Glycosylation of the antibody Fc fragment is essential for Fc receptor–mediated activity. Carbohydrate heterogeneity is known to modulate the activity of effector cells in the blood, in which ...fucosylation particularly affects NK cell–mediated killing. Here, we investigated how the glycosylation profile of 2F8, a human IgG1 monoclonal antibody against epidermal growth factor receptor in clinical development, impacted effector function. Various 2F8 batches differing in fucosylation, galactosylation, and sialylation of the complex-type oligosaccharides in the Fc fragment were investigated. Our results confirmed that low fucose levels enhance mononuclear cell–mediated antibody-mediated cellular cytotoxicity (ADCC). In contrast, polymorphonuclear cells were found to preferentially kill via high-fucosylated antibody. Whole blood ADCC assays, containing both types of effector cells, revealed little differences in tumor cell killing between both batches. Significantly, however, high-fucose antibody induced superior ADCC in blood from granulocyte colony-stimulating factor–primed donors containing higher numbers of activated polymorphonuclear cells. In conclusion, our data demonstrated for the first time that lack of fucose does not generally increase the ADCC activity of therapeutic antibodies and that the impact of Fc glycosylation on ADCC is critically dependent on the recruited effector cell type.
Genetic differences between humans and in vivo model systems, including mice and nonhuman primates, make it difficult to predict the efficacy of immunoglobulin G (IgG) activity in humans and ...understand the molecular and cellular mechanisms underlying that activity. To bridge this gap, we established a small-animal model system that allowed us to study human IgG effector functions in the context of an intact human immune system without the interference of murine Fcγ receptors expressed on mouse innate immune effector cells in vivo. Using a model of B cell depletion with different human IgG variants that recognize CD20, we show that this humanized mouse model can provide unique insights into the mechanism of human IgG activity in vivo. Importantly, these studies identify the bone marrow as a niche with low therapeutic IgG activity.
CD40 is highly expressed on various B-lineage malignancies and represents an attractive immunotherapy target for neoplastic disease. Previous work showed that engineering the Fc domain of an antibody ...for increased binding to Fcγ receptors (FcγRs) significantly enhanced Fc-mediated immune effector function and antitumor activity in vitro and in vivo. We developed a humanized anti-CD40 antibody similarly Fc-engineered for increased FcγR binding (XmAbCD40) and compared its efficacy with that of an anti-CD40 native IgG1 analog and the anti-CD20 antibody rituximab. XmAbCD40 increased antibody-dependent cell-mediated cytotoxicity (ADCC) up to 150-fold relative to anti-CD40 IgG1 against B-lymphoma, leukemia, and multiple myeloma cell lines, and significantly enhanced ADCC against primary tumors. XmAbCD40 was also superior to rituximab in enhancing ADCC (both in cell lines and primary tumors) and in augmenting antibody-dependent cellular phagocytosis. XmAbCD40 significantly inhibited lymphoma growth in disseminated and established mouse xenografts and was more effective than the IgG1 analog or rituximab. An anti-CD40 antibody constructed to abrogate FcγR binding showed no reduction of tumor growth, indicating that the in vivo antitumor activity of XmAbCD40 is primarily mediated via FcγR-dependent mechanisms. These data demonstrate that XmAbCD40 displays potent antitumor efficacy and merits further evaluation for the treatment of CD40+ malignancies.
Ab-dependent polymorphonuclear granulocyte (PMN)-mediated cytotoxicity may play an important role in the control of malignant diseases. However, little is known as to which particular pathways are ...used for the killing of malignant cells by PMN. The production of reactive oxygen intermediates (ROI) has been observed to occur during Ab-dependent, cell-mediated cytotoxicity (ADCC). However, PMN from a patient with chronic granulomatous disease demonstrated strong ADCC against malignant lymphoma cells. Furthermore, the inhibition of ROI production in PMN from healthy donors had no significant effect on ADCC. Therefore, ROI production by the NADPH oxidase of PMN does not appear to be mandatory for PMN-mediated ADCC. Recent data suggest a role for perforins in PMN-mediated cytotoxicity. However, in our assays concanamycin A, an inhibitor of perforin-mediated ADCC by mononuclear cells, had no inhibitory effect on PMN-mediated ADCC. Using electron microscopy we observed that PMN and their target cells intimately interact with the formation of interdigitating membrane protrusions. During PMN and target cell contact there was a mutual exchange of fluorescent membrane lipid dyes that was strongly increased in the presence of tumor-targeting Abs. This observation may be closely related to the recently described process of trogocytosis by lymphocytes. The presence of transient PMN-tumor cell aggregates and the accumulation of PMN with tumor cell-derived membrane lipids and vice versa were associated with effective ADCC as measured by chromium-release or apoptosis induction.
An alternative reduced-toxicity conditioning regimen for allogeneic transplantation, based on treosulfan and fludarabine, has recently been identified. The rationale for this study was to investigate ...the efficacy and safety of this regimen prospectively in patients with a primary myelodysplastic syndrome.
A total of 45 patients with primary myelodysplastic syndromes were conditioned with 3×14 g/m(2) treosulfan and 5×30 mg/m(2) fludarabine followed by allogeneic hematopoietic stem cell transplantation. Subtypes of myelodysplastic syndromes were refractory anemia with excess blasts-2 (44%), refractory cytopenia with multilineage dysplasia (27%), refractory anemia (9%), refractory anemia with ringed sideroblasts (4%), refractory cytopenia with multilineage dysplasia and ringed sideroblasts (4%), refractory anemia with excess blasts-1 (2%), and myelodysplastic syndrome with isolated del (5q) (2%). The myelodysplastic syndrome was unclassified in 7% of the patients. Forty-seven percent of the patients had a favorable karyotype, 29% an unfavorable one, and 18% an intermediate karyotype. Patients were evaluated for engraftment, adverse events, graft-versus-host disease, non-relapse mortality, relapse incidence, overall survival and disease-free survival.
All but one patient showed primary engraftment of neutrophils after a median of 17 days. Non-hematologic adverse events of grade III-IV in severity included mainly infections and gastrointestinal symptoms (80% and 22% of the patients, respectively). Acute graft-versus-host disease grade II-IV developed in 24%, and extensive chronic graft-versus-host disease in 28% of the patients. After a median follow-up of 780 days, the 2-year overall and disease-free survival estimates were 71% and 67%, respectively. The 2-year cumulative incidences of non-relapse mortality and relapse were 17% and 16%, respectively.
Our safety and efficacy data suggest that treosulfan-based conditioning therapy is a promising treatment option for patients with myelodysplastic syndromes. clinicaltrials.gov identifier: NCT01062490.
CD96, a cell surface antigen recently described to be preferentially expressed on acute myeloid leukemia (AML) leukemic stem cells (LSC) may represent an interesting target structure for the ...development of antibody-based therapeutic approaches. The v-regions from the CD96-specific hybridoma TH-111 were isolated and used to generate a CD96-specific single chain fragment of the variable regions (scFv). An affinity maturated variant resulting in 4-fold enhanced CD96-binding was generated by random mutagenesis and stringent selection using phage display. The affinity maturated scFv CD96-S32F was used to generate bivalent mini-antibodies by genetically fusing an IgG1 wild type Fc region or a variant with enhanced CD16a binding. Antibody dependent cell-mediated cytotoxicity (ADCC) experiments revealed that Fc engineering was essential to trigger significant effector cell-mediated lysis when the wild type scFv was used. The mini-antibody variant generated by fusing the affinity-maturated scFv with the optimized Fc variant demonstrated the highest ADCC activity (2.3-fold enhancement in efficacy). In conclusion, our data provide proof of concept that CD96 could serve as a target structure for effector cell-mediated lysis and demonstrate that both enhancing affinity for CD96 and for CD16a resulted in mini-antibodies with the highest cytolytic potential.
We investigated cup-like nuclear morphology of acute myeloid leukemia blasts in 266 randomly selected patients and its association with hematologic findings, disease markers and outcome data. ...Cup-like acute myeloid leukemia was diagnosed in 55 patients (21%). It was associated with female sex, high white blood cell and blast cell counts, normal karyotype, and low CD34 and HLA-DR expression. Mutations of FLT3, NPM1 or both were detected in 84.9% compared with 58.1% in cases without this morphology (p=0.001). There was no influence on response to treatment or survival. Therefore, cup-like nuclear morphology is an indicator of normal karyotype and should guide more specific molecular analyses.
Recent studies in HER-2/neu-targeted immunotherapy demonstrated that polymorphonuclear neutrophils (PMN) mediated Ab-dependent cellular cytotoxicity against HER-2/neu-positive breast cancer cell ...lines. However, the mechanism of cell death remained unclear. We used several assays to analyze the induction of apoptosis in the breast cancer cell line SK-BR-3 via PMN-dependent Ab-dependent cellular cytotoxicity. In the presence of the HER-2/neu Ab 520C9 and PMN from healthy donors, apoptosis occurred as detected by annexin V binding and disappearance of euploid SK-BR-3 nuclei, which can be differentiated from PMN nuclei by their increased DNA contents. Apoptosis induction was observed with E:T cell ratios as low as 10:1. Laser scanning fluorescence microscopy of TUNEL tumor cells or staining for cleaved cytokeratin-18 further confirmed apoptosis of the SK-BR-3 breast cancer cells. Killing via 520C9 was dependent on the interaction with FcR on PMN, because 1) F(ab')(2) fragments of 520C9 mediated no cytotoxicity, 2) target cell death was influenced by a biallelic polymorphism of FcgammaRIIa on the effector cells, and 3) a bispecific Ab against HER-2/neu and the IgA receptor (FcalphaRI) expressed on effector cells significantly induced apoptosis. Thus, PMN induce Ab-dependent apoptosis against human breast cancer cells targeted with HER-2/neu-directed mAbs or FcR directed bispecific Abs.
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