Abstract
HER-1 and HER-2 are tumor-associated antigens overexpressed in several epithelial tumors, and successfully targeted by therapeutic approaches against cancer. Vaccination with their ...recombinant extracellular domains has had encouraging results in the pre-clinical setting. As complex humoral responses targeting multiple epitopes within each antigen are the ultimate goal of such active immunotherapy strategies, molecular dissection of the mixture of antibody specificities is required. The current work exploits phage display of antigenic versions of HER-1 and HER-2 domains to accomplish domain-level epitope mapping. Recognition of domains I, III and IV of both antigens by antibodies of immunized mice was shown, indicating diverse responses covering a broad range of antigenic regions. The combination of phage display and site-directed mutagenesis allowed mutational screening of antigen surface, showing polyclonal antibodies’ recognition of mutated receptor escape variants known to arise in patients under the selective pressure of the anti-HER-1 antibody cetuximab. Phage-displayed HER domains have thus the potential to contribute to fine specificity characterization of humoral responses during future development of anti-cancer vaccines.
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•In cDCD livers, postmortem NRP reduces biliary complications, in particular ITBL.•Postmortem NRP helps improve cDCD liver graft survival.•Use of postmortem NRP facilitates successful ...transplantation of older cDCD livers.
Although there is increasing interest in its use, definitive evidence demonstrating a benefit for postmortem normothermic regional perfusion (NRP) in controlled donation after circulatory death (cDCD) liver transplantation is lacking. The aim of this study was to compare results of cDCD liver transplants performed with postmortem NRP vs. super-rapid recovery (SRR), the current standard for cDCD.
This was an observational cohort study including all cDCD liver transplants performed in Spain between June 2012 and December 2016, with follow-up ending in December 2017. Each donor hospital determined whether organ recovery was performed using NRP or SRR. The propensity scores technique based on the inverse probability of treatment weighting (IPTW) was used to balance covariates across study groups; logistic and Cox regression models were used for binary and time-to-event outcomes.
During the study period, there were 95 cDCD liver transplants performed with postmortem NRP and 117 with SRR. The median donor age was 56 years (interquartile range 45–65 years). After IPTW analysis, baseline covariates were balanced, with all absolute standardised differences <0.15. IPTW-adjusted risks were significantly improved among NRP livers for overall biliary complications (odds ratio 0.14; 95% CI 0.06–0.35, p <0.001), ischaemic type biliary lesions (odds ratio 0.11; 95% CI 0.02–0.57; p = 0.008), and graft loss (hazard ratio 0.39; 95% CI 0.20–0.78; p = 0.008).
The use of postmortem NRP in cDCD liver transplantation appears to reduce postoperative biliary complications, ischaemic type biliary lesions and graft loss, and allows for the transplantation of livers even from cDCD donors of advanced age.
This is a propensity-matched nationwide observational cohort study performed using livers recovered from donors undergoing cardiac arrest provoked by the intentional withdrawal of life support (controlled donation after circulatory death, cDCD). Approximately half of the livers were recovered after a period of postmortem in situ normothermic regional perfusion, which restored warm oxygenated blood to the abdominal organs, whereas the remainder were recovered after rapid preservation with a cold solution. The study results suggest that the use of postmortem normothermic regional perfusion helps reduce rates of post-transplant biliary complications and graft loss and allows for the successful transplantation of livers from older cDCD donors.
Proteolytic enzymes, also known as peptidases, are critical in all living organisms. Peptidases control the cleavage, activation, turnover, and synthesis of proteins and regulate many biochemical and ...physiological processes. They are also involved in several pathophysiological processes. Among peptidases, aminopeptidases catalyze the cleavage of the N-terminal amino acids of proteins or peptide substrates. They are distributed in many phyla and play critical roles in physiology and pathophysiology. Many of them are metallopeptidases belonging to the M1 and M17 families, among others. Some, such as M1 aminopeptidases N and A, thyrotropin-releasing hormone-degrading ectoenzyme, and M17 leucyl aminopeptidase, are targets for the development of therapeutic agents for human diseases, including cancer, hypertension, central nervous system disorders, inflammation, immune system disorders, skin pathologies, and infectious diseases, such as malaria. The relevance of aminopeptidases has driven the search and identification of potent and selective inhibitors as major tools to control proteolysis with an impact in biochemistry, biotechnology, and biomedicine. The present contribution focuses on marine invertebrate biodiversity as an important and promising source of inhibitors of metalloaminopeptidases from M1 and M17 families, with foreseen biomedical applications in human diseases. The results reviewed in the present contribution support and encourage further studies with inhibitors isolated from marine invertebrates in different biomedical models associated with the activity of these families of exopeptidases.
Ovarian cancer (OvCA) remains one of the most devastating malignancies, but treatment options are still limited. We report that amphiregulin (AREG) can serve as an effective and safe pharmacological ...target in a syngeneic murine model. AREG is highly abundant in abdominal fluids of patients with advanced OvCa. In immunocompetent animals, depletion or overexpression of AREG respectively prolonged or shortened animal survival. A new antibody we generated in AREG-knockout mice recognized murine AREG and reproducibly prolonged animal survival in the syngeneic model. The underlying mechanism likely involves binding of wildtype p53 to AREG's promoter and autocrine activation of the epidermal growth factor receptor (EGFR), a step blocked by the antibody. Accordingly, depletion of p53 downregulated AREG secretion and conferred tolerance, whereas blocking an adaptive process involving CXCL1, which transactivates EGFR, might increase therapeutic efficacy. Consistent with these observations, analysis of OvCa patients revealed that high AREG correlates with poor prognosis of patients expressing wildtype TP53. In conclusion, clinical tests of the novel antibody are warranted; high AREG, normal TP53, and reduced CXCL1 activity might identify patients with OvCa who may derive therapeutic benefit.
Defining how epidermal growth factor receptor (EGFR)-targeting therapies influence the immune response is essential to increase their clinical efficacy. A growing emphasis is being placed on immune ...regulator genes that govern tumor - T cell interactions. Previous studies showed an increase in HLA class I cell surface expression in tumor cell lines treated with anti-EGFR agents. In particular, earlier studies of the anti-EGFR blocking antibody cetuximab, have suggested that increased tumor expression of HLA class I is associated with positive clinical response. We investigated the effect of another commercially available anti-EGFR antibody nimotuzumab on HLA class I expression in tumor cell lines. We observed, for the first time, that nimotuzumab increases HLA class I expression and its effect is associated with a coordinated increase in mRNA levels of the principal antigen processing and presentation components. Moreover, using 7A7 (a specific surrogate antibody against murine EGFR), we obtained results suggesting the importance of the increased MHC-I expression induced by EGFR-targeted therapies display higher in antitumor immune response. 7A7 therapy induced upregulation of tumor MHC-I expression
and tumors treated with this antibody display higher susceptibility to CD8
T cells-mediated lysis. Our results represent the first evidence suggesting the importance of the adaptive immunity in nimotuzumab-mediated antitumor activity. More experiments should be conducted in order to elucidate the relevance of this mechanism in cancer patients. This novel immune-related antitumor mechanism mediated by nimotuzumab opens new perspectives for its combination with various immunotherapeutic agents and cancer vaccines.
Background
Antitumor therapies targeting HER1/EGFR and HER2, such as monoclonal antibodies (MAbs) and tyrosine-kinase inhibitors (TKIs), have demonstrated a significant clinical benefit, but the ...emergence of resistance limits long-term efficacy. While secondary HER1 mutations confer tolerance to TKI, compensatory upregulation of HER2 drives resistance to anti-HER1 MAbs, which identifies MAb combinations targeting both receptors as an attractive therapeutic strategy. Nevertheless, toxicity hampers the clinical validation of this approach. Alternatively, cancer vaccines may induce antibodies directed against several antigens with less concern about induced toxicity.
Methods
Polyclonal antibodies (PAbs) targeting HER1 and HER2 were induced in mice or rabbits through immunization. Recognition of different epitopes on targets by PAbs was validated by phage-display technology. Receptor downregulation was evaluated by flow cytometry, immunofluorescence, and Western blot. MTT assays assessed cytotoxicity, while the antitumor effect of PAbs was assayed in nude mice.
Results
PAbs promoted degradation of HER1 and HER2 regarding clinical MAbs or their combinations. As a result, inhibition of cytotoxicity on tumor cell lines was improved, even in the presence of oncogenic mutations in HER1, as well as in cetuximab-insensitive cells. Accordingly, the antitumor effect of vaccination-induced PAbs was observed in lung tumor lines representative of sensitivity or resistance to HER1 targeting therapies.
Conclusions
Immunization against HER1 and HER2 receptors offers an alternative to passive administration of combinations of MAbs, since vaccination-induced PAbs promote the downregulation of both receptors and they have a higher impact on the survival of tumor cells.
As a first step towards a vaccine protecting COVID-19 convalescents from reinfection, we evaluated FINLAY-FR-1A vaccine in a clinical trial.
Thirty COVID-19 convalescents aged 22-57 years were ...studied: convalescents of mild COVID-19, asymptomatic convalescents, both with PCR-positive at the moment of diagnosis; and individuals with subclinical infection detected by viral-specific IgG. They received a single intramuscular injection of the FINLAY-FR-1A vaccine (50 µg of the recombinant dimeric receptor binding domain). The primary outcomes were safety and reactogenicity, assessed over 28 days after vaccination. The secondary outcome was vaccine immunogenicity. Humoral response at baseline and following vaccination was evaluated by ELISA and live-virus neutralization test. The effector T cellular response was also assessed. Cuban Public Registry of Clinical Trials, WHO-ICTRP: https://rpcec.sld.cu/en/trials/RPCEC00000349-En.
No serious adverse events were reported. Minor adverse events were found, the most common, local pain: 3 (10%) and redness: 2 (6·7%). The vaccine elicited a >21 fold increase in IgG anti-RBD antibodies 28 days after vaccination. The median of inhibitory antibody titres (94·0%) was three times greater than that of the COVID-19 convalescent panel. Virus neutralization titres higher than 1:160 were found in 24 (80%) participants. There was also an increase in RBD-specific T cells producing IFN-γ and TNF-α.
A single dose of the FINLAY-FR-1A vaccine against SARS-CoV-2 was an efficient booster of pre-existing natural immunity, with excellent safety profile.
Partial funding for this study was received from the Project-2020-20, Fondo de Ciencia e Innovación (FONCI), Ministry of Science, Technology and the Environment, Cuba.
Las vacunas SOBERANA®02 y SOBERANA® Plus contra el coronavirus tipo 2 causante del síndrome respiratorio agudo severo, recibieron autorización de uso en emergencia por la autoridad reguladora de ...Cuba, y de inmediato aconteció una campaña de vacunación masiva en población pediátrica, lo que devino en una inminente movilización de centros de vacunación y vigilancia de sus eventos adversos. El Centro de Inmunología Molecular realizó un estudio de farmacovigilancia intensiva que evalúo el cumplimiento del esquema heterólogo con ambas vacunas, su seguridad y la incidencia de casos positivos a COVID-19 en niños y adolescentes después de completar el esquema de inmunización. Desde el 15 de septiembre al 31 de diciembre del 2021, participaron 529 sujetos entre 2 y 18 años de edad, de ambos sexos, sin antecedentes de infección por coronavirus tipo 2 del síndrome respiratorio agudo severo, procedentes de 35 municipios y 12 provincias cubanas, quienes recibieron vacuna SOBERANA®02 (dos dosis) y SOBERANA®Plus (una dosis). Se realizó vigilancia de eventos adversos hasta 30 días después de la última dosis recibida. Se consultó la plataforma informática nacional Higia Andariego para identificar los casos positivos al virus del síndrome respiratorio agudo severo coronavirus 2, hasta 3 meses de haber completado la vacunación. El 98,5% de los participantes completó el esquema de vacunación y en el 6,6% se notificó algún evento adverso con relación consistente a la vacunación. Predominaron las reacciones locales (dolor, eritema, inflamación), sobre las reacciones sistémicas (fatiga y febrícula), de intensidad ligera o moderada. Se logró un elevado cumplimiento del esquema de inmunización, con un perfil de seguridad favorable, los sujetos con esquema completo de inmunización no enfermaron de COVID-19.
RESUMEN Introducción: El cáncer constituye la segunda causa principal de muerte en nuestro país y en el mundo. Dentro de los blancos atractivos para el diseño de nuevos agentes anticancerígenos ...encontramos las aminopeptidasas. En particular, dentro de la familia M1, la aminopeptidasa neutra (APN, EC 3.4.11.2) y la aminopeptidasa ácida (APA, EC 3.4.11.7) y dentro de la familia S9 de las peptidasas serino, la enzima dipeptidil peptidasa IV (DPP-IV, EC 3.4.14.5) son blancos establecidos para el desarrollo de nuevos anticancerígenos debido a su sobreexpresión en diferentes tipos de cáncer. Teniendo en cuenta la relevancia biomédica de la inhibición de APN, APA y DPP-IV, el presente trabajo tiene como objetivo la identificación de nuevos inhibidores de estas enzimas con potenciales aplicaciones biomédicas. Métodos: Para la identificación de nuevos inhibidores de las enzimas en estudio se abordaron 2 estrategias de trabajo: a) identificación y caracterización bioquímica de nuevos inhibidores de APN, APA y DPP-IV de baja masa molecular para lo cual se realizaron cribados virtuales en la base de compuestos de baja masa molecular HitFinder, Maybrigde (ThermoFisher) y, b) caracterización bioquímica de la inhibición de APA, APA y DPP-IV por bestatina y bacitracina como nuevos inhibidores de estas enzimas. Posteriormente, se procedió a la validación cinética de los nuevos inhibidores identificados, se realizaron estudios de relación estructura: función in silico, se evaluó la biodistribución en modelo de rata así como se evaluaron sus efectos citotóxicos sobre líneas tumorales APN+/DPP-IV+. Resultados: Se identificaron y validaron cinéticamente 5 nuevos inhibidores duales de APN, APA y DPP-IV con mecanismos de inhibición de tipo no competitivo en la mayoría de los casos. Los estudios in silico de los complejos enzima: inhibidor permitieron la identificación de residuos no conservados dentro de la correspondiente familia y que sientan las bases para el diseño racional de inhibidores de APN, APA y DPP-IV altamente selectivos y con mayores potencialidades biomédicas. Se identificaron por primera vez la presencia de APN y DPP-IV en líneas tumorales de cáncer de alta incidencia y mortalidad en Cuba y el mundo, mediante una metodología establecida en el presente trabajo. Los nuevos inhibidores tienen efectos promisorios sobre estas líneas tumorales APN+/DPP-IV+. Como conclusiones, se informan por primera vez nuevos inhibidores de APN, APA y DPP-IV, con diferentes mecanismos de inhibición, algunos de ellos de tipo no competitivos, que resultan más ventajosos desde el punto de vista terapéutico que inhibidores competitivos, con efectos promisorios sobre líneas tumorales relacionadas con canceres de alta incidencia y mortalidad en Cuba.
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