A T-cell-selective interleukin 2 mutein exhibits potent antitumor activity and is well tolerated in vivo.

Human interleukin 2 (IL-2; Proleukin) is an approved therapeutic for advanced-stage metastatic cancer; however, its use is restricted because of severe systemic toxicity. Its function as a central mediator of T-cell activation may contribute to its efficacy for cancer therapy. However, activation of natural killer (NK) cells by therapeutically administered IL-2 may mediate toxicity. Here we have used targeted mutagenesis of human IL-2 to generate a mutein with approximately 3,000-fold in vitro selectivity for T cells over NK cells relative to wild-type IL-2. We compared the variant, termed BAY 50-4798, with human IL-2 (Proleukin) in a therapeutic dosing regimen in chimpanzees, and found that although the T-cell mobilization and activation properties of BAY 50-4798 were comparable to human IL-2, BAY 50-4798 was better tolerated in the chimpanzee. BAY 50-4798 was also shown to inhibit metastasis in a mouse tumor model. These results indicate that BAY 50-4798 may exhibit a greater therapeutic index than IL-2 in humans in the treatment of cancer and AIDS.

Identification and cloning of human placental bikunin, a novel serine protease inhibitor containing two Kunitz domains.

Interrogation of the public expressed sequence tag (EST) data base with the sequence of preproaprotinin identified ESTs encoding two potential new members of the Kunitz family of serine protease inhibitors. Through reiterative interrogation, an EST contig was obtained, the consensus sequence from which encoded both of the novel Kunitz domains in a single open reading frame. This consensus sequence was used to direct the isolation of a full-length cDNA clone from a placental library. The resulting cDNA sequence predicted a 252-residue protein containing a putative NH2-terminal signal peptide followed sequentially by each of the two Kunitz domains within a 170-residue ectodomain, a putative transmembrane domain, and a 31-residue hydrophilic COOH terminus. The gene for this putative novel protein was mapped by use of a radiation hybrid panel to chromosome 19q13, and Northern analysis showed that the corresponding mRNA was expressed at high levels in human placenta and pancreas and at lower levels in brain, lung, and kidney. An endogenous soluble form of this protein, which was designated as placental bikunin, was highly purified from human placenta by sequential kallikrein-Sepharose affinity, gel filtration, and C18 reverse-phase chromatography. The natural protein exhibited the same NH2 terminus as predicted from the cloned cDNA and inhibited trypsin, plasma kallikrein, and plasmin with IC50 values in the nanomolar range.

Characterization of placental bikunin, a novel human serine protease inhibitor.

We reported previously the cloning of a novel human serine protease inhibitor containing two Kunitz-like domains, designated as placental bikunin, and the subsequent purification of a natural counterpart from human placental tissue (Marlor, C. W., Delaria, K. A., Davis, G., Muller, D. K., Greve, J. M., and Tamburini, P. P. (1997) J. Biol. Chem. 272, 12202-12208). In this report, the 170 residue extracellular domain of placental bikunin (placental bikunin(1-170)) was expressed in baculovirus-infected Sf9 cells using its putative signal peptide. The resulting 21.3-kDa protein accumulated in the medium with the signal peptide removed and could be highly purified by sequential kallikrein-Sepharose and C18 reverse-phase chromatography. To provide insights as to the potential in vivo functions of this protein, we performed an extensive investigation of the inhibitory properties of recombinant placental bikunin(1-170) and both of its synthetically prepared Kunitz domains. All three proteins inhibited a number of serine proteases involved in the intrinsic pathway of blood coagulation and fibrinolysis. Placental bikunin(1-170) formed inhibitor-protease complexes with a 1:2 stoichiometry and strongly inhibited human plasmin (Ki = 0.1 nM), human tissue kallikrein (Ki = 0.1 nM), human plasma kallikrein (Ki = 0.3 nM) and human factor XIa (Ki = 6 nM). Conversely, this protein was a weaker inhibitor of factor VIIa-tissue factor (Ki = 1.6 microM), factor IXa (Ki = 206 nM), factor Xa (Ki = 364 nM), and factor XIIa (Ki = 430 nM). This specificity profile was to a large extent mimicked, albeit with reduced potency, by the individual Kunitz domains. As predicted from this in vitro specificity profile, recombinant placental bikunin(1-170) prolonged the clotting time in an activated partial thromboplastin time assay.

High-performance liquid chromatographic assay for tryptase based on the hydrolysis of dansyl-vasoactive intestinal peptide.

A rapid, sensitive, semiautomated method to measure the activity of mast cell-derived tryptase has been developed. The assay is based on the tryptase-mediated hydrolysis of vasoactive intestinal peptide that was modified to include an N-terminal dansyl reporter group. Tryptase cleaves vasoactive intestinal peptide (VIP) producing two major and two minor products. Full-length VIP was separated from the proteolysis products by reverse-phase (C18) chromatography. The amount of each product as well as the amount of full-length substrate remaining was determined using an in-line fluorescence detector. As little as 0.5 pmol of peptide could be detected. Predominant cleavages were after Arg-14 and Lys-20 with minor cleavages after Lys-15 and Lys-21. The hydrolysis of VIP at Arg-14 was slightly affected by the presence of the dansyl group at the N-terminus. While the Km value was unaffected, the kcat value decreased, yielding a kcat/Km ratio that was eightfold lower. The addition of calcium chloride to the reaction mixture resulted in a slight decrease in the kcat/Km ratio. Cleavage of dansyl-VIP by tryptase was completely inhibited by DesPro2-Arg15-Aprotinin.

Inhibition of cathepsin L-like cysteine proteases by cytotoxic T-lymphocyte antigen-2 beta.

The protein sequence of cytotoxic T-lymphocyte antigen-2 beta (CTLA-2 beta) is 36% identical to the proregion of mouse cathepsin L (Denizot, F., Brunet, J.F., Roustan, P., Harper, K., Suzan, M., Luciani, M. F., Mattei, M. G., and Goldstein, P. (1989) Eur. J. Immunol. 19, 631-635). Here we report the expression, purification, and characterization of recombinant murine CTLA-2 beta. The protein was purified by consecutive gel-filtration, anion-exchange, and reverse-phase (C4) chromatography. Purified CTLA-2 beta exists in solution primarily as a dimer but also as a disulfide-linked tetramer as judged by size exclusion chromatography. Circular dichroism studies suggest that the dimeric form of the protein contains 8% alpha-helix, 67% beta-sheet, and 21% random coil and also indicates that there is a conformational change upon formation of the tetramer. The protein is a competitive inhibitor of certain cysteine proteases including papain (Ki = 25 nM), cathepsins L (Ki = 24 nM) and H (IC50 = 67 nM) but not cathepsin B. CTLA-2 beta forms a noncovalent complex with cathepsin L and has a stoichiometry of binding to papain of 1 mol of CTLA-2 beta/mol of papain. There is no homology between CTLA-2 beta and any of the known cysteine protease inhibitors, including the kininogens and cystatins. Therefore, CTLA-2 beta represents a novel class of cysteine protease inhibitor that is specific for the cathepsin L family of proteases.

An isotope edited classical Raman difference spectroscopic study of the interactions of guanine nucleotides with elongation factor Tu and H-ras p21.

We have measured the Raman spectrum of GDP bound to the elongation factor protein, EF-Tu, and the c-Harvey-ras protein, p21, two proteins of the guanine nucleotide binding family. In order to separate the Raman spectrum of the nucleotide from the much more intense protein spectrum, we investigate the feasibility of "tagging" the normal modes of the nucleotide by isotopic substitution, here by incoporating deuterium-labeled guanine at the C8 position into the active site. A difference spectrum between the labeled and unlabeled protein-nucleotide complex shows the changes in the Raman spectrum of the bound nucleotide that arise from the isotopic exchange. We find that surprisingly good Raman spectra of bound ligands can be obtained with this method and that the method can be easily generalized to other systems. The data show that the guanine amino group of the nucleotide interacts differently with both EF-Tu and p21 than it does with water, showing a change in hydrogen-bonding properties upon binding. On the other hand, no change in hydrogen bonding is observed at guanine's N7. The data strongly suggest that the conformation of the nucleotide when bound to EF-Tu and that p21 is the C2' endo pucker of the ribose ring and anti about the glycosidic bond. These results are compared to previous structural and chemical studies.

Stabilization of the Escherichia coli elongation factor Tu-GTP-aminoacyl-tRNA complex.

The effect of ammonium sulfate on the Escherichia coli elongation factor Tu-GTP-aminoacyl-tRNA complex has been studied. The half-lives of 12 E. coli aminoacyl-tRNA species were determined at 37 degrees C in the presence and absence of an equimolar amount of EF-Tu-GTP and in the presence and absence of 1.5 M ammonium sulfate. The results indicate that the addition of 1.5 M ammonium sulfate to the ternary complex increased the stability of all 12 complexes studied. In addition, the effects of various salts and crystallization agents on the stability of the E. coli EF-Tu-GTP-phenylalanyl-tRNA complex was studied in detail. Binding parameters were also measured under various conditions at 37 degrees C. The results indicate that the stability and the Kassoc of the ternary complex, using phenylalanyl-tRNA, can be increased by the presence of polyethylene glycol or ammonium sulfate.

Three-dimensional models of the GDP and GTP forms of the guanine nucleotide domain of Escherichia coli elongation factor Tu.

Three-dimensional models of the GDP and GTP forms of the guanine nucleotide domain of Escherichia coli elongation factor Tu have been derived from the atomic coordinates of the trypsin-modified form of EF-Tu-GDP and by comparison with the ras p21 structures. The significance of the differences in the guanine nucleotide binding sites of EF-Tu and ras p21 are discussed. Crystallization of the EF-Tu-GMPPNP complex is reported.

Preliminary crystallographic analysis of a complex between tetracycline and the trypsin-modified form of Escherichia coli elongation factor Tu.

Crystals of a complex between the antibiotic tetracycline and the trypsin-modified form of the Escherichia coli protein elongation factor Tu have been grown in a form suitable for high-resolution X-ray diffraction analysis. The crystals belong to space group P2(1), with cell dimensions a = 69.7 A, b = 156.4 A, c = 135.4 A and beta = 95.3 degrees, and contain six molecules of the complex per asymmetric unit. The crystals are well ordered and diffract to a resolution of 2.3 A.

Preparation of Escherichia coli elongation factor Tu-guanosine 5'-triphosphate analogs.

A simple procedure for the bulk preparation of 20 mg of Escherichia coli elongation factor (EF)-Tu-GTP analogs is described. The protocol is based upon the preparation and stabilization of nucleotide-free EF-Tu using an EF-Ts affinity chromatographic resin. The procedure is a general one for the preparation of any GTP analog of EF-Tu.