Biosimilares de anticuerpos monoclonales en enfermedades inflamatorias y cáncer: situación actual, retos y oportunidades / Biosimilars of monoclonal antibodies in inflammatory diseases and cancer: current situation, challenges, and opportunities

El proceso de aprobación de los biosimilares de anticuerpos monoclonales en la Unión Europea está dirigido a descartar la presencia de diferencias significativas con el biológico original en los atributos de calidad, eficacia, inmunogenicidad y seguridad. Proporciona además la justificación para extrapolar la evidencia obtenida con un biosimilar en al menos una indicación al resto de indicaciones aprobadas para su biológico original, simplificando el programa de desarrollo de los biosimilares. Los biosimilares de anticuerpos monoclonales disponibles en la Unión Europea para el tratamiento de enfermedades inflamatorias y del cáncer han cumplido todos los requerimientos establecidos para la aprobación, y en muchos casos disponen de evidencia adicional. Además, los datos de uso en la vida real están confirmando la seguridad y eficacia de estos fármacos en las distintas patologías en las que se están utilizando. En España, varias sociedades médicas avalan el proceso regulatorio de los biosimilares y reconocen su papel en la eficiencia del sistema sanitario. No obstante, todavía existen algunas barreras que limitan su uso. La aplicación de diferentes medidas a nivel de paciente, prescriptor, institucional y nacional podría aumentar la penetración de los biosimilares, liberando recursos que podrían invertirse en otras terapias y, potencialmente, favorecer la innovación

The approval pathway for biosimilars of monoclonal antibodies in the European Union is aimed at ruling out the presence of significant differences with the original biological in quality attributes, efficacy, immunogenicity and safety. It also provides the rationale for extrapolating the evidence obtained with a biosimilar in at least one indication to the rest of the approved indications of its original biological, thus simplifying the deve-lopment programme of biosimilars. Biosimilars of monoclonal antibodies available in the European Union for the treatment of inflammatory diseases and cancer have fulfilled all the requirements for approval, and many of them have additional evidence available. Moreover, real world data confirms the safety and efficacy of these drugs in the indications they are being used for. In Spain, many scientific societies endorse the regulatory pathway of biosimilars and acknowledge their role in the efficiency of the healthcare system. Even so, some barriers remain that limit their use. The implementation of different measures at the patient, prescriber, institutional, and national levels might increase the penetration of biosimilars, freeing up resources that may be invested in other therapies and, potentially, boost innovation

Biosimilars of monoclonal antibodies in inflammatory diseases and cancer: current situation, challenges, and opportunities.

The approval pathway for biosimilars of monoclonal antibodies in the European Union is aimed at ruling out the presence of significant  differences with the original biological in quality attributes, efficacy,  immunogenicity and safety. It also provides the rationale for  extrapolating the evidence obtained with a biosimilar in at least one  indication to the rest of the approved indications of its original  biological, thus simplifying the development programme of biosimilars.  Biosimilars of monoclonal antibodies available in the European Union for  the treatment of inflammatory diseases and cancer have fulfilled all the  requirements for approval, and many of them have additional evidence  available. Moreover, real world data confirms the safety and efficacy of  these drugs in the indications they are being used for. In Spain, many  scientific societies endorse the regulatory pathway of biosimilars and  acknowledge their role in the efficiency of the healthcare system. Even  so, some barriers remain that limit their use. The implementation of  different measures at the patient, prescriber, institutional, and national  levels might increase the penetration of biosimilars, freeing up resources that may be invested in other therapies and, potentially, boost  innovation.

El proceso de aprobación de los biosimilares de anticuerpos  monoclonales en la Unión Europea está dirigido a descartar la presencia  de diferencias significativas con el biológico original en los atributos de  calidad, eficacia, inmunogenicidad y seguridad. Proporciona además la  justificación para extrapolar la evidencia obtenida con un biosimilar en al  menos una indicación al resto de indicaciones aprobadas para su  biológico original, simplificando el programa de desarrollo de los  biosimilares. Los biosimilares de anticuerpos monoclonales disponibles  en la Unión Europea para el tratamiento de enfermedades inflamatorias  y del cáncer han cumplido todos los requerimientos establecidos para la  aprobación, y en muchos casos disponen de evidencia adicional.  Además, los datos de uso en la vida real están confirmando la seguridad  y eficacia de estos fármacos en las distintas patologías en las  que se están utilizando. En España, varias sociedades médicas  avalan el proceso regulatorio de los biosimilares y reconocen su papel  en la eficiencia del sistema sanitario. No obstante, todavía existen  algunas barreras que limitan su uso. La aplicación de diferentes medidas  a nivel de paciente, prescriptor, institucional y  nacional podría aumentar la penetración de los biosimilares, liberando  recursos que podrían invertirse en otras terapias y, potencialmente,  favorecer la innovación.

Fitting a xenobiotic receptor into cell homeostasis: how the dioxin receptor interacts with TGFbeta signaling.

As our knowledge on the mechanisms that control cell function increases, more complex signaling pathways and quite intricate cross-talks among regulatory proteins are discovered. Establishing accurate interactions between cellular networks is essential for a healthy cell and different alterations in signaling are known to underline human disease. Transforming growth factor beta (TGFbeta) is an extracellular cytokine that regulates such critical cellular responses as proliferation, apoptosis, differentiation, angiogenesis and migration, and it is assumed that the latency-associated protein LTBP-1 plays a relevant role in TGFbeta targeting and activation in the extracellular matrix (ECM). The dioxin receptor (AhR) is a unique intracellular protein long studied because of its critical role in xenobiotic-induced toxicity and carcinogenesis. Yet, a large set of studies performed in cellular systems and in vivo animal models have suggested important xenobiotic-independent functions for AhR in cell proliferation, differentiation and migration and in tissue homeostasis. Remarkably, AhR activity converges with TGFbeta-dependent signaling through LTBP-1 since cells lacking AhR expression have phenotypic alterations that can be explained, at least in part, by the coordinated regulation of both proteins. Here, we will discuss the existence of functional interactions between AhR and TGFbeta signaling. We will focus on regulatory and functional aspects by analyzing how AhR status determines TGFbeta activity and by proposing a mechanism through which LTBP-1, a novel AhR target gene, mediates such effects. We will integrate ECM proteases in the AhR-LTBP-1-TGFbeta axis and suggest a model that could help explain some in vivo phenotypes associated to AhR deficiency.

[The most important methodological problems in studies of mortality and disease progression in patients with chronic obstructive pulmonary disease]. / Problemas metodológicos más relevantes en los estudios de mortalidad y progresión de la enfermedad en pacientes con EPOC.

Chronic obstructive pulmonary disease (COPD) is characterized by progressive and incompletely reversible airflow obstruction associated with an anomalous inflammatory response of the lungs, mainly to tobacco smoke. The best indicator of disease progression and severity is measurement of airflow obstruction (forced expiratory volume in 1 second), expressed as a percentage of the predicted value derived from a healthy reference population. Most of the treatments available for COPD have not been shown to clearly affect disease progression or mortality, probably because COPD is a heterogeneous, longstanding process and because there is wide variety in patients' phenotypes, clinical situations, and clinical course. In the last few years, the number of studies on disease progression and mortality in COPD has markedly increased, making survival analysis one of the most important tools for exploiting the resulting data. However, certain methodological factors associated with this type of research, such as the study design, the variables used to measure effect, and determination of the sample, have influenced the conclusions of these studies. Moreover, analysis of disease progression and mortality rates usually entails prolonged follow-up periods and a large number of participants, limiting the funding and continuity of this type of study. In the case of studies of mortality associated with COPD, there are the additional difficulties of identifying the specific cause of death and of identifying the prognostic factors of mortality from the disease, which are the main methodological factors that hamper the possibility of obtaining conclusive data.

Pitx2 regulates gonad morphogenesis.

Organ shape and size, and, ultimately, organ function, relate in part to the cell and tissue spatial arrangement that takes place during embryonic development. Despite great advances in the genetic regulatory networks responsible for tissue and organ development, it is not yet clearly understood how specific gene functions are linked to the specific morphogenetic processes underlying the internal organ asymmetries found in vertebrate animals. During female chick embryogenesis, and in contrast to males where both testes develop symmetrically, asymmetrical gonad morphogenesis results in only one functional ovary. The disposition of paired organs along the left-right body axis has been shown to be regulated by the activity of the homeobox containing gene pitx2. We have found that pitx2 regulates cell adhesion, affinity, and cell recognition events in the developing gonad primordium epithelia. This in turn not only allows for proper somatic development of the gonad cortex but also permits the proliferation and differentiation of primordial germ cells. We illustrate how Pitx2 activity directs asymmetrical gonad morphogenesis by controlling mitotic spindle orientation of the developing gonad cortex and how, by modulating cyclinD1 expression during asymmetric ovarian development, Pitx2 appears to control gonad organ size. All together our observations indicate that the effects elicited by Pitx2 during the development of the female chick ovary are critical for cell topology, growth, fate, and ultimately organ morphogenesis and function.

Problemas metodológicos más relevantes en los estudios de mortalidad y progresión de la enfermedad en pacientes con EPOC / The most important methodological problems in studies of mortality and disease progression in patients with chronic obstructive pulmonary disease

una entidad clínica caracterizada por una limitación progresivay no completamente reversible al flujo en las vías aéreas,que se asocia a una respuesta inflamatoria anómala delos pulmones, principalmente frente al humo del tabaco. Elmejor indicador de la progresión y la gravedad de la enfermedades la medida de la obstrucción al flujo aéreo (volumenespiratorio forzado en el primer segundo), expresadaen porcentaje respecto al valor de referencia.La mayoría de los tratamientos existentes para la EPOCno han mostrado tener un efecto claro en la progresión ymortalidad de la enfermedad, debido probablemente al hechode que la EPOC es un proceso heterogéneo de muy largaduración y con una amplia variedad de fenotipos de pacientes,situaciones clínicas y evolución de la enfermedad.En los últimos años el número de estudios sobre la progresióny mortalidad en la EPOC ha crecido de manera muy significativa,convirtiéndose el análisis de supervivencia en unade las herramientas más importantes de explotación de losdatos resultantes. Sin embargo, ciertos factores metodológicosasociados a este tipo de investigaciones, tales como el diseñodel estudio, las variables de medida del efecto y la determinaciónde la muestra, han influido en las conclusiones finales dedichos estudios. Además, el análisis de la progresión y de latasa de mortalidad suele llevar asociada la necesidad de unalarga duración de seguimiento y un alto número de participantes,lo que limita especialmente la financiación y el seguimientode este tipo de estudios. En el caso de los estudios demortalidad asociada a la EPOC, se suman además las dificultadesa la hora de reconocer la causa específica de la muerte yde identificar los factores pronóstico de mortalidad de la enfermedad,que son los principales factores metodológicos quelimitan la posibilidad de conseguir datos concluyentes(AU)

Chronic obstructive pulmonary disease (COPD) ischaracterized by progressive and incompletely reversibleairflow obstruction associated with an anomalousinflammatory response of the lungs, mainly to tobaccosmoke. The best indicator of disease progression andseverity is measurement of airflow obstruction (forcedexpiratory volume in 1 second), expressed as a percentageof the predicted value derived from a healthy referencepopulation. Most of the treatments available for COPDhave not been shown to clearly affect disease progression ormortality, probably because COPD is a heterogeneous,longstanding process and because there is wide variety inpatients’ phenotypes, clinical situations, and clinical course.In the last few years, the number of studies on diseaseprogression and mortality in COPD has markedlyincreased, making survival analysis one of the mostimportant tools for exploiting the resulting data. However,certain methodological factors associated with this type ofresearch, such as the study design, the variables used tomeasure effect, and determination of the sample, haveinfluenced the conclusions of these studies. Moreover,analysis of disease progression and mortality rates usuallyentails prolonged follow-up periods and a large number ofparticipants, limiting the funding and continuity of thistype of study. In the case of studies of mortality associatedwith COPD, there are the additional difficulties ofidentifying the specific cause of death and of identifying theprognostic factors of mortality from the disease, which arethe main methodological factors that hamper the possibilityof obtaining conclusive data(AU)

Post-transcriptional up-regulation of ADAM17 upon epidermal growth factor receptor activation and in breast tumors.

ADAM17 is a transmembrane metalloprotease involved in the proteolytic release of the extracellular domain of many cell surface molecules, a process known as ectodomain shedding. Despite its likely participation in tumor progression and its current consideration as a therapeutic target, very little is known about the regulation of the expression of ADAM17. Here we show that long term treatment with epidermal growth factor (EGF) leads to a marked increase in the levels of ADAM17. EGF receptor activation does not affect the levels of the mRNA that encodes for, or the rate of synthesis of, ADAM17 but increases its half-life. The effect of EGF is biologically relevant because it increases the shedding of several substrates of ADAM17, including the desmosomal cadherin Dsg-2. Analysis of protein and mRNA levels in mammary tumor samples shows that in vivo the levels of ADAM17 can also be controlled post-transcriptionally. Finally, we show that both the shed extracellular domains of Dsg-2 and ADAM17 are frequently expressed in tumors, further supporting the participation of the metalloprotease in malignant progression.

Biosynthesis of tumorigenic HER2 C-terminal fragments by alternative initiation of translation.

The overactivation of the HERs, a family of tyrosine kinase receptors, leads to the development of cancer. Although the canonical view contemplates HER receptors restricted to the secretory and endocytic pathways, full-length HER1, HER2 and HER3 have been detected in the nucleoplasm. Furthermore, limited proteolysis of HER4 generates nuclear C-terminal fragments (CTFs). Using cells expressing a panel of deletion and point mutants, here we show that HER2 CTFs are generated by alternative initiation of translation from methionines located near the transmembrane domain of the full-length molecule. In vitro and in vivo, HER2 CTFs are found in the cytoplasm and nucleus. Expression of HER2 CTFs to levels similar to those found in human tumors induces the growth of breast cancer xenografts in nude mice. Tumors dependent on CTFs are sensitive to inhibitors of the kinase activity but do not respond to therapeutic antibodies against HER2. Thus, the kinase domain seems necessary for the activity of HER2 CTFs and the presence of these HER2 fragments could account for the resistance to treatment with antibodies.

Proteomic identification of desmoglein 2 and activated leukocyte cell adhesion molecule as substrates of ADAM17 and ADAM10 by difference gel electrophoresis.

In contrast with the early view of metalloproteases as simple extracellular matrix-degrading entities, recent findings show that they are highly specific modulators of different signaling pathways involved, positively or negatively, in tumor development. Thus, before considering a given metalloprotease a therapeutic target, it seems advisable to characterize its function by identifying its repertoire of substrates. Here, we present a proteomic approach to identify ADAM17 substrates by difference gel electrophoresis. We found that the shedding of the extracellular domain of the transferrin receptor and those of two cell-cell adhesion molecules, activated leukocyte cell adhesion molecule (ALCAM) and desmoglein 2 (Dsg-2), is increased in cells overexpressing ADAM17. Genetic evidence shows that while ADAM17 is responsible for the shedding of ALCAM, both ADAM17 and ADAM10 can act on Dsg-2. Activation of the epidermal growth factor receptor leads to the upregulation of the shedding of Dsg-2 and to the concomitant upregulation of ADAM17, but not ADAM10, supporting the ability of overexpressed ADAM17 to shed Dsg-2. These results unveil a role of ADAM10 and ADAM17 in the shedding of cell-cell adhesion molecules. Since loss of cell adhesion is an early event in tumor development, these results suggest that ADAM17 is a useful target in anticancer therapy.

Overexpression of latent transforming growth factor-beta binding protein 1 (LTBP-1) in dioxin receptor-null mouse embryo fibroblasts.

The aryl hydrocarbon receptor (AhR) is a transcriptional regulator of genes involved in xenobiotic metabolism. Increasingly clear is also the role of the AhR in the control of cell growth and proliferation. By analyzing differential patterns of gene expression between wild-type (AhR+/+) and null (AhR-/-) mouse embryo fibroblasts (MEF), we have identified latent transforming growth factor-beta binding protein 1 (LTBP-1) as a negatively AhR-regulated gene in the absence of xenobiotics. Ltbp-1 mRNA and protein expression were markedly increased in AhR-/- MEF. Furthermore, secreted LTBP-1 was elevated in the culture medium and the extracellular matrix of AhR-null MEF. Actinomycin D inhibited Ltbp-1 mRNA overexpression, suggesting regulation at the transcriptional level. AhR activation by dioxin (TCDD) downregulated Ltbp-1, again suggesting an AhR-regulated mechanism. Treatment of AhR+/+ MEF with transforming growth factor-beta(TGF-beta) downregulated AhR and, simultaneously, increased Ltbp-1, further supporting the role of this receptor in LTBP-1 expression. AhR-/- conditioned medium had higher levels of active and total TGF-beta activity, suggesting a role for LTBP-1 in maintaining extracellular TGF-beta concentrations. TGF-beta did not appear to directly regulate Ltbp-1 given that addition of TGFbeta neutralizing antibody or TGFbeta protein to AhR-/- MEF had no effect on Ltbp-1 expression. AhR-/- MEF had lower levels of matrix metalloproteinase 2 (MMP-2) activity, which could not be attributable to MMP-2 mRNA downregulation or MMP-inhibitors Timp-1 and Timp-2 overexpression. These data identify LTBP-1 as one of the few AhR-regulated genes not involved in xenobiotic metabolism and also support the implication of the AhR in controlling TGFbeta activity and cell proliferation.

Down-regulation of CYP1A2 induction during the maturation of mouse cerebellar granule cells in culture: role of nitric oxide accumulation.

Nitric oxide (NO) is responsible for cytochrome P450 (CYP450) loss during isolation and cytokine treatment of primary rat hepatocytes. As P450s mediate the metabolism of toxic chemicals, their inhibition could compromise the cells competence to eliminate toxins, a condition potentially relevant in neurological diseases involving constitutive activation of nitric oxide synthase (NOS) and NO over-production. Here, we have investigated the correlation between NO accumulation and CYP1A2 down-regulation during maturation of mouse cerebellar granule cells (CGC). As neurons matured in culture, the inducible levels of CYP1A2 protein and catalytic activity decreased to almost undetectable values. In parallel, a significant increase in NO concentration was observed. Neuronal NOS remained constitutively active during maturation, thus contributing to NO accumulation. The NOS inhibitor l-NAME, restored CYP1A2 catalytic activity up to 9 days in vitro, supporting a role for NO in the inhibition process. Maturation was also followed by increased NMDA receptor activity and intracellular Ca2+ concentration. We suggest that maintained NOS activity during CGC maturation could lead to NO accumulation and to decreased CYP1A2 inducibility. Increased NMDA receptor activity and Ca2+ entry could contribute to this process. Thus, neurodegeneration could diminish the induction of specific P450s and impair the metabolism of foreign and/or endogenous chemicals in the CNS.

Proteasome inhibition induces nuclear translocation of the dioxin receptor through an Sp1 and protein kinase C-dependent pathway.

The dioxin receptor (AhR), in addition to its role in xenobiotic-induced carcinogenesis, appears to participate in cell proliferation, differentiation and organ homeostasis. Understanding potential mechanisms of activation of this receptor in the absence of exogenous ligands is therefore important to study its contribution to endogenous cellular functions. Using mouse embryo primary fibroblasts, we have previously shown that proteasome inhibition increased AhR transcriptional activity in the absence of xenobiotics. We suggested that proteasome inhibition-dependent AhR activation could involve an increase in the expression of the partner protein dioxin receptor nuclear translocator (ARNT). Since ARNT over-expression induced nuclear translocation of the AhR, and ARNT-deficient cells were unable to translocate this receptor to the nucleus upon proteasome inhibition, we have analyzed the effect of proteasome inhibition on the expression of regulatory proteins controlling ARNT levels. Treatment with the proteasome inhibitor MG132 increased endogenous Sp1 phosphorylation and its DNA-binding activity to the ARNT promoter. Sp1 phosphorylation and binding to the ARNT promoter, ARNT over-expression and AhR nuclear translocation were inhibited by GF109203X, a protein kinase C-specific inhibitor. In addition, MG132 stimulated protein kinase C activity in MEF cells with a pattern similar to that observed for ARNT expression. These data suggest that cellular control of protein kinase C activity, through Sp1 and ARNT, could regulate AhR transcriptional activity in the absence of xenobiotics.

The antiproliferative activity of resveratrol results in apoptosis in MCF-7 but not in MDA-MB-231 human breast cancer cells: cell-specific alteration of the cell cycle.

Resveratrol, a natural phytoalexin, has gained much interest on the basis of its potential chemopreventive activity against human cancer. In this work, using the human breast cancer cell lines MCF-7 and MDA-MB-231, we have analyzed a possible mechanism by which resveratrol could interfere with cell cycle control and induce cell death. Our results show that although resveratrol inhibited cell proliferation and viability in both cell lines, apoptosis was induced in a concentration- and cell-specific manner. In MDA-MB-231, resveratrol (up to 200 microM) lowered the expression and kinase activities of positive G1/S and G2/M cell cycle regulators and inhibited ribonucleotide reductase activity in a concentration dependent manner, without a significant effect on the low expression of tumor suppressors p21, p27, and p53. These cells died by a non-apoptotic process in the absence of a significant change in cell cycle distribution. In MCF-7, resveratrol produced a significant and transient (<50 microM) increase in the expression and kinase activities of positive G1/S and G2/M regulators. Simultaneously, p21 expression was markedly induced in presence of high levels of p27 and p53. These opposing effects resulted in cell cycle blockade at the S-phase and apoptosis induction in MCF-7 cells. Thus, the antiproliferative activity of resveratrol could take place through the differential regulation of the cell cycle leading to apoptosis or necrosis. This could be influenced, among other factors, by the concentration of this molecule and by the characteristics of the target cell.