cancer is a malignant disease caused by an abnormal noncontrolled cellular growth which acts autonomously and is capable of invading either local or distant tissues . these features include sustained proliferative signaling , evading tumor suppressors , resisting cell death , enabling replicative immortality , inducing angiogenesis , and activating invasion and metastasis [ 1 , 2 ] . in fact , more than one and a half million new cancer cases were estimated only in the us in 2016 and almost six hundred thousand of these were estimated to have died of cancer . moreover , the cancer burden around the world is alarmingly growing , so that about twenty - one million of new cancer cases and thirteen million of cancer deaths are expected to occur from now until 2030 . the first - line treatments nowadays against cancer are chemotherapy , radiotherapy , and surgery when tumor is resettable . current conventional cancer treatments are usually not enough in advanced aggressive tumors , as it is reflected in the high number of relapsing rates and the high toxicity associated with current treatments due to their lack of specificity . surgery has the intrinsic technical problem of not removing every single malignant cell , which causes tumor relapses in the majority of the cases , and chemotherapy and radiotherapy are very harmful and usually show serious side effects . due to this limited success of chemotherapy and radiotherapy , new alternative and more specific treatments there is an urge to identify additional treatments to improve patient 's survival and reduce toxicity . immunotherapy has emerged as the most feasible alternative thanks to its high tumor specificity in comparison with chemotherapy . besides the recent encouraging results obtained in clinical trials during the last years , followed by the fda approval of several immune - checkpoint - blockade monoclonal antibodies ( mabs ) [ 611 ] , have pushed cancer immunotherapy back to prominence . the use of these monoclonal antibodies , either as monotherapy or in combination , has achieved very significant results in different kinds of cancer . nevertheless , the use of immunomodulatory monoclonal antibodies has been associated with severe toxicities [ 1316 ] . the 4 - 1bb ab therapy has been correlated with hepatic toxicity and tgn1412 ( cd28 superagonistic monoclonal antibody ) caused a lethal cytokine storm . targeting specific immunomodulatory ligands at the tumor site would increase the antitumor effect of the given therapy and would decrease the severe toxicities associated with dose - limiting and off - target side effects in patients , thereby widening the therapeutic index . thus , in order to fine - tune the antitumor treatments , it is of great importance to find a clinically feasible tool to that effect . several approaches with different protein - derived products , such as engineered antibodies , diabodies , and chimeric receptors , have been described to this end [ 1721 ] . within the last few years , aptamers have appeared to be a promising platform for targeted cancer therapy . aptamers are single - stranded dna or rna oligonucleotides ( odns ) with a tridimensional folding that confers on them a high specificity and affinity for their targets . the selex procedure was developed by tuerk and ellington [ 23 , 24 ] in the early 90s and consists in rounds of iterative selection [ 23 , 24 ] . as can be seen in figure 1 , each round comprises basically three steps : binding , partition , and amplification . in the first step ( binding ) this library consists of a random sequence which can vary between 20 and 100 nucleotides ( nt ) flanked by two constant regions at 5 and 3 end . for the partition step , the bound sequences are removed and target - bound sequences are separated from the target . finally , the target - binding species are amplified by polymerase chain reaction ( pcr ) using primers that anneal at the two constant regions . if the desired aptamer is an rna aptamer , in vitro transcription shall be performed before starting the next round . the selection procedure may usually vary between 9 and 12 rounds of selection , which implies months of work . nowadays , techniques such as high - throughput sequencing which allow us to detect already sequence - enriched families favor the selection procedure drastically reducing the number of rounds [ 25 , 26 ] . the selex procedure has evolved since the first selex protocol new variation of the selection procedures emerged . some of the now used types of selection procedures besides conventional selex are ce - selex , which come from capillary electrophoretic selex ; cell - selex , in which selection is carried out with cells ; or toggle - selex , which on the other hand is used to obtain cross - reactive aptamers [ 26 , 29 ] . a new class of aptamers has been recently described , which are  mirror - image  l - conformed enantiomer aptamers and are called spiegelmers . a novel variation of conventional selex known as tailored - selex   is used to identify ten fixed nucleotide aptamers and no primer binding sites . this approach was validated by identifying a spiegelmer against the migraine - associated target calcitonin gene - related peptide 1 ( alpha - cgrp ) . moreover , the selection procedure which performs the rounds in animals is called in vivo selex , and genomic selex is otherwise used to achieve genomic - encoded functional domains , known as genomic aptamers capable of binding to a specific target . this great variety in aptamer selection procedures allows the technique to effectively isolate aptamers against almost every target and of almost any nature . aptamers can indeed be generated against most targets , including proteins and even small molecules [ 22 , 34 ] . the selex ( systematic evolution of ligands by exponential enrichment ) procedure is an iterative selection of single - stranded oligonucleotides that bind to specific targets . this technique consists of selection rounds which in turn are divided into three steps : binding , partition , and amplification . the first round starts with a randomized library which is incubated with the target during the binding step . afterwards , nonbinding species are discarded and the resulting binding species are separated from the target during the partition step . finally , the binder sequences are amplified by pcr and prepared for the next round . it is noteworthy that if the aptamer of interest is an rna aptamer , in vitro transcription shall be performed before starting the new round . the affinity and specificity are in the majority of cases comparable or even superior to those of antibodies . a dna aptamer that recognizes its target with a dissociation constant ( kd ) of 0.2  nm has been recently described . as mentioned before , aptamers possess several advantages when compared with cell - based products such as antibodies or recombinant proteins . in contrast to what might occur with antibodies , the toxicity or low immunogenicity of specific antigens does not interfere in the aptamer selection process . regarding translation into the clinic , aptamers provide the advantage over antibodies of presenting inherently an antidote [ 51 , 52 ] . the presence of a universal antidote ensures the reversion of its activity if any undesirable effect would arise . short odns show lack or much reduced immunogenicity when compared to antibodies due to the absence of t cell - dependent immunity . moreover , aptamers can be easily modified by chemical processes so as to optimize yield and provide custom - tailored properties . plasma stability can also be significantly enhanced by selective o - methyl or f substitutions for oh residues at the 2 position of the bases [ 22 , 53 ] . in order to widen the sort of aptamer interactions with their targets that can be achieved by including other nonnatural analogous bases [ 22 , 53 ] , there is the addition of aromatic hydrophobic modified nucleotides such as benzyl - du ( bn - du ) and naphthyl - du ( nap - du ) as in the case of somamers . somamers ( slow - off rate modified aptamers ) show protein - like modified side chains . these modifications confer on them the advantage over regular aptamers of exposing fewer hydrogen bonds , fewer charge - charge interactions , and fewer total polar contacts as determined by the summatory of hydrogen bonds and charge - charge interactions . to increase the effective molecular size , conjugation of aptamers to cholesterol led to a significant enhancement of the in vivo half - life of the odns in mice accompanied by a dramatic improvement of biological activity [ 5557 ] . another alternative , clinically compatible carrier is polyethylene glycol ( peg ) , which prevents its exclusion by renal filtration . such postselection modification increases aptamer survival to the maximum rates of sixfold as it has been recently attributed to a pegylated anti - muc1 aptamer - doxorubicin conjugate . thanks to their small size aptamers can be engineered to be multimerized to modulate the immune system [ 35 , 42 , 45 , 46 , 50 ] or to carry very different cargoes such as drugs , radioisotopes , proteins , enzymes , rnas , or even nanostructures [ 6062 ] . they can be chemically synthesized and therefore easy to export to good manufacturing practices ( gmp ) grade . gmp are the summation of practices required to manufacture and sell active pharmaceutical products in this specific case . these guidelines provide the minimum requirements that a pharmaceutical manufacturer must meet to assure that the products are of high quality and do not pose any risk to the consumer or the public . they are cell - based products and that toughens the regulatory approval process and increases the complexity and cost of manufacturing . since the first aptamer isolated in the early 90s , a tremendous amount of new selected aptamers has been published [ 22 , 63 , 64 ] . the anti - pdgf ( platelet - derived growth factor ) and the anti - c5 ( complement component 5 ) rna aptamers are used to treat macular edema and age - related macular degeneration [ 65 , 66 ] . the antifactor ixa of coagulation and the anti - a1 domain of activated von willebrand factor ( vwf ) , among other rna aptamers , are aimed at controlling hemostasis [ 6668 ] . for the treatment of diabetes mellitus , one spiegelmer aptamer that targets the monocyte chemoattractant protein 1 ( mcp-1 also called ccl2 ) the antinucleolin aptamer as1411 and the antistroma cell - derived factor-1 ( sdf-1 also called cxcl12 ) nox - a12 are among the most advanced aptamers for cancer treatment . the first in class was the anti - vegf rna aptamer approved in 2004 by the fda , called macugen , and used for the treatment of age - related macular degeneration . despite the fact that plenty of aptamers have been described in every research field and some of them are used in cancer therapy , in this review we focus on those aptamers used in cancer immunotherapy . as was mentioned above , there is an urge to develop new therapeutic approaches that synergize with conventional chemotherapy and prolong the antitumor effect . we can also envision that the field of cancer immunotherapy is leading towards a multipronged approach to tackle the tumor at different fronts . cancer immunotherapy has come to prominence upon the success of recent clinical trials with immune - checkpoint blockade . lack of costimulation and antigenicity as well as presence of immune - suppressor factors within the tumor microenvironment favors immune escape . thus , major challenges in cancer immunotherapy are ( i ) to activate the immune system to attack the tumor triggering costimulatory signals within the tumor , ( ii ) to counteract negative signals to favor the action of the immune system in the tumor microenvironment , and ( iii ) to enhance tumor antigenicity by the expression of potent tumor neoantigens . following this line , cancer can be attacked from different flanks by pushing the accelerating pedals and releasing the brakes at the same time . moreover , lack of tumor antigenicity is a major bottleneck for the success of immune - checkpoint - blockade antibodies , as it has been recently published [ 6971 ] . a feasible approach to induce the expression of new tumor antigens has been described by pastor et al .   by controlling the nmd inhibition in the tumor which allows for the expression of mutated tumor antigens with premature stop codons that are usually blocked by this mechanism . to date , numerous aptamer constructs have been described that are able to modulate the immune response against cancer . they provide a similar or even superior activity to that of the corresponding monoclonal ab , and their superior targeted delivery capacity confers on them less off - target side effects . thanks to their plasticity , aptamers are a very promising tool as immune - modulatory ligands , since they can be engineered to either activate or block an immune - modulatory receptor [ 45 , 46 , 50 ] . they can be customized to target this immunomodulation to the tumor site and can be engineered for delivering almost any kind of cargo as well . in 2003 the development of first immune - checkpoint - blockade rna aptamer that binds ctla-4 was published by gilboa 's group . the selection of these anti - ctla-4 aptamers was the first being used with immunotherapeutic intentions and opened the door to a new platform in cancer immunotherapy . the aptamer was multivalently engineered ( schematically represented in figure 2(c ) ) and these aptamers showed inhibition of ctla-4 function in vitro and enhancement of tumor immunity in mice . moreover , this assembling of the aptamers into tetrameric forms significantly enhanced their bioactivity in vitro  and  in vivo . indeed , a recently published work demonstrates that ctla-4 delivery strategies are able to target cd8 infiltrated lymphocytes and regulatory t lymphocytes ( tregs ) . this ctla-4 aptamer - based targeting delivery of stat-3 sirna to t lymphocytes resulted in inhibition of tumor growth and of metastasis . stat-3 promotes tumor - cell survival and proliferation in tumor cells , as well as invasion and immunosuppression . it has been widely demonstrated how stat-3 inhibition can be targeted by tlr9 natural ligands such as cpg . this targeted inhibition leads to activation of tumor - associated immune cells and strong antitumor immune responses [ 75 , 76 ] . moreover , stat-3 is persistent in immunosuppressive cells and contributes to the expansion of cd4 tregs . in the work presented by herrmann et al . they show an increase of cd8 t - effector response in vivo due in the first place to the blockade of ctla-4 and subsequently to the stat-3 silencing . silencing stat-3 provided a systemic antitumor response downregulating cd4 tregs which was reflected in inhibition of tumor growth in various cancer cell lines and metastasis too . pd1 is expressed in several cell types including t cells , specifically in cd8 tumor - infiltrating lymphocytes ( tils ) which are in charge of directly eradicating tumor cells . the engagement with pd1 expressed on the surface of lymphocytes within the tumor microenvironment and pdl-1 expressed on tumor cells leads to lymphocyte dysfunction by t cell exhaustion and tumor progression . it has recently been published that a dna aptamer , represented in figure 2(d ) , aimed at blocking the pd1 receptor decreases tumor growth and increases survival in mice tumor models . another exhaustion - associated t cell receptor is tim-3 , which is coexpressed with pd1 by exhausted and dysfunctional t cells . tim-3 has been also identified in a subpopulation of regulatory t cells with a potent immune - suppressive activity which correlates with a bad prognosis in cancer patients . recently our team has published a tim-3 rna antagonistic aptamer ( shown in figure 2(e ) ) able in a therapeutic setting to synergize with suboptimal doses of pdl-1 blockade . this strategy led to an important antitumor activity in vivo at very low doses of both tim-3 aptamer in its monomeric form and pdl-1 blocking antibody . furthermore , several aptamers have been described in an immunotherapy context towards some cytokine blockade . an aptamer known as r5a1 that binds to il-10r has been selected and optimized to block the interaction between il-10 and its receptor on the surface of immune - system cells . il-10 is known to be secreted by tumor cells and promote immune - modulatory responses that favor tumor establishment and growth . the aptamer bound to il-10 receptor on the cell surface and blocked il-10 function in vitro . moreover , the aptamer sequence and therefore the structure were optimized by truncation , discarding putative steric domains increasing aptamer affinity . in addition , systemic administration of the aptamer was capable of inhibiting tumor growth in mice to a comparable level of that of an anti - il-10 receptor monoclonal antibody . the tetrameric form of r5a1 aptamer blocked the il-10 signaling in vitro and the systemic administration of the truncated 48-nucleotide ( schematic representation in figure 2(k ) ) long in vivo blocked il-10 , an action that resulted in inhibition of tumor growth . moreover , an antagonist aptamer against both human and murine il - ra has been recently published by using high - throughput sequencing ( hts ) . this work describes a parallel murine and human specific target selection ( il-10ra and 4 - 1bb ) followed by identification of common sequences . selex allows for the selection of cross - reactive species in a very feasible manner . these aptamers and the majority of them used to date in cancer immunotherapy are summarized in table 1 . il-6 is an immune - suppressive cytokine produced by b cells , t cells , monocytes , and fibroblasts , among other cell types . il-6 secretion by immune cells within the tumor microenvironment leads to accumulation of regulatory cells . thus , the selection of aptamers that block the il-6 action has been of great interest . both selected somamers prevent il-6 signaling by blocking the interaction of il-6 with its receptor ( schematically represented in figure 2(i ) ) and inhibit the in vitro proliferation of tumor cells at levels of efficacy comparable to those of the anti - il-6 mab tocilizumab . another promising strategy is the specific targeting inhibition of interleukin-6 receptor ( il-6r ) and delivery through this molecule . a specific il-6r rna aptamer has been described that recognizes its target with high affinity , as represented in figure 2(j ) . nevertheless , it was able to specifically internalize and deliver cargoes to il-6r expressing cells . a 2012 publication showed that a human and murine rna cross - reactive aptamer against the interleukin 4 receptor alpha was able to trigger apoptosis in myeloid - derived suppressor cells ( mdscs ) . this approach targeted mdscs and tumor - associated macrophages ( tams ) which displayed an increased number of tumor - infiltrating lymphocytes ( tils ) and a reduction in 4t1 mammary carcinoma murine tumor model . in the same year , orava et al .   described a human dna antagonistic aptamer against the tumor necrosis factor alpha ( tnf- ) capable of blocking its activity in vitro baff - r causes proliferation and cell survival upon ligand engagement favoring tumor growth , which enables tumor cells to grow faster than nonmalignant b cells . aptamers that bind baff - r have been selected and showed to inhibit baff - mediated proliferation and survival of malignant b cells . antagonistic aptamers ( as represented in figure 2(h ) ) for baff - r have been selected and demonstrated to inhibit malignant b - cell proliferation . following this line we have recently published a murine aptamer against cd40 as a monomer that acts as a cd40 antagonist , thus blocking the downstream signaling ( figure 2(f ) ) . the cd40 aptamer blockade which is expressed in several b - cell malignancies reduces tumor growth and augments mice survival by 30% . moreover , we have described a cd28 antagonist aptamer ( represented in figure 2(l ) ) . this aptamer is capable of competing for cd28 receptor with its natural ligand b7 , as was shown in vitro in proliferation assays . in these experiments the antagonistic aptamer was able to revert in vitro the costimulation in cd4 t cells induced by b7 ligand . this approach of blocking cd28 would be of great interest in autoimmune diseases or transplants . during transplants acute host versus donor immune responses are developed upon engraftment and immune - suppressor drugs need to be administered to the patient in order to suppress these acute responses . administration of this cd28 antagonist aptamer would suppress immune responses driven by activated t lymphocytes facilitating donor engraftment . two spiegelmers against ccl2 ( nox - e36 ) and cxcl12 ( nox - a12 ) have undergone regulatory safety studies demonstrating good safety profiles in healthy volunteers and today are under phase iia studies in patients . moreover , a human and murine cross - reactive spiegelmer named nox - d20 has been described that specifically antagonizes the complement component c5a . this l - conformed aptamer prevents organ failure and improves survival in a model of sepsis as well as suppressing local and systemic inflammation . activating the positive signals , which corresponds to pushing the gas pedal , has been and remains one key strategy in cancer immunotherapy . apart from the activating signal which comes from the coengagement of mhc - i , tcr , and cd3 , a second signal known as second costimulatory signal is required for the proper t cell activation . this lack of costimulation leads cd8 t cells to become anergic and therefore unable to trigger an immune response . several selected aptamers to major costimulatory receptors ( 4 - 1bb , ox-40 , or cd28 ) have been selected and engineered to costimulate t cells . costimulation of t lymphocytes with odn aptamers has been already well documented to date [ 42 , 45 , 79 , 80 ] . nevertheless , it was not until 2008 that the first aptamer directed to a costimulatory receptor was selected , optimized , and engineered , as represented in figure 2(b ) for costimulation . 4 - 1bb is expressed on t lymphocytes and is one of the major costimulatory receptors that promotes survival and expansion of activated t cells . its ligand 4 - 1bbl is expressed on professional antigen - presenting cells ( apcs ) such as dendritic cells ( dcs ) . that work , published by gilboa 's group , strengthened the idea of using aptamers in cancer immunotherapy . moreover , beyond this murine aptamer , as mentioned above , a murine and human cross - reactive rna aptamer has been described . the engagement with its natural ligand ox-40-l expressed on apcs leads to t cell proliferation , increased cytokine release , and t - lymphocyte survival . ox-40 aptamers with immunotherapeutic intention were published in the year 2008 reinforcing this new platform in cancer immunotherapy . this aptamer was assembled in a two - copy scaffold to yield the costimulatory effect . these two artificial costimulatory aptamers ( 4 - 1bb and ox-40 ) were engineered by two different ways . 4 - 1bb dimers were obtained by adding short complementary sequences at 3 ends which will anneal by pairwise fashion ( schematic representation in figures 2(a ) and 2(b ) ) . on the other hand , the ox-40 dimer was generated by adding an 18 carbon - length polyethylene spacer between the two 3 aptamer - end complementary sequences that will anneal by pairwise fashion [ 42 , 44 ] . in addition , an agonistic aptamer towards human ox-40 has been selected and shown to be dimerized to exert its costimulatory effect , which was mirrored in cellular proliferation and increased inf - gamma production . t cells need at least two signals to be properly activated , as mentioned above , and the most important costimulatory signal comes from the engagement of cd28 and its natural ligand b7 ( cd80 and cd86 ) . various aptamers for targeting and costimulating cd28 have been engineered and they have shown a more potent costimulatory effect than that of the mab 37.51 . in this work published by our group , dimerization was achieved as mentioned above by adding short sequences to the 3 end that will anneal by pairwise fashion , displaying more flexibility and mirroring the average distance between the 2 fv of an igg and guaranteeing a more rigid structure . nevertheless , the highest effect was achieved when generating the dimer by pcr , resulting in a shorter linker , which reduces the distance to the minimum and provides flexibility probably responsible for its augmented costimulatory capacity . any of the dimeric constructs comprised the affinity for its target indicating that the length and flexibility of the linker could be modified to enhance the costimulatory effect . finally , and not less importantly , this work described for the first time a single molecule with the dual role of acting as antagonist in its monomeric form and as agonist by simple dimerization . the main constructs of cd28 agonistic aptamer among others dedicated to costimulating t lymphocytes are schematically represented in figure 2(l ) . furthermore , an idiotype vaccination context triggered a cellular and humoral antitumor immune response more potent than that of the anti - cd28 mab . this humoral and cellular response was reflected in both tumor growth and survival . as has been recently published by our group , two agonistic cd40 aptamer - based constructs are able to recover bone - marrow aplasia while increasing antigen - presenting cells ( apcs ) activation . one of the aptamers described in this work acts as an antagonist , as mentioned above , and again by engineered dimerization we were able to turn it into an agonist . we describe two different cd40 agonistic aptamer constructs able to activate apcs displaying an increased proliferation and expression of costimulatory ligands . recently a dec205 rna murine aptamer able to induce specific antigen cross - presentation has been described ( represented in figure 2(g ) ) . this aptamer - based approach is shown to be efficient for both in vitro and in vivo delivery of specific cargoes for cross - priming . this work demonstrated the potential of this strategy by strongly enhancing t cell - mediated antitumor immunity . finally , a dna aptamer against the fc receptor iii ( cd16 ) was developed to generate a bispecific aptamer to target the antibody - dependent cell - mediated cytotoxicity ( adcc ) to c - met overexpressing tumor cells , as represented in figure 3(c ) . this bispecific aptamer was demonstrated to elicit specific adcc in both human gastric and lung cancer cell lines . the use of agonistic antibodies often leads to off - target toxicity , as happened with some agonistic antibodies described to date with 4 - 1bb or cd28 . targeting costimulation to the tumor site would decrease these side effects , increasing the therapeutic index . immune - checkpoint mabs - based therapy has shown several adverse side effects , such as severe hepatotoxicity , lymphopenia , and thrombocytopenia . the administration of agonistic cd28 antibodies such as tgn1412 displayed multiorgan failure   and made the end of the clinical trial necessary . liver toxicity is one of the major concerns in 4 - 1bb mediated treatment of cancer . in order to increase the therapeutic index , the first bispecific aptamer was generated . bispecific aptamers consisting of both psma-4 - 1bb aptamers have been engineered ( schematically represented in figure 3(d ) ) and showed potent antitumor effects even in low doses when compared with nontargeted costimulation and the correspondent mabs controls . lower levels of psma-4 - 1bb bispecific costimulation were nearly as effective as tenfold increased levels of nontargeted costimulation or the corresponding monoclonal antibody . therefore , as demonstrated by pastor et al . , targeting 4 - 1bb costimulation to the tumor site requires less amount of reagent to achieve a therapeutic effect , exhibiting a superior safety profile . moreover , a work has been recently published in which 4 - 1bb costimulatory aptamer is directed to tumor stroma through the vegf aptamer ( represented in figure 3(b ) ) . this bispecific aptamer has again shown a higher therapeutic index in comparison with the nontargeted costimulation reagents , reaching the same effect with less toxicity . this increase in therapeutic index reduced cd8 t cell hyperplasia as well as spleen , lymph node , lung , and liver weights while displaying a similar antitumor effect . other aptamers such as ox-40 and cd28 have also been shown to be equal to or more potent than their respective mabs [ 35 , 42 , 43 , 45 ] . thus , targeting costimulation to the tumor site seems to be a feasible strategy in antitumor treatments . furthermore , targeting costimulation towards markers implicated in tumor - chemotherapy resistance would act exerting a selection pressure against these types of tumor cells . it is known that cancer - stem cells are responsible for tumor metastasis , chemotherapy resistance , and tumor relapses [ 83 , 84 ] . we have selected a mrp1 aptamer by using a novel combinatory peptide - cell ht selex method . we used this aptamer to isolate a subpopulation of chemotherapy - resistant mrp1-expressing melanoma cell line . we then used this aptamer to generate a new cd28-mrp1 bispecific aptamer , as represented in figure 3(a ) . this bispecific cd28-mrp1 aptamer was able to target in vitro and in vivo costimulation to mrp1-expressing cells and capable of providing a proper costimulatory signal to t cells . in combination with gvax and the foxp3 temporary inhibitor peptide p60 the bispecific aptamer induced higher t cell tumor infiltration , slower tumor growth , and longer survival . we also developed a new strategy to coat ex vivo mrp1-expressing tumor cells to create a new vaccination approach named cd28aptvax . this vaccine consists in irradiated b16-mrp1 cells coated with the bispecific aptamer and its administration in mice significantly delayed mrp1-expressing tumor growth and maintained around 50% survival after 50-day follow - up . the summary of bispecific aptamers is represented in figure 3 . despite the efforts invested in potentiating tumor immunity by activating costimulatory receptors with agonists or inhibiting undesired immunosuppressive responses , absence of antigenicity the enhancement of tumor immunogenicity is one of the most difficult challenges to face in cancer immunotherapy . one of the strategies described to date is to increase the tumor antigenicity by expressing new antigens . the nonsense mediated mrna decay ( nmd ) is in charge of deleting mrnas that encode premature termination codons ( ptcs ) . moreover , the correlation between nmd inhibition and lymphocyte infiltration has been demonstrated . in that work published by el - bchiri et al . , higher accumulation of cd3 cells was inversely correlated with nmd function in colorectal cancer with microsatellite instability ( msi ) . thus , targeting the sirna - mediated inhibition of nmd in tumor cells as was already published indeed induced an antitumor immune - system mediated response . a chimera consisting in psma aptamer coupled with nmd factor - sirnas resulted in an increase of tumor antigenicity in vivo reducing tumor growth . given the fact that activating cd40 signaling would promote cd40-expressing tumor progression , we managed to generate a cd40 agonistic aptamer conjugated with a shrna aimed at inhibiting nmd . as mentioned above [ 72 , 85 , 86 ] , nmd inhibition leads to the expression of new and therefore more potent antigens that renders tumor regression . this optimized chimera was capable of enhancing tumor antigenicity leading to increased lymphocyte infiltration , lowering tumor growth in a b - cell lymphoma tumor model , and increasing mice survival . this treatment significantly reduced tumor growth in a b - cell lymphoma model increasing mice survival . more recently a remarkable study from gilboa 's group   has been published in which the bivalent murine 4 - 1bb aptamer was conjugated with a sirna for raptor , a key factor of the mtorc1 ( mtor complex 1 ) . mtor is an intracellular mediator associated with accumulation of immune - system short - living effector cells . finally , this construct in combination with already established vaccination protocols promoted a potent memory response with cytotoxic effector functions and protective immunity in tumor - bearing mice . although the expression of new and therefore more potent antigens displays an antitumor immune response , in some cases it is not sufficient ( in the majority of tumors due to its immunosuppressive microenvironment ) . in fact , the expression of new antigens induces a higher treg infiltration , indicating that the combination with other antagonist aptamers towards receptors such as il-10r , ctla-4 , pd1 , or tim-3 would be of great interest to optimize the antitumor immune responses . moreover , we have recently published a new work that proves that the target inhibition of foxp3 in tregs can be achieved through cd28 aptamer - foxp3-peptide blocking chimera . as previously described by casares et al . , p60 is a foxp3 inhibitor peptide able to penetrate the membrane of tregs and thereby to inhibit foxp3 . due to lack of specificity , the required doses are very high ; thus , we managed to conjugate this peptide with one of our cd28 described aptamers to deliver p60 to cd28-expressing cells . the cd28-targeted p60-mediated foxp3 inhibition was able to counteract treg immunosuppression by reducing the concentration to 0.5  m . this significant reduction means that concentration can be reduced up to hundreds of times to obtain the same effect . the treatment of ct26 colon carcinoma - bearing mice with the immunodominant ct26 peptide ah1 and incomplete freund 's adjuvant in the presence of 625  pmol of the cd28 aptamer - p60 chimera controlled tumor growth at a similar rate compared to that of 500  nmol of the respective p60 control . aptamers have emerged as a great new therapeutic class of reagents very suitable for cancer immunotherapy . several aptamers have been described to date and some of them have reached clinical grade , but we are still scratching the surface of the potential of this novel therapeutic platform . moreover , this new class of therapeutics has been described as a tool to deliver sirnas or aptamers to the tumor to modulate the immune response . aptamer platforms can be used to tackle the three major challenges in cancer immunotherapy : blockade of immunosuppressive mechanisms , activation of the agonistic immune receptors , and increase of the tumor immunogenicity .
aptamers are single - chained rna or dna oligonucleotides ( odns ) with three - dimensional folding structures which allow them to bind to their targets with high specificity . 
 aptamers normally show affinities comparable to or higher than that of antibodies . they are chemically synthesized and therefore less expensive to manufacture and produce . 
 a variety of aptamers described to date have been shown to be reliable in modulating immune responses against cancer by either blocking or activating immune receptors . 
 some of them have been conjugated to other molecules to target the immune system and reduce off - target side effects . despite the success of first - line treatments against cancer , 
 the elevated number of relapsing cases and the tremendous side effects shown by the commonly used agents hinder conventional treatments against cancer . 
 the advantages provided by aptamers could enhance the therapeutic index of a given strategy and therefore enhance the antitumor effect . 
 here we recapitulate the provided benefits of aptamers with immunomodulatory activity described to date in cancer therapy and the benefits that aptamer - based immunotherapy could provide either alone or combined with first - line treatments in cancer therapy .