Targeting TRAIL death receptor 4 with trivalent DR4 Atrimer complexes.

TRAIL is a trimeric protein that potently induces apoptosis in cancer cells by binding to the trimeric death receptors (DR4 or DR5). Death receptors are attractive therapeutic targets through both the recombinant TRAIL ligand as well as receptor agonist monoclonal antibodies. Although efficacy of the ligand is hampered by its short half-life, agonistic antibodies have a much longer half-life and have shown some clinical efficacy as antitumor agents. However, the efficacy of these antibodies may be limited by their bivalent nature that does not optimally mimic the trimeric ligand. To overcome limitations of currently used death receptor-targeting agents, we engineered trimeric proteins called Atrimer complexes that selectively bind DR4 and potently induce apoptosis in a variety of cancer cells. Atrimer complexes are based on human tetranectin, a trimeric plasma protein of approximately 60 kDa. Loop regions within the tetranectin C-type lectin domains (CTLD) were randomized to create a large phage display library that was used to select DR4-binding complexes. A panel of unique and potent agonist DR4 Atrimer complexes with subnanomolar affinity to DR4 and no detectable binding to DR5 or the decoy receptors was identified. Mechanism of action studies with a selected Atrimer complex, 1G(2), showed that Atrimer complexes induce caspase-dependent and DR4-specific apoptosis in cancer cells while sparing normal human fibroblasts and, importantly, hepatocytes. This proof-of-principle study supports the use of alternative proteins engineered to overcome limitations of therapeutically desirable molecules such as TRAIL.

Brain metastases secondary to breast cancer: symptoms, prognosis and evolution.

AIMS AND BACKGROUND: Brain metastases confer a worse prognosis to breast cancer because they determine a severe increase in mortality. The aim of this study was to identify the early symptoms in patients with brain metastases after breast cancer treatment and to evaluate the median survival rate in women with single and operable brain lesions. PATIENTS AND METHODS: We examined 43 patients with brain metastases secondary to breast cancer treated in the Oncological Institute Prof I Chiricuta, Cluj-Napoca, during the period 2000-2006. RESULTS: The median interval between the breast cancer diagnosis and detection of central nervous metastases was 21 months. The most frequent symptoms were headache, gait disturbance, nausea and vomiting. Patients with a single brain lesion had a median survival of 23 months compared to only 7 months in case of patients with multiple brain metastases. CONCLUSIONS: The prognosis is worse in patients with solitary brain metastases secondary to breast cancer than in patients who present extracranial metastases. Among factors considered favorable in these patients are a single brain lesion, accessibility to surgery, and the absence of associated extracranial metastases.

CD200 expression on tumor cells suppresses antitumor immunity: new approaches to cancer immunotherapy.

Although the immune system is capable of mounting a response against many cancers, that response is insufficient for tumor eradication in most patients due to factors in the tumor microenvironment that defeat tumor immunity. We previously identified the immune-suppressive molecule CD200 as up-regulated on primary B cell chronic lymphocytic leukemia (B-CLL) cells and demonstrated negative immune regulation by B-CLL and other tumor cells overexpressing CD200 in vitro. In this study we developed a novel animal model that incorporates human immune cells and human tumor cells to address the effects of CD200 overexpression on tumor cells in vivo and to assess the effect of targeting Abs in the presence of human immune cells. Although human mononuclear cells prevented tumor growth when tumor cells did not express CD200, tumor-expressed CD200 inhibited the ability of lymphocytes to eradicate tumor cells. Anti-CD200 Ab administration to mice bearing CD200-expressing tumors resulted in nearly complete tumor growth inhibition even in the context of established receptor-ligand interactions. Evaluation of an anti-CD200 Ab with abrogated effector function provided evidence that blocking of the receptor-ligand interaction was sufficient for control of CD200-mediated immune modulation and tumor growth inhibition in this model. Our data indicate that CD200 expression by tumor cells suppresses antitumor responses and suggest that anti-CD200 treatment might be therapeutically beneficial for treating CD200-expressing cancers.

Single amino acid mutations in the cadherin receptor from Heliothis virescens affect its toxin binding ability to Cry1A toxins.

Bacillus thuringiensis Cry protein exerts its toxic effect through a receptor-mediated process. Both aminopeptidases and cadherin proteins were identified as putative Cry1A receptors from Heliothis virescens and Manduca sexta. The importance of cadherin was implied by its correlation with a Cry1Ac resistant H. virescens strain (Gahan, L. J., Gould, F., and Heckel, D. G. (2001) Science 293, 857-860). In this study, the Cry1Ac toxin-binding region in H. virescens cadherin was mapped to a 40-amino-acid fragment, from amino acids 1422 to 1440. This site overlaps with a Cry1Ab toxin-binding site, amino acids 1363-1464 recently reported in M. sexta (Hua, G., Jurat-Fuentes, J. L., and Adang, M. J. (2004) J. Biol. Chem. 279, 28051-28056). Further, feeding of the anti-H. virescens cadherin antiserum or the partial cadherins, which contain the toxin-binding region, in combination with Cry1Ab/Cry1Ac reduced insect mortality by 25.5-55.6% to first instar H. virescens and M. sexta larvae, suggesting a critical function for this cadherin domain in insect toxicity. Mutations in this region, to which the Cry1Ac binds through its loop 3, resulted in the loss of toxin binding. For the first time, we show that the cadherin amino acids Leu(1425) and Phe(1429) are critical for Cry1Ac toxin interaction, and if substituted with charged amino acids, result in the loss of toxin binding, with a K(D) of < 10(-5) m. Mutation of Gln(1430) to an alanine, however, increased the Cry1Ac affinity 10-fold primarily due to an increase on rate. The L1425R mutant can result from a single nucleotide mutation, CTG --> CGG, suggesting that these mutants, which have decreased toxin binding, may lead to Cry1A resistance in insects.

Hydropathic complementarity determines interaction of epitope (869)HITDTNNK(876) in Manduca sexta Bt-R(1) receptor with loop 2 of domain II of Bacillus thuringiensis Cry1A toxins.

In susceptible insects, Cry toxin specificity correlates with receptor recognition. In previous work, we characterized an scFv antibody (scFv73) that inhibits binding of Cry1A toxins to cadherin-like receptor. The CDR3 region of scFv73 shared homology with an 8-amino acid epitope ((869)HITDTNNK(876)) of the Manduca sexta cadherin-like receptor Bt-R(1) (Gomez, I., Oltean, D. I., Gill, S. S., Bravo, A., and Soberón, M. (2001) J. Biol. Chem. 276, 28906-28912). In this work, we show that the previous sequence of scFv73 CDR3 region was obtained from the noncoding DNA strand. However, most importantly, both scFv73 CDR3 amino acid sequences of the coding and noncoding DNA strands have similar binding capabilities to Cry1Ab toxin as Bt-R(1) (869)HITDTNNK(876) epitope, as demonstrated by the competition of scFv73 with binding to Cry1Ab with synthetic peptides with amino acid sequences corresponding to these regions. Using synthetic peptides corresponding to three exposed loop regions of domain II of Cry1Aa and Cry1Ab toxins, we found that loop 2 synthetic peptide competed with binding of scFv73 to Cry1A toxins in Western blot experiments. Also, loop 2 mutations that affect toxicity of Cry1Ab toxin are affected in scFv73 binding. Toxin overlay assays of Cry1A toxins to M. sexta brush border membrane proteins showed that loop 2 synthetic peptides competed with binding of Cry1A toxins to cadherin-like Bt-R(1) receptor. These experiments identified loop 2 in domain II of as the cognate binding partner of Bt-R(1) (869)HITDTNNK(876). Finally, 10 amino acids from beta-6-loop 2 region of Cry1Ab toxin ((363)SSTLYRRPFNI(373)) showed hydropathic pattern complementarity to a 10-amino acid region of Bt-R(1) ((865)NITIHITDTNN(875)), suggesting that binding of Cry1A toxins to Bt-R(1) is determined by hydropathic complementarity and that the binding epitope of Bt-R(1) may be larger than the one identified by amino acid sequence similarity to scFv73.

Heliothis virescens and Manduca sexta lipid rafts are involved in Cry1A toxin binding to the midgut epithelium and subsequent pore formation.

Lipid rafts are characterized by their insolubility in nonionic detergents such as Triton X-100 at 4 degrees C. They have been studied in mammals, where they play critical roles in protein sorting and signal transduction. To understand the potential role of lipid rafts in lepidopteran insects, we isolated and analyzed the protein and lipid components of these lipid raft microdomains from the midgut epithelial membrane of Heliothis virescens and Manduca sexta. Like their mammalian counterparts, H. virescens and M. sexta lipid rafts are enriched in cholesterol, sphingolipids, and glycosylphosphatidylinositol-anchored proteins. In H. virescens and M. sexta, pretreatment of membranes with the cholesterol-depleting reagent saponin and methyl-beta-cyclodextrin differentially disrupted the formation of lipid rafts, indicating an important role for cholesterol in lepidopteran lipid rafts structure. We showed that several putative Bacillus thuringiensis Cry1A receptors, including the 120- and 170-kDa aminopeptidases from H. virescens and the 120-kDa aminopeptidase from M. sexta, were preferentially partitioned into lipid rafts. Additionally, the leucine aminopeptidase activity was enriched approximately 2-3-fold in these rafts compared with brush border membrane vesicles. We also demonstrated that Cry1A toxins were associated with lipid rafts, and that lipid raft integrity was essential for in vitro Cry1Ab pore forming activity. Our study strongly suggests that these microdomains might be involved in Cry1A toxin aggregation and pore formation.