27 research outputs found
Development of Copper-Catalyzed Azide–Alkyne Cycloaddition for Increased in Vivo Efficacy of Interferon β‑1b by Site-Specific PEGylation
The development of protein conjugate therapeutics requires
control
over the site of modification to allow for reproducible generation
of a product with the desired potency, pharmacokinetic, and safety
profile. Placement of a single nonnatural amino acid at the desired
modification site of a recombinant protein, followed by a bioorthogonal
reaction, can provide complete control. To this end, we describe the
development of copper-catalyzed azide–alkyne cycloaddition
(CuAAC, a click chemistry reaction) for site-specific PEGylation of
interferon β-1b (IFNb) containing azidohomoalanine (Aha) at
the N-terminus. Reaction conditions were optimized using various propargyl-activated
PEGs, trisÂ(benzyltriazolylmethyl)Âamine (TBTA), copper sulfate, and
dithiothreitol (DTT) in the presence of SDS. The requirement for air
in order to advance the redox potential of the reaction was investigated.
The addition of unreactive PEG diol reduced the required molar ratio
to 2:1 PEG–alkyne to IFNb. The resultant method produced high
conversion of Aha-containing IFNb to the single desired product. PEG–IFNbs
with 10, 20, 30, and 40 kDa linear or 40 kDa branched PEGs were produced
with these methods and compared. Increasing PEG size yielded decreasing
in vitro antiviral activities along with concomitant increases in
elimination half-life, AUC, and bioavailability when administered
in rats or monkeys. A Daudi tumor xenograft model provided comparative
evaluation of these combined effects, wherein a 40 kDa branched PEG–IFNb
was much more effective than conjugates with smaller PEGs or unPEGylated
IFNb at preventing tumor growth in spite of dosing with fewer units
and lesser frequency. The results demonstrate the capability of site-specific
nonnatural amino acid incorporation to generate novel biomolecule
conjugates with increased in vivo efficacy
Critical roles of CD30/CD30L interactions in murine autoimmune diabetes
CD30/CD30L is a member of tumour necrosis factor (TNF) receptor/TNF superfamily and has been implicated in immune-regulation. A genetic study has also suggested a possible implication of CD30 in spontaneous autoimmune diabetes in NOD mice. In this study, we investigated the involvement of CD30/CD30L in the development of diabetes in NOD mice. Flow cytometric analysis showed that CD30 and CD30L were highly expressed on CD4(+) or CD8(+) T cells in the spleen and pancreatic lymph node of younger NOD mice. In addition, islet-specific CD4(+) or CD8(+) T cell lines expressed CD30 and CD30L. Administration of a neutralizing anti-CD30L monoclonal antibody (mAb) from 2 to 10 week of age completely suppressed the development of spontaneous diabetes in NOD mice. In addition, the treatment with anti-CD30L mAb also inhibited the development of diabetes induced by adoptive transfer of spleen cells from diabetic NOD mice or islet-specific CD4(+) or CD8(+) T cell lines into NOD-SCID mice. Furthermore, anti-CD30L mAb inhibited T cell proliferation in response to islet antigens. These results suggested that CD30/CD30L interaction plays important roles in both induction and effector phases of autoimmune diabetes in NOD mice