David Sánchez-Martín

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

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 Multicompartmental p32/gClqR as a New Target for Antibody-based Tumor Targeting Strategies

Tumor-associated cell surface antigens and tumor-associated vascular markers have been used as a target for cancer intervention strategies. However, both types of targets have limitations due to accessibility, low and/or heterogeneous expression, and presence of tumor-associated serum antigen. It has been previously reported that a mitochondrial/cell surface protein, p32/gC1qR, is the receptor for a tumor-homing peptide, LyP-1, which specifically recognizes an epitope in tumor cells, tumor lymphatics, and tumor-associated macrophages/myeloid cells. Using antibody phage technology, we have generated an anti-p32 human monoclonal antibody (2.15). The 2.15 antibody, expressed in single-chain fragment variable and in trimerbody format, was then characterized in vivo using mice grafted subcutaneously with MDA-MB-231 human breast cancers cells, revealing a highly selective tumor uptake. The intratumoral distribution of the antibody was consistent with the expression pattern of p32 in the surface of some clusters of cells. These results demonstrate the potential of p32 for antibody-based tumor targeting strategies and the utility of the 2.15 antibody as targeting moiety for the selective delivery of imaging and therapeutic agents to tumors.

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.

Tumor-infiltrating myeloid cells activate Dll4/Notch/TGF-β signaling to drive malignant progression.

Myeloid cells that orchestrate malignant progression in the tumor microenvironment offer targets for a generalized strategy to attack solid tumors. Through an analysis of tumor microenvironments, we explored an experimental model of lung cancer that uncovered a network of Dll4/Notch/TGF-β1 signals that links myeloid cells to cancer progression. Myeloid cells attracted to the tumor microenvironment by the tumor-derived cytokines CCL2 and M-CSF expressed increased levels of the Notch ligand Dll4, thereby activating Notch signaling in the tumor cells and amplifying tumor-intrinsic Notch activation. Heightened Dll4/Notch signaling in tumor cells magnified TGF-β-induced pSMAD2/3 signaling and was required to sustain TGF-β-induced tumor cell growth. Conversely, Notch blockade reduced TGF-β signaling and limited lung carcinoma tumor progression. Corroborating these findings, by interrogating RNAseq results from tumor and adjacent normal tissue in clinical specimens of human head and neck squamous carcinoma, we found evidence that TGF-β/Notch crosstalk contributed to progression. In summary, the myeloid cell-carcinoma signaling network we describe uncovers novel mechanistic links between the tumor microenvironment and tumor growth, highlighting new opportunities to target tumors where this network is active.

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.

Generation and characterization of monospecific and bispecific hexavalent trimerbodies

Here, 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.

Lymphocyte Display: A Novel Antibody Selection Platform Based on T Cell Activation

Since their onset, display technologies have proven useful for the selection of antibodies against a variety of targets; however, most of the antibodies selected with the currently available platforms need to be further modified for their use in humans, and are restricted to accessible antigens. Furthermore, these platforms are not well suited for in vivo selections. We present here a novel cell based antibody display platform, which takes advantage of the functional capabilities of T lymphocytes. The display of antibodies on the surface of T lymphocytes, as a part of a chimeric-immune receptor (CIR) mediating signaling, may ideally link the antigen-antibody interaction to a demonstrable change in T cell phenotype, due to subsequent expression of the early T cell activation marker CD69. In this proof-of-concept, an in vitro selection was carried out using a human T cell line lentiviral-transduced to express a tumor-specific CIR on the surface, against a human tumor cell line expressing the carcinoembryonic antigen. Based on an effective interaction between the CIR and the tumor antigen, we demonstrated that combining CIR-mediated activation with FACS sorting of CD69+ T cells, it is possible to isolate binders to tumor specific cell surface antigen, with an enrichment factor of at least 103-fold after two rounds, resulting in a homogeneous population of T cells expressing tumor-specific CIRs.

Selection strategies for anticancer antibody discovery: searching off the beaten path

Antibody-based drugs represent one of the most successful and promising therapeutic approaches in oncology. Large combinatorial phage antibody libraries are available for the identification of therapeutic antibodies and various technologies exist for their further conversion into multivalent and multispecific formats optimized for the desired pharmacokinetics and the pathological context. However, there is no technology for antigen profiling of intact tumors to identify tumor markers targetable with antibodies. Such constraints have led to a relative paucity of tumor-associated antigens for antibody targeting in oncology. Here we review novel approaches aimed at the identification of antibody-targetable, accessible antigens in intact tumors. We hope that such advanced selection approaches will be useful in the development of next-generation antibody therapies for cancer.