Direct Cell Death Research Articles

Development of novel monoclonal antibodies to dog leukocyte antigen DR displaying direct and immune-mediated cytotoxicity toward canine lymphoma cell lines.

Spontaneous canine lymphoma (CL) has become a promising, nonrodent model for advancing the therapeutic strategies of human hematological malignancies. As new resources for veterinary and comparative studies on CL-associated antigens, we developed 2 novel mouse monoclonal antibodies, denoted B5 and E11, that recognized the canine major histocompatibility Class II DR antigens (dog leukocyte antigen DR). Using flow cytometry and solid phase immunoenzymatic assays, we showed that the antigens recognized by B5 and E11 were strongly expressed in several CL cell lines and the ex vivo canine neoplastic cells of B and mixed B/T immunophenotypes. Additionally, we evaluated a minimal cross-reactivity of B5 and E11 with the human B-cell line, Raji. By the ectopic expression of the hybrid murine/canine I-E/DR dimers in the HEK293 cells, we demonstrated that the epitope of B5 was localized to the invariant DRα chain, whereas the epitope of E11 was collectively formed by the DRα and DRβ chains. Both epitopes were conformational and conserved in all the tested unrelated individuals of different dog breeds. In vitro treatment of 2 CL B-cell lines (CLBL1 and CLB70) with B5 and E11 rapidly induced a direct apoptotic cell death. Similarily, both mouse monoclonal antibodies efficiently killed the above cell lines through the mechanisms of complement-dependent and antibody-mediated cellular phagocytosis. Collectively, our data support the further development of B5 and E11 as novel tools for dog leukocyte antigen DR-targeted, preclinical trials involving CL.

The B cell death function of obinutuzumab-HDEL produced in plant (Nicotiana benthamiana L.) is equivalent to obinutuzumab produced in CHO cells.

Plants have attracted attention as bio-drug production platforms because of their economical and safety benefits. The preliminary efficacy of ZMapp, a cocktail of antibodies produced in N. benthamiana (Nicotiana benthamiana L.), suggested plants may serve as a platform for antibody production. However, because the amino acid sequences of the Fab fragment are diverse and differences in post-transcriptional processes between animals and plants remain to be elucidated, it is necessary to confirm functional equivalence of plant-produced antibodies to the original antibody. In this study, Obinutuzumab, a third generation anti-CD20 antibody, was produced in N. benthamiana leaves (plant-obinutuzumab) and compared to the original antibody produced in glyco-engineered Chinese hamster ovary (CHO) cells (CHO-obinutuzumab). Two forms (with or without an HDEL tag) were generated and antibody yields were compared. The HDEL-tagged form was more highly expressed than the non-HDEL-tagged form which was cleaved in the N-terminus. To determine the equivalence in functions of the Fab region between the two forms, we compared the CD20 binding affinities and direct binding induced cell death of a CD20-positive B cells. Both forms showed similar CD20 binding affinities and direct cell death of B cell. The results suggested that plant-obinutuzumab was equivalent to CHO-obinutuzumab in CD20 binding, cell aggregation, and direct cell death via binding. Therefore, our findings suggest that Obinutuzumab is a promising biosimilar candidate that can be produced efficiently in plants.

Low Peripheral Blood NK Cell Count Is Associated with Worse Clinical Outcome in Patients with Follicular Lymphoma (FL) and Diffuse Large B-Cell Lymphoma (DLBCL) Treated with Immunochemotherapy: Results from the Frontline Phase 3 GALLIUM and GOYA Trials

Low Peripheral Blood NK Cell Count Is Associated with Worse Clinical Outcome in Patients with Follicular Lymphoma (FL) and Diffuse Large B-Cell Lymphoma (DLBCL) Treated with Immunochemotherapy: Results from the Frontline Phase 3 GALLIUM and GOYA Trials

Impact of obinutuzumab alone and in combination for follicular lymphoma.

Although rituximab-based chemoimmunotherapy prolongs the survival of patients with follicular lymphoma (FL), this disease is considered incurable in most patients. Thus, new therapies are needed not only for those in the relapsed/refractory setting, but also for initial treatment. Obinutuzumab (G, GA101) is a third-generation, fully humanized type II glycoengineered, anti-CD20 monoclonal antibody that results in increased direct cell death and antibody-dependent, cell-mediated cytotoxicity/phagocytosis compared to rituximab. Obinutuzumab has significant antitumor activity when used alone or in combinations in untreated or relapsed refractory FL patients. Studies have demonstrated its ability to prolong progression-free survival and, in some cases, overall survival, and to eliminate minimal residual disease. Several ongoing trials are investigating combinations with chemotherapy, immunomodulators, targeted drugs, and immunotherapy agents. G is generally well tolerated, with associated adverse effects including infusion-related reactions, neutropenia, thrombocytopenia, and reactivation of hepatitis B virus. Future studies with this antibody should focus on identifying predictive markers and developing chemotherapy-free combinations that will improve the outcome of patients with FL.

Obinutuzumab for the treatment of chronic lymphocytic leukemia and other B-cell lymphoproliferative disorders

ABSTRACTIntroduction: Chemoimmunotherapeutic regimens using the anti-CD20 antibody rituximab improved significantly the survival rates in various B-cell lymphoproliferative disorders (LPDs), including chronic lymphocytic leukemia (CLL). The next-generation CD20 antibody obinutuzumab represents an addition to the drug armamentarium used for the therapeutic management of patients with LPDs.Areas covered: Herein, the authors discuss the biochemical and conformational engineering of obinutuzumab to increase antibody-dependent cell-mediated cytotoxicity and direct cell death. They also describe the available preclinical data on obinutuzumab’s role in B-cell LPDs. Furthermore, the authors summarize the Phase I and II clinical trials of obinutuzumab, focusing on the main pharmacokinetic/pharmacodynamic characteristics, the most common clinically significant adverse events, dose optimization, and clinical outcomes of patients with CLL and other B-cell LPDs treated with obinutuzumab as monotherapy or in combination with other agents. To put these data in perspective, the use of obinutuzumab is compared with that of rituximab in CLL and other B-cell LPDs.Expert opinion: Clinical trials have demonstrated that obinutuzumab is well tolerated. The novel mechanism of action of obinutuzumab is associated with significant efficacy in CLL and other B-cell LPDs. Ongoing clinical trials are expected to determine the optimal use of obinutuzumab in these diseases.

A TLR7 agonist enhances the antitumor efficacy of obinutuzumab in murine lymphoma models via NK cells and CD4 T cells.

This corrects the article DOI: 10.1038/leu.2016.352.

Atezolizumab (A) + cobimetinib (C) + vemurafenib (V) in BRAFV600-mutant metastatic melanoma (mel): Updated safety and clinical activity.

3063 Background: Targeted inhibition of MEK with C and BRAF with V in BRAFV600-mutant mel can lead to both anticancer immune activation and direct tumor cell death. A, an anti–PD-L1 monoclonal antibody, inhibits PD-L1/PD-1 signaling. Combining C + V with A may enhance antitumor activity, potentially leading to improved clinical responses and durability. Preliminary data from this phase Ib study (NCT01656642) showed that A + C + V had a manageable safety profile and promising antitumor activity in patients (pts) with untreated BRAFV600-mutant unresectable/metastatic mel, with increases in CD8-positive T-cell infiltration observed after C + V (Sullivan et al SMR 2016). We present updated safety and efficacy data. Methods: Pts received A + C + V after a 28-day run-in with C + V. A was given at 800 mg q2w, C at 60 mg qd for the first 21 days of each 28-day cycle, and V at 960 mg bid during day 1–21 of run-in and 720 mg thereafter. Safety was evaluated in pts who had ≥1 dose of A; efficacy, in pts who had ≥1 dose of A by the data cutoff date and received ≥1 dose of A, C, or V by the dosed-by date. Results: Thirty-four patients were treated and evaluated for both safety and efficacy. Median survival follow-up was 7.1 months (range 2.5–19.9). Elevated AST/ALT, diarrhea, arthralgia, fatigue, photosensitivity, pyrexia, nausea, flu-like symptoms, maculopapular rash, and pruritus were reported as A- and/or C- and/or V-related in > 20% of pts at any grade (G). A/C/V-related G3–4 adverse events (AEs) were seen in 15 pts (44.1%) with the triple combination (none G5). Three serious AEs were A-related. All AEs were manageable and reversible. Elevated ALT/AST (three pts each) and rash (one pt) led to discontinuation of any drug. Unconfirmed RECIST V1.1 responses were observed in 29 pts (85.3%; six complete, 23 partial). Three pts with confirmed partial responses had resolution of target lesions. Twenty of the 29 responding patients continue to respond at the time of the data cutoff. Conclusions: Updated results confirm preliminary findings that A + C + V has a manageable safety profile and promising antitumor activity in pts with BRAFV600-mutant metastatic mel. Continued exploration of A + C + V is warranted. Clinical trial information: NCT01656642.

Toca 511 gene transfer and treatment with the prodrug, 5-fluorocytosine, promotes durable antitumor immunity in a mouse glioma model.

Background.Toca 511 (vocimagene amiretrorepvec) is a retroviral replicating vector encoding an optimized yeast cytosine deaminase (CD). Tumor-selective expression of CD converts the prodrug, 5-fluorocytosine (5-FC), into the active chemotherapeutic, 5-fluorouracil (5-FU). This therapeutic approach is being tested in a randomized phase II/III trial in recurrent glioblastoma and anaplastic astrocytoma (NCT0241416). The aim of this study was to identify the immune cell subsets contributing to antitumor immune responses following treatment with 5-FC in Toca 511–expressing gliomas in a syngeneic mouse model.Methods.Flow cytometry was utilized to monitor and characterize the immune cell infiltrate in subcutaneous Tu-2449 gliomas in B6C3F1 mice treated with Toca 511 and 5-FC.Results.Tumor-bearing animals treated with Toca 511 and 5-FC display alterations in immune cell populations within the tumor that result in antitumor immune protection. Attenuated immune subsets were exclusive to immunosuppressive cells of myeloid origin. Depletion of immunosuppressive cells temporally preceded a second event which included expansion of T cells which were polarized away from Th2 and Th17 in the CD4+ T cell compartment with concomitant expansion of interferon gamma–expressing CD8+ T cells. Immune alterations correlated with clearance of Tu-2449 subcutaneous tumors and T cell–dependent protection from future tumor challenge.Conclusions.Treatment with Toca 511 and 5-FC has a concentrated effect at the site of the tumor which causes direct tumor cell death and alterations in immune cell infiltrate, resulting in a tumor microenvironment that is more permissive to establishment of a T cell mediated antitumor immune response.

A Review of Obinutuzumab (GA101), a Novel Type II Anti-CD20 Monoclonal Antibody, for the Treatment of Patients with B-Cell Malignancies.

Obinutuzumab (GA101) is a novel, type II, glycoengineered, humanized anti-CD20 monoclonal antibody that has been developed to address the need for new therapeutics with improved efficacy in patients with lymphocytic leukemia and lymphoma of B-cell origin. Obinutuzumab has a distinct mode of action relative to type I anti-CD20 antibodies, such as rituximab, working primarily by inducing direct cell death and antibody-dependent cell-mediated cytotoxicity. Obinutuzumab is under investigation in a wide-ranging program of clinical trials in patients with B-cell malignancies. Efficacy as monotherapy has been reported in patients with relapsed/refractory indolent and aggressive non-Hodgkin lymphoma (NHL) and in chronic lymphocytic leukemia (CLL) of B-cell origin. Improved outcomes have also been noted when obinutuzumab is added to chemotherapy in patients with B-cell NHL, and superiority over rituximab has been reported with combination therapy in patients with CLL. Ongoing research is focusing on developing options for chemotherapy-free treatment and on new combinations of obinutuzumab with novel targeted agents.

Lysosomal Disruption Augments Obinutuzumab-Induced Direct Cell Death

Anti-CD20 mononclonal antibodies (mAbs) have a well-established role in the treatment of B-cell lymphoid malignancies. In addition to classic Fc-dependent mechanisms, including antibody-dependent and complement-mediated cytotoxicity, the so-called type II mAbs induce direct cell death. It has been shown that obinutuzumab, without Fc-crosslinking agents or effector cells, triggers non-apoptotic, lysosomal-dependent programmed cell death (PCD). The mechanism of PCD is characterized by actin reorganization, followed by permeabilization of the lysosomal membrane and subsequent generation of reactive oxygen species (ROS) through NADPH oxidase. Although, mechanisms of PCD are well-described, little is known about factors influencing sensitivity of malignant B-cells to obinutuzumab-mediated direct cell killing. Strategies to improve PCD could be potentially exploited to eliminate malignant cells, which are refractory to conventional immunotherapy. In this study, we aimed to investigate the influence of lysosomotropic agent, chloroquine, on the efficacy of obinutuzumab-mediated cytotoxicity. As PCD is dependent on lysosomal destabilization, we hypothesized that combination of obinutuzumab with lysosome-destabilizing agent would result in increased cell death.In our study, we used a Burkitt lymphoma Raji cell line that is widely employed as a model to assess the efficacy of obinutuzumab. Raji cells were incubated with obinutuzumab, alone or in combination with increasing concentrations of chloroquine, followed by annexin V/PI staining. Chloroquine, significantly increased direct cell death induced by obinutuzumab, without being toxic alone. Those observations were further corroborated by cell staining with other viability dies - TO-PRO-3 iodide and 7-AAD.The efficacy of the tested combination was completely abrogated by cytochalasin D - an inhibitor of actin polymerization and concanamycin A - inhibitor of vacuolar ATPases that activate acidic vacuoles including lysosomes, suggesting that chloroquine and obinutuzumab share a common mechanism of action. Since chloroquine has been reported to promote ROS generation, we analyzed the production of ROS with DCFDA staining. We observed that chloroquine potentiated the oxidative effect of obinutuzumab. Consistently, the effect of the combination was completely abrogated by a cell-permeable ROS scavenger - Tiron. As obinutuzumab has been shown to induce ROS production via activation of NADPH oxidase, we investigated the influence of NADPH oxidase inhibitor - diphenylene iodonium (DPI) on the combination's efficacy. DPI only partially reversed the effect of obinutuzumab, while strongly decreasing the effect of the combination.Altogether, we show for the first time that chloroquine sensitizes cell to lysosomal cell death induced by obinutuzumab. The results of our study provide a strong rationale for combining obinutuzumab with lysosomotropic agents. These findings may have particular importance given the fact that another lysomotropic agent - siramesine has recently been shown to induce selective cytotoxicity in chronic lymphocytic leukemia (CLL). DisclosuresNo relevant conflicts of interest to declare.

Polatuzumab Vedotin Combined with Obinutuzumab for Patients with Relapsed or Refractory Non-Hodgkin Lymphoma: Preliminary Safety and Clinical Activity of a Phase Ib/II Study

Introduction: Previously reported results from this ongoing study showed clinical activity for polatuzumab vedotin (PoV), an antibody drug conjugate (ADC) containing the anti‐mitotic MMAE targeting CD79b, at a dose of 2.4 mg/kg, in combination with rituximab (R) in relapsed/refractory (r/r) diffuse large B‐cell lymphoma (DLBCL) and follicular lymphoma (FL) when patients (pts) were treated until progression (Morschhauser ASH 2014, Advani ASCO 2015). Obinutuzumab (G) is a next generation antiCD20 mAb with enhanced antibody-dependent cellular cytotoxicity.and direct cell death. We report here the safety and efficacy results for this Phase Ib/II study of PoV at 1.8 mg/kg combined with G treated for up to 8 cycles in r/r DLBCL or FL (ClinicalTrials.gov NCT01691898).Methods: This multicenter, open-label Ph Ib/II study evaluated the combination of PoV with G in pts with r/r DLBCL or FL. Primary objective was to assess the anti-tumor activity of PoV + G; secondary endpoints included safety and tolerability and pharmacokinetics. Adult pts (≥18 years) were treated with PoV 1.8 mg/kg intravenously with G every 21 days for a total of 8 cycles. Assessments for anti-lymphoma activity were performed following 4 cycles of study treatment (tx) and at the end of study tx (EOT) by Lugano 2014 criteria.Patients: As of Apr 5, 2016, 70 (38 DLBCL, 32 FL) pts were evaluable for safety. The demographic and baseline characteristics are included in Table 1.Results: Of 70 safety-evaluable patients, 49 (70%) experienced a treatment-related adverse event (AE) of which most were mild (Gr 1/2; 34 of 49). The most common AEs in >20% of pts were Gr 1-3 fatigue (43%), Gr 1-2 diarrhea (34%), Gr 1-3 nausea (30%), Gr 1-2 constipation (21%) and Gr 1 headache (20%).Among the 70 pts who received at least 1 dose of tx, 46% (32/70) pts had Grade 3/4 AEs and 29% (13 DLBCL, 7 FL) had serious AEs (SAEs). The most common Grade 3/4 AE was neutropenia (13%) and the most common SAE occurring in >10% was infections (15%) including pneumonia (n=3), influenza, urinary tract infection (n=2 each), sepsis, sinusitis, and respiratory tract infection (n=1 each). Four pts (2 DLBCL, 2 FL) had Gr 2 peripheral neuropathy (PN, n=2) and peripheral sensory neuropathy (n=2). Onset of PN occurred after cycle 2 and cycle 3 in the DLBCL pts and after cycle 5 in both FL pts. All 4 pts required dose reduction of PoV (1.8 to 1.4 mg/kg). Five pts discontinued PoV due to AE (3 DLBCL, 2 FL), including Gr 3 GI perforation (n=1), Gr 3 malignant neoplasm (n=1), and Gr 3 delirium and urinary tract infection (n=1) in pts with DLBCL, and Gr 5 pneumonia and Gr 2 elevated bilirubin (n=1, each) in FL pts. Eight deaths (1 due to pneumonia in a pt with FL and 7 due to progressive disease in pts with DLBCL) were reported.The median number of treatment cycles received was 2 (0-8) and 5 (0-8) for the pts with DLBCL and FL, respectively. Best objective response rate by PET was available for 44 evaluable (21 DLBCL, 23 FL) pts. The regimen was active in heavily pre-treated pts with an ORR of 78% for FL and 52% for DLBCL (see Table 2).Updated safety and efficacy data will be presented.Conclusions: Early results from this ongoing study show that this novel combination of PoV, at 1.8 mg/kg with fixed duration (≤ 8 cycles), plus G has an acceptable safety profile with evidence of clinical activity in r/r DLBCL or FL pts who were heavily pretreated or refractory to the last prior regimen. [Display omitted] [Display omitted] DisclosuresBrunvand:Millennium / Takeda: Speakers Bureau; Celgene: Speakers Bureau; Genentech: Speakers Bureau. Chen:Seattle Genetics: Consultancy. Press:Roche / Genentech: Consultancy, Research Funding. Chiappella:Pfizer: Speakers Bureau; Celgene: Speakers Bureau; Roche: Speakers Bureau; Janssen-Cilag: Speakers Bureau; Teva: Speakers Bureau; Amgen: Speakers Bureau. Diefenbach:Genentech: Honoraria, Research Funding. Jones:Genentech, Inc.: Employment. Hirata:Genentech: Employment. Flinn:Janssen: Research Funding; Gilead Sciences: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; ARIAD: Research Funding; RainTree Oncology Services: Equity Ownership.

Critical Analysis on the Mechanism of Action (MoA) of the Anti-CD38 Monoclonal Antibody Isatuximab in Multiple Myeloma (MM)

Monoclonal antibodies (mAbs) targeting CD38 are demonstrating remarkable efficacy, particularly when combined with anti-MM agents. Thus, in-depth understanding of the MoA of anti-CD38 mAbs is of utmost importance to design rational treatment combinations. Notably, while there is considerable data about the MoA of Daratumumab, there is virtually no data about the MoA of Isatuximab.Here, we started by analyzing in a selected panel of MM cell lines (RPMI8226, H929, MM1S and OPM2) the effect of Isatuximab on 1) proliferation, 2) direct cell death, 3) complement dependent cytotoxicity (CDC), 4) antibody dependent cell mediated cytotoxicity (ADCC), and 5) antibody dependent cellular phagocytosis (ADPC). Our results show that Isatuximab had no effect on proliferation, and did not directly killed any of the MM cell lines tested. Interestingly, while Isatuximab (similar to drugs such as Cetuximab) binds to C1q, CD38 receptor density in MM cell lines was insufficient to trigger CDC based on the absence of C3 deposition and impact on cell survival. Upon co-culture with human blood leukocytes, Isatuximab induced ADCC particularly on cells showing higher expression of CD38 (P=0.02). Upon co-culture with human-derived M2-like macrophages, we noted a trend (P=.1) towards decreased MM cell expansion; thus, in order to establish the exact contribution of ADCC and ADCP on the efficacy of Isatuximab, we co-cultured RPMI8226 MM cells with human leukocytes and prior to treatment (10µg/mL), used sensitive FACS sorting to remove key immune cell populations such as NK cells, macrophages and T cells from the culture. Our results show that the presence of NK cells was critical for the efficacy of Isatuximab, whereas macrophages and T cell depletion had no effect on MM cell viability in the presence of Isatuximab.We next tested Isatuximab (10µg/mL) in 10 primary patient samples. In the absence of C3 deposition in the majority of samples (ie. no CDC), we observed Isatuximab-induced cell death in all patients tested (P=0.002), along with increased proliferation and activation of NK cells upon Fc recognition. Due to broad expression of CD38 in hematopoietic cells, we also investigated presence of off-target effects of Isatuximab. Importantly, with the exception of CD38bright B cell precursors (P=.0006), there was no toxicity observed in other progenitor and mature hematopoietic cells. We also investigated the potential synergism of Isatuximab in combination with Bortezomib-Dexamethasone; interestingly, the addition of Isatuximab resulted in a (P=0.06) 2.7-fold increment in MM cell death.In order to further understand the direct effect of Isatuximab in both MM and (CD38bright) NK cells, we determined the molecular signature of both subsets in co-culture and upon treatment with Isatuximab. Interestingly (though consistent with the lack of impact in proliferation and direct apoptosis), MM cells showed no gene dysregulation upon treatment with Isatuximab and in the absence of NK cells. By contrast, in the presence of NK cells and respective ADCC, MM cells chemoresistant to Isatuximab showed 184 dysregulated genes, including significant down-regulation of CD38. Noteworthy, Isatuximab binding induced in NK cells up-regulation of genes related to antigen binding, chemokine receptor activity, G-protein coupled peptide receptor or hydrolase activity. One such gene was CD137, which up-regulation was subsequently confirmed at the protein (surface antigen) level.In summary, this critical analysis on Isatuximab's MoA using a comprehensive set of assays, MM cell lines and primary patient samples, suggests that ADCC is the major efficacy contributor for this particular mAb. Thus, drugs that can further stimulate NK cells might be optimal candidates for rational combinations with Isatuximab; in this regard, we showed that Isatuximab induces up-regulation of CD137 which warrants further investigations on the value of combined Isatuximab and anti-CD137 immune therapies. [Display omitted] DisclosuresPaiva:Celgene: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; EngMab: Research Funding; Amgen: Honoraria; Binding Site: Research Funding.

CD20 Antibodies of Human IgA Isotype Mediate CDC, and ADCC By Myeloid Effector Cells

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. DisclosuresCragg:Roche: Consultancy, Research Funding; Gilead Sciences: Research Funding; Baxalta: Consultancy; Bioinvent International: Consultancy, Research Funding; GSK: Research Funding.

Polatuzumab Vedotin Combined with Obinutuzumab, Cyclophosphamide, Doxorubicin, and Prednisone (G-CHP) for Patients with Previously Untreated Diffuse Large B-Cell Lymphoma (DLBCL): Preliminary Results of a Phase Ib/II Dose-Escalation Study

Introduction: Despite the improved outcomes seen with rituximab-CHOP (R-CHOP) in patients (pts) with newly diagnosed DLBCL, a significant number of pts with high risk disease (defined by IPI) are not cured; thus, new therapies are needed. Obinutuzumab (GA101, G) is a glycoengineered type II anti-CD20 monoclonal antibody with improved direct cell death and antibody-dependent cell-mediated cytotoxicity compared to rituximab. In addition, polatuzumab vedotin (PoV) is an antibody drug conjugate (ADC) designed to deliver the potent microtubule inhibitor MMAE to cells expressing CD79b. Thus, PoV has the potential to replace vincristine with a targeted agent. The incorporation of G and PoV would potentially increase the efficacy of the standard R-CHOP regimen. We report preliminary results from the dose-escalation portion of this Phase Ib/II study of PoV combined with obinutuzumab, cyclophosphamide, doxorubicin, and prednisone (G-CHP) (ClinicalTrials.gov NCT01992653).Methods: This multicenter, open-label study will evaluate the safety, tolerability, and pharmacokinetics of escalating doses of PoV with standard doses of G (1000 mg on days 1, 8, and 15 in cycle 1, and 1000 mg on day 1 of each subsequent cycle) and CHP. Adults, aged 18-80 years, with newly diagnosed or relapsed/refractory B-cell NHL were given PoV intravenously 1.4 or 1.8 mg/kg with G-CHP every 21 days for a total of 6 or 8 cycles. Doses were escalated in a 3+3 design based on dose limiting toxicity assessment during cycle 1. Assessments for anti-tumor activity were performed following 4 cycles of study treatment (tx) and at the end of study tx (EOT).Results: As of Feb. 5, 2016, 12 pts (50% male) enrolled in the G-CHP + PoV dose-escalation (esc). The median age was 69.5 years (range 32-77) for all pts. Pts had untreated DLBCL (n=8), relapsed follicular lymphoma (FL, n=2), and untreated mantle cell lymphoma (MCL, n=2). At baseline, pts had stage I/II (n=5), III/IV (n=7) disease and ECOG status of 0-1 (n=12). Of 8 DLBCL pts, 3 had IPI 0-1, 2 had IPI 2, 2 had IPI 3, and 1 had IPI 4. At the time of this report, 8 pts in the dose escalation phase have completed study tx and all 12 had received interim assessments.The most common adverse events (AEs) in > 30% of pts were alopecia (67%), neutropenia (50%), nausea (42%), anemia (33%), fatigue (33%), cough (33%), and rash (33%). Across both dose levels, 10 pts had 30 Gr 3/4 AEs including neutropenia (58%), anemia (25%), thrombocytopenia (25%), febrile neutropenia (17%), pneumonia (8%), C. difficile (8%), sepsis (8%), device related infection (8%), acute kidney injury (8%), hyponatremia (8%), atrial fibrillation (8%), SVT (8%), tachycardia (8%), syncope (8%) and hypertension (8%).Three pts experienced 10 serious AEs (SAEs). One pt in the 1.4 mg/kg cohort had SAEs of febrile neutropenia (also a DLT), thrombocytopenia, and an atrial fibrillation exacerbation. Two pts in the 1.8 mg/kg cohort experienced a total of 7 SAEs. One pt experienced syncope, C. difficile, and febrile neutropenia while the other pt had sepsis, pneumonia, acute kidney injury, and cardiac complications including SVT and 3 vessel bypass surgery. Four (33%) pts experienced Gr 1 peripheral neuropathy (PN) during tx which did not lead to dose modifications. The reported terms included hypoaesthesia, PN, and paresthesia. There was 1 pt with PN in the 1.4 mg/kg cohort and 3 in the 1.8 mg/kg cohort. 3 pts discontinued study treatment: 1 due to recurrent neutropenic fevers (no subsequent tx), 1 due to pneumonia (followed with R-CVP), and 1 due to physician decision due to lack of treatment response (followed with R-ICE). No deaths were reported.Efficacy: PET/CT response for pts with 1L DLBCL as of February 5, 2016 is reported in Table 1.Of the 2 pts with FL, both achieved CR by PET/CT (1 at 1.4 mg/kg and 1 at 1.8 mg/kg). Of the 2 pts with MCL, both achieved CR by PET/CT (1 at 1.4 mg/kg and 1 at 1.8 mg/kg).Conclusions: Early results show that PoV plus G-CHP has an acceptable safety profile in pts with previously untreated DLBCL and other B cell malignancies. Although the protocol-specified MTD was not formally reached, the recommended phase 2 dose of PoV was established at 1.8 mg/kg based on other studies. The study's Phase 2 PoV+R-CHP expansion and PoV+G-CHP dose expansion are ongoing. [Display omitted] DisclosuresForero-Torres:Seattle Genetics: Research Funding; Genentech/Roche: Research Funding; Juno: Research Funding; Incyte: Research Funding; Abbvie: Research Funding; Novartis: Research Funding; Pfizer: Research Funding. Kolibaba:Pharmcyclics: Research Funding; Novartis: Research Funding; Janssen: Research Funding; GSK: Research Funding; Genentech: Research Funding; Cell Therapeutics: Research Funding; Celgene: Research Funding; Amgen: Research Funding; Acerta: Research Funding; Gilead: Consultancy, Research Funding; Seattle Genetics: Research Funding; TG Therapeutics: Honoraria, Research Funding. Jones:Genentech, Inc.: Employment. Lee:Genentech, Inc.: Employment. Sharman:Gilead: Research Funding; Celgene: Research Funding; Pharmacyclics: Research Funding; Acerta: Research Funding; TG Therapeutics: Research Funding; Seattle Genetics: Research Funding.

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.

Obinutuzumab or Rituximab Plus CHOP in Patients with Previously Untreated Diffuse Large B-Cell Lymphoma: Final Results from an Open-Label, Randomized Phase 3 Study (GOYA)

Obinutuzumab or Rituximab Plus CHOP in Patients with Previously Untreated Diffuse Large B-Cell Lymphoma: Final Results from an Open-Label, Randomized Phase 3 Study (GOYA)

A TLR7 agonist enhances the antitumor efficacy of obinutuzumab in murine lymphoma models via NK cells and CD4 T cells

Anti-CD20 monoclonal antibodies (mAb) such as rituximab have been proven to be highly effective at improving outcome in B-cell malignancies. However, many patients ultimately relapse and become refractory to treatment. The glycoengineered anti-CD20 mAb obinutuzumab was developed to induce enhanced antibody-dependent cellular cytotoxicity, antibody-dependent phagocytosis and direct cell death and was shown to lead to improved outcomes in a randomized study in B-CLL. We hypothesized that immune stimulation through Toll-like receptor 7 (TLR7) agonism in combination with obinutuzumab would further enhance lymphoma clearance and the generation of long-term antitumor immune responses. Here we demonstrate, in syngeneic human CD20 (hCD20)-expressing models of lymphoma, that systemic administration of a TLR7 agonist (R848) increases responses when administered in combination with obinutuzumab and protects against disease recurrence. Depletion studies demonstrate that primary antitumor activity is dependent on both NK cells and CD4+ T cells but not on CD8+ T cells. However, both CD4+ and CD8+ T cells appear necessary for the generation of protective immunological memory. Importantly, increased tumor-free survival post obinutuzumab and R848 combination therapy was seen in hCD20 transgenic mice, which express hCD20 on normal B cells. These findings provide a rationale for clinical testing of obinutuzumab in combination with systemically administered TLR7 agonists to further improve outcome.

Prospects in the Application of Photodynamic Therapy in Oral Cancer and Premalignant Lesions.

Oral cancer is a global health burden with significantly poor survival, especially when the diagnosis is at its late stage. Despite advances in current treatment modalities, there has been minimal improvement in survival rates over the last five decades. The development of local recurrence, regional failure, and the formation of second primary tumors accounts for this poor outcome. For survivors, cosmetic and functional compromises resulting from treatment are often devastating. These statistics underscore the need for novel approaches in the management of this deadly disease. Photodynamic therapy (PDT) is a treatment modality that involves administration of a light-sensitive drug, known as a photosensitizer, followed by light irradiation of an appropriate wavelength that corresponds to an absorbance band of the sensitizer. In the presence of tissue oxygen, cytotoxic free radicals that are produced cause direct tumor cell death, damage to the microvasculature, and induction of inflammatory reactions at the target sites. PDT offers a prospective new approach in controlling this disease at its various stages either as a stand-alone therapy for early lesions or as an adjuvant therapy for advanced cases. In this review, we aim to explore the applications of PDT in oral cancer therapy and to present an overview of the recent advances in PDT that can potentially reposition its utility for oral cancer treatment.

Rate-equation modelling and ensemble approach to extraction of parameters for viral infection-induced cell apoptosis and necrosis.

We develop a theoretical approach that uses physiochemical kinetics modelling to describe cell population dynamics upon progression of viral infection in cell culture, which results in cell apoptosis (programmed cell death) and necrosis (direct cell death). Several model parameters necessary for computer simulation were determined by reviewing and analyzing available published experimental data. By comparing experimental data to computer modelling results, we identify the parameters that are the most sensitive to the measured system properties and allow for the best data fitting. Our model allows extraction of parameters from experimental data and also has predictive power. Using the model we describe interesting time-dependent quantities that were not directly measured in the experiment and identify correlations among the fitted parameter values. Numerical simulation of viral infection progression is done by a rate-equation approach resulting in a system of "stiff" equations, which are solved by using a novel variant of the stochastic ensemble modelling approach. The latter was originally developed for coupled chemical reactions.

Elimination of Latently HIV-infected Cells from Antiretroviral Therapy-suppressed Subjects by Engineered Immune-mobilizing T-cell Receptors

Persistence of human immunodeficiency virus (HIV) in a latent state in long-lived CD4+ T-cells is a major barrier to eradication. Latency-reversing agents that induce direct or immune-mediated cell death upon reactivation of HIV are a possible solution. However, clearance of reactivated cells may require immunotherapeutic agents that are fine-tuned to detect viral antigens when expressed at low levels. We tested the antiviral efficacy of immune-mobilizing monoclonal T-cell receptors against viruses (ImmTAVs), bispecific molecules that redirect CD8+ T-cells to kill HIV-infected CD4+ T-cells. T-cell receptors specific for an immunodominant Gag epitope, SL9, and its escape variants were engineered to achieve supraphysiological affinity and fused to a humanised CD3-specific single chain antibody fragment. Ex vivo polyclonal CD8+ T-cells were efficiently redirected by immune-mobilising monoclonal T-cell receptors against viruses to eliminate CD4+ T-cells from human histocompatibility leukocyte antigen (HLA)-A*0201-positive antiretroviral therapy-treated patients after reactivation of inducible HIV in vitro. The efficiency of infected cell elimination correlated with HIV Gag expression. Immune-mobilising monoclonal T-cell receptors against viruses have potential as a therapy to facilitate clearance of reactivated HIV reservoir cells.