ADAPTING PHARMACOKINETIC PROPERTIES OF A HUMANIZED ANTI-INTERLEUKIN-8 ANTIBODY FOR THERAPEUTIC APPLICATIONS USING SITE-SPECIFIC PEGYLATION

Abstract We recently showed that site-specific incorporation of 2′-modifications or neutral linkages in the oligo-deoxynucleotide gap region of toxic phosphorothioate (PS) gapmer ASOs can enhance therapeutic index and safety. In this manuscript, we determined if introducing substitution at the 5′-position of deoxynucleotide monomers in the gap can also enhance therapeutic index. Introducing R- or S-configured 5′-Me DNA at positions 3 and 4 in the oligodeoxynucleotide gap enhanced the therapeutic profile of the modified ASOs suggesting a different positional preference as compared to the 2′-OMe gap modification strategy. The generality of these observations was demonstrated by evaluating R-5′-Me and R-5′-Ethyl DNA modifications in multiple ASOs targeting HDAC2, FXI and Dynamin2 mRNA in the liver. The current work adds to a growing body of evidence that small structural changes can modulate the therapeutic properties of PS ASOs and ushers a new era of chemical optimization with a focus on enhancing the therapeutic profile as opposed to nuclease stability, RNA-affinity and pharmacokinetic properties. The 5′-methyl DNA modified ASOs exhibited excellent safety and antisense activity in mice highlighting the therapeutic potential of this class of nucleic acid analogs for next generation ASO designs.

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Improving Pharmacokinetic Properties of Adrenocorticotropin by Site-Specific Lipid Modification

Journal of Pharmaceutical Sciences ◽

10.1002/jps.10442 ◽

2003 ◽

Vol 92 (9) ◽

pp. 1882-1892 ◽

Cited By ~ 4

Author(s):

Lei Wan ◽

Yu-Hsien Chen ◽

Tse Wen Chang

Keyword(s):

Lipid Modification ◽

Site Specific ◽

Pharmacokinetic Properties ◽

Specific Lipid

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Campylobacter jejuni-Stimulated Secretion of Interleukin-8 by INT407 Cells

Infection and Immunity ◽

10.1128/iai.67.1.88-93.1999 ◽

1999 ◽

Vol 67 (1) ◽

pp. 88-93 ◽

Cited By ~ 82

Author(s):

Thomas E. Hickey ◽

Shihida Baqar ◽

A. Louis Bourgeois ◽

Cheryl P. Ewing ◽

Patricia Guerry

Keyword(s):

Protein Synthesis ◽

Campylobacter Jejuni ◽

Marked Reduction ◽

De Novo ◽

Interleukin 8 ◽

Eukaryotic Cells ◽

Live Cells ◽

Wild Type ◽

Site Specific ◽

De Novo Protein Synthesis

ABSTRACT Incubation of INT407 cells with various clinical isolates ofCampylobacter jejuni resulted in secretion of interleukin-8 (IL-8) at levels ranging from 96 to 554 pg/ml at 24 h. The strains which produced the highest levels of IL-8 secretion were 81-176 and BT44. Induction of IL-8 secretion required live cells of 81-176 and was dependent on de novo protein synthesis. Site-specific mutants of 81-176, which were previously shown to be defective in adherence and invasion, resulted in reduced levels of secretion of IL-8, andcheY mutants of strains 81-176 and 749, which are hyperadherent and hyperinvasive, resulted in higher levels of IL-8 secretion. Another mutant of 81-176, which adheres at about 43% of the wild-type levels but is noninvasive, also showed marked reduction in IL-8 levels, suggesting that invasion is necessary for high levels of IL-8 secretion. When gentamicin was added to INT407 cells at 2 h after infection with 81-176, IL-8 secretion 22 h later was equivalent to that of controls without gentamicin, suggesting that the events which trigger induction and release of IL-8 occur early in the interactions of bacteria and eukaryotic cells.

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Pharmacokinetic properties of mutants of recombinant single chain urokinase-type plasminogen activator obtained by site-specific mutagenesis of Lys158, Ile159 and Ile160

Fibrinolysis and Proteolysis ◽

10.1016/0268-9499(90)90016-d ◽

1990 ◽

Vol 4 (4) ◽

pp. 211-214 ◽

Cited By ~ 4

Author(s):

H.R. Lijnen ◽

L. Nelles ◽

E. Van Houtte ◽

D. Collen

Keyword(s):

Plasminogen Activator ◽

Single Chain ◽

Site Specific ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator ◽

Pharmacokinetic Properties

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Large-cluster and combined fluorescent and gold immunoprobes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166920 ◽

1996 ◽

Vol 54 ◽

pp. 892-893

Author(s):

Richard D. Powell ◽

James F. Hainfeld ◽

Carol M. R. Halsey ◽

David L. Spector ◽

Shelley Kaurin ◽

...

Keyword(s):

Metal Cluster ◽

Sulfonyl Chloride ◽

Gel Filtration ◽

Cross Linking ◽

Site Specific ◽

Fab Fragments ◽

Cluster Label ◽

Covalently Linked ◽

Texas Red ◽

Fold Excess

Two new types of covalently linked, site-specific immunoprobes have been prepared using metal cluster labels, and used to stain components of cells. Combined fluorescein and 1.4 nm “Nanogold” labels were prepared by using the fluorescein-conjugated tris (aryl) phosphine ligand and the amino-substituted ligand in the synthesis of the Nanogold cluster. This cluster label was activated by reaction with a 60-fold excess of (sulfo-Succinimidyl-4-N-maleiniido-cyclohexane-l-carboxylate (sulfo-SMCC) at pH 7.5, separated from excess cross-linking reagent by gel filtration, and mixed in ten-fold excess with Goat Fab’ fragments against mouse IgG (obtained by reduction of F(ab’)2 fragments with 50 mM mercaptoethylamine hydrochloride). Labeled Fab’ fragments were isolated by gel filtration HPLC (Superose-12, Pharmacia). A combined Nanogold and Texas Red label was also prepared, using a Nanogold cluster derivatized with both and its protected analog: the cluster was reacted with an eight-fold excess of Texas Red sulfonyl chloride at pH 9.0, separated from excess Texas Red by gel filtration, then deprotected with HC1 in methanol to yield the amino-substituted label.

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The challenge of detecting modifications on proteins

Essays in Biochemistry ◽

10.1042/ebc20190055 ◽

2020 ◽

Vol 64 (1) ◽

pp. 135-153 ◽

Cited By ~ 1

Author(s):

Lauren Elizabeth Smith ◽

Adelina Rogowska-Wrzesinska

Keyword(s):

Mass Spectrometry ◽

Protein Function ◽

Chemical Properties ◽

Lysine Acetylation ◽

Mass Determination ◽

Post Translational Modifications ◽

Site Specific ◽

The Common

Abstract Post-translational modifications (PTMs) are integral to the regulation of protein function, characterising their role in this process is vital to understanding how cells work in both healthy and diseased states. Mass spectrometry (MS) facilitates the mass determination and sequencing of peptides, and thereby also the detection of site-specific PTMs. However, numerous challenges in this field continue to persist. The diverse chemical properties, low abundance, labile nature and instability of many PTMs, in combination with the more practical issues of compatibility with MS and bioinformatics challenges, contribute to the arduous nature of their analysis. In this review, we present an overview of the established MS-based approaches for analysing PTMs and the common complications associated with their investigation, including examples of specific challenges focusing on phosphorylation, lysine acetylation and redox modifications.

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