Analyses of the Kinetics and Phenotype of Multiple Intraclonal CXCR4/CD5 B Cell Subsets Suggest Differences in Life Cycle Transitioning in CLL

Abstract Chronic lymphocytic leukemia (CLL) is a heterogeneous disease so that defining the dynamic features of the clone and its intraclonal subpopulations are essential to understand disease pathogenesis and to develop novel, effective therapies. For instance, because cell division is linked with new mutations, the ability to preferentially select cells that recently divided allows studying the subpopulation(s) most likely responsible for disease progression and resistance to therapies. The intraclonal kinetics of CLL B cells have been studied in clonal subgroups defined by reciprocal surface levels of CXCR4 and CD5. In that model, three fractions are identified: recently divided "proliferative" (PF; CXCR4 DimCD5 Bright); "intermediate" (IF; CXCR4 IntCD5 Int) and "resting" (RF; CXCR4 BrightCD5 Dim). Here, we have expanded the examination of subpopulations differing for time since last division ("age"). Unmanipulated CLL cells studied ex vivo from 10 patients who drank 2H 2O for 4 weeks were sorted by the relative densities of CXCR4 and CD5 to isolate the formerly identified PF, IF and RF as well as two fractions not previously characterized, "Double Dim" (DDF: CXCR4 DimCD5 Dim) and "Double Bright" (DBF; CXCR4 BrightCD5 Bright). For each fraction, the amount of deuterium incorporated into cellular DNA in vivo was measured. Consistently, the PF contained significantly higher levels of 2H-labeled DNA and higher calculated cell division rates when compared with the RF and IF. Interestingly, the DDF also contained significantly more 2H-labeled DNA compared to the RF; in contrast, the DBF resembled more closely the RF fraction. The overall 2H-incorporation gradient was: PF>DDF>IF>DBF>RF. In CLL, BCR signaling is fundamental, with the amount of membrane (m) IgM associating with signaling competence and disease aggressiveness. Additionally, when engaged independently, mIgM and mIgD can lead to different signaling sequelae. Therefore, we analyzed the 5 subpopulations for the densities of mIgM and mIgD. This showed a distribution similar to that of 2H-DNA incorporation: for IgM: PF=DDF>IF=DBF=RF, and for IgD: PF>DDF>IF=DBF>RF. Accordingly, we next measured 2H-DNA in subpopulations with low, intermediate and high levels of IgM and IgD. This revealed a direct correlation between IG densities and in vivo DNA synthesis, consistent with intraclonal subpopulations with high IGs having divided more recently than those with low IGs. However, these findings are not in line with cell division being primarily initiated by BCR engagement since that would lower mIgM levels. Therefore, we tested if engagement of TLR9 would affect mIG densities on CLL cells. After stimulation of 32 CLL clones with CpG+IL15, anti-IgM+IL4, anti-IgD+IL4, or anti-IgM-IgD+IL4, there was a significant increase in mIGs only after CpG+IL15 activation; each anti-IG stimulation led to downregulation of mIGs. Finally, we questioned the subclonal responsiveness to BTK inhibition in vivo. CLL samples taken from the same patients, before and during ibrutinib treatment, displayed intraclonal changes in mIG densities and cell size, the latter a marker of cellular and metabolic activation also linked with CLL in vivo birth rates. Ibrutinib treatment normalized mIgM and mIgD intraclonal densities and lead to an overall cell size decrease with larger, 2H-enriched and higher mIG density cells being more affected (PF>DDF>IF>DBF>RF). Collectively, these findings suggest that the most recently born cells enter the circulation as the PF from which they transition to either lower CD5 (DDF) or higher CXCR4 (IF and DBF) phenotypes. Each eventually converge as the RF. Moreover, since mIG densities on the more recently divided populations (PF and DDF) are high, the data imply that successful cell division is not solely a consequence of BCR engagement; the involvement of the TLR pathways, concomitantly or in series with BCR signaling, is more consistent with the higher mIG levels. Finally, ibrutinib treatment appears to preferentially target more recently divided cells with high mIG levels. Disclosures Allen: Alexion: Research Funding; Bristol Myers Squibb: Other: Equity Ownership; C4 Therapeutics: Other: Equity Ownership; Sanofi Genzyme: Membership on an entity's Board of Directors or advisory committees.

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Development of a Human Therapeutic L-Cyst(e)Ine-Degrading Enzyme for the Treatment of Hematological Malignancies

Blood ◽

10.1182/blood.v128.22.1587.1587 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1587-1587

Author(s):

Giulia Agnello ◽

Susan Alters ◽

Joseph Tyler ◽

Jinyun Liu ◽

Peng Huang ◽

...

Keyword(s):

Research Funding ◽

Hematological Malignancies ◽

Ex Vivo ◽

Redox Balance ◽

Lymphocytic Leukemia ◽

Employment Equity ◽

Equity Ownership ◽

Antioxidant Mechanisms

Abstract Cancer cells experience higher intrinsic oxidative stress than their normal counterparts and acquire adaptive antioxidant mechanisms to maintain redox balance. This increased antioxidant capacity has been correlated to malignant transformation, metastasis and resistance to standard anticancer drugs. This enhanced antioxidant state also correlates with cancer cells being more vulnerable to additional oxidative insults, therefore disruption of adaptive antioxidant mechanisms may have significant therapeutic implications. Hematological malignancies including Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), Acute Myeloid Leukemia (AML) and Multiple Myeloma (MM) are critically dependent on the cellular antioxidant glutathione (GSH), consistent with the higher intrinsic oxidative stress. L-cysteine is the rate-limiting substrate for GSH biosynthesis and adequate levels of cysteine are critical to maintain the intracellular homeostasis of GSH. CLL and a subset of ALL cells have been reported to rely on the stromal supply of cysteine to increase the synthesis of GSH in order to maintain redox balance, which in turn promotes cell survival and fosters drug resistance. One approach to target this cancer specific dependency is by therapeutic depletion of amino acids via enzyme administration; a clinically validated strategy for the treatment of ALL. Aeglea BioTherapeutics Inc. has developed a bioengineered cysteine and cystine degrading enzyme (Cyst(e)inase, AEB3103) and evaluated its therapeutic efficacy against hematological malignancies in in vitro, ex vivo and in vivo pre-clinical studies. The TCL1-TG:p53 -/- mouse model exhibits a drug resistant phenotype resembling human CLL with unfavorable cytogenetic alterations and highly aggressive disease progression. AEB3103 greatly decreased the viability of TCL1-TG:p53 -/- cells cultured in vitro, whereas the CLL therapeutic, fludarabine, showed minimal cytotoxic effects. In vivo treatment of TCL1-TG:p53 -/- mice with AEB3103 resulted in an increase in median survival time (7 months, p<0.0001) compared to the untreated control group (3.5 months, p<0.001) and a fludarabine treated group (5.3 months, p<0.001). These results indicate a superior therapeutic effect of AEB3103 compared to fludarabine. Additionally, evaluation of AEB3103 in in vitro 2D cultures of patient-derived CLL and MM cells, and in ex vivo 3D cultures of cells derived from ALL and AML PDx models resulted in significant cell growth inhibition with therapeutically relevant IC50 values. Collectively these results demonstrate the sensitivity of hematological malignancies to modulation of GSH levels via AEB3103-mediated cyst(e)ine depletion. Disclosures Agnello: Aeglea BioTherapeutics: Employment. Alters:Aeglea BioTherapeutics: Employment, Equity Ownership. Tyler:Aeglea BioTherapeutics: Employment, Equity Ownership. Huang:Aeglea BioTherapeutics: Research Funding. Stone:Aeglea Biotherapeutics: Consultancy, Equity Ownership, Research Funding; University of Texas at Austin: Employment, Patents & Royalties: I am an inventor of technology related to this abstract. Georgiou:Aeglea Biotherapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Lowe:Aeglea BioTherapeutics: Employment, Equity Ownership. Rowlinson:Aeglea BioTherapeutics: Employment, Equity Ownership.

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Chronic Lymphocytic Leukemia B Cells Display IgM and IgD Isotype-Restricted Features That Affect Association with Co-Receptors, BCR Signaling, and Leukemic B-Cell Growth In Vivo

Blood ◽

10.1182/blood-2018-99-119989 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3124-3124

Author(s):

Andrea Nicola Mazzarello ◽

Marcus Dühren-von Minden ◽

Eva Gentner ◽

Palash Chandra Maity ◽

Gerardo Ferrer ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Board Of Directors ◽

Cell Size ◽

Metabolic Activity ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Advisory Committees ◽

Bcr Signaling

Abstract The leukemic cells in patients with chronic lymphocytic leukemia (CLL) are highly dependent on B-cell receptor (BCR) mediated signaling. Despite this and the fact that >90% of CLL clones co-express IgM and IgD, the composition and molecular mechanisms regulating BCR signaling regarding the two isotypes and the co-receptors with which they associate is lacking. Here we have addressed these issues. First, using Imaging Flow Cytometry, we evaluated BCR organization on the surface membrane of CLL cells from 11 patients who had participated in a 2H2O-labeling study that determined in vivoCLL B-cell birth rates (BR). We found that in all cases mIgM resided in more and larger surface clusters than mIgD. Also, a statistically significant, direct correlation was observed for IgM density and in vivoCLL-cell BR, with patients exhibiting more recently-divided cells having the highest expression of IgM. This was not the case for IgD. BCR signaling requires co-receptors that can co-localize differently with the two isotypes. Thus, we tested co-localization of stimulatory (CD20) and inhibitory (CD22) co-receptors with mIgM and mIgD, using the proximity ligation assay technique that discriminates 10 to 40 nm distances. Higher IgM:CD20 and lower IgD:CD20 co-localization ratios directly associated with in vivo BR. Conversely, patients whose CLL B cells showed greater IgM to CD22 co-localization ratios had lower BRs. Thus, association of IgM with stimulatory versus inhibitory co-receptors correlated with positive or negative regulation of CLL growth in vivo. Next, we questioned the extent that the observed differences in BCR organization affected the entire clone by measuring a marker of single cell metabolic activity - cell size. IgM and BR associated with entire clonal populations that were skewed toward larger, more active cells. Similarly, high BR CLLs displayed an increased mitochondrial maximal respiration and glycolytic activity and capacity, based on measurements of oxygen consumption rate and extracellular acidification rate, respectively. Since our findings supported a link between IgM- but not IgD-BCRs, growth rate in vivoand clonal metabolic activity, we questioned whether intrinsic, constitutive CLL BCR autonomous signaling differed for these two isotypes. To address this, we examined the signaling capacities of CLL-derived BCRs expressed as IgM or IgD isotypes, while maintaining the original IGHV-D-J and IGLV-J rearrangements. We used B cells that do not express endogenous BCR-related molecules but do express an inducible ERT2- SLP-65 fusion protein which enables examining Ca++influx. All BCRs expressed as IgM effectively mobilized Ca++ without need for an external ligand, indicating autonomous signaling. In contrast, BCRs expressed as IgD did not signal autonomously but required crosslinking with anti-BCR. Thus, only mIgM BCRs naturally transduce a signal in the absence of antigen. To determine the extent that BCR signaling influences clonal activity and in vivoBR, we compared cell size of CLL B cells taken from patients before and after 4 weeks of treatment with the Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib (iBTK). Ibrutinib had a strong treatment effect on cell activity, reducing overall cell size in 10/11 patients. A comparison of single cell areas for patients with lower (BR = 0.54%) and higher (BR = 1.42%) BRs showed an overall reduction of the median cell size for both cases. Thus, iBTK treatment leads to an equilibration of the cell size profile among the cases differing in BR, indicating that ibrutinib acts proportionally more potently on more metabolically active CLL B cells. Likewise, these findings are consistent with BCR signaling, transduced through BTK, being responsible for the increased cellular activity of aggressive CLL clones. In conclusion, increased mIgM density and proximity of mIgM to stimulatory receptors is linked to greater metabolic activity clones and increased rate of proliferationin vivo. Conversely, proximity of mIgM to inhibitory receptors has the opposite correlations.Moreover, only mIgM carries out autonomous signaling, providing another biologic trait linking all these features. Thus, our data support a tight, isotype-dependent regulation of BCR signaling and its consequences for CLL B cells. Further understanding these mechanisms should help generate novel therapies to modify the quality of BCR-transduced signaling and thus cell fate. Disclosures Barrientos: Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics/AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees. Rai:Cellectis: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees. Chiorazzi:AR Pharma: Equity Ownership; Janssen, Inc: Consultancy.

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Ex Vivo Mitochondrial Respiration Parallels Biochemical Response to Ibrutinib in CLL Cells

Cancers ◽

10.3390/cancers13020354 ◽

2021 ◽

Vol 13 (2) ◽

pp. 354

Author(s):

Subir Roy Chowdhury ◽

Cheryl Peltier ◽

Sen Hou ◽

Amandeep Singh ◽

James B. Johnston ◽

...

Keyword(s):

Mitochondrial Respiration ◽

Ex Vivo ◽

Standard Dose ◽

Cell Receptor ◽

Lymphocytic Leukemia ◽

Clinical Tool ◽

Potential Biomarker ◽

Apoptotic Pathways ◽

The Impact

Mitochondrial respiration is becoming more commonly used as a preclinical tool and potential biomarker for chronic lymphocytic leukemia (CLL) and activated B-cell receptor (BCR) signaling. However, respiration parameters have not been evaluated with respect to dose of ibrutinib given in clinical practice or the effect of progression on ibrutinib treatment on respiration of CLL cells. We evaluated the impact of low and standard dose ibrutinib on CLL cells from patients treated in vivo on mitochondrial respiration using Oroboros oxygraph. Cytokines CCL3 and CCL4 were evaluated using the Mesoscale. Western blot analysis was used to evaluate the BCR and apoptotic pathways. We observed no difference in the mitochondrial respiration rates or levels of plasma chemokine (C-C motif) ligands 3 and 4 (CCL3/CCL4), β-2 microglobulin (β-2 M) and lactate dehydrogenase (LDH) between low and standard doses of ibrutinib. This may confirm why clinical observations of the safety and efficacy of low dose ibrutinib are observed in practice. Of interest, we also observed that the mitochondrial respiration of CLL cells paralleled the increase in β-2 M and LDH at progression. Our study further supports mitochondrial respiration as a biomarker for response and progression on ibrutinib in CLL cells and a valuable pre-clinical tool.

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The Protein Kinase C Inhibitor MS-553 for the Treatment of Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2019-128871 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2077-2077

Author(s):

Elizabeth M. Muhowski ◽

Amy M. Lehman ◽

Sean D. Reiff ◽

Janani Ravikrishnan ◽

Rose Mantel ◽

...

Keyword(s):

Research Funding ◽

Ohio State University ◽

Lymphocytic Leukemia ◽

State University ◽

Advisory Committees ◽

University Patents ◽

Kinase C ◽

Hla Dr ◽

Bcr Signaling ◽

Equity Ownership

Introduction: Treatment of chronic lymphocytic leukemia (CLL) has been transformed by small molecule inhibitors targeting the B-cell receptor (BCR) signaling cascade. The first-in-class small molecule inhibitor of Bruton's Tyrosine Kinase (BTK), ibrutinib, is FDA approved as a frontline therapy for CLL. However, resistance to BTK inhibition has emerged in patients through acquisition of mutations in BTK or its immediate downstream target, PLCG2, emphasizing the need for alternative targets and therapies. BCR signaling remains intact in the presence of these mutations, making targeted inhibition of proteins downstream of BTK an attractive therapeutic strategy. Protein kinase C-β (PKCβ) is a downstream member of the BCR signaling pathway that we have previously demonstrated as an effective therapeutic target in CLL. MS-553 is a potent, ATP-competitive, reversible inhibitor of several PKC isoforms including PKCβ. Therefore, we evaluated the effects of MS-553 in primary CLL cells. Methods: Primary CLL cells were isolated by negative selection and treated with increasing concentrations of MS-553 to a maximum dose of 10 µM. BCR signaling changes were interrogated by change in target protein phosphorylation by immunoblot following a 24 hour drug incubation with and without phorbol ester stimulation (90 minutes) in CLL samples. Inhibition of CpG-mediated activation of CLL cells was measured using flow cytometry (CD86 and HLA-DR) in ibrutinib refractory patient samples at baseline and post-relapse due to the emergence of the p.C481S BTK mutation. CCL3 and CCL4 expression was measured by ELISA after 24 hours in primary CLL cells in the presence or absence of anti-IgM ligation. TNFα expression was also measured by ELISA in negatively selected, healthy donor T cells treated with MS-553 for 24 hours with or without anti-CD3 and anti-CD28 stimulation. Results: At 24 hours, 5 µM MS-553 inhibited downstream BCR signaling in primary CLL cells, demonstrated by 31% reduced phosphorylation of PKCβ (p=0.08, n=5) and several of its downstream targets including GSK3β (40%, p<.01, n=5) , ERK (46%, p=0.02, n=4) , and IκBα (56%, p=0.04, n=5) compared to vehicle treated, stimulated samples. CpG-mediated TLR9 stimulation increases expression of CD86 and HLA-DR in primary CLL cells. In baseline samples from ibrutinib treated patients, 10 µM MS-553 decreased expression of CD86 by 34% and HLA-DR by 91%. In matched patient samples post-relapse due to ibrutinib resistance, MS-553 (10 µM) maintained the ability to decrease expression of CD86 (49%) and HLA-DR (84%). Pro-inflammatory cytokine expression by primary CLL cells stimulated with anti-IgM decreased in the presence of 5 µM MS-553, with CCL3 decreasing by 36% (p=0.06, n=5) and CCL4 decreasing by 79% (p<.01, n=4) compared to vehicle treated, stimulated controls. TNFα expression by healthy T cells increased with anti-CD3 and anti-CD28 stimulation; 1 µM MS-553 reduced TNFα expression by 97% compared to vehicle treated, stimulated controls (p<.01, n=9). Conclusions: MS-553 is a novel and potent inhibitor of PKC demonstrating in vitro efficacy in CLL. MS-553 is able to inhibit BCR signaling by blocking phosphorylation of PKCβ and its downstream targets. CpG-mediated activation is reduced with MS-553 treatment in ibrutinib refractory patient samples both at baseline and post-relapse. Inflammatory signaling by primary CLL cells is further abrogated by MS-553 in its ability to decrease CCL3 and CCL4 cytokine expression. In an ongoing phase I clinical trial of MS-553, patient samples show a potent and dose dependent decrease in PKCβ activity as measured by a clinical biomarker assay. Together, our results suggest that MS-553 targets PKCβ in primary CLL to inhibit signaling and survival, establishing MS-553 as a potential therapeutic for treating CLL. These data justify continued preclinical and clinical work in the development of MS-553 for the treatment of CLL. Disclosures Niesman: MingSight Pharmaceuticals, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Zhang:MingSight Pharmaceuticals, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Byrd:BeiGene: Research Funding; Ohio State University: Patents & Royalties: OSU-2S; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Ohio State University: Patents & Royalties: OSU-2S; Ohio State University: Patents & Royalties: OSU-2S; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; Genentech: Research Funding; Acerta: Research Funding; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Novartis: Other: Travel Expenses, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Genentech: Research Funding; Acerta: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; BeiGene: Research Funding; BeiGene: Research Funding. Woyach:Verastem: Research Funding; Loxo: Research Funding; Morphosys: Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; AbbVie: Research Funding; Karyopharm: Research Funding.

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Microbial enzymes induce colitis by reactivating triclosan in the mouse gastrointestinal tract

Nature Communications ◽

10.1038/s41467-021-27762-y ◽

2022 ◽

Vol 13 (1) ◽

Author(s):

Jianan Zhang ◽

Morgan E. Walker ◽

Katherine Z. Sanidad ◽

Hongna Zhang ◽

Yanshan Liang ◽

...

Keyword(s):

Gut Microbiota ◽

Molecular Mechanisms ◽

Ex Vivo ◽

Metabolic Activation ◽

Consumer Products ◽

Environmental Chemicals ◽

Intestinal Microbes ◽

Targeted Inhibition

AbstractEmerging research supports that triclosan (TCS), an antimicrobial agent found in thousands of consumer products, exacerbates colitis and colitis-associated colorectal tumorigenesis in animal models. While the intestinal toxicities of TCS require the presence of gut microbiota, the molecular mechanisms involved have not been defined. Here we show that intestinal commensal microbes mediate metabolic activation of TCS in the colon and drive its gut toxicology. Using a range of in vitro, ex vivo, and in vivo approaches, we identify specific microbial β-glucuronidase (GUS) enzymes involved and pinpoint molecular motifs required to metabolically activate TCS in the gut. Finally, we show that targeted inhibition of bacterial GUS enzymes abolishes the colitis-promoting effects of TCS, supporting an essential role of specific microbial proteins in TCS toxicity. Together, our results define a mechanism by which intestinal microbes contribute to the metabolic activation and gut toxicity of TCS, and highlight the importance of considering the contributions of the gut microbiota in evaluating the toxic potential of environmental chemicals.

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A Non-Internalizing Anti-CD40 Antibody, CHIR-12.12, Blocks CD40L-Induced Cytokine Production and Mediates Greater ADCC Than Rituximab in Primary CLL Cells.

Blood ◽

10.1182/blood.v106.11.2964.2964 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2964-2964

Author(s):

Xia Tong ◽

Sharon Lea Aukerman ◽

Karen Lin ◽

Natasha Aziz ◽

Cheryl Goldbeck ◽

...

Keyword(s):

Cell Surface ◽

B Cell ◽

Cytokine Production ◽

Ex Vivo ◽

Lymphocytic Leukemia ◽

Target Cells ◽

Adcc Activity ◽

Effector Cells ◽

Cells Cultured

Abstract CD40 is expressed on chronic lymphocytic leukemia (CLL) cells, and CD40 activation leads to signaling critical for cell survival and proliferation. We have previously described a novel, fully human IgG1 anti-CD40 antagonistic monoclonal antibody, CHIR-12.12, generated in XenoMouse® mice (Abgenix, Inc.), and have demonstrated that it inhibits normal human B cell proliferation and survival and mediates potent antibody-dependent cellular cytotoxicity (ADCC) against primary CLL and non-Hodgkin’s lymphoma cells. In this study, we examined the ability of CHIR-12.12 to modulate cytokine production by primary CLL cells and compared the ADCC activity of CHIR-12.12 with rituximab against primary CLL cells. Primary CLL cells stimulated with CD40L produced a variety of cytokines, including IL-10, TNF-α , IL-8, GM-CSF, IL-6, MCP-1, and MIP-1β. Addition of CHIR-12.12 to primary CLL cells inhibited CD40L-mediated production of these cytokines. Cytokine production by primary CLL cells cultured with CHIR-12.12 alone in the absence of CD40L did not exceed levels produced by CLL cells cultured in medium. These data suggest that CHIR-12.12 is a potent antagonist for CD40L-mediated cytokine production by primary CLL cells and shows no agonistic activity by itself. We next compared the relative ADCC activity of CHIR-12.12 and rituximab against ex vivo primary CLL cells from 8 patients. CHIR-12.12 exhibited greater ADCC than rituximab against CLL cells from all patients. The average percent of maximum lysis by CHIR-12.12 and rituximab were 49 ± 16% and 31 ± 14%, respectively. CHIR-12.12 was greater than 10-fold more potent than rituximab, as measured by ED50 values (14.1 pM versus 155.5 pM, respectively). Quantitative CD20 and CD40 density on CLL cells and the degree of antibody internalization were investigated as potential reasons for the difference in ADCC activity. The greater ADCC potency and efficacy of CHIR-12.12 was not dependent on a higher density of cell surface CD40 molecules, as there were 1.3 to 14-fold higher numbers of CD20 than CD40 molecules on the cell surface. Antibody internalization studies using primary CLL cells conducted by flow cytometry and confocal microscopy show that upon binding to CD40 at 37°C, CHIR-12.12 remains uniformly distributed on the cell surface, even after 3 hours. In contrast, after binding at 37°C, rituximab is redistributed into caps and internalized. These data suggest that the potent ADCC activity of CHIR-12.12 may be partly related to its ability to remain on the surface of target cells uniformly, allowing optimal interaction with effector cells. Taken together, these results suggest that CHIR-12.12 may be effective at mediating potent ADCC against CLL cells in vivo. CHIR-12.12 is currently in Phase I trials for B-cell malignancies.

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A Human Hematopoietic Environment and Autologous T-Cell Growth Support Proliferation of Chronic Lymphocytic Leukemia B Cells in Immune-Deficient Mice

Blood ◽

10.1182/blood.v112.11.26.26 ◽

2008 ◽

Vol 112 (11) ◽

pp. 26-26

Author(s):

Davide Bagnara ◽

Matthew Kaufman ◽

Xiao J. Yan ◽

Kanti Rai ◽

Nicholas Chiorazzi

Keyword(s):

Cell Division ◽

T Cell ◽

B Cells ◽

Cell Growth ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Hematopoietic Microenvironment ◽

Cd34 Cells ◽

T Cell Growth

Abstract B-cell type chronic lymphocytic leukemia (B-CLL), an incurable disease of unknown etiology, results from the clonal expansion of a CD5+CD19+ B lymphocyte. Progress into defining the cell of origin of the disease and identifying a stem cell reservoir has been impeded because of the lack of reproducible models for growing B-CLL cells in vitro and in vivo. To date, attempts to adoptively transfer B-CLL cells into immune deficient mice and achieve engraftment and growth are sub-optimal. At least one possible cause for this is the murine microenvironment’s inability to support B-CLL survival and proliferation. We have attempted to overcome this barrier by creating a human hematopoietic microenvironment by reconstituting the tibiae of NOD/SCID/γcnull mice by intrabone (ib) injection of 1–3 × 105 CD34+ cord blood cells along with ~106 bone marrow-derived human mesenchymal stem cells (hMSCs). When human engraftment of 1–10% CD45+ cells was documented in the blood by immunofluorescence using flow cytometry, a total of 108 PBMCs from individual B-CLL patients were injected into the same bones. Before injection, B-CLL PBMCs were labeled with CFSE to permit distinction of leukemic B cells from normal B cells that might arise from the injected CD34+ cells. CFSE labeling also permitted tracking initial rounds of cell division in vivo. Every two weeks after B-CLL injection, peripheral blood from the mice was examined for the presence of cells bearing human CD45, CFSE, and various human lineage markers by flow cytometry. In the presence of a human hematopoietic microenvironment, CD5+CD19+ leukemic cells underwent at least 6 cell doublings, after which CFSE fluorescence was no longer detectable. Timing of B-CLL cell division varied among patients, occurring between 2 to 6 weeks after the injection of PBMC. In contrast, leukemic cells injected into mice that were not reconstituted by ib injection with hCD34+ cells and hMSCs or were reconstituted with only hMSCs failed to proliferate. Moreover the number of CFSE+CD5+CD19+ cells detected in the blood of mice with a human hematopoietic microenvironment far exceeded that in mice receiving only hMSC. Robust T-cell expansion occurred in several mice receiving CD34+ cells; in some instances T-cell growth was also found without hCD34+ cell injection, although in these cases it was usually less extensive. Based on genome-wide SNP analyses, the T cells were of B-CLL patient origin and not from hCD34+ cells. Furthermore, most of the mice with significant T-cell overexpansion died within 6 weeks of B-CLL cell injection from apparent graft vs. host disease. Therefore in subsequent experiments, we eliminated T cells by injecting an anti-CD3 antibody (OKT3); this treatment led to an inhibition of B-CLL cell proliferation. Moreover the percentage of CD38+ cells in the CFSE+CD5+CD19+ cell fraction was similar to that in the donor patient inoculum only in the mice in which T-cell-mediated B-CLL cell proliferation occurred. The percentage and intensity of CD38− expressing B-CLL cells was higher in the spleen and bone marrow (BM) of mice not treated with OKT3 antibody. Finally, the percentage of CFSE+CD5+CD19+ cells in the spleen far exceeded that in the blood, BM, liver and peritoneum, even when leukemic cells were no longer present in the blood and other organs; these findings suggest that the spleen is better at supporting B-CLL cell viability and proliferation than the other anatomic sites. These studies demonstrate conditions making adoptive xenogeneic transfer and clonal expansion of B-CLL cells into a mouse model possible. Factors conferring an advantage in this model include both a human hematopoietic environment and autologous T cell growth. Increased numbers of CD38+ B-CLL cells, similar to those in the patient, were only found when leukemic B cell division occurred. The optimal site for B-CLL cell growth was murine spleen. Since non-genetic factors promoting B-CLL expansion are not completely known, this model will be useful in discovering these as well as for studying the basic biology of this disease, such as if leukemic stem cells exist and also to conduct preclinical tests on possible therapeutics.

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The Bruton's Tyrosine Kinase Inhibitor, PCI-32765, Is Well Tolerated and Demonstrates Promising Clinical Activity In Chronic Lymphocytic Leukemia (CLL) and Small Lymphocytic Lymphoma (SLL): An Update on Ongoing Phase 1 Studies

Blood ◽

10.1182/blood.v116.21.57.57 ◽

2010 ◽

Vol 116 (21) ◽

pp. 57-57 ◽

Cited By ~ 13

Author(s):

Jan A. Burger ◽

Susan O'Brien ◽

Nathan Fowler ◽

Ranjana Advani ◽

Jeff Porte Sharman ◽

...

Keyword(s):

B Cell ◽

Phase 1 ◽

Study Drug ◽

Lymphocytic Leukemia ◽

Clinical Activity ◽

B Cell Malignancies ◽

Employment Equity ◽

Bcr Signaling ◽

Equity Ownership ◽

Grade 3

Abstract Abstract 57 Introduction: Bruton's tyrosine kinase (Btk) is a downstream mediator of B-cell receptor (BCR) signaling and is not expressed in T-cells or NK-cells. As such, Btk represents an ideal therapeutic target for B-cell malignancies dependent upon BCR signaling. Chronic lymphocytic leukemia (CLL)/small lymphocytic leukemia (SLL) has been reported to have constitutively active BCR signaling. PCI-32765 is a potent, selective, irreversible and orally bioavailable small molecule inhibitor of Btk that has pre-clinical activity in B-cell malignancies (Proc Natl Acad Sci 2010;107(29):13075-80). PCI-32765 was therefore moved forward to a Phase 1 study in B-cell malignancies including patients (pts) with CLL/SLL. A subsequent CLL/SLL-specific Phase 1b study was initiated to further explore safety, pharmacokinetics (PK), pharmacodynamics (PD), and efficacy of PCI-32765. This report includes a composite summary of the CLL/SLL experience in both of these studies. Pts and Methods: Pts with CLL/SLL who had relapsed or refractory disease after >1 prior treatment regimens were eligible for treatment in each of the studies whereas the second Phase 1b study also included a cohort of elderly pts (aged ≥ 65 years) with CLL/SLL who required treatment and were “treatment-naive”. Responses were assessed by the investigator using the International Working Group CLL criteria (Hallek et al, Blood 2008 for pts with CLL) and the International Workshop to Standardize Response Criteria for Non-Hodgkin's Lymphomas (Cheson et al, J Clin Oncol 2007 for pts with SLL). Results: To date, 30 CLL/SLL patients (including 4 treatment-naive) have been enrolled across the 2 studies. Eighty-four percent of subjects are men with an overall median age of 68 (range 44–82) years. Of the subjects with prior therapy for CLL/SLL the median number of prior therapies is 3 (range 1–4). Treatment has been well-tolerated; Grade ≥ 3 toxicities have been infrequent (10/30 pts; 33%). Two study-drug related serious adverse events have been reported: 1 case of viral adenitis (Grade 3) and 1 case of viral infection (Grade 2). Two adverse events have led to discontinuation of study drug: a small bowel obstruction (Grade 3) and exacerbation of chronic obstructive disease (Grade 3); both events were reported as unrelated to study drug. No study-drug related deaths have reported. There has been no change in either NK cell or T cell counts. Target inhibition as measured by a probe of Btk drug occupancy showed inhibition of Btk at PCI-32765 exposure levels of ≥ 245 ng•h/mL. Of the 14 patients currently evaluable for response using the pre-defined criteria, the overall response rate is 64% (1 complete remission [CR], 8 partial remissions [PR], and 4 SD). Both studies are ongoing and open to enrollment. An update on response rate, response duration, safety, and PD information will be presented on enrolled patients based on a November 2010 database cut-off. Conclusion: PCI-32765 is a novel oral and selective “first-in-human” inhibitor of Btk that induces objective partial and complete responses in a substantial proportion of pts with CLL/SLL and has a favorable safety profile. These data support further studies of both monotherapy and also combination treatment with PCI-32765 in CLL/SLL. Disclosures: O'Brien: Pharmacyclics, Inc: Honoraria, PI grant. Fowler:Pharmacyclics: Consultancy, Research Funding. Advani:Pharmacyclics, Inc: Honoraria, PI grant. Sharman:Pharmacyclics, Inc: Honoraria, PI grant. Furman:Pharmacyclics, Inc: PI grant. Izumi:Pharmacyclics, Inc: Employment. Buggy:Pharmacyclics, Inc: Employment, Equity Ownership. Loury:Pharmacyclics: Employment, Equity Ownership. Hamdy:Pharmacyclics, Inc: Employment, Equity Ownership.

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Stroma-Free Ex Vivo Expanded, CD34+ Cord Blood-Derived NK Cells Retain Lytic Activity After Long-Term Engraftment in NSGS Mice

Blood ◽

10.1182/blood.v120.21.580.580 ◽

2012 ◽

Vol 120 (21) ◽

pp. 580-580

Author(s):

Mark Wunderlich ◽

Mahesh Shrestha ◽

Lin Kang ◽

Eric Law ◽

Vladimir Jankovic ◽

...

Keyword(s):

Nk Cells ◽

Research Funding ◽

Nk Cell ◽

Ex Vivo ◽

Employment Equity ◽

Cell Product ◽

Equity Ownership ◽

Cellular Therapeutics

Abstract Abstract 580 Generating a large number of pure, functional immune cells that can be used in human patients has been a major challenge for NK cell-based immunotherapy. We have successfully established a cultivation method to generate human NK cells from CD34+ cells isolated from donor-matched cord blood and human placental derived stem cells, which were obtained from full-term human placenta. This cultivation method is feeder-free, based on progenitor expansion followed by NK differentiation supported by cytokines including thrombopoietin, stem cell factor, Flt3 ligand, IL-7, IL-15 and IL-2. A graded progression from CD34+ hematopoietic progenitor cells (HSC) to committed NK progenitor cells ultimately results in ∼90% CD3-CD56+ phenotype and is associated with an average 10,000-fold expansion achieved over 35 days. The resulting cells are CD16- and express low level of KIRs, indicating an immature NK cell phenotype, but show active in vitro cytotoxicity against a broad range of tumor cell line targets. The in vivo persistence, maturation and functional activity of HSC-derived NK cells was assessed in NSG mice engineered to express the human cytokines SCF, GM-CSF and IL-3 (NSGS mice). Human IL-2 or IL-15 was injected intraperitoneally three times per week to test the effect of cytokine supplementation on the in vivo transferred NK cells. The presence and detailed immunophenotype of NK cells was assessed in peripheral blood (PB), bone marrow (BM), spleen and liver samples at 7-day intervals up to 28 days post-transfer. Without cytokine supplementation, very few NK cells were detectable at any time-point. Administration of IL-2 resulted in a detectable but modest enhancement of human NK cell persistence. The effect of IL-15 supplementation was significantly greater, leading to the robust persistence of transferred NK cells in circulation, and likely specific homing and expansion in the liver of recipient mice. The discrete response to IL-15 versus IL-2, as well as the preferential accumulation in the liver have not been previously described following adoptive transfer of mature NK cells, and may be unique for the HSC-derived immature NK cell product. Following the in vivo transfer, a significant fraction of human CD56+ cells expressed CD16 and KIRs indicating full physiologic NK differentiation, which appears to be a unique potential of HSC-derived cells. Consistent with this, human CD56+ cells isolated ex vivo efficiently killed K562 targets in in vitro cytotoxicity assays. In contrast to PB, spleen and liver, BM contained a substantial portion of human cells that were CD56/CD16 double negative (DN) but positive for CD244 and CD117, indicating a residual progenitor function in the CD56- fraction of the CD34+ derived cell product. The BM engrafting population was higher in NK cultures at earlier stages of expansion, but was preserved in the day 35- cultured product. The frequency of these cells in the BM increased over time, and showed continued cycling based on in vivo BrdU labeling 28 days post-transfer, suggesting a significant progenitor potential in vivo. Interestingly, DN cells isolated from BM could be efficiently differentiated ex vivo to mature CD56+CD16+ NK cells with in vitro cytotoxic activity against K562. We speculate that under the optimal in vivo conditions these BM engrafting cells may provide a progenitor population to produce a mature NK cell pool in humans, and therefore could contribute to the therapeutic potential of the HSC-derived NK cell product. The in vivo activity of HSC-derived NK cells was further explored using a genetically engineered human AML xenograft model of minimal residual disease (MRD) and initial data indicates significant suppression of AML relapse in animals receiving NK cells following chemotherapy. Collectively, our data demonstrate the utility of humanized mice and in vivo xenograft models in characterizing the biodistribution, persistence, differentiation and functional assessment of human HSC-derived cell therapy products, and characterize the potential of HSC-derived NK cells to be developed as an effective off-the-shelf product for use in adoptive cell therapy approaches in AML. Disclosures: Wunderlich: Celgene Cellular Therapeutics: Research Funding. Shrestha:C: Research Funding. Kang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Law:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Jankovic:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Zhang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Herzberg:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Abbot:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hariri:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Mulloy:Celgene Cellular Therapeutics: Research Funding.

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Cytokine and T-Cell Phenotypic Changes Upon In Vivo Ibrutinib Therapy For CLL – Targeting Both CLL Cells and The Tumor-Microenvironment

Blood ◽

10.1182/blood.v122.21.2856.2856 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2856-2856 ◽

Cited By ~ 1

Author(s):

Carsten U Niemann ◽

Angelique Biancotto ◽

Betty Y. Chang ◽

Joseph J. Buggy ◽

J. Philip McCoy ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Inflammatory Cytokines ◽

T Cell Activation ◽

Serum Levels ◽

Lymphocytic Leukemia ◽

T Cell Subsets ◽

Bcr Signaling ◽

Pre Treatment

Abstract Introduction Proliferation of chronic lymphocytic leukemia (CLL) cells is highly dependent on the microenvironment. B-cell receptor (BCR) signaling and interactions of the tumor cells with elements of the tissue microenvironment including T cells and macrophages appear to be of particular importance (Burger et al, Blood 2009; Herishanu at al, Blood 2011; Bagnara at al, Blood 2011). The Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib is highly effective in blocking BCR signaling and leads to impressive clinical responses in CLL (Byrd et al, NEJM 2013). BTK is a member of the TEC kinase family that also includes TEC, IL2-inducible T cell kinase (ITK), and BMX/ETK. BTK is not expressed in T cells; however ITK, which is expressed in T cells, is directly inhibited by ibrutinib, and the drug reduces cytokine secretion from activated T cells without inducing apoptosis (Herman et al, Blood, 2011). Here, we sought to determine the in vivo effect of ibrutinib on T cells and cytokine levels in CLL patients treated with single agent ibrutinib. Methods The effect of ibrutinib on T-cell subsets, T-cell activation, and cytokine profiles was assessed in 10 CLL patients treated with 420mg ibrutinib daily in an ongoing phase II trial (NCT01500733). Matched samples of viably frozen peripheral blood mononuclear cells obtained from patients pre-treatment and after 6 months on ibrutinib were analyzed by flow cytometry. Cytokine levels pre-treatment and on days 1, 28, months 2, and 6 on ibrutinib were measured in the same patients using the Milliplex human cytokine assay. Results Consistent with inhibition of BCR signaling in CLL cells, CCL3 and CCL4 serum levels were rapidly and significantly decreased by ibrutinib as described previously (Ponader et al, Blood, 2012). In addition, serum levels of a number of inflammatory cytokines including IL6, IL8, IFNg, and TNFα were decreased by > 50% by day 28 of ibrutinib treatment and remained so by 6 months. This is of specific interest as “pseudoexhausted” T cells from CLL patients were recently shown to secrete high amounts of IFNg, and TNFα (Riches et al, Blood 2013). Thus, the decreased levels of inflammatory cytokines may reflect a reversal of T cell “pseduoexhaustion”. Furthermore, the immunosuppressive cytokine IL10, a Th1-type cytokine that is secreted by CLL cells and activated T cells, was also rapidly and significantly reduced. These in vivo data are consistent with previous in vitro data showing decreased secretion of IL6 and IL10 from T cells upon exposure to ibrutinib (Herman et al, Blood, 2011). Thus, ibrutinib appears to reduce cytokine and chemokine secretion from both CLL and T cells resulting in an overall decrease in inflammatory cytokines. While absolute T-cell numbers showed little change on treatment, we found that ibrutinib reduced the frequency of activated CD4+ T cells (Table). Furthermore, for 3 out of 4 patients, the percentage of Ki67 positive T cells in the peripheral blood decreased on ibrutinib therapy (mean decrease 63%). The frequency of the Th17 T-cell subset was also diminished. Consistently, a decrease in serum levels of IL17 was seen in the two patients having detectable IL17 levels pre-treatment. While changes in the cytokine pattern (decrease in IFNg and IL10) might suggest inhibition of a Th1-type response, there was no change in the ratio of Th1 to Th2 T-cell subsets by immunophenotyping. Conclusions We here demonstrate a decrease in the levels of inflammatory cytokines and in T-cell activation in CLL patients treated with ibrutinib. Whether this is a direct consequence of BTK inhibition in B-cells or, at least in part, results from inhibition of T-cell signaling remains to be determined. Nevertheless, our data indicate that ibrutinib significantly alters the composition of the tumor microenvironment in CLL, affecting soluble as well as cellular elements. These effects may be important for clinical response and the development of combination therapies and therefore deserve further study. Supported by the Intramural Research Program of NHLBI. We thank our patients for participating and acknowledge Pharmacyclics for providing study drug. Disclosures: Off Label Use: Ibrutinib in chronic lymphocytic leukemia. Chang:Pharmacyclics: Employment, Equity Ownership. Buggy:Pharmacyclics: Employment, Equity Ownership.

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