anti-CD20 Antibody Rituximab Research Articles

Randomised controlled trial comparing ofatumumab to rituximab in children with steroid-dependent and calcineurin inhibitor-dependent idiopathic nephrotic syndrome: study protocol

IntroductionOral steroids induce remission in about 90% of children with idiopathic nephrotic syndrome (INS), which is characterised by severe proteinuria and hypoalbuminaemia. Some children become steroid-dependent (SD) and require addition...

Advanced stage nodular lymphocyte predominant Hodgkin lymphoma in children and adolescents: clinical characteristics and treatment outcome - a report from the SFCE & CCLG groups.

Advanced stage nodular lymphocyte predominant Hodgkin lymphoma (nLPHL) is extremely rare in children and as a consequence, optimal treatment for this group of patients has not been established. Here we retrospectively evaluated the treatments and treatment outcomes of 41 of our patients from the UK and France with advanced stage nLPHL. Most patients received chemotherapy, some with the addition of the anti CD20 antibody rituximab or radiotherapy. Chemotherapy regimens were diverse and followed either classical Hodgkin lymphoma or B non-Hodgkin lymphoma protocols. All 41 patients achieved a complete remission with first line treatment and 40 patients are alive and well in remission. Eight patients subsequently relapsed and 1 patient died of secondary cancer (9 progression-free survival events). The median time to progression for those who progressed was 21months (5·9-73·8). The median time since last diagnosis is 87·3months (8·44-179·20). Thirty-six (90%), 30 (75%) and 27 (68%) patients have been in remission for more than 12, 24 and 36months, respectively. Overall, the use of rituximab combined with multi-agent chemotherapy as first line treatment seems to be a reasonable therapeutic option.

Granulomatose mit Polyangiitis

Granulomatosis with polyangiitis (GPA) is a potentially life-threatening, rare disease. The etiology is unknown. GPA is histomorphologically characterized by extravascular necrotizing granulomatous inflammation and a systemic necrotizing vasculitis of small to medium-sized vessels. Clinically, a pulmonary-renal syndrome with pulmonary infiltrates, alveolar hemorrhage and a rapidly progressive glomerulonephritis is seen in about 80% of the cases with generalized disease. GPA is associated with proteinase 3-specific anti-neutrophil cytoplasmic autoantibodies (PR3-ANCA). Treatment is guided by severity of organ involvement and disease activity. Cytostatic immunosuppressants or the monoclonal anti-CD20 antibody rituximab are applied.

Monitoring the Cellular Binding Events with Quartz Crystal Microbalance (QCM) Biosensors.

Quartz crystal microbalance (QCM) biosensors have been demonstrated as noninvasive and label-free tools for cell based measurements. However, the complexity of biofilms composed of cells with the associated liquid environments is preventive towards forming explicit relationship between the added mass and the change in the frequency output signal. Therefore, the protocols of interface design (surface chemistry, interaction mechanism, and data acquisition), data interpretation, and device fabrication, all need to be finely refined in order to make these biosensors prevail in real life. Especially in the sense of deriving correct inferences from binding events, the fluidic effects (mostly visible in the form of damping resistance of QCM) should be quantitatively excluded from binding measurements. Such strategies can then track even the cellular interactions which are the basis of many physiological functions of life and can be built into smart functional devices for point of care diagnostics. This chapter provides technical details regarding these strategies with a focus on experimental details and procedures of the measurements of anti CD-20 antibody (Rituximab) interactions with B-Lymphoma cancer cells using the QCM method. In addition to a detailed description of specific interactions, we provide mechanisms of data interpretation and device development having potential application to other techniques.

A First-in-Human Study of Autologous T Lymphocytes with Antibody-Dependent Cell Cytotoxicity (ADCC) in Patients with B-Cell Non-Hodgkin Lymphoma (NHL)

Recent trials in patients with B-cell malignancies have demonstrated the clinical potential of chimeric antigen receptor (CAR)-directed T cells. We developed a chimeric receptor (antibody-coupled T-cell receptor, ACTR) consisting of high affinity CD16 V158, CD8α transmembrane domain, 4-1BB and CD3ζ (Kudo et al. Cancer Res 2014). Expression of ACTR conferred ADCC capacity to T cells. In vitro and in immunodeficient mice, ACTR T cells specifically killed B-NHL and chronic lymphocytic leukemia cells tagged with the anti-CD20 antibody rituximab. Trastuzumab induced ACTR-mediated T cell killing of breast and gastric cancer cells, and anti-GD2 of neuroblastoma and osteosarcoma cells. Thus, infusion of ACTR T cells might significantly augment the anti-tumor potential of immunotherapeutic antibodies, and expand options for cell therapy of cancer.To assess the safety of this strategy and explore its efficacy, we began a pilot study in patients with relapsed/refractory CD20+ B-NHL (ATTCK20; ClinicalTrials.gov No. NCT02315118). In this first-in-human study, ACTR expression is achieved by mRNA electroporation and, hence, is transient. In Phase 1a, 3 subjects receive one dose of autologous ACTR T cells (0.5 x 106/kg) 2 days after rituximab (500 mg/m2); a second dose of ACTR T cells at 0.5 x 107/kg 28 days later is allowed if there is no toxicity and evidence of response. Interleukin-2 (IL-2) is given subcutaneously at 1 million IU/m2/d x 3 during the first week after infusion. Phase 1b plans up to 4 infusions of ACTR T cells (0.5 x 107/kg) at 10 day intervals, each preceded by rituximab at 375 mg/m2; IL-2 (as in Phase1a) is given after the first 2 infusions only.Six subjects have been enrolled to date (3 in Phase 1a, 3 in Phase 1b) and received a total of 14 ACTR T cell infusions. All had advanced CD20+ B-NHL (2 diffuse large B cell lymphoma, 2 primary mediastinal B cell lymphoma, 1 mantle cell lymphoma, and 1 mediastinal grey zone lymphoma) and had failed multiple lines of therapy. As a lymphodepleting regimen prior to the first ACTR T cell infusion, 5 of the 6 patients received fludarabine (25 mg/m2/d x 5) plus cyclophosphamide (60 mg/kg/d x 1, n=3; 900 mg/m2/d x 1, n=2); the remaining patient received bendamustine (90 mg/m2/d x 2).After leukapheresis, T cells were cultured under cGMP conditions with anti-CD3 and -CD28 antibodies, and IL-2. After 10 days, cells were electroporated with ACTR mRNA using the MaxCyte GT system. The next day, median ACTR expression in T cells, as shown by flow cytometry, was 95.8% (range, 82.8%-99.3%; n=14). With Rituximab, this produced a median cytotoxicity of 77.5% (67.6%-97.6%; n=14) against CD20+ Daudi cells at a 4:1 effector : target ratio in 4 hours. As expected, ACTR expression in vitro remained high for the first 3-4 days, declining thereafter.We met or exceeded the estimated minimum target levels of CD3+ ACTR+ cells in 13 of the 14 infused cell products. CD3+ ACTR+ cells (mostly CD8+) were detectable in peripheral blood 1 day after infusion and reached their peak on day 3-4 when they represented 0.43-11.20 (median, 1.27) cells per µL. They became undetectable after day 6, reflecting the kinetics of ACTR expression in vitro.As of July 2016, 5 of the 6 patients are evaluable, with a median follow-up of 60 weeks (range, 6-83) after the last ACTR-T cell infusion; the remaining patient has received 3 of the 4 scheduled cycles. The main toxicities were grade 3-4 cytopenias: these were observed in all patients and likely due to the lymphodepleting regimen. There was no non-hematologic toxicity, nor evidence of cytokine release syndrome. In Phase 1a, 2 patients had progressive disease (PD) and 1 had stable disease (SD) on CT scan following the first and second infusions, with PET-CT showing reduced FDG avidity and central areas of photopenia suggestive of necrosis. Of the 2 Phase 1b patients evaluable for response, 1 had PD after the third infusion and was taken off study; the other had SD 1 month after 4 ACTR-T infusions; the 2nd month assessment was interpreted as PD.The initial results of this ongoing trial using autologous T cells with transient ACTR expression are encouraging and warrant additional studies with more potent formulations of this technology. To this end, a clinical trial for patients with relapsed/refractory CD20+ B-NHL in which ACTR is permanently expressed in autologous T cells via viral vector transduction is planned (ClinicalTrials.gov No. NCT02776813). DisclosuresCampana:Unum Therapeutics: Consultancy, Equity Ownership, Patents & Royalties; Juno Therapeutics: Patents & Royalties; Nkarta Therapeutics: Consultancy, Equity Ownership, Patents & Royalties.

Safety and Tolerability of Obinutuzumab (GA101) with Shorter Duration of Infusion in Japanese Patients with Previously Untreated CD20-Positive B-Cell Non-Hodgkin's Lymphoma (NHL)-Comparison with GAO4915g (GATHER) and BO21005 (GOYA) Studies

BackgroundObinutuzumab (GA101) is a novel humanized, glycoengineered Type II anti-CD20 monoclonal antibody. It has greater direct cell death induction and antibody-dependent cell-mediated cytotoxicity and lower complement-dependent cytotoxicity than Type I anti-CD20 antibody rituximab. Studies of obinutuzumab in patients (pts) with previously untreated or relapsed/refractory NHL demonstrated clinical activity with an acceptable safety profile. Infusion-related reactions (IRRs) were the most common adverse event, typically associated with the first infusion. Obinutuzumab 1000 mg is being administered over 4 hrs 15 min for the first infusion on Cycle 1 Day 1, and over 3 hrs 15 min for subsequent infusions. Shortening administration times can result in greater patient convenience and more efficient use of infusion facilities. To evaluate the tolerability of a Shorter Duration of Infusion (SDI), we conducted a Phase II study (JO29737, JapicCTI-152848, 'GATS') of G-CHOP (obinutuzumab in combination with CHOP [cyclophosphamide, doxorubicin, vincristine, prednisolone]) in Japanese pts with previously untreated CD20-positive B-cell NHL.MethodsThis study included adult pts with previously untreated CD20-positive B-cell NHL. Treatment consisted of 8 cycles of obinutuzumab (1000 mg intravenously on Day 1 of each 21-day cycle, with additional doses on Days 8 and 15 of Cycle 1) plus standard CHOP on Day 1 of Cycles 1-6. Obinutuzumab SDI is administered over 1 hr 30 min. Obinutuzumab was administered as an SDI from Cycle 2 to pts who met the following criteria: (1) No ≥ Gr 3 IRR related to obinutuzumab during regular infusions (RI) in Cycle 1, and (2) Lymphocyte count ≤ 5000/µL in peripheral blood prior to SDI. Primary endpoints were tolerability of SDI, pharmacokinetics (PK) of obinutuzumab. Tolerability of SDI was assessed by incidence of ≥ Gr 3 IRR in Cycle 2. Secondary endpoints were overall safety profile and efficacy. IRRs were defined as AEs related to obinutuzumab occurring during infusion or within 24 hrs from the end of infusion. Results were compared to the multicenter, open-label, randomized GOYA study (phase III, RI G-CHOP), and the multicenter, open-label, single-arm GATHER study (phase II, SDI G-CHOP). The GOYA study included 111 Japanese pts.ResultsAt database lock date, 35 pts were enrolled and treated, including 19 diffuse large B-cell lymphoma, 13 follicular lymphoma, and 3 other histologies; median age was 66 yrs (range, 35-78); 12/35 female; Ann Arbor stage I 11.4%, II 25.7%, III 20.0%, IV 42.9%; 7/35 positive bone marrow involvement. In 7 pts, treatment was prematurely discontinued due to AE (n=3; infected dermal cyst, bronchiolitis, and pneumonia aspiration, 1 pt each), progressive disease (n=2), or physician’s decision (n=2). Thirty-three pts received at least one SDI of obinutuzumab, given in Cycle 2 and subsequent cycles in 31 pts. Overall, 48.6% of pts (17/35) experienced IRRs. They were Gr 1/2 in all cases and occurred most commonly on Day 1 Cycle 1 (RI). No SDI-associated IRR was observed in Cycle 2, and very rare cases (2 pts) were noted in Cycles 6, 7, and 8. IRRs occurring in ≥5% pts were pyrexia (25.7%), chills (8.6%), and nausea, blood pressure increased, and headache (5.7%). Gr ≥3 AEs observed in ≥10% of pts were neutropenia (40.0%), neutrophil count decreased (25.7%), febrile neutropenia (17.1%), and leukopenia (14.3%). Obinutuzumab concentration just after Cycle 2 SDI was the same level as the concentration in Cycle 8. This shows PK reached a steady state at Cycle 2 and was not affected by shortening of administration. From PK analysis in the GATS study, AUC7day was 4170±885 µg·day/mL and t1/2 was 15.4±7.0 day, similar to the GATHER values (AUC7day, 3300±1130 µg·day/mL; t1/2, 23.0±15.9 day) after considering individual variability. No ethnic difference was observed. Overall response rate was 82.9% (29/35) (CR 62.9% [22/35], PR 20.0% [7/35]); 2 pts were not evaluable for response.ConclusionsObinutuzumab can be safely administered as SDI. No SDI-associated IRR was observed in Cycle 2. A few IRRs were observed with SDI in later cycles, but all were tolerable and manageable. RI and SDI have comparable safety, PK, and efficacy profiles in Japanese and non-Japanese. DisclosuresHatake:Meiji-Seika: Consultancy; Kyowa Kirin: Honoraria, Research Funding; Otsuka: Consultancy; Chugai: Research Funding. Izutsu:Abbvie: Research Funding; Gilead: Research Funding; Celgene: Research Funding; Janssen Pharmaceutical K.K.: Honoraria; Eisai: Honoraria; Kyowa Hakko Kirin: Honoraria; Chugai Pharmaceutical: Honoraria, Research Funding; Takeda Pharmaceutical: Honoraria; Mundipharma KK: Research Funding. Kinoshita:Janssen: Honoraria; Solasia: Research Funding; Eisai: Honoraria, Research Funding; Ono: Research Funding; Gilead: Research Funding; Kyowa Kirin: Honoraria; Zenyaku: Honoraria, Research Funding; Takeda: Research Funding; Chugai: Honoraria, Research Funding. Tobinai:Kyowa Hakko Kirin: Research Funding; Ono Pharmaceutical: Research Funding; HUYA Bioscience: Honoraria; Chugai Pharma: Research Funding; Eisai: Honoraria, Research Funding; Daiichi Sankyo Co., Ltd.: Consultancy; Celgene: Research Funding; GlaxoSmithKline: Research Funding; Zenyaku Kogyo: Honoraria; SERVIER: Research Funding; Abbvie: Research Funding; Takeda: Honoraria, Research Funding; Mundipharma KK: Honoraria, Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding.

Prognostic Significance of Genomic Alterations in Mantle Cell Lymphoma

BackgroundWhole genome, whole exome, and targeted sequencing have identified various genomic alterations in mantle cell lymphoma (MCL). There remains limited data correlating genomic alterations with clinical outcome in MCL. We describe genomic alterations in MCL using a targeted sequencing platform and correlate these alterations with clinical outcome.MethodsGenomic DNA was isolated from formalin-fixed paraffin-embedded tissue specimens from 55 cases of MCL and screened for clinically relevant mutations by deep-coverage targeted sequencing. Adaptor ligated sequencing libraries were captured by solution hybridization using two custom bait sets targeting 585 biologically significant cancer-related genes for DNA-Seq. Captured libraries were sequenced to high depth (Illumina HiSeq), averaging >300x. Twenty-eight tumor specimens had corresponding matched normal specimens that facilitated identification of somatic mutations with a high degree of confidence. For the remaining 27 unmatched cases, somatic mutations were inferred according to annotated-based rules. Briefly, non-synonymous variants were considered somatic if they met any of the following criteria: 1) previously reported in the COSMIC database at least 5 times or more, 2) previously reported in published MCL genomic data, or 3) truncating mutations including nonsense mutations, frameshift mutations and splicing mutations that are not at the 3’ end of the genes. The sequencing data were also used for detecting copy number changes including amplifications of established oncogenes or homozygous deletions of known tumor suppressor genes. Overall survival (OS) analyses were performed using Kaplan-Meier and log-rank tests.Results55 cases of newly diagnosed MCL treated at MSKCC from 1999 to 2006 with available specimens were analyzed. Median age of diagnosis was 59 years (range 24-79) with a male predominance (76%). Advanced disease (III/IV) was seen in 93% of cases and an elevated Ki-67 (>=30%) was noted in 41% of patients. Initial treatment included intensive therapy (chemotherapy + ASCT/Allo)(55%), radioimmunotherapy (131I-tositumomab followed by chemotherapy) (20%),bendamustine and an anti-CD20 monoclonal antibody (rituximab or ofatumumab) (11%), single agent anti-CD20 monoclonal antibody (5%), initial observation (7%), and other (2%).The most frequent alterations, above a level of 4%, are shown in Figure 1. The most commonly altered gene was ATM, a tumor suppressor involved in DNA damage repair, and observed in 42% of cases. In addition, known driver mutations in MCL were found, such as TP53 (16%) and CCND1 (11%). Also an amplification of CCND1 was observed in one patient. Alterations in chromatin modifying genes (e.g. KMT2D, MEF2B, WHSC1, SMARCA4) were common, occurring collectively in 38% of the cases. Recurrent alterations in NOTCH1 were identified in 9% of cases. Alterations in BIRC3 (7%) and TRAF2 (4%), key components of the alternative NF-ĸB pathway and mediators of apoptosis, were identified. Homozygous deletion of BIRC3 was also noted in one patient. Homozygous deletion of CDNK2A was noted in 9% of cases. Recurrent promoter mutations were observed in TERT (Telomerase Reverse Transcriptase) gene (13%), which encodes an enzyme involved in cell immortality.With a median follow-up of 7 years, the presence of TP53 mutations was significantly associated with inferior survival compared to TP53-unmutated patients (P<0.001), Figure 2. Notably, homozygous deletion of CDNK2A and mutations in NOTCH1 and KMT2D were not prognostically significant in our series.Conclusion:This analysis further describes the mutational landscape in MCL. Our results are congruent with prior data demonstrating the prognostic relevance of p53 mutations. Several of the genomic alterations identified may have therapeutic significance. For example, TRAF2 and BIRC3 mutations, in the alternative NK-κB pathway, are hypothesized to be associated with ibrutinib resistance. Loss of CDNK2A may confer sensitivity to PRMT5 inhibition. In future,we plan to analyze additional cases of MCL to elucidate the biologic and clinical significance of the observed genetic alterations with the aim of developing mechanism-based, biologically-targeted therapy in MCL. [Display omitted] [Display omitted] DisclosuresKumar:Celgene: Honoraria, Other: Scientific Advisory Board; Celgene: Research Funding; Adaptive Biotechnologies: Research Funding; Seattle Genetics: Research Funding; Pharmacyclics: Research Funding. Zelenetz:Gilead Sciences: Research Funding.

CD19 Target Activated Natural Killer (CD19.TaNK) Cellular Therapy: A Novel immunotherapeutic Approach to the Treatment of Non-Hodgkin Lymphoma (NHL)

Background: Continued improvement in the treatment of NHL is desired, especially via the incorporation of 'targeted' immunotherapy agents. This is especially important in B-cell NHL (bNHL) as resistance to rituximab anti-CD20 antibody, and now second-generation antibodies (e.g., obinutuzumab), may occur. Activated NK-92 (aNK-92) is a continuously growing cell line consisting of "pure" (100%) activated NK cells. These cells were subsequently bioengineered to express human anti-CD19 chimeric antigen receptor (CAR) recognizing CD19+ B cells. The goal of this project was to investigate the specificity and the efficacy of a novel 'off the shelf' targeted immunotherapy, CD19.TaNK, in a multitude of B-cell NHL cell lines, including anti-CD20 antibody resistant cell lines.Methods: Using gene expression profiling, Gene Set Enrichment Analysis and Ingenuity Pathway Analysis, we first investigated the expression of NK activation and inhibitory ligands in varied lymphoma cells. The bNHL cell lines, SUDHL10 (DLBCL), L540, L428 (Hodgkin lymphoma), HF1 (follicular), Raji (Burkitt's), and Mino (mantle cell) were purchased from ATCC and maintained in RPMI1640 medium. aNK and CD19.TaNK were supplied by NantKwest, Inc and were maintained in Myelocult supplemented with recombinant human IL-2 (500IU/ml). NK cell mediated cytotoxicity was determined using lactate dehydrogenase (LDH) release glucose-6-phosphate dehydrogenase (G6PD) release (aCella-tox assay). Briefly, 10,000 target bNHL cells were co-cultured with effector NK cells, at clinically relevant effector to target ratios (E:T 1:1-10:1) for 4 hours, and the supernatant was assayed for LDH or G6PD release. Percent cytotoxicity was determined based on the experimental levels of LDH or G6PD release from NK mediated bNHL cell lysis compared to maximum LDH or G6PD release from target cells. To determine if resistance to anti-CD20 antibodies would interfere with sensitivity to CD19.TaNK therapy, rituximab and obinutuzumab resistant bNHL cell lines (SUDHL4, SHUDHL10, and Raji) were established; cells were exposed to incremental increasing concentrations of antibody drugs (5-20μg/ml) over a period of 8 weeks. CD19, CD20 and CD30 expression in bNHL cells was determined by flow cytometry. Additionally, the efficacy of primary NK cells were determined against CD20 monoclonal antibody sensitive and resistant cell lines utilizing droplet microfluidics based assessment.Results: We observed that bNHL cell lines expressed a multitude of ligands associated with stimulating NK cell activity, while expression of inhibitory ligands was minimal. This indicates that NK cell interaction with bNHL cells is predicted to lead to overall robust antitumor immune response (Figure). Using LDH and G6PD release assays in bNHL cell lines, we observed increased cytolytic activity in an E:T ratio dependent manner, with Raji and L428 cells being the most sensitive to CD19.TaNK at 1:1 E:T ratio. Development of resistance to anti-CD20 antibodies (rituximab and obinutuzumab) resulted in significantly decreased down regulation of CD20, but not CD19 or CD30, as detected by flow cytometry. After direct contact with primary NK cells, we observed that rituximab resistant SUDHL10 cells were poorly sensitive (7%), while in rituximab sensitive cells, there was 22% cell loss. Moreover, at 4 hours using CD19.TaNK therapy (1:5 ratios), there was marked cytolytic activity with consistent high LDH release seen across all bNHL cell lines without differences noted regardless of rituximab or obinutuzumab resistance (ie, SUDHL4, SHUDHL10, and Raji). These results were further confirmed using live cell video microscopy measuring the cytolytic activity of CD19.TaNK versus bNHL cells.Conclusion: We identified that bNHL cells contain high expression levels of NK activation ligands and low amounts of inhibitory ligands and that CD19.TaNK immunotherapy had potent single-agent anti-tumor activity against a spectrum of bNHL cells. Furthermore, CD19.TaNK maintained high cytolytic activity in bNHL cells resistant to standard CD20 antibody therapy, which were poorly sensitive to innate NK cells. Ongoing analyses include systems biology studies to determine potential biologic mechanisms of activity of CD19.TaNK therapy as well as well as to help guide optimum combinatorial therapy. [Display omitted] DisclosuresBoissel:NantKwest, Inc.: Employment. Evens:Takeda: Other: Advisory board.

BAFF and CD4 T-Cells Are Major Survival Factors for Splenic Plasma Cells in B Cell Depletion Context: Implications for Autoimmune Diseases

The use of monoclonal anti-CD20 antibody (Rituximab) has greatly improved the treatment of B-cell mediated autoimmune diseases, albeit with variable outcomes. Our previous data in humans suggested that Rituximab induced paradoxically the settlement of splenic long-lived plasma cells (LLPC) in the context of 2 autoimmune cytopenia, immune thrombocytopenia and warm autoimmune hemolytic anemia (1) (2). The presence of splenic autoreactive LLPC explained the failure of Rituximab treatment. To investigate whether this mechanism could have a general relevance and decipher the cellular and molecular mechanism of this process, we used both non auto-immune and auto-immune mouse models.We have taken advantage of the knock-in transgenic mouse model AID-CreERT2-EYFP, which allows the irreversible expression of EYFP in B cells engaged in a germinal center-dependent immune response after tamoxifen regimen, to follow plasma cells (PC) at different times of immunization by sheep red blood cells, and upon anti-CD20 regimen (clone 18B12, Biogen Idec), in the spleen and bone marrow (3).By using a set of diagnostic genes that allowed us to distinguish short-lived and long-lived plasma cells, we compared the transcriptional program by multiplex PCR of EYFP+ B220- PC from controls and anti-CD20 treated mice, immunized and analyzed at the same time, corresponding to the nadir of B-cell depletion. While splenic PC of untreated mice displayed an intermediate profile between short-lived and long-lived plasma cells, splenic PC from anti-CD20 treated mice composed a homogeneous population that displayed a more mature program, similar to the one of natural long-lived bone marrow PC. The absolute number of splenic EYFP+ B220- did not change upon anti-CD20 treatment indicating that B-cell depletion promoted PC differentiation rather than a long-lived PC selection. We identified BAFF (B-cell activating factor) as a major player of this process. Indeed, as described in human spleens, we observed that BAFF level was increased in the supernatants of splenocytes after B-cell depletion. Above all, combination of anti-CD20 and anti-BAFF (clone 10F4, GSK) antibodies dramatically reduced the number of splenic EYFP+B220- LLPC (decrease >5 fold compared with anti-CD20 and control groups, P < 0.001). Targeting BAFF had no major impact on protective long-lived bone marrow PC as IgG1 level in the sera remained unchanged after combination therapy. We identified neutrophils as the main source of BAFF production in the spleen. Finally, CD4+ T-cells also appeared to play a key role in context of B-cell depletion for supporting plasma cell survival in the spleen as they appeared to closely interact with EYFP+ plasma cells by confocal microscopy. Moreover, their depletion (clones YTS 191.1 or GK 1.5, Bioxcell) in vivo induced a significant decrease in the number of splenic LLPCs (decrease > 2 fold compared with anti-CD20 group, P < 0.05). To assess whether B-cell depletion could also modify the splenic plasma cell program in an auto-immune context characterized by an ongoing immune response, we used NZB/NZW mice that spontaneously develop a disease closely resembling human systemic lupus. In line with our previous findings, anti-CD20 treatment also promoted the differentiation of LLPC in the spleen of the NZB/NZW model, while a treatment combining anti-CD20 with anti-BAFF induced a marked reduction in total PC numbers(decrease > 3 fold compared with anti-CD20 group, P < 0.05).In conclusion, the process of PC maturation upon anti-CD20 treatment appeared to be a general mechanism, both in non auto-immune and auto-immune models. We identified BAFF and CD4+ T-cells as key factors in the splenic environment responsible for the emergence of such LLPC. Finally, our results suggest that interfering with the plasma cell survival niche with monoclonal anti-BAFF antibody at the time of B-cell depletion might greatly improve the response rate in B-cell mediated auto-immune cytopenia.(1) Mahevas M, et al, Journal of Clinical Investigation , 2013(2) Mahevas M, et al, Journal of Autoimmunity, 2015(3) Dogan I, et al, Nature Immunology, 2009 DisclosuresNo relevant conflicts of interest to declare.

Cellular Mechanisms Regulating CD20 As a Target of Monoclonal Antibody Therapy in B-Lymphoid Malignancies

Standard treatment of B-lymphoid malignancies nowadays still relies on the administration of monoclonal antibodies (mAbs), with CD20 antigen being the prime target. Although initially effective, repeated cycles of anti-CD20 monoclonal antibody therapy often result in the loss of CD20 on the surface of malignant B cells and consequently in therapy resistance. In spite of the widespread use of CD20 monoclonal antibodies, the exact mechanisms regulating CD20 expression stay largely unrevealed and it mainly remains unclear whether they can be exploited pharmacologically to modulate expression of CD20 in the clinic.Interestingly, application of CD20 mAbs on various B-cell lines in vitro triggers active CD20 signaling within the cells. This signal transmission results in rapid CD20 downregulation at the surface accompanied by dramatically reduced CD20 transcription. Contrary to the prompt initial downmodulation of CD20 transcription, it takes several days upon mAb removal until CD20 transcriptional gene activity and thereafter CD20 surface levels are restored back to normal. To further study cellular mechanisms responsible for regulating CD20 expression, we have mimicked the situation in patients in an in vitro setting by repeated chronic treatments of diverse B-cell lines with gradually increasing doses of clinically used anti-CD20 monoclonal antibodies Rituximab, Ofatumumab and Obinutuzumab. Thereby, we managed to generate permanently resistant lines that no more respond to additional administration of any of the anti-CD20 mAbs. Notably, these cells have permanently strongly downregulated CD20 expression, both on the cell surface as well as already on the level of mRNA gene transcription. In addition, we have utilized the state-of-the-art CRISPR/Cas9 system to fully knock out CD20 gene in B-cell lymphoma (Ramos) and CLL (MEC1) cell lines. As expected, these cells are totally resistant to CD20 monoclonal antibodies. CD20 was originally proposed to function as a calcium channel and to contribute to B-cell receptor signaling, however the exact function of CD20 remains largely elusive yet. Using both experimental systems, we demonstrate that B cells with the loss of CD20 have fairly normal B-cell receptor signaling with no signs of any large defect. Specifically, activation of major signaling proteins like SYK, AKT, ERK, PLCgamma or p38 upon BCR stimulation was equal in CD20 depleted cells when compared to normal cells. Also calcium flux in response to BCR triggering seems to be normal in CD20-deficient cells, thus suggesting that CD20 is dispensable for proper B-cell receptor signaling. Next, we have performed RNA sequencing on these cells in order to better understand intracellular changes on a more global level that are imposed by the deletion or chronic downmodulation of CD20. We have compared the expression of individual genes and gene sets in cells upon CD20 removal and have performed the differential gene set enrichment analysis that will be presented.In summary, analysis of mechanisms regulating CD20 expression and/or function is critically needed for identification of potential novel drug targets that might be applied in the clinic to control CD20 persistence and thereby improve the outcome of anti-CD20 monoclonal antibody therapy.This research has been financially supported by the Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601) and by the research grant AZV-MZ-CR 15-33561AA-4/2015 and grant MUNI/A/1028/2015. DisclosuresNo relevant conflicts of interest to declare.

Primary Central Nervous System Lymphoma and the Blood-Brain Barrier

Recent reports propose high-dose (HD) methotrexate (MTX)-based cytoreduction followed by consolidation with HD chemotherapy (HDC) with or without autologous stem-cell transplantation (ASCT) as first-line treatment for primary central nervous system lymphoma (PCNSL). The main rationale is that HDC-ASCT may improve disease control by achieving higher therapeutic concentrations of cytotoxic chemotherapy in the CNS, thus circumventing the blood-brain barrier (BBB). We have employed an alternative approach to treat PCNSL patients effectively. This first-line treatment enhances delivery of standard dose (SD) MTX-based chemotherapy administered intra-arterially (i.a.) with osmotic BBB opening plus the anti-CD20 monoclonal antibody (mAb) rituximab, followed in complete responders by anti-CD20 mAb maintenance immunotherapy. The main features that distinguish our approach from HDC-ASCT consolidation are to utilize BBB properties to maximize delivery of SD chemotherapy to the CNS sanctuary, and maintenance immunotherapy to delay recurrence. A multi-center study of 149 PCNSL patients (2,079 i.a. BBB treatments) resulted in one death within 48 hrs after treatment, from pulmonary embolism. Prior to routine use of granulocyte colony stimulating factor, granulocytopenic fever occurred in 3% of BBB treatments (Angelov, J Clin Oncol 2009). This contrasts with HDC-ASCT consolidation which carries substantial morbidity and treatment-related mortality (6% to 9%) (Ferrari, Lancet Hemat 2016; Omuro, Blood 2015). Further, HDC-ASCT is used primarily in younger PCNSL patients (< 60-65 yrs) with good performance status and is not feasible in many PCNSL patients given the median age at diagnosis is 60-65 yrs.First-line enhanced BBB delivery of SD MTX-based chemotherapy in 149 patients provided an overall response rate (ORR) of 82% (58% CR rate) and median overall survival (OS) of 37 mo. In low risk patients (age < 60 yrs and KPS ≥ 70) median OS was approximately 14 yrs. Survivors in CR a minimum of 2 yrs after diagnosis (n = 24) were evaluated with standard neuropsychological tests. Median follow-up was 12 yrs (range 2 to 26 yrs) and results indicated long-term preserved or improved cognitive function (Doolittle, J Clin Oncol 2013). Recently we compared efficacy from two first-line MTX-based regimens: HD MTX (intravenous, 4gr/m2) (Thiel, Lancet Oncol 2010), and enhanced BBB delivery of MTX (i.a., 3.5gr/m2). After adjusting for patient characteristics, CR rate and progression free survival were higher in patients < 65 yrs treated with enhanced delivery of MTX (p < 0.001). When first-line rituximab was added to the i.a. enhanced delivery regimen in 24 patients (median age 64 yrs; median KPS 65), the ORR improved to 92% (75% CR) and OS to 61 mo.To prevent recurrence, Ney (Leuk Lymphoma 2009) treated a small group of patients in CR after standard PCNSL treatment, with maintenance rituximab. The addition of maintenance immunotherapy resulted in average disease control of 22 mo or longer. Consistent with the Ney report, we noted prolonged median CR duration of 38 mo (range: 10 to 85 mo) in a small subset treated with maintenance rituximab (n = 9). Increased survival was also seen in translational laboratory studies of a rodent model of intracerebrally implanted MC116 human B-cell lymphoma cells, using single agent rituximab, whether or not delivered with BBB disruption and whether or not MTX was included (Muldoon, Clin Cancer Res 2011). These results suggest that rituximab slowly leaked into main CNS tumor mass even in absence of BBB opening; and given the long half-life, was trapped by binding to CD20+ on tumor cells, attaining sufficient concentration for antitumor efficacy.Our goal is to maximize first-line enhanced BBB delivery of SD chemotherapy to enable less treatment-related morbidity and mortality than is associated with HDC-ASCT in PCNSL, and increase CR duration. Neurocognitive safety has been demonstrated in long-term survivors treated with BBB delivery. Based on the encouraging results from Ney et al, our pilot data using maintenance rituximab, and translational lab studies of mAb delivery to brain, a new randomized multi-center trial is underway to study the effect of maintenance obinutuzumab on CR duration, neurocognition and quality of life in PCNSL patients who achieve CR following first-line MTX-based chemotherapy (NCT02498951). A trial update will be presented during the ASH 2016 meeting. DisclosuresNo relevant conflicts of interest to declare.

The Pan Class-I PI3K Inhibitor Copanlisib Has Preclinical Activity in Mantle Cell Lymphoma, Marginal Zone Lymphoma and Chronic Lymphocytic Leukemia As Single Agent and in Combination with Other Targeted and Conventional Agents

Introduction. Copanlisib is a pan class-I PI3K inhibitor (i) with preferred activity towards PI3Kα and PI3Kδ that is currently in clinical development as single agent and in combination for patients with non-Hodgkin lymphomas. Here, we performed a screening of copanlisib as single agent and in combination with 15 other anti-cancer agents on a panel of 17 lymphoma cell lines derived from mantle cell lymphoma (MCL), marginal zone lymphoma (MZL) and chronic lymphocytic leukemia (CLL).Methods. Cell lines derived from MCL (Jeko1, Rec1, JVM2, Granta519, Maver1, Mino1, SP-49, SP-53, UPN1, Z138), MZL (Karpas1718, VL51, SSK41, ESKOL, HAIR-M, HC-1) and CLL (MEC1) were exposed to increasing doses of copanlisib alone and in combination with increasing doses of other compounds using the fixed ratio set-up. Tested compounds were approved as well as experimental inhibitors of key regulatory pathways: e.g. anti-CD20 monoclonal antibody rituximab, BTK-i ibrutinib, CDK-i roniciclib, DNA damage agent bendamustine, HDAC-i panobinostat, HDAC1/2-i romidepsin, immunomodulatory lenalidomide, JAK1/2-i ruxolitinib, MALT-i MI2, proteasome-i bortezomib. Synergy was assessed with Chou-Talalay combination index (CI): synergism (<0.9), additive (0.9-1.1), antagonism/no benefit (> 1.1). Gene expression profiling (GEP) was done using the Illumina-HumanHT-12 Expression-BeadChips and GSEA (FDR<0.25).Results. Copanlisib showed anti-tumor activity in the vast majority of cell lines with a median IC50 of 22nM (95%C.I.; 15-98) across all the cell lines, 36nM (6-175) in MZL, 22nM (95%C.I., 3-103) in MCL, 23nM in the CLL MEC1. Among all cell lines, the most active other compounds were bortezomib (IC50 5nM; 95%C.I., 5-7), romidepsin (34nM, 2-94), roniciclib (23nM, 18-29), panobinostat (161nM, 11-1263), MI2 (490nM; 224-1000) with remaining having median IC50s >500nM. Among these the only showing higher activity in one histotype was panobinostat, more active in MCL than MZL (IC50 17 vs 557nM, P 0.04). As pattern of activity across the cell lines, copanlisib was positively correlated with ibrutinib (R 0.58, P 0.001) and negatively with ruxolitinib (R -0.55, P 0.021).When copanlisib-containing combinations were tested in the 17 cell lines, different ones frequently appeared synergistic or additive: for example copanlisib plus MI2, 88% (15); plus ibrutinib, 82% (14); plus AKT-i or panobinostat, 76% (13); plus lenalidomide or BET-i, 71% (12); plus rituximab, 65% (11); plus romidepsin, 59% (10); plus roniciclib, 53% (9); plus bortezomib, 47% (8); plus bendamustine, 35% (6); plus ruxolitinib, 12% (2). For combinations with the most promising effect, baseline GEP identified gene-sets associated with different sensitivity to these combinations. High expression of genes involved in interferon signaling, oxidative phosphorylation, fatty acid metabolism, apoptosis, PI3K/AKT/mTOR and IL6/JAK/STAT signaling and low expression of genes involved in cell cycle were associated with synergism to copanlisib combinations with several compounds. Interestingly, largely the opposite was observed for the combination with others, with better synergy in cells with high expression of E2F/MYC targets and genes involved in cell cycle and low expression of transcripts involved in interferon PI3K/AKT/mTOR and IL6/JAK/STAT signaling.Conclusion. Copanlisib was active in cell lines derived from MCL, MZL and CLL. Combinations of copanlisib with a variety of additional anti-cancer compounds were synergistic and/or additive when combined in different cell lines. The combinations of the synergy profiles in the cell lines tested and the specific GEP features might predict lymphomas that could benefit from these regimens. DisclosuresJourdan:Bayer Pharma AG: Employment. Berthold:Bayer Pharma AG: Employment. Rossi:Gilead: Honoraria, Research Funding; Abbvie: Honoraria; Janseen: Honoraria. Lange:Bayer Pharma AG: Employment. Politz:Bayer Pharma AG: Employment.

Sequencing of chronic lymphocytic leukemia therapies.

It is an unprecedented time for the treatment of patients with chronic lymphocytic leukemia (CLL) with the recent approval of several targeted agents for use in frontline, relapsed, refractory, and high-risk disease. Traditionally, frontline management of CLL has been a combination of chemotherapy (fludarabine, cyclophosphamide, bendamustine, or chlorambucil) with an anti-CD20 monoclonal antibody (rituximab, ofatumumab, obinutuzumab). The current landscape is rapidly evolving with the advent of therapies that demonstrate selective inhibition of important pathways necessary for CLL proliferation and survival. Despite considerable progress, much is still unknown and optimal treatment selection and sequence is still debatable. None of the new agents have been compared against each other and the impact of adding an additional agent to monotherapy is not yet fully elucidated. In routine clinical practice, the choice of therapy is based on nonrandomized comparisons, presence of comorbidities, and toxicity considerations. These recently approved drugs (ibrutinib, idelalisib, and venetoclax) are reporting excellent outcomes, including patients with high-risk disease such as 17p deletion (17p-) or TP53 mutations (TP53mut). Ibrutinib and venetoclax have been approved for use in 17p- patients (frontline and relapsed, respectively). Ibrutinib is currently moving into the frontline space given recent regulatory approvals. This review will summarize and interpret the limited therapeutic sequencing data available, highlighting the need for additional studies to optimize combination strategies and treatments after failure or discontinuation of these novel agents.

Transplant-Associated Thrombotic Microangiopathy: Prevalence, Prognostic Factors and Treatment Outcomes in Unrelated Allogeneic Transplant for Hematologic Diseases

Introduction: Transplant-associated thrombotic microangiopathy (TA-TMA) represents a devastating and life-threatening complication occurring more frequently in allogeneic compared to autologous hematopoietic cell transplantation (HCT). Although it has been a well-recognized syndrome for decades, our understanding in terms of epidemiology and more importantly, treatment options remains unclear. Therefore, we aimed to retrospectively investigate the incidence of TA-TMA in patients undergone HCT from unrelated donors and identify prognostic factors and treatment outcomes.Methods: We enrolled consecutive patients who underwent HCT from unrelated donors from 2003 to 2015. TA-TMA diagnosis was based on EBMT (European Bone Marrow Transplantation) criteria: increased (>4%) schistocytes in peripheral blood, thrombocytopenia, increased lactade dehydrogenase, decreased Hb. Anti-thymocyte globulin (ATG, rabbit) was used as standardised part of the conditioning in almost all patients (total dose of 5-7.5mg/kg). Prophylaxis for graft-versus-host disease (GVHD) consisted of cyclosporine or tacrolimus plus methotrexate in myeloablative and mycophenolate mofetil plus cyclosporine in reduced intensity conditioning transplants. Upon identification of TA-TMA, all possible causative factors were fully investigated. The mainstay of treatment strategy was withdrawal of calcineurin inhibitors, plasma infusion and plasma exchange combined with corticosteroid administration and in refractory cases, humanized anti-CD20 monoclonal antibody (Rituximab).Results: In total, we studied 179 patients, 74 male:105 female, aged 37±14 years, who underwent HCT from matched HLA A/B/C/DRB1 (8/8, n=88) and allele or antigen mismatched (n=91) grafts. Grafts derived from peripheral blood (157), bone marrow (18) and umbilical stem cells (4). Conditioning regimens were myeloablative (139), reduced intensity (28) and toxicity (12). Median follow-up was 11.5 (0.1-147.4) months.TA-TMA was diagnosed in 29 (16.2%) patients, 12 male:17 female, aged 34±12 years, 78 (9-721) days post-HCT for acute leukemia (21), Hodgkin (3) and non-Hodgkin (1), lymphoma, MDS (2), aplastic anemia (1) and MPN (1). Conditioning regimens were myeloablative in 24 (12 based on total body irradiation/TBI) and reduced intensity in 5 patients. Among patients with TA-TMA, 10 (34%) presented severe (grade III-IV) acute GVHD, 15 (52%) extensive chronic GVHD, 12 (41%) bacterial, 2 (7%) fungal and 21 (71%) viral (Cytomegalovirus and/or Epstein-Barr viremia) infectious episodes.In univariate analysis, age, gender, diagnosis, disease phase, previous lines of treatment, conditioning (intensity and TBI-based), ABO and HLA incompatibility, infections, acute and chronic GVHD were studied. The presence of TA-TMA was associated with severe acute and extensive chronic GVHD (p=0.001 and p=0.035 respectively) and TBI-based conditioning (p=0.043). In multivariate analysis, severe acute GVHD was the only independent factor associated with TA-TMA (β=3.4, p=0.038).Regarding treatment outcomes, 8 (28%) patients responded to cyclosporine cessation and plasma infusions. The rest 21 out of 29 patients underwent plasma exchange sessions; 14 (48%) of them responded to plasma exchange treatment. One patient that did not respond to plasma exchange received additional Rituximab treatment and the syndrome resolved. The other 6 (20.6%) patients were refractory and eventually succumbed. Treatment-related mortality was 33.3% (9/29) in TA-TMA patients and was directly associated with refractory microangiopathic syndrome in 6/29.Conclusion: Our results suggest that TA-TMA is not rare in unrelated transplant recipients and can be managed in a portion of patients that respond to cyclosporine cessation and plasma exchange. GVHD was the only independent predictor for the development of TA-TMA and therefore, successful prevention and treatment strategies for GVHD need to be timely employed to prevent complications. More importantly, transplanted patients with refractory microangiopathic syndrome showed increased TRM indicating that conventional treatment is inadequate. Recent data of increased complement activation in TA-TMA pave the way for targeted treatment with complement inhibitors in these patients. DisclosuresNo relevant conflicts of interest to declare.

Incidence of Infectious Complications Associated with Bendamustine and Anti-CD20 Monoclonal Antibody Combination at Memorial Sloan Kettering Cancer Center (MSKCC)

Incidence of Infectious Complications Associated with Bendamustine and Anti-CD20 Monoclonal Antibody Combination at Memorial Sloan Kettering Cancer Center (MSKCC)

Phase 1b Results of a Phase 1b/2 Study of Obinutuzmab, Ibrutinib, and Venetoclax in Relapsed/Refractory Chronic Lymphocytic Leukemia (CLL)

BACKGROUNDVenetoclax(VEN), a once daily oral inhibitor of BCL2, has demonstrated high response rates and acceptable toxicity in patients with relapsed or refractory (R/R) CLL both as a single agent and in combination with the anti-CD20 monoclonal antibodies rituximab and obinutuzumab (formerly GA-101, G), where minimal residual disease (MRD) negative responses have been observed in the majority of patients. Ibrutinib (IBR), a once daily oral inhibitor of the Brutontyrosine kinase, likewise induces remissions in the majority of treated patients, but complete response (CR) is uncommon even after prolonged administration. Early genetic studies have demonstrated that BCL2 over-expression rescues BTK deficient XID murine B-cells from spontaneous apoptosis (J Immunol 1996), so we hypothesized that combination therapy would more efficiently achieve deep response endpoints. We report phase 1b results of a single-institution phase 1b/2 study of G, IBR, and VEN to characterize the safety and preliminary efficacy of the combination.METHODSPatients with CLL relapsed after or refractory to ≥1 prior therapy and who required treatment were eligible. Enrolled patients had ECOG ≤1 and preserved end-organ function, including creatinine clearance ≥50 mL/min/m2. Patients with chronic viral hepatitis infection, uncontrolled autoimmunecytopenia, active Richter transformation, and known cysteine-481 BTK mutation or clinical disease progression during treatment with a cysteine-481-binding BTK inhibitor were excluded. G, IBR, and VEN were started sequentially over the first 3 of fourteen 28-day cycles as detailed in the table. To establish the safety of VEN in combination with OBIN and IBR, VEN dose was escalated in 3 x 3 cohorts (100, 200, 400 mg) to a maximum planned dose of 400 mg daily. Dose limiting toxicity (DLT) was defined during the third cycle. Risk assessment for VEN dose ramp-up was conducted according to US prescribing information. Adverse events were assessed and graded using CTCAE v4.03. Response assessment according to IWCLL 2008 criteria, including bone marrow biopsy with 4-colorimmunophenotyping of marrow and peripheral blood (PB) for MRD, occurs after cycles 8 and 14.RESULTSTwelve R/R patients have been treated in the phase 1b portion of the trial. Median age was 57 years (range: 42-70) and median prior therapies was 1 (range: 1-7). Baseline genetic risk features includedunmutatedIGHV in 11 (92%),del(17p) in 1 (8%), del(11q) in 8 (67%), and complex abnormal karyotype in 5 (42%) patients. Tumor lysis (TLS) risk was low in 1 (8%), medium in 7 (58%), and high in 4 (33%) patients at study entry. In general, observed toxicities for the combination were consistent with those reported for the single agents. DLTs were not observed at any VEN dose level, establishing VEN 400 mg daily as safe in combination with standard doses of G and IBR. The most common grade ≥3 adverse events (regardless of attribution) were neutropenia (50%), lymphopenia (33%),hypertension(25%), and fatigue (17%). Grade 1/2 adverse events occurring in over half the patients included bruising (all grade 1, 83%), infusion related reaction (75%), hypertension (67%), headache (67%), hyperuricemia (all grade 1, 75%), hypocalcemia (75%), and diarrhea (all grade 1, 67%), AST and/or ALT elevation (58%), and rash (50%). No cases of either clinical or laboratory TLS were observed. All patients remain on therapy and 6 have reached response assessment after completing 8 cycles of therapy. All 6 have achieved objective response: 5 PR, including 1 MRD-negative in PB (VEN 100) and 1 MRD-negative in both PB and marrow (VEN 100), and 1 CR with MRD-negative PB and marrow (VEN 200).CONCLUSIONSG, IBR, and VEN can be safely administered in combination at doses standard for the treatment of CLL. DLTs were not observed, establishing VEN 400 mg as the recommended phase 2 dose in combination with G and IBR. Adverse events were manageable and largely consistent with those reported in the single agent phase 2 studies. Objective responses, including MRD-negative responses, have been observed among all R/R patients from the first dose cohorts. Accrual continues to parallel phase 2 cohorts of R/R (n=25) and TN (n=25) patients. Updated phase 1b toxicity and response data will be presented. [Display omitted] DisclosuresJones:Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Awan:Pharmacyclics: Consultancy; Novartis Oncology: Consultancy; Innate Pharma: Research Funding.

Upfront Rituximab Maintenance after Induction Therapy Improves Outcome and Reduces the Risk of Histological Transformation in Patients with Follicular Lymphoma - Real World Data from a Danish Population-Based Cohort

Introduction: In Denmark, as in most other Western countries, follicular Lymphoma (FL) is the second most common lymphoma entity. Although still regarded as an incurable condition, FL is characterized by an indolent clinical course, a natural history of recurrent relapses and in some cases transformation to an aggressive histology (HT). The addition of the anti CD20 antibody Rituximab to chemotherapy (r-chemo) has had a profoundly favorable impact on the time to relapse, survival and possibly also transformation rates (N. Wagner-Johnston et al, Blood, 2015; C. Sarkozy et al, JCO, 2016). During the last decade maintenance with Rituximab after initial immuno-chemotherapy, has been introduced with the hopes of further improving outcome for patients with FL.Aims: In this population-based study of real world data, we investigated the effect of upfront Rituximab maintenance (RM) on progression, survival and HT in FL patients.Patients and Methods: Patients diagnosed with histologically verified FL or Diffuse Large B-cell Lymphoma (DLBCL) in Denmark from 2000-2015 were identified through the National Danish Pathology Registry. Pathology reports were reviewed for patients diagnosed with both FL and DLBCL during the study period. HT was defined as initial biopsy proven FL diagnosis followed, in the same patient, by a subsequent biopsy-verified FL grade 3B or DLBCL. The identified patient cohort was cross-linked with the Danish Lymphoma Registry (LYFO). From this population, patients were included if meeting the following criteria: (i) completed first-line induction treatment with r-chemo, (ii) alive and eligible for RM at the time of clinical evaluation after induction treatment, and (iii) no evidence of HT at the same time point. Decision of treatment-free follow up or RM was applied according to local guidelines (RM was introduced at different time-points at different lymphoma-treating tertiary centers in Denmark). The RM-schedule consisted of 375 mg/m2 intravenous or subcutaneous every other month for 2 years.Statistical methods: Patient characteristics were compared using Fisher's exact or Student's t test. All time related end-points were estimated by Kaplan-Meier time-to-event analyses and compared using the log-rank test. Overall survival (OS) was defined from the date of FL diagnosis to the date of death by any cause or censoring; Progression-free survival (PFS) was calculated from the date of FL diagnosis to the date of progression/relapse or censoring. Time-to-transformation (TTT) was calculated from the date of FL-diagnosis to the date of biopsy proven HT. Factors of potential clinical relevance were tested in a multivariate analysis using a Cox proportional Hazards model.Results: 722 patients had completed their first Rituximab-containing induction treatment; 364 patients were consolidated with RM while 358 were not. The two treatment cohorts (no-RM vs. RM) were comparable in terms of sex and age distribution as well as performance score and LDH-elevation. Patients receiving RM had more often advanced Ann Arbor stage (p<0.001), higher FLIPI-score (p<0.001), heavier tumor burden (p<0.001) and bone marrow involvement (p=0.012). As compared to no-RM patients, those consolidated with RM had a significantly improved 5-year OS (83% vs. 90%; p=0.003) and PFS (63% vs. 72%; p<0.001) at univariate level and confirmed in multivariate analysis (fig. 1). Similarly, the rate of HT was reduced in RM treated patients (10% vs. 6%; p=0.067) and the mean TTT was prolonged (1.8 yrs vs. 2.7 yrs; p=0.052).Conclusions: Upfront consolidation with RM after initial treatment with r-chemo in patients with FL results in improved OS and PFS. Furthermore, after RM HT seems to occur less frequently and the event is significantly postponed. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Elimination of FVIII-Specific B Cells By Immunotoxins Comprised of a Single FVIII Domain Fused to Pseudomonas Exotoxin a

The development of inhibitory antibodies (inhibitors) against coagulation factor VIII (FVIII) is the most serious complication for patients with hemophilia A that undergo FVIII replacement therapy. In addition, healthy individuals can spontaneously develop inhibitory anti-FVIII auto-antibodies, which results in acquired hemophilia A. The current standard therapy for patients with hemophilia A and inhibitors, named immune tolerance induction (ITI), is based on frequent and mostly high dose administrations of FVIII. Unfortunately, the eradication of inhibitors can only be achieved in about 70% of patients. Alternative treatment of inhibitor patients with the monoclonal anti-CD20 antibody rituximab results in complete eradication of inhibitors; however, depletion of the entire CD20-positive B cell population is potentially accompanied by severe side effects. Recent studies in hemophilic FVIII knockout mice showed that the application of a FVIII-toxin conjugate resulted in (i) prevention of inhibitor development in naïve mice and (ii) long-term eradication of inhibitors in FVIII-immunized mice.As the use of FVIII for cell targeting of immunotoxins is presumably limited by its high molecular weight (250 kDa) and adhesiveness (off-target reactivity) we explored the potential use of alternative immunotoxins in the current study. The introduced immunotoxins are comprised of a single FVIII domain fused to the Exotoxin A (ETA) from Pseudomonas aeruginosa.The rationale for the use of a single domain instead of full length FVIII as cell-binding component is that immunodominant domains like A2 and C2 might still allow targeting of sufficient amounts of FVIII-specific B-cells by immunotoxins. For proof of concept studies, we generated a histidine-tagged C2 domain-ETA fusion protein (C2-ETA) that was bacterially expressed and purified by affinity chromatography. Purified C2-ETA was recognized by a panel of commercially available monoclonal anti-C2 antibodies in ELISA suggesting proper folding of the C2 domain in the bacterially expressed protein.To test the capacity of C2-ETA to eliminate FVIII-specific B-cells, splenocytes of FVIII-immunized FVIII knockout mice were re-stimulated with FVIII ex vivo in presence and absence of different concentrations of C2-ETA and ETA alone (as control). Re-stimulation of FVIII-specific memory B cells to FVIII- and C2-specific antibody secreting cells (ASC) was analyzed in anEnzyme linked immunospot (ELISPOT) assay using FVIII and C2 as antigens. While differentiation to FVIII-specific ASC was only partially inhibited by C2-ETA, differentiation to C2-specific ASC was completely blocked in a dose-dependent manner. In contrast, the use of ETA alone had no effect. Further analysis of the FVIII domain specificity of antibodies in plasma of FVIII-immunized FVIII knockout mice used for depletion studies revealed a strong contribution of C2-specific antibodies to the overall FVIII-specific immune response.In summary, our results show that the developed C2-ETA immunotoxin is able to specifically eliminate FVIII C2 domain-specific B cells ex vivo. Currently, C2-ETA is tested for its capacity to eliminate FVIII-specific B cells in FVIII knockout mice and additional FVIII domain-ETA immunotoxins are developed. DisclosuresNo relevant conflicts of interest to declare.

Reduction of Mitochondrial Membrane Potential Leads to Enhancement of Type-II CD20-Antibody Cytotoxicity in Diffuse Large B-Cell Lymphoma

Diffuse Large B Cell Lymphoma (DLBCL) is the most prevalent non-Hodgkin lymphoma (NHL) in adults. Since the addition of the Type I anti-CD20 antibody Rituximab to chemotherapy, the overall survival of NHL patients has improved dramatically compared to the pre-Rituximab era. DLBCL however, has the worst survival rates out of all NHLs with an average 5-year survival of 55%. Unfortunately 40% of all DLBCL patients relapse within 2 years, and those that relapse or have refractory disease tend not to respond well to antibody-based salvage therapies. Since the discovery and utilisation of Rituximab, many have tried to enhance the efficacy of anti-CD20 antibodies in order to improve first-line treatment of DLBCL, leading to the evolution of Type II humanised anti-CD20 antibodies.The complete biological role of CD20 remains unclear, however it has been shown to act as part of an ion channel complex that is a component of the store operated calcium (Ca2+) system. This complex has the ability to facilitate mitochondrial membrane permeabilisation, resulting in reduced mitochondrial function.In order to investigate the effect of Type I- and Type II- anti-CD20 antibodies on mitochondrial function, we established a panel of 4 DLBCL cell lines. We used the XF Seahorse Mito Stress Test to reveal bioenergetic profiles of the cell lines before and after treatment with a panel of Type I and Type II anti-CD20 antibodies (2 Type-I and 2 Type-II anti-CD20 antibodies for each cell line). Basal oxidative phosphorylation (OxPhos), ATP production, and maximal and spare respiratory capacity of each sample were calculated as a measure of mitochondrial function. Next we used Metformin, a well-established inhibitor of oxidative phosphorylation to reduce the mitochondrial membrane potential (MMP) across our panel of cell lines. We confirmed MMP reduction by staining cells with JC-1, a chameleon dye used as an indicator of MMP and analysed samples using flow cytometry. We then used the XF Seahorse Mito Stress Test, this time to assess how combining each CD20-antibody with an OxPhos inhibitor effects mitochondrial function (10 conditions for each cell line). Finally, we used the same conditions to conduct clonogenic survival assays to see whether cytotoxicity of Type-I or Type-II anti-CD20 antibodies could be enhanced.We have observed that treatment with anti-CD20 antibodies results in a significant increase in the maximal respiratory capacity of our panel of cell lines. Conversely, pharmacological inhibition of oxidative phosphorylation causes a significant reduction in basal oxidative phosphorylation as well as a reduction in the maximal respiratory capacity of the cell lines in our panel. We also show that treatment combining an OxPhos inhibitor with either Type-I or Type-II CD20-antibodies prevents the increase in maximal respiratory capacity observed with CD20-antibody treatment alone. When analysing the clonogenic survival of cell lines we have found that only the cytotoxicity of Type-II anti-CD20 antibodies is enhanced by simultaneously treating cell lines with Metformin. We also used Annexin V/PI staining to assess cell death and show that inhibiting oxidative phosphorylation in conjunction with CD20-antibody treatment does not result in a significant increase in cell death across our panel of cell lines.Our data indicate for the first time that when cells are treated with CD20-antibodies they increase their maximal mitochondrial respiratory capacity to compensate for reduced basal mitochondrial function. We also show that inhibition of oxidative phosphorylation disables the cells from being able to compensate for the reduced mitochondrial function that is caused by CD20-antibody treatment. Importantly our data show that the reduction of mitochondrial function caused by combining Metformin with Type-II CD20 antibodies leads to a significant reduction in clonogenicity. We believe that understanding the mechanism of the inhibition of mitochondrial function will allow us to establish effective treatment combinations to significantly improve the efficacy of anti-CD20 antibody therapy in DLBCL. DisclosuresNo relevant conflicts of interest to declare.

Rituximab Preferentially Eliminates BCR Signaling Proficient Chronic Lymphocytic Leukemia B Cells In Vivo

The use of anti-CD20 antibody rituximab has significantly improved the outcome of patients with chronic lymphocytic leukemia (CLL). Rituximab has been shown to act through several mechanisms including antibody-dependent cell cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), direct induction of apoptosis, and sensitization to chemotherapy. However, the exact contribution of each of these mechanisms to the clinical efficacy of rituximab in vivo and the exact mechanism of its action remain unclear. Importantly, the levels of cell surface CD20 expression were shown to associate with the efficacy of rituximab.We and others have described that CLL cells that have recently exited the lymph node microenvironment to the peripheral blood express lower cell surface levels of the chemokine receptor CXCR4 and higher levels of the activation marker CD5 (Callisano et al., 2011). We observed that these CLL cells (CXCR4dimCD5bright) have also higher cell surface CD20 expression (~2-fold; P<0.0001) compared to CLL cells from the same patient circulating in the blood stream for a longer time (CXCR4brightCD5dim), and we showed that CD20 is up-regulated by stromal cell interactions through the binding of SDF-1α to CXCR4 (P<0.001). We further hypothesized that higher levels of CD20 on CXCR4dimCD5bright cells will make them the primary target for rituximab in vivo. To investigate this, we obtained peripheral blood samples from CLL patients (N=17) before rituximab administration (day 0) and 24 hours (day 1) after rituximab administration (375 mg/m2, single agent). This showed that rituximab primarily and nearly completely eliminates the CXCR4dimCD5bright subpopulation (8.3 % pre-rituximab vs. 2.1 % post-rituximab, P<0.0001), which harbors the highest levels of CD20 (P<0.005).It has been previously suggested that the CD20 plays a direct role in microenvironmental interactions and especially in B cell receptor signaling (BCR; Uchida et al., 2004). Therefore, we investigated the BCR signaling propensity of CXCR4dimCD5bright subpopulation. We have observed that CXCR4dimCD5bright CLL cells have higher surface immunoglobulin (Ig) expression than CXCR4brightCD5dim cells from the same patient (~2-fold, P<0.005). This was also coupled with higher responsiveness of CXCR4dimCD5bright cells to BCR crosslinking with anti-IgM as measured by calcium flux (P=0.005). Moreover, CXCR4dimCD5bright cells also had higher levels of CD19 (1.8-fold, P<0.0001), which is an important docking molecule in PI3K signaling, and its higher levels lower the threshold for BCR signaling activation.Altogether, this study shows that rituximab primarily and effectively eliminates, at least in the short term, the BCR signaling proficient CLL B cells. It is likely that one of the mechanisms of rituximab action is the indirect inhibition of BCR signaling by eliminating CLL cells with strongly activated BCR pathway. These observations might have important implications for combinatorial therapies with BCR signaling inhibitors such as ibrutinib or idelalisib.This work was supported by: the Ministry of Education, Youth and Sports of the Czech Republic under the project CEITEC 2020 (LQ1601); the European Union's Horizon 2020 research and innovation programme under grant agreement No. 692298; Czech Science Foundation (project No. 16-13334Y); the Ministry of Health of the Czech Republic, grant No. 16-29622A. All rights reserved. This work was financed from the SoMoPro II Programme (project No. 4SGA8684), co-financed by European Union and the South-Moravian Region. This publication reflects only the author's views and the Union is not liable for any use that may be made of the information contained therein. This work was supported by the Ministry of Health of the Czech Republic - conceptual development of research organization (FNBr, 65269705, Sup 3/16), MUNI/A/1028/2015, and G.P. is a city of Ostrava scholarship holder. contact: marek.mraz@email.cz DisclosuresNo relevant conflicts of interest to declare.