The ribosomal incorporation of nonnative amino acids into polypeptides in living cells provides the opportunity to endow therapeutic proteins with unique pharmacological properties. We report here the first clinical study of a biosynthetic protein produced using an expanded genetic code. Incorporation of p-acetylphenylalanine (pAcF) at distinct locations in human growth hormone (hGH) allowed site-specific conjugation with polyethylene glycol (PEG) to produce homogeneous hGH variants. A mono-PEGylated mutant hGH modified at residue 35 demonstrated favorable pharmacodynamic properties in GH-deficient rats. Clinical studies in GH-deficient adults demonstrated efficacy and safety comparable to native human growth hormone therapy but with increased potency and reduced injection frequency. This example illustrates the utility of nonnative amino acids to optimize protein therapeutics in an analogous fashion to the use of medicinal chemistry to optimize conventional natural products, low molecular weight drugs, and peptides.protein engineering | endocrinology | bio-better

Individual T cell receptor (TCR) Valpha elements are expressed preferentially in CD4 or CD8 peripheral T cell subsets. The closely related Valpha3.1 and Valpha3.2 elements show reciprocal selection into CD4 and CD8 subsets, respectively. Transgenic mice expressing site-directed mutants of a Valpha3.1 gene were used to show that individual residues in either the complementarity-determining region 1 (CDR1) or CDR2 were sufficient to change selection from the CD4 subset to the CD8 subset. Thus, the germline-encoded Valpha elements are a major influence on major histocompatibility class complex (MHC) restriction, most likely by a preferential interaction with one or the other class of MHC molecule.

Most CD1d-dependent NKT cells in mice have a canonical Vα14Jα18 TCR rearrangement. However, relatively little is known concerning the molecular basis for their reactivity to glycolipid Ags presented by CD1d. Using glycolipid Ags, soluble forms of a Vα14 NKT cell-derived TCR, and mutant and wild-type CD1d molecules, we probed the TCR/CD1d interaction by surface plasmon resonance, tetramer equilibrium staining, and tetramer staining decay experiments. By these methods, several CD1d α-helical amino acids could be defined that do not greatly alter lipid binding, but that affect the interaction with the TCR. Binding of the Vα14+ TCR to CD1d requires the agonist α-galactosylceramide (α-GalCer), as opposed to the nonantigenic β-galactosylceramide, although both Ags bind to CD1d, indicating that the carbohydrate moiety of the CD1d-bound Ag plays a major role in the TCR interaction. The TCR has a relatively high-affinity binding to the α-GalCer/CD1d complex, with a particularly slow off rate. These unique properties are consistent with the coreceptor-independent action of the Vα14 TCR and may be related to the intense response to α-GalCer by NKT cells in vivo.

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 thymic preference for CD4+ T cells over CD8+ T cells is often attributed to a default pathway favouring CD4+ T cells or to homeostatic mechanisms. It is also clear, however, that T-cell receptor (TCR) preferences for major histocompatibility complex (MHC) class I versus class II binding will strongly influence an individual clone's skewing to the CD4 or CD8 subset. The variable region of each TCR alpha chain (V alpha) studied to date is found to be overrepresented in either CD4+ or CD8+ cells, suggesting that each V alpha element can interact more favourably with either MHC class I or class II molecules. Indeed, TCRs appear to have an intrinsic ability to interact with MHC molecules, and single amino acid residues present in germline-encoded complementarity determining region 1 (CDR1) and CDR2 of the V alpha element can be responsible for determining MHC specificity. Interestingly, the degree of CD4/CD8 skewing is variable among different mouse strains and in human populations. Here, we have shown that polymorphism in CD4/CD8 skewing between B6 and BALB/c mice is determined by the stem cell genotype and not by environmental effects, and that it maps in or near the TCR alpha-chain complex, Tcra. This was confirmed by comparing Tcra(b) with Tcra(a) or Tcra(c) haplotypes in congenic mice. We propose that the array of V alpha genes in various Tcra haplotypes exerts influence over the proportion of CD4 and CD8 subsets generated and may account in part for the observed thymic skewing. Thus, while it has been suggested that the TCR genes have been selected by evolution for MHC binding, our results further indicate selection for class II MHC preference.

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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.