ALBERT

Description: The anti-CD20 mAb rituximab has substantially improved the clinical outcome of patients with a wide range of B-cell malignancies. However, many patients relapse or fail to respond to rituximab, and thus there is intense investigation into the development of novel anti-CD20 mAbs with improved therapeutic efficacy. Although Fc-FcγR interactions appear to underlie much of the therapeutic success with rituximab, certain type II anti-CD20 mAbs efficiently induce programmed cell death (PCD), whereas rituximab-like type I anti-CD20 mAbs do not. Here, we show that the humanized, glycoengineered anti-CD20 mAb GA101 and derivatives harboring non-glycoengineered Fc regions are type II mAb that trigger nonapoptotic PCD in a range of B-lymphoma cell lines and primary B-cell malignancies. We demonstrate that GA101-induced cell death is dependent on actin reorganization, can be abrogated by inhibitors of actin polymerization, and is independent of BCL-2 overexpression and caspase activation. GA101-induced PCD is executed by lysosomes which disperse their contents into the cytoplasm and surrounding environment. Taken together, these findings reveal that GA101 is able to potently elicit actin-dependent, lysosomal cell death, which may potentially lead to improved clearance of B-cell malignancies in vivo.

Description: Abstract 2747 Background: Single-agent immunotherapy with rituximab is a viable treatment option for low risk FL, with limited toxicity and a long duration of response in some patient subsets. We have previously shown that high expression of FcγRIIB promotes rituximab internalisation on various B cell targets, including FL (Blood 2011 118:2530–2540), something not seen with type II anti-CD20 antibodies. The SAKK 35/98 trial examined rituximab monotherapy in FL and now has long-term follow-up data of almost 10 years (JCO 2010 28:4480–4484). We analysed diagnostic tumour samples from this trial to determine the relationship of FcγRIIB expression to responses and clinical outcomes after rituximab treatment in FL. Methods: 202 patients (pts) with newly diagnosed or relapsed FL received induction treatment with rituximab 375 mg/m2 weekly for 4 weeks. Pts with stable or responding disease at week 12 were randomized into 2 groups: no further treatment or prolonged treatment with single infusions of rituximab 375 mg/m2 at weeks 12, 20, 28 and 36. Archived tissue samples from 135 evaluable pts were stained using an anti-human FcγRIIB antibody (clone EP888Y, Abcam) at a dilution of 1:3000 on a Dako autostainer. The samples were pretreated with the Dako EnVisionFLEX target retrieval solution high pH and detection using the Dako AS-Link 48 with Dako EnVision flex plus detection kit. Positive samples were graded into negative/low intensity staining (n=120) versus medium/high (n=13) by an expert lymphoma histopathologist blinded to the clinical outcomes. Data from 2 slides and response at week 12 data for 4 pts were unavailable (1 of whom also has missing slide data), resulting in 130 pts available for analysis. Failure-free survival (FFS) was defined as time from registration until failure to achieve complete/partial response at week 12, progression, relapse, a second cancer or death from any cause. Objective response rate (ORR) was associated with intensity staining levels using Fisher's exact test. All time-to-event endpoints were evaluated using the Kaplan-Meier method; groups were compared using the log-rank test. The hazard ratio (HR) was assessed using Cox proportional hazards models. Results: Registered and randomised pts had very similar baseline characteristics; previously untreated pts had slightly more favourable characteristics but were balanced between the 2 treatment arms. Pts expressing medium/high levels of FcγRIIB were less likely to respond to rituximab by week 12 (ORR 58.1% vs 23.1%, Fisher's exact test, p=0. 02), a finding independent of prior therapy. For FFS, there was a statistically significant difference (p=0.001; HR=0.42; 95% confidence interval (C.I.): 0.23–0.77) between the negative/low staining group (median: 21.4 months; 95% C.I.: 7.0–34.2) and the medium/high staining group (median: 7.0 months; 95% C.I.: Not calculable). The interaction between staining levels and randomised treatment groups for FFS was not statistically significant. There was a non-significant trend towards better overall survival in the low/negative group (median: 140.0 vs 50.0 months; p=0.15; HR=0.57; 95% C.I.: 0.27–1.23); however the event rate was lower (36.8% vs 61.5%). Conclusion: Elevated FcγRIIB expression level is associated with poor response to rituximab in pts with FL. This group may show better results with non-internalising type II antibodies, a hypothesis for validation in future prospective clinical trials. Disclosures: Ghielmini: Roche: Honoraria, Speakers Bureau. Johnson:Roche: Honoraria.

Description: Introduction Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma and is characterized by its genetic and clinical heterogeneity. Patients can develop DLBCL de novo or as a transformation from other lymphoid malignancies, most commonly follicular lymphoma. For patients with relapsed/refractory DLBCL (rrDLBCL), prognosis is extremely poor with 2-year overall survival of 20-40%. While numerous treatments are under investigation to improve patient outcomes, the success of these treatments has been limited as the genetic mechanisms underpinning treatment resistance are largely unknown. Identifying genomic alterations which contribute to relapse may improve salvage therapy for patients with rrDLBCL or allow patients to be stratified prior to frontline treatment. Methods To identify genomic alterations which contribute to R-CHOP resistance, we previously collected samples from patients enrolled in four clinical trials exploring candidate salvage therapies for patients with rrDLBCL as well as a retrospective rrDLBCL cohort, totalling 193 cases (133 de-novo DLBCL, 60 transformed). Plasma samples were collected from each patient upon relapse along with diagnostic tissue biopsies where available. A combination of exome sequencing and target-panel sequencing of lymphoma associated genes was performed on circulating tumour DNA and tissue biopsies (if available). Mutations implicated in R-CHOP resistance were identified through two complimentary strategies. First, the mutation frequency of recurrently mutated genes across de novo rrDLBCL samples was compared to a cohort of unrelated diagnostic DLBCL cases (n=1691) to identify genes enriched for mutations. Second, the genomic landscape and tumor clonal structure was compared prior to and following R-CHOP to identify mutations in each patient that underwent clonal expansion following therapy. Anti-CD20 antibody binding affinity of MS4A1 mutants was evaluated using flow cytometry on transfected CHO-S cells. Results We have identified five genes enriched for mutations in our rrDLBCL cohort relative to diagnostic DLBCL: KMT2D (Mutated in 49%, Q=0.0385), TP53 (47%, Q=1.07x10-9), FOXO1 (11%, Q=0.0727), NFKBIE (11%, Q=0.0385), and MS4A1 (8%, Q=0.0522). Consistent with its characterization as a poor prognostic marker, mutations in TP53 were typically present at diagnosis and remained stable following R-CHOP therapy for both de novo and transformed DLBCL (23/27 cases, 85%). Recurrent mutations affecting Arg248 of TP53 (6.8%, Q=0.0413) were also clonally stable and have previously been associated with poor overall survival across several cancer types. The histone methyltransferase KMT2D is dominated by nonsense and frameshift mutations which were stable or underwent clonal expansion following R-CHOP (17/19, 89%). Recurrent missense mutations in MS4A1 targeted the small loop and adjacent transmembrane domains of CD20, including several patients with a Tyr86 mutation. Transfected cells carrying Tyr86Cys or Leu66Arg mutations were not bound by rituximab or other anti-CD20 antibodies including obinituzumab and ofatumumab. Subclonal populations containing MS4A1 mutations underwent clonal expansion (6 cases) or were stable (1 case) following treatment, including one case with multiple MS4A1 mutations in distinct subclonal populations which both underwent clonal expansion. In another unique case, a series of ctDNA samples were available prior to and following R-CHOP and salvage therapy, where we again observed convergent evolution of two mutually exclusive clonal subpopulations containing MS4A1 mutations. The first subpopulation underwent clonal expansion following frontline therapy but was extinguished following salvage therapy, while the other subpopulation underwent clonal expansion following salvage therapy and harboured a transmembrane domain mutation. Conclusion Mutations in TP53 and truncating mutations in KMT2D are generally present prior to treatment and will be investigated as biomarkers of treatment failure. Additional mutations are not always present at diagnosis, but their emergence can be detected in ctDNA and relapsed tissue, specifically mutations in MS4A1. As mutations in MS4A1 attenuate rituximab binding and are recurrently associated with clonal expansion, they likely impart a selective advantage and lead to resistance against anti-CD20 antibodies. Disclosures Michaud: Epizyme: Employment. Daigle:Epizyme: Employment. Jain:Kite/Gilead: Consultancy. Kuruvilla:Roche: Honoraria; Astra Zeneca: Honoraria; Novartis: Honoraria; Merck: Honoraria; Karyopharm: Honoraria; Gilead: Honoraria; Celgene: Honoraria; BMS: Honoraria; Amgen: Honoraria; Seattle Genetics: Consultancy; Roche: Consultancy; Merck: Consultancy; Karyopharm: Consultancy; Gilead: Consultancy; Janssen: Research Funding; Roche: Research Funding; BMS: Consultancy; Abbvie: Consultancy; Seattle Genetics: Honoraria; Janssen: Honoraria. Assouline:Abbvie: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria, Speakers Bureau. Scott:NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding; Celgene: Consultancy; Roche/Genentech: Research Funding; Janssen: Consultancy, Research Funding. Johnson:BD Biosciences: Other: Provided a significant proportion of the antibodies used in this project free of cost.; Merck: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Abbvie: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy, Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding; Seattle Genetics: Honoraria; Lundbeck: Employment, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel fees, gifts, and others, Research Funding.

Description: The emergence of B cell receptor (BCR) kinase inhibitors has proved effective for the treatment of a number of B-cell malignancies including chronic lymphocytic leukemia (CLL). BTK and PI3K inhibitors have clear efficacy in suppressing tumor progression but have not been curative. A number of patients have developed resistance to these drugs following mutation of the BTK or PLCγ2 gene. Whilst, other patients are unable to tolerate these drugs due to adverse events or progress whilst on therapy for unknown reasons. Thus the development of novel drugs which are still effective once other BCR-kinases inhibitors become ineffective is of paramount importance. Spleen tyrosine kinase (Syk) is essential for B cell receptor signalling pathways as well as a variety of other surface receptors such as MHCII, FC receptors and integrins, all of which have been shown to play a role in CLL biology. Importantly, Syk inhibition has been shown to overcome resistance to ibrutinib, identifying Syk inhibition as a promising strategy to treat these patients. Furthermore, we have previously shown that IL-4 is found in CLL lymph nodes and can promote resistance to ibrutinib and idelalisib by restoring αIgM induced calcium flux and phosphorylated ERK (ASH 2014, abstract #3299). IL-4 signalling is mediated through the JAK/STAT signalling pathways via JAK1 and JAK3, therefore simultaneous inhibition of both Syk and JAK1/3 may be therapeutically beneficial over BCR kinase inhibitors alone. Cerdulatinib (PRT062070) is a dual JAK/Syk inhibitor in a phase I open label dose escalation study and is currently demonstrating clinical activity in patients with relapsed/refractory B cell malignancies including CLL. Our group has now demonstrated in vitro that cerdulatinib, at plasma concentrations achievable in patients, can induce apoptosis of CLL cells in a concentration and time dependent manner with a mean IC50 of 3µM and 1µM at 48 and 72h respectively, defined by annexin V/PI and cleavage of caspase 3 and poly ADP ribose polymerase (PARP). Apoptosis was caspase dependent since treatment with the pan caspase inhibitor ZVAD.fmk significantly inhibited cerdulatinib induced cell death at 24h. Cerdulatinib induced apoptosis coincided with an increase in pro-apoptotic proteins Noxa and Puma and a decrease in the anti-apoptotic protein Mcl-1. Cerdulatinib significantly inhibited IL-4 induced phosphorylation of STAT6 at 300nM (p=.005), BCR induced phosphorylation of AKTS473 with soluble (p=.008) and bead immobilised (BI) (p=.025) αIgM at 30nM and phosphorylation of AKTT308 with BI αIgM at 300nM (p=.008). Furthermore, in patients with CLL, it is thought that CD40L and IL-4 are key factors, which promote survival of CLL cells in proliferation centres within the lymph node microenvironment. Therefore, we cultured CLL cells with a vehicle control or IL-4CD40L, prior to treatment with cerdulatinib. Cerdulatinib alone induced similar levels of apoptosis irrespective of IL-4/CD40L treatment, suggesting cerdulatinib may be able to overcome microenvironmental signals and target cells within the lymph node. Next we explored the possibility of augmenting cerdulatinib induced apoptosis by simultaneous inhibition with the Bcl-2Bcl-XL inhibitor ABT-199. In vitro in the presence of IL-4/CD40L, ABT-199 synergised with cerdulatinib to induce significantly greater cell death than with either agent alone. Therefore these data provide in vitro evidence for the use of cerdulatinib in clinical trials for the treatment of CLL as either a single agent or in combination with other therapies such as ABT-199. Disclosures Strefford: Roche: Research Funding. Davies:Seattle Genetics: Research Funding; Takeda: Honoraria. Coffey:Portola Pharmaceuticals Inc: Employment, Equity Ownership, Research Funding. Steele:Portola Pharmaceuticals: Other: Travel bursary to ASH 2015; Janssen: Other: Travel bursary to EHA 2015.

Description: Chronic lymphocytic leukaemia (CLL) is characterised by an accumulation of B cells which is broadly split into two groups representing a progressive IGHV unmutated (U-CLL) and a more indolent IGHV mutated (M-CLL) disease. Activation of the B cell receptor (BCR) by antigen/autoantigen engagement is crucial for CLL cell survival, disease progression and resistance to therapy, however further research is required to better understand how BCR signalling impacts on CLL biology. Autophagy is known to play a role in tumorigenesis and resistance to therapy in solid tumors, however whether autophagy has a role in CLL biology and how it is regulated has not been fully investigated. Autophagy is important for normal B cell development and is known to be regulated by various drug treatments in vitro in CLL samples. A previous study showed that activation of the BCR on murine splenic B cells with soluble or bead immobilised (BI) anti-IgM induced autophagy and subsequent apoptosis, however, the role of BCR-induced autophagy has not been explored in B cell malignancies and particularly CLL. Firstly, we assessed basal protein expression of key autophagy markers LC3BII, and ATG3 in CLL samples and age-matched normal donor B cells (NDB). CLL cells expressed significantly more LC3BII (p=.014, n=57) and ATG3 (p=.04, n=58) compared with NDB (n=8), with a greater LC3BII protein expression in U-CLL compared to M-CLL (p=.039, n=57), indicating more autophagy occurs in U-CLL. Furthermore basal increases in autophagy markers GABARAPL2 (LC3B family member) (p=.0004, n=34) and ATG4A (p=.04, n=20) at the RNA level were significantly associated with the ability of CLL cells to flux calcium (〉10%) in response to anti-IgM. This indicated a possible role of the BCR in the regulation of autophagy in CLL samples and a possible association with progressive disease. Activation of the CLL BCR with BI anti-IgM significantly induced expression of autophagy markers ATG3 (p=.002, n=22), LC3BII (p

Description: Introduction: Three unconjugated CD20 antibodies have been approved for the treatment of lymphoma patients. All three are of human IgG1 isotype, but nevertheless they differ in their modes of action: type I antibodies (e.g. rituximab and ofatumumab) trigger effective complement-dependent cytotoxicity (CDC), whereas type II antibodies (e.g. obinutuzumab) are potent inducers of direct cell death, while both type I and II antibodies can induce antibody-dependent cellular cytotoxicity (ADCC). Studies in syngeneic mouse models suggest that myeloid cells are the predominant effector cell type for CD20 antibodies (Uchida et al. J Exp Med 199:1659, 2004). However, human myeloid cells, particularly PMN, are activated more effectively by human IgA than by IgG1 antibodies (Dechant et al. Blood 100:4574, 2002) - especially when the latter are engineered for enhanced FcγRIII affinity (Peipp et al. Blood 112:2390, 2008). Antibodies of IgA isotype constitute an integral part of the mucosal immune system, and differ from IgG antibodies in their pharmacokinetic properties and immune effector mechanisms (Boross et al. EMBO Mol Med 5:1213, 2013, Lohse et al. Cancer Res 76:403, 2016). Here, we compared the efficiency of IgG1 and IgA2 isotype variants of the type I CD20 antibody 1F5 in killing lymphoma cells in vitro and in vivo. Methods: Recombinant antibodies against human CD20 were produced by co-transfecting BHK cells with vectors encoding the 1F5 variable, Igα2 or Igγ1 heavy, and κ light chain constant regions, respectively. The resulting isotype variants were compared for their biochemical characteristics as well as Fab- and Fc- mediated effector functions using human lymphoma cell lines as targets. NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice xenotransplanted with human RAJI lymphoma cells were employed to investigate the therapeutic efficacy of human CD20 antibodies. Additionally, in vivo depletion of human CD20 transgenic B cells was evaluated in a syngeneic B cell depletion model using wildtype, human FcαRI transgenic and C3 knock-out mice. Results and Discussion: In vivo studies with xenotransplanted RAJI cells in NSG mice, which lack functional T and NK cells, demonstrated significantly prolonged survival of treated as compared to non-treated mice, indicating that myeloid effector cells may contribute to the therapeutic efficacy of CD20 antibodies against human lymphoma cells. In vitro, neither IgG1 nor IgA2 variants of 1F5 showed efficient Fab-mediated effects such as direct cell death induction and homotypic aggregation compared to type II antibodies. However, human IgA2 but not IgG1 antibody variants against CD20 effectively triggered ADCC by human PMN, the most numerous myeloid effector cell population. Although IgA does not bind C1q, CD20 IgA antibodies also triggered CDC against several lymphoma cell lines. CDC was predominantly mediated by the alternative pathway, as evidenced by the kinetics of lysis, the requirement for higher serum concentrations and inhibition by the C3 inhibitor compstatin. Further in vivo experiments demonstrated that 1F5-IgA2 effectively depleted B cells in a syngeneic human CD20 transgenic B cell depletion model. However, studies in human FcαRI transgenic or C3 knock-out mice indicated that B cell depletion was not mediated by FcαRI or complement - suggesting that other currently undefined mechanisms contribute to the in vivo efficacy of IgA antibodies against CD20. Conclusions: Together, the presented results suggest that CD20 antibodies of human IgA isotype constitute promising immunotherapeutic reagents with unique effector functions. Additional studies are required to further elucidate their effector mechanisms in vitro and in vivo. Disclosures Cragg: Roche: Consultancy, Research Funding; Gilead Sciences: Research Funding; Baxalta: Consultancy; Bioinvent International: Consultancy, Research Funding; GSK: Research Funding.

Description: Radioimmunotherapy using radiolabeled anti-CD20 antibodies (mAb) is an effective new treatment in non-Hodgkin lymphoma with high response rates. However, the molecular mechanisms behind these impressive clinical responses are poorly understood. To elucidate these mechanisms we studied the signaling events evoked in a panel of lymphoma cell lines following treatment with anti-CD20 mAb alone or in combination with irradiation. In all three lymphoma cell-lines tested a synergistic cytotoxic effect was observed when the anti-CD20 mAb B1 was combined with irradiation. The additive effect seen with B1 mAb and radiation was not observed with Rituximab and could be reversed with MEK inhibitors U0126 and PD98059 as well as siRNA targeting MEK1 or 2. Moreover, addition of U0126 reversed the decrease in clonogenic survival triggered by treatment with B1 and irradiation. To further probe the mechanism of this synergistic cell death we used cell lines over-expressing BCL2 or crmA, to block mitochondrial and death receptor pathways, respectively. Although BCL2 and crmA over-expression mediated protection against radiation alone, it had no impact on the increased cytotoxicity induced by B1+irradiation. Morphological studies revealed gross vacuolization of the cytoplasm, yet relatively well preserved nuclei in cells treated with B1+irradiation. Taken together our data indicate that activation of the MAPK cascade is an important factor that contributes to the synergistic effect of anti-CD20 (B1) antibody and irradiation and provides important new insights into how this treatment may work in the clinic.