Targeting antitumoral proteins to breast cancer by local administration of functional inclusion bodies

Biofabrication; Cancer therapy; Functional amyloidsBiofabricación; Terapia contra el cáncer; Amiloides funcionalesBiofabricació; Teràpia contra el càncer; Amiloides funcionalsTwo structurally and functionally unrelated proteins, namely Omomyc and p31, are engineered as CD44-targeted inclusion bodies produced in recombinant bacteria. In this unusual particulate form, both types of protein materials selectively penetrate and kill CD44+ tumor cells in culture, and upon local administration, promote destruction of tumoral tissue in orthotropic mouse models of human breast cancer. These findings support the concept of bacterial inclusion bodies as versatile protein materials suitable for application in chronic diseases that, like cancer, can benefit from a local slow release of therapeutic proteins.This study has been supported by La Fundacio Marato TV3 and NanoCanTri (CIBER-BBN) to E.V. and I.A., and partially by ISCIII (PI15/00272 and PI1702242 co-founded by Fondo Europeo de Desarrollo Regional (FEDER), to E.V. and S.S., respectively), and Agencia Estatal de Investigacion (AEI) and FEDER (BIO2016-76063-R, AEI/FEDER, UE), AGAUR (2017SGR-229) and CIBER-BBN (VENOM4CANCER) granted to A.V. Protein production and DLS have been partially performed by the ICTS "NANBIOSIS," more specifically by the Protein Production Platform of CIBER-BBN/IBB () and the Biomaterial Processing and Nanostructuring Unit (), respectively. Biodistribution and immunohistochemistry assays were performed at the ICTS "NANBIOSIS," specifically by U20/FVPR (). L.S.-G. was supported by predoctoral fellowship from AGAUR (2018FI_B2_00051). L.S. was supported by the European Research Council (CoG #617473) and the Instituto de Salud Carlos III (FIS #PI16/01224). J.S.-F. was supported by an AECC post-doctoral fellowship. A.V. received an ICREA ACADEMIA awar

Targeting antitumoral proteins to breast cancer by local administration of functional inclusion bodies

Altres ajuts de l'Instituto de Salud Carlos III: PI15/00272, PI1702242FIS i #PI16/01224Two structurally and functionally unrelated proteins, namely Omomyc and p31, are engineered as CD44-targeted inclusion bodies produced in recombinant bacteria. In this unusual particulate form, both types of protein materials selectively penetrate and kill CD44 tumor cells in culture, and upon local administration, promote destruction of tumoral tissue in orthotropic mouse models of human breast cancer. These findings support the concept of bacterial inclusion bodies as versatile protein materials suitable for application in chronic diseases that, like cancer, can benefit from a local slow release of therapeutic protein

Study of innate immunity-related proteins for the treatment of bovine infectious diseases

Les malalties infeccioses (MIs) són un repte global que afecta tant a la salut humana com a la salut animal, especialment aquelles causades per microorganismes resistents als antibiòtics. En el cas concret de la indústria bovina, les MIs no només impacten sobre la salut i el benestar animal, sinó també redueixen de forma dràstica la producció i el marge econòmic del sector. L'ús dels antibiòtics permet el control de part de les MIs però l'emergència constant de bacteris resistents a aquests impulsa trobar noves estratègies per substituir als antibiòtics. Per altra banda, l'eficiència de les vacunes existents no és suficient per controlar l'emergència de les MIs fent palès que es necessiten teràpies alternatives pel tractament de les MIs bovines com son la mastitis o la síndrome respiratòria bovina (SRB). La immunitat innata ens proporciona un ampli assortiment de molècules amb un alt potencial terapèutic. Entre elles, les metal·loproteinases, les proteïnes de fase aguda o les citocines s'han estudiat en els últims anys amb resultats interessants. No obstant això, cap d'elles té la versatilitat funcional que tenen els Pèptids de Defensa de l'Hoste (HDPs) ja que tenen activitat antibacteriana, antiviral, antifúngica i immunomoduladora. Aquests pèptids són principalment produïts mitjançant síntesi química, però és un procés que té importants limitacions en termes de costos i escalabilitat que dificulta l'aplicació d'aquestes molècules in vivo. La producció de proteïnes recombinants en bacteris ha emergit com una estratègia ideal per obtenir proteïnes terapèutiques relacionades amb la immunitat innata a baix cost essent possible la seva producció a escala industrial. L'espècie bacteriana més àmpliament usada per produir proteïnes recombinants és Escherichia coli, però Lactococcus lactis també ha mostrat ser una bona alternativa, essent a més un microorganisme lliure de lipopolisacàrid (LPS). En primer lloc, aquesta tesi busca ampliar els sistemes lliures de LPS disponibles per a la producció de proteïnes recombinants relacionades amb la immunitat innata, explorant l'ús de Lactiplantibacillus plantarum. Els resultats mostren que L. plantarum pot ser usat com a fàbrica bacteriana igual que L. lactis, descrivint per primera vegada la formació de cossos d'inclusió (IBs) en aquest sistema d'expressió. En segon lloc, s'han descrit protocols adequats de solubilització per obtenir proteïnes relacionades amb la immunitat innata solubles, pures i actives a partir d'IBs. Finalment, aquesta tesi aconsegueix la producció de HDPs bovins recombinants en sistemes lliures de LPS, essent molècules amb una potent activitat antibacteriana i antiviral contra patògens bovins. En global els resultats d'aquesta tesi suposen l'inici prometedor d'un nou tractament pel SRB i una alternativa viable al ús dels antibiòtics.Las enfermedades infecciosas (EIs) son un desafío global que afecta tanto a la salud humana como a la salud animal, especialmente aquellas causadas por microorganismos resistentes a los antibióticos. En el caso concreto de la industria bovina, las EIs no solo impactan en la salud y el bienestar animal, sino que también reducen de forma drástica la producción y el margen económico del sector. El uso de antibióticos permite el control de parte de las EIs, pero la constante emergencia de bacterias resistentes a estos impulsa a encontrar nuevas estrategias para sustituirlos. Por otra parte, la eficacia de las vacunas existentes no es suficiente para controlar la emergencia de las EIs, lo que pone de manifiesto la necesidad de terapias alternativas para el tratamiento de las EIs bovinas, como la mastitis o el síndrome respiratorio bovino (SRB). La inmunidad innata nos proporciona un amplio surtido de moléculas con un alto potencial terapéutico. Entre ellas, las metaloproteinasas, las proteínas de fase aguda o las citocinas se han estudiado en los últimos años con resultados interesantes. Sin embargo, ninguna de ellas tiene la versatilidad funcional que tienen los Péptidos de Defensa del Huésped (HDPs), ya que poseen actividad antibacteriana, antiviral, antifúngica e inmunomoduladora. Estos péptidos son principalmente producidos mediante síntesis química, pero es un proceso que tiene importantes limitaciones en términos de costes y escalabilidad, lo que dificulta la aplicación de estas moléculas in vivo. La producción de proteínas recombinantes en bacterias ha surgido como una estrategia ideal para obtener proteínas terapéuticas relacionadas con la inmunidad innata a bajo coste, siendo posible su producción a escala industrial. La especie bacteriana más ampliamente utilizada para producir proteínas recombinantes es Escherichia coli, pero Lactococcus lactis también ha demostrado ser una buena alternativa, además de ser un microorganismo libre de lipopolisacáridos (LPS). En primer lugar, esta tesis busca ampliar los sistemas libres de LPS disponibles para la producción de proteínas recombinantes relacionadas con la inmunidad innata, explorando el uso de Lactiplantibacillus plantarum. Los resultados muestran que L. plantarum puede ser utilizado como fábrica bacteriana al igual que L. lactis, describiendo por primera vez la formación de cuerpos de inclusión (IBs) en este sistema de expresión. En segundo lugar, se han descrito protocolos adecuados de solubilización para obtener proteínas relacionadas con la inmunidad innata solubles, puras y activas a partir de IBs. Finalmente, esta tesis logra la producción de HDPs bovinos recombinantes en sistemas libres de LPS, siendo moléculas con una potente actividad antibacteriana y antiviral contra patógenos bovinos. En conjunto, los resultados de esta tesis suponen el inicio prometedor de un nuevo tratamiento para el SRB y una alternativa viable al uso de antibióticos.Infectious diseases (IDs) are a global challenge for human and animal health, particularly those caused by antibiotic-resistant microorganisms. In the specific case of the cattle industry, IDs not only impact on animal health and welfare, but also drastically reduce production and the economic margin of the sector. The use of antibiotics allows the control of part of IDs, but the constant emergence of resistant bacteria encourages finding new strategies to substitute antibiotics. On the other hand, the efficiency of existing vaccines is not sufficient to control the emergence of IDs, making it clear that alternative therapies are needed for the treatment of bovine IDs such as mastitis and bovine respiratory disease (BRD). Innate immunity provides us with an extensive assortment of molecules with high therapeutic potential. Among them, metalloproteinases, acute phase proteins, or cytokines have been studied in the last years with interesting results. Nevertheless, none of them have the functional versatility of Host Defense Peptides (HDPs), which have antibacterial, antiviral, antifungal and immunomodulatory activities. These peptides are mainly produced through chemical synthesis, which has important limitations in terms of cost and scalability to be applied in vivo. Recombinant protein production in bacteria has emerged as an ideal strategy for the obtainment of therapeutic innate immunity-related proteins at lower costs at industrial amounts. The bacterial specie most widely used for recombinant protein production purposes is Escherichia coli, but Lactococcus lactis has also shown to be a good alternative, being a lipopolysaccharide (LPS)-free system. First, this thesis pursues to expand the available systems for the LPS-free recombinant protein production of immune-related protein, exploring the use of Lactiplantibacillus plantarum. The results obtained showed that L. plantarum can be used as a microbial cell factory as it occurs with L. lactis, reporting for the first time the formation of inclusion bodies (IBs) in this expression system. Secondly, suitable solubilization protocols to obtain soluble, pure, and active immune-related proteins from IBs have been developed. Finally, this thesis attains the production of recombinant bovine HDPs in LPS-free recombinant systems which show potent antibacterial and antiviral activities against bovine pathogens. The outcomes derived from this thesis will be the beginning of a new treatment for BRD and a promising alternative to the use of antibiotics in animal production

Sequence edition of single domains modulates the final immune and antimicrobial potential of a new generation of multidomain recombinant proteins

Combining several innate immune peptides into a single recombinant antimicrobial and immunomodulatory polypeptide has been recently demonstrated. However, the versatility of the multidomain design, the role that each domain plays and how the sequence edition of the different domains affects their final protein activity is unknown. Parental multidomain antimicrobial and immunomodulatory protein JAMF1 and several protein variants (JAMF1.2, JAMF2 and AM2) have been designed and recombinantly produced to explore how the tuning of domain sequences affects their immunomodulatory potential in epithelial cells and their antimicrobial capacity against Gram-positive and Gram-negative bacteria. The replacement of the sequence of defensin HD5 and phospholipase sPLA2 by shorter active fragments of both peptides improves the final immunomodulatory (IL-8 secretion) and antimicrobial function of the multidomain protein against antimicrobial-resistant Klebsiella pneumoniae and Enterococcus spp. Further, the presence of Jun and Fos leucine zippers in multidomain proteins is crucial in preventing toxic effects on producer cells. The generation of antimicrobial proteins based on multidomain polypeptides allows specific immunomodulatory and antimicrobial functions, which can be easily edited by modifying of each domain sequence.info:eu-repo/semantics/publishedVersio

Targeting antitumoral proteins to breast cancer by local administration of functional inclusion bodies

Biofabrication; Cancer therapy; Functional amyloidsBiofabricación; Terapia contra el cáncer; Amiloides funcionalesBiofabricació; Teràpia contra el càncer; Amiloides funcionalsTwo structurally and functionally unrelated proteins, namely Omomyc and p31, are engineered as CD44-targeted inclusion bodies produced in recombinant bacteria. In this unusual particulate form, both types of protein materials selectively penetrate and kill CD44+ tumor cells in culture, and upon local administration, promote destruction of tumoral tissue in orthotropic mouse models of human breast cancer. These findings support the concept of bacterial inclusion bodies as versatile protein materials suitable for application in chronic diseases that, like cancer, can benefit from a local slow release of therapeutic proteins.This study has been supported by La Fundacio Marato TV3 and NanoCanTri (CIBER-BBN) to E.V. and I.A., and partially by ISCIII (PI15/00272 and PI1702242 co-founded by Fondo Europeo de Desarrollo Regional (FEDER), to E.V. and S.S., respectively), and Agencia Estatal de Investigacion (AEI) and FEDER (BIO2016-76063-R, AEI/FEDER, UE), AGAUR (2017SGR-229) and CIBER-BBN (VENOM4CANCER) granted to A.V. Protein production and DLS have been partially performed by the ICTS "NANBIOSIS," more specifically by the Protein Production Platform of CIBER-BBN/IBB () and the Biomaterial Processing and Nanostructuring Unit (), respectively. Biodistribution and immunohistochemistry assays were performed at the ICTS "NANBIOSIS," specifically by U20/FVPR (). L.S.-G. was supported by predoctoral fellowship from AGAUR (2018FI_B2_00051). L.S. was supported by the European Research Council (CoG #617473) and the Instituto de Salud Carlos III (FIS #PI16/01224). J.S.-F. was supported by an AECC post-doctoral fellowship. A.V. received an ICREA ACADEMIA awar

Targeting antitumoral proteins to breast cancer by local administration of functional inclusion bodies

Altres ajuts de l'Instituto de Salud Carlos III: PI15/00272, PI1702242FIS i #PI16/01224Two structurally and functionally unrelated proteins, namely Omomyc and p31, are engineered as CD44-targeted inclusion bodies produced in recombinant bacteria. In this unusual particulate form, both types of protein materials selectively penetrate and kill CD44 tumor cells in culture, and upon local administration, promote destruction of tumoral tissue in orthotropic mouse models of human breast cancer. These findings support the concept of bacterial inclusion bodies as versatile protein materials suitable for application in chronic diseases that, like cancer, can benefit from a local slow release of therapeutic protein