Alan J. Bitonti

Monomeric Fc Fusions

The delivery of therapeutic proteins by noninvasive routes of administration has been a challenging goal, hence current modes of delivery generally require injections. However, we have recently shown that a naturally occurring receptor, the neonatal Fc receptor (FcRn) can be utilized to carry aerosolized therapeutic proteins conjugated to a portion of its respective ligand (Fc domain of immunoglobulin G) across epithelial cells of the lung to effectively deliver biologically active molecules to the bloodstream. First-generation dimeric Fc fusion molecules were successfully transported by the pulmonary route and biologic activity was demonstrated in both non-human primates and human volunteers. Continuing efforts to improve transport efficiency have led to the development of an alternate configuration of Fc fusion proteins with improved characteristics. These second generation Fc fusion molecules are monomeric with respect to the therapeutic protein and dimeric with respect to the Fc region, and have been termed Fc fusion ‘monomers’. Several different Fc fusion monomers have demonstrated improved transport efficiency, achieving high bioavailabilities for pulmonary delivery in nonhuman primates. While the traditional dimeric Fc fusion molecule generally increases the half-life compared with the unconjugated effector molecule, the monomer configuration has been shown to result in an even greater extension of the circulating half-life, which improves pharmacokinetic parameters for protein therapeutics, whether administered by pulmonary delivery or injection. Finally, many of the Fc monomer fusions have enhanced biologic activity compared with the dimeric configuration. Because of these many advantages, the monomer configuration promises to be an enabling advance to achieve clinically relevant, noninvasive delivery with potentially less frequent administration regimens for a broad range of protein therapeutics. In addition, molecules that are comprised of heterodimeric subunits or multi-subunit complexes can also be constructed as Fc fusions that result in a molecule with enhanced pharmacokinetics and greater bioactivity. Several examples of novel Fc fusion proteins, both monomer and heterodimer are described herein.

Amelioration of Experimental Autoimmune Myasthenia Gravis in Rats by Neonatal FcR Blockade

The neonatal FcR (FcRn) plays a critical role in IgG homeostasis by protecting it from a lysosomal degradation pathway. It has been shown that IgG has an abnormally short half-life in FcRn-deficient mice and that FcRn blockade significantly increases the catabolism of serum IgG in mice. Therefore, reduction of serum IgG half-life may have therapeutic benefits in Ab-mediated autoimmune diseases. We have studied the therapeutic effects of an anti-rat FcRn mAb, 1G3, in two rat models of myasthenia gravis, a prototypical Ab-mediated autoimmune disease. Passive experimental autoimmune myasthenia gravis was induced by administration of an anti-acetylcholine receptor (AChR) mAb, and it was shown that treatment with 1G3 resulted in dose-dependent amelioration of the disease symptoms. In addition, the concentration of pathogenic Ab in the serum was reduced significantly. The effect of 1G3 was also studied in an active model of experimental autoimmune myasthenia gravis in which rats were immunized with AChR. Treatment with 1G3 significantly reduced the severity of the disease symptoms as well as the levels of total IgG and anti-AChR IgG relative to untreated animals. These data suggest that FcRn blockade may be an effective way to treat Ab-mediated autoimmune diseases.

Reduction of IgG in nonhuman primates by a peptide antagonist of the neonatal Fc receptor FcRn

The neonatal Fc receptor FcRn provides IgG molecules with their characteristically long half-lives in vivo by protecting them from intracellular catabolism and then returning them to the extracellular space. Other investigators have demonstrated that mice lacking FcRn are protected from induction of various autoimmune diseases, presumably because of the accelerated catabolism of pathogenic IgGs in the animals. Therefore, targeting FcRn with a specific inhibitor may represent a unique approach for the treatment of autoimmune disease or other diseases where the reduction of pathogenic IgG will have a therapeutic benefit. Using phage display peptide libraries, we screened for ligands that bound to human FcRn (hFcRn) and discovered a consensus peptide sequence that binds to hFcRn and inhibits the binding of human IgG (hIgG) in vitro. Chemical optimization of the phage-identified sequences yielded the 26-amino acid peptide dimer SYN1436, which is capable of potent in vitro inhibition of the hIgG–hFcRn interaction. Administration of SYN1436 to mice transgenic for hFcRn induced an increase in the rate of catabolism of hIgG in a dose-dependent manner. Treatment of cynomolgus monkeys with SYN1436 led to a reduction of IgG by up to 80% without reducing serum albumin levels that also binds to FcRn. SYN1436 and related peptides thus represent a previously uncharacterized family of potential therapeutic agents for the treatment of humorally mediated autoimmune and other diseases.

Catalytic irreversible inhibition of Trypanosoma brucei brucei ornithine decarboxylase by substrate and product analogs and their effects on murine trypanosomiasis

Ornithine decarboxylase from Trypanosoma brucei brucei was inhibited by several substrate (ornithine) and product (putrescine) analogs both in vitro and in vivo. Since alpha-difluoromethylornithine is effective for the treatment of experimental and clinical African trypanosomiasis, it was possible that the more potent ornithine and putrescine analogs might be more active in treating the disease. However, only alpha-monofluoromethyldehydroornithine methyl ester was more potent than alpha-difluromethylornithine against mouse trypanosomiasis and warrants further study in model infections.

Necessity of antibody response in the treatment of African trypanosomiasis with α-difluoromethylornithine

The role of the immune system in the clearance of Trypanosoma brucei brucei from the bloodstream during alpha-difluoromethylornithine (DFMO) treatment was studied by determining the effects of dexamethasone on immune and therapeutic responses in rats infected with T.b. brucei. Normal DFMO-treated animals exhibited strong antibody responses to trypanosomes and were cured of T. b. brucei infection by a 7-day regimen of 2% DFMO in drinking water. Animals pretreated with dexamethasone were not cured by the same level of DFMO treatment. Nonetheless, in rats pretreated with dexamethasone, trypanosomes were cleared from blood during treatment with DFMO, but all immunocompromised animals eventually succumbed to relapses of the infection. Athymic (nude) mice were cured of T. b. brucei infection by a 72-hr course of treatment with 2% DFMO in their drinking water. These findings suggest that relatively low levels of T-cell-independent, antitrypanosomal antibodies are adequate to clear the bloodstream of parasites during DFMO therapy but that an intact immune response is necessary for cures of the disease to be obtained.

Plasmodium falciparum and Plasmodium berghei: Effects of ornithine decarboxylase inhibitors on erythrocytic schizogony

Five ornithine decarboxylase inhibitors: alpha-difluoromethylornithine (DFMO) (eflornithine); alpha-monofluoromethyl-3,4-dehydroornithine; alpha-monofluoromethyl-3,4-dehydroornithine methyl ester; alpha-monofluoromethyl-3,4-dehydroornithine ethyl ester; and (2R,5R)-delta-methyl-alpha-acetylenic putrescine were shown to inhibit erythrocytic schizogony of Plasmodium falciparum in vitro and reduced spermidine levels in infected erthrocytes. Only DFMO was effective at limiting erythrocytic schizogony of P. berghei in vivo. Administration of DFMO as a 2% solution in the drinking water for 4 days reduced parasitemia in mice by 50% in a 4-day suppression test but did not increase survival time of infected mice. This is the first demonstration of an effect of DFMO on plasmodial erythrocytic schizogony in vivo and suggests that interference with polyamine biosynthesis may, in fact, be a viable chemotherapeutic target in erythrocytic malaria.

Biochemical evidence for the presence of arginine decarboxylase activity in Trypanosoma cruzi.

Trypanosoma cruzi was found to release 14CO2 from radiolabeled arginine, and this effect was inhibited by either DL-alpha-difluoromethylarginine or monofluoromethylagmatine, both specific inhibitors of arginine decarboxylase (ADC). Furthermore, agmatine, which can be derived metabolically only by ADC-mediated arginine decarboxylation, was produced when T. cruzi was incubated with radiolabeled arginine, and agmatine production was inhibited in the presence of DL-alpha-difluoromethylarginine. These results constitute direct biochemical evidence for the presence in T. cruzi of ADC, an enzyme that does not occur in mammalian cells.

Alpha-difluoromethylornithine resistance in Leishmania donovani is associated with increased ornithine decarboxylase activity.

The promastigote form of Leishmania donovani is sensitive to growth inhibition by DL-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC), the first enzyme of the polyamine biosynthetic pathway, with an EC50 value of approximately 30 microM. Exposure of a wild type (DI700) cell population to gradually increasing concentrations of DFMO resulted in the selection of a strain of Leishmania, DFMO-10, which was capable of proliferating in 10 mM DFMO. DFMO-10 cells possessed an EC50 value for DFMO greater than 4 mM, and were cross-resistant to alpha-methylornithine, alpha-monofluoromethyl-3,4-dehydroornithine methyl ester, and delta-methyl-acetylenic putrescine, three other inhibitors of ODC activity. DI700 and DFMO-10 cells accumulated and/or transported [3H]DFMO and a spectrum of basic, neutral, and acidic amino acids at comparative rates. However, the DFMO-resistant Leishmania, if suspended in culture medium in the absence of DFMO for several days, expressed up to 15-fold greater levels of ODC activity than did wild-type cells. The overexpressed ODC in mutant cells appeared kinetically normal, since the ODC activities from DI700 and DFMO-10 cells possessed similar apparent Km values for ornithine and were equally sensitive to inactivation by DFMO. Incubation of extracts of DFMO-10 cells, but not of wild-type parental cells, with [3H]DFMO for 1 h resulted in the labeling of a polypeptide, presumably ODC, which migrated with a molecular weight of 76,000 +/- 4000 on SDS-gel electrophoretograms. As a consequence of the elevated ODC activities, the levels of putrescine in mutant cells released from DFMO exposure were also elevated by about 15-fold over those of wild-type cells, although spermidine levels in DI700 and DFMO-10 cells were similar. In the absence of prolonged selective pressure, the resistance to DFMO, the ODC activity, and the putrescine levels of DFMO-10 cells all returned to those of wild type cells, indicating that the mutant phenotype of DFMO-selected L. donovani was unstable.

Inhibition of bacterial aminopropyltransferases by S-adenosyl-1,8-diamino-3-thiooctane and by dicyclohexylamine

Bacterial aminopropyltransferases from Escherichia coli, Serratia marcescens and Pseudomonas aeruginosa were strongly inhibited by S‐adenosyl‐1,8‐diamino‐3‐thiooctane (AdoDATO) and by dicyclohexylamine. The sensitivity to these drugs in vitro was comparable to that of mammalian spermidine synthase, but AdoDATO was much less potent in reducing spermidine content in the bacteria than in mammalian cells. Although AdoDATO was a stronger inhibitor than dicyclohexylamine in vitro, dicyclohexylamine was more active in reducing bacterial spermidine levels in vivo, suggesting that it is take up better or is more stable in the cell and is the preferable compound for in vivo studies in microorganisms. The strong inhibition of spermidine synthases by AdoDATO which is a transition state analog supports the concept that these enzymes proceed by a single displacement reaction, rather than by a ping‐pong mechanism.

Characterization of spermidine synthase from Trypanosoma brucei brucei

Spermidine synthase from Trypanosoma brucei brucei was characterized and found to be similar to spermidine synthase from other sources. The Km for putrescine was found to be 0.2 mM and the Km for decarboxylated S-adenosylmethionine 0.1 microM. The approximate molecular weight of the enzyme was 74 000 as determined by a combination of molecular sieve chromatography and sucrose density gradient centrifugation. Spermidine synthase activity was markedly inhibited in vitro by dicyclohexylamine (50% inhibition at 3 microM) and cyclohexylamine (50% inhibition at 15 microM); both being competitive inhibitors with respect to putrescine. S-Adenosyl-1,8-diamino-3-thiooctane, a nucleoside bisubstrate analog, was also a potent inhibitor of enzyme activity (50% inhibition at 25 microM). Administration of dicyclohexylamine to mice with trypanosomiasis resulted in no increase in survival time probably due to the lack of effect on trypanosome spermidine concentrations. Other possible inhibitors remain to be tested in vivo.