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Many drugs that target transforming growth factor-β (TGFβ) signalling have disease applications. Preclinical and clinical studies indicate the utility of these agents in fibrosis and oncology, particularly in augmentation of existing cancer therapies, such as radiation and chemotherapy, as well as in tumour vaccines. There are also reports of specialized applications, such as the reduction of vascular symptoms of Marfan syndrome. Here, we consider why the TGFβ signalling pathway is a drug target, the potential clinical applications of TGFβ inhibition, the issues arising with anti-TGFβ therapy and how these might be tackled using personalized approaches to dosing, monitoring of biomarkers as well as brief and/or localized drug-dosing regimens.
The transforming growth factor-β (TGFβ) superfamily of cytokines, which consists of TGFβs, activins, inhibins, Nodal, bone morphogenetic proteins (BMPs), anti-Müllerian hormone (AMH; also known as Müllerian-inhibiting factor) as well as growth and differentiation factors (GDFs), is conserved through evolution and found in all multicellular organisms1. The TGFβs per se are involved in many cellular processes, including growth inhibition, cell migration, invasion, epithelial-mesenchymal transition (EMT), extracellular matrix (ECM) remodelling and immune-suppression2. However, although normally dynamically regulated and involved in maintenance of tissue homeostasis, TGFβs are often chronically over-expressed in disease states, including cancer, fibrosis and inflammation, and this excessive production of TGFβ drives disease progression by modulating cell growth, migration or phenotype. The TGFβ signalling pathway has therefore become a popular target for drug development.
Knowledge about cellular activities gleaned from studying one disease is often applicable to others. For example, inhibition of TGFβ-induced EMT — a process that contributes to cancer progression — is a goal not only of oncologists but also of cardiovascular surgeons to prevent neointimal hyperplasia, and of nephrologists and pneumologists in the treatment of fibrosis3. In addition, the immune-modulatory activities of TGFβ have implications in many diseases, including cancer, cardiovascular disease, asthma, rheumatoid arthritis and multiple sclerosis4.
TGFβ action is highly context-dependent and influenced by cell type, culture conditions, interaction with other signalling pathways, developmental or disease stage in vivo and innate genetic variation among individuals5-9. This makes the pathway a particular challenge for drug development. Nevertheless, over the past decade several drugs targeting the TGFβ signalling pathway have been developed by pharmaceutical companies and biotechnology firms alike. Drug design strategies have been numerous and include the development of small-molecule inhibitors (SMIs) and monoclonal antibodies, as well as the inhibition of gene expression; some drugs have reached Phase III clinical trials for a number of disease applications, particularly fibrosis and oncology. There is an increasing number of preclinical examples of TGFβ inhibitors that are capable of reducing cancer progression and metastasis, and that augment existing cancer therapies (such as radiation therapy in breast cancer) while simultaneously guarding against radiation-induced fibrosis10. Additionally, there are novel reports of targeting TGFβ signalling in less prevalent indications, such as reduction of vascular symptoms of Marfan syndrome (MFS)11,12. Although there have been many reviews on the pleiotropic action of TGFβ during tumorigenesis, which is characterized by tumour-suppressing activity of TGFβ at an early stage of cancer and tumour-promoting activity at later stages13-16, few focus specifically on drug targets, drug classes and possible therapeutic applications beyond the oncology arena. The translation of anti-TGFβ therapies has been pursued most intensively for oncology; however, this Review also discusses the potential of the TGFβ signalling pathway as a target for non-neoplastic disease therapies and addresses the associated challenges in the development and application of these strategies.
The vertebrate genome contains more than 30 pleiotropic ligands that belong to the TGFβ superfamily, including TGFβs, BMPs, GDFs, activins, inhibins, Nodal and AMH1. TGFβ has a conserved motif of nine cysteine residues, eight of which form a tight cysteine knot, with the ninth being crucial for homodimerization2. Aberrant expression and activity of many of the ligands of the TGFβ superfamily are associated with developmental defects and human diseases17. Here we focus on TGFβs as there are currently several clinical trials underway involving therapies targeting TGFβ signalling, whereas other members of the TGFβ superfamily are under-represented in current trials.
Three highly homologous isoforms of TGFβ exist in humans: TGFβ1, TGFβ2 and TGFβ3. They share a receptor complex and signal in similar ways but their expression levels vary depending on the tissue18, and their functions are distinct as demonstrated by the phenotypes of knockout mice19-23. Each TGFβ ligand is synthesized as a precursor, which forms a homodimer that interacts with its latency-associated peptide (LAP) and a latent TGFβ-binding protein (LTBP), forming a larger complex called the large latent complex (LLC). The TGFβ activation process involves the release of the LLC from the ECM, followed by further proteolysis of LAP to release active TGFβ to its receptors2. Matrix metalloproteinase 2 (MMP2) and MMP9 are known to cleave latent TGFβ. In addition to MMPs, thrombospondin 1 (THBS1) is known to activate latent TGFβ24. Alternatively, upon mechanical stretch, αVβ6 integrin can activate TGFβ by binding to the RGD motif present in LAP and inducing the release of mature TGFβ from its latent complex25,26.
Proteolytic cleavage, interaction with integrins or pH changes in the local environment are known to activate latent TGFβ and free active TGFβ for binding to its receptors at the cell membrane. TGFβ superfamily members signal via heteromeric complexes of two related transmembrane type I and type II serine/threonine kinase receptors. Five type II receptors and seven type I receptors (also termed activin receptor-like kinases (ALKs)) have been identified. Auxilliary co-receptors (also known as type III receptors) that regulate the access of TGFβ superfamily members to signalling receptors also exist. Each subfamily of the TGFβ superfamily of ligands binds to type I and type II receptors (Box 1). BMPs can bind to type I receptors alone and, in their absence, can weakly bind to type II receptors, but they show highest affinity when both receptors act together. TGFβ and activin display high affinity only for type II receptors and do not normally interact with isolated type I receptors. Binding to the extracellular domains of type I and type II receptors by the dimeric ligand induces close proximity and a productive conformation for the intracellular serine/threonine kinase domains of the receptors, facilitating the phosphorylation and subsequent activation of the type I receptor. The activation of the type I receptor leads to the propagation of signalling by at least two seemingly independent routes: the SMAD-dependent canonical pathway (Box 1; Fig. 1) and the SMAD-independent or non-canonical pathways (Box 2; Fig. 2).
The basic framework of the canonical signal transduction pathways of three subfamilies of the transforming growth factor-β (TGFβ3) superfamily — TGFβ3s, activins/inhibins/Nodal and bone morphogenetic proteins (BMPs) — is highly conserved. The ligand binds to a specific set of type I and type II receptors, which are both serine/threonine kinases, followed by signal transduction by SMAD proteins31,205. Although each subfamily transmits the signal through a specific signalling pathway, the interaction among the TGFβ, activins/inhibins/Nodal and BMP subfamilies is well recognized during development and in postnatal homeostasis of various organs (Box 3). Upon ligand binding and resultant heterotetrameric receptor complex formation, the constitutively active type II receptor phosphorylates the type I receptor, which in turn propagates a signal by phosphorylating the receptor-specific SMADs (R-SMADs)31,205. Unlike type I and type II receptors, type III receptors do not possess kinase activity and are not required for signal transduction; however, they bind to specific ligands and modulate the signalling pathway either positively or negatively31,205. Phosphorylation of R-SMADs at two serine residues within the extreme carboxyl terminus by type I receptor kinase activity promotes association with the common mediator SMAD (co-SMAD), SMAD4, resulting in nuclear accumulation and sequence-specific binding to DNA in concert with other DNA-binding transcription factors that bind distinct sequences adjacent to the SMAD-binding element (SBE)27, and together these complexes modulate transcription. The inhibitory SMADs (I-SMADs), SMAD6 and SMAD7, antagonize R-SMAD activation by competing with R-SMADs for type I receptor interaction and/or by recruiting specific ubiquitin ligases or phosphatases to the activated receptor complex, thereby targeting it for proteasomal degradation or dephosphorylation, respectively. SMAD7 inhibits signalling from all branches of the TGFβ superfamily, whereas SMAD6 is a specific inhibitor of the BMP signalling pathway. The table indicates the basic molecules in the signal transduction pathway, including three types of receptors and SMADs, for three subfamilies of the TGFβ superfamily of ligands: TGFβs, activins/inhibins/Nodal and BMPs.
|TGFp pathway*
|BMP pathway*
|TGFβ1, TGFβ2, TGFβ3
|Activin A, activin B, inhibin A,
inhibin B, Nodal
|Type I
|TβRI(ALK5), ALK1
|ALK4(ACVR1Bor ACTRIB),
ALK7 (ACVR1C or ACTRIC)
|Type II
|Type III
|TβRIII (betaglycan),
endoglin, CRIPT03
CRIPT03 (TDGF1P3),TβRIII
(HJV or HFE2), endoglin
In addition to activating SMAD proteins, transforming growth factor-β (TGFβ) signalling can regulate the activity of a number of signalling molecules, such as TNF receptor-associated factor 4 (TRAF4), TRAF6, TGFβ-activated kinase 1 (TAK1), p38 mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), JUN N-terminal kinase (JNK), RHO GTPases, phosphoinositide 3-kinase (PI3K)–AKT and nuclear factor-κB (NF-κB), to transmit a signal6. In addition, these non-canonical signals can crosstalk with the SMAD pathways and mutually modulate each other. Both canonical and non-canonical TGFβ signalling can also be influenced by other signalling pathways, such as the RAS, WNT, Hedgehog, Notch, tumour necrosis factor (TNF) and interferon pathways6. The exact nature of the crosstalk with other pathways and biological outcomes is complex and highly context-dependent6. However, some of the crosstalk has been found to modulate the function and stability of SMAD proteins through post-translational modifications, and to define cell type- and context-specific outcomes by inducing other factors that modulate TGFβ activity.
In the SMAD-dependent pathway, activation of TGFβ receptor type I (TβRI; also known as TGFBR1 and ALK5) leads to phosphorylation of receptor-specific SMAD (R-SMAD) proteins. SMAD2 and SMAD3 are substrates of TβRI, whereas type I receptors for BMPs utilize SMAD1, SMAD5 and SMAD8 (Fig. 1). Upon phosphorylation by the receptor, R-SMADs together with the common mediator SMAD4 (co-SMAD) translocate to the nucleus, where they interact with other transcription factors (cofactors) to regulate transcriptional responses27 (Fig. 1). In addition to the canonical role of SMADs as transcription factors, a novel role for R-SMADs in the post-transcriptional regulation of microRNA (miRNA) biogenesis has been identified28 (Fig. 1). Therefore, the canonical TGFβ-SMAD pathway modulates gene expression both transcriptionally and post-transcriptionally to propagate the physiological and pathological activities of TGFβ. In the non-canonical pathway, the activated TGFβ receptor complex transmits a signal through other factors, such as tumour necrosis factor (TNF) receptor-associated factor 4 (TRAF4), TRAF6, TGFβ-activated kinase 1 (TAK1; also known as MAP3K7), p38 mitogen-activated protein kinase (p38 MAPK), RHO, phosphoinositide 3-kinase (PI3K), AKT (also known as protein kinase B), extracellular signal-regulated kinase (ERK), JUN N-terminal kinase (JNK) or nuclear factor-κB (NF-κB) (Box 2; Fig. 2). Thus, cellular responses to TGFβ signalling result from the dynamic combination of canonical and non-canonical signalling cascades. In addition to the complexity generated by the canonical and non-canonical TGFβ signalling pathway, TGFβ signalling can be influenced by different signalling pathways, including the PI3K-AKT, WNT, Hedgehog (HH), Notch, interferon (IFN), TNF and RAS pathways (Box 2; Fig. 2). Interactions with several of these pathways can change the output of TGFβ signalling from suppressing growth to inducing cellular plasticity29. Nuclear accumulation and transcriptional activity of R-SMADs can also be negatively regulated through phosphorylation of multiple Ser-Pro and Thr-Pro residues (in the linker region connecting the MH1 and MH2 domains) by ERK, MAPKs, calcium/calmodulin-dependent protein kinase II and cyclin-dependent kinases (CDKs)30. The mode and outcome of the crosstalk between TGFβ and other signalling pathways vary considerably but are essential to define the activities of TGFβ in propagating spatially and temporally specific outputs6,31,32.
TGFβ is involved in a range of biological processes both during embryogenesis and in adult tissue homeostasis. Although the physiological roles of TGFβ have been extensively reviewed elsewhere16,33-36, the major functions of TGFβ that are relevant to the topic of this Review are briefly outlined below.
TGFβ strongly inhibits the growth of many cell types, including epithelial, endothelial, haematopoietic and immune cells37,38. TGFβ also has pro-apoptotic and differentiation-inducing actions on epithelial cells; together, these actions result in tumour suppression in the context of cancer34. TGFβ in epithelial cells activates transcription of cyclin-dependent kinase inhibitor 1A (CDKN1A) and CDKN2A (which encode p21CIP1 and p15INK4B, respectively) to mediate cell cycle arrest at the G1 phase39. Conversely, TGFβ represses the transcription of MYC, which encodes a potent transcriptional activator of genes that is required for cell proliferation and growth, and inhibitor of DNA binding (ID) family genes, which encode transcription factors that promote cell differentiation and determination40. In oncology, many tumours attenuate TGFβ growth-inhibitory effects but respond to this ligand in a pro-tumorigenic manner. Thus, depending on the tumour type and the stage of tumour progression, TGFβ may provide potent tumour-suppressive or tumour-promoting functions directly on the tumour cell, presumably by mediating differential gene expression programmes (Fig. 3).
Unlike the role of TGFβ signalling during tumorigenesis, the contribution of TGFβ to vascular disease is more complex and confusing. Studies on clinical samples from vascular disorders, such as atherosclerosis, hypertension and pulmonary hypertension, often find signatures of both upregulation and downregulation of TGFβ signalling, as well as complex interactions between this pathway and other ligands of the TGFβ family, such as BMPs (Box 3). This has been confirmed by in vitro studies, demonstrating the contradictory effects of TGFβ in the regulation of vascular cells36,41. Furthermore, the TGFβ pathway often exhibits contrasting effects in different vascular cell types, such as endothelial versus vascular smooth muscle cells36. The promiscuous and cell type-specific action of the TGFβ pathway on vascular cells makes the application of targeted TGFβ signalling therapies for cardiovascular disease a particular challenge.
Within the transforming growth factor-β (TGFβ) superfamily, the crosstalk between three subfamilies — activins/inhibins/Nodal, TGFβ3s and bone morphogenetic proteins (BMPs) — is well established during development and postnatal homeostasis of various organs206-207. In vertebrates, the BMP-SMAD1/SMAD5/SMAD8 and activin–Nodal–TGFβ-SMAD2/SMAD3 signalling pathways execute antagonistic actions in different developmental contexts by inducing the expression of antagonistic factors, such as inhibitory SMADs (I-SMADs: SMAD6 and SMAD7). Some studies have shown that the common mediator SMAD (co-SMAD), SMAD4, is rate-limiting; therefore, when one of the two pathways is activated, it can negatively influence the other pathway by sequestering SMAD4. In endothelial cells, TGFβ can signal not only via canonical TGFβ receptor type I (TβRI)-SMAD2/SMAD3 but also via activin receptor-like kinase 1 (ALK1)-SMAD1/SMAD5/SMAD8 (Ref. 36). In contrast to TGFβ-TβRI signalling-mediated activation of SMAD2 or SMAD3, which leads to endothelium quiescence, TGFβ-ALK1 signalling induces SMAD1/SMAD5/SMAD8 activation and has been shown to stimulate endothelial cell migration, proliferation and tube formation, thus promoting angiogenesis208. BMP9 was shown to induce SMAD2/SMAD3 and SMAD1/SMAD5/SMAD8 phosphorylation via signalling mediated by BMP receptor 2 (BMPR2), activin receptor type II (ACTRII) and ALK1 or ALK2. Cross-activation of TGFβ-specific and BMP-specific receptor-specific SMADs (R-SMADs) by a single ligand is believed to provide a mechanism for the ligand to fine-tune endothelial cell behaviour and function36. In summary, the crosstalk among signalling pathways mediated by different TGFβ family ligands exists in every tissue. However, the mechanism and the biological outcome of this crosstalk are highly species-, tissue- and context-dependent.
TGFβ can induce an EMT of both epithelial and endothelial cells. This has consequences for disease progression in both cancer and fibrosis3. EMT enhances cellular migration and invasive properties, as cell migration requires loss of cell-cell contacts and acquisition of fibroblastic characteristics. E-cadherin is commonly downregulated in many cancers, and its overexpression can suppress invasion by tumour cells. The TGFβ-SMAD pathway mediates the expression of high mobility group AT-hook 2 (HMGA2), which is important for the induction of SNAIL (also known as SNAI1) and SLUG (also known as SNAI2): two zinc-finger transcription factors that are known to repress the E-cadherin gene33. In breast and skin cancer, tumour cell EMT contributes to cancer progression as cells consequently become more migratory and invasive, and they can ultimately transition to a myofibroblastic phenotype3. The myofibroblast further modulates the basic biology of the tumour by increasing ECM elaboration and eliciting a tissue contraction process, which results in increased interstitial fluid pressure (IFP). This has consequences for the efficiency of drug delivery to the tumour42, as drugs cannot penetrate tissue under positive IFP. EMT can also polarize carcinoma cells towards 'stem cell-like' properties, such as increased tumour-initiating capacity and tumour cell drug resistance43. Blocking the TGFβ pathway can thus have a threefold benefit: the reduction of tumour invasion and metastasis; the suppression of cancer stem cell-like properties; and the restoration of negative IFP to enhance chemotherapeutic drug delivery44.
In fibrotic conditions, excessive TGFβ production induced in the diseased state contributes to EMT elaboration, which can further exacerbate fibrosis, as seen in pulmonary45,46, cardiac47 and renal48,49 fibrosis, and in arterial restenosis following surgical trauma50. TGFβ can also promote a proliferative and/or migratory phenotype on smooth muscle cells that can aggravate some vascular diseases, including neointimal formation following vascular surgery51-53.
The ECM is a complex structure that surrounds mammalian cells. It is the major component of connective tissue and is composed of multiple proteins, such as collagen, elastin, fibrillin, fibronectin, lamin and proteoglycans. Fibrosis is characterized by the accumulation of fibroblasts, which secrete excessive amounts of ECM. As TGFβ is widely documented to increase collagen synthesis and deposition by fibroblasts, TGFβ has become a central therapeutic target for different types of fibrosis. TGFβ activity and the synthesis of ECM proteins are mutually regulated. Several genes encoding ECM proteins that are known to be important in driving fibrosis are directly regulated by TGFβ-SMAD signalling pathways. There is a reciprocal regulation of TGFβ by the ECM: latent TGFβ bound to ECM components, such as fibronectin and fibrillin, is inactive until physiological or pathological processes initiate its release. This is seen in MFS, in which the mutation of a fibrillin-encoding gene results in reduced fibrillin levels and a consequent increase in levels of unbound TGFβ; this, in turn, leads to the activation of TGFβ signalling, which is possibly responsible for the aetiology of many Marfanoid features11,12.
The lethal postnatal inflammatory phenotype of Tgfb1-knockout mice19,20,54 demonstrates the important immune-suppressor function of this ligand. The widespread expression profile of TGFβ receptors on all immune cell types suggests that they have broad activities, including responses in cytotoxic CD8+ effector T cells, CD4+ effector T helper 1 (TH1) and TH2 cells, suppressive regulatory T (TReg) cells, natural killer (NK) cells, monocytes, macrophages, neutrophils and eosinophils (Fig. 4). Cell type-specific mouse gene knockout studies with Tgfbr2 demonstrate both direct and indirect actions of TGFβ on effector T cells4.
TGFβ has potent growth-suppressing activity on most precursor cells of the immune system, particularly T and B cells of the adaptive arm. TGFβ is a potent suppressor of T cell proliferation55 and an inducer of B cell apoptosis56. Additionally, the ligand can alter the course of immune cell differentiation. Suppressive TReg cells that are driven by the expression of the transcription factor forkhead box protein P3 (FOXP3) are crucial for maintenance of peripheral immune tolerance as well as regulation of tumour immunity and infection. In CD4+ T cells, Foxp3 expression is positively but indirectly regulated by TGFβ1 through enhanced binding of the SMAD2-induced transcription factor E2A to the Foxp3 gene promoter, and by relief from GATA3-mediated transcriptional inhibition of the Foxp3 promoter by competition with TGFβ-induced Id3 (Ref. 57). TGFβ suppresses inflammatory TH1 and TH2 cell differentiation while stimulating suppressor TReg cells. Overall, TGFβ-mediated suppression of effector CD8+ cytolytic cells and TH cells, together with TGFβ dependence for suppressive TReg cell differentiation, results in the hyper-inflammatory phenotype seen in Tgfb1-/- mice.
During tumour progression, excess TGFβ suppresses immune surveillance by attenuating the antitumour functions of CD8+ T cells, CD4+ T cells and dendritic cells. CD4+ T cell-specific ablation of TGFβ signalling in transgenic mice expressing dominant negative TβRII (DNRII; also known as CD4-ΔTβRII and CD4-ΔTGFBR2) led to the development of autoimmunity58 and enhanced the differentiation of CD8+ cytotoxic T lymphocytes (CTLs). When challenged with tumour cells, these transgenic mice raised a greater tumour-specific CTL response than wild-type littermates58. Tumour-derived TGFβ also blocks the differentiation of antigen-presenting dendritic cells59 and modifies chemokine receptor expression to blunt dendritic cell chemotaxis60, further suppressing immune surveillance.
In addition to having a predominant immune-suppressive function, TGFβ counterintuitively may have a pro-inflammatory role through its effects on TH17 cells and cells of the innate immune system. TGFβ, together with interleukin-6 (IL-6), was reported to be an essential player in driving pro-inflammatory TH17 lineage differentiation61-63. However, there is considerable controversy surrounding this topic. First, different laboratories cannot agree on the specific functions of various TH1, TH2 and TH17 cell types in disease progression. TH17 cells were implicated as major agonists in inflammatory diseases, including inflammatory cancer, asthma and autoimmune disorders55. However, recent studies suggest that the active player in disease progression is in fact a TH17-derived TH1 cell, or a 'TH1-TH17' cell64. Second, the role of TGFβ in regulating the balance between TH1 and TH17 differentiation is in dispute. Despite the widespread acceptance of a role for TGFβ in TH17 differentiation61-63, more recent studies have suggested that TGFβ is totally dispensable for the generation of these cells65,66.
With respect to cells of the innate immune system, TGFβ directly suppresses NK cell-mediated production of IFNγ (which is required for the tumour killing activity of NK cells) through transcriptional effects of SMAD3 on the IFNγ promoter67. It also 'polarizes' macrophages68 and neutrophils69 from a type I, productive phenotype (that evolved to attack and devour foreign agents such as cancer cells) towards a type II phenotype (that has reduced effector function but produces large quantities of inflammatory molecules, such as IL-6, IL-11 and TGFβ). These molecules can exacerbate the local diseased state, resulting in solid tumour progression or inflammation associated with fibrosis or atherosclerosis4.
In summary, the regulation of the immune system by TGFβ is highly complex and context-dependent. It delicately regulates the tolerogenic versus immunogenic arms of the immune system to balance adequate host defence while limiting collateral inflammatory tissue damage. The molecular details of this regulation have been recently reviewed in depth4,55,64.
Virtually every component of the TGFβ pathway has been targeted for drug development (Fig. 5) through numerous design strategies (Fig. 6). Several have been developed through preclinical to clinical trials (Table 1) and many more have been tested only in preclinical systems (Table 2). The drugs that have progressed furthest in clinical development include anti-ligand antisense oligonucleotides (ASOs) from Antisense Pharma70-74, ligand-competitive peptides from Digna Biotech75-78, antibodies that target ligands, receptors or associated proteins spearheaded by Genzyme79-81, and SMIs against TGFβ receptor kinases developed by many companies, with Eli Lilly having an active clinical programme in Phase II development82. The various approaches currently being investigated are discussed in more detail below.
Antisense Pharma uses the strategy of targeting mRNA translation using ASOs to downregulate ligand synthesis70,83. Its focus has been on targeting TGFβ2, which is produced in excessive quantities by glioblastoma and pancreatic carcinoma cells. Trabedersen (AP12009), a synthetic 18-mer phosphorothioate ASO, binds specifically to human TGFB2 mRNA, and this drug has progressed to a Phase III clinical trial for oncology applications (Box 4). One of the challenges of this drug is delivering it directly to the tumour to avoid the off-target toxicity associated with systemic delivery of first-generation ASOs. In the case of glioblastoma, this was achieved using intrathecal catheter delivery directly into the tumour74. More recently, the company has started developing intravenous delivery approaches for pancreatic cancer, which appear to be effective in mouse models73 and were recently shown to be safe in humans84.
A two-part clinical trial of GC-1008 for the treatment of advanced metastatic melanoma (MetM) and renal cell carcinoma (22 patients) found the drug to be safe and well tolerated with no dose-limiting toxicities (DTLs). Five patients achieved at least stable disease as assessed by RECIST (response evaluation criteria in solid tumours) criteria, and therefore received extended treatment. One patient achieved a partial response with a greater than 75% reduction in the target lesion. The only adverse effect was keratoacanthoma-like lesions in sun-damaged skin of two of the patients with MetM. However, these resolved on cessation of drug treatment and were not malignant93. Despite these promising results, the pursuit of GC-1008 for oncology was terminated after Genzyme was acquired by Sanofi in late 2011.
Antisense Pharma has had success with trabedersen (AP12009) in glioblastoma, pancreatic cancer and colon cancer. Preclinical and clinical studies70,71,73 indicate that neutralization of transforming growth factor-β2 (TGFβ2)-mediated immunosuppression, leading to activation of tumour-infiltrating natural killer cells, is the major mode of action. Intra-tumoural administration of trabedersen to glioblastoma led to shrinkage of the targeted tumour as well as tumours elsewhere in the brain. Three Phase I/II studies of trabedersen for recurrent or refractory high-grade glioma (glioblastoma) and anaplastic astrocytoma showed survival benefit compared with conventional chemotherapy209. A randomized, controlled Phase IIb study evaluating the efficacy and safety of two doses (10 and 80 mM) of trabedersen in comparison with standard therapy concluded that patients with glioblastoma on trabedersen had a threefold enhancement in cognitive function 2 and 3 years after therapy compared to standard chemotherapy74. However, questions have been raised about this most recent study210,211. Wick and Weller211 conceded that although trabedersen was clinically safe and that TGFβ inhibitors, in general, show promise for cancer therapy, the conclusions drawn by Bogdahn et al.74 were premature. Because of other advances in both neurosurgical procedures and first-line standard of care for patients with glioblastoma212, the SAPPHIRE Phase III trial of trabedersen was recently halted owing to patient recruitment issues (ClinicalTrials.gov identifier: NCT00761280). Nevertheless, the drug has undergone a Phase I/II trial for patients with advanced pancreatic cancer, MetM or metastatic colorectal carcinoma, and showed excellent safety and encouraging survival results (ClinicalTrials.gov identifier: NCT00844064)84.
Eli Lilly's clinical small-molecule inhibitor LY2157299 was found to be safe and well tolerated in a Phase I glioblastoma trial82. Of 28 patients treated in a dose escalation study (14 days on/14 days off treatment), at least three patients showed antitumour effects with durable responses beyond 1 year. As a result, the Eli Lilly anti-TGFβ signalling programme for oncology continues to be pursued with an ongoing Phase II trial of LY2157299, with or without gemcitabine, for hepatocellular carcinoma, glioblastoma and advanced pancreatic cancer, and with lomustine in patients with treatment-refractory malignant glioma213, plus a new Phase I trial of IMC-TM1, an anti-TGFβ receptor type II (TβRII) antibody.
NovaRx's belagenpumatucel-L (Lucanix) has completed an open-label clinical trial of 75 patients with non-small-cell lung cancer (NSCLC) with a median follow-up of 14.5 months (44 months for patients with stable disease). One-year, two-year and five-year survivals were 55%, 35% and 20%, respectively. Individuals who demonstrated an increase in both cellular and humoral immune reactivity had a significant survival advantage over individuals who showed an increase in only one measure of immunity (32.5 months versus 11.6 months; p = 0.015). On the basis of these findings, an international, randomized Phase III trial to evaluate the efficacy of belagenpumatucel-L in a maintenance setting has been initiated for patients with stage III/IV NSCLC who have stable disease following frontline chemotherapy87.
An anti-TGFB2 antisense strategy has also been used to generate augmented tumour vaccines. Belagenpumatucel-L (Lucanix; NovaRx) is such a drug, in which an ~900-nucleotide TGFB2 antisense construct is transfected into allogeneic non-small-cell lung cancer (NSCLC) cells, which are then used as a tumour vaccine. Here, drug delivery is not an issue as the 'drug' is in fact genetically engineered NSCLC tumour cell lines. This tumour vaccine has superior activity compared to conventional tumour vaccination approaches85,86. A significant dose-related survival difference was seen in patients who received 2.5 × 107 cells per injection, allowing progression to a Phase III clinical trial87.
The advantages of monoclonal antibodies are their specificity and extracellular mechanism of action — an advantage when trying to mop up excess extracellular ligand. This is tempered by the less con venient intravenous mode of delivery. However, prolonged pharmacokinetic stability permits infrequent drug administration. Cambridge Antibody Technologies and Genzyme developed humanized (or murinized for preclinical studies) monoclonal antibodies specific to individual ligands, such as lerdelimumab (CAT-152)88,89 and metelimumab (CAT-192)90, or with pan-ligand specificity, such as fresolimumab (GC-1008)91-93. These antibodies have proceeded through various stages of preclinical and clinical development. Of these three humanized antibodies, fresolimumab has progressed furthest in the clinic for both neoplastic and non-neoplastic applications. This drug was found to be well tolerated and safe at 15 mg per ml in Phase I trials for metastatic melanoma (MetM) plus renal cell carcinoma93 and at 1 mg per ml for the fibrotic disorder focal segmental glomerulosclerosis92. Lerdelimumab88,89 and metelimumab90, despite passing safety tests, failed to show efficacy in fibrotic models of corneal scarring and systemic sclerosis, respectively, and were therefore discontinued90. Despite a promising Phase I oncology trial of fresolimumab, after Genzyme was acquired by Sanofi the company made the decision to focus on fibrotic applications of this drug.
Eli Lilly entered the monoclonal antibody arena with a TGFβ1 ligand-selective blocking antibody, LY2382770, which has progressed to Phase II trials for kidney fibrosis (Table 1). Since merging with ImClone, Eli Lilly has also developed a TβRII-blocking antibody, IMC-TR1 (Ref. 94), which has just entered clinical trials for breast and colon cancer (ClinicalTrials.gov identifier: NCT01646203). In addition, Biogen Idec and Stromedix have developed an anti-integrin β6 antibody that prevents the activation of TGFβ and has been used efficaciously in preclinical studies of fibrosis and cancer95; it is in a Phase II trial for fibrosis (ClinicalTrials.gov identifier: NCT01371305).
Genzyme developed a ligand trap by fusing Fcγ to the extracellular domain of TβRII, but this construct never reached clinical trials96. However, an alternative ligand trap approach, pursued by Digna Biotech, using peptide mimetics of TβRIII (also known as betaglycan and TGFBR3), completed a Phase IIa clinical trial for scleroderma and skin fibrosis, showing safety and efficacy when topically applied to skin (Table 1; Box 4). This company has plans to extend to Phase IIb/III trials in 2013 (J. Dotor, personal communication). A peptide antagonist of TGFβ activation, LSKL (Leu-Ser-Lys-Leu), binds to a conserved sequence in the LAP region of the latent complex and has demonstrated efficacy in reducing TGFβ signalling in vitro97. This antagonist is based on thrombospondin and specifically blocks TGFβ activation. The issue of peptide drug delivery is not a problem for topical application; however, to progress to systemic delivery, Digna Biotech has partnered with Flamel Technologies to investigate proprietary peptide delivery systems.
There are a plethora of SMIs that specifically target the type I receptor of TGFβ to inhibit the phosphorylation of SMAD2 and SMAD3 while keeping at least some non-canonical responses, such as TAK1 activation, intact. These drugs are generally ATP mimetics that bind competitively within the hydrophobic ATP binding pocket of the receptor kinase. The chemistry of these compounds has been extensively reviewed98,99 and some molecular structures are shown in Fig. 6. The obvious advantages of these molecules over most others are their economical production, stability and ease of oral administration, set against a possible disadvantage of cross-inhibition of other kinases. The short half-life of these drugs provides the possibility of rapid drug withdrawal should adverse events arise. Many successful preclinical studies for metastatic cancer have been undertaken with these SMIs, as reviewed previously100,101. However, the only company to continue pursuit of a TβRI-targeted SMI into clinical trials for oncology is Eli Lilly with LY2157299 (Ref. 82) (ClinicalTrials.gov identifier: NCT01373164).
A novel approach to the suppression of ligand production has been the preclinical development of pyrrole-imidazole polyamides that bind with sequence specificity to the TGFB1 gene promoter to attenuate gene expression50,102,103. These large ~17 kDa polymeric molecules (Figs 5,,6)6) bind within the minor groove of DNA to prevent transcription factor binding. Challenges associated with these drugs include the specificity of promoter binding, along with drug delivery issues owing to their large molecular size and the high local concentration required for activity. However, preclinical studies suggest that these molecules might be used in drug-eluting stents for the purpose of reducing restenosis after coronary or carotid artery surgery50.
An alternative approach to suppress TGFβ signalling is gene transfer of antagonizing signalling molecules, such as the inhibitory SMAD7. This approach has been applied in model systems to treat or prevent various pathological conditions, including colonic and hepatic fibrosis, vascular remodelling and diabetic kidney disease104,105. Such an application has the potential to be applied to many other systemic diseases to attenuate the activity of the TGFβ pathway, with the caveat that gene therapy is still far from being widely accepted as a therapeutic approach106.
As an approach to stimulate immune destruction of cancer cells by tumour-infiltrating T cells, human tumour antigen-specific CTLs have been engineered to express DNRII using a clinical grade retrovirus vector. TGFβ-resistant CTLs were found to have a functional advantage over unmodified CTLs in clearing TGFβ-secreting Epstein-Barr virus (EBV)-positive lymphoma in vitro and in vivo107, and this approach to therapy has progressed to a Phase I clinical trial for EBV-positive lymphoma. A further modification of the CTLs, by engineering in an HER2 (also known as ERBB2) chimeric receptor as well as a DNRII, allows the CTLs to target HER2-positive tumour cells108-111. This approach is in a Phase I clinical trial for advanced HER2-positive lung malignancy, labelled the HERCREEM trial (ClinicalTrials. gov identifier: NCT00889954).
Finally, Renova has developed a recombinant TGFβ3 ligand as an anti-scarring agent on the basis of the hypothesis that this ligand has activity that is independent of and antagonistic to TGFβ1 (Ref. 112). The drug, administered by injection around a surgical wound site, progressed to a Phase III clinical trial, but unfortunately it did not reach its primary or secondary efficacy end points.
Pre-existing drugs that have been extensively used for other applications may act, in part, by inhibiting TGFβ. Examples are losartan and candesartan, which are angiotensin type II receptor inhibitors that were originally developed for the treatment of hypertension. Both appear to reduce TGFβ signalling, although the precise molecular mechanisms of this action are still unclear12,113-115. Pirfenidone acts in part by reducing the fibrotic effects of TGFβ116 via unknown targets. It is the first approved drug in Europe for idiopathic pulmonary fibrosis (IPF), and is in a Phase III trial in the United States117,118. On the other side of the coin, some common drugs, including aspirin, elevate circulating TGFβ levels, which — in certain cases such as arteriosclerosis — correlates with disease suppression119.
TGFβ has a biphasic action during tumorigenesis, suppressing tumorigenesis at early stages but promoting tumour progression later on (Fig. 3). This is a paradigm for the action of TGFβ during disease progression in general, including that of fibrosis, inflammation and cardiovascular disease, and it is rooted in the fact that the normal function of this ligand is in the regulation of homeostasis. During disease progression, TGFβ signalling can go into override and, once unharnessed, results in more damage than good. The main goal in cancer therapy is therefore to downmodulate excessive levels of TGFβ ligands.
A major challenge in developing TGFβ inhibitors for cancer therapy has been the fact that these compounds are not cytotoxic or cytostatic to most tumour cells in vitro. They were developed to target properties of the tumour that are required for cancer progression, including migration, invasion and metastasis, as well as effects on the tumour microenvironment (Figs 3,,4).4). Standard cytotoxic screens used by the pharmaceutical industry to identify anticancer drugs were therefore not relevant, and therapeutic utility could only be determined by in vivo efficacy in animal models and ultimately in the clinic.
Two major concerns in TGFβ drug development have been the inadvertent inhibition of the tumour-suppressing arm of TGFβ signalling in cancer120-122 and the development of adverse side effects unrelated to cancer, such as widespread inflammation, autoimmunity or cardiovascular defects that have been revealed by mouse gene knockout studies19-21,123. Preclinical studies suggested that attenuation of TGFβ-mediated growth inhibition would not be a major issue96,124,125. However, clinical trials to date82 have not revealed the cardiac valvulopathy126 or hyperostosis and chondrocyte hypertrophy and hyperplasia127 observed in rat preclinical toxicology studies. Moreover, there has been no widespread evidence of inflammatory complications in clinical trials reported to date54,82. These reassuring safety findings are supported by evidence from patients with the rare disease multiple self-healing squamous epithelioma (MSSE), who have germline-null mutations in the gene encoding TβRI but develop only self-limiting and mostly non-malignant skin lesions128. Intriguingly, in a Phase I clinical trial of GC-1008 for the treatment of MetM, patients developed skin lesions, keratoacanthoma or squamous cell carcinoma (SCC) that were similar to the skin abnormalities reported in MSSE, with the appearance of keratoacanthoma and SCC seemingly influenced by the extent of exposure to GC-1008. These lesions, which appeared on sun-damaged skin, were manifested in approximately 25% of patients who received higher dose levels of GC-1008 and/or longer exposure to the drug, and the lesions resolved on drug withdrawal91,93. To put this toxicity into context, non-melanoma skin cancers, such as SCC and keratoacanthoma, develop in approximately 15-30% of patients with MetM who are treated with BRAF inhibitors such as vemurafenib and dabrafenib129, and therapy with sorafenib and TNF antagonists produced similar findings130,131. Recent data from studies with vemurafenib for MetM therapy suggest that these lesions arise from pre-existent mutant RAS-containing cells within sun-damaged skin132. Intriguingly, one study of keratoacanthoma that appeared in sorafenib-treated patients showed somatic TGFBR1 missense mutations133, one of which was also identified as a causative germline mutation for MSSE128.
Cancer 'stem cells', or tumour-initiating cells (TICs), are defined by their capacity to self-renew and to initiate and persistently propagate the entire tumour. Targeting the cancer stem cell for destruction or irreversible quiescence is therefore the Holy Grail of oncology, especially as these cells are exceedingly resistant to both chemotherapy and radiotherapy, and are responsible for tumour metastasis and recurrence after therapy134. Several groups have now reported the phenomenon that TGFβ-induced EMT can drive tumour cells towards a more 'stem cell-like' phenotype characterized by increased expression of stem cell markers and enhanced tumour-initiating activity in vitro and in vivo43,135. In breast cancer135, the TGFβ and WNT signalling pathways were shown to be the most commonly activated signalling pathways in cancer stem cells that had been fractionated from the bulk tumour on the basis of expression of stem cell markers such as CD44hi and CD24low. In preclinical studies, TGFβ inhibitors have been shown to deplete the stem cell compartment in various cancers — including breast cancer135, glioblastoma136-138 and chronic myeloid leukaemia139 — which leads to increased lifespan in several mouse models of metastatic cancer. Anido et al.137 showed that glioblastoma-initiating cells (GICs, which express the stem cell markers CD44, ID1, ID3, SOX2 and SOX4) responded to LY2109761 by downregulating the expression of 'stem cell' genes. Moreover, patient-derived glioblastoma neurospheres transplanted orthotopically into non-obese diabetic/severe combined immunodeficient mice (NOD/SCID mice, which do not have T cells or B cells) responded to LY2109761 by decreasing in size and reducing their expression of stem cell markers137. The same research team is currently undertaking a Phase I/II clinical trial for glioblastoma using the closely related drug LY2157299 (Ref. 137). Importantly, they showed a reduction of CD44 and ID1 RNA levels after 2 months of LY2157299 treatment in tumour biopsy material from one patient with glioblastoma for whom a salvage surgical resection was performed both before and after 2 months on the trial137. The ability to reduce the number of stem cells in an aggressive tumour such as glioblastoma is a major coup.
It has been argued that TGFβ inhibitors might, however, release isolated and disseminated tumour (stem) cells from dormancy by initiating proliferation and/or disrupting the stem cell niche. A couple of recent studies may give credence to this notion, as systemic TGFβ inhibition resulted in increased numbers of circulating tumours as well as micro- and macro-metastases in mouse models of head and neck SCC and mammary cancer in vivo140,141. It might therefore be wise to use TGFβ inhibitors in combination with cytotoxic drugs to coax tumour cells out of their quiescent niche while simultaneously targeting those that respond proliferatively to TGFβ inhibition using chemotherapy. This strategy may be highly beneficial for 'flushing out' dormant disseminated tumour cells, as alluded to by Carlos Arteaga many years ago142. A further cautionary note is warranted, however, on the basis of two reports indicating that TGFβ may decrease the cancer-initiating cell population of diffuse type gastric carcinoma143 and breast carcinoma144 despite having little or no effect on cellular proliferation. Finally, TGFβ inhibitors might act on the stem cell niche by recruiting bone marrow mesenchymal stem cell-derived myofibroblasts that home in on the primary tumour, contribute to the tumour microenvironment as cancer-associated fibroblasts and consequently promote tumour progression145. Clearly there are tissue- and cell type-specific effects of TGFβ inhibition that can influence the action of TGFβ on the cancer stem cell and its niche146. Understanding the differential molecular mechanisms that elicit these variable responses will be critical to a judicious choice of treatment with TGFβ inhibitors or their derivatives. As TGFβ inhibitors are not directly cytotoxic, the use of these inhibitors in combination with cytotoxic chemotherapeutics may be particularly efficacious. The activation of TGFβ signalling in response to chemotherapeutics may drive the generation of cancer stem cells (via EMT), resulting in their chemoresistance134. This event may be targeted with TGFβ inhibitors, as demonstrated by the synergistic activity of doxorubicin and TGFβ inhibitor combination therapy on breast cancer growth and metastasis147. Studies in multiple myeloma also suggest that TGFβ inhibitors could potentiate the cytotoxic effects of melphalan and dexamethasone148. In vitro, the exposure of multiple myeloma cells to differentiated versus immature MC3T3-E1 pro-osteoblastic cells potentiated chemotherapy-induced multiple myeloma cell death. As TGFβ inhibition acts within the bone microenvironment to elicit osteoblastic differentiation148,149, this combinatorial approach holds great promise for the treatment of multiple myeloma and other bone metastatic cancers. Likewise, in a mouse model of serous gastric cell carcinoma, Ki26894 had an additive effect with a fluorouracil analogue in reducing tumour growth150. Finally, another mechanism whereby TGFβ inhibition can augment conventional therapies is in enhancing drug delivery to the tumour. There are reports that TGFβ inhibition can reduce interstitial tumour pressure44, which enhances the delivery of SMIs, and regulates vascular leakiness, which enhances the delivery of nanoparticle-encapsulated drugs, particularly in highly fibrotic and drug-refractile tumour types such as pancreatic cancer151.
Adoptive T cell therapy involves the harvesting and ex vivo expansion of autologous tumour-specific CTLs followed by their reintroduction into the patient to stimulate tumour killing152. Used most extensively in the treatment of MetM and lung cancer, this therapy often fails owing to the apoptosis of re-grafted CTLs. Preclinical studies suggest that failure may be due to the direct effects of TGFβ on CTLs153, and strategies to prevent such failure include the use of genetically modified CTLs with reduced TGFβ responsiveness. Transduction of CTLs with a virus encoding a DNRII154 has reached Phase I clinical trials, and recent preclinical data indicate that combining CTL therapy with TβRI-targeting SMIs may also significantly improve T cell survival and antitumour T cell cytotoxicity155. Augmenting adoptive T cell therapy with SMIs may be a particularly attractive application of TβRI SMIs as patients need not be exposed to genetically engineered T cells. Moreover, patients might only require short-term exposure to the drug for efficacy in this application, thus avoiding the side effects of long-term SMI drug exposure, such as inflammation19, cardiovascular complications126, bone and/or cartilage problems127, subphyseal hyperostosis as well as chondrocyte hypertrophy and/or hyperplasia, and reducing the risk of developing SMI drug resistance156.
Another clinical application with great promise is augmenting radiotherapy by inhibiting the TGFβ path-way10,81,157. Radiation not only physically activates latent TGFβ in vitro but also induces the biological release of this growth factor as part of a stress response158. Several groups have reported the positive role of TGFβ in supporting the DNA damage repair pathway, particularly through activation of p53 and phosphorylation of ataxia telangiectasia mutated (ATM) after radiation therapy159. Barcellos-Hoff's group demonstrated that LY2109761 and ID11 both attenuate radiation-induced activation of p53 and ATM in breast cancer cells in vitro and in vivo, thus preventing DNA repair and accentuating the cytotoxic effect of radiation81. Even short-term dosing with TβRI inhibitors might provide a considerable therapeutic advantage in potentiating radiotherapy, with the added benefit that the local activation of pro-tumorigenic stroma and tissue fibrosis — a major complication of radiation therapy10 — may also be suppressed by these drugs. In partnership with Genzyme, this group is currently undertaking a Phase I trial of fresolimumab in combination with radiotherapy for metastatic breast cancer. Eli Lilly is also undertaking a Phase I/IIa trial to test the safety and efficacy of LY2157299 in combination with temozolomide-based radiochemotherapy in patients with newly diagnosed malignant glioma157.
Myelodysplastic syndrome (MDS) is characterized by abnormal myeloid and/or erythroid differentiation of bone marrow cells that results in various anaemias and cytopaenias. In one-third of MDS cases, a high-risk group of patients can progress to leukaemia. However, refractory cytopaenias are the major cause of morbidity and mortality in sufferers. It was recently shown that reduced expression of SMAD7, an inhibitor of TβRI, was a common and significant event observed in CD34+ myeloid progenitor cells in the bone marrow of patients with MDS160. Indeed, low levels of myeloid SMAD7 expression were seen in most patients with MDS, regardless of the risk for progression to leukaemia. Downregulation of SMAD7 expression sensitized myeloid precursors to TGFβ such that even very low levels of the ligand elicited an increase in TGFβ responsiveness, as defined by P-SMAD2 levels and enhanced immune-suppressive effect, thus providing another opportunity to utilize TGFβ inhibitors for therapeutic utility in human disease.
Treatment of primary CD34+ haematopoietic stem cells with LY2157299 suppressed the activation of TβRI by its ligand. Moreover, in a liver-specific TGFβ1-overexpressing transgenic mouse model of MDS that exhibits severe anaemia, LY2157299 decreased P-SMAD2 levels in the bone marrow and significantly increased the haematocrit of these mice. Importantly, in ten out of ten primary bone marrow cultures from patients with MDS, administration of LY2157299 significantly increased erythroid (burst-forming unit (BFU-E)) and myeloid (colony-forming unit (CFU); granulocytic monocytic) colony numbers in vitro, harbouring great promise for the treatment of patients with MDS160.
IPF is a progressive, chronic and irreversible lung disease occurring in older adults, and has an unknown cause161. The main histological features of IPF are heterogeneous parenchyma, with areas of fibrosis and honeycombing alternating with areas of less-affected or normal parenchyma. IPF is characterized by a progressive reduction in lung function, with an estimated 20% survival prospect after 5 years, making it more lethal than many cancers. The progressive fibrotic reaction in IPF is associated with an epithelium-dependent fibroblast activation, in which TGFβ plays a major part16. TGFβ1, which is secreted by alveolar epithelial cells in patients with IPF, drives the process by promoting the migration, proliferation and differentiation of resident mesenchymal cells. αVβ6 integrin, which binds and activates latent TGFβ1 and TGFβ3, is highly induced following lung injury or fibrosis162. TGFβ activity163 then promotes activation and differentiation of fibroblasts into myofibroblasts, which are specialized contractile cells that cause aberrant ECM deposition, leading to the destruction of lung architecture, scarring162 and reduced lung function. TGFβ also promotes pulmonary EMT that additionally contributes to the expansion of fibroblasts and myofibroblasts164.
Pirfenidone, a novel compound that inhibits TGFβ activity in vitro, decreased the rate of decline in vital lung capacity and marginally increased progression-free survival in patients with IPF. Pooled data from two concurrent Phase III clinical trials in IPF indicated improvement in pulmonary function in the pirfenidone-treated group165. Currently, there are no US Food and Drug Administration (FDA)-approved drugs for IPF, and pirfenidone is the first such drug to be approved for IPF in Europe. Other approaches to develop TGFβ-based therapies for IPF include gene transfer of a soluble TβRII construct (as a ligand decoy), which attenuated injury and fibrosis in bleomycin-induced IPF in mice166. P144 (disitertide; Digna Biotech), a synthetic peptide that attenuates TGFβ activity and is derived from the extracellular domain of betaglycan, was also shown to reduce carbon tetrachloride-induced liver injury in mice78. P17, another Digna Biotech anti-TGFβ peptide, has been shown to be efficacious in attenuation of injury and fibrosis in bleomycin-induced IPF in mice167. Although P144 has been used clinically for skin fibrosis, drug delivery is an issue for the clinical development of P144 for IPF. However, Digna Biotech has recently partnered with Flamel Technologies to investigate the use of a proprietary drug delivery platform for this application (see the press release: 'Flamel Technologies and Digna Biotech Announce Multiple Product Development Agreement'). Other anti-TGFβ therapies in clinical trials for IPF include the pan TGFβ-neutralizing antibody GC-1008 (Genzyme) and the αVβ6 integrin-blocking antibody STX-100 (Stromedix)168,169.
Renal fibrosis has long been thought to be driven by excess TGFβ, which results in renal scarring and, ultimately, kidney failure170. With the increasing incidence of diabetes and associated kidney damage in affluent countries, this is a clinical application of growing importance. Mice overexpressing an active form of TGFβ1 (from the liver) develop progressive liver and renal fibrosis170. Interestingly, although mice overexpressing active TGFβ1 develop progressive renal injury, latent TGFβ1 also has a protective role in renal fibrosis through negative effects on inflammation49. TGFβ1 mediates progressive renal fibrosis by stimulating the synthesis of ECM production while inhibiting its degradation49. TGFβ1 also mediates renal fibrosis by inducing the transformation of tubular epithelial cells into myofibroblasts through EMT in a similar way to the process seen in IPF171. Blockade of TGFβ1 with neutralizing TGFβ antibodies prevents or ameliorates renal fibrosis in vivo and in vitro, demonstrating the functional role of TGFβ1 in EMT and renal fibrosis. A number of therapeutic interventions that block the action of TGFβ have resulted in various degrees of improvement in kidney structure and function in preclinical studies; such interventions include TGFβ ASOs, a neutralizing anti-TGFβ antibody, a soluble TGFβ receptor, blockade of TGFβ activation by decorin172, an SMI of TGFβ receptors, delivery of the inhibitor protein SMAD7 (Ref. 173) and a THBS1-blocking peptide that interferes with TGFβ activation97. A Phase I/II trial with GC-1008, a pan-TGFβ-neutralizing antibody, exhibited encouraging efficacy in patients with focal segmental glomerulosclerosis92, and Eli Lilly is undertaking trials of its own anti-TGFβ1 monoclonal antibody, LY2382770, in diabetic kidney disease.
Cardiac fibrosis is a pathological feature that is common to a number of forms of heart disease, including myocardial infarction, ischaemic, dilated and hypertrophic cardiomyopathies and congestive heart failure36. The cellular basis of cardiac fibrosis is the aberrant accumulation of collagens and other ECM proteins, which impair ventricular function and predispose to cardiac arrhythmias. Because TGFβ has pleiotropic effects in the cardiovascular system and as cardiac fibrosis is a multifactorial disease, the development of an effective therapy will require a detailed understanding of the role of the TGFβ signalling pathway in this pathogenesis. TGFβ, a potent stimulator of collagen production by cardiac fibroblasts, is induced in response to cardiovascular injury. The TGFβ-SMAD pathway activates the transcription of several key fibrotic genes, such as those encoding connective tissue growth factor (CTGF), fibronectin, collagens and plasminogen activator inhibitor 1 (PAI1)36. TGFβ reduces collagenase production and stimulates the expression of tissue inhibitor of metalloproteinases (TIMPs), resulting in an overall inhibition of ECM degradation and leading to excessive ECM accumulation. P144 has been investigated in a preclinical model of cardiac fibrosis77, and losartan can reverse fibrosis in a mouse model of hypertrophic cardiomyo pathy174; however, no drug targeting the TGFβ pathway has yet reached clinical trials for this application. A recent study demonstrated that miR-21, which is regulated by SMADs upon TGFβ activation, is consistently induced by cardiac stress. As miR-21 plays a part in tumorigenesis by promoting cell proliferation, increased expression of miR-21 might contribute to the progression of fibrotic lesions175. ASOs against miR-21 might therefore become a novel therapeutic approach for treating cardiac fibrosis.
Scleroderma (progressive systemic sclerosis) is a systemic autoimmune disorder characterized by skin sclerosis, calcinosis and changes in microvasculature. Increased expression of TβRI and TβRII in sclerodermal fibroblasts suggests that increased production of type I collagen by autocrine TGFβ signalling leads to aberrant ECM deposition and scarring36. Therapeutic approaches to scleroderma have included inhibition of TGFβ activity in sclerotic tissue. Unfortunately, CAT-192, a TGFβ1-neutralizing antibody, did not show evidence of efficacy in a study on the treatment of patients with early-stage systemic scleroderma90; however, GC-1008 is now in a Phase I clinical trial for patients with diffuse systemic sclerosis. Furthermore, topical application of Digna Biotech's P144 peptide inhibitor of TGFβ1 has shown some efficacy in reducing skin fibrosis in a Phase II clinical trial for systemic sclerosis (see the press release: 'Flamel Technologies and Digna Biotech Announce Multiple Product Development Agreement'), with the caveat that clinical end points for quantifying skin fibrosis have not yet been standardized176.
The development of fibromuscular intimal hyperplasia following angioplasty and coronary artery bypass surgery is a major clinical problem and can lead to coronary artery graft failure. The success of coronary artery reconstructive procedures is limited by the high incidence of restenosis secondary to intimal hyperplasia. TGFβ1 is a major player in the early development of intimal hyperplasia in arteries and peripheral vein grafts. The exact mechanism of action of TGFβ signalling in intimal hyperplasia and subsequent graft failure is unclear, but it is speculated that TGFβ1 contributes at multiple steps, including EMT, promotion of fibroblast, endothelial and vascular smooth muscle cell proliferation, increased collagen synthesis and deposition, and induction of fibrosis177. Soluble forms of the small, leucine-rich proteoglycans decorin and fibromodulin, which possess TGFβ-antagonist activity, exhibit potent intimal hyperplasia-suppressing effects in cultured human saphenous vein, offering the potential for therapeutic benefit after coronary artery bypass surgery178-180. The novel pyrrole-imidazole polyamide drug class, targeted to suppress TGFB1 gene transcription, showed efficacy in reducing neointimal hyperplasia and stimulating re-endothelialization of carotid arteries in a preclinical model of arterial injury50.
MFS is a connective tissue disorder that affects the musculoskeletal, ocular and cardiovascular systems. It is caused by mutations in the gene encoding an ECM protein, fibrillin 1 (FBN1)181. Growing evidence suggests that FBN1 mutations perturb not only the general integrity and elasticity of tissues but also — probably more crucially — local TGFβ signalling11. Normal fibrillin 1-containing microfibrils interact with the large latent TGFβ complex (LLC) to control the release of mature, active TGFβ181. Mutated fibrillin 1 fails to sequester latent TGFβ, leading to the promiscuous activation of TGFβ11. Thus, MFS highlights the critical role of microfibrils in regulating local concentrations of TGFβ and in maintaining the homeostasis, morphogenesis and function of various organs181. Dilation of the aortic root, which leads to aortic rupture and sudden death, is a major clinical issue for patients with MFS and patients of the Loeys-Dietz spectrum who carry mutations in TGFBR1, TGFBR2 and SMAD3. A mouse model of MFS carrying a heterozygous Fbn1 mutation developed an aortic aneurism similar to that of patients with MFS. Administration of TGFβ antagonists, including a TGFβ-neutralizing antibody or the angiotensin II type 1 receptor (AT1) blocker losartan, successfully rescued both cardiovascular and non-cardiovascular manifestations of MFS12,182. As losartan is already in widespread clinical use for hypertension and has shown no adverse effects, this drug is currently being tested in Phase I/II clinical trials for MFS (ClinicalTrials.gov identifiers: NCT00429364, NCT00593710, NCT00683124, NCT00723801, NCT00763893, NCT00782327 and NCT01145612) and may plausibly reduce this life-threatening manifestation of MFS.
Trabeculectomy is a surgical procedure designed to reduce intraocular fluid pressure in patients with glaucoma. However, postoperative scarring and fibrotic blockage of the 'filtering bleb' that drains excess ocular fluid are serious complications of this procedure. In preclinical experiments, administration of neutralizing antibodies against human TGFβ2 (CAT-152) exhibited promising inhibition of scarring after glaucoma surgery in rabbits without having any adverse effects. Initial clinical trials with CAT-152 ameliorated scarring in patients who received trabeculectomy for intractable glaucoma88,183; however, Phase III clinical trials were unable to validate such beneficial effects184. Tranilast, another incidental TGFβ inhibitor, has also been used successfully to reduce re-occurrence of corneal fibrosis, or primary pterygium, following corneal surgery185. It is possible that topical application of more-specific TGFβ inhibitors might also be used in treating corneal haze and conjunctival scarring (Table 1).
Many diseases being tackled with TGFβ inhibitors, including fibrosis, inflammation, autoimmunity and cancer, are complex in nature and show strong genetic predisposition owing to innate genetic variation between individuals. It is well established that there is considerable phenotypic diversity in the range of responses to reduced TGFβ signalling in vivo, which are dictated by differential inheritance of germline genetic variants. This is illustrated by the large spectrum of clinical severity and disease manifestations in individuals with mutations in TGFβ signalling pathway genes8,41,186. Moreover, in mouse models of cancer, asthma and vascular development, outcomes of reduced TGFβ1 levels are strongly influenced by interacting genetic modifier loci5,9,186,187. It is therefore most rational, economical and safe to preselect patient populations before initiating anti-TGFβ drug treatment on the basis of surrogate markers of TGFβ involvement in the disease process (such as increased TGFβ ligand and P-SMAD levels, and specific disease characteristics) and contra-indications of possible adverse side effects (such as susceptibility to inflammation or certain vascular conditions). Peripheral blood may provide non-invasive markers that might be useful in this respect, including the ability to quantitatively screen patient responses to TGFβ inhibition on the basis of measuring P-SMAD2 levels in peripheral blood mononuclear cells (PMNCs)188. Similarly, potential adverse inflammatory effects could be predicted by examining specific immunological responses of PMNCs189-192 to TGFβ inhibition ex vivo.
The simplest biomarker for patient selection for TGFβ inhibitors in oncology is probably high circulating levels of TGFβ, as one major goal of this therapy is to reduce, but not totally ablate, TGFβ signalling193. As indicated above, non-invasive biomarkers for predicting patient responsiveness and efficacy of TGFβ inhibitors have been developed on the basis of measuring P-SMAD2 levels in PMNCs. Indeed, PMNCs can be treated with drugs ex vivo to determine, and thus predict, individual patient responses to SMIs188,193. As TGFβ inhibitors certainly act to reduce tumour metastasis, the assay of circulating tumour cell number may also be a useful indicator for therapeutic response. Moreover, as many of the pro-tumorigenic effects of TGFβ are mediated by immune system modulation, it might be possible to monitor blood cell or plasma protein profiles for patient selection and for surrogate monitoring of patient responses.
In cancer (as well as non-malignant diseases), the outcome of reduced TGFβ signalling may be highly dependent on the innate genetic background of the individual, especially when considering tumour microenvironment effects, such as immune surveillance. Elucidating specifically which genetic variants influence signalling output will not only be useful for dissecting the intricacies of this signalling pathway in vivo, but may also provide predictive markers for the outcome (desired or undesired) of TGFβ signal inhibition. However, for cancer therapeutics, patient selection may be more complex, as the response of both the tumour cell and host (tumour microenvironment and normal patient tissue) to TGFβ blockade needs to be considered. Tumour biopsy and genetic analysis (for example, loss of TGFBR2, SMAD2 or SMAD4)194,195 might predict whether the tumour retains growth sensitivity to TGFβ. Molecular and histological analyses may also contribute to the prediction of tumour responses to TGFβ inhibition. The activation of alternative intracellular signalling pathways and transcription factor profiles has been associated with the switch from tumour suppression — by TGFβ — to tumour progression. These include an increased ratio of liver-enriched inhibitory protein (LIP) to liver-enriched activating protein (LAP), which are isoforms of CCAAT/enhancer-binding protein-β (C/EBPβ), a central transcription factor that binds within the SMAD transcription factor complex to elicit TGFβ-mediated cytostatic responses196. Upregulation of SIX1 in breast cancer has also been shown to be pivotal in the growth-suppressive to tumour-progressive switch197, as has downregulation of DAB2 (Ref. 198). Any of these tumour markers, possibly in combination, may be used in the future to predict tumour responses to TGFβ inhibition.
Finally, the effects of interactions with other anticancer drugs will need to be considered, as some drugs may resurrect the growth-inhibitory arm of the TGFβ signalling pathway, which would then counter-indicate their combinatorial use with TGFβ inhibitors199. Indeed, the ability to specifically target the pro-tumorigenic versus the tumour-suppressive effects of TGFβ on the tumour cell per se will require the development of next-generation drugs focusing on these downstream pathways. In the meantime, much research still remains to be undertaken to make inroads into the area of informed patient selection for oncology applications of TGFβ inhibitors.
In oncology, the development of tumour drug resistance is inevitable134,200,201. It has been documented for standard chemotherapy, pathway-targeted therapies and is even common with anti-angiogenesis inhibitors200,201. Acquired biochemical resistance of tumour cells to LY2109761 has been observed in a preclinical model of SCC and may have adverse consequences in driving a more stem cell-like phenotype156, although this remains to be tested. Carefully restricting TGFβ inhibitors to short-term or intermittent usage should avoid these complications82. Combinatorial and/or sequential treatment with complementary drugs will also be important. It is clear that oncologists will need an arsenal of different anticancer drugs to tackle cancer, in much the same way that antibiotics have been developed to combat infectious diseases.
In conclusion, TGFβ signalling inhibitors are generally safe and may be efficacious in several clinical applications, especially in desperate cases such as end-stage cancer or IPF. The development of these drugs may offer further therapeutic opportunities. Counterintuitively, there have been reports suggesting that inhibition of the TGFβ signalling pathway may be beneficial in autoimmune disorders, such as multiple sclerosis, through downregulation of the TH17 pathway202,203. Recent studies have also suggested that TGFβ-SMAD3 signalling regulates glucose tolerance and energy homeostasis, and that blockade of the pathway may be used for regulation of diabetes and obesity204. The outlook for anti-TGFβ signalling therapy for numerous diseases appears bright. At least four companies are well on their way in clinical drug development, and further scientific and mechanistic studies are warranted in order to optimize patient selection and drug-dosing regimens for each disease application.
The authors give thanks to J. Dotor, J. McPherson and J. Yingling for useful discussions. Work in the authors' laboratories is funded by grants from the US National Cancer Institute, the US National Heart, Lung and Blood Institute (NHLBI), the US National Institute of Arthritis and Musculoskeletal and Skin Diseases, the US National Institute of General Medical Sciences, the Bouque Foundation and the Helen Diller Family Comprehensive Cancer Center to R.J.A., and from the NHLBI, the American Heart Association and the LeDucq Foundation to A.H. We apologize to all colleagues whose work could not be cited owing to space restrictions.
Competing interests statement
The authors declare no competing financial interests.
Rosemary J. Akhurst's homepage: http://cancer.ucsf.edu/research/akhurst-lab Akiko Hata's homepage: http://www.cvri.ucsf.edu/Scientist/Hata.shtml ClinicalTrials.gov website: http://www.clinicaltrials.gov Flamel Technologies and Digna Biotech Announce Multiple Product Development Agreement (27 June 2011 press release): http://www.dignabiotech.com/comun/fichero.asp?id=105
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Bayard School
From Wikipedia, the free encyclopedia
Bayard School
|4830 Hatfield St., Pittsburgh, Pennsylvania
|40°28′37″N 79°57′29″W / 40.47694°N 79.95806°WCoordinates: 40°28′37″N 79°57′29″W / 40.47694°N 79.95806°W
|1 acre (0.40 ha)
|Architectural style
|Pittsburgh Public Schools TR
|NRHP Reference #
|Added to NRHP
|September 30, 1986
The Bayard School in the Upper Lawrenceville neighborhood of Pittsburgh, Pennsylvania is a building from 1874 and is one of Pittsburgh's oldest surviving school buildings. The school was closed in 1938 and sold in 1941. It was later used as a warehouse.[2] It was listed on the National Register of Historic Places in 1986.[1]
- ^ a b National Park Service (2009-03-13). "National Register Information System". National Register of Historic Places. National Park Service.
- ^ Aurand, Martin (1986). "Bayard School" (PDF). Pennsylvania Historic Resource Survey form. Pennsylvania Historical and Museum Commission. Retrieved December 10, 2013.
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|This article about a property in Allegheny County, Pennsylvania on the National Register of Historic Places is a stub. You can help Wikipedia by expanding it.
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Join us as we focus on refinements to the Unified Improvement Planning processes and template for the 2014-15 school year.
What You Need to Know About UIP for the 2014-15 School Year
Hosted by the Colorado League of Charter Schools
Date: Tuesday, September 9
Time: 9:00 AM - 11:00 AM
Location: Online/Webinar
Cost: Free
Description: Attend this webinar to find out what you will need to know about the UIP for the 2014-15 school year, including a few upcoming changes to the UIP. Attendees will learn about:
Please note this event will be recorded for those who are unable to join during the live webinar.
Target Audience: School leaders, assessment staff, or anyone who helps drive school improvement
Registration: Click here to register
7/20/2017Liberty Tree Academy Approved by D49 Board of Education
10/18/2016Renaissance Secondary School Breaks Ground and Opens Enrollment
8/3/2017Implementing Effective Data Teams
8/3/2017PowerSchool: Beginning of Year Readiness
Colorado League of Charter Schools
2696 S. Colorado Blvd., Suite 250
Denver, CO 80222


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Amazing Skincare Tips For Glowing Skin In Monsoon
While you may enjoy getting wet in the rain, your skin surely won't like it. Skin tends to react unstably to the humid and moist monsoon climate. You may notice that on some days it becomes extremely oily and other days it's too dry, making your face look dull. Therefore it is important to give your skin the right treatment to bear with the monsoon climate. Here are a few amazing skincare tips that will help your skin glow even during a downpour.
Monsoon usually causes dirt and pollutants to lodge themselves in your pores. Wash your face at least two to three times a day and especially before you go to bed so that these pollutants don't aggravate your skin.
When you return home after a hectic day, washing your face is sometimes not enough to get all the dirt out. Use a toner if you have dry skin or an astringent is you have oily skin to clean your pores thoroughly.
You may be using a moisturiser during the day but it is also important that you moisturise your face at night too. Your skin naturally repairs itself at night and moisturising will aid it. Regular moisturising before you sleep will also help keep away wrinkles and will ensure that your skin glows.
A good skincare routine should always include exfoliation at least once a week. This will help clear out the dead and dry skin thus keeping your skin supple and glowing.
It is extremely important that you don't skip on your sunscreen even when it is gloomy and dark outside. Even though your skin may not feel the UV rays emitted from the sun, even slight exposure can damage your skin. Remember to use a sunscreen even during monsoon.
Glowing skin is not only a result of external skincare. You must remember to eat healthy vegetables and antioxidant rich fruits to keep your complexion glowing. Make sure to drink plenty of water throughout this season.
*Images courtesy: © Thinkstock photos/ Getty Images
For More:Monsoon
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text: Breadcrumb Home / Dictionary Meaning of industrial life insurance Find definitions for: indus'trial life' insur"ance Pronunciation: [key] life insurance having a relatively low face value in which premiums are paid weekly or monthly to an agent. Random House Unabridged Dictionary, Copyright © 1997, by Random House, Inc., on Infoplease. industrialize industrial park Related ContentDaily Word Quiz: opprobriumAnalogy of the Day: Today's AnalogySpelling Bee: Today's Spelling BeeKids' Word Quiz: preparatoryCrossword: Crossword Puzzle GuideFrequently Misspelled WordsFrequently Mispronounced WordsEasily Confused WordsWriting & Language Related Content Daily Word Quiz: prostrate Analogy of the Day: Today's Analogy Spelling Bee: Today's Spelling Bee Frequently Misspelled Words Frequently Mispronounced Words Easily Confused Words Writing & Language


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Encap Drug Delivery Establishes Hot Melt Extrusion Capability
News Oct 09, 2012
Encap is a contract development and manufacturing organisation (CDMO) which has pioneered the application of liquid and semi-solid filled hard capsules for pharmaceutical products. For more than 20 years the company has successfully applied the technology to a wide range of client new molecular entities (NMEs) which have required solubility and bioavailability enhancement. Since its inception the company has focused on gaining a sound scientific understanding of the formulation strategies which can be tailored to the individual characteristics of the target molecules and more importantly translating these formulations into scalable and commercial liquid filled pharmaceutical products.
It is the company's intention to apply a similar strategy to the development of HME products and Encap will differentiate itself from other development services companies by providing a sound scientific understanding of the technology. This will ensure that products are developed on a rigorous scientific basis and promote robust and scalable manufacturing processes which will enable clients to move quickly and efficiently through clinical development to registration and commercialisation.
Dr Jane Fraser said 'I am delighted to be working with Professor David Jones and Dr Gavin Andrews of QUB which is recognised as a world leader in the field of HME research. Encap is dedicated to providing world-class development and manufacturing services to its global clients and we are confident that we can establish and grow HME as a mainstream technology for poorly soluble molecules. We believe that this will be a valuable addition to the formulator's tool box and will fit well with our other enabling technologies'.
Dr Gavin Andrews reiterated the importance of the grant programme 'Specifically, our intention is that QUB will implement a robust, continuous melt extrusion process capable of engineering solid dispersions. In so doing we aim to complement existing soluble enhancement approaches through the addition of HME and thus bolster Encap's technologies portfolio'
Researchers Develop New Method to Generate Human AntibodiesNews
Researchers hope their approach will help researchers rapidly generate therapeutic antibodies for the treatment of infectious diseases and other conditions such as cancer.READ MORE
Large-Scale Production of Living Brain Cells Enables Entirely New ResearchNews
After performing a biopsy on the patient, the skin cells are transformed into brain cells that effectively imitate the disease and the age of the patient.READ MORE
U.S. Study of Dapivirine Ring in Lactating Women Finds Little Drug Gets Into Breast MilkNews
The antiretroviral drug dapivirine that is released from an experimental vaginal ring to protect against HIV is absorbed in very low concentrations into breastmilk.READ MORE
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10th Pharmacovigilance Congress
Sep 20 - Sep 21, 2017
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Apr 19 - Apr 20, 2018
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Mar 19 - Mar 20, 2018
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1. Drop-Off
Please bring your tagged items during one of these drop-off times. No appointment needed.
- Monday, Sept. 18: 6-8pm
- Tuesday, Sept 19: 9:15am-1pm or 6-8pm
- Wednesday, Sept 20 : 9:15am-12:30 pm
2. Pick-up
- Sunday, Sept 24 – PICK UP: 1-3pm
3. Acceptable Merchandise
- ACCEPTED AT FALL SALE: Gently used (no holes, stains or tears) Fall/Winter clothing (size infant-16 as well as Junior sizes), athletic wear, SMOD (long and short sleeves), jackets/coats, costumes, and Holiday outfits. Sleeveless/ short sleeved holiday attire and sleeveless sweater/corduroy jumpers/dresses will be accepted.
*NOT ACCEPTED AT FALL SALE: Short sleeved shirts and dresses (except for SMOD), sleeveless shirts and dresses (except for holiday/sweater/corduroy), shorts, short sleeved pajamas, sandals, swim suits – Save these for the Spring Sale!
- ACCEPTED AT SPRING SALE: Gently used (no holes, stains or tears) Spring/Summer clothing (size infant-16 plus Junior sizes), athletic wear, swimsuits (no string bikinis, please), and Easter outfits. Jeans, khakis, leggings, lightweight sweaters/cardigans/jackets and rain coats. Long sleeved boys Easter shirts ok. (Shorts must be an appropriate length, acceptance at the discretion of KKS Committee.)
*NOT ACCEPTED AT SPRING SALE: Long sleeved shirts (except for boys Easter shirts), corduroy pants and dresses/jumpers, long sleeved/fleece/flannel pajamas, heavy sweaters and sweater dresses, and sweatshirts – Save these for the Fall Sale!
- Toys, games, children's and young adult books, bikes, baby equipment and furniture, strollers, and car seats.
- Gently worn shoes.
- The following items are accepted only in NEW CONDITION: Underwear (must have tags or come in package), Pacifiers (still in package), Bottle Nipples (still in package)
- Current style, in-season maternity wear.
- Designer or High-End Ladies Handbags – see details here
- We CANNOT ACCEPT stuffed animals, pillow pets, used underwear, bras, used breast pump accessories, toy weapons/guns (aside from nerf and water toys), VHS tapes, adult fiction books, violent/mature video games, or any items that have been recalled by the manufacturer.
4. Instructions:
- Bring all clothes on wire or plastic hangers. Safety pin skirts and shorts/pants to wire hangers. Please do not use clothespins or straight pins.
- Clothing must be clean and pressed with no stains, missing buttons, broken zippers or rips.
- Shoes must be placed in a Ziploc bag (one pair per bag). Each bag must be tagged ON THE OUTSIDE OF THE BAG as indicated in the tagging instructions.
- All toys and baby equipment must be clean and in good working order with all parts secured with clear mailing tape.
- Small items, i.e. diaper covers, small toys, bottles, rattles, etc. may be grouped in a Ziploc bag. Each bag must be tagged as indicated in the tagging instructions.
- Car Seats should be accompanied by the Car Seat Waiver. KKS can only accept car seats that meet the listed standards.
5. Pricing and Tagging
PRICING: You set your own prices. All items must be priced and tagged prior to delivery. No items under $2.00. Please round all prices off to the nearest half dollar.
TAGS: Use one 3" x 5" white index card or 1/4th sheet of card stock as your tag: write your consignor number in the upper left corner, then halfway down the card, write your consignor number again on the left side, list size in the middle section, add a brief description below the size, then list the price below the description (see illustration below). Clip the BOTTOM right corner of the tag if you do NOT want your items reduced to half price during Saturday's half price sale. Tape tags with clear mailing tape on toys, equipment, shoe bags, etc. No masking tape, please.
CORRECT TAGGING IS VERY IMPORTANT! Incorrect tags will not be accepted. Examples of incorrect tags that will NOT be accepted: tags with barcodes, tags with additional information that is not listed on our example, tags with information written in the wrong place, tags written or printed on notebook or printer paper, tags that are smaller than an index card.
***NEW! PRINTABLE TAG TEMPLATES! Just print it out and fill in the blanks! You can print on 3×5 index cards or 8.5 x 11 card stock. (Remember to use card stock instead of printer paper!) DOWNLOAD HERE: 3×5 Index Card Template OR 8.5×11 Cardstock Template
SIZING: Except for maternity clothing, please do not tag clothes "Small" "Medium" or "Large". All clothes must be tagged with a specific numerical size such as 2T, 4, or 8 otherwise they will be rejected. If the manufacturer has tagged the item small, please estimate what numerical size is most appropriate. A note about Junior Items: be sure to mark the tag with the junior size (ex. size 0 or 2 instead of size 14/16).
CARDS MISSING DATA: If a customer would like to buy an item that does not have a price listed on the tag, the worker at the cash register will assign a reasonable price and sell the item. Likewise, if a customer would like to purchase an item that has no consignor number on the tag, We will sell the item and donate the proceeds to charity. Please double check that you give your consignor number and a price on all items.
CHARITY: If you would like to donate your unsold items, please put a "C" in red ink in the bottom LEFT handcorner of your tag, indicating the items goes to charity.
SAFETY PINS: Use a safety pin to secure the tag to the upper right side of the garment, hanger facing left as noted (like a question mark). Entire tag must be in view (i.e. not under the collar). Straight pins will NOT be accepted.
6. Miscellaneous:
- You must have more than 20 items.
- Consignors with over 40 items are invited to a special preview sale. Only the Consignor listed is allowed admission to the special preview sale.
- Items not sold must be picked up on Sunday during the assigned pick up time. All items not picked up will be donated to local charities.
- The Kid'' Konsignment Sale is not responsible for items separated from the tags. These items will not be sold.
- $2.00 will be deducted from the total amount of your check to cover the handling fee.
- We are not responsible for items stolen, tags switched, or items separated from their tags.
- Sort all hanging items by gender and size before drop off.
- Only items meeting the above guidelines will be accepted.
Have a specific question? Email us at email@example.com and we'll try to reply quickly!


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A solid-state imaging device having an arrangement in which well contact is achieved for each pixel is provided. In the solid-state imaging device, a well contact part is formed in an activation region of a photoelectric conversion portion. The well contact part fixes a well in which the photoelectric conversion portion and transistors of the pixel are provided at a predetermined potential.
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Automotive royalty pays a surprise visit to the garage when Allen Grant stops by with his newly restored 1963 Lola Mk6 GT, the prototype vehicle that led to the legendary Ford GT.