Antibody-dependent Cell-mediated Cytotoxicity Assay Research Articles

Abstract B247: Integration of a modularized protein engineering technology and the RheoSwitch Therapeutic System® platform to develop high affinity trastuzumab single chain variable fragment-Fc proteins for gene therapy applications.

Abstract While monoclonal antibodies remain highly effective anticancer agents, their complex post-translational processing preclude their use in multi-effector gene therapy platforms. Breast and ovarian cancers often overexpress HER family receptors and consequently the receptors are targets for effective therapeutics such as the monoclonal antibodies trastuzumab and cetuximab. To address the need for new immune modulating formats that can be encoded in a single therapy, we have designed a platform for rapid conversion and assay of monoclonal antibodies into functional single chain variable fragment-Fc fusion proteins. Here we demonstrate results with trastuzumab and cetuximab as examples of the platform scFv-Fc conversion. The scFv-Fc trastuzumab displays efficient expression from transfected cell lines and high-affinity, high specificity binding to HER2 in plate-based screens with measured KD of 0.15nM compared to commercial trastuzumab's KD of 0.08nM in the same assay. scFv-Fc trastuzumab was shown to bind the HER2+ breast cancer cell lines BT-474 and SKBR3 in FACS-based assays while showing no affinity towards HER2- control cells SHP-77. Antibody-dependent cell-mediated cytotoxicity assays (ADCC) were performed to evaluate ADCC potency of the scFv-Fc trastuzumab. Using NK92/CD16A(158V/V) cells as effectors and BT474 cells as targets, the EC50 of the scFv-Fc trastuzumab was 3.6ng/ml, compared to the EC50 of monoclonal format trastuzumab at 0.94ng/ml. The data provide a direct demonstration of the scFv-Fc format as a viable alternative for achieving molecular targeting in a single chain entity at a level commensurate with KD values of full-length monoclonal antibodies while maintaining ADCC activity. In addition, functional scFv-Fc trastuzumab was expressed in combination with scFv-Fc cetuximab from a single vector. scFv-Fc molecules generated using this modular protein engineering platform were cloned into plasmid DNA vectors controlled by the RheoSwitch Therapeutic System®. Transient transfections into differentiated C2C12 myocytes demonstrated the production of scFv-Fc proteins from muscle cells under control of the RheoSwitch® Activator Ligand. The integrated scFv-Fc optimization platform and inducible multigenic systems described here provides support for future work for the treatment of cancer using multigenic therapeutic antibodies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B247. Citation Format: Charles Reed, Vernon Dailey, Anissa Elayadi, Lindsay Williams, Dina Schneider, Thomas Reed, Jonathan Lewis, Richard Einstein, Samuel Broder. Integration of a modularized protein engineering technology and the RheoSwitch Therapeutic System® platform to develop high affinity trastuzumab single chain variable fragment-Fc proteins for gene therapy applications. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B247.

Preclinical Activity of the Type II CD20 Antibody GA101 (Obinutuzumab) Compared with Rituximab and Ofatumumab In Vitro and in Xenograft Models

We report the first preclinical in vitro and in vivo comparison of GA101 (obinutuzumab), a novel glycoengineered type II CD20 monoclonal antibody, with rituximab and ofatumumab, the two currently approved type I CD20 antibodies. The three antibodies were compared in assays measuring direct cell death (AnnexinV/PI staining and time-lapse microscopy), complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and internalization. The models used for the comparison of their activity in vivo were SU-DHL4 and RL xenografts. GA101 was found to be superior to rituximab and ofatumumab in the induction of direct cell death (independent of mechanical manipulation required for cell aggregate disruption formed by antibody treatment), whereas it was 10 to 1,000 times less potent in mediating CDC. GA101 showed superior activity to rituximab and ofatumumab in ADCC and whole-blood B-cell depletion assays, and was comparable with these two in ADCP. GA101 also showed slower internalization rate upon binding to CD20 than rituximab and ofatumumab. In vivo, GA101 induced a strong antitumor effect, including complete tumor remission in the SU-DHL4 model and overall superior efficacy compared with both rituximab and ofatumumab. When rituximab-pretreated animals were used, second-line treatment with GA101 was still able to control tumor progression, whereas tumors escaped rituximab treatment. Taken together, the preclinical data show that the glyoengineered type II CD20 antibody GA101 is differentiated from the two approved type I CD20 antibodies rituximab and ofatumumab by its overall preclinical activity, further supporting its clinical investigation.

Matrix metalloproteinases inhibition promotes the polyfunctionality of human natural killer cells in therapeutic antibody-based anti-tumour immunotherapy

Activation of human natural killer (NK) cells is associated with the cleavage of CD16 from the cell surface, a process mediated by matrix metalloproteinases (MMPs). In this report, we examined whether inhibition of MMPs would lead to improved NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) function. Using an in-vitro ADCC assay, we tested the anti-tumour function of NK cells with three different therapeutic monoclonal antibodies (mAbs) in the presence of MMPs inhibitor GM6001 or its control. Loss of CD16 was observed when NK cells were co-cultured with tumour targets in the presence of specific anti-tumour antibodies, and was found particularly on the majority of degranulating NK responding cells. Treatment with MMPs inhibitors not only prevented CD16 down-regulation, but improved the quality of the responding cells significantly, as shown by an increase in the percentage of polyfunctional NK cells that are capable of both producing cytokines and degranulation. Furthermore, MMPs inhibition resulted in augmented and sustained CD16-mediated signalling, as shown by increased tyrosine phosphorylation of CD3ζ and other downstream signalling intermediates, which may account for the improved NK cell function. Collectively, our results provide a foundation for combining MMPs inhibitors and therapeutic mAbs in new clinical trials for cancer treatment.

Novel humanized anti‐ CD 20 antibody BM ‐ca binds to a unique epitope and exerts stronger cellular activity than others

Cellular activity of BM-ca, a novel humanized anti-CD20 antibody, was quantitatively compared with that of two other anti-CD20 antibodies used for clinical practice, rituximab and ofatumumab. The results of a complement-dependent cytotoxicity (CDC) assay revealed that the strongest antibody was ofatumumab, followed by BM-ca, with rituximab being the weakest. Ofatumumab and BM-ca were effective not only against rituximab-sensitive SU-DHL-4 cells but also against rituximab-resistant RC-K8 cells. In an antibody-dependent cell-mediated cytotoxicity (ADCC) assay, although the effective concentrations against SU-DHL-4 cells were almost the same among these three antibodies, the maximum cytotoxic level was the highest for BM-ca. In an anti-cell proliferation assay using SU-DHL-4 cells, BM-ca was the most effective and ofatumumab, the weakest. Against RC-K8 cells, only BM-ca was effective. When combined with each of four cancer chemotherapeutics (prednisolone, vincristine, hydroxydaunorubicin, and cisplatin), BM-ca exerted the most effective combinatorial anti-cell proliferation activity. To assess the in vivo effect of BM-ca, we intravenously administered BM-ca into cynomolgus monkeys and found that the peripheral B-cell levels did not decrease in half of the animals. Sequencing of cDNA encoding CD20 of cynomolgus monkeys revealed that the responders and nonresponders had Leu/Pro (hetero) and Leu/Leu (homo) at amino acid (a.a.) position 160, respectively, suggesting that the epitope recognized by BM-ca was around this a.a. By analyzing reactivity to synthetic peptides, the epitope recognized by BM-ca was estimated to be a.a.'s 156–166, not shared with rituximab and ofatumumab. These results suggest BM-ca to be a promising anti-CD20 antibody having superior properties and recognizing a unique epitope.

Abstract B95: MUC16 protects ovarian cancer cells from the cytolytic responses of human and murine natural killer cells and macrophages.

Abstract MUC16 is a membrane-spanning mucin that frequently is overexpressed by ovarian cancer cells. Knockdown of MUC16 is known to increase apoptosis in both ovarian and breast cancer cells. MUC16 also plays an important role in facilitating metastasis and protecting cancer cells from innate immune responses. Here, we demonstrate that MUC16 protects ovarian cancer cells from cytolysis by NK cells and macrophages. Immunodeficient SCID and SCID/beige mice implanted with MUC16-expressing and MUC16 knockdown tumors have a median survival of 78 and 198 days, respectively. In vitro assays demonstrate that better immune recognition of the MUC16 knockdown ovarian cancer cells contributes to the higher median survival of the tumor-bearing immunocompromised animals. NK cells which were unstimulated, activated in vivo with anti-CD40 antibody, and/or stimulated with lipopolysaccharides preferentially lyse MUC16-knockdown cancer cells in standard chromium release assays. Thus the reduction of MUC16 expression leads to a significant increase of the natural cytotoxic responses of NK cells. In addition, MUC16 protects the cancer cells from Antibody-Dependent Cell-mediated Cytotoxicity (ADCC). ADCC of the MUC16 knockdown cancer cells was studied using the anti-EpCAM antibody KS1/4 and its corresponding immunocytokine huKS-IL2. NK cells from immunocompetent C57BL/6 mice exhibited an increase in the cytolysis of MUC16-knockdown ovarian cancer cells in the presence of either KS1/4 or huKS-IL2. Similar effect was also observed when the ADCC assays were conducted using human NK cells isolated from healthy donors. The immune protection provided by MUC16 not only reduces the effectiveness of NK cells, but also murine macrophages, explaining the longer survival observed in the NK cell deficient SCID-beige mice transplanted with the MUC16-knockdown cancer cells. Immunohistochemical analysis of the tumors from the SCID-beige mice showed increased infiltration of macrophages in the tumor nests of MUC16-knockdown tumors. In contrast, macrophages were primarily detected in the peritumoral stroma of MUC16pos tumors. Murine macrophages from unstimulated and anti-CD40 antibody stimulated animals showed a 3-fold increase in tumoristatic activity against the MUC16-knockdown versus the MUC16pos ovarian cancer cells. The results of our study also demonstrate that siRNA-based strategies to knockdown MUC16 can be coupled with immunotherapeutic approaches, such as the use of activated NK cells or specific immunocytokines, to render ovarian tumors especially vulnerable to innate immune responses. Therefore, our results not only indicate that immune protection is a major biological role of MUC16, but also demonstrate that attenuation of MUC16 expression will likely serve as an important strategy to boost the anticancer efficacy of immunotherapeutic agents. Citation Format: Mildred Felder, Arvinder Kapur, Alexander Rakhmilevich, Joseph Connor, Manish S. Patankar. MUC16 protects ovarian cancer cells from the cytolytic responses of human and murine natural killer cells and macrophages. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B95.

Irradiated NK-92 Targets AML Leukemic Stem Cells in Vivo and Gene-Modified CD16+NK-92 Mediates Antibody Dependent Cell Mediated Cytotoxicity (ADCC) Against CD123+ Cells

Abstract 1909 Introduction:Patients with acute myeloid leukemia (AML) in remission relapse frequently from residual leukemic stem cells (LSCs) that are CD34+CD38-CD123+. NK-92 is an infusible CD16- malignant NK cell line cytotoxic against AML and after irradiation for prevention of NK cell malignancy has minimal adverse events in Phase I trials. Here, we tested irradiated NK-92 (iNK-92) against primary AML in a NOD/SCID gamma null (NSG) xenograft model. To optimize killing of LSCs in vitro we utilized a gene modified CD16+NK-92 against the CD123+ leukemia cell line OCI/AML5 treated with and without an anti-CD123 monoclonal antibody (7G3) to facilitate antibody dependent cell mediated cytotoxicity (ADCC). Methods:NSG mice were irradiated with 225 or 325 cGy before infusion with primary AML blasts (1 or 3×10e6 cells). iNK-92 was started 1 or 10 days post AML infusion and given 1–3 times weekly (15 or 20×10e6 cells/dose) to a total dose of 60–75 ×10e6 cells/mouse. In vitro cytotoxicity was measured by the chromium release assay. OCI/AML5 or primary AML was pretreated with 7G3 or anti-Class I antibody respectively at 10 ug/ml 2 hours before ADCC assays. Results:We developed an NSG AML xenograft model using a CD34+CD38+ primary AML sample containing a small fraction of CD34+CD38- cells that were predominantly CD123+. 3×10e6 primary cells induced leukemia in NSG mice by 6 weeks and maintained comparable potency up to quaternary transplantations with a stable immunophenotype. In vitro cytotoxicity against LSCs was assessed by treating 1×10e6 first passage BM derived primary AML +/− 5×10e6 iNK-92 ×4 hours and injecting into 2 cohorts of 5 mice. At 6 weeks mice were sacrificed and bone marrow harvested. Average leukemic engraftment in the iNK-92 group (79.8, 3.48, 92.1, 81.3, 86.1, Av= 68.6%) was less than untreated AML inoculated group (95.0, 93.4, 19.4, 95, 97.3, Av=80.0%), but not statistically significant (p=0.62). Removing one poorly engrafted outlier mouse from each group yielded a higher engraftment in the control (Av=95.1%) versus the treatment group (Av=84.8%) and was significant (p=0.011).To test iNK-92 to treat engrafted leukemia, NSG mice were infused with 3×10e6 AML cells starting day 10 with 20×10e6 iNK-92 weekly ×6 doses. Survival was improved in the treatment group to near statistical significance (p=0.055), but all mice ultimately succumbed to disease. Addition of IL-2 to iNK-92 did not improve outcomes (p=0.13).3×10e6 primary AML cells were also infused into 2 cohorts of 4 mice and treated +/− iNK-92 from day 2 and given 15×10e6 cells twice weekly to 75×10e6 total dose. BM (1×10e6 cells) from each of 4 primary recipients in control and treatment was serially transplanted 1:1 into four new NSG mice. BM engraftment occurred in all AML only cohort secondary mice (80.8, 93.3, 80.4, 96.4 Av=87.7%) while one mouse from iNK-92 group was leukemia free with engraftment at background levels of non-injected mice (96.4, 94.7, 1.8, 95.7 Av=72.2%). Proportion of LSCs in secondary transplanted mice was: AML (8.01, 9.48, 8.66, 5.25, Av=7.85%) and AML + iNK-92 (7.13, 3.46, 0.03, 4.0 Av=3.66%) which was statistically significant (p=0.05).CD16+NK-92 killed CD123+ OCI/AML5 cells at effector:target (E:T) ratios of 25:1, 10:1, 5:1 and 1:1 (% lysis +/−SD: 35.0 +/−4.0, 9.0+/−6.6, -2.0 +/−0.1, 1.7 +/−3.3) and was significantly enhanced (2–6x) when targets were coated with anti-CD123 mAb (% lysis +/−SD: 64.3 +/−3.1, 48.5 +/−4.1, 20.9 +/−0.1, 10.1+/−3.3). Further, CD16+NK-92 also killed primary AML at E:T ratios of 25:1, 10:1, 5:1 and 1:1 (% lysis +/−SD: 34.7 +/−4.6, 15.6 +/− 4.7, 11.5 +/− 2.0, 7.7 +/−1.6) which was significantly enhanced (2–3x) by coating with anti-Class I monoclonal antibodies (% lysis +/−SD: 64.8 +/−10.4, 31.1 +/− 8.9, 30.2 +/− 9.4, 23.9+/−2.8). Conclusion:iNK-92 reduces engraftment of AML and improves survival in a primary AML xenograft model. CD16+NK-92 cytotoxicity against primary AML is enhanced with anti-class I antibodies via ADCC, demonstrating that CD16+NK-92 can be redirected against bulk primary leukemia using a highly expressed cell surface marker. Finally, CD16+NK-92 cytotoxicity can be improved against OCI/AML5 with anti-CD123 antibodies via ADCC, providing the first proof-of-principle for the targeting of leukemic stem cells by combining humoral and cellular approaches. Disclosures:No relevant conflicts of interest to declare.

CSL362: A Monoclonal Antibody to Human Interleukin-3 Receptor (CD123), Optimized for NK Cell-Mediated Cytotoxicity of AML Stem Cells

AbstractAbstract 3598The interleukin-3 receptor alpha chain (IL-3Rα/CD123) is expressed in a variety of hematological malignancies including AML, MDS, B-ALL, Hodgkin’s lymphoma, hairy cell leukemia, systemic mastocytosis, plasmacytoid dendritic cell leukemia and CML. In AML, the majority of AML blasts express CD123 and this receptor is selectively over expressed on CD34+CD38− leukemic stem cells (LSC) compared to normal hematopoietic stem cells. This difference may provide a biological advantage to the leukemic cells given the survival and proliferation-promoting activities of IL-3, whilst at the same time providing an opportunity to target these malignant cells selectively.We have shown previously that 7G3, a mouse monoclonal antibody (mAb) which blocks IL-3 binding to CD123, is capable of eliminating human LSC in a mouse model of human AML by a combination of mechanisms, including engagement of the innate immune system via Fc-dependent mechanisms (Jin et al., 2009 Cell Stem Cell, 5:31). We have subsequently humanised and affinity-matured this antibody and, in addition, have engineered the Fc-domain to optimise potential cytotoxicity against AML cells.The resultant antibody, CSL362, retains the ability to neutralise IL-3 and has enhanced affinity for the FcγRIIIa (CD16) on NK cells. In vitro studies have demonstrated that the increased affinity for CD16 correlates with greater antibody-dependent cell-mediated cytotoxicity (ADCC) against CD123 expressing cell lines compared to CSL360, a non Fc-engineered anti-CD123 mAb. The improved activity was evident as both an increased maximal level of target cell lysis and as a shift in the EC50 of the antibody to lower concentrations. Importantly, both primary AML blasts and CD34+CD38−CD123+LSC were susceptible to CSL362-induced ADCC and this was seen even in samples that were resistant to ADCC by a non Fc-engineered anti-CD123 mAb. In an AML xenograft mouse model, where treatment with the antibody was initiated 4 weeks after engraftment of leukemia cells, CSL362 was more effective in reducing leukemic growth than the non Fc-engineered anti-CD123 mAb.The evaluation of neutrophils, monocytes, macrophages and NK cells in ADCC assays has revealed that the major effector cell responsible for CSL362-mediated cytotoxicity in human peripheral blood is the NK cell. In clinical samples we have been able to demonstrate autologous depletion ex vivo of target AML blasts (collected at diagnosis and cryopreserved) following incubation with CSL362 and peripheral blood mononuclear cells (taken from the same patient at first remission), indicating that NK cell number and function is sufficiently preserved in such patients for CSL362-directed killing of leukemic target cells.The pre-clinical data generated thus far strongly support the clinical development of CSL362 for the treatment of AML in patients with adequate NK cell function. A Phase 1 study of CSL362 in patients with CD123 positive AML in remission is underway (Clinical Trials.gov identifier: NCT01632852). Disclosures:Busfield:CSL Limited: Employment. Biondo:CSL Limited: Employment. Wong:CSL Limited: Employment. Ramshaw:CSL Limited: Research Funding. Lee:CSL Limited: Research Funding. Martin:CSL Limited: Employment. Ghosh:CSL Limited: Employment. Braley:CSL Limited: Employment. Tomasetig:CSL Limited: Employment. Panousis:CSL Limited: Employment. Vairo:CSL Limited: Employment. Roberts:CSL Limited: Research Funding. DeWitte:CSL Behring: Employment. Lock:CSL Limited: Consultancy, Research Funding. Lopez:CSL Limited: Consultancy, Research Funding. Nash:CSL Limited: Employment.

228P - Development of a Novel Quantitative Assay for Predicting ADCC, Antibody Dependent Cell Mediated Cytotoxicity, and Prediction of Pathological Complete Response in Neoadjuvant Chemotherapy for HER-2 Positive Breast Cancer

ABSTRACT Background Antibody-dependent cell-mediated cytotoxicity (ADCC) has been shown to be one of the modes of action for trastuzumab. We have previously reported that FcγRIIIa-158 V/V and FcγRIIa-131 H/H genotypes predicted clinical outcome of trastuzumab in both neoadjuvant and metastatic setting in patients with HER-2 positive breast cancer. The purpose of this study is to directly measure an inter-individual ADCC and to develop a new system for predicting to a clinical effectiveness of trastuzumab. Materials and methods The stability of the inter-individual differences has been confirmed using peripheral blood mononuclear cells (PBMCs) of 11 healthy volunteers (HVs). Next, we adopted an ex vivo gene expression analysis to identify the molecules which correlate with ADCC activity. We examined the expression change of 14 candidate genes in the 8 HVs after ex vivo exposure to heat-aggregated IgG1 for 4 hr using Hem A (+) system. After identification of the molecules predicting ADCC, we evaluated prospectively whether values of fold increase (FIs) of these the molecules are associated with a pathological complete response (pCR) in 18 patients with HER2 positive breast cancer, who received trastuzumab-based neoadjuvant chemotherapy. Results FI in expressions of TNFSF15, IL-6, and CxCL3 are significantly correlated with ADCC activity (R = 0.74, R = 0.85, R = 0.87, respectively). Eligible criteria of the prospective comfort include HER2 positive breast cancer, chemotherapy-naive, measurable disease, PS 0-2 and adequate organ functions. Patients received standard FEC (5-fluorouracil/epirubicin/cyclophosphamide) q3w for 4cycles followed by weekly paclitaxel/trastuzumab for 12 weeks. Patients who achieved a pCR had a higher FI of CXCL-1, CXCL-3, TNFSF-2, and TNFSF-15 than those who did not (p = 0.004, 0.015, =0.0495, and =0.014, respectively). Conclusions These results suggest the novel quantitative ADCC assay have a potential to predict pCR to trastuzumab-based neoadjuvant chemotherapy. Disclosure All authors have declared no conflicts of interest.

A Novel Assay for Antibody-Dependent Cell-Mediated Cytotoxicity against HIV-1- or SIV-Infected Cells Reveals Incomplete Overlap with Antibodies Measured by Neutralization and Binding Assays

The resistance of human immunodeficiency virus type 1 (HIV-1) to antibody-mediated immunity often prevents the detection of antibodies that neutralize primary isolates of HIV-1. However, conventional assays for antibody functions other than neutralization are suboptimal. Current methods for measuring the killing of virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC) are limited by the number of natural killer (NK) cells obtainable from individual donors, donor-to-donor variation, and the use of nonphysiological targets. We therefore developed an ADCC assay based on NK cell lines that express human or macaque CD16 and a CD4(+) T-cell line that expresses luciferase from a Tat-inducible promoter upon HIV-1 or simian immunodeficiency virus (SIV) infection. NK cells and virus-infected targets are mixed in the presence of serial plasma dilutions, and ADCC is measured as the dose-dependent loss of luciferase activity. Using this approach, ADCC titers were measured in plasma samples from HIV-infected human donors and SIV-infected macaques. For the same plasma samples paired with the same test viruses, this assay was approximately 2 orders of magnitude more sensitive than optimized assays for neutralizing antibodies-frequently allowing the measurement of ADCC in the absence of detectable neutralization. Although ADCC correlated with other measures of Env-specific antibodies, neutralizing and gp120 binding titers did not consistently predict ADCC activity. Hence, this assay affords a sensitive method for measuring antibodies capable of directing ADCC against HIV- or SIV-infected cells expressing native conformations of the viral envelope glycoprotein and reveals incomplete overlap of the antibodies that direct ADCC and those measured in neutralization and binding assays.

Development of an ELISA based bridging assay as a surrogate measure of ADCC

Antibody dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action (MoA) for many monoclonal antibody (mAb) therapeutics. As such, quantitative measurement of ADCC activity is key to drug development. Traditional cell lysis based ADCC assays using PBMCs or NK cell lines can be challenging to develop and implement for routine testing. To provide an alternative to the cell lysis based ADCC assay, a non-cell based measure of ADCC activity was developed to determine the ADCC activity of an anti-CD20 mAb, which measures the ability of the mAb to bind to CD20 antigen and FcγRIIIa, simultaneously. The bridging of CD20 and FcγRIIIa is an essential interaction for the initiation of ADCC activity. This ADCC bridging method is simple, offers the ease of use of a standard ELISA, and shows reproducible dose–response curves in the concentration range of 50–1000ng/mL. With interassay variability of 7–10% and recovery of 89–115%, the assay demonstrates acceptable precision and accuracy. The assay is able to detect degradative changes in anti-CD20 mAb samples subjected to light and acid exposure, suggesting that it is suitable for use as a stability-indicating method. The assay is also sensitive to mAb fucose levels. A linear relationship between the ADCC bridging assay and the cell lysis ADCC assay was demonstrated, strongly suggesting that the ADCC bridging assay can be used as a surrogate measure of ADCC.

Humanizing murine IgG3 anti-GD2 antibody m3F8 substantially improves antibody-dependent cell-mediated cytotoxicity while retaining targeting in vivo

Murine IgG3 anti-GD2 antibody m3F8 has shown anti-neuroblastoma activity in Phase I/II studies, where antibody-dependent cell-mediated cytotoxicity (ADCC) played a key role. Humanization of m3F8 should circumvent human anti-mouse antibody (HAMA) response and enhance its ADCC properties to reduce dosing and pain side effect. Chimeric 3F8 (ch3F8) and humanized 3F8 (hu3F8-IgG1 and hu3F8-IgG4) were produced and purified by protein A affinity chromatography. In vitro comparison was made with m3F8 and other anti-GD2 antibodies in binding, cytotoxicity, and cross-reactivity assays. In GD2 binding studies by SPR, ch3F8 and hu3F8 maintained KD comparable to m3F8. Unlike other anti-GD2 antibodies, m3F8, ch3F8 and hu3F8 had substantially slower koff.. Similar to m3F8, both ch3F8 and hu3F8 inhibited tumor cell growth in vitro, while cross-reactivity with other gangliosides was comparable to that of m3F8. Both peripheral blood mononuclear cell (PBMC)-ADCC and polymorphonuclear leukocytes (PMN)-ADCC of ch3F8 and hu3F8-IgG1 were more potent than m3F8. This superiority was consistently observed in ADCC assays, irrespective of donors or NK-92MI-transfected human CD16 or CD32, whereas complement mediated cytotoxicity (CMC) was reduced. As expected, hu3F8-IgG4 had near absent PBMC-ADCC and CMC. Hu3F8 and m3F8 had similar tumor-to-non tumor ratios in biodistribution studies. Anti-tumor effect against neuroblastoma xenografts was better with hu3F8-IgG1 than m3F8. In conclusion, humanizing m3F8 produced next generation anti-GD2 antibodies with substantially more potent ADCC in vitro and anti-tumor activity in vivo. By leveraging ADCC over CMC, they may be clinically more effective, while minimizing pain and HAMA side effects. A Phase I trial using hu3F8-IgG1 is ongoing.

Development and validation of an antibody-dependent cell-mediated cytotoxicity-reporter gene assay

Humanized monoclonal antibodies (mAbs) are the fastest growing class of biological therapeutics that are being developed for various medical indications, and more than 30 mAbs are already approved and in the market place. Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important biological function attributed to the mechanism of action of several therapeutic antibodies, particularly oncology targeting mAbs. The ADCC assay is a complicated and highly variable assay. Thus, the use of an ADCC assay as a lot release test or a stability test for clinical trial batches of mAbs has been a substantial challenge to install in quality control laboratories. We describe here the development and validation of an alternate approach, an ADCC-reporter gene assay that is based on the key attributes of the PBMC-based ADCC assay. We tested the biological relevance of this assay using an anti-CD20 based model and demonstrated that this ADCC-reporter assay correlated well with standard ADCC assays when induced with the drugable human isotypes [IgG1, IgG2, IgG4, IgG4S > P (S228P) and IgG4PAA (S228P, F234A, L235A)] and with IgG1 isotype variants with varying amounts of fucosylation. This data demonstrates that the ADCC-reporter gene assay has performance characteristics (accuracy, precision and robustness) to be used not only as a potency assay for lot release and stability testing for antibody therapeutics, but also as a key assay for the characterization and process development of therapeutic molecules.

Results of a Phase 1 Study of AME-133v (LY2469298), an Fc-Engineered Humanized Monoclonal Anti-CD20 Antibody, in FcγRIIIa-Genotyped Patients with Previously Treated Follicular Lymphoma

AME-133v is a humanized monoclonal antibody engineered to have increased affinity to CD20 and mediate antibody-dependent cell-mediated cytotoxicity (ADCC) better than rituximab. Safety, pharmacokinetics, and efficacy were assessed in a phase 1/2 trial in patients with previously treated follicular lymphoma (FL). AME-133v was characterized in vitro by ADCC and cell binding assays. A phase 1 study was conducted in which 23 previously treated patients with FL were assigned sequentially to one of five dose-escalation cohorts of AME-133v at 2, 7.5, 30, 100, or 375 mg/m(2) weekly × 4 doses. AME-133v showed a 13- to 20-fold greater binding affinity for CD20 and was 5- to 7-fold more potent than rituximab in ADCC assays. Cell binding assays showed AME-133v and rituximab competed for an overlapping epitope on the CD20 antigen, and AME-133v inhibited binding of biotinylated rituximab to CD20 in a concentration-dependent manner. AME-133v was well tolerated by patients and common related adverse events included chills and fatigue. One patient experienced a dose-limiting toxicity of neutropenia. AME-133v showed nonlinear pharmocokinetics with properties similar to rituximab. Selective reduction of B cells during and after AME-133v treatment was shown by flow cytometry of peripheral blood. A partial or complete response was observed in 5 of 23 (22%) patients and the median progression-free survival was 25.4 weeks. AME-133v was safe and well tolerated at the doses tested. AME-133v showed encouraging results as an anti-CD20 therapy in heavily pretreated FL patients with the less favorable FcγRIIIa F-carrier genotype.

Silvestrol Modulates Direct Anti-Tumor Activity Against Epstein-Barr Virus (EBV)-Associated Lymphomas While Sparing Innate and Antigen Specific Adaptive Immunity

Abstract 104Epstein-Barr virus (EBV) is an oncogenic human herpes virus that infects more than 90% of people worldwide and is associated with a broad spectrum of malignant lymphoproliferative disorders (EBV-LPD), and nasopharyngeal and gastric carcinomas. Chemotherapy often leads to prolonged immune suppression, development of opportunistic infections, including EBV reactivation, and increased risk of relapsed disease. The poor prognosis of EBV+ diseases makes it essential to identify novel agents that can deliver direct anti-tumor activity while preserving innate and EBV-specific adaptive immune surveillance.Silvestrol is a cyclopenta[b]benzofuran derived from the plant genus Aglaia and has been shown to possess potent anti-tumor activity against hematologic and solid tumors. Silvestrol interferes with the translation of mRNA with complex 5’ untranslated regions often found on pro-survival oncoproteins. Silvestrol exhibits anti-tumor activity against malignant B-cell lines while causing minimal toxicity to peripheral blood mononuclear cells (PBMC) and resting T cells. To examine the potential selective anti-tumor activity and functionally address the effects of silvestrol on adaptive and innate immune function, we utilized in vitro and in vivo EBV lymphomagenesis models. Fully transformed EBV+ Lymphoblastoid lines (LCL) were derived from EBV-LPD tumors of severe combined immune deficient (SCID) mice engrafted with PBMC from EBV-positive donors (hu-PBL-SCID). EBV-LCLs were plated in the presence of silvestrol (2 – 50nM) and proliferation (MTS assay) and apoptosis (Annexin V/PI) was evaluated (24, 72, 120hr). While silvestrol showed potent anti-proliferative activity at these concentrations, we observed minimal cell death (IC50 = 10nM). The anti-proliferative activity of silvestrol was associated with loss of LMP-1 expression, an EBV oncogene essential for B-cell transformation. Examination of LMP1 induced pathways showed decrease in pAkt levels and an increase in NFkB/p65 levels (total and phospho).To examine the functional consequences of silvestrol on immune surveillance, we utilized a co-culture system where EBV+ LCLs are plated in the presence of autologous PBMCs. Autologous LCL/PBMC co-cultures were plated with silvestrol or DMSO vehicle control and allowed to incubate for 10 days. When EBV+ LCLs are irradiated, expansion of memory EBV-specific CD3+/CD8+ cytotoxic T cells (CTLs), capable of cytotoxicity and IFNg production, is observed. The addition of silvestrol (2–10nM) to co-cultures did not hinder CTL expansion or IFNg production. When unirradiated LCLs were plated in co-cultures, CTL expansion was seen, however, EBV+ LCLs became the dominant population in control conditions by day 10 of culture. Addition of silvestrol to unirradiated co-cultures (2–10nM) led to marked expansion of memory CD3/CD8+ CTLs as well as CD56br NK cells. A dose dependent ablation of viable EBV-LCLs was observed in unirradiated co-cultures supporting the notion that silvestrol allowed for the expansion of both innate and adaptive immune effectors that were capable promoting anti-tumor activity. Immune effector populations that expanded in the presence of silvestrol showed preservation of direct cytotoxicity (adaptive immunity) and antibody dependent cell-mediated cytotoxicity assays (ADCC) against EBV+ LCLs (innate immunity) that was comparable to untreated effector populations.We next examined the efficacy of silvestrol in vivo using the Hu-PBL SCID model depleted of murine NK cells. On d14, mice were randomized to receive treatment with either silvestrol (1.5mg/kg) or vehicle control by IP injection every 48hr. Human cell engraftment was measured by quantifying human IgG in blood. While silvestrol treatment did not affect cell engraftment, all treated mice showed improved survival when compared to mice treated with vehicle control (100% silvestrol vs. 22% control alive at day 140, p ≤0.001). Control mice showed decreased body weight (85% decrease) and splenomegally (spleen wt 421.6 mg vs 72.9 mg for control vs silvestrol) on necropsy at day 140, p≤.0004). There were no toxicities observed in silvestrol treated mice. Low dose silvestrol promotes direct anti-proliferative activity of EBV-transformed LCLs while sparing antigen specific adaptive and innate immune effector function that is capable of delivering potent anti-tumor activity in vitro and in vivo. Disclosures:No relevant conflicts of interest to declare.

Antibody-Dependent Cell-Mediated Cytotoxicity in Simian Immunodeficiency Virus-Infected Rhesus Monkeys

With the recent demonstration in the RV144 Thai trial that a vaccine regimen that does not elicit neutralizing antibodies or cytotoxic T lymphocytes may confer protection against human immunodeficiency virus type 1 (HIV-1) infection, attention has turned to nonneutralizing antibodies as a possible mechanism of vaccine protection. In the current study, we evaluated the kinetics of the antibody-dependent cell-mediated cytotoxicity (ADCC) response during acute and chronic SIVmac251 infection of rhesus monkeys. We first adapted a flow cytometry-based ADCC assay, evaluating the use of different target cells as well as different strategies for quantitation of activated natural killer (NK) cells. We found that the use of SIVmac251 Env gp130-coated target cells facilitates analyses of ADCC activity with a higher degree of sensitivity than the use of simian immunodeficiency virus (SIV)-infected target cells; however, the kinetics of the measured responses were the same using these different target cells. By comparing NK cell expression of CD107a with NK cell expression of other cytokines or chemokine molecules, we found that measuring CD107a expression is sufficient for evaluating the anti-SIV function of NK cells. We also showed that ADCC responses can be detected as early as 3 weeks after SIVmac251 infection and that the magnitude of this antibody response is inversely associated with plasma viral RNA levels in animals with moderate to high levels of viral replication. However, we also demonstrated an association between NK cell-mediated ADCC responses and the amount of SIVmac251 gp140 binding antibody that developed after viral infection. This final observation raises the possibility that the antibodies that mediate ADCC are a subset of the antibodies detected in a binding assay and arise within weeks of infection.

Abstract 3665: Enzymatic depletion of pericellular HA from tumor cell surface sensitizes Erbitux dependent immune cell-mediated cytotoxicity

Abstract Hyaluronan (HA), a ubiquitous high-molar-mass linear glycosaminoglycan (GAG) plays a critical role in cell adhesion, motility, growth and differentiation. HA accumulation is associated with many human cancers. Human tumor cells can form HA rich pericellular matrix which might favor tumor growth by preventing the access of host immune cells to malignant cells. Monoclonal antibody (MAb) based therapeutics are emerging as an important component of therapy for human malignancies. Despite the very high specificity of monoclonal antibodies, resistance to MAb therapy is common. Antibody Dependent Cell-mediated Cytotoxicity (ADCC) is an important effector for the clinical efficacy of MAbs and relies on effective interaction between the Fc domain of antigen bound antibody and FcγRIII positive immune cells. We hypothesize that pericellular HA coating may contribute to resistance of HA-rich malignant cells to MAb mediated therapy by preventing efficient access of effector cells to their tumor targets. Enzymatic depletion of HA with PEGPH20 hyaluronidase could potentially sensitize such tumors to MAb mediated therapy. remove ell surface and ated therapy by preventing tumor cells contact EGFR positive breast carcinoma cell line MDA-MB-231luc and genetically engineered HAS2 over expressing MDA-MB-231HAS2 were used to investigate the role of HA-rich pericellular matrix in inhibition of ADCC. ADCC assay was performed by incubating Calcein AM labeled monolayer cells with monoclonal antibody (1h at 37°C) followed by co-incubation of recombinant human IL-2 (rhIL-2) activated human peripheral blood mononuclear cells (PBMC, 5h at 37°C, 5% CO2) with or without PEGPH20. Live cells from each treatment were assessed by fluorescence readings at Ex/Em 488nm/537nm (SpectraMax M2e plate reader) at the end of the assay. Percentages of cell killing were calculated against control. The student's t test was used to compare the difference between treatments. Particle exclusion assay demonstrated that HAS2 over expressing MDA-MB-231HAS2 cells formed a pericellular coat that inhibited access of the rhIL-2 activated PBMC to tumor cells. Incubation with PEGPH20 removed the HA-rich pericellular matrix and promoted contact of PBMC to tumor cells. Incubation of Erbitux with rhIL-2 activated PBMC induced a dose dependent killing of both MDA-MB-231luc and MDA-MB-231HAS2 cells. PEGPH20 increased ADCC by 10% on MDA-MB-231luc (HAlow) and 25% on MDA-MB-231HAS2 (HAhigh) tumor cells. Our results show that the HA-rich pericellular matrix on adherent tumor cells inhibits ADCC and PEGPH20 depletion of this coat sensitizes the tumor cells to ADCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3665. doi:10.1158/1538-7445.AM2011-3665

Abstract 4565: IMGN529: A therapeutic maytansinoid conjugate of an anti-CD37 antibody with multiple mechanisms of action for B-cell lymphoma and leukemia

Abstract CD37 is a B-cell surface antigen that is an attractive target for antibody and antibody-drug conjugate mediated therapies due to its restricted expression profile. It is expressed on malignant B-cells in NHL and CLL, but on normal tissue its expression is highly restricted to B-cells present in blood and lymphoid tissues. A large panel of anti-CD37 murine monoclonal antibodies were generated and screened for their specific CD37 binding affinity, direct anti-proliferative activity and pro-apoptotic activity against lymphoma cell lines. Selected antibodies were humanized by variable domain resurfacing and one antibody, designated K7153A, demonstrated the best overall activity in terms of direct antibody activity as well as effector function. K7153A demonstrated much stronger pro-apoptotic activity against Ramos and Raji cells than either of two reference compounds, the anti-CD37 SMIP TRU-016 or the anti-CD20 antibody rituximab, and did not require cross-linking to achieve this effect. The antibody-maytansinoid conjugate, IMGN529, was produced by conjugation of K7153A with the potent maytansinoid, DM1, via the non-cleavable linker, SMCC. IMGN529 retains the high specific binding affinity of the K7153A antibody, with an EC50 of 0.5 nM. IMGN529 also demonstrated the same strong pro-apoptotic activity as the K7153A antibody against Ramos cells, with an EC50 of 0.1 nM. Antibody-dependent cell-mediated cytotoxicity (ADCC) assays, using purified human NK cells as effector cells, showed that K7153A and IMGN529 have similar potent ADCC activity against Ramos and Daudi cells with an EC50 of less than 10 pM. In addition, both K7153A and IMGN529 demonstrated comparable complement-dependent cytotoxicity (CDC) in the presence of human complement against Ramos cells. These results indicate that IMGN529 retains the intrinsic functions of the K7153A antibody. IMGN529 was highly cytotoxic in vitro against NHL cell lines such as Daudi, BJAB, Namalwa and SU-DHL-4 with a greater degree of cell killing and lower EC50 value (19 – 36 pM) than the K7153 antibody alone. In contrast, TRU-016 showed no effect on any of these cell lines and rituximab was only active against SU-DHL-4 cells. In vivo, IMGN529 showed markedly higher efficacy against established SU-DHL-4 and BJAB xenograft tumors than the K7153A antibody alone, with significant anti-tumor activity at single doses of 5 mg/kg or lower. Together, these results demonstrate that IMGN529 combines the strong pro-apoptotic activity, CDC and ADCC activity of its anti-CD37 antibody component with the potent cytotoxic activity provided by the targeted delivery of its maytansinoid payload. IMGN529 is a highly active antibody-drug conjugate with a unique combination of anti-tumor activities and is therefore a promising therapeutic candidate for the treatment of CD37-positive lymphomas and leukemias. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4565. doi:10.1158/1538-7445.AM2011-4565

Abstract 1768: A bioluminescent cell-based reporter bioassay alternative to ADCC bioassay

Abstract We have developed a bioluminescent cell-based reporter bioassay with reduced variability and complexity compared with traditional antibody-dependent cell-mediated cytotoxicity (ADCC) bioassays, in order that more accurate and precise quantification of therapeutic antibody Fc functionality can be achieved. Traditional bioassays to quantify ADCC are labor intensive, have high inherent assay variability, and routinely use primary NK cells from blood donors. Our bioluminescent cell-based reporter bioassay is based on the same NFAT pathway-based activation of gene transcription which occurs in NK cells after ligation of FcγRIIIA with target cell-bound antibody in a traditional ADCC assay system. In NK cells this leads to activation of cytokine and FasL gene transcription; FasL participates in lysis of target cells. We generated Jurkat T-cell lines which stably express both human FcγRIIIA and an NFAT-luciferase reporter. These cell lines were evaluated as effector cell replacements of primary NK effector cells in bioassays. The resultant reporter bioassay signal was robust and exhibited appropriate specificity, meaning robust signal above background and appropriate dose-response were achieved and were absolutely dependent on specific target and effector cells, and specific antibody. A “best” effector cell clone was selected based on bioassay performance and stability of the clone. Two types of target systems were evaluated: suspension CD20+ B cell targets with rituximab (anti-CD20) and adherent Her2+ cell targets with trastuzumab (anti-Her2). Our reporter bioassay performed well with both target test systems to quantify biological activity of the therapeutic antibodies. With a goal toward obtaining high inter-assay precision of the bioassay, we evaluated growing and freezing cells under specific conditions to identify those which allowed the cells to be ready for bioassay with minimal handling after thaw and we were able to identify such conditions. Additionally, we identified passage range suitable for banking vials of cells to which we can return time after time, obviating the variability that can arise when using continuously cultured cells for bioassay. We further optimized our bioassay using design of experiments (DoE) and established range of effector to target cell ratios over which the assay gave suitable bioassay performance in test systems. A qualification study was performed for characterization of precision, accuracy, linearity across potency range and stability-indicating ability. The bioluminescent cell-based reporter bioassay provides a robust, low variability and easier-to-perform alternative to traditional ADCC bioassays for quantification of Fc effector functionality of therapeutic antibody candidates. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1768. doi:10.1158/1538-7445.AM2011-1768

Antibody-dependent cell-mediated cytotoxicity is induced by a single-chain Fv-protein III fusion in the presence of a rabbit anti-protein III polyclonal antibody

The use of phage-displayed antibody libraries has enabled the isolation of several thousand cancer-specific monoclonal antibodies. To further select for clones among these antibodies which have therapeutic potential for cancer, several types of in vitro anti-tumor assay, such as an antibody-dependent cell-mediated cytotoxicity (ADCC) assay, are required. The cytotoxic activities of effector cells are triggered by the binding of the Fc portion of IgG to its receptor, necessitating the conversion of a candidate clone with a single-chain variable fragment (scFv) form into a human IgG form. In the laboratory however, this conversion process is expensive and involves laborious steps such as the cloning of mammalian cells that contain an IgG expression vector, the subsequent production of protein, and affinity purification. In our current study, we show that an original fusion of scFv and protein III, a coat protein of the M13 bacteriophage, can induce ADCC activity towards its target cells in the presence of a rabbit anti-protein III polyclonal antibody. Our modified assay method thus enables the more rapid selection of potentially therapeutic clones from phage-displayed antibody libraries.

Towards effective immunotherapy of myeloma: enhanced elimination of myeloma cells by combination of lenalidomide with the human CD38 monoclonal antibody daratumumab

In our efforts to develop novel effective treatment regimens for multiple myeloma we evaluated the potential benefits of combining the immunomodulatory drug lenalidomide with daratumumab. Daratumumab is a novel human CD38 monoclonal antibody which kills CD38+ multiple myeloma cells via antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity and apoptosis. To explore the effect of lenalidomide combined with daratumumab, we first carried out standard antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity assays in which the CD38+ multiple myeloma cell line UM-9 and primary multiple myeloma cells isolated from patients were used as target cells. We also tested the effect of lenalidomide on daratumumab-dependent cell-mediated-cytotoxicity and complement-dependent cytotoxicity of multiple myeloma cells directly in the bone marrow mononuclear cells of multiple myeloma patients. Finally, we determined the daratumumab-dependent cell-mediated cytotoxicity using peripheral blood mononuclear cells of multiple myeloma patients receiving lenalidomide treatment. Daratumumab-dependent cell-mediated cytotoxicity of purified primary multiple myeloma cells, as well as of the UM-9 cell line, was significantly augmented by lenalidomide pre-treatment of the effector cells derived from peripheral blood mononuclear cells from healthy individuals. More importantly, we demonstrated a clear synergy between lenalidomide and daratumumab-induced antibody-dependent cell-mediated cytotoxicity directly in the bone marrow mononuclear cells of multiple myeloma patients, indicating that lenalidomide can also potentiate the daratumumab-dependent lysis of myeloma cells by activating the autologous effector cells within the natural environment of malignant cells. Finally, daratumumab-dependent cell-mediated cytotoxicity was significantly up-regulated in peripheral blood mononuclear cells derived from 3 multiple myeloma patients during lenalidomide treatment. Our results indicate that powerful and complementary effects may be achieved by combining lenalidomide and daratumumab in the clinical management of multiple myeloma.