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Patients with advanced endometrial cancer (EC) show poor outcomes. Thus, the development of new therapeutic approaches to prevent metastasis development in high-risk patients is an unmet need. CXCR4 is overexpressed in EC tumor tissue, epitomizing an unexploited therapeutic target for this malignancy. The in vitro antitumor activity of two CXCR4-targeted nanoparticles, including either the C. diphtheriae (T22-DITOX-H6) or P. aeruginosa (T22-PE24-H6) toxin, was evaluated using viability assays. Apoptotic activation was assessed by DAPI and caspase-3 and PARP cleavage in cell blocks. Both nanotoxins were repeatedly administrated to a subcutaneous EC mouse model, whereas T22-DITOX-H6 was also used in a highly metastatic EC orthotopic model. Tumor burden was assessed through bioluminescence, while metastatic foci and toxicity were studied using histological or immunohistochemical analysis. We found that both nanotoxins exerted a potent antitumor effect both in vitro and in vivo via apoptosis and extended the survival of nanotoxin-treated mice without inducing any off-target toxicity. Repeated T22-DITOX-H6 administration in the metastatic model induced a dramatic reduction in tumor burden while significantly blocking peritoneal, lung and liver metastasis without systemic toxicity. Both nanotoxins, but especially T22-DITOX-H6, represent a promising therapeutic alternative for EC patients that have a dismal prognosis and lack effective therapies.

Through the controlled addition of divalent cations, polyhistidine-tagged proteins can be clustered in form of chemically pure and mechanically stable micron-scale particles. Under physiological conditions, these materials act as self-disintegrating protein depots for the progressive release of the forming polypeptide, with potential applications in protein drug delivery, diagnosis, or theragnosis. Here we have explored the in vivo disintegration pattern of a set of such depots, upon subcutaneous administration in mice. These microparticles were fabricated with cationic forms of either Zn, Ca, Mg, or Mn, which abound in the mammalian body. By using a CXCR4-targeted fluorescent protein as a reporter building block we categorized those cations regarding their ability to persist in the administration site and to sustain a slow release of functional protein. Ca2+ and specially Zn2+ have been observed as particularly good promoters of time-prolonged protein leakage. The released polypeptides result is available for selective molecular interactions, such as specific fluorescent labeling of tumor tissues, in which the protein reaches nearly steady levels.

High rates of relapsed and refractory diffuse large B-cell lymphoma (DLBCL) patients and life-threatening side effects associated with immunochemotherapy make an urgent need to develop new therapies for DLBCL patients. Immunotoxins seem very potent anticancer therapies but their use is limited because of their high toxicity. Accordingly, the self-assembling polypeptidic nanoparticle, T22-DITOX-H6, incorporating the diphtheria toxin and targeted to CXCR4 receptor, which is overexpressed in DLBCL cells, could offer a new strategy to selectively eliminate CXCR4

Current therapy in acute myeloid leukemia (AML) is based on chemotherapeutic drugs administered at high doses, lacking targeting selectivity and displaying poor therapeutic index because of severe adverse effects. Here, we develop a novel nanoconjugate that combines a self-assembled, multivalent protein nanoparticle, targeting the CXCR4 receptor, with an Oligo-Ara-C prodrug, a pentameric form of Ara-C, to highly increase the delivered payload to target cells. This 13.4 nm T22-GFP-H6-Ara-C nanoconjugate selectively eliminates CXCR4+ AML cells, which are protected by its anchoring to the bone marrow (BM) niche, being involved in AML progression and chemotherapy resistance. This nanoconjugate shows CXCR4-dependent internalization and antineoplastic activity in CXCR4+ AML cells in vitro. Moreover, repeated T22-GFP-H6-Ara-C administration selectively eliminates CXCR4+ leukemic cells in BM, spleen and liver. The leukemic dissemination blockage induced by T22-GFP-H6-Ara-C is significantly more potent than buffer or Oligo-Ara-C-treated mice, showing no associated on-target or off-target toxicity and, therefore, reaching a highly therapeutic window. In conclusion, T22-GFP-H6-Ara-C exploits its 11 ligands-multivalency to enhance target selectivity, while the Oligo-Ara-C prodrug multimeric form increases 5-fold its payload. This feature combination offers an alternative nanomedicine with higher activity and greater tolerability than current intensive or non-intensive chemotherapy for AML patients., This work was supported by Instituto de Salud Carlos III (ISCIII, Co-funding from FEDER) [PI18/00650, PIE15/00028, PI15/00378 and EU COST Action CA 17 140 to R.M.; FIS PI17/01246 and RD16/0011/0028 to J.S.; and PI20/00400 to U·U.]; Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) (grant BIO2016-76063-R, AEI/FEDER, UE) to A.V.; (grant PID2019-105416RB-I00) to E.V.; CIBER-BBN [CB06/01/1031 and 4NanoMets to R.M., VENOM4CANCER to A.V., NANOREMOTE to E.V. and NANOLINK to U·U.]; AGAUR [2017 FI_B 00680 to A.F., 2018 FI_B2_00051 to L.S.G.; 2017-SGR-865 to R.M., 2017-SGR-1395 to J.S. and 2017SGR-229 to A.V.]; Josep Carreras Leukemia Research Institute [P/AG to R.M.]; La Marató TV3 [201 941-30-31-32 to J.S. and A.V.]; a grant from the Cellex Foundation, Barcelona [to J.S.]; a grant from La Generalitat de Catalunya (PERIS) [SLT002/16/00433to J.S.]; a grant from the Generalitat de Catalunya CERCA Programme. U.U. is supported by Miguel Servet fellowship (CP19/00028) from Instituto de Salud Carlos III co-funded by Fondo Social Europeo (ESF investing in your future). Finally, A.V. received an ICREA ACADEMIA Award supported by the Catalan Government. We are also indebted to the ISCIII Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) and its ICTS Nanbiosis Platform for their funding. The bioluminescent follow-up of cancer cells and toxicity studies has been performed in the ICTS-141007 Nanbiosis Platform, using its CIBER-BBN Nanotoxicology Unit (http://www.nanbiosis.es/portfolio/u18-nanotoxicology-unit/). Protein production has been partially performed by the ICTS “NANBIOSIS”, more specifically by the Protein Production Platform of CIBER-BBN/IBB (http://www.nanbiosis.es/unit/u1-protein-production-platform-ppp/). Finally, we are very grateful to Servei de Microscopia from UAB for their excellent confocal and electron microscopy services and especially to Alejandro Sánchez-Chardi for its excellent support and contribution in TEM and FESEM images.

Loco-regional recurrences and distant metastases represent the main cause of head and neck squamous cell carcinoma (HNSCC) mortality. The overexpression of chemokine receptor 4 (CXCR4) in HNSCC primary tumors associates with higher risk of developing loco-regional recurrences and distant metastases, thus making CXCR4 an ideal entry pathway for targeted drug delivery. In this context, our group has generated the self-assembling protein nanocarrier T22-GFP-H6, displaying multiple T22 peptidic ligands that specifically target CXCR4. This study aimed to validate T22-GFP-H6 as a suitable nanocarrier to selectively deliver cytotoxic agents to CXCR4

Simple Summary Aimed at minimizing side toxicities cancer therapies require appropriate functional vehicles at the nanoscale, for receptor-mediated tumor-targeted drug delivery. The aim of the present study was to explore the human peptide EPI-X4 as a CXCR4-targeting agent in self-assembled, protein-only nanoparticles. While the systemic tumor biodistribution of EPI-X4-based materials is modest, this peptide shows potent proapoptotic effects on CXCR4+ cancer cells. Interestingly, the in vivo selectivity of EPI-X4 was dramatically improved, once combined into biparatopic nanoparticles, with a second CXCR4 ligand, the peptide T22. Biparatopic nanoparticles promote a highly selective tumor destruction in a mouse model of human colorectal cancer, probably associated to the CXCR4 antagonist role of EPI-X4. This study not only validates a new human ligand of the tumoral marker CXCR4, but it also offers a novel strategy for the combination, in protein nanoparticles, of dual acting ligands of tumoral markers for highly selective drug delivery. Abstract The accumulated molecular knowledge about human cancer enables the identification of multiple cell surface markers as highly specific therapeutic targets. A proper tumor targeting could significantly avoid drug exposure of healthy cells, minimizing side effects, but it is also expected to increase the therapeutic index. Specifically, colorectal cancer has a particularly poor prognosis in late stages, being drug targeting an appropriate strategy to substantially improve the therapeutic efficacy. In this study, we have explored the potential of the human albumin-derived peptide, EPI-X4, as a suitable ligand to target colorectal cancer via the cell surface protein CXCR4, a chemokine receptor overexpressed in cancer stem cells. To explore the potential use of this ligand, self-assembling protein nanoparticles have been generated displaying an engineered EPI-X4 version, which conferred a modest CXCR4 targeting and fast and high level of cell apoptosis in tumor CXCR4+ cells, in vitro and in vivo. In addition, when EPI-X4-based building blocks are combined with biologically inert polypeptides containing the CXCR4 ligand T22, the resulting biparatopic nanoparticles show a dramatically improved biodistribution in mouse models of CXCR4+ human cancer, faster cell internalization and enhanced target cell death when compared to the version based on a single ligand. The generation of biparatopic materials opens exciting possibilities in oncotherapies based on high precision drug delivery based on the receptor CXCR4.

Green fluorescent protein (GFP) is a widely used scaffold for protein-based targeted nanomedicines because of its high biocompatibility, biological neutrality and outstanding structural stability. However, being immunogenicity a major concern in the development of drug carriers, the use of exogenous proteins such as GFP in clinics might be inadequate. Here we report a human nidogen-derived protein (HSNBT), rationally designed to mimic the structural and functional properties of GFP as a scaffold for nanomedicine. For that, a GFP-like β-barrel, containing the G2 domain of the human nidogen, has been rationally engineered to obtain a biologically neutral protein that self-assembles as 10nm-nanoparticles. This scaffold is the basis of a humanized nanoconjugate, where GFP, from the well-characterized protein T22-GFP-H6, has been substituted by the nidogen-derived GFP-like HSNBT protein. The resulting construct T22-HSNBT-H6, is a humanized CXCR4-targeted nanoparticle that selectively delivers conjugated genotoxic Floxuridine into cancer CXCR4+ cells. Indeed, the administration of T22-HSNBT-H6-FdU in a CXCR4-overexpressing colorectal cancer mouse model results in an even more efficient selective antitumoral effect than that shown by its GFP-counterpart, in absence of systemic toxicity. Therefore, the newly developed GFP-like protein scaffold appears as an ideal candidate for the development of humanized protein nanomaterials and successfully supports the tumor-targeted nanoscale drug T22-HSNBT-H6-FdU., Patricia Álamo and Juan Cedano contributed equally to this work. The authors are indebted to Agencia Estatal de Investigación (AEI) and to Fondo Europeo de Desarrollo Regional (FEDER) (grant BIO2016-76063-R, AEI/FEDER, UE), to AGAUR (2017SGR-229) and CIBER-BBN (project NANOPROTHER), granted to AV, to CIBER-BBN (project NANOSCAPE and NANOLINK) and ISCIII (PI20/00400 co-funding FEDER) granted to UU, to ISCIII (PI15/00272 co-founding FEDER) granted to EV and to ISCIII (PIE15/00028 and PI18/00650, co-funding FEDER) and AGAUR (2017 SGR 865 GRC) granted to RM. We are also indebted to CERCA programme (Generalitat de Catalunya) and to the Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) that is an initiative funded by the VI National R&D&I Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III, with assistance from the European Regional Development Fund. We also appreciate the support from the COST-Action Nano2Clinics. Protein production has been partially performed by the ICTS “NANBIOSIS”, more specifically by the Protein Production Platform of CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN)/ IBB, at the UAB sePBioEs scientific-technical service (http://www.nanbiosis.es/portfolio/u1-protein-production-platform-ppp/), and the nanoparticle size analysis by the Biomaterial Processing and Nanostructuring Unit. Synthesis of thiolated oligo-FdU was performed by the ICTS NANBIOSIS Oligonucleotide Synthesis Platform (CIBER-BBN). The in vivo work was performed by the ICTS NANBIOSIS of the CIBER-BBN Nanotoxicology Unit (http://www.nanbiosis.es/portfolio/u18-nanotoxicology-unit/). We are indebted to Servei de Microscopia from UAB for their excellent confocal and electronic microscopy services. We are also indebted to Servei de Cultius Celulars i Anticossos (SCAC) form UAB for their excellent cell culture and flow cytometry facilities and especially to Fran Cortes for his excellent technical support. We are thankful to Dra. Marta Taulés from CCiT-UB for her help in SPR experiments and analysis. We are also thankful to Luis Carlos Navas from Institut d'Investigacions biomèdiques Sant Pau (IIB Sant Pau) for his technical support in immunohistochemistry experiments. UU and LMCD were supported by Miguel Servet (CP19/00028) and PFIS (FI19/00148) contracts respectively from ISCIII co-funded by European Social Fund (ESF investing in your future). NS was supported by a pre-doctoral fellowship from the Government of Navarra and LAC was supported by AECC Scientific Foundation grant postdoctoral fellow. AV received an ICREA ACADEMIA award.

Aïda Falgàs,1– 3 Victor Pallarès,1– 3 Ugutz Unzueta,1– 4 Yáiza Núñez,1,2 Jorge Sierra,2,5 Alberto Gallardo,1 Lorena Alba-Castellón,1,2 Maria Antonia Mangues,3,6 Patricia Álamo,1– 3 Antonio Villaverde,3,4,7 Esther Vázquez,3,4,7 Ramon Mangues,1– 3 Isolda Casanova1– 3 1Biomedical Research Institute Sant Pau (IIB-Sant Pau), Hospital de la Santa Creu i Sant Pau, Barcelona, 08025, Spain; 2Josep Carreras Leukaemia Research Institute (IJC), Barcelona, 08916, Spain; 3CIBER de Bioingeniería Biomateriales y Nanomedicina (CIBER-BBN), Madrid, 28029, Spain; 4Department of Genetics and Microbiology, Universitat Autònoma de Barcelona, Barcelona, 08193, Spain; 5Department of Hematology, Hospital de la Santa Creu i Sant Pau, Barcelona, 08025, Spain; 6Department of Pharmacy, Hospital de la Santa Creu i Sant Pau, Barcelona, 08025, Spain; 7Institute of Biotechnology and Biomedicine (IBB), Universitat Autònoma de Barcelona, Barcelona, 08193, SpainCorrespondence: Esther VázquezInstitute of Biotechnology and Biomedicine (IBB) and Department of Genetics and Microbiology, Universitat Autònoma de Barcelona and CIBER-BBN, Barcelona, SpainTel +34 935812148Email Esther.Vazquez@uab.esRamon ManguesBiomedical Research Institute Sant Pau (IIB-Sant Pau), Josep Carreras Leukaemia Research Institute (IJC) and CIBER-BBN, Barcelona, SpainTel +34 935537918Email RMangues@santpau.catBackground and Purpose: Around 40– 50% of diffuse large-B cell lymphoma (DLBCL) patients suffer from refractory disease or relapse after R-CHOP first-line treatment. Many ongoing clinical trials for DLBCL patients involve microtubule targeting agents (MTAs), however, their anticancer activity is limited by severe side effects. Therefore, we chose to improve the therapeutic window of the MTA monomethyl auristatin E developing a nanoconjugate, T22-AUR, that selectively targets the CXCR4 receptor, which is overexpressed in many DLBCL cells (CXCR4+) and associated with poor prognosis.Methods: The T22-AUR specificity towards CXCR4 receptor was performed by flow cytometry in different DLBCL cell lines and running biodistribution assays in a subcutaneous mouse model bearing CXCR4+ DLBCL cells. Moreover, we determined T22-AUR cytotoxicity using cell viability assays, cell cycle analysis, DAPI staining and immunohistochemistry. Finally, the T22-AUR antineoplastic effect was evaluated in vivo in an extranodal CXCR4+ DLBCL mouse model whereas the toxicity analysis was assessed by histopathology in non-infiltrated mouse organs and by in vitro cytotoxic assays in human PBMCs.Results: We demonstrate that the T22-AUR nanoconjugate displays CXCR4-dependent targeting and internalization in CXCR4+ DLBCL cells in vitro as well as in a subcutaneous DLBCL mouse model. Moreover, it shows high cytotoxic effect in CXCR4+ DLBCL cells, including induction of G2/M mitotic arrest, DNA damage, mitotic catastrophe and apoptosis. Furthermore, the nanoconjugate shows a potent reduction in lymphoma mouse dissemination without histopathological alterations in non-DLBCL infiltrated organs. Importantly, T22-AUR also exhibits lack of toxicity in human PBMCs.Conclusion: T22-AUR exerts in vitro and in vivo anticancer effect on CXCR4+ DLBCL cells without off-target toxicity. Thus, T22-AUR promises to become an effective therapy for CXCR4+ DLBCL patients.Keywords: nanomedicine, targeted drug delivery, MMAE, DLBCL

Nanomedicine has opened an opportunity to improve current clinical practice by enhancing the selectivity in the delivery of antitumor drugs to specific cancer cells. These new strategies are able to bypass toxicity on normal cells increasing the effectiveness of current anticancer treatments. In acute myeloid leukemia (AML) current chemotherapy treatments generate a relevant toxic impact in normal cells and severe side effects or even patient death. In this study, we have designed a self-assembling protein nanoparticle, T22-DITOX-H6, which incorporates a ligand (T22) targeting CXCR4-overexpressing (CXCR4+) cells, and a potent cytotoxic diphtheria toxin domain. CXCR4 is overexpressed in AML leukemic cells and associates with poor prognosis, being, therefore, a relevant clinical target. We demonstrate here that T22-DITOX-H6 induces apoptosis in CXCR4+ leukemic cells through CXCR4-dependent internalization. In addition, repeated T22-DITOX-H6 treatment (10 μg/dose per 10 doses, intravenously injected) in a disseminated AML mouse model (NSG mice intravenously injected with THP-1-Luci cells, n = 10 per group) potently blocks the dissemination of AML cells in bone marrow, spleen and liver of treated mice, without inducing toxicity in healthy tissues. In conclusion, our strategy of selectively ablating CXCR4 positive leukemic cells by administering the T22-DITOX-H6 nanoparticle could be a promising treatment, especially in patients undergoing AML relapse after chemotherapy, in which leukemic cells overexpress CXCR4.

Fluorescent dye labeling is a common strategy to analyze the fate of administered nanoparticles in living organisms. However, to which extent the labeling processes can alter the original nanoparticle biodistribution has been so far neglected. In this work, two widely used fluorescent dye molecules, namely, ATTO488 (ATTO) and Sulfo‐Cy5 (S‐Cy5), have been covalently attached to a well‐characterized CXCR4‐targeted self‐assembling protein nanoparticle (known as T22‐GFP‐H6). The biodistribution of labeled T22‐GFP‐H6‐ATTO and T22‐GFP‐H6‐S‐Cy5 nanoparticles has been then compared to that of the non‐labeled nanoparticle in different CXCR4+ tumor mouse models. We observed that while parental T22‐GFP‐H6 nanoparticles accumulated mostly and specifically in CXCR4+ tumor cells, labeled T22‐GFP‐H6‐ATTO and T22‐GFP‐H6‐S‐Cy5 nanoparticles showed a dramatic change in the biodistribution pattern, accumulating in non‐target organs such as liver or kidney while reducing tumor targeting capacity. Therefore, the use of such labeling molecules should be avoided in target and non‐target tissue uptake studies during the design and development of targeted nanoscale drug delivery systems, since their effect over the fate of the nanomaterial can lead to considerable miss‐interpretations of the actual nanoparticle biodistribution. Fil: Álamo, Patricia. Centro de Investigacion Biomedica En Red.; España. Biomedical Research Institute Sant Pau; España Fil: Pallarès, Victor. Centro de Investigacion Biomedica En Red.; España. Biomedical Research Institute Sant Pau; España Fil: Céspedes, María Virtudes. Centro de Investigacion Biomedica En Red.; España. Biomedical Research Institute Sant Pau; España Fil: Falgàs, Aïda. Centro de Investigacion Biomedica En Red.; España. Biomedical Research Institute Sant Pau; España Fil: Sanchez, Julieta Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina. Universitat Autònoma de Barcelona; España. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Ciencias y Tecnología de los Alimentos; Argentina Fil: Serna, Naroa. Universitat Autònoma de Barcelona; España. Centro de Investigacion Biomedica En Red.; España Fil: Sánchez-García, Laura. Universitat Autònoma de Barcelona; España. Centro de Investigacion Biomedica En Red.; España Fil: Voltà-Duràn, Eric. Universitat Autònoma de Barcelona; España. Centro de Investigacion Biomedica En Red.; España Fil: Morris, Gordon A.. University of Huddersfiel; Reino Unido Fil: Sánchez-Chardi, Alejandro. Universitat Autònoma de Barcelona; España Fil: Casanova, Isolda. Biomedical Research Institute Sant Pau; España. Centro de Investigacion Biomedica En Red.; España Fil: Mangues, Ramón. Biomedical Research Institute Sant Pau; España. Centro de Investigacion Biomedica En Red.; España Fil: Vazquez, Esther. Universitat Autònoma de Barcelona; España. Centro de Investigacion Biomedica En Red.; España Fil: Villaverde Corrales, Antonio. Universitat Autònoma de Barcelona; España. Centro de Investigacion Biomedica En Red.; España Fil: Unzueta, Ugutz. Universitat Autònoma de Barcelona; España. Centro de Investigacion Biomedica En Red.; España

Background and Purpose Around 40–50% of diffuse large-B cell lymphoma (DLBCL) patients suffer from refractory disease or relapse after R-CHOP first-line treatment. Many ongoing clinical trials for DLBCL patients involve microtubule targeting agents (MTAs), however, their anticancer activity is limited by severe side effects. Therefore, we chose to improve the therapeutic window of the MTA monomethyl auristatin E developing a nanoconjugate, T22-AUR, that selectively targets the CXCR4 receptor, which is overexpressed in many DLBCL cells (CXCR4+) and associated with poor prognosis. Methods The T22-AUR specificity towards CXCR4 receptor was performed by flow cytometry in different DLBCL cell lines and running biodistribution assays in a subcutaneous mouse model bearing CXCR4+ DLBCL cells. Moreover, we determined T22-AUR cytotoxicity using cell viability assays, cell cycle analysis, DAPI staining and immunohistochemistry. Finally, the T22-AUR antineoplastic effect was evaluated in vivo in an extranodal CXCR4+ DLBCL mouse model whereas the toxicity analysis was assessed by histopathology in non-infiltrated mouse organs and by in vitro cytotoxic assays in human PBMCs. Results We demonstrate that the T22-AUR nanoconjugate displays CXCR4-dependent targeting and internalization in CXCR4+ DLBCL cells in vitro as well as in a subcutaneous DLBCL mouse model. Moreover, it shows high cytotoxic effect in CXCR4+ DLBCL cells, including induction of G2/M mitotic arrest, DNA damage, mitotic catastrophe and apoptosis. Furthermore, the nanoconjugate shows a potent reduction in lymphoma mouse dissemination without histopathological alterations in non-DLBCL infiltrated organs. Importantly, T22-AUR also exhibits lack of toxicity in human PBMCs. Conclusion T22-AUR exerts in vitro and in vivo anticancer effect on CXCR4+ DLBCL cells without off-target toxicity. Thus, T22-AUR promises to become an effective therapy for CXCR4+ DLBCL patients.

Current therapies fail to eradicate colorectal Cancer Stem Cells (CSCs). One of the proposed reasons for this failure is the selection, by chemotherapy exposure, of resistant cells responsible for tumor recurrence. In this regard, CXCR4 overexpression in tumor associates with resistance and poor prognosis in colorectal cancer (CRC) patients. In this study, the effectiveness of engineered CXCR4-targeted self-assembling toxin nanoparticles has been explored in the selective killing of CXCR4(+) human colon-CSCs compared to 5-Fluorouracil and Oxaliplatin, both classical CRC chemotherapeutic agents. To assess this, 3D spheroid colon-CSCs cultures directly derived from CRC patients and CRC-CSC spheroid-derived tumor mouse models were developed. In these animal models, nanostructured toxins show highly selective induction of pyroptosis in the absence of apoptosis, thus having a great potential to overcome tumor resistance, since the same tumor models show resistance to chemotherapeutics. Results set the basis for further development of more efficient therapies focused on selective CXCR4+ CSCs elimination activating non-apoptotic mechanisms and represent a pre-clinical proof of concept for the use of CSCs-targeted nanostructured toxins as protein drugs for CRC therapy.

Altres ajuts: Fondo Europeo de Desarrollo Regional (FEDER) (grant PID2019-105416RB-I00 to EV. U COST Action CA 17140. AV received an ICREA ACADEMIA award. Miguel Servet (CP19/00028) A functional 29 amino acid-segment of the helix α5 from the human BAX protein has been engineered for production in recombinant bacteria as self-assembling, GFP-containing fluorescent nanoparticles, which are targeted to the tumoral marker CXCR4. These nanoparticles, of around 34 nm in diameter, show a moderate tumor biodistribution and limited antitumoral effect when systemically administered to mouse models of human CXCR4 colorectal cancer (at 300 μg dose). However, if such BAX nanoparticles are co-administered in cocktail with equivalent nanoparticulate versions of BAK and PUMA proteins at the same total protein dose (300 μg), protein biodistribution and stability in tumor is largely improved, as determined by fluorescence profiles. This fact leads to a potent and faster destruction of tumor tissues when compared to individual pro-apoptotic factors. The analysis and interpretation of the boosted effect, from both the structural and functional sides, offers clues for the design of more efficient nanomedicines and theragnostic agents in oncology based on precise cocktails of human proteins. Statement of significance: Several human pro-apoptotic peptides (namely BAK, BAX and PUMA) have been engineered as self-assembling protein nanoparticles targeted to the tumoral marker CXCR4. The systemic administration of the same final amounts of those materials as single drugs, or as combinations of two or three of them, shows disparate intensities of antitumoral effects in a mouse model of human colorectal cancer, which are boosted in the triple combination on a non-additive basis. The superiority of the combined administration of pro-apoptotic agents, acting at different levels of the apoptotic cascade, opens a plethora of possibilities for the development of effective and selective cancer therapies based on the precise cocktailing of pro-apoptotic nanoparticulate agents. ing Research Center on Bioengineering, Biomaterials and Nanomedicine

Background: Novel therapeutic strategies are urgently needed to reduce relapse rates and enhance survival in Diffuse Large B-Cell Lymphoma (DLBCL) patients. CXCR4-overexpressing cancer cells are good targets for therapy because of their association with dissemination and relapse in R-CHOP treated DLBCL patients. Immunotoxins that incorporate bacterial toxins are potentially effective in treating haematological neoplasias, but show a narrow therapeutic index due to the induction of severe side effects. Therefore, when considering the delivery of these toxins as cancer therapeutics, there is a need not only to increase their uptake in the target cancer cells, and their stability in blood, but also to reduce their systemic toxicity. We have developed a therapeutic nanostructured protein T22-PE24-H6 that incorporates exotoxin A from Pseudomonas aeruginosa, which selectively targets lymphoma cells because of its specific interaction with a highly overexpressed CXCR4 receptor (CXCR4+) in DLBCL. Methods: T22-PE24-H6 cytotoxicity and its dependence on the CXCR4 receptor were evaluated in DLBCL cell lines using cell viability assays. Different in vitro experiments (mitochondrial membrane potential, Western Blot, Annexin V and DAPI staining) were conducted to determine T22-PE24-H6 cell death mechanisms. In vivo imaging and therapeutic effect studies were performed in a disseminated DLBCL mouse model that mimics organ infiltration in DLBCL patients. Finally, immunohistochemistry and histopathology analyses were used to evaluate the antineoplastic effect and systemic toxicity. Results: In vitro, T22-PE24-H6 induced selective cell death of CXCR4+ DLBCL cells by activating the apoptotic pathway. In addition, repeated T22-PE24-H6 intravenous administration in a CXCR4+ DLBCL-disseminated mouse model showed a significant reduction of lymphoma burden in organs clinically affected by DLBCL cells (lymph nodes and bone marrow). Finally, we did not observe systemic toxicity associated to the nanoparticle treatment in non-DLBCL-infiltrated organs. Conclusion: We have demonstrated here a potent T22-PE24-H6 antineoplastic effect, especially in blocking dissemination in a CXCR4+ DLBCL model without associated toxicity. Thereby, T22-PE24-H6 promises to become an effective alternative to treat CXCR4+ disseminated refractory or relapsed DLBCL patients.

Bacterial inclusion bodies (IBs) are mechanically stable protein particles in the microscale, which behave as robust, slow‐protein‐releasing amyloids. Upon exposure to cultured cells or upon subcutaneous or intratumor injection, these protein materials secrete functional IB polypeptides, functionally mimicking the endocrine release of peptide hormones from secretory amyloid granules. Being appealing as delivery systems for prolonged protein drug release, the development of IBs toward clinical applications is, however, severely constrained by their bacterial origin and by the undefined and protein‐to‐protein, batch‐to‐batch variable composition. In this context, the de novo fabrication of artificial IBs (ArtIBs) by simple, cell‐free physicochemical methods, using pure components at defined amounts is proposed here. By this, the resulting functional protein microparticles are intriguing, chemically defined biomimetic materials that replicate relevant functionalities of natural IBs, including mammalian cell penetration and local or remote release of functional ArtIB‐forming protein. In default of severe regulatory issues, the concept of ArtIBs is proposed as a novel exploitable category of biomaterials for biotechnological and biomedical applications, resulting from simple fabrication and envisaging soft developmental routes to clinics., Functional microscale protein granules can be obtained by simple physicochemical methods. These materials successfully mimic appealing properties of bacterial inclusion bodies, including amyloidal architecture, biological activity, and the release functional protein in vitro and in vivo. The applicability of these novel materials in biotechnology and in biomedicine is fully supported by their synthetic nature and defined chemical composition.

Current therapies fail to eradicate colorectal Cancer Stem Cells (CSCs). One of the proposed reasons for this failure is the selection, by chemotherapy exposure, of resistant cells responsible for tumor recurrence. In this regard, CXCR4 overexpression in tumor associates with resistance and poor prognosis in colorectal cancer (CRC) patients. In this study, the effectiveness of engineered CXCR4-targeted self-assembling toxin nanoparticles has been explored in the selective killing of CXCR4

Altres ajuts: we are indebted to CIBER de Bioingeniería, Biomateriales y Nanomedicina (projects NANOREMOTE and VENOM4CANCER) to EV and AV respectively, Marató de TV3 foundation (TV32013-132031) and CIBER (NanoMets3) to RM. Protein production has been partially performed by the ICTS "NANBIOSIS", more specifically by the Protein Production Platform of CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN)/IBB, at the UAB SepBioES scientific-technical service (http://www.nanbiosis.es/unit/u1-protein-production-platform-ppp/), whereas the in vivo biodistribution assays were performed in the NANBIOSIS Nanotoxicology platform (http://www.nanbiosis.es/unit/u18-nanotoxicology-unit/). We are also indebted to Fran Cortes from the Cell Culture and Cytometry Units of the Servei de Cultius Cel·lulars, Producció d'Anticossos i Citometria (SCAC), and to the Servei de Microscòpia at the UAB. AV received an ICREA ACADEMIA award. U.U received a Sara Borrell postdoctoral fellowship from ISCIII, MVC was supported by Miguel Servet contract from ISCIII, and JSF received and AECC postdoctoral fellowship. Sustained release of drug delivery systems (DDS) has the capacity to increase cancer treatment efficiency in terms of drug dosage reduction and subsequent decrease of deleterious side effects. In this regard, many biomaterials are being investigated but none offers morphometric and functional plasticity and versatility comparable to protein-based nanoparticles (pNPs). Here we describe a new DDS by which pNPs are fabricated as bacterial inclusion bodies (IB), that can be easily isolated, subcutaneously injected and used as reservoirs for the sustained release of targeted pNPs. Our approach combines the high performance of pNP, regarding specific cell targeting and biodistribution with the IB supramolecular organization, stability and cost effectiveness. This renders a platform able to provide a sustained source of CXCR4-targeted pNPs that selectively accumulate in tumor cells in a CXCR4 colorectal cancer xenograft model. In addition, the proposed system could be potentially adapted to any other protein construct offering a plethora of possible new therapeutic applications in nanomedicine.

Loading capacity and drug leakage from vehicles during circulation in blood is a major concern when developing nanoparticle-based cell-targeted cytotoxics. To circumvent this potential issue it would be convenient the engineering of drugs as self-delivered nanoscale entities, devoid of any heterologous carriers. In this context, we have here engineered potent protein toxins, namely segments of the diphtheria toxin and the Pseudomonas aeruginosa exotoxin as self-assembling, self-delivered therapeutic materials targeted to CXCR4(+) cancer stem cells. The systemic administration of both nanostructured drugs in a colorectal cancer xenograft mouse model promotes efficient and specific local destruction of target tumor tissues and a significant reduction of the tumor volume. This observation strongly supports the concept of intrinsically functional protein nanoparticles, which having a dual role as drug and carrier, are designed to be administered without the assistance of heterologous vehicles.

A single chain polypeptide containing the low density lipoprotein receptor (LDLR) ligand Seq-1 with blood-brain barrier (BBB) crossing activity has been successfully modified by conventional genetic engineering to self-assemble into stable protein-only nanoparticles of 30 nm. The nanoparticulate presentation dramatically enhances in vitro, LDLR-dependent cell penetrability compared to the parental monomeric version, but the assembled protein does not show any enhanced brain targeting upon systemic administration. While the presentation of protein drugs in form of nanoparticles is in general advantageous regarding correct biodistribution, this principle might not apply to brain targeting that is hampered by particular bio-physical barriers. Irrespective of this fact, which is highly relevant to the nanomedicine of central nervous system, engineering the cationic character of defined protein stretches is revealed here as a promising and generic approach to promote the controlled oligomerization of biologically active protein species as still functional, regular nanoparticles. (C) 2016 Elsevier Inc. All rights reserved.

Unliganded drug-nanoconjugates accumulate passively in the tumor whereas liganded nanoconjugates promote drug internalization in tumor cells via endocytosis and increase antitumor efficacy. Whether or not tumor cell internalization associates with enhanced tumor uptake is still under debate. We here compared tumor uptake of T22-GFP-H6, a liganded protein carrier targeting the CXCR4 receptor, and the unliganded GFP-H6 carrier in subcutaneous and metastatic colorectal cancer models. T22-GFP-H6 had a higher tumor uptake in primary tumor and metastatic foci than GFP-H6, with no biodistribution or toxicity on normal tissues. T22-GFP-H6 was detected in target CXCR4

We are grateful to the Protein Production Platform (CIBER-BBN-UAB) for helpful technical assistance and for protein production and purification services (http://​www.​ciber-bbn.​es/​en/​programas/​89-plataforma-de-produccion-de-proteinas-ppp). We are indebted to FIS PI12/01861, Marató 416/C/2013-2030 and NanoMets to RM, MINECO BIO2013-41019-P to AV, AGAUR (2014SGR-132), and CIBER de Bioingeniería, Biomateriales y Nanomedicina (project NANOPROTHER) for funding our research on protein-based therapeutics. We thank the CIBER-BBN Nanotoxicology Unit for fluorescent in vivo follow-up using the IVIS equipment. We are also indebted to the Cell Culture and Citometry Units of the Servei de Cultius Cel·lulars, Producció d'Anticossos i Citometria (SCAC), and to the Servei de Microscòpia, both at the UAB, and to the Soft Materials Service (ICMAB-CSIC/CIBER-BBN). CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. Z. X. and M. P. acknowledge financial support from China Scholarship Council and Universitat Autònoma de Barcelona through pre-doctoral fellowships, respectively. AV received an ICREA ACADEMIA award. Low-density lipoprotein receptors (LDLR) are appealing cell surface targets in drug delivery, as they are expressed in the blood-brain barrier (BBB) endothelium and are able to mediate transcytosis of functionalized drugs for molecular therapies of the central nervous system (CNS). On the other hand, brain-targeted drug delivery is currently limited, among others, by the poor availability of biocompatible vehicles, as most of the nanoparticles under development as drug carriers pose severe toxicity issues. In this context, protein nanoparticles offer functional versatility, easy and cost-effective bioproduction, and full biocompatibility. In this study, we have designed and characterized several chimerical proteins containing different LDLR ligands, regarding their ability to bind and internalize target cells and to self-organize as viral mimetic nanoparticles of about 18 nm in diameter. While the self-assembling of LDLR-binding proteins as nanoparticles positively influences cell penetration in vitro, the nanoparticulate architecture might be not favoring BBB crossing in vivo. These findings are discussed in the context of the use of nanostructured materials as vehicles for the systemic treatment of CNS diseases.

Although all KRas (protein that in humans is encoded by the KRas gene) point mutants are considered to have a similar prognostic capacity, their transformation and tumorigenic capacities vary widely. We compared the metastatic efficiency of KRas G12V (Kirsten rat sarcoma viral oncogene homolog with valine mutation at codon 12) and KRas G13D (Kirsten rat sarcoma viral oncogene homolog with aspartic mutation at codon 13) oncogenes in an orthotopic colorectal cancer (CRC) model. Following subcutaneous preconditioning, recombinant clones of the SW48 CRC cell line [Kras wild-type (Kras WT)] expressing the KRas G12V or KRas G13D allele were microinjected in the mouse cecum. The percentage of animals developing lymph node metastasis was higher in KRas G12V than in KRas G13D mice. Microscopic, macroscopic, and visible lymphatic foci were 1.5- to 3.0-fold larger in KRas G12V than in KRas G13D mice (P < 0.05). In the lung, only microfoci were developed in both groups. KRas G12V primary tumors had lower apoptosis (7.0 ± 1.2 vs. 7.4 ± 1.0 per field, P = 0.02), higher tumor budding at the invasion front (1.2 ± 0.2 vs. 0.6 ± 0.1, P = 0.04), and a higher percentage of C-X-C chemokine receptor type 4 (CXCR4)-overexpressing intravasated tumor emboli (49.8 ± 9.4% vs. 12.8 ± 4.4%, P < 0.001) than KRas G13D tumors. KRas G12V primary tumors showed Akt activation, and ß5 integrin, vascular endothelial growth factor A (VEGFA), and Serpine-1 overexpression, whereas KRas G13D tumors showed integrin ß1 and angiopoietin 2 (Angpt2) overexpression. The increased cell survival, invasion, intravasation, and specific molecular regulation observed in KRas G12V tumors is consistent with the higher aggressiveness observed in patients with CRC expressing this oncogene. © FASEB.

The fully de novo design of protein building blocks for self-assembling as functional nanoparticles is a challenging task in emerging nanomedicines, which urgently demand novel, versatile, and biologically safe vehicles for imaging, drug delivery, and gene therapy. While the use of viruses and virus-like particles is limited by severe constraints, the generation of protein-only nanocarriers is progressively reachable by the engineering of protein-protein interactions, resulting in self-assembling functional building blocks. In particular, end-terminal cationic peptides drive the organization of structurally diverse protein species as regular nanosized oligomers, offering promise in the rational engineering of protein self-assembling. However, the in vivo stability of these constructs, being a critical issue for their medical applicability, needs to be assessed. We have explored here if the cross-molecular contacts between protein monomers, generated by end-terminal cationic peptides and oligohistidine tags, are stable enough for the resulting nanoparticles to overcome biological barriers in assembled form. The analyses of renal clearance and biodistribution of several tagged modular proteins reveal long-term architectonic stability, allowing systemic circulation and tissue targeting in form of nanoparticulate material. This observation fully supports the value of the engineered of protein building blocks addressed to the biofabrication of smart, robust, and multifunctional nanoparticles with medical applicability that mimic structure and functional capabilities of viral capsids.

El pdf del artículo es la versión pre-print.-- et al., Metastases are frequently found during colorectal cancer diagnoses and are the main determinants of clinical outcome. The lack of reliable models of metastases has precluded their mechanistic understanding and our capacity to improve outcome. We studied the effect of E-cadherin and Snail1 expression on metastagenesis in a colorectal cancer model. We microinjected SW480-ADH human colorectal cancer cells, transfected with an empty vector (Mock) or overexpressing Snail1 (Snail1OE) or E-cadherin (E-cadherin OE), in the ceca of nude mice (eight per group) and analyzed tumor growth, dissemination, and Snail1, E-cadherin, β-catenin, and Presenilin1 (PS1) expression in local tumors and/or metastatic foci. Snail1OE cells disseminated only to lymph nodes, whereas Mock or E-cadherinOE cells spread to lymph nodes and peritoneums. Peritoneal tumor foci developed by E-cadherinOE cells presented an increase in E-cadherin proteolysis and nuclear translocation, and enhanced expression of proteolytically active PS1, which was linked to increased tumor growth and shortened mouse survival. Interestingly, local and lymph node tumors in mice bearing E-cadherin OE cells overexpressed E-cadherin, but they did not show Ecadherin proteolysis or nuclear translocation. Remarkably, E-cadherin nuclear translocation and enhanced expression of active PS1 were found in a patient with colorectal signet-ring cell carcinoma. In conclusion, we have established a colorectal cancer metastasis model in which E-cadherin proteolyis and nuclear translocation associates with aggressive foci growth only in the peritoneal microenvironment. Copyright © American Society for Investigative Pathology., Supported by grants from the Ministerio de Ciencia e Innovación (FISPI061294; SAF2008-04702, PS09/00965, CIBER-BBN, CB06/01/1031), and the Generalitat de Catalunya (2009-SGR-1437) (R.M.) and from Ministerio de Ciencia e Innovación (SAF2007-60341; ISCIII-RETIC RD06/0020/0009) and Comunidad de Madrid (S-GEN-0266/2006) (A.M.).

Background: Pancreatic ductal adenocarcinoma is the fourth cause of death in the Western world and, despite the advances in cancer therapies, limited therapeutic benefit (palliating symptoms, better quality of life) may be offered to patients: the overall 5-year survival rate remains < 5%. PM01183 is a new synthetic tetrahydroisoquinoline alkaloid that binds to selected DNA sequences and promotes apoptosis by inducing double-strand breaks at nanomolar concentrations. Confirmed antitumor clinical activity was observed at the recommended dose level of PM01183 in the first-in-human Phase I study in patients with advanced/refractory solid tumors. Currently, PM01183 clinical development includes Phase II single agent trials (pancreas, breast and platinum-resistant/refractory ovarian) as well as Phase I trials in combination (with doxorubicin and gemcitabine) and in advanced acute leukemia. The in vivo antitumor activity of PM01183 was investigated in the human NP9 pancreatic orthotopic model Material and Methods: Athymic nude female mice were orthotopically implanted with luciferase-expressing NP9Luc tumors. Fifteen days after implantation, tumor bearing mice were randomly allocated (N=12 /group) into either PM01183 (at 0.18 mg/kg), gemcitabine (at 180 mg/kg) or control (placebo) groups. Treatments were initiated (Day 0) and administered for 3 consecutive weeks (q7dx3; days 0, 7, 14). Response to treatment was assessed on Day 7 (short-term) by bioluminescence imaging (BLI), and by survival evaluation (long-term): mice were sacrificed when their tumor diameters reached ca. 2 cm or when they became moribund. Statistical differences between groups were determined using two-tailed Mann-Whitney U test and by analysis of the Kaplan-Meier curve. Results: Results showed that PM01183 (q7dx3 at 0.18 mg/kg) induced statistically significant tumor reduction compared to placebo-treated animals by BLI analysis (P= 0.006) on Day 7. Reductions in tumor size were associated to changes in survival median times: PM01183 treatment significantly increased the survival time compared to either gemcitabine- (P= 0.003) or placebo-treated (P= 0.0001) animals. Conclusion: PM01183 demonstrated in vivo antitumor activity in a human pancreatic orthotopic model (NP9), as reflected by metabolic, size-related and survival rate. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1777. doi:1538-7445.AM2012-1777