The interaction between IgE and its high-affinity receptor Fc epsilon RI found on mast cells and basophils is the primary effector pathway in allergic response. To achieve a targeted elimination of cells expressing Fc epsilon RI receptors, we constructed a chimeric protein in which a Fc fragment of mouse IgE is attached to a truncated form of Pseudomonas exotoxin (PE). To prepare the targeting moiety, we used a DNA sequence corresponding to amino acids 301-437, representing 30 residues of domain 2 and domain 3 of the mouse IgE constant region. This sequence was fused at the 5' of a cDNA encoding PE40, a truncated form of PE lacking the cell binding domain. The chimeric protein, termed Fc(2'-3)-PE40, was expressed in Escherichia coli and partially purified. The protein is highly cytotoxic to mouse mast cell lines and bone marrow-derived primary mast cells. This cytotoxicity is specific, as it could be blocked upon addition of whole IgE. Moreover, the protein had no effect on other cell lines of hemopoietic origin. The Fc(2'-3)-PE40 chimeric protein offers a new approach to the treatment of allergic disorders.

SUMMARYThe alarming increase in the incidence of allergic diseases in the past decade has led to a clear call for more effective treatment. Recently, we reported on the construction of a chimeric protein for targeted elimination of cells expressing Fc1 RI receptors. This chimeric protein, designated Fc 2 H -3 -PE 40 , is composed of a Fc fragment of mouse IgE attached to a truncated form of Pseudomonas exotoxin. The Fc 2 H -3 -PE 40 chimeric protein was found to be highly cytotoxic to mouse mast cell lines and primary mouse mast cells. We now demonstrate that Fc 2 H -3 -PE 40 successfully prevents the development of passive cutaneous anaphylaxis reaction (PCA) in mice. Treatment with Fc 2 H -3 -PE 40 for 7 days prevented the PCA reaction in mice by 80% compared with that in control mice given only PBS. Fc 2 H -3 -PE 40M , the mutated, enzymatically inactive analogue of Fc 2 H -3 -PE 40 , did not display this activity . Fc 2 H -3 -PE 40 was also effective when given as a single dose 16 h before antigen exposure, resulting in complete inhibition of the PCA reaction. Moreover, treatment with Fc 2 H -3 -PE 40 did not cause mast cell degranulation, as the serum histamine values of mice treated with Fc 2 H -3 -PE 40 were within the range obtained for control, untreated mice. Thus, the Fc 2 H -3 -PE 40 chimeric protein offers a novel approach to the treatment of allergic disorders.

IL2-PE40 is a chimeric protein composed of human interleukin 2 (IL2) genetically fused to the amino terminus of a truncated form of pseudomonas exotoxin lacking its cell recognition domain (PE40). IL2-PE40 is extremely cytotoxic to IL2 receptor positive cells. This chimeric protein was found to be an effective and selective immunosuppressive agent for IL2 receptor targeted therapy in many models of disorders of the immune response where activated T-cells play a crucial role. In an attempt to produce an improved IL2-PE40 chimeric protein, we constructed new IL2-PE derivatives. This was done by inserting defined DNA sequences within the chimeric gene encoding IL2-PE40. Inserted sequences represent motifs of other proteins known to be targeted and/or sorted to specific compartments inside or outside the cell. One of the proteins, IL2-PE40(LAP+DUP), containing a targeted signal for lysosomal membrane, was 2-3-fold more active than IL2-PE40. The insertion of the LAP sequence also increased the cytotoxicity of another IL2-PE derivative, IL2-PE664Glu. Our results suggest that a selective targeting of IL2-PE chimeric proteins to lysosomes may enable the proteins to reach the cytosol more efficiently, thus improving its specific cytotoxicity. The LAP (lysosomal alkaline phosphatase) sequence may be used as a common motif for increasing the cytotoxicity of other chimeric proteins to be used for targeted immunotherapy.

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