Selective clearance of glycoforms of a complex glycoprotein pharmaceutical caused by terminal N-acetylglucosamine is similar in humans and cynomolgus monkeys

To understand how the carbohydrate moieties of a recombinant glycoprotein affected its pharmacokinetic (PK) properties, the glycan distribution was directly assessed from serial blood samples taken during PK studies in cynomolgus monkeys and humans. The protein studied was an immunoadhesin (lenercept), containing an Fc domain from human immunoglobulin G (IgG-1) and two copies of the extensively glycosylated extra cellular domain of tumor necrosis factor receptor p55. The protein was recovered in pure form using a dual column, immunoaffinity-reversed-phase high-performance liquid chromatography method. The glycans were released and analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Alternatively, trypsin was used to obtain glycopeptides, and these were analyzed by MALDI-TOF. The composition versus time profiles show that the distribution of glycans in the Fc domain was not altered over 10 days of circulation, consistent with their sequestration in the interior of the protein. However, the glycan composition in the receptor domain was changed dramatically in the first 24 h and then remained relatively constant. Analysis of the acidic glycans (derived exclusively from the receptor domain) showed that, in the rapid initial phase of clearance, glycans carrying terminal N-acetylglucosamine (tGlcNAc) were selectively cleared from the circulation. This phenomenon occurred similarly in humans and cynomolgus monkeys. Sialic acid content and terminal galactose showed only small changes. These data confirm the correlation of tGlcNAc and half-life of the molecule, and support the hypothesis that the mannose receptor (which can also bind tGlcNAc) causes the variable clearance of this molecule.     

PK/DB: database for pharmacokinetic properties and predictive in silico ADME models

The study of pharmacokinetic properties (PK) is of great importance in drug discovery and development. In the present work, PK/DB (a new freely available database for PK) was designed with the aim of creating robust databases for pharmacokinetic studies and in silico absorption, distribution, metabolism and excretion (ADME) prediction. Comprehensive, web-based and easy to access, PK/DB manages 1203 compounds which represent 2973 pharmacokinetic measurements, including five models for in silico ADME prediction (human intestinal absorption, human oral bioavailability, plasma protein binding, blood-brain barrier and water solubility). AVAILABILITY: http://www.pkdb.ifsc.usp.br     

Relationship of effective molecular size to systemic clearance in rats of recombinant interleukin-2 chemically modified with water-soluble polymers

We have examined the effects of a variety of chemical modifications to recombinant human interleukin-2 (rIL-2) on its pharmacokinetic behavior in rats. Unmodified rIL-2 is cleared from plasma with half-lives of 3 and 44 min for the alpha and beta phases. Modification of rIL-2 with monomethoxy polyethylene glycol or polyoxyethylated glycerol increased the half-lives as much as 20-fold, although the volume of distribution remained unchanged at 88 +/- 13 ml/kg. The clearance rates correlated with the effective molecular size of the modified protein determined by size exclusion chromatography. Clearance decreased rapidly as the effective molecular size increased from 19.5 to 70 kDa, whereas above 70 kDa the clearance decreased more slowly. This abrupt change at 70 kDa may be related to the permeability threshold of the kidney glomerular membrane which retains proteins larger than albumin in the plasma. Using the relationship between clearance and effective molecular size, the clearance rates of mixtures of modified rIL-2 could be predicted based on their average effective molecular size. Since the effectiveness of rIL-2 therapy is likely to be related to its pharmacokinetic behavior, the ability to design a molecule with a predictable time course in plasma provides a means to study this relationship.     

Correlations between pharmacokinetics of IgG antibodies in primates vs. FcRn-transgenic mice reveal a rodent model with predictive capabilities

Transgenic mice expressing human neonatal Fc receptor (FcRn) instead of mouse FcRn are available for IgG antibody pharmacokinetic (PK) studies. Given the interest in a rodent model that offers reliable predictions of antibody PK in monkeys and humans, we set out to test whether the PK of IgG antibodies in such mice correlated with the PK of the same antibodies in primates. We began by using a single research antibody to study the influence of: (1) different transgenic mouse lines that differ in FcRn transgene expression; (2) homozygous vs. hemizygous FcRn transgenic mice; (3) the presence vs. absence of coinjected high-dose human intravenous immunoglobulin (IVIG), and (4) the presence vs. absence of coinjected high-dose human serum albumin (HSA). Results of those studies suggested that use of hemizygous Tg32 mice (Tg32 hemi) not treated with IVIG or HSA offered potential as a predictive model for PK in humans. Mouse PK studies were then done under those conditions with a panel of test antibodies whose PK in mice and primates is not significantly affected by target binding, and for which monkey or human PK data were readily available. Results from the studies revealed significant correlations between terminal half-life or clearance values observed in the mice and the corresponding values reported in humans. A significant relationship in clearance values between mice and monkeys was also observed. These correlations suggest that the Tg32 hemi mouse model, which is both convenient and cost-effective, can offer value in predicting antibody half-life and clearance in primates.     

An immunochemical study of the human blood group P1, P, and PK glycosphingolipid antigens.

The erythrocyte PK and P blood group antigens have been identified as ceramide trihexoside (CTH), Gal-(alpha, 1 leads to 4)Gal(beta, 1 leads to 4)Glc-Cer, and globoside, GalN-Ac(beta, 1 leads to 3)Gal(alpha, 1 leads to 4)Gal(beta, 1 leads to 4)Glc-Cer, respectively, and the following structure has been proposed for the P1 antigen: Gal(alpha, 1 leads to 4)Gal(beta, 1 leads to 4)GlcNAc(beta, 1 leads to 3)Gal(beta, 1 leads to 4)Glc-Cer. Although the P1 and PK determinants have identical terminal disaccharides, CTH did not inhibit anti-P1. The P1 glycolipid and hydatid cyst glycoprotein inhibited the agglutination of P1K erythrocytes by anti-P1 and unabsorbed anti-P1PPK sera, but neither antigen inhibited a specific anti-PK serum. The P1 and PK glycolipids were equally effective in inhibiting the hemagglutinating activity of a lectin with alpha-galactosyl specificity obtained from ova of Salmo trutta. Anti-P sera were inhibited most effectively by human erythrocyte globoside, and to a lesser extent by Forssman glycolipid and rat kidney globoside. In the latter glycolipid the linkage between the internal galactosyl residues is alpha, 1 leads to 3, rather than alpha, 1 leads to 4, as in erythrocyte globoside. No cross-reactions between P and P1 or PK antigens were detected. New hypotheses are offered to explain the genetic regulation and biosynthesis of the P1, P, and PK antigens.     

The ligand activity of AGE-proteins to scavenger receptors is dependent on their rate of modification by AGEs

The cellular interaction of proteins modified with advanced glycation end-products (AGEs) is believed to induce several different biological responses, which are involved in the development of diabetic vascular complications. We report here that the ratio of protein glycation is implicated in its ligand activity to scavenger receptors. Although highly-modified AGE-bovine serum albumin (high-AGE-BSA) was significantly recognized by human monocyte-derived macrophages and Chinese hamster ovary cells which overexpress such scavenger receptors as CD36, SR-BI (scavenger receptor class B type-I), and LOX-1 (Lectin-like Ox-LDL receptor-1), the mildly-modified-AGE-BSA (mild-AGE-BSA) did not show any ligand activity to these cells. Furthermore, when (111)In-labeled high- or mild-AGE-BSA were injected into the tail vein of mice, the high-AGE-BSA was rapidly cleared from the circulation whereas the clearance rate of the mild-AGE-BSA was very slow, similar to the native BSA. These results demonstrate the first evidence that the ligand activity of the AGE-proteins to the scavenger receptors and its pharmacokinetic properties depend on their rate of modification by AGEs, and we should carefully prepare the AGE-proteins in vitro to clarify the physiological significance of the interaction between the AGE-receptors and AGE-proteins.     

The nematode leucine-rich repeat-containing, G protein-coupled receptor (LGR) protein homologous to vertebrate gonadotropin and thyrotropin receptors is constitutively active in mammalian cells

The receptors for LH, FSH, and TSH belong to the large G protein-coupled, seven-transmembrane protein family and are unique in having a large N-terminal extracellular (ecto-) domain containing leucine-rich repeats important for interactions with the large glycoprotein hormone ligands. Recent studies indicated the evolution of an expanding family of homologous leucine-rich repeat-containing, G protein-coupled receptors (LGRs), including the three known glycoprotein hormone receptors; mammalian LGR4 and LGR5; and LGRs in sea anemone, fly, and snail. We isolated nematode LGR cDNA and characterized its gene from the Caenorhabditis elegans genome. This receptor cDNA encodes 929 amino acids consisting of a signal peptide for membrane insertion, an ectodomain with nine leucine-rich repeats, a seven-TM region, and a long C-terminal tail. The nematode LGR has five potential N-linked glycosylation sites in its ectodomain and multiple consensus phosphorylation sites for protein kinase A and C in the cytoplasmic loop and C tail. The nematode receptor gene has 13 exons; its TM region and C tail, unlike mammalian glycoprotein hormone receptors, are encoded by multiple exons. Sequence alignments showed that the TM region of the nematode receptor has 30% identity and 50% similarity to the same region in mammalian glycoprotein hormone receptors. Although human 293T cells expressing the nematode LGR protein do not respond to human glycoprotein hormones, these cells exhibited major increases in basal cAMP production in the absence of ligand stimulation, reaching levels comparable to those in cells expressing a constitutively activated mutant human LH receptor found in patients with familial male-limited precocious puberty. Analysis of cAMP production mediated by chimeric receptors further indicated that the ectodomain and TM region of the nematode LGR and human LH receptor are interchangeable and the TM region of the nematode LGR is responsible for constitutive receptor activation. Thus, the identification and characterization of the nematode receptor provides the basis for understanding the evolutionary relationship of diverse LGRs and for future analysis of mechanisms underlying the activation of glycoprotein hormone receptors and related LGRs.     

Synthesis and evaluation of a new series of 1'-cyclobutyl-6-(4-piperidyloxy)spiro[benzopyran-2,4'-piperidine] derivatives as high affinity and selective histamine-3 receptor (H3R) antagonists

A novel class of 1'-cyclobutyl-6-(4-piperidyloxy)spiro[benzopyran-2,4'-piperidine] derivatives with low nanomolar affinity for the human and rat histamine-3 receptors (H(3)Rs) are described. The spirobenzopyran piperidine ether analogs demonstrated excellent H(3)R affinity and selectivity against histamine receptor subtypes (H(1)R, H(2)R, and H(4)R), were stable in liver microsomes, and had selectivity against CYP P450 enzymes. Compounds 10, 13, 15, and 16 demonstrated high H(3)R affinity, in vitro liver microsomal stability, selectivity against CYP isoforms, moreover, these ether analogs exhibited acceptable iv pharmacokinetic (PK) properties but had poor oral exposure in rat.     

Human pharmacokinetics of ethynyl estradiol 3-sulfate and 17-sulfate

Pharmacokinetic parameters of ethynyl estradiol 3-sulfate (EE-3) and 17-sulfate (EE-17) were estimated. Each sulfate was administered orally and intravenously to five ovariectomized volunteer women. Blood samples were taken over a period of 24 h. Radioimmunoassay for free and sulfoconjugated ethynyl estradiol (EE) was performed. The analysis of the plasma concentrations obtained after administration of EE-3 and EE-17 indicates significant differences in their pharmacokinetic profiles. EE-3 is cleared more rapidly from the central compartment (systemic circulation), which may indicate that differences in protein binding, tissue binding, metabolism, and distribution exist between EE-3 and EE-17. It has been suggested that these conjugates are a slow-release reservoir for maintenance of blood levels of free EE itself. However, previous studies in baboons have shown that the half-lives of the free and sulfoconjugated EE are similar (ranging from 8.8 to 11.2 h), which is not consistent with this hypothesis. The t1/2 beta (mean 9.28 h) of the 17-sulfate after IV administration was almost identical in women and baboons, and similar to the t1/2 beta of free EE, confirming the previous observation. Only 3.4% of IV and 11.4% of the orally administered 17-sulfate appeared in the blood as free EE; with the 3-sulfate, the conversions were 13.7 and 20.7%, respectively, suggesting that these sulfates are not important slow-release reservoirs. The similarity of pharmacokinetic parameters between women and baboons suggests that this species of nonhuman primate is, in important respects, a suitable animal model for clinical pharmacology.     

Structural and phylogenetic characterization of human SLURP-1, the first secreted mammalian member of the Ly-6/uPAR protein superfamily

Members of the Ly-6/uPAR protein family share one or several repeat units of the Ly-6/uPAR domain that is defined by a distinct disulfide bonding pattern between 8 or 10 cysteine residues. The Ly-6/uPAR protein family can be divided into two subfamilies. One comprises GPI-anchored glycoprotein receptors with 10 cysteine residues. The other subfamily includes the secreted single-domain snake and frog cytotoxins, and differs significantly in that its members generally possess only eight cysteines and no GPI-anchoring signal sequence. We report the purification and structural characterization of human SLURP-1 (secreted mammalian Ly-6/uPAR related protein 1) from blood and urine peptide libraries. SLURP-1 is encoded by the ARS (component B)-81/s locus, and appears to be the first mammalian member of the Ly-6/uPAR family lacking a GPI-anchoring signal sequence. A phylogenetic analysis based on the SLURP-1 primary protein structure revealed a closer relationship to the subfamily of cytotoxins. Since the SLURP-1 gene maps to the same chromosomal region as several members of the Ly-6/uPAR subfamily of glycoprotein receptors, it is suggested that both biologically distinct subfamilies might have co-evolved from local chromosomal duplication events.     

Analysis of role of aromatic residues in extracellular loop 2 of Prokineticin receptor 2 in ligand binding probed with genetically encoded photo-crosslinkers

Prokineticin 2 (PK2) and Prokineticin 2 beta (PK2β), products of alternative splicing of pk2 gene, are chemokine-like proteins. While PK2 mediates its biological activities by signaling with the same efficiency through two homologous G protein coupled receptors, prokineticin receptor 1 (PKR1) and prokineticin receptor 2 (PKR2), PK2β is able to bind specifically PKR1.Extracellular loop 2 (ECL2) of chemokine receptors is a part of a transmembrane (TM) ligand binding site. In the ECL2 of PKR2 is present, as well as in all chemokine receptors, an aromatic residue cluster, involving tryptophan 212 localized four residues after an ECL2 conserved cysteine, and Phenylalanine 198 located in the top of TM 4.In this work, the photoactivatable unnatural amino acid p-benzoyl-L-phenylalanine is incorporated by amber codon suppression technology into PKR2 in position 212. Experiments of photoactivatable cross-linking demonstrated the role of tryptophan in position 212 for binding the ligand contacting Tryptophan in position 24. We also analyzed the role of Phenylalanine 198 in the specificity of PKRs binding. The comparison of TM-bundle binding sites between PKR1 and PKR2 revealed that they are completely conserved except for one residue: valine 207 in human PKR1, which is phenylalanine 198 in human PKR2. The F198V mutation in PKR2 permits to obtain a receptor able to bind more efficiently PK2β, a ligand highly specific for PKR1.     

A novel approach to investigate the effect of methionine oxidation on pharmacokinetic properties of therapeutic antibodies

Preserving the chemical and structural integrity of therapeutic antibodies during manufacturing and storage is a major challenge during pharmaceutical development. Oxidation of Fc methionines Met252 and Met428 is frequently observed, which leads to reduced affinity to FcRn and faster plasma clearance if present at high levels. Because oxidation occurs in both positions simultaneously, their individual contribution to the concomitant changes in pharmacokinetic properties has not been clearly established. A novel pH-gradient FcRn affinity chromatography method was applied to isolate three antibody oxidation variants from an oxidized IgG1 preparation based on their FcRn binding properties. Physico-chemical characterization revealed that the three oxidation variants differed predominantly in the number of oxMet252 per IgG (0, 1, or 2), but not significantly in the content of oxMet428. Corresponding to the increase in oxMet252 content, stepwise reduction of FcRn affinity in vitro, as well as faster clearance and shorter terminal half-life, in huFcRn-transgenic mice were observed. A single Met252 oxidation per antibody had no significant effect on pharmacokinetics (PK) compared with unmodified IgG. Importantly, only molecules with both heavy chains oxidized at Met252 exhibited significantly faster clearance. In contrast, Met428 oxidation had no apparent negative effect on PK and even led to somewhat improved FcRn binding and slower clearance. This minor effect, however, seemed to be abrogated by the dominant effect of Met252 oxidation. The novel approach of functional chromatographic separation of IgG oxidation variants followed by physico-chemical and biological characterization has yielded the first experimentally-backed explanation for the unaltered PK properties of antibody preparations containing relatively high Met252 and Met428 oxidation levels.     

A novel approach to investigate the effect of methionine oxidation on pharmacokinetic properties of therapeutic antibodies

Preserving the chemical and structural integrity of therapeutic antibodies during manufacturing and storage is a major challenge during pharmaceutical development. Oxidation of Fc methionines Met252 and Met428 is frequently observed, which leads to reduced affinity to FcRn and faster plasma clearance if present at high levels. Because oxidation occurs in both positions simultaneously, their individual contribution to the concomitant changes in pharmacokinetic properties has not been clearly established. A novel pH-gradient FcRn affinity chromatography method was applied to isolate three antibody oxidation variants from an oxidized IgG1 preparation based on their FcRn binding properties. Physico-chemical characterization revealed that the three oxidation variants differed predominantly in the number of oxMet252 per IgG (0, 1, or 2), but not significantly in the content of oxMet428. Corresponding to the increase in oxMet252 content, stepwise reduction of FcRn affinity in vitro, as well as faster clearance and shorter terminal half-life, in huFcRn-transgenic mice were observed. A single Met252 oxidation per antibody had no significant effect on pharmacokinetics (PK) compared with unmodified IgG. Importantly, only molecules with both heavy chains oxidized at Met252 exhibited significantly faster clearance. In contrast, Met428 oxidation had no apparent negative effect on PK and even led to somewhat improved FcRn binding and slower clearance. This minor effect, however, seemed to be abrogated by the dominant effect of Met252 oxidation. The novel approach of functional chromatographic separation of IgG oxidation variants followed by physico-chemical and biological characterization has yielded the first experimentally-backed explanation for the unaltered PK properties of antibody preparations containing relatively high Met252 and Met428 oxidation levels.     

Increased serum clearance of oligomannose species present on a human IgG1 molecule

The role of Fc glycans on clearance of IgG molecule has been examined by various groups in experiments where specific glycans have been enriched or the entire spectrum of glycans was studied after administration in pre-clinical or clinical pharmacokinetic (PK) studies. The overall conclusions from these studies are inconsistent, which may result from differences in antibody structure or experimental design. In the present study a well-characterized recombinant monoclonal IgG1 molecule (mAb-1) was analyzed from serum samples obtained from a human PK study. mAb-1 was recovered from serum using its ligand cross-linked to Sepharose beads. The overall purity and recovery of all isoforms were carefully evaluated using a variety of methods. Glycans were then enzymatically cleaved, labeled using 2-aminobenzamide and analyzed by normal phase high performance liquid chromatography. The assays for recovering mAb-1 from serum and subsequent glycan analysis were rigorously qualified at a lower limit of quantitation of 15 μg/mL, thus permitting analysis to day 14 of the clinical PK study. Eight glycans were monitored and classified into two groups: (1) the oligomannose type structures (M5, M6 and M7) and (2) fucosylated biantennary oligosaccharides (FBO) structures (NGA2F, NA1F, NA2F, NA1F-GlcNAc and NGA2F-GlcNAc). We observed that the oligomannose species were cleared at a much faster rate (40%) than FBOs and conclude that high mannose species should be carefully monitored and controlled as they may affect PK of the therapeutic; they should thus be considered an important quality attribute. These observations were only possible through the application of rigorous analytical methods that we believe will need to be employed when comparing innovator and biosimilar molecules.     

Increased serum clearance of oligomannose species present on a human IgG1 molecule

The role of Fc glycans on clearance of IgG molecule has been examined by various groups in experiments where specific glycans have been enriched or the entire spectrum of glycans was studied after administration in pre-clinical or clinical pharmacokinetic (PK) studies. The overall conclusions from these studies are inconsistent, which may result from differences in antibody structure or experimental design. In the present study a well-characterized recombinant monoclonal IgG1 molecule (mAb-1) was analyzed from serum samples obtained from a human PK study. mAb-1 was recovered from serum using its ligand cross-linked to Sepharose beads. The overall purity and recovery of all isoforms were carefully evaluated using a variety of methods. Glycans were then enzymatically cleaved, labeled using 2-aminobenzamide and analyzed by normal phase high performance liquid chromatography. The assays for recovering mAb-1 from serum and subsequent glycan analysis were rigorously qualified at a lower limit of quantitation of 15 μg/mL, thus permitting analysis to day 14 of the clinical PK study. Eight glycans were monitored and classified into two groups: (1) the oligomannose type structures (M5, M6 and M7) and (2) fucosylated biantennary oligosaccharides (FBO) structures (NGA2F, NA1F, NA2F, NA1F-GlcNAc and NGA2F-GlcNAc). We observed that the oligomannose species were cleared at a much faster rate (40%) than FBOs and conclude that high mannose species should be carefully monitored and controlled as they may affect PK of the therapeutic; they should thus be considered an important quality attribute. These observations were only possible through the application of rigorous analytical methods that we believe will need to be employed when comparing innovator and biosimilar molecules.     

Improving the CH1-CK heterodimerization and pharmacokinetics of 4Dm2m,a novel potent CD4-antibody fusion protein against HIV-1

We previously described 4Dm2m, an exceptionally potent broadly neutralizing CD4-antibody fusion protein against HIV-1. It was generated by fusing the engineered single human CD4 domain mD1.22 to both the N and C termini of the human IgG1 heavy chain constant region and the engineered single human antibody domain m36.4, which targets the CD4-induced coreceptor binding site of the viral envelope glycoprotein, to the N terminus of the human antibody kappa light chain constant region via the (G4S)3 polypeptide linkers. However, therapeutic use of 4Dm2m was limited by its short in vivo half-life. Here, we show that a combination of three approaches have successfully increased the persistence of 4Dm2m in mice. First, to stabilize the scaffold, we enhanced heterodimerization between the heavy chain constant domain 1 (CH1) and kappa light chain constant domain (CK) by using structure-guided design and phage-display library technologies. Second, to address the possibility that long polypeptide linkers might render fusion proteins more susceptible to proteolysis, we shortened the (G4S)3 linkers or replaced them with the human IgG1 hinge sequence, which is naturally designed for both flexibility and stability. Third, we introduced two amino acid mutations into the crystallizable fragment (Fc) of the scaffold previously shown to increase antibody binding to the neonatal Fc receptor (FcRn) and prolong half-lives in vivo. Collectively, these approaches markedly increased the serum concentrations of 4Dm2m in mice while not affecting other properties of the fusion protein. The new 4Dm2m variants are promising candidates for clinical development to prevent or treat HIV-1 infection. To our knowledge, this is the first report on stabilized CH1-CK, which is potentially useful as a new heterodimerization scaffold for generation of bispecific and multispecific antibodies or proteins with a more favorable pharmacokinetic profile.     

Effect of chloroquine on the degradation of L-fucose and the polypeptide moiety of plasma membrane glycoproteins

To evaluate the role of lysosomes in the breakdown of the carbohydrate and the polypeptide moiety of plasma membrane glycoproteins, degradation of the plasma membrane glycoprotein gp120 was studied in the liver of rats treated with the lysosomotropic amine chloroquine. Half-lives of degradation of the terminal sugar L-fucose and of L-methionine of gp120 were measured in isolated plasma membranes after pulse-chase experiments in vivo. Chloroquine extended the plasma membrane half-life of the polypeptide moiety of gp120 from 51 h to 143 h. By contrast, L-fucose of gp120 in the plasma membrane was not affected by chloroquine, but decayed with the same short half-lives of 22 h and 23 h in both controls and chloroquine-treated rats. The data suggest that the protein portion of gp120 is degraded within the lysosomes. Conversely, the terminal sugar L-fucose is removed from the glycoprotein independent from proteolysis before segregation of the glycoprotein into the lysosomal compartment.     

A carcinoembryonic antigen (CEA) binding protein from ascites influences CEA uptake by macrophages

A variant of CEA which is less readily endocytosed by macrophages has been isolated from malignant ascites. In vivo, CEA is cleared more slowly by the liver (t1/2 = 15.1 minutes) than CEAs isolated from hepatic metastases (t1/2 = 3.1 minutes). In vitro, rat and human Kupffer cells and rat alveolar macrophages endocytose this CEA less effectively. This slow clearing form of CEA is associated with a smaller (45kD) acidic glycoprotein (CORA) with which it forms a stable complex. CORA can be visualized on reducing gels but not on non reducing gels or by HPLC run under non reducing conditions. This suggests a non-covalent complex between the two glycoproteins. Analysis of protein conformation by circular dichroism revealed changes in the ascites CEA consistent with binding of CORA to the molecule. Western blot showed that CORA crossreacts with antisera to alpha 1-acid glycoprotein and double immunodiffusion demonstrated cross-reactivity but not identify. Sequencing of CNBr peptides showed sequence homology with alpha 1-acid glycoprotein but areas of unique sequence were also found. It is suggested that binding of CORA to CEA blocks the macrophage receptor binding of CEA.     

The prolonged half-lives of new erythropoietin derivatives via peptide addition

Erythropoietin, or Epo, is a hematopoietic cytokine that promotes erythropoiesis, and recombinant human Epo has been used in the treatment of anemia in various chronic diseases. Here, we have constructed novel Epo derivatives with prolonged half-lives by adding peptides to the carboxy terminus of Epo without using linkers. The fused peptides were selected from the carboxy terminal region of human chorionic gonadotropin (hCG) or human thrombopoietin (hTpo), which promote the proper folding, secretion, and stabilization of bioactive glycoproteins. Addition of these peptides did not interfere with secretion or receptor binding, and significantly increased the in vivo half-life of human Epo, as measured by intravenous administration in rats. The plasma half-life of the Epo constructs was longest when the carboxy terminal 28 aa of the beta subunit of hCG was added (Epo-CGC), a half-life that was slightly longer than NESP (Aranesp), which is the most effective Epo product in current clinical use. The transformation of four Ser glycosylation sites to Ala on the CGC sequence also lengthened the plasma half-life of Epo, indicating that the in vivo stabilizing effect of the hCG peptide was due to both structures within the peptide itself and its O-glycosylations. The application of the carboxy terminal half of hTpo also resulted in remarkably reduced elimination of the Epo chimera (Epo-TpC), possibly due to protection by the TpC sequence. The in vivo hematopoietic activity of Epo derivatives in mice was consistent with their pharmacokinetic profiles. Therefore, these derivatives with prolonged half-lives may provide opportunities for developing new Epo therapeutics with less frequent administration.     

The assay design used for measurement of therapeutic antibody concentrations can affect pharmacokinetic parameters

To interpret pharmacokinetic (PK) data of biotherapeutics, it is critical to understand which drug species is being measured by the PK assay. For therapeutic antibodies, it is generally accepted that “free” circulating antibodies are the pharmacologically active form needed to determine the PK/ pharmacodynamic (PD) relationship, safety margin calculations, and dose projections from animals to humans and the eventual characterization of the exposure in the clinic. However, “total” drug may be important in evaluating the dynamic interaction between the drug and the target, as well as the total drug exposure. In the absence of or with low amounts of soluble ligand /shed receptor, total and free drug species are often equivalent and their detection is less sensitive to assay formats or reagent choices. In contrast, in the presence of a significant amount of ligand, assay design and characterization of assay reagents are critical to understanding the PK profiles. Here, we present case studies where different assay formats affected measured PK profiles and data interpretation. The results from reagent characterizations provide a potential explanation for the observed discrepancies and highlight the importance of reagent characterization in understanding which drug species are being measured to accurately interpret PK parameters.