Aptamers Against Live Targets: Is In Vivo SELEX Finally Coming to the Edge?

Targeted therapeutics underwent a revolution with the entry of monoclonal antibodies in the medical toolkit. Oligonucleotide aptamers form another family of target agents that have been lagging behind in reaching the clinical arena in spite of their potential clinical translation. Some of the reasons for this might be related to the challenge in identifying aptamers with optimal in vivo specificity, and the nature of their pharmacokinetics. Aptamers usually show exquisite specificity, but they are also molecules that display dynamic structures subject to changing environments. Temperature, ion atmosphere, pH, and other variables are factors that could determine the affinity and specificity of aptamers. Thus, it is important to tune the aptamer selection process to the conditions in which you want your final aptamer to function; ideally, for in vivo applications, aptamers should be selected in an in vivo-like system or, ultimately, in a whole in vivo organism. In this review we recapitulate the implementations in systematic evolution of ligands by exponential enrichment (SELEX) to obtain aptamers with the best in vivo activity.

ICOS Costimulation at the Tumor Site in Combination with CTLA-4 Blockade Therapy Elicits Strong Tumor Immunity.

Cytotoxic T lymphocyte-associated protein 4 (CTLA-4) blockade therapy is able to induce long-lasting antitumor responses in a fraction of cancer patients. Nonetheless, there is still room for improvement in the quest for new therapeutic combinations. ICOS costimulation has been underscored as a possible target to include with CTLA-4 blocking treatment. Herein, we describe an ICOS agonistic aptamer that potentiates T cell activation and induces stronger antitumor responses when locally injected at the tumor site in combination with anti-CTLA-4 antibody in different tumor models. Furthermore, ICOS agonistic aptamer was engineered as a bi-specific tumor-targeting aptamer to reach any disseminated tumor lesions after systemic injection. Treatment with the bi-specific aptamer in combination with CTLA-4 blockade showed strong antitumor immunity, even in a melanoma tumor model where CTLA-4 treatment alone did not display any significant therapeutic benefit. Thus, this work provides strong support for the development of combinatorial therapies involving anti-CTLA-4 blockade and ICOS agonist tumor-targeting agents.

Identification of LAG3 high affinity aptamers by HT-SELEX and Conserved Motif Accumulation (CMA).

LAG3 receptor belongs to a family of immune-checkpoints expressed in T lymphocytes and other cells of the immune system. It plays an important role as a rheostat of the immune response. Focus on this receptor as a potential therapeutic target in cancer immunotherapy has been underscored after the success of other immune-checkpoint blockade strategies in clinical trials. LAG3 showcases the interest in the field of autoimmunity as several studies show that LAG3-targeting antibodies can also be used for the treatment of autoimmune diseases. In this work we describe the identification of a high-affinity LAG3 aptamer by High Throughput Sequencing SELEX in combination with a study of potential conserved binding modes according to sequence conservation by using 2D-structure prediction and 3D-RNA modeling using Rosetta. The aptamer with the highest accumulation of these conserved sequence motifs displays the highest affinity to LAG3 recombinant soluble proteins and binds to LAG3-expressing lymphocytes. The aptamer described herein has the potential to be used as a therapeutic agent, as it enhances the threshold of T-cell activation. Nonetheless, in future applications, it could also be engineered for treatment of autoimmune diseases by target depletion of LAG3-effector T lymphocytes.

Targeting inhibition of Foxp3 by a CD28 2'-Fluro oligonucleotide aptamer conjugated to P60-peptide enhances active cancer immunotherapy.

The specific inhibition of Treg function has long been a major technical challenge in cancer immunotherapy. So far no single cell-surface marker has been identified that could be used to distinguish Treg cells from other lymphocytes. The only available specific marker mostly expressed in Treg is Foxp3, which is an intracellular transcription factor. A targeting molecule able to penetrate the membrane and inhibit Foxp3 within the cell is needed. P60-peptide is able to do that, but due to lack of target specificity, the doses are extremely high. In this study we have shown as a proof of concept that P60 Foxp3 inhibitor peptide can be conjugated with a CD28 targeting aptamer to deliver the peptide to CD28-expressing cells. The AptCD28-P60 construct is a clinically feasible reagent that improves the efficacy of the unconjugated P60 peptide very significantly. This approach was used to inhibit Treg function in a vaccination context, and it has shown a significant improvement in the induced immune response, entailing a lower tumor load in an antigen-specific cancer vaccine protocol.

MRP1-CD28 bi-specific oligonucleotide aptamers: target costimulation to drug-resistant melanoma cancer stem cells.

In this work we show a clinically feasible strategy to convert in situ the own tumor into an endogenous vaccine by coating the melanoma cancerous cells with CD28 costimulatory ligands. This therapeutic approach is aimed at targeting T-cell costimulation to chemotherapy-resistant tumors which are refractory and been considered as untreatable cancers. These tumors are usually defined by an enrichment of cancer stem cells and characterized by the higher expression of chemotherapy-resistant proteins. In this work we develop the first aptamer that targets chemotherapy-resistant tumors expressing MRP1 through a novel combinatorial peptide-cell SELEX. With the use of the MRP1 aptamer we engineer a MRP1-CD28 bivalent aptamer that is able to bind MRP1-expressing tumors and deliver the CD28 costimulatory signal to tumor-infiltrating lymphocytes. The bi-specific aptamer is able to enhance costimulation in chemotherapy-resistant tumors. Melanoma-bearing mice systemically treated with MRP1-CD28 bivalent aptamer show reduced growth, thus proving an improved mice survival.Besides, we have designed a technically feasible and translational whole-cell vaccine (Aptvax). Disaggregated cells from tumors can be directly decorated with costimulatory ligand aptamers to generate the vaccine Aptvax. CD28Aptvax made of irradiated tumor cells coated with the CD28-agonistic aptamer attached to MRP1 elicits a strong tumor- cell immune response against melanoma tumors reducing tumor growth.

2-fluoro-RNA oligonucleotide CD40 targeted aptamers for the control of B lymphoma and bone-marrow aplasia.

Recent studies have underscored the importance of immunomodulatory antibodies in the treatment of solid and hematological tumors. ODN-Aptamers are rising as a novel class of drugs that can rival therapeutic antibodies. The success of some of the current cancer immunotherapy approaches in oncological patients depends on the intrinsic antigenicity of each tumor as has recently been disclosed, and it is hampered in those patients that are treated with myeloablative chemotherapy or radiotherapy, which induce profound immunosuppression. CD40 agonist and antagonist molecules offer a new therapeutic alternative which has the potential to generate anticancer effects by different mechanisms. HS-SELEX was performed to identify high-affinity aptamers against CD40, and three therapeutic CD40 constructs were engineered as: CD40 agonist aptamer, CD40 antagonist aptamer and CD40 agonistic aptamer-shRNA chimera. It is shown that CD40 agonist aptamers can be used to promote bone-marrow aplasia recovery. CD40 antagonist aptamers are revealed to have a direct antitumor effect on CD40-expressing B-cell lymphoma in vitro and in vivo. Further, in order to identify a therapeutic reagent that will generate the optimal conditions for cancer immunotherapy (antigen-presenting cell activation, tumor antigenicity enhancement and bone-marrow aplasia recovery), CD40 agonist aptamer-shRNA chimera was generated to target the inhibition of the Nonsense mRNA Mediated Decay (NMD) to tumor cells.

BiovaxID®: a customized idiotype vaccine for the treatment of B-cell lymphoma.

Most patients with B-cell lymphoma face an often incurable disease, particularly those diagnosed with an indolent subtype. The addition of passive immunotherapy to old and new chemotherapy regimens has improved both response rates and disease-free survival, leading in many cases to an extended overall survival. However, a cure remains elusive in most cases. For this reason, the patient- and tumor-specific idiotype, that is the collection of epitopes exclusively presented by the tumor clone's surface immunoglobulin, has been extensively studied as a privileged target for vaccine therapy, aiming at preventing disease re-occurrence after standard treatment. BiovaxID(®) (Biovest International, FL, USA), the most clinically advanced among such therapeutic vaccines, finds itself at a crucial turning point when it comes to further development. Both clinical trials in which it has been formally employed have shown intriguing results. Independent studies using slightly different versions of a conceptually identical vaccine provided all proofs of principle required to ascertain the vaccine's value - biological and clinical efficacy as well as clinical benefit. However, all these data have failed to bring an idiotype vaccine to the market owing to reasons that often have very little to do with the product itself. In fact, some successful studies were not conceived with this goal in mind, while others simply did not enroll enough patients to convincingly make their case for regulatory approval. It is likely that one or more new clinical trials will have to be successfully completed to reach the ultimate goal - that is, to make BiovaxID available to most patients and to adequately position it in the very crowded therapeutic algorithm of B-cell lymphoma.