Deregulation of TGF-b superfamily signaling is a causative factor in many diseases. Here we describe a protein engineering strategy for the generation of single-chain bivalent receptor traps for TGF-b superfamily ligands. Traps were assembled using the intrinsically disordered regions flanking the structured binding domain of each receptor as "native linkers" between two binding domains. This yields traps that are approximately threefold smaller than antibodies and consists entirely of native receptor sequences. Two TGF-b type II receptor-based, single-chain traps were designed, termed (TbRII)2 and (TbRIIb)2, that have native linker lengths of 35 and 60 amino acids, respectively. Both single-chain traps exhibit a 100 to 1,000 fold higher in vitro ligand binding and neutralization activity compared with the monovalent ectodomain (TbRII-ED), and a similar or slightly better potency than pan-TGF-b-neutralizing antibody 1D11 or an Fc-fused receptor trap (TbRII-Fc). Despite its short in vivo half-life (<1 hour), which is primarily due to kidney clearance, daily injections of the (TbRII)2 trap reduced the growth of 4T1 tumors in BALB/c mice by 50%, an efficacy that is comparable with 1D11 (dosed thrice weekly). In addition, (TbRII)2 treatment of mice with established 4T1 tumors (100 mm 3 ) significantly inhibited further tumor growth, whereas the 1D11 antibody did not. Overall, our results indicate that our rationally designed bivalent, single-chain traps have promising therapeutic potential.

A unique immunoliposome has been developed as a drug delivery vehicle for immunotherapy. Human recombinant interleukin-2 (IL-2) has been chemically coupled to the external surface of small unilamellar vesicles (SUVs) containing methotrexate as a candidate immunosuppresive agent in order to specifically direct the drug-bearing liposome to activated T-cells expressing the high affinity IL-2 receptor. This drug delivery system is designed to deliver an immunosuppressive agent to those cells that actively participate in disorders such as graft rejection without delivering an effective but potentially toxic drug to all cells of the immune system as well as other healthy tissues. IL-2 was chemically modified with succinimidyl 4-[p-maleidophenyl butyrate](SMPB) while the receptor binding domain on IL-2 was protected by monoclonal anti-IL-2 bound to Protein A-Silica Gel. The antibody recognizes the receptor binding domain of the IL-2 molecule. The IL-2 was derivatized with S-succinimidyl-S-thioacetate (SATA) in order to add an acetyl thioester group to the lipid and create the complex. The derivatized lipid (SATA-PE) was then part of the liposome formulation containing DSPC:cholesterol: SATA-PE at a mole ratio of 1.5:1.0:0.26. SMPB-IL-2 was covalently coupled to the external surface of the SUV after deacetylation of the thioester moiety at pH 7.4 in PBS. Liposomes prepared by sonication or extrusion had an average diameter of 46-50 nm. SUV-IL-2 bound to the high affinity IL-2 receptor as measured by competitive binding assays and Scatchard analysis using 111InCl2-loaded liposomes The preparation exhibited a binding constant of 30 pM, consistent with values for free IL-2 cited in the literature. SUV IL-2 could be used as the sole source of IL-2 for the murine CTLL-2 T-cell line or for human mitogen-activated PBLs. The presence of IL-2 coupled to the surface was absolutely required for delivery of the drug to the cell. When methotrexate was encapsulated within the internal aqueous space, receptor-mediated endocytosis led to the inhibition of proliferation due to delivery of MTX to the cytoplasm of the cell. More than 90% of the methotrexate was retained within the liposome during storage over a 24-h period at 4 degrees C. This immunoliposome represents a new class of cell specific immunoliposomes whose entry into the cell is controlled by a cell surface receptor.

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Two perturbed gamma-gamma directional correlation experiments have been performed to examine internal fields and correlation times for the active region and active site in bovine carbonic anhydrase (BCA-B). A sample of BCA-B with 133Ba implanted in the active region was prepared and time-dependence of the anisotropy of the 356–81 keV cascade of 133Cs was measured at room temperature. Time-differential and time-integrated behavior of the perturbation factor indicates a possible time-dependent interaction with a relaxation constant λ22 = 5×107 sec−1. Using a previously reported value of rotational correlation time of BCA-B, the perturbation factor is found to be caused by a time-dependent electric quadrupole interaction with an electric field gradient having a time-averaged value of 1 V/Å2 at the nuclear site of 57Co. In a second experiment, BCA-B was demetallated and the zinc ion at the active site replaced by 57Co with no appreciable loss of specific activity. Time-differential measurements of the anisotropy of the 122–14 keV case in 57Fe showed that the anisotropy goes rapidly to zero. The time-integrated perturbation factor gives λ22 = 7×107 sec−1 at the active site.

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