13 research outputs found
A TIM-3 Oligonucleotide Aptamer Enhances T Cell Functions and Potentiates Tumor Immunity in Mice
T cell immunoglobulin-3 (TIM-3) is a negative regulator of interferon-γ (IFN-γ) secreting CD4+ T cells and CD8+ T cytotoxic cells. Recent studies have highlighted the role of TIM-3 as an important mediator of CD8+ T cell exhaustion in the setting of chronic viral infections and cancer. In murine tumor models, antibody blockade of TIM-3 with anti-TIM-3 antibodies as monotherapy has no or minimal antitumor activity, suggesting that TIM-3 signaling exerts an accessory or amplifying effect in keeping immune responses in check. Using a combined bead and cell-based systemic evolution of ligands by exponential enrichment (SELEX) protocol, we have isolated nuclease-resistant oligonucleotide aptamer ligands that bind to cell-associated TIM-3 with high affinity and specificity. A trimeric form of the TIM-3 aptamer blocked the interaction of TIM-3 with Galectin-9, reduced cell death, and enhanced survival, proliferation, and cytokine secretion in vitro. In tumor-bearing mice, the aptamer delayed tumor growth as monotherapy and synergized with PD-1 antibody in prolonging the survival of the tumor-bearing mice. Both in vitro and in vivo, the trimeric aptamer displayed superior activity compared to the currently used RMT3-23 monoclonal antibody. This study suggests that multi-valent aptamers could represent an alternative platform to generate potent ligands to manipulate the function of TIM-3 and other immune modulatory receptors.
In murine tumor immunotherapy models, antibody blockade of TIM-3 with anti-TIM-3 antibodies as monotherapy has no or minimal antitumor activity. In this issue of Molecular Therapy, Gefen et al. describe an oligonucleotide TIM-3-binding aptamer that was more effective than anti-TIM-3 antibody in vitro and in vivo
Aptamer-facilitated Protection of Oncolytic Virus from Neutralizing Antibodies
Oncolytic viruses promise to significantly improve current cancer treatments through their tumor-selective replication and multimodal attack against cancer cells. However, one of the biggest setbacks for oncolytic virus therapy is the intravenous delivery of the virus, as it can be cleared from the bloodstream by neutralizing antibodies before it reaches the tumor cells. We have selected DNA aptamers against an oncolytic virus, vesicular stomatitis virus, using a competitive binding approach, as well as against the antigen binding fragment (Fab) of antivesicular stomatitis virus polyclonal antibodies, in order to shield the virus from nAbs and enhance its in vivo survival. We used flow cytometry to identify these aptamers and evaluated their efficiency to shield vesicular stomatitis virus in a cell-based plaque forming assay. These oligonucleotides were then modified to obtain multivalent binders, which led to a decrease of viral aggregation, an increase in its infectivity and an increase in its stability in serum. The aptamers were also incubated in nondiluted serum, showing their effectiveness under conditions mimicking those in vivo. With this approach, we were able to increase viral infectivity by more than 70% in the presence of neutralizing antibodies. Thus, this method has the potential to enhance the delivery of vesicular stomatitis virus through the bloodstream without compromising the patient's immune system
Schematic representation of an electrochemical detection protocol adopted for this study.
<p>A hybrid of a thiol-modified primer and aptamer was self-assembled onto a gold nanoparticles-modified screen-printed carbon electrode (GNPs-SPCE). Binding of the <i>Cryptosporidium parvum</i> oocyst to the immobilized aptamer causes an increase in the redox current, measured by square wave voltammetry.</p
Limit of detection of the aptasensor.
<p>(A) Square wave voltammograms obtained after incubating the R4–6 aptamer-based sensors with (<i>a</i>) 0, (<i>b</i>) 100, (<i>c</i>) 200, (<i>d</i>) 300, (<i>e</i>) 400, (<i>f</i>) 500, (<i>g</i>) 600, (<i>h</i>) 700, and (<i>i</i>) 800 <i>Cryptosporidium parvum</i> oocysts. (B) Calibration plot of the change in current intensity (ΔI) <i>vs</i>. number of oocysts. (C) Calibration plot of the change in potential (ΔE) <i>vs</i>. number of oocysts.</p
Detection of <i>C</i>. <i>parvum</i> in fruit concentrates.
<p>(A) Square wave voltammograms of the selectivity experiments performed by incubating the R4–6 aptamer-based sensor with (<i>a</i>) buffer alone, (<i>b</i>) 300 <i>Cryptosporidium parvum</i> oocysts, and (<i>c</i>) 700 <i>C</i>. <i>parvum</i> oocysts, in pineapple and mango concentrates. (B) Plot of ΔI <i>vs</i>. the tested target. All measurements were repeated three times with separate electrodes (p < 0.005).</p
Anti-Fab Aptamers for Shielding Virus from Neutralizing Antibodies
Oncolytic viruses are promising therapeutics that can
selectively
replicate in and kill tumor cells. However, repetitive administration
of viruses provokes the generation of neutralizing antibodies (nAbs)
that can diminish their anticancer effect. In this work, we selected
DNA aptamers against the antigen binding fragment (Fab) of antivesicular
stomatitis virus polyclonal antibodies to shield the virus from nAbs
and enhance its in vivo survival. For the first time, we used flow
cytometry and electrochemical immunosensing to identify aptamers targeting
the Fab region of antibodies. We evaluated the aptamers in a cell-based
infection assay and found that several aptamer clones provide more
than 50% shielding of VSV from nAbs and thus have the potential to
enhance the delivery of VSV without compromising the patient’s
immune system. In addition, we developed a bifunctional label-free
electrochemical immunosensor for the quantitation of aptamer-mediated
degree of shielding and the amount of vesicular stomatitis virus (VSV)
particles. Electrochemical impedance spectroscopy was employed to
interrogate the level of VSV in a linear range from 5 × 10<sup>4</sup> to 5 × 10<sup>6</sup> PFU mL<sup>–1</sup> with
a detection limit of 10<sup>4</sup> PFU mL<sup>–1</sup>
Electrochemical Sensing of Aptamer-Facilitated Virus Immunoshielding
Oncolytic viruses (OVs) are promising therapeutics that
selectively
replicate in and kill tumor cells. However, repetitive administration
of OVs provokes the generation of neutralizing antibodies (nAbs) that
can diminish their anticancer effects. In this work, we selected DNA
aptamers against an oncolytic virus, vesicular stomatitis virus (VSV),
to protect it from nAbs. A label-free electrochemical aptasensor was
used to evaluate the degree of protection (DoP). The aptasensor was
fabricated by self-assembling a hybrid of a thiolated ssDNA primer
and a VSV-specific aptamer. Electrochemical impedance spectroscopy
was employed to quantitate VSV in the range of 800–2200 PFU
and a detection limit of 600 PFU. The aptasensor was also utilized
for evaluating binding affinities between VSV and aptamer pools/clones.
An electrochemical displacement assay was performed in the presence
of nAbs and DoP values were calculated for several VSV-aptamer pools/clones.
A parallel flow cytometric analysis confirmed the electrochemical
results. Finally, four VSV-specific aptamer clones, ZMYK-20, ZMYK-22,
ZMYK-23, and ZMYK-28, showed the highest protective properties with
dissociation constants of 17, 8, 20, and 13 nM, respectively. Another
four sequences, ZMYK-1, -21, -25, and -29, exhibited high affinities
to VSV without protecting it from nAbs and can be further utilized
in sandwich assays. Thus, ZMYK-22, -23, and -28 have the potential
to allow efficient delivery of VSV through the bloodstream without
compromising the patient’s immune system
Viral Quantitative Capillary Electrophoresis for Counting and Quality Control of RNA Viruses
The world of health care has witnessed an explosive boost
to its
capacity within the past few decades due to the introduction of viral
therapeutics to its medicinal arsenal. As a result, a need for new
methods of viral quantification has arisen to accommodate this rapid
advancement in virology and associated requirements for efficiency,
speed, and quality control. In this work, we apply viral quantitative
capillary electrophoresis (viral qCE) to determine (i) the number
of intact virus particles (ivp) in viral samples, (ii) the amount
of DNA contamination, and (iii) the degree of viral degradation after
sonication, vortexing, and freeze–thaw cycles. This quantification
method is demonstrated on an RNA-based vesicular stomatitis virus
(VSV) with oncolytic properties. A virus sample contains intact VSV
particles as well as residual DNA from host cells, which is regulated
by WHO guidelines, and may include some carried-over RNA. We use capillary
zone electrophoresis with laser-induced fluorescent detection to separate
intact virus particles from DNA and RNA impurities. YOYO-1 dye is
used to stain all DNA and RNA in the sample. After soft lysis of VSV
with proteinase K digestion of viral capsid and ribonucleoproteins,
viral RNA is released. Therefore, the initial concentration of intact
virus is calculated based on the gain of a nucleic acid peak and an
RNA calibration curve. After additional NaOH treatment of the virus
sample, RNA is hydrolyzed leaving residual DNA only, which is also
calculated by a DNA calibration curve made by the same CE instrument.
Viral qCE works in a wide dynamic range of virus concentrations from
10<sup>8</sup> to 10<sup>13</sup> ivp/mL. It can be completed in a
few hours and requires minimum optimization of CE separation
Aptamer-Based Viability Impedimetric Sensor for Viruses
The development of aptamer-based viability impedimetric
sensor
for viruses (AptaVISens-V) is presented. Highly specific DNA aptamers
to intact vaccinia virus were selected using cell-SELEX technique
and integrated into impedimetric sensors via self-assembly onto a
gold microelectrode. Remarkably, this aptasensor is highly selective
and can successfully detect viable vaccinia virus particles (down
to 60 virions in a microliter) and distinguish them from nonviable
viruses in a label-free electrochemical assay format. It also opens
a new venue for the development of a variety of viability sensors
for detection of many microorganisms and spores
Electrochemical Differentiation of Epitope-Specific Aptamers
DNA aptamers are promising immunoshielding agents that
could protect
oncolytic viruses (OVs) from neutralizing antibodies (nAbs) and increase
the efficiency of cancer treatment. In the present Article, we introduce
a novel technology for electrochemical differentiation of epitope-specific
aptamers (eDEA) without selecting aptamers against individual antigenic
determinants. For this purpose, we selected DNA aptamers that can
bind noncovalently to an intact oncolytic virus, vaccinia virus (VACV),
which can selectively replicate in and kill only tumor cells. The
aptamers were integrated as a recognition element into a multifunctional
electrochemical aptasensor. The developed aptasensor was used for
the linear quantification of the virus in the range of 500–3000
virus particles with a detection limit of 330 virions. Also, the aptasensor
was employed to compare the binding affinities of aptamers to VACV
and to estimate the degree of protection of VACV using the anti-L1R
neutralizing antibody in a displacement assay fashion. Three anti-VACV
aptamer clones, vac2, vac4, and vac6, showed the best immunoprotection
results and can be applied for enhanced delivery of VACV. Another
two sequences, vac5 and vac46, exhibited high affinities to VACV without
shielding it from nAb and can be further utilized in sandwich bioassays