Direct Cell Killing Research Articles

The long-acting COX-2 inhibitor mavacoxib (Trocoxil¿) has anti-proliferative and pro-apoptotic effects on canine cancer cell lines and cancer stem cells

The NSAID mavacoxib (Trocoxil™) is a recently described selective COX-2 inhibitor used for the management of inflammatory disease in dogs. It has a long plasma half-life, requiring less frequent dosing and supporting increased owner compliance in treating their dogs. Although the use of NSAIDs has been described in cancer treatment in dogs, there are no studies to date that have examined the utility of mavacoxib specifically. In this study we compared the in vitro activity of a short-acting non-selective COX inhibitor (carprofen) with mavacoxib, on cancer cell and cancer stem cell survival. We demonstrate that mavacoxib has a direct cell killing effect on cancer cells, increases apoptosis in cancer cells in a manner that may be independent of caspase activity, and has an inhibitory effect on cell migration. Importantly, we demonstrate that cancer stem cells derived from osteosarcoma cell lines are sensitive to the cytotoxic effect of mavacoxib. Both NSAIDs can inhibit cancer cell proliferation and induce apoptosis in vitro. Importantly, cancer stem cells derived from an osteosarcoma cell line are sensitive to the cytotoxic effect of mavacoxib. Our results suggest that mavacoxib has anti-tumour effects and that this in vitro anti-cancer activity warrants further study.

Adenovirus E3 protein modulates leukocyte functions

To resist viral infections, leukocytes perform effector functions, such as direct killing of infected cells (elicited by natural killer cells and T lymphocytes) and production of virus-neutralizing antibodies (elicited by B lymphocytes). These effector functions require differentiation of the leukocyte and are preceded by activation of the B- and T-cell receptors and by production of cytokines from multiple cell types. Activation of these effector functions requires a complex signaling network that involves kinases and phosphatases that transfer phosphate groups between each other in the leukocyte cytoplasm. Many viruses have developed systems to resist the effector functions of leukocytes, but (to the best of my knowledge) until now there have been no reports of viral proteins that target these signaling networks. In PNAS, Windheim et al. (1) report a unique resistance system developed by human adenovirus type 19 (HAdV-19). This virus encodes a protein (sec49K) that is secreted from infected cells and targets one of the key regulatory molecules of leukocytes, receptor protein tyrosine phosphatase (RPTP) CD45 (1).

Disease Relapse After TKI Discontinuation In CML Is Related Both To Low Number and Impaired Function Of NK-Cells:Data From Euro-SKI

BackgroundThe inhibition of oncogenic BCR-ABL1 kinase with tyrosine kinase inhibitors (TKIs) has significantly improved the prognosis of CML. Recent reports suggest that approximately 40 % of CML patients who have achieved optimal therapy response (complete molecular remission, CMR) can stop imatinib treatment without recurrence of detectable BCR-ABL1 transcripts. However, no predictive prognostic factors for successful therapy discontinuation have yet been identified. We therefore set up an immunological substudy in the ongoing pan-European EURO-SKI stopping study. We aimed to identify predictive biomarkers for relapse and non-relapse after TKI discontinuation. In addition, we aimed to understand more on the mechanisms of immune surveillance in CML and to study the effects of TKI treatment on the immune system. Materials and methodsPatients in deep molecular remission (MR4, BCR-ABL < 0,01% IS) for at least one year and with TKI treatment for at least 3 years were eligible for the clinical study. Basic lymphocyte immunophenotyping (the proportions and absolute numbers of NK-, T- and B-cells) was performed at the university hospital laboratories at the time of therapy discontinuation, and 1, 6, and 12 months after the TKI discontinuation. In a proportion of patients a more detailed immunophenotypic (analysis of CD45RA, CD57, CD27 and CD62L expressions) and functional analyses were done from fresh blood samples in a central immunology laboratory (Helsinki) at the same time points. The cytotoxicity of NK-cells was studied by measuring the direct killing of target cells (K562) and by the degranulation assay (CD107a/b expression). The secretion of Th1 type of cytokines IFN-γ/TNF-α was studied from both T- and NK-cells. ResultsThus far the basic lymphocyte subclass measurement has been analyzed from 62 patients who have discontinued TKI treatment within the EURO-SKI study. Functional analyses have been performed from 30 patients. 60 patients have used imatinib before treatment discontinuation and 2 patients dasatinib.At baseline, before the treatment discontinuation both CD4+ and CD8+ T-cell counts were within the normal range (median CD4+ 0.73x 109/L, range 0.11-2.4x 109/L; CD8+ 0.35x 109/L, 0.07-1.92 x 109/L). The TKI stop had no significant numerical or functional effect on T-cells, and at 1 month time-point the median T-cell counts were unchanged (CD4+ 0.73x 109/L; CD8+ 0.35x 109/L). Similarly, at the baseline, the median NK-cell count was within a normal range (0.26 x 109/L, range 0.04-1.04 x 109/L), and no significant change was observed 1 month after stopping the treatment (median 0.29 x 109/L). Furthermore, at the baseline and at the 1-month time-point the cytotoxicity of NK-cells and the cytokine secretion of T- and NK-cells did not significantly differ from the healthy controls when all patients were considered as a one group.However, when patients were divided in two groups based on the relapse status, the patients who eventually relapsed had significantly fewer NK-cells already at the baseline (Figure A; absolute count 0.18x 109/L vs. 0.32 109/L, p=0.008; proportions 11% vs. 21%, p=0.001). The phenotype of NK-cells also differed between the two groups, and the patients who relapsed had less NK-cells expressing CD57 (median 58% vs. 69%, p=0.046) and CD16 (median 67% vs. 83%, p=0.018) on the cell surface. Furthermore, the cytotoxicity of NK-cells was impaired in patients who failed to discontinue the TKI treatment successfully and no killing activity was observed in their samples (Figure B; alive K652 cells after co-incubation with effector cells 100% vs. 88%, p=0.07). No clear differences were observed in the function or the numbers of T-cells between relapsing and non-relapsing patients. [Display omitted] ConclusionsThe NK-cell numbers and their function may predict disease relapse after TKI discontinuation. This may have impact on the future stopping trials. In addition, it further illustrates the importance of the immune system in the successful long-term treatment of CML. Disclosures:Ekblom:Novartis: Honoraria; Bristol-Myers Squibb: Honoraria. Hjorth-Hansen:Pfizer, BMS: Honoraria, Travel expenses Other. Porkka:BMS: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Richter:Bristol-Myers Squibb: Consultancy, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau. Mustjoki:Novartis: Honoraria; BMS: Honoraria, Research Funding.

Phase 2 Study Of Otlertuzumab (TRU-016), An Anti-CD37 ADAPTIRTM Protein, In Combination With Bendamustine Vs Bendamustine Alone In Patients With Relapsed Chronic Lymphocytic Leukemia (CLL)

BackgroundCD37 is a tetraspanin protein expressed on the surface of normal and transformed B-cells across a wide range of maturational stages and demonstrates death signaling via SHP1. Otlertuzumab is a CD37-specific therapeutic protein built on the ADAPTIRTM(modular protein technology) platform that has shown significantly greater direct killing of CLL cells than rituximab and higher levels of Fc mediated cellular cytotoxicity of CLL cells than either alemtuzumab or rituximab in pre-clinical models. Preclinical in vitro and in vivo models showed significant activity of otlertuzumab with bendamustine (benda). In phase 1 of this study involving 12 relapsed and/or refractory CLL patients (pts), otlertuzumab plus benda was well tolerated and showed a positive response. This phase 2 trial was conducted to investigate the activity of this combination compared to benda alone. MethodsPts with relapsed CLL who had 1-3 prior treatments, adequate organ function, ECOG performance status ≤2, ANC ≥1200/μL and CrCl >40 mL/min were eligible. Pts were stratified at randomization based on Cumulative Illness Rating Score, CrCl and deletion or mutation of 17p13.1. Pts in the study drug arm received otlertuzumab (20 mg/kg) weekly by IV infusion for two 28-day cycles then every 14 days for four 28-day cycles. In both arms, benda (70 mg/m2) was administered IV on Days 1 and 2 of each cycle for up to six 28-day cycles. Safety was evaluated using CTCAE and IWCLL 2008 Grading scale for Hematologic Toxicity in CLL Studies. Response was determined using the 1996 NCI and 2008 IWCLL Criteria. Flow cytometry was performed on peripheral blood to quantitate and characterize lymphocyte populations. Results65 pts have been treated in the randomized portion of the study, including 32 with otlertuzumab plus benda and 33 with benda alone. Pt characteristics and adverse events (AEs) are shown in the table. To date, 44 patients are evaluable for response assessment by the IWCLL criteria assessed 2.5 months after completion of therapy utilizing investigator assessment of CT scan and bone marrow data. The ORR for the combination of otlertuzumab and benda (n=20) was 80% (16) with a CR rate of 20% (4). For benda alone (n=24) the ORR was 42% (10) with a CR rate of 4% (1). Patients are still in treatment and follow-up but only 10% (2) have progressed on the combination of otlertuzumab and benda while 46% (11) have progressed on benda. The best response at anytime by NCI criteria using available data for the combination of otlertuzumab and benda (n=32) was ORR 78% and CR 16% and for benda alone (n=33) ORR 52% and CR 9%. There were no responses among pts with del17p; among pts with 17p mutation both pts in the combination arm had a response; no pts in the benda alone arm had a response. The frequency of all AEs was similar between treatment arms; hematologic and infection AEs were the most frequent in both arms. There were 13 serious AEs reported by 10 pts in the otlertuzumab+benda arm and 25 reported by 12 pts in the benda alone arm. The half-life of otlertuzumab is 11 days and there was no antidrug antibody in the 14 pts tested to date. ConclusionsPreliminary response rate with otlertuzumab in combination with benda was higher than benda alone by IWCLL or NCI response criteria. The overall incidence of AEs was similar between the 2 treatment cohorts, with a higher incidence of pyrexia, neutropenia and thrombocytopenia with the combination. However, the addition of otlertuzumab did not appear to increase the number of serious adverse events. Pts are in the process of completing treatment and obtaining CT scans and bone marrows; the IWCLL and NCI response rate will be updated. Disclosures:Robak:Gilead Sciences (Advisory board, research support): Consultancy, Honoraria, Research Funding; Emergent BioSolutions (Advisory board, research support): Consultancy, Honoraria, Research Funding; Celgene (Research support): Consultancy, Honoraria, Research Funding; GlaxoSmithKline (Research support): Consultancy, Honoraria, Research Funding; Roche (Advisory board, research support): Consultancy, Honoraria, Research Funding; Johnson & Johnson (Advisory board, research support): Consultancy, Honoraria, Research Funding. Off Label Use: Otlertuzumab is a CD37-specific therapeutic protein that has shown significant activity in CLL in preclinical and clinical studies. Mato:Emergent Product Development: Honoraria. Awan:Lymphoma Research Foundation - Reseach Funding: Research Funding, Speakers Bureau; Spectrum Pharmaceuticals, Inc. - Speakers bureau: Research Funding, Speakers Bureau. Hill:Teva Pharmaceuticals: Honoraria, Research Funding; Emergent Biosolutions: Honoraria, Research Funding. Stromatt:Emergent Product Development: Employment. Jaeger:Emergent: Honoraria, Research Funding.

Phase 1b Study Of Otlertuzumab (TRU-016), An Anti-CD37 ADAPTIR™ Protein, In Combination With Rituximab In Patients With Previously Untreated Chronic Lymphocytic Leukemia (CLL)

BackgroundCD37 is a tetraspanin protein expressed on the surface of normal and transformed B-cells across a wide range of maturational stages and demonstrates death signaling via SHP1. Otlertuzumab is a CD37-specific therapeutic protein built on the ADAPTIR™(modular protein technology) platform that has shown significantly greater direct killing of CLL cells than rituximab and higher levels of Fc-mediated cellular cytotoxicity of CLL cells than either alemtuzumab or rituximab in pre-clinical models. This phase 1b trial was conducted to evaluate the safety and efficacy of otlertuzumab in combination with rituximab in patients with previously untreated CLL. MethodsPatients with untreated CLL that required treatment, had adequate organ function, ECOG ≤2, and absolute neutrophil count ≥800/μL were eligible. Patients were ineligible for chemotherapy as first-line therapy due to patient age, comorbidity, or patient refusal of chemotherapy Patients received otlertuzumab (20 mg/kg) weekly by IV infusion for two 28-day cycles then once a month for 4 months. Rituximab (375 mg/m2 the first dose then 500 mg/m2) was administered after otlertuzumab by IV infusion on the same schedule. Safety was evaluated using CTCAE and IWCLL 2008 Grading scale for Hematologic Toxicity in CLL Studies. Response was determined using the 1996 NCI and the 2008 IWCLL Criteria. Results24 patients have been treated. Patient characteristics and adverse events are shown in the table. The majority of the patients have not completed 6 cycles of therapy. The preliminary response by investigator assessment using NCI criteria at the last assessment is an ORR 88% (21/24); 2 patients have had PD. Six patients have had CT scans and bone marrow assessments 3 months after their last dose of study drug; by IWCLL criteria 1 has a CR, 4 have a PR and 1 has SD. The patient with a CR was MRD negative by five color flow cytometry of bone marrow aspirate. One patient discontinued therapy for a systemic inflammatory response. Severe (Grade 3 or 4) neutropenia was reported in 13% of patients; the incidence of severe infections was low (8%). Serious adverse events were reported for 4 patients: pneumonia, systemic inflammatory response, and deep vein thrombosis in 1 patient; and lymph node pain, worsening sinusitis, and fever in 1 patient each. ConclusionsThe preliminary response rate with otlertuzumab in combination with rituximab is promising. Follow-up is ongoing and the IWCLL response rate will be presented. Disclosures:O'Brien:Emergent Product Development: Research Funding. Stromatt:Emergent Biosolutions: Employment. Awan:Lymphoma Research Foundation - Research Funding: Research Funding, Speakers Bureau; Spectrum Pharmaceuticals, Inc. - Speakers bureau: Research Funding, Speakers Bureau.

Epigallocatechin-3-gallate prevents tumor cell implantation/growth in an experimental rat bladder tumor model

The aim of this study was to determine the efficacy of epigallocatechin-3-gallate (EGCG) (Polyphenon E®) in comparison with mitomycin C (MMC) to prevent tumor cell implantation/growth in an animal model of superficial bladder cancer and search for possible mechanism(s) of action. Female Fisher 344 rats were used to study the effects of EGCG and mitomycin C for the prevention of transitional cell tumor implantation (AY-27). Twenty rats served as a control, tumor implantation and saline wash only. Sixty rats were treated with EGCG (100, 200 and 400 µM) intravesically for 60 or 120 min after tumor implantation. Thirty other rats were divided equally and pretreated with 400 µM EGCG or saline for 120 min before tumor initiation. In a separate series of experiments, 30 rats were treated 2 weeks after tumor initiation with saline or EGCG (400 µM). In a different experiment 39 rats were treated with: saline (n=10) EGCG (n=9) 400 µM, MMC (n=10) 0.5 µM, MMC (n=10) 400 µM. Rats were sacrificed 3 weeks following treatment. Gross and histological analyses were performed on the bladders. EGCG and mitomycin C prevented intravesical tumor growth in a concentration- and time-dependent manner. EGCG pretreatment or treatment 2 weeks post tumor implantation did not have therapeutic effects. Molecular modeling suggests that EGCG inhibits urokinase and matrix metalloproteinase-9. EGCG prevents intravesical tumor implantation/growth with a slightly better efficacy than mitomycin C in this experimental model. The data suggest that EGCG lowers proteolytic activity and lowers probability of cancer cell implantation rather than direct cancer cell killing.

Exopolysaccharides protect Synechocystis against the deleterious effects of Titanium dioxide nanoparticles in natural and artificial waters

We have studied the effect of TiO2 nanoparticles (NPs) on the model cyanobacteria Synechocystis PCC6803. We used well-characterized NPs suspensions in artificial and natural (Seine River, France) waters. We report that NPs trigger direct (cell killing) and indirect (cell sedimentation precluding the capture of light, which is crucial to photosynthesis) deleterious effects. Both toxic effects increase with NPs concentration and are exacerbated by the presence of UVAs that increase the production of Reactive Oxygen Species (hydroxyl and superoxide radicals) by TiO2 NPs. Furthermore, we compared the responses of the wild-type strain of Synechocystis, which possesses abundant exopolysaccharides surrounding the cells, to that of an EPS-depleted mutant. We show, for the first time, that the exopolysaccharides play a crucial role in Synechocystis protection against cell killing caused by TiO2 NPs.

Improved Clearance during Treatment of HPV-Positive Head and Neck Cancer through mTOR Inhibition

Human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) incidence is increasing at a near epidemic rate. We investigated whether the mammalian (or mechanistic) target of rapamycin (mTOR) inhibitor, rapamycin, can be used as a concurrent agent to standard-of-care cisplatin/radiation therapy (CRT) to attenuate tumor lactate production, thus enhancing CRT-induced immune-mediated clearance of this antigenic tumor type. A C57Bl/ 6-derived mouse oropharyngeal epithelial cell line retrovirally transduced with HPV type 16 E6/E7 and human squamous cell carcinoma cell lines were evaluated for their response to rapamycin in vitro with proliferation assays, Western blots, and lactate assays. Clonogenic assays and a preclinical mouse model were used to assess rapamycin as a concurrent agent to CRT. The potential of rapamycin to enhance immune response through lactate attenuation was assessed using quantitative tumor lactate bioluminescence and assessment of cell-mediated immunity using E6/E7-vaccinated mouse splenocytes. Rapamycin alone inhibited mTOR signaling of all cancer cell lines tested in vitro and in vivo. Furthermore, rapamycin administered alone significantly prolonged survival in vivo but did not result in any long-term cures. Given concurrently, CRT/rapamycin significantly enhanced direct cell killing in clonogenic assays and prolonged survival in immunocompromised mice. However, in immunocompetent mice, concurrent CRT/rapamycin increased long-term cures by 21%. Preliminary findings suggest that improved survival involves increased cell killing and enhanced immune-mediated clearance in part due to decreased lactate production. The results may provide rationale for the clinical evaluation of mTOR inhibitors concurrent with standard-of-care CRT for treatment of HPV-positive HNSCC.

In vivo assessment of specific cytotoxic T lymphocyte killing

The direct killing of target cells by cytotoxic T lymphocytes (CTLs) plays a fundamental role in protective immunity to viral, bacterial, protozoan and fungi infections, as well as to tumor cells. In vivo cytotoxic assays take into account the interaction of target and effector cells in the context of the proper microenvironment making the analysis biologically more relevant than in vitro cytotoxic assays. Thus, the development, improvement and validation of in vivo methods are necessary in view of the importance of the results they may provide. We describe and discuss in this manuscript a method to evaluate in vivo specific cytotoxic T lymphocyte killing. We used as model system mice immunized with human recombinant replication-deficient adenovirus 5 (HAd5) containing different transgenes as the trigger of a CTL-mediated immune response. To these mice, we adoptively transferred syngeneic cells labeled with different vital fluorescent dyes. Donor cells were pulsed (target) or not (control non-target) with distinct CD8 T-cell epitopes, mixed in a 1:1 ratio and injected i.v. into immunized or non-immunized recipient mice. After 18–24h, spleen cells are collected and analysed by flow cytometry. A deviation from the 1:1 ratio of control and target cell populations indicates antigen specific lysis of target cells.

Dose-dependent response of tumor vasculature to radiation therapy in combination with Sunitinib depicted by three-dimensional high-frequency power Doppler ultrasound

Large doses of radiation (8-20 Gy) preferentially target tumor vasculature. This vascular response is suggested to regulate tumor response to radiotherapy. Here, we investigate the relative contributions of direct cell killing by radiation versus tumor cell death due to radiation effects on the vasculature. We also examine Sunitinib's mechanism of action as a tumor radiosensitizer. MDA-MB-231 xenografts were treated with radiation doses of 2-16 Gy alone, or in combination with bFGF (endothelial radio-protector) or Sunitinib as pharmacological modulators of the vasculature. Sunitinib was orally administered for 2 weeks at 30 mg/kg before radiotherapy; bFGF was intravenously injected 1 h prior to irradiation. Three-dimensional high-frequency power Doppler ultrasound was used to assess relative changes in tumor vasculature. Immunohistochemistry, clonogenic and tumor growth assays were used to quantify tumor response. Significant reductions in power Doppler signal of up to 50 % were observed for 8 and 16 Gy treatments, along with a dose-dependent increase in cell death. No significant change in power Doppler signal and minimal tumor cell death were noted for tumors treated with radiation and bFGF. Treatments where Sunitinib was combined with radiation demonstrated a significant increase in flow signal at doses equal or greater than 8 Gy. This was accompanied with a significant increase in cell death when compared to radiation or Sunitinib alone. We confirm that tumor response to high doses of radiation is regulated by its vasculature. We also posit that the response observed when radiation is combined with Sunitinib is linked to a vascular "normalization" effect.

Natural Killer cells in Innate Defense against Infective Pathogens

Infectious diseases cause over 300 million illnesses and more than 5 million deaths each year worldwide. Understanding how components of the host immune system function to control disease-causing pathogens is critically important to develop strategies for preventing and controlling these diseases. With the discovery of innate immune receptors, we are beginning to appreciate the important role of innate immunity in the defense against infectious diseases. NK cells are a critical cell population in innate immunity, providing first line of defense against a variety of pathogens. NK cells mediate protection by direct killing of infected target cells and producing cytokines (mainly IFN-γ and TNF) that shape innate and adaptive immune responses. Recent studies have focused on the mechanisms by which NK cells recognize and respond to viruses, bacteria and parasites, and also the role of NK cells in modulating adaptive immune responses.

Glycoengineering of Therapeutic Antibodies

Antibody dependent cellular cytotoxicity (ADCC) is the process of lysis of (tumor) target cells by immune effector cells. It requires the simultaneous binding of an antibody to a cell surface receptor on the target cell and via the Fc region of the antibody to the FcgRIIIa receptor on immune effector cells such as NK cells or macrophages/monocytes. Binding of the antibody to target and immune effector cells results in the cross-linking of FcgRIIIa with subsequent activation of FcgRIIIa signaling and release of proteins mediating the killing of the target cells such as perforin and of proteases such as granzymes. ADCC has been established as an important mode of action of therapeutic antibodies such as rituximab/ MabThera (targeting CD20), cetuximab/Erbitux (targeting EGFR) and trastuzumab/Herceptin (targeting HER2) which are approved for the treatment of various cancers. Translational research on rituximab and cetuximab has established that the response to therapeutic antibodies in patients can be influenced by the FcgRIIIa-158V/F polymorphism. This polymorphism results in the expression of a high affinity FcgRIIIa receptor (158 V) and a low affinity FcgRIIIa receptor (158F) on immune effector cells and impacts the capability of antibodies to mediate ADCC [1–7]. We have recently developed a so-called GlycoMab technology that enhances the affinity of therapeutic antibodies for both the high and low affinity FcgRIIIa receptors through the introduction of a bisecting N-acetyl-glucosamine residue in the carbohydrate chain of the Fc region of the antibodies. The introduction of this bisecting N-acetyl-glucosamine moiety in the carbohydrate chain results in a steric interference with core fucosylation of the carbohydrate. This process and other approaches resulting in the afucosylation of therapeutic antibodies are known as “antibody glycoengineering”. We and others have demonstrated that glycoengineering and lack of the core fucose residue results in an up to 50-fold enhanced affinity of human IgG1 antibodies for the human FcgRIIIa receptors, and this subsequently results in an up to 100-fold enhanced ADCC induction (potency). Recently, we have demonstrated the structural basis of the enhanced affinity of afucosylated Fc regions for FcgRIIIa by X-ray crystallography. These studies provide a structural explanation for the high affinity and FcgRIIIa specificity of glycoengineering as opposed to other technologies used for Fc-engineering such as the introduction ofmutations in the Fc region that also affect binding to other FcgRs [8–15]. Roche is currently investigating three therapeutic glycoengineered antibodies with optimized immune effector function targeting CD20, EGFR and HER3 in different phases of clinical trials. Obinutuzumab (GA101, RG7159) is a type II, anti-CD20 GlycoMab monoclonal antibody that was designed to mediate enhanced direct and immune effector cell-mediated cell killing as compared to rituximab. In addition to enhanced ADCC, due to its type II characteristics, GA101 exhibits enhanced direct cell death induction with a concomitant reduction of CDC. In NHL animal models GA101 demonstrated a superior efficacy in terms of tumor growth inhibition and survival compared to type I CD20 antibodies such as rituximab and ofatumumab, including the induction of complete tumor remission. In non-human primates GA101 induces superior and profound B cell depletion in lymphoid tissues compared to rituximab. In Phase I/II clinical trials GA101 showed promising activity in heavily pretreated NHL patients. GA101 is currently being investigated in pivotal clinical trials in B-CLL, iNHL and DLBCL in combination with chemotherapy, head-to-head with rituximab-based regimens and in Rituximab-refractory iNHL as well as in various Phase II clinical trials. GA101 was the first glycoand Fc-engineered antibody to enter clinical trials and is now the most advanced glycoand Fc-engineered antibody in clinical trials in the world [16–24]. GA201 (RG7160) is a second anti-EGFR GlycoMab monoclonal antibody that is currently being investigated in Phase II clinical trials. It was designed to combine potent EGFR signaling inhibition with enhanced ADCC induction. In xenograft models GA201 demonstrated superior activity compared to cetuximab and panitumumab. Hence, GA201 has the potential to show clinical activity in patients with solid tumors. Phase I clinical testing in heavily pretreated patients with advanced solid tumors demonstrated a manageable toxicity profile. Evidence of anti-tumor activity was also seen, especially in patients with colorectal cancer, including a number of patients who had previously received cetuximab and/or panitumumab (1 CR, 2 PR). Interestingly, one of the partial responses was reported in a tumor-harboring a k-ras mutation. GA201 is currently undergoing Phase II clinical trials in CRC and NSCLC in combination with chemotherapy as well as a neo-adjuvant biomarker study in HNSCC [25].

Functional Characterization of T- and NK-Cells in Chronic Myeloid Leukemia Patients Treated with Interferon-α Monotherapy.

AbstractAbstract 2782 Background:The inhibition of oncogenic BCR-ABL1 kinase with tyrosine kinase inhibitors (TKIs) has significantly improved the prognosis of CML, but the majority of patients need life-long therapy. Before the TKI era, CML patients were treated with interferon-α (IFN-α), and a minor proportion of patients (10–20%) achieved prolonged complete cytogenetic remissions (CCyR). Interestingly, many of the patients in prolonged CCyR were able to discontinue the treatment without imminent disease relapse. As the ultimate goal of current CML therapy is cure, the deeper understanding of the cellular mechanisms behind successful IFN-α discontinuation are of outmost importance. In this study, we analyzed the function of immune effector cells (NK- and T-cells) derived from CML patients in prolonged molecular remission after IFN-α monotherapy in order to understand the putative anti-leukemic effects. METHODS:The study cohort included 13 CML patients treated with IFN-α who were in complete molecular remission (CMR) at the time of blood sampling. Five patients were still on IFN-α therapy (IFN-ON, median duration of the treatment 13.7 years) and 8 had stopped treatment successfully (IFN-OFF) and were off from any treatment (median time without treatment 6.3 years). None of the patients had used any TKIs. Samples from 10 healthy volunteers were used as controls. The cytotoxicity of NK-cells in blood was studied by measuring the direct killing of K562 cells and by degranulation assay (CD107). The cytokine secretion (TNF-α and IFN-g) of NK-cells was measured with flow cytometry after stimulation with K562. The function of T-cells was studied by antibody (OKT3) stimulation and measuring TNF-α and IFN-g production by flow cytometry. In addition, NK-cells were phenotyped by multi-color flow cytometry. RESULTS:IFN-OFF patients had a larger proportion of NK-cells from lymphocytes than IFN-ON patients or healthy controls (IFN-OFF median 23.5 %, IFN-ON 7.0 %, healthy 13.2 %; p=0.0136). Based on the subset analysis, expanded NK-cells in IFN-OFF patients had mature phenotype CD56dimCD62LlowCD27lowCD57high. On the contrary, IFN-ON patients had a larger proportion of immunoregulatory CD56bright NK-cells (20.2% vs. 6.5% in healthy and 3.0% in IFN-OFF, p=0.0035).When the direct killing of K562 cells was studied, NK-cells from healthy controls killed better than NK-cells from either of the patients groups (in healthy 55 % of K562 cells were alive compared to 79% in IFN-ON and 91% in IFN-OFF, p=0.0042). Similar trend was also observed in degranulation assay (median degranulation in healthy controls 8.3 % compared to 4.3 % in IFN-OFF and 3.6 % in IFN-ON group, p= 0.39). The direct killing capability tended to correlate negatively r=-0.60, p=0.07) with the NK-cell proportion (ie. patients with high NK-cell counts had worse killing).Instead of cytotoxicity, NK-cells from IFN-OFF patients seemed to secrete more efficiently cytokines (IFN-γ/TNF-α) than NK-cells from healthy controls, but due to low number of patients no firm conclusions can be made (IFN-OFF median 6.9 %, healthy 0.7 %; p= 0.25).The proportion of potentially cytotoxic CD4+GrB+ T-cells did not significantly differ between the groups, but wide variation was observed between the individual patients (IFN-ON median 9.0 %, IFN-OFF 2.6 %, and healthy controls 3.5 %; p=0.4142). However, the secretion of TNF-α and IFN-γ by CD4+ cells seemed to be increased in IFN-OFF patients when compared to healthy controls (IFN-OFF mean 5.9 %, IFN-ON 3.7 %, healthy 2.5 %; p=0.1055). CONCLUSIONS:CML patients who have been able to discontinue IFN-α therapy (IFN-OFF group) had an expansion of mature CD56dimCD62LlowCD27lowCD57high NK-cells in peripheral blood. Although CD56dim NK-cells have typically been suggested to act as cytotoxic cells, the killing in response to K562 cells was lower in IFN-OFF patients than in controls. It is possible that the expanded NK-cells in IFN-OFF patients represent exhausted terminal stage cells (CD57+) or memory cells that do not react properly against third party target cells (K562). Moreover, they may have an immunoregulatory function instead of cytotoxicity. Further follow-up studies with other patient cohorts (TKI monotherapy and TKI+IFN-α combination therapy treated patients) are ongoing to clarify the issue further. Disclosures:Porkka:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria.

Phase 1b Study of TRU-016, an Anti-CD37 SMIP™ Protein, in Combination with Rituximab and Bendamustine in Relapsed Indolent Lymphoma

AbstractAbstract 3678 Background:CD37 is a tetraspanin protein expressed on the surface of normal and transformed B-cells across a wide range of maturational stages and demonstrates death signaling via SHP1. TRU-016 is a novel humanized anti-CD37 SMIP™ (mono-specific protein therapeutic) that has shown significantly greater direct killing of CLL cells than rituximab and greater Fc mediated cellular cytotoxicity of CLL cells than either alemtuzumab or rituximab in pre-clinical models. In preclinical in vitro and in vivo models of NHL significant activity of TRU-016 against multiple cell lines was observed. In a phase 1 study, TRU-016 showed activity in patients (pts) with CLL and NHL was observed. Given the single-agent clinical activity of TRU-016 and synergistic or additive effect of TRU-016 with multiple agents in preclinical models, this trial of TRU-016 with rituximab and bendamustine was conducted to establish the maximum tolerated dose, overall safety, and clinical activity of TRU-016 in pts with relapsed indolent NHL. Methods:Pts with relapsed or refractory indolent B-cell NHL with adequate organ function, ECOG ≤2, absolute neutrophil count ≥1000/μL, platelets’100,000/μL who were not refractory to bendamustine were eligible. After premedication with acetaminophen, diphenhydramine, and hydrocortisone pts received TRU-016 (10 or 20 mg/kg on Days 1 and 15) over 2–3 hrs combined with rituximab 375 mg/m2 (Day 2) and bendamustine 90 mg/m2 (Days 1 and 2) by IV infusion for up to six 28-day cycles. Safety was evaluated using CTCAE 4.03 and response was determined using the Revised Response Criteria for Malignant Lymphoma (Cheson 2007) after every 2 cycles. Results:12 pts, (9 with follicular lymphoma (FL) and 3 with small lymphocytic lymphoma (SLL)) were treated (6 each dose level). Pt characteristics: median age 57 yrs (range, 51–79), median prior regimens 3 (1–4), 67% ≥ stage III at diagnosis, 4 pts had bulky disease > 5cm. All pts had relapsed after prior rituximab (R) including 3 refractory to their most recent previous treatment. In addition, prior treatments included: CHOP-R (8), RICE (5), single agent R (5), transplant (2). FLIPI scores at study entry (FL pts) were: 8 intermediate, 1 low.The most frequent (>2 pts) adverse events (AEs) were: neutropenia (8), fatigue, nausea, insomnia, and WBC decreased (7 each); anemia, diarrhea, headache, hypophosphatemia, thrombocytopenia, and vomiting (3 each). Grade 3/4 AEs that occurred in >1 pt were neutropenia (6), hypophosphatemia (3), WBC decreased (2). Serious AEs (SAE) included asymptomatic pulmonary thrombosis in 2 pts and febrile neutropenia, pneumonia, myelodysplastic syndrome (pt had prior transplant, CHOP, RICE), deep vein thrombosis, and retinal vein occlusion (last 2 events and pulmonary thrombosis occurred in same pt) in 1 pt each. There was no apparent dose relationship to the SAEs.The best overall response was 10/12 (83%) with 4 CRs (32%). Four responding pts (1 CR, 3 PR) discontinued treatment prior to cycle 6 due to undergoing consolidation with transplant (2), development of myelodysplastic syndrome (1), and delayed neutrophil recovery (1). The 4 discontinuations occurred in the 20 mg cohort and limit the response evaluation of the 20 mg dose. However, after Cycle 2 Day 15, the overall response rate was 67% at 10 mg/kg and 100% at 20 mg/kg. Three of the 4 pts with bulky disease responded to the regimen Conclusions:TRU-016 in combination with rituximab and bendamustine was well-tolerated, induces responses in the majority of patients with relapsed indolent B-NHL. A MTD was not defined. Future combination studies of TRU-016 in NHL are warranted.AgeGenderHistologyBulky Disease (≥5 cm)Rai Staging at dx.Prior Lines of Therapy# of cyclesBest Response (reason for discontinuation)Cohort 1: 10 mg/kg TRU-01656MSLLnoIV16CR55FFLnoIV36CR60MFLyesII16PR51MFLnoI16SD56MSLLyesND32PD65MFLnoIII46PRCohort 2: 20 mg/kg TRU-01655FFLnoIII44PR (transplant)57MFLnoIV32PR (MDS)54FFLnoIII42PR (transplant)74FFLyesIV46PR67MFLnoII12CR (neutropenia)78MSLLyesIV25PR Disclosures:Mato:Celgene, Milennium, Genentech, Seattle Genetics: Speakers Bureau. Stromatt:Emergent Product Development: Employment.

Phase 1b Study of TRU-016, an Anti-CD37 SMIP™ Protein, in Combination with Bendamustine Vs Bendamustine Alone in Relapsed Chronic Lymphocytic Leukemia

Abstract 1795 Background: CD37 is a tetraspanin protein expressed on the surface of normal and transformed B-cells across a wide range of maturational stages and demonstrates death signaling via SHP1. TRU-016 is a novel humanized anti-CD37 SMIP™ (mono-specific protein therapeutic) that has shown significantly greater direct killing of CLL cells than rituximab and greater Fc mediated cellular cytotoxicity of CLL cells than either alemtuzumab or rituximab in pre-clinical models. In preclinical in vitro and in vivo models significant activity of TRU-016 with bendamustine was observed. In a phase 1 study in 57 relapsed and/or refractory CLL patients treated with TRU-016, the maximum tested dose (20 mg/kg) was well tolerated. Given the single-agent clinical activity of TRU-016 and synergistic or additive effect of TRU-016 with multiple agents in preclinical models, this trial of TRU-016 with bendamustine was conducted to establish the maximum tolerated dose, overall safety, and clinical activity of TRU-016 in combination with bendamustine in patients with relapsed CLL. Methods: Patients with relapsed CLL who had 1–3 prior treatments (without prior bendamustine), adequate organ function, ECOG ≤2, and absolute neutrophil count ≥1200/μL were eligible. Patients received TRU-016 (15 or 20 mg/kg) weekly by IV infusion for two 28-day cycles then every 14 days for four 28-day cycles. Bendamustine (70 mg/m2) was administered on Days 1 and 2 of each cycle by IV infusion for up to six 28-day cycles. Safety was evaluated using CTCAE and IWCLL 2008 Grading scale for Hematologic Toxicity in CLL Studies. Response was determined using the 2008 IWCLL Criteria. Results: 12 patients have been treated to date (6 pts at each dose level). Patient characteristics: median age 67 yrs (range, 54–81), 7 patients received 1 and 2 patients received 2 prior regimens, 5 patients had bulky nodes ≥7 cm, 1 had del(17p13.1). All patients were intermediate to high-risk (Rai I/II=75%, III/IV=25%). The most frequent adverse events (&gt;2 pts) were: nausea (8); neutropenia (6); anemia, pyrexia, decreased appetite, hypokalemia, headache, cough (4 each); febrile neutropenia, constipation, diarrhea, dry mouth (3 each); and chills (2). The only grade 3/4 adverse events that occurred in &gt;2 patients were: neutropenia (6) and febrile neutropenia (3). There were 10 serious adverse events reported on 4 patients. Related serious events were febrile neutropenia in 2 patients and autoimmune hemolytic anemia (pre-existing) and urinary tract infection in 1 patient each. There was no apparent dose relationship to adverse event frequency. The best overall response rate at any time according to the investigator was 100% (4 CRs, 2 at each dose) with 9/12 responses occurring at cycle 1. CT scan data, which is necessary to determine IWCLL response, is pending and will be presented. There was no apparent dose relationship to response. Conclusions: TRU-016 in combination with bendamustine was well tolerated and showed a positive response. A randomized trial of TRU-016/bendamustine vs. bendamustine alone is ongoing. Disclosures: Jaeger: Emergent Product Development: Consultancy, Research Funding. Rifkin:Millennium Pharmaceuticals, Inc./Takeda: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; ONYX: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; INCYTE: Speakers Bureau; Amgen: Speakers Bureau. Stromatt:Emergent Product Development: Employment.

Towards CD38-Targeted Immunochemotherapy of Multiple Myeloma: Significant Improvement of Anti-Myeloma Effects of Lenalidomide, Bortezomib and Dexamethason by CD38-Specific Antibody Daratumumab, Even in Chemotherapy Resistant/Intolerant Patients

AbstractAbstract 4988To date, multiple myeloma (MM) remains an incurable malignancy of antibody-producing clonal plasma cells. The introduction of a new generation of immunomodulatory agents, such as lenalidomide (LEN), and the potent proteasome inhibitor bortezomib (BORT), used alone or in combination with steroids (dexamethasone; DEX or prednisone; PRED) has significantly improved the overall survival of MM patients. Nonetheless, all chemotherapy strategies are eventually hampered by the development of drug-resistance. Towards a novel and effective targeted immunotherapy for MM, we have developed daratumumab (DARA), a CD38 human antibody with broad-spectrum killing activity. In vitro, DARA induces substantial anti-MM effects mainly via ADCC (antibody dependent cellular cytotoxicity) and CDC (complement dependent cytotoxicity). In ex vivo assays, which allowed us to address killing of MM cells in bone marrow aspirates isolated from MM patients, enhanced or even synergistic MM cell killing was observed when DARA was combined with LEN, or with cocktails of LEN/BORT/DEX and melphalan/BORT/DEX. We now extended these ex vivo analyses to evaluate whether DARA in combination with LEN, BORT and DEX could improve the lysis of MM cells in bone marrow aspirates derived from 22 patients of whom 9 became refractory for LEN and 6 for LEN and BORT. DARA significantly enhanced the lysis of MM cells when combined with LEN or BORT in virtually all patients, including the LEN- and LEN/BORT-refractory patients. The combination of DARA+BORT and DARA+DEX induced additive killing, suggestive of lysis by independent mechanisms. When combined with LEN, DARA improved the lysis of MM cells in a synergistic manner in both non-refractory and LEN-refractory patients. This is suggestive of killing by at least partly complementary mechanisms. Synergistic activity of LEN and DARA was attributable to LEN-induced activation of effector cells that were involved in DARA-mediated ADCC. In addition, enhanced/synergistic direct killing of MM cells was observed. Experiments are under way to further investigate the mechanism underlying synergistic activity of DARA and LEN. In conclusion, our results provide a rationale for clinical evaluation of DARA in combination with LEN, BORT and DEX including in patients refractory to these drugs. Disclosures:van Bueren:genmab: Employment. de Weers:genmab: Employment. Bakker:genmab: Employment. Parren:genmab: Employment. Lokhorst:genmab: Consultancy, Research Funding. Mutis:genmab: Research Funding.

Transcriptional Profiling of Experimental CD8+Lymphocyte Depletion in Rhesus Macaques Infected with Simian Immunodeficiency Virus SIVmac239

CD8(+) T cells inhibit virus replication in SIV-infected rhesus macaques. However, it is unclear to what extent the viral suppression mediated by CD8(+) T cells reflects direct killing of infected cells as opposed to indirect, noncytolytic mechanisms. In this study, we used functional genomics to investigate noncytolytic mechanisms of in vivo viral suppression mediated by CD8(+) lymphocytes. Eight chronically SIVmac239-infected rhesus macaques underwent CD8(+) lymphocyte depletion, and RNA from whole blood was obtained prior to depletion, during the nadir of CD8(+) cell depletion, and after CD8(+) lymphocyte numbers had rebounded. We observed significant downregulation of the expression of genes encoding factors that can suppress SIV replication, including the CCR5-binding chemokine CCL5/RANTES and CCL4 and several members of the tripartite motif-containing (TRIM) family. Surprisingly, we also noted a strong, widespread downregulation of α- and θ-defensins with anti-HIV activity, which are not expressed by CD8(+) T cells. After cessation of depleting antibody treatment, we observed induction of a transcriptional signature indicative of B lymphocyte activation. Validation experiments demonstrated that animals during this period had elevated levels of B cells coupled with higher expression of the proliferative marker Ki67, indicating that CD8(+) depletion triggered a potent expansion of B cell numbers. Collectively, these data identify antiviral pathways perturbed by in vivo CD8(+) T cell depletion that may contribute to noncytolytic control of SIV replication.

Biologically-Directed Modeling Reflects Cytolytic Clearance of SIV-Infected Cells In Vivo in Macaques

The disappointing outcomes of cellular immune-based vaccines against HIV-1 despite strong evidence for the protective role of CD8+ T lymphocytes (CTLs) has prompted revisiting the mechanisms of cellular immunity. Prior data from experiments examining the kinetics of Simian Immunodeficiency Virus (SIV) clearance in infected macaques with or without in vivo CD8 depletion were interpreted as refuting the concept that CTLs suppress SIV/HIV by direct killing of infected cells. Here we briefly review the biological evidence for CTL cytolytic activity in viral infections, and utilize biologically-directed modeling to assess the possibility of a killing mechanism for the antiviral effect of CTLs, taking into account the generation, proliferation, and survival of activated CD4+ and CD8+ T lymphocytes, as well as the life cycle of the virus. Our analyses of the published macaque data using these models support a killing mechanism, when one considers T lymphocyte and HIV-1 lifecycles, and factors such as the eclipse period before release of virions by infected cells, an exponential pattern of virion production by infected cells, and a variable lifespan for acutely infected cells. We conclude that for SIV/HIV pathogenesis, CTLs deserve their reputation as being cytolytic.

Anti-CD20 IgA can protect mice against lymphoma development: evaluation of the direct impact of IgA and cytotoxic effector recruitment on CD20 target cells

While most antibody-based therapies use IgG because of their well-known biological properties, some functional limitations of these antibodies call for the development of derivatives with other therapeutic functions. Although less abundant than IgG in serum, IgA is the most abundantly produced Ig class in humans. Besides the specific targeting of its dimeric form to mucosal areas, IgA was shown to recruit polymorphonuclear neutrophils against certain targets more efficiently than does IgG1. In this study, we investigated the various pathways by which anti-tumor effects can be mediated by anti-CD20 IgA against lymphoma cells. We found that polymeric human IgA was significantly more effective than human IgG1 in mediating direct killing or growth inhibition of target cells in the absence of complement. We also demonstrated that this direct killing was able to indirectly induce the classical pathway of the complement cascade although to a lesser extent than direct recruitment of complement by IgG. Recruitment of the alternative complement pathway by specific IgA was also observed. In addition to activating complement for lysis of lymphoma cell lines or primary cells from patients with lymphoma, we showed that monomeric anti-CD20 IgA can effectively protect mice against tumor development in a passive immunization strategy and we demonstrated that this protective effect may be enhanced in mice expressing the human FcαRI receptor on their neutrophils. We show that anti-CD20 IgA antibodies have original therapeutic properties against lymphoma cells, with strong direct effects, ability to recruit neutrophils for cell cytotoxicity and even recruitment of complement, although largely through an indirect way.

Abstract 2508: GE-huMab-HER3, a novel humanized, glycoengineered HER3 antibody with enhanced ADCC and superior preclinical in vitro and in vivo efficacy

Abstract HER3 is a member of the Human Epidermal Growth Factor Receptor (HER) family. HER3 is a kinase dead receptor, but by forming heterodimers with other HER family receptors, HER3 works as amplifier for PI3 kinase driven tumorigenesis. It has been reported that tumors treated with EGFR-, HER2- or cMET-targeted therapies can escape via HER3 activation or upregulation. HER3 is expressed in a large variety of tumors for example in non-small cell lung cancer (NSCLC), head and neck, colorectal, gastric, pancreatic, breast, ovarian and prostate cancer. Anti-HER3 antibodies can work via various mechanisms including: (1) blocking ligand (HRGs) binding to the receptor, (2) blocking heterodimerization with other HER family members (HER1, 2 and 4), (3) downregulation of the receptor from the cell surface, and (4) engaging immune effector functions such as antibody-dependent cellular cytotoxicity (ADCC). The first three mechanisms lead to inhibition of HER3 phosphorylation and downstream signaling thereby resulting in tumor cell growth inhibition, while ADCC is a mechanism of direct target cell killing triggered by cross-linking of Fc receptors on immune effector cells (e.g. NK cells, macrophages). GE-huMab-HER3 is a novel humanized and glycoengineered IgG1 antibody that binds to HER3 with high affinity. This antibody prevents ligand binding and receptor heterodimerization thereby blocking receptor phosphorylation. In various tumor xenograft models treatment with this antibody leads to substantial tumor growth inhibition. E.g. GE-huMab-HER3 treatment achieved &amp;gt;50% tumor growth inhibition in 10 out of 17 NSCLC models and in some cases even resulted in complete tumor remission. However, these xenograft experiments only reveal part of this antibody's therapeutic potential. A unique feature of GE-huMab-HER3 that differentiates it from other anti-HER3 antibodies, including AMG 888 and MM-121, is its ability to bind to human FcgRIIIa on immune effector cells with a 50-fold higher affinity than standard IgG1 antibodies, a property conferred by the engineered glycosylation of the antibody Fc region. Consequently, GE-huMab-HER3 exhibits superior potency and efficacy in ADCC, as shown in vitro using recombinant A549 cells and in vivo by its Fc mediated greater anti-tumor effect in A549 orthotopic mouse models compared to a non-glyco-engineered variant of the antibody, WT-huMab-HER3. The combination of strong signaling inhibition and enhanced ADCC capability renders GE-huMabHER3 a highly potent HER3-targeting agent. Phase I clinical testing of this promising novel compound is ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2508. doi:1538-7445.AM2012-2508