Antibody-dependent Cellular Cytotoxicity Effector Cells Research Articles

High-Content Screening Assay for the Identification of Antibody-Dependent Cellular Cytotoxicity Modifying Compounds.

Immunotherapy with antigen-specific antibodies or immune checkpoint inhibitors has revolutionized the therapy of breast cancer. Breast cancer cells expressing the epidermal growth factor receptor HER2 can be targeted by the anti-HER-2 antibody trastuzumab. Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism implicated in the antitumor action of HER-2. Trastuzumab bound to cancer cells can be recognized by the Fc receptors of ADCC effector cells (e.g., natural killer (NK) cells, macrophages, and granulocytes), triggering the cytotoxic activity of these immune cells leading to cancer cell death. We set out to develop an image-based assay for the quantification of ADCC to identify novel ADCC modulator compounds by high-content screening. In the assay, HER2 overexpressing JIMT-1 breast cancer cells are co-cultured with NK-92 cells in the presence of trastuzumab, and target cell death is quantified by automated microscopy and quantitative image analysis. Target cells are distinguished from effector cells based on their EGFP fluorescence. We show how compound libraries can be tested in the assay to identify ADCC modulator drugs. For this purpose, a compound library test plate was set up using randomly selected fine chemicals off the lab shelf. Three microtubule destabilizing compounds (colchicine, vincristine, podophyllotoxin) expected to interfere with NK cell migration and degranulation were also included in the test library. The test screen identified all three positive control compounds as hits proving the suitability of the method to identify ADCC-modifying drugs in a chemical library. With this assay, compound library screens can be performed to identify ADCC-enhancing compounds that could be used as adjuvant therapeutic agents for the treatment of patients receiving anticancer immunotherapies. In addition, the method can also be used to identify any undesirable ADCC-inhibiting side effects of therapeutic drugs taken by cancer patients for different indications.

Determining ADCC Activity of Antibody-Based Therapeutic Molecules using Two Bioluminescent Reporter-Based Bioassays.

Antibody Fc effector function is one of the main mechanisms of action (MoA) for therapeutic monoclonal antibodies. Measurement of antibody-dependent cellular cytotoxicity (ADCC) is critical for understanding the Fc effector function during monoclonal antibody development. This article covers two cell-based ADCC bioassays which can quantitatively measure the antibody potency in ADCC. Basic Protocol 1 describes the ADCC reporter bioassay using engineered ADCC effector cells which measures the FcγRIIIa-mediated luciferase reporter activation upon the binding of antibody-coated target cells. Basic Protocol 2 describes the PBMC ADCC bioassay using primary peripheral blood mononuclear cells (PBMC) as effector cells and engineered HiBiT target cells in an assay that measures the release of HiBiT from target cells upon antibody-mediated target lysis. Optimization of several key assay parameters including cell handling, effector:target (E:T) ratios, assay plate, and plate reader requirement, and how these parameters impact assay performance are discussed. © 2021 Promega Corporation. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: ADCC reporter bioassay using engineered ADCC bioassay effector cells Basic Protocol 2: PBMC ADCC bioassay using primary PBMC and engineered HiBiT target cells.

Abstract 506: A homogenous PBMC ADCC bioassay enables bridging studies with ADCC reporter bioassays in immunotherapy monoclonal antibody development

Abstract Measurement of antibody-dependent cellular cytotoxicity (ADCC) is critical for understanding antibody Fc effector functions during monoclonal antibody development. Classic ADCC assays measure the short-term cytotoxicity of target cells, typically pre-loaded with radioactive or fluorescent dyes, after exposure to antibody bound primary PBMCs or NK cells. These assays are widely used in antibody discovery and characterization during early drug development. However, the reproducibility of these assays is not adequate and they have major issues on biosafety or weak assay sensitivity. Moreover, the use of primary effector cells makes them prone to donor variability, low-throughput and therefore not suitable for implementation in quality-control drug development. We previously developed an ADCC reporter bioassay using engineered ADCC effector cells and demonstrated its specificity and ability to measure ADCC mechanism of action. The assay is prequalified according to ICH guidelines; demonstrates precision, accuracy, linearity, and robustness; and is suitable for product release and stability studies in a quality-controlled environment. In order to enable bridging studies comparing primary PBMC-based ADCC assays and ADCC reporter bioassays, we report the development of an improved PBMC ADCC assay using ADCC-prequalified PBMCs and engineered HiBiT target cells for therapeutic monoclonal antibodies with Fc functions. When target cells expressing HaloTag-HiBiT are incubated with an antibody and PBMCs, the target cells are lysed and release HaloTag-HiBiT, which then bind to LgBiT in the detection reagent to form a functional Nano-luciferase to generate Bioluminescence signal. In addition, it can enable the quantitative measurement of antibody-mediated target cell lysis via specific HaloTag staining using a fluorescent HaloTag ligand by flow cytometry. We demonstrate that the new PBMC ADCC assay is simple, homogenous, highly sensitive, and gives a robust assay window, and is capable of measuring potency of rituximab, cetuximab or trastuzumab. Additionally, it shows antibody potency comparable with the ADCC reporter bioassay in ADCC bridging studies. Citation Format: Pete Stecha, Denise Garvin, Julia Gilden, Jun Wang, Jamison Grailer, Jim Hartnett, Gopal B. Krishnan, Frank Fan, Mei Cong, Zhijie Jey Cheng. A homogenous PBMC ADCC bioassay enables bridging studies with ADCC reporter bioassays in immunotherapy monoclonal antibody development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 506.

Balancing the CD38 Expression on Effector and Target Cells in Daratumumab-Mediated NK Cell ADCC against Multiple Myeloma.

Simple SummaryWe tracked the cytotoxic potential of NK cells towards multiple myeloma cells in daratumumab-mediated antibody-dependent cellular cytotoxicity assays. These cytotoxicity levels could be directly correlated to the expression of the target antigen (CD38) and to the percentage of fratricide between effector cells. Increasing the expression of CD38 on target cells or neutralizing CD38 on effector cells changed the equilibrium between target and effector cell lysis and promoted multiple myeloma cell death. This study highlights the importance of a balanced CD38 expression on target and effector cells and attempts to alter this balance will affect the susceptibility of MM cells towards daratumumab-mediated cellular toxicity. Multiple myeloma (MM) is an incurable cancer characterized by the proliferation and accumulation of monoclonal plasma cells in the bone marrow. The monoclonal anti-CD38 daratumumab has taken a central place in the different treatment regimens for newly diagnosed and relapsed, refractory myeloma. In this study, we correlated the NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) and potential fratricide induced by daratumumab with CD38-expression levels on both effector and target cells. We show that CD38 expression can be modulated by adding all-trans retinoic acid (ATRA) or interferon-α to MM cells to further fine-tune these effects. In addition, we observed that ADCC becomes inefficient when fratricide occurs and both ADCC and fratricide depend on the balance between CD38 expression on effector and target cells. However, the addition of adjuvants (retinoic acid or interferon-α) to myeloma cells or the inhibition of fratricide using a CD38-blocking nanobody on NK-cells can reverse this balance towards ADCC and thus promote lysis of target cells by ADCC. ATRA and interferon-α increased the CD38 expression at the surface of MM cells about three-fold and two-fold, respectively. This increase was of interest for MM cells with low CD38 expression, that became susceptible to daratumumab-mediated ADCC after preincubation. A CD38-blocking nanobody prevented the binding of daratumumab to these NK-cells and blunted the fratricidal effect on effector NK cells. In conclusion, our study highlights the importance of a balanced CD38 expression on target and effector cells and attempts to alter this balance will affect the susceptibility of MM cells towards daratumumab-mediated ADCC.

NKTR-255, a Polymer-Conjugated IL-15 Enhances Antibody-Dependent Cellular Cytotoxicity Mediated By NK Cells in a B Cell Lymphoma Model

Background IL-15 is a cytokine that activates and provides survival benefit to T and NK cells and has great potential as an immunotherapeutic agent for the treatment of cancer. Exploiting the therapeutic value of native IL-15 has been challenging due to its unfavorable pharmacokinetic properties and poor tolerability. NKTR-255 is a polymer-conjugated IL-15 designed to retain binding affinity to the IL-15 receptor alpha chain and have decreased clearance to provide a sustained pharmacodynamics response. NKTR-255 has an enhanced immunotherapeutic effect when combined with certain targeted monoclonal antibodies that mediate tumor killing by antibody dependent cellular cytotoxicity (ADCC). Here we further characterized the effect of NKTR-255 on NK cell-mediated ADCC in a B cell lymphoma model. Methods For in vitro ADCC, NKTR-255 pre-treated human NK cells (effector cells) were co-cultured with RPMI-8226 (target cells) and with daratumumab for 3 hours. The ability of NK cells to lyse target cells was evaluated by detecting 7-AAD stained RPMI-8226. In the Daudi lymphoma model, SCID or SCID beige mice were inoculated IV with 1x107 Daudi cells on Day 0. NKTR-255 (0.3 mg/kg IV) was administered on Days 14, 21 and 28 and a single dose of antibody treatment was given on Day 14 (daratumumab 0.5 mg/kg IP). Survival rate was determined by onset of hind limb paralysis or moribundity as surrogate parameters. Concurrent vs staggered regimen assessment was conducted with NKTR-255 dosed on day 14 and daratumumab on Day 14, 17, or 21. For dose examination, high (0.5 mg/kg) and low (0.05 mg/kg) doses of daratumumab were combined with low (0.03 mg/kg) and high (0.3 mg/kg) doses of NKTR-255 in a concurrent manner. Daudi cells in the bone marrow were assessed 10 days after the NKTR-255 dose by flow cytometry. Results In vitro, NKTR-255 pre-treatment of human purified NK cells enhanced daratumumab-mediated ADCC against RPMI-8226. The ability of NKTR-255 to enhance in vitro ADCC was translated into an enhanced therapeutic efficacy of daratumumab in the Daudi lymphoma model with SCID mice. NKTR-255 combined with daratumumab synergistically provided long-term survival benefit in a NKTR-255 dose-dependent manner. To confirm the contribution of NK cells as effector cells for ADCC in vivo, the therapeutic effect of the combination treatment was assessed in the tumor model in SCID beige mice, which have a selective impairment of NK cell cytotoxic function. The long-term survival benefit by the combination treatment, which was observed in SCID mice (median survival of 27 days in control and 59.5 days in treatment), was attenuated in SCID beige mice (median survival of 23 days in control and 30 days in treatment). Concurrent vs staggered dose regimens and dose ranging for NKTR-255 and daratumumab were evaluated in the Daudi model with SCID mice to understand optimal therapeutic use of NKTR-255 in future clinical studies. Tumor depletion in bone marrow was most effective in concurrent treatment of NKTR-255 and daratumumab and three days-staggered treatment (NKTR-255 first and the antibody 3 days later). In contrast, a 7-day stagger showed no benefit of the combination in tumor depletion. Combination of low dose daratumumab and high dose NKTR-255 was equally effective in tumor depletion as a combination of high dose daratumumab and low dose NKTR-255. Conclusions NKTR-255 was shown to enhance in vivo therapeutic efficacy of daratumumab, a monoclonal antibody that mediates tumor killing by ADCC. The effect was mainly mediated by NK cells. The treatment scheduling experiments demonstrated that antibody should be present during NKTR-255-driven NK cell activation/expansion phase to efficiently deplete Daudi tumor cells in bone marrow. In addition, even low dose of daratumumab treatment showed effective tumor depletion when combined with high dose of NKTR-255. This suggests that NKTR-255 has a potential to expand the therapeutic window of existing tumor-targeted antibodies to patients with, for example, low antibody concentration in target tumor site or low expression of tumor antigen. Disclosures Miyazaki: Nektar Therapeutics: Employment, Equity Ownership. Kivimäe:Nektar Therapeutics: Employment, Equity Ownership. Hennessy:Nektar Therapeutics: Employment, Equity Ownership. Pena:Nektar Therapeutics: Employment, Equity Ownership. Quach:Nektar Therapeutics: Employment, Equity Ownership. Moffet:Nektar Therapeutics: Employment, Equity Ownership. Nieves:Nektar Therapeutics: Employment, Equity Ownership. Zhang:Nektar Therapeutics: Employment, Equity Ownership. Marcondes:Nektar Therapeutics: Employment, Equity Ownership. Madakamutil:Nektar Therapeutics: Employment, Equity Ownership. Zalevsky:Nektar Therapeutics: Employment, Equity Ownership.

Functional Characterization of Gamma Delta T Cells and Allogeneic Activated NK Cells As Effector Cells for Tafasitamab (MOR208)

Introduction Tafasitamab (MOR208) is an Fc-enhanced, humanized, monoclonal antibody that binds to the human B cell surface antigen CD19. CD19 is broadly and homogeneously expressed across different B cell malignancies, including diffuse large B cell lymphoma (DLBCL), and amplifies B cell receptor signaling and induces tumor cell proliferation. Tafasitamab is currently in clinical development in patients with relapsed or refractory DLBCL in combination with the immunomodulatory drug lenalidomide (L-MIND study) and the chemotherapeutic agent bendamustine (B-MIND study). The modes of action of tafasitamab comprise antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and direct cytotoxicity (apoptosis). Tafasitamab carries two amino acid exchanges in the Fc region to increase its affinity to Fcγ receptors, including FcγRIIIa. FcγRIIIa plays a key role in mediating ADCC and is expressed on the surface of natural killer (NK) cells, as well as the majority of γδ T cells. MG4101 (a novel therapeutic agent consisting of cryopreserved, ex vivo-expanded, highly activated NK cells) has demonstrated potent anticancer activity in preclinical in vitro and in vivo studies. Currently, MG4101 is in clinical development in patients with malignant lymphoma and advanced solid tumors. Here, we have characterized two FcγRIIIa receptor-expressing cell types, γδ T cells and NK cells (MG4101), as effector cells for tafasitamab in vitro and explored the concept of supplementing MG4101 during tafasitamab therapy using disseminated in vivo models of non-Hodgkin's lymphoma. Methods γδ T cells (CD3+/γδ T cell receptor+) were derived from different donors by stimulation of peripheral blood mononuclear cells with zoledronate/IL-2 for 9-10 days. These were applied as effector cells in in vitro ADCC assays with tafasitamab in Mino and Jeko-1 mantle cell lymphoma (MCL) cell lines, as well as primary patient-derived chronic lymphocytic lymphoma (CLL) and MCL cells. Further, effector cell activity of MG4101 was assessed using tafasitamab-mediated ADCC assays in Raji and Ramos Burkitt's lymphoma cells. The concept of combining tafasitamab with allogeneic effector cell therapy in vivo was studied in two therapeutic survival models of disseminated lymphoma in SCID mice. In the Raji model, tafasitamab (0.05 µg/mouse) was given on Day 1 after intravenous (IV) tumor inoculation, while MG4101 (2x107 cells/mouse) was given on Days 1, 3, 6, 8 and 10. In the Ramos model, tafasitamab (10 mg/kg) and MG4101 (2x107 cells/mouse) were applied twice weekly for 3 weeks starting on Days 3 and 4, respectively, after IV tumor inoculation. Results Tafasitamab in combination with γδ T cells showed distinctly increased ADCC in Mino and Jeko-1 target cells in vitro, compared with a negative control IgG1 antibody. ADCC assays with patient-derived CLL and MCL target cells confirmed tafasitamab-mediated cytotoxic activity and demonstrated a clear enhancement in activity compared with the non-Fc-enhanced version of tafasitamab that was unable to induce substantial cytotoxicity. In vitro ADCC assays with tafasitamab and MG4101 on Raji and Ramos cell lines confirmed potent effector cell activity of the ex vivo-expanded, cryopreserved, allogeneic NK cells. In the disseminated Raji survival model, combination therapy with a single low dose of tafasitamab (0.05 µg) and MG4101 resulted in a distinct increase in survival of the mice with an increased life span (ILS) of 100% compared with monotherapy (ILS of 57% for tafasitamab and 50% for MG4101). Of note, the combination demonstrated a substantial and more than additive enhancement in survival in the more therapeutic Ramos survival model (Figure 1). Combination therapy with tafasitamab (10 mg/kg) and MG4101 NK cells resulted in superior antitumor activity (ILS of 103%) compared with either tafasitamab monotherapy (ILS of 49%) or MG4101 alone (ILS of 25%). Conclusions FcγRIIIa-expressing immune cell types, including NK cells and γδ T cells, are potent effector cells for tafasitamab-mediated ADCC. Combination therapy with tafasitamab and allogeneic MG4101 NK cells in vivo demonstrated a more than additive survival benefit compared with tafasitamab or MG4101 monotherapy in a disseminated therapeutic lymphoma model. Combination of tafasitamab supplemented with immune effector cells could represent a promising new approach for lymphoma therapy. Disclosures Her: GC LabCell: Employment, Patents & Royalties. Cho:GC LabCell: Employment, Patents & Royalties. Hwang:GC LabCell: Employment, Equity Ownership, Patents & Royalties. Boxhammer:MorphoSys AG: Employment, Patents & Royalties. Steidl:MorphoSys AG: Employment. Patra:MorphoSys AG: Employment. Schanzer:MorphoSys AG: Employment. Endell:MorphoSys AG: Employment, Patents & Royalties.

IVIG induces apoptotic cell death in CD56dim NK cells resulting in inhibition of ADCC effector activity of human PBMC

The mechanism of the efficacy of Intravenous immunoglobulins (IVIG) in autoimmune and inflammatory diseases is not well understood. This study aimed at understanding mechanisms of IVIG-mediated suppression of effector cell activities of peripheral blood mononuclear cells (PBMC) in antibody-dependent cellular cytotoxicity (ADCC). We were particularly interested in CD56dim NK cells, the main ADCC effector cells in PBMC. Exposure of PBMC to IVIG for at least 48 h induced a caspase-3-dependent apoptotic cell death of CD56dim NK cells without affecting CD56bright NK cells. Induction of apoptosis in CD56dim NK cells and concomitant suppression of ADCC effector activities of PBMC was associated with the monomer fraction of IVIG. Moreover, it was independent of IgG sialyation, did not depend on engagement of FcγRIII and could not be mimicked by IVIG (Fab’)2 or IVIG Fc preparations. The described effect could contribute to the reduction of peripheral NK cells observed during IVIG therapy in patients.

Development of Engineered T Cells Expressing a Chimeric CD16-CD3ζ Receptor to Improve the Clinical Efficacy of Mogamulizumab Therapy Against Adult T-Cell Leukemia.

Mogamulizumab (Mog), a humanized anti-CC chemokine receptor 4 (CCR4) mAb that mediates antibody-dependent cellular cytotoxicity (ADCC) using FcγR IIIa (CD16)-expressing effector cells, has recently been approved for treatment of CCR4-positive adult T-cell leukemia (ATL) in Japan. However, Mog failure has sometimes been observed in patients who have accompanying chemotherapy-associated lymphocytopenia. In this study, we examined whether adoptive transfer of artificial ADCC effector cells combined with Mog would overcome this drawback. We lentivirally gene-modified peripheral blood T cells from healthy volunteers and ATL patients expressing the affinity-increased chimeric CD16-CD3ζ receptor (cCD16ζ-T cells). Subsequently, we examined the ADCC effect mediated by those cCD16ζ-T cells in the presence of Mog against ATL tumor cells both in vitro and in vivo cCD16ζ-T cells derived from healthy donors killed in vitro Mog-opsonized ATL cell line cells (n = 7) and primary ATL cells (n = 4) depending on both the number of effector cells and the dose of the antibody. cCD16ζ-T cells generated from ATL patients (n = 3) also exerted cytocidal activity in vitro against Mog-opsonized autologous ATL cells. Using both intravenously disseminated model (n = 5) and subcutaneously inoculated model (n = 4), coadministration of Mog and human cCD16ζ-T cells successfully suppressed tumor growth in xenografted immunodeficient mice, and significantly prolonged their survival (P < 0.01 and P = 0.02, respectively). These data strongly suggest clinical feasibility of the novel combined adoptive immunotherapy using cCD16ζ-T cells and Mog for treatment of aggressive ATL, particularly in patients who are ineligible for allogeneic hematopoietic stem cell transplantation. Clin Cancer Res; 22(17); 4405-16. ©2016 AACR.

NKG2D Acts as a Co-Receptor for Natural Killer Cell-Mediated Anti-HIV-1 Antibody-Dependent Cellular Cytotoxicity.

The utility of antibody-dependent cellular cytotoxicity (ADCC) for eliminating HIV-1-infected cells is of much interest for the design of both prophylactic vaccines for HIV-1 prevention and therapeutics to eliminate latently infected cells following reactivation. Significant research has been conducted to understand the antibody specificities involved in anti-HIV-1 ADCC responses. Perhaps equally important as the identity of the antibodies mediating these responses are factors regulating the ability of ADCC effector cells, in particular, natural killer (NK) cells, to respond to antibody-coated target cells. Indeed, a plethora of activating and inhibitory receptors expressed on the surface of NK cells might act in conjunction with CD16 to influence ADCC. As the expression of NKG2D and its ligands has been linked to HIV-1 disease progression, we evaluated if signals through NKG2D were involved in anti-HIV-1 ADCC. Utilizing assays measuring cytolysis, we provide the first data implicating NKG2D in antibody-dependent NK cell responses against a target cell line either pulsed with gp120 or infected with HIV-1. These observations are highly significant for understanding antibody-dependent NK cell responses against HIV-1 and might be useful for optimizing prophylactics and therapeutics aiming to utilize antibodies and optimally functional NK cells to control HIV-1.

Concomitant Administration of Gene-Modified T Cells Expressing a Chimeric CD16-CD3ζ Receptor with Mogamulizmab Synergistically Suppresses Adult T Cell Leukemia Cells in Vivo

[Background and purpose] Mogamulizumab, a newly developed monoclonal antibody (mAb) targeting the receptor for C-C chemokine 4 (CCR4) has initially demonstrated a promising clinical outcome for the treatment of therapy-resistant Adult T cell leukemia (ATL) of which tumor cells broadly express CCR4. However, in practice, we sometimes encounter unsuccessful cases of ATL treated with mogmulizumab, especially in the setting of combination therapy with multiple anticancer agents. In those cases, ATL tumor cells were still positive for CCR4, but the chemotherapy-induced lymphocytopenia was reproducibly noticeable. Considering the pharmacologic action of mogamulizumab, the defucosylation-enhanced antibody-dependent cellular cytotoxicity (ADCC) exerted by FcγRIIIa (CD16) expressing effector cells in vivo, as observed for Natural Killer (NK) cells, we hypothesized that adoptive transfer of ADCC effector cells might be able to regain the anti-ATL efficacy of mogamulizumab. Accordingly, we examined in vivo the feasibility of T cells gene-modified to express a newly generated chimeric CD16-CD3ζ receptor as a transferable alternative effector cell for mogamulizumab.[Methods] A novel affinity-matured chimeric CD16 with a 158V/V-CD3ζ (cCD16ζ ) gene construct was lentivirally introduced into CD3+ T cells from both healthy individuals (n=4) and patients with adult T cell leukemia (ATL) (n=3) (cCD16ζ-T cells). Using ATL or HTLV-1 infected cell lines variably expressing CCR4 on the cell surface (n=7), and primary ATL tumor cells (n=3), in the context of ADCC activity, functional properties of cCD16ζ-T cells were extensively examined both in vitro and in vivo, compared with those of NK cells from healthy donors (n=3). Next, we examined the in vivo therapeutic efficacy of concomitantly infused cCD16ζ-T cells with mogamulizumab via tail vein using a xenografted mouse model. Finally, we examined the feasibility of double gene-modified T cells to express human telomerase reverse transcriptase (hTERT)-specific TCR and cCD16ζ receptor, because we recently demonstrated that HLA-A*24:02-restricted and hTERT461-469 (VYGFVRACL)-specific TCR gene-modified CD8+ T cells displayed the cytocidal activity against ATL tumor cells (Blood, 2014).[Results] cCD16ζ-T cells were readily expandable in ex vivo culture using anti-CD2/CD3/CD28 beads and recombinant human (rh)IL-2, and they successfully displayed ADCC-mediated tumoricidal activity against CCR4+ MT-4 and ATN-1 (ATL cell lines) cells, but not CCR4- K562 cells with mogamulizumab, in a effector cell number and an antibody dose dependent manner. Pharmacological dose of 0.1μg/ml mogamulizumab could bind to ATL cell line cells expressing variable extent of CCR4 ranging from MFI 1.01 for HUT102 cell line to 12.3 for MT-2 (0.57 for K562 as a negative control), and could mediate the similar degree of ADCC activity exerted by cCD16ζ-T cells. The magnitude of ADCC activity mediated by cCD16ζ-T cells against opsonized ATN-1 with mogamulizumab was almost similar to that by activated NK cells using rhIL-2. This cytotoxicity was inhibited by anti-CD16 mAb. During ADCC, ligation of opsonized cancer cells with mogamulizumab to cCD16ζ receptor stimulated cCD16ζ-T cells to release toxic granules shown by CD107a expression. cCD16ζ-T cells generated from patients with ATL (n=3) successfully displayed ADCC activity against autologous tumor cells in vitro. Human cCD16ζ-T cells infused concomitantly with mogamulizumab synergistically inhibited the growth of disseminated luciferase gene-modified ATN-1 cells in immunodeficient mice, demonstrated using an in vivo bioluminescence assay. Additionally, this tumor suppressive effect contributed to the longer survival of treated mice. Finally, the double-gene modified CD3+ T cells could successfully recognize ATN-1 through both the mogamulizumab-opsonized CCR4 and hTERT epitope/HLA-A24*02 complex on the cell surface. DisclosuresOkamoto:CDM Center, Takara Bio Inc.: Employment. Mineno:Takara Bio Inc.: Employment. Shiku:Takara Bio Inc.: Research Funding.

HIV-Specific Antibody Immunity Mediated Through NK Cells and Monocytes

The partial success of the RV144 trial re-energized the field of HIV vaccine research, which had stalled after vaccines based on neutralizing antibody and cytotoxic T cells had failed to induce protection. A large post-vaccine research effort has focused attention on the role of non-neutralizing antibodies in the protection afforded by the RV144 vaccine. These binding antibodies can initiate immune responses such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) and combine elements of the adaptive and innate immune system in the form of antibodies and effector cells (including NK cells, monocytes and granulocytes). A complex interplay exists between the variable portion of the binding antibody and its HIV antigen target on one hand and the constant region of the antibody and the Fcγ-receptor of the effector cell on the other hand. Technical advances have revolutionized the abilities of scientist to detect the targets of non-neutralizing antibodies, including both envelope and non-envelope epitopes, and their role in forcing escape. Our understanding of the antibody characteristics (including IgG subclasses and Fc glycan profile) is providing valuable insights into their optimal structure and function. We expand on critical research on ADCC effector cells, particularly education of NK cells. We introduce the concept of HIV antibodydependent trogocytosis by monocytes as a potentially important aspect of HIV immunity. In summary, this review highlights recent advances in HIV-specific antibody immunity mediated through NK cells and monocytes.

Natural Killer cells present in gut mucosa as potential ADCC effector cells

Background Data from the RV144 HIV vaccine trial showed a moderate protection from HIV infection in the absence of neutralizing antibodies or CTL activity. In contrast, all vaccinees mounted robust HIV-specific binding antibodies, that may have provided some level of protection through their capacity to recruit antibody dependent cellular cytotoxicity (ADCC). The capacity of an antibody to recruit ADCC relies on its ability to interact with the Fc-receptor, FCgRIII (CD16), expressed on Natural Killer (NK) cells. However, little is known about innate immune effector cells present within mucosa, and whether they have the capacity to be harnessed by ADCC inducing antibodies should they be elicited by a vaccine. Here we hypothesized that abundant numbers of CD16+ NK cells line the gut mucosa, providing a robust effector arm that could be harnessed by ADCC inducing antibodies. Methods The frequency and function of CD16+ cells in the colon was assessed by flow cytometry following enzymatic digestion of intestinal resections from HIV-uninfected subjects.

Murine Antithymocyte Globulin T-Cell Depletion Is Mediated Predominantly by Macrophages, but the Fas/FasL Pathway Selectively Targets Regulatory T Cells

Thymoglobulin is a T-cell-depleting polyclonal rabbit anti-human thymocyte antibody used clinically for immunosuppression in solid organ and hematopoietic stem-cell transplantation. By using a surrogate rabbit anti-mouse thymocyte globulin (mATG), we previously demonstrated that murine regulatory and memory T cells are preferentially spared from mATG depletion in vivo. The current studies were designed to determine whether different effector mechanisms are involved in differential depletion of T-cell subsets by mATG. Complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity (ADCC), and apoptotic mechanisms of depletion by mATG were evaluated in vitro and in vivo. In vitro, there was evidence of differential susceptibility of T-cell subsets by different effector mechanisms where naïve and CD4 effector memory T cells show reduced susceptibility to apoptosis, whereas regulatory T cells are less susceptible to mATG-mediated complement-dependent cytotoxicity and ADCC. However, mATG treatment of mice depleted of ADCC effector cell types (neutrophils, natural killer cells, or macrophages) or deficient in complement C5 or Fas demonstrated that mATG depletion of all T-cell subsets is mediated primarily by macrophages and that the role of neutrophils, natural killer cells, and complement is minimal in vivo. Interestingly, the Fas/FasL pathway does play a role in regulatory T-cell depletion, which is likely a result of increased basal expression of Fas on these cells. These data suggest that macrophages deplete most T cells by mATG in mice, but regulatory T cells are also uniquely susceptible to mATG-mediated Fas-dependent depletion.

A dimeric form of the HIV-1 antibody 2G12 elicits potent antibody-dependent cellular cytotoxicity.

Increasing data support a role for antibody-dependent cellular cytotoxicity (ADCC) in controlling HIV-1 infection. We recently isolated a naturally occurring dimeric form of the anti-HIV-1 antibody 2G12 and found it to be significantly more potent than 2G12 monomer in neutralizing primary virus strains. However, given the unusual structure of dimeric 2G12 with two Fc regions, it was not clear whether 2G12 dimer could bind to the CD16 Fc receptor on ADCC effector cells or trigger ADCC. Here we compared the in-vitro ADCC activities of 2G12 monomer and dimer and investigated the effects of including ADCC-enhancing mutations in both forms of 2G12. An in-vitro ADCC assay using target cells stably expressing gp160 was developed to evaluate the activities of 2G12 monomer and dimer with and without ADCC-enhancing mutations that increase the CD16-binding affinity of the 2G12 Fc region. Both 2G12 monomer and 2G12 dimer elicited ADCC, although the dimer showed increased potency [lower half-maximal concentration (EC(50))] in triggering ADCC, thus confirming its ability to bind CD16 and trigger ADCC. The ADCC-enhancing mutations improved the ADCC activity of 2G12 monomer more than 2G12 dimer such that their EC(50) values were nearly equal. However, no increase in nonspecific ADCC activity was observed using 2G12 IgGs with these mutations. Given the likelihood that ADCC plays a role in protecting against initial infection and/or controlling chronic infection, these data suggest 2G12 dimers and/or addition of ADCC-enhancing mutations could augment the prophylactic and/or therapeutic potential of 2G12.

Defucosylated Anti-CCR4 Monoclonal Antibody Exerts Potent ADCC against Primary ATLL Cells Mediated by Autologous Human Immune Cells in NOD/Shi-Scid, IL-2Rγnull Mice In Vivo.

Abstract 726There are no suitable small animal models to evaluate human antibody-dependent cellular cytotoxicity (ADCC) in vivo, due to species incompatibilities, and it is a current crucial problem in the field of human ADCC research. To overcome this, we have established “humanized mice,” in which human immune cells from healthy individuals function as ADCC effector cells against allogeneic tumor cell lines, using NOD/Shi-scid, IL-2Rγnull (NOG) mice as recipients. In this model, the chimeric anti-CCR4 monoclonal antibody (mAb), KM2760, the Fc region of which is defucosylated to highly enhance ADCC, showed potent antitumor activity by human ADCC against CCR4 expressing tumor cell lines. In addition, KM2760 significantly increased the number of tumor-infiltrating CD56-positive NK cells which mediate ADCC, and reduced the number of tumor-infiltrating FOXP3-positive regulatory T (Treg) cells in the tumor bearing humanized mice. These observations indicate that KM2760 could be an ideal treatment modality for many different cancers, not only to directly kill CCR4-expressing tumor cells, but also to overcome the suppressive effect of Treg cells on the host immune response to tumor cells. Using this humanized mouse model, we now have the opportunity to perform more appropriate preclinical evaluation of many types of mAb based immunotherapy, although in the initial study, we could not completely exclude nonspecific allogeneic immune responses because target and effector cells were obtained from different individuals. In addition, susceptibility to immunotherapy is likely to be different in established cell lines and primary tumor cells isolated directly ex vivo from patients, with the latter certainly being more relevant for evaluation of immunotherapeutic agents. To overcome the subsequent problems, we have established a primary human tumor bearing NOG mouse model, in which autologous human immune cells are engrafted and mediate ADCC but in which endogenous murine cells are unable to mediate ADCC. In the present study, we used NOG mice bearing primary adult T-cell leukemia/lymphoma (ATLL) cells. We report significant antitumor activity in vivo associated with robust ADCC mediated by autologous effector cells from the same patients. The present study is the first to report a mouse model in which a potent antitumor effect of the therapeutic mAb against primary tumor cells is mediated by autologous human immune cells. Human autologous ADCC in mice in vivo was confirmed by the depletion of human immune cells before ATLL PBMC inoculation. In addition, NOG mice bearing primary ATLL cells presented features identical with patients with ATLL. In conclusion, this approach makes it possible to model the human immune system active in mAb based immunotherapy in vivo, and thus to perform more appropriate preclinical evaluations of novel therapeutic mAb. Furthermore, the potent ADCC mediated by defucosylated anti-CCR4 mAb, observed here in vivo in humanized mice, will be exploited in clinical trials in the near future. Disclosures:No relevant conflicts of interest to declare.

Variable Contribution of Monoclonal Antibodies to ADCC in patients with chronic lymphocytic leukemia

Monoclonal antibodies (mAbs) are increasingly used in treatment protocols for chronic lymphocytic leukemia (CLL). Here we determined (i) the extent of antibody-dependent cellular cytotoxicity (ADCC) of four different mAbs against primary CLL cells, (ii) whether ADCC correlates with antigen density on CLL cells, and (iii) whether allogeneic natural killer (NK) cells display superior ADCC than autologous. Effector cells for ADCC were (i) NK-92 cells not expressing FcR, (ii) NK-92 cells transfected with a high-affinity Fc receptor, (iii) autologous NK cells from patients with CLL, (iv) allogeneic NK cells. Results suggest that ADCC contributes to killing of CLL cells by anti-CD20 antibodies (rituximab and veltuzumab), whereas mAbs against CD22 (epratuzumab) and CD23 (lumiliximab) showed minimal ADCC. The magnitude of anti-CD20 mediated ADCC did not correlate with antigen density of CD20. ADCC was not influenced by the FcR genotype expressed by autologous NK cells. Allogeneic NK cells were superior to autologous NK cells in killing primary CLL cells.

Chimeric and humanized anti-HM1.24 antibodies mediate antibody-dependent cellular cytotoxicity against lung cancer cells

HM1.24 antigen (CD317) was originally identified as a cell surface protein that is preferentially overexpressed on multiple myeloma cells. Immunotherapy using anti-HM1.24 antibody has been performed in patients with multiple myeloma as a phase I study. The aim of this study was to evaluate the anti-tumor activity of mouse-human chimeric and humanized anti-HM1.24 monoclonal antibodies (mAbs) against lung cancer cells in vitro. Human peripheral blood lymphocytes and monocytes separated from mononuclear cells (PBMCs) were used as effector cells. Antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) of chimeric and humanized anti-HM1.24 mAbs against lung cancer cells were determined by chromium-release assay. In some experiments, target or effector cells were pretreated with various cytokines. Chimeric and humanized anti-HM1.24 mAbs effectively induced ADCC against lung cancer cells mediated more efficiently by lymphocytes than monocytes. The cytotoxic activity correlated with the level of HM1.24 expression on lung cancer cells. Natural killer cells were identified as the major effector cells in ADCC mediated by the anti-HM1.24 mAb. The treatment of lymphocytes or monocytes with IL-2, IL-12, IL-15, M-CSF, or IFN-gamma significantly increased the ADCC activity. Moreover, the culture of lung cancer cells with IFN-beta or IFN-gamma augmented their susceptibility to ADCC and CDC. PBMCs from patients with lung cancer induced a level of ADCC comparable to that induced by PBMCs from healthy donors. Chimeric or humanized anti-HM1.24 mAbs have potential as a new therapeutic tool in lung cancer, and in combination with interleukins and interferons, could be useful for enhancing ADCC.

Variable Contribution of Different Monocloncal Antibodies to NK Cell-Mediated ADCC Against Primary CLL Cells.

Chronic Lymphocytic Leukemia (CLL) is characterized by the expression of the B-cell antigens CD19, 20 and 22, along with CD5 and CD23. These antigens make the malignant cells an ideal target for monoclonal antibody (mAb) therapy. Although the mechanism of action of mAbs is complex and not fully understood, one well-described action is antibody-dependent cellular cytotoxicity (ADCC). Binding of mAb to its target surface antigen initiates cytotoxicity through the interaction of the Fc portion of the mAb with the Fc receptor (FcR) on natural killer (NK) cells. This triggers release of perforin and granzymes from NK cells, and subsequent killing of the target cells. This study's objectives were to measure ADCC of 4 different mAbs against primary CLL cells, and determine if cytotoxicity is dependent on antigen density.Methods: Mononuclear cells from 16 patients with untreated CLL were separated by density gradient separation and served as targets. The antigen density for CD20, 22 and 23 of each patient sample was quantified by flow cytometry. Effector cells for ADCC were NK-92 cells that do not express FcR, and a high affinity Fc receptor-expressing NK-92 variant (NK92.26.5) that also expresses inhibitory receptors (i.e. KIR). A fluourochrome-based flow cytometric assay determined ADCC by subtracting NK-92 induced cytotoxicity from NK-92.26.5 induced killing. Monoclonal antibodies tested were Rituximab (anti-CD20, Biogen/IDEC), Veltuzumab (anti-CD20, Immunomedics), Epratuzumab (anti-CD22, Immunomedics), and Lumiliximab (anti-CD23, Biogen/IDEC).Results: Mean ADCC of the four antibodies tested against 16 primary CLL cells were: 46.5% for Rituximab; 43.4% for Veltuzumab; 5.8% for Epratuzumab; 8.8% for Lumiliximab. Cytotoxicity of NK-92 and NK-92.26.5 against CLL cells without antibody ranged from 2–6%. Mean antigen density on the 16 CLL patient specimens were: CD20: 27,900 (range: 10,000–56,100); CD22: 820 (600–1300); CD23: 9870 (2340–14,800).Conclusions: We have developed a reliable in vitro assay to measure ADCC of mAbs against CLL cells. Our results indicate that ADCC contributes to cytotoxcity of CLL in vitro, and suggest that the magnitude of ADCC depends upon the surface antigen targeted. Anti-CD20 antibodies had significantly greater ADCC than the anti-CD22 and CD23 antibodies. This pattern was consistent in all patient cells tested. A similar pattern was observed with the surface antigen density on CLL cells tested, suggesting a correlation between cell surface antigen density and ADCC. Our results also suggest that resistance of primary CLL cells toward NK-mediated killing can be overcome by monoclonal antibodies even in the presence of inhibitory KIR expression on NK cells.

FCGR3A gene polymorphisms may correlate with response to frontline R-CHOP therapy for diffuse large B-cell lymphoma

AbstractThe precise mechanism of rituximab plus cyclophosphamide/doxorubicin/vincristine/prednisone (R-CHOP) therapy in diffuse large B-cell lymphoma (DLBCL) is not fully elucidated. Besides overcoming bcl-2-mediated chemoresistance, antibody-dependent cellular cytotoxicity (ADCC), which is activated by effector cells via immunoglobulin G (IgG) fragment C receptors (FcRs), was also proposed as a mechanism of rituximab. The current study evaluated the impact of FcR polymorphism on the response to R-CHOP therapy for DLBCL with the basis that FcR polymorphism can affect rituximab's affinity for ADCC effector cells. The FCGR3A and FCGR2A gene polymorphisms were determined in DLBCL patients receiving R-CHOP (n = 113) compared with CHOP therapy (n = 85). The FCGR3A valine (V) allele was significantly correlated with a higher complete response rate to R-CHOP compared with the phenylalanine (F) allele (88% in V/V vs 79% in V/F vs 50% in F/F; P = .002), while no difference was found between FCGR2A polymorphisms. In addition, V/V allele was associated with faster achievement of response than other alleles. The impact of the FCGR3A gene polymorphism on response rate was not noted in the CHOP group. In terms of overall or event-free survival, no difference was found according to FCGR3A or FCGR2A alleles. The FCGR3A single nucleotide polymorphism (SNP) is predictive of response to R-CHOP, but does not correlate with survival in patients with DLBCL.

Antitumor activity of humanized monoclonal antibody against HM1.24 antigen in human myeloma xenograft models

A humanized monoclonal antibody against HM1.24 antigen (AHM), which is highly expressed on multiple myeloma (MM) cells, induced antibody-dependent cellular cytotoxicity (ADCC) in vitro. In this study, we further characterized AHM and evaluated its potency for clinical application. AHM bound to HM1.24 antigen with a dissociation constant of 0.35 nM, and its epitope resided between Leu116 and Leu127 of the HM1.24 antigen. Single intravenous injection of AHM significantly inhibited tumor growth in both orthotopic and ectopic human MM xenograft models. AHM reduced serum M protein levels and prolonged survival of mice intravenously inoculated with KPMM2 and ARH-77 cells. The number of KPMM2 cells in bone marrow or tumor volume of subcutaneously inoculated RPMI 8226 cells was also inhibited by AHM. The antitumor activity of AHM against tumor cells in bone marrow was diminished when the mice were pretreated with anti-Fcgamma receptor III/II antibody, demonstrating that antitumor activity by AHM requires effector cell functions in vivo. Experiments involving in vitro ADCC assays indicated that NK cells and monocytes/macrophages serve as effector cells for AHM-induced ADCC in mouse and human. Thus, AHM will provide an additional treatment option for MM.