The high-resolution three-dimensional structure of a single immunoglobulin binding domain (B1, which comprises 56 residues including the NH2-terminal Met) of protein G from group G Streptococcus has been determined in solution by nuclear magnetic resonance spectroscopy on the basis of 1058 experimental restraints. The average atomic root-mean-square distribution about the mean coordinate positions is 0.27 angstrom (A) for the backbone atoms, 0.65 A for all atoms, and 0.39 A for atoms excluding disordered surface side chains. The structure has no disulfide bridges and is composed of a four-stranded beta sheet, on top of which lies a long helix. The central two strands (beta 1 and beta 4), comprising the NH2- and COOH-termini, are parallel, and the outer two strands (beta 2 and beta 3) are connected by the helix in a +3x crossover. This novel topology (-1, +3x, -1), coupled with an extensive hydrogen-bonding network and a tightly packed and buried hydrophobic core, is probably responsible for the extreme thermal stability of this small domain (reversible melting at 87 degrees C).

The effects of linker length on binding affinity and degree of aggregation have been examined in the antifluorescein 4-4-20 and anticarcinoma CC49 single-chain Fvs. Longer linkers in the antifluorescein sFvs have higher affinities for fluorescein and aggregate less. A proteolytically susceptible site between Lys8 and Ser9, in the previously reported 212 linker has been identified. A new linker sequence, 218 (GSTSGSGKPGSGEGSTKG) was designed in which a proline was placed at the C-terminal side of the proteolytic clip site in the 212 linker. The CC49 sFv containing the 218 linker showed reduced aggregation and was found to be more stable to proteolysis in vitro, when compared to the CC49/212 sFv. The CC49 sFv with the longer 218 linker had higher affinity than CC49/212 sFv. An aggregated CC49/212 sFv sample had higher affinity than CC49/218 sFv. The CC49/218 and CC49/212 sFvs had similar blood clearances in mice, while the aggregated CC49/212 sFv remained in circulation significantly longer. In mice bearing LS-174T human colon carcinoma xenografts, the CC49/218 sFv showed higher tumor uptake than the CC49/212 sFv and lower tumor uptake than the aggregated CC49/212 sFv. The higher tumor uptake of the CC49/218 is most likely a result of its higher resistance to proteolysis. The higher affinity and higher tumor uptake of the aggregated CC49/212 sFv are most likely due to the repetitive nature of the TAG-72 antigen and the higher avidity of multivalent aggregates. When the sFvs were radiolabeled with a lutetium-chelate the CC49/218 sFv showed a lower accumulation in the liver and spleen compared to the aggregated CC49/212 sFv.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that plays a critical role in angiogenesis, survival, metastasis, drug resistance, and glucose metabolism.

Recombinant interferon-beta-1b (IFN-beta-1b) is used clinically in the treatment of multiple sclerosis. In common with many biological ligands, IFN-beta-1b exhibits a relatively short serum half-life, and bioavailability may be further diminished by neutralizing antibodies. While PEGylation is an approach commonly employed to increase the blood residency time of protein therapeutics, there is a further requisite for molecular engineering approaches to also address the stability, solubility, aggregation, immunogenicity and in vivo exposure of therapeutic proteins. We investigated these five parameters of recombinant human IFN-beta-1b in over 20 site-selective mono-PEGylated or multi-PEGylated IFN-beta-1b bioconjugates. Primary amines were modified by single or multiple attachments of poly(ethylene glycol), either site-specifically at the N-terminus, or randomly on the 11 lysines. In two alternate approaches, site-directed mutagenesis was independently employed in the construction of designed IFN-beta-1b variants containing either a single free cysteine or lysine for site-specific PEGylation. Optimization of conjugate preparation with 12 kDa, 20 kDa, 30 kDa, and 40 kDa amine-selective PEG polymers was achieved, and a comparison of the structural and functional properties of the IFN-beta-1b proteins and their PEGylated counterparts was conducted. Peptide mapping and MALDI-TOF mass spectrometric analysis confirmed the attachment sites of the PEG polymer. Independent biochemical and bioactivity analyses, including antiviral and antiproliferation bioassays, circular dichroism, capillary electrophoresis, flow cytometric profiling, reversed phase and size exclusion HPLC, and immunoassays demonstrated that the functional activities of the designed IFN-beta-1b conjugates were maintained, while the formation of soluble or insoluble aggregates of IFN-beta-1b was ameliorated. Immunogenicity and pharmacokinetic studies of selected PEGylated IFN-beta-1b compounds in mice and rats demonstrated both diminished IgG responses, and over 100-fold expanded AUC exposure relative to the unmodified protein. The results demonstrate the capacity of this macromolecular engineering strategy to address both pharmacological and formulation challenges for a highly hydrophobic, aggregation-prone protein. The properties of a lead mono-PEGylated candidate, 40 kDa PEG2-IFN-beta-1b, were further investigated in formulation optimization and biological studies.

The gene (spg)

We have cloned a full-length 1.6-kilobase cDNA of a human mRNA coding for hypoxanthine phosphoribosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) into a simian virus 40-based expression vector and have determined its full nucleotide sequence. The inferred amino acid sequence agrees with a partial amino acid sequence determined for authentic human HPRTprotein..Transfection of HPRTdeficient mouse IA9 cells with the purified plasmid leads to the expression of human HPRT enzyme activity in cells stably transfected and selected for enzyme activity in hypoxanthine/aminopterin/thymidine medium.Current methods of molecular biology, including the techniques of recombinant -DNA construction and cloning, rapid nucleotide sequence analysis, the design and construction of transducing vectors, and techniques oftransfection ofeukaryotic cells with foreign genes, have made it possible to clone and characterize a large number of eukaryotic genes. One gene of particular interest, not only for basic studies of eukaryotic gene regulation but also for understanding of several important human genetic diseases, is the gene encoding the enzyme hypoxanthine phosphoribosyltransferase (HPRT) (1). This enzyme catalyzes vital steps in the reutilization pathway for purine biosynthesis, and its deficiency leads to forms ofgouty arthritis and to the devastating Lesch-Nyhan disease (2,3). The HPRT locus is known to be X linked in the human and other mammalian genomes, and the availability of a cloned HPRT gene would facilitate studies ofthe organization ofa particularly interesting region of the human X chromosome and of the mechanisms of inactivation of specific and well-mapped regions of the X chromosome. Recently, we succeeded in isolating a human genomic clone containing a portion ofthe HPRT (4) by a combination of gene transfection into enzyme-deficient mouse cells and localization of human sequences by hybridization with probes of middle repetitive human (Alu) sequence, as described (4). A 15-kilobase (kb) EcoRI/BamilI fragment derived from p6B2aE2 and free ofrepetitive human sequences ,has been subcloned into the BamHI and EcoRI-sites ofpBR322 and shown to hybridize to a discrete human cytoplasmic poly(A)+RNA approximately 1.5 kb long, presumably. representing the mRNA for HPRT (4). DNA from this subcloned plasmid, called pBR1.5, was prepared by detergent lysis (5) of chloramphenicol-treated (6) transformed SF8 Escherichia coli (7) followed. by cesium chloride/ethidium bromide ultracentrifugation (8). This DNA was cleaved with EcoRI/BamrHI and the cloned insert was isolated by trough electroelution onto a dialysis membrane (9) The colonies on the replica plates were transferred to Whatman 541 paper, amplified on chloramphenicol plates overnight at 37QC by the method ofGergen et at (13), denatured and fixed to the filters, and screened for HPRT cDNA sequences by hybridization with the gel-purified insert from pBR1.5 containing the HPRT fragment free ofrepeat sequences. The filter replicas of the...

The utility of single-chain Fv proteins as therapeutic agents would be substantially broadened if the circulating lives of these minimal antigen-binding polypeptides were both prolonged and adjustable. Poly(ethylene glycol) (PEG) bioconjugate derivatives of the model single-chain Fv, CC49/218 sFv, were constructed using six different linker chemistries that selectively conjugate either primary amines or carboxylic acid groups. Activated PEG polymers with molecular weights of 2000, 5000, 10 000, 12 000, and 20 000 were included in the sFv bioconjugate evaluation. Additionally, the influence of PEG conjugate geometry in branched PEG strands (U-PEG) and the effect of multimeric PEG-sFv bioconjugates on circulating life and affinity were examined. Although random and extensive PEG polymer conjugations have been achievable in highly active derivatives of the prototypical PEG-enzymes, PEGylation of CC49/218 sFv required stringent adjustment of reaction conditions in order to preserve antigen-binding affinity as measured in either mucin-specific or whole cell immunoassays. Purified bioconjugates with PEG:sFv ratios of 1:1 through 2:1 were identified as promising candidates which exhibit sFv affinity (K(d)) values within 2-fold of the unmodified sFv protein. Interestingly, PEG conjugation to carboxylic acid moieties, using a PEG-hydrazide chemistry, achieved significant activity retention in bioconjugates at a higher PEG:sFv ratio (5:1) than with any of the amine-reactive activated PEG polymers. Prolonged circulating life in mice was demonstrated for each of the PEG conjugates. An increase in PEG polymer length was found to be more effective for serum half-life extension than a corresponding increase in total PEG mass. For example, CC49/218 sFv conjugated to either one strand of PEG-20000, or four strands of PEG-5000, displayed about 20- or 14-fold increased serum half-life, respectively, relative to the unmodified sFv. The demonstrated suitability of established random conjugation chemistries for PEGylation of sFv proteins, in conjunction with innovative site-specific conjugation methods, indicates that production of a panoply of sFv proteins with both engineered affinity and tailored circulating life may now be achievable.

The utility of single-chain Fv proteins as therapeutic agents would be realized if the circulating lives of these minimal antigen-binding polypeptides could be both prolonged and adjustable. We have developed a general strategy for creating tailored monoPEGylated single-chain antibodies. Free cysteine residues were engineered in an anti-TNF-alpha scFv at the C-terminus or within the linker segments of both scFv orientations, V(L)-linker-V(H) and V(H)-linker-V(L). High-level expression of 10 designed variant scFv proteins in Pichia pastoris allowed rapid purification. Optimization of site-specific conjugate preparation with 5, 20 and 40 kDa maleimide-PEG polymers was achieved and a comparison of the structural and functional properties of the scFv proteins and their PEGylated counterparts was performed. Peptide mapping and MALDI-TOF mass spectrometric analysis confirmed the unique attachment site for each PEG polymer. Independent biochemical and bioactivity analyses, including binding affinities and kinetics, antigenicity, flow cytometric profiling and cell cytotoxicity rescue, demonstrated that the functional activities of the 10 designed scFv conjugates are maintained, while scFv activity variations between these alternative assays can be correlated with conjugate and analytical designs. Pharmacokinetic studies of the PEGylated scFv in mice demonstrated up to 100-fold prolongation of circulating lives, in a range comparable to clinical antibodies.