Person: Cuesta Martínez, Ángel
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First Name
Ángel
Last Name
Cuesta Martínez
Affiliation
Universidad Complutense de Madrid
Faculty / Institute
Farmacia
Department
Bioquímica y Biología Molecular
Area
Bioquímica y Biología Molecular
Identifiers
8 results
Search Results
Now showing 1 - 8 of 8
Item Tumor Immunotherapy Using Gene-Modified Human Mesenchymal Stem Cells Loaded into Synthetic Extracellular Matrix Scaffolds(Stem Cells, 2009) Compte, Marta; Cuesta Martínez, Ángel; Sánchez-Martín, David; Alonso-Camino, Vanesa; Vicario, José Luís; Sanz, Laura; Álvarez-Vallina, LuísMesenchymal stem cells (MSCs) are appealing as gene therapy cell vehicles given their ease of expansion and transduction. However, MSCs exhibit immunomodulatory and proangiogenic properties that may pose a risk in their use in anticancer therapy. For this reason, we looked for a strategy to confine MSCs to a determined location, compatible with a clinical application. Human MSCs genetically modified to express luciferase (MSCluc), seeded in a synthetic extracellular matrix (sECM) scaffold (sentinel scaffold) and injected subcutaneously in immunodeficient mice, persisted for more than 40 days, as assessed by bioluminescence imaging in vivo. MSCs modified to express a bispecific α-carcinoembryonic antigen (αCEA)/αCD3 diabody (MSCdAb) and seeded in an sECM scaffold (therapeutic scaffolds) supported the release of functional diabody into the bloodstream at detectable levels for at least 6 weeks after implantation. Furthermore, when therapeutic scaffolds were implanted into CEA-positive human colon cancer xenograft-bearing mice and human T lymphocytes were subsequently transferred, circulating αCEA/αCD3 diabody activated T cells and promoted tumor cell lysis. Reduction of tumor growth in MSCdAb-treated mice was statistically significant compared with animals that only received MSCluc. In summary, we report here for the first time that human MSCs genetically engineered to secrete a bispecific diabody, seeded in an sECM scaffold and implanted in a location distant from the primary tumor, induce an effective antitumor response and tumor regression.Item Generation and characterization of monospecific and bispecific hexavalent trimerbodies(mAbs, 2013) Blanco Toribio, Ana; Sainz Pastor, Noelia; Álvarez Cienfuegos, Ana; Merino, Nekane; Sánchez Martín, David; Bonet, Jaume; Santos Valle, Patricia; Sanz, Laura; Oliva, Baldo; Blanco, Francisco J.; Álvarez Vallina, Luis; Cuesta Martínez, ÁngelHere, we describe a new class of multivalent and multispecific antibody-based reagents for therapy. The molecules, termed “trimerbodies,” use a modified version of the N-terminal trimerization region of human collagen XVIII noncollagenous 1 domain flanked by two flexible linkers as trimerizing scaffold. By fusing single-chain variable fragments (scFv) with the same or different specificity to both N- and C-terminus of the trimerizing scaffold domain, we produced monospecific or bispecific hexavalent molecules that were efficiently secreted as soluble proteins by transfected mammalian cells. A bispecific anti-laminin x anti-CD3 N-/C-trimerbody was found to be trimeric in solution, very efficient at recognizing purified plastic-immobilized laminin and CD3 expressed at the surface of T cells, and remarkably stable in human serum. The bispecificity was further demonstrated in T cell activation studies. In the presence of laminin-rich substrate, the bispecific anti-laminin x anti-CD3 N-/C-trimerbody stimulated a high percentage of human T cells to express surface activation markers. These results suggest that the trimerbody platform offers promising opportunities for the development of the next-generation therapeutic antibodies, i.e., multivalent and bispecific molecules with a format optimized for the desired pharmacokinetics and adapted to the pathological context.Item Inhibition of tumor growth in vivo by in situ secretion of bispecific anti-CEA × anti-CD3 diabodies from lentivirally transduced human lymphocytes(Cancer Gene Therapy, 2007) Compte, Marta; Blanco, Bélen; Serrano, Fernando; Cuesta Martínez, Ángel; Sanz, Laura; Bernad, Antonio; Holliger, Philipp; Álvarez-Vallina, LuisInfiltrating T lymphocytes are found in many malignancies, but they appear to be mostly anergic and do not attack the tumor, presumably because of defective T-cell activation events. Recently, we described a strategy for the tumor-specific polyclonal activation of tumor-resident T lymphocytes based on the in situ production of recombinant bispecific antibodies (bsAbs) by transfected nonhematological cell lines. Here, we have constructed a novel HIV-1-based lentiviral vector for efficient gene transduction into various human hematopoietic cell types. Several myelomonocytic and lymphocytic cell lines secreted the anti-carcinoembryonic antigen (CEA) × anti-CD3 diabody in a functionally active form with CD3+ T-cell lines being the most efficient secretors. Furthermore, primary human peripheral blood lymphocytes (PBLs) were also efficiently transduced and secreted high levels of functional diabody. Importantly gene-modified PBLs significantly reduced in vivo tumor growth rates in xenograft studies. These results demonstrate, for the first time, the utility of lentiviral vectors for sustained expression of recombinant bsAbs in human T lymphocytes. Such T lymphocytes, transduced ex vivo to secrete the activating diabody in autocrine fashion, may provide a promising route for a gene therapy strategy for solid human tumorItem In Vivo Tumor Targeting and Imaging with Engineered Trivalent Antibody Fragments Containing Collagen-Derived Sequences(Plos One, 2009) Cuesta Martínez, Ángel; Sánchez-Martín, David; Sanz, Laura; Bonet, Jaume; Compte, Marta; Kremer, Leonor; Blanco, Francisco J.; Oliva, Baldomero; Álvarez-Vallina, Luis; Christophe EglesThere is an urgent need to develop new and effective agents for cancer targeting. In this work, a multivalent antibody is characterized in vivo in living animals. The antibody, termed “trimerbody”, comprises a single-chain antibody (scFv) fragment connected to the N-terminal trimerization subdomain of collagen XVIII NC1 by a flexible linker. As indicated by computer graphic modeling, the trimerbody has a tripod-shaped structure with three highly flexible scFv heads radially outward oriented. Trimerbodies are trimeric in solution and exhibited multivalent binding, which provides them with at least a 100-fold increase in functional affinity than the monovalent scFv. Our results also demonstrate the feasibility of producing functional bispecific trimerbodies, which concurrently bind two different ligands. A trimerbody specific for the carcinoembryonic antigen (CEA), a classic tumor-associated antigen, showed efficient tumor targeting after systemic administration in mice bearing CEA-positive tumors. Importantly, a trimerbody that recognizes an angiogenesis-associated laminin epitope, showed excellent tumor localization in several cancer types, including fibrosarcomas and carcinomas. These results illustrate the potential of this new antibody format for imaging and therapeutic applications, and suggest that some laminin epitopes might be universal targets for cancer targeting.Item Enhancement of DNA vaccine potency through linkage of antigen to filamentous bacteriophage coat protein III domain I(Immunology, 2006) Cuesta Martínez, Ángel; Suárez Porto, Eduardo; Larsen, Martin; Jensen, Kim Bak; Sanz, Laura; Compte Grau, Marta; Kristensen, Peter; Álvarez‐Vallina, LuisAlthough DNA‐based cancer vaccines have been successfully tested in mouse models, a major drawback of cancer vaccination still remains, namely that tumour antigens are weak and fail to generate a vigorous immune response in tumour‐bearing patients. Genetic technology offers strategies for promoting immune pathways by adding immune‐activating genes to the tumour antigen sequence. In this work, we converted a model non‐immunogenic antigen into a vaccine by fusing it to domain I of the filamentous bacteriophage coat protein III gene. Vaccination with a DNA construct encoding the domain I fusion generated antigen‐specific T helper 1‐type cellular immune responses. These results demonstrate that the incorporation of protein III into a DNA vaccine formulation can modulate the gene‐mediated immune response and may thus provide a strategy for improving its therapeutic effect.Item Enhanced antiangiogenic therapy with antibody‐collagen XVIII NC1 domain fusion proteins engineered to exploit matrix remodeling events(International Journal of Cancer, 2006) Sánchez‐Arévalo Lobo, Víctor J.; Cuesta Martínez, Ángel; Sanz, Laura; Compte, Marta; García, Pascal; Prieto, Jesús; Blanco, Francisco J.; Álvarez‐Vallina, LuisAntiangiogenic therapy is nowadays one of the most active fields in cancer research. The first strategies, aimed at inhibiting tumor vascularization, included upregulation of endogenous inhibitors and blocking of the signals delivered by angiogenic factors. But interaction between endothelial cells and their surrounding extracellular matrix also plays a critical role in the modulation of the angiogenic process. This study introduces a new concept to enhance the efficacy of antibody‐based antiangiogenic cancer therapy strategies, taking advantage of a key molecular event occurring in the tumor context: the proteolysis of collagen XVIII, which releases the endogenous angiogenesis inhibitor endostatin. By fusing the collagen XVIII NC1 domain to an antiangiogenic single‐chain antibody, a multispecific agent was generated, which was efficiently processed by tumor‐associated proteinases to produce monomeric endostatin and fully functional trimeric antibody fragments. It was demonstrated that the combined production in the tumor area of complementary antiangiogenic agents from a single molecular entity secreted by gene‐modified cells resulted in enhanced antitumor effects. These results indicate that tailoring recombinant antibodies with extracellular matrix‐derived scaffolds is an effective approach to convert tumor progression associated processes into molecular clues for improving antibody‐based therapies.Item Factory neovessels: engineered human blood vessels secreting therapeutic proteins as a new drug delivery system(Gene Therapy, 2010) Compte, Marta; Alonso-Camino, Vanesa; Cuesta Martínez, Ángel; Santos-Valle, Patricia; Sánchez-Martín, David; López, Mariola; Vicario, José Luis ; Salas, Clara; Sanz, Laura; Álvarez-Vallina, LuisSeveral works have shown the feasibility of engineering functional blood vessels in vivo using human endothelial cells (ECs). Going further, we explored the therapeutic potential of neovessels after gene-modifying the ECs for the secretion of a therapeutic protein. Given that these vessels are connected with the host vascular bed, we hypothesized that systemic release of the expressed protein is immediate. As a proof of principle, we used primary human ECs transduced with a lentiviral vector for the expression of a recombinant bispecific αCEA/αCD3 antibody. These ECs, along with mesenchymal stem cells as a source of mural cells, were embedded in Matrigel and subcutaneously implanted in nude mice. High antibody levels were detected in plasma for 1 month. Furthermore, the antibody exerted a therapeutic effect in mice bearing distant carcinoembryonic-antigen (CEA)-positive tumors after inoculation of human T cells. In summary, we show for the first time the therapeutic effect of a protein locally secreted by engineered human neovessels.Item Differential transplantability of human endothelial cells in colorectal cancer and renal cell carcinoma primary xenografts(Laboratory Investigation (LI), 2008) Sanz, Laura; Cuesta Martínez, Ángel; Salas, Clara; Corbacho, César; Bellas, Carmen; Álvarez-Vallina, LuísIn vivo models of human tumor vasculature are essential for the study of tumor angiogenesis and validation of therapeutic targets. To date, however, few standardized animal models of human tumor angiogenesis have been characterized. It was recently shown that human renal cell and prostate carcinoma primary xenografts, established from biopsy specimens, contained vessels lined mainly by human endothelial cells 1 month after implantation in immunodeficient mice. We selected colorectal cancer (CRC) as a primary xenograft model and studied the response of the vascular compartment to the new microenvironment during the same lapse of time. Immunohistochemical analysis of the origin of endothelial cells demonstrated that, in contrast to the mentioned study, human endothelial cells were rapidly substituted by their murine counterparts (nearly 50% by day 10 after implantation). Apoptotic human endothelial cells could not be detected 10 days after implantation, suggesting that apoptosis is not the mechanism underlying their replacement. Interestingly, host endothelial cells were found to colocalize with human laminin, suggesting a colonization of human vascular basement membranes after human endothelial cell disappearance. To rule out that the differences observed between the fate of human vasculature in the CRC model and those previously reported were because of methodological aspects, we established renal cell carcinoma (RCC) primary xenografts using the same protocol. In clear contrast with CRC xenografts, vasculature within RCC xenografts was mostly of human origin 35 days after implantation. These results support the notion of angiogenic heterogeneity in malignant neoplasms. Elucidation of the molecular mechanisms that determine persistence or disappearance of human endothelial cells in different tumor contexts can help to shed light on the intimate regulation of the angiogenic process.