Monoclonal antibody 13A to murine CD44 was used to bind the alpha-particle emitter 213Bi to cell surfaces of cultured EMT-6 or Line 1 tumor cells. Data on kinetics and saturation of binding, cell shape and nuclear size were used to calculate the absorbed dose to the nuclei. Treatment of monolayer cells with [213Bi]MAb 13A produced a classical exponential survival curve with no apparent shoulder. Microdosimetry analyses indicated that 1.4-1.7 Gy produced a 37% surviving fraction (D0). Multicellular spheroids were shown to bind [213Bi]MAb 13A mainly on the outer cell layer. Relatively small amounts of activity added to the spheroids resulted in relatively large absorbed doses. The result was that 3-6-fold less added radioisotope was necessary to kill similar fractions of cells in spheroids than in monolayer cells. These data are consistent with the interpretation that the alpha particles from a single 213Bi atom bound to one cell can penetrate and kill adjacent cells. Flow cytometry was used to sort cells originating from the periphery or from the interior of spheroids. Cells from the outside of the [213Bi]MAb 13A exposed spheroids had a lower surviving fraction per administered activity than cells from the interior. Cells were killed efficiently in spheroids up to 20-30 cells in diameter. The data support the hypothesis that alpha-particle emitters should be very efficient at killing cells in micrometastases of solid tumors.

Second generation monoclonal antibodies to the alpha 6 beta 4 subunits of human integrins have been prepared. MAbs 450-9D, 10D, and 11A1 react at different sites on the beta 4 molecule and MAbs 450-30A1 and 33D react at the same site on the alpha 6 subunit. Double determinant (two-site) radioimmunoassays using combinations of these MAbs have been developed. Two assays for beta 4 distinguish between the whole beta 4 molecule and the beta 4 molecule truncated from the C-terminus (form c) while another assay measures the presence of alpha 6 subunits. Data from the two-site assays support the following conclusions: (1) Colon tumors and normal colon mucosa express large amounts of alpha 6 beta 4 although only form c of the beta 4 was detected; (2) There is no evidence for alpha 6 beta 1 expression in colon; however, some of this complex may be present in certain lung tumors. The extracellular domains of alpha 6 and beta 4 can associate with each other even if the cytoplasmic domain of the beta 4 subunit is not present. MAbs to specific domains of the beta 4 molecule may be useful in analyses of forms a and c in normal and malignant tissue. The fact that only the largest beta 4 molecule "a" retains the phosphorylation site may have functional significance.

One of the most promising methods for large-scale studies of protein interactions is isolation of an affinity-tagged protein with its in vivo interaction partners, followed by mass spectrometric identification of the copurified proteins. Previous studies have generated affinity-tagged proteins using genetic tools or cloning systems that are specific to a particular organism. To enable protein-protein interaction studies across a wider range of Gram-negative bacteria, we have developed a methodology based on expression of affinity-tagged "bait" proteins from a medium copy-number plasmid. This construct is based on a broad-host-range vector backbone (pBBR1MCS5). The vector has been modified to incorporate the Gateway DEST vector recombination region, to facilitate cloning and expression of fusion proteins bearing a variety of affinity, fluorescent, or other tags. We demonstrate this methodology by characterizing interactions among subunits of the DNA-dependent RNA polymerase complex in two metabolically versatile Gram-negative microbial species of environmental interest, Rhodopseudomonas palustris CGA010 and Shewanella oneidensis MR-1. Results compared favorably with those for both plasmid and chromosomally encoded affinity-tagged fusion proteins expressed in a model organism, Escherichia coli.