A Detailed Analysis of Parameters Supporting the Engraftment and Growth of Chronic Lymphocytic Leukemia Cells in Immune-Deficient Mice

Patient-derived xenograft models of chronic lymphocytic leukemia (CLL) can be created using highly immunodeficient animals, allowing analysis of primary tumor cells in an in vivo setting. However, unlike many other tumors, CLL B lymphocytes do not reproducibly grow in xenografts without manipulation, proliferating only when there is concomitant expansion of T cells. Here we show that in vitro pre-activation of CLL-derived T lymphocytes allows for a reliable and robust system for primary CLL cell growth within a fully autologous system that uses small numbers of cells and does not require pre-conditioning. In this system, growth of normal T and leukemic B cells follows four distinct temporal phases, each with characteristic blood and tissue findings. Phase 1 constitutes a period during which resting CLL B cells predominate, with cells aggregating at perivascular areas most often in the spleen. In Phase 2, T cells expand and provide T-cell help to promote B-cell division and expansion. Growth of CLL B and T cells persists in Phase 3, although some leukemic B cells undergo differentiation to more mature B-lineage cells (plasmablasts and plasma cells). By Phase 4, CLL B cells are for the most part lost with only T cells remaining. The required B-T cell interactions are not dependent on other human hematopoietic cells nor on murine macrophages or follicular dendritic cells, which appear to be relatively excluded from the perivascular lymphoid aggregates. Notably, the growth kinetics and degree of anatomic localization of CLL B and T cells is significantly influenced by intravenous versus intraperitoneal administration. Importantly, B cells delivered intraperitoneally either remain within the peritoneal cavity in a quiescent state, despite the presence of dividing T cells, or migrate to lymphoid tissues where they actively divide; this dichotomy mimics the human condition in that cells in primary lymphoid tissues and the blood are predominately resting, whereas those in secondary lymphoid tissues proliferate. Finally, the utility of this approach is illustrated by documenting the effects of a bispecific antibody reactive with B and T cells. Collectively, this model represents a powerful tool to evaluate CLL biology and novel therapeutics in vivo.

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Immunomodulatory effects of different intravenous immunoglobulin preparations in chronic lymphocytic leukemia

Scientific Reports ◽

10.1038/s41598-021-92412-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ana Colado ◽

Esteban Enrique Elías ◽

Valeria Judith Sarapura Martínez ◽

Gregorio Cordini ◽

Pablo Morande ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

T Cell Activation ◽

Cell Activation ◽

Lymphocytic Leukemia ◽

Immunomodulatory Effects ◽

Immunoglobulin Preparations

AbstractHypogammaglobulinemia is the most frequently observed immune defect in chronic lymphocytic leukemia (CLL). Although CLL patients usually have low serum levels of all isotypes (IgG, IgM and IgA), standard immunoglobulin (Ig) preparations for replacement therapy administrated to these patients contain more than 95% of IgG. Pentaglobin is an Ig preparation of intravenous application (IVIg) enriched with IgM and IgA (IVIgGMA), with the potential benefit to restore the Ig levels of all isotypes. Because IVIg preparations at high doses have well-documented anti-inflammatory and immunomodulatory effects, we aimed to evaluate the capacity of Pentaglobin and a standard IVIg preparation to affect leukemic and T cells from CLL patients. In contrast to standard IVIg, we found that IVIgGMA did not modify T cell activation and had a lower inhibitory effect on T cell proliferation. Regarding the activation of leukemic B cells through BCR, it was similarly reduced by both IVIgGMA and IVIgG. None of these IVIg preparations modified spontaneous apoptosis of T or leukemic B cells. However, the addition of IVIgGMA on in vitro cultures decreased the apoptosis of T cells induced by the BCL-2 inhibitor, venetoclax. Importantly, IVIgGMA did not impair venetoclax-induced apoptosis of leukemic B cells. Overall, our results add new data on the effects of different preparations of IVIg in CLL, and show that the IgM/IgA enriched preparation not only affects relevant mechanisms involved in CLL pathogenesis but also has a particular profile of immunomodulatory effects on T cells that deserves further investigation.

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Evaluation of vecabrutinib as a model for non-covalent BTK/ITK inhibition for treatment of chronic lymphocytic leukemia

Blood ◽

10.1182/blood.2021011516 ◽

2021 ◽

Author(s):

Billy Michael Chelliah Jebaraj ◽

Annika Müller ◽

Rashmi Priyadharshini Dheenadayalan ◽

Sascha Endres ◽

Philipp M. Roessner ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Chronic Lymphocytic Leukemia ◽

Inhibitory Effect ◽

Lymphocytic Leukemia ◽

Transfer Model ◽

Treatment Benefit ◽

Btk Inhibitors

Covalent Bruton tyrosine kinase (BTK) inhibitors such as ibrutinib have proven to be highly beneficial in the treatment of chronic lymphocytic leukemia (CLL). Interestingly, the off-target inhibition of IL-2-inducible T-cell kinase (ITK) by ibrutinib may also play a role in modulating the tumor microenvironment, potentially enhancing the treatment benefit. However, resistance to covalently binding BTK inhibitors can develop by a mutation in cysteine 481 of BTK (C481S), which prevents the irreversible binding of the drugs. In the present study we performed pre-clinical characterization of vecabrutinib, a next generation non-covalent BTK inhibitor, with ITK inhibitory properties similar to those of ibrutinib. Unlike ibrutinib and other covalent BTK inhibitors, vecabrutinib showed retention of the inhibitory effect on C481S BTK mutants in vitro, similar to that of wildtype BTK. In the murine Eµ-TCL1 adoptive transfer model, vecabrutinib reduced tumor burden and significantly improved survival. Vecabrutinib treatment led to a decrease in CD8+ effector and memory T-cell populations, while the naïve populations were increased. Of importance, vecabrutinib treatment significantly reduced frequency of regulatory CD4+ T-cells (Tregs) in vivo. Unlike ibrutinib, vecabrutinib treatment showed minimal adverse impact on activation and proliferation of isolated T-cells. Lastly, combination treatment of vecabrutinib with venetoclax was found to augment treatment efficacy, significantly improve survival and lead to favourable reprogramming of the microenvironment in the murine Eµ-TCL1 model. Thus, non-covalent BTK/ITK inhibitors such as vecabrutinib may be efficacious in C481S BTK mutant CLL, while preserving the T-cell immunomodulatory function of ibrutinib.

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Defective T cell-mediated, isotype-specific immunoglobulin regulation in B cell chronic lymphocytic leukemia

Blood ◽

10.1182/blood.v71.4.1012.bloodjournal7141012 ◽

1988 ◽

Vol 71 (4) ◽

pp. 1012-1020 ◽

Cited By ~ 1

Author(s):

JS Moore ◽

MB Prystowsky ◽

RG Hoover ◽

EC Besa ◽

PC Nowell

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Cell Activity ◽

Lymphocytic Leukemia ◽

Specific Immunoglobulin ◽

Cell Chronic Lymphocytic Leukemia

The consistent occurrence of T cell abnormalities in patients with B cell chronic lymphocytic leukemia (B-CLL) suggest that the non- neoplastic host T cells may be involved in the pathogenesis of this B cell neoplasm. Because potential defects of immunoglobulin regulation are evident in B-CLL patients, we investigated one aspect of this by studying the T cell-mediated immunoglobulin isotype-specific immunoregulatory circuit in B-CLL. The existence of class-specific immunoglobulin regulatory mechanisms mediated by Fc receptor-bearing T cells (FcR + T) through soluble immunoglobulin binding factors (IgBFs) has been well established in many experimental systems. IgBFs can both suppress and enhance B cell activity in an isotype-specific manner. We investigated the apparently abnormal IgA regulation in a B-CLL patient (CLL249) whose B cells secrete primarily IgA in vitro. Enumeration of FcR + T cells showed a disproportionate increase in IgA FcR + T cells in the peripheral blood of this patient. Our studies showed that the neoplastic B cells were not intrinsically unresponsive to the suppressing component of IgABF produced from normal T cells, but rather the IgABF produced by the CLL249 host T cells was defective. CLL249 IgABF was unable to suppress IgA secretion by host or normal B cells and enhanced the in vitro proliferation of the host B cells. Size fractionation of both normal and CLL249 IgABF by gel-filtration high- performance liquid chromatography (HPLC) demonstrated differences in the ultraviolet-absorbing components of IgABF obtained from normal T cells v that from our patient with defective IgA regulation. Such T cell dysfunction may not be restricted to IgA regulation, since we have found similar expansion of isotype-specific FcR + T cells associated with expansion of the corresponding B cell clone in other patients with B-CLL. These data suggest that this T cell-mediated regulatory circuit could be significantly involved in the pathogenesis of B-CLL.

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The Plant-Derived Agent Silvestrol Has B-Cell Selective Activity In Vitro in Chronic Lymphocytic Leukemia Patient Cells and In Vivo in the Tcl-1 Mouse Model of CLL.

Blood ◽

10.1182/blood.v110.11.3123.3123 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3123-3123 ◽

Cited By ~ 1

Author(s):

David M. Lucas ◽

Ryan B. Edwards ◽

Michael D. De Lay ◽

Derek A. West ◽

Gerard Lozanski ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Tumor Cells ◽

Lymphocytic Leukemia ◽

Pathological Examination ◽

Cell Percentage ◽

Leukocyte Counts

Abstract Chronic Lymphocytic Leukemia (CLL) is an incurable disease with limited therapeutic options, especially for high-risk populations such as the del(17p13) patient subset. Currently available therapies for CLL, even if effective, can have significant detrimental effects on remaining T cells, leaving patients at risk of potentially lethal opportunistic infections. New agents with unique mechanisms of action, independence of key resistance pathways, and selectivity for tumor cells are crucial to make an impact on patient survival. Silvestrol, a structurally unique compound isolated from the plant genus Aglaia, exhibited potent activity against several tumor cell lines and moderate in vivo activity in the P388 mouse leukemia model (J. Org. Chem. 2004, 69:3350; ibid. 69:6156). Based on these results, we tested silvestrol against tumor cells obtained from CLL patients. The LC50 (concentration lethal to 50% of cells relative to untreated control) of silvestrol was 6.5 nM at 72 hours by MTT assay. We performed assays to determine CLL patient cell viability at 72 hours with or without drug washout at various times. In these studies, silvestrol showed up to 50% killing at 72 hours with only a four hour exposure, and reached maximum efficacy with a 24 hour exposure. Silvestrol was similarly effective against cells from CLL patients with or without del(17p13). Furthermore, there was no significant difference in silvestrol-mediated cytotoxicity between lymphoblastic cells with a ten-fold overexpression of Bcl-2 relative to control cells. In MTT assays using isolated CD3+ or CD19+ cells, and in whole blood from healthy volunteers and CLL patients, silvestrol demonstrated substantially more cytotoxicity toward B cells than T cells. We then tested silvestrol using Tcl-1 transgenic mice, which are initially normal but develop a slow-progressing B cell leukemia very similar to human CLL. Lymphocytes obtained from spleens of Tcl-1 mice with leukemia were incubated ex vivo with 80 nM silvestrol and analyzed by flow cytometry. Silvestrol produced an 88% reduction in the B cell percentage after 24 hours with no negative effect on the T cell percentage (8% increase), in contrast to 1 μM fludarabine, which affected both B cell (22% reduction) and T cell (14% reduction) subsets. Non-leukemic mice of the Tcl-1 background strain were treated with 1.0, 1.5 and 2.5 mg/kg/day silvestrol for 5 days to determine a tolerable dose. Three of five mice treated with 2.5 mg/kg/day died at the beginning of the second week of treatment. However, none of the animals treated at 1.0 or 1.5 mg/kg showed signs of toxicity or weight loss even after two full weeks of treatment and were normal at pathological examination. Tcl-1 mice with evidence of leukemia as determined by elevated leukocyte counts and enlarged spleens were then treated with silvestrol at 1.5 mg/kg/day × 5 days for two weeks. Treated mice experienced decreased overall leukocyte counts relative to vehicle controls. Furthermore, CD19+ cell numbers and percentages diminished substantially while the T cells were only mildly affected. Additional leukemic Tcl-1 mice are currently being treated and studies are underway examining the mechanism of action of silvestrol in CLL cells.

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Defective T Cell Migration in Chronic Lymphocytic Leukemia Is Repaired by Lenalidomide

Blood ◽

10.1182/blood.v112.11.3117.3117 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3117-3117

Author(s):

Alan G. Ramsay ◽

Lena Svensson ◽

Nancy Hogg ◽

John G. Gribben

Keyword(s):

T Cells ◽

Cell Migration ◽

T Cell ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Cd8 T Cells ◽

Direct Contact ◽

Immune Surveillance ◽

Lymphocytic Leukemia ◽

T Cell Migration

Abstract We have previously demonstrated that multiple gene expression abnormalities are induced in T cells from chronic lymphocytic leukemia (CLL) patients including defects within the actin cytoskeleton signaling pathways that control immune recognition and motility (Gullu et al. JCI, 2005). T cell immune surveillance requires rapid migratory responses and LFA-1 (CD11a/CD18; αLβ2) is a promigratory receptor that engages the cytoskeleton to control migration. We hypothesized that CLL T cells may exhibit dysfunctional migration in response to ICAM-1, the principal ligand for LFA-1. Using time lapse microscopy, we observed significantly reduced chemokine SDF-1 (CXCL12) induced migration on ICAM-1 of CLL CD4 and CD8 T cells compared to age-matched healthy donor T cells. Healthy T cells tracked for 45 min displayed a random course of migration with an average speed of ~ 8 μm/min, whereas CLL T cells were slower ~ 5 μm/min (n=14, ~ 30% reduction, p<0.01). We further postulated that direct contact of CLL tumor cells with healthy T cells would induce this migratory defect. Healthy CD4 or CD8 T cells were cocultured with either allogeneic CLL B cells or allogeneic healthy B cells and subsequently used in migration assays. Co-culture with CLL cells resulted in significantly reduced T cell migration compared with co-culture with healthy B cells (~ 44% reduction in migration, n=6, p<0.01). Evidence that direct contact was required to induce this migratory defect was shown when no effect was observed when cell-cell adhesion was prevented by pretreatment of CLL cells with anti-ICAM-1 blocking antibody prior to primary co-culture with healthy T cells. This cancer-induced migratory defect was repaired when CLL T cells were pretreated with the immunomodulatory drug Lenalidomide (1μM for 1hr). Treatment with this agent enhanced the migratory potential of CLL T cells to a speed comparable to untreated and treated healthy T cells. The finding that lenalidomide can restore rapid migration in patient T cells provides evidence that this agent may increase immune surveillance in CLL patients.

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Selective Clearance of Chronic Lymphocytic Leukemia Cells in Vivo Following Treatment with UC99961, an Anti-ROR1 Monoclonal Antibody

Blood ◽

10.1182/blood.v120.21.3886.3886 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3886-3886

Author(s):

Eva Hellqvist ◽

Christina C.N. Wu ◽

George F. Widhopf ◽

Alice Shih ◽

Rommel Tawatao ◽

...

Keyword(s):

Monoclonal Antibody ◽

T Cells ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Cord Blood ◽

Peritoneal Lavage ◽

Lymphocytic Leukemia ◽

Leukemia Cells ◽

Human Cord Blood

Abstract Abstract 3886 ROR1 is a receptor-tyrosine kinase like protein expressed on the surface of chronic lymphocytic leukemia (CLL) B cells, but not on normal mature B cells, suggesting that it may be a promising therapeutic target. We have generated a chimeric monoclonal antibody (mAb), UC99961, which binds to an intradomain epitope of human ROR1 (hROR1). UC99961 binds the same epitope as the murine anti-hROR1 mAb, UC D10–001, which has direct cytotoxic effects on hROR1 positive CLL cells. In this study we investigated the in-vivo anti-leukemic activity and tolerability of UC99961 on ROR1+ primary patient CLL cells and human cord-blood-derived B cells and T cells, respectively. For these studies, immunodeficient RAG2−/−γc−/− neonatal mice were reconstituted with a human immune system by intrahepatic xenotransplantation of 1×105 CD34+ human cord blood progenitor cells. Eight to ten weeks post transplantation, cord blood engraftment was verified by peripheral blood screening, at which point the mice received an intraperitoneal transplantation of 2×107 primary patient ROR1+ CLL cells. Twenty-four hours after CLL transplantation, five animals per group were each treated with a single intraperitoneal injection (10mg/kg) of UC99961, UC D10–001, or control IgG. Seven days following mAb treatment, the animals were sacrificed and marrow, spleen, thymus, and peritoneal lavage samples were collected and analyzed by flow cytometry for CLL cells, as well as normal cord-blood-derived B cells and T cells. To confirm mAb administration according to the study design, serial residual ROR1 plasma antibody levels were determined by ELISA. Results from three consecutive experiments using leukemia cells from two different patients showed that the vast majority of CLL B cells remained in the peritoneal cavity of the animals and did not migrate to other hematopoietic organs. Both anti-hROR1 mAbs UC99961 and UC D10–001 significantly reduced the average number of harvested CLL cells in the peritoneal lavage compared to control IgG (99% and 71% reduction respectively), while cord-blood-derived T cells (CD45+3+) in thymus remained unaffected by the mAb treatment. For the majority of cord-blood-derived B cells in marrow and spleen, no significant reduction could be observed after UC99961 or UC D10–001 mAb treatment. A small CD19+ROR1+CD34− cord-blood-derived B cell population was identified in marrow and spleen that was reduced after UC99961 and UC D10–001 mAb treatment. This study demonstrates that the anti-human ROR1 specific mAbs have in vivo anti-leukemic activity with minimal impact on human cord-blood-derived B cells and T cells. From these results, UC99961 appears to be an excellent candidate antibody for future clinical studies for patients with CLL. Disclosures: No relevant conflicts of interest to declare.

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Lenalidomide Promotes The Expansion Of CD8 T Cells With An Effector Memory Phenotype In a Murine Xenograft Model Of Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v122.21.119.119 ◽

2013 ◽

Vol 122 (21) ◽

pp. 119-119

Author(s):

Rita Simone ◽

Sonia Marsilio ◽

Piers E.M. Patten ◽

Gerardo Ferrer ◽

Shih-Shih Chen ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

T Lymphocytes ◽

B Cell ◽

Xenograft Model ◽

Lymphocytic Leukemia ◽

Effector Memory ◽

Cell Engraftment

Abstract Lenalidomide (Revlimid®), a thalidomide analogue, is an orally administered second generation immunomodulator with anti-angiogenic and anti-neoplastic properties. Initial studies treating patients with chronic lymphocytic leukemia (CLL) suggest that lenalidomide can have considerable efficacy and that its mode of action is mainly indirect, affecting non-malignant cells in the microenvironment, in particular T lymphocytes. Because a recently described xenograft model for CLL has highlighted the importance of CLL-derived, autologous T cells in promoting leukemic B-cell engraftment and growth in vivo, we have studied the influence of lenalidomide on the expansion of CLL B- and T-lymphocytes in this model. After an initial 12 day culture of FACS-isolated CLL-derived T cells with or without anti-CD3/CD28 beads plus IL-2 (30 IU/ml), T lymphocytes were transferred into alymphoid NSG mice via the retro-orbital plexus (day 0). On day 7, CLL cells were delivered retro-orbitally. These recipient animals are referred to as “T + PBMC mice”. Mice that did not receive T cells on day 0 but were given CLL PBMCs at day 7, with or without lenalidomide, served as controls (“PBMC only mice”). Recipient mice received lenalidomide (10mg/kg/day) or vehicle control daily by gavage starting at day 0. All mice were sacrificed at day 28 (28 days after T-cell and 21 days after B-cell transfer), and blood, spleen, and bone marrow were collected. On this material, four analyses were performed: [1] level of human CD45+ cell engraftment; [2] numbers and types of CLL-derived T cells; [3] numbers of CLL B cells; and [4] levels of cytokines reflective of Th1 and Th2 immune responses. There was a clear enhancement in human hematopoietic (CD45+) cell engraftment in those mice exposed to lenalidomide. This was most marked for the PBMC only mice (vehicle: 10.64%; lenalidomide: 38.53%), although it was also evident for T + PBMC mice (vehicle: 55.96%; lenalidomide: 69.65%). T-cell phenotyping was carried out, before and after cell culture and also at sacrifice. Prior to culture, CLL samples contained on average ∼96% CD5+CD19+ cells and ∼3% CD5+CD19- cells; for the latter, ∼67% were CD4+ and ∼33% CD8+. After 12-day culture, these percentages remained largely unchanged. However, the numbers and types of T cells recovered from the spleens at sacrifice were quite different after in vivo exposure to lenalidomide. For the PBMC only, the percentages of CD4+ and CD8+ cells in the spleens differed somewhat based on lenalidomide exposure (CD4: Vehicle 86% vs. Lenalidomide 61%; CD8: Vehicle 10% vs. Lenalidomide 28%). However, this change was dramatic for the T + PBMC mice (CD4: Vehicle 64.1% vs. Lenalidomide 28.9%; CD8: Vehicle 34% vs. Lenalidomide 62%). Furthermore, when the CD8+ cells from these animals were subsetted based on antigen-experience and function, it appeared that lenalidomide exposure had led to the outgrowth of a greater number of effector memory (CD45RO+ CD62L-) than central memory (CD45RO+ CD62L+) T-cells. For CLL-derived B cells, the numbers differed, based not only on lenalidomide exposure but also on prior in vitro activation. Specifically, in PBMC only mice, the addition of lenalidomide led to increased numbers of CLL B cells in the spleen (Vehicle: 7.81% vs. Lenalidomide: 14%). Conversely, in the T + PBMC mice, the numbers of B cells decreased (Vehicle: 2.36% vs. Lenalidomide: 0.34%). An analysis of Th1 and Th2-related cytokines in the plasmas of the mice at sacrifice revealed a fall in IL-4, IL-5, and IL-10 and a marked increase in IFNg, consistent with a Th2 to Th1 transition. The above data suggest that administration of lenalidomide permits greater engraftment of human hematopoietic cells in alymphoid mice. Although this enhancement involves all members of the hematopoietic lineage, T cells, in particular CD8+ effector memory T cells, emerge in excess over time. This CD8 expansion is associated with diminished levels of CLL B cells suggesting that the decrease is due to T-cell mediated cytolysis. In contrast, in the absence of prior T-cell activation, CLL T cells appear to support better CLL B-cell growth. These findings suggest that lenalidomide alters B-cell expansion in vivo depending on the activation and differentiation state of the autologous T-cell compartment. They also implicate the generation of cytolytic T cells as one mechanism whereby lenalidomide leads to clinical improvement in CLL. Disclosures: Allen: Celgene Corporation: Honoraria.

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Preclinical Analyses Support Clinical Investigation of Combined Anti-CD19 CAR-T Cell, JCAR017 with Ibrutinib for the Treatment of Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v128.22.3231.3231 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3231-3231 ◽

Cited By ~ 2

Author(s):

Jim Qin ◽

Alex Baturevych ◽

Sherri Mudri ◽

Ruth Salmon ◽

Michael Ports

Keyword(s):

T Cells ◽

T Cell ◽

Chronic Lymphocytic Leukemia ◽

Tumor Burden ◽

Lymphocytic Leukemia ◽

Initial Growth ◽

Car T Cells ◽

Car T Cell ◽

Car T

Abstract Chronic lymphocytic leukemia (CLL) drives systemic immune suppression and T cell dysfunction in patients, highlighting an important consideration in this setting for the manufacturing and efficacy of adoptive cell therapies using autologous T cells. In clinical studies, anti-CD19 CAR-T cells produce durable and complete responses in leukemic and some lymphomatous B cell malignancies. While preconditioning with cyclophosphamide (Cy) and fludarabine (Flu) has improved CAR-T responses in CLL patients, reported complete response rates still have been below 50%; additional therapeutic strategies likely will be required. Ibrutinib, an irreversible inhibitor of BTK, has been approved as a frontline treatment option for patients with CLL. The potent off-BTK activity of ibrutinib on ITK and TEC family kinases could affect CAR T cell biology. Recent work highlighted the ability of ibrutinib to restore CLL patient T cell functionality, enhance CAR-T production and potentially improve clinical efficacy. Additional preclinical work demonstrated improved tumor clearance when anti-CD19 CAR T cells were combined with ibrutinib in several murine tumor models. A preclinical evaluation of the combination between the anti-CD19 CAR-T product, JCAR017, and ibrutinib was performed to determine feasibility for clinical use in CLL. JCAR017 is a second generation CAR-T cell product candidate that contains a 41BB costimulatory endo-domain and is currently in phase 1 trials for non-Hodgkin lymphoma (NHL). A series of in vitro studies assessed the functional activity of JCAR017 cells (derived from 3 healthy donors), in combination with ibrutinib (500-0.05nM), across a dose range covering the cMax and cMin. Cytolytic activity was monitored by co-culturing CAR-T cells with ibrutinib-resistant K562 CD19 tumor cells at an effector-to-target ratio of 2.5:1. Ibrutinib, at concentrations tested, did not inhibit the cytolytic function of JCAR017 cells. For cells derived from some donors, addition of ibrutinib appeared to increase % target killing. To address ibrutinib effects on JCAR017 activation, cell surface markers and cytokines were tracked over 4 days following stimulation with irradiated K562 CD19 cells. We observed no significant effect on JCAR017 surface expression of CD25, CD38, CD39, CD95, CD62L, CCR7, or CD45RO, or of EGFRt, a surrogate transduction marker. With addition of ibrutinib, we observed a modest decrease in the percentage of cells expressing CD69, CD107a and PD-1. With 5 and 50nM of ibrutinib, there was a 19.5% (p<0.01) average increase in IFNγ production. At supraphysiological concentrations (500nM) we observed a 20% (p<0.05) decrease in IL-2 production, suggesting ibrutinib at high concentrations may dampen T cell activation. CAR-T cell expansion after repeated antigen stimulation has been shown to be a predictor of in vivo efficacy. JCAR017 cells stimulated every 3-4 days with irradiated target cells in the presence of ibrutinib showed no inhibition of initial growth. However, after 5 rounds of stimulation, JCAR017 + ibrutinib cells from 1 donor had enhanced proliferation compared to control, untreated cells (p<0.05). Interestingly, after 5 rounds of serial stimulation, we observed an increased proportion of CD4+CXCR3+CRTh2- Th1 cells with 500nM ibrutinib treatment compared to control (p<0.01). We assessed the in vivo anti-tumor activity of JCAR017 in combination with ibrutinib using NSG mice injected with 5x105 Nalm6-luciferase cells. After tumor engraftment, a suboptimal dose (5x105) of JCAR017 cells was transferred to mice and ibrutinib (25 mg/kg qd) was administered for the duration of the study. Ibrutinib treatment alone had no effect on tumor burden compared to vehicle treatment. Mice treated with a suboptimal JCAR017 dose + ibrutinib showed decreased tumor burden (p<0.05) and increased median survival from 44 days to >80 days (p<0.001) compared to the group receiving the suboptimal JCAR017 dose + vehicle. Similar effects were seen in replicate studies using JCAR017 cells produced from multiple donors. Ex vivo evaluation for CAR-T quantitation and immunophenotyping was also performed. Taken together, the results suggest that ibrutinib enhances intrinsic JCAR017 activity and may improve outcomes in CLL patients treated with anti-CD19 CAR T therapy, irrespective of BTK mutational status. A Phase 1b study of JCAR017 in combination with ibrutinib for BTKi R/R CLL is planned. Disclosures Qin: Juno Therapeutics: Employment. Baturevych:Juno Therapeutics: Employment. Mudri:Juno Therapeutics: Employment, Equity Ownership. Salmon:Juno Therapeutics: Employment. Ports:Juno Therapeutics: Employment.

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Leukemia-associated monoclonal and oligoclonal TCR-BV use in patients with B-cell chronic lymphocytic leukemia

Blood ◽

10.1182/blood-2002-03-0746 ◽

2003 ◽

Vol 101 (3) ◽

pp. 1063-1070 ◽

Cited By ~ 50

Author(s):

Mohammad-Reza Rezvany ◽

Mahmood Jeddi-Tehrani ◽

Hans Wigzell ◽

Anders Österborg ◽

Håkan Mellstedt

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

B Cell ◽

Cd8 T Cells ◽

Leukemia Cell ◽

Lymphocytic Leukemia ◽

Cell Chronic Lymphocytic Leukemia

Abstract T-cell receptor–B-variable (TCR-BV) gene usage and the CDR3 size distribution pattern were analyzed by reverse transcription–polymerase chain reaction (RT-PCR) in patients with B-cell chronic lymphocytic leukemia (B-CLL) to assess the T-cell repertoire. The use of TCR-BV families in CD4 and CD8 T cells stimulated with autologous activated leukemic cells was compared with that of freshly obtained blood T cells. Overexpression of individual TCR-BV families was found in freshly isolated CD4 and CD8 T cells. Polyclonal, oligoclonal, and monoclonal TCR-CDR3 patterns were seen within such overexpressed native CD4 and CD8 TCR-BV families. In nonoverexpressed TCR-BV families, monoclonal and oligoclonal populations were noted only within the CD8 subset. After in vitro stimulation of T cells with autologous leukemic B cells, analyses of the CDR3 length patterns showed that in expanded TCR-BV populations, polyclonal patterns frequently shifted toward a monoclonal/oligoclonal profile, whereas largely monoclonal patterns in native overexpressed TCR-BV subsets remained monoclonal. Seventy-five percent of CD8 expansions found in freshly obtained CD8 T cells further expanded on in vitro stimulation with autologous leukemic B cells. This suggests a memory status of such cells. In contrast, the unusually high frequency of CD4 T-cell expansions found in freshly isolated peripheral blood cells did not correlate positively to in vitro stimulation as only 1 of 9 expansions continued to expand. Our data suggest that leukemia cell–specific memory CD4 and CD8 T cells are present in vivo of patients with CLL and that several leukemia cell–associated antigens/epitopes are recognized by the patients' immune system, indicating that whole leukemia cells might be of preference for vaccine development.

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TH2/TH1 Shift Under Ibrutinib Treatment in Chronic Lymphocytic Leukemia

Frontiers in Oncology ◽

10.3389/fonc.2021.637186 ◽

2021 ◽

Vol 11 ◽

Author(s):

Maria Cristina Puzzolo ◽

Ilaria Del Giudice ◽

Nadia Peragine ◽

Paola Mariglia ◽

Maria Stefania De Propris ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Chronic Lymphocytic Leukemia ◽

Complete Response ◽

Relative Increase ◽

Lymphocytic Leukemia ◽

Paired Samples ◽

Cell Cytokine Production ◽

Cell Profile

Ibrutinib may revert the T-helper (Th)2 polarization observed in chronic lymphocytic leukemia (CLL) by targeting the IL-2-inducible kinase, that shows a significant homology with the Bruton tyrosine kinase. In the front-line GIMEMA LLC1114 trial (ibrutinib+rituximab for 6 months, followed by ibrutinib maintenance), we investigated the modulation of T-cell cytokine production in 208 peripheral blood paired samples from 71 CLL patients: 71 samples prior to treatment (Day 0, D0) and at day +14 (D14; n=50), at month +8 (M8; 30), +12 (M12; 25), +18 (M18; 22) and +24 (M24; 10) of treatment. We documented a progressive decrease of CD3+CD4+IL-4+ T cells (Th2), that was significant at M8 and at M12 (p=0.019, p=0.002), a relative increase in the CD3+CD4+IFNγ+ T cells (Th1) and a decrease of CD3+CD4+IL-17+ (Th17) cells that was maintained up to M18 (M8 vs D0 p=0.003, M12 vs D0 p=0.003, M18 vs D0 p=0.004) of ibrutinib treatment. The Th2/Th1 ratio significantly decreased already after 14 days of treatment and was maintained thereafter (D14 vs D0 p=0.037, M8 vs D0 p=0.001, M12 vs D0 p=0.005, M18 vs D0 p=0.002). The Th2/Th1 modulation over time was significant only among patients with unmutated IGHV. The Th2/Th1 ratio below a cut-off of 0.088 at M8 was associated with the achievement of a complete response (CR) (p=0.016). Ibrutinib may shape the CLL T-cell profile, limiting Th2 activation and inducing a shift in the Th2/Th1 ratio. The association between the Th2/Th1 ratio decrease and the CR achievement suggests the in vivo generation of a potential host anti-tumor immune activation induced by ibrutinib.

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