Antibody-dependent Cell-mediated Cytotoxicity Effector Research Articles

Engineering of Trispecific Molecule That Induces Natural Killer Cell Expansion While Mediating CD133-Specific Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

Background: Selective cancer stem cell (CSC) elimination in solid as well as in and hematologic malignancies is a critical goal in immunotherapy since CSC cause drug refractory relapse. Furthermore this small cell group is known for tumor initiation and self-renewal capabilities and was already described to be a valuable target in breast, head and neck as well as in ovarian cancer. CSCs are also important for the development of hematologic malignancies and might be an excellent cell to target, currently not addressed through current therapies, in order to prevent relapse. The design of state-of-art anti-cancer immune engagers should address three important issues including ADCC, CSC targeting, and rapid effector cell expansion that sustains a potent anti-cancer response. In order to improve the current conventional bispecific immune-engager platform, we synthesized a 16133 BiKE consisting of scFvs binding FcγRIII (CD16) on Natural Killer (NK) cells and CD133 on CSC and then introduced a modified IL-15 crosslinker capable of stimulating NK effector cells.Methods: DNA shuffling and ligation techniques were used to assemble and synthesize the 1615133 trispecific NK cell engager (TriKE) (Figure 1A). The construct was tested for specificity using flow cytometry, cytotoxicity using chromium release assays, and lytic degranulation. IL-15-mediated expansion was measured using flow based proliferation assays. Also, Interferon (IFN)-γ release was measured by flow cytometry since it is important in the anti-cancer response.Results: The new TriKE showed specific and efficient induction of NK cell related cytotoxicity as seen in 51chromium release assays with Caco-2 cells, which express high levels of CD133. Activity was superior compared to 16133 BiKE as seen in effector:target ratios of 20:1 (21.9 ± 0.8% vs. 17.9 ± 2.2%), 10:1 (9.4 ± 0.3% vs. 7.9 ± 2.4%) and 5:1 (4.3 ± 0.2% vs. 5.4 ± 1.5%) (Figure 1B). Proliferation and NK expansion with the 1615133 TriKE was far greater than that achieved with the BiKE devoid of IL-15 as tested with purified NK cells exposed to both drugs for 7 days and stained with a reactive dye (Proliferation index 1.7 ± 0.3 vs. 1.2 ± 0.01, p=<0.0001, n=5). Drug binding and induction of cytotoxic degranulation was CD133+ specific as proved with Caco-2 and Raji cells as positive and negative controls (respectively). In Caco-2 cell targets the BiKE as well as the TriKE showed significant superior activity compared to, NCI IL-15, anti-CD16 scFv and anti-CD133 scFv controls (CD107a expression 37.5 ± 0.2% and 36.9 ± 0.2% vs. 19.6 ± 0.1, 18.3 ± 0.7, 12.6 ± 0.4, p<0.001, n=3), (Figure 1C). NK cell related cytokine release measured via IFN-γ detection was higher in the TriKE compared the BiKE (38.3 ± 0.2 % vs. 13.1 ± 0.3 %, p<0.001, n=3) and higher than all other controls. NK cell related cytokine release studies showed that although IFN-γ levels were elevated, they did not approach the levels achieved with IL-12/IL-18 (38.3 ± 0.2 % vs. 60 ± 0.2 %, p<0.001, n=3) indicating that the release induced with the TriKE was not at supraphysiologic levels.Conclusion: 1615133 TriKE showed specific and improved anti-cancer activity over BiKE and provides a self-sustaining mechanism via induction of IL-15 signaling on NK cells. Inclusion of IL-15 might be a promising platform technology since CD133 can be substituted by other promising tumor associated antigens to create a highly specific and efficient drug. By improving NK cell performance, the new TriKE represents a highly active drug against drug refractory relapse inducing CSC with an encouraging safety profile. [Display omitted] DisclosuresMiller:Oxis Biotech Scientific Advisory Board: Membership on an entity's Board of Directors or advisory committees. Vallera:Oxis Biotech: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Combination Therapy with Daratumumab and CAR-NK Targeting CS1 for Multiple Myeloma

Daratumumab (Dara), a targeted therapy utilizing a monoclonal antibody against CD38, and its combination with other are becoming a new standard of care treatment in multiple myeloma (MM). Recently, chimeric antigen receptor (CAR) T cell immunotherapy has been successful in the clinic for the treatment of leukemia and lymphoma. Our preliminary data suggest that both CS1-CAR T cells and CS1-CAR NK cells are effective in eradicating MM cells in vitro and in vivo (Chu et al., 2014, Leukemia and Chu et al., 2014, Clinical Cancer Research). In this study, we investigated the combination therapy with Dara and CS1-CAR NK cells for the treatment of relapsed MM.We first showed that that in MM patients, CD38brightCD138─CD34─CD20+CD27+ MM cancer stem-like cells (CSCs) express CS1 at levels much higher than any other cells, and are susceptible to being eradicated by CS1-CAR NK cells. However, CD34+hematopoietic stem cells from bone marrow of healthy donors do not express CS1. These data suggest that CS1-CAR NK cells can target MM CSCs, and thus may prevent relapse of MM, as ample evidence shows that relapsed or recurrent tumor cells are derived from CSCs.We also demonstrated that CD38 is highly expressed on NK and MM cells. Dara triggered IFN-γ and GZMB expression (p< 0.01) in primary human NK cells, even in the absence of crosslinking with tumor cells. Interestingly, the increase IFN-γ expression can be validated in the CD16 (+) haNK-92 (high-affinity natural killer cells), but not in the parental NK-92 cell line. Blocking the recognition between CD16 and Dara (an IgG1 mAb) with an Fc blocking Ab completely impaired Dara-induced IFN-γ and GZMB expression, indicating that Dara-induced NK cell activation is CD16-dependent. Mechanistically, Dara significantly induced phosphorylation of NFkB and STAT1, indicating that Dara induces IFN-γ and GZMB in NK cells, which may occur through CD16 and be mediated downstream by STAT1 and NFkB. We also found that Dara failed to stimulate GZMB and IFN-γ expression in CD38(-) CD16(+) NK cells, while successful in stimulating CD38(+) CD16(+) NK cells, indicating that Dara induces NK cell activation, which requires not only the binding between CD16 and Fc fragment of Dara, but also the CD38 signaling pathway. Furthermore, we found that Dara mediated cytotoxicity of NK cells against MM cells through antibody-dependent cell-mediated cytotoxicity (ADCC) against CD38-positive (e.g., MM1.S), but not CD38-negative (e.g., U266), which can be blocked by CD16 blocking Ab. Moreover, Dara displays ADCC effects in CD16(+) NK cells but not CD16(-) NK cells. When CD16(+) NK cells were armed with the CS1-CAR, ADCC is still observed against CD38(+) MM cells at low effector to target ratios, i.e., Dara still enhances cytotoxicity of CS1-CAR NK cells, which already have enhanced cytotoxicity.We observed that Dara-induced NK cell ADCC against CD38(+) MM MM1.S cells led to increased T cell proliferation and activation in a co-culture system including dendritic cells. This effect was not observed when MM U266 cells were included as the NK cell target. Out data are consistent with that recent discovery by DiLillo and Ravetch showing that engagement of monoclonal antibody can induce an antitumor vaccine effects (David J et al., Cell, 2015).To tested Dara affects NK cell survival, immunoblotting was performed with anti-cleaved Caspase-3 and anti-cleaved PARP-1 antibodies. We demonstrated that apoptotic activity was increased in both CD16(+)NK cells (primary NK and haNK-92) and parental CD16(-)NK-92 cells treated with Dara for 24 h in a dose-dependent manner. Unlike Dara’s positive effects on CD16(+) NK cells (i.e. stimulating IFN-γ production and ADCC), induction of apoptosis seems to be CD16-independent, as parental NK-92 cells, which are CD16(-), also showed an increased levels of apoptosis induced by Dara. We are testing whether the apoptosis induction is dependent on the antigen for Dara, because as mentioned above, both primary NK cells, and modified as well as unmodified NK-92 cells, that were CD38 (+).In conclusion, our study demonstrates that the combination of Dara and CS1-CAR NK cells, which target two different tumor-associated antigens, both of which have potent anti-MM efficacy, may show additive or synergistic effects; however due to the positive and negative effects of Dara on NK cells, sequential treatment rather than a concomitant treatment modality should be considered. DisclosuresNo relevant conflicts of interest to declare.

Tetraspecific scFv construct provides NK cell mediated ADCC and self-sustaining stimuli via insertion of IL-15 as a cross-linker.

BackgroundThe design of a highly effective anti-cancer immune-engager would include targeting of highly drug refractory cancer stem cells (CSC). The design would promote effective antibody-dependent cell-mediated cytotoxicity (ADCC) and simultaneously promote costimulation to expand and self-sustain the effector NK cell population. Based on our bispecific NK cell engager platform we constructed a tetraspecific killer engager (TetraKE) comprising single-chain variable fragments (scFvs) binding FcγRIII (CD16) on NK cells, EpCAM on carcinoma cells and CD133 on cancer stem cells in order to promote ADCC. Furthermore, an Interleukin (IL)-15-crosslinker enhanced NK cell related proliferation resulting in a highly active drug termed 1615EpCAM133.ResultsProliferation assays showed TetraKE promoted proliferation and enhanced NK cell survival. Drug-target binding, NK cell related degranulation, and IFN-γ production was specific for both tumor related antigens in EpCAM and CD133 bearing cancer cell lines. The TetraKE showed higher killing activity and superior dose dependent degranulation. Cytokine profiling showed a moderately enhanced IFN-γ production, enhanced GM-CSF production, but no evidence of induction of excessive cytokine release.MethodsAssembly and synthesis of hybrid genes encoding the TetraKE were performed using DNA shuffling and ligation. The TetraKE was tested for efficacy, specificity, proliferation, survival, and cytokine production using carcinoma cell lines and functional assays measuring NK cell activity.Conclusion1615EpCAM133 combines improved induction of ADCC with enhanced proliferation, limited cytokine response, and prolonged survival and proliferation of NK cells. By linking scFv-related targeting of carcinoma and CSCs with a sustaining IL-15 signal, our new construct shows great promise to target cancer and CSCs.

Abstract P4-04-18: High serum miR-19a levels correlated with favorable prognosis in patients with metastatic HER2+ breast cancer and might result from effective antibody-dependent cell-mediated cytotoxicity (ADCC) induced by trastuzumab and Th1-mediated antitumor immune response

Abstract Introduction HER2 amplification is found in up to 20% of breast cancer and is associated with poor survival. To date, no predictive biomarker has been validated for clinical practice in patients with HER2+ breast cancer. Type 1 T helper lymphocytes (Th1) are required to activate antitumor cytotoxic T lymphocytes (CTL) necessary for tumor clearance. Particularly, IFN-γ secreted by Th1 cells is necessary for the development of CTL-mediated antitumor response. Trastuzumab can induce antibody-dependent cell-mediated cytotoxicity (ADCC) mediated by natural killer (NK) cells. In turn, trastuzumab-activated NK cells can trigger Th1 cells by driving their differentiation and maturation. Furthermore, NK-mediated ADCC can increase tumor cell membrane permeability through pore formation induced by perforin secreted by NK cells. We found that: 1) high serum miR-19a levels in patients with metastatic HER2+ breast cancer were predictive of favorable prognosis; 2) Th1 cells expressed and secreted high miR-19a levels; 3) and breast cancer tissue expressed higher miR-19a levels than normal adjacent tissue. Therefore, the high serum miR-19a levels in patients with good prognosis may derive from tumor cells killed by NK-mediated ADCC and secretion from Th1 cells. MiR-19a may represent a marker of effective anti-tumor immune response. Results HER2+ metastatic breast cancer patients with high serum miR-19a levels (n=27) had longer PFS (7.9 vs. 4.1 months; p=0.003) and OS (median not reached vs. 13.1 months; p&amp;lt;0.0001) than patients with low serum miR-19a levels (n=24). Locally advanced breast cancer tissue (n=35) expressed higher levels of miR-19a than normal adjacent tissue (n=10) (p=0.048). KPL-4 cells (HER2-amplified) expressed higher miR-19a levels than SKBR3 cells (HER2-amplified) (p=0.010) and MCF-7 cells (non-HER2-amplified, used as control) (p&amp;lt;0.0001). In in vitro ADCC assay, trastuzumab induced an increased tumor cell killing by NK cells and consequent miR-19a release into the supernatants of MCF-7 (p=0.004; p=0.0005), SKBR3 (p=0.001; p&amp;lt;0.0001) and KPL-4 (p=0.0005; p&amp;lt;0.0001), respectively. Th1 cells expressed (p&amp;lt;0.0001) and secreted (p=0.0002) higher miR-19a levels than Th2 cells (Th1 antagonist). Patients with favorable prognosis had higher levels of IFN-γ-secreting Th1 cells (p=0.049) in the peripheral blood than patients with worse prognosis. Conclusion High serum miR-19a levels in patients with metastatic HER2+ breast cancer may result from an increased ability of trastuzumab to induce an effective NK-mediated ADCC and activation of Th1-mediated immune response. This may explain the different clinical outcome between patients with high and low serum miR-19a levels. Our results suggest that miR-19a may potentially represent a novel serum biomarker to evaluate trastuzumab response and Th1-mediated anti-tumor immunity in patients with metastatic HER2+ breast cancer. Citation Format: Anfossi S, Huo L, Woodward WA, Ueno NT, Valero V, Calin GA, Reuben JM. High serum miR-19a levels correlated with favorable prognosis in patients with metastatic HER2+ breast cancer and might result from effective antibody-dependent cell-mediated cytotoxicity (ADCC) induced by trastuzumab and Th1-mediated antitumor immune response. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-18.

An anti-HER2 antibody conjugated with monomethyl auristatin E is highly effective in HER2-positive human gastric cancer

ABSTRACTAntibody-drug conjugate (ADC) is a novel class of therapeutics for cancer target therapy. This study assessed antitumor activity of ADC with an antimitotic agent, monomethyl auristatin E (MMAE) and a humanized monoclonal anti-HER2 antibody, hertuzumab, in gastric cancer. The efficacy of hertuzumab-MC-Val-Cit-PAB-MMAE (hertuzumab-vcMMAE) on human epidermal growth factor receptor 2 (HER2) positive human gastric cancer cells, NCI-N87, was evaluated in vitro and in vivo. The cytotoxicity of hertuzumab was significantly enhanced after conjugation with MMAE. Compared to trastuzumab, hertuzumab had a higher affinity to HER2 and had more potent antibody-dependent cell-mediated cytotoxicity (ADCC) activity in vitro. After conjugation with MMAE, the binding specificity for HER2 was not affected. Furthermore, the internalization of hertuzumab-vcMMAE in HER2 positive gastric cancer cells was verified. Although the conjugation of hertuzumab and MMAE decreased the ADCC effect, the overall cytotoxicity was dramatically increased in HER2 positive gastric cancer cells. In vitro data on this hertuzumab-vcMMAE has exerted much stronger antitumor activity compared to trastuzumab-DM1 in HER2 positive gastric cancer cells. A single administration of hertuzumab-vcMMAE at 5 or 10 mg/kg showed high potency and a sustained tumor inhibitory effect on NCI-N87 xenografts in mice. In conclusion, hertuzumab-vcMMAE conjugate is a highly effective anti-HER2 targeted therapy for HER2-positive gastric cancer.

Phase I/IIa Study of the Human Anti-CD38 Antibody MOR202 (MOR03087) in Relapsed or Refractory Multiple Myeloma

Background: CD38 is a type II transmembrane glycoprotein that is expressed at high levels on multiple myeloma cells. MOR202 is a HuCAL-derived, human, IgG1 anti-CD38 monoclonal antibody showing effective antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and high activity in preclinical models of multiple myeloma.Patients and Methods: Here we reportinterimsafety and preliminary efficacy data from this ongoing, multicenter, MOR202 dose-escalation, phase I/IIa study in patients with relapsed or refractory disease who had failed ≥2 prior therapies for multiple myeloma, including an immunomodulatory drug and a proteasome inhibitor. The objectives are to evaluate the safety, maximum tolerated dose (MTD)/recommended dose and preliminary efficacy of MOR202 when administered as monotherapy or in combination with dexamethasone (DEX); pomalidomide (POM) + DEX; and lenalidomide (LEN) + DEX. Patients received MOR202 as a 2-hour intravenous infusion every 2 weeks (q2w; dose levels 0.01-16 mg/kg), 4 mg/kg weekly (q1w) and 4, 8 and 16 mg/kg q1w + DEX. The combination cohorts receiving MOR202 8 mg/kg with LEN + DEX and POM + DEX have been opened, and the 16 mg/kg q1w with LEN + DEX or POM + DEX, as well as confirmation cohorts, are planned.Results: As of 26 June 2015, 44 patients have been treated; 31 and 13 patients in the q2w and q1w dose levels, respectively. Median age was 69 years (range 44-80); median number of prior therapy lines was 4 (2-11). The MTD has not been reached. The most common treatment-emergent adverse events (TEAEs) at any grade were anemia (15 patients, 34%), fatigue (14 patients, 32%), infusion-related reactions (IRRs) and leukopenia (13 patients, 30% each), lymphopenia and nausea (11 patients, 25% each). Grade ≥3 TEAEs were reported for 28 patients (64%); the most common included lymphopenia (8 patients, 18%), leukopenia (5 patients, 11%) and hypertension (4 patients, 9%). IRRs arose mainly during the first infusion; all were grade 1-2 except for one patient (grade 3); no IRRs occurred in patients receiving MOR202 in combination with DEX. Infections were commonly reported (26 patients, 59%) but in the majority of the cases were not considered to be treatment-related. There have been no treatment-related deaths. Pharmacokinetic (PK) data demonstrated a significant target-mediated drug disposition effect for most patients treated q2w. By contrast, patients treated q1w (4 or 8 mg/kg) showed constant or slightly accumulating MOR202 trough levels, suggesting the potential for full target occupancy at 16 mg/kg. Long-lasting tumor control has already been observed in early monotherapy cohorts, including one partial response and one very good partial response in the weekly cohorts; efficacy analyses are ongoing. First data from the dose escalation of the weekly cohorts with DEX and the combination cohorts with LEN + DEX and POM + DEX will be presented.Conclusions: At doses up to 16 mg/kg,MOR202 was safe and well tolerated. Encouraging preliminary activity of MOR202 was observed, especially with the weekly regimen. PK data show the potential for full target occupancy in patients receiving MOR202 16 mg/kg q1w. This dosing schedule of MOR202 is currently being tested in combination with DEX, LEN + DEX, and POM + DEX. DisclosuresRaab:MorphoSys: Research Funding; Novartis: Consultancy, Research Funding, Speakers Bureau; Celgene: Consultancy; BMS: Consultancy. Goldschmidt:Chugai: Honoraria, Research Funding, Speakers Bureau; Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millenium: Honoraria, Research Funding, Speakers Bureau; Janssen-Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Einsele:Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen/Onyx: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau. Ferstl:Bristol-Myers Squibb: Other: Advisory board; Novartis: Other: Case report presentation. Weisel:Celgene: Consultancy, Honoraria, Other: Travel Support, Research Funding; Amgen: Consultancy, Honoraria, Other: Travel Support; Novartis: Other: Travel Support; Janssen Pharmaceuticals: Consultancy, Honoraria, Other: Travel Support, Research Funding; BMS: Consultancy, Honoraria, Other: Travel Support; Onyx: Consultancy, Honoraria; Noxxon: Consultancy. Klöpfer:MorphoSys: Employment. Weinelt:MorphoSys: Employment. Härtle:MorphoSys: Employment.

A phase I/IIa study of the human CD38 antibody MOR202 (MOR03087) in relapsed or refractory multiple myeloma

Abstract Background: CD38 is a type II transmembrane glycoprotein that is expressed at high levels on multiple myeloma cells. MOR202 is a HuCAL-derived, human, IgG1 anti-CD38 monoclonal antibody showing effective antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and high activity in preclinical models of multiple myeloma. Patients and Methods: Here we reportinterimsafety and preliminary efficacy data from this ongoing, multicenter, MOR202 dose-escalation, phase I/IIa study in patients with relapsed or refractory disease who had failed ≥2 prior therapies for multiple myeloma, including an immunomodulatory drug and a proteasome inhibitor. The objectives are to evaluate the safety, maximum tolerated dose (MTD)/recommended dose and preliminary efficacy of MOR202 when administered as monotherapy or in combination with dexamethasone (DEX); pomalidomide (POM) + DEX; and lenalidomide (LEN) + DEX. Patients received MOR202 as a 2-hour intravenous infusion every 2 weeks (q2w; dose levels 0.01-16 mg/kg), 4 mg/kg weekly (q1w) and 4, 8 and 16 mg/kg q1w + DEX. The combination cohorts receiving MOR202 8 mg/kg with LEN + DEX and POM + DEX have been opened, and the 16 mg/kg q1w with LEN + DEX or POM + DEX, as well as confirmation cohorts, are planned. Results: As of 26 June 2015, 44 patients have been treated; 31 and 13 patients in the q2w and q1w dose levels, respectively. Median age was 69 years (range 44-80); median number of prior therapy lines was 4 (2-11). The MTD has not been reached. The most common treatment-emergent adverse events (TEAEs) at any grade were anemia (15 patients, 34%), fatigue (14 patients, 32%), infusion-related reactions (IRRs) and leukopenia (13 patients, 30% each), lymphopenia and nausea (11 patients, 25% each). Grade ≥3 TEAEs were reported for 28 patients (64%); the most common included lymphopenia (8 patients, 18%), leukopenia (5 patients, 11%) and hypertension (4 patients, 9%). IRRs arose mainly during the first infusion; all were grade 1-2 except for one patient (grade 3); no IRRs occurred in patients receiving MOR202 in combination with DEX. Infections were commonly reported (26 patients, 59%) but in the majority of the cases were not considered to be treatment-related. There have been no treatment-related deaths. Pharmacokinetic (PK) data demonstrated a significant target-mediated drug disposition effect for most patients treated q2w. By contrast, patients treated q1w (4 or 8 mg/kg) showed constant or slightly accumulating MOR202 trough levels, suggesting the potential for full target occupancy at 16 mg/kg. Long-lasting tumor control has already been observed in early monotherapy cohorts, including one partial response and one very good partial response in the weekly cohorts; efficacy analyses are ongoing. First data from the dose escalation of the weekly cohorts with DEX and the combination cohorts with LEN + DEX and POM + DEX will be presented. Conclusions: At doses up to 16 mg/kg,MOR202 was safe and well tolerated. Encouraging preliminary activity of MOR202 was observed, especially with the weekly regimen. PK data show the potential for full target occupancy in patients receiving MOR202 16 mg/kg q1w. This dosing schedule of MOR202 is currently being tested in combination with DEX, LEN + DEX, and POM + DEX. Disclosures Raab: MorphoSys: Research Funding; Novartis: Consultancy, Research Funding, Speakers Bureau; Celgene: Consultancy; BMS: Consultancy. Goldschmidt: Chugai: Honoraria, Research Funding, Speakers Bureau; Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Millenium: Honoraria, Research Funding, Speakers Bureau; Janssen-Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Einsele: Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen/Onyx: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau. Ferstl: Bristol-Myers Squibb: Other: Advisory board; Novartis: Other: Case report presentation. Weisel: Celgene: Consultancy, Honoraria, Other: Travel Support, Research Funding; Amgen: Consultancy, Honoraria, Other: Travel Support; Novartis: Other: Travel Support; Janssen Pharmaceuticals: Consultancy, Honoraria, Other: Travel Support, Research Funding; BMS: Consultancy, Honoraria, Other: Travel Support; Onyx: Consultancy, Honoraria; Noxxon: Consultancy. Klopfer: MorphoSys: Employment. Weinelt: MorphoSys: Employment. Hartle: MorphoSys: Employment.

Origins of a Vaccine-Induced, Human Anti-HIV-1 Antibody

Origins of a Vaccine-Induced, Human Anti-HIV-1 Antibody

Cocrystal Structures of Antibody N60-i3 and Antibody JR4 in Complex with gp120 Define More Cluster A Epitopes Involved in Effective Antibody-Dependent Effector Function against HIV-1.

Accumulating evidence indicates a role for Fc receptor (FcR)-mediated effector functions of antibodies, including antibody-dependent cell-mediated cytotoxicity (ADCC), in prevention of human immunodeficiency virus type 1 (HIV-1) acquisition and in postinfection control of viremia. Consequently, an understanding of the molecular basis for Env epitopes that constitute effective ADCC targets is of fundamental interest for humoral anti-HIV-1 immunity and for HIV-1 vaccine design. A substantial portion of FcR effector function of potentially protective anti-HIV-1 antibodies is directed toward nonneutralizing, transitional, CD4-inducible (CD4i) epitopes associated with the gp41-reactive region of gp120 (cluster A epitopes). Our previous studies defined the A32-like epitope within the cluster A region and mapped it to the highly conserved and mobile layers 1 and 2 of the gp120 inner domain within the C1-C2 regions of gp120. Here, we elucidate additional cluster A epitope structures, including an A32-like epitope, recognized by human monoclonal antibody (MAb) N60-i3, and a hybrid A32-C11-like epitope, recognized by rhesus macaque MAb JR4. These studies define for the first time a hybrid A32-C11-like epitope and map it to elements of both the A32-like subregion and the seven-layered β-sheet of the gp41-interactive region of gp120. These studies provide additional evidence that effective antibody-dependent effector function in the cluster A region depends on precise epitope targeting--a combination of epitope footprint and mode of antibody attachment. All together these findings help further an understanding of how cluster A epitopes are targeted by humoral responses. HIV/AIDS has claimed the lives of over 30 million people. Although antiretroviral drugs can control viral replication, no vaccine has yet been developed to prevent the spread of the disease. Studies of natural HIV-1 infection, simian immunodeficiency virus (SIV)- or simian-human immunodeficiency virus (SHIV)-infected nonhuman primates (NHPs), and HIV-1-infected humanized mouse models, passive transfer studies in infants born to HIV-infected mothers, and the RV144 clinical trial have linked FcR-mediated effector functions of anti-HIV-1 antibodies with postinfection control of viremia and/or blocking viral acquisition. With this report we provide additional definition of the molecular determinants for Env antigen engagement which lead to effective antibody-dependent effector function directed to the nonneutralizing CD4-dependent epitopes in the gp41-reactive region of gp120. These findings have important implications for the development of an effective HIV-1 vaccine.

Trastuzumab enhanced the cytotoxicity of Vγ9Vδ2 T cells against zoledronate-sensitized osteosarcoma cells

Preliminary studies of Vγ9Vδ2 T cells and zoledronate (ZOL) present promising reasons to exploit their immunotherapeutic potential for osteosarcoma treatment (OS). ZOL is a third-generation aminobisphosphonate (ABP) and is well established in the management of cancer-induced bone disease. However, ZOL is characterized by high tropism for bone matrix, and the efficacy of ZOL for sensitizing tumors remains to be optimized. Vγ9Vδ2 T cells are important effectors of antibody-dependent cell-mediated cytotoxicity (ADCC). In this study, we investigated whether Vγ9Vδ2 T cell-mediated killing of ZOL-pretreated OS cells could be increased by the anti-HER-2 monoclonal antibody trastuzumab (TTZ). The cytotoxic activity of Vγ9Vδ2 T cells against osteosarcoma was assessed by an MTS assay in the presence or absence of TTZ. A CD107a assay was used to measure degranulation in cytotoxic Vγ9Vδ2 T cells. Blocking studies were used to determine the effect of relative ligands on Vγ9Vδ2 T cell recognition. TTZ induced an ADCC response in the OS cell line, U2OS; however, it had no effect on another OS cell line HOS with low levels of surface HER2 expression. Although the OS cells pretreated with ZOL for clinically relevant time periods (2hours) stimulated a suboptimal immune response of Vγ9Vδ2 T cells, TTZ could further enhance the cytotoxicity of Vγ9Vδ2 T cells. These results demonstrate that combining TTZ and ZOL significantly increases the cytotoxic potential of Vγ9Vδ2 T cells. This study raises the possibility of utilizing ZOL and TTZ in Vγ9Vδ2 T cell-based immunotherapy for OS.

Abstract P6-14-07: High miR-19a serum levels correlate with favorable prognosis in patients with metastatic HER2+ inflammatory breast cancer and may result from an effective antibody-dependent cell-mediated cytotoxicity induced by trastuzumab

Abstract Background: IBC is a rare but highly aggressive form of locally advanced breast cancer (5-year OS rate: 40.5% IBC vs 85% non-IBC patients) accounting for 10% of all breast cancer deaths. To date, no unique molecular diagnostic or prognostic biomarker has been identified for IBC. Increasing evidence supports the potential value of miRNA as prognostic and predictive serum biomarker in cancer. We found that IBC cells expressed high levels of miR-19a and patients with metastatic IBC HER2+ (MIBC HER2+) and high miR-19a serum levels had better prognosis than patients with MIBC HER2+ and low miR-19a serum levels. As one of the mechanisms of action of trastuzumab is the induction of antibody-dependent cell-mediated cytotoxicity (ADCC), we hypothesized that the increased miR-19a serum levels in MIBC HER2+ patients with favorable clinical outcome could result from an effective ADCC and be used as biomarker to monitor the response to trastuzumab. Methods: Total RNA was isolated using the Total RNA Purification Kit (Invitrogen, Norgen Biotek). MiR-19a levels in tumor tissue, serum, cell lines and supernatants were evaluated by qRT-PCR (Applied Biosystems). ADCC was evaluated by Annexin V-FITC Apoptosis Detection Kit I (BD Pharmingen). Results: Microarray was performed in IBC (n=23) and non-IBC (n=24) tumors and normal tissue (n=12). Microarray showed higher miR-19a expression in IBC compared with non-IBC (p=0.028) and normal tissue (p=0.0002). The two IBC cell lines SUM149 (triple receptor-negative) and KPL-4 (HER2-amplified) expressed higher levels of miR-19a compared with the non-IBC cell lines MDA-231 (triple receptor-negative), SKBR3 (HER2-amplified), and MCF-7 (HER2-non-amplified) (p&amp;lt;0.001, p&amp;lt;0.001, p&amp;lt;0.001 and p&amp;lt;0.05, p&amp;lt;0.05, p&amp;lt;0.01 respectively). To assess whether miR-19a could be released from IBC cells upon NK cell-mediated ADCC, we performed a NK cytotoxicity test using the NK-resistant KPL-4 cells and the NK-sensitive SKBR3 and the NK-sensitive MCF-7 cells as control. Co-incubation with trastuzumab induced increased MCF-7, SKBR3 and KPL-4 cell death (2.1-fold, 2.4-fold and 3.5-fold respectively) and accordingly increased miR-19a levels in their supernatants (MCF-7: 3-fold, p=0.017; SKBR3: 6-fold, p=0.005 and KPL-4: 8-fold, p=0.0001). The pattern of miR-19a levels in the supernatants correlated with that expressed at cellular levels (MCF-7&amp;lt;SKBR3&amp;lt;KPL-4). We measured miR-19a serum levels in patients with MIBC HER2+ (n=27) and metastatic nonIBC HER2+ (n=24). Patients with MIBC HER2+ and high miR-19a level had longer PFS (10.3 vs 3.2 months, p=0.022) and OS (median not reached vs 11.2 months, p=0.003) than patients with low miR-19a levels. Patients with metastatic nonIBC HER2+ and high miR-19a levels had longer OS (32.9 vs 13.3 months; p=0.015) than patients with low miR-19a levels (32.9 vs 13.3 months; p=0.015). Conclusion: High miR-19a serum levels are associated with favorable prognosis in patients with MIBC HER2+ and could result from an effective NK cell-mediated ADCC. MiR-19a may represent a novel serum biomarker to monitor the response to trastuzumab therapy and predict clinical outcome in patients with MIBC HER2+. Citation Format: Simone Anfossi, Antonio Giordano, Lei Huo, Ricardo H Alvarez, Vicente Valero, Gabriel N Hortobagyi, Wendy A Woodward, Naoto T Ueno, George A Calin, James M Reuben. High miR-19a serum levels correlate with favorable prognosis in patients with metastatic HER2+ inflammatory breast cancer and may result from an effective antibody-dependent cell-mediated cytotoxicity induced by trastuzumab [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-14-07.

Role of epidermal growth factor receptor expression level in cetuximab cytotoxicity and antibody-dependent cell-mediated cytotoxicity effect against A549 lung cancer cell line

To investigate the role of epidermal growth factor receptor (EGFR) expression level in cetuximab cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC) effect against A549 lung cancer cell line. A549 cell line and NKTm cells were used as the target cell and the effector cell, respectively. pEGFR-EGFP plasmids were transfected into A549 cells by nucleofector method. EGFR expression levels were measured by immunohistochemistry. The ADCC activity induced by cetuximab was assessed by cell counting kit-8 assay. A549 cells transfected with pEGFR-EGFP plasmids expressed higher level of EGFR protein on membrane and were more sensitive to ADCC activity mediated by cetuximab (P<0.05). The inhibition rate of A549 cells showed no significant difference between transfection group and wild-type group when treated with cetuximab alone (P> 0.05). EGFR expression level influences the sensitivity of A549 lung cancer cell line to ADCC activity mediated by cetuximab but not to cetuximab alone.

Ocaratuzumab, an Fc-engineered antibody demonstrates enhanced antibody-dependent cell-mediated cytotoxicity in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is common in both developed and developing nations where the need for inexpensive and convenient administration of therapy is apparent. Ocaratuzumab is a novel Fc-engineered humanized IgG1 anti-CD20 monoclonal antibody (mAb) designed for effective antibody-dependent cell-mediated cytotoxicity (ADCC) at very low concentrations that may facilitate sub-cutaneous (vs. intravenous) dosing. Here, we report ocaratuzumab’s potency against CLL cells. In vitro assessment of ocaratuzumab’s direct cytotoxicity (DC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and ADCC was performed on CLL cells. Ocaratuzumab induced DC, CDC, and ADCP similarly to rituximab or ofatumumab (anti-CD20 mAbs). However, ocaratuzumab showed an advantage in NK cell-mediated ADCC over these antibodies. In allogeneic ADCC, [E:T (effector:target) ratios = 25:1, 12:1, 6:1], ocaratuzumab (10 µg/mL) improved ADCC by ~3-fold compared with rituximab or ofatumumab (P < 0.001 all tested E:T ratios). Notably, the superiority of ocaratuzumab-induced ADCC was observed at low concentrations (0.1–10 ug/ml; P < 0.03; allogeneic assays). In extended allogeneic ADCC E:T titration, ocaratuzumab (0.1 µg/mL) demonstrated 19.4% more cytotoxicity than rituximab (E:T = 0.38:1; P = 0.0066) and 21.5% more cytotoxicity than ofatumumab (E:T = 1.5:1; P = 0.0015). In autologous ADCC, ocaratuzumab (10 µg/mL) demonstrated ~1.5-fold increase in cytotoxicity compared with rituximab or ofatumumab at all E:T ratios tested (E:Ts = 25:1,12:1,6:1; all P < 0.001). Obinutuzumab, a glyco-engineered anti-CD20 mAb, showed no improvement in ADCC activity compared with ocaratuzumab. The enhanced ADCC of ocaratuzumab suggests that it may be effective at low concentrations. If supported by clinical investigation, this feature could potentially allow for subcutaneous dosing at low doses that could expand the potential of administering chemoimmunotherapy in developing countries.

Gene-modified human α/β-T cells expressing a chimeric CD16-CD3ζ receptor as adoptively transferable effector cells for anticancer monoclonal antibody therapy.

The central tumoricidal activity of anticancer monoclonal antibodies (mAb) is exerted by FcγR IIIa (CD16)-expressing effector cells in vivo via antibody-dependent cell-mediated cytotoxicity (ADCC), as observed for natural killer (NK) cells. In practice, chemotherapy-induced leukopenia and exhaustion of NK cells resulting from ADCC often hamper the clinical efficacy of cancer treatment. To circumvent this drawback, we examined in vivo the feasibility of T cells, gene-modified to express a newly generated affinity-matured (158V/V) chimeric CD16-CD3ζ receptor (cCD16ζ-T cells), as a transferable alternative effector for cancer mAb therapy. cCD16ζ-T cells were readily expandable in ex vivo culture using anti-CD2/CD3/CD28 beads and recombinant human interleukin-2 (rhIL-2), and they successfully displayed ADCC-mediated tumoricidal activity in vitro. During ADCC, ligation of opsonized cancer cells to the introduced cCD16ζ-T cells stimulated the effector cells to produce proinflammatory cytokines and release toxic granules through the activation of the Nuclear factor of activated T cells (NFAT) pathway after phosphorylation of the CD3ζ chain. In parallel, these stimulated cCD16ζ-T cells transiently proliferated and differentiated into effector memory T cells. In contrast, NK cells activated by rhIL-2 displayed similar ADCC activity, but failed to proliferate. Human cCD16ζ-T cells infused concomitantly with anti-CD20 mAb synergistically inhibited the growth of disseminated Raji cells, a CD20(+) lymphoma cell line, in immunodeficient mice, whereas similarly infused rhIL-2-treated NK cells survived for a shorter time and displayed less effective tumor suppression. Our findings strongly suggest the clinical feasibility of cCD16ζ-T cells as adoptively transferable ADCC effector cells that could potentially enhance the clinical responses mediated by currently available anticancer mAbs.

Monoclonal antibody targeting of IL-3 receptor α with CSL362 effectively depletes CML progenitor and stem cells

AbstractDespite the remarkable efficacy of tyrosine kinase inhibitors (TKIs) in eliminating differentiated chronic myeloid leukemia (CML) cells, recent evidence suggests that leukemic stem and progenitor cells (LSPCs) persist long term, which may be partly attributable to cytokine-mediated resistance. We evaluated the expression of the interleukin 3 (IL-3) receptor α subunit (CD123), an established marker of acute myeloid leukemia stem cells, on CML LSPCs and the potential of targeting those cells with the humanized anti-CD123 monoclonal antibody CSL362. Compared with normal donors, CD123 expression was higher in CD34+/CD38– cells of both chronic phase and blast crisis CML patients, with levels increasing upon disease progression. CSL362 effectively targeted CML LSPCs by selective antibody-dependent cell-mediated cytotoxicity (ADCC)–facilitated lysis of CD123+ cells and reduced leukemic engraftment in mice. Importantly, not only were healthy donor allogeneic natural killer (NK) cells able to mount an effective CSL362-mediated ADCC response, but so were CML patients' autologous NK cells. In addition, CSL362 also neutralized IL-3–mediated rescue of TKI-induced cell death. Notably, combination of TKI- and CSL362-induced ADCC caused even greater reduction of CML progenitors and further augmented their preferential elimination over normal hematopoietic stem and progenitor cells. Thus, our data support the further evaluation of CSL362 therapy in CML.

Increasing FcγRIIa affinity of an FcγRIII-optimized anti-EGFR antibody restores neutrophil-mediated cytotoxicity

Antibody-dependent cell-mediated cytotoxicity (ADCC) has been suggested as an essential mechanism for the in vivo activity of cetuximab, an epidermal growth factor receptor (EGFR)-targeting therapeutic antibody. Thus, enhancing the affinity of human IgG1 antibodies to natural killer (NK) cell-expressed FcγRIIIa by glyco- or protein-engineering of their Fc portion has been demonstrated to improve NK cell-mediated ADCC and to represent a promising strategy to improve antibody therapy. However, human polymorphonuclear (PMN) effector cells express the highly homologous FcγRIIIb isoform, which is described to be ineffective in triggering ADCC. Here, non-fucosylated or protein-engineered anti-EGFR antibodies with optimized FcγRIIIa affinities demonstrated the expected benefit in NK cell-mediated ADCC, but did not mediate ADCC by PMN, which could be restored by FcγRIIIb blockade. Furthermore, eosinophils and PMN from paroxysmal nocturnal hemoglobinuria patients that expressed no or low levels of FcγRIIIb mediated effective ADCC with FcγRIII-optimized anti-EGFR antibody. Additional experiments with double FcγRIIa/FcγRIII-optimized constructs demonstrated enhanced PMN-mediated ADCC compared with single FcγRIII-optimized antibody. In conclusion, our data demonstrate that FcγRIIIb engagement impairs PMN-mediated ADCC activity of FcγRIII-optimized anti-EGFR antibodies, while further optimization of FcγRIIa binding significantly restores PMN recruitment.

Vaccine-Induced Plasma IgA Specific for the C1 Region of the HIV-1 Envelope Blocks Binding and Effector Function of IgG

A recently completed Phase III vaccine trial demonstrated a 32% estimated efficacy. An earlier analysis suggested that envelope (Env)-specific antibody responses were associated with a lower risk of infection. Of particular interest, anti-HIV Env immunoglobulin A (IgA) antibodies correlated with increased infection risk. The purpose of this study was to evaluate potential mechanisms of this phenomenon.Phase III controlled evaluation of the RV144 HIV vaccine to prevent HIV infection was conducted in Thailand. The current study represents a substudy in which all participants provided written consent to have plasma and cellular samples stored and subsequently tested.The levels and affinities of IgA and immunoglobulin G (IgG) antibodies for HIV Env proteins were measured. The ability of patient IgA as well as patient-derived IgA monoclonal antibodies to inhibit natural killer cell–mediated, antibody-dependent cell-mediated cytotoxicity (ADCC) was measured.The Env-specific IgA/IgG ratio directly correlated with infection risk, suggesting that the presence of IgA antibody directly inhibited ADCC function. Furthermore, IgA monoclonal antibodies derived from vaccines inhibited binding and blocked ADCC function. The phenomenon of interference of IgG function by IgA antibodies has been reported previously in other settings.Postvaccination polyclonal antibody responses vary among individuals. Those generating an excess of Env-specific IgA seem to be at increased risk for infection secondary to reduced ADCC effector function of natural killer cells.HIV has evolved multiple mechanisms to evade immune detection and elimination. These include impairment of neutralizing antibody generation, emergence of escape mutants, and Env glycosylation and conformational shielding. The vaccine studied in this article resulted in “blocking IgA antibodies” in a number of recipients and thus adds another level of complexity to vaccine design.

Involvement of antibody-dependent cell-mediated cytotoxicity in inflammatory demyelination in a mouse model of neuromyelitis optica

Neuromyelitis optica (NMO) is an inflammatory demyelinating disease of the central nervous system that can cause paralysis and blindness. The pathogenesis of NMO involves binding of immunoglobulin G autoantibodies to aquaporin-4 (AQP4) on astrocytes, which is thought to cause complement-dependent cytotoxicity (CDC) and a secondary inflammatory response leading to oligodendrocyte and neuronal damage. Here, we investigate in vivo the role of antibody-dependent cell-mediated cytotoxicity (ADCC) triggered by AQP4 autoantibodies (AQP4-IgG) in the development of NMO pathology. A high-affinity, human recombinant monoclonal AQP4-IgG was mutated in its Fc region to produce 'NMO superantibodies' with enhanced CDC and/or ADCC effector functions, without altered AQP4 binding. Pathological effects of these antibodies were studied in a mouse model of NMO produced by intracerebral injection of AQP4-IgG and human complement. The original (non-mutated) antibody produced large NMO lesions in this model, with loss of AQP4 and GFAP immunoreactivity, inflammation and demyelination, as did a mutated antibody with enhanced CDC and ADCC effector functions. As anticipated, a mutated AQP4-IgG lacking CDC, but having tenfold enhanced ADCC, produced little pathology. However, unexpectedly, a mutated antibody with ninefold enhanced CDC, but lacking ADCC, produced much less pathology than the original AQP4-IgG. Also, pathology was greatly reduced following administration of AQP4-IgG and complement to mice lacking the FcγIII receptor involved in effector cell activation during ADCC, and to normal mice injected with an Fcγ receptor blocking antibody. Our results provide evidence for the central involvement of ADCC in NMO pathology and suggest ADCC as a new therapeutic target in NMO.

Correlation of ADCC activity with cytokine release induced by the stably expressed, glyco-engineered humanized Lewis Y-specific monoclonal antibody MB314

A major limitation to the application of therapeutic monoclonal antibodies (mAbs) is their reduced in vivo efficacy compared with the high efficacy measured in vitro. Effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) are dramatically reduced in vivo by the presence of high amounts of endogenous IgG in the serum. Recent studies have shown that modification of the glycosylation moieties attached to the Fc part of the mAb can enhance binding affinity to FcγRIIIα receptors on natural killer cells and thus may counteract the reduced in vivo efficacy. In the present study, a humanized IgG1/κ monoclonal antibody recognizing the tumor-associated carbohydrate antigen Lewis Y was stably produced in a moss expression system that allows glyco-engineering. The glyco-modified mAb (designated MB314) showed a highly homogeneous N-glycosylation pattern lacking core-fucose. A side-by-side comparison to its parental counterpart produced in conventional mammalian cell-culture (MB311, formerly known as IGN311) by fluorescence-activated cell sorting analysis confirmed that the target specificity of MB314 is similar to that of MB311. In contrast, ADCC effector function of MB314 was increased up to 40-fold whereas complement dependent cytotoxicity activity was decreased 5-fold. Notably, a release of immunostimulatory cytokines, including interferon γ, monocyte chemotactic protein-1 (MCP-1), interleukin-6 and tumor necrosis factor (TNF) was particularly induced with the glyco-modified antibody. TNF release was associated with CD14+ cells, indicating activation of monocytes.

Abstract 5374: Significance of ADCC for the action of trastuzumab quantified by novel procedures

Abstract Background: The killing of Her2 + breast cancer cells by the antibody trastuzumab is an attractive therapy because of its specificity and limited side effects. However, many Her2 + positive patients have innate resistants or will become resistant to this treatment. Although Her2 status can be determined by immunohisochemistry or FISH, still only 30% of patients positive for high Her2 status will respond to trastuzumab alone. Improved methods to predict sensitivity to trastuzumab are greatly needed. Since trastuzumab is believed to work by antibody-dependent cell-mediated cytotoxicity (ADCC), we have developed a novel and reproducible bioassay to measure trastuzumab-dependent ADCC which may be predictive of patient response. Methods: Using both an xCELLigence platform (Roche Applied Science) and a flow cytometry assay (PanToXiLux from OncoImmunin, Inc), we measured the ability of mononuclear cells (MNCs) to kill four different breast cancer cell lines in the presence or absence of trastuzumab. Two cell lines used have resistance to trastuzumab and two cell lines have lower Her-2 expression. The xCELLigence platform measures killing in real time while the flow cytometry assay measures the transfer of granzyme B and upstream caspase activity from mononuclear cells to the tumor cells and the activation of cell death processes. The ability of mononuclear cells to bind tumor cells in the presence and absence of trastuzumab was evaluated by microscopy. Using miltenyi beads and immunofluroescence, we have measured which MNC population is most effective in ADCC activity. MNCs from patients with breast cancer and from individuals without disease were analyzed. Results: These studies provide a simple and reproducible assay for ADCC activity. Effective ADCC killing is directly related to Her2 expression on the tumor target. The degree of killing is directly related to the number of mononuclear cells added in the study when trastuzumab is added. Addition of trastuzumab alone to these cell lines results in little killing. MNCs from non-diseased individuals as well as those obtained from breast cancer patients display a broad range of ADCC killing efficiency per cell. Finally, our results demonstrate that NK cells are the major MNC population of effect killing. Conclusion: The two assays presented here and other specific studies show that trastuzumab can work with the immune system to increase cell killing of Her2 overexpressing cells by ADCC. A correlation between the results of these assays and the clinical response to trastuzumab will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5374. doi:1538-7445.AM2012-5374