein expressed on the cell surface and within the cytoplasm of transfected TF-1 cells by flow cytometry and western blotting. However, no Flt3Va protein was detected in untransfected TF-1 cells or normal CD34+ bone marrow cells. The 15-7 antibody bound to 26 of 52 primary AML samples and 5 of 10 primagraft samples (PDX models) of human AML. Immunoblotting analyses of PDX models and patient samples confirmed binding to a protein of the expected size (130-160 kDa). Additionally, multi-parameter flow cytometry in 10 PDX models and 52 primary demonstrated that putative AML stem cells (as defined by the CD45dim, CD34, CD38, CD33, c-Kit cell surface expression) co-expressed Flt3Va antigen in 50% samples evaluated. An analysis of Flt3Va protein localization by live cell imaging showed a punctate distribution of Flt3Va on the cell surface. Furthermore, we observed that overexpression of Flt3Va in TF-1 cells led to GM-CSF growth factor independence. Analysis of TF-1 cells in the absence of GM-CSF and Flt3 ligand demonstrated constitutive activation of STAT5, an important mediator of Flt3 signaling, in Flt3Va overexpressing cells. In addition, Erk1/2 phosphorylation was also increased in Flt3Va overexpressing cells, another downstream effector of Flt3. In an effort to determine if Flt3Va+ cells had tumor repopulating ability, we sorted 0.3X10^6 Flt3Va+ and Flt3Va- cells from a PDX sample and injected the sorted populations or unsorted bulk tumor cells into NSG mice. The human cell engraftment in the mice was detected by the expression of human CD45, CD33, CD34, CD38, and c-kit antigens in the peripheral blood. In two experiments, mice injected with Flt3Va+ cells had detectable circulating leukemic cells by ~18 days after injection, while those injected with Flt3Va- cells had detectable circulating leukemic cells after the 4th week. These results suggest both Flt3Va+ and Flt3Va- cell populations are able to reconstitute leukemia after transplantation in NSG mice. However, Flt3Va+ may be expressed by an aggressive AML clone that facilitate early tumor engraftment. Overall, these studies suggest that Flt3Va is a leukemia-specific neoantigen and is an attractive potential immunotherapeutic target in AML. Proteins such as Flt3Va generated by alternative splicing are common in AML and may be targets for of novel blocking antibodies or ADCs, minimizing effects on normal tissues. Disclosures Adamia: Janssen: Research Funding. Nemeth:Janssen: Employment. Attar:Janssen: Employment. Letai:AbbVie: Consultancy, Research Funding; Tetralogic: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Research Funding. Steensma:Millenium/Takeda: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Ariad: Equity Ownership; Genoptix: Consultancy. Weinstock:Novartis: Consultancy, Research Funding. DeAngelo:Novartis: Consultancy; Ariad: Consultancy; Pfizer: Consultancy; Baxter: Consultancy; Celgene: Consultancy; Incyte: Consultancy; Amgen: Consultancy. Stone:Agios: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celator: Consultancy; Juno Therapeutics: Consultancy; Roche: Consultancy; Jansen: Consultancy; Pfizer: Consultancy; ONO: Consultancy; Sunesis Pharmaceuticals: Consultancy; Merck: Consultancy; Xenetic Biosciences: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Amgen: Consultancy; Karyopharm: Consultancy; Seattle Genetics: Consultancy. Griffin:Janssen: Research Funding; Novartis: Consultancy, Research Funding.

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Acute Myeloid Leukemia Stem Cells Cells Are Rare and Heterogeneous in Human Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v114.22.390.390 ◽

2009 ◽

Vol 114 (22) ◽

pp. 390-390 ◽

Cited By ~ 1

Author(s):

Jean-Emmanuel Sarry ◽

Kathleen Murphy ◽

Gwenn-ael Danet-Desnoyers ◽

Martin Carroll

Keyword(s):

Stem Cells ◽

Myeloid Leukemia ◽

Research Funding ◽

Leukemic Stem Cells ◽

Cell Compartment ◽

Nsg Mice ◽

Cd38 Expression ◽

Cell Fractions ◽

Limiting Dilution ◽

Acute Myeloid

Abstract Abstract 390 Human leukemic stem cells are hypothesized to be rare, restricted to phenotypically immature hematopoietic cells and capable of incomplete differentiation. However, recent work in other tumors has challenged this hypothesis. We used a robust xenotransplantation model based on NOD-SCID-IL-2Rγcnull (NSG) mice to better characterize the frequency and heterogeneity of human SCID leukemia initiating cells (SL-IC). We performed an extensive analysis on primary specimens from 11 AML patients. First, we determined the frequency of SL-IC in un-fractionated AML specimens using transplantation (i.v.) in adult NSG mice for 12 weeks and limiting dilution analysis. Our results indicate that SL-IC are rare cells in primary AML and that the frequency of SL-IC varies greatly from patient to patient: one SL-IC per 0.14 to 4.5 × 106 mononuclear cells. Normal hematopoietic stem cells (HSC) are phenotypically characterized as lineage-, CD34+, CD38- and SL-IC were initially described as being restricted to the CD34+38- compartment. To determine in this model if SL-IC are restricted to this immature cell compartment, we sorted AML cells based on surface staining for a lineage cocktail, CD34 and CD38 expression. CD38+ cells were further sorted by expression of CD45RA and CD123. In contrast to previous results, mice injected with cells from multiple different fractions engrafted including fractions with a mature cell phenotype. Although some fractions did not engraft from individual patients, engrafting cells were found in multiple compartments from all individuals studied. For each engrafting fraction, the AML cells found in mice 12 to 16 weeks post-transplant had the same phenotypic heterogeneity (defined by expression of lineage, CD34 and CD38) as observed in primary specimens consistent with either de-differentiation or lineage infidelity for these cell surface markers. Secondary transplant experiments demonstrated that each engrafting fraction contains self-renewing leukemic stem cells. In order to compare the frequencies of SL-IC in each fraction, we sorted 4 different subsets (based on lineage and CD38 expression) from 1 AML patient and performed limiting dilution analysis (LDA) in NSG mice. SL-IC were detected in each subset, but their frequency was 10-fold higher (1 in 38,000 cells) in Lin-CD38- fractions compared to other fractions and un-fractionated samples. However, as Lin-CD38- cells represent only 3% of all leukemic cells, only 34% of SL-IC were present in this fraction. By comparison, the Lin+CD38+ cell compartment has a SL-IC frequency of 1 in 106 cells but 25% of SL-IC are found in this compartment. Overall, this data demonstrate that human AML stem cells are rare but they are not restricted to immature cell fractions. Rather, leukemic stem cells can be found at different frequencies in all cell fractions. These results suggest that efforts to therapeutically target leukemic stem cells specifically may require re-evaluation. Disclosures: Carroll: Cephalon consultancy: Consultancy; Sanofi Aventis Corporation: Research Funding; Kyowa Hakko Kirin Pharmaceutical: Research Funding.

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IL1RAP Antibodies Block IL1-Induced Expansion of Primitive CML Cells and Display Therapeutic Effects in Xenograft Models

Blood ◽

10.1182/blood.v128.22.1118.1118 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1118-1118

Author(s):

Helena Ågerstam ◽

Nils Hansen ◽

Sofia Von Palffy ◽

Carl Sandén ◽

Kristian Reckzeh ◽

...

Keyword(s):

Stem Cells ◽

Research Funding ◽

Effector Cell ◽

Leukemic Cells ◽

Nsg Mice ◽

Xenograft Models ◽

Equity Ownership ◽

In Vivo Effects

Abstract Chronic myeloid leukemia (CML) is currently treated with tyrosine kinase inhibitors (TKIs) but these do not effectively eliminate the CML stem cells. As a consequence, CML stem cells persist and cause relapse in most patients upon drug discontinuation. Furthermore, no effective therapy exists for the advanced stages of the disease. Thus, there is still a need for novel treatment strategies in CML. We have previously shown that Interleukin 1 receptor accessory protein (IL1RAP), a co-receptor of IL1R1, is highly expressed on primitive CML cells and that a polyclonal IL1RAP antibody can direct natural killer (NK) cells to specifically target and destroy CD34+CD38- CML cells in an in vitro-based antibody dependent cell-mediated cytotoxicity (ADCC) assay (Järås et al, PNAS, 2010). The aim of the present study was to investigate the consequences of IL1RAP expression on primitive CML cells and the in vivo therapeutic efficacy of monoclonal IL1RAP antibodies against CML cells. Primary chronic phase (CP) CD34+ CML cells were cultured in medium supplemented with cytokines known to signal through receptor complexes involving IL1RAP. The addition of IL1 to the cultures resulted in a marked cellular expansion specifically for the primitive CD34+CD38- CML cells. Moreover, the CD34+CD38- cells showed phosphorylation of the downstream mediator of IL1-signaling NFKB. RNA-sequencing confirmed the activation of NFKB and of genes involved in cell cycling, indicating that IL1 stimulation of CD34+CD38- CML cells induced proliferation. Upon addition of an IL1RAP antibody capable of blocking IL1-signaling to the suspension cultures, the IL1-induced expansion and NFKB phosphorylation of CD34+CD38- CML cells was suppressed. Interestingly, both the IL1RAP expression and the response to IL1 as measured by NFKB phosphorylation was retained during TKI treatment of the cells. To assess the in vivo effects of IL1RAP antibodies in CML models, we first engrafted NOD/SCID mice with BCR/ABL1 expressing BV173 cells and treated the mice with the monoclonal IL1RAP antibody mAb81.2. Mice receiving treatment with mAb81.2 displayed a prolonged survival compared to controls, accompanied by reduced levels of leukemic cells in the BM. In vitro studies showed that mAb81.2 lacked a direct effect on cellular expansion or apoptosis. Instead, the IL1RAP antibody could direct NK cells to elicit killing of the leukemic cells, thereby suggesting effector cell mediated mechanisms to be an important in vivo mode-of-action. To validate the in vivo effects on primary CML cells, we next engrafted CP or blast phase (BP) CML cells into immunodeficient mice. Following engraftment of CP CD34+ CML cells into NSG mice and subsequent treatment with mAb81.2, a reduction of human myeloid cells was observed, suggesting that the treatment targeted the leukemic graft. Importantly, mAb81.2 treatment also reduced the levels of candidate CD34+CD38-IL1RAP+ CML stem cells. Finally, BP CML cells were engrafted into NOD/SCID mice that have a more intact effector cell function compared to NSG mice. Following treatment with mAb81.2 a significant reduction of leukemic cells in the BM as well as in the periphery was observed compared to control mice. Importantly, secondary transplantations revealed a therapeutic effect also on the BP CML stem cells. In vitro ADCC assays confirmed that CML BP cells, including a sample with the highly TKI-resistant T315I mutation, could be targeted and killed using mAb81.2. We conclude that IL1RAP antibodies can suppress IL1-induced expansion of primitive CML cells and that in vivo administration of IL1RAP antibodies in CML xenograft models has anti-leukemic effects that extend to the CML stem cells. These results show that an antibody-based therapy against IL1RAP can be used to efficiently target CML stem cells. Disclosures Richter: BMS: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Järås:Cantargia AB: Equity Ownership. Fioretos:Cantargia AB: Equity Ownership.

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A Phase I/II Trial of Plerixafor/G-CSF Combined with IV Bu/Flu Conditioning Regimen In AML/MDS Patients Undergoing Allogenic Stem Cell Transplantation

Blood ◽

10.1182/blood.v116.21.2358.2358 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2358-2358 ◽

Cited By ~ 2

Author(s):

Marina Konopleva ◽

Zeng Zhihong ◽

Rui-Yu Wang ◽

Peter F. Thall ◽

Gloria McCormick ◽

...

Keyword(s):

Stem Cells ◽

Phase I ◽

Research Funding ◽

Fold Increase ◽

Leukemic Cells ◽

Leukemic Stem Cells ◽

Preparative Regimen ◽

Relapsed Disease ◽

Fish Cells ◽

Over Time

Abstract Abstract 2358 Allogeneic stem cell transplantation (alloSCT) is an effective treatment for pts with acute myeloid leukemia (AML) in first remission. However, only 10–20% of pts with relapsed disease achieve a durable remission. Microenvironment/leukemia interactions play a major role in chemoresistance of leukemic stem cells residing in the bone marrow niches. In pre-clinical in vivo leukemia models, inhibition of chemokine receptor CXCR4 results in mobilization of leukemic cells into circulation and sensitization to chemotherapy. We hypothesized that mobilization of leukemic stem cells by CXCR4 inhibition and G-CSF will result in improved anti-leukemia activity of a standard preparative regimen followed by alloSCT. In this Phase I/II study, G-CSF is administered at a standard dose beginning on day -9 daily for 6 days, and the CXCR4 inhibitor plerixafor (Mozobil®) from day -7 at one of the 4 dose levels 0 (control), 0.08, 0.16, or 0.24 mg/kg, 8 hours prior of each four daily doses of a standard preparative regimen (Fludarabine, 40mg/m2 and IV Busulfan, 130mg/m2, days -6 through -3). Twenty seven pts have been enrolled in the study to date with a median age of 48 yrs (range 25–65). Baseline characteristics include 13 pts (48%) with de novo AML, 6 (22%) with secondary AML, 5 with MDS and 3 with CML. Among the 24 AML/MDS pts, 14 (58%) had intermediate and 10 (42%) poor risk cytogenetics. Twelve pts (50%) had primary refractory AML, 5 were in 1st or 2nd relapse, 2 were untreated, 3 were in CR1 and 2 in CR2. The source of stem cells was sibling donor in 16 and unrelated donor in 11. After phase I plerixafor dose escalation in 16 pts, 11 pts received 0.24 mg/kg in Phase II. Common grade ≥ 3 adverse events which consisted primarily of neutropenic fever, infections, or rash were seen in 24/27 (89%) pts. There were no toxicities ascribed to the G-CSF/plerixafor component of the regimen. No evidence of significant delays in neutrophil (ANC >500/mm3, median 12.5d, range 10–19) or platelet recovery (plt >20k/mm3, median 12d, range 9–74d) were observed. Grade I-II GVHD was seen in 10/27 pts (37%), with no occurrences of Grade III-IV GVHD. Of the 19 pts with active disease at study entry, 18 achieved a CR. Treatment failure was due to persistent disease in 1 pt (4%), relapsed disease in 10 pts (37%) and early death due to complications from intracranial hemorrhage in 1 pt (4%). Median progression-free survival (PFS) for all pts was 26.6 wks (95%CI: 18.1–33.9 wks) and 15.7 wks (95% CI: 12.1–26.6 wks) in relapsed pts. Median follow-up for all study pts was 19.14 wks (range: 0.7–54.6 wks). Correlative studies analyzed from 16 pts enrolled in the Phase I portion of the trial demonstrate that G-CSF/plerixafor mobilizes CD34+ cells, with the mean fold increase of 5.9-fold at 0.08 mg/kg plerixafor; at 0.16 mg/kg, 13-fold; and at 0.24 mg/kg, 14.2-fold. Based on fitted longitudinal linear mixed models, G-CSF had a significant effect on cell mobilization over time (WBC and CD34+). In contrast, plerixafor at the doses of 0.16 and 0.24 mg/kg was significantly associated with increased cellular CXCR4 expression levels and with mobilization of CXCR4+ cells over time (p<0.02). To determine relative proportion of mobilization of leukemic and non-leukemic cells, we performed FISH analysis on peripheral blood samples from pts with informative cytogenetic abnormalities (n=12). Both, FISH+ and FISH- cell counts increased from day -8 to day -6 and remained relatively stable or decreased thereafter (between day -6 and day -3), with the initial increase much larger for the plerixafor dose level 0.16 mg/kg (mean fold increase FISH+, 24.3; FISH-, 10.3). Over time, the relative increase of FISH+ cells was significantly higher than that of FISH- cells, indicating preferential mobilization of cytogenetically abnormal leukemic over normal cells (p=0.005). In summary, G-CSF/plerixafor is safe in combination with the established IV busulfan/fludarabine preparative regimen for alloSCT in pts with advanced disease. Our data indicate preferential mobilization of clonal leukemic over normal cells. The objective of the ongoing Phase II study is to determine if the combination of G-CSF/plerixafor with busulfan/fludarabine improves PFS compared to historical controls receiving busulfan/fludarabine alone. We hypothesize that interventions disrupting stroma-leukemia interactions may enhance chemosensitivity and therefore the therapeutic efficacy in hematological malignancies. Disclosures: Konopleva: Genzyme: Research Funding. Off Label Use: Plerixafor for transplant in AML. Andreeff: Genzyme: Consultancy, Research Funding. Champlin: Genzyme: Consultancy, Research Funding.

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Selective Targeting of Leukemic Over Normal Stem Cells by the Serotonin Receptor Antagonist SB-216641

Blood ◽

10.1182/blood.v118.21.1884.1884 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1884-1884

Author(s):

Alissa R. Kahn ◽

Kimberly A. Hartwell ◽

Peter G. Miller ◽

Benjamin L. Ebert ◽

Todd R. Golub ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Lines ◽

Serotonin Receptor ◽

Leukemic Cells ◽

Leukemic Stem Cells ◽

Nsg Mice ◽

Cell Kill ◽

Serotonin Receptor Antagonist

Abstract Abstract 1884 Acute myeloid leukemia (AML) is a common and aggressive hematologic malignancy affecting both children and adults which continues to have high mortality rates as well as high morbidity from toxic therapies. New treatments are needed to improve cure rates and decrease morbidity. A niche-based high throughput screen done in a murine system identified candidate small molecules potentially toxic to leukemic stem cells (LSCs) while sparing normal hematopoietic stem cells (HSCs) and bone marrow stroma (Hartwell KA, Miller, PG et al., in preparation). One such compound, SB-216641, demonstrated dose-dependent activity against leukemia in both a cell autonomous and non-autonomous manner, by modifying niche–based support. SB-216641 is a selective serotonin receptor antagonist specific for the 5-HT1B receptor, highlighting a pathway not previously investigated in the context of AML or leukemia stem cell biology. We examined the effects of this candidate small molecule on 7 human primary AML samples. CD34+ cells were isolated from these samples with immunomagnetic beads. Using the colony forming assay to assess kill of progenitor cells, all samples had ≥99% cell kill at 25 μM (10 times the IC-50 found in the murine system). We then assessed the compound's effect on LSCs using the cobblestone area forming cell (CAFC) assay, a standard in vitro stem cell assay. The leukemic cells were pulse treated for 18 hours and washed to remove residual SB-216641 prior to placement on MS-5 murine stroma and therefore only the direct effect on the leukemic cells was measured in this assay. CAFCs were read out at week 5, or week 2 when the sample was FLT3-ITD+ (Chung KY et al, Blood 2005, Vol 105, 77–84). We first tested five samples at 25 μM. All samples formed cobblestone areas in the control setting (46–200 CAFCs/106 cells plated). Four samples had no CAFC formation with SB-216641 and the remaining sample had >95% decrease in CAFC formation. We then performed serial dilutions using the CAFC assay in the human primary samples as well as in HSCs derived from cord blood to obtain the IC-50 for human AML and to ensure that our differential cell kill of LSCs versus normal HSCs held true in the human samples. IC-50 for the human primary leukemias was found to be 630 nanomolar and at 10 μM all leukemic samples were fully killed with 100% survival of normal human HSCs [see figure 1]. As a confirmatory study, using HL60 and U937 human AML cell lines transduced with GFP-luciferase, 500 cells were preincubated with SB-216641 at 25 μM or DMSO control and then injected IV into Nod Scid IL2R-gamma null (NSG) mice and imaged at 5 weeks. In both cell lines, the control mice had engraftment and the mice that received treated cells had no engraftment. HL60 cells were then preincubated with SB-216641 at lower doses (10 and 5 μM) and injected into NSG mice and imaged at 3 weeks. Again, the control mice had engraftment and the mice that received treated cells had no engraftment.Figure 1.Figure 1. 5-HT1B receptor antagonists have not previously been known to be active against AML or leukemic stem cells. Some hematopoietic cells including platelets express serotonin receptors and T-cells specifically have been found to express the 5-HT1b receptor. Selective 5-HT1B receptor antagonists have found to have apoptotic effects in vitro against cell lines of other cancers and may be involved in MAP kinase and P13K/Akt signaling pathways. SB-216641 is a highly promising compound which warrants further investigation. Its high toxicity to LSCs and sparing of normal HSCs make it appealing for possible clinical use in the future. Disclosures: No relevant conflicts of interest to declare.

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Targeting IL3 Receptor in Chronic Myeloid Leukemia.

Blood ◽

10.1182/blood.v114.22.2172.2172 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2172-2172 ◽

Cited By ~ 1

Author(s):

Olga Frolova ◽

Rui-Yu Wang ◽

Borys Korchin ◽

Julie C. Watt ◽

Jorge Cortes ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Clinical Trials ◽

Cell Death ◽

Myeloid Leukemia ◽

Blast Crisis ◽

Leukemic Cells ◽

Leukemic Stem Cells ◽

Induction Of Apoptosis

Abstract Abstract 2172 Poster Board II-149 Despite the great success of imatinib therapy in chronic myeloid leukemia (CML), the presence of a residual leukemic clone is detectable in a proportion of patients with CML. Further, patients with accelerated and blast phase of the disease respond poorly to imatinib. Imatinib and other potent tyrosine kinase inhibitors (TKIs) have limited activity against CD34+38- leukemic stem cells, necessitating the need for novel agents capable of eradicating highly resistant CML stem cells. Expression of IL3 receptor, CD123, was demonstrated on CD34+CD38- leukemic stem cells in AML (Jordan et al., Leukemia, 14: 1777, 2000) and CML (Neering et al., Blood, 110: 2578, 2007; Florian et al., Leuk Lymphoma, 47: 207, 2006) and has been shown to be an effective therapeutic target in pre-clinical AML models (Jin et al,,Cell Stem Cell, 5:31, 2009; Feuring-Buske et al., Cancer Res, 62: 1730, 2002). However, its role in CML stem cells has not been investigated. In this study, we examined expression of CD123 on CML progenitor cells and the therapeutic potential of the CD123 targeting agents, DT388IL3 and DT388K116W, both recombinant IL3-diphtheria toxin (DT) conjugates in in vitro and in vivo CML models. DT388IL3 has been shown to eradicate NOD/Scid-initiating AML stem cells and is currently undergoing Phase I/II clinical trials in AML and MDS. DT388K116W is a new DT fusion protein with high binding affinity to the IL3 receptor that demonstrated high potency anti-leukemic activity. These novel agents are directed to the leukemia stem cell surface, trigger receptor-mediated endocytosis, inhibit protein synthesis, and cause programmed cell death. In a series of nine primary CML samples (five from patients with chronic phase CML and four from patients in blast crisis), CD123 was expressed in 86%±5.7% of CD34+CD38- progenitor cells as determined by flow cytometry. Notably, 86%±3.4% of FACS-sorted CD34+38-123+ cells from 7 primary CML samples were Bcr-Abl(+) by fluorescent in situ hybridization analysis, confirming the leukemic origin of this cell population. We next examined the cytotoxic activity of DT-IL3 agents in KBM5 cells and in primary leukemic blasts. DT388K116W induced a dose-dependent decrease in viability and induction of apoptosis in KBM5 (44.6±4.3% apoptotic cells at 10μg/mL, p≤0.001) and in primary CML cells (69.5±15 % apoptosis, n=4, p=0.04) as determined by viable cell counts and annexin V flow cytometry at 72 hours. DT388K116W induced a greater degree of cell death compared to DT388IL3 in KBM5 cells (44.6% vs 21.3%, p=0.009). In two primary CML samples DT-IL3 agents reduced the absolute numbers of CD34+CD38-CD123+ cells by induction of apoptosis (DT388IL3, by 69% (sample#1) and 21% (sample#2); DT388K116W, by 71% and 62%, respectively). Importantly, combination of imatinib with DT-IL3 further enhanced the apoptotic rate in KBM5 (p=0.0001) and primary leukemic cells (n=3, p=0.035). To examine anti-leukemic activity of these agents in vivo, NOD/Scid/IL2Rγ-KO mice were transplanted with leukemic cells from primary myeloid blast crisis CML. After engraftment of the leukemic cells documented by CD45 flow cytometry in murine blood 20 days post transplantation, mice were left untreated or received 5-day intraperitoneal administration of DT388IL3 or DT388K116W at 0.2mg/kg. These IL3 receptor-targeted agents significantly prolonged survival of treated mice compared to vehicle control (median survival: vehicle= 37, DT388IL3 = 48, DT388K116W = 57 days; p= 0.0005) and reduced leukemia burden as detected by CD45 flow cytometry. These data indicate that the IL3 receptor is highly expressed on CD34+38- Bcr-Abl(+) CML stem cells and represents an exciting new and feasible target for therapeutic intervention. Moreover, DT-IL3 conjugates represent a novel therapeutic modality for selective targeting of highly resistant CML stem cells. DT-IL3 agents, alone or in combination with TKIs, might benefit CML patients by reducing/eliminating leukemic stem cells, a concept to be tested in the future clinical trials. Disclosures: No relevant conflicts of interest to declare.

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The Leukemic Stem Cell (LSC) in Ph+ CML Is a CD34+/CD38−/Lin− Cell That Co-Expresses Dipeptidylpeptidase IV (CD26) and Disrupts LSC-Niche Interactions by Degrading the CXCR4 Ligand SDF-1α

Blood ◽

10.1182/blood.v118.21.961.961 ◽

2011 ◽

Vol 118 (21) ◽

pp. 961-961 ◽

Cited By ~ 1

Author(s):

Harald Herrmann ◽

Irina Sadovnik ◽

Sabine Cerny-Reiterer ◽

Katharina Blatt ◽

Viviane Ghanim ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Progenitor Cells ◽

Research Funding ◽

Leukemic Cells ◽

Regulation Of Expression ◽

Phenotypic Properties ◽

Aberrant Expression ◽

Dipeptidylpeptidase Iv ◽

Nsg Mice

Abstract Abstract 961 In Philadelphia-positive (Ph+) chronic myeloid leukemia (CML), leukemic stem cells (LSC) supposedly reside in a CD34+/CD38−/Lin− fraction of the leukemic clone. However, little is known about phenotypic properties of LSC in CML. We screened for novel LSC markers and targets in CML by gene chip studies and extensive flow cytometry analyses using monoclonal antibodies against various surface antigens (n=50). A total number of 240 bone marrow or peripheral blood samples (CML, n=95; AML, n=103; CMML, n=10, control marrow, n=32) were examined. In common with normal SC, CD34+/CD38− CML LSC were found to co-express the homing-receptor CD44, G-CSF-R (CD114), KIT (CD117), FLT3 (CD135), and CXCR4 (CD184). Similar to LSC in AML and CMML, CML LSC were found to display higher levels of Siglec-3 (CD33) and IL-3RA (CD123). Most significantly, however, we found that in contrast to normal CD34+/CD38− stem cells, CD34+/CD38− CML LSC aberrantly express IL-2RA (CD25), dipeptidylpeptidase IV (DPPIV=CD26), and IL-1RAP. In other myeloid leukemias (AML, CMML), CD34+/CD38− LSC also co-expressed CD25, but usually did not express CD26 or IL-1RAP. Whereas CD26 was expressed almost invariably on CD34+/CD38− cells in all CML patients tested, the surface enzyme was neither detectable in more mature CD34+/CD38+ progenitor cells nor on CD34+/CD38− stem cells in reactive bone marrow or healthy controls. During successful treatment with imatinib or nilotinib (patients examined at CCyR and/or MMR), CD34+/CD38− stem cells invariably showed a ‘normal' phenotype (CD25−, CD26−, IL-1RAP−), whereas in relapsing CML, CD34+/CD38− cells were again found to co-express CD25 and CD26. Sorted Lin−/CD26− stem cells obtained from CML patients (at diagnosis) engrafted irradiated NOD-SCID IL-2Rγ−/− (NSG) mice with normal multilineage BCR/ABL1− hematopoiesis, whereas Lin−/CD26+ stem cells were found to engraft NSG mice with BCR/ABL+ cells. We next examined the regulation of expression of CD25 and CD26 on CML LSC. Whereas expression of CD25 was found to depend on BCR/ABL1 and STAT5-activity, CD26 expression was found to be expressed independent of BCR/ABL1 and independent of STAT5-signaling. In a next step, we examined the potential function of CD26 on CML LSC. In these studies, CD26 was identified as a target-enzyme disrupting the niche-related SDF-1α/CXCR4 axis by degrading SDF-1α. Correspondingly, CD26-targeting gliptins (sitagliptin, 1 μM; vildagliptin, 1 μM) were found to revert recombinant DPPIV/CD26-induced or cellular CD26-induced inhibition of SDF-1α-mediated in vitro migration of CD26+ leukemic cells. Finally, we found that in a CML patient treated with nilotinib, in whom uncontrolled diabetes mellitus required therapy with saxagliptin, BCR/ABL1 levels (in percent of ABL according to IS) that were found to increase before the start of saxagliptin (IS before saxagliptin: 1.6 [-4 months], 2.3 [-3 months], and 2.4 [at therapy-start]), decreased over time during saxagliptin-therapy (IS: 1.0 [+1 month], 1.0 [+3 months], 0.8 [+5 months]). Together, the CML-initiating LSC is a CD34+/CD38− cell that exhibits aberrant expression of IL-1RAP, CD25, and DPPIV/CD26. All three markers may be useful for purification of CML LSC. DPPIV/CD26 appears to be a functionally and pathogenetically relevant antigen that may facilitate niche-independent uncontrolled redistribution and thus extramedullary spread of LSC and LSC-derived progenitor cells in CML. Whether CD26 can be developed as a novel therapeutic target in CML is currently under investigation. Disclosures: Valent: Novartis: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding.

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Inhibiting the Mitochondrial Sulfhydryl Oxidase Alr Reduces Cox17 and Alters Mitochondrial Cristae Structure Leading to the Differentiation of AML and Stem Cells

Blood ◽

10.1182/blood.v130.suppl_1.881.881 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 881-881

Author(s):

Danny V. Jeyaraju ◽

Veronique Voisin ◽

Changjiang Xu ◽

Samir H. Barghout ◽

Dilshad H. Khan ◽

...

Keyword(s):

Stem Cells ◽

Cord Blood ◽

Respiratory Chain ◽

Research Funding ◽

Annexin V ◽

Leukemic Cells ◽

Copper Chaperone ◽

Leukemic Stem Cells ◽

Sulfhydryl Oxidase

Abstract The vast majority of mitochondrial proteins are encoded in the nucleus, translated in the cytoplasm and then imported into the mitochondria. A subset of these imported proteins are folded into their mature and functional forms in the mitochondrial inter-membrane space (IMS) by the Mitochondrial IMS Assembly (MIA) pathway. We found that genes encoding substrates of the MIA pathway are over-expressed in leukemic stem cells compared to bulk AML cells. Therefore, we assessed the effects of inhibiting the MIA pathway in AML. We knocked down the mitochondrial sulfhydryl oxidase ALR, a key regulator of the MIA pathway. Knockdown of ALR with shRNA reduced the growth and viability of OCI-AML2, TEX and NB4 leukemia cells. In addition, knockdown of ALR reduced the engraftment of TEX cells into mouse marrow, demonstrating an effect on the leukemia initiating cells. The small molecule selective ALR inhibitor, MitoBloCK-6, mimicked the effects of ALR knockdown and killed AML cells with an IC50 of 5-10 μM. MitoBloCK-6 preferentially reduced the clonogenic growth of primary AML cells (n=4/5) over normal hematopoietic cells (n=4). However, only 3/10 bulk AML cells were sensitive to MitoBloCK-6 induced cell death by Annexin V/PI staining. Next, we evaluated the efficacy and toxicity of ALR inhibition in vivo . We injected primary AML cells or normal cord blood into the femurs of mice and then treated mice with MitoBloCK-6 (80 mg/kg i.p. 5 of 7 days x 2 weeks). MitoBloCK-6 strongly reduced the engraftment of primary AML samples but did not affect engraftment of cord blood. In secondary transplants, MitoBloCK-6 also targeted leukemic stem cells. No change in mouse body weight, serum chemistries, or organ histology was seen. As expression levels of ALR substrates are increased in AML stem cells, we assessed the effects of ALR inhibition on differentiation in AML. Genetic or chemical inhibition of ALR induced the differentiation of AML cells as evidenced by increased CD surface marker expression and increased non-specific esterase. In addition, ALR inhibition was preferentially cytotoxic towards undifferentiated cells and stem cells over differentiated bulk AML cells. Interrogation of the effects of ALR inhibition on its substrates identified the mitochondrial copper chaperone, Cox17 as the primary downstream target in leukemic cells. Inhibition of ALR selectively reduced levels of Cox17 protein and altered mitochondrial cristae structure. Validating the functional importance of these findings, knockdown of Cox17 phenocopied ALR inhibition and reduced AML proliferation, induced differentiation of AML cells, and altered mitochondrial cristae structure, without changing respiratory chain activity or oxygen consumption. Of note, cristae remodelling independent of respiratory chain function has been recently implicated in cellular differentiation and in yeast, Cox17 regulates the cristae organizing machinery. Thus, we have identified novel mechanisms by which mitochondrial pathways regulate the fate and differentiation of AML cells and stem cells Moreover, inhibition of ALR may be a novel therapeutic strategy to promote the differentiation of AML cells and stem cells. Disclosures Schimmer: Takeda Pharmaceuticals: Research Funding; Medivir: Research Funding; Novartis Pharmaceuticals: Honoraria.

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871 Construction and evaluation of interleukin 3 (IL3)-zetakine redirected cytolytic T Cells for the treatment of CD123 expressing acute myeloid leukemia

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.871 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A912-A912

Author(s):

Rebecca Moeller ◽

Julian Scherer ◽

Sadik Kassim

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Myeloid Leukemia ◽

Cell Activation ◽

Jurkat Cells ◽

Leukemic Stem Cells ◽

Cd69 Expression ◽

Acute Myeloid

BackgroundAcute Myeloid Leukemia (AML) is an aggressive bone marrow malignancy, characterized by the presence of leukemic blasts in the peripheral blood of patients. Poor AML prognoses1 are largely attributable to high rates of disease relapse, of which CD123+ leukemic stem cells (LSCs) are the primary cause.2 3 CD123, the alpha-chain of the IL3 cytokine receptor,6 has been identified as a favorable therapeutic AML target, overexpressed in both LSCs and blasts.4 5 We sought to direct T cells to CD123+ AML cells via cell surface tethered IL3 (termed ”IL3-zetakine”).7 The use of a zetakine instead of a chimeric antigen receptor (CAR) construct enables structure-guided site-directed mutagenesis to increase binding affinity and alter target cell signaling without detrimental T cell hyperactivation.MethodsZetakine constructs were designed using IL3 sequences bound to a transmembrane domain and intracellular costimulatory and CD3z signaling domains. The constructs were transduced into Jurkat cells with lentiviral vectors (LVV). T cell activation via CD69 expression was assessed via flow cytometry of sorted IL3 zetakine-positive Jurkat cells after co-culture with MOLM13 AML cells. Lead constructs were selected based on initial transduction percentage and activation response. In vitro functionality of each IL3 zetakine was tested with LVV transduced primary T cells by flow cytometry.ResultsZetakine constructs yielded a wide range of transduction percentages in Jurkat cells (0 – 98%) prior to sorting. In co-cultures with CD123+ MOLM13 AML cells, Jurkat cells expressing wildtype IL3 constructs lacking a costimulatory domain induced the highest level of CD69 expression (18.7% CD69+ T cells) in an antigen-specific manner (5.3-fold increase of CD69+ T cells over those cultured with MOLM13 CD123KO cells). The K110E mutant IL3 was reported to exhibit a 40-fold increased affinity over wildtype,8 but it showed no detectable zetakine function. However, additional mutant IL3 zetakines increased Jurkat cell activation up to 5.8-fold. Antigen-specific increases in CD69, as well as CD25, surface expression were also observed with zetakine-transduced primary T cells co-cultured with MOLM13 cells, in addition to target cell killing comparable to antibody-based CD123CAR T-cells.ConclusionsThis work establishes IL3 zetakines as a viable alternative to traditional CD123-targeted CAR constructs. Structure-guided IL3 zetakine mutants with altered affinity and activation profiles will further our understanding of CD123-specific cytotoxicity modulation without inducing acute T cell hyperactivation and exhaustion. These results indicate the ability of IL3 zetakine-expressing T cells to kill CD123-expressing AML cells and illustrate the potential of this novel class of therapeutics.ReferencesGanzel C, et al. Very poor long-term survival in past and more recent studies for relapsed AML patients: the ECOG-ACRIN experience. American journal of hematology 2018:10.1002/ajh.25162.Shlush LI, et al. Tracing the origins of relapse in acute myeloid leukaemia to stem cells. Nature 2017;547(7661):104–108.Hanekamp D, Cloos J, Schuurhuis GJ. Leukemic stem cells: identification and clinical application. International Journal of Hematology 2017;105(5):549–557.Bras AE, et al. CD123 expression levels in 846 acute leukemia patients based on standardized immunophenotyping. Cytometry part B: Clinical Cytometry 2019;96(2):134–142.Sugita M, Guzman ML. CD123 as a therapeutic target against malignant stem cells. Hematology/Oncology clinics of North America 2020;34(3):553–564.Mingyue S, et al. CD123: a novel biomarker for diagnosis and treatment of leukemia. Cardiovascular & Hematological Disorders-Drug Targets 2019;19(3):195–204.Kahlon KS, et al. Specific recognition and killing of glioblastoma multiforme by interleukin 13-zetakine redirected cytolytic T cells. Cancer Res 2004;64(24):9160–6.Bagley CJ, et al. A discontinuous eight-amino acid epitope in human interleukin-3 binds the alpha-chain of its receptor. J Biol Chem 1996;271(50):31922–8.

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