Autologous Chronic Lymphocytic Leukemia Cells Research Articles

Impaired T-Cell Proliferation in Chronic Lymphocytic Leukemia Patients Is Linked to Disturbed Lipid Metabolism and Homeostasis

Background Successful T-cell based anti-cancer therapies require proliferation and longevity of effector T cells, features that highly depend on metabolic processes. T cells from chronic lymphocytic leukemia (CLL) patients are characterized by acquired T-cell dysfunction, resulting in impaired activation, proliferation and cytotoxicity upon in-vitro activation (van Bruggen et al, Blood 2019). Whilst the role of glucose metabolism in activated T-cells is extensively studied, the contribution of other energy sources is less clear. Lipid metabolism has only recently come into focus as an important source of energy for immune cells. Aims Molecular and functional analysis of the lipid metabolic pathway in T cells from CLL patients. Methods Peripheral blood mononuclear cells (PBMCs) of untreated CLL patients and, as controls, age-matched healthy donors (HD) were studied. T cells at baseline and after TCR stimulation were analyzed molecularly using transcriptomics and lipidomics, and extensively characterized at protein level by flow cytometry and confocal imaging. Lipid metabolism was manipulated through culturing cells in lipoprotein deficient serum (LPDS), or in the presence of agonists and inhibitors of specific regulatory pathways. Results We found the uptake of lipids, specifically cholesterol, during T-cell activation essential for proliferation, which was evidenced by a nearly absent proliferative capacity of HD T cells in the lack of exogenous lipids which was fully rescued by re-addition of cholesterol-rich low-density lipoproteins (LDL). As CLL T cells portray an altered proliferation pattern even when cultured in normal medium, transcriptomic analysis was performed to investigate the expression of key lipid metabolism genes. CLL T-cells had reduced expression of the master transcription factors SREBP1/2, governing lipid and cholesterol metabolism, when stimulated in the presence of their autologous CLL cells (Figure 1A). Accordingly, downstream protein involved in the synthesis and desaturation of lipids and LDL uptake (specifically FASN, LDL-R and SCD) were significantly reduced in CLL compared to HD T cells. Remarkably, despite reduced capacity for LDL uptake, an accumulation of neutral lipids was found in CLL T cells especially upon stimulation (Figure 1B), a feature earlier described in dysfunctional macrophages. The lipid accumulation observed in CLL T cells was supported by the lack of expression of ATGL, a crucial enzyme for the utilization of neutral lipids for energy production and building of new membranes. In line with this, mitochondrial lipid oxidation was decreased in stimulated CLL T cells compared to HD. To investigate the composition of the accumulated lipids, lipidomics analysis was performed, revealing differential abundance of specific lipid classes in stimulated CLL versus HD T cells. Triglycerides were increased while cholesterol esters and phospholipids were reduced in CLL T cells. Triglycerides likely constitute the neutral lipids accumulation found in these cells. As cholesterol esters and phospholipids are essential for proper membrane formation, reduced levels of these species can contribute to the reduced proliferative capacity of CLL T cells. Summary/conclusion Altogether, the results presented here demonstrate a disturbed lipid composition and utilization in CLL T-cells, which contributes to T-cell dysfunction on at least two levels: decreased bioenergetics and decreased proliferation. Likewise, lipid deprivation due to both decreased uptake and a misbalanced lipidome leads to altered T-cell function. Therefore, strategies to increase lipid uptake, synthesis and utilization in order to rebalance homeostasis could ameliorate CLL T-cell dysfunction and specifically proliferation-defects in CLL.

Dysfunction of T Cells from Chronic Lymphocytic Leukemia (CLL) Patients Is Linked to Disturbed Lipid and Fatty Acid Homeostasis

Background: Successful T cell-based anti-cancer therapies require proliferation and longevity of effector cells, features that highly depend on metabolic processes. A key feature of chronic lymphocytic leukemia (CLL) is acquired T cell dysfunction, resulting in impaired proliferative capacity (van Bruggen, et al Blood Adv 2022). T cell lipid metabolism has only recently come into focus as an important source of energy production and at the same time as essential component for the formation of new membranes upon proliferation. Fatty acid (FA) and lipid uptake in T cells requires several membrane proteins, among which the scavenger FA transporter CD36 and the low-density lipoprotein receptor (LDL-R). Specifically CD36-mediated FA transport has been identified to modulate (intratumoral) Treg and cytotoxic CD8+ T cells (Kolonin, et al, 2021 Immunometab). Whether T cell lipid and FA metabolism are affected in CLL is currently unknown. Methods: Peripheral blood mononuclear cells (PBMCs) of untreated CLL patients and, as control, age-matched healthy donors (HD) were studied. T cells were analyzed by transcriptomics, lipidomics and flow cytometry, either directly after thawing or after a 2-day culture with or without αCD3/αCD28 antibodies. Results: Unbiased transcriptomic analyses of patient-derived T cells following αCD3/αCD28 activation showed reduced expression of the master regulators of FA and cholesterol biosynthesis SREBF1 and SREBF2 in the presence of autologous CLL cells. This was associated with significantly reduced protein abundance of downstream targets of the SREBP pathway, including the key enzymes in FA synthesis and saturation FASN and SCD. In accordance with these data, lipidomic analyses highlighted an altered lipid profile in T cells from CLL patients that clearly demarcated them from HD T cells. In short, unbiased analyses revealed disturbed lipid homeostasis in CLL T cells at multiple levels, which led us to investigate lipid and FA metabolic pathways and their related functional consequences. Studying lipid and FA uptake capacity revealed lower expression of the FA transporter CD36 on CLL T cells. Stimulation of HD T cells resulted in upregulation of CD36 and the main receptor for cholesterol uptake LDL-R, accompanied by increased uptake of long chain fatty acids (LCFA) and increased FA-oxidation, concomitantly with induced expression of the activation marker CD25. In contrast, CLL-derived T cells did not increase CD36 or LDL-R expression, LCFA uptake or oxidation (Fig 1 and 2). Because of a recently suggested role for CD36 in T cell function, we next specifically analyzed CD36-expressing T cells (CD36+), and found these to have higher ability for LCFA and glucose uptake, and higher expression of the glucose transporter GLUT-1 than CD36 negative T cells from the same HD. Accordingly, CD36+ cells were also more likely to increase their CD25 expression and mitochondrial mass upon a 48h stimulation. This CD36+ T cell subpopulation, with potential metabolic and functional advantage, was virtually absent in CLL PBMCs. Impact of FA and lipid metabolism on proliferation was further established by the observation that culturing of HD T cells in absence of exogenous lipids resulted in nearly absent proliferative capacity. However, despite decreased expression of lipid and FA transporters, we observed marked accumulation of lipid droplets (LD) in T cells from CLL patients, suggesting an impaired ability to mobilize lipids towards oxidation pathways or as building blocks for membranes. In line with this, we found that in contrast to HD T cells that increased the expression of the rate-limiting enzyme for lipolysis ATGL, CLL T cells failed to do so. Conclusions: We provide the first data showing a disturbed lipid and FA metabolism of CLL T cells that contributes to T cell dysfunction. This includes decreased expression of FA synthesis enzymes as well as FA and cholesterol transporters. CD36+ T cells are metabolically distinct and are largely absent in CLL. We are investigating strategies to increase FA and lipid metabolism in order to rebalance lipid homeostasis. This could ameliorate CLL T cell dysfunction and specifically proliferation defects in CLL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P618: SECOND-GENERATION CD19 TARGETING TRI-SPECIFIC KILLER ENGAGER DRIVES ROBUST NK CELL FUNCTION AGAINST B CELL MALIGNANCIES

Background: Chronic Lymphocytic Leukemia (CLL) involves uncontrollable clonal expansion of CD5+/CD19+ B lymphocytes. While patients can sometimes coexist with CLL for years, eventually there is progression to bulky adenopathy and pancytopenia from marrow suppression requiring therapy. In recent years, targeted therapies, including utilization of the inhibitors for B cell receptor (BCR) signaling and for B cell lymphoma 2 (Bcl-2) protein, have been effective treatment options. However, these treatments are associated with limitations such as development of drug resistance and the lower treatment efficacy ratio in high-risk patients. Meanwhile, CAR-T cell immunotherapy failed to produce effective treatment outcomes against CLL, mostly due to defects in the effector T cells leading to product failures, as well as being associated with high levels of off-target cytotoxicity. Aims: Thus, approaches to CLL treatment are necessary and NK cell immunotherapy presents a viable and non-toxic option to this problem. Methods: Our group previously demonstrated the immunotherapeutic potential of utilizing first-generation CD19-targeting 161519 Tri-Specific Killer Engager (TriKE®) to drive natural killer (NK) cell cytotoxicity against B-cell malignancies. We have now produced a novel second-generation CD19 TriKE, ‘CAM161519’, utilizing a humanized camelid anti-CD16 VHH single domain antibody (sdAb) ‘CAM16’, a wild-type IL-15 component, and anti-CD19 tumor antigen scFv all linked via short peptide linkers. The TriKE primarily works by generating a cytolytic bridge between NK cells and CD19 expressing B cell malignancies while also providing an expansion and survival signal to the NK cells. Using flow-based assays and imaging based cytolytic assays we explored the capability of CAM161519 to target a variety of B cell lines (Raji, Daudi, and Nalm6) and primary B cell malignancies from patients with CLL and B cell acute lymphoblastic leukemia (B-ALL). Expanded NK cells were incubated with primary CLL targets from 6 different patients and no treatment (NT), rhIL-15 (30 nM), Rituximab (10 ug/mL), or CAM16159 (30 nM). NK cell degranulation and IFNg production was evaluated after 5 hours of co-culture (Figure). CLL killing was evaluated in a similar co-culture, but with a staining mix containing CD5 and CD19, to identify CLL tumor cells in order to determine the percent CLL targets killed normalized to incubation of CLL cells with NK cells alone (Figure Image). Results: The CAM161519 robustly activated NK cell degranulation (CD107a) and inflammatory cytokine production (IFNg) against B cell lines. The activation was specific as it was strongly reduced against CD19 knockout Nalm6 cells. The TriKE also induced specific NK cell proliferation. IncuCyte imaging based cytolytic assays demonstrated strong killing activity against Raji targets. When evaluating activity against primary targets, CAM161519 induced enhanced NK cell degranulation, cytokine production, and cytotoxicity using flow cytometry-based assays against primary CLL (Figure Image) and B-ALL targets when compared to IL-15 treatment. CAM161519 also rescued functionality of NK cells from CLL patients against their autologous CLL cells. Image:Summary/Conclusion: Taken together, these data are indicative of the potential for clinical translation of the CAM161519 TriKE in the CLL and B-ALL settings alone or in combination with cellular products.

Evaluation of allogeneic and autologous membrane-bound IL-21–expanded NK cells for chronic lymphocytic leukemia therapy

AbstractSuccesses with anti-CD20 antibodies in chronic lymphocytic leukemia (CLL) and enhanced activity of Fc-engineered vs unmodified antibody therapy suggest a potentially impactful role for natural killer (NK) cells and other innate immune cells in controlling this disease. Stimulated NK cells have shown promise as a cellular therapy, but their application has been constrained by limited expansion capacity and low cytotoxic activity against CLL cells. Here, we demonstrate that both healthy donor-derived and CLL patient-derived NK cells expand rapidly when stimulated with feeder cells expressing membrane-bound interleukin-21 (mbIL-21) and have potent cytotoxic activity against allogeneic or autologous CLL cells. Combination with anti-CD20 antibodies significantly enhances NK recognition and killing of CLL targets. As any CLL immune therapy would likely be given in combination, we assess commonly used treatments and demonstrate that ibrutinib has mixed suppressive and protective effects on expanded NK cells, whereas expanded NKs are highly resistant to venetoclax. We demonstrate efficacy in vivo in 2 xenograft mouse models of human CLL that support building upon a regimen of venetoclax and obinutuzumab with mbIL-21–expanded NK cells. Collectively, these data support development of mbIL-21–expanded NKs combined with the CD20 antibody obinutuzumab and venetoclax in the treatment of CLL.

Chronic Lymphocytic Leukemia Actively Induces T-Cell Dysfunction By Contact-Dependent Signaling Via CD24 and CD52

IntroductionSuccess rates of autologous T cell-based therapies, such as CAR-T cell therapy, in chronic lymphocytic leukemia (CLL) have been suboptimal and correlate with failure of activation and proliferation of T cells in vitro and in vivo. Previous data showing that impaired CD8 T-cell activation, proliferation and metabolic reprogramming could be restored by purifying CLL T cells via cell-sorting (van Bruggen et al., Blood, 2019) indicating that an as yet unknown, CLL-derived factor is responsible for acquired T-cell dysfunction. In this study we aim to elucidate the mechanistic basis of CLL-mediated T-cell dysfunction.ResultsDynamic analysis of αCD3/CD28 stimulated autologous T cells in presence of CLL cells over a period of 9 days revealed that T-cell activation (CD25, CD71, CD95 and PD-1) in CLL is in fact not impaired but occurs in a delayed fashion. CLL T cells reached peak activation after 5-6 days in contrast to 2-3 days for age-matched healthy donors. (Fig. 1A). This delayed T cell receptor-induced T cell activation was largely normalized with tumor cell depletion by flow-sorting prior to activation. Accordingly, in absence versus presence of autologous CLL cells, CAR-T cells derived from CLL patients showed enhanced proliferation, cytokine production and cytotoxicity, indicating potential clinical relevance. These findings show that T cells in CLL are not (terminally) exhausted but that a CLL-derived factor interferes with proper T-cell activation, leading to a delay in activation and impaired proliferation and cytotoxicity. We attempted to identify the mechanism of action in which CLL cells induce T cell dysfunction and whether these suppressive effects are mediated through a soluble factor secreted by CLL cells or by contact-dependent mechanisms.Previous studies have shown that CD40 activation of CLL cells results in increased expression of key surface-expressed adhesion and costimulatory molecules, but also in alterations of immune-modulatory cytokines secretion. This model was therefore used to decipher mechanisms of CLL-mediated T cell dysfunction. CD40-activation of CLL cells resulted in improved T-cell activation and proliferation upon αCD3/CD28 stimulation in a contact-dependent manner (based on trans-well experiments; Fig. 1B ).Several clinically approved kinase inhibitors were tested to identify signaling cascades involved in CD40-mediated alleviation of T-cell dysfunction. Only pre-treatment of CLL cells with the SRC-inhibitor dasatinib (100nM) abrogated the enhanced T-cell activation induced by CD40-activated CLL cells. Additional control experiments excluded direct effects of dasatinib on T cell function. Dasatinib did not reduce expression of co-stimulatory markers on CD40-activated CLL cells, indicating that lack of co-stimulation was not the sole explanation for CLL-mediated T cell dysfunction. RNA sequencing of CD40-stimulated CLL cells treated with or without dasatinib and filtered for membrane-bound factors revealed the Sialic acid-binding Ig-like lectin 10 (Siglec-10) ligands CD24 and CD52 as potential candidates responsible for inhibiting T-cell function in CLL, which we confirmed at the protein level. We also found increased expression of Siglec-10 on CLL T cells, suggesting a role for Siglec-10 ligation in inhibition of the TCR signaling cascade. Indeed, inhibition of Siglec-10 ligation by blocking CD24, and CD52 antibodies subsequently improved T-cell activation despite presence of CLL cells (Fig. 1C).ConclusionThese results demonstrate that T cells derived from CLL patients are not terminally dysfunctional and can be revived. Our observations indicate that CLL cells actively suppress (CAR) T-cell function in a contact-dependent fashion through CD24- and CD52-mediated Siglec-10 ligation. These proteins might represent targets for therapeutic intervention aimed at enhancing T-cell function in CLL. [Display omitted] DisclosuresKater: Genmab, LAVA: Other: Ad Board, Steering Committee; Abbvie: Honoraria, Other: Ad Board, Research Funding; Janssen, AstraZeneca: Other: Ad Board, steering committee, Research Funding; BMS, Roche/Genentech: Other: Ad Board, , Research Funding.

Daratumumab Decreases Treg-Mediated Immunosuppression and Potentiates CD8+ T-Cell-Induced Killing of Chronic Lymphocytic Leukemia (CLL) Cells Ex Vivo

Introduction: CD38 is expressed on the plasma membrane of lymphoid cells and in chronic lymphocytic leukemia (CLL) pts. increased CD38 expression is associated with a poor clinical outcome. While its role as a prognostic marker has been well established, the approval of daratumumab (Dara, first-in-class anti-CD38 mAb) offers a new role for CD38 as a potential therapeutic target in CLL. Matas-Cespedes et al have reported that Dara induces CLL cell death through antibody-dependent cellular cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP). While CD38 is expressed on CLL cells, it is also expressed on T-cell subsets, such as Tregs. We hypothesized that in addition to its direct immune-mediated activity, Dara modulates the immune cellular environment; skewing T-cell populations from an immunosuppressive towards immune-reactive milieu, thus promoting immune reconstitution for enhanced anti-CLL response.Methods: CD19+ cells were isolated from PBMCs of CLL pts. (n=30). Pts. with ≥30% CD38+ CLL cells were classified as CD38+ (n=22) and pts. with <30% CD38+ cells as CD38- (n=8). Dara-induced ADCC was assessed in Calcein AM labelled CLL cells cocultured with healthy donor PBMCs (E:T ratio, 50:1); CDC using 10% serum from a healthy donor and ADCP using human macrophages (E:T ratio, 2:1). Apoptosis was determined by annexin-V/PI staining followed by flow cytometry. Regulatory T-cells (Treg; CD3+CD4+CD25+CD127loFoxP3+) and cytotoxic effector T-cells (cTL, CD3+CD8+) were identified and isolated by FACS. Data are represented as mean±SEM.Results: Dara (0.1ug/mL) induced significantly greater ADCC in CD38+ (24.63±3.46%) vs. CD38- cells (14.11±1.11%) (p<0.001), whereas CDC (18.22±2.41 vs. 13.58±0.95) and ADCP (9.37±0.77% vs. 8.79±0.92%) were marginally, but insignificantly, increased in CD38+ vs. CD38- CLL cases. We also observed significantly greater apoptosis in CD38+ (36.41±3.34%) vs. CD38- CLL cells (23.39±1.70%) (p<0.005) (Fig 1A). Immune repertoire analysis revealed lower CD3+ T-cell numbers in CLL pts. (11.45±3.25%) compared to those in healthy donor PBMCs (23.64±7.36%); but with a higher percentage of Tregs (10.71±2.47 in CLL pts. vs. 1.96±0.60 in healthy donors). To determine whether Dara could decrease CLL-Tregs, we treated CLL-PBMCs withDara (10µg/ml; 5 days) ex vivo, and noted significant decrease in Treg cells from 12.75±1.33 (Isotype control) to 7.17±0.91, (n=5, p<0.001). As downregulation in Tregs can consequentially lead to a rise in other T-cell subtypes, we observed increased effector cTLs: from 50.08±5.41 to 64.53±3.19 (p<0.02) (Fig 1B). Further analysis on CFSE-labeled CLL-cTLs (primed or unprimed with Dara), showed cTLs proliferated significantly more so when co-cultured with Dara-treated apoptotic CLL (51.75±1.16) cells vs. ibrutinib (31.44±0.36) or rituximab-treated apoptotic CLL cells (36.25±3.77) or live CLL cells (7.04±3.34) (Fig 1C, representative CLL pt.). To determine if Dara-primed cTLs had enhanced cytolytic aptitude towards autologous CLL cells, we cocultured primed cTLs with CLL cells. Compared to unprimed cTLs, specific lysis of target CLL cells was greater in the primed cTL-CLL coculture (12.61±1.75% vs. 28.27±0.82%, respectively). And this effect was noted to be patient-specific as cTLs cultured with JVM13 (CD38+) cells showed lower specific lysis (Fig 1D, E).Conclusion: Here we demonstrate for the first time that Dara modulates CLL-Treg levels and increases effector cTL proliferation. Moreover, Dara-primed cTLs display heightened CLL-specific cytolytic activity than compared to Dara-naïve cTLs. Dara also induces direct apoptosis of CLL cells and exposure of Dara-killed CLL cells to cTLs increases cTL proliferation significantly more so than cTL exposure to CLL cells apoptosed using ibrutinib or rituximab. Altogether, our findings highlight the multiple mechanisms by which Dara (and potentially other anti-CD38 agents, currently being investigated by us) can induce CLL cell death and potentially revert dysfunctional cTL activity from that of a pro-CLL to an anti-CLL composure. [Display omitted] DisclosuresParikh:AstraZeneca: Honoraria; Pharmacyclics: Research Funding; Pharmacyclics: Honoraria. Ding:Merck: Research Funding. Sher:LAM Therapeutics, Inc: Research Funding. Malavasi:Jaansen Pharmaceuticals: Research Funding. Ailawadhi:Amgen: Consultancy, Honoraria; Pharmacyclics: Research Funding; Takeda: Consultancy, Honoraria; Novartis: Consultancy, Honoraria.

Clinical Progression of Chronic Lymphocytic Leukemia (CLL) Is Characterized By a Progressive Increase of the Exhausted T-Cell Phenotype and Immunosuppression Induced By Leukemic Cells

The mechanisms leading to clinical progression of CLL from early asymptomatic stages are not fully elucidated, being the acquisition of molecular alterations an infrequent phenomenon. Abrogation of autologous immune response against neoplastic processes is a key feature defining cancer. In CLL, malignant cells are able to evade immune anti-tumoral responses through inhibitory ligand signaling and defective immune synapse with T cells, which in turn exhibit an impaired proliferation and cytotoxic activity as well as high expression of exhaustion markers. In addition, other immunosuppressive features, such as production of IL-10 by CLL cells, are observed in these patients. Against this background, we hypothesize that evasion from immune surveillance is a mechanism of clinical progression from early stages in CLL that potentially opens a new field of therapeutic opportunities.To study changes in the immune system related to clinical progression, we performed flow cytometry analysis in paired samples from 19 patients with CLL at diagnosis and progression (median time to progression: 2.2 years), and in paired samples from 5 CLL patients without clinical progression (median follow-up: 2.7 years) as controls. At progression, we observed a significant increase in the absolute numbers of the CD45RA-CCR7- effector memory (EM) subset in both CD4+ and CD8+ T cells; CD4+ and CD8+ EM also had increased expression of PD1 at progression. Of note, there was no increase in PD1 in patients that did not progress; pointing out that PD1 plays a relevant role in CLL progression (Figure 1). Moreover, the inhibitory receptors CD244 and CD160 were significantly increased in CD8+ T cells and the co-expression of these receptors was also significantly higher in CD8+ cells at the time of progression. Differential expression of the transcription factors T-bet and Eomes defines the progenitor (T-bethiPD1int) and the terminal (EomeshiPD1hi) exhausted CD8+ T-cell subsets. In CLL, the EomeshiPD1hi CD8+ terminal subpopulation was significantly increased at progression, whereas the T-bethiPD1int CD8+ progenitor subset was stable, indicating the predominance of a more severe exhausted subset. In addition, myeloid-derived suppressor cells (MDSCs) were significantly increased and NK cells were decreased at late stages, while in contrast regulatory T cells remained unchanged. To further evaluate the increase of immunosuppression during progression, we studied IL-10 production in paired CLL samples. After 48 hours of microenvironmental stimuli, expression of IL-10 by CLL cells was significantly higher in samples obtained at the time of progression. In agreement with this, IL-10 plasma levels were higher at progression (n=9 pairs). This enhancement of regulatory B cell properties in CLL cells may also contribute to the increase of exhausted T-cell phenotype observed at clinical progression. To study the influence of B-CLL cells in T cells, we modeled progression in vitro by co-culturing T cells from CLL patients with increasing ratios of autologous CLL cells during 7 days. Under this setting, CLL cells induced a higher expression of PD1 (Figure 2) and CD244 in CD8+ T cells compared to healthy B cells, this increase being dependent on the T:B ratio. To evaluate the contribution of IL-10 to the induction of T-cell exhaustion in vitro we are currently blocking its production from CLL cells and these results will be ready at the time of the meeting. Finally, to rule out the putative contribution of genetic clonal evolution on CLL progression, we analyzed by deep sequencing (median deepness of 16000X) mutations in 8 driver genes (MYD88, NOTCH1, SF3B1, BIRC3, TP53, XPO1, ATM and POT1) and the size of the subclones affected at diagnosis and progression. Out of the 17 cases analyzed, we found driver mutations at diagnosis in 10 patients, from which only one showed clonal evolution at progression (increased allele frequency in SF3B1 and ATM mutations).Taking these results together, we conclude that clinical progression of CLL is potentially driven by an increasingly severe exhausted T-cell phenotype and by an enhancement in immunosuppressive features such as accumulation of MDSCs and elevated IL-10, pointing towards an impaired anti-neoplastic autologous immune response as a main mechanism of clinical progression. These results support the design of new immunotherapeutic strategies for patients in early stages that are likely to progress. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Select Antitumor Cytotoxic CD8+ T Clonotypes Expand in Patients with Chronic Lymphocytic Leukemia Treated with Ibrutinib.

In chronic lymphocytic leukemia (CLL), the T-cell receptor (TCR) repertoire is skewed and tumor-derived antigens are hypothesized as drivers of oligoclonal expansion. Ibrutinib, a standard treatment for CLL, inhibits not only Bruton tyrosine kinase of the B-cell receptor signaling pathway, but also IL2-inducible kinase of the TCR signaling pathway. T-cell polarization and activation are affected by ibrutinib, but it is unknown whether T cells contribute to clinical response. High-throughput TCRβ sequencing was performed in 77 longitudinal samples from 26 patients with CLL treated with ibrutinib. TCRβ usage in CD4+ and CD8+ T cells and granzyme B expression were assessed by flow cytometric analysis. Antitumor cytotoxicity of T cells expanded with autologous CLL cells or with antigen-independent anti-CD3/CD28/CD137 beads was tested. The clonality of the TCR repertoire increased at the time of response. With extended treatment, TCR clonality remained stable in patients with sustained remission and decreased in patients with disease progression. Expanded clonotypes were rarely shared between patients, indicating specificity for private antigens. Flow cytometry demonstrated a predominance of CD8+ cells among expanded clonotypes. Importantly, bulk T cells from responding patients were cytotoxic against autologous CLL cells in vitro and selective depletion of major expanded clonotypes reduced CLL cell killing. In patients with CLL, established T-cell responses directed against tumor are suppressed by disease and reactivated by ibrutinib.See related commentary by Zent, p. 4465.

Patients with Chronic Lymphocytic Leukemia Treated with Ibrutinib Show Expansion of T-Cell Clonotypes Composed of Antitumor Cytotoxic CD8+ T-Cells

We recently showed that ibrutinib induced a more oligoclonal T-cell receptor (TCR) repertoire in patients with chronic lymphocytic leukemia (CLL). Using next generation sequencing (NGS), we found that overrepresented TCRβ clonotypes were mostly patient-specific and that the degree of oligoclonal expansion correlated with CD8+ T-cell counts. Here, we tested the hypothesis that overrepresented clonotypes are CD8+ T-cells that respond to tumor antigens. Flow cytometric profiling of TCR Vβ (TRBV) chain usage, T-cell subset distribution, and granzyme B (GrB) expression was performed on cryopreserved peripheral blood mononuclear cells obtained at baseline and during response to single-agent ibrutinib (n = 5). Antibodies against TRBV were selected to investigate the ten most abundant TCRβ clonotypes identified by NGS. The cumulative frequency of these clonotypes evaluable by flow cytometry increased at the time of response (mean 31.2% ± SEM 3.9% of all T-cells) compared to baseline (mean 24.1% ± SEM 2.3% of all T-cells). Within each TRBV subpopulation, the proportion of CD4+ T-cells decreased (mean fold-change 0.78 ± SEM 0.04), while the proportion of CD8+ T-cells increased (mean fold-change 1.79 ± SEM 0.36). We further observed that the TRBV clonotypes expanding the most during treatment were comprised exclusively of CD8+ T-cells in every patient. These highly expanded CD8+ clonotypes expressed GrB, consistent with a cytotoxic phenotype. To evaluate whether expanding CD8+ clonotypes are tumor-specific, T-cells were expanded ex vivo in two conditions: (1) co-culture with autologous CLL cells, CD40L, IL-2, and IL-7 or (2) with anti-CD3/CD28/CD137 beads, IL-2, and IL-7. Overall, T-cells expanded with autologous CLL cells had a higher proportion of CD8+ GrB+ cells than T-cells expanded with anti-CD3/CD28/CD137 beads. The dominant CD8+ clonotypes detected in each patient at the time of response were preferentially expanded by co-culture with autologous CLL cells compared to expansion with beads. The biased expansion of cytotoxic T-cell clonotypes in the CLL-primed T-cell product supports a TCR mediated response to tumor antigens. Next, cytotoxicity assays were performed by mixing the expanded T-cell products described above with autologous CLL cells at an effector to target ratio of 3:1, 1:1 and 1:3. CLL-primed T-cells killed CLL cells in a dose-dependent fashion. In contrast, bead-expanded T-cells were ineffective at killing CLL cells or even protected tumor cells from spontaneous apoptosis. Thus, tumor-specific cytotoxic T-cells expand during treatment with ibrutinib and are further enriched ex vivo by co-culture with autologous CLL cells. In summary, increased TCR oligoclonality in patients responding to ibrutinib is driven by the expansion of specific CD8+ clonotypes. These clonotypes expand in co-culture with, and are cytotoxic against, autologous CLL cells. Tumor-specific CD8+ T-cell clonotypes may contribute to the overall treatment response with ibrutinib and supports the investigation of therapeutic strategies combining ibrutinib with T-cell directed immunotherapy. Disclosures Baskar: NIH: Patents &amp; Royalties: ROR1 mAb 2A2. Wiestner:Nurix: Research Funding; Acerta: Research Funding; Merck: Research Funding; Pharmayclics: Research Funding.

Ibrutinib Treatment in CLL Patients Improves T Cell Function and Blinatumomab Redirected Cytotoxicity

Introduction:T cell engaging bispecific antibodies (T-BsAb) redirect cytotoxic T cells to tumor associated antigen (TAA)-positive target cells, leading to T-cell activation and lysis of target cells. Blinatumomab is the only T-BsAb approved by FDA. Numerous T-BsAb are undergoing clinical trials, targeting both hematological malignancies (e.g. BCMA, CD123 and CD33) and solid tumors (e.g. CEA, EpCAM, and GPC3). While these T-BsAbs represent a promising novel therapeutic approach, their efficacy remains modest. Ibrutinib is an irreversible inhibitor of Bruton's tyrosine kinase (BTK) and induces durable remissions in chronic lymphocytic leukemia (CLL). Our group has reported that ibrutinib has favorable immunomodulatory effects due to inhibition of interleukin-2 inducible T-cell kinase (ITK). Ibrutinib decreased key immunosuppressive checkpoint molecules and the frequency of regulatory T cells (Treg)(Long, Beckwith et al. 2017). Moreover, Ibrutinib significantly increased the number of the activated T cells by rescuing them from activation induced cell death (AICD). However, the function of ibrutinib 'rescued' T cells and their ability to mediate T-BsAb redirected cytotoxicity is unknown. Therefore we proceeded to study how ibrutinib treatment affects the functional competency of T cells by measuring their capability to mediate Blinatumomab redirected cytotoxicity. To examine T cell function post ibrutinib treatment, T cells were isolated from pre and post treatment PBMC samples by fluorescence activated cell sorting (FACS), and used as effector cells with pre-treatment CLL B-cells as target cells. Blinatumomab redirected T-cell cytotoxicity against tumor cells and T cell survival were assayed by flow cytometry based methods. Effector cells (T cells) were labeled with CFSE. Target (CLL) cells were labeled with CellTrace Violet. They were co-cultured at an E:T ratio of 4:1 with Blinatumomab for 24 hours. Effector cells and target cells can be distinguished by different fluorescence labels. Apoptosis and cell death were evaluated by Annexin-V and PI staining. The number of viable target cells and effector cells were calculated by Countbright counting bead. Results: We observed that post-ibrutinib T cells demonstrated significantly superior cytotoxicity against autologous CLL cells in the presence of blinatumomab in three independent patient samples. Numbers of viable CLL cells co-cultured with post-ibrutinib T cells were lower when compared to those co-cultured with pre-ibrutinib T cells (33.6%, 68% and 55.1% reduction in viable CLL cells for the three patient samples, respectively). We also found that Treg depletion (by depleting CD25high/CD127low CD4+ T cells) further enhanced cytotoxicity for both pre and post-ibrutinib treatment T cells, with the numbers of viable CLL cells reduced another 30-40%. Moreover, compared to pre-ibrutinib T cells, post-ibrutinib treatment T cells again demonstrated superior cytotoxicity when Treg cells were depleted, with decreased numbers of viable CLL cells at the end of co-culture (43%, 69% and 49.4% reduction, respectively). We also observed that the numbers of viable T cells from pre-ibrutinib samples were much lower than post-ibrutinib samples (52.5%, 77.9% and 74% reduction in viable T cells, respectively). Conclusions: Our findings suggest that ibrutinib-rescued T cells are functionally competent. CLL patients' T cells post ibrutinib treatment demonstrated superior cytotoxicity against autologous leukemia cells compared to T cells from treatment baseline. Moreover, we show that increased T cell activity is not solely due to ibrutinib-related Treg depletion. Treg depletion in our experiments further enhanced the cytotoxicity of both post and pre-ibrutinib treatment T cells while post-ibrutinib T cells still demonstrated superior cytotoxicity (Fig 1). Lastly, T cells isolated from post-ibrutinib samples demonstrated significantly improved viability after in-vitro stimulation with blinatumomab, which is likely a result of reduced AICD. Disclosures Bhat: Pharmacyclics: Consultancy; Janssen: Consultancy. Rogers:Janssen: Research Funding; AbbVie: Research Funding; Genentech: Research Funding; Acerta Pharma: Consultancy. Woyach:Janssen: Consultancy, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; AbbVie: Research Funding; Karyopharm: Research Funding; Loxo: Research Funding; Morphosys: Research Funding; Verastem: Research Funding. Muthusamy:Ohio State University: Patents &amp; Royalties: OSU-2S. Byrd:Novartis: Other: Travel Expenses, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; BeiGene: Research Funding; Ohio State University: Patents &amp; Royalties: OSU-2S; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Genentech: Research Funding; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Acerta: Research Funding; Ohio State University: Patents &amp; Royalties: OSU-2S; BeiGene: Research Funding; Novartis: Other: Travel Expenses, Speakers Bureau; TG Therapeutics: Other: Travel Expenses, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Travel Expenses, Research Funding, Speakers Bureau; Genentech: Research Funding; Pharmacyclics LLC, an AbbVie Company: Other: Travel Expenses, Research Funding, Speakers Bureau; Gilead: Other: Travel Expenses, Research Funding, Speakers Bureau.

Eliciting Anti-Tumor T Cell Immunity in Chronic Lymphocytic Leukemia (CLL) with PD-L1/PD-1 Blockade Is Enhanced By Avadomide Immunotherapy through the Triggering of Immunogenic Interferon Signaling

Immune checkpoint blockade has demonstrated potential to reactivate anti-tumor immunity and regress tumors. However, response rates in B-cell non-Hodgkin lymphoma patients have been lower compared to Hodgkin lymphoma, with no activity reported in a recent trial of anti-PD-1 immunotherapy in relapsed CLL. This suggests that certain lymphoma subtypes may harbor non-immunogenic tumor microenvironments (TME).Our preliminary studies revealed that avadomide (CC-122), a cereblon modulator, can enhance T cell immune synapse signaling with autologous CLL cells, resulting in a concomitant increase in PD-1/PD-L1 expression at repaired synapses, indicating that avadomide may represent a complementary treatment partner for checkpoint inhibition. Here, we have extended our pre-clinical studies to investigate how these immunotherapy drugs alter the function and gene signatures of previously exhausted T cells from treatment naïve CLL patients (representing disease heterogeneity).Cytotoxicity assays revealed that treating primary T cells and autologous CLL cells with avadomide (1 μM, 48h) activated anti-tumor T cell killing function (P<.01, n=16), while combining avadomide with anti-PD-1 or anti-PD-L1 treatment significantly enhanced (P<.01) T cell killing function compared to these immunotherapies alone. Notably, anti-PD-1 monotherapy induced comparatively modest improvements of effector function.We next performed RNA sequencing on highly purified T cells from treatment naïve CLL patients, representing extremes of prognosis (n=6 good and n=6 poor including disease harboring TP53 abnormalities), following 18 h treatment with avadomide (100 nM) or anti-PD-1 (nivolumab) or PD-L1 (durvalumab) alone (10 μg/ml) or in combinations. Differential expression pathway analysis revealed that the top functional gene categories common for all the avadomide and combination treated samples (independent of anti-PD-1 or anti-PD-L1 monotherapy) were related to the response to type I and II IFN signaling, as well as inflammatory/stimulatory cytokine TNF-α, proliferative IL-6/JAK/STAT3, and IL-2/STAT5 responses. Type I IFN drives expression of chemokines CXCL10 and CXCL9, which are linked to enhanced tumor-infiltrated lymphocyte recruitment, and these chemoattractant genes were significantly upregulated in the avadomide and combination treated patient samples. In addition to their immunostimulatory roles, type II IFN (IFN-γ) and chronic type I IFN signaling have been linked to T cell resistance/exhaustion. We detected upregulated PD-L1 transcript in avadomide and combination treated T cells - supporting blockade of this inhibitory ligand.To investigate the ability of avadomide to modulate T cell migration, we performed comparative quantitative time-lapse microscopy analysis of pretreated patient T cells (n=12). These assays revealed that avadomide, as well as anti-PD-1 or anti-PD-L1 monotherapies, significantly enhanced (P<.01) T cell migration, and this response was significantly augmented by combination immunotherapy. Luminex protein analysis and cell migration assays confirmed the transcriptome data, highlighting the ability of avadomide and combination immunotherapy to significantly (P<.01) induce the release of CXCL10 (as well as IFN-γ, IL-2, GM-CSF and IL-8) from patient T cells, thereby attracting autologous T cells (CXCR3+). Avadomide as well as its pairing with anti-PD-L1 were also found to increase the proliferation of patient T cells (P<.01, n=6), particularly CD8+ T cells.Patient-derived xenograft models demonstrate that the therapeutic treatment of established tumors (3 weeks post-engraftment) with avadomide treatment (0.5 mg/kg) can activate anti-tumor T cells and significantly reduce disease volume (P<.01) that was associated with increased infiltration of PD-L1+ CD8+ T cells. Pairing avadomide with anti-PD-L1 durvalumab led to maximal activation of anti-tumor T cell immunity.In conclusion, our findings support the concept that PD-L1/PD-1 blockade in CLL could be enhanced when combined with avadomide through the promotion of immunogenic IFN signaling that enhances T cell infiltration and function. We believe these data support the rationale for combination immunotherapy that could convert a non-immunogenic TME into a ‘hot’ T cell inflamed TME which would be sensitive to checkpoint blockade. DisclosuresHagner:Celgene Corporation: Employment, Equity Ownership. Towfic:Celgene Corporation: Employment, Equity Ownership. Gandhi:Celgene Corporation: Employment, Equity Ownership. Stamatopoulos:Abbvie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Patten:L Hoffman La Roche: Honoraria, Research Funding; Gilead Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel, Research Funding; AbbVie Inc: Honoraria, Other: travel; Janssen: Honoraria, Other: travel. Vardi:Gilead: Research Funding; Janssen: Honoraria. Ramsay:MedImmune: Research Funding; Roche Glycart AG: Research Funding; Celgene Corporation: Research Funding.

Chimeric antigen receptor T cells targeting Fc μ receptor selectively eliminate CLL cells while sparing healthy B cells

AbstractAdoptive cell therapy of chronic lymphocytic leukemia (CLL) with chimeric antigen receptor (CAR)–modified T cells targeting CD19 induced lasting remission of this refractory disease in a number of patients. However, the treatment is associated with prolonged “on-target off-tumor” toxicities due to the targeted elimination of healthy B cells demanding more selectivity in targeting CLL cells. We identified the immunoglobulin M Fc receptor (FcμR), also known as the Fas apoptotic inhibitory molecule-3 or TOSO, as a target for a more selective treatment of CLL by CAR T cells. FcμR is highly and consistently expressed by CLL cells; only minor levels are detected on healthy B cells or other hematopoietic cells. T cells with a CAR specific for FcμR efficiently responded toward CLL cells, released a panel of proinflammatory cytokines and lytic factors, like soluble FasL and granzyme B, and eliminated the leukemic cells. In contrast to CD19 CAR T cells, anti-FcμR CAR T cells did not attack healthy B cells. T cells with anti-FcμR CAR delayed outgrowth of Mec-1-induced leukemia in a xenograft mouse model. T cells from CLL patients in various stages of the disease, modified by the anti-FcμR CAR, purged their autologous CLL cells in vitro without reducing the number of healthy B cells, which is the case with anti-CD19 CAR T cells. Compared with the currently used therapies, the data strongly imply a superior therapeutic index of anti-FcμR CAR T cells for the treatment of CLL.

GIFT4 fusokine converts leukemic B cells into immune helper cells.

BackgroundChronic lymphocytic leukemia (CLL) remains incurable with standard therapy, and is characterized by excessive expansion of monoclonal abnormal mature B cells and more regulatory immune properties of T cell compartment. Thus, developing novel strategies to enhance immune function merits further investigation as a possible therapy for CLL.MethodsWe generated a fusion cytokine (fusokine) arising from the combination of human GM-CSF and IL-4 (named GIFT4). Primary CLL cells were treated with GIFT4 or GM-CSG and IL-4 in vitro. GIFT4-triggered STAT5 signaling in CLL cells was examined by Western blot. The phenotype and secretome of GIFT4-treated CLL cells (GIFT4-CLL cells), and the immune stimulatory function of GIFT4-CLL cells on autologous T cells were analyzed by flow cytometry and luminex assay.ResultsGIFT4-CLL up-regulated the expression of co-stimulatory molecules CD40, CD80 and CD86 and adhesion molecule CD54. GIFT4-CLL cells secreted IL-1β, IL-6, ICAM-1 and substantial IL-2 relative to unstimulated CLL cells. GIFT4 treatment led to JAK1, JAK2 and JAK3-mediated hyper-phosphorylation of STAT5 in primary CLL cells, which is essential for GIFT4-triggered conversion of CLL cells. GIFT4-CLL cells directly propelled the expansion of autologous IFN-γ-producing CD314+ cytotoxic T cells in vitro, and that these could lyse autologous CLL cells. Furthermore, administration of GIFT4 protein promoted the expansion of human T cells in NOD-scid IL2Rγnull immune deficient mice adoptively pre-transferred with peripheral blood mononuclear cells from subjects with CLL.ConclusionGIFT4 has potent capability to converts primary CLL cells into APC-like immune helper cells that initiate a T cell driven anti-CLL immune response.

Reactive oxygen species induced by therapeutic CD20 antibodies inhibit natural killer cell-mediated antibody-dependent cellular cytotoxicity against primary CLL cells

The antibody-dependent cellular cytotoxicity (ADCC) of natural killer (NK) cells is assumed to contribute to the clinical efficacy of monoclonal antibodies (mAbs) in chronic lymphocytic leukemia (CLL) and other hematopoietic malignancies of B cell origin. We sought to determine whether reactive oxygen species (ROS)-producing monocytes regulate the ADCC of NK cells against primary CLL cells using anti-CD20 as the linking antibody. The monoclonal CD20 antibodies rituximab and ofatumumab were found to trigger substantial release of ROS from monocytes. Antibody-exposed monocytes induced NK cell apoptosis and restricted NK cell-mediated ADCC against autologous CLL cells. The presence of inhibitors of ROS formation and scavengers of ROS preserved NK cell viability and restored NK cell-mediated ADCC against primary CLL cells. We propose that limiting the antibody-induced induction of immunosuppressive ROS may improve the anti-leukemic efficacy of anti-CD20 therapy in CLL.

Functional Effects Of NOTCH1 Mutations In Chronic Lymphocytic Leukemia Patients

Chronic lymphocytic leukemia (CLL), is characterized by the expansion of mature B lymphocytes present in blood, bone marrow and lymphoid organs. Clinical behavior is highly heterogeneous, thus requiring timely identification of high-risk patients.NOTCH1 encodes a trans-membrane receptor acting as a ligand-activated transcription factor. NOTCH signaling initiates when the ligand, from either the Jagged or Delta families, binds to the receptor and induces successive proteolytic cleavages, resulting in the release and nuclear translocation of the NOTCH intra-cellular domain (NICD). Signaling is terminated by phosphorylation of the PEST domain of NOTCH1, triggering its ubiquitination and proteasomal degradation.Whole exome sequencing approaches have revealed NOTCH1 mutations in 5-10% newly diagnosed CLL cases, with their prevalence increasing to 15-20% in progressive or relapsed patients. The most frequent mutation is 7544-7545delCT frameshift deletion in exon 34, resulting in disruption of the C-terminal PEST domain. Truncation of the PEST domain is predicted to result in NOTCH1 impaired degradation, stabilization of the active NOTCH1, and deregulated signaling.The present study was undertaken with the aim to compare NOTCH1 expression and functional role in CLL patients harboring wild type (WT) or mutated (M) NOTCH1 gene.NOCTH1 mRNA and surface protein were expressed at comparable high levels in peripheral blood (PB) CLL cells obtained from NOTCH1 M and WT patients, consistent with a more general requirement of NOTCH1 signaling in this leukemia. However, at a variance of NOTCH1 WT cases, NOCTH1 M patients displayed remarkable accumulation of both the intermediate molecular species of the activated NOCTH1 receptor, as well as of the active NICD. Consistently, by gene expression profiling NOCTH1 M patients displayed significantly higher levels of HES1 and DTX1, the main NOTCH1 target genes. Overall, these data suggest a more active signaling pathway in NOTCH1 M CLL than in NOTCH1 WT cases.Expression of NOTCH1 and of its target gene (DTX1) varied across disease compartments, being higher in CLL cells obtained from the lymph nodes (LN), as compared to paired samples derived from the PB or the bone marrow (BM). By immunohistochemical analyses of primary LN tissue samples, NOTCH1 M CLL showed an intense nuclear staining as opposed to the more cytoplasmic distribution observed in NOTCH1 WT samples. These data suggest a more active NOTCH1 signaling in CLL residing in the LN microenvironment and confirm the functional effect of NOTCH1 mutations in vivo.When PB CLL cells were cultured in vitro in the absence of any supporting layer or stimulation, they showed a rapid down regulation of the NOTCH pathway, with complete loss of NICD after 24 hours paralleled by a sharp decrease in HES1 and DTX1 transcription. Consistently, levels of presenilin-1 (PSEN1), the catalytic subunit of the g-secretase complex, were also down-regulated offering a partial mechanistic explanation for the NICD loss. NOTCH1 mRNA levels remained unchanged, with accumulation of the receptor at the plasma membrane. These effects were independent of NOTCH1 mutation status and suggested the dependence of NOTCH1 signaling activation upon in vivo microenvironmental interaction, even in NOTCH1 M CLL.Within primary LN biopsies from CLL patients, the NOTCH1 ligand, was highly expressed on CD68+ elements of myeloid origin. This observation prompted the in vitro recreation of a lymphoid niche by co-culturing Jagged1+ nurse-like cells (NLC) with autologous CLL cells. Under these conditions, NOTCH1 activity in CLL cells was sustained over time, as shown by Q-PCR analyses of DTX1 and PSEN1. Moreover, NLCs protect NOTCH1 M CLL cells from fludarabine-induced apoptosis. This microenvironment-induced chemoresistance was prevented by pre-treatment of NOTCH1 M CLL cells with specific g-secretase inhibitors, to block NOTCH1 activation.Taken together, these results show that the 7544-7545delCT mutation in the PEST domain of NOTCH1 has a stabilizing effect on NOTCH1 signaling pathway. They also show that micro-environmental interactions are critical in activating NOTCH1 pathway both in the M and WT patients. Lastly, these results show that NOTCH1 signals micht create local conditions that favour drug resistance, thus making NOTCH1 a potential molecular target in CLL. Disclosures:No relevant conflicts of interest to declare.

Functional impact of NOTCH1 mutations in chronic lymphocytic leukemia

The purpose of this study was to compare the expression and function of NOTCH1 in chronic lymphocytic leukemia (CLL) patients harboring a wild-type (WT) or mutated NOTCH1 gene. NOTCH1 mRNA and surface protein expression levels were independent of the NOTCH1 gene mutational status, consistent with the requirement for NOTCH1 signaling in this leukemia. However, compared with NOTCH1-WT CLL, mutated cases displayed biochemical and transcriptional evidence of an intense activation of the NOTCH1 pathway. In vivo, expression and activation of NOTCH1 was highest in CLL cells from the lymph nodes as confirmed by immunohistochemistry. In vitro, the NOTCH1 pathway was rapidly downregulated, suggesting that signaling relies upon micro-environmental interactions even in NOTCH1-mutated cases. Accordingly, co-culture of Jagged1(+) (the NOTCH1 ligand) nurse-like cells with autologous CLL cells sustained NOTCH1 activity over time and mediated CLL survival and resistance against pro-apoptotic stimuli, both abrogated when NOTCH1 signaling was pharmacologically switched off. Together, these results show that NOTCH1 mutations have stabilizing effects on the NOTCH1 pathway in CLL. Furthermore, micro-environmental interactions appear critical in activating the NOTCH1 pathway both in WT and mutated patients. Finally, NOTCH1 signals may create conditions that favor drug resistance, thus making NOTCH1 a potential molecular target in CLL.

Systematic Identification of Personal Mutated Tumor-Specific Neoantigens in CLL

Abstract 954A major goal of cancer immunotherapy is to target tumor cells with high specificity and minimal toxicity. Tumor neoantigens are attractive targets for immunotherapy as they are generated from cancer-driven gene mutations and are expressed exclusively in tumor cells. Neoantigens have not previously been systematically investigated in human tumors due to technical challenges in identifying them. To address this, and to identify immunogenic epitopes personal to an individual tumor, we sought to exploit results from next-generation DNA sequencing to detect somatic mutations together with algorithms that predict peptide binding to HLA class I. We tested this approach in chronic lymphocytic leukemia (CLL), a B cell malignancy associated with a rich source of mutations (Wang et al, NEJM 2011).In a published study, we sequenced 91 CLL samples using whole-genome (n=3) or whole-exome (n=88; WES) DNA sequencing. To detect mutations, sequences in each tumor sample were compared with corresponding normal sequences, and the frequency of various mutation classes that lead to amino acid alterations were determined. On a per-tumor level, we detected a median of 17 (range: 2–72) missense mutations; 1 (range: 0–6) splice-site mutation; 1 (range: 0–1) read-through mutation, and 1 (range: 0–5) frameshift mutation. Frameshift mutations were predicted to generate potential neo-open reading frames with a median length of 40 amino acids (range: 0–180).Only peptides binding HLA molecules have the potential to elicit immune responses. We therefore focused on mutations generating autologous HLA binding peptides. Using a well-validated MHC class I prediction algorithm, pan-NetMHC, we systematically evaluated the binding potential of 9- and 10-mers tiled around each mutation. For 31 of 91 samples with available HLA typing, a median of 22 strongly binding peptides (range: 9–75) was predicted to be generated from a median of 26 (range: 7–87) mutations per sample. Using a competitive HLA binding assay, we experimentally validated the predicted high-binding capacity of 60 of 112 (53.5%) synthesized peptides, generated from 3 patients.To determine if tumor neoantigens are naturally recognized by cytotoxic T lymphocytes, we focused our analysis on CLL patients who had undergone successful hematopoietic stem cell transplantation (HSCT) followed by repeated vaccinations with irradiated autologous whole CLL cells, as normal donor T cell reconstitution following HSCT can overcome endogenous immune defects of the host and since donor T cells in this setting are already primed against host leukemia cells in vivo. Analysis of one patient identified 31 coding mutations, which generated 47 peptides predicted to bind autologous HLA. Of these, 24 were experimentally confirmed to bind autologous HLA alleles and thereafter were screened for inducing T cell reactivity. Using IFN-g ELISPOT reactivity following 2 ex vivo peptide stimulations, memory CD8+ T cell responses could be detected against a HLA A2-binding peptide derived from mutated FNDC3B (VVMSWAPPV). These cells were preferentially reactive to the mutated (but not wildtype) peptide. Results were confirmed with peptide-reactive T cell clones and staining with HLA-A2+/mutated FNDC3B peptide-specific tetramers. Mutated FNDC3B-reactive T cells also recognized HLA-A2+ expressing cells transfected with a minigene encompassing 300 base pairs surrounding the FNDC3B mutation, consistent with endogenous processing and presentation of the peptide. Ongoing studies focus on exploring the tumor cytolytic potential of mutation-specific T cells, and the kinetics of developing neoantigen-specific T cell reactivity in relation to immune reconstitution.Our studies provide proof-of-concept for systematic identification of tumor neoantigens by integrating information from next-generation sequencing of tumors with predictive HLA binding algorithms. These studies now set the stage for the implementation of clinical studies to explore the therapeutic potential of targeting tumor neoantigens. Disclosures:No relevant conflicts of interest to declare.

PDE4 Inhibitors Block TLR7 and TLR9-Driven Signaling, Proliferation and Cytokine Secretion in CLL Cells As Well As Proliferation Driven by Exposure to Apoptotic Cells

AbstractAbstract 1774A growing body of literature supports the concept that at least a subset of chronic lymphocytic leukemia (CLL) B cell-receptors (BCRs) are reactive with antigens exposed on apoptotic cells. Work done in the autoimmunity field suggests that through their BCRs, autoreactive B cells can internalize RNA and DNA-containing autoantigens, thereby activating endosomal Toll-like receptor (TLR) 7 and TLR9, respectively, with subsequent TLR-mediated signaling, proliferation and cytokine secretion. cAMP signaling modulates inflammatory signaling in immune system cells and our prior studies have shown that PKA, EPAC and CREB-mediated cAMP signaling can be potently induced in CLL cells with inhibitors of the cyclic nucleotide phosphodiesterase (PDE) PDE4 family in the absence of additional exogenous stimulators of adenylate cyclase. We therefore examined the ability of PDE4 inhibitor-mediated cAMP signaling to regulate TLR7 and 9-mediated signaling in CLL cells. Treatment of CLL cells with the TLR7 and 9 agonists R848 and CpG-B oligonucleotide, respectively, induced IRAK1 degradation and activation of three signaling pathways known to be downstream of TLR7 and 9: the transcription factors NFkB and IRF5 as well as the MAP kinase JNK. The prototypic PDE4 inhibitor rolipram inhibited R848-induced TLR7 and CpG-B-induced TLR9 signaling in CLL cells as judged by Western analysis and immunofluorescent studies of NFkB and IRF5 nuclear translocation as well as JNK phosphorylation. Rolipram also blocked R848 and CpG-B-induced CLL proliferation and TNF secretion. In contrast, rolipram augmented R848 and CpG-B-induced CLL IL-10 synthesis, in keeping with prior studies documenting cAMP response elements in the IL-10 promoter. To test whether RNA and/or DNA antigens exposed in apoptotic cells might induce proliferation in CLL cells through BCR-mediated internalization and subsequent activation of TLR7 and TLR9, we irradiated CLL cells and added them in a graded fashion to live autologous CLL cells. Such exposure to irradiated cells undergoing apoptosis drove CLL proliferation in a manner that was sensitive to TLR7 and TLR9 antagonists, IRAK4 kinase inhibitors and the PDE4 inhibitor rolipram. Our work supports the concept that BCR and TLR-driven responses to apoptotic RNA and DNA-containing complexes may drive proliferation in CLL cells and that PDE4 inhibitors and other agents that target TLR signaling may prove to be effective therapeutic agents in this disease. Disclosures:No relevant conflicts of interest to declare.

Impact of Lenalidomide on Gene Expression Profiles of Malignant and Immune Cells in Patients with Chronic Lymphocytic Leukemia

Abstract 976Lenalidomide has recently been demonstrated to have significant activity in chronic lymphocytic leukemia (CLL). Its mechanism of action in this disease is not well understood, but it is thought to act primarily by enhancing anti-tumor immunity and reducing production of pro-tumoral factors in the CLL microenvironment. We have previously demonstrated alterations in the expression of cytoskeletal genes in T-cells from patients with CLL and have subsequently shown that these changes translate into a deficit in T-cell function, due to impaired actin polymerization resulting in defective immunological synapse formation. Treatment of both autologous T-cells and CLL cells with lenalidomide was necessary to repair this defect, suggesting that this may be a key component of this agent's activity in CLL. Therefore we examined the effect of lenalidomide on the global gene expression profiles of isolated B-cells and T-cell subsets from CLL patients and healthy donors. Peripheral blood mononuclear cells from patients with untreated CLL or healthy donors were cultured in the presence of 1 μM lenalidomide or vehicle control for 48 hours. The lymphocyte subsets were isolated, followed by RNA extraction and gene expression profiling using the Affymetrix HGU133Plus2.0 platform. Lenalidomide treatment had similar effects on gene expression in T-cells from both patients with CLL and healthy donors. The most prominent changes in expression were of genes involved in cytoskeletal signaling including a 20-fold increase in WASF1 (Wiskott Aldrich Syndrome protein family, member 1), and greater than 2-fold increases in the expression of Rac-family member RHOC, (Ras homolog gene family, member C), actin binding proteins CORO1B (Coronin 1B), PARVA (Parvin alpha), and the Rho guanine nucleotide exchange factors (GEFs), ARHGEF5 and ARHGEF7. We also observed changes in genes regulating integrin signaling including PXN (Paxilin) and FAK (Focal adhesion kinase), and a shift towards Th1 differentiation with upregulation of TNF, IL-12R, and IL-18R. In addition, we noted increased expression of the transcription factors IKZF1, IKZF4 and IRF4, genes involved in the Ikaros pathways that are essential for hematopoiesis and control of lymphoid proliferation. These changes in gene expression provide further evidence that an important mechanism of action of lenalidomide is the upregulation of the actin cytoskeletal network including Rho-GTPases and integrin activation signaling, and are consistent with our previous observations concerning the functional repair of T-cells in CLL.Initial analysis of the effect of lenalidomide on the gene expression profiles of the CLL B-cells showed similar changes to those previously described in vivo from CLL patients receiving single agent lenalidomide in a clinical trial (Chen et al. JCO 2010). In our system, lenalidomide treatment resulted in a greater than 2-fold upregulation of 189 genes, and a greater than 2-fold downregulation of 85 genes in CLL B-cells. We observed increased expression of several genes belonging to the TNF superfamily including TNF-α, OX40L, and APRIL, and the receptors DR5, DCR2, and OX40. Many of these are known to mediate apoptosis signaling, and we also observed increased expression of pro-apoptotic genes such as FAS, BID (BH3 interacting domain death agonist), HRK (Harakiri), and CFLAR (CASP8 and FADD-like apoptosis regulator), and cell cycle regulators CDKN1A and CDKN1C (Cyclin-dependent kinase inhibitors 1A and 1C). Lenalidomide also upregulated expression of several genes of known importance in the CLL microenvironment, including the chemokines CCL3 and CCL4, CD40, CD274 (PD-L1), CD279 (PD-1), and adhesion molecules LFA3 and ICAM1. The effect of lenalidomide on the gene expression profiles of normal B-cells was less marked, with greater than 2-fold upregulation of 51 genes and downregulation of 12 genes. However, we did observe that lenalidomide treatment induced upregulation of genes involved in cytoskeletal pathways such as RND1 (Rho family GTPase 1), RHOQ (Ras homolog gene family, member Q), and MYO1B (myosin 1B).In conclusion, investigation of the effect of lenalidomide on gene expression profiling in CLL suggests that the drug acts both to enhance T-cell function, and to render the CLL cells more susceptible to immune cell mediated killing. Disclosures:Gribben:Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

Efficient Gene Delivery to Chronic Lymphocytic Leukemia (CLL) Cells with Microbubbles Bearing ROR1 Antibody

Abstract 2857Patients of chronic lymphocytic leukemia (CLL) typically develop progressive immune deficiency, which impairs their response to vaccines. Prior studies showed that infusions of autologous CLL cells transduced ex vivo with adenovirus encoding CD154 could elicit anti-leukemia immune responses. However, the need for high amounts of high-titer adenovirus complicates this approach and handicaps clinical development. A recently developed technology, known as microbubbles, could be activated using safe ultrasound (U/S) energy, potentially providing a new tool with which to affect gene delivery. In this study, we examined whether microbubble-technology could effectively transfect CLL B cells ex vivo or in vivo.To target CLL cells with microbubbles, we used newly generated mAb specific for ROR1, a surface antigen expressed on CLL B cells, but not on normal B cells or most other adult tissues. Anti-ROR1 mAb was incorporated into the lipid shell of microbubble, which was composed with DSPC and DSPE-PEG2000-Maleimide via maleimide chemistry. We examined whether targeting the microbubbles with anti-ROR1 mAb improved our transfection efficiency for ROR1+ CLL cells. For this we generated microbubbles tagged with anti-ROR1 mAb or control Ig and incubated these with fresh whole blood from patients with CLL. We found that targeted bubbles specifically bound to CD19+CD5+ CLL cells, but not to other cells, including RBCs. Next we examined whether anti-ROR1-tagged microbubbles or non-targeted microbubbles could transfect CLL cells with an expression plasmid (pGFP) encoding green-fluorescence protein (GFP). Isolated CLL cells were incubated with pGFP-laden microbubbles for 1 hour at 37° C. Following this the preparations were exposed to ultrasound at 2w/cm2 and 50% duty cycle or left untreated. After 48 hours culture, the cells were collected and stained with propidium iodide (PI) and examined via flow cytometry. Using these conditions, CLL cells in all treatment groups retained high viability (>80%). CLL cells incubated with anti-ROR1 mAb-tagged microbubbles expressed high levels of GFP provided they had also been exposed to ultrasound; CLL cells treated with anti-ROR1-mAb-tagged microbubbles that did not receive ultrasound treatment remained negative for expression of GFP. Similarly, CLL cells treated with non-targeted pGFP-laden microbubbles did not express GFP regardless of whether they also received ultrasound treatment.We also examined whether anti-ROR1-mAb-tagged microbubbles could transfect CLL cells in vivo. For this 107 human CLL B cells were injected into the peritoneum of each RAG-2−/−/ γc−/− mouse. Five minutes later, anti-ROR1-mAb-tagged microbubbles or non-targeted microbubbles laden with expression vectors encoding GFP and luciferase were injected into the peritoneum of each animal. Ten minutes later groups of animals did or did not receive treatment with ultrasound at 2w/cm2 and 50% duty cycle for 1 min. Forty-eight hours later mice were examined for reporter gene expression via whole body imaging. The group of animals that received anti-ROR1-mAb-tagged microbubbles and ultrasound treatment all had high-level expression of luciferase. Cells were recovered from the mice via peritoneal lavage. Immunofluorescence studies confirmed that only ROR1+CD5+ cells expressed GFP. In contrast mice injected with anti-ROR1-mAb-tagged microbubbles but did not receive treatment with ultrasound had no detectable luciferase activity. Groups of mice that were treated with non-targeted microbubbles also had negligible leuciferase activity regardless of whether they received ultrasound treatment. This study reveals that anti-ROR1-mAb tagged microbubbles that are activated by extracorporeal ultrasound treatment can be effective vehicles for specific delivery of genes into CLL cells ex vivo and in vivo. Disclosures:Kipps:Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Abbott Industries: Research Funding; Genentech: Research Funding; GSK: Research Funding; Gilead Sciences: Consultancy, Research Funding; Amgen: Research Funding.