Serotherapy of ovarian cancer

The development of monoclonal antibodies has permitted the identification of several ovarian-tumor-associated antigens which might serve as targets for serotherapy in vivo. With the exception of antibodies directed against growth factor receptors, unmodified monoclonal reagents must activate complement (C') components or bind effector cells to destroy tumor targets. Antibody-dependent cell-mediated cytotoxicity (ADCC) may be particularly important for eliminating tumor cells in vivo. A shortage of functionally active effector cells can limit the efficacy of serotherapy with heteroantisera or monoclonal reagents. The use of immunostimulants such as Corynebacterium parvum has increased the number and activity of effector cells for ADCC within the peritoneal compartment of mice and of patients with ovarian cancer. Intraperitoneal serotherapy can achieve direct contact between antibody and microscopic deposits of ovarian tumor cells which persist following cytoreductive operations and cytotoxic chemotherapy. Conjugation of monoclonal antibodies with radionuclides, drugs or toxins might increase the potency of serotherapy and circumvent the effector shortage. Clinical studies to date have evaluated radionuclide conjugates for imaging and for therapy. Patients with a small volume of disease have responded to treatment. Preclinical models suggest that drug and toxin conjugates might also prove active. Recent studies have demonstrated a synergistic interaction between different immunotoxins. Ovarian carcinoma is likely to be a valuable clinical model for evaluating immunoconjugates which react with epithelial tumor cells.     

NK cells engineered to express a GD2 -specific antigen receptor display built-in ADCC-like activity against tumour cells of neuroectodermal origin

Treatment of high-risk neuroblastoma (NB) represents a major challenge in paediatric oncology. Alternative therapeutic strategies include antibodies targeting the disialoganglioside GD(2) , which is expressed at high levels on NB cells, and infusion of donor-derived natural killer (NK) cells. To combine specific antibody-mediated recognition of NB cells with the potent cytotoxic activity of NK cells, here we generated clonal derivatives of the clinically applicable human NK cell line NK-92 that stably express a GD(2) -specific chimeric antigen receptor (CAR) comprising an anti-GD(2) ch14.18 single chain Fv antibody fusion protein with CD3-ζ chain as a signalling moiety. CAR expression by gene-modified NK cells facilitated effective recognition and elimination of established GD(2) expressing NB cells, which were resistant to parental NK-92. In the case of intrinsically NK-sensitive NB cell lines, we observed markedly increased cell killing activity of retargeted NK-92 cells. Enhanced cell killing was strictly dependent on specific recognition of the target antigen and could be blocked by GD(2) -specific antibody or anti-idiotypic antibody occupying the CAR's cell recognition domain. Importantly, strongly enhanced cytotoxicity of the GD(2) -specific NK cells was also found against primary NB cells and GD(2) expressing tumour cells of other origins, demonstrating the potential clinical utility of the retargeted effector cells.     

Differential responses of human tumor cell lines to anti-p185HER2 monoclonal antibodies

The HER2 protooncogene encodes a receptor tyrosine kinase, p185^HER2. The overexpression of p185^HER2 has been associated with a worsened prognosis in certain human cancers. In the present work we have screened a variety of different tumor cell lines for p185^HER2 expression using both enzyme-linked immunosorbent and fluorescence-activated cell sorting assays employing murine monoclonal antibodies directed against the extracellular domain of the receptor. Increased levels of p185^HER2 were found in breast (5/9), ovarian (1/6), stomach (2/3) and colorectal (5/16) carcinomas, whereas all kidney and submaxillary adenocarcinoma cell lines tested were negative. Some monoclonal antibodies directed against the extracellular domain of p185^HER2 inhibited growth in monolayer culture of breast and ovarian tumor cell lines overexpressing p185^HER2, but had no effect on the growth of colon or gastric adenocarcinomas expressing increased levels of this receptor. The most potent growth-inhibitory anti-p185^HER2 monoclonal antibody in monolayer culture, designated mumAb 4D5 (a murine IgG1 antibody), was also tested in soft-agar growth assays for activity against p185^HER2-overexpressing tumor cell lines of each type, with similar results. In order to increase the spectrum of tumor types potentially susceptible to monoclonal antibody-mediated anti-p185^HER2 therapies, to decrease potential immunogenicity issues with the use of murine monoclonal antibodies for human therapy, and to provide the potential for antibody-mediated cytotoxic activity, a mouse/human chimeric 4D5 (chmAb 4D5) and a humanized 4D5 (rhu)mAb 4D5 HER2 antibody were constructed. Both engineered antibodies, in combination with human peripheral blood mononuclear cells, elicited antibody-dependent cytotoxic responses in accordance with the level of p185^HER2 expression. Since this cytotoxic activity is independent of sensitivity to mumAb 4D5, the engineered monoclonal antibodies expand the potential target population for antibody-mediated therapy of human cancers characterized by the overexpression of p185^HER2.     

Engineered Fc variant antibodies with enhanced ability to recruit complement and mediate effector functions

Engineering the antibody Fc region to enhance the cytotoxic activity of therapeutic antibodies is currently an active area of investigation. The contribution of complement to the mechanism of action of some antibodies that target cancers and pathogens makes a compelling case for its optimization. Here we describe the generation of a series of Fc variants with enhanced ability to recruit complement. Variants enhanced the cytotoxic potency of an anti-CD20 antibody up to 23-fold against tumor cells in CDC assays, and demonstrated a correlated increase in C1q binding affinity. Complementenhancing substitutions combined additively, and in one case synergistically, with substitutions previously engineered for improved binding to Fc gamma receptors. The engineered combinations provided a range of effector function activities, including simultaneously enhanced CDC, ADCC, and phagocytosis. Variants were also effective at boosting the effector function of antibodies targeting the antigens CD40 and CD19, in the former case enhancing CDC over 600-fold, and in the latter case imparting complement-mediated activity onto an IgG1 antibody that was otherwise incapable of it. This work expands the toolkit of modifications for generating monoclonal antibodies with improved therapeutic potential and enables the exploration of optimized synergy between Fc gamma receptors and complement pathways for the destruction of tumors and infectious pathogens.Key words: antibody, Fc, complement, CDC, C1q, ADCC, phagocytosis, CD20, CD19, CD40     

Chemical construction of immunotoxins

Immunotoxins are chimeric proteins consisting of an antibody linked to a toxin. The antibodies most frequently used for the preparation of immunotoxins are murine monoclonal antibodies belonging to IgG isotype. The most used toxins for the chemical construction of immunotoxins are Ricin toxin A chain in its deglycosylated form and recombinant Pseudomonas endotoxin with the cell-binding domain deleted. The linkage of the antibody to the toxin can be accomplished by chemical methods using reagents that crosslink antibody to toxin. The usual crosslinkers attach disulfide groups into the antibody molecule to form a disulfide bond between the antibody and the toxin. Disulfide bonds are susceptible to reduction in the cytoplasm of the targeted cells thereby releasing the toxin so that it can exert its cytotoxic activity only into the cells (e.g., tumor cells) binding the antibody moiety. This article describes various methods to obtain antibodies and toxins and several procedures for their crosslinking as well as “in vitro” and “in vivo” testing of the immunotoxins efficacy.     

Bispecific antibodies targeting cancer cells

In recent years, antibody therapy has become a new treatment modality for tumour patients, although the majority of responses are only partial and not long lasting. Based on evidence that effector-cell-mediated mechanisms significantly contribute to antibody efficacy in vivo, several approaches are currently pursued to improve the interaction between Fc receptor-expressing effector cells and tumour target antigens. These approaches include application of Fc receptor-directed bispecific antibodies, which contain one specificity for a tumour-related antigen and another for a cytotoxic Fc receptor on immune effector cells. Thereby, bispecific antibodies selectively engage cytotoxic trigger molecules on killer cells, avoiding, for example, interaction with inhibitory Fc receptors. In vitro, chemically linked bispecific antibodies directed against the Fc gamma receptors Fc gamma RIII (CD16) and Fc gamma RI (CD64), and the Fc alpha receptor Fc alpha RI (CD89), were significantly more effective than conventional IgG antibodies. Recent animal studies confirmed the therapeutic potential of these constructs. However, results from clinical trials have been less promising so far and have revealed clear limitations of these molecules, such as short plasma half-lives compared with conventional antibodies. In this review, we briefly summarize the scientific background for bispecific antibodies, and describe the rationale for the generation of novel recombinant molecules. These constructs may allow us to more specifically tailor pharmacokinetic properties to the demands of clinical applications.     

Tumor mRNA-loaded dendritic cells elicit tumor-specific CD8(+) cytotoxic T cells in patients with malignant glioma

In this study, we demonstrate that tumor mRNA–loaded dendritic cells can elicit a specific CD8⁺ cytotoxic T-lymphocyte (CTL) response against autologous tumor cells in patients with malignant glioma. CTLs from three patients expressed strong cytolytic activity against autologous glioma cells, did not lyse autologous lymphoblasts or EBV-transformed cell lines, and were variably cytotoxic against the NK-sensitive cell line K-562. Also, DCs-pulsed normal brain mRNA failed to induce cytolytic activity against autologous glioma cells, suggesting the lack of autoimmune response. Two patients' CD8⁺ T cells expressed a modest cytotoxicity against autologous glioma cells. CD8⁺ T cells isolated during these ineffective primings secreted large amounts of IL-10 and smaller amounts of IFN- as detected by ELISA. Type 2 bias in the CD8⁺ T-cell response accounts for the lack of cytotoxic effector function from these patients. Cytotoxicity against autologous glioma cells could be significantly inhibited by anti-HLA class I antibody. These data demonstrate that tumor mRNA–loaded DC can be an effective tool in inducing glioma-specific CD8⁺ CTLs able to kill autologous glioma cells in vitro. However, high levels of tumor-specific tolerance in some patients may account for a significant barrier to therapeutic vaccination. These results may have important implications for the treatment of malignant glioma patients with immunotherapy. DCs transfected with total tumor RNA may represent a method for inducing immune responses against the entire repertoire of glioma antigens.     

Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets

Inhibitory receptors have been proposed to modulate the in vivo cytotoxic response against tumor targets for both spontaneous and antibody-dependent pathways. Using a variety of syngenic and xenograft models, we demonstrate here that the inhibitory FcgammaRIIB molecule is a potent regulator of antibody-dependent cell-mediated cytotoxicity in vivo, modulating the activity of FcgammaRIII on effector cells. Although many mechanisms have been proposed to account for the anti-tumor activities of therapeutic antibodies, including extended half-life, blockade of signaling pathways, activation of apoptosis and effector-cell-mediated cytotoxicity, we show here that engagement of Fcgamma receptors on effector cells is a dominant component of the in vivo activity of antibodies against tumors. Mouse monoclonal antibodies, as well as the humanized, clinically effective therapeutic agents trastuzumab (Herceptin(R)) and rituximab (Rituxan(R)), engaged both activation (FcgammaRIII) and inhibitory (FcgammaRIIB) antibody receptors on myeloid cells, thus modulating their cytotoxic potential. Mice deficient in FcgammaRIIB showed much more antibody-dependent cell-mediated cytotoxicity; in contrast, mice deficient in activating Fc receptors as well as antibodies engineered to disrupt Fc binding to those receptors were unable to arrest tumor growth in vivo. These results demonstrate that Fc-receptor-dependent mechanisms contribute substantially to the action of cytotoxic antibodies against tumors and indicate that an optimal antibody against tumors would bind preferentially to activation Fc receptors and minimally to the inhibitory partner FcgammaRIIB.     

Antibody engineering for therapeutics

With the acceptance of antibodies as therapeutics, a diversity of engineered antibody forms have been created to improve their efficacy, including enhancing the effector functions of full-length antibodies, delivering toxins to kill cells or cytokines in order to stimulate the immune system, and bispecific antibodies to target multiple receptors. After years of in vitro investigation, many of these are now moving into clinical trials and are showing promise. A potential new type of effector function for antibodies, that is, the generation of reactive oxygen species that may effect inflammation or bacterial killing, has been elucidated. In addition, the field has expanded beyond a concentration on immunoglobulin G to include immunoglobulin A antibodies as potential therapeutics.     

Tumor necrosis factor-alpha enhances cytolytic activity of human natural killer cells

The effect of recombinant tumor necrosis factor-alpha (rTNF alpha) on human natural killer (NK) function was examined. Lysis of both the NK-sensitive K562 erythroleukemia line and the relatively insensitive renal carcinoma line Cur by nonadherent peripheral blood lymphocytes was significantly enhanced as a result of an 18-hr preincubation with either rTNF alpha or recombinant interleukin 2 (rIL 2). When cells were preincubated with rTNF alpha and low doses of rIL 2 (1 to 10 U/ml), marked additional augmentation of lysis of both targets was noted which was greater than that caused by either cytokine alone. Similar results were observed when responses of CD16+ large granular lymphocytes selected with the fluorescence-activated cell sorter after staining with the NK-specific monoclonal antibody Leu-11 were examined, indicating that the action of the cytokines was directly on the cytotoxic cells. Augmentation of tumor cell lysis could not be ascribed to a cytolytic activity of rTNF alpha on the targets, because no combination of rIL 2, rTNF alpha, or interferon-gamma caused lysis of K562 or Cur. By flow cytometric analysis, it was found that expression of IL 2 receptors was induced on purified CD16+ large granular lymphocytes by rTNF alpha alone and to an even greater degree by the combination of rTNF alpha and rIL 2. Additional analysis of the expression of surface antigens and blocking studies with monoclonal antibodies showed that enhanced tumor cell lysis was not caused by the augmentation of leukocyte function-associated antigen-1-mediated effector/target interactions. These data indicate that rTNF alpha alone, or in combination with rIL 2, directly augments NK cytotoxic activity.     

Lymphocyte activation in response to melanoma: interaction of NK-associated receptors and their ligands

In recent years, studies on the molecular and cellular mechanisms of immune responses against melanoma have contributed to a better understanding of how these tumours can be recognised by cytotoxic cells and the mechanisms they have developed to escape from innate and adaptive immunity. Lysis of melanoma cells by natural killer (NK) cells and cytolytic T cells is the result of a fine balance between signals transmitted by activating and inhibitory receptors. In addition to the T cell receptor, these were initially described as NK cell-associated receptors (NKRs) and were later also found on subsets of T lymphocytes, particularly effector-memory and terminally differentiated CD8 T cells. An increase of NKR(+)CD8(+) T cells has been found in melanoma patients, correlating with the expansion of differentiated effector CD8(+)CD28(null) CD27(null) T cells. NKRs can regulate the lysis of target cells expressing appropriate ligands. Activating receptors recognise ligands on tumours whereas inhibitory receptors are specific for MHC class I antigens and sense missing self. Altered expression of MHC class I antigens is frequently found on melanoma cells, preventing recognition by specific cytolytic T cells but favouring NK cell recognition. Changes in the expression of NKR-ligands in melanoma contribute in explaining the differences in the capacity of cytotoxic immune cells to control melanoma growth and dissemination.     

Antibody-dependent cell lysis by NK cells is preserved after sarcoma-induced inhibition of NK cell cytotoxicity

Osteosarcoma and Ewing’s sarcoma tumor cells are susceptible to IL15-induced or antibody-mediated cytolytic activity of NK cells in short-term cytotoxicity assays. When encountering the tumor environment in vivo, NK cells may be in contact with tumor cells for a prolonged time period. We explored whether a prolonged interaction with sarcoma cells can modulate the activation and cytotoxic activity of NK cells. The 40 h coculture of NK cells with sarcoma cells reversibly interfered with the IL15-induced expression of NKG2D, DNAM-1 and NKp30 and inhibited the cytolytic activity of NK cells. The inhibitory effects on receptor expression required physical contact between NK cells and sarcoma cells and were independent of TGF-β. Five days pre-incubation of NK cells with IL15 prevented the down-regulation of NKG2D and cytolytic activity in subsequent cocultures with sarcoma cells. NK cell FcγRIIIa/CD16 receptor expression and antibody-mediated cytotoxicity were not affected after the coculture. Inhibition of NK cell cytotoxicity was directly linked to the down-regulation of the respective NK cell-activating receptors. Our data demonstrate that the inhibitory effects of sarcoma cells on the cytolytic activity of NK cells do not affect the antibody-dependent cytotoxicity and can be prevented by pre-activation of NK cells with IL15. Thus, the combination of cytokine-activated NK cells and monoclonal antibody therapy may be required to improve tumor targeting and NK cell functionality in the tumor environment.     

Targeting of tumor cells by lymphocytes engineered to express chimeric receptor genes

Adoptive cellular immunotherapy of cancer has been limited to date mostly due to the poor immunogenicity of tumor cells, the immunocompromised status of cancer patients in advanced stages of their disease, and difficulties in raising sufficient numbers of autologous tumor-specific T lymphocytes. On the other hand, the slow tumor penetration and short half-life of exogenously administered tumor-specific monoclonal antibodies have provided major obstacles for an effective destruction of tumor cells by the humoral effector arm of the immune system. Attempts to improve the efficacy of adoptive cellular cancer immunotherapy have led to the development of novel strategies that combine advantages of T cell-based (i.e., efficient tumor penetration, cytokine release and cytotoxicity) and antibody-based (high specificity for tumor-associated antigens) immunotherapy by grafting cytotoxic T lymphocytes (CTLs) with chimeric receptors composed of antibody fragments (which recognize tumor-cell antigens) and a cellular activation motif. Antigen recognition is therefore not restricted by major histocompatibility genes, as the physiological T-cell receptor, but rather is directed to native cell surface structures. Since the requirements of major histocompatibility complex (MHC) restriction in the interaction of effector cells with target cells are bypassed, the tumor cell-binding of CTLs grafted with chimeric receptors is not affected by down-regulation of HLA class I antigens and by defects in the antigen-processing machinery. Ligand binding by the chimeric receptor triggers phosphorylation of immunoglobulin tyrosine activation motifs (ITAMs) in the cytoplasmic region of the molecule and this activates a signaling cascade that is required for the induction of cytotoxicity, cytokine secretion and proliferation. Here, the authors discuss the potential of lymphocytes grafted with chimeric antigen receptors in the immunotherapy of malignant disease.     

How can we improve antibody-based cancer therapy?

Monoclonal antibodies (mAbs) as a class of novel oncology therapeutics are demonstrating clinical efficacy as measured by tumor response (shrinkage in tumor size), and prolongations in progression-free survival (PFS) and overall survival (OS). However, clinical benefits are often limited to when antibodies are used in combination with chemotherapy or radiation modalities, with tumor responses only seen in a fraction of patients, and improvements in PFS and OS are incremental.¹ The potential of mAbs and mAb constructs has yet to be fully exploited for maximal clinical benefit. New approaches to further improve the effectiveness of these mAb therapies include (1) selection of patients who may derive the most benefit based on the molecular characteristics of their tumors; (2) improvements in biodistribution to maximize delivery of mAbs to susceptible tumor cells; and (3) optimization of antibody immune effector mechanisms such as antibody-dependent cellular cytotoxicity (ADCC).Key words: monoclonal antibodies, solid tumors, cancer, pharmacogenomics, biodistribution, bioengineeringMonoclonal antibodies (mAbs) as a class of novel oncology therapeutics are demonstrating clinical efficacy as measured by tumor response (shrinkage in tumor size), and prolongations in progression-free survival (PFS) and overall survival (OS). However, clinical benefits are often limited to when antibodies are used in combination with chemotherapy or radiation modalities, with tumor responses only seen in a fraction of patients, and improvements in PFS and OS are incremental.¹ The potential of mAbs and mAb constructs has yet to be fully exploited for maximal clinical benefit. New approaches to further improve the effectiveness of these mAb therapies include (1) selection of patients who may derive the most benefit based on the molecular characteristics of their tumors; (2) improvements in biodistribution to maximize delivery of mAbs to susceptible tumor cells; and (3) optimization of antibody immune effector mechanisms such as antibody-dependent cellular cytotoxicity (ADCC) (Fig. 1).Open in a separate windowFigure 1Efficacy of monoclonal antibodies may be improved by selecting responding patient subpopulations, improving biodistribution and delivery of antibody to the tumor and maximizing antibody-mediated immune responses through application of protein and glyco-engineering.     

Molecular Control of B Cell Activation and Immunological Synapse Formation

B cells form an essential part of the adaptive immune system by producing specific antibodies that can neutralize toxins and target infected or malignant cells for destruction. During B cell activation, a fundamental role is played by a specialized intercellular structure called the immunological synapse (IS). The IS serves as a platform for B cell recognition of foreign, often pathogenic, antigens on the surface of antigen‐presenting cells (APC). This recognition is elicited by highly specific B cell receptors (BCR) that subsequently trigger carefully orchestrated intracellular signaling cascades that lead to cell activation. Furthermore, antigen internalization, essential for full B cell activation and differentiation into antibody producing effector cells or memory cells, occurs in the IS. Recent developments especially in various imaging‐based methods have considerably advanced our understanding of the molecular control of B cell activation. Interestingly, the cellular cytoskeleton is emerging as a key player at several stages of B cell activation, including the initiation of receptor signaling. Here, we discuss the functions and molecular mechanisms of the IS and highlight the multifaceted role of the actin cytoskeleton in several aspects of B cell activation.      

Listeriolysin O as cytotoxic component of an immunotoxin

Monoclonal antibodies (mAbs) have been developed over the past years as promising anticancer therapeutics. The conjugation of tumor specific mAbs with cytotoxic molecules has been shown to improve their efficacy dramatically. These bifunctional immunotoxins, consisting of covalently linked antibodies and protein toxins, possess considerable potential in cancer therapy. Many of them are under investigation in clinical trials. As a result of general interest in new toxic components, we describe here the suitability of the bacterial protein Listeriolysin O (LLO) as cytotoxic component of an immunotoxin. Unique characteristics of LLO, such as its acidic pH optimum and the possibility to regulate the cytolytic activity by cysteine‐oxidation, make LLO an interesting toxophore. Oxidized LLO shows a substantially decreased cytolytic activity when compared with the reduced protein as analyzed by hemolysis. Both oxidized and reduced LLO exhibit a cell‐type‐unspecific toxicity in cell culture with a significantly higher toxicity of reduced LLO. For cell‐type‐specific targeting of LLO to tumor cells, LLO was coupled to the dsFv fragment of the monoclonal antibody B3, which recognizes the tumor‐antigen Lewis Y. The coupling of LLO to dsFv‐B3 was performed via cysteine‐containing polyionic fusion peptides that act as a specific heterodimerization motif. The novel immunotoxin B3‐LLO could be shown to specifically eliminate antigen positive MCF7 cells with an EC₅₀ value of 2.3 nM, whereas antigen negative cell lines were 80‐ to 250‐fold less sensitive towards B3‐LLO.     

Antigen forks: bispecific reagents that inhibit cell growth by binding selected pairs of tumor antigens

Bispecific antibodies of a new category, termed antigen forks, were constructed by crosslinking antibodies that recognized pairs of distinct tumor cell surface antigens. At concentrations of 1–100 nM, several such forks inhibited the growth of human tumor cell lines bearing both relevant antigens. The same cells were not inhibited by unconjugated component antibodies, and the active conjugates did not inhibit the growth of human cell lines that expressed lower levels of relevant antigens. The three most active antigen forks all contained monoclonal antibody 454A12, which recognizes human transferrin receptor. This antibody was conjugated respectively to antibodies 113F1 (against a tumor-associated glycoprotein complex), 317G5 (against a 42-kDa tumor-associated glycoprotein), or 520C9 (against the c-erbB-2 protooncogene product). The 317G5-454A12 fork strongly inhibited the HT-29 and SW948 human colorectal cancer cell lines, while the 113F1-454A12 fork was also effective against SW948. By designing forks against antigens of incompatible function that are co-expressed at high levels on tumor cells but not on normal tissues, it may be possible to generate reagents that inhibit tumor growth with enhanced selectivity.     

Engineered Fc variant antibodies with enhanced ability to recruit complement and mediate effector functions

Engineering the antibody Fc region to enhance the cytotoxic activity of therapeutic antibodies is currently an active area of investigation. The contribution of complement to the mechanism of action of some antibodies that target cancers and pathogens makes a compelling case for its optimization. Here we describe the generation of a series of Fc variants with enhanced ability to recruit complement. Variants enhanced the cytotoxic potency of an anti-CD20 antibody up to 23-fold against tumor cells in CDC assays, and demonstrated a correlated increase in C1q binding affinity. Complement-enhancing substitutions combined additively, and in one case synergistically, with substitutions previously engineered for improved binding to Fc gamma receptors. The engineered combinations provided a range of effector function activities, including simultaneously enhanced CDC, ADCC, and phagocytosis. Variants were also effective at boosting the effector function of antibodies targeting the antigens CD40 and CD19, in the former case enhancing CDC over 600-fold, and in the latter case imparting complement-mediated activity onto an IgG1 antibody that was otherwise incapable of it. This work expands the toolkit of modifications for generating monoclonal antibodies with improved therapeutic potential, and enables the exploration of optimized synergy between Fc gamma receptors and complement pathways for the destruction of tumors and infectious pathogens.