Marta Compte

Lipopolysaccharide Activates Toll-like Receptor 4 (TLR4)-mediated NF-κB Signaling Pathway and Proinflammatory Response in Human Pericytes

Background: Endothelial cells are well known inflammatory effectors, but TLR4 expression and function in human pericytes have not been addressed. Results: Pericytes express TLR4, and stimulation with LPS or HMGB1 promotes cytokine and chemokine secretion and overexpression of adhesion molecules via NF-κB. Conclusion: TLR4 activation in pericytes triggers a proinflammatory and proangiogenic program. Significance: Pericytes are active players in inflammatory responses beyond their homeostatic role. Pericytes and mesenchymal stem cells (MSCs) are ontogenically related, and in fact, no significant phenotypic differences could be observed by flow cytometry. Transcriptome analysis of human pericytes and MSCs revealed that 43 genes were up-regulated more than 10-fold in pericytes compared with MSCs. Identification of Toll-like receptor 4 (TLR4) as one of the most abundant RNA species in pericytes with respect to MSCs and confirmation of TLR4 expression on the cell surface led us to obtain a comprehensive overview of the expression program of lipopolysaccharide (LPS)-stimulated pericytes. Transcriptional profiling of LPS-treated cells revealed that 22 genes were up-regulated more than 5-fold. Of them, 10 genes encoded chemokines and cytokines (CXCL10, CCL20, IL8, CXCL1, IL6, CCL2, IL1B, CXCL2, IL1A, and CXCL6), and three genes encoded adhesion molecules (ICAM1, VCAM1, and SELE). LPS induced nuclear translocation of the transcription factor NF-κB in stimulated pericytes. Moreover, inhibition of NF-κB activation by SC-514 blocked LPS-induced up-regulation of a subset of chemokine genes, confirming the key role of NF-κB in LPS signaling in pericytes. At the protein level, we assessed the secretion of the proinflammatory cytokines and chemokines IL-6, IL-8, CXCL1, CXCL2, CXCL3, and CCL2 not only after LPS treatment but also in HMGB1-stimulated pericytes. Up-regulation of the adhesion molecules ICAM-1 and VCAM-1 resulted in an increased adhesion of peripheral blood leukocytes to an LPS-treated pericyte monolayer. The role of pericytes in the inflammatory context has been scarcely addressed; according to these results, pericytes should be considered as active players in the inflammatory cascade with potential physiopathological implications.

Long-term in vivo imaging of human angiogenesis: Critical role of bone marrow-derived mesenchymal stem cells for the generation of durable blood vessels

Angiogenesis is a multistep process that encompasses complex molecular and cellular interactions that can not be recapitulated in vitro. Here, we demonstrate that vasculature generated from lentivirally transduced human primary endothelial cells expressing firefly luciferase and co-implanted with human bone marrow mesenchymal stem cells in immunodeficient mice can be assessed quantitatively by in vivo whole body bioluminescence imaging for more than 120 days. Luciferase activity correlated with the formation of a network of functional, mature blood vessels of human nature inside the implant that critically depend on the presence of mesenchymal stem cells. In summary, our study offers an unprecedented opportunity to perform long-term serial analysis of the molecular events involved in the angiogenic process and monitoring responses to anti-angiogenic agents.

Tumor Immunotherapy Using Gene-Modified Human Mesenchymal Stem Cells Loaded into Synthetic Extracellular Matrix Scaffolds†‡§

Mesenchymal 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. STEM CELLS 2009;27:753–760

Antibody engineering: facing new challenges in cancer therapy

AbstractAntibody-based therapeutics are beginning to realize the promise enclosed in their early denomination as “magic bullets”. Initial disappointment has turned into clinical and commercial success, and engineered antibodies currently represent over 30% of biopharmaceuticals in clinical trials. Recent structural and functional data have allowed the design of a new generation of therapeutic antibodies, with strategies ranging from complement-mediated and antibody-dependant cellular cytotoxicity enhancement to improved cytotoxic payloads using toxins, drugs, radio nuclides and viral delivery. This review considers the structure of different types of recombinant antibodies, their mechanism of action and how their efficacy has been increased using a broad array of approaches. We will also focus on the additional benefits offered by the use of gene therapy methods for the in vivo production of therapeutic antibodies.

In Vivo Tumor Targeting and Imaging with Engineered Trivalent Antibody Fragments Containing Collagen-Derived Sequences

There 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.

The coming of age of engineered multivalent antibodies.

The development of monoclonal antibody (mAb) technology has had a profound impact on medicine. The therapeutic use of first-generation mAb achieved considerable success in the treatment of major diseases, including cancer, inflammation, autoimmune, cardiovascular, and infectious diseases. Next-generation antibodies have been engineered to further increase potency, improve the safety profile and acquire non-natural properties, and constitute a thriving area of mAb research and development. Currently, a variety of alternative antibody formats with modified architectures have been generated and are moving fast into the clinic. In fact, the bispecific antibody blinatumomab was the first in its class to be approved by the US Food and Drug Administration (FDA) as recently as December 2014. Here, we outline the fundamental strategies used for designing the next generation of therapeutic antibodies, as well as the most relevant results obtained in preclinical studies and clinical trials.

Selection of functional human antibodies from retroviral display libraries

Antibody library technology represents a powerful tool for the discovery and design of antibodies with high affinity and specificity for their targets. To extend the technique to the expression and selection of antibody libraries in an eukaryotic environment, we provide here a proof of concept that retroviruses can be engineered for the display and selection of variable single-chain fragment (scFv) libraries. A retroviral library displaying the repertoire obtained after a single round of selection of a human synthetic scFv phage display library on laminin was generated. For selection, antigen-bound virus was efficiently recovered by an overlay with cells permissive for infection. This approach allowed more than 103-fold enrichment of antigen binders in a single selection cycle. After three selection cycles, several scFvs were recovered showing similar laminin-binding activities but improved expression levels in mammalian cells as compared with a laminin-specific scFv selected by the conventional phage display approach. Thus, translational problems that occur when phage-selected antibodies have to be transferred onto mammalian expression systems to exert their therapeutic potential can be avoided by the use of retroviral display libraries.

Enhanced antiangiogenic therapy with antibody-collagen XVIII NC1 domain fusion proteins engineered to exploit matrix remodeling events

Antiangiogenic 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.

Factory neovessels: engineered human blood vessels secreting therapeutic proteins as a new drug delivery system

Several 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.

CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors

A human single-chain variable fragment (scFv) antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs). The repertoire was fused to a first-generation T cell receptor ζ (TCRζ)-based chimeric antigen receptor (CAR). We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2) bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR) and the selection context (cell synapse), which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells.