Antagonizing leptin: current status and future directions.

DOI 10.1515/hsz-2013-0283 Biol. Chem. 2014; 395(5): 499–514

Review Lennart Zabeau, Frank Peelman and Jan Tavernier*

Antagonizing leptin: current status and future directions Abstract: The adipocyte-derived hormone/cytokine leptin acts as a metabolic switch, connecting the body’s nutritional status to high energy consuming processes such as reproduction and immune responses. Inappropriate leptin responses can promote autoimmune diseases and tumorigenesis. In this review we discuss the current strategies to modulate leptin signaling and the possibilities for their use in research and therapy. Keywords: antagonism; autoimmunity; leptin. *Corresponding author: Jan Tavernier, Flanders Institute for Biotechnology (VIB), Department of Medical Protein Research, Faculty of Medicine and Health Sciences, Ghent University, A. Baertsoenkaai 3, B-9000 Ghent, Belgium, e-mail: [email protected] Lennart Zabeau and Frank Peelman: Flanders Institute for Biotechnology (VIB), Department of Medical Protein Research, Faculty of Medicine and Health Sciences, Ghent University, A. Baertsoenkaai 3, B-9000 Ghent, Belgium

Introduction: a little history… In 1950, the first obese mouse strain, the ob/ob mouse, was discovered at The Jackson Laboratory. These animals were not only morbidly obese and hyperphagic, but also insulin resistant, infertile, and mildly diabetic (Coleman, 2010). Fifteen years later, a second obese strain was identified with a very comparable phenotype. These animals develop severe and life-shortening diabetes, and were called diabetic or db/db. A series of elegant parabiosis experiments showed that the db/db mutant mouse overproduced a blood-borne satiety factor but could not respond to it, whereas the ob/ob mutant recognized and responded to the factor but could not produce it (Coleman, 1973, 2010). It took over 40 years before the obese gene was positionally cloned by Friedman and colleagues and was found to encode a hormone that they called leptin (after the Greek ‘leptos’ for thin) (Zhang et al., 1994). One year later, Tartaglia and colleagues were able to isolate

and identify the leptin receptor gene using an expression cloning strategy (Tartaglia et al., 1995).

Leptin and its receptor Leptin is a non-glycosylated 16 kDa hormone with cytokine-like characteristics. The mature protein adopts the 4-α-helical bundle structure, making it a member of the class I family of cytokines (Madej et al., 1995), and is further characterized by an intra-molecular disulphide bond that is necessary for its biological activity (Rock et al., 1996; Haglund et al., 2012). The hormone is mainly, but not exclusively, produced by adipocytes, and its serum levels positively correspond with the energy stored in the fat mass (Frederich et al., 1995; Halaas et al., 1995; Considine et al., 1996). Low leptin expression could also be shown in the placenta, stomach, mammary epithelium and skeletal muscle (Señarís et al., 1997; Bado et al., 1998; Wang et al., 1998). The leptin receptor (LR) is a single membrane-spanning receptor belonging to the class I cytokine receptor family (Tartaglia et al., 1995). At present, six LR isoforms produced by alternative splicing or ectodomain shedding have been identified: LRa-f. All forms have an identical extracellular domain consisting of two so-called cytokine receptor homology domains (CRH1 and CRH2), an immunoglobulin-like domain (IGD) and two additional, membrane-proximal fibronectin type III (FN III) domains (Figure 1A). The isoforms differ in the length of the cytoplasmic tail: LR long form (LRb or LRlo) is the only variant capable of efficient signaling; LRe is an extracellular soluble variant; while the rest are called short forms. LRb is highly expressed in certain nuclei of the hypothalamus, a region of the brain involved in energy homeostasis (Mercer et al., 1996; Schwartz et al., 1996; Fei et al., 1997), but also in a broad range of other cell types (see below). The precise role of the short forms remains elusive, but involvement in transport of leptin over the blood brain barrier (BBB) (Hileman et al., 2000) or in renal clearance (Tartaglia et al., 1995) has been suggested. The soluble LR

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500 L. Zabeau et al.: Antagonizing leptin: current status and future directions

Figure 1 LR activation mechanism illustrated by homology models for the leptin:LR complex. (A) The extracellular part of the leptin receptor contains five domains. The CRH2 and IGD are the domains that contact leptin in the activated receptor complex. (B) Leptin binds with high affinity to the CRH2 domain (top) and subsequently binds the IGD of a second leptin receptor. This leads to a 2:2 leptin:LR complex (bottom). The green and purple spheres indicate the position of the leptin-binding site III mutations S120A-T121A and 39LDFI-AAAA42, that block interaction with the IGD. (C) In the activated receptor complex, the FN III domains approach each other.

is the main binding protein for leptin in the blood and modulates the leptin bioavailability (Ge et al., 2002).

LR activation Profound insights into the LR activation mechanisms are a prerequisite for the rational design and development of leptin and LR antagonists. Crystal structure information for the leptin:LR complex is still limited. Until now, only two crystallography studies have been published: the structure of the leptin W100E mutant (a leptin mutant with wild-type activity, but with dramatically improved solubility and propensity to crystalize) (Zhang et al., 1997), and the leptin-binding CRH2 domain in complex with the Fab fragment of a leptin-blocking monoclonal antibody (Carpenter et al., 2012). A series of studies showed the importance of the CRH2, IGD and FN III subdomains in LR activation. The CRH2 domain is the major leptin-binding determinant (Fong et al., 1998; Zabeau et al., 2004). Two independent mutagenesis studies identified a region of four consecutive hydrophobic residues in CRH2 involved in this interaction, and mutation of these both affects binding and signaling (Iserentant et al., 2005; Niv-Spector et al., 2005b). The IGD has no detectable binding affinity for leptin, but is nonetheless crucial for LR activation. LR variants lacking this domain show unaltered leptin-binding, but are unable to signal via STAT3 (Fong et al., 1998; Zabeau et al., 2004). We identified residues L370, A407, Tyr409, His417 and

His418 in a conserved surface patch in the β-sheet formed by β-strands 3, 6 and 7 as the center of the leptin-binding site in this domain (Peelman et al., 2006). The membraneproximal FN III domains also lack any binding affinity for leptin, but are also indispensable for LR activation and can by themselves position the cytoplasmic tails, promoting signaling (Fong et al., 1998; Zabeau et al., 2005). Combined mutation of two conserved FN III cysteine residues resulted in a receptor completely devoid of biological activity, but with unaltered leptin-binding characteristics (Zabeau et al., 2005). Like other class I cytokine receptors, the LR forms preformed oligomers on the cellular membrane (Couturier and Jockers, 2003; Zabeau et al., 2004). We used a complementation of signaling strategy to show that leptin can induce higher-order clustering of its receptor (i.e., more than two receptors) (Zabeau et al., 2004). Detailed mutagenesis of leptin identified three potential receptor-binding sites (I, II and III), analogous to highly similar interleukin-6 (IL-6)-related cytokines (Peelman et al., 2004). Mutations in binding site I (located at the C terminus of helix D) moderately affect binding and signaling. Binding site II residues (at the surface of helices A and C) are crucial for binding to the CRH2 domain, but mutations in this region have only limited effect on signaling. Finally, site III mutations (located at the N terminus of helix D) impair receptor activation without affecting binding to CRH2. These studies and more recent single particle electron microscopy (Mancour et al., 2012) and small-angle X-ray scattering experiments (Moharana et al., submitted) were


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used to propose a working model for the activation of the LR. In this model, leptin binds to the CRH2 domain of a first receptor via its site II and engages a second receptor via the site III – IGD interaction, leading to a 2:2 quaternary signaling complex (Figure 1). Additional receptorreceptor interactions may lead to the formation of 4:2 and/or 4:4 complexes, but this is still under investigation.

LR signaling Like all members of the class I cytokine receptor family, the LR has no intrinsic kinase activity, and uses associated JAKs (Janus kinases) for intracellular signaling. A wellconserved membrane-proximal proline-rich box1 motif in the receptor is essential for this association, while a less well-defined box2 motif (which is absent in the short isoforms) also contributes to kinase activation (Bahrenberg et al., 2002; Kloek et al., 2002). JAK2 activation allows LR signaling via the JAK/ STAT (signal transducers and activators of transcription), SHP2/ MAPK (mitogen-activated protein kinase), PI3K and AMP-activated protein kinase (AMPK) pathways [for an extensive review see (Wauman and Tavernier, 2011)]. In the JAK/STAT pathway, ligand induced receptor clustering leads to activation of JAK2 by cross-phosphorylation. Activated JAK2 then rapidly phosphorylates tyrosine residues in the cytosolic domain of the receptor, providing docking sites for signaling molecules such as STATs. JAKs subsequently phosphorylate the STATs, which then translocate as dimers to the nucleus to modulate transcription of target genes. The STAT molecule primarily involved in leptin signaling is STAT3 (Vaisse et al., 1996), but also activation of STAT1, STAT5 and STAT6 could be shown in cultured cells (Baumann et al., 1996; Ghilardi et al., 1996; Rosenblum et al., 1996). Knock-in mice expressing a LR variant deficient in STAT3 activation are hyperphagic and obese, thereby underscoring the essential role of this pathway in leptin-regulated energy metabolism (Bates et al., 2003). Recruitment of SH2-containing protein tyrosine phosphatase 2 (SHP2) and growth factor receptor-bound protein 2 (Grb2) to the activated LR induces JAK2-dependent activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) MAPK (Carpenter et al., 1998; Li and Friedman, 1999; Banks et al., 2000; Bjørbaek et al., 2001). This leads to up-regulation of the immediate early genes egr-1 and c-fos in cell culture and in vivo in the hypothalamus (Elias et al., 1999; Bjørbaek et al., 2001; Cui et al., 2006). The physiological importance of this SHP2/MAPK pathway

is underscored by the observations that neuron-specific deletion of SHP2 results in early-onset obesity and leptin resistance and that pharmacological inhibition of ERK1/2 in the hypothalamus prevents the anorectic and weightreducing effects of leptin (Zhang et al., 2004; Rahmouni et al., 2009). LR activation induces phosphorylation of several members of the insulin receptor substrate (IRS) family, the recruitment of the regulatory p85 subunit and activation of phosphatidylinositol 3-kinase (PI3K), leading to the accumulation of its product phosphatidylinositol 3,4,5-triphosphate (PIP3) (Bjørbaek et al., 1997; Kellerer et al., 1997; Duan et al., 2004; Wauman et al., 2008). Downstream effects are the activation of 3-phosphoinositide-dependent protein kinase 1 (PDK1), Akt and cyclic nucleotide phosphodiesterase 3B (PDE3B), a cAMPdegrading enzyme (Zhao et al., 2002). Of note, IRS2-/- mice are hyperphagic and obese (Withers et al., 1998; Burks et al., 2000). Leptin regulates 5′-AMPK activity in a tissue-specific manner. In muscle tissue and hepatocytes, leptin activates AMPK, thereby inhibiting the acetyl-CoA carboxylase (ACC) activity (Minokoshi et al., 2002; Uotani et al., 2006; Miyamoto et al., 2012), while in the hypothalamus AMPK and ACC are respectively inhibited and stimulated by the hormone, which contributes to the anorectic effect of leptin (Zhao et al., 2003; Andersson et al., 2004; Minokoshi et al., 2004). In a hepatocyte cell line, AMPK requires JAK kinase activity, but this does not seem to depend on intracellular phosphotyrosine motifs in the LR (Uotani et al., 2006).

Body weight regulation and obesity The crucial role of leptin in long-term regulation of body weight by balancing food intake and energy expenditure is well established. The hormone is mainly produced by adipocytes thereby signaling the body’s energy stores and functions as a negative feedback adipostat, an efferent satiety signal and an anti-obesity hormone. It plays a role in the adaptive response to fasting and starvation. Food restriction is followed by a decrease in circulating leptin levels, resulting in a set of neuroendocrine responses that favor survival in periods of limited energy supplies. These actions include the up- or down-regulation of specific neuropeptides in the orexigenic neuropeptide Y (NPY) and anorexigenic POMC neurons, located in the ARC nucleus of the hypothalamus. The anorexigenic effects of leptin are mediated by the proteolytic processing of POMC


502 L. Zabeau et al.: Antagonizing leptin: current status and future directions and the subsequent liberation of α-MSH (melanocytestimulating hormones). The antagonistic neuropeptides Agouti-related transcript (AgRP) and NPY are negatively regulated by leptin. Obesity can be defined as a non-malignant, complex medical condition in which excess body fat increases the risk of obesity-related conditions like type 2 diabetes, heart disease, obstructive sleep apnea, certain types of cancer and osteoarthritis (Haslam and James, 2005). People are defined as obese when they have a body mass index (individual’s body mass divided by the square of their height) greater than 30 kg/m2. Obesity, with increasing prevalence in adults and children, is believed to be a leading preventable cause of death worldwide and one of the most serious public health problems of the 21st century (Barness et al., 2007). Obesity, like many other medical conditions, is the result of the complex interplay between both environmental (with excessive food intake and the lack of physical activity as the most important) and genetic and even epigenetic factors (Barness et al., 2007). Loss-of-function mutations in leptin or LR genes (see above), or genetic ablation of leptin’s central signaling (Wauman and Tavernier, 2011) results in an obese phenotype. Genome-wide association studies further identified almost 50 genes that relate to an increased obesity risk in humans [reviewed in (Xu et al., 2013b)]. Epigenetic factors that might further influence the prevalence of obesity include differential DNA methylation and certain histone modifications (Campión et al., 2010; Xu et al., 2013a). In the last decade it became clear that leptin is more than a satiety signal, and rather acts as a ‘metabolic switch’ (Matarese et al., 2002). Indeed, leptin or LR deficiency not only causes severe obesity, but also abnormalities in lipid and glucose metabolism (Friedman and Halaas, 1998), hematopoiesis (Bennett et al., 1996), immunity (Lord et al., 1998), reproduction (Chehab et al., 1996), angiogenesis (Sierra-Honigmann et al., 1998), vascular remodeling (Konstantinides et al., 2001), blood pressure (Mark et al., 1999), and bone formation (Ducy et al., 2000).

Leptin in innate and adaptive immune responses Ozata and colleagues were the first to postulate a role of leptin in the control of human immunity. They reported that seven obese members of a Turkish family with congenital leptin deficiency died during childhood because of infections (Ozata et al., 1999). Daily subcutaneous

injections of recombinant leptin in three morbidly obese leptin deficient children not only had beneficial effects on appetite, fat mass, hyperinsulinemia, and hyperlipidemia but also reversed the reduction in numbers of circulating CD4+ T cells and impaired T cell proliferation and cytokine release (Farooqi et al., 2002). Similarly, mice lacking leptin or a functional LR appear to be impaired in both cellmediated and humoral immunity (Mandel and Mahmoud, 1978). It became clear that leptin affects almost all cells of the innate immunity, the nonspecific first line defense of the immune response (Figure 2). Dendritic cells, the major antigen presenting cells, from ob/ob mice appear less potent in stimulation of allogenic T cells in vitro, most likely because of the increased secretion of immunosuppressive cytokines like transforming growth factorb (TGF-b) (Macia et al., 2006). In human dendritic cells, leptin promotes survival and TH1 priming (Mattioli et al., 2005) and migration of immature cells by enhanced cytoskeleton dynamics and increased chemotactic responsiveness (Mattioli et al., 2008). In macrophages/ monocytes, leptin influences phagocytosis (Mancuso et al., 2004), pro-inflammatory cytokine secretion (Gainsford et al., 1996), proliferation, up-regulation of activation markers (Zarkesh-Esfahani et al., 2001), and it acts as a potent chemoattractant for these cells (Gruen et al., 2007). Leptin can also promote chemotaxis of neutrophils and the release of reactive oxygen species (Caldefie-Chezet et al., 2001, 2003), although it cannot be excluded that these are indirect effects of tumor necrosis factor-α (TNFα released by monocytes (Zarkesh-Esfahani et al., 2004). Finally, leptin plays a role in natural killer cell development, differentiation, activation, and cytotoxicity (Tian et al., 2002; Zhao et al., 2003). Leptin and LR deficiency or malnutrition/starvation has a great impact on the cellularity of the thymus, both on B and T cells. Ob/ob mice have 70% fewer B cells, and significant lower counts of pre-B and immature B cells (Claycombe et al., 2008). In the T cell compartment, double positive CD4+CD8+ immature thymocytes are most affected, as these cells require leptin as a survival, antiapoptotic factor (Howard et al., 1999). Leptin administration profoundly restores these defects (Howard et al., 1999; Claycombe et al., 2008; Tanaka et al., 2011), but the cellular targets of leptin in restoring this thymic cellularity and whether the lack of leptin or its receptor specifically affects subpopulations of T lymphocytes in the periphery remains unclear. At least in vitro, several studies point to direct effects of leptin on T cells. It can stimulate proliferation and the increase in expression of adhesion molecules in CD4+ T cells (Lord et al., 1998). However, leptin



Figure 2 Effects of leptin on innate and adaptive immune responses. See text for more details.

has different effects on proliferation and cytokine secretion by human naïve (CD45RA) and memory (CD45RO) CD4+ T cells. On naïve cells, leptin stimulates proliferation and IL-2 secretion; while in memory cells it promotes the switch towards TH1 immune response with up-regulation of interferon-γ (IFN-γ) and TNF-α (Lord et al., 2002). De Rosa and colleagues showed that Foxp3+CD4+CD25HIGH T regulatory (TReg) cells, a cellular subset that is known to suppress autoreactive responses mediated by other CD4+ cells, produce leptin and this blocks their own proliferation (De Rosa et al., 2007). In vitro neutralization with an anti-leptin antibody, together with anti-CD3/CD28 co-stimulation, resulted in TReg proliferation. More recently it was reported that TH17 cell numbers are reduced in ob/ob mice, while leptin administration restored TH17 cell numbers (Yu et al., 2013). Leptin promoted TH17 responses in normal human CD4+ T cells and in mice, both in vitro and in vivo, by inducing RORγt transcription. It is of special note that leptin alone is unable to induce most (if not all) above mentioned effects on T cells, but needs co-administration

of other nonspecific immune-stimulants, and this in contrast to the effects on macrophages/monocytes and dendritic cells. Given its pro-inflammatory characteristics, it is not surprising that leptin can contribute to the onset and progression of autoimmune diseases (La Cava and Matarese, 2004). Indeed, increased peripheral leptin secretion in humans is associated with chronic inflammation and autoimmunity whereas decreased leptin levels generally inhibit autoimmune disease onset and progression. Likewise, the balance between TH1 (IL-2, IFN-γ, TNF-α and IL-18) and TH2 (IL-4 and IL-10) cytokines is dysregulated in favor of the latter in leptin deficient mice, making the animals more resistant to experimental induced autoimune diseases. Examples include experimentally autoimmune encephalomyelitis, a model for multiple sclerosis (Matarese et al., 2001), antigen-induced arthritis (Busso et al., 2002), experimentally induced (with concanavalin A or Pseudomonas aeruginosa exotoxin A) hepatitis (Faggioni et al., 2000; Siegmund et al., 2002), experimentally induced colitis


504 L. Zabeau et al.: Antagonizing leptin: current status and future directions (Siegmund et al., 2002), and experimentally induced glomerulonephritis (Tarzi et al., 2004). Leptin administration to ob/ob mice restores sensitivity in these disease models, while treatment of wild-type animals even worsened the clinical outcome of the disease.

Leptin in cancer Clinical reports unequivocally link elevated serum leptin levels (caused by obesity) to an increased risk of certain cancers including prostate (Garofalo and Surmacz, 2006), breast (Cirillo et al., 2008), colerectal (Pais et al., 2009), renal cancers (Liao et al., 2013) and myeloma (Gogas et al., 2008). The etiological factors linking obesity and cancer development include insulin resistance, chronic hyperinsulinemia, greater biodisposition of steroidal hormones, local inflammation, and adipokine (with leptin and adiponectin as the most important ones) secretion by adipose tissue (Calle, 2007). In vitro and preclinical in vivo data suggest that leptin acts as a mitogenic agent to promote proliferation and survival of prostate, breast, and ovarian cancer cells and/or enhances cancer angiogenesis, migration and invasion (Choi et al., 2004; Frankenberry et al., 2006; Ray and Cleary, 2010). These aspects are briefly discussed in the context of breast cancer cell lines, but are applicable to cells originating from other types of cancer. The proliferative effect of leptin on cancer cell lines is mediated by different signaling pathways, including the activation of STAT3, ERK, and activator protein 1 (AP-1) (Hu et al., 2002). This leads to the induction of expression of c-myc, steroid receptor coactivator-1 (SCR-1) (Yin et al., 2004), aromatase (Catalano et al., 2003), and genes involved in cell cycle progression, including cyclin D1, A2, G, and cyclin-dependent kinase 2 (CDK2), CDKN1A and CDKN2A (Saxena et al., 2007; Perera et al., 2008). Unique in breast cancer cells is the leptin-mediated transactivation of the HER2/Neu (ErbB2) receptor, an epidermal growth factor family receptor that is overexpressed in 20–30% of cases of breast and cervix cancers (Nair, 2005), leading to proliferation (Okumura et al., 2002; Soma et al., 2008). Leptin induces the expression of anti-apoptotic genes B-cell lymphoma 2 (bcl-2) and Survivin (Artwohl et al., 2002), and suppresses insulin-like growth factor 1 receptor (IGF1R) and TNF receptor type 1-associated DEATH domain protein (TRADD) (Perera et al., 2008). Multiple studies suggest that leptin alters the tumor microenvironment to favor tumor growth and progression. For example, leptin can increase the secretion of

fibroblast growth factor 9 (FGF-9) (Perera et al., 2008) or matrix metalloproteinases (MMPs), enzymes involved in the invasion and metastasis of tumors (McMurtry et al., 2009). Likewise, leptin promotes the adhesion-dependent expression of E-cadherin, an intracellular adhesion molecule related to epithelial tumors (Mauro et al., 2007), and mediates the overexpression of extracellular matrix genes, including connective tissue growth factor (CTGF), villin 2 and basigin (Perera et al., 2008). Finally, leptin promotes angiogenesis through the expression of vascular endothelial growth factor (VEGF) and VEGFR2 (Gonzalez et al., 2006), and Notch expression and activation (Guo and Gonzalez-Perez, 2011). Cachexia or wasting syndrome is the medical condition characterized by weight loss, muscle atrophy, fatigue, weakness, and significant loss of appetite. This loss of body mass cannot be reversed nutritionally. Cachexia is seen in 80% of patients with advanced cancer, and may account for up to 20% of cancer deaths (Tisdale, 2002). Leptin levels are significantly decreased in cachexia patients (Smiechowska et al., 2010) and they appear resistant to the orexigenic effects of the observed hypoleptinemia. This is probably a result of an increased LR expression in the hypothalamus (Bing et al., 2001). Proinflammatory cytokines like TNF-α, IL-1, and IL-6, are more likely to cause cachexia.

Leptin and LR antagonists In some physiological or pathological situations like uncontrolled immune responses in autoimmune diseases, tumorigenesis, elevated blood pressure, and certain cardiovascular diseases, it is desirable to block leptin activity. At present, four strategies are being used to antagonize leptin signaling: (i) leptin antagonistic mutants that bind to, but do not activate the receptor; (ii) leptin peptide antagonists that consist of parts of the leptin sequence; (iii) leptin and LR specific (monoclonal) antibodies or nanobodies that prevent productive binding of leptin; and (iv) soluble LR variants that trap free leptin in the circulation. A schematic representation of these strategies is shown in Figure 3.

Leptin mutants The ideal leptin antagonist binds the LR with an affinity comparable to or higher than wild-type leptin but is completely devoid of biological activity. In this respect,



Figure 3 Leptin and LR antagonists. Schematic representation of different strategies to antagonize leptin or its receptor, including leptin mutants, leptin peptide antagonists, neutralizing leptin and LR antibodies or nanobodies, and soluble LR variants.

mutants in binding site III are of special interest (see above). Before any structural insights in the LR activation mechanisms, Verploegen and colleagues developed the first leptin antagonist (Verploegen et al., 1997). They showed that an arginine to glutamine substitution at position 128 in human leptin abolished biological activity (on Ba/F3 cells expressing a LR chimera) without affecting

binding. In vivo administration in C57BL/6J mice resulted in weight gain and hyperinsulinemia. Although the R128 residue is part of a sequence conserved in mammalian and non-mammalian leptins, the antagonistic properties of the substitution appeared to be species-specific since similar mutations in ovine and chicken did not result in antagonism (Raver et al., 2002). Based on a detailed mutagenesis study, we identified the serine and threonine residues on position 120 and 121 as crucial for the interaction with the LR IGD domain. Mutation of these residues to alanines in human and mouse leptin (resulting in leptin S120A-T121A) creates potent leptin antagonists both in vitro and in vivo (Peelman et al., 2004) (Figure 1B). In a parallel approach, Niv-Spector et al. used a sensitive, bidimensional hydrophobic cluster analysis to identify a hydrophobic stretch in leptin’s A-B loop (39LDFI-42) as part of the binding site III (Niv-Spector et al., 2005a) (Figure 1B). Mutation of these residues resulted in a potent antagonist in vitro on LR expressing Ba/F3 cells (Niv-Spector et al., 2005a), neonatal rat ventricular cardiomyocytes (Rajapurohitam et al., 2006), rat and human intestinal mucin-producing cells (El Homsi et al., 2007), and androgen sensitive and insensitive prostate cancer cells (Samuel-Mendelsohn et al., 2011). The halflife in circulation could be increased by pegylation and this illustrates that mice administered with the pegylated antagonist showed rapid and dramatic increase in food intake with resulting weight gain (Elinav et al., 2009b). The antagonist was further optimized by an additional D23L substitution, resulting in the so-called superactive leptin antagonist (SLA). This mutein exhibits an over 60-fold increased binding to the LR, and 14-fold higher antagonistic activity in vitro (Shpilman et al., 2011; Niv-Spector et al., 2012). The SLA was further used to prove that the resistance to obesity and the altered locomotor activity of transgenic mice brain specifically overexpressing urokinase-type plasminogen activator is caused by high leptin levels (Chapnik et al., 2013). More recently, it was reported that the antagonist ameliorated the chronic kidney disease-associated cachexia (Cheung et al., 2014). In autoimmune disease models the SLA also proved its effectiveness. Administration of the antagonist markedly improved the clinical outcome of chronic thioacetamide (TAA) fibrosis (Elinav et al., 2009a) and chronic colitis in IL-10-/- mice (Singh et al., 2013). Finally, the antagonist was proved to be a valuable tool to study the role of pre- and neonatal leptin in the predisposition to obesity (Attig et al., 2008), organ development (Attig et al., 2011), and hypothalamic development in males and females (Mela et al., 2012).


506 L. Zabeau et al.: Antagonizing leptin: current status and future directions

Leptin peptide antagonists Gonzalez and colleagues were the first to describe the design and utilization of leptin peptide antagonists (LPAs). Based on the suggested role of helices A and C in leptin, they designed two LPAs corresponding to amino acids 3–34 (LPA-1) and 70–95 (LPA-2) of human leptin (26 and 32 residues, respectively). LPA-2 binds specifically and with high affinity to the LR and potently inhibits leptindependent increases in the concentration of b3-integrin, IL-1, leukemia inhibitory factor, and their corresponding receptors in rat endometrial cells (Gonzalez and Leavis, 2003). In mice, a pegylated variant of LPA-2 attenuates leptin-induced growth of mouse mammary tumor cells in immunodeficient mice (Gonzalez et al., 2009). The mechanism of action of these peptides has yet to be clarified. An alternative design of LPAs was described by Otvos and colleagues. They synthesized four proposed receptor-binding fragments (site I, IIa, IIb, and III) and tested their effect on cellular proliferation. Agonistic/antagonistic properties greatly depended on the presence of leptin in the assay: the combined site II and site III LPAs selectively blocked leptin-driven growth, while a glycopeptide site III analog proved to be a full agonist in the absence of the hormone (Otvos et al., 2008). The site III peptide was further modified to a 12-residue glycosylated peptomimetic E1/6-amino-hexanoic acid (the resulting peptide was called Allo-Aca) variant and proved to be sufficient to reduce weight and restore fertility in high-fat diet-induced obese mice (Kovalszky et al., 2010). In two models of breast cancer, Allo-Aca clearly suppressed the growth of human breast cancer xenografts when administered intraperitoneally or subcutaneously and extended the average survival by two weeks (Otvos et al., 2011a,b). In both mild and more aggressive rheumatoid arthritis models, Allo-Aca reduced the extent of joint swelling and the number of arthritic joints (Otvos et al., 2011c). More recently, the LPA was reported to inhibit leptin-induced angiogenesis and signaling in monkey retinal and bovine corneal endothelial cells (Scolaro et al., 2013) and the formation of tubes and mitogenesis of HUVECs (Ferla et al., 2011).

Leptin and LR antibodies and nanobodies Neutralizing antibodies directed against a ligand or its receptor are a classical and effective way to interfere with cytokine signaling. Iversen and colleagues were the first to successfully use a neutralizing anti-LR monoclonal antibody in vivo (Iversen et al., 2002). They tested the antibody

in an acute myelocytic leukemia transplant model, and showed that treatment more than halved the number of bone marrow leukemic cells and significantly decreased angiogenesis. Administration in fa/fa rats that express a defective LR had no effect on either leukemic cell growth or angiogenesis. Anti-leptin or LR antibodies were shown to be a powerful tool to study the role of leptin in multiple sclerosis. In experimental autoimmune encephalomyelitis (EAE)-susceptible mice (Sanna et al., 2003) or relapsingremitting multiple sclerosis patients (Matarese et al., 2005), activated T cells produce leptin and this sustains their own proliferation in an autocrine fashion in vitro. Both antibodies directed against leptin or its receptor are able to effectively block this proliferation. In vivo administration of an anti-leptin antibody improved the clinical outcome, slowed disease progression, reduced disease relapses and inhibited the antigen-specific T cell proliferation in proteolipid protein peptide (PLP)-induced EAE (De Rosa et al., 2006). Similarly, antibody neutralization inhibited leptin-mediated TH17 responses in lupus-prone mice (Yu et al., 2013). Finally, the direct hypertrophic effects of leptin on cultured neonatal rat ventricular myocytes are blocked by antibodies directed against the receptor (Rajapurohitam et al., 2006). In a rat coronary artery ligation (CAL) model for myocardial infarction, a neutralizing LR antibody prevented cardiac left ventricular post-infarction hypertrophy. This resulted in a clear improvement in haemodynamic parameters (Purdham et al., 2008). Based on these results, the myocardial LR was proposed as a target for prevention of post-infarction remodeling. Nanobodies are a relative new alternative for classical antibodies. A nanobody is the cloned and isolated variable domain of the heavy-chain antibodies uniquely found in members of the Camelidae family (llama, dromedary and camel) (Hamers-Casterman et al., 1993; Van der Linden et al., 2000). Major advantages over classical antibodies are their tissue penetration, stability, easier genetic manipulation and expression in bacteria. We recently generated and evaluated a panel of nanobodies targeting the LR, and identified neutralizing nanobodies targeting the CRH2, IGD and FN III domains (Zabeau et al., 2012). Only nanobodies directed against the CRH2 domain inhibited leptin-binding, supporting the fact that this domain is the major leptin-binding determinant. We could show that a nanobody that targets the IGD domain potently interfered with leptin-dependent regulation of hypothalamic NPY expression, and that daily intraperitoneal injection increased body weight, body fat content, food intake, liver size and serum insulin levels. More recently, we demonstrated that a CRH2- or IGD-specific



nanobody exacerbated concanavalin A (Con A) induced hepatitis in wild-type mice but not in mice that are deficient for invariant natural killer T (iNKT) cells, supporting a new concept of leptin protecting towards T-cell-mediated hepatitis via modulation of iNKT cells (Venken et al., 2014). Finally, local administration of the CRH2-specific nanobody at low dose adjacent to a tumor decreased tumor mass with no visible effects on body weight or food intake in a mouse model of melanoma (Xiao et al., submitted).

Soluble LR variants The last strategy to inhibit LR signaling utilizes the extracellular part of the receptor that competes for leptin-binding with the membrane bound receptor. This is commercially available as a fusion protein with the human IgG1 Fc domain. To our knowledge, only two studies used this type of leptin antagonism. In the first, De Rosa et al. showed that this way of leptin blockade ameliorates the clinical outcome of PLP-induced EAE (De Rosa et al., 2006). Similar results were obtained with a neutralizing anti-leptin antibody (see above). In the second study, administration of the LR:Fc fusion protein protected against sepsis-mediated morbidity and mortality (Shapiro et al., 2010).

Future challenges and directions Leptin and LR antagonists have a clear potential as therapeutics for the treatment of autoimmune diseases and cancer. However, the major drawback of most, if not all, current leptin and LR antagonistic strategies is that they also give rise to undesirable weight gain upon administration (with the exception of the anorexia from aggressive cancers). On average, 1-week treatments result in a weight gain of 10–15%. Deeper insights in the LR activation mechanism, sig naling and physiology will help to further optimize these antagonists. Three strategies can be envisaged: (i) avoiding transport of leptin antagonist to the hypothalamic nuclei by blocking transport of the antagonist through the BBB, (ii) inhibition of selective pathways, (iii) and/or cell or tissue-specific antagonism. The BBB is the physical boundary between leptin’s central weight regulating effects and the peripheral immune and cancer promoting activities. The BBB might therefore be part of a strategy to design selective leptin

and LR antagonists. The precise mechanism by which leptin is transported over the BBB remains controversial. There is evidence that the LR short variants are involved in this transport: the receptors are highly expressed in brain microvessels (Bjorbaek et al., 1998), transfection in Madin-Darby canine kidney cells allows directed transport of labeled leptin (Hileman et al., 2000) and rats lacking any membrane-anchored LR show marked decrease in the transport (Kastin et al., 1999). In contrast, several reports suggest that leptin-sensing neurons of the arcuate nucleus behave differently from neurons in other sites of the hypothalamus and that they make direct contact with the blood circulation. For example, diet-induced-obesity results in a decrease in leptin sensitivity caused by overexpression of SOCS3 (suppressor of cytokine signaling 3; a negative regulator of leptin signaling) in ARC neurons, but not in other regions of the brain (Munzberg et al., 2004). Furthermore, it is only possible to detect basal STAT3 phosphorylation in the ARC neurons, and these respond more rapidly and sensitively to exogenous administrated leptin (Faouzi et al., 2007). Finally, leptin-responsive neurons that express the LR or show STAT3 activation can be labeled by BBB-impermeable fluorescent tracers (Cheunsuang and Morris, 2005; Faouzi et al., 2007). If these peripheral blood-ARC neuron contacts are crucial in leptin’s metabolic function, creation of selective peripheral leptin antagonists by avoiding their transport via the BBB may not be a viable strategy. The observation that LR signaling is not strictly dependent on JAK2 activation might open the possibility to uncouple pathways and thus functions of leptin. For example, leptin can induce STAT3 phosphorylation in γ2A JAK2-null cells (Jiang et al., 2008), or FAK phosphorylation in human colon carcinoma cell lines in the presence of a JAK2 inhibitor (Ratke et al., 2010). The same inhibitor was also reported to be insufficient to block leptinmediated inhibition of thymic apoptosis in rats (Mansour et al., 2006). It was suggested in all three examples that members of the Src kinase family can allow JAK2-independent LR signaling. Uncoupling of signaling pathways has been reported for two other long-chain α-helical bundle cytokines. Erythropoietin (Epo) can, in addition to its hematopoietic role, exert tissue-protective effects on non-hematopoietic cells (Brines, 2010). While Epo receptor (EpoR) dimerisation is sufficient for red blood cell production, the tissueprotective hetero-receptor complex may contain the βc chain (Brines et al., 2004). As a consequence, Epo derivatives or peptides could be developed that are tissue protective without erythropoietic effects both in vitro and in vivo


508 L. Zabeau et al.: Antagonizing leptin: current status and future directions (Leist et al., 2004; Brines et al., 2008). In the case of growth hormone (GH), X-SCID patients lacking the yc chain were shown to display impaired GH signaling in B lymphocytes (Adriani et al., 2006). Alternatively, mutations in the GHR itself were identified that can uncouple the JAK2/STAT5 and ERK pathways (Rowlinson et al., 2008) indicating that different ligand binding modi may activate different pathways from the same receptor complex. A third and final option is to selectively target leptin or LR antagonist to certain (cancer or immune) cell types or sites of inflammation through coupling to antibodies/ nanobodies or drug conjugates (Simon et al., 2013).

Uncoupling of central and peripheral functions of leptin and the design of selective antagonist remains one of the big challenges in the leptin field for the future. Acknowledgments: We apologize to our colleagues that space limitations did not allow us to cite all the relevant literature. This work was funded by IU, A.P. (P6/36) and Research Foundation-Flanders (FWO-V, Project G.0521.12N). Received November 21, 2013; accepted February 5, 2014; previously published online February 11, 2014

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After finishing his biotechnology studies at Ghent University in 1998, Lennart Zabeau joined the CRL to study the mechanisms underlying cytokine receptor clustering and activation. He started to work on the interleukin-5 receptor, but later on the leptin receptor became his main research interest. He obtained a PhD in 2004. With an FWO fellowship he is currently involved in the design and evaluation of leptin and leptin receptor antagonists in vitro and in mouse models for certain autoimmune diseases.

Jan Tavernier founded the Cytokine Receptor Laboratory (CRL) in 1996. He obtained his PhD in 1984 in the early days of recombinant DNA on the cloning of several interferon and interleukin genes. In the same year he moved to industry, first Biogen, later Roche, where he continued cytokine research and demonstrated for the first time the shared use of cytokine receptor subunits. He became full professor at Ghent University in 1996 and currently heads the CRL as part of the VIB Department of Medical Protein Research.

After graduating as a biologist in 1993, Frank Peelman obtained his PhD in 1999 on the structure-function relationships of lecithin:cholesterol acyltransferase at the Biochemistry department of Ghent University. In 2002, he joined the Cytokine Receptor Lab to investigate the properties of leptin binding to its receptor. In 2006 he became a full professor at Ghent University, and his current research focuses on the molecular dissection of proteinprotein interactions, with focus on cytokines and Toll-like receptor signaling.


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