Targeting Cell-Bound MUC1 on Myelomonocytic and Monocytic Leukemias and Leukemic Stem Cells: Therapeutic Implications

Abstract Monocytic neoplasms comprise a heterogeneous group of hematologic malignancies including chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), acute myelomonocytic and monocytic leukemia (AML-M4 and AML-M5), and monocytic sarcoma. Monocytic or granulomonocytic hyperplasia is a finding frequently-if not invariably-shared by these different entities, as is a poor therapeutic outcome in the absence of hematopoietic stem cell transplantation. Cell surface molecules aberrantly expressed or overexpressed by leukemic cells represent potential disease-specific therapeutic targets. MUC1, a polymorphic type I high molecular weight glycoprotein represents such a molecule. MUC1 consists of an extracellular domain containing 20 to 125 tandem repeats of a 20 amino acid-long sequence, followed by a transmembrane domain and a short cytoplasmic tail leading to intracellular signaling. Cleavage of MUC1 yields two unequal chains: a large extracellular alpha subunit containing the tandem repeat array bound in a strong non-covalent interaction to a smaller beta subunit containing the transmembrane and cytoplasmic domains. Essentially all anti-MUC1 antibodies reported to date target the highly immunogenic tandem repeat of the MUC1 alpha chain. Because the alpha chain binds the cell-bound domains of MUC1 only intermittently in an 'on-and-off' manner, agents directed against the alpha chain will not effectively target MUC1+ cells. In contrast, the MUC1 SEA domain represents a stable structure fixed to the cell surface at all times. We therefore generated mAbs that specifically recognize the cell-bound MUC1 SEA domain. One of them, a partially humanized murine mAb termed DMB-5F3 was used to examine the expression of MUC1 on AML cells by flow cytometry. A series of twenty-two AML samples (blood-derived n=12; bone marrow-derived n=10; AML0=2, AML1=2, AML2=10, AML4=1, AML5=5, AML6=2) collected either at the time of diagnosis or at relapse were analysed for MUC1 expression by flow cytometry. A murine mammary tumor cell line stably transfected with human MUC1 DNA served as control. Blasts cells from 5 AML samples highly expressed MUC1, and significantly, all were of monocytic or myelomonocytic lineage (AML4=1, AML5=4). Leukemic stem cells (CD34pos or CD34neg linneg) from the MUC1+ AMLs were examined and likewise found to express MUC1. In addition, AML cell lines MV411, MOLM14, and SHI-1 derived from monocytic leukemic lineage clearly expressed cell surface MUC1, while non- monocytic leukemic cell lines U937, K562, and HL60 had little or no expression. Normal monocytes and monocytes derived from patients with activated monocytosis were also found to express MUC1. Based on these findings we examined MUC1 expression in a series of myelomonocytic leukemia (CMML and JMML). In fifteen CMML samples examined (type 1 n=11, type 2 n=4) (blood n=7, BM n=7) 92%-100% (median 99.7%) of CD14+CD56+ CMML cells bound mAb DMB-5F3 to cell-surface MUC1. CD14+CD16+CD56+ blast cells from 2 pts with JMML were also found to express MUC1 (between 64% and 71 % positive). Based on these findings we conclude that expression of MUC1 is restricted to monocytic and myelomonocytic leukemias and that MUC1 represents an effective target for leukemic immunotherapy. Significantly, anti-MUC1 mAb also targets monocytic leukemic stem cells, reinforcing its therapeutic potential. The fact that the anti-MUC1 antibody DMB-5F3 can enter cells and thereby ferry Ab-bound toxin opens the way for us to demonstrate leukemic cell killing with anti-MUC1 mAb-immunotoxin conjugates. Disclosures Moreau: Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Leukemic Cell Expressed CTLA-4 Suppresses T Cells Via Down-Modulation of CD80 By Trans-Endocytosis

Blood ◽

10.1182/blood.v128.22.3221.3221 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3221-3221 ◽

Cited By ~ 1

Author(s):

Priscilla Do ◽

Kyle A. Beckwith ◽

Larry Beaver ◽

Brittany G. Griffin ◽

Xiaokui Mo ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

B Cells ◽

Cell Surface ◽

Board Of Directors ◽

Tumor Cells ◽

Research Funding ◽

Leukemic Cell ◽

Advisory Committees

Abstract The function of CTLA-4 on non-T cells is largely ignored and currently ill defined despite rapidly growing interest in targeting this immune checkpoint protein in several cancers. While anti-CTLA-4 therapy is proposed to work through inhibition of the immunosuppressive effect of CTLA-4 on T cells, multiple examples of non-T cell expressed CTLA-4 have been reported. These cells include tumor cells of hematological and non-hematological origin and normal B cells. In this study, we have defined a novel immune suppressive role for non-T cell, tumor expressed CTLA-4 in Chronic Lymphocytic Leukemia (CLL). We have detected by microarray that CTLA-4 is in the top 5 most differentially expressed genes between pooled samples of healthy donor normal B cells (N=6) and pooled CLL leukemic B cells (N=5). Upregulation of CTLA-4 by CLL B cells compared to normal B cells was validated by RT-qPCR and flow cytometry. CTLA-4 was predominantly intracellular (42/46 CTLA-4+) and not on the cell surface (2/48 CTLA-4+) in primary CLL samples. B cell activating factors (CD40L, PMA/Ionomycin, LPS, IL4, LPS+IL4, CD40L+IL4, CpG, and anti-IgM) could not induce surface expression of CTLA-4; however, co-culture with anti-CD3/anti-CD28 or ConA activated T cells (autologous or allogeneic) resulted in detectable CTLA-4 on the cell surface of leukemic B cells. This induction did not occur with resting T cells. This finding suggests a role for CTLA-4+ tumor cells in sites of T cell activation, such as the lymph node, a site of leukemic cell proliferation in CLL. To mechanistically study leukemic B cell expressed CTLA-4, we generated CLL-derived Mec1 and OSU-CLL that inducibly express CTLA-4 upon doxycycline (dox) treatment. Mec1 and OSU-CLL cells highly express the ligands for CTLA-4, CD80 and CD86. Dox-induction of CTLA-4 resulted in decreased expression of Mec1 and OSU-CLL expressed CD80, a critical T cell co-stimulatory protein (N=3). Blockade of CTLA-4 using the anti-CTLA-4 therapeutic antibody, Ipilimumab, could restore CD80 on Mec1 and OSU-CLL cells (N=3). Because T cell-expressed CTLA-4 has been previously shown by others to down-modulate CD80 via trans-endocytosis, we co-cultured CTLA-4+ Mec1 and CTLA-4+ primary CLL cells with stably transfected CD80-GFP or CD86-GFP Hek293 cell lines to assess uptake of CD80/CD86 into CTLA-4 expressing tumor cells as the mechanism of CD80 down-modulation. Transfer of CD80-GFP and CD86-GFP was detected by flow cytometry in primary CLL cells and the Mec1 cell line, consistent with the ability of T cell expressed CTLA-4 to trans-endocytose CD80 and CD86. Furthermore, uptake of CD80-GFP or CD86-GFP by primary tumor cells was CTLA-4 dependent, demonstrated by inhibition of GFP uptake in the presence of Ipilimumab. Following determination of decreased CD80, we found that co-culture of primary T cells with Mec1 CTLA-4+ cells resulted in decreased IL2 production measured by Cytokine Bead Array. The loss of IL2 signified decreased co-stimulation as a result of tumor expressed CTLA-4. Studies are ongoing regarding dependence on CD80 or CD86. A minor subset of T cells, Tregs, are known to exert profound immunosuppressive effects through their expression of CTLA-4. Due to our results, tumor expressed CTLA-4 has an overlapping function with Treg CTLA-4, and it is imperative that we define the immunosuppressive effects as, in patients, the leukemic cells may comprise a much larger proportion of white blood cells than T cells. Efforts are now underway to address the effect of tumor expressed CTLA-4 in suppressing anti-tumor immunity in vivo utilizing a novel mouse model. Suppression of T cells by tumor expressed CTLA-4 is a novel finding that is broadly applicable to fields within and outside of cancer research as the pathway and mechanism described here are potentially applicable to CTLA-4 in diverse disease contexts and to the general biology of CTLA-4. [Funding: This work was supported by P01 CA95426. PD received the Pelotonia Graduate Fellowship. Any opinions, findings, and conclusions expressed in this material are those of the author(s) and do not necessarily reflect those of the Pelotonia Fellowship Program] Disclosures Jones: AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics, LLC, an AbbVie Company: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Leukemic Stem Cells Persistence Measured By Multiparametric Flow Cytometry Is a Biomarker of Poor Prognosis in Adult Patients with Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2019-128025 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2688-2688

Author(s):

Francesco Buccisano ◽

Raffaele Palmieri ◽

Maria Irno Consalvo ◽

Alfonso Piciocchi ◽

Luca Maurillo ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Poor Prognosis ◽

Leukemic Stem Cells ◽

Advisory Committees ◽

Multiparametric Flow Cytometry ◽

Significant Difference ◽

Acute Myeloid

Introduction: Despite the recent advances in chemotherapy regimens, relapse still substantially affects prognosis of intensively treated adult acute myeloid leukemia (AML) patients. There is growing evidence that a residual populations of leukemic cells may survive chemotherapy and outgrow, eventually causing relapse. These chemo-resistant cells are particularly abundant in the fraction of leukemic stem cells (LSC), which are endowed with pronounced self-renewal properties allowing to initiate and maintain leukemic clone. These cells can be detected, by high sensitivity multiparametric flow-cytometry (MFC), in the CD34+/CD38- fraction of the leukemic populations and can be distinguished from normal hematopoietic stem cells by the expression of specific markers. In recent clinical trials, LSC have been demonstrated to represent a biomarker of poor prognosis when detected at diagnosis but also during treatment course. Moreover, the combined estimate of measurable residual disease (MRD) and LSC refines the prognostic assessment as determined by the sole application of MRD detection. Aim: We analyzed a series of patients (pts) treated in the context of GIMEMA trials, in whom the LSC frequency was assessed by MFC at diagnosis. Pts with measurable levels of LSC were tested again after the consolidation cycle. At the same timepoint "standard" MRD was also determined. The purpose of the study was to demonstrate a correlation between LSC burden at baseline and prognosis in terms of overall (OS) and disease-free survival (DFS). Furthermore, we wanted to investigate the relationship between LSC and "standard" MRD persistence (>0.035%) after consolidation, and possible correlation with outcome. Methods: LSC were evaluated by MFC as described elsewhere (Terwijn, PLoS 2014). LSC were quantified exploiting the expression of the C-type lectin-like molecule-1 (CLL1) and applying a sequential gating strategy that contained the CD34+/CD38- population. Pts were defined as LSC negative (LSCneg) in case of zero LSC count, LSClow or LSChigh when LSC were >0<0,03% or >0.03%, respectively. After consolidation, any level >0 was considered as a LSC persistence. Methods of analysis and thresholds were set according to previous publications (Zeijlemaker, Leukemia 2019). Results: We analyzed 130 pts with de novo AML, in whom LSC determination was available at the baseline. Fifty-nine (45,4%) pts were LSCneg, 49 (37,7%) LSClow, 22 (16,9%) LSChigh. We did not observe any correlation between baseline LSC level and genetic/cytogenetic risk at diagnosis. There was not a significant difference in terms of OS duration according to the 3 LSC levels, however, pts who were LSChigh had the shortest OS (36-month estimate OS of 71.5% vs. 65.4 % vs 52.4 % for the LSCneg, LSClow and LSChigh categories respectively; p=0.21). A statistically significant difference, regardless of the belonging to the LSClow or LSChigh category was observed when we focus on the subgroup of 30 pts with intermediate-risk AML, with a 36-month estimate OS of 76% vs. 77.8% vs 25% for the LSCneg, LSClowand LSChigh categories respectively (p=0.023) (Figure 1A). In 19 patients, LSC persistence was assessed at the post-consolidation time-point. Nine LSChigh pts who failed to eradicate residual LSC at this timepoint had a worse outcome as compared to those belonging to the same category but achieving a LSC clearance or those who were LSClow (36-month OS of 62.5% vs. 59.2% vs. 66.7% vs. 25% for the LSClow converted into LSCneg, LSClow not converted into LSCneg, LSChigh converted into LSCneg and LSChigh not converted into LSCneg categories, respectively; P=0.062) (Figure 1B). In 27 pts LSC and "standard" MRD determination was available. LSC persistence determined a worse 3-years OS both in MRD negative (66.7% vs 85.7%, p=0.44) and MRD positive pts (<20% vs 75.0%, p=0.041). Conclusions: In line with the experience of other European groups, we demonstrated that MFC monitoring of LSC is feasible and provides prognostic information when performed at diagnosis and during treatment course. MFC assessment of LSC also offers the opportunity to monitor pts who lack aberrant phenotypes suitable for "standard" MRD investigation. When the 2 approaches - standard "MRD" and LSC assessment - are combined together, the prognosis prediction of AML can be further refined. Finally, LSC assessment can potentially represent an effective tool to monitor the effect of LSC targeting agents. Disclosures Buccisano: Astellas: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Venditti:Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy.

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Multiparametric Flow-Cytometry Is a Reliable Tool for Measurable Residual Disease Assessment and Risk-Stratification of FLT3-Mutated AML Patients

Blood ◽

10.1182/blood-2019-129184 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5083-5083

Author(s):

Raffaele Palmieri ◽

Luca Maurillo ◽

Alfonso Piciocchi ◽

Maria Ilaria Del Principe ◽

Valentina Arena ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Board Of Directors ◽

Residual Disease ◽

Cell Transplant ◽

Advisory Committees ◽

Npm1 Mutation ◽

Multiparametric Flow Cytometry ◽

Reliable Tool ◽

Measurable Residual Disease

Background: Mutations of the gene encoding Fms Related Tyrosine Kinase 3 (FLT3), at the juxta-membrane level (ITD), represent the most common lesions found in Acute Myeloid Leukemia (AML), identifying a subgroup of patients (pts) with unfavorable prognosis. FLT3-ITD mutations are considered an unreliable tool for measurable residual disease (MRD) monitoring, due to their intraclonal heterogeneity and instability during the course of disease. Instead, multiparametric flow cytometry (MFC) may represent an alternative to monitor MRD in this molecular subset. In fact, through the recognition and monitoring of leukemia associated immunophenotypes, MFC is applicable to > 90% of AML patients with a sensitivity of 10-4. Aims: The aim of our study was to investigate the reliability of MFC in MRD assessment of 72 FLT3-ITD positive pts whose treatment allocation was prospectively decided according to the genetic/cytogenetic profile at diagnosis and post consolidation MRD. FLT3-ITD pts were to receive, after induction and consolidation, allogeneic stem cell transplant (ASCT), whatever the source of stem cells. In this subgroup analysis, we investigated if FLT3-ITD mutated pts have a different propensity to achieve high quality (e.g. MRD negative) complete remission as compared to FLT3 wildtype ones. Furthermore, we seek for a correlation between different levels of MRD and overall (OS) and disease-free survival (DFS). Methods: We included in the analysis 72 pts with de novo AML carrying FLT3-ITD mutations whose MRD assessment at the post-consolidation timepoint was available. Pts were defined as MRD-negative, when obtaining a residual leukemic cells count below the threshold of 3.5x10-4 (0.035%). MRD positive pts (with MRD ≥ 3.5x10-4 RLC) were stratified into 3 classes according to the levels of MRD (0.035%-0.1%; >0.1%-1%; >1%). We compared the MRD status and clinical outcome with a matched group of FLT3 wildtype AML (n = 203) treated in the same protocol. Results: Overall median age was 49 (range 18-60.9). The 2 cohorts were balanced in terms of age and sex distribution. In the FLT3-ITD group, 80/126 (64%) cases carried a concomitant NPM1 mutation vs 107/374 (28.6%) of FLT3 wildtype ones (p <0.001). Furthermore, FLT3 mutated pts had a median WBC count of 35x109/L vs 9.5x109/L of those FLT3 wildtype (p < 0.001). MRD determination after consolidation cycle was available in 72/126 FLT3-ITD pts (57%) and in 203/374 FLT3 wildtypeones (54.3%), respectively. After having received induction and consolidation course, 47/72 FLT3-ITD pts (65,2%) were submitted to allogenic stem cells transplantation (ASCT). At the post-consolidation time-point, MRD negativity rate was significantly lower in FTL3-ITD pts (27/72, 37.5%) as compared to those FLT3 wildtype (94/203, 46.3%). Furthermore, 38/72 (52.8%) and 10/72 (13.9%) FLT3-ITD pts had a level of MRD > 0.1% and > 1%, respectively as compared to 65/203 (33.0%) and 15/203 (7.4%) of FLT3 wildtypeones, respectively (p=0.017). When considering the different MRD stratification levels of FLT3-ITD pts, OS probability at 24 months was 57.2% (27 pts), 71.4% (7 pts), 53.6% (28 pts) and 20% (10 pts), for the MRD categories <0.035%, 0.035%-0.1%, >0.1%-1%, >1%, respectively (p=0.028). DFS probability at 24 months was 53.8% (27 pts), 71.4% (7 pts), 34.9% (27 pts) and 20% (10 pts), for the MRD categories <0.035%, 0.035%-0.1%, >0.1%-1%, >1%, respectively (p=0.038). Summary/Conclusion: We demonstrated that MRD determination by MFC is a reliable tool to assess remission quality and prognosis in FLT-ITD positive patients. This subpopulation shows a lower propensity to obtain a MRD negative CR, with the majority of pts maintaining an amount of MRD > 0.1% after standard treatment. Even though most of these pts were addressed to ASCT, post-consolidation MRD maintained its negative impact on OS and DFS, particularly for those pts with MRD >1%. In the attempt to improve the quality of response, prevent leukemia recurrence and pursue a durable remission, delivery of FLT3 inhibitors as a maintenance after transplant may represent a promising option. Disclosures Venditti: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy; Astellas: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees. Buccisano:Janssen: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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ABT-737 Targets Leukemic Stem Cells In Mouse Models of Mutant NRASD12/hBCL-2- Mediated Acute Myeloid Leukemia Progression with Increased Survival

Blood ◽

10.1182/blood.v116.21.3308.3308 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3308-3308

Author(s):

Rose Ann Padua ◽

Stephanie Beurlet ◽

Patricia Krief ◽

Nader Omidvar ◽

Carole Le Pogam ◽

...

Keyword(s):

Stem Cells ◽

Disease Progression ◽

Board Of Directors ◽

Research Funding ◽

Leukemic Stem Cells ◽

Spleen Cells ◽

Employment Equity ◽

Advisory Committees ◽

Equity Ownership ◽

Bh3 Mimetic

Abstract Abstract 3308 Background: Animal models enable us to understand disease progression and provide us with reagents to test various therapeutic strategies. We have previously developed a mouse model of myelodysplasia/acute myelogenous leukemia (MDS/AML) progression using mutant NRASD12 and overexpression of human hBCL-2 (Omidvar et al Cancer Res 67:11657-67, 2007). Expanded leukemic stem cells (LSC) were identified as Lin-/Sca1+/KIT+ (LSK) populations, with increased myeloid colony growth and were transplantable. Increased hBCL-2 and RAS-GTP complex were observed in both MDS/AML diseases. The MDS-like disease had increased apoptosis, whilst the AML-like mice had liver apoptosis patterns similar to wild type. The single NRASD12 line also had increased apoptosis. In this present study using a BCL-2 homology domain 3 (BH3) mimetic ABT-737 (Abbott), we have evaluated the effects of targeting BCL-2 in our preclinical models. Methods & Results: Treatment with the inhibitor shows a reduction of LSK cells, reduced progenitor numbers in colony assays and clearance of the liver infiltrations in both MDS and AML models. Gene expression profiling of the MDS mice shows regulation of 399 genes upon treatment including 58 genes expressed by the single mutant RAS mice and not expressed in the untreated AML mice. 78 genes were shared between single NRASD12 and diseased mice and not the treated mice. These studies potentially identify the contribution of NRASD12 genes to disease progression. By confocal microscopy we observed that in the MDS mice the majority of the RAS and BCL-2 co-localized to the plasma membrane, where active pro-apoptotic RAS is normally located, whereas in the AML disease RAS and BCL-2 co-localized in the mitochondria, where BCL-2 is normally found (Omidvar et al 2007). After treatment with the inhibitor the AML co-localization of RAS and BCL-2 shifted to the plasma membrane where single NRASD12 is normally localized. Furthermore, increased RAS-GTP levels was detected in both Sca1+ and Mac1+ enriched spleen cells and interestingly an increase in BCL-2 expression was observed in peripheral blood and in spleen cells after treatment; this increase in BCL-2 was associated with a decrease in the phosphorylation of serine 70 and an increase in phosphorylation of threonine 56 of BCL-2. ABT-737 treatment led to increased phosphorylated ERK resembling RAS and reduced MEK and AKT phosphorylation, changes detected by western blots and the nanoimmunoassay (NIA, NanoPro, Cell Biosciences) that might account for the increased apoptosis, measured by TUNEL and In vivo imaging by single-photon emission computed tomography (SPECT) using Tc-99m-labelled AnnexinV (SPECT). In contrast, although treated MDS mice had increased apoptosis they did not have an increase in overall expression of BCL-2 or in RAS-GTP levels. Treatment of both MDS and AML models with this inhibitor significantly extended lifespan from diagnosis with mean survival of 28 days untreated vs 80 days treated (p=0.0003) and mean survival from birth of 39 untreated vs 85 days treated (p<0.0001) respectively Conclusions: Genomics, proteomics and imaging have been employed in the MDS/AML models to characterize disease progression and follow response to treatment to the BH3 mimetic ABT-737 in order to gain molecular insights in the evaluation of the efficacy. ABT-737 appears to target LSCs, induce apoptosis, regulating RAS and BCL-2 signalling pathways, which translated into significantly increased survival. Disclosures: Padua: Vivavacs SAS: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Auboeuf:GenoSplice technology: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. de la Grange:GenoSplice technology: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Fenaux:Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen Cilag: Honoraria, Research Funding; ROCHE: Honoraria, Research Funding; AMGEN: Honoraria, Research Funding; GSK: Honoraria, Research Funding; Merck: Honoraria, Research Funding; Cephalon: Honoraria, Research Funding. Tu:Cell Biosciences Inc;: Employment. Yang:Cell Biosciences Inc;: Employment. Weissman:Amgen, Systemix, Stem cells Inc, Cellerant: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Felsher:Cell Bioscience:. Chomienne:Vivavacs SAS: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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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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Secreted Mutant Calreticulins As Rogue Cytokines Trigger Thrombopoietin Receptor Activation Specifically in CALR Mutated Cells: Perspectives for MPN Therapy

Blood ◽

10.1182/blood-2018-99-118348 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4-4 ◽

Cited By ~ 15

Author(s):

Christian Pecquet ◽

Thomas Balligand ◽

Ilyas Chachoua ◽

Anita Roy ◽

Gaelle Vertenoeil ◽

...

Keyword(s):

Electron Microscopy ◽

Flow Cytometry ◽

Cell Surface ◽

Board Of Directors ◽

Research Funding ◽

Advisory Committees ◽

Allele Burden ◽

In Trans ◽

Equity Ownership ◽

In Cells

Abstract Background Mutant calreticulins carrying the sequence translated after a +1 frameshift at the C-terminus are major drivers of myeloproliferative neoplasms (MPNs). These mutant CALRs bind and activate TpoR/MPL in cells co-expressing TpoR and mutant CALRs, resulting in persistent JAK2-STAT5 signaling. Whether mutant CALR proteins are secreted, thus acting in trans on other cells, is not known. Aims Our objectives were to: 1) assess the direct TpoR-mutant CALR interaction both when expressed in the same or in different cells; 2) determine whether mutant CALRs are secreted; and 3) determine whether mutant CALR can act as extracellular cytokines. Methods Engineered CALR and TpoR mutants were analyzed by a combination of biochemical approaches (bioluminescence resonance energy transfer, recombinant protein production), functional assays (cell growth and transcriptional assays, flow cytometry, primary megakaryocytic clonogenic assay, analysis of CALR del52 knock-in mice) and cell imaging (confocal microscopy, flow cytometry and immuno-gold electron microscopy). Secreted CALRs were determined by ELISA using mutant specific antibodies. Results 1) Two systems provided evidence that mutant CALRs and TpoR directly interact. First, using Nano-BRET in cells co-expressing N-terminally fused TpoR or EpoR with Nano-luciferase and mutant or WT CALR C-terminally tagged with HaloTag that is bound to the 618-ligand fluorophore, we show that TpoR and mutant CALRs interact in a complex whether the two proteins are within 10 nm. The interaction does not occur between TpoR and WT CALR, or between EpoR and mutant or WT CALRs. Second, expressing mutant CALR and TpoR extracellular domain in S2 Drosophila Schneider cells showed that stable complex formation requires immature high mannose structure on TpoR. Lastly, we could detect surface expression of the TpoR/CALRdel52 complex using proximity ligation assay with anti-TpoR and anti-mutant CALR antibodies in CRISPR/Cas9 engineered UT7/Tpo cells that express endogenous mutant CALR and TpoR levels. 2) We used flow cytometry, confocal immunofluorescence and immunogold electron microscopy and could show that mutant CALRs are trafficking via cis-, medial- and trans-Golgi to the cell-surface and are secreted, independently from TpoR expression. Importantly, mutant CALRs are also secreted in CALR mutated MPN patients as determined by mutant CALR-specific ELISA assay in patient plasma (mean plasma level 24.6 ng/ml, range 0-156.5 ng/ml). In the 113 evaluated CALR mutated patients from different centers the plasma mutant CALR levels correlated with CALR mutant allele burden (P<0.001). Secreted mutant CALR can also be found in plasma from knock-in CALR del52 mice. 3) We show that recombinant mutant CALR can act as a cytokine and specifically stimulate JAK2-STAT5 pathway in cells that carry the TpoR at the surface. Using Nano-BRET, we could demonstrate that extracellular mutant Halo-tagged CALR can specifically bind in trans to the cell-surface TpoR fused with Nano-luciferase, but not to EpoR fused with Nano-luciferase. This binding and the subsequent JAK2 activation were obtained at levels of around 100-150 ng/ml only in cells exposing at the cell-surface TpoR with at least one immature N-linked sugar. This can be accomplished by co-expressing in the reporter cells non-tagged mutant CALR, which will promote cell-surface localization of partially immature TpoR. The effect of exogenous mutant CALR could involve both stabilization of the endogenous cell-surface mutant CALR-TpoR complexes and binding to unoccupied immature TpoRs. Conclusion We show that mutant CALRs directly interact with TpoR and also are secreted and can act as rogue cytokines, leading to activation of cells carrying TpoR. Activation of TpoR in trans is efficient at mutant CALR levels similar to those detected in patients when target cells co-express heterozygous mutant CALR and TpoR, where endogenous mutant CALR transports to the surface TpoR with immature glycosylation. Thus, secreted mutant CALRs is predicted to expand the MPN clone. Given that cell-surface mutant CALR in TpoR expressing cells is crucial for oncogenicity, and that mutant CALRs are also secreted correlating with allele burden, we discuss how antibodies and other immunotherapy approaches could specifically target the mutant CALR MPN clone. Disclosures Xu: MyeloPro Research and Diagnostics GmBH: Employment. Hug:MyeloPro Diagnostics and Research GmbH: Employment. Gisslinger:Janssen: Consultancy, Honoraria; AOP Orphan: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Shire: Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Kralovics:MyeloPro Diagnostics and Research GmbH: Equity Ownership. Constantinescu:Personal Genetics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees; AlsaTECH: Equity Ownership; Novartis: Honoraria; MyeloPro Research and Diagnostics GmbH: Equity Ownership.

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Identification of Novel Surface Markers and Targets In Neoplastic Stem Cells In AML and CML: a Flow-Gene-Flow Screen Approach.

Blood ◽

10.1182/blood.v116.21.1575.1575 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1575-1575 ◽

Cited By ~ 2

Author(s):

Harald Herrmann ◽

Sabine Cerny-Reiterer ◽

Irina Sadovnik ◽

Viviane Winter ◽

Katharina Blatt ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Stem Cell ◽

Cell Surface ◽

Progenitor Cells ◽

Gene Chip ◽

Cytokine Receptors ◽

Stem Cell Markers ◽

Surface Markers ◽

Leukemic Stem Cells

Abstract Abstract 1575 The concept of leukemic stem cells (LSC) is increasingly employed to explain the biology of various myeloid neoplasms and to screen for pivotal targets, with the hope to improve drug therapy through elimination of disease-initiating cells. Although the stem cell hypothesis may apply to all neoplasms, leukemia-initiating cells have so far only been characterized in some detail in myeloid leukemias. In an attempt to identify novel cell surface markers and targets on leukemic stem cells (LSC) in acute (AML) and chronic myeloid leukemia (CML), we examined CD34+/CD38- and CD34+/CD38+ populations of leukemic cells in a cohort of patients with AML (n=55) and CML (n=20). In a first step, cell surface antigen profiles were determined by multicolor flow cytometry. In this screen, we were able to show that CD34+/CD38- LSC in AML and CML consistently express certain cytokine receptors, including G-CSFR (CD114), SCFR/KIT (CD117), and IL-3RA (CD123). The low affinity IL-2R (CD25) was detectable on CD34+/CD38- stem cells in patients with CML, and in a subset of AML patients. Other cytokine receptors (R) such as FLT3, IGF-1R, endoglin (CD105), GM-CSFRA (CD116), and OSMR were expressed variably on CD34+/CD38- progenitor cells, whereas the EPOR was not detectable on LSC. We were also able to detect several established therapeutic targets on LSC, including CD33 and CD44. Whereas CD44 was consistently expressed on all LSC in all donors, CD33 was found to be expressed variably on subpopulations of LSC in AML and CML, depending on the phase and type of disease. By using cytokine ligands (G-CSF, IL-3, SCF, EPO) and targeted drugs, we were also able to confirm that identified cytokine receptors and targets were functionally active molecules and potentially relevant targets. In a next step, highly enriched (purity >98%) sorted CD34+/CD38- cells, CD34+/CD38+ cells, and CD34- cells were collected in patients with AML and CML, and in 3 cord blood samples as controls. Purified cells were subjected to gene chip analyses, qPCR, and functional analyses. The identity of leukemic progenitors was confirmed by FISH, and expression of markers and targets in CML stem cells and AML stem cells was confirmed by qPCR. In gene chip analyses, we screened for novel LSC markers and targets. Candidate genes were selected based and their specific expression in progenitor cell fractions and surface membrane location, which was confirmed by antibody staining experiments. Novel stem cell markers identified so far include ROBO4, NPDC-1, and CXCR7. The previously described surface markers MDR-1 and CLL-1 were also identified by flow cytometry, but were also found to be expressed on more mature hematopoietic cells. By contrast, ROBO4 was found to be expressed preferentially on CD34+/CD38- stem cells, but less abundantly on CD34+/CD38+ progenitor cells in CML. Interestingly, whereas ROBO4 was expressed on CD34+/CD38- stem cells in most patients with CML, ROBO4 expression on leukemic stem cells was only found in a subset of AML patients. By contrast, CD34+/CD38- stem cells in AML frequently expressed CLL-1 and NPDC-1 on their surface. In conclusion, we have identified novel markers and targets in CD34+/CD38- progenitor cells in AML and CML. These markers may be useful for the identification and isolation of leukemic stem cells in AML and CML, and for the validation of drug effects on these cells. Disclosures: De Angelis: Biopharm R&D, GSK: Employment. Holmes:Biopharm R&D, GSK: Employment. Valent:Domantis: Research Funding.

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Antibody-Targeting of IL-3 Receptor-α Increases the Susceptibility of CD34+ CML Progenitors to Dasatinib-Induced Cell Death,

Blood ◽

10.1182/blood.v118.21.3745.3745 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3745-3745

Author(s):

Eva Nievergall ◽

Deborah L. White ◽

Hayley Ramshaw ◽

Angel F. Lopez ◽

Timothy P. Hughes ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Progenitor Cells ◽

Board Of Directors ◽

Myeloid Leukemia ◽

Research Funding ◽

Blast Crisis ◽

Chronic Phase ◽

Advisory Committees ◽

Cd34 Cells

Abstract Abstract 3745 Despite the remarkable efficacy of tyrosine kinase inhibitors (TKIs) in the treatment of chronic myeloid leukemia (CML), Ph+ CD34+ progenitor cells remain detectable even in patients with stable complete cytogenetic response. Over 40% of patients in stable complete molecular remission will develop molecular relapse within 6 months of stopping imatinib. While the exact causes are largely unknown, one of the proposed mechanisms is the protection of leukemic stem and early progenitor cells by the paracrine or autocrine production of cytokines, such as IL-3, GM-CSF and G-CSF, which activate survival pathways that bypass TKI-induced cytocidal effects. In acute myeloid leukemia (AML), the IL-3 receptor α chain (CD123) is recognized as a specific marker for CD34+/CD38− stem cells and therefore is attracting increasing interest as a therapeutic target. However, the function of CD123 in CML remains to date mostly unexplored. The aim of this study is to investigate potential synergy between TKIs and CSL362 (a humanized antibody version of 7G3 against CD123) in targeting CML progenitor and stem cells. CD34+ and CD34+/CD38− cells were isolated from mononuclear cells of newly diagnosed CML chronic phase and blast crisis patients. Flow cytometry studies indicated significantly increased CD123 expression on CD34+/CD38− cells of CML patients in both chronic phase and blast crisis when compared to normal hematopoietic stem cells (p<0.01 and p<0.001 for chronic phase and blast crisis, respectively; Figure A). A functional relevance of increased CD123 expression was demonstrated by IL-3-dependent increase in STAT5 phosphorylation (260.5% of baseline with 20 ng/ml IL-3; n=12; p<0.001) in CML CD34+ cells. Dasatinib inhibits STAT5 phosphorylation by blocking BCR-ABL signaling but only in the absence of IL-3 (62.5% of baseline for dasatinib alone vs. 130.8% for dasatinib + IL-3; n=3; p<0.01). In agreement, IL-3 effectively rescues dasatinib-induced cell death, as evaluated by AnnexinV/7-AAD staining (103.3% vs. 72.45%, n=5; p<0.01) and CFU-GM colony forming assays (69.39% vs. 46.13% relative to no treatment control; n=4; p<0.05). CSL362, in turn, revokes IL-3-mediated STAT5 phosphorylation (37.12% vs. 130.8%; n=3; p<0.001) and cytoprotection (45.05% vs. 69.39% CFC; n=4; p<0.01). In order to further elucidate the role of CSL362, CML CD34+ cells were cultured with increasing concentrations of dasatinib in the presence of IL-3 and CSL362 or BM4 isotype-matched control antibody. Even at very low dasatinib concentrations, CSL362 significantly reduces CML CD34+ colony forming cells (p<0.05; Figure B). Together these results substantiate a relevant role for IL-3-mediated resistance in CML progenitor cells and additionally confirming the ability of CSL362 to effectively bind to CD123 and impede IL-3 function. CSL362 furthermore has been optimized to mediate antibody dependent cell cytotoxicity (ADCC). CSL362 causes specific cell lysis of CML CD34+ progenitor cells in co-culture with allogeneic Natural killer cells as determined by increased lactate dehydrogenase release (ADCC activity of 42.4% ± 8.1%; n=3) and a decrease in the number of CFU-GM colonies by 74.1 % ± 12.2% (n=3). Collectively, our results indicate that a combination of dasatinib and CSL362 inhibits CML progenitor cell survival more effectively in vitro. Therefore, targeting IL-3 receptor α with CSL362 in chronic phase and blast crisis CML patients might provide a novel specific treatment approach aiding the elimination of refractory chronic myeloid leukemic stem and progenitor cells. A: Flow cytometry analysis reveals that CD123 expression is significantly higher in CD34+/CD38− cells of CML patients in chronic phase (CML-CP) and blast crisis (BC-CML) as compared to normal patients (NP), as previously documented for AML patients. ** p<0.01, *** p<0.001 by unpaired, two-tailed Student's t-test. B: In the presence of IL-3, CSL362 significantly reduces the number of colony forming cells. CD34+ cells of de novo CML-CP patients were cultured with dasatinib (0 to 10 nM) +IL-3 (1 ng/ml) ± CSL362 or BM4 (isotype control for CSL362). After 72 hours of culture live cells were plated for CFU-GM assay and colonies were counted after 2 weeks. Mean ± SE of three independent experiments is shown, n=4, p<0.05 by two-way ANOVA. Disclosures: Nievergall: CSL: Research Funding. White:CSL: Research Funding. Lopez:CSL: Research Funding. Hughes:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hiwase:CSL: Research Funding.

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Phenotypic Characterization of Leukemia-Initiating Stem Cells in Chronic Myelomonocytic Leukemia (CMML)

Blood ◽

10.1182/blood-2019-122530 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4223-4223

Author(s):

Gregor Eisenwort ◽

Alexandra Keller ◽

Michael Willmann ◽

Irina Sadovnik ◽

Greiner Georg ◽

...

Keyword(s):

Stem Cells ◽

Board Of Directors ◽

Research Funding ◽

Advisory Board ◽

Chronic Myelomonocytic Leukemia ◽

Multicolor Flow Cytometry ◽

Advisory Committees ◽

Cd34 Cells ◽

Myelomonocytic Leukemia

Chronic myelomonocytic leukemia (CMML) is a stem cell-derived hematopoietic neoplasm characterized by dysplasia, uncontrolled expansion of monocytic (progenitor) cells in the bone marrow (BM) and in the peripheral blood (PB), and an increased risk of progression to secondary acute myeloid leukemia (sAML). Patients with advanced CMML and sAML are often highly resistant to therapy and their prognosis is dismal. It is thought that drug resistance in myeloid malignancies is a quality of leukemia stem cells (LSC), but little is known about CMML-initiating and propagating LSC. We investigated the phenotype and functional behavior of putative CMML-initiating cells in 15 patients with CMML (7 females, 8 males; median age 73 years; range 45-82 years) and 6 with sAML following CMML (1 female, 5 males; median age 67.5 years; range 66-76 years). BM and/or PB samples were examined by multicolor flow cytometry using antibodies against CD34, CD38 and various additional surface markers and target antigens. In a subset of patients, putative stem and progenitor cells (CD34+ cells, CD34+/CD38─ cells and CD34+/CD38+ cells) were FACS-sorted to high purity (>95%) and were employed in xenotransplantation experiments or in drug testing experiments. We found that CMML-initiating and propagating LSC reside within the CD34+/CD38─ fraction of the malignant clone. Whereas highly purified CD34+ cells engrafted NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG) 1Eav/MloySzJ (NSGS) mice with full-blown CMML (engraftment rate 44.8±26.0%), no CMML was produced by the bulk of CD34- monocytic cells (engraftment rate 0.8±0.5%; p=0.002). CMML engraftment was also detectable when transplanting unselected mononuclear cells (engraftment rate 19.7±10.9%). By contrast, no leukemic engraftment was produced by CD38+ CMML fractions (engraftment rate 0.1±0.1%; p=0.003), indicating that the NSGS-repopulating CMML LSC reside specifically in a CD34+/CD38- fraction of the clone. In sAML, both the CD34+/CD38- cell fraction (engraftment rate 92.2±6.2%) and the CD34+/CD38+ fraction (engraftment rate 80.5±7.2%) produced engraftment with AML blasts in NSGS mice. In a next step, we established the cell surface phenotype of CD34+/CD38- LSC in CMML and sAML. As assessed by multicolor flow cytometry, CD34+/CD38- CMML cells invariably expressed CD33/Siglec-3, CD117/KIT, CD123/IL3RA, CD133/AC133, CD135/FLT3, and IL-1RAP. In a subset of patients, CMML LSC also expressed CD52 (9/11 patients; 81%), CD114 (3/7 patients; 43%), CD184 (9/12 patients; 75%), CD221 (8/11 patients; 73%) and/or CLL-1 (7/13 patients; 54%). CMML LSC did not express CD25 or CD26. However, in patients with sAML, LSC also displayed CD25 (median fluorescence intensity, MFI: CMML: 0.9 vs. sAML: 23.0; p<0.001). Compared to hematopoietic stem cells in normal BM (NBM), CMML LSC displayed slightly increased levels of CD117/KIT (MFI CMML: 32.5 vs. MFI NBM: 15.0; p=0.019), CD135/FLT3 (MFI CMML: 1.9 vs. MFI NBM: 0.8; p=0.001), CD184/CXCR4 (MFI CMML: 1.6 vs. MFI NBM 0.9; p=0.027), and IL-1RAP (MFI CMML: 1.6 vs. MFI NBM: 0.8; p=0.004). No correlations between surface-marker expression on LSC and the type of CMML (CMML-0/1/2 or dysplastic vs. proliferative CMML) or the clinical course were found. To confirm the clinical relevance of expression of surface target antigens on CMML LSC, we applied the CD33-targeted drug gemtuzumab-ozogamicin (GO). As assessed by combined staining for LSC (CD34+/CD38-) and AnnexinV/DAPI, incubation of CMML LSC with GO (0.001-1 µg/ml) resulted in dose-dependent apoptosis in all donors tested, and the same result was obtained in the monoblastic cell lines THP-1 (GO at 1 µg/ml: 94.2±1.5% vs. control: 12.7±2.2%, p<0.05) and Mono-Mac-1 (GO at 1 µg/ml: 56.4±12.1% vs. control: 10.1±0.6% p<0.05). In conclusion, LSC in CMML and sAML reside within CD34+/CD38─ cell populations that express distinct profiles of surface markers and target antigens. During progression of CMML into sAML, LSC apparently acquire CD25. Characterization of CMML LSC and LSC in sAML should facilitate their enrichment and the development of LSC-eradicating therapies. Disclosures Hoermann: Novartis: Honoraria; Roche: Honoraria. Sperr:Celgene: Consultancy, Honoraria; Novartis: Honoraria. Sill:Astex: Other: Advisory board; Novartis: Other: Advisory board; AbbVie: Other: Advisory board; Astellas: Other: Advisory board. Geissler:Novartis: Honoraria; AOP: Honoraria; Roche: Honoraria; Amgen: Honoraria; AstraZeneca: Honoraria; Ratiopharm: Honoraria; Celgene: Honoraria; Abbvie: Honoraria; Pfizer: Honoraria. Deininger:Blueprint: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Research Funding; Ascentage Pharma: Consultancy, Honoraria; Fusion Pharma: Consultancy; TRM: Consultancy; Sangoma: Consultancy; Adelphi: Consultancy; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Humana: Honoraria; Incyte: Honoraria; Novartis: Honoraria; Sangamo: Consultancy. Valent:Pfizer: Honoraria; Blueprint: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Honoraria; Deciphera: Honoraria, Research Funding.

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Targeted Killing of AML Cells By Bispecific Antibody Armed Activated T Cells

Blood ◽

10.1182/blood-2019-132003 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 5151-5151

Author(s):

Lawrence G. Lum ◽

Ewa Kubicka ◽

Bliemeister T Edwin ◽

Archana Thakur ◽

Manley Huang

Keyword(s):

Stem Cells ◽

Monoclonal Antibody ◽

Flow Cytometry ◽

T Cells ◽

Cell Expansion ◽

Bispecific Antibody ◽

Leukemic Cell ◽

Gemtuzumab Ozogamicin ◽

Leukemic Stem Cells ◽

Activated T Cells

Background: Acute myeloid leukemia (AML) is a heterogeneous disease characterized by the accumulation of hematopoietic stem/progenitor cells that poses a significant therapeutic challenge. Recurrence of the disease is attributed to leukemia-initiating cells, also referred to as leukemic stem cells (LSCs), which are thought to be spared from chemotherapy and capable of reinitiating the disease. Thus, novel non-toxic therapeutic strategies to target and eradicate LSCs and AML blasts are urgently needed. Immunotherapy (IT) is an attractive strategy to improve outcomes for patients with AML, as it does not rely on the cytotoxic mechanisms employed by conventional chemotherapies. Methods: Bispecific antibody was produced by chemical hetero-conjugation of anti-CD33 monoclonal antibody (gemtuzumab ozogamicin [GO]) linked to calicheamicin to anti-CD3 monoclonal antibody (anti-CD3 x GO bispecific antibody, CD33Bi) or anti-CD3 and anti-CD123 (CD123Bi). We tested four AML cell lines (EOL-1, KG-1, NoMo-1 and TF-1) as targets for cytotoxicity by CD33Bi and CD123Bi armed ATC using flow cytometry-based cytotoxicity assays. Cytokines and chemokines released during CD33Bi or CD123Bi armed ATC mediated killing of targets were analyzed by multiplex luminex assay. A xenogeneic NOD/SCID/gamma chain KO (NSG) mouse model was used for the evaluation of in vivo activity of Bispecific antibody (50ng/million cells) Armed activated T cells (BATs). Engraftment of AML cells-KG1 was tracked by quantifying the human CD45+ AML cells using flow cytometry. Two patient samples were analyzed for blast and LSC population before and after incubation with CD123-BATs. Results: In a non-radioactive quantitative flow cytometry-based cytotoxicity assay Bispecific antibody (50ng/million cells) Armed activated T cells (BATs) show substantial killing by both CD33-BATs and CD123-BATs against AML cell lines (EOL1, KG1, NoMo1 and TF-1) at 1:1 and 2:1 E/T ratios ranging from 60-90% cytotoxicity and produced increased levels of Th1 cytokines (TNF-alpha, GM-CSF and IFN-gamma). The KG1 engrafted 6- to 8-week-old NSG mice (n=8 mice/group) injected IV with 20 x 106 CD33-BATs (3x/week for 4 weeks; 100ml/injection) was able to inhibit leukemic cell expansion significantly compared to 0.06 mg/kg gemtuzumab ozogamicin [GO] antibody (p<0.0006); likewise significant inhibition of leukemic cell expansion was observed with CD123-BATs compared to PBS control mice (p<0.009). One patient sample incubated with CD123-BATs showed significant reduction in blast after CD123-BATs treatment compared to before treatment (13.2% vs. 30%) or CD34+CD38- LSC after CD123-BATs treatment (5.2%) compared to before treatment (20%). Second patient sample also showed 50% reduction in blast population, no LSC were detected in this sample. Conclusions: ATC armed with CD33Bi or CD123Bi at 50 ng/106 cells showed comparable cytotoxicity directed at either high or low leukemic blast populations, able to inhibit leukemic cell expansion significantly compared to the control mice. This approach may provide a potent and non-toxic strategy to target leukemic blasts and leukemic stem cells. Disclosures Huang: TransTarget Inc.: Other: Co-Founder.

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