The impact of co-expression of wild-type EGFR and its ligands determined by immunohistochemistry for response to treatment with cetuximab in patients with metastatic colorectal cancer

Anti-EGFR mAbs cetuximab and panitumumab are routinely used for the treatment of patients with KRAS-wild type metastatic colorectal cancer (mCRC). However, in some patients their efficacy remains modest and with no clear association between the EGFR protein expression determined by PharmDx™ kit, and response to anti-EGFR therapies. Therefore, we investigated the relative expression and predictive value of wild-type EGFR (wtEGFR), mutated EGFRvIII and EGFR ligand proteins in mCRC patients treated with cetuximab. The expression levels of wtEGFR, EGFRvIII, and EGFR ligand were determined by immunohistochemistry (IHC) in 60 tumour specimens using specific antibodies. Sections were scored according to the percentage of positive tumour cells, intensity and cellular location of staining, and these were associated with response, overall survival (OS) and progression-free survival (PFS). At cut-off value > 5%, wtEGFR, and EGFRvIII were present in 44%, and 41%, betacellulin (BTC) in 72%, followed by epigen (67%), TGFα (58%), amphiregulin (34%), EGF (31%) of the cases, respectively and 96% of the wtEGFR positive cases had co-expression of at least one ligand. We found a significant association between the expression of wtEGFR and poor response to cetuximab. In addition, the co-expression of wtEGFR with one ligand at a cut-off value of > 5% and > 10% was associated with worse response to cetuximab (P = 0.021, and P = 0.005 respectively). We found a 3-fold and 5-fold increased risk of shorter OS with expression of BTC and epigen. Interestingly, the expression of wtEGFR and its co-expression with one or two ligands was associated with shorter PFS but not with OS. The relative expression of wtEGFR and its competing ligands, which is the target for therapeutic interventions with anti-EGFR antibodies, could serve as a more reliable predictive biomarker of response to therapy with anti-EGFR mAbs in mCRC patients and warrants further investigation in large prospective studies

Developing a model of evacuation after an earthquake in Lebanon

This article describes the development of an agent-based model (AMEL, Agent-based Model for Earthquake evacuation in Lebanon) that aims at simulating the movement of pedestrians shortly after an earthquake. The GAMA platform was chosen to implement the model. AMEL is applied to a real case study, a district of the city of Beirut, Lebanon, which potentially could be stricken by a M7 earthquake. The objective of the model is to reproduce real life mobility behaviours that have been gathered through a survey in Beirut and to test different future scenarios, which may help the local authorities to target information campaigns.Comment: 8 pages, 11 figures, ISCRAM Vietnam Conference, November 201

Nuevas metodologías para la producción de anticuerpos recombinantes en plantas

Genetic engineering has allowed the design and production of recombinant antibodies (rmAbs) in plants. Nowadays, rmAbs are used in the treatment of a wide range of pathologies such as infectious diseases, inflammatory diseases and cancer, making rmAbs an important group of biomolecules within the pharmaceutical and biotechnology industry. By the time this study was started, the immunoglobulin G (IgG) was the antibody isotype predominantly expressed in plants. In recent years Modular DNA cloning technology has facilitated antibody engineering, with the development and expression of new rmAbs formats. However, there is hardly any study where different antibody formats are produced and compared in terms of yield and neutralizing capacity. Therefore, the starting point of the first chapter of this thesis is a comparative study where five different formats of the same commercial rmAb (Infliximab) against the human cytokine Tumor Necrosis Factor (TNF-¿) were expressed and compared. The results obtained in Chapter 1 demonstrate that both the isotype and the structure of the chosen rmAb influence the yield and the neutralizing capacity of rmAb. The expression of new antibody formats not only refers to the antibody isotype or structure; the format also refers to the combination of antibody idiotypes, leading to the production of oligo or polyclonal antibodies. Therefore, the possibility of co-expressing different monoclonal antibodies simultaneously in plants (creating oligoclonal or polyclonal formats) was raised. In the second chapter of this thesis, the expression of three rmAbs against the Ebola virus glycoprotein was studied. The three rmAbs were transiently expressed in N. benthamiana individually, by establishing separated production lines; in parallel, all three rmAbs were also co-expressed simultaneously in the same production line. The results obtained in this chapter demonstrated that the individual expression of rmAbs is feasible. However, when all three rmAbs are co-expressed, a drastic decrease in the binding of the antibody to the antigen was observed due to chain shuffling, as each heavy chain (HC) can be bound to any light chain (LC) other than its cognate chain, giving rise to an antibody cocktail with lower activity. With the objective of developing a method that allows co-expression of several rmAb in a single production line, we next proposed to exploit the viral interference phenomenon (also known as superinfection exclusion, SE). The results shown in Chapter three demonstrate that the production of an oligoclonal cocktail composed of 36 rmAbs in plants was possible using a viral expression system showing SE. The data obtained in this chapter showed that the resulting oligoclonal cocktail was active and capable of neutralizing toxic activities of the venom of the snake Bothrops asper in vitro and in vivo, wich was used as a model for studying the efficacy of the oligoclonal antibodies produced. The results of this thesis confirm and support the use of plants as platforms for the expression of alternative formats of antibodies.La ingeniería genética ha permitido el diseño y la producción de anticuerpos recombinantes (rmAbs) en plantas. Hoy en día, los rmAbs se utilizan en el tratamiento de un amplio rango de patologías como enfermedades infecciosas, enfermedades inflamatorias y cáncer, convirtiéndose en un importante grupo de biomoléculas dentro de la industria farmacéutica y biotecnológica. Hasta la fecha de este estudio, en plantas se ha producido mayoritariamente la inmunoglobulina del tipo G (IgG). Gracias al desarrollo de la ingeniería del ADN recombinante y de la ingeniería de anticuerpos, es posible diseñar y producir nuevos formatos de rmAbs. Sin embargo, apenas existen estudios comparativos donde se demuestre si el formato de anticuerpo elegido es el idóneo en términos de rendimiento y capacidad neutralizante. Por tanto, el punto de partida del primer Capítulo de esta tesis consistió en la realización de un estudio comparativo de la expresión en plantas de cinco formatos distintos de un mismo rmAb comercial (Infliximab) frente a la citoquina humana Tumor Necrosis Factor (TNF-¿). Los resultados obtenidos en el Capítulo 1 demuestran que tanto el isotipo como la estructura del rmAb elegido influye en los niveles de rendimiento y en la capacidad neutralizante del rmAb. La expresión de nuevos formatos de anticuerpos no solo afecta al isotipo o a la estructura de las regiones constantes, sino que también se puede incluir en este término la expresión conjunta de distintos idiotipos de anticuerpos recombinantes, dando lugar a anticuerpos policlonales u oligoclonales recombinantes. Por tanto en esta tesis se planteó la posibilidad de co-expresar simultáneamente distintos anticuerpos monoclonales en plantas formando un cóctel oligoclonal. En el segundo Capítulo de esta tesis se diseñaron tres rmAbs frente a la glicoproteína de la cubierta del virus del Ébola. Los tres rmAbs se expresaron transitoriamente en N. benthamiana de manera individual mediante el establecimiento de líneas paralelas de producción y también se co-expresaron los tres rmAbs simultáneamente en una misma línea de producción. Los resultados obtenidos en este Capítulo demostraron que la expresión de los rmAbs de manera individual es factible. Sin embargo, cuando se co-expresan los tres rmAbs se observa una drástica disminución en la unión del anticuerpo al antígeno debido al barajado de cadenas, fenómeno por el cual cada cadena pesada (HC) se puede unir con cualquier cadena ligera (LC) distinta de su acompañante, dando lugar a un anticuerpo con una baja actividad. Finalmente, con el objetivo de desarrollar un método que permita co-expresar en una misma línea de producción varios rmAbs de forma reproducible se propuso explotar el fenómeno de la exclusión viral, un característica propia de los virus de plantas. Los resultados mostrados en el Capítulo 3 demuestran que es posible la producción de un cóctel oligoclonal compuesto por 36 rmAbs en N. benthamiana aprovechando el fenómeno de la exclusión viral. Los datos obtenidos en este capítulo muestran que el cóctel oligoclonal producido de esta forma mantiene intactas las actividades de los anticuerpos individuales y es capaz de neutralizar las actividades tóxicas del veneno de la serpiente Bothrops asper en ensayos in vitro e in vivo. Los resultados de esta tesis confirman y avalan el uso de las plantas como plataformas de expresión de formatos alternativos de anticuerpos.El desenvolupament de l'enginyeria genètica ha permès el disseny i la producció d'anticossos recombinants (rmAbs) en plantes. Hui en dia, els rmAbs s'utilitzen en el tractament d'un ampli rang de patologies com malalties infeccioses, malalties inflamatòries i càncer convertint-se en un important grup de biomolècules dins de les indústries farmacèutiques i biotecnològiques. Fins a la data, s'han expressat majoritàriament la immunoglobulina del tipus G. Gràcies al desenvolupament de l'enginyeria de l'ADN recombinant i l'enginyeria dels anticossos s'han desenvolupat i expressat formats alternatius de rmAbs. Tanmateix, hi ha molts pocs estudis comparatius on es demostra si el format de l'anticòs elegit influeix en el rendiment i en la capacitat neutralitzant. Per tant, el punt de partida del primer Capítol d'esta Tesi és la realització d'un estudi comparatiu on s'expressen cinc formats diferents d'un mateix anticòs comercial (Infliximab) front a la citocina humana Tumor Necrosis Factor (TNF-¿). Els resultats obtesos demostren que tant l'isotip com l'estructura del rmAb elegit influeix en el rendiment i en la capacitat neutralitzant del rmAb. L'expressió de nous formats d'anticossos no sols afecta a l'isotip o a l'estructura del rmAb sinó que també pot incloure's dins d'aquest concepte l'expressió individual i l'expressió conjunta de diferents rmAbs. Partint d'aquesta hipòtesi, es va plantejar la possibilitat de co-expressar diferents rmAbs (còctel oligoclonal) en plantes. En el segon Capítol d'esta tesi es dissenyaren tres rmAbs front a la glicoproteïna del virus de l'Ébola. Els tres rmAbs s'expressaren transitòriament en N. benthamiana de manera individual mitjançant l'establiment de línies paral·leles de producció i també es co-expressaren els tres rmAbs en la mateixa línia de producció. Els resultats obtesos en este Capítol demostraren que l'expressió dels rmAbs de manera individual és factible. Tanmateix, quan es co-expressaren els tres rmAbs s'observà una dràstica disminució en la unió de l'anticòs a l'antigen com a conseqüència del shuffling chain, pel qual la cadena pesada (HC) s'uneix amb qualsevol cadena lleugera (LC) diferent a la seua acompanyant, formant un anticòs amb una baixa capacitat d'unió a l'antigen. Amb l'objectiu de desenvolupar un mètode que permeta co-expressar, en una mateixa línia de producció, un còctel oligoclonal es proposà explotar el fenomen de l'exclusió viral. Els resultats obtesos en el Capítol 3 demostren que l'expressió d' un còctel oligoclonal format per 36 rmAbs en plantes és possible. Els resultats mostren que el nostre còctel oligoclonal es capaç de neutralitzar activitats tòxiques del verí de la serp Bothrops asper en assaigs in vitro i in vivo. Els resultat obtesos en aquesta Tesi confirmen i avalen l'ús de les plantes com plataformes d'expressió de formats alternatius d'anticossos.Huet Trujillo, E. (2017). Nuevas metodologías para la producción de anticuerpos recombinantes en plantas [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90469TESI

Classification of current anticancer immunotherapies

© 2014. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.During the past decades, anticancer immunotherapy has evolved from a promising therapeutic option to a robust clinical reality. Many immunotherapeutic regimens are now approved by the US Food and Drug Administration and the European Medicines Agency for use in cancer patients, and many others are being investigated as standalone therapeutic interventions or combined with conventional treatments in clinical studies. Immunotherapies may be subdivided into "passive" and "active" based on their ability to engage the host immune system against cancer. Since the anticancer activity of most passive immunotherapeutics (including tumor-targeting monoclonal antibodies) also relies on the host immune system, this classification does not properly reflect the complexity of the drug-host-tumor interaction. Alternatively, anticancer immunotherapeutics can be classified according to their antigen specificity. While some immunotherapies specifically target one (or a few) defined tumor-associated antigen(s), others operate in a relatively non-specific manner and boost natural or therapy-elicited anticancer immune responses of unknown and often broad specificity. Here, we propose a critical, integrated classification of anticancer immunotherapies and discuss the clinical relevance of these approaches.info:eu-repo/semantics/publishedVersio

A glycosylated Fc-Fused GLP-1 Exhibits an Equivalent Glucose-Lowering Effect, but Lesser Gastrointestinal Side Effects than Dulaglutide

학위논문 (박사) -- 서울대학교 대학원 : 의과대학 임상의과학과, 2020. 8. 장학철.GLP-1 수용체 작용제 (GLP-1 RA)는 매력적인 항 당뇨병 치료제임에도 불구하고 주로 메스꺼움, 구토 및 복통과 같은 부작용에 따른 낮은 복약 순응도로 인해 제한된 치료 효과를 보여왔다. 본 시험에서는 새롭게 개발된 glycosylated Fc 융합 GLP-1 RA (GLP-1-gFc)의 효능 및 부작용을 dulaglutide와 비교 평가하였다. 기기 및 세포-기반 시험관내 분석을 통해 GLP-1-gFc가 dulaglutide보다 10 배 적은 결합 친화도 및 4 배 적은 역가를 가짐을 확인하였다. 역가를 고려한 용량 (dulaglutide 용량의 4 배)에서 GLP-1-gFc는 dulaglutide와 비슷한 혈당 저하 효과를 나타냈다. 그러나, dulaglutide와 동등한 효능을 보이는 용량 및 그보다 더 높은 용량에서 dulaglutide와는 다르게 쥐의 구역질 / 구토 반응 또는 원숭이의 QT 간격 변화를 유발하지 않았다. 이러한 경향은 건강한 피험자에 대한 임상 1상 시험에서 재확인되었다; 경구 포도당 내성 시험 연구에서 포도당 조절 효과는 위장 장애 및 맥박 변화의 정도보다 훨씬 더 극 적으로 나타났다. 이러한 결과들은 GLP-1-gFc이 시판 및 개발중인 높은 활성의 GLP-1 RA보다 더 나은 안전성을 가짐으로서 당뇨병 환자에 대한 치료 혜택을 극대화할 수 있는 새로운 GLP-1 RA로 사용될 수 있음을 시사한다.Despite their attractiveness as novel antidiabetic agents, GLP-1 receptor agonists (GLP-1 RAs) have provided limited therapeutic benefits due to common drug nonadherence, due mainly to side effects such as nausea, vomiting, and abdominal pain. Considering different GLP-1 receptor density throughout the organs, binding modulation to change receptor binding mechanism could be tried for the invention of novel GLP-1 RAs with better safety profile. I constructed a novel glycosylated Fc-fused GLP-1 RA (GLP-1-gFc) and determined binding affinity and potency using in-vitro instrumental and cell-based analyses followed by in-vivo comparison of glucose-lowering and side effects between GLP-1-gFc and dulaglutide. A Phase 1 clinical trial was conducted to confirm the efficacy and safety profile of GLP-1-gFc. GLP-1-gFc showed 10 times less binding affinity and 4 times less potency than dulaglutide in in-vitro. A potency-adjusted dose delayed HbA1c increase comparable to that of dulaglutide (Change % for 6 weeks: 2.4 mg/kg GLP-1-gFc, 4.34 ± 0.40 versus 0.6 mg/kg dulaglutide, 4.26 ± 0.22; n.s.). However, the equivalent efficacy dose and higher dose did not induce malaise-related responses (Blueberry bar consumption, g/mouse: 2.4 mg/kg GLP-1-gFc, 0.15 ± 0.03 versus 0.6 mg/kg dulaglutide, 0.04 ± 0.01; p<0.01) or QT interval changes (mean at 14h – 20h, mSc: 0.28 mg/kg GLP-1-gFc, 0.0 – 8.0 versus 0.07 mg/kg dulaglutide, 8.0 – 27.7; n.s.), observed as safety parameters in rats and monkeys, compared to those of dulaglutide. Glucose reductions in an oral glucose tolerance test were significant at day 3 post-dose without severe gastrointestinal adverse events and pulse rate changes in healthy subjects. These results suggest that GLP-1-gFc could be used as a novel GLP-1 RA with better safety than dulaglutide to maximize therapeutic benefits in subjects with type 2 diabetes.INTRODUCTI 1 RESEARCH DESIGN AND METHODS 5 RESULT 18 DISCUSSION 23 REFERENCES 28 FIGURES 35 TABLES 55 요약 (국문초록) 62Docto

Intrinsic NLRP3 inflammasome activity is critical for normal adaptive immunity via regulation of IFN-γ in CD4+ T cells

The NLRP3 inflammasome controls interleukin-1b maturation in antigen-presenting cells, but a direct role for NLRP3 in human adaptive immune cells has not been described.We found that the NLRP3 inflammasome assembles in human CD4+ Tcells and initiates caspase-1–dependent interleukin-1b secretion, thereby promoting interferon-g production and T helper 1 (TH1) differentiation in an autocrine fashion. NLRP3 assembly requires intracellular C5 activation and stimulation of C5a receptor 1 (C5aR1), which is negatively regulated by surface-expressed C5aR2. Aberrant NLRP3 activity in Tcells affects inflammatory responses in human autoinflammatory disease and in mouse models of inflammation and infection. Our results demonstrate that NLRP3 inflammasome activity is not confined to “innate immune cells” but is an integral component of normal adaptive TH1 responses

Development of bioanalytical devices for the detection of ciguatoxins and the ciguatoxin producing genera Gambierdiscus and Fukuyoa

La ciguatera (CFP) és una intoxicació alimentària que provoca símptomes gastrointestinals, cardíacs i neurològics que poden durar setmanes, mesos o fins i tot anys. És causada per la ingestió de peixos que contenen ciguatoxines (CTXs), compostos produïts per microalgues dels gèneres Gambierdiscus i Fukuyoa, les quals s'acumulen en els peixos i a través de les xarxes tròfiques. Aquesta tesi té com a objectiu proporcionar eines biotecnològiques per a la caracterització del risc de CFP i així garantir la seguretat alimentària i protegir la salut humana. Per aconseguir aquest objectiu, s'han desenvolupat tècniques d' extracció ràpida d'ADN i CTXs mitjançant l'ús de dispositius portàtils. A continuació, d'una banda, s'han utilitzat l'amplificació isotèrmica per polimerasa recombinasa (RPA) i la PCR amb cebadors amb cues per amplificar ADN dels gèneres Gambierdiscus i Fukuyoa i de les espècies G. australs and G. excentricus, i així detectar els productes de l'amplificació amb assajos i biosensors d'hibridació tipus sàndwich. Por una altra banda, s'han desenvolupat immunoassaigs i immunosensors a partir d'anticossos contra quatre CTXs pertanyents a dos grups de congèneres (CTX1B i CTX3C). Finalment, els sistemes desenvolupats s'han aplicat amb èxit a l'anàlisi de mostres naturals.La ciguatera (CFP) es una intoxicación alimentaria que provoca síntomas gastrointestinales, cardíacos y neurológicos que pueden durar semanas, meses o incluso años. Es causada por la ingestión de peces que contienen ciguatoxinas (CTXs), compuestos producidos por microalgas de los géneros Gambierdiscus y Fukuyoa, las cuales se acumulan en los peces y a través de las redes tróficas. Esta tesis tiene como objetivo proporcionar herramientas biotecnológicas para la caracterización del riesgo de CFP y así garantizar la seguridad alimentaria y proteger la salud humana. Para conseguir este objetivo, se han desarrollado técnicas de extracción rápida de ADN y CTXs mediante el uso de dispositivos portátiles. A continuación, por un lado, se han utilizado la amplificación isotérmica por polimerasa recombinasa (RPA) y la PCR con cebadores con colas para amplificar ADN de los géneros Gambierdiscus y Fukuyoa y de las especies G. australes and G. excentricus, y así detectar los productos de la amplificación con ensayos y biosensores de hibridación tipo sándwich. Por otro lado, se han desarrollado inmunoensayos e inmunosensores a partir de anticuerpos contra cuatro CTXs pertenecientes a dos grupos de congéneres (CTX1B y CTX3C). Por último, los sistemas desarrollados se han aplicado con éxito al análisis de muestras naturales.Ciguatera fish poisoning (CFP) is a disease that causes gastrointestinal, cardiological and neurological symptoms that can last weeks, months or even years. It is caused by the ingestion of fish containing ciguatoxins (CTXs), compounds produced by microalgae of the genera Gambierdiscus and Fukuyoa, which accumulate into fish flesh and through the food webs. This thesis aims at providing biotechnological tools for the characterization of the CFP risk in order to guarantee food safety and protect human health. To achieve this objective, fast extraction techniques for DNA and CTXs with the use of portable devices have been developed. Then, on the one hand, isothermal recombinase polymerase amplification (RPA) and PCR with tailed primers have been used to amplify DNA from the genera Gambierdiscus and Fukuyoa and from the species G. australes and G. excentricus, amplicons that were subsequently detected with sandwich hybridization assays and biosensors. On the other hand, antibodies that target four main CTXs belonging to two groups of congeners (CTX1B and CTX3C) have been used in the development of immunoassays and immunosensors. Finally, the developed systems were successfully applied to the analysis of natural samples

ESAT-6 targeting to DEC205+ antigen presenting cells induces specific-T cell responses against ESAT-6 and reduces pulmonary infection with virulent Mycobacterium tuberculosis

Airways infection with Mycobacterium tuberculosis (Mtb) is contained mostly by T cell responses, however, Mtb has developed evasion mechanisms which affect antigen presenting cell (APC) maturation/recruitment delaying the onset of Ag-specific T cell responses. Hypothetically, bypassing the natural infection routes by delivering antigens directly to APCs may overcome the pathogen\u27s naturally evolved evasion mechanisms, thus facilitating the induction of protective immune responses. We generated a murine monoclonal fusion antibody (α-DEC-ESAT) to deliver Early Secretory Antigen Target (ESAT)-6 directly to DEC205+ APCs and to assess its in vivo effects on protection associated responses (IFN-γ production, in vivo CTL killing, and pulmonary mycobacterial load). Treatment with α-DEC-ESAT alone induced ESAT-6-specific IFN-γ producing CD4+ T cells and prime-boost immunization prior to Mtb infection resulted in early influx (d14 post-infection) and increased IFN-γ+ production by specific T cells in the lungs, compared to scarce IFN-γ production in control mice. In vivo CTL killing was quantified in relevant tissues upon transferring target cells loaded with mycobacterial antigens. During infection, α-DEC-ESAT-treated mice showed increased target cell killing in the lungs, where histology revealed cellular infiltrate and considerably reduced bacterial burden. Targeting the mycobacterial antigen ESAT-6 to DEC205+ APCs before infection expands specific T cell clones responsible for early T cell responses (IFN-γ production and CTL activity) and substantially reduces lung bacterial burden. Delivering mycobacterial antigens directly to APCs provides a unique approach to study in vivo the role of APCs and specific T cell responses to assess their potential anti-mycobacterial functions