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.

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.

The catalytic role of the active site aspartic acid in serine proteases.

The role of the aspartic acid residue in the serine protease catalytic triad Asp, His, and Ser has been tested by replacing Asp102 of trypsin with Asn by site-directed mutagenesis. The naturally occurring and mutant enzymes were produced in a heterologous expression system, purified to homogeneity, and characterized. At neutral pH the mutant enzyme activity with an ester substrate and with the Ser195-specific reagent diisopropylfluorophosphate is approximately 10(4) times less than that of the unmodified enzyme. In contrast to the dramatic loss in reactivity of Ser195, the mutant trypsin reacts with the His57-specific reagent, tosyl-L-lysine chloromethylketone, only five times less efficiently than the unmodified enzyme. Thus, the ability of His57 to react with this affinity label is not severely compromised. The catalytic activity of the mutant enzyme increases with increasing pH so that at pH 10.2 the kcat is 6 percent that of trypsin. Kinetic analysis of this novel activity suggests this is due in part to participation of either a titratable base or of hydroxide ion in the catalytic mechanism. By demonstrating the importance of the aspartate residue in catalysis, especially at physiological pH, these experiments provide a rationalization for the evolutionary conservation of the catalytic triad.

Selective alteration of substrate specificity by replacement of aspartic acid-189 with lysine in the binding pocket of trypsin.

To test the role of Asp-189 which is located at the base of the substrate binding pocket in determining the specificity of trypsin toward basic substrates, this residue was replaced with a lysine residue by site-directed mutagenesis. Both rat trypsinogen and Lys-189 trypsinogen were expressed and secreted into the periplasmic space of Escherichia coli. The proteins were purified to homogeneity and activated by porcine enterokinase, and their catalytic activities were determined on natural and synthetic substrates. Lys-189 trypsin displayed no catalytic activity toward arginyl and lysyl substrates. Further, there was no compensatory change in specificity toward acidic substrates; no cleavage of aspartyl or glutamyl bonds was detected. Additional studies of substrate specificity involving gas-phase sequence analyses of digested natural substrates revealed an inherent but low chymotrypsin-like activity of trypsin. This activity was retained but modified by the Asp to Lys change at position 189. In addition to hydrolyzing phenylalanyl and tyrosyl peptide bonds, the mutant enzyme has the unique property of cleaving leucyl bonds. On the basis of computer graphic modeling studies of the Lys-189 side chain, it appears that the positively charged NH2 group is directed outside the substrate binding pocket. The resulting hydrophobic cavity may explain the altered substrate specificity of the mutant enzyme. The relatively low chymotrypsin-like activity of both recombinant enzymes may be due to distorted positioning of the scissile bond with respect to the catalytic triad rather than to the lack of sufficient interaction between the hydrophobic side chains and the substrate binding pocket of the enzyme.

Redesigning trypsin: alteration of substrate specificity.

A general method for modifying eukaryotic genes by site-specific mutagenesis and subsequent expression in mammalian cells was developed to study the relation between structure and function of the proteolytic enzyme trypsin. Glycine residues at positions 216 and 226 in the binding cavity of trypsin were replaced by alanine residues, resulting in three trypsin mutants. Computer graphic analysis suggested that these substitutions would differentially affect arginine and lysine substrate binding of the enzyme. Although the mutant enzymes were reduced in catalytic rate, they showed enhanced substrate specificity relative to the native enzyme. This increased specificity was achieved by the unexpected differential effects on the catalytic activity toward arginine and lysine substrates. Mutants containing alanine at position 226 exhibited an altered conformation that may be converted to a trypsin-like structure upon binding of a substrate analog.

A kinetic study of interactions of (Rp)- and (Sp)-adenosine cyclic 3',5'-phosphorothioates with type II bovine cardiac muscle adenosine cyclic 3',5'-phosphate dependent protein kinase.

The stereoselectivity of the adenosine cyclic 3',5'-phosphate (cAMP) binding sites on the regulatory subunit of the type II bovine cardiac muscle cAMP-dependent protein kinase was investigated by examining the interactions of (Rp)- and (Sp)-adenosine cyclic 3',5'-phosphorothioates (cAMPS) with these sites. While activation of the holoenzyme and binding to the regulatory subunit of the type II kinase were observed for both of these diastereomers, there were significant differences between the interactions of the cAMPS isomers with the enzyme. In particular, the Sp isomer is more potent than the Rp species not only in the activation of reconstituted, as well as directly isolated, holoenzyme but also in the inhibition of [3H]cAMP binding to the regulatory subunit. A marked preference for the binding of the Sp isomer to site 2 in the regulatory subunit exists. Hydrogen bonding of a functional group on the regulatory subunit with preferential orientation toward the exocyclic oxygen rather than the sulfur of the thiophosphoryl residue may be involved in the observed selectivity of cAMPS binding and activation. In addition to our findings on the stereoselectivity of the binding of cAMPS to cAMP-dependent protein kinase, we have established a method for the reconstitution of holoenzyme from the purified subunits without subjecting the regulatory protein to denaturing conditions.

Prostaglandin synthesis by isolated rat glomeruli: effect of angiotensin II.

Prostaglandins play a role in the regulation of renal blood flow and glomerular filtration. In the presence of [14C]arachidonate the pattern of prostaglandins produced by isolated glomeruli was PGF2 alpha > PGE2 > PGD2 = TXB2 = 6-keto-PGF1 alpha (a metabolite of prostacyclin). Glomeruli prelabeled with [14C]arachidonate showed an additional labeled prostaglandin that co-chromatographs with 6,15-diketo-13,13-dihydro-PGF1 alpha and may represent breakdown product of prostacyclin. Thus, prostacyclin, judged by its breakdown products, was the second most abundant prostaglandin produced. These results were confirmed by specific radioimmunoassays for PGF2 alpha, PGE2, and 6-keto-PGF1 alpha. Isolated glomeruli produced 1,740 pg x 10 min-1 x mg protein-1 of PGF2 alpha, 798 of 6-keto-PGF1 alpha, and 266 od PGE2. In prelabeled glomeruli angiotensin II causes a small but significant increase in 14C-labeled prostaglandins. Radioimmunoassay for 6-keto-PGF1 alpha showed that the angiotensin stimulation was specific for prostacyclin. Angiotensin II also affected the glomerular handling of [14C]arachidonate. It decreased the uptake of extracellular [14C]arachidonate and increased the incorporation of intracellular [14C]arachidonate into glomerular phospholipids. Based on these results, we propose that in the glomerulus angiotensin increases prostaglandin synthesis and stimulates deacylation and reacylation of phospholipids.