Anti-lymphoma Agent Research Articles

Oxadiazole Derivatives of Diclofenac as an Anti-proliferative Agent for B-cell Non-Hodgkin Lymphoma: An In vitro and In Silico Studies.

Non-Hodgkin lymphoma of B cell origin is the common type of lymphoma- related malignancy with poor response rate with conventional front-line therapies. The aim of the present study was to investigate the potential of new anti-inflammatory oxadiazole derivatives of Diclofenac as an anti-lymphoma agent through in vitro and in silico approaches. Anti-lymphoma potential was evaluated by alamar blue technique. MTT assay employed for cytotoxicity. Gene and protein expression studies was performed by qRT-PCR and ELISA respectively. Docking studies was performed by using MOE program. Among five diclofenac derivatives, (II) showed promising anti-lymphoma effects, where it inhibited the expression of BCL-2, p-38 MAPK and TGF-β in both follicular and Burkitt's lymphoma cells and was non-toxic against normal human fibroblast cells. The in silico studies against BCL-2 revealed that the unsubstituted Sulphur group in (II) is involved in the crucial interactions with the binding site residue. The compound (II) can be a potential therapeutic candidate for B-cell non-Hodgkin lymphoma and deserves further development as a novel anti-lymphoma agent.

How to improve RCHOP as frontline therapy for diffuse large B-cell lymphoma: a systematic review and meta-analysis of 21 randomized controlled trials.

RCHOP is the standard of care for patients with diffuse large b-cell lymphoma (DLBCL) but failures occur in approximately 40% of them. We performed a meta-analysis of 21 randomized controlled trials (RCTs) comparing experimental regimens with RCHOP. We searched the database of PubMed with proper criteria, and data of efficacy (Progression Free Survival-PFS) in the ITT population were extracted and analyzed. Cross comparisons of RCTs were performed by using the CINEMA software. Odds ratio (OR) and 95% confidence intervals (95%, CI) are reported. The literature search yielded 21 RCTs including 5785 patients in the RCHOP arm and 5648 patients in the experimental arm. Odds ratio (OR) for PFS in the total cohort was OR (95%, CI): 0.87 (0.76-0.99), p=0.02. Among different strategies to improve RCHOP, addition of a novel agent on RCHOP improved PFS. In total 1740 patients in the RCHOP arm were compared with 1755 in the RCHOP plus a novel agent arm, and the OR (95% CI) for PFS was 0.84 (0.71-0.97), p=0.02. Indirect comparisons of nine studies adding a novel agent on RCHOP does not give prominence to any agent. Subgroup analysis according to cell of origin was performed for non-GC DLBCL patients. In this subgroup, 1546 patients treated with RCHOP were compared with 1538 patients treated with experimental regimens. The OR (95% CI) for PFS was 0.86 (0.73-1.02), p=0.34. Overall survival data extracted from 18 studies showed no superiority of experimental regimens over RCHOP. Efficacy of RCHOP backbone is marginally improved when adding a novel anti-lymphoma agent.

Vincristine Sulfate Liposome Injection (VSLI) with Bendamustine and Rituximab As First-Line Therapy for Indolent B-Cell Lymphomas: A Phase 1 Study

Vincristine Sulfate Liposome Injection (VSLI) with Bendamustine and Rituximab As First-Line Therapy for Indolent B-Cell Lymphomas: A Phase 1 Study

R-Nanochop Incorporating a TFR1-Targeted Doxorubicin Nanocarrier Is Superior to R-CHOP in a PDX Model of Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) comprises a third of non-Hodgkin lymphoma (NHL), with incidence steadily rising in what is already the most common hematologic malignancy. The backbone of potentially curative frontline chemoimmunotherapy combinations like R-CHOP is doxorubicin (Dox), still considered the most active anti-lymphoma agent. Relapsed or refractory (rel/ref) disease after frontline treatment carries persistently poor prognosis, even with recent advances in immunotherapy. Development and implementation of new therapeutic strategies remain urgent priorities to address unmet clinical needs in both high-risk previously untreated and rel/ref patients. We have studied transferrin receptor (TFR1) expression as a marker of high-risk DLBCL revealing significantly worse outcomes following frontline therapy associated with high expression in diagnostic samples. TFR1 is therefore a rational target for treatments aimed specifically at high-risk DLBCL. We used third-generation carbon-nitride dot (CND) nanocarriers we have developed conjugated to Dox and holo-transferrin (TF) to develop a chemotherapeutic-nanocarrier (NanoDox) designed to deliver Dox to TFR1-expressing tumors while sparing non-malignant tissues. Dox's mechanism is primarily through nuclear DNA damage or induction of reactive oxygen species (ROS) H202. We treated DLBCL cell lines with NanoDox or Dox for 24-hours and observed induction of nuclear DNA damage marker γ-H2Ax at drastically lower doses (10 nM NanoDox vs. 100 nM Dox). Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay results yielded similar findings in BJAB cells, as observed under γ-H2Ax analysis, with TUNEL induction at significantly lower NanoDox concentrations. Treatment with NanoDox did not generate significant changes in ROS H202 when compared to Dox at 24-hours. Dox is known for a strong cytotoxic profile that extends past its terminal half-life of 24-36 hours. Therefore, we treated BJAB, Farage and DHL4 cells for 24-hours with a range of NanoDox and Dox doses, followed by washout and tracked cell viability for 6 additional days. We found as low as 10 nM NanoDox induced rapid and irreversible cytotoxicity, an effect only seen at much higher doses of Dox. These results combined with fluorescent confocal microscopy studies confirmed NanoDox works primarily through rapid nuclear Dox accumulation, causing DNA damage and apoptosis. Building on initial NanoDox dosing studies in NOD Scid Gamma (NSG) mice, we constructed R-nanoCHOP as an alternate to R-CHOP by replacing Dox with NanoDox at a working dose (WD) of 33.0 mg/kg. We next engrafted patient-derived xenograft (PDX) tumors derived from a previously untreated patient with germinal center B-cell (GCB) subtype DLBCL. We randomized NSG animals to two groups at tumor engraftment and treated with 21-day cycles of R-nanoCHOP (n=10) or R-CHOP (n=11). The treatments controlled tumor volume (TV) similarly, but R-nanoCHOP significantly improved overall survival associated with greatly reduced toxicities to host animals, which tolerated an average of 2 additional cycles of R-nanoCHOP. Studies with murine cells in vitro, including A20 B-cell lymphoma cells, confirmed these effects were not due to species differences, with human holo-TF alone and as part of NanoDox binding mouse and human TFR1 with equal affinity. This work establishes proof of principle in DLBCL for targeting TFR1, a specific marker of clinically high-risk disease. CND nanocarriers provide a flexible platform to exploit this, with our initial studies enhancing the safety profile of Dox, which remains the most active frontline DLBCL agent more than 50 years after it entered use. We are now optimizing NanoDox by substituting the holo-TF with an anti-TFR1 single chain variable fragment (scFv), decreasing size of the overall nanoconjugate by >50%. We believe dosing optimization from this change and additional optimizations to Dox delivery intracellularly will permit investigational new drug (IND) studies of absorption, distribution, metabolism and excretion (ADME). In sum, we provide compelling clinically relevant evidence for targeting TFR1 in DLBCL as a new therapeutic approach. Disclosures No relevant conflicts of interest to declare.

Abstract PO-48: Cytotoxic mechanism of a novel transferrin receptor-targeting chemotherapeutic nanocarrier for use in diffuse large B-cell lymphoma

Abstract Diffuse large B-cell lymphoma (DLBCL), the most common hematologic malignancy, is an aggressive form of non-Hodgkin lymphoma. Approximately 60% of DLBCL patients achieve long-term disease-free survival from frontline R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone), but patients with relapsed or refractory disease have poor prognosis. Doxorubicin (Dox) remains the most active anti-lymphoma agent, but its use is limited by well-characterized toxicities, including potentially irreversible cardiac damage. Targeted and specific delivery of Dox to tumors to overcome these limitations has been an area of active research. Targeted delivery of Dox via the transferrin receptor (TFR1) has preclinical efficacy in solid malignancies but has yet to be explored in lymphoma. We have uncovered for the first time a significant association between TFR1 expression and poor prognosis in DLBCL. Carbon-nitride dots (CNDs) are third-generation nanocarriers with inherent photoluminescence, which are easily conjugated with a wide variety of substrates. We conjugated Dox and transferrin (TF), the ligand for TFR1, to CNDs to develop CND-Dox-TF (CDT). CDT is uniquely designed to enhance Dox delivery to TFR1-expressing DLBCL tumors while limiting effects on nonmalignant tissues. BJAB and Farage cell lines treated with CDT for 24 hours underwent apoptosis at a significantly lower concentration when compared to Dox (50 nM vs. 1000 nM). Overall, CDT was 1-2 Log10 more potent than Dox against DLBCL cell lines. Co-incubation with 250 uM of antagonist holo-transferrin significantly decreased BJAB and Farage sensitivity to CDT, confirming activity through TFR1. Confocal microscopy of BJAB cells incubated up to 24 hours with 250 nM Dox and 30 nM CDT reveled enhanced nuclear colocalization of Dox at a significantly lower concentration. Prior dose-finding experiments in non-tumor bearing mice identified a CDT safe working dose of 33.0 mg/kg containing 16% moles of Dox in comparison to Dox maximum-tolerated dose (MTD) 3.3 mg/kg. We engrafted 2 groups of 10 NSG mice with a Dox-sensitive high-TFR1 expressing patient-derived xenograft (PDX) tumor. Mice were dosed with CDT and Dox on day 0, 14, and 24. CDT demonstrated similar anti-lymphoma efficacy with reduced toxicity: CDT-treated mice displayed no significant decline in body weight, which is characteristic and was observed following each single-agent Dox treatment. Histology analyses revealed little to no obvious damage to CDT-treated nonmalignant tissues, including myocardium. With a working dose of CDT identified, we now have under way clinically relevant R-CHOP vs. R-nanoCHOP (CDT substituted for Dox) assessments in our PDX mouse model. Capitalizing on the unique design, we expect to observe improved anti-lymphoma efficacy, with decreased toxicities and an improvement in overall survival. In sum, we provide mechanistic insight for novel DLBCL nano-chemotherapy and illustrate preclinical efficacy for a promising new therapeutic approach. Citation Format: Artavazd Arumov, Piumi Y. Liyanage, Asaad Trabolsi, Evan R. Roberts, Braulio Ferreira, Daniel Bilbao, Roger M. LeBlanc, Jonathan H. Schatz. Cytotoxic mechanism of a novel transferrin receptor-targeting chemotherapeutic nanocarrier for use in diffuse large B-cell lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr PO-48.

Smart Biomimetic Nanocomposites Mediate Mitochondrial Outcome through Aerobic Glycolysis Reprogramming: A Promising Treatment for Lymphoma

Toxicity and drug resistance caused by chemotherapeutic drugs have become bottlenecks in treating tumors. The delivery of anticancer drugs based on nanocarriers is regarded as an ideal way to solve the aforementioned problems. In this study, a new antilymphoma nanodrug CD20 aptamer-RBCm@Ag-MOFs/PFK15 (A-RAMP) is designed and constructed, and it consists of two parts: (1) metal-organic frameworks Ag-MOFs (AM) loaded with tumor aerobic glycolysis inhibitor PFK15 (P), forming a core part (AMP); (2) targeted molecule CD20 aptamer (A) is inserted into the red blood cell membrane (RBCm) to form the shell part (A-R). A-RAMP under the guidance of CD20 aptamer actively targets B-cell lymphoma both in vitro and in vivo. As a result, A-RAMP not only significantly inhibits the effect on tumor growth but also shows no obvious side effects on the treated nude mice, indicating that A-RAMP can accurately target tumor cells, reprogram aerobic glycolysis, and exert synergistic antitumor effect by Ag+ and PFK 15. Furthermore, the antitumor mechanism of A-RAMP in vivo by apoptotic pathway and targeting metabonomics are explored. These results suggest that A-RAMP has a promising application prospect as an smart, safe, effective, and synergistic antilymphoma agent.

Design and synthesis of novel 1-substituted 3-(6-phenoxypyridin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine analogs as selective BTK inhibitors for the treatment of mantle cell lymphoma.

Ibrutinib (IBN), a first-in-class BTK-inhibitor, was approved by the FDA for the treatment of mantle cell lymphoma (MCL). Although IBN shows excellent performance as an anti-lymphoma agent, it has some undesirable side effects due to its off-target activities. Our studies yielded a novel series of 3-(6-phenoxypyridin-3-yl)-4-amine-1H-pyrazolo[3,4-d]pyrimidine derivatives capable of potent inhibition of Bruton's tyrosine kinase (BTK). Notably, compound 13e explained potent BTK inhibitory activity and could completely inhibit the phosphorylation of BTK and PLCγ2 in Z138 cells at low micromolar concentration. Compared with IBN, compound 13e improved anti-proliferative activities 3-40 folds in MCL cell lines with IC50 values lower than 1 μM. Low micromolar doses of 13e could induce strong cell apoptosis in Jeko-1 and Z138 cells. In addition, compound 13e showed greater BTK selectivity and higher stability in human liver microsomes than IBN and potential safety improvement for the treatment of MCL.

The SMAC mimetic LCL-161 displays antitumor activity in preclinical models of rituximab-resistant B-cell lymphoma

AbstractClinical observations suggest the existence of shared resistance pathways between rituximab and chemotherapy agents. To explore the mechanisms of rituximab resistance, our group created rituximab-resistant cell lines (RRCLs), which display altered expression of several inhibitor of apoptosis (IAP) family proteins. Here, we provide evidence to support pharmacologically targeting IAPs in lymphoma with LCL-161, a small molecule mimetic of the second mitochondria-derived activator of caspases (SMAC). The antitumor effect of LCL-161 was determined using luminescent adenosine triphosphate assays, flow cytometry, SCID mouse xenografts, and ex vivo patient biopsy sample studies. In vitro exposure to LCL-161 also resulted in a dose-dependent decrease in IAP levels, along with synergistic enhancement of the antitumor effect of cytotoxic chemotherapy, in rituximab-sensitive cell lines and RRCLs. In addition, LCL-161 increased the cytotoxic effect of the proteasome inhibitor carfilzomib in ex vivo lymphoma patient samples. The combination of LCL-161 with the chemotherapy regimen rituximab, gemcitabine, and vinorelbine (RGV) improved in vivo survival compared with RGV alone in severe combined immunodeficient mice implanted with RRCLs but not in animals implanted with rituximab-sensitive cell lines. In summary, LCL-161 exhibits synergistic antitumor activity in both in vitro and in vivo models of resistant lymphoma. Our data support further preclinical investigation of LCL-161 as a novel antilymphoma agent.

Abstract 799: The novel histone deacetylase inhibitor pracinostat is an effective anti-lymphoma agent

Abstract Background. Currently, 3 histone deacetylase inhibitors (HDACi) are clinically approved for the treatment of T-cell lymphomas: vorinostat, romidepsin and belinostat. Pracinostat (SB939) is a class I, II and IV HDACi (Novotny-Diermayr et al, Mol Cancer Ther 2010), in phase 3 in combination with azacitidine for acute myeloid leukemia patients who are unfit to receive intensive remission induction chemotherapy (NCT03151408). Here, we tested it on a large panel of lymphoma cell lines alone or in combination with a BCL2 inhibitor, venetoclax and with 5-azacitidine. Methods. 60 cell lines derived from T-cell lymphoma (10), diffuse large B cell lymphoma (DLBCL, 25), mantle cell lymphoma (10), marginal zone lymphoma (5), Hodgkin lymphoma (4), chronic lymphocytic leukemia (2), primary mediastinal B-cell lymphoma (1), murine (2) and canine (1) lymphomas were exposed to increasing doses of pracinostat and vorinostat alone or to pracinostat in combination with venetoclax or 5-azacitidine for 72h. Cell proliferation was assessed using the MTT assay. Chou-Talalay index was used to determine synergism. For apoptosis and cell cycle analyses, cells were treated with 250 nM pracinostat for 72h then stained with Annexin V and 7-AAD (apoptosis) or fixed then stained with 7-AAD (cell cycle) before flow cytometry. Results. All lymphoma subtypes responded well to pracinostat (median IC50 250 nM; 95%C.I., 171-324 nM). Pracinostat compared favorably with vorinostat (306 nM; 95%C.I., 241-363 nM), and the anti-proliferative activity of the two compounds was correlated (R=0.8, P<0.0001). Comparison of consensus cluster classification DLBCL subgroups showed that BCR DLBCL were more sensitive to HDACi than OxPhos DLBCL (pracinostat median IC50 BCR: 171 nM, OxPhos: 865 nM, P 0.001; vorinostat median IC50 BCR: 246 nM, OxPhos: 829 nM, P 0.01. No differences were observed between the DLBCL subtypes based on the cell of origin. Pracinostat mostly provoked cell cycle arrest in the S and G2/M phases with S-phase arrest associated with OxPhos and G2/M arrest with BCR DLBCL. Pracinostat caused pronounced apoptosis in 2/8 DLBCL cell lines. Functional annotation analysis to compare DLBCL with IC50 values < 200 nM (n=12) and DLBCL with IC50 > 400 nM (n=7) revealed an enrichment of mitochondrial metabolic pathways, DNA repair and cell cycle regulation in DLBCL with IC50 >400 nM. Combination with venetoclax was beneficial in 3/3 cell lines, while the addition of 5-azacitidine benefited 2 out of 3 cell lines. Conclusions. Pracinostat robustly inhibits the proliferation of lymphoma cells with a similar IC50 range to vorinostat. Its anti-proliferative activity can be cytotoxic or cytostatic. Combination of pracinostat with other compounds further inhibits lymphoma cell proliferation. In DLBCL, the OxPhos phenotype and an enrichment of mitochondrial metabolism, DNA repair and cell cycle pathways are associated with a poorer response to pracinostat. Citation Format: Afua A. Mensah, Filippo Spriano, Eugenio Gaudio, Chiara Tarantelli, Luciano Cascione, Luca Aresu, Emanuela Lovati, Emanuele Zucca, Anastasios Stathis, Claudio Pietra, Francesco Bertoni. The novel histone deacetylase inhibitor pracinostat is an effective anti-lymphoma agent [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 799.

Triptonide acts as a novel potent anti-lymphoma agent with low toxicity mainly through inhibition of proto-oncogene Lyn transcription and suppression of Lyn signal pathway

Lyn is a proto-oncogene overexpressed and constitutively activated in lymphoma, and plays an important role in lymphoma initiation and malignant progression. Hence, the oncogenic Lyn has recently been targeted for novel anti-lymphoma drug discovery; however, the effective Lyn-targeted drug for lymphoma treatment with low toxicity is absent in the clinical setting. The goal of this study is to explore powerful and low toxic Lyn-targeted anti-lymphoma agent. Here we show that triptonide, a small molecule purified from the herb Tripterygium wilfordii Hook F, potently inhibits the proliferation of human B-lymphoma Raji and T-lymphoma Jurkat cells with IC50 of 5.7nM and 4.8nM, respectively. Strikingly, triptonide at a dose of 5mg/kg/day almost completely inhibited the lymphoma growth in human lymphoma cells-xenografted mice without obvious side effects, particularly; the tumors in 6 mice among the 8 xenografted mice were completely eradicated in vivo. Cell biological studies showed that triptonide at the doses of 2.5–10nM notably suppressed B-lymphoma cell colony-forming capability, and that triptonide at the dose of 20nM promoted apoptosis through activation of PARP and caspase 3, but reduction of BCL2 protein levels in the lymphoma cells. Molecular studies revealed that triptonide markedly inhibited oncogenic Lyn transcription through suppressing the promoter activity of the gene, and that it remarkably reduced both total and phosphorylated Lyn proteins, and diminished Lyn downstream ERK and ATK signal pathways. Additionally, triptonide significantly enhanced p38 phosphorylation. Together, triptonide exerts potent anti-lymphoma effect with low toxicity mainly through inhibition of proto-oncogene Lyn transcription and suppression of Lyn downstream ERK and ATK signal pathways, providing an attractive drug candidate for development of novel anti-lymphoma therapeutics.

Human Umbilical Cord Wharton's Jelly Stem Cell Conditioned Medium Induces Tumoricidal Effects on Lymphoma Cells Through Hydrogen Peroxide Mediation.

Several groups have reported that human umbilical cord Wharton's jelly stem cells (hWJSCs) possess unique tumoricidal properties against many cancers. However, the exact mechanisms as to how hWJSCs inhibit tumor growth are not known. Recent evidence suggests that exposure of cancer cells to high hydrogen peroxide (H2 O2 ) levels from H2 O2 -releasing drugs causes their death. We therefore explored whether the tumoricidal effect of hWJSCs on lymphoma cells was mediated via H2 O2 . We first exposed lymphoma cells to six different molecular weight cut-off (MWCO) concentrates of hWJSC-conditioned medium (hWJSC-CM) (3, 5, 10, 30, 50, 100 kDa) for 48 h. Since, the 3 kDa-MWCO concentrate showed the greatest cell inhibition we then investigated whether the tumoricidal effect of the specific 3 kDa-MWCO concentrate on two different lymphoma cell lines (Ramos and Toledo) was mediated via accumulation of H2 O2 . We used a battery of assays (MTT, propidium iodide, mitochondria membrane potential, apoptosis, cell cycle, oxidative stress enzymes, hydrogen peroxide, mitochondrial superoxide, hydroxyl radical, peroxynitrile anion, and lipid peroxidation) to test this mechanism. The hWJSC-CM-3 kDa MWCO concentrate significantly decreased cell viability and mitochondrial membrane potential and increased cell death and apoptosis in both lymphoma cell lines. There were significant increases in superoxide dismutase with concomitant decreases in glutathione peroxidase, catalase, and thioredoxin peroxidase activities. H2 O2 levels, mitochondrial superoxide, hydroxyl radical, peroxynitrile anion, and lipid peroxidation were also significantly increased in both lymphoma cell lines. The results suggested that the hWJSC-CM-3 kDa MWCO concentrate regulates cellular H2 O2 leading to a tumoricidal effect and may thus be a promising anti-lymphoma agent. J. Cell. Biochem. 117: 2045-2055, 2016. © 2016 Wiley Periodicals, Inc.

Three Steps to Breaking Immune Tolerance: A Microparticle Approach

Lymphoma immunotherapy can result in durable immune and clinical responses. Nevertheless, the clinical impact of such therapy remains suboptimal and there is still a need to apply our growing understanding of cancer immunity to the design and testing of rational combination approaches to lymphoma immunotherapy. This includes consideration of the three steps necessary to generate an antitumor response: (1) providing tumor antigens to dendritic cells (DCs) in a manner that enhances their ability to process and present antigens to T cells; (2) enhancing T cell activation; and (3) overcoming immunosuppression so that activated anti-lymphoma T cells can proceed unrestrained.In situ immunization generates systemic immune responses through local injection of agents into the tumor, thus providing DCs with the patient’s own tumor antigens. We evaluated a three-step approach to in situ immunization against lymphoma using Doxorubicin, anti-CTLA-4 and anti-OX40: (1) Doxorubicin (Dox) to induce the expression of “eat-me” signals by dying tumor cells, facilitating their phagocytosis by DCs; (2) anti-OX40 antibody to augment OX40-mediated stimulation of T cells; (3) anti-CTLA-4 antibody to block immunosuppression imposed by CTLA-4 on T cells. While Dox is highly effective as a systemic anti-lymphoma agent and has been reported to induce immunogenic cell death, intratumoral injection of soluble Dox is not clinically feasible due to its strong vesicant effect. Poly(lactide-co-glycolide) (PLGA) is an FDA-approved polymer that is used in biodegradable surgical sutures and microparticles (MPs) with sustained-release properties. Thus, PLGA MPs loaded with Dox (Dox MPs) represent a clinically translatable approach for delivering Dox, as its slow release would decrease likelihood of vesication. In addition, PLGA MPs at an optimal size of 1-µm enhance antigen-specific immune responses by activating the NALP3 inflammasome in DCs.While both tumor cells and DCs are exposed to Dox released by MPs in the tumor, we found in vitro that Dox MPs (1-µm) are less cytotoxic to DCs than to A20 B-lymphoma cells and do not require internalization for their cytotoxic activity. Dox MPs significantly enhanced phagocytosis of A20 by DCs as compared to soluble Dox. In vivo, we used the two-tumor mouse model to assess immune responses. This allowed us to monitor the local effect (on the tumor injected with MP) and the systemic effect (on the distant tumor that was not injected with MP) of therapy. Dox MPs injected intratumorally do not induce vesication even at doses as high as 100 µg Dox. Using a low dose of Dox MPs (2 µg Dox) and of antibody to limit systemic toxicity, we found that three-step therapy induced CD4- and CD8- T cell-dependent systemic immune responses that enhanced T cell infiltration into distant tumors. This led to their eradication and significantly improved survival as compared to antibody-only therapy (87% of mice treated with all three components became tumor-free). Moreover, all three components were required for maximum efficacy (Figure 1). These results demonstrate that systemic antitumor immune responses can be generated locally by three-step therapy. The potential value of this approach to immunotherapy is not limited to lymphoma and merits further evaluation in lymphoma and other cancers. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

High Anti-Lymphoma Activity of Bendamustine/Mitoxantrone/Rituximab (BMR) in Rituximab Pretreated Relapsed or Refractory Indolent Lymphomas. A Multicentre Phase II Study of the German Low Grade Lymphoma Study Group (GLSG).

Purpose: Bendamustine is a new anti-lymphoma agent with promising activity. Based on a preceeding phase I study the current trial explored Bendamustine in combination with Mitoxantrone and Rituximab (BMR) in patients with relapsed or refractory indolent lymphomas.Patients and Methods: Patients with relapsed or refractory symptomatic stage III/IV indolent lymphomas with or without prior treatment with Rituximab were eligible. Therapy consisted of Bendamustine 90 mg/m2 days 1+2, Mitoxantrone 10 mg/m2 day 1, Rituximab 375 mg/m2 day 8. Treatment was repeated on day 29 for a total of 4 cycles.Results: Between 04/03 and 07/04 62 patients were recruited from 24 participating institutions, 40% of whom had received prior Rituximab therapy. Median age was 67 years (40–83). Lymphoma subtypes were 30 follicular (FL), 18 mantle cell (MCL), 4 B-CLL with plasmacytic differentiation, 3 lymphoplasmacytic (LPL), 3 marginal zone (MZL), 2 diffuse large B-cell (DLBCL), 1 high grade lymphoma not otherwise specified and 1 hairy cell leukemia. The overall response rate (ORR) was 88% with 36% CR and 53% PR. ORR in Rituximab pretreated patients was 75% (38% CR, 38% PR). After a median observation time of 17 months (1 – 33), the estimated median progression free survival is 19 months. Treatment related toxicities of grade 3/4 comprized a reversible myelosuppression (10 % anemia, 78 % leukocytopenia, 46 % granulocytopenia, 16 % thrombocytopenia). However, unexpected hospitalisations were necessary after 10 of 231 cycles (4%) only.Conclusion: BMR is a very effective new outpatient immuno-chemotherapy with low toxicity for patients with relapsed/refractory indolent lymphoma.Response ratesAll patientsFLMCLpretreated with Rituximabn62301825end of therapy without staging3301evaluable patients59271824CR21 (36%)13 (48%)6 (33%)9 (38%)PR31 (53%)12 (44%)8 (44%)9 (38%)MR1 (2%)0 (0%)1 (6%)1 (4%)SD0 (0%)0 (0%)0 (0%)0 (0%)PD6 (10%)2 (7%)3 (17%)5 (21%)EX0 (0%)0 (0%)0 (0%)0 (0%)CR+PR52 (88%)25 (93%)14 (78%)18 (75%) [Display omitted]

Initial evaluation of 227Th- p-benzyl-DOTA-rituximab for low-dose rate α-particle radioimmunotherapy

Radioimmunotherapy has proven clinically effective in patients with non-Hodgkin's lymphoma. Radioimmunotherapy trials have so far been performed with β-emitting isotopes. In contrast to β-emitters, the shorter range and high linear energy transfer (LET) of α particles allow for more efficient and selective killing of individually targeted tumor cells. However, there are several obstacles to the use of α-particle immunotherapy, including problems with chelation chemistry and nontarget tissue toxicity. The α-emitting radioimmunoconjugate 227Th-DOTA- p-benzyl-rituximab is a new potential anti-lymphoma agent that might overcome some of these difficulties. The present study explores the immunoreactivity, in vivo stability and biodistribution, as well as the effect on in vitro cell growth, of this novel radioimmunoconjugate. To evaluate in vivo stability, uptake in balb/c mice of the α-particle-emitting nuclide 227Th alone, the chelated form, 227Th- p-nitrobenzyl-DOTA and the radioimmunoconjugate 227Th-DOTA- p-benzyl-rituximab was compared in a range of organs at increasing time points after injection. The immunoreactive fraction of 227Th-DOTA- p-benzyl-rituximab was 56–65%. During the 28 days after injection of radioimmunoconjugate only, very modest amounts of the 227Th had detached from DOTA- p-benzyl-rituximab, indicating a relevant stability in vivo. The half-life of 227Th-DOTA- p-benzyl-rituximab in blood was 7.4 days. Incubation of lymphoma cells with 227Th-DOTA- p-benzyl-rituximab resulted in a significant antigen-dependent inhibition of cell growth. The data presented here warrant further studies of 227Th-DOTA- p-benzyl-rituximab.

Methylseleninic Acid Results in Rapid Changes in Intracellular Selenium Species and Sensitises Human Lymphoma Cells to Doxorubicin.

Background: We have previously shown that selenium compounds such as methylseleninic acid (MSA) demonstrate cytostatic and cytotoxic effects in human lymphoma cell lines (Last et al., ASH 2004). We have now further investigated the activity of MSA in combination with standard cytotoxic agents and set up a novel method to measure total intracellular selenium concentration after incubation with MSA, and the appearance of other selenium species. Additionally, we have examined whether NF-κ B may be involved in any interaction seen.Procedures: The effects of MSA alone, and in combination with doxorubicin, on cell number and viability were investigated in a panel of MSA sensitive (CRL-2261 and SUD-4) and less sensitive (DHL-4 and DoHH2) diffuse large B-cell lymphoma cell lines using a Viacount assay on an automated cell analyzer system (GUAVA PCA-96). MSA activity in normal cells was investigated in PBMC's, separated from whole blood using a ficoll density gradient method. To investigate MSA uptake into cells the combination of flow injection analysis with quadrupole inductively coupled plasma mass spectrometry (ICP-MS) was used. An ion-pair reverse phase HPLC method, with ICP-MS, was developed for the determination of intracellular selenium species. NF-κ B activity was studied following TNF-α induction using a luciferase based reporter gene assay (pLUC-NFκ B).Results: MSA inhibited the growth of human lymphoma cells in a concentration and time-dependent manner, but had a reduced effect in normal PBMC's. MSA increased the potency of doxorubicin in all lymphoma cell lines when used in combination for 48h. Doxorubicin (1–3μ M) showed no effect in terms of cell kill, but the addition of sub-toxic concentrations of MSA resulted in a marked increase in cytotoxicity, with an 80–90% reduction in cell viability in MSA sensitive cells at 1μ M MSA, and a 60–70% reduction in less sensitive MSA cells at 20μ M MSA. It was notable that cell lines that were less sensitive to MSA also had lower basal intracellular selenium levels. Cell lines also varied in their ability to take up MSA after a 4h and 24h exposure. Preliminary data suggest that MSA was mainly converted to methylselenocysteine (70%) at 4h, with a smaller amount appearing as selenomethionine (20%). We also investigated whether MSA exposure altered NF-κ B activity, and observed a concentration-dependent inhibition of NF-κ B in the TNF-α induced system used, resulting in 35% and 75% reduction at 5μ M and 10μM MSA, respectively, in DoHH-2 and DHL-4 cells.Conclusions: Our continuing investigations in lymphoma cell lines confirmed the cytotoxic activity of selenium species as a single agent, but importantly, and for the first time in the lymphoma setting, showed a synergistic effect in terms of cell kill when used in combination with a standard anti-lymphoma agent. The novel ICP-MS technique used will prove useful in our ongoing studies of selenium in combination with chemotherapeutics in an animal model by characterising intracellular changes in selenium species. The effect of MSA on NF-κ B activity suggests a molecular mechanism that may be mediating the interaction observed. In conclusion these findings support the use of selenium compounds in the treatment of lymphomas.

Ureteral obstruction caused by L-asparaginase-induced pancreatitis in a child with acute lymphoblastic leukemia.

L-asparaginase, an effective antileukemia and antilymphoma agent, is toxic to many organ systems. We report a case of ureteral obstruction caused by L-asparaginase via the inflammatory complication of acute pancreatitis. The patient was an 11-year-old boy with acute lymphoblastic leukemia. Six days after completing a 4-week induction therapy containing 9 doses of L-asparaginase, severe left abdominal pain developed. Abdominal computed tomography showed phlegmon formation anterior to the pancreatic head and in the left posterior pararenal space. The strands of inflammatory soft tissues encased the upper third of the left ureter, causing left hydroureter and left hydronephrosis. The ureteral obstruction resolved after insertion of a double-J catheter that remained in place for 66 days. This case suggests that L-asparaginase may play a role in the pathogenesis of ureteral obstruction in children receiving chemotherapy.

Characterization of the effects of asparaginase from escherichia coli and a glutaminase‐free asparaginase from vibrio succinogenes on specific cell‐mediated cytotoxicity

AbstractAsparaginases isolated from Escherichia coli and Erwinia carotovora are effective in the treatment of acute lymphoblastic leukemia in man. During treatment with either of these enzymes, patients frequently experience pronounced toxicity, including liver and pancreatic dysfunction and immunosuppression. The capabilities of these enzymes to hydrolyze L‐glutamine, an important amino acid in mammalian intermediary metabolism, have led investigators to suggest that the glutaminase activity may be responsible for the observed toxicities. Unlike other asparaginases, an enzyme isolated in our laboratory from Vibrio succinogenes, has been shown to be a potent antilymphoma agent and to be highly specific for L‐asparagine. Our previous work has established that the glutaminase‐free asparaginase from V. succinogenes does not suppress the in vivo humoral or cell‐mediated immune responses of mice to sheep red blood cells, whereas the E. coli enzyme abolishes these responses. In this study, we describe the mechanisms by which E. coli asparaginase suppresses specific antibody‐dependent cell‐mediated cytotoxicity. An analysis of the kinetics of appearance of the specific cell‐mediated cytotoxic response of spleen cells after immunization with sheep red blood cells revealed that it paralleled the IgG plaque‐forming cell response. In addition, 18‐h culture supernatants from immune spleen cells conferred upon non‐immune cells the ability to specifically lyse sheep red blood cells. Pretreatment of immune supernatants with protein A‐Sepharose 4B absorbed out the soluble arming factor. An analysis of E. coli asparaginase‐induced suppression of specific antibody‐dependent cell‐mediated cytotoxicity revealed that a decreased synthesis of specific IgG was responsible for the observed reduction in cytotoxic reactivity. When culture supernatants from spleen cells of immunized animals not treated with asparaginase were added to spleen cells from E. coli asparaginase‐treated mice, however, there was an increased cytotoxic response. This suggested that there was an increase in the number or ratio of effector cells for the specific antibody‐dependent cell‐mediated cytotoxic response in spleens of enzyme‐treated animals. Through the characterization of the immunosuppressive effects of two asparaginases—one having the catalytic capability to hydrolyze L‐glutamine and the other lacking this catalytic activity—we now have evidence that the immunosuppressive effects of E. coli asparaginase on specific antibody‐dependent cell‐mediated cytotoxicity cannot be the result of asparagine depletion alone. Our data demonstrate that the glutaminase‐free asparaginase from V. succinogenes is not immunosuppressive and suggest that an asparaginase with high substrate specificity for L‐asparagine may be a safer treatment for leukemia patients.

Evidence that the L-asparaginase of guinea pig serum is responsible for its antilymphoma effects. I. Properties of the L-asparaginase of guinea pig serum in relation to those of the antilymphoma substance.

A number of the properties of the L-asparaginase present in guinea pig serum have been examined and shown to be indistinguishable from those of the agent responsible for inhibiting cells of lymphoma 6C3HED in vivo. The patterns of instability of the enzyme to changes in temperature and pH were found to parallel closely those of the antilymphoma agent. L-Asparaginase activity was essentially absent from the serum of newborn guinea pigs and this failed to inhibit 6C3HED cells. On separating guinea pig serum proteins by salt precipitation, electrophoresis, and chromatography on DEAE cellulose, antilymphoma activity was found only in fractions which contained L-asparaginase.