Targeting c-MYC with T-Cells

Over-expression of the proto-oncogene c-MYC is frequently observed in a variety of tumors and is a hallmark of Burkitt´s lymphoma. The fact that many tumors are oncogene-addicted to c-MYC, renders c-MYC a powerful target for anti-tumor therapy. Using a xenogenic vaccination strategy by immunizing C57BL/6 mice with human c-MYC protein or non-homologous peptides, we show that the human c-MYC protein, despite its high homology between mouse and man, contains several immunogenic epitopes presented in the context of murine H2^b haplotype. We identified an MHC class II-restricted CD4⁺ T-cell epitope and therein an MHC class I-restricted CD8⁺ T-cell epitope (SSPQGSPEPL) that, after prime/boost immunization, protected up to 25% of mice against a lethal lymphoma challenge. Lymphoma-rejecting animals contained MHC multimer-binding CD8⁺ cell within the peripheral blood and displayed in vivo cytolytic activity with specificity for SSPQGSPEPL. Taken together these data suggest that oncogenic c-MYC can be targeted with specific T-cells.     

Epstein-Barr Viruses That Express a CD21 Antibody Provide Evidence that gp350's Functions Extend beyond B-Cell Surface Binding

The gp350 glycoprotein encoded by BLLF1 is crucial for efficient Epstein-Barr virus (EBV) infection of resting B cells. Gp350 binds to CD21, but whether this interaction sums up its functions remains unknown. We generated gp350-null EBVs that display CD19-, CD21-, or CD22-specific antibodies at their surface (designated as ΔBLLF1-Ab). Gp350-complemented (ΔBLLF1-C) and ΔBLLF1-Ab were found to bind equally well to B cells. Surprisingly, ΔBLLF1 binding was reduced only 1.7-fold relative to its complemented counterparts. Furthermore, B cells exposed to ΔBLLF1-Ab or ΔBLLF1 viruses presented structural antigens with comparable efficiency and achieved 25 to 80% of the T-cell activation elicited by ΔBLLF1-C. These findings show that the gp350-CD21 interaction pair plays only a modest role during virus transfer to the endosomal compartment. However, primary B cells or Raji B cells infected with ΔBLLF1-C viruses displayed a 35- to 70-fold higher infection rates than those exposed to ΔBLLF1, ΔBLLF1-CD22Ab, or ΔBLLF1-CD19Ab viruses. Complementation of the gp350 knockout phenotype with CD21Ab substantially enhanced infection rates relative to ΔBLLF1 but remained sevenfold (Raji B-cell line) to sixfold (primary B cells) less efficient than with gp350. We therefore infer that gp350 mainly exerts its functions after the internalization step, presumably during release of the viral capsid from the endosomal compartment, and that CD21-dependent but also CD21-independent molecular mechanisms are involved in this process. The latter appear to be characteristic of B-cell infection since transfection of CD21 in 293 cells improved the infection rates with both ΔBLLF1-CD21Ab and ΔBLLF1-C to a similar extent.The Epstein-Barr virus (EBV) is a B lymphotropic virus that infects its target cells through multiple interactions between envelope glycoproteins and their cognate cellular receptors (for a review, see reference 18). gp350 is the most abundant viral protein in the viral envelope. This large protein is encoded by the BLLF1 gene, is heavily glycosylated and localizes to various subcellular compartments (cytoplasm, endoplasmic reticulum, Golgi, and plasma membrane) of replicating cells (11, 17, 38, 44). Multiple reports provided solid evidence that EBV binds to primary B cells through its interaction with CD21, the complement receptor 2 (CR2) (23, 30-32, 47, 48). Several gp350 domains appear to be involved in the formation of a stable complex with CD21, one of which has been identified as the receptor-binding site (amino acids [aa] 142 to 161) (46). This glycan-free domain is also recognized by the neutralizing gp350-specific antibody 72A1 (15, 46). The introduction of specific mutations has allowed identification of further residues that contribute to gp350-CR2 binding (54). Virions treated with antibodies against gp350 have an enhanced ability to infect epithelial cells (50). Binding of gp350 to CD21 activates several pathways, in particular NF-κB, which results in interleukin-6 (IL-6) upregulation (6, 45). The addition of free gp350 to primary B cells leads to CD19 tyrosine phosphorylation and the subsequent recruitment of the phosphatidylinositol 3-kinase (41). Both of these kinase activities are necessary for efficient EBNA-LP and EBNA2 production shortly after infection, which suggests that gp350 contributes to this process. More generally, preincubation of resting B cells with gp350 markedly enhances the expression of transfected expression plasmids (42). Deletion of gp350 reduces B-cell infection efficiency by 2 orders of magnitude (19). Reciprocally, introduction of CR2 in nonlymphoid cells massively enhances infection rates in nonlymphoid cells (22). However, gp350-null mutants retain a residual infectious potential (19). This contrasts with other viral proteins such as gp110, gp85, or gp42 that are absolutely required for infection (18). This might reflect the fact that these glycoproteins are mainly involved in fusion with the target cell membrane, whereas gp350 appears to be required solely for target cell binding (13, 34, 52). However, it is unclear whether gp350 serves additional functions during viral infection in addition to its clear implication in virus binding. We also wanted to learn whether the gp350-CD21 binding sums up gp350''s interactions with cellular proteins. We posited that if this were the case, a CD21-specific antibody should be able to fully complement the gp350-null phenotype. We therefore exchanged gp350 against antibodies directed against CD19, CD21, or CD22 within the viral envelope and investigated the ability of these proteins to confer full infectious potential to mature virions.     

Retargeting polymer-coated adenovirus to the FGF receptor allows productive infection and mediates efficacy in a peritoneal model of human ovarian cancer

BACKGROUND: Transductional targeting of adenovirus following systemic or regional delivery remains one of the most difficult challenges for cancer gene medicine. The numerical excess and anatomical advantage of normal (non-cancer) cells in vivo demand far greater detargeting than is necessary for studies using single cell populations in vitro, and this must be coupled with efficient retargeting to cancer cells. METHODS: Adenovirus (Ad5) particles were coated with reactive poly[N-(2-hydroxypropyl)methacrylamide] copolymers, to achieve detargeting, and retargeting ligands were attached to the coating. Receptor-mediated infection was characterised in vitro and anticancer efficacy was studied in vivo. RESULTS: Polymer coating prevented the virus binding any cellular receptors and mediated complete detargeting in vitro and in vivo. These fully detargeted vectors were efficiently retargeted with the model ligand FGF2 to infect FGFR-positive cells. Specific transduction activity was the same as parental virus, and intracellular routing appeared unaffected. Levels of transduction were up to 100-fold greater than parental virus on CAR negative cells. This level of specificity permitted good efficacy in intraperitoneal cancer virotherapy, simultaneously decreasing peritoneal adhesions seen with parental virus. Following intravenous delivery FGF2 mediated unexpected binding to erythrocytes, improving circulation kinetics, but preventing the targeted virus from leaving the blood stream. CONCLUSIONS: Polymer cloaking enables complete adenovirus detargeting, providing a versatile platform for receptor-specific retargeting. This approach can efficiently retarget cancer virotherapy in vivo. Ligands should be selected carefully, as non-specific interactions with non-target cells (e.g. blood cells) can deplete the pool of therapeutic virus available for targeting disseminated disease.     

Sulindac derivatives inhibit cell growth and induce apoptosis in primary cells from malignant peripheral nerve sheath tumors of NF1-patients

Background  

Malignant peripheral nerve sheath tumors (MPNSTs) are neoplasms leading to death in most cases. Patients with Neurofibromatosis type 1 have an increased risk of developing this malignancy. The metabolites of the inactive prodrug Sulindac, Sulindac Sulfide and Sulindac Sulfone (Exisulind) are new chemopreventive agents that show promising results in the treatment of different cancer types. In this study we examined the antineoplastic effect of these compounds on primary cells derived from two MPNSTs of Neurofibromatosis type 1 patients.     

Somatic mitochondrial DNA mutations in neurofibromatosis type 1-associated tumors

Neurofibromatosis type 1 is an autosomal dominantly inherited disease predisposing to a multitude of tumors, most characteristically benign plexiform neurofibromas and diffuse cutaneous neurofibromas. We investigated the presence and distribution of somatic mitochondrial DNA (mtDNA) mutations in neurofibromas and in nontumor tissue of neurofibromatosis type 1 patients. MtDNA alterations in the entire mitochondrial genome were analyzed by temporal temperature gradient gel electrophoresis followed by DNA sequencing. Somatic mtDNA mutations in tumors were found in 7 of 19 individuals with cutaneous neurofibromas and in 9 of 18 patients with plexiform neurofibromas. A total of 34 somatic mtDNA mutations were found. All mutations were located in the displacement loop region of the mitochondrial genome. Several plexiform neurofibromas from individual patients had multiple homoplasmic mtDNA mutations. In cutaneous neurofibromas, the same mtDNA mutations were always present in tumors from different locations of the same individual. An increase in the proportion of the mutant mtDNA was always found in the neurofibromas when compared with nontumor tissues. The somatic mtDNA mutations were present in the Schwann cells of the analyzed multiple cutaneous neurofibromas of the same individual. The observed dominance of a single mtDNA mutation in multiple cutaneous neurofibromas of individual patients indicates a common tumor cell ancestry and suggests a replicative advantage rather than random segregation for cells carrying these mutated mitochondria.