Antibody-dependent Cell Cytotoxicity Assays Research Articles

P871: ISB 1442, A FIRST-IN-CLASS CD38 AND CD47 BISPECIFIC ANTIBODY INNATE CELL MODULATOR FOR THE TREATMENT OF CD38 POSITIVE HEMATOLOGICAL MALIGNANCIES

Background: ISB 1442 is a first in class 2 + 1 biparatopic bispecific antibody targeting CD38 x CD47 using the BEAT® 2.0 (Bispecific Engagement by Antibodies based on the TCR) platform. The CD38 binding arm consists of bi-paratopic Fabs that strongly bind to CD38 in two different epitopes on tumor cells and do not functionally compete with daratumumab. The anti-CD47 arm comprises a single Fab arm designed to block the interaction between CD47 and the signal-regulatory protein alpha (SIRPα) receptor present on phagocytes (including macrophages, monocytes and dendritic cells). With this design, the high affinity anti-CD38 Fab arms preferentially drive binding to tumor cells and enables blocking of proximal CD47 receptors on the same cell via avidity-induced binding. The Fc portion of ISB 1442 is engineered to enhance antibody dependent cell phagocytosis (ADCP), antibody dependent cell cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). Aims: ISB 1442 was developed for the treatment of hematologic malignancies that express CD38, including multiple myeloma (MM), acute myeloid leukemia (AML), and T-acute lymphoblastic leukemia (T-ALL), with the intention of overcoming mechanisms of resistance to CD38-targeted therapies such as daratumumab and isatuximab, and minimizing hemagglutination/hemolysis on red blood cell (RBC) observed with anti-CD47 monoclonal antibodies (mAb) such as magrolimab. Methods: ISB 1442 was tested for its capacity in vitro to induce ADCP, ADCC and CDC relative to daratumumab and magrolimab across a broad range of MM, AML and T-ALL cell lines expressing different levels of CD38 and CD47. To assess the complex mechanisms of action of ISB 1442 in a single system, a multiple mode of action of killing (MMoAK) assay was established to allow for simultaneous killing by natural killer cells (ADCC), autologous macrophages (ADCP), and complement from human serum (CDC). In vivo, ISB 1442 was assessed in a therapeutic model of subcutaneously established Raji tumor xenograft in CB17/SCID mice compared to daratumumab and magrolimab. On-target specificity was evaluated in vitro in human and monkey whole blood assays. Results: In vitro, ISB 1442 exhibited higher killing potency compared to daratumumab across a range of CD38-expressing MM, AML and T-ALL tumor cells. Additionally, ISB 1442 showed in vitro tumor killing potency through phagocytosis comparable to magrolimab, acting mostly through ADCP. In the CDC, ADCC and MMoAK assays, ISB 1442 exhibited tumor cell killing that was twice as high as daratumumab in MM cell lines. In vivo, ISB 1442 induced higher tumor growth inhibition than daratumumab and comparable tumor regression to magrolimab. ISB 1442 did not cause any detectable hemolysis, RBC depletion or platelet aggregation and showed markedly lower hemagglutination relative to magrolimab, suggesting a more favorable on-target specificity profile in humans. In monkeys, ISB 1442 showed more pronounced binding on RBC than magrolimab due to higher expression of CD38, suggesting that monkey is a more sensitive species than human for toxicological evaluation of CD38-targeted therapies. Summary/Conclusion: In summary, we report a novel approach for the treatment for CD38 positive hematologic malignancies by co-targeting CD38 and CD47 using a first in class multispecific antibody. Based on its unique design and multiple mechanisms of action, ISB 1442 is anticipated to enhance antitumor activity in patients relative to anti-CD38 mAbs by overcoming primary and acquired tumor escape mechanisms of resistance.

Autologous T Cells Modified to Co-Express CD33-Specific Chimeric Antigen Receptor and a Kill Switch for Treatment of CD33+ Acute Myeloid Leukemia

Relapsed/refractory acute myeloid leukemia (AML) is an aggressive malignancy with poor outcomes underscoring the need to implement new therapies. Adoptive transfer of genetically modified T cells with specificity redirected through a chimeric antigen receptor (CAR) has resulted in clinical responses, particularly for B-cell malignancies. CD33 is an attractive target for immunotherapy of AML as this transmembrane protein is expressed on majority of AML blasts. On target, but off tumor effects are anticipated as this target is also expressed on normal myeloid cells and on a subset of activated T and NK cells. Therefore, we generated a lentiviral vector co-expressing a CD33-specific CAR with a kill switch, HER1t, for the generation of CD33-specific CAR+HER1t+ T (CD33-CAR-T) cells. HER1t improves the safety profile by providing a mechanism for selective in vivo depletion of these genetically modified T cells upon addition of the clinically-available monoclonal antibody, cetuximab. Co-expression of CAR and HER1t on transduced T cells was confirmed by flow cytometry and western blot analyses. The redirected specificity was demonstrated by the specific killing of CD33+ tumor cells as well as significant release of cytokines IFNγ, TNFα, IP-10, IL-13, IL-18, and LIF upon co-culture with AML tumor cells. The ability to eliminate AML in vivo was determined in immunocompromised (NSG) mice bearing MOLM-13 (CD33+CD19neg) tumor. A single administration of human CD33-CAR-T cells resulted in a significant reduction in tumor burden (Figure) and improvement in overall survival with an overall median survival time of 46 days compared to 15 days for control treated mice (saline only, untransduced (CARneg) T cells, or CD19-specific CAR+ T (CD19-CAR-T) cells). Assessment from the plasma of CD33-CAR-T treated mice revealed production of cytokines consistent with observations from in vitro activation of genetically modified T cells. CD33-CAR-T cells were effectively eliminated in the presence of cetuximab both in vitro in an antibody dependent cell cytotoxicity assay and in an in vivo adoptive T cell transfer model in immunocompromised NOD/SCID mice. In preparation for a clinical trial, we manufactured CD33-CAR-T cells from an individual with relapsed AML. As with T cells from healthy volunteers, transduced T cells from the patient co-expressed CAR and HER1t and were effective at specifically eliminating autologous CD33+ tumor as well as producing cytokines in response to CAR signaling. These pre-clinical data provide a strong rationale to evaluate CD33-CAR-T therapy in a clinical trial for treatment of AML. The FDA has approved an investigational new drug application to initiate phase 1 clinical trial at MD Anderson Cancer Center for treatment of CD33+relapsed/refractory AML with autologous CD33-CAR-T cells. [Display omitted] DisclosuresSong:Intrexon Corporation: Employment. Tian:Intrexon Corporation: Employment. Carvajal-Borda:Intrexon Corporation: Employment. Plummer:Intrexon Corporation: Employment. Shah:Intrexon Corporation: Employment. Wierda:Emergent: Consultancy, Honoraria, Research Funding; Merck: Consultancy, Honoraria; Karyopharm: Research Funding; Gilead: Consultancy, Honoraria, Research Funding; GSK/Novartis: Consultancy, Honoraria, Research Funding; Genentech/Roche: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding; Kite: Research Funding; Genzyme: Consultancy, Honoraria; The University of Texas MD Anderson Cancer Center: Employment; Pharmacyclics: Consultancy, Honoraria, Research Funding; Janssen: Research Funding; Sanofi: Consultancy, Honoraria; Acerta: Research Funding; Juno: Research Funding. Cooper:Organovo Holdings: Equity Ownership; Sangamo Biosciences: Patents & Royalties; Miltenyi Biotec: Honoraria; Ampliphi Biosciences: Equity Ownership; Argos Therapeutics: Equity Ownership; Intrexon Corporation: Equity Ownership, Patents & Royalties; Procter & Gamble: Equity Ownership; Targazyme, Inc.: Equity Ownership; Ferring: Consultancy; Immatics: Equity Ownership, Patents & Royalties, Research Funding; Ziopharm: Employment, Equity Ownership, Research Funding. Chan:Intrexon Corporation: Employment.

Abstract PR15: A novel NK cell-targeted therapeutic strategy against pancreatic cancer

Abstract Recent years have witnessed an increased incidence of pancreatic adenocarcinoma (PDAC). Pancreatic tumors are poorly immunogenic; current immunotherapeutic strategies largely focus on boosting adaptive immunity while ignoring immunosuppressed innate immune players such as natural killer cells (NKs). NK cells respond to tumor insults by generating IFN-γ for T-cell activation. Multiple studies suggest that tumor cells evade NK cell-mediated killing by 1) developing “escape variants” and 2) regulating inhibitory and activating receptors on NK cells, imparting anergic/exhaustive phenotype inside the pancreatic tumor microenvironment. We posit that a therapeutic combination that not only overturns tumor-mediated NKs dysfunction but also boosts cytotoxic CD8 T cells will provide long-lasting therapeutic benefits in PDAC. Given this, we explored the efficacy of a unique therapeutic combination, tumor antigen-targeted IgE antibody (humanized anti-MUC1.IgE) in combination of anti-PD-L1 (for relieving T-cell exhaustion) and PolyICLC (for dendritic cell maturation) in a preclinical model of pancreatic cancer using mice transgenic for human MUC1 and FcϵRI (hMUC1/hFcϵRI). This therapeutic combination induced MUC1 specific rejection of two different human MUC1-expressing pancreatic tumor cell lines (Panc02.MUC1, KPC.MUC1) and prolonged the overall survival of mice challenged with subcutaneous and orthotopic tumors as compared to control counterparts. Additionally, this combination generated CD8 T-cell memory response as evidenced by MUC1 specific rejection/delays of tumors in mice rechallenged with MUC1-expressing tumors. Cytokine/chemokine profiling of anti-MUC1.IgE+anti-PD-L1+PolyICLC treated tumors further demonstrates a reduction in proinflammatory cytokines as compared to control counterparts. Most importantly, NK and CD8 T cells were involved in cell-mediated antitumor responses, as in vivo depletion of these subtypes abrogated the tumor-protective benefits in mice bearing orthotopic tumors. Anti-MUC1.IgE+anti-PD-L1+PolyICLC combination appears to increase circulating NKs and reverse NK cell exhaustion inside pancreatic tumor microenvironment. Additional data suggest that this therapeutic combination boosts tumor cell killing by NK cells in antibody-dependent cell cytotoxicity assays (ADCC). In sum, this is the first study to show that specific stimulation of IgE/FcϵRI axis in combination with PolyICLC and anti-PD-L1 can activate both CD8 T and NK cell effector pathways and provide long-lasting tumor-protective benefits against pancreatic cancer. This abstract is also being presented as Poster A48. Citation Format: Kamiya Mehla, Thomas C. Caffrey, Kelly A. O'Connel, Raghupathy Madiyalakan, Christopher F. Nicodemus, Michael A. Hollingsworth. A novel NK cell-targeted therapeutic strategy against pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR15.

Daratumumab-225Actinium conjugate demonstrates greatly enhanced antitumor activity against experimental multiple myeloma tumors

ABSTRACT Daratumumab is an anti-CD38 directed monoclonal antibody approved for the treatment of multiple myeloma (MM) and functions primarily via Fc-mediated effector mechanisms such as complement-dependent cytotoxicity (CDC), antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cellular phagocytosis, and T-cell activation. However, not all patients respond to daratumumab therapy and management of MM remains challenging. Radioimmunotherapy with alpha particle-emitting radionuclides represents a promising approach to significantly enhance the potency of therapeutic antibodies in cancer treatment. Here we report the results of mechanistic and feasibility studies using daratumumab radiolabeled with an alpha-emitter 225Actinium for therapy of MM. CD38-positivelymphoma Daudi cell line and MM cell lines KMS-28BM and KMS-28PE were treated in vitro with 225Ac-daratumumab. 225Ac-daratumumab Fc-functional properties were assessed with C1q binding and ADCC assays. The pharmacokinetics and tumor uptake of 111In-daratumumab in Daudi tumor-bearing severe combined immunodeficiency (SCID) mice were measured with microSPECT/CT. The therapeutic effects of 225Ac-daratumumab on Daudi and KSM28BM tumors in mice and treatment side effects were evaluated for 50 days posttreatment. The safety of 225Ac-labeled antimurine CD38 mAb in immunocompetent mice was also evaluated. 225Ac-daratumumab efficiently and specifically killed CD38-positive tumor cells in vitro, while its complement binding and ADCC functions remained unaltered. MicroSPECT/CT imaging demonstrated fast clearance of the radiolabeled daratumumab from the circulation and tissues, but prolonged retention in the tumor up to 10 days. Therapy and safety experiments with 225Ac-daratumumab showed a significant increase in the antitumor potency in comparison to naked antibody without any significant side effects. Our results highlight the potential of targeting alpha-emitters to tumors as a therapeutic approach and suggest that 225Ac-daratumumab may be a promising therapeutic strategy for the treatment of hematologic malignancies.

A Novel Anti-IgM/HLA-DR Bispecific Antibody for Treatment of Refractory B Cell Malignancies

Introduction: IgM composes a part of the B cell receptor, and its expression is restricted to B cell lineage cells. There have been several reports that refractory B cell malignant cells frequently express IgM. For example, activated B cell-like (ABC) diffuse large cell lymphoma (DLBCL) is a subtype with a poor prognosis following R-CHOP treatment (Lenz et al., N Engl J Med, 2008), and the tumor cells generally express IgM-type immunoglobulin (Ruminy et al., Leukemia, 2011). In addition, IgM-expressing chronic lymphocytic leukemia (CLL) was also found to have a poor prognosis (D'Avola et al., Blood, 2016). Therefore, IgM is a promising therapeutic target for refractory B cell malignancies. However, anti-IgM antibodies may not function fully in the body due to the presence of soluble IgM. To improve the selectivity to the IgM expressing cells, we produced a bispecific antibody bound to IgM and HLA-DR, which are both expressed by B cells, and it was able to bind IgM+HLA-DR+ cells in the presence of soluble IgM. Thus, the aim of the present study was to examine the in vitro anti-tumor activity of the anti-IgM/HLA-DR bispecific antibody against B cell lymphoma, and to clarify its efficacy and tolerability in non-human primates (NHP).Methods: BTA106 is the bispecific antibody that can bind to IgM and HLA-DR, and BTA111 is glycoengineered BTA106. To investigate their direct inhibitory effects and Fc-mediated effects against B cell lymphoma cell lines, a growth inhibition assay, antibody-dependent cell cytotoxicity (ADCC) assay and complement dependent cytotoxicity (CDC) assay were performed. In the growth inhibition assay and CDC assay, cell proliferation was measured by WST-8. In the ADCC assay, human peripheral blood mononuclear cells were used as effector cells. Dead cells were stained with propidium iodide and measured by flow cytometry. JeKo-1 (Mantle cell lymphoma, MCL), Ramos (Burkitt's lymphoma, BL), RL (DLBCL), B104 (B cell lymphoma) and two rituximab-resistant cell lines, RL-4RH (Czuczman et al., Clin Cancer Res., 2008) and RRBL1 (generously provided by Dr. Tomita, Tomita et al., Int J Hematol., 2007), were used. To clarify the efficacy and tolerability of BTA106 in NHP, a dose escalation study and single-dose administration study were performed using cynomolgus monkeys.Results: BTA106 and BTA111 demonstrated direct growth inhibition activity, and CDC and ADCC activity, although their intensities differed among cell lines. In the case of rituximab-resistant cell lines, RRBL1 and RL-4RH, BTA106 and BTA111 exhibited weak or no growth inhibitory effects, but their CDC and ADCC activity was superior to that of rituximab. Development of resistance to rituximab is also a poor prognostic factor and remains problematic. Thus, BTA106 and BTA111 are expected as therapeutic antibodies to overcome rituximab resistance.A dose escalation study and single dose study were performed in cynomolgus monkeys. BTA106 greatly reduced peripheral B cells. Moreover, after the administration of BTA106, lymph follicles disappeared in axillary lymph nodes. In these studies, decreased locomotor activity and vomiting were observed; however, these symptoms resolved 2 hrs after injection. Other BTA106-related clinical toxicities or changes in body weight were not observed.Conclusions: In the in vitro studies, BTA106 and BTA111 demonstrated significant anti-tumor effects on rituximab-sensitive and -resistant B cell lymphoma cell lines. The efficacy and toxicity studies in cynomolgus monkeys revealed that BTA106 depletes peripheral and lymph node B cells, and it was well tolerated. BTA111 was considered to be significantly more effective than BTA106 due to glycoengineering, which induced high ADCC activity. These data suggest that BTA111 is an effective and potent therapeutic antibody for B cell malignancies, especially rituximab-resistant B cell lymphoma. Currently, a repeated administration study of BTA111 with cynomolgus monkeys to evaluate its efficacy and toxicity is ongoing. DisclosuresOhashi:Zenyaku Kogyo Co., Ltd.: Employment. Miyashita:Zenyaku Kogyo Co., Ltd.: Employment. Nagata:Zenyaku Kogyo Co., Ltd.: Employment. Tatebe:Zenyaku Kogyo Co., Ltd.: Employment. Otsuka:Zenyaku Kogyo Co., Ltd.: Employment. Suzuki:Zenyaku Kogyo Co., Ltd.: Employment. Sasaki:Zenyaku Kogyo Co., Ltd.: Employment. Enami:Zenyaku Kogyo Co., Ltd.: Employment. Tsukada:Zenyaku Kogyo Co., Ltd.: Employment. Fukushima:Zenyaku Kogyo Co., Ltd.: Employment.

DNA Methyltransferase Inhibitors Increase Expression of CD38 and Enhance Daratumumab Efficacy in Multiple Myeloma

Introduction: The anti-CD38 monoclonal antibody Daratumumab is highly effective against multiple myeloma, is well tolerated, and has high single agent activity as well as combination effects with lenalidomide-dexamethasone as well as bortezomib-dexamethasone. Patient response to daratumumab monotherapy is highly correlated with pretreatment levels of CD38 expression on MM plasma cells (Nijhof et al, Leukemia (2015) 29:2039) and CD38 loss is correlated with daratumumab resistance (Nijhof et al, Blood (2016) 128:959). As a result, there is significant interest in elucidating the regulation and role of CD38 in MM. Recently, All Trans Retinoic Acid (ATRA), a known small molecule inducer of CD38 in myeloid cells, as well as the FDA-approved histone deacetylase inhibitor panobinostat, were both demonstrated to induce CD38 in MM plasma cells leading to increased lysis by daratumumab. Examining ENCODE data, we found the presence of a CpG island at the first exon of CD38. We hypothesized that removing methylation sites from this CpG island may de-repress CD38 transcription and lead to increased CD38 protein at the cell surface in MM plasma cells. Therefore, here we studied the role of DNA methyl-transferase inhibitors (DNMTis), currently FDA-approved for treatment of myelodysplastic syndrome, as agents to potentiate daratumumab therapy.Methods: We treated MM cell lines (RPMI-8226, MM.1S, XG-1, KMS12-PE) with two different DNMTis, 5-Azacytidine and decitabine, and assessed CD38 cell surface expression by flow cytometry. Similarly, we treated MM patient bone marrow aspirates ex vivo and assessed induction of CD38 expression in the CD138 positive population by flow cytometry. We analyzed CD38 mRNA levels and total CD38 protein levels by qRT-PCR and western blotting respectively. ATRA was used as a positive control in all experiments. We further tested the functional effect of DNMTi treatment on MM cell lines using an Antibody Dependent Cell Cytotoxicity (ADCC) assay. Briefly, live treated cells were incubated overnight with daratumumab and NK92-CD16 transgenic cells at and E:T ratio of 20:1, and lysis was measured using CytoTox-Glo (Promega).Results: Flow analysis revealed that DNMTi treatment induces a 1.2-2 fold increase in CD38 surface protein expression in a dose-dependent manner across MM cell lines. DNMTi treatment consistently yielded similar or higher increases in CD38 expression than that seen in ATRA- or panobinostat-treated cells. Despite significantly lower single-agent cytotoxicity, we found that decitabine led to similar surface CD38 induction as 5-Azacytidine. By RT-qPCR, 5-Azacytidine increased CD38 mRNA expression ~3 fold versus DMSO control, compared to ~2 fold mRNA increase with ATRA. In functional ADCC assays, DNMTi treatment also led to greater lysis than ATRA. Furthermore, the combination of both DNMTi and ATRA was additive, leading to the greatest lysis by NK cells. In contrast, in ex vivo-treated patient samples, ATRA induced greater CD38 expression than 5-Azacytidine on malignant plasma cells. However, this result is expected since MM plasma cells from patients typically do not proliferate in standard ex vivo culture, and active DNA replication is a requirement for successful DNMT inhibition based on known mechanism of action. In patients, however, we anticipate that continual plasma cell proliferation will lead to effective increases in CD38 after DNMTi treatment, as found in MM cell lines here.Summary and Conclusions: Our results here demonstrate that CD38 expression in MM cells is regulated by DNA methylation and targeting DNMTs with small molecule inhibitors leads to increased vulnerability to Daratumumab treatment. We propose that combination treatment with DNMTi and Daratumumab can lead to higher efficacy of daratumumab in daratumumab-naïve MM, as well as reversal of daratumumab-resistance. These combinations should be tested in clinical trials. DisclosuresWiita:Sutro Biopharma: Research Funding; TeneoBio: Research Funding.

Abstract B57: Effect of ADAM17 inhibition on the release of IFN-y during natural killer cell-mediated antibody dependent cellular cytotoxicity in cancer

Abstract Introduction: Antibody-dependent cell cytotoxicity (ADCC) by NK cells is a key mechanism in the anti-cancer effects of therapeutic antibodies. CD16a is an IgG Fc receptor (FcRIII) present on the surface of NK cells and plays essential role in recognizing tumor-bound antibodies to initiate ADCC. NK cells are triggered to release cytokines like IFN-y during ADCC which has been reported to have a number of antitumor activities. A disintegrin and metalloproteinase 17 (ADAM17) plays key role in downregulating CD16a in the tumor microenvironment and facilitate tumor induced anergy through the inactivation of cell cytotoxic function. Downregulation of CD16a on NK cells and decreased levels of IFN-y have been reported in ovarian cancer patients. We hypothesize that by blocking the function of ADAM17 we can enhance IFN-y release during ADCC by maintaining higher CD16a levels on the NK cells. To test this hypothesis, we used ADAM17 function blocking antibody MEDI3622 (MedImmune), HER2 receptor positive ovarian (SKOV-3 and MA148) and CD20 receptor positive lymphoblastoid (Raji) cancer cell lines. To further study the role of CD16a in this process we used monoclonal function block anti-CD16 antibody (clone: 3G8) and cleavage resistant CD16a NK92 cells (CD16a/S197P) where we substituted serine with proline at the location of 197 in the membrane proximal region. Description: NK cells derived from peripheral blood (PBNK) were isolated through negative selection EasySepTM (StemCell, Cat: 19055). These PBNK cells were incubated with target cells in the presence of a therapeutic antibody to initiate ADCC with or without blocking ADAM17 by MEDI3622. The samples were incubated at 37°C in CO2 incubator and the supernatant were collected after 4 hrs of incubation by briefly spinning them at 1500 rpm for 5 minutes. IFN-y was measured through CBA (BD Bioscience, Cat: 560111). Summary: The blocking of ADAM17 by MEDI3622 in the presence of herceptin showed an increase in the IFN-y release in comparison to herceptin alone group during ADCC with two ovarian cancer cell lines i.e. SKOV-3 (p<0.0001) and MA148 (p<0.0001). Furthermore, this increase was consistent when we performed the ADCC assay with a different lymphoblastoid related cancer cell line (Raji) with a dissimilar target (CD20 receptor) and therapeutic antibody (rituximab) where we again found a significant increase in IFN-y release in MEDI3622 with rituximab group in comparison to rituximab alone group (p<0.0001). Next we investigated the role of CD16a receptor in the release of IFN-y. We blocked the CD16a receptor with a monoclonal antibody (clone: 3G8) during herceptin mediated ADCC by PBNK cells in SKOV-3 cancer cell line with or without MEDI3622 and found a substantial decrease in the IFN-y release (P<0.0001) in both the groups (herceptin+MEDI3622 and herceptin alone groups). In another experiment, where we expressed a cleavage resistant CD16a (CD16a/S197P) in human NK cells line (NK92), we compared the IFN-y release by CD16a/S197P to CD16a/WT NK92 cells during herceptin mediated ADCC in SKOV-3 tumor. In this experiment, NK92a/S197P showed higher release of IFN-y in comparison to NK92aWT (p<0.0001). Upon blocking ADAM17 on CD16a/WT there was a significant increase in the IFN-y (p<0.0003) release. However, NK92a/S197P did not show any further increase in the presence of MEDI3622 (p=ns) signifying the key role of CD16a in this process. Conclusion: Our results indicate that the inhibition of ADAM17 enhances the IFN-y release by NK cells during ADCC by blocking the shedding of CD16a. Based on these results, ADAM17 may be considered a checkpoint target to improve tumor killing and its utility can be exploited in cancer immunotherapy. Citation Format: Hemant K. Mishra, Wu Jianming, Nabendu Pore, Bruce Walcheck. Effect of ADAM17 inhibition on the release of IFN-y during natural killer cell-mediated antibody dependent cellular cytotoxicity in cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B57.

Glypican-1 as a novel immunotherapeutic target in prostate cancer.

174 Background: New effective therapies for men with prostate cancer are desperately needed. Recently, cancer immunotherapy has emerged as an important new treatment strategy for prostate cancer and for castrate resistant prostate cancer (CRPC). Multiple studies have identified the heparan sulfate proteoglycan-1 Glypican 1 (GPC-1) as being overexpressed in different cancers, and also as being a possible marker of poor prognosis in several solid tumor cancers. GPC-1 has been recently identified as a potential marker for prostate cancer. The MIL-38 monoclonal antibody detects GPC-1 and an IgG1 chimeric version of this antibody has been developed for preclinical studies. Here we sought to examine MIL-38 binding to a panel of prostate cancer cell lines and examine its feasibility as a novel immunotherapeutic agent targeting GPC-1 in prostate cancer Methods: Expression of GPC-1 in CRPC cell lines was examined by Flow cytometry and Western Blotting using MIL-38 as the detector antibody. The competency of GPC-1 as an immunotherapeutic target was assessed via chimeric MIL-38 induced Antibody Dependent Cell Cytotoxicity (ADCC) using high affinity Natural Killer cells (haNKs) in vitro . Results: Flow cytometry and Western blot assessments of normal prostatic epithelial cells (i.e. RWPE-1) and cells from prostate cancer cell lines (i.e. PC-3, 22RV1, DU-145, VCaP, LNCaP, CWR-R1, and LAPC-4) revealed that only cancer cells expressed GPC-1. Enzalutamide resistant cell lines demonstrated higher expression of GPC-1 than their respective parental line. ADCC assays demonstrated enhanced haNK – prostate cancer cell cytotoxicity in the presence of chimeric MIL-38 anti-GPC-1 antibody, while the IgG1 isotype control had no effect. Conclusions: GPC-1 protein was expressed by most prostate cancer cell lines, including enhanced expression by enzalutamide resistant cells. Preliminary in vitro ADCC assay results revealed the potential utility of GPC-1 as an immunotherapeutic target in prostate cancer.

ACP-196 Is a Second Generation Inhibitor of Bruton Tyrosine Kinase (BTK) with Enhanced Target Specificity

Background: Signaling through the B-cell receptor (BCR) results in cell survival and proliferation, and this pathway is critical for the progression of many B-cell malignancies, such a chronic lymphocytic leukemia (CLL). BTK, a TEC family kinase, is essential to the BCR signaling cascade. The recent approval of the first-in-class BTK inhibitor for the treatment certain B-cell malignancies, ibrutinib, has validated BTK as a clinical target. ACP-196 is a novel, second generation inhibitor of BTK with an IC50of 3 nM against purified BTK and EC50 of 8 nM in a human whole-blood CD69 B-cell activation assay. In vivo, oral administration of ACP-196 inhibits BCR-induced CD69 expression in murine splenic lymphocytes with an ED50 of 1.3 mg/kg compared with 2.9 mg/kg for ibrutinib. ACP-196 also has improved target specificity over ibrutinib with 323-,94-, 19- and 9-fold selectivity over the other TEC kinase familymembers (ITK, TXK, BMX, and TEC, respectively) and no activity against EGFR (Covey AACR, 2015). TXK and ITK are critical for T-cell receptor activation and development. ITK is also necessary for natural killer (NK)-cell mediated antibody dependent cell cytotoxicity (ADCC), which is important for the efficacy of monoclonal antibody therapy, eg, obinutuzumab. Additionally, therapeutic blockade of EGFR signaling is employed for treatment of epithelial cancers and is associated with adverse events, such as diarrhea and skin rash. Here, we describe results from signaling and functional assays to further elucidate the differential effects of ACP-196 on primary CLL cells compared with T-cells, NK-cells and epithelial cells.Methods: CLL patient samples were evaluated for pathway activation in the presence and absence of ACP-196 using phospho-protein immunoblotting. Helper T-cell (Th) skewing assays were performed using purified naive CD4+ T-cells from C57BL/6, Itk knock out (KO) and Itk/Txk double KO mice in the presence ACP-196 or ibrutinib. CD8+ T-cell activity was assessed by cytolytic activity (CTL) assays using T-cells isolated from C57BL/6 mice. The CTL response was initiated with various effector:T-cell ratios using C57BL/6 T-cells and murine BALB/c lymphoblasts. To evaluate NK-cell ADCC function, standard chromium release assays were done using purified NK-cells isolated from healthy volunteer peripheral blood mononuclear cells and cultured with obinutuzumab-coated (10 µg/mL) CLL cells in the presence and absence of ACP-196. Immunoblotting of H460 lung cancer cells for phospho-EGFR was used to evaluate the effect of ACP-196 on EGFR signaling.Results: ACP-196 treatment of primary human CLL cells showed a dose-dependent (0.01μM-1μM) decrease in p-BTK (Y223), p-ERK (T202/Y204) and p-AKT (T308) and p-IκB-α (S32), without impact on total protein levels. These findings demonstrate ACP-196 inhibits autophosphorylation of BTK as well as down-stream effectors of BCR signaling.Phospho-protein immunoblotting of primary human T-cells and Jurkat cells showed that ACP-196 does not inhibit T-cell receptor signaling molecules downstream of ITK and TXK such as IκBα, NFAT, or JunB. In in vitro helper T cell skewing assays, ACP-196, unlike ibrutinib, showed no effect on differentiation of Th subsets (Th1/Th2/Th17) or regulatory T cells (Tregs). The effect of ibrutinib on Th and Treg differentiation was similar to that observed in cells from Itk KO and Itk/Txk KO mice. In addition, ACP-196 had no effect on CD8+ T-cell viability or cytotoxicity as compared with ibrutinib-treated CD8+ T cells (46% and 36% reduction, respectively).In the in vitro ADCC assay, ACP-196 had no effect on NK cell function (chromium release of 34.8% vs 39.6% for the vehicle control). Lastly, at clinically relevant concentrations ACP-196 did not attenuate autophosphorylation of EGFR in H460 lung epithelial cells demonstrating no effect of ACP-196 on EGFR signaling.Conclusions: ACP-196 selectively inhibits BTK and BCR signaling at pharmacologic concentrations without off-target effects on T-cells, NK-cells and lung epithelial cells. ACP-196 is currently in Phase 3 trials for the treatment of CLL (ClinicalTrials.gov NCT0247568 and NCT02477696).This work was supported by the NIH NCI R01CA197870 and Acerta Pharma. DisclosuresGulrajani:Acerta Pharma: Employment. Covey:Acerta Pharma BV: Employment, Equity Ownership, Patents & Royalties. Kaptein:Acerta Pharma: Employment. Van Lith:Acerta Pharma: Employment. Izumi:Acerta Pharma: Employment. Hamdy:Acerta Pharma BV: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Ulrich:Acerta Pharma: Employment. Lannutti:Acerta Pharma: Employment. Johnson:Acerta Pharma: Research Funding.

Effects of Ibrutinib on Rituximab and GA-101 Induced Antibody-Dependent Cell Cytotoxicity (ADCC) in Lymphoma Cells in Vitro

Introduction: Ibrutinib is a first-in-class, oral, once-daily, small molecule that covalently binds to and inhibits Bruton’s tyrosine kinase (BTK), an essential enzyme in the B-cell receptor signaling pathway. Ibrutinib has shown single-agent efficacy and good tolerability in patients with various B-cell malignancies, and has been approved for treatment of previously treated chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). In addition, ibrutinib is also being evaluated in combination with several other anticancer agents, including the anti-CD20 monoclonal antibodies rituximab and ofatumumab, as well as obinutumumab (GA-101), a next-generation anti-CD20 antibody recently approved to treat CLL. A previous report suggested that the combination of ibrutinib and rituximab under certain conditions results in antagonistic effects on ADCC, a possible mechanism of action of rituximab (Kohrt HE, et al. Blood. 2014;123:1957-1960). However, clinical data in high-risk CLL in combination with rituximab showed high response rates compared with available data on the single agents (Burger JA, et al. Blood. 2012;120 (21):187). As the variance could be due to experimental conditions that do not replicate well the unique pharmacokinetics (PK; such as short half-life and lower Cmax; Advani RH, et al. JCO. 2013;31:88-94) and selectivity profile of ibrutinib under clinical dosing conditions, we modified the assay to better mimic the physiological PK profile, and further, to test these possible effects of ibrutinib in combination with the newer anti-CD20 antibody obinutumumab.Methods:Peripheral blood mononuclear cells (PBMC) isolated from different donors were used as effector cells in this study, in addition to the purified natural killer (NK) cells used previously, and were treated as follows: (1) no treatment for 24 hrs (control), (2) pretreatment with 200nM ibrutinib for 2hrs (2 hr T), (3) pretreatment with 200nM ibrutinib for 2 hours with a 24-hour washout period (2hr T/24 hr WO) which mimics the physiological PK profile of ibrutinib, (4) pretreatment with 200nM ibrutinib for 24 hours (24 hr C). ADCC assays were then conducted on target lymphoma cell lines (Ramos and Daudi) with rituximab (10 µg/ml) or obinutuzumab (10 µg/ml) alone or in combination with 200nM ibrutinib for 3 hours. Percent-specific lysis was measured using the Calcein AM assay. BTK occupancy was measured as described previously (Honigberg LA, et al. PNAS. 2010;107:13075-13080). Cytokine levels were measured using the 10-plex Proinflammatory Panel 1 from Meso Scale Discovery per recommended procedures.Results: Continuous exposure to ibrutinib reduced rituximab-mediated ADCC in the studied lymphoma cell lines. However, under physiologically-relevant conditions where cells were exposed to ibrutinib for 2 hrs and then washed off, the antagonistic effect on rituximab-mediated ADCC was minimized. Even with a 2 hr exposure, BTK was still fully occupied at 24 hrs after washoff, suggesting that the chronic effect may be due at least in part to other targets of ibrutinib (Honigberg LA, et al. PNAS. 2010;107:13075-13080) not continuously inhibited under physiological conditions. Obinutuzumab induced a higher level of ADCC than seen with rituximab in the same cell lines, and ibrutinib pretreatment had minimal effects on obinutuzumab-mediated ADCC, especially at 2 hr exposure with or without ibrutinib washout. Figure 1 shows typical data in Daudi cells, derived from a Burkitt’s patient and not appreciably killed by ibrutinib at 200nM in 24 hrs. Cytokine measurements showed that interferon-gamma was reduced by chronic exposure to ibrutinib but this effect was also mitigated by 2 hr ibrutinib exposure followed by 24 hr washoff. [Display omitted] Conclusions: These results demonstrate that, when administered under physiologically-relevant conditions in vitro, the effects of ibrutinib on anti-CD20 antibody-induced ADCC are substantially mitigated, particularly with obinutuzumab (GA-101). DisclosuresDavis:Janssen: Employment. Syed:Janssen R&D: Employment. Axel:Janssen: Employment. Hall:Janssen: Employment. Sasser:Janssen R&D: Employment. Balasubramanian:Janssen R&D: Employment, Equity Ownership.

Development of RTXM83 (a potential rituximab biosimilar): In vitro and in vivo comparability with MabThera.

e14020 Background: RTXM83, a proposed biosimilar to rituximab, has an identical amino acid sequence and similar physicochemical and in vitro functional properties to reference drug, MabThera (sourced from EU). This study was designed to demonstrate comparability between RTXM83 and MabThera, both in vitro and in vivo. Methods: Mass fingerprinting and mass spectrometry analysis; UV CD spectroscopy; Glycan and charge variant analysis; SDS-PAGE analysis and SE HPLC analysis; CDC and ADCC assays; FcRIII and c1q assays; PK/PD study. Results: The results indicate similar primary structural attributes, as evident from peptide mass fingerprinting and mass spectrometry analysis. Far and near UV CD spectroscopy indicates comparable secondary and tertiary structure between the two molecules. Glycan and charge variant analysis also indicate similar post translational modifications. The product related impurity profile, as indicated by SDS-PAGE analysis and SE HPLC analysis, indicates similar purity. Both molecules demonstrated similar binding affinity to CD20 receptor and their ability to recruit the complements, c1q and finally demonstrated similar potency in in vitro complement dependent cytotoxicity (CDC) assay. The similarity in effector function was determined by evaluating the binding with Fc?RIII followed by in vitro antibody dependent cell cytotoxicity (ADCC). Both molecules showed similar potency in in vitro apoptosis assay. The in vitro binding affinities to neonatal receptors are also similar, predicting similar half-life in vivo. The in vivo PK/PD study was conducted in cynomologus monkeys and the results indicate that the AUC, T1/2, Cmax of RTXM83 are within 80-120% range of the reference drug, MabThera. While evaluating the PD parameters, it was observed that the time of onset, magnitude and duration of PD response (CD20 and CD40 cell depletion) was similar between RTXM83 and Mabthera. Conclusions: herefore, all data generated to date are establishing a high comparability (high similarity) between Mabthera and RTX83, meeting all requirements for clinical approval of our product.

Antitumor Activity of An RNA-Based Agonist of TLR7 and 8 In Preclinical Models of Hematological Malignancies

AbstractAbstract 421Treatment of hematological malignancies, non-Hodgkins lymphoma in particular, has evolved greatly in the past decade with the advent of rituximab, proteasome inhibitors and other new agents. Nonetheless, the need remains for effective agents with novel mechanisms of action, which lack cross-resistance to current standards of care. Agents that regulate host immune function may contribute to the control of hematological tumors by affecting innate and acquired immune responses targeting cancer cells. In this context, Toll-like receptors (TLR) are ideal targets for combination therapy as they play a key role in modulating immune responses and can influence the tumor microenvironment through induction of cytokines/chemokines and enhance antibody-dependent cell cytotoxicity (ADCC), a key mechanism involved in rituximab antitumor effects. We have shown that a novel class of synthetic oligoribonucleotide compounds, acting as TLR7 and 8 agonists, induce potent T helper 1-type immune responses in preclinical models (Proc Natl. Acad. Sci. USA, 104: 13750, 2007) and exert significant antitumor effects (Mol Cancer Ther. 9: 1788, 2010). In this study, we have evaluated a combination strategy involving a TLR7/8 dual agonist with rituximab, bortezomib or cyclophosphamide, in human xenograft and murine syngeneic lymphoma models. We performed ADCC assays using human peripheral blood mononuclear cells (PBMCs) stimulated with the agonist as effectors and rituximab-coated Raji, a B cell lymphoma, or Granta-519, a mantle cell lymphoma tumor cell line, as targets. Combining agonist-treated PBMCs with uncoated Raji cells or untreated PBMCs with rituximab-coated Raji cells resulted in about 35% cytotoxicity. When agonist-treated PBMCs were combined with rituximab-coated Raji cells, cytotoxicity was enhanced to 65%. Agonist treatment of PBMCs alone did not induce killing of uncoated Granta-519 cells whereas treatment with rituximab caused 25% cytotoxicity. Combining agonist treatment with rituximab coating of Granta-519 cells increased cytotoxicity to 51%. Thus, the agonist significantly enhances the ADCC activity of rituximab. NOD-SCID mice implanted i.v. with Raji cells were treated with 50 mg/kg agonist (s.c., twice weekly for 5 weeks), 10 mg/kg rituximab (i.p., twice weekly for 5 weeks), the combination of the two agents or PBS starting at day 3. The control group of mice that received PBS developed a disseminated systemic disease with a median survival 18.5 days and died within 24 days. Treatment with agonist or rituximab alone lead to moderate tumor growth inhibition with a median survival of 24 days (p = 0.043) or 36 days (p < 0.01), respectively. However, all mice in both groups developed lymphoma and died within 74 days. Treatment of mice with combination of agonist and rituximab resulted in long term tumor-free survival of 63% of the treated mice (p = 0.0005 compared to antibody alone group) with a median survival of 82 days. Similar antitumor effects were observed in HBL-2 mantle cell lymphoma xenografts in Rag2 mice. Flow cytometry analysis of human CD20+, CD19+ and CD20−CD19+ cells in the peripheral blood of mice bearing HBL-2 tumors showed that treatment with the agonist or rituximab lead to a 19% and 70% reduction of lymphoma cells, respectively. In contrast, treatment with agonist and rituximab increased clearance of total human cells to 97%. Interestingly, although rituximab was very effective in clearing CD20+ cells, it did not impact the number of CD20−CD19+ cells which accumulated over time and account for 8% of the total lymphoma cell population in the blood stream. Treatment with agonist and rituximab, significantly reduced the number of circulating CD20−CD19+ cells. The agonist also showed synergistic antitumor activity when combined with either cyclophosphamide or bortezomib in murine B-cell lymphoma xenografts from Em-myc transgenic mice and A20 lymphoma in BALB/c mice, respectively. Combination treatment in both models resulted in significant tumor growth inhibition compared to cyclophosphamide (p = 0.035) or bortezomib (p = 0.0001) alone. Our data suggest that this novel dual agonist of TLR7 and TLR8 could enhance the antitumor activity of rituximab as well as other standard agents in the treatment of non-Hodgkins lymphoma and other hematological malignancies. Based on these data we have selected the dual agonist IMO-4200 as lead candidate for clinical development. Disclosures:No relevant conflicts of interest to declare.

Preclinical Antilymphoma Activity of a Humanized Anti-CD40 Monoclonal Antibody, SGN-40

SGN-40 is a humanized IgG1 antihuman CD40 that is currently in a phase I clinical trial for the treatment of multiple myeloma. As surface CD40 expression on B-lineage cells is maintained from pro-B cells to plasma cells, SGN-40 may be applicable to treatment of other B-cell neoplasias, including non-Hodgkin's lymphoma. In this study, we examined potential in vitro and in vivo anti-B-lineage lymphoma activity of SGN-40. Recombinant SGN-40 was expressed and purified from Chinese hamster ovary cells and characterized based on binding affinity, specificity, and normal B-cell stimulation. The ability of SGN-40 to target neoplastic B cells was examined in vitro by proliferation inhibition, cytotoxicity, and antibody-dependent cell cytotoxicity assays and in vivo by human lymphoma xenograft models. Recombinant SGN-40 showed high affinity, Kd of approximately 1 nmol/L, and specific binding to CD40. Whereas SGN-40 was a weak agonist in stimulating normal B-cell proliferation in the absence of IL-4 and CD40L, it delivered potent proliferation inhibitory and apoptotic signals to, and mediated antibody-dependent cytotoxicity against, a panel of high-grade B-lymphoma lines. These in vitro antilymphoma effects were extended to disseminated and s.c. xenograft CD40 tumor models. In these xenograft models, the antitumor activity of SGN-40 was comparable with that of rituximab. The preclinical in vitro and in vivo antilymphoma activity of SGN-40 observed in this study provides a rationale for the clinical testing of SGN-40 in the treatment of CD40+ B-lineage lymphomas.

Increased parasiticide activity against Trichinella spiralis newborn larvae during pregnancy.

To evaluate whether pregnancy has a synergetic effect on the host's immune response against Trichinella spiralis infection, immunological and parasitological parameters relating to the infection were assessed in pregnant rats and compared to those observed in virgin infected rats. The muscle parasite load was lower in pregnant infected rats but no differences were found in the intestinal worm burdens or the fecundity of female worms. The ability of sera to mediate death in newborn larvae (NBL) in an antibody-dependent cell cytotoxicity assay was higher for pregnant rats, even in the absence of specific anti-NBL antibodies. High levels of total and anti-NBL IgE were found in both groups, however, these levels were higher in the group of pregnant infected animals. No differences were found in anti-NBL IgGAM titers, nevertheless in some pregnant infected rats these antibodies were found earlier. No differences were found in peritoneal or blood eosinophil counts. Offspring born to infected dams were found to be infected. The results obtained in this model demonstrate that during pregnancy there is an enhanced helminthotoxic effect towards the NBL. Despite this immunoactivation, vertical transmission of the parasite is possible.

Interleukin-2 enhances the natural killer cell response to Herceptin-coated Her2/neu-positive breast cancer cells.

The Her2/neu (c-erbB-2) oncogene encodes a 185-kDa protein tyrosine kinase which is overexpressed in 20% of breast adenocarcinomas and is recognized by a humanized anti-Her2/neu monoclonal antibody (mAb) (rhu4D5 or Herceptin). Natural killer (NK) cells are capable of mediating antibody-dependent cell cytotoxicity (ADCC) against antibody-coated targets via their expression of a low-affinity receptor for IgG (FcgammaRIII or CD16). NK cells can be expanded in cancer patients via the administration of low-dose interleukin-2 (IL-2) and become potent cytotoxic effectors following exposure to high doses of IL-2. We tested IL-2-activated NK cells against Her2/neu+ (MCF-7Her2/neu) and Her2/neu- (MDA-468) breast cancer cell lines in a 4-h 51Cr-release cytotoxicity assay in the presence or absence of rhu4D5 mAb (effector : target ratio = 10 : 1). Specific lysis of rhu4D5-coated MCF-7Her2/neu and MDA-468 target cells by IL-2-activated NK cells was 35% and 3%, respectively (p < 0.05). Lysis was less than 5% when targets were treated with either the non-humanized mu4D5 mAb or control huIgG. Lysis of rhu4D5-coated MCF-7Her2/neu cells was inhibited by 80 % when NK cells were pre-treated with an anti-Fc receptor antibody prior to use in the cytotoxicity assay. Enhanced ADCC of MCF-7Her2/neu target cells was seen when the effector cells consisted of mononuclear cells obtained from a patient demonstrating significant expansion of NK cells secondary to therapy with low-dose IL-2. Serum from patients receiving infusions of rhu4D5 mAb could substitute for exogenous antibody in the ADCC assay. NK cells activated by rhu4D5-coated tumor cells in the presence of IL-2 also produced large amounts of IFN-gamma with concomitant up-regulation of cell-surface activation markers CD25 and CD69. These results lend support to the concurrent use of rhu4D5 mAb and IL-2 therapy in patients with cancers that express the Her2/neu oncogene.

Cytotoxicity-blocking antibodies in human chronic trichinellosis

Antisurface newborn larva (NBL) antibodies (Abs) were found in sera from individuals chronically infected with Trichinella spiralis. These Abs were incapable of inducing NBL death by activation of normal human leukocytes of peripheral blood as determined by in vitro assays of antibody-dependent cell cytotoxicity (ADCC). Besides, such sera blocked the cytotoxic reaction mediated by Abs produced a few weeks after infection. The blocking activity could not be attributed to any particular isotype by the indirect immunofluorescence technique. Purified antisurface NBL Abs obtained from sera from chronically infected patients recognized antigens of muscle-larva excretory-secretory products (ML-ESP) in an enzyme-linked immunosorbent assay (ELISA) and an immunoelectrotransfer blot assay. Likewise, as did chronic sera, a monoclonal Ab raised against ML-ESP blocked NBL death in ADCC assays. These results suggest that during the course of an infection by T. spiralis, Abs related to ML-ESP provide an immunoevasive mechanism for avoidance by NBL of an important anti-NBL host effector mechanism.

Generation of phagocytic MAK and MAC-DC for therapeutic use: Characterization and in vitro functional properties

Phagocytic cells with macrophage or dendritic cell phenotype, able to capture and ingest tumor cells, were derived in large numbers from peripheral blood mononuclear cells using two different activation procedures. Peripheral blood mononuclear cells were stimulated in nonadherent conditions in the presence of human AB serum with either granulocyte-macrophage colony-stimulating factor and dihydroxy-vitamin D 3 for 7 days and with interferon-gamma for the last 18 hours to obtain activated macrophages (MAK) or with granulocyte-macrophage colony-stimulating factor and interleukin-13 for 7 days (with fresh interleukin-13 added on day 4) to obtain macrophage-dendritic cells (MAC-DC). A strong ability of MAC-DC to phagocytose yeasts was observed, in contrast to a low–intermediate phagocytosis capacity by MAK. Both CD14 + FCγR + (FcγRI/CD64, FcγRII/CD32, FcγRIII/CD16) MAK and CD1a + /CD86 + , CD14 − MAC-DC were able to phagocytose whole tumor cells. However, only MAK phagocytosis was enhanced by FcγR engagement. MAK but not MAC-DC could lyse tumor cell in antibody-dependent cell cytotoxicity assays, via FcγRI. Thus, MAK as well as MAC-DC may represent valuable tools for different in vivo therapy strategies that do or do not include the use of monoclonal antibodies.

Effect of tunicamycin and monensin on biosynthesis, transport, and maturation of bovine herpesvirus type-1 glycoproteins.

The effect of tunicamycin and monensin on the biosynthesis, intracellular transport, and maturation of bovine herpesvirus type-1 (BHV-1) glycoproteins was examined. Tunicamycin completely inhibited the production of infectious virus particles and significantly reduced the incorporation of [3H]glucosamine into viral glycoproteins. In the presence of monensin, reduced amounts of infectious virus particles were produced, which was mainly due to inhibition of virus release, rather than virus production. Monensin only slightly inhibited viral glycoprotein synthesis. The effects of these compounds on infectivity indicated that glycosylation is required for the production of infectious virus, though complete processing of the glycoproteins is not essential. In addition, egress of the virions from infected cells probably requires a functional Golgi complex. In the presence of tunicamycin or monensin various degrees of glycosylation of the major glycoproteins occurred, consequently their rates of migration differed from that of the normal glycoproteins. Tunicamycin completely blocked glycosylation of GVP 6/11a/16 and GVP 7. In contrast, GVP 3/9 and GVP 11b were partially glycosyaated in the presence of tunicamycin. These results indicated that GVP 6/11a/16 and GVP 7 are N-linked glycoproteins, but GVP 3/9 and GVP 11b contain both N- and O-linked oligosaccharide side chains. Tunicamycin blocked the transport of all viral glycoproteins to the cell surface, suggesting that glycosylation is required for this process. In the presence of monensin, the viral glycoproteins were transported and expressed on the cell surface indicating that transport does not require complete processing of the glycoproteins and may occur via a Golgi-independent pathway. In addition, monensin-treated BHV-1 infected cells could act as target cells in an antibody-dependent cell cytotoxicity assay. Thus, complete glycosylation may not be essential for maintenance of antigenicity and participation in immune destruction.

Antibody-dependent cell cytotoxicity in chemical-induced rat pancreas and colon cancer

Antibody-dependent cell cytotoxicity (ADCC) was measured in rats having 1,2-dimenthylhydrazine (DMH)-induced colon and 7,12-dimethylbenz[ a]anthracene (DMBA)-induced pancreas adenocarcinomas. The ADCC assay consisted of determining the levels of cytotoxicity induced in noncommitted peripheral blood lymphoid cells (PBLC) by the serum of its components as measured by the increased release of radioiodinated peripheral and integral membrane proteins from injured and killed X-ray-induced rat small bowel adenocarcinoma target cells. The serum component responsible for the ADCC appeared to be an immunoglobulin of the IgG class based upon solubility in ammonium sulfate, molecular weight, inactivation by pepsin and reactivity with Protein A. These immune responses were specific for entodermal tissue since serum obtained from those rats having ectodermal (spontaneous-occurring mammary and transplanted prostate) as well as mesodermal (asbestos-induced) lesions did not demonstrate ADCC in the assay. This serum which was lytic in the presence of the PBLC obtained from the rats having the chemically induced lesions was found not to be cytotoxic in itself to the cultured cells of these tumors or in the presence of PBLC to normal kidney epithelial or skin fibroblast cells. The findings suggest that identical or common antigens do exist in cancer cells arising in entodermally derived tissue that are induced by ionizing radiation, and aliphatic and polyaromatic carcinogens which can then stimulate a common antitumor immunity. These results support the concept that measurement of such immune responsiveness will permit sensitive and specific in vivo determination of those environmental substances which induce cancer cells.