Orchestrating a Symphony on a Single Conjugate: Aptamer Targeting, Gene Silencing, and Immunomodulation to Enhance Antitumor Response
Authors: Brandon D. Ng, Dan Peer
Journal: Molecular Therapy (2017)

Over the past decade, immunotherapy has rapidly changed the landscape of medicine, especially oncology. Checkpoint inhibitors and monoclonal antibodies capable of deploying the body’s own immune functions against diseased cells have been heralded as a soon-to-be standard of care for cancer. The excitement around immunotherapy has been accompanied by impressive results, although some are tailored only to a limited number of patients and others are accompanied by substantial toxicity profiles.1Morrissey K.M. Yuraszeck T.M. Li C.-C. Zhang Y. Kasichayanula S. Immunotherapy and novel combinations in oncology: Current landscape, challenges, and opportunities.Clin. Transl. Sci. 2016; 9: 89-104Crossref PubMed Scopus (125) Google Scholar Therefore, there are still unmet needs within the field. In this issue of Molceular Therapy, Rajagopalan et al.2Rajagopalan A. Berezhnoy A. Schrand B. Puplampu-Dove Y. Gilboa E. Aptamer-targeted attenuation of IL-2 signaling in CD8+ T cells enhances antitumor immunity.Mol. Ther. 2017; 25 (Published online January 4, 2017)https://doi.org/10.1016/j.ymthe.2016.10.021Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar performed a study aimed at using an aptamer-small interfering RNA (siRNA) conjugate to modulate the function and differentiation of activated CD8+ T cells. Their work combines gene silencing, aptamer targeting, and immunomodulation to produce a therapeutic effect. Strategies such as the one used in this study may pave the way for more selective and effective control of therapeutic immunomodulation. RNA interference (RNAi) technology has been steadily developing alongside immunotherapy but is not yet at the stage of market approval. This therapeutic strategy has great potential to target a variety of cellular functions with either short hairpin (shRNA) or siRNAs and can also produce immunomodulating effects by halting the expression of important signaling molecules. A major challenge of gene silencing is safe and efficient delivery of RNAi payloads to specific target cells. As a further obstacle, leukocytes are notoriously hard to transfect.3Hazan-Halevy I. Landesman-Milo D. Rosenblum D. Mizrahy S. Ng B.D. Peer D. Immunomodulation of hematological malignancies using oligonucleotides based-nanomedicines.J. Control. Release. 2016; (Published online August 2, 2016)https://doi.org/10.1016/j.jconrel.2016.07.052Crossref PubMed Scopus (16) Google Scholar, 4Ramishetti S. Kedmi R. Goldsmith M. Leonard F. Sprague A.G. Godin B. Gozin M. Cullis P.R. Dykxhoorn D.M. Peer D. Systemic gene silencing in primary T lymphocytes using targeted lipid Nanoparticles.ACS Nano. 2015; 9: 6706-6716Crossref PubMed Scopus (120) Google Scholar, 5Weinstein S. Toker I.A. Emmanuel R. Ramishetti S. Hazan-Halevy I. Rosenblum D. Goldsmith M. Abraham A. Benjamini O. Bairey O. et al.Harnessing RNAi-based nanomedicines for therapeutic gene silencing in B-cell malignancies.Proc. Natl. Acad. Sci. USA. 2016; 113: E16-E22Crossref PubMed Scopus (66) Google Scholar Recent advances in targeted delivery strategies for RNAi have mitigated some of the challenges, one example being the use of aptamers as targeting moieties. Oligonucleotide aptamers can be either RNA or DNA based. The secondary and tertiary structures of aptamers allow them to recognize specific target molecules. They have found application in molecular sensors, inhibitors or activators, and targeting moieties.6Lakhin A.V. Tarantul V.Z. Gening L.V. Aptamers: problems, solutions and prospects.Acta Naturae. 2013; 5: 34-43Crossref PubMed Scopus (294) Google Scholar Aptamers have become attractive targeting ligands due to their ease of production and low immunogenicity. The aptamer generation process comprises an extensive selection of oligonucleotide libraries for increasingly specific binding to the target molecule or cell. By conjugating a specific aptamer to siRNA, both targeting and therapeutic capabilities can be combined in a single small structure. CD8+ T cell differentiation is currently known to be mediated by the interaction of its surface CD25 receptors with interleukin 2 (IL-2). Binding of IL-2 results in differentiation into short-lived effector cells (SLECs), as opposed to memory precursor effector cells (MPECs). After eradicating antigen-presenting cells, SLECs undergo cell apoptosis, whereas MPECs can survive and recognize antigens again upon subsequent encounters. Rajagopalan et al.2Rajagopalan A. Berezhnoy A. Schrand B. Puplampu-Dove Y. Gilboa E. Aptamer-targeted attenuation of IL-2 signaling in CD8+ T cells enhances antitumor immunity.Mol. Ther. 2017; 25 (Published online January 4, 2017)https://doi.org/10.1016/j.ymthe.2016.10.021Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar designed a 4-1BB aptamer conjugated to an siRNA targeted to CD25 (siCD25) that binds to the 4-1BB receptor preferentially on CD8+ activated T cells to deliver siCD25. 4-1BB is a stimulatory receptor that is upregulated in activated T cells, promoting their survival and expansion. While activation of this 4-1BB receptor has a crucial role in influencing T cell function, the monovalent aptamer does not activate the 4-1BB signaling cascade. Previous work has indicated the requirement for 4-1BB to be cross-linked by a multivalent aptamer to induce receptor activation.7McNamara J.O. Kolonias D. Pastor F. Mittler R.S. Chen L. Giangrande P.H. Sullenger B. Gilboa E. Multivalent 4-1BB binding aptamers costimulate CD8+ T cells and inhibit tumor growth in mice.J. Clin. Invest. 2008; 118: 376-386Crossref PubMed Scopus (246) Google Scholar Hence, the monovalent nature of the 4-1BB aptamer selected here allows it to function solely as a siRNA targeting and delivery agent without target stimulation. The delivered siRNA leads to reduced CD25 expression, which impedes its interaction with IL-2, causing the CD8+ T cells to differentiate into MPECs instead of SLECs. From a therapeutic perspective, the study showed modest indications of breast cancer tumor infiltration by the T cells and a reasonable increase in tumor susceptibility to secondary treatment implemented in parallel with the conjugate. The work by Rajagopalan et al.2Rajagopalan A. Berezhnoy A. Schrand B. Puplampu-Dove Y. Gilboa E. Aptamer-targeted attenuation of IL-2 signaling in CD8+ T cells enhances antitumor immunity.Mol. Ther. 2017; 25 (Published online January 4, 2017)https://doi.org/10.1016/j.ymthe.2016.10.021Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar exemplifies a successful approach of using an aptamer-based targeting moiety to deliver siRNAs and silence a receptor to modulate immune function (Figure 1). Inducing cytotoxic T cell differentiation to MPECs yields a stronger prolonged immune response to antigen presenting cells. Additional investigation may demonstrate this avenue as a key advance in the immunotherapy field. The use of aptamers is an exciting approach for targeting of siRNAs and, with the appropriate oligonucleotide structure, the binding affinity can be much higher than that afforded by immunoglobulin G (IgG) antibodies.8Gilboa E. Berezhnoy A. Schrand B. Reducing toxicity of immune therapy using Aptamer-Targeted drug delivery.Cancer Immunol. Res. 2015; 3: 1195-1200Crossref PubMed Scopus (26) Google Scholar Unlike antibodies, the entire production process can be carried out using chemical synthesis.9Yan A.C. Levy M. Aptamers and aptamer targeted delivery.RNA Biol. 2009; 6: 316-320Crossref PubMed Scopus (66) Google Scholar Additionally, aptamer-siRNA conjugates may have an advantage over conventional siRNA introduction because of their processing by Dicer substrates.10McNamara 2nd, J.O. Andrechek E.R. Wang Y. Viles K.D. Rempel R.E. Gilboa E. Sullenger B.A. Giangrande P.H. Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras.Nat. Biotechnol. 2006; 24: 1005-1015Crossref PubMed Scopus (872) Google Scholar It has been demonstrated that shRNAs and RNA-aptamer conjugates must undergo cleavage within the cell by Dicer before they are suitable for gene silencing. This natural cleavage reaction within the cell allows for higher effectiveness in reaching and silencing the desired target gene. An important concern with aptamers is the specificity of oligonucleotide candidates for the intended target. Aptamer selection is not at the standard of the protein engineering approach used for antibody selection. Additionally, it is laborious and time consuming. While SELEX and other technologies have become much more efficient in recent years, they still fail at times to produce aptamer candidates suitable for their intended purpose.11Blind M. Blank M. Aptamer selection technology and recent advances.Mol. Ther. Nucleic Acids. 2015; 4: e223Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar This gap in selection quality may be closed with new advances in bioinformatics, next-generation sequencing, improved chemistry, and stricter separation methods.12Darmostuk M. Rimpelova S. Gbelcova H. Ruml T. Current approaches in SELEX: An update to aptamer selection technology.Biotechnol. Adv. 2015; 33: 1141-1161Crossref PubMed Scopus (408) Google Scholar Another challenge for aptamer development is to ensure retention of a stable and functional structure in vivo. RNA-based aptamers can exhibit greater structural diversity than their DNA-based counterparts, but they are exceptionally prone to degradation not only by natural alkaline hydrolysis, but also by nucleases.6Lakhin A.V. Tarantul V.Z. Gening L.V. Aptamers: problems, solutions and prospects.Acta Naturae. 2013; 5: 34-43Crossref PubMed Scopus (294) Google Scholar While both RNA- and DNA-based aptamers have a short circulation half-life, they can rapidly penetrate tissues to arrive at their target site because of their small size.13Jo H. Ban C. Aptamer-nanoparticle complexes as powerful diagnostic and therapeutic tools.Exp. Mol. Med. 2016; 48: e230Crossref PubMed Scopus (114) Google Scholar Nevertheless, it is unclear whether the delivery and clearance kinetics are favorable for controlling the release schedule of a therapeutic payload. Recent progress includes chemical modifications to the sugar moieties of the oligonucleotides and PEGylation to increase half-life in the body.14Bruno J.G. A review of therapeutic aptamer conjugates with emphasis on new approaches.Pharmaceuticals (Basel). 2013; 6: 340-357Crossref PubMed Scopus (88) Google Scholar, 15Dassie J.P. Liu X.Y. Thomas G.S. Whitaker R.M. Thiel K.W. Stockdale K.R. Meyerholz D.K. McCaffrey A.P. McNamara 2nd, J.O. Giangrande P.H. Systemic administration of optimized aptamer-siRNA chimeras promotes regression of PSMA-expressing tumors.Nat. Biotechnol. 2009; 27: 839-849Crossref PubMed Scopus (478) Google Scholar The extraordinary potential of RNAi has, in turn, sparked efforts to discover more effective methods for delivery of these therapeutic nucleic acids. Antibody protamine conjugates and polymers are among some of the approaches.16Song E. Zhu P. Lee S.-K. Chowdhury D. Kussman S. Dykxhoorn D.M. Feng Y. Palliser D. Weiner D.B. Shankar P. et al.Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors.Nat. Biotechnol. 2005; 23: 709-717Crossref PubMed Scopus (908) Google Scholar One exciting strategy involves encapsulating siRNAs into protective nanoparticles that can be surface functionalized for cell-specific targeting in vivo. Recent studies have shown effective gene silencing using siRNA-loaded LNPs: one specific for anti-CD4 to target T lymphocytes and, more recently, anti-CD38 for B cell malignancies.4Ramishetti S. Kedmi R. Goldsmith M. Leonard F. Sprague A.G. Godin B. Gozin M. Cullis P.R. Dykxhoorn D.M. Peer D. Systemic gene silencing in primary T lymphocytes using targeted lipid Nanoparticles.ACS Nano. 2015; 9: 6706-6716Crossref PubMed Scopus (120) Google Scholar, 5Weinstein S. Toker I.A. Emmanuel R. Ramishetti S. Hazan-Halevy I. Rosenblum D. Goldsmith M. Abraham A. Benjamini O. Bairey O. et al.Harnessing RNAi-based nanomedicines for therapeutic gene silencing in B-cell malignancies.Proc. Natl. Acad. Sci. USA. 2016; 113: E16-E22Crossref PubMed Scopus (66) Google Scholar Combinations of nanoparticle-based therapies with aptamer targeting are also being explored.13Jo H. Ban C. Aptamer-nanoparticle complexes as powerful diagnostic and therapeutic tools.Exp. Mol. Med. 2016; 48: e230Crossref PubMed Scopus (114) Google Scholar Rajagopalan and colleagues2Rajagopalan A. Berezhnoy A. Schrand B. Puplampu-Dove Y. Gilboa E. Aptamer-targeted attenuation of IL-2 signaling in CD8+ T cells enhances antitumor immunity.Mol. Ther. 2017; 25 (Published online January 4, 2017)https://doi.org/10.1016/j.ymthe.2016.10.021Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar execute a successful attempt in developing a target-specific aptamer without co-stimulatory effects, silencing of the desired target, and modulation of CD8+ T cell differentiation. Their work highlights the importance of selecting and tuning an aptamer that functions precisely. A plethora of additional studies are being done in the field to improve the technologies surrounding aptamer selection, specificity, and stability. This strategy may open new avenues in selective immunomodulation, which results in potent antitumor responses and might ultimately become a novel therapeutic modality in treating cancers. Aptamer-Targeted Attenuation of IL-2 Signaling in CD8+ T Cells Enhances Antitumor ImmunityRajagopalan et al.Molecular TherapyDecember 8, 2016In BriefRajagopalan et al. show that aptamer-targeted downregulation of IL-2 receptor or Axin-1 in 4-1BB-expressing cells promotes the differentiation of antigen-activated 4-1BB-expressing CD8+ T cells into long lasting memory cells and inhibits tumor growth in mice. Full-Text PDF Open Archive