Cell-penetrating Alphabody protein scaffolds for intracellular drug targeting.

The therapeutic scope of antibody and nonantibody protein scaffolds is still prohibitively limited against intracellular drug targets. Here, we demonstrate that the Alphabody scaffold can be engineered into a cell-penetrating protein antagonist against induced myeloid leukemia cell differentiation protein MCL-1, an intracellular target in cancer, by grafting the critical B-cell lymphoma 2 homology 3 helix of MCL-1 onto the Alphabody and tagging the scaffold's termini with designed cell-penetration polypeptides. Introduction of an albumin-binding moiety extended the serum half-life of the engineered Alphabody to therapeutically relevant levels, and administration thereof in mouse tumor xenografts based on myeloma cell lines reduced tumor burden. Crystal structures of such a designed Alphabody in complex with MCL-1 and serum albumin provided the structural blueprint of the applied design principles. Collectively, we provide proof of concept for the use of Alphabodies against intracellular disease mediators, which, to date, have remained in the realm of small-molecule therapeutics.

Monoclonal TCR-redirected tumor cell killing.

T cell immunity can potentially eradicate malignant cells and lead to clinical remission in a minority of patients with cancer. In the majority of these individuals, however, there is a failure of the specific T cell receptor (TCR)­mediated immune recognition and activation process. Here we describe the engineering and characterization of new reagents termed immune-mobilizing monoclonal TCRs against cancer (ImmTACs). Four such ImmTACs, each comprising a distinct tumor-associated epitope-specific monoclonal TCR with picomolar affinity fused to a humanized cluster of differentiation 3 (CD3)-specific single-chain antibody fragment (scFv), effectively redirected T cells to kill cancer cells expressing extremely low surface epitope densities. Furthermore, these reagents potently suppressed tumor growth in vivo. Thus, ImmTACs overcome immune tolerance to cancer and represent a new approach to tumor immunotherapy.

Infection of mature dendritic cells with herpes simplex virus type 1 dramatically reduces lymphoid chemokine-mediated migration.

Herpes simplex virus type 1 (HSV-1) is able to establish latency in infected individuals. In order to characterize potential new immune-escape mechanisms, mature dendritic cells (DCs) were infected with HSV-1 and total cellular RNA was isolated from infected and mock-infected populations at different time points. RNA profiling on Affymetrix Human Genome U133A arrays demonstrated a dramatic downregulation of the migration-mediating surface molecules CCR7 and CXCR4, an observation that was further confirmed by RT-PCR and fluorescence-activated cell sorting analyses. Furthermore, migration assays revealed that, upon infection of mature DCs, CCR7- and CXCR4-mediated migration towards the corresponding CCL19 and CXCL12 chemokine gradients was strongly reduced. It is noteworthy that the infection of immature DCs with HSV-1 prior to maturation led to a failure of CCR7 and CXCR4 upregulation during DC maturation and, as a consequence, also induced a block in their migratory capacity. Additional migration assays with a Deltavhs mutant virus lacking the virion host shutoff (vhs) gene, which is known to degrade cellular mRNAs, suggested a vhs-independent mechanism. These results indicate that HSV-1-infected mature DCs are limited in their capacity to migrate to secondary lymphoid organs, the areas of antigen presentation and T-cell stimulation, thus inhibiting an antiviral immune response. This represents a novel, previously unrecognized mechanism for HSV-1 to escape the human immune system.

Deletion of the virion host shutoff protein (vhs) from herpes simplex virus (HSV) relieves the viral block to dendritic cell activation: potential of vhs- HSV vectors for dendritic cell-mediated immunotherapy.

Herpes simplex virus (HSV) infects dendritic cells (DC) efficiently but with minimal replication. HSV, therefore, appears to have evolved the ability to enter DC even though they are nonpermissive for virus growth. This provides a potential utility for HSV in delivering genes to DC for vaccination purposes and also suggests that the life cycle of HSV usually includes the infection of DC. However, DC infected with HSV usually lose the ability to become activated following infection (M. Salio, M. Cella, M. Suter, and A. Lanzavecchia, Eur. J. Immunol. 29:3245-3253, 1999; M. Kruse, O. Rosorius, F. Kratzer, G. Stelz, C. Kuhnt, G. Schuler, J. Hauber, and A. Steinkasserer, J. Virol. 74:7127-7136, 2000). We report that for DC to retain the ability to become activated following HSV infection, the virion host shutoff protein (vhs) must be deleted. vhs usually functions to destabilize mRNA in favor of the production of HSV proteins in permissive cells. We have found that it also plays a key role in the inactivation of DC and is therefore likely to be important for immune evasion by the virus. Here, vhs would be anticipated to prevent DC activation in the early stages of infection of an individual with HSV, reducing the induction of cellular immune responses and thus preventing virus clearance during repeated cycles of virus latency and reactivation. Based on this information, replication-incompetent HSV vectors with vhs deleted which allow activation of DC and the induction of specific T-cell responses to delivered antigens have been constructed. These responses are greater than if DC are loaded with antigen by incubation with recombinant protein.

Herpes simplex virus infection of dendritic cells: balance among activation, inhibition, and immunity.

Several lines of evidence suggest that dendritic cells (DCs), the most potent antigen-presenting cells known, play a role in the immunological control of herpes simplex virus (HSV) infections. HSV infection of DCs induced submaximal maturation, but DCs failed to mature further in response to lipopolysaccharide (LPS). LPS induced interleukin (IL)-12 secretion, and the induction of primary and secondary T cell responses were impaired by infection. Ultimately, DC infection resulted in delayed, asynchronous apoptotic cell death. However, infected DCs induced HSV recall responses in some individuals. Furthermore, soluble factors secreted by DCs after infection induced DC maturation and primed for IL-12 secretion after LPS stimulation. These data support a pathogenetic model of HSV infection, in which initial delay in the generation of immune responses to HSV at peripheral sites is mediated by disruption of DC function but is overcome by bystander DC maturation and cross-presentation of HSV antigens.