Volatiles and Refractories in Surface-Bounded Exospheres in the Inner Solar System.

Volatiles and refractories represent the two end-members in the volatility range of species in any surface-bounded exosphere. Volatiles include elements that do not interact strongly with the surface, such as neon (detected on the Moon) and helium (detected both on the Moon and at Mercury), but also argon, a noble gas (detected on the Moon) that surprisingly adsorbs at the cold lunar nighttime surface. Refractories include species such as calcium, magnesium, iron, and aluminum, all of which have very strong bonds with the lunar surface and thus need energetic processes to be ejected into the exosphere. Here we focus on the properties of species that have been detected in the exospheres of inner Solar System bodies, specifically the Moon and Mercury, and how they provide important information to understand source and loss processes of these exospheres, as well as their dependence on variations in external drivers.

Aptamer-mediated transcriptional gene silencing of Fox p 3 inhibits regulatory T cells and potentiates antitumor response.

The inhibition of immunosuppressive mechanisms may switch the balance between tolerance and surveillance, leading to an increase in antitumor activity. Regulatory T cells play an important role in the control of immunosuppression, exhibiting the unique property of inhibiting T cell proliferation. These cells migrate to tumor sites or may be generated at the tumor site itself from the conversion of lymphocytes exposed to tumor microenvironment signaling. Because of the high similarity between regulatory T cells and other lymphocytes, the available approaches to inhibit this population are nonspecific and may antagonize antitumor response. In this work we explore a new strategy for inhibition of regulatory T cells based on the use of a chimeric aptamer targeting a marker of immune activation harboring a small antisense RNA molecule for transcriptional gene silencing of Fox p 3, which is essential for the control of the immunosuppressive phenotype. The silencing of Fox p 3 inhibits the immunosuppressive phenotype of regulatory T cells and potentiates the effect of the GVAX antitumor vaccine in immunocompetent animals challenged with syngeneic tumors. This novel approach highlights an alternative method to antagonize regulatory T cell function to augment antitumor immune responses.

Development and Preliminary Clinical Activity of PD-1-Guided CTLA-4 Blocking Bispecific DART Molecule.

Combination immunotherapy with antibodies directed against PD-1 and CTLA-4 shows improved clinical benefit across cancer indications compared to single agents, albeit with increased toxicity. Leveraging the observation that PD-1 and CTLA-4 are co-expressed by tumor-infiltrating lymphocytes, an investigational PD-1 x CTLA-4 bispecific DART molecule, MGD019, is engineered to maximize checkpoint blockade in the tumor microenvironment via enhanced CTLA-4 blockade in a PD-1-binding-dependent manner. In vitro, MGD019 mediates the combinatorial blockade of PD-1 and CTLA-4, confirming dual inhibition via a single molecule. MGD019 is well tolerated in non-human primates, with evidence of both PD-1 and CTLA-4 blockade, including increases in Ki67+CD8 and ICOS+CD4 T cells, respectively. In the ongoing MGD019 first-in-human study enrolling patients with advanced solid tumors (NCT03761017), an analysis undertaken following the dose escalation phase revealed acceptable safety, pharmacodynamic evidence of combinatorial blockade, and objective responses in multiple tumor types typically unresponsive to checkpoint inhibitor therapy.

Tumor-targeted silencing of the peptide transporter TAP induces potent antitumor immunity.

Neoantigen burden is a major determinant of tumor immunogenicity, underscored by recent clinical experience with checkpoint blockade therapy. Yet the majority of patients do not express, or express too few, neoantigens, and hence are less responsive to immune therapy. Here we describe an approach whereby a common set of new antigens are induced in tumor cells in situ by transient downregulation of the transporter associated with antigen processing (TAP). Administration of TAP siRNA conjugated to a broad-range tumor-targeting nucleolin aptamer inhibited tumor growth in multiple tumor models without measurable toxicity, was comparatively effective to vaccination against prototypic mutation-generated neoantigens, potentiated the antitumor effect of PD-1 antibody or Flt3 ligand, and induced the presentation of a TAP-independent peptide in human tumor cells. Treatment with the chemically-synthesized nucleolin aptamer-TAP siRNA conjugate represents a broadly-applicable approach to increase the antigenicity of tumor lesions and thereby enhance the effectiveness of immune potentiating therapies.

Time-of-day-dependent global distribution of lunar surficial water/hydroxyl.

A new set of time-of-day-dependent global maps of the lunar near-infrared water/hydroxyl (H2O/OH) absorption band strength near 2.8 to 3.0 µm constructed on the basis of Moon Mineralogy Mapper (M3) data is presented. The analyzed absorption band near 2.8 to 3.0 µm indicates the presence of surficial H2O/OH. To remove the thermal emission component from the M3 reflectance spectra, a reliable and physically realistic mapping method has been developed. Our maps show that lunar highlands at high latitudes show a stronger H2O/OH absorption band in the lunar morning and evening than at midday. The amplitude of these time-of-day-dependent variations decreases with decreasing latitude of the highland regions, where below about 30°, absorption strength becomes nearly constant during the lunar day at a similar level as in the high-latitude highlands at midday. The lunar maria exhibit weaker H2O/OH absorption than the highlands at all, but showing a smaller difference from highlands absorption levels in the morning and evening than at midday. The level around midday is generally higher for low-Ti than for high-Ti mare surfaces, where it reaches near-zero values. Our observations contrast with previous studies that indicate a significant concentration of surficial H2O/OH at high latitudes only. Furthermore, although our results generally support the commonly accepted mechanism of H2O/OH formation by adsorption of solar wind protons, they suggest the presence of a more strongly bounded surficial H2O/OH component in the lunar highlands and parts of the mare regions, which is not removed by processes such as diffusion/thermal evaporation and photolysis in the course of the lunar day.

Aptamer-Targeted Attenuation of IL-2 Signaling in CD8+ T Cells Enhances Antitumor Immunity.

Immune responses elicited against cancer using existing therapies such as vaccines or immune stimulatory antibodies are often not curative. One way to potentiate antitumor immunity is to enhance the long-term persistence of anti-tumor CD8+ T cells. Studies have shown that the persistence of activated CD8+ T cells is negatively impacted by the strength of interleukin 2 (IL-2) signaling. Here, we used small interfering RNAs (siRNAs) against CD25 (IL-2Rα) to attenuate IL-2 signaling in CD8+ T cells. The siRNAs were targeted to 4-1BB-expressing CD8+ T cells by conjugation to a 4-1BB-binding oligonucleotide aptamer. Systemic administration of the 4-1BB aptamer-CD25 siRNA conjugate downregulated CD25 mRNA only in 4-1BB-expressing CD8+ T cells promoting their differentiation into memory cells. Treatment with the 4-1BB aptamer-CD25 siRNA conjugates enhanced the antitumor response of a cellular vaccine or local radiation therapy. Indicative of the generality of this approach, 4-1BB aptamer-targeted delivery of an Axin-1 siRNA, a rate-limiting component of the ß-catenin destruction complex, enhanced CD8+ T cell memory development and antitumor activity. These findings show that aptamer-targeted siRNA therapeutics can be used to modulate the function of circulating CD8+ T cells, skewing their development into long-lasting memory CD8+ T cells, and thereby potentiating antitumor immunity.

Reducing Toxicity of Immune Therapy Using Aptamer-Targeted Drug Delivery.

Modulating the function of immune receptors with antibodies is ushering in a new era in cancer immunotherapy. With the notable exception of PD-1 blockade used as monotherapy, immune modulation can be associated with significant toxicities that are expected to escalate with the development of increasingly potent immune therapies. A general way to reduce toxicity is to target immune potentiating drugs to the tumor or immune cells of the patient. This Crossroads article discusses a new class of nucleic acid-based immune-modulatory drugs that are targeted to the tumor or to the immune system by conjugation to oligonucleotide aptamer ligands. Cell-free chemically synthesized short oligonucleotide aptamers represent a novel and emerging platform technology for generating ligands with desired specificity that offer exceptional versatility and feasibility in terms of development, manufacture, and conjugation to an oligonucleotide cargo. In proof-of-concept studies, aptamer ligands were used to target immune-modulatory siRNAs or aptamers to induce neoantigens in the tumor cells, limit costimulation to the tumor lesion, or enhance the persistence of vaccine-induced immunity. Using increasingly relevant murine models, the aptamer-targeted immune-modulatory drugs engendered protective antitumor immunity that was superior to that of current "gold-standard" therapies in terms of efficacy and lack of toxicity or reduced toxicity. To overcome immune exhaustion aptamer-targeted siRNA conjugates could be used to downregulate intracellular mediators of exhaustion that integrate signals from multiple inhibitory receptors. Recent advances in aptamer development and second-generation aptamer-drug conjugates suggest that we have only scratched the surface.

Identifying high-affinity aptamer ligands with defined cross-reactivity using high-throughput guided systematic evolution of ligands by exponential enrichment.

Oligonucleotide aptamers represent a novel platform for creating ligands with desired specificity, and they offer many potentially significant advantages over monoclonal antibodies in terms of feasibility, cost, and clinical applicability. However, the isolation of high-affinity aptamer ligands from random oligonucleotide pools has been challenging. Although high-throughput sequencing (HTS) promises to significantly facilitate systematic evolution of ligands by exponential enrichment (SELEX) analysis, the enormous datasets generated in the process pose new challenges for identifying those rare, high-affinity aptamers present in a given pool. We show that emulsion PCR preserves library diversity, preventing the loss of rare high-affinity aptamers that are difficult to amplify. We also demonstrate the importance of using reference targets to eliminate binding candidates with reduced specificity. Using a combination of bioinformatics and functional analyses, we show that the rate of amplification is more predictive than prevalence with respect to binding affinity and that the mutational landscape within a cluster of related aptamers can guide the identification of high-affinity aptamer ligands. Finally, we demonstrate the power of this selection process for identifying cross-species aptamers that can bind human receptors and cross-react with their murine orthologs.

Large scale analysis of the mutational landscape in HT-SELEX improves aptamer discovery.

High-Throughput (HT) SELEX combines SELEX (Systematic Evolution of Ligands by EXponential Enrichment), a method for aptamer discovery, with massively parallel sequencing technologies. This emerging technology provides data for a global analysis of the selection process and for simultaneous discovery of a large number of candidates but currently lacks dedicated computational approaches for their analysis. To close this gap, we developed novel in-silico methods to analyze HT-SELEX data and utilized them to study the emergence of polymerase errors during HT-SELEX. Rather than considering these errors as a nuisance, we demonstrated their utility for guiding aptamer discovery. Our approach builds on two main advancements in aptamer analysis: AptaMut-a novel technique allowing for the identification of polymerase errors conferring an improved binding affinity relative to the 'parent' sequence and AptaCluster-an aptamer clustering algorithm which is to our best knowledge, the only currently available tool capable of efficiently clustering entire aptamer pools. We applied these methods to an HT-SELEX experiment developing aptamers against Interleukin 10 receptor alpha chain (IL-10RA) and experimentally confirmed our predictions thus validating our computational methods.

Targeting 4-1BB costimulation to the tumor stroma with bispecific aptamer conjugates enhances the therapeutic index of tumor immunotherapy.

Despite the recent successes of using immune modulatory Abs in patients with cancer, autoimmune pathologies resulting from the activation of self-reactive T cells preclude the dose escalations necessary to fully exploit their therapeutic potential. To reduce the observed and expected toxicities associated with immune modulation, here we describe a clinically feasible and broadly applicable approach to limit immune costimulation to the disseminated tumor lesions of the patient, whereby an agonistic 4-1BB oligonucleotide aptamer is targeted to the tumor stroma by conjugation to an aptamer that binds to a broadly expressed stromal product, VEGF. This approach was predicated on the premise that by targeting the costimulatory ligands to products secreted into the tumor stroma, the T cells will be costimulated before their engagement of the MHC-peptide complex on the tumor cell, thereby obviating the need to target the costimulatory ligands to noninternalizing cell surface products expressed on the tumor cells. Underscoring the potency of stroma-targeted costimulation and the broad spectrum of tumors secreting VEGF, in preclinical murine tumor models, systemic administration of the VEGF-targeted 4-1BB aptamer conjugates engendered potent antitumor immunity against multiple unrelated tumors in subcutaneous, postsurgical lung metastasis, methylcholantrene-induced fibrosarcoma, and oncogene-induced autochthonous glioma models, and exhibited a superior therapeutic index compared with nontargeted administration of an agonistic 4-1BB Ab or 4-1BB aptamer.

Aptamer-targeted inhibition of mTOR in T cells enhances antitumor immunity.

Recent studies have underscored the importance of memory T cells in mediating protective immunity against pathogens and cancer. Pharmacological inhibition of regulators that mediate T cell differentiation promotes the differentiation of activated CD8(+) T cells into memory cells. Nonetheless, pharmacological agents have broad targets and can induce undesirable immunosuppressive effects. Here, we tested the hypothesis that aptamer-targeted siRNA inhibition of mTOR complex 1 (mTORC1) function in CD8(+) T cells can enhance their differentiation into memory T cells and potentiate antitumor immunity more effectively than the pharmacologic inhibitor rapamycin. To specifically target activated cells, we conjugated an siRNA targeting the mTORC1 component raptor to an aptamer that binds 4-1BB, a costimulatory molecule that is expressed on CD8(+) T cells following TCR stimulation. We found that systemic administration of the 4-1BB aptamer-raptor siRNA to mice downregulated mTORC1 activity in the majority of CD8(+) T cells, leading to the generation of a potent memory response that exhibited cytotoxic effector functions and enhanced vaccine-induced protective immunity in tumor-bearing mice. In contrast, while treatment with the general mTORC1 inhibitor rapamycin also enhanced antigen-activated CD8(+) T cell persistence, the cytotoxic effector functions of the reactivated memory cells were reduced and the alloreactivity of DCs was diminished. Consistent with the immunological findings, mice treated with rapamycin, but not with 4-1BB aptamer-raptor siRNA, failed to reject a subsequent tumor challenge.

AptaCluster - A Method to Cluster HT-SELEX Aptamer Pools and Lessons from its Application.

Systematic Evolution of Ligands by EXponential Enrichment (SELEX) is a well established experimental procedure to identify aptamers - synthetic single-stranded (ribo)nucleic molecules that bind to a given molecular target. Recently, new sequencing technologies have revolutionized the SELEX protocol by allowing for deep sequencing of the selection pools after each cycle. The emergence of High Throughput SELEX (HT-SELEX) has opened the field to new computational opportunities and challenges that are yet to be addressed. To aid the analysis of the results of HT-SELEX and to advance the understanding of the selection process itself, we developed AptaCluster. This algorithm allows for an efficient clustering of whole HT-SELEX aptamer pools; a task that could not be accomplished with traditional clustering algorithms due to the enormous size of such datasets. We performed HT-SELEX with Interleukin 10 receptor alpha chain (IL-10RA) as the target molecule and used AptaCluster to analyze the resulting sequences. AptaCluster allowed for the first survey of the relationships between sequences in different selection rounds and revealed previously not appreciated properties of the SELEX protocol. As the first tool of this kind, AptaCluster enables novel ways to analyze and to optimize the HT-SELEX procedure. Our AptaCluster algorithm is available as a very fast multiprocessor implementation upon request.

Isolation and optimization of murine IL-10 receptor blocking oligonucleotide aptamers using high-throughput sequencing.

Interleukin-10 (IL-10) is a key suppressor of inflammation in chronic infections and in cancer. In mice, the inability of the immune system to clear viral infections or inhibit tumor growth can be reversed by antibody-mediated blockade of IL-10 action. We used a modified selection protocol to isolate RNA-based, nuclease-resistant, aptamers that bind to the murine IL-10 receptor. After 5 rounds of selection high-throughput sequencing (HTS) was used to analyze the library. Using distribution statistics on about 11 million sequences, aptamers were identified which bound to IL-10 receptor in solution with low K(d). After 12 rounds of selection the predominant IL-10 receptor-binding aptamer identified in the earlier rounds remained, whereas other high-affinity aptamers were not detected. Prevalence of certain nucleotide (nt) substitutions in the sequence of a high-affinity aptamer present in round 5 was used to deduce its secondary structure and guide the truncation of the aptamer resulting in a shortened 48-nt long aptamer with increased affinity. The aptamer also bound to IL-10 receptor on the cell surface and blocked IL-10 function in vitro. Systemic administration of the truncated aptamer was capable of inhibiting tumor growth in mice to an extent comparable to that of an anti- IL-10 receptor antibody.

Thermal Stability of siRNA Modulates Aptamer- conjugated siRNA Inhibition.

Oligonucleotide aptamer-mediated in vivo cell targeting of small interfering RNAs (siRNAs) is emerging as a useful approach to enhance the efficacy and reduce the adverse effects resulting from siRNA-mediated genetic interference. A current main impediment in aptamer-mediated siRNA targeting is that the activity of the siRNA is often compromised when conjugated to an aptamer, often requiring labor intensive and time consuming design and testing of multiple configurations to identify a conjugate in which the siRNA activity has not been significantly reduced. Here, we show that the thermal stability of the siRNA is an important parameter of siRNA activity in its conjugated form, and that siRNAs with lower melting temperature (T(m)) are not or are minimally affected when conjugated to the 3' end of 2'F-pyrimidine-modified aptamers. In addition, the configuration of the aptamer-siRNA conjugate retains activity comparable with the free siRNA duplex when the passenger strand is co-transcribed with the aptamer and 3' overhangs on the passenger strand are removed. The approach described in this paper significantly reduces the time and effort necessary to screening siRNA sequences that retain biological activity upon aptamer conjugation, facilitating the process of identifying candidate aptamer-siRNA conjugates suitable for in vivo testing.Molecular Therapy - Nucleic Acids (2012) 1, e51; doi:10.1038/mtna.2012.41; published online 16 October 2012.