Current Immunology Reviews (v.8, #2)

Disease modifying, anti-inflammatory strategies often target pathogenic cells once they are localized within the site of inflammation. The prototypes of biologic drugs such as anti-TNFα or anti-p40 (IL-12 and IL-23) therapies act on cytokines produced by tissue macrophages for example in the synovium in Rheumatoid Arthritis (RA) or in the intestinal mucosa in Intestinal Bowel Disease (IBD). Furthermore, immunosuppressant drugs such as cyclosporin, steroids, cyclophosphamide and mycophenolate mofetil (MMF) similarly act on intracellular targets in cells already localized in the target organ. A similar phenomenon applies to cancer, where chemotherapies address the cells locally in the tumour, such as anti-epidermal growth factor receptor and all commonly used cytotoxic agents. However while effective for a certain subpopulation of patients, a significant unmet medical need exists for most diseases where dysregulation of the immune system is the fundamental cause. An attractive alternative strategy is to prevent aberrantly activated cells from accessing the site of inflammation in the first place, in other words targeting the capacity of cells to migrate through the endothelium. Cell migration is a well orchestrated and tightly coordinated succession of steps depicted below. The first event is the activation of the endothelium by proinflammatory cytokines. This results in the upregulation of selectins, molecules which mediate rolling of leukocytes on the endothelial surface. Therefore, prevention of the release of soluble TNF from its tethered transmembrane form by the inhibition of the enzyme catalyzing this shedding, would be a relevant target. This is covered in Chapter 1. Chapters 2 and 3 address the second stage of the cell recruitment process, the activation of integrins on the leukocyte mediated by the activation of chemokine receptors by their respective ligand. The chemokine family is a large family of small proteins, whose major, but not only, function is the control of cellular migration. Chemokine receptors belong to the class of sevenmembrane spanning G protein-coupled receptors, which have proved highly tractable targets for many pharmaceutical drugs. Therefore, the identification of the chemokine family over a quarter of a century ago gave great promise to the pharmaceutical industry as potentially tractable anti-inflammatory targets. We have decided not to include a chapter on the small molecular weight inhibitors of chemokine receptors, as this subject has been addressed extensively by a number of recent reviews, and the reader is referred to these [1-11]. However, overall the success of this approach has been disappointing, and again the reader is referred to recent opinion articles discussing several hypotheses addressing why success has been so difficult [12, 13]. But we do not exclude the future success of this approach, as the first anti-inflammatory program to meet its endpoints in Phase II, inhibition of CCR9 for Crohn's disease, is currently in Phase III, and if successful, will dispel the reputation of chemokine receptors being “undruggable”....

Leucocyte recruitment from the bloodstream into tissues depends on a coordinated sequence of adhesive interactions between leucocytes and the vascular wall. It is tightly regulated, both spatially and temporally, such that leucocyte recruitment is efficient and the integrity of the vascular wall is not impaired. Although the cell adhesion molecules and chemokines that mediate adhesion have been identified, the signalling events that control extravasation are just starting to be understood. Ectodomain shedding by the ADAMs family of metalloproteinases is emerging as an important regulatory step in leucocyte-endothelial cell interactions. The evidence for ADAMs involvement in leucocyte recruitment will be reviewed with particular emphasis on L-selectin and ADAM17. The regulation of ADAM catalytic activity is complex and controlled by intracellular signaling pathways and cellular localization. ADAM activity is also regulated by substrate availability and the roles of extracellular and intracellular domains of L-selectin in regulating ADAM dependent ectodomain shedding will also be reviewed.

The α4β7 integrin is a leukocyte homing receptor with selective tissue tropism for the gastrointestinal tract through its interaction with MAdCAM-1, an adhesion receptor expressed on the endothelium of the gut mucosa. Crohn’s disease (CD) and ulcerative colitis (UC), two inflammatory bowel diseases resulting from intestinal immunedysregulation, are associated with pronounced infiltration of α4β7 positive lymphocytes. This has triggered the development of inhibitors of the α4β7/MAdCAM-1 homing pathway. Vedolizumab is a humanized monoclonal antibody selective for α4β7 that demonstrated efficacy in early clinical studies for the treatment of CD and UC and is currently in phase 3 clinical trials. Targeting of α4β7 is also achieved by less selective therapeutic modalities which also block one of the two other leukocyte integrins that share a subunit with α4β7, namely, α4β1 and αEβ7. Natalizumab is an anti-α4 monoclonal antibody and dual α4β1 and α4β7 antagonist approved for the treatment of multiple sclerosis and CD. Other therapies in development include antibodies targeting the β7 subunit of α4β7 and αEβ7, MAdCAM-1, and dual α4 small molecule antagonists. This review will focus on the mechanism of action, pharmacology, efficacy and safety properties as well as future opportunities that may arise from this unique class of leukocyte anti-adhesion antagonists.

Blocking the action of alpha4 integrin would be expected to be of therapeutic benefit in the management of autoimmune diseases. Although this has been successfully demonstrated in the clinic with a monoclonal antibody for the treatment of multiple sclerosis, there are no small molecule alpha4 integrin antagonists on the market despite significant endeavour over the last 15-20 years. We review our efforts in this area, starting from a cyclic peptide based on an integrin recognition sequence and culminating in the low molecular weight clinical candidate, CDP323. We include a discussion on pre-clinical pharmacological data for CDP323.

Chemokines cannot be easily antagonized by low molecular weight (LMW) compounds and are therefore more suitable for targeting clinically via ‘biologics’. Significant beneficial features of mAbs as compared to LMW compounds include a high selectivity for their target, reducing the risk of off-target side effects, as well as the prolonged pharmacokinetics, with half-lives ranging from days to weeks, necessitating less frequent dosing. In this HOT TOPIC, our aim is to focus on reviewing available information regarding anti-chemokine mAbs that have reached clinical development stage and discuss not only the target relevance and the clinical outcomes, but also describe the characteristics of the therapeutic antibodies. Indeed, as clinical efficacy - or lack thereof - is highly dependent on the biology of the target, so are the properties and administration modality of the drug which will also impact on the clinical outcome and thus on the validity of targeting chemokines in disease settings.

Monoclonal antibodies (mAbs) serve as research reagents, for dissecting the biology of chemokine and chemoattractant receptors. However mAbs are also an attractive class of therapeutic, because they are able to block large protein-protein interactions, and are generally easier and more predictable for clinical development. mAbs are also capable of depleting leukocyte subsets, such as pathogenic cell types, through processes such as phagocytosis, complement fixation and ADCC. Chemokine receptors are probably one of the best classes of molecules for marking different leukocyte subsets, such as Th1, Th2, Th17 and Tfh cells, and hence should provide a means for selective depletion. This short review will discuss the uses of mAbs to chemoattractant receptors, as research reagents and as therapeutics.

Phosphoinositide 3-kinase (PI3K) has been positioned at the heart of an evolutionarily conserved cellular compass and/or the biochemical mechanisms that facilitate cell migration. PI3K has therefore, become a popular drug target for inhibition of leukocyte migration in response to inflammatory chemoattractant mediators including members of the chemokine family. PI3K has also been implicated as a key regulator in novel mechanisms mediated by the T cell antigen receptor and the costimulatory molecule CD28 that guide the access and retention of specific T cells into antigenrich non-lymphoid tissue. The precise role of PI3K in the regulation of cell migration remains open to refinement, as numerous examples of PI3K-independent leukocyte migration (particularly with respect to T lymphocytes), have been described. The best studied leukocytes in terms of their migratory response and the role of PI3K are neutrophils and macrophages (that constitute the innate immune response), T lymphocytes (key elements of the adaptive immune response) and eosinophils (typical infiltrating cells at the sites of allergen-IgE reactions that constitute a link between innate and adaptive systems). Genetic and pharmacological approaches have been employed to assess the contribution of individual class 1 PI3K isoforms to migratory response of these leukocytes and will be considered in this article.

Targeting Chemokines in Cancer by Raffaella Bonecchi (161-169).
The chemokine system is now recognized as a key element in cancer-related inflammation because it can affect tumor progression acting on tumor-stroma and also directly on tumor cells. Chemokines are produced by both tumor cells and the tumor microenvironment and modulate not only leukocyte infiltration and angiogenesis but also senescence, cell survival and metastasis. Here, we review available information in preclinical and clinical settings that suggest that the chemokine system represents a valuable target for the development of innovative therapeutic strategies.

S1P Receptor Modulators in Cell Trafficking and Therapeutics by Pedro J. Gonzalez-Cabrera (170-180).
FTY720 (fingolimod, Gilenya®) is a sphingosine analog prodrug that induces lymphocyte sequestration in secondary lymphoid organs, resulting in peripheral lymphopenia. Lymphopenia by FTY720 is characterized by a dosedependent and sustained reduction of circulating T and B lymphocytes within hours of administration, accounting for a 70% reduction of CD4+ T cells, 90% reduction of CD8+ T-cells, and > 50% reduction of CD19% B-cells, with a single administration. Mechanistically, lymphocyte sequestration following FTY720 administration in vivo is due to blockade of lymphocyte egress into efferent lymph. The induction of lymphopenia by FTY720 requires the generation of its active phosphate intermediate, FTY720-P, a step catalyzed by phosphorylation of FTY720 by the ubiquitous sphingosine-kinase 2. In turn, FTY720-P becomes a potent subnanomolar agonist on four of the five sphingosine-1 phosphate (S1P) receptors (S1P1,3,4,5). Clinically, FTY720 has recently become the first oral therapy to be approved for the treatment of relapsingremitting multiple sclerosis. As a research tool, FTY720 has been critical in providing mechanistic insights into lymphocyte trafficking in physiology and disease. Although FTY720-P displays similar potency across S1P receptors, it is the S1P1 subtype, a class-A-type Gi-coupled GPCR, that is the sole mediator of FTY720-P’s ability to induce lymphopenia. S1P1 is broadly expressed in mammals, with relatively high expression found in neuronal, immune and endothelial cells, and has proven to be critical for vascular development in rodents. The aim of this review is to highlight FTY720’s efficacy as an immunosuppressant, examine its mechanisms of action, and the emergence of mono-selective S1P1 agonist therapies for autoimmune diseases.

Aberrant cell recruitment is a hallmark of inflammatory responses, a complex process orchestrated by a unique interplay of adhesion, chemotactic and pro-inflammatory molecules. To date there are only two marketed drugs that block cell migration, interestingly both for Multiple Sclerosis (MS). Tysabri/Natalizumab, a humanized monoclonal antibody against the cellular adhesion molecule VLA-4 was the first to be approved for the treatment of MS. A second inhibitor of migration, Gilenya/fingolimod entered the market last year, having received FDA approval in 2010. There are many more drugs targeting cellular recruitment in preclinical and clinical development, reflecting the promise that lies behind inhibiting this process as treatment for a range of inflammatory diseases. Confirmation of efficacy of the compound/drug in vivo is an important part of the drug development process and is essential for drugs that have a novel mechanism of action. Additionally, toxicological tests to determine the safety of the drug are a pre-requisite for successful IND filing. In pre-clinical development studies, drugs are preferentially tested for efficacy in rodent species such as mice, however in order for these tests to be relevant, the compound has to show cross-reactivity with the target in the test species, which can be a major hurdle for some target classes including chemokines. Not only do such tests rely on compound cross-reactivity, but differences in biology, in particular of the immune system between rodents and humans, can cause additional problems for drug development. In this review, we discuss, in the context of cell migration and inflammation, the problems that arise during pre-clinical testing in vivo, including strategies to mitigate these risks.