A functional mammalian display screen identifies rare antibodies that stimulate NK cell-mediated cytotoxicity.

Therapies that boost the antitumor immune response have shown a great deal of success. Although most of these therapies have focused on enhancing T cell functions, there is a growing interest in developing therapies that can target other immune cell subsets. Like T cells, natural killer (NK) cells are cytotoxic effector cells that play a key role in the antitumor response. To advance the development of NK-based therapies, we developed a functional screen to rapidly identify antibodies that can activate NK cells. We displayed antibodies on a mammalian target cell line and probed their ability to stimulate NK cell-mediated cytotoxicity. From this screen, we identified five antibodies that bound with high affinity to NK cells and stimulated NK cell-mediated cytotoxicity and interferon-γ (IFN-γ) secretion. We demonstrate that these antibodies can be further developed into bispecific antibodies to redirect NK cell-mediated cytotoxicity toward CD20+ B cell lymphoma cells and HER2+ breast cancer cells. While antibodies to two of the receptors, CD16 and NCR1, have previously been targeted as bispecific antibodies to redirect NK cell-mediated cytotoxicity, we demonstrate that bispecific antibodies targeting NCR3 can also potently activate NK cells. These results show that this screen can be used to directly identify antibodies that can enhance antitumor immune responses.

Negative impact of high body mass index on cetuximab-mediated cellular cytotoxicity against human colon carcinoma cells.

This study assessed the relationship between the ability of Natural Killer (NK) cells to activate antibody-dependent cellular cytotoxicity against human HT29 colorectal cancer cells exposed to cetuximab and the body mass index of the human subjects from whom the NK cells had been obtained. NK cells obtained from 73 human donors were co-incubated with HT-29 human colon cancer cells in the presence or absence of cetuximab. Antibody-dependent cellular cytotoxicity was assessed by measuring LDH release. A significant negative correlation was observed between body mass index and cetuximab-induced antibody-dependent cellular cytotoxicity. NK cells obtained from subjects who were overweight or with obesity were less efficient in killing cetuximab-treated HT29 cells than those derived from normal weight donors. Our results suggest that the success of cetuximab-containing regimens might be impaired in overweight and obese patients with colorectal cancer.

ß2 Integrin Signaling Cascade in Neutrophils: More Than a Single Function.

Neutrophils are the most prevalent leukocytes in the human body. They have a pivotal role in the innate immune response against invading bacterial and fungal pathogens, while recent emerging evidence also demonstrates their role in cancer progression and anti-tumor responses. The efficient execution of many neutrophil effector responses requires the presence of ß2 integrins, in particular CD11a/CD18 or CD11b/CD18 heterodimers. Although extensively studied at the molecular level, the exact signaling cascades downstream of ß2 integrins still remain to be fully elucidated. In this review, we focus mainly on inside-out and outside-in signaling of these two ß2 integrin members expressed on neutrophils and describe differences between various neutrophil stimuli with respect to integrin activation, integrin ligand binding, and the pertinent differences between mouse and human studies. Last, we discuss how integrin signaling studies could be used to explore the therapeutic potential of targeting ß2 integrins and the intracellular signaling cascade in neutrophils in several, among other, inflammatory conditions in which neutrophil activity should be dampened to mitigate disease.

ADCC can improve graft vs leukemia effect after T- and B-cell depleted haploidentical stem cell transplantation in pediatric B-lineage ALL.

Posttransplant relapsed B-cell precursor ALL can be cured by 2nd hematopoietic stem cell transplantation (HSCT) in 20% of patients. The major cause of death after second HSCT is leukemic relapse. One reliable predictor for survival after 2nd-HSCT are posttransplant MRD levels. Patients with detectable or increase of MRD are likely to relapse. Patients in complete molecular remission show the best leukemia-free survival and lowest cumulative incidence (CI) of relapse. As patients who undergo second or subsequent HSCT are high-risk patients, we evaluated the prophylactic use of the chimeric Fc-optimized CD19-4G7SDIE-mAb. Posttransplant relapsed CD19+ BCP-ALL patients, who underwent a second or subsequent haplo-HSCT from a T- and B-cell depleted graft received posttransplant prophylactic CD19-4G7SDIE-mAb treatment on compassionate use in complete molecular remission, to increase the antileukemic activity of the new reconstituting immune system by recruiting Fc-expressing effector cells. NK cells recovered early and robust. The 3 year overall survival in 15 evaluable patients was 56%, the 3 year event-free survival was 55% and the CI of relapse 38%. Compared to a historical control group, the CI of relapse was markedly lower and consecutively the EFS higher. Posttransplant-targeted therapy may overcome the need for unspecific GvL effect of undesired GvHD, that can cause severe morbidity and mortality. Due to a low adverse event profile the CD19-4G7SDIE-mAb may be suitable for broad administration to consolidate posttransplant MRD negativity.

Identification of HIV gp41-specific antibodies that mediate killing of infected cells.

Antibodies that mediate killing of HIV-infected cells through antibody-dependent cellular cytotoxicity (ADCC) have been implicated in protection from HIV infection and disease progression. Despite these observations, these types of HIV antibodies are understudied compared to neutralizing antibodies. Here we describe four monoclonal antibodies (mAbs) obtained from one individual that target the HIV transmembrane protein, gp41, and mediate ADCC activity. These four mAbs arose from independent B cell lineages suggesting that in this individual, multiple B cell responses were induced by the gp41 antigen. Competition and phage peptide display mapping experiments suggested that two of the mAbs target epitopes in the cysteine loop that are highly conserved and a common target of HIV gp41-specific antibodies. The amino acid sequences that bind these mAbs are overlapping but distinct. The two other mAbs were competed by mAbs that target the C-terminal heptad repeat (CHR) and the fusion peptide proximal region (FPPR) and appear to both target a similar unique conformational epitope. These gp41-specific mAbs mediated killing of infected cells that express high levels of Env due to either pre-treatment with interferon or deletion of vpu to increase levels of BST-2/Tetherin. They also mediate killing of target cells coated with various forms of the gp41 protein, including full-length gp41, gp41 ectodomain or a mimetic of the gp41 stump. Unlike many ADCC mAbs that target HIV gp120, these gp41-mAbs are not dependent on Env structural changes associated with membrane-bound CD4 interaction. Overall, the characterization of these four new mAbs that target gp41 and mediate ADCC provides evidence for diverse gp41 B cell lineages with overlapping but distinct epitopes within an individual. Such antibodies that can target various forms of envelope protein could represent a common response to a relatively conserved HIV epitope for a vaccine.

Immune Checkpoint Inhibition Overcomes ADCP-Induced Immunosuppression by Macrophages.

Antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) critically contribute to the efficacy of anti-tumor therapeutic antibodies. We report here an unexpected finding that macrophages after ADCP inhibit NK cell-mediated ADCC and T cell-mediated cytotoxicity in breast cancers and lymphomas. Mechanistically, AIM2 is recruited to the phagosomes by FcγR signaling following ADCP and activated by sensing the phagocytosed tumor DNAs through the disrupted phagosomal membrane, which subsequently upregulates PD-L1 and IDO and causes immunosuppression. Combined treatment with anti-HER2 antibody and inhibitors of PD-L1 and IDO enhances anti-tumor immunity and anti-HER2 therapeutic efficacy in mouse models. Furthermore, neoadjuvant trastuzumab therapy significantly upregulates PD-L1 and IDO in the tumor-associated macrophages (TAMs) of HER2+ breast cancer patients, correlating with poor trastuzumab response. Collectively, our findings unveil a deleterious role of ADCP macrophages in cancer immunosuppression and suggest that therapeutic antibody plus immune checkpoint blockade may provide synergistic effects in cancer treatment.

Monoclonal antibody-mediated killing of tumour cells by neutrophils.

Neutrophils represent the most abundant population of circulating cytotoxic effector cells. Moreover, their number can be easily increased by treatment with granulocyte-colony stimulating factor or granulocyte macrophage-colony stimulating factor, without the need for ex vivo expansion. Because neutrophils express Fc receptors, they have the potential to act as effector cells during monoclonal antibody therapy of cancer. Additionally, as neutrophils play a role in the regulation of adaptive immune responses, exploiting neutrophils in mAb therapy may result in long-term antitumour immunity. There is limited evidence that neutrophils play a prominent role in current immunoglobulin G-based immunotherapy. However, as IgA induces neutrophil recruitment, novel therapeutic strategies that aim to target the IgA Fc receptor FcαRI may fully unleash the potential of enlisting neutrophils as cytotoxic effector cells in antibody therapy of cancer.

High-throughput cytotoxicity and antigen-binding assay for screening small bispecific antibodies without purification.

The cytotoxicity of T cell-recruiting antibodies with their potential to damage late-stage tumor masses is critically dependent on their structural and functional properties. Recently, we reported a semi-high-throughput process for screening highly cytotoxic small bispecific antibodies (i.e., diabodies). In the present study, we improved the high-throughput performance of this screening process by removing the protein purification stage and adding a stage for determining the concentrations of the diabodies in culture supernatant. The diabodies were constructed by using an Escherichia coli expression system, and each diabody contained tandemly arranged peptide tags at the C-terminus, which allowed the concentration of diabodies in the culture supernatant to be quantified by using a tag-sandwich enzyme-linked immunosorbent assay. When estimated diabody concentrations were used to determine the cytotoxicity of unpurified antibodies, results comparable to those of purified antibodies were obtained. In a surface plasmon resonance spectroscopy-based target-binding assay, contaminants in the culture supernatant prevented us from conducting a quantitative binding analysis; however, this approach did allow relative binding affinity to be determined, and the relative binding affinities of the unpurified diabodies were comparable to those of the purified antibodies. Thus, we present here an improved high-throughput process for the simultaneous screening and determination of the binding parameters of highly cytotoxic bispecific antibodies.

Glycoengineering of antibody (Herceptin) through yeast expression and in vitro enzymatic glycosylation.

Monoclonal antibodies (mAbs) have been developed as therapeutics, especially for the treatment of cancer, inflammation, and infectious diseases. Because the glycosylation of mAbs in the Fc region influences their interaction with effector cells that kill antibody-targeted cells, and the current method of antibody production is relatively expensive, efforts have been directed toward the development of alternative expressing systems capable of large-scale production of mAbs with desirable glycoforms. In this study, we demonstrate that the mAb trastuzumab expressed in glycoengineered P. pastoris can be remodeled through deglycosylation by endoglycosidases identified from the Carbohydrate Active Enzymes database and through transglycosylation using glycans with a stable leaving group to generate a homogeneous antibody designed to optimize the effector functions. The 10 newly identified recombinant bacterial endoglycosidases are complementary to existing endoglycosidases (EndoA, EndoH, EndoS), two of which can even accept sialylated tri- and tetraantennary glycans as substrates.

The strong in vivo anti-tumor effect of the UIC2 monoclonal antibody is the combined result of Pgp inhibition and antibody dependent cell-mediated cytotoxicity.

P-glycoprotein (Pgp) extrudes a large variety of chemotherapeutic drugs from the cells, causing multidrug resistance (MDR). The UIC2 monoclonal antibody recognizes human Pgp and inhibits its drug transport activity. However, this inhibition is partial, since UIC2 binds only to 10-40% of cell surface Pgps, while the rest becomes accessible to this antibody only in the presence of certain substrates or modulators (e.g. cyclosporine A (CsA)). The combined addition of UIC2 and 10 times lower concentrations of CsA than what is necessary for Pgp inhibition when the modulator is applied alone, decreased the EC50 of doxorubicin (DOX) in KB-V1 (Pgp+) cells in vitro almost to the level of KB-3-1 (Pgp-) cells. At the same time, UIC2 alone did not affect the EC50 value of DOX significantly. In xenotransplanted severe combined immunodeficient (SCID) mice co-treated with DOX, UIC2 and CsA, the average weight of Pgp+ tumors was only ∼10% of the untreated control and in 52% of these animals we could not detect tumors at all, while DOX treatment alone did not decrease the weight of Pgp+ tumors. These data were confirmed by visualizing the tumors in vivo by positron emission tomography (PET) based on their increased 18FDG accumulation. Unexpectedly, UIC2+DOX treatment also decreased the size of tumors compared to the DOX only treated animals, as opposed to the results of our in vitro cytotoxicity assays, suggesting that immunological factors are also involved in the antitumor effect of in vivo UIC2 treatment. Since UIC2 binding itself did not affect the viability of Pgp expressing cells, but it triggered in vitro cell killing by peripheral blood mononuclear cells (PBMCs), it is concluded that the impressive in vivo anti-tumor effect of the DOX-UIC2-CsA treatment is the combined result of Pgp inhibition and antibody dependent cell-mediated cytotoxicity (ADCC).

[Therapeutic monoclonal antibodies for hematological diseases].

Over the past decade, monoclonal antibodies (mAbs) have been demonstrated to be powerful therapeutic options for hematological diseases. MAbs exert selective therapeutic effects through binding specific molecules, which can minimize the frequency and magnitude of their adverse effects. The major mechanisms of the cytotoxic activity by mAbs against hematological malignancies are complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC). To enhance the ADCC activity, the defucosylated version of mAbs have been developed to enhance their binding ability to Fcgamma receptor IIIa. Antibody-drug conjugates and radiolabeled mAbs are also designed to selectively kill tumor cells. Owing to the recent innovation of mAb generation and modification techniques, we can expect more benefical mAbs with diverse functions as important therapeutic strategies.

Cytotoxicity and DNA damage in the neutrophils of patients with sickle cell anaemia treated with hydroxyurea

Hydroxyurea (HU) is the most important advance in the treatment of sickle cell anaemia (SCA) for preventing complications and improving quality of life for patients. However, some aspects of treatment with HU remain unclear, including their effect on and potential toxicity to other blood cells such as neutrophils. This study used the measurement of Lactate Dehydrogenase (LDH) and Methyl ThiazolTetrazolium (MTT) and the comet assay to investigate the cytotoxicity and damage index (DI) of the DNA in the neutrophils of patients with SCA using HU.In the LDH and MTT assays, a cytoprotective effect was observed in the group of patients treated, as well as an absence of toxicity. When compared to patients without the treatment, the SS group (n=20, 13 women and 07 men, aged 18-69 years), and the group of healthy individuals (AA) used as a control group (n=52, 28 women and 24 men, aged 19-60 years), The SSHU group (n=21, 11 women and 10 men, aged 19-63 years) showed a significant reduction (p<0.001) in LDH activity and an increase in the percentage of viable cells by the MTT (p<0.001). However, the SSHU group presented significantly higher DI values (49.57±6.0 U/A) when compared to the AA group (7.43 ± 0,94U/A) and the SS group (22.73 ±5.58 U/A) (p<0.0001), especially when treated for longer periods (>20 months), demonstrating that despite the cytoprotective effects in terms of cell viability, the use of HU can induce DNA damage in neutrophils.

A hidroxiuréia (HU) constitui o avanço mais importante no tratamento da anemia falciforme (AF) por prevenir complicações e aumentar a qualidade de vida dos pacientes. Entretanto, alguns aspectos do tratamento com HU permanecem obscuros, incluindo a sua ação e potencial toxicidade em outras células sanguíneas, tais como neutrófilos. Este estudo utilizou a mensuração da lactato desidrogenase (LDH) e do metil tiazoltetrazólio (MTT) e o ensaio do cometa para investigar a citotoxicidade e índice de dano (ID) ao DNA em neutrófilos de pacientes com AF em uso do medicamento. Nos ensaios de LDH e MTT, observou-se além de ausência de toxicidade, uma ação citoprotetora no grupo de pacientes tratados, Grupo SSHU (n=21, 11 mulheres e 10 homens, com idades entre 19-63 anos), quando comparados aos pacientes sem tratamento, Grupo SS (n=20, 13 mulheres e 07 homens, 18-69 anos), e grupo de indivíduos saudáveis (AA) usado como controle (n=52, 28 mulheres e 24 homens, 19-60 anos), com redução significativa (p<0,001) na atividade de LDH e aumento no percentual de células viáveis pelo MTT (p<0,001). Entretanto, o grupo SSHU apresentou valores de ID significativamente elevados (49,57±6,0 U/A), quando comparados ao grupo AA (7,43 ± 0,94U/A) e grupo SS (22,73 ±5,58 U/A) (p<0,0001), especialmente quando tratados por períodos mais longos (>20 meses), demonstrando que apesar dos efeitos citoprotetores quanto à viabilidade celular, o uso da HU pode induzir lesão ao DNA de neutrófilos.

Real time assays for quantifying cytotoxicity with single cell resolution.

A new live cell-based assay platform has been developed for the determination of complement dependent cytotoxicity (CDC), antibody dependent cellular cytotoxicity (ADCC), and overall cytotoxicity in human whole blood. In these assays, the targeted tumor cell populations are first labeled with fluorescent Cell Tracker dyes and immobilized using a DNA-based adhesion technique. This allows the facile generation of live cell arrays that are arranged arbitrarily or in ordered rectilinear patterns. Following the addition of antibodies in combination with serum, PBMCs, or whole blood, cell death within the targeted population can be assessed by the addition of propidium iodide (PI) as a viability probe. The array is then analyzed with an automated microscopic imager. The extent of cytotoxicity can be quantified accurately by comparing the number of surviving target cells to the number of dead cells labeled with both Cell Tracker and PI. Excellent batch-to-batch reproducibility has been achieved using this method. In addition to allowing cytotoxicity analysis to be conducted in real time on a single cell basis, this new assay overcomes the need for hazardous radiochemicals. Fluorescently-labeled antibodies can be used to identify individual cells that bear the targeted receptors, but yet resist the CDC and ADCC mechanisms. This new approach also allows the use of whole blood in cytotoxicity assays, providing an assessment of antibody efficacy in a highly relevant biological mixture. Given the rapid development of new antibody-based therapeutic agents, this convenient assay platform is well-poised to streamline the drug discovery process significantly.

CD19 targeting of chronic lymphocytic leukemia with a novel Fc-domain-engineered monoclonal antibody.

CD19 is a B cell-specific antigen expressed on chronic lymphocytic leukemia (CLL) cells but to date has not been effectively targeted with therapeutic monoclonal antibodies. XmAb5574 is a novel engineered anti-CD19 monoclonal antibody with a modified constant fragment (Fc)-domain designed to enhance binding of FcgammaRIIIa. Herein, we demonstrate that XmAb5574 mediates potent antibody-dependent cellular cytotoxicity (ADCC), modest direct cytotoxicity, and antibody-dependent cellular phagocytosis but not complement-mediated cytotoxicity against CLL cells. Interestingly, XmAb5574 mediates significantly higher ADCC compared with both the humanized anti-CD19 nonengineered antibody it is derived from and also rituximab, a therapeutic antibody widely used in the treatment of CLL. The XmAb5574-dependent ADCC is mediated by natural killer (NK) cells through a granzyme B-dependent mechanism. The NK cell-mediated cytolytic and secretory function with XmAb5574 compared with the nonengineered antibody is associated with enhanced NK-cell activation, interferon production, extracellular signal-regulated kinase phosphorylation downstream of Fcgamma receptor, and no increased NK-cell apoptosis. Notably, enhanced NK cell-mediated ADCC with XmAb5574 was enhanced further by lenalidomide. These findings provide strong support for further clinical development of XmAb5574 as both a monotherapy and in combination with lenalidomide for the therapy of CLL and related CD19(+) B-cell malignancies.

Complement-dependent tumor cell lysis triggered by combinations of epidermal growth factor receptor antibodies.

Therapeutic monoclonal antibodies against the epidermal growth factor receptor (EGFR) have advanced the treatment of colon and head and neck cancer, and show great promise for the development of treatments for other solid cancers. Antibodies against EGFR have been shown to act via inhibition of receptor signaling and induction of antibody-dependent cellular cytoxicity. However, complement-dependent cytotoxicity, which is considered one of the most powerful cell killing mechanisms of antibodies, seems inactive for such antibodies. Here, we show a remarkable synergy for EGFR antibodies. Combinations of antibodies against EGFR were identified, which resulted in potent complement activation via the classic pathway and effective lysis of tumor cells. Studies on a large panel of antibodies indicated that the observed synergy is a general mechanism, which can be activated by combining human IgG1 antibodies recognizing different, nonoverlapping epitopes. Our findings show an unexpected quality of therapeutic EGFR antibodies, which may be exploited to develop novel and more effective treatments for solid cancers.

The role of molecular markers in predicting response to therapy in patients with colorectal cancer.

Advances in systemic therapy for colorectal cancer have dramatically improved prognosis. While disease stage has traditionally been the main determinant of disease course, several molecular characteristics of tumor specimens have recently been shown to have prognostic significance. Although to date no molecular characteristics have emerged as consistent predictors of response to therapy, retrospective studies have investigated the role of a variety of biomarkers, including microsatellite instability, loss of heterozygosity of 18q, type II transforming growth factor beta receptor, thymidylate synthase, epidermal growth factor receptor, and Kirsten-ras (KRAS). This paper reviews the current literature, ongoing prospective studies evaluating the role of these markers, and novel techniques such as gene profiling, which may help to uncover the more complex molecular interactions that will predict response to chemotherapy in patients with colorectal cancer.

Role of IgE receptors in IgE antibody-dependent cytotoxicity and phagocytosis of ovarian tumor cells by human monocytic cells.

Antibodies directed against tumor-associated antigens are emerging as effective treatments for a number of cancers, although the mechanism(s) of action for some are unclear and still under investigation. We have previously examined a chimeric IgE antibody (MOv18 IgE), against the ovarian tumor-specific antigen, folate binding protein (FBP), and showed that it can direct human PBMC to kill ovarian cancer cells. We have developed a three-color flow cytometric assay to investigate the mechanism by which IgE receptors on U937 monocytes target and kill ovarian tumor cells. U937 monocytes express three IgE receptors, the high-affinity receptor, FcepsilonRI, the low-affinity receptor, CD23, and galectin-3, and mediate tumor cell killing in vitro by two mechanisms, cytotoxicity, and phagocytosis. Our results suggest that CD23 mediates phagocytosis, which is enhanced by upregulation of CD23 on U937 cells with IL-4, whereas FcepsilonRI mediates cytotoxicity. We show that effector : tumor cell bridging is associated with both activities. Galectin-3 does not appear to be involved in tumor cell killing. U937 cells and IgE exerted ovarian tumor cell killing in vivo in our xenograft model in nude mice. Harnessing IgE receptors to target tumor cells suggests the potential of tumor-specific IgE antibodies to activate effector cells in immunotherapy of ovarian cancer.

[Targeted therapy: what does it mean? A physician point of view]. / Qu'est-ce qu'une thérapie ciblée? Le point de vue du clinicien.

Over the past few years, the development of novel antitumoral strategies considered as a more appropriate molecularly drug target has replaced the more empiric screening way of research for new cytotoxic drugs. The better understanding of the molecular mechanisms underlying carcinogenesis has provided rapidly this emerging development of a huge patterns of targeted drugs that can inhibit specifically cancer pathways and key molecules in tumor cells or environment, implicated in tumor growth, progression, apoptosis, angiogenesis and metastases. Some of these new targeted agents have already demonstrated their significant efficacy in cancer patients and recently been approved in US and Europe. The recent technological advances in pharmacogenomics and proteomics have led to improve the identification of biomarkers predictive of response and thereby to identify the patients that would be more likely to respond from such a therapy. However, even though some of these new compounds have shown high progress and have become new standards of care and cure for cancer patients, failure rates are still common especially in oncology and deserve a greater attention to the type of these new molecular target, diagnostic criteria to tailor patient and to the different surrogate markers that can be used as outcome measures.

Comparison of biological activity among nonfucosylated therapeutic IgG1 antibodies with three different N-linked Fc oligosaccharides: the high-mannose, hybrid, and complex types.

The structure of asparagine-linked oligosaccharides attached to the antibody constant region (Fc) of human immunoglobulin G1 (IgG1) has been shown to affect the pharmacokinetics and antibody effector functions of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). However, it is still unclear how differences in the N-linked oligosaccharide structures impact the biological activities of antibodies, especially those lacking core fucose. Here, we succeeded in generating core fucose-lacking human IgG1 antibodies with three different N-linked Fc oligosaccharides, namely, a high-mannose, hybrid, and complex type, using the same producing clone, and compared their activities. Cultivation of an alpha-1,6-fucosyltransferase (FUT8) knockout Chinese hamster ovary cell line in the presence or absence of a glycosidase inhibitor (either swainsonine or kifunensine) yielded antibody production of each of the three types without contamination by the others. Two of three types of nonnaturally occurring atypical oligosaccharide IgG1, except the complex type, reduced the affinity for both human lymphocyte receptor IIIa (FcgammaRIIIa) and the C1q component of the complement, resulting in reduction of ADCC and CDC. The bulky structure of the nonreducing end of N-linked Fc oligosaccharides is considered to contribute the CDC change, whereas the structural change in the reducing end, i.e. the removal of core fucose, causes ADCC enhancement through improved FcgammaRIIIa binding. In the pharmacokinetic profile, although no significant difference of human neonatal Fc receptor (FcRn)-binding affinity was observed among the three types, the complex type showed longer serum half-lives than the other types irrespective of core fucosylation in mice, which also suggests the contribution of the nonreducing end structure. The present study provides basic information on the effects of core fucose-lacking N-linked Fc oligosaccharides on antibody biological activities.

Establishment and functional characterization of novel natural killer cell lines derived from a temperature-sensitive SV40 large T antigen transgenic mouse.

Natural killer (NK) cells belong to an important lymphocyte population that eliminates transformed cells and invading pathogens without any prior sensitization. NK cells possess not only natural killing activity against non-self and altered-self cells but also exhibit cytokine production and antibody-dependent cell-mediated cytotoxicity (ADCC). Despite their important roles in the innate immune system, little is known about the details of NK cell biology. In spite of that several murine NK cell clones have been established, studies have mainly focused on their natural killing activity but not their cytokine production or ADCC. In this study, we established and characterized eight novel, immortalized murine NK cell clones derived from a temperature-sensitive SV40 large-T antigen transgenic mouse. These NK cell lines continuously proliferated for more than 30 months in a culture medium supplemented with interleukin 2. All cell lines contained azurophilic granules in the cytoplasm, and a few clones retained the NK cell functions, such as natural killing activity, cytokine production, and ADCC. In addition, one clone could serve as a host for transient as well as stable gene transfection. Taken together, these findings indicate that the cell lines could constitute useful tools for detailed analysis of murine NK cell biology.