The effects of linker length on binding affinity and degree of aggregation have been examined in the antifluorescein 4-4-20 and anticarcinoma CC49 single-chain Fvs. Longer linkers in the antifluorescein sFvs have higher affinities for fluorescein and aggregate less. A proteolytically susceptible site between Lys8 and Ser9, in the previously reported 212 linker has been identified. A new linker sequence, 218 (GSTSGSGKPGSGEGSTKG) was designed in which a proline was placed at the C-terminal side of the proteolytic clip site in the 212 linker. The CC49 sFv containing the 218 linker showed reduced aggregation and was found to be more stable to proteolysis in vitro, when compared to the CC49/212 sFv. The CC49 sFv with the longer 218 linker had higher affinity than CC49/212 sFv. An aggregated CC49/212 sFv sample had higher affinity than CC49/218 sFv. The CC49/218 and CC49/212 sFvs had similar blood clearances in mice, while the aggregated CC49/212 sFv remained in circulation significantly longer. In mice bearing LS-174T human colon carcinoma xenografts, the CC49/218 sFv showed higher tumor uptake than the CC49/212 sFv and lower tumor uptake than the aggregated CC49/212 sFv. The higher tumor uptake of the CC49/218 is most likely a result of its higher resistance to proteolysis. The higher affinity and higher tumor uptake of the aggregated CC49/212 sFv are most likely due to the repetitive nature of the TAG-72 antigen and the higher avidity of multivalent aggregates. When the sFvs were radiolabeled with a lutetium-chelate the CC49/218 sFv showed a lower accumulation in the liver and spleen compared to the aggregated CC49/212 sFv.

A number of studies have clearly demonstrated that flagellin is a potent adjuvant that promotes robust immune responses when it is given with a protein antigen. In view of the potential biological and practical benefits of a recombinant protein vaccine composed of a single fusion protein containing flagellin and antigen, we have evaluated the efficacy of a fusion protein composed of flagellin and two protective antigens of Yersinia pestis (F1 and V) in eliciting protection against respiratory challenge with Y. pestis. Flagellin-F1-V was produced and purified in high yield under good manufacturing practices conditions. The fusion protein retains full Toll-like receptor 5-stimulating activity in vitro. Using a prime-boost immunization protocol, we found that flagellin-F1-V elicits robust antigen-specific humoral immunity in mice and two species of nonhuman primates. Immune mice were fully protected against intranasal challenge with 150 mean tolerated doses of Y. pestis CO92. In immune mice, the bacteria were completely cleared within 3 days after challenge. Flagellin-F1-V exhibited full stability for at least 297 days at 4°C and at least 168 days at 25°C. At between 29 and 84 days at 37°C, the protein exhibited a loss of biological activity that appeared to be associated with a substantial change in protein diameter, possibly due to oligomerization. On the basis of our results, we believe that flagellin-F1-V is an outstanding candidate for evaluation in studies with humans.Yersinia pestis, a gram-negative coccobacillus, causes an acute and often fatal disease that may appear in one of three major manifestations: bubonic, septicemic, or pneumonic disease. Transmission to humans most commonly occurs via a bite by infectious fleas and is associated with regional lymphadenopathy or bubo. In the United States, bubonic plague accounts for 80 to 90% of the cases of Y. pestis infection. Pneumonic plague is the most dangerous form of the disease and is generally fatal if an individual does not receive appropriate treatment within 18 h after the onset of respiratory symptoms. Pneumonic plague would be the most likely outcome in the case of a bioterrorism attack.In view of the seriousness of infection with Y. pestis, a substantial effort has been focused on the development of protective vaccines. Killed bacteria have been used in plague vaccines for over 100 years, and a formalin-inactivated bacterial vaccine has been licensed in the United States. However, its efficacy has not been properly evaluated in well-controlled studies. Nonetheless, a retrospective study of armed services personnel who served in Vietnam provided indirect evidence for efficacy in the prevention of bubonic plague (8). However, no evidence in support of a protective effect against the pneumonic form of the disease was obtained. Although a live attenuated strain of Y. pestis (strain EV76) has been evaluated (31), more recent studies have focused on the development of acellular vaccines, in particular, vaccines containing the fraction 1 capsular antigen ...

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