Cell Antibody-dependent Cellular Cytotoxicity Research Articles

Comparative Assessment of Different Clinically Utilized CD20 Directed Antibodies in Chronic Lymphocytic Leukemia (CLL) Cells Reveals Divergent NK-Cell, Monocyte and Macrophage Properties,

Abstract 3717The anti-CD20 antibody rituximab represents the first therapy to contribute to prolongation of survival in CLL yet controversy exists as to how it promotes tumor elimination. Pre-clinical studies in CLL have suggested that innate immune cells, complement, and direct antibody killing might contribute to anti-CD20 antibody efficacy. Murine models of B-cell depletion by CD20 antibodies have shown monocytes to be the most important effector cell, although significant controversy around this point exists. Given the success of rituximab, second generation anti-CD20 antibodies (Ofatumumab and GA101 are now in phase III testing in CLL with reported improved direct (GA101), complement (Ofatumumab), or NK cell effector cell killing (GA101). To date, direct comparison of these three antibodies for direct, complement, and effector cell engagement has not been performed. Additionally, the impact of afucosylation engineering of GA101 on monocyte and macrophage function has not been reported. As a Type II anti-CD20 antibody, GA101 mediated significantly increased cell death (∼25%) without Fc crosslinking when compared to Ofatumumab or rituximab (5–8%). Direct cytotoxicity assessment of 19 pts with cross-linking demonstrated GA101 (25%) to mediate significantly greater (p=0.0003) killing than rituximab (15%) but not Ofatumumab (20%). Complement mediated killing was significantly increased over media with Ofatumumab (∼30%), whereas an average of only 5–10% killing was observed with rituximab or GA101. Immobilized GA101 significantly increased NK cell activation as detected by IFNg production and CD107a induction (p=0.005) more than rituximab or Ofatumumab. In addition, GA101 mediates 2 to 3 fold greater NK cell-mediated Antibody Dependent Cellular Cytotoxicity (ADCC) compared to Ofatumumab or rituximab at higher antibody concentrations, but not at concentrations less than 0.05 mg/ml. This enhanced ADCC with GA101 is seen with normal NK cells as well as NK cells from CLL patients. Ofatumumab mediated significantly greater (p=0.0001) NK cell ADCC than rituximab. Given the importance of macrophages in depletion of anti-CD20 tumors in mouse models of lymphoma, we next focused on this. The three anti-CD20 antibodies show Antibody Dependent Cellular Phagocytosis (ADCP) capability with Monocyte Derived Macrophages (MDM) against CLL B cells, with Ofatumumab exhibiting the greatest ADCP compared to GA101 (60% vs 40%; p=0.0036). In addition, primary monocytes stimulated with immobilized GA101 show less TNFa release, when compared to rituximab or Ofatumumab. To investigate this mechanistically, monocytes were stimulated with plate bound rituximab, Ofatumumab or GA101. Compared to the non Fc engineered antibodies (rituximab and Ofatumumab) Fc engineered GA101 induces reduced pan tyrosine phosphorylation and phosphorylation of ERK. No differences in phosphorylation of FcgRIIa or FcgRIIb was observed between the engineered and non-engineered antibodies. Further mechanistic studies to elucidate these differences in monocytes based upon antibody afucosylation changes, or differential phosphorylation of FcgR are ongoing. Collectively, our data indicate that GA101 and Ofatumumab are both superior to rituximab against CLL cells and have differential properties with respect to apoptosis, CDC, and effector cell-mediated killing. GA-101 mediates the most potent NK cell mediated killing at high concentrations whereas Ofatumumab has the greatest monocyte activation and phagocytosis despite absence of any engineering. These findings have relevance in the choice of the optimal CD20 antibody for treatment of CLL and combination strategies used. Disclosures:No relevant conflicts of interest to declare.

NK Cell Function and Antibodies Mediating ADCC in HIV-1-Infected Viremic and Controller Patients

Natural killer (NK) cells have been suggested to play a protective role in HIV disease progression. One potent effector mechanism of NK cells is antibody-dependent cellular cytotoxicity (ADCC) mediated by antiviral antibodies binding to the FcγRIIIa receptor (CD16) on NK cells. We investigated NK cell-mediated ADCC function and the presence of ADCC antibodies in plasma from 20 HIV-1-infected patients and 10 healthy donors. The HIV-positive patients were divided into two groups: six who controlled viremia for at least 8 y without treatment (controllers), and 14 who were persistently viremic and not currently on treatment. Plasma from both patient groups induced NK cell IFN-γ expression and degranulation in response to HIV-1 envelope (Env) gp140-protein-coated cells. Patient antibodies mediating ADCC were largely directed towards the Env V3 loop, as identified by a gp140 protein lacking the V3 loop. Interestingly, in two controllers ADCC-mediating antibodies were more broadly directed to other parts of Env. A high viral load in patients correlated with decreased ADCC-mediated cytolysis of gp140-protein-coated target cells. NK cells from both infected patients and healthy donors degranulated efficiently in the presence of antibody-coated HIV-1-infected Jurkat cells. In conclusion, the character of ADCC-mediating antibodies differed in some controllers compared to viremic patients. NK cell ADCC activity is not compromised in HIV-infected patients.

The broadly neutralizing HIV-1 IgG 2F5 elicits gp41-specific antibody-dependent cell cytotoxicity in a FcγRI-dependent manner

A role for antibody-dependent cellular cytotoxicity (ADCC) in controlling initial development of HIV-1 infection is supported by a growing number of studies. 2F5, a broadly HIV-1-neutralizing IgG specific for HIV-1 envelope gp41, has been extensively studied in vitro and in vivo for its neutralizing and transcytosis-blocking activities. In the present paper, we have studied the in vitro ADCC potential of 2F5. We have developed an ADCC model based on either monocytic cell line THP1 or monocytes, both FcγRI(+) FcγRIII(-) as effector cells, and natural killer resistant-CEM (NKr-CEM) either coated with HIV envelope subunit, or stably expressing an X4 tropic HIV-1 envelope as target cells. Finally, in order to better simulate the in vivo situation, we used R5-tropic JR-CSF HIV-1-infected NKr-CEM as targets. ADCC was monitored using a fluorescently based, nonradioactive, easy to use assay. 2F5 triggered ADCC of HIV-1 envelope subunit coated cells. Remarkably, 2F5 at ng/ml concentration elicited ADCC of both X4-tropic HIV-1 envelope-expressing cells, and R5-HIV-infected cells. ADCC relied on binding to the FcγRI on effector cell and was abolished by preincubation of 2F5 with its cognate epitope ELDKWA. The capacity of the broadly neutralizing 2F5 to elicit ADCC, and thereby linking adaptive and innate immunity, expands its prophylactic potential. Raising antibodies to the membrane proximal region of HIV-1 envelope with similar ADCC properties, in addition to neutralization, should be taken into account in HIV-1 vaccine design.

Antibody-Dependent Natural Killer Cell–Mediated Cytotoxicity Engendered by a Kinase-Inactive Human HER2 Adenovirus-Based Vaccination Mediates Resistance to Breast Tumors

Cancer vaccines may have applications in the therapy and prevention of mammary carcinoma. To investigate such applications, we constructed a recombinant adenoviral vaccine expressing a kinase-inactive mutant form of human HER2 and introduced this into BALB/c wild-type (WT) or HER2 transgenic mice. Here, we report contributions by antibody responses and natural killer (NK) cells in tumor protection in this model. One i.p. vaccination protected WT mice from the HER2-expressing mouse carcinoma D2F2/E2. Half of the HER2 transgenic mice were protected fully and long term after preventive vaccination. Tumor growth in mice that eventually developed neoplastic lesions was delayed. Protection in WT and HER2 transgenic mice was associated with high or low levels of IgG2a antibodies, respectively, whereas CTLs were observed in WT but not in HER2 transgenic mice. Depleting CD4(+) or CD8(+) cells in vaccinated WT mice had limited effects, suggesting that protection was largely independent of CD4(+) or CD8(+) T cells. In contrast, antibody-mediated tumor rejection seemed to contribute significantly based on a loss of protection in mice deficient for Fc-γ RI/III or B cells. Further, a role for antibody-dependent cellular cytotoxicity (ADCC) mediated by NK cells was indicated by evidence that vaccine protection could be abolished by in vivo depletion of NK cells. Lastly, NK cells and immune sera purified from WT or HER2 transgenic mice exhibited efficient ADCC of HER2-expressing tumor cells in vitro. Our findings define a critical requirement for NK cells in vaccine-induced protection against HER2-expressing tumors.

A recombinant trispecific single‐chain Fv derivative directed against CD123 and CD33 mediates effective elimination of acute myeloid leukaemia cells by dual targeting

Two trivalent constructs consisting of single-chain Fv antibody fragments (scFvs) specific for the interleukin-3 receptor alpha chain (CD123), CD33 and the Fcgamma-receptor III (CD16) were designed and characterized for the elimination of acute myeloid leukaemia (AML) cells. The dual targeting single-chain Fv triplebody (sctb) [123 x ds16 x 33] and the mono targeting sctb [123 x ds16 x 123] both specifically bound their respective target antigens and were stable in human serum at 37 degrees C for at least 5 d. Both constructs induced potent antibody-dependent cellular cytotoxicity (ADCC) of two different AML-derived CD33- and CD123 double-positive cell lines in the low picomolar range using isolated mononuclear cells (MNCs) as effector cells. In these experiments the dual targeting molecule produced significantly stronger lysis than the mono targeting agent. In addition, the sctbs showed a high potency in mediating ADCC of primary leukaemia cells isolated from peripheral blood or bone marrow of seven AML patients. Hence, these novel molecules displayed potent anti-leukaemic effects against AML cells in vitro and represent attractive candidates for further preclinical development.

Effective Elimination of Acute Myeloid Leukemic Cells by Recombinant Bispecific Antibody Derivatives Directed Against CD33 and CD16

Single-chain Fv triplebodies (sctb), consisting of a single polypeptide chain with 3 single-chain antibody variable fragments connected in tandem, were generated as antileukemic agents. A CD19-specific sctb of this format has previously been shown to be superior to a bispecific single-chain Fv antibody fragment (bsscFv) for the elimination of leukemic B-lineage cells, but corresponding targeted agents for the treatment of acute myeloid leukemia are still lacking. For this purpose, both a bsscFv and a sctb specific for CD33 and the trigger molecule CD16 (FcgammaRIII) were produced. The sctb displayed 3.5-fold greater avidity for CD33 than the bsscFv 33xds16, whereas both had close to equal affinity for CD16. In antibody-dependent cellular cytotoxicity (ADCC) reactions with human mononuclear cells as effectors, both the bsscFv 33xds16 and the sctb induced lysis of tumor cells with half maximum effective concentrations (EC50) in the low picomolar range. It is interesting to note that the sctb promoted equal lysis of human leukemia-derived cell lines at 10 to 200-fold lower concentrations than the bsscFv. Both molecules mediated ADCC of primary patient cells. In conclusion, both the bsscFv 33xds16 and the sctb 33xds16x33 eliminated acute myeloid leukemia cells in ADCC reactions, but the novel sctb format showed significantly greater specific activity.

Influence of NKG2D/DNAM-1 interaction with tumor cell ligands on the ability of lenalidomide to enhance ADCC of antibody-coated solid tumor cells.

e21008 Background: Lenalidomide has been shown to enhance the antibody- dependent cellular cytotoxicity (ADCC) of rituximab-treated B cell chronic lymphocytic leukemia (B-CLL) and non-Hodgkin lymphoma (NHL) tumor cells. The combination appears to be especially active in patients so we sought to build on these observations by determining lenalidomide's ability to enhance ADCC of solid tumor cells. Methods: Purified NK cells were used as effector cells and tumor cell lines were used as targets after pre-treatment with cetuximab or trastuzumab. 23 tumor cell lines were used; 11 colorectal (CRC), 3 breast, 3 ovary, 2 lung, 2 head and neck and 2 bone sarcoma. Cytotoxicity was assessed using a standard lactate dehydrogenase release assay and specific lysis calculated. NK cell expression of activating receptors from 30 donors and tumor cell expression of NK cell ligands were assessed by flow cytometry. Results: Lenalidomide increased NK cell-specific lysis of cetuximab-coated CRC cells; HCT- 116, from 19% to 39% (p < 0.01), HT-29, 32% to 50% (p < 0.05) and also of trastuzumab-coated breast cancer cells; SKBR-3, 32% to 44% (p < 0.01), MCF-7, 30% to 70% (p < 0.01). Variable sensitivity of CRC cell lines was dependent on EGFR expression (range 5%-96%) but independent of either KRAS or BRAF mutational status or FCγRIIIa genotype. CRC cell line expression of NK cell ligands, PVR, MIC-A, ULBP2 and ULBP3 correlated with sensitivity to ADCC. NK cell expression of DNAM-1, NKG2D, but not NKp46, correlated with the extent of ADCC and its enhancement by lenalidomide. Antibody blocking of NKG2D, and to a lesser extent DNAM-1, inhibited CRC cell killing from 70% to 31% and 48% respectively (both p < 0.05), whereas blocking of individual cognate ligands had minimal effect. Lenalidomide enhanced NK cell production of gmcsf, IL-8, MIP-1α/βm RANTES, MCP-1 and IP-10 but reversed phosphorylation of Src family kinases induced by antibody alone. Conclusions: Lenalidomide enhances the ADCC of solid tumor cells, the extent of which is largely dependent on NKG2D/NKG2D ligand interactions, providing a rationale for exploratory clinical studies and assessment of potential biomarkers predictive lenalidomide-mediated ADCC. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Celgene Celgene

In vivo Cytotoxicity of Type I CD20 Antibodies Critically Depends on Fc Receptor ITAM Signaling

Antibody-Fc receptor (FcR) interactions play an important role in the mechanism of action of most therapeutic antibodies against cancer. Effector cell activation through FcR triggering may induce tumor cell killing via antibody-dependent cellular cytotoxicity (ADCC). Reciprocally, FcR cross-linking of antibody may lead to the induction of apoptotic signaling in tumor cells. The relative importance of these bisecting pathways to in vivo antibody activity is unknown. To unravel these roles, we developed a novel mouse model with normal FcR expression but in which FcR signaling was inactivated by mutation of the associated gamma-chain. Transgenic mice showed similar immune complex binding compared with wild-type mice. In contrast, ADCC of cells expressing frequently used cancer targets, such as CD20, epidermal growth factor receptor, Her2, and gp75, was abrogated. Using the therapeutic CD20 antibodies ofatumumab and rituximab, we show that FcR cross-linking of antibody-antigen immune complexes in the absence of gamma-chain signaling is insufficient for their therapeutic activity in vivo. ADCC therefore represents an essential mechanism of action for immunotherapy of lymphoid tumors.

Depletion of the C3 component of complement enhances the ability of rituximab-coated target cells to activate human NK cells and improves the efficacy of monoclonal antibody therapy in an in vivo model

AbstractGrowing evidence indicates antibody-dependent cellular cytotoxicity (ADCC) contributes to the clinical response to monoclonal antibody (mAb) therapy of lymphoma. Recent in vitro analysis suggests C3b can inhibit mAb-induced natural killer (NK)–cell activation and ADCC. Further studies were conducted to assess the effect of C3 depletion on mAb-induced NK activation and therapy of lymphoma. Normal human serum inhibited the ability of rituximab-coated lymphoma cells to activate NK cells as previously reported. Serum did not inhibit NK-cell activation when it was preincubated with cobra venom factor (CVF) to deplete C3. Similar results were found when transudative pleural fluid or nonmalignant ascites was used as surrogates for extravascular fluid, suggesting the inhibitory effect of complement may be present in the extravascular compartment, in which many malignant lymphocytes reside. In vivo, C3 was depleted before mAb treatment in a syngeneic murine model of lymphoma. Survival of lymphoma-bearing mice after treatment with CVF plus mAb and with a human C3 derivative with CVF-like functions (HC3-1496) plus mAb was both superior to that of mAb alone. These studies show that complement depletion enhances NK-cell activation induced by rituximab-coated target cells and improves the efficacy of mAb therapy in a murine lymphoma model.

Anti-PR1/HLA-A2 Antibody (8F4, A TCR-Mimic) Mediates Specific Lysis of AML Stem Cells but Not Normal Hematopoietic Stem Cells.

Abstract 15PR1 (VLQELNVTV) is an HLA-A2-restricted peptide derived from endogenous myeloid leukemia-associated antigens (LAA) proteinase 3 and neutrophil elastase. PR1/HLA-A2 is targeted by PR1-specific cytotoxic T lymphocytes (PR1-CTL) that contribute to cytogenetic remission in patients with CML after allogeneic stem cell transplantation or interferon therapy, and PR1 peptide vaccination induces specific CD8 immunity in AML, MDS, and CML patients. Because clinical effects after PR1 vaccination are associated with a low amount of leukemia and because the effects on leukemia stem cells (LSCs) are unknown, we sought to develop a PR1/HLA-A2-specific MAB that could be more effective in high disease burden cases and that might also target LSC. We previously reported a mouse IgG2A antibody (8F4) with PR1/HLA-A2 specificity. We now used 8F4 to study PR1 expression by FACS and confocal mircroscopy on leukemia subsets and normal peripheral blood mononuclear cells (PBMC). Circulating blasts from peripheral blood (PB) or leukapheresis products (LP) from AML patients (5 HLA-A2+, 2 control HLA-A2-,) were studied (see Table 1). PR1 expression (MFI±SEM) was higher in AML (23.7±5.18) compared to control (4.0±2.42) and normal PBMC (13.6±0.23; p = 0.046). Confocal microscopy confirmed bright heterogeneous surface expression of PR1 on AML, but was nearly absent on healthy donor PB. Interestingly, PR1 expression was higher on LSC (CD34+CD38-lin-) (76±44) compared to healthy donor HSC (CD34+CD38-lin-) (13.4), which suggests that only differentiated myeloid cells might be more susceptible to 8F4 toxicity. To study whether 8F4 mediated specific lysis of PR1+ cells, we determined cell-mediated cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) of 8F4 compared to isotype control. 8F4 induced 51% specific lysis by CDC and 15% specific lysis by ADCC of PR1-pulsed T2 cells, which was dose-dependent on both 8F4 and PR1, with no lysis of target cells by isotype control. Moreover, 8F4 induced CDC-mediated lysis of blasts from 5/5 HLA-A2+ AML patients, but not blasts from an HLA-A2- AML patient (see Table 1), or mononuclear cells from HLA-A2+ healthy donors. To study the in vivo effects of 8F4 on AML, we used an established xenogeneic model of human AML in NOD/SCID mice. 1 × 106 blasts from 4 HLA-A2+ AML patients (including the HLA-A2+ patients susceptible to 8F4-mediated lysis in vitro) and 1 HLA-A2- patient were mixed for one hour under serum-free conditions with 8F4 or isotype control antibody and adoptively transferred to NOD/SCID mice. Engraftment of AML was compared on day 14 and AML growth was confirmed on day 28 by comparing FACS analysis and histopathology of BM from sacrificed animals. 8F4 treatment of AML cells, but not isotype treatment, abrogated engraftment of 4/4 HLA-A2+ AML patients, but not of 1 HLA-A2- AML patient on d14. By day 28, AML expanded by 150% in one surviving isotype-treated animal. Thus, 8F4-mediated specific lysis of AML and LSC by CDC and ADCC mechanisms in vitro were confirmed by in vivo results of AML engraftment. Taken together, these results show that 8F4, a novel TCR-mimic antibody that targets only PR1/HLA-A2+ cells, mediates lysis of AML blasts and LSC, but not total normal BM cells or HSC. Therefore, 8F4 may be useful as an immunotherapeutic agent in the treatment of HLA-A2+ myeloid leukemia, and as the first antibody therapy that may target and eliminate LSC.Table 1Patients' characteristics and 8F4 effectsUPNDiagnosis+Disease statusHLA-A2SexAgeSample*Blast8F4 FCMIn vitro CDC lysisIn vivo Engraftmentiso8F4AML1AML-M1 secondary MyelofibrosisUntreated+M79LP87%++++−AML2AML-M2Relapsed+M61PB66%++++Low*−AML3AML-M5Refractory+F73PB64%+++++−AML4AML-M1Untreated+F55LP92%+++N/DN/DAML5AML-M2Refractory relapsed+M33PB98%N/DN/D+−AML6AML-M5Untreated+M53LP96%++N/DN/DN/DAML7AML-M1Untreated+M24LP85%++−−AML8AML-M1Untreated−F41PB91%−−LowLow+French-American-British AML subtypeFCM flowcytometry staining intensity (+ indicates staining intensity close to those of normal PBMC, ++ and +++ indicate increased intensity)N/D The experiment was not performed due to low viability of sample (less than 50%) or sample was not availableLow* - indicates engraftment <0.5% of viable mCD45- cells, effect of treatment is inaccessible.*Peripheral blood (PB) or leukopheresis (LP) Disclosures:No relevant conflicts of interest to declare.

Macrophage Polarization and Acquired Resistance to Rituximab in CNS Lymphoma.

Abstract 3749Poster Board III-685Macrophages are a critical component of anti-tumor immunity but may be subverted from the classically-activated, or M1 phenotype, which mediates tumor elimination, to an alternatively-activated, M2 phenotype, which promotes tumor progression. Interleukin-4 (IL-4) signaling is a pivotal regulator of macrophage polarization to the M2 phenotype.The efficacy of rituximab appears to be mediated predominantly by antibody-dependent cellular cytotoxicity (ADCC) with tumor-associated macrophages being the dominant effector cell. We hypothesize that the phenotype of tumor-associated macrophages (M1 vs. M2) may be a potential determinant of rituximab efficacy or resistance. Relevant to this hypothesis is our demonstration of the intratumoral expression of IL-4 by CNS lymphoma (Blood, 2006; Clin Cancer Res., 2009) as well as our observation that the M2 polarization of macrophages by IL-4 treatment in vitro (18 h) results in an 8-fold decrease in rituximab-mediated ADCC of Raji lymphoma cells compared to ADCC-mediated by naïve or M1 polarized macrophages (p<0.001).There is an extreme paucity of molecular information regarding the role and phenotype of tumor macrophages in non-Hodgkin lymphoma in general, including CNS lymphoma. While macrophages from the cerebrospinal fluid (CSF) are routinely quantified in cytospin analyses, to date there is no established information regarding the phenotype and state of differentiation of macrophages within the leptomeningeal compartment. We have developed a novel flow-cytometry-based protocol for the isolation and phenotypic characterization of macrophages from the CSF of patients with CNS lymphoma. We have used candidate markers of M2 differentiation based upon our gene expression studies of CD14+ activated CSF macrophages, including CD206 and Factor XIII (each of which have previously been shown to be induced in macrophages upon IL-4 stimulation). We have identified M1 differentiation of CSF CD14+ macrophages by high fluorescence when incubated with DAF-FM diacetate, a cell-permeable marker of nitric oxide synthase (iNOS). Using flow-cytometry to evaluate the relative expression of DAF-FM and CD206, we demonstrate the presence of at least four subpopulations of activated macrophages in the CSF of CNS lymphoma patients. M1 macrophages, which highly express iNOS are denoted as DAF-FM(+)/CD206(-). M2 macrophages, which weakly express iNOS but which highly express CD206 are denoted as DAF-FM (-)/CD206(+). In addition, we have detected a mixed phenotype with features of both M1 and M2 macrophages, DAF-FM(+)/CD206(+), which we have termed dual-activated. A fourth subpopulation of activated macrophages within the CSF do not express iNOS or CD206.Thus far, CSF macrophage subpopulations have been characterized and sorted from thirty-five subjects: 18 patients with CNS lymphoma and 17 control subjects with non-neoplastic conditions. We demonstrate for the first time the association of M2 macrophages within the CSF with the pathogenesis of CNS lymphoma: there was a greater than six-fold increase in the proportion of macrophages with M2 features in immunocompetent subjects with CNS lymphoma compared to controls (p<0.001). By contrast, the proportion of macrophages with M1 features was similar between lymphoma and controls.In addition, we reproducibly detected an increase in the ratio of M1:M2 macrophages which correlated with therapeutic response to intrathecal methotrexate or cytarabine (within 94 h) in CNS lymphoma patients. By contrast, the intraventricular administration of rituximab was reproducibly associated with a greater than three-fold increase in the relative ratio of M2:M1 macrophages which was sustained compared to macrophages analyzed pre-intrathecal rituximab (p<0.001). In each of four cases, increases in M2 macrophage polarization anticipated the onset of intrathecal rituximab resistance and tumor progression.We believe this to be the first application of flow-cytometry to define the polarization states of intratumoral macrophages in non-Hodgkin lymphoma as well as the first description of dynamic changes in macrophage phenotypes during the evolution of resistance to rituximab therapy. The elucidation of distinct macrophage subpopulations based upon the expression of candidate markers of M1 vs. M2 phenotype may provide insight into tumor pathogenesis and prognosis. Disclosures:Off Label Use: While we describe the use of intrathecal administration of rituximab in patients with recurrent CNS and intraocular lymphomaa, the focus of our study is on the relationship between M2 macrophage polarization and the pathogenesis of CNS lymphoma as well as the potential relationship of macrophage polarization to acquired resistance to rituximab in CNS lymphoma patients.

Ciamexone suppressed anti-islet ADCC of mononuclear cells and serum from type 1 (insulin-dependent) diabetic patients.

ADCC (Antibody-dependent cellular cytotoxicity) against xenogenic islets has frequently been found in newly diagnosed Type 1 (insulin-dependent) diabetics suggesting that when combined with autologous serum in vitro, the destruction of islet cells caused by mononuclear cells (MNC) reflects islet destruction in vivo. In this study the ability of Ciamexone to suppress the anti-islet ADCC in vitro was investigated. We selected both ADCC positive and ADCC negative subjects from a group of Type 1 diabetics. The targets, islets from neonatal rats, were incubated for one hour with the probands serum. The ADCC was then measured by the 51Cr-release of the serum-treated islets after a 6h-incubation with MNC of the same donor. Results both with and without co-incubation of Ciamexone were compared. Ciamexone does not influence the spontaneous 51Cr-release from neonatal rat islets treated with the probands serum but significantly suppresses the anti-islet ADCC. The fact that Ciamexone suppresses anti-islet ADCC may explain its possible effectiveness in Type 1 diabetes.

Depletion of the C3 Component of Complement Enhances the Ability of Rituximab-Coated Target Cells to Activate Human NK Cells and Improves the Efficacy of Monoclonal Antibody Therapy in An in Vivo Model

Antibody-dependent cellular cytotoxicity (ADCC) is felt to play an important role in mediating the anti-tumor effects of Rituximab (R). We previously reported that C3b deposition on R-coated target cells interferes with the binding of the Fc portion of R to NK cell CD16. This interaction inhibits the activation of NK cells and NK cell-mediated ADCC of 51Cr-labeled R-coated target cells. The current studies were designed to determine whether C3 depletion enhances the ability of mAb-coated targets to activate NK cells in vitro and improves mAb therapy in vivo. Normal human serum inhibited the ability of R-coated lymphoma cells to activate NK cells as previously reported. NK activation was increased when serum was pre-incubated with cobra venom factor (CVF) to deplete C3. Similar results were found when non-malignant ascites or transudative pleural fluid, as a surrogate for extravascular fluid, was used as the source of complement. For in vivo analysis, we utilized a syngeneic, immunocompetent murine model in which ADCC has been previously demonstrated to be a key mechanism of action. CVF or a human C3 derivative with CVF-like functions (HC3-1496) was used to deplete C3 in vivo. In this model, C3H/HeN mice were inoculated with murine 38C13 lymphoma cells (day 0) and treated with 2 doses of CVF or HC3-1496 (day 3 and day 5). Four hours after the initial dose of CVF or HC3-1496, mice were treated with a single dose of an anti-lymphoma mAb directed against the 38C13 idiotype (MS11G6). Untreated mice all developed tumor and died with a median survival of 28 days. All mice treated with mAb alone eventually developed tumor and died with a median survival of 42 days. Survival following treatment with CVF plus mAb was superior to that of mAb alone (Fig 1, p=0.0312) with 50% of mice remaining tumor free. Survival following treatment with HC3-1496 plus mAb was also superior to that of mAb alone (Fig 2, p=0.0002) with 80% of mice remaining tumor free. In summary, depletion of the C3 component of complement enhanced the ability of R-coated target cells to activate human NK cells, and improved the efficacy of mAb therapy in an in vivo model of lymphoma. Furthermore, these studies suggest the inhibitory effects of complement on NK activation and ADCC may be seen in the extravascular compartment such as within involved lymph nodes. We conclude that depletion of complement through use of agents such as CVF or HC3-1496 could be considered as an approach to enhancing the efficacy of R therapy. [Display omitted] [Display omitted]

Characterization of MDX-1401, a Human Anti-CD30 Antibody with Enhanced Effector Function, for Therapy of Malignant Lymphoma.

Therapeutic monoclonal antibodies kill target cells by multiple mechanisms including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent phagocytosis (ADP), complement-dependent cytotoxicity (CDC), and direct induction of apoptosis. Antibody binding to FcgRIIIA (CD16), which is expressed on NK cells, macrophages, and CD8+ cells, is essential for ADCC. MDX-1401 is a fully human monoclonal antibody of IgG1 (kappa) isotype that binds to human CD30. This second generation anti-CD30 antibody was derived from parental antibody MDX-060 but differs in that the oligosaccharides of the IgG1 lack fucose, a property that conveys increased antibody affinity for Fc receptor CD16 (FcgRIIIa) and thus stimulates both more potent and efficacious ADCC than a fucosylated IgG1. The KD of the parental antibody for CD30 expressed on L540 Hodgkin's lymphoma cells is approximately 1.4 nM while the KD of MDX-1401 is 1.9 nM, demonstrating that binding affinity for antigen are nearly identical. However, the EC50 for MDX-1401 binding to CD16-transfected cells is approximately 3 nM compared to barely detectable binding by parental antibody, confirming that the absence of fucose conveys increased affinity for FcgRIIIa. Data is presented that demonstrates MDX-1401 has significantly improved potency and efficacy in an ADCC assay as evidenced by a decrease in EC50 as well as an increase in maximum specific lysis. Importantly, the enhanced ADCC activity is observed among a large panel of cell lines with varying numbers of CD30 receptors per cell, including one cell line with low antigen expression in which parental antibody failed to induce ADCC. MDX-1401 enhances ADCC activity with all polymorphic variants including lower affinity FcgRIIIa Phe/Phe158 and FcgRIIIa Phe/Val158 effector cells. Furthermore, MDX-1401 is efficacious in inhibiting growth of CD30+ tumor xenografts in mice. The low doses of antibody required for ADCC activity irrespective of donor genotype, the capacity to mediate ADCC of target cells expressing low levels of CD30, and increased in vivo efficacy support the development of MDX-1401 for treatment of malignant lymphoma. Phase 1 studies in subjects with Hodgkin's lymphoma are currently underway.

TGF-β Utilizes SMAD3 to Inhibit CD16-Mediated IFN-γ Production and Antibody-Dependent Cellular Cytotoxicity in Human NK Cells

TGF-beta can be a potent suppressor of lymphocyte effector cell functions and can mediate these effects via distinct molecular pathways. The role of TGF-beta in regulating CD16-mediated NK cell IFN-gamma production and antibody-dependent cellular cytotoxicity (ADCC) is unclear, as are the signaling pathways that may be utilized. Treatment of primary human NK cells with TGF-beta inhibited IFN-gamma production induced by CD16 activation with or without IL-12 or IL-2, and it did so without affecting the phosphorylation/activation of MAP kinases ERK and p38, as well as STAT4. TGF-beta treatment induced SMAD3 phosphorylation, and ectopic overexpression of SMAD3 resulted in a significant decrease in IFN-gamma gene expression following CD16 activation with or without IL-12 or IL-2. Likewise, NK cells obtained from smad3(-/-) mice produced more IFN-gamma in response to CD16 activation plus IL-12 when compared with NK cells obtained from wild-type mice. Coactivation of human NK cells via CD16 and IL-12 induced expression of T-BET, the positive regulator of IFN-gamma, and T-BET was suppressed by TGF-beta and by SMAD3 overexpression. An extended treatment of primary NK cells with TGF-beta was required to inhibit ADCC, and it did so by inhibiting granzyme A and granzyme B expression. This effect was accentuated in cells overexpressing SMAD3. Collectively, our results indicate that TGF-beta inhibits CD16-mediated human NK cell IFN-gamma production and ADCC, and these effects are mediated via SMAD3.

Lenalidomide Enhances Natural Killer Cell and Monocyte-Mediated Antibody-Dependent Cellular Cytotoxicity of Rituximab-Treated CD20+ Tumor Cells

Lenalidomide has significant activity in myelodysplastic syndromes, multiple myeloma, and non-Hodgkin's lymphoma (NHL). In previous studies, natural killer (NK) cell expansion by lenalidomide was shown to enhance the cytotoxic effect of rituximab. This study assessed the ability of lenalidomide to enhance antibody-dependent cellular cytotoxicity (ADCC) in rituximab-treated NHL cell lines and primary tumor cells from patients with B-cell chronic lymphocytic leukemia (B-CLL) in vitro. An in vitro ADCC system was used to assess the ability of lenalidomide to enhance human NK cell and monocyte function in response to rituximab. Lenalidomide directly enhanced IFN-gamma production via Fc-gamma receptor-mediated signaling in response to IgG. It was also a potent enhancer of NK cell-mediated and monocyte-mediated tumor cell ADCC for a variety of rituximab-treated NHL cell lines in vitro, an effect that was dependent on the presence of antibody and either interleukin-2 or interleukin-12. Lenalidomide also enhanced the ability of NK cells to kill primary tumor cells derived from three patients with B-CLL who have been treated previously with fludarabine plus cyclophosphamide. Enhanced NK cell ADCC was associated with enhanced granzyme B and Fas ligand expression and could be inhibited by a granzyme B inhibitor and partially inhibited by antibody to FasL. Enhanced NK cell Fc-gamma receptor signaling is associated with enhanced phosphorylated extracellular signal-related kinase levels leading to enhanced effector function. These findings suggest that lenalidomide has the potential to enhance the rituximab-induced killing of NHL cell lines and primary B-cell chronic lymphocytic leukemia cells via a NK cell-mediated and monocyte-mediated ADCC mechanism in vitro, providing a strong rationale for the combination of lenalidomide with IgG1 antibodies to target tumor-specific antigens in patients with cancer.

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

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

The Activation of Natural Killer Cell Effector Functions by Cetuximab-Coated, Epidermal Growth Factor Receptor–Positive Tumor Cells is Enhanced By Cytokines

Natural killer (NK) cells express an activating Fc receptor (FcgammaRIIIa) that mediates antibody-dependent cellular cytotoxicity (ADCC) and production of immune modulatory cytokines in response to antibody-coated targets. Cetuximab is a therapeutic monoclonal antibody directed against the HER1 antigen. We hypothesized that the NK cell response to cetuximab-coated tumor cells could be enhanced by the administration of NK cell-stimulatory cytokines. Human NK cells stimulated with cetuximab-coated tumor cells and interleukin-2 (IL-2), IL-12, or IL-21 were assessed for ADCC and secretion of IFN-gamma and T cell-recruiting chemokines. IL-21 and cetuximab were given to nude mice bearing HER1-positive xenografts. Stimulation of human NK cells with cetuximab-coated tumor cells and IL-2, IL-12, or IL-21 resulted in 3-fold to 10-fold higher IFN-gamma production than was observed with either agent alone. NK cell-derived IFN-gamma significantly enhanced monocyte ADCC against cetuximab-coated tumor cells. Costimulated NK cells also secreted elevated levels of chemokines (IL-8, macrophage inflammatory protein-1alpha, and RANTES) that could direct the migration of naive and activated T cells. IL-2, IL-12, and IL-21 enhanced NK cell ADCC against tumor cells treated with cetuximab. The combination of cetuximab, trastuzumab (an anti-HER2 monoclonal antibody), and IL-21 mediated greater NK cell cytokine secretion and ADCC than any agent alone. Furthermore, administration of IL-21 enhanced the effects of cetuximab in a murine tumor model. These results show that cetuximab-mediated NK cell activity can be significantly enhanced in the presence of NK cell-stimulatory cytokines. These factors, therefore, may be effective adjuvants to administer, in combination with cetuximab, to patients with HER1-positive malignancies.

Augmented Antitumor Activity against B-Cell Lymphoma by a Combination of Monoclonal Antibodies Targeting TRAIL-R1 and CD20

Mapatumumab and lexatumumab are fully humanized, high-affinity immunoglobulin G(1 lambda) monoclonal antibodies (mAb) that target/activate the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 1 (TRAIL-R1) and receptor 2 (TRAIL-R2), respectively, triggering the extrinsic apoptotic pathway. Theoretically, synergistic antitumor activity should be observed by combining TRAIL-R mAbs with agents (e.g., rituximab) that activate the intrinsic apoptotic pathway. To this end, targeted antigen expression in a NHL-cell panel was evaluated by flow cytometry. NHL cells were exposed to mapatumumab or lexatumumab followed by rituximab, isotype, or RPMI. DNA synthesis was quantified by [(3)H]-thymidine incorporation assays. Induction of apoptosis was detected by flow-cytometric analysis. For antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CMC) studies, standardized (51)Cr-release assays were done. We inoculated severe combined immunodeficiency (SCID) mouse with Raji cells i.v. The animals then were treated with various combinations of rituximab, mapatumumab, lexatumumab, and isotype alone or in combination. In vitro exposure to mapatumumab resulted in significant apoptosis (30-50%) and decreased DNA synthesis in sensitive lymphoma cells. Mapatumumab/rituximab combination resulted in a significant inhibition of cell proliferation (90% reduction) when compared with mapatumumab (60% reduction) or rituximab (5% reduction). In vivo, the median survival time of animals treated with mapatumumab and rituximab was longer (not reached) than those treated with rituximab monotherapy [33 days (95% confidence interval, 29-37), log-rank test, P = 0.05]. Mapatumumab induces apoptosis, cell growth arrest, ADCC, and CMC. The combination of mapatumumab plus rituximab is more effective in controlling lymphoma growth in vivo than either antibody. Rituximab and mapatumumab warrant further evaluation against B-cell lymphoma.

Lenalidomide and pomalidomide strongly enhance tumor cell killing in vitro during antibody-dependent cellular cytotoxicity (ADCC) mediated by trastuzumab, cetuximab and rituximab

3023 Background: Potential mechanisms of action of lenalidomide and pomalidomide (CC-4047) include anti-angiogenic, anti- proliferative and immunomodulatory activities, e.g., enhancement of T cell and NK cell function. Both drugs appear to enhance T cell activation and Th1-type cytokines in cancer patients. Also, both drugs have been shown to enhance rituximab-mediated protection in a mouse lymphoma model. Methods: We have utilized an in vitro ADCC system to assess the ability of these drugs to enhance human NK cell function in response to the approved therapeutic antibodies trastuzumab, cetuximab and rituximab. Results: Pre-treatment of NK cells with either pomalidomide or lenalidomide greatly enhanced IFN-γ production by NK cells in response to IgG in the presence of either IL-2 or IL- 12. In a series of functional ADCC assays, Her2/neu overexpressing breast cancer cells (SKBR3 &amp; MCF-7) pre-coated with trastuzumab, EGFR positive colorectal cancer cells (HCT-116) pre-coated with cetuximab and NHL cell lines (Namalwa, Farage &amp; Raji) pre-coated with rituximab, were exposed to NK cells pre-treated with pomalidomide, lenalidomide or thalidomide. Both pomalidomide and lenalidomide synergistically increased (up to 6-fold) NK cell-mediated killing of antibody coated tumor cells in a dose-dependent manner. Thalidomide had no effect in this system. There was minimal tumor cell killing by antibody alone or in the absence of antibody. The presence of IL-2 or IL-12 was required to see enhanced killing by either drug. Enhanced ADCC was associated with increased signaling in NK cells, specifically with inhibition of the negative regulator SHIP-1. Other downstream effects on signaling pathways are also being investigated. Monocyte-mediated tumor cell ADCC was also enhanced by both drugs. Conclusions: We have shown that pomalidomide and lenalidomide (but not thalidomide) strongly enhance the ability of therapeutic antibodies to induce ADCC via NK cell/monocyte-mediated killing of tumor cells in vitro. These results provide a strong rationale for combination of either lenalidomide or pomalidomide with antibodies to tumor-specific surface antigens in cancer patients. No significant financial relationships to disclose.