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Brain fatty acid-binding protein (B-FABP) is normally expressed in radial glial cells, where it plays a role in the establishment of the radial glial fiber network required for neuronal migration. B-FABP is also expressed in astrocytoma tumors and in some malignant glioma cell lines. To address the role of B-FABP in malignant glioma, we have studied the growth properties of clonal populations of malignant glioma cells modified for B-FABP expression. Here, we demonstrate that expression of B-FABP in B-FABP-negative malignant glioma cells is accompanied by the appearance of radial glial-like properties, such as increased migration and extended bipolar cell processes, as well as reduced transformation. Conversely, B-FABP depletion in B-FABP-positive malignant glioma cells results in decreased migration, reduction in cell processes, and a more transformed phenotype. Moreover, expression of B-FABP in astrocytomas is associated with regions of tumor infiltration and recurrence. Rather than being a direct manifestation of the tumorigenic process, we propose that the ability of high-grade astrocytoma cells to migrate long distances from the primary tumor reflects properties associated with their cell of origin. Thus, targeting B-FABP-expressing cells may make a significant impact on the treatment of these tumors.

Murine monoclonal antibody 1A4A1 has been shown to recognize a conserved neutralizing epitope of envelope glycoprotein E2 of Venezuelan equine encephalitis virus. It is a potential candidate for development of a second generation antibody for both immunodiagnosis and immunotherapy. In order to minimize the immunogenicity of murine antibodies and to confer human immune effector functions on murine antibodies, a recombinant gene fusion was constructed. It encoded a human IgG1 heavy chain constant region and a single-chain fragment variable antibody of 1A4A1. After expression in bacteria as inclusion bodies, the recombinant antibody was purified and refolded in vitro. The recombinant soluble antibody was demonstrated to retain high antigen-binding affinity to Venezuelan equine encephalitis virus and to possess some human IgG crystallizable fragment domain functions, such as recognition by protein G and human complement C1q binding. On non-reducing and reducing gel electrophoresis analysis of proteolytic fragments of the recombinant antibody, disulfide bond formation was found in the hinge region of the antibody. From these data, it was concluded that the recombinant antibody was capable of antigen recognition, and retained several functional activities. This work forms the basis for characterization of the recombinant antibody as to efficacy in vivo.

Previously cloned recombinant A116 single chain fragment variable (scFv) antibody gene has been re-engineered for enhanced reactivity to Venezuelan equine encephalitis virus (VEE) successfully. A PCR-based site-directed mutagenesis approach was adopted to re-introduce the three single-base deletions in the 5' region of the V(L) gene of A116, corresponding to the framework-1 region. The mutagenized A116 was designated as MA116. The introduction of these three bases corrected a localized frame-shift to a consensus framework-1 amino acid sequence. Four MA116 clones (MA116-4, MA116-14, MA116-15, and MA116-16) have been analysed in detail for their reactivity to VEE antigen, and all showed varying degrees of reactivity to VEE antigen. ScFv antibody expressed by MA116-14, MA116-15, and MA116-16 clones showed three to five-fold enhanced enzyme-linked immunosorbant assay reactivity to VEE antigen over the parental A116 clone, while scFv antibody from MA116-4 was less reactive than A116 clone. MA116-15 purified scFv protein showed comparable reactivity to the parental 1A4A-1 monoclonal antibody in recognizing VEE antigen. Sequence analysis revealed that only MA116-15 had incorporated the three intended base insertions. The varying degrees of reactivity of MA116 clones are discussed in light of their molecular changes.

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