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1. Dendritic Cell-Mediated Cross-Priming by a Bispecific Neutralizing Antibody Boosts Cytotoxic T Cell Responses and Protects Mice against SARS-CoV-2

Author

European Commission, Fundación BBVA, Ministerio de Ciencia e Innovación (España), Instituto de Salud Carlos III, Asociación Española Contra el Cáncer, Fundación CRIS contra el Cáncer, La Caixa, Comunidad de Madrid, Atresmedia, Banco Santander, Consejo Superior de Investigaciones Científicas (España), Universidad Francisco de Vitoria, Fundación Merck Salud, Lázaro-Gorines, Rodrigo [0000-0002-6885-3486], Pérez, Patricia [0000-0001-8983-6784], Heras-Murillo, Ignacio [0000-0002-8797-8786], Adán-Barrientos, Irene [0000-0003-4442-9645], Albericio, Guillermo [0000-0003-0190-4848], Astorgano, David [0000-0002-2969-1840], Luczkowiak, Joanna [0000-0001-6950-9372], Labiod, Nuria [0000-0001-7297-1162], Harwood, Seandean Lykke [0000-0003-4654-8832], Segura-Tudela, Alejandro [0000-0002-5506-0153], Rubio-Pérez, Laura [0000-0002-2877-6092], Nugraha, Yudhi [0000-0003-1186-4093], Shang, Xiaoran [0000-0002-3986-4557], Li, Yuxing [0000-0001-9785-2960], Alfonso, Carlos [0000-0001-7165-4800], Abeyawardhane, Dinendra L. [0000-0003-3002-7792], Navarro, Rocío [0000-0002-0083-7711], Compte, Marta [0000-0002-7138-9266], Sanz, Laura [0000-0002-3119-3218], Weber, David J. [0000-0002-8824-1110], Blanco, Francisco J. [0000-0003-2545-4319], Esteban, Mariano [0000-0003-0846-2827], Pozharski, Edwin [0000-0001-7012-5376], Godoy-Ruiz, Raquel [0000-0003-4569-0781], Muñoz, Inés G. [0000-0001-6732-4059], Delgado, Rafael [0000-0002-6912-4736], Sancho, David [0000-0003-2890-3984], García-Arriaza, Juan [0000-0002-5167-5724], Álvarez-Vallina, Luis [0000-0003-3053-6757], Lázaro-Gorines, Rodrigo, Pérez Ramírez, Patricia, Heras-Murillo, Ignacio, Adán-Barrientos, Irene, Albericio, Guillermo, Astorgano, David, Flores, Sara, Luczkowiak, Joanna, Labiod, Nuria, Harwood, Seandean Lykke, Segura-Tudela, Alejandro, Rubio-Pérez, Laura, Nugraha, Yudhi, Shang, Xiaoran, Li, Yuxing, Alfonso, Carlos, Adipietro, Kaylin A., Abeyawardhane, Dinendra L., Navarro, Rocío, Compte, Marta, Yu, Wenbo, MacKerell Jr., Alexander D., Sanz, Laura, Weber, David J., Blanco, Francisco J., Esteban, Mariano, Pozharski, Edwin, Godoy-Ruiz, Raquel, Muñoz, Inés G., Delgado, Rafael, Sancho, David, García-Arriaza, Juan, Álvarez-Vallina, Luis, European Commission, Fundación BBVA, Ministerio de Ciencia e Innovación (España), Instituto de Salud Carlos III, Asociación Española Contra el Cáncer, Fundación CRIS contra el Cáncer, La Caixa, Comunidad de Madrid, Atresmedia, Banco Santander, Consejo Superior de Investigaciones Científicas (España), Universidad Francisco de Vitoria, Fundación Merck Salud, Lázaro-Gorines, Rodrigo [0000-0002-6885-3486], Pérez, Patricia [0000-0001-8983-6784], Heras-Murillo, Ignacio [0000-0002-8797-8786], Adán-Barrientos, Irene [0000-0003-4442-9645], Albericio, Guillermo [0000-0003-0190-4848], Astorgano, David [0000-0002-2969-1840], Luczkowiak, Joanna [0000-0001-6950-9372], Labiod, Nuria [0000-0001-7297-1162], Harwood, Seandean Lykke [0000-0003-4654-8832], Segura-Tudela, Alejandro [0000-0002-5506-0153], Rubio-Pérez, Laura [0000-0002-2877-6092], Nugraha, Yudhi [0000-0003-1186-4093], Shang, Xiaoran [0000-0002-3986-4557], Li, Yuxing [0000-0001-9785-2960], Alfonso, Carlos [0000-0001-7165-4800], Abeyawardhane, Dinendra L. [0000-0003-3002-7792], Navarro, Rocío [0000-0002-0083-7711], Compte, Marta [0000-0002-7138-9266], Sanz, Laura [0000-0002-3119-3218], Weber, David J. [0000-0002-8824-1110], Blanco, Francisco J. [0000-0003-2545-4319], Esteban, Mariano [0000-0003-0846-2827], Pozharski, Edwin [0000-0001-7012-5376], Godoy-Ruiz, Raquel [0000-0003-4569-0781], Muñoz, Inés G. [0000-0001-6732-4059], Delgado, Rafael [0000-0002-6912-4736], Sancho, David [0000-0003-2890-3984], García-Arriaza, Juan [0000-0002-5167-5724], Álvarez-Vallina, Luis [0000-0003-3053-6757], Lázaro-Gorines, Rodrigo, Pérez Ramírez, Patricia, Heras-Murillo, Ignacio, Adán-Barrientos, Irene, Albericio, Guillermo, Astorgano, David, Flores, Sara, Luczkowiak, Joanna, Labiod, Nuria, Harwood, Seandean Lykke, Segura-Tudela, Alejandro, Rubio-Pérez, Laura, Nugraha, Yudhi, Shang, Xiaoran, Li, Yuxing, Alfonso, Carlos, Adipietro, Kaylin A., Abeyawardhane, Dinendra L., Navarro, Rocío, Compte, Marta, Yu, Wenbo, MacKerell Jr., Alexander D., Sanz, Laura, Weber, David J., Blanco, Francisco J., Esteban, Mariano, Pozharski, Edwin, Godoy-Ruiz, Raquel, Muñoz, Inés G., Delgado, Rafael, Sancho, David, García-Arriaza, Juan, and Álvarez-Vallina, Luis

Abstract

Administration of neutralizing antibodies (nAbs) has proved to be effective by providing immediate protection against SARS-CoV-2. However, dual strategies combining virus neutralization and immune response stimulation to enhance specific cytotoxic T cell responses, such as dendritic cell (DC) cross-priming, represent a promising field but have not yet been explored. Here, a broadly nAb, TNT, are first generated by grafting an anti-RBD biparatopic tandem nanobody onto a trimerbody scaffold. Cryo-EM data show that the TNT structure allows simultaneous binding to all six RBD epitopes, demonstrating a high-avidity neutralizing interaction. Then, by C-terminal fusion of an anti-DNGR-1 scFv to TNT, the bispecific trimerbody TNTDNGR-1 is generated to target neutralized virions to type 1 conventional DCs (cDC1s) and promote T cell cross-priming. Therapeutic administration of TNTDNGR-1, but not TNT, protects K18-hACE2 mice from a lethal SARS-CoV-2 infection, boosting virus-specific humoral responses and CD8+ T cell responses. These results further strengthen the central role of interactions with immune cells in the virus-neutralizing antibody activity and demonstrate the therapeutic potential of the Fc-free strategy that can be used advantageously to provide both immediate and long-term protection against SARS-CoV-2 and other viral infections.

Published
2023

2. Structural analysis of ING3 protein and histone H3 binding

Author

Ministerio de Ciencia, Innovación y Universidades (España), Ministerio de Universidades (España), European Commission, Ferreras-Gutiérrez, Mariola [0000-0003-4421-3158], Chaves-Arquero, Belén [0000-0002-2761-2336], González-Magaña, Amaia [0000-0002-6007-8390], Merino, Nekane [0000-0003-0721-4813], Amusategui-Mateu, Ignacio [0000-0001-5427-1808], Huecas, Sonia [0000-0002-6419-441X], Medrano, Francisco Javier [0000-0002-8185-9751], Blanco, Francisco J. [0000-0003-2545-4319], Ferreras-Gutiérrez, Mariola, Chaves-Arquero, Belén, González-Magaña, Amaia, Merino, Nekane, Amusategui-Mateu, Ignacio, Huecas, Sonia, Medrano, Francisco Javier, Blanco, Francisco J., Ministerio de Ciencia, Innovación y Universidades (España), Ministerio de Universidades (España), European Commission, Ferreras-Gutiérrez, Mariola [0000-0003-4421-3158], Chaves-Arquero, Belén [0000-0002-2761-2336], González-Magaña, Amaia [0000-0002-6007-8390], Merino, Nekane [0000-0003-0721-4813], Amusategui-Mateu, Ignacio [0000-0001-5427-1808], Huecas, Sonia [0000-0002-6419-441X], Medrano, Francisco Javier [0000-0002-8185-9751], Blanco, Francisco J. [0000-0003-2545-4319], Ferreras-Gutiérrez, Mariola, Chaves-Arquero, Belén, González-Magaña, Amaia, Merino, Nekane, Amusategui-Mateu, Ignacio, Huecas, Sonia, Medrano, Francisco Javier, and Blanco, Francisco J.

Abstract

Proteins belonging to the ING family regulate the transcriptional state of chromatin by recruiting remodeling complexes to sites with histone H3 trimethylated at Lysine 4 (H3K4me3). This modification is recognized by the Plant HomeoDomain (PHD) present at the C-terminal region of the five ING proteins. ING3 facilitates acetylation of histones H2A and H4 by the NuA4-Tip60 MYST histone acetyl transferase complex, and it has been proposed to be an oncoprotein. The crystal structure of the N-terminal domain of ING3 shows that it forms homodimers with an antiparallel coiled-coil fold. The crystal structure of the PHD is similar to those of its four homologs. These structures explain the possible deleterious effects of ING3 mutations detected in tumors. The PHD binds histone H3K4me3 with low-micromolar, and binds the non-methylated histone with a 54-fold reduced affinity. Our structure explains the impact of site directed mutagenesis experiments on histone recognition. These structural features could not be confirmed for the full-length protein as solubility was insufficient for structural studies, but the structure of its folded domains suggest a conserved structural organization for the ING proteins as homodimers and bivalent readers of the histone H3K4me3 mark.

Published
2023

3. A PD-L1/EGFR bispecific antibody combines immune checkpoint blockade and direct anti-cancer action for an enhanced anti-tumor response

Author

Ministerio de Ciencia e Innovación (España), Instituto de Salud Carlos III, Fundación CRIS contra el Cáncer, Asociación Española Contra el Cáncer, Fundación la Caixa, Fundación para la Investigación Biomédica del Hospital Universitario 12 de Octubre, Universidad Francisco de Vitoria, Comunidad de Madrid, Rubio-Pérez, Laura [0000-0002-2877-6092], Lázaro-Gorines, Rodrigo [0000-0002-6885-3486], Harwood, Seandean Lykke [0000-0003-4654-8832], Compte, Marta [0000-0002-7138-9266], Navarro, Rocío [0000-0002-0083-7711], Tapia-Galisteo, Antonio [0000-0002-0507-8435], Bonet, Jaume [0000-0001-5210-4387], Blanco, Belén [0000-0001-5085-7756], Lykkemark, Simon [0000-0001-8920-6926], Ramírez-Fernández, Ángel [0000-0002-3265-6878], Ferreras-Gutiérrez, Mariola [0000-0003-4421-3158], Domínguez-Alonso, Carmen [0000-0002-0446-9629], Díez-Alonso, Laura [0000-0002-9545-6910], Segura-Tudela, Alejandro [0000-0002-5506-0153], Hangiu, Oana [0000-0002-2641-8531], Erce-Llamazares, Ainhoa [0000-0002-3656-1913], Blanco, Francisco J. [0000-0003-2545-4319], Santos, Cruz [0000-0001-5164-5050], Rodríguez-Peralto, J. L. [0000-0002-6578-7153], Sanz, Laura [0000-0002-3119-3218], Alvarez-Vallina, Luis [0000-0003-3053-6757], Rubio-Pérez, Laura, Lázaro-Gorines, Rodrigo, Harwood, Seandean Lykke, Compte, Marta, Navarro, Rocío, Tapia-Galisteo, Antonio, Bonet, Jaume, Blanco, Belén, Lykkemark, Simon, Ramírez-Fernández, Ángel, Ferreras-Gutiérrez, Mariola, Domínguez-Alonso, Carmen, Díez-Alonso, Laura, Segura-Tudela, Alejandro, Hangiu, Oana, Erce-Llamazares, Ainhoa, Blanco, Francisco J., Santos, Cruz, Rodríguez-Peralto, J. L., Sanz, Laura, Álvarez-Vallina, Luis, Ministerio de Ciencia e Innovación (España), Instituto de Salud Carlos III, Fundación CRIS contra el Cáncer, Asociación Española Contra el Cáncer, Fundación la Caixa, Fundación para la Investigación Biomédica del Hospital Universitario 12 de Octubre, Universidad Francisco de Vitoria, Comunidad de Madrid, Rubio-Pérez, Laura [0000-0002-2877-6092], Lázaro-Gorines, Rodrigo [0000-0002-6885-3486], Harwood, Seandean Lykke [0000-0003-4654-8832], Compte, Marta [0000-0002-7138-9266], Navarro, Rocío [0000-0002-0083-7711], Tapia-Galisteo, Antonio [0000-0002-0507-8435], Bonet, Jaume [0000-0001-5210-4387], Blanco, Belén [0000-0001-5085-7756], Lykkemark, Simon [0000-0001-8920-6926], Ramírez-Fernández, Ángel [0000-0002-3265-6878], Ferreras-Gutiérrez, Mariola [0000-0003-4421-3158], Domínguez-Alonso, Carmen [0000-0002-0446-9629], Díez-Alonso, Laura [0000-0002-9545-6910], Segura-Tudela, Alejandro [0000-0002-5506-0153], Hangiu, Oana [0000-0002-2641-8531], Erce-Llamazares, Ainhoa [0000-0002-3656-1913], Blanco, Francisco J. [0000-0003-2545-4319], Santos, Cruz [0000-0001-5164-5050], Rodríguez-Peralto, J. L. [0000-0002-6578-7153], Sanz, Laura [0000-0002-3119-3218], Alvarez-Vallina, Luis [0000-0003-3053-6757], Rubio-Pérez, Laura, Lázaro-Gorines, Rodrigo, Harwood, Seandean Lykke, Compte, Marta, Navarro, Rocío, Tapia-Galisteo, Antonio, Bonet, Jaume, Blanco, Belén, Lykkemark, Simon, Ramírez-Fernández, Ángel, Ferreras-Gutiérrez, Mariola, Domínguez-Alonso, Carmen, Díez-Alonso, Laura, Segura-Tudela, Alejandro, Hangiu, Oana, Erce-Llamazares, Ainhoa, Blanco, Francisco J., Santos, Cruz, Rodríguez-Peralto, J. L., Sanz, Laura, and Álvarez-Vallina, Luis

Abstract

Immune checkpoint blockade (ICB) with antibodies has shown durable clinical responses in a wide range of cancer types, but the overall response rate is still limited. Other effective therapeutic modalities to increase the ICB response rates are urgently needed. New bispecific antibody (bsAb) formats combining the ICB effect and a direct action on cancer cells could improve the efficacy of current immunotherapies. Here, we report the development of a PD-L1/EGFR symmetric bsAb by fusing a dual-targeting tandem trimmer body with the human IgG1 hinge and Fc regions. The bsAb was characterized in vitro and the antitumor efficacy was evaluated in humanized mice bearing xenografts of aggressive triple-negative breast cancer and lung cancer. The IgG-like hexavalent bsAb, designated IgTT-1E, was able to simultaneously bind both EGFR and PD-L1 antigens, inhibit EGF-mediated proliferation, effectively block PD-1/PD-L1 interaction, and induce strong antigen-specific antibody-dependent cellular cytotoxicity activity in vitro. Potent therapeutic efficacies of IgTT-1E in two different humanized mouse models were observed, where tumor growth control was associated with a significantly increased proportion of CD8+ T cells. These results support the development of IgTT-1E for the treatment of EGFR+ cancers.

Published
2023

4. Molecular insight into the PCNA-binding mode of FBH1

Author

National Institutes of Health (US), Ministerio de Ciencia e Innovación (España), Chaves-Arquero, Belén [0000-0002-2761-2336], Ruiz-Albor, Antonio [0000-0001-8931-8856], Zhang, Gongyi [0000-0003-3010-3804], Blanco, Francisco J. [0000-0003-2545-4319], Kutateladze, Tatiana G., Liu, Jiuyang, Chaves-Arquero, Belén, Wei, Pengcheng, Tencer, Adam H., Ruiz-Albor, Antonio, Zhang, Gongyi, Blanco, Francisco J., National Institutes of Health (US), Ministerio de Ciencia e Innovación (España), Chaves-Arquero, Belén [0000-0002-2761-2336], Ruiz-Albor, Antonio [0000-0001-8931-8856], Zhang, Gongyi [0000-0003-3010-3804], Blanco, Francisco J. [0000-0003-2545-4319], Kutateladze, Tatiana G., Liu, Jiuyang, Chaves-Arquero, Belén, Wei, Pengcheng, Tencer, Adam H., Ruiz-Albor, Antonio, Zhang, Gongyi, and Blanco, Francisco J.

Abstract

F-box DNA helicase 1 (FBH1) is involved in the regulation of cell responses to replicative stress. FBH1 is recruited to stalled DNA replication fork by PCNA where it inhibits homologous recombination and catalyzes fork regression. Here, we report the structural basis for the molecular recognition of two distinctly different motifs of FBH1, FBH1PIP and FBH1APIM, by PCNA. The crystal structure of PCNA in complex with FBH1PIP and analysis of NMR perturbations reveal overlapped FBH1PIP and FBH1APIM binding sites of PCNA and the dominant contribution of FBH1PIP in this interaction.

Published
2023

5. Impaired function of solute carrier family 19 leads to low folate levels and lipid droplet accumulation in hepatocytes

Author

Programa Iberoamericano de Ciencia y Tecnología para el Desarrollo, Fundación la Caixa, European Commission, Ministerio de Economía y Competitividad (España), Institut National de la Santé et de la Recherche Médicale (France), Instituto de Salud Carlos III, Eusko Jaurlaritza, Fundación BBVA, Cano, Ainara [0000-0001-6019-8926], Vazquez-Chantada, Mercedes [0000-0002-5665-2548], Conde-Vancells, Javier [0000-0003-2791-2678], Mosen-Ansorena, David [0000-0003-4874-7931], Blanco, Francisco J. [0000-0003-2545-4319], Cleḿent, Karine [0000-0002-2489-3355], Aron-Wisnewsky, Judith [0000-0002-3104-9769], Gual, Philippe [0000-0001-7393-8356], García-Monzón, Carmelo [0000-0002-2118-8706], Martínez-Chantar, María Luz [0000-0002-6446-9911], Mato, José M. [0000-0003-1264-3153], Aransay, Ana M. [0000-0002-0013-3052], Cano, Ainara, Vazquez-Chantada, Mercedes, Conde-Vancells, Javier, Gonzalez-Lahera, Aintzane, Mosen-Ansorena, David, Blanco, Francisco J., Cleḿent, Karine, Aron-Wisnewsky, Judith, Tran, Albert, Gual, Philippe, García-Monzón, Carmelo, Caballería, Joan, Castro, Azucena, Martínez-Chantar, María Luz, Mato, José M., Zhu, Huiping, Finnell, Richard H., Aransay, Ana M., Programa Iberoamericano de Ciencia y Tecnología para el Desarrollo, Fundación la Caixa, European Commission, Ministerio de Economía y Competitividad (España), Institut National de la Santé et de la Recherche Médicale (France), Instituto de Salud Carlos III, Eusko Jaurlaritza, Fundación BBVA, Cano, Ainara [0000-0001-6019-8926], Vazquez-Chantada, Mercedes [0000-0002-5665-2548], Conde-Vancells, Javier [0000-0003-2791-2678], Mosen-Ansorena, David [0000-0003-4874-7931], Blanco, Francisco J. [0000-0003-2545-4319], Cleḿent, Karine [0000-0002-2489-3355], Aron-Wisnewsky, Judith [0000-0002-3104-9769], Gual, Philippe [0000-0001-7393-8356], García-Monzón, Carmelo [0000-0002-2118-8706], Martínez-Chantar, María Luz [0000-0002-6446-9911], Mato, José M. [0000-0003-1264-3153], Aransay, Ana M. [0000-0002-0013-3052], Cano, Ainara, Vazquez-Chantada, Mercedes, Conde-Vancells, Javier, Gonzalez-Lahera, Aintzane, Mosen-Ansorena, David, Blanco, Francisco J., Cleḿent, Karine, Aron-Wisnewsky, Judith, Tran, Albert, Gual, Philippe, García-Monzón, Carmelo, Caballería, Joan, Castro, Azucena, Martínez-Chantar, María Luz, Mato, José M., Zhu, Huiping, Finnell, Richard H., and Aransay, Ana M.

Abstract

Low serum folate levels are inversely related to metabolic associated fatty liver disease (MAFLD). The role of the folate transporter gene (SLC19A1) was assessed to clarify its involvement in lipid accumulation during the onset of MAFLD in humans and in liver cells by genomic, transcriptomic, and metabolomic techniques. Genotypes of 3 SNPs in a case-control cohort were initially correlated to clinical and serum MAFLD markers. Subsequently, the expression of 84 key genes in response to the loss of SLC19A1 was evaluated with the aid of an RT2 profiler-array. After shRNA-silencing of SLC19A1 in THLE2 cells, folate and lipid levels were measured by ELISA and staining techniques, respectively. In addition, up to 482 amino acids and lipid metabolites were semi-quantified in SLC19A1-knockdown (KD) cells through ultra-high-performance liquid chromatography coupled with mass spectrometry. SNPs, rs1051266 and rs3788200, were significantly associated with the development of fatty liver for the single-marker allelic test. The minor alleles of these SNPs were associated with a 0.6/−1.67-fold decreased risk of developing MAFLD. When SLC19A1 was KD in THLE2 cells, intracellular folate content was four times lower than in wild-type cells. The lack of functional SLC19A1 provoked significant changes in the regulation of genes associated with lipid droplet accumulation within the cell and the onset of NAFLD. Metabolomic analyses showed a highly altered profile, where most of the species that accumulated in SLC19A1-KD-cells belong to the chemical groups of triacylglycerols, diacylglycerols, polyunsaturated fatty acids, and long chain, highly unsaturated cholesterol esters. In conclusion, the lack of SLC19A1 gene expression in hepatocytes affects the regulation of key genes for normal liver function, reduces intracellular folate levels, and impairs lipid metabolism, which entails lipid droplet accumulation in hepatocytes.

Published
2023

6. Structural analisis of the black-legged tick saliva protein Salp15

Author

Ministerio de Ciencia e Innovación (España), Chaves-Arquero, Belén [0000-0002-2761-2336], Merino, Nekane [0000-0003-0721-4813], Tomás-Cortázar, Julen [0000-0002-0812-0395], Rojas, Adriana L. [0000-0002-7358-3505], Anguita, Juan [0000-0003-2061-7182], Blanco, Francisco J. [0000-0003-2545-4319], Chaves-Arquero, Belén, Persson, Cecilia, Merino, Nekane, Tomás-Cortázar, Julen, Rojas, Adriana L., Anguita, Juan, Blanco, Francisco J., Ministerio de Ciencia e Innovación (España), Chaves-Arquero, Belén [0000-0002-2761-2336], Merino, Nekane [0000-0003-0721-4813], Tomás-Cortázar, Julen [0000-0002-0812-0395], Rojas, Adriana L. [0000-0002-7358-3505], Anguita, Juan [0000-0003-2061-7182], Blanco, Francisco J. [0000-0003-2545-4319], Chaves-Arquero, Belén, Persson, Cecilia, Merino, Nekane, Tomás-Cortázar, Julen, Rojas, Adriana L., Anguita, Juan, and Blanco, Francisco J.

Abstract

Salp15 is one of the proteins in the saliva of the tick Ixodes scapularis. Together with other biomolecules injected into the mammalian host at the biting site, it helps the tick to sustain its blood meal for days. Salp15 interferes with the cellular immune response of the mammalian host by inhibiting the activation of CD4+ T-lymphocytes. This function is co-opted by pathogens that use the tick as a vector and invade the host when the tick bites, such as Borrelia burgdorferi, the causative agent of Lyme borreliosis. Because of the immunity-suppressing role of Salp15, it has been proposed as a candidate for therapeutic applications in disorders of the immune system. The protein is produced as a 135-residue long polypeptide and secreted without its N-terminal signal 1-21 sequence. Detailed structural studies on Salp15 are lacking because of the difficulty in producing large amounts of the folded protein. We report the production of Salp15 and its structural analysis by NMR. The protein is monomeric and contains a flexible N-terminal region followed by a folded domain with mixed + secondary structures. Our results are consistent with a three-dimensional structural model derived from AlphaFold, which predicts the formation of three disulfide bridges and a free C-terminal cysteine.

Published
2022

7. Trispecific T-cell engagers for dual tumor-targeting of colorectal cancer

Author

Instituto de Salud Carlos III, Comunidad de Madrid, Ministerio de Economía y Competitividad (España), Ministerio de Ciencia e Innovación (España), Asociación Española Contra el Cáncer, Fundación CRIS contra el Cáncer, Tapia-Galisteo, Antonio [0000-0002-0507-8435], Sánchez Rodríguez, Íñigo [0000-0002-6440-0922], Aguilar-Sopeña, Óscar [0000-0002-2435-8598], Harwood, Seandean Lykke [0000-0003-4654-8832], Ferreras-Gutiérrez, Mariola [0000-0003-4421-3158], Navarro, Rocío [0000-0002-0083-7711], Corbacho, Cesáreo [0000-0002-6644-3475], Compte, Marta [0000-0002-7138-9266], Lacadena, Javier [0000-0002-7314-0333], Blanco, Francisco J. [0000-0003-2545-4319], Chames, Patrick [0000-0002-6104-6286], Roda-Navarro, Pedro [0000-0003-3799-8823], Álvarez-Vallina, Luis [0000-0003-3053-6757], Sanz, Laura [0000-0002-3119-3218], Tapia-Galisteo, Antonio, Sánchez Rodríguez, Íñigo, Aguilar-Sopeña, Óscar, Harwood, Seandean Lykke, Narbona, Javier, Ferreras-Gutiérrez, Mariola, Navarro, Rocío, Martín-García, Laura, Corbacho, Cesáreo, Compte, Marta, Lacadena, Javier, Blanco, Francisco J., Chames, Patrick, Roda-Navarro, Pedro, Álvarez-Vallina, Luis, Sanz, Laura, Instituto de Salud Carlos III, Comunidad de Madrid, Ministerio de Economía y Competitividad (España), Ministerio de Ciencia e Innovación (España), Asociación Española Contra el Cáncer, Fundación CRIS contra el Cáncer, Tapia-Galisteo, Antonio [0000-0002-0507-8435], Sánchez Rodríguez, Íñigo [0000-0002-6440-0922], Aguilar-Sopeña, Óscar [0000-0002-2435-8598], Harwood, Seandean Lykke [0000-0003-4654-8832], Ferreras-Gutiérrez, Mariola [0000-0003-4421-3158], Navarro, Rocío [0000-0002-0083-7711], Corbacho, Cesáreo [0000-0002-6644-3475], Compte, Marta [0000-0002-7138-9266], Lacadena, Javier [0000-0002-7314-0333], Blanco, Francisco J. [0000-0003-2545-4319], Chames, Patrick [0000-0002-6104-6286], Roda-Navarro, Pedro [0000-0003-3799-8823], Álvarez-Vallina, Luis [0000-0003-3053-6757], Sanz, Laura [0000-0002-3119-3218], Tapia-Galisteo, Antonio, Sánchez Rodríguez, Íñigo, Aguilar-Sopeña, Óscar, Harwood, Seandean Lykke, Narbona, Javier, Ferreras-Gutiérrez, Mariola, Navarro, Rocío, Martín-García, Laura, Corbacho, Cesáreo, Compte, Marta, Lacadena, Javier, Blanco, Francisco J., Chames, Patrick, Roda-Navarro, Pedro, Álvarez-Vallina, Luis, and Sanz, Laura

Abstract

Retargeting of T lymphocytes toward cancer cells by bispecific antibodies has demonstrated its therapeutic potential, with one such antibody approved for the treatment of acute lymphoblastic leukemia (blinatumomab) and several other in clinical trials. However, improvement of their efficacy and selectivity for solid tumors is still required. Here, we describe a novel tandem T-cell recruiting trispecific antibody for the treatment of colorectal cancer (CRC). This construct, termed trispecific T-cell engager (TriTE), consists of a CD3-specific single-chain Fv (scFv) flanked by anti-epidermal growth factor receptor (EGFR) and anti-epithelial cell adhesion molecule (EpCAM) single-domain VHH antibodies. The TriTE was well expressed in mammalian and yeast cells, bound the cognate antigens of the three parental antibodies, and enabled the specific cytolysis of EGFR- and/or EpCAM-expressing cancer cells, without inducing T cell activation and cytoxicity against double-negative (EGFR−EpCAM−) cancer cells. Bivalent bispecific targeting of double-positive HCT116 cells by TriTE improved in vitro potency up to 100-fold compared to single-positive cells and significantly prolonged survival in vivo. In addition, it was less efficient at killing single-positive target cells than the corresponding bispecific controls, leading to potentially enhanced tumor specificity. Moreover, dual targeting of two tumor-associated antigens may contribute toward preventing the tumor escape by antigen loss caused by selective pressures from conventional single-targeting T-cell engagers, and may help to overcome antigenic heterogeneity.

Published
2022

8. Identification of exosomal microrna signature by liquid biopsy in hereditary hemorrhagic telangiectasia patients

Author

Ministerio de Economía y Competitividad (España), European Commission, Junta de Andalucía, Pozo-Agundo, Ana [0000-0003-2188-6296], Martorell-Marugán, Jordi [0000-0002-5186-0735], Soriano, Olga [0000-0001-7666-7969], Jodar, Ana-Belén [0000-0002-6136-7477], Botella, Luisa María [0000-0002-6310-2245], Carmona-Sáez, Pedro [0000-0002-6173-7255], Blanco, Francisco J. [0000-0003-2545-4319], Pozo-Agundo, Ana, Villaescusa, Nerea, Martorell-Marugán, Jordi, Soriano, Olga, Leyva, Socorro, Jodar, Ana-Belén, Botella, Luisa María, Carmona-Sáez, Pedro, Blanco, Francisco J., Ministerio de Economía y Competitividad (España), European Commission, Junta de Andalucía, Pozo-Agundo, Ana [0000-0003-2188-6296], Martorell-Marugán, Jordi [0000-0002-5186-0735], Soriano, Olga [0000-0001-7666-7969], Jodar, Ana-Belén [0000-0002-6136-7477], Botella, Luisa María [0000-0002-6310-2245], Carmona-Sáez, Pedro [0000-0002-6173-7255], Blanco, Francisco J. [0000-0003-2545-4319], Pozo-Agundo, Ana, Villaescusa, Nerea, Martorell-Marugán, Jordi, Soriano, Olga, Leyva, Socorro, Jodar, Ana-Belén, Botella, Luisa María, Carmona-Sáez, Pedro, and Blanco, Francisco J.

Abstract

Hereditary hemorrhagic telangiectasia (HHT) is a rare autosomal dominant vascular dysplasia characterized by epistaxis, mucocutaneous telangiectases, and arteriovenous malformations (AVM) in the visceral organs. The diagnosis of HHT is based on clinical Curaçao criteria, which show limited sensitivity in children and young patients. Here, we carried out a liquid biopsy by which we isolated total RNA from plasma exosome samples. A cohort of 15 HHT type 1 patients, 15 HHT type 2 patients, and 10 healthy relatives were analyzed. Upon gene expression data processing and normalization, a statistical analysis was performed to explore similarities in microRNA expression patterns among samples and detect differentially expressed microRNAs between HHT samples and the control group. We found a disease-associated molecular fingerprint of 35 miRNAs over-represented in HHT vs. controls, with eight being specific for HHT1 and 11 for HHT2; we also found 30 under-represented, including nine distinct for HHT1 and nine for HHT2. The analysis of the receiver operating characteristic (ROC) curves showed that eight miRNAs had good (AUC > 75%) or excellent (AUC > 90%) diagnosis value for HHT and even for type HHT1 and HHT2. In addition, we identified the cellular origin of these miRNAs among the cell types involved in the vascular malformations. Interestingly, we found that only some of them were incorporated into exosomes, which suggests a key functional role of these exosomal miRNAs in the pathophysiology of HHT.

Published
2021

9. The immunosuppressive effect of the tick protein, Salp15, is long-lasting and persists in a murine model of hematopoietic transplant

Author

European Commission, Eusko Jaurlaritza, Ministerio de Economía y Competitividad (España), Instituto de Salud Carlos III, European Research Council, Barriales, Diego [0000-0002-6433-3379], Blanco, Francisco J. [0000-0003-2545-4319], Sutherland, James D. [0000-0003-3229-793X], Barrio, Rosa [0000-0002-9663-0669], Rojas, Adriana [0000-0002-7358-3505], Carracedo, Arkaitz [0000-0001-5957-1260], Abecia, L. [0000-0003-4097-8903], Aransay, Ana M. [0000-0002-8271-612X], Anguita, Juan [0000-0003-2061-7182], Tomás-Cortázar, Julen, Martín-Ruiz, Itziar, Barriales, Diego, Pascual-Itoiz, Miguel Ángel, Gutiérrez de Juan, Virginia, Caro-Maldonado, Alfredo, Merino, Nekane, Marina, Alberto, Blanco, Francisco J., Flores, Juana María, Sutherland, James D., Barrio, Rosa, Rojas, Adriana, Martínez-Chantar, María Luz, Carracedo, Arkaitz, Simó, Carolina, García-Cañas, Virginia, Abecia, Leticia, Lavín, José Luis, Aransay, Ana M., Rodríguez, Héctor, Anguita, Juan, European Commission, Eusko Jaurlaritza, Ministerio de Economía y Competitividad (España), Instituto de Salud Carlos III, European Research Council, Barriales, Diego [0000-0002-6433-3379], Blanco, Francisco J. [0000-0003-2545-4319], Sutherland, James D. [0000-0003-3229-793X], Barrio, Rosa [0000-0002-9663-0669], Rojas, Adriana [0000-0002-7358-3505], Carracedo, Arkaitz [0000-0001-5957-1260], Abecia, L. [0000-0003-4097-8903], Aransay, Ana M. [0000-0002-8271-612X], Anguita, Juan [0000-0003-2061-7182], Tomás-Cortázar, Julen, Martín-Ruiz, Itziar, Barriales, Diego, Pascual-Itoiz, Miguel Ángel, Gutiérrez de Juan, Virginia, Caro-Maldonado, Alfredo, Merino, Nekane, Marina, Alberto, Blanco, Francisco J., Flores, Juana María, Sutherland, James D., Barrio, Rosa, Rojas, Adriana, Martínez-Chantar, María Luz, Carracedo, Arkaitz, Simó, Carolina, García-Cañas, Virginia, Abecia, Leticia, Lavín, José Luis, Aransay, Ana M., Rodríguez, Héctor, and Anguita, Juan

Abstract

Salp15, a salivary protein of Ixodes ticks, inhibits the activation of naïve CD4 T cells. Treatment with Salp15 results in the inhibition of early signaling events and the production of the autocrine growth factor, interleukin-2. The fate of the CD4 T cells activated in the presence of Salp15 or its long-term effects are, however, unknown. We now show that Salp15 binding to CD4 is persistent and induces a long-lasting immunomodulatory effect. The activity of Salp15 results in sustained diminished cross-antigenic antibody production even after interruption of the treatment with the protein. Transcriptionally, the salivary protein provokes an acute effect that includes known activation markers, such as Il2 or Cd44, and that fades over time. The long-term effects exerted by Salp15 do not involve the induction of either anergy traits nor increased populations of regulatory T cells. Similarly, the treatment with Salp15 does not result in B cell anergy or the generation of myeloid suppressor cells. However, Salp15 induces the increased expression of the ectoenzyme, CD73, in regulatory T cells and increased production of adenosine. Our study provides a profound characterization of the immunomodulatory activity of Salp15 and suggests that its long-term effects are due to the specific regulation of CD73. Introduction

Published
2017

10. Tumor targeted 4-1BB agonist antibody-albumin fusions with high affinity to FcRn induce anti-tumor immunity without toxicity

Author

Oana Hangiu, Marta Compte, Anders Dinesen, Rocio Navarro, Antonio Tapia-Galisteo, Ole A. Mandrup, Ainhoa Erce-Llamazares, Rodrigo Lázaro-Gorines, Daniel Nehme-Álvarez, Carmen Domínguez-Alonso, Seandean L. Harwood, Carlos Alfonso, Belen Blanco, Laura Rubio-Pérez, Anaïs Jiménez-Reinoso, Laura Díez-Alonso, Francisco J. Blanco, Laura Sanz, Kenneth A. Howard, Luis Álvarez-Vallina, Ministerio de Ciencia e Innovación (España), European Commission, Instituto de Salud Carlos III, Asociación Española Contra el Cáncer, Fundación CRIS contra el Cáncer, Fundación BBVA, La Caixa, Novo Nordisk Foundation, Center for Multifunctional Biomolecular Drug Design (Denmark), Comunidad de Madrid, Ministerio de Ciencia, Innovación y Universidades (España), Hangiu, Oana, Compte, Marta, Navarro, Rocío, Tapia-Galisteo, Antonio, Mandrup, Ole A., Lázaro-Gorines, Rodrigo, Nehme-Álvarez, Daniel, Domínguez-Alonso, Carmen, Harwood, Seandean Lykke, Alfonso, Carlos, Jiménez-Reinoso, Anaïs, Díez-Alonso, Laura, Blanco, Francisco J., Sanz, Laura, Alvarez-Vallina, Luis, Hangiu, Oana [0000-0002-2641-8531], Compte, Marta [0000-0002-7138-9266], Navarro, Rocío [0000-0002-0083-7711], Tapia-Galisteo, Antonio [0000-0002-0507-8435], Mandrup, Ole A. [0000-0002-9700-4328], Lázaro-Gorines, Rodrigo [0000-0002-6885-3486], Nehme-Álvarez, Daniel [0000-0001-5054-5373], Domínguez-Alonso, Carmen [0000-0002-0446-9629], Harwood, Seandean Lykke [0000-0003-4654-8832], Alfonso, Carlos [0000-0001-7165-4800], Jiménez-Reinoso, Anaïs [0000-0001-8229-1881], Díez-Alonso, Laura [0000-0002-9545-6910], Blanco, Francisco J. [0000-0003-2545-4319], Sanz, Laura [0000-0002-3119-3218], and Alvarez-Vallina, Luis [0000-0003-3053-6757]

Subjects
Multidisciplinary, Immunology, Immunological methods, Immune response, Cancer
Abstract

17 p.-4 fig.-1 tab.-1 grph. abst., Costimulation of tumor-infiltrating T lymphocytes by anti-4-1BB monoclonal antibodies (mAbs) has shown anti-tumor activity in human trials, but can be associated with significant off-tumor toxicities involving FcγR interactions. Here, we introduce albumin-fused mouse and human bispecific antibodies with clinically favorable pharmacokinetics designed to confine 4-1BB costimulation to the tumor microenvironment. These Fc-free 4-1BB agonists consist of an EGFR-specific VHH antibody, a 4-1BB-specific scFv, and a human albumin sequence engineered for high FcRn binding connected in tandem (LiTCo-Albu). We demonstrate in vitro cognate target engagement, EGFR-specific costimulatory activity, and FcRn-driven cellular recycling similar to non-fused FcRn high-binding albumin. The mouse LiTCo-Albu exhibited a prolonged circulatory half-life and in vivo tumor inhibition, with no indication of 4-1BB mAb-associated toxicity. Furthermore, we show a greater therapeutic effect when used in combination with PD-1-blocking mAbs. These findings demonstrate the feasibility of tumor-specific LiTCo-Albu antibodies for safe and effective costimulatory strategies in cancer immunotherapy., Financial support for this work was obtained from the MCIN/AEI/10.13039/501100011033 (SAF2017-89437-P and PDC2021-121711-100 to LA-V, PID2019-104544GB-I00 to CA, and PID2020-113225GB-I00 to FJB), partially supported by the European Regional Development Fund (ERDF); the Carlos III Health Institute (ISCIII) (PI19/00132 to LS; PI20/01030 to BB), partially supported by the ERDF; the ISCIII-RICORS within the Next Generation EU program (plan de Recuperación, Transformación y Resilencia); the Spanish Association Against Cancer (AECC 19084 to LA-V); the CRIS Cancer Foundation (FCRIS-2018-0042 and FCRIS-2021-0090 to LA-V), the BBVA Foundation (Ayudas Fundación BBVA a Equipos de Investigación Científica SARS-CoV-2 years COVID-19 to LA-V); and the Fundació “La Caixa” (HR21-00761 project IL7R_LungCan to LA-V). AD, OAM, and KAH were funded by the Novo Nordisk Foundation, Grant; CEMBID (Center for Multifunctional Biomolecular Drug Design, Grant Number: NNF17OC0028070). OH was supported by an industrial PhD fellowship from the Comunidad de Madrid (IND2020/BMD-17668). AE-L was supported industrial PhD fellowship from the Carlos III Health Institute (IFI18/00045). CD-A was supported by a predoctoral fellowship from the Spanish Ministry of Science Innovation and Universities (PRE2018-083445). LR-P was supported by a predoctoral fellowship from the Immunology Chair, Universidad Francisco de Vitoria/Merck. LD-A was supported by a Rio Hortega fellowship from the Carlos III Health Institute (CM20/00004).

Published
2022
Full Text
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11. Trispecific T-cell engagers for dual tumor-targeting of colorectal cancer

Author

Antonio Tapia-Galisteo, Íñigo Sánchez Rodríguez, Oscar Aguilar-Sopeña, Seandean Lykke Harwood, Javier Narbona, Mariola Ferreras Gutierrez, Rocío Navarro, Laura Martín-García, Cesáreo Corbacho, Marta Compte, Javier Lacadena, Francisco J. Blanco, Patrick Chames, Pedro Roda-Navarro, Luis Álvarez-Vallina, Laura Sanz, Hospital Universitario Puerta de Hierro-Majadahonda [Madrid, Spain], Universidad Complutense de Madrid = Complutense University of Madrid [Madrid] (UCM), Aarhus University [Aarhus], Centro de Investigaciones Biológicas Margarita Salas, Leadartis Sl, Centre de Recherche en Cancérologie de Marseille (CRCM), Aix Marseille Université (AMU)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU), Hospital Universitario 12 de Octubre [Madrid], Chames, Patrick, Instituto de Salud Carlos III, Comunidad de Madrid, Ministerio de Economía y Competitividad (España), Ministerio de Ciencia e Innovación (España), Asociación Española Contra el Cáncer, Fundación CRIS contra el Cáncer, Tapia-Galisteo, Antonio, Sánchez Rodríguez, Íñigo, Aguilar-Sopeña, Óscar, Harwood, Seandean Lykke, Ferreras-Gutiérrez, Mariola, Navarro, Rocío, Corbacho, Cesáreo, Compte, Marta, Lacadena, Javier, Blanco, Francisco J., Roda-Navarro, Pedro, Álvarez-Vallina, Luis, Sanz, Laura, Tapia-Galisteo, Antonio [0000-0002-0507-8435], Sánchez Rodríguez, Íñigo [0000-0002-6440-0922], Aguilar-Sopeña, Óscar [0000-0002-2435-8598], Harwood, Seandean Lykke [0000-0003-4654-8832], Ferreras-Gutiérrez, Mariola [0000-0003-4421-3158], Navarro, Rocío [0000-0002-0083-7711], Corbacho, Cesáreo [0000-0002-6644-3475], Compte, Marta [0000-0002-7138-9266], Lacadena, Javier [0000-0002-7314-0333], Blanco, Francisco J. [0000-0003-2545-4319], Chames, Patrick [0000-0002-6104-6286], Roda-Navarro, Pedro [0000-0003-3799-8823], Álvarez-Vallina, Luis [0000-0003-3053-6757], and Sanz, Laura [0000-0002-3119-3218]

Subjects
Mammals, cancer immunotherapy, T-Lymphocytes, [SDV]Life Sciences [q-bio], single-domain antibodies, Immunology, Neoplasms. Tumors. Oncology. Including cancer and carcinogens, colorectal cancer, RC581-607, Epithelial Cell Adhesion Molecule, Lymphocyte Activation, scFv, scfv, ErbB Receptors, singledomain antibodies, [SDV] Life Sciences [q-bio], Oncology, Trispecific antibodies, tandem antibodies, Animals, Immunology and Allergy, Immunologic diseases. Allergy, Colorectal Neoplasms, trispecific antibodies, RC254-282
Abstract

14 p.-6 fig., Retargeting of T lymphocytes toward cancer cells by bispecific antibodies has demonstrated its therapeutic potential, with one such antibody approved for the treatment of acute lymphoblastic leukemia (blinatumomab) and several other in clinical trials. However, improvement of their efficacy and selectivity for solid tumors is still required. Here, we describe a novel tandem T-cell recruiting trispecific antibody for the treatment of colorectal cancer (CRC). This construct, termed trispecific T-cell engager (TriTE), consists of a CD3-specific single-chain Fv (scFv) flanked by anti-epidermal growth factor receptor (EGFR) and anti-epithelial cell adhesion molecule (EpCAM) single-domain VHH antibodies. The TriTE was well expressed in mammalian and yeast cells, bound the cognate antigens of the three parental antibodies, and enabled the specific cytolysis of EGFR- and/or EpCAM-expressing cancer cells, without inducing T cell activation and cytoxicity against double-negative (EGFR−EpCAM−) cancer cells. Bivalent bispecific targeting of double-positive HCT116 cells by TriTE improved in vitro potency up to 100-fold compared to single-positive cells and significantly prolonged survival in vivo. In addition, it was less efficient at killing single-positive target cells than the corresponding bispecific controls, leading to potentially enhanced tumor specificity. Moreover, dual targeting of two tumor-associated antigens may contribute toward preventing the tumor escape by antigen loss caused by selective pressures from conventional single-targeting T-cell engagers, and may help to overcome antigenic heterogeneity., This study was funded by grants from Instituto de Salud Carlos III PI16/00357, PI19/00132), partially supported by the European Regional Development Fund (ERDF), Comunidad Autónoma de Madrid (S2010-BMD-2312), and Ministerio de Economía y Competitividad (RTC-2016-5118-1) to L.S.; and from Ministerio de Ciencia e Innovación (SAF2017-89437-P and PID2020-117323RB-I00), partially supported by ERDF, the Spanish Association Against Cancer (AECC 19084) and the CRIS Cancer Foundation FCRIS-2018-0042, FCRIS-2021-0090 (FCRIS-2018-0042 and FCRIS-2021-0090) to L.A-V. A.T-G. was supported by a predoctoral fellowship from Comunidad Autónoma de Madrid (PEJD-2018-PRE/BMD-8314);Spanish Ministry of Science and Innovation [SAF2017-89437-P, PID2020-117323RB-I00].

Published
2022
Full Text
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12. Identification of exosomal microrna signature by liquid biopsy in hereditary hemorrhagic telangiectasia patients

Author

Nerea Villaescusa, Olga Soriano, Socorro Leyva, Francisco J. Blanco, Ana Pozo-Agundo, Jordi Martorell-Marugán, Luisa María Botella, Ana Belén Jódar-Reyes, Pedro Carmona-Sáez, Ministerio de Economía y Competitividad (España), European Commission, Junta de Andalucía, Pozo-Agundo, Ana [0000-0003-2188-6296], Martorell-Marugán, Jordi [0000-0002-5186-0735], Soriano, Olga [0000-0001-7666-7969], Jodar, Ana-Belén [0000-0002-6136-7477], Botella, Luisa María [0000-0002-6310-2245], Carmona-Sáez, Pedro [0000-0002-6173-7255], Blanco, Francisco J. [0000-0003-2545-4319], Pozo-Agundo, Ana, Martorell-Marugán, Jordi, Soriano, Olga, Jodar, Ana-Belén, Botella, Luisa María, Carmona-Sáez, Pedro, Blanco, Francisco J., [Pozo-Agundo,A, Villaescusa,N, Soriano,O, Blanco,FJ] Department of Biochemistry and Molecular Biology (III) and Inmunology, School of Medicine, University of Granada, Granada, Spain. [Pozo-Agundo,A, Blanco,FJ] Centre for Biomedical Research, Biopathology and Regenerative Medicine Institute (IBIMER), University of Granada, Granada, Spain. [Martorell-Marugán,J, Carmona-Sáez,P] Bioinformatics Unit, Pfizer, Andalusian Government Centre of Genomics and Oncological Research (GENYO), University of Granada, Granada, Spain. [Martorell-Marugán,J, Carmona-Sáez,P] Department of Statistics and OR, University of Granada, Granada, Spain. [Leyva,S] San Cecilio Clinic Universitary Hospital, Granada, Spain. [Jódar-Reyes,AB] Biocolloid and Fluid Physics Group, Excellence Research Unit Modeling Nature (MNat), Department of Applied Physics, School of Sciences, University of Granada, Granada, Spain. [Botella,LM] Department of Molecular Biomedicine, Centro de Investigaciones Biológicas (CSIC), Madrid, Spain. [Botella,LM] Centro de Investigación Biomédica en Red, CIBERER, Instituto de Salud Carlos III, Madrid, Spain., This work was funded by the Spanish Program for Young Investigators of the Ministerio de Economía y Competitividad and Fondo Europeo de Desarrollo Regional (grant number SAF2015-74313JIN, MINECO/FEDER, UE) to FJB, and and 'Programa Operativo FEDER 2014–2020 and Consejería de Economía y Conocimiento de la Junta de Andalucía' (grant number B1-FQM-112-UGR18) to ABJR

Subjects
Telangiectasia hemorrágica hereditaria, Pathology, Phenomena and Processes::Genetic Phenomena::Phenotype [Medical Subject Headings], Phenomena and Processes::Genetic Phenomena::Genetic Processes::Gene Expression Regulation [Medical Subject Headings], Gene Expression, Telangiectases, Exosomes, Phenomena and Processes::Genetic Phenomena::Genetic Processes::Gene Expression [Medical Subject Headings], Exosomas, Organisms::Eukaryota::Animals::Chordata::Vertebrates::Mammals::Primates::Haplorhini::Catarrhini::Hominidae::Humans [Medical Subject Headings], Cohort Studies, Arteriovenous malformations, hemic and lymphatic diseases, Biology (General), Telangiectasia, Spectroscopy, MicroARNs, Endoglin, General Medicine, Computer Science Applications, Chemistry, Phenotype, Hereditary hemorrhagic telangiectasia, miRNAs, Telangiectasia, Hereditary Hemorrhagic, Phenomena and Processes::Genetic Phenomena::Genotype [Medical Subject Headings], Anatomy::Cells::Cellular Structures::Intracellular Space::Cytoplasm::Cytoplasmic Structures::Organelles::Cytoplasmic Vesicles::Transport Vesicles::Exosomes [Medical Subject Headings], medicine.symptom, congenital, hereditary, and neonatal diseases and abnormalities, medicine.medical_specialty, Phenomena and Processes::Genetic Phenomena::Genetic Variation::Mutation [Medical Subject Headings], Genotype, QH301-705.5, Phenomena and Processes::Genetic Phenomena::Genetic Processes::Gene Expression::Transcription, Genetic::Transcriptome [Medical Subject Headings], Mucocutaneous zone, Analytical, Diagnostic and Therapeutic Techniques and Equipment::Investigative Techniques::Genetic Techniques::Gene Expression Profiling [Medical Subject Headings], Exosome, Article, Catalysis, Inorganic Chemistry, Antigens, CD, Diseases::Cardiovascular Diseases::Vascular Diseases::Arteriovenous Malformations [Medical Subject Headings], microRNA, otorhinolaryngologic diseases, medicine, Humans, Diseases::Hemic and Lymphatic Diseases::Hematologic Diseases::Hemorrhagic Disorders::Hemostatic Disorders::Telangiectasia, Hereditary Hemorrhagic [Medical Subject Headings], Physical and Theoretical Chemistry, Liquid biopsy, QD1-999, Molecular Biology, Receiver operating characteristic, business.industry, Gene Expression Profiling, Organic Chemistry, medicine.disease, Health Care::Health Care Quality, Access, and Evaluation::Quality of Health Care::Health Care Evaluation Mechanisms::Epidemiologic Study Characteristics as Topic::Epidemiologic Studies::Cohort Studies [Medical Subject Headings], Chemicals and Drugs::Nucleic Acids, Nucleotides, and Nucleosides::Antisense Elements (Genetics)::RNA, Antisense::MicroRNAs [Medical Subject Headings], MicroRNAs, Gene Expression Regulation, Dysplasia, Biopsia líquida, Mutation, Malformaciones arteriovenosas, Chemicals and Drugs::Biological Factors::Antigens::Antigens, Surface::Antigens, Differentiation::Antigens, CD [Medical Subject Headings], business, Transcriptome
Abstract

15 p.-5 fig.-3 tab., Hereditary hemorrhagic telangiectasia (HHT) is a rare autosomal dominant vascular dysplasia characterized by epistaxis, mucocutaneous telangiectases, and arteriovenous malformations (AVM) in the visceral organs. The diagnosis of HHT is based on clinical Curaçao criteria, which show limited sensitivity in children and young patients. Here, we carried out a liquid biopsy by which we isolated total RNA from plasma exosome samples. A cohort of 15 HHT type 1 patients, 15 HHT type 2 patients, and 10 healthy relatives were analyzed. Upon gene expression data processing and normalization, a statistical analysis was performed to explore similarities in microRNA expression patterns among samples and detect differentially expressed microRNAs between HHT samples and the control group. We found a disease-associated molecular fingerprint of 35 miRNAs over-represented in HHT vs. controls, with eight being specific for HHT1 and 11 for HHT2; we also found 30 under-represented, including nine distinct for HHT1 and nine for HHT2. The analysis of the receiver operating characteristic (ROC) curves showed that eight miRNAs had good (AUC > 75%) or excellent (AUC > 90%) diagnosis value for HHT and even for type HHT1 and HHT2. In addition, we identified the cellular origin of these miRNAs among the cell types involved in the vascular malformations. Interestingly, we found that only some of them were incorporated into exosomes, which suggests a key functional role of these exosomal miRNAs in the pathophysiology of HHT., This work was funded by the Spanish Program for Young Investigators of the Ministerio de Economía y Competitividad and Fondo Europeo de Desarrollo Regional (grant number SAF2015-74313JIN; MINECO/FEDER, UE) to FJB; and “Programa Operativo FEDER 2014–2020 and Consejería de Economía y Conocimiento de la Junta de Andalucía” (grant number B1-FQM-112-UGR18) to ABJR.

Published
2021

13. Running title: Non-toxic broad anti-tumor activity of an EGFR×4-1BB bispecific trimerbod

Author

Compte, Marta, Harwood, Seandean Lykke, Erce-Llamazares, Ainhoa, Tapia-Galisteo, Antonio, Romero, Eduardo, Ferrer, Irene, Garrido-Martin, Eva M., Enguita, Ana Belén, Ochoa, Maria Carmen, Blanco, Belén, Oteo, Marta, Merino, Nekane, Nehme-Álvarez, Daniel, Hangiu, Oana, Domínguez-Alonso, Carmen, Zonca, Manuela, Ramírez-Fernández, Ángel, Blanco, Francisco J., Morcillo, Miguel Ángel, Muñoz, Inés G., Melero, Ignacio, Rodríguez-Peralto, J. L., Paz-Ares, Luis, Sanz, Laura, Álvarez-Vallina, Luis, European Commission, Ministerio de Ciencia, Innovación y Universidades (España), Ministerio de Economía y Competitividad (España), Instituto de Salud Carlos III, Fundación CRIS contra el Cáncer, Fundación Científica Asociación Española Contra el Cáncer, Compte, Marta [0000-0002-7138-9266], Harwood, Seandean Lykke [0000-0003-4654-8832], Tapia-Galisteo, Antonio [0000-0002-0507-8435], Garrido-Martin, Eva M. [0000-0002-8094-2668], Ochoa, Maria Carmen [0000-0001-9920-2144], Oteo, Marta [0000-0002-2855-3403], Merino, Nekane [0000-0003-0721-4813], Nehme-Álvarez, Daniel [0000-0001-5054-5373], Hangiu, Oana [0000-0002-2641-8531], Domínguez-Alonso, Carmen [0000-0002-0446-9629], Zonca, Manuela [0000-0003-3868-1975], Blanco, Francisco J. [0000-0003-2545-4319], Muñoz, Inés G. [0000-0001-6732-4059], Rodríguez-Peralto, J. L. [0000-0002-6578-7153], Paz-Ares, Luis [0000-0002-1947-3364], Sanz, Laura [0000-0002-3119-3218], and Alvarez-Vallina, Luis [0000-0003-3053-6757]

Subjects
PD-L1, Trimerbody, Checkpoint blockade, Bispecific antibody, T cells, 4-1BB
Abstract

32 p.-4 fig. Purpose: The induction of 4-1BB signaling by agonistic antibodies can drive the activation and proliferation of effector T cells and thereby enhance a T-cell–mediated antitumor response. Systemic administration of anti-4-1BB–agonistic IgGs, although effective preclinically, has not advanced in clinical development due to their severe hepatotoxicity. Experimental Design: Here, we generated a humanized EGFR-specific 4-1BB-agonistic trimerbody, which replaces the IgG Fc region with a human collagen homotrimerization domain. It was characterized by structural analysis and in vitro functional studies. We also assessed pharmacokinetics, antitumor efficacy, and toxicity in vivo. Results: In the presence of a T-cell receptor signal, the trimerbody provided potent T-cell costimulation that was strictly dependent on 4-1BB hyperclustering at the point of contact with a tumor antigen-displaying cell surface. It exhibits significant antitumor activity in vivo, without hepatotoxicity, in a wide range of human tumors including colorectal and breast cancer cell-derived xenografts, and non–small cell lung cancer patient-derived xenografts associated with increased tumor-infiltrating CD8+ T cells. The combination of the trimerbody with a PD-L1 blocker led to increased IFNγ secretion in vitro and resulted in tumor regression in humanized mice bearing aggressive triple-negative breast cancer. Conclusions: These results demonstrate the nontoxic broad antitumor activity of humanized Fc-free tumor-specific 4-1BB-agonistic trimerbodies and their synergy with checkpoint blockers, which may provide a way to elicit responses in most patients with cancer while avoiding Fc-mediated adverse reactions. This work was supported by grants from the European Union [IACT Project (602262), H2020-iNEXT (1676)]; the Spanish Ministry of Science, Innovation and Universities and the Spanish Ministry of Economy and Competitiveness (SAF2017-89437-P, CTQ2017-83810-R, RTC-2016-5118-1, RTC-2017-5944-1), partially supported by the European Regional Development Fund; the Carlos III Health Institute (PI16/00357), co-founded by the Plan Nacional de Investigación and the European Union; the CRIS Cancer Foundation (FCRIS-IFI-2018); and the Spanish Association Against Cancer (AECC, 19084). C. Domínguez-Alonso was supported by a predoctoral fellowship from the Spanish Ministry of Science, Innovation and Universities (PRE2018-083445). M. Zonca was supported by the Torres Quevedo Program from the Spanish Ministry of Economy and Competitiveness, co-founded by the European Social Fund (PTQ-16-08340).

Published
2021
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14. Structural Analysis of the Black-Legged Tick Saliva Protein Salp15

Author

Belén Chaves-Arquero, Cecilia Persson, Nekane Merino, Julen Tomás-Cortazar, Adriana L. Rojas, Juan Anguita, Francisco J. Blanco, Ministerio de Ciencia e Innovación (España), Chaves-Arquero, Belén [0000-0002-2761-2336], Merino, Nekane [0000-0003-0721-4813], Tomás-Cortázar, Julen [0000-0002-0812-0395], Rojas, Adriana L. [0000-0002-7358-3505], Anguita, Juan [0000-0003-2061-7182], Blanco, Francisco J. [0000-0003-2545-4319], Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Chaves-Arquero, Belén, Merino, Nekane, Tomás-Cortázar, Julen, Rojas, Adriana L., Anguita, Juan, and Blanco, Francisco J.

Subjects
Mammals, Lyme Disease, Ixodes, Organic Chemistry, General Medicine, Tick protein, NMR, Catalysis, Computer Science Applications, Inorganic Chemistry, Borrelia burgdorferi, Protein structure, Animals, Salivary Proteins and Peptides, Physical and Theoretical Chemistry, Saliva, Salp15, Molecular Biology, Spectroscopy, tick protein, protein structure
Abstract

9 p.-3 fig.-1 tab., Salp15 is one of the proteins in the saliva of the tick Ixodes scapularis. Together with other biomolecules injected into the mammalian host at the biting site, it helps the tick to sustain its blood meal for days. Salp15 interferes with the cellular immune response of the mammalian host by inhibiting the activation of CD4+ T-lymphocytes. This function is co-opted by pathogens that use the tick as a vector and invade the host when the tick bites, such as Borrelia burgdorferi, the causative agent of Lyme borreliosis. Because of the immunity-suppressing role of Salp15, it has been proposed as a candidate for therapeutic applications in disorders of the immune system. The protein is produced as a 135-residue long polypeptide and secreted without its N-terminal signal 1–21 sequence. Detailed structural studies on Salp15 are lacking because of the difficulty in producing large amounts of the folded protein. We report the production of Salp15 and its structural analysis by NMR. The protein is monomeric and contains a flexible N-terminal region followed by a folded domain with mixed α + β secondary structures. Our results are consistent with a three-dimensional structural model derived from AlphaFold, which predicts the formation of three disulfide bridges and a free C-terminal cysteine., This research was funded by the Spanish Ministry of Science and Innovation, grant number PID2020-113225GB-I00.

Published
2022
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15. The immunosuppressive effect of the tick protein, Salp15, is long-lasting and persists in a murine model of hematopoietic transplant

Author

Miguel Angel Pascual-Itoiz, Itziar Martín-Ruiz, Virginia Gutiérrez-de Juan, Francisco J. Blanco, Leticia Abecia, Julen Tomás-Cortázar, Ana M. Aransay, Arkaitz Carracedo, Virginia García-Cañas, Diego Barriales, Alfredo Caro-Maldonado, José Luis Lavín, Nekane Merino, María L. Martínez-Chantar, Adriana L. Rojas, Juan Anguita, Juana M. Flores, Rosa Barrio, Carolina Simó, Alberto Marina, James D. Sutherland, Héctor Rodríguez, European Commission, Eusko Jaurlaritza, Ministerio de Economía y Competitividad (España), Instituto de Salud Carlos III, European Research Council, Barriales, Diego [0000-0002-6433-3379], Blanco, Francisco J. [0000-0003-2545-4319], Sutherland, James D. [0000-0003-3229-793X], Barrio, Rosa [0000-0002-9663-0669], Rojas, Adriana [0000-0002-7358-3505], Carracedo, Arkaitz [0000-0001-5957-1260], Abecia, L. [0000-0003-4097-8903], Aransay, Ana M. [0000-0002-8271-612X], Anguita, Juan [0000-0003-2061-7182], Barriales, Diego, Blanco, Francisco J., Sutherland, James D., Barrio, Rosa, Rojas, Adriana, Carracedo, Arkaitz, Abecia, L., Aransay, Ana M., and Anguita, Juan

Subjects
0301 basic medicine, CD4-Positive T-Lymphocytes, Myeloid, Transcription, Genetic, medicine.medical_treatment, Graft vs Host Disease, lcsh:Medicine, Hematopoietic stem cell transplantation, Lymphocyte Activation, T-Lymphocytes, Regulatory, Immunoglobulin G, Article, law.invention, Immune tolerance, Immunomodulation, 03 medical and health sciences, Mice, 0302 clinical medicine, law, medicine, Immune Tolerance, Animals, Salivary Proteins and Peptides, lcsh:Science, health care economics and organizations, Multidisciplinary, biology, CD44, lcsh:R, Hematopoietic Stem Cell Transplantation, Hematopoietic Stem Cells, Adenosine, 3. Good health, Cell biology, Hematopoiesis, Haematopoiesis, 030104 developmental biology, medicine.anatomical_structure, Immunology, biology.protein, Suppressor, lcsh:Q, Immunosuppressive Agents, 030215 immunology, medicine.drug
Abstract

Salp15, a salivary protein of Ixodes ticks, inhibits the activation of naïve CD4 T cells. Treatment with Salp15 results in the inhibition of early signaling events and the production of the autocrine growth factor, interleukin-2. The fate of the CD4 T cells activated in the presence of Salp15 or its long-term effects are, however, unknown. We now show that Salp15 binding to CD4 is persistent and induces a long-lasting immunomodulatory effect. The activity of Salp15 results in sustained diminished cross-antigenic antibody production even after interruption of the treatment with the protein. Transcriptionally, the salivary protein provokes an acute effect that includes known activation markers, such as Il2 or Cd44, and that fades over time. The long-term effects exerted by Salp15 do not involve the induction of either anergy traits nor increased populations of regulatory T cells. Similarly, the treatment with Salp15 does not result in B cell anergy or the generation of myeloid suppressor cells. However, Salp15 induces the increased expression of the ectoenzyme, CD73, in regulatory T cells and increased production of adenosine. Our study provides a profound characterization of the immunomodulatory activity of Salp15 and suggests that its long-term effects are due to the specific regulation of CD73. Introduction, Supported by grants from the Department of Education of the Basque Government (PI2013-49 to JA and PI2012-42 to RB). JA is funded by the European Union (Grant Agreement number 602272). AMA and JLL’s work was supported by the Basque Department of Industry, Tourism and Trade (Etortek and Elkartek Programs), the Innovation Technology Department of Bizkaia and the CIBERehd Network. The work of AC is supported by a Ramón y Cajal award, the Basque Department of Industry, Tourism and Trade (Etortek), ISCIII (PI13/00031), FERO VIII Fellowship, the BBVA foundation, MINECO (SAF2016-79381-R) and the European Research Council Starting Grant (336343). CIBERonc was co-funded with FEDER funds. AC-M was funded by a Juan de la Cierva program award and the European Union MSCA program (CIG 660191). RB was funded by MINECO grants BFU2011-25986 and BFU2014-52282-P and the Consolider Program (BFU2014-57703-REDC). FJB was funded by a MINECO grant (CTQ2014-56966-R). D.B. is funded by a MINECO FPI fellowship. We thank the MINECO for the Severo Ochoa Excellence accreditation (SEV-2016-0644).

Published
2017
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16. A tumor-targeted trimeric 4-1BB-agonistic antibody induces potent anti-tumor immunity without systemic toxicity

Author

Ángel M. Cuesta, Eduardo Caleiras, Francisco J. Blanco, Inés G. Muñoz, Jorge Bernardino de la Serna, M. Angela Aznar, Ainhoa Erce-Llamazares, Ignacio Melero, Natalia Nuñez-Prado, Manuela Zonca, Antonio Tapia-Galisteo, Jorge L. Martínez-Torrecuadrada, Simon Lykkemark, Juan M. Zapata, Gema Perez-Chacon, Nekane Merino, Marta Compte, Laura Sanz, Seandean Lykke Harwood, Luis Álvarez-Vallina, Kasper Mikkelsen, Rocío Navarro, Marie Curie Career Integration Grant, Ministerio de Economía y Competitividad (España), Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF), Instituto de Salud Carlos III, Danish Council for Independent Research, Agencia Estatal de Investigación (España), Ministerio de Ciencia, Innovación y Universidades (España), UK Innovation Research Centre, European Commission, Federación Española de Enfermedades Raras, Merino, Nekane [0000-0003-0721-4813], Blanco, Francisco J. [0000-0003-2545-4319], Bernardino de la Serna, Jorge [0000-0002-1396-3338], Sanz, Laura [0000-0002-3119-3218], Merino, Nekane, Blanco, Francisco J., Bernardino de la Serna, Jorge, and Sanz, Laura

Subjects
0301 basic medicine, STIMULATION, Cytotoxicity, Immunologic, Skin Neoplasms, medicine.medical_treatment, General Physics and Astronomy, Adaptive Immunity, CD8-Positive T-Lymphocytes, Epitope, Induction, Epitopes, Mice, Cancer immunotherapy, CD137, lcsh:Science, 4-1BB COSTIMULATION, IN-VIVO, Cancer, Mice, Inbred BALB C, Multidisciplinary, biology, Chemistry, INDUCTION, Acquired immune system, CANCER, 3. Good health, Multidisciplinary Sciences, ErbB Receptors, Science & Technology - Other Topics, Epitopes, B-Lymphocyte, Female, Immunotherapy, Antibody, Anti-cd137 mab, medicine.drug_class, Science, Monoclonal-antibodies, Cd137, Mice, Nude, Monoclonal antibody, Article, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Tumor Necrosis Factor Receptor Superfamily, Member 9, Immune system, Lymphocytes, Tumor-Infiltrating, In-vivo, Cell Line, Tumor, medicine, 4-1bb costimulation, Animals, Humans, IMMUNOTHERAPY, ANTI-CD137 MAB, Science & Technology, T-cells, General Chemistry, Xenograft Model Antitumor Assays, 030104 developmental biology, Immunoglobulin G, Stimulation, biology.protein, Cancer research, T-CELLS, lcsh:Q, MONOCLONAL-ANTIBODIES, Epitope Mapping, Single-Chain Antibodies
Abstract

The costimulation of immune cells using first-generation anti-4-1BB monoclonal antibodies (mAbs) has demonstrated anti-tumor activity in human trials. Further clinical development, however, is restricted by significant off-tumor toxicities associated with FcγR interactions. Here, we have designed an Fc-free tumor-targeted 4-1BB-agonistic trimerbody, 1D8N/CEGa1, consisting of three anti-4-1BB single-chain variable fragments and three anti-EGFR single-domain antibodies positioned in an extended hexagonal conformation around the collagen XVIII homotrimerization domain. The1D8N/CEGa1 trimerbody demonstrated high-avidity binding to 4-1BB and EGFR and a potent in vitro costimulatory capacity in the presence of EGFR. The trimerbody rapidly accumulates in EGFR-positive tumors and exhibits anti-tumor activity similar to IgG-based 4-1BB-agonistic mAbs. Importantly, treatment with 1D8N/CEGa1 does not induce systemic inflammatory cytokine production or hepatotoxicity associated with IgG-based 4-1BB agonists. These results implicate FcγR interactions in the 4-1BB-agonist-associated immune abnormalities, and promote the use of the non-canonical antibody presented in this work for safe and effective costimulatory strategies in cancer immunotherapy., This study was supported by grants from the European Union [IACT Project (602262), iNEXT Project (1676) and a Marie Curie Career Integration Grant (PCIG13-GA-2013-618914)], the Ministerio de Economía y Competitividad (CTQ2017-83810-R, RTC-2016-5118-1, SAF2017-83267-C2-1-R, SAF2017-89437-P, and PTQ-16-08340), the Fondo de Investigación Sanitaria/Instituto de Salud Carlos III (PI16/00357 and PI16/00895), the UK Research and Innovation (18130023), and the Danish Council for Independent Research (DFF-6110-0053). The CIC bioGUNE is a Severo Ochoa Center of Excellence (Ministerio de Economia y Competitividad award SEV-2016-0644). This study was also funded by FEDER.

Published
2018
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