Bacillus thuringiensisto 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 3 CGG, suggesting that these mutants, which have decreased toxin binding, may lead to Cry1A resistance in insects.

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.

Lipid rafts are characterized by their insolubility in nonionic detergents such as Triton X-100 at 4°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 glycosylphosphatidylinositolanchored proteins. In H. virescens and M. sexta, pretreatment of membranes with the cholesterol-depleting reagent saponin and methyl-␤-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.

In susceptible lepidopteran insects, aminopeptidase N and cadherin-like proteins are the putative receptors for Bacillus thuringiensis (Bt) toxins. Using phage display, we identified a key epitope that is involved in toxin-receptor interaction. Three different scFv molecules that bind Cry1Ab toxin were obtained, and these scFv proteins have different amino acid sequences in the complementary determinant region 3 (CDR3). Binding analysis of these scFv molecules to different members of the Cry1A toxin family and to Escherichia coli clones expressing different Cry1A toxin domains showed that the three selected scFv molecules recognized only domain II. Heterologous binding competition of Cry1Ab toxin to midgut membrane vesicles from susceptible Manduca sexta larvae using the selected scFv molecules showed that scFv73 competed with Cry1Ab binding to the receptor. The calculated binding affinities (K d ) of scFv73 to Cry1Aa, Cry1Ab, and Cry1Ac toxins are in the range of 20 -51 nM. Sequence analysis showed this scFv73 molecule has a CDR3 significantly homologous to a region present in the cadherin-like protein from M. sexta (Bt-R 1 ), Bombyx mori (Bt-R 175 ), and Lymantria dispar. We demonstrated that peptides of 8 amino acids corresponding to the CDR3 from scFv73 or to the corresponding regions of Bt-R 1 or Bt-R 175 are also able to compete with the binding of Cry1Ab and Cry1Aa toxins to the Bt-R 1 or Bt-R 175 receptors. Finally, we showed that synthetic peptides homologous to Bt-R 1 and scFv73 CDR3 and the scFv73 antibody decreased the in vivo toxicity of Cry1Ab to M. sexta larvae. These results show that we have identified the amino acid region of Bt-R 1 and Bt-R 175 involved in Cry1A toxin interaction.

In susceptible insects, Cry toxin specificity correlates with receptor recognition. In previous work, we charac