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XTENs are unstructured, nonrepetitive protein polymers designed to prolong the in vivo half-life of pharmaceuticals by introducing a bulking effect similar to that of poly(ethylene glycol). While XTEN can be expressed as a recombinant fusion protein with bioactive proteins and peptides, therapeutic molecules of interest can also be chemically conjugated to XTEN. Such an approach permits precise control over the positioning, spacing, and valency of bioactive moieties along the length of XTEN. We have demonstrated the attachment of T-20, an anti-retroviral peptide indicated for the treatment of HIV-1 patients with multidrug resistance, to XTEN. By reacting maleimide-functionalized T-20 with cysteine-containing XTENs and varying the number and positioning of cysteines in the XTENs, a library of different peptide-polymer combinations were produced. The T-20-XTEN conjugates were tested using an in vitro antiviral assay and were found to be effective in inhibiting HIV-1 entry and preventing cell death, with the copy number and spacing of the T-20 peptides influencing antiviral activity. The peptide-XTEN conjugates were also discovered to have enhanced solubilities in comparison with the native T-20 peptide. The pharmacokinetic profile of the most active T-20-XTEN conjugate was measured in rats, and it was found to exhibit an elimination half-life of 55.7 ± 17.7 h, almost 20 times longer than the reported half-life for T-20 dosed in rats. As the conjugation of T-20 to XTEN greatly improved the in vivo half-life and solubility of the peptide, the XTEN platform has been demonstrated to be a versatile tool for improving the properties of drugs and enabling the development of a class of next-generation therapeutics.

A novel β-lactamase scaffold library in which the target-binding moiety is built into the enzyme was generated using phage display technology. The binding element is composed of a fully randomized 8 amino acid loop inserted at position between Y34 and K37 on the outer surface of Enterobacter cloacae P99 cephalosporinase (β-lactamase, E.C. 3.5.2.6) with all library members retaining catalytic activity. The frequency and diversity of amino acids distributions in peptide inserts from library clones were analyzed. The complexity of the randomized loop appears consistent with standards of other types of phage display library systems. The library was panned against SKBR3 human breast cancer cells in 1 round using rolling circle amplification of phage DNA to recover bound phage. Individual β-lactamase clones, independent of phage, were rapidly assessed for their binding to SKBR3 cells using a simple high throughput screen based on cell-bound β-lactamase activity. SKBR3 cell-binding β-lactamase enzymes were also shown to bind specifically using an immunochemical method. Selected β-lactamase clones were further studied for their protein expression, enzyme activity and binding to nontumor cell-lines. Overall, the approach outlined here offers the opportunity of rapidly selecting targeted β-lactamase ligands that may have a potential for their use in enzyme prodrug therapy with cephalosporin-based prodrugs. It is expected that a similar approach will be useful in developing tumor-targeting molecules of several other enzyme candidates of cancer prodrug therapy. © 2007 Wiley-Liss, Inc.

XTEN™ is a class of unstructured hydrophilic, biodegradable protein polymers designed to increase the half-lives of therapeutic peptides and proteins. XTEN polymers and XTEN fusion proteins are typically expressed in Escherichia coli and purified by conventional protein chromatography as monodisperse polypeptides of exact length and sequence. Unstructured XTEN polypeptides have hydrodynamic volumes significantly larger than typical globular proteins of similar mass, thus imparting a bulking effect to the therapeutic payloads attached to them. Since their invention, XTEN polypeptides have been utilized to extend the half-lives of a variety of peptide- and protein-based therapeutics. Multiple clinical and preclinical studies and related drug discovery and development efforts are in progress. This review details the most current understanding of physicochemical properties and biological behavior of XTEN and XTENylated molecules. Additionally, the development path and status of several advanced drug discovery and development efforts are highlighted.

XTEN, unstructured biodegradable proteins, have been used to extend the in vivo half-life of genetically fused therapeutic proteins and peptides. To expand the applications of XTEN technology to half-life extension of other classes of molecules, XTEN protein polymers and methods for chemical XTENylation were developed. Two XTEN precursors were engineered to contain enzymatically removable purification tags. The proteins were readily expressed in bacteria and purified to homogeneity by chromatography techniques. As proof-of-principle, GLP2-2G peptide was chemically conjugated to each of the two XTEN protein polymers using maleimide-thiol chemistry. The monodisperse nature of XTEN protein polymer enabled reaction monitoring as well as the detection of peptide modifications in the conjugated state using reverse phase-high performance liquid chromatography (RP-HPLC) and electrospray ionization mass spectrometry. The resulting GLP2-2G-XTEN conjugates were purified by preparative RP-HPLC to homogeneity. In comparison with recombinantly fused GLP2-2G-XTEN, chemically conjugated GLP2-2G-XTEN molecules exhibited comparable in vitro activity, in vitro plasma stability and pharmacokinetics in rats. These data suggest that chemical XTENylation could effectively extend the half-life of a wide spectrum of biologically active molecules, therefore broadening its applicability.

The substrate spectrum of Vsr DNA mismatch endonuclease of Escherichia coli K-12 was investigated using fluorescence-labelled oligonucleotide substrates and a DNA sequencer for detection and quantification of substrates and reaction products. Fourteen substrates were found to be processed by the enzyme, which differ in one or two positions from the canonical pentanucleotide sequence CTA/TGG (T mismatched to G). Relative second-order rate constants of these substrates were determined in groups of four by multiple substrate kinetics and compared to the underresentation of the corresponding pentanucleotides in the E. coli K-12 genome. The high quality of correlation further establishes active mutagenesis by VSP repair as a significant driving force of the evolution of the E. coli K-12 genome and provides clues to its possible selective value.

The S' subsite specificity of four homologous serine proteases, rat chymotrypsin, rat trypsin, alpha-lytic protease, and cercarial protease from Schistosoma mansoni, was studied by measuring acyl-transfer reactions to 100 pentapeptide nucleophiles. Peptides of the general structures H-Xaa-Ala-Ala-Ala-Ala-NH2, H-Ala-Xaa-Ala-Ala-Ala-NH2, and H-Ala-Ala-Xaa-Ala-Ala-NH2 were synthesized, where Xaa is D-Ala, Cit, and all natural amino acids except Cys. The variable residues of these nucleophiles occupy the P'1, P'2, and P'3 positions in acyl-transfer reactions. The P'1 and P'2 residues were found to influence the efficiency of the nucleophiles by more than 2 orders of magnitude, whereas the S'3 subsite shows a lower specificity in all four enzymes. We synthesized consensus peptides of the general structure H-aa1-aa2-aa3-Ala-Ala-NH2, in which two or three positions were occupied by amino acids that showed the highest specificity in the first series of nucleophiles. Peptides with optimal amino acid residues in the P'2 and P'3 positions show a very high efficiency in chymotrypsin- and trypsin-catalyzed reactions. Otherwise, large specific side chains in the P'1 and P'3 positions of the nucleophiles show less than additive binding contributions due to steric hindrance. Comparison of chymotrypsin-catalyzed acyl-transfer reactions to nucleophiles of the structures H-Xaa-Leu-Arg-Ala-Ala-NH2 and H-Xaa-Ala-Ala-Ala-Ala-NH2 reveals a significantly different P'1 specificity for both series which confirms steric hindrance between large P'1 and P'3 residues.(ABSTRACT TRUNCATED AT 250 WORDS)

In spite of the enormous progress in the synthesis of peptides and proteins using commercial peptide synthesizers and the immense technological possibilities of recombinant DNA technology, a CN ligase is an indispensable tool for the racemization-free fragment condensation of peptides. Since activation of the C-terminal α-carboxyl group of a peptide segment could cause partial racemization, chemical condensations of peptide fragments are prone to racemization. For the synthesis of the huge number of peptides and proteins, however, nature has only developed the ribosomal peptidyltransferase, which exhibits its full catalytic function independent of the side-chain functions of the amino acids being coupled. However, its function requires coordination with numerous other ribosomal factors. Besides the limited possibilities of using multienzyme complexes of bacterial peptide synthesis systems, the only alternatives to peptidyltransferase are proteases, which, based on their in vivo function as hydrolases, cannot act as ideal ligases. However, by exploiting the intrinsic reversibility of hydrolytic reactions and by adjusting appropriate physicochemical reaction parameters, the protease acitivity can be used in the direction of ligation. Undoubtedly, the course of kinetically controlled, serine and cysteine protease-catalyzed reactions can be more efficiently influenced than the equilibrium-controlled protease-catalyzed synthesis. This article describes the influence of the enzyme specificity on the efficiency of kinetically controlled synthesis and points the way toward a broad exploitation of serine and cysteine proteases for the catalysis of CN bond formation.

The published theory of kinetically controlled enzymatic peptide synthesis needed experimental verification. We carried out the measurement of the peptide yield and estimation of the key parameters α and β for papain-catalyzed synthesis of Mal-L-Phe-L-Ala-LLeuNH2 from Mal-L-Phe-L-AlaOMe and L-LeuNH2. The experimental results demonstrate that this theory adequately describes the real process. © 1992 John Wiley & Sons, Inc.

Increasing the in vivo residence times of protein therapeutics could decrease their dosing frequencies. We show that genetic fusion of an unstructured recombinant polypeptide of 864 amino acids, called XTEN, to a peptide or protein provides an apparently generic approach to extend plasma half-life. Allometric scaling suggests that a fusion of XTEN to the exenatide peptide should increase exenatide half-life in humans from 2.4 h to a projected time of 139 h. We confirmed the biological activity of the exenatide-XTEN fusion in mice. As extended stability might exacerbate undesirable side effects in some cases, we show that truncating the XTEN sequence can regulate plasma half-life. XTEN lacks hydrophobic amino acid residues that often contribute to immunogenicity and complicate manufacture. Based on data on XTEN fusions to exenatide, glucagon, GFP and human growth hormone, we expect that XTEN will enable dosing of otherwise rapidly cleared protein drugs at up to monthly intervals in humans.

In spite of the enormous progress in the synthesis of peptides and proteins using commercial peptide synthesizers and the immense technological possibilities of recombinant DNA technology, a CN ligase is an indispensable tool for the racemization-free fragment condensation of peptides. Since activation of the C-terminal α-carboxyl group of a peptide segment could cause partial racemization, chemical condensations of peptide fragments are prone to racemization. For the synthesis of the huge number of peptides and proteins, however, nature has only developed the ribosomal peptidyltransferase, which exhibits its full catalytic function independent of the side-chain functions of the amino acids being coupled. However, its function requires coordination with numerous other ribosomal factors. Besides the limited possibilities of using multienzyme complexes of bacterial peptide synthesis systems, the only alternatives to peptidyltransferase are proteases, which, based on their in vivo function as hydrolases, cannot act as ideal ligases. However, by exploiting the intrinsic reversibility of hydrolytic reactions and by adjusting appropriate physicochemical reaction parameters, the protease acitivity can be used in the direction of ligation. Undoubtedly, the course of kinetically controlled, serine and cysteine protease-catalyzed reactions can be more efficiently influenced than the equilibrium-controlled protease-catalyzed synthesis. This article describes the influence of the enzyme specificity on the efficiency of kinetically controlled synthesis and points the way toward a broad exploitation of serine and cysteine proteases for the catalysis of CN bond formation.

Increase in KC1 concentration from 0.1 m to 3 m enhances the chymotrypsin-catalyzed acyl-transfer to a series of dipeptides, H2N—CH(R1)C(O)NHCH(R2)C(O)O, by factors between 15 and 44. The observed positive salt effect seems to be a screening of the unfavorable electrostatic interaction between the dipeptide carboxyl group and a negatively charged area in the S' subsite on the enzyme surface. The effect is of practical use in preparative peptide synthesis—in 3 m KCl solutions the analytical yields of tripeptides of up to 99 per cent have been obtained.

INTRODUCTION More than 95% of circulating clotting factor VIII (FVIII) exists in a non-covalent complex with von Willebrand Factor (VWF). While VWF stabilizes and protects FVIII from its clearance pathways, it also subjects FVIII to VWF-mediated clearance. Thus, interaction with VWF imposes a limitation on the extent of FVIII half-life extension achieved by current technologies (Fc fusion, PEGylation etc.). Recombinant FVIIIFc-VWF-XTEN (rFVIIIFc-VWF-XTEN) is a novel fusion protein, consisting of the FVIII binding D'D3 domains of VWF fused to a single chain rFVIIIFc (scFVIIIFc). Appending the domains of VWF to FVIII provides the protection and stability of endogenous VWF, while avoiding the limitation imposed by VWF clearance. Besides D'D3 domains, it also contains two XTEN linkers. XTEN is an unstructured polypeptide consisting of six amino acids repeats (Gly, Ala, Pro, Glu, Ser, Thr). Fusion of XTEN to a protein reduces the rate of clearance and degradation of the fusion protein. In rFVIIIFc-VWF-XTEN, one XTEN linker replaces the B-domain of FVIII and other is attached to the D'D3 domains. In preclinical studies, this protein has shown >4-fold prolonged half-life and similar in vivo acute efficacy compared to rFVIII. In the current study, we examined the impact of various modifications on the in vitro plasma stability of rFVIIIFc-VWF-XTEN protein. MATERIALS AND METHODS rFVIIIFc-VWF-XTEN is a fusion protein which is expressed as a dual chain molecule. One chain expresses the D'D3 domains linked to a Fc monomer through an XTEN linker. This polypeptide is co-expressed with a single chain rFVIIIFc monomer to generate a dimer, via the disulfide-bond between the Fc domains. To assess the in vitro plasma stability, fusion proteins were expressed in HEK293 cells, purified and incubated with plasma from FVIII KO (Hem A) or FVIII/VWF DKO mice, for various time periods at 37 degree centigrade. After the desired incubation time, plasma stability of the recombinant proteins was determined by FVIII chromogenic activity assay. Results and Conclusions rFVIIIFc-VWF-XTEN fusion protein showed significantly enhanced in vitro plasma stability compared to rFVIII. In FVIII KO plasma, rFVIII started losing activity by 4 hours, and by 24 hours it lost more than 80% of its activity. The decline in activity was more pronounced and rapid when rFVIII was incubated with FVIII/VWF DKO plasma, mainly due to the absence of protection provided by VWF. Conversely, in the case of rFVIIIFc-VWF-XTEN, there was no significant drop in activity even after 6 hours (in both FVIII KO and DKO plasma). By 24 hours, only 10-15% activity reduction was observed in FVIII KO plasma and about a 35% decrease in DKO plasma. Further studies were conducted to evaluate various parameters which contributed to the improved stability of this fusion protein. Our results suggest that there are multiple factors which contribute to the overall stability of rFVIII-VWF-XTEN protein. These include: presence of covalently attached D'D3 domains, enhanced stability of single chain FVIII isoform used in the fusion protein and presence of the XTEN linker in the B-domain of FVIII. These data suggest that superior plasma stability of this novel fusion protein might be a contributing factor to its prolonged in vivo half-life and efficacy. Disclosures Seth Chhabra: Biogen: Employment, Equity Ownership. Moore:Biogen: Employment, Equity Ownership. Furcht:Biogen: Employment, Equity Ownership. Holthaus:Biogen: Employment. Liu:Biogen: Employment, Equity Ownership. Liu:Biogen: Employment, Equity Ownership. Schellenberger:Amunix Operating Inc: Employment. Kulman:Biogen: Employment. Salas:Biogen: Employment, Equity Ownership. Peters:Biogen: Employment.

Antibody-directed enzyme prodrug therapy (ADEPT) delivers chemotherapeutic agents in high concentration to tumor tissue while minimizing systemic drug exposure. β-Lactamases are particularly useful enzymes for ADEPT systems due to their unique substrate specificity that allows the activation of a variety of lactam-based prodrugs with minimal interference from mammalian enzymes. We evaluated the amino acid sequence of β-lactamase from Enterobacter cloacae for the presence of human T-cell epitopes using a cell-based proliferation assay using samples from 65 community donors. We observed a low background response that is consistent with a lack of preexposure to this enzyme. β-Lactamase was found to contain four CD4+ T-cell epitopes. For two of these epitopes, we identified single amino acid changes that result in significantly reduced proliferative responses while retaining stability and activity of the enzyme. The β-lactamase variant containing both changes induces significantly less proliferation in human and mouse cell assays, and 5-fold lower levels of IgG1 in mice were observed after repeat administration of β-lactamase variant with adjuvant. The β-lactamase variant should be very suitable for the construction of ADEPT fusion proteins, as it combines high activity toward lactam prodrugs, high plasma stability, a monomeric architecture, and a relatively low risk of eliciting an immune response in patients.

Penicillin acylase from E. coli is able to catalyze both the introduction and the removal of the phenylacetyl group. We have established that phenylacetyl derivatives of amino acids and peptides can be used in protease-catalyzed peptide synthesis. Here the synthesis of leucine-enkephalin using enzymes for N-terminal amino group protection, peptide bond formation and deprotection is described.

The S′-subsite specificity of endoproteinase Glu-C (V8 proteinase) was studied by acyl transfer reactions using Z-Glu-OMe as acyl donor and a series of amino acid- and peptide-derived nucleophiles. The partition constant, which characterizes specificity, was determined by a method based on the integrated rate equation. V8 proteinase prefers amino acid residues with hydrophobic side chains in the P1′ position. Di- and tripeptide amides are more efficient nucleophilic amino components than amino acid amides.

We have developed a rapid and convenient procedure for the characterization of the S' subsite specificity of serine proteases. A mixture of peptide nucleophiles is incubated with the enzyme in the presence of excess of a specific ester substrate. The decrease in each nucleophile concentration is monitored by high-performance liquid chromatography analysis of the dansylated mixture. Relative kinetic parameters for each nucleophile in the mixture are then calculated using a new statistical algorithm that relates all pairs of nucleophiles. As a first application, we investigated the S'1 subsite specificity of chymotrypsin, trypsin, and a recently described trypsin mutant, Tr-->Ch[S1 + L1 + L2] with chymotrypsin-like primary specificity [Hedstrom, L., Szilagyi, L., & Rutter, W. J. (1992) Science 255, 1249-1253]. For this purpose 21 peptide nucleophiles of the general structure H-Xaa-Ala-Ala-Ala-Ala-NH2 were prepared by multiple solid-phase synthesis, where Xaa represents D-alanine, citrulline, and all natural amino acids except cysteine. Relative second-order rate constants for the enzyme-catalyzed acyl transfer to these nucleophiles were determined over a range of 10(2). Chymotrypsin and trypsin have markedly different S'1 specificities. The order of preference in chymotrypsin-catalyzed acyl transfer reactions is positively charged > aliphatic > aromatic >> negatively charged, D-Ala, Pro P'1 side chain. Trypsin prefers hydrophobic residues, but like chymotrypsin aliphatic residues are better than aromatic residues in P'1 position. The S'1 specificity of the mutant Tr-->Ch[S1 + L1 + L2] is similar to the specificity of trypsin; however, P'1 aromatic residues have low reactivity characteristic of chymotrypsin.

We describe a new approach for the production of peptides using a combination of recombinant DNA technology, chemical synthesis, and proteinase-catalyzed processing. An artificial substance P-precursor is produced as a beta-galactosidase (1-459) fusion protein containing nine copies of the decapeptide sequence Arg-Leu-Arg-Arg-Pro-Lys-Pro-Gln-Gln-Phe. The fusion protein accumulates in E. coli as insoluble inclusion bodies which are easily isolated and purified. The decapeptide blocks are selectively cleaved from the insoluble fusion protein by alpha-chymotrypsin. Alternatively, a dodecapeptide ester is produced when a dipeptide ester is included in the chymotrypsin reaction mixture. This peptide ester is converted converted to substance P by papain-catalyzed acyl transfer and subsequent tryptic cleavage. These results demonstrate that peptides can be readily produced by a combination of recombinant DNA technology and proteinase-catalyzed conversion. The approach allows incorporation of groups other than natural amino acids into oligo- and polypeptides.

From the literature we collected all available quantitative data on the chymotrypsin-catalyzed hydrolysis of series of amino acid and peptide substrates. Utilizing this data base, we performed calculations on their quantitative structure/activity relationship (QSAR). The substrates were considered to be composed of fragments; log(kcat/Km) values for the substrates resulted from additive contributions of their fragments. Despite the fact that the kinetic constants in the data base were determined by different authors under various reaction conditions, the data are well described by the simple additivity model. Obviously, the intrinsic specificity of chymotrypsin dominates the influence of varying reaction conditions.

We investigated the deacylation of two acyl-alpha-chymotrypsins by added nucleophiles. The nucleophile binding site of the enzyme shows a strong preference for positively charged compounds. Most of our data can be explained by direct electrostatic interaction between the ionic nucleophiles and two negatively charged residues which are located close to the active site of the enzyme molecule. The influence of inorganic salts on the acyl transfer includes the following effects: (1) reduction of electrostatic interactions between the acyl-enzyme and the nucleophile by addition of salts; (2) binding of divalent cations to the nucleophile binding site of the acyl-enzyme leading to a significantly changed specificity; and (3) linear dependence of the activity coefficients of the added nucleophiles on salt concentration.

The S'-subsite specificity of bovine pancreatic alpha-chymotrypsin was investigated by acyl transfer reactions using a series of amino-acid- and peptide-derived nucleophiles. The nucleophilic efficiency covers a range of more than three orders of magnitude, reflecting the specificity of the acyl transfer process. Positively charged H-Arg-NH2 was the most efficient nucleophile of the series while peptides with free carboxyl groups show poor nucleophilic behaviour. This is explained by electrostatic interactions with the residues Asp35 and Asp64 of the enzyme. These negatively charged groups, which are localized near the appropriate S' binding sites, repel carboxylate groups of the nucleophiles. There is a good correlation between the nucleophile efficiencies found for different acyl enzymes. An investigation of a series of 14 water-soluble acyl donor esters, differing both in the P1 residue and in the number of amino acids, revealed that the nature of the acyl group affected the acyl-enzyme partitioning between water and added nucleophile in the range of one order of magnitude.

The partitioning of maleylphenylalanine-α-chymotrypsin formed using maleylphenylalanine methyl ester as acyl donor between amino acid-derived nucleophiles and water was determined spectrophotometrically. The interpretation of the results obtained from graphical analysis gave evidence of a high conformational specificity of α-chymotrypsin towards basic amino acid derivatives in the P′ 1 position including significant differences between the amide and methyl ester of arginine. Amides of neutral amino acids with large side-chains show higher nucleophile reactivity in comparison with glycine amide.

The papain-catalyzed reaction of esters of Z-alanine with various arginine esters was investigated. Using H-Arg-OPr i as a nucleophile the expected dipeptide product results in high yield. Otherwise, reactions with arginine esters of primary alkohols provide products undergoing further reactions. This allows the synthesis of N-protected peptide esters containing two or more arginine residues in a one-step reaction. The influence of reaction conditions on the process was investigated.

A number of maleyl peptide p-nitrobenzyl esters have been synthesized to study elastase-catalyzed hydrolysis reactions. These new substrates were used as acyl donors to investigate the S'-subsite specificity of porcine pancreatic elastase by partitioning of the acyl enzyme between various added nucleophiles and water. The following results were obtained. 1. Porcine pancreatic elastase prefers amino acid residues with small side chains in the P'1 position. 2. The nucleophile binding is improved by a positively charged P'1 side chain, whereas a negatively charged function results in a very low binding tendency. 3. Elongation of the nucleophile to the P'2 position leads to higher aminolysis rates. 4. S' specificity is substantially influenced by the P1 residue of the acyl enzyme.