Chimeric antigen receptors for the retargeting of cytotoxic effector cells.

T lymphocytes recognize specific antigens through interaction of the T cell receptor (TCR) with short peptides presented by major histocompatibility complex (MHC) class I or II molecules. For initial activation and clonal expansion, naïve T cells are dependent on professional antigen-presenting cells (APCs) that provide additional co-stimulatory signals. TCR activation in the absence of co-stimulation can result in unresponsiveness and clonal anergy. To bypass immunization, different approaches for the derivation of cytotoxic effector cells with grafted recognition specificity have been developed. Chimeric antigen receptors have been constructed that consist of binding domains derived from natural ligands or antibodies specific for cell-surface antigens, genetically fused to effector molecules such as the TCR alpha and beta chains, or components of the TCR-associated CD3 complex. Upon antigen binding, such chimeric receptors link to endogenous signaling pathways in the effector cell and generate activating signals similar to those initiated by the TCR complex. Since the first reports on chimeric antigen receptors, this concept has steadily been refined and the molecular design of chimeric receptors has been optimized. Aided by advances in recombinant antibody technology, chimeric antigen receptors targeted to a wide variety of antigens on the surface of cancer cells and of cells infected by human immunodeficiency virus (HIV) have been generated. In initial clinical studies, infusion of such cells into patients proved to be safe and transient therapeutic effects have been observed.

DNA-carrier proteins for targeted gene delivery.

The development of vectors for cell-specific gene delivery is a major goal of gene therapeutic strategies. Significant progress has been made in the construction of non-viral vectors that combine different functions required for gene transfer in an artificial complex. To some extent this can be achieved by complexing plasmid DNA with synthetic compounds such as lipids and polycations. Alternative approaches rely on the activities of natural or recombinant DNA-carrier proteins to achieve uptake and intracellular delivery of plasmid DNA. Nuclear proteins such as histones and members of the high mobility group protein family have been shown to condense DNA and transfect cultured cells. Some structural proteins of DNA viruses spontaneously assemble with plasmid DNA and form transfection-competent pseudocapsids. In addition, chimeric fusion proteins have been engineered that incorporate in a single polypeptide chain heterologous protein domains which facilitate binding to plasmid DNA, specific recognition of target cells, induction of receptor-mediated endocytosis, and DNA transport through intracellular compartments.

A chimeric fusion protein containing transforming growth factor-alpha mediates gene transfer via binding to the EGF receptor.

Fusion proteins engineered to incorporate distinct functions which co-operate in mediating the cell-type specific uptake and intracellular delivery of DNA present an attractive approach for the development of self-assembling vector systems for targeted gene transfer. Here we have chosen the EGF receptor overexpressed in many human tumors of epithelial origin as a target for a novel modular fusion protein. We have fused a cDNA fragment of the human EGF receptor ligand TGF-alpha to sequences encoding the translocation domain of Pseudomonas exotoxin A as an endosome escape activity, and the DNA-binding domain of the yeast GAL4 transcription factor. Upon bacterial expression, this TEG fusion protein displayed specific binding to EGF receptors. Complexes of the chimeric protein and plasmid DNA carrying a luciferase reporter gene, after condensation with poly-L-lysine resulted in an up to 150-fold increase in reporter gene expression in EGF receptor expressing cells in comparison to poly-L-lysine-DNA complexes alone. While in COS-1 cells no additional endosome escape activity was required, in A431 cells gene delivery was dependent on the simultaneous presence of the endosome destabilizing reagent chloroquine indicating that cell-type specific factors such as different intracellular routing of protein-DNA complexes greatly influence transfection efficiency.

A modular DNA carrier protein based on the structure of diphtheria toxin mediates target cell-specific gene delivery.

Modular fusion proteins that combine distinct functions required for cell type-specific uptake and intracellular delivery of DNA present an attractive approach for the development of self-assembling vectors for targeted gene delivery. Here, we describe a novel DNA carrier protein termed GD5 that mimics the structure of the bacterial diphtheria toxin (DT) and facilitates target cell-specific gene transfer via receptor-mediated endocytosis. GD5 carries at the N terminus the DNA-binding domain of the yeast transcription factor Gal4, which is connected to a C-terminal antibody fragment specific for the tumor-associated ErbB2 antigen via an internal DT translocation domain as an endosome escape activity. Bacterially expressed GD5 protein specifically bound to ErbB2-expressing cells and formed protein-DNA complexes with a luciferase reporter gene construct. These complexes, after compensation of excess negative charge with poly-L-lysine, served as a specific transfection vector for ErbB2-expressing cells. Inhibitors of endosomal acidification drastically reduced GD5-mediated transfection, indicating that the DT translocation domain of GD5, similar to the parental toxin, is strictly dependent on the transit through an acidic environment. Our results suggest that fusion proteins that employ the natural endosome escape mechanism of bacterial toxins might aid in the development of efficient nonviral vectors for applications in gene therapy.

CD31/PECAM-1 is a ligand for alpha v beta 3 integrin involved in adhesion of leukocytes to endothelium.

To protect the body efficiently from infectious organisms, leukocytes circulate as nonadherent cells in the blood and lymph, and migrate as adherent cells into tissues. Circulating leukocytes in the blood have first to adhere to and then to cross the endothelial lining. CD31/PECAM-1 is an adhesion molecule expressed by vascular endothelial cells, platelets, monocytes, neutrophils, and naive T lymphocytes. It is a transmembrane glycoprotein of the immunoglobulin gene superfamily (IgSF), with six Ig-like homology units mediating leukocyte-endothelial interactions. The adhesive interactions mediated by CD31 are complex and include homophilic (CD31-CD31) or heterophilic (CD31-X) contacts. Soluble, recombinant forms of CD31 allowed us to study the heterophilic interactions in leukocyte adhesion assays. We show that the adhesion molecule alpha v beta 3 integrin is a ligand for CD31. The leukocytes revealed adhesion mediated by the second Ig-like domain of CD31, and this binding was inhibited by alpha v beta 3 integrin-specific antibodies. Moreover alpha v beta 3 was precipitated by recombinant CD31 from cell lysates. These data establish a third IgSF-integrin pair of adhesion molecules, CD31-alpha v beta 3 in addition to VCAM-1, MadCAM-1/alpha 4 integrins, and ICAM/beta 2 integrins, which are major components mediating leukocyte-endothelial adhesion. Identification of a further versatile adhesion pair broadens our current understanding of leukocyte-endothelial interactions and may provide the basis for the treatment of inflammatory disorders and metastasis formation.