Rituximab is a mainstay in the therapy for a broad variety of B-cell malignancies. Despite its undeniable therapeutic value, we still do not fully understand the mechanisms of action responsible for rituximab's anti-tumor effects. Direct signaling, complement dependent cellular cytotoxicity and antibody dependent cellular cytotoxicity all appear to play a role in rituximab efficacy. In vitro, animal model and clinical data addressing each of these mechanisms of action are reviewed, as are data speaking to the complexity of interactions between these mechanisms. Taken together, these data suggest different mechanisms are likely important in different scenarios. Study of the complex mechanisms of action that contribute to the clinical efficacy of rituximab have led to novel clinical trials including novel combinations, schedules, and generation of additional antibodies designed to have even greater effect. Such studies need to be accompanied by rigorous correlative analysis if we are to understand the importance of various mechanisms of action of rituximab and use that information to improve on what is already an indispensable approach to therapy.

For 20 years, monoclonal antibodies (mAbs) have been a standard component of cancer therapy, yet there is still much room for improvement. Efforts continue to build better cancer therapeutics based on mAbs. Anti-cancer mAbs function via a variety of mechanisms including directly targeting the malignant cells, modifying the host response to the malignant cells, delivering cytotoxic moieties to the malignant cells or retargeting cellular immunity towards the malignant cells. Characteristics of mAbs that affect their efficacy include antigen specificity, overall structure, affinity for the target antigen and how a mAb component is incorporated into a construct that can trigger target cell death. This article reviews the various approaches to using mAb-based therapeutics to treat cancer, the strategies used to take advantage of the unique potential of each approach, and provides examples of current mAb-based treatments.

Cancer immunotherapy comprises a variety of treatment approaches, incorporating the tremendous specificity of the adaptive immune system (T cells and antibodies) as well as the diverse and potent cytotoxic weaponry of both adaptive and innate immunity. Immunotherapy strategies include antitumor monoclonal antibodies, cancer vaccines, adoptive transfer of ex vivo activated T and natural killer cells, and administration of antibodies or recombinant proteins that either costimulate immune cells or block immune inhibitory pathways (so-called immune checkpoints). Although clear clinical efficacy has been demonstrated with antitumor antibodies since the late 1990s, other immunotherapies had not been shown to be effective until recently, when a spate of successes established the broad potential of this therapeutic modality. These successes are based on fundamental scientific advances demonstrating the toleragenic nature of cancer and the pivotal role of the tumor immune microenvironment in suppressing antitumor immunity. New therapies based on a sophisticated knowledge of immune-suppressive cells, soluble factors, and signaling pathways are designed to break tolerance and reactivate antitumor immunity to induce potent, long-lasting responses. Preclinical models indicate the importance of a complex integrated immune response in eliminating established tumors and validate the exploration of combinatorial treatment regimens, which are anticipated to be far more effective than monotherapies. Unlike conventional cancer therapies, most immunotherapies are active and dynamic, capable of inducing immune memory to propagate a successful rebalancing of the equilibrium between tumor and host.

DNA molecules containing unmethylated CpG-dinucleotides in particular base contexts (''CpG motifs'') are excellent adjuvants in rodents, but their effects on human cells have been less clear. Dendritic cells (DCs) form the link between the innate and the acquired immune system and may inf luence the balance between T helper 1 (Th1) and Th2 immune responses. We evaluated the effects of CpG oligodeoxynucleotides alone or in combination with granulocytemacrophage colony-stimulating factor (GMCSF) on different classes of purified human DCs. For primary dendritic precursor cells isolated from human blood, CpG oligonucleotides alone were superior to GMCSF in promoting survival and maturation (CD83 expression) as well as expression of class II MHC and the costimulatory molecules CD40, CD54, and CD86 of DCs. Both CD4-positive and CD4-negative peripheral blood dendritic precursor cells responded to CpG DNA which synergized with GMCSF but these DCs showed little response to lipopolysaccharide (LPS). In contrast, monocyte-derived DCs did not respond to CpG, but they were highly sensitive to LPS, suggesting an inverse correlation between CpG and LPS sensitivity in different subsets of DCs. Compared with GMCSF, CpG-treated peripheral blood DCs showed enhanced functional activity in the mixed lymphocyte reaction and induced T cells to secrete increased levels of Th1 cytokines. These findings demonstrate the ability of specific CpG motifs to strongly activate certain subsets of human DCs to promote Th1-like immune responses, and support the use of CpG DNA-based trials for immunotherapy against cancer, allergy, and infectious diseases.The vertebrate immune system has the ability to recognize the presence of bacterial DNA on the basis of recognition of so-called CpG motifs, unmethylated cytidine-guanosine dinucleotides within a specific pattern of flanking bases (1). It is known from the literature that CpG oligonucleotides are excellent adjuvants in murine models. CpG DNA is as potent as the complete Freund's adjuvant regarding the induction of B cell and T cell responses, but it is less toxic and it induces a T helper 1 (Th1) response (2, 3). Alum, the adjuvant that is used routinely in human vaccination, induces the less favorable Th2 response. CpG is more effective than alum, and the combination of CpG and alum shows synergy in mice (4, 5). CpG oligonucleotides enhance the efficacy of immunization with tumor antigen in a murine B cell lymphoma model (2), induce antigen-specific cytotoxic T cell responses (5, 6), and have utility in the immunotherapy of allergy, infectious disease, and cancer disease models (7-9). Furthermore, the presence of immunostimulatory DNA sequences in plasmids has been shown to be required for effective intradermal gene immunization (10).Dendritic cells (DCs) form the link between the innate and the acquired immune system by presenting antigens and by their expression of pattern recognition receptors that detect microbial molecules in their local environment. The use of DCs as a cellular adju...

Recent advances in our understanding of the immune response are allowing for the logical design of new approaches to cancer immunization. Bacterial DNA is capable of inducing activation of B cells, NK cells, and monocytes (1-5). In addition, bacterial DNA can induce production in vitro and in vivo of a variety of proinflammatory cytokines (6-8). In contrast, vertebrate DNA does not induce lymphocyte activation. Bacterial DNA contains a much higher frequency of unmethylated CpG dinucleotides than does vertebrate DNA due to (i) CpG suppression (the under representation of CpG in vertebrate genomes) and (ii) methylation of 80% of the CpG in vertebrates. It is possible that lymphocyte activation by the CpG motif in bacterial DNA represents an immune defense mechanism that can distinguish bacterial from host DNA (1). Select synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODN) have immunologic effects similar to those seen with bacterial DNA. CpG ODN can stimulate monocytes, macrophages, and dendritic cells that then produce several cytokines, including the TH1 cytokine interleukin 12. This effect synergizes with CpG ODN to induce NK cell production of interferon ␥ (6). Both human and murine leukocytes respond to this novel pathway of immune activation, although individual CpG ODN differ somewhat in their ability to activate various immune cell populations and induce cytokine production in human and murine systems.The molecular mechanisms responsible for CpG ODNinduced immune cell activation are still under investigation. We have recently reported that CpG ODN trigger the production of reactive oxygen species that activate NF-B (9). This activation, in turn, leads to cellular activation. Irrespective of the mechanism involved, it is clear that select CpG ODN can have powerful immunologic effects and might be useful therapeutic agents in a number of circumstances, including cancer immunotherapy. For example, we have demonstrated that CpG ODN can enhance antibody-dependent cellular cytotoxicity and improve the in vivo efficacy of monoclonal antibody therapy in a syngeneic murine lymphoma model (10).CpG ODN can induce activation of antigen-presenting cells and enhance production of cytokines known to participate in the development of an active immune response. They also enhance B cell activation, particularly when the B cell receptor is cross-linked (1). These effects are likely to promote antigenspecific responses. Indeed, Branda et al. (11) demonstrated that an anti-sense ODN, which in retrospect is noted to contain the CpG motif, enhances antibody response to antigen. We therefore used a well-established animal model to assess whether CpG ODN can function as an immune adjuvant in antitumor immunization.

Giving prophylactic platelets at a threshold of < or = 10,000/microL compared with < or = 20,000/microL can decrease the total utilization of platelets with only a small adverse effect on bleeding, and no statistically significant effect on morbidity.

Cancer immunotherapy has proven to be challenging as it depends on overcoming multiple mechanisms that mediate immune tolerance to self-antigens. A growing understanding of immune tolerance has been the foundation for new approaches to cancer immunotherapy. Adoptive transfer of immune effectors such as antitumor monoclonal antibodies and Chimeric Antigen Receptor T cells bypasses many of the mechanisms involved in immune tolerance by allowing for expansion of tumor specific effectors ex vivo. Vaccination with whole tumor cells, protein, peptide, or dendritic cells has proven challenging, yet may be more useful when combined with other cancer immunotherapeutic strategies. Immunomodulatory approaches to cancer immunotherapy include treatment with agents that enhance and maintain T cell activation. Recent advances in the use of checkpoint blockade to block negative signals and so maintain the antitumor response are particularly exciting. With our growing knowledge of immune tolerance and ways to overcome it, combination treatments are being developed, tested and have particular promise. One example is in situ immunization that is designed to break tolerance within the tumor microenvironment. Progress in all these areas is continuing based on clear evidence that cancer immunotherapy designed to overcome immune tolerance can be useful for a growing number of cancer patients.

Cancers often arise as the end stage of inflammation in adults, but not in children. As such there is a complex interplay between host immune cells during neoplastic development, with both an ability to promote cancer as well as limit or eliminate it, most often complicit with the host. In humans, defining inflammation and the presence of inflammatory cells within or surrounding the tumor is a critical aspect of modern pathology. Groups defining staging for neoplasms are strongly encouraged to assess and incorporate measures of the presence of apoptosis, autophagy, and necrosis as well as the nature and quality of the immune infiltrate. Both environmental as well as genetic factors enhance the risk of cigarette smoking, H. pylori, hepatitis B/C, human papilloma virus, solar irradiation, asbestos, pancreatitis, or other causes of chronic inflammation. Identifying suitable genetic polymorphisms in cytokines, cytokine receptors, and Toll-like receptors among other immune response genes is also seen as high value as genomic sequencing becomes less expensive. Animal models which incorporate and assess not only the genetic anlagen but also the inflammatory cells and the presence of microbial pathogen [PAMPs] and damage associated molecular pattern molecules [DAMPs] are necessary. Identifying micro-RNAs involved in regulating the response to damage or injury are seen as highly promising. Although no therapeutic strategies to prevent or treat cancers based on insights into inflammatory pathways are currently approved for the common epithelial malignancies, there remains substantial interest in agents targeting COX2 or PPARγ, ethyl pyruvate, as well as steroids and several novel agents on the horizon.